Synthesis and evaluation of novel 4H-pyrazole and thiophene derivatives derived from chalcone as potential anti-proliferative agents, Pim-1 kinase inhibitors, and PAINS

Article abstract of DOI:10.1002/jhet.3928

The chalcone derivatives 3a-d reacted with either malononitrile or ethyl cyanoacetate in ethanol in the presence of catalytic amount of ammonium acetate in an oil bath at 120°C to give the Knowevenagel condensation products 5a-h. The latter compounds reac

Full text of DOI:10.1002/jhet.3928

Received: 6 December 2019
DOI: 10.1002/jhet.3928
Revised: 12 January 2020
Accepted: 28 January 2020
A R T I C L E
Synthesis and evaluation of novel 4H-pyrazole
and thiophene derivatives derived from chalcone
as potential anti-proliferative agents, Pim-1 kinase
inhibitors, and PAINS
Nadia Y. Megally Abdo1
| Eman M. Samir2 | Rafat M. Mohareb3
1Chemistry Department, Faculty of
Education, Alexandria University,
Alexandria, A. R. Egypt
Abstract
The chalcone derivatives 3a-d reacted with either malononitrile or ethyl
cyanoacetate in ethanol in the presence of catalytic amount of ammonium ace-
tate in an oil bath at 120ꢀC to give the Knowevenagel condensation products
5a-h. The latter compounds reacted with hydrazine hydrate and afforded the
4H-pyrazole derivatives 7a-h, respectively. The reaction of compounds 7a-h
with ethyl cyanoacetate in dimethylformamide under refluxing condition
afforded the cyanoacetamido derivatives 8a-h, respectively. When compounds
8a-h reacted with elemental sulfur and either of malononitrile or ethyl
cyanoacetate in ethanol containing triethylamine, the thiophene derivatives
9a-h and 10a-h, respectively, were obtained. The structure of the newly syn-
thesized compounds was established by the analytical and spectral data. All
the newly synthesized compounds were evaluated against the six cancer cell
lines: A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460. Com-
pounds 3c, 5h, 7g, 7h, 8f, 9e, 9g, and 10g were selected to examine their Pim-1
kinase inhibition activity as these compounds showed high inhibition toward
the c-Met kinase and the tested cancer cell lines. Furthermore, compounds 3b,
3c, 5g, 5h, 7f, 7g, 7h, 8e, 8f, 8g, 8h, 9e, 9f, 9g, 9h, 10g and 10h were selected
to be tested for pan-assay interference compounds analysis (PAINS). Almost
all the tested compounds showed zero PAINS alert and can be used as drug
compounds in the future.
2Department of Organic
Chemistry, National Organization for
Drug Control & Research (NODCAR),
P.O. 29, Cairo, A. R. Egypt
3Department of Chemistry, Faculty of
Science, Cairo University, Cairo,
A. R. Egypt
Correspondence
Nadia Y. Megally Abdo, Chemistry
Department, Faculty of Education,
Alexandria University, Alexandria 21526,
Egypt.
Email: nadiamegally@yahoo.com;
nadiamegally@gmail.com
1 | INTRODUCTION
synthetically by condensing aryl ketones with aromatic
aldehydes in the presence of a catalyst or condensing
Because of the impressive anti-infective activities[1] of
chalcones, they are considered as important classes of
compounds. Benzylidene acetophenone scaffold is the
1,3-diphenyl-2E-propene-1-one (chalcones), which is con-
sidered as one of the most important classes of natural
products across the plant kingdom. It contains two aromatic
nuclei that are joined by a three-carbon α,β-unsaturated
carbonyl bridge. Generally, chalcones can be obtained
agents.[2] Chalcone compounds were used in many hetero-
cyclic reactions to produce a variety of biological active target
molecules.[3–5] The produced molecules showed analgesic,[6]
arthritis,[7] anti-inflammatory,[8] antimalarial,[9,10]
antipyretic,[11] antibacterial,[12] antiviral,[13,14] and
anticancer[15–17] effects. Many reported works also
showed that chalcones have anti-proliferative and cyto-
toxic effects.[18–20] There are many studies through
J Heterocyclic Chem. 2020;1–17.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
MEGALLY ABDO ET AL.
which chalcones are characterized by high cytotoxicities
and inhibitions toward different cancer cell lines.[21–23]
Originally, chalcones were first isolated from flavonoid
biosynthesis in plants[24]; afterwards, they were struc-
turally modified through the synthesis of different
polyfunctionally substituted compounds.[25] Basically,
it has been shown that the removal of α,β-unsaturated
carbonyl system could decrease their biological activi-
ties.[26] A number of synthetic modifications, such as fused
heterocyclic compounds,[27] biphenyl-based[28] coumarin-
based chalcones,[29] or other substitutions,[30–34] have also
been reported to affect the biological activities including
anticancer activities of chalcones.[35–37]
In the present work, because of the high pharmaceu-
tical activities of chalcones[38] as described before, we
tried to make different heterocyclization reactions of
some chalcones, which leads to the preparation of
pyrazole and thiophene rings together with their anti-
tumor evaluations.
presence of ammonium acetate in an oil bath at 120ꢀC
gave the Knowevenagel condensation products 5a-h
(Scheme 1). The structures of compounds 5a-h were
confirmed on the basis of their respective analyses.
1
Thus, the H NMR spectrum of compound 5a showed
the presence of two doublets at δ = 6.38, 6.41 ppm
corresponding to the two vinyl protons beside multi-
plet at δ = 7.26 to 7.39 ppm for the two C6H5 protons.
Moreover, the 13C NMR spectrum revealed the pres-
ence of signals at 89.3, 90.5 (CH═CH), 116.5, 116.9
(2CN), and 131.2, 134.2 (C═C) beside the signals
corresponding to the two phenyl carbons. Further con-
firmation for these structures were obtained through
the studying of their reactivities toward some chemical
reagents.
Thus, the reaction of any of compounds 5a-h with
hydrazine hydrate gave the pyrazole derivatives 7a-h,
respectively. The analytical and spectral data of the latter
products were consistent with their respective structures
(see Section 4). The 2-amino group of the pyrazole deriva-
tives 7a-h showed interesting reactivity toward amide for-
mation. Thus, the reaction of any of compounds 7a-h with
one fold of ethyl cyanoacetate in dimethylformamide
under refluxing condition gave the monocyanoacetamido
derivatives 8a-h, respectively (Scheme 2). However, the
use of two folds of ethyl cyanoacetate in case of 7a, 7b, 7c,
and 7d gave unidentified mixture of products, and our tri-
als to separate the bis product failed. Our explanation for
such findings is that for compounds 7a, 7b, 7c, and 7d, it
seemed that there is a tautomeric structure through which
the second NH2 group exist in the imino form instead of
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
In this work, we demonstrated a series of reactions
starting from different charlcones that underwent a series
of heterocyclization reaction to produce biologically
active target molecules. Thus, the reaction of either of
acetophenone (1a) or 4-chloroacetophenone (1b) with
either of benzaldehyde (2a) or 4-methoxybenzaldehyde
(2b) in ethanol containing sodium hydroxide gave the
chalcone derivatives 3a-d.[39–41] It has been reported the
reaction of chalcone derivatives with either malononitrile
or ethyl cyanoacetate in varieties of reaction conditions
to produce each time specific product. Thus, carrying the
reaction in toluene containing Al(OiPr)3, the product
mainly according to Michael addition[42] followed by
cyclization to pyridine in ethanolic sodium ethoxide
solution. In other report, carrying the reaction in MW
and in the presence of morphiline the product is the
dicyanobenzene derivative.[43] Moreover, carrying the
reaction in ethanol containing piperidine gave pyran
derivative.[44] Our result for this reaction is clearly in
conflict with previous results as the result of using dif-
ferent reaction conditions. These significant discrepan-
cies and our extensive work on the synthesis of the
Knoevenagel condensation products prompted us to
carefully reinvestigate the product of this reaction
using ammonium acetate. Thus, the reaction of any of
the chalcone derivatives 3a, 3b, 3c, or 3d with either of
malononitrile (4a) or ethyl cyanoacetate (4b) in the
1
pure NH2 group. The H NMR spectrum of compound 8a
(as an example) revealed the presence of two singlets at
δ = 3.80 and 8.26 ppm corresponding to the CH2 and NH
(D2O exchangeable) groups of the cyanoacetamido moiety.
In addition to the presence of a singlet at δ = 4.56 ppm for
the two protons of the NH2 (D2O exchangeable), two dou-
blets at δ = 6.34, 6.43 ppm indicating the presence of the
CH═CH group and a multiplet at δ = 7.25 to 7.39 ppm for
the two C6H5 protons. Moreover, the 13C NMR spectrum
showed the presence of signals at 27.2 (CH2), 89.3, 90.5
(CH═CH), 117.1 (CN), 131.5, 134.4 (C═C), 168.1 (CO),
and 172.4, 176.2 (2C═N) beside signals corresponding to
the two phenyl carbons.
The cyanomethyl moiety present in compounds 8a-h
showed interesting reactivity toward the Gewald's thio-
phene synthesis.[45–47] Thus, the reaction of any of com-
pounds 8a-h with elemental sulfur and malononitrile
(4a) in ethanol containing triethylamine gave the thio-
phene derivatives 9a-h. The analytical and spectral data
of 9a-h were in agreement with their respective structures.
It is important to note that in these reactions, we used the

MEGALLY ABDO ET AL.
3
SCHEME 1 Synthesis of
compounds 3a-d and 5a-h
ideal conditions of Gewald's thiophene reaction because of
reacting two molecules of different cyanomethylene
compounds with elemental sulfur in the presence of
triethylamine as a catalyst and the possibility of Micheal
addition reaction occurred through intermediate steps.
Thus, the 1H NMR spectrum of 9a (as an example)
showed the presence of three singlets at δ = 4.46, 4.56,
5.21 ppm corresponding to the presence of the three
NH2 groups beside another singlet at δ = 8.29 ppm (D2O
exchangeable) for the NH proton. In addition to that the
presence of two doublets at δ = 6.32, 6.48 ppm
corresponding to the two vinyl protons and multiplet at
δ = 7.22 to 7.36 ppm for the two C6H5 protons. More-
over, the 13C NMR spectrum revealed the presence of
signals at 89.1, 90.8 (CH═CH), 117.0 (CN), 131.1, 134.7
(C═C), 167.4 (CO), 172.1, 176.6 (2C═N) beside
twelve signals corresponding to the two phenyl and
thiophene carbons. Similarly, the reaction of any of
compounds 8a-h with elemental sulfur and ethyl
cyanoacetate (4b) in ethanol containing triethylamine
gave the thiophene derivatives 10a-h (Scheme 3).
2.2 | Biology
2.2.1 | Cell proliferation assay
The newly synthesized compounds were screened for
anti-proliferative activities (Table 1) against the six can-
cer cell lines A549, HT-29, MKN-45, U87MG, and
SMMC-7721 and H460 by applying the standard MTT
assay in vitro, using foretinib as the positive control.[48–50]
The cancer cell lines were cultivated in a minimum
essential medium (MEM) supplemented with 10% fetal
bovine serum (FBS). In the MEM medium, 4 × 103 cells
were plated onto each well of a 96-well plate and kept in
5% CO2 at 37ꢀC for 24 hours. At the specified final con-
centrations, the compounds tested were added to the cul-
ture medium, and the cell cultures were sustained for
72 hours. To each well fresh, MTT was added at a termi-
nal concentration of 5 μg/mL and kept in dark with the
cells at 37ꢀC for 4 hours. An enzyme-linked immunosor-
bent assay (ELISA) reader was used to measure the
absorbance (the absorbance of MTT formazan at 492 nM

