Morphological changes of some pathogenic microbial strains induced by novel thiadiazole derivatives

Article abstract of DOI:10.1007/s00044-013-0779-x

In the quest for potent antimicrobial agents, some new 1,3,4-thiadiazole derivatives (2-11) were synthesized from the starting compound (1) 5-amino-1,3,4- thiadiazole-2(3H)-thione, with an aim to evaluate their antimicrobial effect on different pathogenic organisms isolated from microbial-infected cancer patients who had not yet received their radiation dose, compared to reference antibiotics. The titled compounds 5, 6, 10, and 11 were found to possess comparable or more potent activity than the reference compounds amikacin and sulperazone. Compound 5 was the most distinctive derivative identified in the present study because of its remarkable antibacterial activity exhibited against the Gram +ve strains, it has been scanned under electron microscope to determine the morphological changes that has taken place in the bacterial cells of Bacillus cereus and non-irradiated and irradiated Staphylococcus aureus. MIC of compound 5 and/or gamma irradiation affected the morphology of the tested strains causing; membrane damage, deformity on the surface of some cells, disappearance of the septum, elongation of some cells, and shortening of others. It may be hoped that the present study will encourage efforts toward the development of novel antibacterial agents that could be better in terms of efficacy and safety. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0779-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1844–1854
DOI 10.1007/s00044-013-0779-x
ORIGINAL RESEARCH
Morphological changes of some pathogenic microbial strains
induced by novel thiadiazole derivatives
•
M. G. El-Gazzar N. H. Zaher S. Y. El-Tablawy
•
Received: 18 June 2013 / Accepted: 31 August 2013 / Published online: 22 September 2013
Ó Springer Science+Business Media New York 2013
Abstract In the quest for potent antimicrobial agents,
some new 1,3,4-thiadiazole derivatives (2–11) were syn-
thesized from the starting compound (1) 5-amino-1,3,4-
thiadiazole-2(3H)-thione, with an aim to evaluate their
antimicrobial effect on different pathogenic organisms
isolated from microbial-infected cancer patients who had
not yet received their radiation dose, compared to reference
antibiotics. The titled compounds 5, 6, 10, and 11 were
found to possess comparable or more potent activity than
the reference compounds amikacin and sulperazone.
Compound 5 was the most distinctive derivative identified
in the present study because of its remarkable antibacterial
activity exhibited against the Gram ?ve strains, it has been
scanned under electron microscope to determine the mor-
phological changes that has taken place in the bacterial
cells of Bacillus cereus and non-irradiated and irradiated
Staphylococcus aureus. MIC of compound 5 and/or gamma
irradiation affected the morphology of the tested strains
causing; membrane damage, deformity on the surface of
some cells, disappearance of the septum, elongation of
some cells, and shortening of others. It may be hoped that
the present study will encourage efforts toward the devel-
opment of novel antibacterial agents that could be better in
terms of efficacy and safety.
Keywords Thiadiazole Á Antimicrobial Á
Gamma irradiation Á Scanning electron microscope (SEM)
Introduction
Dealing with infectious diseases still considered a critical
challenging issue. This is attributed to the emerging of new
infectious diseases and increasing number of multidrug
resistant (MDR) microbial pathogens. And this problem is
more obvious for the Gram ?ve bacteria (Tenover and
McDonald, 2005; Pfeltz and Wilkinson, 2004). It is well-
thought-out that this therapeutic problem is a crucial part in
immunocompromised persons as cancer patients receiving
gamma irradiation as therapy. They are more susceptible
for different microbial infections (Sander and Bottger,
1999) and are in need to use effective antimicrobial agents.
Besides Farrag and co-authors (Farrag et al., 2002) had
reported that the low doses of gamma irradiation had an
effect on the resistance of some bacterial isolates to dif-
ferent antibiotics resulted in increasing difficulty in treating
infections caused by these organisms.
The need to design new compounds to deal with the
evolving resistance to old and newfangled antimicrobials
has become one of the most important areas of research
today. A possible approach to overwhelmed MDR problem
is to design novel agents with different mechanisms of
action so that cross resistance with existing drugs cannot
occur (Khan et al., 2005).
M. G. El-Gazzar Á N. H. Zaher (&)
Drug Chemistry Lab, Drug Radiation Research Department,
National Center for Radiation Research and Technology, PO
Box 29, Nasr City, Cairo, Egypt
Organic compounds integrating heterocyclic ring sys-
tems continue to draw considerable attention due to their
wide range of biological activities. Among different five-
membered heterocyclic systems thiadiazoles which act as
H-binding domain with two electron donor system also as a
constrained pharmacophores. The chemistry of 1,3,
e-mail: nashwazah@hotmail.com
S. Y. El-Tablawy
Drug Microbiology Lab, Drug Radiation Research Department,
National Center for Radiation Research and Technology, Cairo,
Egypt
123

Med Chem Res (2014) 23:1844–1854
1845
Fig. 1 Commercially available
1,3,4-thiadiazole drugs
S
N
N
HN
O S O
N
N
O
H
N
H2N
S
S
S NH2
-
O
+
N
O
O
N
N
O
O
Desaglybuzole
Acetazolamide
Furidiazine
4-thiadiazole derivatives has been of increasing interest as
a versatile moiety, identified for its usefulness in medicine
to be included in commercially available drugs as desa-
glybuzole, acetazolamide, and furidiazine (Guirgis et al.,
1976; Bashir et al., 1990; Cohen et al., 1975) (Fig. 1).
