Targeting hepatocellular carcinoma: Synthesis of new pyrazole-based derivatives, biological evaluation, DNA binding, and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2019.04.011

A new series of pyrazole derivatives was prepared in this work, including pyrazolopyrimidines, pyrazolotriazines, pyrazolylthienopyridines, and 2-(pyrazolylamino)thiazol-4-ones, utilizing 3-amino-5-methyl-1H-pyrazole as a synthetic precursor. Their in vit

Full text of DOI:10.1016/j.bioorg.2019.04.011

Bioorganic Chemistry 88 (2019) 102917
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Targeting hepatocellular carcinoma: Synthesis of new pyrazole-based
derivatives, biological evaluation, DNA binding, and molecular modeling
studies
a
a,⁎
b
c
Dina M. Omran , Mariam A. Ghaly , Shahenda M. El-Messery , Farid A. Badria ,
d
a
Ehab Abdel-Latif , Ihsan A. Shehata
a
b
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of pyrazole derivatives was prepared in this work, including pyrazolopyrimidines, pyrazolo-
triazines, pyrazolylthienopyridines, and 2-(pyrazolylamino)thiazol-4-ones, utilizing 3-amino-5-methyl-1H-pyr-
azole as a synthetic precursor. Their in vitro anticancer activity was tested on hepatocellular carcinoma cell line,
HepG2. The results revealed that the pyrazolylhydrazonoyl cyanide 8, the pyrazolopyrimidine 3, and the pyr-
azolylaminothiazolone 17 were the most active with IC50 values of 2, 7, and 7 µM respectively in comparison
with 5.5 µM for cisplatin as a reference drug. Interestingly, all the synthesized compounds showed higher se-
lectivity index than cisplatin. DNA binding assay was also carried out for the synthesized compounds to ratio-
nalize their mechanism of action. Molecular modeling studies, including docking into DNA minor groove,
flexible alignment, and surface mapping, were conducted. Results obtained proved the superior DNA-binding
affinity of the most active anticancer compounds.
Pyrazole
Synthesis
Anticancer activity
DNA binding
Molecular docking
1
. Introduction
considered the most abundant binding modes of small ligands to DNA
[
5,6]. One of the prototype AT-selective DNA minor groove binders is
Cancer is ongoing to be a serious health trouble worldwide.
distamycin (I). It is a natural tripeptide antibiotic containing three
According to GLOBOCAN 2018 estimates, cancer new cases and deaths
have risen to 18.1 million and 9.6 million respectively in 2018 [1].
Hepatocellular carcinoma is considered as one of the most prevalent
cancers and the second leading cause of cancer deaths. Although the
mortality rate from other widespread cancers, e.g. lung and breast
cancers, is diminishing, the death rate from liver cancer elevated by
methylpyrrole units [7,8].
2
.8% in men and 3.4% in women per year [2]. Despite the great ad-
vances made in the chemotherapeutic management of cancer patients,
discovering novel efficient anticancer agents, selective on cancerous
and less toxic to normal cells, is still one of the hottest areas in med-
icinal chemistry research. DNA has long been proven to be the most
important target in cancer therapy. Generally, DNA interactive drugs
include DNA-alkylating agents, DNA intercalators, and DNA groove
binders [3]. Interaction of a small molecule with DNA leads to inter-
ruption in replication, transcription, and repair, and ultimately killing
the fast growing cells [4]. Intercalation and minor groove binding are
The majority of clinically used anticancer drugs are derived from
nitrogen heterocyclic compounds. Considerable attention has been paid
to the pyrazole scaffold as a highly flexible drug-like building block that
is widely used in the development of anticancer agents [9–11]. Rux-
olitinib and crizotinib (Fig. 1) are examples of pyrazole-containing
drugs used for treating myeloproliferative neoplasm and non-small cell
lung carcinoma respectively [12]. In addition, Reddy et al. [13]
⁎
Corresponding author.
E-mail address: mariamaghaly@mans.edu.eg (M.A. Ghaly).
https://doi.org/10.1016/j.bioorg.2019.04.011
Received 3 February 2019; Received in revised form 22 March 2019; Accepted 6 April 2019
Available online 08 April 2019
0045-2068/ © 2019 Published by Elsevier Inc.

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
flexible alignment, and surface mapping were also performed in order
to rationalize the experimental data obtained on the basis of computer-
aided tools.
2
. Results and discussion
2.1. Chemistry
The reaction of 3-amino-5-methyl-1H-pyrazole (1) with ethyl 2-
cyano-3,3-bis(methylthio)acrylate (2) [16] has been carried out by
heating in DMF to furnish the corresponding 5-amino-7-methylthio-
pyrazolo[1,5-a]pyrimidine derivative 3 (Scheme 1). The structure of
compound 3 was elucidated based on its compatible spectroscopic data.
−
1
Fig. 1. Examples of pyrazole-containing anticancer drugs.
The absorption of ester carbonyl, at lower frequency (1670 cm ) ra-
−1
ther than the usual absorption in the region 1735 cm , is attributed to
its formation of intramolecular hydrogen bonding with the next amino
reported the antiproliferative activities of a series of pyrazolylamide-
containing compounds, where compound II showed potent cytotoxic
activity (Fig. 2). In another study, the in vitro cytotoxic activities of a
series of substituted thiazolylpyrazole derivatives were reported, of
which compound III (Fig. 2) was the most potent, showing an IC50
value of 0.2 µM against HepG2 cell line [14]. In a third study, the an-
ticancer activity of a series of trisubstituted pyrazoles against hepato-
cellular carcinoma (HCC) cell lines was tested. Compound IV, bearing a
thiazolyl moiety, was the most active against HCC cell lines used, in-
cluding HepG2, and had a better activity than the reference drug sor-
afenib [12].
