Mononuclear late first row transition metal complexes of ONO donor hydrazone ligand: Synthesis, characterization, crystallographic insight, in vivo and in vitro anti-inflammatory activity

Article abstract of DOI:10.1016/j.molstruc.2017.10.022

Air and moisture stable coordination compounds of late first row transition metal ions, viz., Co(II), Ni(II), Cu(II) and Zn(II) with a newly designed ligand, (E)-2-amino-N'-(1-(2-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)ethylidene)benzohydrazide (H₂L) were prepared and extensively characterized using various spectro-analytical techniques. The ligand acts both in mono as well as doubly deprotonated manner. The ligand to metal stoichiometry was found to be 1:2 in case of complexes using chloride salts, whereas 1:1 in case of copper (II) complex using its acetate salt. The molecular structures of H₂L, nickel and copper complexes were unambiguously determined by single-crystal X-ray diffraction studies reveal that H₂L exists in a zwitterionic form while copper complex has copper centre in a distorted square planar environment. On the other hand, cobalt, nickel and zinc complexes display distorted octahedral coordination around the metal ion. In case of [Ni(HL)₂].H₂O, intramolecular C–H?π stacking interaction were observed between the centroid of five membered chelate ring and phenyl proton C5–H5 and intermolecular C–H?π stacking interaction between the centroid of phenyl ring, dehydroacetic acid (DHA) ring and phenyl protons. The [Cu(L)DMF] complex is stabilized by intramolecular hydrogen bonding N1H?N2 and by intermolecular hydrogen bonding N1H?O4. Intermolecular interactions were investigated by Hirshfeld surfaces. Further, H₂L and its metal complexes were screened for their in vivo and in vitro anti-inflammatory activities. The activity of the ligand has enhanced on coordination with transition metals. The tested compounds have shown excellent activity, which is almost equipotent to the standard used in the study.

Full text of DOI:10.1016/j.molstruc.2017.10.022

Accepted Manuscript
Mononuclear late first row transition metal complexes of ONO donor hydrazone
ligand: Synthesis, characterization, crystallographic insight, in vivo and in vitro anti-
inflammatory activity
Umashri Kendur, Geeta H. Chimmalagi, Sunil M. Patil, Kalagouda B. Gudasi,
Christopher S. Frampton, Chandrashekhar V. Mangannavar, Iranna S. Muchchandi
PII:
S0022-2860(17)31353-4
10.1016/j.molstruc.2017.10.022
MOLSTR 24393
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 13 July 2017
Revised Date: 5 October 2017
Accepted Date: 6 October 2017
Please cite this article as: U. Kendur, G.H. Chimmalagi, S.M. Patil, K.B. Gudasi, C.S. Frampton,
C.V. Mangannavar, I.S. Muchchandi, Mononuclear late first row transition metal complexes of ONO
donor hydrazone ligand: Synthesis, characterization, crystallographic insight, in vivo and in vitro anti-
inflammatory activity, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.10.022.
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ACCEPTED MANUSCRIPT
Graphical abstract
Hirshfeld surfaces
80
60
40
20
0
[Cu(L)2.H2O]
[Zn(HL)2]
[Cu(HL)2]
[Ni(HL)2].H2O
[Co(HL)2]
H2L
Aceclofenac
Concentration (µg/ml)
Anti-inflammatory activity
ORTEP projections

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Mononuclear late first row transition metal complexes of ONO donor hydrazone ligand:
Synthesis, characterization, crystallographic insight, in vivo and in vitro anti-inflammatory
activity
a
a
a
a,*
Umashri Kendur , Geeta H. Chimmalagi , Sunil M. Patil , Kalagouda B. Gudasi
,
Christopher S. Frampton b , Chandrashekhar V. Mangannavar c, Iranna S. Muchchandi c
a Department of studies in Chemistry, Karnatak University, Dharwad-580003, Karnataka, India
b
Institute of Materials & Manufacturing, Wolfson Centre for Materials Processing, Brunel
University, London, Uxbridge, United Kingdom
c H.S.K. College of Pharmacy, Bagalkot-587101, Karnataka, India
Abstract
Air and moisture stable coordination compounds of late first row transition metal ions, viz.,
Co(II), Ni(II), Cu(II) and Zn(II) with a newly designed ligand, (E)-2-amino-N'-(1-(2-hydroxy-6-
methyl-4-oxo-4H-pyran-3-yl)ethylidene)benzohydrazide (H2L) were prepared and extensively
characterized using various spectro-analytical techniques. The ligand acts both in mono as well
as doubly deprotonated manner. The ligand to metal stoichiometry was found to be 1:2 in case of
complexes using chloride salts, whereas 1:1 in case of copper (II) complex using its acetate salt.
The molecular structures of H2L, nickel and copper complexes were unambiguously determined
by single-crystal X-ray diffraction studies reveal that H2L exists in a zwitterionic form while
copper complex has copper centre in a distorted square planar environment. On the other hand,
cobalt, nickel and zinc complexes display distorted octahedral coordination around the metal ion.
In case of [Ni(HL)2].H2O, intramolecular C-H···π stacking interaction were observed between
the centroid of five membered chelate ring and phenyl proton C(5)-H(5) and intermolecular C-
H···π stacking interaction between the centroid of phenyl ring, dehydroacetic acid (DHA) ring
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and phenyl protons. The [Cu(L)DMF] complex is stabilized by intramolecular hydrogen bonding
N(1)H···N(2) and by intermolecular hydrogen bonding N(1)H···O(4). Intermolecular
interactions were investigated by Hirshfeld surfaces. Further, H2L and its metal complexes were
screened for their in vivo and in vitro anti-inflammatory activities. The activity of the ligand has
enhanced on coordination with transition metals. The tested compounds have shown excellent
activity, which is almost equipotent to the standard used in the study.
Keywords
•
(E)-2-amino-N'-(1-(2-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)ethylidene)
benzohydrazide
•
•
•
•
•
Transition metal complexes
Single crystal X-ray diffraction study
Mono-deprotonated and Doubly deprotonated
Hirshfeld surface analysis
Anti-inflammatory activity
1. Introduction
The coordination chemistry of N-acyl hydrazone (NAH) ligands has captivated
considerable assiduity in past few decades, due to their chelating capability, structural flexibility,
variable bonding modes towards transition metal ions and a wide range of biological applications
[1-4]. NAH has also been used as the bedrock for designing new analgesic and anti-
inflammatory compounds, because the NAH subunit is a primal pharmacophore for binding to
and inhibiting cyclooxygenases (COXs) [5]. A number of transition metal complexes of NAH
have been reported to exhibit DNA interactions, as well as antimicrobial and antitumor
properties [6,7,8a]. The biological activity associated with these compounds is attributed to the
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presence of –CONHN=CH– moiety. On coordination, the activity of hydrazone increases.
