Mononuclear Co(III), Ni(II) and Cu(II) complexes of tridentate di-tert-butylphenylhydrazone: Synthesis, characterization, X-ray crystal structures, Hirshfeld surface analysis, molecular docking and in vivo anti-inflammatory activity

Article abstract of DOI:10.1002/aoc.4337

A new hydrazone (LH₂) derived from the condensation of 2-(4-fluorobenzamido)benzohydrazide with 3,5-di-tert-butyl-2-hydroxybenzaldehyde was used to synthesize Co(III), Ni(II) and Cu(II) complexes. These were characterized using various physicochemical, thermal, spectroscopic and single-crystal X-ray diffraction techniques. All the complexes crystallize in a monoclinic crystal system with P2₁/n space group and Z = 4. Structural studies of [Co(L)(LH)]?H₂O indicate the presence of both amido and imidol tautomeric forms of the ligand, resulting in a distorted octahedral geometry around the Co(III) ion. On the other hand, in the [Ni(L)(DMF)] and [Cu(L)(H₂O)] complexes, the ligand coordinates to the metal through imidol form resulting in distorted square planar geometry, in which the fourth position is occupied by the oxygen of coordinated DMF in [Ni(L)(DMF)] and by a water molecule in [Cu(L)(H₂O)]. Hirshfeld surface calculations were performed to explore hydrogen bonding and C―H???π interactions. Molecular docking studies were carried out to study the interaction between the synthesized compounds and proteins (cyclooxygenase-2 and 5-lipoxygenase). The complexes along with the parent ligand were screened for their in vivo anti-inflammatory activity, using the carrageenan-induced rat paw oedema method. The complexes show significant anti-inflammatory potencies.

Full text of DOI:10.1002/aoc.4337

Received: 5 January 2018
DOI: 10.1002/aoc.4337
Revised: 31 January 2018
Accepted: 3 February 2018
F U L L P A P E R
Mononuclear Co(III), Ni(II) and Cu(II) complexes of
tridentate di‐tert‐butylphenylhydrazone: Synthesis,
characterization, X‐ray crystal structures, Hirshfeld surface
analysis, molecular docking and in vivo anti‐inflammatory
activity
Geeta H. Chimmalagi1 | Umashri Kendur1 | Sunil M. Patil1 | Kalagouda B. Gudasi1
Christopher S. Frampton2 | Mahantesh B. Budri1 | Chandrashekhar V. Mangannavar3 |
Iranna S. Muchchandi3
|
1 Department of Chemistry, Karnatak
A new hydrazone (LH2) derived from the condensation of 2‐(4‐fluorobenzamido)
benzohydrazide with 3,5‐di‐tert‐butyl‐2‐hydroxybenzaldehyde was used to syn-
thesize Co(III), Ni(II) and Cu(II) complexes. These were characterized using var-
ious physicochemical, thermal, spectroscopic and single‐crystal X‐ray diffraction
techniques. All the complexes crystallize in a monoclinic crystal system with
P21/n space group and Z = 4. Structural studies of [Co(L)(LH)]⋅H2O indicate
the presence of both amido and imidol tautomeric forms of the ligand, resulting
in a distorted octahedral geometry around the Co(III) ion. On the other hand, in
the [Ni(L)(DMF)] and [Cu(L)(H2O)] complexes, the ligand coordinates to the
metal through imidol form resulting in distorted square planar geometry,
in which the fourth position is occupied by the oxygen of coordinated DMF in
[Ni(L)(DMF)] and by a water molecule in [Cu(L)(H2O)]. Hirshfeld surface calcu-
lations were performed to explore hydrogen bonding and C―H⋅⋅⋅π interactions.
Molecular docking studies were carried out to study the interaction between the
synthesized compounds and proteins (cyclooxygenase‐2 and 5‐lipoxygenase).
The complexes along with the parent ligand were screened for their in vivo
anti‐inflammatory activity, using the carrageenan‐induced rat paw oedema
method. The complexes show significant anti‐inflammatory potencies.
University, Dharwad‐580003, Karnataka,
India
2 Institute of Materials and Manufacturing,
Wolfson Centre for Materials Processing,
Brunel University, London, UK
3 H. S. K. College of Pharmacy, Bagalkot‐
587101, Karnataka, India
Correspondence
Kalagouda B. Gudasi, Department of
Chemistry, Karnatak University, Dharwad
– 580003, Karnataka, India.
Email: kbgudasi@gmail.com
Funding information
UGC, UPE‐FAR‐I Program, Grant/Award
Number: 14‐3/2012
KEYWORDS
anti‐inflammatory activity, Hirshfeld surface analysis, molecular docking, transition metal complexes,
X‐ray diffraction study
1 | INTRODUCTION
metabolites of arachidonic acid (AA) such as prostaglan-
dins and leukotrienes (generated by cyclooxygenase
Over the past several decades, inflammation has been
renowned as a devastating burden and the prime basis
(COX) and 5‐lipoxygenase (5‐LOX) enzymatic pathways,
respectively) have been associated as mediators in an
assortment of diseases, including asthma, inflammation
of
various
inflammatory‐related
diseases.
The
Appl Organometal Chem. 2018;e4337.
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https://doi.org/10.1002/aoc.4337

