Studies of Newly Synthesized Octahedral Copper Complexes Based on Coumarins Along With Their...

ISSN No:APPLY FOR, V – 1, I – 1, 2014 Journal Club for Applied Sciences (JCAS)

Manuscript No: JCAS/RES/2014/10, Received On: 02/08/2014 , Revised On : 05/08/2014, Accepted On: 10/08/2014

RESEARCH ARTICLE

© All Rights Reserved by “Journals Club & Co.” 1

Studies of Newly Synthesized Octahedral Copper Complexes Based on Coumarins Along

With Their Antioxidant, Anti-Tubercular And Antimicrobial Activity Evaluation

Patel KA1, Patel SK2, Patel CJ1, Dholariya RH1, Patel DK* 1 Chemistry Department, V. P. & R. P. T. P. Science College, Sardar Patel University,

Vallabh Vidhyanagar – 388 120, Gujarat – India 2 Chemistry Department, Shree P. M. Patel Institute of PG studies and research in science, Anand – 388 001, Gujarat – India

ABSTRACT

Some newly heterochelates synthesized by reflux of 3-acetyl 4-hydroxy coumarin, Enrofloxacin

and transition metal. 1H, 13C, IR and ESI Mass confirm the formation of ligand. The

heterochelates were characterized on the basis of different spectroscopic techniques like IR

studies and elemental analysis while the geometry of complexes was octahedral which is

confirmed by electronic spectra analysis. The compounds were subjected to antimicrobial,

antioxidant and anti-tubercular activity viewing using serial broth dilution method and Minimum

Inhibitory Concentration (MIC) is determined.

KEYWORDS

Biological aspect, Enrofloxacin, Metal Chelates

INTRODUCTION Coumarins comprise a really giant category

of compounds found throughout the

kingdom Plantae. they're found at high

levels in some essential oils, notably

cinnamon bark oil (7,000 ppm), cassia leaf

oil (up to eighty seven,300 ppm) and

lavender oil. Coumarin is additionally found

in fruits (e.g. bilberry, cloudberry), tea and

different foods like chicory [1]. Most

coumarins occur in higher plants, with the

richest sources being the rue family and

carrot family. though distributed throughout

all elements of the plant, the coumarins

occur at the best levels within the fruits,

followed by the roots, stems and leaves.

*Address for Correspondence:

Atul K. Patel, Chemistry Department, V. P. & R. P. T. P. Science College, Sardar Patel University, Vallabh Vidhyanagar, Gujarat, India.


Environmental conditions and seasonal

changes will influence the incidence in

numerous elements of the plant. Recently

six new minor coumarins are isolated from

the fruits and also the stem bark of genus

Calophyllum dispar (Clusiaceae). The genus

Calophyllum, that includes two hundred

species, is cosmopolitan within the tropical

rain forest wherever many species square

measure employed in people drugs [2].

though most of the natural coumarins alive

are isolated from the upper plants, some

members are discovered in microorganisms.

Some vital coumarin members are isolated

from microorganism sources e.g. antibiotic

and coumermycin from actinomycete, and

aflatoxins from Aspergillus species.

Quinolones (quinolonecarboxylic acids or 4-

quinolones) are antibacterial drugs and are

commonly used as treatment for many

infections [3] since they can effectively

inhibit DNA replication. Enrofloxacin

(=Herx, Fig. 1) is a typical second-

generation quinolone antimicrobial drug

presenting a broad spectrum of activity

against a wide range of Gram-negative and

Gram-positive bacteria, including those

resistant to β-lactam antibiotics and

sulfonamid. Enrofloxacin is the first

fluoroquinolone developed for veterinary

application and is usually used for the

treatment of some urinary tract, respiratory

tract and skin infectious diseases in pets and

livestock.

Experimental

Materials

All reagents were of analytical reagent (AR)

grade purchased commercially from Spectro

chem. Ltd., Mumbai-India and used without

further purification. Solvents employed were

distilled, purified and dried by standard

procedures prior to use[4]. Clioquinol was

purchased from Agro Chemical Division,

Atul Ltd., Valsad-India. The metal nitrates

used were in hydrated form.

