Reduction, Mannich reaction, and antimicrobial activity evaluation of some new 1,2,4-triazol-3-one derivatives

Article abstract of DOI:10.3906/kim-1103-23

Ethyl[4-arylmethyleneamino-3-(4-metylphenyl)-5-oxo-4,5-dihydro-1H -1,2,4-triazole-1-yl]acetates (3a-e and 10a-d) were obtained starting from 4-amino-2,4-dihydro-3H -1,2,4-triazol-3-ones (1 and 9) in 2 steps. The treatment of 3a-e with NaBH₄ resulted in the formation of 3 kinds of product incorporating carboxcilic acid (4a-c) or alcohol (5a-e) functionality. [4-{[(4-Methoxyphenyl)methylene]amino}- and 4-{[pyridin-4- ylmethylene]amino}-3- (4-methylphenyl)-5-oxo-4,5-dihydro-1H -1,2,4-triazole-1-yl] acetic acids (6a, 6e) were obtained by the hydrolysis of the corresponding esters (5a-5e). The treatment of 6a with NaBH₄ caused the reduction of only the imine bond; the carboxyl group remained unchanged. Then this carboxylic acid was converted to the corresponding hydrazide (8) in 2 steps by reaction with ethanol and hydrazine hydrate, respectively. The synthesis of Mannich bases was performed by the reaction of the corresponding 1,2,4- triazoles containing an imine group (10a-d) with several primary or secondary amines including morpholine or piperazine nucleus. All newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial activity study revealed that the Mannich bases (11-16) showed good activity against the test microorganisms as compared with ampicillin. TUeBITAK.

Full text of DOI:10.3906/kim-1103-23

Turk J Chem
36 (2012) , 567 – 582.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1103-23
Reduction, Mannich reaction, and antimicrobial activity
evaluation of some new 1,2,4-triazol-3-one derivatives
1
1
2
1
˘
1,∗
Seda FANDAKLI , Serap BAS¸OGLU , Hakan BEKTAS¸ , Meltem YOLAL ,
3
˙
˘
Ahmet DEMIRBAS¸ , S¸engu¨l ALPAY KARAOGLU
1Department of Chemistry, Faculty of Sciences, Karadeniz Technical University,
61080 Trabzon-TURKEY
e-mail: ndemirbas651@mynet.com
2Department of Chemistry, Faculty of Sciences, Giresun University,
28100 Giresun-TURKEY
3Department of Biology, Faculty of Sciences, Rize University,
53100 Rize-TURKEY
Received: 11.03.2011
Ethyl[4-arylmethyleneamino-3-(4-metylphenyl)-5-oxo-4,5-dihydro-1H -1,2,4-triazole-1-yl]acetates (3a-e
and 10a-d) were obtained starting from 4-amino-2,4-dihydro-3H -1,2,4-triazol-3-ones (1 and 9) in 2 steps.
The treatment of 3a-e with NaBH4 resulted in the formation of 3 kinds of product incorporating carboxcilic
acid (4a-c) or alcohol (5a-e) functionality. [4-{[(4-Methoxyphenyl)methylene]amino}- and 4-{[pyridin-4-
ylmethylene]amino}-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1H -1,2,4-triazole-1-yl] acetic acids (6a, 6e) were
obtained by the hydrolysis of the corresponding esters (5a-5e). The treatment of 6a with NaBH4 caused
the reduction of only the imine bond; the carboxyl group remained unchanged. Then this carboxylic acid
was converted to the corresponding hydrazide (8) in 2 steps by reaction with ethanol and hydrazine hydrate,
respectively. The synthesis of Mannich bases was performed by the reaction of the corresponding 1,2,4-
triazoles containing an imine group (10a-d) with several primary or secondary amines including morpholine
or piperazine nucleus.
All newly synthesized compounds were screened for their antimicrobial activity. The antimicrobial
activity study revealed that the Mannich bases (11-16) showed good activity against the test microorganisms
as compared with ampicillin.
Key Words: 1,2,4-Triazole-3-one, reduction, hydrolysis, Mannich base, antimicrobial activity
∗Corresponding author
567

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
Introduction
The synthesis of high nitrogen-containing heterocyclic systems has been attracting increasing interest over
the past decade due to their utility in various applications, such as propellants, explosives, pyrotechnics, and
especially chemotherapy. In medicinal chemistry, azoles are a widely used and studied class of antimicrobial
agent due to their safety profile and high therapeutic index.1−7
The 1,2,4-triazole nucleus has been incorporated into a wide variety of therapeutically important agents.
Ribavirin (antiviral), rizatriptan (antimigraine), alprazolam (anxiolytic), vorozole, letrozole, and anastrozole
(antitumoral) are some examples of drugs containing 1,2,4-triazole moiety.8−12 Conazoles constitute another
class of azole-based drugs such as itraconazole, fluconazole, voriconazole, and ravuconazole.2−5 Some of the
other major applications of conazoles are for crop protection. As pharmaceuticals, they have been used for
the treatment of local and systemic fungal infections, which are important problems in phytopathology and
especially in medicine.
Substituted piperazines are important pharmacophores that constitutes a part of some important drugs
such as crixivan (an HIV protease inhibitor) and siprofloxacin (a potent antibacterial agent).13,14 The drugs
prazosin, lidoflazine, and urapidil, which contain a piperazine nucleus in their structures, have been used as
cardiovascular agents.15,16
Several compounds that contain a piperazine or morpholine nucleus possessing antimicrobial activity
have been synthesized, some of which contains an azole ring as well.17−22 For example, while eperezolid, which
is an oxazolidinone class antibiotic, consists of morpholine and oxazolidinone rings linked to each other via a
fluorophenilene linkage; another antibiotic, linezolid, contains a piperazine ring instead of morpholine.23,24 On
the other hand, itraconazole, posaconazole, and ketoconazole contain a piperazine and one or more azole rings
in their structures.25
Multi-component reactions (MCRs) have been considered an important method in organic synthesis with
the advantages ranging from lower reaction times, increased reaction rates to higher yields, and reproducibility.26
The Mannich reaction is a 3-component condensation reaction involving an active hydrogen containing com-
pound, formaldehyde, and a primary or secondary amine.26 The aminoalkylation of aromatic substrates by
Mannich reaction constitutes a major strategy for the preparation of several modifications of biologically active
compounds.27 Mannich bases are physiologically reactive due to the basic function making the molecule soluble
in aqueous solvents when it is transformed into aminium salt.15 Mannich bases have been reported as potential
biological agents. They find application as antitubercular,28 antimalarial,29 vasorelaxing, 30 anticancer,31 and
analgesic drugs.32 They have also found several applications in the polymer industry as paints and surface
active reagents.33
Mannich bases derived from 1,2,4-triazole compounds carryingN -methyl piperazine moiety have been
reported to possess protozocidal and antibacterial activity. The drugs prazosin, lidoflazine, and urapidil, which
contain a piperazine nucleus in their structures, have been used as cardiovascular agents.15,16,34
Imine compounds constitute another intensively synthesized and studied class of organic molecules due to
their different applications, such as biological activities,35−39 investigation of their ability to make a coordination
complex with transition metal cations, and improvement of their properties for analytical applications.40−42
Some Schiff base derivatives of 1,2,4-triazoles have been synthesized as antimicrobial and/or antitumoral agents
in our laboratories.36,43−50
568

