Synthesis, structures and the biological activity study on the metal complexes of 2-(4-aminophenyl)benzothiazole derivative

Article abstract of DOI:10.1016/j.ica.2011.10.004

(2-(4-(Benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5) was synthesized and then its three metal complexes were prepared with structures determined by ¹H NMR or X-ray. In all complexes, ZL-5 is deprotonated to generate the neutral

Full text of DOI:10.1016/j.ica.2011.10.004

Inorganica Chimica Acta 382 (2012) 35–42
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Inorganica Chimica Acta
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Synthesis, structures and the biological activity study on the metal complexes of
2-(4-aminophenyl)benzothiazole derivative
a
a
b
a
b
Guo-Wu Lin a, Yue Wang a, , Qiao-Mei Jin , Tao-Tao Yang , Jie-Mei Song , Yi Lu , Qing-Jie Huang ,
⇑
Ke Song b, Jun Zhou b, Tao Lu a,c,
⇑
a College of Basic Science, China Pharmaceutical University, Nanjing 210009, China
b School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
(2-(4-(Benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5) was synthesized and then its
three metal complexes were prepared with structures determined by 1H NMR or X-ray. In all complexes,
ZL-5 is deprotonated to generate the neutral complex unit and acts as a tetradentate ligand, providing
iminodiacetic N atom and three O atoms to bond with metal ions. Meantime we studied on the interac-
tion of complexes with DNA by ultraviolet spectrum and fluorescent spectrum. Also the cleavage abilities
of the complexes on plasmid pBR 322 DNA were studied by gel electrophoresis. We deduce the Co(II)
complex 1 with the largest planar structure has the strongest DNA binding ability mainly by the interca-
lation mode, while the Cu(II) complex 2 with the coordinated water cut the DNA most effectively. And in
the oxidative cleavage process of all complexes, the hydroxyl radical is produced which can be captured
by DMSO.
Received 11 November 2010
Received in revised form 12 July 2011
Accepted 1 October 2011
Available online 12 October 2011
Keywords:
Benzothiazole
Cu(II) complex
Crystal structure
DNA binding
DNA cleavage
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
possible means, these prodrugs can be hydrolyzed into two parts
which have biological activity, respectively, thus may greatly en-
Nowadays, health is one of the most important domains we
human beings have focused on in our society. However, tumor is
the biggest killer of our lives, so there has been steadily increasing
research in the field of anticancer therapy over recent years.
2-(4-Aminophenyl)benzothiazole (CJM 126) and its analogues
comprise a novel mechanistic class of antitumor agents [1,2]. This
nucleus comes from the related structure polyhydroxylated
2-phenylbenzothiazoles, flavone quercetin and the isoflavone
genistein, which are tyrosine kinase inhibitors bearing potent anti-
tumor activity [3,4]. It is well known that the compounds with the
basic structure of 2-(4-aminophenyl)benzothiazole or its deriva-
tives which have high selectivity and strong activity on tumor cells
are a new type of antitumor drugs both in vivo and vitro, especially
on women’s breast cancer and ovarian cancer [5,6]. They can also
be transformed into prodrugs with a famous example lysyl-amide
(NSC 710305) (Fig. 1), which acts as a prodrug to exhibit the ability
through hydrolysis to form two parts [7–9] to emit the active phar-
maceutical ingredient, and then promote the bioavailability.
So if some kind of ligands coordinated with various metal
ions to be potent prodrugs, the body absorbs them through all
hance the antineoplastic activity of the chemotherapeutics and
reduce the toxicity as well as resistance of drugs. From this
point, we focus on the synthesis the (2-(4-(benzothia-
zole)phenyl)carbamoylmethyl)iminodiacetic acid together with
the transition metal complexes. In the recent study, it is reported
that various transition metal ions (such as Cu2+, Zn2+, Ni2+ and
other ions) can bind to the imidazole group of the histidine res-
idues in the vicinity of the enzyme’s active site pockets. It pro-
vides a novel strategy for enhancing the binding affinity of an
active site-directed inhibitor by attaching an iminodiacetic acid
(IDA)-conjugated Cu2+, which could interact with one of the sur-
face-exposed histidine (His-4) residues in carbonic anhydrase or
matrix metalloproteinase inhibitors [10,11]. And a weak inhibitor
of carbonic anhydrase (viz., benzenesulfonamide) can be con-
verted into a strong inhibitor by attaching an iminodiacetate
IDA–Cu2+ group via a suitable spacer. In such a ‘two-prong’
inhibitor, whereas the benzenesulfonamide group binds at the
active site pocket of the enzyme, the IDA–Cu2+ moiety loops
around and interacts with one of the surface exposed histidine
residues [12].
In our work, the series of transition metal complexes of (2-(4-
(benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid were
studied and the schemes were shown in Fig. 2. The title complex
⇑
Corresponding authors. Fax: +8625 86185163 (Y. Wang), +8625 86185180
(T. Lu).
E-mail addresses: zwy_1115@yahoo.com.cn (Y. Wang), lut163@163.com (T. Lu).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.10.004

36
G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
intermedium sodium (2-(4-(benzothiazole)phenyl)carbamoylm-
H2N
(CH2)4
F
N
S
N
ethyl)iminodiacetate (ZL-4) was synthesized by condensation
between equimolar amounts of obtained (ZL-3) with iminodiacetic
acid (IDA) in the presence of Na2CO3 in ethanol solution (75%), after
acidation of ZL-4, the title ligand (ZL-5) was achieved. At last,
reaction with transition metals can produce corresponding metal
coordination complexes.
