Synthesis and antimicrobial activities of 2-(5-mercapto)-1,3-oxadiazol-2- ylmethyl-1,2,4-triazol-3-one derivatives

Article abstract of DOI:10.3906/kim-0911-10

The synthesis of 4-amino-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-5-(4- methylphenyl)-2,4-dihydro-3H - 1,2,4-triazol-3-one (2) and 4-amino-2-[(4-amino- 5-mercapto-4H-1,2,4-triazol-3-yl)methyl]-5-(4-methylphenyl)-2,4-dihydro-3H-1,2, 4-triazol-3-one (5)

Full text of DOI:10.3906/kim-0911-10

Turk J Chem
34 (2010) , 347 – 358.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-0911-10
Synthesis and antimicrobial activities of 2-(5-mercapto)-
1,3-oxadiazol-2-ylmethyl-1,2,4-triazol-3-one
derivatives
1,∗
1
1
˙
˙
1
˙
3
Ahmet DEMIRBAS¸ , Deniz S¸AHIN , Neslihan DEMIRBAS¸
˘
S¸engu¨l Alpay KARAOGLU , Hakan BEKTAS¸
1Karadeniz Technical University, Department of Chemistry 61080 Trabzon-TURKEY
e-mail: ndemirbas651@mynet.com
2Rize University, Department of Biology, 53100 Rize-TURKEY
3Giresun University, Department of Chemistry, 28100 Giresun-TURKEY
Received 04.11.2009
The synthesis of 4-amino-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-5-(4-methylphenyl)-2,4-dihydro-3H -
1,2,4-triazol-3-one (2) and 4-amino-2-[(4-amino-5-mercapto-4H -1,2,4-triazol-3-yl)methyl]-5-(4-methylphenyl)-
2,4-dihydro-3H -1,2,4-triazol-3-one (5) was performed starting from 2-[4-amino-3-(4-methylphenyl)-5-oxo-
4,5-dihydro-1H -1,2,4-triazol-1-yl]acetohydrazide (1). The treatment of 2 and 5 with 4-fluorobenzaldehyde
(for 8) or salicylaldehyde (for 6) afforded the corresponding Schiff bases (6 and 8). The condensa-
tion of 5 with phenacyl bromide produced 4-amino-5-(4-methylphenyl)-2-[(6-phenyl-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazin-3-yl)methyl]-2,4-dihydro-3H -1,2,4-triazol-3-one (7). The alkylation of 2 and 8 was car-
ried out by using methyl iodide in basic media.
Compounds 3 and 10a,b were prepared by aminoalkylation of 2 and 8 with 4-fluorophenyl piperazine,
morpholine, or 4-methyl piperazine in the presence of formaldehyde.
The synthesized compounds were screened for their antimicrobial activities and, with the exception of 2,
4, 7, and 9, were found to possess good or moderate activities against the screened bacterial strains except
Candida tropicalis and Candida albicans.
Key Words: 1,2,4-Triazole, 1,3,4-oxadiazole, Mannich base, antimicrobial activity.
Introduction
The treatment of infectious diseases remains an important and challenging problem due to a combination
of factors including emerging infectious diseases and the increasing number of multidrug resistant microbial
∗Corresponding author
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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
pathogens. In spite of the wide range of antimicrobial drugs with different mechanisms of action used to
treat microbial infections, either alone or in combination, and the existence many compounds used in different
phases of clinic trials, microbial infections are becoming a worldwide problem. There is already evidence that
antimicrobial resistance is associated with an increase in mortality. The problem with clinically used drugs
is not only the increasing microbial resistance, but also that they are accompanied by toxic side effects that
are often dose limiting.1−5 Due to these reasons, it is important to search for and synthesize new classes of
compounds that preferably consist of chemical characteristics that clearly differ from those of existing agents.