1
group. The H NMR spectrum clearly indicated triplet at δ 1.32 ppm
and quartet at δ 4.26 ppm for the ethoxy group (eOCH
2
CH ). The two
3
singlet signals at δ 2.42 and 2.47 were attributed to the protons of two
methyl groups (pyrazole-CH
3
and -SCH ). The proton of pyrazole-C3
3
resonated as singlet at δ 6.10 ppm while the protons of amino function
resonated as two singlet signals at δ 6.92 and 8.65 ppm. N,N-Dimethyl-
N′-pyrazolylformimidamide derivative 4 has been synthesized by re-
fluxing aminopyrazole
1 with dimethylformamide-dimethylacetal
(
DMF-DMA) in toluene [17]. Heating of N,N-dimethyl-N′-pyr-
azolylformimidamide derivative 4 with malononitrile in ethyl alcohol
induced further heterocyclization reaction to afford 7-amino-6-cyano-
pyrazolo[1,5-a]pyrimidine derivative 5 via elimination of dimethyla-
The incorporation of the pyrazole ring with other biologically active
heterocycles, e.g. pyrimidine, has been successfully employed by El-
Naggar and co-workers [15] who reported the remarkable activity of
the pyrazolopyrimidine derivative V against HepG2 cancer cell lines
showing an IC50 value less than that of doxorubicin (Fig. 2).
mine molecule HN(CH
3
2
) . The structure of compound 5 was secured
based on its compatible spectroscopic data. The infrared spectrum re-
−1
vealed absorptions at 3293, 3178 and 2222 cm
to indicate the ex-
1
istence of amino (NH
2
) and nitrile (C^N) groups, respectively. The H
Taking all the above considerations into account, the research re-
ported herein involves the synthesis of a new series of polysubstituted
pyrazole-based derivatives, including pyrazolopyrimidines and their
isosteric pyrazolotriazines, pyrazolylamides, and 2-(pyrazolylamino)
thiazol-4-ones (Fig. 2), their in vitro anticancer evaluation against
HepG2 cell line, and their DNA-binding evaluation. Furthermore, mo-
lecular modeling studies, including docking into DNA minor groove,
NMR spectrum indicated a singlet for one proton at δ 8.25 ppm, which
refers to the proton of pyrimidine at the fifth position.
3
-Amino-5-methyl-1H-pyrazole (1) was employed as an important
precursor for the preparation of pyrazolo[5,1-c]triazine derivatives
through its diazotization by sodium nitrite and HCl followed by cou-
pling with various activated nitriles. Thus, the diazocoupling reaction
of the pyrazolyl diazonium chloride 6 with 3-iminobutanenitrile (7)
Fig. 2. Examples of pyrazole-containing anticancer compounds and the designed target compounds.
2

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Scheme 1. Synthesis of pyrazolopyrimidines 3 and 5.
Scheme 2. Synthesis of pyrazolotriazine derivative 9.
was carried out in ethanol and sodium acetate to produce the corre-
sponding hydrazonoyl cyanide 8, which upon warming in acetic acid
gave the corresponding pyrazolo[5,1-c]triazine derivative 9 (Scheme
spectral data. The presence of infrared absorptions at υ = 3249, 3182
−1
and 1761 cm
referred to the functional groups NeH and C]O, re-
1
spectively. The H NMR signal at δ 4.22 ppm that integrated for two
protons indicated the presence of methylene group (eCOCH -Cl). The
2
). The infrared spectrum displayed the absorptions of NeH stretching
2
−
1
vibrations at 3392 and 3235 cm conforming to the amino function, in
nucleophilic replacement of chlorine atom from the pyrazolyl-chlor-
oacetamide derivative 13 by different sulfur nucleophiles was in-
vestigated. Firstly, nucleophilic substitution of chlorine was tested by
the reaction of 13 with 2-mercapto-nicotinonitrile compound 14; the
reaction proceeded in acetone containing anhydrous potassium carbo-
nate to furnish the conforming sulfide compound 15. This sulfide
compound 15 was used as a precursor for the building of its corre-
sponding thieno[2,3-b]pyridine derivative 16 through the successful
intramolecular cyclization in hot ethanolic sodium ethoxide (Scheme
4). The formation of this thieno[2,3-b]pyridine derivative 16 may be
interpreted through the nucleophilic addition of the methylene group to
nitrile function according to the mechanistic consideration. The struc-
tures of these synthesized compounds 15 and 16 were established by
agreeable IR and NMR data. The IR spectrum of 15 displayed the ab-
−1
1
addition to the absorption of one carbonyl group at 1661 cm . The H
NMR signals resonated as singlet at δ 2.50 ppm (pyrazole-CH ), singlet
at δ 2.73 (COCH ), singlet at δ 6.90 (pyrazole-H8) and two singlet
signals at δ 9.11 and 9.30 (NH
3
3
2
).
In addition, the diazotized aminopyrazole compound 6 has been
coupled with benzothiazolyl-cyanoacetamide derivative 10 by stirring
in cold ethanol containing sodium acetate to furnish the conforming
hydrazonoyl cyanoacetamide 11. This hydrazone was readily cyclized
into its pyrazolotriazine derivative 12 by heating in acetic acid (Scheme
3
). The chemical structure of 12 was confirmed by spectroscopic
techniques including IR and NMR analyses. The infrared absorption at
−
1
1
663 cm
clearly indicated the presence of carbonyl-amidic (CONH).
), singlet at δ
1
The H NMR signals were singlet at δ 2.30 ppm (eCH
3
−1
6
.95 ppm (pyrazole-H4), two singlet signals at δ 8.83 and 9.34 (NH
2
)
sorption band of nitrile group at 2215 cm that disappeared from the
1
and singlet at δ 12.19 ppm (NH).
IR spectrum of 16 as an indication for the cyclization progress. The H
Chloroacetylation of 3-aminopyrazole 1 was achieved by stirring
with chloroacetyl chloride in the presence of DMF and triethylamine to
afford the pyrazolyl-chloroacetamide derivative 13 (Scheme 4). The
chemical structure of 13 was confirmed because of its agreeable
NMR spectrum of compound 15 exhibited the two protons of methylene
group as singlet signal at δ 4.12 ppm; this signal disappeared from
spectrum of the product 16 as additional evidence for the successful
cyclization reaction.