Physical Measurements

All reactions were monitored by thin-layer

chromatography (TLC on alluminium plates

coated with silica gel 60 F254, 0.25 mm

thickness, E. Merck, Mumbai-India) and

detection of the components were measured

under UV light or explore in Iodine

chamber. Carbon, hydrogen and nitrogen

were estimated by elemental analyzer

PerkinElmer, USA 2400-II CHN analyzer.

Metal ion analyses was carry out by the

dissolution of solid complex in hot

concentrated nitric acid, further diluting with

distilled water and filtered to remove the

precipitated organic ligands. Remaining

solution was neutralized with ammonia

solution and the metal ions were titrated


against EDTA. 1H and 13C NMR

measurements were carried out on Advance-

II 400 Bruker NMR spectrometer, SAIF,

Chandigarh. The chemical shifts were

measured with respect to TMS which used

as internal standard and DMSO-d6 used as

solvent.Infrared spectra of solids were

recorded in the region 4000-400 cm−1 on a

Nicolet Impact 400D Fourier-Transform

Infrared Spectrophotometer using KBr

pellets.The FAB mass spectrum of the

complex was recorded at SAIF, CDRI,

Lucknow with JEOL SX-102/DA-6000

mass spectrometer. Melting point of the

ligands and metal complexes were measured

by open capillary tube method. Solid state

magnetic susceptibility measurements were

carried out at room temperature using a

Gouy’s magnetic susceptibility balance with

mercury tetrathiocyanato cobaltate(II) being

used as a reference standard (g = 16.44×10−6

c.g.s. units). The electronic spectra were

collected using LAMBDA 19 UV/Vis/NIR

spectrophotometer in the region 200-1200

nm.

Synthesis of 4-hydroxy 3-acetyl coumarin

A mixture of 6-bromo salicylaldehyde (12.2

g, 0.1mol), ethyl acetoacetate (13.0 g,

0.1mol) and 3 to 4 drop piperidine were

stirred for 10 min. at room temperature in a

100 mL round bottom flask. After 10 min. it

was heated for 30 min in water bath. A

yellow solid obtained was taken out and

washed with cold ether. It was recrystallized

from chloroform-hexane. Yield: 92%;

M.p.119.5 °C.

Synthesis of Ligands (L1-L5)

The neutral bidentate ligands were

synthesized using Claisen-Schmidt

condensation. General procedure for

synthesis of the ligands (L) is shown in

Scheme 1. The ligands were characterized

using elemental analysis, FT-IR, Mass and

NMR (1H &13C) spectroscopy

.

Scheme 1: General Procedure for Synthesis of Ligands (L)


Synthesis of Metal Complexes

[Cu(L1)(En)(H2O)OH](C1) ]

An aqueous solution of Cu(NO3)2•3H2O salt

(10 mmol) was added into ethanolic

solution of ligand (L1) (10 mmol) and

subsequently an ethanolic solution of

ciprofloxacin (10 mmol) was added with

continuous stirring. Then the pH was

adjusted in between 4.5-6.0 by addition of

diluted NH4OH solution. The resulting

solution was refluxed for 5 h and then

heated over a steam bath to evaporate up to

half of the volume. The reaction mixture

was kept overnight at room temperature. A

fine coloured crystalline product was

obtained. The obtained product was washed

with ether and dried over vacuum

desiccators.

Complexes C2-C5 was prepared according to

same method and their physicochemical

parameters are summarized in Table 1. The

synthetic protocol of complexes is shown in

Scheme 2,

SCHEME 2 : General Procedure for Synthesis of Complexe (C)

Antimicrobial Activity

The synthesized ligand and corresponding

metal(II) complexes were screened in vitro

for their antibacterial activity against two

Gram(+ve)Streptococcus pyogenes, Bacillus

subtilis and two Gram(−ve) Escherichia coli,

Pseudomonas aeruginosa, where antifungal

againstCandida albicansand Aspergillus

nige rusing the broth dilution method. All

the ATCC culture was collected from

institute of microbial technology, Bangalore.

