Synthesis and bio-evaluation of 2-imino-4-amino thiazole capped silver nanoparticles

Article abstract of DOI:10.14233/ajchem.2016.19457

Silver nanoparticles capped with N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)benzamide is synthesized by simple chemical reduction method, prepared by treatment of aqueous silver ions with hot ethanolic solution of N-(4-amino-3,5-diphenyl-3H-thiazol-2-yl

Full text of DOI:10.14233/ajchem.2016.19457

Asian Journal of Chemistry; Vol. 28, No. 3 (2016), 658-662
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19457
Synthesis and Bio-evaluation of 2-Imino-4-amino thiazole Capped Silver Nanoparticles
1,*
1
2
R. SARADA , V. JAGANNADHARAO and B. SYAMA SUNDAR
1Department of Chemistry, Anil Neerukonda Institute of Technology and Sciences, Sangivalasa-531 162, India
2Vice-Chancellor, Yogi Vemana University, Kadapa-516 216, India
*Corresponding author: E-mail: ramarajusrd@gmail.com
Received: 11 July 2015;
Accepted: 13 August 2015;
Published online: 5 December 2015;
AJC-17670
Silver nanoparticles capped with N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)benzamide is synthesized by simple chemical reduction
method, prepared by treatment of aqueous silver ions with hot ethanolic solution of N-(4-amino-3,5-diphenyl-3H-thiazol-2-
ylidine)benzamide. The size and shape of obtained silver nanoparticles are spherical and uniform with average size of 34 nm and are
characterized by using UV-visible, FTIR, XRD, SEM and TEM studies. The capped silver nanoparticle showed significant inhibitory
activity against various human pathogenic bacteria with different doses of 25, 50, 75 and 100 µg. The synthesized silver nanoparticle
capped compound was potent inhibitory agent when compared with standard antibiotic ciprofloxacin.
Keywords: Antimicrobial, Capping agent, Silver nanoparticles, Triazole derivatives.
not only seen as building blocks in natural products, but are
also considered to be extraordinarily useful scaffolds, especially
in combinatorial and medicinal chemistry [11-15]. Further-
more, thiazolines have interesting applications in agriculture
as acaricides, insecticides and plant growth regulators [16,17].
In general the synthesis of 2-iminothiazoline derivatives
was achieved by the reaction of thiourea derivatives with
various substituted α-bromoketone in the presence of suitable
base [18,19]. Here also the similar reaction procedure was
adopted for the synthesis of 4-amino-2-iminothiazole from
the reaction of aroylthiourea with bromo benzyl cyanide.
As it was known from the literature that triethylamine
works as best base for this kind of transformation [20], the
same base was used in this method. The thiourea derivatives
were synthesized using the previous standard procedure. The
product was fully characterized using all spectroscopic analysis
INTRODUCTION
2-Iminothiazoline derivatives are important 5-membered
heterocyclic compounds have proven to be a structural feature
providing a broad spectrum of biological activity. These are
playing key role in the development of drugs for hypertension
[1], inflammation [2], cancer therapies [3], etc. Due to their
inherent low toxicities and good pharmacokinetic profile, these
compounds have been recognized as privileged structural
motif in medicinal chemistry. Recently, 2-imino-thiazolines
were found to have antifungal activity [4] and skin whitening
properties [5]. The pifithrin (Pft-α) was isolated by screen of
chemical libraries having 2-iminothiazoline skeleton is the lead
compound of p53 in activators and have received increasing
attention due to their possible applications in several major neuro-
degenerative disorders such asAlzheimer’s disease, Parkinson’s
disease, stroke, cancers therapy and other pathologies related
to various signaling pathways [6-9]. In this present work the
derivative of 2-imino thiozoline i.e., N-(4-amino-3,5-diphenyl-
3H-thiazol-2-ylidine)benzamide is used as a capping agent to
control the size of silver nanoparticles and stabilize them. The
capping agent also enhances the biological activity of silver
nanoparticles
1
(IR, H NMR, 13C NMR and mass). In the IR spectrum, the
characteristic peak for NH2 was observed at 3340-3222 cm-1
and C=N was observed at 1590-1550 cm-1. In 1H NMR, NH2
appeared as broad singlet at δ 3.5 ppm.
