Synthesis of some (E)-6-[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5-ones and their biological evaluation as antitumor agents

Article abstract of DOI:10.1007/s00044-012-0192-x

The synthesis of some new (E)-6-[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5- ones directly linked to either pyrazole, pyrazoline, pyrazolidine counterparts, or to substituted thio and hydrazono functionalities is described. Six of the newly synthesized compounds were selected by the National Cancer Institute (NCI) to be evaluated for their in vitro antitumor activity according to the protocol of the NCI in vitro disease-oriented human cells screening panel assay. The results revealed that the pyrazole derivative 5c was found to be the most active member in this screen as evidenced by its ability to exert potential growth inhibitory activity against most of the tested subpanel tumor cell lines with selective influence on leukemia subpanel tumor cell lines (GI₅₀ values 2.01-3.03 μM). Moreover, a comparative study for log GI₅₀ values of both compound 5c and 5-fluorouracil (5-FU) revealed that compound 5c showed higher potency than 5-FU against most of the tested subpanel tumor cell lines. Thus compound 5c could be considered as a suitable lead towards the design of broad spectrum antitumor active agents targeting various human tumor cell lines.

Full text of DOI:10.1007/s00044-012-0192-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1909–1924
DOI 10.1007/s00044-012-0192-x
ORIGINAL RESEARCH
Synthesis of some (E)-6-[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5-
ones and their biological evaluation as antitumor agents
•
Hayam M. Ashour Marwa H. El-Wakil
•
•
Mounir A. Khalil Khadiga A. Ismail
•
Ibrahim M. Labouta
Received: 20 February 2012 / Accepted: 6 August 2012 / Published online: 21 August 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The synthesis of some new (E)-6-[2-(furan-2-
yl)ethenyl]-1,2,4-triazin-5-ones directly linked to either
pyrazole, pyrazoline, pyrazolidine counterparts, or to
substituted thio and hydrazono functionalities is described.
Six of the newly synthesized compounds were selected by
the National Cancer Institute (NCI) to be evaluated for
their in vitro antitumor activity according to the protocol of
the NCI in vitro disease-oriented human cells screening
panel assay. The results revealed that the pyrazole deriv-
ative 5c was found to be the most active member in this
screen as evidenced by its ability to exert potential growth
inhibitory activity against most of the tested subpanel
tumor cell lines with selective influence on leukemia sub-
panel tumor cell lines (GI50 values 2.01–3.03 lM). More-
over, a comparative study for log GI50 values of both
compound 5c and 5-fluorouracil (5-FU) revealed that
compound 5c showed higher potency than 5-FU against
most of the tested subpanel tumor cell lines. Thus com-
pound 5c could be considered as a suitable lead towards the
design of broad spectrum antitumor active agents targeting
various human tumor cell lines.
Introduction
Cancer poses a serious human health problem despite much
progress in understanding its biology and pharmacology.
Although cancer research has led to a number of new and
effective solutions, the medicines used as treatments have
clear limitations due to lack of selectivity leading to tox-
icity, metastatic spreading and the intrinsic or acquired
resistance developed after few therapeutic cycles (Bran˜a
and Ramos, 2001; Cozzi, 2003). At the same time, random
screening remains one of the main routes to discover new
leads with antineoplastic activity and the National Cancer
Institute (NCI), Bethesda, USA, is still playing an articular
role in this field, with special emphasis on novel chemical
structures that have not had extensive clinical evaluation
(Cocco et al., 2000).
Among the wide variety of heterocycles that have been
explored for developing pharmaceutically important mol-
ecules, the 1,2,4-triazines have received great attention as
chemotherapeutic agents. Azanucleosides (6-azacytosine
and 6-azauracil), structurally based on the 1,2,4-triazine
scaffold were proved to display antitumor (Creasey et al.,
1963), antiviral (Sidwell et al., 1968), and antifungal
activities (Sangshetti and Shinde, 2010). In addition, some
1,2,4-triazin-6(1H)-ones were reported to display signifi-
cant broad spectrum antitumor activity against lympho-
blastic leukemia CEM, myeloid leukemia K562, and lung
adenocarcinoma A549 cancer cell lines (Gucky et al.,
2009) while, some 1,2,4-triazine 5-one derivatives were
found to exhibit strong antiproliferative effect on human
leukemia K-562 cell line (Krauth et al., 2010). Moreover,
particular interest has been focused on 6-azaisocytosine
(3-amino-1,2,4-triazin-5(2H)-one), an isosteric isomer of
6-azacytosine and 6-azauracil as potential transcription
inhibitor (Pal chykovska et al., 2004). On the other hand, a
Keywords Synthesis ꢀ 1,2,4-Triazin-5-ones ꢀ Pyrazole ꢀ
Antitumor activity
H. M. Ashour (&) ꢀ M. H. El-Wakil ꢀ M. A. Khalil ꢀ
K. A. Ismail ꢀ I. M. Labouta
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria 21521, Egypt
e-mail: hayamashour@ymail.com
123

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Med Chem Res (2013) 22:1909–1924
H
H
N
literature survey revealed that some pyrazoles and pyrazole
containing compounds have been implemented as antileu-
kemic (Daidone et al., 2004a; Chou et al., 2007; Manetti
et al., 2008), antitumor (Li et al., 2006; Xia et al., 2007; Xia
et al., 2008; Farag et al., 2008), and antiproliferative agents
(Schenone et al., 2004; Daidone et al., 2004b), beside their
capability to exert remarkable anticancer effects through
inhibiting different types of enzymes that play important
roles in cell division (Warshakoon et al., 2006; Huang et al.,
2007; Zhu et al., 2007). Furthermore, some thioethers were
found to show enhanced antimicrobial and antitumor
activities (Gulerman et al., 2001; Khalil et al., 2003) beside
being a common structural subunit in SDABOs (dihydro-
alkylthio-benzyl-oxopyrimidines) which possess antipro-
liferative as well as antiviral activities (Manetti et al.,
2005). Addditionally, hydrazono derivatives with their
effective contribution as antineoplastic agents (Remers,
2004) were not far of our attention.
O
N
O
N
N
N
R1HN
H3CO
R
N
R
N
OCH3
H3CO
OCH3
OCH3
OCH3
N
N
O
N
NH
NH2
N
N
O
N
Cl
6-azaisocytosine
N
N
N
O
NH
O
R1
O
S
S
N
R
O
N
R
A
B
R= NH2, 4-nitrobenzylideneamino
R= NH2, 4-nitrobenzylideneamino; R1= alkyl
Inspired by the above-mentioned facts and as a contin-
uation of an ongoing research program aimed at the dis-
covery of novel chemotherapeutic agents (Rostom et al.,
2009; Ashour and Abdel Wahab, 2009; Rostom et al.,
2011), it seemed worthwhile to synthesize new structure
hybrids (A–C) incorporating the 1,2,4-triazin-5-one scaf-
fold linked to a pyrazole, pyrazoline, pyrazolidine ring or
substituted thioether and hydrazono moieties (Fig. 1)
which are believed to be responsible for the biological
significance of some relevant natural and synthetic che-
motherapeutic agents. This combination was suggested in
an attempt to investigate the possible synergistic influence
of such structure hybridization on the anticipated biological
activity hoping to discover a new lead structure that would
have a significant antitumor potential. In addition, variation
in the nature and size of substituents was also attempted, as
it would offer variable electronic, lipophilic, and steric
environment that would influence the targeted biological
activity. The present work reports the synthesis and the
results of preliminary antitumor screening of compounds
selected by the NCI.
N
N
O
NH
R
O
C
R= substituted thio, hydrazono moiety, pyrazole, pyrazoline, pyrazolidine counterparts
Fig. 1 The structure of some reported antitumor 1,2,4-triazines and
the newly synthesized compounds
compounds 2a, b and two doublets at 3.67–3.87 ppm for
compounds 2c–e attributed to SCH2 protons. In addition,
investigation of 1H-NMR spectra for compounds
2a–e revealed presence of two doublets for ethenyl C1 and
C2 protons at 6.65–6.81 and 7.56–7.75 ppm with coupling
constant 16.05–16.7 Hz indicating their existence as E-iso-
mers (Williams and Fleming, 1980). 13C-NMR spectrum of
compound 2e as an example showed a signal at 50.55 ppm
corresponding to SCH2 carbon and other signals were
observed at their expected chemical shifts. Condensation of
the hydrazine derivative 3 with 4-substituted benzaldehydes,
furfural, 5-nitro-2-furfural, isatin, and N-methyl isatin in
boiling ethanol resulted in the formation of the corre-
sponding hydrazones 4a–g. 1H-NMR spectra of compounds
4a–e showed singlets for the N=CH protons in the range
7.82–8.08 ppm indicating existence of these compounds in
the E configuration around the N=C (Pretsch et al., 2000,
pp 211–212). 13C-NMR spectrum of compound 4a provided
further confirmation of the chemical structure. On the other
hand, reaction of 3 with phenacyl or 4-substituted phenacyl
cyanides in ethanol/acetic acid mixture furnished the target
Chemistry
Synthesis of the intermediate and target compounds was
accomplished according to the steps depicted in Schemes 1
and 2. In Scheme 1, the starting thione 1 and the hydrazine
intermediate 3 were prepared according to previously
reported reaction conditions (Slouka, 1962; Osman
et al., 2007). Heating 1 with phenacyl or 4-substituted
phenacyl bromide in ethanol gave rise to the substituted
2-oxoethylsulfanyl analogs 2a–e. 1H-NMR spectra of
compounds 2a–e revealed singlets at 4.45 and 4.38 ppm for
1
5-aminopyrazoles 5a–d. Inspection of H-NMR spectra of
compounds 5a–d indicated presence of a singlet at
5.88–5.92 ppm corresponding to pyrazole C4-proton in
addition to D2O exchangeable singlets at 7.06–7.10 ppm
123