Moreover, coordination suppresses the polarity of the metal ion because of the partial sharing of
its positive charge with the donor atom inside the chelate ring system, formed during
coordination. This process consecutively increases the lipophilic nature of the central metal
atom/ion, which in turn favours its pervasion more efficiently through the lipid layers of the
microorganism [8b,c].
Further, transition metal complexes of NAH have also shown high catalytic activities in
various chemical reactions, to name a few, epoxidation of olefins [9-10], polymerization of
ethylene [11], and transamidation of carboxamides with amines [12], etc. Aggoun et al.,
described the electrocatalytic behaviour of the copper complex of hydrazone ligand prepared by
condensation of dehydroacetic acid on 1,2-diaminopropane towards electro-reduction of alkyl
and aryl halides [13].
Dehydroacetic acid (DHA), a pyranone derivative is a herculean material which is
involved in the synthesis of many heterocyclic compounds. It is prepared by the base-catalyzed
(tert-amines; imidazole; 1,4-diazabicyclo[2.2.2]octane (DABCO); pyridine) dimerization of
diketene. These compounds have occupied a dominating position in coordination chemistry that
explores therapeutic and pharmacological activities [14,15]. Studies on metal chelates with
Schiff base of dehydroacetic acid have been reported due to their excellent chelating capacity in
modern coordination chemistry. Recently, Edward et.al., reported the design and synthesis of a
novel class of diaryl heterocyclic with a central six-membered lactone (pyran-2-one) ring which
exhibited good in vitro COX-2 inhibitory potency and selectivity [16a-h]. Cobalt, nickel, copper
and zinc being bio-essential elements, it is conceived that their complexes may find more
applications in numerous biological processes [17].
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Considering the above-mentioned facts and in continuation of our ongoing research on
the study of pharmacological properties of transition metal complexes, the present work was
undertaken as an attempt to explore the structural relationship of DHA derived hydrazone with
late first row transition metals. Here we report the synthesis and characterization of the DHA
derived ligand, its transition metal complexes. A preliminary screening for in vivo and in vitro
anti-inflammatory activity of these new compounds was also studied.
2. Experimental
2.1. General procedures
All the reagents used in this study were purchased from Sigma Aldrich and used as
rendered. The synthesis of precursor, o-aminobenzoylhydrazide the precursor was synthesized
according to the procedure reported earlier [18, 19]. Infrared spectra of the ligand and its metal
complexes were recorded in the region 4000–400 cm-1 on a Nicolet 170 SX FT-IR spectrometer
1
(KBr disc matrix). The H NMR and 13C NMR spectra were recorded on a Bruker AV400 II
spectrometer at 400 MHz in DMSO-d6/CDCl3 at room temperature using TMS as an internal
reference. All the compounds were analyzed for carbon, hydrogen and nitrogen using a Thermo
quest elemental analyzer and metal complexes were analyzed for their metal content by standard
methods [20]. The UV–Vis spectra of all the compounds in DMF were recorded on a Varian
Cary 50 Bio UV–Vis spectrophotometer. Thermograms of the metal complexes were recorded in
nitrogen atmosphere on a SDT Q600 Analyzer, keeping the final temperature at 1000°C with a
heating rate of 10°C/min heating rate. The molar conductivity measurements of 1 mM solutions
in DMF were carried out on equiptronics EQ-665 conductivity bridge. EPR spectra were
recorded both at room temperature (300K) and liquid nitrogen temperature (77K) on a Varian E-
112 X-band spectrometer using TCNE (tetracyanoethylene) as ‘g’ marker (g = 2.00277) at a
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frequency of 9.65 GHz under the magnetic strength of 3000 G. The EI mass spectrum of the
ligand was obtained with a Shimadzu GCMS-QP2010S spectrometer. The ESI mass spectral data
for all the complexes were obtained from Agilent Technologies India Pvt. Ltd. The X-ray
diffraction data of nickel complex were collected at 293 K on a Bruker SMART APEX2 CCD
area-detector diffractometer using a graphite monochromater Mo-Kꢀ (λ = 0.71073 Å) radiation
source. The frames were integrated with the Bruker SAINT Software package using a narrow-
frame algorithm. In the absence of anomalous scattering, Friedel pairs were merged. The H
atoms were all located in a difference map, but those attached to carbon atoms were repositioned
geometrically. The H atoms were initially refined with soft restraints on the bond lengths and
angles to regularize their geometry, after which the positions were refined with riding constraints
[21]. All non-hydrogen atoms were refined anisotropically. Structure solution and refinement
were performed using Crystals [22]. Molecular graphics were generated using Cameron [23];
structure figures were generated with ORTEP-III [24a]. The X-ray diffraction data of ligand and
copper complex were collected on an Oxford Diffraction (Agilent Technologies), SuperNova X-
ray diffractometer equipped with an Oxford Cryosystems Cobra Low temperature device using
Cu-Kꢀ radiation (λ = 1.54184 & 1.54178 Å ) from a Super Nova Cu X-ray micro source and
focusing mirror optics. The structures were solved by direct methods and refined against F2 by
fullmatrix least-squares using the program SHELXTL [24b].
2.2. Synthesis of (E)-2-amino-N'-(1-(2-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)ethylidene)benzo
hydrazide (H2L)
A methanolic solution of o-aminobenzoylhydrazide (2 g; 0.01 mol) was added dropwise
to a solution of 2-acetyl-5-hydroxy-3-oxo-4-hexenoic acid δ-lactone (2.23 g; 0.01 mol) in
methanol (20 mL) and the mixture was stirred for 0.5 h at room temperature on mechanical
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shaker. The progress of the reaction was monitored using TLC. The precipitate formed was
filtered off, washed with methanol and then dried in vacuo. Pale yellow crystals obtained from
slow evaporation of its methanolic solution were suitable for single crystal X-ray diffraction
(SC-XRD) analysis. Schematic representation for the synthesis of ligand is given in (Scheme 1).