4
MEGALLY ABDO ET AL.
SCHEME 2 Synthesis of
compounds 7a-h and 8a-h
and for the reference wavelength at 630 nM) after dis-
solving the formazan crystals in 100 μL of DMSO
each well.
compound 3c (X═H, Y═OCH3) was of the highest cyto-
toxicity. For the reaction of compounds 3a-d with either
malononitrile or ethyl cyanoacetate to give compounds
5a-h, it is obvious that compounds 5f (R═COOEt, X═Cl,
Y═OCH3), 5g (R═COOEt, X═H, Y═OCH3), and 5h
(R═COOEt, X═Cl, Y═H) were the most inhibit com-
pounds. Similarly, for the pyrazole derivatives 7a-h, it is
clear that compounds 7f (R0═OH, X═Cl, Y═OCH3), 7g
(R0═OH, X═H, Y═OCH3), and 7h (R0═OH, X═Cl, Y═H)
were the most cytotoxic compounds. On the other hand,
the N-cyanoacetamido derivatives 8a-h, it is of great
value to note that compounds 8e (R0═OH, X═Y═H), 8f
(R0═OH, X═Cl, Y═OCH3), 8g (R0═OH, X═H, Y═OCH3),
and 8h (R0═OH, X═Cl, Y═H) were of the highest inhibi-
tions among the eight compounds. Moreover, for the
thiophene derivatives 9a-h, compounds 9d-h were the
most active compounds. The cytotoxicity of such com-
pounds was attributed to the presence of the pyrazole
ring together with the thiophene moieties. However, the
thiophene derivatives 10a-h, compounds 10e, 10g, and
10h were of the highest cytotoxicity. It is obvious that the
presence of the OH group through the three compounds
The mean values of three independent experiments,
expressed as IC50 values (inhibitory concentration 50%),
were calculated by using the Bacus Laboratories Incorpo-
rated Slide Scanner (Bliss) software and presented in
Table 1. Most of the synthesized compounds exhibited
potent anti-proliferative activity with IC50 values less
than 12.00 μM. Generally, the variations of substituents
within the pyrazole and thiophene moieties have a nota-
ble influence on the anti-proliferative activity.
2.2.2 | Structure activity relationship
It is clear from Table 1 that compounds 3b, 3c, 3d, 5f, 5g,
5h, 7f, 7g, 7h, 8e, 8f, 8g, 8h, 9d-9h, 10e, 10g, and 10h
were the most cytotoxic compounds against the six can-
cer cell lines. Considering the chalcones 3a-d, it is clear
that compounds 3b, 3c, and 3d were the most active com-
pounds through this series of compounds, in addition

MEGALLY ABDO ET AL.
5
SCHEME 3 Synthesis of
compounds 9a-h and 10a-h
together with the thiophene and pyrazole moieties was
the reason for such activities.
kinase proteins diluted in 39 μL was added to the kinase
reaction, which was initiated by the kinase buffer
solution.
In summary, compounds 3b, 3c, 3d, 5g, 5h, 7f, 7g,
7h, 8e, 8f, 8g, 8h, 9e, 9f, 9g, 9h, 10g, and 10h were of the
higher cytotoxicity than the reference foretinib against
U87MG cell line. On the other hand, compounds 3c, 8g,
and 9h with IC50's 0.23 μM, 0.26 μM, and 0.32 μM were
higher cytotoxic than the reference foretinib against
SMMC-7721 cell line.
Compounds 3b, 3c, 5g, 5h, 7f, 7g, 7h, 8e, 8f, 8g, 9e,
9f, 9g, 9h, 10g, and 10h were selected for inhibition of
the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR,
and PDGFR. It clear from Table 2 that compounds 3c,
5h, 7g, 7h, 8f, 9e, 9g, and 10g were the most potent of
the tested compounds toward the five tyrosine kinases.
Compound 5g showed high potency toward the two
kinases Flt-3 and PDGFR with IC50's 0.62 nM and
0.73 nM, respectively. In addition, compound 7f showed
activity toward the three kinases VEGFR-2, EGFR, and
PDGFR with IC50's 0.72 nM, 0.59 nM, and 0.31 nM,
respectively. Compounds 3b and 10h showed the lowest
potency among the tested compounds.
2.2.3 | Inhibition of the most active
compounds against tyrosine kinases
(Enzyme IC50 [nM])
The most active compounds 3b, 3c, 5g, 5h, 7f, 7g, 7h, 8e,
8f, 8g, 8h, 9e, 9f, 9g, 9h, 10g, and 10h were further evalu-
ated against the five tyrosine kinase (c-Kit, Flt-3, VEGFR-
2, EGFR, and PDGFR) using the screening method
(Table 2). In summary, 20 μg/mL poly(Glu, Tyr) 4:1
(Sigma) was precoated as a substrate in 384-well plates.
To each well, 50 μL of 10 M ATP (Invitrogen) solution
diluted in kinase reaction buffer (50μM HEPES, Ph 7.0,
1M DTT, 1M MgCl2, 1M MnCl2, and 0.1% NaN3) was
added. The concentrations of compounds diluted in
10 μL of 1% DMSO (v/v) were used. Purified tyrosine
2.2.4 | Inhibition of selected compounds
toward Pim-1 kinase
Furthermore, compounds 3c, 5h, 7g, 7h, 8f, 9e, 9g, and
10g were selected to examine their Pim-1 kinase inhibi-
tion activity (Table 3) as these compounds showed high
inhibition toward the c-Met kinase and the tested cancer
cell lines at a range of 10 concentrations, and the IC50

6
MEGALLY ABDO ET AL.
TABLE 1 In vitro growth inhibitory effects IC50 (μM) of the selected compounds against cancer cell lines
IC50 SEM, μM
Compound No
A549
H460
HT29
MKN-45
U87MG
SMMC-7721
3.57 1.96
1.36 0.86
0.23 0.08
1.24 0.89
8.27 3.17
5.41 1.28
2.86 1.06
5.43 2.42
5.42 2.27
0.48 0.21
0.58 0.21
0.86 0.29
6.80 2.49
6.14 2.35
5.49 2.24
3.18 0.49
2.76 0.92
0.69 0.13
0.69 0.28
0.64 0.23
6.59 1.28
5.74 1.93
5.24 2.69
3.25 1.68
0.69 0.23
1.20 0.53
0.26 0.19
0.57 0.29
8.39 2.52
6.80 2.71
7.63 2.17
1.05 0.36
0.44 0.16
0.49 0.26
0.46 0.20
0.32 0.17
9.32 2.57
7.09 2.42
7.93 2.31
7.49 2.53
0.49 0.16
(Continues)
3a
3b
3c
3d
5a
5b
5c
5d
5e
5f
2.61 1.23
1.65 0.81
0.22 0.13
0.75 0.34
8.42 2.43
6.16 2.32
1.64 0.84
3.21 1.48
6.24 3.21
0.79 0.24
0.24 0.18
0.37 0.19
9.29 2.39
6.28 2.79
2.68 1.34
4.21 1.83
1.28 0.82
0.53 0.25
0.52 0.31
0.62 0.39
7.32 2.18
6.82 1.48
4.56 1.24
3.46 1.42
1.21 0.86
1.09 0.53
0.82 0.44
0.71 0.32
10.58 4.25
5.69 1.04
12.28 4.06
1.06 0.59
0.88 0.25
0.68 0.21
0.37 0.09
0.28 0.08
8.42 2.65
5.85 1.69
9.42 2.64
6.20 2.53
1.08 0.64
4.26 0.09
1.29 0.78
0.39 0.21
0.53 0.19
8.52 2.79
6.27 2.31
2.87 1.26
5.73 1.69
5.39 2.28
0.53 0.22
0.56 0.31
0.53 0.26
8.28 2.68
5.78 1.28
2.73 1.94
3.62 1.61
2.61 1.39
0.38 0.19
0.59 0.23
0.72 0.31
8.62 3.62
6.74 2.84
3.36 1.63
3.24 1.80
0.93 0.46
1.21 0.69
0.39 0.21
0.83 0.46
12.49 4.28
7.40 2.16
8.48 2.53
1.15 0.68
0.69 0.17
0.73 0.26
0.26 0.18
0.29 0.05
8.69 2.43
8.59 2.69
8.69 2.43
7.08 2.35
0.85 0.28
2.43 1.28
1.29 0.81
0.43 0.18
0.28 0.09
7.56 2.69
6.46 2.29
2.39 0.84
6.42 2.42
6.39 2.24
0.83 0.39
0.83 0.53
0.31 0.19
6.33 1.28
6.35 2.58
5.28 1.84
3.68 1.32
1.59 0.39
0.63 0.35
0.69 0.31
0.53 0.36
6.23 1.82
8.23 2.63
4.63 1.36
2.29 1.02
0.62 0.31
1.32 0.68
0.64 0.41
0.71 0.32
10.57 2.73
6.39 2.42
8.62 2.70
2.79 1.15
0.46 0.23
1.05 0.42
0.57 0.31
0.80 0.24
7.80 2.54
8.73 2.80
6.84 2.48
8.04 3.54
0.94 0.42
3.65 1.26
0.68 0.29
0.63 0.34
0.26 0.07
8.37 2.49
4.63 1.28
1.93 1.08
5.68 1.32
8.18 2.90
1.08 0.55
0.58 0.22
0.68 0.28
4.32 2.83
5.29 2.80
6.26 1.79
4.58 1.39
2.53 1.06
0.72 0.31
1.03 0.68
0.64 0.29
5.32 1.62
7.68 1.84
5.53 1.35
3.60 1.86
1.46 0.87
0.92 0.44
0.27 0.08
0.33 0.17
9.52 2.84
8.29 2.63
8.31 3.70
0.89 0.27
0.92 0.26
0.87 0.30
0.48 0.14
0.42 0.28
9.17 2.74
8.59 2.49
9.42 2.56
9.22 3.69
0.85 0.32
2.26 1.05
0.72 0.38
0.28 0.09
0.35 0.21
6.46 2.31
6.53 2.41
2.62 1.12
4.28 1.28
8.27 2.73
1.22 0.86
0.28 0.16
0.35 0.28
6.74 1.38
6.84 1.82
6.23 1.46
2.92 0.73
3.63 1.23
0.58 0.24
0.53 0.23
0.58 0.31
4.28 1.62
8.39 2.59
4.96 1.48
4.59 1.34
0.79 0.35
0.79 0.38
0.49 0.28
0.28 0.07
11.62 2.93
8.26 2.92
9.66 2.17
0.92 0.37
0.56 0.29
0.61 0.24
0.28 0.16
0.38 0.18
8.36 2.48
9.36 3.41
8.52 2.39
8.95 2.70
0.92 0.37
5g
5h
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
9g
9h
10a
10b
10c
10d
10e