Med Chem Res (2013) 22:1909–1924
1911
due to NH2 protons. Additionally, condensation of 3 with an
equimolar amount of acetyl acetone in ethanol gave rise to
at 3.44 ppm attributed to pyrazoline C4 proton, while its MS
spectrum revealed a molecular ion peak at m/z 285 (21 %)
which is in accordance with its molecular formula. It should
be noted that the pyrazolinone derivative 9 could be directly
prepared from the hydrazine intermediate 3 by fusing the
latter compound with ethyl acetoacetate in an oil bath at
160 °C.
Referring to Scheme 2, stirring 10 (Rohmer, 1898) with
thiocarbohydrazide in ethanol containing a catalytic
amount of glacial acetic acid resulted in the formation of
the respective thiocarbohydrazone 11. IR spectra of the
latter compound displayed absorption band characteristic
for C=O group at low frequency; 1661 cm-1, while its 1H-
NMR spectrum showed a D2O exchangeable singlet for the
NH proton at a high chemical shift; 10.3 ppm confirming
presence of a hydrogen bond between the C=O and NH
groups (Pretsch et al., 2000, pp. 290–291). This led to the
suggestion that the configuration around the C=N is Z.
Heating 11 in 1 N sodium hydroxide gave rise to the cor-
1
the requisite 3,5-dimethyl pyrazole 6. H-NMR spectrum
revealed presence of three singlets at 2.22, 2.46, and
6.24 ppm interpreted for two CH3 groups and pyrazole
C4-proton. Heating the same intermediate 3 with diethyl
malonate in a mixture of ethanol/glacial acetic acid afforded
1
the pyrazolidine-3,5-dione 7. H-NMR spectrum for this
compound showed a singlet for the pyrazolidine C4-protons
at 2.45 ppm in addition to a D2O exchangeable singlet at
8.70 ppm due to pyrazolidine NH proton. Reaction of 3 with
an equimolar amount of ethyl acetoacetate in boiling ethanol
1
yielded the ethyl butanoate ester 8. H-NMR spectrum of
compound 8 revealed a singlet for the C-3 methyl group at a
high chemical shift (1.94 ppm) confirming that the config-
uration at C-3 is E (Pretsch et al., 2000, pp 211–212).
Attempts to cyclize the ethyl butanoate ester to the corre-
sponding pyrazolinone 9 using high boiling point solvents
were unsuccessful; however fusion of the ethyl butanoate
ester in an oil bath at 160 °C afforded the respective pyr-
azolinone 9 in a good yield. 1H-NMR spectrum of 9 showed
a singlet at 2.47 ppm assigned for CH3 protons and a singlet
responding
4-amino-3-thioxo-3,4-dihydro-1,2,4-triazin-
5(2H)-one 12. IR spectrum for compound 12 lacked
absorption bands for OH group and showed absorption
N
N
O
ii
iv
O
NH
NH
N
N
R1
O
S
O
N
N
H
1
5a-d
H2N
i
N
N
N
N
O
O
O
O
O
NH
v
NHNH2
NH
NH
R1
N
O
N
S
O
6
N
O
N
3
2a-e
O
N
N
N
NH
NH
iii
vi
H
N
N
R2
N
O
N
N
O
O
H
4a-g
7
O
N
N
N
O
O
NH
NH
N
OC2H5
ix
N
O
N
N
N
O
H
CH3
O
9
8
O
R1 = C6H5, 4-CH3C6H4, 4-ClC6H4, 4-BrC6H4, 4-NO2C6H4; R2 = 4-CH3OC6H4CH, 4-ClC6H4CH, 4-NO2C6H4CH, 2-furylidene, 5-
nitro-2-furylidene, isatinylidene, N-methyl isatinylidene.
Scheme 1 Reagents and reaction conditions: i R1COCH2Br, absolute
ethanol, reflux, ii hydrazine 98 %, absolute ethanol, reflux, iii
aldehydes or ketones, absolute ethanol, glacial acetic acid, reflux, iv
R1COCH2CN, ethanol, glacial acetic acid, reflux, v acetylacetone,
absolute ethanol, reflux, vi diethyl malonate, glacial acetic acid,
reflux, vii ethyl acetoacetate, absolute ethanol, reflux, viii ethyl
acetoacetate, fusion, oil bath, 160 °C, ix fusion, oil bath, 160 °C
123

1912
Med Chem Res (2013) 22:1909–1924
S
N
N
i
COOH
N
O
C
ii
O
O
O
NH
S
C
O
C
N
NHNH2
H
O
HO
NH2
12
10
11
iv
iii
N
N
N
O
O
O
N
N
NH
R1
SR2
O
N
O
N
N
S
N
N
S
O
NH2
v
15a-c
14
13a-c
NO2
NO2
R1 = morpholino, piperidino, pyrrolidino; R2 = CH3, C2H5, C6H5CH2
Scheme 2 Reagents and reaction conditions: i thiocarbohydrazide,
ethanol, glacial acetic acid, stir, r.t., ii 1 N sodium hydroxide, iii
methyl iodide or ethyl iodide or benzyl chloride, dry dimethyl
formamide, anhydrous potassium carbonate, stir, r.t., iv 4-nitro-
benzaldeyhde, absolute ethanol, glacial acetic acid, reflux,
v ClCH2CH2R1.HCl, potassium hydroxide, stir, r.t.
bands characteristic for NH2 and N-CS moieties whereas,
its 1H-NMR spectrum verified its structure. Stirring the
aminothione 12 with an equivalent amount of methyl
iodide, ethyl iodide or benzyl chloride in dry DMF con-
taining anhydrous potassium carbonate afforded the S-alkyl
In vitro antitumor screening
Primary in vitro one-dose assay
Out of the newly synthesized compounds, six derivatives
namely: 2c, 5c, 7, 12, 14, and 15a were selected by the
National Cancer Institute (NCI) in vitro disease-oriented
human cells screening panel assay to be evaluated for their
in vitro antitumor activity.
1
derivatives 13a-c in good yields. IR, H-NMR, and 13C-
NMR spectral data confirmed the chemical structure of
these derivatives. Additionally, synthesis of the azomethine
derivative 14 was achieved by condensing the thione 12
with 4-nitrobenzaldehyde in boiling ethanol containing few
drops of glacial acetic acid. IR spectrum of 14 lacked
absorption bands characteristic for NH2 group, while its
1H-NMR spectrum revealed singlets attributed to the
nitrophenyl group in addition to a singlet for the N=CH
proton at 8.89 ppm indicating that the configuration around
the C=N is E (Pretsch et al., 2000, pp 211–212). Moreover,
the MS spectrum of 14 showed a molecular ion peak at m/
z 369 (20.5 %) which matches with its molecular formula.
Finally, alkylation of the azomethine 14 with hydrochloride
salts of 2-substituted ethyl chloride derivatives in aqueous
KOH furnished the corresponding 2-substituted ethyl-
An effective one-dose assay has been added to the
NCI-60 cell screen in order to increase compound
throughput and reduce data-turnaround time to suppliers
while maintaining efficient identification of active com-
pounds (Weislow et al., 1989; Monks et al., 1991; Boyd
and Paull, 1995). All compounds submitted to the NCI-60
cell screen are tested initially at a single high dose (10 lM)
in the full NCI-60 cell panel including leukemia, non-small
cell lung, colon, CNS melanoma, ovarian, renal, prostate,
and breast cancer cell lines. Only compounds which satisfy
pre-determined threshold inhibition criteria would proceed
to the five-dose screen. The threshold inhibition criteria for
proceeding to the five-dose screen was designed to effi-
ciently capture compounds with anti-proliferative activity,
and it is based on careful analysis of historical Develop-
ment Therapeutic Program (DTP) screening data. Data are
reported as a mean graph of the percent growth of treated
cells, and presented as percentage growth inhibition (GI%)
caused by the test compounds (Table 1).
1
sulfanyl derivatives 15a–c. Inspection of H-NMR spectra
of the latter compounds indicates presence of signals for
SCH2 and NCH2 protons at their expected chemical shifts.
Furthermore, 13C-NMR spectrum of compound 15b dis-
played signals at 28 and 58.20 ppm corresponding to SCH2
and NCH2 in addition to signals of piperidine carbons
which appeared at their expected values.
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Med Chem Res (2013) 22:1909–1924
1913
Table 1 Mean growth percent, delta values, and in vitro percentage growth inhibition (GI%) caused by the selected compounds against some
tumor cell lines at the single-dose assay
Comp. no.
NSC-number
746136/1
Mean growth percent
97.13
Delta
77.96
Panel
Subpanel cell lines (growth inhibition percent)
2c
Breast cancer
Ovarian cancer
Leukemia
MCF7 (80.83), T-47D (38.32), MDA-MB-48 (57.34)
IGROV 1 (80.44)
K-562 (38.06)
Renal cancer
Melanoma
RXF 393 (62.53), UO-31 (33.27)
SK-MEL-2 (42.31)
5c
748494/1
49.14
48.11
Non-small cell
lung cancer
A549/ATCC (62.66), EKVX (58.26), HOP-92 (39.68),
NCI-H226 (52.63), NCI-H23 (41.17), NCI-H322 M
(75.57), NCI-H460 (79.63)
Colon cancer
HCC-2998 (51.49), HCT-116 (59.00), HCT-15 (61.94),
HT29 (55.77), KM12 (44.01), SW-620 (50.75)
Breast cancer
MCF7 (54.06), T-47D (37.26)
Ovarian cancer
IGROV 1 (64.98), OVCAR-3 (62.13), OVCAR-4
(70.9), OVCAR-5 (39.07), OVCAR-8 (75.15), NCI/
ADR-RES (94.27), SK-OV-3 (39.34)
Leukemia
CCRF-CEM (66.06), HL-60(TB) (4.83), K-562 (58.29),
MOLT-4 (55.63), RPMI-8226 (56.67), SR (61.94)
Renal cancer
786-O (53.06), A498(66.15), ACHN (70.57), CAKI-1
(98.97), RXF 393 (32.23), SN 12C (31.83), TK-10
(48.80), UO-31 (96.08)
Melanoma
LOX IMVI(50.13), M14 (42.64), MDA-MB-435
(37.18), SK-MEL-28 (33.64), SK-MEL-5
(54.04),UACC-257 (30.42), UACC-62 (47.52)
Prostate cancer
CNS cancer
PC-3 (47.44), DU-145 (36.73)
SF-268 (47.48), SF-295 (90.1), SF-539 (44.65), SNB-19
(42.23), SNB-75 (32.65), U251 (49.39)
7
752242/1
746140/1
105.76
102.26
60.02
79.29
Leukemia
HL-60(TB) (54.26)
12
Breast cancer
Ovarian cancer
Melanoma
MCF7 (72.68)
IGROV 1 (77.03), OVCAR-8 (59.10)
SK-MEL-2 (70.66)
14
746142/1
748493/1
96.42
92.86
71.98
Breast cancer
Ovarian cancer
Melanoma
MCF7 (73.08)
IGROV 1 (75.56), OVCAR-8 (47.79)
SK-MEL-2 (70.32)
15a
113.30
Breast cancer
Leukemia
MCF7 (69.03), T-47D (31.84)
CCRF-CEM (120.44, lethality), RPMI-8226 (32.99),
SR(66.41)
Data obtained from NCI in vitro disease-oriented human tumor cell screen at 10 lM concentration
The obtained results revealed that compound 2c was
able to exhibit promising broad spectrum anticancer
activity particularly against breast cancer MCF7, MDA-
MB-48, Ovarian cancer IGROV 1, and renal cancer RXF
393 cell lines (GI% values 80.83, 57.34, 80.44, and 62.53,
respectively). However, its overall antitumor profile did not
meet the pre-determined threshold inhibition criteria and
therefore was not sufficient to proceed to the five-dose
screen. Compounds 12 and 14 displayed remarkable
growth inhibitory activity towards breast cancer MCF7,
ovarian cancer IGROV 1, and melanoma SK-MEL-2 cell
lines (GI% range 70.32–77.03). Moreover, compound 15a
exhibited significant activity against breast cancer MCF7
and SR cell lines (GI% values 69.03, 120.44, and 66.41,
respectively). It should be noted here that compound 15a
showed lethal effect towards the CCRF-CEM cell line with
20.44 %. On the other hand, compound 7 was proved to be
the weakest anticancer member in this screen owing to its
low potency and narrow margin of activity, which was
against only leukemia HL-60(TB) and with GI% value of
54.26. Whereas, compound 5c was found to be the most
active member in this preliminary screen as evidenced by
its ability to exert potential growth inhibitory activity
against most of the tested subpanel tumor cell lines.
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Med Chem Res (2013) 22:1909–1924
Consequently, it passed successfully this assay and was
carried over to the five-dose screen against a panel of about
60 different tumor cell lines.
Table 2 GI50, TGI, and LC50 of some selected in vitro tumor cell
lines (lM) for compound 5c
Panel-cell line
Compound 5c
GI50
TGI
LC50
In vitro full panel (five-dose) 60-cell line assay
for compound 5c
Leukemia
CCRF-CEM
HL-60(TB)
K-562
2.88
2.83
2.79
2.01
3.03
16.8
8.19
23
[100
[100
[100
[100
[100
About 60 cell lines of nine tumor subpanels, including
leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate, and breast cancer cell lines, were
incubated with five concentrations (0.01–100 lM) for each
compound and were used to create log concentration %
growth inhibition curves. Three response parameters (GI50,
TGI, and LC50) were calculated for each cell line. The GI50
value (growth inhibitory activity) corresponds to the con-
centration of the compounds causing 50 % decrease in net
cell growth, the TGI value (cytostatic activity) is the con-
centration of the compounds resulting in total growth
inhibition and the LC50 value (cytotoxic activity) is the
concentration of the compounds causing net 50 % loss of
initial cells at the end of the incubation period (48 h).
Subpanel and full panel mean-graph midpoint values (MG-
MID) for certain agents are the average of individual real
and default GI50, TGI, or LC50 values of all cell lines in the
subpanel or the full panel, respectively.
MOLT-4
18.5
23.2
RPMI-8226
Non-small cell lung cancer
A549/ATCC
EKVX
3.48
3.57
5.77
1.61
12.2
5.13
1.47
1.88
5.12
34.2
31.1
76.8
9.28
75.6
53.9
38.2
13.9
36.8
[100
[100
[100
[100
[100
[100
[100
[100
[100
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
3.44
5.59
2.92
1.89
7.82
3.19
5.02
18.4
[100
15.6
[100
[100
[100
[100
[100
[100
[100
In the present study, compound 5c exhibited potential
antitumor activities against most of the tested subpanel
tumor cell lines (GI50 and TGI values \100 lM). This
compound showed a distinctive pattern of sensitivity
against some individual cell lines (Table 1), as well as a
broad spectrum (MG-MID) of antitumor activity (Table 2).
A deep insight into the obtained results (Table 2)
revealed that compound 5c exhibited remarkably high
activity against renal cancer CAKI-1 cell line with GI50
value of 0.37 lM. It also displayed a distinguished sensi-
tivity profile towards all leukemia cell lines with GI50
range of 2.01–3.03 lM beside a potential activity against
seven cell lines with GI50 range of 1.23–1.63 lM. In
addition, compound 5c showed appreciable growth inhib-
itory potential against 58 cell lines with GI50 range of
1.88–18.7 lM. Further interpretation of the obtained data
revealed that compound 5c was able to totally inhibit the
growth of 50 cell lines at 4.6–84.8 lM. Moreover, 5c was
cytotoxic against three cell lines; ovarian cancer OVCAR-
3, NCI/ADR-RES, and prostate cancer DU-145 (LC50
values 31.8, 96.4, and 55.4 lM, respectively).
[100
[100
32
HT29
KM12
SW-620
[100
CNS cancer
SF-268
1.99
1.58
5.49
5.81
10.6
4.47
25.1
24.3
26.1
69.4
37.2
84.8
[100
[100
[100
[100
[100
[100
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3 M
M14
6.69
11
73.4
53.1
31.7
[100
77.9
82.3
24.8
71.8
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
2.27
5.22
15.6
4.06
3.83
6.59
5.07
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
The results also revealed that compound 5c displayed
high growth inhibitory potential (GI50 MG-MID 3.98 lM)
(Table 3), together with reasonable cytostatic (TGI MG-
MID 35.48 lM) and mild cytotoxic (LC50 MG-MID
97.72 lM) activities (Table 4).
4.89
1.48
3.45
5.49
37.8
5.2
[100
31.8
OVCAR-3
OVCAR-4
OVCAR-5
28.8
82
[100
[100
The ratio obtained by dividing the compound full panel
MG-MID (lM) by its individual subpanel MG-MID (lM)
123

