Novel 2-aminothiazonaphthalimides as visible light activatable photonucleases: Effects of intercalation, heterocyclic-fused area and side chains

Article abstract of DOI:10.1016/j.bmcl.2005.02.053

A new family of 2-aminothiazonaphthalimides with different side chains as novel intercalative and visible light activatable photonucleases, was designed, synthesized and quantitatively evaluated. The order of their photocleaving abilities was parallel to that of their intercalative properties. The compound with linear heterocyclic-fused chromophore could intercalate into and photocleave DNA more efficiently than the one with angular heterocyclic-fused chromophore. B₂, the most efficient compound, caused obvious DNA damage at 1 μM. Mechanism experiment showed that superoxide anion was involved.

Full text of DOI:10.1016/j.bmcl.2005.02.053

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1769–1772
Novel 2-aminothiazonaphthalimides as visible light
activatable photonucleases: effects of intercalation,
heterocyclic-fused area and side chains
Zhigang Li,a Qing Yangb and Xuhong Qiana,c,
*
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, PO Box 158, Zhongshan Road, Dalian 116012, China
bDepartment of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116012, China
cShanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237, China
Received 24 December 2004; revised 15 February 2005; accepted 17 February 2005
Abstract—A new family of 2-aminothiazonaphthalimides with different side chains as novel intercalative and visible light activatable
photonucleases, was designed, synthesized and quantitatively evaluated. The order of their photocleaving abilities was parallel to
that of their intercalative properties. The compound with linear heterocyclic-fused chromophore could intercalate into and photo-
cleave DNA more efficiently than the one with angular heterocyclic-fused chromophore. B2, the most efficient compound, caused
obvious DNA damage at 1 lM. Mechanism experiment showed that superoxide anion was involved.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Photonucleases hold great promise in molecular biology
and medicinal chemistry. Some of them have been pro-
ven to be valuable tools in biotechnology including thera-
peutic agents,1 structural probes and photofootprinting
agents as well.2 Triggered by ultra-violet or visible light,
such agents can cause significant damage on DNA with
or without site selectivity.3
cleic acid to form a stable DNA–drug complex with the
aid of hydrogen bonds formed between the amino group
and the sugar–phosphate chain. Herein, a new family of
2-aminothiazonaphthalimides, A1–2 and B1–2 (Fig. 1),
was synthesized by fusing a 2-aminothiazole group to
different positions of the naphthalimide skeleton. Two
aminoalkyl side chains varying in length were incorpo-
rated into the parent structures not only to increase
the affinity interaction with DNA4,6 but also to elucidate
the influence of them on biological activities. Further-
more, the comparison of effects of structural features
of such agents on photocleaving activities could offer
novel insights for rational molecular design.
Many naphthalimide derivatives were well-known
photonucleases3 or anticancer drugs.4 Our previous
study has demonstrated that the presence of sulfur
rather than its oxo-counterpart promoted the DNA
intercalating and photocleaving ability.3f Based on this
knowledge, we expected fusing novel thio-heterocyclic
group into the naphthalimide skeleton would be an effi-
cient strategy.
R
N
R
N
O
O
O
O
2-Aminothiazole moiety, proven its value in medicinal
chemistry, has been successfully applied in dopamine
agonists, such as B-HT920, PD118440 and pramipexole,
the widely used anti-Parkinsonian agent.5 In these cases,
2-aminothiazole group was assumed to interact with nu-
S
N
NH2
N
S
A1-2
B1-2
NH2
A1, B1: R=CH2CH2N(CH3)2
A2, B2: R=CH2CH2CH2N(CH3)2
Keywords: Plasmid DNA; Photocleaver; Intercalation; Heterocycles.
*
Corresponding author. Tel.: +86 411 83673466; fax: +86 411
83673488; e-mail addresses: xhqian@dlut.edu.cn; xhqian@ecust.edu.cn
Figure 1. Structures of novel 2-aminothiazonaphthalimides.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.053

1770
Z. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1769–1772
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
N
c
b
d
a
+
B
A
NH2
+
N
NO2
NH2
S
NH2
Scheme 1. Synthesis of 2-aminothiazonaphthalimides. Reagents and conditions: (a) NaNO3, H2SO4, 0 ꢁC, 1 h, 85% yield; (b) SnCl2, concentrated
HCl, 80 ꢁC, 2 h, 80% yield; (c) KSCN, Br2, AcOH, 48 h, 95% yield; (d) corresponding amine, ethanol, reflux 2 h.
