Illustration for series of new metal ion complexes extracted from pyrazolone derivative, spectral, thermal, QSAR, DFT/B3LYP, docking and antitumor investigations

Article abstract of DOI:10.1016/j.molliq.2016.11.035

New VO(II), Mn(II), Co(II), Ni(II) and Cu(II) - pyrazolone complexes were isolated and deliberately characterized. The analytical and spectral tools emphasis on the isolation of bi-nuclear complexes except Mn(II) one. Spectral parameters and reduced magnetic moments are the bases for proposing bi-nuclear complexes. Variable coordination nos. are observed with isolated complexes in-between four, five or six coordination around central atoms. TG analysis and kinetic parameters are concerned in study to examine the thermal stability of complexes. Implementing Gaussian09 program to optimize the structural formula for the investigated compounds. Applying DFT/B3LYP method, the frontier energy gapes were calculated and other important theoretical parameters. QSAR calculations proposes a distinguish biological activity for pyrazolone ligand. Molecular docking using Auto Dock tools were utilized to explain the experimental behavior of the organic compound towards the microorganisms through theoretical visualization. The docked complexes of 2YXS, 2CGY and 2JW2 protein receptors for prostate, colon and liver carcinoma were investigated and the complexation energies were calculated. The estimated energies display best expectation for excellence antitumor activity for pyrazolone compound towards variable carcinoma. IC₅₀ calculated represent an excellent priority for Cu(II) complex in overcoming liver and prostate carcinoma cell lines in comparing with reference drug. Moreover, the Mn(II) complex displays high comparative impact also with colon carcinoma.

