Synthesis and evaluation of 2-(5-(aryl)-1,3,4-oxadiazol-2-ylthio)-N-(3- (trifluoromethyl)phenyl)acetamides and N-(4-chloro-3-fluorophenyl)-2-(5-(aryl)- 1,3,4-oxadiazol-2-ylthio)acetamides as antimicrobial agents

Article abstract of DOI:10.1007/s12039-014-0625-9

A series of 2-mercapto-5-phenyl-1,3,4-oxadiazole derivatives have been condensed with different phenyl acetamide derivatives possessing fluorine atom at meta position; resulting in the formation of 2-(5-aryl-1,3,4-oxadiazol-2- ylthio)-N-(3-(trifluoromethyl)phenyl)acetamide (5a-j) and N-(4-chloro-3- fluorophenyl)-2-(5-aryl-1,3,4-oxadiazol-2-ylthio)acetamide (5k-t) derivatives. The antimicrobial properties of the synthesized entities (5a-t) measured as their MIC (Minimum Inhibitory Concentration) values were evaluated by using the broth dilution method against Gram-positive bacteria (S. aureus and E. faecalis), Gram-negative bacteria (E. coli and P. aeruginosa) and fungi (C. albicans and A. niger). The results of antimicrobial activities (in μg/ml) revealed the fact that the compounds 5a and g bearing a maximum number of fluorine atoms showed the highest potency among the synthesized compounds against the broad panel of bacterial and fungal strains. The presence of fluorine atom at the meta position in the phenyl ring of final derivatives (5a-t) brought about an enhancement of their antimicrobial properties to a notable extent.

Full text of DOI:10.1007/s12039-014-0625-9

c
J. Chem. Sci. Vol. 126, No. 3, May 2014, pp. 827–835. ꢀ Indian Academy of Sciences.
Synthesis and evaluation of 2-(5-(aryl)-1,3,4-oxadiazol-2-ylthio)-N-(3-
(trifluoromethyl)phenyl)acetamides and N-(4-chloro-3-fluorophenyl)-2-
(5-(aryl)-1,3,4-oxadiazol-2-ylthio)acetamides as antimicrobial agents
KALPESH PARIKH and DEEPKUMAR JOSHI∗
Chemistry Division, Chemistry Research Laboratory, Sheth MN Science College, Patan 384 265, India
e-mail: deepjoshi359@yahoo.co.in
MS received 9 June 2013; revised 6 January 2014; accepted 11 January 2014
Abstract. A series of 2-mercapto-5-phenyl-1,3,4-oxadiazole derivatives have been condensed with different
phenyl acetamide derivatives possessing fluorine atom at meta position; resulting in the formation of 2-(5-aryl-
1,3,4-oxadiazol-2-ylthio)-N-(3-(trifluoromethyl)phenyl)acetamide (5a–j) and N-(4-chloro-3-fluorophenyl)-2-
(5-aryl-1,3,4-oxadiazol-2-ylthio)acetamide (5k–t) derivatives. The antimicrobial properties of the synthesized
entities (5a–t) measured as their MIC (Minimum Inhibitory Concentration) values were evaluated by using the
broth dilution method against Gram-positive bacteria (S. aureus and E. faecalis), Gram-negative bacteria (E.
coli and P. aeruginosa) and fungi (C. albicans and A. niger). The results of antimicrobial activities (in μg/ml)
revealed the fact that the compounds 5a and g bearing a maximum number of fluorine atoms showed the high-
est potency among the synthesized compounds against the broad panel of bacterial and fungal strains. The
presence of fluorine atom at the meta position in the phenyl ring of final derivatives (5a–t) brought about an
enhancement of their antimicrobial properties to a notable extent.
Keywords. 1,3,4-Oxadizoles; antifungal activity; antibacterial activity; MIC; SAR study.
antifungal,13,14 antiproliferative,15 anticancer,16,17 tyro-
sinase inhibitor18 and tiodazosin possessing oxadia-
zole nucleus is widely used as antihypertensive.19
Other biological properties associated with 1,3,4-
oxadiazole skeletal includes anti-inflammatory20,21 as
well as analgesic activity.22,23 Some derivatives con-
taining 1,3,4-oxadiazole as a core, are utilized as
muscle relaxants,24 tranquillizers25 and cathepsin K
inhibitors.26 The molecule is also useful as angiogene-
sis inhibitor and HIV integrase inhibitor.27 In the con-
text of medicinal significance; a number of routes have
been developed for synthesizing 1,3,4-oxadiazoles.28 –32
Several synthetic methods under harsh conditions using
different reagents viz. Cu (II)33 and phosphorous oxy-
chloride34 have been established.
1. Introduction
Fluorine containing compounds play a very important
role for their applications in medicinal chemistry. Fluo-
rine being the most electronegative atom with small
atomic radius helps to enhance the biological acti-
vity of newly designed molecules.1 Fluorinated com-
pounds have been widely used as important antifungal,2
anticancer3 and antibacterial drugs.4 Some of the fluori-
nated motifs have been utilized for treating cardiovascu-
lar and obesity related diseases.5 A new series of potent
GPR119 agonists, possessing fluorine atom as a sub-
stituent have been reported.6 Henceforth, fluorine sub-
stitution remains a lucrative aspect in the development
of bio-active drug molecules.
