Chapter 4 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/28515/11/11...Chapter 4 Reactivity...
Transcript of Chapter 4 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/28515/11/11...Chapter 4 Reactivity...
83
Chapter 4
Reactivity of M(acac)2 (M = Cu, Ni, Co, Mn) towards
iminodiacetic acid in presence of α-diimine:
Synthesis, X-ray crystallography, spectroscopic
characterization and biological studies
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INTRODUCTION
Functionalized dicarboxylic acids and their metal complexes have potential
uses for developing novel pharmaceutical agents [1]. The coordination chemistry of
iminodiacetic acid (H2imda) has been a subject of continuous investigations due to its
tridentate chelating behaviour towards metal ions resulting in complexes which show
structural diversities [2]. In aqueous biological pH range (pH = 6–7), the carboxylic
acid protons of H2imda dissociate to give iminodiacetate (imda2–
) dianion which is
quite reactive to transition metal ions forming metal iminodiacetate complexes [3].
Reactions of metal iminodiacetate with an α-diimine viz. 1,10-phenanthroline (phen)
or 2,2'-bipyridine (bipy) produce mixed-ligand complexes, some of which have been
successfully exploited as model compounds to explain binding modes and structural
features in several metalloenzymes [4]. Several mixed-ligand complexes have been
found to exhibit superoxide dismutase (SOD) activity [5,6] which is related to the
flexibility of the geometric transformations around the metal centres [7]. Moreover,
some of these complexes play a decisive role not only in activation of enzymes but
also in the storage and transport of active substances through biological membranes
[8]. Transition metal ternary complexes have received particular focus and have been
employed in mapping protein surfaces [9], as probes for biological redox centers [10]
and in protein capture for purifications [11].
The study of the structure of model ternary complexes provides information
about how biological systems achieve their specificity and stability, as well as
strategies to improve these features for biotechnological applications. Ternary
complexes of oxygen donor ligands and heteroaromatic N-bases have been of special
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interest as they can display exceptionally high stability [12]. X-ray structural data of
the ternary complexes [2,3] with iminodiacetate (imda2–
) or its substituted analogues
as primary ligand and N-donors as secondary ligand have revealed the influence of the
secondary (auxiliary) ligand on the conformations of the primary iminodiacetate
ligands. Studies of such complexes have also explained the observed changes in the
protein conformations at the metal-protein centers in the absence as well as in the
presence of appropriate substrates [13]. The structure of the ternary complexes bearing
the composition M/H2imda/N-heterocycle in the mole ratio 1/1/2 display a
hexacoordinate elongated octahedral (4+1+1) geometry where imda is in a fac-
tridentate conformation [14]. Complexes having the composition M/H2imda/N-
heterocycle in 1/1/1 mole ratio, invariably adopt a five coordinate square pyramidal
(4+1) coordination geometry.
Prompted by the biological activities exhibited by the transition metal ternary
complexes and also the possible structural diversities, it was contemplated to carry out
the syntheses of mixed-ligand metal iminodiacetate complexes, which were
characterized by spectral and X-ray crystallographic technique.
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EXPERIMENTAL
Materials
The reagents metal salts (Merck), 2, 2’-bipyridine (Merck) and iminodiacetic
acid (Aldrich) used were of synthetic grades. Ultrapure water was used for all
experiments. The commercially available solvents were distilled and then used for
synthesis.
Instrumentation
Melting points were determined by open capillary method and are uncorrected.
The IR spectra as KBr pellets were recorded on a Shimadzu FT-IR 157
spectrophotometer. 1H and
13C NMR spectra were recorded on a Varian Mercury Plus
300 spectrometer using TMS as an internal standard. The X-band EPR spectra were
taken at room temperature as well as liquid nitrogen temperature on a Varian E-112
spectrometer operating at 9.1 GHz. FAB-Mass spectra on Jeol SX-102/DA-6000 mass
spectrometer using argon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was
10 kV and m-nitrobenzyl alcohol (NBA) was used as matrix.
X-ray crystallographic data collection
Single-crystal X-ray diffraction of complex (1) and (2) was performed on a
BRUKER SMART 1000 CCD diffractometer equipped with a graphite crystal
monochromator situated in the incident beam for data collection. The structure of the
complex (1) and (2) was solved by direct methods and refined against F2
using the
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SHELXL-97 software [15]. The X-ray data of the complex was recorded at IIT,
Kanpur.
Synthesis of M(acac)2 (M = Cu, Ni, Co, Mn)
Synthesis of bis(acetylacetonato) copper(II), [Cu(acac)2]
To a 25 mL aqueous solution of CuCl2·2H2O (24 mmol, 4 gm) was slowly
added a solution of 5 mL methanolic solution of with rapid stirring. Now sodium
acetate (84 mmol, 6.8 gm) in 15 mL distilled water was added to the resulting mixture
dropwise. The mixture was then heated to about 80 0C for 15 minutes while the rapid
stirring was maintained. The reaction mixture was first cooled to room temperature &
then cooled further in ice bath. The blue grey product was collected by vacuum
filteration and washed with 100 mL of cold distilled water. The product was dried at
110 0C in an oven. The crude product was recystallised from hot methanol.