Full text of DOI:10.1016/j.molliq.2016.11.035

Illustration for series of new metal ion complexes extracted from pyrazolone
derivative, spectral, thermal, QSAR, DFT/B3LYP, docking and antitumor
investigations
Fawaz A. Saad, Nashwa M. El-Metwaly, Thoraya A. Farghaly, Marwa
G. El-Ghalban, Gamil A. Al-Hazmi, Kamel. A. Saleh, Mohammad Y. Alfaifi
PII:
DOI:
Reference:
S0167-7322(16)33393-1
doi:10.1016/j.molliq.2016.11.035
MOLLIQ 6579
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
29 October 2016
7 November 2016
9 November 2016
Please cite this article as: Fawaz A. Saad, Nashwa M. El-Metwaly, Thoraya A. Farghaly,
Marwa G. El-Ghalban, Gamil A. Al-Hazmi, Kamel. A. Saleh, Mohammad Y. Alfaifi,
Illustration for series of new metal ion complexes extracted from pyrazolone derivative,
spectral, thermal, QSAR, DFT/B3LYP, docking and antitumor investigations, Journal
of Molecular Liquids (2016), doi:10.1016/j.molliq.2016.11.035
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ACCEPTED MANUSCRIPT
Illustration for series of new metal ion complexes extracted from pyrazolone
derivative, spectral, thermal, QSAR, DFT/ B3LYP, docking and antitumor
investigations
Fawaz A. saad1, Nashwa M. El-Metwaly1,2*, Thoraya A. Farghaly1,3, Marwa G. El-Ghalban1,2, Gamil A. Al-
Hazmi4,5, Kamel. A. saleh6, Mohammad Y. Alfaifi6
1Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah Almukkarramah, Saudi Arabia.
2Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt
3Department of Chemistry, Faculty of Science, University of Cairo, Giza, 12613, Egypt
4Chemistry Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha, Saudi Arabia
5Chemistry Department, Faculty of Applied Sciences, Taiz University, P.O. Box 82, Taiz, Yemen
6Biology Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha, Saudi Arabia
Abstract
New VO(II), Mn(II), Co(II), Ni(II) and Cu(II) - pyrazolone complexes were isolated and
deliberately characterized. The analytical and spectral tools emphasis on the isolation of bi-nuclear
complexes except Mn(II) one. Spectral parameters and reduced magnetic moments are the bases for
proposing bi-nuclear complexes. Variable coordination nos. are observed with isolated complexes
in-between four, five or six coordination around central atoms. TG analysis and kinetic parameters
are concerned in study to examine the thermal stability of complexes. Implementing Gaussian09
program to optimize the structural formula for the investigated compounds. Applying DFT/B3LYP
method, the frontier energy gapes were calculated and other important theoretical parameters.
QSAR calculations proposes a distinguish biological activity for pyrazolone ligand. Molecular
docking using Auto Dock tools were utilized to explain the experimental behavior of the organic
compound towards the microorganisms through theoretical visualization. The docked complexes of
2YXS, 2CGY and 2JW2 protein receptors for prostate, colon and liver carcinoma were investigated
and the complexation energies were calculated. The estimated energies display best expectation for
excellence antitumor activity for pyrazolone compound towards variable carcinoma. IC50 calculated
represent an excellent priority for Cu(II) complex in overcoming liver and prostate carcinoma cell
lines in comparing with reference drug. Moreover, the Mn(II) complex displays high comparative
impact also with colon carcinoma.
Keywords: DFT/B3LYP, Molecular Docking, Spectral, TEM, Antitumor
*Corresponding author: Prof. Nashwa El-Metwaly
E mail address: n_elmetwaly00@yahoo.com
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Telephone No. : +9660538553123
1. Introduction
Pyrazolone compounds refer to class of 5- membered ring structures include two nitrogen atoms
and one carbonyl group. Their chemistry is significantly interesting because their extensive
applications in therapeutic field [1-3] and anti-inflammatory[4]. The coordination chemistry will be
enriched with the use of such important compound. The former studies introduces Zn(II) and Hg(II)
metal ion complexes derived from pyrazolone derivatives with potential antitumor activity[6]. Also,
in agriculture, they are used as pesticides [7]. Pyrazoles are potential appreciate to form complexes,
they are suitable agents for investigating active sites of bio-molecules [8] and for modeling bio-
systems for oxygen transfer [9]. Metal ions are usually bonded to the imidazole moiety of hystidine,
which is a part of proteins in living organisms. From the literature reports [10], both of pyrazole and
imidazole are proper to mimic enzymatic reactions. The coordination chemistry of oxovanadium
and copper ions are highly concerned because of their important role in biological and catalytic
systems [11-13]. This is due to their electronic configuration are easily specified by EPR study. The
aim of the work is the use of synthesized pyrazolone derivative includes multi-donor centers to give
a great chance for poly-nuclear complexes. Such poly-nuclear complexes will serve Excellency in
the biological field based on the intensive effect of the metallic role and pyrazolone ring. All
possible spectral and analytical tools will be used to inspect the structural formula of the complexes.
Computational studies will be utilized to deepen the study, Gaussian09 molecular modeling, QSAR
and Autodock tools. Antitumor activity will be concerned against three carcinoma cell lines to give
a clear insight about the expected biological efficiency for the poly-nuclear complexes in comparing
with original ligand and referenced drug.
2. Experimental
2.1. Reagents
The chemicals used in this study to prepare pyrazolone ligand; cyanoacetic hydrazide, sodium
