Antiproliferative Activity, Multikinase Inhibition, Apoptosis- Inducing Effects and Molecular Docking of Novel Isatin–Purine Hybrids

The traditional single-treatment strategy for cancer is frequently unsuccessful due to the complexity of cellular signaling. However, suppression of multiple targets is vital to defeat tumor cells. In this research, new compounds for the treatment of cancer were developed successfully as novel hybrid anticancer agents. Based on a molecular hybridization strategy, we designed hybrid agents that target multiple protein kinases to fight cancer cells. The proposed hybrid agents combined purine and isatin moieties in their structures with 4-aminobenzohydrazide and hydrazine as different linkers. Having those two moieties in one molecule enabled the capability to inhibit multiple kinases, such as human epidermal receptor (EGFR), human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 2 (CDK2). Anticancer activity was evaluated by performing cytotoxicity assays, kinase inhibition assays, cell cycle analysis, and BAX, Bcl-2, Caspase 3 and Caspase 9 protein level determination assays. The results showed that the designed hybrids tackled the cancer by inhibiting both cell proliferation and metastasis. A molecular docking study was performed to predict possible binding interactions in the active site of the investigated protein kinase enzymes.


Introduction
Cancer is one of the most serious diseases, and the second leading cause of death. It is described by overexcited and uncontrolled cell proliferation [1]. Around one third of the population worldwide is expected to be diagnosed with cancer during their lifetimes [2]. According to the International Agency for Research on Cancer, in 2018 about 18.1 million people were diagnosed with cancer and around 9.6 million people died of the disease. It is estimated that the morbidity and mortality of cancer will continue to rise, with cancer cases growing to 24.1 million new cases and 13.0 million deaths by 2030 [3]. When an appropriate treatment strategy is provided, around two thirds of human cancers can be treated effectively [4]. The development of treatment resistance is the major obstacle to successful cancer therapy. The most effective therapies frequently fail to result in a thorough tumor response, and eventually end up with treatment resistance and tumor deterioration [5]. Therefore, continuous exploration of novel anticancer agents with low toxicity and high efficiency against both drug-sensitive and drug-resistant cancers are still imperative goals for the research and development of anticancer drugs [6]. Additionally, treating cancer patients with more than one drug has a number of negative consequences. These consequences can be avoided by using a single drug with multiple molecular targets, which nowadays is a much-preferred therapeutic strategy [7][8][9].

Aim of the Study
Each cell represents various genetic makeups, due to differences in the active oncogenes and inactive tumor suppressor genes. A single drug may successfully destroy cells holding a specific alteration while cells driven by other stimulants persist [28]. The complexity of cellular signaling frequently makes the traditional single-treatment strategy unsuccessful, and suppression of multiple targets is vital for defeating tumor cells [29,30]. Therefore, the aim of this study is to develop new drugs with inhibiting effects on cancer cell overexpression. Hybrid compounds were designed ( Figure 2) that combine distinct parts from two anticancer agents: isatin-bearing (indole-2,3-dione) compounds and purinecontaining compounds. Both parts have been used with FDA-approved drugs. Indeed, isatin-purine hybrid molecules may have the potential to suppress tumor activity, and therefore may provide novel anticancer agents with several molecular targets.

Aim of the Study
Each cell represents various genetic makeups, due to differences in the active oncogenes and inactive tumor suppressor genes. A single drug may successfully destroy cells holding a specific alteration while cells driven by other stimulants persist [28]. The complexity of cellular signaling frequently makes the traditional single-treatment strategy unsuccessful, and suppression of multiple targets is vital for defeating tumor cells [29,30]. Therefore, the aim of this study is to develop new drugs with inhibiting effects on cancer cell overexpression. Hybrid compounds were designed ( Figure 2) that combine distinct parts from two anticancer agents: isatin-bearing (indole-2,3-dione) compounds and purine-containing compounds. Both parts have been used with FDA-approved drugs. Indeed, isatin-purine hybrid molecules may have the potential to suppress tumor activity, and therefore may provide novel anticancer agents with several molecular targets.

Chemistry
In the design of isatin-purine hybrid compounds 4-9, isatin and purine were linked using 4-aminobenzohydrazide as a linker, while isatin-purine hybrid compounds 11-16 were linked using hydrazine as a linker.

