New pyrimidine and pyrazole-based compounds as potential EGFR inhibitors: Synthesis, anticancer, antimicrobial evaluation and computational studies

Article abstract of DOI:10.1016/j.bioorg.2021.105078

This study was focused on the synthesis of new pyrimidines 4a,b, 5a,b and pyrazoles 6a, b as ATP mimicking tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR). The new compounds were assessed as cytotoxic candidates against human breast cancer cells (MCF-7) and hepatocellular carcinoma cells (HepG-2). All the new compounds appeared as more potent cytotoxic agents than erlotinib, while only compound 4a exhibited more potency than 5-flourouracil and 4b analogue was equipotent to it. Accordingly, the kinase suppression effect of 4a and 4b was further evaluated against EGFR^WT, EGFR^L858R and EGFR^T790M. Both pyrimidine analogues 4a and 4b displayed outstanding inhibitory activity against EGFR^WT and its two mutated isoforms EGFR^L858R and EGFR^T790M in comparing to erlotinib and osimertinib as reference drugs. Additionally, all the new analogues were subjected to antimicrobial assay. Interestingly, both 4a and 4b represented the most promising activity of wide spectrum antimicrobial effect against the examined microbes in comparison to gentamycin and ketoconazole as standard drugs. Moreover, docking results proved the good binding interactions of the compounds 4a and 4b with EGFR^WT and EGFR^T790M which were in accordance with the results of the in vitro enzyme assay. Additional in silico ADMET studies were performed for the new derivatives which represented their good oral absorption, good drug-likeness properties and low toxicity risks in human.

Full text of DOI:10.1016/j.bioorg.2021.105078

Bioorganic Chemistry 114 (2021) 105078
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
New pyrimidine and pyrazole-based compounds as potential EGFR
inhibitors: Synthesis, anticancer, antimicrobial evaluation and
computational studies
Ismail M.M. Othmana, Zahra M. Alamshanyb, Nada Y. Tashkandib, Mohamed A.
,
M. Gad-Elkareema, Manal M. Anwarc *, Eman S. Nossierd
a Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt
b Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 21551, P.O. Box 42805, Saudi Arabia
c Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt
d Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study was focused on the synthesis of new pyrimidines 4a,b, 5a,b and pyrazoles 6a, b as ATP mimicking
tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR). The new compounds were assessed as
cytotoxic candidates against human breast cancer cells (MCF-7) and hepatocellular carcinoma cells (HepG-2). All
the new compounds appeared as more potent cytotoxic agents than erlotinib, while only compound 4a exhibited
more potency than 5-flourouracil and 4b analogue was equipotent to it. Accordingly, the kinase suppression
effect of 4a and 4b was further evaluated against EGFRWT, EGFRL858R and EGFRT790M. Both pyrimidine ana-
logues 4a and 4b displayed outstanding inhibitory activity against EGFRWT and its two mutated isoforms
EGFRL858R and EGFRT790M in comparing to erlotinib and osimertinib as reference drugs. Additionally, all the new
analogues were subjected to antimicrobial assay. Interestingly, both 4a and 4b represented the most promising
activity of wide spectrum antimicrobial effect against the examined microbes in comparison to gentamycin and
ketoconazole as standard drugs. Moreover, docking results proved the good binding interactions of the com-
pounds 4a and 4b with EGFRWT and EGFRT790M which were in accordance with the results of the in vitro enzyme
assay. Additional in silico ADMET studies were performed for the new derivatives which represented their good
oral absorption, good drug-likeness properties and low toxicity risks in human.
Pyrimidines
Pyrazoles
Cytotoxic
EGFR inhibition
Antibacterial
Antifungal
Molecular docking
ADMET prediction
MCF-7
HepG-2
1. Introduction
suppression of EGFR downstream PI3K/mTOR signaling pathway,
leading to increase the lack of oxygen to improve the sensitivity of the
Epidermal growth factor receptor (EGFR) is a receptor tyrosine ki-
nase (RTK), belongs to ErbB family and performs a central role in
signaling pathways of the cell such as; cell division, growth, differenti-
ation, metabolism, adhesion, motility and death [1,2]. It is upregulated
in multiple human malignancies, including non-small cell lung cancer
(NSCLC), hepatocellular, colorectal, head and neck squamous cell car-
cinoma and breast cancers [3–5]. Different studies in the chemothera-
peutic field showed that suppression of EGFR-TK introduces a rational
basis for the development of a class of targeted therapies, EGFR-TK in-
hibitors (EGFR-TKIs). They act by competing with ATP for ATP-binding
site on EGFR, thus blocking the signaling pathway leading to significant
resistance to cancer cells, as well as angiogenesis, in addition to
cancer cells to radiation [6–8].
Despite the significant benefits of the first-generation EGFR in-
hibitors (gefitinib [9] and erlotinib [10]) in cancer treatment, the
mutation in the ATP binding pocket of EGFR exon 20 leading to a
threonine-to-methionine substitution at the amino acid position 790
(T790M) led to predominant drug resistance in 50% of cancer patients.
At the same time, L858R mutation at exon 20 was detected as another
mechanism of resistance to the first-generation EGFR-TKIs [8,11–13].
The second generation, irreversible TKIs including afatinib and daco-
mitinb containing a Michael acceptor functional group were developed
to overcome these resistant tumor clones. However, due to low kinase
selectivity between EGFRT790M mutation and EGFRWT (wild type EGFR),
* Corresponding author.
E-mail address: manal.hasan52@live.com (M.M. Anwar).
https://doi.org/10.1016/j.bioorg.2021.105078
Received 26 February 2021; Received in revised form 11 May 2021; Accepted 7 June 2021
Available online 10 June 2021
0045-2068/© 2021 Published by Elsevier Inc.

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
serious undesirable effects had occurred such as; skin rash and gastro-
intestinal toxicity which limited the clinical use of these inhibitors
[14–17]. Accordingly, the third-generation EGFR inhibitors with py-
rimidine core structures such as WZ4002, rociletinib and osimertinib
(AZD9291) were created [18–21] (Fig. 1). Osimertinib is the only US
used by ATP and may be exploited for inhibitor specificity, and (v) a
phosphate binding pocket which is largely solvent exposed and can be
utilized for improving the pharmacokinetics of the inhibitor [26,27]
(Fig. 2).
Different structure-activity relationship studies (SAR) represented
that there are four common pharmacophoric features are shared by
EGFR-TKIs: (i) a flat heterocyclic-aromatic ring system which interact
with the adenine binding site and participates in H-bonding interactions
with the corresponding amino acids, (ii) a terminal hydrophobic head
for insertion in the hydrophobic pocket I, (iii) NH-linker that creates H-
bonding with amino acid residues in the linker area, (iv) a hydrophobic
tail which inserts into the hydrophobic area II [28–31] (Fig. 3).