NH
O
NH2
S
NH2
CJM 126
NSC 710305
Fig. 1. The structure of 2-(4-aminophenyl)benzothiazole (CJM 126) and 2-(4-
amino-3-methylphenyl)-5-fluorobenzothiazole (NSC 710305).
2.2.1. 2-(4-Nitrophenyl)benzothiazole (ZL-1)
is mainly consisting of two parts: the 2-(4-aminophenyl)benzothi-
azole and the iminodiacetic complex, linked by –CO–CH2– group.
Also their structures and the binding and cleavage ability with
DNA were studied.
4-Nitrobenzoic acid (6.7 g, 0.04 mol) was dissolved in polyphos-
phoricacid (100 mL) at 180 °C. 2-Aminothiophenol (5.0 g, 0.04 mol)
was added and the resulting solution was stirred at 180 °C for 5 h.
After cooling, the reaction mixture was poured into ice-cold aque-
ous ammonia (100 mL). The precipitate was collected by filtration
and washed with water (50 mL ꢀ 2). The product was dried and
crystallized from acetoacetate to give the 2-(4-nitrophenyl)benzo-
thiazoles (ZL-1) as a white solid (8.3 g, 0.03 mol, yield 81%), m.p.:
160–161 °C; MS m/z 258.0 (M+1).
2. Experimental
2.1. Apparatus and reagents
All solvents and other reagents were all of chemical grade and
used as received. Water for solution preparation was distilled
water. Calf Thymus DNA (CT DNA) and pBR 322 plasmid DNA were
purchased from Sigma.
2.2.2. 2-(4-Aminophenyl)benzothiazole (ZL-2)
4-Aminobenzoic acid (6.6 g, 0.048 mol) was dissolved in
polyphosphoricacid (120 mL) at 200 °C. 2-Aminothiophenol
(6.0 g, 0.048 mol) was added and the resulting solution stirred at
200 °C for 5 h. After cooling, the reaction mixture was poured into
ice-cold aqueous ammonia (100 mL). The precipitate was collected
by filtration and washed with water (50 mL ꢀ 2). The product was
dried and crystallized from acetoacetate to afford the correspond-
ing adduct 2-(4-aminophenyl)benzothiazoles (ZL-2) as a yellow
solid (9.9 g, 0.043 mol, yield 90%), m.p.: 155–157 °C; MS m/z
227.3 (M+1).
Reaction progress was monitored using analytical thin layer
chromatography (TLC) on percolated Merck silica gel Kiesegel 60
F254 plates, and the spots were detected under UV light
(254 nm). Melting points were determined with a digital melting
point apparatus and are reported uncorrected. 1H NMR spectra
was recorded at 300 MHz on a Bruker ARX 300 spectrometers. IR
spectra were measured on a Jasco FT/IR-430 spectrophotometer.
Mass spectra were recorded on an a Quattro microMS Micromass
UK mass spectrometer, and was recorded on an electrospray ioni-
zation mass spectrometer as the value m/z. The X-ray measure-
ments were made on a Rigaku RAXIS RAPID diffractometer with
2.2.3. 2-(4-Chloroacetamidophenyl)benzothiazole (ZL-3)
In a 500 mL single-necked, round-bottomed flask equipped
with a magnetic stirrer, the following were placed: 2-(4-amino-
phenyl)benzothiazoles (ZL-2) (8.0 g, 0.035 mol), triethylamine
(14.8 mL, 0.106 mol) and anhydrous acetone (200 mL), then the
chloroacetyl chloride (8.5 mL, 0.106 mol) in anhydrous acetone
(10 mL) was added drop wise to the stirred mixture. After the mix-
ture was boiled for 4 h, the precipitate was collected and washed
with water (50 mL ꢀ 2) to give 2-(4-chloroacetamidophenyl)ben-
zothiazoles (ZL-3) as pale-yellow powder (9.2 g, 0.03 mol, yield
86%). m.p.: 214–215 °C; MS m/z 303.5 (M+1), dH 1H NMR (DMSO-
d6) 10.80 (1H, s, –CO–NH–), 8.20–7.44 (8H, m, Ar–H ꢀ 8), 4.33
(2H, s, –CH2Cl).
a graphite monochromatised MoK
a radiation (k = 0.71069 Å) using
x
scan mode. All the UV absorption spectra were recorded on a
Shimadzu UV-2100 spectrophotometer after through mixing for
5 min at 25 °C. Electronic absorption spectra and fluorescence
spectra were performed with a Shimadzu UVW-2101PC spectro-
photometer and
a Shimadzu FR-5301 spectrofluorometer at
25 °C, respectively. The gel was photographed on a capturing sys-
tem gel printer plus TDI. Bands were quantified using the Scion Im-
age for Windows software program based on NHI Image.
2.2. Synthesis of the (2-(4-(benzothiazole)phenyl)carbamoylmethyl)
iminodiacetic acids (ZL-5) and its complexes 1–3
Synthesis of the (2-(4-(benzothiazole)phenyl)carbamoylmeth-
yl)iminodiacetic acids (ZL-5) was outlined in Scheme 1. For the
first, the 2-(4-nitrophenyl)benzothiazole (ZL-1) and 2-(4-amino-
phenyl)benzothiazole (ZL-2) were obtained by the condensation
in polyphosphoric acid (PPA) at 180–200 °C, (ZL-2) also can get
form (ZL-1) by reduction of nitro; Secondly, reaction of amines
with chloroacetyl chloride in refluxing dry acetone gave the
2-(4-chloroacetamidophenyl)benzothiazoles (ZL-3). Then, the key
2.2.4. (2-(4-(Benzothiazole)phenyl)carbamoylmethyl)iminodiacetic
acid (ZL-5)
In a 150 mL single-necked, round-bottomed flask equipped
with a magnetic stirrer, the following were placed: 2-(4-chloro-
acetamidophenyl)benzothiazoles (ZL-3) (5.0 g 0.017 mol), imino-
diacetic acid (IDA) (2.2 g 0.018 mol), sodium carbonate (3.0 g
0.028 mol) and 75% ethanol (200 mL). The stirred mixture was
heated under reflux for 10 h, then cooled to room temperature to
H
O
H3C
H3C
H
O
H3C
N
N
OH2
H3C
N
N
Cu
Cu
Co
2H2O
O
O
O
O
O
.5H2O
O
C
O
C
S
N
O
C
S
N
O
S
N
O
O
N
N
N
O
N
N
H
N
H
H
O
O
O
Fig. 2. The chemical structure of complex 1–3.

