Uses of dimedone to synthesis pyrazole, isoxazole and thiophene derivatives with antiproliferative, tyrosine kinase and Pim-1 kinase inhibitions

Article abstract of DOI:10.1007/s00044-020-02579-4

We are aiming in this work to synthesize target molecules not only possess antitumor activities but also kinase inhibitors. The target molecules were obtained from dimedone, which reacted with triethoxymethane to produce a product that is capable for many heterocyclization reactions to give fused pyrazole, thiophene and isoxazole derivatives. Compounds 7b, 7c, 7d, 9b, 11, 12c, 12d, 14b, 16b, 17c, 17d, 18c, 18d, and 18e were the most cytotoxic compounds, their further tests toward the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR and Pim-1 kinase showed that compounds 7b, 7d, 11, 12c, 14b, 16b, 17d, 18d, and 18e were the most potent of the tested compounds toward the five tyrosine kinases and compounds 7b, 7d, 14b, 16b, and 18e were of the highest inhibitions toward Pim-1 kinase. PAINS the most cytotoxic compounds showed zero PAINS alert, therefore, these compounds can be used as useful drugs in the future.

Full text of DOI:10.1007/s00044-020-02579-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02579-4
ORIGINAL RESEARCH
Uses of dimedone to synthesis pyrazole, isoxazole and thiophene
derivatives with antiproliferative, tyrosine kinase and Pim-1 kinase
inhibitions
1
Fatma Mohamed Manhi2 Mahmoud Ali Abdelaziz Mahmoud3 Amal Abdelwahab2
●
●
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Rafat Milad Mohareb
Received: 13 February 2020 / Accepted: 30 May 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
We are aiming in this work to synthesize target molecules not only possess antitumor activities but also kinase inhibitors.
The target molecules were obtained from dimedone, which reacted with triethoxymethane to produce a product that is
capable for many heterocyclization reactions to give fused pyrazole, thiophene and isoxazole derivatives. Compounds 7b,
7c, 7d, 9b, 11, 12c, 12d, 14b, 16b, 17c, 17d, 18c, 18d, and 18e were the most cytotoxic compounds, their further tests
toward the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR and Pim-1 kinase showed that compounds 7b,
7d, 11, 12c, 14b, 16b, 17d, 18d, and 18e were the most potent of the tested compounds toward the five tyrosine kinases and
compounds 7b, 7d, 14b, 16b, and 18e were of the highest inhibitions toward Pim-1 kinase. PAINS the most cytotoxic
compounds showed zero PAINS alert, therefore, these compounds can be used as useful drugs in the future.
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●
●
●
Keywords Pyrazole Isoxazole Thiophene Cytotoxicity Tyrosine kinase
Introduction
Chun-Bao 2006; James et al. 2006; Majumdar and Samanta
2001, 2002) and ready availability of cyclohexane-1,3-
diones (Ilkhanizadeh et al. 2019; Bonsignore et al. 1993;
Deng et al. 1999) and its derivatives make them suitable
precursors for the preparation of divergent organic com-
pounds, for example chromene derivatives, which possess
anticancer, antioxidant, spasmolytic, antianaphylactic, anti-
HIV and antibacterial activities (Ilkhanizadeh et al. 2019;
Sonderegger et al. 2003), oxozolidinones with antibacterial
activity (Cui et al. 2005; Jae-Min et al. 1997; Jo et al. 2004;
Pae et al. 1999) substituted xanthene derivatives with sev-
eral uses in dyes (Banerjee and Mukherjee 1981; Menchen
et al. 2003), laser technology (Ahmad et al. 2002), fluor-
escent compounds (Knight and Stephens 1989), and more
importantly, have been reported to show a variety of bio-
logical activity (Hideo and Teruomi 1981; Poupelin et al.
1978; Lambert et al. 1997). In addition, acridine and its
derivatives display antimalarial, anticancer, antibacterial,
and mutagenic properties, while phenylbutazones exhibit
unique pharmacological uses for pain treatment associated
with Tietze’s syndrome and rheumatoid arthritis (Dewse
and Potter 1975). On the other hand, with the increasing
environmental concerns, green chemistry has attracted
major scientific and commercial interest in recent years.
Multicomponent one-pot reactions are an efficient and
Cyclohexane-1,3-dione, commonly known as dimedone,
belongs to the family of cyclic 1,3-diketones class of
organic compounds. A wide range of applications of
cyclohexan-1,3-dione includes its use as reagent for various
analytical techniques (Bodini et al. 1990; Benitez and
Allison 1974). In addition, they are used as a versatile
synthon for of several spiro and heterocyclic compounds
(Ashry et al. 2009), such as xanthene derivatives, which
have emerged as an important class of compound because
of their industrial importance (Saitoh et al. 2002) and other
synthetic applications (Cremlyn and Saunders 1993). The
versatile chemistry (Greenhill et al. 1977; Guan-Wu and
* Rafat Milad Mohareb
raafat_mohareb@yahoo.com
1
Department of Chemistry, Faculty of Science, Cairo University,
Giza, Arab Republic of Egypt
2
National Organization for Drug Control & Research,
P.O. 29, Cairo, Arab Republic of Egypt
3
Department of Chemistry, Faculty of Science, University of
Tabuk, P.O. Box 741, Tabuk 71491, Kingdom of Saudi Arabia

Medicinal Chemistry Research
economical procedure, with wide uses in the preparation of
heterocycles molecules (Ramazani and Rezaei 2010;
Ramazani et al. 2011, 2012). On the other hand, organic
transformation takes place in aqueous media gives a clean,
environmentally safe, and cost-effective approach.
Recently, Barakat et al. reported examples for MCR (one-
pot fission) for example reaction of substituted cyclohex-
anedione with alkanal mediated by diethylamine in water
(Al-Majid et al. 2013, 2017; Barakat et al. 2013, 2014a,
2014b; Al-Najjar et al. 2014).
Recently our research group reported several reactions of
dimedone to produce thaizoles and hydrazone derivatives.
The produced compounds showed high antiproliferative
activities against cancer cell lines together with high inhi-
bitions toward tyrosine kinases (Mohareb et al. 2019a,
2019b, 2018). This encouraged us to continue this goal
through the reaction of dimedone with triethoxymethane
and using the produced molecule as a suitable starting
material for subsequent heterocyclization to produce a
variety of fused derivatives. The antiproliferative activities
of the synthesized compounds and their inhibitiory activity
toward tyrosine kinases were demonstrated.
Scheme 1 Synthesis of compounds 5a, b; 7a–d, and 9a, b
Results and discussion
hydrochloride (8) in 1,4-dioxane containing sodium acetate
gave the isoxazole derivative 11.
The synthetic route to prepare a new class of biologically
active heterocycles compounds corporated dimetone moiety
starting from 2-ethoxymythyleno was discussed. The syn-
thetic pathways employed to prepare the new target deri-
vatives are depicted in Schemes 1–4. Thus, the reaction of
dimedone (1) with triethoxymethane in an oil bath at 120 °C
gave the 2-(ethoxymethylene)-5,5-dimethylcyclohexane-
1,3-dione (3). Compound 3 reacted with either mal-
ononitrile (4a) or ethyl cyanoacetate (4b) to afford the
alkylated products 5a and 5b, respectively. The structures
of compounds 5a, b were based on their respective analy-
tical and spectral data (see “Experimental” section). Com-
pounds 5a, b were used to synthesize pyrazole derivatives
through the reaction of either 5a or 5b with either hydrazine
hydrate (6a) or phenylhydrazine (6b) to give the pyrazole
derivatives 7a–d, respectively. On the other hand,
the reaction of either compound 5a or 5b with hydro-
xylamine hydrochloride (8) in 1,4-dioxane containing
sodium acetate gave the isoxazole derivatives 9a and 9b,
respectively (Scheme 1).
The reaction of either 10a or 10b with elemental sulfur
and either malononitrile (4a) or ethyl cyanoacetate (4b) in
1,4-dioxane solution containing triethylamine gave the
thieno[3,2-e]indazole derivatives 12a–d, respectively. The
chemical structures of the latter products were assured by
1
1
spectral data (IR, H, 13C NMR, MS). Thus, the H NMR
spectrum of compound 12a (as an example) showed the
presence of one NH2 group at
δ 4.83 ppm (D2O
exchangeable), presence of one NH group at δ 8.29 (D2O
exchangeable) and a singlet at δ 2.36 ppm indicating one
CH2 group. In addition, the 13C NMR spectrum revealed,
beside the expected signals, a signal at δ 118.2 due to
presence CN group, two signals at δ 136.9, 132.6 for pyr-
azole C-4, C-5 and four signals at δ 138.4, 135.2, 132.8,
130.2 for thiophene carbons. Similarly, the reaction of the
isoxazle derivative 11 with elemental sulfur and either
malononitrile (4a) or ethyl cyanoacetate (4b) in 1,4-dioxane
solution containing triethylamine gave the dihydrothieno
[3′,2′:3,4]benzo[1,2-c]isoxazole derivatives 13a and 13b,
respectively (Scheme 2).
The reaction of compound 3 with elemental sulfur and
either malononitrile (4a) or ethyl cyanoacetate (4b) in 1,4-
dioxane solution containing triethylamine gave the 6,7-
dihydrobenzo[b]thiophene derivatives 14a and 14b,
respectively. On the other hand, compound 3 reacted
with either aniline (15a), 4-chloroaniline (15b), or
The high yields of compounds 7a–d and 9a, b encour-
aged us to continue such series of reactions but using 2-
(ethoxymethylene)-5,5-dimethylcyclohexane-1,3-dione (3).
Thus, the reaction of compound 3 with either hydrazine
hydrate (6a) or phenylhydrazine (6b) gave the 1H-indazole
derivatives 10a and 10b, respectively. On the other hand,
the reaction of compound
3
with hydroxylamine