acetate tri-hydrate and sodium nitrite were purchased from Fulka and employed as it is. The metal
salts used for complexation are VOSO4.XH2O, MnCl2.4H2O, CoCl2(H2O)6, NiCl2(H2O)6 and
CuCl2.2H2O, were commercially available from Sigma-Aldrich. The solvents used in synthesis
process are utilized without previous purification.
2.2. Synthesis
2.2.1. Synthesis of (Z)-3-amino-4-(2-phenylhydrazono)-1-H-pyrazol-5(4H)-one
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Pyrazolone derivative was prepared as reported previously [14]. Freshly prepared cyanoacetic
hydrazide (0.99g, 0.01 mol) in ethanol (50 ml) was stirred with sodium acetate trihydrate (1.36 g,
0.01 mol) for 15 min. The mixture was put in ice bath to 0-5 C. While, the solution was cooled,
arenediazonium chloride was prepared by diazotizing the appropriate aniline derivative (0.01 mol)
in 6 M hydrochloric acid (6 mL) with cold sodium nitrite solution (10 ml, 1M) as usual. Diazonium
salt solution was added drop wisely (20 min.) to a cold solution of cyanoacetic hydrazide. While
stirring and keeping the temperature below 5C, the resulting mixture was left for 3 h in a
refrigerator. The synthesized arylhydrazone was filtered off and exsiccated. Refluxing of ethanolic
arylhydrazone for 2hr gives, 3-amino-4-(2phenylydrazono)-4,5-dihydropyrazol-5-ones (HL) ligand
[scheme 1].
Scheme1.Synthesis of pyrazolone ligand(HL)
2.2.2. Synthesis of metal ion complexes
Synthesis process was implemented at normal conditions of mixtures pHs. Equi-molar ratios (1 : 1)
were mixed from pyrazolone ligand (HL) to each metal salt. 1.016g (5 mmol) of pyrazolone ligand
was dissolved in ethanol. Then, added drop wisely to each metal salt solution attributed to,
VOSO4.XH2O(0.815g), MnCl2.4H2O(0.990g), CoCl2(H2O)6 (1.190g); NiCl2(H2O)6 (1.190g) and
CuCl2.2H2O(0.853g). The mixtures refluxed for 3 – 4 h, the colored precipitates were filtered off
washed with ethanol, Et2O and finally dried in a vacuum desiccators. The influence of ligand charge
transfer character is clearly affecting on colors of complexes.
2.3.Cytotoxicity against tumor cells
The toxicity of isolated complexes as well as their original salts were tested against HCT-116,
HEPG-2 and PC-3 tumor cell lines by sulfo-rhodamine B (SRB). Cells were preserved in RPMI
media appended with streptomycin (100 µg/mL), penicillin (100 units/mL) and 10% heat-
inactivated fetal bovine serum in a humidified, 5% (v/v) carbon dioxide atmosphere at 37 ºC, the
cells were sub-cultured tow times in a week. Exponentially resurgent cells were collected using
0.25% tryps in–EDTA and plated in 96-wellplates at 1000cells/well. Cells were exposed to the
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extracts for 72h and subsequently fixed with TCA (10%) for 1h at 4°C. After cells were washed
several times, they were exposed to 0.4% SRB solution for 10 min in a dark place and then washed
with 1% AcOH. After drying over night, Tris–HCl was used to dissolve the SRB-stained cells and
the color intensity was measured at 540 nm. The cell viability was measured by Trypan blue (0.4)
stain, permit us to distinguish viable (non-stained) and dead (stained) cells from each other. Viable
and dead cells were counted with Nikon microscope with the assistance of hemocytometer. Viable
cell percentage = [1.00–(Number of blue cells÷Number of total cells)] × 100. The statistical
analysis was accomplished through the use of Sigma Plot version 12.0
2.4. Equipments
Carbon, H and N were analyzed at Micro analytical unit. The content of metal and the conjugated
anions were estimated by utilizing standard methods [15]. The infrared spectra, as KBr discs, were
recorded on JASCO FT-IR-4100 Spectrophotometer (400–4000cm-1). The electronic and H1NMR
(200 MHz) spectra were recorded on UV2 Unicam UV/Vis, and a Varian Gemini
Spectrophotometers. The effective magnetic moments were estimated at the ordinary temperature
x`MT
by applying eff = 2.828
, where XM is the molar susceptibility corrected using Pascal’s
constants for diamagnetism of all atoms in the ligand using a Johnson Matthey magnetic
susceptibility balance. Thermal analysis was carried out on a Shimadzu thermogravimetric analyzer
at a heating rate of 10oC min-1 under nitrogen. X-ray diffraction types (XRD) were acquired on
Pikagu diffractometer by utilizing Cu / Kα radiation. Scanning electron microscopy (SEM) images
were possessed in Joel JSM-6390 tools. ESR spectra of VO(II) and Cu(II) powdered complexes
were obtained on a Bruker EMX Spectrometer working in the X-band (9.60 GHz) with 100 kHz
modulation frequency. The microwave power was set at 1 mW, and modulation amplitude was set
at 4 Gauss. The low field signal was obtained after 4 scans with a 10 fold increase in the receiver
gain. A powder spectrum was obtained in a 2 mm quartz capillary at ordinary temperature.
2.5. Theoretical calculations
2.5.1. Kinetic studies
All the kinetic parameters assigned to thermal analysis were abstracted from TG curves and
tabulated. The order (n) and the energy of activation (E) were determined for suitable
decomposition stages. Formerly, different researchers [16-24] were established equations and
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discussing their advantages. The rate of decomposition is the product of two separate functions of
temperature and conversion using:
d
dt
= k(T ) f (α)
(1)
where, α is the fraction decomposed at time t, k(T) is the temperature dependent function and f(α) is
the conversion function. The rate constant and dependent function k(T) is of Arrhenius type.
K = A e-E*/ RT
(2)
where R, is the gas constant in (J mol-1 k-1) substituting equation (2) into equation (1) we get this
equation :
A
d
 