[Yield 85%; m.p.284-288 C (dec)] Anal Calc. (%) for Cu(C5H7O2)2: C, 30.96
; H, 3.61 , Found (%), C, 30.68; H, 3.60. FT-IR ( as KBr disc, cm-1
): ν (CH3), 2922,
2854 cm-1
; ν(C C) + ν (C O), 1576 cm-1
; ν(C O) + ν (C C), 1529 cm-1
;
п(CH), 783 cm-1
; ν(MO) + ν(C-CH3), 454 cm-1
; ν(C C) + ν (C O), 939 cm-1
; Pr
(CH3), 1017 cm-1
; δ(CH) + ν(C-CH3), 1190 cm-1
; ν(C-CH3) + ν (C C), 1273 cm-1
;
δs(CH3), 1357 cm-1
; δd(CH3), 1414 cm-1
; п(CH3-C(O)-C), 652 cm-1
.
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Synthesis of bis(acetylacetonato) nickel(II), [Ni(acac)2]
To a solution of NiCl2.6H2O (0.25 mol, 59.4 gm) in 250 mL of water was
added a solution of acetylacetone (0.5 mol, 50 g) in 100 mL of methanol, while
stirring. A solution of 0.5 mol of sodium acetate in 150 mL of water was added to the
resulting mixture. The mixture was heated, cooled to room temperature, and placed in
the refrigerator for several hours. The green solid was filtered off, washed with water,
and dried in vacuum desiccator.
[Yield 80%; m.p. 235 C ] Anal Calc. (%) for Ni(C5H7O2)2: C, 31.3; H, 3.65,
Found (%), C, 31.28; H, 3.61. FT-IR ( as KBr disc, cm–1
): ν (CH), 3081 cm-1
; ν
(CH3), 2994, 2927 cm-1
; ν(C C) + ν (C O), 1523 cm-1
; ν(C O) + ν (C C), 1529
cm-1
; п(CH), 767 cm-1
; ring def.+ ν(MO), 593 cm-1
; ν(C C) + ν (C O), 933 cm-1
;
Pr (CH3), 1024 cm-1
; δ(CH) + ν(C C), 1463 cm-1
; ν(C-CH3) + ν (C C), 1265 cm-1
;
δd(CH3), 1404 cm-1
; п(CH3-C(O)-C), 668 cm-1
.
Synthesis of diaquobis(acetylacetonato) cobalt(II), [Co(acac)2·2H2O]
To a solution of 80 mg NaOH in 1 mL water, was slowly add distilled
acetylacetone (2 mmol, 200 mg), with swirling. The yellow solution is added
dropwise, with stirring, to a solution of CoCl2·6H2O (1 mmol, 238 mg) in 1.5 mL of
water. The product was isolated by suction filteration and washed with water until the
washings are nearly colourless. After drying, the crude product was dissolved in a hot
mixture of 2 mL ethanol and 1.3 mL chloroform (do not boil the resulting solution).
The resulting mixture was filtered by vacuum pump and dried in desiccator.
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[Yield 85%; m.p. 167.5 C (dec)] Anal Calc. (%) for Co(C5H7O2)2·2H2O: C,
28.64; H, 4.29, Found (%), C, 28.60; H, 3.61. FT-IR ( as KBr disc, cm–1
): ν (OH),
3424 cm–1
; ν (CH3), 2994, 2927 cm-1
; ν(C C) + ν (C O), 1521 cm-1
; ν(C O) + ν
(C C), 1529 cm-1
; п(CH), 770 cm-1
; ring def.+ ν(MO), 575 cm-1
; ν(C C) + ν (C
O), 933 cm-1
; Pr (CH3), 1020 cm-1
; δ(CH) + ν(C –CH3), 1203 cm-1
; ν(C-CH3) + ν (C
C), 1263 cm-1
; δd(CH3), 1402 cm-1
; п(CH3-C(O)-C), 675 cm-1
; ν(MO) + ν (C-CH3),
424 cm-1
; δ(CH) + ν(C C), 1461 cm-1
.
Synthesis of bis(acetylacetonato) manganese, [Mn (acac)2]
To a solution of MnCl2.4H2O (0.25 mol, 49.5 g) in 250 mL of water at room
temperature is added a solution of acetylacetone (0.5 mol, 50g) in 100 mL methanol.
The mixture is stirred during the addition with magnetic stirrer. A solution of sodium
acetate tri-hydrate (0.5 mol, 68.1g) in 150 mL water is then added slowly with stirring,
followed by addition of 21 mL of concentrated aqueous ammonia. The mixture is
stirred about ten minutes at room temperature and then placed for several hours in a
refrigerator. The light yellow solid that forms is fltered off on a buchner funnel under
a stream of nitrogen and washed with several portions of cold water. The solid is dried
in vacuum desiccator at room temperature for 6 to 8 hours.
