Discovery of new pyridine-quinoline hybrids as competitive and non-competitive PIM-1 kinase inhibitors with apoptosis induction and caspase 3/7 activation capabilities

Abstract New quinoline-pyridine hybrids were designed and synthesised as PIM-1/2 kinase inhibitors. Compounds 5b, 5c, 6e, 13a, 13c, and 14a showed in-vitro low cytotoxicity against normal human lung fibroblast Wi-38 cell line and potent in-vitro anticancer activity against myeloid leukaemia (NFS-60), liver (HepG-2), prostate (PC-3), and colon (Caco-2) cancer cell lines. In addition, 6e, 13a, and 13c significantly induced apoptosis with percentage more than 66%. Moreover, 6e, 13a, and 13c significantly induced caspase 3/7 activation in HepG-2 cell line. Furthermore, 5c, 6e, and 14a showed potent in-vitro PIM-1 kinase inhibitory activity. While, 5b showed potent in-vitro PIM-2 kinase inhibitory activity. Kinetic studies using Lineweaver–Burk double-reciprocal plot indicated that 5b, 5c, 6e, and 14a behaved as competitive inhibitors while 13a behaved as both competitive and non-competitive inhibitor of PIM-1 kinase enzyme. Molecular docking studies indicated that, in-silico affinity came in coherence with the observed in-vitro inhibitory activities against PIM-1/2 kinases.


Introduction
Human Proviral Integration Moloney (PIM) kinases are a family of serine/threonine kinases includes PIM-1, PIM-2, and PIM-3 isoforms.The PIM-1 kinase has direct role in tumorigenesis in various haematological malignancies 1 , such as leukaemia and lymphoma [2][3][4][5] .Also, PIM-1 kinase is overexpressed in several solid tumours such as prostate 6 , colon 7 , hepatic 8 , and breast cancers 9 .In addition, it is involved in many biological processes such as cell cycle, cell proliferation, apoptosis, and drug resistance 10 .PIM-1 kinase has unusual adenosine triphosphate (ATP) binding pocket having proline 123 in the hinge region which is specific to PIM-1 kinase over other serine/threonine kinases 11 .Therefore, ATP could not bind to the hinge region by a second hydrogen bond as other protein kinases 12,13 .In addition, most PIM-1 kinase inhibitors are ATP-competitive inhibitors 14 .PIM-1 kinase competitive inhibitors are classified into non-ATP mimetics and ATP-mimetics.ATP-mimetics bind directly to the hinge region via Glu121 while non-ATP mimetics bind to Lys67 15,16 .PIM-1 kinase inhibition is an attractive target to overcome PIM-1 kinase induced chemotherapy resistance caused by induction of hypoxia in tumour cells 17 .X-ray crystallographic study of the lead scaffold I showed on interaction to ATPbinding site of PIM-1 kinase through prominent Hydrogen bond interaction of the carbonyl group on the pyridone ring with the Lys67.Another weak H-bond was observed with the hinge region between the main chain carbonyl of Glu121 and an aromatic hydrogen (C-3 position and C-4-position) on 6-phenyl moiety.Besides, the hydrophobic interactions with aryl moieties at 4 and 6 positions 18 (Figure 1).
Pyridine-quinoline hybrid structure (compound II) showed potent inhibition of PIM-1 kinase, increase anticancer activity against human prostatic PC-3 cancer cell line as well as induction apoptosis 19 .In addition, structure based drug design of PIM-1 kinase inhibitors revealed that electron deficient quinolone core, as in compound III, was able to form strong interactions with the hinge region of PIM-1 kinase 20 (Figure 2).
In continuation of our previously published work 21 , the structure-based design strategy was rationalised via tethering the pyridine moiety with the quinoline moiety with keeping the common respective pharmacophoric features of the lead scaffold I (Figure 1) to improve the PIM kinase inhibitory activity.It must be pointed out that, the six-position of pyridine ring was selected to be substituted with the bioactive pharmacophore 3-hydroxyphenyl group which was reported to enhance the anticancer activity through hydrogen bond interactions.In addition, the designed scaffold was modified at numerous positions, where the pyridine ring was substituted with either oxygen or its isostere sulphur or its isostere amino group at position 2.Moreover, pyridine was also hybridised with electron deficient quinolone or quinoline substituted at position 2 with either piperidine or morpholine and substituted at six-position with either hydrophilic or hydrophobic groups to impart variety of enzyme interactions hoping to enhance binding affinity hence potency (Figure 3).

General
All chemicals and solvents were purchased from commercial suppliers.Melting points were determined in open-glass capillaries using a Griffin melting point apparatus and were uncorrected.The progress of the reactions was monitored by thin-layer chromatography (TLC) on commercially available precoated silica gel aluminium-backed plates and the spots were visualised by exposure to iodine vapours or UV-lamp at k 254 nm for few seconds.Infra-red spectra (IR) were recorded, for KBr discs, on a PerkinElmer RXIFT-IR.Nuclear magnetic resonance spectra, 1 H-NMR and 13 C-NMR, were recorded on Bruker spectrometer (400 MHz) using DMSO-d6 as solvent using deuterated dimethyl sulfoxide as solvents.The data were reported as chemical shifts or d values (ppm) relative to tetramethyl silane (TMS) as internal standard.Signals were indicated by the following abbreviations: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartette and m ¼ multiplet, dd ¼ doublet of doublet and br.¼ broad.Electron impact mass spectra (EIMS) were run on a gas chromatograph/mass spectrophotometer Shimadzu GCMS/QP-2010 plus (70 eV).Relative intensity % corresponding to the most characteristic fragments is recorded.Elemental microanalyses were performed at the microanalytical unit, the Regional Centre of Microbiology and Biotechnology, Al-Azhar University, Egypt.

