Synthesis and antimicrobial activities of some new biheterocyclic compounds containing 1, 2, 4-triazol-3-one and 1, 3, 4-thiadiazole moieties

Article abstract of DOI:10.3906/kim-0909-271

2-(4-Amino-3-(4-chlorophenyl)-5-oxo-4, 5-dihydro-1 H-1, 2, 4-triazol-1-yl)-N'-[(2, 6 dihalogenophenyl)-methylene]acetohydrazides (3a, b) was obtained via the formation of 2-(4-amino-3-(4-chlorophenyl)-5-oxo-4, 5-dihydro-1 H-1, 2, 4-triazol-1-yl)acetohydrazide (2), which was obtained starting from 4-amino-5-(4-chlo-rophenyl)-2, 4-dihydro-3H-1, 2, 4-triazol-3-one (1) in 2 steps. 2-{[4-amino-3-(4-chlorophenyl)-5-oxo-4, 5-dihydro-1H-1, 2, 4-triazol-1-yl]acetyl}-N-phenylhydra-zine carbothioamide (4), which was prepared starting from 2, was converted to the corresponding 1, 3, 4-thiadiazole derivative (5) in acidic media. Moreover, the basic treatment of 4 resulted in the formation of 4-amino-5-(4-chlorophenyl)-2-[(4-phenyl-5-thioxo-4, 5-dihydro-1H-1, 2, 4-triazol-3-yl)methyl]-2, 4-dihydro-3H-1, 2, 4-triazol-3-one (7). The reactions of compounds 5 and 7 with methyl iodide in the presence of sodium ethoxide afforded the corresponding N-methyl (6) and S-methyl (8) derivatives, respectively. The synthesis of Mannich bases (10a and 10b) was performed from the reaction of 7 with morpholine or piperazine in the presence of formaldehyde. All the newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial activity study revealed that compounds 3a, 3b, and5 showed good antimicrobial activities against the test microorganisms as compared with ampicillin. tuebitak.

Full text of DOI:10.3906/kim-0909-271

Turk J Chem
34 (2010) , 517 – 527.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-0909-271
Synthesis and antimicrobial activities of some new
biheterocyclic compounds containing 1,2,4-triazol-3-one
and 1,3,4-thiadiazole moieties
1
2,∗
2
˙
˙
Hakan BEKTAS¸ , Ahmet DEMIRBAS¸ , Neslihan DEMIRBAS¸ ,
2
3
˘
Hacer BAYRAK , Sengu¨l Alpay KARAOGLU
1Giresun University, Faculty of Sciences, Department of Chemistry,
28100 Giresun-TURKEY
2Karadeniz Technical University, Faculty of Sciences, Department of Chemistry,
61080 Trabzon-TURKEY
e-mail: ndemirbas651@mynet.com
3
Rize University, Faculty of Sciences, Department of Biology,
53100 Rize-TURKEY
Received 15.09.2009
2-(4-Amino-3-(4-chlorophenyl)-5-oxo-4,5–dihydro-1H -1,2,4-triazol-1-yl)-Nꢀ -[(2,6-dihalogenophenyl)-
methylene]acetohydrazides (3a,b) was obtained via the formation of 2-(4-amino-3-(4-chlorophenyl)-5-oxo-
4,5-dihydro-1H -1,2,4-triazol-1-yl)acetohydrazide (2), which was obtained starting from 4-amino-5-(4-chlo-
rophenyl)-2,4-dihydro-3H -1,2,4-triazol-3-one (1) in 2 steps. 2-{[4-amino-3-(4-chlorophenyl)-5-oxo-4,5–dihydro-
1H -1,2,4-triazol-1-yl]acetyl}-N -phenylhydra-zine carbothioamide (4), which was prepared starting from 2,
was converted to the corresponding 1,3,4-thiadiazole derivative (5) in acidic media. Moreover, the basic
treatment of 4 resulted in the formation of 4-amino-5-(4-chlorophenyl)-2-[(4-phenyl-5-thioxo-4,5-dihydro-
1H -1,2,4-triazol-3-yl)methyl]-2,4-dihydro-3H -1,2,4-triazol-3-one (7). The reactions of compounds 5 and 7
with methyl iodide in the presence of sodium ethoxide afforded the corresponding N-methyl (6) and S-
methyl (8) derivatives, respectively. The synthesis of Mannich bases (10a and 10b) was performed from
the reaction of 7 with morpholine or piperazine in the presence of formaldehyde.
All the newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial
activity study revealed that compounds 3a, 3b, and 5 showed good antimicrobial activities against the test
microorganisms as compared with ampicillin.
