Synthesis and anticancer evaluation of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, 1,2,4-triazoles and mannich bases

Article abstract of DOI:10.1248/cpb.c15-00059

A series of 5-(pyridin-4-yl)-N-substituted-1,3,4-oxadiazol-2-amines (3a-d), 5-(pyridin-4-yl)-N-substituted- 1,3,4-thiadiazol-2-amines (4a-d) and 5-(pyridin-4-yl)-4-substituted-1,2,4-triazole-3-thiones (5a-d) were obtained by the cyclization of hydrazinecarbothioamide derivatives 2a-d derived from isonicotinic acid hydrazide. Aminoalkylation of compounds 5a-d with formaldehyde and various secondary amines furnished the Mannich bases 6a-p. The structures of the newly synthesized compounds were confirmed on the basis of their spectral data and elemental analyses. All the compounds were screened for their in vitro anticancer activity against six human cancer cell lines and normal fibroblast cells. Sixteen of the tested compounds exhibited significant cytotoxicity against most cell lines. Among these derivatives, the Mannich bases 6j, 6m and 6p were found to exhibit the most potent activity. The Mannich base 6m showed more potent cytotoxic activity against gastric cancer NUGC (IC₅₀=0.021 μM) than the standard CHS 828 (IC₅₀=0.025 μM). Normal fibroblast cells WI38 were affected to a much lesser extent (IC₅₀>10 μM).

Full text of DOI:10.1248/cpb.c15-00059

Vol. 63, No. 5
Chem. Pharm. Bull. 63, 369–376 (2015)
369
Regular Article
Synthesis and Anticancer Evaluation of 1,3,4-Oxadiazoles,
1,3,4-Thiadiazoles, 1,2,4-Triazoles and Mannich Bases
,a
Nadia Youssef Megally Abdo* and Mona Monir Kamelb
a Chemistry Department, Faculty of Education, Alexandria University; Alexandria 21526, Egypt: and b Pharmaceutical
Organic Chemistry Department, Faculty of Pharmacy, Cairo University; Cairo 11562, Egypt.
Received January 20, 2015; accepted March 2, 2015
A series of 5-(pyridin-4-yl)-N-substituted-1,3,4-oxadiazol-2-amines (3a–d), 5-(pyridin-4-yl)-N-substi-
tuted-1,3,4-thiadiazol-2-amines (4a–d) and 5-(pyridin-4-yl)-4-substituted-1,2,4-triazole-3-thiones (5a–d)
were obtained by the cyclization of hydrazinecarbothioamide derivatives 2a–d derived from isonicotinic acid
hydrazide. Aminoalkylation of compounds 5a–d with formaldehyde and various secondary amines furnished
the Mannich bases 6a–p. The structures of the newly synthesized compounds were confirmed on the basis
of their spectral data and elemental analyses. All the compounds were screened for their in vitro anticancer
activity against six human cancer cell lines and normal fibroblast cells. Sixteen of the tested compounds
exhibited significant cytotoxicity against most cell lines. Among these derivatives, the Mannich bases 6j, 6m
and 6p were found to exhibit the most potent activity. The Mannich base 6m showed more potent cytotoxic
activity against gastric cancer NUGC (IC50=0.021µM) than the standard CHS 828 (IC50=0.025µM). Normal
fibroblast cells WI38 were affected to a much lesser extent (IC50>10 µM).
Key words anticancer activity; Mannich base; 1,2,4-triazole; 1,3,4-oxadiazole; 1,3,4-thiadiazole; isonicotinic
acid hydrazide
About 13% deaths of human beings throughout the world by in vitro and in vivo studies. High expectations for therapies
are caused by cancer, which is characterized by uncontrolled are attached to the extensive ongoing research conducted on
cell growth, metastasis, and invasion.1) Among the existing the group of 2-amino-1,3,4-thiadiazole substituted with 2,4-di-
cancer therapies, chemotherapy has turned out to be one of the hydroxyphenyl group.15–17)
most significant treatments in cancer management.2) However,
The small and simple triazole nucleus is present in com-
the most important clinical problem related to the use of new pounds aimed at evaluating new entities that possess antimi-
therapeutic strategies is the high toxicity of new potentially crobial, antitubercular, anticonvulsant, antidepressant, anti-
cytotoxic compounds. Thus in the light of existing problems malarial and antiinflammatory activities.18,19) A large number
in cancer therapy, discovery of novel, efficient, safe and selec- of 1,2,4-triazoles have been incorporated into a wide variety
tive anticancer agents is a thrust area for medicinal chemists.3) of therapeutically interesting drug candidates possessing anti-
The ring-closure reactions of carbohydrazides to prepare cancer activities.20,21) Literature survey reveals that important
1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles are chemotherapeutics, such as Vorozole, Letrozole and Anas-
well-known and have been thoroughly studied. 1,3,4-Oxadia- trozole (Fig. 1) that consist of substituted 1,2,4-triazole ring,
zoles are an important class of heterocyclic compounds with are currently being used for the treatment of breast cancer.22)
a wide range of biological activities.4–6) Among these, a few Moreover, the aminoalkylation of aromatic substrates by Man-
different substituted 1,3,4-oxadiazoles have exhibited potent nich reaction is of considerable importance for the synthesis
antitumor activities.7–10) Recently, a series of new 1,3,4-oxa- and modification of biologically active compounds.23) Mannich
diazole derivatives incorporating a pyridine moiety were bases are common pharmacophores in the design and devel-
synthesized and developed as potential telomerase inhibi- opment of anticancer chemotherapeutic agents.24–27) Some
tors.11) On the other hand, the literature presents the variously Mannich bases are reported to exhibit activity in vitro against
substituted 1,3,4-thiadiazoles as compounds with a broad Maurine P388 lymphocytic leukemia cells.28)
spectrum of biological activity.12–14) The anticancer potential
In the design of new compounds, development of hybrid
of 2-amino-1,3,4-thiadiazole derivatives has been documented molecules through the combination of different pharmaco-
Fig. 1. Chemical Structures of Breast Cancer Drugs
*To whom correspondence should be addressed. e-mail: nadiamegally@yahoo.com
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 5 (2015)
Chart 1. Synthesis of Compounds 3a–d and 4a–d
Chart 2. Synthesis of Compounds 5a–d and 6a–p
phores in one structure may lead to compounds with increased thesis of the title compounds is shown in Charts 1 and 2.