Many compounds consisting of 5-membered heterocyclic rings represent important building blocks in
organic and medicinal chemistry. In addition, they are interesting in their own right, due to their pharma-
cological properties. Therefore, the synthesis of new derivatives of triazoles, oxadiazoles, and thiadiazoles has
been attracting considerable attention for the past few decades.6−19 Among the heterocycles, the 1,2,4-triazole
nucleus has been incorporated into a wide variety of therapeutically important agents. Ribavirin, Rizatriptan,
and Alprazolam are the best examples of drugs containing 1,2,4-triazole moiety (Chart 1).11−13Some of the
other drugs being used for the treatment of fungal disease and including a 1,2,4-triazole ring in their structures
are Ravuconazole, Fluconazole, Posaconazole, Voriconazole, and Itraconazole (Chart 1).14−17 Furthermore,
Mannich bases of 1,2,4-triazole derivatives have been reported to possess protozocidal and antibacterial activ-
ity. Drugs such as Prazosin, Lidoflazin, and Urapidil carrying a piperazine nucleus have been used as good
cardiovascular agents (Chart 2).17 Furthermore, compounds containing a 1,3,4-oxadiazole ring have also been
reported to possess a broad spectrum of biological activities including insecticidal, antibacterial, anticancer, and
anti-inflammatory activities.18−20
In addition, several compounds that contain a piperazine or morpholine nucleus with antimicrobial
activity have been synthesized; some of which contain an azole ring as well. For instance, while Eperezolide,
which is a member of the oxazolidinone class of antibiotics, consists of morpholine and oxazolidinone rings linked
each other via a fluorophenylene linkage, another antibiotic, Linezolide, contains a piperazine ring instead of
morpholine.21 Some Mannich bases derived from 1,2,4-triazole derivatives carrying a methyl piperazine or
morpholine ring have been described as possessing protozocidal, antimicrobial, or anticancer activities.17−22
As part of our continuing study on the synthesis of antitumoral and antimicrobial compounds, we re-
ported a series of Schiff base derivatives containing 1,2,4-triazol-5-one ring as antitumor agents.10,23−27 In the
library screening of our new compounds as antimicrobial assay system, several biheterocyclic compounds con-
sisting of 1,2,4-triazol-5-one and 1,3,4-thiadiazole rings provided inhibitory activity on tested microorganisms.10
Moreover, we have reported some compounds consisting of a triazolothiadiazine or triazolothiadiazole ring as
antimicrobial agents.28 According to these observations, in the design of new bioactive compounds, the devel-
opment of hybrid molecules through the combination of different pharmacophores in one frame may lead to
compounds with interesting biological profiles.
In the present study, prompted by these observations, the synthesis and antimicrobial screening of new
1,2,4-triazole derivatives incorporating different pharmacophores as hybrid molecules possessing antimicrobial
activity were aimed.
348

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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
N
NH2
CH3
N
N
N
H
N
O
N
N
N
O
HOCH2
N
Cl
Alprazolam
N
N
CH3
N
OH
OH
CH3
Rizatriptan
OH
Ribavirin
F
N
OH
N
N
N
N
N
N
CN
N
N
N
N
F
F
N
S
F
N
N
N
OH
F
F
F
Fluconazole
Voriconazole
Ravuconazole
CH3
N
N
O
N
N
N
N
N
O
OH
N
F
O
Posaconazole
F
CH3
N
N
O
O
N
O
N
N
N
N
N
Cl
O
Itraconazole
Cl
Chart 1.
Experimental
Melting points were determined on a Bu¨chi B-540 melting point apparatus and are uncorrected. 1 H-NMR and
13 C-NMR spectra were recorded on a Varian-Mercury 200 MHz spectrometer. The IR spectra were measured
on potassium bromide pellets using a Perkin-Elmer 1600 series FTIR spectrometer. Combustion analysis was
performed on a Costech Elemental Combustion System CHNS-O elemental analyzer. All the chemicals were
349

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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
O
O
F
F
HO
O
O
N
O
N
N
N
N
O
NHAC
NHAC
Linezolid
Eperezolid
F
O
.HCl
CH3O
CH3O
N
N
N C
H
N
CH3
O
N
N
O
N
NH2
CH3
Prazosin
Lidoflazin
F
O
N
CH3
N
CH3
N
N
NH
O
CH3
Urapidil
Chart 2.
obtained from Fluka Chemie AG Buchs (Switzerland). Compound 1 was obtained in the way reported earlier.10
General Method for the Synthesis of 4-Amino-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-
5-(4-methyl phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2). 2-[4-amino-3-(4-methylphenyl)-5-oxo-4,5-
dihydro-1H -1,2,4-triazol-1-yl]acetohydrazide (1) (0.01 mol) and CS2 (0.60 mL, 0.01 mol) were added to a
solution of KOH (0.56 g, 0.01 mol) in 50 mL of H2 O and 50 mL of ethanol. The reaction mixture was
refluxed for 3 h. After evaporating the solvents under reduced pressure to dryness, a solid was obtained. It
was dissolved in 300 mL of H2 O and acidified with conc. HCl. The precipitate was filtered off, washed with
H2 O, and recrystallized from ethanol to afford the desired compound. Yield 72%, mp 225-227 ◦ C; IR (KBr,
υ, cm−1): 3323 and 3213 (NH2), 2750 (SH), 1676 (C=O), 1519, 1505 and 1601 (C=N); Anal. Calcd (%) for
C
12 H12 N6 O2 S: C, 47.36; H, 3.97; N, 27.62. Found; C, 47.56; H, 4.02; N, 27.43; 1 H-NMR (DMSO-d6δ ppm):
2.35 (3H, s, CH3), 5.12 (2H, s, CH2), 5.56 (2H, bs, NH2), 7.30 (2H, d, j =8.0 Hz, arH ), 7.88 (2H, d, j =8.0
Hz, arH), 14.65 (H, bs, SH); 13 C-NMR (DMSO-d6δ ppm): 20.81 (CH3), 40.58-38.48 (CH2 + DMSO d6), arC:
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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
[123.23 (C), 127.45 (2CH), 128.78 (2CH), 139.88 (C)], 145.73 (C-3, triazole), 153.06 (C-5, triazole), 158.85 (C-2,
oxadiazole), 177.86 (C-5, oxadiazole).
