Design, Synthesis, Anticancer Activity and Docking Studies of Thiazole Linked Phenylsulfone Moiety as Cyclin-Dependent Kinase 2 (CDK2) Inhibitors

Abstract Inhibition of the cyclin dependent kinase 2 (CDK2) is a well-known cancer treatment approach. Based on the molecular docking simulation analysis, two new series of 2-(1-(4-methoxyphenyl)-2-(phenylsulfonyl)ethylidene)hydrazineylidene)-2,5-dihydrothiazole as CDK-2 inhibitors were designed. The lead compound IV (5-benzoyl-N2-phenyl-1,3-thiazole-2,4-diamine) was modified and optimized prior to this analysis. In contrast to Roniciclib (−8.6 kcal/mol), docking results of CDK2 hits showed high affinity and docking scores ranging from −9.1 to −10.3 kcal/mol. All of the compounds studied were evaluated in vitro for CDK2 inhibitory activity. In comparison to Roscovitine's IC50 of 0.432 µM, compound 11b had a maximum IC50 of 0.416 µM. Additionally, all hits were tested for antiproliferative activities against PC-3 human prostate cancer cells, HEPG-2 human hepatocellular carcinoma, and MCF-7 breast adenocarcinoma cell lines. The arylhydrazine moiety of the tested compounds 11a–e demonstrated high potency against HEPG-2, with 11b (IC50 = 0.11 µM) being more active than doxorubicin (IC50 = 0.12 µM). In addition, compound 11b (IC50 = 0.245 µM) was nearly as effective as doxorubicin (IC50 = 0.246 µM) against MCF-7 cancer cells. 11d showed superior antiproliferative activity against PC-3 cell line (IC50= 0.23 µM) relative to doxorubicin (IC50 = 0.29 µM).


Introduction
Cancer is the leading cause of death globally, with almost 10 million deaths expected by 2020. Breast cancer (2.26 million new cases) was the most prevalent cancer in 2020. 1 As a result, discovering and developing a drug with novel mechanisms of action has become a major focus for researchers seeking to discover and improve scaffolds for potential anticancer effects with higher selectivity and lower toxicity. 2 A variety of drugs based on the heterocyclic framework have been identified and are being tested as chemotherapeutic agents. Many natural products and synthetic medicines contain thiazole derivatives, which have been shown to have important anticancer, [3][4][5] anticonvulsant, antimicrobial, antidiabetic, anti-inflammatory, and antioxidant activity. 6,7 Several antitumor drugs and active molecules based on the thiazole scaffold have been discovered as represented in Figure 1. Compound I had antitumor activity in vitro and was selective for breast cancer cells MCF-7 (IC 50 ¼ 22.8 mM). 8 Moreover, molecules II and III showed excellent inhibition against prostatic carcinoma PC-3 (IC 50 ¼ 0.3 mM) and hepatic cancer cell HEPG-2 (IC 50 ¼ 1.98 mM), 9,10 respectively. In addition, as shown in Figure 1B, thiazole is a common core in several active compounds with a wide range of significant biological effects, particularly the inhibition of CDK2. 11 CDK2 is a cyclin dependent kinase (CDK) that belongs to the CDK family, which is in control of both cell cycle initiation and progression. Furthermore, the activity of CDK family members is altered in many tumor cells, where they are required for the phosphorylation of key components for cell proliferation. [12][13][14][15] CDK2 inhibition can successfully prevent the proliferation of breast cancer cells and hepatocellular carcinoma. 16,17 In addition, it has been reported to have a crucial role in the progression of the prostate cell cycle. 18 The purpose of this research was to design and synthesize a number of new CDK2 inhibitors with anti-cancer activity.
Thus, relying on the above points and in order to achieve our target the thiazole nucleus was chosen as the main structure of the proposed compounds, and compound IV was chosen as our lead molecule. The new CDK2 inhibitors were designed based on a molecular docking analysis which was preceded by the modification and optimization of the lead IV. This could be represented in Figure 2 as follows: Maintenance of the thiazole moiety since it associates hydrophobic interactions with the hinge region 19 at CDK2's ATP binding site. Chain extensions at position 2 and 5 of the thiazole ring as 11a-f and 15 or position 2 only as 5a,b, and 7 facilitate the best interaction with the catalytic region. Substituted arylhydrazine participates in hydrophobic interactions primarily with the gatekeeper Phe80 residue. 20 Extension of the structure by phenylsulfonyl moiety, which forms a hydrogen bond with Lys89 residue and hydrophobic interaction with Leu298 in the hinge region. Lys89 helps to determine the selectivity of CDK-2 inhibitors. 21 The antitumor activity of the derivatives was an important part of our research, so the newly synthesized molecules that showed high docking scores and excellent binding mode were tested as CDK2 inhibitors and antiproliferative agents against human breast, liver, and prostate cancer cell lines.