Key Words: 1,2,4-triazol-3-one, 5-thioxo-1,2,4-triazole, 1,3,4-thiadiazole, Mannich base, antimicrobial
activity
∗Corresponding author
517

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
Introduction
The synthesis of new compounds that are used to treat infections effectively and that lack the side effects
associated with current therapies remains a major challenge in medicinal chemistry. In some cases, especially in
patients with impaired liver or kidney functions, the use of antimicrobial drugs to treat infections causes several
problems. Moreover, from the pharmacoeconomic cost-efficiency point and seeking better patient compliance,
antimicrobial agents with high therapeutic effect, high safety, and minimum adverse effects are considerably
desirable.1,2
The Mannich reaction is a 3-component condensation reaction involving an active hydrogen containing
compound, formaldehyde, and a secondary amine. The aminomethylation of aromatic substrates by the Mannich
reaction is of considerable importance for the synthesis and modification of biologically active compounds.3 In
recent years, Mannich bases have gained importance due to their application in pharmaceutical chemistry. They
have been found to possess antibacterial, antifungal, anticancer, antitubercular, analgesic, and antiinflammatory
properties.3
1,2,4-Triazoles are an important class of heterocycles, and have been the subject of great interest due
to their pharmacological properties.4−12 Very promising therapeutic applications have been obtained using
the 1,2,4-triazole system. There are a number of drugs containing 1,2,4-triazole nucleus, such as itraconazole,
fluconazole, and voriconazole (antifungal), that have been used for the treatment of fungal infections.13−16
Some other dugs including this heterocycle are ribavirin (antiviral), rizatriptan (antimigraine), alprazolam
(anxiolytic), vorozole (antitumoral), letrozole (antitumoral), and anastrozole (antitumoral).17−21
Another azole ring, 1,3,4-thiadiazole, is incorporated into various biologically active compounds.22−24
Among these, acetazolamide is a carbonic anhydrase inhibitor and has been used for the treatment of glaucoma,
epilepsy, fluid retention, and altitude sickness.25 The other 1,3,4-thiadiazole containing drug, cefazolin, is a first-
generation cephalosporin antibiotic, and has been used in the treatment of a variety of infections caused by
gram-positive bacteria such as pharyngitis, uncomplicated skin infections, and endocarditis. It can also be used
for the treatment of several bacterial infections involving the lung, bone, joint, stomach, blood, heart valve, and
urinary tract.26−28
In view of the above facts and in continuation of our interest in the synthesis of 1,2,4-triazole derivatives
as antimicrobial agents,4,8,10−12,29−33 herein we report the synthesis and antimicrobial activities of new 1,2,4-
triazole derivatives.
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
as potassium bromide pellets using a Perkin-Elmer 1600 series FT-IR spectrometer. Mass spectra were run
on a Quattro LC-MS (ESI, 70 eV) Instrument. Combustion analysis was performed on a Costech Elemental
Combustion System CHNS-O elemental analyzer. All the chemicals were obtained from Fluka Chemie AG
Buchs (Switzerland).
518

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
Synthesis of 2-(4-amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)acetohyd-
razide (2). Compound 1 (10 mmol) was refluxed with an equivalent amount of sodium in absolute ethanol
for 2 h. Then ethyl bromoacetate (10 mmol) was added and the mixture refluxed for an additional 8 h. After
evaporating the solvent under reduced pressure, a solid appeared. This product was refluxed with hydrazine
hydrate (25 mmol) in n-butanol for 4 h. After cooling to room temperature, a white solid appeared. This crude
product was recrystallized from ethanol to afford the desired product. Yield 98%, mp = 215-218 ˚C; Anal.
Calcd. (%) for: C10 H11 N6 ClO2 : C, 42.49, H, 3.92, N, 29.73. Found: C, 42.50, H, 3.90, N, 29.65; IR (KBr, ν ,
cm−1): 3302-3213 (NH+2NH2), 1717 (C=O), 1668 (C=O), 1577 (C=N); 1 H-NMR (DMSO-d6)δ (ppm): 4.34
(s, 2H, NCH2), 4.46 (s, 2H, NHNH2), 5.55 (s, 2H, NH2), 7.52-7.65 (m, 2H, ar-H), 7.82-8.05 (m, 2H, ar-H), 9.27
(s, NHNH2); 13 C-NMR (DMSO-d6)δ (ppm): 48.69 (CH2), arC: [126.82 (C), 127.38 (C), 133.82 (C), 134.93
(C), 138.47(C), 138.97 (C)], 151.79 (triazole C-3), 158.55 (triazole C-5), 167.45 (C=O); MS (ESI): m/z (%)
283.69 (M+1, 32), 305 (100), 273 (34), 229 (48).
General Method for the synthesis of compounds 3
A solution of compound 2 (0.01 mol) in ethanol was refluxed with 2,6-dichlorobenzaldehyde (for 3a) or 2-chloro-
6-fluorobenzaldehyde (for 3b) (0.01 mol) for 3 h. On cooling the reaction mixture to room temperature, a white
solid appeared. This crude product was recrystallized from dimethyl sulfoxide–water (1:2) to afford the desired
product.
