Synthesis and spectral characterization of Zn(II) microsphere series for antimicrobial application

Article abstract of DOI:10.1016/j.molstruc.2014.06.009

Microsphere series have been synthesized by reacting zinc(II) acetate dihydrate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole/oxadiazole/triazole with salicylaldehyde. Elemental analysis suggests that the complexes have 1:2 and 1:1 stoichiometry of the type [Zn(L)₂(H₂O)₂] and [Zn(L′)(H ₂O)₂]; LH = Schiff bases derived from 2-hydrazino-5- [substituted phenyl]-1,3,4-thia/oxadiazole with salicylaldehyde; L′H ₂ = Schiff bases derived from 3-(substituted phenyl)-4-amino-5- hydrazino-1,2,4-triazole and salicylaldehyde and were characterized by elemental analyses, IR, ¹H NMR and ¹³C NMR spectral data. Scanning electron microscopy (SEM) showed that synthesized materials have microsphere like structure and there EDX analysis comparably matches with elemental analysis. For the antimicrobial application Schiff bases and their zinc(II) complexes were screened for four bacteria e.g. Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus pyogenes and four fungi e.g. Cyrtomium falcatum, Aspergillus niger, Fusarium oxysporium and Curvularia pallescence by the reported method. Schiff base and Zn(II) compounds showed significant antimicrobial activities. However, activities increase upon chelation. Thermal analysis (TGA) data of compound (10) showed its stability up to 300 °C.

Full text of DOI:10.1016/j.molstruc.2014.06.009

Journal of Molecular Structure 1074 (2014) 376–383
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis and spectral characterization of Zn(II) microsphere series
for antimicrobial application
⇑
Ajay K. Singh, Sarvesh K. Pandey, O.P. Pandey, S.K. Sengupta
Department of Chemistry, DDU Gorakhpur University, Gorakhpur 273009, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
ꢀ
ꢀ
Nano-sized zinc(II) complexes were
synthesized and well characterized.
Schiff base and Zn(II) complexes
showed significant antimicrobial
activities.
ꢀ
Zn(II) complexes look like a
microsphere and their sizes are
1–2 lm.
Zinc mapping
Mix mapping
a r t i c l e i n f o
a b s t r a c t
Article history:
Microsphere series have been synthesized by reacting zinc(II) acetate dihydrate with Schiff bases derived
from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole/oxadiazole/triazole with salicylaldehyde. Ele-
mental analysis suggests that the complexes have 1:2 and 1:1 stoichiometry of the type [Zn(L)2(H2O)2]
and [Zn(L0)(H2O)2]; LH = Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thia/oxadi-
azole with salicylaldehyde; L0H2 = Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydra-
zino-1,2,4-triazole and salicylaldehyde and were characterized by elemental analyses, IR, 1H NMR and
13C NMR spectral data. Scanning electron microscopy (SEM) showed that synthesized materials have
microsphere like structure and there EDX analysis comparably matches with elemental analysis. For
the antimicrobial application Schiff bases and their zinc(II) complexes were screened for four bacteria
e.g. Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus pyogenes and four fungi e.g.
Cyrtomium falcatum, Aspergillus niger, Fusarium oxysporium and Curvularia pallescence by the reported
method. Schiff base and Zn(II) compounds showed significant antimicrobial activities. However, activities
increase upon chelation. Thermal analysis (TGA) data of compound (10) showed its stability up to 300 °C.
Ó 2014 Elsevier B.V. All rights reserved.
Received 14 March 2014
Received in revised form 3 June 2014
Accepted 3 June 2014
Available online 12 June 2014
Keywords:
Schiff bases
Zinc(II)
SEM
TGA
EDX
Antimicrobial activity
Introduction
particular, zinc salicylate has been approved by the US Food and
Drug Administration for use as an anti-oxidant, carbonless copying
Zinc has been known to be an essential trace element in eukary-
otes for more than a century [1]. There is 2–3 g of zinc in adult
humans, making it one of the most prevalent ‘‘trace’’ elements. In
paper and stabiliser for polymers [2]. Metal zinc complexes
became attractive for their interesting properties such as antimi-
crobial [3], enzymatic activity [4], personal care [5], anti-HIV drug
[6], and photoluminescence as well as electroluminescence, etc.
[7]. In other hand 1,3,4-thiadiazoles and their Schiff bases have
received significant importance because of their diverse
⇑
Corresponding author. Tel.: +91 551 2203621.
E-mail address: sengupta@hotmail.co.uk (S.K. Sengupta).
http://dx.doi.org/10.1016/j.molstruc.2014.06.009
0022-2860/Ó 2014 Elsevier B.V. All rights reserved.