Various literatures investigated that pyrimidine scaffold is an
important building block in many antineoplastic agents via multi-
suppression of different protein kinases including EGFR-TKs
[4,32–34]. In addition, pyrazole and fused pyrazole systems were
investigated as promising motifs in many antiproliferative agents
against a broad range of cancers targeting the kinase inhibition activity
of EGFR [35–37]. Various molecular simulation studies indicated that
many pyrazole analogues fitted perfectly in the active pocket of EGFR
kinase resulting in potent anticancer activity [38]. Accordingly, the
FDA approved third-generation inhibitor, exhibiting
a significant
selectivity toward the drug-resistant mutant EGFRT790M over the wild-
type EGFR (EGFRWT), rendering it an important medical regimen for
patients with EGFRT790M mutation [22]. Despite the excellent charac-
teristics of osimertinib, it exhibited several adverse effects in clinical
application, including diarrhea, rash, decreased appetite and car-
diotoxicity [23–25]. Accordingly, there is still a great demand to
develop new EGFR inhibitors with high selectivity against the
EGFRT790M and EGFRL858R mutation to overcome the undesirable
drawbacks and drug resistance.
The ATP binding active site of EGFR-TK is composed of five major
regions (i) an adenine binding pocket which bears the key amino acids
that create the H-bonding with the adenine ring, (ii) a sugar region
which constitutes the hydrophilic pocket, (iii) hydrophobic region I,
which is not exploited by ATP but plays a crucial role in inhibitor
selectivity and binding affinity, (iv) hydrophobic region II which is not
Fig. 1. The chemical structures of various drugs of EGFR tyrosine kinase inhibiting activity.
2

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
Fig. 2. The structure of ATP-binding site of EGFR-TK.
Fig. 3. Proposed hypothetic model of the new pyrimidine and pyrazole compounds as EGFR TKIs.
3

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
strategy of this work was focused on design and synthesis of new de-
rivatives bearing pyrimidine-2-one(thione) or pyrazole motif having the
essential pharmacophoric features of EGFR-TKIs. The first position was
pyrimidine or pyrazole moieties (hetero-aromatic system) to occupy the
adenine binding pocket. The second position was the terminal 2-methox-
yphenyl ring (hydrophobic head). NH linker was utilized in the third
position as a hydrogen bond donor. The fourth position was either
phenyl or 4-tolyl rings incorporated at pyrimidine-C6 or pyrazole-C5 to
occupy the hydrophobic region II of the ATP binding pocket (Fig. 3). its
biological isostere pyrazole
leading to the formation of the key intermediate 2-((2-methoxyphenyl)
amino) acetonitrile (2). The latter compound 2 was treated with
different aromatic aldehydes namely; benzaldehyde and/or 4-methyl-
benzaldehyde in glacial acetic acid to produce the corresponding acry-
lonitrile derivatives 3a, b, which were subsequently reacted with urea
and/or thiourea in refluxing ethanol containing a catalytic amount of
HCl to afford the pyrimidin-2-ones 4a, b and pyrimidine-2-thiones 5a, b
respectively. On the other hand, the treatment of 3a, b with hydrazine
hydrate in refluxing ethanol for 8 h accomplished the target pyrazole
derivatives 6a, b. The molecular structures of the new compounds were
confirmed depending on their elemental analyses, and spectral data. IR
spectra of the target pyrimidine derivatives 4a, b and 5a, b showed
absorption stretching bands at the regions 3489–3356 cmꢀ 1 represent-
All the newly synthesized compounds were evaluated as anticancer
agents against human breast cancer cells (MCF-7) and human liver
carcinoma cell line (HepG2). The compounds exhibited the most
promising cytotoxic activity were evaluated for their ability to suppress
the wild-type EGFRWT and the mutated isoforms EGFRL858R and
EGFRT790M. Furthermore, molecular docking study was performed for
the promising derivatives to rationalize and emphasize their modes of
action as EGFR-TKIs. In addition, in silico ADMET prediction was carried
out for the new derivatives to explore their drug-likeness properties.
It is known that different types of cancers are essentially caused by
various factors including certain types of infections [39]. Study of
various antibiotic cytotoxic pharmaceutics including Actinomycin,
Adriamycin/Doxorubicin and some other candidates revealed that in
addition to DNA intercalating or DNA damaging effects, the stimulation
of the existing host defense mechanism is considered as one of the ap-
proaches that these chemotherapeutics exert their antimicrobial effects
[40]. Based on these evidences, in comparison to gentamycin and ke-
toconazole as standard drugs, all the new pyrimidine and pyrazole de-
rivatives were also evaluated as antimicrobial agents against various
gram-positive, gram-negative bacterial and fungal strains.
ing NH2, NH groups, at 1676, 1684 cmꢀ 1 due to C O groups of com-
–
–
pounds 4a, b and at 1176, 1159 cmꢀ 1 representing C S groups of
–
–
compounds 5a, b. On the other hand, 1H NMR chemical shifts repre-
sented the protons of the new pyrimidines 4a, b, 5a, b at the expected
regions. Singlet signals were presented at δ 3.76–3.85 ppm due to the
three protons of –OCH3 functionalities, δ 2.02, 2.15 ppm due to CH3
protons of compounds 4b, 5b, δ 8.36–10.42 representing NH, NH2
protons as well as the signals of the aromatic protons appeared at their
expected regions δ 7.04–8.10 ppm. Also, 13C NMR spectra of the com-
pounds 4a, b, 5a, b exhibited singlet signals at the range δ 55.94–58.12
ppm contributing to OCH3 carbons, δ 17.84, 20.95 ppm due to CH3
carbons of compounds 4b, 5b, as well as at δ 160.27–179.21 ppm for
–
–
–
C
–
O and C S carbons. Different signals appeared at δ 106.18–153.77
ppm due to the aromatic carbons. Additionally, 1H NMR spectra of the
target pyrazole derivatives 6a, b represented the methoxy group protons
at δ 3.82, 3.78 ppm, the D2O exchangeable protons of NH2 and NH at δ
5.76, 6.38, 9.71–12.16 ppm as well as the aromatic protons at δ
7.04–8.08 ppm. Also, 13C NMR spectra of the compounds 6a, b dis-
played signals at δ 59.3, 58.15 ppm for OCH3 carbons, δ 21.88 ppm
referring to CH3 carbon of 6b and at δ 107.56–147.91 ppm contributing
to the aromatic carbons.