Literature study reveals the importance of nitro-
gen containing heterocyclic compounds as essential
antibacterial and antifungal drugs.7 Among several
different five-membered heterocyclic rings; 1,3,4-
oxadiazoles are the class of heterocyclic compounds
with a wide range of pharmaceutical and biologi-
cal action.8–10 The different biological activity posses-
sed by 1,3,4-oxadiazoles includes antimicrobial,11,12
Furthermore, it is observed that the fluorine atom, if
substituted with oxadiazole nucleus results in the for-
mation of potent bio-active molecules.35,36 Encouraged
by the above mentioned findings, it was rationalized
to design newer 1,3,4-oxadiazole motifs with fluorine
atom substituted as -CF3 and –F at the meta position (C-
15 in the main structure). Strategically placed –F and
-CF3 at the meta position with respect to –NHCOR-
group in the phenyl ring makes them easily removable
due to the effect of partial positive charge on meta posi-
tion generated by the presence of partial electronegative
∗For correspondence
827

828
Kalpesh Parikh and Deepkumar Joshi
charge at ortho and para position in the resonating 2.2a 5-(4-Fluorophenyl)-1,3,4-oxadiazole-2-thiol (2a):
structure. Thus, easy removal of fluorine substituent Yield–77%; m.p.: 210◦ C; IR (ATR, cm−1): 1055,
exhibits remarkable impact in altering various biologi- 1253 (-C-O-C- str.), 1423 (-C=C- str. aromatic ring),
cal steps; which includes binding with enzymes or 1549 (-C=N- str. sec. amine), 2581 (-S-H str.), 3078
1
receptor and clearance of exogenous substance.37 The (CH str. aromatic ring); H NMR (400 MHz, DMSO-
action of mechanism of several molecules containing d6, δ, ppm): 7.16–8.15 (4H, m, Ar-H), 11.82 (1H, s,
fluoro substituent at meta position is explained in a -SH); 13C NMR (100 MHz, DMSO-d6, δ, ppm): 116.2
review by Begue and Bonnet-deplon.4
(C2), 116.2 (C4), 122.5 (C6), 129.4 (C1), 129.4 (C5),
163.2 (C3), 164.8 (C7), 170.9 (C8); LCMS (m/z): 197
(M+); Anal. calcd. for C8H5FN2OS: C-48.97, H-2.57,
N-14.28, S-16.34; Found: C-49.02, H-2.61, N-14.31,
S-16.37%.
2. Experimental
2.1 General
2.3 Synthesis of 2-chloro-N-phenylacetamide
derivatives (4A and B)39
All the chemicals and solvents required for the syn-
thesis were purchased from Merck Ltd., Sdfine Chem-
icals, LOBA Chemie and HIMEDIA. The majority of
the reactions were carried out by standard procedure for
the exclusion of moisture. The composition (in %) of
elements (CHN) for the synthesized compounds were
determined by using Perkin-Elmer 2400 CHN ana-
lyzer. Melting points were determined on electro ther-
mal melting point apparatus using the open end capil-
lary method and were reported uncorrected. IR spectra
of all compounds were recorded on Bruker alpha-t FT-
Amines (0.01 mol) were made soluble in appropriate
quantity of DMF containing a few drops of TEA (3-4
drops) and stirred well for 10 min. In this well-stirred
mixture chloroacetylchloride (0.015 mol, 113 g/mol,
1.19 ml) was added slowly within 15 min maintain-
ing the temperature below 5◦C. The mixture thus
obtained was stirred for 4 h at room temperature and
then the solution containing precipitates was poured
onto crushed ice. The solid separated out was well
stirred, kept overnight and filtered. The product was
crystallized from acetone. The purity of the synthe-
sized derivatives and the extent of completion of reac-
tion were monitored using TLC with mobile phase
n-hexane: Ethyl acetate (6:4). The process was repeated
for differently substituted fluorine derivatives.
1
IR spectrophotometer in ATR. The H NMR and 13C
NMR were obtained using Bruker Spectrophotometer-
400 MHz where DMSO-d6 was used as solvent and
TMS as reference. Mass spectral analysis was con-
ducted on Schimadzu mass spectrophotometer LC-MS
2010. TLC plates used for monitoring the completion
of the reaction were purchased from Merck (TLC Silica
gel 60 F254).
2.3a 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide
(4A): Yield–87%; m.p.: 88◦ C; IR (ATR, cm−1): 1113
(-C-F, str.), 1667 (C=O str. carbonyl group), 2726 (CH2
1
2.2 Synthesis of 5-Aryl-1,3,4-oxadiazole-2-thiol
(2a–j)38
str. methylene), 3313 (NH str. sec. amine); H NMR
(400 MHz, DMSO-d6, δ, ppm): 4.06 (2H, s, -CH2),
7.26-8.06 (4H, m, Ar-H), 10.71 (1H, s, -NH); 13C
NMR (100 MHz, DMSO-d6, δ, ppm): 42.4 (C9), 121.2
(C14), 124.9 (C12), 125.8 (C16), 129.6 (C13), 131.7
(C15), 139.0 (C11), 165.0 (C10); 19F NMR (376 MHz,
DMSO-d6, δ, ppm): −61.36 (3F, s, -CF3); LCMS (m/z):
238(M+); Anal. calcd. for C9H7ClF3NO: C-45.49,
H-2.97, N-5.89 Found: C-45.54, H-3.01, N-5.92%.