Chemistry
In the design of isatin-purine hybrid compounds 4-9, isatin and purine were linked using 4-aminobenzohydrazide as a linker, while isatin-purine hybrid compounds 11-16 were linked using hydrazine as a linker.
First, ethyl 4-((9H-purin-6-yl)amino)benzoate 2 (Scheme 1) was obtained from a reaction of ethyl 4-aminobenzoate with a solution of 6-chloro-9H-purine in absolute ethanol. The reaction mixture was stirred during reflux for 7 h and the desired product was filtered and washed with cold water. The intermediate (2) was obtained with a yield of 97.38% and fully characterized by 1 H NMR and LCMS to be used for the next step.

Cytotoxicity Assay
The in vitro antiproliferative activity of the synthesized hybrid compounds 4-9 (isatin-purine hybrid compounds with 4-aminobenzohydrazide spacer) and 11-16 (isatin-purine hybrid compounds with hydrazine spacer) was preliminarily assessed using an MTT colorimetric assay against four human cancer cell lines: hepatocellular carcinoma (HepG2), mammary gland cancer (MCF-7), breast cancer (MDA-MB-231) and epithelioid cervix carcinoma (HeLa). The cytotoxic activities of the evaluated compounds are expressed as IC50 values (µM), as shown in (Table 1). Sunitinib, a well-known multikinase inhibitor, was used as a reference compound. The tested compounds showed  Next, ethyl 4-((9H-purin-6-yl)amino)benzoate 2 (Scheme 1) was refluxed in an excess of hydrazine hydrate for 3 h. The mixture was cooled and poured into ice-cold water. The resultant precipitate was filtered, washed thoroughly with water and dried to obtain the desired product, 4-((9H-purin-6-yl)amino)benzohydrazide 3. The intermediate 3 was obtained with a yield of 84.15% and fully characterized by 1 H NMR and LCMS to be used for the next step.
Synthesis of the final compounds 4-9 (Scheme 1) started with mixing 4-((9H-purin-6-yl)amino)benzohydrazide 3, the appropriate isatin, and glacial acetic acid in absolute ethanol and refluxing for about 8-12 h. The reaction mixture was cooled and added to ice water, and the formed precipitate was filtered off, washed with water and recrystallized from the proper solvent to obtain the desired compounds. The final compounds were obtained with a yield of 70.72 to 97.08% and fully characterized by 1 H NMR and LCMS.
For the second series, 6-hydrazinyl-9H-purine 10 was synthesized first (Scheme 1) by refluxing 6-chloro-9H-purine in an excess of hydrazine hydrate for 1 h. The mixture was cooled and poured into ice cold water. The resultant precipitate was filtered, washed thoroughly with water and dried to obtain the intermediate 10. The intermediate 10 was obtained with yield 80.44% and fully characterized by 1 H NMR and LCMS to be used for the next step.
Compounds 11-16 (Scheme 1) were synthesized by following the same procedure as for compounds 4-9, using 6-hydrazinyl-9H-purine 10, the appropriate isatin, and glacial acetic acid in absolute ethanol. The reaction was refluxed for about 3-7 h, then cooled and added to ice water. The formed precipitate was filtered off, washed with water and recrystallized from the proper solvent to obtain the desired compounds. The final compounds were obtained with a yield of 72.46 to 99.5% and fully characterized by 1 H NMR and LCMS (Supplementary Data).

