Pharmacological investigation of new niclosamide-based isatin hybrids as antiproliferative, antioxidant, and apoptosis inducers

Materials

All solvents were freshly distilled and purified according to standard procedures⁴⁶. The used chemicals, 5-chlorosalicylic acid and 2-chloro-4-nitroaniline were received from Sigma Aldrich, while isatin from Panreac, EU. Also, the solvents such as methanol were obtained from El Salam Company, Egypt, Ethanol (International Company, Egypt), Acetic acid from El Nasr Company. Inaccurate melting points (M.Ps) of the novel designed compounds are determined and reported on the digital Gallen Kamp MFB-595 instrument. All reactions were routinely checked with thin-layer chromatography (TLC) of Merck Silica Gel 60 F254 (0.25 mm thick) and visualization with a UV lamp. A Shimadzu 440 spectrophotometer is used to measure the IR spectra at the range, 400–4000 cm⁻¹. A Bruker (400 and 101 MHz) spectrometer is used to evaluate the ¹H and ¹³C signals in the NMR spectra and are recorded relative to deuterated solvent signals only in dimethyl sulfoxide (DMSO-d₆). Values of the chemical shift were listed as δ ppm units. Mass spectra were performed on a Shimadzu GS/MSQP 2010 plus spectrometer at 70 eV. The substituted indoline-2,3-dione, 5-(piperidin-1-ylsulfonyl)indoline-2,3-dione and 5-((4-methylpiperazin-1-yl)sulfonyl)indoline-2,3-dione were prepared according to the previous work^47,48,49 and used as reagents. Also, Niclosamide was synthesized by the reaction of 5-chloros-2-hydroxybenzoic acid and 2-chloro-4-nitroaniline according to the reported method⁵⁰.

Synthesis of New Hybrids

Synthesis of N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide (Niclosamide amine)

This intermediate was synthesized according to literature⁵¹, a solution of Niclosamide (2 g, 5 mmol) and Zn dust (0.46 g, 7.08 mmol) in MeOH (12.5 mL) were treated with AcOH (12.5 mL) slowly. A slight exotherm was noted during the early heating. Precipitate was observed in the reaction mixture, after 5 min, and Me OH (5 mL) was added every 30 min for 2 h to facilitate stirring. After 3 h, the reaction mixture was filtered through a plug of Celite to remove the zinc and concentrated to afford the desired solid powder. M.p. 220–222 °C; Pale yellow powder; Yield = 100%; IR: υ/cm^-1: 3437, 3358 ((NH₂, OH), 3283 (NH amide), 3040 (CH-aromatic), 1680 (C=O). Anal. Calc. for C₁₃H₁₀Cl₂N₂O₂ (297.14): C, 52.55; H, 3.39; N, 9.43. Found: C, 52.53; H, 3.38; N, 9.45.

Synthesis of Niclosamide-Isatin Hybrids (X_o, X₁, and X₂)

General procedure

Niclosamide amine (0.3 g, 1 mmol) is added to an acetic acid-catalyzed solution of indoline-2,3-dione, 5-(piperidin-1-ylsulfonyl) indoline-2,3-dione, or 5-((4-methyl piperazine-1-yl)sulfonyl) indoline-2,3-dione in EtOH absolute. The resulting mixture was heated under reflux (monitored by TLC) for 3–6 h until the condensed reaction was completed. To acquire the desired Schiff’s bases, the new precipitation was collected by filtering out and recrystallizing from EtOH / dioxane (1:2) to obtain the pure desired products.

5-Chloro-N-(2-chloro-4-((2-oxoindolin-3-ylidene)amino)phenyl)-2-hydroxybenzamide (Xo)

M.p. 250–252 °C; Light orange powder; Yield = 84% (0.36 g); IR: υ/cm^-1: 3220, 3182, 3159 (br. OH, NH-amide), 3086 (CH.aromatic), 1708 (C=O), 1631 (C=N). ¹H NMR (DMSO-d₆) δ/ppm: 6.56 (d, J = 8.0 Hz, 1H, Ar–H), 6.78 (t, J = 7.4 Hz, 1H, Ar–H), 6.89 (d, J = 8.0 Hz, 1H, Ar–H), 7.04 (s, 1H, Ar–H), 7.06 (s, 1H, OH; D₂O exchangeable), 7.27 (d, J = 6.8 Hz, 1H, Ar–H), 7.36 (t, J = 7.8 Hz, 1H, Ar–H), 7.48 (d, J = 8.0 Hz, 2H, Ar–H), 7.97 (d, J = 6.8 Hz, 1H, Ar–H), 8.45 (s, 1H, Ar–H), 10.91, 11.02 (2s, 2H, 2NH; D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ/ppm: 112.7, 113.3, 116.2, 117.8, 119.1, 119.9, 122.4, 123.3, 125.2, 125.9 (C–Cl), 129.4, 130.1, 132.6, 133.9, 135.3 (C–Cl), 138.9, 147.6, 151.2 (C–OH), 156.3 (C=N), 163.4 (C=O), 163.9 (C=O); MS m/z (%): 427 (M⁺² , 18.68), 425 (M⁺, 39.48), 380 (100.00), 331 (89.10), 264 (48.02). Anal. Calc. for C₂₁H₁₃Cl₂N₃O₃ (426.25): C, 59.17; H, 3.07; N, 9.86. Found: C, 59.83; H, 3.01; N, 9.37. Found: C, 59.43; H, 3.04; N, 9.47.