Chemistry
Our research program is focused on the synthesis of bioactive heterocyclic compounds [22][23][24][25][26][27][28][29][30][31][32] and in continuation of this program we started with synthesis of the starting active methylene derivative, 2-benzenesulfonyl-1-(4-methoxy-phenyl)-ethanone (1) from the reaction of 2-bromo-1-(4-methoxy-phenyl)-ethanone with sodium-benzene-sulfinate under reflux for 6 h as illustrated in Scheme 1. The proposed structure 1 was confirmed by checking its spectral data. The IR spectrum of derivative 1 showed the absorption band of the carbonyl group (C ¼ O) vibrating at ¼ 1664 cm À1 . The 1 H NMR spectrum of such derivative 1 revealed the distinguished signal of the CH 2 group which distinctly appeared at d ¼ 5.23 ppm in addition to the nine aromatic protons that are resonating at their expected values (see "Experimental" section).
The synthesis of the multilateral hitherto unreported 2-(1-(4-methoxyphenyl)-2-(phenylsulfonyl)ethylidene)hydrazine-1-carbothioamide 3 was accomplished through the condensation reaction of the sulfone derivative 1 with thiosemicarbazide 2 in acidic ethanol, as sketched in Scheme 1. The synthesized phenylsulfonyl-carbothioamide derivative 3 was established using spectroscopic techniques. The 1 H NMR spectrum for phenylsulfonyl-carbothioamide derivative 3 is described in Figure 3. The registered spectrum of phenylsulfonyl-carbothioamide derivative 3 revealed the presence of the two nonequivalent protons of NH 2 group at two different chemical shift values (d ¼ 7.91 and 8.25 ppm) and this difference was attributed to the intramolecular-hydrogen bond of one proton of NH 2 with C ¼ N moiety. 33,34 Further evidence, the 13 C NMR spectrum of the  phenylsulfonyl-carbothioamide derivative 3 confirms its chemical structure as it is characterized with the accurate number of nonequivalent-carbons, the carbon chemical shift of the C ¼ S group was appeared at d ¼ 179.11 ppm and the value for d of the C ¼ N was 139.27 ppm.
The carbothioamides were known with their activity for synthesis of bioactive thiazole ring. [33][34][35][36] Reaction between 2-(2-(phenylsulfonyl)-1-(p-methoxyphenyl)ethylidene)hydrazine-1-carbothioamide (3) and a-bromocarbonyl derivatives 4a,b in dioxane as well as the presence of Et 3 N furnished the respective derivatives of phenylsulfonyl-thiazole 5a,b as depicted in Scheme 2. The proposed structure for such novel phenylsulfonyl-thiazoles has been confirmed on the basis of their spectral and elemental analyses. The 1 H NMR spectrum of thiazole derivative 5b showed four singlet signals in the regions d 2.32 (CH 3 ), 3.86 (OCH 3 ), 4.62 (CH 2 ), and 5.16 (CH 2 ) ppm in addition to the multiplet signals of the aromatic protons for thirteen protons appeared in the region of d 6.68-8.25 ppm. All molecular weights (m/z) for the two phenylsulfone-thiazole derivatives 5a,b which have been extracted from their mass spectra were found identical with the calced values.
All these thiazole products 11a-f spectroscopically characterized. All IR spectra of derivatives 11a-f revealed the demise of NH 2-absorption bands of the starting thiosemicarbazone or the carbonyl (C ¼ O) group of the reacted hydrazonoyl chlorides. In comparison between the two 1 H NMR of the starting phenylsulfone-thiosemicarbazone 3 and the thiazole derivative 11f (taken as  an example), we noted the disappearance of the nonequivalent two protons of NH 2 (supplementary file). The whole 1 H NMR spectrum of arylazothiazole derivative 11f has the remarkable four singlet signals at d ¼ 2.51, 3.85, 5.21, 10.43, for CH 3 , OCH 3 , CH 2 , and NH protons, respectively in addition to thirteen aromatic protons in the range d ¼ 6.99-7.87 ppm.
In a similar manner, the reactivity of phenylsulfonyl-carbothioamide derivative 3 toward C-1-(ethoxycarbonyl)-N-4-(4-chlorophenyl)hydrazonoyl chloride 12 was investigated. Thus, reaction of phenylsulfonyl-carbothioamide derivative 3 with the halogenated reagent 12 in dioxane afforded the corresponding arylazothiazolone derivative 15 as shown in Scheme 4. The suggested structure of the product 15 of the previous reaction was assured and confirmed based on the observation of the data extracted from its spectral analyses.

