Chapter 4 - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/28515/11/11...Chapter 4 Reactivity...

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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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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.

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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

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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

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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

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Fig. 1. ORTEP view of the copper complex (1) with 50% probability of thermal ellipsoids.

Fig. 2. Packing diagram of the complex (1)

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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

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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)

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Fig. 3. ORTEP view of the Nickel complex (2) with 50% probability of thermal

ellipsoids.

Fig. 4. Packing diagram of the complex (2).

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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

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(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.

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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

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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

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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

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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.

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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.

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