Cytotoxicity Assay
The in vitro antiproliferative activity of the synthesized hybrid compounds 4-9 (isatinpurine hybrid compounds with 4-aminobenzohydrazide spacer) and 11-16 (isatin-purine hybrid compounds with hydrazine spacer) was preliminarily assessed using an MTT colorimetric assay against four human cancer cell lines: hepatocellular carcinoma (HepG2), mammary gland cancer (MCF-7), breast cancer (MDA-MB-231) and epithelioid cervix carcinoma (HeLa). The cytotoxic activities of the evaluated compounds are expressed as IC 50 values (µM), as shown in (Table 1). Sunitinib, a well-known multi-kinase inhibitor, was used as a reference compound. The tested compounds showed varying degrees of antiproliferative activities against the examined cell lines compared to the reference compound. In general, compounds 11-16 showed better cytotoxic activity profiles (IC 50 ≥ 8.93 ± 0.8) than compounds 4-9 (IC 50 ≥ 19.05 ± 1.4 µM) against all tested cancer cell lines except for compound 6 (R=F), which demonstrated better activity than the corresponding compound in the second series (compounds 11-16).  methoxy) was very potent and displayed highly promising cytotoxic activity against all tested cell lines, with IC50 values ranging from 8.93 to 14.89 µM. Additionally, compound 15 demonstrated cytotoxic activity comparable to the reference drug sunitinib against the HepG2, MCF-7, MDA-MB-231 and HeLa cell lines, with IC50 values of 9.61, 10.78, 14.89 and 8.93 µM, respectively. On the other hand, the remaining compounds revealed only modest inhibitory activities against the tested cell lines.

Structure-Activity Relationship (SAR)
The structure-activity relationships of the tested isatin-purine hybrid compounds towards HepG2, MCF-7, MDA-MB-231 and HeLa cell lines revealed that most of the compounds 11-16 with a hydrazine linker showed more potent cytotoxic activity against all tested cell lines than compounds 4-9 with a 4-aminobenzohydrazide linker. This may be because the shorter linker gives compounds better fit and interaction with the targeted kinases. However, compound 6, with 4-aminobenzohydrazide and R=F, showed more potent activity against the MDA-MB-231 cell line than compound 13, which has the same R substitution and hydrazine. However, introducing a F group into both 4aminobenzohydrazide and hydrazine compounds led to better cytotoxicity compared to most other compounds in each series. This may be because the F group was able to form a hydrogen bond during the interaction with the receptors.

Structure-Activity Relationship (SAR)
The structure-activity relationships of the tested isatin-purine hybrid compounds towards HepG2, MCF-7, MDA-MB-231 and HeLa cell lines revealed that most of the compounds 11-16 with a hydrazine linker showed more potent cytotoxic activity against all tested cell lines than compounds 4-9 with a 4-aminobenzohydrazide linker. This may be because the shorter linker gives compounds better fit and interaction with the targeted kinases. However, compound 6, with 4-aminobenzohydrazide and R=F, showed more potent activity against the MDA-MB-231 cell line than compound 13, which has the same R substitution and hydrazine. However, introducing a F group into both 4-aminobenzohydrazide and hydrazine compounds led to better cytotoxicity compared to most other compounds in each series. This may be because the F group was able to form a hydrogen bond during the interaction with the receptors.
In addition, compound 15, with a hydrazine linker and R=OCH 3 , was the most potent cytotoxic agent against all tested cancer cell lines. The potency of compound 15 may be due to the fact that hydrazine is a short linker and the methoxy group was able to form a hydrogen bond with the targeted site in the kinase enzymes.

In Vitro Kinase Inhibitory Activity
The newly prepared compound 15 was further assayed for its inhibitory activity against EGFR, HER2, VEGFR2 and CDK2. The results were reported as 50% inhibition concentration values (IC 50 , expressed as µM) in comparison to erlotinib, roscovitine, sorafenib and lapatinib as reference drugs (Table 2). Interestingly, the reported kinase inhibition results for compound 15 were compatible with that of the cytotoxicity assays (Table 1). This may explain the possible mechanism of cytotoxic action for the designed isatin-purine hybrid compounds. Furthermore, compound 15 exhibited inhibitory activities in the nanomolar ranges comparable to the positive controls against the four investigated protein kinases, with Her2 being the most sensitive, followed by EGFR, VEGFR2 and CDK2. The IC 50 values of the kinase inhibitory activities were 0.15, 0.143, 0.534 and 0.192 µM, respectively.

