Synthesis, structure elucidation and antitumour activity of N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid

Article abstract of DOI:10.1016/j.ejmech.2009.04.034

New N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid (2-12) were designed and prepared by the condensation reaction of exo-S-ethyl-7-oxabicyclo-[2.2.1]-hept-5-ene-2,3-dicarbonyl isothiosemicarbazide (1) with primary amines. The che

Full text of DOI:10.1016/j.ejmech.2009.04.034

European Journal of Medicinal Chemistry 44 (2009) 3788–3793
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, structure elucidation and antitumour activity of N-substituted amides
of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid
,
b
c
d
Anna Pachuta-Stec a , Jolanta Rzymowska , Liliana Mazur , Ewaryst Mendyk ,
*
a
c
´
Monika Pitucha , Zofia Rza˛czynska
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 6 Staszica Street, 20-081 Lublin, Poland
b Chair and Department of Biology and Genetics, Medical University, 6 Staszica Street, 20-081 Lublin, Poland
c Department of General and Coordination Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
d Analytical Laboratory, Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
New N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid (2–12) were designed and
prepared by the condensation reaction of exo-S-ethyl-7-oxabicyclo-[2.2.1]-hept-5-ene-2,3-dicarbonyl
isothiosemicarbazide (1) with primary amines. The chemical structure of all compounds was confirmed
by IR, 1H NMR, 13C NMR spectra, the X-ray crystallography (for compounds 8, 11, 12) and elemental
analysis.
Received 22 January 2009
Received in revised form
17 April 2009
Accepted 23 April 2009
Available online 3 May 2009
Moreover, compounds 9–11 were screened for their anticancer activity. Compounds 9 (in concen-
trations of 0.32 mM and 0.16 mM), 10 (in concentrations of 0.28 mM and 0.14 mM), and 11 (in concen-
trations of 0.35 mM and 0.17 mM) were found to be evidently effective in vitro against lung cell line
(IC50). The distinctly marked antiproliferative effect of compounds 9 and 10 in breast carcinoma cells in
vitro was ascertained. Moreover, the lowest cytotoxicity of compound 9 in concentrations of 0.16 mM and
0.03 mM against the normal skin fibroblast cell line and breast carcinoma cell in vitro after 24- and 48-h
periods of incubation was noticed in this study.
Keywords:
N-Substituted amides
1,2,4-Triazole
Anticancer activity
X-ray crystal structure analysis
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Prompted by these biological data we synthesized some
unknown derivatives of 1,2,4-triazole, containing the amide and
1,2,4-Triazole and its derivatives are an important group of
compounds in modern heterocyclic chemistry. From scientific
literature it is known that depending on the type of substituents
derivatives of 1,2,4-triazole show a wide range of pharmacological
activities. Some of them were found to possess antifungal [1,2],
antimicrobial [3,4], anti-inflammatory [5–7], antidepressant [8]
and antiviral [9,10] properties. Certain compounds containing
a 1,2,4-triazole skeleton have shown anticonvulsant [11,12] and
antitumour activity [13–19].
In addition they have a wide range of therapeutic properties and
are used as drugs in modern medicine. Vorozole, Letrozole and
Anastrozole, having triazole moieties, are very effective nonste-
roidal aromatase inhibitors. They are useful for preventing breast
cancer [20–22]. Therefore triazoles, particularly 1,2,4-triazoles, are
perspective scaffolds for designing anticancer drugs.
ethylthio group, to explore their possible biological activity. In this
paper we present the discoveries, synthesis and testing results of
possible anticancer activity of N-substituted amides of 3-(3-ethyl-
thio-1,2,4-triazol-5-yl)propenoic acid.
2. Chemical part
In the present work exo-S-ethyl-7-oxabicyclo-[2.2.1]-hept-5-
ene-2,3-dicarbonyl isothiosemicarbazide (1) was used as the
starting material. This compound was prepared by a previously
reported method [23] by the direct condensation of exo-7-oxabi-
cyclo-[2.2.1]-hept-5-ene-2,3-dicarboxylic anhydride with S-ethyl
isothiosemicarbazide hydrobromide in glacial acetic acid at room
temperature. N-Substituted amides of 3-(3-ethylthio-1,2,4-triazol-
5-yl)propenoic acid (2–12) were obtained by heating of starting
compound with aliphatic and aromatic primary amines in boiling
glacial acetic acid. The reaction conditions were established
experimentally. The general synthetic pathway is presented in
Scheme 1.
* Corresponding author. Tel.: þ48 81 5357373.