Ethanol (40 ml) and potassium hydroxide (0.01 mol,
56.11 g/mol, 0.56 g in 2 ml H2O) were taken in a
dry round bottom flask. Various aryl substituted ben-
zoic acid hydrazide (0.01 mol) were added to it and
stirred well to get a clear solution. Carbon disulphide
(0.02 mol, 76.14 g/mol, 1.2 ml) was added to the clear
solution obtained above and refluxed for 12–15 h. The
ethanol was distilled off and then cooled to room tem-
perature. The content was poured into water and acid- 2.3b 2-Chloro-N-(4-chloro-3-fluorophenyl)acetamide
ified with diluted HCl till the precipitates were sepa- (4B): Yield–78%; m.p.: 122◦ C; IR (ATR, cm−1):
rated. The separated solid was washed with cold water 1153 (-C-F, str.), 1671 (C=O str. carbonyl group),
and dried to get the desired product. The completion of 2731 (CH2 str. methylene), 3397 (NH str. sec. amine);
the reaction was monitored on TLC using ethyl acetate: 1H NMR (400 MHz, DMSO-d6, δ, ppm): 4.06 (2H, s,
Benzene (6:4) as mobile phase. All the derivatives were -CH2), 7.30-7.80 (3H, m, Ar-H), 10.51(1H, s, -NH);
obtained using the above stated procedure.
13C NMR (100 MHz, DMSO-d6, δ, ppm): 42.4 (C9),

Antimicrobial evaluation of 1,3,4-oxadiazoles
829
111.8 (C16), 116.8 (C14), 124.9 (C12), 129.6 (C13), 139.0 the liberated hydrochloric acid during the reaction.
(C11), 163.2 (C15), 165.0 (C10); 19F NMR (376 MHz, This mixture was allowed to stir for 4 h at room
DMSO-d6, δ, ppm): -115.69 (1F, s, -F); LCMS (m/z): temperature. The resulting material was poured onto
222(M+); Anal. calcd. for C8H6Cl2FNO: C-43.27, crushed ice and stirred well for 30 min. The solid
H-2.72, N-6.31 Found: C-43.31, H-2.76, N-6.35%.
separated out was filtered and washed with cold
water. The product obtained was dried and recrys-
tallized from alcohol. The completion of the reac-
tion was confirmed by using TLC with mobile phase
n-hexane: Ethyl acetate (6:4). All the entitled deriva-
tives were obtained by the above stated procedure.
2.4 Synthesis of final compounds (5a–j)
In a round bottom flask (moisture-free) contain-
ing 2-chloro-N-(3-(trifluoromethyl)phenyl)acetamide
(0.01 mol, 237 g/mol, 2.37 g) in acetone, various aryl
substituted 1,3,4-oxadiazole-2-thiols (0.01 mol) were
added. K2CO3 (0.02 mol, 138 g/mol, 2.76 g) was added
to neutralize the liberated hydrochloric acid during the
reaction. This mixture was allowed to stir for 4 h at
room temperature. The resulting material was poured
onto crushed ice and stirred well for 30 min. The solid
separated out was filtered and washed with cold water.
The product obtained was dried and recrystallized from
alcohol. All the targeted derivatives were obtained by
the above stated procedure. The completion of the
reaction was confirmed by using TLC with mobile
phase n-hexane: Ethyl acetate (8:2) and (7:3) (as per
the resolution).
2.5a N-(4-Chloro-3-fluorophenyl)-2-(5-(4-fluoro-
phenyl)-1,3,4-oxadiazol-2-ylthio)acetamide (5k):
Yield–69%; m.p.: 186◦ C; IR (ATR, cm−1): 765 (-C-Cl
str.), 1035, 1243 (-C-O-C- str.), 1169 (-C-F, str.), 1341
(-C-N- str. sec. amine), 1452 (-C=C- str. aromatic
ring), 1683 (C=O str. carbonyl group), 2725 (CH2 str.
methylene), 3035 (CH str. aromatic ring), 3407 (NH str.
1
sec. amine); H NMR (400 MHz, DMSO-d6, δ, ppm):
4.24 (2H, s, -CH2), 7.07–8.02 (7H, m, Ar-H), 10.46
(1H, s, -NH); 13C NMR (100 MHz, DMSO-d6, δ, ppm):
38.9 (C9), 111.8 (C16), 116.2 (C4), 116.2 (C2), 116.8
(C14), 122.5 (C6), 124.9 (C12), 129.4 (C5), 129.4 (C1),
129.6 (C13), 139.0 (C11), 163.2 (C3), 163.2 (C15), 164.8
(C7), 168.4 (C10), 171.1 (C8); 19F NMR (376 MHz,
DMSO-d6, δ, ppm): -115.69 (1F, s, -F (c−15)), -108.64
(1F, s, p-F); LCMS (m/z): 382 (M+); Anal. calcd. for
C16H10ClF2N3O2S: C-50.34, H-2.64, N-11.01, S-8.40
Found: C-50.38, H-2.66, N-11.04, S-8.42%.