Molecular Docking
Compound 15 was docked in the active sites of EGFR, VEGFR2 and Her2 to rationalize its biological activity and to predict the possible types of drug-receptor interactions. Erlotinib, sorafenib and lapatinib were used as reference compounds, as they are the cocrystallized ligands in EGFR, VEGFR2 and Her2, respectively. Erlotinib was stabilized by a hydrogen bond with Met769 in the active site of EGFR. In addition, erlotinib had several hydrophobic interactions with Lys721, Val702, Ala719, Leu820 and Leu694, and two van der Waals interactions with Thr830 and Met769. On the other hand, compound 15 was stabilized by two hydrogen bonds between Met769 and the amide of the isatin moiety. Furthermore, compound 15 had multiple hydrophobic interactions with Ala719, Leu764, Thr766, Lys721, Thr830, Val702, Leu694 and Leu820 (Figure 3). The binding affinities for erlotinib and compound 15 with the EGFR were −9.5 and −8.9 Kcal/mol, respectively. Sorafenib, the reference compound for the docking experiment for VEGFR2, produced key interactions in the active site of VEGFR2 by forming hydrogen bonds with Cys919 (two hydrogen bonds), Asp1046 and Glu885, and several other types of interactions with the pocket's amino acid residues. The hydrazine spacer of compound 15 formed a hydrogen bond with Asp1046, while fifteen hydrophobic interactions stabilized the compound in the active site of VEGFR2 (Figure 4). The binding affinities for sorafenib and compound 15 with VEGFR2 were −11.9 and −9.3 Kcal/mol, respectively. Regarding Her2, lapatinib formed three hydrogen bonds with amino acid residues, Met801, Thr798 and Thr862, and three halogen bonds with Ser783 (two) and Arg784 (one), and also had hydrophobic interactions with Leu800, Leu852, Met801, Ala751, Gln799, Ser783, Phe864, Arg784, Leu785, Leu796, Val 734 and Lys753. In contrast, Compound 15 bound the active site with two hydrogen bonds with Lys753 and The862, and had hydrophobic interactions with Ala751, Leu796, Lys753, Thr862, val734 and Leu 852 ( Figure 5). The binding affinities for lapatinib and compound 15 with Her2 were −9.8 and −8.7 Kcal/mol, respectively. and also had hydrophobic interactions with Leu800, Leu852, Met801, Ala751, Gln799, Ser783, Phe864, Arg784, Leu785, Leu796, Val 734 and Lys753. In contrast, Compound 15 bound the active site with two hydrogen bonds with Lys753 and The862, and had hydrophobic interactions with Ala751, Leu796, Lys753, Thr862, val734 and Leu 852 ( Figure 5). The binding affinities for lapatinib and compound 15 with Her2 were −9.8 and −8.7 Kcal/mol, respectively.

Cell Cycle Analysis and Apoptosis Rate
In addition, the cell cycle progression assay as described by Wang et al. [31,32] was used to investigate the effect of compound 15 in HepG2 cells. The biological effect of compound 15 was measured by treating HepG2 cells with 9.61 µM of the compound for 24 h. The compound's effect on cell cycle distribution was analyzed by comparing with untreated control cells (Table 3, Figure 6). The results showed a massive accumulation of the treated HepG2 cells at the pre-G1 (21.47%, 10-fold) compared to the control cells (2.02%), revealed by flow cytometric analysis.

Cell Cycle Analysis and Apoptosis Rate
In addition, the cell cycle progression assay as described by Wang et al. [31,32] was used to investigate the effect of compound 15 in HepG2 cells. The biological effect of compound 15 was measured by treating HepG2 cells with 9.61 µM of the compound for 24 h. The compound's effect on cell cycle distribution was analyzed by comparing with untreated control cells (Table 3, Figure 6). The results showed a massive accumulation of the treated HepG2 cells at the pre-G1 (21.47%, 10-fold) compared to the control cells (2.02%), revealed by flow cytometric analysis.   On the other hand, an Annexin V/propidium iodide (PI) double staining assay was conducted to identify the mode of cell death promoted by compound 15 (9.61 µM) following 24 h incubation with HepG2 cells. The results, in Table 4 and Figure 7, showed a notable increase in apoptotic cells in early-stage (0.38% to 1.86%) and late-stage (0.19% to 12.58%) apoptosis in the HepG2 cell line as compared to the controls. These results of the apoptosis induction effect, along with the previous results of cell cycle arrest, indicated the potential anti-cancer effect of compound 15.  On the other hand, an Annexin V/propidium iodide (PI) double staining assay was conducted to identify the mode of cell death promoted by compound 15 (9.61 µM) following 24 h incubation with HepG2 cells. The results, in Table 4 and Figure 7, showed a notable increase in apoptotic cells in early-stage (0.38% to 1.86%) and late-stage (0.19% to 12.58%) apoptosis in the HepG2 cell line as compared to the controls. These results of the apoptosis induction effect, along with the previous results of cell cycle arrest, indicated the potential anti-cancer effect of compound 15.   On the other hand, an Annexin V/propidium iodide (PI) double staining assay was conducted to identify the mode of cell death promoted by compound 15 (9.61 µM) following 24 h incubation with HepG2 cells. The results, in Table 4 and Figure 7, showed a notable increase in apoptotic cells in early-stage (0.38% to 1.86%) and late-stage (0.19% to 12.58%) apoptosis in the HepG2 cell line as compared to the controls. These results of the apoptosis induction effect, along with the previous results of cell cycle arrest, indicated the potential anti-cancer effect of compound 15.

