Solvatochromism, DNA binding, antitumor activity and molecular modeling study of mixed-ligand copper(II) complexes containing the bulky ligand: Bis[N-(p-tolyl)imino]acenaphthene

Article abstract of DOI:10.1016/j.ejmech.2007.02.014

Four mixed-ligand copper(II) complexes of the nitrogen ligand bis[N-(p-tolyl)imino]acenaphthene 1 (p-Tol-BIAN). These complexes, namely [Cu(p-Tol-BIAN)₂](ClO₄)₂ 2, [Cu(p-Tol-BIAN)(acac)](ClO₄) 3, [Cu(p-Tol-BIAN)

Full text of DOI:10.1016/j.ejmech.2007.02.014

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 42 (2007) 1325e1333
http://www.elsevier.com/locate/ejmech
Original article
Solvatochromism, DNA binding, antitumor activity and
molecular modeling study of mixed-ligand copper(II)
complexes containing the bulky ligand:
Bis[N-( p-tolyl)imino]acenaphthene
c
b
Usama El-Ayaan a,b, , Alaa A.-M. Abdel-Aziz , Shar Al-Shihry
*
a Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
b Department of Chemistry, College of Science, King Faisal University, P.O. Box 1759, Hofuf 31982, Saudi Arabia
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Received 3 December 2006; received in revised form 26 January 2007; accepted 12 February 2007
Available online 2 March 2007
Abstract
Four mixed-ligand copper(II) complexes of the nitrogen ligand bis[N-( p-tolyl)imino]acenaphthene 1 ( p-Tol-BIAN). These complexes,
namely [Cu( p-Tol-BIAN)2](ClO4)2 2, [Cu( p-Tol-BIAN)(acac)](ClO4) 3, [Cu( p-Tol-BIAN)Cl2] 4 and [Cu( p-Tol-BIAN)(AcOH)2](ClO4)2 5,
were prepared and characterized. Solvatochromism of the novel copper complexes in various solvents has been studied. Molecular mechanics
(MMþ) and molecular dynamic simulations have been performed to learn more about the solvatochromic behaviour and the DNA binding af-
finity of these complexes.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Solvatochromism; Bis[N-( p-tolyl)imino]acenaphthene; DNA binding; Molecular modeling
1. Introduction
Moreover, mixed-ligand complexes are observed in biological
systems or in the intermediate chemical reactions with metal
ions, which are important to understand the respective chemis-
try. Investigations concerning diimine mixed-ligand chelate
systems would provide toward understanding the driving forces
that led to the formation of such mixed-ligand complexes [4e6].
The investigation of solvatochromic behaviour [7] of mixed-
ligand metal complexes has been of importance, because it pro-
vides a quantitative approach to recognize the solvent behaviour
and the role of the solvent in physico-chemical studies [8].
Moreover, it is very helpful for developing environmental sensor
materials, which are chromotropic and exhibit color change
when exposed to solvent or pollutant molecules [9].
Organometallic chemistry has been developed, in the last
four decades, to be the largest and important branch as a link
connecting the fields of organic and inorganic chemistry [1].
One of the major applications of the transition metal complexes
is its medical testing as antibacterial and antitumor agents
aiming toward the discovery of an effective and safe ther-
apeutic regimen for the treatment of bacterial infections and
cancers [2]. In addition, a great many Schiff’ base complexes
with metals have also provoked wide interest because they
possess a diverse spectrum of biological and pharmaceutical
activities, including antitumor and antioxidative activities [3].
In the present study, we report the solvatochromism, DNA
binding affinity and cytotoxic activity of four mixed-ligand
copper(II) complexes of the nitrogen ligand bis[N-(p-tolyl)imi-
no]acenaphthene 1 ( p-Tol-BIAN). These complexes are [Cu
( p-Tol-BIAN)2](ClO4)2 2, [Cu(p-Tol-BIAN)(acac)](ClO4) 3,
* Corresponding author. Department of Chemistry, College of Science, King
Faisal University,P.O. Box 1759, Hofuf 31982, Saudi Arabia. Tel.: þ966 03
5800000; fax: þ966 03 5886437.