2. Results and discussion
2.1. Chemistry
The new pyrimidine and pyrazole derivatives were efficiently syn-
thesized as outlined in Scheme 1 starting with compound 2-methoxyani-
line (1) which was allowed to react with chloroacetonitrile in ethanol
Scheme 1. The synthetic approach for synthesis of the target pyrimidine and pyrazole derivatives.
4

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
2.2. Biological activity
Table 2
Inhibitory assessment of the tested compounds 4a and 4b in comparison with
erlotinib and osimertinib against EGFRWT, EGFRL858R and EGFRT790M
Compound No.
.
2.2.1. In vitro cytotoxic activity
The in vitro growth inhibitory potency of the newly prepared com-
pounds was scrutinized against breast (MCF-7) and hepatocellular
(HepG-2) cancer cell lines as well as lung fibroblast (WI38) normal cells
in comparing with 5-flourouracil and erlotinib as two standard drugs
using the colorimetric MTT assay [41,42]. The concentrations of the
IC50 (mean SEM) (µM)
EGFRWT
EGFRL858R
EGFRT790M
Erlotinib
Osimertinib
4a
0.096 ± 0.021
0.510 ± 0.01
0.087 ± 0.013
0.110 ± 0.014
0.041 ± 0.20
0.020 ± 0.03
0.044 ± 0.15
0.058 ± 0.12
0.550 ± 0.10
0.028 ± 0.05
0.026 ± 0.01
0.038 ± 0.04
compounds that induced 50% inhibition of cell viability (IC50
detected and tabulated in Table 1.
, μM) were
4b
SEM: Standard error mean; each value is the mean of three values.
Interestingly, all of the tested derivatives displayed significant
cytotoxic activity against both types MCF-7 and HepG2 cancer cell lines
of IC50 range 0.01–0.35 µM that was much more potent than that ob-
tained by the reference drug erlotinib of IC50; 0.42, 0.82 µM, respec-
tively. On the other hand, while comparing the resultant activities of the
compounds with 5-flourouracil, compound 6-phenylpyrimidin-2-one 4a
represented 2 and 3 folds more potent activity than that of the reference
investigate their potential mechanism of action. Based on the cytotoxic
data, compounds 4a and 4b were selected to study their EGFRWT,
EGFRL858R and EGFRT790M inhibitory activity [44], utilizing erlotinib
and osimertinib as standard drugs and the results were expressed as IC50
(µM) (Table 2).
Both compounds 4a and 4b demonstrated significant EGFRWT
inhibitory activity (IC50; 0.087 ± 0.013 and 0.110 ± 0.014 µM) that was
approximately equal to that obtained by erlotinib (IC50 = 0.096 ± 0.021
µM) and about 5.8 and 4.6 times, respectively higher than that obtained
drug against MCF-7 and HepG-2 cancer cell lines, respectively (IC50
;
0.01 ± 0.03 µM, IC50, 5-FU; 0.02 ± 0.01, 0.03 ± 0.01 µM, respectively). A
mild decrease in the activity was determined by the analogue 6-(p-tolyl)
pyrimidin-2-one 4b to be approximately equipotent to 5-FU of IC50
;
by the reference drug osimertinib (IC50 = 0.510 ± 0.011 μM). Further-
0.02 ± 0.01 µM. Further remarkable reduction in the cytotoxic activity
was detected by the pyrimidin-2-thione analogues 5a, b by 2–4 folds
against both cancer cell types of IC50 values ranging from 0.04 to 0.08
µM. This result could be explained due to the tendency of the
pyrimidine-2-oxygen atom to form additional H-bonds with different
amino acid residues of the target protein stronger than the sulfur atom
[43], thus representing more potent inhibitory effect. Also, the cytotoxic
potency was weakened by 4.5–8 folds upon replacement of the pyrim-
idine moiety with the five-membered pyrazole core as compounds 6a b
exhibiting IC50 values ranging from 0.09 ± 0.03–0.16 ± 0.03 µM.
Although the key intermediates 2 and 3a, b represented more potent
cytotoxic activity against the tested cell lines comparing to erlotinib as a
reference drug, they showed 12–17 folds less cytotoxic activity while
comparing to 5-flourouracil of IC50 values ranging from 0.24 ± 0.02 to
0.35 ± 0.03 µM. Thus, cyclization of 2 and 3a, b to establish their py-
rimidine and the pyrazole analogues 4a, b–6a, b is essential to enhance
the cytotoxic potency.
more, both compounds 4a and 4b employed potent inhibitory activity
against EGFRL858R that was close to that obtained by erlotinib (IC50
=
0.044 ± 0.15, 0.058 ± 0.12 µM, respectively, IC50, Erlotinib = 0.041 ±
0.020), but slightly less than the potency obtained by osimertinib by
2–2.5 folds. Also, 21 and 14 times more significant activity against
EGFRT790M was gained by 4a and 4b, respectively in comparing to
erlotinib (IC50 = 0.026 ± 0.15, 0.038 ± 0.12 µM, respectively, IC50
,
Erlotinib = 0.550 ± 0.10) and approximate equal activity while comparing
to osimertinib (IC50, Erlotinib = 0.028 ± 0.05). Fortunately, both com-
pounds 4a and 4b exhibited more potent inhibitory activity against the
mutant forms of EGFR over the wild-type form. Taking together, com-
pounds 4a and 4b could be considered more advantageous than other
EGFR-TKIs since they exhibited promising multi-kinase inhibiting ac-
tivity (with the highest selectivity toward the mutants EGFRT790M and
EGFRL858R) that might overcome EGFR-TKIs drug resistance by inter-
fering with intracellular signaling pathways or by suppression the
mutated binding sites or genes. Thus, both compounds 4a and 4b could
be used as promising basic templates for generation of new drugs used
for the treatment of different cancer types harboring EGFRT790M and
EGFRL858R mutations.
Referring to the results of cytotoxicity against WI38 normal cells
(Table 1), the nine tested derivatives showed lower cytotoxicity against
WI38 (IC50; 39.26–68.01 μM) than 5-FU (IC50; 10 ± 0.056 μM). They
exhibited promising safety profile to be safer antitumor agents than the
reference drug 5-FU.
2.2.3. Antimicrobial activity
It has been employed that the pyrimidine and pyrazole motifs are
versatile lead molecules in the field of antimicrobial discovery and
development [45–47], so it was of interest to assess the antimicrobial
activity of the newly prepared compounds alongside their cytotoxic
evaluation aiming to gain new pyrimidine and pyrazole candidates of
dual potent anticancer and antimicrobial activities. The antimicrobial
potency of the tested compounds was in vitro screened versus two
pathogenic gram-positive bacteria viz. Streptococcus pneumoniae RCMB
010010 and Staphylococcus epidermidis RCMB010024, two pathogenic
gram-negative bacteria viz. Proteus vulgaris RCMB 010085, E. Coli RCMB
010052 and two fungal strains viz. Aspergillus fumigatus RCMB 02568 and
Syncephalastrum racemosum RCMB 05922. Gentamycin and ketocona-
zole were utilized as reference drugs for antibacterial and antifungal
activity, respectively [48].
