Synthesis and anticancer activity evaluation of new 1,2,3-triazole-4- carboxamide derivatives

Article abstract of DOI:10.1007/s00044-013-0841-8

Anticancer screening of several novel 1,2,3-triazoles has been performed. The 1,2,3-triazole derivatives were synthesized from available starting materials according to the convenient synthetic procedures using a multicomponent reaction which gave a wide

Full text of DOI:10.1007/s00044-013-0841-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2426–2438
DOI 10.1007/s00044-013-0841-8
ORIGINAL RESEARCH
Synthesis and anticancer activity evaluation of new 1,2,3-triazole-
4-carboxamide derivatives
•
Nazariy Pokhodylo Olga Shyyka Vasyl Matiychuk
•
Received: 3 June 2013 / Accepted: 9 October 2013 / Published online: 20 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Anticancer screening of several novel 1,2,3-
triazoles has been performed. The 1,2,3-triazole derivatives
were synthesized from available starting materials
according to the convenient synthetic procedures using a
multicomponent reaction which gave a wide access to tri-
azole derivatives production. The synthesized compounds
were tested for their anticancer activity in NCI60 cell lines.
It was observed that some compounds showed remarkable
anticancer activity. Two of them possessed a significant
activity on leukemia, melanoma, non-small cell lung, CNS,
ovarian, renal, and breast cancer. 5-Amino-1-p-tolyl-1H-
[1,2,3]triazole-4-carboxylic acid (2,5-dichloro-phenyl)-
amide showed a significant correlation in COMPARE
analysis.
synthase kinase-3 (Olesen et al., 2003), antagonists of
GABA receptors (Bascal et al., 1996; Biagi et al., 1993),
agonists of muscarinic receptors (Moltzen et al., 1994),
neuroleptic (Chakrabarti et al., 1989), as well as com-
pounds showing anti-HIV-1 (Alvarez et al., 1994), cyto-
toxic (Sanghvi et al., 1990), antihistaminic (Buckle et al.,
1986), and antiproliferative activities (Hupe et al., 1991).
Moreover, the triazole fragment includes an antibiotic
Cefatricin (Fass and Prior, 1978; Bohm and Karow, 1981)
and anticonvulsant drug Rufinamide (Portmann et al.,
1998). Thus, design and synthesis of novel triazole deriv-
atives are a perspective direction of medicinal chemistry
for the scientists working in this field.
Epidermal growth factor receptor (EGFR) is the first
growth factor receptor to be proposed as a target for cancer
therapy (Ciardiello and Tortora, 2008). The development
of specific antagonists targeting the EGFR has proved to be
a promising therapeutic concept. Although EGFR plays a
few roles in human body, its overexpression or overactivity
leads to a number of cancers as well (Zhang et al., 2007).
Actually, the majority of human epithelial cancers,
including lung cancer, breast cancer, and ovarian cancer,
are marked by functional activation of growth factors and
receptors of the EGFR family. Therefore, selective block-
ade of EGFR has been shown to be an effective therapeutic
approach against multiple epithelial cancers (Pao and
Chmielecki, 2010). EGFR plays an important role in car-
cinogenesis and is therefore an intriguing target for cancer
therapy. The present study is devoted to the synthesis and
evaluation of anticancer activity of new 1,2,3-triazole-4-
carboxamides. The aim of the research was to perform a
molecular docking of some 1,2,3-triazoles into EGFR
tyrosine kinase active site for purposeful searching of
EGFR tyrosine kinase inhibitors as potential anticancer
agents.
Keywords Anticancer activity ꢀ
1,2,3-Triazole-4-carboxamide ꢀ COMPARE analysis ꢀ
Molecular docking
Introduction
Triazoles and their derivatives are of great importance in
medicinal chemistry and can be used for the synthesis of
numerous heterocyclic compounds with different biologi-
cal activities. It is well known that triazoles are very
attractive targets for combinatorial library synthesis
because of their broad range of pharmaceutical activities.
They have been reported to be inhibitors of glycogen
N. Pokhodylo (&) ꢀ O. Shyyka ꢀ V. Matiychuk
Organic Chemistry Department, Ivan Franko National University
of Lviv, Kyryla and Mefodiya Str., 6, Lviv 79005, Ukraine
e-mail: pokhodylo@gmail.com
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Med Chem Res (2014) 23:2426–2438
2427
Scheme 1 Synthesis of 1,2,3-
triazole-4-carboxamide
derivatives
O
R3
R1 = 4-NO2
O
N
OH
R2
N
N
N3
OH
NH2
N
R1
R1
N
R2
N
R2COCH2CO2Et
NaOH
EtOH
O
2a-j
1a-j
MeONa
MeOH
N
4a-c
R1
O
3a-d
7a-d
N
R3
O
O
NH2
N
S
N
Cl
R4
N
S
N
N
N
R5
H
N
7a, b
R2
6a, b
N
H
R5
N
Me
8a-c
O
6, 7: R5 = H (a), Cl (b)
N
5a-f
R5
R1
N
O
N
N3
O
R1
MeONa
MeOH
N
N
R4
+
R4
N
H
R1
N
NH2
10a-l
H
9a-k
2a-s
N3
O
N
R1
N
R4
DBU
+
N
+
H2N
Me
MeCN
2a-s
Me
1a-k
11
N
O
N
N
O
N3
MeONa
MeOH
NH2
N
+
N
H
13
N
H
2s
12
Results and discussion
It has previously been shown that 3-aryl-2-chloroprop-
anals 6a, b were easily obtained by the Meerwein reaction
of arenediazonium chlorides with acrolein. Such reactions
were carried out in aqueous acetone solution in the pre-
sence of CuCl2 as catalyst. Aldehydes 6a, b were subjected
to heterocyclization with thiourea on heating in alcohol to
yield corresponding 2-amino-5-benzyl-1,3-thiazoles 7a,
b (Obushak et al., 2004). The reaction of 2-amino-5-ben-
zyl-1,3-thiazoles 7a, b with 1H-1,2,3-triazole acid chlo-
rides led to the corresponding amides 8a–c in good yields.
The reaction of azides with acetonitriles with activated
methylene group is a convenient method for 5-amino-1H-
1,2,3-triazoles synthesis. Using cyanacetic acid amides and
aryl azides, we obtained a series of new 5-amino-1H-1,2,3-
triazole-4-carboxamides 10a–l in good yields. Moreover,
recently, we have proposed a new multicomponent strategy
for 1-(R1-phenyl)-5-methyl-N-R2-1H-1,2,3-triazole-4-car-
boxamides in high yields by the reaction of arylazides with
dicetene and appropriate amines produced (Pokhodylo
et al., 2009a). In the current study, we have found that
multicomponent methods were suitable for preparation of
5-amino-1H-1,2,3-triazole-4-carboxamides 10a–l from
arylazides, amines, and 3-(3,5-dimethyl-1H-pyrazol-1-yl)-
3-oxopropanenitrile as donor of cyanoacetic acid fragment.
Chemistry
The compounds presented in the article were obtained in
a simple and convenient synthetic path (Scheme 1). The
1H-1,2,3-triazole 4-carboxylic acids 3a–d were synthe-
sized by base-catalyzed cyclocondensation of aryl azides
2a–c (prepared from sodium azide and diazonium salts
obtained from anilines 1a–c) with ethyl acetoacetate
according to the procedure described in the literature
(Pokhodylo et al., 2009c). It was found that the reaction of
1H-1,2,3-triazolyl-4-nitroarenes 3a–d with arylacetonitr-
iles in ethanol in the presence of an excess of sodium
hydroxide formed compounds 4a–c in good yields. The
1H-1,2,3-triazole amides 5a–f were synthesized by in situ
simple activation of acids 3a–d using CDI, carbodiimides
(EDC and DCC) or ready conversion to the corresponding
chlorides with thionyl or oxalyl chloride, in the presence
of a tertiary amine, in dichloromethane or acetonitrile
used as the reaction solvent. Overall yield for the amides
5a–f was 51–73 % that allowed quickly and without
chromatographic purification creating a library of such
compounds.