Yield: 87 %; m.p.: 193°C; Color: Pale yellow .Anal. Calcd for C15H15N3O4 (%): C, 59.79; H,
5.02; N, 13.95. Found (%): C, 59.82; H, 4.98; N, 13.92. IR (cm-1): ꢁ = 3323 (m, NH2,sym.);
3357 (m, NH2 asym.); 3447 (m, N-H); 1675 (s, pyranone C=O); 1645 (s, amide C=O); 1619 (m,
C=N).1H NMR (DMSO-d6, 400 MHz) ꢂ(ppm) :15.90 (s, 1H, O3H), 8.065 (s, 2H, N1H2), 5.838
(s, 1H, C11H), 6.772 (d, J=8.4Hz, 1H, C4H). 13C NMR (DMSO-d6, 100 MHz) ꢂ(ppm): 162.9
(azomethine C8=N), 170.8 (C7=O), 106.2 (C11H), 16.5 (C14H3), 19.2 (C15H3). ꢃmax (nm): 270
π → π*, 351
n → π*.
2.3. Synthesis of metal complexes
Methanolic solutions of NiCl2.6H2O (0.19 g, 0.8 mmol), CuCl2.2H2O (0.14 g, 0.8 mmol)
and Cu(CH3COO)2.H2O (0.16 g, 1.6 mmol) were added dropwise to the methanolic solution of
H2L (0.50 g, 1.6 mmol) and stirred for 2 h on mechanical shaker. Co(II) and Zn(II) complexes
were obtained by refluxing methanolic solution of CoCl2.6H2O (0.19 g, 0.8 mmol) and
anhydrous ZnCl2 (0.11 g, 0.8 mmol) with methanolic solution of H2L for 3 h on water bath. The
products obtained were filtered off, washed with hot methanol and dried in vacuo. Slow
evaporation of methanolic solution of Ni(II) complex and DMF solution of Cu (II) complex
which was obtained by using Cu(CH3COO)2.H2O salt yielded dark green color single crystals at
room temperature suitable for XRD study. Attempts to grow single crystals of the remaining
metal complexes were unsuccessful for now.
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2.3.1. [Co(HL)2]
Yield: 52.2 %, Color: purple. Anal. Calcd for C30H28CoN6O8 (%): C, 54.63; H, 4.28; N, 12.74;
Co, 8.94. Found (%): C, 54.68; H,4.25; N, 12.78; Co, 8.90. IR (cm-1): ꢁ = 1674 (s, pyranone
C=O); 1620 (s, amide C=O); 1571 (m, C=N). ꢃmax (nm): 273 π → π*, 373 n → π*, 610 & 674
(d–d transitions). Molar conductivity : 3.06 ohm-1 cm2 mol-1
2.3.2. [Ni(HL)2].H2O
Yield: 53.6 %, Color: green. Anal. Calcd for C30H30N6NiO9 (%): C, 53.20; H, 4.46; N, 12.41; Ni,
8.67. Found (%): C, 53.16; H, 4.49; N, 12.39; Ni, 8.69. IR (cm-1):ꢁ = 1670 (s, pyranone C=O);
1615 (s, amide C=O); 1574 (m, C=N). ꢃmax(nm): 274 π → π*, 395 n → π*, 554 & 940 (d–d
transitions). Molar conductivity: 2.54 ohm-1 cm2 mol-1
2.3.3. [Cu(HL)2]
Yield: 52.8 %, Color: dark green. Anal. Calcd for C30H28CuN6O8 (%): C, 54.26; H, 4.25; N,
12.65; Cu, 9.57. Found(%): C, 54.23; H,4.21; N, 12.62; Co, 9.53. IR (cm-1): ꢁ = 1677 (s,
pyranone C=O); 1637 (s, amide C=O); 1599 (m, C=N). ꢃmax(nm): 275 π → π*, 386 n → π*, 714
(d–d transitions). Molar conductivity: 1.98 ohm-1 cm2 mol-1
2.3.4. [Zn(HL)2]
Yield: 44.8 %, Color: Yellow. Anal. Calcd for C30H28ZnN6O8 (%): C, 51.54; H, 4.97 ; N, 13.03;
Zn, 3.38. Found (%): C, 51.59; H, 4.95 ; N, 13.07; Zn, 3.41. IR (cm-1): ꢁ = 1682 (s, pyranone
1
C=O); 1648 (m, amide C=O); 1590 (m, C=N). H NMR (DMSO-d6, 400 MHz) ꢂ(ppm) : 6.935
(s, 2H, N1H2), 5.737 (s, 1H, C11H), 6.633 (d, J=8.4Hz, 1H, C4H). 13C NMR (DMSO-d6, 100
MHz) ꢂ(ppm): 160.1(azomethine C8=N), 169.6 (C7=O), 107.2 (C11H), 18.9 (C14H3), 19.4
(C15H3).ꢃmax(nm): 276 π → π*, 384 n → π*. Molar conductivity: 3.19 ohm-1 cm2 mol-1
2.3.5. [Cu(L)(H2O)]
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Yield: 63.4 %, Color: green. Anal. Calcd for C15H15CuN3O5 (%): C, 47.31; H, 3.97; N, 11.03;
Cu, 16.69. Found(%):C, 47.28; H, 4.00; N, 11.09; Cu, 16.72. IR (cm-1): ꢁ = 1670 (s, pyranone
C=O); 1608 (s, amide C=O); 1575 (m, C=N); 1357 (m, C-O). ꢃmax(nm): 273 π → π*, 390 n →
π*, 630 & 635 (d–d transitions). Molar conductivity: 1.98 ohm-1 cm2 mol-1
2.4. Pharmacology
Ligand and its metal complexes were evaluated for in vivo anti-inflammatory activity by
carrageenan-induced rat paw edema model [25,26] and for in vitro anti-inflammatory activity by
protein denaturation method [27].
2.4.1. In vivo anti-inflammatory activity: Carrageenan-induced rat paw edema model
In vivo anti-inflammatory activity was measured as described by Winter et al. [25]. One
hour after the administration of the test compounds, the paw edema was induced by injecting 1%
carrageenan lambda (a pro-inflammatory agent; prepared in 0.9% NaCl) hypodermically in the
sub-plantar region of right hind paw. The test compounds were suspended in 0.5% sodium
carboxy methyl cellulose (Na–CMC) and administered at dose of 5 and 10 mg/kg per body
weight and declofenac, an anti-inflammatory drug in use was administered orally at a dose of 10
mg/kg, p.o. as the standard. The control group received 0.5% Na–CMC in distilled water. The
paw volume was measured at different intervals of time (0.5, 1, 3 and 5 h) using a digital
plethysmometer (UGO Basile, Italy), before and after injection of 1% carrageenan lambda.