Medicinal Chemistry Research
Scheme 3 Synthesis of compounds 14a, b; 16a–c, and 17a–f
Scheme 2 Synthesis of compounds 10a, b; 11; 12a–d, and 13a, b
4-methylaniline (15c) to produce the arylaminomethylene
cyclohexane derivatives 16a–c, respectively. With the
optimized reaction conditions for thiophene formation in
hand the scope of this reaction was examined. This protocol
was successful for one pot formation of benzo [b] thiophene
derivatives. Therefore, the reaction of either 16a, 16b, or
16c with elemental sulfur and either malononitrile or ethyl
cyanoacetate in 1,4-dioxane solution containing triethyla-
mine gave the arylaminomethylene-6,7-dihydrobenzo[b]
thiophene derivatives 17a–f, respectively (Scheme 3). The
analytical and spectral data of the latter product were in
heterocyclic compounds derived from dimedone and their
evaluation through cancer cell lines gave results that can
encourage continuing work in the future as these com-
pounds can be used as synthons for the production of
anticancer agents.
Biology section
Cell proliferation assay
1
agreement with their respective structures. Thus, the H
The antiproliferative activities of the newly synthesized
compounds were evaluated against the five c-Met-
dependent cancer cell lines namely A549 (nonsmall cell
lung cancer), HT29 (human colon cancer), MKN-45
(human gastric cancer), U87MG (human glioblastoma),
SMMC-7721 (human liver cancer), and one c-Met-
independent cancer cell line H460 (human lung cancer)
using the standard MTT assay in vitro with foretinib as the
positive control. The results expressed as IC50 were sum-
marized in Table 1 and the values are the average of at least
three independent experiments. The MTT Cell Proliferation
Assay measures the cell proliferation rate and conversely,
when metabolic events lead to apoptosis or necrosis, the
reduction in cell viability. The number of assay steps has
been minimized as much as possible to expedite sample
processing. The MTT reagent yields low background
absorbance values in the absence of cells. For each cell type
the linear relationship between cell number and signal
NMR spectrum of compound 17a (as an example) showed,
beside the expected signals, the presence of one NH2 group
at δ 4.88 ppm (D2O exchangeable), the presence of one NH
group at δ 8.40 (D2O exchangeable), a singlet at δ 2.35 ppm
indicating one CH2 group In addition, 13C NMR showed
signals at δ 138.1, 136.7, 132.4, 130.8 due to the thiophene
carbons and a signal at δ 116.5 indicating the presence of
CN group.
Continuing heterocyclizations of compounds were
adopted through the reaction of either 16a, 16b, or 16c with
either hydrazine hydrate or phenylhydrazine in 1,4-dioxane,
which produced the indazole 18a–f, respectively. On the
other hand, the reaction of either 16a, 16b, or 16c with
hydroxylamine hydrochloride (8) in 1,4-dioxane containing
sodium acetate gave the corresponding benzo[c]isoxazole
derivatives 19a–c, respectively (Scheme 4). Basically,
in this work we synthesized
a
series of fused

Medicinal Chemistry Research
Scheme 4 Synthesis of
compounds 18a–f and 19a–c
produced is established, thus allowing an accurate quanti-
fication of changes in the rate of cell proliferation. The MTT
reagent is ready to use and stable at 4 °C in the dark for up
to 18 months, provided there is no contamination. Care
should be taken not to contaminate the MTT Reagent with
cell culture medium during pipetting. We recommend that
the appropriate volume required for each experiment be
removed and aseptically placed into a separate clean tube
and the stock bottle returned to 4 °C in the dark. Plate cells
at 1000–100,000 per well, incubate for 6–24 h to which add
10 μL MTT reagent incubate for 2–4 h until purple pre-
cipitate is visible then add 100 μL detergent reagent. Finally
leave at room temperature in the dark for 2 h and record
absorbance at 570 nm. The mean values of three indepen-
dent experiments, expressed as IC50 values, were presented
in Table 1. HTRF assay utilizes the signal generated by the
fluorescence resonance energy transfer between donor and
acceptor molecules in close proximity. Dual-wavelength
detection helps to eliminate media interference, and the final
signal is proportional to the extent of product formation.
Thus far, the reported applications of this technology for
in vitro kinase assays have mainly focused on high-
throughput screening (HTS). The MTT assay is a colori-
metric assay for assessing cell metabolic activity. NAD(P)
H-dependent cellular oxidoreductase enzymes may, under
defined conditions, reflect the number of viable cells pre-
sent. These enzymes are capable of reducing the tetrazolium
dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide to its insoluble formazan, which has a
purple color. Other closely related tetrazolium dyes
including XTT, MTS and the WSTs, are used in conjunc-
tion with the intermediate electron acceptor, 1-methoxy
phenazine methosulfate. With WST-1, which is cell-
impermeable, reduction occurs outside the cell via plasma
membrane electron transport. However, this traditionally
assumed explanation is currently contended as proof has
also been found of MTT reduction to formazan in lipidic
cellular structures without apparent involvement of oxi-
doreductases. Tetrazolium dye assays can also be used to
measure cytotoxicity (loss of viable cells) or cytostatic
activity (shift from proliferation to quiescence) of potential
medicinal agents and toxic materials. MTT assays are done
in the dark since the MTT reagent is sensitive to light.
Within this protocol, replace serum-containing media with
serum-free media and MTT reagent in cell cultures incubate
for 3 h at 37 °C add MTT solvent and incubate for 15 min
analyze with microplate reader.
All the synthesized compounds were tested to know their
inhibitory activities against the six cancer cell lines. From
the data listed in Table 1 it was noticed that about 30% of
the synthesized compounds showed high cytotoxicities
against the six cancer cell lines. Compounds were 7b, 7d,
9b, 11, 12c, 12d, 14b, 16b, 17c, 17d, 18c, 18d, and 18e
were the most cytotoxic compounds among the tested
compounds. On the other hand, compounds 5b, 7c, 9a, 12a,
13b, 14a, 16c, and 17e showed moderate cytotoxicity
against the six cancer cell lines.
Structure activity relationship
From Table 1 it is clear that many compounds of the newly
synthesized products showed high cytotoxicity against the
six cancer cell lines. The ethoxymethylene derivative 3
showed low inhibitions toward the six cancer cell lines. On
the other hand, the alkylated products 5a (R=CN) and 5b
(R=COOEt) revealed that compound 5b was of moderate
cytotoxicities against the six cancer cell lines but it is higher
than that of compound 5a. Considering the pyrazole deri-
vatives 7a–d, it is clear that 7a (Y=NH2 and R=H) which is
of low inhibitions and compounds 7b (Y=OH, R=H) and

Medicinal Chemistry Research
Table 1 In vitro growth
inhibitory effects IC50 SEM
(μM) of the newly synthesized
compounds against cancer
cell lines
Compound no IC50 SEM (μM)
A549 H460
HT29
MKN-45
U87MG
SMMC-77217721
3
6.89 1.70 8.63 2.42 8.38 2.08
4.23 1.25 5.64 1.83 6.31 2.51
2.19 0.86 2.82 1.06 3.14 1.07
6.35 2.40 5.69 1.39 7.28 2.61
0.38 0.21 0.29 0.08 0.36 0.20
1.62 0.93 1.47 0.83 0.96 0.29
0.25 0.16 0.25 0.08 0.42 0.19
1.14 0.32 1.27 0.68 1.38 0.83
0.56 0.28 0.61 0.28 0.72 0.25
3.42 1.39 2.80 1.04 3.39 1.82
5.28 2.31 6.36 2.34 5.48 1.39
0.25 0.06 0.29 0.13 0.43 0.15
1.43 0.69 1.82 0.96 1.95 0.43
4.43 1.31 4.71 1.25 5.69 2.38
0.43 0.18 0.36 0.18 0.35 0.29
0.63 0.31 0.72 0.35 0.43 0.25
4.80 1.46 5.86 1.39 4.69 1.08
1.28 0.72 1.63 0.96 0.79 0.19
1.62 0.84 0.83 0.39 1.64 0.81
0.91 0.52 0.41 0.28 0.52 0.13
4.51 1.62 5.29 1.28 6.48 2.25
0.34 0.19 0.63 0.35 0.64 0.65
1.40 0.28 2.26 1.06 2.28 1.01
8.52 2.68 7.39 2.48 8.30 3.62
6.29 2.59 5.49 1.73 6.92 1.68
0.42 0.20 0.37 0.18 0.43 0.16
0.32 0.26 0.63 0.18 0.35 0.16
3.28 1.19 2.73 1.09 4.61 1.06
6.38 2.52 6.59 1.36 8.64 2.60
4.62 1.54 3.26 1.03 4.52 1.41
5.34 1.38 2.56 1.27 4.38 1.42
1.03 0.84 0.36 0.24 0.64 0.39
0.28 0.13 0.36 0.13 0.29 0.11
0.32 0.18 0.35 0.08 0.42 0.24
4.29 2.59 3.39 1.39 5.42 2.38
6.26 2.68 4.53 2.15 5.24 2.31
9.52 2.51 6.32 2.82 5.18 1.49
4.28 1.23 5.68 1.72 6.69 2.17
6.49 1.29
6.52 2.46
2.83 1.02
6.93 2.72
0.38 0.16
0.93 0.41
0.47 0.28
0.96 0.36
0.36 0.13
2.31 1.26
6.27 2.42
0.37 0.15
2.49 1.84
4.93 1.39
0.29 0.18
0.62 0.19
5.36 1.93
0.83 0.41
1.69 0.80
1.30 0.86
8.43 2.25
0.41 0.63
1.46 0.85
9.30 2.69
8.71 2.59
0.29 0.14
0.49 0.25
1.36 0.84
8.36 1.28
5.36 1.64
3.46 1.26
0.56 0.37
0.36 0.15
0.43 0.24
7.32 2.42
4.22 2.08
7.64 2.46
1.38 0.49
5.41 1.38 6.80 1.59
6.19 2.38 5.19 1.29
3.63 1.27 1.59 0.83
6.35 1.80 5.39 1.68
0.42 0.18 0.36 0.231
1.83 0.79 0.74 0.36
0.39 0.26 0.73 0.29
1.42 0.83 1.61 0.73
0.53 0.17 0.49 0.25
2.94 1.25 2.77 1.26
5.29 1.63 4.09 2.17
0.26 0.12 0.52 0.12
1.24 0.96 1.73 0.49
4.80 2.27 2.93 1.39
0.52 0.17 0.43 0.29
0.41 0.26 0.83 0.15
6.29 2.73 8.06 2.53
1.63 0.69 0.92 0.63
0.83 0.42 1.80 0.93
1.02 0.85 0.63 0.36
6.38 2.42 6.59 1.73
0.53 0.26 0.39 0.24
1.25 0.68 0.92 0.53
8.53 2.80 7.36 2.98
6.83 2.15 8.76 2.41
0.58 0.19 0.36 0.12
0.46 0.22 0.29 0.15
2.45 0.69 3.26 1.30
7.83 1.69 8.42 2.65
4.26 1.21 5.26 1.51
5.28 1.26 4.48 1.62
0.64 0.15 0.48 0.25
0.31 0.29 0.24 0.13
0.28 0.08 0.36 0.28
4.74 2.28 1.70 0.73
2.35 2.31 4.28 1.29
5.83 1.58 8.72 2.60
4.22 1.53 6.42 2.18
5a
5b
7a
7b
7c
7d
9a
9b
10a
10b
11
12a
12b
12c
12d
13a
13b
14a
14b
16a
16b
16c
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
19a
19b
19c
Foretinib
0.08 0.01 0.18 0.03 0.15 0.023 0.03 0.005 0.90 0.13 0.44 0.062
7d (Y=OH, R=Ph) are of high cytotoxicities while com-
pound 7c (Y=NH2, R=Ph) showed moderate inhibitions.
Considering the isoxazole derivatives 9a, b where com-
pound 9b (Y=OH) was of higher cytotoxicities than com-
pound 9a (Y=NH2). On the other side, compound 9a gave
high inhibitions toward MKN-45 cell line with IC50
0.92 μM. Considering the fused pyrazole derivatives 10a
and 10b, it was obvious from Table 1 that both of them
showed low inhibitions. On the other hand, the fused iso-
xazole derivative 11 showed high inhibitions toward the six
cancer cell lines. On the other side, investigations of the
thieno[3,2-e]indazole derivatives 12a–d, surprisingly,
compound 12a (R=CN, R′=H) showed slight decline in the
activity observed and compound 12b (R=COOEt, R′=H)