(3)

 e-E* RT
dT
where φ, is the linear heating rate (dT/dt). From the integration and approximation, this equation
can be obtained in the following form:
E *
RT
 AR 
ln g () 
ln


E *


(4)
where g(α) is a function depends on the mechanism of reaction. The right hand side is known as
temperature integral and has no close for solution. So, several techniques have been used to
evaluate the temperature integral. The kinetic parameters for the ligand and its complexes are
evaluated using Coat-Redfern [18] and Horowitz-Metzger methods [23].
2.5.2. Molecular modeling methodology
The optimized structural geometry of pyrazolone and its complexes were executed by DFT/B3LYP
method with different base sets using Gaussian09 software [25]. Gauss-View molecular
visualization program are used to display Gaussian files [26]. In agreement with the numerical
pattern appeared in the view of the compounds in gas phase, DFT/B3LYP quantum chemical
parameters are calculated from HOMO–LUMO energies. Also, significant bond lengths, oscillator
strength, excitation energy and effective charges for coordinating groups in optimized structures
will be deducted. QSAR computations were performed for the ligand to expect its biological
activity.
2.5.3. Molecular docking
Implementing Autodock tools 4.2, docking computations applying Gasteiger partial charges added
to ligand (designed drug) atoms, were executed. On the ligand- protein pattern, the calculations
were executed. Non-polar hydrogen atoms were conjoined, and rotatable bonds were clarified. After
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the addition of; fundamental hydrogen atoms, Kollman united atom type charges and salvation
parameters the Auto Dock tools were applied [27]. Autogrid program were used to accomplish
affinity (grid) maps of ×× Å grid points and 0.375 Å spacing [28]. Determining Vander Waals and
electrostatic terms were performed by Auto Dock parameter set- and distance-dependent dielectric
functions, respectively. Simulative docking were executed by Solis & Wets local search method and
Lamarckian genetic algorithm (LGA) [29]. Incidentally, initial position, orientation and torsions of
ligand molecule were set. All rotatable torsions were emitted during docking. 10 different runs were
used to derive each docking experiment, that were set to block after a ultimate of 250000 energy
estimations. The size was established to 150. Throughout the study, a translational step of 0.2 Å,
quaternion and torsion steps of 5 were utilized.
3. Results and discussion
3.1. General
Essential physical and analytical data for pyrazolone and its VO(II), Mn(II), Co(II), Ni(II) and
Cu(II) complexes are displayed in Table 1. All the investigated complexes have high melting points,
insoluble in all organic solvents except DMSO and DMF are sparingly soluble. 2:1 (M:L) molar
ratio is the universal formularization proposed for all investigated complexes except Mn(II)
complex displays 1:1 ratio. The divergence of Mn(II) ratio may refer to its relative big size which
may instable the bi-nuclear complex. Whereas, the VO(II) complex is different due to the trans
effect of V=O bond which facilitates the poly-nuclear formation. The molar conductivity
measurements cannot be performed due to sparingly soluble feature of the complexes which mainly
attached with neutral coordination spheres. This is anticipated behavior with chloride and sulphate
anions which favor their covalent attachments with metal ions.
3.2. IR and 1HNMR
Essential vibrations of pyrazolone and its complexes were presented in Table 2. Free pyrazolone
spectrum represents the following bands: 3410, 3333; 3185; 1671; 1586; 1625; 1442,1493
attributing to υNH2; υNH; υC=O; υC=N; δNH2; δNHs [30]. Its 1H NMR (DMSO-d6) spectrum (Fig.
1S)displays: 5.8 (s, 2H, NH2), 7.1-7.53 (m, 6H, NH, Ar-H), 10.53 (s, 1H, NH). Its MS m/z (%) 204
(M++1, 12), 203 (M+, 100), 126 (67), 93 (38), 77 (60), 65 (61). These varification tools introducing
the best structural formual of the free organic compound which beeing confirmed with molecular
modeling implementation.
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Fig. 1. Optimized and occupied geometries of pyrazolone ligand (HL)(1 and 2, respectively)
The coordination manner across central atoms was proposed from IR spectral data. Neutral tetra-
dentate mode was suggested with bi-nuclear complexes whereas, bi-dentate mode with mono-
nuclear Mn(II) one. Neutral mode appeared, reflects the relative acidity of complexation media
yielded from using acidic salts for transition metals. The lower shift observed generally with υNH2;
υNH; υC=O; υC=N; δNH2; δNH bands in VO(II), Co(II), Ni(II) and Cu(II) complexes, verifies the
coordination manner suggested by NNNO donors. While, the lower shift for υC=O; υNH and δNH
bands in Mn(II) complex spectrum represents NO as coordinating sites. An observable broadness
with bands assigned for υNHs and υC=N prohibits the exact determination for non-coordinating
twins groups. VO(II) complex spectrum displays new bands at 1387.7, 1319.98 and 968.8 cm-1
attributing to υas(SO4), υs(SO4) and υ(V=O) vibrations. The broadness appeared at the upper region
may suggest of crystal or coordinating hydrating molecules accompanied with the synthesized
complexes[31]. Also, the presence of new bands assign for δr(H2O) and δw(H2O) vibrations [32]
verifies their presence in coordinating or crystal forms. New week bands abstracted are attributing
to υM-N and υM-O vibrations to verify the bonding manner. υM-Cl vibration band cannot easily
detected in the scan range applied by the used IR equipment.
3.3. Electronic spectra and magnetic measurements
In DMSO solvent, significant electronic transition bands recorded for 0.1 mmol/L as well as some
spectral data were recorded and tabulated( Table 3). Intra-ligand transition bands are: 31,250;
