Thiadiazole: A new family of intercalative photonuclease with electron transfer and radical mechanisms

Article abstract of DOI:10.1016/S0960-894X(03)00737-6

A new family of photonuclease, thiadiazole-naphthalimide were synthesized and evaluated. Thiadiazole group was incorporated for the first time. These compounds intercalated into DNA efficiently and damaged DNA as low as 10 μM photochemically. Mechanism experiment showed that electron transfer and radicals were involved.

Full text of DOI:10.1016/S0960-894X(03)00737-6

Bioorganic & MedicinalChemistry Letters 13 (2003) 3513–3515
Thiadiazole: A New Family of Intercalative Photonuclease with
Electron Transfer and Radical Mechanisms
Yonggang Li,a,c Yufang Xu,a,c Xuhong Qianb,* and Baoyuan Qua,c
aEast China University of Science and Technology, Shanghai 200237, China
bState Key Lab. Fine Chemicals, Dalian University of Technology, Dalian 116012, China
cShanghai Key Lab. Chem. Biology, Shanghai 200237, China
Received 1 May 2003; accepted 10 July 2003
Abstract—A new family of photonuclease, thiadiazole-naphthalimide were synthesized and evaluated. Thiadiazole group was
incorporated for the first time. These compounds intercalated into DNA efficiently and damaged DNA as low as 10 mM photo-
chemically. Mechanism experiment showed that electron transfer and radicals were involved.
# 2003 Elsevier Ltd. All rights reserved.
Photonucleases are of great interest in biology, chem-
istry and medicine. Triggered by the ultra-violet or visi-
ble light, they can initiate significant damage on DNA
without externalchemicalinitiators via a wide variety of
mechanism such as radicals, electron transfer or singlet
oxygen.1,2 It is an effective strategy to incorporate new
functionalgroups to design novelDNA photocelavers.
Our group have reported severalseries of photo-
nucleases containing hydroperoxide, N-aroyloxy or
thiono groups, which cleaved DNA via oxygen-centered
radicals or electron transfer.3ꢀ6 Herein, a new functional
group, thiadiazole, is incorporated into the area of
photodamage of DNA for the first time and a new
family of photonucleases based on this group are
designed and assessed.
been reported to possess the 1,2,3-thiadiazole ring as
functionalgroup. Thus, it was chosen as a photo-trig-
gered active part.
In our research, naphthalene ring was conjugated to the
thiadiazole moiety to shift the absorption batho-
chromically, which was safe to manipulate and quite
matched with the photoirradiation wavelength. More-
over, planar rigid structure would facilitate the drug to
intercalate into DNA efficiently as it was known that
enhanced DNA affinity was helpful for the photo-
damage of DNA.3 N,N-dimethylaminoethyl or similar
groups were also chosen for the enhanced DNA affinity
because these groups appeared commonly in clinically
usefulanti-cancer drugs that interacted with DNA such
as amonafide and mitonafide.17,18 Hence, a new family
of thiadiazole photonuclease A1, A2 and A3 (Fig. 1) was
designed.
Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl
ester (BTH), a plant activator, induced accumulation of
SAR (Systematic Acquired Resistance) gene and
expression of PR-1 (Pathogensis-Related) gene in
plant.7ꢀ10 It was possibly implied that thiadiazole com-
pounds had some potentialinteraction with nucleic acid.
Also, 1,2,3-thiadiazoles would produce several active
intermediates photochemically, such as thioketocarbene,
thiocarbondiradicaland thioketene ( Scheme 1).11ꢀ13 We
expected that these intermediates should be able to react
with DNA similarly based on the fact that triazole could
be used as a photonuclease.14ꢀ16 Few photocleaver had
These compounds were synthesized from 4-bromo-3-
nitronaphthalic anhydride shown in Scheme 2. After
separation with column chromatography their structures
1
were confirmed by IR, H NMR, HRMS and element
analysis.19
*Corresponding author. Tel.: +86-411-367-3466; fax: +86-411-367-
3488; e-mail: xhqian@dlut.edu.cn
Scheme 1. Photolysis of typical thiadiazole compound.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00737-6