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Med Chem Res (2014) 23:2426–2438
3. Conformers generating (Omega2). Further program
Fred2 will choose minimum energy conformation for
each molecule.
Using commercially available or easily prepared azides and
amines, 5-amino-1H-1,2,3-triazole-4-carboxamides 10a–
l were obtained in a one-pot system in high yields and in
short time. The 5-aminotriazole 13 was prepared by the
cycloaddition reactions of arylazides with cyanacetyl in-
doles (Pokhodylo et al., 2009b).
4. 3D molecular docking (Fred2).
As a result, we obtained values of seven scoring func-
tions (Chemgauss2, Chemscore, PLP, Screenscore,
Shapegauss, Zapbind, and Consensus). Consensus scoring
function was selected to analyze the results because of its
property to result in ranking (compound ranking), which
includes values of all scoring functions.
Molecular docking
Research was performed by the method of molecular
docking as a computer approach in the search of molecules
with affinity to certain biotargets. Docking studies were
conducted with OpenEye Scientific Software program
package that includes Fred Receptor, Vida, Flipper,
Babel3, Omega2, and Fred2. Chrystallographic model of
EGFR tyrosine kinase was obtained from Protein Data
Bank (www.rcsb.org). The obtained 1,2,3-triazole deriva-
tives and well-known selective EGFR inhibitors, such as
Erlotinib, Lapatinib, Gefitinib, and others, were selected as
research objects. The obtained seven scoring function
values (Chemgauss2, Chemscore, PLP, Screenscore,
Shapegauss, Zapbind, and Consensus) were used for in
silico estimation of EGFR tyrosine kinase-compound
binding. Consensus (cumulative) scoring function ranking
allowed us to select compounds, which could prospectively
be selective EGFR tyrosine kinase inhibitors at the level of
erlotinib for future (in-depth) pharmacological studies as
well as could be used as templates for the synthesis of
various related analogs. With the help of Fred receptor, the
active site (biotarget) of EGFR tyrosine kinase was
obtained from crystallographic model for performing
molecular docking.
Analysis and ranking of the molecular docking results
were obtained using the selected compounds and crystal-
lographic model of EGFR tyrosine kinase with cumulative
scoring function (consensus) that allowed selecting 22
compounds for further evaluation of in vitro anticancer
activity. The interactions between EGFR tyrosine kinase-
active site and the most active compounds 10d and 10h are
shown in the Fig. 1.
Evaluation of anticancer activity in vitro
The synthesized 1,2,3-triazoles (5a–f, 8a–c, 10a–l, and 13)
were submitted and evaluated at the single concentration of
10-5 M toward panel of the *60 cancer cell lines. The
human tumor cell lines were derived from the nine different
cancer types: leukemia, non-small cell lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast cancers.
Primary anticancer assays were performed according to the
US NCI protocol, as described elsewhere (http://dtp.nci.nih.
gov; Monks et al., 1991; Boyd and Paull, 1995; Boyd, 1997;
Shoemaker, 2006). The results for each compound are
reported as the percent growth (GP) (Table 1). Range of
growth (%) shows the lowest and the highest growths that
were found among different cancer cell lines.
Molecular docking studies include the following stages:
1. Generating R-, S-, and cys-trans isomers of ligands
using program Flipper.
The synthesized 1,2,3-triazoles (5a–f, 8a–c, 10a–l, 13)
displayed moderate (5b, c, d, 8b, 10a, d, i, h) or low (5a, e,
f, 8a–c, 10b, c, e–g, j–l) activity in the in vitro screen on
tested cell lines. However, a selective influence of some
compounds on several cancer cell lines was observed
2. 3D optimization of isomers using program Hyper
Chem 7.5 (www.hyper.com) (molecular mechanics
method MM? and semi-empirical quantum-mechani-
cal method PM3).
Fig. 1 Compounds 10h (a) and
10d (b) docked in the active site
of EGFR tyrosine kinase in
comparison with selective
inhibitor Gefitinib
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Med Chem Res (2014) 23:2426–2438
2429
Table 1 Anticancer screening
data in concentration 10-5
Compound Mean Range of
growth (%) growth (%)
The most sensitive cell lines
Growth of the
most sensitive
cell lines
M
5a
5b
103.19
80.29
78.54–131.66 UO-31 (renal cancer)
78.54
89.82
46.61
49.91
54.10
56.59
61.49
62.03
52.03
56.21
64.20
65.30
68.04
58.37
60.62
74.23
76.16
77.59
81.74
82.59
67.83
83.61
85.12
88.90
80.00
K-562 (leukemia)
46.61–108.20 SNB-75 (CNS Cancer)
IGROV1 (ovarian cancer)
SF-539 (CNS cancer)
MDA-MB-468 (breast cancer)
HOP-92 (non-small cell lung cancer)
NCI-H522 (non-small cell lung cancer)
52.03–121.05 UO-31 (renal cancer)
A498 (renal cancer)
5c
89.85
97.13
UACC-62 (melanoma)
RPMI-8226 (leukemia)
MDA-MB-468 (breast cancer)
58.37–138.35 HOP-92 (non-small cell lung cancer)
PC-3 (prostate cancer)
5d
HOP-62 (non-small cell lung cancer)
RPMI-8226 (leukemia)
5e
5f
103.81
102.87
77.59–160.15 SR (leukemia)
MCF7 (breast cancer)
UO-31 (renal cancer)
67.83–116.41 UO-31 (renal cancer)
UACC-62 (melanoma)
SR (leukemia)
NCI-H522 (non-small cell lung cancer)
80.00–124.71 CAKI-1 (renal cancer)
8a
8b
100.04
92.49
A549/ATCC (non-small cell lung cancer) 80.30
53.93–138.01 SNB-75 (CNS Cancer)
53.93
54.81
68.04
73.35
75.49
75.63
62.67
63.27
70.75
74.94
76.62
55.94
56.67
59.59
60.87
68.23
75.78
69.40
74.28
HOP-62 (non-small cell lung cancer)
OVCAR-8 (Ovarian Cancer)
UO-31 (renal cancer)
SF-268 (CNS Cancer)
U251 (CNS Cancer)
8c
92.82
86.31
62.67–113.87 RPMI-8226 (leukemia)
CCRF-CEM (leukemia)
UO-31 (renal cancer)
EKVX (non-small cell lung cancer)
UACC-62 (melanoma)
10a
55.94–143.12 SK-MEL-5 (melanoma)
EKVX (non-small cell lung cancer)
RPMI-8226 (leukemia)
T-47D (breast cancer)
10b
10c
102.26
96.20
68.23–154.14 SR (leukemia)
MOLT-4 (leukemia)
69.40–149.48 HOP-92 (non-small cell lung cancer)
PC-3 (Prostate Cancer)
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Med Chem Res (2014) 23:2426–2438
Table 1 continued
Compound Mean Range of
growth (%) growth (%)
The most sensitive cell lines
Growth of the
most sensitive
cell lines
10d
53.33
-13.42–98.42
RXF 393 (renal cancer)
UO-31 (renal cancer)
-13.42
-7.62
-3.42
1.32
HOP-92 (non-small cell lung cancer)
SNB-75 (CNS Cancer)
786-0 (renal cancer)
2.96
10e
10f
95.78
106.99
95.24
71.45–119.00 HOP-92 (non-small cell lung cancer)
SNB-75 (CNS Cancer)
71.45
76.15
77.40
78.93
83.26
83.60
64.01
66.68
75.89
76.75
78.69
-27.30
-9.89
-4.64
-2.61
-1.64
56.31
60.93
62.57
87.03
89.06
81.40
81.69
82.49
71.71
77.35
78.38
73.67
78.51
T-47D (breast cancer)
78.93–154.29 SF-539 (CNS Cancer)
MCF7 (breast cancer)
SR (leukemia)
10 g
64.01–140.52 CCRF-CEM (leukemia)
SR (leukemia)
EKVX (non-small cell lung cancer)
HOP-92 (non-small cell lung cancer)
A498 (renal cancer)
10h
10i
64.36
88.03
-27.30–126.14 SNB-75 (CNS Cancer)
U251 (CNS Cancer)
SF-539 (CNS Cancer)
T-47D (breast cancer)
786-0 (renal cancer)
56.31–139.48 SK-MEL-5 (melanoma)
RPMI-8226 (leukemia)
MDA-MB-468 (breast cancer)
87.03–145.64 NCI-H522 (non-small cell lung cancer)
A498 (renal cancer)
10j
104.92
98.82
10k
81.40–127.82 RPMI-8226 (leukemia)
A498 (renal cancer)
MCF7 (breast cancer)
10l
13
100.31
98.00
71.71–135.21 K-562 (leukemia)