G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
37
NH2
N
a
COOH
O2N
H2N
NO2
NH2
+
S
SH
ZL-1
b
NH2
N
S
N
S
c
d
COOH
NHCOCH2Cl
+
SH
ZL-2
ZL-3
CH2COONa
CH2COONa
CH2COOH
CH2COOH
N
S
N
S
e
f
NHCOCH2N
NHCOCH2N
ZL-5
ZL-4
g
Complexes
1-3
Scheme 1. Reagents and conditions: (a) polyphosphoric acid, 180 °C, 5 h; (b) Fe powder, NH4Cl, 75% C2H5OH, reflux 2 h; (c) polyphosphoric acid, 200 °C, 5 h; (d) chloroacetyl
chloride; acetone, reflux 4 h, then 10% Na2CO3; (e) iminodiacetic acid (IDA), Na2CO3, 75% C2H5OH, reflux 2 h; (f) 4% HCl; (g) CuCl2ꢁ2H2O or CoCl2ꢁ2H2O, DMF, several months.
give the key intermediate (ZL-4). The reaction mixture was precip-
itated at pH 2–3 with 5% HCl, and collected by filtration in vacuum,
washed with water and methanol, dried and crystallized from
absolute methanol to afford the corresponding adduct (2-(4-(ben-
zothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5)
(6.1 g, 0.015 mol, yield 88.3%)as a white precipitate. m.p.: 223–
226 °C; MS m/z 400.4 (M+1); dH 1H NMR (DMSO-d6) d 12.61 (2H,
s, –COOH ꢀ 2), d 10.62 (1H, s, –CO–NH–), 8.20–7.40 (m, 8H, Ar–
H), 4.33 (2H, s, –CH2Cl), d 3.54 (4H, s, –CH2– ꢀ 2).
buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH
7.4) and complex solution with ascorbate at a 10-fold molar excess
relative to the complex to yield a total volume of 18 lL [15]. The
complex with different concentration was dissolved in DMF and
MeOH with volume ratio 4:1. After mixing, the sample was incu-
bated at 37 °C for 90 min. The reaction was quenched by the addi-
tion of sodium ethylene diamine tetraacetic acid (EDTA-2 Na) and
5 lL of loading buffer (0.25% bromphenol blue, 50% glycerol). Then
the solution was subjected to electrophoresis. Electrophoresis was
achieved at 80 V for 70 min in apt TAE buffer (40 mM Tris acetate/
1 mM EDTA). The gel was photographed on a capturing system gel
printer plus TDI. Bands were quantified using the Scion Image for
Windows software based on NHI Image.
2.2.5. Metal coordination compounds
Orange platelet crystals of ZL-5-Co-Phen (complex 1) were ob-
tained by slow evaporation of a DMF solution of (ZL-5), 1, 10-phe-
nanthroline and CoCl2.6H2O (molar ratio 1:1:1) at room
temperature for several weeks.
2.6. Research on the mechanism of cleavage of pBR 322
Blue prism crystals of ZL-5-Cu-1 (complex 2) were obtained by
slow evaporation of a DMF solution of (ZL-5) and CuCl2. 2H2O (mo-
lar ratio 1:1) at room temperature for several weeks.
Blue needle crystals of ZL-5-Cu-2 (complex 3) were obtained by
slow evaporation of a DMF solution of (ZL-5) and CuCl2ꢁ2H2O (mo-
lar ratio1:1) at pH 3–4 at room temperature for several weeks.
Reactions were carried out by the method mentioned above.
Also, to study the cleavage mechanism, DMSO, KI and NaN3 was
added respectively as comparison. After mixing, the sample was
incubated at 37 °C for 90 min. The reactions were quenched at
appropriate time by the addition of sodium ethylene diamine tet-
raacetic acid (EDTA-2 Na) and 5 lL of loading buffer (0.25% brom-
phenol blue, 50% glycerol). Then the solution was subjected to
electrophoresis on 0.8% agarose gel in TAE buffer (40 mM Tris ace-
tate/1 mM EDTA) at 80 V and visualized by ethidium bromide
staining.
2.3. Study with UV spectroscopy
The absorption spectra of the interaction of complexes with CT
DNA have been recorded for a constant complex concentration
with increasing amount of CT DNA. Complexes were dissolved in
a DMF-methanol mixture (4:1 v/v).
3. Results and discussion
2.4. Study with fluorescence spectroscopy
3.1. X-ray crystal analysis
Ethidium bromide (EB) emits intense fluorescence light in the
presence of DNA, due to strong intercalation between the adjacent
DNA base pairs. The competitive binding of the complexes to DNA
reduces the emission intensity of EB with DNA [13,14]. In the bind-
ing study, the fluorescence spectra (kex = 520 nm, kem = 590 nm)
were recorded at room temperature and complexes solution with
original concentration of 2.0 ꢀ 10ꢂ4 M were added to a volume of
2 mL of the EB–DNA solution to get the final concentration of
DNA and EB being 4.0 ꢀ 10ꢂ5 M and 4.0 ꢀ 10ꢂ6 M, respectively.