Biological evaluation
Cdk2/cyclin A2 assay Table 1 shows the IC 50 results for the target compounds. Many of the new compounds had a significant inhibitory effect on CDK2/A2 kinase, with IC 50 values varying from 0.41 to 15.36 mM, as compared to Roscovitine's IC 50 of 0.432 mM. Compound 11b had a maximum IC 50 of 0.416 mM. On the other hand, the lowest inhibitory compounds were 5b and 11c (IC 50 of 15.36 and 12.22 mM, respectively). We further investigated cytotoxic activities against HEPG-2, PC-3, and MCF-7 cell lines after finding that most of the tested compounds had promising inhibitory activities against CDK2.
In vitro antiproliferative activity screening The obtained molecules were evaluated for antiproliferative activities with the application of the MTT method using human prostate carcinoma PC-3, breast adenocarcinoma MCF-7 and human  hepatocellular carcinoma HEPG-2 cell lines. Doxorubicin was employed as a positive control. The results are shown in Table 1. The most potent thiazoles 11b and 11d were more active than doxorubicin, and other compounds are good to moderately active cytotoxic agents. The tested compounds 11a-e, which contain an arylhydrazine moiety in position 5 of the thiazole ring, exhibited strong potency against HEPG-2 with an IC 50 of less than 1 mM. The electron withdrawing Cl atom at position 3 of aryl hydrazine of compound 11f (IC 50 ¼ 3.28 mM) decreases the activity against the hepatocellular carcinoma cell line. Molecules 3, 5b, 11a,b,e,f, and 15 have high inhibitory activity against the MCF-7 cell line, with IC 50 values ranging from 0.245 to 1.00 mM. As compared to doxorubicin (IC 50 ¼ 0.246 mM), compound 11b was the most active (IC 50 ¼ 0.245 mM). On the other hand, compounds 5a, 7, 11c, and 11d demonstrated moderate inhibitory activity with IC 50 values of 2.02, 3.35, 1.20, and 1.54 mM, respectively. 11d showed superior antiproliferative activity against PC-3 cell line (IC 50 ¼ 0.23 mM) relative to doxorubicin (IC 50 ¼ 0.29 mM). Thiazoles 5a, b, 11a, and 11f were more potent than thiazolidin-4-ones 7 and 15 against PC-3 cell line.