General method for the synthesis of compounds 6 and 8
Salicyl aldehyde (for 6) or 4-fluorobenzaldehyde (for 8) (10 mmol) was added to the solution of the corresponding
compound 2 or 5 (10 mmol) in absolute ethanol, and the mixture was heated until a clear solution was obtained.
Then a few drops of concentrated sulfuric acid were added as a catalyst and the solution was allowed to reflux
for 3-4 h. On cooling of the reaction mixture to room temperature, a solid appeared. The product was filtered
off and recrystallized from dimethylsulfoxide-water (1:1) to obtain the desired compound.
4-{[(2-Hydroxyphenyl)methylene]amino}-2-[(4-{[(2-hydroxyphenyl)methylene] amino}-5-
mercapto-4H-1,2,4-triazol-3-yl)methyl]-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (6).
Yield: 94%; mp 238-239 ◦ C; IR (KBr , υ, cm−1): 3414 (2OH), 2712 (SH), 1713 (C=O), 1620, 1603 and 1582
(3C=N), Anal. Calcd (%) for C26 H22 N8 O3 S: C, 59.30; H, 4.21, N, 21.28. Found: C, 59.46; H, 4.28; N, 21.06;
1 H-NMR (DMSO-d6δ ppm): 2.36 (3H, s, CH3), 4.41 (2H, s, NCH2), 6.72-6.99 (4H, m, arH), 7.29-7.42 (3H,
m, arH), 7.63-7.68 (3H, m, arH), 7.87 (2H, m, arH), 9.84 (1H, s, N=CH), 10.08 (1H, s, N=CH), 10.38 (2H, s,
2OH), 14.07 (1H, s, SH).
4-{[(4-Fluorophenyl)methylene]amino}-2-[(5-mercapto-1,3,4-oxadiazol-2-yl)methyl]-5-(4-
methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (8). Yield: 87%; mp 224-225 ◦ C; IR (KBr, υ, cm−1):
2723 (SH), 1696 (C=O), 1613, 1601 and 1508 (3C=N); Anal. Calcd (%) for C19 H15 FN6 O2 S: C, 55.60; H,
3.68, N, 20.48. Found: C, 55.76; H, 3.73; N, 20.46; 1 H-NMR (DMSO-d6δ ppm): 2.12 (3H, s, CH3), 4.72 (2H,
s, NCH2), 7.30-7.38 (4H, m, arH), 7.58 (2H, d, j =8.0 Hz, arH), 8.49 (2H, d, j =8.0 Hz), 9.03 (1H, s, N=CH),
13.28 (1H, bs, SH); 13 C-NMR (DMSO-d6δ ppm): 20.45 (CH3), 40.53-38.66 (CH2 + DMSO-d6), arC: [114.93
and 114.24 (2CH-C-F), 158.15 and 162.47 (C-F), 124.17 (2CH), 125.27 (2CH), 133.02 (2CH), 128.98 (C), 139.84
(C), 145.12 (C)], 136.42 (N=CH), 143.65 (C-3, triazole), 155.83 (C-5, triazole), 159.43 (C-2, oxadiazole), 178.17
(C-5, oxadiazole).
General method for the synthesis of compounds 3 and 10a,b
Into a solution of the corresponding compound 2 or 8 (10 mmol) in dimethyl formamide were added 4-(4-
fluorophenylpiperazine (for 3), morpholine (for 10a) or methylpiperazine (for 10b) (10 mmol), and formaldehyde
(40%, 1.5 mL) and the mixture was stirred at room temperature for 2 h. Then distilled water was added and
the resulting solution was kept overnight in the cold. The separated solid was collected by filtration and
recrystallized from ethanol to yield the target compounds.