3

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Scheme 3. Synthesis of pyrazolotriazine derivative 12.
Moreover, the reaction of pyrazolyl chloroacetamide derivative 13
with ammonium thiocyanate has been carried out in boiling ethanol to
afford 2-((5-methyl-1H-pyrazol-3-yl)amino)thiazol-4(5H)-one (17)
which finds support from its correct spectral data (Scheme 5). The in-
2.2. Biological evaluation
2.2.1. Cytotoxicity assay
Fourteen compounds were tested in vitro against hepatocellular
carcinoma cell line (HepG2). Interestingly, all the tested compounds
revealed a higher selectivity index (SI) than cisplatin as a reference
drug (SI = 0.91). In particular, compounds 3, 8, and 17 exhibited no-
table cytotoxicity against HepG2 with IC50 values of 7, 2, and 7 µM,
respectively, in comparison with 5.5 µM for cisplatin, in addition to
SI > 2 (Table 1).
−
1
frared absorption at 1722 cm
assigned the carbonyl group of the
thiazol-4-one ring. The cyclic methylene group of the thiazol-4-one ring
1
resonated as singlet ( H NMR spectrum) for two protons at δ 3.91 ppm.
The constructed thiazol-4-one derivative 17 was condensed with three
benzaldehyde derivatives 18 (namely, 4-toulaldehyde, 4-anisaldehyde
and 4-nitrobenzaldehyde) in glacial CH COOH and anhydrous
3
CH
3
COONa (Knoevenagel condensation reaction) furnished the corre-
sponding
5-arylidene-2-((5-methyl-1H-pyrazol-3-yl)amino)thiazol-
2
.2.2. DNA-binding assay
4
(5H)-one derivatives 19a-c. The infrared absorption of these thiazo-
−1
DNA represents the target of many small molecules that bind it and
lones 19a-c in the region 1700–1707 cm
clearly indicated the pre-
proved to be effective anticancer drugs. Various strategies have been
used to study the interaction of these small molecular weight com-
pounds with DNA. Methyl green in particular works by reversibly
binding to DNA, producing a colored complex stable at neutral pH,
while unbound methyl green fades. The DNA-binding active candidate
displaces methyl green from its DNA complex. A spectroscopic assay
1
sence of carbonyl-thiazolone ring. The H NMR signals of 19b (as an
example) were singlet at δ 2.23 ppm (pyrazole-CH
.81 ppm (eOCH ), singlet at δ 5.89 ppm (pyrazole-H4), two doublet at
δ 7.10 and 7.65 ppm (aromatic-H) and singlet for two protons at δ
2.24 ppm (2NH).
3
), singlet at δ
3
3
1
Scheme 4. Synthesis of pyrazolyltheinopyridine derivative 16.
4

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Scheme 5. Synthesis of 5-arylidene-2-(pyrazolylamino)-thiazol-4-one derivatives 19a-c.
Table 1
Table 2
The cytotoxic activity and selectivity of the synthesized compounds against
hepatocellular carcinoma (HepG2) cell line.
Effect of synthesized compounds on the displacement activity for methyl green
from DNA (DNA-binding assay).
*
*
VERO-B (IC50, µM)**
SI*
*% of Methyl Green Displacement
Compound
HepG2 (IC50, µM)
Compound
3
4
5
8
9
7 ± 0.40
40 ± 0.50
40 ± 0.50
2 ± 0.03
40 ± 0.50
12 ± 1.5
40 ± 0.50
40 ± 0.50
11.8 ± 0.20
12 ± 1.5
7 ± 0.40
50 ± 0.30
50 ± 0.30
40 ± 0.50
5.5 ± 1.5
15.001 ± 0.10
50.007 ± 0.32
50.003 ± 0.40
9.000 ± 0.05
50.002 ± 0.07
30.001 ± 0.02
50.008 ± 0.15
50.007 ± 0.02
26.000 ± 0.03
30 ± 0.20
2.14
1.25
1.25
4.50
1.25
2.50
1.25
1.25
2.20
2.50
2.14
1.20
1.20
1.25
0.91
3
80.98 ± 0.40
47.77 ± 0.03
49.91 ± 0.12
90.41 ± 0.31
48.64 ± 0.24
66.63 ± 0.14
56.57 ± 0.11
49.52 ± 0.27
69.33 ± 0.110
66.24 ± 0.20
80.27 ± 0.13
30.50 ± 0.07
39.53 ± 0.18
50.31 ± 0.32
97.22 ± 0.01
4
5
8
9
1
1
1
1
1
1
1
1
1
1a
2a
3
11a
12a
13
5
15
6
16
7
15 ± 0.17
17
9a
9b
9c
60 ± 0.25
19a
60 ± 0.04
19b
50 ± 0.03
19c
Positive Control
5 ± 0.11
Doxorubicin (Standard)
*
*
SI: Selectivity Index.
* Values represent the concentration (mean ± SD, n = 3–5 separate de-
terminations) required for a 50% decrease in the initial absorbance of the DNA
methyl green solution.
* Values represent the concentration (mean ± SD, n = 3–5 separate de-
terminations).
could determine this displacement through measuring the absorbance
decrease at 630 nm [18].
analysis of the target compounds were obtained using the MM force-
field [20] followed by (calculations in vacuo, bond dipole option for
electrostatics using PolakeRibiere algorithm, and RMSD gradient of
Table 2 showed that compounds 3, 8, and 17 displaced methyl
green from DNA and bound to DNA with % of displacement at 80%,
0
.15 kcal/Å mol) implemented in MOE, 2009.10 [21].
9
0%, and 80% respectively.