Biological evaluation
All chemicals, solvents, media and kits were purchased from commercial suppliers.Biological evaluation procedures were performed in Medical Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), Egypt.

Cytotoxicity screening
Cytotoxicity of the tested compounds was evaluated using normal human lung fibroblast Wi-38 cell line compared to currently used anticancer drug (doxorubicin) using MTT method was used for assaying cell viability 26 (Supplementary Page S55).

Determination of the anticancer activity
Anticancer effect of the above-mentioned compounds was evaluated using four human cancer cell lines.Colon cancer cells (Caco-2), myeloid leukaemia cell line (NFS-60), liver cancer cell line (HepG-2) and prostate cancer cell line (PC-3) using MTT method was used for assaying cell viability 26 (Supplementary Page S55).

Flow cytometric analysis of apoptosis
The apoptosis-dependent c active compounds was determined by quantification of annexin-stained apoptotic cells using the FITC signal detector (FL1) against the phycoerythrin emission signal detector (FL2) (Supplementary Page S55).

Caspase 3/7 activation assay
The percentage of caspase 3/7 activation was quantified using the Apo-ONE V R caspase 3/7 kit following the manufacturer's instructions 27 (Supplementary Page S56).

Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM).Statistical significance was estimated by the multiple comparisons Tukey post-hoc analysis of variance (ANOVA) using the SPSS16 program 28 (Supplementary Page S56).

PIM-1 and PIM-2 kinase inhibitory activity
The most active anticancer compounds were tested for their ability to in-vitro inhibit PIM-1 and PIM-2 kinase utilising PIM-1 and PIM-2 Kinase Assay Kit -Promega Corporation catalogue #V4032, following the manufacturer's instructions 29 .

Molecular docking study
The X-ray structures of PIM-1 and PIM-2 enzymes were retrieved from the Protein Data Bank (PDB) with PDB IDs: 2OBJ 18 and 4X7Q 30 , respectively.The PDB structures were prepared using OpenEye's MakeReceptor GUI of OEDOCKING 3.5.0.4 of OpenEye Scientific Software 31,32 at default levels.Redundant chains, nonessential ions, water molecules and ligands were discarded.The structural water molecule of PIM-1 kinase was kept.The search box around the X-ray co-crystal ligand was set 15.33 Å x 17.00 Å x 19.33 Å (5039 Å 3 ) as box dimensions (box volume) for PIM-1 kinase, and 19.00 Å x 16.67 Å x 16.00 Å (5066 Å 3 ) for PIM-2 kinase.Site shape potentials were calculated via setting the binding site contours in a balance between the protein and the solvent.The protein files were saved as OEDU format for FRED docking.
The studied structures were built via Marvin Sketch 17.2.6.0ChemAxon (http://www.chemaxon.com),the lowest energy conformer was generated using MMFF94 Force Field, with normal optimisation limit at default settings.Then different conformations were generated for FRED docking via OpenEye OMEGA conformer generation software 33,34 .The docking was carried out using OpenEye's FRED docking software at default levels 31,32 .
The 3 D and 2 D depictions of the docking poses in PIM-1 and PIM-2 binding sites were generated via Molecular Operating Environment (MOE) 35 .

In-silico prediction of the pharmacokinetics properties
The in-silico predictions of the pharmacokinetics properties were performed using SwissADME3 6 .

