beni-suef university journal of basic and applied sciences 5 (2016) 85–96

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Full Length Article

Synthesis, anticancer activity and molecular

docking study of Schiff base complexes
containing thiazole moiety

Mokhles M. Abd-Elzaher a,*, Ammar A. Labib a, Hanan A. Mousa a,

Samia A. Moustafa a, Mamdouh M. Ali b, Ahmed A. El-Rashedy c
a
Inorganic Chemistry Department, National Research Centre, 33 El Beehouth St., Dokki, PO 12622, Giza, Egypt
b
Biochemistry Department, National Research Centre, 33 El Beehouth St., Dokki, PO 12622, Giza, Egypt
c
Natural and Microbial Product Research Department, Division of Pharmaceutical and Drug Industries,
National Research Centre, 33 El Beehouth St., Dokki, PO 12622, Giza, Egypt

A R T I C L E I N F O A B S T R A C T

Article history: A Schiff base ligand 1 was prepared from condensation of salicyaldehyde with 2-amino-4-
Received 14 November 2015 phenyl-5-methyl thiazole. The ligand forms complexes with CoII, NiII, CuII, and ZnII in good
Received in revised form 20 January yield. The synthesized compounds were characterized by elemental analysis, magnetic sus-
2016 ceptibility, molar conductance, infrared spectra, 1H and 13C NMR, mass, electronic absorption
Accepted 23 January 2016 and ESR spectroscopy. The anticancer activity of the synthesized compounds was studied
Available online 18 February 2016 against different human tumor cell lines: breast cancer MCF-7, liver cancer HepG2, lung car-
cinoma A549 and colorectal cancer HCT116 in comparison with the activity of doxorubicin
Keywords: as a reference drug. The study showed that ZnII complex showed potent inhibition against
Schiff bases human TRK in the four cell lines (HepG2, MCF7, A549, HCT116) by the ratio 80, 70, 61 and
Metal complex 64% respectively as compared to the inhibition in the untreated cells. Moreover, the mo-
Anticancer lecular docking into TRK (PDB: 1t46) was done for the optimization of the aforementioned
Spectral characterization compounds as potential TRK inhibitors.
Docking © 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

base ligands are considered “privileged ligands” because they

1. Introduction are mainly prepared by condensation between aldehydes and
primary amines (Chang et al., 2016). These ligands are able to
The chemistry of the Schiff base ligands and their metal com- coordinate many different metals and to stabilize them in
plexes has expanded enormously and encompasses a vast various oxidation states (Ejidike and Ajibade, 2015). They are
area of organometallic compounds and various aspects of used also as pigments and dyes, catalysts, intermediates in
bioinorganic chemistry (Abu-Diefa and Mohamed, 2015). Schiff organic synthesis, and as polymer stabilizers (Menati et al.,

* Corresponding author. Inorganic Chemistry Department, National Research Center, Giza, Egypt. Tel.: +202 3334748; fax: +202 33370931.
E-mail address: Mokhlesm20@yahoo.com (M.M. Abd-Elzaher).
http://dx.doi.org/10.1016/j.bjbas.2016.01.001
2314-8535/© 2016 Beni-Suef University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
86 beni-suef university journal of basic and applied sciences 5 (2016) 85–96