E-mail address: anna.pachuta@am.lublin.pl (A. Pachuta-Stec).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.034

A. Pachuta-Stec et al. / European Journal of Medicinal Chemistry 44 (2009) 3788–3793
NH NH2
3789
H2N
S
C2H5Br
O
N
S
CH3
H2N
O
O
+
.
HBr
NH2
O
AcOH
NH3
CH3
NH2
O
N
S
O
R
NH2
+
N
O
1
O
O
9
R
3
8
NH
R
7
6
NH
4
O
N
N
5
S
S
CH3
11
NH
N
2
10
CH3
1
NH
N
2-12
2
3
4
R= -CH2CH2CH3
R= -CH2CH2CH2CH3
-Br
CH3
6
R= -CH2-
10 R= -
11 R= -
12 R= -
7
8
R= -CH2CH2-
o
i
m
p
15
14
12
13
R= -
R= -CH2CH2CHCH3
12
CH3
16
Cl
R= -
-OCH3
9
5
R= -
Scheme 1.
3. Results and discussion
3.1. Chemistry
3.1.2. Crystal structure analysis
Molecular structures of compounds 8, 11 and 12, together with
the atom-labelling schemes are shown in Figs. 1–3, respectively. An
X-ray analysis elucidates that compounds 8, 11 and 12 adopt the cis
(Z) configuration of the olefin C8]C9 atoms. The bond distances of
the triazole and arylamide fragments are within typical ranges [24],
moreover, the main structural parameters in all three molecules are
very similar. As indicated from the torsion angles (Table 1) the
central propenoamide part in all molecules is almost perfectly
planar and essentially coplanar with triazole and phenyl rings. A
small distortion from planarity is observed in 11; the dihedral angle
between propenoamide and phenyl best planes amounts to 5.9(5)ꢀ,
which may be caused by intermolecular hydrogen bonding. A cis
3.1.1. Synthesis
During our investigations we expected to obtain N-substituted
amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)bicyclo[2.2.1]hept-5-
ene-2-carboxylic acid. To our surprise nucleophilic ring opened and
decomposed. The reaction of compound 1 with aliphatic and
aromatic primary amines in boiling glacial acetic acid produced
unexpected N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-
yl)propenoic acid (2–12). The chemical structure of obtained
compounds was elucidated on the basis of 1H and 13C NMR.

3790
A. Pachuta-Stec et al. / European Journal of Medicinal Chemistry 44 (2009) 3788–3793
Fig. 1. Molecular structure of 8 with the atom-labelling scheme. The displacement ellipsoids are drawn at the 50% probability level. Dashed lines indicate hydrogen bonds.
arrangement of O1 with respect to the protonated N1 atom allows
formation of strong intramolecular hydrogen bond N1–H1/O1
[with N1/O1 distance of 2.619(5), 8, 2.610(4), 11 and 2.567(3) Å,
12]. This interaction, together with weak C17–H17/O1 hydrogen
bond [with C17/O1 distances of 2.902(5), 8, 2.849(3), 11,
2.867(4) Å, 12], may control the conformation of the 3-(1,2,4-
triazole)arylamide part of molecules in the solid state.
5. Experimental protocol
5.1. Chemistry
Melting points were determined using Fischer–Johns block
and presented without any corrections. Elemental analyses were
performed on a Perkin–Elmer 2400 CHN Analyser and were in
range of ꢂ0.4% for each element analyzed. IR spectra were
recorded in KBr on a Perkin–Elmer 1725X FTIR spectrometer. NMR
spectra (1H and 13C ) were recorded on a Bruker Avance 300 MHz
spectrometer in solution noted and with TMS as an internal
standard. Chemicals were purchased from Sigma–Aldrich or
Lancaster Synthesis and used without further purification. S-Ethyl
isothiosemicarbazide hydrobromide was obtained by the method
described in Ref. [25].
3.2. Biological evaluation
Some synthesized compounds were studied for their antitumour
activity (Table 2). Compounds 9–11 were evaluated for their cytotoxic
effect against two cancer cell lines: human lung cancer cell line A549
and human breast carcinoma cell line T47D. These compounds were
examined against human lung tumour cells in vitro. They were
unfortunately toxic at the higher doses (100 and 50 m
g mLꢁ1) for
normal fibroblast cultures. The growth of inhibition activity in breast
carcinoma cell line was observed for compound 9 in concentration of
0.32 mM and 0.16 mM after 72 h of incubation.