E-mail address: uelayaan@kfu.edu.sa (U. El-Ayaan).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.014

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U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
[Cu( p-Tol-BIAN)Cl2] 4 and [Cu( p-Tol-BIAN)(AcOH)2](ClO4)2
5. Molecular mechanics (MMþ) and molecular dynamic simula-
tion have been performed to learn more about the solvatochromic
behaviour and DNA binding affinity of the described complexes.
of interaction. They are (i) minor groove binding, (ii) major
groove binding, (iii) intercalation (between two base pairs)
and (iv) surface binding [14]. Metal complexes having a planar
aromatic heterocyclic functionality (that can insert and stack
between the base pairs of double helical DNA) have contrib-
uted to our understanding of fundamental nucleic acid recog-
nition. These metal complexes have been applied in the
development of DNA cleavage reagents, have assisted in the de-
velopment of low molecular weight drugs, and as stand-alone
metal complexes and have been used as agents to understand
fundamental complex interactions with DNA [15]. A variety
of methods have been utilized for the interaction of small
molecular weight compounds with DNA, such as equilibrium
dialysis [16].
Briefly [17], a fixed amount of ligand is spotted on the RP-
18 TLC plates followed by addition of known amount of DNA
on the same spot. The plate was then developed and the posi-
tion of the DNA was determined by spraying the plates with
anisaldehyde reagent. It is important to establish if the re-
sponse of the test system is dependent on the dose of the
test substance. In the presence of increasing quantities of
DNA intercalators, a greater portion of DNA is bound to
form a complex, and consequently, the free DNA was detected
as a blue spot (MeOHeH2O, 8:2) on RP-18 TLC after spray-
ing with anisaldehyde reagent. On the other hand, compounds
with high binding affinity to DNA retained on the base line.
However, when the DNA was mixed with compounds with
which it is known to interact (ethidium bromide), the complex
was retained at the origin when MeOHeH2O (8:2) was used
for elution. On the other hand, inactive compounds did not
cause the DNA to be retained at the origin.
Moreover, methyl green reversibly binds polymerized DNA
forming a stable complex at neutral pH. The maximum absorp-
tion for the DNAemethyl green complex is 642e645 nm. This
colorimetric assay [17b,18] was used to measure the displace-
ment of methyl green from DNA by compounds having the
ability to bind with DNA. The degree of displacement was deter-
mined spectrophotometrically by measuring the change in the
initial absorbance of the DNAemethyl green solution in the
presence of reference compound.
Results from DNA binding assay (Table 3) revealed that
compounds 2 and 5 showed the highest affinity to DNA, which
was demonstrated by retaining the complex at the origin or by
migrating for very short distances, and by measuring IC50
(concentration required for 50% decrease in the initial absor-
bance of the DNAemethyl green solution). Compounds 3
and 4 showed moderate activity while the original ligand 1
showed weak activity.
2. Results and discussion
2.1. Electronic spectra of complexes in various solvents
The absorption spectra of complexes 2e5 were measured in
different organic solvents with different donor numbers (DN).
The characteristic properties of these complexes are (i) their
high solubility in various organic solvents, and (ii) the change
in the color of the solution on going from one solvent to an-
other, that is, strong solvatochromism of their solutions [10].
The spectra show one broad band attributed to the promotions
of the electron in the lower energy orbitals to the hole in dx2ꢀy2
orbital of the copper(II) ion (d9). The position of this band
is shifted to longer wavelength (red shift) as the donor number
of solvent increases (Table 1). Conductivity data (Table 2) of
studied complexes suggest that ClOꢀ4 acts only as a counter
ion [11]. Therefore, the complex structure depends only on
the donor properties of the solvent used (Gutmann’s donor
number DN) [12], and only soluteesolvent interactions can
be considered.
Solvatochromic behaviour was studied quantitatively by
applying the linear solvation free energy relationship [13].
Linearity of the nmax vs. DN, confirms the solvatochromic be-
haviour in perchlorate complexes. The slope value is the
smallest in case of 2, in which two diimine ligands are coor-
dinated to the copper(II) centre imposing a large steric hin-
drance to the axial coordination of incoming solvent leading
to less solvatochromic behaviour.