25,000 and 23,809 cm-1 assign for π → π* and n→ π* transitions, respectively. The molar
absorptivity value for n→ π* transition band is ε1 = 454 mol-1dm3cm-1 suitable for first excitation
process inside pyrazolone. A moderate conjugation observed for chromophor groups in the
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pyrazolone may affect on charge transfer (CT) position which close to visible region and deepen the
compound color. The molar absorptivity value was elevated with the complexes first transition
bands. This may refer to d-d transitions which have high oscillator strength values especially for the
introductory transition step inside splitting d orbital's. [(VO)2(SO4)2(C9H9N5O)] 2H2O complex
2
2
spectrum shows a significant band at 13,150 cm-1 assigns for B2→2E transition while, B2→2B1
transition is strongly occluded with CT band at 26,521cm-1 in square-pyramidal configuration
(Fig.2A) [33]. The reduced magnetic moment value (1.34BM) is supporting a strong interaction
inside the bi-nuclear complex. [MnCl2(C9H9N5O).2(H2O)] complex spectrum shows bands at:
6
18,420; 23,529; 27,027 and 31,250 cm-1. The bands are corresponding to A1g→4T1g(G4),
6
6
6A1g→4Eg,4A1g(G4)(10B+5C), A1g→4Eg,(D4)(17B+5C), A1g→4T1g(P4) (7B+7C) transitions,
respectively in octahedral configuration (Fig. 2B). The magnetic moment value (5.96BM) is within
the normal range of high spin low field mono-nuclear d5 complexes. The 10Dq, Racah and
nephelauxetic parameters values (8442.5 cm-1, 767.5 cm-1 and 0.892) were calculated and
displaying a soft shift from elemental shape and a significant ionic attachment may be proposed
based on the β value near the upper limit (β=1) for pure ionic interaction.
[Co2Cl4(C9H9N5O).4(H2O)] complex spectrum shows d-d transition bands at: 16,255 and 19,560
4
cm-1 correspond to4T1g→4A2g(υ2) and T1g→4T1g(p)(υ3) transitions, respectively. The calculated
spectral parameter values; 8704.9 cm-1, 870.5 cm-1 and 0.896 attributing to 10Dq, B and β,
respectively are within the range for octahedral configuration (Fig. 2C)[34]. The high β value
(0.896) indicates a strong ionic interaction around metal centers [35]. The reduced magnetic
moment value (4.31BM) indicates a strong metal-metal interaction inside the bi-nuclear complex.
[Ni2Cl4(C9H9N5O)]2H2O complex spectrum shows bands at 19,450 and 20,833 cm-1 correspond to
1A1g→1A2g(υ1) and 1A1g→1B1g(υ2), transitions, respectively[36,37] in square-planar configuration.
The diamagnetic appearance of the complex goes attached with square planar of d8 systems.
[Cu2Cl4(C9H9N5O)]2H2O complex spectrum shows a significant d-d transition band at 18,590 cm-1
2
2
corresponds to B1g → Eg in square-planar configuration [38]. Also, band at 20,000 cm-1 is
attributing to LMCT. The reduced magnetic moment value (1.46BM) is also an indicative for bi-
nuclear complex. Generally, Intra-ligand transition bands, n→π*and π →π* expose to little shifted
appearance in all complexes spectra. Changing Z and q values for 3d transition metal complexes,
the inter-electronic repulsion parameter (Racah) values are varied. Whereas, Z is the functional
charge of cation and q is the filling up number of dq shell. The Racah parameter is well- expressed
by the relation: B (cm-1) = 384 + 58q + 124(z+1) - 540/(z+1). The calculated value of manganese
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ion is +1.498 and for cobalt ion is +1.436, they are considerably below than the formal oxidation
states(II) this reduction is suitable for M-N or M-O bonds [39].
Fig. 2. The optimized geometries for, VO(II), Mn(II), Co(II), Ni(II) and Cu(II) complexes
(A,B,C,D and E, respectively)
3.4. ESR spectral analysis
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Spin Hamiltonian operators and the G values of solid Cu(II) and VO(II) complexes are calculated
(Table 4) from their ESR spectra (Fig. 2S). The axially symmetric g tensor parameters are g11 > g >
2
2.0023 indicating that the dx −y2 orbital is a ground stat [40]. The G - factor is expressed by, G = (g11 -
2.0023) / (g- 2.0023) = 4, which measures the exchange interaction inside the complex. According to
Hathaway [41], G ˃ 4, negligible exchange interaction between copper(II) centers, while, G ˂ 4, is vice
verse. Here, the highly reduced value(1.825) introduces a strong interaction between neighbor copper
centers which express their presence inside one sphere [42,43]. This agrees with determined magnetic
moment. The g11(2.336) value is matched with highly covalent O-M and N-M bonds [44]. The tendency
of A11 to decrease with increasing g11 is an index for tetrahedral distortion (f =g11/A11) [45,46].
Whenever, the calculated value is presented in the range for square-planar configuration. The
σ2(covalence of in-plane σ-bonding) and β2 (covalence of in plane π-bonding) were accomplished by
2
using: .2 = ((A/0.036) + (g – 2.0023) + 3/7(g – 2.0023) + 0.04 and  = (g – 2.0023) E/ –82,
–1
Where  (spin- orbital coupling) = -828 cm for free copper ion and E (18,590 cm-1) is the electronic
2
transition energy. The  value indicates that, in-plane -bonding are highly ionic, while, 2 value
indicates that in-plane σ-bonding is highly covalent [47].
The spectrum of VO+2 complex demonstrates an eight- line manner attributing to the analogous and
vertical ingredients of g- and hyperfine (hf) A- tensors. The analogous and vertical ingredients are well
resolved. Nitrogen super-hyperfine splitting is not monitored in the complex, which points out that the
single electron is in the dxy orbital. The pattern suggests that g and A are axially symmetric in nature.
The factors A and g are appeared to be in covenant with the values commonly known for vanadyl
complex in square-pyramidal geometry. The molecular orbital coefficient 2 and 2 were executed by.
A11
P
A
P
7
6
5
9
 2