5.1.1. Procedure for synthesis of exo-S-ethyl-7-oxabicyclo-[2.2.1]-
hept-5-ene-2,3-dicarbonyl isothiosemicarbazide (1)
2 g (0.01 mol) of S-ethyl isothiosemicarbazide hydrobromide
and 1.66 g (0.01 mol) of exo-7-oxabicyclo-[2.2.1]-hept-5-ene-2,3-
dicarboxylic anhydride in 5 mL of glacial acetic acid were left for
72 h at room temperature. The reaction mixture was neutralized
with 25% aq. ammonia solution and left for crystallization for 2 h.
The crystalline precipitate was filtered off and crystallized from
ethanol–water. Yield 89%, m.p. 191–193 ꢀC. Spectroscopic data for
4. Conclusion
In this paper we reported an efficient and convenient method
of obtaining biologically active N-substituted amides of 3-(3-
ethylthio-1,2,4-triazol-5-yl)propenoic acid by the condensation
reaction of primary amines with the isothiosemicarbazide. The
reactions were carried out under moderately drastic conditions
and the solid products were isolated in medium and good yields.
Interestingly, during cyclization reaction nucleophilic ring opened
and decomposed, which has not been reported so far.
C11H13N3O3S: 1H NMR (
d, ppm, DMSO-d6, TMS): 1.28 (t, 3H,
J ¼ 7.2 Hz, –SCH2CH3), 2.78 (s, 2H, CH–CH), 2.92 (q, 2H, J ¼ 7.2 Hz,
–SCH2CH3), 5.09 (s, 2H, CH–O–CH), 6.54 (s, 2H, CH]CH), 7.01 (s, 2H,
NH2); 13C NMR (DMSO-d6): 14.77 (–SCH2CH3), 23.98 (–SCH2CH3),
45.67 (CH–CH), 80.08 (CH–O–CH), 136.23 (CH]CH), 163.86
(C–SCH2CH3), 173.30 (2C]O).
Compounds 9–11 were found effective in vitro against lung cell
line. The most effective were two examined doses 100 and
50 m
g mLꢁ1 (IC50). The cytotoxicity of compounds 9–11 against
breast carcinoma cell line was weak. Moreover, the lowest
cytotoxicity of compound 9 in concentrations of 0.16 mM and
0.03 mM against the normal skin fibroblast cell line was noticed in
this study. Unfortunately, compound 10 (in concentrations of
0.28 mM and 0.14 mM), and compound 11 (in concentrations of
0.35 mM and 0.17 mM) have distinctly marked cytotoxicity.
5.1.2. General procedure for synthesis of N-substituted amides
of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid (2–12)
1.34 g (0.005 mol) of 1 and 0.005 mol of n-propyl-, n-butyl-, iso-
pentyl-, cyclohexyl-, benzyl-, 2-phenylethyl-, phenyl-, 2-chlorophenyl-,
4-bromophenyl-, 3-methylphenyl-, 4-methoxyphenyl-amine in 3 mL
of glacial aceticacidwere refluxedfor 2 h. Aftercooling and standing for
Fig. 2. Molecular structure of 11.

A. Pachuta-Stec et al. / European Journal of Medicinal Chemistry 44 (2009) 3788–3793
3791
Fig. 3. Molecular structure of 12.
a few hours at room temperature the precipitate was filtered off and
crystallized from ethanol–water.
J ¼ 12.5 Hz, –CH]CHCONH), 6.78 (d, 1H, J ¼ 12.8 Hz, –CH]
CHCONH), 9.04 (bs, 1H, NHamide), 14.94 (bs, 1H, NHtriazole); 13C NMR
(DMSO-d6): 15.01 (–SCH2CH3), 22.21 (–CH2CH2CH(CH3)2), 25.19
(–CH2CH2CH(CH3)2), 25.50 (–SCH2CH3), 37.29 (–CH2CH2CH(CH3)2),
37.49 (–CH2CH2CH(CH3)2), 123.23 (–CH]CHCONH), 127.13
(–CH]CHCONH), 155.10 (Ctriazole), 169.90 (C–SCH2CH3), 165.14
(Camide).