2.2.2. Kinase inhibitory assessment
In the present work, the analogues that represented the most potent
cytotoxic efficacy were subjected to Kinase inhibitory assessment to
Table 1
In vitro cytotoxic efficiency of the new derivatives against MCF-7 as well as
HepG-2 cell lines.
Compound No.
IC50
(μ
M)a
IC50
(μ
M)a
IC50
(
μ
M)a
MCF-7
HePG-2
WI-38
2
0.24 ± 0.02
0.33 ± 0.01
0.35 ± 0.02
0.01 ± 0.03
0.02 ± 0.01
0.04 ± 0.04
0.08 ± 0.02
0.09 ± 0.03
0.15 ± 0.01
0.02 ± 0.01
0.42 ± 0.2
0.26 ± 0.04
0.35 ± 0.03
0.27 ± 0.02
0.01 ± 0.01
0.02 ± 0.01
0.04 ± 0.02
0.07 ± 0.03
0.10 ± 0.03
0.16 ± 0.03
0.03 ± 0.01
0.82 ± 0.4
57.31 ± 3.6
51.32 ± 3.1
68.01 ± 2.21
66.27 ± 2.21
52.08 ± 3.23
39.98 ± 2.6
47.75 ± 2.0
48.75 ± 2.19
52.32 ± 3.1
10 ± 0.05
3a
3b
4a
The antimicrobial activity of all synthesized compounds was carried
out using agar well diffusion method [49] and the obtained results were
recorded for each tested compound as the average diameter of the in-
hibition zones in mm for the bacterial or fungal growth around the discs
(Table 3). Furthermore, two-fold serial dilution method [50,51] was
utilized to determine the Minimum Inhibitory Concentration (MIC)
4b
5a
5b
6a
6b
5-FUb
Erlotinib
–
values (expressed in
μg/mL) for the compounds and the results were
a
b
IC50 values = mean ± SD of three independent determinations.
recorded in (Table 4).
5-FU: 5-fluorouracil.
5

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
Table 3
In vitro antimicrobial activities of the synthesized compounds using well diffusion agar assay and expressed as mean diameter of inhibition zones (mm)
Compd. No.
Mean diameter of inhibition zones (Mean ± SEM) (mm)
Gram þ ve Bacteria
Gram-ve Bacteria
Fungi
Streptococcus
pneumoniae RCMB
010,010
Staphylococcus
epidermidis
Proteus vulgaris
RCMB 010,085
E. coliRCMB
010,052
Aspergillus
fumigatus RCMB
02,568
SyncephalastrumracemosumRCMB
05,922
RCMB010024
2
16.4 ± 0.16
NA
NA
14.3 ± 0.24
18.4 ± 0.36
NA
11.2 ± 0.14
NA
NA
NA
3a
13.9 ± 0.29
NA
NA
NA
3b
NA
NA
NA
NA
4a
27.0 ± 0.11
25.0 ± 0.33
23.2 ± 0.15
19.8 ± 0.24
15.5 ± 0.32
18.6 ± 0.15
27.2 ± 0.80
–
25.1 ± 0.15
24.5 ± 0.14
21.9 ± 0.26
20.1 ± 0.18
10.8 ± 0.16
NA
19.7 ± 0.14
18.6 ± 0.15
17.3 ± 0.43
16.5 ± 0.13
13.7 ± 0.16
NA
25.3 ± 0.18
26.2 ± 0.01
24.1 ± 0.13
18.2 ± 0.44
12.1 ± 0.25
22.0 ± 0.14
26.3 ± 0.15
–
22.8 ± 0.17
22.2 ± 0.10
21.4 ± 0.12
12.1 ± 0.18
17.1 ± 0.15
NA
18.9 ± 0.50
20.3 ± 0.14
19.1 ± 0.22
16.2 ± 0.46
21.7 ± 0.15
NA
4b
5a
5b
6a
6b
Gentamycin
Ketoconazole
25.4 ± 0.180
–
19.9 ± 0.30
–
–
–
23.7 ± 0.10
22.8 ± 0.10
NA: No activity under the screening conditions; -: Not tested; SEM = standard error mean; each value is the mean of different values.
Table 4
Minimal inhibitory concentrations (MICs) of the synthesized compounds against the tested pathogenic bacteria and fungi.
Compound
No.
MIC (Mean ± SEM) (μg/mL)
Gram þ ve Bacteria
Gram -ve Bacteria
Fungi
Streptococcus
pneumoniae RCMB
010,010
Staphylococcus
epidermidis
Proteus vulgaris
RCMB 010,085
E. ColiRCMB
010,052
Aspergillus
fumigatus RCMB
02,568
Synce-phalastrumracemosum
RCMB 05,922
RCMB010024
2
31.25 ± 0.50
NA
NA
125 ± 0.72
7.81 ± 0.11
0.98 ± 0.35
7.81 ± 0.27
15.63 ± 0.76
31.25 ± 0.45
125 ± 0.85
NA
500 ± 0.83
NA
NA
NA
3a
125 ± 0.25
0.06 ± 0.32
0.06 ± 0.22
0.49 ± 0.09
1.95 ± 0.35
500 ± 0.53
NA
NA
NA
4a
0.03 ± 0.55
0.06 ± 0.11
0.12 ± 0.33
0.98 ± 0.22
62.5 ± 0.11
7.81 ± 0.13
0.24 ± 0.66
–
0.06 ± 0.15
0.03 ± 0.25
0.12 ± 0.28
62.5 ± 0.78
250 ± 0.37
0.49 ± 0.70
0.03 ± 0.82
–
0.12 ± 0.45
0.49 ± 0.11
0.24 ± 0.18
250 ± 0.92
7.81 ± 0.71
NA
0.98 ± 0.62
0.98 ± 0.17
31.25 ± 0.34
31.25 ± 0.34
0.24 ± 0.05
NA
4b
5a
5b
6a
6b
Gentamycin
Ketoconazole
0.06 ± 0.72
–
1.95 ± 0.15
–
–
–
0.12 ± 0.68
0.49 ± 0.07
On the basis of the MIC values in Table 4, it was exhibited that there
is a wide variability in the antimicrobial potency of the tested de-
rivatives. It has been detected that the pyrimidine-2-one derivatives 4a
and 4b were 8–6 folds more potent antibacterial candidates against
2.3. Computational studies
2.3.1. Molecular docking
To furnish a deep insight into the binding modes of the most potent
new derivatives 4a and 4b within the active sites of EGFRWT and
EGFRT790M enzymes, molecular docking simulation was done using
Molecular Operating Environment software (MOE®) 2008.10 [52].