Med Chem Res (2013) 22:1909–1924
1915
is considered as a measure of compound selectivity. Ratios
between 3 and 6 refer to moderate selectivity, ratios [6
indicate high selectivity towards the corresponding cell
line, while compounds meeting neither of these criteria are
rated non-selective (Acton et al., 1994). In this context,
compound 5c was found to be non-selective with broad
spectrum antitumor activity against the nine tumor sub-
panels tested with selectivity ratios ranging between 0.59
and 1.47 at the GI50 MG-MID level. Moreover, log GI50
values for compound 5c was illustrated with respect to
5-fluorouracil as a comparative study for anticancer
potency (Table 5), where values of -0.4 and less are
considered to be of high anticancer activity. The compar-
ative study indicated that compound 5c displayed anti-
tumor activity against all human tumor cell lines higher
than 5-fluorouracil.
Table 2 continued
Panel-cell line
Compound 5c
GI50
TGI
LC50
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-O
4.47
1.23
5.58
65.8
8.11
7.43
[100
96.4
[100
3.98
1.62
3.8
21.2
6.62
24.8
4.6
[100
[100
[100
[100
[100
[100
[100
[100
A498
ACHN
CAKI-1
0.37
13.1
18.7
3.64
1.63
RXF 393
SN12C
54.9
[100
23.1
13.7
TK-10
UO-31
The above results revealed that this compound could be
an appropriate candidate for further derivatization in order
to explore the scope and limitations of its potential hoping
to find more selective and active anticancer agents.
Prostate cancer
PC-3
5.6
56.2
18.3
[100
55.4
DU-145
3.01
Breast cancer
MCF7
4.05
13.9
4.42
4.37
3.08
50.4
[100
43.8
44.2
22.2
[100
[100
[100
[100
[100
MDA-MB-231/ATCC
BT-549
Experimental
T-47D
All reagents and solvents were purchased from commercial
suppliers and were dried and purified when necessary by
standard techniques. Melting points were determined in
MDA-MB-468
Data obtained from NCI in vitro disease-oriented human cell screen
Table 3 Median growth inhibitory concentrations (GI50, lM) of in vitro subpanel tumor cell lines for compound 5c
MG-MID
Subpanel tumor cell lines GI50 MG-MID (lM) (SI)
A
B
C
D
E
F
G
H
I
3.98
2.71 (1.47)
4.47 (0.89)
4.27 (0.93)
4.99 (0.80)
6.70 (0.59)
3.80 (1.0)
5.85 (0.68)
4.30 (0.92)
6.39 (0.62)
Median values calculated according to the data obtained from NCI’s in vitro disease-oriented human tumor cell screen
A Leukemia, B non-small cell lung cancer, C colon cancer, D CNS cancer, E Melanoma, F ovarian cancer, G renal cancer, H prostate cancer,
I breast cancer; GI50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent; SI
selectivity index
Table 4 Median total growth inhibitory concentrations (TGI, lM) and lethal concentration (LC50, lM) of in vitro subpanel tumor cell lines for
compound 5c
MG-MID
Subpanel tumor cell lines
A
B
C
D
E
F
G
H
I
35.48
17.94
a
–
41.08
–
22.0
–
44.48
–
59.29
–
43.14
(89.7)b
37.35
–
40.15
(50.0)
48.12
–
(97.72)
Median values calculated according to the data obtained from NCI’s in vitro disease-oriented human tumor cell screen. For subpanel tumor cell
lines, see footnote of Table 3
TGI (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent
a
LC50 (MG-MID) value [ 100 lM
b
LC50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent
123