2.2. Biological activity
2.2.1. DNA as an affinity probe for evaluation of
biologically active compounds
DNA is the pharmacologic target of many drugs currently
in clinical use or in advanced clinical trials. Small molecules
that bind genomic DNA have proven to be effective antican-
cer, antibiotic, and antiviral therapeutic agents. It is known
that a small molecule can bind to DNA through various modes
Table 1
Absorption maxima lmax (nm) of complexes, 2e5, in different solvents
Solvent
DNa
lmax (3 l/mol cm)b
2
3
4
5
CH2Cl2
CH3NO2
CH3CN
CH3OH
HCON(CH3)2
CH3SOCH3
a
0
2.7
656 (429)
661 (387)
693 (195)
714 (150)
740 (272)
763 (65)
610 (338)
630 (255)
N.d.c
620 (215)
N.d.c
607 (429)
N.d.c
2.2.2. Antitumor activity against Ehrlich ascite carcinoma
cells in mice [19,20]
14.1
19.1
26.6
29.8
720 (200)
750 (153)
769 (206)
868 (56)
N.d.c
720 (209)
763 (331)
830 (44)
745 (272)
763 (472)
845 (67)
2.2.2.1. Effect on survival time. The first measure that can be
used to compare the anti-neoplastic activities for the tested
compounds is the increase in survival time for each treated
group over the control group [19]. The mean survival time
(MST) for each group was calculated by dividing the total
Donor number of solvents, from Ref. [12].
b
c
The extinction coefficient values are given in parenthesis.
N.d., not detected.

U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
1327
Table 2
Analytical and physical data of complexes 2e5
Compound, empirical formula
Lm cm2/U mol
Found (calcd.) %
C
meff B.M.
F. wt
AN
MN
H
N
[Cu( p-Tol-BIAN)2](ClO4)2 (2) C52H40Cl2CuN4O8
[Cu( p-Tol-BIAN)(acac)](ClO4) (3) C31H28ClCuN2O6
[Cu( p-Tol-BIAN)Cl2] (4) C26H20Cl2CuN2
983.35
623.56
494.9
330
180
25
192
100
5
62.28 (63.51)
60.07 (59.71)
62.47 (63.10)
48.1 (48.5)
4.15 (4.10)
4.54 (4.53)
4.24 (4.10)
3.66 (3.80)
5.44 (5.69)
4.27 (4.49)
5.27 (5.66)
3.60 (3.77)
1.39
1.56
1.88
1.98
[Cu( p-Tol-BIAN)(AcOH)2](ClO4)2 (5) C30H28Cl2CuN2O12
743.0
320
188
survival times for all the mice in that group by the number of
mice in the same group, then the percent increase in lifespan
for each group over the control group was calculated as fol-
lows [19]:
each one of the treated groups and from the control group. A
20-fold dilution was made for the taken cells in saline. The cells
in the final dilution were stained with Giemsa stain, and the
number of the viable cells was counted under the microscope.
As shown in Table 6, both compounds 2 and 5 showed sig-
nificant reduction in viable cell count even higher than that
produced by 5-FU. However, the highest degree of reduction
in viable cell count was demonstrated by compound 2.
% Increase in lifespan over control
MST of treated group
¼
ꢁ 100 ꢀ 100:
MST of control group
2.2.2.4. Effect on body weight [19]. Both compounds 2 and
5-FU showed minimal ascites and slight increase in body
weight unlike the control group, in which large ascites were
produced and a marked increase in body weight was observed.
Comparing the % increase in lifespan of the control group
in each treated group (Table 4) revealed that the compound 3
has shown the same increase in lifespan produced by the stan-
dard drug 5-FU. For compound 5, the increase in lifespan was
even higher than that of 5-FU. The maximum increase in
lifespan was obtained by compound 2, which nearly cured
the animals, expanding their lifespan to be approached to the
normal non-diseased group.
2.3. Molecular modeling studies
2.3.1. Solvation energy calculations
An attempt to gain a better insight on the molecular struc-
tures of the solvated complex 4, and the free ligand bis[N-( p-
tolyl)imino]acenaphthene 1, conformational analysis of the
target compounds has been performed by MMþ [21] force field
as implemented in HyperChem 5.1 [22]. The starting atomic
coordinates of the target compounds were obtained from the
X-ray data of the structure analogues [23]. The PM3 semi-
empirical [24] calculations performed on free ligand bis[N-
( p-tolyl)imino]acenaphthene showed that the diaryl groups
were arranged itself perpendicular to the plane of acenaphthene
with face to edge interaction and are not in the plane of the
imine N-bond but make an angle :C2eN2eC6eC7 of 61.4ꢂ
to it. This non-coplanar orientation of p-tolyl substituent is
caused by the presence of naphthalene backbone, which pre-
vents rotation of p-tolyl substituent in the plane formed by
the imine function as we mentioned in our previous report
[2a]. Moreover, it is noteworthy to say that the MMþ and
PM3 calculations give results which are in good agreement
with the X-ray data of the structure analogues [2a,23].
