Effect of microwave irradiation on the synthesis of 1,2,4-triazol-3-one derivatives and their antimicrobial activities

Article abstract of DOI:10.3184/174751912X13400138806685

An easy efficient method for the synthesis of N-(3,5-dichlorophenyl)-5- alkyl/aryl-3H-1,2,4-triazol-3-one derivatives under microwave irradiation has been developed using the reaction of 3,5-dichloroaniline and ethoxycarbonylhydrazones. This reaction occu

Full text of DOI:10.3184/174751912X13400138806685

484 RESEARCH PAPER
AUGUST, 484–488
JOURNAL OF CHEMICAL RESEARCH 2012
Effect of microwave irradiation on the synthesis of 1,2,4-triazol-3-one
derivatives and their antimicrobial activities
Bahittin Kahvecia*, Fatih Yılmazb, Emre Menteşeb and Fatih Şaban Berişc
aNutrition and Dietetics, Faculty of Health Sciences, KaradenizTechnical University, Trabzon 61080, Turkey
bDepartment of Chemistry, Art and Science Faculty, RecepTayyip Erdog˘an University, Rize 53100, Turkey
Department of Biology, Art and Science Faculty, RecepTayyip Erdog˘an University, Rize 53100, Turkey
An easy efficient method for the synthesis of N-(3,5-dichlorophenyl)-5-alkyl/aryl-3H-1,2,4-triazol-3-one derivatives
under microwave irradiation has been developed using the reaction of 3,5-dichloroaniline and ethoxycarbonylhydra-
zones. This reaction occurs more efficiently and faster than the conventional heating method. We obtained 5-
mercapto-1,3,4-oxadiazol derivatives by this method. The newly synthesised compounds have been tested for their
antimicrobial activity against Enterobacter cloaceae, Escherichia coli, Klebsiella pneumonie, Pseudomonas aerugi-
nosa, Proteus vulgaris, Salmonella typhimurium, Yersinia pseudotuberculosis, Bacillus subtilis and Staphylococcus
aereus.
Keywords: microwave irradiation, triazoles, 3,5-dichloroaniline, 1,3,4-oxadiazole, antimicrobial activity
Since the publication of the first report in 1986 on microwave-
assisted organic synthesis, the use of microwave heating has
become so popular that it has been termed the Bunsen burner
of 21st century.1,2 In many respects, microwave irradiation is
superior to classical heating because it reduces the reaction
time and provides higher efficiency and purity. Using this
technique, a large number of new drug candidates have been
synthesised by synthetic organic chemists.3,4
studies using the reaction of 3,5-dichloroaniline and ethoxy-
carbonylhydrazones. However, by using a mono-mode
microwave reactor with pressure and temperature control, this
reaction has been achieved.2,12 A microwave procedure enabled
us to prepare oxadiazole derivatives of 3,5-dichloroaniline
(Scheme 2). The yields of compounds 2a–d with microwave
heating and conventional method are shown in Table 1.
The triazole ring is found in several biologically–active
compounds. Recent examples include Fluconazole, an antifun-
gal drug,5,6 and Letrozole, an effective aromatase inhibitor,7
which are shown in Fig. 1. These compounds have been
synthesised by various methods. One of the most effective and
common methods is the reaction of an aromatic amine and
ethoxycarbonylhydrazone using conventional heating. Recently,
this technique has been modified by using microwave heating4
and significant improvements to the yield and purity have been
obtained (Scheme 1).
These compounds containing triazole and oxadiazole
moieties have led medicinal chemists to synthesise new drug
candidates because of their diverse pharmaceutical activity. In
particular, compounds including both these heterocycles in
their structure have been found as an effective antimicrobial
compounds.8–10
Antimicrobial activity
All of the synthesised compounds were tested against three
Gram-positive bacteria in accordance with published proto-
cols13,14. The results were compared with the standard drug
ampicillin (Table 2). As can be seen, all newly synthesised
compounds showed anti-bacterial effect, ranging from good
to moderate, with a minimum inhibitory concentration of
31.25–1500 µg mL−1 in dimethyl sulfoxide.
