New approaches for the synthesis of pyrazole, thiophene, thieno[2,3-b]pyridine, and thiazole derivatives together with their anti-tumor evaluations

Article abstract of DOI:10.1007/s00044-013-0664-7

The reaction of cyanoacetylhydrazine (1) with acetylchloride (2) gave the N-acyl derivative 3. The latter underwent ready cyclization in sodium ethoxide to give the pyrazole derivative 4 which was the key compound for the synthesis of thiophene, thieno[2,

Full text of DOI:10.1007/s00044-013-0664-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0664-7
ORIGINAL RESEARCH
New approaches for the synthesis of pyrazole, thiophene,
thieno[2,3-b]pyridine, and thiazole derivatives together
with their anti-tumor evaluations
•
Rafat M. Mohareb Amira E. M. Abdallah
•
Mahmoud A. Abdelaziz
Received: 25 February 2013 / Accepted: 29 May 2013
Ó Springer Science+Business Media New York 2013
Abstract The reaction of cyanoacetylhydrazine (1) with
acetylchloride (2) gave the N-acyl derivative 3. The latter
underwent ready cyclization in sodium ethoxide to give the
pyrazole derivative 4 which was the key compound for the
synthesis of thiophene, thieno[2,3-b]pyridine, and thiazole
derivatives. The anti-tumor evaluations of the newly syn-
thesized products against the three human tumor cell lines,
namely, breast adenocarcinoma (MCF-7), non-small cell
lung cancer (NCI-H460), and CNS cancer (SF-268), were
studied. Some of these compounds were found to exhibit
much higher inhibitory effects toward the three tumor cell
lines than the reference doxorubicin. Molecular modeling
of the four compounds 12c, 12f, 16a, and 16d, which
showed the maximum inhibitory effect, were done.
Introduction
The pyrazole scaffold represents a common motif in many
pharmaceutical active and remarkable compounds demon-
strating a wide range of pharmacological activities; the most
important activities are the anti-inflammatory (Elguero
et al., 2002; Szabo et al., 2008); antibacterial and antifungal
(Tanitame et al., 2005; Tanitame et al., 2004); hypoglyce-
mic (Cho et al., 2009; Dugi et al., 2009); anti-hyperlipi-
demic (Momose et al., 2001); inhibition of cyclooxigenase-
2 (Rida et al., 2009), p38 MAP kinase (Regan et al., 2002),
and CDK2/Cyclin A (Brasca et al., 2007; Pevarello et al.,
2006); and antiangiogenic activities (Abadi et al., 2003).
Heterocyclic rings and, in particular, the pyrazole ring
represent an advantageous choice for the synthesis of
pharmaceutical compounds with different activities and
good safety profiles (Michaelides, 2010; Hubbard and Till,
2000). Different pyrazole derivatives have also been tested
for their antiproliferative activities in vitro and anti-tumor
activity in vivo, often resulting in promising lead com-
pounds (Ludwig et al., 2004; Perchellet et al., 2006; Insu-
asty et al., 2009; Labbozzetta et al., 2009; Pevarello et al.,
2004; Sanchez and Cobo, 2010). In the view of the facts
mentioned above and as part of our initial efforts to discover
potentially active new agents, we have synthesized some
new pyrazole derivatives. The novel derivatives were
characterized by spectral data and elemental analysis. These
compounds were screened against the three cancer cell
lines, breast adenocarcinoma (MCF-7), non-small cell lung
cancer (NCI-H460), and CNS cancer (SF-268). Our data
indicate that these novel pyrazole derivatives target the
tumor cells. In particular, four compounds 12c, 12f, 16a,
and 16d emerged as potent anti-tumor compounds, which
may be used as lead compounds, but deserve further study in
order to obtain insight into their mechanism of action.
Keywords Pyrazole Á Thiophene Á
Thieno[2,3-b]pyridine Á Thiazole Á Anti-tumor
R. M. Mohareb (&)
Chemistry Department, Faculty of Science, Cairo University,
Cario, Egypt
e-mail: raafat_mohareb@yahoo.com
A. E. M. Abdallah
Chemistry Department, Faculty of Science, Helwan University,
Cario, Egypt
M. A. Abdelaziz
Preparatory Year Department, AL-Ghad International Colleges
for Applied Medical Sciences, Tabuk Male, Saudi Arabia
M. A. Abdelaziz
Basic Science Department, Modern Academy For Engineering
and Technology in Maadi, Cairo, Egypt
123