Chemistry
In our concernment for the synthesis of biologically active compounds, the synthetic strategies used for the preparation of the target compounds were outlined in three schemes.We illustrated herein the study for the synthesis of two new series of 4,6-diaryl À2-oxo or thioxo-1,2-dihydropyridine-3-carbonitriles 5a-f and 6a-f outlined in Scheme 1.The first step in this work was acetylation of aromatic amines 1a-c with acetic anhydride gave the corresponding substituted N-phenylacetamides 2a-c, which after treatment with Vilsmeier's reagent afforded the substituted 2chloro-3-formylquinolines 3a-c.After that, 2-chloro-3-formylquinolines 3a-c were reacted with morpholine or piperdine to give 2-(morpholine/piperdine-4-yl)quinoline-3-carbaldehyde 4a-f according to reported reaction conditions [22][23][24] .2-(morpholine/piperdine-4-yl)quinoline-3-carbaldehyde 4a-f was then subjected to one pot reaction with the 3-hydroxyacetophenone and ethyl cyanoacetate or cyanothioacetamide in the presence of excess ammonium acetate to give the corresponding 3-cyanopyridones 5a-f or 3-cyanothiopyridines 6a-e, respectively.Structures of the synthesised compounds 5a-f and 6a-e were confirmed by elemental microanalysis, IR, 1 H NMR for all compounds, beside EI/MS for compounds 5b,e and 6b,e and 13 C NMR spectra for compound 5b,d and 6c,e.The IR spectra of all synthesised compounds showed bands at a stretching frequency () around 3400 cm À1 corresponding to the N-H group and around 2200 cm À1 corresponding to CN group.The 1 H-NMR spectra of the pyridone derivatives showed a methine proton as a deshielded singlet peak at the downfield region d (6.9 À 7.3) ppm while for thiopyridine derivatives appeared around 8.25 ppm. 13C-NMR spectra revealed signals for CN and a highly deshielded signal corresponding to C¼O or C¼S.EI-MS of 5b, 5e, and 6e showed [M þ ] peak at 458, 456, and 468, respectively in addition to [M þ þ2] peak for 5b at 460 and for 5e at 458.Scheme 1, the obtained results declared that, compounds 5a, 5b, 5c, 5d, and 6e had the most potent activity against Caco-2 cell line (Figure 5) even superior to doxorubicin.While compounds 5e, 5f, and 6b illustrated nearly equipotent activity.Also, compounds 5b and 5c exhibited high sensitivity against PC-3 cell line compared to doxorubicin (Figure 8).Moreover, compounds 5b and 5c exerted higher anticancer activity than doxorubicin against HepG-2 cell line.While compound 6e displayed promising anticancer activity nearly equal to doxorubicin (Figure 6).Furthermore, compound 5b represented high potency against NFS-60 cell line rather than doxorubicin.Whereas compound 5c showed nearly equipotent activity (Figure 7).Consequently, compounds 5b and 5c exerted the highest anticancer activity against all tested human cancer cell lines, correlated to other compounds and doxorubicin.Indeed, compound 6e demonstrated higher anticancer activity against Caco-2 and HepG-2 compared to doxorubicin and nearly equipotent activity against NFS-60 and PC-3 cell lines.The selectivity index (SI) was calculated for the tested compounds as shown (Table 1 in Supplementary Material).The results indicated that, compounds 5b, 5c, and 6e showed the highest SI values.Compound 5c was found to have the highest SI values against all cancer lines.Consequently, it was the most selective compound towards all cancer cell lines as well as the most non-toxic towards Wi-38.Additionally, morphological changes on the four cancer cell lines (HepG-2, Caco-2, NFS-60, and PC-3) were investigated before and after treatment with the most active and safe compounds 5b, 5c, and 6e in comparison with cells treated with the reference doxorubicin.As shown, these compounds caused severe shrinking and collapsing in the normal shape of four studied cancer cell lines as shown in (Figure 9).
The synthesis of 2-O-substitutedpyridine derivatives is very straightforward and demonstrated in Scheme 2. The key intermediates, phenyl acetate derivatives 7a-c, were synthesised via heating compounds 5a-c with excess acetic anhydride.IR spectra of newly synthesised compounds 7a-c displayed absence of the band characteristic to OH function and presence of the absorption band for carbonyl moiety of the acetyl group (COOCH 3 ) at 1760-1763 cm À1 . 1 H-NMR spectra for compounds 13a-c revealed presence of shielded singlet signal attributed to CH 3 protons at a range of d 2.30-2.31ppm and in addition, absence of D 2 O exchangeable signal corresponding to OH. 13 C-NMR spectrum for compound 13c showed signals assigned for a highly shielded signal corresponding to acetyl CH 3 at d 21.29 ppm and highly deshielded signal corresponding to carbonyl group of the ester at d 174.50 ppm.First alkylation takes place by treatment of intermediated 7a-c with ethyl bromoacetate in presence of potassium carbonate to give compounds 8a-c.IR spectra of compounds 8a-c displayed a characteristic two absorbing bands for two esters C¼O at 1750-1780 cm À1 . 1 H-NMR spectrum for compound 8a revealed disappearance of D 2 O exchangeable signal corresponding to NH.In addition, it showed shielded triplet signal integrated for three protons assigned for CH 3 at d 1.21 ppm, a singlet signal for two protons attributed to CH 2 at d 2.32 ppm and a quartette band integrated for two protons corresponding to CH 2 at d 4.20 ppm.Compounds 8a-c were hydrolysed using sodium bicarbonate in methanol\water (1:1) to produce the acid derivatives 9a-c.IR spectra of compounds 9a-c lacked the absorption bands of the carbonyl groups of the starting compounds.Moreover, a broad band characteristic to carboxylic (OH) appeared at 3000-3600 cm À1 in addition to an absorption band assigned for acidic carbonyl function observed at 1598-1732 cm À1 and an absorbing band for OH function was obtained at 3403-3448 cm À1 . 1 H-NMR spectra for compounds 9a-c revealed absence of triplet and quartette signals corresponding to C 2 H 5 of ester and singlet attributed to CH 3 of acetyl group.Instead, spectra showed two D 2 O exchangeable signal resonated at downfield corresponding to 2OH protons at range of d 9.77-9.84and 13.10-13.22ppm. 13C-NMR spectrum for compound 9a showed appearance of highly deshielded signal corresponding to C¼O for acid at d 170.09 ppm.EI/MS for 9b showed [M þ þ2] at m/z 518, molecular ion peak [M þ ] at m/z 516 and base peak at m/z 413.Furthermore, the intermediates 7a-c were alkylated with N-aryl-2-chloroacetamides in the presence of anhydrous potassium carbonate in dry acetone to yield the intermediates 10a-i which were further deprotected by sodium bicarbonate in aqueous methanol (1:1) to prepare the final compounds 11a-i.IR spectra of the target compounds 10a-i displayed stretching absorption bands for amide NH and C¼O functions at 3407-3447 and 1667-1680 cm À1 , respectively.The synthesis of the target pyridine-quinolone hybrids was outlined in Scheme 3.This scheme discussed the chemical pathway used to prepare quinolone aldehydes 12a-c via hydrolysis of 2chloro-6-substitutedquinoline-3-carbaldehydes 3a-c which were prepared by the action of Vilsmeier's reagent on acetanilide derivatives 2a-c 25 .Those target quinolone aldehydes 12a-c were reacted with 3-hydroxyacetophenone, ammonium acetate and ethyl cyanoacetate or cyanothioacetamide or malononitrile to give the corresponding 3-cyanopyridones 13a-c or 3-cyanothiopyridines 14a-c, or 3-cyano-2-aminopyridines 15a-c, respectively.The IR spectra of compounds 13a-c showed absorption bands at 3304-3400 cm À1 attributed to NH, in addition to absorption bands at 2217-2221 cm À1 and 1645-1647 cm À1 assigned to CN and C¼O functions, respectively.Moreover, IR of compounds 14a-c showed characteristic SH band at 2540-2600 cm À1 .Compounds 15a-c displayed the presence of two strong bands of asymmetrical and symmetrical stretching frequency of NH 2 function at 3380-3418 and 3290-3356 cm-1, respectively.The 1 H-NMR spectra of the all-synthesised compounds showed a methine proton as a deshielded singlet peak at the downfield region d 6.76-7.50ppm.In addition, 1 H-NMR spectra of compounds 14a-c showed highly deshielded D 2 O exchangeable SH proton appeared at a range d 14.17-14.25 ppm which confirm the presence of all derivatives in thiol tautomer, while compounds 15a-c showed D 2 O singlet for two protons corresponding to NH 2 protons at d 6.81-6.94ppm. 13C-NMR spectra showed signals for CN and highly deshielded signal corresponding to C¼O, C¼S or C-NH 2 determined to be in accordance with the recommended molecule structures.EI-MS of 13c and 14a showed [M þ ] peak at 385 and 371, respectively.