Compound 2a–b, 3d, 4a, 4d, 5a and 5c were found to
have moderate activity against all bacteria with an MIC value
of 500 µg mL−1. Compound 2c has an MIC of 93.75 µg mL−1
against S. typhymirium (ATCC 14028, and 375 µg mL−1 against
E. cloaceae (ATCC 13047), P. vulgaris (ATCC 13315),
B. subtilis (ATCC 66333), S. aureus (ATCC 25923), and
750 µg mL−1 against the other bacteria. Compound 3d has MIC
values of 375 µg mL−1 against E. cloaceae (ATCC 13047),
P. vulgaris (ATCC 13315), Y. pseudotuberculosis (ATCC 911),
and S. typhymirium (ATCC 14028), and 750 µg mL−1 against
the others. Among the others, compound 4d showed the second
best activity, with an MIC value of 46.88 against E. coli (ATCC
25922). It also has a good activity against another gram-
negative bacterium, E. cloaceae (ATCC 13047), with MIC
value of 187.5 µg mL−1. The MIC of this compound against
other bacteria is 375 µg mL−1 with the exception of S. aureus
(ATCC 25923) for which the MIC was 750 µg mL−1. Com-
pound 5d has shown good activity against S. typhymirium
(ATCC 14028) with an MIC of 93.75 µg mL−1, while the MIC
value for all other bacteria was 375 µg mL−1, except for
P. aeruginosa (ATCC 27853) and S. aureus (ATCC 25923), for
which the MIC was 750 µg mL−1. Compound 4b has shown an
MIC values of 375 µg mL−1 against P. vulgaris (ATCC 13315),
750 µg mL−1 against E. cloaceae (ATCC 13047), E. coli (ATCC
25922), K. pneumonie (ATCC 13883), Y. pseudotuberculosis
(ATCC 911), and S. aureus (ATCC 25923), and 1500 µg mL−1
against P. aeruginosa (ATCC 27853), B. subtilis (ATCC
66333), and S. typhymirium (ATCC 14028). Compound 5b has
MIC values of 187.5 µg mL−1 against B. subtilis (ATCC 66333)
and S. typhymirium (ATCC 14028), 750 µg mL−1 against
E. cloaceae (ATCC 13047), E. coli (ATCC 25922), K. pneumonie
(ATCC 13883), P. vulgaris (ATCC 13315), and Y. pseudotu-
berculosis (ATCC 911), and 1500 µg mL−1 against P. aerugi-
nosa (ATCC 27853) and S. aureus (ATCC 25923).
The purpose of present study was to synthesise N-(3,5-
dichlorophenyl)-1,2,4-triazol-3-one derivatives including
1,3,4-oxadiazol-2-thiol moiety under microwave irradiation.
Results and discussion
Our research has revealed that electron–withdrawing groups
on aromatic amines have an important negative effect on the
reaction of ethoxycarbonylhydrazones and aromatic amines.11
Because it is hard to overcome the electron–withdrawing
effect of 3,5-dichloroaniline, there have been no reports of any
Fig. 1 Structures of fluconazole and letrozole.
* Correspondent. E-mail: bahittin@yahoo.com.tr

JOURNAL OF CHEMICAL RESEARCH 2012 485
Scheme 1 Triazole synthesis with microwave irradiation.
Scheme 2 Synthetic route of compounds 2a–d, 3a–d, 4a–d and 5a–d.
Table 1 Comparison of yields and times for compounds 2a–d
93.75, and 93.75 µg mL−1, respectively. Compounds 2a, 2c, 3a,
3c, 4a, 4c, 5a and 5c had moderate activity against all bacteria
tested with an MIC value of 500 µg mL−1 when compared to
the all compounds. The antibacterial activity of compounds
against two Gram positive bacteria, S. aureus (ATCC 25923)
and B. subtilis (ATCC 66333), are shown in Table 2. The best
activity against the gram positive bacterium, B. subtilis (ATCC
66333), is 187.50 µg mL−1. The other compounds showed
moderate activity against the other gram positive bacteria.