MEGALLY ABDO ET AL.
7
TABLE 1 (Continued)
IC50 SEM, μM
Compound No
A549
H460
HT29
MKN-45
U87MG
SMMC-7721
1.48 0.83
0.74 0.25
0.62 0.36
0.44 0.062
10f
2.57 1.04
1.02 0.28
0.36 0.12
0.08 0.01
2.75 0.92
1.27 0.82
0.48 0.16
0.18 0.03
1.69 0.73
0.58 0.30
0.69 0.25
0.15 0.023
2.68 1.49
0.87 0.16
0.56 0.23
0.03 0.0055
1.69 0.77
0.55 0.21
0.49 0.21
0.90 0.13
10g
10h
Foretinib
TABLE 2 Inhibition of tyrosine kinases enzyme IC50 (nM) of compounds 3b, 3c, 5g, 5h, 7f, 7g, 7h, 8e, 8f, 8g, 9e, 9f, 9g, 9h, 10g,
and 10h
Compound
c-Kit
2.58
0.68
1.05
0.35
1.16
0.26
0.33
1.15
0.26
2.65
0.26
1.36
0.40
1.36
0.27
4.68
Flt-3
1.69
0.37
0.62
0.62
1.05
0.35
0.51
1.27
0.19
1.37
0.72
1.27
0.37
1.80
0.17
5.77
VEGFR-2
2.42
0.92
1.15
0.49
0.72
0.72
1.08
1.08
0.42
1.22
0.31
1.47
0.29
1.27
1.97
6.80
EGFR
2.80
0.47
1.62
0.81
0.59
0.29
0.28
0.59
0.38
1.59
0.47
1.09
0.18
1.00
0.81
4.29
PDGFR
1.63
0.58
0.73
0.27
0.31
0.83
0.52
1.42
0.61
1.26
0.33
0.96
0.42
0.66
1.05
5.39
3b
3c
5g
5h
7f
7g
7h
8e
8f
8g
9e
9f
9g
9h
10g
10h
TABLE 3 The inhibition activity of compounds 3c, 5h, 7g, 7h,
8f, 9e, 9g, and 10g toward Pim-1 Kinase
were less effective (IC50 > 10 μM). SGI-1776 was used as
positive control with IC50 0.048 μM in the assay. These
profiles in combination with cell growth inhibition data
of compounds 3c, 5h, 7g, 7h, 8f, 9e, 9g, and 10g were
listed in Table 3 indicated that Pim-1 was a potential tar-
get of these compounds.
Compound
Inhibition Ratio At 10μM
IC50, μM
>10
3c
5h
7g
7h
8f
21
96
86
18
16
92
89
24
0.24
0.30
>10
>10
2.2.5 | PAINS analysis
9e
9g
10g
0.23
0.32
Compounds 3b, 3c, 5g, 5h, 7f, 7g, 7h, 8e, 8f, 8g, 8h, 9e,
9f, 9g, 9h, 10g, and 10h were selected to be tested for
pan-assay interference compounds analysis (PAINS). Suc-
cessful drug discovery process involves high-throughput
screening (HTS). During HTS, it is predicted that suc-
cesses from HTS operations include primarily false posi-
tives between the actual hits if it is found.[51,52]
Compounds can be regarded as false positives because of
>10
values were calculated. Compounds 5h, 7g, 9e, and 9g
were the most potent to inhibit Pim-1 activity with IC50
value of 0.24 μM, 0.30 μM, 0.23 μM, and 0.32 μM, respec-
tively. On the other hand, compounds 3c, 7h, 8f, and 10g

8
MEGALLY ABDO ET AL.
TABLE 4 Drug like the character of different compounds and standard drugs foretinib and SGI-1776
Compound Druglikeness Rule Medicinal Chemistry Rules
Lvio.a/No. of vio.a
None
Vvio.b/No. of vio.b
None
Gvio.c/No. of vio.c
None
Lead likeliness /No.
PAINS alertd of vio.
3b
None
0
0
1
0
0
0
0
0
1
0
0
0
0
1
3c
None
None
None
None
5g
None
None
None
1
5h
None
None
None
2
7f
None
None
None
None
7g
None
None
None
None
7h
None
None
None
None
8e
None
None
None
1
8f
None
None
None
3
8g
None
None
None
1
8h
None
None
None
None
9e
None
None
None
None
9f
None
None
None
None
9g
None
None
None
2
9h
None
None
None
1
10g
10h
Foretinib
SGI-1776
None
None
None
None
0
0
0
0
None
None
None
1
1
2
None
None
None
None
None
None
Abbreviations: Gvio., Ghose filter; Lvio., Lipinski's rule; Vvio., Veber rules; PAINS, pan-assay interference compounds analysis.
a number of reasons like binding interactions by forming
aggregates,[53–55] by being protein-reactive entities[56–58]
or by directly interfering with assay signaling. PAINS are
chemical entities that are frequently false positives in
HTS. PAINS have a tendency to nonspecifically react
with several biological targets moderately, then specifi-
cally disturbing one preferred.[59] A number of disorderly
functional groups are collected by numerous PAINS.[60]
Unwanted compounds may negatively influence not only
enzyme assays but also phenotypic screens and show bio-
logical activity for the wrong reason.[61] The PAINS viola-
tions of proposed compounds and reference drugs are
given in Table 4. Almost all the compounds showed zero
PAINS alert and can be used as lead compounds.
data. All the newly synthesized compounds were evalu-
ated against the six cancer cell lines A549, HT-29, MKN-
45, U87MG, and SMMC-7721 and H460. Compounds 3c,
5h, 7g, 7h, 8f, 9e, 9g, and 10g were selected to examine
their Pim-1 kinase inhibition activity as these compounds
showed high inhibition toward the c-Met kinase and the
tested cancer cell lines. Furthermore, compounds 3b, 3c,
5g, 5h, 7f, 7g, 7h, 8e, 8f, 8g, 8h, 9e, 9f, 9g, 9h, 10g, and
10h were selected to be tested for PAINS. Almost all the
tested compounds showed zero PAINS alert and can be
used as lead compounds.
4 | EXPERIMENTAL
4.1 | Chemistry
3 | CONCLUSION
The obtained compounds showed their melting points
using electrothermal digital melting point apparatus and
are uncorrected. IR spectra (KBr discs) were measured
on a Fourier transform infrared (FTIR) plus 460 or Pye
Unicam SP-1000 spectrophotometer (Pye Unicam, UK,
Cambridge). 1H NMR spectra were obtained using Varian
Gemini-300 (300 MHz, Varian UK) using DMSO-d6 as a
The well-known chalcones 3a-d were considered as
the key starting material through their reactions with dif-
ferent reagents to synthesize the 4H-pyrazole derivatives
7a-h and the thiophene derivatives 9a-h and 10a-h,
respectively. The structure of the newly synthesized
compounds was established by the analytical and spectral

MEGALLY ABDO ET AL.
9
solvent and tetramethylsilane @@(TMS) as internal stan-
dard chemical shifts are expressed as δ = ppm. The
mass spectra were measured with Hewlett Packard
5988 A GC/MS system (Hewlett Packard, Agilent,
USA) instrument. Analytical data were obtained from
on Vario EL III Elemental CHNS analyzer. The anti-
tumor evaluation has been carried out through the
National Cancer Research Center in Cairo, Egypt,
where the IC50 values were calculated. Compounds
3a-d were synthesized according to the reported
procedures.[39–41]
2-(3-(4-Methoxyphenyl)-1-phenylallylidene)
malononitrile (5c)
Yellow crystals from ethanol, yield (1.97 g, 77%), mp:
203ꢀC to 206ꢀC. IR (KBr) υmax cm−1: 3053 (CH, aro-
1
matic), 2222, 2220 (2CN), 1631 (C═C); H NMR (DMSO-
d6, 200 MHz): δ = 3.66 (s, 3H, OCH3), 6.30, 6.43 (2d, 2H,
J = 10.17 Hz, CH═CH), 7.24-7.46 (m, 9H, C6H5, C6H4);
13C NMR (DMSO-d6, 75 MHz): δ = 52.4 (OCH3), 89.6,
90.7 (CH═CH), 116.6, 116.8 (2CN), 120.3, 122.6, 122.8,
123.5, 125.9, 126.3, 127.4, 128.2 (C6H5, C6H4), 131.1, 134.6
(C═C). Anal. calculated for C19H14N2O: C, 79.70; H,
4.93; N, 9.78. Found: C, 79.59; H, 5.22; N, 10.02. MS: m/e
286 (M+, 42%).
4.1.1 | General procedure for the
synthesis of the penta-2,4-dienenitrile
derivatives 5a-h
2-(1-[4-Chlorophenyl]-3-phenylallylidene)
malononitrile (5d)
Pale yellow crystals from ethanol, yield (1.97 g, 68%), mp:
155ꢀC to 157ꢀC. IR (KBr) υmax cm−1:3053 (CH, aromatic),
2225, 2220 (2CN), 1631 (C═C); 1H NMR (DMSO-d6,
200 MHz): δ = 6.34, 6.45 (2d, 2H, J = 10 Hz, CH═CH),
7.22-7.48 (m, 9H, C6H5, C6H4); 13C NMR (DMSO-d6,
75 MHz): δ = 89.1, 90.7 (CH═CH), 116.6, 116.8 (2CN),
120.3, 121.9, 122.8, 123.6, 124.1, 126.2, 127.1, 127.8 (C6H5,
C6H4), 131.2, 134.3 (C═C). Anal. calculated for
C18H11ClN2: C, 74.36; H, 3.81; N, 9.63. Found: C,
74.52; H, 4.04; N, 9.82. MS: m/e 290 (M+, 28%).
Either of malononitrile (0.66 g, 0.01 mol) or ethyl
cyanoacetate (1.07 g, 0.01 mol) and ammonium acetate
(1.00 g) were added to any of compounds 3a (2.08,
0.01 mol), 3b (2.72 g, 0.01 mol), 3c (2.38 g, 0.01 mol), or
3d (2.42 g, 0.01 mol). The reaction mixture in each case
was heated in an oil bath at 120ꢀC for 1 hour then was
left to cool. The solid product, formed in each case, upon
trituration with diethyl ether was collected by filtration.
2-(1,3-Diphenylallylidene)malononitrile (5a)
Pale yellow crystals from ethanol, yield (1.81 g, 71%),
mp: 180ꢀC to 183ꢀC. IR (KBr) υmax cm−1: 3055 (CH,
aromatic), 2223, 2220 (2CN), 1632 (C═C); 1H NMR
Ethyl 2-cyano-3,5-diphenylpenta-2,4-dienoate (5e)
Pale yellow crystals from ethanol, yield (2.30 g, 76%), mp:
196ꢀC to 198ꢀC. IR (KBr) υmax cm−1: 3056 (CH, aro-
matic), 2221 (CN), 1710 (CO), 1630 (C═C); 1H NMR
(DMSO-d6, 200 MHz): δ = 1.13 (t, 3H, J = 7.18 Hz, CH3),
4.22 (q, 2H, J = 7.18 Hz, CH2), 6.32, 6.41 (2d, 2H,
J = 10 Hz, CH═CH), 7.29-7.38 (m, 10H, 2C6H5); 13C
NMR (DMSO-d6, 75 MHz): δ = 16.3 (OCH2CH3), 50.4
(OCH2CH3), 89.3, 90.5 (CH═CH), 116.8 (CN), 120.1,
121.6, 123.0, 123.8, 125.6, 126.8, 127.4, 128.2 (2C6H5),
131.1, 134.5 (C═C), 168.9 (CO). Anal. calculated for
C20H17NO2: C, 79.19; H, 5.65; N, 4.62. Found: C,
79.08; H, 5.48; N, 4.80. MS: m/e 303 (M+, 42%).
(DMSO-d6, 200 MHz):
δ = 6.38, 6.41 (2d, 2H,
J = 9.89 Hz, CH═CH), 7.26-7.39 (m, 10H, 2C6H5); 13C
NMR (DMSO-d6, 75 MHz): δ = 89.3, 90.5 (CH═CH),
116.5, 116.9 (2CN), 120.6, 122.4, 123.5, 124.1, 124.8,
125.4, 127.3, 128.3 (2C6H5), 131.2, 134.2 (C═C). Anal.
calculated for C18H12N2: C, 84.35; H, 4.72; N, 10.93.
Found: C, 84.66; H, 5.01; N, 11.23. MS: m/e
256 (M+, 28%).
2-(1-(4-Chlorophenyl)-3-(4-methoxyphenyl)allylidene)
malononitrile (5b)
Pale yellow crystals from ethanol, yield (2.11 g, 66%), mp:
Ethyl 3-(4-chlorophenyl)-2-cyano-5-(4-methoxyphenyl)
penta-2,4-dienoate (5f)
166ꢀC to 169ꢀC. IR (KBr) υmax cm−1: 3056 (CH, aro-
1
matic), 2221, 2220 (2CN), 1634 (C═C); H NMR (DMSO-
Pale yellow crystals from ethanol, yield (2.93 g, 80%), mp:
233ꢀC to 235ꢀC. IR (KBr) υmax cm−1: 3055 (CH, aro-
matic), 2220 (CN),1714 (CO), 1630 (C═C); 1H NMR
(DMSO-d6, 200 MHz): δ = 1.14 (t, 3H, J = 6.72 Hz, CH3),
3.70 (s, 3H, OCH3), 4.23 (q, 2H, J = 6.72 Hz, CH2), 6.31,
6.44 (2d, 2H, J = 9.95 Hz, CH═CH), 7.25-7.49 (m, 8H,
2C6H4); 13C NMR (DMSO-d6, 75 MHz): δ = 16.6
(OCH2CH3), 50.5 (OCH3), 52.8 (OCH2CH3), 89.1, 90.6
(CH═CH), 116.9 (CN), 119.8, 120.2, 121.8, 122.7, 124.9,
d6, 200 MHz): δ = 3.69 (s, 3H, OCH3), 6.32, 6.40 (2d, 2H,
J = 9.91 Hz, CH═CH), 7.23-7.42 (m, 8H, 2C6H4); 13C
NMR (DMSO-d6, 75 MHz): δ = 52.8 (OCH3), 89.3, 90.5
(CH═CH), 116.5, 116.9 (2CN), 120.4, 121.8, 122.9, 123.6,
126.2, 127.8, 128.1, 129.7 (2C6H4), 131.1, 134.6 (C═C).
Anal. calculated for C19H13ClN2O: C, 71.14; H, 4.08; N,
8.73. Found: C, 71.29; H, 3.82; N, 8.90. MS: m/e
320 (M+, 38%).