Results were expressed in terms of edema volume as Mean ± SEM and mean percent inhibition
according to the following equation,
Edema Volume = Vt - Vc
where, Vt – Paw volume in mL, at time t, after carrageenan administration.
Vc – Paw volume in mL, before carrageenan administration
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Percent inhibition = Ec - Et / Ec
where, Ec – Edema volume of rat of control group, at time t
Et – Edema volume of rat paw, at time t
2.4.2. In vitro anti-inflammatory activity: Egg albumin denaturation method
A mixture contained 0.2 mL of egg albumin (from fresh hen's egg), 2.8 mL of phosphate
buffered saline (PBS, pH 6.4) and 2 mL of various concentrations of ligand and its metal
complexes so that final concentrations become 31.25, 62.5, 125, 250, 500,1000 µg/mL and
similar volume of double-distilled water served as control. Later the mixtures were incubated at
(37 ± 2) °C in an incubator (Bio-technics, India) for about 15 min and then heated at 70 °C for 5
min. After cooling, their absorbance was measured at 660 nm (UV-1800 Spectrophotometer,
SHIMADZU) by using vehicle as blank. Aceclofenac sodium was used as reference drug and
treated similarly for determination of absorbance. The percentage inhibition of protein
denaturation was calculated by using the following equation,
% Inhibition = 100 × (Abs of control - Abs of sample) / Abs of control.
2.5. Hirshfeld surface analysis
Hirshfeld surfaces (HSs) and 2D fingerprint plots (FPs) were generated using Crystal
Explorer 3.1 [28] based on results of SC-XRD studies. The function dnorm is a ratio encompassing
the distances of any surface point to the nearest interior (di) and exterior (de) atom and the van
der Waals radii of the atoms [29 a,b]. The negative value of dnorm indicates the sum of di and de is
shorter than the sum of the relevant van der Waals radii, which is measured to be a closest
contact and is visualized as red colour in HSs. The white colour represents intermolecular
distances close to van der Waals contacts with dnorm equal to zero, whereas contacts longer than
the sum of van der Waals radii with positive dnorm values are coloured with blue. A plot of di
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versus de is a 2D fingerprint plot which recognizes the existence of different types of
intermolecular interactions.
3. Results and discussion
3.1. Single crystal X-ray diffraction and Hirshfeld surface analyses of H2L, [Ni(HL)2].H2O and
[Cu(L)DMF]
Summaries of the crystallographic data, selected bond lengths and bond angles of H2L,
[Ni(HL)2].H2O and [Cu(L)DMF] are given in Tables S1 and 1, respectively. ORTEP
representations (asymmetric unit) of the same showing 50% displacement ellipsoids are shown in
Figures 1-3.
The single-crystal X-ray structure of H2L was determined at 100 K and is shown to be
monoclinic with space group P21/n. Asymmetric unit of H2L consists of 3 molecules namely A,
B and C with a small trace of crystal held water molecule, (O1W) refines to just under 5% per 3
molecules of H2L. These three molecules A, B and C differ marginally from one another in their
bond distances and bond angles. Such crystallographically independent and chemically similar
molecules are known as bond stretch isomers [30 a,b]. All the three conformers of the ligand is
found to exist in zwitterionic form and formally a neutral species as evident by presence of acidic
proton (H3AB,H3BB,H3CB) on N3A,N3B,N3C respectively, rather than O2A,O2B,O2C where
there exists a single bond character for C11-O2 and double bond character for C8-N3 [30c].
Azomethine and ˃C=O (mean of O1-C7-N2-N3-C8) form a dihedral angle of 24.03° with the
phenyl ring (mean of C1–C6) and 17.09° with DHA ring (mean C10-C11-C12-C13-O3-C15)
respectively. Furthermore, the azomethine ˃C=N bond distance (1.309(16) Å) agrees well with
the values for double bond character confirming the formation of imine bond. The torsion angles
-3.92(17) to 10.8(17)° exhibited by N3–N2–C7–O1, -5.41(17) to 11.49(17)° by N3-C8-C10-C11
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and -4.19(18) to 2.10(18)° by C8–C10–C11–O2 indicate that N3,O1 ; N3,C11 and C8,O2 are cis
to each other respectively, while the torsion angle -159.44(12) to 158.01(11)° exhibited by C7–
N2–N3–C8 indicates that C7 and C8 are trans to each other. The structure of H2L is stabilized
by intramolecular N3-H···O2 (2.481(13) Å); N1-H···O1(2.712(15) Å) and intermolecular N1-
H···O2 (2.909(14) Å); N2-H···O4 (2.916(13) Å) hydrogen bonding (Table 2).
The crystal structure of [Ni(HL)2].H2O contains 0.5 molecule in the asymmetric unit and
structure dimerises across the inversion center. Hence, results in a pair of tridentate ligands
coordinated in mer fashion, through an amide carbonyl oxygen, azomethine nitrogen and oxygen
of -OH (pyranone ring) via deprotonation. According to International Tables for
Crystallography, [Ni(HL)2].H2O having space group P31, the nickel ion occupies a general
position 3a. The bonds Ni–O1(2.082(2) Å), Ni–N3 (2.022(2) Å) and Ni–O2 (1.987(2) Å)
forming one five-membered CN2ONi and other six-membered C3NONi chelate rings with bite
angles of 78.75° and 88.08°, respectively, suggest distortion from an ideal octahedral geometry
[31]. The bond lengths in [Ni(HL)2].H2O for: C7–O1 = 1.240(4), N3–C8= 1.289(4) and C11–
O2= 1.276(4) Å, which are longer or shorter than those of the corresponding distances in the free
ligand H2L, C7–O1 = 1.231(15), N3–C8= 1.309(16) and C11–O2= 1.264(15) Å is due to the
coordination of ligand to the central Ni(II) atom. The crystal is stabilized by intramolecular N1-
H···Ow (3.207(8) Å) and intermolecular N1-H···O4 (3.312(5) Å); N2-H···O4 (2.978(4) Å); Ow-
H···O2 (2.853(4) Å) hydrogen bonding (Table 2). In addition, the molecule is stabilized by
intramolecular C-H···π stacking interactions between the centroid of five membered chelate ring
and phenyl proton C5-H5, with the contact distance of 3.400 Å and two intermolecular C-H···π
stacking interactions, one between the centroid of phenyl ring and proton of DHA ring C12-
H14A, another between the centroid of DHA ring and phenyl proton C4-H4, with the contact
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distances of 3.685 and 3.228 Å respectively (Fig. S1). The C-H···π interactions are important
noncovalent intermolecular forces similar to hydrogen bonding.