biological activity. However, reviewing the literature reveals The reaction of isonicotinic acid hydrazide 1 with various
that the potential anticancer activity of isonicotinic acid hy- aryl isothiocyanates afforded the corresponding hydrazin-
drazide derivatives and Mannich bases derived from 1,2,4-tri- ecarbothioamide derivatives30,31) 2a–d. Oxidative cyclization
azoles have not yet been thoroughly investigated. Prompted by of the latter compounds in 4N NaOH using a mixture of I2/
the aforementioned findings and in continuation of our previ- KI (5%) furnished the corresponding 5-(pyridin-4-yl)-N-sub-
ous work on discovering the anticancer potential of triazole stituted-1,3,4-oxadiazol-2-amines32,33) (3a–d) via elimination
derivatives,29) in the present study it was planned to synthesize of H2S. On the other hand, 5-(pyridin-4-yl)-N-substituted-
hybrid compounds that comprise pyridine ring and the variant 1,3,4-thiadiazol-2-amines32,34) (4a–d) were obtained by cy-
heterocyclic ring systems in order to identify new candidates clization of 2a–d upon reaction with cold conc. H2SO4. The
that may be of value in designing new, potent, selective and structure of the synthesized compounds was confirmed on
less toxic anticancer agents. All the synthesized compounds the basis of their spectral data and elemental analyses. The
were evaluated for their in vitro cytotoxicity against six IR spectra of the hydrazinecarbothioamide derivatives 2a–d
human cancer cell lines and normal fibroblast cells and their displayed the presence of signals in the range of 1672–1684
structure–activity relationship (SAR) was investigated.
and 1249–1255cm−1 corresponding to C=O and C=S, respec-
1
tively. Meanwhile, the H-NMR spectra of these compounds
showed three singlet signals corresponding to the NH groups
Results and Discussion
Chemistry The reaction sequence employed for the syn- in the range of δ 9.71–10.82ppm. The carbothioamides 2a–d

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
371
underwent cyclization upon heating under reflux with 4N aq. cancer (MCF) and normal fibroblast cells (WI38). For com-
NaOH to afford 5-(pyridin-4-yl)-4-substituted-1,2,4-triazole- parison purposes, CHS 828, a pyridyl cyanoguanidine, was
3-thiones35–37) (5a–d). The mercapto triazole derivatives are used as standard antitumor drug39) (Fig. 2). The IC50 values in
reported to be present in thione-thiol tautomeric forms in micro molar (µM) are listed in Table 1. Sixteen of the tested
solutions.38) The structure elucidation of the triazoles 5a–d re- compounds showed cytotoxic activity with IC50 values <1µM.
vealed their presence in the thione form predominantly which The Mannich bases 6j, 6m and 6p were found to be the most
was further confirmed by their conversion to their Mannich potent derivatives. All the synthesized compounds were tested
bases. Thus treatment of compounds 5a–d with each of N- for their cytotoxicity against normal fibroblast cells as many
methylpiperazine, morpholine, diethyl amine and benzylpi- anticancer drugs are toxic not only against cancer cells but
peridine in the presence of formaldhyde solution afforded the also normal ones. The results obtained showed that normal
1
corresponding Mannich bases 6a–p. The H-NMR spectra of fibroblast cells (WI38) were affected to a much lesser extent
the Mannich bases 6a–p displayed additional signals due to (IC50>10 µM).
–N–CH2–N– groups in the range of δ 5.25–5.78 ppm integrat-
Structure Activity Relationship In the present study, on
ing to two protons, beside signals belonging to methylpipra- correlating the structures of the synthesized oxadiazoles,
zine, morpholine, diethylamine or benzylpiperdine moieties. thiadiazoles and triazoles with their anticancer activity, it has
Furthermore, the 13C-NMR spectra of compounds 6a–p exhib- been observed that the electronegativity of the substituents
ited signals in the range of δ 69.77–77.35ppm (N–CH2–N) and at the aromatic rings manipulated the magnitude of activity.
δ 169.41–171.68ppm (C=S).
In Vitro Cytotoxicity Effect on the Growth of Human
Cancer Cell Lines The heterocyclic compounds, prepared in
this study, were evaluated according to standard protocols for
their in vitro cytotoxicity against six human cancer cell lines
including cells derived from human gastric cancer (NUGC),
human colon cancer (DLD1), human liver cancer (HA22T and
HEPG2), nasopharyngeal carcinoma (HONE1), human breast Fig. 2. Chemical Structure of CHS 828
b)
Table 1. Cytotoxicity of Compounds 3a–d, 4a–d, 5a–d and 6a–p against a Variety of Cancer Cell Linesa) [IC50 (µM)]
Cytotoxicity (IC50 in µM)
Compound No.
NUGC
DLDI
HA22T
HEPG2
HONE1
MCF
WI38
3a
3b
3c
2.312
2.116
0.890
1.220
1.142
1.165
0.418
0.028
2.286
1.249
0.338
0.320
2.230
1.150
3.230
1.288
1.135
2.340
2.218
0.380
0.482
0.040
0.860
1.127
0.021
0.328
1.350
0.028
0.025
1.262
2.765
0.725
1.833
2.222
1.322
0.135
0.229
1.245
2.220
0.538
0.749
2.598
1.065
3.028
1.092
2.160
1.792
1.346
0.410
0.139
0.066
0.120
0.269
0.044
0.246
1.160
0.036
2.315
3.274
2.838
1.372
2.257
1.348
2.350
0.753
0.180
2.631
3.218
0.820
0.194
2.188
2.160
3.212
1.739
2.160
2.826
2.620
0.128
0.280
0.219
0.543
0.324
0.320
0.234
2.290
0.128
2.067
1.439
3.220
1.184
1.277
1.328
2.221
0.970
0.264
2.128
1.550
0.444
0.499
2.772
2.349
2.026
1.488
0.814
2.646
3.837
0.089
0.520
0.028
0.390
1.093
0.066
0.185
2.120
0.439
1.245
1.330
2.440
1.964
2.129
1.820
2.152
0.849
0.887
2.112
2.466
1.528
2.871
2.280
1.059
2.129
2.879
0.780
1.174
2.341
1.890
1.179
1.090
1.160
0.549
0.880
0.129
1.126
0.018
0.015
2.296
2.239
1.758
1.320
2.242
1.224
2.072
2.290
3.140
2.693
0.818
0.840
1.265
2.828
0.188
1.702
1.286
3.932
3.280
0.082
1.108
0.320
1.051
0.125
0.338
1.267
0.048
1.120
0.018
na
na
na
na
na
na
na
3d
4a
4b
4c
4d
5a
1.330
na
5b
5c
0.120
na
5d
6a
na
480
6b
6c
na
0.48
na
6d
6e
na
6f
na
6g
na
6h
6i
2.199
na
6j
na
6k
6l
2.170
na
6m
6n
6o
na
na
na
6p
CHS 828
2.219
na
a) NUGC, gastric cancer; DLDI, colon cancer; HA22T and HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; MCF, breast cancer; WI38, normal fibroblast cells.
b) The sample concentration that produces a 50% reduction in cell growth.