HOP-92 (non-small cell lung cancer)
HL-60(TB) (leukemia)
73.67–126.74 MCF7 (breast cancer)
MDA-MB-468 (breast cancer)
(Table 1). The compound 10d was highly active on renal
cancer RXF 393 cell line (GP = -13.42 %), while the
compound 10h was highly active on the CNS cancer SNB-
75 cell line (GP = -27.30 %). Compounds 10a, 10i were
quite active on the melanoma SK-MEL-5 cell line
(GP = 55.94 % and GP = 56.31 %, respectively) and
leukemia RPMI-8226 cell line (GP = 59.59 % and
GP = 60.93 %, respectively). Compounds 5b, 8b were
quite active on the CNS cancer SNB-75 cell line
(GP = 46.61 % and GP = 53.93 %, respectively). The
majority of the tested compounds displayed growth
inhibition on non-small cell lung cancer cell line HOP-92
(5b, d, 10c, d, e, g, l) and different cell lines of Leukemia
(5a, c, e, f, 8c, 10a, b, i, f, k, l). Finally, compounds 10d,
10h were selected for in vitro testing against a full panel of
about 60 tumor cell lines at tenfold dilutions of five con-
centrations (100, 10, 1, 0.1, and 0.01 lM). Based on the
cytotoxicity assays, three antitumor activity dose–response
parameters were calculated for each experimental agent
against each cell line: GI50—molar concentration of the
compound that inhibits 50 % net cell growth; TGI—molar
concentration of the compound leading to total inhibition;
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Med Chem Res (2014) 23:2426–2438
2431
and LC50—molar concentration of the compound leading
to 50 % net cell death. Values were calculated for each of
these parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the
value was expressed as greater or less than the maximum or
minimum concentration tested. Mean graph midpoints
(MG_MID) were calculated for each of the parameters,
giving an averaged activity parameter over all the cell lines
for each compound. For the calculation of the MG_MID,
insensitive cell lines were included with the highest con-
centration tested.
standard agents. Similarity of pattern to that of the seed is
expressed quantitatively as a Pearson correlation coeffi-
cient (PCC). The results obtained with the COMPARE
algorithm indicate that compounds high in this ranking
may possess a mechanism of action similar to that of the
seed compound. We used accessible online tool—NCI
COMPARE analysis—to discover the similarity of com-
pounds10d, 10h with the seed one (Table 4). Correlations
with PCC[0.6 were selected as significant. The compound
10h did not yield any significant activity correlation with
any of the standard agents. This may indicate that it has a
unique mode of anticancer action. The compound 10d
showed a significant correlation with nitroestrone and some
The most potent inhibition of human tumor cells was
found for compounds 10d and 10h (Table 2) (MG_MID
GI50 -4.52 and -4.12, respectively). The tested com-
pounds showed a broad spectrum of growth inhibition
activity against human tumor cells, as well as some dis-
tinctive patterns of selectivity. In general, compounds 10d,
10h selectively inhibited the growth of CNS cancer cell
lines. We found that the compounds 10d possessed sig-
nificant activity on non-small cell lung cancer cell line
HOP-92 (Log GI50 = -6.23); ovarian cancer cell lines,
OVCAR-4 and SK-OV-3 (Log GI50 = -5.79 and Log
GI50 = -5.55); and melanoma cell line MALME-3 M
(Log GI50 = -5.61). The compound 10h demonstrated
high growth inhibition on breast cancer line MDA-MB-
231/ATCC (Log GI50 = -5.19) and on ovarian cancer line
SK-OV-3 (Log GI50 = -4.86). Furthermore, these com-
pounds 10d, 10h appeared to be the most active against
CNS cancer cell lines SNB-75 (Log GI50 = -5.86 and Log
GI50 = -5.41, respectively), and the compound l pos-
sessed high activities on SF-539 and U251 cell lines (CNS)
(Log GI50 = -4.75 and Log GI50 = -4.93, respectively).
The most potent and selective cytotoxic activities against
separate tumor cell lines are shown in Table 3 and Fig. 2.
Table 3 The influences of compounds 10d, 10h on the growth of
individual tumor cell lines
Compound Disease
Cell line
Log
GI50
Log
TGI
10d
Leukemia
Leukemia
K-562
SR
-5.11 -4.00
-5.60 -4.22
-6.23 -5.23
Non-small cell
lung cancer
HOP-92
CNS cancer
CNS cancer
CNS cancer
CNS cancer
CNS cancer
Melanoma
SF-268
SF-295
SF-539
SNB-75
U25
-5.26 -4.00
-5.30 -4.00
-5.37 -4.00
-5.86 -5.22
-5.22 -4.00
-5.61 -4.00
-5.79 -4.00
-5.55 -4.00
-5.46 -4.30
-5.23 -4.00
-5.35 -4.00
MALME-3M
Ovarian cancer OVCAR-4
Ovarian cancer SK-OV-3
Renal cancer
Renal cancer
Breast cancer
Breast cancer
CNS cancer
CNS cancer
CNS cancer
Melanoma
RXF 393
TK-10
HS 578T
MDA-MB-231/ATCC -5.21 -4.00
10h
SF-539
-4.75 -4.00
-5.41 -4.00
-4.93 -4.00
-4.89 -4.00
-4.86 -4.00
-4.43 -4.00
-4.52 -4.00
SNB-75
U25
COMPARE analysis
MALME-3M
NCI’s COMPARE algorithm (Paull et al., 1989; Zaharevitz
et al., 2002; http://dtp.nci.nih.gov/docs/compare/compare.
html; Weinstein et al., 1997) allows us to suppose bio-
chemical mechanisms of action of novel compounds on the
basis of their in vitro activity profiles compared to those of
Ovarian cancer SK-OV-3
Renal cancer
Renal cancer
Breast cancer
RXF 393
TK-10
MDA-MB-231/ATCC -5.19 -4.00
Table 2 Summary of anticancer screening data at dose-dependent assay
Compound
N
Log GI50
N1
Log TGI
Range
MG_MID
N2
Range
MG_MID
10d
10h
59
59
20
8
-6.23 to -4.05
-5.41 to -4.01
-4.52
-4.12
4
0
-5.22 to -4.22
-4.05
-4.00
–
N number of human tumor cell lines tested at the 2nd stage assay, Ni number of sensitive cell lines (parameters Log GI50 and Log TGI\-4.00)
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Med Chem Res (2014) 23:2426–2438
Fig. 2 Anticancer activities of
the most active compounds 10d,
10h
6,50
6,00
5,50
5,00
4,50
4,00
3,50
Non-small cell lung cancer
(HOP-92)
CNS cancer (SF-539 )
CNS cancer (SNB-75)
CNS cancer (U25)
Melanoma (MALME-3M)
Ovarian cancer (SK-OV-3)
Renal cancer (RXF 393)
Renal cancer (TK-10)
Breast cancer (MDA-MB-
231/ATCC)
10d
10h
compound
Table 4 COMPARE analysis results for the tested compounds
Compound PCC Target
Target
vector
NSC
Count
common
cell lines
Seed Target Target mechanism of action
Stdev Stdev
10d
0.832 Nitroestrone
S321803 55
0.232 0.065 Inhibits estrogen sulfotransferase (EST), a progesterone-induced
secretory endometrial enzyme which affects estrogen receptor
levels
0.685 Flavoneacetic
acid
S347512 56
0.23 0.088 Flavone acetic acid exhibits an antiproliferative effect on
endothelial cells as a result of a superoxide-dependent
mechanism, which induces changes in permeability of the
vasculature of the tumor
0.663 Piperazine
alkylator
S344007 56
0.23 0.146 Alkylating agent, alkylate DNA at the N7 position of guanine
0.635 Spirohydantoin S172112 46
mustard
0.252 0.186 Bifunctional alkylating agent
10h
0.336 Nitroestrone
S321803 54
0.331 0.265 Inhibits estrogen sulfotransferase (EST), a progesterone-induced
secretory endometrial enzyme which affects estrogen receptor
levels
alkylating agents. This molecular target should be consid-
ered as the first priority and be explored for the hit-to-lead
optimization.
cell line HOP-92. The obtained results of antitumor
activities of such derivatives can be interesting with the
promise to obtain more selective and active anticancer
agents among 1,2,3-triazoles and prove the necessity of
further investigation.