Cytotoxicity screening
Cytotoxicity test is one of the biological evaluations and screening tests that use human normal and cancer cells in-vitro to observe the effect of the studied compounds on their growth using MTTbased cytotoxicity.Microculture MTT method 26  against normal human lung fibroblast (Wi-38) using doxorubicin (Dox) as standard anticancer drug.The highest IC 50 and EC 100 values illustrate that the compounds were safe on the proliferation of normal human cells.All the tested compounds showed significant noncytotoxic effect on human lung fibroblasts (Wi-38) with IC 50 ranging from 0.0255 to 0.759 mM compared to doxorubicin (IC 50 ¼ 0.0266 mM).Compounds 5b, 5c, 6a-e, 7a-c, 9a-c, 11a-d, 11f-i, 14a-c, and 15a-c showed the highest IC 50 values > 0.099 mM which revealed their safety on normal human cells (Table 1 in Supplementary Material, Figure 4).

In-vitro anticancer screening
All final compounds were evaluated against four human cancer cell lines namely; liver cancer (HepG2) (ATCCV R HB-8065 TM ), colon cancer (Caco-2) (ATCC V R HTB-37 TM ), leukaemia cancer (NFS-60) (ATCC V R CRL-1838 TM ), and prostate cancer (PC-3) (ATCC V R CRL-1435 TM ) using doxorubicin (Dox) as the reference drug being widely used in tumour management utilising microculture MTT assay, declared by Mosmann 26 (Table 1 in Supplementary Material, Figures 5-8).The provided data revealed that most of the tested compounds exerted remarkable broad antitumor activity against all tested cancer cell lines.
As a result, it can be concluded that pyridone moiety is important for high anticancer activity Moreover, compounds bearing 4-morpholinyl moiety are more crucial than that having 1-piperdinyl moiety.Therefore, new compounds were designed to keep morpholinyl À 2-oxopyridine scaffold basic structure with various substitutions (Scheme 3) aiming to potentiate the anticancer activity and hence their PIM kinase inhibitory activity.Compounds 7a-c were designed to acetylate 3-hydroxyphenyl group to study the effect of free OH on the activity.Such structural modification was found to abolish the anticancer activity compared with the hydroxyl derivatives 5a-c which confirmed the importance of the free hydroxyl group for maintaining high anticancer activity.Our study was excreted to include carboxylic acid moiety on 2-oxopyridine aiming to increase its solubility and hence its bioavailability.Such structural modification doesn't possess superior anticancer activity.On the other hand, compound 9b showed low anticancer activity but with equipotent with the reference drug against HepG-2 cell line with IC 50 ¼ 0.0454 mM.The O-(N-aryl acetamide) derivatives 11d, 11e, and 11f showed potent anticancer activity even more potent than doxorubicin against HepG-2 cell line with IC 50 ¼ 0.0458, 0.0416 and 0.0363 mM, respectively (Figure 6).Those compounds showed reasonable anticancer activity against NFS-60, PC-3 and Caco-2 cell lines with IC 50 < 0.1 mM (Figures 5, 7, and 8).This activity may be due to an increased lipophilicity (6-chloroquinoline) that might enhance the binding pattern to protein hydrophobic moieties.Moreover, compound 11i showed nearly equipotent activity to the reference drug against HepG-2 cell line with IC 50 ¼ 0.0486 mM.Finally, compounds 11b, 11g, and 11h displayed moderate anticancer activity against the four cancer cell lines.Another modification was performed through tethering the pyridine moiety with quinolone to study their effect on anticancer activity (Scheme 3).The results indicated that most of the tested compounds 13a-c, 14a-c, and 15a-c exerted potent broad antitumor activity against all tested cancer cell lines.Especially, the unsubstituted pyridone derivative 13a that displayed extraordinary increase in anticancer activity.It was twenty-one-fold more active against HepG-2 cell line (IC 50 ¼0.00238lM) and with threefold against NFS-60 cell line (IC50 ¼ 0.009 lM) compared to the reference (IC 50 ¼0.0504and 0.0332 lM, respectively) (Figures 5-8).In addition, 6-methoxy derivative 13c exhibited thirteen-fold high in the activity against HepG-2 cell line and with three-fold high against NFS-60 cell line.Furthermore, the 6-chloro derivative 13b was equipotent activity with the reference drug against HepG-2 and NFS-60 cell lines with IC 50 ¼ 0.0309, 0.03373 lM, respectively, and with pronounced anticancer activity against PC-3 and Caco-2 cell lines.In addition, replacing of 2-oxo group on pyridine ring with 2-mercapto function 14a-c resulted in slightly decreasing in the anticancer activity.It was found that, the unsubstituted derivative 14a was the most active one against HepG-2 and NFS-60 cell lines with IC 50 ¼ 0.0127and 0.0232 lM, respectively, and showed moderate activity against PC-3 and Caco-2 cell lines.Moreover, the 6-methoxy derivative 14c possessed anticancer activity against HepG-2 with IC 50 ¼0.0557lM, in a manner that is equipotent to doxorubicin.On the other hand, a dramatic decline in the anticancer activity was recorded with replacement of 2-mercapto group with 2-amino group against the four cancer cell lines.Furthermore, the SI results (Table 1 in Supplementary Material) revealed that, compounds 13a, 13c, and 14a showed the highest SI values than the reference drug doxorubicin.They were the least cytotoxic and highest anticancer activity with high SI value compared to the Dox.These results indicated a successful discovery of new anticancer drug candidates and confirmed that they could be safe drugs against tumours.Additionally, morphological changes on the four cancer cell lines (HepG-2, Caco-2, NFS-60 and PC-3) were demonstrated before and after treatment with the most active and safe compounds 13a, 13b, and 14a in comparison with cells treated with the reference doxorubicin.those compounds caused severe shrinking and collapsing in the normal shape of four studied cancer cell lines as shown in Figure 9.