Med Chem Res
Results and discussion
bath to give the 6-hydroxythieno[2,3-b]pyridine-5-carbo-
nitrile derivatives 10a and 10b, respectively. Such cycli-
zation reactions will not proceed if the amide is being
formed through the 2-aminopyrazole moiety present in
both of compounds 8a and 8b.
Herein, in order to extend our research on anticancer het-
erocyclic derivatives with high inhibitory effects toward
some cancer cell lines, we report the synthesis of new
pyrazole derivatives starting from the N-acetyl-2-cyanoa-
cetohydrazide 3; the latter is formed through acetylation of
the 2-cyanohydrazide (1). Thus, compound 3 underwent
cyclization in sodium ethoxide solution to give the pyra-
zole derivative 4. The structure of compound 4 was based
on analytical and spectral data. Thus, the 1H-NMR showed
a singlet at d 2.77 ppm indicating one CH3 group, a singlet
at d 4.79 ppm (D2O exchangeable) indicating the presence
of one NH2 group, a singlet at d 6.86 ppm for the pyrazole
H-4, and a singlet at d 10.22 ppm indicating one OH group.
Moreover, the 13C-NMR spectrum showed the presence of
d 29.6 (CH3), 104.8, 152.3, 154.6 (pyrazole C), and 168.9
(C=O). Compound 4 reacted with bromine in acetic acid
solution to give the 1-(5-amino-3-hydroxy-1H-pyrazol-1-
yl)-bromoethanone 5. It is of great value to note that the
reaction occurred in acetic acid solution; thus, the acetyl
group is an active moiety toward bromination (Sunil
Kumar et al., 2007). However, one might expect that the
C-4 of the pyrazole derivative 4 is activated by the 3-OH
and the 5-NH2 group; to our knowledge, such activity
requires basic catalyzed conditions.
Based on the optimized reaction conditions established
above, a series of novel aryl hydrazono derivatives were
obtained with the aim of improving the inhibitory effect
against the tested cancer cell lines. Moreover, in order to
study the relationship between the structure and activity for
this type of compounds, we have studied the affects of
substitution on the aryl azo compounds as well as the effect
of CN group in the case of 9a and the ester group in the
case of 9b. Thus, the reaction of either 9a or 9b with any of
the aryldiazonium salts, namely, the benzenediazonium
chloride (11a), the 4-methylphenyldiazonium salt (11b), or
the 4-chlorophenyl diazonium salts (11c), in 0–5 °C gave
the aryl hydrazono derivatives 12a–f (Scheme 2).
Encouraged by the excellent results, we next investi-
gated the ability of compounds 9a,b to form thiazoles and
thiophene derivatives. Several studies (Bondock et al.,
2010; Al-Said et al., 2011) have described the synthesis of
thiazole and thiophene derivative, their importance, and
applications as intermediates for the synthesis of hetero-
cyclics (Gouda et al., 2010; Fadda et al., 2009). The
present study aimed to contribute with the chemical and
pharmacological studies of such group of compounds.
Thus, the reaction of compound 9a or 9b with phenyliso-
thiocyanate (13) in the presence of basic dimethylform-
amide gave the intermediate potassium sulfide salts 14a,b,
respectively. Heterocyclization of the latter intermediate
with a-halocarbonyl compounds gave, interestingly, either
thiazole or thiophene derivatives depending on the nature
of the a-halocarbonyl compound. Thus, their reaction with
either phenacyl bromide (15a) or chloroacetone (15b) gave
The N-acetyl group in compound 4 showed interesting
reactivity towards Gewald’s thiophene synthesis. Thus, its
reaction with either malononitrile (6a) or ethyl cyanoace-
tate (6b) and elemental sulfur in the presence of triethyl-
amine gave the thiophene derivatives 8a and 8b,
respectively (Scheme 1). Their respective 1H-NMR and
13C-NMR spectra were the tools of their structural eluci-
dation. The 2-amino group of compounds 8a and 8b was
condensed with ethyl cyanoacetate (6b) in dimethylform-
amide to give the thiophen-2-acetamide derivatives 9a and
9b, respectively. The IR spectrum of compound 9a showed
the presence of two CN groups stretching at t 2,227 and
2,220 cm-1 and the presence of one C=O stretching at t
1,688 cm-1. Moreover, the 1H-NMR spectrum showed d at
4.77 ppm (D2O exchangeable) indicating the presence of
one NH2 group, a singlet at d 4.81 ppm indicating the
presence of CH2 group, two singlets at d 6.83 and
6.84 ppm corresponding to the pyrazole H-4 and the thio-
phene H-5, and two singlets at d 8.21 and 10.08 ppm for
the NH and the OH groups, respectively. The amide for-
mation occurred at the 2-aminothiophene moiety, not at the
3-aminopyrazole moiety; this was established not only via
the spectral data, indicated above, but also through the
ready cyclization of the products 9a and 9b through the
Michael addition of the CH2 group to the 3-cyano group.
Thus, both of compounds 9a and 9b underwent ready
cyclization in sodium ethoxide solution in a boiling water
1
the thiophene derivatives 16a–d. Their H-NMR and 13C-
NMR data are in agreement with their respective structures.
Thus, the 1H-NMR spectrum of 16a (as an example)
showed the presence of a singlet at d 2.88 indicating the
CH3 group, two singlets of d 4.77 and 4.82 ppm (D2O
exchangeable) indicating the presence of the two NH2
groups, two singlets at d 6.80 and 6.85 ppm corresponding
to the pyrazole H-4 and the thiophene H-5, respectively, a
multiplet at d 7.29–7.38 ppm corresponding to the phenyl
group, and three singlets at d 8.26, 8.80, and 10.20 ppm
(D2O exchangeable) corresponding to the two NH groups
and the OH group, respectively. Moreover, its 13C-NMR
spectrum showed d: 31.2 (CH3), 104.3, 153.7, 154.3 (pyr-
azole C), 116.9 (CN), 119.0, 120.4, 120.9, 121.8, 124.8,
130.9, 133.6, 134.0, 139.4, 141.5, 143.8, 144.6 (two thio-
phene, C6H5), 163.9, 164.8 (2C=O). On the other hand, the
reaction of the intermediate potassium sulfide salts 14a or
14b with ethyl chloroacetate (17) gave the thiazole
123

Med Chem Res
H2C
NC
CONHNH2
CH2
CN
CONHNHCOCH3
H3C
COCl
+
1
2
3
OH
OH
Br2
NaOEt
N
N
H2N
AcOH
3
H2N
N
N
COCH2Br
COCH3
4
5
OH
N
H2N
N
X
CN
Et3N
1.4-dioxan
H2C
+
4
CH2
S8
+
COCH2S
X
NH
7a, X = CN
b, X = COOC2H5
6a, X = CN
b, X = COOC2H5
NH2
N
X
N
HO
NH2
S
8a, X = CN
b, X = COOC2H5
Scheme 1 Synthesis of compounds 3–8a,b
derivatives 18a and 18b, respectively. The analytical and
spectral data of the latter products are in agreement with
their respective structures.
was from Cambrex (New Jersey, USA). Dimethyl sulfox-
ide (DMSO), doxorubicin, penicillin, streptomycin, and
sulforhodamine B (SRB) were from Sigma Chemical Co.
(Saint Louis, USA).
Anti-tumor activity tests
Cell cultures
Reagents
Three human tumor cell lines, MCF-7 (breast adenocarci-
noma), NCI-H460 (non-small cell lung cancer), and SF-
268 (CNS cancer), were used. MCF-7 was obtained from
Fetal bovine serum (FBS) and L-glutamine were from
Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium
123

Med Chem Res
NH2
NH2
N
X
DMF
H2C
CN
COOC2H5
N
N
X
+
HO
NH2
N
S
HO
NHCOCH2CN
S
8a, X = CN
b, X = COOC2H5
6b
9a, X = CN
b, X = COOC2H5
NaOEt
NH2
X
CN
N
N
OH
HO
N
S
10a, X = NH2
b, X = OH
NH2
-
+
N
X
EtOH/NaOH
0-5 oC
9a,b
+
NCl
Y
N
N
HO
NHCOC-CN
NNH
9a, X = CN
b, X = COOC2H5
11a, Y = H
b, Y = CH3
c, Y = Cl
S
X
Y
12
a
CN
H
CH3
Cl
Y
b
c
CN
CN
COOC2H5
COOC2H5
COOC2H5
d
e
f
H
CH3
Cl
Scheme 2 Synthesis of compounds 9a,b–12a–f
the European Collection of Cell Cultures (ECACC, Salis-
bury, UK), and NCI-H460 and SF-268 were kindly pro-
vided by the National Cancer Institute (NCI, Cairo, Egypt).
They grow as a monolayer and routinely maintained in
RPMI-1640 medium supplemented with 5 % heat-inacti-
vated FBS, 2 mM glutamine, and antibiotics (penicillin
100 U/mL, streptomycin 100 lg/mL) at 37 °C in a
humidified atmosphere containing 5 % CO2. Exponentially
growing cells were obtained by plating 1.5 9 105 cells/mL
for MCF-7 and SF-268 and 0.75 9 104 cells/mL for NCI-
H460, followed by 24 h of incubation. The effect of the
vehicle solvent (DMSO) on the growth of these cell lines
123

