Design and synthesis of novel pyridopyrimidine derivatives with anchoring non-coplanar aromatic extensions of EGFR inhibitory activity

Article abstract of DOI:10.1016/j.bioorg.2021.105318

The present study describes the synthesis of three series of 4-substituted pyridopyrimidin derivatives 4a-h, 5a-d. 6a-d, starting from 2-amino-6-(4-methoxyphenyl)-4-(4-(substituted) phenyl)nicotinonitrile 2a-d via the reaction with N,N-dimethyl-N-' substi

Full text of DOI:10.1016/j.bioorg.2021.105318

Bioorganic Chemistry 116 (2021) 105318
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel pyridopyrimidine derivatives with anchoring
non-coplanar aromatic extensions of EGFR inhibitory activity
,
,*
Amal A.M. Eissaa, Kholoud F.M. Aljamala, Hany S. Ibrahimb *, Heba Abdelrasheed Allama
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt
A R T I C L E I N F O
A B S T R A C T
Keyword:
The present study describes the synthesis of three series of 4-substituted pyridopyrimidin derivatives 4a-h, 5a-d.
6a-d, starting from 2-amino-6-(4-methoxyphenyl)-4-(4-(substituted) phenyl)nicotinonitrile 2a-d via the reaction
with N,N-dimethyl-N-’ substituted phenyl formimidamide to obtain 4a-h or with either phenyl isothiocyanate 1:1
and 1:2 to obtain 5a-d, 6a-d respectively. The synthesized compounds were evaluated for their effectiveness as
EGFR inhibitors against Gefitinib. Six compounds; 4b,g,h, 5c and 6a,d prompted significantly higher EGFR
inhibitory activity relative to that of Gefitinib. While two compounds 4d and 4f showed IC50 values non-
significantly different from that of the reference drug. Furthermore, compounds 4a, 4 h, 6a and 6d were cho-
sen to be assessed in vitro for their cytotoxicity against two EGFR-overexpressing cell lines; two human cancer
cell lines namely: MCF7 and MDA-MB-361. Moreover, cell cycle analysis and apoptotic assay was applied for
compound 4b that showed most potent inhibitory activity on EGFR, and the highest cytotoxicity against MCF7
and MDA-MB-361, where cell cycle arrest was achieved at pre G and S phases with increased apoptosis. Addi-
tionally, a molecular docking study was achieved to inspect the interaction of this compound with the active site
of EGFR-TK.
Pyrido-pyrimidin
EGFR
Cytotoxicity
Apoptosis
1. Introduction
expression and tumor resistance to chemotherapy and radiation therapy
has been described. [8,9]
Cancer is considered as a life-threatening malignant disease, with
high morbidity and mortality rate [1]. The side effects, systemic toxicity
and resistance of the traditional non-selective cytotoxic chemothera-
peutic drugs makes finding of effective nontoxic selective new anti-
cancer agent is a must [2,3]. Targeted chemotherapeutic agents
selectively target cancer cells or the tumor microenvironment that
maintain cancer growth with greater efficiency and negligible side ef-
fects on normal cells [3]. Targeted chemotherapies attack signaling
pathways that regulates tumor cell cycle and its microenvironment
provoking cell apoptosis, affecting tumor cell proliferation and/or hin-
dering tumor mass growing [4,5]. Recent genomic studies have shown
abnormal activations in cancer cells in the genes encoding Receptor
Tyrosine Kinases (RTKs) such as EGFR, HER2/ErbB2, and MET, among
many others [6]. Since development of small molecules targeting
Epidermal Growth Factor Receptor (EGFR) is a well-recognized
approach in designing antitumor agents, EGFR signaling pathway has
been widely considered for its important role in the development of
many malignant tumors [7]. Also, a positive correlation between EGFR
Lapatinib (I) was developed by GSK as the first dual EGFR/HER2
inhibitor and was approved in 2007 by the FDA for treating of breast
cancer. [10]
According to the ATP binding mode inside EGFR, several 4-amino-
quinazoline derivatives were designed as a competitive inhibitor for
ATP. For example, Gefitinib (II) and Erlotinib (III) (Fig. 1), 4-anilino-
quinazoline derivatives, were approved by FDA for the treatment of
NSCLC [11,12]. To overcome the resistance of more advance cancer
cells for EGFR inhibitors, Lapatinib (I) (Fig. 1) was designed as dual
EGFR/HER2 inhibitor by targeting the additional back pocket in ATP
binding site through extended 3- fluorobenzyloxy moiety. This extra
binding region allowed Lapatinib to has an extra time during its irre-
versible inhibitory activity compared to Gefitinib and Erlotinib
[10,13]. Opposing the extended moiety of Lapatinib, compound (IV)
was found to have a proper inhibitory activity against EGFR without the
presence of extended moiety targeting the back pocket in the ATP
binding site. This activity was justified due the hydrophobic effect of 4-
trifluoromethylsalicyl moiety (Fig. 1) [11].
* Corresponding authors.
E-mail addresses: hany.s.ibrahim@gmail.com (H.S. Ibrahim), heba.abdelkhalek@pharma.cu.edu.eg (H. Abdelrasheed Allam).
https://doi.org/10.1016/j.bioorg.2021.105318
Received 2 August 2021; Received in revised form 25 August 2021; Accepted 29 August 2021
Available online 1 September 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
Modifications of the main scaffold of the previously mentioned lead
compounds took place by performing changes in the fused benzene ring
of 4-amino quinazoline moiety to different aromatic rings. This concept
was achieved in TAK-285 (V) which is a potent EGFR inhibitor having a
pyrrolo[3,2-d]pyrimidine scaffold (Fig. 2) [12]. Additional replacement
of the fused benzene ring of 4-amino quinazoline moiety was repre-
sented in compound (VI) with a hinge binder region of 1H-pyrazolo[3,4-
d]pyrimidine scaffold. This compound was characterized by a very
potent inhibitory activity till 1 digit nanomolar range (IC50 = 5 nM)
[13]. Even this concept was supported by using a neighboring pseudo
fused ring as illustrated in compound (VII) where the intramolecular
hydrogen bond so that the hinge binding appeared as pyrimido[4,5-d]
pyrimidine moiety (Fig. 2). The intramolecular hydrogen bond was
approved by the co-crystal structure of compound (VII) co-crystalized
with EGFR (PDB code 3BEL) [14].
hydroxide gave the chalcone intermediate 1a-d in about 91–99% yield.
2-Amino-3-cyano-4,6-diaryl pyridines 2a–d were synthesized by the
reaction of the different chalcones with malononitrile in the presence of
ammonium acetate with a slight modification of the reported procedure
[16–18]. IR spectra of compounds 2b and 2c revealed the presence of a
forked band at 3383–3209 cmꢀ 1 assigned to (NH2) and strong charac-
teristic bands at 2212 and 2198 cmꢀ 1 corresponding to the (C N) group
–
–
–
respectively. 1H NMR spectra of 2b and 2c exhibited D2O exchangeable
singlet signals at 6.77 and 6.92 ppm, respectively, corresponding to NH2
protons. In addition, a singlet signals observed at 2.55 and 2.99 ppm
corresponding to methyl and dimethyl protons. Also, a characteristic
singlet signals observed at the aromatic range due to the pyrimidine
proton at 7.16 and 7.21 ppm, respectively.
In scheme 2, aiming to get the target compounds 4a-h, compounds
2a-d were reacted with DMF/DMA giving the formimidamide in-
termediates 3a-d. These derivatives were prepared to react with various
anilines hoping to obtain our target compounds 4a-h but unfortunately,
the start compounds 2a-d were obtained again. Therefore, we tried to
get the target compounds by another pathway. The pyridine derivatives
2a-d were reacted directly with the N,N-dimethyl-N’ subistituted phenyl
formimidamide (prepared by reacting the substituted anilines with
DMF/DMA) through Dimroth rearrangement as reported for similar
derivatives Fig. 4 [19].
With respect to the previous data, in this research we will report the
design, synthesis and biological evaluation of EGFR inhibitors. The
design is based on the utilization of 4-anilinopyrido[2,3-d]pyridine
scaffold as a hinge binding moiety as in compounds 4a-h, 5a-d and 6a-
d (Fig. 3). This scaffold was extended with a substituted phenyl group
which is non coplanar to the pyrido[2,3-d]pyridine scaffold to target the
non-covalent hydrophobic part of the active site of EGFR as planned
before [15]. Also, on 6-position of pyrido[2,3-d]pyridine, we will add 4-
methoxyphenyl substituent to add an extended methoxy group analo-
gous to Gefitinib (I), Erlotinib (II) and compound (VII) through an ar-
omatic linker. In this study, we will discuss another series 6a-d, similar
to compounds 4a-h and 5a-d, but we add phenyl thiourea extension to
4-aminopyrido[2,3-d]pyridine scaffold. The extra extension will be
addressed aiming to target the back pocket of the ATP binding site as
shown in Fig. 3.