Five minutes later after adding the complexes, the fluorescence
intensity of the mixture was recorded.
The X-ray measurements were made on a Rigaku RAXIS RAPID
diffractometer with a graphite monochromatised MoK
a radiation
(k = 0.71069 Å) using scan mode. A summary of the crystallo-
x
graphic data and structure refinements is given in Table 1. The data
were corrected for Lorentz and polarization effects. The structure
was solved by direct methods [16] using the crystal structure
[17] software package. The refinement was performed using SHEL-
XL-97 [18]. All H atoms were located from difference Fourier maps
and treated as riding, with C–H distance of 0.93, phenol N–H dis-
tance of 0.86 and Uiso(H) values equal to 1.2 Ueq(C) and Uiso(water
H) values equal to 1.5 Ueq(C) (Ueq is the equivalent isotropic dis-
placement parameter for the pivot atom).
P
2.5. Research on the cleavage of pBR 322 by complexes
The function of
w (Fo2–Fc2)2 was minimized by using the
weight scheme of w = 1/[
r
2(Fo2) + (aP)2 + bP], where P = (Fo2 +
P
P
P
Reactions were carried out in a total volume of 18
l
L by mixing
2Fc2)/3. Final R [= (|Fo|ꢂ|Fcj)/ |Fo|], Rw [=( w(|Fo| ꢂ |Fc|)2/
P
P
1
l
L of supercoiled (SC) pBR 322 plasmid DNA (0.25
l
g/lL), a
w|Fo|2)1/2] and S (goodness of fit) [=( w(|Fo| ꢂ |Fc|)2/(MꢂN)1/2),

38
G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
Table 1
Crystal data and structure refinement for complexes 1–3.
Complex
1
2
3
Formula
C
31H33CoN5O10
S
C22H24CuN4O7S
552.05
monoclinic
P21/c
25.160(5)
7.1510(14)
13.609(3)
90.00
93.43(3)
90.00
2444.1(8)
4
C22H24CuN4O7.50
560.05
orthorhombic
Pbca
S
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
726.63
triclinic
P1
ꢀ
8.9990(7)
12.7766(7)
14.0652(8)
92.661(2)
102.770(2)
97.389(2)
1559.39(17)
2
22.359(5)
6.7830(14)
39.112(8)
90.00
90.00
90.00
a
(°)
b (°)
(°)
c
V (Å)
Z
5932(2)
8
Crystal size (mm)
Shape
0.5 ꢀ 0.5 ꢀ 0.2
platelet
orange
0.4 ꢀ 0.2 ꢀ 0.1
0.2 ꢀ 0.2 ꢀ 0.1
needle
blue
prism
Color
blue
1.500
Dcalc (g cmꢂ3
)
1.548
1.254
Reflections collected
Unique reflections
Absorption coefficient (mmꢂ1
F(000)
7094
6151
0.662
646.00
0.0409
0.1176
1.118
4771
2980
1.029
1140
0.0800
0.1919
1.028
5294
2100
0.850
2312
0.0826
0.1913
1.011
)
R (F2 > 2
wR (F2)
r
(F2))
Goodness of fit (GOF) n F2
Number of variables
434
281
301
Table 2
Bond distances (Å) and angles (°) for compounds.
Complex
Bond lengths
Bond angles
1
Co–O2 2.073(2)
Co–O4 2.058(2)
Co–O5 2.094(14)
Co–N3 2.197(16)
Co–N4 2.101(2)
Co–N5 2.168(16)
O2–Co–O4 154.49(5)
O2–Co–O5 94.28(5)
O2–Co–N3 79.76(5)
O2–Co–N4 104.19(5)
O2–Co–N5 85.51(5)
O4–Co–O5 95.28(5)
O4–Co–N3 78.63(5)
O4–Co–N4 98.96(5)
O4–Co–N5 88.92(5)
O5–Co–N3 80.30(5)
O5–Co–N4 92.51(6)
O5–Co–N5 170.28(6)
N3–Co–N4 172.11(6)
N3–Co–N5 109.17(6)
N4–Co–N5 78.15(6)
2
Cu–O4 1.949(5)
Cu–O6 1.952(5)
Cu–O2 1.962(4)
Cu–N1 2.020(5)
Cu–O7 2.361(5)
Cu–O5 2.398(4)
O4–Cu–O6 95.9(2)
O4–Cu–O2 167.25(18)
O6–Cu–O2 96.2(2)
O4–Cu–N1 85.48(19)
O6–Cu–N1 169.20(19)
O2–Cu–N1 83.38(18)
O4–Cu–O7 86.04(18)
O6–Cu–O7 96.56(19)
O2–Cu–O7 88.62(17)
N1–Cu–O7 94.22(18)
O4–Cu–O5 86.52(19)
O6–Cu–O5 88.79(18)
O2–Cu–O5 97.71(18)
N1–Cu–O5 80.58(16)
O7–Cu–O5 171.24(17)
3
Cu–O2 1.927(5)
Cu–O5 1.934(5)
Cu–O3 1.960(5)
Cu–N2 2.029(6)
Cu–O1 2.308(5)
O2–Cu–O5 96.5(2)
O2–Cu–O3 94.4(2)
O5–Cu–O3 166.1(2)
O2–Cu–N2 178.0(2)
O3–Cu–N2 86.9(2)
O2–Cu–O1 99.8(2)
O5–Cu–O1 96.9(2)
O3–Cu–O1 89.88(19)
where M = no. of reflections and N = no. of variables used for the
refinement] are given in Table 1. Anisotropic displacement coeffi-
cients were refined for all non-hydrogen atoms. Selected bond dis-
tances and angles are listed in Table 2.