Med Chem Res
was evaluated in all the experiments by exposing untreated
control cells to the maximum concentration (0.5 %) of
DMSO used in each assay.
obvious that compound 16a with the X = CN, R = CH3
showed the highest inhibitory effect among the four com-
pounds and 16d with X = CO2Et, R = Ph showed the
maximum inhibitory effect toward breast adenocarcinoma
(MCF-7) among the four compounds. The cytotoxicity
effects of the newly synthesized compounds toward the three
cancer cell lines are indicated through Figs. 1, 2, and 3.
Moreover, the data described through Table 1 were
studied through statistical ANOVA program. The statistical
data are presented through Tables 2 and 3 and the statis-
tical ANOVA diagram Fig. 4.
Tumor cell growth assay
The effects of synthesized compounds 4–18a,b on the in vitro
growth of human tumor cell lines were evaluated according to
the procedure adopted by the National Cancer Institute (NCI,
USA)inthe‘‘InvitroAnticancerDrugDiscoveryScreen’’that
uses the protein-binding dye sulforhodamine B to assess cell
growth. Briefly, exponentially, cells growing in 96-well plates
were then exposed for 48 h to five serial concentrations of
each compound, starting from a maximum concentration of
150 lM. Following this exposure period, adherent cells were
fixed, washed, and stained. The bound stain was solubilized
and the absorbance was measured at 492 nm in a plate reader
(Bio-Tek Instruments Inc., Powerwave XS, Wincoski, USA).
For each test compound and cell line, a dose–response curve
was obtained and the growth inhibition of 50 % (GI50), cor-
responding to the concentration of the compounds that
inhibited 50 % of the net cell growth, was calculated as
described elsewhere. Doxorubicin was used as a positive
control and tested in the same manner.
Experimental
All melting points were determined on an electrothermal
apparatus (Bu¨chi 535, Switzerland) in an open capillary
Table 1 Effects of the synthesized compounds 4–18a,b on the
growth of three human tumor cell lines
Compound
GI50 (lMmol/L)
MCF-7
NCI-H460
SF-268
4
8.6 1.4
55.4 12.2
38.1 0.6
22.6 12.2
0.6 0.04
11.8 0.6
4.3 0.8
4.9 0.8
42.1 8.0
17.3 1.4
12.6 8.6
0.3 0.08
14.5 0.8
2.0 0.8
3.8 0.8
38.9 8.8
24.3 1.5
51.4 14.6
0.1 0.08
16.7 1.6
1.5 0.1
5
7a
Effect on the growth of human tumor cell lines
7b
8a
The effect of compounds 4–18a,b was evaluated on the
in vitro growth of three human tumor cell lines representing
different tumor types, namely, breast adenocarcinoma
(MCF-7), non-small cell lung cancer (NCI-H460), and CNS
cancer (SF-268), after a continuous exposure for 48 h. The
results are summarized in Table 1. All of the tested com-
pounds were able to inhibit the growth of the tested human
tumor cell lines in a dose-dependant manner (data not
shown). The results indicated through Table 1 revealed that
‘‘compounds 8a, 9a, 10a, 10b, 12a, 12c, 12f, 16a, 16b, 16d
and 18a showed the highest inhibitory effect against all the
three tumor cell lines.’’ On the other hand, compounds 12c,
12f, 16a, and 16d showed high inhibitory effects against all
the three human tumor cell lines, which were much higher
than the reference doxorubicin. Compounds 5, 7a, 9b, 12b,
12d, 12e, 16c, and 18b showed the lowest inhibitory effect.
The rest of the compounds 4, 7b, and 8b showed a moderate
growth inhibitory effect. Comparing compound 8a, 8b and
9a, 9b, it is obvious that the presence of the CN groups in 8a,
9a showed a higher inhibitory effect than the CO2Et groups
in 8b, 9b, and 10b. Comparing compounds 12a, 12b, 12c,
12d, 12e, and 12f, it is obvious that compound 12c with
X = CN and Y = Cl showed the highest inhibitory effect
among the six compounds. Moreover, 12f with X = CO2Et
and Y = Cl showed a high inhibitory effect, but it is still
lower than 12c. Similarly, for compounds 16a–d, it is
8b
9a
9b
20.0 0.2
0.7 0.50
2.0 0.6
22.6 1.4
0.2 0.08
2.0 0.4
28.4 0.6
1.0 0.02
1.5 8.0
10a
10b
12a
12b
12c
12d
12e
12f
16a
16b
16c
16d
18a
18b
Doxorubicin
0.9 0.2
0.1 0.02
44.0 0.8
0.02 0.001
32.6 1.4
22.2 12.8
0.02 0.008
0.02 0.008
3.6 1.4
0.3 0.05
20.5 1.1
0.01 0.001
34.4 0.6
30.0 9.0
0.01 0.001
0.01 0.004
2.4 0.8
38.0 1.8
0.01 0.003
20.0 0.2
26.7 17.5
0.07 0.05
0.03 0.007
2.0 1.2
32.0 1.8
0.01 0.006
4.2 2.6
12.0 0.8
0.03 0.002
2.2 0.04
18.6 2.6
0.09 0.008
14.5 4.1
0.06 0.005
0.5 0.2
20.9 4.9
0.04 0.008
18.3 2.6
0.09 0.007
Results are given in concentrations that were able to cause 50 % of
cell growth inhibition (GI50) after a continuous exposure of 48 h and
show mean SEM of three independent experiments performed in
duplicate
CELL NO 1 MCF-7
CELL NO 2 NCI-H460
CELL NO 3 SF-268
123

Med Chem Res
55.4
60
Fig. 1 Cytotoxicity of
compounds 4–18a,b toward
MCF-7
50
38
38.1
40
32
MCF-7 Data
26.7
30
22.6
20.9
20
20
20
10
0
11.8
8.6
4.3
4.2
2
2
0.9
0.7
0.6
0.04
0.01
0.03 0.07
0.01
5
4
9b
9a
8b
8a
7b
7a
12f
18b 18a 16d 16c 16b 16a
12e 12d 12c 12b 12a 10b 10a
Doxorubicin
50
45
40
35
30
25
20
15
10
5
Fig. 2 Cytotoxicity of
compounds 4–18a,b toward
NCI-H460
44
42.1
32.6
22.2
NCI-H460
22.6
18.6
17.3
14.5
12.6
12
4.9
3.6
2.2
2
2
0.2
0.3
0.09
0.03
0.02 0.02
0.02
0.1
0
5
4
9b
9a
8b
8a
7b
7a
12f
16c
12c
18b
18a 16d
16b
16a
12e
12d
12b
12a 10b
10a
Doxorubicin
60
50
40
30
20
10
0
Fig. 3 Cytotoxicity of
compounds 4–18a,b toward
SF-268
51.4
38.9
34.4
30
28.4
SF-268
24.3
20.5
18.3
16.7
14.5
3.8
2.4
1.5
0.3
1.5
1
0.5
0.09
0.06
0.01 0.01
0.01
0.1
5
4
9b
9a
8b
8a
7b
7a
12f
18b 18a 16d 16c 16b 16a
12e 12d 12c 12b 12a 10b 10a
Doxorubicin
Table 2 ANOVA
Cells
Sum of squares df Mean square
F
Sig.
Between groups
Within groups
Total
67.220
15018.954
15086.174
2
33.610
0.148 0.863
66 227.560
68
Table 3 Test of homogeneity of variances
Levene statistic
df1
df2
Sig.
Cells
0.551
2
66
0.579
tube and are uncorrected. Elemental analysis was performed
on a Yanaco CHNS Corder elemental analyzer (Japan). IR
spectra (t, cm-1) were recorded in KBr pellets on a PA-9721
IR spectrophotometer (Shimadzu, Japan). 1H-NMR and 13C-
NMR spectra were obtained on a Jeol 300 MHz (Japan)
spectrometer with DMSO-d6 as a solvent using TMS as
internal reference, and chemical shifts (d) are expressed in
ppm. Mass spectra were recorded on Kratos (75 eV) MS
Fig. 4 Statistics ANOVA diagram
equipment (Germany). Synthetic pathways are presented in
Schemes 1, 2, and 3. Theanti-tumor evaluations of the newly
synthesized compounds on the growth of three human tumor
cell lines are given in Table 1.
123

