Anticancer evaluation and molecular modeling of multi-targeted kinase inhibitors based pyrido[2,3-d]pyrimidine scaffold

Article abstract of DOI:10.1080/14756366.2018.1437729

An efficient synthesis of substituted pyrido[2,3-d]pyrimidines was carried out and evaluated for in vitro anticancer activity against five cancer cell lines, namely hepatic cancer (HepG-2), prostate cancer (PC-3), colon cancer (HCT-116), breast cancer (MC

Full text of DOI:10.1080/14756366.2018.1437729

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Anticancer evaluation and molecular modeling of
multi-targeted kinase inhibitors based pyrido[2,3-
d]pyrimidine scaffold
Heba S. A. Elzahabi, Eman S. Nossier, Nagy M. Khalifa, Rania A. Alasfoury &
May A. El-Manawaty
To cite this article: Heba S. A. Elzahabi, Eman S. Nossier, Nagy M. Khalifa, Rania A. Alasfoury
& May A. El-Manawaty (2018) Anticancer evaluation and molecular modeling of multi-targeted
kinase inhibitors based pyrido[2,3-d]pyrimidine scaffold, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 546-557, DOI: 10.1080/14756366.2018.1437729
To link to this article: https://doi.org/10.1080/14756366.2018.1437729
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 27 Feb 2018.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 546–557
https://doi.org/10.1080/14756366.2018.1437729
RESEARCH PAPER
Anticancer evaluation and molecular modeling of multi-targeted kinase inhibitors
based pyrido[2,3-d]pyrimidine scaffold
Heba S. A. Elzahabia, Eman S. Nossiera, Nagy M. Khalifab , Rania A. Alasfourya and May A. El-Manawatyc
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt; Drug Exploration & Development Chair
c
(DEDC), Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of
Pharmacognosy, Pharmaceutical Science Division, National Research Centre, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 1 January 2018
Revised 5 February 2018
Accepted 5 February 2018
An efficient synthesis of substituted pyrido[2,3-d]pyrimidines was carried out and evaluated for in vitro
anticancer activity against five cancer cell lines, namely hepatic cancer (HepG-2), prostate cancer (PC-3),
colon cancer (HCT-116), breast cancer (MCF-7), and lung cancer (A-549) cell lines. Regarding HepG-2, PC-3,
HCT-116 cancer cell lines, 7-(4-chlorophenyl)-2-(3-methyl-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-5-(p-tolyl)- pyr-
ido[2,3-d]pyrimidin-4(3H)-one (5a) exhibited strong, more potent anticancer (IC50: 0.3, 6.6 and 7 mM) relative
to the standard doxorubicin (IC50: 0.6, 6.8 and 12.8 mM), respectively. Kinase inhibitory assessment of 5a
showed promising inhibitory activity against three kinases namely PDGFR b, EGFR, and CDK4/cyclin D1 at
two concentrations 50 and 100 mM in single measurements. Further, a molecular docking study for com-
pound 5a was performed to verify the binding mode towards the EGFR and CDK4/cyclin D1 kinases.
KEYWORDS
Pyrido[2,3-d]pyrimidine
derivatives; anticancer
activity; EGFR; CDK4/cyclin
D1; molecular docking
CH
3
O
NH
H
N
N
N
N
CH
3
Cl
O
Compound 5a
Introduction
cancer and normal cells9,10. The advances in molecular biology
and genetics improve identification of molecular targets that are
unique to cancer cells or overexpressed on them. The design of
agents affecting these targets promises the development of more
selective anticancer drugs with less toxic side effects11. Pyrido[2,3-
d]pyrimidines were reported to display antitumor properties,
which may be attributed to inhibition of different enzymes that
involved in carcinogenesis cases. The prominent examples were
pyrido[2,3-d]pyrimidines A–E that exhibited a potent inhibitory
activity against various kinases, e.g. TKs, PI3K, and CDK4/612,13
(Figure 1). Based on the structural features of the previous pyr-
ido[2,3-d]pyrimidines, we aimed to prepare a new group of pyr-
ido[2,3-d]pyrimidinone congeners, which were screened for their
inhibitory activity against TKs, CDK4/6, and PI3K enzymes.
Simultaneously, they were tested for their anticancer activity
against cancer cells expressing the previous enzymes. Further,
molecular modeling study was performed to explore the most
Cancer is one of the leading causes of death in the world, charac-
terized by the loss of control of cell proliferation, leading almost
invariably to death, in untreated patients1,2. Chemotherapy, alone
or in combination with surgery, is commonly the most efficient
anti-cancer remedy. However, the use of available chemotherapeu-
tics is limited mainly due to drug resistance and toxicities3.
Developing resistance to chemotherapy belongs to many reasons
like poor uptake of the drug, alternative metabolic paths, and
increased production of the target protein, mutations that block
the drug binding to its target or efflux systems that expel drugs
from the cell4–8. So, combination of chemotherapies with different
targets increases efficiency, antagonizes the resistance, and
decreases toxicity as well. Traditional anticancer drugs work by dis-
rupting the function of DNA. Some of these drugs may affect DNA
directly or inhibit the enzymes controlling DNA synthesis. These
drugs are mostly nonselective and having cytotoxicity to both appropriate binding modes of the most potent target compounds
CONTACT Nagy M. Khalifa
nagykhalifa@hotmail.com
Drug Exploration & Development Chair (DEDC), Department of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
547
Cl
O
O
N
N
N
Cl
O
N
N
X
O
N
N
X
N
NH
X=NH
A
X=NH
B
O
N
O
N
F
N
NH
N
HO
H3CO
N
N
N
N
X
O
S
X=NH
C
D
O
N
COCH3
N
N
N
N
S
E
SO2NH2
Lead optimization
R1
O
NH
Ar
N
N
X
R2
Figure 1. Reported and proposed pyrido[2,3-d]pyrimidine conjugates with anticancer and tyrosine kinase inhibitory activity.
that matched ligands binding modes. The applied modeling pro- of the theoretical values). Mass spectra were recorded on a DI-50
R
V
gram was Molecular Operating Environment (MOE ) 2008.10.
unit of Shimadzu GC/MS-QP 2010 plus Spectrometer (Livingston,
West Lothian, UK) or on single quadrupole Mass Spectrometer ISQ
LT (Thermo Scientific) (Austin, TX, USA).
Materials and methods
Chemistry
General procedure for the synthesis of 2,3-dihydro-2-thioxo-5,7-
diarylpyrido[2,3-d]pyrimidin-4(1H)-one (3a–c, e–g)
Melting points by using electro thermal apparatus on open capil-
lary tubes were recorded. IR spectra (KBr) were performed on a
Shimadzu 435 IR Spectrophotometer (Shimadzu Bruker, Tokyo,
Japan) (ꢀ cmꢀ1). 1H and 13 C NMR NMR spectra were recorded on
Varian Mercury VX-300 NMR 300 MHz spectrophotometer
(Stoughton, USA) or on Agilent Technologies 400 MHz NMR spec-
trophotometer (Santa Clara, CA, USA) using DMSO-d6 as a solvent
300 MHz oron Agilent Technologies 400 MHz NMR spectrophotom-
eter and the chemical shifts were estimated in ppm, relative to
TMS as an internal standard. Physical data (C, H, and N) agreed
2-Thioxopyrimidine
1
(1.43 g, 0.01 mol) and
a
series of
a,b-unsaturated ketones 2a–g (0.01 mol) in dry DMF (20 ml) was
refluxed for 10–15 h and the reaction progress was monitored by
TLC. The solid mass created on cooling was filtered and crystal-
lized from (DMF) to give the title products.