[Yield 85%; m.p. 187.6 C (dec)].Anal Calc. (%) for Mn(C5H7O2)2: C, 31.6; H,
3.69, Anal. Cal. (%) Found (%), C, 31.5; H, 3.64. FT-IR ( as KBr disc, cm–1
): ν (CH3),
2980, 2965 cm-1
; ν(C C) + ν (C O), 1595, 1516 cm-1
; ν(C O) + ν (C C), 1528
cm-1
; п(CH), 780 cm-1
; ring def.+ ν(MO), 605 cm-1
; ν(C C) + ν (C O), 926 cm-1
;
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Pr (CH3), 1017 cm-1
; δ(CH) + ν(C –CH3), 1192 cm-1
; ν(C-CH3) + ν (C C), 1259 cm-
1; δs(CH3), 1350 cm
-1; п(CH3-C(O)-C), 669 cm
-1; ν(MO) + ν (C-CH3), 463 cm
-1.
Synthesis of the complexes
Synthesis of [Cu(imda)(bipy)]·6H2O (1)
To an aqueous solution of iminodiacetic acid (2 mmol, 0.266 g), 2,2’-bipyridne
(2 mmol, 0.312 g) was added in small batches and stirred for 1 h at room temperature
forming a homogeneous clear solution. A blue methanolic solution of Cu(acac)2 (2
mmol, 0.523 g) was added drop wise to the above mixture. The stirring was continued
overnight followed by refluxing for 24 h. which produced a stable blue colour
solution. It was left standing in air in a beaker at room temperature. Beautiful blue
coloured crystals of (1) were collected after few days.
Yield 70%, m.p. >300 C. Anal Calc. (%) for C14H24CuN3O10: C, 36.72; H,
5.28; N, 9.18, Found (%): C, 36.69; H, 5.26; N, 9.18. IR (KBr, υ cm–1
): (COO-, asym
),1614 cm-1
; (COO-, sym
), 1381 cm
-1; (Cu–N), 420 cm
-1; (C=C and C=N), 1590–1445
cm-1
; (H2O), 3422 cm-1
. 1H NMR ( ppm, D2O) (300 MHz): 3.42, (2H, s, CH2);
10.22, (1H, s, iminic NH); 7.6–8.7, (8H, m, aromatic protons). 13
C NMR ( ppm, D2O)
(75 MHz): 35, 123, 126, 128, 134, 137 and 178.
Synthesis of [Ni(imda)(bipy)(H2O)]·4H2O (2)
To an aqueous solution of iminodiacetic acid (3 mmol, 0.399 g), 2,2’-bipyridne
(3 mmol, 0.468g) was added in small batches and stirred for 1 h at room temperature
forming a homogeneous clear solution. A green methanolic solution of Ni(acac)2 (3
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mmol, 0.771 g) was added drop wise to the above mixture. The colour of the solution
is changed to light purple colour. The stirring was continued overnight followed by
refluxing for 24 h. which produced a stable blue colour solution. Solution was left
standing in air in a beaker at room temperature. Beautiful blackish grey coloured
crystals of (2) were obtained after few days.
Yield 62%, m.p. 316 C (dec). Anal Calc. (%) for C14H22NiN3O9: C, 38.65; H,
5.10; N, 9.66, Found (%): C, 38.10; H, 5.10; N, 9.65. IR (KBr, υ cm–1
): (COO-, asym),
1618 cm-1
; (COO-, sym), 1343 cm
-1; (Ni–N), 409 cm
-1; (C=C and C=N), 1522–1443
cm-1
; (H2O), 3430 cm-1
. 1H NMR ( ppm, D2O) (300 MHz): 3.54, (2H, s, CH2); 11.08,
(1H, s, iminic NH); 7.6–8.8, (8H, m, aromatic protons). 13
C NMR ( ppm, D2O) (75
MHz): 35, 121, 125, 127, 138, 141 and 179.
Synthesis of [Co(imda)(bipy)(H2O)]·4H2O (3)
To an aqueous solution of iminodiacetic acid (2 mmol, 0.266 g), 2,2’-bipyridne (2
mmol, 0.312 g) was added in small batches and stirred for 1 h at room temperature
forming a homogeneous clear solution. A methanolic solution of Co(acac)2(H2O)2 (2
mmol, 0.586 g) was added drop wise to the above mixture. The stirring was continued
overnight followed by refluxing for 24 h. Solution was left standing in air in a beaker
at room temperature. Beautiful crystals of (3) were collected as above.