1916
Med Chem Res (2013) 22:1909–1924
Table 5 Log GI50 values (lM) of compound 5c with respect to 5-FU
Table 5 continued
Panel/cell line
Log GI50
5c
Panel/cell line
Log GI50
5-FU
5c
5-FU
Leukemia
CCRF-CEM
HL-60(TB)
K-562
Renal cancer
786-O
-5.54
-5.55
-5.56
-5.7
-4.5
-4.7
-4.7
-4.9
-5.3
-5.4
-4.9
-5
A498
-5.79
-5.42
-6.43
-4.88
-4.73
-5.44
-5.79
ACHN
-5
MOLT-4
CAKI-1
-5.4
-4.3
-4.6
-3.9
-5.3
RPMI-8226
Non-small cell lung cancer
A549/ATCC
EKVX
-5.52
RXF 393
SN12C
-5.46
-5.45
-5.24
-5.79
-4.91
-5.29
-5.83
-5.73
-5.29
-5.7
-3.5
-4.7
-3.8
-3.6
-4.9
-4.7
-6
TK-10
UO-31
HOP-62
Prostate cancer
PC-3
HOP-92
-5.25
-5.52
-4.3
-5
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
DU-145
Breast cancer
MCF7
-5.39
-4.86
-5.07
-5.35
-5.36
-5.8
-3.3
-3.6
-4
MDA-MB-231/ATCC
HS 578T
BT-549
-4.4
-5.46
-5.25
-5.54
-5.72
-5.11
-5.5
-5.2
-5.8
-5.4
-5.2
-5.2
-5
T-47D
-4.1
HT29
open glass capillaries using Stuart capillary melting point
apparatus (Stuart Scientific Stone, Staffordshire, UK) and
are uncorrected. Infrared (IR) spectra were recorded on
Perkin-Elmer 1430 infrared spectrophotometer (Perkin
Elmer, Beaconsfield, UK) and measured by t9 cm-1 scale
KM12
SW-620
-5.3
-4.6
CNS cancer
SF-268
-5.7
-4.3
-4.3
-5.9
-3.9
-3.7
-4.4
1
using KBr cell. H-NMR spectra were scanned on Jeol-
SF-295
-5.8
500 MHz spectrometer (Jeol, Tokyo, Japan) and Varian
Mercury VX-300 using tetramethylsilane (TMS) as internal
standard and DMSO-d6 as the solvent (chemical shifts are
given in d ppm). Splitting patterns were designated as
follows: s: singlet; brs: broad singlet; d: doublet; dd: dou-
blet of doublet; t: triplet; m: multiplet. 13C-NMR proton
decoupled spectra were recorded on a Varian Mercury VX-
300 spectrometer in DMSO-d6 and measured in d scale.
Mass spectra were run on a Finnigan mass spectrometer
model SSQ/7000 (70 eV) or on a gas chromatograph/mass
spectrometer Schimadzu GCMS-QP 2010 Plus (70 eV).
Elemental analyses were performed on Elementar Vario E1
and were found within 0.4 % of the theoretical values.
Follow up of the reactions and checking the purity of the
compounds was made by thin layer chromatography (TLC)
on silica gel-precoated aluminum sheets (Type 60 GF254;
Merck; Germany) and the spots were detected by exposure
to UV lamp at k 254 nm for few seconds. Compounds 1
(Slouka, 1962), 3 (Osman et al., 2007), and 10 (Rohmer,
1898) were prepared according to previously reported
reaction conditions.
SF-539
-5.26
-5.24
-4.97
-5.35
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
-5.17
-4.96
-5.64
-4.81
-5.39
-5.42
-5.18
-5.3
-5.2
-4.7
-4.3
-3.4
-4.3
-4.9
-4
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
-4.9
-5.31
-5.83
-5.46
-5.26
-5.35
-5.25
-4.9
-4.6
-4.2
-3.8
-4.7
-3.8
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
123

Med Chem Res (2013) 22:1909–1924
1917
3-[(2-Aryl-2-oxoethyl)sulfanyl]-6-[(E)-2-(furan-2-
yl)ethenyl]-1,2,4-triazin-5(2H)-ones (2a–e)
7.48, 7.57 (2 9 d, J = 8.4 Hz, each 2H, chlorophenyl
C3,5-H and chlorophenyl C2,6-H), 7.69–7.74 (m, 2H, eth-
enyl C2-H and furan C5-H), 8.32 (s, 1H, NH, D2O
exchangeable); Anal. Calcd for C17H12ClN3O3S (373.81):
C, 54.62; H, 3.24; N, 11.24; S, 8.58; found: C, 54.62; H,
3.13; N, 11.06; S, 8.59.
A mixture of the thione 1 (2.2 g, 10 mmol) and the
appropriate phenacyl bromide (10 mmol) in absolute eth-
anol (10 ml) was heated under reflux for 2 h. The reaction
mixture was cooled to room temperature and the separated
product was filtered, washed with ethanol, dried and
crystallized from dioxane/water.
(E)-3-[{2-(4-Bromophenyl)-2-oxoethyl}sulfanyl]-6-[2-
(furan-2-yl)ethenyl]-1,2,4-triazin-5(2H)-one (2d)
Yellow solid (80 %); m.p.: 218–220 °C; IR (KBr, cm-1):
3164 (NH), 3018 (CH furan), 2964, 2901 (CH2), 1673
(C=O), 1608 (C=N), 1582, 1506, 1478 (C=C, d NH), 1287,
1069 (C–S–C), 1255, 1011 (C–O–C), 732 (oop furan);
1H-NMR (500 MHz, DMSO-d6) d: 3.67, 3.77 (2 9 d,
J = 12.2 Hz, each 1H, S-CH2), 6.51–6.56 (m, 1H, furan
C4-H), 6.67 (d, J = 16.05 Hz, 1H, ethenyl C1-H), 6.79 (d,
J = 3.1 Hz, 1H, furan C3-H), 7.50, 7.61 (2 9 d,
J = 8.0 Hz, each 2H, bromophenyl C3,5-H and bromo-
phenyl C2,6-H), 7.69–7.75 (m, 2H, ethenyl C2-H and furan
C5-H), 8.32 (s, 1H, NH, D2O exchangeable); Anal. Calcd
for C17H12BrN3O3S (418.26): C, 48.82; H, 2.89; N, 10.05;
S, 7.67; found: C, 49.09; H, 2.52; N, 9.87; S, 7.67.
(E)-6-[2-(Furan-2-yl)ethenyl]-3-[(2-phenyl-2-
oxoethyl)sulfanyl]-1,2,4-triazin-5(2H)-one (2a)
Brown solid (76 %); m.p.: 192–194 °C; IR (KBr, cm-1):
3146 (NH), 3095 (CH furan), 2964, 2916 (CH2), 1692,
1659(C=O), 1625 (C=N), 1552, 1526, 1482 (C=C, d NH),
1290, 1076 (C–S–C), 1232, 1014 (C–O–C), 744 (oop
furan); 1H-NMR (300 MHz, DMSO-d6) d: 4.45 (s, 2H,
SCH2), 6.54–6.59 (m, 1H, furan C4-H), 6.77 (d,
J = 3.9 Hz, 1H, furan C3-H), 6.81 (d, J = 16.7 Hz, 1H,
ethenyl C1-H), 7.23–7.34 (m, 5H, phenyl-H), 7.56 (d,
J = 16.7 Hz, 1H, ethenyl C2-H), 7.78 (s, 1H, furan C5-H),
12.40 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C17H13N3O3S (339.37): C, 60.17; H, 3.86; N, 12.38; found:
C, 59.78; H, 3.47; N, 12.73.
(E)-6-[2-(Furan-2-yl)ethenyl]-3-[{2-(4-nitrophenyl-2-
oxoethyl}sulfanyl]-1,2,4-triazin-5(2H)-one (2e)
(E)-6-[2-(Furan-2-yl)ethenyl]-3-[{2-(4-methylphenyl)-2-
oxoethyl}sulfanyl]-1,2,4-triazin-5(2H)-one (2b)
Orange solid (60 %); m.p.: [300 °C; IR (KBr, cm-1):
3113 (NH), 3025 (CH furan), 2900, 2856 (CH2), 1675
(C=O), 1621 (C=N), 1575, 1472 (C=C, d NH), 1519, 1347
(NO2), 1281, 1077 (C–S–C), 1219, 1019 (C–O–C), 754
Yellow solid (63 %); m.p.: 218–220 °C; IR (KBr, cm-1):
3275 (NH), 3075 (CH furan), 2921, 2850 (CH2, CH3),
1705, 1653 (C=O), 1628 (C=N), 1529, 1473 (C=C, d NH),
1281, 1087 (C–S–C), 1194, 1017 (C–O–C), 750 (oop
furan); 1H-NMR (300 MHz, DMSO-d6) d: 2.34 (s, 3H,
CH3), 4.38 (s, 2H, SCH2), 6.57–6.62 (m, 1H, furan C4-H);
6.75 (d, J = 3.9 Hz, 1H, furan C3-H), 6.79 (d,
J = 16.7 Hz, 1H, ethenyl C1-H), 7.33 (d, J = 8.4 Hz, 2H,
methylphenyl C3,5-H), 7.56 (d, J = 16.7 Hz, 1H, ethenyl
C2-H), 7.75 (s, 1H, furan C5-H), 7.85 (d, J = 8.4 Hz, 2H,
methylphenyl C2,6-H), 12.39 (s, 1H, NH, D2O exchange-
able); Anal. Calcd for C18H15N3O3S (353.39): C, 61.18; H,
4.28; N, 11.89; found: C, 61.53; H, 3.88; N, 11.53.
1
(oop furan); H-NMR (300 MHz, DMSO-d6) d: 3.74, 3.87
(2 9 d, J = 12.6 Hz, each 1H, S-CH2), 6.51–6.57 (m, 1H,
furan C4-H), 6.68 (d, J = 16.2 Hz, 1H, ethenyl C1-H), 6.81
(d, J = 3.3 Hz, 1H, furan C3-H), 7.72 (d, J = 16.2 Hz, 1H,
ethenyl C2-H), 7.75 (s, 1H, furan C5-H), 7.89, 8.29 (2 9 d,
J = 8.7 Hz, each 2H, nitrophenyl C2,6-H and C3,5-H), 8.59
(s, 1H, NH, D2O exchangeable); 13C-NMR (300 MHz,
DMSO-d6) d 50.55 (CH2), 122.11 (furan C4), 122.99 (furan
C3), 127.26 (ethenyl C1), 132.90 (nitrophenyl C3,5), 134.49
(ethenyl C2), 137.64 (nitrophenyl C2,6), 152.0 (nitrophenyl
C1), 154.54 (furan C5), 156.33 (furan C2), 157.21 (nitro-
phenyl C4), 158.5 (triazine C6), 161.01 (triazine C3),
175.18 (triazine C5), 175.98 (C=O); Anal. Calcd for
C17H12N4O5S (384.37): C, 53.12; H, 3.15; N, 14.58; S,
8.34; found: C, 52.79; H, 2.89; N, 14.84; S, 8.22.
(E)-3-[{2-(4-Chlorophenyl)-2-oxoethyl}sulfanyl]-6-[2-
(furan-2-yl)ethenyl]-1,2,4-triazin-5(2H)-one (2c)
Yellow solid (78 %); m.p.: 228–230 °C; IR (KBr, cm-1):
3171 (NH), 3097 (CH furan), 2962, 2915 (CH2), 1672
(C=O), 1615 (C=N), 1588, 1533, 1483 (C=C, d NH), 1286,
6-[(E)-2-(Furan-2-yl)ethenyl]-3-(2-substituted
hydrazono)-1,2,4-triazin-5(2H)-ones (4a–g)
1
1091 (C–S–C), 1257, 1013 (C–O–C), 748 (oop furan); H-
NMR (500 MHz, DMSO-d6) d: 3.67, 3.78 (2 9 d,
J = 12.2 Hz, each 1H, S-CH2), 6.51–6.54 (m, 1H, furan
C4-H), 6.65–6.80 (m, 2H, ethenyl C1-H and furan C3-H),
A mixture of the hydrazine 3 (0.48 g, 2.2 mmol) and the
proper aldehyde or ketone (2.2 mmol), in absolute ethanol
(10 ml) containing a catalytic amount of glacial acetic acid,
123