The lowest energy conformers of the most active DNA binders 8, 3,
and 17, were represented in Fig. 3a,b,c while Fig. 3d,e showed the
lowest conformers of inactive candidates 19a and 19b respectively. The
X-ray crystallographic structure of DNA dodecamer d(CGCAAATTT
GCG) conjugated with a bifurcated hydrogen-bonded conformation of
the AT base pairs with its complexed distamycin A was obtained from
the Protein Data Bank (PDB code: 2DND) and used for the docking
study [22].
2.3. Molecular modeling studies
2
.3.1. Molecular docking study
Molecular modeling studies have been introduced in this work as a
valuable tool to rationalize the DNA binding results obtained experi-
mentally. Binding modes of ligands to DNA usually include intercala-
tion between adjacent base pairs or intrusion into the minor or the
major groove [19].
Fig. 4 showed the binding mode of distamycin reference. It was
bound to DNA bases adenine, cytosine, and thiamine via hydrogen
bonding network interactions by 79%, 67% and 88% respectively with
a calculated binding energy of −11.3452 kcal/Å mol.
To get a better image, the molecular structures of both the active
compounds 8, 3, and 17, and inactive counterparts 19a and 19b, in a
comparative modeling study style, were constructed and overviewed at
DNA binding site. At the beginning of the work, conformational
Compounds 8, 3, and 17 were selected in our modeling study as
active DNA binding agents. Alignment of these derivatives against
5

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Fig. 3. Lowest energy conformers of active compounds (a) 8, (b) 3, (c) 17, and the least active compounds (d) 19a and (e) 19b in balls and cylinders.
distamycin reference was conducted. The results were shown
−9.9987 kcal/Å mol (Fig. 6c) (DNA base pairs were omitted in previous
figures for simplicity). On the contrary, docking study of inactive DNA
binders represented by 19a, 19b obviously exhibited a different
binding mode into DNA minor groove. Both of them were not well
hosted into the DNA bases, and did not show any hydrogen bonding to
them (Fig. 7a,b).
(
Fig. 5a–c) where it was obvious the high alignment between the active
DNA binders and distamycin. Moreover, their overall shape oriented
along the minor groove in a curved manner which in major part in-
terprets their DNA binding activity.
The binding mode of these compounds were also under focus, where
a remarkable note was detected. These compounds simply manifested a
binding mode (hydrogen bonding) similar to distamycin which may
account for their higher activity. Compound 8 showed triad hydrogen
bonding mode via 16%, 91%, and 78% with binding energy of
Such clear contrast between the tested active and inactive candi-
dates has interpreted the difference in biological response of these
compounds into DNA double strand and showed the importance of
hydrogen bonding in stabilizing the binding interactions.
−
10.4489 kcal/Å mol (Fig. 6a). Compound 3 had the hydrogen
bonding in bi mode by 42% and 66% with a binding affinity of
10.2834 kcal/Å mol (Fig. 6b). Compound 17 binding mode occurred
via double interaction by 13% and 94% with binding affinity of
2
.3.2. Flexible alignment
−
Alignment of compounds at the active site is a known technique to
structurally analyse protein ligand complexes. A good alignment is
Fig. 4. (a) Distamycin, shown in yellow space filled form lying in between DNA base pairs, DNA shown in ribbon form. (b) DNA minor groove binding of distamycin
(
yellow) with DNA in 2D form. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
6

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Fig. 5. Sketches of minimized and docked structures in the DNA (blue ribbon) revealing overlay of distamycin (cyan) in duplex together with: (a) 8 (orange), (b) 3
pink) and (c) 17 (brown). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(
achieved when the strain energy of each molecule is small along with
occurrence of similar shape and aromatic atoms overlapping between
compounds [23]. Triple flexible alignment experiments have been
performed. Firstly, that experiment was conducted using three active
candidates 8, 3, and 17 against each other through generation of the
lowest energy of 200 conformers for each compound, minimization by a
distance dependent dielectric model, then selection of a low energy set
of 100 for further analysis. In Fig. 8a, a higher alignment score was
calculated with the least strain energy which represents a better
alignment score. Secondly, the same experiment was done using the
two chosen inactive derivatives 19a and 19b which again showed
higher matching, Fig. 8b. Finally, the alignment experiment was re-
peated using both active and inactive candidates represented by 8 and
19b which, as expected, showed recognized deviation from alignment
pattern ensuring and illustrating that their biological activity are
different.
2.3.3. Surface mapping
For further investigating the different activity patterns of the tested
compounds, a surface mapping study was conducted [24]. At first,
surface mapping of active candidate 8 was created (Fig. 9a) followed by
surface hydrophobic mapping of inactive counterpart compound 19b
(Fig. 9b). Compound 8 showed more greener areas on the map in-
dicating more hydrophobic regions, while the inactive compound 19b
showed a different surface mapping distribution with less green col-
oration indicating higher hydrophilic feature of the whole molecule.
Fig. 6. DNA minor groove binding of (a) 8 (orange), (b) 3 (pink), and (c) 17 (brown). DNA is shown in ribbon form. Investigated compounds are shown in ball and
cylinder form. Hydrogen-bonding (HB) interactions with the DNA bases are shown in dashed lines. Numbers indicate the percent of HB. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
7

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Fig. 7. (a) 2D binding mode and residues involved in the recognition of 19a. (b) Close-up view of inactive candidate 19b binding in the DNA minor groove,
highlighting the conformation of DNA active out of minor groove.
Fig. 8. (a) Flexible alignment of active DNA binders 8 (orange), 3 (pink), and 17 (brown). (b) Flexible alignment of inactive DNA binders 19a (yellow) and 19b
(
green). (c) Flexible alignment of active DNA binders 8 (orange) and inactive one 19b (yellow). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Comparing the two figures (Fig. 9a,b) obviously confirms the different
patterns of the compounds of interest providing additional explanation
for their different DNA binding activity.
particularly, compound 8, making it a promising anticancer candidate
that could be combined with commonly used chemotherapeutic drugs
aiming to improve the tumor response. A more strict in vivo study could
be undertaken to reveal more preclinical data such as bioavailability,
pharmacokinetics, and oral stability with expectation of good activity
and high safety.
