Synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-oxadiazoles

Article abstract of DOI:10.1016/j.ejmech.2013.06.029

A series of thiazole clubbed 1,3,4-oxadiazole derivatives (5a-l) have been synthesized and characterized by IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Synthesized compounds were evaluated for their antimicrobial and cytotoxic ac

Full text of DOI:10.1016/j.ejmech.2013.06.029

European Journal of Medicinal Chemistry 67 (2013) 54e59
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antimicrobial and cytotoxic activities of some novel thiazole
clubbed 1,3,4-oxadiazoles
*
N.C. Desai , Nayan Bhatt, Hardik Somani, Amit Trivedi
Division of Medicinal Chemistry, Department of Chemistry (DST-FIST Sponsored), Mahatma Gandhi Campus,
Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar 364 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiazole clubbed 1,3,4-oxadiazole derivatives (5ael) have been synthesized and characterized
by IR, 1H NMR, 13C NMR and mass spectral analysis. Synthesized compounds were evaluated for their
antimicrobial and cytotoxic activities. The results indicated that, compounds 5c and 5i exhibited the most
potent antibacterial activity. Compound 5f was found to be the most potent antifungal agent. The
structure activity relationship revealed that the presence of electron withdrawing groups at para position
of phenyl ring remarkably enhanced the antibacterial activity of synthesized compounds. Further, the
results of preliminary MTT cytotoxicity studies on HeLa cells suggested that potent antimicrobial activity
of 5b, 5c, 5f, 5h and 5i is accompanied by low cytotoxicity.
Received 26 March 2013
Received in revised form
3 June 2013
Accepted 10 June 2013
Available online 19 June 2013
Keywords:
Thiazole
Ó 2013 Elsevier Masson SAS. All rights reserved.
1,3,4-Oxadiazole
Thiazole clubbed 1,3,4-oxadiazoles
Antibacterial activity
Antifungal activity
Cytotoxicity
1. Introduction
class of potent antimicrobial agents, which are reported to inhibit
bacteria by blocking the biosynthesis of certain bacterial lipids and/
The treatment of infectious diseases still remain a challenging
task because of combination of factors such as an alarming increase
in number of multi-drug-resistant microbial pathogens and advent
of newer infectious diseases such as severe acute respiratory
syndrome (SARS), and avian influenza. Despite the availability of
or by additional mechanisms [17,18].
On the other hand, 1,3,4-oxadiazole heterocycles are very good
bioisosteres of amides and esters, which can contribute substantially
in enhancing pharmacological activity by participating in hydrogen
bonding interactions with the receptors [19]. Potent pharmacological
activity of 1,3,4-oxadiazoles can be attributed to the presence
of toxophoric eN]CeOe linkage which may react with the nucle-
ophilic centers of the microbial cell [20]. Further, the widespread use
of 1,3,4-oxadiazoles as a scaffold in medicinal chemistry established
this moiety as a member of the privileged structures class and its
derivatives have exhibited a wide range of biological activities such
as antibacterial [21], antitubercular [22], vasodialatory [23], anti-
fungal [24], cytotoxic [25], anti-inflammatory and analgesic [26,27],
hypolipidemic [28], anticancer [29] and ulcerogenic [30]. Oxadiazole
derivatives have been found to possess broad spectrum antimicro-
bial activity and therefore are useful substructures for further
molecular exploration [30]. The most prominent examples of pre-
scribed agents featuring the 1,3,4-oxadiazoles nucleus include the
antiretroviral raltegravir [31], antihypertensive nesapidil [32] and
the antibiotic furamizole [33] (Fig. 1).
a
large number of antibiotics and chemotherapeutics, the
increasing clinical importance of drug-resistant microbial patho-
gens have lent additional urgency in microbiological and antifungal
research [1e4]. A potential solution to the antibiotic resistance is to
design and explore innovative heterocyclic agents with novel mode
of actions. In this context, thiazole derivatives have been playing a
crucial role in medicinal chemistry. Thiazole nucleus constitutes an
integral part of all the available penicillins, which have transformed
the bacterial diseases therapy [5]. They display quite a broad
spectrum of biological activities [6], which have found applications
in the treatment of allergies [7], hypertension [8], inflammation [9],
schizophrenia [10], microbial infections [11,12], HIV infections [13],
hypnotics [14] and pain [15]. They are also used as new inhibitors of
bacterial DNA gyrase B [16]. Further, thiazoles have emerged as new
Prompted by above-mentioned observations and in continua-
tion of our search for new, potent, selective, and less toxic anti-
microbial agents [34e39], we report herein the synthesis of some
* Corresponding author. Tel./fax: þ91 278 2439852.
