Synthesis of pro-apoptotic indapamide derivatives as anticancer agents.

Abstract 4-Chloro-3-({[(substitutedamino)carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide (1–20) and 4-chloro-3-({[3-(substituted)-4-oxo-1,3-thiazolidine-2-ylidene]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide derivatives (21–31) were synthesized from 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)-3-sulfamoylbenzamide (indapamide). 4-Chloro-3-({[(4-chlorophenyl) amino) carbonothioyl]amino}sulfonyl)-N-(2-methyl-2,3-dihydro-1H-indole-1-yl)benzamide 12 demonstrated the highest proapoptotic activity among all synthesized compounds on melanoma cell lines MDA–MB-435 with 3.7% growth inhibition at the concentration of 10 µM. Compound 12 (SGK 266) was evaluated in vitro using the MTT colorimetric method against melanoma cancer cell line MDA–MB435 growth inhibition for different doses and exhibited anticancer activity with IC50 values of 85–95 µM against melanoma cancer cell line MDA–MB435. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I, II, IX and XII. The compund inhibited these enzymes with IC50 values ranging between 0.72 and 1.60 µM.


Introduction
In the progress of novel drug discovery, the easiest and most effective way is to work with drug substances with known activity and molecular structure. Basic method is to synthesize the new analogs and homologs of drug substance chosen as precursor with proven biological activity and molecular structure. In this way, it is possible to approach novel drug substances with the same or different biological activity. Moleculer modification offers the researchers the opportunity of improvement in properties of biological efficiency, mechanism of action, administration pathway, toxicity and stability. If it is not possible to obtain drug candidates from precursor drugs, the knowledge will be gained for the synthesis of novel drug substances which could be a key role in research area. Molecular modification methods give the chance to obtain active pharmaceutical ingredients with several pharmacological activities. One of the most significant examples is sulfonamide derivatives which have several biological activities ( Figure 1). Sulfonamides represent one of the classical chemotypes associated with potent carbonic anhydrase (CA) inhibition 1 . CA I and II, CA isoforms, are rather abundant in many tissues and participate in important physiological processes 2,3 . Indapamide, 4-chloro-N-(2-methylindolin-1-yl)-3-sulphamoylbenzamide, have been discovered in the 1960s-1970s, when little was known about the various CA isozymes. This drug was a much weaker one (KI of 2520 nM) inhibitor against CA II 4 .

General
All chemicals were purchased from Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, MO) or Fluka (Buchs, Switzerland). Melting points were determined with a Barnstead melting point apparatus (Barnstead/Electrothermal 9200). Infrared (IR) spectra (KBr disc) were obtained with a Perkin Elmer Spectrum One (Waltham, MA). 1 H NMR and 13 C NMR spectra in DMSO-d 6 were recorded on a BRUKER AVANCE-DPX (Billerica, MA) spectrometer (400 MHz) and chemical shifts are given in ppm downfield from tetramethylsilane (TMS) as an internal standard using DMSO-d 6 as solvent. Data are reported as follows: chemical shift, multiplicity (br.: broad singlet, d: dublet; m: multiplet, s: singlet and t: triplet), coupling constants (Hz), integration. Elemental analyses were performed on Flash EA 1112 series elemental analyzer (Thermo Finnigan, Italy). Mass spectra were measured on a JMS-700 double-focusing mass spectrometer (JEOL, Akishima, Tokyo, Japan). Follow up of the reactions and checking the purity of the compounds were made by TLC on silica gel protected aluminium sheets (Type 60 F 254 , Merck 1.05550.0001) (Darmstadt, Germany), and the spots were detected by means of UV lamp at ¼ 254 nm.

Biology
Primary anticancer assay was performed in accordance with the protocol of the Drug Evaluation Branch, National Cancer Institute, Bethesda [8][9][10][11] . The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, 100 mL of cells were inoculated into 96-well microtiter plates at plating densities ranging from 5000 to 40 000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 C, 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. The cytotoxic and/or growth inhibitory effects of the compounds were tested in vitro against the full panel of 60 human tumor cell lines derived from nine neoplastic diseases at 10-fold dilutions. The percentage of growth was evaluated spectrophotometrically versus controls which were not treated with the test agents. Briefly, effect of the compounds on the growth parameters of the different cancer cell lines was evaluated relative to equivalent amounts of DMSO treated controls and expressed as percent growth rate. The compounds were added at 10 À5 M concentration for 48 h.  15 . The MTT metabolic assay was carried out at the seeding density of 1 Â 10 4 cells/well in 96-well flat-bottom cell culture plates with 100 mL of opti-MEM (invitrogen, USA). Following 24-h incubation at 37 C, 5% CO 2 , media was aspirated, compounds were dissolved in DMSO and diluted with medium before addition to the cell cultures at the concentrations of 5 and 10 mg/mL. Cells were incubated for 48 h at 37 C, 5% CO 2 . After the incubation period 10 mL of the MTT labeling reagent [final concentration 0.5 mg/mL (Cell proliferation kit MTT, Roche, Germany)] was added to each well. Samples were incubated for 4-12 h in a humidified atmosphere (e.g. 37 C, 5.0% CO 2 ) and 100 mL of the solubilization buffer was added into each well. The plate was allowed to stand overnight in the incubator in a humidified atmosphere (e.g. 37 C, 5% CO 2 ) and the formazan precipitates were then solubilized. Absorbance of the formazan product was measured spectrophotometrically at 550 and 690 nm. Statistical analyses were done using unpaired Student's t-test using Prism 3.0 (GraphPad Software, San Diego, CA).

TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining was performed on MDA-MB-435 cell line. Cells were cultured in DMEM supplemented with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, 100 units of penicillin/ ml and 100 mg of streptomycin/ml at 37 C in a humidified atmosphere of 5% CO 2 in air. Cells were seeded into 6-well plates at a density of 1.5 Â 10 5 cells/well. Following one-day incubation, medium was replaced and adjacent wells have been inoculated with different concentrations of synthesized compound. Apoptosis was detected after 24 and 48 h, using ApopTag Plus in situ apoptosis detection kit peroxidase (Chemicon) following manufacturer's protocol with minor modifications. Apoptotic cells were observed brown after the color reaction with DAB (3,3 0diaminobenzidine), while counter staining was managed using methyl green to observe the living cells. Apoptotic cells were detected by standard light microscopy. Live and death cells were counted by two independent observers. TUNEL assay identifies early stage apoptosis by enzymatically labeling 3 0 -OH end of DNA strand breaks with modified nucleotides. Since late stage apoptotic and necrotic cells were detached in the adherent cell culture, given counts merely reflects cells in the early apoptotic stage.

Flow-cytometric analysis for apoptotic cell rate by Annexin V-FITC
Test was performed using ''Annexin V-FITC apoptosis detection kit'' (eBioscience) as described previously 16 . Briefly, MDA-MB-435 cells (1 Â 10 6 ) both untreated and treated with indapamide derivatives, were harvested, washed with PBS twice and suspended with binding buffer. The cells were double-stained with Annexin-V-FITC and propidium iodide for 10 min in the dark at room temperature.

CA inhibition studies
Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na 2 SO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with distilleddeionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature (prior to assay, in order to allow for the formation of the E-I complex). The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier 17 and represent the mean from at least three different determinations. CA isoforms were recombinant ones obtained in house as reported earlier [17][18][19] .
The 1 H NMR spectra of the sulfonylthiourea derivatives (1-20) revealed that CH 3 protons at indoline ring came out as a doublet with the integration three protons at 1.09-1.32 ppm.   was obtained using electron impact ionization technique. The molecular ion peak observed at m/z 492.0680 Da was within the acceptable limit for molecular weight and the empirical formula of compound 22. The characteristic fragmentations for 4-thiazolidinone were also observed. The main fragmentation product was observed as 1-imino-2-methyl-2,3-dihydro-1H-indolium cation, giving the base peak at m/z 147.0935 Da 35,36 .
It was also investigated whether the effect of compound 12 was mediated via the apoptotic pathway; and if so which apoptotic pathway was responsible for this process. This part of study was performed at the Department of Biophysics, School of Medicine, Marmara University. Adherent cell population was determined by cell count and number of apoptotic cells was detected by TUNEL assay and FACS analysis 16 . Annexin-V, a well-established technique to determine apoptosis, binds and detects translocation of phosphatidyl serines to the outer membrane, an indication of the beginning of apoptosis 37 . Flow cytometric analysis of the cell lines was performed using FITC-labeled Annexin-V and propidium iodide, a DNA-binding dye used as an indicator of DNA damage. Our results were introduced in Figure 7, where Q4 and Q2 were regions of early and late stages of apoptosis, respectively, and Q1 involved necrotic cells.
In this study, compound 12 was added to the cell cultures at indicated concentrations (0, 10, 50, 80 and 100 mM) and apoptosis was determined at 24 and 48 h following inoculation. A dosedependent increase in the number of apoptotic cells can be seen as the concentration rises, which reaches to its maximum at 80 mM for 24-h period. The cell numbers were substantially reduced at 100 mM at the end of 24-h period and at 80 and 100 mM at the end of 48-h period, as serious indicators of cytotoxicity ( Figures 5  and 6). Addition of 100 mM substance was completely destructive for the cells which prevented apoptotic analyses due to extremely low cell number and completely distorted morphology (Figure 7).
In addition, compound 12 have further been investigated for CA inhibition (Table 1). There was no inhibition up to 50 mM inhibitor against hCA I, whereas hCA II, IX and XII was inhibited with IC 50 of in the range of 0.72-1.60 mM, being thus a low micromolar inhibitor with a potency similar to clinically used sulfonamide acetazolamide (AAZ) in Supporting information (Figures 8 and 9).

Conclusion
The objective of this study was to synthesize and investigate the anticancer activity of new sulfonylthiourea or 4-thiazolidinone derived from indapamide with the hope of discovering new structure leads serving as anticancer agents. Our aim has been verified by the synthesis of two different groups of structure hybrids comprising basically the indole moiety attached to either sulfonylthiourea or 4-thiazolidinone counterparts through various linkages for synergistic purpose. From the preliminary results of cell growth inhibition study, we could conclude that the selected by NIH-NCI compounds 16, 22, 26, 28 and 30 show no significant  and apoptotic cells were detected by TUNEL assay and FACS analysis. The apoptotic effect of the compound 12 was started at 50 mM following 24-h incubation. At 48-h incubation of compound 12 displayed highly toxic effects on cells as demonstrated by dramatically reduced cells number and clear deformations in shape. The obtained results clearly revealed that compound 12 derived from the indapamide exhibited better growth inhibition and apoptotic effect than their 4-thiazolidinone and other compounds. In addition, this compound was investigated as inhibitors of four physiologically relevant human carbonic anhydrase isoforms, hCA I and II (cytosolic idoforms) as well   as hCA IX and XII (transmembrane, tumor-associated isoforms).
As seen from data of Table 1, hCA I was not inhibited, whereas the remaining isoforms were inhibited with IC 50 -s in the range of 0.72-1.60 mM. Finally, the broad spectrum anticancer activity displayed by compound 12 will be of interest for future derivatization in the hope of finding more active and selective anticancer agents.