The synthesis of these compounds was outlined in
Scheme 1. Nitration of the starting material, 1,8-naph-
thalic anhydride with sodium nitrate in concentrated
H2SO4 at 0 ꢁC for 1 h, 3-nitro-1,8-naphthalic anhydride
was obtained and then reduced by Tin(II) chloride
dehydrate in concentrated hydrochloric acid to give
3-amino-1,8-naphthalic anhydride. Upon cyclization of
3-amino-1,8-naphthalic anhydride by a reported proce-
dure,7 in which 3-amino-1,8-naphthalic anhydride was
treated with potassium thiocyanate and bromine in ace-
tic acid, two 2-aminothiazonaphthalic anhydride iso-
Figure 2. Fluorescence spectra before and after interaction of com-
pound B2 and calf thymus DNA. Curves F and F-CT corresponded to
mers were obtained by filtration in high yield. Without
further purification, the obtained anhydride mixtures
were condensed with the corresponding amine in etha-
nol to form the corresponding naphthalimide mixtures,
compound B2 before and after being mixed with DNA. Numbers 1–4
indicated the concentration of B2, 5, 10, 20, 40 lM, respectively. DNA
applied was 50 lM (bp).
A1–2 and B1–2, with the ratio of 1:10. After separation
with careful column chromatography, each pure tar-
geted product was obtained. All of their structures were
X2 were stronger than those of X1 (X = A,B), suggesting
the importance of the length of aminoalkyl chain serving
as DNA groove binder and/or external electrostatic bin-
der. In our cases, the chain with three methylene units
between two nitrogen atoms significantly enhanced the
intercalating abilities of the aminothiazonaphthalimides.
That might be the reason accounting for the order of
intercalating ability: A2 > A1, B2 > B1.
1
confirmed by H NMR, HRMS and IR.8
The UV–vis and fluorescent data for these compounds
were measured and shown in Table 1. It was found that
they had slight differences in both absorption wave-
length (around 420 nm) and emission wavelength
(around 475 nm). The activating factor was visible light
(420 nm) rather than UV light, indicating the safe
manipulation towards them and thus in turn their preva-
lent advantage as biological tools.
The photocleavage activities of A1, A2, B1, B2, were evalu-
ated using closed supercoiled pBR322 DNA under
photo-irradiation with a transluminator (400 nm) at a
distance of 20 cm at 0 ꢁC for 3 h under aerobic condi-
tions and analyzed on a 1% agarose gel. The cleavage
efficiency was defined by the degree of conversion of
the supercoiled pBR322 DNA (form I) to relaxed circu-
lar DNA (form II). It was apparent that these com-
pounds photocleaved the supercoiled DNA with
different activities by the order: B2 > A2 > B1 > A1
(Fig. 3a), which was parallel to that of their intercalative
properties. Further the experiment showed that B2, the
most efficient compound, nicked supercoiled DNA
(form I, DNA in 200 lM base pair) to give a 29% re-
laxed circular form (form II) at a concentration as low
as 1 lM, while the concentration for giving 100% form
II (Fig. 3b) was 100 lM under identical conditions. Also
the cleaving activity of B2 increased remarkably with
prolonging photo-irradiation time (Fig. 3c), indicating
it was a time-dependent process. As no damage was ob-
served in the absence of light (lane 3), it was thus be-
lieved that the visible light functioned as a trigger to
initiate the DNA strand scission. These newly designed
naphthalimides possessing 2-aminothiazole showed
much high photobiological activities by comparison
with their parent compound without 2-aminothiazole.3f
The Scatchard binding constants between compounds,
A1, A2, B1, B2 and CT-DNA (in 30 mM Tris–HCl buffer,
pH 7.5) were determined using fluorescence technique
method (Fig. 2)9 and were shown in Table 1. The inter-
calating abilities of Bn were stronger than those of their
isomers, An (n = 1,2), indicating that the compound
with linear heterocyclic-fused chromophore was more
easily intercalated into DNA than the one with angular
chromophore. Furthermore, the intercalating abilities of
Table 1. Spectral data,a,b Scatchard binding constants of these
compounds
Compds
UV kmax
(lge)
FL kmax (U)
Scatchard
binding constants
(·104 MÀ1
)
A1
A2
B1
B2
421 (3.50)
422 (3.73)
421 (3.72)
421 (3.81)
475.5 (0.003)
478.3 (0.004)
475.5 (0.003)
478.4 (0.004)
1.98
2.87
2.56
4.73
a In absolute DMSO.
b With quinine sulfate in sulfuric acid as quantum yield standard
(U = 0.55).