5-(2,4-Dichlorophenyl)-2,3-dihydro-2-thioxo-7-p-tolylpyrido[2,3-
d]pyrimidin-4(1H)-one (3a)
with the proposed structures and obtained by using a Vario Yield: 67%; mp: 309–311 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3402 (2NH), 1697
1
Elemental analyzer (Yamashitacho, Yokohama, Japan) (within 0.4% (C¼O); HNMR (400 MHz, DMSO-d6): d, 12.13 & 13.02 (2s, 2H, 2NH),

548
H. S. A. ELZAHABI ET AL.
8.12 (d, 2H, J ¼ 8.4 Hz), 7.92 (s, 1H, C-6 pyridine), 7.41 (s, 1H), 7.38 5-(2,4-Dichlorophenyl)-2-hydrazinyl-7-p-tolylpyrido[2,3-d]pyrimidin-
(d, 2H, J ¼ 8.4 Hz), 7.33 (d, 2H, J ¼ 8.4 Hz), 2.35 (s, 3H, CH3); MS: [m/
4(3H)-one (4a)
z (%), 417 (0.84, Mþþ4), 415 (1.96, Mþþ2), 413 (2.28, Mþ), 119
Yield: 27%; mp: 167–169 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3410, 3190 (2NH,
NH2), 1678 (C¼O); 1H NMR (400 MHz, DMSO-d6): d 10.13 (1s, 1H,
(100)].
NH), 7.98 (s, 1H, C-6 pyridine), 7.55 (d, 2H, J ¼ 8 Hz), 7.42 (d, 1H,
J ¼ 6.8 Hz), 7.38 (d, 2H, J ¼ 10 Hz), 7.34 (d, 2H, J ¼ 8 Hz), 6.17 (s, 1H,
7-(4-Bromophenyl)-2,3-dihydro-2-thioxo-5-p-tolylpyrido[2,3-d]pyri-
midin-4(1H)-one (3b)
NH), 4.32 (s, 2H, NH2), 2.36 (s, 3H, CH3); MS: [m/z (%), 415 (1.69,
Mþþ4), 413 (2.00, Mþþ2), 411 (4.86, Mþ)].
Yield: 88%; mp: 310–312 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3410 (2NH), 1701
(C¼O); 1HNMR (400 MHz, DMSO-d6): d12.13, 13.02 (2s, 2H, 2NH),
8.17 (d, 2H, J ¼ 8 Hz), 7.92 (s, 1H, C-6 pyridine), 7.72 (d, 2H,
J ¼ 8.8 Hz), 7.41 (d, 2H, J ¼ 8.8 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz), 2.06 (s,
3H, CH3); MS: [m/z (%), 425 (2.57, Mþþ2), 423 (2.04, Mþ), 180
(100)].
7-(4-Chlorophenyl)-2-hydrazinyl-5-p-tolylpyrido[2,3-d]pyrimidin-
4(3H)-one (4b)
Yield: 54%; mp: 233–234 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421, 3190 (2NH,
1
NH2), 1716 (C¼O); HNMR (400 MHz, DMSO-d6): d8.29, 8.28 (2s, 3H,
NH, NH2), 8.19 (d, 2H, J ¼ 8.8 Hz) , 8.08 (s, 1H, NH), 7.93 (s, 1H, C-6
pyridine), 7.53 (d, 2H, J ¼ 8 Hz), 7.25 (d, 2H, J ¼ 8 Hz), 7.17 (d, 2H,
J ¼ 8 Hz), 2.30 (s, 3H, CH3); MS: [m/z (%), 379 (2.32, Mþ þ2), 367
(100)].
7-(4-Chlorophenyl)-2,3-dihydro-2-thioxo-5-p-tolylpyrido[2,3-d]pyri-
midin-4(1H)-one (3c)
Yield: 75%; mp: 302–304 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3394 (2NH), 1705
(C¼O); 1HNMR (300 MHz, DMSO-d6): d 12.25, 13.00 (2s, 2H, 2NH),
8.25 (d, 2H, J ¼ 8.7 Hz), 7.94 (s, 1H, C-6 pyridine), 7.59 (d, 2H,
J ¼ 8.7 Hz), 7.34 (d, 2H, J ¼ 9 Hz), 7.22 (d, 2H, J ¼ 8.1 Hz), 2.37 (s, 3H,
CH3); MS: [m/z (%), 381 (7.05, Mþ þ2), 379 (14.24, Mþ), 40 (100)].
2-Hydrazinyl-5-(4-methoxyphenyl)-7-p-tolylpyrido[2,3-d]pyrimidin-
4(3H)-one (4d)
Yield: 73%; mp: 260–262 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3425, 3290 (2NH,
NH2), 1728 (C¼O); 1H NMR (400 MHz, DMSO-d6): d 10.17 (s, 1H,
NH), 8.26 (s,1H, NH), 8.06 (d, 2H, J ¼ 8 Hz), 7.30 (s, 1H, C-6 pyridine),
7.33 (d, 2H, J ¼ 8.4 Hz), 7.29 (d, 2H, J¼ 8.8 Hz), 6.92 (d, 2H,
J ¼ 8.8 Hz), 4.20 (s, 2H, NH2), 3.79 (s, 3H, OCH3), 2.38 (s, 3H, CH3);
MS: [m/z (%), 373 (100, Mþ)].
2,3-Dihydro-5-(4-methoxyphenyl)-2-thioxo-7-p-tolylpyrido[2,3-
d]pyrimidin-4(1H)-one (3e)
Yield: 75%; mp: 361–363 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1678
(C¼O); 1HNMR (400 MHz, DMSO-d6): d 12.28, 12.95 (2s, 2H, 2NH),
8.11 (d, 2H, J ¼ 8 Hz), 7.55 (s, 1H, C-6 pyridine), 7.39 (d, 2H,
J ¼ 8.4 Hz), 7.38 (d, 2H, J ¼ 8.8 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz), 3.82 (s,
3H, OCH3), 2.36 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6): d
175.71, 159.97, 153.79, 153.21, 141.19, 134.27, 130.88, 130.84,
129.93, 127.94, 119.23, 113.31, 108.02, 55.64, 21.42; MS: [m/z (%),
375 (14.85, Mþ), 91 (100)].
7-(4-Chlorophenyl)-2-hydrazinyl-5-(3,4,5-trimethoxyphenyl)pyr-
ido[2,3-d]pyrimidin-4-(3H)-one (4e)
Yield: 37%; mp: 279–281 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421, 3111 (2NH,
NH2), 1716 (C¼O); 1H NMR (400 MHz, DMSO-d6): d 10.03 (s, 2H,
NH), 8.30 (d, 2H, J ¼ 8 Hz), 7.93 (s, 1H, C-6 pyridine), 6.79 (d, 2H,
J ¼ 8 Hz), 7.62 (d, 2H, J ¼ 8 Hz), 6.16 (s, 2H, NH2), 3.77, 3.75 (2s, 9H,
3OCH3); MS: [m/z (%), 454 (1.33, Mþþ1), 453 (3.52, Mþ), 77 (100)].
7-(4-Chlorophenyl)-2,3-dihydro-5-(3,4,5-trimethoxyphenyl)-2-thioxo-
pyrido[2,3-d]pyrimidin-4(1H)-one (3f)
Yield: 74%; mp: 306–308 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3402 (2NH), 1708
(C¼O); 1HNMR (400 MHz, DMSO-d6): d 12.31, 13.22 (2s, 2H, 2NH),
8.26 (d, 2H, J ¼ 8 Hz), 7.93 (s, 1H, C-6 pyridine), 7.60 (d, 2H,
J ¼ 8.8 Hz), 6.76 (s, 2H), 3.70, 3.76 (2s, 9H, 3OCH3); MS: [m/z (%),
457 (0.1, Mþ þ2), 455 (1.6, Mþ), 95 (100)].
