Synthesis and anticancer evaluation of some new 4-amino-3-(p-methoxybenzyl) -4,5-dihydro-1,2,4-triazole-5-one derivatives

Article abstract of DOI:10.1515/znb-2008-1109

4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-triazol-1-yl-acetic acid ethyl ester (2) was prepared from 4-amino-4,5-dihydro-1H-1,2,4-triazole-5-one (1) and ethyl bromoacetate. Compound 3 was synthesized by the condensation of 2 with hydrazine hydrate. The treatment of compound 3 with various aromatic aldehydes resulted in the formation of arylidene hydrazides as cis-trans conformers 4a-g. Thiosemicarbazide derivative 5 was prepared by the reaction of compound 3 with phenylisothiocyanate. Cyclization of 5 with sodium hydroxide resulted in the formation of compound 6. Treatment of 6 with benzyl bromide gave compound 7. Four of the newly synthesized compounds were screened for their anticancer activity against a panel of 60 cell lines derived from nine cancer types, namely, non-small cell lung, colon, breast, ovarian, leukemia, renal, melanoma, prostate and CNS cancers at a fixed dose of 10 μM.

Full text of DOI:10.1515/znb-2008-1109

Synthesis and Anticancer Evaluation of Some New 4-Amino-3-
(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
Olcay Bekircana, Murat Kucuka, Bahittin Kahvecib, and Hakan Bektasa
a Department of Chemistry, Faculty of Arts & Sciences, Karadeniz Technical University,
Trabzon, Turkey
b Department of Chemistry, Faculty of Arts & Sciences, Rize University, Rize, Turkey
Reprint requests to Dr. Olcay Bekircan. E-mail: obekircan@gmail.com
Z. Naturforsch. 2008, 63b, 1305 – 1314; received July 25, 2008
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-triazol-1-yl-acetic acid ethyl ester (2) was
prepared from 4-amino-4,5-dihydro-1H-1,2,4-triazole-5-one (1) and ethyl bromoacetate. Com-
pound 3 was synthesized by the condensation of 2 with hydrazine hydrate. The treatment of com-
pound 3 with various aromatic aldehydes resulted in the formation of arylidene hydrazides as cis-
trans conformers 4a – g. Thiosemicarbazide derivative 5 was prepared by the reaction of compound 3
with phenylisothiocyanate. Cyclization of 5 with sodium hydroxide resulted in the formation of com-
pound 6. Treatment of 6 with benzyl bromide gave compound 7. Four of the newly synthesized
compounds were screened for their anticancer activity against a panel of 60 cell lines derived from
nine cancer types, namely, non-small cell lung, colon, breast, ovarian, leukemia, renal, melanoma,
prostate and CNS cancers at a fixed dose of 10 µM.
Key words: 1,2,4-Triazoles, E/Z Geometrical Isomers, cis/trans Amide Conformers,
Benzylidenehydrazides, Anticancer Activity
Introduction
benzylidene derivatives of acid hydrazides are also as-
sociated with diverse biological activities [27, 28]. In
an attempt to obtain new compounds with possible
anticancer properties, some novel 1,2,4-triazole-5-one
derivatives were designed and synthesized, and their
anticancer properties were evaluated.
Cancer has always been among the most incurable
diseases, and man has been prone to this deadly illness
for ages. Even today, we lack the effective medicine or
treatment that can be applied to most cancer sufferers.
Thus, the quest for certain effective and less harmful
cancer drugs is the priority of science [1].
It was reported that compounds having triazole
moieties, such as vorozole, letrozole and anastrozole
appeared to be very effective aromatase inhibitors,
Results and Discussion
Chemistry
The synthetic pathway leading to the title com-
which in turn prevented breast cancer [2 – 4]. Some pounds is given in Scheme 1. The starting compound,
1,2,4-triazole compounds are considered interesting 3-(4-methoxybenzyl)-4-amino-4,5-dihydro-1,2,4-tri-
heterocycles since they possess important pharma- azole-5-one (1), was prepared according to the litera-
cological activities such as antifungal and antivi- ture [29]. Treatment of 1 with ethyl bromoacetate in
ral activities. For example, fluconazole [5], itracona- Na/absolute ethanol gave 1,2,4-triazol-1-yl-acetic acid
zole [6], ravuconazole [7], ICI 153066 [8], posacona- ethyl ester 2. The IR spectrum of compound 2 showed
zole [9], and voriconazole [10] are antifungal drugs. absorption bands at 3291 and 3210 cm−1 correspond-
Furthermore, certain 1,2,4-triazole derivatives have ing to the NH2 and at 1755 and 1709 cm−1 correspond-
been reported as anticonvulsants [11], antimicro- ing to ester C=O and triazole C=O groups, respec-
bial [12], antimalarial [13], antihypertensive[14], anal- tively. 1,2,4-Triazol-1-yl-acetic acid hydrazide 3 was
gesic [15], antiviral [16], antiinflammatory [17], an- prepared by the reaction of 2 and hydrazine hydrate in
tioxidant [18, 19], antitumor [20, 21], anti HIV [22], butanol.
fungicidal [23], insecticidal [24], herbicidal [25],
and pesticidal agents [26]. In addition to these, N- aldehydes in ethanol to give the corresponding Schiff
Compound 3 was condensed with various aromatic
c
0932–0776 / 08 / 1100–1305 $ 06.00 ꢀ 2008 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
Scheme 1. Synthesis of the compounds.
bases 4a – g in very good yield. In the 1H NMR spec- drazides may exist as E/Z geometrical isomers about
tra of compounds 4, NCH2, N=CH, and NH proton C=N double bonds and cis/trans amide conformers
signals were recorded as double singlets, and in the (Scheme 2) [30 – 33]. It is known that the N=CH
13C NMR spectra of these compounds NCH2, tri- double bond restricts rotation and gives rise to the
azole C-3, triazole C-5, and N=CH carbon signals formation of E and Z isomers with the E isomer
were also recorded as double peaks. According to dominating, and that the equilibration rate is rather
the literature, N-benzylidene derivatives of acid hy- low [30– 33]. It has been reported that when acid
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
1307
Table 1. 1H NMR and
13C NMR chemical shifts
and percentage of cis/trans
conformersa.