1,3,4-Thiadiazole is a widespread scaffold in a number of
synthetic molecules exhibiting attractive biological conse-
quences as antiviral, anticancer and cytotoxic, anticonvul-
sant, antihyperlipedemic, anti-inflammatory and analgesic,
antidepressant, antioxidant, immuno-modulator, amebic,
and antileishmanial (Hranjec et al., 2011; Noolvi et al.,
2012; Zhang et al., 2012; Guan et al., 2012; Dogan et al.,
Results and discussion
Chemistry
Considering the 5-amino-3H-[1,3,4]thiadiazole-2-thione
(1) system as scaffold, we synthesized new derivatives. The
synthesis of our target compounds (2–7), (8–11) is outlined
in Schemes 1, 2 respectively. The starting compound (1)
was prepared according to the method reported by (Cho
and Kim, 1993), depending on the reaction between thio-
semicarbazide and carbon disulfide in alkaline medium
followed by acidification.
¨
2002; Mougenot et al., 2012; Ozadal et al., 2012; Chida-
All structures of newly synthesized compounds were
supported by physical, micro analytical, and spectral data.
The target compounds 2-substituted-(5-thioxo-4, 5-dihy-
dro[1, 3, 4]thiadiazol-2-yl) (2–7) were obtained through the
reaction with different electrophilic reagents. Compound
(1) was converted into (2-chloro-N-(5-thioxo-4,5-dihydro-
[1, 3, 4]thiadiazol-2-yl)acetamide) (2), by nucleophilic
acylation of amino group with chloroacetyl chloride in
DMF stirred at room temperature. Where IR spectrum
revealed characteristic strong intensity bands at 2,922,
1,682, and 825 cm-1 for the introduced (CH aliph, C=O
and C–Cl stretching), respectively confirming the forma-
tion of the chloro acyl derivative. In addition, it showed
absorption band at 3,187 cm-1 (N–H stretching) due to
conversion of NH2 into amide. 1H-NMR spectrum dis-
played up-field signal at 4.34 ppm for the introduced CH2
group and downfield shifted signal appearing at 7.90 ppm,
due to the conversion of the free amino into amide.
13C-NMR exhibited new up-field signal at 40.3 ppm for
CH2 and new signal at 167.19 ppm ascribed to C=O. Up-
field shift experienced from 161(C-2 start compound 1) to
154.1 ppm.
nanda et al., 2012; Jubie et al., 2012; Khan et al., 2010;
Aguilar et al., 2012; Siddiqui et al., 2012; Ardestani et al.,
2012). Many of thiadiazole compounds have found a lot of
useful chemotherapeutic application as antimicrobial,
antibacterial against many pathogenic bacteria and resistant
mycobacterium, and also as antifungal agents (Sumangala
et al., 2012; Muralikrishna et al., 2012; Plech et al., 2012;
Zoumpoulakis et al., 2012; Almajan et al., 2010; Barbu-
ceanu et al., 2012; Kolavi et al., 2006).
Inspired by these findings, and in continuation of our
ongoing efforts on the synthesis of heterocycles with
potential antimicrobial activities (Ghorab et al., 2011). The
aim of this work to synthesize via simple convenient
methods, a new series of 1,3,4-thiadiazoles having different
radicals linked to amino group from 2 position, for eval-
uation of their antimicrobial profile. Also to study their
effect on the morphology of some pathogenic microor-
ganisms before and after irradiation to investigate the
susceptibility of the tested bacterial strains to the synthe-
sized compounds and studying the synergistic effect of
gamma radiation. The target compounds were rationalized
so as to comprise some pharmacophores that are believed
to be responsible for the pharmacological activity of some
pertinent chemotherapeutic agents such as sulfonamide,
thiazole, pyrrole, aniline, acetophenone, and Schiff’s base
functionalities (Ghorab et al., 2013; Sadek et al., 2011;
Mohamed et al., 2009; Sivakumar et al., 2008; Sabry et al.,
2013).
Compound (2) was cyclized into (2-(5-thioxo-4,
5-dihydro-[1, 3, 4]thiadiazol-2-ylamino)thiazol-4-one) (3)
by treatment with ammonium thiocyanate in ethanol. The
reaction proceeded via an intermolecular cyclization and
liberation of hydrogen chloride molecule then rearrange-
ment (Siavosh et al., 2006). Where IR spectrum showed the
disappearance of the peak at 825 cm-1 as a result of
123

1846
Med Chem Res (2014) 23:1844–1854
Scheme 1 Synthetic pathways
for compounds 2–7
HN
N
S
S
N C
H
6
Cl
CHO
Cl
HN
N
HN
N
HN
N
O
PhCOCH2Br
EtOH
ClCOCH2Cl
DMF
S
S
N
H
CH2Cl
NH2
S
S
1
S
S
4
N
H
Ph
2
O
NCS
CN
CN
NH4SCN
N
H2C
NaOEt, EtOH
SO2NH2
HN
N
HN
N
O
HN
N
S
S
S
3
N
S
S
N
NH
Ph
S
S
N
H
H
S
H2N
CN
7
5
SO2NH2
Scheme 2 Synthetic pathways
for compounds 8–11
HN
N
N
H
C O
S
S
H
C O
10
H
COO
H
HN
N
O
HN
N
N
N
O
O
N
H
NHPh
S
S
PhN
CO
PhN
CO
NH2
S
S
N
NHPh
NHPh
S
S
PhHN
:
1 1
1
8
O
9
A
S
C2O
HN
N
O
O
N
S
11
cyclization and loss of HCl molecule. The 2 (NH)
stretching bands (3,190 and 3,187 cm-1) were shifted to
lower frequency regions 3,167 and 3,034 cm-1, respec-
of compound 1 with phenacyl bromide in refluxing ethanol
with loss of one mole of hydrogen bromide. Where IR
spectrum revealed the presence of characteristic absorption
peaks at 3,011 and 2,932, 2,848 cm-1 for (CH aromatic and
CH aliphatic stretching), respectively, and a strong intensity
stretching band at 1,690 cm-1 ascribed for the (C=O) group
introduced, confirming the ethanone derivative formation.