1918
Med Chem Res (2013) 22:1909–1924
was heated under reflux for 1 h. The reaction mixture was
cooled and the precipitated product was filtered, washed
with ethanol, dried and crystallized from the proper
solvent.
6.69 (d, J = 3.05 Hz, 1H, furan C3-H), 6.84 (d, J = 20 Hz,
1H, ethenyl C1-H), 7.82 (s, 1H, CH = N), 7.84 (d,
J = 20 Hz, 1H, ethenyl C2-H), 8.11 (d, J = 8.4 Hz, 2H,
nitrophenyl C2,6-H), 8.14 (s, 1H, furan C5-H), 8.22 (d,
J = 8.4 Hz, 2H, nitrophenyl C3,5-H), 12.24, 13.40 (2 9 s,
each 1H, 2 NH, D2O exchangeable); Anal. Calcd for
C16H12N6O4.1/2 H2O (361.31): C, 53.19; H, 3.63; N,
23.26; found: C, 53.44; H, 3.50; N, 23.16.
6-[(E)-2-(Furan-2-yl)ethenyl]-3-[(E)-2-(4-methoxy-
benzylidene)hydrazono]-1,2,4-triazin-5(2H)-one (4a)
Yellow solid (98 %); m.p.: 282–284 °C (ethanol); IR (KBr,
cm-1): 3350, 3250 (NH), 3050 (CH furan), 2912, 2838
(CH3), 1663 (C=O), 1620 (C=N), 1558, 1505, 1464 (C=C,
6-[(E)-2-(Furan-2-yl)ethenyl]-3-[(E)-2-{(furan-2-
yl)methylene}hydrazono]-1,2,4-triazin-5(2H)-one (4d)
1
d NH), 1243, 1025 (C–O–C), 738 (oop furan); H-NMR
(300 MHz, DMSO-d6) d: 3.82 (s, 3H, OCH3), 6.54–6.58
(m, 1H, furan C4-H), 6.73 (d, J = 3.3 Hz, 1H, furan C3-H),
6.89 (d, J = 16.2 Hz, 1H, ethenyl C1-H), 6.99 (d,
J = 8.7 Hz, 2H, methoxyphenyl C3,5-H), 7.74 (s, 1H, furan
C5-H), 7.75–7.85 (m, 3H, ethenyl C2-H and methoxy-
phenyl C2,6-H), 8.05 (s, 1H, N = CH), 11.6, 12.9 (2 9 s,
each 1H, 2 NH, D2O exchangeable). 13C-NMR (300 MHz,
DMSO-d6) d 64.76 (OCH3), 121.38 (furan C4), 121.85
(furan C3), 123.55 (methoxyphenyl C3,5), 128.95 (ethenyl
C1), 132.12 (ethenyl C2), 136.01 (methoxyphenyl C1),
138.58 (methoxyphenyl C2,6), 152.80 (N = CH), 153.63
(furan C5), 154.51 (furan C2), 160.87 (triazine C6), 161.56
(methoxyphenyl C4), 170.21 (triazine C3), 174.0 (C=O);
Anal. Calcd for C17H15N5O3 (337.33): C, 60.53; H, 4.48;
N, 20.76. Found: C, 60.19; H, 4.17; N, 20.52.
Yellow orange solid (90 %); m.p.: 264–266 °C (dimethyl
formamide); IR (KBr, cm-1): 3325, 3182 (NH), 3025 (CH
furan), 1663 (C=O), 1615 (C=N), 1563, 1509 (C=C, d NH),
1275, 1016 (C–O–C), 745 (oop furan); 1H-NMR
(500 MHz, DMSO-d6) d: 6.55-6.56 (m, 1H, furan C4-H),
6.62–6.63 (m, 1H, furan C4-H), 6.72 (d, J = 3.1 Hz, 1H,
furan C3-H), 6.83 (d, J = 16.1 Hz, 1H, ethenyl C1-H), 7.03
(d, J = 3.05 Hz, 1H, furan C3-H), 7.73 (s, 1H, furan C5-H),
7.78 (d, J = 16.05 Hz, 1H, ethenyl C2-H), 7.82 (d,
J = 1.6 Hz, 1H, furan C5-H), 7.97 (s, 1H, CH = N), 11.71,
12.88 (2 9 s, each 1H, 2 NH, D2O exchangeable); Anal.
Calcd for C14H11N5O3. 2H2O (333.30): C, 50.45; H, 4.54;
N, 21.01; found: C, 50.06; H, 4.32; N, 21.13.
6-[(E)-2-(Furan-2-yl)ethenyl]-3-[(E)-2-{(5-nitrofuran-2-
yl)methylene}hydrazono]-1,2,4-triazin-5(2H)-one (4e)
3-[(E)-2-(4-Chlorobenzylidene)hydrazono]-6-[(E)-
2-(furan-2-yl)ethenyl]1,2,4-triazin-5(2H)-one (4b)
Orange solid (96 %); m.p.: [300 °C (dioxane); IR (KBr,
cm-1): 3400, 3150 (NH), 3025 (CH furan), 1663 (C=O),
1628 (C=N), 1595, 1497 (C=C, d NH), 1567, 1349 (NO2),
1249, 1018 (C–O-C), 738 (oop furan); 1H-NMR
(500 MHz, DMSO-d6) d: 6.56–6.57 (m, 1H, furan C4-H),
6.74 (d, J = 3.8 Hz, 1H, furan C3-H), 6.84 (d,
J = 16.1 Hz, 1H, ethenyl C1-H), 7.42 (d, J = 3.8 Hz, 1H,
nitrofuran C3-H), 7.73 (s, 1H, furan C5-H), 7.78 (d,
J = 16.1 Hz,1H, ethenyl C2-H), 7.80 (d, J = 3.8 Hz, 1H,
nitrofuran C4-H), 8.01 (s, 1H, CH = N), 12.28, 13.14
(2 9 s, each 1H, 2 NH, D2O exchangeable); Anal. Calcd
for C14H10N6O5.1H2O: C, 46.67; H, 3.36; N, 23.33; found:
C, 46.29; H, 3.07; N, 23.43.
Yellow solid (97 %); m.p.: 276–278 °C (ethanol); IR (KBr,
cm-1): 3393, 3300 (NH), 3050 (CH furan), 1663 (C=O),
1636 (C=N), 1553, 1505 (C=C, d NH), 1273, 1017 (C–O–
1
C), 741 (oop furan); H-NMR (300 MHz, DMSO-d6) d:
6.57–6.62 (m, 1H, furan C4-H), 6.76 (d, J = 3.6 Hz, 1H
furan C3-H), 6.88 (d, J = 15.9 Hz, 1H, ethenyl C1-H), 7.51
(d, J = 8.4 Hz, 2H, chlorophenyl C3,5-H), 7.76 (s, 1H,
furan C5-H), 7.84 (d, J = 15.9 Hz, 1H ethenyl C2-H), 7.98
(d, J = 8.4 Hz, 2H, chlorophenyl C2,6-H), 8.08 (s, 1H,
N=CH), 11.9, 13.1 (2 9 s, each 1H, 2 NH, D2O
exchangeable); Anal. Calcd for C16H12ClN5O2 (341.75): C,
56.23; H, 3.54; N, 20.49; found: C, 56.08; H, 3.54; N,
20.35.
3-[2-{6-((E)-2-(Furan-2-yl)ethenyl)-5-oxo-2,5-dihydro-
1,2,4-triazin-3-yl}hydrazono]indolin-2-one (4f)
6-[(E)-2-(Furan-2-yl)ethenyl]-3-[(E)-2-(4-nitroben-
zylidene)hydrazono]-1,2,4-triazin-5(2H)-one (4c)
Orange solid (80 %); m.p.:[300 °C (dimethyl formamide/
water); IR (KBr, cm-1): 3357, 3181, 3105 (NH), 3042 (CH
furan), 1688 (C=O), 1624 (C=N), 1556, 1491 (C=C, d NH),
1231, 1046 (C–O–C), 741 (oop furan); 1H-NMR (500 MHz,
DMSO-d6) d: 6.56–6.59 (m, 1H, furan C4-H), 6.76–6.82 (m,
3H, furan C3-H, isatin C4-H and ethenyl C1-H), 6.94–7.23
(2 9 t, J = 7.5 Hz, each 1H, isatin C5,6-H), 7.62 (d,
Yellow solid (95 %); m.p.: [300 °C (dimethyl formam-
ide); IR (KBr, cm-1): 3361 (NH), 3045 (CH furan), 1675
(C=O), 1621 (C=N), 1587, 1503 (C=C, d NH), 1558, 1338
1
(NO2), 1250, 1016 (C–O–C), 738 (oop furan); H-NMR
(500 MHz, DMSO-d6) d: 6.52–6.56 (m, 1H, furan C4-H),
123