Compounds 2b and 3b did not show significant antibacterial
activities.
Compd
MWI
Conventional heating
(180–190 °C)
(185 °C)
Time/min
Yield/%
Time/h
Yield/%
2a
2b
2c
2d
10
10
10
10
70
65
70
50
8
8
8
8
6
7
7
5
Conclusions
Experimental
A rapid, simple and efficient method for the synthesis of 1,2,4-
triazol-3-one and its oxadiazole derivatives has been devel-
oped. Compounds 2a–d could not be synthesised efficiently by
conventional heating. The reaction of ethoxycarbonylhydra-
zones and 3,5-dichloroaniline was achieved by microwave
heating and further reactions of compounds 2a–d have been
achieved. The antibacterial activity of the triazole derivatives
varied for the bacterial strains tested. Of the 16 compounds,
compound 4d showed the best antibacterial activity against
Gram negative bacteria. Compounds 2d, 4b, and 5d showed
better activity against Escherichia coli (ATCC 25922) and
Salmonella typhimurium (ATCC 14028), with MICs of 46.88,
All the chemicals were supplied by Merck, Aldrich and Fluka.
Ethoxycarbonyl hydrazones (1a–d) were prepared according to the
Pinner method.12–14 Melting points were determined in capillary tubes
on a Büchi oil-heated melting point apparatus and uncorrected.
1H NMR and 13C NMR spectra were performed on a Varian-Mercury
200 MHz spectrometer in DMSO-d6 using TMS as internal. Elemental
analyses were performed on a Carla Erba 1106 CHN analyser. Mass
spectra were recorded on Thermo Scientific Quantum Access max
LC-MS spectrophotometer. A mono-mode CEM-Discover microwave
was used to carry out microwave reactions in 30 mL microwave
process vials with temperature control by IR detection temperature
sensor. All reactions were monitored by TLC using precoated
aluminum sheets (silica gel 60 F 2.54 0.2 mm thickness).

486 JOURNAL OF CHEMICAL RESEARCH 2012
Table 2 In vitro antibacterial activity data of compounds
Compd
Stock solution
/mg mL−1
Microorganisims and minimal inhibition concentration value
Gram-negative bacteria Gram-positive bacteria
Eclo
Ec
Kp
Pae
Pv
St
Yp
Bs
Sau
2a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
500
1500
500
375
500
1500
500
375
500
750
500
187.5
500
750
500
375
NE
500
1500
500
750
500
1500
500
750
500
750
500
500
1500
500
750
500
1500
500
750
500
750
500
375
500
750
500
375
NE
500
1500
500
750
500
1500
500
750
500
1500
500
375
500
1500
500
750
NE
500
1500
500
375
500
1500
500
375
500
375
500
375
500
750
500
375
NE
500
1500
500
500
1500
500
750
500
1500
500
375
500
750
500
375
500
750
500
375
NE
500
1500
500
500
1500
500
375
500
1500
500
750
500
750
500
750
500
1500
500
750
NE
2b
2c
2d
93.75
375
3a
500
1500
500
375
500
1500
500
375
500
500
1500
500
750
500
1500
500
375
500
187.5
500
375
NE
–
3b
3c
3d
4a
4b
4c
4d
46.88
5a
500
750
500
375
NE
–
5b
187.5
5c
500
93, 75
NE
5d
DMSO
PC + amp.
100
–
–
–
–
–
–
–
µg mL−1
PC
+
+
+
+
+
+
+
+
+
Eclo, Enterobacter cloaceae ATCC 13047; Ec, Escherichia coli ATCC 25922; Kp, Klebsiella pneumonie ATCC 13883; Pae, Pseudomo-
nas aeruginosa ATCC 27853; Pv, Proteus vulgaris ATCC 13315; St, Salmonella typhimurium ATCC 14028; Yp, Yersinia pseudotuber-
culosis ATCC 911; Bs, Bacillus subtilis ATCC 66333; Sau, Staphylococcus aereus ATCC 25923. PC, positive control as broth medium
without chemical component; amp, ampicilline; NE, not effective for growing.