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CHIMMALAGI ET AL.
and cell proliferation.[1,2] Classical non‐steroidal anti‐
inflammatory drugs (NSAIDs) which are non‐selective
COX inhibitors have been widely used for the treatment
of inflammation.[3,4] In the treatment of arthritis,
NSAIDs offer an efficient treatment, but safety is signifi-
cantly compromised, mainly due to an up‐regulation of
the AA metabolism by the 5‐LOX pathway, increasing
the formation of pro‐inflammatory leukotrienes and con-
tributing to gastrointestinal ulcerations and atherosclero-
sis.[5] Since the selective inhibition of one pathway in
the AA cascade appears to cause undesired effects, more
recently several clinically effective NSAIDs have been
structurally modified to yield potent dual COX‐2/5‐LOX
inhibitors.[6,7]
Aroylhydrazones are an extremely versatile group of
compounds in the family of Schiff bases. Transition
metal complexes of aroylhydrazones have been widely
studied for several decades due to their structural and
electronic properties and copious applications in various
fields. Hydrazones exhibit amido–imidol tautomerism in
solution, and various modes of coordination are found
in their metal complexes.[8,9] The tautomeric forms of
the ligands in their metal complexes are dependent on
temperature, pH of the medium, nature of the substitu-
ents and the metal ions.[10] The possibility of tautomer-
ism in this class of compounds has led to an interest
in the field of pharmacology and catalysis.[11,12]
Hydrazones and their metal complexes evince a variety
of biological and pharmacological activities, such as
anti‐inflammatory,[13] anti‐hypertensive,[14] anti‐micro-
bial,[15] anti‐cancer,[16] anti‐tuberculous[17] and antioxi-
dant[18] activities.
The di‐tert‐butylphenols represent a potent class of
anti‐fungal, anti‐oxidant[19] and well‐known anti‐inflam-
matory agents which are dual COX/LOX inhibitors.[6,20]
Recently, Ghatak and co‐workers have investigated
the application of di‐tert‐butylphenylhydrazones as
inhibitors of pro‐inflammatory agents, such as COX‐2
and 5‐LOX enzymes.[21] A literature survey reveals
that di‐tert‐butylphenolhydrazone derivatives could be
considered as successful pharmacophores in the design
of effective anti‐inflammatory drugs with a superior
safety profile. The coordination of bioactive organic mol-
ecules and anti‐inflammatory drugs with metal ions is a
common approach for enhancing the therapeutic
potency and reducing the toxicity of the organic mole-
cules.[22,23] Previous studies have shown that the metal
complexes of anti‐inflammatory drugs available on the
market exhibited more potent anti‐inflammatory activity
than the drug itself in rats or mice, with fewer adverse
effects.[24,25]
structures, Hirshfeld surface analysis and anti‐inflamma-
tory activity of first row transition metal complexes derived
from tridentate (E)‐N′‐(3,5‐di‐tert‐butylsalicylidene)‐2‐(4‐
fluorobenzamido)benzohydrazide (LH2). The potential
binding interactions between the synthesized compounds
and proteins (COX‐2 and 5‐LOX) were explored using
molecular docking studies.
2 | EXPERIMENTAL PROTOCOLS
2.1 | Materials and Physical
Measurements
The chemicals used were of analytical reagent grade and
used without further purification. Hydrated metal salts
were used as supplied. Carbon, hydrogen and nitrogen
were determined using a Thermoquest CHN analyser.
Metal contents of the complexes were determined accord-
ing to a literature procedure.[26] Infrared (IR) spectra
were recorded with a Nicolet‐6700 FT‐IR spectrometer
1
in the 400–4000 cm−1 region using KBr discs. H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were
recorded with Bruker spectrometer, in deuterated
dimethylsulfoxide (DMSO‐d6) with tetramethylsilane as
an internal standard. Mass spectra were recorded with a
Waters XEVO TQS micro mass spectrometer and
a Shimadzu QP 2010S GC mass spectrometer. Electron
paramagnetic resonance (EPR) spectra of [Cu(L)(H2O)]
were recorded at both room temperature and 77 K with a
Varian E‐4 X‐band spectrometer using tetracyanoethylene
as the g‐marker. UV–visible spectra were recorded with a
JASCO V‐670 UV–visible spectrophotometer in the
200–1100 nm range using dimethylformamide (DMF) as
the solvent. Conductance measurements of complexes
(1 mM) were recorded in DMF using an ELICO‐CM‐82
conductivity bridge. Thermogravimetric (TG)/differential
thermogravimetric analysis (DTA) studies of the metal
complexes were carried out over the temperature
range 25–1000 °C using
a Universal V4.5A (TA
Instruments).
2.2 | Synthesis of LH2
A schematic of the synthesis of LH2 is shown in Scheme 1.
In the first step, 4‐fluorobenzoyl chloride (1.51 g, 10 mmol)
was added dropwise to a solution of methyl anthranilate
(I; 1.58 g, 10 mmol) in benzene (200 ml) and stirred for
3 h at room temperature to afford methyl 2‐(4‐
fluorobenzamido)benzoate (II; yield: 89%). In the second
step, 99% hydrazine hydrate (5 g, 0.1 mol) was added to
a methanolic solution of II (2.73 g, 10 mmol) and refluxed
for 4 h to afford 2‐(4‐fluorobenzamido)benzohydrazide
(III; yield: 74%). Finally, a methanolic solution of 3,5‐di‐
Encouraged by earlier reports, the work presented here
focused on the synthesis, characterization, crystal

CHIMMALAGI ET AL.
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SCHEME 1 Synthetic route for preparation of LH2
tert‐butyl‐2‐hydroxybenzaldehyde (2.34 g, 10 mmol) was
added to a methanolic suspension of III (2.73 g, 10 mmol).
The reaction mixture along with a catalytic amount of gla-
cial acetic acid (five drops) was continuously stirred and
further refluxed for 2 h. Progress of the reaction was mon-
itored by TLC. The resulting solid was filtered off, washed
with cold methanol and dried in air.
Colour: reddish brown; yield: 74%. Anal. Calcd for
C58H63CoF2N6O7 (%): C, 66.15; H, 6.03; Co, 5.60; N, 8.58.
Found (%): C, 66.22; H, 5.91; Co, 5.52; N, 7.83. IR (KBr,
cm−1): 3433 (O―H, broad), 3367 (N1―H), 1680
(C7=O1), 1586 (C=N), 1615 (C=N, new), 1235 (C―O).
ESI‐MS (m/z): 1035 [M − H2O + H]+. UV–visible: λmax
(DMF): 271, 325, 439 nm. Molar conductance
(Ω−1 cm2 mol−1): 3.43.
LH2. Colour: white; yield: 86%; m.p. 264–266 °C. Anal.
Calcd for C29H32FN3O3 (%): C, 71.14; H, 6.59; N, 8.58.
Found (%): C, 70.90; H, 6.40; N, 8.46. IR (KBr, cm−1):
3443 (O―H, broad), 3319, 3215 (N1―H, N2―H), 1680
2.3.2 | Synthesis of [Ni(L)(DMF)]
1
(C7=O1), 1634 (C14=O2), 1596 (C=N), 1279 (C―O). H
A mixture of LH2 (0.490 g, 1.0 mmol), sodium acetate
(0.164 g, 2.0 mmol) and NiCl2⋅6H2O (0.24 g, 1.0 mmol)
in 30 ml of methanol was refluxed for 6 h. The reddish
brown precipitate obtained was filtered, washed with
hot methanol and dried in air. Further, the precipitate
was dissolved in 30 ml of DMF. Single crystals suitable
for X‐ray diffraction studies were obtained by slow evapo-
ration of the solution over a period of 10–11 days.
Colour: reddish brown; yield: 59%. Anal. Calcd for
C32H37FN4NiO4 (%): C, 62.06; H, 6.02; Ni, 9.48; N, 9.05.
Found (%): C, 61.97; H, 5.94; Ni, 9.35; N, 8.91. IR (KBr,
cm−1): 3446 (N1―H), 1671 (C7=O1), 1588 (C=N), 1621
(C=N, new), 1235 (C―O). ESI‐MS (m/z): 619 [M + H]+.
UV–visible: λmax (DMF): 272, 361, 420 nm. Molar conduc-
tance (Ω−1 cm2 mol−1): 7.55.
NMR (400 MHz, DMSO‐d6, δ, ppm): 1.25 (9H, s, tert‐
Bu), 1.38 (9H, s, tert‐Bu), 7.12 (1H, d, C23H, J = 2 Hz),
7.31–7.28 (2H, m, C2H & C4H), 7.44–7.40 (2H, m, C11H
& C29H), 7.63 (1H, t, C10, J = 8 Hz), 7.87 (1H, d, C9, J
= 8 Hz), 8.01–7.98 (2H, m, C1H & C5H), 8.42 (1H, d,
C12H, J = 8 Hz), 8.56 (1H, s, C15H), 11.65 (1H, s, O3H,
D2O exchange), 12.14 (1H, s, N2H, D2O exchange), 12.38
(1H, s, N1H, D2O exchange). 13C NMR (100 MHz,
DMSO‐d6, δ, ppm): 31.20 ((CH3)3), 33.82 ((CH3)3), 29.25
(C―(CH3)3), 34.58 (C―(CH3)3), 115.78 (C2,C4), 116
(C16), 121.59 (C29), 125.85 (C23), 129.73 (C1, C5),
130.93 (C6), 138.96, 116.79, 132.63, 123.38, 128.62, 120.78
(C8‐13, aromatic), 135.68 (C18), 140.52 (C24), 152.33
(C15), 154.65 (C17), 163.67 (C14), 164.36 (C7). UV–visible:
λmax (DMF): 272, 300 nm.
2.3.3 | Synthesis of [Cu(L)(H2O)]
2.3 | Synthesis of Metal Complexes
A
mixture of LH2 (0.490 g, 1.0 mmol) and
A schematic of the synthesis of the complexes is shown in
Scheme 2.
Cu(CH3COO)2⋅H2O (0.20 g, 1.0 mmol) in 30 ml of meth-
anol was stirred for 3 h at room temperature. The resul-
tant solution on slow evaporation afforded green crystals
of [Cu(L)(H2O)], suitable for X‐ray diffraction studies.
Colour: green; yield: 77%. Anal. Calcd for
C29H32CuFN3O4 (%): C, 61.20; H, 5.67; Cu, 11.17; N,
7.38. Found (%): C, 61.15; H, 5.55; Cu, 11.03; N, 7.25. IR
(KBr, cm−1): 3439 (O―H, broad, N1―H), 1673
(C7=O1), 1584 (C=N), 1612 (C=N, new), 1238 (C―O).
ESI‐MS (m/z): 569 [M + H]+. UV–visible: λmax (DMF):
272, 323, 413, 635 nm. Molar conductance (Ω−1 cm2 mol−1):
6.32.
2.3.1 | Synthesis of [Co(L)(LH)]⋅H2O
A
mixture of LH2 (0.490 g, 1.0 mmol) and
Co(CH3COO)2⋅4H2O (0.124 g, 0.5 mmol) in 30 ml of
methanol was refluxed for 6 h. The reddish brown precip-
itate obtained was filtered off, washed with cold methanol
and dried in air. Single crystals suitable for X‐ray diffrac-
tion studies were obtained by slow evaporation of the
filtrate.