2-Hydrazinyl-5-(3,4,5-trimethoxyphenyl)-7-p-tolylpyrido[2,3-d]pyri-
midin-4(3H)-one (4f)
Yield: 68%; mp: 301–303 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3348, 3103 (2NH,
NH2), 1716 (C¼O); 1HNMR (400 MHz, DMSO-d6): d 10.12 (s, 2H,
2NH), 8.19 (d, 2H, J ¼ 8 Hz), 7.99 (s, 1H, C-6 pyridine), 7.36 (d, 2H,
J ¼ 8 Hz), 6.76 (d, 2H, J ¼ 8 Hz), 6.12 (s, 2H, NH2), 3.76, 3.77 (2s, 9H,
3OCH3), 2.36 (s, 3H, CH3); MS: [m/z (%), 433 (1.49, Mþ), 115 (100)].
2,3-Dihydro-5-(3,4,5-trimethoxyphenyl)-2-thioxo-7-p-tolylpyr-
ido[2,3-d]pyrimidin-4-(1H)-one (3g)
Yield: 55%; mp: 318–320 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1701
(C¼O); 1HNMR (300 MHz, DMSO-d6): d12.13, 12.98 (2s, 2H, 2NH),
8.15 (d, 2H, J ¼ 8.4 Hz), 7.94 (s, 1H, C-6 pyridine), 7.34 (d, 2H,
J ¼ 8.1 Hz), 6.76 (s, 2H), 3.77, 3.72 (2s, 9H, 3OCH3), 2.38 (s, 3H, CH3);
MS: [m/z (%), 435 (12.70, Mþ), 77.07 (100)].
General procedure for the synthesis of2–(3-methyl-5-oxo-2,3-
dihydro-1H-pyrazol-1-yl)-5,7-diarylpyrido[2,3-d]pyrimidin-4(3H)-
one (5a–d), 2-(3-amino-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-5,7-
diarylpyrido[2,3-d]pyrimidin-4(3H)-one (6a–d) and 1-(4-oxo-5,7-
diaryl-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)pyrazolidine-3,5-
dione (7a–c)
General procedure for the synthesis of 2-hydrazinyl -5,7-
diarylpyrido[2,3-d]pyrimidin-4(3H)-one (4a–f)
2-Hydrazinyl derivatives (0.01 mol) and active methylene (ethyl
acetoacetate, ethyl cyanoacetate, or diethylmalonate) (0.01 mol)
Derivatives 3a,c–g (0.004 mol) and hydrazine reagent 99% (3 ml, was refluxed in glacial acetic acid for 4–6 h. After completion of
0.006 mol) was refluxed in absolute ethyl alcohol (20 ml) for the reaction, the precipitate created on pouring into ice-water was
10–15 h. On cooling, the residue created was filtered and purified filtered and purified from (acetic acid) to give the title compounds
from (DMF).
5a–d, 6a–d and 7a–c, respectively.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
549
7-(4-Chlorophenyl)-2-(3-methyl-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-
5-(p-tolyl)pyrido-[2,3-d]pyrimidin-4(3H)-one (5a)
2-(3-Amino-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-7-(4-chlorophenyl)-
5-(3,4,5-trimethoxyphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (6c)
Yield: 68%; mp: 339–341 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3433 (br, 2NH, NH2),
1726, 1678 (2C¼O); 1HNMR (400 MHz, DMSO-d6): d 13.68 (s, 2H,
2NH), 8.31 (d,2H), 8.20 (s, 1H, C-4 pyrazolone), 7.79 (s, 1H, C-6 pyri-
dine), 7.60 (d, 2H), 6.78 (d, 2H), 3.96 (s, 2H, NH2), 3.79, 3.77 (2s, 9H,
3OCH3); MS: [m/z (%), 424 (2, Mþ), 175 (100)].
Yield: 67%; mp: 385–387 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3422 (2NH), 1716
(2C¼O); 1HNMR (400 MHz, DMSO-d6): d 11.16, 11.66 (2s, 2H, 2NH),
8.21 (d, 2H), 8.18 (s, 1H, C-4 pyrazolone), 7.57 (d, 2H, J ¼ 8 Hz), 7.50
(s, 1H, C-6 pyridine), 7.30 (d, 2H, J ¼ 8 Hz), 7.19 (d, 2H, J ¼ 8 Hz),
2.35 (s, 3H, CH3), 1.89 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6): d
172.44, 161.77, 157.81, 154.58, 153.98, 137.86, 136.26,
136.07,135.88, 129.63, 129.34, 129.09, 128.42, 118.39, 106.54, 21.50,
21.32; MS: [m/z (%), 446 (22, Mþþ2), 444 (50, Mþ), 186 (100)].
2-(3-Amino-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-7-(p-tolyl)-5-(3,4,5-
trimethoxy phenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (6d)
Yield: 52%; mp: 350–352 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3390 (br, 2NH, NH2),
1716, 1675 (2C¼O); 1H NMR (400 MHz, DMSO-d6): d 11.46, 13.61
(2s, 2H, 2NH), 8.19 (d, 2H), 8.17 (s, 1H, C-4 pyrazolone), 7.72 (s, 1H,
C-6 pyridine), 7.36 (d, 2H, J ¼ 8 Hz), 6.78 (s, 2H), 3.96 (s, 2H, NH2),
3.73, 3.77 (2s, 9H, 3OCH3), 2.38 (s, 3H, CH3); 13 C NMR (100 MHz,
DMSO-d6): d 171.74, 163.73, 159.72, 155.23, 154.45, 153.36, 152.68,
150.89, 144.52, 141.52, 141.35, 137.86, 135.06, 134.46, 130.03,
128.11, 118.25, 106.80, 60.49, 56.47, 21.44; MS: [m/z (%), 500 (Mþ,
0.7); 107 (100)].
2-(3-Methyl-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-5,7-di-p-tolylpyr-
ido[2,3-d]pyrimidin-4-(3H)-one (5b)
Yield: 53%; mp: 342–344 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3429 (2NH), 1727,
1651 (2C¼O); 1H NMR (400 MHz, DMSO-d6): d 11.93, 13.59 (2s, 2H,
2NH), 8.28 (d, 2H, J ¼ 8 Hz), 8.17 (s, 1H, C4-pyrazolone), 7.68 (s, 1H,
C6-pyridine), 7.59 (d, 2H, J ¼ 8 Hz), 7.37 (d, 2H, J ¼ 8 Hz), 7.24 (d,
2H, J ¼ 8 Hz), 2.35, 2.39 (2s, 6H, 2CH3), 1.88 (s, 3H, CH3); MS: [m/z
(%), 423 (0.79, Mþ), 367 (100)].
1-(7-(4-Chlorophenyl)-4-oxo-5-(p-tolyl)-3,4-dihydropyrido[2,3-d]pyri-
midin-2-yl) pyrazolidine-3,5-dione (7a)
7-(4-Chlorophenyl)-2-(3-methyl-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-
5-(3,4,5-trimethoxyphenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (5c)
Yield: 42%; mp: 323–325 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421(2NH), 1719
(2C¼O); 1HNMR (400 MHz, DMSO-d6): d 11.97, 13.71 (2s, 2H, 2NH),
8.31 (d, 2H, J ¼ 8 Hz), 8.20 (s, 1H, C-4 pyrazolone), 7.78 (s, 1H, C-6
pyridine), 7.62 (d, 2H, J ¼ 8 Hz), 6.78 (s, 2H), 3.73, 3.77 (2s, 9H,
3OCH3), 1.88 (s, 3H, CH3); 13CNMR (100 MHz, DMSO-d6): d175.81,
162.13, 158.37, 155.58, 154.61, 152.70, 137.94, 136.27, 135.92,
134.87, 129.94, 129.49, 118.54, 106.85, 105.95, 60.50, 56.49; MS: [m/
z (%), 521 (0.83, Mþþ2), 519 (3.84, Mþ), 376 (100)].