The microelemental analyses and spectroscopic data of the products
were in accordance with the assigned structures. Where, the IR spectra
of compounds 4a-h revealed the presence of sharp bands at 3221–3313
cmꢀ 1 of the two NH groups, disappearance of (C N) band around 2200
–
–
–
range was noticed. 1H NMR spectra of 4a-h exhibited D2O exchangeable
singlet signals with smaller integration corresponding to one NH proton
rather than the two NH2 protons in the start compound 2a-d. New
singlet signals appear 8–9 ppm corresponding to the new proton found
at the pyrimidine ring. In addition to, 1H NMR of compounds 4a,c,e,g
showed the presence of one singlet band around 2.2 ppm corresponding
to three protons of the m-toluidine ring at position 4. 13C NMR spectra of
4a,g revealed characteristic signals at 20, 23.59 attributed to the methyl
of the m-toluidine ring at position 4. Increase in number of the bands
caused by the additional aromatic ring.
2. Results and discussion
2.1. Chemistry
The synthetic pathways applied for the synthesis of the intermediates
and target pyridopyrimidine are shown in Schemes 1, 2 and 3.
In scheme 1, reaction of the appropriate 4- substituted benzaldehyde
with 4-methoxy acetophenone in presence of alcoholic sodium
Scheme 3 shows the reaction of compounds 2a-d with phenyl iso-
thiocyanate 1:1 or 1:2 in pyridine furnished the target compounds 5a-
Fig. 1. Lead compounds Lapatinib (1) Gefitinib (2), Erlotinib (3), and compound (4) and their inhibitory activity against EGFR.
2

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
Fig. 2. Examples of EGFR inhibitors with a core analogous to 4-aminoquinazoline core in the aforementioned lead compounds.
Fig. 3. The design of the targeted compounds 4a-h, 5a-d and 6a-d to target the important regions of the binding pocket of EGFR.
Scheme 1. Reagents and conditions: (i) alcoholic NaOH 40%, stand overnight at RT (ii) Ammonium acetate in absolute ethanol, Reflux 10 h.
d and 6a-d respectively. Structures assigned to the products were in
agreement with the micro-elemental and spectral analyses. 1H NMR
spectra of compounds 5a-d exhibited two D2O exchangeable singlet
signals rather than the one singlet was in the start corresponding to NH2
protons. One of these exchangeable singlet signals appears near 13 ppm
corresponding to NH at the pyrimidine ring. In addition to extra five
aromatic protons appear due to extra phenyl ring. 13C NMR spectra of 5a
and 5d revealed characteristic signals at 181.97 and 181.91 attributed
to C=S respectively. On the other hand, 1H NMR spectra ascertained the
presence of extra aromatic ring indicated by the increase in the
integration of the aromatic protons, along with the disappearence of
singlet signal of the NH at the pyrimidine ring near 13 ppm. Along with
the 13C NMR spectra that showed the appearance of two new signals
around 160–170 attributed to C=S groups.
2.2. Biological evaluation
The novel synthesized targeted compounds were screened for their
EGFR-TK inhibitory assay and the most active four compounds were
evaluated for their in vitro cytotoxicity against two cancer cell lines,
3

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
Scheme 2. Reagent and condition: (i) equimolar amount of DMF/DMA, heat to 80–90 ◦C for 4 hr (ii) CH3COOH, reflux 24 h. (iii) N,N-dimethyl-N’ substituted phenyl
formimidamide in acetic acid, reflux 5–7 hrs.
which are; the breast cancer cell line MCF-7 and non-small cell lung
cancer cell line MDA-MB. Moreover, cell cycle analysis and apoptotic
assay was applied for compound 4b to inspect its effect on the cell cycle
progress and the percentage of apoptosis induced by it.
inhibitory effect compared to Gefitinib. Additionally, The results
revealed that thiourea derivatives 6a,d and 4-anilino derivative 4b, g
were more significantly potent EGFR inhibitors than Gefitinib by nearly
1.5 fold.
2.2.1. EGFR - TK inhibitory assay
2.2.2. In vitro cytotoxic activity
The sixteen newly synthesized target compounds, 4a-h, 5a-d and 6a-
d were examined in vitro for their EGFR inhibition activity. Table 1
displays the inhibition data (IC50 values) of the tested compounds to-
wards EGFR using Gefitinib as a reference compound. As shown in
Table 1, all the examined compounds exhibited EGFR inhibitory activity
with IC50 values in the range of (31.24– 233.3 nM). Upon statistical
analysis of the results, it was found that the activity of six compounds;
namely, 4-anilinopyridopyrimidine derivatives 4b,g,h, one of 4-anilino-
thionpyridopyrimidine 5c and two thiourea derivatives 6a,d was
significantly higher than that of Gefitinib. While compounds 4d and 4f
showed IC50 values non– significantly different from that of the refer-
ence drug.
Four compounds 4b, g and 6a, d, out of the target compounds that
provoked greater EGFR inhibitory activity (31.24–39.31 nM), were
chosen to be assessed in vitro for their cytotoxicity against two cancer
cell lines; The Human Caucasian breast adenocarcinoma cell line MDA-
MB-361 [20,21] and the breast cancer cell line MCF-7. These cell lines
are well-known to be EGFR-overexpressing cell lines, [22–27] to pro-
voke their cellular efficiency using MTT assay [28]. Gefitinib was used
as a reference standard. The in vitro cytotoxic results of the examined
compounds are presented in Table 2.
The data presented in Table 2 that, compound 4b displayed the most
selective anticancer activity against MCF-7 and MDA-MB-361 cell lines
with IC50 of 4.82 and 4.43 nM, respectively, relative to Gefitinib (IC50
= 4.97 nM).
The results obtained displayed that, the 4-anilinopyridopyrimidine
derivatives can be considered the most active targeted compounds,
meanwhile the 4-anilinothionpyridopyrimidine derivatives 5a-d are less
active. Noticeably, all the active compounds except 5c contain halogen
either in position 4 or 5, or in both positions. Interestingly, fluorine
containing compounds 4b, d and h are equipotent or more potent than
Gefitinib. All bromine-containing compounds 4 g, 4 h and 6d except 5d
have more significant EGFR inhibitory activity than Gefitinib. The two
thiourea derivatives 6a and 6d containing halogen in position 5 were
more active; while those free of halogen have significantly lower EGFR
2.2.3. Cell cycle Analysis
cell cycle analysis and apoptotic assay was achieved for the 5-(4-
Chlorophenyl)-N-(4-fluorophenyl) pyrido[2,3-d]pyrimidin-amine 4b
that displayed a superior EGFR inhibitory activity, anticancer activity
against MCF-7 and MDA-MB-361 cell line in order to explore the effect
of this compound on cell cycle progression and the apoptosis percentage
made by the compound as shown in Table 3 and Fig. 5.
The results acquired from Table 3 and Fig. 5 showed that after the
4

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
Scheme 3. Reagent and condition: (i) equimolar amount of phenyl isothiocyanate in pyridine heated to 80–90 ◦C for 6–8 hr (ii) phenyl isothiocyanate in pyridine,
reflux 2–3 hr.
Fig. 4. proposed mechanism of Dimroth rearrangement for the preparation of pyridopyrimidine derivatives 4a-h.
addition of compound 4b the percentage of DNA content was increased
(54.11 %) in comparison to the control cells (37.21 %). This means
increasing pre G1 phase and cell growth arrest at S phase in MDA-MB-
361 cells after 24 h. This data is a sign for its apoptotic activity.
membrane integrity and allows measuring the apoptotic death kinetics
with regard to the cell cycle.
The apoptotic induction effect of compound 4b on in MDA-MB-361
cells is presented in Table 4 and Fig. 6.
The results achieved from Table 4 and Fig. 6 revealed an increase in
the total apoptotic cells percentage in MDA-MB-361 cell line after using
compound 4b (36.71 %) relative to control cells (2.55 %) which rep-
resents a noticeable marker of apoptosis.
2.2.4. Apoptotic Assay
To inspect the apoptotic effect and to calculate apoptosis percentage
prompted by compound 4b in MDA-MB-361 cells, Annexin V-FITC/
propidium iodide dual staining assay was used [29,30]. The Annexin V
assay can identify the early phases of apoptosis before the losing the cell
5

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
docking and the pose with the best score (-7.9567 Kcal/mol). In the
binding mode displayed in Figs. 4 and 5, compound 4b achieved two
main critical interactions that were reported for Gefitinib [32] which are
a hydrogen bond with Met793 (2.5 Å) and water mediated hydrogen
bond with Asp855 in the hinge region with the N1 and N3 atoms of
pyridine fused ring, respectively (Fig. 7). Additionally, the non-coplanar
phenyl ring with 4-chloro substitution was embedded in the hydro-
phobic region inside the non-covalent binding pocket. This pocket is
surrounded by Arg841, Gly 721 and Asp855 (Fig. 8).