4-Amino-5-(4-methyphenyl)-2-[(4-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-5-thioxo-4,5-
dihydro-1,3,4-oxadiazol-2-yl}methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (3). Yield: 64%; mp 142-
144 ◦ C (from butyl acetate–petroleum ether 1:1); IR (KBr, υ, cm−1): 3413 and 3230 (NH2), 1710 (C=O),
1509 and 1617 (C=N), 1162 (C=S); Anal. Calcd (%) for C23 H25 FN8 O2 S: C, 55.63; H, 5.07, N, 22.57. Found:
C, 55.81; H, 5.13; N, 22.44; 1 H-NMR (DMSO-d6δ ppm): 2.07 (3H, s, CH3), 2.45-2.58 (4H, m, morpholine-
2CH2), 3.71-3,86 (4H, m, morpholine-2CH2), 4.72 (2H, s, NCH2), 5.28 (2H, NCH2 NH), 5.37 (2H, s, NH2),
6.79 (2H, d, arH, j = 8.8 Hz), 6.94 (2H, d, arH, j = 8.8 Hz), 7.19 (2H, d, arH, j = 8.0 Hz), 8.64 (2H, d,
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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
arH, j =8.0 Hz); 13 C-NMR (DMSO-d6δ ppm): 21.07 (CH3), 44.63 (NCH2), 48.24 (morpholine 2CH2), 55.76
(morpholine 2CH2), 66.26 (NCH2 N), arC: [114.97 and 114.24 (2CH-C-F), 158.13 and 162.46 (C-F), 124.14
(2CH), 124.73 (2CH), 125.64 (C), 128.26 (CH), 136.79 (CH), 140.13 (C), 145.12 (C)], 144.16 (C-3, triazole),
152.61 (C-5, triazole), 160.07 (C-2, oxadiazole), 177.38 (C-5, oxadiazole).
4-{[(4-Fluorophenyl)methylene]amino}-5-(4-methylphenyl)-2-{[4-(morpholin-4-ylmethyl)-5-
thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (10a). Yield:
78%, mp 217-220 ◦ C (from ethanol); IR (KBr, υ, cm−1): 1713 (C=O), 1600 and 1508 (2C=N), 1328 (C=S);
Anal. Calcd (%) for C24 H24 FN7 O3 S: C, 56.57; H, 4.75, N, 19.24. Found: C, 56.58; H, 4.70; N, 19.22; 1 H-NMR
(DMSO-d6)δ ppm: 2.39 (3H, s, CH3), 3.21-3.58 (8H, m, morpholine-4CH2), 4.96 (2H, s, CH2), 5.22 (2H, s,
CH2), 7.26 (2H, d, arH, j =9.4 Hz), 7.75-7.91 (6H, m, arH), 9.65 (1H, s, N=CH); 13 C-NMR (DMSO-d6)δ
ppm: 20.18 (CH3), 44.23 (NCH2), 48.24 (morpholine 2CH2), 55.76 (morpholine 2CH2), 65.68 (NCH2 N), arC:
[115.04 and 115.24 (2CH-C-F), 158.10 and 162.73 (C-F), 123.18 (2CH), 125.33 (2CH), 125.62 (C), 129.37 (CH),
137.41 (CH), 139.82 (C), 145.18 (C)], 129.71 (N=CH), 145.06 (C-3, triazole), 153.15 (C-5, triazole), 163.36 (C-2,
oxadiazole), 178.13 (C-5, oxadiazole).
4-{[(4-Fluorophenyl)methylene]amino}-5-(4-methylphenyl)-2-({4-[(4-methyl piperazin-1-yl)
methyl]-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (10b).
Yield: 72%, mp 157-159 ◦ C (from ethanol); IR (KBr, υ, cm−1): 1717 (C=O), 1624, 1601 and 1585 (C=N),
1223 (C=S); Anal. Calcd (%) for C25 H27 FN8 O2 S: C, 57.46; H, 5.21, N, 21.44. Found: C, 57.61; H, 5.26; N,
21.37; 1 H-NMR (DMSO-d6δ ppm): 2.23 (3H, s, CH3), 2.41 (8H, bs, morpholine-4CH2), 2.75 (3H, s, CH3),
4.96 (2H, s, CH2), 5.23 (2H, s, CH2), 7.30-7.36 (4H, m, arH), 7.78 (2H, d, j = 8.4 Hz, arH), 7.91 (2H, t, arH, j =
5.2 Hz), 9.64 (1H, s, N=CH); 13 C-NMR (DMSO-d6δ ppm): 21.34 (CH3), 23.20 (N-CH3) 46.28 (NCH2), 51.97
(morpholine 2CH2), 54.42 (morpholine 2CH2), 67.48 (NCH2 N), arC: [158.65 and 165.24 (C-F), 115.44 and
116.28 (2CH-C-F), 129.10 (CH), 129.70 (CH), 128.58 (C), 124.48 (2CH), 125.64 (C), 136.50 (2CH), 141.37 (C)],
136.71 (N=CH), 142.12 (C-3, triazole), 157.18 (C-5, triazole), 163.96 (C-2, oxadiazole), 180.17 (C-5, oxadiazole).