Yield: 23%; mp: 358–360 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1716,
1730 (3C¼O); 1HNMR (400 MHz, DMSO-d6): d 12.39 (s, 2H, 2NH),
8.29 (d, 2H, J ¼ 8 Hz), 7.89 (s, 1H, C-6 pyridine), 7.61 (d, 2H,
J ¼ 8 Hz), 7.33 (d, 2H, J ¼ 8 Hz), 7.22 (d, 2H, J ¼ 8 Hz), 2.95 (s, 2H, C-
4 pyrazolidinone), 2.37 (s, 3H, CH3); MS: [m/z (%), 447 (14,Mþþ2),
445 (2,Mþ), 77 (100)].
1-(7-(4-Chlorophenyl)-4-oxo-5-(3,4,5-trimethoxyphenyl)-3,4-dihydro-
pyrido[2,3-d]pyrimidin-2-yl)pyrazolidine-3,5-dione (7b)
Yield: 66%; mp: 370–372 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1732,
1660 (3C¼O); 1HNMR (400 MHz, DMSO-d6): d 13.95 (s, 2H, 2NH),
8.20 (d, 2H, J ¼ 8 Hz), 7.70 (s, 1H, C-6 pyridine), 7.33 (d, 2H,
J ¼ 8 Hz), 6.65 (d, 2H, J ¼ 8 Hz), 3.73, 3.77 (2s, 9H, 3OCH3), 2.95 (s,
2H, C4-pyrazolidinone); MS: [m/z (%), 521 (4, Mþþ2), 523 (0.8, Mþ),
95 (100)].
2-(3-Methyl-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-7-(p-tolyl)-5-(3,4,5-
trimethoxy phenyl)pyrido[2,3-d]pyrimidin-4(3H)-one (5d)
Yield: 43%; mp: 357–359 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3435 (2NH), 1719
(2C¼O)); 1H NMR (400 MHz, DMSO-d6): d 11.94, 13.67 (2s, 2H,
2NH), 8.20 (d, 2H), 8.18 (s,1H, C4-pyrazolone), 7.36 (d, 2H, J ¼ 8 Hz),
7.72 (s, 1H, C6-pyridine), 6.76 (d, 2H, J ¼ 8 Hz), 3.73, 3.77 (2s, 9H,
3OCH3), 2.38 (s, 3H, CH3), 1.88 (s, 3H, CH3); MS: [m/z (%), 500 (1.59,
Mþþ1), 106 (100)].
1-(4-Oxo-7-(p-tolyl)-5-(3,4,5-trimethoxyphenyl)-3,4-dihydropyr-
ido[2,3-d]pyrimidin-2-yl)pyrazolidine-3,5-dione (7c)
Yield: 34%; mp: 352–354 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3448 (2NH), 1732,
1
2-(3-Amino-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-7-(4-chlorophenyl)-
5-(p-tolyl)pyrido [2,3-d]pyrimidin-4(3H)-one (6a)
1690 (3C¼O); HNMR (400 MHz, DMSO-d6): d 12.92 (br s, 2H, 2NH),
8.31 (d, 2H, J ¼ 8 Hz), 7.93 (s, 1H, C6-pyridine), 7.37 (d, 2H,
J ¼ 8 Hz), 6.78 (d, 2H, J ¼ 8 Hz), 3.72, 3.76 (2s, 9H, OCH3), 2.94 (s,
Yield: 65%; mp: 382–384 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3430 (br, 2NH, NH2),
1726, 1644 (2C¼O); 1HNMR (400 MHz, DMSO-d6): d 13.90 (s, 2H, 2H, C-4 pyrazolidinone), 2.36 (s, 3H, CH3); 13CNMR (100 MHz,
DMSO-d6): d 170.45, 167.54, 167.16, 151.59, 147.78, 141.47, 134.15,
2NH), 8.30 (d, 2H, J ¼ 8 Hz), 8.18 (s, 1H, C-4 pyrazolone), 7.69 (s, 1H,
130.22, 128.01, 121.34, 106.85, 90.72, 60.47, 56.47; MS: [m/z (%),
C-6 pyridine), 7.61 (d, 2H, J ¼ 8 Hz), 7.37 (d, 2H, J ¼ 8 Hz), 7.25 (d,
501 (20, Mþ), 55 (100)].
2H, J ¼ 8 Hz), 3.96 (s, 2H, NH2), 2.38 (s, 3H, CH3); MS: [m/z (%), 446
(22, Mþþ2), 444 (50, Mþ), 186 (100)].
General procedure for the synthesis of2-(2-(aryl-2-
ylmethylene)hydrazinyl)-5,7-diarylpyrido[2,3-d]pyrimidin-4(3H)-
one (8a–f)
2-(3-Amino-5-oxo-2,3-dihydro-1H-pyrazol-1-yl)-5,7-di-p-tolylpyr-
ido[2,3-d]pyrimidin-4-(3H)-one (6b)
Yield: 42%; mp: 380–382 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3429 (br, 2NH, NH2),
1727, 1680 (2C¼O); 1H NMR (400 MHz, DMSO-d6): d 12.48 (s, 2H,
2NH), 8.16 (d,2H, J ¼ 8 Hz), 8.13 (s, 1H, C-4 pyrazolone), 7.80 (s, 1H,
C-6 pyridine), 7.36 (d, 2H, J ¼ 8 Hz), 7.32 (d, 2H, J ¼ 8 Hz), 7.22 (d,
2H, J ¼ 8 Hz), 3.97 (s, 2H, NH2), 2.37, 2.38 (2s, 6H, 2CH3); MS: [m/z
(%), 424 (2, Mþ), 175 (100)].
2-Hydrazinylpyrido[2,3-d]pyrimidines 4a,d (0.01 mol) and aromatic
aldehydes namely, benzaldehyde, 4-chlorobenzaldehyde, 4-anisal-
dehyde, or thiophene-2-carbaldehyde (0.02 mol) in acetic acid
(10 ml) was refluxed for 4 h. The mixture was poured onto ice-
water and the residue formed was filtered and purified from
(acetic acid).

550
H. S. A. ELZAHABI ET AL.
7-(4-Chlorophenyl)-2-(2-(thiophen-2-ylmethylene)hydrazinyl)-5-(p-
tolyl)pyrido[2,3-d]pyrimidin-4(3H)-one (8a)
Biological evaluation
In-vitro antitumor assay
Yield: 65%; mp: 360–362 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1728
(C¼O); 1HNMR (400 MHz, DMSO-d6): d 13.5 (s, 2H, 2NH), 8.29 (d,
2H), 8.18 (s, 1H, N¼CH), 7.70 (s, 1H, C-6 pyridine), 7.61–7.65 (m,
3H), 7.45 (d, 2H, J ¼ 8 Hz), 7.32 (d, 2H, J ¼ 12.3 Hz), 7.00 (d, 2H,
J ¼ 8 Hz), 2.38 (s, 3H, CH3); MS: [m/z (%) 473 (15, Mþþ2), 471
(10, Mþ), 43 (100)].
Anticancer screening (In vitro bioassay on human cancer cell lines)
was determined by the Bioassay-Cell Culture Laboratory, National
Research Centre, Cairo, Egypt. It was adopted against five cancer
cell lines (HepG2, PC-3, HCT116, MCF-7, and A549); doxorubicin
was used as a reference standard according to a previously
reported methods14–16
.
2-(4-Chlorobenzylidene)hydrazinyl)-7-(4-chlorophenyl)-5-p-tolylpyr-
ido[2,3-d]pyrimidin-4(3H)-one (8b)
Kinase inhibition assay
The in-vitro enzyme inhibition determination for compound 5a
(which showed promising anticancer activity against HePG-2, PC-3,
HCT-116 cancer cell lines in comparison with doxorubicin was car-
ried out in KINEXUS Corporation, Vancouver, British Columbia,
Canada. Kinexus has developed an open-access, on-line resource
called DrugKiNET, www.drugkinet.ca. The evaluation performed
profiling of the compound 5a against a range of five protein kin-
ases [(PDGFR beta, EGFR, CDK4/Cyclin D1, PI3K (p100b/p85a, PI3K
(p100a/p85a)] according to a previously reported method16.
