Kinase Inhibitors of Novel Pyridopyrimidinone Candidates : Synthesis and In Vitro Anticancer Properties

Drug Exploration & Development Chair (DEDC), Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Department of 'erapeutical Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Department of Chemistry, Faculty of Science and Technology, Al-Neelain University, Khartoum, Sudan


Introduction
Cancer is a great public health issue characterized by an uncontrolled increase of cancer cells through cell division and the cells undergo modification by their DNA, leading to death [1][2][3].Due to drug resistance and the serious effects of treatment by chemotherapy, the use of available chemotherapeutics is often limited [4].e combination of chemotherapies with several targets increases selectivity, reduces the resistance, and lowers toxicity towards infected and noninfected cells.Heterocycles have emerged as strong scaffolds for numerous biological considerations [5] and represent a significant part in the designing and detection of novel pharmacologically active entities [6].e pyridopyrimidine compounds are a group of fused heterocycles that possess various pharmacological applications as antitumor, topoisomerase I inhibitor, adenosine kinase inhibitor, growth regulator, antihepatitis C virus, antiinflammatory, antileishmanial, antiviral, antimicrobial, anticonvulsant, antimycobacterial, CNS depressant, antihypertensive, antiallergic, diuretic, tyrosine kinase inhibitor, and calcium channel antagonist [7][8][9][10][11][12][13][14][15][16].Among them, pyrido [2,3-d] pyrimidin-4-ones (A-C) were found to lower cell proliferation in various cancer cell lines through inhibition of various kinases, e.g., TKs, PI3K, and CDK4/6 (Figure 1) [17][18][19].In continuation of our earlier studies that involved synthesis of different other substituted pyridopyrimidine compounds [20][21][22] and based on the structural features of pyrido [2,3-d]pyrimidine, this study is designed to synthesize various groups containing different substituents in the phenyl ring at position 5 of the parent compound pyrido [2,3-d] pyrimidinone to further improve the SAR-relationship for their cytotoxicity and also for their inhibitory activity against TKs, CDK4/6, and PI3K enzymes.

Results and Discussion
Treatment of the 2-hydrazinyl intermediate 4 with active methylene, namely, ethyl acetoacetate, ethyl cyanoacetate, diethyl malonate, or ammonium isothiocyanate afforded the corresponding 5-substituted pyrazolones and triazolopyrimidines 6-9 (Scheme 1).e new pyrazolone ring linked to the pyridopyrimidine backbone in compound 6 was proved with the appearance of strong bands at 3214 and 1725 cm −1 referred to NH and CO functions of the pyrazole ring in IR spectrum and existence of two singlet peaks at δ 1.87 and 10.87 ppm due to methyl attached to the pyrazole ring and -NH protons.e molecular formula C 18 H 14 N 6 O of 6 was confirmed by the presence of molecular ion peak [M + ] at m/z 330 in MS.On the contrary, the new pyrazolidine-3,5-dione ring in compound 8 was proved by the existence of strong bands at 3210, 1705, and 1682 cm −1 referred to NH and 2 CO functions in the IR spectrum.e methylene group of the new ring appeared as a singlet peak at δ 2.98 ppm in 1 H NMR spectrum and signal at 90.21 ppm in 13

Journal of Chemistry
Finally, the new 3-aminotriazole ring fused with pyridopyrimidine moiety in 9 was proved in the IR spectra by the existence of bands at 3460 and 3317 cm −1 referred to NH 2 and NH functions besides other band at 1669 cm −1 due to carbonyl of pyridopyrimidine moiety. 1 H NMR spectrum displayed two singlets at 5.60 and 12.55 attributed to NH 2 and NH protons of the new fused ring. 13C NMR spectrum displayed the carbons at their expected regions, and MS gave a [M + ]-ion peak at m/z 355 equivalents to the molecular formula of 9.

In Vitro Cytotoxic Screening against HepG2, PC-3, and
HCT-116 Cell Lines.Anticancer evaluation of the newly obtained products represented in Figure 2 was screened against three human cancer cell lines (HepG2, PC3, or HCT-116) [23].
e tested compounds that displayed inhibitory effect more than 90% referring doxorubicin as a standard drug (IC 50 0.6 μM) were chosen for IC 50 examination (concentrations required for 50% inhibition of cell viability).e in vitro screening of compounds 3-9 at 100 μM (Figure 3) exhibited remarkable anticancer activities, and compound 6 showed promising potency against hepatic cancer cell line (anti-HepG2) with (IC 50 � 0.5 μM).