General method for the synthesis of compounds 4 and 9
Into a solution of the corresponding compound 2 or 8 (10 mmol) in absolute ethanol was added an equivalent
amount of methyl iodide, and the reaction mixture was refluxed in the presence of sodium ethoxide (10 mmol)
for 3-4 h (the completion of the reaction was checked with TLC). After the removal of the reaction solvent
under reduced pressure, a solid was obtained. It was recrystallized from ethyl acetate-petroleum ether (comp.
4) or ethanol (comp. 9) to obtain the desired compound.
4-Amino-5-(4-methylphenyl)-2-{[5-(methylthio)-1,3,4-oxadiazol-2-yl]methyl}-2,4-dihydro-
3H-1,2,4-triazol-3-one (4). Yield: 74%, mp 143-145 ◦ C, IR (KBr, υ, cm−1): 3326 and 3202 (NH2), 1721
(C=O), 1629, 1584 and 1507 (C=N), Anal. Calcd (%) for C13 H14 N6 O2 S: C, 49.05; H, 4.43, N, 26.40. Found;
C, 49.21; H, 4.48; N, 26.24; 1 H-NMR (DMSO-d6δ ppm): 2.02 (3H, s, CH3), 2.52 (3H, s, CH3), 4.70 (2H, s,
NCH2), 5.33 (2H, s, NH2), 7.23 (2H, d, j = 8.0 Hz, arH), 7.98 (2H, d, j =8.0 Hz, arH); 13 C-NMR (DMSO-d6δ
ppm): 20.45 (CH3), 22.18 (CH3), 44.27 (NCH2 + DMSO-d6), arC: [125.16 (2CH), 127.66 (C), 134.40 (2CH),
139.84 (C)], 142.94 (C-3, triazole), 154.73 (C-5, triazole), 162.43 (C-2, oxadiazole), 181.05 (C-5, oxadiazole).
4-{[(4-Fluorophenyl)methylene]amino}-5-(4-methylphenyl)-2-{[5-(methylthio)-1,3,4-oxa-
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Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
diazol-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (9). Yield: 71%, mp 175-177 ◦ C, IR (KBr, υ,
cm−1): 1715 (C=O), 1601, 1518 and 1509 (3C=N); Anal. Calcd (%) for C20 H17 FN6 O2 S: C, 56.59; H, 4.04; N,
19.80. Found; C, 56.71; H, 4.12; N, 19.74; 1 H-NMR (DMSO-d6δ ppm): 2.41 (3H, s, CH3), 2.73 (3H, s, CH3),
5.31 (2H, s, CH2), 7.21-7.33 (4H, m, arH), 7.76-7.91 (4H, m, arH), 9.67 (1H, s, N=CH); 13 C-NMR (DMSO-d6δ
ppm): 20.22 (CH3), 24.76(S -CH3 ) 46.67 (NCH2), arC: [115.12 and 115.47 (2CH-C-F), 157.12 and 162.34
(C-F), 123.43 (2CH), 125.33 (2CH), 125.65 (C), 129.23 (CH), 138.73 (CH), 138.57 (C), 145.36 (C)], 130.53
(N=CH), 144.25 (C-3, triazole), 154.57 (C-5, triazole), 163.67 (C-2, oxadiazole), 178.69 (C-5, oxadiazole).
Synthesis of 4-Amino-2-[(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)methyl]-5-(4-methylp-
henyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (5). Method 1: To a solution of the compound 2 (10 mmol)
in 1-butanol was added hydrazine hydrate and the reaction mixture was allowed to reflux for 5 h (completion of
the reaction was checked with TLC). After cooling of the reaction mixture in the cold, a solid appeared. This
was recrystallized from ethanol to obtain the desired compound.