1H-NMR spectrum displayed signals at 4.77 and 8.75 ppm
which disappeared upon D2O exchange, confirming the
presence two (NH) groups, while the protons of the aromatic
ring introduced showed sharp peaks at 7.42–7.99 ppm.
1
tively. H-NMR spectrum displayed an up-field shift for
CH2, appearing at 2.98 ppm due to its inclusion in the 1,3
thiazole-2-yl ring formed. 13C-NMR spectrum exhibited a
new signal at 167.9 ppm ascribed to C-2 of thiazole ring
formed, along with the up-field shifted values for C-5 thi-
azole at 36.4 ppm due to rearrangement.
Similarly, (1-phenyl-2-(5-thioxo-4,5-dihydro-1,3,4-thia-
diazol-2-ylamino)-ethanone) 4 was prepared by the reaction
123

Med Chem Res (2014) 23:1844–1854
1847
13C-NMR spectrum showed new signals at 40.05 and
176.25 ppm corresponding to the introduced (CH2), and
(C=O), respectively. Whereas the aromatic carbon atoms
appeared in the range of 129.3–135.6 ppm. C-2 thiadiazole
exhibited up-field shifted frequency at 146.3 ppm. Com-
pound (4) was cyclized into (2-amino-4-phenyl-1-(5-thioxo-
4, 5-dihydro[1, 3, 4]thiadiazol-2-yl)-1H-pyrrole-3-carboni-
trile) 5 by treatment with malononitrile in ethanol. Where IR
spectrum revealed absorption bands at 3,245, 3,187 for the
(NH2 stretching) group formed and a characteristic medium
intensity band at 2,222 cm-1 confirming the presence of
carbonitrile group introduced. 1H-NMR spectrum displayed
signals at 3.52 ppm for the NH2 group which disappeared
upon D2O exchange, and at 4.73 ppm ascribed for the (CH)
group of the pyrrole ring. 13C-NMR spectrum showed the
absence of the signal ascribed for (C=O), instead new signals
appeared at 108 and 111 ppm ascribed for C-3 pyrrole and
C:N, respectively. While C-2 and 4-pyrrole signals
appeared with the aromatic ones.
3,031 cm-1 due to the aromatic CH and strong intensity
absorption band at 1,685 cm-1 assigned for C=O stretch-
ing. 1H-NMR spectrum displayed signals at 7.28–7.48 ppm
attributed to the aromatic protons of the phenyl ring
introduced. 13C-NMR spectrum showed additional signal
for C=O at 169.69 ppm. While the aromatic carbon atoms
appeared in the range of 118.16–139.69 ppm.
The polysubstituted target compound (2-di-(N-phenyl-
formamide) amino-5-(N-phenyl-sulfinic amide) [1,3,4-
thiadiazole]) 9 was obtained by the reaction of the starting
compound 1 with excess phenylisocyanate in refluxing
pyridine. Where IR spectrum revealed bands at 3,245 and
3,278 cm-1 ascribed for 3NH-stretching, 3,028 cm-1 due
to CH aromatic-stretching and characteristic strong inten-
sity absorption peaks at 1,654 and 1,676 cm-1 assigned for
the three (C=O stretching) groups. The disappearance of
the signal ascribed for (C=S), confirmed the polysubstitu-
tion. 1H-NMR spectrum displayed signals for the three NH
groups at 3.42 and 8.67 ppm which disappeared upon
exchange with D2O and the aromatic protons of the three
phenyl rings introduced appeared as multiplets at range
6.97–7.48 ppm. 13C-NMR spectrum revealed a great up-
field shift for (C=S) at 165.43. An additional bands for
(3C=O) introduced appeared at 149.6 and 152.48 ppm. The
C-2 of thiadiazole encountered downfield shift at 170 ppm.
The target compound (N-formyl-N-(5-thioxo-4,5-dihy-
dro-1,3,4-thiadiazol-2-yl)-formamide) 10 was attained by
the neat reaction of the starting compound 1 with formic
acid for 10 h. Where IR spectrum showed absorption peaks
at 3,331 cm-1 assigned for NH-stretching. New absorption
peaks appeared at 2,879 and 1,658 cm-1 due to stretching
The target compound (5-[(2-chloro-benzylidene)-
amino]-3H-[1,3,4]-thiadiazole-2-thione) 6 is formed upon
reaction of compound 1 with 2-chlorobenzaldehyde in re-
fluxing ethanol through Schiff’s base formation. Where IR
spectrum revealed absorption bands at 3,022 and 2,956,
2,854 cm-1 for (CH aromatic and CH aliphatic stretching),
respectively, and a characteristic strong intensity band at
1
834 cm-1 attributed to (C–Cl stretching). H-NMR spec-
trum displayed signals at 7.32 ppm for the CH group of
Schiff’s base. The protons of the aromatic ring introduced
showed sharp peaks at 7.34–7.78 ppm ascribed for chlo-
robenzene ring introduced. 13C-NMR spectrum showed the
presence of a new signal at 155.2 ppm assigned for (CH
Schiff’s base group), whereas the aromatic carbon atoms
appeared in the range of 126.9–137.9 ppm.