E-mail address: dnisheeth@rediffmail.com (N.C. Desai).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.029

N.C. Desai et al. / European Journal of Medicinal Chemistry 67 (2013) 54e59
55
Fig. 1. Commercially available drugs containing thiazole and 1,3,4-oxadiazole nucleus.
novel structural hybrids by combining thiazole and 1,3,4-
oxadiazole pharmacophores in single molecular framework in or-
der to investigate their in vitro antimicrobial activity. In addition,
cytotoxicity studies were also conducted in HeLa cell lines to
evaluate the ability of these compounds to inhibit the cell growth.
The substitution pattern of 1,3,4-oxadiazole ring was carefully
selected in order to confer different electronic environment to the
molecules.
Formation of titled compounds 5ael was confirmed by charac-
teristic IR spectrum absorption bands in the range of 3330e
3350 cmꢀ1, 3200e3250 cmꢀ1, 2810e2840 cmꢀ1, 2750e2800 cmꢀ1
and 1660e1690 cmꢀ1 corresponding to eNH stretching of sec-
ondary amine, eNH stretching of amide, eCH3, eCH2e and >C]O
of amide respectively. Singlets at around
d 2.03e2.10, 2.40e2.50,
4.15e4.25, 4.35e4.45 and 9.10e9.20 ppm in 1H NMR were due to e
CH3 in anilide group, HeteCH3, secondary amine, eCH2e and eNH
amide group respectively. The aromatic ring protons were observed
at
substitution pattern on phenyl ring. Characteristic peaks at around
168.5e168.9 and 177.0e177.3 ppm in 13C NMR confirmed the
presence of >C]O and >C]S groups respectively. The mass
spectrum of 5ael revealed that observed molecular ion peaks were
in agreement with molecular weight of respective compound.
d 6.20e8.05 ppm and J value were found to be in accordance with
2. Results and discussion
d
2.1. Chemistry
The reaction sequences employed for synthesis of the target
compounds 5ael are outlined in Scheme 1. Ethyl 2-amino-4-
methylthiazole-5-carboxylate (1) was taken as starting material
and reacted with acetic anhydride to afford ethyl 2-acetamido-4-
methylthiazole-5-carboxylate (2), which on further reaction with
hydrazine hydrate in absolute ethanol yielded intermediate N-(5-
(hydrazine carbonyl)-4-methylthiazol-2-yl)acetamide (3). The in-
termediate obtained thus, was refluxed with carbon disulfide in the
presence of potassium hydroxide in ethanol (99.5%) to yield inter-
mediate N-(4-methyl-5-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-
yl)thiazol-2-yl)acetamide (4). Mannich condensation of interme-
diate 4 with 36% formaldehyde and an appropriately substituted
aniline derivative in ethanol (99.5%) furnished the desired com-
pounds 5ael. Both analytical and spectral data of the synthesized
compounds 5ael were fully in agreement with proposed structures.
2.2. Antimicrobial studies
2.2.1. Antibacterial studies
All the synthesized compounds 5ael were evaluated for their
in vitro antibacterial activity against Staphylococcus aureus MTCC
96, Streptococcus pyogenes MTCC 442 (Gram-positive), Escherichia
coli MTCC 443 and Pseudomonas aeruginosa MTCC 1688 (Gram-
negative) by conventional broth microdilution method using
chloramphenicol as a control drug for antibacterial activity [40].
The results of the antimicrobial studies are presented in Table 1. In
general, compounds 5ael demonstrated better antibacterial activ-
ity than antifungal activity. Compounds 5c and 5i emerged as the
Scheme 1. Synthetic routes for the compounds 5ael. Reagents and conditions: (a) (CH3CO)2O, reflux, 1 h; (b) NH2NH2.H2O, EtOH, reflux, 5 h; (c) CS2, KOH, EtOH, reflux, 12 h; (d)
R-C6H4NH2, HCHO, reflux, 3h.

56
N.C. Desai et al. / European Journal of Medicinal Chemistry 67 (2013) 54e59
Table 1
Table 2
Antimicrobial screening results of compounds 5ael.
Levels of cytotoxicity induced by selected compounds
on HeLa cells at 24 h of drug exposure.