2-(4-Amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N ’-[(2,6-dichlorop-
henyl)-methylene]acetohydrazide (3a). Yield 85%, mp 258-259 ◦ C; Anal. Calcd. (%) for: C17 H13 N6
O2 Cl3 : C, 46.44, H, 2.98, N, 19.11, Found; C, 46.42, H, 2.95, N, 19.15; IR (KBr, ν , cm−1): 3206, 3067
(NH+NH2), 1714, 1687 (C=O), 1585 (C=N); 1 H-NMR (DMSO-d6) δ (ppm): 4.91 and 5.00 (s, 2H, benzyl-
CH2 , cis/trans amide conformers), 5.62 (s, 2H, NH2), 7.39-7.66 (m, 4H, ar-H), 7.82-8.03 (m, 2H, ar-H),
8.28-8.30 (m, 1H, ar-H), 9.66 (s, 1H, N=CH), 12.01 (s, 1H, NH);13 C-NMR (DMSO-d6)δ (ppm): 46.87 (CH2),
arC: [115.56 (C), 118.68 (C), 124.70 (C), 125.96 (C), 127.88 (C), 128.23 (C), 131.45 (C), 132.88 (C), 136.56
(C), 137.82 (C), 143.34 (C), 151.11 (C)], 154.65 (N=CH), 156.24 (triazole C-3), 162.86 (triazole C-5), 167.48
(C=O); MS (ESI): m/z (%) 439 (M, 20), 396 (52), 328 (68), 229 (100), 215 (50).
2-(4-Amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-N ’-[(2-chloro-6- flu-
orophenyl)methylene]acetohydrazide (3b). Yield 92%, mp 267-268 ◦ C; Anal. Calcd. (%) for: C17 H13 N6
O2 Cl2 : C, 48.24, H, 3.10, N, 19.86, Found; C, 48.20, H, 3.12, N, 19.82; IR (KBr, ν , cm−1): 3208, 3131 and
3070 (NH+NH2), 1716, 1693 (C=O), 1566 (C=N); 1 H-NMR (DMSO-d6)δ (ppm): 4.91 and 5.00 (s, 2H, benzyl-
CH2 , cis/trans amide conformers), 5.65 (s, 2H, NH2), 7.31-7.66 (m, 4H, ar-H), 7.84-8.08 (m, 2H, ar-H), 8.28 (s,
1H, ar-H), 9.66 (s, 1H, N=CH), 12.07 (s, 1H, NH);13 C-NMR (DMSO-d6)δ (ppm): 46.52 (CH2), arC: [115.27
(C), 119.78 (C), 124.70 (C), 125.96 (C), 127.98 (C), 128.74 (C), 131.45 (C), 132.88 (C), 135.09 (C), 137.06 (C),
142.46 (C), 150.09 (C)], 153.81 (N=CH), 156.88 (triazole C-3), 162.87 (triazole C-5), 167.63 (C=O); MS (ESI):
m/z (%) 423 (M, 8), 445 (40), 229 (100).
2-{[4-Amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetyl}-N -pheny-
lhydrazinecarbothioamide (4). A mixture of acid hydrazide (2) (0.01 mol) and phenyl isothiocyanate (0.015
mol) was allowed to reflux in ethanol for 2 h. Then the solution was cooled to room temperature and a white
519

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
solid appeared. This product was filtered and recrystallized from dimethyl sulfoxide–water (1: 2) to yield the
target product.
Yield 91%, mp 192-193 ◦ C; Anal. Calcd. (%) for: C17 H16 ClN7 O2 S: C, 48.86, H, 3.86, N, 23.46, S,
7.67, Found; C, 48.85, H, 3.83, N, 23.47, S, 7.64; IR (KBr, ν , cm−1): 3269 (3NH+NH2), 1714 (triazole-C=O),
1689 (exocyclic-C=O), 1598 (C=N), 1352 (C=S); 1 H-NMR (DMSO-d6)δ (ppm): 4.66 (s, 2H, NCH2), 5.63 (s,
NH2), 7.18-7.31 (m, 1H, ar-H), 7.35-7.45 (m, 3H, ar-H), 7.53-7.67 (m, 3H, ar-H), 7.82-8.05 (m, 2H, ar-H), 9.64
(s, NH), 9.78 (s, NH), 10.41 (s, NH); 13 C-NMR (DMSO-d6)δ (ppm): 51.12 (CH2), arC: [128.78 (C), 128.86
(2C), 129.29 (2C), 129.75 (2C), 130.48 (2C), 133.55 (C), 135.51 (C), 139.55 (C)], 150.78 (triazole C-3), 154.51
(triazole C-5), 171.88 ( C=O), 183.12 (C=S).