Medicinal Chemistry Research
Table 2 Inhibition of tyrosine kinases (Enzyme IC50 (μM)) and WI38
by compounds 7b, 7c, 7d, 9b, 11, 12c, 12d, 14b, 16b, 17c, 17d, 18c,
18d, and 18e
exhibited low inhibitions. Interestingly, compounds 12c
(R=CN, R′=Ph) and 12d (R=COOEt, R′=Ph) was found
to be the most potent analog of this series toward the six
cancer cell lines. Considering the dihydrothieno[3′,2′:3,4]
benzo[1,2-c]isoxazole derivatives 13a and 13b, it is clear
from Table 1 that compound 13a (R=CN) showed lesser
activity than compound 13b (R=COOEt). Surprisingly, the
6,7-dihydrobenzo[b]thiophene derivatives 14a, b showed
from moderate to high inhibitions. On the other hand, for
the arylaminomethylene cyclohexane derivatives 16a–c
compound 16b (X=Cl) exhibited the highest cytotoxicities
among the three compounds. The reaction of compounds
16a–c with elemental sulfur and either malononitrile or
ethyl cyanoacetate to form the 4-(arylaminomethylene)-
benzo[b]thiophene derivatives 17a–f where compounds 17c
(R=CN, X=Cl) and 17d (R=COOEt, X=Cl) exhibited the
highest cytotoxicities among the six compounds. For the N-
aryl-tetrahydro-indazol-3-amine derivatives 18a–f, it is
clear from Table 1 that compounds 18c (R=H, X=Cl), 18d
(R=Ph, X=Cl), and 18e (R=H, X=Me) are of the highest
cytotoxicities among the six compounds. Surprisingly, all
compounds of the 6,7-dihydrobenzo[c]isoxazole derivatives
19a–c exhibited noticeable decline of inhibitions toward the
six cancer cell lines. It is of great value to mention that some
compounds showed inhibitions higher than that of foretinib
(IC50 0.90 μM) against the U87MG cell line compounds 7b,
9b, 11, 12c, 12d, 14a, 17c, 17d, 18c, 18d, and 18e showed
higher inhibition than foretinib with IC50’s 0.42, 0.39, 0.53,
0.26, 0.52, 0.53, 0.58, 0.46, 0.64, 0.31, and 0.28 μM. On the
other hand, compounds 7b, 12c, 16b, 17d, 18d, and 18e
with IC50’s 0.36, 0.43, 0.39, 0.36, 0.29, and 0.24 μM,
respectively, showed higher inhibitions than foretinib
againat (IC50 0.44 μM) the SMMC-7721 cell line.
Compound c-Kit Flt-3 VEGFR-2 EGFR PDGFR WI38
7b
0.28
1.30
0.26
1.61
0.62
0.82
1.08
0.36
0.46
1.03
0.16
1.09
0.26
1.42
0.32 0.28
1.24 0.53
0.42 0.30
1.25 2.25
0.42 0.24
0.43 0.63
1.37 2.73
0.52 0.41
0.26 0.29
0.24 0.53
0.28 0.17
2.85 1.41
0.45 0.19
0.39 0.59
0.61
0.62
0.62
1.08
0.38
0.29
2.61
0.68
0.37
1.28
0.29
1.38
0.35
1.60
0.43
0.71
0.40
0.93
0.29
0.43
1.59
0.39
0.32
1.22
0.34
2.77
0.28
0.52
28.21
36.27
44.02
62.49
28.17
34.58
42.63
58.70
39.05
23.41
2.80
7c
7d
9b
11
12c
12d
14b
16b
17c
17d
18c
18d
18e
33.72
42.83
38.52
expressed in most human cancers such as breast, kidney,
and colon where patients with tumors showing elevated
VEGF expression have a poor prognosis (Ferrara 2002). It
clear from Table 2 that compounds 7b, 7d, 11, 12c, 14b,
16b, 17d, 18d, and 18e were the most potent of the tested
compounds toward the five tyrosin kinases. Compound 9b
showed high potency toward PDGFR kinase with IC50’s
0.93 μM. In addition compound 18e showed high inhibi-
tions toward Flt-3, VEGFR-2, and PDGFR tyrosine kinases
with IC50’s 0.39, 0.59, and 0.52 μM. It is clear from Table 2
that all the tested compounds have low toxicity against the
WI38, normal fibroblast cells except compound 17d has
moderate cytoxicity.
The above activity pattern showed that that chloro
group was playing a crucial role in the activity. In sum-
mary, compounds 7b, 7c, 7d, 9b, 11, 12c, 12d, 14b, 16b,
17c, 17d, 18c, 18d, and 18e were the most cytotoxic
compounds.
Inhibition of selected compounds toward Pim-1
kinase
Furthermore, compounds 7b, 7d, 11, 12c, 14b, 16b, 17d,
18d, and 18e were selected to examine their Pim-1 kinase
inhibition activity (Table 3) as these compounds showed
high inhibition toward the tested cancer cell lines at a range
of ten concentrations and the IC50 values were calculated.
Compounds 7b, 7d, 14b, 16b, and 18e were the most potent
to inhibit Pim-1 kinase with IC50 value of 0.26, 0.23, 0.21,
0.28, and 0.28 μM, respectively. On the other hand, com-
pounds 11, 12c, 17d, and 18d were of decline potencies
(IC50 > 10 μM). Through this assay, SGI-1776 was used as
positive control with IC50 0.048 μM. These profiles in
combination with cell growth inhibition data of compounds
7b, 7d, 11, 12c, 14b, 16b, 17d, 18d, and 18e were listed in
Table 3 indicated that Pim-1 was a potential target of these
compounds.
Inhibition of tyrosine kinases (Enzyme IC50 (nM))
and normal cell line WI38
Compounds 7b, 7c, 7d, 9b, 11, 12c, 12d, 14b, 16b, 17c,
17d, 18c, 18d, and 18e were selected for inhibition of the
five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and
PDGFR and evaluation toward WI38, the normal fibroblast
cells due to their high inhibitions toward the six cancer cell
lines. These receptor tyrosine kinases have been implicated
in vascular development by affecting the proliferation and
migration of endothelial cells or pericytes. Among them,
VEGF is a major regulator of tumor angiogenesis via
endothelial cell proliferation and blood vessel permeability
(DiSalvo et al. 1995; Senger et al. 1983). VEGF is

Medicinal Chemistry Research
Table 3 The inhibitor activity of compounds 7b, 7d, 11, 12c, 14b,
16b, 17d, 18d, and 18e toward Pim-1 kinase
Table 4 PAINS evaluation of selected compounds using standard
drugs foretinib and SGI-1776
Compound
Inhibition ratio At 10 μM
IC50 (μM)
Compound Drug likeness rule
Medicinal chemistry rules
Lvio.a/ Vvio.b/ Gvio.c/ Lead
PAINS
alertd
7b
90
92
24
20
94
88
26
19
88
–
0.26
0.23
>10
No. of No. of
No. of
likeliness/
No. of vio
7d
vioa
viob
vioc
11
7b
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
1
0
0
1
0
1
0
0
1
0
0
0
1
0
1
0
0
12c
>10
7c
14b
16b
17d
18d
18e
0.21
0.28
>10
7d
9b
11
3
>10
12c
12d
14b
16b
17c
17d
18c
18d
18e
None
3
0.28
0.048
SGI-1776
2
1
3
None
None
1
Pan Assay Interference Compounds (PAINS)
Successful drug discovery process involves HTS. During
HTS it is predicted that successes from HTS operations
include primarily false positives between the actual hits if it
is found (Jonathan and Georgina 2010; McGovern 2002).
Compounds can be regarded as false positives due to a
number of reasons like binding interactions by forming
aggregates (McGovern and Shoichet 2003; Feng and
Shoichet 2006; Feng et al. 2005) by being protein-reactive
entities (Metz et al. 2007; Huth et al. 2007; Jonathan and
Michael 2014) or by directly interfering with assay signal-
ing. PAINS are chemical entities that are frequently false
positive in HTS. PAINS have a tendency to nonspecifically
react with several biological targets moderately, then spe-
cifically disturbing one preferred target (Jayme and Michael
2014). A number of disorderly functional groups are col-
lected by numerous PAINS (Martin and Stephane 2016).
Unwanted compounds may negatively influence not only
enzyme assays but also phenotypic screens and show bio-
logical activity for the wrong reason (Cheng and Li 2012).
PAINS violations of proposed compounds and reference
drugs are given in Table 4. Almost all the compounds
showed zero PAINS alert and thus can be used in the future
as good antitumor agents.
2
Foretinib None
None
None
SGI-1776 None
aLvio. Lipinski’s rule
bVvio. Veber rules
cGvio. Ghose filter
dPAINS Pan Assay Interference Compounds Analysis
(Shimadzu) and ESI-esquire 3000 Bruker Daltonics instru-
ment. Elemental analyses were measured using the Micro-
Analytical Data center at Cairo University. All reactions
were monitored by TLC on 2 × 5 cm precoated silica gel 60
F254 plates of thickness of 0.25 mm (Merck) for getting
complete reactions
General procedure for the synthesis of
2-(ethoxymethylene)-5,5-dimethylcyclohexane-1,
3-dione (3)
To a dry solid of dimedone (1.40 g, 0.01 mol) triethox-
ymethane (1.48 g, 0.01 mol) was added. The reaction mix-
ture was heated in an oil bath at 120 °C for 1 h then left to
cool. The solidified product was triturated with diethyl ether
and the participated crystals were collected by filtration.
White crystals from ethanol; m.p. 215–217 °C; yield
Experimental
1
Dry solvents were used through this work and all melting
points of the synthesized compounds were recorded on
Buchi melting point apparatus D-545. The IR spectra
(KBr disks) were recorded on Bruker Vector 22 instrument.
72%. IR (KBr) cm−1: 3055, 2986, 1689, 1688, 1632. H
NMR (300 MHz, DMSO-d6) δ 6.63 (s, 1H, CH), 3.46 (q,
2H, J = 6.35 Hz, CH2), 2.31, 2.28 (2s, 4H, 2CH2), 1.12 (t,
3H, J = 6.35 Hz, CH2), 1.09, 1.06 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 166.6, 164.4 (C-1, C-3),
108.8, 103.5 (CH=C), 58.4 (OCH2CH3), 50.6 (C-4), 36.2
(C-5), 28.6 (C-4), 24.4 (2CH3), 16.2 (OCH2CH3); EIMS:
13C NMR and H NMR spectra were measured on Bruker
1
DPX200 instrument in DMSO-d6 with TMS as internal
standard. Mass spectra were measured using EIMS