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Y. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3513–3515
II. All of these compounds were found to cleave DNA
efficiently into form II as shown in Figure 3a and A2
acted more efficiently than the other two under this
condition. Further experiment showed that A2 could
damage the supercoiled DNA into the relaxed circular
form as low as 10 mM (Fig. 3b). Its DNA photodamage
ability was sensitive to pH value. With the elongation of
irradiation time, it photo-nicked DNA more intensely.
However, no DNA damage was observed in the absence
of light.
Figure 1. Novelphotonucelases A1–A3.
Mechanism experiment was performed with the addi-
tion of histidine (singlet oxygen quencher), dithio-
threitol(DTT, superoxide radicalscavenger),
superoxide dismutase (SOD, superoxide radical killer)
and DMSO (radicalquencher) ( Fig. 4). Obviously,
singlet oxygen was not involved in the DNA damage.
However, DMSO inhibited its reactivity a little and
DTT did greatly (Fig. 4, lanes 1, 2 and 7). Thus, it was
believed that electron transfer from nucleobase to com-
pound and then to oxygen to form superoxide played a
major role in the photodamage of DNA besides radicals
produced upon photoirradiation. As for the similar tri-
azole derivatives, Wender group proposed the electron
transfer mechanism.15,16 However, Armitage figured
that the spontaneous nature of the cleavage and a dis-
tinct preference for cleavage at the 30-G of the GG steps
by the triazole argued against cleavage by an electron
transfer pathway,1 and Manfredini reported the possible
radicalmechanism for the pyrazol-trizoesl DNA
Scheme 2. Synthesis of target compounds: (a) benzylmercaptan,
K2CO3, DMF, Ar, 8 h, 93% yield; (b) SnCl2, HCl, 2 h, 98% yield; (c)
NaNO2, HCl, 0 ꢁC, 3.5 h, 86% yield; (d) RNH2, ethanol, 2.5 h, yield:
A1: 65%; A2: 70%; A3: 58%.
It was found that the absorption wavelengths of com-
pounds A1–A3 were at 362 nm, which was quite matched
with the photoirradiation wavelength of transluminator
(366 nm). Also, their weak fluorescence (È<0.0003)
implied that their excitation energy was easily trans-
ferred from the excited singlet state to lead to the clea-
vage of the N–S bond,11,13 besides the dissipation in
internaltransfer (data not shown). Meanwhiel, with A2
as an example, a study of the intercalation of A2 to calf
thymus DNA was carried out using UV–vis spectra
technique instead of the fluorescence quenching
method,4,20 as A1–A3 had very weak fluorescence. By
titration a solution of A2 (in 30 mM Tris–HClbuffer,
pH 7.5) with an increased CT–DNA it appeared hypo-
chromic and bathochromic shift in the UV spectrum of
A2 (Fig. 2), in accordance with the intercalation
model.21
The cleavage activities of compounds A1ꢀ3 were eval-
uated using closed supercoiled pBR322 DNA under
photoirradiation with a transluminator (366 nm) at a
distance of 20 cm at 0 ꢁC for 2 h and analyzed on a 1%
agarose gel. The photocleavage efficiency was defined by
the degree of the relaxation of supercoiled DNA,
relaxed circular DNA (single-strand cleavage) as form
Figure 3. Photocleavage of closed supercoiled DNA by photo-
nucleases in 10% acetonitrile in Tris–HCl buffer. (a) photoirradiation:
2 h; lane1: DNA alone (no hn); lane 2: DNA alone; lanes 3–5: DNA
and compound A1–A3 at concentration of 100 mM. (b) lane 1: DNA
alone (no hn); lane 2: DNA alone (hn, 120 min); lanes 3–7: DNA and
A2 at concentration of 10, 20, 50, 100, 200 mM, respectively.
Figure 4. Effect of additives on the photocleavage of DNA in 10%
acetonitrile in Tris–HCl buffer. photoirradiation: 2 h; lane 3: DNA
alone (no hn); lane 4: DNA alone; lanes 1 and 5: DNA and compound
A2 at concentration of 50 mM; lane 6: DNA and A2 in the presence of
histidine (6 mM); lane 7: DNA and A2 in the presence of dithiothreitol
(DTT, 30 mM); lane 8: DNA and A2 in the presence of superoxide
dismutase (SOD, 100 mg/mL); lane 2: DNA and A2 with addition of
DMSO (1.4 M).
Figure 2. Interaction of A2 and calf thymus DNA. Absorption chan-
ges of A2 during addition of calf thymus DNA (0, 50, 100, 200 mM) in
DMSO–10 mM Tris–HCl(pH 7.5) soul tion (1:4, v/v).