Synthesis of compound 2a–d
56.27; H, 3.46; N, 13.12. Found: C, 56.31; H, 3.45; N, 13.10%,
LC-MS: 347/345 [M+Na], 322/320 [M+1].
Conventional method: A mixture of the corresponding ethoxycar-
bonylhydrazone (1a–d) (0.01 mol) and 3,5-dichloroaniline (0.01 mol)
was heated in an oil bath for 6 h at 180 °C. After cooling it to room
temperature, an oily solid was obtained. This crude product was
extracted into ethyl acetate (4 x 10 mL) and the mixture was cooled to
room temperature. A white solid product appeared. This crude product
was filtered and recrystalllised from ethylacetate.
Microwave method: 3,5-Dichloroaniline (0.01 mol) and ethoxycar-
bonylhydrazone (0.01 mol) in a microwave process vial was sealed
into the microwave reactor and allowed to stir for 1 minute. Then,
the suspension was heated under microwave irradiation at 100 W at
185 ºC, with stirring and air-jet cooling. Completion of reaction was
achieved in 10 min. as indicated by TLC. The entire product was
cooled and extracted into ethyl acetate (4 x 10 mL). The mixture
was cooled to room temperature. A white solid appeared. This crude
product was filtered and recrystallised from ethylacetate.
Synthesis of compounds 3a–d
Conventional method: Dry K2CO3 (0.025 mol) was added to a solu-
tion of compound 2a–d (0.01 mol) in acetone (30 mL), and stirred for
30 min. Then, ethyl bromoacetate (0.012 mol) was added and stirred
for 1 night. After the reaction was complete (monitored by TLC, ethyl
acetate: hexane, 3:1), the product was precipitated by the addition of
water. It was filtered, dried and recrystallised by ethanol to obtain the
desired product.
Microwave method: Dry K2CO3 (0.025 mol) was added to a solu-
tion of compound 2a–d (0.01 mol) in acetone (10 mL) was taken in
a closed vessel. The mixture was irradiated in a microwave oven at
90 ºC, 5 min (hold time) with pressure control. Then, the mixture was
cooled to room temperature and ethyl bromoacetate (0.012 mol) was
added. Again, it was irradiated in microwave at 90 ºC for10 min (hold
time) at 300 W maximum power. After the reaction was complete
(monitored as stated above), the mixture was cooled, transferred to a
beaker and the product was precipitated by the addition of water. The
product was purified as described above.
Ethyl [4-(3,5-dichlorophenyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetate (3a): Yield: 85% (for conventional method),
95% (for microwave method), m.p. 154–155 °C, 1H NMR (200 MHz,
CDCl3) δ 7.51 (s, 1H), 7.28 (s, 2H), 4.58 (s, 2H), 4.22 (q, 2H, J =
7.0 Hz), 1.31 (t, 3H, J = 7.0); 13C NMR (50 MHZ, DMSO-d6) δ 169.3,
152.1, 146.7, 135.0, 134.6, 128, 7, 126.3, 60.8, 44.3, 20.5;Anal. Calcd
for C13H13Cl2N3O3: C, 47.29; H, 3.97; N, 12.73. Found: C, 47.32; H,
3.99; N, 12.71%, LC-MS: 355/353 [M+Na], 332/330 [M+1].
Ethyl [4-(3,5-dichlorophenyl)-3-ethyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetate (3b): Yield: 88% (for conventional method),
97% (for microwave method), m.p. 121–122 °C, 1H NMR (200 MHz,
CDCl3) δ 7.47 (s, 1H), 7.27 (s, 2H), 4.59 (s, 2H), 4.24 (q, 2H, J =
7.0 Hz), 2.53 (q, 2H, J = 7.4 Hz), 1.31 (t, 3H, J = 7.4 Hz), 1.24 (t, 3H,
J = 7.0 Hz); 13C NMR (50 MHZ, DMSO-d6) δ 168.5, 153.3, 147.5,
135.6, 135.3, 129, 5, 126.9, 61.9, 47.1, 19.8, 14.72, 9.85; Anal. Calcd
for C14H15Cl2N3O3: C, 48.85; H, 4.39; N, 12.21. Found: C, 48.83; H,
4.42; N, 12.23%, LC-MS: 369/367 [M+Na], 346/344 [M+1].