asymmetric unit (Fig. 3). In complex 2 the Cu2+ ion is bonded to
the amide O atom of DMF ligand and the O atoms of water molecules,
one iminodiacetic N atom and three O atoms from ZL-5 ligand. Here,
each ZL-5 acts as a tetradentate ligand to generate a slightly dis-
torted octahedral coordination geometry, with the amide O6 atom
of DMF, the iminodiacetic N1 atom of ZL-5 ligand and two carboxyl-
ate O2 and O4 atom from iminodiacetate occupying the equatorial
plane, leaving two apex positions for carboxyl O5 atom from ZL-5 li-
gandand theactivatingwaterO7 atomoccupying the axialdirection.
The bond length of Cu–O5 and Cu–O7 are 2.398(4) Å and
2.361(5) Å, respectively in axial direction, significantly longer than
its equatorial location on the other bond length (from 1.949(5) to
2.020(5) Å), which can be explained by John–Teller effect. The
whole structure is stabilized by the intermolecular hydrogen bond,
3.2. Structure studies
3.2.1. The structure of complex 1 (ZL-5-Co-phen)
Complex 1 (ZL-5-Co-phen) contains an [Co(ZL-5)(phen)] com-
plex moiety (phen is 1,10-phenanthroline) and five water mole-
cules in the asymmetric unit. The detailed structure description
was published in our previous work [19].
3.2.2. The structure of complex 2 (ZL-5-Cu-1)
together with the
p–p interaction between the adjacent (2-
Complex
2
(ZL-5-Cu-1) contains an neutral [Cu(ZL-
phenyl)benzothiazole planes.
5)(DMF)(H2O)] moiety (DMF is dimethylformamide) in the

G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
39
Fig. 4. ORTEP drawing of complex 3.
(2-phenyl)benzothiazole fracture extending and forming
interaction to make the whole structure more stable.
p–p
3.3. Study with UV spectroscopy
Solutions of CT DNA in 50 mM NaCl/5 mM Tris–HCl (pH 7.4)
gave a ratio of UV absorbance at 260 and 289 nm of 1.8–1.9:1, indi-
cating that the DNA was sufficiently free of protein [20]. The con-
centration of CT DNA was determined spectrophotometrically by
employing an extinction coefficient of 6600 Mꢂ1 cmꢂ1 at 260 nm
[21].
The absorption spectra of complex 1 with increasing amount of
CT DNA at a constant concentration of the complex were given in
Fig. 5. As the DNA concentration is increased, the main band of this
complex at 331 nm exhibit hypochromism with 3 nm red shift,
which suggest the complex 1 can strongly bind to DNA by interca-
lating interaction. Because of structural similarity, a similar result
was found for complex 2 and 3. As the DNA concentration increas-
ing, the maximum absorption bands of these two complexes at
331 nm exhibit hyperchromism with 3 nm blue shift. The hyper-
chromism effects observed suggest that the contacts between the
molecule of the complex and groove of DNA are very important.
From the absorption of three complexes, we can deduce the max-
Fig. 3. ORTEP drawing of complex 2.
3.2.3. The structure of complex 3 (ZL-5-Cu-2)
Complex 3 (ZL-5-Cu-2) contains an [Cu(ZL-5)(DMF)] unit and
two free water molecules (Fig. 4). Each Cu2+ ion is bonded to the
amide O atom of DMF ligand and one iminodiacetic N atom of
ZL-5 ligand, and three O atoms from ZL-5 ligand. Here, ZL-5 also
acts as a tetradentate ligand through two iminodiacetic O, one
iminodiacetic N, and one amide O atom, while each DMF ligand
act as monodentate ligand through N atoms. It generated a slightly
distorted square pyramid geometry, with the amide O2 atom of
DMF, the iminodiacetic N2 atom, O3 atom and O5 atom from ZL-
5 ligand occupying the equatorial plane, while carboxylate O1
atoms of ZL-5 ligand occupying the axial plane.
By comparison, in all three complexes, ZL-5 is deprotonated to
generate the neutral complex unit. The part of the phenyl benzo-
thiazole is planar, indicating by the mean dihedral angle of phenyl
and benzothiazole 3.394 Å, with the mean deviation from the plane
by 0.0229 Å. The part of the iminodiacetic acid together with –CO–
CH2– is twisted to provide four coordination atoms, thus forming
three twisted five-member chelated rings, and leaving the basic
imumabsorption at 331 nm may attributedto the p–
p⁄ transition by
the ligand ZL-5. In all cases, as the result of complex formation, the
strong band at 331 nm as well as the other weak bands at near
347 nm remarkably changed in their intensity with a little red-shift
orblue-shift withequal absorptionpoint, indicatingtheformationof
new DNA-complexes compounds, but three complexes had a differ-
ent binding mode due to their different structures.
3.4. Study with fluorescence spectroscopy
Fluorescent emission of EB bound to DNA in the presence of
complex 1 is shown in Fig. 6. With the concentration of DNA

40
G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
Fig. 5. UV–Vis absorption spectra of complex 1–3 (from left to right) with increasing amount of CT DNA. The ratio of CDNA/Ccomplex in each curve was increasing along arrow
direction.
lane
1
2
3
4
Form II
Form III
Form I
Fig. 8a. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA (0.25 g/ L) by complex 1 in the presence of 10-fold excess of ascorbate for
l
l
90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1, DNA control; lane 2, complex 1 (5 ꢀ 10ꢂ6 M); lane 3, complex 1
(5 ꢀ 10ꢂ5 M); lane 4, complex 1 (5 ꢀ 10ꢂ4 M).
increasing. In three cases, the emission band of the DNA–EB system
decreased in intensity with the increase of the concentration of the
complexes results from all complexes replacing EB from the DNA–
EB system. Such a characteristic change is often observed in the
intercalative DNA interaction. In comparison, it is apparent from
the plot shown Fig. 7 that EB bound DNA was efficiently quenched
by complex 1, in which displacement process of complex occurred.