2% Luria broth solution was prepared in


distilled water while, pH of the solution was

adjusted to 7.4±0.2 at room temperature and

sterilized by autoclaving at 15 lb pressure

for 25 min. The tested bacterial and fungal

strains were prepared in the luria broth and

incubated at 37 °C and 200 rpm in an orbital

incubator for overnight. Sample solutions

were prepared in DMSO for concentration

200, 150, 100, 50, 25, 12.5, 6.25 and 3.125,

µg/mL. The standard drug solution of

Streptomycin (antibacterial drug) and

Nystatin (antifungal drug) were prepared in

DMSO. Serial broth micro dilution was

adopted as a reference method. 10 µl

solution of test compound was inoculated in

5 mL luria broth for each concentration

respectively and additionally one test tubes

was kept as control. Each of the test tubes

was inoculated with a suspension of

standard microorganism to be tested and

incubated at 35 °C for 24 h. At the end of

the incubation period, the tubes were

examined for the turbidity. Turbidity in the

test tubes indicated that microorganism

growth has not inhibited by the antibiotic

contained in the medium at the test

concentration. The antimicrobial activity

tests were run in triplicate.

Anti-tubercular Activity

Test compounds were evaluated for in vitro

antimycobacterial activity. The MICs were

determined and interpreted for M.

tuberculosis H37Rv according to the

procedure of the approved microdilution

reference method of antimicrobial

susceptibility testing [5]. Compounds were

taken at concentrations of 100, 50, 25, 12.5

6.25 and 3.125 µg/mL in 2% DMF. M.

tuberculosis H37Rv strain was used in

Middle brook 7H-9 broth which was

inoculated with standard as well as test

compounds and incubated at 37 °C for 4

weeks. The bottles were inspected for

growth twice a week for a period of 3

weeks. Readings were taken at the end of

fourth week. The appearance of turbidity

was considered as bacterial growth and

indicates resistance to the compound. The

growth was confirmed by making a smear

from each bottle and performing a ZN stain.

Test compounds were compared to reference

drugs Ethambutol (MIC=3.25µg/mL) and

the antimicrobial and anti-tubercular activity

tests were run in triplicate.

Antioxidant Studies

Ferric reducing antioxidant power (FRAP)

was measured by a modified method of

Benzie and Strain [6]. The antioxidant

potentials of the compounds were estimated

as their power to reduce the TPTZ-Fe(III)

complex to TPTZ-Fe(II) complex (FRAP

assay), which is simple, fast, and


reproducible. FRAP working solution was

prepared by mixing a 25.0 mL, 10 mM

TPTZ solution in 40 mM HCl, 20 mM

FeCl3.6H2O and 25 mL, 0.3 M acetate buffer

at pH 3.6. A mixture of 40.0 mL, 0.5 mM

sample solution and 1.2 mL FRAP reagent

was incubated at 37 °C for 15 min.

Absorbance of intensive blue colour [Fe(II)-

TPTZ] complex was measured at 593 nm.

The ascorbic acid was used as a standard

antioxidant compound. The results are

expressed as ascorbic equivalent (mmol/100

g of dried compound). All the tests were run

in triplicate and are expressed as the mean

and standard deviation (SD) .

RESULT AND DISCUSSION The synthesized Cu(II) complexes were

characterized by elemental analysis, FTIR

and mass spectra, The metal ion in their

complexes were determined after

mineralization. The metal content in

chemical analysis was estimated by

complexometrically[7], while geometry of

the complexes was confirmed from

electronic spectra.

Elemental Analysis

The analytical and physiochemical data of

the complexes are summarized in Table

1.The experimental data were in very good

agreement with the calculated ones. The

complexes were colored, insoluble in water

and commonly organic solvents while

soluble in DMSO as well as stable in air.

The structure of the complexes is assumed

according to the chemical reaction as shown

below;

