Synthesis and biological activities of methylenebis-4H-1,2,4-triazole derivatives

Article abstract of DOI:10.3906/kim-1212-66

5,5 ′ -Methylenebis(4{phenyl-4H-1,2,4-triazole-3-thiol) (2) was synthesized starting from hydrazinecarbothioamide compound (1). Treatment of compound 2 with ethyl bromoacetate produced diethyl 5,5 ′ -fmethylenebis[(4- phenyl-4H-1,2,4-triazole-5,3-diyl)thio]gdiacetate (3), which was converted to the corresponding diacetohydrazide derivative (4) by treatment with hydrazine hydrate. The reaction of compound 4 with several aldehydes produced the corresponding arylidene hydrazides, 5a{d. Syntheses of Mannich bases 6a{c were carried out by the treatment of compound 2 with several amines in the presence of formaldehyde. (4f[5-(f5-[(4-Amino-2-chlorophenyl)thio]-4- phenyl-4H-1,2,4- Triazol-3-ylgmethyl)-4-phenyl-4H-1,2,4-triazol-3-yl]thiog-3- chlorophenyl)amine (8) was prepared by reduction of 2 nitro groups of 3,3 ′ -methylenebisf5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4H-1,2,4- triazoleg (7) that were obtained from the condensation of 2 with 3,4-dichloronitrobenzene. The newly synthesized compounds were screened for their antimicrobial activities; some of them were found to be active towards the test microorganisms as the results demonstrated that the synthesized compounds exhibited a broad spectrum of activity with minimum inhibitory concentration (MIC) values of 31.3{500 μg/mL against gram-positive and gram-negative bacteria, Candia albicans and Saccharomyces cerevisiae. All compounds displayed lower activity in this series against the microorganisms with MIC values of 31.3{500 μg/mL than did the compared control drugs of ampicillin, streptomycin, and uconazole. TUeBITAK.

Full text of DOI:10.3906/kim-1212-66

Turk J Chem
2013) 37: 812 – 823
Turkish Journal of Chemistry
(
http://journals.tubitak.gov.tr/chem/
¨
˙
⃝
c TUBITAK
Research Article
doi:10.3906/kim-1212-66
Synthesis and biological activities of methylenebis-4H -1,2,4-triazole derivatives
∗
¨
Yıldız UYGUN, Hacer BAYRAK, Havva OZKAN
Department of Chemistry, Faculty of Sciences, Karadeniz Technical University, Trabzon, Turkey
Received: 27.12.2012
•
Accepted: 10.05.2013
•
Published Online: 16.09.2013
•
Printed: 21.10.2013
′
Abstract: 5,5 -Methylenebis(4–phenyl-4H -1,2,4-triazole-3-thiol) (2) was synthesized starting from hydrazinecarboth-
′
ioamide compound (1). Treatment of compound 2 with ethyl bromoacetate produced diethyl 5,5 -{methylenebis[(4-
phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}diacetate (3), which was converted to the corresponding diacetohydrazide deriva-
tive (4) by treatment with hydrazine hydrate. The reaction of compound 4 with several aldehydes produced the corre-
sponding arylidene hydrazides, 5a–d. Syntheses of Mannich bases 6a–c were carried out by the treatment of compound
2
with several amines in the presence of formaldehyde. (4{[5-({5-[(4-Amino-2-chlorophenyl)thio]-4-phenyl-4H -1,2,4-
triazol-3-yl}methyl)-4-phenyl-4H -1,2,4-triazol-3-yl]thio}-3-chlorophenyl)amine (8) was prepared by reduction of 2 nitro
groups of 3,3 -methylenebis{5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4H -1,2,4-triazole} (7) that were obtained from the
condensation of 2 with 3,4-dichloronitrobenzene.
′
The newly synthesized compounds were screened for their antimicrobial activities; some of them were found to be
active towards the test microorganisms as the results demonstrated that the synthesized compounds exhibited a broad
spectrum of activity with minimum inhibitory concentration (MIC) values of 31.3–500 µg/mL against gram-positive
and gram-negative bacteria, Candia albicans and Saccharomyces cerevisiae. All compounds displayed lower activity in
this series against the microorganisms with MIC values of 31.3–500 µg/mL than did the compared control drugs of
ampicillin, streptomycin, and fluconazole.
Key words: 1,2,4-Triazole, morpholine, furan, Schiff base, Mannich base, antimicrobial activity
1
. Introduction
1
,2,4-Triazole moiety has been incorporated into a number of therapeutically important agents.1,2 Itraconazole,
fluconazole, voriconazole,3,4 triazolam,5 alprazolam,6 etizolam and furacilin,7 ribavirin,8 hexaconazole,9 tria-
dimefon,10 myclobutanil,11 rizatriptan,12 and fluotrimazole13 are known drugs containing 1,2,4-triazole rings.
It is well known that one of the main aims of therapeutic treatment is the control of serious infections,
along with the prevention and treatment of some infectious complications due to other therapeutic procedures
such as cancer chemotherapy and surgery. In some cases such as tuberculosis, cancer, or AIDS, and in cases of
organ transplantation, fungal infections became an important complication and a major cause of morbidity and
mortality in immune-compromised individuals.14 However, in recent years, much attention has been focused on
the treatment of infections caused by multidrug-resistant bacteria and fungi resulting from the widespread use
and misuse of the known antimicrobial agents.15 This serious global health problem requires efforts toward the
design and synthesis of new antimicrobial agents that are effective against pathogenic microorganisms resistant
to currently available drugs.
∗
Correspondence: h.bayrak@ktu.edu.tr
812