5.1.2.1. N-Propyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic
acid (2). Yield 65%, m.p. 73–75 ꢀC. Spectroscopic data for
C10H16N4OS: IR (KBr) (
n
, cmꢁ1): 3239 (NH), 2931 (CH), 1659 (C]O),
1558 (C]N), 1444 (CH), 1255 (C–N); 1H NMR (
d, ppm, DMSO-d6,
TMS): 0.89 (t, 3H, J ¼ 7.4 Hz, –CH2CH2CH3), 1.30 (t, 3H, J ¼ 7.3 Hz,
–SCH2CH3), 1.52 (hex., 2H, J ¼ 7.2 Hz, –CH2CH2CH3), 3.08 (q, 2H,
J ¼ 7.1 Hz, –SCH2CH3), 3.17 (q, 2H, J ¼ 6.8 Hz, –CH2CH2CH3), 6.33
(d, 1H, J ¼ 13.2 Hz, –CH]CHCONH), 6.81 (d, 1H, J ¼ 12.7 Hz,
–CH]CHCONH), 9.02 (bs, 1H, NHamide), 14.98 (bs, 1H, NHtriazole);
13C NMR (DMSO-d6): 11.40 (–CH2CH2CH3), 15.02 (–SCH2CH3),
21.89 (–CH2CH2CH3), 25.47 (–SCH2CH3), 40.84 (–CH2CH2CH3),
123.32 (–CH]CHCONH), 126.99 (–CH]CHCONH), 152.19 (Ctriazole),
159.90 (C–SCH2CH3), 165.27 (Camide).
5.1.2.4. N-Cyclohexyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)-
propenoic acid (5). Yield 78%, m.p. 107–109 ꢀC. Spectroscopic data
for C13H20N4OS: 1H NMR (
d, ppm, DMSO-d6, TMS): 1.14–1.27 (m, 5H,
cyclohexyl), 1.30 (t, 3H, J ¼ 7.3 Hz, –SCH2CH3), 1.56–1.84 (m, 5H,
cyclohexyl), 3.09 (q, 2H, J ¼ 7.3 Hz, –SCH2CH3), 3.65–3.74
(m, 1H, CHcyclohexyl), 6.29 (d, 1H, J ¼ 11.8 Hz, –CH]CHCONH), 6.78
(d, 1H, J ¼ 12.6 Hz, –CH]CHCONH), 8.95 (bs, 1H, NHamide), 14.93
(bs, 1H, NHtriazole).
5.1.2.2. N-Butyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic
5.1.2.5. N-Benzyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)prope-
acid (3). Yield 70%, m.p. 65–67 ꢀC. Spectroscopic data for
noic acid (6). Yield 74%, m.p. 95–97 ꢀC. Spectroscopic data for
C11H18N4OS: IR (KBr) (
n
, cmꢁ1): 3234 (NH), 2960 (CH), 1659 (C]O),
C14H16N4OS: 1H NMR (
d, ppm, DMSO-d6, TMS): 1.27 (t, 3H,
1555 (C]N), 1445 (CH), 1257 (C–N); 1H NMR (
d
, ppm, DMSO-d6,
J ¼ 7.3 Hz, –SCH2CH3), 3.04 (q, 2H, J ¼ 7.3 Hz, –SCH2CH3), 4.44
(d, 2H, J ¼ 5.9 Hz, CH2–Ph), 6.34 (d, 1H, J ¼ 12.9 Hz, –CH]CHCONH),
6.82 (d, 1H, J ¼ 12.9 Hz, –CH]CHCONH), 7.25–7.38 (m, 5H, Ph), 9.65
(bs, 1H, NHamide), 14.68 (bs, 1H, NHtriazole); 13C NMR (DMSO-d6):
15.01 (–SCH2CH3), 25.56 (–SCH2CH3), 42.71 (CH2–Ph), 123.66
(–CH]CHCONH), 126.90 (–CH]CHCONH), 127.06 (p-PhC), 127.52
(m-Ph2C), 128.41 (o-Ph2C), 138.34 (i-PhC), 152.98 (Ctriazole), 159.8
(C–SCH2CH3), 165.27 (Camide).
TMS): 0.89 (t, 3H, J ¼ 7.2 Hz, –CH2CH2CH2CH3), 1.28–1.38 (m, 5H,
–SCH2CH3 þ –CH2CH2CH2CH3), 1.48 (hex., 2H, –CH2CH2CH2CH3),
3.09 (q, 2H, J ¼ 7.2 Hz, –SCH2CH3), 3.26 (q, 2H, J ¼ 6.5 Hz,
–CH2CH2CH2CH3), 6.30 (d, 1H, J ¼ 11.9 Hz, –CH]CHCONH), 6.78
(d, 1H, J ¼ 12.4 Hz, –CH]CHCONH), 9.04 (bs, 1H, NHamide), 14.94
(bs, 1H, NHtriazole); 13C NMR (DMSO-d6): 13.51 (–CH2CH2CH2CH3),
15.00 (–SCH2CH3), 19.64 (–CH2CH2CH2CH3), 25.51 (–SCH2CH3),
30.67 (–CH2CH2CH2CH3), 38.76 (–CH2CH2CH2CH3), 123.27
(–CH]CHCONH), 127.11 (–CH]CHCONH), 152.63 (Ctriazole), 160.00
(C–SCH2CH3), 165.22 (Camide).
