5. Materials and Methods
5.1. General
Acetonitrile was distilled twice from P2O5 and CaH2. Commercially available reagents were used without further purification. The Boc protection of 4-aminopyridine was carried out following the literature [25].
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP. To a solution of di-tert-butyl dicarbonate (3 mmol) in acetonitrile (3 cm3) at room temperature 4-aminopyridine (3 mmol) was slowly added. This mixture was then allowed to stir for 3 h at room temperature. The solvent was evaporated and the crude 4-[N-(tert-butoxycarbonyl)amino]pyridine (>95%) was used in the electrolyses without further purification. Rf (30% ethyl acetate in light petroleum ether) 0.20; 1H NMR (200 MHz, CDCl3) δ 1.53 (s,9H), 6.9 (bs, 1H), 7.32 (dd, J = 4.8, 1.6 Hz, 2H), 8.45 (dd, J = 4.8, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.2, 81.7, 112.3, 145.6, 150.3, 151.9; EIMS, m/z: 194 (M.+, 1%), 137 (2%), 121 (5%), 120 (8%), 94 (50%), 78 (4%), 57 (100%).
5.2. Electrochemical ALkylation of N-Boc-4AP
Constant current electrolyses (I = 25 mA cm−2) were performed under a nitrogen atmosphere, at 20°C, using an Amel Model 552 potentiostat equipped with an Amel Model 731 integrator. All the experiments were carried out in a divided glass cell separated through a porous glass plug filled up with a layer of gel (i.e., methyl cellulose 0.5% volume dissolved in DMF-Et4NPF6 1.0 mol dm−3); Pt spirals (apparent areas 0.8 cm2) were used both as cathode and anode. MeCN-Et4NPF6 0.1 mol dm−3 was used as solvent-supporting electrolyte system (catholyte: 20 cm3; anolyte: 5 cm3). 1 mmol of N-Boc-4-aminopyridine was present in the catholyte. After 145 C were passed, the current was switched off and 1 mmol of alkylating agent was added to the catholyte. The solution was kept under stirring at room temperature for 2 hours; then the solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography, using a mixture of ethyl acetate/light petroleum ether 2/8 in volume, obtaining the pure products.
Flash column chromatography was carried out using Merck 60 kieselgel (230–400 mesh) under pressure. GC-MS measurements were carried out on SE 54 capillary column using a Fisons 8000 gas chromatograph coupled with a Fisons MD 800 quadrupole mass selective detector. 1H and 13C NMR spectra were recorded at room temperature using a Bruker AC 200 spectrometer using CDCl3 as internal standard.
tert-Butyl (octyl)pyridin-4-ylcarbamate
1a. Rf (80% ethyl acetate in dichloromethane) 0.60; 1H NMR (200 MHz, CDCl3) δ 0.88 (t, J=6.5 Hz,3H), 1.20–1.30 (m, 10H), 1.49–1.86 (m, 3H), 1.50 (s, 9H), 3.69 (app t, J=7.6 Hz,2H), 7.24 (dd, J = 6.2, 1.6 Hz, 2H), 8.51 (dd, J=6.2,1.6 Hz,2H); 13C NMR (50 MHz, CDCl3) δ 14.0, 22.6, 26.7, 28.2, 28.4, 29.1, 31.7, 48.7, 81.4, 118.8, 150.0, 150.1, 153.4.
tert-Butyl (3-phenylpropyl)pyridin-4-ylcarbamate
1b. Rf (50% ethyl acetate in light petroleum ether) 0.46; 1H NMR (200 MHz, CDCl3) δ 1.48 (s, 9H), 1.86–2.02 (m, 2H), 2.64 (t,J=7.6 Hz, 2H), 3.74 (app t, J=7.6 Hz,2H), 7.12–7.33 (m, 7H), 8.49 (dd, J=4.8,1.6 Hz,2H); 13C NMR (50 MHz, CDCl3) δ 28.2, 30.0, 33.0, 48.3, 81.6, 118.9, 126.1, 128.3, 128.5, 141.0, 149.7, 150.3, 153.4; EIMS, m/z: M.+ absent, 212 (5%), 107 (100%), 105 (5%), 91 (25%), 78 (27%), 77 (12%).