BAX and Bcl-2, Caspase 3 and Caspase 9 Level Protein Assays
We also investigated the effect of compound 15, which was the most active compound, on protein expression levels of BAX, Bcl-2 Caspase 3 and Caspase 9. In these assays, compound 15 was used at its cytotoxic concentration (9.61 µM) and incubated with HepG2 cells for 24 h. The results showed an increase in the level of BAX, Caspase 3 and Caspase 9 proteins, with 3.36-, 3.90-and 3.11-fold increases, respectively, compared to the control cells (Table 5). In addition, Bcl-2 protein levels in the HepG2 cell significantly decreased, by 2.30-fold, after treatment with compound 15, which affected the apoptosis pathway compared to the control.

Chemistry and Analysis
The 1 H NMR spectrum was recorded with a Bruker 700 MHz NMR spectrometer in deuterated dimethyl sulfoxide (DMSO-d 6 ) using tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm, and J values are reported in Hz. The following abbreviations were used in reporting spectrum data: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), and m (multiplet). An Agilent 6320 Ion Trap mass spectrometer (Agilent Technology, La Jolla, CA, USA) was used to obtain the mass spectra for all compounds.

General Procedure A for Synthesis of Compounds 4-9
A mixture of 4-((9H-purin-6-yl)amino)benzohydrazide (0.2 gm, 0.7 mmol), the appropriate isatin (0.7 mmol), and glacial acetic acid (1 mL) in absolute ethanol (20 mL) was refluxed for about 8 h. The reaction was then cooled and added to ice water. Afterwards, the resultant solid was filtered, rinsed with water and recrystallized from a proper organic solvent to achieve the desired compounds [19].

General Procedure B for Synthesis of Compounds 11-16
A mixture of 6-hydrazinyl-9H-purine 0.3 gm, 2 mmol), the appropriate isatin (0.7 mmol), and glacial acetic acid (1 mL) in absolute ethanol (20 mL) was refluxed for about 3-7 h. The reaction was then cooled and added to ice water. Afterwards, the resultant solid was filtered, rinsed with water and recrystallized from a proper organic solvent to obtain the desired compounds [19,33,34].

6-hydrazineyl-9H-purine (10)
6-chloro-9H-purine (10 mmol) was refluxed in an excess of hydrazine hydrate for 1 h. The mixture was cooled and poured into ice-cold water. The resultant precipitate was filtered, washed thoroughly with water and dried to obtain the product [ Compound 15 was evaluated for inhibitory activity against EGFR, VEGFR-2, Her2 and CDK2. A human ELISA kit (Enzyme-Linked Immunosorbent Assay) for each of the abovementioned protein kinase enzymes was used in this assay. First, a specific antibody for each enzyme was added separately to a 96-well plate, and 100 µL of the standard solution or the compound 15 was added. The mixture was then incubated at room temperature for 2.5 h, after which the wells were washed and 100 µL of the prepared biotin antibody was added and incubated for 1 h at room temperature. After another washing step, 100 µL of streptavidin solution was added and incubated for 45 min at room temperature. The wells were then washed for a third time and 100 µL of TMB Substrate reagent was added and incubated for 30 min at room temperature. Finally, 50 µL of the stop solution was added, and the absorbance was read immediately at 450 nm. A standard curve was plotted showing concentration on the X-axis and absorbance on the Y-axis [14,[36][37][38][39].

