Development of newly synthesised quinazolinone-based CDK2 inhibitors with potent efficacy against melanoma

Abstract Inhibiting Cyclin-dependent kinase 2 (CDK2) has been established as a therapeutic strategy for the treatment of many cancers. Accordingly, this study aimed at developing a new set of quinazolinone-based derivatives as CDK2 inhibitors. The new compounds were evaluated for their anticancer activity against sixty tumour cell lines. Compounds 5c and 8a showed excellent growth inhibition against the melanoma cell line MDA-MB-435 with GI% of 94.53 and 94.15, respectively. Cell cycle analysis showed that compound 5c led to cell cycle cessation at S phase and G2/M phase revealing that CDK2 could be the plausible biological target. Thus, the most cytotoxic candidates 5c and 8a were evaluated in vitro for their CDK2 inhibitory activity and were able to display significant inhibitory action. The molecular docking study confirmed the obtained results. ADME study predicted that 5c had appropriate drug-likeness properties. These findings highlight a rationale for further development and optimisation of novel CDK2 inhibitors.


Introduction
Cancer prevalence and mortality are promptly increasing worldwide. The growing prominence of cancer; particularly with fast population growth and ageing in many parts of the world partially reveals a marked decline in mortality rates caused by coronary heart disease and stroke, with respect to cancer. Accordingly, cancer rapidly emerges to be the principal cause of death worldwide 1 .
Cyclin-dependent kinases (Cdks) are serine/threonine kinases. The mammalian cells contain 13 CDKs, among them CDK1, CDK2, CDK3, CDK4, and CDK6 which are involved in regulating the cell cycle checkpoints via initiation of proteinaceous substrates phosphorylation, using adenosine triphosphate (ATP) 2,3 . Abnormalities in the cell cycle regulation induce unrestricted cell growth, which is usually considered a hallmark of cancer 4 . Disruption of a deregulated cell cycle, as a result of overactivation of a CDK, has been accepted as a strategy for the treatment of proliferative diseases, especially cancer 5 .
Cyclin-dependent kinase 2 (CDK2) is a key regulator of the cell cycle transition and progression. CDK2 performs a critical role in controlling various events of the cell division cycle, such as DNA repair, gene transcription, G1-S transition, and modulation of G2 progression [6][7][8] . Additionally, the altered expression of cyclins whose association with CDKs is essential for their catalytic activity can drive aberrant proliferation in cancer. In the same vein, the overexpression of CDK2 regulatory subunits cyclin A and/or E is a key oncogenic process in many types of cancers 9,10 . Owing to the crucial role of CDK2 in cell cycle regulation, transcription activity, and tumours epigenetic modifications, CDK2 inhibition may confer a therapeutic benefit against certain cancers viz breast cancer 11 , colo-rectal cancer 12,13 , glioblastoma [14][15][16] , and melanoma 17,18 .
Several potent CDK2 inhibitors are under clinical evaluation, such as dinaciclib (I) 19 (an inhibitor of several CDKs), milciclib (II) 20 , and roniciclib (III) 21 (Figure 1). Nevertheless, some adverse effects have been observed during clinical trials, revealing that the discovery of new CDK2 inhibitors is still an active field of research 22 .
In the course of finding several chemical substances which may serve as leads for developing new anticancer agents, we are particularly interested in the present study with quinazolinone derivatives in an attempt to achieve novel CDK2 inhibitors serving as chemotherapeutic agents. Continuing our studies on quinazolinone analogues as attractive candidates as antitumor agents 30,31 , we have designed some new quinazolinone derivatives containing phenoxy methyl group at position 2 and bearing different moieties which are known to enhance the antitumor activity attached to the 3-amino group viz un/substituted phenyl acetamides 32 , un/ substituted benzene sulfonamides 33 , benzylidenes 34 , and acetyl   (Figure 1). In this work, the substitution pattern at the 2 and 3 positions of the quinazolinone scaffold was selected to provide a different electronic environment that would affect the lipophilicity, and hence the activity of the target molecules. The aim of constructing these congeners is an attempt to attain active CDK2 inhibitors with potentiated activity against cancerous cells in which CDK2 is overexpressed.
All the newly synthesised quinazolinones were subjected to in vitro anticancer screening by the National Cancer Institute (USA) against 60 different human cell lines. The most potent compounds 5c and 8a were selected to be further studied through the determination of their half-maximal inhibitory concentration (IC 50 ) values against melanoma MDA-MB-435 (5c and 8a) and glioblastoma SNB-75 cell lines (5c). To investigate the mechanistic pathways of the anticancer activity of the most active representatives of this class, they were subjected to in vitro CDK2 inhibition assay. Furthermore, the effect of 5c on the induction of apoptosis in MDA-MB-435 and SNB-75 cell lines was also investigated using flow cytometry and molecular docking study was carried out for the most active compound 5c to identify the binding features required for its antitumor activity.