2.4a 2-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-ylthio)-
N-(3-(trifluoromethyl)phenyl)acetamide (5a): Yield–
68%; m.p.: 166◦ C; IR (ATR, cm−1): 1055, 1253
(-C-O-C- str.), 1113 (-C-F, str.), 1349 (-C-N- str. sec.
amine), 1401 (-C=C- str. aromatic ring), 1667 (C=O
str. carbonyl group), 2726 (CH2 str. methylene), 3096
(CH str. aromatic ring), 3313 (NH str. sec. amine);
1H NMR (400 MHz, DMSO-d6, δ, ppm): 4.32 (2H, s,
-CH2), 7.26–8.06 (8H, m, Ar-H), 10.71 (1H, s, -NH);
13C NMR (100 MHz, DMSO-d6, δ, ppm): 39.0 (C9),
116.2 (C2), 116.2 (C4), 120.9 (C14), 122.5 (C6), 124.9
(C12), 125.9 (C16), 129.4 (C1). 129.4 (C5), 129.6 (C13),
131.7 (C15), 139.0 (C11), 163.2 (C3), 164.8 (C7), 168.4
(C10), 171.1 (C8); 19F NMR (376 MHz, DMSO-d6, δ,
ppm): -61.36 (3F, s, -CF3), -108.56 (1F, s, 4-F); LCMS
(m/z): 398(M+); Anal. calcd. for C17H11F4N3O2S:
C-51.39, H-2.79, N-10.58, S-8.07; Found: C-51.43,
H-2.83, N-10.63, S-8.10%.
2.6 Antimicrobial assay
The newly synthesized entities (5a–t) were screened
for their antimicrobial assay against a broad panel of
Gram-positive bacteria S. aureus (ATCC No. 25923),
E. faecalis (ATCC No. 29212), Gram-negative bacteria
E. coli (ATCC No. 25922), P. aeruginosa (ATCC No.
27853) and fungi C. albicans (ATCC No. 10231), A.
niger (ATCC No. 1015). The samples for antifungal as
well as antibacterial evaluation were tested by standard
protocols like Micro dilution/Broth titer method. The
stock solution (1000 μg/ml) for each compound was
prepared and screening for antimicrobial activity was
carried out by diluting the stock solution and preparing
the sets consecutively from 1000, 500, 250, 125, 62.5,
31.25, 15.62 up to 7.8 μg/ml. The tubes along with the
control were then kept for incubation at 37◦C for 24 h.
2.5 Synthesis of final compounds (5k–t)
In a round bottom flask (moisture free); 2-chloro-N- Suspensions were further inoculated on an appropriate
(4-chloro-3-fluorophenyl)acetamide (0.01 mol, 222 g/ media and growth was noted after 48 h. The obtained
mol, 2.22 g) was made soluble in appropriate amount of results (MIC) in μg/ml by observing the highest dilu-
acetone. To this solution, various aryl substituted 1,3,4- tions (low turbidity) were recorded and compared with
oxadiazole-2-thiols (0.01 mol) were added. K2CO3 the MIC value of standard drugs ciprofloxacin and
(0.02 mol, 138 g/mol, 2.76 g) was added to neutralize fluconazole.

830
Kalpesh Parikh and Deepkumar Joshi
as a neutralizing agent by condensing 2a–j with 4A and
3. Results and discussion
4B respectively.
3.1 Chemistry
Synthesis of the desired compounds and interme-
diates was accomplished according to the steps
depicted in scheme 1. The use of two very simple but
most important fluorine substituted anilines played
an important role in the enhancement of biologi-
cal results of the synthesized compounds (5a–t).
The compounds 3-(trifluoromethyl)aniline (3A) and
4-chloro-3-fluoroaniline (3B) were used as a start-
ing material for the synthesis of the targeted com-
pounds. These two fluoro substituted aniline were
treated with chloroacetyl chloride (CAC), triethylamine
(TEA) and dimethylformamide (DMF) resulting in
the formation of useful intermediates; 2-chloro-N-(3-
(trifluoromethyl)phenyl)acetamide (4A), and 2-chloro-
N-(4-chloro-3-fluorophenyl)acetamide (4B), respec-
tively. On the other hand, differently substituted benzo-
hydrazide derivatives (1a–j) were treated with CS2 and
KOH in an alcoholic medium to undergo cyclization
resulting in the formation of potent 1,3,4-oxadiazole
derivatives (2a–j). The oxadiazole containing fluoro
substituent derivatives (5a–t) were synthesized under
mild conditions using acetone as a solvent and K2CO3
3.2 Characterization
3.2a IR data: IR spectral data obtained here lead
to the confirmation about the formation of targeted
compounds. The broad stretching band appearing at
3313 cm−1 and 3407 cm−1 indicated the presence of
secondary amine in compounds 5a and 5k, respectively.