UYGUN et al./Turk J Chem
In our recent studies, we obtained some bitriazolyl compounds containing another heterocyclic group
responsible for biological activity such as morpholine, thiomorpholine, or a furan nucleus.16 Moreover, some
4-arylidenamino-1,2,4-triazoles including morpholine or a piperazine nucleus synthesized in our laboratory were
found to possess antimicrobial activity.17
In organic syntheses, the compounds incorporating amino and mercapto groups together have been ac-
cepted as useful intermediates for further reactions. In this connection, the synthesis of some triazolothiadiazoles
or triazolothiadiazines having antimicrobial activity has been performed starting from 4-amino-5-mercapto-
1
,2,4-triazoles in our laboratories. The amino and mercapto groups, which can also react with electrophiles,
are considered as ready-made nucleophilic centers for the synthesis of condensed heterocyclic rings. Some
,2,4-triazole derivatives containing N -methylpiperazine moiety, which were obtained by Mannich reactions,
1
have been reported as antimicrobial agents. More recently, some Mannich bases possessing antimicrobial activ-
ity have been synthesized in our laboratory between 4-arylidenamino-1,2,4-triazoles and methyl piperazine or
morpholine.18
In the present study, as an extension of our previous studies on the synthesis of nitrogenated heterocycles
with potential chemotherapeutic activities, synthesis and antimicrobial activity screening studies of some new
1,2,4-triazole derivatives have been performed.
2
. Results and discussion
′
The synthetic route of the compounds is shown in Figures 1 and 2. Synthesis of 5,5 -methylenebis(4-phenyl-
′
4
H -1,2,4-triazole-3-thiol) (2) was performed by cyclocondensation of 2,2 -(1,3-dioxopropane-1,3-diyl)bis(N -
phenylhydrazinecarbothioamide) (1) in basic media. In the IR and NMR spectra of compound 2, the absence
of any signals pointing toward a carbonyl group supported the ring cyclization. In addition, in the 1 H NMR
spectrum, the presence of the signal pointing toward 2 –SH groups that was observed at 13.89 ppm as a D2 O
exchangeable singlet integrating 2 protons pointed toward a cyclic form for this compound. The reaction of 2
′
with ethyl bromoacetate in the presence of sodium ethoxide produced diethyl 5,5 -{methylenebis[(4-phenyl-4H -
1
13
1
,2,4-triazole-5,3-diyl)thio]}diacetate (3). In the H and C NMR spectra of compound 3, additional signals
originating from ester moiety were recorded at the related chemical shift values, while the signal due to the –SH
function disappeared. Compound 3 was converted to a hydrazide derivative with hydrazine hydrate in good
yield. The IR and 1 H NMR spectra of the hydrazide compound (4) exhibited signals belonging to the -NHNH2
function. Several aldehydes were treated with compound 4 to give the corresponding Schiff bases (5a–d) in
good yields. In the 1 H NMR spectra of compounds 5a–d, additional signals originating from arylidene moiety
were observed at the aromatic region between 8.04 and 8.34 ppm. The signal originating from the –NHNH2
group of parent compound 4 disappeared in the 1 H NMR and IR spectra of compounds 5a–d.
It was reported that arylidene hydrazides may exist as E/Z geometrical isomers about the -N=CH
double bond and as cis/trans amide conformers.1
8−20
A literature survey19 revealed that dimethyl-d6 sulfoxide
solution causes the emergence of the geometrical E isomer in higher percentages, whereas less polar solvents
support the Z isomer by an intramolecular hydrogen bond. In the present study, the NMR spectral data were
taken in dimethyl-d6 sulfoxide solution and no signals pointing to the Z isomer were present. However, due
to the cis/trans conformers of the E geometrical isomer of compounds 5a–d, certain signals were recorded
as 2 sets in the NMR spectra. The signals observed as 2 sets at 7.96–8.36 ppm and 10.08–12.05 ppm were
attributed to the -N=CH bond and 2 –NH functions of cis- and trans-conformers, respectively. The upfield
lines of -N=CH and 2NH protons were attributed to the cis-conformer of the amide structure and downfield
8
13

UYGUN et al./Turk J Chem
O
O
S
O
O
S
i
H NNH
2
NHNH2
NH
HNNH
NHNH
NH
1
ii
N
N
N
N
N
N
N N
O
O
S
N
N
S
iii
HS
N
N
SH
EtO
OEt
3
2
iv
N
N
N N
O
O
S
N
N
S
H NNH
2
NHNH2
Comp
Ar
OCH3
5
a
4
5b
v
HO
Cl
N
N
N N
O
O
5c
S
N
N
S
Cl
ArCH NNH
NHN CHAr
Br
5d
5
a-d
Figure 1. Synthetic pathway for the preparation of compounds 1–5. i :PhNCS; ii: NaOH; iii: Na(k) , EtOH,
BrCH COOEt; iv: H NNH ; v: ArCHO.
2
2
2
814

UYGUN et al./Turk J Chem
O
O
N
N
NH
N
N
N
N
NH
S
N
S
N
6a
NH
i
N
N
N
N
NH
O
O
N
N
N N
S
N
S
N
HS
N
SH
N
ii
6b
iii
2
O
O
N
N
iv
N
N
N N
S
N
N
S
Cl
Cl
N
N
N N
6c
O N
2
S
N
S
NO2
N
7
v
Cl
Cl
N
N
N N
H N
2
S
N
S
NH2
N
8
Figure 2. Reaction and conditions for the synthesis of compounds 6a–c, 7, and 8. i: 2-morpholinoethanamine, HCHO;
2 2
ii: furan-2-ylmethanamine, HCHO; iii: morpholine, HCHO; iv: Na(k), EtOH, 3,4-dichloronitrobenzene; v: H NNH ,
Pd-C.
8
15