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
Over 10 years our interest has focused on the synthesis of novel heterocyclic systems that have antimicro-
bial activity. In the present study, as a part of our ongoing study, we synthesized new 1,2,4-triazole derivatives
as possible antimicrobial agents, some of which contain different heterocyclic rings such as piperazine and
morpholine moieties.
Experimental
All the chemicals were purchased from Fluka Chemie AG Buchs (Switzerland) and were used without further
purification. Melting points of the synthesized compounds were determined in open capillaries on a Bu¨chi B-
540 melting point apparatus and are uncorrected. The reactions were monitored by thin-layer chromatography
(TLC) on silica gel 60 F254 aluminum sheets. The mobile phase was ethanol and ethyl acetate (1:1) and detection
was performed using UV light. IR spectra were recorded as potassium bromide pellets using a PerkinElmer
1600 series FTIR spectrometer. 1 H-NMR and 13 C-NMR spectra were recorded on a BRUKER AVANCE
II 400 MHz NMR Spectrometer (chemical shift in ppm downfield from TMS as an internal reference). The
elemental analysis was performed on a Costech Elemental Combustion System CHNS-O elemental analyzer.
All the compounds gave C, H and N analysis within 0.4% of the theoretical values. The mass spectra were
obtained on a Quattro LC-MS (70 eV) instrument.
General method for the preparation of compounds 2a-e and 10a-d
The corresponding compound 1 or 9 (10 mmol) was heated in an oil bath with a suitable aromatic aldehyde
(10 mmol) at 130-140 ◦ C for 2 h. On cooling the mixture to room temperature, a solid was obtained. This
crude product was recrystallized from ethanol solvent to obtain the desired compound.
4-{[(4-Methoxyphenyl)methylene]amino}-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-
3-one (2a). Yield: 2.52 g, 81%; mp 200-202 ◦ C; IR (KBr, cm−1): 3214 (NH), 1697 (C=O), 1605 (C=N);
1 H-NMR (DMSO-d6 δ ppm): 2.32 (s, 3H, CH3), 3.38 (s, 3H, OCH3), [ar-H: 7.02 (dd, 2H, J = 8.8 Hz), 7.28
(d, 2H, J = 8.2 Hz), 7.69-7.76 (m, 4H)], 9.46 (s, 1H, CH), 12.26 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm):
156.86 (N=CH), 151.74 (triazole C5), 144.84 (triazole C3), ar-C: [114.81 (2CH), 124.25 (C), 126.11 (C), 128.04
(2CH), 129.34 (2CH), 129 (2CH), 140.06 (C), 162.56 (C)], 55.67 (OCH3), 21.24 (CH3); Anal. calcd (%) for
C17 H16 N4 O2 : C, 66.22; H, 5.23; N, 18.17. Found: C, 66.28; H, 5.21; N, 18.10.
4-{[2-Furylmethylene]amino}-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (2b).
Yield: 1.81 g, 67%; mp 211-213 ◦ C; IR (KBr cm−1): 3162 (NH), 1698 (C=O), 1609 (C=N); 1 H-NMR (DMSO-
d6 δ ppm): 2.37 (s, 3H, CH3), [ar-H: 6.74 (dd, 1H, J1 = 4.0 Hz, J2 = 1.8 Hz), 7.20 (d, 1H, J = 3.4 Hz),
7.33 (d, 2H, J = 8.2 Hz), 7.77 (d, 2H, J = 8.4 Hz), 7.95 (d, 1H, J = 1.2 Hz)], 9.54 (s, 1H, N=CH), 12.35
(s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 146.77 (N=CH), 151.28 (triazole C5), 144.40 (triazole C3), ar-C:
[112.53 (CH), 118.00 (CH), 123.68 (C), 127.69 (2CH), 128.93 (2CH), 139.75 (C), 145.03 (CH), 148.18 (C)], 20.85
(CH3); LC-MS: m/z (%) [M]+ (%) 291 (100) [M+Na]+ , 269 (46) [M+1]+ ; 254 (16), 176 (35), 146 (22), 138
(16); Anal. calcd (%) for C14 H12 N4 O2 : C, 62.68; H, 4.51; N, 20.88. Found: C, 62.77; H, 4.63; N, 20.71.
5-(4-Methylphenyl)-4-{[phenylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-one (2c).
Yield: 0.82 g, 68%; mp 195-196 ◦ C; IR (KBr cm−1): 3176 (NH), 1695 (C=O), 1612 (C=N ); 1 H-NMR (DMSO-
d6 δ ppm): 2.38 (s, 3H, CH3), [ar-H: 7.36 (d, 2H, J = 8.4 Hz), 7.46 (m, 3H), 7.70-7.85 (m, 4H)], 9.58 (s,
569

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
1H, N=CH), 12.31 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 156.23 (N=CH), 151.24 (triazole C5), 144.56
(triazole C3), ar-C: [123.71(C), 127.70 (4CH), 129.00 (4CH), 131.55 (CH), 133.27 (C), 139.78 (C)], 20.88 (CH3);
Anal. calcd (%) for C16 H14 N4 O: C, 69.05; H, 5.07; N, 20.13. Found: C, 69.12; H, 5.18; N, 20.07.
5-(4-Methylphenyl)-4-{[pyridin-2-ylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-one
(2d). Yield: 1.81 g, 67%; mp 209-210 ◦ C; IR (KBr, cm−1): 3165 (NH), 1719 (C=O), 1584 (C=N); 1 H-NMR
(DMSO-d6 δ ppm): 2.39 (s, 3H, CH3), [ar-H: 7.35(d, 2H, J = 8.0 Hz), 7.48-7.58 (m, 1H), 7.81 (d, 2H, J = 8.0
Hz), 7.94 (d, 2H, J = 4.0 Hz), 8.71 (d, 1H, J = 4.8 Hz)], 9.75 (s, 1H, N=CH), 12.37 (s, 1H, NH); 13 C-NMR
(DMSO-d6 δ ppm): 149.82 (N=CH), 151.14 (triazole C5), 144.60 (triazole C3), ar-C: [120.31 (CH), 123.50 (C),
125.51 (CH), 127.92 (2CH), 128.99 (2CH), 137.14 (CH), 139.85 (C), 152.26 (C), 154.885 (CH)], 20.86 (CH3);
LC-MS: m/z (%) [M]+ 302 (66) [M+Na]+ , 280 (100) [M+1]+ ; 299 (12), 291 (9), 281 (17), 105 (17); Anal.
calcd (%) for C15 H13 N5 O: C, 64.51; H, 4.69; N, 25.07. Found: C, 64.73; H, 4.85; N, 24.86.
5-(4-Methylphenyl)-4-[(pyridin-4-ylmethylene)amino]-2,4-dihydro-3H-1,2,4triazol-3-one (2e).
Yield: 1.18 g, 43%; mp 271-273 ◦ C ; IR (KBr, cm−1): 3003 (NH), 1721 (C=O), 1603 (C=N); 1 H-NMR (DMSO-
d6 δ ppm): 2.39 (s, 3H, CH3), [ar-H: 7.36 (d, 2H, J = 7.8 Hz), 7.73-7.80 (m, 4H), 8.73 (d, 2H, J = 5.0 Hz)],
9.79 (s, 1H, N=CH), 12.44 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 152.72 (N=CH), 151.07 (triazole C5),
144.63 (triazole C3), ar-C: [121.33 (2CH), 123.412 (C), 127.944 (2CH), 129.042 (2CH), 139.458 (C), 140.536
(C), 150.47 (2CH)], 20.89 (CH3); LC-MS: m/z (%) [M]+ 302 (17) [M+Na]+ , 280 (32) [M+1]+ ; 280 (31), 229
(100), 146 (21),129 (70), 108 (15); Anal. calcd (%) for C15 H13 N5 O: C, 64.51; H, 4.69; N, 25.07. Found: C,
64.66; H, 4.65; N, 25.14.
4-{[2-Furylmethylene]amino}-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (10a). Yield: 1.63
g, 72%; mp 209-210 ◦ C ; IR (KBr, cm−1): 3182 (NH), 1701 (C=O), 1613 (C=N); 1 H-NMR (DMSO-d6
δ
ppm): 2.28 (s, 3H, CH3), [ar-H: 6.72 (d, 1H, J1 = 4.0 Hz), 7.02 (d, 1H, J = 4.0 Hz), 7.90 (d, 1H, J = 1.2
Hz)], 9.40 (s, 1H, N=CH), 12,18 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 148.63 (N=CH), 150.62 (triazole
C5), 143.88 (triazole C3), ar-C: [112.23 (CH), 118.68 (CH), 145.90 (CH), 149.42 (C)], 20.12 (CH3); Anal. calcd
(%) for C8 H8 N4 O2 : C, 50.00; H, 4.20; N, 29.15. Found: C, 50.12; H, 4.58; N, 29.22.
5-Methyl-4-{[phenylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-one (10b). Yield: 1.12
g, 75%; mp 201-202 ◦ C; IR (KBr, cm−1): 3198 (NH), 1710 (C=O), 1616 (C=N); 1 H-NMR (DMSO-d6
δ
ppm): 2.22 (s, 3H, CH3), [ar-H: 7.12-7.46 (m, 3H), 7.74-7.8846 (m, 2H)], 9.45 (s, 1H, N=CH), 12.28 (s, 1H,
NH); 13 C-NMR (DMSO-d6 δ ppm): 155.56 (N=CH), 150.65 (triazole C5), 144.28 (triazole C3), ar-C: [128.68
(2CH), 129.84 (2CH), 132.68 (C), 135.72 (C)], 21.42 (CH3); Anal. calcd (%) for C10 H10 N4 O: C, 59.40; H,
4.98; N, 27.71. Found: C, 59.35; H, 4.88; N, 27.65.
4-{[(2-Methoxyphenyl)methylene]amino}-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (10c).
Yield: 1.96 g, 80%; mp 189-190 ◦ C; IR (KBr, cm−1): 3222 (NH), 1701 (C=O), 1615 (C=N); 1 H-NMR (DMSO-
d6 δ ppm): 2.20 (s, 3H, CH3), 3.52 (s, 3H, OCH3), [ar-H: 7.04 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J = 8.4
Hz)], 9.32 (s, 1H, CH), 12.32 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 152.46 (N=CH), 150.74 (triazole C5),
143.18 (triazole C3), ar-C: [114.38 (2CH), 122.46 (C), 127.26 (2CH), 160.34 (C)], 55.88 (OCH3), 22.12 (CH3);
Anal. calcd (%) for C11 H12 N4 O2 : C, 56.89; H, 5.21; N, 24.12. Found: C, 56.75; H, 5.32; N, 24.22.
4-{[(4-Hydroxyphenyl)methylene]amino}-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (10d).
Yield: 2.31 g, 82%; mp 211-212 ◦ C; IR (KBr, cm−1): 3232 (NH), 1705 (C=O), 1618 (C=N); 1 H-NMR (DMSO-
570