Deducing from the slope of the quenching plot with the complex
concentration, the DNA binding propensity can be reflected as
the relative order: complex 1 > complex 2 > complex 3. This obser-
vation suggests that the complex competes with the intercalative
EB binding to DNA in a different degree which is coinciding with
their solid structure in which complex 1 has a largest plane coordi-
nated to ancillary phen ligand. Thus the degree of intercalation
interaction was further testified by the fluorescence quenching
propensity besides the absorption spectrum results.
Fig. 6. Fluorescence emission spectra (excited at 520 nm) in the EB-CT DNA system
with increasing amounts of 2.0 ꢀ 10ꢂ4 M of complex 1.
3.5. pBR 322 DNA cleavage promoted by three complexes
3.5.1. Cleavage ability of pBR 322 by the complexes at different
concentration
The metal complexes cause random cuts to one of the DNA
strands. As a consequence, the supercoiled form opens to form
an open circular after one nick and subsequently to a linear form
if two nicks on complementary strands are within a short distance.
Finally, the DNA degraded into small pieces of different size which
cannot be detected in our assay. The cleavage products were sub-
jected to gel electrophoretic separation and the gels analyzed after
ethidium bromide staining [22–24].
Fig. 7. The emission intensity of CT DNA-bonded EB(DNA/EB = 10/1) when adding
different concentrations of three complexes in 50 mM Tris–HCl buffer (pH 7.4) at
room temperature on addition of complex 1 (d), complex 2 (s), complex 3 (.).
The oxidative DNA cleavage activity of the complexes was stud-
ied by gel electrophoresis using SC pBR 322 DNA in Tris–HCl buffer
(pH 7.4). Control experiment done under the same conditions does
not show any apparent cleavage activity. The conversion of the
supercoiled pBR 322 DNA (SC DNA, Form I) to the nicked DNA
(NC DNA, Form II) and linear DNA (Form III) becomes more effi-
cient when increasing the concentration of the complexes. With
increased, the fluorescence intensity was largely decreased. The
fluorescence intensities were plotted against the concentration of
three complexes to get slopes in Fig. 7.
The emission intensity of the DNA–EB system (Ex = 520 nm) de-
creased apparently with the concentration of each complex

G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
41
lane
1
2
3
4
5
Form II
Form III
Form I
Fig. 9. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA(0.25 lg/lL) by ligand ZL-5 in the presence of 10-fold excess of ascorbate for
90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1, DNA control; lane 2, ZL-5 (5 ꢀ 10ꢂ6 M); lane 3, ZL-5
(5 ꢀ 10ꢂ5 M); lane 4, ZL-5 (5 ꢀ 10ꢂ4 M), lane 5, ZL-5 (2.5 ꢀ 10ꢂ4 M).
Fig. 8b. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA(0.25 g/ L) by complex 2 in the presence of 10-fold excess of ascorbate for
90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1, DNA control; lane 2, complex 2 (2.5 ꢀ 10ꢂ7 M); lane 3, complex 2
(2.5 ꢀ 10ꢂ6 M); lane 4, complex 2 (2.5 ꢀ 10ꢂ5 M); lane 5, complex 2 (2.5 ꢀ 10ꢂ4 M).
l
l
lane
1
2
3
4
5
Table 6
Oxidation cleavage data of SC pBR 322 by ligand ZL-5.
Form II
Form III
Form I
Lane
Concentration
Supercoiled (%)
Nicked
Linear
1
2
3
4
5
DNA control
5 ꢀ 10ꢂ6
100
100
100
100
100
5 ꢀ 10ꢂ5
5 ꢀ 10ꢂ4
Fig. 8c. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA(0.25 g/ L) by complex 3 in the presence of 10-fold excess of ascorbate for
2.5 ꢀ 10ꢂ4
l
l
90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1, DNA control; lane 2, complex 3 (5 ꢀ 10ꢂ7 M); lane 3, complex 3
(5 ꢀ 10ꢂ6 M); lane 4, complex 3 (5 ꢀ 10ꢂ5 M); lane 5, complex 3 (5 ꢀ 10ꢂ4 M).
Table 7
Oxidation cleavage data of SC pBR 322 by complex 1.
Lane Compounds
Supercoiled
(%)
Nicked
(%)
Linear
Table 3
Oxidation cleavage data of SC pBR 322 by complex 1.
1
2
3
4
5
DNA control
DNA + complex 1
DNA + complex 1 + 2
DNA + complex 1 + 10 mM KI
DNA + complex 1 + 10 mM
NaN3
100
Lane Concentration Supercoiled
(Form I) (%)
Nicked (Form
II) (%)
Linear (Form
III) (%)
44.80
59.65
47.35
46.55
55.20
40.35
52.65
53.45
lL DMSO
1
2
3
4
DNA control
100
85.89
8.48
5 ꢀ 10ꢂ6
5 ꢀ 10ꢂ5
5 ꢀ 10ꢂ4
M
M
M
14.11
91.52
57.96
42.04
lane
1
2
3
4
5
Form II
Form I
Table 4
Oxidation cleavage data of SC pBR 322 by complex 2.