In the present article, new derivatives of 1,2,3-triazole-
4-carboxamides were described. Molecular docking was
performed to search molecules with affinity to EGFR
tyrosine kinase as potent target for cancer therapy. In vitro
anticancer activity for the synthesized compounds was
evaluated. These preliminary results allowed identifying
the most active compounds: 22 of the synthesized com-
pounds were tested, and two of them, 10d, 10h, displayed
considerable antitumor activity against leukemia, mela-
noma, lung, CNS, ovarian, renal, and breast cancer cell
lines. In particular, the compound 10d can be defined as a
prospective antitumor agent with the values of logGI50 and
logTGI being -6.23 and -5.23, respectively, for NSCLC
Experimental
Materials and methods
1H NMR spectra were recorded on a Varian Mercury 400
1
instrument (400 MHz for H and 100 MHz for 13C) with
TMS or deuterated solvent as an internal reference. Mass
spectra were run using Agilent 1100 series LC/MSD with
an API-ES/APCI ionization mode. Satisfactory elemental
123

Med Chem Res (2014) 23:2426–2438
2433
analyses were obtained for new compounds (C 0.17,
HAr-2,6), 7.88 (dd, J = 9.4, 0.8 Hz, 1H, HBis-7), 7.68–7.55
(m, 4H, HAr-3,5 ? HBis-6 ? HArNH-2), 7.45 (d, J = 8.1 Hz,
1H, HArNH-6), 7.19 (t, J = 8.1 Hz, 1H, HArNH-5), 6.61 (d,
J = 8.1 Hz, 1H, HArNH-4), 3.79 (d, J = 0.9 Hz, 3H, HMeO),
2.70 (d, J = 0.8 Hz, 3H, HMe). 13C NMR (100 MHz,
DMSO-d6) d 165.4 (CBis-3), 160.1 (CArNH-3), 160.0 (CO),
157.1 (CBis-7a), 141.7 (CTr-4), 140.2 (CAr-1), 139.3 (CTr-5),
132.3 (CBis-5), 131.9 (CHBis-6), 134.5 (CAr-4), 129.4
(29CHAr-2,6), 129.6 (CHArNH-5), 126.7 (29CHAr-3,5),
126.4 (CAr-1), 120.7 (CBis-3a), 117.6 (CHBis-4), 113.5
(CHBis-7), 112.7 (CHArNH-6), 109.7 (CHArNH-4), 106.3
(CHArNH-2), 55.4 (CMeO), 10.4 (CMe). MS m/z 460, 462
(M?H)?. Calcd. for C24H18ClN5O3: C, 62.68; H, 3.95; N,
15.23; Found: C, 62.61; H, 3.92; N, 15.32 %.
H
0.21, and N 0.19). All compounds are in the form
of white solids. In NMR spectra data, the abbreviations Bis
means 2,1-benzisoxazole cycle; Tr—triazole; Tz—thia-
zole; Td—thiadiazole; and Fur—furane correspondently.
Chemistry
General procedure for the synthesis of 5-alkyl-1H-1,2,3-
triazole-4-carboxamides 5a–f and 8a–c
To the suspension of 1H-1,2,3-triazole-4-carboxylic acid
(1 mmol) in dry dichloromethane (DCM, 15 mL), oxalyl
chloride (0.17 mL, 2 mmol) and two drops of DMF were
added at 0 °C. The mixture was allowed to be stirred for
15 min at 0 °C and for *3 h at room temperature, until no
gas was observed. The solvent and the excess of oxalyl
chloride were then distilled off to dryness. The obtained acyl
chloride, without further purification, was dissolved in dry
DCM (20 mL), cooled at 0 °C, and Et3N (0.42 mL, 3 mmol)
was added under stirring followed by addition of appropriate
amines (1 mmol) in DCM (5 mL). The resulting mixture
was allowed to stir at room temperature for an appropriate
time, until complete conversion, as indicated by TLC. The
solution was washed with water, a 5 % hydrocloric acid
solution, sat. solution of sodium hydrocarbonate, and brine,
dried over anhydrous Na2SO4, concentrated in vacuo to
yield carboxamides 5a–f and 8a–c.
1-[3-(4-Chlorophenyl)-2,1-benzisoxazol-5-yl]-N-(furan-2-
ylmethyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (5c)
Yield 89 %, mp 144–145 °C. IR [cm-1]: 1688 (CO); 3371
1
(NH). H NMR (400 MHz, DMSO-d6): d 8.84 (t, J = 6.
0 Hz, 1H, NH), 8.41 (d, J = 0.8 Hz, 1H, HBis-4), 8.18 (d,
J = 8.7 Hz, 2H, HAr-2,6), 7.86 (dd, J = 9.4, 0.8 Hz, 1H,
H
Bis-7), 7.60 (d, J = 8.7 Hz, 2H, HAr-3,5), 7.57 (dd, J = 9.
4, 1.7 Hz, 1H, HBis-6), 7.45 (dd, J = 1.8, 0.8 Hz, 1H,
Fur-5), 6.33 (dd, J = 3.2, 1.8 Hz, 1H, HFur-4), 6.24 (d,
H
J = 3.2 Hz, 1H, HFur-3), 4.49 (d, J = 6.0 Hz, 2H, CH2), 2.
64 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6) d 165.4
(CBis-3), 160.1 (CO), 157.1 (CBis-7a), 149.7 (CFur-2), 141.7
(CTr-4), 141.3 (CHFur-5), 139.3 (CTr-5), 132.3 (CBis-5),
131.9 (CHBis-6), 134.5 (CAr-4), 129.4 (29CHAr-2,6), 126.7
(29CHAr-3,5), 126.4 (CAr-1), 120.7 (CBis-3a), 117.6
(CHBis-4), 113.5 (CHBis-7), 110.5 (CHFur-4), 108.7 (CHFur-3),
37.2 (CMeO), 10.4 (CMe). MS m/z 434, 436 (M?H)?. Calcd.
for C22H16ClN5O3: C, 60.91; H, 3.72; N, 16.14; Found: C, 60.
99; H, 3.75; N, 16.21 %.
5-Methyl-1-(3-phenyl-2,1-benzisoxazol-5-yl)-N-[3-(trifluo-
romethyl)phenyl]-1H-1,2,3-triazole-4-carboxamide (5a)
Yield 94 %, mp 167–168 °C. IR [cm-1]: 1665 (CO); 3330
1
(NH). H NMR (400 MHz, DMSO-d6): d 10.93 (s, 1H,
NH), 8.51 (s, 1H, HBis-4), 8.38 (s, 1H, HAr-2), 8.16 (d, J =
7.6 Hz, 2H, HPh-2,6), 8.12 (d, J = 9.0 Hz, 1H, HBis-7), 7.
93 (d, J = 9.0 Hz, 1H, HBis-6), 7.67–7.53 (m, 5H,
HPh-3,4,5 ? HAr-5,6), 7.43 (d, J = 7.6 Hz, 1H, HAr-3), 2.