MOE program. Validation of the docking procedures were firstly done
via re-docking of the original co-crystallized ligands, erlotinib and
AZD9291 within the active sites of EGFRWT and EGFRT790M enzymes
(PDB code: 1 M17 and 6JX0) and revealed energy scores ꢀ 12.25 and
ꢀ 11.75 kcal/mol at root mean square deviation (RMDS) values equal
0.75 and 0.88 Å, respectively [53,54].
Streptococcus pneumoniae (MIC; 0.03, 0.06
(MIC; 0.24
gentamicin against Staphylococcus epidermidis exhibiting MIC values;
μ
g/mL) than gentamicin
μ
g/mL). Also, they were equipotent to the reference drug
0.06
μg/mL. Furthermore, both 4a and 4b represented approximate
equal potency to that obtained by gentamicin against both tested gm-
negative bacteria Proteus vulgaris and E. Coli (MIC values; 0.98, 0.06,
0.03 μg/mL) (MIC Gentamycin; 1.95, 0.03 μg/mL). Compound 4b was 4
folds less potent than gentamicin against E. Coli of MIC value; 7.81 μg/
mL. On the other hand, the antifungal activity of the 4a and 4b was
slightly less than that of the reference drug Ketoconazole representing
The docking results of 4a and 4b within the ATP-binding pocket of
EGFRWT illustrated that they interact with EGFRWT through similar
hydrogen bonding interactions with energy scores of ꢀ 13.22 and
ꢀ 12.90 kcal/mol, respectively. The 4-aminopyrimidine-2-one ring was
fitted through H-bond acceptors between the carbonyl oxygen and the
backbone of the key amino acid Met769 (distance in 4a and 4b: 2.88 and
2.58 Å, respectively). The protons of the amino groups formed H-bond
donors with the sidechain of Gln767 in case of 4a and 4b (distance: 1.66
and 2.21 Å, respectively) and additional H-bond donor with the side-
chain of Gln766 in case of 4a (distance: 2.38 Å). Moreover, the side
chain of Thr766 afforded two H-bonds with the proton of NH and the
oxygen of methoxy group (Figs. 4 and 5).
MIC values ranging from 0.12 to 0.98 μg/mL, MIC Ketoconazole; 0.12, 0.49
μ
g/mL.
Conversely, the pyrimidine-2-thione derivatives 5a and 5b appeared
to be less potent as antibacterial agents against the tested gm-positive of
MIC values range 0.49–1.95 g/mL except compound 5a which repre-
sented more potent activity against Streptococcus pneumoniae RCMB
010010 of MIC value 0.12 g/mL, while comparing to gentamicin. Un-
fortunately, both 5a and 5b exhibited moderate activity against the
tested gm-negative bacteria of MIC range; 0.12–62.5 g/mL and mod-
erate to weak potency against the tested fungal strains of MIC range;
μ
μ
μ
31.25–250 μg/mL, comparing to Ketoconazole.
Depending on the resultant data the pyrimidine-2-one is a promising
scaffold in producing antibacterial activity more than pyrimidine-2-
thione nucleus.
On the other hand, the promising targets 4a and 4b bound nicely
within the ATP-active site of EGFRT790M in a similar manner with energy
scores of ꢀ 12.65 and ꢀ 11.88 kcal/mol, respectively. The 4-
6

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
Fig. 5. A & B images representing 2D & 3D binding views of compound 4b into
the active site of EGFRWT (PDB ID: 1 M17).
Fig. 4. A & B images representing 2D & 3D binding views of compound 4a into
that they are candidates for good solubility, capacity for penetrating cell
membranes and intestinal absorption and are basically recognized as
good indicators of drug transport in the intestines, Caco-2 monolayers
penetration, and blood-brain barrier crossing [56–58]. Their nOHNH
value (H-bond donors) are less than 5 giving them higher solubility in
cellular membranes with also nON values (H-bond acceptors) in the
range of 3–6 and molecular weight in the range of 162.19–338.44.
Molecular volumes of the target compounds in this series increased as
increasing MW and ranged from 155.65 to 301.35 Å3. The milog P
(octanol/water partition coefficient) values for all compounds under
inspection are less than 5, especially for compounds 2, 4a and 4b out-
lining them a good lipophilicity character. The great lipophilicity of
these compounds may protect against ROS (Reactive Oxygen species)
damage and is due in part to their smaller polar surface areas.
the active site of EGFRWT (PDB ID: 1 M17).
aminopyrimidine-2one scaffold was fixed tightly via two hydrogen bond
donors between N-3 and the carbonyl oxygen with the backbone of
Met793. Furthermore, the proton of the amino group exhibited H-bond
donors with the backbone of Gln791 in case of 4a and 4b (distance: 1.87
and 1.65 Å, respectively) (Figs. 6 and 7).
Finally, and referring to the superimposition models in Fig. 8, the
two compounds 4a and 4b bearing 4-aminopyrimidine-2-one scaffold
were well embedded in the active pockets of EGFRWT and EGFRT790M
through various H-bond interactions. Furthermore, the illustrated
binding pattern explained the excellent EGFRWT and EGFRT790M inhib-
itory activity of these derivatives comparing with the co-crystalized
inhibitors erlotinib and AZD9291, respectively.
2.3.2.1. In silico toxicity potential. Toxicity risks and physicochemical
properties of the newly synthesized compounds were estimated via the
methodology developed by Osiris [59]. All compounds were predicted
for their toxicity including mutagenicity, tumorgenicity, skin irritancy
and reproductive effect. From the results displayed in Table 6, all
compounds would be safe and were predicted to have no side effects.
The best results were obtained by compounds 4a, 4b, 5a, 5b, 6a and 6b
indicating that they contain predominantly active fragments which are
frequently presented in commercial drugs [60]. Drug score is a measure
of compound’s potential to have drug-conform behavior. The highest
drug-score value was ascribed to compound 4a justifying its both
effectiveness and potentiality as a new drug candidate.