3
. Conclusion
In this study, a new series of polysubstituted pyrazole-based deri-
vatives was synthesized and evaluated for anticancer activity against
hepatocellular carcinoma. DNA binding assay was also performed for
the synthesized compounds to rationalize their mechanism of action.
Results obtained proved the superior DNA-binding affinity of com-
pounds 8, 3, and 17. Molecular modeling studies, including docking
into DNA minor groove, flexible alignment, and surface mapping were
carried out. The results were in accordance with the experimental re-
sults, providing additional explanation for the DNA-binding activity of
the most active anticancer compounds. The results obtained proved the
unique character, high selectivity and promising activity of several
tested compounds against aggressive liver cancer cell line HepG2,
4. Experimental protocols
4.1. Chemistry
Melting points are uncorrected and measured on Gallenkamp elec-
tric apparatus. The IR spectra were recorded on a Thermo Scientific
Nicolet iS10 FTIR spectrometer. The NMR spectra were determined
1
using a Bruker WP 300 spectrometer at 300 MHz for H NMR or
1
3
100 MHz for C NMR. The MS analysis was carried on a Quadrupole
GC/MS Thermo Scientific Focus/DSQII at 70 eV. Elemental analyses (C,
H and N) were determined on Perkin-Elmer 2400 analyzer.
8

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
Fig. 9. (a) Surface map for the most active compound 8 in the pocket side. (b) Surface map for the least active compound 19b in the pocket side. Green: hydrophobic,
blue: mild polar, pink: hydrophilic.
4
.1.1. Synthesis of 5-amino-6-ethoxycarbonyl-2-methyl-7-methylthio-pyrazolo
ethyl alcohol for 2 hrs. The solid obtained upon cooling was picked up
by filtration and then dried.
[1,5-a]pyrimidine (3)
−
1
To a suspension of 3-amino-5-methyl-1H-pyrazole (1) (2 mmol,
White solid, yield = 68%, m.p. = 240–242 °C. IR (νmax/cm ):
1
0
.19 g) in 15 ml DMF, ethyl 2-cyano-3,3-bis(methylthio)acrylate (2)
3293, 3178 (NH
2
), 2222 (C^N), 1674 (C]N). H NMR (DMSO‑d
6
): δ
(
2 mmol, 0.43 g) was added and boiled under reflux for 4 hrs. The solid
2.41 (s, 3H), 6.39 (s, 1H, pyrazole-H3), 8.25 (s, 1H, pyrimidine-H5),
13
that separated upon dilution with 20 ml cold water was picked up by
filtration. Recrystallization of the filtered solid was achieved by heating
in ethanol.
8.81 (s, 2H, NH
149.51, 151.42, 155.85.MS (m/z, %): 173 (100.00), 120 (18.03), 108
(8.53). Analysis for C (173.18): Calcd: C, 55.48; H, 4.07; N,
40.44%. Found: C, 55.34; H, 4.10; N, 40.55%.
2
). C NMR (DMSO‑d
6
): δ 14.75, 73.14, 97.71, 116.67,
8
H N
7 5
−1
Yellow solid, yield = 64%, m.p. = 191–193 °C. IR (νmax/cm ):
1
3
374, 3263 (eNH
2
), 1670 (C]O). H NMR (CDCl
3
): δ 1.32 (t,
J = 7.15 Hz, 3H), 2.42 (s, 3H), 2.47 (s, 3H, SCH
3
), 4.26 (q, J = 7.15 Hz,
4
.1.4. Synthesis of N-(5-methyl-1H-pyrazol-3-yl)-hydrazonoyl cyanides 8
and 11
A well-stirred suspension of 1 (10 mmol, 0.97 g) in conc. HCl (3 ml)
was cooled in ice bath and diazotized with NaNO solution (10 mmol,
1
3
2
H), 6.10 (s, 1H, pyrazole-H3), 6.92 (s) and 8.65 (s) (2H, NH
2
).
): δ 9.66, 9.90, 10.04, 56.55, 84.38, 90.88, 142.75,
S (266.32):
C
NMR (DMSO‑d
6
1
44.31, 151.59, 157.87, 162.43. Analysis for C11
H
14
N
4 2
O
2
Calcd: C, 49.61; H, 5.30; N, 21.04%. Found:C, 49.43; H, 5.36; N,
0
.70 g). The obtained cold solution was slowly added to a well-stirred
2
1.15%.
solution of 3-iminobutanenitrile 7 (10 mmol, 0.82 g) and/or ben-
zothiazolyl-cyanoacetamide derivative 10 (10 mmol) in 30 ml ethanol
and 3 g of sodium acetate. The reaction mixture was stirred for an hour
and then kept in refrigerator for 12 h. The solid obtained was filtered,
washed with 5 ml cold ethanol and dried to pick the hydrazonoyl cy-
anides 8 and 11.
4
.1.2. Synthesis of N,N-dimethyl-N'-(5-methyl-1H-pyrazol-3-yl)-formimidamide
(4)
To a solution of 3-amino-5-methyl-1H-pyrazole (1) (10 mmol,
0
.97 g) in 15 ml toluene, DMF-DMA (10 mmol, 1.2 ml) was added and
refluxed for 4 hrs. The solid obtained upon cooling was filtered and
recrystallized from ethyl alcohol.
4
.1.5. 2-Imino-N-(5-methyl-1H-pyrazol-3-yl)propanehydrazonoyl cyanide
White solid, yield = 86%, m.p. = 122–123 °C, lit. m.p. 120–122 °C
(
8)
−1
1
−
1
[
17]. IR (νmax/cm ): 3204 (NeH), 1636 (C]N). H NMR (DMSO‑d ): δ
6
Yellow solid, yield = 46%, m.p. = 128–130 °C. IR (νmax/cm ):
1
2
1
.19 (s, 3H), 2.86 (s, 3H), 2.97 (s, 3H), 6.22 (s, 1H, pyrazole-H4), 8.12 (s,
13
3
324, 3135 (NeH), 2235 (C^N). H NMR (CDCl
3
): δ 2.68 (s, 3H), 3.05
H, N]CH), 10.39 (s, 1H, NH). C NMR (DMSO-d
6
7
): δ 11.82, 34.41
(
s, 3H), 7.20 (s, 1H, pyrazole-H4), 7.28 (s, 1H, NH). Analysis for
(190.21): Calcd: C, 50.52; H, 5.30; N, 44.18%. Found: C,
0.31; H, 5.36; N, 44.30%.