62 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6): d 167.2
(CBis-3), 160.6 (CO), 157.1 (CBis-7a), 140.2 (CArNH-1),
139.6 (CTr-4), 138.4 (CTr-5), 132.5 (CArNH-3), 132.1
(CBis-5), 131.9 (CHBis-6), 130.5 (CHArNH-5), 130.4
(29CHPh-2,6 ? CPh-4), 127.5 (29CHPh-3,5), 127.4 (CPh-1),
126.0 (d, J = 274.3 Hz, CF3), 124.7 (CHArNH-6), 120.7
(CBis-3a), 120.1 (CHArNH-4), 117.7 (CHArNH-2), 117.2
(CBis-4), 113.6 (CBis-7), 10.2 (CMe). MS m/z 464 (M?H)?.
Calcd. for C24H16F3N5O2: C, 62.20; H, 3.48; N, 15.11;
Found: C, 62.21; H, 3.56; N, 15.18 %.
N-(2-chlorophenyl)-5-ethyl-1-(3-phenyl-2,1-benzisoxazol-
5-yl)-1H-1,2,3-triazole-4-carboxamide (5d) Yield 85 %,
1
mp 120–121 °C. IR [cm-1]: 1662 (CO); 3354 (NH). H
NMR (400 MHz, DMSO-d6): d 9.79 (s, 1H, NH), 8.47 (s,
1H, HBis-4), 8.39 (d, J = 7.9 Hz, 1H, HAr-2), 8.16 (d, J =
7.0 Hz, 2H, HPh-2,6), 7.90 (d, J = 9.5 Hz, 1H, HBis-7), 7.
64–7.48 (m, 5H, HPh-3,4,5 ? HBis-6 ? HAr-6), 7.37 (t,
J = 7.4 Hz, 1H, HAr-5), 7.17 (t, J = 7.4 Hz, 1H, HAr-4), 3.
13 (q, J = 7.4 Hz, 2H, CH2), 1.18 (t, J = 7.4 Hz, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6) d 167.3 (CBis-3),
160.3 (CO), 157.1 (CBis-7a), 144.2 (CTr-4), 138.1 (CTr-5),
140.8 (CAr-1), 132.4 (CBis-5), 132.3 (CHBis-6), 130.7 (CPh-4),
130.5 (29CHPh-2,6), 129.6 (CHAr-3), 129.1 (29CHPh-3,5),
128.1 (CPh-1), 127.4 (CAr-5), 120.9 (CBis-3a), 120.5 (CAr-2),
119.7 (CHAr-4), 117.7 (CHBis-4), 114.3 (CHAr-6), 113.5
(CHBis-7), 17.2 (CH2), 13.4 (CH3). MS m/z 444, 446
(M?H)?. Calcd. for C24H18ClN5O2: C, 64.94; H, 4.09; N, 15.
78; Found: C, 64.99; H, 4.13; N, 15.61 %.
1-(3-(4-Chlorophenyl)benzo[c]isoxazol-5-yl)-N-(3-methoxy-
phenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide
(5b)
Yield 91 %, mp 209–210 °C. IR [cm-1]: 1675 (CO); 3347
(NH). 1H NMR (400 MHz, DMSO-d6): d 10.29 (s, 1H, NH),
8.47 (d, J = 0.9 Hz, 1H, HBis-4), 8.20 (d, J = 8.1 Hz, 2H,
123

2434
Med Chem Res (2014) 23:2426–2438
1H NMR (400 MHz, DMSO-d6): d 11.72 (s, 1H, NH), 7.61
(t, J = 7.9 Hz, 1H, HAr-5), 7.42 (d, J = 7.6 Hz, 1H, HAr-4),
7.33 –7.20 (m, 7H, HPhC ? HAr-6 ? HTz), 7.16 (t, J = 7.
7 Hz, 1H, HAr-3), 4.11 (s, 2H, HCH2), 3.85 (s, 3H, HMeO), 2.
42 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6) d 159.0
(CO), 156.6 (CTz-2), 153.8 (CAr-2), 140.1 (CPh-1), 140.0
(CTr-4), 136.5 (CTr-5), 134.9 (CTz-5), 132.4 (CHAr-4), 131.6
(CTz-4), 128.6 (29CHPh-2,6), 128.6 (29CHPh-3,5), 128.6
(CHAr-6), 126.6 (CHPh-4), 123.8 (CAr-1), 121.1 (CHAr-5),
112.9 (CHAr-3), 56.1 (CH3O), 32.7 (CH2), 9.2 (CMe). MS
m/z 406 (M?H)?. Calcd. for C21H19N5O2S: C, 62.21; H, 4.
72; N, 17.27; Found: C, 62.22; H, 4.79; N, 17.33 %.
N-(3-chlorophenyl)-5-ethyl-1-(3-phenyl-2,1-benzisoxazol-
5-yl)-1H-1,2,3-triazole-4-carboxamide (5e) Yield 85 %,
1
mp 193–194 °C. IR [cm-1]: 1672 (CO); 3367 (NH). H
NMR (400 MHz, DMSO-d6): d 10.61 (s, 1H, NH), 8.45 (s,
1H, HBis-4), 8.22–8.07 (m, 3H, HPh-2,6 ? HAr-2), 7.89 (d,
J = 9.6 Hz, 1H, HBis-7), 7.83 (d, J = 7.9 Hz, 1H, HAr-6),
7.67–7.49 (m, 4H, HPh-3,4,5 ? HBis-6), 7.30 (t, J = 7.
5 Hz, 1H, HAr-5), 7.07 (d, J = 8.0 Hz, 1H, HAr-4), 3.13 (q,
J = 7.2 Hz, 2H, CH2), 1.18 (t, J = 7.2 Hz, 3H, CH3). 13C
NMR (100 MHz, DMSO-d6) d 167.3 (CBis-3), 160.2 (CO),
157.1 (CBis-7a), 144.2 (CTr-4), 138.1 (CTr-5), 139.2 (CAr-1),
134.8 (CAr-3), 132.4 (CBis-5), 132.3 (CHBis-6), 130.7
(CPh-4), 130.5 (29CHPh-2,6), 129.1 (29CHPh-3,5), 128.1
(CPh-1), 130.0 (CAr-5), 123.4 (CHAr-4), 120.9 (CBis-3a),
118.6 (CHAr-2), 117.7 (CHBis-4), 116.5 (CHAr-6), 113.5
(CHBis-7), 17.2 (CH2), 13.4 (CH3). MS m/z 444, 446
(M?H)?. Calcd. for C24H18ClN5O2: C, 64.94; H, 4.09; N,
15.78; Found: C, 65.11; H, 4.18; N, 15.83 %.
N-[5-(3-chlorobenzyl)-1,3-thiazol-2-yl]-5-methyl-1-(4-
methylphenyl)-1H-1,2,3-triazole-4-carboxamide (8c)
Yield 92 %, mp 210–211 °C. IR [cm-1]: 1655 (CO); 3392
(NH). 1H NMR (400 MHz, DMSO-d6): d 12.26 (s, 1H, NH),
7.49 (d, J = 8.2 Hz, 2H, HAr-2,6), 7.41 (d, J = 8.1 Hz, 2H,
HAr-3,5), 7.37–7.29 (m, 3H, HArC ? HTz), 7.29–7.21 (m,
2H, HArC), 4.11 (s, 2H, HCH2), 2.46 (s, 3H, HMe), 2.38 (s, 3H,
N-(4-chlorophenyl)-5-ethyl-1-(3-phenyl-2,1-benzisoxazol-
5-yl)-1H-1,2,3-triazole-4-carboxamide (5f) Yield 91 %,
mp 190–191 °C. IR [cm-1]: 1624 (CO); 3395 (NH). 1H NMR
(400 MHz, DMSO-d6): d 10.73 (s, 1H, NH), 8.53 (s, 1H,
H
MeAr). 13C NMR (100 MHz, DMSO-d6) d 159.9 (CO), 157.
3 (CTz-2), 143.6 (CAr-1), 140.7 (CArC-1), 139.3 (CArC-3),
137.4 (CTr-4), 135.4 (CTr-5), 133.8 (CTz-5), 133.3 (CAr-4),
131.6 (CTz-4), 131.2 (CArC-2), 130.8 (29CHAr-3,5), 128.9
(CArC-2), 127.8 (CArC-6), 127.2 (CArC-4), 125.9 (29CHAr-2,6),
32.1 (CH2), 21.4 (CMeAr), 10.2 (CMe). MS m/z 424 (M?H)?.