Y. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3513–3515
3515
cleaving agents.14 Here, thiadiazole-naphthalimide pho-
tocleaved DNA via combined electron transfer and
radicalmechamism. (The tripelt state of naphthailmide
was also possibly involved in the photocleavage of
DNA.22,23) It should be pointed out that SOD did not
slow the rate of DNA-cleaving reaction because the
hydrogen peroxide produced by SOD from superoxide
could lead to DNA damage photochemically.
13. Krauss, P.; Zeller, K.-P.; Meier, H.; Muller, E. Tetra-
hedron 1971, 27, 5953.
14. Manfredini, S.; Vicentini, C. B.; Manfrini, M.; Bianchi,
N.; Rutigliano, C.; Mischiati, C.; Gambari, R. Bioorg. Med.
Chem. 2000, 8, 2343.
15. Wender, P. A.; Touami, S. M.; Alayrac, C.; Philipp, U. C.
J. Am. Chem. Soc. 1996, 118, 6522.
16. Touami, S. M.; Poon, C. C.; Wender, P. A. J. Am. Chem.
Soc. 1997, 119, 7611.
17. Malviya, V. K.; Liu, P. Y.; Alberts, D. S.; Surwit, E. A.;
Craig, J. B.; Hanningan, E. V. Am. J. Clin. Oncol. 1992, 15, 41.
18. Rosell, R.; Carles, J.; Abad, A.; Ribelles, N.; Barnadas,
A.; Benavides, A.; Martin, M. Invest. New Drugs 1992, 10,
171.
In summary, the present work demonstrated the design
and evaluation of novel intercalative photonucleases,
thiadiazole fused naphthalimides A1–A3. Thiadiazole is
incorporated as a photoactive moiety in the photo-
nuclease study for the first time. These compounds
could cleave circular supercoiled pBR322 efficiently
under the irradiation of long-wavelength UV light (366
nm) via electron transfer and radical mechanism. The
anti-tumor research of these compounds are in progress.
19. A1: mp 101–102 ꢁC. H NMR (CDCl3) d (ppm): 1.9 (m,
1
2H, NCH2), 2.22 (s, 6H, NCH3), 2.43 (t, J1=7.16 Hz, J2=7.27
Hz, 2H, CH2), 4.23 (t, J1=7.49 Hz, J2=7.66 Hz, 2H,
CONCH2), 7.91 (t, J1=7.74 Hz, J2=7.75 Hz, 1H, 2-H), 8.4 (t,
J1=7.86, J2=0.86, 1H, 1-H), 8.73 (t, J1=7.16, J2=0.81, 1H,
3-H), 9.58(s, 1H, 7-H). HRMS: C17H16N4O2S calculated:
340.0994; found: 340.0991. m/z (%): 340.0991 (M+) (10.41),
240.0145 (2.96), 210.0064 (10.85), 157.0130 (5.39), 84.0832
(71.88), 58.0655 (100). IR (KBr): 2960, 2870, 1710, 1670, 1300
cmꢀ1. element analysis: C17H16N4O2S calculated: C59.98,
H4.74, N16.46; found: C59.74, H4.48, N16.65. A2: mp 178–
179 ꢁC. 1H NMR (DMSO-d6) d (ppm): 2.25 (s, 6H, NCH3),
2.57 (t, J1=6.87 Hz, J2=6.97 Hz, 2H, NCH2), 4.19 (t,
J1=6.86 Hz, J2=6.99 Hz, 2H, CONCH2), 8.06 (t, J1=8.06
Hz, J2=7.72 Hz, 1H, 2-H), 8.64 (d, J=7.44 Hz, 1H, 1-H),
8.85(d, J=8.04 Hz, 1H, 3-H), 9.38 (d, J=1.52 Hz, 1H, 7-H).
HRMS: C16H14N4O2S calculated: 326.0837; found: 326.0788.
m/z (%): 326.0788 (M+) (25.29), 254.0261 (14.92), 209.9968
(39.54), 182.0024 (45.43), 155.9992 (42.73), 71.0683 (84.29),
Acknowledgements
NationalNaturalScience Foundation of China, The
Ministry of Education of China and Shanghai Founda-
tion of Science and Technology financially supported
this study.
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