Table 1
EGFR inhibitory values of the target compounds with respect to Gefitinib as a
reference standard.
Cpd. No.
EGFR IC50 (nM)
Cpd. No.
EGFR IC50 (nM)
4a
87.04 ± 1.87
31.72 ± 0.69
213.60 ± 4.33
47.89 ± 1.04
107.01 ± 1.02
48.27 ± 2.4
5a
5b
5c
5d
6a
6b
6c
6d
58.74 ± 1.27
112.10 ± 2.59
39.98 ± 0.84
140.40 ± 3.11
35.44 ± 0.75
58.36 ± 1.26
233.30 ± 4.66
31.24 ± 0.65
4b
4c
4d
4e
4f
4 g
4 h
Gefitinib
38.73 ± 0.81
46.12 ± 1.3
3. Conclusion
52.37 ± 1.09
Sixteen novel pyrido-pyrimidine derivatives were designed, synthe-
sized and screened for their In vitro EGFR inhibitory activity. The results
elicited that six compounds have significant superior EGFR inhibitory
activity relative to Gefitinib. Two compounds showed IC50 values non–
significantly different from that of the reference drug. Compounds 4b, g
and 6a, d were further screened against human cancer cell lines, MCF7
and MDA-MB-361 and they furnished interesting results. Compound 4b
was additionally subjected to cell cycle analysis and apoptotic assay.
Furthermore, cell cycle analysis of compound 4b revealed increasing pre
G1 phase with cell growth arrest at S phase in addition to its apoptotic
effect. The binding mode of compound 4b in the active site of EGFR was
investigated and achieved two main critical interactions that were re-
ported for Gefitinib
Table 2
Cytotoxicity of the tested compounds against cell lines MCF-7 and MDA-MB-
361 using Gefitinib as a reference standard.
Cpd. No.
Cytotoxicity
IC50 M)
(μ
MCF-7
MDA-MB-361
4b
4.82 ± 0.2
4.43 ± 0.23
11.90 ± 0.61
29.80 ± 1.52
22.20 ± 1.13
7.27 ± 0.49
4h
12.70 ± 0.52
32.70 ± 1.34
10.80 ± 0.44
4.97 ± 0.24
6a
6d
Gefitinib
4. Experimental
Table 3
Cell cycle analysis results of compound 4b after 24 h in MDA-MB-361 cell line.
4.1. Chemistry
Cpd.
No.
%G0-
G1
%S
%G2/
M
%Pre-
G1
Comment
The requirements and specifications of all the devices used in the
synthesis and analysis of the prepared compounds were supplied in the
supplementary data. Compounds 1a [33], 1b [34], 1c [35] and 1d [36]
were . While compounds 2a, d [37,38] were synthesized according to
the reported method with some modifications.
4b
39.81
54.11
37.21
6.08
36.71
Pre G1 apoptosis and Cell
growth arrest at S phase.
Control
54.29
8.5
2.55
2.3. Molecular modeling study
Table 4
In this study, investigation of the binding mode of compound 4b in
the active site of EGFR took place through docking studies which were
performed on the crystal structure 1XKK retrieved from protein data
bank and which co-crystalized with Gefitinib.[31] Re-docking score was
calculated for the prepared gefitinib to assess the docking protocol and
the RMSD value were calculated to give 1.7530 Å with docking score
(-10.8279 Kcal/mol). Two given poses were given as a result of the
Effect of compound 4b on induction of apoptosis compared to the control cells
after 24 h.
Cpd. No.
Apoptosis
Total
Necrosis
Early
Late
4b
36.71
2.55
1.75
0.64
22.65
0.28
12.31
1.63
Control
Fig. 5. Effect of compound 4b on the cell cycle of MDA-MB-361 cells after 24 h.
6

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
Control/ MDA-MB-361
4b/ MDA-MB-361after 24 h
Fig. 6. Effect of compound 4b on apoptosis induction of MDA-MB-361cells after 24 h.
Fig. 7. Docking pose of compound 4b in the active site of EGFR (pdb:1XKK).
Binding mode showing main hydrogen bonds with Met793 and water mediated
hydrogen bond with Asp855.
Fig. 8. Binding mode of compound 4b to the active site of EFGR with hydro-
phobic surface showed the role of the non-coplanar aromatic ring with 4-chlor-
osubstitution (Blue; hydrophobic and red; hydrophilic). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web
version of this article.)
4.1.1. General procedure for the preparation of compounds 2b,c
A mixture of the appropriate chalcone 1b, c (0.05 mol), malononi-
trile (0.05 mol) and ammonium acetate (0.5 mol) in ethanol (80 mL) was
refluxed for 10–16 hr. The solid obtained was filtered off while hot,
washed several times with portions of boiling ethyl alcohol, dried to give
compounds 2b and c respectively.
νmax/cmꢀ 1): 3483,3387 (–NH2), 3074,3047 (arom.CH), 2970, 2893
–
1
–
(aliph.CH), 2199 (C N), 1608 (C=C); H NMR (DMSO‑d6) δ ppm: 2.55
–
(s,3H, SCH3), 3.83 (s,3H, OCH3), 6.92 (s, 2H, NH2 D2O exchange), 7.03
(d, 2H, J = 8.0 Hz, arom.H), 7.21 (s,1H, pyrid.H), 7.41 (d, 2H, J = 8.3
Hz, arom.H), 7.62 (d, 2H, J = 8.3 Hz, arom.H), 8.10 (d, 2H, J = 8.8 Hz,
arom.H). 13C NMR (DMSO‑d6) δ ppm: 23.59, 55.87, 100.82, 114.80,
116.55, 126.10, 129.64, 129.74, 132.56, 141.76, 153.90, 154.61,
158.62, 161.91, 169.48. MS, m/z: 347.28 [M+], 350.16 [M + 3]+ Anal.
Calcd for C20H17N3OS (347.44): C, 69.14; H, 4,93; N, 12.09; Found: C,
69.32; H, 4,79; N, 12.20; %.
4.1.1.1. 2-Amino-4-(4-dimethylaminophenyl)-3-cyano-6-(4-methox-
yphenyl)pyridine (2b). White crystals, yield 56%, m.p. 205–207 ◦C; IR
(KBr, νmax/cmꢀ 1): 3460, 3363 (–NH2), 3176 (arom.CH), 2951, 2924
1
–
–
(aliph.CH), 2212 (C N), 1635 (C=C); H NMR:(DMSO‑d6) δ ppm: 2.99
–
(s,6H, N(CH3)2), 3.83 (s,3H, OCH3), 6.77 (s, 2H, NH2, D2O exchange),
6.84 (d, 2H, J = 9.8 Hz, arom.H), 7.03 (d, 2H, J = 8.4 Hz, arom.H), 7.17
(s,1H, pyrid.H), 7.58 (d, 2H, J = 9.2 Hz, arom.H), 8.09 (d, 2H, J = 8.8
Hz, arom.H). 13C NMR (DMSO‑d6) δ ppm: 40.08, 55.84, 104.65, 114.22,
118.59, 122.87, 128.23, 129.73, 130.90, 131.87, 137.36, 148.18,
158.18, 159.75, 161.38, 162.02. Anal. Calcd. for C21H20N4O (344.42):
C, 73.23; H, 5.85; N, 16.27; Found: C, 73.46; H, 5.91; N, 16.20.
4.1.2. General procedure for the preparation of compounds (3a-d)
A mixture of appropriate 2-amino-6-(4-methoxyphenyl)-4-(sub)nico-
tinonitrile 2a-d (0.01 mol) and DMF/DMA (0.01 mol) in the least
amount possible of DMF was heated to 100–110 ◦C for 4 hr. After con-
centration and cooling, the separated solid was filtered, washed with
boiling ethanol and purified by recrystallization from chloroform-
4.1.1.2. 2-Amino4-(4-methylthiophenyl)-3-cyano-6-(4-methoxyphenyl)
pyridine (2c). White crystals, yield 67%, m.p. 207–209 ◦C; IR (KBr,
7

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
ethanol mixture (1:2) to afford compounds 3a-d.
C27H21ClN4O: (452.94): C, 71.60; H, 4.67 ; N, 12.37; Found: C, 71.72; H,
4.71; N, 12.28%.
4.1.2.1. N’-(4-(4-chlorophenyl)-3-cyano-6-(4-methoxyphenyl)pyridin-2-
yl)-N,N-dimethylformimidamide (3a). White crystals, yield 93%, m.p.