Calcd. for C21H18ClN5OS: C, 59.50; H, 4.28; N, 16.52; Found:
C, 59.53; H, 4.49; N, 16.62 %.
HBis-4), 8.15 (s, J = 7.6 Hz, 2H, HPh-2,6), 7.88-7.96 (m, 3H,
HBis-7 ? HAr-2,6), 7.65-7.55 (m, 4H, HPh-3,4,5 ? HBis-6), 7.
37 (d, J = 8.4 Hz, 2H, HAr-3,5), 3.03 (q, J = 7.6 Hz, 2H,
CH2), 1.05 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6) d 167.3 (CBis-3), 160.0 (CO), 157.1 (CBis-7a), 144.
4 (CTr-4), 138.3 (CTr-5), 138.1 (CAr-1), 132.5 (CBis-5), 132.1
(CHBis-6), 130.5 (CPh-4), 130.3 (29CHPh-2,6), 129.1
(29CHPh-3,5), 128.1 (CPh-1), 127.6 (29CHAr-3,5), 127.3
(CAr-4), 122.7 (29CHAr-2,6), 120.8 (CBis-3a), 117.7 (CHBis-4),
113.6 (CHBis-7), 17.2 (CH2), 13.5 (CH3). MS m/z 444, 446
(M?H)?. Calcd. for C24H18ClN5O2: C, 64.94; H, 4.09; N,
15.78; Found: C, 64.87; H, 4.11; N, 15.75 %.
General procedure for the synthesis of amides 10a, c–e,
g–i, k, l
An appropriate cyanacetamide 9 (1 mmol) was added to
the solution of sodium methoxide (54 mg, 1 mmol) in dry
methanol (10 mL). The solution of appropriate arylazide 2
(10.0 mmol) in dry methanol (5 mL) was added dropwise.
The mixture was stirred for 1 h. The resulting suspension
was filtered, and the solid product was washed with water
and methanol to give triazole 10 as a white solid.
N-(5-benzyl-1,3-thiazol-2-yl)-5-methyl-1-phenyl-1H-1,2,3-
triazole-4-carboxamide
(8a) Yield
92 %,
mp
1
198–199 °C. IR [cm-1]: 1677 (CO); 3315 (NH). H NMR
(400 MHz, DMSO-d6): 11.88 (s, 1H, NH), 7.82–7.49 (m,
5H, HPh), 7.37–7.16 (m, 6H, HPhC ? HTz), 4.11 (s, 2H,
CH2), 2.62 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6):
d 159.0 (CO), 156.7 (CTz-2), 140.1 (CPhC-1), 138.4 (CTr-4),
137.2 (CTr-5), 135.5 (CPh-1), 134.9 (CTz-5), 131.6 (CTz-5),
130.1 (CHPh-4), 129.8 (29CHPh-2,6), 128.6 (29CHPhC-2,6),
128.6 (29CHPhC-3,5), 126.6 (CHPhC-4), 125.5 (29CHPh-
3,5), 32.7 (CH2), 9.8 (CMe). MS m/z 376 (M?H)?. Calcd.
for C20H17N5OS: C, 63.98; H, 4.56; N, 18.65; Found: C, 64.
08; H, 4.79; N, 18.59 %.
General procedure for the synthesis of amides 10b, f, j
An appropriate amine 1 (1 mmol), arylazide 1 (1 mmol),
and DBU (0.15 mL, 1 mmol) were added to the solution of
3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile
(1 mmol) in dry acetonitrile (20 mL). The mixture was
heated under reflux during 30 min. Then, it was cooled to
room temperature, and the solid started to sediment. The
product was filtered and washed with methanol to give
triazole 10 as a white solid.
N-(5-benzyl-1,3-thiazol-2-yl)-1-(2-methoxyphenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxamide (8b) Yield
92 %, mp 210–211 °C. IR [cm-1]: 1689 (CO); 3393 (NH).
5-Amino-N-(2,4-dichlorophenyl)-1-(2-methylphenyl)-1H-1,
2,3-triazole-4-carboxamide (10a) Yield 88 %, mp
123

Med Chem Res (2014) 23:2426–2438
2435
138–139 °C. IR [cm-1]: 1671 (CO), 3028, 3335 (NH). H
NMR (400 MHz, DMSO-d6): d 9.29 (s, 1H, NH), 8.39 (d,
J = 8.9 Hz, 1H, HArNH-6), 7.55–7.39 (m, 4H, HAr), 7.37–
7.31 (m, 2H, HAr), 6.35 (s, 2H, NH2), 2.16 (s, 3H, HMe).
13C NMR (100 MHz, DMSO-d6): d 160.2 (CO), 146.2
(CTr-5), 135.9 (CAr-1), 134.2 (CAr-2), 133.4 (CArNH-1),
131.4 (CHAr-3), 130.5 (CHAr-6), 128.8 (CHAr-4), 128.0
(CTr-4), 127.8 (29CHArNH-3,5), 127.2 (CHAr-5), 124.0
(CHArNH-4), 122.6 (CHArNH-6), 120.6 (CArNH-2), 17.6
(CMe). MS m/z 362, 363, 364 (M?H)?. Calcd. for
C16H13Cl2N5O: C, 53.05; H, 3.62; N, 19.33; Found: C, 53.
15; H, 3.56; N, 19.37 %.
(CArNH-1), 132.5 (CAr-4), 131.4 (CHArNH-3), 130.9
(29CHAr-2,6), 129.7 (CHArNH-5), 125.6 (CTr-4), 125.1
(29CHAr-3,5), 123.7 (CHArNH-4), 123.0 (CHArNH-6), 116.
1 (CArNH-2), 21.2 (CMe). MS m/z 362, 363, 364 (M?H)?.
Calcd. for C16H13Cl2N5O: C, 53.05; H, 3.62; N, 19.33;
Found: C, 53.01; H, 3.67; N, 19.25 %.
1
5-Amino-N-(5-ethyl-1,3,4-thiadiazol-2-yl)-1-(4-methyl-
phenyl)-1H-1,2,3-triazole-4-carboxamide (10e) Yield
78 %, mp 259–260 °C. IR [cm-1]: 1628 (CO), 3025, 3341
(NH). H NMR (400 MHz, DMSO-d6): d 7.46 (d, J = 7.
9 Hz, 2H, HAr-3,5), 7.37 (d, J = 7.9 Hz, 2H, HAr-2,6), 6.
1
79 (s, 2H, NH2), 2.79 (q, J = 7.6 Hz, 2H), 2.36 (s, 3H,
Me), 1.21 (t, J = 7.6 Hz, 3H, HMe). 13C NMR (100 MHz,
5-Amino-N-benzyl-1-(4-methylphenyl)-1H-1,2,3-triazole-4-
carboxamide (10b) Yield 97 %, mp 197–198 °C. IR
[cm-1]: 1678 (CO), 3010, 3334 (NH). 1H NMR (400 MHz,
DMSO-d6): d 8.60 (t, J = 6.3 Hz, 1H, NH), 7.46 (d, J = 8.
4 Hz, 2H, HAr-2,6), 7.37 (d, J = 8.4 Hz, 2H, HAr-3,5), 7.
34 (d, J = 7.2 Hz, 2H, HPh-2,6), 7.28 (t, J = 7.2 Hz, 2H,
HPh-3,5), 7.20 (t, J = 7.2 Hz, 1H, HPh-4), 6.23 (s, 2H,
NH2), 4.47 (d, J = 6.3 Hz, 2H, CH2), 2.44 (s, 3H, HMe).
13C NMR (100 MHz, DMSO-d6) d 160.1 (CO), 144.8
(CTr-5), 139.8 (CAr-1), 138.5 (CPh-1), 132.5 (CAr-4), 130.9
(29CHAr-2,6), 128.4 (29CHPh-3,5), 127.5 (29CHPh-2,6),
127.1 (CHPh-4), 125.1 (29CHAr-3,5), 124.5 (CTr-4), 43.5
(CH2), 21.20 (CMe). MS m/z 308 (M?H)?. Calcd. for
C17H17N5O: C, 66.43; H, 5.58; N, 22.79; Found: C, 66.52;
H, 5.67; N, 22.73 %.