Cu(NO3)2 • 3 H2O + L + EN ----------->

[Cu(L)(EN)(H2O)OH] • 3H2O + 2HNO3

FT-IR spectra

A comparison of the IR spectra of the

complexes with those of the free ligands

reveals interesting features relating to the

metal-ligand interactions. IR spectroscopy

has been used in order to confirm the

deprotonation and binding mode of

enrofloxacin. The bands at ~1732 cm−1 and

~1253 cm−1 attributed to the stretching

vibrations ν(C=O)carboxylic and ν(C-O)carboxylic

respectively, of the carboxylic moiety (–

COOH) of enrofloxacin, have been shifted

in the range ~1580-1600 cm−1 and ~1370-

1385 cm−1 assigned as antisymmetric,

ν(C=O)asym, and symmetric, ν(C=O)sym,

stretching vibrations of the carboxylato

group, respectively. The difference

Δ=[ν(C=O)asym−ν(C=O)sym], a useful

characteristic tool for determining the

coordination mode of the carboxylato

ligands, reaches a value of ~220–240 cm−1

indicative of a monodentate coordination

mode [8]. Whereas ν(C=O)p is shifted from

~1620–1645 cm−1 upon bonding. The


overall changes of the IR spectrum suggest

that enrofloxacin and ligands are

coordinated to the copper via the ketone

oxygen and carboxylato oxyge. These

changes in the IR spectra suggest that

enrofloxacin is coordinated to metal via

pyridone and one carboxylate oxygen atoms.

These data are further supported by ν(Cu–O)

which appearat ∼515 cm−1 [9]. The IR

spectra of the coumarin derivatives shows

~1612 and ~1745 cm−1bands corresponding

to α, β-unsaturated ketone and lactone

carbonyl ketone respectively, on

complexation these peaks shifted to a lower

frequency ~1600 and ~1730 cm−1due to

complex formation.

Table 2 : FT-IR data of Synthesized Compounds

Comp.

υ(O–

H)br cm-

1

υ(COO)sy υ(COO)asy

α, β-

unsaturated

υ(C=O) s cm-1

lactone

carbonyl

υ(C=O) s

cm-1

υ(C=O)

of

pyridone

υ(M–O)w

cm-1

C1 3432 1378 1593 1606 1727 1623 513

C2 3436 1374 1585 1613 1736 1622 531

C3 3421 1373 1587 1603 1725 1639 525

C4 3444 1373 1581 1605 1714 1627 538

C5 3443 1376 1576 1601 1722 1636 532 s = strong, w = weak, br = broad

Electronic Spectra and Magnetic

Measurement

Electronic spectral data along with magnetic

susceptibility measurements gave sufficient

support to determine the geometry of metal

complexes. The electronic spectra of

complexes were recorded in DMF solution

with scan range 200-1200 nm. Usually

octahedral geometry was found for Cu(II)

complexes[10], then again, several copper

compounds show temperature dependent

geometric distortions and single copper ions

in a host lattice of regular symmetry may

reveal interesting spectroscopic properties.

The electronic spectra of hexa coordinate

Cu(II) complexes were either D4h or C4v

symmetry, the Eg and T2g level of 2D free

ion term will split into B1g, A1g, B2g and Eg

levels, respectively under the influence of

the distortion, Which cause the two

transitions such as 2B1g→2B2g and 2B1g→2A1g. This promotes the distorted

octahedral Cu(II) complex which was usual

in the d9 system. The electronic spectra of


Cu(II) complexes (C1-C5) display three

prominent bands. Low intensity broad band

in the region 16,800-17,700 cm−1 was

assigned as 10 Dq band corresponding to 2Eg→2T2g transition. In addition, there was a

high intensity band in the region 22,610-

27,000 cm−1. This band was due to

symmetry forbidden ligand → metal charge

transfer transition. The band above 27,000

cm−1 was assigned as ligand band. Therefore

distorted octahedral geometry around Cu(II)

ion was suggested on the basis of electronic

spectra, which was further revealed by its

magnetic moment of 1.79-1.86 BM falls

within the range generally observed for

octahedral Cu(II) complexes. Therefore the

electronic spectral data and magnetic

moment data support the octahedral

geometry of the all complexes

Table 3 : Electronic Spectral Data of the Complexes

Compounds Transition band observed (cm-1) effB.M Geometry

C1 17,265 25,220 1.82 Octahedral C2 17,340 25,240 1.79 Octahedral C3 17,370 25,300 1.86 Octahedral C4 17,290 25,345 1.93 Octahedral C5 17,230 25,310 1.85 Octahedral

Antimicrobial Bioassay

All synthesized compounds were evaluated

for their antimicrobial activity against

various microorganisms such as E. coli, P.

aeruginosa,S. pyogenes, B. subtilis, A. niger

and C. albicans using Kirby- Bauer disk

diffusion method and results were compared

with standard drugs as summarized in (table

4). Kolokolov et al. have suggested that the

transition metal complexes with biologically

active ligands frequently exhibit higher

biological activity and lower toxicity than

initial ligands, which makes possible their

use in medicine and biochemistry. Raman et

al. have reported that complex exhibit

higher antimicrobial activity than free

ligands. An increased in activity of metal

chelates can be explained on the basis of

chelation theory, according to which

polarities of ligands and central metal atoms

are reduced through charge equilibration

over whole chelate ring. This increases

lipophilic character of metal chelate and

favours its permeation through lipid layer of

bacterial membranes [11]. .