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CHIMMALAGI ET AL.
SCHEME 2 Synthetic route for preparation of complexes
Crystal Explorer 3.1[30] based on the pertinent CIF files.
Three‐dimensional HS maps generated with dnorm using
a red, white and blue colour scheme give a precise picture
of close contacts, van der Waals contacts and longer con-
tacts.[31] The combination of de (distance from any surface
point to the nearest exterior atom) and di (distance from
any surface point to the nearest interior atom) in the form
of a 2D fingerprint plot provides summary of intermolec-
ular contacts in the crystal.[32,33]
2.4 | Single‐Crystal X‐ray Crystallographic
Studies
Single‐crystal X‐ray data of all the complexes were col-
lected at 100 K with a Rigaku SuperNova Dualflex AtlasS2
diffractometer using Cu Kα radiation (λ = 1.54184 Å).
Integration, absorption correction and determination of
unit cell parameters were performed using the
CrysAlisPro program package.[27] The structures were
solved by a direct method with SHELXD‐2014/6 and
refined against F2 by the full‐matrix least‐squares tech-
nique in the anisotropic approximation (except hydrogen
atoms) using the SHELXL‐2014/6 package.[28] Hydrogen
atom positions were calculated geometrically and refined
using the riding model. Mercury CSD 2.0 program[29]
was used for molecular graphics.
2.6 | Molecular Docking Simulation
High‐resolution crystallographic structures of celecoxib‐
bound COX‐2 (PDB ID: 3LN1) and human 5‐LOX (PDB
ID: 3O8Y) were retrieved from the RSC Protein Data
Bank. AutoDock Tools and AutoDock Vina[34] were
employed to set up and perform docking calculations of
LH2 and its complexes in their binding to proteins. The
metal complexes were taken from their crystal structures
as a CIF file and were converted to the PDB format using
Mercury software. The geometrical optimization of LH2
2.5 | Hirshfeld Surface (HS) Analysis
The molecular HSs were mapped with dnorm, and two‐
dimensional (2D) fingerprint plots were generated using

CHIMMALAGI ET AL.
5 of 16
was done using density functional theory with the ORCA
computational chemistry package.[35] All calculations
were performed using the hybrid functional BP in combi-
nation with the Ahlrichs split‐valence double‐ξ basis set
def2‐SVP[36] for all the atoms. The output of the calcula-
tions was visualized using the molecular visualizer tool
Avogadro 1.1.1.[37]
3 | RESULTS AND DISCUSSION
LH2 and its complexes are soluble in chloroform, DMF
and DMSO. Analytical data for all the compounds are in
good agreement with their proposed formulae. Analytical,
physicochemical and spectral parameters are compiled in
Section 2.
In docking analysis, the binding site was assigned to
include the entire protein, which was enclosed in a grid
box with dimensions 60 × 60 × 60 Å3 and a grid spacing
of 0.765 Å. The genetic algorithm population size and
the maximum number of evaluations were 150 and 2
500 000, respectively. A total of 50 runs were carried out.
A maximum of 50 conformers were considered for each
molecule and the root‐mean‐square cluster tolerance
was set to 2.0 Å in each run. Discovery Studio 4.1.0 and
Python Molecule Viewer[38] were used to explore the
results obtained.
3.1 | IR Spectral Data
The numbering scheme followed for LH2 is given in
Scheme 1. The tert‐butyl substituent groups in LH2
and its complexes are conspicuous by their typical
absorption patterns between 2870 and 2955 cm−1 [39]
.
The IR spectrum of LH2 (Figure S1) shows a broad
band at 3443 cm−1 attributed to free ν(O―H). Absence
of this band in the spectra of all the complexes indi-
cates deprotonation of the phenolic oxygen and subse-
quent coordination to the metal. In addition, the
ν(C―O) band observed at 1279 cm−1 in the spectrum
of LH2 shows a blue shift by 30–40 cm−1 suggesting
the coordination of phenolic oxygen to the metal ion.
The ν(C=N) band appearing at 1596 cm−1 in the LH2
spectrum is shifted towards lower frequency upon com-
plexation, indicating the involvement of azomethine
nitrogen in coordination.
2.7 | Anti‐inflammatory Screening
Male Sprague Dawley rats (H. S. K. College of Pharmacy,
Bagalkot, India) weighing 160–220 g housed at 25 2 °C
were fasted with free access to water at least 16–22 h prior
to experiments. A paw oedema was induced by injecting
1% λ‐carrageenan (0.2 ml in 0.9% NaCl) subcutaneously
in the sub‐plantar region of right hind paw. Animals were
divided into groups of six each. The rat paw thickness was
measured with a digital plethysmometer (UGO Basile
7140) before and 1 h after carrageenan injection to detect
the carrageenan‐induced inflammation. The test com-
pounds were suspended in 0.5% sodium carboxymethyl-
cellulose (Na‐CMC) and administered at doses of 5 and
10 mg kg−1 of body weight and diclofenac was adminis-
tered orally at a dose of 10 mg kg−1 to all groups of rats 1 h
after carrageenan injection. The control groups received
0.5% Na‐CMC in distilled water. Changes in paw volume,
in millilitres, were recorded at 0.5, 1, 3 and 5 h after injec-
tion of the test compounds, reference drug and control.
For statistical analysis, we used GraphPad Prism 3.0.
Results were expressed in terms of oedema volume as
mean SEM and mean percent inhibition. The oedema
inhibition was calculated according to the following
equation:
The sharp bands at 1680 and 1634 cm−1 in the LH2
spectrum are assigned to ν(C7=O1) and ν(C14=O2),
respectively.[40] The band due to ν(C7=O1) remains
almost unaltered in the spectra of all the complexes
suggesting its non‐involvement in coordination. The
absence of bands due to ν(C14=O2) and ν(N2―H)
(3215 cm−1) and the appearance of a new band in
the region 1610–1620 cm−1 due to the stretching vibra-
tion of the conjugated ―C=N―N=C― moiety in the
complexes[41] indicate the enolization and subsequent
coordination of oxygen atom to the central metal ion.
In addition, the ν(C14=O2) band at 1634 cm−1
undergoes a substantial red shift to 1624 cm−1 in the
spectrum of [Co(L)(LH)]⋅H2O (Figure S2) indicating
the presence of both imidol (L2−) and amido (LH−)
tautomers of the ligand in the complex. The strong
absorption band at 1639 cm−1 in the spectrum of
[Ni(L)(DMF)] (Figure S3) is attributed to the character-
istic stretching mode of >C=O present in the coordi-
nated DMF molecule.[42] A medium intensity band at
3319 cm−1 is assigned to ν (N1H) of LH2. This band
is not observed for the complexes and might be
obscured by the broad band in the region 3433–
3446 cm−1, due to the water molecules present in the
complexes.
Vc−Vt
Oedema inhibition ð%Þ ¼
× 100
Vc
where Vc is the oedema volume of rat of control group, at
time t, and Vt is the oedema volume of rat of test
compound group, at time t.