Medicinal Chemistry Research
m/z 196 [M]+ (32%); analysis Calcd for C11H16O3 (196.24):
C, 67.32; H, 8.22%. Found: C, 67.27; H, 8.36%.
2-((3,5-Diamino-1H-pyrazol-4-yl)methylene)-5,
5-dimethylcyclohexane-1,3-dione (7a)
General procedure for the synthesis of the 2,6-
dioxocyclohexylidene derivatives 5a, b
Orange crystals from 1,4-dioxane; m.p. 270–273 °C; yield
68%. IR (KBr) cm−1: 3489–3341, 3055, 2985, 1690, 1687,
1630. H NMR (300 MHz, DMSO-d6) δ 8.26 (s, 1H, D2O
1
To a solution of compound 3 (1.96 g, 0.01 mol) in 1,4-
dioxane (40 mL) containing triethylamine (0.50 mL) either
malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate
(1.07 g, 0.01 mol) was added. The reaction mixture was
heated under reflux for 1 h then left to cool and the formed
solid product was collected by filtration, dried and crystal-
lized from ethanol to give 5a, b, respectively.
exchangeable, NH), 6.68 (s, 1H, CH), 4.87, 5.21 (2s, 4H,
D2O exchangeable, 2NH2), 2.36, 2.24 (2s, 4H, 2CH2), 1.07,
1.03 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ =
171.8 (C=N), 166.8 (C-1), 164.3 (C-3), 136.0, 132.4
(pyrazole C-4, C-5), 108.2 (CH=C), 56.8 (C-2), 36.3 (C-5),
28.5 (C-4), 24.8 (2CH3); EIMS: m/z 248 [M]+ (32%);
analysis Calcd for C12H16N4O2 (248.28): C, 58.05; H, 6.50;
N, 22.57%. Found: C, 57.86; H, 6.37; N, 22.38%.
2-((4,4-Dimethyl-2,6-dioxocyclohexylidene)methyl)
malononitrile (5a)
2-((5-Amino-3-hydroxy-1H-pyrazol-4-yl)methylene)-
5,5-dimethylcyclohexane-1,3-dione (7b)
Yellow crystals from ethanol; m.p. 233–236 °C; yield 62%.
IR (KBr) cm−1: 3055, 2985, 2223, 2220, 1690, 1688, 1630.
1H NMR (300 MHz, DMSO-d6) δ 4.92, 6.61 (2d, 2H,
CH=CH), 2.36, 2.23 (2s, 4H, 2CH2), 1.08, 1.04 (2s, 6H,
2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 166.6 (C-1),
116.8, 117.0 (2CN), 109.7, 60.8 (CH=CH), 56.6 (C-2),
36.5 (C-5), 28.8 (C-4), 24.8 (2CH3); EIMS: m/z 216 [M]+
(40%); analysis Calcd for C12H12N2O2 (216.24): C, 66.65;
H, 5.59; N, 12.96%. Found: C, 66.72; H, 5.45; N, 13.16%.
Reddish brown crystals from 1,4-dioxane; m.p.
237–240 °C; yield 70%. IR (KBr) cm−1: 3548–3327, 3055,
1
2984, 1693, 1687, 1630. H NMR (300 MHz, DMSO-d6) δ
10.31 (s, 1H, D2O exchangeable, OH), 8.28 (s, 1H, D2O
exchangeable, NH), 6.67 (s, 1H, CH), 4.73 (s, 2H, D2O
exchangeable, NH2), 2.36, 2.26 (2s, 4H, 2CH2), 1.08, 1.03
(2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 171.6
(C=N), 166.5 (C-1), 164.1 (C-3), 136.0, 132.6 (pyrazole C-
4, C-5), 108.4 (CH=C), 56.6 (C-2), 36.3 (C-5), 28.4 (C-4),
24.8 (2CH3); EIMS: m/z 249 [M]+ (36%); analysis Calcd
for C12H15N3O3 (249.27): C, 57.82; H, 6.07; N, 16.86%.
Found: C, 57.62; H, 6.27; N, 16.73%.
Ethyl 2-cyano-3-(4,4-dimethyl-2,
6-dioxocyclohexylidene)propanoate (5b)
Yellow crystals from ethanol; m.p. 247–250 °C; yield 73%.
1
IR (KBr) cm−1: 3054, 2983, 2220, 1705–1687, 1630. H
2-((3,5-Diamino-1-phenyl-1H-pyrazol-4-yl)
NMR (300 MHz, DMSO-d6) δ 4.90, 6.63 (2d, 2H,
CH=CH), 4.22 (q, 2H, J = 7.25 Hz, OCH2CH3), 2.38, 2.21
(2s, 4H, 2CH2), 1.13 (t, 3H, J = 7.23 Hz, OCH2CH3), 1.09,
1.03 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ =
166.8 (C-1), 164.6, 163.8 (C-3, ester CO), 116.8 (CN),
109.6, 60.5 (CH=CH), 56.8 (C-2), 52.3 (OCH2CH3), 36.3
(C-5), 28.6 (C-4), 24.7 (2CH3), 16.5 (OCH2CH3); EIMS: m/
z 263[M]+ (32%); analysis Calcd for C14H17NO4 (263.29):
C, 63.87; H, 6.51; N, 5.32%. Found: C, 64.02; H, 6.39;
N, 5.52%.
methylene)-5,5-dimethylcyclo-hexane-1,3-dione (7c)
Red crystals from 1,4-dioxane; m.p. 215–217 °C; yield 72%.
IR (KBr) cm−1: 3474–3362, 3055, 2985, 1689, 1687, 1630.
1H NMR (300 MHz, DMSO-d6) δ 7.28–7.41 (m, 5H, C6H5),
6.64 (s, 1H, CH), 4.89, 5.24 (2s, 4H, D2O exchangeable,
2NH2), 2.38, 2.23 (2s, 4H, 2CH2), 1.08, 1.02 (2s, 6H, 2CH3);
13C NMR (DMSO-d6, 75 MHz): δ = 171.6 (C=N), 166.7
(C-1), 164.6 (C-3), 136.3, 132.6 (pyrazole C-4, C-5), 127.8,
125.4, 122.8, 120.4 (C6H5), 108.8 (CH=C), 56.8 (C-2), 36.2
(C-5), 28.8 (C-4), 24.6 (2CH3); EIMS: m/z 324 [M]+ (38%);
analysis Calcd for C18H20N4O2 (324.38): C, 66.65; H, 6.21;
N, 17.27%. Found: C, 66.80; H, 6.36; N, 17.42%.
General procedure for the synthesis of the pyrazole
derivatives 7a–d
To a solution of either 5a (2.16 g, 0.01 mol), 5b (2.63 g,
0.01 mol) in ethanol (40 mL) either of hydrazine hydrate
(0.50 mL, 0.01 mol) or phenylhydrazine (1.80 g, 0.01 mol)
was added. The reaction mixture, in each case, was heated
under reflux for 3 h then was left to cool and the formed solid
product was collected by filtration upon pouring onto ice/
water mixture containing a few drops of hydrochloric acid.
2-((5-Amino-3-hydroxy-1-phenyl-1H-pyrazol-4-yl)
methylene)-5,5-dimethylcyclo-hexane-1,3-dione (7d)
Orange crystals from ethanol; m.p. 257–259 °C; yield 78%.
IR (KBr) cm−1: 3483-3352, 3055, 2983, 1688, 1687, 1630.
1H NMR (300 MHz, DMSO-d6) δ 10.24 (s, 1H, D2O
exchangeable, OH), 7.25–7.43 (m, 5H, C6H5), 6.66 (s, 1H,

