Design, Synthesis and Preliminary Biological Evaluation of Novel Benzyl Sulfoxide 2-Indolinone Derivatives as Anticancer Agents

In this work, a series of novel benzyl sulfoxide 2-indolinone derivatives was designed and synthesized as potent anticancer agents. Tyrosine kinase inhibitory activity assay indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was further investigated against five human cancer cell lines (HeLa, HepG2, MCF-7, SCC-15, and A549). Several compounds exhibited evident activities. Among them, (Z)-3-(((4-bromobenzyl)sulfinyl)methylene)indolin-2-one (6j) and (Z)-3-((benzylsulfinyl)methylene)-5-bromoindolin-2-one (6o) were found to be effective tyrosine kinase inhibitors (IC50 = 1.34 and 2.69 μM, respectively) in addition to having noteworthy antitumor potential (the average IC50 value of 6j or 6o was less than 40 μM). This class of novel derivatives has promising potential for further development as anticancer agents.

The reaction mixture was extracted with CH 2 Cl 2 (100 mL). The organic phase was dried over Na 2 SO 4 and concentrated to give the corresponding benzylic thiols (4a~4n) as a colorless liquid.
Benzyl mercaptane (4a). Benzyl thiol was purchased from commercial suppliers was of reagents grade and used without further purification.
2-fluorobenzyl mercaptane (4b). Obtained in 59.0% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [4] 3-fluorobenzyl mercaptane (4c). Obtained in 64.0% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [5] 4-fluorobenzyl mercaptane (4d). Obtained in 83.5% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [6] 2-chlorobenzyl mercaptane (4e). Obtained in 100% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [7] 3-chlorobenzyl mercaptane (4f). Obtained in 90.0% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [7] 4-chlorobenzyl mercaptane (4g). Obtained in 91.0% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [8] 2-bromobenzyl mercaptane (4h). Obtained in 92.0% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [9] 3-bromobenzyl mercaptane (4i). Obtained in 92.1% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [7] 4-bromobenzyl mercaptane (4j). Obtained in 100% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. [9] 3-methoxybenzyl mercaptane (4k). Obtained in 88.8% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. 2-methylbenzyl mercaptan (4l). Obtained in 89.5% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. 3-methylbenzyl mercaptan (4m). Obtained in 100% yield, colorless liquid. It was identified by comparison with its physical data with the literature data. 4-methylbenzyl mercaptan (4n). Obtained in 97.3% yield, colorless liquid. It was identified by comparison with its physical data with the literature data.

Extraction of tissue extract PTK:
the mouse brain tissue was quickly removed and weighed, followed by addition of five-fold volumes of pre-cooled homogenization buffer for homogenization. After centrifugation at 4℃, 1,000 g for 10 min, nuclei and cell debris were removed. The supernatant S1 was collected and centrifuged at 4℃, 10,000 g for 20 min, and then the supernatant S2 was collected. The precipitate P2, which represents the crude membrane protein fraction, was retained. S2 included cytoplasmic protein, and was used for testing protein tyrosine kinase (PTK) activity. In the detection of the membrane protein fraction, two-fold volumes of lysis buffer was added to P2, and the resultant was placed on ice for 10 min and centrifuged at 4℃, 10,000 g for 10 min. The supernatant S3, which represents the crude membrane protein including soluble membrane protein, was collected for testing PTK activity. Protein content in cytoplasm or membrane protein was detected by using BCA protein concentration kit (purchased from Beyotime Institute of Biotechnology). The tissue extract was stored at -70℃.

2.1.4.
Coating of 96-well plate: PTK substrate was dissolved and added in an amount of 100μL to each well. The plate was covered with its lid, and incubated at 4℃ overnight (10-12 hours). Then it was washed with 200μL elution buffer once, and dried at 37℃ for 2 h. After that, it was washed with 10mM PBS once, dried at room temperature, and then stored at 4℃ for further use.
2.1.5. Groups: Blank control: 60μL 1×tyrosine kinase buffer + 50μL 1×tyrosine kinase buffer containing ATP; Negative control: 10μL 1×tyrosine kinase buffer + 50μL 1×tyrosine kinase buffer containing tissue extract + 50μL 1×tyrosine kinase buffer containing ATP; Positive control: 10μL 1×tyrosine kinase buffer containing L029 + 50μL 1×tyrosine kinase buffer containing tissue extract + 50μL 1×tyrosine kinase buffer containing ATP; Vehicle control: 10μL 1×tyrosine kinase buffer containing DMSO + 50μL 1×tyrosine kinase buffer containing tissue extract + 50μL 1×tyrosine kinase buffer containing ATP; Compounds to be screened: 10μL 1×tyrosine kinase buffer containing compounds to be tested + 50μL 1× tyrosine kinase buffer containing tissue extract + 50μL 1×tyrosine kinase buffer containing ATP. (First, L029 and the compounds were allowed to interact with the tyrosine kinase tissue extract at room temperature for 10 min in advance, and did the same to the Blank control and the Vehicle control, then 50μL 1×tyrosine kinase buffer containing ATP was added to respective groups) 2.1.6. Tyrosine kinase inhibitory activity assay: 1×tyrosine kinase buffer was prepared by evenly mixing 1 mL 10× tyrosine kinase buffer with 9 mL DDW. The tissue extract was diluted with 1×tyrosine kinase buffer appropriately, mixed with it gently and evenly, and placed on ice. ATP stock solution was mixed with 1×tyrosine kinase buffer at the final concentration of 3mM , and placed on ice. The microplate was added in respective wells with the groups formulated as above, covered with the lid, and incubated at 37℃ for 30 min. The microplate was washed with 200μL elution buffer and tapped until the residual buffer was removed, and this procedure was repeated for five times. To each well, 100μL antibody diluent (antibody elution buffer diluted at a ratio of 1:2000) was added. The microplate was covered with the lid, and incubated at room temperature for 30 min. OPD solution was prepared by addition of 4 mg OPD into a mixture of 4.86 mL 0.1 mol/L citric acid solution and 5.14 mL 0.2 mol/L Na 2 HPO 4 solution, followed by addition of 50μL 30% H 2 0 2 to allow for complete dissolution, and then was protected from light. The antibody solution was removed. The microplate was washed with 200μL elution buffer and tapped until the residual buffer was removed, and this procedure was repeated for five times. 100μL freshly prepared OPD was added, and reacted at room temperature light tight for precisely 7 min. Negative wells showed an orange color. 100μL 2.5 N H 2 SO 4 was added to terminate the reaction. OD was measured at 492 nm.
2.1.7. Sample screening: Samples were screened preliminarily by using triplicate assay. The effect of the samples on tyrosine kinase inhibitory activity was tested, and the inhibition rate was calculated. Inhibition rate (%)=(OD negative control -OD sample ) / (OD negative control* -OD blank ) × 100% ; *While calculating inhibition rate of positive drugs, OD negative control in the equation was replaced with OD vehicle control to exclude the influence of DMSO on tyrosine kinase activity.