2.2.2.2. Effect on biochemical and haematological parameters
[19]. On the day 14, the biochemical and haematological
parameters, as regard to haemoglobin level, erythrocytes and
leucocytes counts, were compared in the group treated with
the standard drug 5-FU and the group treated with the newly
synthesized compound 2, which has shown the highest % in-
crease in lifespan over control, with values obtained from nor-
mal and control groups.
As shown in Table 5, the biochemical and haematological
parameters in the group treated with the compound 2 have
been nearly recovered completely to be within the normal
values. The values are comparable to those obtained from
the group treated with 5-FU, and they are even better in
some of the aspects, as regard to leucocyte count.
2.2.2.3. Effect on viable cell count of Ehrlich ascite carcinoma
cells [19]. After five days of treatment, 100 ml samples were
taken from Ehrlich ascite carcinoma cells from three mice in
Table 3
DNA binding activity of compounds 1e5 using DNAemethyl green displace-
ment assay
Table 4
The % increase in lifespan for each treated group over the control group in
EAC test
Compd. no.
IC50 (mg/ml)a
1
78 ꢃ 2
36 ꢃ 3
56 ꢃ 4
63 ꢃ 2
48 ꢃ 1
28 ꢃ 3
Group no.
Treatment (I.P.)
% Increase in lifespan
2
1
2
3
4
5
6
Normal (untreated)
Control (Ehrlich only)
71.4
0.0
3
4
2
55.1
42.0
44.1
42.8
5
Daunomycin
3
5
5-FU
a
Values represent the concentration (mean ꢃ SD, n ¼ 3 ꢀ 5) required for
50% decrease in the initial absorbance of DNAemethyl green solution.

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U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
Table 5
Effect of the treatment on biochemical and haematological parameters
Table 7
Experimental, calculated solvation energies, van der Waals energies and elec-
trostatic energies of complex 4 with various solvents
Group type
(Hb/g) % RBCs/106 mm3 Total WBCs/103 mm3
Solvent
lmax(3 l/mol cm)a Esolvationb, Evan der Eelectrostaticd,
Normal
Control (Ehrlich only)
13.4
5.7
5.1
4.35
47.4
(kcal/mol)
Waalsc,
(kcal/mol)
(kcal/mol)
3.15
4.96
5.87
2
5-FU
9.1
15.6
12.45
14.7
CH2Cl2
CH3CN
CH3OH
620
720
750
17.01
8.43
2.90
12.70
12.77
8.04
4.41
0.81
1.26
ꢀ16.22
1.01
HCON(CH3)2 769
(CH3)2SO
ꢀ2.31
ꢀ6.00
11.48
1.05
The correlation between the solvatochromism as expressed
by the absorption spectra of compound 4 measured in different
organic solvents and the calculated solvation energy was stud-
ied using molecular dynamic calculation as shown in Table 7
and Fig. 1. However, each of the solvated complexes has been
subjected to molecular dynamic (MD) simulations followed by
energy minimization of the snap shots collected at regular time
intervals during the MD simulations. After energy minimiza-
tion all the energy minima obtained have been compared
with each other and the one with the lowest energy has been
picked as the representative of the solvated complex
(Fig. 1). The least energy complex thus picked up has been se-
lected to calculate the solvation energy as a measure of solva-
tochromism. The solvation energies between compound 4 and
solvents are given in Table 7. The complexes are characterized
by the presence of a number of non-bonded interactions
formed between compound 4 and the solvent molecules in
addition to the covalent linkage formed between the metal
centre and solvent molecules. The solvents (DMSO, DMF,
and MeOH) offer more favourable non-bonded interactions
as compared to the CH2Cl2 and MeCN, this is evident from
their respective non-bonded energy terms (Table 7), which
are about 3e20 kcal/mol less as compared to CH2Cl2. Among
the studied solvated complex, 4eDMSO and 4eDMF com-
plexes are associated with the lowest non-bonded energy
term and are stabilized by the CH/p interaction of alkyl group
868
a
Data were taken from Table 1.