A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
377
Erba 1108 models. The thermal degradation processes and stabili-
ties of the zinc complexes was investigated using a thermo-gravi-
metric analyzer (Mettler Toledo TGA/SDTA851 with Star software)
under a nitrogen atmosphere with a heating rate of 10 °C/min from
30 to 500 °C. For scanning electron microscopy (SEM), gold sputter
coating were carried out on desired zinc(II) complexes samples at
pressure ranging in between 1 and 0.1 Pa. Sample was loaded in
the machine, which was operated at 10ꢁ2 to 10ꢁ3 Pa with EHT
15.00 kV with 300 V collector bias using Leo microscope SEMs
were recorded. Transmission electron microscope (TEM) analysis
for zinc complexes was performed by a JEOL 1200EX. The JEOL
1200EX TEM with tungsten, operated at an accelerating voltage
of up to 120 kV. The optical densities of microbial solutions were
evaluated by using a VARIAN 50 bio UV–Vis spectrophotometer
instruments. Zinc was estimated gravimetrically as its dioxide
ZnO2 [32]. A known weight of the compound was decomposed
by concentrated nitric acid and the mass was extracted with dis-
tilled water, sodium carbonate solution was added. The precipitate
obtained was filtered by Whatmann filter paper No. 41, and finally
ignited in silica crucible to zinc oxide. Elemental analysis (C, H, N,
Zn) indicates that the found and calculated values were within
acceptable limits ( 0.5). Molar conductance of 10ꢁ3 M solutions
of the complexes in DMSO was recorded on a Hanna EC 215 con-
ductivity meter by using 0.01 M KCl water solution as calibrant.
The purity of compounds was checked by thin layer chromatogra-
phy on silica gel plate using ether and ethyl acetate as a solvent
system. Iodine chamber was used as a developing chamber.
(a)
(b)
Fig. 1. Oxadiazole derived compound patented for; (a) anti-HIV drugs; (b) anti-
cancer drugs.
biochemical, antifungal and antibacterial application [8,9].
Oxadiazole derivatives have been found to possess tuberculostatic
[10], antiviral [11], antibacterial [12,13], antimalarial [14], fungi-
cidal [15], anticonvulsant [16], anti-inflammatory [17], analgesic
[18,19] and insecticidal [20] activities. 7-(-5-{4-fluorobenzyl}-1,3,
General procedure for the synthesis of zinc(II) complexes
The route for the preparation of zinc(II) complexes is shown in
Fig. 2.
4-oxadiazole-2-yl)-8-hydroxy-N-(2-hydroxym
ethyl)-1,6-naph-
thyri-dine-5-caboxamide (Fig. 1a) have been patented for the HIV
integrase inhibition [21] and 2-{5-(3-florophenyl)-1,3,4-oxadiaz-
ole-2-yl}-N-(pyridin-4-ylmethyl) aniline (Fig. 1) patented as anti-
cancer agent [22]. Triazoles and their derivatives are found to be
associated with various biological activities, such as anticonvul-
sant, antifungal, anticancer, anti-inflammatory and antibacterial
properties [23–27]. Several compounds containing 1,2,4-triazole
ring are well known for drugs and there amino derived Schiff bases
well known for antimicrobial properties [28–30]. Salicylaldehyde
isonicotinoyl hydrazone was patented for protection against reti-
nal disease [31].
Synthesis of Schiff base hydrazone derived from 5-(substituted aryl)-2-
hydrazino-1,3,4-(thiadiazole, oxadiazole) and salicylaldehyde
(L1H–L8H)
A mixture of 5-[substituted aryl]-2-hydrazino-1,3,4-(thia/oxa-
diazole) and salicylaldehyde in 1:1 M ratio respectively was
refluxed in ethanol (30 cm3) containing few drops of concentrated
sulphuric acid for 5–6 h [33]. The product separated on evapora-
tion of the ethanol was recrystallized from ethanol–ether mixture
(1:1).
Synthesis of Schiff base hydrazone derived from 5-(substituted aryl)-3-
hydrazino-1,2,4-(triazole) and salicylaldehyde (L09H2–L012H2)
In this paper, we report the synthesis, spectroscopic, micro-
scopic, thermal study and antimicrobial activities of zinc(II) com-
plexes with Schiff bases derived from substituted heterocycles
(thiadiazole, oxadiazole and triazole) with salicylaldehyde.
A
mixture of 3-(substituted phenyl)-4-amino-5-hydrazino-
1,2,4-triazole and salicylaldehyde in 1:2 M ratio in an ethanol
(30 cm3) containing a few drops of concentrated sulphuric acid
was refluxed for ca. 4–7 h [34]. On concentrating the solution up
to ꢂ10 cm3, crystals appeared which were filtered and recrystal-
lized from ethanol–ether mixture (1:1).
Experimental
Reagents
Synthesis of zinc(II) complexes (1–8)
An ethanolic solution (30 cm3) of zinc(II) acetate dihydrate
(0.01 mol) was added to a refluxing solution of appropriate Schiff
base (L1H–L8H) (0.02 mol) in ethanol (30 cm3) containing alcoholic
solution of sodium acetate (0.02 mol). The reaction mixture was
refluxed for about 8–13 h. The coloured complex was obtained.