2.3.2. In silico ADMET prediction and drug likeness study
Based on the Lipinski’s rule (Rule of five) using molinspiration
software [55,56] (Table 5), all tested compounds satisfy the “Rule of
five” and meet all criteria for good permeability and bioavailability
displaying rotatable bonds number in the range of 3–4, with the lowest
value allowed to compound 2, therefore, obviously exhibiting small
conformational flexibility. The percentage of absorption (%ABS) which
is deduced from the TPSA values according to the following equation: %
ABS = 109–0.345 × TPSA was also determined. All candidate com-
pounds exhibited a great %ABS ranging from 76.90 to 93.45% sug-
gesting their efficient oral absorption. Topological polar surface area
(TPSA) results are < 140 Å2 for all the analyzed compounds suggesting
7

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
Fig. 7. A & B images representing 2D & 3D binding views of compound 4b into
the active site of EGFRT790M (PDB ID: 6JX0).
to EGFR-TKIs. Molecular docking study rationalized the potent sup-
pression effect of the pyrimidine ꢀ 2-ones 4a and 4b due to their good
fitting with the best binding energy in the active site of EGFRWT and its
mutated isoforms EGFRL858R and EGFRT790M. As a trial to gain new
pyrimidine and pyrazole – based derivatives of dual anticancer and
antimicrobial activity, all the new analogues were assessed as antibac-
terial and antifungal agents against a number of pathogenic gram-
positive, gram-negative bacterial and fungal strains using gentamycin
and ketoconazole as standard drugs. The results proved that 4a and 4b
among the tested derivatives were the most promising agents repre-
senting a wide spectrum antimicrobial activity against the examined
microbes. Furthermore, Lipinski rule of five and ADME profile suggested
strongly that all the synthesized pyrazole and pyrimidine compounds
were fulfilling the criteria of drug likeness approach. Accordingly, the
pyrimidine scaffold is considered as a promising template in the field of
development and optimization of new drugs of dual anticancer and
antimicrobial activities.
Fig. 6. A & B images representing 2D & 3D binding views of compound 4a into
the active site of EGFRT790M (PDB ID: 6JX0).
3. Conclusions
In summary, new pyrimidine-2-one(thione) and pyrazole derivatives
bearing the essential pharmacophoric features of EGFR-TKIs were
designed, synthesized and evaluated as cytotoxic candidates against
human breast (MCF-7) and hepatocellular (HepG2) cancer cell lines,
where 5-flourouracil and erlotinib were serving as standard drugs. All
the new compounds revealed more potent cytotoxic activity than erlo-
tinib. On the other hand, only the pyrimidine-2-one compound 4a
exhibited more potent anticancer activity than 5-flourouracil, while 4b
analogue was equipotent to it. The inhibitory profiles against the three
isoforms EGFRWT, EGFRL858R and EGFRT790M were evaluated for the
most promising compounds 4a, 4b, utilizing erlotinib and osimertinib as
reference drugs. Both analogues 4a, 4b exhibited more potent sup-
pression effect against the mutant forms EGFRL858R and EGFRT790M over
the wild-type form EGFRWT and hence they might overcome resistance
8

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
4. Experimental
4.1. Chemistry
The instruments used for measuring the melting points, spectral data
(IR, Mass, 1H NMR and 13C NMR) and elemental analysis are provided in
details in Supplementary material.
4.1.1. 2-((2-Methoxyphenyl) amino) acetonitrile (2)
A mixture of 2-methoxyaniline (1) (1.2 g, 10 mmol) and chlor-
oacetonitrile (0.75 g, 10 mmol) in absolute ethanol (20 mL) was refluxed
for 5 h. The solid product formed after cooling was collected by filtration
and recrystallized from ethanol to afford the title compound 2.
Yield 82%, colorless; m.p. 160-162˚C; IR (νmax/cmꢀ 1): 3328 (NH),
3039 (CH-arom.), 2972 (CH-aliph.), 2223 (CN), 1602 (C C); 1H NMR
–
–
(DMSO‑d6): δ 3.85 (s, 3H, OCH3), 4.53 (s, 2H, CH2), 7.17–7.37 (m, 3H,
Ar-H), 7.53 (d, 1H, J = 10.00 Hz, Ar-H), 9.43 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6): δ 48.20 (–CH2), 56.19 (OCH3),
112.77, 115.32, 116.82, 120.09, 123.99, 136.43; Anal. for C9H10N2O
(162.19): Calcd. C, 66.65; H, 6.21; N, 17.27; Found: C, 66.87; H, 6.43; N,
17.49%.
4.1.2. General procedure for the synthesis of 2-((2-methoxyphenyl)
amino)-3-(substituted) acrylonitrile 3a, b
A mixture of the acetonitrile derivative 2 (1.6 g, 10 mmol), appro-
priate aromatic aldehydes namely; benzaldehyde and/or 4-methylben-
zaldehyde (10 mmol) and anhydrous sodium acetate (10 mmol) in
glacial acetic acid (15 mL) was refluxed for 12 h then allowed to cool.
The solid product was collected, dried and recrystallized from dioxane.
4.1.2.1. 2-((2-Methoxyphenyl) amino)-3-phenylacrylonitrile (3a). Yield
71%; brown crystals; m.p. 222-224˚C; IR (νmax/cmꢀ 1): 3374 (NH), 3035
(CH-arom.), 2967 (CH-aliph.), 2222 (CN), 1611 (C C); 1H NMR
–
–
(DMSO‑d6): δ 3.75 (s, 3H, OCH3), 7.33–7.37 (m, 2H, Ar-H), 7.54–7.57
(m, 2H, Ar-H), 7.64–7.66 (m, 3H, Ar-H), 8.19 (d, 2H, J = 10.00 Hz, Ar-
H), 8.34 (s, 1H, -C = CH), 9.87 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6): δ 56.36 (OCH3), 111.45, 113.27, 115.57, 117.85, 120.05,
122.79, 123.69, 125.12, 126.97, 127.80, 132.44, 136.35, 141.80; Anal.
for C16H14N2O (250.30): Calcd. C, 76.78; H, 5.64; N, 11.19; Found: C,
76.55; H, 5.86; N, 11.41.