The presence of –C-O-C- bond in 1,3,4-oxadizole ring
was confirmed by two stretching bands at 1055 and
1253 cm−1 in compound 5a. Also the presence of
stretching vibration bands at 1035 and 1243 cm−1 in
compound 5k confirmed the presence of –C-O-C- bond
in form of oxadiazole nucleus. A weak absorption band
at 2726 (5a) and 2725 cm−1 (5k) helped to conclude the
presence of ‘–CH2’ methylene functional group in the
targeted compounds. The presence of –C=O group in
vicinity to –NH linkage can be predicted by the stretch-
ing frequency of 1667 and 1683 cm−1 in compounds
5a and k, respectively. The presence of -C=C- in aro-
matic ring was confirmed due to the visibility of a band
at 1401 and 1452 cm−1 in 5a and k, respectively. –C-N-
linkage in the structure can be proved by a stretching
l
l
l
l
l
l
l
l
Scheme 1. Synthetic route for the preparation of compounds 5a–t.

Antimicrobial evaluation of 1,3,4-oxadiazoles
831
frequency obtained at 1349 cm−1 in molecule 5a and at
1341 cm−1 in compound 5k.
carbon had a field shift to 139.8 ppm, inspite of having
a direct contact to –OCH3 due to the presence of two
methoxy groups each at C-4 and C-2. Similarly, the car-
bon C-2 (5j) and C-3 (5h) having a –NO2 substitution
exerted a signal at 149.0 and 148.2, respectively. The
carbon numbering to the structure is given in figure 1.
3.2b 1H NMR data: The formation of the targeted
compounds can be easily confirmed by interpreting
the obtained absorption peaks in the proton spec-
tral data. If we take into consideration compound
2-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-ylthio)-N-(3-
(trifluoromethyl)phenyl)acetamide (5a); a sharp peak
observed at δ = 4.32 confirmed the presence of two pro-
tons of the methylene group in the targeted molecule.
Absorption peak at δ = 10.71 executed the presence
of a proton of secondary amine in the structure. The
protons present on the aromatic nucleus showed the
absorption details between δ = 7.26 and 8.06 ppm.
The compound N-(4-chloro-3-fluorophenyl)-2-(5-(4-
fluorophenyl)-1,3,4-oxadiazol-2-ylthio)acetamide (5k)
3.2d 19F NMR: The chemical shifts values derived
from the 19F NMR spectra clearly confirmed the pres-
ence of fluorine atoms in the synthesized molecules. In
molecule 5a, the δ value obtained at −61.36 ppm con-
firmed the presence of –CF3 as a substituent. Similarly,
a shift at δ value −115.69 ppm helped to prove the
presence of –F (attached to C-15) in the structure 5k.
Furthermore, the presence of fluoro substituents in 5a
and k at the para position (C-3) was proved by obtain-
ing δ value at −108.56 and −108.64 ppm, respectively.
Thus, fluorine spectral details added more support to the
formation of titled compounds.
1
with the 3-fluoro substituent when screened for H
NMR spectra, the following absorption details were
observed. The absorption spectra at δ = 4.24 exhi-
bited the presence of protons of methylene group. The
presence of a proton of secondary amine proved its
presence by showing an absorption peak at δ = 10.46.
The presence of seven protons on two phenyl rings
were confirmed by δ = 7.07 to 8.02.
3.3 Antimicrobial activity
Broad panel of Gram-positive bacteria (S. aureus
and E. faecalis), Gram-negative bacteria (E. coli and
P. aeruginosa) and fungal strains (C. albicans and
A. niger) were utilized for testing the antimicrobial
properties of the targeted compounds (5a–t). The
results of the antibacterial and antifungal activities in
MIC (μg/ml) have been reported in tables 1 and 2,
respectively. It was observed that most of the com-
pounds exhibited excellent activity as compared to the
standards. A very few of them exhibited moderate or
poor activity. The standard drug used against antibac-
terial strains was ciprofloxacin and fluconazole was
employed as a standard against fungal strains. The MIC
value of ciprofloxacin was recorded to be 62.5 μg/ml
against Gram-positive bacterial strain S. aureus, where
as the same standard responded to MIC 125 μg/ml;
when used against E. faecalis, E. coli and P. aeruginosa.
Fluconazole, when used as a standard drug against fun-
gal strains C. albicans and A. niger, MIC value was
3.2c 13C NMR data: The 13C NMR added more con-
firmation to the formation of the targeted motifs. The δ
values were seen to vary between 38.9 and 168.4. The
chemical shifts were assigned by taking into consider-
ation the spectral information produced in an article by
Aranda et al.40 The carbon atom ‘10’ in contact with
electronegative atom oxygen corresponding to a car-
bonyl group showed a maximum downfield shift in the
spectrum at δ = 168.4. The 13C NMR signal obtained
at 38.9 ppm can be assigned to the carbon ‘9’ of the
methylene group present in between the sulphur and the
carbonyl group. The carbon ‘7’ present in between the
nitrogen and the oxygen atom of the oxadiazole ring
and in contact to the phenyl ring with different substitu-
tion exhibited a shift at 164.8 ppm. The chemical shift
details of the carbon in the phenyl ring with the dif-
ferent substituent was seen to vary between 104.8 and
163.2 ppm, where the δ = 104.8 corresponded to the
C−1 and C−5 carbon of the compounds 5e and o due
to the presence of a methoxy group at C−2 and C−4.