1
CH aliphatic and C=O, respectively. H-NMR spectrum
displayed only two signals for the NH group at 2.5 ppm
which disappeared upon exchange with D2O and signal
attributed to CHO groups at 8.4 ppm. 13C-NMR spectrum
revealed three signals: an up-field shift for (C-2 thiadia-
zole) at 150.63 ppm, whereas the formyl carbon atoms
appeared at 160.35 ppm and C-5 (C=S) appeared at
183.26 ppm.
The preparation of (4-[3-(5-thioxo-4,5-dihydro-[1,3,4]
thiadiazol-2-yl)-thioureido]-benzenesulfonamide)
7
was
attained by the reaction of compound 1 with 4-iso-
thiocyanatobenzenesulfonamide in refluxing dimethylform-
amide. Where IR spectrum revealed absorption bands at
3,022, 1,387, 1,145 cm-1 assigned for CH aromatic and SO2
groups-stretching, respectively. 1H-NMR spectrum displayed
signals at 5.50 ppm for the NH2 and signals at 6.50, 6.70,
8.25 ppm for the 3NH groups which disappeared upon
exchange with D2O. The aromatic protons for the AB system
of the benzene ring introduced appeared at 7.66 and
7.81 ppm. 13C-NMR spectrum exhibited additional signal for
the most de-shielded carbon atom at 200.01 ppm attributed to
the thiocyanate C introduced. Whereas the aromatic carbon
atoms appeared in the range of 122.2–139.4 ppm.
Similarly, the preparation of disubstituted compound
(N-acetyl-N-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)
acetamide) 11 was achieved through the neat reaction of
compound 1 in acetic anhydride. Where IR spectrum dis-
played signals at 2,897 cm-1 for aliphatic CH-stretching
assigned for the introduced acetyl groups together with
strong intensity absorption bands at 1,664 cm-1 attributed
1
to the carbonyl groups of the acetyl moieties. H-NMR
spectrum displayed only two signals for the acetyl groups
and NH at 2.02 and 12.29 ppm, respectively. 13C-NMR
spectrum revealed new up-field signal assigned for CH3
groups at 22.35 ppm. C-2 thiadiazole encountered up-field
shift at 152.21 ppm, whereas the carbon atoms of the acetyl
groups appeared at 169.19 ppm.
The target compound (1-phenyl-3-(5-thioxo-4,5-dihy-
dro-[1,3,4]thiadiazol-2-yl)-urea) 8 was obtained by the
unimolar reaction of compound 1 with phenylisocyanate in
pyridine. Where IR spectrum revealed absorption band at
123

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Med Chem Res (2014) 23:1844–1854
Further, the EIMS of all the synthesized compounds are
group by formylation and acetylation in compounds 10 and
11 considerably increased the activity.
in conformity with the assigned structures along with the
elemental analyses which were found within the limit of
0.4 % of theoretical values for all the synthesized
compounds.
Effect of c-irradiation on the susceptibility of Gram
?ve bacterial strains to the synthesized compounds
Antimicrobial screening
The results in (Table 2) revealed that the most potent
pyrrole derivative 5 revealed good activity against normal
control and irradiated S. aureus with inhibition zones equal
to that of Amikacin. While in case of normal control of B.
cereus, it showed higher activity rather than the reference
antibiotic Amikacin. For this, the minimum inhibitory
concentration (MIC) and the effect of compound 5 on the
morphology of S. aureus and B. cereus using scanning
electron microscope (SEM) were carried out. Similarly, for
the rest of the tested compounds, it was shown that irra-
diation did not affect the susceptibility of the bacterial
strains and acted synergistically with the tested com-
pounds. These results constitute a value for cancer patients
receiving gamma radiotherapy. They are more susceptible
for bacterial infection and in need for antimicrobial without
affecting the bacteria susceptibility especially the Gram
?ve ones. In this case compound 5 adds a merit of syn-
ergism with gamma radiation as antibacterial.
Structure activity relationship (SAR)
From the results obtained in (Table 1) we can observe that,
the preliminary screening for the antimicrobial activity of
the synthesized compounds affects Gram ?ve bacteria and
does not affect Gram -ve ones or the tested fungal strain.
The most potent compound in this study was the thiadia-
zole derivative 5 bearing a pyrrole ring showing the highest
antibacterial activity among the tested compounds and is
more potent or nearly equal to that of the standard anti-
bacterial agents. It was found to be nearly as potent as
Amikacin and Sulperazone especially against Staphylo-
coccus aureus and Bacillus cereus with inhibition zones
nearly equal to that of the reference antibiotic amikacin.
This comes in accordance that compounds bearing pyrrole
ring posses potent antibacterial activity (Ghorab et al.,
2011; Mohamed et al., 2009). In addition, it showed higher
activity than compound 4 which proves the efficacy of
cyclization and introduction of pyrrole ring on activity.
Chloroacetylation of amino group in compound 2 resulted
in a drastic drop in the activity which was improved by
cyclization and formation of thiazolone ring in compound
3. The Schiff’s base derivative 6, the thioureido derivative
7, the mono- and polysubstituted ureido derivatives 8 and 9
showed moderate activity, while, disubstitution on amino
Scanning electron microscope study (SEM)
A control culture typical to the normal control S. aureus is
shown in (Fig. 2a) as spherical cells with normal shape,
size and complete septum (magnification 15,000), while
cells treated with MIC of compound 5, revealed membrane
damage by the appearance of projecting cellular material
and bleb formation. Furthermore, disappearance of septum
Table 1 Antimicrobial
screening of thiadiazole
derivatives (2–10)
Tested compounds Inhibition zones (mm)a
P. aeruginosa K. pneumonia S. aureus S. epidermidis B. cereus C. albicans
2
–
–
–
–
–
–
3
–
–
10
20
25
20
9
7
9
–
4
–
–
15
17
12
–
18
20
22
7
–
5
–
–
–
6
–
–
–
NT not tested
a
Slight activity: 12–14 mm
7
–
–
–
(?); moderate activity:
15–18 mm (??); high activity:
C19 mm (???)
