3.1. Synthesis
Melting points (°C) were determined in a Stuart SMP3 and are uncorrected. 1H- and 13C-NMR spectra were recorded on a Varian Unity Plus at 300 and 75.4 MHz, respectively or on a Bruker Avance III at 400 and 100.6 MHz, respectively. Two dimensional 1H-13C correlations were performed to attribute some signals. HRMS using EI was performed by the mass spectrometry service of the University of Vigo, C.A.C.T.I., Spain. The reactions were monitored by thin layer chromatography (TLC) using Macherey-Nagel pre-coated aluminium silica gel 60 sheets (0.20 mm) with UV254 indicator. Dry-flash cromatography was performed on Panreac, Silica Gel 60, 40–63 microns. Ether refers to diethylether. Xantphos refers to 8,9-dimethyl-4,5-bis(diphenylphosphane)xanthene.
3.1.1. General Procedure for the Synthesis of Nitrodiarylamines <bold>2a–c</bold>
A dry Schlenk tube was charged under Ar with dry dioxane (3 mL), the bromonitrobenzene (1.1 equiv.), Pd(OAc)2 (15 mol%), Xantphos (18 mol%), Cs2CO3 (2 equiv.) and the methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate (1), and the mixture was heated under Ar at 120 °C for 2 h. After cooling water (5 mL) and ethyl acetate (5 mL) were added. The phases were separated and the aqueous phase was extracted with more ethyl acetate (2 × 5 mL). The organic phase was dried (MgSO4) and filtered. Removal of the solvent gave a brown solid which was submitted to dry flash using ether and a solid was obtained.
Methyl 3-(4-nitrophenylamino)thieno[3,2-b]pyridine-2-carboxylate (2a): From thienopyridine 1 (150 mg, 0.724 mmol) and 1-bromo-4-nitrobenzene (161 mg, 0.796 mmol), compound 2a was isolated as a brownish solid (195 mg, 82%), m.p. 203–205 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 3.96 (s, 3H, OMe), 7.08 (d, J = 9.2 Hz, 2H, 2′ and 6′-H), 7.43 (dd, J = 8.2 and 4.2 Hz 1H, 6-H), 8.13 (d, J = 9.2 Hz, 2H, 3′ and 5′-H), 8.19 (dd, J = 8.2 and 1.6 Hz, 1H, 7-H), 8.67 (dd, J = 4.2 and 1.6 Hz, 1H, 5-H), 8.76 (br s, 1H, NH) ppm. 13C-NMR (100.6 MHz, DMSO-d6): δ = 52.5 (OMe), 113.8 (C-2), 118.2 (2′ and 6′-CH), 122.1 (6-CH), 124.9 (3′ and 5′-CH), 131.5 (7-CH), 134.2 (C), 141.7 (C), 141.8 (C), 147.2 (5-CH), 147.6 (C-3), 148.0 (C-1’), 164.5 (C=O) ppm. HRMS (EI) Calcd. for C15H11N3O4S [M+] 329.0470; found 329.0479.
Methyl 3-(2-nitrophenylamino)thieno[3,2-b]pyridine-2-carboxylate (2b): From thienopyridine 1 (150 mg, 0.724 mmol) and 1-bromo-2-nitrobenzene (161 mg, 0.796 mmol), compound 2b was isolated as a reddish solid (190 mg, 80%), m.p. 202–204 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 3.97 (s, 3H, OMe), 6.93–7.00 (m, 2H, 6′ and 5′-H), 7.32–7.37 (m, 1H, 4′-H), 7.42 (dd, J = 8.2 and 4.4 Hz, 1H, 6-H), 8.19-8.22 (m, 2H, 3′ and 7-H), 8.67 (dd, 1H, J = 4.4 and 1.6 Hz, 5-H), 10.59 (br s, 1H, NH) ppm. 13C-NMR (100.6 MHz, DMSO-d6): δ = 52.7 (OMe), 118.6 (C-2), 119.9 (5′-CH), 120.1 (6′-CH), 121.8 (6-CH), 125.9 (3′-CH), 131.4 (7-CH), 133.9 (4′-CH), 134.00 (C-1′), 135.9 (C), 139.2 (C), 139.3 (C), 147.4 (5-CH), 148.3 (C), 163.3 (C=O) ppm. HRMS (EI) Calcd. for C15H11N3O4S [M+] 329.0470; found 329.0478.