Medicinal Chemistry Research
CH), 4.86 (s, 2H, D2O exchangeable, NH2), 2.39, 2.25 (2s,
4H, 2CH2), 1.08, 1.02 (2s, 6H, 2CH3); 13C NMR (DMSO-
d6, 75 MHz): δ = 171.8 (C=N), 166.4 (C-1), 164.8 (C-3),
136.7, 132.2 (pyrazole C-4, C-5), 126.9, 125.1, 123.5,
120.2 (C6H5), 108.6 (CH=C), 56.6 (C-2), 36.4 (C-5), 28.6
(C-4), 24.8 (2CH3); EIMS: m/z 325 [M]+ (26%); analysis
Calcd for C18H19N3O3 (325.36): C, 66.45; H, 5.89; N,
12.91%. Found: C, 66.63; H, 6.15; N, 12.68%.
compound 3 (2.63 g, 0.01 mol) in ethanol (40 mL). The
reaction mixture, in each case, was heated under reflux for
3 h then left to cool and the formed solid product was
collected by filtration upon pouring onto ice/water mixture
containing a few drops of hydrochloric acid.
6,6-Dimethyl-6,7-dihydro-2H-indazol-4(5H)-one
(10a)
General procedure for the synthesis of the isoxazole
derivatives 9a, b
Brown crystals from 1,4-dioxane; m.p. 160–163 °C; yield
60%. IR (KBr) cm−1: 3462–3359, 3052, 2985, 1688, 1630.
1H NMR (300 MHz, DMSO-d6) δ 8.26 (s, 1H, D2O
exchangeable, NH), 6.73 (s, 1H, pyrazole H-3), 2.39, 2.27
(2s, 4H, 2CH2), 1.09, 1.03 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 173.6, 172.5 (2C=N), 165.8 (C-
4), 136.6, 132.2 (pyrazole C-4, C-5), 56.8 (C-2), 36.3 (C-6),
28.6 (C-7), 24.8 (2CH3); EIMS: m/z 164 [M]+ (36%);
analysis Calcd for C9H12N2O (164.20): C, 65.83; H, 7.37;
N, 17.06%. Found: C, 65.69; H, 715; N, 17.28%.
To a solution of either compound 5a (2.16 g, 0.01 mol), 5b
(2.63 g, 0.01 mol) in ethanol (40 mL) containing sodium
acetate (1.00 g) hydroxylamine hydrochloride (0.78 g,
0.01 mol) was added. The reaction mixture was heated
under reflux for 2 h then poured onto ice/water and the
formed solid product was collected by filtration.
2-((3,5-Diaminoisoxazol-4-yl)methylene)-5,
5-dimethylcyclohexane-1,3-dione (9a)
6,6-Dimethyl-2-phenyl-6,7-dihydro-2H-indazol-4
(5H)-one (10b)
White crystals from 1,4-dioxane; m.p. 270–273 °C; yield
75%. IR (KBr) cm−1: 3487–3328, 3055, 2985, 1690, 1688,
1630. H NMR (300 MHz, DMSO-d6) δ 6.66 (s, 1H, CH),
Pale yellow crystals from 1,4-dioxane; m.p. 98–100 °C;
1
1
yield 67%. IR (KBr) cm−1: 2984, 1688, 1630. H NMR
4.89, 5.23 (2s, 4H, D2O exchangeable, 2NH2), 2.37, 2.23 (2s,
4H, 2CH2), 1.08, 1.05 (2s, 6H, 2CH3); 13C NMR (DMSO-d6,
75 MHz): δ = 171.4 (C=N), 166.5 (C-1), 164.5 (C-3), 132.6,
136.3 (isoxazole C-4, C-5), 108.6 (CH=C), 56.5 (C-2), 28.9
(C-4), 36.5 (C-5), 24.7 (2CH3); EIMS: m/z 249 [M]+ (42%);
analysis Calcd for C12H15N3O3 (249.27): C, 57.82; H, 6.07;
N, 16.86%. Found: C, 57.63; H, 6.15; N, 16.69%.
(300 MHz, DMSO-d6) δ 7.26–7.38 (m, 5H, C6H5), 6.74 (s,
1H, pyrazole H-3), 2.37, 2.23 (2s, 4H, 2CH2), 1.09, 1.06
(2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 173.5,
172.8 (2C=N), 165.5 (C-4), 136.3, 132.5 (pyrazole C-4, C-
5), 128.4, 126.2, 125.3, 123.6, 121.2 (C6H5), 56.7 (C-2),
36.6 (C-6), 28.3 (C-7), 24.8 (2CH3); EIMS: m/z 240 [M]+
(24%); analysis Calcd for C15H16N2O (240.30): C, 74.97;
H, 6.71; N, 11.66%. Found: C, 74.83; H, 6.48; N, 11.80%.
2-((5-Amino-3-hydroxyisoxazol-4-yl)methylene)-5,
5-dimethylcyclohexane-1,3-dione (9b)
6,6-Dimethyl-6,7-dihydrobenzo[c]isoxazol-4(5H)-one
(11)
White crystals from 1,4-dioxane; m.p. 240–243 °C; yield
78%. IR (KBr) cm−1: 3567–3342, 3052, 2987, 1692, 1688,
1630. 1H NMR (300 MHz, DMSO-d6) δ 10.30 (s, 1H, D2O
exchangeable, OH), 6.66 (s, 1H, CH), 4.86 (s, 2H, D2O
exchangeable, NH2), 2.37, 2.21 (2s, 4H, 2CH2), 1.08, 1.06
(2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 171.8
(C=N), 166.3 (C-1), 164.2 (C-3), 132.8, 136.1 (isoxazole
C-4, C-5), 108.3 (CH=C), 56.7 (C-2), 36.3 (C-5), 28.6 (C-
4), 24.8 (2CH3); EIMS: m/z 250 [M]+ (36%); analysis
Calcd for C12H14N2O4 (250.25): C, 57.59; H, 5.64; N,
11.19%. Found: C, 57.73; H, 5.72; N, 11.30%.
Hydroxylamine hydrochloride (1.40 g, 0.01 mol) was added
to a solution of compound 3 (1.93 g, 0.01 mol) in 1,4-
dioxane (40 mL) containing sodium acetate (1.00 g). The
reaction mixture was heated under reflux for 2 h then poured
onto ice/water and the formed solid product was collected by
filtration.
Pale yellow crystals from 1,4-dioxane; m.p.
207–210 °C; yield 68%. IR (KBr) cm−1: 3054, 2985,
1
1688, 1630. H NMR (300 MHz, DMSO-d6) δ 7.03 (s,
1H, isoxazole H-5), 2.37, 2.25 (2s, 4H, 2CH2), 1.09, 1.03
(2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ =
176.3 (C=N), 166.4 (C-4), 138.3, 133.1 (isoxazole C-4,
C-5), 56.8 (C-2), 36.3 (C-6), 32.5 (C-5), 28.6 (C-7), 24.8
(2CH3); EIMS: m/z 165 [M]+ (28%); analysis Calcd for
C9H11NO2 (165.19): C, 65.44; H, 6.71; N, 8.48%. Found:
C, 65.73; H, 6.54; N, 8.69%.
General procedure for the synthesis of the indazole
derivatives 10a, b
Either of hydrazine hydrate (0.1 mL, 0.02 mol) or phe-
nylhydrazine (3.16 g, 0.02 mol) was added to a solution of

Medicinal Chemistry Research
General procedure for the synthesis of the thieno
[3,2-e]indazole derivatives 12a–d
136.6, 132.4 (pyrazole C-4, C-5), 138.4, 135.2, 132.8,
130.2 (thiophene C), 128.3, 127.4, 125.4, 122.1 (C6H5),
36.8 (C-4), 28.3 (C-7), 24.5 (2CH3); EIMS: m/z 320 [M]+
(38%); analysis Calcd for C18H16N4S (320.41): C, 67.47; H,
5.03; N, 17.49; S, 10.01%. Found: C, 67.68; H, 5.25; N,
17.68; S, 10.25%.
Each of elemental sulfur and either malononitrile (0.66 g,
0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) were
added to a solution of either 10a (1.49 g, 0.01 mol) or 10b
(2.40 g, 0.01 mol) in 1,4-dioxane (40 mL) containing trie-
thylamine (0.50 mL). The reaction mixture, in each case,
was heated under reflux for 2 h then left to cool. The formed
solid product was collected by filtration, dried and crystal-
lized from ethanol to give 12a–d, respectively.
Ethyl 7-amino-5,5-dimethyl-2-phenyl-4,5-dihydro-
2H-thieno[3,2-e]indazole-8-carboxylate (12d)
Orange crystals from 1,4-dioxane; m.p. 86–88 °C; yield
1
66%. IR (KBr) cm−1: 3483–3326, 3053, 2985, 1630. H
7-Amino-5,5-dimethyl-4,5-dihydro-2H-thieno[3,2-e]
indazole-8-carbonitrile (12a)
NMR (300 MHz, DMSO-d6) δ 7.42–7.25 (m, 5H, C6H5),
6.66 (s, 1H, pyrazole H-3), 4.80 (s, 2H, D2O exchangeable,
NH2), 4.23 (q, 2H, J = 6.56 Hz, OCH2CH3), 2.37 (s, 2H,
CH2), 1.12 (t, 3H, J = 6.56 Hz, OCH2CH3), 1.09, 1.07 (2s,
6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 174.6
(C=N), 136.9, 132.4 (pyrazole C-4, C-5), 138.8, 135.6,
132.2, 130.5 (thiophene C), 127.3, 125.6, 124.1, 122.0
(C6H5), 52.6 (OCH2CH3), 36.9 (C-4), 28.4 (C-7), 24.6
(2CH3), 16.2 (OCH2CH3); EIMS: m/z 367 [M]+ (48%);
analysis Calcd for C20H21N3O2S (367.46): C, 65.37; H,
5.76; N, 11.44; S, 8.73%. Found: C, 65.48; H, 5.49; N,
11.59; S, 9.02%.
Orange crystals from 1,4-dioxane; m.p. 200–204 °C; yield
72%. IR (KBr) cm−1: 3489–3337, 3054, 2985, 2220, 1630.
1H NMR (300 MHz, DMSO-d6) δ 8.29 (s, 1H, D2O
exchangeable, NH), 6.65 (s, 1H, pyrazole H-3), 5.21 (s, 2H,
D2O exchangeable, NH2), 2.36 (s, 2H, CH2), 1.09, 1.06 (2s,
6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 173.8
(C=N), 118.2 (CN), 136.9, 132.6 (pyrazole C-4, C-5),
138.4, 135.2, 132.8, 130.2 (thiophene C), 36.6 (C-4), 28.6
(C-7), 24.8 (2CH3); EIMS: m/z 244 [M]+ (48%); analysis
Calcd for C12H12N4S (244.32): C, 58.99; H, 4.95; N, 22.93;
S, 13.12%. Found: C, 59.76; H, 5.19; N, 23.26; S, 13.26%.
General procedure for the synthesis of the
dihydrothieno[3′,2′:3,4] benzo[1,2-c]isoxazol-
derivatives 13a, b
Ethyl 7-amino-5,5-dimethyl-4,5-dihydro-2H-thieno
[3,2-e]indazole-8-carboxylate (12b)
To a solution of compound 11 (1.65 g, 0.01 mol) in 1,4-
dioxan (40 mL) containing triethylamine (0.50 mL) each of
elemental sulfur and either malononitrile (0.66 g, 0.01 mol)
or ethyl cyanoacetate (1.07 g, 0.01 mol) were added. The
reaction mixture was heated under reflux for 2 h then left to
cool. The formed solid product was collected by filtration,
dried and crystallized from ethanol to give 13a, b,
respectively.
Orange crystals from 1,4-dioxane; m.p. > 300 °C; yield
1
78%. IR (KBr) cm−1: 3474–3348, 3054, 2985, 1630. H
NMR (300 MHz, DMSO-d6)
δ 8.32 (s, 1H, D2O
exchangeable, NH), 6.68 (s, 1H, pyrazole H-3), 4.82 (s, 2H,
D2O exchangeable, NH2), 4.22 (q, 2H, J = 7.26 Hz,
OCH2CH3), 2.38 (s, 2H, CH2), 1.12 (t, 3H, J = 7.26 Hz,
OCH2CH3), 1.09, 1.06 (2s, 6H, 2CH3); 13C NMR (DMSO-
d6, 75 MHz): δ = 174.3 (C=N), 136.6, 132.4 (pyrazole C-4,
C-5), 138.4, 135.2, 132.8, 130.2 (thiophene C), 52.3
(OCH2CH3), 36.6 (C-4), 28.6 (C-7), 24.8 (2CH3), 16.2
(OCH2CH3); EIMS: m/z 291 [M]+ (28%); analysis Calcd
for C14H17N3O2S (291.37): C, 57.71; H, 5.88; N, 14.42; S,
11.00%. Found: C, 57.98; H, 5.73; N, 14.59; S, 10.83%.
7-Amino-5,5-dimethyl-4,5-dihydrothieno[3′,2′:3,4]
benzo[1,2-c]isoxazole-8-carbonitrile (13a)
Pale yellow crystals from 1,4-dioxane; m.p. 170–173 °C;
yield 70%. IR (KBr) cm−1: 3447–3328, 3054, 2985, 2220,
1630. 1H NMR (300 MHz, DMSO-d6) δ 6.67 (s, 1H,
isoxazole H-5), 4.90 (s, 1H, D2O exchangeable, NH2),
2.38 (s, 2H, CH2), 1.09, 1.07 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 173.5 (C=N), 116.4 (CN),
138.3, 133.2 (isoxazole C-4, C-5), 140.1, 138.1, 136.4,
134.2, 131.6, 130.0 (thiophene, isoxazole C), 36.6 (C-4),
28.4 (C-7), 24.8 (2CH3); EIMS: m/z 245 [M]+ (36%);
analysis Calcd for C12H11N3OS (245.30): C, 58.76; H,
4.52; N, 17.13; S, 13.07%. Found: C, 58.93; H, 4.66; N,
17.29; S, 13.38%.
7-Amino-5,5-dimethyl-2-phenyl-4,5-dihydro-2H-
thieno[3,2-e]indazole-8-carbonitrile (12c)
Orange crystals from 1,4-dioxane; m.p. 76–78 °C; yield
75%. IR (KBr) cm−1: 3492-3332, 3054, 2985, 2220, 1630.
1H NMR (300 MHz, DMSO-d6) δ 7.43–7.26 (m, 5H, C6H5),
6.68 (s, 1H, pyrazole H-3), 4.82 (s, 2H, D2O exchangeable,
NH2), 2.38 (s, 2H, CH2), 1.09, 1.06 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 174.3 (C=N), 118.3 (CN),