Method 2: A solution of compound 1 (10 mmol) was treated with a solution of potassium hydroxide
(15 mmol) dissolved in absolute alcohol at 0-5 ◦ C by stirring. Then 15 mmol of carbon disulfide was added
slowly and the reaction mixture was stirred overnight at room temperature. The separated solid product was
filtered, washed with cold absolute ethanol, and finally dried. It was directly used for the next step without
purification. The solid product was mixed with a mixture of water (80 mL) and hydrazine hydrate (20 mmol)
and refluxed for 5 h. During the progress of the reaction, the reaction mixture turned green with evolution of
hydrogen sulfide and finally it became homogeneous. It was diluted with a small amount of cold water and then
treated with concentrated hydrochloric acid. The white precipitate was filtered, washed with cold water, and
recrystallized from dimethyl sulfoxide-water (1/1). Yield: 77%, mp 227-228 ◦ C, IR (KBr, υ, cm−1): 3301-3170
(2NH2), 2747 (SH), 1700 (C=O), 1614, 1577 and 1535 (3C=N); Anal. Calcd (%) for C12 H14 N8 OS: C, 45.27;
H, 4.43; N, 35.20. Found; C, 45.41; H, 4.52; N, 35.14; 1 H-NMR (DMSO-d6δ ppm): 2.40 (3H, s, CH3), 4.87
(2H, s, NCH2), 5.33 (4H, brs, 2NH2), 7.33 (2H, d, arH, j =8.0 Hz), 7.82 (2H, d, arH, j =8.0 Hz), 13.70 (1H,
s, SH); 13 C-NMR (DMSO-d6δ ppm): 20.27 (CH3), 45.64 (NCH2), arC: [125.47 (2CH), 126.96 (C), 135.12
(2CH), 139.67 (C)], 142.94 (C-3, triazole), 154.73 (C-5, triazole), 154.76 (C-2, triazole), 155.07 (C-5, triazole).
4-Amino-5-(4-methylphenyl)-2-[(6-phenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-zin-3-yl)
methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (7). A mixture of compound 5 (10 mmol) and phenacyl
bromide (12 mmol) in absolute ethanol was refluxed for 6 h. The reaction mixture was slowly quenched with
crushed ice by stirring and was neutralized with solid K2 CO3 . The solid that separated after standing overnight
was filtered, washed with cold water, dried and recrystallized from dimethyl sulfoxide-water (1:1) to afford the
desired compound. Yield 98%, mp 262-263 ◦ C; IR (KBr, υ, cm−1): 3277-3200 (NH2), 1708 (C=O), 1644,
1506 and 1463 (4C=N); Anal. Calcd (%) for C20 H18 N8 OS: C, 57.40; H, 4.34; N, 26.78. Found; C, 57.41; H,
4.42; N, 26.54; 1 H-NMR (DMSO-d6δ ppm): 2.34 (3H, s, CH3), 4.42 (2H, s, CH2), 5.33 (2H, s, NH2), 5.58
(2H, bs,SCH2), 7.27 (2H, d, j =7.8 Hz, arH), 7.54-7.61 (3H, m, arH), 7.87 (2H, j =7.8 Hz, arH), 8.06 (2H, d,
j =7.4 Hz, arH); 13 C-NMR (DMSO-d6δ ppm): 21.73 (CH3), 33.39 (CH2 , thiadiazine) 47.43 (NCH2), arC:
[125.69 (2CH), 129.79 (2CH), 130.16 (C), 131.37 (3CH), 133.22 (2CH), 140.34 (C), 141.07 (C)], 148.63 (C-8a,
triazolothiadiazine), 152.78 (C-3, triazole), 154.49 (C-5, triazole), 159.24 (C-3, triazolothiadiazine), 161.17 (C-6,
triazolothiadiazine).
353

˙
Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
Microbiology
Antimicrobial activity assessment
All test microorganisms, gram negative bacteria: Escherichia coli ATCC 35218, Klebsiella pneumoniae ATCC
13883, Yersinia pseudotuberculosis ATCC 911, Enterobacter aerogenes ATCC 13048, Pseudomonas aeruginosa
ATCC 10145; gram positive bacteria: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212,
Bacillus cereus 709 Roma, and yeast-like fungi: Candida tropicalis ATCC 13803, Candida glabrata 66032, and
Candida albicans ATCC 60193) were obtained from the Refik Saydam Hifzissihha Institute (Ankara, Turkey).
All newly synthesized compounds were weighed and dissolved in dimethylsulphoxide to prepare extract stock
solution of 10,000 μg/mL.
The antimicrobial effects of the substances were tested quantitatively in respective broth media by
using double dilution and the minimal inhibition concentration (MIC) values (μg/mL) were determined.30
The antibacterial and antifungal assays were performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI,
USA) at pH 7.3 and buffered Yeast Nitrogen Base (Difco) at pH 7.0, respectively. The MIC was defined as the
lowest concentration that showed no growth. Ampicillin (10 μg) and fluconazole (5 μg) were used as standard
antibacterial and antifungal drugs, respectively. Dimethylsulphoxide at a dilution of 1:10 was used as solvent
control. The results are shown in the Table.
Table. Antimicrobial activities of the newly synthesized compounds (μg/mL).
Comp.
No.
