Copper complexes with phosphonium containing hydrazone ligand: Topoisomerase inhibition and cytotoxicity study

Article abstract of DOI:10.1016/j.ejmech.2014.02.049

Four new copper(II) complexes containing phosphonium substituted hydrazone (L) with the formulations [CuL]Cl(3), [Cu(phen)L]Cl(4), [Cu(bpy)L]Cl(5), [Cu(dbpy)L]Cl(6), (where L = doubly deprotonated hydrazone; phen = 1,10′-phenanthroline; bpy = 2,2′-bipyridine; dbpy = 5,5′-dimethyl-2,2′-bipyridine) have been synthesized. The compounds were characterized by elemental analysis, spectroscopic methods and in the case of crystalline products by X-ray crystallography. The cytotoxicity and topoisomerase I (topo I) inhibition activities of these compounds were studied. It is noteworthy that the addition of N,N-ligands to the copper(II) complex lead to the enhancement in the cytotoxicity of the compounds, especially against human prostate adenocarcinoma cell line (PC-3). Complex 4 exhibits the highest activity against PC-3 with the IC₅₀ value of 3.2 μΜ. The complexes can also inhibit topo I through the binding to DNA and the enzyme.

Full text of DOI:10.1016/j.ejmech.2014.02.049

European Journal of Medicinal Chemistry 76 (2014) 397e407
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Copper complexes with phosphonium containing hydrazone ligand:
Topoisomerase inhibition and cytotoxicity study
,
a
a
b
Shin Thung Chewa, Kong Mun Lo a , Sze Koon Lee , Mok Piew Heng , Wuen Yew Teoh ,
*
,
Kae Shin Sim b, Kong Wai Tan a
*
a Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
b Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new copper(II) complexes containing phosphonium substituted hydrazone (L) with the formula-
tions [CuL]Cl(3), [Cu(phen)L]Cl(4), [Cu(bpy)L]Cl(5), [Cu(dbpy)L]Cl(6), (where L ¼ doubly deprotonated
hydrazone; phen ¼ 1,100-phenanthroline; bpy ¼ 2,20-bipyridine; dbpy ¼ 5,50-dimethyl-2,20-bipyridine)
have been synthesized. The compounds were characterized by elemental analysis, spectroscopic
methods and in the case of crystalline products by X-ray crystallography. The cytotoxicity and topo-
isomerase I (topo I) inhibition activities of these compounds were studied. It is noteworthy that the
addition of N,N-ligands to the copper(II) complex lead to the enhancement in the cytotoxicity of the
compounds, especially against human prostate adenocarcinoma cell line (PC-3). Complex 4 exhibits the
Received 11 October 2013
Received in revised form
17 February 2014
Accepted 18 February 2014
Available online 19 February 2014
Keywords:
Hydrazones
Cytotoxicity
Topoisomerase I inhibitor
Copper(II) complexes
highest activity against PC-3 with the IC50 value of 3.2
the binding to DNA and the enzyme.
m
M. The complexes can also inhibit topo I through
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
enhanced interaction with DNA [19,20]. Cytotoxic activities of
several copper(II) complexes have already been evaluated for their
potential antitumour activities on different cell lines [21,22]. Top-
oisomerases have been identified as important targets in cancer
chemotherapy and are among the most widely clinically used
anticancer drugs [23e25]. Yet, very few metal complexes have been
reported to inhibit topoisomerases in contrast to organic molecules
[26,27]. Based on the above findings, we attempt to develop a new
series of copper(II) complexes containing 5-(triphenylphospho-
niummethyl)-salicylaldehyde benzoylhydrazone] chloride ligand
(2) and heterocyclic bases such as 1,100-phenanthroline (phen),
2,20-bipyridine (bpy) and 5,50-dimethyl-2,20-bipyridine (dbpy) as
copper based anticancer agents. In order to gain better insight into
the mode of action of these potentially cytotoxic compounds, we
report herein the first example for the interaction of copper com-
plexes of hydrazone ligand with topoisomerase I.
Success of cisplatin as an anticancer agent has drawn the
attention of many chemists towards the development of new metal
based drugs in order to overcome the drawbacks of cisplatin [1e4].
Copper is well known as a bio-essential element for human and its
complexes have proved to be excellent for biological applications
which include the treatment of cancers [5e8]. Over the last decade,
Schiff bases are gaining prominence in medicinal chemistry due to
its great chemotherapeutic application [9,10]. It is believed that the
type of coordinated ligands and the geometrical orientation of the
ligands are crucial factors in promoting the interaction of a given
metal complex with DNA.
Recently, Krishnamoorthy and co-workers have shown that
copper hydrazones complexes have better potential than other
metal complexes in the conversion of DNA from supercoiled form
(form I) to the nicked circular form (form II) [11]. Apart from that,
there are also numerous copper complexes of hydrazone which
possess anticancer properties [12e18]. Therefore, copper hydra-
zones complexes are one of the important candidates in metal
based drugs research. On the other hand, heterocyclic bases play a
pivotal role in many biological application and significantly
2. Results and discussion
2.1. Syntheses and characterization
The reaction scheme for the synthesis of copper complexes was
presented in Scheme 1. The complexes were highly soluble in
DMSO, DMF and methanol. They are non-hygroscopic and stable
both in solid and solution phases. The elemental analyses data for
* Corresponding authors.
http://dx.doi.org/10.1016/j.ejmech.2014.02.049
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

398
S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
Scheme 1. Reaction scheme and proposed structures.
the Cu(II) complexes are in good agreement with the molecular
formulae of the complexes. Compound 1 is white in colour while
Schiff base ligand 2 is yellow and all the metal complexes are green.
presented in Table 1 and the spectra are shown in Figs. S1 and S2.