The energy of interactions between complex 4 and solvents.
Evan der Waals term describes the repulsive forces keeping two non-bonded
b
c
atoms apart at close range and the attractive force drawing them together at long
range.
d
Eelectrostatic describes the classical non-bonded electrostatic interactions
of charge distributions.
potent 4 as a representative example. Each of the DNAetarget
complexes has been subjected to molecular dynamic (MD)
simulations followed by energy minimization of the snap shots
collected at regular time intervals during the MD simulations.
After energy minimization all the energy minima obtained
have been compared with each other and the one with the low-
est energy has been picked as the representative of the DNAe
target complex (Fig. 2). The least energy complex thus picked
up has been selected to calculate the interaction energy as
a measure of stability of complex. The interaction energies
between DNA (AT) base pairs and compounds 2 and 4 are
given in Table 8. It can be seen from this table that the dimer
DNAe2B renders more stability to the complex compared
to the monomers DNAe2A and DNAe4. This property corre-
lates well with the experimentally determined values of the
DNA binding in these complexes. The complexes are charac-
terized by the presence of a number of non-bonded interactions
(Table 8) formed between DNA and copper complexes. The di-
meric 2B offers more favourable non-bonded interactions as
compared to the monomeric 2A or 4 and this is evident from
their respective non-bonded energy terms (Table 8), which
are about 18e25 kcal/mol less as compared to 2A and 4. The
dimeric 2B is associated with the lowest non-bonded energy
term and is stabilized by pep interaction among the p-tolyl
groups, p-stacking interaction between each pair of p-tolyl
aromatic ring and CH/p interaction among the p-tolyl groups
and aliphatic frame of pair bases (AT) [2a,25]. The binding en-
ergies calculated in HyperChem for the three models, dimeric
2B, monomeric 2A and compound 4, were found to be ꢀ70.9,
ꢀ34.2 and ꢀ30.7 kcal/mol, respectively, correlating well with
the experimental DNA binding values. The more negative the
relative binding energy, the more potent the binding is between
DNA and target molecules.
˚
with acenaphthene ring (w2.9 A) [2a,25]. This non-bonded
interaction may be strong enough to compensate the steric re-
pulsion between the solvent molecule and the compound 4.
The relative solvation energies calculated in HyperChem for
complex 4 with DMSO, DMF, MeOH, MeCN and CH2Cl2
were found to be ꢀ6.0, ꢀ2.31, 2.9, 8.43 and 17.01 kcal/mol,
respectively, correlating well with the experimental solvato-
chromism values. The more negative the solvation energy,
the more stable the 4-solvent complex.
2.3.2. DNA binding energy calculations
Docking studies were also performed to investigate the
DNA binding differences of the most potent 2 and the least
Table 6
Viable cells count of EAC after 5 days of treatment with tested compounds and
5-FU
Group no.
Group type
Count (cells)/100 ml
2
3
4
5
6
Control (Ehrlich only)
2
192.8 ꢁ 106
37.6 ꢁ 106
50.2 ꢁ 106
45.6 ꢁ 106
83.6 ꢁ 106
2.4. Conclusion
3
5
5-FU
The solvatochromic behaviour of four mixed-ligand cop-
per(II) complexes containing the bidentate nitrogen ligand

U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
1329
Fig. 1. Docking of various solvents with the energy minimized complex 4 with ball and cylinder rendering.
bis[N-( p-tolyl)imino]acenaphthene 1 was studied. These
complexes are [Cu( p-Tol-BIAN)2](ClO4)2 2, [Cu( p-Tol-BIA-
N)(acac)](ClO4) 3, [Cu( p-Tol-BIAN)Cl2] 4 and [Cu( p-Tol-
BIAN)(AcOH)2](ClO4)2 5. However, the relative solvation
energies calculated for complex 4 with DMSO, DMF,
MeOH, MeCN and CH2Cl2 were found to be ꢀ6.0, ꢀ2.31,
2.9, 8.43 and 17.01 kcal/mol, respectively, which correlated
well with the experimental solvatochromism values. In vivo
antitumor and in vitro DNA binding activities were performed
showing that complexes 2, 3 and 5 gave the highest DNA bind-
ing affinity and antitumor activity which are more active than
the parent ligand bis[N-( p-tolyl)imino]acenaphthene 1. Mo-
lecular dynamics simulation has been performed to study the
DNA binding affinity of the most and least active complexes

1330
U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
Fig. 2. Docking studies of the most potent DNA binding compound, 2 (A, B; upper panel), and least potent, 4 (lower panel), into DNA base pairs (AT) with CPK
rendering. The compounds 2 and 4 are shown in violet, to distinguish them from the DNA base pairs and hydrogen atoms were omitted for clarity (For interpre-
tation of the references to color in this figure legend, the reader is referred to the web version of this article.).