4-Amino-2-[(5-anilino-1,3,4-thiadiazol-2-yl)methyl]-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-
triazol-3-one (5). A mixture of the carbothioamide (4) (0.01 mol) in cold concentrated sulfuric acid (28 mL)
was stirred for 10 min. Then the mixture was allowed to reach room temperature. After stirring for an addi-
tional 30 min, the resulting solution was poured into ice-cold water and made alkaline to pH 8 with ammonia.
The precipitated product was filtered and recrystallized from ethanol to obtain the desired product. Yield 92%,
mp 206-208 ◦ C; Anal. Calcd. (%) for: C17 H14 ClN7 OS: C, 51.06, H, 3.53, N, 24.52, S, 8.02, Found; C, 51.02,
H, 3.50, N, 24.55, S, 8.08; IR (KBr, ν , cm−1): 3324-3115 (NH+NH2), 1711 (C=O), 1601 (C=N); 1 H-NMR
(DMSO-d6)δ (ppm): 5.31 (s, 2H, NCH2), 5.60 (s, NH2), 6.94-7.02 (m, 1H, ar-H), 7.28-7.36 (m, 2H, ar-H),
7.51-7.64 (m, 4H, ar-H), 7.81-8.05 (m, 2H, ar-H), 10.36 (s, NH);13 C-NMR (DMSO-d6)δ (ppm): 45.93 (CH2),
arC: [119.37 (2C), 123.97 (C), 129.91 (2C), 130.35 (2C), 130.95 (2C), 131.99 (C), 137.20 (C), 148.32 (C)], 154.97
(triazole C-3), 155.35 (triazole C-5), 159.42 (thiadiazole C-2), 167.42 (thiadiazole C-5); MS (ESI): m/z (%) 400
(M, 75), 422 (92), 229 (38), 190 (100).
General method for the synthesis of compounds 6 and 8
To a solution of compound 5 (for 6) or compound 7 (for 8) in absolute ethanol was added an equivalent amount
of sodium and the mixture was stirred at room temperature for 30 min. Then methyl iodide (0.01 mol) was
added and refluxed for an additional 4 h. After evaporating the solvent under reduced pressure a solid appeared.
This crude product was recrystallized from ethanol to obtain the target compound.
4-Amino-5-(4-chlorophenyl)-2-({5-[methyl(phenyl)amino]-4,5-dihydro-1,3,4-thiadiazol-2-yl}
methyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (6). Yield 74%, mp 187-188 ◦ C; Anal. Calcd. (%) for:
C18 H16 ClN7 OS: C, 52.24, H, 3.90, N, 23.69, S, 7.75, Found; C, 51.84, H, 3.45, N, 24.22, S, 7.84; IR (KBr,
ν , cm−1): 3233 (NH2), 1725 (C=O), 1610 (C=N); 1 H-NMR (DMSO-d6)δ (ppm): 3.24 (s, 3H, CH3), 5.38
(s, 2H, NCH2), 5.58 (s, 2H, NH2), 6.90-7.18 (m, 1H, ar-H), 7.26-7.46 (m, 2H, ar-H), 7.58-7.72 (m, 4H, ar-H),
7.80-8.15 (m, 2H, ar-H);13 C-NMR (DMSO-d6)δ (ppm): 32.14 (CH3), 42.65 (CH2), arC: [118.26 (2C), 122.92
(C), 128.98 (2C), 130.36 (2C), 130.88 (2C), 132.39 (C), 136.68 (C), 149.56 (C)], 154.96 (triazole C-3), 156.68
(triazole C-5), 159.65 (thiadiazole C-2), 167.48 (thiadiazole C-5); MS (ESI): m/z (%) 414 (M, 70), 229 (65),
190 (100).
4-Amino-5-(4-chlorophenyl)-2-{[5-(methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl]methyl}-2,4-
dihydro-3H-1,2,4-triazol-3-one (8). Yield 67%, mp 148-149 ◦ C; Anal. Calcd. (%) for: C18 H16 ClN7 OS:
C, 52.24, H, 3.90, N, 23.69, S, 7.75, Found; C, 52.25, H, 3.92, N, 23.65, S, 7.70; IR (KBr, ν , cm−1): 3297
520

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
(NH2), 1709 (C=O), 1499 (C=N); 1 H-NMR (DMSO-d6)δ (ppm): 3.37 (s, 3H, CH3), 5.06 (s,2H, NCH2), 5.37
(s, 2H, NH2), 7.36-7.49 (m, 7H, ar-H), 7.86-7.89 (d, 2H, ar-H,J =7.2 Hz);13 C-NMR (DMSO-d6)δ (ppm): 14.18
(CH3), 38.09-40.59 (DMSO-d6 + NCH2), arC: [124.97 (C), 126.73 (2C), 128.39 (2C), 129.12 (2C), 129.57 (2C),
129.95 (C), 132.12 (C), 134.65 (C)], 143.72 (triazole C-3), 143.75 (triazole C-3’), 150.88 (triazole C-5), 152.52
(triazole C-5’); MS (ESI): m/z (%) 414 (M,100), 416 (M+2, 42), 271 (22), 204 (28), 153 (82).