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Chem. Pharm. Bull.
Vol. 63, No. 5 (2015)
Thus, compounds bearing 4-chlorophenyl and 4-bromophenyl ity against six human cancer cell lines and normal fibroblast
pharmacophores were found to be the only active compounds. cells and their SAR was investigated. The screening results
Among the oxadiazole derivatives 3a–d, only compound 3c revealed that the presence of the 4-chlorophenyl and 4-bro-
showed selective moderate activity against gastric and colon mophenyl substituents was essential for activity. The Mannich
cancer cell lines. On the other hand, 1,3,4-thiadiazole deriva- bases demonstrated promising cytotoxic activity. Most of the
tives 4c and 4d exhibited broad spectrum activity. Compound compounds that exhibited anticancer activity were nontoxic to
4d demonstrated almost equipotent activity against gastric normal fibroblast cells suggesting that they may serve as lead
cancer cell line NUGC (IC50=0.028µM) compared to the compounds for further development of new drugs.
standard CHS 828, 4d also showed almost four fold higher
activity against liver cancer cell lines compared to its 4-chlo-
Experimental
rophenyl substituted analog 4c. The superior cytotoxic activity
Chemistry All melting points were determined on a
of the 1,3,4-thiadiazole derivatives compared to those of their Stuart apparatus and the values given are uncorrected. IR
bioisosters 1,2,4-oxadiazoles and 1,3,4-oxadiazoles was previ- spectra (KBr, cm−1) were determined on a Shimadzu IR 435
ously reported.3) On the other hand, the 4-bromophenyl substi- spectrophotometer (Faculty of Pharmacy, Cairo University,
1
tuted mercaptotriazole 5d showed more potent activity against Egypt). H- and 13C-NMR spectra were recorded on Bruker
liver cancer HA22T than 5c.
Ascend 400MHz spectrophotometers (Microanalytical Unit,
To verify the effects of structural modification on the anti- Faculty of Pharmacy, Cairo University, Egypt) using tetra-
cancer activity, sixteen Mannich bases 6a–p incorporating methylsilane (TMS) as internal standard. Chemical shift val-
methylpiperazine, morpholine, diethylamine and benzylpi- ues are recorded in ppm on δ scale. The electron impact (EI)
peridine moieties were synthesized starting from the 1,2,4-tri- mass spectra were recorded on a Hewlett Packard 5988 spec-
azoles 5a–d. The screening results again revealed the superior trometer (Microanalysis Center, Cairo University, Egypt). El-
anticancer activity of the 4-chlorophenyl and 4-bromophenyl emental analyses were carried out at the Microanalysis Center,
substituted derivatives compared to their p-tolyl and 4-me- Cairo University, Egypt; found values were within 0.35% of
thoxyphenyl congeners. The p-tolyl derivatives proved to be the theoretical ones. Progress of the reactions was monitored
almost devoid of activity, while only one 4-methoxyphenyl using thin layer chromatography (TLC) sheets precoated with
derivative was active.
UV fluorescent silica gel Merck 60F 254 and were visualized
Methylpiperazine and morpholine derived Mannich bases using UV lamp.
were found to display significant cytotoxic activity. The 4-bro-
General Method for Preparation of 2a–d A mixture of
mophenyl derivative 6m incorporating a methylpiperazine isonicotinic acid hydrazide 1 (0.01mol) and the appropriate p-
moiety, showed more potent cytotoxic activity against gastric substituted phenyl isothiocyanate (0.01mol) in ethanol (40mL)
cancer NUGC (IC50=0.021µM) compared to the standard CHS was heated under reflux for 1h. The reaction mixture was
828 and exhibited significant activity against colon cancer cooled and the obtained solid was filtered, washed and crystal-
DLDI and liver cancer HEPG2. Meanwhile, the 4-chloro- lized from ethanol.
phenyl derivative 6j incorporating a morpholine moiety, was
2-Isonicotinyl-N-(4-methylphenyl)hydrazinecarbothio-
found to be the most potent among all the tested Mannich amide30,31) (2a) Yield: 95%; mp: 176–178°C; IR (KBr,
1
bases against liver cancer HEPG2 (IC50=0.028µM).
cm−1): 3275–3111 (3NH), 1681 (C=O), 1255 (C=S); H-NMR
Considering the Mannich bases bearing a diethylamino (DMSO-d6) δ: 2.48 (s, 3H, CH3), 7.11–7.28 (m, 4H, Ar-H), 7.83
moiety only the 4-chlorophenyl derivative 6k showed cyto- (d, 2H, J=5.9Hz, pyridine H-3, H-5), 8.75 (d, 2H, J=5.9Hz,
toxic activity against four cell lines while 6c and 6o exhibited pyridine H-2, H-6), 9.72, 9.78, and 10.81 (3s, 3H, NH, D2O
selective activity against breast cancer MCF.
exchangeable); MS (EI): m/z (%) 286 (M+, 29). Anal. Calcd for
Among the 4-methoxyphenyl Mannich bases, cytotoxic ac- C14H14N4OS: C, 58.72; H, 4.93; N, 19.57. Found: C, 58.94; H,
tivity was displayed only by the benzyl piperidine derivative 4.63; N, 19.25.
6h. This dramatic increase in activity may be attributed to the
2-Isonicotinyl-N-(4-methoxyphenyl)hydrazinecarbothio-
presence of the benzyl moiety. Moreover, the 4-bromophenyl amide30,31) (2b) Yield: 92%; mp: 170–172°C; IR (KBr,
derivative 6p exhibited almost equipotent cytotoxic activity cm−1): 3259–3109 (3NH), 1674 (C=O), C=S (1249); H-NMR
1
against gastric NUGC and nasopharyngeal carcinoma HONE1 (DMSO-d6) δ: 3.73 (s, 3H, OCH3), 6.88–7.38 (m, 4H, Ar-H),
compared to the standard CHS 828 as well as the highest po- 7.84 (d, 2H, J=5.9Hz, pyridine H-3, H-5), 8.75 (d, 2H,
tency against colon cancer.