2013). Large numbers of Schiff bases have also been shown to IR spectra were recorded in the 4000-400 cm−1 on a spectrom-
exhibit a broad range of biological activities, including anti- eter (Jasco FTIR- 6100 Japan), using KBr pellets. 1H and 13C NMR
tumor, anti-bacterial, fungicidal and anticarcinogenic properties were recorded on a Bruker DPX 300, δ values relative to the deu-
(Nagesh et al., 2015; Salehi et al., 2015; Shukla et al., 2013; terated DMSO. Mass spectra: Jeol JMS-700 using FAB technique
Zaltariov et al., 2015; Zayed and Zayed, 2015). Metal com- with a 3-nitrobenzyl alcohol NBA matrix. Magnetic suscepti-
plexes of Schiff bases with heterocyclic compounds also find bilities were measured at 20 °C by the Gouy method at the
applications as potential drugs, (Andersen, 1999; Konstantinović Faculty of Science, Cairo University. The molar conductance
et al., 2003) due to the presence of multifunctional groups measurements were measured in solution of the metal com-
(Chohan et al., 2004; Joseyphus et al., 2006; Vashi and Naik, 2004; plexes in DMF (10−3) using Metrohem 660 conductivity meter.
Venugopala and Jayashree, 2003). The excessive attention of Electronic absorptions were recorded on a automatic spectro-
synthesizing determined broad range of N and S chelating photometer (PG Instruments ltd., +80 + UV–Vis) in DMSO. ESR
ligands as thiazole molecule have attracted significant inter- measurements were made at approximately 298K with a Bruker
est. This is because thiazoles have a great pharmacological E500, X-band spectrometers operating at a frequency of 9.5 GHz
activity. Besides these atoms play an important role in the co- at National Institute for Standards, Giza.
ordination of metals at the active sites of various metal
biomolecules that have a therapeutic activity or serving as study
2.2. Synthesis of the Schiff base ligand C17H14N2OS (1)
models for metallo-enzymes (Chen et al., 2012; Venkatraman
and metal complexes (2–5)
et al., 2010; Yenilmez et al., 2013). Thiazoles are very impor-
tant building blocks in medicinal chemistry and can be found 2-amino-4-phenyl-5-methyl thiazole (1 mmol, 2.95 g) dis-
in numerous natural products (e.g. epothilone) and biologi- solved in about 20 mL absolute ethanol was added slowly to
cally important compounds including the anticancer drug a magnetically stirred solution of salicyladehyde (1 mmol, 1.22 g),
dasatinib, antiviral clinical candidate TMC435350 and anti- in the presence of few drops of glacial acetic acid. The mixture
diabetic drug candidate MB06322 (Dang et al., 2008; Doggrell, was refluxed for four hours. Then the solution was concen-
2005; Erion et al., 2005; Lin et al., 2009). Recently, thiazoles found trated to its half volume then cooled; n-hexane was added to
application in drug development for the treatment of aller- the reaction mixture drop wise until a product began to pre-
gies (Brzezińska et al., 2003), hypertension (Mishra et al., 2015), cipitate. The formed product was filtered off, washed several
inflammation (Sharma et al., 1998), schizophrenia (Jaen et al., times with n-hexane, and recrystallized from ethanol. The dif-
1990), bacterial (Suzuki et al., 1994), HIV infections (Bell et al., ferent complexes were prepared by addition of 1 mmol of
1995), hypnotics (Ergenc et al., 1999), as fibrinogen receptor an- CoCl2.6H2O, NiCl2.6H2O, CuCl2.2H2O and ZnCl2, dissolved in about
tagonist with antithrombotic activity (Badorc et al., 1997) and 20 mL ethanol, into a solution of the ligand (2 mmol in 20 mL
as new inhibitors of bacterial DNA gyrase B (Rudolph et al., ethanol). The mixture was refluxed for three hours, then the
2001). solution was concentrated. The obtained solid products were
Following all these observations and as a part of our con- filtered, washed twice with cold n-hexane and dried.
tinuing research on the coordination chemistry of multidentate
ligands (Abd-Elzaher, 2004a, 2004b; Abd-Elzaher et al., 2005, 2006,
2010, 2012a, 2012b; Fouda et al., 2008a, 2008b), we report here 2.3. Anticancer activity
the preparation and characterization of a Schiff base ligand
derived from condensation of salicyaldehyde with 2-amino- 2.3.1. Chemicals
4-phenyl-5-methyl thiazole. The study has been extended to Fetal bovine serum (FBS) and L-glutamine were obtained from
synthesize CuII, CoII, NiII and ZnII complexes with the pre- Gibco Invitrogen Company (Scotland, UK). Dulbecco’s modi-
pared ligand. All the prepared complexes have been fied Eagle’s medium (DMEM) was provided from Cambrex (New
characterized by IR, 1H and 13C NMR, mass spectra, ESR, UV– Jersey, USA). Dimethyl sulfoxide (DMSO), doxorubicin, penicil-
Vis, in addition to elemental analysis, molar conductivity and lin, and streptomycin were obtained from Sigma Chemical
magnetic susceptibility. Company (Saint Louis, MO, USA). Human tyrosine kinase (TRK)
In the same direction and in continuing effort to find more ELISA kit was purchase from Glory Science Co., Ltd (Del Rio,
potent and selective anticancer compounds, herein, antican- TX 78840, USA).
cer activity of the prepared compounds was carried out against
four different human tumor cell lines including breast cancer 2.3.2. Cell lines and culturing
cell line MCF-7, liver cancer cell line HepG2, lung carcinoma A549 Anticancer activity screening for the tested compounds uti-
and colorectal cancer HCT116 that may act through tyrosine lizing 4 different human tumor cell lines including breast cancer
kinase (TRK) inhibition. Molecular Docking has been done to cell line MCF-7, liver cancer cell line HepG2, lung carcinoma
evaluate the binding affinity of the Ni and Zn complexes to TRK. A549 and colorectal cancer HCT116 were obtained from the
American Type Culture Collection (Rockville, MD, USA) through
LGC Standards GmbH, Wesel, Germany. The tumor cells were
2. Experimental maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat inactivated fetal calf serum
2.1. Materials and methods (GIBCO), penicillin (100 UmL−1) and streptomycin (100 μgmL−1)
at 37 °C in humidified atmosphere containing 5% CO2. Cells at
All chemicals were obtained from Merck. Elemental analyses a concentration of 0.50 × 106 were grown in a 25 cm2 flask in
were determined at the micro analytical center, Cairo University. 5 mL of complete culture medium.
 beni-suef university journal of basic and applied sciences 5 (2016) 85–96 87