5.1.2.6. N-(Ethylphenyl) amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)-
propenoic acid (7). Yield 71%, m.p. 93–95 ꢀC. Spectroscopic data for
C15H18N4OS: IR (KBr) (
n
, cmꢁ1): 3437 (NH), 2972 (CH), 1655 (C]O),
5.1.2.3. N-Isopentyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)-
1566 (C]N), 1439 (CH), 1263 (C–N); 1H NMR (
d, ppm, DMSO-d6,
propenoic acid (4). Yield 72%, m.p. 90–92 ꢀC. Spectroscopic data for
TMS): 1.28 (t, 3H, J ¼ 7.1 Hz, –SCH2CH3), 2.79–2.84 (m, 2H,
CH2CH2Ph), 3.07 (q, 2H, J ¼ 7.1 Hz, –SCH2CH3), 3.33–3.46 (m, 2H,
CH2CH2–Ph), 6.28 (d, 1H, J ¼ 11.8 Hz, –CH]CHCONH), 6.76 (d, 1H,
J ¼ 13.1 Hz, –CH]CHCONH), 7.23–7.29 (m, 5H, Ph), 9.17 (bs, 1H,
NHamide), 14.87 (bs, 1H, NHtriazole); 13C NMR (DMSO-d6): 15.04
(–SCH2CH3), 25.51 (–SCH2CH3), 34.59 (CH2CH2–Ph), 40.67
(CH2CH2–Ph), 123.45 (–CH]CHCONH), 126.17 (p-PhC), 127.11
(–CH]CHCONH), 128.32 (m-Ph2C), 128.59 (o-Ph2C), 139.11 (i-PhC),
155.11 (Ctriazole), 159.90 (C–SCH2CH3), 165.32 (Camide).
C12H20N4OS: IR (KBr) (
n
, cmꢁ1): 3235 (NH), 2956 (CH), 1657 (C]O),
1558 (C]N), 1441 (CH), 1259 (C–N); 1H NMR (
d
, ppm, DMSO-d6,
TMS): 0.89 (s, 3H, CH3isopentyl), 0.91 (s, 3H, CH3isopentyl), 1.28 (t, 3H,
J ¼ 7.3 Hz, –SCH2CH3), 1.38 (q, 2H, J ¼ 7.2 Hz, –CH2CH2CH(CH3)2),
1.57–1.66 (m, 1H, –CH2CH2CH(CH3)2), 3.09 (q, 2H, J ¼ 7.3 Hz,
–SCH2CH3), 3.27 (q, 2H, J ¼ 6.7 Hz, –CH2CH2CH(CH3)2), 6.29 (d, 1H,
Table 1
Selected torsion angles (ꢀ).
5.1.2.7. N-Phenyl amide of 3-(3-ethylthio-1,2,4-triazol-5-yl)prope-
noic acid (8). Yield 79%, m.p. 205–207 ꢀC. Spectroscopic data for
8
11
12
C13H14N4OS: IR (KBr) (
n
, cmꢁ1): 3439 (NH), 2964 (CH), 1664 (C]O),
N1–C5–C8–C9
4.2(7)
ꢁ0.5(8)
0.8(7)
2.2(4)
0.4(4)
4.8(6)
0.3(6)
0.5(6)
1555 (C]N), 1444 (CH), 1257 (C–N); 1H NMR (
d
, ppm, DMSO-d6,
C5–C8–C9–C10
C8–C9–C10–O1
C8–C9–C10–N11
C9–C10–N11–C12
C10–N11–C12–C17
TMS): 1.24 (t, 3H, J ¼ 7.2 Hz, –SCH2CH3), 3.05 (q, 2H, J ¼ 7.3 Hz,
–SCH2CH3), 6.44 (d, 1H, J ¼ 13.0 Hz, –CH]CHCONH), 6.85 (d, 1H,
J ¼ 12.4 Hz, –CH]CHCONH), 7.09–7.16 (m, 1H, p-Ph), 7.29–7.39
(m, 2H, m-Ph), 7.68–7.70 (m, 2H, o-Ph), 10.94 (bs, 1H, NHamide),
ꢁ0.2(4)
179.0(2)
179.2(2)
6.9(4)
178.6(4)
ꢁ179.5(3)
179.6(3)
ꢁ2.8(5)
ꢁ179.1(4)
0.8(7)

3792
A. Pachuta-Stec et al. / European Journal of Medicinal Chemistry 44 (2009) 3788–3793
14.40 (bs, 1H, NHtriazole); 13C NMR (DMSO-d6): 15.02 (–SCH2CH3),
25.56 (–SCH2CH3),119.89 (o-Ph2C),123.20 (–CH]CHCONH),124.25
(p-PhC), 127.81 (–CH]CHCONH), 128.79 (m-Ph2C), 138.35 (i-PhC),
151.80 (Ctriazole), 159.90 (C–SCH2CH3), 163.80 (Camide).