2 and 4, respectively. The relative binding energies calculated
for the deduced models, dimeric 2B, monomeric 2A and com-
pound 4, were found to be ꢀ70.9, ꢀ34.2 and ꢀ30.7 kcal/mol,
respectively, correlating well with the experimental DNA
binding values.
3. Experimental
3.1. Instrumentation and materials
All starting materials were purchased from Sigmae
Aldrich Co., USA, and used without further purification.
Elemental analyses (C, H, N) were performed on a Perkin-
Elmer 2400 Series II Analyzer. Electronic spectra were re-
corded on a UV-UNICAM 2001 spectrophotometer using
10 mm pass length quartz cells at room temperature. Mag-
netic susceptibility was measured with a Sherwood Scien-
tific magnetic susceptibility balance at 297 K. Infrared
spectra were recorded on a Perkin-Elmer FT-IR Spectro-
meter 2000 as KBr pellets and as Nujol mulls in the
Table 8
The values of energy of interaction calculated for the DNA-monomer and
dimer complexes
Compd. No.
Binding energy Evan der
(kcal/mol)
Eelectrostatic IC50 (mg/ml)a
(kcal/mol)
Waals
(kcal/mol)
4
ꢀ30.7
ꢀ7.34
ꢀ0.067
ꢀ18.40
ꢀ22.38
ꢀ17.57
ꢀ42.14
63 ꢃ 2
36 ꢃ 3
36 ꢃ 3
2A (monomer) ꢀ34.2
2B (dimer)
ꢀ70.8
Data were taken from Table 3.
4000e370 cmꢀ1 spectral range. H and 13C NMR measure-
ments at room temperature were obtained on a Jeol JNM
1
a

U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
1331
LA 300 WB spectrometer at 400 MHz, using a 5 mm probe
3.4. Biological screening
head in CDCl3. Chemical shifts are given in ppm relative to
internal TMS (tetramethylsilane); Giemsa stain was from
Fisher Scientific Co, Fair Lawn, NY, USA, TLC plates
from RP-18F254; 0.25 mm, Merck, DNA, calf thymus type
I, Sigma, 100 mg/ml, and DNAemethyl green was from
Sigma, St. Louis, MO, USA.
3.4.1. Evaluation of the degree of DNA binding
3.4.1.1. DNA binding assay on TLC plates
3.4.1.1.1. Methods. The TLC plates used in the assay were
pre-developed first using methanolewater (8:2). The tested
compounds were then applied (5 mg/ml in methanol) at the or-
igin, followed by the spotting of DNA (1 mg/ml in methanole
water mixture (8:2)) at the same positions at the origin. Ethid-
ium bromide was used as a positive control. After complete
spotting, the plates were developed with the same solvent
system, and the positions of DNA were visualized by spraying
the plates with anisaldehyde, which produces a blue color with
DNA. The intensity of the color was proportional to the quan-
tity of DNA added to the plate.
3.2. Synthesis of bis[N-( p-tolyl)imino]acenaphthene
( p-Tol-BIAN), 1
Preparation of the ligand was carried out in two steps as
follow.
3.2.1. Step 1: preparation of ( p-Tol-BIAN)ZnCl2
A mixture of 3.0 g acenaphthenequinone (16.5 mmol),
2.5 g anhydrous ZnCl2 (18.0 mmol) and 3.75 g p-toluidine
(35.0 mmol) in 100 ml acetic acid was heated under reflux
(80 ꢂC) for 1 h. Then the mixture was cooled to room tem-
perature and the solid product was filtered off to give an
orange solid that was washed with acetic acid followed by
diethyl ether and air dried, yield 7.5 g of ( p-Tol-BIAN)ZnCl2
(91%).