Flow cytometric analysis of apoptosis
Flow cytometric annexin V/propidium iodide analysis 37 was used for detecting the apoptotic effects of the most active and nontoxic anticancer compounds 5b, 5c, 6e, 13a, 13c, and 14a.Results (Figures 10 and 11 and Table 1) showed that all tested compounds except 5c possessed apoptosis-dependent death by over 42% in the treated HepG-2, Caco-2, NFS-60, and PC-3 cancer cell lines higher than doxorubicin which showed less than 39% apoptotic cell.It is worth mentioning that, compounds 6e, 13a, and 13c exerted the highest percentage of annexin-stained population cells (>66%) in comparison with the other most effective studied compounds.

Caspase 3/7 activation assay
It's a fluorescence assay that measures caspase-3 and À7 activities.Caspase 3/7 activators are well known as apoptotic inducers.In view of this, the apoptotic induction mechanism displayed by the most active compounds 5b, 5c, 6e, 13a, 13c, and 14a was explained by estimating the fold increase in caspase 3/7 activation in the treated cancer cell line relative to the untreated HepG-2 cells.Results (Table 2) revealed that compounds 6e and 13a exhibited the highest caspase 3/7 up regulation by more than 3 folds.In addition, compounds 5b, 5c, and 13c significantly induced caspase 3/7 activation by more than two folds in HepG-2 cancer cell lines.

In-vitro PIM-1 and PIM-2 kinase inhibitory activity
The active synthesised compounds 5b, 5c, and 6e, were evaluated for their PIM-1 and PIM-2 kinase inhibitory activities using Quercetin, as reference drug (Table 3).All compounds showed potent PIM-1 kinase inhibitory activity comparable to the reference quercetin.The PIM-2 kinase inhibitory activity results showed that, compound 5b showed the highest inhibitory activity among other compounds compared to quercetin.While compound 5c and 6e excreted slightly less activity than the reference.Therefore, PIM-1 kinase inhibitory activities were tested for the other active synthesised compounds 13a, 13c, and 14a using Quercetin, as reference drug (Table 3).Compounds 13a and 14a showed potent PIM-1 kinase inhibitory activity.

Mechanism of PIM-1 kinase inhibition
To investigate the mechanism of inhibition of the most active compounds 5b, 5c, 6e, 13a, and 14a on PIM-1 kinase activity, enzyme kinetic parameters (maximum activity "V max " and substrate concentration at half V max "K m ") were assessed by Lineweaver-Burk double-reciprocal plot.As it was shown in this Figures 12 and 13.Lineweaver-Burk double-reciprocal plot for compounds 5b, 5c, 6e, and 14a showed the same Y-intercept with the control and quercetin (the same V max in comparison with control while the X-intercept has the negative sign (right to the control) which indicated an increasing in K m value from 7.24 mM (in case of control) to 14.16, 24.69, 27.84, 8.92, and 19.59 mM, for 5b, 5c, 6e, 14a, and quercetin.respectively.These results indicated that, those compounds are competitive inhibitors for PIM-1 kinase enzyme.In addition, the highest K was recorded in 6e and 5b comparing to 5c, 14a and reference PIM kinase inhibitor (quercetin) indicates the strongest inhibition effect of 6e and 5b via lowering the binding affinity of PIM kinase to its substrate by more than three folds.On the other hand, compound 13a revealed V max at a higher position on the axis (reduced in V max value) and also has the negative sign on the X-intercept (right to control) which indicated increase in K m value therefore, this compound is both competitive and non-competitive inhibitor for PIM-1 kinase enzyme.