4-Amino-5-(4-chlorophenyl)-2-[(4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]-
2,4-dihydro-3H-1,2,4-triazol-3-one (7). A solution of carbothioamide (4) (0.01 mol) in 2N NaOH was
refluxed for 3 h. Then the resulting solution was cooled to room temperature and acidified to pH 3-4 with 37%
HCl. The precipitate formed was filtered, washed with water, and recrystallized from ethanol–water (1:1) to
afford the desired compound. Yield 93%, mp 177-178 ◦ C; Anal. Calcd. (%) for: C17 H14 ClN7 OS: C, 51.06,
H, 3.53, N, 24.52, S, 8.02, Found; C, 51.00, H, 3.57, N, 24.52, S, 8.06; IR (KBr, ν , cm−1): 3307-3098 (NH2),
2667 (SH), 1698 (C=O), 1492 (C=N), 1342 (C=S); 1 H-NMR (DMSO-d6)δ (ppm): 4.97 (s, 2H, NCH2), 5.38 (s,
NH2), 7.35-7.65 (m, 7H, ar-H), 7.00-7.91 (m, 2H, ar-H), 14.00 (s, SH);13 C-NMR (DMSO-d6)δ (ppm): 38.07-
40.83 (DMSO-d6 + CH2), arC: [124.89 (C), 126.53 (C), 127.47 (C), 127.96 (C), 128.43 (C), 129.18 (C), 129.46
(C), 129.78 (C), 132.80 (C), 134.76(C), 143.99 (C), 147.21 (C)], 148.50 (triazole C-3), 152.54 (triazole C-3’),
156.79 (triazole C-5), 168.31 (triazole C-5’); MS (ESI): m/z (%) 400 (M, 75), 402 (M+2, 32), 423 (M+Na, 92),
229 (95), 211 (40).
5-(4-Chlorophenyl)-4-{[(4-methoxyphenyl)methylene]amino}-2-[(4-phenyl-5-thioxo-4,5-dih-
ydro-1H-1,2,4-triazol-3-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (9). A mixture of compound
7 (0.01 mol) with anisaldehyde (0.01 mol) was heated in an oil bath at 140 ◦ C for 2 h. On cooling it to room
temperature, a solid appeared. The crude product recrystallized from ethanol to afford the desired compound.
Yield 83%, mp 138-140 ◦ C; Anal. Calcd. (%) for: C25 H20 ClN7 O2 S: C, 57.97, H, 3.89, N, 18.93, S, 6.19,
Found; C, 57.92, H, 3.85, N, 18.95, S, 6.21; IR (KBr, ν , cm−1): 3155 (NH), 2726 (SH), 1712 (C=O), 1490
(C=N), 1204 (C=S); 1 H-NMR (DMSO-d6)δ (ppm): 3.84 (s, 3H, OCH3), 5.09 (s, 2H, NCH2), 7.05-7.09 (d, 2H,
ar-H, J =8.6 Hz), 7.33-7.48 (m, 5H, ar-H), 7.57- 7.61 (d, 2H, ar-H, J =8.6 Hz), 7.72-7.81 (m, 4H, ar-H), 9.18 (s,
N=CH), 14.08 (s, SH);13 C-NMR (DMSO-d6)δ (ppm): 43.50 (CH2), 55.37 (OCH3), arC: [110.17 (C), 111.48
(C), 114.40 (C), 117.38 (C), 121.14 (C), 123.27 (C), 124.42 (C), 126.83 (C), 127.52 (C), 128.39 (C), 129.67 (C),
131.61 (C), 132.91 (C), 133.53 (C), 135.22 (C), 140.43 (C), 142.9(C), 157.02 (C)], 147.28 (CH), 148.64 (triazole
C-3), 152.99 (triazole C-3’), 162.23 (triazole C-5), 168.49 (triazole C-5’); MS (ESI): m/z (%) 518 (M, 100), 520
(M+2, 60), 486 (34), 365 (42), 313 (44).
General method for the synthesis of compounds 10a and 10b
To a solution of compound 8 (0.01 mol) in dichloromethane was added morpholine (for 9a) or piperazine (for
9b) in the presence of formaldehyde (40%, 1.5 mL) and the mixture was stirred at room temperature for 2 h.
Then water was added and kept overnight in cold conditions. The solid separated was collected by filtration
and recrystallized from ethylacetate–petroleum ether (1:2) to yield the target compounds.