2a
3a
4a
5a
6a
7a
8b
Microorganisms and Minimal Inhibition Concentration Values (μg/mL)
Ec
Kp
Yp
En
Pa
Sa
Ef
Bc
500
0.49
Ca
Ct
> 500 > 500 > 500 > 500 > 500 > 500 > 500
> 500 > 500
> 500 > 500
< 0.12 < 0.12
> 500 > 500 > 500
< 0.12 0.24 < 0.12
0.24
0.49
125
0.49
0.24
125
125
0.98
> 500
0.98
0.49
125
0.98
125
> 500 > 500 > 500
1.95
0.49
125
1.95
0.98
125
1.95
0.49
> 500 > 500
> 500 > 500
< 0.12 < 0.12 < 0.12
> 500 > 500 > 500
0.98
> 500
125
> 500 > 500 > 500
3.9 > 500 > 500
125
125
125
31.3
31.3
9a
> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500
10aa
10ba
Amp.
Flu.
125
62.5
10
125
62.5
125
62.5
18
125
62.5
125
62.5
18
62.5
62.5
35
62.5
62.5
10
15.6
62.5
15
> 500 > 500
> 500 > 500
> 128
> 128
< 1
8
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Pa: Pseudomonas aeruginosa ATCC
27853, Ef: Enterococcus faecalis ATCC 29212, Sa: Staphylococcus aureus ATCC 25923, Kp: Klebsiella pneumoniae
ATCC 13883, En: Enterobacter aerogenes ATCC 13048; Bc: Bacillus cereus 702 Roma, Ct: Candida tropicalis ATCC
13803, Ca: Candida albicans ATCC 60193, a : solvent is ethanol, b : solvent is dimethyl sulfoxide, Amp: Ampicillin, Flu:
Fluconazole .
354

˙
Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
Results and discussion
In the present study, a series of 10 compounds was synthesized. The Scheme illustrates the method used for the
preparation of target compounds. The structures of all the newly synthesized compounds were elucidated by
IR, 1 H-NMR, and 13 C-NMR spectroscopic methods and elemental analysis. In the IR spectra of all compounds
C=N bands were observed at about 1629-1463 cm−1 . According to the IR spectroscopic data of compounds 2,
5, 6, and 8, which have a mercapto structure, the observation of -SH function at 2750-2723 and the absence of
an absorption at about 1300-1100 cm−1 region cited for the C=S stretching band showed that these compounds
were in mercapto form.
N
N
N
N
S
O
N
N
ii
O
N
NH2
3
O
N
N
F
NHNH2
SH
O
CH3
N
N
N
N
N
N
O
N
O
N
NH2
2
i
SCH3
NH2
O
1
N N
CH3
CH3
N
O
N
NH2
4
iv
iii
v
CH3
N
viii
SH
N
N
N
NH2
N
N
SH
O
N
N
O
N
NH2
O
N
N
C
CH3
5
CH3
H
vi
vii
F
8
N
N
N
N
S
SH
N
N
N
N
N
N
N
N
ix
iii
R
O
N
NH2
O
N
N
CH
N
N
N
N
CH3
SCH3
OH
S
CH3
O
O
7
CH
N
N
N
N
HO
O
O
N
N
N
C
N
C
CH3
CH3
6
H
H
F
F
10a:
R=
N
O
9
10b:R=
N
N CH3
Scheme. i= CS2 /KOH, ii= HCHO and 4-aminomorpholine or 1-(4-fluorophenyl)piperazyne, iii= 1) CS2 /KOH, room
temp. 2) H2 NNH2 , v= H2 NNH2 (reflux), vi= salicyl aldehyde, vii= phenacylbromide, viii= CHO2 H4 F(p-), ix=
HCHO and morpholine or 4-methyl piperazyne.
355

˙
Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
The treatment of hydrazides with CS2 in the presence of KOH is a general method leading to the
formation of 5-thioxo-1,3,4-oxadiazole derivatives.28,29 As the starting material 2-[4-amino-3-(4-methylphenyl)-
5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]aceto-hydrazide (1) was used to produce synthesis of 4-amino-2-[(5-
mercapto-1,3,4-oxadiazol-2-yl)methyl]-5-(4-methyl phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2) (Scheme). In
contrast to compound 1, the 1 H-NMR spectrum of compound 2 displayed a signal at 1.65 ppm belonging to
the –SH group, while the -NHNH2 signals disappeared. Compound 5 was synthesized starting from compound
1 or 2. The IR spectra of compound 5 showed 2 sharp absorption bands at 3301 and 3170 cm−1 representing
2 –NH2 groups. The signal integrating 4 protons seen at 5.33 ppm in the 1 H-NMR spectrum of compound 5
were attributed to the 2 –NH2 groups (exch. with D2 O). Furthermore, the 1 H-NMR spectra of 5 displayed
an additional singlet due to the –SH group at 13.70 ppm integrating 1 proton, while no signal representing a
hydrazide structure appeared.