(

 g11  g  ge )
14 14

2.0023 - g
2 = 
8 2 
where g = ( g - g) x 10-3
The negative values of 2 command to negative 2 values. Since hyperfine conjunction disciplinarians
are negative, calculations were done taking A11 and A as negative, which gave positive values of 2
and 2. The calculated, 2 and 2 indicate the highly ionic of in – plane - and- bonding. The
2
spectrum of the complex shows band at 13,150 cm-1 (E2) which assigned to B2→2E, transition.
Assuming pure d-orbital's by using first- and second- order perturbation theory. The parameters
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attributed to transition energy are called Spin Hamiltonian and executed by the following expression: g
= ge – (2 ⁄ E2)
Where ge is the free- electron g value (2.0023). Using E2 value, spin orbital coupling constant ()is
evaluated(110.62). A value for  of 250 cm-1 is reported [48] for free V+4 ion. The high reduction in the
magnitude of  for the double bonded oxovanadium complex (V=O)+2 is attributed to substantial -
bonding. While, the value is inside the logical borders announced. The orbital reduction factors viz.,
Kand K are also calculated using: 2K = (g - 2.00277) E /8 λ and 2K = (g- 2.00277) E / 2 λ. For pure
σ- bonding K ≈ K ≈ -0.77 while, 2K ˃ 2K signifies in plane σ- bonding, with 2K<2K accounting for out
of plane π- bonding [49].
Calculation of dipole term(p),
Dipolar term of Cu(II) complex will be determined by: P = 2 γCu βo βN (r-3), where γCu is the magnetic
moment of copper, βo is the Bohr magneton, βN is the nuclear magneton and r is the distance from
nucleus to outer electron shells. While, in the VO(II) complex the value can be determined by :
7(A11- A)
P = 
6 + 3/2 (/E1)
If A11 is taken to be negative and A positive, the value of p will be more than 270 G, which is far from
the expected value. Thus, the signs of both A11 and A are used as negative and are indicated in the
form of isotropic hf constant (Ao). McGarvey theoretically accomplished p value as +136 G for
vanadyl complexes and the value (118.28) of this complex don't deviate much from this expected value
Calculation of MO coefficients and bonding parameters
The g values observed are different from electronic value (2.0023). This assigns to spin orbit interaction
of dxy ground state level. The isotropic and anisotropic, g and A parameters were calculated from Eqs;
Ao = (A11 + 2 A) ⁄ 3 and go = (g11 + 2 g ) ⁄ 3. Taking A11 and A to be negative values, K expression
of; K = - (Ao/ p) – ( ge - go)
Thus K (Fermi – contact term) can be determined. The Fermi contact term, k, is a sense of polarization
exerted by the uneven apportionment of d-electron density on the inner core s-electron.
3.5. X – Ray diffraction
The patterns of pyrazonone ligand and its VO(II), Mn(II), Co(II), Ni(II) and Cu(II) complexes were
obtained at 10˚ < 2θ < 80˚ range [Fig. 3,3S]. XRD analysis is used to give a clear insight about
lattice dynamics of solid compounds. The clear patterns from peaks attributed to reactants reflect
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the obscure of any reactant pullution [50]. Ligand, Mn(II) and Cu(II) complexes patterns show
crystalline diffraction peaks suitable for nano-crystalline feature, while the others are in amorphous
states [51,52]. The crystallinity appeared reflects the isolation of a strictly known irregular
crystallizes. While, the amorphous appearance reflects the indiscriminate orientation of atoms inside
3D space. The 2θ, d values, full width at half maximum (FWHM) of main intensity peak, intensity
(%) and particle size were presented in Table 5. The crystallite size was determined by using
FWHM for intense peaks by Deby–Scherrer equation; B = 0.94 λ/(S Cos θ), where S is the
crystallite size, θ is the diffraction angle, B is FWHM, Cu/Kα (λ) = 1.5406 Ao. D-spacing values are
the inner crystal plane which determined by Bragg equation: nλ = 2dsin(θ) at n = 1. The calculated
sizes are present excellently in nanometer range (below 35nm).
3.6. Scanning electron microscopy
SEM was used to study the habit and the surface morphology of all investigated compounds(Fig.
4S). The images of highly magnetic compounds are not strictly resolved may be due to insufficient
electron beam used to provide required resolution. Also, the micrographs are elongated rod-like and
needle-shaped with numerous territorial patches. So the accurate determination for the particle sizes
is completely absent [53-55]. It is obvious from this study that all the complexes crystals, were
grown up from just a single molecule to several accumulated distribution with particle sizes starting
from few nanometers to multi- hundred. The isolation of extended rod like crystals may happen by
two nucleation process by distribution and piling up of layers growth. It is suggested that if rate of
growth is faster along C-axis and a great number of growth nuclei maintain active across C-axis in
comparison with vertical to C-axis, extended crystals result [56]. Habit exhibited by different
crystals is slightly due to the growth along the strength bond due to anisotropy included in crystal
structure bonding. When the fullness overrides a definite limit, the abnormal growth in the needles,
rods and platelets is the normal result. It is reasonable to believe that the environmental conditions
do change in nature and variable features of morphologies. These shapes may reflect their excellent
property and thermal stability with mild catalytic activity. The spherical micrographs are observed
with VO(II) and Cu(II) complexes by size ranges of: 0.11-0.34 and 0.15-0.64μm, respectively.
Generally, the micrographs indicate that the isolation of strict-defined crystals free from any impact
for metal ions on external surface.
3.7. Thermal analysis
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The distractive feature of the investigated compounds was translated to data displayed in Table 6.
The compounds display instability towards the TG analysis except Co(II) complex. This behavior is
mainly attached with the existence of occluded water molecules with metal ion complexes. Two to
o
three endothermic degradation steps are sufficient to degrade them completely till 600-820 C. The
residual parts of complexes are mainly metal oxide completely pure from carbons. The limited
degradation steps are mainly associated with polymeric structures which may expel a bulky
molecule in a step. This behavior may expected with most investigated complexes. The high
conformity between calculated and found weight losses is referring to exact frontier barriers of each
degradation steps.
3.8. Theoretical calculations
3.8.1. Kinetic studies
In order to clarify the influence of occluded metal ions with the organic compounds on the thermal
behavior, the order n and the heat of activation E for defiant degradation steps were determined
using TG and DTG.
3.8.1.1 Coats – Redfern equation
The equation is an integral method, represented as :

