New pyridazine-based binuclear nickel(II), copper(II) and zinc(II) complexes as prospective anticancer agents

Article abstract of DOI:10.1039/c5nj02739j

A new class of pyridazine-based binuclear nickel(II), copper(II) and zinc(II) complexes of the type [M₂(L^1-3)₂](NO₃)₂ (1-9) with tridentate Schiff base ligands 3-chloro-6-(salicylidenehydrazinyl)pyrid

Full text of DOI:10.1039/c5nj02739j

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1
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Page 1 of 44
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NEW PYRIDAZINE-BASED BINUCLEAR NICKEL(II), COPPER(II) AND ZINC(II)
COMPLEXES AS PROSPECTIVE ANTICANCER AGENTS
a
a
a
Ummer Muhammed Rafi , Dharmasivam Mahendiran , Azees Khan Haleel ,
b
b
a
Rakesh Pandeet Nankar , Mukesh Doble and Aziz Kalilur Rahiman* .
a
PostꢀGraduate and Research Department of Chemistry, The New College (Autonomous),
Chennaiꢀ600 014, India.
Bioengineering and Drug Design Lab, Department of Biotechnology, Indian Institute of
Technology Madras, Chennaiꢀ600 036, India.
b
Abstract
A
new class of pyridazineꢀbased binuclear nickel(II), copper(II) and
1
ꢀ3
zinc(II) complexes of the type
[M
2
(L )
2
](NO
3
)
2
(1-9) with tridentate
1
Schiff
base
ligands
3ꢀchloroꢀ6ꢀ(salicylidenehydrazinyl)pyridazine
(HL ),
2
3ꢀchloroꢀ6ꢀ(5ꢀnitrosalicylidenehydrazinyl)pyridazine
(HL )
and
3ꢀchloroꢀ6ꢀ(4ꢀ
3
diethylaminosalicylidenehydrazinyl)pyridazine (HL ) were synthesized and characterized.
3
The molecular structure of the ligand HL was determined by the single crystal XRD method.
The geometry optimization and HOMOꢀLUMO energy level calculations were carried out
using DFT studies. The electrochemical studies of nickel(II) (1-3) and copper(II) (4-6)
1
complexes exhibit two irreversible oneꢀelectron reduction waves at Epc = −0.323 to −0.463 V
2
and E = −0.634 to −0.790 V in cathodic potential region. The nickel(II) complexes (1-3)
pc
1
2
exhibit two irreversible oneꢀelectron oxidation waves at Epa = 1.009 to 1.025 V and Epa
=
1.140 to 1.153 V in the anodic potential region. The spectroscopic data indicate that the
ligands behave as a monoanionic tridentate ligand through the deprotonated phenolic oxygen
and nitrogen atoms of azomethine group and pyridazine ring. The emission studies of the
complexes at room temperature indicate the enhancement of fluorescence intensity than the
ligands due to chelationꢀenhanced fluorescence effect (CHEF) suggesting the possible
fluorescent probes of these Schiff bases for metal ions. In vitro cytotoxic activity of all the
complexes were assessed against one breast cancer (MDAꢀMBꢀ231) and one healthy
1

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myoblast nonꢀcancer (Lꢀ6) cell lines. The IC50 values of complexes 3 and 9 indicate their
high cytotoxicity against MDAꢀMBꢀ231 cells when compared to the standard drug cisplatin
suggesting that these complexes may act as potential antitumor agents. Nuclear chromatin
cleavage have also been observed with AO/EB staining assay. Flow cytometry analysis of
complexes 3 and 9 revealed cell cycle arrest in the S phase. Molecular docking studies were
also carried out for the complexes 3 and 9 to find their binding affinity with protein EGFR
kinase.
Keywords: Schiff base, Xꢀray crystal structure, Emission properties, Electrochemical studies,
Anticancer activity, Apoptosis, Cell cycle arrest.
*
Corresponding author. Tel.: +91 44 2835 0297; Fax: + 91 44 2835 2883.
Eꢀmail: akrahmanjkr@gmail.com
. Introduction
Transition metal complexes with bi, tri and tetradentate Schiff base ligands containing
1
oxygen and nitrogen donor atoms have the ability to possess unusual configuration and
remarkable biomedical activities that could be used to develop new metalꢀbased anticancer
agents [1]. Pyridazine, a type of heterocyclic diazine has wide applications in drug design [2],
agrochemical activities [3] and has also been used as ligands in coꢀordination chemistry.
There have been numerous systems such as acyclic ligands [4], coordination polymers [5],
cryptands [6] and also macrocycles [7] in which pyridazine units have been incorporated.
Over the past years, pyridazine derivatives have been extensively studied and have shown a
wide range of pharmacological activities such as antidepressant [8], antibacterial [9], COXꢀ2
inhibitor [10] and anticancer activities [11]. The introduction of new substituent or functional
groups at various positions on aromatic or heteroaromatic fragments of a potential drug might
lead to changes in its molecular shape that allows optimum binding to the receptor, as well as
its physicoꢀchemical properties that affect drug distribution and metabolism [12].
2

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Pyridazineꢀbased transition metal complexes have received immense attention in coꢀ
ordination chemistry due to their great structural diversity [13, 14]. A unique chemical
behavior can be expected due to the different electronic ligand properties when compared to
pyridine systems. When compared to pyridine, the presence of two Nꢀdonor atoms in
pyridazine leads to a decrease in electron donor abilities, which is reflected in significant
lower pKa values (2.24 for parent pyridazine whereas 5.25 for pyridine) [15]. In many cases,
pyridazine-based ligands are capable of coordinating two metal atoms when they are in close
proximity through their diazine fragments and are substituted in 3ꢀ and 6ꢀ position with two
donors to exploit the chelate effect and thus increase the binding tendency of the electronꢀ
poor second nitrogen donor. However, these systems have been observed only in pyridazine
ligands with preferred compartmental or in macrocyclic systems [16, 17]. Thus, Pyridazine
without adjacent coordination sites act mostly as monodentate ligands comparable to pyridine
[
18]. Pyridazine with only one additional donor next to a ring nitrogen atom leading to biꢀ or
tridentate ligands have been less investigated in coordination chemistry [19].
Cancer is the second leading cause of human deaths after cardiovascular diseases
[
20]. Though chemotherapy is the mainstay of cancer treatment, the use of available
chemotherapeutics still suffers from major limitations such as lack of selectivity of cancer
tissues, bringing unwanted side effects and the acquisition by cancer cells of multipleꢀdrug
resistance [21]. Therefore, the search for new drugs, which can selectively target the tumor
cells is today’s goal of cancer therapy. Introduction of appropriate heterocycles into a lead
compound is a common strategy in the drug discovery process. Hence, our strategy was to
synthesize three new pyridazineꢀbased Schiff base ligands that contain additional donor sites
at the 3ꢀposition of the heterocycles, whereas the 6ꢀposition was substituted by a nonꢀ
coordinating group. The three Schiff base ligands were prepared by the condensation of
3ꢀchloroꢀ6ꢀhydrazinopyridazine
with
salicylaldehyde,
5ꢀnitrosalicylaldehyde
or
3

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4ꢀ(diethylamino)salicylaldehyde. Using these ligands, a new series of nickel(II), copper(II)
and zinc(II) complexes were synthesized. In vitro anticancer activities of the complexes have
been screened against one human breast cancer (MDAꢀMBꢀ231) and one healthy nonꢀcancer
myoblast (Lꢀ6) cell lines by MTT assay. The cell death or apoptotic studies was carried out
by using AO/EB staining method and the cell cycle arrest was performed by flow cytometry
analysis.