J=5.9Hz, pyridine H-2, H-6), 9.71, 9.76 and 10.82 (3s, 3H,
From the previous findings, it might be concluded that the NH, D2O exchangeable); MS (EI): m/z (%) 302 (M+, 39). Anal.
presence of the bioactive methylpiprazine, morpholine and Calcd for C14H14N4O2S: C, 55.61; H, 4.67; N, 18.53. Found: C,
benzyl piperidine moieties play an important role in display- 55.42; H, 4.33; N, 18.79.
ing cytotoxic activity. Thus it is obvious that while some of
2-Isonicotinyl-N-(4-cholorophenyl)-2-isonicotionylhydra-
the compounds were not the most potent, their specific activ- zinecarbothioamide30,31) (2c) Yield: 94%; mp: 172–174°C;
ity against particular cell lines makes them of interest for fur- IR (KBr, cm−1): 3244–3122 (3NH), 1674 (C=O), 1255 (C=S);
ther development as anticancer drugs.
1H-NMR (DMSO-d6) δ: 7.17–7.47 (m, 4H, Ar-H), 7.85 (d, 2H,
J=5.9Hz, pyridine H-3, H-5), 8.77 (d, 2H, J=5.9Hz, pyridine
H-2, H-6), 9.92 (brs, 2H, NH, D2O exchangeable), 10.88 (s,
Conclusion
This study presented the synthesis and characterization of a 1H, NH, D2O exchangeable); MS (EI): m/z (%) 306 (M+, 67).
series of oxadiazoles, thiadiazoles, triazoles and sixteen Man- Anal. Calcd for C13H11ClN4OS: C, 50.90; H, 3.61; N, 18.26.
nich bases derived from 1,2,4-triazoles. All the synthesized Found: C, 50.65; H, 3.52; N, 17.98.
compounds were evaluated for their in vitro anticancer activ-
2-Isonicotinyl-N-(4-bromophenyl)-2-isonicotinoylhydra-

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
373
zinecarbothioamide30,31) (2d) Yield: 93%; mp: 175–177°C; C14H12N4S: C, 62.66; H, 4.51; N, 20.88. Found: C, 62.45; H,
IR (KBr, cm−1): 3244–3120 (3NH), 1672 (C=O), 1255 (C=S); 4.32; N, 20.94.
1H-NMR (DMSO-d6) δ: 7.46–7.54 (m, 4H, Ar-H), 7.86 (d,
N-(4-Methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-thiadiazol-
2H, J=5.9Hz, pyridine H-3, H-5), 8.78 (d, 2H, J=5.9Hz, 2-amine32) (4b) Yield: 80%; mp: >300°C; IR (KBr, cm−1):
1
pyridine H-2, H-6), 9.92, 9.96, 10.88 (3s, 3H, NH, D2O ex- 3261 (NH), 1635, 1612 (C=N); H-NMR (DMSO-d6) δ: 3.73 (s,
changeable); MS (EI): m/z (%) 351 (M+, 100). Anal. Calcd for 3H, OCH3), 6.96–7.58 (m, 4H, Ar-H), 8.02 (br s, 2H, pyridine
C13H11BrN4OS: C, 44.46; H, 3.16; N, 15.95. Found: C, 44.31; H, H-3, H-5), 8.77 (br s, 2H, pyridine H-2, H-6), 10.70 (s, 1H,
3.12; N, 16.15.
NH, D2O exchangeable); MS (EI): m/z (%) 284 (M+, 42). Anal.
General Method for Preparation of 3a–d A solution of Calcd for C14H12N4OS: C, 59.14; H, 4.25; N, 19.70. Found: C,
I2/KI (5%) was added to the appropriate carbothioamide 2a–d 58.99; H, 4.15; N, 19.96.
(0.01mol) in 4N NaOH (20mL), until persistent yellow color.
N-(4-Chlorophenyl)-5-(pyridin-4-yl)-1,3,4-thiadiazol-
The reaction mixture was refluxed for 4h, cooled and poured 2-amine32,34) (4c) Yield: 77%; mp: 238–240°C; IR (KBr,
onto ice cold water. The obtained solid was filtered, washed cm−1): 3257 (NH), 1633, 1614 (C=N); H-NMR (DMSO-d6) δ:
1
with water, dried and crystallized from ethanol.
7.40–7.71 (m, 4H, Ar-H), 8.18 (br s, 2H, pyridine H-3, H-5),
5-(Pyridin-4-yl)-N-p-tolyl-1,3,4-oxadiazol-2-amine32)
(3a) 8.86 (br s, 2H, pyridine H-2, H-6), 11.01 (s, 1H, NH, D2O ex-
Yield: 83%; mp: 298–300°C; IR (KBr, cm−1): 3481 (NH), 1604 changeable); MS (EI): m/z (%) 288 (M+, 36). Anal. Calcd for
1
(C=N); H-NMR (DMSO-d6) δ: 2.38 (s, 3H, CH3), 7.24 (d, 2H, C13H9ClN4S: C, 54.07; H, 3.14; N, 19.40. Found: C, 54.26; H,
J=5.9Hz, pyridine H-3, H-5), 7.27–7.34 (m, 4H, Ar-H), 8.57 3.02; N, 19.61.
(d, 2H, J=5.9Hz, pyridine H-2, H-6), 14.34 (s, 1H, NH, D2O
N-(4-Bromophenyl)-5-(pyridin-4-yl)-1,3,4-thiadiazol-
exchangeable); MS (EI): m/z (%) 252 (M+, 36). Anal. Calcd for 2-amine34) (4d) Yield: 82%; mp: 242–244°C; IR (KBr,
C14H12N4O: C, 66.65; H, 4.79; N, 22.21. Found: C, 66.84; H, cm −1): 3267 (NH), 1633, 1610 (C=N); H-NMR (DMSO-d6)
4.63; N, 22.43.
1
δ: 7.52–7.63 (m, 4H, Ar-H), 7.99 (br s, 2H, pyridine H-3, H-5),
N-(4-Methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol- 8.77 (br s, 2H, pyridine H-2, H-6), 10.88 (s, 1H, NH, D2O
2-amine32) (3b) Yield: 85%; mp: >300°C; IR (KBr, cm−1): exchangeable); MS (EI): m/z (%) 333 (M+, 10). Anal. Calcd for
1
3471 (NH), 1608 (C=N); H-NMR (DMSO-d6) δ: 3.81 (s, 3H, C13H9BrN4S: C, 46.86; H, 2.72; N, 16.81. Found: C, 46.95; H,
OCH3), 7.05 (d, 2H, J=5.9Hz, pyridine H-3, H-5), 7.25–7.34 2.56; N, 16.64.