180–182 ◦C; IR (KBr, νmax/cmꢀ 1): 3039 (CH arom), 2970 (CH aliph); 1H
NMR (DMSO‑d6) δ ppm: 2.47 (s,6H, N(CH3)2), 3.81 (s,3H, OCH3), 6.93
(d, 2H, J = 8.9 Hz, ArH), 7.44(s,1H,formim,H), 7.44 (d, 2H, J = 5.2 Hz,
ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.93 (d, 2H, J = 8.8 Hz, ArH), 7.97
(s,1H, Pyrid. H). Anal. Calcd for C22H19ClN4O (390.87): C, 67.60; H,
4.90; N, 14.33; Found: C, 66.81; H, 4.09; N, 13.99; %.
4.1.3.2. 5-(4-Chlorophenyl)-N-(4-fluorophenyl)-7-(4-methoxyphenyl)pyr-
ido[2,3-d] pyrimidin-4-amine (4b). White crystals, yield 35%, m.p.
200–202 C; IR (KBr, νmax/cmꢀ 1): 3402 (NH), 3039 (CH.arom), 2954
◦
(CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 3.87 (s, 3H, OCH3),
7.11–7.17 (m, 4H, ArH), 7.62–7.74 (m, 7H, ArH and NH D2O exchange),
7.98 (s, 1H, Pyrido. H), 8.34 (d, 2H, J = 8.3 Hz, ArH), 8.72 (s, 2H,
Pyrimid. H); MS, m/z: 456.38 [M]+, 457.74 [M + 1]+. Anal. Calcd for
C
26H18ClFN4O (456.91): C, 68.35; H, 3.97; N, 12.26; Found: C, 68.45; H,
4.05; N, 12.36; %.
4.1.2.2. N’-(3-cyano-6-(4-methoxyphenyl)-4-(4-(methylthio)phenyl)pyr-
idin-2-yl)-N,N-dimethylformimidamide (3b). Yellowish white crystals,
yield 95%, m.p. 200–202 ◦C; IR (KBr, νmax/cmꢀ 1): 3070, 3016 (CH
arom), 2966 (CH aliph); 1H NMR (DMSO‑d6) δ ppm: 2.97 (s,6H, N
(CH3)2), 3.41 (s,6H, N(CH3)2), 3.90 (s,3H, OCH3),), 7.03 (d, 2H, J = 8.8
Hz, ArH), 7.14(s,1H,formim,H), 7.36 (d, 2H, J = 8.4 Hz, ArH), 7.72 (d, J
= 8.4 Hz, 2H, ArH), 8.04 (d, 2H, J = 8.8 Hz, ArH), 8.66 (s,1H, Pyrid. H);
Anal. Calcd for C24H25N5O (399.50): C, 72.16; H, 6.31; N, 17.53. ;
Found: C, 71.23; H, 6.34; N, 17.50. %.
4.1.3.3. 5-(4-(Dimethylamino)phenyl)-N-(m-tolyl)-7-(4-methoxyphenyl)
pyrido[2,3-d] pyrimidin-4-amine (4c). Yellowish white crystals, yield
65%, m.p. 228–230 ◦C; IR (KBr, νmax/cmꢀ 1): 3344 (NH), 3040 (CH.
arom), 2997, 2968 ,2931 (CH aliphatic); 1H NMR (DMSO‑d6) δ ppm:
2.21 (s, 3H, CH3), 3.02 (s, 6H, 2 CH3), 3.85 (s, 3H, OCH3), 6.30–6.94 (m,
4H, ArH), 7.09–7.24 (m, 4H, ArH), 7.39–7.47 (m, 2H, ArH), 7.70 (s, 1H,
NH, D2O exchange), 7.86 (s, 1H, Pyrid. H), 8.28–7.32 (m ,2H, ArH), 8.73
(s, 1H, Pyrimid. H) .MS, m/z: 461.25 [M]+. Anal. Calcd for C29H27N5O
(461.57): C, 75.46; H, 5.90; N, 15.17; Found: C, 75.58; H, 6.01; N, 15.29
%.
4.1.2.3. N’-(3-cyano-4-(4-(dimethylamino)phenyl)-6-(4-methoxyphenyl)
pyridin-2-yl)-N,N-dimethylformimidamide (3c). White crystals, yield
94%, m.p. 198–200 ◦C; IR (KBr, νmax/cmꢀ 1): 30,974 (CH .arom), 2924
(CH. aliph); 1H NMR (DMSO‑d6) δ ppm: 2.16 (s, 3H, SCH3), 2.55 (s,6H, N
(CH3)2), 3.84 (s, 3H, OCH3), 7.09 (d, 2H, J = 8.8 Hz, ArH), 7.46 (d, 2H, J
= 8.4 Hz, ArH), 7.68 (d, J = 8.4 Hz, 2H, ArH), 7.92(s,1H,formim,H),
8.02 (d, 2H, J = 8.8 Hz, ArH), 8.68 (s,1H, Pyrid. H);.13C NMR
(DMSO‑d6) δ ppm: 14.73, 23.59, 55.87, 100.82, 114.80, 116.55, 126.10,
129.64, 129.74, 132.56, 141.76, 149.66, 153.90, 154.61, 158.62,
161.91, 169.48 Anal. Calcd for C23H22N4OS (402.52) : C, 68.63; H, 5.51;
N, 13.92; Found: C, 67.39; H, 4.69; N, 13.69%.
4.1.3.4. 5-(4-(Dimethylamino)phenyl)-N-(4-fluorophenyl)-7-(4-methox-
yphenyl)pyrido [2,3-d]pyrimidin-4-amine (4d). White crystals, yield
65%, m.p. 232–234 ◦C; IR (KBr, νmax/cmꢀ 1): 3348 (NH), 3032 (CH.
arom), 2962, 2912 (CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 3.31
(s,6H, N(CH3)2), 3.86 (s, 3H, OCH3), 7.09 (d, 2H, J = 9.8 Hz, ArH), 7.18
(s, 1H, NH, D2O exchange), 7.58 (d, 2H, J = 8.4 Hz, ArH), 7.63 (d, 2H, J
= 9.6 Hz, ArH), 7.77–7.82 (m, 2H, ArH), 7.96–7.99 (m, 2H, J = 8.9 Hz,
ArH), 8.29 (d, 2H, J = 9.9 Hz, ArH), 8.55 (s, 1H, Pyrid. H), 8.74 (s, 1H,
Pyrimid. H); MS, m/z: 465.59 [M]+. Anal. Calcd for C28H24FN5O:
(465.53): C, 72.24; H, 5.20; N, 15.04; Found: C, 72.86; H, 5.16; N, 14.77;
%.
4.1.2.4. (Z)-N’-(4-(4-bromophenyl)-3-cyano-6-(4-methoxyphenyl)pyr-
idin-2-yl)-N,N-dimethylformimidamide (3d). White crystals, yield 95%,
m.p. 180–182 ◦C; IR (KBr, νmax/cmꢀ 1): 3078, 3024 (CH.arom), 2956
(CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 2.87 (s,6H, N(CH3)2), 3.85
(s,3H, OCH3), 7.09 (d, 2H, J = 7.2 Hz, ArH), 7.68 (d, 2H, J = 8.4 Hz,
ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH), 7.97(s,1H,formim,H), 8.02 (d, 2H, J
= 8.4 Hz, ArH), 8.70 (s,1H, Pyrid. H); Anal. Calcd for C22H19BrN4O
(435.33): C, 60.70; H, 4.40; N, 12.87; Found: C, 61.48; H, 4.71; N,
11.40%.
4.1.3.5. 7-(4-methoxyphenyl)-5-(4-(methylthio)phenyl)-N-(m-tolyl)pyr-
ido[2,3-d] pyrimidin-4-amine (4e). White crystals, yield 35%, m.p.
203–205 C; IR (KBr, νmax/cmꢀ 1): 3314 (NH), 3051, 3024 (CH.arom),
◦
2997, 2962, 2935 (CH aliphatic); 1H NMR (DMSO‑d6) δ ppm:2.14 (s, 3H,
CH3), 2.55 (s, 3H, SCH3), 3.83 (s, 3H, OCH3), 6.30–6.38 (m, 2H, ArH),
6.86 (t, 1H, J = 7.6 Hz, ArH), 6.93 (s, 1H, NH, D2O exchange), 7.03 (d,
2H, J = 8.8 Hz, ArH), 7.21 (s, 1H, Pyrid. H), 7.41 (d, 2H, J = 8.4 Hz,
ArH), 7.62 (d, 2H, J = 8.4 Hz, ArH), 8.10 (d, 2H, J = 8.8 Hz, ArH), 8.77
(s,1H, Pyrimid. H); 13C NMR (DMSO‑d6) δ ppm: 14.79, 21.67, 55.77,
85.88, 108.70, 111.65, 114.45, 115.03. 117.05, 117.80, 126.05, 129.13,
129.26, 129.29, 130.39, 133.72, 138.16, 140.98, 148.96, 154.49,
158.76, 161.35, 161.45. ; MS, m/z: 461 [Mꢀ 3]+. Anal. Calcd for
C28H24N4OS (464.16): C, 72.39; H, 5.21 ; N, 12.06; Found: C, 71.72; H,
5.71; N, 12.28%.