Methyl 3-(4-methoxy-2-nitrophenylamino)thieno[3,2-b]pyridine-2-carboxylate (2c): From thieno-pyridine 1 (150 mg, 0.724 mmol) and 4-bromo-3-nitroanisole (185 mg, 0.796 mmol), compound 2c was isolated as a reddish solid (220 mg, 82%), m.p. 213–214 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 3.82 (s, 3H, OMe), 3.87 (s, 3H, OMe), 7.02 (d, J = 9.2 Hz, 1H, 6′-H), 7.14 (dd, J = 9.2 and 2.8 Hz, 1H, 5′-H), 7.53–7.58 (m, 2H, 3′ and 6-H), 8.55 (dd, J = 8.4 and 1.6 Hz, 1H, 7-H), 8.60 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H), 9.72 (br s, 1H, NH) ppm. 13C-NMR (100.6 MHz, DMSO-d6): δ = 52.5 (OMe), 55.8 (OMe), 107.2 (3′-CH), 114.3 (C-2′), 122.5 (6-CH), 122.9 (5′-CH), 123.5 (6′-CH), 132.1 (C), 132.4 (7-CH), 133.5 (C), 136.8 (C-1′), 139.9 (C), 147.1 (C), 147.4 (5-CH), 153.2 (C-2), 163.3 (C=O) ppm. HRMS (EI) Calcd. for C16H13N3O5S [M+] 359.0576; found 359.0559.
3.1.2. General Procedure for the Synthesis of Aminodiarylamines <bold>3a–c</bold>
In a round bottom flask with 4.5 mL EtOH/THF/H2O (3:1:0.5), compounds 2a–c, NH4Cl (1 equiv.), Fe (8 equiv.) were heated at 100 °C for 2 h. After cooling the mixture was evaporated to give a dark solid. This was submitted to dry-flash using ether and the product was obtained as a yellow or greenish solid.
Methyl 3-(4-aminophenylamino)thieno[3,2-b]pyridine-2-carboxylate (3a): From nitrodiarylamine 2a (100 mg, 0.304 mmol) compound 3a was isolated as a greenish solid (88.0 mg, 97%), m.p. 200–202 °C. 1H-NMR (300 MHz, DMSO-d6): δ = 3.77 (s, 3H, OMe), 4.84 (br s, 2H, NH2), 6.45 (d, J = 8.4 Hz, 2H, 3′ and 5′-H), 6.77 (d, J = 8.4 Hz, 2H, 2′ and 6′-H), 7.46 (dd J = 8.2 and 4.2 Hz, 1H, 6-H), 8.39 (dd, J = 8.2 and 1.6 Hz, 1H, 7-H), 8.51 (dd, J = 4.2 and 1.6 Hz, 1H, 5-H), 8.60 (br s, 1H, NH) ppm. 13C-NMR (75.4 MHz, DMSO-d6,): δ = 51.9 (OMe), 103.6 (C), 113.7 (3′ and 5′-CH), 122.3 (6-CH), 123.0 (2′ and 6′-CH), 130.9 (C), 131.8 (7-CH), 133.7 (C), 144.9 (C-4′), 145.3 (C), 146.4 (5-CH), 147.2 (C), 164.4 (C=O) ppm. HRMS (EI) Calcd. for C15H13N3O2S [M+] 299.0728; found 299.0732.