The complex was filtered off, washed thoroughly with ethanol
and dried under vacuo.
All reagents were purchased from Aldrich, solvents used were
purified by proper methods and Milli-Q water was used for all
experiments.
Instruments
Melting points were determined on a Buchi 530 apparatus in
open capillary tubes. IR spectra were recorded on a Shimadzu
8201 PC model FT IR spectrophotometer as KBr disks. 1H and 13C
NMR spectra were recorded on a Bruker DRX-300 spectrometer
using DMSO-d6 as solvent. Chemical shifts (d) are reported in parts
per million (ppm) relative to an internal standard of Me4Si.
Elemental analyses were recorded by Elementar Vario EL III Carlo
Complex (1). Yield 70%; colour: cream; decomp. temp. 210–212 °C;
IR (KBr) cmꢁ1: 3490 (AOH), 3165 (ANH), 2950 (ArAH), 1620 (C@N
azomethine), 1580 (C@N thiadiazole), 1545 (C@C), 1348 (CAO),
1155 (NAN), 1050 (CASAC), 805, 745 (AOH wagging and rocking),
485 (ZnAO), 435 (ZnAN); 1H NMR (DMSO-d6): d 12.45 (s, 2H,

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A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
Fig. 2. Reagents and conditions (a) CS2 + KOH, ice cold stirring/conc. H2SO4; for the preparation of thiadiazole (b) CS2 + KOH/refluxed; for the preparation of oxadiazole; (c)
NH2NH2ꢃ2H2O, refluxed ca. 5–7 h (d) salicylaldehyde, refluxed ca. 5–6 h (e) zinc acetate dihydrate/CH3COONa (f) CS2 + KOH/NH2NH2ꢃ2H2O, refluxed ca. 5–8 h (g)
NH2NH2ꢃ2H2O, refluxed ca. 5–6 h (h) salicylaldehyde, refluxed ca. 4–7 h.
ANH), 6.95–7.40 (m, 18H, ArAH), 5.55 (s, 4H, H2O), 8.25 (s, 2H); 13
C
Complex (3). Yield 70%; colour: dark yellow; decomp. temp. 244–
246 °C; IR (KBr) cmꢁ1: 3450 (AOH), 3200 (ANH), 3050 (ArAH),
1605 (C@N azomethine), 1560 (C@N thiadiazole), 1540 (C@C),
1345 (CAO), 1140 (NAN), 1046 (CASAC), 822, 748 (AOH wagging
and rocking), 770 (CACl), 488 (ZnAO), 435 (ZnAN); 1H NMR
(DMSO-d6): d 12.52 (s, 2H, ANH), 7.06–7.65 (m, 16H, ArAH), 5.45
(s, 4H, H2O), 8.31 (s, 2H); 13C NMR DMSO-d6): d 175.8, 174.0,
166.5, 158.3, 135.1, 132.2, 131.8, 131.3, 130.7, 129.6, 122.1,
116.6, 115.5; C30H24N8O4S2Cl2Zn: C, 47.35; H, 3.18; N, 14.72; S,
8.41; Zn, 8.60; Found: C, 47.27; H, 3.14; N, 14.61; S, 8.40; Zn, 8.52.
NMR (DMSO-d6): d 175.0, 174.7, 166.8, 158.6, 134.3, 132.8, 131.8,
130.7, 130.3, 129.3, 121.9, 116.9, 115.0; % Anal. Cal. for C30H26N8
O4S2Zn: C, 52.60; H, 3.79; N, 16.19; S, 9.27; Zn, 9.45; Found: C,
52.51; H, 3.79; N, 16.25; S, 9.10; Zn, 9.33.
Complex (2). Yield 63%; colour: light yellow; decomp. temp. 228–
232 °C; IR (KBr) cmꢁ1: 3475 (AOH), 3182 (ANH), 2980 (ArAH),
1605 (C@N azomethine), 1570 (C@N thiadiazole), 1542 (C@C),
1350 (CAO), 1142 (NAN), 1060 (CASAC), 815, 736 (AOH wagging
and rocking), 770 (CACl), 490 (ZnAO), 445 (ZnAN); 1H NMR
(DMSO-d6): d 12.46 (s, 2H, ANH), 7.00–7.55 (m, 16H, ArAH), 5.45
(s, 4H, H2O), 8.17 (s, 2H); 13C NMR (DMSO-d6): d 175.5, 174.6,
166.2, 158.0, 138.2, 133.7, 131.9, 131.6, 130.7, 130.0, 129.5,
128.8, 121.1, 116.0, 115.7; % Anal. Cal. for C30H24N8O4S2Cl2Zn: C,
47.35; H, 3.18; N, 14.72; S, 8.41; Zn, 8.60; Found: C, 47.23; H,
3.13; N, 14.67; S, 8.43; Zn, 8.53.