tert-Butyl (benzyl)pyridin-4-ylcarbamate
1c. Rf (60% ethyl acetate in light petroleum ether) 0.58; 1H NMR (200 MHz, CDCl3) δ 1.45 (s, 9H), 4.94 (s, 2H), 7.20–7.37 (m, 7H), 8.46 (dd, J=4.8, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 52.5, 82.1, 118.2, 126.3, 127.3, 128.7, 137.7, 150.2, 150.1, 153.5; EIMS, m/z: M.+ absent, 227 (4%), 183 (14%), 91 (100%), 78 (7%), 57 (51%).
tert-Butyl (2,6-dichlorobenzyl)pyridin-4-ylcarbamate
1d. Rf (50% ethyl acetate in light petroleum ether) 0.60; 1H NMR (200 MHz, CDCl3) δ 1.48 (s, 9H), 5.29 (s, 2H), 7.02–7.21 (m, 5H), 8.41 (dd, J=4.8,1.6 Hz,2H); 13C NMR (50 MHz, CDCl3) δ 28.2, 46.7, 81.7, 121.5, 128.6, 129.5, 131.7, 136.1, 148.1, 149.9, 153.3; EIMS, m/z: 352 (M.+, 1%), 252 (3%), 163 (6%), 161 (30%), 159 (42%), 78 (76%), 51 (100%).
tert-Butyl (4-fluorobenzyl)pyridin-4-ylcarbamate
1e. Rf (50% ethyl acetate in light petroleum ether) 0.49; 1H NMR (200 MHz, CDCl3) δ 1.45 (s, 9H), 4.89 (s, 2H), 6.97–7.22 (m, 6H), 8.47 (app d, J=6.0 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 51.8, 82.2, 116.6 (d, J = 21.5 Hz), 118.4, 128.1 (d, J=8.0 Hz), 133.4 (d, J = 3.2 Hz), 149.9, 150.3, 153.4, 162.2 (d,J=245.4 Hz); EIMS, m/z: 302 (M.+, 1%), 245 (4%), 201 (53%), 108 (100%), 78 (42%), 57 (100%).
tert-Butyl (4-trifluoromethylbenzyl)pyridin-4-ylcarbamate
1f. Rf (50% ethyl acetate in light petroleum ether) 0.41; 1H NMR (200 MHz, CDCl3) δ 1.46 (s, 9H), 4.99 (s, 2H), 7.20–7.36 (m, 4H), 7.61 (app d, J=8.4 Hz, 2H), 8.49 (app d, J=6.2 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 52.2, 82.5, 118.1, 124.0 (q, J=271.9 Hz), 125.7 (q, J = 3.7 Hz), 126.6, 129.7 (q, J = 32.3 Hz), 141.9, 149.8, 150.4, 153.3; EIMS, m/z: M.+ absent, 251 (9%), 158 (34%), 145 (2%), 78 (25%), 69 (9%), 57 (100%).
tert-Butyl (2-oxo-2-phenylethyl)pyridin-4-ylcarbamate
1 g. Rf (60% ethyl acetate in light petroleum ether) 0.50; 1H NMR (200 MHz, CDCl3) δ 1.46 (s, 9H), 5.09 (s, 2H), 7.26 (d, J=6.4 Hz, 2H), 7.48–7.68 (m, 3H), 7.99 (d, J=8.2 Hz, 2H), 8.47–8.55 (m, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 55.5, 82.5, 118.7, 127.9, 128.9, 133.9, 134.7, 150.2, 153.2, 193.7.
tert-Butyl (2-(4-fluoropheny)-2oxoethyl)pyridin-4-ylcarbamate
1 h. Rf (50% ethyl acetate in light petroleum ether) 0.50; 1H NMR (200 MHz, CDCl3) δ 1.46 (s, 9H), 5.05 (s, 2H), 7.15–7.27 (m, 4H), 7.99–8.06 (m, 2H), 8.52 (dd, J = 5.0, 1.4 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 55.4, 82.6, 116.2 (d, J=22.0 Hz), 118.8, 130.6 (d, J=9.4 Hz), 131.1 (d, J=3.2 Hz), 150.1, 150.2, 153.2, 166.2 (d, J=256.1 Hz), 192.2.
