Synthesis of 3-Arylidene and 3-Arylimine Oxindole Derivatives and Evaluation of Their Src Kinase Inhibitory and Antiproliferative Activities

Shaya Mokhtari1§, Amir Nasrolahi Shirazi2,3§, Rakesh Kumar Tiwari2,3, Keykavous Parang2,3,* and Farzad Kobarfard4* 1Central Research Laboratories, Shaheed Beheshti University of Medical Sciences, Tehran, Iran 2Chapman University School of Pharmacy, Irvine, CA, 92618, United States of America 3Chao Family Comprehensive Cancer Center, School of Medicine, University of California, Irvine, Shanbrom Hall, 101 The City Drive, Orange, CA 92868, United States of America 4Department of Medicinal Chemistry, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran


Introduction
Protein tyrosine kinases (PTKs) are a group of enzymes, which catalyze the transfer of the γ-phosphate group of ATP to tyrosine residues of proteins. PTKS play critical roles in signal transduction and cellular biochemical pathways [1]. The level of cell tyrosine phosphorylation in different proteins is normally controlled by PTKs and tyrosine phosphatases. c-Src (Src kinase) is a non-receptor PTK and an early upstream signal transduction enzyme that is activated or overexpressed in several human cancers, such as breast, lung, colon, esophagus, skin, cervix, and gastric tissues [2,3]. Thus, inhibition of c-Src kinase has become a strategy for therapeutic intervention for different types of cancer.
Several studies have provided compelling evidence that Src kinase plays a crucial role in osteoclast function [4]. Thus, Src kinase is also a potential pharmacologic target for the treatment of bone loss diseases, such as osteoporosis [5,6].
Based on the mechanism of action, current available Src kinase inhibitors can be classified into two major groups [7]: Inhibitors that compete with ATP for its binding pocket and inhibitors that work by interfering protein-protein interactions between the enzyme and its protein substrate. Competitive ATP binding site Src kinase inhibitors have shown to be more promising in terms of their potency and therapeutic applications. Several heterocyclic compounds have been used as competitive ATP binding site inhibitors, such as pyrazolo (3,4-d) pyrimidine (PP1), pyrrolo(2,3-d)pyrimidine (CGP76030), pyrido(2,3-d)pyrimidines (PP-166285), quinolinecarbonitrile (compound I), and indolinone derivatives ( Figure 1) [8][9][10][11].
Pyrazolopyrimidine derivatives including PP1 and PP2 were found to be highly potent Src kinase inhibitors with IC 50 values in the nanomolar range. Indole derivatives such as SU6656 and SU6657 have been also reported as selective and potent Src-inhibitors with IC 50 values in the nanomolar range [12]. Recently, a number of 1,3-dihydroindole-2-one derivatives were reported to show Src and Yes tyrosine kinase inhibitory potency [13]. Olgen et al. have previously discovered 1-benzylindole-2-piperidinoethyl carboxylate, as a potent inhibitor of Src with IC 50 value of 1.4 µM [14]. They have also reported a series of 3-(substituted-benzylidene)-1,3-dihydroindoline-2-thione derivatives and the corresponding indoline-2-one congeners for their ability to inhibit Src kinase [15].
In continuation of our efforts to synthesize Src kinase inhibitors using new scaffolds and to investigate novel chemical structures as Src kinase inhibitors [17][18][19][20][21], a group of 3-arylilidene substituted oxindoles (a) and 3-arylimine substituted oxindoles (b) (Figure 2) were synthesized and evaluated for their inhibitory activity against Src kinase. We investigated the effect of various substituents in arylilidene and arylimine moieties at position 3 of the indole-2-one scaffold.

Cell proliferation assay
Antiproliferative assay was performed using Cell Titer 96 aqueous one solution cell proliferation assay kit (Promega, USA). In a brief, after reaching 75-80% confluency of cells under microscope, cells (5000 cells/well) were seeded in 96-well microplates in media (100 μL). After 24 h, compounds (50 μM) were added to wells in triplicate. Doxorubicin (10 μM) and DMSO were tested in the assay as positive and negative controls. After 72 h incubation, CellTiter 96 aqueous solution (20 μL) was added into wells. The plate was kept at 37°C for 1-2 h. The formazan product absorbance at 490 nm was measured by 96-well plate reader. The blank control was recorded by measuring the absorbance at 490 nm with wells containing medium mixed with CellTiter 96 aqueous solution but no cells. Results were expressed as the percentage of the control (without compound set at 100%). The results of the inhibition of MCF-7, SK-OV-and HT-9 cells by compounds (a 1 -a 42 ) and (b 1 -b 27 ) Series (50 μM) after 72 h incubation is demonstrated in Table 1. All the experiments were performed in triplicate.