Yield 65%, m.p. >300 C (dec). Anal Calc. (%) for C14H22CoN3O9: C, 38.63;
H, 5.09; N, 9.65, Found (%): C, 38.62; H, 5.08; N, 9.63. IR (KBr, υ cm–1
): (COO-,
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asym), 1625 cm-1
; (COO-, sym), 1350 cm
-1; (Co–N), 419 cm
-1; (C=C and C=N), 1573–
1439 cm-1
; (H2O), 3423 cm-1
. 1H NMR ( ppm, D2O) (300 MHz): 3.45, (2H, s, CH2);
10.42, (1H, s, iminic NH); 7.3–8.4, (8H, m, aromatic protons). 13
C NMR ( ppm, D2O)
(75 MHz): 37, 121, 126, 129, 134, 138, and 172.
Synthesis of [Mn(imda)(bipy)(H2O)]·4H2O (4)
It was prepared in the same manner as discussed above using Iminodiacetic
acid (2 mmol, 0.266 g), Mn(acac)2 (2 mmol, 0.253g) and 2,2’-bipyridine (2 mmol,
0.312 g). Crystals of (4) were obtained in an identical manner which however, were
not of good quality to X-ray crystallography.
Yield 69%, m.p. >300 C. Anal Calc. (%) for C14H22MnN3O9: C, 38.99; H,
5.14; N, 9.74, Found (%): C, 38.91; H, 5.12; N, 9.72. IR (KBr, υ cm–1
): (COO-, asym),
1620 cm–1
; (COO-, sym), 1346 cm
–1; (Mn–N), 415 cm
–1; (C=C and C=N), 1577–1425
cm–1
; (H2O), 3429 cm–1
. 1H NMR ( ppm, D2O) (300 MHz): 3.53, (2H, s, CH2);
10.36, (1H, s, iminic NH); 7.9–8.3, (8H, m, aromatic protons). 13
C NMR ( ppm, D2O)
(75 MHz): 34, 124, 126, 132, 135, 139 and 174.
Superoxide dismutase (SOD) mimic activity
In vitro SOD activity was measured using alkaline DMSO as a source of
superoxide radical ion (O2.-) and nitrobluetetrazolium (NBT) as (O2
.-) scavenger
[16,17]. In general, 400 µL of the sample to be assayed was added to a solution
containing 2.1 mL of 0.2 M potassium phosphate buffer (pH 7.6) and 1 mL of 56 µM
NBT. The tubes were kept in ice for 20 min and then 1.5 mL of alkaline DMSO
solution was added while stirring. The absorbance was then monitored at 540 nm
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against a sample prepared under similar condition except that NaOH was absent in
DMSO. The measure of SOD activity, expressed as IC50 is the concentration of the
substrate or complex which causes 50% inhibition of the reduction of NBT.
Microbial Analyses
Antibacterial screening was performed by the established disc diffusion
method and ciprofloxacin was used as the standard. Antifungal screening was done by
agar diffusion method and Greseofulvin was used as standard. The stains used were
Escherichia coli (K-12), Bacillus subtilis (MTCC-121), Staphylococcus aureus (IOA-
SA-22), Salmonella Typhymurium (MTCC-98), Candida albicans, Aspergillus
fumigatus and Penicillium marneffei.
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RESULTS & DISCUSSION
The meatl precursors M(acac)2 (M = Cu, Ni, Co and Mn) exhibited ample
reactivity with the reaction mixture containing 1:1 mole equivalent of the
iminodiacetic acid and α-diimine (2, 2’-bipyridine) in water-methanol medium at
refluxing condition. This has afforded quite good yield of crystalline products. The
analytical data of the final products are in good aggrement with the stoichiometry
[Cu(imda)(bipy)]·6H2O (1), [Ni(imda)(bipy)(H2O)]·4H2O (2),
[Co(imda)(bipy)(H2O)]·4H2O (3) and [Mn(imda)(bipy)(H2O)]·4H2O (4). The
formation of the ternary system can be represented as following reaction sequences:
N
RT
Stirred
Clear colourlesssolution
M(acac)2
[M(imda)(2,2'-bipy)].6H2O
H2O
N
M= Cu(1)
Or
[M(imda)(2,2'-bipy)(H2O)].4H2O
M= Ni(2), Co (3), Mn(4)
+
HO
NH
OH
O O
Scheme 1. Synthetic procedure of the complex (1-4)
FAB-Mass spectra
The peaks corresponding to the isotope of highest abundance for each metal
i.e. (Cu, Ni, Co and Mn) has been considered in the FAB-Mass specra. The FAB-Mass
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spectra of the compounds recorded in m-nitrobenzylalcohol (NBA) matrix contained
several strong intensity peaks. The m /z values of the peaks are attributed to the
corresponding molecular ions as well as the species generated from the step-wise
fragmentations of the molecular ions. The peaks at m/z = 351 for (1), m/z = 350 for
(2), m/z = 349 for (3) and m/z = 343 for (4) are consistent with the generation of
molecular ions [Cu(imda)(bipy)-H]+
, [Ni(imda)(bipy+H)]+, [Co(imda)(bipy)+H]
+ and
[Mn(imda)(bipy)-H]+, respectively. However, stepwise fragmentation of the molecular
ion was also indicated to produce [Cu(bipy)]+ (m/z = 219), [Cu(bipy)–H]
+ (m/z = 218)
for (1), [Ni(bipy)]+ (m/z = 215), [(imda)+H]
+ (m/z = 134) for
(2), [Co(bipy)]
+ (m/z =
215) for (3) and [Mn(bipy)]+ (m/z = 211) for (4). The dissociation of the auxiliary
ligand i.e. 2, 2’-bipyridine has produced [M(bipy)]+ as the ultimate product in all the
cases suggesting that the ultimate fragmentation product in each case is quite stable
even under the FAB-Mass condition. The FAB-Mass studies, therefore corroborate
that the imda2–
anion binds metal ions with stronger coordinate-covalent bonds
compared to that provided by the auxiliary ligand 2,2’-bipyridine, albeit, 2,2’-
bipyridine is a well known strong chelating agent. The chelation from carboxylate
group of imda2–
towards metal ion is apparently stronger owing to the fact that the M–
O bonds have significant covalent character compared to the M–N
coordination/chelation.