2.3.3. In vitro cytotoxicity assay sample were positively correlated and the optical density was
The antiproliferative activity was measured in vitro using the determined at 450 nm. The level of TRK in samples was cal-
Sulfo-Rhodamine-B stain (SRB) assay according to the previ- culated (pmol mL−1) as duplicate determinations from the
ously reported standard procedure (Skehan et al., 1990). Cells standard curve.
were inoculated in 96-well microtiter plate (104 cells per well)
for 24 hours before treatment with the tested compounds to 2.4. Molecular docking study
allow attachment of cell to the wall of the plate. Test com-
pounds were dissolved in DMSO at 1 mg mL−1 immediately Molecular Operating Environment (MOE) 2008.10 (Moe source:
before use and diluted to the appropriate volume just before Chemical Computing Group Inc., Quebec, Canada, 2008) program
addition to the cell culture. Different concentration of tested was used in molecular docking studies. Firstly, a Gaussian
compounds and doxorubicin (2, 5, 10 or 20 μg mL−1) were added Contact surface around the binding site was sketched, then the
to the cells. Triplicate wells were prepared for each indi- surface enclosed the van Waals surface (we made filling in
vidual dose. Monolayer cells were incubated with the prepared solvent in accessible gaps). Finally docking studies were done
compounds for 48 hours at 37 °C and in atmosphere of 5% CO2. to assess the binding free energy of the inhibitor inside the
After 48 hours, cells were fixed, washed, and stained for 30 min macromolecule. The Dock scoring in MOE software was done
with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye utilizing London dG scoring function and has been upgrading
was removed by four washes with 1% acetic acid, and the at- by using two unrelated refinement methods, the Grid-Min pose
tached stain was recovered by Tris-EDTA buffer. Color intensity and the Force-filed have been improved to check that refined
was measured in an ELISA reader. The relation between both poses meet the specified conformations. Auto rotatable bonds
the surviving fraction and the drug concentration was plotted were allowed; the best ten binding poses were directed to analyze
to get the survival curve for each cell line after the specified for achieving the best score. To compare the docking poses to
time. The concentration required for 50% inhibition of cell vi- the ligand in the co-crystallized structure and to obtain RMSD
ability (IC50) was calculated and the results are given in Table 1. of the docking pose database browser was used.
The results were compared to the antiproliferative effects of
the reference control doxorubicin. 2.4.1. Preparation of ligands and target protein-tyrosine
kinase
2.3.4. In vitro human tyrosine kinase (TRK) concentration The compound contributed in this study as ligands was studied
assay for their binding affinity into protein-tyrosin kinase (TRK). To
The effect of tested compounds on the level of human tyro- build a three dimensional model of the structures, molecular
sine kinase (TRK) was determined in the four different human builder tool in MOE was used. Energy minimization was carried
tumor cell lines (MCF-7, HepG2, A549 and HCT116). The cells out through Force-Filed MMFF94X. Optimization was carried
at a concentration of 0.50 × 106 were grown in a 25 cm2 flask out using gradient of 0.001 for determining the lowest energy
in 5 mL of DMEM culture medium and were treated with 20 μl confirmation with most favorable geometry. The crystal struc-
of IC50 values of the compounds or the standard reference drug, tures of c-kit receptor protein-tyrosine kinase in complex with
Doxorubicin dissolved in DMSO, then incubated for 24 h at 37 °C, STI-571 (Imatinib or Gleevec) were picked up from the Protein
in a humidified 5% CO2 atmosphere. The cells were harvested Date Bank (PDB) (http://www.rcsb.org/pdb/explore/explore.do
and homogenates were prepared in saline using a tight pestle ?structureId=1T46) (PDB code: 1t46) (Mol et al., 2004). Partial
homogenizer until complete cell disruption. charges and Hydrogen atom were put on to the protein with
To determine the level of TRK in samples, a double-antibody the protonation 3d application in MOE. This application is
sandwich enzyme-linked immunosorbent assay (ELISA) was carried out to assign position hydrogen atom in the macro-
used. In brief, samples containing TRK were added to mono- molecule structures and ionization states.
clonal antibody enzyme well which is pre-coated with human
TRK monoclonal antibody, incubation; then, add TRK antibod- 2.4.2. Molecular modeling and analysis of the docked results
ies labeled with biotin, and combined with Streptavidin-HRP To rank the binding affinity of the synthesized compounds to
to form immune complex; then carry out incubation and wash TRK protein the binding free energy and hydrogen bonds
again to remove the uncombined enzyme. Then add Chromo- between the ligand and amino acid in TRK were used. Evalu-
gen solution A, B, the color of the liquid changes into blue, and ation of the hydrogen bonds were done by measuring the
at the effect of acid, the color finally becomes yellow. The hydrogen bond length, which does not exceed 3.7 A°. In ad-
chroma of color and the concentration of the human TRK of dition, RMSD of the co-crystal ligand position compared to the