The bands in the region of 312e328 nm and 228e276 nm of
complexes 3e6 are due to the n /
the hydrazone ligands and the coordinated diimine ligands
[19,29]. The n / * transition, which appears in the region of
289e300 nm in the spectrum of the uncomplexed hydrazone
ligand was slightly shifted to higher wavelength upon
p* and p / p* transitions of
p
2.2. Infrared spectra
The IR spectra of the complexes in the region 4000e400 cmꢀ1
were analyzed. In comparison with the free ligand, the data gave
evidence of coordination of ligand 2 to the copper metal ion via
phenolate oxygen, azomethine nitrogen and enolate oxygen. In the
IR spectrum of 2, a strong band at 1679 cmꢀ1 has disappeared in all
the IR spectra of complexes which is assigned to the stretching
vibration of carbonyl [v(C]O)] [19,28]. The infrared spectra of 3e6
display IR absorption bands at 1613, 1612, 1612, and 1612 cmꢀ1
respectively which are ascribed to the v(C]N) stretching fre-
quencies of the complexes, whereas for the free ligand the same
band was observed at 1619 cm-1. The shift of this band on
complexation towards lower wavenumbers indicates the coordi-
nation of azomethine nitrogen v(C]N) to the copper centre
[11,18,29]. The appearance of the bands in the 1500e1503 cmꢀ1
range in the complexes are due to the asymmetric stretching vi-
bration of the newly formed N]C bond as a result of the enoliza-
tion of ligand (2) [11,30]. In addition, the characteristic v(NeN)
stretching of the free ligand 2 (observed at 1028 cm-1) undergoes a
positive shift to a higher wavenumber upon complexation which is
attributed to the diminished repulsion between the lone pairs of
adjacent nitrogen atoms [31e33]. The formation of complexes 3e6
have been confirmed by the presence of bands around w489 and
w517 cmꢀ1 corresponding to v(CueN) and v(CueO) respectively
[19,28,30].
a
complexation. This is an indication of the enolization followed by
the deprotonation of the ligand during complexation [30]. The
broad bands observed at approximately 400 nm are attributed to
the ligand-to-metal-charge transfer (LMCT) transition [11,29,33].
Their broadness may be due to the overlapping of the LMCT
transitions of O / Cu and N / Cu. The complexes with N,N donor
ligands (4e6) displayed ded bands in the 649e655 nm range,
which tend to be indicative of a distorted square pyramidal ge-
ometry [19] while complex 3 that displays a square planar coor-
dination, absorbs at 636 nm [34].
2.4. 1H- and 13C- NMR
Additional structural information can be deduced from the 1H
NMR and 13C NMR spectra and relevant chemical shifts are pre-
sented in Tables S1 and S2. Since copper(II) complexes are para-
magnetic in nature, its NMR spectrum could not be obtained. In the
1H NMR spectrum of compound 1, the chemical shift for the alde-
hydic proton appears at 10.10 ppm. Upon the formation of Schiff
base ligand 2, the aldehydic proton (eCHO) is replaced by azome-
thine proton (N]CH) which is shifted upfield to 8.46 ppm. The
formation of ligand 2 is further corroborated by the presence of e
NH proton at 12.24 ppm. The multiplets that appear at the region of
7.62e7.89 ppm are ascribed to the aromatic protons from triphe-
nylphosphine, whereas the aromatic protons of the benzhydrazide
appear in the region of 7.49e7.59 ppm and 7.91e7.93 ppm. The
sharp doublets signal around 5.00 ppm is assigned to the methy-
lene proton.
2.3. Electronic spectra
The significant electronic absorption bands in the spectra of the
ligand 2 and all the complexes recorded in methanol solution are

S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
l
/nm (ε/Lcmꢀ1 molꢀ1) for ligand and its copper(II) complexes in methanol.
399
Table 1
Electronic spectral assignments,
Compounds
ded
LMCT
n /
p
*
p / p*
2
3
4
5
6
e
e
289 (54,521.6), 300 (51,044.8)
313 (39,570.4), 328 (36,924.0)
312 (43,261.6), 327 (37383.2)
312 (41,484.0), 328 (37,324.0)
312 (48,252), 327 (37,284.8)
277 (51,250.4)
228 (92,246.4), 269 (55,742.4)
228 (153,222.4), 268 (103,222.4)
229 (111,669.6), 276 (76,757.6)
229 (106,865.6), 270 (71,025.6)
636 (158.57)
655 (165.16)
649 (162.23)
655 (161.01)
392 (34,970.4)
391 (34,043.2)
392 (35,322.4)
392 (34,365.6)
From 13C NMR spectrum of compound 1, the chemical shift at
189.41 ppm and 160.83 ppm is due to C]O and CeOH respectively.
It is noteworthy that when compound 1 condensed with benzhy-
drazide, the chemical shift of carbonyl carbon (C]O) is replaced by
azomethine carbon (C]N) in ligand 2 with 146.80 ppm. Addi-
tionally, the signal at 162.95 ppm is ascribed to the carbonyl carbon
from the benzhydrazide. The doublet that falls around 27.00 ppm is
assigned to the methylene carbon for both 1 and 2.
counterions linked to the cations via an OeH/Cl hydrogen bonding
ꢀ
interaction
[O1eH1/Cl2
¼
2.983(2)
A,
symmetry
ꢀ
operator: ꢀx, ꢀy þ 1, ꢀz; O3eH3/Cl1 ¼ 2.996(2) A, symmetry
operator: ꢀx, ꢀy þ 1, ꢀz] (Table 4). The condensation of 1 with
benzhydrazide gave the Schiff base ligand, [5-(triphenylphospho-
niummethyl)-salicylaldehyde benzohydrazone] chloride mono-
hydrate (2). The unit cell of compound 2 as depicted in Fig. 2 also
consists of two molecules which are linked together by hydrogen
bonding between the water molecule and imino hydrogen with the
chloride anion. In addition, the presence of intramolecular
hydrogen bonding between the phenolate hydroxyl group and the
azomethine nitrogen stabilizes the molecules. The relatively short
2.5. X-ray crystal structures
Compounds 1e5 were structurally characterized by single
crystal X-ray crystallography. Selected crystallographic data are
summarized in Table 2. Figs. 1e5 show the ORTEP plots of com-
pounds 1e5. Selected bond lengths and angles are given in Table 3.
The chloromethylation of salicylaldehyde followed by the addition
of triphenylphosphine yielded (3-formyl-4-hydroxybenzyl)triphe-
nylphosphonium chloride (1). Its asymmetric unit (Fig. 1) consists
of two crystallographically independent cations and two chloride
ꢀ
ꢀ
bond distances of C60eO4 [1.223(3) A] and C27eO2 [1.222(3) A]
ꢀ
ꢀ
compared to those of C56eO3 [1.356(3) A] and C23eO1 [1.354(3) A]
(Table 3) indicate that the Schiff base exists predominantly in keto
configuration in the solid phase.