tert-Butyl (2-(4-chloropheny)-2oxoethyl)pyridin-4-ylcarbamate
1i. Rf (20% ethyl acetate in dichloromethane) 0.30; 1H NMR (200 MHz, CDCl3) δ 1.45 (s, 9H), 5.04 (s, 2H), 7.21 (dd, J=4.6,1.6 Hz, 2H), 7.50 (d, J=8.8 Hz, 2H), 7.93 (d, J=8.0 Hz, 2H), 8.51 (dd, J=4.6,1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 55.4, 82.6, 118.8, 129.3, 133.0, 140.5, 149.9, 150.3, 153.1, 192.7.
tert-Butyl (2-(4-methoxypheny)-2oxoethyl)pyridin-4-ylcarbamate
1j. Rf (40% ethyl acetate in dichloromethane) 0.50; 1H NMR (200 MHz, CDCl3) δ 1.46 (s, 9H), 3.90 (s, 3H), 5.04 (s, 2H), 6.99 (d, J=9.0 Hz, 2H), 7.24 (dd, J=4.8,1.6 Hz, 2H), 7.97 (d, J=9.0 Hz, 2H), 8.50 (dd, J=4.8, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 55.1, 55.5, 82.3, 114.1, 118.7, 127.8, 130.2, 150.2, 153.3, 164.1, 192.0.
5.3. Deprotection of Compounds 1a–j
To a solution of 1 (1 mmol) in CH2Cl2 (5 cm3), kept at 0°C, 1 cm3 of CF3COOH was added. This mixture was allowed to stir for 3 h at 0°C. The solution was then mixed with aqueous sodium carbonate till pH 8 and extracted with ethyl acetate. The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 2.
N-(Octyl)pyridin-4-amine
2a. Rf (20% dichloromethane in ethyl acetate) 0.16; 1H NMR (200 MHz, CDCl3) δ 0.89 (t, J=7.2 Hz, 3H), 1.25–1.34 (6H), 1.61–1.70 (m, 2H), 2.89–3.12 (m, 2H), 3.15–3.24 (m, 2H), 5.43–5.48 (m, 2H), 6.57 (d, J=5.2 Hz, 2H), 8.11 (d, J=5.2 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 14.1, 22.6, 27.0, 28.8, 29.2, 29.2, 31.8, 42.9, 107.4, 155.2.
N-(3-Phenylpropyl)pyridin-4-amine
2b. Rf (ethyl acetate) 0.46; 1H NMR (200 MHz, CD3CN) δ 1.91–2.06 (m, 2H), 2.71 (t, J=7.4 Hz, 2H), 3.17–3.27 (m, 2H), 4.9 (bs, 1H), 6.62–6.66 (m, 2H), 7.14–7.46 (m, 5H), 7.45–7.97 (m, 2H); 13C NMR (50 MHz, CD3CN) δ 29.8, 32.5, 42.0, 107.3, 125.9, 128.3, 128.4, 141.0, 141.5, 157.8; EIMS, m/z: 212 (M.+, 1%), 107 (100%), 91 (43%), 78 (13%).
N-(Benzyl)pyridin-4-amine
2c. Rf (ethyl acetate) 0.57; 1H NMR (200 MHz, CDCl3) δ 4.46 (d, J=6.0 Hz, 2H), 6.7 (bs, 2H), 7.1 (bs, 1H), 7.21–7.41 (m, 5H), 8.0 (bs, 2H); 13C NMR (50 MHz, CDCl3) δ 46.8, 107.4, 127.2, 127.6, 128.8, 137.7, 148.6, 154.0; EIMS, m/z: 184 (M.+, 15%), 183 (15%), 107 (5%), 91 (100%), 78 (16%).
