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