Table 1 – Analytical and some physical characteristics for the prepared compounds.
No. Ligand/complexes Color M. wt. Calcd. (found) (%) AM Yield (%)
C H N
1 HL (C17H15N2SO) Yellow 295 69.13 (68.91) 5.12 (5.26) 9.48 (9.21) – 82
2 (C34H28CoN4O2S2) Greenish blue 647.6 63.05 (62.85) 4.36 (4.43) 8.65 (8.41) 16 65
3 (C34H28N4NiO2S2) Light brown 647.4 63.07 (62.85) 4.36 (4.32) 8.65 (8.52) 18 63
4 (C34H28CuN4O2S2) Brown 652.2 62.61 (62.26) 4.33 (4.2) 8.59 (8.48) 14 70
5 (C34H28N4O2S2Zn) Light yellow 654.1 62.43 (62.19) 4.31 (4.45) 8.57 (8.42) 11 64
88 beni-suef university journal of basic and applied sciences 5 (2016) 85–96

Scheme 1 – Preparation of the ligand and its complexes.

docking pose was used in ranking. Both RMSD as well as the the metal. The elemental analyses of the compounds 1–5 were
mode of interaction of the native ligand within the crystal struc- consistent with the calculated results from the empirical
ture of c-kite tyrosin kinase receptor were used as standard formula of each compound (Table 1).
docked model.
3.1. IR spectra

The IR spectra of the compounds 1–5 were represented in

3. Results and discussion Table 2. The IR spectra of the prepared ligand 1, which showed
a strong band at 1632 cm−1, were assigned to the formation of
The Schiff base ligand 1 was prepared from condensation of the azomethine (C=N) group (Abd-Elzaher, 2004a, 2004b;
salicyaldehyde with 2-amino-4-phenyl -5-methyl thiazole Abd-Elzaher et al., 2005). The band at 1567 cm−1 was attrib-
(Scheme 1). The ligand and its metal complexes (1–5) were found uted to υcyclic(C=N) of thiazole ring (Venkatraman et al., 2010).
to be stable in air and light. All compounds 1–5 were soluble Phenolic C—O stretch band is observed at 1278 cm−1; another
in common organic solvents such as ethanol, methanol, medium band appeared at 753 cm−1, which was assigned to
acetone, and chloroform. The elemental analysis confirmed that C—S—C (ring) stretching vibration (Abd-Elzaher, 2004a, 2004b).
all complexes 2–5 have 2:1 molar ratio between the ligand and Another band was observed centered at 2987 cm−1, which

Table 2 – The IR spectral (cm−1) assignment for the ligand and its complexes.
No. Ligand/complexes (OH)hydrogen-bond C=N (C=N)thiazole C—O C—S—C M—O M—N
1 HL(C17H15N2OS) 2987 1632 1567 1278 753 – –
2 (C34H28CoN4O2S2) – 1611 1517 1261 758 502 424, 443
3 (C34H28N4NiO2S2) – 1615 1522 1256 754 500 421, 454
4 (C34H28CuN4O2S2) – 1606 1562 1257 749 492 428
5 (C34H28N4O2S2Zn) – 1612 1523 1263 757 529 422, 449
 beni-suef university journal of basic and applied sciences 5 (2016) 85–96 89