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
d6 δ ppm): 2.19 (s, 3H, CH3), [ar-H: 7.08 (s, 1H), 7.10 (s, 1H), 7.62-7.78 (m, 2H)], 9.38 (s, 1H, CH), 10.36
(s, 1H, OH), 12.46 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 153.84 (N=CH), 152.62 (triazole C5), 145.92
(triazole C3), ar-C: [117.02 (CH), 119.42 (C), 121.62 (CH), 128.96 (CH), 142.60 (CH), 159.48 (C)], 23.56 (CH3).
Anal. calcd (%) for C10 H10 N4 O2 : C, 55.04; H, 4.62; N, 25.68. Found: C, 55.12; H, 4.58; N, 25.75.
General method for the synthesis of compounds 3a-e
The solution of the corresponding compound 2 (10 mmol) in absolute ethanol was stirred under reflux with
an equivalent amount of sodium for 2 h. Then ethyl bromoacetate (10 mmol) was added and the mixture was
refluxed for an additional 8 h. After evaporating the solvent under reduced pressure, a solid appeared. This
was recrystallized from ethanol/water (1:2) to afford the desired compound.
Ethyl [4-{[(4-methoxyphenyl)methylene]amino}-3-(4-metylphenyl)-5-oxo-4,5-dihydro-1H-1,
2,4-triazol-1-yl]acetate (3a). Yield: 2.52 g, 81%; mp 115-116 ◦ C; IR (KBr, cm−1): 1704, 1751 (C=O),
1606 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 1.24 (s, 3H, CH3), 3.79 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.19
(q, 2H, OCH2 , J = 7.0 Hz), 4.76 (s, 2H, N=CH2), [ar-H: 6.94 (d, 2H, 9Hz), 7.27 (d, 2H, 7.8 Hz), 7.72 (d,
2H, 8.6 Hz), 7.85 (d, 2H, 7.8 Hz)], 9.68 (s, 1H, N=CH); 13 C-NMR (DMSO-d6 δ ppm): 168.18 (C=O), 156.07
(N=CH), 151.04 (triazole C5), 145.43 (triazole C3), ar-C: [114.81 (2CH), 124.25 (C), 126.11 (C), 128.04 (2CH),
129.34 (2CH), 129 (2CH), 140.06 (C), 162.56 (C)], 61.21 (OCH2), 55.20 (OCH3), 46.58 (NCH2), 20.83 (CH3),
13.88 (ester CH3); Anal. calcd (%) for C21 H22 N4 O4 : C, 63.95; H, 5.62; N, 14.20. Found: C, 64.18; H, 5.77;
N, 14.26.
Ethyl (3-(4-methylphenyl)-5-oxo-4-{[2-furylmethylene]amino}-4,5-dihydro-1H-1,2,4-triazol-
1-il) acetate (3b). Yield: 3.3 g, 96.7%; mp 124-125 ◦ C; IR (KBr, cm−1): 1705, 1752 (C=O), 1609 (C=N);
1 H-NMR (DMSO-d6 δ ppm): 1.29 (t, 3H, CH3 , J = 7.2 Hz), 2.38 (s, 3H, CH3), 4.26 (q, 2H, OCH2 , J = 6.6
Hz), 4.66 (s, 2H, N=CH2), [ar-H: 6.53 (dd, 1H, J1 = 5 Hz, J2 = 1.2 Hz), 6.90 (d, 1H, J = 3.4 Hz), 7.27 (d,
2H, J = 8.4 Hz), 7.59 (s ,1H), 7.88 (d, 2H, J = 8.0 Hz)], 9.69 (s, 1H, N=CH); 13 C-NMR (DMSO-d6 δ ppm):
167.37 (C=O), 145.86 (N=CH), 150.72 (triazole C5), 144.46 (triazole C3), ar-C: [112.22 (CH), 116.82 (CH ),
123.22 (C), 128.40 (2CH), 129.15 (2CH), 140.69 (C), 145.01 (CH), 148.92 (C)], 61.92 (OCH2), 46.83 (NCH2),
21.51 (CH3), 14.16 (CH3); LC-MS: m/z (%) [M]+ 377 (100) [M+Na]+ , 355 (70) [M+1]+ ; 356 (11), 333 (12),
281 (30), 144 (47), 146 (28); Anal. calcd (%) for C18 H18 N4 O4 : C, 61.01; H, 5.12; N, 15.81. Found: C, 61.27;
H, 5.33; N, 15.69.
Ethyl (3-(4-methylphenyl)-5-oxo-4-{[pyridin-2-ylmethylene]amino}-4,5-dihydro-1H-1,2,4-
triazol-1-yl) acetate (3d): Yield: 1.72 g, 70%; mp 130-131 ◦ C; IR (KBr, cm−1): 1713, 1749 (C=O), 1508
(C=N); 1 H-NMR (DMSO-d6 δ ppm): 1.24 (t, 3H, CH3 , J = 7.0 Hz), 2.39 (s, 3H, CH3), 4.20 (q, 2H, OCH2 ,
J = 7.2 Hz), 4.77 (s, 2H, NCH2), [arH: 7.37 (d, 2H, J = 8.0 Hz), 7.44 (dd, 1H, J1 = 7.0 Hz, J2 = 4.4 Hz),
7.81 (d, 2H, J = 8.0 Hz), 7.96 (d, 2H, J = 3.6 Hz), 8.72 (d, 1H, J = 4.6 Hz)], 9.70 (s, 1H, NH); 13 C-NMR
(DMSO-d6 δ ppm): 167.46 (C=O), 149.88 (N=CH), 151.91 (triazole C5), 143.82 (triazole C3), ar-C: [120.56
(CH), 122.69 (C), 125.73 (CH), 128.0 (2CH), 129.13 (2CH), 137.22 (C), 140.37 (C), 149.66 (CH), 155.58 (CH)],
61.29 (OCH2), 46.58 (NCH2), 20.88 (CH3), 13.89 (CH3); LC-MS: m/z (%) [M]+ 388 (100) [M+Na]+ , 366
(50) [M+1]+ ; (367 (10), 153 (25), 105 (17); Anal. calcd (%) for C19 H19 N5 O3 : C, 62.46; H, 5.24; N, 19.17.
Found: C, 62.63; H, 5.48; N, 19.19.
Ethyl (3-(4-methylphenyl)-5-oxo-4-{[pyridin-4-ylmethylene]amino}-4,5-dihydro-1H-1,2,4-
571