General
Starting materials and solvents were purchased from commercial sources and were used without further purification. Melting points were determined by open capillary tube method using Electrothermal 9100 melting point apparatus (Staffordshire, UK) and were uncorrected. Elemental analyses and Mass spectra were carried out at the Regional Centre for Mycology and Biotechnology, Al-Azhar University, Egypt. Mass spectral data were given as m/z (relative abundance %). NMR and Infra-red spectra were performed at the Microanalytical Unit-Faculty of Pharmacy, Cairo University. NMR spectra were recorded on a Bruker Ascend 400/R ( 1 H: 400, 13 C: 100 MHz) spectrometer and were run in deuterated chloroform (CDCl 3 ) or deuterated dimethylsulphoxide (DMSO-d6). Chemical shift values (d) were given in parts per million (ppm). Coupling constants were given in hertz (Hz) and spin multiplicities were given as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), or m (multiplet). Infra-red spectra (IR) were recorded as Potassium bromide discs on Schimadzu FT-IR 8400S spectrophotometer (Kyoto, Japan) and expressed in wavenumber (t max , cm À1 ). TLC was performed on TLC plates UV fluorescent silica gel (60 FZSU), using chloroform: methanol 9:1 as the eluting system, and the spots were visualised by the ultraviolet lamp. The starting compounds 1 36 , 2 37,38 , and the key intermediates 3 37,38 and 7 37,38 were prepared as reported.
2.1.2. General procedure for the synthesis of compounds 4a-c and 5a-c A mixture of equimolar amounts of the amino derivative 3 and the appropriate 2-chloro-N-phenylacetamide or p-substituted benzenesulfonyl chloride in dry DMF (10 mL) containing anhydrous potassium carbonate (1.5 mmol, 0.2 g) were refluxed for 20 h. The mixture was poured over crushed ice and the precipitated solid was filtered off, washed with water, dried, and recrystallized from the suitable solvent.

General procedure for the synthesis of compounds 6a and 6b
A mixture of equimolar amounts of compound 3 and the corresponding aromatic aldehyde was refluxed in glacial acetic acid (15 ml) for 8 h. The reaction mixture was poured over crushed ice and the precipitated solid was filtered off, washed with water, dried, and recrystallized from ethanol affording 6a and 6b, respectively.  2.1.4. General procedure for the synthesis of compounds 8a-c Equimolar amounts of the chloro derivative 8 and the appropriate piperazine in dry DMF (10 ml) containing anhydrous potassium carbonate (1.5 mmol, 0.2 g), were refluxed for 20 h. The reaction mixture was cooled, poured over crushed ice and the yielded solid was crystallised from methanol to give the corresponding 8a-c.

Biological assays
All experimental procedures applied in the mentioned biological assays were conducted as reported earlier; NCI-60 human tumour cell lines screening [39][40][41] , determination of IC 50 in MDA-MB-435 and SNB-75 cell lines 42,43 , Cell cycle analysis 44 , Apoptosis determination 45 , CDK2 inhibition assay 46 and have been provided in the Supplementary Materials.