Table 3 – 1H and 13C NMR data of the ligand and its ZnII complex.
Ligand/ complex Ligand ZnII complex
1
H NMR (DMSO-d6), 2.38(s, CH3 in thiazole ring), 6.93–7.80 (m,9 H,2Ph), 9.19(s, 2.25 (s, CH3 in thiazole), 6.58–7.78 (m,18H, 4Ph), 9.08 (s, 2H,
δ (ppm) 1H, CH=N), 11.51(s, 1H, OH) 2CH=N)
13
C NMR (DMSO-d6), 16.3 (CH3 attached to thiazole rings), 120.2, 141.6, 153.7 16.5 (CH3 attached to thiazole rings), 120.3, 141.6, 153.9
δ (ppm) (thiazole rings), 148.2 (C=N), 117.4–164 (aromatic (thiazole rings), 149.1 (C=N), 118.4–164.3 (aromatic
carbons) carbons)

indicate the presence of an intramolecular hydrogen bond in- appeared in the range of 117.4–164 ppm (Yıldız et al., 2010). The
13
teraction between phenolic group with imine-nitrogen atom C NMR signals of the ZnII complex were shifted slightly
(Nazır et al., 2000; Yıldız et al., 1998). downfield compared with that of the ligand, which may be due
By comparing the IR spectra of complexes 2–5 with the spec- to coordination of the ligand with the metal ions (Abd-Elzaher
trum of the free ligand, the disappearance of the intramolecular et al., 2012a, 2012b).
hydrogen bond signal of the free ligand was observed, which
indicates the removal of the proton of hydroxyl group during 3.3. Molar conductivity measurements
the chelation. This is further supported by the shift of C—O
band from 1278 cm−1 (in ligand) to 1256–1263 cm−1 (in com- The molar conductivities of 10−3 M of the complexes (dis-
plexes) (Abd-Elzaher et al., 2005). However, the band due to the solved in DMF) at room temperature were measured (Table 1).
azomethine (C=N) group in the free ligand (at 1632 cm−1) was The results were in the range 11–18 Ω−1.mol−1.cm2 for CoII, NiII,
shifted to a lower frequency (1606–1615 cm−1) in the com- CuII and ZnII complexes. Conductivity measurements reveal that
plexes. This shift indicates coordination of the azomethine all the metal complexes have conductivity values in the range
nitrogen to the metal ions in the complexes. In addition, the characteristic for non-electrolytic nature (Andersen, 1999;
band due to υcyclic(C=N) of thiazole ring (in ligand) was shifted Chohan et al., 2004; Golcu et al., 2005; Joseyphus et al., 2006;
in all complexes except Cu-complex; this shift indicates the Vashi and Naik, 2004; Venugopala and Jayashree, 2003).
involvement of the thiazole nitrogen atom in the complex for-
mation (2,3,5) and not coordinated in the case of complex 4
3.4. Electronic spectra and magnetic moment
(Bolos et al., 1999). The C—S—C band of the free ligand, which
appears at 753 cm−1, showed a very little effect by complex-
The electronic absorption spectrum of the ligand showed three
ation indicating that the sulfur atom is not involved in the
bands at 272, 338 and 453 nm (Table 4). The first one may be
chelation (Nakamoto, 1998; Neelakantan et al., 2008; Omar and
assigned to intra-ligand π–π* transition, which is nearly un-
Mohamed, 2005). Two new bands in the range 484–502 and 421–
changed on complexation, whereas the second and third bands
454 cm−1 were also observed (Table 2). These two bands were
may be assigned to the n–π* and charge transfer transition of
observed in the complexes and not found in the free ligand
the azomethine group and nitrogen atom of the thiazole ring
and they are attributed to M—O and M—N bonds in the com-
(Sreeja et al., 2004).
plexes respectively (Rana et al., 1982).
The electronic spectra of the CoII complex showed two bands
at 572 and 484 nm. These bands are assigned to the transi-
3.2. NMR spectra tions 4T1g(F) →4A2g(F) and 4T1g(F) →4T2g (P), respectively; the CoII
complex also showed magnetic moment at 4.6 B.M., which in-
The 1H and 13C NMR spectra of the ligand and its ZnII complex dicates the presence of CoII complex in octahedral geometry
were recorded at room temperature using DMSO-d6 as a solvent (Abd-Elzaher et al., 2012a, 2012b; Fouda et al., 2008a, 2008b). The
(Table 3). The 1H NMR spectra of the ligand showed a singlet electronic absorption of NiII complex showed three bands at 595,
at δ2.38 ppm attributed to methyl group attached to the thia- 387 and 841 nm (Table 4). These bands are assigned to the tran-
zole ring, and multiplet at δ 7.80–6.93 ppm due to aromatic sitions 3A2g (F) →3T1g (F), 3A2g→3T1g (p) and 3A2g→3T2g respectively
protons. The spectra showed also azomethine proton (CH=N) (Abd-Elzaher et al., 2006; El-Shiekh et al., 2006; Lever, 1984); the
as singlet at δ 9.19 ppm. The OH proton is observed as singlet magnetic moment of NiII complex was 3.2 B.M. These results sug-
at δ 11.51 ppm. A comparison of the 1H NMR spectra of the ZnII gested the presence of octahedral geometry for NiII complex. The
complex and the free ligand indicates that the proton signal electronic spectra of the CuII complex showed two bands at 642,
corresponding to OH group of ligand has disappeared in ZnII
complex, which may be due to deprotonation. The CH=N proton
in the ligand is shifted to 9.08 ppm in the ZnII complex, which
suggested that the azomethine nitrogen is involved in the co- Table 4 – The electronic spectra and magnetic moments
ordination (Maurya et al., 2005). for the ligand and its complexes.
The 13C NMR of the ligand showed a signal at 16.3 ppm which No. Ligand/complexes Bands in DMSO μeff (BM)
was assigned to the methyl group attached to the thiazole rings, 1 HL(C17H15N2OS) 453, 338, 272 –
and three signals at 120.2, 141.4 and 159.4 ppm were as- 2 (C34H28CoN4O2S2) 572, 484, 453, 340, 270 4.6
signed to the thiazole rings (Abd-Elzaher et al., 2012a, 2012b). 3 (C34H28N4NiO2S2) 841, 595, 450, 387, 271 3.2
The signal that appeared at 152.2 ppm was assigned to the 4 (C34H28CuN4O2S2) 624, 508, 452, 356, 270 1.81
5 (C34H28N4O2S2Zn) 450, 350, 270 Dia.
carbon of the azomethine group. Signals due to phenyl carbons
90 beni-suef university journal of basic and applied sciences 5 (2016) 85–96