Med Chem Res (2013) 22:1909–1924
1919
J = 16.1 Hz, 1H, ethenyl C2-H), 7.75 (d, J = 1.6 Hz, 1H,
furan C5-H), 8.40–8.45 (m, 1H, isatin C7-H), 10.50, 12.90,
13.9 (3 9 s, each 1H, 3 NH, D2O exchangeable); Anal.
Calcd for C17H12N6O3ꢀ2H2O (384.35): C, 53.12; H, 4.20; N,
21.87; found: C, 52.98; H, 3.87; N, 21.94.
1680 (C=O), 1624 (C=N), 1548, 1486 (C=C, d NH), 1248,
1012 (C–O–C), 743 (oop furan); 1H-NMR (300 MHz,
DMSO-d6) d: 2.35 (s, 3H, CH3), 5.88 (s, 1H, pyrazole C4-
H), 6.56-6.62 (m, 1H, furan C4-H), 6.88 (d, J = 3.6 Hz, 1H
furan C3-H), 6.93 (d, J = 16.2 Hz, 1H, ethenyl C1-H), 7.1
(br.s, 2H, NH2, D2O exchangeable), 7.27 (d, J = 8.2 Hz,
2H, methylphenyl C3,5-H), 7.82–7.89 (m, 4H, furan C5-H,
ethenyl C2-H and methylphenyl C2,6-H), 13.9 (br.s, 1H,
NH, D2O exchangeable); Anal. Calcd for C19H16N6O2
(360.37): C, 63.32; H, 4.48; N, 23.32; found: C, 63.65; H,
4.27; N, 23.13.
3-[2-{6-((E)-2-(Furan-2-yl)ethenyl)-5-oxo-2,5-dihydro-
1,2,4-triazin-3-yl}hydrazono]-1-methylindolin-2-one (4g)
Orange solid (87 %); m.p.: 260–262 °C (dimethyl form-
amide/water); IR (KBr, cm-1): 3344, 3310(NH), 3024 (CH
furan), 2932, 2875 (CH3), 1676 (C=O), 1620 (C=N), 1550,
1520 (C=C, d NH), 1245, 1042 (C–O–C), 741 (oop furan);
1H-NMR (300 MHz, DMSO-d6) d: 2.7 (s, 3H, CH3),
6.54–6.60 (m, 1H, furan C4-H), 6.78–6.84 (m, 3H, furan
C3-H, isatin C4-H and ethenyl C1-H), 6.93–7.21 (2 9 t,
J = 7.5 Hz, each 1H, isatin C5,6-H), 7.65 (d, J = 16.2 Hz,
1H, ethenyl C2-H), 7.76 (d, J = 1.6 Hz, 1H, furan C5-H),
8.37–8.42 (m, 1H, isatin C7-H), 10.40, 14.0 (2 9 s, each
1H, 2NH, D2O exchangeable); Anal. Calcd for
C18H14N6O3 (362.34): C, 59.67; H, 3.89; N, 23.19; found:
C, 59.45; H, 3.64; N, 23.04.
(E)-3-[5-Amino-3-(4-chlorophenyl)-1H-pyrazol-1-yl]-
6-[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5-(2H)-one (5c)
Pale yellow solid (85 %); m.p.: 280–282 °C; IR (KBr,
cm-1): 3415, 3300, 3177 (NH), 3025 (CH furan), 1658
(C=O), 1620 (C=N), 1546, 1488 (C=C, d NH), 1250, 1016
1
(C–O–C), 762 (oop furan); H-NMR (500 MHz, DMSO-
d6) d: 5.89 (s, 1H, pyrazole C4-H), 6.58–6.61 (m, 1H, furan
C4-H), 6.81 (d, J = 3.8 Hz, 1H, furan C3-H), 6.89 (d,
J = 16.8 Hz, 1H, ethenyl C1-H), 7.07 (br.s, 2H, NH2, D2O
exchangeable), 7.48 (d, J = 8.4 Hz, 2H, chlorophenyl
3-(5-Amino-3-aryl-1H-pyrazol-1-yl)-6-[(E)-2-(furan-2-
yl)ethenyl]-1,2,4-triazin-5-(2H)-ones (5a–d)
C
3,5-H), 7.76 (s, 1H, furan C5-H), 7.87 (d, J = 16.8 Hz,
1H, ethenyl C2-H), 7.94 (d, J = 7.3 Hz, 2H, chlorophenyl
2,6-H), 13.98 (s, 1H, NH, D2O exchangeable); 13C-NMR
C
A solution of the hydrazine 3 (0.48 gm, 2.2 mmol) and the
selected phenacyl cyanide (2.2 mmol) in ethanol/glacial acetic
acid mixture (10 ml) (4:1) was refluxed for 3 h. The reaction
mixture was cooled and the precipitated product was filtered,
washed with ethanol, dried and crystallized from dioxane.
(500 MHz, DMSO-d6) d: 85 (pyrazole C4), 112.51 (furan
C4), 112.92 (ethenyl C1), 118.41 (furan C3), 124.11(ethenyl
C2), 127.83 (chlorophenyl C2,6), 128.56 (chlorophenyl
C3,5), 130.70 (chlorophenyl C1), 133.60 (chlorophenyl C4),
140.01 (triazine C6), 144.65 (furan C5), 145.58 (pyrazole
C3), 149.93 (furan C2), 151.84 (pyrazole C5), 152.02 (tri-
azine C3), 153.02 (C=O); Anal. Calcd for C18H13ClN6O2
(380.79): C, 56.78; H, 3.44; N, 22.07; found: C, 56.75; H,
3.74; N, 22.02.
(E)-3-(5-Amino-3-phenyl-1H-pyrazol-1-yl)-6-[2-
(furan-2-yl)ethenyl]-1,2,4-triazin-5-(2H)-one (5a)
Pale yellow solid (80 %); m.p.: 262–264 °C; IR (KBr,
cm-1): 3418, 3344, 3306, 3178 (NH), 3058 (CH furan),
1660 (C=O), 1617(C=N), 1576, 1546 (C=C, d NH), 1245,
1016 (C–O–C), 752 (oop furan); 1H-NMR (300 MHz,
DMSO-d6) d: 5.92 (s, 1H, pyrazole C4-H), 6.60–6.65 (m,
1H, furan C4-H), 6.86 (d, J = 3.6 Hz, 1H furan C3-H), 6.93
(d, J = 16.2 Hz, 1H, ethenyl C1-H), 7.1 (br.s, 2H, NH2,
D2O exchangeable), 7.40–7.50 (m, 3H, phenyl C3,4,5-H),
7.81 (d, J = 1.5 Hz, 1H, furan C5-H), 7.90–7.97 (m, 3H,
ethenyl C2-H and phenyl C2,6-H), 13.9 (br.s, 1H, NH, D2O
exchangeable); Anal. Calcd for C18H14N6O2 (346.34): C,
62.42; H, 4.07; N, 24.27; found: C, 62.27; H, 3.77; N, 24.0.
(E)-3-[5-Amino-3-(4-bromophenyl)-1H-pyrazol-1-yl]-
6-[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5-(2H)-one (5d)
Yellowish white solid (78 %); m.p.: 288–290 °C; IR (KBr,
cm-1): 3396, 3361, 3294 (NH), 3023 (CH furan), 1662
(C=O), 1624(C=N), 1548, 1488 (C=C, d NH), 1249, 1012
1
(C–O–C), 753 (oop furan). H-NMR (500 MHz, DMSO-
d6) d: 5.89 (s, 1H, pyrazole C4-H), 6.58–6.62 (m, 1H, furan
C4-H), 6.83 (d, J = 3.8 Hz, 1H, furan C3-H), 6.90 (d,
J = 16.4 Hz, 1H, ethenyl C1-H), 7.06 (br.s, 2H, NH2, D2O
exchangeable), 7.43 (d, J = 7.3 Hz, 2H, bromophenyl
(E)-3-[5-Amino-3-(4-methylphenyl)-1H-pyrazol-1-yl]-6-
[2-(furan-2-yl)ethenyl]-1,2,4-triazin-5-(2H)-one (5b)
C
3,5-H), 7.75 (s, 1H, furan C5-H), 7.89 (d, J = 16.4 Hz,
1H, ethenyl C2-H), 7.90 (d, J = 7.3 Hz, 2H, bromophenyl
2,6-H), 13.95 (s, 1H, NH, D2O exchangeable); Anal.
C
Yellow solid (60 %); m.p.: 272–274 °C; IR (KBr, cm-1):
3398, 3347, 3301 (NH), 3023 (CH furan), 2918 (CH3),
Calcd for C18H13BrN6O2 (425.24): C, 50.84; H, 3.08; N,
19.76; found: C, 50.93; H, 2.82; N, 19.62.
123