As can be seen from Fig. 3, the copper ion is coordinated to the
tridentate hydrazone ligand, through the phenolate oxygen (O1),
azomethine nitrogen (N1) and enolate oxygen (O2) and with a
Table 2
Crystallographic data for compounds 1e5.
Compounds
1
2
3
4
5
Chemical formula
Mr
C26H22ClO2P
432.86
C33H30ClN2O3P
569.01
C33H26ClCuN2O2.38
618.52
P
C47H42ClCuN4O4P
856.81
C45H42ClCuN5O4P
846.80
Crystal system
Unit cell dimension
Triclinic, P-1
Triclinic, P-1
Monoclinic, P2 (1)/c
Triclinic, P-1
Triclinic, P-1
ꢀ
a (A)
9.7734 (1)
13.0679 (2)
17.6675 (2)
105.527 (1)
92.536 (1)
90.629 (1)
2171.36 (5)
4
10.2722 (2)
16.1112 (3)
19.5664 (4)
68.629 (1)
78.208 (1)
73.093 (1)
2867.6 (1)
4
8.6309 (3)
23.3370 (8)
14.3886 (5)
10.4487 (2)
13.5982 (5)
15.4688 (5)
110.199 (3)
100.089 (2)
90.576 (2)
2024.9 (1)
2
12.3972 (2)
12.9623 (2)
14.3509 (2)
97.526 (1)
112.581 (1)
104.634 (1)
1992.05 (5)
2
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢁ)
(ꢁ)
(ꢁ)
106.307 (2)
3
ꢀ
V (A )
2781.6 (2)
4
Z
F (000)
904
1.324
2.4e28.3
0.27
1192
1.318
2.5e26.9
0.23
1272
1.477
2.6e23.5
0.98
890
1.405
2.5e29.3
0.70
880
1.412
2.6e28.4
0.71
Dx (Mg mꢀ3
)
q
(ꢁ)
m
(mmꢀ1
)
T (K)
100 (2)
Colourless, Block
0.10 ꢂ 0.05 ꢂ 0.04
0.974
100 (2)
Yellow, Block
0.10 ꢂ 0.10 ꢂ 0.05
0.978
100 (2)
Green, Block
0.20 ꢂ 0.15 ꢂ 0.08
0.829
100 (2)
Green, Block
0.20 ꢂ 0.05 ꢂ 0.02
0.873
100 (2)
Green, Block
0.1 ꢂ 0.08 ꢂ 0.05
0.649
Crystal colour, habit
Crystal size (mm)
Tmin
Tmax
0.989
0.989
0.926
0.986
0.746
Observed data [I > 2
s(I)]
7215
7573
3711
6328
8202
Rint
0.029
26.5
1.2
0.042
25.5
1.1
0.071
25.5
2.3
0.022
25.0
2.5
0.016
27.5
1.9
qmax (ꢁ)
qmin (ꢁ)
h
k
l
ꢀ12 to 12
ꢀ16 to 16
ꢀ22 to 22
0.040
0.104
1.05
ꢀ12 to 12
ꢀ19 to 19
ꢀ23 to 23
0.046
0.121
1.02
ꢀ10 to 10
ꢀ28 to 26
ꢀ17 to 17
0.047
0.123
1.02
ꢀ12 to 12
ꢀ16 to 11
ꢀ17 to 18
0.036
0.099
1.07
ꢀ16 to 16
ꢀ16 to 16
ꢀ18 to 18
0.035
0.102
1.04
R [F2 > 2
wR (F2)
S
s
(F2)]
ꢀꢀ3
Drmax (e A
Drmin (e A
CCDC number
)
0.45
0.40
0.96
0.70
0.67
ꢀꢀ3
)
ꢀ0.31
ꢀ0.30
ꢀ0.51
ꢀ0.57
ꢀ0.30
957,702
957,703
957,704
957,705
957,706

400
S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
ꢀ
C24eO2 [1.295(4) A] shows that the Schiff base adopts the enol
configuration when coordinated to the copper ion. There is a water
molecule (O3) that persists in the molecular structure which was
found to be disordered and was refined with a chemical occupancy
of 0.38.
On the other hand, the reaction of copper(II) ion with hydrazone
ligand 2 in the presence of 1,100-phenanthroline and ethanol
afforded the copper complex 4 (Fig. 4) and 5 (Fig. 5), which has a
five coordinated copper centre, with a distorted square pyramidal
bonding pattern. The basal plane of the distorted square pyramidal
coordination is formed by the dianionic tridentate ONO-donor
ligand and the N4 atom of phen, while the axial position is occu-
pied by N3 of phen. One of the reasons for the deviation from an
ideal stereochemistry is the restricted bite angle imposed both by
the ONO-donor hydrazone ligand [O1eCu1eN1 ¼ 92.67(7)ꢁ, N1e
Cu1eO2
¼
81.23(7)ꢁ] and the phen ligand [N3eCu1e
N4 ¼ 78.84(8)ꢁ] [30]. As with complex 3, the small differences
ꢀ
between the bond distances of C23eO1 [1.315(3) A] and C24eO2
[1.288(3) A] and those of Cu1eO1 [1.919(2) A] and Cu1eO2
[1.946(2) A] also indicate that the copper(II) ion is coordinated to
ꢀ
ꢀ
ꢀ
the enolized form of the Schiff base ligand. The presence of an
ethanol and a water molecule links the chloride ion to the cationic
copper complex via the H-bonding interactions with the enolate
oxygen of the ligand [O3eH3A/Cl1
H4A/O1 ¼ 2.833(3) A].
¼
3.097(2), O4e
ꢀ
In complex 5, the Schiff base is situated on the basal plane and
bpy displays axial (N3)-equatorial (N4) coordination. Furthermore,
it is solvated by an acetonitrile and two water molecules in which
the hydrogen atoms of the water molecules are H bond donors to
the chloride ion and the enolate oxygen atoms of an inversion
related molecule, forming a dimeric unit in its crystal packing. In
addition, there is an appreciable pseudo Jahn-Teller effect high-
Fig. 1. An ORTEP view of 1 showing the atom labelling scheme (50% probability
thermal ellipsoids).
fourth bond formed between the copper metal and a chloride ion.