3.4.1.2. Colorimetric assay for the degree of DNA binding
3.4.1.2.1. Methods. DNAemethyl green complex (20 mg)
was suspended in 100 ml of 0.05 M triseHCl buffer (pH
7.5) containing 7.5 mM MgSO4 and stirred at 37 ꢂC with
a magnetic stirrer for 24 h. The calculated amounts of sam-
ples were placed in Eppendorf tubes, and 200 ml of the
DNAemethyl green solution was added to each tube. The
samples were incubated in dark at ambient temperature
and after 24 h, the final absorbance of each sample was de-
termined at 642e645 nm. The results were recorded in form
of the IC50 of each compound, which is the sample concentra-
tion required to produce 50% decrease in the initial absor-
bance of the DNAemethyl green complex. Daunomycin
was used as a positive control.
3.2.2. Step 2: removal of ZnCl2
( p-Tol-BIAN)ZnCl2 (6.7 g) was added to a solution of 4 g
K2CO3 in 150 ml water and the mixture was heated under re-
flux with continuous stirring. The mixture was then filtered
and the product was washed repeatedly with water. The prod-
uct was dissolved in boiling ethanol while the solid zinc car-
bonate was removed by filtration. Ethanolic solution of the
ligand was evaporated to the quarter and set aside. After one
day the product was filtered and dried in vacuo.
3.4.2. In vivo antitumor activity against Ehrlich ascite
carcinoma cells in mice
The prolongations of lifespan of Ehrlich ascite carcinoma
cells (EAC) bearing hosts, the recovery of normal biochemical
and haematological profiles and the reduction in viable tumor
cell count are three important measures that have been used in
this in vivo testing for the evaluation of the anti-neoplastic ac-
tivity for three of the newly synthesized compounds, selected
on the basis of the results obtained from the previous evalua-
tion method, where they showed the highest DNA-affinity.
These compounds are 2, 3 and 5.
Yield: 3.50 g (72%). Found: C, 86.60; H, 5.50; N, 7.66.
Calc. for C26H20N2 (360.45): C, 86.64; H, 5.59; N, 7.77%.
1H NMR (CDCl3, recorded at 400 MHz at 23.6 ꢂC) d ¼ 2.44
(s, p-Me), 6.92 (d, H3), 7.03 (d, H9), 7.26 (d, H10), 7.37
(pst, H4), 7.86 (d, H5). 13C NMR (CDCl3, 400 MHz,
24.5 ꢂC): d ¼ 21.06 ( p-Me), 161.06 (C1), 128.49 (C2),
123.68 (C3), 127.38 (C4), 128.62 (C5), 131.0 (C6), 141.49
(C7), 149.0 (C8), 118.04 (C9, C13), 129.77 (C10, C12),
133.68 (C11).
3.4.2.1. Materials. Ehrlich ascite carcinoma cells (EACs): the
cells of Ehrlich ascites tumor were obtained from National
Cancer Institute, Cairo, Egypt. After harvesting and prepara-
tion of the cells, their total number and viability were deter-
mined by counting using Trypan blue. The desired
concentration of tumor cells (2 ꢁ 106 cells/0.2 ml) was ob-
tained by dilution with saline (0.9% sodium chloride solution).
The viability of tumor cells obtained and used in this experi-
ment was always higher than 90%. Below this percentage,
the cells were discarded and the entire procedure was repeated.
5-Fluorouracil was obtained from SigmaeAldrich Co., USA.
Adult Swiss male albino mice (20e25 g) of both sexes were
used in this experiment. They were purchased from
3.3. Synthesis of copper(II) complexes
Mixed-ligand copper complexes (2e5) containing the
rigid ligand bis[N-( p-tolyl)imino]acenaphthene were prepared
by adding the appropriate amount of copper perchlorate or
copper chloride (in case of 4) to an ethanolic solution of
the p-Tol-BIAN ligand. To this mixture acac (in case of 3)
or acetic acid (in case of 5) was added. The mixtures were
stirred (while heating) for 1e3 h. The resulting green solid
product was filtered, washed with ethanol and air dried.
The analytical and physical data of complexes are listed in
Table 2.