Complex (4). Yield 66%; colour: brown; decomp. temp. 248–250 °C;
IR (KBr) cmꢁ1: 3425 (AOH), 3190 (ANH), 2982 (ArAH), 1610 (C@N
azomethine), 1565 (C@N thiadiazole), 1540 (C@C), 1515 (CANO2),
1345 (CAO), 1150 (NAN), 1055 (CASAC), 805, 750 (AOH wagging
and rocking), 490 (ZnAO), 445 (ZnAN); 1H NMR (DMSO-d6): d
12.44 (s, 2H, ANH), 6.90–7.40 (m, 16H, ArAH), 5.46 (s, 4H, H2O),

A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
379
8.22 (s, 2H); 13C NMR (DMSO-d6): d 175.0, 174.6, 166.8, 158.0,
150.1, 140.2, 131.2, 131.0, 129.7, 124.6, 122.1, 116.8, 115.3; Anal.
Cal. for C30H24N10 O10S2Zn: C, 46.07; H, 3.09; N, 17.91; S, 8.20;
Zn, 8.36; Found: C, 46.00; H, 3.11; N, 17.81; S, 8.04; Zn, 8.12.
129.0, 127.5, 121.7, 116.9, 114.9; Anal. Cal. for C22H20N6O4Zn: C,
53.08; H, 4.05; N, 16.88; Zn, 13.14; Found: C, 53.12; H, 4.01; N,
16.73; Zn, 13.03.
Complex (10). Yield 75%; colour: light yellow; decomp. temp. 255–
258 °C; IR (KBr) cmꢁ1: 3460 (AOH), 3180 (ANH), 3040 (ArAH),
1612 (C@N azomethine), 1545 (C@N triazole), 1540 (C@C), 1145
(NAN), 815, 720 (AOH wagging and rocking), 762 (CACl), 485
(ZnAO), 440 (ZnAN); 1H NMR (DMSO-d6): d 12.56 (s, 1H, ANH),
6.90–7.55 (m, 12H, ArAH), 5.45 (s, 4H, H2O), 8.27 (s, 2H); 13C
NMR (DMSO-d6): d 166.4, 159.0, 158.1, 157.2, 152.7, 139.7, 133.4,
130.8, 130.5, 130.0, 129.6, 128.7, 122.6, 115.7, 114.8; Anal. Cal.
for C22H19N6O4ClZn: C, 49.64; H, 3.60; N, 15.79; Zn, 12.29; Found:
C, 49.55; H, 3.53; N, 15.70; Zn, 12.31.
Complex (5). Yield 78%; colour: light orange; decomp. temp. 218–
222 °C; IR (KBr) cmꢁ1: 3420 (AOH), 3180 (ANH), 2955 (ArAH),
1615 (C@N azomethine), 1585 (C@N oxadiazole), 1545 (C@C),
1340 (CAO), 1170 (NAN), 1080 (CAOAC), 810, 740 (AOH wagging
and rocking), 492 (ZnAO), 435 (ZnAN); 1H NMR (DMSO-d6): d
12.50 (s, 2H, ANH), 7.00–7.60 (m, 18H, ArAH), 5.45 (s, 4H, H2O),
8.30 (s, 2H); 13C NMR (DMSO-d6): d 170.2, 168.6, 165.5, 158.6,
131.6, 131.4, 131.9, 130.7, 129.4, 127.8, 122.6, 115.9, 114.0; Anal.
Cal. for C30H26N8O6Zn: C, 54.60; H, 3.97; N, 16.98; Zn, 9.91; Found:
C, 54.38; H, 3.83; N, 16.90; Zn, 9.77.
Complex (11). Yield 72%; colour: light yellow; decomp. temp. 256–
260 °C; IR (KBr) cmꢁ1: 3465 (AOH), 3195 (ANH), 3030 (ArAH),
1608 (C@N azomethine), 1575 (C@N triazole), 1540 (C@C), 1365
(CAO), 1160 (NAN), 815, 748 (AOH wagging and rocking), 777
(CACl), 495 (ZnAO), 435 (ZnAN); 1H NMR (DMSO-d6): d 12.55 (s,
1H, ANH), 6.95–7.45 (m, 12H, ArAH), 5.48 (s, 4H, H2O), 8.31 (s,
2H); 13C NMR (DMSO-d6): d 166.7, 159.8, 158.0, 157.5, 152.0,
135.6, 131.7, 131.4, 130.8, 129.6, 129.4, 128.3, 122.4, 115.7,
114.7; Anal. Cal. for C22H19N6O4ClZn: C, 49.64; H, 3.60; N, 15.79;
Zn, 12.29; Found: C, 49.58; H, 3.51; N, 15.85; Zn, 12.22.