Yield: 23%; mp: 366–368 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3423 (2NH), 1719
(C¼O); 1H NMR (400 MHz, DMSO-d6): d 13.71 (s, 2H, 2NH), 8.29 (d,
2H, J ¼ 8 Hz), 8. 18 (s,1H, N¼CH), 7.91 (d, 2H, J ¼ 8 Hz), 7.59 (d, 2H,
J ¼ 8 Hz), 7.50 (s, 1H, C-6 pyridine), 7.39–7.34 (m, 4H), 7.25 (d, 2H,
J ¼ 8 Hz), 2.38 (s, 3H, CH3); MS: [m/z (%) 503 (2.14, Mþþ2), 499
(0.75, Mþ), 214 (100)].
7-(4-Chlorophenyl)-2-(2-(4-methylbenzylidene)hydrazinyl)-5-(p-tol-
yl)pyrido[2,3-d]pyrimidin-4(3H)-one (8c)
Yield: 58%; mp: 375–377 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3427 (2NH), 1728
(C¼O); 1H NMR (400 MHz, DMSO-d6): d 13.5 (br s, 2NH), 8.28 (d,
2H, J ¼ 8 Hz) , 8.18 (s, 1H, N¼CH), 7.80 (d, 2H, J ¼ 8 Hz), 7.60 (d, 2H,
J ¼ 8 Hz), 7.49 (s, 1H, C6-pyridine), 7.37 (d, 2H, J ¼ 8 Hz), 7.33–7.23
(m, 4H), 2.28, 2.25 (2s, 6H, 2CH3); MS: [m/z (%) 481 (12, Mþþ2), 64
(100)].
Molecular modeling study
All the molecular modeling calculations and docking simulation
studies were performed utilizing Molecular Operating Environment
R
(MOEV) 2008.1017. The three-dimensional X-ray structures of EGFR
(PDB code: 1M17)18,19 and CDK6 was used instead of CDK4 (PDB
code: 2EUF)20,21 were obtained from the Protein Data Bank
through the internet.
2-(2-Benzylidenehydrazinyl)-5–(4-methoxyphenyl)-7-(p-tolyl)pyr-
ido[2,3-d]pyrimidin-4-(3H)-one (8d)
Results and discussion
Yield: 53%; mp: 351–353 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3427 (2NH), 1728
Chemistry
(C¼O); 1H NMR (400 MHz, DMSO-d6):
d 13.60 (s, 2H, 2NH),
New group of 5,7-diaryl pyrido[2,3-d]pyrimidinones 3a–g were cre-
ated through reaction of the starting precursor 2-mercapto-4-
hydroxy-6-aminopyrimidine 1 with a, b unsaturated ketones 2a–g
in dry DMF according to the applied method reported for ana-
logue 3d22–24. Compound 3c as an example showed absorption
band at 3394 cmꢀ1 assigned for NH group in IR spectrum and
three singlet signals at d 7.94, 12.25, and 13.00 ppm attributed to
C6 proton of pyridopyrimidine ring and 2NH protons in 1HNMR
spectrum. Further, the MS revealed molecular ion peak at m/z 379
(Mþ) agreed with the molecular weight of the assigned structure.
Also, 13 C-NMR spectrum of 3e analogue showed signal at
175.71 ppm for C¼S group.
8.13–8.18 (m, 3H), 7.64 (s, 1H, C-6 pyridine), 7.41–7.45 (m, 4H), 7.35
(d, 2H, J ¼ 8 Hz), 6.97 (d, 2H, J ¼ 8.8 Hz), 3.82 (s, 3H, OCH3), 2.37 (s,
3H, CH3); MS: [m/z (%) 461 (4, Mþ), 43 (100)].
2-(4-Chlorobenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-p-tolyl-
quinazolin-4(3H)-one (8e)
Yield: 33%; mp: 361–363 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3422 (2NH), 1670
(C¼O); 1H NMR (400 MHz, DMSO-d6): d 11.72, 11.28 (2s, 2H, 2NH),
8.25 (s, 1H, N¼CH), 8.11 (d, 2H, J ¼ 8 Hz), 8.00 (d, 2H, J ¼ 8 Hz), 7.43
(s, 1H, C-6 pyridine), 7.41–7.50 (m, 4H), 7.30 (d, 2H, J ¼ 8 Hz), 6.94
(d, 2H, J ¼ 8 Hz), 3.81 (s, 3H, OCH3), 2.36 (s, 3H, CH3); MS: [m/z (%)
497 (26, Mþþ2), 495 (84, Mþ), 384 (100)].
Nucleophilic attack22–25 of hydrazine hydrate on thioxo deriva-
tives 3a,c–g yielded the corresponding 2-hydrazinopyrido[2,3-
d]pyrimidin-4(3H)-ones 4a–f (Scheme 1). IR spectrum of compound
4a showed existence of three absorption bands at 3410, 3190, and
1678 cmꢀ1 corresponding to NH2, NH, and C¼O groups, respect-
ively. 1H NMR spectrum of 4d revealed singlet signals at d2.38,
3.79, 4.20, 8.26, and 10.17 ppm assignable for CH3, OCH3, NH2, and
two NH protons, respectively.
5-(4-Methoxyphenyl)-2-(2-(4-methylbenzylidene)hydrazinyl)-7-(p-
tolyl)pyrido[2,3-d]pyrimidin-4(3H)-one (8f)
Yield: 27%; mp: 380–382 ꢁC; IR (KBr, cmꢀ1) ꢀ: 3421 (2NH), 1670
(C¼O); 1H NMR (400 MHz, DMSO-d6): d 11.82,11.73 (2s, 2H, 2NH),
8.10 (s, 1H, N¼CH), 7.84 (d, 2H, J ¼ 8 Hz), 7.37–7.45 (m, 4H), 7.33
(d, 2H, J ¼ 8 Hz), 7.21 (d, 2H, J ¼ 8 Hz), 6.95 (d, 2H, J ¼ 8 Hz), 3.81 (s,
3H, OCH3), 2.35, 2.33 (s, 6H, 2CH3); 13 C NMR (100 MHz, DMSO-d6):
161.20, 159.91, 140.73, 140.44, 135.38, 134.64, 133.87, 132.21,
130.66, 129.56, 129.85, 128.24, 127.98, 127.90, 127.70, 127.21,
117.49, 113.46, 113.24, 55.61, 21.51, 21.38; MS: [m/z (%) 475 (9,
Mþ), 42 (100)].
Derivatives 4 b,c,e,f were condensed with active methylene
compounds (ethylacetoacetate, ethylcyanoacetate, or diethylmalo-
nate) in glacial acetic acid26 to give the corresponding three-sub-
stituted pyrazolone derivatives 5a–d, 6a–d, 7a–c, respectively.