UYGUN et al./Turk J Chem
lines of the protons of the same group to the trans-conformer.21 Furthermore, the -OH group of compound 5b
was seen as 2 different singlets due to the existence of cis/trans-amide conformers. The treatment of 2 with
several primary and secondary amines in the presence of formaldehyde afforded the corresponding Mannich
bases (6a–c). With this conversion, additional signals derived from amine moiety and methylene linkage were
observed at the related chemical shift values in the 1 H- and 13 C NMR spectra of compounds 6a–c, while no
signal pointing toward the –SH function is present. Treatment of compound 2 with 3,4-difluoronitrobenzene
′
resulted in the formation of 3,3 -methylenebis{5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4H -1,2,4-triazole} (7),
and in the NMR spectra there are additional signals of aromatic protons instead of the –SH protons. The nitro
groups of this compound were then reduced to amine groups using hydrazine hydrate as the hydrogen source in
the presence of Pd/C catalyst. In the 1 H NMR spectrum of compound 8, the presence of a D2 O exchangeable
signal at 5.78 ppm integrating 4 protons confirmed the reduction. The absorption bands due to amino groups
−
1
were observed at 3337 and 3210 cm
in the IR spectrum of 8.
All of the newly synthesized compounds gave elemental analysis results consistent with the proposed
structures. In addition, molecular masses of the newly synthesized compounds were confirmed by the appearance
+
+
+
+
of [M] , [M + 1] , [M + 2] , or [M + Na] ion peaks at corresponding m/z values in the mass spectra of
these compounds.
All of the newly synthesized compounds were tested for their antimicrobial activities, and only the posi-
tive results are presented in the Table. According to the results obtained, compound 1 displayed better activity
against Staphylococcus aureus and Enterococcus faecalis, which are gram-positive cocci, and Bacillus cereus,
which is a gram-positive spore bacillus, than against enteric bacteria Escherichia coli and Yersinia pseudotuber-
culosis and gram-negative bacillus Pseudomonas aeruginosa. This compound (1) displayed complete inactivity
towards Mycobacterium smegmatis, an atypical tuberculosis factor, and Candida albicans and Saccharomyces
cerevisiae, which are yeast-like fungi. According to the obtained results, it can be concluded that the con-
version of carbothioamide moiety into the 1,2,4-triazole nucleus in compound 2 and the further substitution
reactions leading to the formation of ester (3) and hydrazide (4) derivatives resulted in the inactivity against
the test microorganisms. Schiff base derivatives (5a–c) of compound 4 exhibited antibacterial activity against
the test microorganisms with minimum inhibitory concentration (MIC) values varying between 62.5 and 250
µg/mL. Moreover, marginal antifungal activities were observed for compounds 5a–c against Candida albicans
and Saccharomyces cerevisiae. Among the Mannich bases (6a–c), compound 6c was found to have the most
potent antibacterial activity against the test microorganisms, except for Mycobacterium smegmatis, with MIC
values between 62.5 and 125 µg/mL. Furthermore, compound 6c displayed slight antifungal activity against
Saccharomyces cerevisiae with a MIC value of 250 µg/mL.
Motivated by these findings and in continuation of our ongoing efforts, we would like to report here the
synthesis and investigation of biological activities of new bitriazolyl derivatives incorporating various heterocyclic
rings responsible for biological activity in a single structure.
3
. Experimental
All of the chemicals were purchased from Fluka Chemie AG (Buchs, Switzerland) and used without further
purification. Melting points of the synthesized compounds were determined in open capillaries on a B u¨ chi
B-540 melting point apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography
(
TLC) on silica gel 60 F254 aluminum sheets. The mobile phase was acetone and diethyl ether (1:2), and
detection was done using UV light. IR spectra were recorded as potassium bromide pellets using a PerkinElmer
816

UYGUN et al./Turk J Chem
Table. Screening for antimicrobial activity of the compounds 1, 5a–d, and 6a–c.
Minimal inhibition concentration values (µg/mL)
Comp. no.
Ec
Yp
Pa
62.5
62.5
62.5
62.5
-
500
500
62.5
> 128
Sa
Ef
Bc
Ms
-
Ca
-
Sc
-
1
62.5 62.5
62.5 125
62.5 125
62.5 125
250 250
250 250
250 250
62.5 62.5
31.3 31.3 31.3
5
a
125 31.3 62.5 62.5 500 500
125 31.3 62.5 62.5 500 500
125 31.3 62.5 62.5 250 250
125 125 250 125 500 500
500 500 250
500 500 250
125 62.5 62.5
5
b
5
c
5
d
6
a
-
-
-
-
-
-
-
-
6
b
6
c
250
Amp.
Strep.
Flu.
2
32
2
2
< 1
4
< 8 < 8
Ec: E. coli ATCC 35218, Yp: Y. pseudotuberculosis ATCC 911, Pa: P. aeruginosa ATCC 10145, Sa: S. aureus ATCC
2
6
5923, Ef: E. faecalis ATCC 29212, Bc: B. cereus 709 Roma, Ms: M. smegmatis ATCC607, Ca: C. albicans ATCC
0193, S. cerevisiae RSKK 251, Amp.: ampicillin, Strep.: streptomycin, Flu.: fluconazole, (-): no activity at test
concentrations.
600 series FT-IR spectrometer. H NMR and 13 C NMR spectra were recorded on a BRUKER AVANCE II
1
1
4
00 MHz NMR spectrometer (chemical shift in ppm downfield from TMS as an internal reference). The mass
spectra were obtained with a Quattro LC-MS (70 eV) instrument. The elemental analysis was performed on a
Costech Elemental Combustion System CHNS-O elemental analyzer. All compounds gave C, H, and N analysis
values within ꢀ0.4% of the theoretical values.
′
3
.1. 2,2 -(1,3-Dioxopropane-1,3-diyl)bis(N -phenylhydrazinecarbothioammide) (1)
The mixture of malonohydrazide (10 mmol) and phenyl isothiocyanate (20 mmol) in absolute ethanol was
refluxed for 3 h. On cooling the reaction content to room temperature, a white solid formed. This crude
product was filtered off and recrystallized from ethanol to afford the desired compound. Yield 91%, mp 181–182
◦
−1
C. IR (KBr, υ, cm ): 3220 (6NH), 3120 (Ar CH). 1675 (C=O), 1352 (2C=S), 1521 (Ar C=C), Anal.
1
Calcd. (%) for C17 H18 O2 S2 N6 : C, 50.73; N: 20.88; H: 4.51. Found: C, 50.77; N: 20.84; H: 4.47. H NMR
(
DMSO-d6 , δ ppm): 2.86 (2H, s, CH2), 6.52–6.74 (2H, brs, arH), 6.84–7.11 (8H, m, arH), 9.20 (2H, s, 2NH),
.40 (2H, s, 2NH), 9.91 (2H, s, 2NH); 13 C NMR (DMSO-d6 , δ ppm): 44.33 (CH2), arC: [125.60 (4CH), 129.30
4CH), 136.11 (2CH), 137.07 (2C)], 167.12 (2C=O), 169.22 (2C=S); EI MS m/z (%): 101.13 (31), 168.11 (25),
9
(
+
+
+
4
03.39 ([M + 1] , 100), 404.33 ([M + 2] , 19), 425.36 ([M + Na] , 18).
′
3
.2. 5,5 -Methylenebis(4-phenyl-4H -1,2,4-triazole-3-thiol) (2)
A solution of compound 1 (10 mmol) in water was refluxed in the presence of 2 N NaOH (20 mmol) for 2 h; the
resulting solution was then cooled to room temperature and acidified to pH 4 with 37% HCl. The precipitate
formed was filtered off, washed with water, and recrystallized from ethanol to afford the desired compound.
◦
−1
Yield 91%, mp 82–83 C. IR (KBr, υ, cm ): 3054 (Ar CH), 2727 (SH), 1574 and 1590 (C=N), 1442 (Ar
C=C). Anal. Calcd. (%) for C17 H14 N6 S2 : C, 55.72; N, 22.93; H, 3.85. Found: C, 55.70; N, 22.96; H, 3.80.
1
H NMR (DMSO-d6 , δ ppm): 3.87 (2H, s, CH2), 7.06 (4H, d, J =4.8 Hz, arH), 7.5 (6H, d, J =4.8 Hz, arH),
8
17