Table 4 : Antimicrobial and Antioxidant Activities of Synthesized Compounds

Antimicrobial Activity (Minimal Inhibition Concentration, in µg/mL) Antioxidant

Activity Anti-

tubercular

activity a

Entry

Gram negative bacteria Gram positive

bacteria Fungus

FRAP

value(mmol/10

0 g)

E. coli

P.

aeruginosa

S.

pyogenes

B.

subtili

s

C.

albicans

A.

niger

L1 400 200 200 600 200 200 NT 25

L2 200 200 400 400 200 400 NT 25

L3 100 200 100 200 200 200 NT 25

L4 400 400 400 >600 400 200 NT 25

L5 100 100 100 200 200 200 NT 25

C1 100 200 100 200 100 100 76.86 6.25

C2 40 70 70 40 100 100 83.94 12.5

C3 70 100 70 100 70 100 83.33 25

C4 100 100 100 200 100 100 55.02 12.5

C5 70 100 100 100 100 100 62.90 6.25

Ciprofloxacin 20 10 20 05 NT NT NT NT

Ethambutol NT NT NT NT NT NT NT 3.25

Norfloxacin 10 10 10 10 NT NT NT NT

Flucanazole NT NT NT NT 10 10 NT NT

Nystatin NT NT NT NT 100 100 NT NT

E. Coli= ATCC25922; P. aeruginosa= ATCC25619; S. pyogenes= ATCC12384 ; B. subtilis= ATCC11774 ;C.albicans= ATCC 66027; A.niger= ATCC 64958

NT= Not tested

The results were compared with those of the

standard drug. All the metal complexes were

more potent bactericides than the ligand. C1

complex was much less microbially active

than the other complexes.

Antioxidant Studies

From Table 4, it can be seen that the highest

inhibition of growth occurred on C2 complex

against the microorganism, while C3, C4 and


C5 shows enhance activity than C1 but less

potent than C2. A capacity to transfer a

single electron i.e. the antioxidant power of

all compounds was determined by a FRAP

assay. The FRAP value was expressed as an

equivalent of standard antioxidant ascorbic

acid (mmol/100 g of dried compound).

FRAP values indicate that all the

compounds have a ferric reducing

antioxidant power. The compounds C2 and

C3 showed relatively high antioxidant

activity while compound C1, C4 and C5

shows poor antioxidant power (Table 4).

In conclusion, the antimicrobial testing

results reveal that complexes possess higher

activity at lower concentration compared to

parent ligand. It is known that chelation

tends to make the Schiff bases more

powerful and potent bacteriostatic agents

CONCLUSION Here elucidate the synthesis of biological

active coumarin derivatives (L1-L5) and their

Cu(II) complexes (C1-C5). The structures of

the ligands were investigated and confirmed

by the elemental analysis, FT-IR, 1H-NMR, 13C-NMR and mass spectral studies.

Complexes shows momentous effective

antioxidant activities compared to their

ligands employed for complexation.

ACKNOWLEDGEMENT

We are grateful to The Principal, and

management of V. P. & R. P. T. P. Science

College, Vallabh Vidyanagar and authors

are also thnksfull to shree P.M. Patel

institute of P.G. Studies and research in

science, anand for providing infrastructures

and obligatory facilities for research. We are

thankful to The Director, SICART, Vallabh

Vidyanagar for analytical facilities. Authors

are also grateful to The Director, SAIF-

RSIC, Panjab University, Chandigarh for

NMR analysis.

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HOW TO CITE THIS ARTICLE

Patel, K, A., Patel, S, K., Patel, C, J., Dholariya, R, H., Patel, D, K. (2014). Studies of Newly Synthesized Octahedral Copper Complexes Based on Coumarins Along With Their Antioxidant, Anti-Tubercular And Antimicrobial Activity Evaluation. Journal Club for Applied Sciences (JCAS), 1(I), 1-11

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