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Ethyl 7-amino-5,5-dimethyl-4,5-dihydrothieno
[3′,2′:3,4]benzo[1,2-c]isoxazole-8-carboxylate (13b)
2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 138.9,136.7,
133.2, 130.4 (thiophene C), 51.6, 52.3 (two OCH2CH3),
36.7 (C-4), 28.8 (C-7), 24.8 (2CH3), 16.2, 16.3 (two
OCH2CH3); EIMS: m/z 323 [M]+ (40%); analysis Calcd for
C16H21NO4S (323.41): C, 59.42; H, 6.54; N, 4.33; S,
9.91%. Found: C, 59.58; H, 6.80; N, 4.52; S, 10.16%.
Orange crystals from 1,4-dioxane; m.p.180–182 °C; yield
60%. IR (KBr) cm−1: 3474-3352, 3055, 2985, 1630. 1H NMR
(300 MHz, DMSO-d6) δ 6.68 (s, 1H, isoxazole H-5), 4.85 (s,
2H, D2O exchangeable, NH2), 4.21 (q, 2H, J = 6.30 Hz,
OCH2CH3), 2.39 (s, 2H, CH2), 1.13 (t, 3H, J = 6.30 Hz,
OCH2CH3), 1.09, 1.08 (2s, 6H, 2CH3); 13C NMR (DMSO-d6,
75 MHz): δ = 174.8 (C=N), 136.5, 132.6 (pyrazole C-4, C-5),
138.9, 137.3, 134.5, 130.2 (thiophene C), 52.3 (OCH2CH3),
36.7 (C-4), 28.6 (C-7), 24.7 (2CH3), 16.1 (OCH2CH3); EIMS:
m/z 292 [M]+ (28%); analysis Calcd for C14H16N2O3S
(292.35): C, 57.52; H, 5.52; N, 9.58; S, 10.97%. Found: C,
57.47; H, 5.63; N, 9.80; S, 9.68%.
General procedure of the 2-((phenylamino)
methylene)cyclohexane derivatives 16a–c
To a solution of compound 3 in 1,4-dioxane (40 mL) either
aniline (0.93 g, 0.01 mol), 4-chloroaniline (1.27 g, 0.01 mol) or
4-methylaniline (1.07 g, 0.01 mol) was added. The reaction
was heated under reflux for 3 h then poured onto ice/water
mixture containing a few drops of hydrochloric acid and the
precipitated solid product was collected by filtration.
General procedure for the synthesis of the 6,
7-dihydrobenzo[b]thiophene derivatives 14a, b
5,5-Dimethyl-2-((phenylamino)methylene)
cyclohexane-1,3-dione (16a)
Each of elemental sulfur and either malononitrile (0.66 g,
0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) were added
to a solution of compound 3 (1.96 g, 0.01 mol) in 1,4-dioxane
(40 mL) containing triethylamine (0.50 mL). The reaction
mixture was heated under reflux for 2 h then left to cool. The
formed solid product was collected by filtration, dried and
crystallized from ethanol to give 14a, b, respectively.
Orange crystals from ethanol; m.p. 165–167 °C; yield 78%. IR
1
(KBr) cm-1: 3480–3226, 3055, 2986, 1689, 1688, 1632. H
NMR (200 MHz, DMSO-d6) δ 8.40 (s, 1H, D2O exchange-
able, NH), 7.28–7.39 (m, 5H, C6H5), 6.65 (s, 1H, CH), 2.36,
2.28 (2s, 4H, 2CH2), 1.09, 1.06 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 166.8, 164.5 (C-1, C-3), 128.6,
127.3, 125.0, 122.3 (C6H5), 108.4, 103.5 (CH=C), 50.6 (C-4),
36.2 (C-5), 28.8 (C-4), 24.5 (2CH3); EIMS: m/z 243 [M]+
(38%); analysis Calcd for C15H17NO2 (243.30): C, 74.05; H,
7.04; N, 5.76%. Found: C, 73.84; H, 7.32; N, 5.90%.
2-Amino-4-(ethoxymethylene)-7,7-dimethyl-5-oxo-
4,5,6,7-tetrahydrobenzo-[b]-thiophene-3-
carbonitrile (14a)
Yellow crystals from 1,4-dioxane; m.p. 166–168 °C; yield
65%. IR (KBr) cm−1: 3473–3341, 3054, 2985, 2220, 1630.
1H NMR (300 MHz, DMSO-d6) δ6.59 (s, 1H, CH=C), 4.92
(s, 2H, D2O exchangeable, NH2), 3.89 (q, 2H, J = 7.01 Hz,
CH2), 2.36 (s, 2H, CH2), 1.13 (t, 3H, J = 7.01 Hz, CH3),
1.09, 1.07 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz):
δ = 116.8 (CN), 138.1,134.2, 131.6, 130.0 (thiophene C),
52.6 (OCH2CH3), 36.9 (C-4), 28.4 (C-7), 24.8 (2CH3), 16.0
(OCH2CH3); EIMS: m/z 276 [M]+ (24%); analysis Calcd
for C14H16N2O2S (276.35): C, 60.85; H, 5.84; N, 10.14; S,
11.60%. Found: C, 60.94; H, 5.71; N, 10.26; S, 11.83%.
2-(((4-Chlorophenyl)amino)methylene)-5,
5-dimethylcyclohexane-1,3-dione (16b)
Orange crystals from ethanol; m.p. 185–188 °C; yield 65%.
IR (KBr) cm−1: 3472–3346, 3055, 2986, 1689, 1688, 1630.
1H NMR (200 MHz, DMSO-d6) δ 8.35 (s, 1H, D2O
exchangeable, NH), 7.49–7.25 (m, 4H, C6H4), 6.65 (s, 1H,
CH), 2.36, 2.28 (2s, 4H, 2CH2), 1.09, 1.07 (2s, 6H, 2CH3);
13C NMR (DMSO-d6, 75 MHz): δ = 167.3, 164.8 (C-1, C-
3), 128.8, 126.1, 124.2, 122.3 (C6H4), 108.6, 104.1
(CH=C), 50.4 (C-4), 36.6 (C-5), 28.3 (C-4), 24.6 (2CH3);
EIMS: m/z 277 [M]+ (24%); analysis Calcd for
C15H16ClNO2 (277.75): C, 64.87; H, 5.81; N, 5.04%.
Found: C, 64.64; H, 5.79; N, 5.19%.
Ethyl 2-amino-4-(ethoxymethylene)-7,7-dimethyl-5-
oxo-4,5,6,7-tetrahydro-benzo[b]-thiophene-3-
carboxylate (14b)
5,5-Dimethyl-2-((p-tolylamino)methylene)
cyclohexane-1,3-dione (16c)
Orange crystals from 1,4-dioxan; m.p. 225–228 °C; yield
1
77%. IR (KBr) cm−1: 3496–3331, 3054, 2985, 1630. H
NMR (300 MHz, DMSO-d6) δ 6.65 (s, 1H, CH=C), 4.87 (s,
2H, D2O exchangeable, NH2), 4.21, 3.87 (q, 4H, J = 6.93,
5.42 Hz, two OCH2CH3), 2.34 (s, 2H, CH2), 1.11, 1.14 (2t,
6H, J = 6.93, 5.42 Hz, two OCH2CH3), 1.09, 1.08 (2s, 6H,
Orange crystals from ethanol; m.p. 190–194 °C; yield 74%.
IR (KBr) cm−1: 3484–3353, 3055, 2985, 1689, 1687, 1630.
1H NMR (200 MHz, DMSO-d6) δ 8.38 (s, 1H, D2O
exchangeable, NH), 7.48–7.23 (m, 4H, C6H4), 6.68 (s, 1H,