This gives rise to a zwitterion species with a positive charge at the
phosphorus centre. The transoid angles N1eCu1eCl1 [174.63(9)ꢁ]
and O1eCu1eO2 [172.8(1)ꢁ] which deviate from the values of 180ꢁ
[34] and the sum (S) of the six inter-bond angles for complex 3 is
707ꢁ, which deviates from the ideal angle of 720ꢁ, suggesting that
the copper centre adopts a distorted square planar geometry
[35,36]. Further evidence can be seen from the small dihedral angle
of 4.6(1)ꢁ formed between the two near planar rings Cu1eN1eN2e
ꢀ
lighted by Cu1eN3 distance [2.237(2) A] which is significantly
longer than that observed for the equatorial Cu1eN4 distance
ꢀ
[2.018(2) A] [37]. It has long been proven that Jahn-Teller effect is
operative in the case of d9 transition metal complexes. Thus, one
trans pair of coordination bonds are elongated while the remaining
four are shortened [11]. Akin to complex 4, the angle formed be-
tween copper metal center and diimine ligand bpy [77.55(6) o] is
ꢀ
C24eO2 (RMS deviation ¼ 0.0013 A) and Cu1eN1eC31eC32e
known to be a standard bite angle [19,29]. The trigonality index,
calculated using the equation )/60 [38] (for perfect square
¼ (
pyramidal and trigonal bipyramidal geometries the values of are
zero and unity, respectively) [16,30,39]. The value of for 4 and 5
s is
ꢀ
C23eO1 rings (RMS deviation ¼ 0.0065 A). The relative small dif-
s
b
ꢀ
a
ꢀ
ference between the bond distances of C23eO1 [1.317(4) A] and
s
s
are 0.20 and 0.27 respectively which further affirm the geometry of
distorted square pyramidal [38].
Fig. 2. An ORTEP view of 2 showing the atom labelling scheme (50% probability
Fig. 3. An ORTEP view of 3 showing the atom labelling scheme (50% probability
thermal ellipsoids).
thermal ellipsoids).

S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
401
Fig. 4. An ORTEP view of 4 showing the atom labelling scheme (50% probability thermal ellipsoids).
3.6. Cytotoxic activity
lowest IC50 value of 3.2 mM for compound 4. The cytotoxicity of the
ternary complexes (4e6) decreases by the following order:
4 > 6 > 5. The observed order of decrease reflects the important
role of extended aromatic ring and hydrophobicity of the diimines
in improving cytotoxicity.
Toxicity is a common limitation in terms of the introduction of
new compounds into the pharmaceutical industry. In order to
determine the in vitro cytotoxic activities of the compounds, ex-
periments were carried out using human lung carcinoma cell line
(A549), human prostate adenocarcinoma cell line (PC-3) and non-
cancer human fibroblast cell line (MRC-5). Cell viability was
determined by MTT assay after 72 h of treatment with increasing
concentration of the compounds. The compounds were dissolved in
DMSO and blanks samples containing same concentration of DMSO
were taken as controls to identify the activity of solvents in this
MTT assay. The results were analyzed by mean of cell viability
expressed as IC50 values are shown in Table 5. Compound 1 is found
In order to determine the selectivity of the compounds, the
cytotocities of the compounds were tested on MRC-5 non-
cancerous cell line. It is notably that complex 3 is non toxic,
whereas ligand 2 is toxic towards normal cell line. Therefore,
complexation of ligand 2 by copper leads to an enhancement in
cytoselectivity. By comparing both tumour cell lines, all the com-
pounds seems to be more toxic against PC-3 cell line than A549 cell
line. Researchers point out that there is an increase of 2- to 10-fold
of topo I enzyme in prostate tumours, compared to benign hyper-
plastic prostate tissue from the same patients [40]. Therefore,
prostate carcinoma cells are good models to study compounds that
are designed to target topo I. Complex 4 and 5 possess similar
selectivity towards PC-3 cell line with the selectivity index (SI) of
1.59 and 1.58 respectively. Complexes bearing phen and bpy co-
ligands are more selective towards cancerous cells were reported
to be less cytotoxic (IC50 > 30
mM) to cancerous and normal cells. It
becomes cytotoxic when it is in the form of Schiff base ligand 2.
Surprisingly, complex 3 (IC50 ¼ 16.6 ꢃ 3.1
cytotoxicity with ligand 2 (IC50 ¼ 14.4 ꢃ 2.6
m
M) possess similar
m
M) against PC-3 cell
line. However, the addition of N, N ligands to the copper complex
leads to an increase in cytotoxicity of compounds 4e6 with the
Fig. 5. An ORTEP view of 5 showing the atom labelling scheme (50% probability thermal ellipsoids).

402
S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
Table 3
enzymatic activity of topo I, like that of other topoisomerases,
breaks the DNA backbone reversibly by replacing a DNA phos-
phodiester bond with a bond between a phosphate at one end of
the DNA break and a tyrosine residue of the topo I protein [40,52e
54]. In the case of topo I, the tyrosine links to the phosphate at the
30-hydroxyl end of the DNA break. DNA relaxation takes place by
rotation of the free end of the broken DNA around the intact DNA
strand [55].
As a preliminary investigation of the topo I inhibition, we report
herein the study by DNA relaxation assay. This assay provides a
direct means of determination whether the drugs affect the un-
winding of a supercoiled duplex DNA to a nicked open circular and
relaxed DNA [28]. One unit of topo I could fully convert the super-
coiled pBR322 DNA to fully relaxed topoisomers (Fig. 6(A), Lane 5).
After the incubation of DNA with topo I, the appearance of the
slowest moving bands of more relaxed DNA can be seen on the gel
image (Fig. 6(AeE), Lane 5) The disappearance or the decrease in the
intensity of the slowest moving band upon incubation of DNA, topo I
and the complexes is the indicationof topoI inhibition. The degreeof
inhibition of Topo I increases with increasing concentration (5e
ꢁ
ꢀ
Selected bond lengths (A) and bond angles ( ) for compounds 1e5.