Entry
R
Minimum inhibitory concentration (MIC), mg/mL
Compounds
IC50 (mM)
Gram-positive Gram-
Fungic
bacteriaa
negative
5b
5c
5f
5h
>100
>100
98.60
>100
96.48
3.24
bacteriab
Sa
Sp
Ec
Pa
Ca An
Ac
5i
5a
5b
5c
5d
5e
5f
5g
5h
5i
5k
5k
5l
e2-F
e3-F
e4-F
250
50
100 250 500
100 50 50
500 >1000 500
Doxorubicin
500
500
500
250
25
250
500
500
500
500
500 >1000
500 >1000
250 250
100 500
25 62.5
500 500
100 250
250 100
500 250
500 500
250 250
e
12.5
25 12.5 12.5
>1000 500 250
500 500 500
>1000 500 500
250 500 250
100 50 50
25 12.5 12.5
250 250 100
500 250 500
The known numbers of cells (1.0 ꢁ 104) were incu-
bated for 24 h in a 5% CO2 incubator at 37 ꢂC in the
presence of different concentrations of test com-
pounds. After 24 h of drug incubation the MTT solu-
tion was added and supernatant was discarded and
100 mL DMSO was added in each well and absorbance
was recorded at 540 nm by ELISA reader.
e2-OCH3 500
e3-OCH3 >1000
e4-OCH3 500
e2-NO2 100
e3-NO2 50
e4-NO2 25
e2-CH3 500
e3-CH3 500
e4-CH3 500
>1000 500 >1000 500
50 50 50 e
broth microdilution method. The results indicated that compound 5f
substituted with methoxy group at para position of the phenyl ring
was found to be the most promising agent against both C. albicans
Chloramphenicol
Ketoconazole
e
50
e
e
e
e
e
e
50
50 50
a
Sa (Staphylococcus aureus MTCC 96); Sp (Staphylococcus pyogenes MTCC 442).
Ec (Escherichia coli MTCC 443); Pa (Pseudomonas aeruginosa MTCC 1688).
Ca (Candida albicans MTCC 227); An (Aspergillus niger MTCC 282); Ac (Aspergillus
and A. niger having 2-fold higher MIC (25 mg/mL) in comparison with
b
c
control drug ketoconazole. The enhanced activity of compound 5f
may be attributed to the presence of electron releasing group at para
position. The contrasting nature of substitution pattern at para
position of the phenyl ring of most active antibacterial and antifungal
agents indicate that the structural requirements are different for
binding of drug to bacterial or fungal targets, respectively [43]. All
other compounds showed less inhibition against the tested micro-
organisms as compared to the standard drug.
clavatus MTCC 1323).
most effective antibacterial agents with 2- to 4-fold higher MIC
(12.5e25 mg/mL) than the reference drug chloramphenicol. While,
compounds 5b and 5h exhibited comparable antibacterial activity
with the standard drug. From the results of the antimicrobial ac-
tivity of the synthesized compounds 5ael, the following structure
activity relationships can be derived: the antibacterial activity was
considerably affected by substitution pattern on the phenyl ring
and the most active compounds contain electron withdrawing
substituent at para and meta positions of the phenyl ring
(p > m > o). In contrast, the presence of electron releasing groups
on the phenyl ring witnessed a substantial decrease in antibacterial
activity for compounds 5def and 5kel. The role of electron with-
drawing group in improving antimicrobial activity is very well
supported by previous studies [41,42]. Compounds 5c and 5i,
substituted with inductively electron withdrawing fluoro and nitro
groups, respectively at para position showed the highest antibac-
terial activity (F > NO2). It is a very well-known fact that electron
withdrawing substitution such as fluoro/nitro at the para position
of the aromatic ring increases the lipophilicity of molecules. This
property is directly related to antimicrobial activity as it facilitates a
compound to diffuse through the biological membranes and reach
to its site of action. The presence of lipophilic substituent at para
position of phenyl ring provided a positive influence on antibac-
terial activity. On the other hand, presence of the same functional
groups at meta position resulted in slight decrease in the antibac-
terial activity (5b and 5h) but, still produced significant inhibitory
action compared to the standard drug chloramphenicol (F > NO2).
While, substituting the phenyl ring with fluoro and nitro group at
ortho position resulted in noticeable decrease in the antibacterial
activity of compounds 5a and 5g respectively. On the basis of MIC
values, it may be concluded that electron withdrawing atom/group
such as fluoro and nitro at the para position of phenyl ring induced
a positive effect while electron donating groups such as methyl and
methoxy induced a negative effect on antibacterial activity of
compounds 5ael.
2.2.3. Cytotoxicity studies
In vitro cytotoxicity of compounds 5b, 5c, 5f, 5h and 5i were
evaluated against human cervical cancer cell line (HeLa) by the 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay [44], which measures the reduction of tetrazolium
bromide salt into a formazan dye by mitochondrial dehydrogenases
in treated versus untreated cells. The results are summarized in
Table 2. It was observed that none of the tested compounds exhibited
any significant cytotoxic effect on HeLa cells, suggesting a great
potential for their in vivo use as antimicrobial agents.