5-(4-Chlorophenyl)-4-{[(4-methoxyphenyl)methylene]amino}-2-{[4-phenyl-1-(morpholin-4-
ylmethyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]methyl}-2,4-dihydro- 3H -1,2,4-triazol-3-one
(10a). Yield 68%, mp 133-135 ◦ C; Anal. Calcd. (%) for: C30 H29 ClN8 O3 S: C, 58.39, H, 5.74, N, 18.16, S,
521

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
5.20, Found; C, 58.35, H, 5.72, N, 18.13, S, 5.15; IR (KBr, ν , cm−1): 1712 (C=O), 1492 (C=N), 1313 (C=S);
1 H-NMR (DMSO-d6)δ (ppm): 2.74 (t, 2H, CH2 , J =2.2 Hz), 3.60 (t, 2H, CH2 , J =2.4 Hz), 3.85 (s, 3H,
OCH3), 5.12 (s, 4H, CH2), 5.49 (brs, 2H, NCH2), 5.52 (brs, 2H, NCH2), 7.08-7.11 (m, 2H, ar-H), 7.35-7.50
(m, 4H, ar-H), 7.54-7.63 (m, 4H, ar-H), 7.74-7.78 (m, 3H, ar-H), 9.19 (s, N=CH); 13 C-NMR (DMSO-d6)δ
(ppm): 45.54 (CH2), 57.30 (OCH3), 58.12 (2CH2), 59.45 (2CH2), 65.23 (CH2), arC: [116.45 (C), 125.15 (C),
127.05 (2C), 129.38 (2C), 130.57 (2C), 131.14 (2C), 131.38 (2C), 131.56 (C), 131.76 (C), 135.12 (2C), 137.10
(C), 157.02 (C)], 146.73 (CH), 148.64 (triazole C-3), 151.34 (triazole C-5), 164.56 (triazole C-3’), 167.49 (triazole
C-5’); MS (ESI): m/z (%) 618 (M+1, 18), 606 (28), 518 (46), 229 (100).
5-(4-Chlorophenyl)-4-{[(4-methoxyphenyl)methylene]amino}-2-{[4-phenyl-1-(piperazine-1-
ylmethyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl]methyl}-2,4-dihydro- 3H -1,2,4-triazol-3-one
(10b). Yield 78%, mp 169-170 ◦ C; Anal. Calcd. (%) for: C31 H32 ClN9 O2 S: C, 59.09, H, 5.12, N, 20.00, S,
5.09, Found; C, 59.02, H, 5.10, N, 19.90, S, 5.13; IR (KBr, ν , cm−1): 1716 (C=O), 1489 (C=N), 1296 (C=S);
1 H-NMR (DMSO-d6)δ (ppm): 2.44 (s, 3H, NCH3), 2.74 (t, 2H, CH2 , J = 2.4 Hz), 3.56 (brs, 2H, CH2), 3.84
(s, 3H, OCH3), 4.97 (s, 4H, CH2), 5.21 (brs, 2H, NCH2), 5.42 (brs, 2H, NCH2), 7.06-7.10 (m, 2H, ar-H), 7.59-
7.63 (m, 4H, ar-H), 7.77-7.82 (m, 4H, ar-H), 7.90-7.95 (m, 3H, ar-H), 9.49 (s, N=CH);13 C-NMR (DMSO-d6)δ
(ppm): 43.55 (CH3), 45.36 (CH2), 56.48 (OCH3), 58.72 (2CH2), 59.83 (2CH2), 66.31 (CH2), arC: [115.35
(C), 124.15 (C), 126.05 (2C), 129.49 (2C), 130.89 (2C), 131.14 (2C), 131.38 (2C), 131.92 (C), 131.77 (C), 134.73
(2C), 137.10 (C), 157.65 (C)], 146.71 (CH), 148.45 (triazole C-3), 151.55 (triazole C-5), 164.66 (triazole C-3’),
167.54 (triazole C-5’); MS (ESI): m/z (%) 631 (M+1, 56), 606 (34), 518 (46), 229 (100).
Antimicrobial activity
All test microorganisms were obtained from the Refik Saydam Hıfzıssıhha Institute (Ankara, Turkey) and were as
follows: Escherichia coli ATCC 35218, Klebsiella pneumoniae ATCC 13883, Yersinia pseudotuberculosis ATCC
911, Enterobacter aerogenes ATCC 13048, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC
25923, Enterococcus faecalis ATCC 29212, Bacillus cereus 709 Roma, Candida tropicalis ATCC 13803, Candida
glabrata 66032, and Candida albicans ATCC 60193. All the 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.34
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) was used as standard antibacterial and
antifungal drugs. Dimethylsulphoxide at a dilution of 1:10 was used as solvent control.