IR spectra
The characteristic peak of the neutral carboxylate group ν(COOH) observed at
3322 cm-1
in the FT-IR spectra of ligand H2imda was absent in the present spectra of
the complexes. This suggests that the chelating ligand H2imda binds to metal in the
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deprotonated forms. The type of mode of coordination of the COO- group in the
complexes has been compared from the sepration of νsym(COO-) and νasym(COO
-)
bands. The strong intensity absorption bands near 1600-1650 cm–1
and 1300-1350 cm–
1 are assignable to νasym(COO
-) str. and νsym(COO
-) str. vib., respectively [18]. The
separation of the νasym and νsym(COO-) str. vib. [∆ν = νasym(COO
-)–νsym(COO
-) ~250
cm–1
] is very large indicating a chelation from the two anionic COO– group of imda
2–
moiety to the metal ion in all the complexes [18]. The ν(C=N) and ν(C=C) str. vib.
bands were observed in 1580–1427 cm–1
region. The presence of a medium intensity
band appearing at ~430 cm–1
is consistent with the formation of M–N bond(s) in the
molecule. The observed negative shift in ν(C–N) str. vib. (~100 cm–1
) relative to that
observed in free uncoordinated H2imda moiety further corroborates the additional
coordination from the iminic nitrogen of the iminodiacetate moiety. The M-O
coordination has also been indicated as a weak peak around at ~450 cm-1
in the FT-IR
spectra of the complexes. The presence of the coordinated /or lattice water molecules
was also indicated as a broad band in 3500–3200 cm–1
region [18].
1H and
13C NMR spectra
The 1H NMR spectra of the complexes recorded in D2O showed a singlet at
3.40–3.52 ppm attributable to CH2 protons. Iminic NH proton in the complexes
resonates as a singlet at ~10.20–11.10 ppm. The aromatic protons characteristic of
bipyridine appeared as multiplets at 7.2–8.9 ppm. In 13
C NMR spectra, the resonance
signals due to carbons of the aromatic ring of the α-diimine and the CH2 group of
the imda2-
skeleton were indicated at 120-140 and 42 ppm range, respectively.
97
However, the signal observed at the most downfield 175 ppm is attributed to the –
COO- functions of the imda
2- anion.
EPR spectra
The X-band EPR of the mononuclear complex [Cu(imda)(bipy)]·6H2O (1) is
anisotropic giving well splitted signals for g1 = 1.8, g2 = 2.09 and g3 = 2.20. This
anisotropy is known to arise due to presence of a gross distortion in the molecular
geometry [19] from the perfect Oh symmetry. Five coordinate trigonal bipyramidal
(tbp) or square pyramidal (sq. py.) geometry [20] around Cu2+
ion, effectively
experiences greater extent of distortions compared to that observed in the Oh
symmetry. However, there is not much difference between the energy of the two
former coordinatively unsaturated geometrical configurations. The ground state energy
level for the tbp and sq-py structures are dx2_
y2 and dz
2 respectively [21]. The
magnitude of the rhombic distortion parameter (R) defined as (g2 – g1)/ (g3 – g2), is
helpful to ascertain the type as well as the extent of distortion and the related ground
energy state. An observed R>1 reflects sq-py geometry with dz2 ground energy state,
whereas R<1.0 is indicative of dx2-y
2 energy state of the tbp geometry [22]. The
magnitude of R for the complex (1) comes out 2.6 (i.e. R>1) and is consistent with a
five coordinated sq-py structure, which is corroborated from X-ray crystallographic
structural data.