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
triazol-1-yl) acetate (3e). Yield: 2.5 g, 69%; mp 157-160 ◦ C ; IR (KBr cm−1): 1709, 1740 (C=O), 1508
(C=N); 1 H-NMR (DMSO-d6 δ ppm): 1.32 (t, 3H, CH3 , J = 7.2 Hz), 2.44 (s, 3H, CH3), 4.28 (q, 2H,
OCH2 , J = 7.2 Hz), 4.67 (s, 2H, NCH2), [ar-H: 7.31 (d, 2H, J = 7.8 Hz), 7.63 (d, 2H, J = 4.6 Hz), 7.82
(d, 2H, J = 8.4 Hz), 8.73 (d, 2H, J = 5.0 Hz)], 9.93 (s, 1H, NH); 13 C-NMR (DMSO-d6 δ ppm): 167.20
(C=O), 149.98 (N=CH), 150.50 (triazole C5), 145.17 (triazole C3), ar-C: [121.80 (2CH), 122.90 (C), 128.58
(2CH), 129.24 (2CH), 141.06 (C), 141.60 (C), 151.84 (2CH), 62.06 (OCH2), 46.87 (NCH2), 21.57 (CH3), 14.25
(CH3); LC-MS: m/z (%) [M]+ 388 (100) [M+Na]+ , 366 (19) [M+1]+ ; 283 (20), 105 (51); Anal. calcd (%) for
C19 H19 N5 O3 : C, 62.46; H, 5.24; N, 19.17. Found: C, 62.48; H, 5.29; N, 19.11.
General method for the synthesis of compounds 4a-c and 5a-e
The solution of NaBH4 (10 mmol) in absolute ethanol was added to the solution of the corresponding compound
3 (10 mmol) in absolute ethanol, and the reaction mixture was allowed to reflux for 3 h. Then 50 mL of water
was added and the separated solid was collected by filtration and recrystallized from ethanol/water to afford
compound 5a-e. After neutralization of the filtrate by HCl, a solid appeared. This was filtered off, washed with
water, and recrystallized from (0.9 g, 24%) ethanol/water (1:2) to give compounds 4a-c.
[4-[(4-Methoxybenzyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihidro-1H-1,2,4-triazol-1-yl]
acetic acid (4a). Yield: 0.9 g, 24%; mp 197-198 ◦ C; IR (KBr, cm−1): 3285 (NH), 1666, 1763 (C=O), 1613
(C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.40 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 4.11 (d, 2H, NHCH2 , J = 4.8
Hz), 4.56 (s, 2H, NCH2), 5.57 (t, 1H, NH, J = 4.8 Hz), [ar-H: 6.70 (d, 2H, J = 8.8 Hz), 7.11 (d, 2H, J = 8.8
Hz), 7.16 (d, 2H, J = 8.4 Hz), 7.73 (d, 2H, J = 8.4 Hz)], 11.24 (s, 1H, OH); 13 C-NMR (DMSO-d6 δ ppm):
169.10 (C=O), 153.26 (triazole C5), 144.98 (triazole C3), ar-C: [113.48 (2CH), 123.33 (C), 127.45 (2CH), 127.54
(C), 128.62 (2CH), 130.56 (2CH), 139.86 (C), 158.95 (C)], (55.02 (OCH3), 53.26 (NCH2), 46.45 (NHCH2),
21.33 (CH3); LC-MS: m/z (%) 391 (100) [M+Na]+, 369 [M+1]+; 377 (25), 369 (30), 229 (98), 191 (13); Anal.
calcd (%) for C19 H20 N4 O4 : C, 61.95; H, 5.47; N, 15.21. Found: C, 62.17; H, 5.53; N, 15.20.
[4-[(2-Furylmethyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazo-1-yl]acetic
acid (4b). Yield: 1.26 g, 37%; mp 138-141 ◦ C ; IR (KBr, cm−1): 3290 (NH) 1696, 1737 (C=O), 1506 (C=N);
1 H-NMR (DMSO-d6 δ ppm): 2.31 (s, 3H, CH3), 4.53 (s, 2H, NHCH2), 4.18 (d, 2H, NCH2 , J = 3.6 Hz),
6.76 (t, 1H, J = 3.8 Hz), [ar-H: 6.13 (d, 1H, J = 3.2 Hz), 6.23 (dd, 1H, J1 = 4H, J2 = 2.2 Hz), 7.19 (d, 2H,
J = 8.2 Hz), 7.40 (d, 1H, J = 1.8 Hz), 7.65 (d, 2H, J = 8.0 Hz)], 10.95 (s, 1H, OH); 13 C-NMR (DMSO-d6
δ ppm): 169.90 (C=O), 153.57 (triazole C5), 145.30 (triazole C3), ar-C: [109.99 (CH), 111.05 (CH), 123.99
(C), 127.74 (2CH), 129.39 (2CH), 140.26 (C), 143.40 (CH), 150.81 (C)], 47.48 (NCH2), 45.38 (NHCH2), 21.64
(CH3); LC-MS: m/z (%) 351 (100) [M+Na]+, 329 (87) [M+1]+; M/Z 316 (9), 315 (59), 220 (13), 176 (20),
153 (32), 152 (36), 146 (53), 138 (38), 123 (32); Anal. calcd (%) for C16 H16 N4 O4 : C, 58.53; H, 4.91; N, 17.06.
Found: C, 58.57; H, 5.14; N, 17.09.
[4-Benzylamino-3-(4-methylphfenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetic acid (4c).
Yield: 1.1 g, 31%; mp 185-186 ◦ C; IR (KBr cm−1): 3265 (NH), 1677, 1737 (C=O), 1503 (C=N); 1 H-NMR
(DMSO-d6 δ ppm): 2.40 (s, 3H, CH3), 4.20 (d, 2H, NHCH2 , J = 4.8 Hz), 4.56 (s, 2H, NCH2), 5.77 (t, 1H,
NH, J = 4.8 Hz), [ar-H: 7.17-7.23 (m, 7H), 7.75 (d, 2H, J = 8.0 Hz)], 10.98 (s, 1H, OH); 13 C-NMR (DMSO-d6
δ ppm): 169.18 (C=O), 153.24 (triazole C5), 144.90 (triazole C3), ar-C: [123.36 (C), 127.43 (2CH), 127.56
572

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
(CH), 128.14 (2CH), 128.66 (2CH), 129.20 (2CH), 135.61 (C), 139.85 (C)], 53.74 (NCH2), 46.82 (NHCH2),
21.30 (CH3); LC-MS: m/z (%) 361 (100) [M+Na]+ , 339 (90) [M+1]+ ; 340 (19), 149 (18), 106 (15); Anal.
calcd (%) for C18 H18 N4 O3 : C, 63.90; H, 5.36; N, 16.56. Found: C, 64.16; H, 5.41; N, 16.54.
2-(2-Hydroxyethyl)-4-[(4-methoxybenzyl)amino]-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-
triazol-3-one (5a). Yield: 2.5 g, 66%; mp 104-105 ◦ C; IR (KBr, cm−1): 3263 (NH), 1699 (C=O), 1613
(C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.40 (s, 3H, CH3), 3.48 (brs, 1H, OH), 3.77 (s, 3H, OCH3), 4.09 (d, 2H,
NHCH2 , J = 5.4 Hz), 3.93-4.10 (m, 4H, NCH2 CH2), 4.98 (t, 1H, NH, J = 5.6 Hz), [ar-H: 7.75 (d, 2H, J = 7.6
Hz), 7.10-7.25 (m, 4H), 7.80 (d, 2H, J = 7.5 Hz)]; 13 C-NMR (DMSO-d6 δ ppm): 153.57 (triazole C5), 144.71
(triazole C3), ar-C: [113.77 (2CH), 123.23 (C), 127.29 (C), 127.33 (2CH), 128.87 (2CH), 130.61 (2CH), 140.39
(C), 159.31 (C)], 61.24 (CH2 OH), 55.20 (OCH3), 54.00 (NCH2), 48.92 (NHCH2), 21.47 (CH3); LC-MS: m/z
(%) 378 (100) [M+ Na]+ , 355 (9) [M+1]+ ; 355 (13), 229 (13); Anal. calcd (%) for C19 H22 N4 O3 : C, 64.39;
H, 6.26; N, 15.81. Found: C, 64.56; H, 6.41; N, 15.77.
4-[(2-Furylmethyl)amino]-2-(2-hydroxyethyl)-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-tri-
azol-3-one (5b). Yield: 2.3 g, 71%; mp 70-74 ◦ C; IR (KBr, cm−1): 3292 (NH), 1697 (C=O), 1507 (C=N);
1 H-NMR (DMSO-d6 δ ppm): 2.39 (s, 3H, CH3), 3.76 (brs, 1H, OH), 4.01-4.06 (m, 4H, NCH2 CH2), 4.22 (d,
2H, NHCH2 , J = 4.8 Hz), 5.09 (t, 1H, NH, J = 4.8 Hz), [ar-H: 6.15-6.22 (m, 2H), 7.19 (s, 1H), 7.24 (d, 2H,
J = 7.2 Hz), 7.79 (d, 2H, J = 8.4 Hz)]; 13 C-NMR (DMSO-d6 δ ppm): 153.76 (triazole C5), 145.03 (triazole
C3), ar-C: [110.08 (CH), 110.53 (CH), 123.23 (C), 127.52 (2CH), 129.23 (2CH), 140.62 (C), 143.00 (CH), 149.50
(C)], 61.10 (CH2 OH), 49.13 (NCH2), 46.88 (NHCH2), 21.69 (CH3); LC-MS: m/z (%) 337 (100) [M+Na]+ ,
315 (8) [M+1]+ ; 338 (19), 315 (8); Anal. calcd (%) for C16 H18 N4 O3 : C, 61.14; H, 5.77; N, 17.82. Found: C,
61.33; H, 5.92; N, 17.67.
2-(2-Hydroxyethyl)-4-benzylamino-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one
(5c). Yield: 2.03 g, 62%; mp 128-129 ◦ C; IR (KBr, cm−1): 3236, 3285 (NH), 1703 (C=O), 1528 (C=N);
1 H-NMR (DMSO-d6 δ ppm): 2.40 (s, 3H, CH3), 4.17 (d, 2H, NHCH2 , J = 5.6 Hz), 3.95-4.10 (m, 4H,
NCH2 CH2), 4.97 (t, 1H, NH, J = 5.8 Hz), [ar-H: 7.20-7.30 (m, 7H), 7.75 (d, 2H, J = 8.0 Hz)], 3.45 (brs.,
OH); 13 C-NMR (DMSO-d6 δ ppm): 153.24 (triazole C5), 144.90 (triazole C3), ar-C: [123.36 (C), 127.43
(2CH), 127.56 (CH), 128.14 (2CH), 128.66 (2CH), 129.20 (2CH), 135.61(C), 139.85 (C)], 60.93 (CH2 OH), 53.74
(NCH2), 46.82 (NHCH2), 21.30 (CH3); LC-MS: m/z (%) 347 (100) [M+Na]+ , 325 (58) [M+1]+ ; 149 (9), 119
(7); Anal. calcd (%) for C18 H20 N4 O2 : C, 66.65; H, 6.21; N, 17.27. Found: C, 66.78; H, 6.27; N, 17.19.
2-(2-Hydroxyethyl)-5-(4-methylphenyl)-4-[(pyridin-2-ylmethyl)amino]-2,4-dihydro-3H-1,2,4-
triazol-3-one (5d). Yield: 1.93 g, 62%; mp 112-114 ◦ C; IR (KBr, cm−1): 3269 (NH), 1699 (C=O), 1596
(C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.33 (s, 3H, CH3), 4.29 (d, 2H, NHCH2 , J = 5.5 Hz), 3.62-3.83 (m,
4H, NCH2 CH2), 6.83 (t, 1H, J = 5.2 Hz, NH), [ar-H: 7.17-7.27 (m, 4H), 7.61-7.75 (m, 1H), 7.728 (d, 2H,
J = 8.4 Hz), 8.42 (d, 1H, J = 4.6 Hz)], 4.86 (brs., OH); 13 C-NMR (DMSO-d6 δ ppm): 152.65 (triazole C5),
143.79 (triazole C3), ar-C: [122.30 (CH), 122.79 (CH), 123.54 (C), 127.05 (2CH), 128.56 (2CH), 136.19 (CH),
139.23 (C), 148.61 (CH), 156.24 (C)], 47.50 (CH2 OH), 58.29 (NCH2), 54.12 (NHCH2), 20.74 (CH3); LC-MS:
m/z(%) 327 (9), 326 (38), 229 (13), 345 (6); Anal. calcd (%) for C17 H19 N5 O2 : C, 62.76; H, 5.89; N, 21.52.
Found: C, 62.91; H, 5.95; N, 21.44.
573