Statistical analysis
The presented data and results were confirmed by at least three independent experiments. The data have been expressed as means ± SD and analysed with GraphPad Prism 8.0 software. Scheme 1. Preparation of the key starting compound 3 and the target compounds 4a-c, 5a-c, and 6a,b. Reagents and solvents: (a) Methyl anthranilate, dry benzene, (b) Hydrazine hydrate, n-butanol, (c) appropriate 2-chloro-N-phenylacetamide, dry DMF, anhydrous K 2 CO 3 , (d) p-substituted benzenesulfonyl chloride, dry DMF, anhydrous K 2 CO 3 , (e) cinnamaldehyde or vanillin, glacial acetic acid.
Statistical comparisons were made by Student's t-test. p-Values <0.05 (p < 0.05) was considered statistically significant.

Molecular docking study
The molecular docking study of the most active compound was achieved using Molecular Operating Environment (MOE, 2010.10) software. The X-ray crystal structure of CDK2 enzyme co-crystallised with the quinazoline derivative DIN-234325 as an inhibitor (PDB code: 2B53) was downloaded from the protein data bank 25,47 . The enzyme was prepared for docking by removal of all ligands and water molecules that are not involved in the binding. One conserved water molecule involved in the co-crystalized ligand binding to the CDK2 binding site was kept ( Figure 5). The protein structure was prepared for docking study using Protonate 3 D protocol in MOE with default options. The Triangle Matcher placement method and London dG scoring function were used for the evaluation of the binding pattern and binding affinity of the ligands. Docking protocol was first validated by redocking of the co-crystallised ligand (DIN-234325) in the active site of the enzyme with an energy score (S) ¼ À18.24 kcal/mol and root mean square deviation (RMSD) of 0.485 Å. The validated docking protocol was then used to study the ligand-target interactions in the active site for the most potent newly synthesised compounds 5c to predict its binding mode and binding affinity to justify its promising potency.

Drug likeness properties
The free Swiss ADME web tool available from the Swiss Institute of Bioinformatics (SIB) was used for the calculation of the physicochemical descriptors as well as to predict the ADME parameters, pharmacokinetic properties, and drug-like nature of the most active newly synthesised candidate 5c [48][49][50] . The SMILES of the tested compound were inserted directly on the webpage followed by running the prediction process.

Chemistry
The target quinazolin-4(3H)-one derivatives were synthesised as outlined in Schemes 1, 2. The starting compounds 1 36 , 2 37,38 , and the key intermediates 3 37,38 and 7 37,38 were prepared according to the reported procedures. As depicted in Scheme 1, stirring of 3-amino-2-(phenoxymethyl)quinazolin-4(3H)-one 3 with the appropriate 2-chloro-N-phenylacetamide or various 4-substituted benzene sulphonyl chloride in dry Dimethyl formamide at 70 C in the presence of anhydrous Potassium carbonate afforded compounds 4a-c and 5a-c, respectively. The chemical structures of the new phenyl acetamide derivatives 4a-c were confirmed by the appearance of an additional singlet signal in 1 H-NMR spectra around 5.30 ppm assigned to the new methylene group. Furthermore, the 1 H-NMR showed characteristic signals in the aromatic region attributed to the additional aromatic protons in compounds 4a-c and 5a-c. 13

NCI-60 human tumour cell lines screening
In this research, all of the newly synthesised quinazolinones were subjected to in vitro anticancer screening by the National Scheme 2. Synthesis of the target derivatives 8a-c. Reagents and solvents: (a) Chloroacetyl chloride, dry DMF, (b) appropriate piperazine, dry DMF, anhydrous K 2 CO 3 .       respectively. Other tested compounds exhibited weak activity against most investigated cell lines. Thus, compounds 5c and 8a were subjected to further testing to determine their IC 50 against the most sensitive cell lines. Furthermore, 5c and 8a were evaluated for their cytotoxic activity in normal fibroblast cells WI-38 to explore the compounds' selectivity towards tumour cells. As shown in Table 4, the tested compounds were found to be more selective to the cancer cells. Interestingly, the quinazolinone 5c showed superior selectivity towards melanoma MDA-MB-435 cell line with the highest selectivity index (SI ¼ 20.63).