Compound Conformer NMR NCH2 N=CH OH
NH Triazole Triazole Percentage of
C-3
–
147.81
–
C-5
–
154.38
–
trans/cis
71.4
4a
4b
4c
4d
4e
4f
trans
cis
H
C
H
C
4.84
47.24 143.01
4.42 8.15
47.64 146.19
4.82 7.97
46.33 142.68
4.40 8.17
46.67 146.00
4.88 8.07
46.39 142.21
4.46 8.28
46.87 146.77
4.85 8.31
46.39 138.81
4.42 8.52
46.92 142.05
4.84
46.45 139.10
4.44 8.37 11.02 11.93
46.85 144.76
4.81 8.26
46.40 140.84
4.41 8.39 10.38 11.80
46.77 146.65
4.79 8.16
46.36 141.37
4.37 8.35
46.85 142.66
7.95
–
–
–
–
11.72
–
11.77
–
28.7
148.10
164.07
trans
cis
H
C
H
C
–
–
–
–
11.64
–
11.64
–
–
–
72.7
27.3
146.96
–
147.21
153.53
–
163.00
trans
cis
H
C
H
C
–
–
–
–
11.86
–
11.86
–
–
–
70.9
29.2
147.08
–
147.32
153.56
–
163.38
trans
cis
H
C
H
C
–
–
–
–
11.83
–
11.91
–
–
–
73.2
26.9
147.07
–
147.34
153.54
–
163.29
trans
cis
H
C
H
C
8.22 10.37 11.63
–
–
61.0
39.0
–
–
146.94
–
153.57
–
–
–
147.36
163.11
trans
cis
H
C
H
C
9.56 11.53
–
–
–
56.8
43.2
–
146.95
–
153.54
–
–
–
147.31
162.90
4g
trans
cis
H
C
H
C
–
–
–
–
11.50
–
11.59
–
–
–
69.6
30.4
146.98
–
147.26
153.58
–
162.68
a
200 MHz spectrometer; all
spectra in [D6]DMSO.
Scheme 2. E/Z Isomers and cis/trans amide conformers for compounds 4.
1
hydrazide derivatives are dissolved in polar solvents ratios can easily be determined by H NMR integra-
such as [D6]DMSO, the geometrical E isomers of tion.
these compounds undergo a rapid cis/trans amide equi-
librium, in which the trans conformer predominates
[30 – 33]. The E isomers and the cis/trans conformer
The chemical shift values of cis/trans conformers
belonging to protons of NCH2, N=CH, OH, and NH in
the 1H NMR and to carbons of NCH2, triazole C-3, tri-
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
Table 2. Sixty cell line in vitro anticancer screening (Percent
azole C-5, and N=CH in the 13C NMR spectra of com-
pounds 4 and the percentage ratios of cis/trans con-
formers are given in Table 1. These data prove the E
isomers and trans conformer structures (I) to be dom-
inant forms among the four possible structures [30–
33]. The proton signals of N=CH, NH, and OH of the
trans conformer are found at higher field than those of
the cis conformer. In contrast, the N-CH2 proton signal
of the trans conformer is found downfield compared to
the cis conformer, because of steric hindrance [30].
The condensation of the acid hydrazide 3 with
phenyl isothiocyanate resulted in the formation of
thiosemicarbazide 5. The IR spectra of 5 exhibited a
strong C=S absorption at 1246 cm−1. The 13C NMR
signal of this group was observed at δ = 180.71 ppm.
Growth, PG) results for compounds 2, 3, 6 and 7.
Panel/cell line
Percent growtha
2
3
6
7
Mean
Non-small cell lung
Cancer
A549/ATCC
EKVX
HOP-62
114.67 106.98 100.43 113.72 109
86.90 96.77 110.39 89.65 96
108.56 104.23 114.40 97.75 106
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
55.63
103.39
54.55
–
47.17
52
99
97.73 101.21 94.39
101.48 103.85 102.94 96.20 101
110.20 121.03 95.83 83.27 103
139.00 121.41 119.90 119.78 125
110.51
98.48 102.39 82.88
99
103.37 100.56 105.94 91.65 100.2b
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
107.29 113.97 121.36 114.60 114
104.55 107.00 131.84 94.77 110
98.71 108.04 108.19 99.94 104
101.82 105.47 100.23 110.19 104
109.21 109.69 106.34 111.44 109
1
The H NMR spectrum displayed three singlets due
to three different –NH groups at δ = 9.61, 9.73
and 10.29 ppm. Cyclization of 5 with sodium hydrox-
ide resulted in the formation of 1-(5-thioxo-1,2,4-tri-
azol-3-yl)-5-oxo-4,5-dihydro-1,2,4-triazole 6. The IR
spectrum of compound 6 showed a strong C=S absorp-
tion at 1358 cm−1, and the 13C NMR signal of this
group was observed at δ = 168.14 ppm. The IR spec-
trum of 6 displayed bands at 3448 and 1358 cm−1 as-
sociated with the N-H and C=S functionalities. In the
1H NMR spectrum of compound 6, the NH signal was
observed as a singlet at δ = 13.90 ppm. Therefore, the
compound was proven to be in the thionic form [34].