Structure activity relationship
The obtained results explained that most of the tested compounds exerted remarkable broad antitumor activity against all tested cancer cell lines.Compounds 5a, 5b, 5c, and 5d that contain pyridone moiety hybridised with quinoline derivatives and compound 6e which comprises thiopyridine moiety showed the most potent activity against Caco-2 cell line superior to doxorubicin which confirm the importance of the presence of 2-oxo or 2thiopyridine core in the structure.Also, compounds 5b and 5c exerted the highest anticancer activity against all tested human cancer cell lines, correlated to other compounds and doxorubicin.Therefore, compounds containing morpholino moiety showed higher activity compared to piperdine-1-yl moiety.Except for, compound 6e which substituted with 2-piperidine and 6-methoxy group on quinoline ring demonstrated also higher anticancer activity against Caco-2 and HepG-2 than doxorubicin and nearly equipotent activity against NFS-60 and PC-3 cell lines.Finally, substitution at 6-position of quinoline with either lipophilic chloro group or hydrophilic methoxy group showed higher activity rather than unsubstituted one.On the other hand, acetylation of 3hydroxyphenyl group was found to abolish the anticancer activity compared with the hydroxyl derivatives 5a-c which confirmed the importance of the free hydroxyl group for maintaining high anticancer activity.Furthermore, SAR study on different alkyl group on 2-oxopyridine revealed that, insertion of N-aryl acetamide showed potent anticancer activity rather than presence of carboxylic acid group which may be due to an increased lipophilicity that might enhance the binding pattern to the enzyme active site by hydrophobic interaction.In addition, linking pyridine to the electron deficient quinolone moiety achieved the goal and exhibited broad in-vitro antitumor activity against all tested cancer cell lines.Moreover, pyridone derivatives (13a and 13c) and thiopyridine derivative 14a showed the highest in-vitro anticancer activity against HepG-2 and NFS-60 cell lines superior to reference doxorubicin.Also, compounds 13a and 14a revealed potent PIM-1 kinase competitive inhibitory activity.These findings confirmed the importance of substitution at position 2 of pyridine with either oxo or thioxo rather than amino group in anticancer and PIM-1 kinase inhibitory activity.Besides, substitution at position 6 of quinolone with hydrophilic methoxy group or unsubstituted derivatives showed the highest activity rather than the hydrophobic chloro derivatives which could be due to increased polar interaction between the quinolone and PIM-1 kinase hinge region.

Ligand efficiency (LE)
A correlation between the potency of a compound (IC 50 ) and the number of its heavy atoms (non-hydrogen atoms) is called Ligand Efficiency.It is a measurement of the binding energy which related to the potency of the compound (pIC 50 ) with consider its molecular size 38 .The suggested LE value for lead-likeness should be in the range of 0.3 while the acceptable LE value for drug-likeness should be higher than 0.3 (Table 4).

Ligand lipophilic efficiency (LLE)
LLE is a criterion that evaluate the potency in terms of lipophilicity.In other words, it measures the binding of a ligand to a given target in respect to its lipophilicity 38 .LLE value should be !3 for lead compound to be acceptable and recommended to be LLE value ! 5 for drug like candidate (Table 4).
According to the results, compounds 5b and 5c obeyed acceptable ranges of LE (>0.3) and LLE values (> 3).Therefore, these compounds fulfilled the criteria of lipophilicity and ligand efficacy to be lead-like compounds.On the other hand, compounds 13a, 13c, and 14a showed drug like criteria (LE >0.3, LLE >5).Therefore, these compounds could impart improved potency along with convenient lipophilicity which make these compounds promising drug-like candidates in anticancer drug discovery.