UYGUN et al./Turk J Chem
1
3.89 (2H, s, 2SH); 13 C NMR (DMSO-d6 , δ ppm): 24.29 (CH2), arC: [128.54 (4CH), 130.10 (4CH), 130.38
(
2CH), 133.57 (2C)], 147.48 (triazole 2C-3), 168.50 (triazole 2C-5); EI MS m/z (%): 118.87 (50), 145.90 (22),
+
+
+
1
60.98 (25), 176.93 (23), 335.17 (39), 367.21 ([M + 1] , 100), 368.21 ([M + 2] , 25), 405.19 ([M + K] , 18).
′
3
.3. Diethyl 5,5 -{methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}diacetate (3)
Metallic sodium (20 mmol) was added to a solution of compound 2 (10 mmol) in absolute ethanol, and the
mixture was refluxed for 2 h. After cooling the reaction content to room temperature, ethyl bromoacetate (20
mmol) was added and this mixture was refluxed for another 3 h (the progress of the reaction was monitored by
TLC). The precipitated salt was removed by adding water and a white solid appeared. This crude product was
◦
recrystallized from ethanol and water (1:2) to afford the desired product. Yield 93%, mp 133–134 C. IR (KBr,
υ, cm 1): 3068 (Ar CH), 1747 and 1735 (2C=O), 1596 (2C=N), 1439 and 1496 (Ar C=C). Anal. Calcd. (%)
for C25 H26 N6 O4 S2 : C, 55.75; N, 15.60; H, 4.87. Found: C, 55.71; N, 15.65; H, 4.83. 1 H NMR (DMSO-d6 , δ
ppm): 1.15 (6H, t,J =7.1 Hz, 2CH3), 4.01 (6H, s, 3CH2), 4.12 (4H, q, 2CH2), 7.13 (4H, d, J = 2.4 Hz, arH),
−
7
.55 (6H, s, arH); 13 C NMR (DMSO-d6 , δ ppm): 14.66 (2CH3), 22.66 (2CH2), 34.51 (2CH2), 61.89 (2CH2),
arC: [127.62 (2CH), 128.86 (2CH), 130.38 (2CH), 130.60 (2CH), 130.89 (2CH), 132.85 (2C)], 151.80 (triazole
2
1
C-3), 154.70 (triazole 2C-5), 168.68 (2C=O); EI MS m/z (%): 409.27 (72), 410.21 (16), 539.30 ([M + 1]+ ,
+
00), 540.36 ([M + 2] , 29), 541.36 (12).
′
3
.4. 2,2 -{Methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}diacetohydrazide (4)
Hydrazine hydrate (60 mmol) was added to a solution of compound 3 (10 mmol) in absolute ethanol, and the
mixture was allowed to reflux for 3 h. On cooling the reaction mixture to room temperature, a white solid
appeared. The crude product was filtered off and recrystallized from ethanol and water (1:2) to give the desired
◦
−1
compound 4. Yield 50%, mp 210–211 C. IR (KBr, υ, cm ): 3048 (Ar CH), 1665 (C=O), 1623 and 1597
C=N), 1541 and 1498 (Ar C=C). Anal. Calcd. (%) for C21 H22 N10 O2 S2 : C, 49.40; N, 27.43; H, 4.34.
Found: C, 49.37; N, 27.44; H, 4.30. 1 H NMR (DMSO-d6 , δ ppm): 3.80 (4H, s, 2CH2), 3.96 (2H, s, CH2), 4.28
(
(
(
(
(
4H, s, 2NH2), 7.11 (4H, d, J = 7.1 Hz, arH), 7.52 (6H, d, J = 7.0 Hz, arH), 9.31 (2H, s, 2NH); 13 C NMR
DMSO-d6 , δ ppm): 34.50 (CH2), 38.08–40.62 (DMSO + 2CH2), arC: [127.47 (4CH), 130.69 (4CH), 131.15
2CH), 132.32 (2C)], 151.13 (triazole 2C-3), 151.66 (triazole 2C-5), 167.15 (2C=O); EI MS m/z (%): 479.25
+
95), 480.32 (25), 533.31 ([M + Na] , 18), 551.30 (50), 573.33 (100), 574.40 (30), 575.28 (24).
3
.5. General method for the synthesis of compounds 5a–d
A mixture of compound 4 (10 mmol) and an appropriate aldehyde (20 mmol) in glacial acetic acid was stirred
at room temperature for 15 min. The mixture was then refluxed for 3 h, controlled by TLC. After the solvent
was evaporated under reduced pressure, a solid was obtained. This crude product was recrystallized from an
appropriate solvent.
′
′
3
.5.1. 2,2 -{Methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}bis{N -[(4-methoxy phenyl)
methylene]acetohydrazide} (5a)
◦
−1
Recrystallized from ethyl acetate. Yield 82%, mp 204–205 C. IR (KBr, υ, cm ): 3192 (NH), 3115 (Ar CH),
1687 and 1667 (C=O), 1606 and 1572 (C=N), 1512 and 1497 (Ar C=C), 1251 (C-O). Anal. Calcd. (%) for
818