Z. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1769–1772
1771
Figure 4. Effect of additives on the photocleavage of closed super-
coiled pBR322 DNA (200 lM per base pair) by compound B2 (70 lM)
in the buffer of Tris–HCl (20 mM, pH 7.5) for 3 h. Lane 1, DNA alone
(no hm); lane 2, DNA alone (hm 3 h); lane 3 DNA and compound B2;
lanes 4–7, DNA and compound B2 in the presence of histidine (6 mM),
dithiothreitol (DTT, 30 mM), ethanol (1.7 M), superoxide dismutase
(SOD, 100 mg/mL), respectively.
under photo-irradiation. However, it should be pointed
out that addition of SOD to the reaction mixture did not
slow the rate of DNA-cleaving reaction. This observa-
tion does not rule out the possibility that superoxide an-
ion is involved in the DNA-cleaving process. It is
because the hydrogen peroxide produced by SOD from
superoxide anion under photo-irradiation or in the pres-
ence of trace amount of metallic cations, could itself lead
to DNA cleavage which then contradicts the inhibition
of superoxide anion by SOD.
Figure 3. Photocleavage of supercoiled pBR322 DNA (200 lM base
pair) in the buffer of Tris–HCl (20 mM, pH 7.5). (a) Photocleavage of
pBR322 DNA by different compounds (100 lM) for 3 h. Lane 1, DNA
alone (no hm); lane 2, DNA alone (hm 3 h); lanes 3–6, DNA and
compound A1, B1, A2, B2 (hm 3 h), respectively. (b) Photocleavage of
pBR322 DNA by B2 at various concentrations for 3 h. Lane 1, DNA
alone (no hm); lane 2, DNA alone (hm 3 h); lanes 3–7, B2 at
concentration of 1, 5, 10, 50, 100 lM, respectively. (c) Photocleavage
of pBR322 DNA by B2 (100 lM) at various time intervals. Lane 1,
DNA alone (no hm); lane 2, DNA alone (hm 3 h); lanes 3–7, 0, 30, 60,
120, 180 min, respectively.
With AM1-semi-empirical quantum calculations
(Hyperchem 7.0), the electron densities of these com-
pounds were calculated, showing slight difference be-
tween the ground state (figure not shown) and excited
(singlet or triplet) state for the same compound. How-
ever, the electron densities between these compounds
were very different at either the ground state or excited
state. Electron densities of these compounds at excited
triplet state were shown in Figure 5 as examples: both
nitrogen and sulfur atoms in the thiazole groups of Bn
(n = 1, 2) were ꢀnakedꢁ, while An (n = 1,2) had obviously
higher electron densities in these two atoms. It was in-
ferred that the nitrogen and sulfur atoms in the thiazole
groups of Bn could attract the electron more strongly,
then the electron might be easier to transfer from
the side chains to the thiazole cycles and then to oxygen
to form superoxide anions. The mechanism experiment
also implied that electron transfer might play an
important role in the course of photocleavage. Therefore,
compared to those of An, the higher electron transfer
abilities of Bn were inferred to confer them higher
activities.
In order to establish the reactive species responsible for
cleavage of the plasmid DNA, B2 was chosen as an
example to perform mechanistic experiments by addi-
1
tion of histidine (singlet oxygen O2 quencher), dithio-
threitol (DTT, superoxide anion OÁ2À scavenger),
ethanol (hydroxyl free radical OHÆ scavenger) and
superoxide dismutase (SOD, superoxide anion OÁ2À killer),
respectively (Fig. 4). It was clear that histidine and
ethanol had no obvious effects on the cleavage reaction,
indicating that singlet oxygen and hydroxyl free radical
were not likely to be the cleaving agents. The DNA-
cleaving activity of B2 decreased greatly in the presence
of DTT (Fig. 4, lane 5), indicating the superoxide anion
was likely to be involved. In our cases, compounds pos-
sibly bound with DNA by aminoalkyl side chains which
then transfer electrons from nucleobases to the chro-
mophores, then to oxygen to generate superoxide anions
Figure 5. Electron densities at the excited triplet state of A1, A2, B1, B2 simulated with AM1 semi-empirical calculation.