lung and breast carcinoma cells. These studies were carried out on
A549 (ECACC 86012804 human lung epithelial) and T47D (ECACC
85102201 human breast epithelial). The primary cell line of normal
human skin fibroblasts was used in this experiment. The studies
were carried out for the purpose to choose the compounds having
promising antiproliferative and anticancer properties. The influ-
ence of new synthesized amides on normal human skin fibroblast
cells was also determined.
5.1.2.8. N-(2-Chlorophenyl) amide of 3-(3-ethylthio-1,2,4-triazol-5-
yl)propenoic acid (9). Yield 81%, m.p. 112–114 ꢀC. Spectroscopic data
for C13H13N4OSCl: IR (KBr) (
n
, cmꢁ1): 3202 (NH), 2973 (CH), 1671
(C]O),1544 (C]N),1444 (CH),1256 (C–N); 1H NMR (
d, ppm, DMSO-
The cell lines were incubated at 104 cells per mL density on
microtiter plates. Tested compounds were then added at three
d6, TMS): 1.24 (t, 3H, J ¼ 7.2 Hz, –SCH2CH3), 3.08 (q, 2H, J ¼ 7.3 Hz,
–SCH2CH3), 6.50 (bs, 1H, –CH]CHCONH), 6.90 (d, 1H, J ¼ 12.9 Hz,
–CH]CHCONH), 7.24 (t, 1H, J ¼ 7.1 Hz, p-Ph), 7.36 (t, 1H, J ¼ 7.3 Hz,
m-Ph), 7.53 (d, 1H, J ¼ 7.2 Hz, m-Ph), 7.83 (d, 1H, J ¼ 7.5 Hz, o-Ph),
11.08 (bs,1H, NHamide),14.44 (bs,1H, NHtriazole); 13C NMR (DMSO-d6):
14.93 (–SCH2CH3), 25.66 (–SCH2CH3), 123.93 (–CH]CHCONH),
126.53 (o-PhC), 126.69 (o-PhC–Cl), 126.82 (p-PhC), 127.36 (m-PhC),
127.62 (–CH]CHCONH), 129.52 (m-PhC), 134.43 (i-PhC), 154.82
(Ctriazole), 159.90 (C–SCH2CH3), 163.89 (Camide).
examined concentrations: 10, 50 and 100 m
g mLꢁ1, and cultures
were incubated at standard conditions (37 ꢀC, 5% CO2 and 90%
humidity) for 24, 48 and 72 h.
Determinations were made with 5-bromo-20-deoxy-uridine
(BrdU) labelling and detection kit (Roche) on Elisa reader (BIO-TEC
Instruments, USA). Cell viability of normal and carcinoma cells was
evaluated spectrophotometrically. Results of every spectrophoto-
metrical measurement were noticed as percent of growth inhibi-
tion or growth stimulation.
5.1.2.9. N-(4-Bromophenyl) amide of 3-(3-ethylthio-1,2,4-triazol-5-
All experiments were repeated in triplicates.
yl)propenoic acid (10). Yield 78%, m.p. 230–232 ꢀC. Spectroscopic
data for C13H13N4OSBr: IR (KBr) (
n
, cmꢁ1): 3432 (NH), 2929 (CH),
5.3. X-ray structure analyses
1664 (C]O), 1547 (C]N), 1490 (CH), 1259 (C–N); 1H NMR (
d
, ppm,
DMSO-d6, TMS): 1.22 (t, 3H, J ¼ 7.3 Hz, –SCH2CH3), 3.02 (q, 2H,
J ¼ 7.3 Hz, –SCH2CH3), 6.41 (d, 1H, J ¼ 12.7 Hz, –CH]CHCONH), 6.83
(d, 1H, J ¼ 12.7 Hz, –CH]CHCONH), 7.51–7.56 (m, 2H, o-, m-,
p-Ph), 7.64–7.68 (m, 2H, o-, m-, p-Ph), 10.98 (bs, 1H, NHamide), 14.28
(bs, 1H, NHtriazole); 13C NMR (DMSO-d6): 15.00 (–SCH2CH3),
25.69 (–SCH2CH3), 115.65 (p-PhC), 121.64 (o-Ph2C), 123.60
(–CH]CHCONH), 127.79 (–CH]CHCONH), 131.58 (m-Ph2C), 137.97
(i-PhC), 154.90 (Ctriazole), 159.90 (C–SCH2CH3), 164.00 (Camide).