1332
U. El-Ayaan et al. / European Journal of Medicinal Chemistry 42 (2007) 1325e1333
Pharmacology Department, Faculty of pharmacy, Mansoura
University, Egypt. They were housed in microlon boxes in
a controlled environment (temperature 25 ꢃ 2 ꢂC and 12 h
dark/light cycle) with standard laboratory diet and water
regimen.
shots was subjected to final molecular mechanics relaxation.
The PolakeRibiere minimization method was applied with
a gradient value of 0.01 kcal to test for convergence, to generate
the refined complex. The energy of the complexesolvent
(Ecomplexesolvent) and the energies of solvent (Esolvent) and com-
plex (Ecomplex) individually after separating from the complex
were calculated. Energy of solvation (Esolvation) between com-
plex and solvent molecules was calculated using the following
formula: Esolv ¼ Ecomplexesolvent ꢀ (Ecomplex þ Esolvent), where
Esolv ¼ total energy of interaction of complex and solvent,
Ecomplex ¼ total energy of the complex alone, Esolvent ¼ total
energy of the two molecules of solvent.
3.4.2.2. Methods. Animals were divided into 6 groups with
10 animals for each including normal (non-treated), control
group (Ehrlich only, non-treated), treated group with the tar-
get compounds (5 mg/kg) and positive control group treated
with 5-fluorouracil (20 mg/kg). All the animals in the treated
groups (from 2 to 6) were inoculated with 2 ꢁ 106 Ehrlich
ascite carcinoma cells/mouse on the day zero. Treatment
started 24 h after inoculation by intra-protenial injection of
the drug. The animals in groups 3e5 were injected by the
tested compound in a dose of 100 mg/mouse, which is nearly
equivalent to 5 mg/kg of body weight, while the standard
group (group 6) has received I.P. treatment with 20 mg/kg
of body weight of 5-fluorouracil. The control group (group
2) was treated with the same volume of 0.9% sodium chlo-
ride solution. All the treatments were given for nine succes-
sive days.
3.5.2. DNA binding calculation using molecular dynamic
docking
Molecular dynamic studies were carried out using the fol-
lowing protocol: heating phase (equilibration) ¼ 30 ps and
sampling phase ¼ 100 ps. Intermittent structures of the com-
plex formed at every 10 ps of simulation were collected and
subjected to EM. The minima of all the snap shots were exam-
ined in order to select the lowest energy conformation as a rep-
resentative of DNAetarget complex for further studies. The
energy of the DNAetarget complex (Ecomplex) and the energies
of DNA (EAT) and target molecules (Etarget) individually after
separating from the complex were calculated. Energy of inter-
action (Eint) between DNA (AT) and target molecule was
3.5. Molecular modeling
3.5.1. Modeling of solvatochromism through solvation
energy calculations
calculated using the following formula: Eint ¼ Ecomplex
ꢀ
(EAT þ Etarget), where Eint ¼ energy of interaction of the
complex, Ecomplex ¼ total energy of the complex, EAT and
Etarget are the individual total energies of the DNA (AT) and
the target molecules calculated after they are separated from
each other, respectively.
Initial structures for the complex 4 as a representative
example and the solvents (Table 7) used in this study were
constructed using the HyperChem version 5.1 program, which
was also used for manipulation and interactive solvation en-
ergy calculation protocol. The conformational searching in
torsional space was performed using the multi-conformer
method [17b]. Energy minima for 1, 2 and 4 were determined
by a semi-empirical method PM3 (as implemented in Hyper-
Chem 5.1). The conformations thus obtained were confirmed
as minima by vibrational analysis. Atom-centred charges for
each molecule were computed from the PM3 wavefunctions
(HyperChem 5.1) which provides derived charges that closely
resemble those obtainable from ab initio 6-31G* calculations.
The MMþ force field (as implemented in HyperChem 5.1)
was used for all interactive docking of the hosteguest molec-
ular dynamic calculations. For reasons of computational
expense, solvent effect was simulated using a simple dis-
tance-dependent dielectric constant and two molecules of a sol-
vent were used for docking into vicinity of complex 4. The
distance between the metal core of complex and the docked
Acknowledgements
The financial support by the Deanship of Scientific Re-
search (Project Number 7035) King Faisal University, Saudi
Arabia is gratefully acknowledged. Authors would like to
thank Prof Farid Badria (Department of Pharmacognosy, Fac-
ulty of Pharmacy, Mansoura University, Egypt) for providing
the biological data.
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