Med Chem Res
NH2
N
X
DMF/KOH
+
Ph-NCS
9a,b
N
HO
NH CO
CN
SK
9 a, X = CN
b, X = COOC2H5
S
13
PhHN
14a,b
ClCH2COOC2H5
17
RCOCH2Y
15a, R = CH3, Y= Cl
b, R = Ph, Y= Br
NH2
NH2
N
X
N
N
X
HO
NHCO
CN
S
N
S
HO
NH CO
NH2
S
Ph
N
PhHN
COR
S
HO
16
a
X
R
18a, X = CN
b, X = COOC2H5
CN
CH3
Ph
b
c
CN
COOC2H5
CH3
d
COOC2H5
Ph
Scheme 3 Synthesis of compounds 14a,b–18a,b
42.43; H, 4.81; N, 30.01. MS: m/e = 141 (M?, 10 %). IR,
1-(5-Amino-3-hydroxy-1H-pyrazol-1-yl)ethanone 4
1
t: 3577–3323 (OH, NH2), 1690 (C=O), 1653 (C=N). H-
A suspension of compound 3 (1.41 g, 0.01 mol) in sodium
ethoxide solution [prepared by dissolving metallic sodium
(0.64 g, 0.01 mol) in absolute ethanol (30 mL)] was boiled
in a boiling water bath for 8 h. The reaction mixture was
left to cool and then poured onto ice/water containing few
drops of hydrochloric acid. The formed solid product was
collected by filtration.
NMR, d: 2.77 (s, 3H, CH3), 4.79 (s, 2H, NH2), 6.86 (s, 1H,
pyrazole H-4), 10.22 (s, 1H, OH). 13C-NMR, d: 29.6
(CH3), 104.8, 152.3, 154.6 (pyrazole C), 168.9 (C=O).
1-(5-Amino-3-hydroxy-1H-pyrazol-1-yl)-
bromoethanone 5
To a solution of compound 4 (1.41 g, 0.01 mol) in acetic
acid (40 mL) at 50 °C, bromine (1.80 g, 0.01 mol) was
added dropwise. The reaction mixture was kept at room
temperature for 1 h with continuous stirring. The solid
product was formed upon pouring onto ice/water and the
formed solid product was collected by filtration.
Compound 4
Crystallized from ethanol to give white crystals, yield
1.27 g (90 %), m.p. 210–213 °C. Anal. Calculated for
C5H7N3O2 (141.13): C, 42.55; H, 5.00; N, 29.77. Found: C,
123

Med Chem Res
Compound 5
N-(4-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-
cyanothiophen-2-yl)-2-cyano-acetamide 9a and ethyl
2-(2-cyanoacetamido)-4-(5-amino-3-hydroxy-1H-
pyrazol-1-yl)thiophene-3-carboxylate 9b
Crystallized from acetic acid to give white crystals, yield
1.49 g (68 %), m.p. 160–163 °C. Anal. Calculated for
C5H6BrN3O2 (220.02): C, 27.29; H, 2.75; N, 19.10. Found:
C, 27.39; H, 2.88; N, 19.01. MS: m/e = 220 (M?, 23 %),
222 (M? ? 2, 12 %). IR, t: 3547–3363 (OH, NH2), 1688
General procedure
1
(C=O), 1650 (C=N). H-NMR, d: 4.07 (s, 2H, CH2), 4.81
A solution of either 8a (2.21 g, 0.01 mol) or 8b (2.68 g,
0.01 mol)indimethylformamide(30 mL),ethylcyanoacetate
(6b) (1.13 g, 0.01 mol) was added. The reaction mixture, in
each case, was heated under reflux for 3 h and then left to cool
onreachingroomtemperature. Thesolidproductformedupon
pouring onto ice/water was collected by filtration.
(s, 2H, NH2), 6.82 (s, 1H, pyrazole H-4), 10.02 (s, 1H,
OH). 13C-NMR, d: 38.2 (CH2), 104.3, 153.0, 154.8 (pyr-
azole C), 167.3 (C=O).
2-Amino-4-(5-amino-3-hydroxy-1H-pyrazol-1-
yl)thiophene-3-carbonitrile 8a and ethyl 2-amino-4-(5-
amino-3-hydroxy-1H-pyrazol-1-yl)thiophene-3-
carboxylate 8b
Compound 9a Crystallized from 1,4-dioxan to give orange
crystals, yield 2.27 g (79 %), m.p. 266–269 °C. Anal. Calcu-
lated for C11H8N6O2S (288.29): C, 45.83; H, 2.80; N, 29.15; S,
11.12. Found: C, 45.73; H, 3.18; N, 29.05; S, 11.33. MS: m/
e = 288 (M?, 68 %). IR, t: 3572–3366 (OH, NH, NH2), 2227,
2220 (2 CN), 1688 (C=O), 1636 (C=N). 1H-NMR, d: 4.77 (s,
2H, NH2), 4.81 (s, 2H, CH2), 6.83, 6.84 (2s, 2H, pyrazole H-4,
thiophene H-5), 8.21 (s, 1H, NH), 10.08 (s, 1H, OH). 13C-NMR,
d: 39.2 (CH2), 116.5, 117.9 (2 CN), 104.4, 152.9, 154.8 (pyra-
zole C), 118.0, 124.9, 134.5, 138.8 (thiophene C), 164.5 (C=O).
General procedure
To a solution of compound 4 (1.41 g, 0.01 mol) in 1,4-
dioxan containing triethylamine (0.50 mL) and elemental
sulfur (0.32 g, 0.01 mol), either malononitrile (6a) (0.66 g,
0.01 mol) or ethyl cyanoacetate (6b) (1.13 g, 0.01 mol)
was added. The reaction mixture in each case was heated
under reflux for 1 h, then poured onto ice/water containing
few drops of hydrochloric acid, and the formed solid
product, in each case, was collected by filtration.
Compound 9b Crystallized from 1,4-dioxan to give orange
crystals, yield 2.88 g (86 %), m.p. 230–233 °C. Anal. Cal-
culated for C13H13N5O4S (335.34): C, 46.56; H, 3.91; N,
20.88; S, 9.56. Found: C, 46.63; H, 4.02; N, 20.92; S, 9.63.
MS: m/e = 335 (M?, 46 %). IR, t: 3576–3337 (OH, NH,
Compound 8a Crystallized from acetic acid to give pale
yellow crystals, yield 1.49 g (68 %), m.p. 160–163 °C.
Anal. Calculated for C8H7N5OS (221.24): C, 43.43; H,
3.19; N, 31.66; S, 14.49. Found: C, 43.29; H, 3.28; N,
31.85; S, 14.57. MS: m/e = 221 (M?, 33 %). IR, t:
3529–3342 (OH, 2NH2), 2222 (CN), 1632 (C=N). 1H-
NMR, d: 4.55, 4.83 (2s, 4H, 2NH2), 6.86, 6.99 (2s, 2H,
pyrazole H-4, thiophene H-5), 10.12 (s, 1H, OH). 13C-
NMR, d: 116.8 (CN), 104.2, 153.3, 154.6 (pyrazole C),
117.8, 125.6, 133.2, 138.1 (Thiophene C).
1
NH2), 1693, 1684 (2C=O), 2220 (CN), 1648 (C=N). H-
NMR, d: 1.12 (t, 3H, J = 7.26 Hz, CH3), 4.24 (q, 2H,
J = 7.26 Hz, CH2), 4.73 (s, 2H, NH2), 4.88 (s, 2H, CH2),
6.79, 6.92 (2s, 2H, pyrazole H-4, thiophene H-5), 8.22 (s, 1H,
NH), 10.19 (s, 1H, OH). 13C-NMR, d: 16.3 (CH3), 39.7, 42.6
(2CH2), 104.3, 153.2, 154.6 (pyrazole C), 117.9 (CN), 118.4,
125.0, 133.46, 138.74 (thiophene C), 164.0, 166.3 (2C=O).
4-Amino-3-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-6-
hydroxythieno[2,3-b]-pyridine-5-carbonitrile 10a
and 3-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-4,6-
dihydroxythieno[2,3-b]pyridine-5-carbonitrile 10b
Compound 8b Crystallized from acetic acid to give yel-
low crystals, yield 2.09 g (78 %), m.p. 148–151 °C. Anal.
Calculated for C10H12N4O3S (268.29): C, 44.77; H, 4.51;
N, 20.88; S, 11.95. Found: C, 44.69; H, 4.58; N, 20.95; S,
11.83. MS: m/e = 268 (M?, 18 %). IR, t: 3544–3337 (OH,
2NH2), 1688 (C=O), 1644 (C=N). 1H-NMR, d: 1.14 (t, 3H,
J = 7.02 Hz, CH3), 4.22 (q, 2H, J = 7.02 Hz, CH2), 4.53,
4.86 (2s, 4H, 2NH2), 6.84, 6.92 (2s, 2H, pyrazole H-4,
thiophene H-5), 10.23 (s, 1H, OH). 13C-NMR, d: 16.2
(CH3), 42.3 (CH2), 104.0, 153.1, 154.4 (pyrazole C), 118.3,
125.2, 133.6, 138.4 (Thiophene C), 164.2 (C=O).
General procedure
A suspension of either 9a (2.88 g, 0.01 mol) or 9b (3.35 g,
0.01 mol) in sodium ethoxide solution [prepared by dis-
solving metallic sodium (0.64 g, 0.01 mol) in absolute
ethanol (30 mL)] was boiled in a boiling water bath for 8 h.
The reaction mixture was left to cool and then poured onto
ice/water containing few drops of hydrochloric acid. The
formed solid product was collected by filtration.
123