(
2C), 96.15, 155.02, 158.86, 162.68. Analysis for C
H
12 4
N
(152.20):
C
8
H N
10 6
Calcd: C, 55.24; H, 7.95; N, 36.81%. Found:C, 55.40; H, 7.91; N, 36.89%.
5
4
.1.3. Synthesis of 7-amino-6-cyano-2-methylpyrazolo[1,5-a]pyrimidine (5)
A suspension of N,N-dimethyl-N'-pyrazol-3-yl-formimidamide
3 mmol, 0.45 g) and malononitrile (3 mmol, 0.20 g) was refluxed in
4.1.6. 2-(Benzothiazol-2-ylamino)-N-(5-methyl-1H-pyrazol-3-yl)-2-
oxoacetohydrazonoyl cyanide (11)
4
(
Reddish brown solid, yield = 66%, m.p. = 156–157 °C. IR (νmax/
9

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
−
1
1
cm ): 3332, 3293, 3199 (NeH), 2219 (C^N), 1662 (C]O). H NMR
poured into cold water and neutralized with HCl. The precipitate that
formed was filtered, dried and recrystallized from 30 ml ethanol to give
the conforming thieno[2,3-b]pyridine compound 16.
(
CDCl
3
): δ 2.60 (s, 3H), 6.89 (s, 1H, pyrazole-H4), 7.36–7.88 (m, 4H),
8
.70 (s, 1H), 8.79 (s, 1H). Analysis for C14
H
11
N OS (325.35): Calcd: C,
7
−
1
5
1.68; H, 3.41; N, 30.14%. Found: C, 51.54; H, 3.37; N, 30.20%.
White solid, yield = 48%, m.p. = 210–211 °C. IR (νmax/cm ):
1
3
341, 3187 (NH
2
and NeH), 1658 (C]O). H NMR (DMSO‑d
6
): δ 2.21
4
.1.7. Synthesis of 4-amino-7-methylpyrazolo[5,1-c][1,2,4]triazines 9 and
(s, 3H), 2.56 (s, 3H), 2.68 (s, 3H), 6.25 (s, 1H, pyrazole-H4), 6.92 (s,
1
3
12
1H, NH
2
), 7.01 (s, 1H, pyridine-H5), 9.82 (s, 1H). C NMR (DMSO‑d
6
):
A suspension of hydrazonoyl cyanides 8 and/or 11 (3 mmol) was
δ 11.71, 19.87, 24.15, 103.83, 121.02, 122.68, 125.38, 137.83, 143.62,
heated in 10 ml acetic acid for half an hour. The solid obtained upon
147.41, 154.44, 156.02, 159.00, 160.04. Analysis for C14
15 5
H N OS
cooling was filtered and dried to furnish the pyrazolotriazines 9 and/or
(301.37): Calcd: C, 55.80; H, 5.02; N, 23.24%. Found: C, 55.68; H, 5.08;
1
2.
N, 23.35%.
4
.1.8. 3-Acetyl-4-amino-7-methylpyrazolo[5,1-c][1,2,4]triazine (9)
4.1.13. Synthesis of 2-((5-methyl-1H-pyrazol-3-yl)amino)thiazol-4(5H)-
−
1
Brown solid, yield = 42%, m.p. = 280–281 °C. IR (νmax/cm ):
one (17)
1
3
392, 3235 (NH
2
), 1661 (C]O). H NMR (DMSO‑d
6
): δ 2.50 (s, 3H),
To a suspension of chloroacetamido-pyrazole derivative 13
1
3
2
.73 (s, 3H), 6.90 (s, 1H, pyrazole-H8), 9.11 (s, 1H), 9.30 (s, 1H).
C
(10 mmol, 1.73 g) in 20 ml ethanol, ammonium thiocyanate (15 mmol,
1.14 g) was added and boiled under reflux for 5 hrs. The solid obtained
upon cooling was filtered and recrystallized from ethanol to furnish the
thiazolone derivative 17.
NMR (DMSO‑d
6
): δ 14.74, 26.98, 98.33, 125.21, 141.00, 144.17,
1
49.67, 157.20. Analysis for C
8
H
10
N (191.19): Calcd: C, 50.26; H,
6
4
.74; N, 36.63%. Found: Calcd: C, 50.45; H, 4.71; N, 36.76%.
−
1
Yellow solid, yield = 67%, m.p. = 225–226 °C. IR (νmax/cm ):
1
4
.1.9. 4-Amino-N-(benzo[d]thiazol-2-yl)-7-methylpyrazolo[5,1-c][1,2,4]-
3276, 3136 (NeH), 1722 (C]O). H NMR (DMSO‑d
6
): δ 2.19 (s, 3H,
triazine-3-carboxamide (12)
CH
3
), 3.91 (s, 2H, CH
2
), 5.78 (s, 1H, pyrazole-H4), 11.54 (s, 1H), 11.94
−1
13
Brown solid, yield = 56%, m.p. = 258–259 °C. IR (νmax/cm ):
(s, 1H). C NMR (DMSO‑d
6
): δ 11.36, 35.98, 97.84, 139.55, 152.19,
1
3
3
1
443, 3346 (NH and NH
2
), 1663 (C]O). H NMR (DMSO‑d
6
): δ 2.30 (s,
161.92, 177.40. Analysis for C
H
7 8
N OS (196.23): Calcd: C, 42.85; H,
4
H, CH
H), 8.03 (d, 1H), 8.83 (s, 1H), 9.34 (s, 1H), 12.19 (s, 1H, NH).