Lane
Concentration
Supercoiled (%)
Nicked (%)
Linear (%)
1
2
3
4
5
DNA control
100
45.79
37.52
2.5 ꢀ 10ꢂ7 M
42.96
49.88
92.39
11.25
12.60
7.61
Fig. 10a. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA (0.25 g/ L) by complex 1 in the presence of 10-fold excess of ascorbate for
2.5 ꢀ 10ꢂ6
2.5 ꢀ 10ꢂ5
2.5 ꢀ 10ꢂ4
M
M
M
l
l
90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1, DNA control; lane 2, DNA + complex 1 (2.5 ꢀ 10ꢂ5 M); lane 3,
DNA + complex
1
(2.5 ꢀ 10ꢂ5 M) + 2
lL
DMSO; lane 4, DNA + complex
1
(2.5 ꢀ 10ꢂ5 M) + 10 mM KI; lane 5, DNA + complex 1 (2.5 ꢀ 10ꢂ5 M) + 10 mM NaN3.
Table 5
Oxidation cleavage data of SC pBR 322 by complex 3.
lane
1
2
3
4
5
Lane Concentration Supercoiled
(Form I) (%)
Nicked (Form
II) (%)
Linear (Form
III) (%)
Form II
Form III
1
2
3
4
5
DNA control
100
93.84
90.20
3.84
5 ꢀ 10ꢂ7
5 ꢀ 10ꢂ6
5 ꢀ 10ꢂ5
5 ꢀ 10ꢂ4
M
M
M
M
6.16
9.80
57.83
45.76
Form I
38.33
54.24
Fig. 10b. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA (0.25 g/ L) by complex 2 in the presence of 10-fold excess of ascorbate in a
l
l
buffer solution for 90 min (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1: DNA control; lane 2: DNA + complex 2 (2.5 ꢀ 10ꢂ5 M); lane 3:
the increasing concentration of the complex, the DNA cleavage
propensity is apparently enhanced, as is shown in Figs. 8a–c.
Especially the best cleavage result is obtained on the concentration
DNA + complex
2
(2.5 ꢀ 10ꢂ5 M) + 2
lL
DMSO; lane 4: DNA + complex
2
(2.5 ꢀ 10ꢂ5 M) + 10 mM KI; lane 5: DNA + complex 2 (2.5 ꢀ 10ꢂ5 M) + 10 mM NaN3.
of 5 ꢀ 10ꢂ5 (complex 1), 2.5 ꢀ 10ꢂ7 M (complex 2) and 5 ꢀ 10ꢂ5
M
(complex 3), when degradation to the linear form was observed.
With more high concentration, the linear form of complex 2 was
almost disappearing to footprint for it is furthermore degraded to
small pieces, while the Form II and form III in complex 1 and 3
are still coexisting. The oxidation cleavage data of SC pBR 322 by
all complexes were listed in Table 3–5. By comparison, complex

42
G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42
Table 8
with the complexes alone. But it is apparent that DMSO plays as
an effective free catcher to capture the hydroxyl radical during
the oxidative cleavage process.
Oxidation cleavage data of SC pBR 322 by complex 2.
Lane
Compounds
Supercoiled
100
Nicked
Linear
1
2
3
4
5
DNA + control
DNA + complex 2
DNA + complex 2 + 2
DNA + complex 2 + 10 mM KI
DNA + complex 2 + 10 mM NaN3
53.42
57.31
50.41
61.14
46.58
29.75
49.59
38.86
4. Conclusion
l
L DMSO
12.94
Herein, we built a new way to make the bioactive carriers (2-(4-
aminophenyl)benzothiazole) linked to the small metal complexes
for the research of the complexes bearing possible biological activ-
ity. On this base, we study on the interaction of coordination com-
pounds and DNA systematically through two methods: gel
electrophoresis and optical spectrum which includes ultraviolet
spectrum and fluorescent spectrum. By fluorescence titration
method, we deduce the complex 1 with the largest planar structure
has the strongest DNA binding ability mainly by the intercalation
mode by its largest planar structure. And the electrophoresis result
indicates the ability of the complex 2 cutting the DNA most effec-
tively for the coordinated water. In the nuclear oxidative cleavage
process of all complexes, the hydroxyl radical is produced which
can be captured by DMSO. The study above will be helpful on
the complexes design and the chemical drug development.
lane
1
2
3
4
5
Form II
Form I
Fig. 10c. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid
DNA (0.25 g/ L) by complex 3 in the presence of 10-fold excess of ascorbate in a
l
l
buffer solution for 90 min (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at
37 °C. Key: lane 1: DNA control; lane 2: DNA + complex 3 (2.5 ꢀ 10ꢂ5 M); lane 3:
DNA + complex
3
(2.5 ꢀ 10ꢂ5 M) + 2
lL
DMSO; lane 4: DNA + complex
3
(2.5 ꢀ 10ꢂ5 M) + 10 mM KI; lane 5: DNA + complex 3 (2.5 ꢀ 10ꢂ5 M) + 10 mM NaN3.
Appendix A. Supplementary material
CCDC 670230, 669578, 741991 contain the supplementary crys-
tallographic data for complex 1–3. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Table 9
Oxidation cleavage data of SC pBR 322 by complex 3.
Lane Compounds
Supercoiled
(%)
Nicked
(%)
Linear
1
2
3
4
5
DNA + control
DNA + complex 3
DNA + complex 3 + 2
DNA + complex 3 + 10 mM KI
DNA + complex 3 + 10 mM
NaN3
100
References
77.88
91.40
60.68
74.46
22.12
8.60
39.32
25.54
lL DMSO
[1] E. Kashiyama, I. Hutchinson, M.S. Chua, S.F. Stinson, L.R. Phillips, G. Kaur, E.A.