1772
Z. Li et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1769–1772
Lett. 2004, 45, 1247–1251; (m) Xu, Y.; Qian, X., et al.
Bioorg. Med. Chem. 2003, 11, 5427–5433.
In summary, a new family of 2-aminothiazonaphthal-
imides as visible light activatable photonucleases was
synthesized and determined to have different DNA
photocleaving activities. The order of their photocleav-
ing abilities was parallel to that of their intercalative
properties. The compound with linear heterocyclic-fused
chromophore exhibited higher DNA intercalating and
DNA photocleaving activities than the one with angular
chromophore. B2 showed the highest activity. Mecha-
nism experiment showed that superoxide anion was
involved.
4. (a) Bran˜a, M. F.; Ramos, A. Curr. Med. Chem.: Anticancer
Agents 2001, 1, 237–255; (b) Bran˜a, M. F.; Cacho, M.;
Garcia, M. A., et al. J. Med. Chem. 2002, 45, 5813–5816;
(c) Sami, S. M.; Dorr, R. T.; Alberts, D. S.; Iyengar, B. S.;
Remers, W. A. J. Med. Chem. 1996, 39, 4978–4987; (d)
Sami, S. M.; Dorr, R. T.; Alberts, D. S.; Solyom, A. M.;
Remers, W. A. J. Med. Chem. 2000, 43, 3067–3073.
5. (a) Zhang, A.; Xiong, W.; Hilbert, J. E.; DeVita, E. K.;
Bidlack, J. M.; Neumeyer, J. L. J. Med. Chem. 2004, 47,
1886–1888; (b) van Vliet, L. A.; Rodenhuis, N.; Wikstrom,
H.; Pugsley, T. A.; Serpa, K. A., et al. J. Med. Chem. 2000,
43, 3549–3557.
6. (a) Malviya, V. K.; Liu, P. Y.; Alberts, D. S.; Surwit, E. A.;
Craig, J. B.; Hanningan, E. V. Am. J. Clin. Oncol. 1992, 15,
41–44; (b) Rosell, R.; Carles, J.; Abad, A.; Ribelles, N.;
Barnadas, A.; Benavides, A.; Martin, M. Invest. New Drugs
1992, 10, 171–175.
Acknowledgments
Financial support by the National Key Project for Basic
Research (2003CB114400) and under the auspices of
National Natural Science Foundation of China is
greatly appreciated.
7. Zhang, A.; van Vliet, S., et al. Tetrahedron Lett. 2003, 44,
6459–6462.
8. Compound A1: mp >300 ꢁC. 1H NMR (DMSO-d6) d
(ppm): 2.64 (s, 6H, NCH3), 3.10 (s, 2H, NCH2), 4.32 (t,
J1 = 6.0 Hz, J2 = 6.0 Hz, 2H, CONCH2), 7.85 (t,
J1 = 7.8 Hz, J2 = 7.8 Hz, 1H, 2-H), 8.48 (t, J = 7.6 Hz,
3H, 1-H, NH2), 8.72 (d, J = 8 Hz, 1H, 3-H), 8.87 (s, 1H, 7-
H). HRMS: calcd for C17H17N4O2S (M+H)+: 341.1072.
Found: 341.1070. IR (KBr): 2927, 2857, 1730, 1646, 1351,
779 cmÀ1. Compound A2: mp 260–261 ꢁC. 1H NMR
(DMSO-d6) d (ppm): 1.62 (s, 2H, CH2), 2.59 (s, 6H,
NCH3), 2.97 (s, 2H, NCH2), 4.11 (s, 2H, CONCH2), 7.84 (t,
J1 = 7.8 Hz, J2 = 7.8 Hz, 1H, 2-H), 8.46 (d, J = 7.8 Hz, 1H,
1-H), 8.50 (s, 2H, NH2), 8.73 (d, J = 8.4 Hz, 1H, 3-H), 8.85
(s, 1H, 7-H). HRMS: calcd for C18H19N4O2S (M+H)+:
355.1229. Found: 355.1211. IR (KBr): 2920, 2850, 1720,
1650, 1330, 779 cmÀ1. Compound B1: mp 290–291 ꢁC 1H
NMR (DMSO-d6) d (ppm): 2.50 (s, 6H, NCH3), 2.91 (s, 2H,
NCH2), 4.26 (s, 2H, CONCH2), 7.84 (t, J1 = 8 Hz,
J2 = 8 Hz, 1H, 2-H), 8.03 (s, 2H, NH2), 8.28 (d,
J = 8.2 Hz, 1-H), 8.36 (d, J = 6 Hz, 1H, 3-H), 8.40 (s, 1H,
10-H). HRMS: calcd for C17H17N4O2S (M+H)+: 341.1072.