UYGUN et al./Turk J Chem
1
C37 H34 N10 O4 S2 : C, 59.50; N, 18.75; H, 8.57. Found: C, 59.53; N, 18.76; H, 8.53. H NMR (DMSO-d6 , δ
ppm): 3.77 (6H, s, 2CH3), 4.00 (4H, s, 2CH2), 4.40 (2H, s, CH2), 6.98 (2H, d, J = 7.8 Hz, arH), 7.15 (6H, s,
arH), 7.57 (10H, m, arH), 8.11 and 7.93 (2H, s, 2N=CH, cis/trans conformers), 11.58 and 11.63 (2H, s, 2NH
cis/trans conformers); 13 C NMR (DMSO-d6 , δ ppm): 22.57 (CH2), 35.61 (2CH2), 55.99 (2CH3), arC: [115.02
(
2CH), 127.19 (2C), 127.20 (3CH), 127.72 (3CH), 129.15 (3CH), 129.46 (2CH), 130.57 (3CH), 130.81 (2CH),
33.02 (2C), 150.75 (2C)], 144.43 (2CH), 147.69 (2CH), 161.44 (triazole 2C-3), 163.68 (triazole 2C-5), 168.83
1
(
(
2C = O); EI MS m/z (%): 335.17 (39), 686.66 (55), 687.72 (100), 701.66 (58), 702.66 (28), 708.73 (20), 721.73
+
19), 722.79 (15), 743.80 (35), 744.74 ([M - 2] , 22).
′
′
3
.5.2. 2,2 -{Methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}bis{N -[(2-hydroxy phenyl)
methylene]acetohydrazide} (5b)
◦
−1
Recrystallized from ethanol. Yield 80%, mp 193–194 C. IR (KBr, υ, cm ): 3400 (OH), 3200 (NH), 3051 (Ar
CH), 1681 (C=O), 1621 and 1596 (C=N), 1497 (C=C). Anal. Calcd. (%) for C35 H30 N10 O4 S2 : C, 58.48; N,
1
1
9.49; H, 4.21. Found: C, 58.47; N, 19.40; H, 4.22. H NMR (DMSO-d6 , δ ppm): 4.01 (4H, s, 2CH2), 4.40
(
2H, s, CH2), 6.90 (4H, d, J = 7.2 Hz, arH), 7.19 (6H, d, J = 7.4 Hz, arH), 7.60 (8H, t, J = 7.6 Hz, arH), 8.29
and 8.39 (2H, s, 2N=CH, cis/trans conformers), 10.08 and 10.97 (2H, s, 2NH cis/trans conformers), 11.60 and
1.97 (2H, s, 2OH, cis/trans conformers); 13 C NMR (DMSO-d6 , δ ppm): 22.74 (CH2), 35.31 (CH2), 56.74
1
(
(
2CH2), arC: [116.85 (CH), 117.05 (CH), 120.10 (3CH), 127.69 (3CH), 126.96 (2CH), 129.87 (2CH), 130.87
3CH), 131.98 (3CH), 132.86 (2C), 150.67 (2C), 151.66 (2C)], 142.03 (2CH), 147.93 (2CH), 157.97 (triazole
+
2
C-3), 163.86 (triazole 2C-5), 168.76 (2C=O); EI MS m/z (%): 717.44 (28), 719.39 ([M + 1] , 89), 720.39
(
38), 741.35 (100), 742.36 (38), 757.42 (42).
′
′
3
.5.3. 2,2 -{Methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}bis{N -[(2,6-dichloro phenyl)
methylene]acetohydrazide} (5c)
Recrystallized from ethanol and water (1:2). Yield 75%, mp 187–188 C. IR (KBr, υ, cm ): 3202 (NH),
◦
−1
3064 (Ar CH), 1685 (C=O), 1556 and 1582 (C=N), 1498 (C=C). Anal. Calcd. (%) for C35 H26 N10 O2 S2 Cl2 :
1
C, 50.98; N, 17.20; H, 3.18. Found: C, 51.00; N, 17.22; H, 3.15. H NMR (DMSO-d6 , δ ppm): 3.95 (4H,
brs, 2CH2), 4.35 (2H, s, CH2), 7.13 (4H, s, arH), 7.42 (12H, q, arH), 8.22 and 8.36 (2H, s, 2N=CH, cis/trans
conformers), 11.90 and 12.05 (2H, s, 2NH, cis/trans conformers); 13 C NMR (DMSO-d6 , δ ppm): 22.74 (CH2),
3
1
1
8
8.19–40.71 (2CH2 + DMSO), arC: [127.47 (2CH), 129.89 (2CH), 128.94 (2CH), 130.58 (2CH), 131.02 (2CH),
31.89 (2CH), 132.43 (2CH), 132.55 (2CH), 133.53 (4C), 134.57 (4C), 139.97 (2CH)], 150.92 (triazole 2C-3),
64.46 (triazole 2C-5), 169. 59 (2C=O); EI MS m/z (%): 118.80 (31), 301.23 (44), 413.37 (53), 845.24 (69),
+
+
47.20 ([M + Na] , 100), 849.21 ([M + Na + 2] , 60).
′
′
3
.5.4. 2,2 -{Methylenebis[(4-phenyl-4H -1,2,4-triazole-5,3-diyl)thio]}bis{N -[(4-bromo phenyl)met-
hylene]acetohydrazide} (5d)
Recrystallized from dimethyl sulfoxide and water (1:3). Yield 78%, mp 196–197 C. IR (KBr, υ, cm ): 3200
NH), 1692 (C=O), 1610 and 1591 (C=N), 1497 (Ar C=C). Anal. Calcd. (%) for C35 H28 N10 O2 S2 Br2 : C,
7.77; N, 16.58; H, 3.34. Found: C, 49.79; N, 16.55; H, 3.33. 1 H NMR (DMSO-d6 , δ ppm): 4.01 (4H, d, J =
◦
−1
(
4
8
19