Compounds 5a–d were confirmed by the existence of an add-
itional band at 1651 cmꢀ1 for C¼O group of the newly created
pyrazolone nucleus in IR spectrum of compound 5b. Additionally,
1
the HNMR spectrum of 5a showed signals at d 1.89 and 8.18 ppm

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
551
R1
S
O
O
NC
H2N
NH2
O
+
NH
H
N
NaOEt
N
N
N
CH3
R2
O
O
O
5a-d
a, R1 = 4-CH3
b, R1 = 4-CH3
; R2 = 4-Cl
; R2 = 4-CH3
c, R1 = 3,4,5-(OCH3)3 ; R2 = 4-Cl
d, R1 = 3,4,5-(OCH3)3 ; R2 = 4-CH3
NH
S
R1
R2
+
H2N
N
H
1
2a-g
DMF
O
O
AcOH
R1
O
R1
O
N
O
NH
N2H4.H2O
NH
NH2
N
N
N
N
H
S
R2
R2
H
4a-f
3a-g
a, R1 = 2,4-(Cl)2
b, R1 = 4-CH3
c, R1 = 4-CH3
d, R1 = 4-OCH3
e, R1 = 3,4,5-(OCH3)3 ; R2 = 4-Cl
f, R1= 3,4,5-(OCH3)3 ; R2 = 4-CH3
; R2 = 4-CH3
; R2 = 4-Cl
; R2 = 4-CH3
; R2 = 4-CH3
a, R1 = 2,4-(Cl)2
b, R1 = 4-CH3
c, R1 = 4-CH3
d, R1 = 4-CH3
e, R1 = 4-OCH3
; R2 = 4-CH3
; R2 = 4-Br
; R2 = 4-Cl
; R2 = 4-CH3
; R2 = 4-CH3
f, R1 = 3,4,5-(OCH3)3 ; R2 = 4-Cl
g, R1= 3,4,5-(OCH3)3 ; R2 = 4-CH3
4b,c,e,f
AcOH
O
O
O
4b,d
ArCHO
NC
Et
Et
O
O
O
R1
R1
R1
O
O
O
N
NH
NH
NH
H
H
N
N
Ar
N
N
N
N
N
N
N
N
N
NH2
O
R2
R2
R2
H
O
O
6a-d
8a-f
7a-c
a, R1 = 4-CH3
b, R1 = 4-CH3
; R2 = 4-Cl
; R2 = 4-CH3
c, R1 = 3,4,5-(OCH3)3 ; R2 = 4-Cl
d, R1 = 3,4,5-(OCH3)3 ; R2 = 4-CH3
a, R1 = 4-CH3
; R2 = 4-Cl
b, R1 = 3,4,5-(OCH3)3 ; R2 = 4-Cl
c, R1 = 3,4,5-(OCH3)3 ; R2 = 4-CH3
a, R1 = 4-CH3 ; R2 = 4-Cl
b, R1 = 4-CH3 ; R2 = 4-Cl
c, R1 = 4-CH3 ; R2 = 4-Cl
; Ar = 2-thienyl
; Ar = 4-(Cl)-C6H4
; Ar = 4-(CH3)-C6H4
d, R1 = 4-OCH3 ; R2 = 4-CH3 ; Ar = C6H5
e, R1 = 4-CH3 ;R2 = 4-OCH3 ; Ar = 4-(Cl)-C6H4
f , R1 = 4-CH3 ;R2 = 4-OCH3 ; Ar = 4-(CH3)-C6H4
Scheme 1. Synthesis of Pyrido[2,3-d]pyrimidine derivatives.
assignable for CH3 group and C4 proton of pyrazole moiety. In 106.80 ppm assigned for the carbon of the CH3, OCH3, and C4 of
addition, 13 C NMR spectra of 5a revealed signals at d 21.32, the pyrazole moiety. The carbonyl signals were recorded at d
106.54, 161.77, and 172.44 ppm assigned for CH3 group, C-4 of 163.37 and 171.74 ppm.
pyazole ring, C¼O of pyridine ring and C¼O of pyrazolone
Compounds 7a–c showed strong band for C¼O group in pyra-
moiety, respectively.
zole moiety at 1690 cmꢀ1 in the IR spectrum of 7c and presence
On the other hand, 6a–d derivatives displayed broad band at of singlet signal equivalent to one proton at d 2.94 ppm represents
3429 cmꢀ1 for NH2 and two NH groups in IR spectrum of com- the CH2 protons of the pyrazolyl moiety in 1H NMR spectrum.
pound 6b. Additionally, 1H NMR spectrum showed three singlet 13 C NMR spectra of compound 7c showed signals at d 56.47,
signals at d 3.97, 8.13, and 12.48 ppm related to NH2 group, C4 of 60.47, 90.72, 167.16, 167.54 and 170.45 ppm corresponding to the
pyrazole moiety, and two NH groups, respectively. 13CNMR spec- carbon of OCH3, C4 of pyrazole ring and three C¼O carbons,
trum for compound 6d showed signal at d 21.44, 56.47, 60.49, and respectively.

552
H. S. A. ELZAHABI ET AL.
Table 1. Percentage of growth inhibition activity of compounds 3a–c,e–g, 4a,b,
d–f, 5a–d, 6a–d, 7a–c, 8a–f against HepG-2, PC-3, HCT-116, MCF-7, and A-549
cell lines at (100 lM) dose.
Condensation of the hydrazinyl derivatives 4 b,d with various
aromatic aldehydes22,25 afforded the corresponding 2-(2-(Aryl-2-
ylmethylene)
hydrazinyl)-5,7-diarylpyrido[2,3-d]pyrimidin-4(3H)-
Growth inhibition (%)
ones 8a–f. Structures of compounds 8a–f were confirmed by the
presence of signals corresponding to the –N¼CH proton at d
8.10–8.25 ppm. 13CNMR spectrum for 8f revealed signals at d
21.38, 21.51, 55.61, 159.91, and 161.20 ppm assigned for CH3,
OCH3, –NH ¼ CH and C¼O carbons, respectively. MS of compound
8 b showed the molecular ion peak [Mþ] at m/z 499 and m/z 503
corresponding for the chlorine isotopes.
Compounda
HepG-2
PC-3
HCT-116
MCF-7
A-549
3a
3b
3c
3e
3f
3g
4a
4b
4d
4e
4f
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
8a
8b
92
26
38
14
69
39
90
96
96
81
24
99
35
70
99
92
63
74
85
92
80
42
64
94
99
54
35
91.5
100
96
53
6
49
92
91
30
67
40
44
44
90
99
96
64
40
99
60
63
93
90
69
56
88
91
52
49
66
91
93
65
57
70
100
86
41
39
51
86
92
37
0
100
81
61
98
85
99
100
99
86
78
99
34
49
95
1
45
91
100
0
84
9
76
100
100
100
94
66
99
23
81
98
100
4
59
99
100
75
72
98
100
99
94
68
82
0
Biological evaluation
52
54
54
93
100
34
77
88
97
73
66
83
89
96
77
23
86
91
Invitro antitumor assay
All the synthesized compounds 3–8 were tested for their anti-
cancer activity against hepatic cancer (HepG-2), prostate cancer
(PC-3), colon cancer (HCT-116), breast cancer (MCF-7), and lung
cancer (A-549) cell lines. Preliminary screening against the cancer
cell lines was performed, using doxorubicin as a reference drug at
doses of 100 lM. Variable results were recorded for the test com-
pounds 3–8 (Table 1). Pyridopyrimidine derivatives that exhibited
inhibitory activity >90% compared to doxorubicin were selected
for IC50 and IC90 screening (Tables 2 and 3).
Almost, hepatic cancer cell line (HepG2) showed remarkable
sensitivity towards the test compounds 3–8 compared to doxo-
rubicin. Pyrazolylpyrido[2,3-d]pyrimidines 5a and 7a were equipo-
tent, possessing twice activity relative to doxorubicin (IC50 of
0.3 lM each and 0.6 lM respectively). Pyrazolyl analogue 5d exhib-
ited remarkable activity of IC50 0.9 lM. Hydrazino precursor 4a was
almost equipotent to doxorubcin of IC50 0.7 lM. Compounds 3a,
4d and 8c displayed moderate activity of IC50 1.7 lM, 1.2 lM, and
1 lM, respectively. Noticeably, the test compounds showed lower
anticancer activity than doxorubicin, concerning IC90 measurement
(Table 2).
Regarding the PC-3 cell line, hydrazino precursor 4d and pyra-
zolyl analogue 5a were more potent than doxorubicin as a refer-
ence standard (IC505.47 lM, 6.6 lM, and 6.8 lM respectively). Also,
pyrazolyl analogue 6d as well as the hydrazone derivative 8a dis-
played remarkable activity of IC50 7.9 lM and 7.97 lM respectively.
