Synthesis and in vitro activity of novel 2-(benzylthio)-4-chloro-5-(1,3,4-oxadiazol-2-yl)benzenesulfonamide derivatives

Abstract Two series of novel 4-chloro-2-(benzylthio)-5-(1,3,4-oxadiazol-2-yl)benzenesulfonamides and their N-aroyl derivatives have been synthesized and evaluated for in vitro anticancer activity against the full NCI-60 cell line panel. Most of the compounds exhibited antiproliferative activity. Among them a compound bearing an N-(thien-2-ylcarbonyl) moiety showed broad-spectrum activity with 50% growth inhibition (GI50) values in the range of 2.02–7.82 μM over 50 cell lines. Graphical abstract .

This led us to an assumption that expansion of the series of 2-mercapto-N-acylbenzenesulfonamide potential anticancer agents, in which groups of varying size and electronic properties are placed at positions 2, 5, and N-of the benzenesulfonamide ring, may shed light on the structural features contributing to the biological activities.

In vitro biological activity
Compounds 2a-2d and 4a-4j submitted to National Cancer Institute (NCI) were evaluated for their in vitro anticancer activity. Sulfonamides 2a and 2c showed significant selectivity toward leukemia cell line CCRF-CEM (Fig. 4), whereas 2d appears to be substantially inactive. HOP-92, non-small cell lung cancer, and renal cancer A498 cell lines reveal some insight into structure-activity relationship (SAR). Cytostatic activity of 2a-2c toward those cell lines increases when CLogP and calculated molar refractivity (CMR) of the compound increase (Table 2).
Compound 4c (NSC 754633) which satisfied predetermined threshold inhibition criteria was selected for the NCI five-dose (0.01-100 lM) assay and exhibited remarkable anticancer activity against most of the tested cell lines representing nine different subpanels (Table 3). Only NCI/ ADR-RES (adriamycin-resistant cell line) expressing high levels of MDR1 and Pgp-170 glycoprotein [36,37] was found to be insensitive at the highest tested concentration (100 lM). The obtained data revealed some subpanel sensitivity toward renal, central nervous system (CNS), and breast cancer cell lines (subpanel selectivity ratio: 1.04-1.46). The CNS cancer subpanel showed highest sensitivity with mean GI 50 value of 3.24 lM and mean concentration causing total growth inhibition at 12.68 lM level. It is worth mentioning that the cytotoxic effect of 4c was less pronounced in the leukemia subpanel [50% lethal concentration (LC 50 ) for all tested leukemia cell lines [100 lM]. A relatively large difference in mean cytostatic (mean-graph GI 50 = 4.27 lM) and cytotoxic (mean-graph LC 50 = 58.88 lM) indicators could be projected to potential low toxicity against normal cells resulting in a broad therapeutic index.

Conclusions
We designed a new and efficient method of obtaining substituted 2-mercaptobenzenesulfonamides from readily available 2,4-dichlorobenzenesulfonamides under optimized mild phase-transfer catalysis conditions. This approach offers easy and quick isolation of the products and preparative-scale synthesis. Novel 2-mercaptobenzenesulfonamides and their structurally diverse N-(hetero)aroyl derivatives were evaluated for in vitro antiproliferative activity. The discovered N-acylbenzenesulfonamide 4c shows promising anticancer activity toward 50 human cancer cell lines and could be considered as a lead for further optimization.

Experimental
Melting points were determined with a Boëtius apparatus. Infrared (IR) spectra were taken using a Thermo Mattson Satellite FTIR spectrophotometer, 1 H and 13 C nuclear magnetic resonance (NMR) were taken with a Varian Gemini 200 MHz or Varian Unity Plus 500 MHz spectrometer. Chemical shifts are reported in ppm (d). The results of elemental analyses for C, H, and N were in agreement with the calculated values within ±0.4% range.
General procedure for the synthesis of 1a, 1b A mixture of 2.84 g 2,4-dichloro-5-sulfamoylbenzhydrazide (10 mmol) and the appropriate orthoester (60 mmol) in 30 cm 3 glacial AcOH was refluxed for 7-12 h. After cooling to room temperature, stirring was continued overnight. The precipitate was filtered off, washed with cold EtOH and petroleum ether, and purified by crystallization from EtOH.    General procedure for the synthesis of 2a-2d To a suspension of the appropriate 2,4-dichlorobenzenesulfonamide 1a, 1b (5 mmol) in 30 cm 3 MeCN and 0.1 cm 3 water, 1.52 g K 2 CO 3 (11 mmol) and 0.016 g TBAB (0.05 mmol) were added. The obtained reaction mixture was vigorously stirred under an argon atmosphere, and slowly the appropriate mercaptan (5 mmol) was added dropwise. After 24 h of stirring at room temperature, the reaction mixture was concentrated under reduced pressure to dryness, and 15 cm 3 EtOH was added. The precipitate was filtered off and suspended in 30 cm 3 water, stirred for 30 min, and filtered off. The crude product was purified by crystallization from EtOH.  General procedure for the synthesis of N-acylbenzenesulfonamides 4a-4c To a suspension of the appropriate pyridinium salt 3a-3c (0.5 mmol) in 5 cm 3 EtOH, 2 cm 3 10% p-TSA solution in EtOH was added and stirred at room temperature for 1 h. The precipitate was filtered off and washed with EtOH and water.

NCI in vitro anticancer screen
As of early 2007 all compounds submitted to the NCI-60 cell screen are tested initially at a single high dose (10 lM) in the full NCI-60 cell panel representing human leukemia, melanoma and lung, colon, brain, breast, ovary, kidney, and prostate cancers. Briefly, the compounds were solubilized in DMSO and added at a single concentration, and the cell culture was incubated for 48 h at 37°C, 5% CO 2 , 95% air, and 100% relative humidity. End points were determined by colorimetric sulforhodamine B (SRB) assay [40]. Results for each compound were reported as a mean-graph of the percent growth of the treated cells relative to the nodrug control, and relative to the time-zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0) [41]. According to Developmental Therapeutics Program (DTP) anticancer screening paradigm, after obtaining the results for one-dose assay, careful analysis of DTP screening data was performed and compound 4c (NSC 754633) which satisfied predetermined threshold inhibition criteria was selected for the NCI five-dose (0.01-100 lM) assay. The results were used to create dose-response curves (log 10 of sample concentration versus % growth), and three response parameters (GI 50 , TGI, and LC 50 ) were calculated for each cell line. GI 50 measures the growth inhibitory power of the test agent, TGI signifies a cytostatic effect, and LC 50 signifies a cytotoxic effect.