(m, 4H, Ar-H), 8.57 (d, 2H, J=5.9Hz, pyridine H-2, H-6),
General Method for Preparation of 5a–d A solution of
14.31 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO) δ: the appropriate carbothioamide 2a–d (0.01mol) in 1N NaOH
55.92, 115.12, 122.00, 122.30, 127.11, 130.30, 133.74, 149.01, (30mL) was heated under reflux for 2h. The solution was
150.55, 160.27, 169.99; MS (EI): m/z (%) 269 (M+H, 16). Anal. cooled and acidified to pH 5–6 with acetic acid. The precipi-
Calcd for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, tate formed was filtered, washed with water and crystallized
62.88; H, 4.64; N, 21.08.
from ethanol.
N-(4-Chlorophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-2-
5-(Pyridin-4-yl)-4-p-tolyl-2H-1,2,4-triazole-3(4H)-thione35)
amine32) (3c) Yield: 86%; mp >300°C; IR (KBr, cm−1): (5a) Yield: 85%; mp: 224–226°C; IR (KBr, cm−1): 2654
1
1
3446 (NH), 1610 (C=N); H-NMR (DMSO-d6) δ: 7.26 (d, 2H, (SH), 1604 (C=N), 1284 (C=S); H-NMR (DMSO-d6) δ: 2.34
J=5.9Hz, pyridine H-3, H-5), 7.47–7.67 (m, 4H, Ar-H), 8.59 (s, 3H, CH3), 7.21–7.31 (m, 6H, 4 Ar-H and pyridine H-3, H-5),
(d, 2H, J=5.9Hz, pyridine H-2, H-6), 14.39 (s, 1H, NH, D2O 8.52 (s, 2H, pyridine H-2, H-6); MS (EI): m/z (%) 268 (M+,
exchangeable); MS (EI): m/z (%) 272 (M+, 30). Anal. Calcd for 78). Anal. Calcd for C14H12N4S: C, 62.66; H, 4.51; N, 20.88.
C13H9ClN4O: C, 57.26; H, 3.33; N, 20.55. Found: C, 56.99; H, Found: C, 62.79; H, 4.71; N, 20.62.
3.01; N, 20.67.
4-(4-Methoxyphenyl)-5-(pyridin-4-yl)-2H-1,2,4-triazole-
N-(4-Bromophenyl)-5-(pyridin-4-yl)-1,3,4-oxadiazol- 3(4H)-thione36) (5b) Yield: 82%; mp: 218–220°C; IR (KBr,
2-amine33) (3d) Yield: 81%; mp: 223–225°C; IR (KBr, cm−1): cm−1): 2561 (SH), 1608 (C=N), 1261 (C=S); 1H-NMR
3436 (NH), 1606 (C=N); H-NMR (DMSO-d6) δ: 7.26 (d, 2H, (DMSO-d6) δ: 3.80 (s, 3H, OCH3), 7.03–7.25 (m, 4H, Ar-H),
1
J=5.9Hz, pyridine H-3, H-5), 7.39–7.82 (m, 4H, Ar-H), 8.60 7.29 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.56 (d, 2H, J=6Hz,
(d, 2H, J=5.9Hz, pyridine H-2, H-6), 14.38 (s, 1H, NH, D2O pyridine H-2, H-6); MS (EI): m/z (%) 284 (M+, 100). Anal.
exchangeable); MS (EI): m/z (%) 317 (M+, 17). Anal. Calcd for Calcd for C14H12N4OS: C, 59.14; H, 4.25; N, 19.70. Found: C,
C13H9BrN4O: C, 49.23; H, 2.86; N, 17.67. Found: C, 49.45; H, 58.94; H, 4.02; N, 19.89.
2.76; N, 17.54.
4-(4-Chlorophenyl)-5-(pyridin-4-yl)-2H-1,2,4-triazole-
General Method for Preparation of 4a–d The appropri- 3(4H)-thione37) (5c) Yield: 86%; mp: 261–263°C; IR (KBr,
ate carbothioamide 2a–d (0.01mol) was added portion wise to cm−1): 2551 (SH), 1614 (C=N), 1280 (C=S); 1H-NMR
cold conc. H2SO4 (10mL) with stirring. The solution was kept (DMSO-d6) δ: 7.26–7.49 (m, 4H, Ar-H), 7.60 (d, 2H, J=6Hz,
at room temperature for 2h and then poured onto ice-cold pyridine H-3, H-5), 8.59 (d, 2H, J=6Hz, pyridine H-2, H-6);
water. The reaction mixture was stirred and allowed to stand MS (EI): m/z (%) 288 (M+, 100). Anal. Calcd for C13H9ClN4S:
for 1h. The separated orange solid was filtered, washed with C, 54.07; H, 3.14; N, 19.40. Found: C, 54.32; H, 3.34; N, 19.67.
water and crystallized from ethanol.
4-(4-Bromophenyl)-5-(pyridin-4-yl)-2H-1,2,4-triazole-
5-(Pyridin-4-yl)-N-p-tolyl-1,3,4-thiadiazol-2-amine32,34) (4a) 3(4H)-thione37) (5d) Yield: 84%; mp: 298–300°C; IR
Yield: 78%; mp: 227–229°C; IR (KBr, cm−1): 3263 (NH), (KBr, cm−1): 2559 (SH), 1608 (C=N), 1290 (C=S); H-NMR
1
1
1633, 1608 (C=N); H-NMR (DMSO-d6) δ: 2.26 (s, 3H, CH3), (DMSO-d6) δ: 7.26–7.42 (m, 4H, Ar-H), 7.73 (d, 2H, J=6Hz,
7.17–7.53 (m, 4H, Ar-H), 8.28 (brs, 2H, pyridine H-3, H-5), pyridine H-3, H-5), 8.60 (d, 2H, J=6.0Hz, pyridine H-2, H-6);
8.89 (brs, 2H, pyridine H-2, H-6), 10.94 (s, 1H, NH, D2O ex- MS (EI): m/z (%) 333 (M+, 28). Anal. Calcd for C13H9BrN4S:
changeable); MS (EI): m/z (%) 268 (M+, 56). Anal. Calcd for C, 46.86; H, 2.72; N, 16.81. Found: C, 46.66; H, 2.55; N; 16.64.

374
Chem. Pharm. Bull.
