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

Abstract 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)- pyrido[2,3-d]pyrimidin-4(3H)-one (5a) exhibited strong, more potent anticancer (IC50: 0.3, 6.6 and 7 µM) relative to the standard doxorubicin (IC50: 0.6, 6.8 and 12.8 µM), respectively. Kinase inhibitory assessment of 5a showed promising inhibitory activity against three kinases namely PDGFR β, EGFR, and CDK4/cyclin D1 at two concentrations 50 and 100 µM in single measurements. Further, a molecular docking study for compound 5a was performed to verify the binding mode towards the EGFR and CDK4/cyclin D1 kinases.


Introduction
Cancer is one of the leading causes of death in the world, characterized by the loss of control of cell proliferation, leading almost invariably to death, in untreated patients 1,2 . Chemotherapy, alone or in combination with surgery, is commonly the most efficient anti-cancer remedy. However, the use of available chemotherapeutics is limited mainly due to drug resistance and toxicities 3 . 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 cell [4][5][6][7][8] . So, combination of chemotherapies with different targets increases efficiency, antagonizes the resistance, and decreases toxicity as well. Traditional anticancer drugs work by disrupting 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 cancer and normal cells 9,10 . 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 effects 11 . Pyrido [2,3d]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 CDK4/6 12,13 ( Figure 1). Based on the structural features of the previous pyrido [2,3-d]pyrimidines, we aimed to prepare a new group of pyrido [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 appropriate binding modes of the most potent target compounds that matched ligands binding modes. The applied modeling program was Molecular Operating Environment (MOEV R ) 2008. 10.

Chemistry
Melting points by using electro thermal apparatus on open capillary tubes were recorded. IR spectra (KBr) were performed on a Shimadzu 435 IR Spectrophotometer (Shimadzu Bruker, Tokyo, Japan) ( cm À1 ). 1 H 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 spectrophotometer (Santa Clara, CA, USA) using DMSO-d6 as a solvent 300 MHz oron Agilent Technologies 400 MHz NMR spectrophotometer and the chemical shifts were estimated in ppm, relative to TMS as an internal standard. Physical data (C, H, and N) agreed with the proposed structures and obtained by using a Vario Elemental analyzer (Yamashitacho, Yokohama, Japan) (within ±0.4% of the theoretical values). Mass spectra were recorded on a DI-50 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).

Biological evaluation
In-vitro antitumor assay 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 methods [14][15][16] .

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 carried 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 kinases [(PDGFR beta, EGFR, CDK4/Cyclin D1, PI3K (p100b/p85a, PI3K (p100a/p85a)] according to a previously reported method 16 .

Molecular modeling study
All the molecular modeling calculations and docking simulation studies were performed utilizing Molecular Operating Environment (MOE V R ) 2008.10 17 . 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.

Results and discussion
Chemistry New group of 5,7-diaryl pyrido[2,3-d]pyrimidinones 3a-g were created through reaction of the starting precursor 2-mercapto-4hydroxy-6-aminopyrimidine 1 with a, b unsaturated ketones 2a-g in dry DMF according to the applied method reported for analogue 3d [22][23][24] . Compound 3c as an example showed absorption band at 3394 cm À1 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 1 HNMR spectrum. Further, the MS revealed molecular ion peak at m/z 379 (M þ ) 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.
Nucleophilic attack 22-25 of hydrazine hydrate on thioxo derivatives 3a,c-g yielded the corresponding 2-hydrazinopyrido[2,3d]pyrimidin-4(3H)-ones 4a-f (Scheme 1). IR spectrum of compound 4a showed existence of three absorption bands at 3410, 3190, and 1678 cm À1 corresponding to NH 2 , NH, and C¼O groups, respectively. 1 26 to give the corresponding three-substituted pyrazolone derivatives 5a-d, 6a-d, 7a-c, respectively. Compounds 5a-d were confirmed by the existence of an additional band at 1651 cm À1 for C¼O group of the newly created pyrazolone nucleus in IR spectrum of compound 5b. Additionally, the 1 HNMR spectrum of 5a showed signals at d 1.89 and 8.18 ppm assignable for CH 3 group and C4 proton of pyrazole moiety. In addition, 13 C NMR spectra of 5a revealed signals at d 21.32, 106.54, 161.77, and 172.44 ppm assigned for CH 3 group, C-4 of pyazole ring, C¼O of pyridine ring and C¼O of pyrazolone moiety, respectively.

Biological evaluation
Invitro antitumor assay All the synthesized compounds 3-8 were tested for their anticancer 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 compounds 3-8 (Table 1). Pyridopyrimidine derivatives that exhibited inhibitory activity >90% compared to doxorubicin were selected for IC 50 and IC 90 screening (Tables 2 and 3).
Regarding the PC-3 cell line, hydrazino precursor 4d and pyrazolyl analogue 5a were more potent than doxorubicin as a reference standard (IC 50 5.47 lM, 6.6 lM, and 6.8 lM respectively). Also, pyrazolyl analogue 6d as well as the hydrazone derivative 8a displayed remarkable activity of IC 50 7.9 lM and 7.97 lM respectively. On the other hand, pyridopyrimidine derivatives 3a, 4b, 8 b-d exhibited moderate activity with IC 50 ranged from 9.2 lM to 12 lM. Interestingly, IC 90 showed promising anti-prostate cancer activity for pyrazolyl analogue 5a versus doxorubicin (IC 90 14.1 lM and 13.8 lM, respectively). The rest of the test compounds showed IC 90 ranged from moderate to poor activity compared to doxorubicin (Table 2).
Potent activity against HCT-116 cell line was recorded for three of the test compounds, e.g. 2-hydraino derivative 4d and 3methyl-5-oxo-pyrazolyl 5a,d. They were almost twice the activity of doxorubicin exerting IC 50 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 IC 90 21 lM vs. 51.7 lM, respectively. Simultaneously, compounds 3a and 5a exhibited higher activity than doxorubicin of IC 90 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).

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-CH 3 -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-CH 3 -phenyl) at C-5 and C-7, respectively. Replacing methypyrazolone moiety in 5d with aminopyrazolone 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-CH 3 -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 displayed upon adopting the same replacement in 3e to afford hydrazide analogue 4d. The later was more potent than 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. doxorubicin against prostate and colon cancers. Hydrophilic electron rich nature of the hydrazide moiety enabled the electronic factor 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 doxorubicin. So, it was subjected for in vitro inhibition assessment to measure its inhibitory activity against a panel of five different protein and lipid kinases. Two concentrations of 5a were applied (50 lM and 100 lM) in single measurement compared to blank control.
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 applying 100 lM of 5a to 75%. As a conclusion, the inhibitory effect of 5a against both PDGFR beta and EGFR was remarkable and directly 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 concentration 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, doubling 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 experimental. 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.

Molecular docking results
Molecular docking technique represents the pattern of the interaction 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  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 resolved 27 . On the other hand, Xray 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 binding energy comparable to the reference, erlotinib (Docking score¼ À7.95 and À5.35 Kcal/mol, respectively). The orientation of 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 A o ) 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).

Docking study on CDK6
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 pyridopyrimidine ring and N2 of pyrazolone moiety to the main chain amide of Asp163 (distance ¼1.44, 1.43 A o ). Moreover, para-chlorophenyl moiety projected on the opposite side, sequestered into a hydrophobic 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]pyrimidinone scaffold and the side chain substituent. Both moieties involved in binding mode interaction. Binding pose covered different 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 erlotinib. Furthermore, similar results were predicted for the binding pose of CDK6 with both PD0332991 and compound 5a.

Disclosure statement
No potential conflict of interest was reported by the authors.