Treatment of 6 with benzyl bromide in Na/absolute
KM12
96.37 114.76
99.61 95.44 102
SW-620
Mean:
101.57 111.59 111.10 111.61 109
102.79 110.07 111.24 105.43 107.4b
Breast cancer
BT-549
HS 578T
MCF7
129.99 107.96 137.86 94.39 118
115.44 116.39 112.05 122.04 116
130.23 144.87 120.12 129.31 131
MDA-MB-231/ATCC 99.38
93.23 102.10 95.17
119.70 108.70 109.98 94.85 108
99.00 106.22 99.72 99.58 101
97
MDA-MB-435
NCI/ADR-RES
T-47D
104.46 110.88 103.06 110.94 107
Mean:
114.03 112.61 112.13 106.61 111.3b
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
1
ethanol gave the S-benzyl derivative 7. The H NMR
120.68 120.40 112.47 30.37
106.67 112.65 114.79 106.21 110
113.36 107.26 122.22 118.54 115
96
spectrum of compound 7 showed no signal belonging
to an NH group. Instead, the S-CH2 proton signals ap-
peared at δ = 4.88 ppm as a singlet, and the S-CH2
carbon signal at δ = 35.98 ppm.
91.67
97.17
91.41 96.08
94
102.44 104.88 105.41 104.24 104
103.79 127.62 106.23 108.51 112
106.44 111.66 108.76 93.99 105.2b
Mean:
Leukemia
CCRF-CEM
HL-6O(TB)
K-562
MOLT-4
RPMI-8226
SR
Anticancer activity
66.95 −13.82 −66.63 73.73
15
108
85
107
75
114.26 101.95
93.13 82.05
–
–
–
–
The tumor growth inhibition properties of com-
pounds 2, 3, 6, and 7 with the NCI codes NSC 741870,
NSC 741871, NSC 741872, and NSC 741873, selected
among compounds 1 – 7 by the National Cancer In-
stitute (NCI), USA, were screened on 60 human tu-
mor cell lines, derived from nine cancer types, namely,
non-small cell lung, colon, breast, ovarian, leukemia,
renal, melanoma, prostate, and CNS cancers [35, 36].
The results are expressed as percentage growth (PG) at
the single dose of test compounds of 10 µM. Smaller
80.60
93.41 109.27 118.54
85.88
4.45
76.35
70.12
92.55
73.69
75.16 68.53
–
–
49
Mean:
51.92 71.13 69.5b
tivity on the whole cell panel, although they did not
prove cytotoxic or cytostatic at the tested concentration
(10 µM). The screening data are presented in Table 2.
The performances of the four test compounds were
values represent better growth inhibitory activity. Neg- evaluated individually on each cell type as well as all
ative values show the tumoricidal activity. The com- cell lines. The sensitivity orders of the cancer cell types
pounds, in general, showed mild antiproliferative ac- to individual compounds were in the following orders
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
1309
Table 2 (continued).
Panel/cell line
cancer (108.76) > colon cancer (111.24) > breast can-
cer (112.13) > prostate cancer (127.37).
Percent growtha
2
3
6
7
Mean
Compound 7: leukemia (71.13) > non-small cell
lung cancer (91.65) > prostate cancer (92.83) > ovar-
ian cancer (93.99) > CNS cancer (102.05) > renal
cancer (102.36) > melanoma (103.64) > colon can-
cer (105.43) > breast cancer (106.61).
Renal cancer
786-0
A498
ACHN
CAKI-I
RXF393
SN12C
TK10
108.53
130.75
104.97
76.07
111.03
94.31
116.48
81.24
102.92
103.92
115.06
110.18
99.87
102.18
93.22
117.49
91.20
104.14
110.48 98.53 105
124.05 106.33 119
104.89 113.93 108
106.26 90.42
103.19 112.18 107
95.54 103.54 97
103.60 115.12 113
95.92 78.83 87
105.49 102.36 103.7b
93
The overall sensitivity order of the cell types to all
four test compounds is leukemia (69.5) > non-small
cell lung cancer (100.2) > renal cancer (103.7) >
CNS cancer (105.16) > ovarian cancer (105.21) >
melanoma (106.6) > colon cancer (107.4) > prostate
cancer (109.0) > breast cancer (111.3).
UO-31
Mean:
Melanoma
LOX IMVI
M14
MALME-3M
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Mean:
93.86
109.69
114.42
105.68
116.19
106.93
107.82
93.79
92.31
119.63
142.34
103.51
115.78
104.65
103.71
93.32
93.93 101.73
95
125.46 108.83 116
100.12 93.23 113
100.53 100.31 103
125.39 124.19 120
108.61 102.10 106
107.31 106.32 106
The order of overall effectiveness of the test com-
pounds against all tested cell lines is 7 (99.26) >
3 (103.40) > 2 (103.57) > 6 (104.66).
The highest antiproliferative activity was observed
against the CCRF-CEM leukemia cell line with aver-
age PG value of 15. Tumoricidal activity was observed
only with compounds 3 and 6 against CCRF-CEM cell
line with PG values of −13.82 and −66.63, respec-
tively. Because all four compounds showed promising
antitumor potential against the CCRF-CEM leukemia
cell line among all cancer cell lines, and compounds 3
and 6 were even tumoricidal, synthesis and anticancer
evaluation of structural analogs and various derivatives
of these compounds deserve attention.