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
2-(2-Hydroxymethyl)-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (5e). Yield: 1.39
g, 63%; mp 243-245 ◦ C; IR (KBr, cm−1): 3383 (NH), 1694 (C=O), 1513 (C=N); 1 H-NMR (DMSO-d6 δ ppm):
2.34 (s, 3H, CH3), 3.66-3.74 (m, 4H, NCH2 CH2), 12.11 (s, 1H, NH), [ar-H: 7.67 (d, 2H, 8.2 Hz), 7.29 (d, 2H, 7.3
Hz)], 3.42 (brs., OH); 13 C-NMR (DMSO-d6 δ ppm): 154.46 (triazole C5), 143.31 (triazole C3), ar-C: [123.89
(C), 124.56 (2CH), 129.40 (2CH), 139.56 (C)], 46.68 (CH2 OH), 58.50 (NCH2), 20.84 (CH3); LC-MS: m/z(%)
342 (41), 328 (38), 230 (24); Anal. calcd (%) for C11 H13 N3 O2 : C, 60.26; H, 5.98; N, 19.17. Found: C, 60.32;
H, 5.86; N, 19.22.
General method for the synthesis of compounds 6a, 6e
The suspension of compound 2a (10 mmol, 3.94 g) in 3 M HCl (50 mL) was refluxed for 3 h. On cooling the
reaction mixture to room temperature, a white solid appeared. This was recrystallized from ethanol to give the
pure compound.
[4-{[(4-Methoxyphenyl)methylene]amino}-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl] acetic acid (6a). Yield: 2.96 g, 80%; mp 218-222 ◦ C; IR (KBr, cm−1): 1691, 1758 (C=O),
1573 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.37 (s, 3H, CH3), 3.49 (brs, OH), 3.83 (s, 3H, OCH3), 4,63 (s,
2H, NCH2), [ar-H: 7.07 (d, 2H, J = 8.6 Hz), 7.33 (d, 2H, J = 8.2 Hz), 7.76-7.81 (m, 4H)], 9.49 (s, 1H, N=CH);
13 C-NMR (DMSO-d6 δ ppm): 169.01 (C=O), 162.11 (triazole C5), 143.44 (triazole C3), ar-C: [114.48 (2CH),
123.15 (C), 125.42 (C), 127.73 (2CH), 129.76 (2CH), 130.56 (2CH), 140.10 (C), 149.91 (C)], 157.19 (N=CH),
46.68 (NCH2), 55.32 (OCH3), 20.87 (CH3); LC-MS: m/z (%) 389 (9) [M+Na]+ , 367 (100) M+1)+ ; 133 (30),
187 (61), 233 (70), 321 (13), 368 (25); Anal. calcd (%) for C19 H18 N4 O4 : C, 62.29; H, 4.95; N, 15.29. Found:
C, 62.43; H, 5.16; N, 15.12.
(3-(4-Methylphenyl)-5-oxo-4-{[pyridin-4-ylmethylene]amino}-4,5-dihydro-1H-1,2,4-triazol-
1-yl) acetic acid (6e). Yield: 3 g, 90%; mp > 290 ◦ C; IR (KBr, cm−1): 1707 (C=O), 1617 (C=N); 1 H-NMR
(DMSO-d6 δ ppm): 2.39 (s, 3H, CH3), 3.43 (brs, OH), 4.65 (s, 2H, NCH2), [ar-H: 7.37 (d, 2H, J = 7.8 Hz),
7.74-7.81 (m, 4H), 8.74 (d, 2H, J = 6.0 Hz)], 9,75 (s, 1H, N=CH); 13 C-NMR (DMSO-d6 δ ppm): 168.86
(C=O), 149.56 (triazole C5), 147.76 (triazole C3), ar-C: [121.45 (2CH), 122.69 (C), 128.00 (2CH), 129.16 (2CH)
139.21 (C), 140.39 (C), 150.42 (2CH)], 153.53 (N=CH), 46.67 (NCH2), 20.88 (CH3); LC-MS: m/z (%) 360
(8) [M+Na]+ , 338 (66) [M+1]+ ; 152 (15), 187 (9), 273 (31), 339 (14); Anal. calcd (%) for C17 H15 N5 O3 : C,
60.53; H, 4.48; N, 20.76. Found: C, 60.58; H, 4.57; N, 20.62.
{4-[(4-Methoxybenzyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}
acetate (7). The solution of compound 4a in absolute ethanol was stirred under reflux conditions in the
presence of a catalytic amount of conc. sulfuric acid for 4 h. On cooling the reaction mixture to room
temperature a white solid was obtained. This crude product was collected by filtration, washed with water, and
recrystallized from ethanol to give the pure compound. Yield: 3.25 g, 79%; mp 119-120 ◦ C; IR (KBr, cm−1):
3406, 3293 (NH), 1704, 1751 (C=O), 1617 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 1.30 (t, 3H, J = 7.6 Hz,
CH3), 2.40 (s, 3H, CH3), 3.77 (s, 3H, CH3), 4.12 (s, 2H, NHCH2), 4.25 (q, 2H, OCH2 , J = 7.0 Hz), 4.63 (s,
2H, NCH2), [ar-H: 6.75 (m, 3H, J = 8.6 Hz + NH), 7.15-7.26 (m, 4H), 7.78 (d, 2H, J = 8.2 Hz)]; 13 C-NMR
(DMSO-d6 δ ppm): 167.51 (C=O), 153.38 (triazole C5), 145.23 (triazole C3), ar-C: [113.82 (2CH), 123.33 (C),
127.31 (2CH), 127.58 (C), 128.95 (2CH), 130.77 (2CH), 140.43 (C), 159.38 (C)], 61.85 (OCH2), 55.26 (OCH3),
574