Complex (6). Yield 71%; colour: orange; decomp. temp. 200–
202 °C; IR (KBr) cmꢁ1: 3485 (AOH), 3205 (ANH), 2970 (ArAH),
1605 (C@N azomethine), 1560 (C@N oxadiazole), 1542 (C@C),
1355 (CAO), 1140 (NAN), 1080 (CAOAC), 825, 758 (AOH wagging
and rocking), 772 (CACl), 485 (ZnAO), 440 (ZnAN); 1H NMR
(DMSO-d6): d 12.48 (s, 2H, ANH), 7.00–7.55 (m, 16H, ArAH), 5.45
(s, 4H, H2O), 8.26 (s, 2H); 13C NMR (DMSO-d6): d 170.9, 168.1,
165.0, 158.3, 137.8, 133.4, 130.8, 130.4, 130.0, 129.1, 128.6,
127.8, 122.6, 116.7, 115.8; Anal. Cal. for C30H24N8O6Cl2 Zn: C,
49.44; H, 3.32; N, 15.37; Zn, 8.97; Found: C, 49.31; H, 3.38; N,
15.25; Zn, 8.90.
Complex (12). Yield 66%; colour: yellow; decomp. temp. 248–
250 °C; IR (KBr) cmꢁ1: 3425 (AOH), 3210 (ANH), 2950 (ArAH),
1615 (C@N azomethine), 1515 (C@N triazole), 1550 (C@C), 1520
(CANO2), 1350 (CAO), 1140 (NAN), 825, 760 (AOH wagging and
rocking), 480 (ZnAO), 435 (ZnAN); 1H NMR (DMSO-d6): d 12.44
(s, 2H, ANH), 7.04–7.44 (m, 12H, ArAH), 5.46 (s, 4H, H2O), 8.30
(s, 2H); 13C NMR (DMSO-d6): d 166.5, 159.3, 158.8, 157.3, 152.6,
148.6, 137.4, 131.7, 130.5, 128.1, 124.4, 122.6, 116.7, 115.3; Anal.
Cal. for C22H24N10O6Zn: C, 48.68; H, 3.53; N, 18.06; Zn, 12.05;
Found: C, 48.59; H, 3.53; N, 18.16; Zn, 12.00.
Complex (7). Yield 65%; colour: light brown; decomp. temp. 216–
218 °C; IR (KBr) cmꢁ1: 3455 (AOH), 3195 (ANH), 3040 (ArAH),
1612 (C@N azomethine), 1570 (C@N oxadiazole), 1530 (C@C),
1340 (CAO), 1145 (NAN), 1075 (CAOAC), 820, 755 (AOH wagging
and rocking), 770 (CACl), 495 (ZnAO), 448 (ZnAN); 1H NMR
(DMSO-d6): d 12.52 (s, 2H, ANH), 7.04–7.55 (m, 16H, ArAH), 5.50
(s, 4H, H2O), 8.41 (s, 2H); 13C NMR (DMSO-d6): d 170.6, 168.2,
165.6, 158.4, 135.6, 130.8, 130.6, 129.7, 129.0, 125.3, 123.4,
115.7, 114.0; Anal. Cal. for C30H24N8O6Cl2 Zn: C, 49.44; H, 3.32;
N, 15.37; Zn, 8.97; Found: C, 49.28; H, 3.34; N, 15.20; Zn, 8.80.
Biological activity
Complex (8). Yield 66%; colour: brown; decomp. temp. 214–260 °C;
IR (KBr) cmꢁ1: 3430 (AOH), 3210 (ANH), 2950 (ArAH), 1605 (C@N
azomethine), 1565 (C@N oxadiazole), 1555 (C@C), 1510 (CANO2),
1360 (CAO), 1155 (NAN), 1087 (CAOAC), 830, 720 (AOH wagging
and rocking), 480 (ZnAO), 435 (ZnAN); 1H NMR (DMSO-d6): d
12.54 (s, 2H, ANH), 7.04–7.60 (m, 16H, ArAH), 5.51 (s, 4H, H2O),
8.32 (s, 2H); 13C NMR (DMSO-d6): d 170.9, 168.1, 165.1, 158.4,
148.6, 133.4, 131.9, 131.5, 130.1, 129.4, 122.6, 115.7, 114.3; Anal.
Cal. for C30H24N10O10Zn: C, 48.04; H, 3.23; N, 18.68; Zn, 8.72;
Found: C, 48.00; H, 3.01; N, 18.47; Zn, 8.55.
The antimicrobial properties of the Schiff bases (L1H–L8H and
L09H2–L012H2) and there Zn(II) complexes were tested against four
fungal strains Cyrtomium falcatum, Aspergillus niger, Fusarium oxys-
porium and Curvularia pallescence and four bacteria namely Bacillus
subtilis, Pseudomonas Aeruginosa, Salmonella typhi and Streptococcus
pyogenes. Bacteria/fungi are potentially hazardous and care has
been taken while working with them. Standard bio safety lab tech-
niques were followed while handling bacteria/fungi and various
media. Gloves were used during all experimentation, and any acci-
dental spills were immediately sterilized using 70% isopropanol/
water followed by bleach. The work area was also sterilized with
70% isopropanol/water after completion of work. Unused media
and bacterial suspensions were first deactivated with commercial
bleach for 1 h before being disposed in bio safety bags. All material
that had come in contact with bacteria (e.g., pipette tips, tubes,
agar plates, etc.) was also thrown in bio safety bags in tightly
closed bins. Bio safety bags were autoclaved for 2 h before final
disposal.