The synthesis of the corresponding Schiff base derivatives (6 and 8) of compounds 2 and 5 was performed
by the reaction of compounds 2 and 5 with 4-fluorophenylbenzaldehyde (for 8) or salicylaldehyde (for 6) in
the presence of the catalytic amount of H2 SO4 . In the 1 H-NMR spectra of the compounds 6 and 8, no signal
representing the presence of an amino group in the structure appeared; instead, additional signals due to a
4-fluorophenyl or salicyl nucleus were recorded at the aromatic region. Due to slight solubility in dimethyl
sulfoxide-d6 solution, a satisfactory 13 C-NMR spectrum was not recorded for compound 6. The absorption
bands due to the 2 –OH groups of 2-hydroxyphenyl moiety were recorded at 3414 cm−1 in the IR spectra of 6.
Moreover, the signal recorded at 10.38 ppm in the 1 H-NMR spectra of 6 was assigned to the 2 –OH groups.
Both compounds 2 and 8 were converted to their corresponding S-methyl derivatives (4 and 9) by the
reaction with methyl iodide in the presence of sodium ethoxide. The absence of the signal corresponding to
the –SH group in the IR and 1 H-NMR spectra of compounds 4 and 9, and the appearance of an additional
singlet signal at 2.52 (for 4) and 2.73 (for 9) ppm in the 1 H-NMR spectra indicated the S-methyl derivatives
of compounds 2 and 8. In the 13 C-NMR spectra, these methyl groups resonated at 22.18 and 24.76 ppm, for
4 and 9, respectively.
The aminoalkylation of compounds 2 and 8 was performed in the presence of 4-fluorophenyl piperazine
(for 3), morpholine (for 10a) or 4-methyl piperazine (for 10b), and formaldehyde. In the 1 H- and 13 C-NMR
spectra of compounds 3, 10a, and 10b, the presence of additional signals due to morpholine, 4-fluorophenyl
morpholine, or methyl piperazine moiety confirmed the conversion of compounds 2 and 8 into the corresponding
Mannich bases.
The treatment of compound 5 with phenacyl bromide facilitated the conversion of 4-amino-5-mercapto-
1,2,4-triazole ring 5 into [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine system; thus, compound 7 was obtained. In
contrast to compound 5, the 1 H-NMR spectra of compound 7 exhibited no signal due to the –SH group,
whereas signals that originated from thiadiazine C-6 and phenyl group at position 5 of the thiadiazine ring
were present. The disappearance of the signal at 13.70 ppm, corresponding to the –SH proton of the parent
compound 5, in the 1 H-NMR spectrum of product 7 confirmed the involvement of the –SH proton in the
condensation reaction.
All the synthesized compounds gave elemental analysis data consistent with the assigned structures.
All the compounds were tested for their antimicrobial activities. MICs were recorded as the minimum
concentrations of compounds that inhibit the growth of tested microorganisms. Based on the obtained results,
356

˙
Synthesis and antimicrobial activities of..., A. DEMIRBAS¸, et al.,
it can be concluded that the conversion of a 1,3,4-oxadiazole ring (compound 2) into an amino-1,2,4-triazole
ring (compound 5) afforded good antimicrobial activity with MIC values in the range of 0.12-1.95 μg/mL.
Moreover, the 2-hydroxyphenyl methylenamino derivative (6) of compound 5 was found to be active against the
test microorganisms (except Candida tropicalis and Candida albicans), with MIC values between 0.12 and 0.98
μg/mL. The other compound having a Schiff base structure, 8, displayed moderate antimicrobial activity against
the gram negative bacterial strain and good activity against the gram positive bacteria used in the study. On
the other hand, the S-methyl derivative (9) of compound 8 was found to possess no activity. Similarly, the other
S-methyl compound, 4, was found to be inactive toward the test microorganisms. Among the Mannich bases
(3, 10, and 10b) synthesized in the study, the most active compound was 3, with MIC values in the range of
0.12-0.98 μg/mL. Compound 10a was found to be active against the microbial strains used in the study (except
Candida tropicalis and Candida albicans), with the MIC value 62.5 μg/mL. On the other hand, compound 10b
exhibited activity with the MIC value of 125 μg/mL against Escherichia coli, Klebsiella pneumoniae, Yersinia
pseudotuberculosis, Enterobacter aerogenes, and Pseudomonas aeruginosa, while it displayed good activity with
the MIC value of 62.5 μg/mL towards Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus.
Acknowledgements
¨
˙
The support provided by the Scientific and Technological Research Council of Turkey (TUBITAK, Project
no: 107T333) and Karadeniz Technical University, BAP, Turkey (Ref. No. 2005.111.002.4), is gratefully
acknowledged.
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358

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