The single-crystal X-ray structure of [Cu(L)DMF] was determined at 100 K and is shown
to be Triclinic with space group P-1. Asymmetric unit of [Cu(L)DMF] consists of one molecule.
The crystal structure of [Cu(L)DMF] contains a tridentate ligand coordinated through an amide
carbonyl oxygen via deprotonation after enolisation, azomethine nitrogen and oxygen of -OH
(pyranone ring) via deprotonation and also contains a molecule of coordinated DMF. The
structure forms a dimer across the inversion centre via a long, (2.685Å), Cu-N bond. The ONO
donor sites of the tridentate ligand coordinate to the Cu(II) centre forming one five-membered
CN2OCu and other six-membered C3NOCu chelate rings with bite angles of 82.98° and 92.10°,
respectively, represents distortion from an ideal square planar geometry. The average bond
lengths in the complex are: C7–O1 = 1.299(19), N3–C8= 1.308(2) and C11–O2= 1.281(19) Å,
which are longer or shorter than those of the corresponding distances in the ligand, suggesting
considerable delocalization of the charge on the chelate rings [32a]. [Cu(L)DMF] is stabilized
by intramolecular hydrogen bonding N1-H···N2 (2.691(19) Å) and also by intermolecular
hydrogen bonding N1-H···O4 (2.997(18) Å) (Table 2). In addition, the molecule is stabilized by
three intermolecular C-H···π stacking interactions acting between the centroid of chelate ring
and phenyl proton C2-H2, the centroid of DHA ring and phenyl protons C2-H2 & C3-H3, with
the contact distances of 3.549, 3.758, 3.577 Å respectively (Fig. S2). The π ···π interactions can
play an important role in packing. In this structure the π ···π interaction is an offset stacking, i.e.
the rings (DHA and phenyl) are parallel slipped. The ring normal and the vector between the ring
centroids form an angle of 20.91° up to centroid-centroid distance of 3.896 Å (Fig. S3) [32b].
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Hirshfeld surface analysis is a technique which reproduces the results of X-ray crystal
structure analysis and helps to elucidate all the intermolecular interactions in a novel visual
manner [33]. This method uses visual recognition of properties of atom contacts through
mapping of a range of functions (dnorm, shape index, curvedness, etc.) onto this surface [34]. The
increasing popularity of this tool comes from the fact that it allows for recognition of less
directional contacts, for instance, H···H dispersion forces. Another essential advantage is that all
(di, de) contacts created by a molecule of interest can be expressed in the form of a two-
dimensional (2D) plot, known as the 2D fingerprint plot. The de and di are defined, respectively,
as the distance from the Hirshfeld surface to the nearest atom in the molecule itself and the
distance from the surface to the nearest nucleus outwards from the surface. The shape of this
plot, which is unique for each molecule, is determined by dominating intermolecular contacts
[35]. The Hirshfeld surface mapped with a dnorm function for the H2L, [Ni(HL)2].H2O and
[Cu(L)(DMF)] (Fig. 4a-4c) clearly shows the red spots derived from hydrogen bonding
interactions. The plots of 2D fingerprint for the H2L, [Ni(HL)2].H2O and [Cu(L)(DMF)] (Fig.
4a-4c) shows the most significant H···H interactions with contribution of 37.9%, 41.8% and
47.0% respectively, other C···H interaction characterized by winglike peripheral spikes (21.1%,
24.2% and 15.4% respectively), N···H (4.6%, 4.7% and 2.8% respectively) and O···H
characterized by longer and thinner spikes (26.3%, 26.7% and 20.9% respectively) (Fig. 4d). The
non-directional H···H contacts are characterized by broader spikes in [Cu(L)(DMF)] and
relatively sharper spikes in H2L, [Ni(HL)2].H2O. And, the percentage contribution of H···H
contact is also a measure of strength of the crystal lattice [36]. The measureables like volume
(VH), area (SH), globularity (G) and asphericity (V) can also be calculated using HSs. The term,
globularity [37] is found to be < 1 for H2L, [Ni(HL)2].H2O and [Cu(L)(DMF)] which indicates
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that the molecular surface is more structured, not a sphere. The asphericity [38a,b] is a measure
of anisotropy which decreases in the ensuing order for the compounds: [Cu(L)(DMF)] > H2L >
[Ni(HL)2].H2O. 2D fingerprint plots of the H2L, [Ni(HL)2].H2O and [Cu(L)(DMF)] of different
intermolecular contacts are displayed in Figures 4d and S22.
3.2. Spectral studies
The IR spectrum of the ligand (H2L) showed two highest frequency bands of medium
intensity at 3357 and 3323 cm-1 ascribed to asymmetric and symmetric modes of the ꢄ(-NH2)
group, respectively. These frequencies have appeared slightly at lower wave number compared
to reported compound (2-aminobenzyolhydrazone of 1,3-dione) [39], probably due to the
involvement of free amino group in intramolecular hydrogen bonding with the oxygen of C=O of
hydrazide moiety as confirmed by crystallographic studies. The presence of symmetric and
asymmetric bands in all the metal complexes due to –NH2 group indicate the non-involvement of
this group in coordination to the metal centres. A medium intense band at 3447 cm-1 in the free
ligand is ascribed to ꢄ(–OH) (pyranone ring). A strong band at 1645 cm-1 in H2L is assigned to
amide ꢄ(C=O). This band shows a negative shift in all the complexes except [Cu(L)(H2O)]
indicating its coordination to the metal ion. The absence of –OH (pyranone ring) stretching band
in all the complexes, clearly indicates the involvement of pyranone oxygen in coordination with
the metal ion via deprotonation [40]. The strong band at 1619 cm-1 due ꢄ(C=N) in the ligand has
shifted to lower wave number i.e. ~1599 cm-1 in the spectra of all the complexes indicating its
coordination to the metal ion. The presence of strong band at 1675 cm-1 assigned to ꢄ(C=O) of
pyranone ring in ligand and all the complexes indicates its non-involvement in coordination. The
above assignments suggest that the ligand has coordinated through amide carbonyl oxygen,
azomethine nitrogen and oxygen of –OH group (pyranone ring) via deprotonation in all the
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complexes except [Cu(L)(H2O)]. In case of [Cu(L)(H2O)], amide carbonyl stretching ν(C=O) is
absent while new bands have been observed at 1608 and 1357 cm-1. These are ascribed to newly
formed ꢄ(C=N) and ꢄ(C-O) groups respectively. This indicates the involvement of amide
carbonyl oxygen in coordination via enolisation and deprotonation [41a]. Thus, the IR spectral
data clearly reveals that the ligand acts as monobasic for all complexes except [Cu(L)(H2O)]
where it behaves as doubly deprotonated, tridentate ligand. The IR spectra of H2L, [Cu(HL)2]
and [Cu(L)(H2O)] (Fig. S4, S5 and S6) are provided as the supplementary material.