6 of 16
CHIMMALAGI ET AL.
and 2B1g → 2Eg transitions as for a square planar configu-
ration around the metal ion.[45] All the complexes show
an intense band at 410–440 nm, which can be assigned
to charge transfer transitions.
X‐band EPR measurements were carried out in
powder form as well as in frozen solution of [Cu(L)
(H2O)] in DMSO. The EPR spectrum of powder sample
exhibits isotropic intense broad signal with giso = 2.04
with no hyperfine splitting. From solution EPR measure-
ments, it was possible to resolve the hyperfine pattern
13
3.2 | 1H NMR and C NMR Studies
The 1H NMR spectrum of LH2 was recorded in the range
0–20 ppm and is presented in Figure S4. The signals at
12.38, 12.14 and 11.65 ppm are attributed to the D2O‐
exchangeable N2H, N1H and O3H protons, respectively.
The two singlets at 1.25 and 1.38 ppm correspond to two
sets of magnetically non‐equivalent tert‐butyl groups.[43]
The singlet at 8.56 ppm is assigned to the azomethine
proton (C15H), which confirms the formation of
hydrazone. The aromatic protons resonate in the range
(Figure S11) with g║ = 2.30, g⊥ = 2.05, G = 6.51, A║
=
1
7.12–8.42 ppm. The D2O exchange H NMR spectrum
of LH2 is provided in the supporting information
182 × 10−4 cm−1 and A⊥ = 57 × 10−4 cm−1. From the
observed g values, g║ > g⊥ > ge (2.0023), it is evident that
(Figure S5).
2
2
the unpaired electron is localized in dx −y orbital of the
Cu(II) ion and the spectrum is characteristic of axial
symmetry.[46] The quotient g║/A║ measures the degree
of tetrahedral distortion. This quotient ranges from
approximately 105 to 135 cm for square planar structures.
The g║/A║ value of 126 cm for [Cu(L)(H2O)] is in agree-
ment with the crystallographic data.[47] Further, it is
expected that there is no exchange coupling between
two copper centres in the solid state, as the axial symme-
try parameter G = g║ − 2/g⊥ − 2 is found to be more than
4 for the complexes.[48]
The 13C NMR spectrum of LH2 (Figure S6) shows
signals at 164.36 and 163.67 ppm and are assigned to the
carbonyl (C7 and C14) carbons, respectively. A singlet at
152.32 ppm ascribed to the azomethine carbon (C15)
confirms the formation of hydrazone functionality. The
two intense bands observed at 31.20 and 33.82 ppm
correspond to methyl carbons (―(CH3)3) of two non‐
equivalent tert‐butyl groups.
3.3 | Mass Spectral Studies
The ESI mass spectrum of LH2 (Figure S7) shows a
molecular ion [M]+ peak at 489. That of [Co(L)(LH)]
⋅H2O (Figure S8) shows a molecular ion [M − H2O + H]
+ peak at 1035. This assignment is in good agreement with
the ascribed +3 oxidation state for cobalt. ESI mass spec-
tral studies of [Ni(L)(DMF)] and [Cu(L)(H2O)] (Figure S9)
show their molecular ion [M + H]+ peaks at 619 and 569,
respectively. Apart from this, spectra show additional
peaks, which are due to molecular cations of various
fragments of the complexes and isotopes.
3.5 | Thermal Analysis
Thermal behaviour of all the complexes was studied over
the temperature range 25–1000 °C under nitrogen atmo-
sphere. TG/DTA curves of [Co(L)(LH)]⋅H2O (Figure S12)
show the first exothermic weight loss (1.96%, calcd
1.71%) at 150 °C which is consistent with the removal of
hydrogen‐bonded lattice‐held water molecule. In the
second stage, part of the ligand is lost in the range 230–
330 °C with an exothermic DTA curve at 270 °C. Further
mass loss (43%) in the range 330–430 °C is ascribed to the
decomposition of the remaining part of the ligand.
The plateau obtained above 430 °C corresponds to the
formation of stable metal oxide with residual weight of
7.8%. The first weight loss of 12.02% (calcd 11.80%)
between 230 and 330 °C exhibited by [Ni(L)(DMF)]
(Figure S13) is accounted for by the loss of a coordinated
DMF molecule. The second weight loss of 75.7% between
220 and 410 °C corresponds to the loss of one ligand mol-
ecule. The corresponding DTA peak at 400 °C for the
complex signifies the exothermic process. The plateau
obtained above 410 °C corresponds to the formation of
stable NiO. [Cu(L)(H2O)] shows an initial weight loss of
3.32% (calcd 3.16%) between 130 and 150 °C and can be
accounted for by the loss of a coordinated water molecule.
The dehydrated complex then decomposes in a single
3.4 | Electronic and EPR Spectral Studies
Electronic spectra of LH2 and its complexes (Figure S10)
were measured in DMF. The free ligand exhibits strong
absorptions at 272 and 300 nm. The former is assigned
to π → π* transition while the latter to n → π* transi-
tion.[44] The band at 272 nm remains unchanged in the
spectra of the complexes. The absorption at 300 nm
undergoes a red shift upon complexation. This indicates
the donation of a lone pair of electrons to the metal ion
and hence the involvement of azomethine nitrogen in
coordination. No d–d transitions could be observed in
the case of [Co(L)(LH)]⋅H2O and [Ni(L)(DMF)]. Absence
of any electronic transition at longer wavelength indicates
a large crystal‐field splitting. A broad band in the elec-
tronic spectrum of [Cu(L)(H2O)] with peak maximum at
2
2
635 nm is assigned to the combination of B1g → A1g