10
MEGALLY ABDO ET AL.
1
126.5, 128.3, 128.8 (2C6H4), 131.4, 134.2 (C═C), 167.1
(CO). Anal. calculated for C21H18ClNO3: C, 68.57; H,
4.93; N, 3.81. Found: C, 68.32; H, 5.08; N, 4.02. MS: m/e
367 (M+, 38%).
3054 (CH, aromatic), 1653 (C═N), 1632 (C═C); H NMR
(DMSO-d6, 200 MHz): δ = 4.59, 5.03 (2s, 4H, D2O
exchangeable, 2NH2), 6.35, 6.43 (2d, 2H, J = 9.88 Hz,
CH═CH), 7.28-7.36 (m, 10H, 2C6H5); 13C NMR (DMSO-
d6, 75 MHz): δ = 89.1, 90.7 (CH═CH), 120.8, 121.5, 123.8,
124.7, 125.3, 126.8, 127.8, 128.1 (2C6H5), 131.8, 134.6
(C═C), 172.8, 176.2 (2C═N). Anal. calculated for
C18H16N4: C, 74.98; H, 5.59; N, 19.43. Found: C, 75.03; H,
5.38; N, 19.39. MS: m/e 288 (M+,36%).
Ethyl 2-cyano-5-(4-methoxyphenyl)-3-phenylpenta-
2,4-dienoate (5g)
Pale yellow crystals from ethanol, yield (2.09 g, 63%), mp:
205ꢀC to 208ꢀC. IR (KBr) υmax cm−1: 3055 (CH, aro-
matic), 2220 (CN), 1709 (CO), 1632 (C═C); 1H NMR
(DMSO-d6, 200 MHz): δ = 1.12 (t, 3H, J = 7.02 Hz, CH3),
3.68 (s, 3H, OCH3), 4.22 (q, 2H, J = 7.02 Hz, CH2), 6.30,
6.45 (2d, 2H, J = 10.1 Hz, CH═CH), 7.23-7.44 (m, 9H,
C6H5, C6H4); 13C NMR (DMSO-d6, 75 MHz): δ = 16.7
(OCH2CH3), 50.1 (OCH3), 52.4 (OCH2CH3), 89.2, 90.4
(CH═CH), 116.5 (CN), 119.9, 120.8, 121.3, 123.5, 124.4,
125.8, 128.1, 128.6 (C6H5, C6H4), 131.3, 134.5 (C═C),
168.9 (CO). Anal. calculated for C21H19NO3: C, 75.66.; H,
5.74; N, 4.20. Found: C, 75.42; H, 5.63; N, 4.31. MS: m/e
333 (M+, 41%).
4-(1-(4-Chlorophenyl)-3-(4-methoxyphenyl)allylidene)-
4H-pyrazole-3,5-diamine (7b)
Yellow crystals from 1,4-dioxan, yield (2.28 g, 65%), mp:
196ꢀC to 198ꢀC. IR (KBr) υmax cm−1: 3498, 3346 (2NH2),
1
3055 (CH, aromatic), 1650 (C═N), 1631 (C═C); H NMR
(DMSO-d6, 200 MHz): δ = 3.69 (s, 3H, OCH3), 4.73, 5.08
(2s, 4H, D2O exchangeable, 2NH2), 6.38, 6.41 (2d, 2H,
J = 9.97 Hz, CH═CH), 7.25-7.48 (m, 8H, 2C6H4); 13C
NMR (DMSO-d6, 75 MHz): δ = 52.6 (OCH3), 89.4, 90.9
(CH═CH), 120.3, 122.8, 122.9, 123.1, 124.6, 126.5, 127.3,
129.4 (2C6H4), 131.3, 134.2 (C═C), 172.5, 176.2 (2C═N).
Anal. calculated for C19H17ClN4O: C, 64.68; H, 4.86; N,
15.88. Found: C, 64.70; H, 4.57; N, 16.16. MS: m/e
352 (M+, 24%).
Ethyl 3-(4-chlorophenyl)-2-cyano-5-phenylpenta-
2,4-dienoate (5h)
Dark yellow crystals from ethanol, yield (2.46 g, 73%),
mp: 148ꢀC to 151ꢀC. IR (KBr) υmax cm−1: 3055 (CH, aro-
matic), 2220 (CN), 1710 (CO), 1633 (C═C); 1H NMR
(DMSO-d6, 200 MHz): δ = 1.12 (t, 3H, J = 7.38 Hz, CH3),
4.22 (q, 2H, J = 7.38 Hz, CH2), 6.32, 6.43 (2d, 2H,
J = 10.21 Hz, CH═CH), 7.21-7.49 (m, 9H, C6H5, C6H4);
13C NMR (DMSO-d6, 75 MHz): δ = 16.7 (OCH2CH3), 50.3
(OCH2CH3), 89.3, 90.2 (CH═CH), 116.4 (CN), 120.6,
121.93, 122.5, 123.8, 124.4, 126.8, 127.1, 127.9 (C6H5,
C6H4), 131.6, 134.2 (C═C), 169.5 (CO). Anal. calculated
for C20H16ClNO2: C, 71.11; H, 4.77; N, 4.15%. Found: C,
70.92; H, 4.80; N, 4.31%. MS: m/e 337 (M+, 24%).
4-(3-(4-Methoxyphenyl)-1-phenylallylidene)-4H-
pyrazole-3,5-diamine (7c)
Yellow crystals from 1,4-dioxan, yield (2.01 g, 70%), mp:
222ꢀC to 225ꢀC. IR (KBr) υmax cm−1: 3439, 3372 (2NH2),
1
3055 (CH, aromatic), 1650 (C═N), 1630 (C═C); H NMR
(DMSO-d6, 200 MHz): δ = 3.67 (s, 3H, OCH3), 4.71, 5.12
(2s, 4H, D2O exchangeable, 2NH2), 6.36, 6.43 (2d, 2H,
J = 9.93 Hz, CH═CH), 7.23-7.46 (m, 9H, C6H5, C6H4);
13C NMR (DMSO-d6, 75 MHz): δ = 52.4 (OCH3), 89.2,
90.5 (CH═CH), 120.1, 121.6, 122.7, 123.5, 123.9, 126.2,
128.1, 128.6 (C6H5, C6H4), 131.5, 134.7 (C═C),172.8, 176.2
(2C═N). Anal. calculated for C19H18N4O: C, 71.68; H,
5.70; N, 17.60. Found: C, 71.83; H, 5.92; N, 17.41. MS: m/
e 318 (M+, 65%).
4.1.2 | General procedure for the
synthesis of the pyrazole derivatives 7a-h
Hydrazine hydrate (0.50 mL, 0.01 mol) was added to a
solution of any of compounds 5a (2.56 g, 0.01 mol), 5b
(3.20 g, 0.01 mol), 5c (2.86 g, 0.01 mol), 5d (2.90 g,
0.01 mol), 5e (3.03 g, 0.01 mol), 5f (3.67 g, 0.01 mol), 5g
(3.33 g, 0.01 mol) or 5h (3.37 g, 0.01 mol) in 1,4-dioxan
(40 mL). The reaction mixture in each case was heated
under reflux for 3 hours then poured onto a solution of
ice/water mixture containing a few drops of hydrochloric
acid and the formed precipitate was collected by filtration.
4-(1-(4-Chlorophenyl)-3-phenylallylidene)-4H-pyrazole-
3,5-diamine (7d)
White crystals from 1,4-dioxan, yield (2.54 g, 79%), mp:
177ꢀC to 179ꢀC. IR (KBr) υmax cm−1: 3457, 3338 (2NH2),
1
3055 (CH, aromatic), 1650 (C═N), 1632 (C═C); H NMR
(DMSO-d6, 200 MHz): δ = 4.56, 5.02 (2s, 4H, D2O
exchangeable, 2NH2), 6.36, 6.45 (2d, 2H, J = 9.87 Hz,
CH═CH), 7.24-7.48 (m, 9H, C6H5, C6H4); 13C NMR
(DMSO-d6, 75 MHz): δ = 89.3, 90.2 (CH═CH), 120.5,
121.6, 123.2, 124.3, 126.2, 126.9, 127.5, 129.9 (C6H5, C6H4),
131.2, 134.3 (C═C), 172.8, 176.0 (2C═N). Anal. calculated
for C18H15ClN4: C, 66.98; H, 4.68; N, 17.36. Found: C,
67.25; H, 4.36; N, 17.48. MS: m/e 322 (M+, 42%).
4-(1,3-Diphenylallylidene)-4H-pyrazole-3,5-diamine (7a)
White crystals from 1,4-dioxan, yield (2.01 g, 70%), mp:
266ꢀC to 268ꢀC. IR (KBr) υmax cm−1: 3487, 3368 (2NH2),