The 13C NMR displaying a signal at 163.2 ppm in 5a
and k was due to the presence of fluorine atom to the
carbon C-3 by a single bond. The 13C NMR spectra for
the compounds 5e and o exhibited sharp peak at 153.5
indicating the presence of -OCH3 group in direct con-
tact with the carbon C-2 and C-4. The signal of C-3
t
Figure 1. Carbon numbering of the final derivatives 5a–t.

832
Table 1. Antimicrobial screening of the compounds 5a–j.
Minimum inhibitory concentration (MIC) in μg/ml
Kalpesh Parikh and Deepkumar Joshi
Gram-positive bacteria Gram-negative bacteria
E. faecalis E. coli P. aeruginosa
ATCC 25923 ATCC 29212 ATCC 25922 ATCC 27853 ATCC 10231 ATCC 1015
Fungi
C. albicans
A. niger
S. aureus
Compounds
5a (R:p-F, X:CF3, Y:H)
5b (R:o-Cl, X:CF3, Y:H)
5c (R:p-CH3, X:CF3, Y:H)
5d (R:o-Br, X:CF3, Y:H)
5e (R:tri-OCH3, X:CF3, Y:H)
5f (R:o-CH3, X:CF3, Y:H)
5g (R:H, X:CF3, Y:H)
5h (R:p-NO2, X:CF3, Y:H)
5i (R:p-Cl, X:CF3, Y:H)
5j (R:m-NO2, X:CF3, Y:H)
Fluconazole
62.5
62.5
62.5
62.5
62.5
31.25
31.25
31.25
31.25
31.25
–
62.5
62.5
62.5
62.5
31.25
31.25
31.25
31.25
62.5
62.5
–
31.25
15.63
31.25
62.5
62.5
62.5
31.25
31.25
31.25
15.63
–
62.5
62.5
62.5
62.5
62.5
62.5
125
125
62.5
62.5
125
125
31.25
125
–
62.5
62.5
62.5
125
125
125
62.5
62.5
62.5
62.5
62.5
–
31.25
31.25
31.25
31.25
15.63
31.25
31.25
–
Ciprofloxacin
62.5
125
125
125
Std.: Standard drug fluconazole for antifungal and ciprofloxacin for antibacterial tests
observed at 125 and 62.5 μg/ml, respectively. In gene- No. 25923). Other compounds viz. 5a (p-F), 5b
ral, it was observed that most of the compounds have (o-Cl), 5c (p-CH3), 5d (o-Br), 5e (3,4,5-tri-OCH3), 5k
resulted to be potent antibacterial, and a very few of (p-F), 5l (o-Cl), 5n (o-Br), 5o (3,4,5-tri-OCH3) and 5r
them were found to exhibit good antifungal activity.
(p-NO2) were observed to exhibit MIC value equal to
that of the standard drug (MIC 125 μg/ml). Only two
compounds 5p (o-CH3) and 5s (p-Cl) were exhibiting
poor activity (MIC 250 μg/ml). A very few exhibited
excellent activity (MIC 31.25 μg/ml) against E. faecalis
3.4 Antibacterial activity
The targeted compounds were screened for their such as 5e (3,4,5-tri-OCH3), 5f (o-CH3), 5g (H) and 5h
antibacterial activity and their MIC (μg/ml) values (p-NO2). The other bio-chemical motifs 5a (p-F), 5b
were recorded. It was observed that compounds 5f (o- (o-Cl), 5c (p-CH3), 5d (o-Br), 5i (p-Cl), 5j (m-NO2) and
CH3), 5g (H), 5h (p-NO2), 5i (p-Cl) and 5j (m-NO2) 5l (o-Cl) showed good activity. It was observed that the
showed excellent activity (MIC = 31.25 μg/ml), when targeted molecules 5s (p-Cl), 5r (p-NO2), 5p (o-CH3),
tested against Gram-positive bacteria S. aureus (ATCC 5o (3,4,5-tri-OCH3) and 5k (p-F) exhibited activity
Table 2. Antimicrobial screening of the compounds 5k–t.