N-(2,6-Dichlorobenzyl)pyridin-4-amine
2d. Rf (ethyl acetate) 0.38; 1H NMR (200 MHz, CD3CN) δ4.59 (s, 2H), 6.0 (bs, 1H), 6.68 (dd, J=5.2, 1.4 Hz, 2H), 7.27–7.47 (m, 3H), 8.41 (app d, J=5.4 Hz, 2H); 13C NMR (50 MHz, CD3CN) δ 42.2, 107.4, 128.7, 130.4, 132.8, 136.0, 147.3, 154.6; EIMS, m/z: 256 (M.+ + 4, 1%), 254 (M.+ + 2, 7%), 252 (M.+, 14%), 162 (10%), 160 (67%), 158 (100%), 78 (37%).
N-(4-Fluorobenzyl)pyridin-4-amine
2e. Rf (ethyl acetate) 0.27; 1H NMR (200 MHz, CDCl3) δ 4.38 (d, J = 5.4 Hz, 2H), 5.1 (bs, 1H), 6.5 (bs, 2H), 7.02–7.10 (m, 2H), 7.30–7.34 (m, 2H), 8.2 (bs, 2H); 13C NMR (50 MHz, CDCl3) δ 46.3, 107.8, 115.8 (d, J=21.5 Hz), 129.0 (d, J=8.1 Hz), 133.1, 148.5, 153.8, 166.8 (d, J=205.9 Hz),; EIMS, m/z: 202 (M.+, 5%), 107 (16%), 109 (100%), 78 (16%).
N-(4-Trifluoromethylbenzyl)pyridin-4-amine
2f. Rf (ethyl acetate) 0.25; 1H NMR (200 MHz, CDCl3) δ 4.51 (d, J=6.0 Hz, 2H), 6.2 (bs, 1H), 6.6 (bs, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.67 (d, J=8.0 Hz, 2H), 8.1 (bs, 2H); 13C NMR (50 MHz, CDCl3) δ 45.4, 107.8, 124.4 (q, J=271.0 Hz), 125.4 (q, J=3.9 Hz), 128.4 (q, J = 31.9 Hz), 127.7, 143.2, 146.7, 155.0; EIMS, m/z: 252 (M.+, 80%), 183 (11%), 159 (100%), 107 (52%), 78 (31%).
1-(4-Methoxyphenyl)-2-(pyridin-4-ylamine)ethan-1-one
2 g. Rf (50% ethyl acetate in ethanol) 0.15; 1H NMR (200 MHz, CDCl3) δ 3.90 (s, 3H), 4.57 (d, J = 3.8 Hz, 2H), 4.6 (bs, 1H), 6.58 (d, J = 6.2 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 7.98 (d, J = 9.0 Hz, 2H), 8.19 (d, J = 6.2 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 48.2, 55.6, 108.1, 114.1, 127.2, 130.2, 150.8, 153.6, 164.5, 191.6.
5.4. Alkylation of Compounds 2a,c,e
To a solution of 2 (1 mmol) in anhydrous DMSO (2 cm3), kept at rt under N2, 1.5 mmol of t-BuOK was added. This mixture was allowed to stir for 20 min at rt; then 1 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. The solution was then mixed with water and extracted with dichloromethane. The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 3.
N-Benzyl-N-octylpyridin-4-amine
3ac
. Rf (80% ethyl acetate in ethanol) 0.38; 1H NMR (200 MHz, CDCl3) δ 0.86–0.92 (m, 3H), 1.20–1.40 (m, 10H), 1.63–1.70 (m, 2H), 3.42 (app t, J = 7.6 Hz, 2H), 4.59 (s, 2H), 6.51 (d, J = 5.2 Hz, 2H), 7.14–7.38 (m, 5H), 8.18 (d, J = 5.2 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 14.1, 22.6, 26.9, 27.0, 29.2, 29.4, 29.7, 31.8, 50.7, 53.4, 106.9, 126.2, 127.3, 128.8, 136.8, 148.9, 153.6.