4.1.3. Synthetic procedures of compounds (4a-h)
A mixture of the appropriate 2-amino-6-(4-methoxyphenyl)-4-(sub)
nicotinonitrile 2a-d (0.01 mol) and N,N-dimethyl-N’subistituted phe-
nylformimidamide (0.01 mol) (prepared by refluxing equivalent amount
of the required aniline with DMA/DMF for 2 hrs in DMF) was refluxed in
acetic acid (7 mL) for 5–7 hrs. After cooling, neutralization was per-
formed using 10% alcoholic sodium hydroxide solution. The separated
solid was filtered, washed with water several times, separated by pre-
parative chromatography using chloroform–methanol mixture (9.5:0.5)
as eluent.
4.1.3.6. N-(3,4-dichlorophenyl)-7-(4-methoxyphenyl)-5-(4-(methylthio)
phenyl)pyrido [2,3-d]pyrimidin-4-amine (4f). White crystals, yield 62%,
m.p. 229–231 ◦C; IR (KBr, νmax/cmꢀ 1): 3410 (NH), 3051, 3020 (CH.
arom), 2997, 2962 (CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 2.57 (s,
3H, SCH3), 3.87 (s, 3H, OCH3), 7.13 (d, 1H, J = 8.7 Hz, ArH), 7.17 (d,
2H, J = 8.6 Hz, ArH), 7.43 (d, 2H, J = 8.4 Hz, ArH), 7.51 (s,1H, ArH),
7.71 (s,1H, Pyrid. H), 7.74 (d, 2H, J = 8.7 Hz, ArH), 7.88 (s, 1H, NH, D2O
exchange), 7.94 (d, 1H, J = 8.4 Hz, ArH), 8.19 (s, 1H, Pyrimidin. H),
8.28 (d, 2H, J = 8.4 Hz, ArH). MS, m/z: 519.55 [M]+, 521[M + 2]+.
Anal. Calcd for C27H20Cl2N4OS: (519.44): C, 62.43; H, 3.88; N, 10.79;
Found: C, 62.52; H, 3.85; N, 10.79%.
4.1.3.1. 5-(4-Chlorophenyl)-7-(4-methoxyphenyl)-N-(m-tolyl)pyrido[2,3-
d]pyrimidin-4-amine (4a). White crystals, yield 55%, m.p. 208–210 ◦C;
IR (KBr, νmax/cmꢀ 1): 3383 (NH), 3032, 3005 (CH.arom), 2970, 2935
(CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 2.25 (s, 3H, CH3), 3.87 (s, 3H,
OCH3), 6.87 (d, 1H, J = 8.4 Hz, ArH), 6.99 (s, 1H, ArH), 7.06–7.19 (m,
4H, ArH), 7.54 (s, 1H, NH, D2O exchange), 7.65 (d, 2H, J = 8.5 Hz, ArH),
7.71 (d, 2H, J = 8.5 Hz, ArH), 7.98 (s, 1H, Pyrid. H), 8.34 (d, 2H, J = 8.9
Hz, ArH), 8.76 (s, 1H, Pyrimid. H); 13C NMR (DMSO‑d6) δ ppm: 14.79,
21.58, 55.84, 106.51, 114.78, 118.10, 120.64. 121.48, 124.92, 129.06,
129.27, 129.87, 130.09, 131.80, 134.74, 137.05, 138.43, 138.69,
148.02, 157.78, 158.36, 160.18, 160.57, 162.01.: . Anal. Calcd for
4.1.3.7. 5-(4-bromophenyl)-N-(m-tolyl)-7-(4-methoxyphenyl)pyrido[2,3-
d]pyrimidin-4-amine (4g). White crystals, yield 30%, m.p. 211–13 ◦C; IR
8

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
(KBr, νmax/cmꢀ 1): 3406 (NH), 3051 (CH.arom), 2996,2935 (CH
ArH), 7.29–7.37 (m,4H, ArH), 7.50 (d, 2H, J = 9.8 Hz, ArH), 7.58 (s, 1H,
Pyrid. H), 7.63 (d, 2H, J = 9.8 Hz, ArH), 7.72 (s, 1H, NH, D2O, ex-
change), 8.27 (d, 2H, J = 6.1 Hz, ArH), 13.03 (s, 1H, NH, D2O ex-
change); MS, m/z : 482.25 [M]+. Anal. Calcd for C27H22N4OS2 (482.62) :
C, 67.20; H, 4.59; N, 11.61; Found: C, 67.29; H, 4.69; N, 11.69%.
1
aliphatic); H NMR (DMSO‑d6) δ ppm: 2.17 (s, 3H, CH3), 3.85 (s, 3H,
OCH3), 6.99–7.11 (m, 3H, ArH), 7.23 (s, 1H, ArH), 7.62 (d, 1H, J = 8.4
Hz, ArH), 7.68 (d, 2H, J = 8.4 Hz, ArH), 7.76 (d, 1H, J = 8.5 Hz, ArH),
7.82 (d, 2H, J = 8.5 Hz, ArH), 7.96 (s, 1H, Pyrid. H), 8.11 (d, 1H, J = 8.8
Hz, ArH), 8.21 (d, 2H, J = 8.9 Hz, ArH, PyrimidH), 10.89 (s, 1H, NH,
D2O exchange) ; 13C NMR (DMSO‑d6) δ ppm: 23.59, 55.84, 79.63, 85.89,
100.86, 108.75, 114.65, 116.40, 117.52, 123.58, 124.184,0.129.50,
130.25, 130.95, 131.42, 132.20, 135.68, 136.76, 153.90, 158.82,
161.23, 161.98, 169.46 .Anal. Calcd for C27H21BrN4O (497.40): C,
65.20; H, 4.26; N, 11.26; Found: C, 65.72; H, 4.71; N, 11.28%.
4.1.4.4. 5-(4-Bromophenyl)-7-(4-methoxyphenyl)-4-(phenylamino)pyrido
[2,3-d] pyrimidine-2(1H)-thione (5d). Creamy white crystals, yield 58%,
m.p. 208–210 ◦C; IR (KBr, νmax/cmꢀ 1): 3487, 3317 (NHs), 3078, 3024
(CH.arom), 2956 (CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 3.87 (s,3H,
OCH3), 6.98 (t, 1H, J = 7.2 Hz, ArH), 7.04 (d, 2H, J = 8.9 Hz, ArH), 7.11
(d, 2H, J = 8.6 Hz, ArH), 7.62 (s, 1H, Pyrid. H), 7.65 (t, 2H, J = 8.2 Hz,
ArH), 7.76–7.82 (m, 4H, ArH), 8.11 (d, 2H, J = 8.9 Hz, ArH), 8.31 (s,1H,
NH, D2O, exchange), 13.06 (s, 1H, ,D2O, exchange NH); MS, m/z: 514.78
[M]+. Anal. Calcd for C26H19BrN4OS (514.05): C, 60.59; H, 3.72; N,
10.87; Found: C, 60.48; H, 3.71; N, 11.40%.
4.1.3.8. 5-(4-bromophenyl)-N-(4-fluorophenyl)-7-(4-methoxyphenyl)pyr-
ido[2,3-d] pyrimidin-4-amine (4h). White crystals, yield 25%, m.p.
220–222 C; IR (KBr, νmax/cmꢀ 1): 3379 (NH), 3051 (CH.arom), 2931
◦
(CH aliphatic); 1H NMR (DMSO‑d6) δ ppm: 3.83 (s, 3H, OCH3),
7.09–7.13 (m, 4H, ArH), 7.26 (s, 1H, NH, D2O exchange), 7.59–7.91 (m,
6H, ArH), 8.06 (s, 1H, Pyrid. H), 8.28 (d, 2H, J = 8.0 Hz, ArH), 8.67 (s,
1H, Pyrimid. H). 13C NMR (DMSO‑d6) δ ppm: 55.37, 102.15, 114.10,
117.20, 120.95, 121.52, 123.26, 124.66, 128.75, 128.88, 129.39,
129.87, 130.29, 136.18, 137.01, 137.58, 138.82, 146.72, 158.10,
158.57, 160.98, 161.84. Anal. Calcd for C26H18BrClN4O (501.36): C,
60.31; H, 3.50; N, 10.82; Found: C, 60.45; H, 3.05; N, 10.36%.