3. Conclusion
In conclusion, some new structural hybrids of thiazole and 1,3,4-
oxadiazole were synthesized and investigated for their antimicro-
bial property with an anticipation of generating new structural
leads serving as potent antimicrobial agents. Many of the synthe-
sized motifs (5b, 5c, 5h and 5i), possessing electron withdrawing
atom/group such as fluoro and nitro at para and meta positions
were identified as the most potent antibacterial agents. Albeit, it
was observed that para position was more favorable for enhancing
the antibacterial activity. While, compound 5f with electron
releasing group at para position came out as the most promising
antifungal agent. The potent antimicrobial activity of most active
compounds 5b, 5c, 5f, 5h and 5i were accompanied with relatively
low level of cytotoxicity. The results described here, merits further
investigations in our laboratories using a forward chemical genetic
approach for finding lead molecules as antimicrobial agents.
4. Experimental
2.2.2. Antifungal studies
4.1. Chemistry
The in vitro antifungal activity of synthesized compounds 5ael
were determined against Candida albicans MTCC 227, Aspergillus
niger MTCC 282 and Aspergillus clavatus MTCC 1323 by conventional
All reactions except those in aqueous media were carried out by
standard techniques for the exclusion of moisture. Melting points

N.C. Desai et al. / European Journal of Medicinal Chemistry 67 (2013) 54e59
57
were determined on an electrothermal melting point apparatus
and were reported uncorrected. TLC on silica gel plates (Merck, 60,
254) was used for purity checking and reaction monitoring.
4.5.1. N-(5-(4-(((2-fluorophenyl)amino)methyl)-5-thioxo-4,5-dihydro-
1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl)acetamide (5a)
F
Yield: 64%, m.p. 178e180 ꢂC; IR (KBr): v/cmꢀ1 3331 (secondary
amine NH), 3211 (secondary amide NH), 3028 (aromatic ring CH),
2831 (CH3), 2762 (CH2), 1681 (CO), 1113 (CF). 1H NMR (DMSO-d6),
Column chromatography on silica gel (Merck, 70e230 mesh and
230e400 mesh ASTH for flash chromatography) was applied when
necessary to isolate and purify the reaction products. Elemental
analysis (% C, H, N) was carried out by a PerkineElmer 2400 CHN
analyzer. IR spectra of all compounds were recorded on a Perkine
Elmer FT-IR spectrophotometer in KBr. 1H NMR spectra were
recorded on Varian Gemini 300 MHz and 13C NMR spectra on
Varian Mercury-400 100 MHz in DMSO-d6 as a solvent and tetra-
methylsilane (TMS) as an internal standard. Mass spectra were
scanned on a Shimadzu LC-MS 2010 spectrometer. All reactions
requiring anhydrous conditions were carried out under nitrogen
atmosphere using oven-dried glassware.
d
ppm: 2.05 (s, 3H, eNHCOCH3), 2.44 (s, 3H, eNeC(CH3)eCe), 4.11
(s, 1H, AreNHeCH2), 4.44 (s, 2H, HeteCH2eNH), 6.56e7.10 (m, 4H,
AreH), 9.17 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
d
ppm: 17.0 (CH3), 24.1 (COCH3), 70.6 (CH2), 115.2, 116.3, 121.3, 125.1,
130.6, 132.4, 154.3 (CeF), 156.6 (CeCH3), 157.1, 162.9, 168.9 (amide
C]O) and 177.1 (C]S); MS (EI): m/z 379.1 (Mþ). Anal. Calcd. For
C15H14FN5O2S2 C-47.48, H-3.72, N-18.46; Found: C-47.34, H-3.68, N-
18.57%.
4.5.2. N-(5-(4-(((3-fluorophenyl)amino)methyl)-5-thioxo-4,5-dihydro-
1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl)acetamide (5b)
Yield: 61%, m.p. 166e168 ꢂC; IR (KBr): v/cmꢀ1 3330 (secondary
amine NH), 3217 (secondary amide NH), 3023 (aromatic ring CH),
2830 (CH3), 2769 (CH2), 1669 (CO), 1111 (CF). 1H NMR (DMSO-d6),
4.2. Synthesis of intermediate ethyl 2-acetamido-4-methylthiazole-
5-carboxylate (2)
d
ppm: 2.04 (s, 3H, eNHCOCH3), 2.46 (s, 3H, eNeC(CH3)eCe), 4.10
Ethyl 2-amino-4-methylthiazole-5-carboxylate (0.01 mol) was
taken in a round bottom flask and acetic anhydride (0.02 mol) was
added. The reaction mixture was refluxed at 140e150 ꢂC for 1 h and
then poured into cold water under constant stirring to get solid
product. The mixture was heated to boiling to decompose excess of
acetic anhydride and cooled. The solid obtained was filtered,
washed with water and dried. The ethyl 2-(acetyl amino)-4-
methyl-1,3-thiazole-5-carboxylate was collected and recrystal-
lized from ethanol to get pure white crystals. Yield: 74%; m.p.
158 ꢂC; Anal. obs. C, 47.08%; H, 5.48%; N, 12.44%. Calcd. for
C9H12N2O3S: C, 47.35%; H, 5.30%; N, 12.27%.