Methyl 3-(2-aminophenylamino)thieno[3,2-b]pyridine-2-carboxylate (3b): From nitrodiarylamine 2b (100 mg, 0.304 mmol) compound 3b was isolated as a greenish solid (86.0 mg, 95%), m.p. 204–206 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 3.79 (s, 3H, OMe), 4.97 (br s, 2H, NH2), 6.39 (m, 1H, ArH), 6.63–6.83 (m, 3H, ArH), 7.48 (m, 1H, 6-H), 8.12 (br s, 1H, NH), 8.42–8.52 (m, 2H, 7 and 5-H) ppm. 13C-NMR (100.6 MHz, DMSO-d6): δ = 52.0 (OMe), 105.9 (C-2), 114.9 (CH), 115.9 (CH), 122.2 (CH), 122.3 (6-CH), 124.1 (CH), 127.9 (C), 131.8 (7-CH), 133.6 (C), 141.2 (C), 145.2 (C), 146.7 (5-CH), 147.2 (C), 164.2 (C=O) ppm. HRMS (EI) Calcd. for C15H13N3O2S [M+] 299.0728; found 299.0726.
Methyl 3-(2-amino-4-methoxhyphenylamino)thieno[3,2-b]pyridine-2-carboxylate (3c): From nitro-diarylamine 2c (100 mg, 0.278 mmol) compound 3c was isolated as a greenish solid (83.0 mg, 90%), m.p. 172–174 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 3.65 (s, 3H, OMe), 3.79 (s, 3H, OMe), 5.00 (br s, 2H, NH2), 5.99 (dd, J = 8.8 and 2.4 Hz, 1H, 5′-H), 6.31 (d, J = 2.4 Hz, 1H, 3′-H), 6.61 (d, J = 8.8 Hz, 1H, 6′-H), 7.43 (dd, J = 8.2 and 4.4 Hz, 1H, 6-H), 8.09 (br s, 1H, NH), 8.38 (dd, J = 8.2 and 1.6 Hz, 1H, 7-H), 8.47 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C-NMR (100.6 MHz, DMSO-d6): δ = 51.9 (OMe), 54.8 (OMe), 100.0 (3′-CH), 101.2 (5′-CH), 103.4 (C), 120.9 (C), 122.2 (6-CH), 124.9 (6′-CH), 131.7 (7-CH), 133.68 (C), 143.7 (C), 146.5 (C), 146.5 (5-CH), 147.0 (C), 157.2 (C-4’), 164.6 (C=O) ppm. HRMS (EI) Calcd. for C16H15N3O3S [M+] 329.0834; found 329.0827.
3.2. Cell Growth Inhibitory Activity and Toxicity to Non-Tumor Cells
3.2.1. Standards and Reagents
DMSO (dimethyl sulfoxide) was analytical grade from Fisher Scientific (Paris, France). Fetal bovine serum (FBS), L-glutamine, hank’s balanced salt solution (HBSS), trypsin-EDTA (ethylenediaminetetraacetic acid), penincillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively) and RNAse A were from Gibco Invitrogen Co. (Paisley, UK). RPMI-1640 medium was from Cambrex (East Rutherford, NJ, USA) and DMEM medium was from Hyclone (Logan, UT, USA). Acetic acid, ellipticine, doxorubicin, sulforhodamine B (SRB), trypan blue, trichloroacetic acid (TCA), Tris and propidium iodide were from Sigma Chemical Co. (St. Louis, MO, USA). Water was treated in a Mili-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).
3.2.2. Solutions of the Compounds
Stock solutions of the tested aminodiarylamines were prepared in DMSO and kept at −20 °C. Appropriate dilutions were prepared in the corresponding solvent prior to the assays. The effect of DMSO on the growth of the cell lines was evaluated by treating cells with the maximum concentration of DMSO used in the assays (0.25%). No influence was found (data not shown).