97.14 92.37
94
106.05
109.41
107.31 103.64 106.6b
Prostate cancer
DU-145
PC-3
130.53
89.38
109.96
110.11
101.56
105.84
135.38 99.34 119
119.36 86.31
99
Mean:
127.37 92.83 109.0b
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
165.75
111.88
91.16
108.21
100.50
85.94
107.86
100.36
102.03
100.82
103.40
114.75 110.93 125
107.52 94.17 104
103.43 94.88
103.18 104.04 104
107.55 107.78 105
96.28 100.52 100
105.45 102.05 105.2b
104.66 99.29
94
99.85
104.27
101.03
112.32
Mean:
Overall mean: 103.57
Conclusion
a
Percent growth values represent the percentage of the cell growth
In conclusion, a series of 4-amino-3-(p-methoxy-
benzyl)-4,5-dihydro-1,2,4-triazole-5-one derivatives
was synthesized as potential anticancer agents.
Benzylidenehydrazide derivatives 4 appear as both
configurational (E/Z) and conformational (cis/trans)
isomers. The E geometrical isomers and trans amide
conformers are dominant forms based on 1H and
13C NMR data. Compounds 2, 3, 6, and 7 were
selected for a full 60-cell panel anticancer screening
at a fixed dose of 10 µM, where they showed mild
activity. The highest activity was observed against
leukemia cell types, with the lowest PG values. The
leukemia subtype CCRF-CEM was the most suscepti-
ble to anticancer activity of the test compounds, and
compounds 3 and 6 were even tumoricidal against this
cell line with negative PG values. The synthesis of
with respect to the initial cell count at a single compound concentra-
tion (10 µM); b these values are averages of all the percent growth
values for all four test compounds but for a single organ specific
cancers, e. g. non-small cell lung cancers.
(average PG values are provided in parantheses):
Compound 2: leukemia (76.35) > colon can-
cer (102.79) > renal cancer (102.92) > non-small cell
lung cancer (103.37) > melanoma (106.05) > ovar-
ian cancer (106.44) > prostate cancer (109.96) > CNS
cancer (112.32) > breast cancer (114.03).
Compound 3: leukemia (73.69) > non-small cell
lung cancer (100.56) > CNS cancer (100.82) > re-
nal cancer (104.14) > prostate cancer (105.84) >
melanoma (109.41) > colon cancer (110.07) > ovar-
ian cancer (111.66) > breast cancer (112.61).
Compound 6: leukemia (51.92) > CNS can- new analogs or derivatives of these compounds and
cer (105.45) > renal cancer (105.49) > non-small cell testing of their anticancer activity especially against
lung cancer (105.94) > melanoma (107.31) > ovarian leukemia cancers should provide explanation in terms
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
of structure – activity relationships and will probably δ = 29.41 (benzyl CH2), 46.27 (NCH2), 54.93 (OCH3), Ar-
C: [113.70 (2C), 127.56, 129.79 (2C), 157.91], 147.14 (tri-
azole C-3), 153.28 (triazole C-5), 165.92 (hydrazide C=O). –
Anal. for C12H16N6O3 (292.30): calcd. C 49.31, H 5.52,
N 28.75; found C 49.29, H 5.52, N 28.74.
yield better anticancer compounds.
Experimental Section
Chemistry
All chemicals used in this study were of high purity
and purchased from Merck (Darmstadt, Germany) and Fluka
(Buchs, Switzerland). Melting points were determined on a
Bu¨chi oil heated melting point apparatus (Bu¨chi Labortech-
General method for the synthesis of compounds 4
Compound 3 (0.01 mol) was refluxed with 0.01 mol of
the appropriate aldehyde in ethanol for 4 h. The mixture was
cooled, and the solid that separated was recrystallized from
an appropriate solvent to afford the desired compound.
1
nik, Flawil, Switzerland) and are uncorrected. H NMR and
13C NMR spectra (δ, ppm) were recorded on a Varian-
Mercury 200 MHz spectrometer (Varian Inc., Palo Alto,
CA, USA) using tetramethylsilane as an internal reference.
The IR spectra (λ, cm−1) were run on a Perkin-Elmer
1600 FTIR spectrophotometer (Perkin-Elmer, Beaconfields,
England) by using KBr pellets. Microanalyses were per-
formed on a Carlo Erba 1106 elemental analyzer (Carlo Erba,
Milan, Italy). Starting compound 1 was synthesized by a pub-
lished method [29].
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 3-bromo-benzylidenehydrazide (4a)
M. p. 202 – 203 ◦C (DMSO-water). Yield 87 %. – IR
(KBr): ν = 3339 (NH), 3220, 3186 (NH2), 1724 (triazole
C=O), 1672 (hydrazide C=O), 1635, 1589 (C=N) cm−1. –
1H NMR ([D6]DMSO): δ = 3.70 (s, 3H, OCH3), 3.81 (s,
2H, benzyl CH2), 4.84 and 4.42 (s, 2H, NCH2, trans and
cis conformers), 5.31 (s, 2H, NH2), Ar-H: [6.84 (d, 2H,
J = 8.4 Hz), 7.18 (d, 2H, J = 8.4 Hz), 7.33 – 7.43 (m, 1H),
7.57 – 7.71 (m, 2H)], 7.95 and 8.15 (s, 1H, N=CH, trans and
cis conformers), 11.72 and 11.77 (s, 1H, NH, trans and cis
conformers). – 13C NMR ([D6]DMSO): δ = 30.20 (benzyl
CH2), 47.24 and 47.64 (NCH2, trans and cis conformers),
55.71 (OCH3), Ar-C: [114.50 (2C), 122.91, 126.87, 128.38,
129.73, 130.52 (2C), 131.60, 133.20, 137.09 and 137.21
(trans and cis conformers), 158.70], 143.01 and 146.19
(N=CH, trans and cis conformers), 147.81 and 148.10 (tri-
azole C-3, trans and cis conformers), 154.38 and 164.07
(triazole C-5, trans and cis conformers), 167.92 (hydrazide
C=O). – Anal. for C19H19BrN6O3 (459.30l): calcd. C 49.69,
H 4.17, N 18.30; found C 49.69, H 4.15, N 18.32.