8
–
–
10
15
20
20
20
30
NT
–
9
7
–
9
–
–
7
7
–
10
–
–
20
13
25
30
NT
–
22
20
20
28
NT
–
–
b
Inhibition of protein synthesis
(Amikacin sensitive C17 mm).
11
–
–
–
Amikacinb
Sulperazonc
Nystatined
DMF
15
30
NT
–
11
27
NT
–
NT
NT
7
Reference antibiotics
c
Inhibition of cell wall.
Reference antibiotics
d
Manufactured by Bristol–
Myers Squibb, Giza, Egypt
–
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Med Chem Res (2014) 23:1844–1854
1849
Table 2 Effect of gamma
irradiation on the susceptibility
of tested Gram ?ve strains to
the biologically active
compounds
Tested compounds Inhibition zones (mm)a
S. aureus
S. epidermidis
B. cereus
Non-irradiated Irradiated Non-irradiated Irradiated Non-irradiated Irradiated
3
4
5
6
7
8
9
12
20
20
16
10
7
15
20
19
16
10
10
7
7
12
12
13
6
7
20
25
13
10
10
12
13
15
25
35
11
15
25
20
7
11
15
25
16
10
9
7
7
7
8
7
9
10
20
20
20
40
16
17
17
26
13
11
24
30
13
11
20
28
20
20
20
32
a
11
Slight activity: 12–14 mm
(?); moderate activity:
15–18 mm (??); high activity:
C19 mm (???)
Amikacin
Sulperazon
Fig. 2 a–e Scanning electron micrographs of untreated and treated
S. aureus, with or without c-irradiation. a Normal control S. aureus.
b, c Cells treated with MIC of compound 5. d S. aureus cells exposed
to gamma radiation. e The synergistic effect of gamma irradiation and
compound 5
was noticed in some cells Fig. 2b, c (magnification
15,000).
(Fig. 3b; treated cells), displayed many morphological
changes including elongation in some cells, shortening in
others, disappearance of the septum, and aggregation of the
cellular material at the poles of the cell (magnification
7,500).
Figure 2d showed S. aureus cells exposed to gamma
radiation appeared longer and irregularly shaped than that
of the normal control ones (magnification 15,000). Mean-
while, the synergistic effect of gamma irradiation and
compound 5, caused deformity of some cells’ surface and
elongation of others as shown in Fig. 2e (magnification
10,000).
Our results were in agreement with many investigators
(Sanyal and Greenwood, 1993), when staphylococcal cells
were treated with vancomycin. Also, Eltablawy and El-
hifnawi (2010) found that there were morphological
changes in Pseudomonas aeruginosa treated with garlic oil.
It was reported (Rhayour et al., 2003; Bennis et al., 2004)
As shown in (Fig. 3a; normal control B. cereus), cells
are normal in shape and size (magnification 10,000), while
123

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Med Chem Res (2014) 23:1844–1854
Fig. 3 a, b Scanning electron
micrographs of untreated and
treated B. cereus. a Normal
control B. cereus. b Treated
cells with MIC of compound 5
that structures like blebs in the outside surface of treated
cells, may represent collection of cytoplasmic constituents
which were pushed through cracks produced in the cell
wall.
recorded on ABB Bomem FT-IR spectrometer MB 104
with KBr pellets. 1H-NMR and 13C-NMR spectra were
recorded using a Bruker 300 NMR spectrometer operating
at 400.13 and 100.77 MHz, respectively. Microanalyses
were obtained with an Elemental analyses system GmbH
VarioEL V300 element analyzer. The purity of the com-
pounds was checked by TLC on pre-coated SiO2 gel
(HF254, 200 mesh) aluminum plates (E Merk) and visu-
alized in UV chamber. Mass spectra were run on HP Model
MS-5988 (Hewlett Packard, Palo, Alto, California, USA).
IR, 1H-NMR, 13C-NMR, Mass and elemental analysis were
consistent with the assigned structures.
Conclusions
The antimicrobial activity of the synthesized compounds
may be due to the presence of versatile pharmacophore
which might increase the lipophilic character of the mol-
ecules, by which it facilitates the crossing through the
biological membrane of the microorganism and thereby
inhibit their growth. Or acting additionally by different
mechanisms of action for the hybrids. For this reason, we
can see that compound 5 2-amino-4-phenyl-1-(5-thioxo-
4,5-dihydro[1,3,4]thiadiazol-2-yl)-1H-pyrrole-3-carboni-
trile bearing the pyrrole moiety was found to exhibit the
most potent antimicrobial activity against Gram ?ve S.
aureus and B. cereus when compared with Amikacin. At
the same time it acts synergistically with gamma radiation.
We here in recommend further pharmacological studies
to be conducted in future research to deal with the toxicity
profiling and to perform the exact mechanistic assays to
reveal the exact mechanism of action.