2. Experimental
2.1. General methods and materials
The compound 3ꢀchloroꢀ6ꢀhydrazinopyridazine was prepared by following the
procedure described in the literature [22]. 3,6ꢀdichloropyridazine, salicylaldehyde,
ꢀnitrosalicylaldehyde, 4ꢀ(diethylamino)salicylaldehyde and metal(II) nitrates were
5
purchased from Aldrich and used without further purification. Solvents used in the synthesis
were dried and purified before being used according to standard procedure [23]. The melting
points were determined using electro thermal capillary apparatus and reported as uncorrected
values. Elemental analyses (CHN) were performed on a Carlo Erba model 1106 elemental
analyzer. IR spectra were recorded on a PerkinꢀElmer 297 spectrophotometer using KBr
−
1
pellets in the range of 4000−400 cm . NMR spectra were recorded on a Bruker Avance
1
13
6
spectrometer using DMSOꢀd as solvent (400 MHz for H and 100 MHz for C NMR). ESI
mass spectra were recorded on a Thermo Scientific Orbitrap Elite Mass spectrometer. UVꢀ
Vis spectra were recorded on a Cary 100 version 11.1 spectrophotometer using DMSO as
solvent. The luminescence spectra were recorded on FluoroMax 4CꢀTCSPC fluorescence
spectrofluorometer at room temperature using DMSO as solvent. XꢀBand EPR spectra were
recorded at 25 °C on a Varian EPRꢀE 112 spectrometer using 2,2′ꢀdiphenylꢀ1ꢀpicrylhydrazyl
(
DPPH) as the reference. The room temperature magnetic moment was measured on a PAR
modelꢀ155) vibrating sample magnetometer. Molar conductivity was measured with an Elico
(
4

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digital conductivity bridge model CMꢀ88, using a freshly prepared DMF solution of the
complexes. Cyclic voltammograms were obtained on CHI 602D (CH Instruments Co., USA)
electrochemical analyzer under oxygen free conditions using a threeꢀelectrode cell with a
DMF solution of TBAP (0.1 M) as the supporting electrolyte. A Pt wire, glassy carbon and
the Ag/AgCl electrodes (saturated KCl solution) were used as counter, working and reference
+
electrodes, respectively. A ferrocene/ferrocenium (Fc/Fc ) couple was used as an internal
standard. The reported potentials were relative to the Ag/AgCl electrode and E1/2 of the
+
−3
Fc/Fc couple. The concentration of complex solutions was taken around 1.0 × 10 M and
−
1
the scan rate was 100 mVs .
1-3
2
.2. Synthesis of pyridazine-based ligands (HL )
All the ligands were synthesized by the [1+1] Schiff base condensation reaction.
1
3
-chloro-6-(salicylidenehydrazinyl)pyridazine (HL )
An ethanolic solution (30 mL) of salicylaldehyde (0.61 mL, 5 mmol) was slowly
added to an ethanolic solution (30 mL) of 3ꢀchloroꢀ6ꢀhydrazinopyridazine (0.72 g, 5 mmol)
with constant stirring in the presence of acetic acid (3 drops) as a catalyst under the nitrogen
atmosphere. The stirring was continued for 1 h and refluxed for 2 h. The product was
collected by filtration, washed with ethanol and dried in vacuo.
Yield: 1.02 g (82.04%), mp: 252 °C. Colour: yellow. Anal. calcd. for C11
9 4
H N OCl,
(FW: 248.67): C, 53.13; H, 3.65: N, 22.53. Found: C, 53.08; H, 3.62; N, 22.52%. Selected IR
−1
data (KBr, cm ): 3452 ν(−OH), 3218 ν(−NH), 1628 ν(−C=N), 1283 ν(Ar−O), 1533
−1
−1
1
ν(−N=N). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 339 (19,616). H NMR (400 MHz,
DMSOꢀd ) : δ 11.63 (s, 1H, phenolic proton), 10.17 (s, 1H, NH), 8.43 (s, 1H, azomethine
CH=N), 7.72 ( d, 1H, Ar−H, J = 7.6 Hz), 7.66 (d, 1H, Arpdz−H, J = 9.6 Hz), 7.55 (d, 1H,
6
–
1
3
Ar −H, J = 9.6 Hz), 7.22 (t, 1H, Ar−H, J = 7.6 Hz), 6.89 (m, 2H, Ar−H). C NMR (400
pdz
MHz, DMSOꢀd ): δ 158.86, 156.44, 147.75, 140.76, 130.88, 130.41, 126. 99, 119.84, 116.57,
6
5

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1
3
1
1
3
16.31. C (DEPTꢀ135) NMR (400 MHz, DMSOꢀd
6
): δ 140.75, 130.89, 130.41, 126.98,
19.85, 116.57, 116.31 (methine carbons).
2
-chloro-6-(5-nitrosalicylidenehydrazinyl)pyridazine (HL )
2
1
The solid ligand HL was prepared by following the procedure similar to that of HL
using 5ꢀnitrosalicylaldehyde (0.84 g, 5 mmol) instead of salicylaldehyde.
Yield: 1.27 g (86.49%), mp: 260 °C. Colour: yellow. Anal. calcd. for C11
H
8
N
5
O
3
Cl,
(FW: 293.67): C, 44.99; H, 2.75: N, 23.85. Found: C, 44.95; H, 2.72; N, 23.84%. Selected IR
−1
data (KBr, cm ): 3452 ν(−OH), 3208 ν(−NH), 1629 ν(−C=N), 1290 ν(Ar−O), 1518
−1
−1
1
ν(−N=N). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 336 (20,838), 465 (2,110). H NMR
(
400 MHz, DMSOꢀd ) : δ 11.80 (s, 1H, phenolic proton), 11.60 (s, 1H, NH ), 8.51 ( s, 1H,
6
Ar−H ), 8.33 (s, 1H, azomethine –CH=N), 8.04 (d, 1H, Ar−H, J = 9.2 Hz), 7.62 (d, 1H,
Ar−H, J = 9.2 Hz), 7.57 (d, 1H, Arpdz−H, J = 9.6 Hz), 7.00 (d, 2H, Arpdz−H, J = 9.2 Hz).
1
3
6
C NMR ( 400 MHz, DMSOꢀd ): δ 161.79, 158.92, 148.21, 140.56, 137.08, 130.66, 126.16,
1
3
1
1
3
22.15, 121.65, 117.07, 116.56. C (DEPTꢀ135) NMR (400 MHz, DMSOꢀd ): δ 137.07,
6
30.66, 126.16, 121.06, 117.07, 116.58 (methine carbons).
3
-chloro-6-(4-(diethylamino)salicylidenehydrazinyl)pyridazine (HL )
3
1
The solid ligand HL was prepared by following the procedure similar to that of HL
using 4ꢀ(diethylamino)salicylaldehyde (0.97 g, 5 mmol) instead of salicylaldehyde.