Sausville, T.D. Bradshaw, A.D. Westwell, M.F.G. Stevens, J. Med. Chem. 42
(1999) 4172.
[2] T.D. Bradshaw, S. Wrigley, D.F. Shi, R.J. Schultz, K.D. Paull, M.F.G. Stevens, Br. J.
Cancer 77 (1998) 745.
[3] M.F.G. Stevens, C.J. McCall, P. Lelieveld, P. Alexander, A. Richter, D.E. Davies, J.
Med. Chem. 37 (1994) 1689.
[4] P.C. Yates, C.J. McCall, M.F.G. Stevens, Tetrahedron 47 (1991) 6493.
[5] D. Raghvendra, K.S. Prabhat, K.B. Pawan, B. Snehendu, S.H.N.M. Narayana,
Mini-Rev. Med. Chem. 6 (2006) 633.
[6] B. Eileen, A. Smitha, K. Glenda, M. LingHua, A. Keli, F.S. Sherman, A.S. Edward, P.
Yves, Cancer Chemother. Pharmacol. 58 (2006) 62.
[7] I. Hutchinson, S.A. Jennings, B.R. Vishnuvajjala, A.D. Westwell, M.F.G. Stevens, J.
Med. Chem. 45 (2002) 744.
[8] T.D. Bradshaw, A.D. Westwell, Curr. Med. Chem. 11 (2004) 1241.
[9] F. Iduna, M. Anne, H. Curtis, M.F.G. Stevens, T.D. Bradshaw, Breast Cancer Res.
Treat. 87 (2004) 97.
2 shows the highest cleavage ability for its coordinated water may
produce the OH radical in the oxidizing process which will acceler-
ate the cleavage reaction. By comparison with the reported other
copper complexes [25–27], complex 2 shows the most effective
DNA cleavage probability. We also studied the interaction between
the ligand alone and DNA by electrophoresis method. The result
got in the same system and the same concentration (Fig. 9 and Ta-
ble 6) showed that the ligand alone does not cause the nuclear oxi-
dative cleavage process of plasmid PBR 322.
[10] A.L. Banerjee, M. Swanson, B.C. Roy, X.G. Jia, M.K. Haldar, S. Mallik, D.K.
Srivastava, J. Am. Chem. Soc. 126 (2004) 10875.
[11] A.L. Banerjee, S. Tobwala, M.K. Haldar, M. Swanson, B.C. Roy, S. Mallik, D.K.
Srivastava, Chem. Commun. (2005) 2549.
[12] B.C. Roy, A.L. Banerjee, M. Swanson, X.G. Jia, M.K. Haldar, S. Mallik, D.K.
Srivastava, J. Am. Chem. Soc. 126 (2004) 13206.
3.5.2. Mechanism of pBR 322 DNA cleavage
[13] J.B. Le Pecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87.
[14] M.J. Waring, J. Mol. Biol. 13 (1965) 269.
[15] M.E. Reichman, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047.
[16] A. Altomare, M. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, A. Guagliardi, A.
Moliterni, G. Polidori, P. Spagna, J. Appl. Crystallogr. 32 (1999) 115.
[17] Crystal Structure Analysis Package, Rigaku and Rigaku/MSC, 9009 New Trails
Dr. The Woodlands TX 77381 USA, 2000–2004.
[18] G.M. Sheldrick, SHELXL97, University of Gttingen, Germany, 1997.
[19] G.W. Lin, Y. Wang, T. Lu, Acta Crystallogr. 64 (2008) 179.
[20] J. Marmur, J. Mol. Biol. 3 (1961) 208.
[21] M.E. Reichmann, S.A. Rice, C.A. Thomas, P.J. Doty, J. Am. Chem. Soc. 76 (1954)
3047.
[22] L.F. Povirk, W. Wübker, W. Köhnlein, F. Hutchinson, Nucleic Acids Res. 4 (1977)
3573.
[23] J. Drak, N. Iwasawa, S. Danishefsky, D.M. Crothers, Proc. Natl. Acad. Sci. USA 88
(1991) 7464.
[24] M.E. Branum, L. Que Jr., JBIC 4 (1999) 593.
To verify the reactive oxygen species are responsible for the
cleavage, reactions were carried out in the presence of typical scav-
engers for single oxygen (NaN3), for superoxide (KI), and for hydro-
xyl radical (DMSO). As the data of complex 1 showed in Fig. 10a
and Table 7, the lane 3 adding DMSO is different from others.
The Form I is more than that of the lane 2, lane 4, lane 5. So the
DMSO plays as an important inhibitor. The data of complex 2
shown in Fig. 10b and Table 8 and those of complex 3 shown in
Fig. 10c and Table 9, the lane adding DMSO still have more Form
I than the others, verifying that the DMSO plays as an inhibitor
in complexes. However, for the Form II and Form I the lane 2, lane
4, lane 5 stay almost in the same level. So, both KI and NaN3 do not
have the evident effect on inhibiting the metal complex cutting
DNA.
[25] C. Tu, Y. Shao, N. Gan, Q. Xu, Z.J. Guo, Inorg. Chem. 43 (2004) 4761.
[26] Y. Jin, J.A. Cowan, J. Am. Chem. Soc. 127 (2005) 8408.
[27] B.K. Santra, P.A.N. Reddy, G. Neelakanta, S. Mahadevan, M. Nethajia, A.R.
Chakravarty, J. Inorg. Biochem. 89 (2002) 191.
That means the cleavage isn’t influenced by N3ꢂ, neither is it
influenced by normal ions, such as iodine ion for the lane adding
NaN3 or KI does not present essentially differences from the lane