Experimental protocol
2-Chloro-N-(5-thioxo-4,5-dihydro-[1,3,4]thiadiazol-2-yl)
acetamide (2) A mixture of 5-amino-3H-[1,3,4]thiadia-
zole-2-thione 1 (0.5 g, 0.003 mol) and chloroacetyl chlo-
ride (0.3 g, 0.003 mol) in DMF was stirred at room
temperature for 2 h, the reaction mixture was poured onto
ice water and the solid obtained was recrystallized from
ethanol to give 2. Yield, 88 %, mp 280 °C. IR (KBr,
cm-1): 3,190, 3,187 (2NH), 2,922, 2,848 (CH aliph.), 1,682
(C=O), 1,230 (C=S), 825 (C–Cl). 1H-NMR (DMSO-d6,
ppm): 4.34 [s, 2H, CH2], 7.90, 9.10 [2 s, 2H, 2NH,
exchangeable with D2O]. 13C-NMR (DMSO-d6, ppm): 40.
3 (CH2), 154.1 (C-thiadiazole), 167.19 (C=O), 183.0 (C=
S). MS: m/z 209 (M?, 4.2 %), 76 (100 % base peak).
Analysis calculated for C4H4N3OS2Cl C, 22.91; H, 1.92;
N, 20.04; found: C, 22.78; H, 1.62; N, 20.23.
In conclusion, we reported here a simple and convenient
route for the synthesis of some new 1,3,4-thiadiazole
derivatives for antimicrobial, gamma synergistic and bac-
terial morphological evaluation.
Materials and methods
Chemistry
2-(5-Thioxo-4,5-dihydro-[1,3,4]thiadiazol-2-ylamino)thia-
zol-4-one (3) A mixture of 2 (0.5 g, 0.002 mol) and
ammonium thiocyanate (0.17 g, 0.002 mol) was refluxed
in ethanol for 1 h. The reaction mixture was cooled, poured
onto ice water and the solid obtained was recrystallised
from dioxane to give 3. Yield, 70 %, mp 280 °C. IR (KBr,
cm-1): 3,167, 3,034 (2NH), 1,682 (C=O), 1,230 (C=S). 1H-
NMR (DMSO-d6, ppm): 2.98 [s, 2H, CH2-thiazole], 4.03,
Measurement
The melting points were taken in an open capillary tube
and are uncorrected. The IR spectra of the compounds were
123

Med Chem Res (2014) 23:1844–1854
1851
7.86 [2 s, 2H, 2NH, exchangeable with D2O]. 13C-NMR
(DMSO-d6, ppm): 36.4 (C-thiazole), 154.9, 167.9, 175.01,
183.2. MS: m/z 232 (M?, 8.76 %), 58 (100 % base peak).
Analysis calculated for C5H4N4OS3: C, 25.85; H, 1.74; N,
24.12, found: C, 25.65; H, 1.56; N, 24.45.
Analysis calculated for C9H6ClN3S2: C, 42.27; H, 2.36;
N, 16.43, found: C, 42.47; H, 2.12; N, 16.71.
4-[3-(5-Thioxo-4,5-dihydro-[1,3,4]thiadiazol-2-yl)-thio-
ureido]-benzenesulfonamide (7) A mixture of 1 (0.5 g,
0.003 mol) and 4-isothiocyanatobenzenesulfonamide (0.
72 g, 0.003 mol) was refluxed in DMF containing 3 drops
of TEA for 5 h. the reaction mixture was cooled, poured
onto ice water and the solid obtained was recrystallised
from ethanol to give 7. Yield, 73 %, mp 138–140 °C. IR
(KBr, cm-1): 3,367, 3,266, 3,176 (NH, NH2), 3,022 (CH
arom.), 1,230 (C=S), 1,387, 1,145 (SO2). 1H-NMR
(DMSO-d6, ppm): 5.50 [s, 2H, NH2, exchangeable with
D2O], 6.50, 6.70, 8.25 [3 s, 3H, 3NH, exchangeable with
D2O], 7.66, 7.81 [2d, 4H, AB system, J = 4.5]. 13C-NMR
(DMSO-d6, ppm): 122.2, 125.6, 133.6, 139.4, 152.1, 181.4
(C=S), 200.01. MS: m/z 347 (M?, 5.1 %), 63 (100 % base
peak). Analysis calculated for C9H9N5O2S4: C, 31.11; H, 2.
61; N, 20.16, found: C, 31.34; H, 2.48; N, 20.37.
1-Phenyl-2-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)
ethanone (4) A mixture of 1 (0.5 g, 0.003 mol) and
phenacyl bromide (0.7 g, 0.003 mol) was refluxed in eth-
anol for 3 h. the reaction mixture was cooled, and the
precipitated solid was recrystallised from dioxane to give 4.
Yield, 95 %, mp 204–205 °C. IR (KBr, cm-1): 3,269,
3,124 (2NH), 3,011 (CH arom.), 2,932, 2,848 (CH aliph.),
1
1,690 (C=O), 1,230 (C=S). H-NMR (DMSO-d6, ppm): 4.
87 [s, 2H, CH2], 4.77, 8.75 [2 s, 2H, 2NH, exchangeable
with D2O], 7.42-7.99 [m, 5H, Ar–H]. 13C-NMR (DMSO-
d6, ppm): 40.05 (CH2), 129.3, 129.7, 130.02, 135.6, 146.3
(C-thiadiazole), 176.25 (C=O), 183.1 (C=S). MS: m/z 251
(M?, 1.76 %), 79 (100 % base peak). Analysis calculated
for C10H9N3OS2: C, 47.79; H, 3.61; N, 16.72, found: C,
47.56; H, 3.81; N, 16.52.