Compound 1
O1eC23
O2eC26
O3eC49
O4eC52
1.346 (2)
1.218 (3)
1.349 (2)
1.217 (3)
Compound 2
N1eC26
N1eN2
N2eC27
N3eC59
N3eN4
N4eC60
O1eC23
O2eC27
O3eC56
O4eC60
1.290 (3)
1.374 (3)
1.372 (3)
1.282 (3)
1.369 (3)
1.366 (3)
1.354 (3)
1.222 (3)
1.356 (3)
1.223 (3)
Compound 3
Cl1eCu1
Cu1eO1
Cu1eN1
Cu1eO2
N1eC31
N1eN2
N2eC24
O1eC23
O2eC24
2.229 (1)
1.883 (3)
1.930 (3)
1.936 (3)
1.287 (5)
1.404 (4)
1.314 (5)
1.316 (5)
1.295 (4)
O1eCu1eN1
N1eCu1eO2
O1eCu1eCl1
O2eCu1eCl1
N1eCu1eCl1
O1eCu1eO2
92.7 (1)
81.3 (1)
92.60 (9)
93.46 (8)
174.6 (1)
172.8 (1)
160
not fully inhibited even up to 160
Among the compounds, compound 4 is slightly more active than
the others because it started to inhibit topo I at 40 M (Fig. 6(C),
Lane 10). On the other hand, the least active compound 5, shows no
inhibition at 40 M (Fig. 6(D), Lane 10). It is obvious that the
mM) of the complexes (Fig. 6(BeE)). But, the function of topo I is
mM of the complexes.
m
Compound 4
Cu1eO1
Cu1eN1
Cu1eO2
Cu1eN4
Cu1eN3
N1eC31
N1eN2
m
1.919 (2)
1.926 (2)
1.946 (2)
2.003 (2)
2.270 (2)
1.293 (3)
1.397 (3)
1.329 (3)
1.315 (3)
1.288 (3)
O1eCu1eN1
N1eCu1eO2
O1eCu1eN4
O2eCu1eN4
O1eCu1eN3
N1eCu1eN3
O2eCu1eN3
N4eCu1eN3
92.67 (7)
81.21 (7)
95.07 (7)
93.57 (7)
96.72 (7)
94.56 (7)
104.70 (7)
78.84 (8)
addition of phen into the copper (II) hydrazone complex causes a
lowering in the inhibitory concentration of topo I. By comparing the
results of cytotoxicity and topo I inhibition assay for the ternary
complexes, it seem likely that the cytotoxicities of the compounds
are directly proportional to the topo I inhibition ability. Complex 4
with the lowest IC50 value of 3.2 mM appeared to be the most active
N2eC24
O1eC23
O2eC24
topo I inhibitor (Fig. 6(C)). However, there is no direct correlation
between the topo I inhibition activity of complex 3 (without N, N
ligand) with its cytotoxicity. Of primary concern is the inevitable
interaction between all the compounds with DNA without addition
of topo I that would lead to the decline in rate of DNA migration
(Fig. 6(AeE), Lane 3). To this end, it is logical to assume that the
Compound 5
Cu1eN1
Cu1eO1
Cu1eO2
Cu1eN4
Cu1eN3
N1eC31
N1eN2
1.921 (2)
1.948 (1)
1.976 (1)
2.018 (2)
2.237 (2)
1.289 (2)
1.391 (2)
1.321 (2)
1.320 (2)
1.293 (2)
N1eCu1eO1
N1eCu1eO2
O1eCu1eN4
O2eCu1eN4
N1eCu1eN3
O1eCu1eN3
O2eCu1eN3
N4eCu1eN3
92.64 (6)
80.98 (6)
92.85 (5)
96.22 (6)
95.82 (6)
100.49 (5)
103.54 (6)
77.55 (6)
Table 4
Hydrogen bonds for compounds 1, 2, 4 and 5.
N2eC24
O1eC23
O2eC24
ꢀ
ꢀ
ꢀ
D-h/a
d (D-H) (A)
d (H/A) (A)
d (D/A) (A)
<DHA (ꢁ)
Compound 1
O3eH3/Cl1
O1eH1/Cl2_#1
0.84
0.84
2.17
2.14
2.996 (2)
2.983 (2)
169.8
176.3
earlier [41e44]. Complex 6 is somehow lower in terms of selectivity
with SI of 1.07. Delocalized lipophilic cations (DLC) are known to
selectively accumulate in mitochondria of cancerous cells. There-
fore, complexes with lipophilic cation moiety are known to be more
sensitive towards cancer cells [45]. A general elevation in the
mitochondrial membrane potential (DJm) has been linked to
malignant transformation [46]. Compounds having lipophilic
cation such as triphenylphosphonium moieties have been shown to
dissipate the mitochondrial membrane potential upon uptake into
mitochondria and lead to cell death [47,48]. This could be one of the
possible explanations of better cytoselectivity of complex 4 and 5.
However, we have yet to come out with a plausible explanation for
the lack of selectivity of complex 6 with a lipophilic cation moiety.