Ethyl [4-(3,5-dichlorophenyl)-3-phenyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetate (3c): Yield: 82% (for conventional method),
94% (for microwave method), m.p. 102–103 °C, 1H NMR (200 MHz,
CDCl3) δ 7.40 (s, 1H), 7.35 (s, 2H), 7.26–7.19 (m, 5H) 4.69 (s, 2H),
4.29 (q, 2H, J = 7.8 Hz), 1.32 (t, 3H, J = 7.8 Hz); 13C NMR (50 MHz,
4-(3,5-Dichlorophenyl)-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one
1
(2a): Cas no: 1269125-43-0, m.p. 192–193 °C, H NMR (200 MHz,
DMSO-d6) δ 11.73 (s, 1H), 7.73 (s, 1H), 7.62 (s, 2H), 2.09 (s, 3H); 13
C
NMR (50 MHZ, DMSO-d6) δ 152.6, 145, 7, 134.6, 134.2, 128, 1, 125,
3, 13.5; Anal. Calcd for C9H7Cl2N3O: C, 44.29; H, 2.89; N, 17.22.
Found: C, 44.33; H, 2.93; N, 17.20%, LC-MS: 268/266 [M+Na],
246/244 [M+1].
4-(3,5-Dichlorophenyl)-5-ethyl-2,4-dihydro-3H-1,2,4-triazol-3-one
1
(2b): M.p. 203–204 °C, H NMR (200 MHz, DMSO-d6) δ 11.75 (s,
1H), 7.73 (s, 1H), 7.62 (s, 2H), 2.45 (q, 2H, J = 7), 1.03 (t, 3H, J = 7)
;
13C NMR (50 MHz, DMSO-d6) δ 154.9, 149.7, 136.8, 134.9, 129.7,
127.2, 32.01, 15.5; Anal. Calcd for C10H9Cl2N3O: C, 46.53; H, 3.51;
N, 16.28. Found: C, 46.52; H, 3.54; N, 16.29%, LC-MS: 282/280
[M+Na], 260/258 [M+1].
4-(3,5-Dichlorophenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one
1
(2c): M.p. 234–235 °C, H NMR (200 MHz, DMSO-d6) δ 12.28 (s,
1H), 7.71 (s, 1H), 7.44 (s, 2H), 7.42–7.32 (m, 5H); 13C NMR(50 MHZ,
DMSO-d6) δ 155.1, 148.1, 137.5, 136.8, 129.7, 129.5 127.8, 127.0,
124.1, 122.5; Anal. Calcd for C14H9Cl2N3O: C, 54.92; H, 2.96; N,
13.73. Found: C, 54.90; H, 2.98; N, 13.75%, LC-MS: 330/328
[M+Na], 308/306 [M+1].
4-(3,5-Dichlorophenyl)-5-benzyl-2,4-dihydro-3H-1,2,4-triazol-3-one
1
(2d): M.p. 151–152 °C, H NMR (200 MHz, DMSO-d6) δ 10.43
(s, 1H), 7.40 (s, 1H), 7.30 (s, 2H), 7.26–7.00 (m, 5H), 3.82 (s, 2H); 13
C
NMR (50 MHZ, DMSO-d6) δ 155.2, 150.0, 136.3, 135.8, 129.7, 125.5
123.8, 122.8, 121.9, 120.2, 31.3; Anal. Calcd for C15H11Cl2N3O: C,

JOURNAL OF CHEMICAL RESEARCH 2012 487
DMSO-d6) δ 166.9, 151.5, 147.8, 135.1, 134.8 129.3, 128.2, 126.5,
125.6, 123.1, 60.2,47.5, 14.9; Anal. Calcd for C18H15Cl2N3O3:
C, 55.12; H, 3.85; N, 10.71. Found: C, 55.13; H, 3.88; N, 10.70%,
LC-MS: 417/415 [M+Na], 394/392 [M+1].