EXPERIMENTAL
All the reagents used were of AR grade. Silver nitrate
was obtained from National Refinery Pvt. Ltd. and a 0.1M
aqueous solution was used as stock solution. Sodium boro-
hydroxide was obtained from Merck, India. Organic-free water
was used throughout the experiment.
Synthesis and characterization of N-(4-amino-3,5-
diphenyl-3H-thiazol-2-ylidine)benzamide: 2-Iminothiazo-
line derivatives are important class of 5-membered heterocyclic
compounds, because of their wide applications [10]. They are

Vol. 28, No. 3 (2016)
Synthesis and Bio-evaluation of 2-Imino-4-amino thiazole Capped Silver Nanoparticles 659
The UV-visible spectra were recorded on a Shimadzu UV-
visible spectrophotometer and the solutions were taken in a
1 cm well stoppered quartz cuvette. Fourier transform infrared
(FTIR) spectral characteristics of the samples were collected
on a Shimadzu FTIR spectrometer with the samples as KBr
pellets. The FTIR spectrum was recorded over 45 scans of
each sample and the background spectrum was automatically
subtracted. The formation of single-phase compound was
checked by X-ray diffraction (XRD) technique. The XRD
pattern was taken with X-ray diffractometer (XPERT-PRO) at
room temperature, using CuKα radiation λ = 1.5406 Å over a
wide range of Bragg angles (30° ≤ 2θ ≤ 85°). SEM micrograph
of N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)benzamide
capped silver nanoparticles was obtained on a NOVA-230 with
an operating voltage of 10 KV. For SEM analysis, the specimen
was suspended in distilled water, dispersed ultrasonically to
separate individual particles and one or two drop of the suspen-
sion deposited onto holey-carbon coated copper grids and dried
under infrared lamp.
Synthesis: To 10 mmol of thiourea derivative in acetoni-
trile, triethyl amine (10.50 eqiv.) was added and stirred at room
temperature (25-30 °C) for 10 min until the solution turns to
pale yellow. To that 10.10 mmol of bromobenzylcyanide
(which was dissolved in 1 mL acetonitrile) was added drop
wise for 10 min, then a yellow precipitate was obtained after
15 min. The solvent was concentrated under reduced pressure
to separate solid material; it was filtered and washed with
hexane to get rid of the excess triethyl amine. The obtained
yellow coloured crystalline compound was purified and charac-
terized (Scheme-I).
The formation, morphology, photo physical and biological
activity of the N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)-
benzamide capped silver nanoparticles are reported. The silver
nanoparticles with uniform shapes and sizes can be obtained by
a simple chemical reaction of silver idodide (AgI) and sodium
borohydride. The advantage of the method is ease of preparation,
convenience in use and especially the obtained silver nanopar-
ticles are uniform in their shapes and sizes. The shapes and sizes
of the metal nanoparticles are significant parameters for their
bio-evaluation measurements. Furthermore, UV-visible spectro-
scopy was employed to determine the optimum conditions for
the preparation of stable silver colloids. Time-dependent UV-
visible spectroscopy and Scanning electron microscopy (SEM) are
employed to monitor the process of silver nanoparticles forma-
tion. There is a change in the shape of the silver nanoparticles
during the formation of the nanoparticles was observed. This may
be helpful in understanding the growth of the nanoparticles and
creates a new dimension in controlling the shapes of nanoparticles.
Antimicrobial studies of 2-imino-4-amino thiazole and their
silver nanoparticles
Human pathogenic bacteria: Human pathogenic
bacteria species Salmonella typhi, Vibrio cholera, Shigella
dysenteriae, Staphylococcus aureus are used in this study.
These were collected from Department of Microbiology,
Andhra Medical College, Visakhapatnam, India.
Preparation of bacterial inoculums: The microorga-
nisms were inoculated into Muller Hinton broth and incubated
at 35 2 °C for 4 h. The turbidity of the resulting suspensions
was diluted with MH broth to obtain a transmittance of 25 %
at 580 nm. That percentage was found spectrophotometrically
comparable to 1 McFarland turbidity standard. This level of
turbidity is equivalent to approximately 3.0 × 108 CFU/mL.