Fig. 8. 3D superimposition binding views of compound 4a (yellow) and 4b
(blue) with erlotinib (red) into the active site of EGFRWT (PDB ID: 1 M17) (A)
and with AZD9291 (red) into the active site of EGFRT790M (PDB ID: 6JX0) (B),
respectively. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Table 5
Calculated molecular properties of the newly synthesized compounds for assessment of the drug likeness.
miLogPa<5
% ABSb
TPSAc
Natoms
MWe<500
M.Vol.f
nONg<10
nOHNHh g<5
nviol
.
nrotbj(<10)
d
i
Compd No.Rule
2
1.59
4.05
4.50
3.08
3.53
3.42
3.87
3.53
3.98
ꢀ 4.21
3.77
ꢀ 0.59
93.45
93.45
93.45
76.90
76.90
82.79
82.79
82.79
82.79
40.08
85.16
86.32
45.05
45.05
45.05
93.04
93.04
75.97
75.97
75.97
75.97
199.74
69.08
65.72
12
19
20
23
24
23
24
21
22
33
36
9
162.19
250.30
264.33
308.34
322.37
324.41
338.44
280.33
294.36
477.60
531.44
130.08
155.65
237.68
254.24
275.91
292.47
284.79
301.35
256.93
273.49
450.66
452.47
96.91
3
3
3
6
6
5
5
5
5
12
8
4
1
1
1
4
4
4
4
4
4
11
0
2
0
0
0
0
0
0
0
0
0
2
1
0
3
4
4
4
4
4
4
4
4
7
7
0
3a
3b
4a
4b
5a
5b
6a
6b
Gentamycin
Ketoconazole
5-FU
a
Octanol-water partition coefficient, calculated by the methodology developed by Molinspiration.
% ABS: percentage of absorption.
b
c
d
e
f
TPSA topological polar surface area.
Number of non-hydrogen atoms.
Molecular weight.
molecular volume.
g
h
i
Number of hydrogen-bond acceptors (O and N atoms).
Number of hydrogen-bond donors (OH and NH groups).
Number of ‘‘Rule of five” violations
j
Number of rotatable bonds.
9

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
Table 6
Toxicity risks, solubility, drug-likeness, and drug score of the synthesized compounds.
Compound No.
Toxicity risks
Mutagen-icity
Solubility
Drug-likeness
DrugScore
Tumorigen-icity
Irritancy
Reproductive effect
2
green
green
green
green
green
green
green
green
green
green
green
red
green
green
green
green
green
green
green
green
green
green
green
red
green
green
green
green
green
green
green
green
green
green
green
red
green
green
green
green
green
green
green
green
green
green
green
red
ꢀ 2.14
ꢀ 4.12
ꢀ 4.46
ꢀ 3.93
ꢀ 4.27
ꢀ 4.01
ꢀ 4.35
ꢀ 4.16
ꢀ 4.51
ꢀ 1.18
ꢀ 2.99
ꢀ 1.76
ꢀ 3.72
ꢀ 4.36
ꢀ 4.69
2.24
0.49
0.39
0.35
0.78
0.67
0.72
0.57
0.76
0.66
0.77
0.61
0.06
3a
3b
4a
4b
1.01
5a
1.33
5b
0.11
6a
2.60
6b
1.35
Gentamycin
Ketoconazole
5-FU
4.88
11.18
ꢀ 4.50
Green; low risk, red: high risk
4.1.2.2. 2-((2-Methoxyphenyl) amino)-3-(p-tolyl) acrylonitrile (3b).
exchangeable), 9.92, 10.42 (2 s, 2H, 2NH, D2O exchangeable); 13C NMR
(DMSO‑d6): δ 56.28 (OCH3), 106.14, 109.20, 115.32, 121.01, 122.32,
124.98, 126.41, 127.62, 129.85, 134.25, 136.30, 141.84, 151.45,
Yield 68%; brown crystals; m.p. 217-219˚C; IR (νmax/cmꢀ 1): 3279 (NH),
3062 (CH-arom.), 2933 (CH-aliph.), 2205 (CN) 1602 (C C); 1H NMR
–
–
–
(DMSO‑d6): δ 2.01 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 7.11–7.18 (m, 2H,
Ar-H), 7.27–7.32 (m, 1H, Ar-H), 7.47 (d, 1H, J = 10.00 Hz, Ar-H), 7.84
(d, 2H, J = 10.00 Hz, Ar-H), 7.99 (d, 2H, J = 10.00 Hz, Ar-H), 8.26 (s,
1H, -C = CH), 9.65 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO‑d6):
δ 20.08 (CH3), 55.23 (OCH3), 111.81, 112.90, 114.81, 116.43, 120.85,
121.02, 122.36, 127.37, 129.71, 130.74, 132.42, 135.58, 139.90,
143.80; Anal. for C17H16N2O (264.32): Calcd. C, 77.25; H, 6.10; N,
10.60; Found: C, 77.46; H, 6.31; N, 10.82.
178.21 (C S); Anal. for C17H16N4OS (324.40): Calcd. C, 62.94; H, 4.97;
–
N, 17.27; S, 9.88; Found: C, 62.73; H, 4.75; N, 17.48; S, 9.67.
4.1.3.3. 4-Amino-5-((2-methoxyphenyl) amino)-6-(p-tolyl) pyrimidine-2
(1H)-thione (5b). Yield 69%; yellow crystals; m.p. 291-293˚C; IR (νmax
/
cmꢀ 1): 3489, 3369 (NH2), 3244, 3153 (2NH), 3043 (CH-arom.), 2930
1
–
(CH-aliph.), 1159 (C S); H NMR (DMSO‑d ): 2.02 (s, 3H, CH ), 3.85 (s,
–
6
3
3H, OCH3), 7.11 (d, 2H, J = 10.00 Hz, Ar-H), 7.20–7.60 (m, 6H, Ar-H),
7.89 (s, 2H, NH2, D2O exchangeable), 9.16, 9.94 (2 s, 2H, 2NH, D2O
exchangeable); 13C NMR (DMSO‑d6) δ: 20.95 (CH3), 58.12 (OCH3),
4.1.3. General procedure for the preparation of 4-amino-5-((2-methoxy-
phenyl) amino)-6-(substituted phenyl) pyrimidin-2(1H)-one (thione)
derivatives 4a, b and 5a, b
105.80, 110.12, 117.74, 121.64, 123.77, 125.09, 127.65, 128.79,
–
130.79, 133.72, 136.42, 142.35, 153.78, 179.11 (C S); Anal. for
–
A mixture of the compounds 3a, b (10 mmol), urea and/ or thiourea
(10 mmol) in absolute ethanol (20 mL) containing (1 mL) hydrochloric
acid was heated under reflux for 7–9 h. The solid product produced on
hot was collected by filtration and recrystallized from ethanol to give the
target products 4a, b and 5a, b, respectively.
C
18H18N4OS (338.43): Calcd. C, 63.88; H, 5.36; N, 16.56; S, 9.47; Found:
C, 63.65; H, 5.58; N, 16.75; S, 9.68.
4.1.4. General procedure for the preparation of compounds N4-(2-
methoxyphenyl)-5-substituted phenyl-1H-pyrazole-3,4-diamine derivatives
6a, b
4.1.3.1. 4-Amino-5-((2-methoxyphenyl)
amino)-6-phenylpyrimidin-2
A mixture of compounds 3a, b (10 mmol) and hydrazine hydrate (2
mL) in ethanol (30 mL) was refluxed for 8 h. After cooling, the obtained
precipitate was filtered, dried and crystallized from dioxane to give the
corresponding derivatives 6a, b.