5-Chloro-N-(2-chloro-4-((2-oxo-5-(piperidin-1-ylsulfonyl)indolin-3-ylidene)amino)phenyl)-2-hydroxybenzamide (X₁)

M.p. 290–292 °C; Light red powder; Yield = 94% (0.53 g); IR: υ/cm^-1: 3456, 3438, 3309 (br. OH, NH-amide), 2940, 2856 (CH. aliphatic), 1734 (C=O), 1616 (C=N). ¹H NMR (DMSO-d₆) δ/ppm: 1.32 (s, 2H, CH₂-pipridinyl), 1.46 (br. s, 4H, 2CH₂-pipridinyl), 2.65 (br. s, 4H, 2CH₂-pipridinyl), 6.75 (s, 1H, Ar–H), 7.05 (s, 1H, OH; D₂O exchangeable), 7.13 (dd, J = 8.0, 4.2 Hz, 1H, Ar–H), 7.35–7.48 (m, 2H, Ar–H), 7.64 (d, J = 5.6 Hz, 1H, Ar–H), 7.70 (d, J = 8.0 Hz, 1H, Ar–H), 7.86–7.87 (m, 1H, Ar–H), 7.97 (s, 1H, Ar–H), 8.49 (s, 1H, Ar–H), 11.36, 11.47 (2s, 2H, 2NH; D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ/ppm: 23.4 (CH₂-pipridinyl), 25.3 (2CH₂-pipridinyl), 47.1 (2CH₂-pipridinyl), 112.7, 112.9, 116.2, 118.5, 119.7, 124.0, 128.8, 129.0 (C–Cl), 130.4, 133.5, 134.4 (C–Cl), 137.0, 138.0, 147.2, 151.1, 155.4 (C–OH), 159.9 (C=N), 163.0 (C=O), 164.2 (C=O); MS m/z (%): 574 (M⁺² , 8.14), 572 (M⁺, 3.27), 380 (100.00), 306 (82.33), 278 (19.54). Anal. Calc. for C₂₆H₂₂Cl₂N₄O₅S (573.45): C, 54.46; H, 3.87; N, 9.77. Found: C, 54.13; H, 3.98; N, 9.98.

5-Chloro-N-(2-chloro-4-((5-((4-methylpiperazin-1-yl)sulfonyl)-2-oxoindolin-3ylidene)amino) phenyl)-2-hydroxybenzamide (X₂)

M.p. 300–302 °C; Deep red powder; Yield = 92% (0.54 g); IR: υ/cm^-1: 3415, 3296 (br. OH, NH-amide), 3099 (CH.aromatic), 2942, 2855 (CH. aliphatic), 1741 (C=O), 1613 (C=N). ¹H NMR (DMSO-d₆) δ/ppm: 2.02 (s, 3H, N-CH₃), 2.31–2.47 (m, 4H, 2CH₂-piperazinyl), 3.05–3.09 (m, 4H, 2CH₂-piperazinyl), 6.32–6-52 (m, 1H, Ar–H), 6.68 (d, J = 6.2 Hz, 1H, Ar–H), 7.01 (d, J = 6.4 Hz, 1H, Ar–H), 7.11 (s, 1H, OH; D₂O exchangeable), 7.36 (s, 1H, Ar–H), 7.45 (d, J = 8.0 Hz, 1H, Ar–H), 7.64 (d, J = 8.0 Hz, 1H, Ar–H), 7.72 (s, 1H, Ar–H), 7.91 (d, J = 8.0 Hz, 1H, Ar–H), 7.97 (s, 1H, Ar–H), 10.37, 11.54 (2s, 2H, 2NH; D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ/ppm: 44.1 (N-CH₃), 47.1 (2 CH₂-piperazinyl), 52.6 (2 CH₂-piperazinyl), 113.3, 114.0, 117.1, 119.3, 119.6, 123.2, 123.6, 124.1, 126.7 (C–Cl), 127.2, 129.4, 130.1, 133.6 (C–Cl), 137.6, 139.6, 148.0, 154.6, 157.0 (C–OH), 159.9 (C=N), 164.3 (C=O), 165.8 (C=O). Anal. Calc. for C₂₆H₂₃Cl₂N₅O₅S (588.46): C, 53.07; H, 3.94; N, 11.90. Found: C, 53.44; H, 3.84; N, 11.98.