Synthesis of 4-amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-
1,2,4-triazol-1-yl-acetic acid ethyl ester (2)
A mixture of 1 (0.01 mol) and Na (0.01 mol) was re-
fluxed in absolute ethanol for 2 h. Then, ethyl bromoacetate
(0.01 mol) was added and refluxed for an additional 6 h.
After evaporating, the solid◦was recyrstallized from ethanol-
water (1 : 2). M. p. 97 – 98 C. Yield 65 %. – IR (KBr): ν =
3291, 3210 (NH2), 1755 (ester C=O), 1709 (triazole C=O),
1
1654 (C=N), 1240 (C–O) cm−1. – H NMR ([D6]DMSO):
δ = 1.22 (t, 3H, J = 6.0 Hz, CH3), 3.75 (s, 3H, OCH3),
3.85 (s, 2H, benzyl CH2), 4.18 (q, 2H, J = 6.0 Hz, OCH2),
4.55 (s, 2H, NCH2), 5.39 (s, 2H, NH2), Ar-H [6.90 (d, J =
7.4 Hz), 7.22 (d, J = 7.4 Hz)]. – 13C NMR ([D6]DMSO):
δ = 13.90 (CH3), 29.35 (benzyl CH2), 46.42 (NCH2), 54.91
(OCH3), 60.94 (OCH2), Ar-C: [113.70 (2C), 127.43, 129.72
(2C), 157.92], 147.53 (triazole C-3), 153.17 (triazole C-
5), 167.86 (ester C=O). – Anal. for C14H18N4O4 (306.32):
calcd. C 54.89, H 5.92, N 18.29; found C 54.81, H 5.95,
N 18.34.
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 4-fluoro-benzylidenehydrazide (4b)
M. p. 267 – 268 ◦C (DMSO-water). Yield 84 %. – IR
(KBr): ν = 3324 (NH), 3266, 3206 (NH2), 1719 (triazole
C=O), 1695 (hydrazide C=O), 1676, 1610 (C=N) cm−1. –
1H NMR ([D6]DMSO): δ = 3.70 (s, 3H, OCH3), 3.81 (s, 2H,
benzyl CH2), 4.82 and 4.40 (s, 2H, NCH2, trans and cis con-
formers), 5.31 (s, 2H, NH2), Ar-H: [6.84 (d, 2H, J = 8.4 Hz),
Synthesis of 4-amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-
1,2,4-triazol-1-yl-acetic acid hydrazide (3)
A solution of 2 (0.01 mol) and hydrazine hydrate 7.18 (d, 2H, J = 8.8 Hz), 7.28 (d, 2H, J = 8.8 Hz), 7.57 (d,
(0.01 mol) was refluxed in butanol for 4 h. The mixture was 2H, J = 8.4 Hz)], 7.97 and 8.17 (s, 1H, N=CH, trans and cis
cooled, and the solid that◦separated was recrystallized from conformers), 11.64 (s, 1H, NH). – 13C NMR ([D6]DMSO):
ethanol. M. p. 200 – 201 C. Yield 70 %. – IR (KBr): ν = δ = 29.36 (benzyl CH2), 46.33 and 46.67 (NCH2, trans and
3306 – 3166 (NH + 2NH2), 1726 (triazole C=O), 1668 (hy- cis conformers), 54.87 (OCH3), Ar-C: [113.65 (2C), 115.47
1
drazide C=O), 1613 (C=N), 1249 (C–O) cm−1. – H NMR (2C), 127.54, 128.90 (2C), 129.67 (2C), 130.46, 157.86,
([D6]DMSO): δ = 3.70 (s, 3H, OCH3), 3.77 (s, 2H, benzyl 165.13], 142.68 and 146.00 (N=CH, trans and cis conform-
CH2), 4.18 (s, 2H, NCH2), 4.25 (s, 2H, hydrazide NH2), 5.24 ers), 146.96 and 147.21 (triazole C-3, trans and cis conform-
(s, 2H, NH2), Ar-H: [6.85 (d, 2H, J = 7.8 Hz), 7.16 (d, 2H, ers), 153.53 and 163.00 (triazole C-5, trans and cis conform-
J = 7.8 Hz)], 9.16 (s, 1H, NH). – 13C NMR ([D6]DMSO): ers), 167.92 (hydrazide C=O). – Anal. for C19H19FN6O3
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1311
(398.40): calcd. C 57.28, H 4.81, N 21.09; found C 57.21, (C=N) cm−1. – 1H NMR ([D6]DMSO): δ = 3.70 (s, 3H,
H 4.73, N 21.03.
OCH3), 3.81 (s, 2H, benzyl CH2), 4.84 and 4.44 (s, 2H,
NCH2, trans and cis conformers), 5.32 (s, 2H, NH2), Ar-
H: [6.83 – 6.87 (m, 3H), 7.18 (d, 2H, J = 7.6 Hz), 7.34 –
7.42 (m, 1H), 7.86 and 7.75 (s, 1H, trans and cis conform-
ers)], 8.22 and 8.37 (s, 1H, N=CH, trans and cis conform-
ers), 10.37 and 11.02 (s, 1H, OH, trans and cis conform-
ers), 11.63 and 11.93 (s, 1H, NH, trans and cis conform-
ers). – 13C NMR ([D6]DMSO): δ = 29.41 (benzyl CH2),
46.45 and 46.85 (NCH2, trans and cis conformers), 54.88
(OCH3), Ar-C: [110.77 and 110.35 (trans and cis conform-
ers), 113.66 (2C), 118.20 and 118.47 (trans and cis conform-
ers), 122.39 and 121.07 (trans and cis conformers), 129.71
(2C), 130.02, 133.32, 137.02, 155.40 and 156.14 (trans and
cis conformers), 157.87], 139.10 and 144.75 (N=CH, trans
and cis conformers), 146.94 and 147.36 (triazole C-3, trans
and cis conformers), 153.57 and 163.11 (triazole C-5, trans
and cis conformers), 167.90 (hydrazide C=O). – Anal. for
C19H19BrN6O4 (475.30): calcd. C 48.01, H 4.03, N 17.68;
found C 48.05, H 4.03, N 17.67.