Ethyl [4-(3,5-dichlorophenyl)-3-benzyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetate (3d): Yield: 78% (for conventional method),
91% (for microwave method), m.p. 129–130 °C, 1H NMR (200 MHz,
CDCl3) δ 7.38 (s, 1H), 7.26 (s, 2H), 7.1–6.9 (m, 5H) 4.62 (s, 2H), 4.29
(q, 2H, J = 7.8 Hz), 3.83 (s, 2H), 1.32 (t, 3H, J = 7.8 Hz); 13C NMR
(50 MHZ, DMSO-d6) δ 166.1, 150.3, 147.7, 135.3, 134.6 129.9,
128.5, 127.9, 125.2, 123.9, 63.2,47.5, 30.3 14.9; Anal. Calcd for
C19H17Cl2N3O3: C, 56.17; H, 4.22; N, 10.34. Found: C, 56.15; H, 4.24;
N, 10.36%, LC-MS: 431/429 [M+Na], 408/406 [M+1].
mixture and it was then heated again 300 W at 100 ºC. Completion
of reaction was achieved in 10 min. as indicated by TLC. Then, the
mixture was neutralised with 4 N HCl and left to cool. The precipi-
tated product was filtered, washed with H2O and recrystallised from
Ethanol.
4-(3,5-Dichlorophenyl)-2-[5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-
5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (5a): Yield: 63% (for
conventional method), 77% (for microwave method), m.p. 210–
211 °C, 1H NMR (200 MHz, DMSO-d6) δ 14.42 (s, 1H), 7.78 (s, 1H),
7.68 (s, 2H), 5.08 (s, 2H), 2.49 (s, 3H); 13C NMR (50 MHZ, DMSO-
d6) δ 178.8, 159.5, 152.6, 144.6, 135.2, 129.4, 127.5, 126.9, 40.3,
12.8; Anal. Calcd for C12H9Cl2N5O2S: C, 40.24; H, 2.53; N, 19.55.
Found: C, 40.22; H, 2.55; N, 19.57%, LC-MS: 383/381 [M+Na],
360/358 [M+1].
Synthesis of compounds 4a–d
4-(3,5-Dichlorophenyl)-2-[5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-
5-ethyl-2,4-dihydro-3H-1,2,4-triazol-3-one (5b): Yield: 69% (for
conventional method), 82% (for microwave method), m.p. 160–
161 °C, 1H NMR (200 MHz, DMSO-d6) δ 14.62 (s, 1H), 7.79 (s, 1H),
7.70 (s, 2H), 5.11 (s, 2H), 2.46 (q, 2H, J = 7.0 Hz), 1.03 (t, 3H,
J = 7.0 Hz); 13C NMR (50 MHZ, DMSO-d6) δ 178.7, 159.6, 152.9,
144.4, 135.5, 135.3, 129.6, 127.2,40.4, 19.8, 9.8; Anal. Calcd for
C13H11Cl2N5O2S: C, 41.95; H, 2.98; N, 18.81. Found: C, 41.96; H,
2.99; N, 18.83%, LC-MS: 397/395 [M+Na], 374/372 [M+1].
4-(3,5-Dichlorophenyl)-2-[5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-
5-phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one (5c): Yield: 65% (for
conventional method), 80% (for microwave method), m.p.181–
183 °C, 1H NMR (200 MHz, DMSO-d6) δ 14.75 (s, 1H), 7.76 (s, 1H),
7.53 (s, 2H), 7.45–7.38 (m, 5H), 5.23 (s, 2H); 13C NMR (50 MHZ,
DMSO-d6) δ 178.6, 159.6, 153.9, 144.7, 136.6, 135.8, 135.1, 129.9,
127.2, 125.6, 123.2,40.2; Anal. Calcd for C17H11Cl2N5O2S: C, 48.58;
H, 2.64; N, 16.66. Found: C, 48.61; H, 2.63; N, 16.68%, LC-MS:
445/443 [M+Na], 422/420 [M+1].