Flow Cytometry Analysis for Cell Cycle
Flow cytometry analysis was carried out using an ab139418 Propidium Iodide flow cytometry kit/BD to detect the effect of the synthesized compound 15 on cell cycle distribution. First, HepG2 cell were seeded in 6-well plates at a density of 2 × 10 5 /well and incubated for 24 h. HepG2 cells were then treated with 9.61 µM of compound 15 for 24 h. The treated cells were then fixed in 70% ethanol for 12 h at 4 • C, rinsed with cold PBS, incubated with 100 µL RNase A for 0.5 h at 37 • C, and stained with Propidium Iodide (400 µL) in the dark at RT for an extra 0.5 h. The stained cells were determined utilizing Epics XLMCL™ flow cytometer equipment (Beckman Coulter, Apeldoorn, Netherlands), and the results were collected and analyzed using Flowing software (version 2.5.1, Turku Centre for Biotechnology, Turku, Finland).

Flow Cytometry Analysis for Apoptosis
Flow cytometry cell apoptosis analysis was conducted to examine the apoptotic effect of the synthesized compounds using the Annexin V-FITC cell apoptosis detection kit (Biovision, K101-100). First, HepG2 cells in a density of 2 × 10 5 were seeded in 6-well plates and incubated for 24 h. The cells were then treated with 9.61 µM compound 15 for 24 h. The treated cells were then detached with trypsin (5 min, 37 • C), collected by centrifugation (5 min, 300× g), washed twice with PBS, and resuspended in 0.1 mL of a 1X binding buffer. After that, the cells were double-stained with 5 µL Annexin V-FITC and 5 µL PI in the dark at room temperature for 15 min. The stained cells were evaluated using an Epics XL-MCL™ Flow Cytometer (Beckman Coulter, Apeldoorn, Netherlands) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm, and the data were interpreted using the Flowing software (version 2.5.1, Turku Centre for Biotechnology, Turku, Finland).

Molecular Docking
The PDB Data Bank (http://www.rcsb.org, (accessed on 25 December 2022) was utilized to obtain the X-ray crystal structures of the EGFR kinase domain in complex with erlotinib (PDB ID: 4HJO), the VEGFR2 kinase domain in complex with sorafenib (PDB ID:4ASD) and the kinase domain of human HER2 (PDB ID: 3RCD) in complex with lapatinib. The Discovery Studio, AutoDock Tools, Vina, and PyRx (The Scripps Research Institute, La Jolla, CA) software programs were used for docking simulations. First, the protein crystal structures were prepared by removing all additional molecules such as water, ligands, and sulfate. The generated data was saved in PDB file format. Second, AutoDock Tools was used to add polar hydrogens to the previous file saved in the PDB format, and the data were saved in PDBQT format. Third, using Discovery Studio, the co-crystallized ligands were separated from the protein structure and saved in a PDB file. Compound 15 was also saved in a PDB file. A grid box was employed to eliminate nonspecific binding interactions and minimize docking time. Finally, PyRx was used to perform the docking simulations, while the lowest energy pose of compound 15 superimposed with the cocrystallized ligands was used to study the interaction with the kinase enzyme.

Conclusions
Isatin-purine hybrid compounds were designed, synthesized and biologically evaluated successfully. Some of these compounds showed potency comparable to the reference compounds in inhibiting cell proliferation against HepG2, MCF-7, MDA-MB-231 and HeLa cell lines. In particular, compound 15, with a methoxy substitution and a hydrazine linker, retained good activity in the nanomolar range in the inhibition of EGFR, Her2, VEGFR2 and CDK2 compared to the reference compounds. In addition, cell cycle analysis and BAX, Bcl-2, Caspase 3 and Caspase 9 protein level determination assays indicated the apoptosis-inducing effect of compound 15. Overall, this work presents the isatin-purine hybrid compounds as novel compounds targeting multi-kinases that may prove useful in the discovery of new anticancer therapeutics.