Vol. 63, No. 5 (2015)
General Method for Preparation of 6a–p A mixture of 69.77, 115.24, 121.56, 127.10, 129.26, 132.94, 146.85, 150.32,
each of triazole 5a–d (0.01mol), formaldehyde (40%, 1.5mL) 160.59, 171.48; MS (EI): m/z (%) 396 (M+, 72). Anal. Calcd for
and the appropriate substituted secondary amine (0.01mol) in C20H24N6OS: C, 60.58; H, 6.10; N, 21.20. Found: C, 60.77; H,
dioxane (20mL) was stirred for 1h and left overnight at room 6.32; N, 21.43.
temperature. The obtained solid was filtered, dried and crys-
4-(4-Methoxyphenyl)-2-(morpholinomethyl)-5-(pyridin-4-
tallized from ethanol.
yl)-2H-1,2,4-triazole-3(4H)-thione (6f) Yield: 75%; mp:
2-((4-Methylpiperazin-1-yl)methyl)-5-(pyridin-4-yl)-4-p- 182–184°C; IR (KBr, cm−1): 1610 (C=N), 1255 (C=S);
tolyl-2H-1,2,4-triazole-3(4H)-thione (6a) Yield: 78%; mp: 1H-NMR (DMSO-d6) δ: 2.93 (s, 4H, 2CH2 morpholine), 3.74
179–181°C; IR (KBr, cm−1): 1604 (C=N), 1274 (C=S); (s, 4H, 2CH2 morpholine), 3.89 (s, 3H, OCH3), 5.28 (s, 2H,
1H-NMR (DMSO-d6) δ: 2.31 (s, 3H, CH3), 2.42 (s, 3H, CH3), CH2), 7.04–7.22 (m, 4H, Ar-H), 7.24 (d, 2H, J=6Hz, pyridine
2.47 (s, 4H, 2CH2 of piperazine), 3.01 (s, 4H, 2CH2 of pipera- H-3, H-5), 8.60 (d, 2H, J=6Hz, pyridine H-2, H-6); 13C-NMR
zine), 5.77 (s, 2H, CH2), 7.19–7.35 (m, 4H, Ar-H), 7.37 (d, 2H, (DMSO-d6) δ: 50.84, 55.59, 66.91, 77.35, 115.28, 121.57,
J=6Hz, pyridine H-3, H-5), 8.60 (d, 2H, J=6Hz, pyridine 127.09, 129.29, 132.91, 147.06, 150.40, 160.67, 171.68; MS (EI):
H-2, H-6); 13C-NMR (DMSO-d6) δ: 21.42, 46.80, 54.72, 57.62, m/z (%) 383 (M+, 84). Anal. Calcd for C19H21N5O2S: C, 59.51;
77.03, 121.63, 127.86, 130.73, 130.83, 131.4, 132.80, 140.88, H, 5.52; N, 18.26. Found: C, 59.78; H, 5.36; N, 18.42.
147.29, 150.35, 169.72; MS (EI): m/z (%) 380 (M+, 40). Anal.
Calcd for C20H24N6S: C, 63.13; H, 6.36; N, 22.09. Found: C, (pyridin-4-yl)-2H-1,2,4-triazol-3(4H)-thione (6g) Yield: 70%;
2-((Diethylamino)methyl)-4-(4-methoxyphenyl)-5-
63.37; H, 6.32; N, 21.83.
mp: 179–181°C; IR (KBr, cm−1); 1608 (C=N), 1261 (C=S);
2-(Morpholinomethyl)-5-(pyridin-4-yl)-4-p-tolyl-2H-1,2,4- 1H-NMR (DMSO-d6) δ: 1.04 (t, 6H, J=7.2Hz, 2 CH2–CH3),
triazole-3(4H)-thione (6b) Yield: 75%; mp: 178–180°C; IR 2.85 (q, 4H, J=7.2Hz, 2 CH2–CH3), 3.81 (s, 3H, OCH3), 5.57
(KBr, cm−1): 1597 (C=N), 1247 (C=S); H-NMR (DMSO-d6) (s, 2H, CH2), 7.06–7.14 (m, 4H, Ar-H), 7.29 (d, 2H, J=6Hz,
1
δ: 2.44 (s, 3H, CH3), 2.92 (s, 4H, 2CH2 morpholine), 3.66 (s, pyridine H-3, H-5), 8.06 (d, 2H, J=6Hz, pyridine H-2, H-6);
4H, 2CH2 morpholine), 5.27 (s, 2H, CH2), 7.17–7.32 (m, 4H, 13C-NMR (DMSO-d6) δ: 13.21, 45.43, 60.34, 76.72, 119.90,
Ar-H), 7.35 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.56 (d, 2H, 126.41, 131.61, 134.05, 137.73, 152.05, 155.04, 165.28, 174.66;
J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6) δ: 21.41, MS (EI): m/z (%) 369 (M+, 64). Anal. Calcd for C19H23N5OS:
50.81, 66.88, 70.12, 121.59, 127.83, 130.75, 132.00, 132.29, C, 61.76; H, 6.27; N, 18.95. Found: C, 61.98; H, 6.13; N, 19.01.
140.64, 146.92, 150.34, 171.47; MS (EI): m/z (%) 367 (M+,
2-((4-Benzylpiperidin-1-yl)methyl)-4-(4-methoxyphenyl)-
54). Anal. Calcd for C19H21N5OS: C, 62.10; H, 5.76; N, 19.06. 5-(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione (6h) Yield:
Found: C, 62.32; H, 5.46; N, 19.12.