A broad singlet at 15.76 ppm in the free ligand, ascribed to OH proton has disappeared in
1
the H NMR spectrum of [Zn(HL)2], indicating the coordination of oxygen of –OH group
(pyranone ring) via deprotonation to the metal ion. A broad singlet at 8.06 ppm is assigned to
two protons of N1. This signal has shifted upfield by 1.13 ppm, revealing the breakdown of
intramolecular hydrogen bonding upon complexation. The signals due to OH proton and two
protons of N1 are D2O exchangeable (Fig. S8). A singlet appeared at 5.84 ppm in free ligand
ascribed to C12-H corresponds to one proton. A doublet and triplet at 6.77 and 6.57 ppm in free
ligand are assigned to C2-H and C4-H, respectively. The aromatic protons resonated as
multiplets in the region 7.57-7.24 ppm in the spectrum of the free ligand have shifted slightly
downfield in the spectrum of [Zn(HL)2]. The C9-H3 and C14-H3 protons observed as singlets at
2.11 and 2.61 ppm in uncoordinated ligand have shifted to 2.06 and 2.49 ppm, respectively on
1
complexation. The H NMR spectra of H2L (Fig. S7) and [Zn(HL)2] (Fig. S9) are provided as
the supplementary material. In the 13C NMR spectrum of the ligand, the signals at 166.7 and 94.7
ppm correspond to C11 and C9, respectively, and have shifted to 164.4 and 98.0 ppm in the 13C
NMR spectrum of [Zn(HL)2] indicating the coordination of oxygen of –OH group (pyranone
ring) via deprotonation. The peaks at 162.9, 106.2, 16.5 and 19.2 ppm are assigned to C8, C12,
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C14 and C9, respectively. The azomethine carbon suffers a downfield shift in the spectrum of
[Zn(HL)2] indicating the involvement of azomethine nitrogen in coordination to the metal ion.
Methyl carbon C14 also showed a downfield shift in [Zn(HL)2], indicating the coordination of
oxygen of –OH group (pyranone ring) via deprotonation to the metal ion. The 13C NMR spectra
of H2L (Fig. S10) and [Zn(HL)2] are provided as the supplementary material (Fig. S11).1H NMR
and 13C NMR spectra of [Zn(HL)2] suggests coordination of the ligand through oxygen of –OH
group (pyranone ring) via deprotonation, azomethine nitrogen and amide carbonyl oxygen.
The mass spectrum of the ligand (Fig. S12) has shown the molecular ion peak [M] + at
m/z 301. The ESI mass spectra of [Co(HL)2], [Ni(HL)2].H2O and [Zn(HL)2] complexes are given
as supplementary material (Fig. S13, S14 and S15). ESI mass spectral studies of [Co(HL)2],
[Ni(HL)2].H2O, [Cu(HL)2], [Zn(HL)2] and [Cu(L)(H2O)] show their molecular ion peaks
[M+H]+ at 659, 659, [M+K]+ at 700, [M+Na]+ at 689 and [M+H]+ at 380 respectively. Apart
from this, spectra also show some additional peaks, which are due to molecular cations of
various fragments of the complexes. By comparing the analytical and spectral data of all the
complexes relevant structures were assigned.
The X-band EPR spectra of paramagnetic [Cu(HL)2] complex was measured in the solid
state at room temperature and in solution state (DMF) at liquid nitrogen temperature i.e. 77 K.
EPR spectra is recorded only for copper complexes, due to the magnetic interactions between the
paramagnetic copper centers [41b]. EPR spectra of [Cu(HL)2] are shown in (Fig. S16 and S17).
The EPR spectrum at 300 K show a broad absorption band, which is isotropic due to tumbling
motion of molecule. The ‘giso’ value at 300 K is 2.06. The g∥ and gꢅ values observed for the
complex at 77K are 2.27 and 2.04, respectively. The trend observed, g∥ > gꢅ > 2.02, for the
present copper complex is typical of a copper (II) (d9) ion in axial symmetry with the unpaired
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electron residing in dx2-y2 orbital [42 a,b]. The g∥ value (2.27 < 2.3) indicates a larger percentage
of covalency of metal-ligand bonding [43a]. In axial symmetry the g-values are related by the
expression [43 b,c].
G = g∥ − 2/g⊥ − 2
where, G is a measure of the exchange interaction between copper centers in the
polycrystalline solid. According to Hathway, if the values of the G are greater than the 4, the
exchange interaction is negligible, whereas when the value of G is less than 4 considerable
interaction is indicated in the solid complex [42, 43d]. In present investigation the axial
symmetry parameter G being more than 4, rules out the exchange interaction between the copper
centers.
3.3. Electronic Spectral Studies
The electronic spectra of the ligand, as well as complexes, were recorded in DMF
solvent. The ligand exhibit two absorption bands in the UV–visible region around 270 and 351
nm. The first intense band around 270 nm is assigned to a ligand π → π* transition. This band
remains almost unchanged in the spectra of all the complexes. The second broad band around
351 nm assigned to n→π* transition, has suffered bathochromic shift upon complexation [44a].