Yield: 1.26 g (78.80%), mp: 250 °C. Colour: Yellow. Anal. calcd. for C H N OCl,
15
18
5
(FW: 319.79): C, 56.34; H, 5.67: N, 21.90. Found: C, 56.29; H, 5.63; N, 21.89%. Selected IR
−1
data (KBr, cm ): 3468 ν(−OH), 3194 ν(−NH), 1631 ν(−C=N), 1282 ν(Ar−O), 1535
−1
−1
1
ν(−N=N). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 371 (38,063). H NMR ( 400 MHz,
DMSOꢀd ) : δ 11.27 (s, 1H, phenolic proton), 10.18 (s, 1H, NH ), 8.23 (s, 1H, azomethine
CH=N), 7.60 (d, 1H, Ar −H, J = 9.2 Hz), 7.35 (d, 2H, Ar−H, J = 8.8 Hz), 6.25 (d, 1H,
6
–
pdz
Ar −H, J = 8.8 Hz), 6.12 (s, 1H, Ar−H), 3.36 (q, 4H, CH , J = 6.8 Hz), 1.18 (t, 6H, CH ,
pdz
2
3
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13
6
J = 6.8 Hz). C NMR (400 MHz, DMSOꢀd ): δ 158.53, 158.42, 150.07, 146.98, 143.89,
1
3
1
30.21, 129.56, 115.88, 108.11, 104.33, 97.95, 44.26, 13.03. C (DEPTꢀ135) NMR (400
6
MHz, DMSOꢀd ): δ143.88, 130.21, 129.55, 115.88, 104.33, 97.94 (methine carbons), 44.26
(methylene carbons), 13.03 (methyl carbons).
2.3. General procedure for the synthesis of pyridazine-based binuclear metal(II)
complexes
The binuclear nickel(II), copper(II) and zinc(II) complexes were prepared by
following the same procedure as given below: An ethanolic solution (30 mL) of
Ni(NO .6H O (0.29 g, 1.2 mmol), Cu(NO .3H O (0.29 g, 1.2 mmol) or Zn(NO .6H
0.36 g, 1.2 mmol) was added to an equimolar amount of appropriate ligand (HL , 1.2
3
)
2
2
3
)
2
2
3
)
2
2
O
1
ꢀ3
(
mmol) in ethanol (30 mL) with constant stirring while a colour change was immediately
observed. The stirring was continued for 30 min and refluxed on a water bath for additional
3
h, filtered while hot, and the filtrate was allowed to stand at room temperature for few days.
The solid complexes obtained were recrystallized using hot ethanol.
1
[Ni
2
(L )
2
3 2
](NO ) (1)
Yield: 0.51 g (58%). Colour: Brown. Anal. calcd. for C H N O Cl Ni ,
22
16 10
8
2
2
(FW: 736.72): C, 35.87; H, 2.19: N, 19.01. Found: C, 35.83; H, 2.17; N, 19.00%. Selected IR
−
1
data (KBr, cm ): 3220 ν(−NH), 1610 ν(−C=N), 1264 ν(Ar−O), 1555 ν(−N=N), 1383
−
−1
−1
ν(NO
3
) . UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 263 (15,007), 298 (14,001), 418 (5,336),
1
+
1 +
5
88 (970). ESIꢀMS (m/z): 609.959 ([NiL ] ; 60%), 304.972 ([NiL ] ; 100%). Conductance
2
−1
2
−1
(
Λ , ꢁ cm mol ) in DMF: 161.
M
2
[
Ni (L ) ](NO ) (2)
2
2
3 2
14 12 2 2
Yield: 0.58 g (58%). Colour: Brown. Anal. calcd. for C22H N O12Cl Ni ,
(FW: 826.71): C, 31.96; H, 1.71: N, 20.33. Found: C, 31.93; H, 1.69; N, 20.32%. Selected IR
−
1
data (KBr, cm ): 3219 ν(−NH), 1601 ν(−C=N), 1257 ν(Ar−O), 1552 ν(−N=N), 1384
−
−1
−1
ν(NO ). UVꢀVis (DMSO) λ
(nm) (ε (M cm )): 233 (11,220), 301 (15,239), 412
max
3
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2
+
2 +
(
2
14,509), 594 (677). ESIꢀMS (m/z): 699.925 ([NiL ] ; 53%), 349.962 ([NiL ] ; 100%).
−
1
2
−1
Conductance (Λ
M
, ꢁ cm mol ) in DMF: 131.
(3)
Yield: 0.55 g (52%). Colour: Brown. Anal. calcd. for C H N O Cl Ni ,
3
[Ni
2
(L )
2
](NO
3
)
2
30
34 12
8
2
2
(FW: 878.96): C, 40.99; H, 3.89: N, 19.12. Found: C, 40.96; H, 43.87%; N, 19.11. Selected
−1
IR data (KBr, cm ): 3220 ν(−NH), 1604 ν(−C=N), 1243 ν(Ar−O), 1565 ν(−N=N), 1384
−
−1
−1
ν(NO
3
). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 311 (10,928), 423 (9,115), 572 (778).
3
+
3 +
ESIꢀMS (m/z): 754.095 ([NiL ]
2
; 67%), 376.052 ([NiL ] ; 100%). Conductance (Λ
M
,
−1
2
−1
ꢁ
cm mol ) in DMF: 148.
1
[
Cu (L ) ](NO ) (4)
2
2
3 2
16 10 8 2 2
Yield: 0.58 g (65%). Colour: Green. Anal. calcd. for C22H N O Cl Cu
,
(FW: 746.42): C, 35.40; H, 2.16: N, 18.77. Found: C, 35.37; H, 2.14; N, 18.76%. Selected IR
−
1
data (KBr, cm ): 3224 ν(−NH), 1605 ν(−C=N), 1250 ν(Ar−O), 1545 ν(−N=N), 1384
−
−1
−1
ν(NO ). UVꢀVis (DMSO) λ (nm) (ε (M cm )): 275 (15,044), 398 (8,370), 437 (2,504),
3
max
1
+
2
1 +
6
55 (734). ESIꢀMS (m/z): 620.930 ([CuL ] ; 64%), 309.967 ([CuL ] ; 100%). Conductance
−1
2
−1
(Λ
M
⊥
, ꢁ cm mol ) in DMF: 152. g|| = 2.254, g = 2.092. ꢂeff = 1.59 B.M.
2
[Cu
2
(L )
2
](NO
3
)
2
(5)
Yield: 0.60 g (60%). Colour: Green. Anal. calcd. for C22
14 12 2 2
H N O12Cl Cu ,
(FW: 836.42): C, 31.60; H, 1.69: N, 20.10. Found: C, 31.56; H, 1.67; N, 20.08%. Selected IR
−
1
data (KBr, cm ): 3115 ν(−NH), 1612 ν(−C=N), 1240 ν(Ar−O), 1546 ν(−N=N), 1387
−
−1
−1
ν(NO ). UVꢀVis (DMSO) λ
(nm) (ε (M cm )): 263 (20,602), 372 (23,928), 441
max
3
2
+
2 +
(
2
16,571), 650 (436). ESIꢀMS (m/z): 710.902 ([CuL ] ; 40%), 354.955 ([CuL ] ; 100%).
−1
2
−1
Conductance (Λ
M
, ꢁ cm mol ) in DMF: 172. g|| = 2.262, g = 2.088. μeff = 1.63 B.M.