The Bausch & Lomb® spectrophotometer, Model Spectronic
20 was used to adjust the transmittance of the working suspen-
sions. This suspension used as inoculums.
Characterization of N-(4-Amino-3,5-diphenyl-3H-
thiazol-2-ylidine)benzamide (1a): m.p.: 78-80 °C; IR (KBr,
ν
max, cm-1): 3220, 1670,1598,1560,1219; 1H NMR (CDCl3 400
MHz): δ 6.0 (S, 2H, NH2), 6.98.0 (m, 15H, aromat); 13 C NMR
(CDCl3 100 MHz): δ 95.3, 124.1, 126.7, 127.2, 129.8, 131.2,
133.7, 135.5, 136.8, 137.6, 167.0, 174.4, 178.5.
Agar-well diffusion assay: The modified agar-well
diffusion method of Perez et al. [21] was employed. Each selec-
tive medium was inoculated with the microorganism suspended
in Muller Hinton broth. Once the agar was solidified, it was
punched with a 6 mm diameter wells and filled with required
concentration of compounds. Ciprofloxacin (antibiotic) used as
standard for positive control while pure solvents were used as
negative control. Results were determined based on size of the
inhibitory zone surrounding the wells containing the extract, on
comparing with standard and blank. The diameter of zones of
inhibition was measured in mm using Hi-Media zone reader.
Minimum inhibitory concentration:The minimum inhibi-
tory concentration of synthesized silver-nanoparticle was deter-
mined using broth dilution assay. The medium containing
different concentrations of compounds viz., 100 mg to 100 µg
per mL prepared by serial dilution (10-1 dilution).After inocu-
lation of culture, the tubes were incubated for 24 h at 37 °C.
The MIC of each sample was determined by measuring the
optical density in the spectrophotometer (Electronics India) at
580 nm and compared the result with those of the non-inocu-
lated broth used as blank. Control was prepared with media
and inoculums only without compounds. The experiment was
conducted according to NCCLS standards.
Br
H2N
TEA
N
S
NH NH
CN
N
MeCN, rt
+
S
O
O
1a
Scheme-I: Synthetic approach
Synthesis of silver nanoparticle assembly
N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)-
benzamide capped silver nanoparticles: A total of 2.5 mL
of 10-2 MAgNO3 was added to 75 mL of triply distilled organic-
free water, to this 5 mL of 10-2 M N-(4-amino-3,5-diphenyl-
3H-thiazol-2-ylidine)benzamide (dissolved in hot EtOH) was
added as stabilizer with stirring. After 10 min of mixing, 2.5
mL of 10-2 M KI was added drop wise into the solution slowly,
until a green yellowAgI colloid was obtained. The silver colloid
was finally obtained when 20 mg of NaBH4 was added to the
AgI colloidal solution and the reaction mixture was continually
stirred for about 20 min. During the whole reaction, the colour
of the colloidal solution changed from green-yellow to nut-
brown at the beginning, then to brown and finally to black.

660 Sarada et al.
RESULTS AND DISCUSSION
Asian J. Chem.
are noticed under SEM. The difference in size is possibly due
to the fact that the nanoparticles are being formed at different
times. Scanning electron microscopic (SEM) measurement of
the synthesized nanoparticles shows that they are in spherical
shape.
X-ray diffraction: XRD spectra of silver nanoparticles
measured in the small and wide angle region are shown in
Fig. 3. The XRD spectrum confirms the tendency of nano-
particles to form the organized structures, as seen from the
peaks in the small angle XRD spectrum. The peaks are
broadened because of the nano crystalline nature of silver
nanoparticles. By comparing with standard database values,
all the peaks can be indexed to face-centered cubic (fcc) silver
crystal structure. Three peaks at 2y values of 38.299, 64.853
and 77.542, correspond to the (1 1 1), (2 0 0) and (2 2 0) planes
of silver nanoparticle, respectively.
UV-visible spectroscopy: Fig. 1 shows UV-visible spectra
of the obtained silver colloids. The surface plasmon resonance
(SPR) band is broad indicating poly-dispersed nanoparticles.