): δ 14.75, 98.58, 116.24, 117.90, 121.13, 122.18,
23.72, 126.70, 147.37, 149.11, 151.18, 157.14, 161.22, 172.50. MS
m/z, %): 325 (7.31), 297 (4.04), 216 (27.21), 176 (100.00), 149
3
), 6.95 (s, 1H, pyrazole-H4), 7.34 (t, 1H), 7.47 (t, 1H), 7.80 (d,
4.11; N, 28.55%. Found: C, 42.98; H, 4.06; N, 28.46%.
1
3
C
NMR (DMSO‑d
6
4.1.14. Synthesis of 2-((5-methyl-1H-pyrazol-3-yl)amino)-5-arylidenethiazol-
4(5H)-one derivatives 19a-c
1
(
(
To a suspension of thiazol-4-one derivative 17 (3 mmol, 0.59 g) in
53.73), 122 (58.77). Analysis for C14
H
11
N
7
OS (325.35): Calcd: C,
15 ml glacial CH COOH, the appropriate para-substituted benzaldehyde
3
5
1.68; H, 3.41; N, 30.14%. Found: C, 51.84; H, 3.47; N, 30.03%.
derivative (3 mmol) and 0.5 g of fused CH COONa were added and then
3
boiled under reflux for 4 h. The reaction mixture was poured onto ice-
water, the obtained solid was picked up by filtration and recrystallized
from ethyl alcohol.
4.1.10. Synthesis of 3-chloroacetamido-5-methyl-1H-pyrazole (13)
To a cold suspension of 1 (10 mmol, 0.97 g) and anhydrous K
2
CO
3
(
(
10 mmol, 1.38 g) in 20 ml dry acetone, chloroacetyl chloride
15 mmol, 1.2 ml) was added drop by drop. The reaction mixture was
4.1.15. 2-((5-Methyl-1H-pyrazol-3-yl)amino)-5-(4-methylbenzylidene)-
stirred for 4 h and then diluted with 50 ml ice-cold water. The solid
formed was filtered and recrystallized from ethyl alcohol.
thiazol-4(5H)-one (19a)
−
1
Yellow solid, yield = 59%, m.p. = 144–145 °C. IR (νmax/cm ):
−1
1
White solid, yield = 82%, m.p. = 118–120 °C. IR (νmax/cm ):
3218, 3138 (NeH), 1707 (C]O). H NMR (DMSO‑d
6
): δ 2.23 (s, 3H),
1
3
249, 3182 (NeH), 1761 (C]O), 1665 (C]N). H NMR (CDCl
3
): δ 2.62
2.36 (s, 3H), 5.89 (s, 1H, pyrazole-H4), 7.15 (d, 1H, NH), 7.36 (d, 2H),
13
(
s, 3H), 4.22 (s, 2H), 5.85 (s, 1H), 6.82 (s, 1H, pyrazole-H4), 11.95 (s,
7.50 (d, 2H), 7.62 (s, 1H, olefinic C]CH), 12.36 (s, 2H, 2NH). C NMR
(DMSO‑d ): δ 11.29, 21.52, 116.88, 125.29, 126.94, 129.64, 129.78,
1
3
1
1
2
H). C NMR (DMSO-d
6
): δ 14.51, 42.49, 104.07, 148.63, 155.36,
6
65.91. Analysis for C
6
H
8
ClN
3
O (173.60): Calcd: C, 41.51; H, 4.65; N,
130.43 (4C), 131.47, 133.28, 140.28, 165.21. Analysis for C15
H
14
4
N OS
4.21%. Found: C, 41.58; H, 4.62; N, 24.29%.
(298.36): Calcd: C, 60.38; H, 4.73; N, 18.78%. Found: C, 60.26; H, 4.77;
N, 18.68%.
4
.1.11. Synthesis of 2-((3-cyano-4,6-dimethylpyridin-2-yl)thio)-N-(5-
methyl-1H-pyrazol-3-yl)acetamide (15)
4.1.16. 5-(4-Methoxybenzylidene)-2-((5-methyl-1H-pyrazol-3-yl)amino)-
To a stirred suspension of pyridine-2-thiol derivative 14 (10 mmol
thiazol-4(5H)-one (19b)
−
1
1
.64 g) and anhydrous K
2
CO
3
(10 mmol, 1.38 g) in 40 ml acetone, 3-
Yellow solid, yield = 44%, m.p. = 161–163 °C. IR (νmax/cm ):
1
chloroacetamido-5-methyl-1H-pyrazole (13) (10 mmol, 1.73 g) was
added. The reaction mixture was refluxed for two hrs and then allowed
to cool to room temperature. The solid, obtained after dilution by 20 ml
cold water, was filtered and dried. Recrystallization of the crude pro-
duct was achieved by heating in ethyl alcohol.
3214, 3135 (NeH), 1706 (C]O). H NMR (DMSO‑d
6
): δ 2.23 (s, 3H,
CH
(d, 2H), 7.65 (s, 1H, olefinic C]CH), 12.24 (s, 2H, 2NH). C NMR
(DMSO‑d ): δ 11.26, 55.92, 98.63, 115.29 (2C), 126.72, 129.81, 131.88
(2C), 137.41, 147.73, 151.74, 156.07, 160.90, 168.36. Analysis for
3
), 3.81 (s, 3H, OCH
3
), 5.89 (s, 1H, pyrazole-H4), 7.10 (d, 2H), 7.55
13
6
−1
Yellow solid, yield = 60%, m.p. = 144–145 °C. IR (νmax/cm ):
C
15
H
14
N
4
O S (314.36): Calcd: C, 57.31; H, 4.49; N, 17.82%. Found: C,
2
1
3
3
7
329 (NeH), 2215 (C^N), 1728 (C]O). H NMR (DMSO-d
6
): δ 2.17 (s,
57.47; H, 4.54; N, 17.73%.