Med Chem Res
Compound 10a Crystallized from 1,4-dioxan to give
orange crystals, yield 2.47 g (86 %), m.p. 170–173 °C.
Anal. Calculated for C11H8N6O2S (288.29): C, 45.83; H,
2.80; N, 29.15; S, 11.12. Found: C, 45.62; H, 2.92; N, 29.32;
S, 11.33. MS: m/e = 288 (M?, 16 %). IR, t: 3576–3333
temperature for an additional 1 h and the formed solid
product was collected by filtration.
Compound 12a Crystallized from ethanol to give reddish
brown crystals, yield 3.21 g (82 %), m.p. 133–135 °C.
Anal. Calculated for C17H12N8O2S (392.39): C, 52.03; H,
3.08; N, 28.56; S, 8.17. Found: C, 51.93; H, 3.18; N, 28.45;
S, 8.23. MS: m/e = 392 (M?, 42 %). IR, t: 3537–3321
(OH, 2NH, NH2), 2225, 2220 (2 CN), 1685 (C=O), 1638
(C=N). 1H-NMR, d: 4.82 (s, 2H, NH2), 6.82, 6.88 (2s, 2H,
pyrazole H-4, thiophene H-5), 7.29–7.38 (m, 5H, C6H5),
8.20–825 (2s, 2H, 2NH), 10.18 (s, 1H, OH). 13C-NMR, d:
116.8, 117.3 (2 CN), 104.5, 153.2, 154.6 (pyrazole C),
118.2, 122.0, 123.4, 124.6, 130.8, 133.9, 138.9 (thiophene,
C6H5, C), 164.8 (C=O), 173.8 (C=N).
1
(2OH, 2NH2), 2222 (CN), 1640 (C=N). H-NMR, d: 4.79,
4.86 (2s, 4H, 2NH2), 6.75, 6.89 (2s, 2H, pyrazole H-4,
thiophene H-5), 10.16, 10.22 (2s, 2H, 2OH). 13C-NMR, d:
117.8 (CN), 104.5, 153.6, 154.9 (pyrazole C), 118.2, 124.7,
133.3, 138.8, 140.2, 143.8, 148.2 (thiophene, pyridine C).
Compound 10b Crystallized from 1,4-dioxan to give pale
brown crystals, yield 1.90 g (66 %), m.p. 180–183 °C.
Anal. Calculated for C11H7N5O3S (289.27): C, 45.67; H,
2.44; N, 24.21; S, 11.08. Found: C, 45.86; H, 2.66; N; 24.02;
S, 10.83. MS: m/e = 289 (M?, 100 %). IR, t: 3555–3320
(3OH, NH2), 2227 (CN), 1630 (C=N). 1H-NMR, d: 4.83 (s,
2H, NH2), 6.86, 6.94 (2s, 2H, pyrazole H-4, thiophene H-5),
10.09, 10.27, 10.29 (3s, 3H, 3OH). 13C-NMR, d: 117.8
(CN), 104.3, 152.6, 155.7 (pyrazole C), 118.8, 124.8, 134.0,
138.8, 140.7, 141.5, 149.6 (thiophene, pyridine C).
Compound 12b Crystallized from ethanol to give red
crystals, yield 3.57 g (88 %), m.p. 190–193 °C. Anal. Cal-
culated for C18H14N8O2S (406.42): C, 53.19; H, 3.47; N,
27.57; S, 7.89. Found: C, 52.92; H, 3.32; N, 27.62; S, 7.93.
MS: m/e = 406 (M?, 18 %). IR, t: 3555–3340 (OH, 2NH,
1
NH2), 2222, 2220 (2 CN), 1689 (C=O), 1643 (C=N). H-
NMR, d: 2.88 (s, 3H, CH3), 4.79 (s, 2H, NH2), 6.80, 6.84 (2s,
2H, pyrazole H-4, thiophene H-5), 7.28–7.36 (m, 4H, C6H4),
8.22, 8.25 (2s, 2H, 2NH), 10.23 (s, 1H, OH). 13C-NMR, d:
24.8 (CH3), 116.6, 117.8 (2 CN), 104.6, 154.6, 155.0 (pyr-
azole C), 118.8, 122.2, 123.6, 125.6, 128.9, 133.6, 138.7
(thiophene, C6H4, C), 164.6 (C=O), 173.4 (C=N).
N-(4-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-
cyanothiophen-2-yl)-a-phenyl-hydrazono-2-
cyanoacetamide 12a, N-(4-(5-amino-3-hydroxy-1H-
pyrazol-1-yl)-3-cyanothiophen-2-yl)-a-(p-
methylphenylhydrazono)-2-cyanoacetamide 12b, N-(4-
(5-amino-3-hydroxy-1H-pyrazol-1-yl)-3-
cyanothiophen-2-yl)-a-(p-chlorophenylhydrazono)-2-
cyanoacetamide 12c, ethyl 2-(a-phenylhydrazono-2-
cyanoacetamido)-4-(5-amino-3-hydroxy-1H-pyrazol-1-
yl)thiophene-3-carboxylate 12d, ethyl 2-a-(p-
methylphenylhydrazono)-2-cyanoacetamido)-4-(5-
amino-3-hydroxy-1H-pyrazol-1-yl)thiophene-3-
carboxylate 12e and ethyl 2-a-(p-
chlorophenylhydrazono)-2-cyanoacetamido)-4-(5-
amino-3-hydroxy-1H-pyrazol-1-yl)thiophene-3-
carboxylate 12f
Compound 12c Crystallized from ethanol to give Orange
crystals, yield 3.07 g (72 %), m.p. 144–147 °C. Anal.
Calculated for C17H11ClN8O2S (426.84): C, 47.84; H, 2.60;
N, 26.25; S, 7.51. Found: C, 47.97; H, 2.52; N, 26.32; S,
7.63. MS: m/e = 426 (M?, 8 %). IR, t: 3571–3320 (OH,
2NH, NH2), 2226, 2220 (2 CN), 1687 (C=O), 1642 (C=N).
1H-NMR, d: 4.80 (s, 2H, NH2), 6.78, 6.82 (2s, 2H, pyrazole
H-4, thiophene H-5), 7.26–7.39 (m, 4H, C6H4), 8.24, 8.26
(2s, 2H, 2NH), 10.33 (s, 1H, OH). 13C-NMR, d: 116.8,
117.9 (2 CN), 104.2, 154.4, 155.2 (pyrazole C), 118.3,
122.0, 123.9, 125.1, 129.2, 132.8, 139.5 (thiophene, C6H4,
C), 163.8 (C=O), 172.8 (C=N).
General procedure
To a cold solution (0–5 °C) of either 9a (2.88 g, 0.01 mol)
or 9b (3.35 g, 0.01 mol) in ethanol (40 mL) containing
sodium hydroxide (5 mL, 10 %),benzenediazonium chlo-
ride (11a), p-methylbenzene-diazonium chloride (11b), or
p-chlorobenzenediazonium chloride (11c) [prepared by
adding sodium nitrite (0.70 g, 0.01 mol) solution to a cold
solution (0–5 °C) of aniline (0.94 g, 0.01 mol), p-methy-
laniline (1.04 g, 0.01 mol), or p-chloroaniline (1.24 g,
0.01 mol) in the appropriate quantity of concentrated
hydrochloric acid with continuous stirring] was added. The
whole reaction mixture, in each case, was stirred at room
Compound 12d Crystallized from 1,4-dioxan to give red
crystals, yield 2.89 g (66 %), m.p. 196–199 °C. Anal. Cal-
culated for C19H17N7O4S (439.45): C, 51.93; H, 3.90; N,
22.31; S, 7.30. Found: C, 52.23; H, 4.11; N, 22.42; S, 7.60.
MS: m/e = 439 (M?, 12 %). IR, t: 3571–3332 (OH, 2NH,
1
NH2), 1689, 1682 (2C=O), 2223 (CN), 1638 (C=N). H-
NMR, d: 1.11 (t, 3H, J = 6.53 Hz, CH3), 4.23 (q, 2H,
J = 6.53 Hz, CH2), 4.80 (s, 2H, NH2), 6.74, 6.98 (2s, 2H,
pyrazole H-4, thiophene H-5), 7.28–7.41 (m, 5H, C6H5),
8.20, 8.31 (2s, 2H, 2NH), 10.21 (s, 1H, OH). 13C-NMR, d:
16.1 (CH3), 42.5 (CH2), 104.6, 153.8, 154.0 (pyrazole C),
123