78%; mp: 161–163°C; IR (KBr, cm−1): 1600 (C=N), 1257
1
2-((Diethylamino)methyl)-5-(pyridin-4-yl)-4-p-tolyl-2H-1,2,4- (C=S); H-NMR (DMSO-d6) δ: 1.24–1.71 (m, 5H, 2 CH2+CH
triazole-3(4H)-thione (6c) Yield: 70%; mp: 168–170°C; IR piperidine), 2.49 (s, 4H, 2CH2 piperidine), 3.23 (s, 2H, CH2–
(KBr, cm−1); 1602 (C=N), 1278 (C=S); H-NMR (DMSO-d6) C6H5), 3.89 (s, 3H, OCH3), 5.27 (s, 2H, CH2), 7.02–7.25 (m,
1
δ: 1.23 (t, 6H, J=7.2Hz, 2 CH2–CH3), 2.45 (s, 3H, CH3), 2.92 9H, Ar-H), 7.28 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.58 (d,
(q, 4H, J=7.2Hz, 2 CH2–CH3), 5.78 (s, 2H, CH2), 7.19–7.28 2H, J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6) δ:
(m, 4H, Ar-H), 7.35 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.56 32.15, 37.44, 43.15, 51.32, 55.58, 70.37, 115.22, 121.56, 125.82,
(d, 2H, J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6): 127.20, 128.16, 129.08, 129.34, 133.06, 140.52, 146.78, 150.32,
δ: 13.03, 21.24, 45.66, 71.91, 121.51, 127.76, 130.52, 131.65, 160.60, 171.45; MS (EI): m/z (%) 471 (M+, 29). Anal. Calcd for
132.81, 140.40, 147.05, 150.10, 170.60; MS (EI): m/z (%) 353 C27H29N5OS: C, 68.76; H, 6.20; N, 14.85. Found: C, 68.54; H,
(M+, 55). Anal. Calcd for C19H23N5S: C, 64.56; H, 6.56; N, 6.32; N, 14.63
19.81. Found: C, 64.78; H, 6.33; N, 19.72.
4-(4-Chlorophenyl)-2-((4-methylpiperazin-1-yl)methyl)-
2-((4-Benzylpiperidin-1-yl)methyl)-5-(pyridin-4-yl)-4-p- 5-(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione
(6i) Yield:
tolyl-2H-1,2,4-triazole-3(4H)-thione (6d) Yield: 78%; mp: 78%; mp: 159–161°C; IR (KBr, cm−1): 1606 (C=N), 1269
1
171–173°C; IR (KBr, cm−1): 1600 (C=N), 1267 (C=S); (C=S); H-NMR (DMSO-d6) δ: 1.27 (s, 3H, CH3), 2.48 (s, 4H,
1H-NMR (DMSO-d6) δ: 1.29–1.71 (m, 5H, 2 CH2+CH pi- 2CH2 of piperazine), 3.12 (s, 4H, 2CH2 of piperazine), 5.76
peridine), 2.46 (s, 3H, CH3), 2.47 (s, 4H, 2CH2 piperidine), (s, 2H, CH2), 7.23–7.28 (m, 4H, Ar-H), 7.52 (d, 2H, J=6Hz,
3.24 (s, 2H, CH2–C6H5), 5.27 (s, 2H, CH2), 7.13–7.29 (m, pyridine H-3, H-5), 8.63 (d, 2H, J=6Hz, pyridine H-2, H-6);
9H, Ar-H), 7.34 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.57 (d, 13C-NMR (DMSO-d6) δ: 46.01, 49.68, 54.55, 71.95, 121.58,
2H, J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6) δ: 129.47, 130.12, 132.55, 132.75, 136.09, 146.68, 150.26, 169.51;
21.44, 32.17, 37.44, 43.16, 51.32, 70.70, 121.56, 125.83, 127.90, MS (EI): m/z (%) 400 (M+, 52). Anal. Calcd for C19H21ClN6S:
128.16, 129.09, 130.72, 132.13, 133.04, 140.53, 140.56, 146.67, C, 56.92; H, 5.28; N, 20.96. Found: C, 56.67; H, 5.32; N, 21.03.
150.31, 171.28; MS (EI): m/z (%) 455 (M+, 29). Anal. Calcd
for C27H29N5S: C, 71.18; H, 6.42; N, 15.37 Found: C, 71.27; H, yl)-2H-1,2,4-triazole-3(4H)-thione (6j) Yield: 75%; mp:
6.32; N, 15.43.
139–141°C; IR (KBr, cm−1): 1597 (C=N), 1278 (C=S);
4-(4-Chlorophenyl)-2-(morpholinomethyl)-5-(pyridin-4-
4-(4-Methoxyphenyl)-2-((4-methylpiperazin-1-yl)methyl)-5- 1H-NMR (DMSO-d6) δ: 2.90 (s, 4H, 2CH2 morpholine), 3.67
(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione (6e) Yield: 78%; (s, 4H, 2CH2 morpholine), 5.25 (s, 2H, CH2), 7.20–7.28 (m,
mp: 188–190°C; IR (KBr, cm−1): 1600 (C=N), 1266 (C=S); 4H, Ar-H), 7.50 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.60 (d,
1H-NMR (DMSO-d6) δ: 2.31 (s, 3H, CH3), 2.49 (s, 4H, 2CH2 2H, J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6) δ:
of piperazine), 2.99 (s, 4H, 2CH2 of piperazine), 3.89 (s, 3H, 50.81, 66.87, 70.26, 121.58, 129.51, 130.37, 132.57, 133.02,
OCH3), 5.32 (s, 2H, CH2), 7.03–7.21 (m, 4H, Ar-H), 7.23 (d, 136.47, 146.70, 150.52, 171.33; MS (EI): m/z (%) 387 (M+, 54).
2H, J=6Hz, pyridine H-3, H-5), 8.58 (d, 2H, J=6Hz, pyridine Anal. Calcd for C18H18ClN5OS: C, 55.74; H, 4.68; N, 18.06.
H-2, H-6); 13C-NMR (DMSO-d6) δ: 46.02, 50.25, 54.93, 55.56, Found: C, 55.98; H, 4.36; N, 18.32.

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
375
4-(4-Chlorophenyl)-2-((diethylamino)methyl)-5-(pyridin-4- H-2, H-6); 13C-NMR (DMSO-d6) δ: 32.14, 37.44, 43.13, 51.33,
yl)-2H-1,2,4-triazole-3(4H)-thione (6k) Yield: 70%; mp: 77.05, 121.57, 124.53, 125.85, 128.17, 129.58, 132.7, 133.31,
178–180°C; IR (KBr, cm−1); 1606 (C=N), 1269 (C=S); 133.44, 133.65, 140.49, 146.39, 150.49, 150.58, 171.05; MS (EI):
1H-NMR (DMSO-d6) δ: 1.24 (t, 6H, J=7.2Hz, 2 CH2–CH3), m/z (%) 520 (M+, 47). Anal. Calcd for C26H26BrN5S: C, 60.00;
2.92 (q, 4H, J=7.2Hz, 2 CH2–CH3), 5.77 (s, 2H, CH2), H, 5.03; N, 13.46. Found: C, 60.27; H, 5.32; N, 13.43.