Biological assays in vitro studies

Human cancer cell line

Human Colon cancer cell line HCT-116, Human breast Cancer Cell line MCF-7 and Liver Cancer cell line HEPG-2, in addition to normal monkey kidney cell line Vero were obtained frozen in liquid nitrogen (− 180 °C) obtained from VACSERA (the Egyptian Company for Production of Vaccines, Sera and Drugs). The tumor cell line was maintained as monolayer cultures in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. The treatment protocol has been approved by the research ethics committee of the NCRRT, Cairo, Egypt (2A/22).

Cytotoxicity assay

The cytotoxicity of the novel hybrids was evaluated using sulforhodamine-B (SRB) method according to that of Skehan et al.⁵². In 96-well microtiter plates, cells were seeded at a concentration of 3 × 10³ cells/well. The cells were treated for 48 h with different concentrations (0, 6.25, 12.5, 25, and 50 μg/ml) of synthesized compounds and parent as control. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm using an ELISA microplate reader (TECAN Sunrise TM, Germany). The mean values estimated as the percentage of cell viability as follows:

O.D (treated cells)/O.D (control cells) × 100. The IC₅₀ value (the concentration that produces 50% inhibition of cell growth) of each drug was calculated using dose–response curve-fitting models (Graph-Pad Prism software, version 5). The compound X1 was highly selective for the colon cancer cell line (HCT-116), so the colon cancer cell line was chosen for further investigations.

Preparation of cell-free media and cell lysate

Cells of the HCT-116 cell line were cultured in T75 flasks, left for 24 h, and then treated with IC₅₀ concentration of the compounds Niclosamide as apparent and its analog X1 for 48 h. The medium was collected and used for the determination of NOx level. Cell pellets were prepared by removing the cells from the flasks by trypsinization and used for the determination of glutathione and MDA content. The treated and control cell pellets were collected, washed, and suspended in cold lysis buffer, then sonicated and centrifuged, and the clear supernatant was taken into another Eppendorf.

Determination of protein concentration

The Bradford method⁵³ was used to determine the protein concentration in the medium and cell lysate. The procedure was based on the Coomassie brilliant blue G-250 dye creating a compound with the protein and being detectable spectrophotometrically at 595 nm. The concentration was then calculated using a standard calibration curve.

Determination of non-protein reduced thiols content (glutathione content)

Reduced glutathione (GSH) in cell lysate was determined according to the method of Ellman⁵⁴, it is based on the reduction of Ellman’s reagent [5,5′-dithio-bis-(2-nitrobenzoic acid)] by SH groups to form 1 mol of 2-nitro-5-mercaptobenzoic acid per mole of SH. The optical density was measured at 412 nm against a reagent blank and the results were expressed as μmol/mg protein.

Determination of total nitrate/nitrite (NOx)

Estimation of the total nitrate/nitrite (NOx) in cell culture media as a stable end product, nitrite was evaluated following Miranda et al. method⁵⁵. The nitrate reduction by vanadium trichloride and detection by the acidic Griess reaction are the fundamentals of the test. A brightly colored product was produced after the diazotization of sulfanilic acid with nitrite at an acidic pH and coupling with N-(10-naphthyl) ethylenediamine and measured spectrophotometrically at 540 nm and expressed as nmol/mg protein.

Determination of lipid peroxidation

Lipid peroxidation products were determined by measuring malondialdehyde (MDA) levels in cell lysate using the method of Buege and Aust⁵⁶. The principle mainly depends on the formation of thiobarbituric acid reactive substances, which have a pink color with absorption in spectrophotometry at 535 nm wavelength. The results were expressed as nmol/ mg protein.

Determination of mRNA expression of apoptotic genes

Using quantitative real-time PCR, the expression of BAX, Bcl-2, PAR-4, and BCL-xl genes (Table 4) in cells was quantified. The quality and amount of the total RNA were assessed using nanodrop (Thermo Fisher, UK) after it had been extracted from the control and treated cells with Trizol Reagent (Invitrogen, Carlsbad, CA). cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA) was used to reverse-transcribe single-stranded RNA into complementary DNA. Using a thermocycler (Biometra, Germany), thermal cycling was started under the following conditions: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C for ∞. Real-time PCR analysis was conducted using the thermo-cycler Step OneTM (Applied Biosystems). Each RT-reaction served as a template in a 20 μL PCR reaction containing 0.2 μmol/L of each primer and SYBR green master mix (Thermo Fisher Scientific, UK). Real-time PCR reactions were performed at 50 °C for 2 min, 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 min and 56 °C for 1 min. The mRNA levels of these genes were normalized to GAPDH (ΔCT). The ΔCT was calibrated against an average of the control samples.