MEGALLY ABDO ET AL.
11
5-Amino-4-(1,3-diphenylallylidene)-4H-pyrazol-3-ol (7e)
Pale yellow crystals from ethanol, yield (1.93 g, 67%), mp:
210ꢀC to 212ꢀC. IR (KBr) υmax cm−1: 3527-3368 (OH,
D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 89.5, 90.3 (CH═CH), 120.2, 120.8, 121.5, 122.8, 123.9,
125.8, 126.2, 128.9 (C6H5, C6H4), 131.1, 134.6 (C═C) 172.5,
176.3 (2C═N). Anal. calculated for C18H14ClN3O: C,
66.77; H, 4.36; N, 12.98. Found: C, 66.92; H, 4.60; N, 12.72.
MS: m/e 323 (M+, 38%).
1
NH2), 3055 (CH, aromatic), 1650 (C═N), 1632 (C═C); H
NMR (DMSO-d6, 200 MHz): δ = 4.58 (s, 2H, D2O
exchangeable, NH2), 6.38, 6.42 (2d, 2H, J = 9.87 Hz,
CH═CH), 7.29-7.38 (m, 10H, 2C6H5), 10.31 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 89.6, 90.6 (CH═CH), 119.3, 120.8, 122.6, 125.9, 127.2,
127.8, 128.1, 129.3 (2C6H5), 131.4, 134.6 (C═C), 172.6,
176.2 (2C═N). Anal. calculated for C18H15N3O: C,
74.72; H, 5.23; N, 14.52. Found: C, 74.39; H, 5.41; N,
14.73. MS: m/e 289 (M+, 27%).
4.1.3 | General procedure for the
synthesis of the 2-cyano-N-(4H-pyrazol-3-yl)
acetamide derivatives 8a-h
Ethyl cyanoacetate (1.07 g, 0.01 mol) was added to a solu-
tion of any of compounds 7a (2.88, 0.01 mol), 7b (3.52 g,
0.01 mol), 7c (3.18 g, 0.01 mol), 7d (3.22 g, 0.01 mol), 7e
(2.89 g, 0.01 mol), 7f (3.53 g, 0.01 mol), 7g (3.19 g,
0.01 mol) or 7h (3.23 g, 0.01 mol) in dimethylformamide
(40 mL). The reaction mixture, in each case, was heated
under reflux for 3 hours then was left to cool, and the
solid product produced upon pouring onto ice/water was
collected by filtration.
5-Amino-4-(1-(4-chlorophenyl)-3-(4-methoxyphenyl)
allylidene)-4H-pyrazol-3-ol (7f)
Yellow crystals from ethanol, yield (2.64 g, 75%), mp:
177ꢀC to 179ꢀC. IR (KBr) υmax cm−1: 3548-3373 (OH,
1
NH2), 3055 (CH, aromatic), 1651 (C═N), 1630 (C═C); H
NMR (DMSO-d6, 200 MHz): δ = 3.68 (s, 3H, OCH3), 4.56
(s, 2H, D2O exchangeable, NH2), 6.36, 6.45 (2d, 2H,
J = 10.13 Hz, CH═CH), 7.22-7.48 (m, 8H, 2C6H4), 10.33
(s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6,
75 MHz): δ = 52.6 (OCH3), 89.4, 90.4 (CH═CH), 120.8,
120.9, 121.2, 123.6, 125.8, 126.3, 129.6, 130.1 (2C6H4),
131.2, 134.8 (C═C), 172.8, 176.2 (2C═N). Anal. calculated
for C19H16ClN3O2: C, 64.50; H, 4.56; N, 11.88. Found: C,
64.81; H, 4.69; N, 11.64. MS: m/e 353 (M+, 32%).
N-(5-Amino-4-(1,3-diphenylallylidene)-4H-pyrazol-
3-yl)-2-cyanoacetamide (8a)
Yellow crystals from 1,4-dioxan, yield (2.55 g, 72%), mp:
201ꢀC to 204ꢀC. IR (KBr) υmax cm−1: 3469-3331 (NH2,
NH), 3055 (CH, aromatic), 2258 (CN), 1681 (CO), 1651
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.80 (s, 2H, CH2), 4.56 (s, 2H, D2O exchangeable,
NH2), 6.34, 6.43 (2d, 2H, J = 10.07 Hz, CH═CH),
7.25-7.39 (m, 10H, 2C6H5), 8.26 (s, 1H, D2O exchange-
able, NH); 13C NMR (DMSO-d6, 75 MHz): δ = 27.2
(CH2), 89.3, 90.5 (CH═CH), 117.1 (CN), 120.4, 121.7,
122.2, 123.4, 125.9, 126.1, 127.3, 128.5 (2C6H5), 131.5,
134.4 (C═C),168.1 (CO), 172.4, 176.2 (2C═N). Anal.
calculated for C21H17N5O: C, 70.97; H, 4.82; N, 19.71.
Found: C, 71.08; H, 4.69; N, 19.53. MS: m/e
355 (M+,36%).
5-Amino-4-(3-(4-methoxyphenyl)-1-phenylallylidene)-
4H-pyrazol-3-ol (7g)
Pale yellow crystals from ethanol, yield (2.23 g, 70%), mp:
231ꢀC to 234ꢀC. IR (KBr) υmax cm−1: 3561-3358 (OH,
1
NH2), 3056 (CH, aromatic), 1648 (C═N), 1632 (C═C); H
NMR (DMSO-d6, 200 MHz): δ = 3.69 (s, 3H, OCH3), 4.59
(s, 2H, D2O exchangeable, NH2), 6.34, 6.42 (2d, 2H,
J = 9.84 Hz, CH═CH), 7.24-7.49 (m, 9H, C6H5, C6H4),
10.31 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-
d6, 75 MHz): δ = 52.4 (OCH3), 89.2, 90.7 (CH═CH),
120.6, 121.3, 122.8, 123.2, 124.6, 127.1, 127.8, 129.5
(C6H5, C6H4), 131.4, 134.5 (C═C), 172.9, 175.9 (2C═N).
Anal. calculated for C19H17N3O2: C, 71.46; H, 5.37; N,
13.16. Found: C, 71.38; H, 5.49; N, 12.92. MS: m/e
319 (M+, 58%).
N-(5-Amino-4-(1-(4-chlorophenyl)-3-(4-methoxyphenyl)
allylidene)-4H-pyrazol-3-yl)-2-cyanoacetamide (8b)
Yellow crystals from 1,4-dioxan, yield (2.84 g, 68%), mp:
180ꢀC to 183ꢀC. IR (KBr) υmax cm−1: 3485-3359 (NH2,
NH), 3055 (CH, aromatic), 2250 (CN), 1685 (CO),1650
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.68 (s, 3H, OCH3), 3.83 (s, 2H, CH2), 4.71 (s, 2H,
D2O exchangeable, NH2), 6.35, 6.39 (2d, 2H, J = 10 Hz,
CH═CH), 7.21-7.49 (m, 8H, 2C6H4), 8.26 (s, 1H, D2O
exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz):
δ = 27.6 (CH2), 52.3 (OCH3), 89.2, 90.7 (CH═CH), 117.0
(CN), 120.1, 121.5, 122.6, 123.8, 124.4, 125.6, 126.8, 127.3
(2C6H4), 131.6, 134.5 (C═C),167.9 (CO), 172.5, 176.6
(2C═N). Anal. calculated for C22H18ClN5O2: C, 62.93; H,
5-Amino-4-(1-(4-chlorophenyl)-3-phenylallylidene)-4H-
pyrazol-3-ol (7h)
Yellowish brown crystals from 1,4-dioxan, yield (2.51 g,
79%), mp: 188ꢀC to 191ꢀC. IR (KBr) υmax cm−1: 3536-3331
(OH, NH2), 3054 (CH, aromatic), 1642 (C═N), 1630
1
(C═C); H NMR (DMSO-d6, 200 MHz): δ = 4.53 (s, 2H,
D2O exchangeable, NH2), 6.36, 6.41 (2d, 2H, J = 10 Hz,
CH═CH), 7.22-7.48 (m, 9H, C6H5, C6H4), 10.33 (s, 1H,