H
DMSO-d6) d 166.4 (CTd-1), 165.2 (CTd-2), 161.5 (CO),
144.1 (CTr-5), 138.6 (CArN-1), 134.0 (CAr-4), 130.7
(29CHAr-2,6), 124.0 (29CHAr-3,5 ? CTr-4), 24.2 (CEt),
21.4 (CMe), 15.0 (CEt). MS m/z 330 (M?H)?. Calcd. for
C14H15N7OS: C, 51.05; H, 4.59; N, 29.77; Found: C, 51.
17; H, 4.51; N, 29.83 %.
5-Amino-1-(3,5-dimethylphenyl)-N-(1-methylethyl)-1H-1,2,
3-triazole-4-carboxamide
(10f) Yield
85 %,
mp
182–183 °C. IR [cm-1]: 1667 (CO), 3030, 3321 (NH). H
NMR (400 MHz, DMSO-d6): d 7.60 (d, J = 8.3 Hz, 1H,
NH), 7.17 (s, 2H, HPh-2,6), 7.10 (s, 1H, HPh-4), 6.22 (s, 2H,
NH2), 4.14 (dq, J = 13.2, 6.5 Hz, 1H, HCH), 2.40 (s, 6H,
1
H
Me), 1.22 (d, J = 6.6 Hz, 6H, HMe). 13C NMR (100 MHz,
DMSO-d6) d 159.7 (CO), 146.2 (CTr-5), 139.5 (CArN-1), 139.
8 (29CAr-3,5), 130.3 (CHAr-4), 124.4 (CTr-4), 123.1
(29CHAr-2,6), 41.5 (CH), 23.2 (29CMe), 21.20 (29CMe).
MS m/z 274 (M?H)?. Calcd. for C14H19N5O: C, 61.52; H, 7.
01; N, 25.62; Found: C, 61.54; H, 7.11; N, 25.58 %.
5-Amino-N-(4-methoxyphenyl)-1-(4-methylphenyl)-1H-1,2,
3-triazole-4-carboxamide (10c) Yield 74 %, mp
1
219–220 °C. IR [cm-1]: 1639 (CO), 3038, 3345 (NH). H
NMR (400 MHz, DMSO-d6): d 9.85 (s, 1H, NH), 7.72 (d,
J = 9.1 Hz, 2H, HAr-2,6), 7.48 (d, J = 8.4 Hz, 2H,
H
ArNH-2,6), 7.38 (d, J = 8.4 Hz, 2H, HArNH-3,5), 6.82 (d,
J = 9.1 Hz, 2H, HAr-3,5), 6.34 (s, 2H, NH2), 3.75 (s, 3H,
MeO), 2.45 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6)
5-Amino-N-(2,6-dimethylphenyl)-1-(4-fluorophenyl)-1H-1,
2,3-triazole-4-carboxamide (10g) Yield 74 %, mp
1
H
206–207 °C. IR [cm-1]: 1657 (CO), 3061, 3338 (NH). H
d 160.1 (CO), 157.1 (CArNH-4), 144.8 (CTr-5), 139.8
(CAr-1), 132.7 (CArNH-1), 132.5 (CAr-4), 130.9 (29CHAr-2,6),
122.1 (29CHArNH-2,6), 124.5 (CTr-4), 125.1 (29CHAr-3,5),
114.5 (29CHArNH-3,5), 55.3 (CMeO), 21.20 (CMe). MS
m/z 324 (M?H)?. Calcd. for C17H17N5O2: C, 63.15; H, 5.30;
N, 21.66; Found: C, 63.23; H, 5.31; N, 21.72 %.
NMR (400 MHz, DMSO-d6): d 9.46 (s, 1H, NH), 7.65 (dd,
J = 8.8, 4.8 Hz, 2H, HAr-2,6), 7.37 (t, J = 8.8 Hz, 2H,
HAr-3,5), 7.06 (br.s, 3H, HArNH-3,4,5), 6.35 (s, 2H, NH2),
2.24 (s, 6H, HMe). 13C NMR (100 MHz, DMSO-d6) d 162.
5 (d, J = 247.5 Hz, CAr-4), 160.81 (CO), 146.5 (CTr-5),
135.4 (29CArNH-2,6), 134.2 (CArNH-1), 130.7 (CAr-1), 128.
0 (29CHArNH-3,5), 127.7 (d, J = 9.0 Hz, 29CHAr-2,6),
122.0 (CTr-5), 117.1 (d, J = 23.4 Hz, 29CHAr-3,5), 124.5
(CHArNH-4), 21.20 (CMe), 18.4 (29CMe). MS m/z 326
(M?H)?. Calcd. for C17H16FN5O: C, 62.76; H, 4.96; N,
21.53; Found: C, 62.82; H, 4.93; N, 21.67 %.
5-Amino-N-(2,5-dichlorophenyl)-1-(4-methylphenyl)-1H-1,
2,3-triazole-4-carboxamide (10d) Yield 79 %, mp
1
233–234 °C. IR [cm-1]: 1646 (CO), 3057, 3305 (NH). H
NMR (400 MHz, DMSO-d6): d 9.49 (s, 1H, NH), 8.24 (s,
1H, HArNH-6), 7.56 (d, J = 8.5 Hz, 1H, HArNH-3), 7.45 (d,
J = 8.2 Hz, 2H, HAr-3,5), 7.39 (d, J = 8.2 Hz, 2H,
HAr-2,6), 7.22 (d, J = 8.5 Hz, 1H, HArNH-4), 6.57 (s, 2H,
NH2), 2.38 (s, 3H, HMe). 13C NMR (100 MHz, DMSO-d6)
d 160.8 (CO), 146.0 (CTr-5), 139.8 (CArN-1), 132.7
5-Amino-N-(2,4-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
1,2,3-triazole-4-carboxamide (10h) Yield 94 %, mp
1
216–217 °C. IR [cm-1]: 1664 (CO), 3009, 3359 (NH). H
123

2436
Med Chem Res (2014) 23:2426–2438
NMR (400 MHz, DMSO-d6): 8.92 (s, 1H), 8.18 (dd, J = 8.
8, 1.2 Hz, 1H), 7.64 (ddd, J = 8.8, 4.8, 1.3 Hz, 2H), 7.38
(td, J = 8.8, 1.3 Hz, 2H), 6.61 (dd, J = 2.4, 1.3 Hz, 1H),
6.52–6.39 (m, 3H), 3.95 (d, J = 1.2 Hz, 3H), 3.79 (d, J =
1.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) d 162.5 (d,
J = 247.5 Hz, CAr-4), 159.9 (CO), 156.2 (CArNH-4), 149.5
(CArNH-2), 145.2 (CTr-5), 130.7 (CAr-1), 127.5 (d, J = 9.
0 Hz, 29CHAr-2,6), 122.2 (CTr-4), 121.1 (CArNH-1), 120.2
(CArNH-6), 117.3 (d, J = 23.4 Hz, 29CHAr-3,5), 104.1
(CArNH-5), 98.7 (CArNH-3), 56.1 (CMeO), 55.4 (CMeO). MS
m/z 358 (M?H)?. Calcd. for C17H16FN5O3: C, 57.14; H, 4.
51; N, 19.60; Found: C, 57.21; H, 4.47; N, 19.70 %.
122.2 (CTr-4), 120.8 (CHAr-2), 115.8 (d, J = 24.2 Hz,
29CHAr-3,5). MS m/z 366 (M?H)?. Calcd. for
C16H11F4N5O: C, 52.61; H, 3.04; N, 19.17; Found: C, 52.69;
H, 3.22; N, 19.11 %.