Src kinase activity assay
The effect of synthesized compounds on the activity of c-Src kinase was assessed by Transcreener® ADP 2 FI Assay, from Bell Brook Labs, Madison, WI, (catalogue no. 3013-1K) according to manufacturer's protocol. 384-well Low volume Black non-binding surface round bottom microplate was purchased from Corning (#3676). In summary, the kinase reaction was started in 384-well low volume black microplate with the incubation of the 2.5 μL of the reaction cocktail (0.7 nM of His 6 -Src kinase domain in kinase buffer) with 2.5 μL of prediluted compounds (dissolved in 10% DMSO, 4X target concentration) for 10 min at room temperature using microplate shaker. The reaction cocktail was made using the kinase buffer HEPES (200 mM, pH 7.5), MgCl 2 (16 mM), EGTA (8 mM), DMSO (4%), Brij-35 (0.04%), and N H O R 2-mercaptoethanol (43 mM). Kinase reaction was started by adding 5 μL of ATP/substrate (40 μM/600 μM) cocktail and incubated for 30 min at room temperature on microplate shaker. Src optimal peptide (AEEEIYGEFEAKKKK) was used as the substrate for the kinase reaction. Kinase reaction was stopped by adding 10 μL of the 1X ADP Detection Mixture to the enzyme reaction mixture and mixed using a plate shaker. The mixture was incubated at room temperature for 1 h, and the fluorescence intensity was measured. The 1X ADP Detection Mixture was prepared by adding ADP2 Antibody-IRDye® QC-1 (10 μg/mL) and ADP Alexa594 Tracer (8 nM) to Stop and Detect Buffer B (1X). Fluorescence Intensity measurements were performed using fluorescence intensity optical module using the excitation of 580 nm and emission of 630 nm with band widths of 10 nm by Optima, BMG Labtechmicroplate reader. IC 50 of the compounds were calculated using ORIGIN 6.0 (origin lab) software. IC 50 is the concentration of the compound that inhibited enzyme activity by 50%. All the experiments were carried out in triplicate.
In the case of compound a 30 , the aldehyde was synthesized in three steps starting with bromination of 4-tolunitrile, with N-bromosuccinimide (NBS), reaction with 4-methylpiperazine, followed by conversion of nitrile group to aldehyde in the presence of Raney Nickel and formic acid, respectively (Scheme 2).
The synthesis of 3-arylimino-2-oxindoles was achieved by the reaction of an appropriate isatin with different aryl amines in the presence of catalytic amount of acetic acid in absolute ethanol (Scheme 3, Table 2).
Both arylilidene and arylimine derivatives were obtained as colored crystalline or powdered products, and they were purified by crystallization. Attempts to separate the cis/trans isomers were unsuccessful due to interconvention of cis and trans isomers during the dissolution of the separated compounds in ethanol and other polar solvents as the extracting solvents. The structure of all the synthesized compounds was confirmed by using IR, 1 H NMR, 13 C NMR, ESI-Mass spectra, and CHNS elemental analysis.
H-1 hydrogen of indole ring was proved to be exchangeable in the presence of few drops of deuterium oxide in 1 H NMR spectra. Scrutinizing the 1 H NMR for the compounds studied in the present study revealed that for all 3-arylilidene-2-oxindoles and 3-arylimine-2-oxindoles, H-4 hydrogen of indole ring appears at around 6 ppm in E isomers and around 6.8-7 ppm in Z isomers. This phenomenon can be explained by the anisotropic effect of aryl ring on H-4 hydrogen of indole ring in E isomers ( Figure 3).
All 69 compounds were evaluated for their inhibitory activity against Src tyrosine kinase and antiproliferative activities. IC 50 values of the compounds against Src kinase were determined using a fluorescence intensity assay. The results are shown in Tables 3  and 4. The most potent compounds against Src kinase were among 3-arylimine-2-oxindole derivatives. Among all compounds, b 11 , b 16 , and b 26 showed IC 50 values of 5.3, 10.4 and 17 µM, respectively, against Src kinase (Figure 4). All the compounds were among 3-arylimine-2oxindoles. Only one 3-arylilidene-2-oxindole (compound a 1 ) showed modest Src kinase inhibitory activity (IC 50 =12.9 µM). Both 3-arylilidene-2-oxindoles and 3-arylimine-2-oxindoles were also tested for their cytotoxic effects against three tumor cell lines: human ovarian adenocarcinoma (SK-OV3), breast adenocarcinoma (MCF-7), and colon adenocarcinoma (HT-29) cell lines at 50 µM concentration, and the results were obtained in a percentage of inhibition of proliferation (Tables 3 and 4). As it is shown in Table  3, a number of the 3-arylilidene-2-oxindole derivatives showed the inhibitory potency higher than 50% in cells Among the three cancer cell lines used in this study, HT-29 was found to be the most sensitive cell line. Nineteen compounds showed greater than 50% proliferation inhibition in this cell line. Thirteen out of these nineteen compounds are among arylidene derivatives. Thus, it appears that arylidenes are more potent cytotoxic agents against colon cancer cell lines.  