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
54.19 (NCH2), 47.10 (NHCH2), 21.53 (CH3), 20.12 (CH3); Anal. calcd (%) for C21 H24 N4 O4 : C, 63.62; H,
6.10; N, 14.13. Found: C, 63.70; H, 6.18, N, 14.11.
Synthesis of 2-[4-[(4-methoxybenzyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetohydrazide (8). Hydrazine hydrate (25 mmol) was added to the solution of compound 7 in
ethanol and the reaction mixture was allowed to reflux for 3 h. Then the resulting solution was kept overnight
in cold conditions. The separated solid was collected by filtration and recrystallized from ethanol to yield the
target compounds. Yield: 3.2 g, 87%; mp 173-174 ◦ C; IR (KBr, cm−1): 3264, 3318 (NH), 1691, 1710, 1746
(C=O), 1611 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.34 (s, 3H, CH3), 3.68 (s, 3H, OCH3), 4.08 (d, 2H, J =
4.0 Hz, NHCH2), 4.35 (s, 2H, NCH2), 6.74 (t, 3H, NH+NH2), 9.30 (s, NH), [ar-H: 6.74 (d, 2H, J = 9.0 Hz),
7.09 (d, 2H, J = 8.6 Hz), 7.22 (d, 2H, J = 8.2 Hz), 7.66 (d, 2H, J = 8.2 Hz)]; 13 C-NMR (DMSO-d6 δ ppm):
165.70 (C=O), 152.92 (triazole C5), 145.03 (triazole C3), ar-C: [113.38 (2CH), 119.50 (C), 127.13 (2CH), 128.17
(C), 128.57 (2CH), 130.19 (2CH), 137.50 (C), 159.70 (C)], 54.86 (OCH3), 51.84 (NCH2), 41.82 (NHCH2),
20.85 (CH3); Anal. calcd (%) for C19 H22 N6 O3 : C, 59.67; H, 5.80; N, 21.98. Found: C, 59.81; H, 5.81; N,
21.91.
General method for the synthesis of compounds 11-16
The suitable amine (10 mmol) was added to the solution of the corresponding compound 10 (10 mmol) in water
and the reaction content was stirred at room temperature in the presence of formaldehyde solution (20 mmol)
for 5 h. Then, the separated solid was filtered off and recrystallized from benzene-petroleum ether (1:2) (for
11-14) or acetone (for 15 and 16) to yield the target compound.
2-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-4-[(2-furylmethylene)amino]-5-methyl-2,4-
dihydro-3H-1,2,4-triazol-3-one (11). Yield: 3.92 g, 85%; mp 150 ◦ C; IR (KBr, cm−1): 1712 (C=O), 1508
(C=N), 1316 (C-N); 1 H-NMR (DMSO-d6 δ ppm): 2.38 (s, 3H, CH3), 2.81 (brs, 4H, 2CH2), 3.08 (brs, 4H,
2CH2), 4.75 (s, 2H, CH2), [ar-H: 6.73-6.75 (m, 1H), 6.89-7.07 (m, 1H), 7.24-7.26 (m, 1H), 7.32-7.37 (m, 2H),
7.78-7.82 (m, 2H)], 9.51 (s, 1H, N=CH); 13 C-NMR (DMSO-d6 δ ppm): 21.69 (CH3), 49.75 (2CH2), 50.20
(2CH2), 66.77 (CH2), arC: [113.42 (CH), 116.13 (CH), 118.01 (CH), 119.23 (CH), 124.00 (C), 127.49 (CH),
128.69 (CH), 140.85 (C), 146.49 (CH), 148.67 (C)] 151.16 (triazole C5), 154.40 (N=CH), 159.09 (triazole C3);
MS (ESI): m/z (%) 100.78 (100), 116.77 (70), 180.91 (78), 399.22 (13); Anal. calcd (%) for C19 H21 FN6 O2 :
C, 59.37; H, 5.51; N, 21.86. Found: C, 59.48; H, 5.63; N, 21.74.
2-[(4-(4-Fluorophenyl)piperazin-1-yl)methyl]-4-[(phenylmethylene)amino]-5-Methyl-2,4-
dihydro-3H-1,2,4-triazol-3-one (12). Yield: 4.34 g, 90% ; mp 136 ◦ C; IR (KBr, cm−1): 1702 (C=O),
1508 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.48 (s, 3H, CH3), 2.86 (brs, 4H, 2CH2), 3.14 (brs, 4H, 2CH2),
4.65 (s, 2H, CH2), [ar-H: 6.72 (s, 2H), 6.82-7.12 (m, 2H), 7.34-7.46 (m, 3H), 7.53-7.74 (m, 2H)], 9.62 (s, 1H,
N=CH); 13 C-NMR (DMSO-d6 δ ppm): 20.47 (CH3), 48.20 (2CH2), 51.56 (2CH2), 68.47 (CH2), arC: [112.18
(CH), 115.58 (2CH), 120.85 (CH), 123.76 (C), 125.78 (CH), 130.60 (2CH), 143.56 (C), 145.92 (CH), 147.34
(CH), 148.46 (C)] 151.22 (triazole C5), 155.64 (N=CH), 159.03 (triazole C3); MS (ESI): m/z (%) 100.78 (100),
116.77 (70), 180.91 (78), 394.22 [M+ ], (13); Anal. calcd (%) for C21 H23 FN6 O: C, 63.94; H, 5.88; N, 21.31.
Found: C, 63.82; H, 5.78; N, 21.28.
4-{[(4-Methoxyphenyl)methylene]amino}-5-methyl-2-(morpholin-4-ylmethyl)-2,4-dihydro-3
575

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
H -1,2,4-triazol-3-one (13). Yield: 2.28 g, 68%; mp 152 ◦ C; IR (KBr cm−1): 1701 (C=O), 1438 (C=N),
1116 (C-O); 1 H-NMR (DMSO-d6 δ ppm): 2.28 (s, 3H, CH3), 2.56 (brs, 4H, 2CH2), 3.27 (brs, 4H, 2CH2),
3.81 (s, 3H, OCH3), 4.54 (s, 2H, CH2), [ar-H: 7.02-7.06 (m, 2H), 7.72-7.75 (m, 2H)], 9.53-9.54 (s, 1H, N=CH);
13 C-NMR (DMSO-d6 δ ppm): 20.21 (CH3), 50.61 (2CH2), 56.10 (OCH3), 62.36 (CH2), 66.69 (2CH2), arC:
[116.42 (CH), 128.56 (CH), 129.14 (CH), 130.33 (CH), 132.38 (C), 145.23 (C)], 151.01 (triazole C5), 155.04
(N=CH), 162.70 (triazole C3); MS (ESI): m/z (%) 100.09 (56), 127.80 (100), 128.80 (28), 148.81 (20), 383.18
(35), 399.20 (59); Anal. calcd (%) for C16 H21 N5 O3 : C, 57.99; H, 6.39; N, 21.13. Found: C, 57.82; H, 6.43; N,
21.18.
4-{[(2-Hydroxyphenyl)methylene]amino}-5-methyl-2-(morpholin-4-ylmethyl)-2,4-dihydro-3
H -1,2,4-triazol-3-one (14). Yield: 1.16 g, 38%; mp 170 ◦ C IR (KBr, cm−1): 1702 (C=O), 1419 (C=N),
1112 (C-O); 1 H-NMR (DMSO-d6 δ ppm) 2.42 (s, 3H, CH3), 2.51 (brs, 4H, 2CH2), 3.48 (brs, 4H, 2CH2),
4.46 (s, 2H, CH2), 4.73 (s, 1H, OH), [ar-H: 6.88 (m, 2H) 7.26 (brs, 1H), 7.77 (brs, 1H)], 9.86 (m, 1H, N=CH);
13 C-NMR (DMSO-d6 δ ppm): 11.63 (CH3), 50.22 (2CH2), 52.20 (CH2), 66.48 (2CH2), arC: [116.92 (CH),
120.22 (CH), 127.03 (CH), 133.71 (CH), 143.80 (C), 150.94 (C)], 152.11 (triazole C5), 158.08 (N=CH), 163.26
(triazole C3); LC-MS: m/z (%) 127.73 (28), 131.80 (18), 155.82 (23), 178.84 (100), 187.84 (52), 227.87 (91),
228.94 (18), 318.01 ([M+1]+ , 21), 356.04 ([M+K]+ , 12); Anal. Calcd (%) for C15 H19 N5 O3 : C, 56.77; H, 6.03;
N, 22.07. Found: C, 56.62; H 6.17; N, 22.12.
4-{[(2-Hydroxyphenyl)methylene]amino}-5-methyl-2-{[(2-morpholin-4-ylethyl)amino]met-
hyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (15). Yield: 1.28 g, 25%; mp 131 ◦ C; IR (KBr, cm−1): 1707
(C=O), 1419 (C=N), 1114 (C-O); 1 H-NMR (DMSO-d6 δ ppm): 2.12 (s, 3H, CH3), 2.16 (brs, 4H, 2CH2), 2.44
(brs, 2H, CH2), 3.19 (brs, 4H, 2CH2), 3.45 (brs, 2H, CH2), 4.74 (s, 2H, NCH2 N), 4.94 (s, 1H, OH), [ar-H:
6.87 (brs, 2H), 7.29 (brs, 1H), 7.75 (brs, 1H)], 9.87 (s, 1H, N=CH), 10.27 (s, 1H, OH); 13 C-NMR (DMSO-d6
δ
ppm): 11.77 (CH3), 53.71 (2CH2), 57.71 (CH2), 66.62 (2CH2), 67.70 (2CH2), arC: [117.08 (CH), 120.05 (C),
127.03 (CH), 130.44 (CH), 133.80 (CH), 143.90 (C)], 151.74 (triazole C-5), 152.15 (N=CH), 158.18 (triazole
C-3); MS (ESI): m/z (%) 100.71 (76), 113.72 (75), 130.80 (100), 154.82 (25), 170.83 (21), 186.84 (40), 373.05
(40); Anal. calcd (%) for C17 H24 N6 O3 : C, 56.65; H, 6.71; N, 23.32. Found: C, 56.63; H, 6.48; N, 23.47.
4-{[(2-Hydroxyphenyl)methylene]amino}-5-methyl-2-[(4-methylpiperazin-1-yl)methyl]-2,4-
dihydro-3H-1,2,4-triazol-3-one (16). Yield: 2.62 g, 78%; mp 154 ◦ C; IR (KBr, cm−1): 1698 (C=O),
1429 (C=N); 1 H-NMR (DMSO-d6 δ ppm): 2.11 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.48 (brs, 4H, 2CH2), 2.63
(s, 2H, CH2), 3.37 (brs, 4H, 2CH2), 4.64 (s, 1H, OH), [ar-H: 6.92-6.96 (m, 1H), 7.31-7.35 (m, 1H), 7.73-7.77
(m, 2H)], 9.85 (s, 1H, N=CH); 13 C-NMR (DMSO-d6 δ ppm): 21.69 (CH3), 46.42 (CH3), 50.06 (2CH2),
55.20 (2CH2), 66.79 (CH2), arC: [127.11 (CH), 128.76 (CH), 129.83 (CH), 119.95 (CH), 133.38 (C), 140.80
(C)], 151.16 (triazole C5), 154.41 (N=CH), 158.49 (triazole C3); MS (ESI): m/z (%) 113.83 (54), 152.78 (85),
228.95 (25), 246.14 (100), 284.24 (53), 370.15 ([M+1+K]+ , 10), 373.22 (93), 447.36 (31); Anal. calcd (%) for
C16 H22 N6 O2 : C, 58.17; H, 6.71; N, 25.44. Found: C, 58.22; H, 6.68; N, 25.57.
Antimicrobial activity
All test microorganisms were obtained from the Refik Saydam Hygiene Institute (Ankara, Turkey) and were
as follows: E. coli ATCC35218, E. aerogenes ATCC13048, Y. pseudotuberculosis ATCC911, P. aeruginosa
576