4-(3,5-Dichlorophenyl)-2-[5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-
5-benzyl-2,4-dihydro-3H-1,2,4-triazol-3-one (5d): Yield: 62% (for
conventional method), 75% (for microwave method), m.p.121–
122 °C, 1H NMR (200 MHz, DMSO-d6) δ 13.17 (s, 1H), 7.71 (s, 1H),
7.40 (s, 2H), 7.20–7.03 (m, 5H), 4.63 (s, 2H), 4.12 (2, 2H); 13C NMR
(50 MHZ, DMSO-d6) δ 169.8, 153.5, 153.3, 145.7, 137.3, 135.5,
135.4, 135.1, 129.4, 129.1, 127.6, 127.0, 47.2, 32.4; Anal. Calcd for
C18H13Cl2N5O2S: C, 49.78; H, 3.02; N, 16.13. Found: C, 49.76; H,
3.04; N, 16.14%, LC-MS: 445/443 [M+Na], 422/420 [M+1].
Conventional method: hydrazine monohydrate (0.025 mol) was
added to a solution of compound 3a–d (0.01 mol) in dry ethanol
(25 mL), and refluxed for 6 hours (monitored by TLC, ethyl acetate:
hexane: 3:1). After cooling the mixture to room temperature, a white
solid appeared. This crude product was filtered, dried and recrystal-
lised from ethanol to yield the pure product.
Microwave method: Solutions of compound 3a–d (0.01 mol) in dry
ethanol (10 mL) and hydrazine monohydrate (0.025 mol) were placed
in a closed vessel. The mixture was irradiated in a microwave oven at
130 ºC for 10 min (hold time) at 300W maximum power. After the
reaction was complete (monitored as stated above), the mixture was
cooled to room temperature, transferred to a beaker and a white solid
appeared. This crude product was filtered and purified as before.
2-[4-(3,5-Dichlorophenyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl] acetohydrazide (4a): Yield: 72% (for conventional
1
method), 83% (for microwave method), m.p.204–205 °C, H NMR
(200 MHz, DMSO-d6) δ 9.35 (s, 1H), 7.52 (s, 1H), 7.27 (s, 2H), 4.30
(s, 4H), 2.22 (s, 3H); 13C NMR (50 MHZ, DMSO-d6) δ 168.4, 153.9,
147.8, 136.1, 135.9 129.3, 126.9, 47.2, 20.2 ; Anal. Calcd for
C11H11Cl2N5O: C, 41.79; H, 3.51; N, 22.15. Found: C, 41.80; H, 3.53;
N, 22.16%, LC-MS: 321/319 [M+Na], 318/316 [M+1].
2-[4-(3,5-Dichlorophenyl)-3-ethyl-5-oxo-4,5-dihydro-1H-1,2,4-tri-
azol-1-yl] acetohydrazide (4b): Yield: 69% (for conventional method),
86% (for microwave method), m.p. 183–184 °C, 1H NMR (200 MHz,
DMSO-d6) δ 9.45 (s, 1H), 7.81 (s, 1H), 7.63 (s, 2H), 4.30 (s, 4H), 2.49
(q, 2H, J = 7.4 Hz), 1.03 (t, 3H, J = 7.4 Hz); 13C NMR (50 MHZ,
DMSO-d6) δ 168.5, 153.3, 147.3, 135.9, 135.2, 129.3, 127.0, 47.2,
19.8 ; Anal. Calcd for C12H13Cl2N5O: C, 43.65; H, 3.97; N, 21.21.
Found: C, 43.70; H, 3.96; N, 21.24%, LC-MS: 355/353 [M+Na],
332/330 [M+1].
2-[4-(3,5-Dichlorophenyl)-3-phenyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetohydrazide(4c):Yield:76%(forconventionalmethod),
89% (for microwave method), m.p. 166–167 °C, 1H NMR (200 MHz,
DMSO-d6) 10.15 (s, 1H), 7.95 (s, 1H), 7.71 (s, 2H), 7.42–7.17 (m,
5H), 4.63 (s, 4H); 13C NMR (50 MHZ, DMSO-d6) δ 166.5, 156.3,
147.1, 134.9, 130.2, 129.1, 127.3, 127.0, 124.5, 123.8, 47.2; Anal.