Medicinal Chemistry Research
CH), 2.78 (s, 3H, CH3), 2.38, 2.26 (2s, 4H, 2CH2), 1.09,
1.07 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ =
167.7, 164.6 (C-1, C-3), 127.3, 125.6, 123.8, 121.7 (C6H4),
108.8, 104.5 (CH=C), 50.6 (C-4), 36.8 (C-5), 30.2 (CH3),
28.4 (C-4), 24.8 (2CH3); EIMS: m/z 257 [M]+ (36%);
analysis Calcd for C15H19NO2 (257.33): C, 74.68; H, 7.44;
N, 5.55%. Found: C, 74.51; H, 7.67; N, 5.63%.
for C20H22N2O3S (370.47): C, 64.84; H, 5.99; N, 7.56; S,
8.66%. Found: C, 64.69; H, 5.74; N, 7.69; S, 8.73%.
2-Amino-4-(((4-chlorophenyl)amino)methylene)-7,
7-dimethyl-5-oxo-4,5,6,7-tetrahydro-benzo[b]
thiophene-3-carbonitrile (17c)
Orange crystals from 1,4-dioxane; m.p. 123–125 °C; yield
78%. IR (KBr) cm−1: 3469–3346, 3054, 2985, 2220, 1688,
1630. H NMR (200 MHz, DMSO-d6) δ 8.36 (s, 1H, D2O
General procedure for the synthesis of the 6,
7-dihydrobenzo[b] thiophene derivatives 17a–f
1
exchangeable, NH), 7.49–7.21 (m, 4H, C6H4), 6.82 (s, 1H,
C=CH), 4.84 (s, 2H, D2O exchangeable, NH2), 2.38 (s, 2H,
CH2), 1.09, 1.06 (2s, 6H, 2CH3); 13C NMR (DMSO-d6,
75 MHz): δ = 138.5, 135.2, 133.1, 130.4 (thiophene C),
127.5, 125.2, 123.3, 122.1 (C6H4), 116.8 (CN), 36.6 (C-4),
28.3 (C-7), 24.8 (2CH3); EIMS: m/z 357 [M]+ (26%);
analysis Calcd for C18H16ClN3OS (357.86): C, 60.41; H,
4.51; N, 11.74; S, 8.96%. Found: C, 60.58; H, 4.73; N,
11.90; S, 9.25%.
Each of elemental sulfur and either malononitrile (0.66 g,
0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) were
added to a solution of either 16a (2.43 g, 0.01 mol), 16b
(2.77 g, 0.01 mol), or 16c (2.42 g, 0.01 mol) in 1,4-dioxane
(40 mL) containing triethylamine (0.50 mL). The reaction
mixture was heated under reflux for 4 h then left to cool.
The formed solid product was collected by filtration, dried,
and crystallized from ethanol to give 17a–f, respectively.
2-Amino-7,7-dimethyl-5-oxo-4-((phenylamino)
methylene)-4,5,6,7-tetrahydrobenzo-[b]thiophene-
3-carbonitrile (17a)
Ethyl 2-amino-4-(((4-chlorophenyl)amino)
methylene)-7,7-dimethyl-5-oxo-4,5,6,
7-tetrahydrobenzo[b]thiophene-3-carboxylate (17d)
Orange crystals from 1,4-dioxane; m.p.127–129 °C; yield
75%. IR (KBr) cm−1: 3487–3326, 3054, 2985, 2220, 1688,
1630. H NMR (200 MHz, DMSO-d6) δ 8.40 (s, 1H, D2O
Pale brown crystals from 1,4-dioxane; m.p. 118–121 °C;
yield 62%. IR (KBr) cm−1: 3492–3329, 3054, 2984, 2220,
1701, 1687, 1630. H NMR (200 MHz, DMSO-d6) δ 8.40
1
1
exchangeable, NH), 7.46–7.26 (m, 5H, C6H5), 6.80 (s, 1H,
C=CH), 4.88 (s, 2H, D2O exchangeable, NH2), 2.35 (s, 2H,
CH2), 1.09, 1.08 (2s, 6H, 2CH3); 13C NMR (DMSO-d6,
75 MHz): δ = 136.6, 132.4 (pyrazole C-4, C-5), 138.1,
136.7, 132.4, 130.8 (thiophene C), 128.3, 126.1, 123.8,
122.1 (C6H5), 116.5 (CN), 36.8 (C-4), 28.6 (C-7), 24.5
(2CH3); EIMS: m/z 323 [M]+ (40%); analysis Calcd for
C18H17N3OS (323.41): C, 66.85; H, 5.30; N, 12.99; S,
9.91%. Found: C, 66.73; H, 5.28; N, 13.30; S, 10.16%.
(s, 1H, D2O exchangeable, NH), 7.49–7.23 (m, 4H, C6H4),
6.83 (s, 1H, C=CH), 4.83 (s, 2H, D2O exchangeable, NH2),
4.23 (q, 2H, J = 7.11 Hz, CH2), 2.37 (s, 2H, CH2), 1.12 (t,
3H, J = 7.11 Hz, CH2), 1.09, 1.08 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 138.1, 136.8, 135.6, 130.1
(thiophene C), 129.8, 124.8, 122.6, 120.1 (C6H4), 52.3
(OCH2CH3), 36.6 (C-4), 28.1 (C-7), 24.9 (2CH3), 16.2
(OCH2CH3); EIMS: m/z 404 [M]+ (32%); analysis Calcd
for C18H21ClN2O3S (404.91): C, 59.33; H, 4.23; N, 6.92; S,
7.92%. Found: C, 59.28; H, 4.41; N, 7.36; S, 8.25%.
Ethyl 2-amino-7,7-dimethyl-5-oxo-4-((phenylamino)
methylene)-4,5,6,7-tetrahydrobenzo-[b]thiophene-
3-carboxylate (17b)
2-Amino-7,7-dimethyl-5-oxo-4-((p-tolylamino)
methylene)-4,5,6,7-tetrahydro-benzo[b]thiophene-
3-carbonitrile (17e)
Orange crystals from 1,4-dioxane; m.p. 90–92 °C; yield
70%. IR (KBr) cm−1: 3472–3341, 3054, 2985, 2220, 1702,
1688, 1630. H NMR (200 MHz, DMSO-d6) δ 8.38 (s, 1H,
Orange crystals from 1,4-dioxane; m.p. 120–123 °C; yield
58%. IR (KBr) cm−1: 3496–3352, 3054, 2985, 2220, 1688,
1630. H NMR (200 MHz, DMSO-d6) δ 8.34 (s, 1H, D2O
exchangeable, NH), 7.46–7.26 (m, 4H, C6H4), 6.80 (s, 1H,
C=CH), 4.87 (s, 2H, D2O exchangeable, NH2), 2.87 (s, 3H,
CH3), 2.36 (s, 2H, CH2), 1.09, 1.07 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 137.3, 136.1, 132.8, 130.9
(thiophene C), 127.5, 123.9, 122.6, 120.8 (C6H4), 116.6
(CN), 36.6 (C-4), 28.6 (C-7), 24.7 (2CH3); EIMS: m/z 337
[M]+ (30%); analysis Calcd for C19H19N3OS (337.44):
1
1
D2O exchangeable, NH), 7.44–7.25 (m, 5H, C6H5), 6.82 (s,
1H, C=CH), 4.85 (s, 2H, D2O exchangeable, NH2), 4.22 (q,
2H, J = 6.55 Hz, CH2), 2.37 (s, 2H, CH2), 1.12 (t, 3H, J =
6.55 Hz, CH2), 1.09, 1.06 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 138.3, 136.5, 133.8, 130.3
(thiophene C), 127.4, 125.6, 123.3, 121.5 (C6H5), 52.1
(OCH2CH3), 36.8 (C-4), 28.4 (C-7), 24.7 (2CH3), 16.2
(OCH2CH3); EIMS: m/z 370 [M]+ (24%); analysis Calcd

Medicinal Chemistry Research
C, 67.63; H, 5.68; N, 12.45; S, 9.50%. Found: C, 67.58; H,
5.73; N, 12.60; S, 9.25%.
1630. 1H NMR (200 MHz, DMSO-d6) δ 8.28, 8.34 (2s, 2H,
D2O exchangeable, 2NH), 7.24–7.42 (m, 15H, 3C6H5),
2.43, 2.25 (2s, 4H, 2CH2), 1.09, 1.02 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 173.6, 172.4 (2C=N),
136.2, 132.5 (pyrazole C-4, C-5), 120.0, 121.3, 122.7,
123.4, 123.6, 124.1, 124.5, 124.8, 125.0, 125.6, 126.4,
127.1 (3C6H5), 56.4 (C-5), 36.3 (C-6), 28.9 (C-7), 24.6
(2CH3); EIMS: m/z 421 [M]+ (26%); analysis Calcd for
C27H27N5 (421.54): C, 76.93; H, 6.46; N, 16.61%. Found:
C, 76.83; H, 6.70; N, 16.84%.
Ethyl 2-amino-7,7-dimethyl-5-oxo-4-((p-tolylamino)
methylene)-4,5,6,7-tetrahydro-benzo[b]thiophene-
3-carboxylate(17f)
Pale brown crystals from 1,4-dioxane; m.p. 115–117 °C; yield
70%. IR (KBr) cm−1: 3472–3331, 3054, 2984, 2220, 1705,
1
1687, 1630. H NMR (200 MHz, DMSO-d6) δ 8.43 (s, 1H,
D2O exchangeable, NH), 7.52–7.21 (m, 4H, C6H4), 6.85 (s,
1H, C=CH), 4.82 (s, 2H, D2O exchangeable, NH2), 4.21 (q,
2H, J = 7.38 Hz, CH2), 2.78 (s, 3H, CH3), 2.37 (s, 2H, CH2),
N-(4-Chlorophenyl)-4-hydrazono-6,6-dimethyl-
4,5,6,7-tetrahydro-2H-indazol-3-amine (18c)
1.12 (t, 3H, J = 7.38 Hz, CH2), 1.08, 1.06 (2s, 6H, 2CH3); 13
C
NMR (DMSO-d6, 75 MHz): δ = 137.8, 136.6, 134.9, 131.6
(thiophene C), 127.1, 125.6, 123.2, 120.7 (C6H4), 52.6
(OCH2CH3), 36.8 (C-4), 30.3 (CH3), 28.0 (C-7), 24.8 (2CH3),
16.1 (OCH2CH3); EIMS: m/z 384 [M]+ (24%); analysis Calcd
for C21H24N2O3S (384.49): C, 65.60; H, 6.29; N, 7.29; S,
8.34%. Found: C, 65.42; H, 6.09; N, 7.41; S, 8.50%.
Pale yellow crystals from 1,4-dioxane; m.p. 240–244 °C;
yield 68%. IR (KBr) cm−1: 3474–3358, 3052, 2985, 1650,
1630. 1H NMR (200 MHz, DMSO-d6) δ 8.27, 8.30 (2s, 2H,
D2O exchangeable, 2NH), 7.23–7.40 (m, 4H, C6H4), 4.84
(s, 2H, D2O exchangeable, NH2), 2.41, 2.27 (2s, 4H, 2CH2),
1.07, 1.04 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz):
δ = 173.1, 172.5 (2C=N), 136.8, 132.5 (pyrazole C-4, C-5),
120.1, 123.8, 124.3, 125.6 (C6H4), 56.4 (C-5), 36.9 (C-6),
28.6 (C-7), 24.9 (2CH3); EIMS: m/z 303 [M]+ (42%);
analysis Calcd for C15H18ClN5 (303.13): C, 59.30; H, 5.97;
N, 23.05%. Found: C, 59.53; H, 6.15; N, 22.92%.
General procedure for the synthesis of the 4,5,6,
7-tetrahydro-2H-indazole derivatives 18a–f
To a solution of either 16a (2.43 g, 0.01 mol), 16b (2.77 g,
0.01 mol), or 16c (2.42 g, 0.01 mol) in 1,4-dioxane (40 mL)
either of hydrazine hydrate (0.1 mL, 0.02 mol) or phe-
nylhydrazine (3.16 g, 0.02 mol) was added. The reaction
mixture, in each case, was heated under reflux for 3 h then
was left to cool and the formed solid product was collected
by filtration upon pouring onto ice/water mixture containing
a few drops of hydrochloric acid.
N-(4-Chlorophenyl)-6,6-dimethyl-2-phenyl-4-(2-
phenylhydrazono)-4,5,6,7-tetrahydro-2H-indazol-3-
amine (18d)
Yellow crystals from 1,4-dioxane; m.p. 107–110 °C; yield
78%. IR (KBr) cm−1: 3486–3348, 3050, 2985, 1655, 1632.
1H NMR (200 MHz, DMSO-d6) δ 8.26, 8.32 (2s, 2H, D2O
exchangeable, 2NH), 7.26–7.48 (m, 14H, 2C6H5, C6H4),
2.40, 2.28 (2s, 4H, 2CH2), 1.08, 1.02 (2s, 6H, 2CH3); 13C
NMR (DMSO-d6, 75 MHz): δ = 173.8, 172.5 (2C=N),
136.5, 132.3 (pyrazole C-4, C-5), 120.1, 120.8, 121.6,
123.7, 123.9, 124.2, 124.6, 125.2, 125.4, 125.9, 126.6,
127.3 (2C6H5, C6H4), 56.6 (C-5), 36.2 (C-6), 28.5 (C-7),
24.6 (2CH3); EIMS: m/z 455 [M]+ (36%); analysis Calcd
for C27H26ClN5 (455.98): C, 71.12; H, 5.75; N, 15.36%.
Found: C, 71.30; H, 5.93; N, 15.41%.
4-Hydrazono-6,6-dimethyl-N-phenyl-4,5,6,
7-tetrahydro-2H-indazol-3-amine (18a)
Pale yellow crystals from 1,4-dioxane; m.p. 225–228 °C;
yield 68%. IR (KBr) cm−1: 3484–3331, 3052, 2985, 1655,
1630. 1H NMR (200 MHz, DMSO-d6) δ 8.29, 8.31 (2s, 2H,
D2O exchangeable, 2NH), 7.26–7.39 (m, 5H, C6H5), 4.86
(s, 2H, D2O exchangeable, NH2), 2.42, 2.29 (2s, 4H, 2CH2),
1.07, 1.03 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz):
δ = 173.3, 172.8 (2C=N), 136.6, 132.8 (pyrazole C-4, C-5),
120.3, 122.5, 124.6, 125.1 (C6H5), 56.6 (C-5), 36.8 (C-6),
28.3 (C-7), 24.8 (2CH3); EIMS: m/z 269 [M]+ (33%);
analysis Calcd for C15H19N5 (269.34): C, 66.89; H, 7.11; N,
26.00%. Found: C, 66.74; H, 6.94; N, 25.80%.
4-Hydrazono-6,6-dimethyl-N-(p-tolyl)-4,5,6,
7-tetrahydro-2H-indazol-3-amine (18e)
Pale yellow crystals from 1,4-dioxane; m.p. 235–236 °C;
yield 70%. IR (KBr) cm−1: 3474–3358, 3052, 2985, 1650,
1630. 1H NMR (200 MHz, DMSO-d6) δ 8.29, 8.33 (2s, 2H,
D2O exchangeable, 2NH), 7.25–7.46 (m, 4H, C6H4), 4.86
(s, 2H, D2O exchangeable, NH2), 2.80 (s, 3H, CH3), 2.43,
2.29 (2s, 4H, 2CH2), 1.09, 1.04 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 173.3, 172.6 (2C=N), 136.5,
6,6-Dimethyl-N,2-diphenyl-4-(2-phenylhydrazono)-
4,5,6,7-tetrahydro-2H-indazol-3-amine (18b)
Pale yellow crystals from 1,4-dioxane; m.p. 110–112 °C;
yield 73%. IR (KBr) cm−1: 3476–3353, 3050, 2985, 1653,