Structure-Activity Relationships.
e cytotoxic screening results revealed that thioxo precursor 3 displayed poor anticancer activity against all cancer cell lines.Upon converting the thioxo group in 3 to hydrazide in 4, antihepatic cancer (HepG2) effect was greatly increased as a result of the presence of the hydrophilic electron-rich nature in compound 4 which causes the electron factor to give a positive impact on the antiproliferative properties.Attachment of the pyrazole ring as a substituent at position 2 of the backbone moiety as in compounds 6, 7, and 8 afforded the highest potency of anticancer activity.Compound 6 that carried 5-methyl-3oxopyrazole exhibited the highest activity against all the tested cell lines.e activity was reduced and shifted toward the PC-3 cell line after replacement of the 5-methyl-3-oxopyrazole nucleus at 6 by 3-amino-5-oxopyrazol in 7. Upon replacing the mentioned pyrazole moiety in 6 by 3,5-dioxopyrazole in 8, or by fused triazolo [4,3-a]pyrimidine in 9, the activity profile was changed.

Kinase Inhibition Screening.
According to the data of cytotoxic assay, the highly potent derivative 6 was chosen for in vitro inhibition assessment against a list of different protein, AKT1, AKT2, BRAF (V600E), CDK2/cyclin A1, CHK1, EGFR, PDGFRβ, and c-RAF kinases at 100 μM utilized the radiometric or ADP-Glo assay procedure.ree of the tested kinases (BRAF V600E, EGFR, and PDGFRβ)  6 Journal of Chemistry were highly inhibited by more than 90% with the highest inhibition recorded with EGFR at 97%.On the contrary, compound 6 appeared to partially activate the c-RAF kinase with a rise in counts of 37% over the control substrate rates (Table 1, Figure 4).

Molecular Docking Study.
Molecular docking was used to analyze the supposed binding mode of the designed compound with CDK6 and EGFR to better understand the mechanism of inhibition.For the docking studies, the crystal structure of the complex CDK6 and EGFR has been chosen  (PDB code 5L2I [24] and PDB code 4HJO [25]).e most active compound 6 in the current study was docked into the CDK6 and EGFR kinase's putative active site.For the receptor preparation, the Molecular Operating Environment software package MOE ® 2015 [26] has been used by means of the removal of water molecules and the addition of hydrogen atoms.MOE has also been used for the graphic structure of ligands 3D and then saved on data lists after minimizing structure and geometries energy.For each receptor, the pockets were then used to dock ligands after setting London dG to scoring function and GBVI/WSA dG to re scoring function.erefore, the scoring and RMSD (root-mean-square deviation) values for the best conformation of each ligand with different receptors are shown in Table 2, as well as 2D and 3D figures of each selected conformation are shown in Figures 3 and 4. Docking simulation of compound 6 into kinase domain of CDK6 and EGFR postulated the pivotal function of both the backbone moiety and the side chain substituent (Figures 5  and 6).A library of substituted pyridopyrimidines 3-9 were designed and screened for their cytotoxicity.ere are potent growth inhibitory effects against hepatic, prostate, and colon cancer cells lines, in comparison with doxorubicin as positive control.Regarding HepG2 cell line, compound 6 showed the greatest inhibitory activities against hepatic cancer (HepG2) with inhibition percent (IC 50 � 0.5 μM) more potent than doxorubicin (IC 50 � 0.6 μM).A molecular docking study of compound 6 into the ATP binding site of EGFR exhibited identical binding as erlotinib.

Figure 1 :
Figure 1: Reported and suggested pyridopyrimidines integrated with kinase inhibitors and anticancer properties.

Figure 3 :
Figure 3: IC 50 of the tested compounds against cancer cell lines.
MS revealed [M + ] at m/z 330 agreed with the MF C 18 H 14 N 6 O.
3.1.Chemistry.A group of substituted pyridopyrimidines were obtained via treatment of starting 1 with α,β-unsaturated ketone 2 in dry DMF to afford 2-thioxo derivative 3. IR spectrum of 3 revealed four strong bands at 3327, 3234, 1668, and 1178 cm −1 due to 2NH, CO, and CS functions, respectively.In the same time, 1 HNMR spectrum displayed two singlets at δ 12.26 and 13.01 ppm assignable for 2NH protons.Also,13CNMR spectrum showed signal at 178.01 ppm for C�S carbon.Treatment of hydrazine hydrate with thioxo derivative 3 afforded the corresponding 2-hydrazinyl derivative 4. IR spectrum of 4 revealed four peaks at 3446, 3362, 3205, and 1671 cm −1 attributed to amino, two amides, and carbonyl functions, respectively.Moreover, 1 H NMR spectrum confirmed the presence of amino and amide protons by existence of three singlet peaks at δ 4.80, 11.38, and 12.76 ppm.e H NMR spectrum.MS gave a [M + ]-ion peak at m/z 398 equivalents to the molecular formula C 21 H 14 N6 6 O 3 .

Table 1 :
Percentage of kinase inhibition of derivative 6 at 100 μM.

Table 2 :
Molecular simulation results for ACV/PNV interactions with HAS.