Crystal data for 8: C13H14N4OS, orthorhombic, space group Pna21,
a ¼ 10.693(2), b ¼ 24.154(5), c ¼ 5.105(2) Å, V ¼ 1318.5(6) Å3, Z ¼ 4,
dx ¼ 1.382 g cmꢁ3, T ¼ 295(2) K. 3039 data were collected up to
q
¼ 45ꢀ for a yellow crystal with dimensions 0.54 ꢃ 0.40 ꢃ 0.09 mm.
Final R indices for 1475 reflections with I > 2s(I) and 173 refined
parameters are: R1 ¼0.0590, wR2 ¼ 0.1078 (R1 ¼0.1633, wR2 ¼ 0.1431
for all 3039 data).
Crystal data for 11: C14H16N4OS, monoclinic, space group P21/c,
a ¼ 5.067(1),
b ¼ 25.433(4),
c ¼ 10.879(4) Å,
b
¼ 91.38(2)ꢀ,
V ¼ 1401.6(6) Å3, Z ¼ 4, dx ¼ 1.367 g cmꢁ3, T ¼ 295(2) K. 6465 data
5.1.2.10. N-(3-Methylphenyl) amide of 3-(3-ethylthio-1,2,4-triazol-5-
yl)propenoic acid (11). Yield 67%, m.p. 184–186 ꢀC. Spectroscopic
were collected up to
q
¼ 45ꢀ for a yellow crystal with dimensions
data for C14H16N4OS: IR (KBr) (
n
, cmꢁ1): 3435 (NH), 3037 (CH), 1674
0.59 ꢃ 0.32 ꢃ 0.17 mm. Final R indices for 1854 reflections with
(C]O), 1565 (C]N), 1449 (CH), 1253 (C–N); 1H NMR (
d
, ppm,
I > 2
s
(I) and 174 refined parameters are: R1 ¼0.0476, wR2 ¼ 0.1059
DMSO-d6, TMS): 1.24 (t, 3H, J ¼ 7.2 Hz, –SCH2CH3), 2.31 (s, 3H,
CH3–Ph), 3.06 (q, 2H, J ¼ 7.3 Hz, –SCH2CH3), 6.43 (d, 1H, J ¼ 12.5 Hz,
–CH]CHCONH), 6.84 (d, 1H, J ¼ 12.5 Hz, –CH]CHCONH), 6.93
(d, 1H, J ¼ 7.5 Hz, p-Ph), 7.21 (t, 1H, J ¼ 7.8 Hz, m-Ph), 7.46 (d, 1H,
J ¼ 8.5 Hz, o-Ph), 7.54 (s, 1H, o-Ph), 10.92 (bs, 1H, NHamide), 14.43 (bs,
1H, NHtriazole); 13C NMR (DMSO-d6): 15.01 (–SCH2CH3), 21.14 (CH3–
Ph), 25.66 (–SCH2CH3), 117.03 (o-PhC), 120.35 (o-PhC), 123.60
(–CH]CHCONH), 124.80 (p-PhC), 128.05 (–CH]CHCONH), 128.61
(m-PhC), 138.01 (m-Ph–CH3), 138.40 (i-PhC), 152.50 (Ctriazole),
159.90 (C–SCH2CH3), 163.70 (Camide).
(R1 ¼0.1099, wR2 ¼ 0.1280 for all 3242 data).
Crystal data for 12: C14H16N4O2S, monoclinic, space group P21/c,
a ¼ 5.523(2),
b ¼ 23.314(7),
c ¼ 11.667(7) Å,
b
¼ 102.82(5)ꢀ,
Table 2
Inhibition (GI) of normal and tumour cells growth in vitro of the tasted compounds.