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 4-(trifluoromethyl)-benzylidenehydraz-
ide (4c)
M. p. 248 – 249 ◦C (DMSO-water). Yield 75 %. – IR
(KBr): ν = 3333 (NH), 3270, 3212 (NH2), 1716 (triazole
C=O), 1693 (hydrazide C=O), 1616, 1569 (C=N) cm−1. –
1H NMR ([D6]DMSO): δ = 3.73 (s, 3H, OCH3), 3.84 (s,
2H, benzyl CH2), 4.88 and 4.46 (s, 2H, NCH2, trans and
cis conformers), 5.35 (s, 2H, NH2), Ar-H: [6.85 (d, 2H,
J = 8.4 Hz), 7.20 (d, 2H, J = 8.4 Hz), 7.78 (d, 2H, J =
8.2 Hz), 7.93 (d, 2H, J = 8.2 Hz)], 8.07 and 8.28 (s, 1H,
N=CH, trans and cis conformers), 11.86 (s, 1H, NH). –
13C NMR ([D6]DMSO): δ = 29.40 (benzyl CH2), 46.39
and 46.87 (NCH2, trans and cis conformers), 54.88 (OCH3),
125.56 (CF3), Ar-C: [113.66 (2C), 126.67, 127.44 (2C),
127.55 (2C), 128.08, 129.72 (2C), 137.79, 157.88], 142.21
and 146.77 (N=CH, trans and cis conformers), 147.08 and
147.32 (triazole C-3, trans and cis conformers), 153.56 and
163.38 (triazole C-5, trans and cis conformers), 168.25 (hy-
drazide C=O). – Anal. for C20H19F3N6O3 (448.40): calcd.
C 53.57, H 4.27, N 18.74; found C 53.78, H 4.29, N 18.78.
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 2-hydroxy-5-methoxybenzylidene-
hydrazide (4f)
M. p. 197 – 198 ◦C (DMSO-water). Yield 95 %. – IR
(KBr): ν = 3340 (NH), 3334 (OH), 3275, 3191 (NH2),
1717 (triazole C=O), 1676 (hydrazide C=O), 1610, 1580
(C=N) cm−1. – 1H NMR ([D6]DMSO): δ = 3.69 (s, 3H,
OCH3), 3.70 (s, 3H, OCH3), 3.81 (s, 2H, benzyl CH2),
4.81 and 4.42 (s, 2H, NCH2, trans and cis conformers),
5.31 (s, 2H, NH2), Ar-H: [6.82 – 6.86 (m, 4H), 7.19 (d,
2H, J = 8.4 Hz), 7.25 (bs, 1H)], 8.26 and 8.39 (s, 1H,
N=CH, trans and cis conformers), 9.56 and 10.38 (s, 1H,
OH, trans and cis conformers), 11.53 and 11.80 (s, 1H, NH,
trans and cis conformers). – 13C NMR ([D6]DMSO): δ =
29.38 (benzyl CH2), 46.40 and 46.77 (NCH2, trans and cis
conformers), 54.88 (OCH3), 55.34 (OCH3), Ar-C: [111.71,
113.67 (2C), 117.10, 118.06 and 118.27 (trans and cis con-
formers), 120.17 and 118.72 (trans and cis conformers),
127.56, 129.72 (2C), 150.42 and 151.18 (trans and cis con-
formers), 151.99 and 152.13 (trans and cis conformers),
157.87], 140.84 and 146.65 (N=CH, trans and cis conform-
ers), 146.95 and 147.31 (triazole C-3, trans and cis con-
formers), 153.54 and 162.90 (triazole C-5, trans and cis con-
formers), 167.67 (hydrazide C=O). – Anal. for C22H26N6O5
(470.49): calcd. C 56.33, H 5.20, N 19.71; found C 56.34,
H 5.35, N 19.58.
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 2,4-dichlorobenzylidenehydrazide (4d)
M. p. 270 – 271 ◦C (DMSO-water). Yield 92 %. – IR
(KBr): ν = 3336 (NH), 3287, 3225 (NH2), 1711 (triazole
C=O), 1695 (hydrazide C=O), 1615, 1589 (C=N) cm−1. –
1H NMR ([D6]DMSO): δ = 3.71 (s, 3H, OCH3), 3.81 (s,
2H, benzyl CH2), 4.85 and 4.42 (s, 2H, NCH2, trans and
cis conformers), 5.30 (s, 2H, NH2), Ar-H: [6.84 (d, 2H,
J = 8.4 Hz), 7.18 (d, 2H, J = 8.4 Hz), 7.43 – 7.51 (m, 1H),
7.69 (bs, 1H), 7.90 – 8.04 (m, 1H)], 8.31 and 8.52 (s, 1H,
N=CH, trans and cis conformers), 11.83 and 11.91 (s, 1H,
NH, trans and cis conformers). – 13C NMR ([D6]DMSO):
δ = 29.38 (benzyl CH2), 46.39 and 46.91 (NCH2, trans and
cis conformers), 54.88 (OCH3), Ar-C: [113.65 (2C), 127.82,
128.14, 129.20, 129.71 (2C), 130.15, 133.52, 134.86, 135.85,
157.87], 138.81 and 142.05 (N=CH, trans and cis conform-
ers), 147.07 and 147.34 (triazole C-3, trans and cis conform-
ers), 153.54 and 163.29 (triazole C-5, trans and cis conform-
ers), 168.18 (hydrazide C=O). – Anal. for C19H18Cl2N6O3
(449.30): calcd. C 50.79, H 4.04, N 18.70; found C 50.76, H
4.11, N18.70.