Single crystal X-ray diffraction studies
The crystal data with structure refinements of complexes
[Cu(imda)(bipy)]·6H2O (1) and [Ni(imda)(bipy)(H2O)]·4H2O (2) are given in Table
98
1 and 2. The ORTEP view (50% probability of thermal ellipsoid) and packing
diagrams of complex (1) are shown in Figs. 1 & 2 and for complex (2) are shown in
Figs. 3 & 4, respectively. The structure of (1) contains an arrangement of a discrete
triclinic unit such that each Cu(II) ion acquires a distorted square pyramidal
coordination geometry. However, in complex (2) the Ni(II) ion acquires an un-
symmetrically elongated hexagonal coordination geometry. It is apparent from the
molecular structures that in both the complexes the chelator (2, 2’-bipyridine) satisfies
the two coordination sites while the chelating iminodiacetate anion (imda)2-
provides
three coordination sites which include the imino-N and two oxygens from
unsymmetric coordination from the two carboxylato functions i.e. [N,O,O] behaving
as a tridentate ligand.
In complex (1) the Cu (II) adopts a five coordinate geometry, preferably square
pyramidal type. The basal plane of the square pyramidal structure consists of two
donor sites (N2,N3) of the α- chelator (2, 2’ bipyridine) and two donor sites (N1,O3)
from the iminic-N and carboxylate- O fonctions of the imda2-
moiety. The axial
coordination is from the second oxygen site (O1) of the carboxylate function in the
imda2-
anion (Fig. 1). The bond distances between the donor to Cu(II) ion i.e. binding
sites to the metal ion are [Cu1-O1 = 2.233(2), Cu1-N1 = 2.024(2), Cu1-N2 = 1.992(2),
Cu1-N3 = 2.017(2), Cu1-O3 = 1.956(2) Å]. The important bond distances and bond
angles are summerised in Table 3. However, the structural motif of the molecular unit
of the complex (2) is rather different from that of (1) [Ni1-O2 = 2.059(3), Ni1-N1 =
2.068(4), Ni1-N2 = 2.047(4), Ni-N3 = 2.072(4), Ni-O4 = 2.055(3) Å] (Table 3). Here
the Ni(II) adopts a coordinatively saturated hexacoordinate geometry (Fig. 3). Here
99
too, the basal plane is formed from two donor sites (N2, N3) of the α-diimine chelator
i.e. 2, 2’- bipyridine and imino nitrogen (N1) as well as one of the carboxylato oxygen
(Unsymmetrically coordinated O4). The oxygen of the second carboxylate atom (O2)
from the another unsymmetically carboxylate group of imda2-
along with H2O(5) form
the axial bonds of the octahedral structure.
Table 1. Crystal data and refinement parameters of (1).
CCDC deposit No. 783595
Empirical formula C14H24CuN3O10
Formula weight 457.90
Crystal system Triclinic
Colour/ Shape Blue/ Cubic
Space group P-1
Unit cell dimensions a (Å) – 7.835(5)
b (Å) – 10.581(5)
c(Å) – 12.341(5)
α(°) – 83.364(5)
β(°) – 77.146(5)
γ(°) – 72.254(5)
V ( Å3) 948.7(8)
F_ 000 476
Z 2
T(K) 293(2)
(Mo, K) (Å) 0.71069
range () 2.57–28.21
Limiting indices -9≤h≤9
-12 ≤k ≤13
-15 ≤l ≤13
Structure refinement ‘SHELXL-97(Sheldrick, 1997)’
Total reflections 3768
Reflections gt. 3392
No. of refined parameters 301
Goodness of fit on F2 1.061
Final R indices [I>2(I)] R1 = 0.0407, wR2 = 0.0.0965
R indices (all data) R1 = 0.0468, wR2 = 0.1067
100
Fig. 1. ORTEP view of the copper complex (1) with 50% probability of thermal ellipsoids.
Fig. 2. Packing diagram of the complex (1)
101
Table 2. Crystal data and refinement parameters.