(s, 1H, AreNHeCH2), 4.42 (s, 2H, HeteCH2eNH), 6.54e7.22 (m, 4H,
AreH), 9.15 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
d
ppm: 17.2 (CH3), 24.1 (COCH3), 70.7 (CH2),102.2,108.9,109.3,131.1,
132.6, 149.2, 156.6 (CeCH3), 157.1, 162.9, 163.7 (CeF), 168.9 (amide
C]O) and 177.1 (C]S); MS (EI): m/z 379.1 (Mþ). Anal. Calcd. For
C
15H14FN5O2S2 C-47.48, H-3.72, N-18.46; Found: C-47.31, H-3.73, N-
18.51%.
4.5.3. N-(5-(4-(((4-fluorophenyl)amino)methyl)-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl)acetamide (5c)
Yield: 59%, m.p. 132e134 ꢂC; IR (KBr): v/cmꢀ1 3329 (secondary
amine NH), 3210 (secondary amide NH), 3022 (aromatic ring CH),
2839 (CH3), 2760 (CH2), 1678 (CO), 1117 (CF). 1H NMR (DMSO-d6),
4.3. SynthesisofintermediateN-(5-(hydrazinecarbonyl)-4-methylthiazol-
2-yl)acetamide (3)
d
ppm: 2.04 (s, 3H, eNHCOCH3), 2.41 (s, 3H, eNeC(CH3)eCe), 4.13
(s, 1H, AreNHeCH2), 4.44 (s, 2H, HeteCH2eNH), 7.02e7.05 (m, 4H,
Intermediate (2) (0.01 mol) and 99% hydrazine hydrate
(0.015 mol) were taken in a round bottom flask and mixture was
refluxed for 10 min. Alcohol was added till both the layers were
miscible and refluxing was continued for 5 h. Excess of alcohol and
unreacted hydrazine hydrate was distilled out and the contents
were poured into a beaker. The solid was recrystallized from
ethanol to get pure crystalline product. Yield: 64%; m.p. 172 ꢂC;
Anal. obs. C, 38.98%; H, 4.82%; N, 26.37%. Calcd. for C7H10N4O2S: C,
39.24%; H, 4.70%; N, 26.15%.
AreH), 9.16 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
d
ppm: 17.1 (CH3), 24.1 (COCH3), 70.6 (CH2), 116.3 (2C), 118.9 (2C),
132.5, 143.4, 155.7 (CeF), 156.5 (CeCH3), 157.1, 162.9, 168.9 (amide
C]O) and 177.2 (C]S); MS (EI): m/z 379.1 (Mþ). Anal. Calcd. For
C15H14FN5O2S2 C-47.48, H-3.72, N-18.46; Found: C-47.31, H-3.64, N-
18.54%.
4.5.4. N-(5-(4-(((2-methoxyphenyl)amino)methyl)-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl)acetamide (5d)
Yield: 58%, m.p. 139e141 ꢂC; IR (KBr): v/cmꢀ1 3331 (secondary
amine NH), 3208 (secondary amide NH), 3020 (aromatic ring CH),
2831 (CH3), 2766 (CH2), 1672 (CO), 1211e1103 (CeOeC). 1H NMR
4.4. Synthesis of intermediate N-(4-methyl-5-(5-thioxo-4,5-dihydro-
1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (4)
(DMSO-d6),
d ppm: 2.04 (s, 3H, eNHCOCH3), 2.44 (s, 3H, eNe
A mixture of intermediate (3) (0.01 mol), potassium hydroxide
(0.01 mol), carbon disulfide (0.02 mol) and ethanol (99.5%)
(100 mL) was refluxed for 12 h. The excess solvent was removed by
vacuum evaporation, and the residue was dissolved in water and
acidified with acetic acid to get solid product. It was filtered, dried
and recrystallized from watereethanol (60e40). Yield: 64%; m.p.
198 ꢂC; Anal. obs. C, 37.65%; H, 3.32%; N, 22.06%. Calcd. for
C8H8N4O2S2: C, 37.49%; H, 3.15%; N, 21.86%.
C(CH3)eCe), 4.13 (s, 1H, AreNHeCH2), 3.71 (s, 3H, AreOCH3), 4.44
(s, 2H, HeteCH2eNH), 6.66e7.02 (m, 4H, AreH), 9.16 (s, 1H, Hete
NHeCO); 13C NMR (400 MHz, DMSO-d6),
d ppm: 17.1 (CH3), 24.0
(COCH3),55.7 (OCH3), 71.2 (CH2), 113.5, 114.4, 121.1, 121.7, 132.4,
138.4, 144.5 (CeOCH3), 156.4 (CeCH3), 162.9, 168.7 (amide C]O)
and 177.1 (C]S); MS (EI): m/z 391.1 (Mþ). Anal. Calcd. For
C16H17N5O3S2 C-49.09, H-4.38, N-17.89; Found: C-49.23, H-4.25, N-
18.30%.