UYGUN et al./Turk J Chem
4
.6 Hz, 2CH2), 4.41 (2H, s, CH2), 7.16 (4H, s, arH), 7.58 (14H, d, J = 16.2 Hz, arH), 7.96 and 8.15 (2H, s,
N=CH, cis/trans conformers), 11.71 and 11.81 (2H, s, 2NH, cis/trans conformers); 13 C NMR (DMSO-d6 ,
2
δ ppm): 22.73 (CH2), 35.52 (2CH2), arC: [124.92 (2C), 127.68 (2CH), 129.42 (2CH), 129.65 (2CH), 130.58
(
(
2CH), 130.83 (2CH), 132.52 (4CH), 132.97 (C), 134.01 (C), 143.34 (2CH), 146.51 (2CH), 150.94 (2C)], 151.68
triazole 2C-3), 164.09 (triazole 2C-5), 169.16 (2C=O); EI MS m/z (%): 701.20 (23), 803.77 (100), 804.72 (53),
+
8
45.18 ([M] , 37).
3
.6. General method for the synthesis of compounds 6a–c
The mixture of compound 2 (10 mmol) and the corresponding amine (20 mmol) in DMF was stirred in the
presence of formaldehyde (37%, 10 mmol) at room temperature for 2.5 h (the progress of the reaction was
monitored by TLC). Water was added and this mixture was kept overnight in cold conditions. The solid
that separated was collected by filtration and recrystallized from DMSO and water (1:3) to yield the target
compound.
′
3
.6.1. 5,5 -Methylenebis(2-{[(2-morpholin-4-ylethyl)amino]methyl}-4-phenyl-2,4-dihydro-3H -
1
,2,4-triazole-3-thione) (6a)
◦
−1
Recrystallized from dimethyl sulfoxide and water (1:3). Yield 83%, mp 193–194 C. IR (KBr, υ, cm ): 3049
(
Ar CH), 1591 and 1575 (Ar C=C), 1641 (C=N), 1114 (C=S). Anal. Calcd. (%) for C31 H42 N10 O2 S2 : C,
5
8
7
6
2
5
7.21; N, 21.52; H, 6.50. Found: C, 57.17; N, 21.50; H, 6.47. 1 H NMR (DMSO-d6 , δ ppm): 2.32–2.42 (16H, m,
CH2), 2.98 (4H, brs, 2CH2), 3.52 (4H, s, 2CH2), 4.01 (2H, brs, CH2), 5.33 (4H, brs, 2CH2), 7.17 (4H, s, arH),
.52 (6H, s, arH), 11.24 (2H, s, 2NH); 13 C NMR (DMSO-d6 , δ ppm): 38.92–41.42 (6CH2), 54.13 (2NCH2),
6.94 (2NCH2 + OCH2), 66.93 (NCH2 N), arC: [128.61 (4CH), 130.29 (6CH), 133.87 (2C)], 146.33 (triazole
C-3), 168.41 (triazole 2C-5); EI MS m/z (%): 335.17 (39), 533.32 (30), 561.43 (23), 585.54 (37), 587.68 (100),
88.56 (100), 596.68 (25), 603.60 (37), 603.72 (21), 604.60 (15), 663.63 (48), 664.64 (22).
′
3
.6.2. 5,5 -Methylenebis(2-{[(2-furylmethyl)amino]methyl}-4-phenyl-2,4-dihydro-3H -1,2,4-tria-
zole-3-thione) (6b)
◦
−1
Recrystallized from dimethyl sulfoxide and water (1:3). Yield 95%, mp 191–192 C. IR (KBr, υ, cm ): 3062
(
Ar CH), 1146 (C=S), 1591 and 1574 (Ar C=C), 1667 (C=N). Anal. Calcd. (%) for C29 H28 N8 O2 S2 : C,
5
4
7
9.57; N, 19.16; H, 4.83. Found: C, 59.60; N, 19.14; H, 4.80. 1 H NMR (DMSO-d6 , δ ppm): 3.39 (2H s, CH2),
.04 (4H, brs, 2CH2), 5.33 (4H, brs, CH2), 6.20 (2H, s, furan-arH), 6.36 (4H, s, furan-arH), 7.17 (4H, a, arH),
.51 (6H, s, arH), NH was not observed. 13 C NMR (DMSO-d6 , δ ppm): 38.22–40.73 (DMSO + CH2), 47.20
(
CH2), 72.50 (CH2), arC: [108.57 (CH), 110.55 (2CH), 128.15 (2CH), 129.79 (CH), 130.20 (CH), 133.33 (C),
142.52 (CH), 145.60 (C)], 151.98 (triazole 2C-3), 168.09 (triazole 2C-5); EI MS m/z (%): 539.35 (100), 540.35
+
(
(
38), 540.48 (31), 541.42 (20), 547.59 (18), 553.63 (60), 561.37 (38), 569.74 (22), 585.66 ([M + 1] , 31), 587.80
30).
820