Symmetry operation#1: ꢀx, ꢀy þ 1, ꢀz
Compound 2
O3eH3A/N3
0.84
1.79
2.529
2.576
144.9
145.5
O1eH1/N1
0.84
1.84
O5eH5A/Cl2
O5eH5B/Cl1_#1
O6eH6A/Cl1_#1
O6eH6B/Cl2
N4eH4A/Cl1_#1
N2eH2A/Cl2
1.04 (4)
0.92 (4)
0.88 (4)
0.78 (4)
0.97 (3)
0.86 (3)
2.07 (4)
2.30 (4)
2.45 (4)
2.40 (4)
2.33 (3)
2.29 (3)
3.105 (2)
3.211 (2)
3.276 (2)
3.178 (2)
3.272 (2)
3.138 (2)
178 (3)
170 (3)
158 (3)
172 (4)
162 (2)
173 (3)
Symmetry operation#1: x þ 1, y, z
Compound 4
O3eH3A/Cl1_#1
O4eH4A/O1
0.84
0.84
2.28
1.96
3.097 (2)
2.833
162.9
175.6
Symmetry operation#1: x, y þ 1, z
Compound 5
2.7. Topoisomerase I inhibition
O3eH3A/Cl1
0.71 (4)
0.80 (4)
0.74 (3)
0.87 (4)
2.51 (4)
2.01 (4)
2.50 (3)
2.35 (4)
3.219 (2)
2.796
3.243 (2)
3.205 (2)
179 (4)
170 (4)
178 (3)
170 (3)
O4eH4A/O1_#1
O3eH3B/Cl1_#2
O4eH4B/Cl1_#2
DNA topoisomerase I (Topo I), an essential enzyme in metazoans
has been identified as a target among the most widely used clinical
drugs for treatment of cancer [49e51]. Topoisomerases have
pivotal roles in DNA replication and transcription [28,40]. The
Symmetry operation#1: ꢀx þ 1, ꢀy, ꢀz þ 1
Symmetry operation#2: ꢀx þ 1, ꢀy þ 1, ꢀz

S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
403
Table 5
(Fluka) were used as received without further purification. All the
solvents were of reagent grade. The pBR322, gene ruler 1 kb DNA
ladder, 6ꢂ loading buffer and Tris-(hydroxymethyl)aminomethane
(Tris) were procured from BioSyn Tech (Fermentas). Analytical
grade agarose powder was obtained from Promega. Sodium chlo-
ride, human DNA topoisomerase I and ethidium bromide were
purchased from Sigma Chemical Co. (USA). MTT (Methyl-
thiazolyldiphenyl-tetrazolium bromide), RPMI 1640 medium,
EMEM (Eagle’s Minimum Essential Medium), sodium bicarbonate,
cis-platin, carboxymethyl cellulose, EDTA, DMSO were purchased
from SigmaeAldrich company. Foetal bovine serum, penicillin/
The cytotoxicity activity for compounds 1-6 on A549, PC-3 and MRC-5 cells
respectively.
Compounds
Cytotoxicity IC50 (mM)
A549
PC-3
MRC-5
Selectivity index (SI)
1
2
3
4
5
6
>30
>30
>30
NA
0.85
NA
1.59
1.58
1.07
>30
>30
14.4 ꢃ 2.6
16.6 ꢃ 3.1
3.2 ꢃ 0.2
8.1 ꢃ 0.5
4.6 ꢃ 0.5
12.2 ꢃ 0.6
>30
4.2 ꢃ 0.8
5.1 ꢃ 0.3
12.8 ꢃ 1.5
4.9 ꢃ 0.1
>30
6.7 ꢃ 1.7
SI ¼ IC50 of MRC-5/IC50 of PC-3.
streptomycin (100ꢂ), amphotericin B (250
mg/mL) and sodium
pyruvate (100 mM) were from PAA Laboratories.
cationic complexes bind to DNA at high concentration and cause
the DNA to travel slower because of the formation of DNA and metal
complex aggregate with higher molecular weight or the decrease in
the negative charge of the DNA due to the binding with the cationic
complexes [42,56e58]. Therefore, we could not determine the
concentration of compounds that lead to full inhibition of topo I.
In an attempt to obtain more insight into the inhibition activity
of the topo I in this work, we used three variations of mixing the
4.2. Physical measurements
IR spectra were recorded as KBr pellets by using a PerkineElmer
Spectrum RX-1 FTIR spectrophotometer. NMR spectra were recor-
ded in deuterated DMSO-d6 on a JEOL JNM GX-270 FT NMR System
Spectrometer. Elemental analyses were performed on a Perkine
Elmer EA2400 CHNS elemental analyzer. UVeVis spectroscopic
measurements were carried out on a PerkineElmer Lambda 40
spectrophotometer.
DNA, topo I and compounds 2e6 (60 mM) respectively for topo I
inhibition assay. It is possible to predict a generalized mechanism of
action of topo I inhibition based on the three variations of mixing.
When the three components are mixed simultaneously, there is
slight inhibition of topo I as can seen by the presence of the fastest
moving band with low intensity (Form I) which consists of super-
coiled DNA and poorly relaxed DNA (Fig. 7, Lane 5). Similar band can
be observed by incubating DNA and compound 4 before the addi-
tion of topo I (Fig. 7, Lane 6). Interestingly, when compound 4 is
incubated with topo I before the addition of DNA (Fig. 7, Lane 7), the
intensity of the fastest moving bands are the highest (Form I).
However, the slowest moving band still remains with lower in-
tensity (Form II). These observations suggest that the mechanism of
action for topo I inhibition is based on two pathways, one involving
the binding of complex to DNA and the other comprise the binding
of complex to topoisomerase. In light of these results, we can
deduce that binding of complex to topo I is a preferred inhibition
pathway regardless of the structures of the complexes (Fig. 7, S3e
S5 Lane 7). Nevertheless, continued research on this will hope-
fully lead to a better assessment of the usefulness of these com-
pounds as anticancer drugs.
4.3. Preparation of ligand and complexes
4.3.1. Synthesis of 5-(triphenylphosphoniummethyl)-
salicylaldehyde (1)
The 5-chloromethylsalicylaldehyde was prepared according to
the standard chloromethylation method by Huang and co-workers
[59]. It was further reacted with triphenylphosphine to form a
phosphonium salt by minor modification of the procedure reported
by Wang and co-workers [60]. 5-chloromethylsalicylaldehyde
(0.171 g, 1 mmol) and triphenylphosphine (0.262 g, 1 mmol) was
refluxed in toluene (40 mL) for 5 h. The white solid formed was
filtered, washed with toluene and air-dried. Crystals were formed
by recrystallize using ethanol.
Yield: 78%, white solid, m.p.: 275e276 ꢁC. Anal. Calc for
C
26H22ClO2P: C, 72.14; H, 5.12. Found: C, 72.06; H, 5.15%. IR (KBr
disc, cmꢀ1): v(C]O): 1676, v(CeO): 1112.
Characteristic: 1H NMR (DMSO-d6, TMS, ppm 400 MHz, s,
singlet; d, doublet; t, triplet; m, multiplet): 11.10 (s, 1H, OH); 10.10
(s, 1H, CHO); 7.61e7.88 (m, 15H, aromatic CH); 6.91e7.18 (m, 3H,
aromatic CH); 5.07e5.11 (d, J ¼ 16 Hz, 2H, CH2). 13C NMR (DMSO-d6,
TMS, ppm 100 MHz): 189.41 (C]O); 160.83 (CeOH); 117.20e138.01
(Ar); 26.98, 27.44 (CH2).