CCDC deposit No. 857475
Empirical formula C14H22NiN3O9
Formula weight 452.69
Crystal system Triclinic
Colour/ Shape Blackish grey/ Cubic
Space group P-1
Unit cell dimensions a (Å) – 10.7439(10)
b (Å) – 12.6888(2)
c(Å) – 14.293(3)
α(°) – 74.921(3)
β(°) – 77.928(3)
γ(°) – 89.343(3)
V ( Å3) 1837.9(6)
F_ 000 872
Z 4
T(K) 293(2)
(Mo, K) (Å) 0.71073
range () 2.50–25.93
Limiting indices -12≤h≤13
-15 ≤k ≤8
-17≤l ≤17
Structure refinement ‘SHELXL-97(Sheldrick, 1997)’
Total reflections 6656
Reflections gt. 5157
No. of refined parameters 495
Goodness of fit on F2 1.092
Final R indices [I>2(I)] R1 = 0.0610, wR2 = 0.1678
R indices (all data) R1 = 0.0773, wR2 = 0.1927
102
Table 3. Selected bond distances (Å) and bond angles (0) of complexes (1) and (2)
Complex (1)
Bond lengths
Cu1- O3 1.956(2)
Cu1-N2 1.992(2)
Cu1-N3 2.017(2)
Cu1-N1 2.024(2)
Cu1-O1 2.233(2)
Bond angles
O3-Cu-N2 94.42(9)
O3-Cu1-N3 166.78(9)
N2-Cu1-N3 81.48(10)
O3-Cu1-N1 85.71(9)
N3-Cu1-N1 99.34(10)
O3-Cu1-O1 94.43(8)
Complex (2)
Bond lengths
Ni1-O4 2.055(3)
Ni1-N1 2.068(4)
Ni1-O2 2.059(3)
Ni1-N2 2.047(4)
Ni1-N3 2.072(4)
Ni1-O5 2.094(3)
Bond angles
O4-Ni1-N1 82.85(14)
O2-Ni1-N1 82.55(14)
N2-Ni1-N3 79.91(15)
N2-Ni1-O4 96.01(13)
103
Fig. 3. ORTEP view of the Nickel complex (2) with 50% probability of thermal
ellipsoids.
Fig. 4. Packing diagram of the complex (2).
104
Biological activities
Antibacterial studies
The newly prepared compounds were screened for their antibacterial activity
against Escherichia coli (K-12), Bacillus subtilis (MTCC-121), Staphylococcus aureus
(IOA-SA-22) and Salmonella Typhymurium (MTCC-98) bacterial stains by disc
diffusion method [23, 24]. The discs measuring 5mm in diameter were prepared from
whatman no.1 filter paper sterilized by dry heat at 142C for 1h. The sterile discs
previously soaked in a concentration of the test compounds were placed in a nutrient
agar medium. The plates were inverted and kept in an incubator at 31±1 C. The
inhibition zone thus formed was measured (in mm) after 24 h (Table 4). The screening
was performed at two different concentrations i.e. 1 µg/mL and 100 µg/mL and
ciprofloxacin was used as the standard. Minimum inhibitory concentrations (MICs)
were determined by broth dilution technique. The nutrient broth, which contained
logarithmic serially two fold diluted amount of test compounds and controls were
inoculated within approximately 5 × 105
c.f.u. of actively dividing bacteria cells.
Table 4. Zone of inhibition (mm) of the complexes.
The cultures were incubated for 24 h at 38 C and the growth was monitored visually
and spectrophotometrically. The lowest concentration (highest dilution) required to
arrest the growth of bacteria was regarded as minimum inhibitory concentration
Complex Escherichia
coli
Bacillus
subtilis
Staphylococcus
aureus
Salmonella
Typhymurium
Conc. (µg/ml) 1 100 1 100 1 100 1 100
(1) 35 12 31 17 34 16 27 15
(2) 10 5 10 13 0 0 0 0
(3) 16 12 16 13 10 6 14 12
(4) 0 0 14 12 13 4 0 0
105
(MIC). To obtain the minimum bacterial concentration (MBC), 0.1 mL volume was
taken from each tube and spread on agar plates. The number of c.f.u. was counted after
17–24 h of incubation at 35 C. MBC was defined as the lowest drug concentration at
which 99.9% of the inoculum was killed. The minimum inhibitory concentration and
minimum bacterial concentration are given in Table 5. The investigation of
antibacterial screening data revealed that all the tested compounds showed moderate to
good bacterial inhibition. Copper complex showed good inhibition against all bacteria
at 1 µg/mL concentration. Cobalt complex showed slightly less activity than that of
copper complex. Nickel complex was found to be inactive against staphylococcus
aureus and salmonella typhymurium while Manganese complex showed no activity
towards bacillus subtilis and salmonella typhymurium.
106
Table 5. MIC and MBC results of the complexes.
Ciprofloxacin is used as the standard. MIC (µg/ml) = minimum inhibitory concentration, i.e.
the lowest concentration to completely inhibit bacterial growth; MBC (µg/ml) = minimum
bactericidal concentration, i.e., the lowest concentration to completely kill bacteria.
Antifungal studies
The newly prepared complexes were screened for their antifungal activity
against Candida albicans, Aspergillus fumigatus and Penicillium marneffei by agar
diffusion method [25,26]. Sabourands agar media was prepared by dissolving peptone
(1g), D-glucose (4 g) and agar (2 g) in distilled water (100 mL) and adjusting pH to
5.9. Normal saline water was used to make suspension spore of fungal stain lawning.
A loopful of particular fungal stain was transferred to 3 mL saline to get suspension of
corresponding species. Twenty milliliters of agar media was poured into each petri
dish. Excess of suspension was decanted and plates were dried by placing in an
incubator at 38 C for 1h. Using an agar punch, wells were made and each well was
labeled. A control was also prepared in triplicate and maintained at 38 C for 3-6 days.