Synthesis of zinc(II) complexes (9–12)
An ethanolic solution (30 cm3) of zinc(II) acetate dihydrate
(0.01 mol) was added to a refluxing solution of appropriate Schiff
base (L09H2–L012H2) (0.01 mol) in ethanol (30 cm3) containing alco-
holic solution of sodium acetate (0.02 mol). The reaction mixture
was refluxed for about 8–13 h. The coloured complex was
obtained. The complex was filtered off, washed thoroughly with
ethanol and dried under vacuo.
All newly prepared Schiff bases and Zn(II) complexes were
screened for their antimicrobial activity against four fungi and four
bacteria by reported method [35,36]. Microbial activity of each
compound was evaluated at three different concentrations, i.e.,
Complex (9). Yield 70%; colour: cream; decomp. temp. 260–262 °C;
IR (KBr) cmꢁ1: 3450 (AOH), 3165 (ANH), 2940 (ArAH), 1612 (C@N
azomethine), 1555 (C@N triazole), 1545 (C@C), 1330 (CAO), 1155
(NAN), 800, 732 (AOH wagging and rocking), 490 (ZnAO), 420
(ZnAN); 1H NMR (DMSO-d6): d 12.45 (s, 1H, ANH), 7.05–7.60 (m,
13H, ArAH), 5.45 (s, 4H, H2O), 8.25 (s, 2H); 13C NMR (DMSO-d6):
d 166.0, 159.4, 158.7, 157.4, 151.6, 132.3, 130.8, 130.4, 129.3,
10, 100 and 200 lg/ml. Petridishes of equal diameter were steril-
ised at 180 °C. Solution of 1 ml of each concentration was poured
in presterilised petridishes and 9 ml of LB agar was added immedi-
ately. Each dish was rotated on the table top in order to achieve
thorough mixing of medium with the compound. After this,

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A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
bacterial strains were inoculated in the dishes (diameter 5 mm).
These set were then inoculated at appropriate temperature. The
colony diameter of the test organism was measured with mm scale
after 6 days. The percentage inhibition of the growth of the test
organism was calculated by following formula
Results and discussion
All the final compounds (1–12) were obtained in good yields.
The physical properties of these compounds are mentioned in
experimental section and the synthetic route is given in Fig. 2.
% Inhibition ¼ 100ðCd ꢁ TdÞ=Cd
Infrared spectra
where Cd is the colony diameter of control set; and Td is the colony
diameter of treated set. To ensure that solvent had no effect on fun-
gal and bacterial growth, a control test was performed with test
medium supplemented with DMSO at the same dilutions as used
in the experiment.
The IR spectra provide valuable information regarding the nat-
ure of the functional groups attached to the metal atom. The spec-
tra of Schiff bases of type (L1H–L8H) show a medium band at ca.
m(NAH) which remains almost at the
same position in the complexes indicating the non-involvement
3210–3165 cmꢁ1 due to
(A)
(B)
2 µ
m
1 µ
m
(C)
(D)
200 nm
µ
m
10
Element
C K
Weight%
49.77
Atomic%
62.16
20.84
13.32
3.00
(E)
O K
17.16
NK
16.89
Zn K
Totals
13.08
100.00
Fig. 3. SEM image of zinc(II) compound 10 and EDX performance; (A) low magnification SEM image; (B) high magnification image; (C) zinc mapping shown by red colour dot;
(D) carbon, oxygen, nitrogen mix mapping represent by blue, red, and green colour dot; (E) EDX performed results shown in table and also present in bar diagram. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
381
of (NAH) group in bond formation [29]. The ligands show one med-
Proton magnetic resonance spectra
ium intensity band at ca. 1635 cmꢁ1 assignable to
m(C@N) which
shifts to lower frequency (ca. 20–15 cmꢁ1) in the complexes [36].
This shift indicates the coordination of azomethine nitrogen to
metal ion [37–39]; the band at ca. 448–420 cmꢁ1 is assigned to
The proton magnetic resonance spectra of these complexes
have been recorded in DMSO-d6. The intensities of all the reso-
nance lines were determined by planimetric integration. The fol-
lowing conclusions can be derived by comparing the spectra of
ligands with their corresponding complexes. The spectra of Schiff
bases exhibit signals at 12.50 and 8.05 ppm due to NH and azome-
thine protons, respectively. In zinc(II) complexes (1–12), the first
signal remains almost at the same position but the second signal
shifts to downfield. The downfield shift indicates the deshielding
effect due to the co-ordination of azomethine nitrogen to central
metal ion. The Schiff bases shows ca. 10.40 ppm signal due to phe-
nolic AOH these signals disappears in their corresponding com-
plexes (1–12), this confirms that the hydroxyl group reacted with
metal ion via deprotonation. Schiff bases and their corresponding
Zn(II) complexes show multiplet at 6.90–7.60 ppm due to aromatic
protons. Zinc(II) complexes (1–12) show new signal at about
5.5 ppm due to the water protons.