(1H)-one (4a). Yield 63%; yellow crystals; m.p. 276-278˚C; IR (νmax
/
cmꢀ 1): 3445, 3356 (NH2), 3282, 3180 (2NH), 3086 (CH-arom.), 2973
(CH-aliph.), 1676 (C O), 1621 (C C); 1H NMR (DMSO‑d6): 3.76 (s,
–
–
–
–
3H, OCH3), 7.41–7.45 (m, 4H, Ar-H, NH2, D2O exchangeable),
7.55–7.75 (m, 5H, Ar-H), 8.14 (d, 2H, J = 10.00 Hz, Ar-H), 9.07, 10.2 (2
s, 2H, 2NH, D2O exchangeable); 13C NMR (DMSO- d6): δ 57.87 (OCH3),
4.1.4.1. N4-(2-Methoxyphenyl)-5-phenyl-1H-pyrazole-3,4-diamine (6a).
Yield 59%; brown crystals; m.p. 300-302˚C; IR (νmax/cmꢀ 1): 3481, 3308
(NH2), 3278, 3155 (2NH), 3063 (CH-arom.), 2925 (CH-aliph.), 1607
108.89, 110.75, 113.88, 116.41, 122.22, 122.39, 125.16, 127.9, 128.60,
1
–
–
–
133.76, 136.71, 153.77, 160.27 (C O); Anal. for C17H16N4O2 (308.33):
(C C); H NMR (DMSO‑d ): 3.82 (s, 3H, OCH ), 5.76 (s, 2H, NH , D O
–
2
2
exchangeable), 7.04–7.106 (m, 2H, Ar-H), 7.335–7.45 (m, 2H, Ar-H),
7.55–7.62 (m, 3H, Ar-H), 7.85 (d, 2H, Ar-H, J = 10.00 Hz), 9.71,
12.16 (s, 2H, 2NH, D2O exchangeable); 13C NMR (DMSO- d6): δ 59.3
(OCH3), 107.56, 112.03, 115.03, 120.77, 123.27, 125.18, 127.87,
128.35, 131.24, 134.75, 139.88, 141.25, 147.91. Anal. for C16H16N4O
(280.32): Calcd. C, 68.55; H, 5.75; N, 19.99; Found: C, 68.76; H, 5.93; N,
19.77.
Calcd. C, 66.22; H, 5.23; N, 18.17; Found: C, 66.45; H, 5.43; N, 18.38.
4.1.3.1.1. 4-Amino-5-((2-methoxyphenyl) amino)-6-(p-tolyl) pyr-
imidin-2(1H)-one (4b). Yield: 66%; yellow crystals; m.p. 263-265˚C; IR
(
νmax/cmꢀ 1): 3466, 3386 (NH2), 3298, 3170 (2NH), 3055 (CH-arom.),
1
–
–
–
–
2969 (CH-aliph.), 1684 (C O), 1609 (C C); H NMR (DMSO‑d ): 2.15
6
(s, 3H, CH3), 3.85 (s, 3H, OCH3), 7.15–7.33 (m, 4H, Ar-H, NH2, D2O
exchangeable), 7.48 (d, 1H, J = 10.00 Hz, Ar-H), 7.82 (d, 1H, J = 10.00
Hz, Ar-H), 8.01 (d, 2H, J = 10.00 Hz, Ar-H), 8.27 (d, 2H, J = 10.00 Hz,
Ar-H), 9.72, 10.39 (2 s, 2H, 2NH, D2O exchangeable); 13C NMR (DMSO-
d6): δ 17.84 (CH3), 55.94 (OCH3), 106.18, 109.12, 111.84, 116.34,
4.1.4.2. N4-(2-Methoxyphenyl)-5-(p-tolyl)-1H-pyrazole-3,4-diamine
(6b). Yield 56%; brown crystals; m.p. 296-298˚C; IR (νmax/cmꢀ 1): 3428,
3332 (NH2), 3185, 3129 (2NH), 3032 (CH-arom.), 2966, 2912 (CH-
121.96, 122.06, 125.19, 129.15, 130.64, 134.24, 137.72, 142.13,
–
1
–
153.64, 161.24 (C O); Anal. for C18H18N4O2 (322.36): Calcd. C, 67.07;
–
aliph.), 1620 (C C); H NMR (DMSO‑d ): 2.31 (s, 3H, CH ), 3.78 (s, 3H,
–
3
OCH3), 6.38 (s, 2H, NH2, D2O exchange6able), 7.07 (d, 1H, J = 10.00 Hz,
Ar-H), 7.43–7.74 (m, 3H, Ar-H), 7.99 (d, 2H, J = 10.00 Hz, Ar-H), 8.08
(d, 2H, J = 10.00 Hz, Ar-H), 9.98, 12.30 (2 s, 2H, 2NH, D2O
exchangeable); 13C NMR (DMSO- d6): δ 21.88 (CH3), 58.15 (OCH3),
111.92, 113.90, 115.81, 120.65, 120.71, 123.96, 126.40, 129.48,
130.35, 132.57, 138.15, 143.21, 147.87; Anal. for C17H18N4O (294.35):
H, 5.63; N, 17.38; Found: C, 67.28; H, 5.85; N, 17.59.
4.1.3.2. 4-Amino-5-((2-methoxyphenyl)
amino)-6-phenylpyrimidine-2
(1H)-thione (5a). Yield 71%; yellow crystals; m.p. 287-289˚C; IR
(
νmax/cmꢀ 1): 3432, 3379 (NH2), 3271, 3108 (2NH), 3042 (CH-arom.),
1
–
–
–
2924 (CH-aliph.), 1607 (C C), 1176 (C S); H NMR (DMSO‑d ): δ 3.77
–
(s, 3H, OCH3), 7.23–8.01 (m, 9H, Ar-H), 8.36 (s, 2H, N6H2, D2O
10

I.M.M. Othman et al.
Bioorganic Chemistry 114 (2021) 105078
˙
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Calcd. C, 69.37; H, 6.16; N, 19.03; Found: C, 69.55; H, 6.37; N, 19.25.
4.2. In vitro anticancer screening
The screening was performed using MTT assay [41,42] at Regional
Center for Mycology and Biotechnology, Al- Azhar University. More
details were provided in Supplementary material.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgments
The authors are thankful to the Faculty of Pharmacy-Al-Azhar Uni-
versity / Egypt, for performing the spectral and biological data of the
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