Found: 341.1073. IR (KBr): 2921, 2850, 1696, 1654, 1330,
777 cmÀ1. Compound B2: mp 240–241 ꢁC. 1H NMR
(DMSO-d6) d (ppm): 1.82 (t, J1 = 6.4 Hz, J2 = 7.2 Hz, 2H,
CH2), 2.28 (s, 6H, NCH3), 3.16 (s, 2H, NCH2), 4.06 (t,
J1 = 7.2 Hz, J2 = 7.2 Hz, 2H, CONCH2, 7.79 (t,
J1 = 7.8 Hz, J2 = 8 Hz, 1H, 2-H), 8.01 (s, 2H, NH2), 8.20
(d, J = 8.4 Hz, 1-H), 8.31 (d, J = 7.2 Hz, 1H, 3-H), 8.34 (s,
1H, 10-H). HRMS: calcd for C18H19N4O2S (M+H+):
355.1229. Found: 355.1219. IR (KBr): 2960, 2823, 1695,
References and notes
1. (a) Parrish, J. A.; Fitzpatrick, T. B.; Tannenbaum, L.;
Pathak, M. A. N. Eng. J. Med. 1974, 291, 1207–1211; (b)
Jori, G.; Reddi, E. In Photodynamic Therapy of Neoplastic
Disease; Kessel, D., Ed.; CRC: Boston, 1990; pp 117–130;
(c) Henderson, B. W.; Dougherty, T. J. Photochem.
Photobiol. 1992, 55, 145–157.
2. (a) Cimino, D. G.; Gamper, H. B.; Isaacs, S. T.; Hearst, J.
E. Annu. Rev. Biochem. 1985, 54, 1151–1193; (b) Bailly, C.;
Mollegard, N. E.; Nielsen, P. E.; Waring, M. J. EMBO J.
1995, 9, 2121–2131; (c) Briener, K. M.; Daugherty, M. A.;
Oas, T. G.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117,
11673–11679.
3. (a) Armitage, B. Chem. Rev. 1998, 98, 1171–1200; (b) Saito,
I.; Nakatani, K. Bull. Chem. Soc. Jpn. 1996, 69, 3007–3019;
(c) Satio, I.; Takayama, M.; Sakurai, T. J. Am. Chem. Soc.
1994, 116, 2653–2654; (d) Satio, I.; Takayama, M. J. Am.
Chem. Soc. 1995, 117, 5590–5591; (e) Satio, I.; Takayama,
M.; Sugiyama, H.; Nakatani, K. J. Am. Chem. Soc. 1995,
117, 6406–6407; (f) Qian, X.; Huang, T., et al. J. Chem.
Soc., Perkin Trans. 2 2000, 715–718; (g) Qian, X.; Yao, W.,
et al. Tetrahedron Lett. 2001, 42, 6175–6178; (h) Qian, X.;
Mao, P.; Yao, W.; Guo, X. Tetrahedron Lett. 2002, 43,
2995–2998; (i) Yao, W.; Qian, X.; Hu, Q. Tetrahedron Lett.
2000, 41, 7711–7715; (j) Li, Y.; Xu, Y.; Qian, X., et al.
Bioorg. Med. Chem. Lett. 2003, 13, 3513–3515; (k) Qian, X.;
Li, Y.; Xu, Y., et al. Bioorg. Med. Chem. Lett. 2004, 14,
2665–2668; (l) Li, Y.; Xu, Y.; Qian, X., et al. Tetrahedron
1651, 1335, 779 cmÀ1
9. Gupta, M.; Ali, R. J. Biochem. 1984, 95, 1253–1257.
.