2
d
A T
E *
RT




exp
dt (5)



n


0 (1)
 T
1
For convenience of integration initial limit T1, is usually taken as zero. This equation on integration
gives :
ln 1 





AR
E *
ln
 ln


(6)


2
T
E * RT




ln 1 


A plot of ln
(LHS) versus 1/T, E* is the energy of activation in Jmol-1 extracted from


2
T


the slop and A is (S-1) from the value of intercept (Fig. 5S). The activation entropy( ∆S*) in (J K-
1mol-1) was executed by:
Ah


S *  R ln
(7)




K
B
T
s
Where kB is the Boltzmann constant, h is the Plank’s constant and Ts is the DTG peak temperature
[18]
3.8.1.2. Horowitz – Metzger equation
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Using derived relation [28] i:
E
(8)

ln ln 1  




RTm
Where , is the fraction decomposed at time t and T Tm
Plot, ln ln 1  versus (Fig. 5S), is a linear relation, the slope of which E ,is evaluated
can be extracted from the relation :
.





and
Z






E
RTm
E
RTm
(9)
Z 
exp
2
Where
n  33.64758182.295m 435.9073m2 551.157m3 357.3703m4 93.4828m5 (10)
Where m is the fraction of the substance decomposed at Tm

is rate of heating, n, is the reaction order which can be determined from:
.
Enthalpy of activation ∆H* and Gibbs free energy(∆G* ) were deduced from ∆H* = E* -RT and
∆G* = ∆H* - T∆S*, equations. The entropy of activation(∆S*) was abstracted by conducting
equation 7 and the data were displayed in table 7. The following significant observations were
listed: (i) the activation energy values are reduced(E) which may reflect the soft elongated bonds
inside the coordination sphere (ii) Negative ∆S* values may reflect the ordered state of fragments
which exceeded across the degradation process. (iii) The endothermic decomposition behavior was
reflected on the positive ∆H* values. (iv) The positive ∆G* values represent that the decomposition
free energy for residue is higher than that of initial compound and non – spontaneous. The upraising
of T∆S*values (by negative singe) than the ligand lead to override ∆G* values due to the lower
decomposition rate for the complexes[57,58]
3.8.2. Molecular modeling study
Molecular modeling theoretical tool implementing Gaussian09 program was employed for
pyrazolone ligand and its metal ion complexes to abstract essential quantum parameters and
optimized geometries. The border orbital energy gap between EHOMO & ELUMO is an essential
parameter used to recognize the electronic structure of molecules and also to define their relativities
[59]. MO's images of free pyrazolone (Fig. 4A,B), an observable presence of HOMO and LUMO
mainly centered on donor atoms, nitrogen's and carbonyl oxygen, which are considered the location
for donors and acceptors. MO's images of metal ion complexes display an observable location for
HOMO and LUMO levels with donor sites beside Mn(II) and Co(II) centers. while, the ruling out of
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VO(II), Ni(II) Cu(II) centers, which may reflect the flexibility of charge transfer with big sizes
central atoms. Electronegativity (χ), chemical potential (μ), global of hardness (η), global of softness
(S), global of electrophilicity index (ω) and the absolute softness (ϭ) were evaluated according to
definite equations[60,61]. One in-between important quantum parameters is the electrophilicity
index (ω,) which reflects the relative toxicity and reactivity of different selective sites. Whenever,
electrophilicity may define the biological activity of proposed drugs. Also, indicates the
stabilization energy while acquiring extra negative charge from the environment by drug systems. η
and ϭ indicators, are measuring the molecular stability and reactivity. Also, their concepts are
belonging to each others. The softness indicators are the vice verse character for global hardness
[62].
3.8.2.1 quantum chemical parameters
The quantum parameters calculated are tabulated (Table 8) and the comparative investigation
introduces the following notice; concerning free ligand. I) Soft character of the ligand expects its
flexible reactivity towards the metal atoms. II) Positive electrophilicity indicator (χ) and negative
electronic chemical potential (μ) values indicate that the compound is capable for capturing
electrons from environment and its energy decrease coherently. Therefore, the electrochemical
potential must be negative. While, the quantum parameters calculated for visualized structures are
introducing the following important notice; I) The reduction of energy gapes (ΔE) in comparing
with free ligand reflects the high softness of complexes and an expectation for distinguish biological
activity. II) The reduction for EHOMO than the free ligand may be accompanied with the strength of
metallic bonds.III) The dipole moment values ordered by: Mn(II) ˃ Co(II) ˃ VO(II) ˃ Ni(II) ˃ HL ˃
Cu(II). This order of arrangement agrees with the bigger the metal size the relatively ionic
interaction bonds. This may clarify the interaction of donor ionic sites with Mn(II) and Co(II) ions
than the others. This is considerably agrees with the spectral Racah parameter values estimated for
Mn(II) and Co(II) complexes.
3.8.2.2. The bond lengths, atomic charges and oscillator strengths
According to numbering scheme and the data log file, essential bond lengths and effective charge
density on coordinating sites were deducted and tabulated in Table 9. The chosen bonds relative to
coordinating sites suffer an elongation than the genuine form of the ligand. This is supporting the
mode of coordination. Also, the reduction of charges over O(10), N(6), N(7) and N(8) atoms in
comparing with free ligand, verify their contribution in the mode. Whenever, the relatively reduced
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charges over free N and O atoms, may refer to their participation in conjugation and presence of
intra-ligand H-bonding. Oscillator strength is a quantity that indicates the permeability for the
absorption or emission of radiation by the molecule consumed for transitions between energy levels.
Its value can range from 0 to 1. A strong transition will have value close to 1. The calculated values
reflect the relative lower electronic transition efficiency for such electronically condensed metal
atoms.
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Fig. 4A. The frontier molecular orbital's of HOMO pictures for HL, VO(II), Mn(II), Co(II),
Ni(II) and Cu(II) complexes(A,B,C,D,E and F, respectively)
Fig.4B . The frontier molecular orbital's of LUMO pictures for HL, VO(II), Mn(II), Co(II),
Ni(II) and Cu(II) complexes(A,B,C,D,E and F, respectively)
3.8.2.3 QSAR calculations
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A interconnection between the anticancer activity of pyrazolone and its partition coefficient (log P=
0.36) is an inverse relationship[63]. A very low log P level reflects an expected distinguish activity.
Log P of the compound was determined for the optimized structures by HyperChem software.
Whereby, the structure was slightly optimized by molecular mechanics force field (MM+) and
followed by precision adjustment with semi-empirical AM1. The structure optimized without
confirming any indexes, leads to the geometry equilibrium state data (Table 10). The energy
minimization protocol employed the Polake-Ribiere conjugated gradient algorithm.
3.8.3. Molecular docking
Recently, computer-encouraged drug design, different computational tools were enhanced
dramatically by using more sophisticated and computationally intense methods. Autodock tools
used to clarify the biological features of candidate drug or emphasis on experimental results. This
study interested in using new designed drug to be used for antitumor investigation. The molecular
docking was carried out for pyrazolone (Guest, fig 5) with different carcinoma protein receptors
(Host) as: prostate carcinoma (2YXS, Crystal Structure of N-terminal domain of human galectin-8