Docking with PIM-1 and PIM-2 kinases
The aim of this section in our study is to provide in-silico insights to rationalise the observed in-vitro PIM-1 and PIM-2 inhibition activity based on our design approach.
For this we performed molecular docking experiments utilising the high-resolution X-ray structures for PIM-1 and PIM-2 kinases, namely, PDB IDs: 2OBJ and 4X7Q, respectively.To validate the docking setup, a pose-retrieval docking experiment was conducted for the co-crystal ligand against its respective X-ray coordinates.All pose-retrieval experiments were successful (Figures 14A and 16 Concerning PIM-1 kinase, the docking score distribution (Table 5) exhibits narrow range of scores for 5b, 5c, and 6e.This indicates comparable in-silico affinity which came in coherence with the observed in-vitro inhibitory activities (IC 50 range: 1 À 1.31 mM) against PIM-1 kinase.However, the score distribution of the series of 13a, 13c, and 14a displayed superior scores compared to the 5 and 6 candidates, as well as the reference quercetin.One explanation for this is the topological differences between both series that affects pose orientations (as discussed later).It is obvious that 14a exhibited the best score among all compounds which agrees with the observed in-vitro inhibitory activities (14a is the best active one).
Regarding PIM-2 kinase, the docking score of 5b and Quercetin indicate superior in-silico binding compared to 5c and 6e, as shown in Table 5.Again, this observation is in consistency with the measured in-vitro data where both 5b and Quercetin are the most active against PIM-2 kinase with a sub-micromolar range of activity compared to 5c and 6e (low micromolar range).It is worth mentioning that 5c, 6e, and Quercetin (except 5b) display superior docking score for PIM-1 compared to PIM-2 rationalising their observed in-vitro activities.The docking experiments for 13a, 13c, and 14a against PIM-2 were omitted due to the lack of PIM-2 inhibitory activity data.
Based on our analysis on the crystal PIM-1 protein structure, a structural water molecule appears to mediate a H-bonding bonding interaction of the pyridone scaffold of the co-crystal ligand with the side chain of Glu89.Therefore, we kept the structural water molecule in our docking setup.The pose retrieval of the cocrystal ligand was successful implying an acceptable accuracy of the docking setup.
Although both the co-crystal ligand and the selected candidates (5b, 5c, and 6e) share pyridone/thiopyridine moiety, the docking pose of the core pyridone scaffold of the selected candidates showed a flip towards Glu171.This is attributable to the topology difference between the co-crystal ligand which is 4,6-   diaryl pyridone derivative and our quinolinylpyridone/thiopyridine hybrids.
Elucidating the docking poses of 5b, 5c, and 6e, they all produce comparable interactions in the binding site of PIM-1 kinase, as seen in Figure 14B.For instance, the core pyridone/thiopyridine demonstrate a favourable hydrophobic interaction with Glu171.Besides, the hydroxyphenyl group shows H-bonding interaction with the backbone of Phe49, e.g. as seen in Figure 14C for 5c.The methoxy/chloro quinoline moiety is well packed in the hydrophobic area (green area in Figure 14) indicating hydrophobic interactions with Leu174 and Leu44.Interestingly, the morpholine/ piperidine moiety appeared to be partially included in a hydrophobic interaction with Ile104 and Ala45, and partially solventexposed.Overall, these findings exhibited comparable docking score for 5b, 5c, and 6e, as shown in Table 5.These results are consistent with observed in-vitro inhibitory activity against PIM-1 kinase since these compounds exhibited comparable IC 50 values.On the other hand, the postulated binding poses of the hybrids of pyridone/thiopyridine with 2-quinolone derivatives, namely 13a, 13c, and 14a, displayed flipped poses compared to 5 and 6 candidates, as exhibited in Figure 15.This is attributable  to the differences of topology and substitution pattern between both series.For instance, unlike 5b, 5c, and 6e poses, the pyridone/thiopyridine poses of 13a, 13c, and 14a point towards the structural water molecule (Figure 15A).This orientation enables the 2-quinolone moiety to have the advantage of making H-bonding interactions with the side chain of Asp128, and to be packed between the hydrophobic side chains of Val126 and Leu174 implying favourable hydrophobic interactions (Figure 15A,B).Likewise, the pyridone/thiopyridine moiety of 13a, 13c, and 14a is packed between the hydrophobic side chains of Val52 and Ile185.Overall, this postulated orientation produced superior in-silico binding particularly for 13a and 14a, as observed in Table 5.
The postulated binding pose of 5b illustrates favourable interactions in the binding site of PIM-2 kinase as shown in Figure 16(B,C).The pyridone scaffold demonstrates H-bonding interaction with the side chain of Asp182 and hydrophobic interaction with the side chain of Ile181.The chloroquinoline moiety is packed in the hydrophobic area (green area in Figure 16B) formed by Leu38, Ala59, Val46 and Asp124, while the morpholine moiety is directed towards the polar area (red area in Figure 16B) formed by the side chains of Glu167 and Lys40.The hydroxyphenol group appeared to show hydrophobic interactions with the side chains of Val46.Like 5b, the docking poses of 5c and 6e showed similar interactions in the binding site of PIM-2 kinase.Interestingly, the chloroquinoline moiety of 5b appeared to be well packed in the hydrophobic area avoiding the possible steric bumps of the bigger methoxyquinoline moiety of the congeneric 5c and 6e.Globally, these observations contribute to the superior score of 5b towards the binding site of PIM-2 kinase compared to 5c and 6e.

In-silico physicochemical predictions
The purpose of this section is to deliver hints about the drug-likeness and pharmacokinetic properties of the active compounds 5b, 5c, and 6e.Explicating these properties can be supportive in the context of medicinal chemistry.Typically, a molecule that fails to fulfil the drug-likeness standards also fail as a clinical candidate due to poor bioavailability, adverse effects or other concerns.We employed the automated SwissADME 36 for the pharmacokinetics and drug-likeness predictions.The evaluations against passive human gastrointestinal absorption (GIA) and blood-brain barrier (BBB) permeation were extracted from the BOILED-Egg model 36,39 , which represent some parameters for the pharmacokinetics predictions.The compounds presented high gastrointestinal absorption except 6e and 14a, as seen in Table 6.Also, these molecules were suggested to fail to permeate through BBB, and hence, they are expected to show low incidence for central nervous system (CNS) adverse effects.Permeability glycoprotein (P-gp) is proposed to be the most important member among ATP-binding cassette transporters or ABC-transporters 36 since it is part of the resistance mechanisms against drugs 36,40 .5b, 5c, and 6e molecules were foreseen to be substrate for the P-gp which introduce a chance of being resistant against the human biological membranes.However, 13a, 13c, and 14a were predicted to escape the P-gp indicating low incidence of developing resistance against human biological membranes.
Commonly, 50-90% of therapeutic molecules are judged to be a substrate of at least one of the five major isoforms of Cytochrome P (CYP) enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4) 36,42,43 .Inhibition of these isoenzymes is unquestionably one cause of pharmacokinetics-related drug-drug interactions leading to toxic or adverse effects 44,45 .As shown in  Table 6, all molecules were predicted to display inhibition of at least one of the CYP isoforms, with exception of 13a.This recommends administering these candidates in a sole regime and not in combination with other therapeutic agents.Fortunately, all molecules showed no alert to be a possible PAINS (pan-assay interference compounds) 46 , as shown in Table 6.This stresses that their chemical structures would not interfere in protein assays denoting the in-vitro results to be robust with minimum artefacts.Lastly, drug-likeness properties underlined that all molecules do not display violations for the Lipinski and Veber rules 47 highlighting that these candidates would demonstrate good bioavailability profiles.