Docking study
The docking analyses were carried out using Autodock 4.2 and Biovia Discovery Studio 4.5 to dock the target compounds in the CDK2 enzyme's binding site. To explain the biological findings and gain further insight into binding orientations and interactions, a docking review on the target compounds was conducted, as well as an overview of their binding modes. The selected docking pose was chosen from a list of ten potential docking poses based on its binding mode's similarity to that of the co-crystallized ligand. The Protein Data Bank provided the crystal structures of the CDK2 enzyme (PDB ID: 5iev). 37 The lead compounds were re-docked in the CDK2 kinase active site to validate the docking. The RMSD was 0.43 Å, indicating a valid docking process. Two types of inhibitors can be designed based on the activity state of the protein kinase: type I and type II inhibitors. The first type of inhibitors engage the active kinase's ATP binding pocket, while type II occupies the inactive kinase's ATP binding pocket. 38 All compounds bind the ATP binding site of CDK2 enzyme as reference compound roniciclib Figure 4 and form H-bond between one of the key binding residues Lys89 and sulfonyl group. Figure 5 showed the superimposition of Roniciclib and 11b in the active site of CDK2 kinase. Table 2 lists the compounds and their interacting residues, as well as the forms of interactions, docking scores, and 2D binding mode for each. The arylhydrazine moiety at position 5 of thiazoles of 11a-f, 15 and aryl group at position 4 of thiazoles in 5a,b fit in the hinge region 39 of CDK2 and interact hydrophobically with important residues such as Val18, Lys33, Ala31, Val64, and Ala144. All docked molecules bind with gatekeeper residue Phe80 via Pi-alkyl or Pi-sigma interaction except 5b, 7, 11a,e,f. The 3D receptor interaction of roniciclib, IV, and 11b, which docked in the active site of CDK2, is shown in Figure 6. At the binding site, all compounds had lower energy than roniciclib and compound IV, enhancing affinity and suggesting that they are effective as CDK2 inhibitors. Molecule 11b formed one hydrogen bond with Lys89 and 14 hydrophobic interactions in the active site of CDK2 ( Figure 6) and showed strong antitumor activity against (HEPG-2 and MCF-7) and good anticancer activity against PC-3 cancer cell lines with high inhibitory activity against CDK2A (Table 1) and could be used as a lead for further research.  With an IC 50 of less than 1 mM, the examined compounds 11a-e, which contain an arylhydrazine moiety, showed high potency against HEPG-2. The inhibitory activity of molecules 3, 5b, 11a,b,e,f, and 15 against the MCF-7 cell line is high, with IC 50 values ranging from 0.245 to 1.00 mM. The anticancer activity of 11d was superior in the PC-3 cell line (IC 50 ¼ 0.23 mM). In this preliminary analysis, 11b performed the best among the active hits and can be regarded as a promising lead compound for the development of more potent drugs.

Chemistry
All pictures as well as the characterization of each device that were utilized for recording the spectral data were illustrated in the supplementary file.

Biological assays
Cell viability (MTT assay) VACSERA-Cell Culture Unit, Cairo, Egypt, provided cancer cell lines, including liver cancer cell lines (HEPG-2), breast adenocarcinoma cell line (MCF-7), and prostate carcinoma cell lines (PC-3). Doxorubicin was used as a standard reference drug for comparison. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to monitor the cytotoxicity of the tested samples. Human cancer cell lines were grown at a density of 10 5 cells per well in 96-well plates and incubated for 24 h at 37 C in a 5% CO 2 incubator. The experiment was then incubated for 2 days after the growth media was replaced with 0.1 ml of new medium containing graded concentrations of the test chemicals to be or equivalent DMSO in each well. The cells were then incubated for the next 4 h with a 10 ll MTT solution (5 lg/ml) in each well. MTT-formazan crystals were treated with 100 ll DMSO, and the absorbance of each well was measured at 570 nm with an automated ELISA reader device (TECAN, CHE). The nonlinear regression fitting models were used to measure the IC 50 values (Graph Pad, Prism Version 5). A one-way ANOVA test with p less than 0.05 was used to assess the statistical differences.
Inhibitory test for cyclin dependent kinase 2 (CDK2) The CDK-2/cyclin A2 protein kinase assays were carried out at the Egyptian Company for the Production of Vaccines, Sera, and Drugs (VACSERA) in Dokki, Egypt. CDK2 quantitative analysis in human tissue homogenates and other body fluid in vitro is performed using a sandwich enzyme immunoassay kit (Cloud-Clone.Corp.). A CDK2 specific antibody has been pre-coated on the microtiter plate. A biotin conjugated antibody specific to CDK2 is applied to the appropriate microtiter plate wells after the standards or samples have been added. Then, in each individual microplate well, Avidin conjugated to Horseradish Peroxidase (HRP) is added and incubated. Only those wells containing CDK2, enzyme conjugated Avidin, and biotin conjugated antibody can change color after adding TMB substrate solution. The sulfuric acid solution completes the enzyme-substrate reaction, and the color variation is measured spectrophotometrically at 450 nm ±10 nm wavelength.