7.22–7.29 (m, 4H, Ar-H), 7.53 (d, 2H, J=6Hz, pyridine H-3,
In Vitro Cytotoxic Assay Chemicals Fetal bovine
H-5), 8.64 (d, 2H, J=6Hz, pyridine H-2, H-6); MS (EI): m/z serum (FBS) and L-glutamine, were purchased from Gibco
(%) 373 (M+, 29). Anal. Calcd for C18H20ClN5S: C, 57.82; H, Invitrogen Co. (Scotland, U.K.). RPMI-1640 medium was
5.39; N, 18.73. Found: C, 57.65; H, 5.08; N, 18.91.
purchased from Cambrex (NJ, U.S.A.). Dimethyl sulfoxide
2-((4-Benzylpiperidin-1-yl)methyl)-4-(4-chlorophenyl)-5- (DMSO), CHS 828, penicillin, streptomycin and sulforho-
(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione (6l) Yield: 78%; damine B (SRB) were purchased from Sigma Chemical Co.
mp: 166–168°C; IR (KBr, cm−1): 1598 (C=N), 1267 (C=S); (Saint Louis, MO, U.S.A.).
1H-NMR (DMSO-d6) δ: 1.46–1.71 (m, 5H, 2 CH2 + CH
Cell Cultures Cell cultures were obtained from the Eu-
piperidine), 2.50 (s, 4H, 2CH2 piperidine), 3.23 (s, 2H, CH2– ropean Collection of cell Cultures (ECACC, Salisbury, U.K.)
C6H5), 5.27 (s, 2H, CH2), 7.12–7.29 (m, 9H, Ar-H), 7.51 (d, and human gastric cancer (NUGC), human colon cancer
2H, J=6Hz, pyridine H-3, H-5), 8.60 (d, 2H, J=6Hz, pyridine (DLD1), human liver cancer (HA22T and HEPG2), human
H-2, H-6); 13C-NMR (DMSO-d6) δ: 32.14, 37.44, 43.13, 51.33, breast cancer (MCF), nasopharyngeal carcinoma (HONE1)
77.06, 121.56, 125.85, 128.17, 129.08, 129.56, 132.72, 133.13, and normal fibroblast cells (WI38) were kindly provided by
136.40, 140.49, 146.44, 150.48, 150.57, 171.12; MS (EI): m/z the National Cancer Institute (NCI, Cairo, Egypt). They were
(%) 476 (M+, 65). Anal. Calcd for C26H26ClN5S: C, 65.60; H, grown as monolayer and routinely maintained in RPMI-1640
5.51; N, 14.71. Found: C, 65.42; H, 5.39; N, 14.64.
4-(4-Bromophenyl)-2-((4-methylpiperazin-1-yl)methyl)-5- glutamine and antibiotics (penicillin 100U/mL, streptomycin
(pyridin-4-yl)-2H-1,2,4-triazole-3(4H)-thione (6m) Yield: 100mg/mL), at 37°C in a humidified atmosphere containing
medium supplemented with 5% heat inactivated FBS, 2mM
80%; mp: 170–172°C; IR (KBr, cm−1): 1606 (C=N), 1271 5% CO2. Exponentially growing cells were obtained by plat-
(C=S); 1H-NMR (DMSO-d6) δ: 2.19 (s, 3H, CH3), 2.43 (s, ing 1.5×105 cells/mL for the six human cancer cell lines fol-
4H, 2CH2 piperazine), 2.74 (s, 4H, 2 CH2 piperazine), 5.77 lowed by 24h of incubation. The effect of the vehicle solvent
(s, 2H, CH2), 7.20–7.28 (m, 4H, Ar-H), 7.69 (d, 2H, J=6Hz, (DMSO) on the growth of these cell lines was evaluated in
pyridine H-3, H-5), 8.64 (d, 2H, J=6Hz, pyridine H-2, H-6); all the experiments by exposing untreated control cells to the
13C-NMR (DMSO-d6) δ: 45.63, 49.82, 54.63, 72.22, 121.61, maximum concentration (0.5%) of DMSO used in each assay.
124.44, 129.70, 129.76, 132.56, 133.22, 146.88, 150.37, 169.52;
MS (EI): m/z (%) 445 (M+, 43). Anal. Calcd for C19H21BrN6S:
Conflict of Interest The authors declare no conflict of
C, 51.24; H, 4.75; N, 18.87. Found: C, 51.44; H, 4.61; N, 18.99. interest.
4-(4-Bromophenyl)-2-(morpholinomethyl)-5-(pyridin-4-
yl)-2H-1,2,4-triazole-3(4H)-thione (6n) Yield: 82%; mp:
138–140°C; IR (KBr, cm−1): 1598 (C=N), 1276 (C=S);
1H-NMR (DMSO-d6) δ: 2.91 (s, 4H, 2CH2 morpholine), 3.73
(s, 4H, 2CH2 morpholine), 5.27 (s, 2H, CH2), 7.20–7.23 (m,
4H, Ar-H), 7.68 (d, 2H, J=6Hz, pyridine H-3, H-5), 8.63 (d,
2H, J=6Hz, pyridine H-2, H-6); 13C-NMR (DMSO-d6) δ:
50.81, 66.86, 70.25, 121.60, 124.59, 129.77, 132.57, 133.35,
133.54, 146.64, 150.51, 171.25; MS (EI): m/z (%) 432 (M+, 53).
Anal. Calcd for C18H18BrN5OS: C, 50.01; H, 4.20; N, 16.20.
Found: C, 50.29; H, 4.40; N, 16.42.
2-((Diethylamino)methyl)-4-(4-bromophenyl)-5-(pyridin-4-
yl)-2H-1,2,4-triazole-3(4H)-thione (6o) Yield: 75%; mp:
178–180°C; IR (KBr, cm−1): 1606 (C=N), 1271 (C=S);
1H-NMR (DMSO-d6) δ: 1.11 (t, 6H, J=7.2Hz, 2 CH2–CH3),
3.33 (q, 4H, J=7.2Hz, 2 CH2–CH3), 5.57 (s, 2H, CH2),
7.14–7.44 (m, 4H, Ar-H), 7.75 (d, 2H, J=6Hz, pyridine H-3,
H-5), 8.61 (d, 2H, J=6Hz, pyridine H-2, H-6); 13C-NMR
(DMSO-d6) δ: 13.02, 45.82, 71.93, 121.57, 124.24, 129.72,
132.50, 133.10, 133.27, 146.79, 150.28, 169.41; MS (EI): m/z
(%) 417 (M+, 90). Anal. Calcd for C18H20BrN5S: C, 51.68; H,
4.82; N, 16.74. Found: C, 51.93; H, 4.61; N, 16.43.
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