12
MEGALLY ABDO ET AL.
4.32; N, 16.68. Found: C, 63.28; H, 4.49; N, 16.53. MS: m/e
N-(4-(1-(4-Chlorophenyl)-3-(4-methoxyphenyl)
allylidene)-5-hydroxy-4H-pyrazol-3-yl)-
419 (M+, 30%).
2-cyanoacetamide (8f)
N-((5-Amino-4-(3-(4-methoxyphenyl)-
1-phenylallylidene)-4H-pyrazol-3-yl)-
2-cyanoacetamide (8c)
Yellow crystals from 1,4-dioxan, yield (2.89 g, 69%), mp:
113ꢀC to 115ꢀC. IR (KBr) υmax cm−1: 3568-3360 (OH,
NH), 3055 (CH, aromatic), 2255 (CN), 1684 (CO),1651
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.68 (s, 3H, OCH3), 3.76 (s, 2H, CH2), 6.36, 6.45 (2d,
2H, J = 9.96 Hz, CH═CH), 7.22-7.48 (m, 8H, 2C6H4), 8.33
(s, 1H, D2O exchangeable, NH), 10.33 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 27.6 (CH2), 52.1 (OCH3) 89.2, 90.4 (CH═CH), 117.2
(CN), 120.8, 120.9, 121.2, 123.6, 125.8, 126.3, 129.6, 130.1
(2C6H4), 131.4, 134.5 (C═C), 168.3 (CO), 172.8, 176.2
(2C═N). Anal. calculated for C22H17ClN4O3: C, 62.79; H,
4.07; N, 13.31. Found: C, 62.59; H, 4.28; N, 13.52. MS: m/e
420 (M+, 28%).
Yellow crystals from 1,4-dioxan, yield (3.08 g, 80%), mp:
166ꢀC to 168ꢀC. IR (KBr) υmax cm−1: 3461-3349 (NH2,
NH), 3055 (CH, aromatic), 2253 (CN), 1681 (CO), 1650
(C═N), 1632 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.69 (s, 3H, OCH3), 3.84 (s, 2H, CH2), 4.74, (s, 2H,
D2O exchangeable, NH2), 6.36, 6.46 (2d, 2H, J = 9.94 Hz,
CH═CH), 7.21-7.49 (m, 9H, C6H5, C6H4), 8.29 (s, 1H,
D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz):
δ = 27.6 (CH2), 52.4 (OCH3), 89.1, 90.8 (CH═CH), 116.9
(CN), 120.4, 121.3, 122.9, 124.1, 125.3, 126.7, 127.8, 128.9
(C6H5, C6H4), 131.3, 134.8 (C═C), 168.4 (CO), 172.5,
176.6 (2C═N). Anal. calculated for C22H19N5O2: C,
68.56; H, 4.97; N, 18.17. Found: C, 68.73; H, 5.21; N,
18.26. MS: m/e 385 (M+, 42%).
2-Cyano-N-(5-hydroxy-4-(3-(4-methoxyphenyl)-
1-phenylallylidene)-4H-pyrazol-3-yl)acetamide (8g)
Pale yellow crystals from ethanol, yield (2.54 g, 66%),
mp: 177ꢀC to 179ꢀC. IR (KBr) υmax cm−1: 3583-3339
(OH, NH), 3054 (CH, aromatic), 2255 (CN), 1687
N-(5-Amino-4-(1-(4-chlorophenyl)-3-phenylallylidene)-
4H-pyrazol-3-yl)-2-cyanoacetamide (8d)
1
Yellowish brown crystals from 1,4-dioxan, yield (2.76 g,
71%), mp: 201ꢀC to 204ꢀC. IR (KBr) υmax cm−1: 3472-3358
(NH2, NH), 3055 (CH, aromatic), 2248 (CN), 1680 (CO),
(CO), 1643 (C═N), 1630 (C═C); H NMR (DMSO-d6,
200 MHz): δ = 3.67 (s, 3H, OCH3), 3.80 (s, 2H, CH2),
6.32, 6.45 (2d, 2H, J = 10.11 Hz, CH═CH), 7.21-7.47
(m, 9H, C6H5, C6H4), 8.25 (s, 1H, D2O exchangeable,
NH), 10.33 (s, 1H, D2O exchangeable, OH); 13C NMR
(DMSO-d6, 75 MHz): δ = 27.4 (CH2), 52.3 (OCH3),
89.5, 90.9 (CH═CH), 116.6 (CN), 119.8, 121.6, 122.3,
123.9, 125.3, 126.8, 127.3, 128.2 (C6H5, C6H4), 131.4,
134.7 (C═C), 167.8 (CO),172.3, 176.6 (2C═N). Anal.
calculated for C22H18N4O3: C, 68.38; H, 4.70; N, 14.50.
Found: C, 68.52; H, 4.93; N, 14.68. MS: m/e
386 (M+, 38%).
1
1650 (C═N), 1631 (C═C); H NMR (DMSO-d6, 200 MHz):
δ = 3.72 (s, 2H, CH2), 4.58 (s, 2H, D2O exchangeable, NH2),
6.33, 6.48 (2d, 2H, J = 10.1 Hz, CH═CH), 7.21-7.47 (m, 9H,
C6H5, C6H4), 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR
(DMSO-d6, 75 MHz): δ = 27.8 (CH2), 89.5, 90.6 (CH═CH),
116.9 (CN), 120.8, 122.9, 123.1, 125.8, 126.5, 127.3, 127.8,
128.2 (C6H5, C6H4), 131.6, 134.5 (C═C), 168.5 (CO), 172.8,
176.4 (2C═N). Anal. calculated for C21H16ClN5O: C,
64.70; H, 4.14; N, 17.96. Found: C, 64.52; H, 4.38; N, 17.58.
MS: m/e 389 (M+, 36%).
N-(4-(1-(4-Chlorophenyl)-3-phenylallylidene)-5-hydroxy-
4H-pyrazol-3-yl)-2-cyanoacetamide (8h)
2-Cyano-N-(4-(1,3-diphenylallylidene)-5-hydroxy-4H-
pyrazol-3-yl)acetamide (8e)
Pale brown crystals from 1,4-dioxan, yield (2.61 g,
Yellow crystals from 1,4-dioxan, yield (2.77 g, 78%), mp:
189ꢀC to 191ꢀC. IR (KBr) υmax cm−1: 3558-3342 (OH,
NH), 3056 (CH, aromatic), 2254 (CN), 1679 (CO), 1650
(C═N), 1632 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.75 (s, 2H, CH2), 6.34, 6.46 (2d, 2H, J = 10.09 Hz,
CH═CH), 7.26-7.42 (m, 10H, 2C6H5), 8.19 (s, 1H, D2O
exchangeable, NH), 10.28 (s, 1H, D2O exchangeable,
OH); 13C NMR (DMSO-d6, 75 MHz): δ = 27.9 (CH2),
89.4, 90.9 (CH═CH), 116.8 (CN), 120.9, 121.8, 122.3,
123.7, 125.8, 126.5, 128.5, 128.8 (2C6H5), 131.3, 134.4
(C═C), 167.7 (CO), 172.6, 176.1 (2C═N). Anal. calculated
for C21H16N4O2: C, 70.77; H, 4.53; N, 15.72. Found: C,
70.42; H, 4.68; N, 15.80. MS: m/e 356 (M+, 42%).
67%), mp: 232ꢀC to 235ꢀC. IR (KBr) υmax cm−1
:
3547-3361 (OH, NH), 3055 (CH, aromatic), 2248 (CN),
1
1684 (CO), 1642 (C═N), 1630 (C═C); H NMR (DMSO-
d6, 200 MHz): δ = 3.80 (s, 2H, CH2), 6.34, 6.45 (2d, 2H,
J = 9.87 Hz, CH═CH), 7.20-7.42 (m, 9H, C6H5, C6H4),
8.28 (s, 1H, D2O exchangeable, NH), 10.33 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 27.8 (CH2), 89.5, 90.6 (CH═CH), 116.9 (CN), 120.4,
120.3, 121.8, 122.6, 123.7, 125.3, 125.8, 128.3 (C6H5,
C6H4), 131.3, 134.7 (C═C), 168.2 (CO),172.3, 176.8
(2C═N). Anal. calculated for C21H15ClN4O2: C,
64.54; H, 3.87; N, 14.34. Found: C, 64.73; H, 4.13; N,
14.28. MS: m/e 390 (M+, 26%).

MEGALLY ABDO ET AL.
13
4.1.4 | General procedure for the
synthesis of the thiophene derivatives 9a-h
NH), 3055 (CH, aromatic), 2223 (CN), 1687 (CO), 1648
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.67 (s, 3H, OCH3), 4.48, 4.74, 5.28 (3s, 6H, D2O
exchangeable, 3NH2), 6.35, 6.48 (2d, 2H, J = 9.89 Hz,
CH═CH), 7.23-7.48 (m, 9H, C6H5, C6H4), 8.26 (s, 1H, D2O
exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz):
δ = 52.2 (OCH3), 89.3, 90.6 (CH═CH), 116.6 (CN), 120.2,
122.6, 122.5, 123.6, 125.1, 126.5, 127.3, 128.4, 130.2, 132.6,
134.9, 136.1 (C6H5, C6H4, thiophene C), 131.6, 134.9 (C═C),
167.1 (CO), 172.2, 176.8 (2C═N). Anal. calculated for
C25H21N7O2S: C, 62.10; H, 4.38; N, 20.28; S, 6.63. Found: C,
61.89; H, 4.66; N, 20.41; S, 7.01. MS: m/e 483 (M+, 68%).
Elemental sulfur (0.32 g, 0.01 mol) and malononitrile (0.66 g,
0.01 mol) were added to any of 8a (4.55 g, 0.01 mol), 8b
(4.19 g, 0.01 mol), 8c (3.85 g, 0.01 mol), 8d (3.89 g, 0.01 mol),
8e (3.56 g, 0.01 mol), 8f (4.20 g, 0.01 mol), 8g (3.86 g,
0.01 mol) or 8h (3.90 g, 0.01 mol) in 1,4-dioxan (50 mL) con-
taining triethylamine (1.0 mL). The reaction mixture, in each
case, was heated under reflux for 2 hours then poured onto
ice/water containing a few drops of hydrochloric acid, and
the formed solid product was collected by filtration.
3,5-Diamino-N-(5-amino-4-(1,3-diphenylallylidene)-4H-
pyrazol-3-yl)-4- cyano-thiophene-2-carboxamide (9a)
Pale brown crystals from ethanol, yield (2.71 g, 60%), mp:
158ꢀC to 160ꢀC. IR (KBr) υmax cm−1: 3483-3329 (NH2,
NH), 3055 (CH, aromatic), 2220 (CN), 1690 (CO), 1648
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 4.46, 4.56, 5.21 (3s, 6H, D2O exchangeable, 3NH2),
6.32, 6.48 (2d, 2H, J = 10.07 Hz, CH═CH), 7.22-7.36 (m,
10H, 2C6H5), 8.29 (s, 1H, D2O exchangeable, NH); 13C
NMR (DMSO-d6, 75 MHz): δ = 89.1, 90.8 (CH═CH),
117.0 (CN), 120.8, 121.0, 121.6, 122.8, 124.3, 126.8, 127.5,
128.2, 130.5, 132.4, 135.9, 137.0 (2C6H5, thiophene C),
131.1, 134.7 (C═C),167.4 (CO),172.1, 176.6 (2C═N). Anal.
calculated for C24H19N7OS: C, 63.56; H, 4.22; N, 21.62; S,
7.07. Found: C, 63.70; H, 4.39; N, 21.80; S.7.28. MS: m/e
453 (M+, 26%).
3,5-Diamino-N-(5-amino-4-(1-(4-chlorophenyl)-
3-phenylallylidene)-4H-pyrazol-3-yl)-4-cyanothiophene-
2-carboxamide (9d)
Brown crystals from 1,4-dioxan, yield (3.01 g, 62%), mp: 166ꢀC
to 169ꢀC. IR (KBr) υmax cm−1: 3482-3337 (NH2, NH), 3055
(CH, aromatic), 2222 (CN), 1688 (CO), 1650 (C═N), 1631
1
(C═C); H NMR (DMSO-d6, 200 MHz): δ = 4.39, 4.58, 5.28
(3s, 6H, D2O exchangeable, 3NH2), 6.30, 6.48 (2d, 2H,
J = 10.13 Hz, CH═CH), 7.22-7.53 (m, 9H, C6H5, C6H4), 8.26
(s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6,
75 MHz): δ = 89.5, 90.4 (CH═CH), 116.7 (CN), 120.3, 121.8,
122.6, 125.5, 126.3, 127.1, 128.4, 129.0, 130.6, 132.7, 134.8, 138.2
(C6H5, C6H4, thiophene C), 131.3, 134.7 (C═C), 166.9 (CO),
172.3, 176.6 (2C═N). Anal. calculated for C24H18ClN7OS: C,
59.07; H, 3.72; N, 20.09; S, 6.57%. Found: C, 58.84; H, 3.69; N,
19.82; S, 6.39. MS: m/e 487 (M+, 26%).
3,5-Diamino-N-(5-amino-4-(1-(4-chlorophenyl)-
3-(4-methoxyphenyl)-allylidene)-4H-pyrazol-3-yl)-
4-cyanothiophene-2-carboxamide (9b)
3,5-Diamino-4-cyano-N-(4-(1,3-diphenylallylidene)-
5-hydroxy-4H-pyrazol-3-yl)thiophene-
2-carboxamide (9e)
Yellow crystals from 1,4-dioxan, yield (3.87 g, 75%), mp:
155ꢀC to 158ꢀC. IR (KBr) υmax cm−1: 3449-3326 (NH2, NH),
3055 (CH, aromatic), 2220 (CN), 1683 (CO), 1650 (C═N),
Yellow crystals from 1,4-dioxan, yield (3.49 g, 77%), mp:
133ꢀC to 136ꢀC. IR (KBr) υmax cm−1: 3548-3329 (OH, NH2,
NH), 3053 (CH, aromatic), 2220 (CN), 1685 (CO), 1650
(C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 4.38, 4.58 (2s, 4H, D2O exchangeable, 2NH2), 6.31, 6.48
(2d, 2H, J = 10.12 Hz, CH═CH), 7.29-7.39 (m, 10H, 2C6H5),
8.42 (s, 1H, D2O exchangeable, NH), 10.26 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 89.1, 90.9 (CH═CH), 116.7 (CN), 120.5, 121.6, 122.6,
123.9, 124.5, 127.3, 128.1, 128.6, 130.4, 132.7, 134.5, 138.0
(2C6H5, thiophene C), 131.6, 134.8 (C═C),167.5 (CO), 172.3,
176.4 (2C═N). Anal. calculated for C24H18N6O2S: C,
63.42; H, 3.99; N, 18.49; S, 7.05. Found: C, 63.58; H, 4.05; N,
18.39; S, 6.88. MS: m/e 454 (M+, 30%).
1
1630 (C═C); H NMR (DMSO-d6, 200 MHz): δ = 3.68 (s,
3H, OCH3), 4.58, 4.71, 5.22 (3s, 6H, D2O exchangeable,
3NH2), 6.37, 6.38 (2d, 2H, J = 10.07 Hz, CH═CH), 7.21-7.52
(m, 8H, 2C6H4), 8.28 (s, 1H, D2O exchangeable, NH); 13C
NMR (DMSO-d6, 75 MHz): δ = 52.1 (OCH3), 89.2, 90.7
(CH═CH), 116.8 (CN), 120.4, 122.3, 122.6, 123.8, 124.4,
125.9, 126.3, 127.3, 130.3, 132.6, 135.9, 137.4 (2C6H4, thio-
phene C), 131.6, 134.5 (C═C), 167.7 (CO), 172.2, 176.8
(2C═N). Anal. calculated for C25H20ClN7O2S: C, 57.97; H,
3.89; N, 18.93; S, 6.19. Found: C, 58.27; H, 4.02; N, 19.18; S,
6.27. MS: m/e 517 (M+, 26%).
3,5-Diamino-N-(5-amino-4-(3-(4-methoxyphenyl)-
1-phenylallylidene)-4H-pyrazol-3-yl)-4-cyanothiophene-
2-carboxamide (9c)
3,5-Diamino-N-(4-(1-(4-chlorophenyl)-
3-(4-methoxyphenyl)allylidene)-5-hydroxy-4H-pyrazol-
3-yl)-4-cyanothiophene-2-carboxamide (9f)
Yellow crystals from 1,4-dioxan, yield (3.38 g, 70%), mp:
Yellow crystals from 1,4-dioxan, yield (3.57 g, 69%), mp:
185ꢀC to 188ꢀC, IR (KBr) υmax cm−1: 3478-3353 (NH2,
148ꢀC to 150ꢀC. IR (KBr) υmax cm−1: 3563-3352 (OH,