Flow cytometric analysis and apoptotic studies
Compound 5c showed the highest antiproliferative activity against MDA-MB-435 and SNB-75 cancer cell lines; consequently, flow cytometric analysis was carried out to study its effect on the cell cycle phases in the same cancer cell lines and thus identify its plausible biological target. Besides, apoptotic activity was measured by Annexin V-based flow cytometric analysis.    Table 5 and Figure 2.
The obtained results revealed that compound 5c led to pre G1 apoptosis with a cell growth arrest at S phase in melanoma MDA-MB-435 cells after 24 h. which was confirmed by an increase in the percentage of DNA content (40.97%) after addition of the compound compared to control cells (34.28%). For glioblastoma SNB-75 cells, the results showed that the quinazolinone 5c induced pre G1 apoptosis with a cell growth arrest at G2/M phase which was confirmed by an increase in the percentage of DNA content (31.78%) after the addition of the compound compared to the to that of the untreated control cells (11.59%). Statistical analysis of the different stages of the cell cycle demonstrated the ability of 5c to induce significant levels of pre G1 apoptosis and cell cycle arrest in S phase and G2/M compared to the control.
Thus, cell cycle arrest induced by the quinazolinone 5c at S phase and G2/M phase suggests that CDK2 could be the biological target for the newly synthesised compounds.

Apoptosis determination.
The apoptotic activity of quinazolinone 5c in melanoma MDA-MB-435 and glioblastoma SNB-75 cell lines was further studied using Annexin V-FITC assay. As shown in Table 6

In vitro CDK2 inhibitory activity
It was reported that CDK2 is overexpressed in melanoma 44,45 . Accordingly, the most potent congeners on MDA-MB-435 the melanoma cancer cell line; 5c and 8a were selected as representatives to study the CDK2 inhibitory activity of the newly synthesised compounds. The quinazolinones 5c and 8a were further investigated in vitro for their kinase inhibitory activity on CDK2 and were compared to the CDK2 kinase inhibitor roscovitine as a reference standard. The results were presented in Table 7. The anti-proliferative activity of the quinazolinone 5c appeared to correlate well with its ability to inhibit CDK2 at sub-micromolar range with IC 50 value 0.63 mM which is twice as potent as roscovitine (IC 50 ¼ 1.28 mM). Additionally, Compound 8a exhibited IC 50 value equal to 1.74 mM which is slightly higher than that of the reference drug.

Molecular docking study
A molecular docking technique was performed to interpret the observed promising activity of compound 5c on CDK2. Therefore, docking of 5c into the three-dimensional X-ray crystallographic structure of the cyclin-dependent kinase2 (PDB code: 2B53) 25 in complex with the quinazoline DIN-234325 (IC 50 ¼ 0.6 ± 0.1 mM) was carried out using Molecular Operating Environment (MOE) 2010.10. The inhibitor binds into the ATP binding pocket located in the deep cleft between the upper and lower domains of CDK2. Through two hydrogen bond interactions, the N1 of the quinazoline ring interacts with the key amino acids Glu81 and Leu83 via a water molecule. It is worth mentioning that the two previously mentioned interactions are consistently observed in the hinge region for ATP-competitive CDK2 inhibitors 54,55 . Stacking interactions were shaped through the quinazoline ring of the ligand and Val18 and Leu134 residues. Additionally, the aniline ring attached at position 6 of the quinazoline ring forms pi staking interaction with Gln 85.
To confirm the accuracy of the docking procedure, validation was performed by re-docking of the co-crystallised ligand into the catalytic domain of CDK2. The validation results revealed that the used docking protocol is suitable for the proposed docking study. This is demonstrated by the perfect alignment between the coordinates of DIN-234325 and its self-docked pose with root mean square deviation (RMSD) of 0.485 Å, docking score (S) of À18.24 Kcal mol À1 . Additionally, the self-docked pose was able to achieve the same binding interactions depicted by the co-crystallised ligand with the main amino acids in the CDK2 binding site (Figures 4, 5).
The outcomes of the molecular docking study showed the binding mode of compound 5c within the binding pocket of CDK2 kinase ( Figure 6; binding score (S) of À13.8 Kcal mol À1 ) indicating that two hydrogen bonds are formed between the quinazolinone moiety and the hinge region. Specifically, the quinazolinone carbonyl oxygen forms two water-mediated hydrogen bonds with the Leu83-NH and Glu81-CO. Moreover, the planar aromatic core makes extensive hydrophobic contacts with Ala31, Phe80, Ile10, Leu134, Val18, and Ala144 in a similar mode to that of the co-crystallised ligand. Additionally, other hydrogenpi interactions were observed between the phenyl rings attached to the C2 and N2 of the quinazolinone ring and Glu12 and Gln85, respectively.