⊥
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3
[Cu
2
(L )
Yield: 0.49 g (46%). Colour: Green. Anal. calcd. for C30
FW: 888.66): C, 40.55; H, 3.86: N, 18.91. Found: C, 40.51; H, 3.82; N, 18.90%. Selected IR
2 3 2
](NO ) (6)
34 12 8 2 2
H N O Cl Cu ,
(
−
1
data (KBr, cm ): 3212 ν(−NH), 1608 ν(−C=N), 1249 ν(Ar−O), 1568 ν(−N=N), 1384
−
−1
−1
ν(NO ). UVꢀVis (DMSO) λ (nm) (ε (M cm )): 308 (13,725), 411 (10,200), 611 (820).
3
max
3
+
3 +
ESIꢀMS (m/z): 764.095 ([CuL ]
2
; 61%), 381.041 ([CuL ] ; 100%). Conductance (Λ
M
,
−1
2
−1
ꢁ
⊥
cm mol ) in DMF: 166. g|| = 2.249, g = 2.058. ꢂeff = 1.62 B.M.
1
[Zn
2
(L )
2
](NO
3
)
2
(7).
16 10 8 2 2
Yield: 0.51 g (57%). Colour: Yellow. Anal. calcd. for C22H N O Cl Zn
,
(FW: 750.11): C, 35.23; H, 2.15: N, 18.67. Found: C, 35.19; H, 2.13; N, 18.66%. Selected IR
−
1
data (KBr, cm ): 3217 ν(−NH), 1615 ν(−C=N), 1277 ν(Ar−O), 1551 ν(−N=N), 1380
−
−1
−1
ν(NO
3
, uncoordinated). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 338 (22,461). ESIꢀMS
1
+
1 +
−1
2
−1
(
m/z): 625.932 ([ZnL ] ; 58%), 310.968 ([ZnL ] ; 100%). Conductance (Λ , ꢁ cm mol )
2
M
in DMF: 153.
2
[Zn
2
(L )
2
](NO
3
)
2
(8)
Yield: 0.61 g (60%). Colour: Yellow. Anal. calcd. for C H N O Cl Zn ,
22
14 12 12
2
2
(FW: 840.11): C, 31.45; H, 1.68: N, 20.01. Found: C, 31.42; H, 1.67; N, 19.99%. Selected IR
−
1
data (KBr, cm ): 3208 ν(−NH), 1613 ν(−C=N), 1251 ν(Ar−O), 1554 ν(−N=N), 1384
−
−1
−1
ν(NO
3
, uncoordinated). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 326 (16,285). ESIꢀMS
2
+
2 +
−1
2
−1
(
m/z): 715.962 ([ZnL ] ; 49%), 355.954 ([ZnL ] ; 100%). Conductance (Λ , ꢁ cm mol )
2
M
in DMF: 159.
3
[Zn
2
(L )
2
](NO
3
)
2
(9)
Yield: 0.57 mg (53%). Colour: Yellow. Anal. calcd. for C30
34 12 8 2 2
H N O Cl Zn ,
(FW: 892.35): C, 40.38; H, 3.84: N, 18.84. Found: C, 40.34; H, 3.81; N, 18.83%. Selected IR
−1
data (KBr, cm ): 3220 ν(−NH), 1603 ν(−C=N), 1243 ν(Ar−O ), 1562 ν(−N=N), 1382
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−
−1
−1
ν(NO
m/z): 768.081 ([ZnL ]
in DMF: 147.
.4. X-Ray crystallographic analysis
Single crystals suitable for Xꢀray analysis were obtained by the slow evaporation of
HL in DMSO at room temperature. The yellow coloured crystal was cut to suitable size
0.35 × 0.30 × 0.20 mm) and mounted on Kappa Apex2 CCD diffractometer equipped with
3
, uncoordinated). UVꢀVis (DMSO) λmax (nm) (ε (M cm )): 319 (19,240). ESIꢀMS
3
+
3 +
−1
2
−1
(
2 M
; 65%), 382.043 ([ZnL ] ; 100%). Conductance (Λ , ꢁ cm mol )
2
3
(
graphite monochromated Mo Kα radiation in the wavelength (λ) of 0.71073 Å at room
°
temperature. The intensity data were collected using ω and φ scans with frame width of 0.5 .
The frame integration and data reduction were performed using Bruker SAINTꢀPlus (Version
7.06a) software. The multiꢀscan absorption corrections were applied to the data using
SADABS program [24]. The structure was solved by using SIR92 [25] and the full matrix
2
least squares refinement on F was performed using SHELXLꢀ97 program [26]. The
molecular graphics, hydrogen bonding and packing figures were generated by using the
software ORTEP 3.0 [27]. The final Rꢀvalue was 0.0873, and the final electrodensity map
−
3
contains maximum and minimum peak heights of 0.341 and −0.409 eÅ , respectively.
2.5. Computational details
Gaussian 03 [28] software package was used for theoretical calculations. The
quantum chemical calculations were performed applying DFT method, with Beckeꢀ3ꢀLeeꢀ
YangꢀParr (B3LYP) supplemented with the standard 6ꢀ31G(d) basis set [29]. The optimized
geometry corresponding to the minimum on the potential energy surface has been obtained
by solving selfꢀconsistent field equation iteratively.
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2
.6. Anticancer activity
.6.1. Cell viability assay
2
The rat myoblast nonꢀcancer (Lꢀ6) and human breast cancer (MDAꢀMBꢀ231) cell
lines were purchased from National Center for Cell Science (NCCS) Pune, India. Both the
cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Himedia, India)
containing 10% fetal bovine serum (FBS) (Gibco, Life Technologies Corporation, Grand
Island, NY, USA) and 1% antibioticꢀantimycotic solution 100X (Himedia) in a humidified
2
atmosphere with 5% of CO at 37 °C. Stock solutions of all the compounds were prepared in
cell culture grade DMSO (Himedia).
Effect of treatment of compounds on cell viability was determined using tetrazolium
dye, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) assay as reported
earlier [30]. Cells were seeded in a 96 well plate at a density of 5000 cells/well. When cells
reached 60% confluency, they were treated with increasing concentrations of complexes (3.9,
7.8, 15.6, 31.25, 62.5, 125, 250 and 500 ꢂM) for 24 h. After 24 h, the medium was replaced
with 0.5 mg/mL of MTT and incubated for 4 h. Live cells have the ability to reduce yellow
soluble MTT dye to blue insoluble formazan crystals. The medium was removed and
formazan crystals were dissolved by adding DMSO (200 ꢂL/well). The optical density of
each well at 570 nm was measured in a multimode plate reader (Enspire, Perkin Elmer,
Waltham, MA, USA). The % cell viability for each treatment was calculated using the
following equation,
A
570 nm of treated cells
570 nm of control cells
Cell viability (%) =
× 100
A
IC50, the concentration of compounds required to attain 50% of cell viability (relative to
control), for each compound was estimated by nonꢀlinear fitting of curve obtained by plotting
%
cell viability verses log concentration (GraphPad Prism, Version 5.04).
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2.6.2. Cell clonogenic assay
Cell clonogenic assay was preformed as described earlier [31]. MDAꢀMBꢀ231 cells
were seeded in a 6 well plate at a density of 400 cells/well/2 mL. Cells were grown for 7 days
until the colonies were formed. After 7 days, cells were treated with 75 ꢂM of complex 3 or
3
7 ꢂM of complex 9 (IC concentrations of complexes). Treatment was stopped after 24 h
50
and cells were allowed to grow for another 7 days. The media was aspirated and cells were
washed with PBS (pH 7.4) to remove the detached cells. Colonies of cells were fixed in ice
cold methanol for 15 min and stained with 0.5% crystal violet solution.