4.5. General procedure for the synthesis of N-(5-(4-(((aryl)amino)
methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-4-methylthiazol-
2-yl)acetamide (5ael)
4.5.5. N-(5-(4-(((3-methoxyphenyl)amino)methyl)-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl) acetamide (5e)
Yield: 58%, m.p. 139e141 ꢂC; IR (KBr): v/cmꢀ1 3344 (secondary
amine NH), 3223 (secondary amide NH), 3020 (aromatic ring CH),
2833 (CH3), 2784 (CH2), 1674 (CO), 1204e1093 (CeOeC). 1H NMR
A mixture of intermediate (4) (0.01 mol) and an appropriately
substituted aniline derivatives (0.01 mol) was refluxed in ethanol
(50 mL) with 36% formaldehyde (0.02 mol) for 3 h. The resulting
solid was crystallized from a suitable solvent.
(DMSO-d6),
d ppm: 2.07 (s, 3H, eNHCOCH3), 2.44 (s, 3H, eNe
C(CH3)eCe), 3.75 (s, 3H, AreOCH3), 4.15 (s, 1H, AreNHeCH2), 4.44
(s, 2H, HeteCH2eNH), 6.20e7.30 (m, 4H, AreH), 9.20 (s, 1H, Hete

58
N.C. Desai et al. / European Journal of Medicinal Chemistry 67 (2013) 54e59
NHeCO); 13C NMR (400 MHz, DMSO-d6),
(COCH3), 55.7 (OCH3), 70.9 (CH2), 105.5, 105.8, 109.2, 110.3, 132.4,
148.5,156.4 (CeCH3), 157.0,161.4 (CeOCH3), 162.9,168.7 (amide C]
O) and 177.1 (C]S); MS (EI): m/z 391.1 (Mþ). Anal. Calcd. For
d
ppm: 17.1(CH3), 24.0
2953 (AreCH3), 2840 (HeteCH3), 2771 (CH2), 1681 (CO). 1H NMR
(DMSO-d6), ppm: 2.04 (s, 3H, eNHCOCH3), 2.12 (AreCH3), 2.46 (s,
d
3H, eNeC(CH3)eCe), 4.09 (s, 1H, AreNHeCH2), 4.42 (s, 2H, Hete
CH2eNH), 6.63e7.04 (m, 4H, AreH), 9.16 (s, 1H, HeteNHeCO); 13C
C
16H17N5O3S2 C-49.09, H-4.38, N-17.89; Found: C-49.25, H-4.49, N-
NMR (400 MHz, DMSO-d6), d ppm: 17.1 (HeteCH3), 17.6 (AreCH3),
17.78%.
24.1 (COCH3), 71.2 (CH2), 110.6, 121.8 (AreCeCH3), 122.1, 126.5, 127.0,
132.3,146.5,156.5 (HeteCeCH3),157.0,162.9,168.9 (amide C]O) and
177.1 (C]S); MS (EI): m/z 375.08 (Mþ). Anal. Calcd. For C16H17N5O2S2
C-51.18, H-4.56, N-18.65; Found: C-51.30, H-4.40, N-18.52%.
4.5.6. N-(5-(4-(((4-methoxyphenyl)amino)methyl)-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl)-4-methylthiazol-2-yl)acetamide (5f)
Yield: 59%, m.p. 150e152 ꢂC; IR (KBr): v/cmꢀ1 3339 (secondary
amine NH), 3228 (secondary amide NH), 3011 (aromatic ring CH),
2832 (CH3), 2780 (CH2), 1670 (CO), 1211e1099 (CeOeC). 1H NMR
4.5.11. N-(4-methyl-5-(5-thioxo-4-((m-tolylamino)methyl)-4,5-
dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5k)
(DMSO-d6),
d
ppm: 2.04 (s, 3H, eNHCOCH3), 2.45 (s, 3H, eNe
Yield: 57%, m.p. 151e153 ꢂC; IR (KBr): v/cmꢀ1 3330 (secondary
amine NH), 3229 (secondary amide NH), 3027 (aromatic ring CH),
2951 (AreCH3), 2830 (HeteCH3), 2775 (CH2), 1680 (CO). 1H NMR
C(CH3)eCe), 3.83 (s, 3H, AreOCH3), 4.21 (s,1H, AreNHeCH2), 4.42 (s,
2H, HeteCH2eNH), 6.77e7.18 (m, 4H, AreH), 9.15 (s, 1H, HeteNHe
CO); 13C NMR (400 MHz, DMSO-d6),
d
ppm: 17.1(CH3), 24.0(COCH3),
(DMSO-d6), d ppm: 2.03 (s, 3H, eNHCOCH3), 2.34 (AreCH3), 2.42 (s,
55.8 (OCH3), 70.8 (CH2), 115.1 (2C), 115.8 (2C), 132.4, 139.9, 151.7 (Ce
OCH3),156.4 (CeCH3),157.0,162.9,168.7 (amide C]O) and 177.1 (C]
S); MS (EI): m/z 391.1 (Mþ). Anal. Calcd. For C16H17N5O3S2 C-49.09, H-
4.38, N-17.89; Found: C-49.17, H-4.52, N-17.98%.