Med Chem Res
116.8 (CN), 118.2, 122.3, 123.9, 125.0, 133.46, 134.8, 138.7
(thiophene, C6H5, C), 164.0, 166.6 (2C=O), 172.5 (C=N).
potassium hydroxide (0.56 g, 0.01 mol), phenylisothiocy-
anae (13) (1.30 g, 0.01 mol) was added. The whole reac-
tion mixture was stirred at room temperature overnight.
The next day, chloroacetone (15a) (0.92 g, 0.01 mol),
phenacylbromide (15b) (2.0 g, 0.01 mol), or ethyl chloro-
acetate (17) (1.22 g, 0.01 mol) was added with continuous
stirring overnight at room temperature and then poured
onto ice/water containing few drops of hydrochloric acid
(till pH 6). The solid product, formed in each case, was
collected by filtration.
Compound 12e Crystallized from 1,4-dioxan to give brown
crystals, yield 3.17 g (70 %), m.p. 120–122 °C. Anal. Calcu-
lated for C20H19N7O4S (453.47): C, 52.97; H, 4.22; N, 21.62; S,
7.07. Found: C, 52.73; H, 4.30; N, 21.82; S, 7.14. MS: m/
e = 453 (M?, 33 %). IR, t: 3562–3330 (OH, 2NH, NH2),
1688, 1684 (2C=O), 2223 (CN), 1634 (C=N). 1H-NMR, d: 1.13
(t, 3H, J = 6.88 Hz, CH3), 2.89 (s, 3H, CH3), 4.22 (q, 2H,
J = 6.88 Hz, CH2), 4.76 (s, 2H, NH2), 6.76, 6.98 (2s, 2H,
pyrazole H-4, thiophene H-5), 7.28–7.37 (m, 4H, C6H4), 8.19,
8.29 (2s, 2H, 2NH), 10.25 (s, 1H, OH). 13C-NMR, d: 16.4
(CH3), 27.5 (CH3), 42.6 (CH2), 104.2, 152.9, 153.8 (pyrazole
C), 116.9(CN), 118.4, 122.7, 123.6, 125.0, 133.46, 134.3, 137.5
(thiophene, C6H4, C), 163.6, 166.8 (2C=O), 172.2 (C=N).
Compound 16a Crystallized from ethanol to give yellow
crystals, yield 3.64 g (76 %), m.p. 120–122 °C. Anal. Calcu-
lated for C21H17N7O3S2 (479.53): C, 52.60; H, 3.57; N, 20.45;
S, 13.37. Found: C, 52.43; H, 3.41; N, 20.49; S, 13.26. MS: m/
e = 479 (M?, 15 %). IR, t: 3555–3312 (OH, 2NH, 2NH2),
1
2223, 2227 (2CN), 1683 (C=O), 1634 (C=N). H-NMR, d:
Compound 12f Crystallized from 1,4-dioxan to give
brown crystals, yield 3.17 g (70 %), m.p. 120–122 °C. Anal.
Calculated for C19H16ClN7O4S (473.89): C, 48.16; H, 3.40;
N, 20.69; S, 6.77. Found: C, 48.01; H, 3.52; N, 20.88; S, 6.93.
MS: m/e = 473 (M?, 20 %). IR, t: 3551–3322 (OH, 2NH,
2.88 (s, 3H, CH3), 4.77, 4.82(2s, 4H, 2NH2),6.80,6.85(2s, 2H,
pyrazole H-4, thiophene H-5), 7.29–7.38 (m, 5H, C6H5), 8.26,
8.80 (2s, 2H, 2NH), 10.20 (s, 1H, OH). 13C-NMR, d: 31.2
(CH3), 116.9 (CN), 104.3, 153.7, 154.3 (pyrazole C), 119.0,
120.4, 120.9, 121.8, 124.8, 130.9, 133.6, 134.0, 139.4, 141.5,
143.8, 144.6 (two thiophene, C6H5, C), 163.9, 164.8 (2C=O).
1
NH2), 1688, 1684 (2C=O), 2223 (CN), 1634 (C=N). H-
NMR, d: 1.11 (t, 3H, J = 6.88 Hz, CH3), 4.26 (q, 2H,
J = 6.88 Hz, CH2), 4.88 (s, 2H, NH2), 6.72, 6.89 (2s, 2H,
pyrazole H-4, thiophene H-5), 7.24–7.36 (m, 4H, C6H4),
8.20, 8.30 (2s, 2H, 2NH), 10.25 (s, 1H, OH). 13C-NMR, d:
27.8 (CH3), 42.8 (CH2), 104.5, 152.8, 153.9 (pyrazole C),
116.9 (CN), 118.6, 122.2, 123.8, 124.8, 133.8, 134.6, 137.7
(thiophene, C6H4, C), 163.4, 166.9 (2C=O), 172.0 (C=N).
Compound 16b Crystallized from ethanol to give pale
yellow crystals, yield 3.35 g (62 %), m.p. 190–193 °C.
Anal. Calculated for C26H19N7O3S2 (541.60): C, 57.66; H,
3.54; N, 18.10; S, 11.84. Found: C, 57.48; H, 3.42; N,
18.29; S, 11.66. MS: m/e = 541 (M?, 20 %). IR, t:
3512–3357 (OH, 2NH, 2NH2), 2220, (CN), 1685 (C=O),
1
1636 (C=N). H-NMR, d: 4.78, 4.84 (2s, 4H, 2NH2), 6.83,
5-Acetyl-4-amino-N-(4-(5-amino-3-hydroxy-1H-
pyrazol-1-yl)-3-cyanothiophen -2-yl)-2-
6.89 (2 s, 2H, pyrazole H-4, thiophene H-5), 7.26–7.39 (m,
10H, 2C6H5), 8.25, 8.86 (2s, 2H, 2NH), 10.24 (s, 1H, OH).
13C-NMR, d: 117.2 (CN), 104.6, 153.8, 154.2 (pyrazole C),
118.8, 120.2, 120.9, 121.8, 124.8, 126.0, 128.5, 129.9,
133.6, 134.2, 139.4, 142.1, 143.8, 144.8 (two thiophene,
2C6H5, C), 163.2, 166.2 (2C=O).
(phenylamino)thiophene-3-carboxamide 16a, 4-amino-
5-benzoyl-N-(4-(5-amino-3-hydroxy-1H-pyrazol-1-yl)-
3-cyanothiophen-2-yl)-2-(phenylamino)thiophene-3-
carboxamide 16b, 5-acetyl-4-amino-N-(4-(5-amino-3-
hydroxy-1H-pyrazol-1-yl)-3-ethoxycarbonyl-thiophen-
2-yl)-2-(phenylamino)thiophene-3-carboxamide 16c,
4-amino-5-benzoyl-N-(4-(5-amino-3-hydroxy-1H-
pyrazol-1-yl)-3-ethoxycarbonylthiophen-2-yl)-2-
(phenylamino)thiophene-3-carboxamide 16d, N-(4-(5-
amino-3-hydroxy-1H-pyrazol-1-yl)-3-cyanothiophen-2-
yl)-2-cyano-2-(4-hydroxy-3-phenylthiazol-2(3H)-
ylidene)acetamide 18a and ethyl 2-(2-cyano-2-(4-
hydroxy-3-phenylthiazol-2-(3H)-ylidene)acetamido)-4-
(5-amino-3-hydroxy-1H-pyrazol-1-yl)thiophene-3-
carboxylate 18b
Compound 16c Crystallized from ethanol to give pale
brown crystals, yield 4.67 g (89 %), m.p. 190–193 °C.
Anal. Calculated for C23H22N6O5S2 (526.59): C, 52.46; H,
4.21; N, 15.96; S, 12.18. Found: C, 52.59; H, 4.50; N,
16.22; S, 12.28. MS: m/e = 526 (M?, 22 %). IR, t:
3524–3318 (OH, 2NH, 2NH2), 1693, 1683,1680 (3C=O),
1
1636 (C=N). H-NMR, d: 1.14 (t, 3H, J = 6.89 Hz, CH3),
2.69 (s, 3H, CH3), 4.22 (q, 2H, J = 6.89 Hz, CH2), 4.79,
4.86 (2s, 4H, 2NH2), 6.80, 6.85 (2s, 2H, pyrazole H-4,
thiophene H-5), 7.29–7.38 (m, 5H, C6H5), 8.26, 8.80 (2s,
2H, 2NH), 10.24 (s, 1H, OH). 13C-NMR, d: 16.8, 26.8
(2CH3), 42.8 (CH2), 104.5, 152.9, 154.8 (pyrazole C),
118.3, 120.4, 121.6, 121.8, 124.8, 130.9, 134.8, 133.6,
139.4, 141.5, 143.8, 144.6 (two thiophene, C6H5, C), 164.1,
165.4, 168.0 (3C=O).
General procedure
To a solution of either 9a (2.88 g, 0.01 mol) or 9b (3.35 g,
0.01 mol) in dimethylformamide (30 mL) containing
123