5-Amino-N-(2,4-dimethoxyphenyl)-1-[3-(trifluoromethyl)
phenyl]-1H-1,2,3-triazole-4-carboxamide (10l) Yield
86 %, mp 163–164 °C. IR [cm-1]: 1694 (CO), 3013, 3397
(NH). 1H NMR (400 MHz, DMSO-d6): d 8.93 (s, 1H, NH),
8.19 (d, J = 8.8 Hz, 1H, HArNH-5), 7.91 (br.s, 2H, HArNH),
7.87–7.77 (m, 2H, HAr), 6.69–6.55 (m, 3H, HAr ? NH2), 6.
47 (d, J = 8.7 Hz, 1H, HArNH-6), 3.96 (s, 3H, HMeO), 3.79
(s, 3H, HMeO). 13C NMR (100 MHz, DMSO-d6) d 159.7
(CO), 156.2 (CArNH-4), 149.5 (CArNH-2), 145.2 (CTr-5),
135.8 (CAr-1), 131.3 (q, J = 33.0 Hz, CAr-3), 131.0
(CHAr-5), 127.8 (CHAr-6), 125.6 (29CHAr-4), 123.7 (q,
J = 272.9 Hz, CF3), 122.2 (CTr-4), 121.1 (CArNH-1), 120.8
(CHAr-2), 120.2 (CArNH-6), 104.1 (CArNH-5), 98.8
(CArNH-3), 56.1 (CMeO), 55.4 (CMeO). MS m/z 408
(M?H)?. Calcd. for C18H16F3N5O3: C, 53.07; H, 3.96; N,
17.19; Found: C, 53.02; H, 3.87; N, 17.29 %.
5-Amino-N-(4-ethylphenyl)-1-(4-methoxyphenyl)-1H-1,2,3-
triazole-4-carboxamide (10i) Yield 92 %, mp 183–184
1
°C. IR [cm-1]: 1655 (CO), 3017, 3360 (NH). H NMR
(400 MHz, DMSO-d6): d 9.79 (s, 1H, NH), 7.69 (d, J = 8.
4 Hz, 2H, HArNH-3,5), 7.48 (d, J = 8.8 Hz, 2H, HArNH-3,5),
7.13–7.04 (m, 4H, HAr), 6.28 (s, 2H, NH2), 3.86 (s, 3H,
HMeO), 2.59 (q, J = 7.6 Hz, 2H, HEt), 1.21 (t, J = 7.6 Hz,
3H, HEt). 13C NMR (100 MHz, DMSO-d6) d 160.8 (CO),
159.9 (CArNH-4), 145.4 (CTr-4), 138.5 (CArN-4), 136.9
(CArNH-1), 128.0 (CAr-1), 127.7 (29CHAr-2,6), 126.0
(29CHArNH-2,6), 122.0 (CTr-5), 120.3 (29CHAr-3,5), 114.9
(29CHArNH-3,5), 55.7 (CMeO), 28.2 (CEt), 16.1 (CEt). MS
m/z 338 (M?H)?. Calcd. for C18H19N5O2: C, 64.08; H, 5.
68; N, 20.76; Found: C, 64.11; H, 5.75; N, 20.85 %.
Pharmacology
A primary anticancer assay was performed at *60 human
tumor cell lines panel derived from nine neoplastic dis-
eases, in accordance with the protocol of the Drug Evalu-
ation Branch, National Cancer Institute, Bethesda. The
tested compounds were added to the culture at a single
concentration (10-5 M). and the cultures were incubated
for 48 h. End-point determinations were made with a
protein binding dye, sulforhodamine B (SRB). Results for
each tested compound were reported as the percent of
growth of the treated cells compared with the untreated
control cells. The percentage growth was evaluated spec-
trophotometrically versus controls not treated with test
agents. The cytotoxic and/or growth inhibitory effects of
the most active selected compounds were tested in vitro
against the full panel of about 60 human tumor cell lines at
tenfold dilutions of five concentrations ranging from 10-4
to 10-8 M. The 48-h continuous drug exposure protocol
was followed, and an SRB protein assay was used to
estimate cell viability or growth.
5-Amino-N-cyclopentyl-1-[3-(trifluoromethyl)phenyl]-1H-1,
2,3-triazole-4-carboxamide (10j) Yield 93 %, mp
1
152–153 °C. IR [cm-1]: 1679 (CO), 3026, 3348 (NH). H
NMR (400 MHz, DMSO-d6): 7.95–7.87 (m, 2H, HAr), 7.
85–7.78 (m, 3H, HAr ? NH), 6.50 (s, 2H, NH2), 4.25 (h,
J = 7.3 Hz, 1H, CH), 2.00–1.83 (m, 2H, CH2), 1.81–1.68
(m, 2H, CH2), 1.67–1.50 (m, 4H, CH2). 13C NMR
(100 MHz, DMSO-d6) d 159.9 (CO), 145.2 (CTr-5), 135.8
(CAr-1), 131.3 (q, J = 32.5 Hz, CAr-3), 131.0 (CHAr-5),
127.8 (CHAr-6), 125.6 (29CHAr-4), 123.7 (q, J = 271.
3 Hz, CF3), 122.2 (CTr-4), 120.8 (CHAr-2), 50.5 (CH), 32.7
(29CH2), 24.2 (29CH2). MS m/z 340 (M?H)?. Calcd. for
C15H16F3N5O: C, 53.10; H, 4.75; N, 20.64; Found: C, 53.
19; H, 4.63; N, 20.53 %.
5-Amino-N-(4-fluorophenyl)-1-[3-(trifluoromethyl)phenyl]-
Using the seven absorbance measurements [time zero,
(Tz), control growth in the absence of drug, (C), and test
growth in the presence of drug at the five concentration
levels (Ti)], the percentage growth was calculated at each
of the drug concentration levels. Percentage growth inhi-
bition was calculated as
1H-1,2,3-triazole-4-carboxamide (10k) Yield 95 %, mp
1
211–212 °C. IR [cm-1]: 1668 (CO), 3022, 3383 (NH). H
NMR (400 MHz, DMSO-d6): d 10.19 (s, 1H, NH), 8.07–7.
65 (m, 6H, HAr), 7.04 (t, J = 8.8 Hz, 2H, HArNH-3,5), 6.69
(s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6) d 159.68
(CO), 159.7 (d, J = 250.1 Hz, CArNH-4), 145.19 (CTr-5),
135.8 (CAr-1), 131.3 (q, J = 32.7 Hz, CAr-3), 131.0 (CHAr-5),
127.9 (CArNH-1), 121.5 (d, J = 9.0 Hz, 29CHAr-2,6), 127.8
(CHAr-6), 125.6 (29CHAr-4), 123.7 (q, J = 272.1 Hz, CF3),
[(Ti - Tz)/(C - Tz)] 9 100 for concentrations for
which Ti C Tz
[(Ti - Tz)/Tz] 9 100 for concentrations for which
Ti \ Tz.
123

Med Chem Res (2014) 23:2426–2438
2437
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Three dose–response parameters were calculated for
each compound. Growth inhibition of 50 % (GI50) was
calculated from [(Ti - Tz)/(C - Tz)] 9 100 = 50, which
is the drug concentration resulting in a 50 % lower net
protein increase in the treated cells (measured by SRB
staining) compared to the net protein increase seen in the
control cells. The drug concentration resulting in total
growth inhibition (TGI) was calculated from Ti = Tz. The
LC50 (concentration of drug resulting in a 50 % reduction
in the measured protein at the end of the drug treatment
compared to that at the beginning) indicating a net loss of
cells following treatment was calculated from [(Ti - Tz)/
Tz] 9 100 = -50. Values were calculated for each of
these three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the
value for that parameter was expressed as more or less than
the maximum or minimum concentration tested. The log-
GI50, logTGI, and logLC50 were then defined as the mean
of the logs of the individual GI50, TGI, and LC50 values,
respectively. The lowest values were obtained with the
most sensitive cell lines. Compounds having these values
B4 were declared to be active.
Acknowledgments The authors are grateful to Dr. V. L. Narayanan
from the Drug Synthesis and Chemistry Branch, the National Cancer
Institute, Bethesda, MD, USA, for his help in in vitro evaluation of
anticancer activity.
Conflict of interest The authors have declared no conflict of
interest.
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4-carboxylic acid derivatives. A New Class of Selective GSK-3
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