A smooth and narrow absorption bands at 434 nm is observed
for N-(4-amino-3,5-diphenyl-3H-thiazol-2-ylidine)benzamide
capped silver nanoparticles. The optical absorption spectra of
metal nanoparticles are dominated by surface plasmon reso-
nances (SPR), which shift to longer wavelengths with increase
of particle size. The position and shape of plasmon absorption
of silver nanoclusters are strongly dependent on the particle
size, dielectric medium and surface-adsorbed species. The
surface plasmon absorption of silver nanoparticles have the short
wavelength band in the visible region around 409 nm is due to
the transverse electronic oscillation.
434 nm
0.5
Silver nanoparticle
600
0.4
0.3
0.2
0.1
0
400
200
300
340
380
420
460
500
540
580
620
Wavelength (nm)
Fig. 1. UV-visible absorbance spectra of 2-imino-4-amino thiazole capped
silver nanoparticles in DMSO
Scanning electron microscopy: The SEM images of
synthesized 2-imino-4-amino thiazole capped silver nanopar-
ticles shown in Fig. 2, indicates that the particles are nearly
crystalline and spherical.
20
30
40
50
(°)
60
70
80
2
θ
Fig. 3. XRD patterns 2-imino-4-amino thiazole capped silver nanoparticles
From this Fig. 3, it can be noticed that the silver particles
capped with 2-imino-4-amino thiazole appeared basically
amorphous and abroad. Size-dependent and structure-specific
features in diffraction patterns can be quite striking in nano
meter-sized particles. Small particles have fairly distinct diff-
raction patterns, both as a function of size and as a function
of structure type. In general, regardless of structure, there is a
steady evolution in the aspect of diffraction profiles, as particles
become larger no abrupt changes occur but more details will
be resolved when features grow continuously from the diff-
raction profile. These observations form the basis for a direct
technique of diffraction pattern analysis that can be used to
obtain structural information from experimental diffraction
data. The silver particles are almost crystalline with the appea-
rance of diffraction peaks at the scattering angles (2y) at 38-
39, which could be indexed to the scattering from the planes
(111), (200), (220) and (311), respectively. It is well known
that with diminishing crystallite size the measured XRD pattern
exhibits broadening and very often overlapping reflections.
The broadening of the reflections is inversely proportional to
the crystallite size (i.e. size of coherently diffracting domains).
The relation is known as Scherrer’s equation where “y” is the
diffraction angle of a particular reflection. The total diffracted
intensity for a given Bragg reflection from a crystallite is the
Fig. 2. SEM images of synthesized 2-imino-4-amino thiazole capped silver
nanoparticles
The Scherrer rings, characteristic of fcc silver is clearly
observed and showing that the structure seen in the SEM image
is nanocrystalline in nature. It is also observed that the silver
nanoparticles are scattered over the surface and no aggregates

Vol. 28, No. 3 (2016)
Synthesis and Bio-evaluation of 2-Imino-4-amino thiazole Capped Silver Nanoparticles 661
sum of independently diffracted intensities by each of the unit-
cell columns making-up the crystallite. It means that the
calculated size distribution is in fact a distribution of diffraction
column lengths in a given crystallographic direction perpen-
dicular to the diffraction planes and not of crystallite (coherently
diffracting domains) sizes. Theoretical considerations show
that the interference function of a polycrystalline or nano
crystalline solid is identical to that of an arrangement of isolated
particles with the same size or size distribution as those of the
polycrystalline or nano crystalline solid. Thus the values of
Scherrer’s formula are solely an estimate of a volume-weighted
average column length. This explains the difference between
the experimental and theoretical values and the values from
the Scherrer’s formula is termed ‘‘apparent crystallite size’’.
FT-IR analysis: The IR spectra shown in Fig. 4. the silver
nanoparticles capped by 2-imino-4-amino thiazole and the free
2-imino-4-amino thiazole molecule are similar to one another,
indicating that the organic molecules have indeed become a
part of the nanoparticles. However, there is remarkable diffe-
rence in the peak intensity found between the peaks of IR
spectra of free 2-imino-4-amino thiazole and 2-imino-4-amino
thiazole capped silver nanoparticles. The reason for this inten-
sity difference between the spectra is believed to be the amino
molecules on the nano particle, forming a relatively closely
packed amino layer and thereby the molecular motion being
constrained. Thus, this steric constraining effect on the trans-
verse mode (rocking mode, wagging mode, etc.) is stronger
than the longitudinal mode (stretching mode, etc). Therefore,
the change of the peak intensity of the longitudinal modes is
smaller than that of the transverse mode. The C–N stretching
mode is due to the position of the C–N bond nearest to the
surface of the silver particle and a chemical bond can form
between N and Ag atoms.