14
MEGALLY ABDO ET AL.
NH2, NH), 3055 (CH, aromatic), 2223 (CN), 1686 (CO),
1650 (C═N), 1630 (C═C); 1H NMR (DMSO-d6,
200 MHz): δ = 3.68 (s, 3H, OCH3), 4.58, 4.79 (2s, 4H,
D2O exchangeable, 2NH2), 6.34, 6.47 (2d, 2H,
J = 10.05 Hz, CH═CH), 7.25-7.46 (m, 8H, 2C6H4), 8.31
(s, 1H, D2O exchangeable, NH), 10.34 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 52.5 (OCH3) 89.2, 90.6 (CH═CH), 117.0 (CN), 120.6,
120.3, 121.2, 124.2, 125.8, 126.7, 128.6, 128.7, 130.2,
132.6, 133.9, 135.1 (2C6H4, thiophene C), 131.2, 134.7
(C═C), 167.8 (CO), 172.8, 176.6 (2C═N). Anal. calcu-
lated for C25H19ClN6O3S: C, 57.86; H, 3.69; N, 16.19; S,
6.18. Found: C, 57.62; H, 3.49; N, 16.05; S, 6.27. MS: m/e
518 (M+, 55%).
4.1.5 | General procedure for the
synthesis of the thiophene derivatives 10a-h
Elemental sulfur (0.32 g, 0.01 mol) and ethyl cyano-
acetate (1.07 g, 0.01 mol) were added to any of 8a
(4.55 g, 0.01 mol), 8b (4.19 g, 0.01 mol), 8c (3.85 g,
0.01 mol), 8d (3.89 g, 0.01 mol), 8e (3.56 g, 0.01 mol), 8f
(4.20 g, 0.01 mol), 8g (3.86 g, 0.01 mol) or 8h (3.90 g,
0.01 mol)
in
1,4-dioxan
(50 mL)
containing
triethylamine (1.0 mL). The reaction mixture, in each
case, was heated under reflux for 2 hours then poured
onto ice/water containing a few drops of hydrochloric
acid and the formed solid product was collected by
filtration.
3,5-Diamino-4-cyano-N-(5-hydroxy-
Ethyl 2,4-diamino-5-(5-amino-
4-(3-(4-methoxyphenyl)-1-phenylallylidene)-4H-pyrazol-
3-yl)thiophene-2-carboxamide (9g)
4-(1,3-diphenylallylidene)-4H-pyrazol-3-yl)carbamoyl)
thiophene-3-carboxylate (10a)
Yellow crystals from ethanol, yield (2.90 g, 60%), mp:
199ꢀC to 202ꢀC. IR (KBr) υmax cm−1: 3569-3335 (OH,
NH2, NH), 3054 (CH, aromatic), 2223 (CN), 1686 (CO),
1640 (C═N), 1630 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 3.69 (s, 3H, OCH3), 4.58, 4.68 (2s, 4H, D2O exchange-
able, 2NH2), 6.31, 6.46 (2d, 2H, J = 9.89 Hz, CH═CH),
7.23-7.49 (m, 9H, C6H5, C6H4), 8.23 (s, 1H, D2O
exchangeable, NH), 10.35 (s, 1H, D2O exchangeable,
OH); 13C NMR (DMSO-d6, 75 MHz): δ = 52.3 (OCH3),
89.5, 90.9 (CH═CH), 116.6 (CN), 119.2, 121.4, 122.8,
123.9, 125.3, 126.8, 127.3, 128.5, 131.8, 132.4, 133.8, 135.1
(C6H5, C6H4, thiophene C), 131.6, 134.8 (C═C), 166.8
(CO), 172.4, 176.2 (2C═N). Anal. calculated for
C25H20N6O3S: C, 61.97; H, 4.16; N, 17.34; S, 6.62.
Found: C, 62.08; H, 4.26; N, 17.59; S, 6.81. MS: m/e
484 (M+, 38%).
Pale brown crystals from ethanol, yield (3.60 g, 72%), mp:
223ꢀC to 226ꢀC. IR (KBr) υmax cm−1: 3469-3342 (NH2, NH),
3055 (CH, aromatic), 1718, 1681 (2CO),1643 (C═N), 1630
1
(C═C); H NMR (DMSO-d6, 200 MHz): δ = 1.13 (t, 3H,
J = 7.22 Hz, CH3), 4.23 (q, 2H, J = 7.22 Hz, CH2), 4.45,
4.51, 5.28 (3s, 6H, D2O exchangeable, 3NH2), 6.30, 6.46 (2d,
2H, J = 10.13 Hz, CH═CH), 7.26-7.39 (m, 10H, 2C6H5),
8.27 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6,
75 MHz): δ = 16.8 (OCH2CH3), 58.6 (OCH2CH3), 89.3, 90.6
(CH═CH), 120.6, 120.8, 121.3, 122.5, 124.8, 126.8, 127.2,
128.3, 130.8, 132.6, 133.9, 135.7 (2C6H5, thiophene C), 131.6,
134.8 (C═C), 167.9, 169.1 (2CO),172.9, 177.1 (2C═N). Anal.
Calculated for C26H24N6O3S: C, 62.38; H, 4.83; N, 16.79; S,
6.41. Found: C, 62.48; H, 4.65; N, 16.83; S, 6.29. MS: m/e
500 (M+, 42%).
Ethyl 2,4-diamino-5-(5-amino-4-(1-(4-chlorophenyl)-
3-(4-methoxyphenyl)-allylidene)-4H-pyrazol-3-yl)
carbamoyl)thiophene-3-carboxylate (10b)
3,5-Diamino-N-(4-(1-(4-chlorophenyl)-
3-phenylallylidene)-5-hydroxy-4H-pyrazol-3-yl)-
4-cyanothiophene-2-carboxamide (9h)
Yellow crystals from 1,4-dioxan, yield (4.62 g, 82%),
mp: 208ꢀC to 210ꢀC. IR (KBr) υmax cm−1: 3468-3351 (NH2,
NH), 3054 (CH, aromatic), 1720, 1684 (2CO), 1650 (C═N),
Orange crystals from 1,4-dioxan, yield (2.83 g, 58%), mp:
177ꢀC to 180ꢀC. IR (KBr) υmax cm−1: 3537-3379 (OH,
NH2, NH), 3055 (CH, aromatic), 2222 (CN), 1681 (CO),
1
1630 (C═C); H NMR (DMSO-d6, 200 MHz): δ = 1.12 (t,
1
1641 (C═N), 1628 (C═C); H NMR (DMSO-d6, 200 MHz):
3H, J = 7.09 Hz, CH3), 3.69 (s, 3H, OCH3), 4.22 (q, 2H,
J = 7.09 Hz, CH2), 4.54, 4.68, 5.20 (3s, 6H, D2O exchange-
able, 3NH2), 6.37, 6.38 (2d, 2H, J = 9.87 Hz, CH═CH),
7.21-7.52 (m, 8H, 2C6H4), 8.29 (s, 1H, D2O exchangeable,
NH); 13C NMR (DMSO-d6, 75 MHz): δ = 16.8 (OCH2CH3),
52.3 (OCH3), 58.4 (OCH2CH3), 89.0, 90.5 (CH═CH), 119.6,
122.8, 123.4, 124.2, 124.8, 126.3, 126.6, 127.7, 130.5, 134.2,
135.7, 137.8 (2C6H4, thiophene C), 131.4, 134.1
(C═C),167.4, 168.9 (2CO), 172.8, 176.4 (2C═N). Anal. cal-
culated for C27H25ClN6O4S: C, 57.39; H, 4.46; N, 14.87; S,
5.67. Found: C, 57.41; H, 4.65; N, 14.68; S, 5.83. MS: m/e
565 (M+, 42%).
δ = 4.55, 4.80 (2s, 4H, D2O exchangeable, 2NH2), 6.34,
6.48 (2d, 2H, J = 10.09 Hz, CH═CH), 7.22-7.47 (m, 9H,
C6H5, C6H4), 8.26 (s, 1H, D2O exchangeable, NH), 10.32
(s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6,
75 MHz): δ = 89.5, 90.6 (CH═CH), 117.0 (CN), 120.8,
122.6, 124.2, 124.6, 126.1, 125.3, 125.8, 128.7, 129.0, 130.8,
132.5, 134.2 (C6H5, C6H4, thiophene C), 131.1, 134.5
(C═C), 167.3 (CO), 172.4, 176.8 (2C═N). Anal. calculated
for C24H17ClN6O2S: C, 58.95; H, 3.50; N, 17.19; S, 6.56.
Found: C, 58.72; H, 3.78; N, 17.05; S, 6.73. MS: m/e
488 (M+, 18%).