On the other hand, pyridopyrimidine derivatives 3a, 4b, 8 b–d
exhibited moderate activity with IC50 ranged from 9.2 lM to
12 lM. Interestingly, IC90 showed promising anti-prostate cancer
activity for pyrazolyl analogue 5a versus doxorubicin (IC90 14.1 lM
and 13.8 lM, respectively). The rest of the test compounds showed
IC90 ranged from moderate to poor activity compared to doxorubi-
cin (Table 2).
Potent activity against HCT-116 cell line was recorded for three
of the test compounds, e.g. 2-hydraino derivative 4d and 3-
methyl-5-oxo-pyrazolyl 5a,d. They were almost twice the activity
of doxorubicin exerting IC50 6.9 lM, 7 lM, and 5.9 lM vs. 12.8 lM,
respectively. Excepting pyridopyrimidines 4d and 5a,d, none of
the test compounds showed high potency against HCT-116 colon
cancer. Preferentially, the hydrazide derivative 4d was also more
potent than doxorubicin by two folds, exerting IC90 21 lM vs.
51.7 lM, respectively. Simultaneously, compounds 3a and 5a
exhibited higher activity than doxorubicin of IC90 48 lM and
36 lM, respectively (Table 2). In contrary, breast and lung cancer
cell lines exhibited remarkable resistance towards all of the test
compounds 3–8 (Table 3).
78
100
83
78
32
54
100
8c
8d
8e
40
97
100
8f
Doxorubicina
aConcentrations of the test compounds and positive control (doxorubicin) were
100 lM.
Structure-activity relationship
In general, the structure–activity relationships of the screened
products indicated that, existence of pyrazolyl moiety at C-2 of
pyrido[2,3-d]pyrimidines 5a,d, 6d, and 7a derivatives afforded the
maximum potency of anticancer activity. Noticeably, 5a and 7a
shared the same pyridopyrimidine scaffold with (4-CH3-phenyl)
and (4-chlorophenyl) at C-5 and C-7 respectively. Compound 5a
linked 3-methyl-5-oxopyrazolyl moiety showed broad anticancer
effect against hepatic, prostate, and colon cancers. Replacing the
methyl group of pyrazole moiety in 5a with carbonyl group in 7a
afforded 3,5-dioxopyrazole where the activity profile was changed.
Comparatively, it was retained against hepatic cancer but
diminished against prostate and colon cancers in 7a. Moreover,
compounds 5d and 6d shared pyridopyrimidine scaffold with
(3,4,5-trimethoxyphenyl) and (4-CH3-phenyl) at C-5 and C-7,
respectively. Replacing methypyrazolone moiety in 5d with amino-
pyrazolone in 6d shifted the anticancer activity from anti-colon to
anti-prostate cancer, respectively. Noticeably, the steric factor
potentially affected the anticancer activity in 5a–d. The data
recorded remarkable decrease in the activity when (4-CH3-phenyl)
group in 5a was exchanged by (3,4,5-trimethoxyphenyl) in 5c.
Similarly, introduction of steric bulky group in 7b–c diminished
the anticancer activity. In contrary, steric factor did not affect the
anticancer activity in 6a–d, except 6d derivative that carried the
bulky (3,4,5-trimethoxyphenyl) group at C-5 (Figure 2).
Thioxo precursors 3a–c,e–g displayed poor anticancer activity
against all cancer cell lines. Anti-hepatic cancer effect was greatly
increased upon converting thioxo group in 3c to hydrazide moiety
in 4b (equipotent to doxorubicin). Also, great records were dis-
played upon adopting the same replacement in 3e to afford
hydrazide analogue 4d. The later was more potent than

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
553
Table 2. IC50 and IC90 of the test compounds 3a,f,g, 4a,b,d,e, 5a,d, 6a–d,f against HepG-2, PC-3 and HCT-116 cell lines.
IC50/IC90 (lM)
HepG-2
PC-3
HCT-116
Compound
IC50
IC90
IC50
IC90
IC50
IC90
48
–
3a
3f
1.7 0.56
–
19.27 2.11
9.2 0.9
17.5 1.9
92.98
24 3.2
–
6
–
55.69
4
9
3g
4a
4b
4d
4e
5a
5d
6a
6c
6d
7a
8a
8b
8c
8d
–
–
–
–
–
–
86
–
21 1.5
36
53 3.6
13 2.1
0.7 0.09
1.2 0.5
–
0.3 0.02
0.9 0.06
4.8 1.03
–
80.8 4.6
17.6
19 1.4
–
14.96 0.3
10.44
37.15 2.1
18.46
38 2.1
24
6.9 0.9
–
5
1
1
2
2
5.47 0.4
32.52 0.8
6.6 0.5
26.7 1.1
20.7 1.3
–
7.9 0.4
24.7 0.8
7.97 0.2
12 0.9
15.57 1.6
78.29 4.9
14.1 0.8
89 4.2
79.9 3.6
–
16
77.9
53.35 3.7
23.5 1.9
2.1 0.3
18.5 2.5
7
0.5
2
–
5.9 0.9
28 1.2
–
21.6 3.69
–
–
–
24 2.5
–
22.3 3.5
19.27 1.1
–
2
4
–
0.3 0.01
–
3.07 1.4
25 3.6
–
23 1.8
28 1.1
53 5
60 9.1
59 8.9
1
0.09
11.65 0.95
9.5 0.99
85.82 3.6
–
–
8f
40.7 8.9
0.6 0.05
84.6 9.9
1.8 0.2
47.9
6.8 1.2
2
91.6 6.1
13.8 0.8
–
12.8
Doxorubicin
1
51.7 0.7
IC50: Compound concentration required to inhibit the cell viability by 50%.
SEM: Standard error mean; each value is the mean of three values.
Table 3. IC50 and IC90 of the test compounds 3a,f,g, 4a,b,d,e, 5a,d, 7a, 8a–d,f
against MCF-7and A549 cell lines.
Three of the tested enzymes are protein kinases, e.g. platelet-
derived growth factor receptor (PDGFR) and epidermal growth fac-
tor receptor (EGFR) are tyrosine kinases. The third enzyme was
cyclin-dependent kinase (CDK4/CyclinD) that phosphorylates ser-
ine/threonine residues. The remaining tested enzymes are lipid
kinases that belong to phosphoinositide 3-kinases (PI3Ks), e.g. PI3K
(p100b/p85a) and PI3K (p100a/p85a).
IC50/IC90 (lM)
MCF-7
A-549
Compound
IC50
IC90
IC50
IC90
3a
3f
–
–
-–
–
3.52 0.5
25
–
4
28.79
4
Maximum inhibitory percentage of 5a was recorded (Table 4)
against PDGFR beta of 82% and 94% at concentrations of 50 lM
and 100 lM, respectively. Also, remarkable inhibitory percentage
was exhibited against EGFR of 81% and 86% at the previously
mentioned concentrations. On the other hand, 5a at 50 lM
recorded low inhibition percent against CDK4/Cyclin D1 of 35%.
The inhibition of the previous enzyme was retrieved upon apply-
ing 100 lM of 5a to 75%. As a conclusion, the inhibitory effect of
5a against both PDGFR beta and EGFR was remarkable and dir-
ectly proportional to the concentration. Interestingly, the inhibitory
effect of 5a toward PDGFR beta was more prominent and more
concentration sensitive than EGFR. For example, increase the con-
centration of 5a by two-fold from 50 lM to 100 lM afforded 10%
increase of the activity against PDGFR. Concerning EGFR kinase,
applying twice concentration of 5a showed 5% increase of the
activity. Although the effect of 5a against CDK4/CyclinD1 was less
prominent, but it was more concentration dependent. So, dou-
bling the concentration of 5a enhanced the inhibitory effect by
almost two-folds (Table 4) (Figure 3). Unexpectedly, compound 5a
showed activation effect toward the two lipid kinases, PI3K
(p100b/p85a) and PI3K (p100a/p85a) above the control experimen-
tal. The activation percentages were recorded in (Table 4). So, the
potent anticancer activity of 5a against HepG-2, PC-3, and HCT-
116 could be attributed to its remarkable inhibitory activity against
protein kinase enzymes.