3H, eNeC(CH3)eCe), 4.07 (s, 1H, AreNHeCH2), 4.42 (s, 2H, Hete
CH2eNH), 6.52e7.11 (m, 4H, AreH), 9.16 (s, 1H, HeteNHeCO); 13C
NMR (400 MHz, DMSO-d6),
d ppm: 17.1 (HeteCH3), 21.2 (AreCH3),
24.1 (COCH3), 70.8 (CH2), 110.5, 113.2117.4, 129.4, 132.4, 132.3, 139.3
(AreCeCH3), 147.5, 156.5 (HeteCeCH3), 157.0, 162.9, 168.9 (amide
C]O) and 177.1 (C]S); MS (EI): m/z 375.08 (Mþ). Anal. Calcd. For
4.5.7. N-(4-methyl-5-(4-(((2-nitrophenyl)amino)methyl)-5-thioxo-
4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamide (5g)
Yield: 52%, m.p. 155e157 ꢂC; IR (KBr): v/cmꢀ1 3327 (secondary
amine NH), 3224 (secondary amide NH), 3014 (aromatic ring CH),
2833 (CH3), 2784 (CH2), 1671 (CO), 1533 (NO2). 1H NMR (DMSO-d6),
C16H17N5O2S2 C-51.18, H-4.56, N-18.65; Found: C-51.00, H-4.48, N-
18.76%.
4.5.12. N-(4-methyl-5-(5-thioxo-4-((p-tolylamino)methyl)-4,5-
dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamꢀid1e (5l)
d
ppm: 2.03 (s, 3H, eNHCOCH3), 2.46 (s, 3H, eNeC(CH3)eCe), 4.24
(s, 1H, AreNHeCH2), 4.42 (s, 2H, HeteCH2eNH), 7.34e8.05 (m, 4H,
Yield: 61%, m.p. 163e165 ꢂC; IR (KBr): v/cm 3327 (secondary
amine NH), 3228 (secondary amide NH), 3029 (aromatic ring CH),
2947 (AreCH3), 2839 (HeteCH3), 2775 (CH2), 1687 (CO). 1H NMR
AreH), 9.14 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
d
ppm: 17.0 (CH3), 24.0 (COCH3), 69.7 (CH2), 114.3, 118.1, 126.1, 131.8,
132.4, 135.7, 146.7, 156.4 (CeCH3), 157.0, 162.9, 168.7 (amide C]O)
(DMSO-d6), d ppm: 2.07 (s, 3H, eNHCOCH3), 2.34 (AreCH3), 2.43 (s,
and 177.1 (C]S); MS (EI): m/z 406.05 (Mþ). Anal. Calcd. For
3H, eNeC(CH3)eCe), 4.10 (s, 1H, AreNHeCH2), 4.41 (s, 2H, Hete
C
16H17N5O3S2 C-44.33, H-3.47, N-20.68; Found: C-44.44, H-3.32, N-
CH2eNH), 6.48e7.01 (m, 4H, AreH), 9.16 (s, 1H, HeteNHeCO); 13C
20.78%.
NMR (400 MHz, DMSO-d6), d ppm: 17.1 (HeteCH3), 21.3 (AreCH3),
24.0 (COCH3), 70.8 (CH2), 113.4 (2C), 129.6 (AreCeCH3), 129.8 (2C),
132.3,144.6,156.5 (HeteCeCH3), 157.1, 162.9, 168.9 (amide C]O) and
177.1 (C]S); MS (EI): m/z 375.08 (Mþ). Anal. Calcd. For C16H17N5O2S2
C-51.18, H-4.56, N-18.65; Found: C-51.42, H-4.44, N-18.72%.