2.6.3. Apoptosis studies
Cell apoptosis was assessed using the AO/EB fluorescent staining method. MDAꢀ
4
MBꢀ231 cells were seeded in a 12 well plate at a density of 4 × 10 cells/well/mL. Cells were
allowed to grow for 24 h. Cells were treated with 75 ꢂM of complex 3 or 37 ꢂM of complex
9
(IC50 concentration of complexes) for 24 h. After 24 h, cells were harvested with trypsinꢀ
EDTA solution and resuspended in 100 ꢂL of PBS (pH 7.4). The morphological changes in
the cells after treatment with complexes 3 and 9 were assessed by fluorescence microscopy
after staining with AO/EB. 10 ꢂL of cell suspension was mixed with 10 ꢂL of freshly
prepared AO/EB staining solution (50 ꢂg/mL) and obseved under Olympus 1Xꢀ51 inverted
fluoroscent microscope at 10X magnification and images were taken using OptikaView
Version 2.8.3.0 software. The cells were counted within 20 min and the percentage of
necrotic and apoptotic cells were calculated using the following formula [32],
Apoptotic cells (%) = (VA + NVA) ꢃ (VN + VA + NVN + NVA) × 100
Necrotic cells (%) = (NVN) ꢃ (VN + VA + NVN + NVA) × 100
where
VNꢄ=ꢄViable cells with normal nuclei (bright green chromatin having organized structure),
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VAꢄ=ꢄViable cells with apoptotic nuclei (highly condensed or fragmented bright green
chromatin)
NVNꢄ=ꢄNonviable cells with normal nuclei (bright orange chromatin having organized
structure) and
NVAꢄ=ꢄNonviable cells with apoptotic nuclei (highly condensed or fragmented bright orange
chromatin).
2.6.4. Flow cytometry for cell cycle analysis
5
MDAꢀMBꢀ231 cells were seeded in a 6 well plate at a density of 2 × 10 cells/well/2
mL. When reached 60% confluency, cells were treated with 75 ꢂM of complex 3 or 37 ꢂM of
complex 9 (IC concentration of complexes) for 24 h. Then, the cells were trypsinised,
5
0
washed with phosphate buffer saline (pH 7.4) and incubated with RNAase A (10 ꢂg/mL) at
room temperature for 30 min followed by staining with propidium iodide (5 ꢂg/mL) for 10
min. The percentage of cells in different phases of cell cycle was determined using BD
FACSVerse flow cytometer and BD FACSuite software (BD Biosciences) [32].
2.7. Molecular docking studies with protein EGFR kinase
Molecular docking studies were carried out using the AutoDock Tools (ADT) version
.5.6 and AutoDock version 4.2.5.1 docking programmes. The structures of the complexes 3
1
and 9 were converted into PDB format from mol format by OPENBABEL. The crystal
structure of the epidermal growth factor receptor (EGFR) protein tyrosine kinase (PDB ID:
1M17) was downloaded from the protein data bank (http://www.rcsb.org./pdb). The receptor
(EGFR) and the ligand (complexes) files were prepared using AutoDock Tools. First, all the
heteroatoms including water molecules were deleted while polar hydrogen atoms and
Kollman charges were added to the receptor molecule, then rotatable bonds in ligands were
assigned. All other bonds were allowed to rotate. The EGFR was enclosed in a box with
number of grid points in x × y × z directions, 60 × 60 × 60 and a grid spacing of 0.4 Å.
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Lamarckian genetic algorithms, as implemented in AutoDock, was employed to perform
docking calculations. All other parameters were default settings. For each of the docking
cases, the lowest energy docked conformation, according to the Autodock scoring function,
was selected as the binding mode. Visualization of the docked position has been done by
using PyMOL molecular graphics program.
3. Results and discussion
3.1. Synthesis of ligands and complexes
1
ꢀ3
The pyridazineꢀbased ligands (HL ) were synthesized in a two step procedure, in
which 3ꢀchloroꢀ6ꢀhydrazinopyridazine was prepared by refluxing 3,6ꢀdichloropyridazine with
hydrazine hydrate in boiling aqueous ammonia followed by condensation with substituted
salicylaldehyde (salicylaldehyde, 5ꢀnitrosalicylaldehyde or 4ꢀ(diethylamino)salicylaldehyde).
1
ꢀ3
The binuclear metal(II) complexes were prepared by the direct reaction of ligands (HL )
with an equimolar amount of metal(II) nitrates in ethanol (Scheme 1). All the complexes
were soluble in polar solvents like ethanol, DMSO and DMF. The elemental and spectral
analyses agree well with the proposed structure of the complexes.
3
3.2. Crystal structure of ligand HL
3
The crystal structure of ligand HL has been authenticated by single crystal Xꢀray
1
diffraction method, which crystallizes in a monoclinic crystal system with P2 /n space group.
3
The ORTEP diagram of ligand HL with the atomic labeling scheme is shown in Fig. 1 while
a summary of crystallographic data collection and refinement parameters are compiled in
Table 1. The selected bond lengths and bond angles are depicted in Table S1.
1
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R2
R1
O
OH
Cl
NH NH2
N
OH
N
N
HN
Cl
R2
R1
N
N
(
HL1ꢀ3
)
1
2
3
HL HL
HL
R1 = H
= H
M = Ni(II), Cu(II) or Zn(II)
H
NEt2
M(NO ) nH O
3 2.
2
R
2
NO
2
H
2
R2
R1
Cl
N
N
N
H
N
O
O
N
M
M
2NO3
N
N
N
H
Cl
R1
R2
1
ꢀ3
Scheme 1 Schematic route for the synthesis of ligands (HL ) and binuclear metal(II)
complexes (1-9).
In the crystal structure, the chloro substituted pyridazine A (C1−C4/N4/N5/Cl1), the
central part B (C5/N2/N3), the phenyl ring C (C6ꢀC11), the two diethylamino group D
(C12/C13/N1) and E (C14/C15/N1) are planar with root mean square (r.m.s) deviation of
0.0056, 0.0000, 0.0080, 0.0000 and 0.0000 Å, respectively. The dihedral angle between A/B,
A/C and B/C are 10.08 (0.15)°, 14.55 (0.07)° and 5.89 (0.17)°, respectively, indicating that
they are conjugated to allow extensive π overlap. The bond length of C5−C6 (1.445 (3) Å) is
intermediate between single and double carbonꢀcarbon bond while the bond lengths of
C4−N3 (1.364(3) Å) and C9−N1 (1.389(3) Å) are intermediate between the single and double
carbonꢀnitrogen bond, which indicates the partial delocalization of electron in the molecule.
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3
Fig. 1 The ORTEP diagram of the ligand HL with the atomic numbering scheme.