This is an indication of coordination of azomethine nitrogen to the metal ions. For [Co(HL)2],
4
4
two spin-allowed transitions observed at 915 and 756 nm were assigned to T1g(F) → T2g (νꢆ)
and 4T1g(F) → 4A2g (νꢇ) transitions indicating an octahedral geometry around Co(II) ion [44a,b].
The electronic spectrum of [Ni(HL)2].H2O displays d–d transitions around 554 and 940 nm,
3
3
3
3
assignable to A2g(F) → T1g(F) (νꢇ) and A2g(F) → T2g(F) (νꢆ) transitions, respectively,
indicating an octahedral geometry [45]. A broad band at 714 nm appearing as an envelope in the
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2
2
[Cu(HL)2], assigned to the Eg → T2g transition reveals an octahedral geometry [46a,b]. Broad
band in the electronic spectrum of [Cu(L)(H2O)] with peak maxima at 620 nm was assigned to
2
the combination of 2B1g →2A1g and B1g → 2Eg transitions suggest a square-planar configuration
around the Cu(II) ion [47,48]. The π → π*, n→π* and d–d transition bands exhibited by the
ligand and its metal complexes are shown in Figures S18 and S19.
3.4. Thermal Studies
Thermal studies (TG and DT analaysis) of all the complexes have been undertaken to
know the presence/absence of coordinated/lattice held solvent/water molecules, to confirm their
composition and to understand the thermal stability. The thermogram of [Ni(HL)2].H2O showed
a weight loss of 2.70% (Calc. 2.66%) between the temperature range 50 and 100°C indicating the
presence of one lattice held water molecule. This is clearly evidenced in DTA curve in the form
of an exothermic peak at 75°C. Weight loss of 88.82% (Calc. 88.75%) around 200–527°C
corresponds to the loss of two ligand molecules. This process is further supported by two
exothermic peaks in DTA curve at 299 and 440°C. The plateau obtained after heating above
450°C corresponds to the formation of stable nickel oxide and the metal content (8.60%)
calculated from this residue tallies with the metal analysis (8.57%).
[Co(HL)2] has remained thermally stable up to a temperature of 200°C, showing the
absence of any lattice held molecules. The weight loss of about 91.70% (Calc. 91.06%) in the
range of 200–620°C is due to the loss of a two ligand molecules. This is clearly evidenced in
DTA curve in the form of two exothermic peaks at 276 and 590°C. The plateau obtained after
heating above 600°C corresponds to the formation of stable cobalt oxide.
The thermogram of [Cu(L)(H2O)] showing a weight loss of 4.80 % (Calc. 4.73 %)
between the temperature range 180-200°C correspond to the loss of one coordinated water
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molecule. Weight loss of 78.76% (Calc. 78.68%) around 250–540°C correspond to the loss of a
ligand molecule. The plateau obtained after heating above 470°C corresponds to the formation of
stable copper oxide. In case of [Cu(HL)2] and [Zn(HL)2] complexes, no weight loss was
observed up to the temperature 260°C and indicates the absence of any lattice held/coordinated
solvent molecule. The weight loss of 85.27 and 90.03% (Calc. 85.22% and 90.22%) around 265–
650°C correspond to the loss of two ligand molecule. Weight of the residue obtained after
heating the complex above 650°C corresponds to the formation of stable metal oxides.
Thus, thermal studies support the suggested composition for the complexes. As
representatives, thermograms of [Ni(HL)2].H2O and [Co(HL)2] are given in Figures S20 and S21
respectively.
3.5. Anti-inflammatory activity
In the present investigation, the control group showed a significant increase of edema in the right
hind paw of the animals in the plantar region. This is due to the release of inflammatory
mediators such as histamine, serotonin, kinins, and prostaglandins (PGs). The edema
development after carrageenan injection has been described as a biphasic event in which various
mediators operate in sequence to produce this inflammatory response. The initial phase of edema
(0-1 h), which is not inhibited by non-steroidal anti-inflammatory drugs, has been attributed to
the release of histamine, 5-hydroxytryptamine (5-HT) and bradykinin. In contrast, the second
accelerating phase of swelling (1-6 h), has been correlated with the elevated production of
prostaglandins [49], and more recently has been attributed to the induction of inducible cyclo-
oxygenase (COX-2) in the hind paw. Local neutrophil infiltration and activation also contribute
to this inflammatory response by producing, among other mediators, oxygen-derived free
radicals such as superoxide anion (O2-) and hydroxyl radicals. Among them, edema is one of the
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most fundamental actions of acute inflammation and is an essential parameter to be considered
when evaluating compounds with potential anti-inflammatory activities [50a].
The coordination of bioactive molecules to metal ions is a promising approach to improve the
therapeutic potency and/or to decrease the toxicity of drug molecules. The resulting metal complexes
possess greater lipophilicity profiles compared to the free ligands, allowing them to more easily pass
through cell membranes to exert their biological effects [8b,c].
3.5.1. In vivo anti-inflammatory activity
The anti-inflammatory activity of the newly synthesized compounds were evaluated by applying
the carrageenan-induced paw edema bioassay in rats using diclofenac as reference drug [50b-d].
In the present investigation, the ligand and its metal complexes exhibited a dose-dependent
response (Table 3). The ligand, H2L showed a consistent and significant decrease in paw edema
at 0.5, 1, 3 and 5 h after drug administration, while complexes at higher dose gave a good
response up to the fifth hour but at a lower dose, the significant response decreased as time
advanced. Further, the copper complexes ([Cu(HL)2] and [Cu(L)(H2O)]) showed significant
inhibition of paw edema at a lower concentration of 5 mg/kg. The percentage inhibition (22-
82%) shown by [Cu(HL)2] over the time period of 0.5–1 h was significant and comparable with
the standard compound, viz. Diclofenac (90-98%) (Fig. 5a-5b).In comparison with H2L, all the
complexes showed excellent effect at 0.5 and 1 h, [Co(HL)2], [Zn(HL)2] and [Cu(L)(H2O)]
retained their activity up to fifth hour, while [Ni(HL)2].H2O and [Cu(HL)2] showed a gradual
decrease in activity. The enhanced anti-inflammatory activity of the complexes compared to the
free ligand may be explained on the basis of enhanced pervasion through cellular membrane on
complexation by Tweedy’s Chelation theory and Overtone’s concept [51a]. According to
Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favors the
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passage of only the lipid-soluble materials in which liposolubility as an important factor, which
controls the biological activity. On chelation, the polarity of the metal ion will be reduced due to
the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with
donor groups [51b]. It is also expected that the more extensive heteroaromatic ring like DHA and
the presence of lipophilic group C=N would bestow greater lipophilicity on complexes and
enable it to penetrate the cell wall and promote adverse intracellular interactions [51c]. Hence,
the enhanced activity of the complexes can be related to reduced overall polarity of the molecule,
which increases the lipophilic nature of the complex, favoring the efficient permeation through
lipid layer.