3.2.3. Cell Culture of Cell Lines and of Porcine liver Primary Cells
Four human tumor cell lines, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-small cell lung cancer) and HepG2 (hepatocellular carcinoma) were used. MCF-7, A375-C5 and HepG2 were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and NCI-H460 was kindly provided by the National Cancer Institute (NCI, Bethesda, MD, USA). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium supplemented with 5% FBS (MCF-7, NCI-H460 and A375-C5 cells) or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (HepG2 cells), at 37 °C in a humidified atmosphere containing 5% CO2.
Porcine liver primary cell culture (PLP1) was prepared from freshly harvested porcine liver, obtained from a local slaughter house. Briefly, the liver tissue was rinsed in HBSS solution containing 100 U/mL penicillin, 100 µg/mL streptomycin and divided into 1 × 1 mm3 explants. Some of these explants were placed in 25 cm2 tissue flasks in DMEM medium supplemented with 10% FBS, 2 mM nonessential amino acids, 100 U/mL penicillin and 100 mg/mL streptomycin. This cell culture was maintained according to the procedure described previously by us [13].
3.2.4. Cell Growth Inhibition Assay in Tumour Cell Lines and Primary Porcine Liver Cells
The effects of the compounds on the in vitro growth of human tumor cell lines and non-tumor porcine liver primary cells were evaluated according to the procedure adopted by the NCI (USA) in their “In vitro Anticancer Drug Discovery Screen”, using the SRB assay to assess cell growth [5,6,7].
For the cell lines, exponentially growing cells were obtained by plating 1.5 × 105 cells/mL for MCF-7, 0.75 × 105 cells/mL for A375-C5 and NCI-H460, and 5.0 × 105 cells/mL for HepG2, followed by 24 h of incubation. For the primary porcine liver cells, cells were incubated in 96-well plates at a density of 5.0 × 105 cells/mL, and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin.
Following a 24 h period to allow cells to adhere, cells were treated for 48 h with six serial dilutions of each test compound, starting from a maximum concentration of 125 µM. Following the 48 h treatment period, adherent cells were fixed in situ, washed and stained with SRB. The bound stain was solubilized and the absorbance was measured at 492 nm in the case of MCF-7, NCI-H460 and A375-C5 cells and at 515 nm in the case od HepG2 and PLP1 primary cells, in a plate reader (Biotek Instruments Inc., Powerwave XS, Bedfordshire, UK). Dose response curves were obtained and the concentration of the diarylamine that inhibited 50% of the net cell growth (GI50) was calculated as described elsewhere [6,7,8]. Doxorubicin and ellipticine were used as positive controls of cell growth inhibition.
3.2.5. Analysis of Cell Cycle Distribution Profile
NCI-H460 cells were plated in 6-well plates at a final density of 1.5 × 105 cells/well and incubated at 37 °C for 24 h. Cells were then treated with compound 3c at its GI50 concentration (1.4 µM). As controls, cells were also incubated with medium only (blank) and with the compound’s vehicle (DMSO). Following 48 h of treatment, cells were fixed overnight in ice-cold 70% ethanol and re-suspended in PBS containing 0.1 mg/mL RNase A and 5 µg/mL propidium iodide. Cellular DNA content was analysed by flow cytometry using an Epics XL-MCL Coulter Flow cytometer (Brea, CA, USA) plotting at least 20,000 events per sample, as previously described [6,7]. The analysis of cell cycle distribution was subsequently performed using the FlowJo 7.2 software (Tree Star, Inc., Ashland, USA) after cell debris and aggregates exclusion.
3.2.6. Analysis of Apoptosis
Following 48 h treatment of NCI-H460 cells with compound 3c, as described above, the levels of apoptosis were analysed by flow cytometry using the Human Annexin V-FITC/PI apoptosis kit (Bender MedSystems, Vienna, Austria), according to the manufacturer’s instructions. Flow cytometry was carried out using an Epics XL-MCL Coulter flow cytometer plotting at least 20,000 events per sample, as previously described [6,7]. The data was analysed using the FlowJo 7.2 software (Tree Star, Inc.).