Drug likeness properties
The drug-likeness profile for the most potent candidate 5c was predicted using SwissADME server 48 . The results of the Swiss ADME prediction of drug-likeness of this compound revealed no alerts for Pan Assay Interfering Substances (PAINS) and no violation to Lipinski and other pharmacokinetics filters (for more details see supporting materials, Figure S1). Moreover, the selected quinazolinone 5c showed a predicted log Po/w value in the range of 3.25-4.06 with moderate water solubility, high GIT absorption, and no BBB permeability and thus minimal predicted CNS adverse effects. Furthermore, 5c is not P-glycoprotein substrates (PGPÀ) so it is not liable to the efflux by this transporter and thus avoiding the drug-resistance mechanism used by some tumour cell lines 56 (for boiled egg chart of compound 5c see supporting materials, Figure S2). The oral bioavailability radar chart of compound 5c demonstrated its good predicted oral bioavailability (Figure 7). Collectively the tested compound possesses promising physicochemical and pharmacokinetic properties.

Conclusion
In this study, three series of quinazolin-4(3H)-one derivatives were designed and synthesised as CDK2 inhibitors. All the newly synthesised compounds were screened for their antiproliferative activity against a panel of sixty cancer cell lines. The quinazolinones 5c and 8a displayed excellent growth inhibition against the melanoma cell line MDA-MB-435 (GI% ¼ 94.53 and 94.15, respectively). The quinazolinone 5c also exhibited promising activity against the glioblastoma cell line SNB-75 (GI% ¼ 63.09), while 8a showed potent to moderate activity against leukaemia cell lines SR (GI% ¼ 69.55) and K-562 (GI% ¼ 67.80). Consequently, the IC 50 of 5c and 8a against the most sensitive cell line (MDA-MB-435) in this research work was detected. Compound 5c has emerged as the most active member against MDA-MB-435 with IC 50 value of 3.03 mM which is 1.7-folds more potent than roscovitine, the reference standard (IC 50 ¼ 5.26 mM). Compound 8a also showed good potency on MDA-MB-435 cell line with IC 50 value of 6.22 mM which is comparable to that of the reference drug. The quinazolinone 5c triggered cell cycle arrest in the S phase in melanoma MDA-MB-435 cells and in the G2/M phase in glioblastoma SNB-75 cells suggesting that CDK2 could be the potential biological target for the newly synthesised congeners. Furthermore, compound 5c induced  apoptosis in MDA-MB-435 and SNB-75 cell lines as indicated by the percent of the total apoptosis of 28.65 and 44.22%, respectively in comparison to the positive control, annexin V (% apoptosis 1.91, 2.53). Accordingly, the most active compounds 5c and 8a were tested for their CDK2 inhibitory activities. Interestingly, the outcomes of CDK2 inhibitory assay were consistent with that of the cytotoxicity data. The quinazolinones 5c and 8a displayed CDK2 IC 50 values of 0.63 mM and 1.74 mM, sequentially in comparison with the used reference roscovitine (IC 50 ¼ 1.28 mM). The molecular docking study confirmed the biological results via prediction of the possible binding mode of the most active compound 5c with CDK2 active site indicating its ability to form a pair of crucial hydrogen bonds with the CDK2 hinge region (Leu83 and Glu81). Moreover, the most active compound 5c possessed promising pharmacokinetic properties as depicted by the computational ADME study. These outcomes endorsed that the most active candidates 5c and 8a may serve as useful lead compounds in the search for promising anti-melanoma agents acting through CDK2 inhibition.