Medicinal Chemistry Research
132.8 (pyrazole C-4, C-5), 120.9, 123.2, 125.1, 126.8
(C6H4), 56.4 (C-5), 38.1 (CH3), 36.9 (C-6), 28.8 (C-7), 24.9
(2CH3); EIMS: m/z 283 [M]+ (36%); analysis Calcd for
C16H21N5 (283.37): C, 67.82; H, 7.47; N, 24.71%. Found:
C, 67.59; H, 7.53; N, 24.65%.
1H NMR (200 MHz, DMSO-d6) δ 10.23 (s, 1H, D2O
exchangeable, OH), 8.28 (s, 1H, NH, D2O exchangeable,
NH), 7.21–7.47 (m, 4H, C6H4), 2.36, 2.27 (2s, 4H, 2CH2),
1.08, 1.06 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz):
δ = 176.8 (C=N), 166.1 (C-4), 138.8, 133.1 (isoxazole C-4,
C-5), 120.8, 121.9, 124.2, 125.6 (C6H4), 56.5 (C-2), 36.3
(C-6), 32.3 (C-5), 28.6 (C-7), 24.7 (2CH3); EIMS: m/z 305
[M]+ (23%); analysis Calcd for C15H16ClN3O2 (305.76): C,
58.92; H, 5.27; N, 13.74%. Found: C, 59.21; H, 5.39; N,
13.53%.
6,6-Dimethyl-2-phenyl-4-(2-phenylhydrazono)-N-(p-
tolyl)-4,5,6,7-tetrahydro-2H-indazol-3-amine (18f)
Yellow crystals from 1,4-dioxane; m.p. 87–90 °C; yield
65%. IR (KBr) cm−1: 3492–3326, 3050, 2985, 1653, 1630.
1H NMR (200 MHz, DMSO-d6) δ 8.28, 8.31 (2s, 2H, D2O
exchangeable, 2NH), 7.28–7.53 (m, 14H, 2C6H5, C6H4),
2.84 (s, 3H, CH3), 2.40, 2.28 (2s, 4H, 2CH2), 1.08, 1.03 (2s,
6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ = 173.8,
172.5 (2C=N), 136.5, 132.3 (pyrazole C-4, C-5), 120.1,
120.8, 120.9, 121.7, 121.9, 122.2, 122.4, 123.6, 124.4,
124.9, 125.6, 128.8 (2C6H5, C6H4), 56.8 (C-5), 38.3 (CH3),
36.4 (C-6), 28.3 (C-7), 24.8 (2CH3); EIMS: m/z 435 [M]+
(28%); analysis Calcd for C28H29N5 (435.56): C, 77.21; H,
6.71; N, 16.08%. Found: C, 77.40; H, 6.52; N, 16.36%.
6,6-Dimethyl-3-(p-tolylamino)-6,7-dihydrobenzo[c]
isoxazol-4(5H)-one oxime (19c)
Pale yellow crystals from 1,4-dioxane; m.p. 180–183 °C;
yield 70%. IR (KBr) cm−1: 3559–3348, 3055, 2983, 1630.
1H NMR (200 MHz, DMSO-d6) δ 10.26 (s, 1H, D2O
exchangeable, OH), 8.28 (s, 1H, NH, D2O exchangeable,
NH), 7.25–7.44 (m, 4H, C6H4), 2.79 (s, 3H, CH3), 2.39,
2.23 (2s, 4H, 2CH2), 1.06, 1.03 (2s, 6H, 2CH3); 13C NMR
(DMSO-d6, 75 MHz): δ = 176.8 (C=N), 166.2 (C-4),
138.8, 133.4 (isoxazole C-4, C-5), 120.1, 121.4, 123.8,
124.6 (C6H4), 56.3 (C-2), 36.5 (C-6), 32.6 (C-5), 30.3
(CH3), 28.2 (C-7), 24.6 (2CH3); EIMS: m/z 285 [M]+
(42%); analysis Calcd for C16H19N3O2 (285.34): C, 67.35;
H, 6.71; N, 14.73%. Found: C, 67.40; H, 6.39; N, 14.62%.
General procedure for the synthesis of the isoxazole
derivatives 19a–c
Hydroxylamine hydrochloride (0.78 g, 0.01 mol) was added
to a solution of compound either 16a (2.43 g, 0.01 mol),
16b (2.77 g, 0.01 mol), or 16c (2.42 g, 0.01 mol) in 1,4-
dioxane (40 mL) containing sodium acetate (1.00 g). The
reaction mixture, in each case, was heated under reflux for
2 h then poured onto ice/water and the formed solid product
was collected by filtration.
Conclusion
In summary, in order to develop potent antitumor agents,
we have designed and synthesized a series of new het-
erocyclic compounds using dimedone. The produced
compounds were screened through six cancer cell lines
where compounds 7b, 7c, 7d, 9b, 11, 12c, 12d, 14b, 16b,
17c, 17d, 18c, 18d, and 18e were the most cytotoxic
compounds. Their further tests toward the five tyrosine
kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR and
Pim-1 kinase showed that compounds 7b, 7d, 14b, 16b,
and 18e were the most potent of the tested compounds
toward the five tyrosin kinases and compounds 7b, 7d,
14b, 16b, and 18e were of the highest inhibitions toward
Pim-1 kinase. PAINS the most cytotoxic compounds
showed zero PAINS alert thus these compounds are
considered anticancer agents without restrictions. There-
fore, the results laid a foundation for further improving
the potency and the selectivity of this series of
compounds.
6,6-Dimethyl-3-(phenylamino)-6,7-dihydrobenzo[c]
isoxazol-4(5H)-one oxime (19a)
Pale yellow crystals from 1,4-dioxane; m.p. 252–255 °C; yield
1
63%. IR (KBr) cm−1: 3580–3328, 3054, 2985, 1630. H
NMR (200 MHz, DMSO-d6) δ 10.22 (s, 1H, D2O exchange-
able, OH), 8.25 (s, 1H, NH, D2O exchangeable, NH),
7.22–7.39 (m, 5H, C6H5), 2.39, 2.23 (2s, 4H, 2CH2), 1.06,
1.05 (2s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz): δ =
176.6 (C=N), 166.2 (C-4), 138.6, 133.3 (isoxazole C-4, C-5),
119.3, 121.6, 123.6, 124.8 (C6H5), 56.8 (C-2), 36.7 (C-6), 32.2
(C-5), 28.4 (C-7), 24.8 (2CH3); EIMS: m/z 271 [M]+ (34%);
analysis Calcd for C15H17N3O2 (271.31): C, 66.40; H, 6.32; N,
15.49%. Found: C, 66.27; H, 6.48; N, 15.27%.
3-((4-Chlorophenyl)amino)-6,6-dimethyl-6,
7-dihydrobenzo[c]isoxazol-4(5H)-one oxime (19b)
Compliance with ethical standards
Pale yellow crystals from 1,4-dioxane; m.p. 230–233 °C;
yield 72%. IR (KBr) cm−1: 3559–3341, 3056, 2983, 1630.
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
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jurisdictional claims in published maps and institutional affiliations.
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