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
ATCC43288, S. aureus ATCC25923, E. faecalis ATCC29212, B. cereus 709 Roma, C. albicans ATCC60193,
C. tropicalis ATCC 13803, A. niger RSKK 4017, and S. cerevisiae RSKK 251. All the newly synthesized
compounds were weighed and dissolved in dimethylsulphoxide to prepare extract stock solution of 5.000 mg/mL.
A screening test using agar-well diffusion51 as adapted earlier52 was used for all newly synthesized compounds.
Each microorganism was suspended in Mueller Hinton (MH) (Difco, Detroit, MI, USA) broth and diluted
approximately to 106 colony forming units (cfu)/mL. They were flood-inoculated onto the surface of MH agar
and Sabouraud Dextrose Agar (SDA) (Difco) and then dried. For C. albicans and C. tropicalis, SDA was used.
Five millimeter diameter wells were cut from the agar using a sterile cork-borer, and 50 mL of the extract
substances was delivered into the wells. The plates were incubated for 18 h at 35 ◦ C. Antimicrobial activity
was evaluated by measuring the zone of inhibition against the test organism. Ampicillin (10 mg) and fluconazole
(5 mg) were the standard drugs. Dimethyl sulfoxide was used as solvent control. The antimicrobial activity
results are summarized in the Table.
Table. Screening for antimicrobial activity of compounds 11-16 (mm).
Compound
Microorganisms and inhibition zone (mm)
no.
11
Ec Ea
18 15
16 12
15 13
12 12
20 18
16 16
Yp
16
15
11
12
20
14
Pa
15
13
10
12
20
14
Sa Ef Bc Ca Sc
16 12 16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12
15
10
14
8
6
-
15
12
-
13
14
15
20 16 22
12 15
16
8
Amp.
Flu
10 10 > 18 > 18 35 10 15
25 25
Ec: Escherichia coli ATCC 25922, Ea: Enterobacter aeruginosa ATCC 13048, Yp: Yersinia pseudotuberculosis ATCC
911, Pa: Pseudomonas aeruginosa ATCC 43288, Sa: Staphylococcus aureus ATCC 25923, Ef: Enterococcus faecalis
ATCC 29212, Bc: Bacillus cereus 702 Roma, Ca: Candida albicans ATCC 60193, Saccharomyces cerevisiae RSKK 251,
4017, Amp.: Ampicillin, Flu.: Flukonazol, (-): No Activity.
Result and discussion
Synthesis of the intermediate and target compounds was performed according to the reactions outlined in
Schemes 1-3. The starting compounds 1, 2c, 3c, and 9 were prepared following a previously reported literature
procedure.53 Ethoxycarbonylmethylation of 2,4-dihydro-3H -1,2,4-triazole-3-one derivatives (2a-e) with ethyl
bromoacetate by refluxing in absolute ethanol in the presence of sodium ethoxide afforded the ethyl acetate
derivatives (3a-e) in good yields. The 1 H- and 13 C-NMR spectra of compounds 3a-e exhibited additional
signals derived from the –CH2 CO2 Et group at the related chemical shift values. Moreover, compounds 3a and
3b gave a stable M+1 ion peak.
577

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
O
N NH
N NH
O
N
N
EtO-, BrCH2CO2Et
H3C
O
N
NH2
O
N
N
RCHO
O
N
N
H3C
H3C
H
R
H
R
1
2a-e
3a-e
2a,3a,4a,5a,6a R=4-metoxyphenyl
2b,3b,4b,5b R=furan-2-yl
2c,3c,4c,5c
R=phenyl
NaBH4
2d,3d,4d,5d R=pyridin-2-yl
2e,3e,4e,6e
R=pyridin-4-yl
O
OH
OH
OH
N N
N N
N
N
O
O
O
N
NH
N
NH
N
H
+
+
H3C
H3C
H3C
R
R
5e
4a-c
5a-d
Scheme 1. Synthesis of compounds 2-5.
O
O
O
OH
O
OH
N
N
N
N
N
N
3M HCl
NaBH4
O
O
O
N
NH
N
N
N
N
H3C
H3C
H3C
R
H
R
H
R
2a, 2e
4a
6a, 6e
C2H5OH
H2SO4
O
O
O
NHNH2
N N
N
N
H2NNH2.H2O
O
O
N
N
NH
NH
H3C
H3C
O
O
8
7
Scheme 2. Synthesis of compounds 6-8.
578

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
N NH
N
N
F
H3C
O
N
NH2
N N
H3C
O
N
N
9
H
R
RCHO
11: R= furan-2-yl 12: R= phenyl
N NH
N
O
N
N
H3C
O
N
N
amine
H3C
O
N
N
H
R
10a-d
CH3
O
a: R= furan-2-yl
b: R = phenyl
13
c: R = 4-metoxhyphenyl
d: R = 2-hydroxyphenyl
R
N
N
14: R =
N
O
H3C
O
N
N
N
O
15: R =
16: R =
N
H
N
N CH3
HO
Scheme 3. Synthesis of compounds 10-16.
The treatment of compounds 3a-d with NaBH4 resulted in the formation of 2 kinds of product: one of
which was formed from the reduction of the ester groups of compounds 3a-d to alcohol functionality while the
other corresponded to the hydrolyzed derivatives of esters to carboxylic acids.
The 1 H- and 13 C-NMR spectra of 4 and 5 type compounds displayed no signals belonging to the -
OCH2 CH3 group; instead, new signals derived from the carboxylic acid functionality or ethyl alcohol moiety
appeared at 3.62-4.10 ppm (-NCH2 -NCH2) and 3.45-4.86 ppm (-OH), respectively. Moreover, spectroscopic
data showed that NaBH4 reduced imine functionality of compounds 3a-d to the corresponding arylmethylamino
substituent (controlled by changing with D2 O). Furthermore, compounds 4a and 4b gave a relatively stable
M+1 ion peak and all compounds 4 gave reasonable elemental analysis data.
On the other hand, under the same reaction conditions, compound 3e produced 2-(2-hydroxyethyl)-
5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-one (5e), in which the pyridine-4-ylmethylamino moiety is
removed from the structure.
The synthesis of carboxylic acids (6a and 6b) was performed from the treatment of 2a and 2b with 3 M
HCl or 3 M NaOH without any reduction in the imine group. The treatment of 6a with NaBH4 afforded the
579