3. Conclusion
Mononuclear Cu(II) complexes of hydrazone ligand containing
triphenylphosphonium moiety were synthesized and characterized.
Their cell viability assay and topo I inhibition activity have been
intensively studied. Crystal structure of 3 shows a distorted square
planar geometry while 4 and 5 reveal similar distorted square py-
ramidal geometry. As previously mentioned, complex 4 gives rise to
the lowest IC50 value against PC-3 cell lines. However, it can be seen
that complex 5 has better selectivityamong the compounds. In brief,
DNA relaxation assay shows favourable results that all the com-
pounds can inhibit topo I upon complexation and the binding
through the enzyme seems to be the preferable inhibition pathway.
4.3.2. Synthesis of ligand [5-(triphenylphosphoniummethyl)-
salicylaldehyde benzoylhydrazone] chloride monohydrate (2)
The ligand was synthesized by condensing benzhydrazide
(0.136 g, 1 mmol) with compound 1 (0.433 g, 1 mmol) in ethanol
(30 mL) for 4 h. Slow evaporation of the solvent yielded yellow
crystals. The crystals were filtered, washed with cold ethanol and
air-dried.
Yield: 85%, yellow solid, m.p.: 276e277 ꢁC. Anal. Calc for
C
33H28ClN2O2P$H2O: C, 69.95; H, 5.31; N, 4.92. Found: C, 69.67; H,
5.41; N, 5.06%. IR (KBr disc, cmꢀ1): v(C]O): 1679, v(C]N): 1619,
v(N N): 1028.
Characteristic: 1H NMR (DMSO-d6, TMS, ppm 400 MHz, s,
4. Experimental
]
4.1. Materials
singlet; d, doublet; t, triplet; m, multiplet): 12.24 (s, 1H, NH); 11.39
(s, 1H, OH); 8.46 (s, 1H, CH]N); 7.91e7.93 (d, J ¼ 8 Hz, 2H, aromatic
CH); 7.86e7.89 (t, J ¼ 7.2 Hz, 3H, aromatic CH); 7.63e7.72 (m, 12H,
aromatic CH); 7.57e7.59 (d, J ¼ 8 Hz,1H, aromatic CH); 7.49e7.53 (t,
J ¼ 7.2 Hz, 2H, aromatic CH); 7.14 (s, 1H, aromatic CH); 6.76e6.83
(m, 2H, aromatic CH); 5.05e5.08 (d, J ¼ 12 Hz, 2H, CH2). 13C NMR
Paraformaldehyde (BDH Limited poole Endland), salicyladehyde
(Merck), triphenylphosphine (Merck), benzhydrazide (Sigmae
Aldrich), 1,100-phenanthroline (Acros), 2,20-bipyridine, 5,50-
dimethyl-2,20-bipyridine and copper(II) acetate monohydrate

404
S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
Fig. 6. Human topoisomerase I inhibition assay by gel electrophoresis. The gel images from AeE are for compounds 2e6, respectively. Electrophoresis results of incubating human
topoisomerase I (0.25 unit/20 L) with pBR322 in the absence or presence of 5e160 M of compound: Lane 1 and 13, gene ruler 1 Kb DNA ladder; Lane 2, DNA alone (control); Lane
3, DNA þ 160 M compound (control); Lane 5, DNA þ 1 unit human topoisomerase I (control); Lane 7e12, DNA þ 1 unit human topoisomerase I þ varying concentration of
compound; Lane 7, 5 M; Lane 8, 10 M; Lane 9, 20 M; Lane 10, 40 M; Lane 11, 80 M; Lane 12, 160 M.
m
m
m
m
m
m
m
m
m
(DMSO-d6, TMS, ppm 100 MHz): 162.95 (C]O); 157.14 (CeOH);
146.80 (C]N); 116.82e135.12 (Ar); 27.09, 27.56 (CH2).
for 2 h followed by addition of ligand (2) (0.551 g, 1 mmol) in
ethanol (20 mL) and the mixture was refluxed for another 2 h. The
green complex formed was extracted with hexane and recrystal-
lized from DMF-ethanol mixture.
4.3.3. Synthesis of chlorido[5-(triphenylphosphoniummethyl)-
salicylaldehyde benzoylhydrazonato] copper(II) monohydrate (3)
Copper(II) acetate monohydrate (0.200 g,1 mmol) and ligand (2)
(0.551 g, 1 mmol) were refluxed in ethanol (30 mL) for 3 h. The
green complex formed was filtered off and rinsed with cold
ethanol. Crystals were formed by recrystallization using methanol.
Yield: 60%, green solid, m.p.: 244e245 ꢁC. Anal. Calc for
Yield: 69%, green solid, m.p.: 178e180 ꢁC. Anal. Calc for
C
45H36ClCuN4O2P$2H2O 1 EtOH: C, 64.38; H, 5.29; N, 6.39. Found: C,
63.84; H, 4.78; N, 6.11%. IR (KBr disc, cmꢀ1): v(C]N): 1612, v(N]C):
1501, v(N N): 1046.
]
4.3.5. Synthesis of (2,20-bipyridine)[5-
(triphenylphosphoniummethyl)-salicylaldehyde
C
33H27ClCuN2O2P$H2O: C, 62.86; H, 4.48; N, 4.44. Found: C, 63.37;
H, 3.90; N, 4.58%. IR (KBr disc, cmꢀ1): v(C]N): 1613, v(N]C): 1503,
v(N N): 1047.
benzoylhydrazonato] copper(II) dihydrate acetonitrile (5)
Same as complex 4 but using 2,20-bipyridine instead of 1,100-
phenanthroline. The green complex formed was extracted with
hexane and purified by recrystallization from acetonitrile-
chloroform mixture.