Results and discussion
The synthesis of the intermediate and target compounds was performed according to the reactions outlined in
the Scheme. The starting compound, 4-amino-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (1), was
prepared following a previously reported literature procedure.35 The reaction of compound 1 with hydrazine
522

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
hydrate followed by the treatment of ethyl bromoacetate in basic media led to the formation of compound 2 in
a good yield. The 1 H-NMR spectrum of compound 2 exhibited 3 different signals integrating one proton that
are exchangeable with D2 O. The first one, which was observed at 4.46 ppm, belongs to the amino group of the
hydrazide structure (-NHNH2); the second one, which was recorded at 5.55 ppm, represents the amino group
at position 4 of the 1,2,4-triazole ring; and the third one, which was observed at 9.29 ppm, points to hydrazide
-NHNH2 . The condensation of compound 2 with some aromatic aldehydes, namely 2,6-dichlorobenzaldehyde
and 2-chloro-6-fluorobenzaldehyde, in absolute ethanol yielded the corresponding 2-(4-amino-3-(4-chlorophenyl)-
5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-Nꢀ-(2,6-dihalogenophenylmethylene)-acetohydrazides (3a, 3b). The
evidence for the formation of arylidenamino derivatives (3a, 3b) can be achieved by 1 H-NMR, 13 C-NMR, IR,
and mass spectroscopic methods and elemental analysis. In the 1 H-NMR spectra of compounds 3a and 3b,
additional signals that originated from arylidene moiety was observed in the aromatic region, and an additional
signal was recorded at 9.66 ppm derived from the –N=CH- bond, whereas the signal due to the –NHNH2 group
amino group disappeared. The presence of a peak at 5.62 (for 3a) and 5.65 (for 3b) ppm in the 1 H-NMR
spectra of compounds 3a and 3b indicated that only hydrazide-NH2 condensed with aromatic aldehydes in
the applied reaction conditions and the other amino group at position 4 of the 1,2,4-triazole ring remained
unchanged. In the 13 C-NMR spectra, a new signal belonging to the –N=CH group appeared at 154.65 (3a)
ppm or 153.81 (3b) ppm. Moreover, compounds 3a and 3b exhibited mass spectra and elemental analysis data
consistent with the assigned structures. The compounds having an arylidene-hydrazide structure may exist as
E/Z geometrical isomers about a –C=N double bond and cis/trans amide conformers.36−38 According to the
literature, the compounds containing an imine bond are present in higher percentages in dimethyl-d6 sulfoxide
solution in the form of geometrical E isomer about a –C=N double bond.36−38 The Z isomer can be stabilized
in less polar solvents by an intramolecular hydrogen bond. In the present study, the spectral data were obtained
in dimethyl-d6 sulfoxide solution and no signal belonging to Z isomer was observed. On the other hand, the
cis-trans conformers of E isomer were present in the dimethyl-d6 sulfoxide solution of compounds 3a and 3b.
In the 1 H-NMR spectra of compounds 3a,b, 2 sets of signals each belonging to the benzylic CH2 group of cis-
and trans- conformers were observed at 4.91 ppm and 5.00 ppm, respectively. The upfield lines of benzylic-CH2
protons were assigned to cis-conformer of the amide structure and downfield lines of the protons of the same
group to trans-conformer of the amide structure.
2-{[4-amino-3-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetyl}-N -phenylhydrazinecarbot-
hioamide (4) was obtained in high yield and purity by the nucleophilic addition of hydrazide-NH2 of compound
3 to phenylisothiocyanate. The reaction was carried out at reflux temperature in ethanol and afforded the
desired cabothioamide derivative (4), which was the starting material for further cyclizations. The IR spectrum
of compound 4 displayed a broad signal at 3269 cm−1 due to 3 NH and 1 NH2 absorptions. In the 1 H-NMR
spectra of compound 4, these groups were observed at 9.64, 9.78, 10.41, and 5.63 ppm, respectively.
The acidic treatment of compound 4 in cold conditions and then at room temperature resulted in the for-
mation of 4-amino-2-[(5-anilino-1,3,4-thiadiazol-2-yl)methyl]-5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazol-3-
one (5) in good yield. According to the literature, it is expected that the treatment of carbothioamides with
aqueous NaOH resulted in a cyclization leading to the formation of a 5-thioxo-1,2,4-triazole ring.38−42 Beside
this method, in another study performed in our laboratory,8 type 7 compounds were prepared by the heating
of the corresponding carbohioamides to 130 ◦ C in an oil bath for 2 h or refluxing type 4 compounds in ethanol
523

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
O
O
N NH
N
N
NHNH2
ArCHO
N
N
NH N CH Ar
O
OEt-, BrCH2CO2Et
N
NH2
1
1)
O
N
O
N
NH2
2)H2NNH2
NH2
CI
CI
2
CI
3a: Ar=
3b: Ar=
-C6H3Cl2(2,6-)
-C6H3ClF(2,6-)
PhNCS
O
NH
S
N
N
N
H
N
N
NHNH
S
N
N
NH
S
N
N
N
N
O
N
NaOH
H SO
2
4
O
NH2
N
O
N
CI
NH2
NH2
CI
7
CI
4
5
EtO-
EtO-, CH3I
(4-)CH3OC6H4CHO
N
CH3I
NH
N
N
N
N
N
N
S
CH3
N
SCH3
N
N
S
N
N
N
N
O
N
N
C
O
N
NH2
O
N
NH2
CI
H
CI
CI
6
8
9
OCH3
morpholine or
piperazine,
HCHO
R
N
N
S
N
N
N
O
N
N
C
N
N
O
10a: R=
10b: R=
CI
H
NH
OCH3
Scheme. Synthetic pathway for the preparation of compounds 2-10.