electron microscopy. A sample of silver nanoparticles from a
freshly synthesized clear yellow sol was prepared by drying a
small drop on a carbon-coated 200-mesh copper grid. The TEM
image of one region of the sample is shown in Fig. 5. The
TEM image shows the silver particles are spherical with sizes
of 34 2 nm.
Fig. 5. Transmission electron micrographs of the silver nanoparticles used
in this work (a) The bar marker represents 20 nm
Antibacterial activities: The synthesized 2-imino-4-
amino thiazole capped silver nanoparticles showed significant
inhibitory activity against various human pathogenic bacteria
species, like S. typhi, V. cholera, S. dysenteriae and E. faecalis.
It was found that, the synthesized silver nanoparticle compound
was potent inhibitory agent when compared with standard
antibiotic. 13mm was the highest zone of inhibition showed
by compound against S. typhi and S. dysenteriae (Table-1). E.
faecaulis and V. cholerae showed sensitivity to compounds
when compared with compounds inhibitory potential against
S. typhi and S. dysenteriae. From Table-2, synthesized silver
nanoparticle compound showed dose dependent inhibitory
activity. Zone of inhibition was increases with concentration
of compound. 1mg/mL is the lowest MIC of compound against
V. cholera. The results are comparable with antibiotic cipro-
floxacin.
4500
4000
3500
3000 2500
2000
1500
1000
500
Wavelength (cm–1)
Fig. 4. FT-IR spectra of 2-imino-4-amino thiazole capped silver nanoparticles
TABLE-1
From the spectrum of free 2-imino-4-amino thiazole, it
can be noticed that the azo group appeared in 1620-1597 cm-1
region, respectively. The two bands appeared between 1384
and 1319 cm-1 are assigned to the stretching vibration of C-N
group. The band appeared at 767 cm-1 is assigned to C–S stret-
ching mode. Generally, the vibrational spectrum of silver
nanoparticles capped with 2-imino-4-amino thiazole molecule
is similar to the spectrum of 2-imino-4-amino thiazole.
Particle size measurement using TEM: Silver nano-
particles that produced were examined using transmission
ANTIBACTERIAL ACTIVITY OF COMPOUNDS
AGAINST HUMAN PATHOGENS
Zone of inhibition (mm)*
S.
typhi
V.
cholera
S.
E.
dysenteriae faecaulis
6
13
8
8
11
7
6
13
7
7
12
7
Compound 1a
AgNPs+
DMSO
Ciprofloxacin
17
17
15
15
+50 µg of compound (1 µg/µL concentrated), *6 mm is the well size

662 Sarada et al.
Asian J. Chem.
TABLE-2
DOSE DEPENDENT INHIBITORY EFFECT OF AgNPs+ ON VARIOUS HUMAN PATHOGENIC BACTERIA SPECIES
Zone of inhibition (mm) at different doses
Type of human pathogenic bacterial sps.
Causing disease
25 µg
11
15
50 µg
14
14
75 µg
16
16
100 µg
17
18
Salmonella typhi
Vibrio cholera
Typhiod fever
Cholera
Shigella dysenteriae
Enterococcus faecalis
Dysentery
Gastro, intestinal infections
12
13
14
12
15
13
13
15
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Conclusion
N-(4-Amino-3,5-diphenyl-3H-thiazol-2-ylidine)-benzamide
capped silver nanoparticles with uniform size and shape are
synthesized by easy and convenient method and the morpho-
logy, photo physical and biological activities were studied.
The synthesized 2-imino-4-amino thiazole capped silver nano-
particles showed significant inhibitory activity against various
human pathogenic bacteria species, like S. typhi, V. cholera,
S. dysenteriae and E. faecalis. This compound showed dose
dependent inhibitory activity. Zone of inhibition was increases
with concentration of compound. It was found that compound
was potent inhibitory agent when compared with standard
antibiotic.
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