4.1.5. Method for the preparation of compounds (6a-d)
To a mixture of appropriate 2-amino-6-(4-methoxyphenyl)-4-(sub)
nicotinonitrile 2a-d (0.01 mol) in pyridine (7 mL), phenyl isothiocya-
nate (0.02 mol) was added, refluxed for 2–3 hr. After cooling, the re-
action mixture was added to ethanol and the separated solid was
filtered, washed with water and purified by boiling with EtOH and
filtering while hot.
4.1.4. General procedure for the synthesis of compounds (5a-d)
To a mixture of appropriate 2-amino-6-(4-methoxyphenyl)-4-(sub)
nicotinonitrile 2a-d (0.01 mol) in pyridine (7 mL), phenyl isothiocya-
4.1.5.1. 4.1.5.1. 1-(5-(4-Chlorophenyl)-7-(4-methoxyphenyl)-3-phenyl-2-
thioxo-2,3-dihydro pyrido[2,3-d]pyrimidin-4-yl)-3-phenylthiourea (6a).
◦
Brownish yellow solid, yield 95%, m.p.240–242 ◦C; IR (KBr, νmax
/
nate (0.01 mol) was added, heated to 80–90 C for 6–8 hr. After con-
cmꢀ 1): 3294, 3255 (NHs), 3055 (CH.arom), 2924 (CH aliphatic); 1H
NMR (DMSO‑d6) δ ppm: 3.86 (s,3H, OCH3), 6.98 (t, 1H, J = 7.3 Hz, ArH),
7.02–7.12 (m, 3H, ArH), 7.28–7.34 (m, 6H, ArH), 7.39 (s, 1H, NH, D2O
exchange), 7.62 (s, 1H, Pyrid. H), 7.66 (d, 2H, J = 8.4 Hz, ArH), 7.73 (d,
2H, J = 8.4 Hz, ArH), 7.88–7.91 (m, 2H, ArH), 8.28 (d, 2H, J = 8.8 Hz,
ArH), 9.59 (s, 1H, NH,D2O exchange) . MS, m/z: 605.07 [M]+ ; Anal.
Calcd for C33H24 Cl N5OS2 (605.11): C, 65.39; H, 3.99; N,11.55; Found:
C, 65.45; H, 404; N, 11.45; %.
centration and cooling, the reaction mixture was added to ethanol and
the separated solid was filtered, washed with boiling ethanol and puri-
fied by recrystallization from chloroform-EtOH mixture (1:2) to afford
compounds 5a-d.
4.1.4.1. 5-(4-Chlorophenyl)-7-(4-methoxyphenyl)-4(phenylamino)pyrido
[2,3d] pyrimidine-2-(1H)-thione (5a). Yellowish white crystals, yield
92%, m.p. 212–214 C; IR (KBr, νmax/cmꢀ 1): 3375, 3345 (NHs), 3039
◦
(CH arom), 2970 (CH aliph); 1H NMR (DMSO‑d6) δ ppm: 3.86 (s,3H,
OCH3), 7.10 (d, 3H, J = 8.7 Hz, ArH), 7.31 (d, 3H, J = 7.3 Hz, ArH), 7.66
(d, J = 8.4 Hz, 2H, ArH), 7.73 (d, 2H, J = 7.6 Hz, ArH), 7.75 (s,1H, Pyrid.
H), 7.78 (s, 1H, NH, D2O exchange), 8.29 (d, 2H, J = 8.8 Hz, ArH), 13.05
(s, 1H, NH,D2O exchange); 13C NMR (DMSO‑d6) δ ppm: 55.89, 102.33,
114.79, 118.22, 121.58, 125.14, 129.16, 129.25, 129.32, 129.89,
131.94, 135.11, 136.00, 138.04, 148.31, 153.27, 154.19, 159.68,
162.25, 181.97.Anal. Calcd for C26H19ClN4OS (470.10): C, 66.31; H,
4.07; N, 11.90; Found: C, 66.41; H, 4.00; N, 11.99; %.
4.1.5.2. 1-(5-(4-Dimethylaminophenyl)-7-(4-methoxyphenyl)-3-phenyl-2-
thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-phenylthiourea (6b).
Brown solid, yield 95%, m.p. 262–263 ◦C; IR (KBr, νmax/cmꢀ 1): 3302,
3263 (NHs), 3194, 3124, 3070 (CH.arom), 2993, 2924 (CH aliphatic);
1H NMR (DMSO‑d6) δ ppm: 3.01 (s, 6H, 2 CH3), 3.87 (s, 3H, OCH3),
6.95–7.10 (m, 6H, - ArH), 7.25–7.32 (m, 6H, - ArH), 7.49 (s, 1H, Pyrid.
H), 7.51 (d, 2H, J = 6.8 Hz, ArH), 7.59 (s, 1H, NH, D2O exchange),
7.91–7.94 (m, 2H, ArH), 8.24 (d, 2H, J = 9.8 Hz, ArH), 9.52 (s, 1H, NH,
D2O exchange); MS, m/z: 614.83 [M]+ , 616.64 [M + 2]+ . Anal. Calcd
for C35H30N6OS2 (614.19): C, 68.38; H, 4.90; N,13.67; Found: C, 68.40;
H, 4.89; N, 13.68; %.
4.1.4.2. 5-(4-Dimethylaminophenyl)-7-(4-methoxyphenyl)-4-(phenyl-
amino)pyrido [2,3d] pyrimidine-2(1H)-thione (5b). Yellow crystals, yield
75%, m.p. 230–232 ◦C; IR (KBr, νmax/cmꢀ 1): 3456, 3360 (NHs), 3070,
3016 (CH arom), 2966 (CH aliph); 1H NMR (DMSO‑d6) δ ppm: 3.04
(s,6H, N(CH3)2), 3.86 (s,3H, OCH3), 6.95 (d, 2H, J = 8.7 Hz, ArH), 7.10
(d, 3H, J = 8.8 Hz, ArH), 7.29–7.33 (m, 4H, ArH), 7.53 (d, 2H, J = 8.5
Hz, ArH), 7.67 (s,1H, Pyrid. H), 7.88 (s, 1H, NH, D2O exchange), 8.27 (d,
2H, J = 8.7 Hz, ArH), 12.96 (s, 1H, NH, D2O exchange);. 13C NMR
(DMSO‑d6) δ ppm: 40.00, 55.84, 102.42, 112.77, 114.75, 118.34,
121.41, 123.31, 125.10, 129.25, 129.75, 130.66, 137.86, 146.39,
150.38, 151.81, 153.05, 159.37, 160.53, 162.07, 181.91. Anal. Calcd for
4.1.5.3. 1-(7-(4-Methoxyphenyl)- 5-(4-methylthiophenyl)-3-phenyl-2-thi-
oxo-2,3-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-phenylthiourea
(6c).
Brownish solid, yield 90%, m.p. 257–259 ◦C; IR (KBr, νmax/cmꢀ 1): 3302,
3263 (NHs), 3074 (CH.arom), 2997, 2931 (CH aliphatic); 1H NMR
(DMSO‑d6) δ ppm: 2.51 (s,3H, SCH3), 3.85 (s, 3H, OCH3), 6.91 (t, 1H, J
= 7.2 Hz, ArH), 7.00–7.10 (m, 6H, - ArH), 7.15 (t, 1H, J = 7.4 Hz, ArH),
7.28 (d, 2H, J = 6.8 Hz, ArH), 7.32 (d, 2H, J = 7.00 Hz, ArH), 7.50 (s,1H,
pyrid. H), 7.53 (d, 2H, J = 7.2 Hz, ArH), 7.65 (s, 1H, NH, D2O exchange),
7.91–7.94 (m, 2H, ArH), 8.27 (d, 2H, J = 8.7 Hz, ArH), 9.57 (s, 1H, NH,
D2O exchange); 13C NMR (DMSO‑d6) δ ppm: 15.41, 55.37, 102.15,
112.43, 114.10, 117.20, 120.950, 121.52, 123.26, 124.66, 128.75,
128.88, 129.39, 129.87, 130.29, 136.18, 137.01, 137.58, 138.82,
146.72, 158.10, 158.57, 160.98, 161.84, 162.44, 178.01 . Anal. Calcd
for C34H27N5OS3: (617.14): C, 66.10; H, 4.41; N, 11.34; Found: C, 66.23;
H, 4.48; N, 11.25; %.
C
28H25N5OS (479.18): C, 70.12; H, 5.25; N, 14.60. ; Found: C, 70.23; H,
5.34; N, 14.50. %.