for 8 h. The yields of these 3 methods were very close to each other. In the 1 H-NMR spectra of compounds
5 and 7 and 8a,c,d,f the presence of only 1 signal for the –NH group integrating for 1 proton (exch. with
D2 O) confirmed the cyclization at the side chain of the triazole ring in compound 4. In the 13 C-NMR spectra
of compound 5, no signal due to the –C=S group was observed, while the signal derived from this group was
resonated at 168.31 ppm (triazole C-5) in the spectrum of compound 7. Furthermore, the 1 H- NMR spectrum
of compound 7 exhibited a singlet peak at 14.00 representing the presence of a –SH proton, while the 1 H-NMR
524

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
spectrum of compound 5 displayed a signal at 10.36 ppm pointing to an exocyclic –NH- group. These values
are consistent with the literature.4,19,32,38,42,43In addition, elemental analyses were consistent with the assigned
structures for compounds 5 and 7, and the mass spectra of the cyclization products (5, 7) showed molecular
ion peaks consistent with their molecular formulae.
It is interesting to note that compounds 7 are present in their thioxo-mercapto tautomeric forms in
solids as indicated by their IR spectra (presence of an absorption in the region of 2667 cm−1 for -SH stretch-
ing and presence of two absorption maxima at 1342 cm−1 characteristic of -C=S group in these type of
compounds.4,20,33,44 These tautomers were present also in dimethyl sulfoxide as suggested by NMR spectral
data.
The newly synthesized compounds 2-10 were evaluated in vitro for their antimicrobial activities. The
results are presented in the Table. The compounds having a substituted phenylmethylene-acetohydrazide struc-
ture, 3a and 3b, and 5, which contains a 1,3,4-thiadiazole nucleus, showed the most potent antimicrobial ac-
tivities against the test microorganisms. The replacement of the –NH- group in compound 5 by a hydrophobic
methyl group leading to compound 6 diminished the antimicrobial activity. Compound 7 demonstrated slight ac-
tivity against Enterobacter aerogenes (En), Staphylococcus aureus (Sa), Enterococcus faecalis (Ef), and Bacillus
careus (Bc). When compound 7 was converted into S-methyl derivative (8) no change was observed in antimicro-
bial activity. Similarly, for compound 9, the conversion of amino group into 4-methoxyphenylmethyleneamino
group caused no change in the antimicrobial activity. Comparison of the antimicrobial activity of 10a,b with
compound 9 indicates that the replacement of triazole-NH- proton by bulky morpholine or piperazine ring
resulted in no change in antimicrobial activity except for Bacillus careus (Bc).
Table. Antimicrobial activity of the compounds (μg/mL).
Compounds Microorganisms and Minimal Inhibition Concentration
No
2
Ec.
Kp.
Yp.
En.
Pa.
Sa.
Ef.
Bc.
> 250
< 1.95
< 1.95
125
> 500 > 500 > 500 > 250 > 500
250
250
3a
3b
4
< 1.95 < 1.95 < 1.95 < 1.95 < 1.95 < 1.95 < 1.95
< 1.95 < 1.95 < 1.95 < 1.95 < 1.95 < 1.95 < 1.95
> 500 > 500 > 500
< 1.95 < 1.95 < 1.95
62.5
3.90
7.81
125
> 500
< 1.95
500
125
1.95
15.63
125
125
125
500
500
35
125
7.81
125
125
125
125
500
500
10
5
3.90
1.95
125
6
> 500
500
250
7
> 500 > 500 > 500
> 500 > 500 > 500
> 500 > 500 > 500
> 500 > 500 > 500
> 500 > 500 > 500
> 500
> 500
> 500
> 500
> 500
18
8
125
125
9
125
62.5
125
10a
10b
Amp.
250
250
125
10
> 128
18
> 128
15
Ec.: Escherichia coli ATCC 35218, Kp.: Klebsiella pneumoniae ATCC 13883, Yp.: Yersinia pseudotuberculosis ATCC
911, En.: Enterobacter aerogenes ATCC 13048, Pa.: Pseudomonas aeruginosa ATCC 10145, Sa.: Staphylococcus aureus
ATCC 25923, Ef.: Enterococcus faecalis ATCC 29212, Bc.: Bacillus cereus 709 Roma, Amp.: Ampicillin.
525

Synthesis and antimicrobial activities of..., H. BEKTAS¸, et al.,
Acknowledgement
¨
˙
This work supported by the Scientific and Technological Research Council of Turkey (TUBITAK, Project no:
107T333).
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