CHIMMALAGI ET AL.
7 of 16
step, leaving behind stable CuO above 460 °C with a resi-
due of 12.2%.
of the refinement process are summarized in Table 1.
Perspective ORTEP views of [Co(L)(LH)]⋅H2O, [Ni(L)
(DMF)] and [Cu(L)(H2O)] along with the atom number-
ing schemes are depicted in Figures 1–3, respectively. A
summary of the bond lengths and bond angles is given in
Table 2. Relevant hydrogen bond interactions are
compiled in Table 3.
3.6 | Molecular Structures of [Co(L)(LH)]
⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)]
The asymmetric unit of [Co(L)(LH)]⋅H2O contains a
neutral [Co(L)(LH)] and one lattice‐held water of crystal-
lization. The two inequivalent ONO tridentate ligands
The solid‐state structures of all the complexes were
analysed using single‐crystal X‐ray studies. Details of
the crystallographic data collection and the parameters
TABLE 1 Crystal data and structure refinement details of complexes [Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)]
[Co(L)(LH)]⋅H2O
[Ni(L)(DMF)]
[Cu(L)(H2O)]
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C58H63CoF2N6O7
1053.07
C32H37FN4NiO4
619.36
C29H32CuFN3O4
569.11
100 (1)
100 (1)
100 (1)
1.54184
1.54184
1.54184
Monoclinic
P21/n
Monoclinic
P21/n
Monoclinic
P21/n
16.1762 (7)
23.4349 (9)
14.7596 (6)
90
13.58381 (15)
9.45350 (10)
23.9224 (2)
90
14.1773 (3)
9.43391 (18)
21.2097 (4)
90
b (Å)
c (Å)
α (°)
β (°)
98.447 (4)
90
92.6059 (9)
90
104.267 (2)
90
γ (°)
Volume (Å3)
5534.5 (4)
4
3068.81 (6)
4
2749.25 (9)
4
Z
Density (calculated) (g cm−3
)
1.264
1.341
1.375
Absorption coefficient (mm−1
)
2.950
1.312
1.503
F(000)
2216.0
1304.0
1188.0
Crystal size (mm3)
0.249 × 0.125 × 0.107
5.522 to 148.986
0.426 × 0.408 × 0.207
7.344 to 148.978
0.136 × 0.115 × 0.056
6.8 to 148.982
2θ range for data collection (°)
Index ranges
−20 ≤ h ≤ 13,
−27 ≤ k ≤ 29,
−17 ≤ l ≤ 18
−16 ≤ h ≤ 16,
−11 ≤ k ≤ 11,
−29 ≤ l ≤ 23
−17 ≤ h ≤ 17,
−8 ≤ k ≤ 11,
−26 ≤ l ≤ 19
Reflections collected
25 876
14 274
12 172
Independent reflections
11 321 [Rint = 0.0286,
Rsigma = 0.0366]
6250 [Rint = 0.0140,
Rsigma = 0.0167]
5623 [Rint = 0.0169,
Rsigma = 0.0230]
Completeness
99.9% (to θ = 74.493°)
Gaussian
99.9% (to θ = 74.489°)
Gaussian
100% (to θ = 74.491°)
Multi‐scan
Absorption correction
Data/restraints/parameters
Goodness‐of‐fit on F2
11321/0/699
6250/0/391
5623/0/361
1.020
1.014
1.001
Final R indexes [I ≥ 2σ(I)]
Final R indexes [all data]
Largest diff. peak and hole/e Å−3
R1 = 0.0414, wR2 = 0.0995
R1 = 0.0555, wR2 = 0.1072
0.33/−0.40
R1 = 0.0309, wR2 = 0.0836
R1 = 0.0323, wR2 = 0.0846
0.27/−0.43
R1 = 0.0308, wR2 = 0.0822
R1 = 0.0367, wR2 = 0.0856
0.31/−0.45

8 of 16
CHIMMALAGI ET AL.
(differing in their protonation state, Scheme 2) are coordi-
nated to Co(III) in an octahedral field preserving electro-
neutrality of the molecule as
a whole. In the
coordination sphere, both the ligands (L2− and LH−) are
almost perpendicular to each other. The mean planes of
the two ligands have a dihedral angle of 84.08°.
Azomethine nitrogens (N3 and N6) of the two ligands
reside trans to each other whereas the other two donor
sites ((O2, O5) and (O3, O6)) remain cis to each other.
The imino nitrogen atoms are axially positioned
(Co1―N3 1.877(15) Å and Co1―N6 1.855(15) Å) and four
oxygen atoms constitute the equatorial plane of the octa-
hedron. The bite angles for the ligands (L2− and LH−)
lie in the range 82.84–94.20°, indicating a distortion from
an ideal octahedral geometry,[49] with the trans‐donor
bond angles in the range 174.9(3)–177.7(3)° and the cis‐
donor bond angles in the range 87.0(3)–94.7(3)°. The
Co1―Ophenolate bond distances of 1.864(13) and
1.857(13) Å are for Co1―O3 and Co1―O6 bonds, respec-
tively. The Co1―O2amido and Co1―O5imidol bond dis-
tances are 1.957(13) and 1.930(13) Å, respectively.
By comparing the bond distances, the O5 atom of the
doubly deprotonated ligand (L2−) is found to be more
strongly bound to the Co(III) ion than the O2 atom of
the singly deprotonated ligand (LH−). The coexistence
of both the tautomeric forms of ligand within a complex
is substantiated by the bond distances in the region of
five‐membered chelate rings. The C14―O2amido (1.267(2)
Å) and C43―O5imidol (1.309(2) Å) differ in their lengths.
The N2―C14 (1.326(2) Å) is more of σ in character
compared to N5―C43 (1.306(2) Å). [Co(L)(LH)]⋅H2O
FIGURE 2 ORTEP projection of [Ni(L)(DMF)] showing 50%
probability ellipsoids
N2―H2⋅⋅⋅O7(W) (2.63(2) Å), N1―H1⋅⋅⋅O2 (2.73(2) Å)
and N4―H4⋅⋅⋅N5 (2.63(2) Å) and intermolecular O7(W)
―H7A⋅⋅⋅O4 (2.721(2) Å) and O7(W)―H7B⋅⋅⋅O5 (2.77(2)
Å) hydrogen bonds (Figure S14). Lattice‐held water mole-
cule is involved in three different hydrogen interactions.
O7 of lattice water acts as donor to the carbonyl oxygens
O4 and O5 of two adjacent molecules and also as an
acceptor to amide nitrogen N2. In addition, the complex
exhibits C―H⋅⋅⋅π and Cg⋅⋅⋅Cg interactions. Intramolecu-
lar C―H⋅⋅⋅π interactions are observed between tert‐butyl
hydrogens (H56A, H27A) and six‐membered chelate rings
is stabilized by
a
number of intramolecular
((Co1/O3/C29/C16/C15/N3),
(Co1/O6/C58/C45/C44/
N6)), respectively. An intermolecular stacking occurs
between the centroids of two phenyl rings ((C45/C46/
FIGURE 1 ORTEP projection (drawn at 30% probability level) of
[Co(L)(LH)]⋅H2O with partial atom labelling scheme
FIGURE 3 ORTEP projection of [Cu(L)(H2O)] showing 50%
probability ellipsoids