MEGALLY ABDO ET AL.
15
Ethyl 2,4-diamino-5-(5-amino-4-(3-(4-methoxyphenyl)-
1-phenylallylidene)-4H-pyrazol-3-yl)carbamoyl)
thiophene-3-carboxylate (10c)
128.1, 130.6, 133.9, 134.1, 138.8 (2C6H5, thiophene C),
131.3, 134.1 (C═C), 168.1, 169.3 (2CO), 172.5, 176.2
(2C═N). Anal. calculated for C26H23N5O4S: C, 62.26; H,
4.62; N, 13.96; S, 6.39. Found: C, 62.48; H, 4.80; N,
14.27; S, 6.47. MS: m/e 501 (M+, 28%).
Yellow crystals from 1,4-dioxan, yield (3.49 g, 66%), mp:
233ꢀC to 235ꢀC, IR (KBr) υmax cm−1: 3459-3342 (NH2,
NH), 3054 (CH, aromatic), 1722, 1690 (2CO), 1644 (C═N),
1
1630 (C═C); H NMR (DMSO-d6, 200 MHz): δ = 1.13 (t,
Ethyl 2,4-diamino-5-(4-(1-(4-chlorophenyl)-
3H, J = 6.77 Hz, CH3), 3.69 (s, 3H, OCH3), 4.22 (q, 2H,
J = 6.77 Hz, CH2), 4.43, 4.78, 5.27 (3s, 6H, D2O exchange-
able, 3NH2), 6.31, 6.48 (2d, 2H, J = 9.96 Hz, CH═CH),
7.25-7.50 (m, 9H, C6H5, C6H4), 8.28 (s, 1H, D2O exchange-
able, NH); 13C NMR (DMSO-d6, 75 MHz): δ = 16.6
(OCH2CH3), 52.4 (OCH3), 58.1 (OCH2CH3), 89.3, 90.8
(CH═CH), 120.5, 122.9, 123.5, 124.9, 125.7, 126.8, 127.3,
128.4, 130.2, 132.3, 134.5, 135.8 (C6H5, C6H4, thiophene
C), 131.8, 135.3 (C═C), 167.7, 168.7 (2CO), 172.5, 176.6
(2C═N). Anal. calculated for C27H26N6O4S: C, 61.12; H,
4.94; N, 15.84; S, 6.04. Found: C, 61.36; H, 4.79; N,
15.69; S, 6.29. MS: m/e 530 (M+, 48%).
3-(4-methoxyphenyl)-allylidene)-5-hydroxy-4H-pyrazol-
3-yl)carbamoyl)thiophene-3-carboxylate (10f)
Yellow crystals from 1,4-dioxan, yield (4.13 g, 73%), mp:
240ꢀC to 244ꢀC. IR (KBr) υmax cm−1:3570-3362 (OH, NH2,
NH), 3055 (CH, aromatic), 1724, 1685 (2CO), 1649
(C═N), 1626 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 1.12 (t, 3H, J = 7.69 Hz, CH3), 3.66 (s, 3H, OCH3),
4.24 (q, 2H, J = 7.69 Hz, CH2), 4.56, 4.73 (s, 4H, D2O
exchangeable, 2NH2), 6.34, 6.49 (2d, 2H, J = 9.96 Hz,
CH═CH), 7.25-7.54 (m, 8H, 2C6H4), 8.29 (s, 1H, D2O
exchangeable, NH), 10.33 (s, 1H, D2O exchangeable,
OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 16.6
(OCH2CH3), 52.8 (OCH3), 58.5 (OCH2CH3), 89.2, 90.9
(CH═CH), 120.8, 121.0, 121.2, 123.6, 125.2, 126.9, 128.3,
128.4, 130.8, 132.3, 133.6, 135.5 (2C6H4, thiophene C),
131.5, 134.6 (C═C), 168.1, 169.3 (2CO), 172.3, 176.2
(2C═N). Anal. calculated for C27H24ClN5O5S: C,
57.29; H, 4.27; N, 12.37; S, 5.66. Found: C, 57.51; H,
4.08; N, 12.45; S, 5.80. MS: m/e 566 (M+, 36%).
Ethyl 2,4-diamino-5-(5-amino-4-(1-(4-chlorophenyl)-
3-phenylallylidene)-4H-pyrazol-3-yl)carbamoyl)
thiophene-3-carboxylate (10d)
Brown crystals from 1,4-dioxan, yield (3.21 g, 60%), mp:
244ꢀC to 247ꢀC. IR (KBr) υmax cm−1: 3479-3341 (NH2, NH),
3056 (CH, aromatic), 1723, 1687 (2CO), 1650 (C═N), 1631
1
(C═C); H NMR (DMSO-d6, 200 MHz): δ = 1.12 (t, 3H,
J = 6.52 Hz, CH3), 4.22 (q, 2H, J = 6.52 Hz, CH2), 4.34,
4.58, 5.25 (3s, 6H, D2O exchangeable, 3NH2), 6.32, 6.46 (2d,
2H, J = 9.91 Hz, CH═CH), 7.22-7.48 (m, 9H, C6H5, C6H4),
8.24 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6,
75 MHz): δ = 16.8 (OCH2CH3), 58.1 (OCH2CH3), 89.2, 90.6
(CH═CH), 120.6, 122.0, 122.9, 124.1, 125.9, 126.8, 127.1,
128.7, 130.9, 132.4, 134.2, 138.9 (C6H5, C6H4, thiophene C),
131.6, 134.5 (C═C), 167.5, 169.2 (2CO), 172.1, 176.8
(2C═N). Anal. calculated for C26H23ClN6O3S: C, 58.37; H,
4.33; N, 15.71; S, 5.99%. Found: C, 58.51; H, 4.63; N,
15.80; S, 6.24. MS: m/e 535 (M+, 34%).
Ethyl 2,4-diamino-5-(5-hydroxy-4-(3-(4-methoxyphenyl)-
1-phenylallylidene)-4H-pyrazol-3-yl)carbamoyl)
thiophene-3-carboxylate (10g)
Yellow crystals from ethanol, yield (3.71 g, 70%), mp:
155ꢀC to 158ꢀC. IR (KBr) υmax cm−1: 3541-3357 (OH, NH2,
NH), 3054 (CH, aromatic), 1718, 1692 (2CO), 1640 (C═N),
1
1632 (C═C); H NMR (DMSO-d6, 200 MHz): δ = 1.14 (t,
3H, J = 6.80 Hz, CH3), 3.68 (s, 3H, OCH3), 4.22 (q, 2H,
J = 6.80 Hz, CH2), 4.54, 4.66 (2s, 4H, D2O exchangeable,
2NH2), 6.35, 6.46 (2d, 2H, J = 10.06 Hz, CH═CH),
7.21-7.55 (m, 9H, C6H5, C6H4), 8.26 (s, 1H, D2O exchange-
able, NH), 10.33 (s, 1H, D2O exchangeable, OH); 13C NMR
(DMSO-d6, 75 MHz): δ = 16.6 (OCH2CH3), 52.5 (OCH3),
58.7 (OCH2CH3), 89.2, 90.7 (CH═CH), 120.8, 121.6, 122.3,
123.9, 125.8, 126.5, 127.3, 128.7, 131.9, 132.7, 133.2, 135.9
(C6H5, C6H4, thiophene C), 131.3, 134.6 (C═C), 167.9,
168.7 (2CO), 172.1, 176.5 (2C═N). Anal. calculated for
C27H25N5O5S: C, 61.00; H, 4.74; N, 13.17; S, 6.03.
Found: C, 60.84; H, 4.86; N, 13.49; S, 6.29. MS: m/e
531 (M+, 42%).
Ethyl 2,4-diamino-5-(4-(1,3-diphenylallylidene)-
5-hydroxy-4H-pyrazol-3-yl)carbamoyl)thiophene-
3-carboxylate (10e)
Yellow crystals from 1,4-dioxan, yield (3.40 g, 68%), mp:
158ꢀC to 161ꢀC. IR (KBr) υmax cm−1: 3559-3372 (OH, NH2,
NH), 3058 (CH, aromatic), 1718, 1691 (2CO),1650 (C═N),
1629 (C═C); 1H NMR (DMSO-d6, 200 MHz): δ = 1.12
(t, 3H, J = 7.11 Hz, CH3), 4.22 (q, 2H, J = 7.11 Hz, CH2),
4.38, 4.58 (2s, 4H, D2O exchangeable, 2NH2), 6.35, 6.48
(2d, 2H, J = 9.96 Hz, CH═CH), 7.27-7.42 (m, 10H, 2C6H5),
8.46 (s, 1H, D2O exchangeable, NH), 10.28 (s, 1H, D2O
exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz):
δ = 16.8 (OCH2CH3), 58.3 (OCH2CH3), 89.3, 90.9
(CH═CH), 120.1, 121.9, 122.6, 123.9, 125.9, 127.6, 127.8,
Ethyl 2,4-diamino-5-(4-(1-(4-chlorophenyl)-
3-phenylallylidene)-5-hydroxy-4H-pyrazol-3-yl)
carbamoyl)thiophene-3-carboxylate (10h)
Yellow crystals from 1,4-dioxan, yield (4.07 g, 76%), mp:
177ꢀC to 180ꢀC. IR (KBr) υmax cm−1: 3562-3359 (OH,

16
MEGALLY ABDO ET AL.
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NH2, NH), 3055 (CH, aromatic), 1720, 1689 (2CO), 1644
(C═N), 1624 (C═C); 1H NMR (DMSO-d6, 200 MHz):
δ = 1.12 (t, 3H, J = 6.38 Hz, CH3), 4.21 (q, 2H,
J = 6.38 Hz, CH2), 4.55, 4.83 (2s, 4H, D2O exchangeable,
2NH2), 6.32, 6.45 (2d, 2H, J = 10.09 Hz, CH═CH),
7.24-7.49 (m, 9H, C6H5, C6H4), 8.24 (s, 1H, D2O
exchangeable, NH), 10.30 (s, 1H, D2O exchangeable,
OH); 13C NMR (DMSO-d6, 75 MHz): δ = 16.8 (OCH2CH3),
58.5 (OCH2CH3), 89.1, 90.4 (CH═CH), 120.4, 121.3, 124.7,
126.9, 127.3, 125.0, 125.2, 128.1, 129.4, 131.6, 133.3, 134.8
(C6H5, C6H4, thiophene C), 131.4, 134.7 (C═C), 167.5,
168.6 (2CO), 172.2, 176.5 (2C═N). Anal. calculated for
C26H22ClN5O4S: C, 58.26; H, 4.14; N, 13.07; S, 5.98.
Found: C, 58.47; H, 4.26; N, 12.98; S, 6.15. MS: m/e
536 (M+, 26%).
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ORCID
Nadia Y. Megally Abdo https://orcid.org/0000-0003-
0130-5060
Rafat M. Mohareb https://orcid.org/0000-0003-3922-
803X
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How to cite this article: Megally Abdo NY,
Samir EM, Mohareb RM. Synthesis and evaluation
of novel 4H-pyrazole and thiophene derivatives
derived from chalcone as potential anti-
proliferative agents, Pim-1 kinase inhibitors, and
PAINS. J Heterocyclic Chem. 2020;1–17. https://doi.
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