Compounds a 8 , a 20 , a 38 , and b 15 showed consistently>50% proliferation inhibition against all three cancer cell lines. Among 3-arylidene-2oxindole derivatives, compounds a 22 , a 38 , and a 15 were the most potent compounds against HT-29, SK-OV-3, and MCF-7 cells, respectively.
Src is a protein tyrosine kinase that is involved in the regulation of multiple signal transduction pathways that are critical to cell survival and proliferation. Here, the Src kinase inhibition assay revealed that four compounds a1, b11, b16, and b26 showed the highest inhibitory activity by IC 50 values of 12.9, 5.3, 10.4, and 17 µM, respectively. A comparison among the chemical structures of b11, b16, and b26 showed that all these compounds carry an electron withdrawing group, such as nitro, dichloro, and SO 3 Na, as a substituents R group. Moreover, the presence of an electron donating groups like hydroxyl, methyl, or amine functional groups on the aryl ring was found to be facilitating the   interaction with the binding site. However, as it was described above, there are additional factors such as molecular flexibility, the orientation of chemical functional groups, and proximity to binding sites that contribute to kinase inhibitory potency. Thus, further modeling investigations are required to determine the appropriate functional groups for generating more optimal Src kinase inhibition potency.
Furthermore, the antiproliferative activity of compounds in three different cancer cell lines including HT-29, SK-OV-3, and MCF-7 showed that the activity was cell-dependent. Among all compounds, a8, a38, a20, b15, a22, a36, and a15 showed the highest antiproliferative potency by 70%, 67%, 70%, 76%, 76%, 76%, and 77%, respectively, in various types of cells. However, a8 and a38 were more potent in SK-OV-3 cells compared to other types of cells. A similar pattern was observed for compounds a20 and a22 in HT-29 cells and a15 and b15   in MCF-7 cells. Comparing the chemical structures of compounds revealed that the majority of them carry electron withdrawing groups including Br, Cl, and NO 2 either as the R substituent or on the aryl ring. Several factors contribute to the antiproliferative activity of a compound, such as cellular uptake and mechanism of action. Further investigations are needed to determine the mechanism of action like intercalating ability with DNA, radical generating property, apoptosis pathway, and/or cell necrosis.
A direct correlation between Src kinase inhibitory potency and cytotoxicity of all compounds individually was not discovered. However, comparing the results obtained in Src kinase inhibitory and cytotoxic studies revealed the following different trends: In general, arylilidenes were more cytotoxic agents than arylimines, possibly due to the presence of α,β-unsturated amide and a different cytotoxicity mechanism. On the other hand, arylimines exhibited higher Src kinase inhibitory activity than arylilidenes. We postulate that the arylimines are modestly active against Src kinase and less active in antiproliferative assays possibly because of limited cellular permeability. Compound a 1 was the only arylilidene derivative with high potency against Src kinase along with modest activities against HT-29 and SK-OV-3 cell lines. Further studies are required to optimize the Src kinase inhibitory and antiproliferative activities of these compounds to find an optimized one that works both as Src kinase inhibitor and antiproliferative agent for potential cancer therapy.

Conclusions
In conclusion, a number of novel 3-arylilidene-and 3-arylimine-2-oxindole [30] derivatives were synthesized and evaluated for Src kinase inhibitory and antiproliferative activities. In general, arylilidenes exhibited higher antiproliferative activity than arylimines. Compound b 11 in 3-arylimine-2-oxindoles showed IC 50 values of 5.3 µM against Src kinase. These data suggest that 3-arylilidene and 3-arylimine-2oxindole chemical scaffolds can be used as new scaffolds for further structure optimization for generating compounds with higher antiproliferative or Src kinase inhibitory activities, respectively.