The crystal lattice packing showing the internuclear hydrogen bonding is shown in
Fig 2. The crystal packing diagram projecting along the crystallographic bꢀaxis is depicted in
Fig. S1 and the hydrogen bonding parameters are shown in Table S2. The presence of orthoꢀ
hydroxyl group in Schiff bases favors the intramolecular hydrogen bonding. Two types of
intramolecular hydrogen bonds, either O−HꢅꢅꢅN (enolꢀimine form) or N−HꢅꢅꢅO (ketoꢀamine
form) are possible in 2ꢀhydroxy Schiff bases. The Schiff bases derived from salicylaldehyde
always form the N−HꢅꢅꢅO type of hydrogen bonding, regardless of the nature of the
3
Nꢀsubstituent [33]. In the crystal structure of HL , the molecular conformation has been
stabilized by intramolecular O1−H1ꢅꢅꢅN2 hydrogen bond resulting in the formation of a six
membered ring. This indicates that only enol–imine form has been favored than the keto
form. These are evident from the observed C11−O1 bond distance of 1.361 Å, which is
consistent with a single bond and the N2=C5 bond distance of 1.292 Å, which is indicative of
3
a double bond. Each molecule of ligand HL has three intermolecular hydrogenꢀbonding with
bond length of 2.48, 2.67 and 2.054 Å.
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3
Table 1 Crystal data and structure refinement details for ligand HL .
Empirical Formula
Formula weight
Temperature (K)
Wavelength (Ǻ)
Crystal system
Space group
a (Å)
15 18 5
C H N OCl
319.79
293 (2)
0.71073
Monoclinic
P21/n
6.7997(5)
9.5387(8)
24.3565(19)
90
b (Å)
c (Å)
α (°)
β (°)
95.512(8)
90
γ (°)
3
Volume, V (Å )
1572.5(2)
4
Z
3
Density, ρcalc (mg/m )
1.351
−
1
Absorption coefficient (mm )
F(000)
0.252
672
Crystal size (mm)
0.35 × 0.30 × 0.20
2.29 to 24.99
θ Range for data collection (°)
Limiting indices
−8
≤
h
≤
7, −11
≤
k
≤
11, −28
≤
l ≤ 28
Reflections collected
Independent reflections
Max. and min transmission
Refinement method
22501
22501 [R(int) = 0.0000]
0.9593 and 0.9129
2
Fullꢀmatrix leastꢀsquares on F
22501/2/205
Data/restraints/parameters
2
Goodness of fit on F
1.085
Final R indices
R1 = 0.0873, wR2 = 0.1891
R1 = 0.1530, wR2 = 0.2150
R indices (all data)
−3
Largest diff. peak and hole (e.Å ) 0.341 and −0.409
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Fig. 2 View of crystal lattice packing showing the internuclear hydrogen bonding of ligand
3
HL .
3.3. Spectral characterization
IR spectra provide valuable information regarding the mode of coordination of the
ligand to the metal ion (Table S3). Coordination of the Schiff base ligands to the metal ions
through the nitrogen atom is expected to reduce electron density in the azomethine link and
lower the ν(C=N) absorption frequency [34]. The ν(C=N) stretching vibration (1628−1631
−1
−1
cm ) of azomethine group is shifted to lower wavenumber side (1601–1615 cm ) with a
−
1
frequency difference ꢆν of 13–28 cm indicating the involvement of azomethine nitrogen in
coordination to the metal ion. Similarly, involvement of phenolic −OH group of ligands in
bonding with the metal ions can be inferred from the appearance of ν(Ar−O) stretching
−1
−1
vibration in the lower frequency (1240−1277 cm ) with respect to ligands (1283−1290 cm )
−1
with a frequency difference ꢆν of 6–50 cm [35]. The band assigned to ν(N=N) stretching
−1
vibration of the unsymmetrical pyridazine ring was located at 1518–1535 cm for all the
−
1
ligands, which was shifted to 1539–1568 cm in the complexes suggesting the participation
of the pyridazine nitrogen to the metal coordination. Appearance of new weak bands in the
−1
region 512–568 and 421–454 cm in the spectra of all the complexes were assigned to the
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stretching frequencies of ν(M−O) and ν(M−N) bonds, respectively [36]. The presence of
−1
anions is indicated by the typical stretching vibration of nitrate at 1380–1387 cm for all the
complexes. All these features were consistent with the proposed structure of the complexes
indicating that all the ligands are coordinated to the central metal ion as monoanionic
tridentate ligand through a nitrogen atom of the azomethine group, one of the nitrogen atom
of the pyridazine ring and an oxygen atom of the phenolic group.
1
ꢀ3
1
13
13
The formation of ligands (HL ) was further authenticated by H, C and C DEPTꢀ
135 NMR spectra (Fig. S2-S10). The main chemical shifts of the proton signals in the
1
13
H NMR spectra and related chemical shifts of the carbon signals in C NMR of the ligands
1
ꢀ3
1
(
HL ) with their assignments are listed in Table S4. H NMR spectroscopy provided
evidence for the monosubstitution as the singlet assignable to the equivalent aromatic protons
of the 3,6ꢀdichloropyridazine (starting material) at 7.51 ppm splits into two doublets at 7.45
2
and 7.11 ppm, while the primary amine (NH ) and secondary amine (NH) protons appears at
1
4
.40 and 8.23 ppm, respectively in the H NMR spectrum of 3ꢀchloroꢀ6ꢀhydrazinopyridazine.
After condensation of 3ꢀchloroꢀ6ꢀhydrazinopyridazine with appropriate salicylaldehyde, the
1
ꢀ3
ligands (HL ) formed were confirmed by the imine formation evidenced by a signal around
8.50–8.23 ppm. The primary amine peak at 4.40 ppm was disappeared and the secondary
amine peak was shifted downfield to 11.60–10.17 ppm. A signal around 11.80−11.27 ppm
indicates the presence of phenolic hydroxyl group, which was shifted downfield because of
the intramolecular hydrogen bonding between it and the azomethine nitrogen. The peaks
corresponding to the aromatic proton were observed in the region 6.89–8.04 ppm. The
3
presence of diethylamino substituent in the ligand HL is evidenced by a quartet peak at 3.36
ppm corresponding to four methylene hydrogens and a triplet peak at 1.18 ppm due to six
methyl hydrogens.
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13
1ꢀ3
The C NMR spectra of the ligands (HL ) were characterized by the presence of
characteristic signals at 143–137 ppm for the azomethine carbon (C=N). The spectra were
further characterized by the absence of the CHO proton signal at 190 ppm from the
corresponding aldehydes and the carbons of phenyl ring were observed in the range of
3
130–104 ppm. The peaks corresponding to methylene and methyl carbons for the ligand HL
1
3
were observed at 44 and 13 ppm, respectively. The C NMR signals of the synthesized
13
ligands have further been substantiated by
C DEPTꢀ135 spectra (Distortionless
1
13
Enhancement by Polarization Transfer), a technique for Hꢀdecoupledꢀ C spectra used as a
means of enhancing signal intensity, which can show positive CH and CH, and negative
3
CH signals while nonꢀprotonꢀbearing carbons are not seen in DEPT spectra because the
2
technique relies on polarization transfer. The DEPTꢀ135 spectra exhibited signals for
1
ꢀ3
azomethine carbon of the Schiff bases (HL ) in the range of 143ꢀ137 ppm. The quaternary
1
3
1ꢀ3
carbon atom signals seen in the C NMR spectra for the ligands (HL ) have been
3
disappeared in DEPTꢀ135. The DEPTꢀ135 spectra of ligand HL shows a negative signal for
1
ꢀ3
methylene carbon at 44 ppm. Hence, the formation of the ligands (HL ) has been clearly
authenticated by the NMR spectroscopy.