1920
Med Chem Res (2013) 22:1909–1924
(E)-3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-[2-(furan-2-yl)
ethenyl]-1,2,4-triazin-5(2H)-one (6)
furan), 2986, 2901 (CH2, CH3), 1732, 1685 (C=O), 1628
(C=N), 1572, 1550, 1500 (C=C, d NH), 1185, 1016 (C–O–
1
C), 742 (oop furan); H-NMR (500 MHz, DMSO-d6) d:
A mixture of the hydrazine 3 (0.2 g, 0.9 mmol) and ace-
tylacetone (0.1 g, 0.11 ml, 1 mmol) in absolute ethanol
was heated under reflux for 3 h. The reaction mixture was
cooled, and the formed precipitate was filtered, washed
with ethanol, dried and crystallized from ethanol. Shiny
yellow crystals (68 %); M.p. 180–182 °C; IR (KBr, cm-1):
3288, 3116 (NH), 3069 (CH furan), 2988, 2923 (CH3),
1676 (C=O), 1619 (C=N), 1588, 1558, 1540, 1493 (C=C, d
NH), 1239, 1018 (C–O–C), 746 (oop furan); 1H-NMR
(500 MHz, DMSO-d6) d: 2.22, 2.46 (2 9 s, each 3H,
pyrazole CH3), 6.24 (s, 1H, pyrazole C4-H), 6.54–6.57 (m,
1H, furan C4-H), 6.80 (d, 1H, J = 2.3 Hz, furan C3-H),
6.86 (d, J = 16.1 Hz, 1H, ethenyl C1-H), 7.76 (s, 1H, furan
C5-H), 7.86 (d, J = 16.1 Hz, 1H, ethenyl C2-H), 14.16 (s,
1H, NH, D2O exchangeable); Anal. Calcd for C14H13N5O2
(283.29): C, 59.36; H, 4.63; N, 24.72. Found: C, 59.27; H,
4.92; N, 24.42.
1.16 (t, J = 6.9 Hz, 3H, CH2-CH3), 1.94 (s, 3H, CH3), 4.07
(q, J = 6.9 Hz, 2H, CH2-CH3), 6.50–6.54 (m, 1H, furan
C4-H), 6.70 (d, J = 3.1 Hz, 1H, furan C3-H), 6.81 (d,
J = 16.1 Hz, 1H, ethenyl C1-H), 7.71 (s, 1H, furan C5-H),
7.77 (d, J = 16.1 Hz, 1H, ethenyl C2-H), 10.72, 12.74
(2 9 s, each 1H, 2 NH, D2O exchangeable); Anal. Calcd
for C15H17N5O4 (331.33): C, 54.38; H, 5.17; N, 21.14
Found: C, 54.04; H, 5.51; N, 20.78.
(E)-6-[(2-(Furan-2-yl)ethenyl]-3-(3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl)-1,2,4-triazin-5(2H)-one (9)
Method A Compound 8 (0.5 g, 1.5 mmol) was heated in an
oil bath at 160 °C for 20 min. The reaction mixture was
left to cool and the obtained product was triturated with
EtOH, filtered, dried, and crystallized from dimethyl
formamide/water. Reddish brown solid (72 %).
Method B The hydrazine derivative 3 (0.2 g, 0.9 mmol)
was heated with ethyl acetoacetate (0.13 g, 0.12 ml,
1 mmol) in an oil bath at 160 °C for 1 h. The reaction
mixture was left to cool and the solidified residue was
triturated with ethanol, filtered, dried, and crystallized from
dimethyl formamide/water. (65 %); m.p.: 190–192 °C; IR
(KBr, cm-1): 3433 (NH), 3066 (CH furan), 2923 (CH3),
1679 (C=O), 1637 (C=N), 1542, 1487 (C=C, d NH), 1210,
1012 (C–O–C), 743 (oop furan); 1H-NMR (500 MHz,
DMSO-d6) d: 2.47 (s, 3H, pyrazoline C3-CH3), 3.44 (s, 2H,
pyrazoline C4-H), 6.58–6.61 (m, 1H, furan C4-H), 6.81 (d,
J = 3.1 Hz, 1H, furan C3-H), 6.86 (d, J = 16.1 Hz, 1H,
ethenyl C1-H), 7.77 (s, 1H, furan C5-H), 7.89 (d,
J = 16.1 Hz, 1H, ethenyl C2-H), 13.38 (s, 1H, NH, D2O
exchangeable); MS (m/z, %): 285 (M?, 21.0), 98 (100.0);
Anal. Calcd for C13H11N5O3ꢀ‘H2O (294.27): C, 53.06; H,
4.11; N, 23.80; found: C, 52.91; H, 4.29; N, 23.96.
(E)-3-(3,5-Dioxopyrazolidin-1-yl)-6-[2-
(furan-2-yl)ethenyl]-1,2,4-triazin-5(2H)-one (7)
A mixture of the hydrazine 3 (0.2 g, 0.9 mmol) and diethyl
malonate (0.16 g, 0.15 ml, 1 mmol) in a mixture of etha-
nol/glacial acetic acid (10 ml) (1:1) was heated under
reflux for 8 h and then cooled. The precipitated solid was
filtered, washed with ethanol, dried and crystallized from
glacial acetic acid. Red crystals (57 %); m.p.: 165–167 °C;
IR (KBr, cm-1): 3341, 3101 (NH), 3059 (CH furan), 2903
(CH2), 1707, 1662 (C=O), 1628 (C=N), 1583, 1552, 1500
1
(C=C, d NH), 1205, 1029 (C–O–C), 761 (oop furan); H-
NMR (500 MHz, DMSO-d6) d: 2.45 (s, 2H, pyrazolidine
C4-H), 3.36 (br.s., 1H, pyrazolidine NH), 6.56–6.60 (m,
1H, furan C4-H), 6.87 (d, J = 3.8 Hz, 1H,furan C3-H), 7.0
(d, J = 16.3 Hz, 1H, ethenyl C1-H), 7.79 (s, 1H, furan C5-
H), 7.86 (d, J = 16.3 Hz, 1H, ethenyl C2-H), 8.70 (s, 1H,
NH, D2O exchangeable), 13.59 (s, 1H, triazine NH, D2O
exchangeable); Anal. Calcd for C12H9N5O4 (287.23): C,
50.18; H, 3.16; N, 24.38; found: C, 49.77; H, 3.49; N,
24.56.
(2Z, 3E)-4-(Furan-2-yl)-2-[2-
(thiocarbohydrazono)]butenoic acid (11)
A mixture of the 2-furylidene pyruvic acid 10 (5 g,
30 mmol) and thiocarbohydrazide (3.2 g, 30 mmol) in a
mixture of ethanol (20 ml) and glacial acetic acid (0.5 ml)
was stirred at room temperature for 15 min. It was filtered,
washed with water, dried and crystallized from dimethyl
formamide/water. Orange red solid (79 %); m.p.:
238–240 °C; IR (KBr, cm-1): 3437, 3290, 3200 (OH, NH),
3078 (CH furan), 1661 (C=O), 1626 (C=N), 1568, 1551,
1501 (C=C, d NH), 1534, 1277, 1119, 965 (N–C=S), 1232,
1016 (C–O–C), 753 (oop furan); 1H-NMR (500 MHz,
DMSO-d6) d: 6.50 (s, 2H, NH2, D2O exchangeable),
6.55–6.59 (m, 1H, furan C4-H), 6.78–6.83 (m, 2H, furan
(E)-Ethyl 3-[2-{6-(2-(furan-2-yl)ethenyl)-5-oxo-2,5-
dihydro-1,2,4-triazin-3-yl} hyrazono]butanoate (8)
A mixture of the hydrazine 3 (0.2 g, 0.9 mmol) and ethyl
acetoacetate (0.13 g, 0.12 ml, 1 mmol) in absolute ethanol
was heated under reflux for 3 h. The reaction mixture was
left to cool to room temperature and the obtained product
was filtered, washed with ethanol, dried and crystallized
from ethanol. Pale yellow crystals (76 %); m.p.:
156–158 °C; IR (KBr, cm-1): 3296, 3100 (NH), 3038 (CH
123

Med Chem Res (2013) 22:1909–1924
1921
(E)-4-Amino-3-(ethylsulfanyl)-6-[2-(furan-2-yl)
ethenyl]-1,2,4-triazin-5(4H)-one (13b)
C3-H and ethenyl C1-H), 7.64 (d, J = 16.1 Hz, 1H, ethenyl
C2-H), 7.77 (s, 1H, furan C5-H), 9.68, 10.30 (2s, each 1H,
2NH, D2O exchangeable); Anal. Calcd for C9H10N4O3S
(254.27): C, 42.51; H, 3.96; N, 22.03; found: C, 42.74; H,
3.95; N, 22.35.
Brown solid (55 %); m.p.: 118–120 °C; IR (KBr, cm-1):
3293, 3201 (NH2), 3075 (CH furan), 2973, 2926 (CH2,
CH3), 1676 (C=O), 1624 (C=N), 1536, 1486 (C=C, d NH),
1287, 1072 (C–S-C), 1231, 1018 (C–O–C), 737(oop furan);
1H-NMR (500 MHz, DMSO-d6) d: 1.33 (t, J = 7.0 Hz,
3H, CH2–CH3), 4.45 (q, J = 7.0 Hz, 2H, CH2–CH3), 6.59
(dd, J = 3.1, 1.5 Hz, 1H, furan C4-H), 6.67 (s, 2H, NH2,
D2O exchangeable), 6.82–6.85 (m, 2H, ethenyl C1-H and
furan C3-H), 7.71 (d, J = 16.8 Hz, 1H, ethenyl C2-H), 7.78
(d, J = 1.6 Hz, 1H, furan C5-H); Anal. Calcd for
C11H12N4O2S.1/2 H2O (273.31): C, 48.34; H, 4.79; N,
20.50; S, 11.73; found: C, 48.66; H, 4.39; N, 20.17; S,
11.64.
(E)-4-Amino-6-[2-(furan-2-yl)ethenyl]-3-thioxo-3,4-
dihydro-1,2,4-triazin-5(2H)-one (12)
A solution of the thiocarbohydrazone 11 (2.5 g, 10 mmol)
in 1 N NaOH (20 ml) was heated for 20 min. After cool-
ing, the solution was acidified with dil. H2SO4 to pH 6. The
formed precipitate was filtered, washed with water, dried,
and crystallized from dioxane.
Pale brown crystals (93 %); m.p.: 230–232 °C; IR (KBr,
cm-1): 3437, 3254 (NH), 3078 (CH furan), 1660 (C=O),
1625 (C=N), 1593, 1517, 1499 (C=C, d NH), 1542, 1278,
1104, 961 (N–C=S), 1235, 1013 (C–O–C), 754 (oop furan);
1H-NMR (500 MHz, DMSO-d6) d: 6.51 (s, 2H, NH2, D2O
exchangeable), 6.55–6.59 (m, 1H, furan C4-H), 6.78–6.83
(m, 2H, furan C3-H and ethenyl C1-H), 7.64 (d,
J = 16.1 Hz, 1H, ethenyl C2-H), 7.76 (s, 1H, furan C5-H),
13.90 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C9H8N4O2S (236.25): C, 45.75; H, 3.41; N, 23.72; found:
C, 46.02; H, 3.64; N, 23.47.
(E)-4-Amino-3-(benzylsulfanyl)-6-[2-(furan-2-yl)
ethenyl]-1,2,4-triazin-5(4H)-one (13c)
Pale yellow solid (60 %); m.p.: 142–144 °C; IR (KBr,
cm-1): 3290, 3190 (NH2), 3025 (CH furan), 2947(CH2),
1676 (C=O), 1629 (C=N), 1563, 1530, 1484 (C=C, d NH),
1291, 1081 (C–S–C), 1253, 1015 (C–O–C), 730 (oop
furan); 1H-NMR (500 MHz, DMSO-d6) d: 5.65 (s, 2H,
S-CH2), 6.59 (dd, J = 3.4, 1.93 Hz, 1H, furan C4-H), 6.66
(s, 2H, NH2, D2O exchangeable), 6.82–6.86 (m, 2H, eth-
enyl C1-H and furan C3-H), 7.24–7.37 (m, 5H, phenyl-H),
7.71 (d, J = 16.1 Hz, 1H, ethenyl C2-H), 7.78 (s, 1H, furan
C5-H); 13C-NMR (300 MHz, DMSO-d6) d: 44.21 (CH2),
123.01 (furan C4), 124.97 (furan C3), 127.0 (ethenyl C1),
128.50 (ethenyl C2), 134.29 (phenyl C4), 137.14 (phenyl
4-Amino-6-[(E)-2-(furan-2-yl)ethenyl]-3-(substituted
sulfanyl)-1,2,4-triazin-5(4H)-ones (13a–c)
A mixture of the thione 12 (0.38 g, 1.6 mmol), anhydrous
K2CO3 (0.33 g, 2.4 mmol) and dimethylsulfate, ethyl
iodide or benzyl chloride (2.4 mmol) in dry dimethyl
formamide (4 ml) was stirred at room temperature for 12 h.
The reaction mixture was then diluted with water and the
obtained product was filtered, washed with water, dried,
and crystallized from ethanol.
C3,5), 139.13 (phenyl C2,6), 143.8 (phenyl C1), 145.71
(furan C5), 152.85 (furan C2), 154.48 (triazine C3), 158.0
(triazine C6), 163.0 (C=O); Anal. Calcd for C16H14N4O2S
(326.37): C, 58.88; H, 4.32; N, 17.17; found: C, 58.64; H,
4.07; N, 16.88.
6-[(E)-2-(Furan-2-yl)ethenyl]-4-[(E)(4-
nitrobenzylidene)amino]-3-thioxo-3,4-dihydro-1,2,4-
triazin-5(2H)-one (14)
(E)-4-Amino-6-[2-(furan-2-yl)ethenyl]-3-(methylsulfanyl)-
1,2,4-triazin-5(4H)-one (13a)
Yellow solid (72 %); m.p.: 230–232 °C; IR (KBr, cm-1):
3242, 3180 (NH2), 3075 (CH furan), 2947 (CH3),
1661(C=O), 1621 (C=N), 1544, 1494 (C=C, d NH), 1286,
A mixture of the amino thione 12 (0.5 g, 2.1 mmol) and
4-nitrobenzaldehyde (0.32 g, 2.1 mmol) in absolute etha-
nol (20 ml) containing few drops of glacial acetic acid was
heated under reflux for 1 h. The reaction mixture was left
to cool to room temperature and the formed precipitate was
filtered, washed with ethanol, dried and crystallized from
glacial acetic acid. Yellow solid (61 %); m.p.: 256-258 °C.
IR (KBr, cm-1): 3207 (NH), 3094 (CH furan), 1701 (C=O),
1620 (C=N), 1595, 1525, 1482 (C=C, dNH), 1520, 1260,
1137, 973 (N–C=S), 1525, 1342 (NO2), 1260, 1015 (C–O–
1
1078 (C–S–C), 1231, 1010 (C–O–C), 755 (oop furan); H-
NMR (500 MHz, DMSO-d6) d 3.32 (s, 3H, CH3, under
DMSO), 6.50 (s, 2H, NH2, D2O exchangeable), 6.57 (dd,
J = 3.8, 1.55 Hz, 1H, furan C4-H), 6.80 (d, J = 3.8 Hz,
1H, furan C3-H), 6.81, 7.64 (2 9 d, J = 16.05 Hz, each
1H, ethenyl C1-H and ethenyl C2-H), 7.77 (s, 1H, furan C5-
H); Anal. Calcd for C10H10N4O2S (250.28): C, 47.99; H,
4.03; N, 22.39; S, 12.81. Found: C, 47.65; H, 4.39; N,
22.07; S, 13.12.
1
C), 747 (oop furan); H-NMR (500 MHz, DMSO-d6) d:
123