]
4.3.4. Synthesis of (1,100-phenanthroline)[5-
(triphenylphosphoniummethyl)-salicylaldehyde
benzoylhydrazonato] copper(II) monohydrate ethanol (4)
Copper(II) acetate monohydrate (0.200 g, 1 mmol) and 1,100-
phenanthroline (0.180 g, 1 mmol) were heated in ethanol (20 mL)
Yield: 67%, green solid, m.p.: 108e110 ꢁC. Anal. Calc for
C43H36ClCuN4O2P$3H2O: C, 62.62; H, 5.13; N, 6.79. Found: C, 63.02;
H, 4.67; N, 6.30%. IR (KBr disc, cmꢀ1): v(C]N): 1612, v(N]C): 1502,
v(N]N): 1043.
4.3.6. Synthesis of (5,50-dimethyl-2,20-bipyridine)[5-
(triphenylphosphoniummethyl)-salicylaldehyde
benzoylhydrazonato] copper(II) trihydrate (6)
Same as complex 4 but using 5,50-dimethyl-2,20-bipyridine
instead of 1,100-phenanthroline. The green powder formed was
extracted with hexane and purified by methanol.
Yield: 71%, green solid, m.p.: 186e187 ꢁC. Anal. Calc for
C
43H36ClCuN4O2P$3H2O: C, 63.37; H, 5.44; N, 6.57. Found: C, 63.38;
H, 5.10; N, 6.13%. IR (KBr disc, cmꢀ1): v(C]N): 1612, v(N]C): 1500,
v(N N): 1045.
]
4.4. X-ray crystallography
The unit cell parameters and the intensity data were collected
on a Bruker SMART APEX CCD diffractometer, equipped with a Mo
Fig. 7. Effect of sequence of mixing for the human topoisomerase I inhibition assay of
complex 4. Electrophoresis results of incubating human topoisomerase I (0.25 unit/
ꢀ
20
(control); Lane 3, DNA þ 1 unit human topoisomerase I (control); Lane 5, DNA þ 60
4 þ 1 unit human topoisomerase I, all components mixed at the same time; Lane 6,
DNA þ 60 M 4 þ 1 unit human topoisomerase I, DNA þ complex 4 incubated for
30 min first before the addition of topo I; Lane 7, DNA þ 60 M 4 þ 1 unit human
topoisomerase I, complex 4 þ topo I incubated for 30 min first before DNA is added.
mL) with pBR322: Lane 1, 4 and 8, gene ruler 1 Kb DNA ladder; Lane 2, DNA alone
Ka
X-ray source (
l
¼ 0.71073 A). The APEX2 software was used and
mM
the SAINT software for cell refinement and data reduction. Ab-
sorption corrections on the data were made using SADABS. The
structures were solved and refined by SHELXL97 [61]. Molecular
graphics were drawn by using XCEED [62]. The structures were
m
m

S.T. Chew et al. / European Journal of Medicinal Chemistry 76 (2014) 397e407
405
solved by direct-methods and refined by a full-matrix least-squares
4.8. Statistical analysis
procedure on F2 with anisotropic displacement parameters for non-
hydrogen atoms.
The IC50 values for cytotoxic activity were obtained by non-
linear regression using GraphPad Prism statistical software.
Acknowledgement
4.5. Eschrichia coli topoisomerase I inhibition assay
We thank UM for financial support (RG148-11AFR). STC would
like to thank UM for scholarship and financial assistance (PV089/
2012A).
The human DNA topoisomerase I inhibition activity was deter-
mined by measuring the relaxation of supercoiled plasmid DNA
pBR322. For measurement of human topoisomerase I activity, the
reaction mixtures were comprised of 10 mM TriseHCl, pH 7.5,
List of abbreviations
100 nM NaCl, 1 mM phenylmethylsulfonyl fluoride,
sulfonyl fluoride, PMSF, and 1 mM 2-mercatoethanol, 0.25
plasmid DNA pBR322, 1 unit of human topoisomerase I, and the test
compound with final concentration of 160 M. All reactions were
conducted at a final volume of 20 L and were prepared on ice.
Upon enzyme addition, reaction mixtures were incubated at 37 ꢁ
for 30 min. The reactions were terminated by addition of 2 L of 10%
SDS, followed by 3 L of dye solution comprising 0.02% bromo-
a-Toluene-
mg
cisplatin cis-diamminedichloroplatinum(II)
MTT
DNA
phen
bpy
(3-(4,5-2-yl)-2,5-ditetrazolium bromide
DeoxyriboNucleic Acid
m
m
1,100-phenanthroline
C
2,20-bipyridine
m
dbpy
5,50-dimethyl-2,20-bipyridine
m
DMSO dimethylsulfoxide
phenol blue and 50% glycerol. SDS is required to observed a linear
DNA fragment and to denature topoisomerase I, preventing further
functional enzymatic activity. The mixtures were applied to 1.25%
agarose gel and electrophoresed for 2 h at 80 V with running buffer
of Tris-acetate EDTA (TAE) at pH 8.1. The gel was stained, destained,
and photographed under UV light using a Syngene Bio Imaging
system and the digital image was viewed with Gene Flash software.
In human topoisomerase I inhibition condition study, the same
protocol was applied. This study is designed to deduce the mode of
action of complexes in the human DNA topoisomerase I inhibition
study. The sequence of addition of the main components (human
DNA topoisomerase I, plasmid DNA pBR322, and metal complexes)
was varied. For the first condition, human DNA topoisomerase I
with the metal complex was incubated at 37 ꢁC for 30 min before
the addition of DNA. The mixture was incubated for another 30 min
at the same temperature after the addition of DNA. As for the
second condition, the metal complex and DNA was incubated for
30 min at 37 ꢁC first, followed by the addition of topoisomerase I.
This mixture was incubated for another 30 min at 37 ꢁC after the
addition of topoisomerase I.
DMF
Tris
dimethylformamide
tris-(hydroxymethyl)aminomethane
EMEM Eagle’s Minimum Essential Medium
EDTA
IR
KBr
NMR
LMCT
TAE
SI
ethylenediaminetetraacetic acid
infrared
potassium bromide
nuclear magnetic resonance
ligand-to-metal-charge transfer
tris-acetate EDTA
selectivity index
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.049.
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4.7. MTT Cytotoxicity assay
The MTT cytotoxicity assay was carried out as previously
described by Mosmann [63]. All samples were dissolved in DMSO to
form stock solution before cytotoxicity testing. The final concen-
tration of DMSO in each well was 0.5%. The cytotoxicity of each
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