UYGUN et al./Turk J Chem
′
3
.6.3. 5,5 -Methylenebis[2-(2-morpholin-4-ylmethyl)-4-phenyl-2,4-dihydro-3H -1,2,4-triazole-3-
thione] (6c)
◦
−1
Recrystallized from dimethyl sulfoxide and water (1:3). Yield 86%, mp 196–197 C. IR (KBr, υ, cm ): 3050
(
Ar CH), 1592 and 1579 (C=N), 1498 (Ar C=C), 1113 (C=S). Anal. Calcd. (%) for C27 H32 N8 O2 S2 : C,
1
5
7.42; N, 19.84; H, 5.71. Found: C, 57.39; N, 19.81; H, 5.69. H NMR (DMSO-d6 , δ ppm): 2.64 (8H, s,
4CH2), 3.56 (8H, s, 4CH2), 4.01 (2H, s, CH2), 5.00 (4H, s, 2CH2), 7.12 (4H, d, J = 5.4 Hz, arH), 7.51 (6H, s,
arH); 13 C NMR (DMSO-d6 , δ ppm): 24.01 (CH2), 50.74 (2CH2), 66.77 (4CH2), 69.31 (4CH2), arC: [128.59
(
4CH), 130.17 (6CH), 133.89 (2C)], 146.02 (triazole 2C-3), 169.51 (triazole 2C-5); EI MS m/z (%): 367.28 (81),
+
+
4
66.46 (100), 467.46 (31), 565.52 ([M + 1] , 75), 566.58 ([M + 2] , 25).
′
3
.7. 3,3 -Methylenebis{5-[(2-chloro-4-nitrophenyl)thio]-4-phenyl-4H -1,2,4-triazole} (7)
The mixture of compound 2 (10 mmol) and metallic sodium (20 mmol) in ethanol was refluxed for 2 h. 3,4-
Dichloronitrobenzene (20 mmol) was then added and this mixture was refluxed for an additional 22 h (the
progress of the reaction was monitored by TLC). The solvent was evaporated under reduced pressure, and the
◦
solid obtained by adding water was filtered off and recrystallized from ethyl acetate. Yield 71%, mp 77–78 C.
IR (KBr, υ, cm 1): 3096 (Ar CH), 1537, 1574, and 1598 (C=N), 1453 (C=C), 1520 and 1349 (NO2). Anal.
−
Calcd. (%) for C29 H18 N8 O4 S2 Cl2 : C, 51.41; N, 16.54; H, 2.68. Found: C, 51.42; N, 16.55; H, 2.66. 1 H NMR
(
(
DMSO-d6 , δ ppm): 4.27 (2H, s, CH2), 7.00 (2H, d, J = 8.6 Hz, arH), 7.12 (4H, d, J = 6.6 Hz, arH), 7.45
7H, d, J =6.6 Hz, arH), 8.05 (2H, d, J =9.0 Hz, arH), 8.27 (1H, s, arH); 13 C NMR (DMSO-d6 , δ ppm):
24.04 (CH2), arC: [123.53 (nitrophenyl 2CH), 125.30 (nitrophenyl 2CH), 127.74 (4CH), 128.88 (4CH), 130.88
(
2CH), 131.06 (2CH), 132.76 (4C), 142.13 (2C), 144.94 (2C)], 147.12 (triazole 2C-3), 153.84 (triazole 2C-5);
+
EI MS m/z (%): 553.43 (21), 569.68 (28), 587.63 (34), 601.63 (22), 607.57 ([M + 1 - 2Cl] , 20), 663.59 (79),
664.71 (44), 685.72 (100), 686.60 (50).
3
.8. (4{[5-({5-[(4-amino-2-chlorophenyl)thio]-4-phenyl-4H -1,2,4-triazol-3-yl}methyl)-4-phenyl-4H -
,2,4-triazol-3-yl]thio}-3-chlorophenyl)amine (8)
1
Pd-C (20 mmol) catalyst was added to a solution of compound 7 (10 mmol) in butanol, and the mixture was
allowed to reflux in the presence of hydrazine hydrate (60 mmol) for 6 h (the progress of the reaction was
monitored by TLC). The catalyst was removed by filtration. After evaporating the solvent under reduced
pressure, a white solid appeared. This crude product was recrystallized from ethyl acetate and ether (1:2) to
◦
−1
afford the desired compound. Yield 31%, mp 111–113 C. IR (KBr, υ, cm ): 3337 and 3210 (NH2), 3100
Ar CH), 1629 and 1596 (C=N), 1498 and 1478 (Ar C=C). Anal. Calcd. (%) for C29 H22 N8 S2 Cl2 : C, 56.40;
N, 18.14; H, 3.59. Found: C, 56.42; N, 18.11; H, 3.62. 1 H NMR (DMSO-d6 , δ ppm): 4.00 (2H, s, CH2), 5.78
4H, s, 2NH2), 6.40 (2H, d, J =8.4 Hz, arH), 6.60 (2H, s, arH), 6.90 (6H, d, J =10.06 Hz, arH), 7.42 (6H,
m, arH); 13 C NMR (DMSO-d6 , δ ppm): 24.07 (CH2), arC: [123.28 (nitrophenyl 2CH), 125.27 (nitrophenyl
CH), 127.65 (4CH), 128.79 (4CH), 130.45 (2CH), 131.09 (2CH), 132.66 (4C), 142.09 (2C), 145.00 (2C)], 147.48
triazole 2C-3), 153.87 (triazole 2C-5); EI MS m/z (%): 337.24 (31), 346.25 (75), 355.20 (62), 356.26 (47),
85.23 (100), 386.23 (81).
(
(
2
(
3
8
21