with D-lactose, classification: sugar binding protein), colorectal carcinoma (2CGY, structure of
helix pomatum agglutinin with Forssmann antigen) and hepatic carcinoma (2JW2, Validation of
inter-helical orientation of the sterile-alpha-motif of human deleted in liver cancer 2 by residual
dipolar couplings). The calculated energies for docking procedure are presented in Table 11. The
binding free energy, constant of inhibition, energy of electrostatic, energy of total intercooled and
receptor-inhibitor complexes interact surface were deduced. So, the reduction in binding energy due
to mutation, will increase the inhibitor interaction affinity towards receptors [64]. The tabulated data
display the best inhibition activity against 2YXS and 2JW2 protein receptors carcinoma. Also,
reflect the best interaction stability for docked complexes (Fig. 6,6S). Accordance to computation,
HB plot curves (Fig. 7,7S) clarify a high interaction with all receptors by comparable results. So, an
expectation for its inhibition activity towards all tested carcinoma by excellent efficiency especially
with prostate and liver cancers. This is referring to variable capability of multi-central groups for H-
bonding interaction inside docking complexes. Inter-hydrogen bonding was appeared with all
complexes excellently. 2D- plot curves (Fig. 8,8S) verify the mode of interaction inside the docking
complex. Finally the anticancer efficiency of pyrazolone ligand will be substantially anticipated
towards prostate and liver carcinoma cells.
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Fig. 5. The docked pyrazolone compound
3.9. Antitumor activity
A long the coordination chemistry a stationary increase of chemical products at various levels for
different purposes. The coordination chemistry is considered one of most effective fields in
anticancer drug design and development. Cancer, defines as uncontrolled cell division, and also
characterized by days regulation of apoptosis, proliferation, invasion, angiogenesis and metastasis
[65]. Therefore, the expansion of drug scan prevents or keeps the aforementioned cancer properties
under control considered a victory. In this study all the prepared complexes beside the original
pyrazolone ligand were investigated towards HEPG2, PC3 and HCT116 cell lines in comparing
with doxorubicin drug. HEPG2 cell viability and IC50 showed different responses depending on the
complex. In comparing with doxorubicin referenced drug, the Cu(II) complex, displays the highest
and significant effects towards hepatic and prostate carcinoma, while Mn(II) and VO(II) complexes
display best impact towards colon and hepatic carcinoma cells, respectively. Contrary to
expectations, Cu(II) complex, showed two folded impact than VO(II) complex, despite the known
anticancer efficiency of VO(II) complexes in comparing with Cu(II) complexes. This may be due to
the roll of copper in regeneration of infected cell [66,67]. Likewise, the influence of Cu(II) complex
presents linear effect, started with 0.01 doses which is seems as a small dose. Ni(II) complex was
exhibited a significant impact also against HEPG2 (Table 12). While other elements present curved
effect continued on a regular basis with the increase of the dose (Fig. 9). The ligand displays a very
low influence on HepG2, while presented a high effect on both PC3 and HCT116. Co(II) complex
exhibited different effect on different cell lines. While, HepG2 presented moderate response, PC3
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ACCEPTED MANUSCRIPT
displays very low response, but HCT116 was react more strongly. In the light of the outcomes,
together with the results of previous in vivo and in vitro studies, suggest that the administration of
Cu(II) complex may has a roll in prevention of liver and prostate cancers and [68]. PC-3 and
HCT116 cell lines exhibit different response against the same complexes. Also, the main goal in our
study is to introduce permissible complexes serve excellently as new designed drugs after other
intensive examination from specialists. The complexes need further studies to investigate the
pathway that may activate in different cancer cells. Here, the interaction mechanism may be
discussed based on chelation theory [69]. On chelation, the charge of metal ion will be reduced by
great magnitude due to the overlapping with donor orbital's. Further, over the whole chelate ring the
increases in delocalization of p-electrons enhances the complex lipophilicity. The lipophilicity
enhances the permeation into lipid membranes and closing the binding sites of infected cell
enzymes.
4. Conclusion
The synthesized complexes using the pyrazolone ligand were investigated and characterized using
all possible tools. The bi-nuclear complexes in the main feature except Mn(II) one. The spectral
techniques have introduced the best molecular and structural formulas for the complexes. TG
analysis and kinetic parameters are in importance to examine their thermal stability. Moreover, the
theoretical investigation implementing different programs to emphasis on the experimental results
may strengthen the study at all. As; Gaussian09 program optimizes the structural formulas applying
DFT/B3LYP method. QSAR calculations expects the best inhibition of pyrazolone towards infected
cells beside molecular docking outcomes. In comparing with referenced drug, IC50 calculated
represented an excellent impact for Cu(II) complex in overcoming liver and prostate carcinoma,
while the Mn(II) complex displays a high comparative impact against colon carcinoma cell line.
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Fig. 3 . X-Ray manners of pyrazolone and VO(II) complex, A and B respectively
Fig. 5. Protein- inhibitor complex for 2JW2 receptor
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Fig. 6. HB plot of 2JW2 receptor with HL inhibitor
Fig. 7. 2D plot of 2JW2 receptor with HL inhibitor
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Fig. 8. Dose response curves against HEPG2, HCT116 and PC3, (A, B, C) respectively.
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Fig. 6. Protein- inhibitor complex for 2JW2 receptor
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Table 1. Analytical and physical data of pyrazolone ligand and its complexes.
Compound
Color
Elemental analysis (%) Calcd (Found)
Empirical formula (Calcd)
C
H
N
Cl /SO4
M
1) [C9H9N5O](203.204)
53.20(53.49) 4.46(4.34) 34.46(34.75) ----
-----
33.99(34.11) 18.03(18.00)
Deep orange
Greenish-Brown
Orange
2)[(VO)2(SO4)2(C9H9N5O)]2H2O(565.24)
3)[MnCl2(C9H9N5O).2(H2O)](365.08)
4)[Co2Cl4(C9H9N5O).4(H2O)](534.94)
5)[Ni2Cl4(C9H9N5O)]2H2O(498.43)
6)[Cu2Cl4(C9H9N5O)]2H2O(508.14)
19.12(19.20) 2.32(2.31) 12.39(12.41)
29.61(29.56) 3.59(3.60) 19.18(19.20)
9.71(9.71)
15.05(15.10)
22.03(22.02)
23.55(23.53)
20.21(20.16) 3.20(3.20) 13.09(13.11) 26.51(26.49)
21.69(21.70) 2.63(2.60) 14.05(13.98) 12.02(12.10)
Dark-Brown
Pale-Brown
Pale-Brown
21.27(21.26) 2.58(2.57) 13.78(13.80)
11.98(11.97) 25.01(25.11)
Table 2. Significant IR spectral bands (cm-1) of pyrazolone ligand and its complexes
δr(H2O),
δw(H2O)
Compound
δNH2 δNHs
NH2+OH
NH C=O
C=N
M-O M-
N
1442,1493
1437,1485
1439,1496
1436,1473
1432,1498
1366,1496
3410,3333
3398
3185 1671
3173 1652
3165 1683
3160 1650
---- 1640
3177 1682
1586 1625
1551 1595
1597 1637
1552 1600
1559 1590
1574 1597
---
---- ----
592 490
461 449
586 527
533 490
575 467
1) [C9H9N5O]
875,662
781,683
860,668
860,668
880,684
2)[(VO)2(SO4)2(C9H9N5O)]2H2O
3)[MnCl2(C9H9N5O).2(H2O)]
4)[Co2Cl4(C9H9N5O).4(H2O)]
5)[Ni2Cl4(C9H9N5O)]2H2O
6)[Cu2Cl4(C9H9N5O)]2H2O
3336,3352
3320,3331
3331(center)
3443, 3309
Table 3. Spectroscopic , covalence parameters and magnetic moment values
*
Compound υ1(cm-1),υ2
*ε 1, ε 2, (X10-5)**ʄ1,
Intraligand and charge
transfer(cm-1)
Z
μeff
**
ʄ
2,
(BM)
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