Minimum inhibitory concentration (MIC) in μg/ml
Gram-positive bacteria
S. aureus E. faecalis
ATCC 25923 ATCC 29212 ATCC 25922 ATCC 27853 ATCC 10231 ATCC 1015
Gram-negative bacteria
E. coli P. aeruginosa
Fungi
C. albicans
A. niger
Compounds
5k (R:p-F, X:F, Y:Cl)
5l (R:o-Cl, X: F, Y:Cl)
5m (R:p-CH3, X:F, Y:Cl)
5n (R:o-Br, X:F, Y:Cl)
5o (R:tri-OCH3, X:F, Y:Cl)
5p (R:o-CH3, X:F, Y:Cl)
5q (R:H, X:F, Y:Cl)
5r (R:p-NO2, X:F, Y:Cl)
5s (R:p-Cl, X:F, Y:Cl)
5t (R:m-NO2, X:F, Y:Cl)
Fluconazole
62.5
62.5
–
125
62.5
–
62.5
31.25
–
125
125
–
250
250
–
250
125
–
62.5
62.5
125
62.5
62.5
125
31.25
–
125
125
125
125
125
125
125
–
62.5
62.5
31.25
62.5
62.5
62.5
125
–
125
62.5
62.5
125
62.5
62.5
62.5
–
125
250
125
125
125
125
125
125
–
125
250
125
250
125
125
125
62.5
–
Ciprofloxacin
62.5
125
125
125
Std.: Standard drug fluconazole for antifungal and ciprofloxacin for antibacterial tests

Antimicrobial evaluation of 1,3,4-oxadiazoles
833
of similar MIC value to that of a standard (MIC 250 μg/ml) against C. albicans. When the synthesized
125 μg/ml). The compound 5m (p-CH3) was observed compounds were tested against A. niger (ATCC No.
to show zero activity against both S. aureus and 1015); very few compounds 5a (p-F), 5b (o-Cl), 5c (p-
E. faecalis.
CH3), 5g (H), 5h (p-NO2), 5i (p-Cl) and 5j (m-NO2)
The targeted series of compounds (5a–t); when intro- exhibited moderate activity (MIC 62.5 μg/ml) as com-
duced against Gram-negative bacteria E. coli; other pared to the standard drug. Other compounds did not
than 5m (p-CH3) and 5t (m-NO2), all compounds exhi- turn up to be good antifungal against A. niger as they
bited excellent to good activity as compared to the stan- exhibited poor activity as compared to the fluconazole
dard drug. The compounds 5b (o-Cl) and 5j (p-NO2) drug.
have shown good activity and far better than the stan-
dard drug with the MIC value of 15.63 μg/ml. The
compounds 5a (p-F), 5c (p-CH3), 5g (H), 5h (p-NO2),
3.6 Structure-activity relationship
5i (p-Cl), 5l (o-Cl) and 5p (o-CH3) showed excellent
activity (MIC 31.25 μg/ml). The MIC values of the
compounds 5d (o-Br), 5e (3,4,5-tri-OCH3), 5f (o-CH3),
5k (p-F), 5n (o-Br), 5o (3,4,5-tri-OCH3), 5q(H), 5r(p-
NO2) and 5s (p-Cl) were good as compared to standard
with a MIC value of 62.5 μg/ml. Thus, all the com-
pounds were showing better activity than the standard
ciprofloxacin (MIC 125 μg/ml), when treated against
The study of SAR helped in revealing that the use
of different substitution and their effect on various
microbial strains. Different electronic environments
were made possible on the phenyl ring as to con-
fer the change in activity due to different func-
tional groups. Both electron-withdrawing and electron-
donating groups were chosen as substitutions during the
designing and synthesis of target molecules as indicated
Gram-negative bacteria E. coli. Another Gram-negative
bacteria P. aeruginosa was employed to check the acti-
in figure 2. The use of different substitution in the tar-
get entities proved to be the most effective parameter in
vity of the series of compounds synthesized. Compound
the synthesis of bio-active molecules. From the antimi-
5h (p-NO2) exhibited best MIC value (15.63 μg/ml)
crobial activity data available in the report, it can be
concluded that majority of compounds with electron-
withdrawing functional groups like –NO2, -F, -Cl and
–Br exhibited better activity than the compounds with
among all the synthesized compounds. Several com-
pounds such as 5d (o-Br), 5e (3,4,5-tri-OCH3), 5f (o-
CH3), 5g (H), 5k (p-F) and 5l (o-Cl) also showed excel-
lent activity (MIC 31.25 μg/ml) compared to the stan-
electron-donating groups like –CH3 and -OCH3. The
dards against P. aeruginosa. Chemical entities 5a (p-
compounds 5a (p-F), 5b (o-Cl), 5d (o-Br), 5h (p-NO2),
F), 5b (o-Cl), 5c (p-CH3), 5o (3,4,5-tri-OCH3), 5r (p-
5i (p-Cl) and 5j (m-NO2) possessing electron with-
drawing groups showed excellent activity against all the
bacterial strains and a moderate activity against fungal
NO2), 5s (p-Cl) and 5t (m-NO2) responded to Gram-
negative bacteria P. aeruginosa up to 62.5 μg/ml. Com-
pounds 5k (p-F), 5l (o-Cl), 5n (o-Br) and 5q (H)
exhibited activity similar to that of the standard drug
ciprofloxacin (MIC 125 μg/ml) against P. aeruginosa.
Overall, amongst the targeted molecules, only com-
pound 5m did not respond to any of the bacterial
strain, where as compounds 5f (o-CH3), 5g (H), 5h (p-
NO2), 5j (m-NO2) and 5b (o-Cl) have exhibited excel-
lent activity against all the bacterial strains utilized for
conducting the MIC value tests.