m
(ZnAN). Schiff bases show a broad band at ca. 2660 cmꢁ1 due to
intramolecular H-bonded OH group This band disappears in their
corresponding Zn(II) complexes indicating the coordination of phe-
nolic oxygen to metal ion through deprotonation. This is further
supported by shift in phenolic CAO band from ca. 1290 cmꢁ1 (in
the free ligands) to ca. 1350 cmꢁ1 in the complexes. The coordina-
tion through phenolic oxygen is further confirmed by the appear-
ance of band at ca. 480 cmꢁ1 assignable to
[40]. The Schiff bases type (L1H–L4H) shows strong band at ca.
1050 cmꢁ1 due to the
(CASAC) vibration; this position remains
m(ZnAO) phenolic
m
unchanged in their corresponding Zn(II) complexes type (1–4),
indicating non-coordination of thiadiazole ring sulphur to metal
atom. The
m(CAOAC) vibration appears as a strong band at ca.
1080 cmꢁ1 in the free ligands type (L5H–L8H) [41]; the position
of which also remains the same in their corresponding complexes
type (5–8), indicating non-coordination of oxadiazole ring oxygen
to metal atom. The presence of coordinated water molecules in
these complexes is indicated by a broad band in the region
3490–3420 cmꢁ1 and two weaker bands in the region ca. 830–
13C NMR spectra
The 13C NMR spectra were recorded in DMSO-d6. Schiff bases
derived from thiadiazole and oxadiazole (L1H–L8H) show signals
at ca. d 164 for their azomethine carbon which shift downfield in
their corresponding zinc(II) complexes (1–8) due to coordination
through the azomethine nitrogen. Schiff bases of type (L1H–L4H)
show signals at ca. d 175, 174 due to the thiadiazole ring carbon
and oxadiazole derived Schiff bases (L5H–L8H) show signals at d
170, 168. These signals remain unchanged in their corresponding
complexes indicating that thiadiazole and oxadiazole ring nitrogen
are not participated in bond formation.
805 and 760–720 cmꢁ1 due to
m(AOH) rocking and wagging modes
of vibrations, respectively [42].
100
TGA
DTG
IV
0.05
Schiff bases derived from hydrazino triazole (V–VI) show signal
at d 164 and 157 for azomethine carbon and which shift downfield
in zinc metal complexes due to coordination through the azome-
thine nitrogen. For triazole ring carbons, two signals appear at ca.
d 158, 157 which remain unchanged in their corresponding com-
plexes (9–12) indicating that triazole ring carbons are not partici-
pated in bond formation. For aromatic ring, a number of signals
were appeared.
I
II
III
0.00
90
80
70
60
50
-0.05
-0.10
-0.15
-0.20
-0.25
-0.30
Microscopic characterization
The compound (10) was dissolved in DMSO and solution with
concentration 100 lg/ml was made. One drop of sample was paste
in aluminium grid and dried under IR lamp. Typical SEM image of
the compound 10 was taken and result obtained is shown in Fig. 3A
and B. The compound (10) looks like microsphere. For the
100
200
300
400
500
Temperature (oC)
Fig. 4. TGA and DTGA curve for zinc(II) complex 10.
Table 1
Antifungal results of Zn(II) complexes (1–12) and standard drug at concentration (
l
g/ml).
Compound
% Fungicidal inhibition in
lg/ml concentration
C. falcatum
A. niger
F. oxysporum
C. pallescence
200
100
10
44
200
100
10
46
200
100
10
43
200
100
10
42
1
2
3
4
5
6
7
8
9
10
11
12
72
82
77
79
74
79
77
75
74
85
78
80
100
64
73
67
69
60
68
65
56
67
75
69
70
100
80
86
82
84
75
83
79
78
81
89
84
83
100
64
75
69
66
65
67
64
63
66
73
70
72
100
77
82
80
76
70
76
75
72
81
87
83
82
100
64
72
66
67
58
68
64
69
63
72
66
69
100
75
84
82
83
72
79
76
75
75
86
78
77
100
62
68
63
65
57
62
60
63
62
69
65
66
100
55
49
51
41
52
45
49
46
62
55
57
100
55
52
54
44
50
46
47
48
59
53
55
54
48
50
45
51
47
49
44
56
57
58
100
51
47
44
37
47
43
44
43
54
49
51
100
Fluconazole
100

382
A.K. Singh et al. / Journal of Molecular Structure 1074 (2014) 376–383
elemental visualization EDX mapping were performed and results
obtained are shown in Fig. 3C and D. Zinc elemental mapping of
compound (10) show that Zn element equivalently distributed in
microsphere (Fig. 3C). For the confirmation of other element avail-
ability in the microsphere mix elemental mapping were performed
and results obtained are systematized in Fig. 3D. EDX performed
results are systematically arranged in Fig. 3E, and these results
are closely comparable to elemental analysis results.