3.5.2. In vitro anti-inflammatory activity
In vitro pharmacological evaluation of the title compounds was carried out to evaluate
their anti-inflammatory activity. All the synthesized compounds were subjected to anti-
inflammatory effect against denaturation of hen's egg albumin method [52a,b] at the
concentration (31.25-1000 µg/mL) with standard aceclofenac drug. The outcome of anti-
inflammatory screening of the title compounds are summarized in Table 4. The percentage
inhibition of all the synthesized compounds showed higher activity against the denaturation of
protein. Among the compounds, [Ni(HL)2].H2O, [Cu(HL)2] and [Zn(HL)2] exhibited an
excellent inhibition of heat induced protein denaturation 80.6%, 55.3% and 45.3% respectively
and these compounds are more active compared to standard aceclofenac drug (43.2%). All the
metal complexes showed good inhibitory anti-inflammatory activity than the free ligand against
the denaturation of the protein.
4. Conclusion
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In the present work, Co(II), Ni(II), Cu(II) and Zn(II) complexes of a new ligand, (E)-2-
amino-N'-(1-(2-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl)ethylidene)
benzohydrazide
were
designed and synthesized in good yield. Analytical and spectroscopic data for the metal
complexes indicate a 1:1 (M: L) stoichiometry for [Cu(L)(H2O)] adopting a square planar
geometry and 1:2 for the [Co(HL)2], [Ni(HL)2].H2O, [Cu(HL)2] and [Zn(HL)2] adopting an
octahedral geometry around the metal ion. The organic motif, H2L coordinated in monoanionic
i.e. coordinating through oxygen of –OH group (pyranone ring) via deprotonation in [Co(HL)2],
[Ni(HL)2].H2O, [Cu(HL)2], [Zn(HL)2] and in dianionic form i.e. deprotonation after enolisation
of amide carbonyl & deprotonation of –OH group (pyranone ring) in [Cu(L)(H2O)]. Thus, the
ligand acts in mono as well as doubly deprotonated manner. The tentative structures for metal
complexes are depicted in figure 6. All interactions in crystal structures of H2L, [Ni(HL)2].H2O
and [Cu(L)DMF] have also been studied by Hirshfeld surface analysis. H2L and its metal
complexes have also been screened for their in vivo and in vitro anti-inflammatory activities. The
results showed that activity of ligand has enhanced on complexation. The increase in activity of
the metal complexes is probably due to the greater lipophilic nature of the complexes. Among
the complexes tested, [Cu(HL)2] has shown highest activity. The difference in activity among the
tested compounds may be attributed to the electrostatic nature of ligand and central metal ion.
5. Significance and justification
The present research reports in vivo and in vitro anti-inflammatory activity of late first row
transition metal complexes of novel ligand. The results showed that activity of ligand has
enhanced on complexation. The results of the present study are quite inspiring and warrant
further, extensive testing toward transfer into the clinical arena.
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Appendix A. Supplementary data
Crystallographic data for the structural analysis have been deposited with the Cambridge
Crystallographic Data Centre, CCDC reference number: 1534307 (H2L), 1534306
([Ni(HL)2].H2O) and 1534309 ([Cu(L)DMF]). Copies of this information may be obtained free
of the charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (Fax: +44-
1223-336033; E-mail: deposit@ ccdc.cam.ac.uk or http://www.ccd.cam.ac.uk).
Acknowledgements
Authors thank USIC, Karnatak University, Dharwad for IR spectral analyses, TGA and SC-XRD
studies and CDRI Lucknow for ESI mass analyses. Recording of 1H and 13C NMR spectra from
IISC-Bangalore and EPR spectra from IIT-Bombay are gratefully acknowledged. Authors are
also thankful to Dr. M. Netaji for his help in solving the crystal data of Nickel complex. One of
the author, Geeta H. Chimmalagi acknowledges UGC for providing RFSMS scholarship.
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O
O
H
N
O
N
H
O
NH2
Zwitterionic structure
O
O
NH2
OCH3
NH2
(ii)
(i)
N
H
O
NH2
O
HO
O
O
OH
N
N
O
H
O
NH2
Neutral structure
H2L
Reagents and Conditions : (i) NH2.NH2.H2O, MeOH, Reflux (8-9 h)
(ii) MeOH, RT (stirring, 0.5 h)
Scheme 1. Synthetic protocol.
Fig. 1. ORTEP projection of H2L showing 50% probability ellipsoids.
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Fig. 2. ORTEP projection of [Ni(HL)2].H2O showing 50% probability ellipsoids.
Fig. 3. ORTEP projection of [Cu(L)(DMF)] showing 50% probability ellipsoids.
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Fig. 4a. Hirshfeld surfaces mapped with dnorm and 2D fingerprint plot of H2L depicting all
intermolecular contributions.
Fig. 4b. Hirshfeld surfaces mapped with dnorm and 2D fingerprint plot of [Ni(HL)2].H2O
depicting all intermolecular contributions.
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Fig. 4c. Hirshfeld surfaces mapped with dnorm and 2D fingerprint plot of [Cu(L)(DMF)] depicting
all intermolecular contributions.
0
20
40
60
80
100
O•••H
H•••O
H•••H
C•••H
H•••C
C•••C
N•••H
H•••N
Other
H2L
[Ni(HL)2].H2O
[Cu(L)(H2O)]
Fig. 4d. Distribution of individual intermolecular interactions on the basis of Hirshfeld surface
analysis of H2L, [Ni(HL)2].H2O and [Cu(L)DMF].
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0.5 h 1 h 3 h 5 h
100
80
60
40
20
0
Fig. 5a. Anti-inflammatory activity of H2L and its metal complexes at different time intervals
(0.5 to 5 h) with 5 mg/kg dose.
0.5 h 1 h 3 h 5 h
100
80
60
40
20
0
Fig. 5b. Anti-inflammatory activity of H2L and its metal complexes at different time intervals
(0.5 to 5 h) with 10 mg/kg dose.
28