3.5 Antifungal activity
From the antifungal activity results, it can be evalu-
ated that the compound 5j (m-NO2) exhibited excel-
lent activity (MIC 31.25 μg/ml) from the complete
series, when employed against C. albicans (ATCC No.
10231) as compared to the standard drug flucona-
zole (125 μg/ml). Other compounds 5a (p-F), 5b (o-
Cl), 5c (p-CH3), 5f (o-CH3) and 5g (H) showed good
activity (62.5 μg/ml). Compounds 5k (p-F), 5l (o-Cl)
and 5o (3,4,5-tri-OCH3) resulted in poor activity (MIC
Figure 2. General structures of targeted bioactive com-
pounds 5a–t.

834
Kalpesh Parikh and Deepkumar Joshi
Table 3. Antimicrobial screening of the compounds 5g, 5q and N-phenyl-2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)acetamide
(indicating the correlation between fluorine atom and biological activity).
Minimum inhibitory concentration (MIC) in μg/ml
Gram-positive bacteria
S. aureus E. faecalis
ATCC 25923 ATCC 29212 ATCC 25922
Gram-negative bacteria
Fungi
C. albicans
ATCC 10231 ATCC 1015
E. coli
P. aeruginosa
ATCC 27853
A. niger
Compounds
5* (R:H, X: H, Y:H)
5g (R:H, X:CF3, Y:H)
5q (R:H, X:F, Y:Cl)
125
31.25
62.5
125
31.25
125
250
31.25
62.5
250
31.25
125
250
62.5
125
125
62.5
250
5*= N-phenyl-2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)acetamide
strains. It was also observed that the compounds 4. Conclusion
5c (p-CH3), 5e (3,4,5-tri-OCH3), 5f (o-CH3), 5m (p-
In the present work, more emphasis was given on deve-
lopment of new bio-active heterocyclic derivatives bear-
ing fluorine atoms. The presence of hydrophobic group
fluorine at position 15 (in the final structure as shown
in figure 1) helped to demonstrate its importance lead-
ing to higher activity of the synthesized scaffolds, even
higher than the standard drugs used. The SAR study
reveals that the electronic effect of the various sub-
stituents plays a very important role in identifying the
targeted compounds with electron with-drawing groups
(NO2, F, Cl and Br) as potent microorganism growth
inhibitors. Also the importance of fluorine atom (quan-
titatively) is proved to be of much importance in the
enhancement of biological properties of the derived
compounds as indicated in the SAR study. The results
prompted the titled oxadiazole compounds as interest-
ing leads for further physiochemical optimization and
are also interesting for future scope, to be used as tem-
plates for generating better antibacterial and antifungal
lead molecules.
CH3), 5o (3,4,5-tri-OCH3) and 5p (o-CH3) possessing
electron- donating groups showed moderate activity or
even less than the standards used.
The antibacterial as well as antifungal MIC values for
compounds 5g (H) well supports even in the absence
of any substitutions the antimicrobial activity which
seems to be an excellent quality with respect to some
electron-donating groups against each bacterial and
fungal strains used. This has helped to confirm that the
fluorine substitution on the main structure at position X
(attached to C-15) in the form of –CF3 shoots up the
potency of target molecules as an effective antimicro-
bial agent. The effect of decrease in the potency of tar-
get molecule due to reduction of electron-withdrawing
functional group ‘fluorine’, can be proved by compar-
ing the biological activity data between the compounds
5g (R:H, X:CF3, Y:H) and 5q (R:H, X:F, Y:Cl). Also
if a compound (5*) with no fluorine substituent or no
electron-withdrawing group is taken, activity is found
to be diminishing as shown in table 3. The compound
5g exerted excellent activity (31.25 μg/ml) against both
the Gram-positive and Gram-negative bacterial strains,
where as the compound 5q showed comparatively
low activity (62.5 μg/ml) against the same bacterial
strains.
Supplementary information
Supplementary information (spectral data) of com-
pounds, can be seen in the website of Journal of Chem-
ical Sciences (www.ias.ac.in/chemsci).
Along with the presence of electron-withdrawing
groups, the presence of fluorine substitution at position
X in each compound has helped to enhance the antimi-
crobial potency across the panel of Gram-positive,
Gram-negative and fungal strains. Overall, the presence
Acknowledgements
of electron-withdrawing groups like -F, -Cl, -Br and Authors would like to express their sincere thanks to
-NO2 in the chemical entities as substitution and fluo- Sheth M. N. Science College, Patan for providing
rine atom (-CF3 or –F) in the heterocyclic skeleton laboratory facilities. We are highly thankful to SAIF
have helped to increase the antimicrobial potency. Thus, Chandigarh and Indian Institute of Science (IISc),
in the future, the combination of different electron- Bangalore, for analytical and instrumentation facilities.
withdrawing groups will be performed on oxadiazole One of our author DSJ is highly indebted to Department
ring for generating better lead entities as potent antibac- of Science and Technlogy (DST), India for awarding
terial and antifungal agents.
INSPIRE fellowship (DST-JRF).

Antimicrobial evaluation of 1,3,4-oxadiazoles
835
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