508 nm, which are assigned to 2B1g→2A1g and 2B1g →2Eg transi- 3.7. Anticancer activity
tions respectively (Abd-Elzaher, 2004a, 2004b; Fouda et al., 2008a,
2008b), and the magnetic moment of CuII complex was 1.81 B.M. Chemotherapy is the major approach for both localized and
Both the electronic spectra and the magnetic values proposed metastasized cancer. Therefore, the synthesized compounds
the presence of the square planar geometry for CuII complex. were screened for their in vitro cytotoxicity and growth inhibi-
On the basis of the above observation and spectral data, it is tory activities against 4 different human tumor cell lines
suggested that the CoII, NiII and ZnII complexes show octahe- including breast cancer cell line MCF-7, liver cancer cell line
dral geometry structures; however, the CuII complex shows HepG2, lung carcinoma A549 and colorectal cancer HCT116. The
square-planar geometry structure (Lever, 1984). results were compared with the activity of the known anti-
cancer doxorubicin as a reference drug. The cytotoxicity of the
3.5. Mass spectra tested compounds was expressed by median growth inhibi-
tory concentration (IC50) which required producing 50% cytotoxic
The mass spectrum supports the proposed empirical formula effect against cancer cells after 48 hours exposure to tested
of the ligand. It reveals the molecular ion peak m/z at 294 con- compounds. The screening results are given in Table 1.
sistent with the molecular weight of the ligand. While the It is evident that for the HepG2 and MCF-7 cells, all the
fragments at m/z = 214, 201, 188, and 83 correspond to C12H12N3O, tested compounds showed anticancer activity with IC50 values
C12H12N2O, C11H11N2O, and C4H3S, respectively. The mass spectra that ranged from 6.20 to 9.22 and from 6.00 to 10.00 μg mL−1
of the complexes 2–5 show molecular ion peaks m/z at 648, respectively (Table 5). It is clear from the data that the order
647, 652 and 654, consistent with the molecular weight of these of cytotoxicity activity against the two cancer cell lines of
complexes, respectively. the tested compounds was ZnII complex, NiII complex, CuII
complex, CoII complex and (L)1 in descending order. While in
3.6. Electron spin resonance spectra case of A549 cells, only ZnII complex and NiII complex showed
anticancer activity at IC50 of 5.30 and 9.10 μg mL−1 respec-
The solid state ESR spectra of 4 exhibit axially symmetric tively. In addition, HCT116 revealed anticancer activity for
g-tensor parameters with gǁ <= g⊥ > 2.0023 indicating that the L, CuII complex and ZnII complex with IC50 of 9.50, 6.70 and
copper site has a dx2-y2 ground-state characteristic of square 6.20 μgmL−1 respectively. It is clear from the results that in all
planar stereochemistry (El-Sonbati et al., 2011; Speie et al., 1996). four cell lines the cytotoxicity of ZnII complex showed a
The spin Hamiltonian parameters of the complex at gǁ, g⊥, and strong activity compared with doxorubicin. Furthermore, to
Aǁ have the values 2.157, 2.064, 2.095 and 207 × 10−4 cm−1, with elucidate the mechanism by which the prepared compounds
exchange interaction parameter (G) 4.6. A good indication of exert their antitumor activities, we estimated the level of
square planar coordination geometry for the copper complex human TRK in the cancer cells treated with the prepared
moiety according to the gǁ and |Aǁ| values has been shown. compounds.
In addition, in axial symmetry, the g-values are related by Tyrosive kinases (TRKs) are indispensable for numerous pro-
the expression G = (gǁ − 2)/(g⊥ − 2) = 4, where G is the ex- cesses in the cell. These enzymes catalyze phosphorylation of
change interaction parameter. According to Hathaway and different cellular substrates. Phosphorylation in turn regu-
Billing (Diab et al., 2010; El-Sonbati et al., 2011; Hathaway and lates various cellular functions. Normally, their activity is
Billing, 1970), if the value of G is greater than 4, the exchange stringently regulated. However, under pathological condi-
interaction between CuII centers in the solid state is negli- tions, TRKs can be deregulated, leading to alterations in the
gible, whereas when it is less than 4, a considerable exchange phosphorylation and resulting in uncontrolled cell division, in-
interaction is indicated in the solid complex. The calculated hibition of apoptosis, and other abnormalities and consequently
G values for 4 is 4.6 suggesting that there are no copper– to diseases (Shchemelinin et al., 2006a, 2006b). Various cancers
copper exchange interactions (Diab et al., 2010; El-Sonbati et al., and other diseases are known to be caused or accompanied
2011; Hathaway and Billing, 1970; Speie et al., 1996). by deregulation of the phosphorylation. Inhibition of TRKs has