4.1.4.3. 7-(4-Methoxyphenyl)-5-(4-methylthiophenyl)-4-(phenylamino)
pyrido[2,3-d] pyrimidine-2(1H)-thione (5c). Yellow crystals, yield 64%,
m.p. 220–222 ◦C; IR (KBr, νmax/cmꢀ 1): 3471, 3302 (NHs), 30,974 (CH .
arom), 2924 (CH. aliph); 1H NMR (DMSO‑d6) δ ppm: 2.58 (s, 3H, SCH3),
3.85 (s, 3H, OCH3), 6.98 (t, 1H, J = 7.3 Hz, ArH), 7.08 (d, 2H, J = 8.7 Hz,
9

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
4.1.5.4. 1-(5-(4-Bromophenyl)-7-(4-methoxyphenyl)-3-phenyl-2-thioxo-
2,3-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-phenylthiourea,. (6d). Brow
nish solid, yield 88%, m.p. 238–240 ◦C; IR (KBr, νmax/cmꢀ 1): 3305,3263
(NH), 3194,3128,3074 (CH.arom), 2997 (CH aliphatic); 1H NMR
(DMSO‑d6) δ ppm: 3.85 (s, 3H, OCH3), 6.96 (t, 1H, J = 7.4 Hz, ArH),
6.98–7.12 (m, 4H, ArH), 7.28–7.32 (m, 6H, ArH), 7.58 (s, 1H, Pyrid. H),
7.64–7.71 (m, 5H, ArH), 8.25 (d, 2H, J = 9.8 Hz, ArH), 9.54 (s, 1H, NH,
D2O exchange), 13.00 (s, 1H, NH, D2O exchange); 13C NMR (DMSO‑d6) δ
ppm: 55.37, 102.11, 114.10, 117.06, 120.93, 121.49, 123.22, 124.61,
125.76, 128.74, 128.88, 129.37, 129.91, 130.53, 132.82, 137.53,
137.60, 138.86, 146.70, 158.19 ,158.56 ,160.96, 161.62, MS, m/z: 649
[M]+,651[M + 2]+.. Anal. Calcd for C33H24BrN3OS2 (649.06): C, 60.92;
H, 3.72; N, 10.76; Found: C, 61.01; H, 3.88; N, 10.86%.
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.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105318.
References
[1] G.S. Hassan, Synthesis and antitumor activity of certain new thiazolo[2,3-b]
quinazoline and thiazolo[3,2-a]pyrimidine analogs, Med. Chem. Res. 23 (1) (2014)
388–401, https://doi.org/10.1007/s00044-013-0649-6.
4.2. Biological evaluation
[2] V.T. DeVita, E. Chu, A history of cancer chemotherapy, Cancer Res. 68 (21) (2008)
8643–8653, https://doi.org/10.1158/0008-5472.CAN-07-6611.
[3] V.V. Padma, An overview of targeted cancer therapy, Biomedicine 5 (2015) 19,
https://doi.org/10.7603/s40681-015-0019-4.
Biological evaluation was achieved in the laboratory of the Egyptian
company for the production of vaccines, sera, and drugs (VACSERA,
Giza, Egypt).
[4] F. Broekman, E. Giovannetti, G.J. Peters, Tyrosine kinase inhibitors: Multi-targeted
or single-targeted, World J. Clin. Oncol. 2 (2011) 80–93, https://doi.org/10.5306/
wjco.v2.i2.80.
[5] M. Topcul, I. Cetin, Endpoint of cancer treatment: targeted therapies, Asian Pac. J.
Cancer Prev. 15 (11) (2014) 4395–4403, https://doi.org/10.7314/
APJCP.2014.15.11.4395.
4.2.1. EGFR-TK inhibitory assay
The EGFR-TK inhibitory assay applied for all of the synthesized
compounds was explained in details in the supplementary data.
[6] Z. Du, C.M. Lovly, Mechanisms of receptor tyrosine kinase activation in cancer,
Mol. Cancer 17 (58) (2018) 1–13, https://doi.org/10.1186/s12943-018-0782-4.
[7] R.S.M. Ismail, S.M. Abou-Seri, W.M. Eldehna, N.S.M. Ismail, S.M. Elgazwi, H.
A. Ghabbour, M.S. Ahmed, F.T. Halaweish, D.A. Abou El Ella, Novel series of 6-(2-
substitutedacetamido)-4-anilinoquinazolines as EGFR-ERK signal transduction
inhibitors in MCF-7 breast cancer cells, Eur. J. Med. Chem. 155 (2018) 782–796,
https://doi.org/10.1016/j.ejmech.2018.06.024.
4.2.2. In vitro cytotoxic activity
Four compounds 4b, g and 6a, d, that showed good EGFR inhibitory
activity (31.24–39.31 nM) after being examined for their EGFR inhibi-
tory activity, were chosen to be assessed for their in vitro cytotoxicity
against two EGFR-overexpressing cell lines, two human tumor cell lines,
MCF-7 and MDA-MB-361, (breast carcinoma cell lines). The procedure
was described in details in the supplementary file.
[8] K. Wosikowski, D. Schuurhuis, G.J. Kops, M. Saceda, S.E. Bates, Altered gene
expression in drug-resistant human breast cancer cells, Clin. Cancer Res. 3 (1997)
2405–2414.
[9] K.K. Ang, B.A. Berkey, X. Tu, H.Z. Zhang, R. Katz, E.H. Hammond, K.K. Fu, L. Milas,
Impact of epidermal growth factor receptor expression on survival and pattern of
relapse in patients with advanced head and neck carcinoma, Cancer Res. 62 (24)
(2002) 7350–7356.
4.2.3. Cell cycle analysis
[10] B. Moy, P. Kirkpatrick, S. Kar, P. Goss, Lapatinib, Nat. Rev. Drug Discov. 6 (6)
(2007) 431–432, https://doi.org/10.1038/nrd2332.
Cell cycle analysis assay was achieved for compound 4b in order to
inspect its effect on cell cycle progression. The experimental assay was
explained in the supplementary data.
[11] D.-D. Li, Y.-J. Qin, J. Sun, J.-R. Li, F. Fang, Q.-R. Du, Y. Qian, H.-B. Gong, H.-L. Zhu,
Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual
EGFR/HER2 Tyrosine Kinase Inhibitors, PLOS ONE 8 (2013) 69427, https://doi.
org/10.1371/journal.pone.0069427.
[12] T. Ishikawa, M. Seto, H. Banno, Y. Kawakita, M. Oorui, T. Taniguchi, Y. Ohta,
T. Tamura, A. Nakayama, H. Miki, H. Kamiguchi, T. Tanaka, N. Habuka, S. Sogabe,
J. Yano, K. Aertgeerts, K. Kamiyama, Design and Synthesis of Novel Human
Epidermal Growth Factor Receptor 2 (HER2)/Epidermal Growth Factor Receptor
(EGFR) Dual Inhibitors Bearing a Pyrrolo[3,2-d]pyrimidine Scaffold, J. Med.
Chem. 54 (23) (2011) 8030–8050, https://doi.org/10.1021/jm2008634.
[13] R. Ducray, P. Ballard, B.C. Barlaam, M.D. Hickinson, J.G. Kettle, D.J. Ogilvie, C.
B. Trigwell, Novel 3-alkoxy-1H-pyrazolo[3,4-d]pyrimidines as EGFR and erbB2
receptor tyrosine kinase inhibitors, Bioorg. Med. Chem. Lett. 18 (3) (2008)
959–962, https://doi.org/10.1016/j.bmcl.2007.12.035.
4.2.4. Apoptotic assay
To inspect the apoptosis induction effect and to calculate the
apoptosis percentage prompted by compound 4b in MDA-MB-361 cells,
Annexin V-FITC/propidium iodide dual staining assay was done and the
details were presented in the supplementary data.
4.3. Molecular modeling study
[14] G. Xu, L.L. Searle, T.V. Hughes, A.K. Beck, P.J. Connolly, M.C. Abad, M.P. Neeper,
G.T. Struble, B.A. Springer, S.L. Emanuel, R.H. Gruninger, N. Pandey, M. Adams,
S. Moreno-Mazza, A.R. Fuentes-Pesquera, S.A. Middleton, L.M. Greenberger,
Discovery of novel 4-amino-6-arylaminopyrimidine-5-carbaldehyde oximes as dual
inhibitors of EGFR and ErbB-2 protein tyrosine kinases, Bioorg. Med. Chem. Lett.
18 (12) (2008) 3495–3499, https://doi.org/10.1016/j.bmcl.2008.05.024.
[15] S.N. Milik, D.S. Lasheen, R.A.T. Serya, K.A.M. Abouzid, How to train your
inhibitor: Design strategies to overcome resistance to Epidermal Growth Factor
Receptor inhibitors, Eur. J. Med. Chem. 142 (2017) 131–151, https://doi.org/
10.1016/j.ejmech.2017.07.023.