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
corresponding compound 4a, as expected. [4-[(4-Methoxybenzyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihydro-
1H -1,2,4-triazole-1-yl]acetate (7) was obtained by Fisher esterification of 4a; then the ester was converted
to the corresponding hydrazide, 2-[4-[(4-methoxybenzyl)amino]-3-(4-methylphenyl)-5-oxo-4,5-dihidro-1H-1,2,4-
triazole-1-yl]acetohydrazide (8) by treatment with hydrazine hydrate. The structures of compounds 7 and 8
were confirmed on the basis of spectroscopic methods.
The aminoalkylation of imine compounds, 2a-c, 2f was carried out by Mannich reaction using sev-
eral amines containing a morpholine or piperazine nucleus. The obtained 2-alkyltriazole derivatives (11-16)
exhibited spectroscopic and elemental analysis data consistent with the proposed structures.
All the newly synthesized compounds were tested for their antimicrobial activities and only the positive
results are presented in the Table. Among them, compounds 11-16, which possess a Mannich base structure
and carry a morpholine or piperazine nucleus in the position 2 of the 1,2,4-triazole scaffold, demonstrated
good antimicrobial activities against gram-positive and gram-negative bacterial strains, while they exhibited no
activity against the yeast-like fungi Candida albicans and Saccharomyces cerevisiae.
Acknowledgments
The authors are grateful for the support provided by the Scientific and Technological Research Council of Turkey
¨
˙
(TUBITAK, Project no: 107T333) and Karadeniz Technical University, BAP, Turkey (Ref. No. 2007.111.002.6).
References
1. Amir, M.; Kumar, H.; Javed, S.A. Eur. J. Med. Chem. 2008, 43, 2056-2066.
2. Navidpour, L.; Shafaroodi, H.; Abdi, K.; Amini, M.; Ghahremani, M. H.; Dehpourd, A. R.; Shafiee, A. Bioorg.
Med. Chem. 2006, 14, 2507-2517.
3. Tehranchian, S.; Akbarzadeh, T.; Fazeli, M. R.; Jamalifar, H.; Shafiee, A. Bioorg. Med. Chem. Lett. 2005, 15,
1023-1025.
4. Holla, B.S.; Poorjary, N. K.; Rao, S. B.; Shivananda, M. K. Eur. J. Med. Chem. 2002, 37, 511-517.
5. Rostom, A. F. S.; Ashour, H. M. A.; Abd El Razik, H. A.; Abd El Fattah, H.; A, El Fattah.; El-Din, N. N. Bioorg.
Med. Chem. 2009, 17, 2410-2422.
6. Rostom, S. A. F.; El-Ashmawy; I. M.; Abd El Razik, H. A.; Badr, M. H.; Ashour, H. M. A. Bioorg. Med. Chem.
2009, 17, 882-895.
7. Bektas, H.; Karaali, N.; Sahin, D.; Demirbas, A.; Karaoglu, S.A.; Demirbas, N. Molecules 2010, 15, 2427-2438.
8. Cai, S.; Li, Q. S.; Borchardt, R.T.; Kuczera, K.; Schowen, R. L.; Bioorg. Med. Chem. 2007, 15, 7281-7287.
9. Rao, B. M.; Sangaraju, S.; Srinivasu, M. K.; Madhavan, P.; Devi, M. L.; Kumar, P. R.; Candrasekhar, K. B.;
Arpitha, C.; Balaji, T. S. J. Pharm. Biomed. Anal. 2006, 41, 1146-1151.
10. Hancu, G.; Gaspar, A.; Gyeresi, A. J. Biochem. Biophys. Methods 2007, 69, 251-259.
11. Bajetti, E.; Zilembo, N.; Bichisao, E.; Pozzi, P.; Toffolatti, L. Crit. Rev. Oncol. Hematol. 2000, 33, 137-142.
12. Demirbas, A.; Ceylan, S.; Demirbas, N. J. Heterocyclic Chem. 2007, 44, 1271-1280.
580

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
13. Chaudhary, P.; Kumar, R.; Verma, A. K.; Singh, D.; Yadav, V.; Chhillar, A. K.; Sharma, G. L.; Chandra, R.
Bioorg. Med. Chem. 2006, 14, 1819-1826.
14. Tomar, V.; Bhattacharjee, G.; Kumar, K. A. Bioorg. Med. Chem. Lett. 2007, 17, 5321-5324.
15. Karthikeyan, M. S.; Prasad, D. J.; Poojary, B.; Bhat, K. S.; Holla B. S.; Kumari, N. S. Bioorg. Med. Chem. 2006,
14, 7482-7489.
16. Ridley, J. M.; Dooley, P. C.; Milnes, C. T.; Witchel, H. J.; Hancox, J. C. J. Mol. Cell. Cardiol. 2004, 36, 701-705.
17. Rawal, R.K.; Phabhakar, Y. S.; Kati, S. B.; De Clercq, E. Bioorg. Med. Chem. 2005, 13, 6771-6776.
18. Bonde, C. G.; Gaikwad, N. J. Bioorg. Med. Chem. 2004, 12, 2151-2161.
19. Rawal, R. K.; Tripathi, R.; Kati, S. B.; Pannecouque, C.; De Clercq, E. Bioorg. Med. Chem. 2007, 15, 1725-1731.
20. Dixit, P. P.; Patil, V. J.; Nair, P. S.; Jain, S.; Sinha, N.; Arora, S. K. Eur. J. Med. Chem. 2006, 41, 423-428.
21. Cui, Y.; Dang, Y.; Yang, Y.; Zhang, S.; Ji, R. Bioorg. Med. Chem. Lett. 2005, 40, 209-214.
22. Weidinger-Wells, M.; Boggs, C. M.; Foleno, B. D.; Melton, J.; Bush, K.; Goldschmitdt, R. M.; Hlasta, D. J. Bioorg.
Med. Chem. 2002, 10, 2345-2351.
23. Philips, O. A.; Udo, E. E.; Ali, A. A. M.; Samuel, S. M. Eur. J. Med. Chem. 2007, 42, 214-225.
24. Dixit, P. P.; Nair, P. S.; Patil, V. J.; Jain, S.; Arora, S. K.; Sinha, N. Bioorg. Med. Chem. Lett. 2005, 15, 3002-3005.
25. Gupta, A.; Unadkat, J. D.; Mao, Q. Drug Discovery Interface 2007, 96, 3226-3235.
26. Sujith, K. V.; Jyothi, N.; Rao, A.; Prashanth, S.; Balakrishna, K. Eur. J. Med. Chem. 2009, 44, 3697-3702.
27. Tramontini, M.; Angliolini, L. Tetrahedron 1990, 46, 1791-1837.
28. Joshi, S.; Khosla, N.; Tiwari, P. Bioorg. Med. Chem. 2004, 12, 571-576.
29. Lopes, F.; Capela, R.; Goncaves, J. O.; Horton, P. N.; Hursthouse, M. B.; Iley, J.; Casimiro, C. M.; Bom, J.;
Moreira, R. Tetrahedron Lett. 2004, 45, 7663-7666.
30. Ferlin, M. G.; Chiarelotto, G.; Antonucci, F.; Caparrotta, L.; Froldi, G. Eur. J. Med. Chem. 2002, 37, 427-434.
31. Holla, B. S.; Veerandra, B.; Shivanada, M. K.; Poojary, B. Eur. J. Med. Chem. 2003, 38, 759-767.
32. Malinka, W.; Swiatek, P.; Filipek, B.; Sapa, J.; Jezierska, A.; Koll, A. Farmaco 2005, 60, 961-968.
33. Tramontini, A. L.; Ghedeni, N. Polymer 1988, 29, 771-775.
34. Liu, L.; Zhu, S. Carbohydr. Polymer 2007, 68, 472-476.
35. Li, Z.; Junfeng, S. Talanta 2007, 73, 943-947.
36. Demirba¸s, N.; U˘gurluog˘lu, R.; Demirba¸s, A. Bioorg. Med. Chem., 2002, 10, 3717-3723.
37. Todeschini, A. R.; Miranda, A. N.; Silva, K. C. M.; Parrini S. C.; Barreiro, E. Eur. J. Med. Chem. 1998, 33,
189-199.
38. Rando, D. G.; Sato, D. N.; Siqueira, L.; Malvezzi, A.; Leite, C. Q. F.; Amaral, A. T.; Ferreira F. I.; Tavares, L. C.
Bioorg. Med. Chem. 2002, 10, 557-560.
39. Mamolo, M. G.; Falagiani, V.; Zampieri, D.; Vio L.; Ban, E. Farmaco, 2001, 56, 587-592.
40. Galic, N.; Peric, B.; Kojic-Prodic, B.; Cimerman, Z. J. Mol. Stuc. 2001, 559, 187-194.
41. Wyrzykiewicz, E.; Prukah, D. J. Heterocyclic Chem. 1998, 35, 381-387.
42. Reddy, H.; Lirgappa, Y. Ind. J. Heterocyclic Chem. 1994, 33, 919-923.
581

Reduction, Mannich reaction, and antimicrobial activity..., S. FANDAKLI, et al.
43. Demirba¸s, N.; U˘gurluog˘lu, R. Turk. J. Chem., 2004, 28, 559-571.
44. Demirba¸s, N.; U˘gurluog˘lu, R.; Turk. J. Chem. 2004, 28, 679-690.
45. Demirbas, N.; Demirbas, A.; Karao˘glu, S. A. Russian J. Bioorg. Chem. 2005, 31, 387-397.
46. Bayrak, H.; Demirbas, A.; Alpay Karaoglu, S.; Demirbas, N. Eur. J. Med. Chem. 2009, 44, 1057-1066.
47. Demirbas, A.; Sahin, D.; Demirbas, N.; Alpay Karaoglu, S. Eur. J. Med. Chem. 2009, 44, 2896-2903.
48. Bayrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S. A. Eur. J. Med. Chem. 2009, 44, 4362-4366.
49. Bayrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S. A. Eur. J. Med. Chem. 2010, 45, 4726-4732.
50. Bektas, H.; Demirbas, A.; Demirbas, N.; Bayrak, H.; Karaoglu, S. A. Turk J Chem. 2010, 34, 517-527.
51. Perez, C.; Pauli, M.; Bazerque, P.; Acta Biologia Med. Experimentalis 1990, 15, 113-115.
52. Ahmad, I.; Mehmood, Z.; Mohammed, F. J. Ethnopharmacology 1998, 62, 183-193.
53. Milcent, R.; Redeuilh, C. J. Heterocyclic Chem. 1997, 16, 403-407.
582