1-Phenyl-3-(5-thioxo-4,5-dihydro-[1,3,4]thiadiazol-2-yl)-
urea (8) A mixture of 1 (0.5 g, 0.003 mol) and phenyl-
isocyanate (0.36 g, 0.003 mol) was refluxed in pyridine for
8 h. The reaction mixture was cooled, poured onto ice
water and the solid obtained was recrystallised from acetic
acid to give 8. Yield, 92 %, mp 241–243 °C. IR (KBr,
cm-1): 3,252, 3,198 (3NH), 3,031 (CH arom.), 1,685 (C=
2-Amino-4-phenyl-1-(5-thioxo-4, 5-dihydro[1, 3, 4]thia-
diazol-2-yl)-1H-pyrrole-3-carbonitrile (5) A mixture of 4
(0.5 g, 0.001 mol) and malononitrile (0.13 g, 0.001 mol)
was refluxed in ethanol containing sodium ethoxide (0.
04 g, 0.001 mol) for 3 h. The reaction mixture was cooled,
poured onto ice water, acidified with dil. HCl and the solid
obtained was recrystallised from dioxane to give 5. Yield,
80 %, mp 110–112 °C. IR (KBr, cm-1): 3,245, 3,187
(NH2), 3,072 (CH arom.), 2,222 (CN), 1,230 (C=S).
1H-NMR (DMSO-d6, ppm): 3.52 [s, 2H, NH2, exchange-
able with D2O], 4.73 [s, 1H, CH-pyrrole], 7.30 [s, 1H, NH,
exchangeable with D2O], 7.46–7.93 [m, 5H, Ar–H]. 13C-
NMR (DMSO-d6, ppm): 108, 111 (C-pyrrole), 118 (CN),
120.2, 127, 128.4, 128.7, 133.6, 133.72, 169.77, 193.26,
MS: m/z 299 (M?, 0.19 %), 54 (100 % base peak). Ana-
lysis calculated for C13H9N5S2: C, 52.16; H, 3.03; N,
23.39, found: C, 52.45; H, 3.23; N, 23.67.
1
O), 1,230 (C=S). H-NMR (DMSO-d6, ppm): 7.28–7.48
[m, 5H, Ar–H], 8.70 [s, 1H, NH, exchangeable with D2O],
8.96 [s, 2H, 2NH, exchangeable with D2O], 13C-NMR
(DMSO-d6, ppm): 118.16, 121.78, 128.75, 139.69 (C-aro-
matic), 152.52 (C-thiadiazole), 169.69 (C=O), 181 (C=S).
MS: m/z 252 (M?, 23.4 %), 253 (M??1, 8.1 %), (65,
100 % base peak). Analysis calculated for C9H8N4OS2: C,
42.84; H, 3.20; N, 22.21, found: C, 42.56; H, 3.48; N,
22.61.
2-Di-(N-phenylformamide)amine-5-(N-phenylsulfinic amide)
[1,3,4-thiadiazole] (9) A mixture of 1 (0.5 g, 0.003 mol)
and phenylisocyanate (1 g, excess) was refluxed in pyri-
dine for 8 h. The reaction mixture was cooled, poured onto
ice water and the solid obtained was recrystallised from
acetic acid to give 9. Yield, 90 %, mp 229–230 °C. IR
(KBr, cm-1): 3,245, 3,278 (3NH), 3,028 (CH arom.),
1,685, 1,674 (C=O). 1H-NMR (DMSO-d6, ppm): 6.97–7.48
[m, 15H, Ar–H], 8.67 [s, 1H, NH, exchangeable with D2O].
13C-NMR (DMSO-d6, ppm): 181.29, 121.72, 128.69, 139.
64 (C-aromatic), 149.6 (2 N–C=O), 152.48 (S–C=O), 165.
43 (C=S), 170.0 (C-thiadiazole). MS: m/z 490 (M?, 4 0.
14 %), 491 (M??1, 2.1 %), (65, 100 % base peak). Ana-
lysis calculated for C23H18N6O3S2: C, 56.31; H, 3.70;
N, 17.13, found: C, 56.55; H, 3.51; N, 17.49.
5-[(2-Chloro-benzylidene)-amino]-3H-[1,3,4]thiadiazole-
2-thione (6) A mixture of 1 (0.5 g, 0.003 mol) and
2-chlorobenzaldehyde (0.47 g, 0.003 mol) was refluxed in
ethanol containing 1 ml glacial acetic acid for 5 h. the
reaction mixture was cooled, poured onto ice water and the
solid obtained was recrystallised from dioxane to give 6.
Yield, 75 %, mp 200–202 °C. IR (KBr, cm-1): 3,156
(NH), 3,022 (CH arom.), 2,956, 2,854 (CH aliph.), 1,230
1
(C=S), 834 (C–Cl). H-NMR (DMSO-d6, ppm): 7.32 [s,
1H, CH], 7.34–7.78 [m, 4H, Ar–H], 8.77 [s, 1H, NH,
exchangeable with D2O]. 13C-NMR (DMSO-d6, ppm): 126.
9, 129.2, 131.3, 134.7, 135.4, 137.9, 165.7 (CH), 155.2, 181.
7. MS: m/z 255 (M?, 23.4 %), 76 (100 % base peak).
123

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Med Chem Res (2014) 23:1844–1854
N-Formyl-N-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)
formamide (10) A solution of 1 (0.5 g, 0.003 mol) in
formic acid was refluxed for 10 h. The reaction mixture
was cooled where needle shaped crystals were filtered,
dried and recrystallized from dioxane to give 10. Yield,
87 %, mp 188–190 °C. IR (KBr, cm-1): 3,331 (NH), 2,879
(CH aliph.), 1,658 (2C=O) and 1,230 (C=S). 1H-NMR
(DMSO-d6, ppm): 2.50, [s, 1H, NH, exchangeable with
D2O], 8.42 [s, 2H, 2CHO], 13C-NMR (DMSO-d6, ppm):
150.63 (C-thiadiazole), 160.35 (2CHO), 183.26 (C=S).