Med Chem Res
Compound 16d Crystallized from ethanol to give yel-
low crystals, yield 2.94 g (56 %), m.p. 110–113 °C.
Anal. Calculated for C28H24N6O5S2 (588.66): C, 57.13;
H, 4.11; N, 14.28; S, 10.89. Found: C, 57.42; H, 4.38;
N, 14.30; S, 10.93. MS: m/e = 588 (M?, 52 %). IR, t:
3583–3338 (OH, 2NH, 2NH2), 1688, 1685, 1680
(3C=O), 1636 (C=N). 1H-NMR, d: 1.12 (t, 3H,
J = 6.29 Hz, CH3), 4.24 (q, 2H, J = 6.29 Hz, CH2),
4.73, 4.88 (2s, 4H, 2NH2), 6.82, 6.84 (2s, 2H, pyrazole
H-3, thiophene H-5), 7.26–7.41 (m, 10H, 2C6H5), 8.28,
8.82 (2s, 2H, 2NH), 10.26 (s, 1H, OH). 13C-NMR, d:
16.6 (CH3), 42.3 (CH2), 117.0 (CN), 104.6, 152.6, 154.3
(pyrazole C), 118.0, 120.6, 121.8, 124.8, 126.0, 126.9,
127.4, 131.6, 133.6, 134.5, 139.9, 141.8, 142.0, 143.8,
Docking of compound 12c:
12c
energy = -22.5
Tyr
Gly
B15
B147
Asn
B132
Leu
B134
Tyr
Asn
A132
Leu
A134
A15
Leu
B148
Gly
Asp
A147
Asp
B145
A145
S
H
H
N
Leu
A148
N
H
O
N
N
N
N
H
Cl
O
NH
N
H
N
Lys
A129
Gln
A131
Asp
Lys
Asp
B86
B129
A86
Gln
B131
Fig. 5 Docking of compound 12c
123