Thermal analysis
Thermal stability of compound (10) was analysed by thermo-
gravimetric analysis (TGA) (Fig. 4) and which shows four steps
weight losses. The first decomposition step ranges from 50 to
100 °C showing loss of 11.18 percentage mass involving losing
bound water (moisture) and second step ranges from 100 to
220 °C with loss of 2.5 weight percent indicating that compound
has coordinated water [43]. Subsequently, in stage III and IV (a
range from 220 to 366 °C and 366 to 500 °C) weight losses are
22.63% and 16.13%, respectively. Third and four step weight losses
indicate the decomposition of Schiff bases. At 500 °C temperature,
47.56 mass percentage remained.
Fig. 5. In vitro antifungal spectrum of compounds 2, 6, 10 and fluconazole (Std.) at
200 g/ml concentration.
l
Antimicrobial activity
The Schiff bases are found to be biologically active and their
corresponding Zn(II) complexes show significantly enhanced anti-
fungal (Table 1) and antibacterial (Table 2) activities. As chelation
increases, bacterial and fungal growth inhibition also increases.
Actual mechanism of increased activity of complexes is not certain
but factors like solubility, dipole moment and cell permeability
mechanism and their enzymatic action may be the possible reason.
According to Overtone’s concept of cell permeability, the lipid
membrane surrounding the cell favours the passage of lipid-solu-
ble materials, making the solubility an important factor controlling
the antimicrobial activity [44]. Tweedy’s chelation theory the
polarity of the metal ion will be reduced to a greater extent due
to the overlap of the ligand orbital and partial sharing of the posi-
tive charge of the metal ion with donor groups. Further, it increases
the delocalization of
p-electrons over the whole chelate ring and
enhances the lipophilicity of the hetero chelates. The increased
lipophilicity enhances the penetration of the hetero chelates into
lipid membranes and blocks the metal binding sites in the enzymes
of microorganisms. These hetero chelates also disturb the respira-
tion process of the cell and block the synthesis of proteins, which
actually restricts further growth of the organisms. Furthermore,
the mode of action comprising the compounds may involve the
Fig. 6. In vitro antibacterial spectrum of compounds 2, 6, 10 and streptomycin (Std.)
at 200
l
g/ml concentration.
Table 2
Antibacterial results of Zn(II) complexes (1–12) and standard drug at concentration (lg/ml).
Compounds
% Antibacterial inhibition in
l
g/ml concentration
P. aeruginosa
200
B. subtilis
S. pyogenes
S. typhi
200
100
10
27
100
10
20
200
100
10
25
200
100
10
31
1
2
3
4
5
6
7
8
9
10
11
12
69
77
71
74
66
75
72
68
72
83
79
77
100
48
55
51
50
43
50
46
45
51
56
53
52
100
63
71
69
66
60
69
64
68
64
74
72
76
100
54
59
57
58
47
54
51
50
54
63
59
57
100
69
75
69
73
61
65
60
65
68
78
72
70
100
47
58
55
54
40
52
45
48
54
60
58
54
100
76
86
79
81
70
81
76
79
78
88
83
81
100
54
62
59
57
49
55
52
53
62
66
63
63
100
30
27
26
23
27
25
24
29
35
30
29
100
30
26
24
22
27
25
23
26
33
29
27
100
29
27
26
20
25
23
22
26
32
30
29
100
40
35
36
27
33
29
31
37
43
39
37
100
Ciprofloxacin

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Conclusions
Schiff bases (L1H–L8H) are monobasic, bidentate ligands coordi-
nating through azomethine nitrogen and phenolate oxygen atom
(NO donor). Schiff bases (L09H2–L012H2) are dibasic, tetradentate
ligands coordinating through azomethine nitrogen atoms and phe-
nolate oxygen atoms (N2O2 donor). The complexes are insoluble in
all common organic solvent except DMF and DMSO. The structures
of Schiff bases and complexes have been established by elemental
analysis and spectral studies IR, 1H NMR and 13C NMR. All these
data puts together leads us to propose the structure of zinc(II)
complexes shown in Fig. 2. The presence of coordinated water mol-
ecules in complexes has been confirmed by TGA data. Scanning
electron microscope image showed that zinc complexes look like
a microsphere and their sizes are 1–2 lm. Antifungal and antibac-
terial activities of the ligands and corresponding complexes have
also been evaluated which showed that the activities increase on
chelation.
Acknowledgements
The authors thank to University Grants Commission, New Delhi,
India for financial support. Authors are also thankful to Department
of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur, for help
in evaluating antibacterial activities.
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