The fungal activity of each compound was compared with Greseofulvin as standard
Complex Escherichia
coli
Bacillus
subtilis
staphylococeus
aureus
Solmonella
typhimurium
MIC MBC MIC MBC MIC MBC MIC MBC
(1) 6.25 12.5 6.25 6.25 25 12.5 12.5 25
(2) 6.25 12.5 6.25 6.25 0 0 0 0
(3) 12.5 6.25 12.5 6.25 6.25 6.25 6.25 12.5
(4) 0 0 0 0 12.5 6.25 6.25 6.25
Standard 6.25 12.5 6.25 12.5 6.25 12.5 12.5 12.5
107
drug. Inhibition zones were measured and compared with controls. The fungal zones
of inhibition are given in Table 6.
Table 6. Zones of inhibition (mm) of the complexes
The cultures were incubated for 50 h at 35 C and the growth was monitored.
The lowest concentration (highest dilution) required to arrest the growth of fungus was
regarded as minimum inhibitory concentration (MIC). To obtain minimum fungicidal
concentration (MFC), 0.1 mL volume was taken from each tube and spread on agar
plates. The number of c.f.u. was counted as the lowest drug concentration at which
99% of the inoculum was killed. The minimum inhibitory concentration and minimum
fungicidal concentration are given in Table 7. The antifungal screening data showed
that only copper and cobalt complexes exhibited good activity. Nickel and Manganese
complexes are inactive towards Aspergillus fumingatus and Penicillium marneffei
respectively.
Complex Candida albicans Aspergillus
fumingatus
Penicillium
marneffei
(1) 29 30 21
(2) 17 15 16
(3) 1 13 17
(4) 13 10 0
108
Table 7. MIC and MFC results of the complexes
Greseofulvin is used as the standard. MIC (µg/ml) = minimum inhibitory concentration, i.e. the
lowest concentration to completely inhibit fungal growth; MFC (µg/ml) = minimum fungicidal
concentration, i.e., the lowest concentration to completely kill fungus.
Superoxide dismutase (SOD) activity
In order to test the application of the present compounds as possible
catalytic/scavangers for the toxic O2-. radicals which cause oxidative stress in human
body [27], the complex was examined for SOD mimic activity. The complex (1) was
tested for SOD mimic activity by the well known method [28]. It is stablished [29]
that mixed ligand ternary complexes analogous to complexes (1) show pronounced
SOD mimic activities. The present complex (1) exhibited considerable dismutation
activity [IC50 = 55.5 µmolar dm-3
]. This high activity can be rationalized in terms of
the ligand environment and coordination geometry around the Cu2+
ion.
Thus we found, from the biological screening data that the present complexes
(1-4) were screened for their antimicrobial properties and compared with that of
ciprofloxacin and greseofulvin as standared for antibacterial and antifungal screening,
respectively using a disc diffusion method for antibacterial screening and agar
diffusion method for antifungal screening. MICs were recorded as minimum
concentration of the compound, which inhibits the growth of tested microorganism.
Complex Candida albicans Aspergillus
fumingatus
Penicillium
marneffei
MIC MFC MIC MFC MIC MFC
(1) 6.25 6.25 6.25 12.5 6.25 12.5
(2) 6.25 6.25 0 0 6.25 12.5
(3) 6.25 12.5 6.25 6.25 6.25 6.25
(4) 6.25 6.25 6.25 12.5 0 0
Standard 6.25 12.5 6.25 12.5 6.25 12.5
109
Complexes (1) and (3) showed good activities while (2) and (4) were found to be less
active or inactive against some of the bacterial and fungal stains (Tables 5 and 7). The
copper complex (1) also exhibited considerable superoxide dismutase (SOD) activity.
110
CONCLUSION
Mixed-ligand complexes [M(imda)(bipy)]·6H2O (M = Cu),
[M(imda)(bipy)(H2O)]·4H2O (M = Ni, Co and Mn) where H2imda is the primary
ligand and α- diimine (2,2’-bipyridine) is the auxiliary chelator were synthesized and
variously characterized employing physico-chemical and spectral methods. The FT-IR
spectral data indicate the involvement of both the –COO- function in coordination to
metal ion but in asymmetric manner such that a five coordinate geometry is adopted
around Cu(II) while a saturated six coordinate geometry around the [Ni(II), Co(II) and
Mn(II)] ion due to the additional coordination from water. The EPR data indicate an
anisotropic g (i.e. g1, g2 and g3) values in complex (1). The single crystal X-ray data
for Cu(II) and Ni(II) complexes have confirmed the above described geometries
around the metal ion. The microbial screening data of the complexes suggest that
Cu(II) and Co(II) have good activity compared to Ni(II) and Mn(II) complexes. The
SOD activity measured for the Cu(II) complex indicate that it can be exploited as
antioxidant.
111
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