Table 5 – The IC50 values and the percent of TRK of the tested compounds in 4 different human cancer cell lines.
Compound IC50 (μgmL−1) % of TRK inhibition*
HepG2 MCF-7 A549 HCT116 HepG2 MCF-7 A549 HCT116
L (ligand) 9.22 10.00 – 9.50 19 6.5 – 18
CoII complex 8.16 9.11 – – 33 19 – –
NiII complex 7.20 7.30 9.10 – 73 55 38.5 –
CuII complex 7.50 8.70 – 6.70 67 33 – 43
ZnII complex 6.20 6.00 5.30 6.20 80 70 61 64
DMSO – – – – – – – –
Doxorubicin 4.20 4.40 4.70 5.25 81 84 83.5 83
Data were expressed as average of three independent experiments.
The percentage changes as compared with control untreated cells (DMSO treated).
 beni-suef university journal of basic and applied sciences 5 (2016) 85–96 91

been shown to be a promising therapeutic strategy. Many TRK inhibition. It is clear that ZnII complex showed potent inhibi-
inhibitors (TRKs) have been produced and tested in clinic by tion against human TRK in the four cell lines (HepG2, MCF7, A549,
now. These molecules have a low molecular weight and most HCT116) by the ratio 80, 70, 61 and 64% respectively as com-
of them bind to protein kinases competing with ATP for the pared to the inhibition in the untreated cells as listed in Table 5.
ATP-binding site (Bogoyevitch et al., 2005). The availability of The highest activity of the ZnII complex than the others may
newer inhibitors will be the main target of many researchers be attributed to the function of the ZnII complex as a com-
in the future. petitive inhibitor of hemeoxygenase (HMOX1), which is
The results in Table 5 showed that most of the tested com- produced in large amounts in solid tumors (Huang et al., 2005),
pounds exhibited inhibitory potential against human TRK. In case in humans and animal tumor models. HMOX1 has been adapted
of human liver cancer HepG2 NiII, CuII and ZnII complexes re- to defend against oxidative and other cellular stresses (Huang
vealed inhibition to TRK by 73, 67 and 80% as compared with et al., 2005).
the untreated cancer cells, while doxorubicin showed 81% in-
hibition. In case of human breast cancer MCF7 NiII and ZnII 3.8. Molecular modeling: docking study
complexes were found to be potent inhibition against human
TRK by 55% and 70% respectively, while doxorubicin showed 84% 3.8.1. Molecular modeling: docking study
inhibition. Moreover, in case of human lung cancer A549, only The prepared compounds were analyzed for the binding af-
ZnII complex was found to be potent inhibition against human finity of tyrosine kinases receptor (PDB 1t46) for the purpose
TRK by 61% while doxorubicin showed 83.5% inhibition. Fur- of both find out the interaction between studied compounds
thermore, in case of colorectal cancer HCT116 CuII and ZnII and c-kit receptor and for lead optimization. Molecular mod-
complexes were found to be potent inhibition against human eling calculation was carried out to investigate the binding free
TRK by 43 and 64% respectively, while doxorubicin showed 83% energies of these inhibitor inside the target c-kit kinase receptor.