Time of
GI (%)
incubation (h)
10
11
9
Compound
Doses
I
II
III
I
II
III
I
II
III
Cell line
Normal cell line
HSF
5.1.2.11. N-(4-Methoxyphenyl) amide of 3-(3-ethylthio-1,2,4-triazol-
5-yl)propenoic acid (12). Yield 71%, m.p. 218–220 ꢀC. Spectroscopic
24
48
72
75 75
75 75
50 75
0
0
0
5
50
0
0
0
25
50
75
0
0
0
0
0
5
data for C14H16N4O2S: IR (KBr) (
n
, cmꢁ1): 3431 (NH), 2963 (CH),1663
50 50
50 50
(C]O),1558 (C]N),1442 (CH),1245 (C–N); 1H NMR (
d
, ppm, DMSO-
d6, TMS): 1.25 (t, 3H, J ¼ 7.2 Hz, –SCH2CH3), 3.07 (q, 2H, J ¼ 7.3 Hz,
–SCH2CH3), 3.75 (s, 3H, CH3O–Ph), 6.42 (d, 1H, J ¼ 12.7 Hz,
–CH]CHCONH), 6.85 (d, 1H, J ¼ 12.7 Hz, –CH]CHCONH), 6.93
(d, 2H, J ¼ 9.0 Hz, m-Ph), 7.60 (d, 2H, J ¼ 9.0 Hz, o-Ph), 10.84 (bs, 1H,
NHamide), 14.50 (bs, 1H, NHtriazole); 13C NMR (DMSO-d6): 15.03
(–SCH2CH3), 25.63 (–SCH2CH3), 55.17 (CH3OPh), 113.94 (m-Ph2C),
121.44 (o-Ph2C), 123.59 (–CH]CHCONH), 127.89 (–CH]CHCONH),
131.45 (p-PhC),133.50 (i-PhC),155.92 (Ctriazole),159.90 (C–SCH2CH3),
163.33 (Camide).
Cancer cell line
A549
24
48
72
50 50 25 50 50 20 50 50 25
50 50 25 50 50
5
0
50 50 25
75 50 25
75 50
0
50 25
T47D
24
48
72
5
5
0
0
0
0
0
5
5
0
5
5
0
0
0
0
5
20 10
25 25
10 10
90 50
25 25
GI – growth inhibition factor; HSF – human skin fibroblasts; A549 – human lung
cancer cell line; T47D human breast cancer cell line; examined doses of
compounds: I – concentration of 100
g mLꢁ1, which corresponds to concentration
of 0.32 mM for compound 9, 0.28 mM for compound 10, and 0.35 mM for compound
11; II – concentration of 50
g mLꢁ1, which corresponds to concentration of 0.16 mM
for compound 9, 0.14 mM for compound 10, and 0.17 mM for compound 11; III –
concentration of 10
g mLꢁ1, which corresponds to concentration of 0.03 mM for
compound 9, 0.03 mM for compound 10, and 0.03 mM for compound 11.
–
m
5.2. Tumour cells proliferation assay
m
The newly synthesized compounds 9–11 were evaluated for
their anticancer activity in human tumour cell lines derived from
m

A. Pachuta-Stec et al. / European Journal of Medicinal Chemistry 44 (2009) 3788–3793
3793
V ¼ 1465(1) Å3, Z ¼ 4, dx ¼ 1.380 g cmꢁ3, T ¼ 295(2) K. 6730 data
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were collected up to
q
¼ 45ꢀ for a yellow crystal with dimensions
0.50 ꢃ 0.42 ꢃ 0.08 mm. Final R indices for 1181 reflections with
I > 2
s
(I) and 192 refined parameters are: R1 ¼0.0607, wR2 ¼ 0.0966
(R1 ¼0.2250, wR2 ¼ 0.1350 for all 3375 data).
Single-crystal diffraction data were measured at room temper-
ature in the
tometer using graphite-monochromated Mo Ka radiation
¼ 0.71073). The stability of intensities was monitored by
u/2q mode on an Oxford Diffraction Xcalibur diffrac-
(l
measurement of 3 standards every 100 reflections. Crystal struc-
tures were solved by direct methods using SHELXS97 [26] and
refined by the full-matrix least-squares on F2 using the SHELXL97
[27]. All non-hydrogen atoms were refined with anisotropic
displacement parameters. The H-atoms were positioned geomet-
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Ueq(C, N).
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The CCDC 704272 (compound 8), CCDC 704273 (compound 11)
and CCDC 704274 (compound 12) contain the supplementary
crystallographic data for this paper. These data can be obtained free
of charge at www.ccdc.ac.uk/conts/retrieving.html or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: þ44-1223-336-033; e-mail: deposit@ccdc.cam.
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