Calcd for C16H13Cl2N5O: C, 50.81; H, 3.46; N, 18.52. Found: C, 50.83;
H, 3.50; N, 18.55%, LC-MS: 403/401 [M+Na], 380/378 [M+1].
2-[4-(3,5-Dichlorophenyl)-3-benzyl-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl] acetohydrazide (4d): Yield: 65% (for conventional method),
82% (for microwave method), m.p. 141–142 °C, 1H NMR (200 MHz,
DMSO-d6) δ 9.23 (s, 1H), 7.7 (s, 1H), 7.37 (s, 2H), 7.19–7.04 (m, 5H),
4.32 (s, 4H), 3.91 (s, 2H); 13C NMR (50 MHZ, DMSO-d6) δ 163.2,
153.9, 145.5, 136.9, 136.1, 134.2, 129.3, 128.4, 127.1, 124.8, 45.2,
30.8; Anal. Calcd for C17H15Cl2N5O: C, 52.06; H, 3.85; N, 17.85.
Found: C, 51.10; H, 3.88; N, 17.83%, LC-MS: 417/415 [M+Na],
394/392 [M+1].
Anti-microbial activity
The qualitative screening of the susceptibility of different microbial
strains to the compounds was performed by the quantitative assay of
minimal inhibitory concentration (MIC, µg mL−1) based on liquid
medium serial microdilutions.13,14 The MIC assays were performed in
LB medium at pH 7.2. The stock solutions of the compounds were
prepared in dimethyl sulfoxide (DMSO). The dilution series of the
chemical compounds to be tested were prepared from 1500 to 0.24 µg
mL−1 concentrations in 100 µL medium. The plates were incubated at
37.0 1 °C for 18–24 h. Dimethylsulphoxide, LB medium with or
without antibiotic, ampicillin, were used as solvent control, positive,
and negative controls, respectively. The MIC was taken to be the last
well in the dilution series that did not exhibit growth as determined on
the basis of turbidity.
The determination of minimum inhibitory concentration14 was done
with two of Gram-positive bacterial strains, namely Bacillus subtilis
(ATCC 66333) and Staphylococcus aureus (ATCC 25923), and seven
Gram-negative bacterial strains which were Enterobacter cloaceae
(ATCC 13047), Escherichia coli (ATCC 25922), Klebsiella pneumo-
nie (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Proteus
vulgaris (ATCC 13315), Salmonella typhymirium (ATCC 14028), and
Yersinia pseudotuberculosis (ATCC 911). These were inoculated into
a Luria broth medium containing 1% tryptone, 0.5% yeast extract,
0.5% sodium chloride. The pH of the medium was adjusted to 7.2 and
incubated at 37 ºC for 18–24 h. The optical density of the bacteria
from mid-log phase of growth was measured at 600 nm and diluted in
fresh medium so as to get an optical density of 0.004 (corresponding
to 5 × 105 colony forming units mL−1). The results of these compounds
are shown in Table 2.
Synthesis of compounds 5a–d
Conventional method: Solutions of KOH (0.01mol) in water
(20 mL) and CS2 (0.01 mol) were added to a solution of 4a–d
(0.01 mol) in ethanol (20 mL) and then, the mixture was refluxed for
8 hours. After the reaction was complete, (monitored by TLC ethyl
acetate: hexane, 3:1), the mixture was cooled to room temperature
and neutralised with diluted HCl (4N). The mixture was left to cool
and the precipitated product was filtered, washed with H2O and
recrystallised from ethanol.
Microwave method: Solutions of 4a–d (0.01 mol) in ethanol
(10 mL) and KOH (0.01 mol) in water (5 mL) were placed in a micro-
wave process vial. Then, the mixture was heated under microwave
irradiation at 300 W at 100 ºC, with stirring and air-jet cooling for
5 min. After the mixture was cooled, CS2 (0.01 mol) was added to the
Received 12 March 2012; accepted 23 May 2012
Paper 1201208 doi: 10.3184/174751912X13400138806685
Published online: 8 August 2012

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