The crystal structure of EGFR (1XKK) was downloaded from protein
data bank and prepared by MOE.2019.102 using quick preparation
protocol. [39] All waters with removed except the critical water mole-
cules (4 and 22) in the active site that were involved in water mediated
hydrogen bond. The docking process was restricted by pharmacophore
features assigned to the two critical hydrogen bonds of Gefitinib with
Met793 and Asp855. Compound 4b and Gefitinib was prepared ac-
cording to ligand preparation protocol. Docking simulations were run
with Induced fit Refinement and Triangle matcher replacement with the
respect of the binding site determination according to the co-crystallized
ligand.
[16] S. Bhargava, L.K. Rajwanshi, Synthesis of some novel pyrido[2,3-d] pyrimidine
derivatives and their antimicrobial investigations, Indian J. Chem. 52B (2013)
448–452, https://doi.org/10.1002/CHIN.201327164.
[17] R. Sabour, M.F. Harras, A.B.M. Mehany, Design, synthesis, cytotoxicity screening
and molecular docking of new 3-cyanopyridines as survivin inhibitors and
apoptosis inducers, Bioorg. Chem. 94 (2020) 103358, https://doi.org/10.1016/j.
bioorg.2019.103358.
[18] N. Kumar, S. Tiwari, A.K. Yadav, Pyrido[2,3-d]pyrimidines and their
ribofuranosides: Synthesis and antimicrobial investigations, Indian J. Chem. 46B
(2007) 702–706.
4.4. IC50 calculations and statistical analysis
´
[19] A. Foucourt, C. Dubouilh-Benard, E. Chosson, C. Corbiere, C. Buquet, M. Iannelli,
B. Leblond, F. Marsais, T. Besson, Microwave-accelerated Dimroth rearrangement
for the synthesis of 4-anilino-6-nitroquinazolines. Application to an efficient
synthesis of a microtubule destabilizing agent, Tetrahedron 66 (2010) 4495–4502,
https://doi.org/10.1016/j.tet.2010.04.066.
All IC50 calculations and statistical comparison of the results were
carried out GraphPad Prism 9.00 software using the One-way ANOVA
followed by Bonferroni test for multiple comparisons at P < 0.05. The
results presented herein are expressed as mean ± SD.
10

A.A.M. Eissa et al.
Bioorganic Chemistry 116 (2021) 105318
[20] A. Sultan, B. Ling, H. Zhang, B. Ma, D. Michel, J. Alcorn, J. Yang, Synergistic Effect
between Sphingosine-1-Phosphate and Chemotherapy Drugs against Human
Brainmetastasized Breast Cancer MDA-MB-361 cells, J. Cancer 4 (2013) 315–319,
https://doi.org/10.7150/jca.5956.
[31] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson,
B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, D.W. Rusnak, T.M. Gilmer,
L. Shewchuk, A Unique Structure for Epidermal Growth Factor Receptor Bound to
GW572016 (Lapatinib): Relationships among Protein Conformation, Inhibitor Off-
Rate, and Receptor Activity in Tumor Cells, Cancer Res. 64 (2004) 6652–6659,
https://doi.org/10.1158/0008-5472.CAN-04-1168 Published September 2004.
[32] N. Verma, A. Rai, V. Kaushik, D. Brünnert, K.R. Chahar, J. Pandey, P. Goyal,
Identification of gefitinib off-targets using a structure-based systems biology
approach; their validation with reverse docking and retrospective data mining, Sci.
Rep. 6 (2016) 33949, https://doi.org/10.1038/srep33949.
[21] A. Ray, K.J. Nkhata, M.P. Cleary, Effects of leptin on human breast cancer cell lines
in relationship to estrogen receptor and HER2 status, Int. J. Oncol. 30 (6) (2007)
1499–1509.
[22] J.M. Gee, J.F. Robertson, E. Gutteridge, I.O. Ellis, S.E. Pinder, M. Rubini, R.
I. Nicholson, Epidermal growth factor receptor/HER2/insulin-like growth factor
receptor signalling and oestrogen receptor activity in clinical breast cancer,
Endocr. Relat. Cancer 12 (2005) S99–S111, https://doi.org/10.1677/erc.1.01005.
[23] G.D. Lewis, J.A. Lofgren, A.E. McMurtrey, A. Nuijens, B.M. Fendly, K.D. Bauer, M.
X. Sliwkowski, Growth regulation of human breast and ovarian tumor cells by
heregulin: Evidence for the requirement of ErbB2 as a critical component in
mediating heregulin responsiveness, Cancer Res. 56 (1996) 1457–1465.
[24] R. Mass, The role of HER-2 expression in predicting response to therapy in breast
cancer, Semin. Oncol. 27 (2000) 46–52.
[33] S. Sebti, A. Solhy, R. Tahir, S. Boulaajaj, J.A. Mayoral, J.M. Fraileb, A. Kossirc,
H. Oumimounc, Calcined sodium nitrate/natural phosphate: an extremely active
catalyst for the easy synthesis of chalcones in heterogeneous media, Tetrahedron
Lett. 42 (45) (2001) 7953–7955, https://doi.org/10.1016/S0040-4039(01)01698-
7.
[34] S.S. Rao, U.S. Gahlot, S.S. Dulawat, R. Vyas, K.L. Ameta, B.L. Verma, Microwave-
induced improved synthesis and antibacterial activities of some chalcones and
their 1-acyl-3,5-diaryl-2-pyrazolines, Afinidad 60 (505) (2003) 271–276.
[35] P.M. Sivakumar, S.P. Seenivasan, V. Kumar, M. Doble, Synthesis,
antimycobacterial activity evaluation, and QSAR studies of chalcone derivatives,
Bioorganic Med. Chem. Lett. 17 (6) (2007) 1695–1700, https://doi.org/10.1016/j.
bmcl.2006.12.112.
[25] R.I. Nicholson, R.A. McClelland, P. Finlay, C.L. Eaton, W.J. Gullick, A.R. Dixon, J.F.
R. Robertson, R.W. Blamey, I.O. Ellis, Relationship between EGF-R, c-erbB-2
protein expression and Ki67 immunostaining in breast cancer and hormone
sensitivity, Eur. J. Cancer. 29 (7) (1993) 1018–1023, https://doi.org/10.1016/
S0959-8049(05)80215-1.
[26] M. Moerkens, Y. Zhang, L. Wester, B. van de Water, J.H. Meerman, Epidermal
growth factor receptor signalling in human breast cancer cells operates parallel to
[36] R.P. Barnes, T.C. Goodwin, T.W. Cotton, A Study of the Direction of enolization of
p-methoxy-p’-bromodibenzoylmethane and p-methoxy-p’-
estrogen receptor
α
signalling and results in tamoxifen insensitive proliferation,
ethoxydibenzoylmethane. J, J. Am. Chem. Soc. 69 (1947) 3135–3138, https://doi.
org/10.1021/ja01204a059.
BMC Cancer. 14 (2014) 283, https://doi.org/10.1186/1471-2407-14-283.
[27] K. Subik, J.F. Lee, L. Baxter, T. Strzepek, D. Costello, P. Crowley, L. Xing, M.
C. Hung, T. Bonfiglio, D.G. Hicks, P. Tang, The expression patterns of ER, PR,
HER2, CK5/6, EGFR, KI-67 and AR by immunohistochemical analysis in breast
cancer cell lines, Breast Cancer (Auckl) 4 (2010) 35–41.
[37] Z. kheilkordi, G. Mohammadi Ziarani, S. Bahar, A. Badiei, Badiei The green
synthesis of 2-amino-3-cyanopyridines using SrFe12O19 magnetic nanoparticles as
efficient catalyst and their application in complexation with Hg2+ ions, J. Iranian
Chem. Soc. 16 (2) (2019) 365–372.
[28] H. Takeuchi, M. Baba, S. Shigeta, An application of tetrazolium (MTT) colorimetric
assay for the screening of anti-herpes simplex virus compounds, J. Virol. Methods.
33 (1991) 61–71, https://doi.org/10.1016/0166-0934(91)90008-N.
[29] R. Hingorani, J. Deng, J. Elia, C. McIntyre, D. Mittar, Detection of apoptosis using
the BD Annexin V FITC assay on the BD FACSVerseTM system, BD Biosci. (2011)
1–12.
[38] T. Akbarpoor, A. Khazaei, J.Y. Seyf, N. Sarmasti, M.M. Gilan, One-pot synthesis of
2-amino-3-cyanopyridines and hexahydroquinolines using eggshell-based nano-
magnetic solid acid catalyst via anomeric-based oxidation, Res Chem. Intermed. 46
(2) (2020) 1539–1554, https://doi.org/10.1007/s11164-019-04049-y.
[39] Molecular Operating Environment (MOE), 2019.01; Chemical Computing Group:
1010 Sherbooke St. West, Suite 910, Montreal, QC, Canada, H3A 2R7; 2019.
[30] W. Gorczyca, Cytometric analyses to distinguish death processes, Endocr. Relat.
Cancer. 6 (1999) 17–19, https://doi.org/10.1677/erc.0.0060017.
11