The ESI mass spectra provide additional structural information about the
stereochemistry of the studied complexes, which was characterized by moderate to high
relative intensity molecular ion peaks. The observed molecular ion peaks for the binuclear
complexes were consistent with the proposed empirical molecular formulae. Fig. 3 shows the
+
ESI mass spectra of complexes 4 and 5, which show a molecular ion (M ) peak at
m/z = 620.93 and 710.90, respectively, suggesting that the complexes are binuclear in nature.
They also exhibit an additional peak at m/z = 622.92 and 712.89, respectively, corresponds to
37
(
M+2) peak attributed to the isotope peaks due to Cl. The base peaks at m/z = 309.96 and
1
+
2 +
3
54.95 for the complexes 4 and 5 are due to [CuL ] and [CuL ] , respectively.
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Fig. 3 ESIꢀmass spectra of the copper(II) complexes 4 (a) and 5 (b).
1
ꢀ3
The electronic spectra of the ligands (HL ) and metal(II) complexes (1-9) were
recorded in DMSO in the range of 200ꢀ800 nm (Fig. S11-S13), and the data are presented in
1
&3
Table S5. The ligands HL exhibit a single absorption peak at 336 and 371 nm respectively
2
attributed to πꢀπ* intraꢀligand transition whereas the ligand HL exhibits peaks at 336 and 465
nm assigned to πꢀπ* and nꢀπ* intraꢀligand transitions, respectively. The high hyperchromic
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3
1&2
shift exhibited by the ligand HL when compared to ligands HL
may be due to the
3
presence of electronꢀreleasing –N(Et)
2
group in the ligand HL . The metal(II) complexes
show less intense πꢀπ* intraꢀligand transition than their respective free ligands with
considerable blue shift except for zinc(II) complexes. The binuclear nickel(II) complexes
(1ꢀ3) exhibit LMCT band at 412−423 nm and a weak dꢀd band around 572–594 nm due to
2
2
A
1g → A1g, which suggest square planar geometry for the complexes [37]. The electronic
spectra of copper(II) complexes (4ꢀ6) show an intense ligand to metal charge transfer
transition around 411−441 nm and the dꢀd band in the range 611–655 nm which can be
2
2
assigned to B → A transition suggesting a square planar geometry [38]. The zinc(II)
1
g
1g
complexes (7ꢀ9) exhibit a single peak at 319−338 nm due to πꢀπ* intraꢀligand transition and
no dꢀd transition was observed for these complexes due to the lack of metal to ligand charge
10
transfer transition because of d electronic configuration of zinc(II) complexes, and
tetrahedral geometry is assigned for these complexes.
1
ꢀ3
The emission properties of the ligands (HL ) and metal(II) complexes (1-9) were
−5
studied at room temperature in 1 × 10 M DMSO (Fig. 4, 5 & S14) and the data are
presented in Table S6. The process such as magnetic perturbation, redox activity, electronic
energy transfer etc., may quench the fluorescence of a ligand by transition metal ions during
complexation. However, enhancement of fluorescence through complexation is much of
interest due to the photochemical applications of these complexes [39, 40].
1
ꢀ3
The ligands HL exhibit the photoluminescence with weak intense emission at 415,
439 and 481 nm, respectively. However, the nickel(II) complexes 1 and 2 show an intense
broad band at 341 and 473 nm, respectively, and the copper(II) complexes 4 and 5 exhibit an
intense band at 331 and 346 nm, respectively whereas the zinc(II) complexes 7 and 8 exhibit
intense band at 395 and 322 nm, respectively. These emissions are neither metalꢀtoꢀligand
charge transfer (MLCT) nor ligandꢀtoꢀmetal charge transfer (LMCT), but they may be
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assigned to intraꢀligand (nꢀπ* or πꢀπ*) fluorescence. However, the factor of chelation induced
rigidity also plays an important role in causing a change in the fluorescence behavior of the
free ligand on complexation with the metal ions. The rigidity induced in the complexes is in
turn dependent on the coordination geometry that the ligand assumes around the metal center
[
41]. The Schiff bases synthesized here have imine moiety, which exhibits poor fluorescence
in part due to isomerization of the C=N double bond in the excited state and in part due to
excitedꢀstate proton transfer (ESPT) involving the phenolic protons. Upon stable chelation
with metal ions, the C=N isomerization was inhibited; additionally, the coordination of the
ligands with the respective metal(II) ions prevents ESPT, leading to fluorescence
enhancement. Moreover, the reaction of a metal ion with a chelating agent induces rigidity in
the resulting molecule and tends to produce a large chelationꢀenhanced fluorescence effect
(CHEF), which induces the large enhancement of fluorescence intensity [42]. Hence, these
1ꢀ3
Schiff bases ligands (HL ) may also be used as a fluorescent probes for metal ions.
60000
40000
20000
0
1
4
7
3
00
350
400
450
500
Wavelength (λ), nm
Fig. 4 Photoluminescence (PL) spectra of binuclear metal(II) complexes (1, 4 & 7) of ligand
1
HL .
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30000
25000
20000
15000
10000
2
5
8
5
000
0
350
400
450
500
550
Wavelength (λ), nm
Fig. 5 Photoluminescence (PL) spectra of binuclear metal(II) complexes (2, 5 & 8) of ligand
2
HL .
EPR studies of polycrystalline samples were recorded at room temperature (Fig. 6).
The g|| and g
and 2.058−2.092, respectively, while the gav value was determined to be 2.121−2.146 from
the relation gav = (g|| + 2g )/3. These calculated data are in agreement with an orbitally
⊥
values for the copper(II) complexes (4ꢀ6) have been calculated as 2.249−2.262
⊥
non degenerate state for the fourꢀcoordinate copper(II) complexes under investigation.
According to Hathaway et al. [43] axial symmetry parameter G, determined as
⊥
G = (g||−2.0023)/(g −2.0023), measures the exchange interaction between the metal centers in
a polycrystalline solid. If G > 4, the exchange interaction is negligible, but if G < 4 indicates
considerable exchange interaction in the solid complexes. The copper(II) complexes have G
values lesser than 4 indicating the presence of exchange interaction in the solid complexes.
The value of g|| > g
⊥
> 2.0023 and G value within the range 2.5–4.0 are consistent with a
ground state, and a square based geometry can be proposed for the complexes [44]. The
|| value, a moderately sensitive function is used for the indication of covalency. The g|| > 2.3
d
g
x y
2ꢀ 2
is characteristics of an ionic environment and g|| < 2.3 is of covalent environment in M−L
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bonding [45]. The observed g|| values for the complexes was less than 2.3, in agreement with
the covalent character of the M−L bond which has been further supported by the G value,
which was less than 4.0 [46].
4
000
000
0
2
-
-
-
2000
4000
6000
2600
2800
3000
3200
3400
3600
3800
Magnetic Field (G)
Fig. 6 EPR spectrum of the copper(II) complex 4.
The magnetic moment values of binuclear copper(II) complexes (4ꢀ6) at room
temperature was found in the range of 1.59ꢀ1.63 B.M, which is below the ‘spin only’ value of
1.73 B.M. The lower value of magnetic moment at room temperature is consistent with
square planar geometry around the metal ions and are attributed to the antiꢀferromagnetic
exchange interaction between two copper(II) metal ions, which indicates the formation of
binuclear complexes [47].