CHIMMALAGI ET AL.
9 of 16
TABLE 2 Selected bond lengths and angles of complexes [Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)]
[Co(L)(LH)]⋅H2O
Bond lengths (Å)
Co1–N6
1.855(15)
1.857(13)
1.864(13)
1.877(15)
1.930(13)
Co1–O2
O2–C14
O3–C29
O5–C43
O6–C58
1.957 (13)
1.267 (2)
1.314 (2)
1.309 (2)
1.320 (2)
N2–C14
N3–C15
N5–C43
N6–C44
1.326 (2)
Co1–O6
1.290 (2)
1.306 (2)
1.292 (2)
Co1–O3
Co1–N3
Co1–O5
Bond angles (°)
N6–Co1–O6
N6–Co1–O3
N6–Co1–N3
O6–Co1–N3
O3–Co1–N3
N6–Co1–O5
[Ni(L)(DMF)]
94.20 (6)
88.57 (6)
176.56 (7)
88.30 (6)
93.69 (6)
83.20 (6)
O6–Co1–O5
O3–Co1–O5
N3–Co1–O5
N6–Co1–O2
O6–Co1–O2
174.99 (6)
91.53 (6)
94.13 (6)
94.87 (6)
87.94 (6)
O3–Co1–O2
N3–Co1–O2
O5–Co1–O2
N6–Co1–O6
N6–Co1–O3
176.45 (5)
82.84 (6)
88.02 (5)
94.20 (6)
88.57 (6)
Bond lengths (Å)
Ni1–N3
1.805 (10)
1.809 (8)
1.832 (9)
Ni1–O4
O2–C14
N2–N3
1.895 (9)
1.303 (15)
1.406 (13)
N3–C15
O3–C29
O4–C30
1.300 (16)
1.314 (15)
1.259 (15)
Ni1–O3
Ni1–O2
Bond angles (°)
N3–Ni1–O3
N3–Ni1–O2
[Cu(L)(H2O)]
95.99 (4)
84.61 (4)
O3–Ni1–O2
N3–Ni1–O4
175.60 (4)
175.57 (4)
O3–Ni1–O4
O2–Ni1–O4
86.86 (4)
92.81 (4)
Bond lengths (Å)
Cu1–O3
1.869 (11)
1.897 (13)
1.9191 (11)
Cu1–O4
O2–C14
O3–C17
1.960 (12)
1.292 (19)
1.317 (19)
N2–C14
N2–N3
N3–C15
1.322 (2)
1.394 (17)
1.291 (2)
Cu1–N3
Cu1–O2
Bond angles (°)
O3–Cu1–N3
O3–Cu1–O2
94.61 (5)
N3–Cu1–O2
O3–Cu1–O4
82.36 (5)
93.49 (5)
N3–Cu1–O4
O2–Cu1–O4
171.78 (6)
89.57 (5)
176.70 (5)
C47/C52/C53/C58) and (C1―C6)) with a Cg⋅⋅⋅Cg distance
of 3.832 Å (Figure S15).
(84.61° in [Ni(L)(DMF)] and 82.36° in [Cu(L)(H2O)])
undergo compression whereas the N3―Ni1―O3 and
N3―Cu1―O3 angles (95.99° in [Ni(L)(DMF)] and
94.61° in [Cu(L)(H2O)]) undergo expansion from their
ideal 90° value,[50] and this behaviour is anticipated
because the compressed and expanded bite angles are
enclosed by five‐membered and six‐membered chelate
rings, respectively.
The molecular structure of [Ni(L)(DMF)] is stabi-
lized by an intramolecular hydrogen bonding
N1―H1A⋅⋅⋅N2 (2.71(14) Å) (Figure S16). [Cu(L)(H2O)]
is stabilized by strong intramolecular hydrogen bond
N1―H1B⋅⋅⋅N2 (2.63(18) Å). In an extended crystal
[Ni(L)(DMF)] and [Cu(L)(H2O)] have a distorted
square planar geometry around the Ni(II) and Cu(II)
ions in which the base plane is occupied by two oxygen
atoms and one nitrogen atom of the doubly
deprotonated ONO tridentate hydrazone (L2−), while
the fourth position is occupied by the oxygen atom
(O4) of coordinated DMF and water molecule in [Ni(L)
(DMF)] and [Cu(L)(H2O)], respectively. The square
planar geometry around Ni1 and Cu1 suffers some
distortion which is evident from the chelate bite angles
made by the ONO donor set of the ligand in both the
complexes. The N3―Ni1―O2 and N3―Cu1―O2 angles
structure,
an
intermolecular
hydrogen
bond

10 of 16
CHIMMALAGI ET AL.
TABLE 3 Hydrogen bond distances and bond angles in complexes [Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)]
D―H⋅⋅⋅A
d(D―H) (Å)
d(H―A) (Å)
d(D―A) (Å)
D―H―A (°)
[Co(L)(LH)]⋅H2O
N1―H1⋅⋅⋅O2
0.74 (2)
0.91 (3)
0.86 (3)
0.75 (3)
0.94 (3)
2.15 (2)
1.73 (3)
1.88 (3)
1.98 (3)
1.85 (3)
2.736 (2)
2.632 (2)
2.633 (2)
2.721 (2)
2.777 (2)
137 (2)
171 (2)
145 (2)
170 (3)
167 (3)
N2―H2⋅⋅⋅O7
N4―H4⋅⋅⋅N5
O7―H7A⋅⋅⋅O4#1
O7―H7B⋅⋅⋅O5#2
[Ni(L)(DMF)]
N1―H1A⋅⋅⋅N2
[Cu(L)(H2O)]
O4―H4C⋅⋅⋅O1#3
N1―H1B⋅⋅⋅N2
0.841 (18)
2.015 (18)
2.7158 (14)
140.3 (16)
0.85 (3)
0.84 (2)
1.74 (3)
2.5829 (16)
2.6355 (18)
177 (3)
146 (2)
1.90 (2)
3
Symmetry codes: #1: +X, +Y, −1 + Z; #2: 1 − X, 1 − Y, 1 − Z; #3: ½ + X,
/2 − Y, ½ + Z.
O4―H4C⋅⋅⋅O1 is formed by O4 of the coordinated water
molecule as a hydrogen bond donor towards the car-
bonyl oxygen atom O1 (2.58(16) Å) (Figure S17). In
addition, [Cu(L)(H2O)] is also stabilized by
intermolecular Cg⋅⋅⋅Cg interaction between centroid of
five‐membered chelate ring (Cu1/O2/C14/N2/N3) and
centroid of phenyl ring (C1―C6) with a Cg⋅⋅⋅Cg dis-
tance of 3.609 Å (Figure S18).
FIGURE 4 Hirshfeld surfaces mapped with dnorm of [Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)]
FIGURE 5 2D fingerprint plots of [Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)] showing all intermolecular interactions