Conclusion
New pyridine-quinoline hybrids were designed and synthesised as PIM-1 kinase inhibitors.They were evaluated against four human cancer cell lines namely; HepG2, Caco-2, NFS-60, and PC-3 using Dox as the reference.Compounds 5b, 5c, 13a, 13c, and 14a showed the highest anticancer activity against all tested human cancer cell lines, correlated to other compounds and doxorubicin.Also compound 6e demonstrated higher anticancer activity against Caco-2 and HepG-2 compared to Dox and nearly equipotent activity against NFS-60 and PC-3 cell lines.On the other hand, flow cytometric annexin V/propidium iodide analysis observed that, compound 6e revealed the highest percentage of annexin-stained population cells (>67%) in the four tested cancer cell lines in comparison with the other most effective studied compounds.In addition, caspase 3/7 activation assay elicited that, all tested compounds significantly induced caspase 3/7 activation in HepG-2 cancer cell lines.Furthermore, compounds 5b, 5c, 6e, 13a, and 14a showed potent PIM-1 kinase inhibitory activity comparable to the reference quercetin.Besides, kinetic studies using Lineweaver-Burk double-reciprocal plot for the most active compounds on PIM-1 kinase proved that, compounds 5b, 5c, 6e, and 14a and quercetin behaved as competitive inhibitors for PIM-1 kinase.While, compound 13a was both competitive and non-competitive inhibitor of PIM-1 kinase enzyme.In addition, molecular modelling study indicated that, compounds 5b and 5c fulfilled the criteria of lipophilicity and ligand efficacy to be lead-like compounds.Moreover, drug-likeness properties underlined that all molecules do not display violations for the Lipinski and Veber rules highlighting that these candidates would demonstrate good bioavailability profiles.Molecular docking studies indicated that, in-silico affinity came in coherence with the observed in-vitro inhibitory activities against PIM-1/2 kinases.

Figure 1 .
Figure 1.A schematic representation of pyridone derivative I complexed with PIM-1 kinase.Dashed lines indicated hydrogen bonds.

Figure 9 .Scheme 2 .
Figure 9. Morphological alterations of the most active compounds-treated cancer cells lines in comparison with the untreated cancer cells.
(A) with RMSD value < 2 Å indicating high docking accuracy for the docking setup for pose prediction purposes.

Figure 10 .
Figure 10.Flow charts of Annexin-PI analysis of 5b, 5c, and 6etreated cancer cell lines in comparison with the untreated cancer cells.

Figure 11 .
Figure 11.Flow charts of Annexin-PI analysis of 13a, 13c, and 14atreated cancer cell lines in comparison with the untreated cancer cells.

Figure 14 .
Figure 14.(A) Overlay of the X-ray co-crystal (simon sticks) on its docked pose (cyan sticks) in the binding site of PIM-1 kinase (PDB ID: 2OBJ).(B) Overlay of the docking poses of 5b, 5c, and 6e as green, cyan, and magenta sticks, respectively, in the binding site of PIM-1 kinase.(C) Interaction pattern of 5c with PIM-1 residues in 2D depictions.Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively.Dashed lines indicate favourable interactions.Non-polar hydrogen atoms were omitted for clarity.

Figure 15 .
Figure 15.(A) Overlay of the docking poses of 13a, 13c, and 14a as magenta, green, and cyan sticks, respectively, in the binding site of PIM-1 kinase.(B) Interaction pattern of 13a, 13c, and 14a with PIM-1 residues in 2D depictions.Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively.Dashed lines indicate favourable interactions.Non-polar hydrogen atoms were omitted for clarity.

Figure 16 .
Figure 16.(A) Overlay of the X-ray co-crystal (grey sticks) on its docked pose (cyan sticks) in the binding site of PIM-2 kinase (PDB ID: 4X7Q).(B) Overlay of the docking pose of 5b, 5c, and 6e as green, cyan, and magenta sticks, respectively, in the binding site of PIM-2 kinase.(C) Interaction pattern of 5b with PIM-2 residues in 2D depictions.Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively.Dashed lines indicate favourable interactions.Non-polar hydrogen atoms were omitted for clarity.
was used to determine cytotoxic activity of the newly synthesised compounds Figure 5.In vitro anticancer activity, IC 50 (lM), of the tested compounds against Caco-2.Figure 6.In vitro anticancer activity, IC 50 (lM), of the tested compounds against HepG-2.

Table 1 .
The total percentage of the apoptotic cell population in the most effective compounds-treated cancer cells lines.All values were expressed as mean ± SEM.

Table 2 .
Illustrates relative fold increase in caspase activity by the most effective compounds relative to untreated HepG2 cancer cells.All values were expressed as mean ± SEM.

Table 3 .
In-vitro PIM-1 and PIM-2 kinase inhibition data of the most active compounds.
aThe docking score of FRED.b ND stands for not determined.