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 5-bromo-2-hydroxybenzylidenehydraz-
ide (4e)
M. p. 234 – 235 ◦C (DMSO-water). Yield 78 %.
IR (KBr): ν = 3348 (NH+OH), 3275, 3191 (NH2),
4-Amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azol-1-yl-acetic acid 2,3,4-trimethoxybenzylidenehydrazide
(4g)
–
M. p. 179 – 180 ◦C (DMSO-water). Yield 78 %. – IR
1719 (triazole C=O), 1670 (hydrazide C=O), 1610, 1588 (KBr): ν = 3334 (NH), 3287, 3173 (NH2), 1708 (triazole
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O. Bekircan et al. · New 4-Amino-3-(p-methoxybenzyl)-4,5-dihydro-1,2,4-triazole-5-one Derivatives
C=O), 1687 (hydrazide C=O), 1615, 1593 (C=N) cm−1. – 1H NMR ([D6]DMSO): δ = 3.70 (s, 3H, OCH3), 3.72 (s, 2H,
1H NMR ([D6]DMSO): δ = 3.36 (s, 3H, OCH3), 3.70 benzyl CH2), 4.63 (s, 2H, NCH2), 5.04 (s, 2H, NH2), Ar-
(s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), H: [6.87 (d, 2H, J = 8.4 Hz), 7.05 – 7.10 (m, 2H), 7.14 (d,
3.82 (s, 2H, benzyl CH2), 4.79 and 4.37 (s, 2H, NCH2, 2H, J = 8.4 Hz), 7.27 – 7.28 (m, 3H)], 13.90 (s, 1H, NH). –
trans and cis conformers), 5.31 (s, 2H, NH2), Ar-H: [6.82 – 13C NMR ([D6]DMSO): δ = 29.35 (benzyl CH2), 40.60
6.92 (m, 3H), 7.18 (d, 2H, J = 8.4 Hz), 7.51 – 7.60 (m, (NCH2), 54.98 (OCH3), Ar-C: [113.71 (2C), 127.25, 127.61,
1H)], 8.16 and 8.35 (s, 1H, N=CH, trans and cis conform- 127.85 (2C), 128.13 (2C), 129.80 (2C), 136.29, 157.93],
ers), 11.50 and 11.59 (s, 1H, NH, trans and cis conform- 145.78 (triazole C-3), 146.83 (triazole C-3, thioxo ring),
ers). – 13C NMR ([D6]DMSO): δ = 29.41 (benzyl CH2), 152.34 (triazole C-5), 168.14 (triazole C-5, thioxo ring). –
46.36 and 46.85 (NCH2, trans and cis conformers), 54.90 Anal. for C19H19N7O2S (409.47): calcd. C 55.73, H 4.68,
(OCH3), 55.88 (OCH3), 60.37 (OCH3), 61.63 (OCH3), Ar- N 23.95; found C 55.73, H 4.66, N 23.93.
C: [108.56, 113.68 (2C), 119.96, 120.52, 127.60, 129.74
(2C), 139.71, 152.36 and 152.47 (trans and cis conform-
ers), 154.94 and 155.13 (trans and cis conformers), 157.90],
141.37 and 142.66 (N=CH, trans and cis conformers),
146.98 and 147.26 (triazole C-3, trans and cis conformers),
153.58 and 162.68 (triazole C-5, trans and cis conformers),
167.63 (hydrazide C=O). – Anal. for C22H26N6O5 (470.49):
calcd. C 56.16, H 5.57, N 17.86; found C 56.16, H 5.59,
N 17.63.
Synthesis of 1-(4-phenyl-5-benzylthio-1,2,4-triazol-3-yl)
methyl-4-amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-
1,2,4-triazole (7)
Compound 6 (0.01 mol) was refluxed with an equivalent
amount of sodium in absolute ethanol for 1 h. Then, benzyl
bromide (0.01 mol) was added and the mixture refluxed for
an additional 5 h. After evaporating, the solid was recyrstal-
lized from ethanol-water (1 : 1). M. p. 82 – 83 ◦C. Yield
67 %. – IR (KBr): ν = 3321, 3210 (NH2), 1707 (triazole
Synthesis of 1-(4-amino-3-p-methoxybenzyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl)-acetyl-4-phenyl thiosemicarbaz-
ide (5)
1
C=O), 1610, 1584 (C=N) cm−1. – H NMR ([D6]DMSO):
δ = 3.70 (s, 2H, benzyl CH2), 3.72 (s, 3H, OCH3), 4.32
(s, 2H, NCH2), 4.88 (s, 2H, SCH2), 5.06 (s, 2H, NH2), Ar-
H: [6.86 (d, 2H, J = 6.4 Hz), 7.10 – 7.14 (m, 4H), 7.25 – 7.29
(m, 4H), 7.39 – 7.45 (m, 4H)]. – 13C NMR ([D6]DMSO): δ =
29.30 (benzyl CH2), 35.98 (SCH2), 46.25 (NCH2), 54.97
(OCH3), Ar-C: [113.71 (2C), 12.72 (2C), 127.42, 128.39
(2C), 128.86 (2C), 128.97, 129.43 (2C), 129.83 (2C), 129.91,
131.99, 136.87, 157.74], 147.52 (triazole C-3), 148.47 (tri-
azole C-3, benzylthio ring), 152.21 (triazole C-5), 155.92
(triazole C-5, benzylthio ring). – Anal. for C26H25N7O2S
(499.59): calcd. C 62.51, H 5.04, N 19.63; found C 62.51,
H 5.00, N 19.76.