CHIMMALAGI ET AL.
11 of 16
TABLE 4 Relative contributions of various intermolecular interactions to the Hirshfeld surface area of complexes [Co(L)(LH)]⋅H2O, [Ni(L)
(DMF)] and [Cu(L)(H2O)]
H⋅⋅⋅H
(%)
C⋅⋅⋅H/H⋅⋅⋅C
(%)
N⋅⋅⋅H/H⋅⋅⋅N
(%)
O⋅⋅⋅H/H⋅⋅⋅O
(%)
F⋅⋅⋅H/H⋅⋅⋅F
(%)
C⋅⋅⋅C
(%)
Other
(%)
Interaction
[Co(L)(LH)]⋅H2O
[Ni(L)(DMF)]
[Cu(L)(H2O)]
59.6
53.3
50.9
14.2
18.0
18.8
2.5
3.5
3.4
7.7
9.3
6.4
7.7
6.8
7.9
3.1
3.4
2.6
2.1
4.4
8.4
O⋅⋅⋅H/H⋅⋅⋅O interactions comprise 7.7, 9.3 and 6.4% of
the total HS area for each molecule of [Co(L)(LH)]
⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)], respectively. In
all the complexes, the spikes in the bottom left (donor)
and bottom right (acceptor) area of the fingerprint plots
represent the O⋅⋅⋅H and H⋅⋅⋅O interactions, respectively.
The ‘wings’ seen in the fingerprint plot of [Co(L)(LH]
⋅H2O belong to signature C―H⋅⋅⋅π interactions, with
the ‘wings’ in the lower right and lower left of the fin-
gerprint plot representing C―H⋅⋅⋅π acceptor and
C―H⋅⋅⋅π donor interactions, respectively. The propor-
tion of C⋅⋅⋅H/H⋅⋅⋅C interactions comprise 14.2, 18 and
18.8% of the total HS area for each molecule of
[Co(L)(LH)]⋅H2O, [Ni(L)(DMF)] and [Cu(L)(H2O)],
respectively. The C⋅⋅⋅H interactions in all the complexes
are mainly due to neighbouring tert‐butyl groups
3.7 | Hirshfeld Surface Analysis
HS analysis is a powerful technique for understanding
the nature of intermolecular interactions within a crys-
tal structure using a fingerprint plot.[51] HSs of the
metal complexes are illustrated in Figure 4 showing
surfaces mapped with dnorm. The information regarding
intermolecular interactions which are summarized in
Table 3 is visible by the spots on the Hirshfeld surfaces.
The dominant O⋅⋅⋅H interactions are highlighted by the
deep red area of dnorm surface. Light red spots are due
to N―H⋅⋅⋅O and O―H⋅⋅⋅O interactions. Other visible
spots in the surfaces are due to H⋅⋅⋅H contacts.[52] The
H⋅⋅⋅H, O⋅⋅⋅H/H⋅⋅⋅O, C⋅⋅⋅H/H⋅⋅⋅C and N⋅⋅⋅H/H⋅⋅⋅N inter-
molecular interactions appear as distinct spikes in the
2D fingerprint plot (Figure 5). The proportion of
FIGURE 6 Docking figures of LH2 and its metal complexes in COX‐2 protein cavity

12 of 16
CHIMMALAGI ET AL.
FIGURE 7 Docking figures of LH2 and its metal complexes in 5‐LOX protein cavity
pointing towards the aromatic ring of the phenol moi-
3.8 | Molecular Docking Studies
ety. No significant C―H⋅⋅⋅π acceptor or donor interac-
tions are observed for [Ni(L)(DMF)] and [Cu(L)(H2O)].
The majority of contacts present in all the three com-
plexes are due to H⋅⋅⋅H interactions. These interactions
make up 50.9 to 59.6% of the Hirshfeld surface of the
molecules. These contacts are mainly due to the tert‐
butyl groups present in the complexes.[53] The relative
contributions of various intermolecular interactions to
the Hirshfeld surface area are summarized in Table 4.
Molecular docking studies were performed to investigate
interactions between the synthesized compounds and
pro‐inflammatory targets COX‐2 and 5‐LOX enzymes.
These are the key enzymes involved in regulating the
AA metabolic pathway and the production of pro‐
inflammatory prostaglandins and leukotrienes. There-
fore, they have been validated as selective targets of
anti‐inflammatory drugs. LH2 and its Co(III), Ni(II)
TABLE 5 Molecular docking results of LH2 and its complexes in COX‐2 and 5‐LOX protein cavities
COX‐2 5‐LOX
No. of
No. of
Binding energy hydrogen Interacting Distance Binding energy hydrogen Interacting Distance
Molecule
(kcal mol−1
)
bonds
residues
(Å)
(kcal mol−1
)
bonds
residues
(Å)
LH2
−10.1
1
Cys32
2.24
−9.3
2
Glu287
Leu288
2.77
2.78
[Co(L)(LH)]⋅H2O −12.6
1
Arg46
2.54
−10.8
5
Arg246
Glu287
2.21, 2.21
1.84, 2.45
2.83
[Ni(L)(DMF)]
[Cu(L)(H2O)]
−10.5
−11.8
1
1
Asn19
Asn19
2.98
2.8
−9.6
−9.4
1
1
Asn328
Ala453
2.50
3.62