4.5.8. N-(4-methyl-5-(4-(((3-nitrophenyl)amino)methyl)-5-thioxo-
4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)aceꢀta1mide (5h)
Yield: 55%, m.p. 165e167 ꢂC; IR (KBr): v/cm 3329 (secondary
amine NH), 3225 (secondary amide NH), 3010 (aromatic ring CH),
2838 (CH3), 2781 (CH2), 1672 (CO), 1538 (NO2). 1H NMR (DMSO-d6),
4.6. Biological assay
d
ppm: 2.03 (s, 3H, eNHCOCH3), 2.44 (s, 3H, eNeC(CH3)eCe), 4.18 (s,
1H, AreNHeCH2), 4.43 (s, 2H, HeteCH2eNH), 7.20e7.60 (m, 4H, Are
H), 9.14 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
ppm:
4.6.1. In vitro evaluation of antimicrobial activity
d
All MTCC cultures were collected from Institute of Microbial
Technology, Chandigarh. The MICs of synthesized compounds were
carried out by broth microdilution method against the standard
bacterial strains S. aureus MTCC 96, S. pyogenes MTCC 442, E. coli
MTCC 443 and P. aeruginosa MTCC 1688 and antifungal activity
against the standard fungal strains C. albicans MTCC 227, A. niger
MTCC 282 and A. clavatus MTCC 1323. DMSO was used as diluents
to get desired concentration of compounds to test upon standard
bacterial strains. Serial dilutions were prepared in primary and
secondary screening. The control tube containing no antibiotic was
immediately subcultured (before inoculation) by spreading a
loopful evenly over a quarter of plate of medium suitable for the
growth of the test organism and put for incubation at 37 ꢂC over-
night. The tubes were then incubated overnight. The MIC of the
control organism was read to check the accuracy of the compound
concentrations. The MIC was defined as the lowest concentration of
the antibiotic or test sample allowing no visible growth. All the
tubes showing no visible growth (same as control tube) were
subcultured and incubated overnight at 37 ꢂC. The amount of
growth from the control tube before incubation (which represents
the original inoculum) was compared. Subcultures might show:
similar number of colonies indicating bacteriostatic; a reduced
number of colonies indicating a partial or slow bactericidal activity
17.1 (CH3), 24.0 (COCH3), 70.7 (CH2), 106.8, 112.1, 119.5, 130.5, 132.4,
148.5, 148.7, 156.4 (CeCH3), 157.0, 162.9, 168.7 (amide C]O) and
177.1 (C]S); MS (EI): m/z 406.01 (Mþ). Anal. Calcd. For C16H17N5O3S2
C-44.44, H-3.47, N-20.68; Found: C-44.20, H-3.39, N-20.62%.
4.5.9. N-(4-methyl-5-(4-(((4-nitrophenyl)amino)methyl)-5-thioxo-
4,5-dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)aceꢀta1mide (5i)
Yield: 60%, m.p. 159e161 ꢂC; IR (KBr): v/cm 3331 (secondary
amine NH), 3227 (secondary amide NH), 3010 (aromatic ring CH),
2830 (CH3), 2782 (CH2), 1671 (CO), 1524 (NO2). 1H NMR (DMSO-d6),
d
ppm: 2.03 (s, 3H, eNHCOCH3), 2.45 (s, 3H, eNeC(CH3)eCe), 4.11 (s,
1H, AreNHeCH2), 4.41 (s, 2H, HeteCH2eNH), 6.72e8.04 (m, 4H, Are
H), 9.15 (s, 1H, HeteNHeCO); 13C NMR (400 MHz, DMSO-d6),
ppm:
d
17.1 (CH3), 24.0 (COCH3), 70.9 (CH2), 114.4 (2C), 127.5 (2C), 132.4,
136.4, 153.7, 156.4 (CeCH3), 157.0, 162.9, 168.9 (amide C]O) and
177.1 (C]S); MS (EI): m/z 406.01 (Mþ). Anal. Calcd. For C16H17N5O3S2
C-44.44, H-3.47, N-20.68; Found: C-44.56, H-3.60, N-20.75%.
4.5.10. N-(4-methyl-5-(5-thioxo-4-((o-tolylamino)methyl)-4,5-
dihydro-1,3,4-oxadiazol-2-yl)thiazol-2-yl)acetamꢀid1e (5j)
Yield: 62%, m.p. 142e144 ꢂC; IR (KBr): v/cm 3331 (secondary
amine NH), 3220 (secondary amide NH), 3022 (aromatic ring CH),

N.C. Desai et al. / European Journal of Medicinal Chemistry 67 (2013) 54e59
59
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primary screening 500, 250 and 200
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second set of dilution against all microorganisms. The compounds
found active in primary screening were similarly diluted to obtain
m
g/mL concentration as a stock solution. In
g/mL concentrations of the
m
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of 100 M/well. Cells in the control wells received the same volume
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mM) in the volume
m
removed and cell cultures were incubated with 100 ml MTT reagent
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We would like to express our sincere gratitude to the Depart-
ment of Chemistry, Mahatma Gandhi Campus, Maharaja Krishna-
kumarsinhji Bhavnagar University, Bhavnagar for providing
research and library facilities.
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