3.4. Electrochemical properties
The conductivity measurements in nonꢀaqueous solutions can be used in structural
studies of metal complexes within the limits of their solubility and provide a method of
testing the degree of ionization of the complexes. The molar conductivity values in DMF
−
3
−1
2
−1
solution (10 M) at 25 °C for all the complexes were in the range 131–172 ꢁ cm mol
indicating 1:2 electrolytic behavior [48], implying the nonꢀcoordination of nitrate anions to
the central metal(II) ion.
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3.4.1. Reduction process at cathodic potential
The redox properties of the binuclear nickel(II) and copper (II) complexes (1-6) were
studied by cyclic voltammetry in the range 0 to –1.2 V in DMF using TBAP as the supporting
electrolyte (Fig. S15) and the data are summarized in Table S7. The binuclear complexes in
the cathodic region display two step irreversible one electron transfer reduction process. The
1
first reduction wave appeared in the potential range ( Epc) −0.323 to −0.463 V while the
2
second reduction potential ( Epc) was observed at −0.634 to −0.790 V, which can be
attributed to the redox couple species generally as:
II
I
II
I
M → M M → M
2
2
3.4.2. Oxidation process at anodic potential
The cyclic voltammograms of binuclear nickel(II) complexes (1ꢀ3) in the anodic
region are shown in Fig. S16 and the data are summarized in Table S7. In the anodic
potential region, the nickel(II) complexes show two step irreversible one electron oxidation
process. The first oxidation wave was obtained in the range from +1.009 V to +1.024 V,
while the second oxidation wave obtained in the region from +1.140 V to +1.153 V. The two
oxidation processes for the nickel(II) complexes were assigned as follows:
II
III
II
III
Ni
2 2
→ Ni Ni → Ni
3.5. Computational details
3.5.1. Geometry optimization
In the absence of crystal data, it was thought worthwhile to obtain structural
information through molecular modeling. Molecular mechanics, which provides the energy
minimized conformation is a tool of increasing importance for the structural investigation of
coordination and organometallic compounds. Complexes 1, 3, 5 and 9 were optimized at
B3LYP/LANL2DZ and B3LYP/GEN levels in the gas phase. The optimized ground state
geometry structures of these complexes are shown in Fig. S17, and the calculated structural
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parameters (bond lengths and bond angles) are given in Table S8 along with the reported
experimental values [14, 38]. The acceptable results were obtained by DFT/B3LYP method
with LANL2DZ and GEN basis sets for the complexes 1, 3, 5 and 9. The calculated values of
bond angles obtained by using B3LYP/LANL2DZ level are more appropriate than the
B3LYP/GEN level. Therefore, DFT/B3LYP/LANL2DZ level was preferred to B3LYP/GEN
level for determining the molecular structure. The B3LYP method in combination with the
LANL2DZ basis gives an excellent estimation of M–N bond distances, M–N2, M–N4,
M–O1, M–O2, M–N7 and M–N9. Molecular geometries can be predicted by calculating τ
values from the following equation [49]:
β–α
τ =
60
where α and β are the equatorial and axial bond angles, respectively. Generally, if the
molecular geometry is square planar, τ value is close to 0, while the τ value close to 1
indicates tetrahedral geometry. If τ value is between 0 and 0.5, the molecular geometry is
distorted tetrahedral. For the complex 9, α (N(2)−Zn(1)−N(4)) value is 79.64° and β
(
(
N(9)−Zn(1)−O(1)) value is 88.39°. For complexes 1, 3 and 5 α (N(2)−Ni(1)−N(4)),
N(2)−Ni(1)−N(4)) and (N(2)−Cu(1)−N(4)) values are 78.00°, 77.47° and 78.88°,
respectively and β (N(9)−Ni(1)−O(1)), (N(9)−Ni(1)−O(1)) and (N(9)−Cu(1)−O(1)) values
are 84.02°, 83.09° and 84.15°, respectively. Based on the above results, distorted tetrahedral
geometry is adopted by the complex 9 as their τ values lies between 0 and 0.5. In the case of
complexes 1, 3 and 5, the value of τ is equal to 0, which indicate the square planar geometry
for the nickel(II) and copper(II) centers. Generally, the calculated bond lengths are longer
than the experimental data because the former are optimized in the gas phase and the latter
are in a tight crystal lattice. The calculated bond lengths and bond angles agree reasonably
with the reported experimental values.
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3.5.2. Frontier molecular orbital (FMO) analysis
FMOs (HOMO and LUMO) play an important role in the study of electrical and
optical properties. Additionally, the HOMOꢀLUMO energy gap values help to exemplify the
chemical reactivity, kinetic stability and electrical transport properties of the molecule [50].
Recently, the HOMOꢀLUMO energy gap has been used to prove the bioactivity from
intramolecular charge transfer and this energy gap values also correlated with various
biological systems like antibacterial, antioxidant and DNA binding studies [51−53]. A variety
of applications are available for the use of the HOMOꢀLUMO gap as a quantum descriptor in
establishing correlation in various chemical and biochemical systems [54, 55]. Molecules
with a lower HOMOꢀLUMO energy gap show a noteworthy degree of intramolecular charge
transfer (ICT) from the electronꢀdonor groups to the electronꢀaccepter groups through πꢀ
conjugated path [56]. This explains the extent of ICT from the electronꢀdonor groups to
electronꢀacceptor groups through conjugated path. A large HOMOꢀLUMO gap implies high
stability for the molecule in the sense of its lower reactivity in chemical reactions [55].
Systems having high EHOMO are good electron donors while those having low ELUMO energy
are good electron acceptors. The orbital energy level analysis for complexes 3, 5, 6, 7 and 9
showed that EHOMO values are –2.49, –1.76, –2.47, –2.21 and –2.56 eV, respectively while
E
LUMO values are −1.54, –0.34, –1.24, –0.86 and –1.91 eV, respectively. As a result,
complexes 3, 6, 7 and 9 are better electron donors while complex 5 is a better electron
acceptor. The ꢆE values of complexes 3 (0.95 eV), 5 (1.42 eV), 6 (1.23 eV), 7 (1.35 eV) and
9
(0.65 eV) calculated at the DFT level reflects the higher stability of 3 and 9 compared to
other complexes (Fig. 7 & S18). It could be noted that 3 and 9 has higher ability towards the
intramolecular charge transfer than the other complexes. The energy gap value indicates the
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eventual charge transfer interactions within the molecule, and also the obtained lower band
gap value supports the observed trend of biological activity.
Fig. 7 Frontier molecular orbitals of the complexes 3 (a), 5 (b), 6 (c) and 9 (d).
Generally, the electronic properties of the molecules are calculated from the total
energies and the Koopman’s theorem. In this context, ionization potential was calculated
from IP = –EHOMO while the electron affinity is computed from EA = –ELUMO. Further,
important quantities such as electronegativity (χ), hardness (η), softness (ζ), and
electrophilicity index (ψ) were deduced from ionization potential and electron affinity values.
IP + EA
Electronegativity (χ) µ = –X =
2
IP – EA
Chemical hardness (η) =
2
1
Softness (ζ) =
2
η
2
µ
Electrophilicity index (ψ) =
2
η
2
9