MS: m/z 189 (M?, 5.43 %), 190 (M? ? 1, 3.0 %), (161,
100 % base peak). Analysis calculated for C4H3N3O2S2:
C, 22.35; H, 1.88; N, 26.07, found: C, 22.61; H, 1.67; N,
26.46.
Effect of c-irradiation on the susceptibility of Gram
?ve bacterial strains to the synthesized compounds
This study was conducted to evaluate the effect of c-irra-
diation on the susceptibility of the tested Gram ?ve bac-
terial strains to the synthesized compounds. Irradiation
process was carried out by using cesium 137 gamma cell,
40, Atomic energy of Canada Limited, Commercial prod-
uct located at NCRRT and the dose rate was 0.775 rad/s. at
the time of experiment. The Gram ?ve bacterial strains
were subjected to a dose level 24.4 Gray (Gy) which is
biologically equivalent to in vitro fractionated multiple
therapeutic dose of some cancer patients (Barton, 1995) in
order to study the effect of irradiation to their susceptibility
to the biologically active compounds.
N-Acetyl-N-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)
acetamide (11) A solution of 1 (0.5 g, 0.003 mol) in
acetic anhydride was refluxed for 10 h. Where a solid
formed on hot which was filtered, dried and recrystallized
from dioxane to give 11. Yield, 89 %, mp 175–177 °C. IR
(KBr, cm-1): 3,325 (NH-stretching), 2,897 (CH aliph.),
1,660 (2C=O) and 1,230 (C=S). 1H-NMR (DMSO-d6,
ppm): 2.02 [s, 6H, 2CH3], 12.29 [s, 1H, NH exchangeable
with D2O]. 13C-NMR (DMSO-d6, ppm): 144.12 (C-thia-
diazole), 168.65 (C=O), 183.38 (C=S). Analysis calculated
for C6H7N3O2S2: C, 33.17; H, 3.25; N, 19.34;, found: C,
33.21; H, 3.59; N, 19.62.
The MIC determination for compound 5
The MIC of the most promising compound 5 was deter-
mined by agar streak dilution method (Yousif et al., 2011).
A stock solution of the compound in DMF was prepared
and graded quantities were incorporated in a specified
quantity of molten sterile nutrient agar medium, then
poured into a petri dish to give a depth of 3–4 mm and
allowed to solidify. Suspension of the microorganism was
prepared to contain approximately 10 5 CFU ml-1, applied
to the plates and incubated at 37 °C for 24 h. The MIC was
considered to be the lowest concentration of the test
compound exhibiting no visible growth of bacteria. MIC
values were 125, 250, and 250 ll ml-1 for S. aureus (non-
irradiated and irradiated) and B. cereus (non-irradiated),
respectively, which are the most sensitive bacterial strains.
Antimicrobial screening
Five bacterial isolates representing Gram -ve and Gram
?ve bacteria namely; P. aeruginosa, Klebsiella pneumonia
(K. pneumonia), S. aureus, Staphylococcus epidermidis (S.
epidermidis), and B. cereus, were recovered on Nutrient
and MacConkey agar. One yeast fungus Candida albicans
(C. albicans) was isolated on Sabouraud agar (oxoid). They
were obtained from the Drug Microbiology Laboratory—
Drug Radiation Research department—National center for
radiation research and Technology (NCRRT).
Scanning electron microscope (SEM) study
The effect of compound 5, the most promising biologically
active compound, on the morphology of the tested bacterial
strains was studied using SEM (JSM-5400 JEOL) located
at NCRRT. Tested bacterial strains were subcultured in
nutrient broth (Difco). They were incubated for 24 h at
37 °C under static stirring. Compound 5 at its MIC was
added directly in the medium after 3 h incubation. In case
of S. aureus, the effects of irradiation at 24.4 Gy and
synergistic effect of irradiation with MIC of compound 5
were also investigated. At the end of incubation, the cul-
tures were prefixed with 2 % glutaraldehyde for 2 h at
4 °C. Post-fixation was carried out using a 2 % osmium
tetroxide solution for 30 min at 30 °C. After each fixation
the cells were washed twice with 0.1 M sodium cacodylate
and the bacterial cells were prepared for electron micros-
copy according to the recommended method Philipp
(1981).
The antimicrobial susceptibility testing was performed
in vitro using the agar diffusion disk method (Barry, 1981).
The tested compounds (10 mg) were dissolved in dimeth-
ylformamide (DMF, 1 ml) which showed no inhibition
zone and used as negative control. Antimicrobial potenti-
alities of the tested compounds were estimated by placing
presterilized filter paper disks (6 mm in diameter) impre-
genated with the tested compound and placed on the
solidified medium. Inhibition zones were measured after
24–48 h of incubation at 37 °C. Reference antibiotic disks
(6 mm in diameter), Amikacin (AN 30 lg disk-1), Sulpr-
azon (Scf 105 lg disk-1), and fungicide Nystatine (NS
30 lg disk-1), were used as standards.
123

Med Chem Res (2014) 23:1844–1854
1853
hydroxamic acid derivatives as novel histone deacetylase
inhibitors. Bioorg Med Chem 20:3865–3872
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