3g
4a
4b
4d
4e
5a
6a
6c
6d
7a
8a
8b
8c
8d
8f
30 2.3
–
–
–
–
–
21 2.1
–
–
33 4.6
–
–
95 11.2
–
–
–
-–
–
–
–
–
10.94 1.1
8.7 0.9
26.42 3.1
24.8
2
–
–
9.6 1.1
–
83.44 5.9
7.19 1.6
27.8 3.1
69
6
–
39.65 2.1
–
37.57 2.8
60 9.7
–
–
69 8.1
–
–
–
–
–
–
14.6 1.9
31.1 2.5
17 2.1
–
–
–
–
–
–
–
–
Doxorubicin
2.2 3.1
5.2 1.9
0.087 0.9
0.35 0.7
IC50: Compound concentration required to inhibit the cell viability by 50%; SEM
Standard error mean; each value is the mean of three values.
doxorubicin against prostate and colon cancers. Hydrophilic elec-
tron rich nature of the hydrazide moiety enabled the electronic fac-
tor to afford positive impact on the anticancer activity. Compounds
4b and 4d lacked their activity by extending the hydrazide moiety
to benzylidene hydrazide in 8b,d. Obviously, 8a–f failed to record
any potent activity against all cancer cell lines.
Kinase inhibition assay
Upon cellular screening on HepG-2, PC-3, and HCT-116, compound
5a exhibited higher anticancer activity in comparison with doxo-
rubicin. So, it was subjected for in vitro inhibition assessment to
measure its inhibitory activity against a panel of five different pro-
tein and lipid kinases. Two concentrations of 5a were applied
(50 lM and 100 lM) in single measurement compared to blank
control.
Molecular docking results
Molecular docking technique represents the pattern of the inter-
action between a small molecule and a protein at the atomic level.
This approach can explore the behavior of small molecules in the
binding site of the target proteins. So, docking simulation was

554
H. S. A. ELZAHABI ET AL.
Figure 2. IC50 of compounds 4d, 5a, 5d, 7a against HepG-2, PC-3, HCT-116, MCF-7, and A-549 cell lines.
Table 4. Percentage of kinases inhibition of compound 5a at (50 and 100 lM).
score¼ ꢀ7.95 and ꢀ5.35 Kcal/mol, respectively). The orientation of
Compound 5a
pyrido[2,3-d]pyrimidinone scaffold of 5a was parallel to the hinge
region due to the two bulky aryl rings. The scaffold posed in the
adenine binding region of the ATP binding site. The pyrazolyl
C¼O carbon of 5a formed a hydrogen bond acceptor (distance:
2.44 Ao) with the backbone NH of Met769 in the hinge region.
Para-tolyl moiety shared hydrophobic interaction with Gly695 and
para-chlorophenyl ring formed arene-arene interaction with
Phe699 (Figure 4).
% Inhibition
Kinase
50 lM
100 lM
CDK4/CyclinD1
EGFR
PDGFRb
PI3K (p110a/p85a)
PI3K (p110b/p85a)
ꢀ35
ꢀ81
ꢀ82
181
ꢀ75
ꢀ86
ꢀ94
182
473
504
Negative (ꢀ) values: Inhibition of target activity by the compound; Positive (þ)
value: activation of target activity.
Docking study on CDK6
performed in this work using Molecular Operating Environment
R
(MOEV)17 2008.10. All the interaction energies and different calcu-
Compound 5a bound in ATP binding pocket of CDK6 displayed
good fitting and minimum binding energy as well as the reference
PD0332991 (Docking score¼ -5.187 and -4.145, respectively). Also,
it formed bidentate hydrogen bonding between N3 of pyridopyri-
midine ring and N2 of pyrazolone moiety to the main chain amide
of Asp163 (distance ¼1.44, 1.43 Ao). Moreover, para-chlorophenyl
moiety projected on the opposite side, sequestered into a hydro-
phobic region (Figure 5).
So, docking simulation of compound 5a into kinase domain of
EGFR and CDK6 postulated the vital role of both pyrido[2,3-d]pyri-
midinone scaffold and the side chain substituent. Both moieties
involved in binding mode interaction. Binding pose covered differ-
ent macromolecular interactions, e.g. H-bonding, arene-arene, and
hydrophobic interactions. Compound 5a could be anticipated to
bind efficiently to the ATP binding site of EGFR compared to erlo-
lations were automatically calculated.
Kinase inhibitory screening assay promoted compound 5a that
showed promising inhibitory activity against three kinases, namely
PDGFR b, EGFR, and CDK4/Cyclin D1. The X-ray crystallography of
PDGFR b structure was not fully resolved27. On the other hand, X-
ray crystallography structure was reported for EGFR (pdb code:
1M17)18,19 with erlotinib. Simultaneously, the structure of CDK4/
CyclinD1 with PD0332991 was reported (PDB ID: 2EUF)20,21. So the
docking study was achieved for both EGFR and CDK4/Cyclin D1
kinases to predict the binding modes, affinities, and orientations
of compound 5a at the active sites of them.
Docking study on EGFR
Docking of 5a into the active site of EGFR explored minimum tinib. Furthermore, similar results were predicted for the binding
binding energy comparable to the reference, erlotinib (Docking pose of CDK6 with both PD0332991 and compound 5a.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
555
Figure 3. Percentage of kinases inhibition of compound 5a against EGFR, CDK4 at (50 and 100lM).
Figure 4. The proposed binding mode of compound 5a docked in the active site of EGFR. A and B showing 2D and 3D ligand-receptor interactions (hydrogen bonds
are illustrated as arrows; C atoms are colored gray, N blue, and O red).
cancer cells in comparison to doxorubicin. Regarding HepG-2 cell
line, compounds 5a, 7a were more potent (IC50¼ 0.3 mM) than
doxorubicin. Also, compound 4 b was nearly equipotent
(IC50¼ 0.7 mM) to doxorubicin (IC50¼ 0.6 mM). Upon cellular screen-
ing on PC-3, compounds 4d, 5a exhibited higher activity than
doxorubicin (IC50¼ 5.47, 6.6, 6.8 mM respectively). For HCT-116 cell
Conclusions
A novel group of substituted pyrido[2,3-d]pyrimidinones 3–8, were
synthesized and evaluated for in vitro anticancer activity. There
was potent growth inhibitory activity against HepG-2, PC-3, and
HCT-116 cell lines and weak activity against MCF-7 and A-549

556
H. S. A. ELZAHABI ET AL.
Figure 5. The proposed binding mode of compound 5a docked in the active site of CDK6. A and B showing 2D and 3D ligand-receptor interactions (hydrogen bonds
are illustrated as arrows; C atoms are colored gray, N blue, and O red.
line, 4d, 5a, and 5d derivatives showed two-folds the activity of
doxorubicin (IC50¼ 6.9, 7, 5.9, and 12.8 mM, respectively). The
highly potent anticancer compound 5a exhibited promising
inhibitory activity against PDGFR b, EGFR, and CDK4/Cyclin D1
kinases at two concentrations of 50 lM and 100 lM in single
measurement. Furthermore, the molecular docking study for
compound 5a into the ATP binding site of EGFR and CDK6
explored an efficient binding as that of erlotinib and
PD0332991, respectively.
ORCID
Nagy M. Khalifa
http://orcid.org/0000-0001-9019-925X
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