A mixture of 3 (0.01 mol) and phenyl isothiocyanate
(0.01 mol) was refluxed in ethanol for 2 h. The mixture was
cooled, and the solid that◦separated was recrystallized from
ethanol. M. p. 179 – 180 C. Yield 78 %. – IR (KBr): ν =
3338 – 3202 (3NH + NH2), 1717 (triazole C=O), 1681 (exo-
cyclic C=O), 1611 (C=N), 1246 (C=S) cm−1. – 1H NMR
([D6]DMSO): δ = 3.70 (s, 3H, OCH3), 3.79 (s, 2H, benzyl
CH2), 4.46 (s, 2H, NCH2), 5.31 (s, 2H, NH2), Ar-H: [6.84
(d, 2H, J = 7.8 Hz), 7.18 (d, 2H, J = 7.8 Hz), 7.29 – 7.43
(m, 5H)], 9.64 (s, 1H, NH), 9.73 (s, 1H, NH), 10.29 (s, 1H,
NH). – 13C NMR ([D6]DMSO): δ = 29.59 (benzyl CH2),
46.68 (NCH2), 55.10 (OCH3), Ar-C: [113.85 (2C), 125.34,
126.02, 127.59 (2C), 128.19 (2C), 129.97 (2C), 139.02,
158.08], 147.49 (triazole C-3), 153.58 (triazole C-5), 166.64
(exocyclic C=O), 180.71 (C=S). – Anal. for C19H21N7O3S
(427.48): calcd. C 53.38, H 4.95, N 22.94; found C 53.39,
H 4.95, N 22.73.
Pharmacology
Sixty human cancer cell line anticancer screening
The human tumor cell lines of the cancer screening panel
were grown in RPMI 1640 medium containing 5 % fetal
bovine serum and 2 mM L-glutamine. Cells were inoculated
into 96 well microtiter plates in 100 µL samples at plat-
ing densities ranging from 5000 to 40000 cells/well depend-
ing on the doubling time of individual cell lines. After cell
Synthesis of 1-(4-phenyl-5-thioxo-1,2,4-triazol-3-yl)methyl-
4-amino-3-p-methoxybenzyl-5-oxo-4,5-dihydro-1,2,4-tri-
azole (6)
◦
inoculation, the microtiter plates were incubated at 37 C,
5 % CO2, 95 % air and 100 % relative humidity for 24 h prior
to addition of the test compounds.
Solid thiosemicarbazide 5 (0.01 mol) was added portion-
wise to 20 mL of a 2 N NaOH solution. The reaction mix-
ture was refluxed for 2 h. The solution was cooled to r. t.
After 24 h, two plates of each cell line were fixed in
and acidified to pH 3 – 4 with 37 % HCl. The precipitated situ with TCA, to represent a measurement of the cell pop-
solid was filtered, washed thoroughly with water, dried, and ulation for each cell line at the time of compound addition
◦
recrystallized from ethanol-water (1 : 2). M. p. 219 – 220 C. (Tz). The compounds were solubilized in DMSO and diluted
Yield 83 %. – IR (KBr): ν = 3448 (NH), 3275, 3132 (NH2), to 20 µM, the complete medium containing 50 µg/mL gen-
1731 (triazole C=O), 1613, 1516 (C=N), 1358 (C=S) cm−1. – tamicin. Aliquots of 100 µL of the compound solution were
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1313
added to the appropriate microtiter wells already containing
100 µL of medium, resulting in the required final compound
concentration (10 µM).
If (MeanODtest −MeanODtzero) < 0, then
PG = 100×(MeanODtest −MeanODtzero
/MeanODtzero
)
Where:
Following compound addition, the plates were incubated
for an additional 48 h at 37 ◦C, 5 % CO2, 95 % air, and
100 % relative humidity. For adherent cells, the assay was ter-
minated by the addition of cold TCA. Cells were fixed in situ
by the gentle addition of 50 µL of cold 50 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 min at 4 ◦C.
The supernatant was discarded, and the plates were washed
five times with tap water and air dried. Sulforhodamine B
(SRB) solution (100 µL), 0.4 % (w/v) in 1 % acetic acid, was
added to each well, and plates were incubated for 10 min
at r. t. After staining, unbound dye was removed by washing
five times with 1 % acetic acid, and the plates were air dried.
Bound stain was subsequently solubilized with 10 mM trizma
base, and the absorbance was read on an automated plate
reader at a wavelength of 515 nm.
MeanODtzero = The average of optical density measure-
ments SRB-derived color just before exposure of cells to the
test compound.
MeanODtest = The average of optical density measure-
ment of SRB-derived color after 48 h exposure of cells to the
test compound.
MeanODctrl = The average of optical density measure-
ments of SRB-derived color after 48 h with no exposure of
cells to the test compound.
Smaller positive PG values are indicative of better
antiproliferative activity, and negative values show that
the compound is tumoricidal, killing already existing
cells.
The measured effect of the compound on a cell line was
calculated according to one or the other of the following two
expressions and expressed as percent growth (PG):
Acknowledgement
The authors thank the National Cancer Institute (NCI) at
Bethesda, USA, for the anticancer screening tests of the se-
lected compounds.
If (MeanODtest −MeanODtzero) ≥ 0, then
PG = 100×(MeanODtest −MeanODtzero
)
/(MeanODctrl −MeanODtzero
)
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