CHIMMALAGI ET AL.
13 of 16
The docked poses in COX‐2 protein, displayed in
Figure 6, reveal that LH2 displays hydrogen bond con-
tact with Cys32 amino acid (>N―H⋅⋅⋅O=C< (amino
acid), 2.24 Å). In addition, it forms some weak hydro-
phobic interactions with Pro139, Leu138, Lys 454,
Cys21 and Arg455 amino acids. The most notable inter-
actions were observed in the case of the Co(III) complex
with the highest binding affinity of −12.6 kcal mol−1
comprising hydrogen bonding interactions with Arg46
amino acid (>N―H⋅⋅⋅O=C< (amino acid), 2.54 Å) and
hydrophobic interactions with Arg29 (π–σ, 3.86 Å) and
Tyr108 (π–σ (3.77 Å) and π–π (5.1 Å)). The Ni(II) com-
plex acts as hydrogen bonding acceptor for Asn19
(>C=O⋅⋅⋅H―N (amino acid), 2.98 Å) and Gln313(C6H4
π cloud⋅⋅⋅H―N(amino acid), 3.24 Å). The carbonyl oxy-
gen of the Cu(II) complex is a classical hydrogen bond
acceptor for Asn19 (>C=O⋅⋅⋅H―N (amino acid),
2.8 Å). In addition, van der Waals interactions with
Asp144 (3.7 Å) and weak hydrophobic interactions
(4.6–5.0 Å) are observed in the docked model for the
Cu(II) complex.
The docked poses in 5‐LOX protein, displayed in
Figure 7, reveal that LH2 forms a hydrogen bonding
interaction with the protein with 9.3 kcal mol−1. The
Co(III) complex exhibited highest binding affinity of
−10.8 kcal mol−1, which stems from the excellent five
hydrogen bonding contacts made with the protein.
The carbonyl oxygens of the amide groups act as
hydrogen bond acceptors for Arg246 (>C=O⋅⋅⋅H―N<
(amino acid), 1.84–2.45 Å) and hydrogen bond donors
for Glu287 (>N―H⋅⋅⋅O=C< (amino acid), 2.83 Å).
The higher affinity of the Co(III) complex is attributed
to the octahedral structure. The Ni(II) and Cu(II) com-
plexes show binding affinity of −9.6 and
FIGURE 8 Effect of LH2 and its metal complexes on inhibition of
carrageenan‐induced rat paw oedema at a dose of (a) 5 mg kg−1 and
(b) 10 mg kg−1
and Cu(II) complexes were docked at the active sites of
COX‐2 and 5‐LOX proteins, and various interactions
have been laid out.
TABLE 6 Anti‐inflammatory activity of LH2 and its metal complexesa
Paw volume in ml (% oedema inhibition)
Groups
0.5 h
1 h
3 h
5 h
Control
1.097 0.044
1.197 0.014
1.170 0.062
1.343 0.028
Diclofenac (10 mg kg−1
)
0.030 0.027*** (97.27%) 0.030 0.027*** (97.49%) 0.108 0.040*** (90.74%) 0.025 0.013*** (98.14%)
LH2 (5 mg kg−1
LH2 (10 mg kg−1
[Co(L)(LH)]⋅H2O (5 mg kg−1
[Co(L)(LH)]⋅H2O (10 mg kg−1
[Ni(L)(DMF)] (5 mg kg−1
[Ni(L)(DMF)] (10 mg kg−1
[Cu(L)(H2O)] (5 mg kg−1
[Cu(L)(H2O)] (10 mg kg−1
)
0.330 0.037*** (69.92%) 0.273 0.030*** (77.19%) 1.233 0.055 (5.38%)
0.233 0.031*** (78.73%) 0.320 0.019*** (73.27%) 0.960 0.066 (17.95%)
0.323 0.031*** (75.93%)
0.137 0.042*** (89.82%)
)
)
0.157 0.015*** (85.69%) 0.297 0.076*** (75.19%) 0.357 0.032*** (69.51%) 0.233 0.033*** (82.63%)
0.073 0.039*** (93.32%) 0.097 0.002*** (91.92%) 0.287 0.030*** (75.50%) 0.063 0.080*** (95.28%)
0.173 0.006*** (84.23%) 0.230 0.061*** (80.79%) 0.583 0.052*** (50.15%) 0.390 0.044*** (70.96%)
0.157 0.013*** (85.72%) 0.070 0.084*** (94.15%) 0.357 0.063*** (69.51%) 0.0967 0.056*** (92.80%)
)
)
)
)
0.250 0.041*** (84.23%) 0.217 0.043*** (81.87%) 1.093 0.046 (6.58%)
0.187 0.027*** (82.98%) 0.050 0.004*** (95.82%) 0.883 0.100 (24.50%)
0.243 0.035*** (81.88%)
0.090 0.050*** (93.30%)
)
aResults expressed in mean SEM (n = 6); ANOVA followed by Dunnett's test.
***p < 0.001, when compared to control group.

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CHIMMALAGI ET AL.
−9.4 kcal mol−1, respectively. The former is in hydro-
gen bonding contacts with Asn328 (>C=O⋅⋅⋅H―N
(amino acid), 2.50 Å) and the latter forms a non‐classi-
cal hydrogen bond with Ala453. The molecular docking
results lead to the conclusion that LH2 and its com-
plexes effectively bind to the proteins which substanti-
ate the observed activity with the Co(III) complex
showing the highest tendency towards protein binding.
Molecular docking results of all the synthesized anti‐
inflammatory models are listed in Table 5.
Co(III) complex. While in the Ni(II) and Cu(II)
complexes, ligand coordinates to metal through imidol
tautomeric form resulting in distorted square planar
geometry. HS analysis was undertaken to explore the
detailed intermolecular contacts. Fingerprint plots
generated from HSs are employed for analysing and
comparing intermolecular interactions. In vivo anti‐
inflammatory activities of the free ligand and the
complexes revealed that complexation enhanced the
anti‐inflammatory potency of the ligand. Molecular
docking analyses of the compounds with COX‐2 and
5‐LOX proteins demonstrated a good fit of these
compounds in both the protein cavities. The Co(III)
complex showed a best fit pose in the protein cavities
with the lowest binding energy of −12.6 (in COX‐2)
and −10.8 kcal mol−1 (in 5‐LOX). The results of docking
studies strongly correlate with in vivo anti‐inflammatory
activity results.
3.9 | Anti‐inflammatory Activity
All the synthesized compounds were screened for their
in vivo anti‐inflammatory activity using the carra-
geenan‐induced rat paw oedema model which is a
well‐known model of acute inflammation that includes
biphasic phases and a number of mediators participate
in the inflammatory response evoked by carra-
geenan.[54] The early phase of the inflammatory
response is presumably mediated by the release of his-
tamine and 5‐hydroxytryptamine for 90 min followed
by the kinin‐mediated increased vascular permeability
up to 2.5 h. The later phase involves neutrophil infiltra-
tion and the release of prostaglandins and prostaglan-
ACKNOWLEDGEMENTS
The authors thank USIC, Karnatak University, Dharwad
for IR and electronic spectral analyses, CDRI Lucknow for
ESI mass analysis, and Indian Institute of Science,
Bangalore and IIT Bombay for recording NMR and ESR
spectra. The authors sincerely acknowledge the financial
support of UGC, New Delhi under UPE‐FAR‐I Program
(no. 14‐3/2012). One of the authors (G.H.C.) is grateful to
UGC for awarding a RFSMS fellowship.
din‐associated leukocytes into the site of oedema[55]
;
thus the feet rapidly became swollen, reaching close
to the control's level by 3.5 h. The experimental results
demonstrated that LH2 and its metal complexes signifi-
cantly reduced both phases of the carrageenan‐induced
oedema (Figure 8). Comparison of LH2 with its com-
plexes indicates that the metal complexes exhibit better
activity than the ligand itself. This is due to the
increased lipophilic nature of the complexes.[56,57]
Among the complexes, [Co(L)(LH)]⋅H2O exhibited sig-
nificant inhibition of paw oedema (95.5%) at a concen-
tration of 10 mg kg−1. Anti‐inflammatory activity of the
synthesized metal complexes is greater as compared to
corresponding metal salts. The results indicating
oedema volume and percentage inhibition of inflamma-
tion of synthesized compounds and metal salts at vari-
ous time intervals are summarized in Table 6 and
Table S1, respectively.
ORCID
Kalagouda B. Gudasi
http://orcid.org/0000-0002-0063-
7656
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How to cite this article: Chimmalagi GH,
Kendur U, Patil SM, et al. Mononuclear Co(III),
Ni(II) and Cu(II) complexes of tridentate di‐tert‐
butylphenylhydrazone: Synthesis, characterization,
X‐ray crystal structures, Hirshfeld surface analysis,
molecular docking and in vivo anti‐inflammatory
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