H), 2.40 (s, 3H), 2.43 (s, 3H), 4.12 (s, 2H), 6.18 (s, 1H, pyrazole-H4),
.09 (s, 1H, pyridine-H5), 10.64 (s, 1H, NH), 11.59 (s, 1H, NH).
4.1.17. 2-((5-Methyl-1H-pyrazol-3-yl)amino)-5-(4-nitrobenzylidene)
thiazol-4(5H)-one (19c)
Analysis for C14
H
15
N OS (301.37): Calcd: C, 55.80; H, 5.02; N, 23.24%.
5
Found: C, 55.91; H, 5.06; N, 23.15%.
Reddish brown solid, yield = 56%, m.p. = 204–205 °C. IR (νmax
/
−
1
1
cm ): 3364, 3148 (NeH), 1700 (C]O). H NMR (DMSO‑d
6
): δ 2.22 (s,
4
.1.12. Synthesis of 3-amino-4,6-dimethyl-N-(5-methyl-1H-pyrazol-3-yl)-
3H, CH
2H), 8.33 (d, 2H), 12.48 (s, 2H, 2NH). C NMR (DMSO‑d
99.44, 119.12, 124.73 (2C), 127.19, 130.72, 130.84, 131.06 (2C), 140.67,
147.46, 148.57, 172.44. Analysis for C14 S (329.33): Calcd: C,
51.06; H, 3.37; N, 21.27%. Found: C, 51.28; H, 3.30; N, 21.14%.
3
), 5.89 (s, 1H, pyrazole-H4), 7.65 (s, 1H, olefinic C]CH), 7.81 (d,
13
thieno[2,3-b]pyridine-2-carboxamide (16)
6
): δ 11.22,
The prepared acetamide derivative 15 (3 mmol, 0.90 g) was re-
fluxed for an hour in sodium ethoxide solution (prepared by dissolving
11 5 3
H N O
0
.10 g sodium metal in 20 ml ethanol). The reaction mixture was
10

D.M. Omran, et al.
Bioorganic Chemistry 88 (2019) 102917
4
.2. Biological evaluation
AM1 (as implemented in MOE, 2009.10). Polake–Ribiere algorithm and
Root Mean Square Deviation (RMSD) gradient of 0.01 kcal/mol con-
formational searching in torsional space was carried out using the
multiconformer method.
4.2.1. Cytotoxicity assay
The cytotoxic activity of the synthesized compounds was tested
against hepatocellular carcinoma cell line (HepG2). African green
monkey kidney cells (Vero-B) were used as normal cells to examine the
selectivity toward cancer cells. HepG2 cell line was obtained from the
American Type Culture Collection (ATCC). The cells were cultivated at
4.3.2. Docking study
The 3D structures in their neutral forms of the selected derivatives,
which represented the most and least active compounds, were built
using the MOE of Chemical Computing Group Inc software. The global-
minimum (the lowest energy conformer) of each of them was docked
into DNA double strands code ID 2DND, obtained from the Protein Data
Bank of Brookhaven National Laboratory.
3
1
1
7 °C and 10% CO in DMEM (Lonza, Germany) medium supplied with
2
0% fetal bovine serum (Lonza, Germany), 100 IU/ml penicillin and
00 µg/ml streptomycin (Lonza, Germany). Cisplatin (cis-diaminepla-
tinum (II) dichloride) was obtained from Sigma-Aldrich and used as a
positive control. It was dissolved in 0.9% saline and stored as an 8 mM
stock solution at −20 °C. The tested compounds were dissolved in
DMSO and stored at −20 °C. The cells' viability was quantified by
measuring the activity of mitochondrial succinate dehydrogenase in
viable cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) [25,26].
All the hydrogens were added to a refinement protocol in which the
constraints on the enzyme were gradually removed and minimized till
reaching a 0.01 kcal/mol/Å RMSD gradient. Energy minimization was
performed using ‘AMBER’ molecular mechanics force field. For each
ligand, energy minimizations were carried out using 1000 steps of
steepest descent, followed by conjugate gradient minimization to a
RMSD energy gradient of 0.01 Kcal/mol/Å. Energy of binding was
calculated [27,28]. The alpha triangle placement method and the
London dG scoring method were used for docking. DNA-ligand com-
plexes were minimized using the MMFF94x force field, till reaching a
0.1 kcal mol/A˚ RMSD gradient.
The cells were seeded in 96-well plates at a concentration of
4
5
× 10 cells/ml (100 µl/well) and incubated overnight at 37 °C and 5%
CO . A serial dilution of the tested compounds or cisplatin was then
2
added. DMSO (0.1%) was used as a negative control. The cells were
incubated for 48 h, followed by addition of 15 µl of MTT (5 mg/ml in
PBS) to each well and further incubation for additional 4 h. Formazan
crystals were dissolved by 100 µl acidified SDS solution (10% SDS/
4.3.3. Flexible alignment and surface mapping
0
.01 N HCl in PBS). After 14 h of incubation at 37 °C and 5% CO
2
, the
The investigated compounds underwent flexible alignment and
surface mapping experiments using ‘Molecular Operating Environment’
software (MOE of Chemical Computing Group Inc., on a Core i7
workstation). Optimization of their geometry was achieved using the
MMFF94x forcefield followed by a flexible alignment using systematic
conformational search to identify the lowest energy aligned con-
formation(s) [29,30].
absorbance was measured using a BioTek microplate reader at 570 nm.
Every experiment was repeated three times and a standard deviation
(
SD ± ) was calculated. IC50 was calculated as the concentration that
caused 50% inhibition of cell growth. The growth of the cells was
monitored and the images were obtained by Gx microscopes
(
GXMGXD202 Inverted Microscope) at 10x magnification.
Selectivity index was calculated by the following equation:
SI = IC50 normal/ IC50 cancer, where IC50 normal and IC50 cancer are
the concentration of the tested compound that killed 50% of normal
cells and of cancer cells respectively.
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MG) stock solution was prepared by using methylene chloride ex-
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Bioorganic Chemistry 88 (2019) 102917
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