Western blotting of Caspase-3

The ReadyPrep TM protein extraction kit (total protein) provided by Bio-Rad Inc (Catalog #163-2086) was employed according to manufacturer instructions and was added to each sample of the homogenized tissues of all different groups. Bradford Protein Assay Kit (SK3041) for quantitative protein analysis was provided by Bio Basic Inc (Markham Ontario L3R 8T4 Canada). A Bradford assay was performed according to manufacturer instructions to determine protein concentration in each sample. 20 μg protein concentration of each sample was then loaded with an equal volume of 2 × Laemmli sample buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris HCl. The pH was checked and brought to 6.8. Each previous mixture was boiled at 95 °C for 5 min to ensure denaturation of protein before loading on polyacrylamide gel electrophoresis. Polyacrylamide gels were performed using TGX Stain-Free™ FastCast™ Acrylamide Kit (SDS-PAGE), which was provided by Bio-Rad Laboratories Inc Cat # 161-0181. The SDS-PAGE TGX Stain-Free FastCast was prepared according to manufacturer instructions. The gel was assembled in a transfer sandwich as follows from below to above (filter paper, PVDF membrane, gel, and filter paper). The sandwich was placed in the transfer tank with 1 × transfer buffer, which is composed of 25 mM Tris, 190 mM glycine, and 20% methanol. Then, the blot was run for 7 min at 25 V to allow protein bands to transfer from the gel to the membrane using BioRad Trans-Blot Turbo. The membrane was blocked in tris-buffered saline with Tween 20 (TBST) buffer and 3% bovine serum albumin (BSA) at room temperature for 1 h. The components of the blocking buffer were as follows; 20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20, and 3% bovine serum albumin (BSA). Primary antibodies of caspase-3 (sc-56053, Santa Cruz Biotechnology, USA) were purchased. Primary antibodies were diluted in TBST according to manufactured instructions. Incubation was done overnight in each primary antibody solution, against the blotted target protein, at 4 °C. The blot was rinsed 3–5 times for 5 min with TBST. Incubation was done in the HRP-conjugated secondary antibody (Goat anti-rabbit IgG-HRP-1mg Goat mab-Novus Biologicals) solution against the blotted target protein for 1 h at room temperature. The blot was rinsed 3–5 times for 5 min with TBST.

The chemiluminescent substrate (Clarity TM Western ECL substrate Bio-Rad cat#170-5060) was applied to the blot according to the manufacturer’s recommendation. Briefly, equal volumes were added from solution A (Clarity western luminal/enhancer solution) and solution B (peroxidase solution). The chemiluminescent signals were captured using a CCD camera-based imager. Image analysis software was used to read the band intensity of the target proteins against control sample beta-actin (housekeeping protein) by protein normalization on the ChemiDoc MP imager.

Annexin V assay for the assessment of apoptosis

HCT-116 cells were plated (1 × 10⁶ cells/well) in six-well plates, allowed to attach overnight, and treated with the IC₅₀ of the parent and synthesized analog for 24 h of treatment. The adherent and floating cells were collected, washed twice with (4 °C) PBS, and resuspended in 400 μL binding buffer. Annexin V-FITC apoptosis detection kit (Beckman Coulter, Brea, CA) was used as the manufacturer’s recommendation using a Beckman Coulter Epics XL Flow Cytometer.

Cell cycle assay by flow cytometry

Cell cycle DNA index kit (Beckman Coulter, California, USA) was used as the manufacturer’s recommendation using a Beckman Coulter Epics XL Flow Cytometer.

Docking studies

The crystal structure of the XIAP BIR2 domain complexes with a benzodiazepinone-based inhibitor (ligand) was retrieved from the protein data bank (PDB ID: 4KJU). Auto-Dock (MGL-tools) was used to determine the grid box dimension of targeted proteins. The grid box was exported in text format. In the meanwhile, the target enzyme was exported in PDQT format.

Statistical analysis

Results were expressed as mean ± SD, Data were analyzed by one-way analysis of variance ANOVA followed by Tukey’s multiple comparisons tests using software Prism 5.0 (Graph Pad, San Diego, CA, USA) and two-way ANOVA followed by Bonferroni test (a) Significantly different from the control group, (b) significantly different from Niclosamide at P˂0.05.