UYGUN et al./Turk J Chem
4
. Antimicrobial activity assessment
All test microorganisms were obtained from the Refik Saydam Hıfzıssıhha Institute (Ankara, Turkey) and
were as follows: E. coli ATCC35218, Y. pseudotuberculosis ATCC911, P. aeruginosa ATCC43288, S. aureus
ATCC25923, E. faecalis ATCC29212, B. cereus 709 Roma, M. smegmatis ATCC607, C. albicans ATCC60193,
and S. cerevisiae RSKK 251. All of the newly synthesized compounds were weighed and dissolved in dimethyl
sulfoxide to prepare extract stock solutions of 5 mg/mL.
5
. MIC method
The antimicrobial activities of synthesized compounds were tested quantitatively in respective broth media
by using double dilution and the MIC values (µg/mL) were determined.22 The antibacterial and antifungal
assays were performed in Mueller-Hinton broth (Difco, Detroit, MI, USA) at pH 7.3 and in 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 the standard antibacterial and antifungal
drugs, respectively. Dimethyl sulfoxide at a dilution of 1:10 was used as the solvent control. The results are
shown in the Table.
Acknowledgments
This project was supported by Karadeniz Technical University (Ref. 8623 and 8663). The authors thank S¸ eng u¨ l
Alpay Karao ˘g lu for performing the antimicrobial screening studies.
References
1
2
.
.
Heindel, N. D.; Reid, I. R. J. Heterocycl. Chem. 1980, 17, 1087–1088.
Holla, B. S.; Kalluraya, B.; Sridhar, K. R.; Drake, E.; Thomas, L. M.; Bhandary, K. K.; Levine, M. S. Eur. J. Med.
Chem. 1994, 29, 301–308.
3
4
5
6
.
.
.
.
Merk and Co. The Merk Index, 12th ed., Merk and Co. Inc., Whitehouse Station, NJ, USA, 1996.
Haber, J. Cas. Lek. Cesk. 2001, 140, 596–604.
Lee, S.; Kamide, T.; Tabata, H.; Takahashi, H.; Shiro, M.; Natsugari, H. Bioorg. Med. Chem. 2008, 16, 9519–9523.
Chaytor, A. In xPharm: The Comprehensive Pharmacology Reference; Enna, S. J.; Bylund, D. B., Eds.; Elsevier,
Amsterdam, 2007.
7
8
.
.
Subrahmanya, B. K.; Poojary, B.; Jagadeesh P. D.; Naik, P.; Holla, B. S. Eur. J. Med. Chem. 2009, 44, 5066–5070.
Kumarapperuma, S. C.; Sun, Y.; Jeselnik, M.; Chung, K.; Parker, W. B.; Jonsson, C. B.; Arterburn, J. B. Bioorg.
Med. Chem. Lett. 2007, 17, 3203–3207.
9
.
Prasad, D. J.; Ashok, M.; Karegoudar, P.; Poojary, B.; Holla, B. S.; Kumari, N. S. Eur. J. Med. Chem. 2009, 44,
5
51–557.
1
1
1
1
1
1
1
0. Jin, Z.; Hu, Y.; Huo, A.; Tao, W.; Shao, L.; Liu, J.; Fang, J. J. Organometallic Chem. 2006, 691, 2340–2345.
1. Bhat, K. S.; Poojary, B.; Prasad, D. J.; Naik, P.; Holla, B. S. Eur. J. Med. Chem. 2009, 44, 5066–5070.
2. Dahlof, C. G. H.; Rapoport, A. M.; Sheftell, F. D.; Lines, C. R. Clin. Ther. 1999, 21, 1823–1836.
3. Faidallah, H. M.; Khan, K. A.; Asiri, A. M. J. Fluorine Chem. 2011, 132, 870–877.
4. Turan-Zitouni, G.; Kaplancikli, Z. A.; Yildiz, M. T.; Chevallet, P.; Kaya, D. Eur. J. Med. Chem. 2005, 40, 607–613.
5. Akbas, E.; Berber, I. Eur. J. Med. Chem. 2005, 40, 401–405.
6. Bayrak, H.; Demirba ¸s , A.; Belkta ¸s , H.; Karao ˘g lu, S¸ . A.; Demirba ¸s , N. Turk. J. Chem. 2010, 34, 835–846.
822

UYGUN et al./Turk J Chem
1
1
1
2
2
7. Bayrak, H.; Demirbas, A.; Demirbas, N.; Karaoglu, S. A. Eur. J. Med. Chem. 2009, 44, 4362–4366.
8. Bayrak, H.; Demirbas, A.; Karaoglu, S. A.; Demirbas, N. Eur. J. Med. Chem. 2009, 44, 1057–1066.
9. Wyrzykiewicz, E.; Prukala, D. J. Heterocyclic Chem. 1998, 35, 381–387.
0. Galic, N.; Peric, B.; Kojic-Prodic, B.; Cimerman, Z. J. Mol. Struct. 2001, 559, 187–194.
1. Rando, D.; Sato, D. N.; Siqueira, L.; Malvezzi, A.; Leite, C. Q. F.; Amaral, A. T.; Ferreira, E. I.; Tavares, L. C.
Bioorg. Med. Chem. 2002, 10, 557–560.
2
2. NCCLS. NCCLS Document M7-A3, NCCLS, Villanova, PA, USA, 1993.
8
23

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