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Med Chem Res (2013) 22:1909–1924
6.56–6.59 (m, 1H, furan C4-H), 6.81 (d, J = 3.8 Hz, 1H,
furan C3-H), 6.84, 7.63 (2 9 d, J = 16.1 Hz, each 1H,
ethenyl C1-H and ethenyl C2-H), 7.78 (s, 1H, furan C5-H),
6.60–6.66 (m, 1H, furan C4-H), 6.87 (d, J = 3.0 Hz, 1H,
furan C3-H), 7.18 (d, J = 16.5 Hz, 1H, ethenyl C1-H), 7.82
(s, 1H, furan C5-H), 7.87 (d, J = 16.5 Hz, 1H, ethenyl
C2-H), 8.20, 8.42 (2 9 d, J = 8.7 Hz, each 2H, nitro-
phenyl C2,6-H and C3,5-H), 9.58 (s, 1H, N=CH); 13C-NMR
(300 MHz, DMSO-d6) d: 28.0 (SCH2), 30.71 (piperidine
C4), 31.92 (piperidine C3,5), 58.20 (NCH2), 61.65 (piperi-
dine C2,6), 122.01 (furan C4), 122.80 (furan C3), 133.92
(nitrophenyl C3,5), 135.6 (ethenyl C1), 138.3 (ethenyl C2),
139.57 (nitrophenyl C2,6), 144.30 (nitrophenyl C1), 148.5
(N=CH), 152.0 (furan C5), 154.0 (nitrophenyl C4), 155.0
(furan C2), 158.32 (triazine C3), 161.46 (triazine C6),
165.29 (C=O); Anal. Calcd for C23H24N6O4S (480.54): C,
57.49; H, 5.03; N, 17.49; S, 6.67; found: C, 57.19; H, 4.79;
N, 17.46; S, 6.58.
8.18, 8.39 (2 9 d, J = 8.4 Hz, each 2H, nitrophenyl C2,6
-
H and nitrophenyl C3,5-H), 8.89 (s, 1H, N = CH), 14.06 (s,
1H, NH, D2O exchangeable); MS (m/z, %): 369 (M?,
20.5), 119 (100.0). Anal. Calcd for C16H11N5O4S (369.35):
C, 52.03; H, 3.00; N, 18.96; found: C, 52.35; H, 3.06; N,
18.62.
6-[(E)-2-(Furan-2-yl)ethenyl]-4-[(E)(4-
nitrobenzylidene)amino]-3-[(2-substituted
ethyl)sulfanyl]-1,2,4-triazin-5(4H)-ones (15a-c)
To a suspension of compound 14 (0.48 g, 1.3 mmol) in
aqueous KOH solution (0.22 g, 2 ml, 4 mmol), the
appropriate alkylating agent (1.3 mmol) was added. The
reaction mixture was stirred at room temperature for 24 h
then acidified with glacial acetic acid till pH 6. The sepa-
rated solid product was filtered, dried, and crystallized
from the proper solvent.
6-[(E)-2-(Furan-2-yl)ethenyl]-4-[(E)-(4-
nitrobenzylidene)amino]-3-[(2-(pyrrolidin-1-
yl)ethyl)sulfanyl]-1,2,4-triazin-5(4H)-one (15c)
Orange brown solid (40 %); m.p.: 120–122 °C (methanol/
water); IR (KBr, cm-1): 3050 (CH furan), 2926, 2854
(CH2), 1687 (C=O), 1620 (C=N), 1591, 1558, 1493 (C=C),
1523, 1343 (NO2), 1260, 1075 (C–S–C), 1224, 1014 (C–
O–C), 746 (oop furan); 1H-NMR (500 MHz, DMSO-d6) d:
1.87–1.89 (m, 4H, pyrrolidine C3,4-H2), 2.97–3.20 (m, 4H,
pyrrolidine C2,5-H2), 3.37 (t, J = 6.9 Hz, 2H, S-CH2), 3.38
(t, J = 6.9 Hz, 2H, N-CH2), 6.60–6.63 (m, 1H, furan C4-
H), 6.84 (d, 1H, J = 3.1 Hz, furan C3-H), 7.08 (d,
J = 16.1 Hz, 1H, ethenyl C1-H), 7.80 (s, 1H, furan C5-H),
7.83 (d, J = 16.1 Hz, 1H, ethenyl C2-H), 8.18, 8.40
(2 9 d, J = 8.4 Hz, each 2H, nitrophenyl C2,6-H and
nitrophenyl C3,5-H), 9.50 (s, 1H, N=CH); Anal. Calcd for
C22H22N6O4S (466.51): C, 56.64; H, 4.75; N, 18.01; S,
6.87; found: C, 56.29; H, 4.79; N, 17.70; S, 7.12.
6-[(E)-2-(Furan-2-yl)ethenyl]-3-[(2-(morpholin-1-
yl)ethyl)sulfanyl]-4-[(E)-(4-nitrobenzylidene)amino]-
1,2,4-triazin-5(4H)-one (15a)
Orange solid (52 %); m.p.: 132–134 °C (dimethylform-
amide); IR (KBr, cm-1): 3068 (CH furan), 2924, 2853,
(CH2), 1686 (C=O), 1620 (C=N), 1591, 1557, 1491 (C=C),
1523, 1343 (NO2), 1262, 1069 (C–S–C), 1220, 1012
(morpholine and furan C–O–C), 745 (oop furan); 1H-NMR
(500 MHz, DMSO-d6) d: 2.63 (t, J = 6.9 Hz, 2H, S-CH2),
3.16 (t, J = 6.9 Hz, 2H, N-CH2), 3.54–3.58 (m, 8H, mor-
pholine C2,3,5,6-H2), 6.56–6.59 (m, 1H, furan C4-H), 6.74
(d, J = 3.1 Hz, 1H, furan C3-H), 6.85, 7.54 (2 9 d,
J = 16.1 Hz, each 1H, ethenyl C1-H and ethenyl C2-H),
7.74 (s, 1H, furan C5-H), 8.12, 8.34 (2 9 d, J = 8.4 Hz,
each 2H, nitrophenyl C2,6-H and nitrophenyl C3,5-H), 8.99
(s, 1H, N = CH); Anal. Calcd for C22H22N6O5S (482.51):
C, 54.76; H, 4.60; N, 17.42; S, 6.65; found: C, 54.39; H,
4.79; N, 17.69; S, 6.75.
In vitro antitumor screening
Preliminary in vitro one-dose antitumor screening
Anti-tumor activity screening for compounds 2c, 5c, 7, 12,
14, and 15a at a dose of 10 lM utilizing 55 different
human tumor cell lines, representing leukemia, melanoma
and cancers of the lung, colon, brain, ovary, breast, pros-
tate, and kidney was carried out according to standard
procedure (Skehan et al., 1990; Rubinstein et al., 1990).
The human tumor cell lines of the cancer screening panel
are grown in RPMI 1640 medium containing 5 % fetal
bovine serum and 2 mmol L-glutamine. For a typical
screening experiment, the tumor cells were inoculated into
96-well microtiter plates in 100 ll at plating densities
ranging from 5,000 to 40,000 cells/well. Density of the
6-[(E)-2-(Furan-2-yl)ethenyl]-4-[(E)-(4-
nitrobenzylidene)amino]-3-[(2-(piperidin-1-
yl)ethyl)sulfanyl]-1,2,4-triazin-5(4H)-one (15b)
Brown solid (60 %); m.p.: 141–143 °C (ethanol/water); IR
(KBr, cm-1): 3035 (CH furan), 2933, 2850 (CH2), 1690
(C=O), 1619 (C=N), 1591, 1523, 1497, (C=C), 1523, 1344
(NO2), 1280, 1073 (C–S–C), 1226, 1016 (C–O–C), 757
1
(oop furan); H-NMR (500 MHz, DMSO-d6) d: 1.39–1.50
(m, 6H, piperidine C3,4,5-H2), 1.73–1.79 (m, 4H, piperidine
C2,6-H2), 2.67 (m, 2H, SCH2), 2.89 (m, 2H, NCH2),
123

Med Chem Res (2013) 22:1909–1924
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Acknowledgments The authors are deeply thankful to the staff
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