Fig. 1 – The binding mode of the native ligand STI with C-kit exhibited one H-bond donor with THR 670 at distance 2.08 and
one H-bond donor with ILE 789 at distance 2.19 and one H-bond donor with CYS 673 at distance 2.85 and one H-bond
acceptor with HOH 1105 at distance 2.82 its score was −20.04 k.calmol−1.
92 beni-suef university journal of basic and applied sciences 5 (2016) 85–96

3.8.2. Validation of the docking performance and accuracy 3.8.3. The binding affinities of the synthesized compounds
Docking of the native co-crystallized STI-571 ligand (Imatinib into c-kit kinase receptor
or Gleevec) was used to validate the docking accuracy of the To compare affinity and to find out the interaction between
program. The docked ligand was exactly superimposed on the ligand and receptor, molecular docking study was done (Fig. 1).
native co-crystallized one with RMSD being 0.40 Å´ and binding For the docking calculation, firstly, the protein structure (PDB
free energies of −20.04 kcal.mol−1. The hydrogen bonds between code: 1t46) was detached from the inhibitor and hydrogen atoms
the amino acids and the docked ligand were the same as those were added. The binding free energy, hydrogen bond and RMSD
between the amino acid and the native ligand. were used to determine the binding affinity. All the prepared

Fig. 2 – The binding mode of the Zn-complex with C-kit receptor exhibited one H-bond acceptor with HOH 1105 at distance
2.64; and one H-bond acceptor with HOH 1105 at distance 2.63 and one H-bond acceptor with HOH 1106 at distance 3.32
and its score was −15.77 k.cal mol−1.
 beni-suef university journal of basic and applied sciences 5 (2016) 85–96 93

Fig. 3 – The binding mode of the Ni-complex with C-kit exhibited one H-bond acceptor with HOH 1033 at distance 3.16 and
its score was −11.98 kcalmol−1.
94 beni-suef university journal of basic and applied sciences 5 (2016) 85–96

compounds were docked into the same binding site of the thiophenol and its coordination with some transition metals.
native co-crystallized ligand. Both ZnII and NiII 5,3 respec- Monatsh Chem 2012b;143:909–13.
tively, gave the best docking score (Figs. 1–3). Abu-Diefa AM, Mohamed IMA. A review on versatile applications
of transition metal complexes incorporating Schiff bases.
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The Schiff-base ligand 1 (E)-2-(((5-methyl-4-phenylthiazol-2-
antagonists: identification of ethyl 3-[N-[4-[4-
yl)imino)methyl)phenol was synthesized and determined with [amino[(ethoxycarbonyl) imino]methyl]phenyl]- 1,3-thiazol-2-
different spectroscopic techniques, followed by synthesis of four yl]-N-[1-[(ethoxycarbonyl) methyl] piperid-4-yl] amino]
novel cobalt, nickel, copper and zinc complexes. The new com- propionate (SR 121787) as a potent and long-acting
pounds were investigated for inhibition against human TRK antithrombotic agent. J Med Chem 1997;40:3393–401.
in vitro cytotoxicity for four human tumor cell lines. The ZnII Bell FW, Cantrell AS, Hogberg M, Jaskunas SR, Johansson NG,
Jordon CL, et al. Phenethyl-Thiazolethiourea (PETT)
complex showed potent inhibition against human TRK in the
compounds, a new class of HIV-1 reverse transcriptase
four cell lines (HepG2, MCF7, A549, HCT116) by the ratio 80, 70, inhibitors. 1. Synthesis and basic structure-activity
61 and 64% respectively as compared to the inhibition in the relationship studies of PETT analogs. J Med Chem
untreated cells in comparison with the reference drug (doxo- 1995;38:4929–36.
rubicin); also most of the tested compounds exhibited inhibitory Bogoyevitch MA, Barr RK, Ketterman AJ. Peptide inhibitors of
potential against human protein tyrosine kinase (PTK). The syn- protein kinases-discovery, characterization and use. Biochim
thesized compounds were investigated for the binding affinity Biophys Acta 2005;1754:79–99.
Bolos CA, Fanourgakis PV, Christidis PC, Nikolov G. Crystal and
of tyrosine kinases receptor (PDB1t46). MOE (molecular mod-
molecular structures of [(2-amino-5-methyl-1,3-
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We are grateful for financial support from the National Re- reduce Schiff base ligand based transition metal complexes. J
search Center, Cairo, Egypt (project no. 10070102). Mol Struct 2016;1106:366–72.
Chen P, Horton LB, Mikulski RL, Deng L, Sundriyal S, Palzkill T,
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