Med Chem Res
144.8 (two thiophene, two C6H5, C), 163.7, 165.4, 166.2
(3C=O).
Anal. Calculated for C20H13N7O3S2 (463.49): C, 51.83; H,
2.83; N, 21.15; S, 13.84. Found: C, 52.03; H, 2.61; N,
20.89; S, 13.76. MS: m/e = 463 (M?, 35 %). IR, t:
3571–3349 (2OH, NH, NH2), 2226, 2221 (2CN), 1687
Compound 18a Crystallized from 1,4-dioxan to give
yellow crystals, yield 3.38 g (73 %), m.p. 266–269 °C.
1
(C=O), 1632 (C=N). H-NMR, d: 4.79 (s, 2H, NH2), 6.78,
Fig. 6 Docking of compound
12f
Docking of compound 12f:
12f
energy = -26.5
Asn
132
Gln
131
Asp
145
Val
18
Ile
10
N
Ala
144
O
H
S
H
N
N
NH
N
H
HN
N
N
O
O
H
O
Cl
Leu
83
Leu
134
Phe
80
His
84
123

Med Chem Res
6.83 6.88 (3s, 3H, pyrazole H-4, thiophene H-5, thiazole
H-5), 7.31–7.42 (m, 5H, C6H5), 8.29 (s, 1H, NH), 10.26,
10.29 (2s, 2H, 2OH). 13C-NMR, d: 116.6, 117.3 (2CN),
119.0, 120.3 (C=C), 104.6, 153.8, 154.8 (pyrazole C),
118.0, 121.3, 121.7, 121.8, 126.8, 130.0, 130.9, 139.4,
142.8, 143.8, 148.0, 150.0, 151.6 (thiophene, thiazole,
C6H5, C), 164.0 (C=O).
Compound 18b Crystallized from ethanol to give orange
crystals, yield 3.06 g (60 %), m.p. 188–190 °C. Anal.
Docking of compound 16a
16a
energy = -30.2
Lys
33
Asp
145
Thr
47
Tyr
15
N
H
O
H
N
N
S
H
N
N
Asp
86
S
NH2
O
O
NH2
Asn
132
Gln
131
Fig. 7 Docking of compound 16a
123

Med Chem Res
Calculated for C22H18N6O5S2 (510.55): C, 51.76; H,
3.55; N, 16.46; S, 12.56. Found: C, 51.61; H, 3.40; N,
16.86; S, 12.72. MS: m/e = 510 (M?, 30 %). IR, t:
3563–3388 (2OH, NH, NH2), 2222 (CN), 1690, 1688
(2C=O), 1634 (C=N). 1H-NMR, d: 1.13 (t, 3H,
J = 7.07 Hz, CH3), 4.26 (q, 2H, J = 7.07 Hz, CH2),
4.76 (s, 2H, NH2), 6.83, 6.86, 7.01 (3s, 3H, pyrazole
H-4, thiophene H-5, thiazole H-5), 7.29–7.40 (m, 5H,
C6H5), 8.35 (s, 1H, NH), 10.24, 10.28 (2 s, 2H, 2OH).
13C-NMR, d: 16.6 (CH3), 42.6 (CH2), 117.2 (CN), 104.6,
152.2, 154.8 (pyrazole C), 118.3, 120.4, 121.4, 122.5,
124.8, 126.6, 127.4, 131.6, 133.6, 139.9, 142.0, 143.8,
144.8, 148.9, 152.0, 153.3 (thiophene, thiazole, C6H5, C),
162.3, 163.8 (2C=O).
Docking of compound 16d:
16d
energy = -35.5
Asn
132
Gln
131
Glu
51
O
O
O
H
NH
N
S
H
N
O
N
Asp
145
S
Val
64
NH2
O
NH2
Lys
89
Phe
80
Ile
10
Val
18
His
84
Lys
33
Gln
85
Asp
86
Ala
144
Ala
31
Leu
134
Phe
82
Glu
8
Leu
83
Fig. 8 Docking of compound 16d
123

Med Chem Res
Preparation for docking
rings. The anti-tumor evaluations of the newly synthesized
products showed that compounds 12c, 12f, 16a, and 16d
were the most active compounds toward the three cancer
cell lines.
Docking was carried out on an Intel Pentium 1.6 GHz pro-
cessor, 512 MB memory, with windows XP operating sys-
tem and Molecular Operating Environment (MOE 2008.10;
Chemical Computing Group, Canada) as the computational
software. All the minimizations were performed with MOE
until a root mean square deviation (RMSD) gradient of
Acknowledgments R. M. Mohareb would like to express his
deepest thank to the Alexander von Humboldt Foundation in Bonn for
affording his a fellowship in Germany, Mu¨nchen during summer,
2012 for doing research and completing this work.
˚
0.05 kcal/mol/A with MMFF94x force field was reached and
the partial charges were automatically calculated. The 3D
structure of the Protein Cyclin-Dependent Kinase2 (CDK2)
complexes with (Thiophene Carboxamide) was obtained
from the Protein Data Bank (PDB ID: 1EVE) at Research
Collaboration for Structural Bioinformatics (RCSB), protein
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In this work, we succeeded in synthesizing a series of
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