N-(4-Fluorobenzyl)-N-octylpyridin-4-amine
3ae. Rf (ethyl acetate) 0.40; 1H NMR (200 MHz, CDCl3) δ 0.86–0.92 (m, 3H), 1.23–1.35 (m, 10H), 1.62–1.72 (m, 2H), 3.42 (app t, J = 7.8 Hz, 2H), 4.58 (s, 2H), 6.56 (app d, J = 6.4 Hz, 2H), 6.99–7.16 (m, 4H), 8.17 (app d, J = 6.4 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 14.1, 19.2, 22.6, 26.9, 29.2, 29.3, 31.7, 51.3, 53.3, 107.4, 116.1 (d, J = 21.7 Hz), 127.9 (d, J = 8.1 Hz), 130.9 (d, J = 3.5 Hz), 144.9, 155.3, 162.3 (d, J = 246.5 Hz).
N-(4-Fluorobenzyl)-N-(3-phenylpropyl)pyridin-4-amine
3be. Rf (ethyl acetate: n-hexane: methanol 50 : 33 : 17) 0.48; 1H NMR (200 MHz, CDCl3) δ 1.91–2.03 (m, 2H), 2.68 (t, J = 7.4 Hz, 2H), 3.43 (app t, J = 7.8 Hz, 2H), 4.54 (s, 2H), 6.44 (app d, J = 5.4 Hz, 2H), 6.96–7.36 (m, 9H), 8.15 (bs, 2H); 13C NMR (50 MHz, CDCl3) δ 28.1, 33.0, 49.9, 52.9, 106.8, 115.8 (d, J = 21.6 Hz), 126.3, 127.9 (d, J = 8.0 Hz), 128.3, 128.6, 132.1 (d, J = 3.2 Hz), 140.7, 147.9, 153.7, 162.1 (d, J = 245.8 Hz).
5.5. Dialkylation of 4-Aminopyridine
To a solution of 4AP (1 mmol) in anhydrous DMSO (2 cm3), kept at rt under N2, 2 mmol of t-BuOK was added. This mixture was allowed to stir for 20 min at rt; then 2 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. The solution was then mixed with water and extracted with dichloromethane. The solvent was removed under reduced pressure and the mixture was purified by flash chromatography, yielding pure compound 3.
N,N-Di(3phenylpropyl)pyridin-4-amine
3bb
. Rf (80% ethyl acetate in dichloromethane) 0.20; 1H NMR (200 MHz, CD3CN) δ 1.84–1.99 (m, 4H), 2.65 (t, J = 7.5 Hz, 4H), 3.29 (t, J = 7.5 Hz, 4H), 6.30 (d, J = 5.6 Hz, 2H), 7.17–7.31 (m, 10H), 8.13 (d, J = 5.6 Hz, 2H); 13C NMR (50 MHz, CD3CN) δ 28.3, 33.1, 49.5, 106.4, 126.2, 128.3, 128.5, 141.1, 140.7, 152.4.
N,N-Dibenzylpyridin-4-amine
3cc
. Rf (80% ethyl acetate in ethanol) 0.32; 1H NMR (200 MHz, CDCl3) δ 4.67 (s, 4H), 6.58 (dd, J = 4.8, 1.6 Hz, 2H), 7.19–7.40 (m, 10H), 8.20 (dd, J = 4.8, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 53.2, 107.1, 126.4, 127.4, 128.9, 136.8, 150.2, 153.9.
N,N–Di(4-fluorobenzyl)pyridin-4-amine
3ee
. Rf (80% ethyl acetate in dichloromethane) 0.40; 1H NMR (200 MHz, CDCl3) δ 4.61 (s, 4H), 6.56 (dd, J = 5.0, 1.6 Hz, 2H), 6.99–7.19 (m, 8H), 8.22 (dd, J = 5.0, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 52.5, 107.1, 115.8 (d, J = 21.6 Hz), 128.1 (d, J = 8.0 Hz), 132.3 (d, J = 3.2 Hz), 150.3, 153.6, 162.2 (d, J = 245.8 Hz).
N,N-Di(4-trifluoromethylbenzyl)pyridin-4-amine
3ff
. Rf (80% ethyl acetate in dichloromethane) 0.25; 1H NMR (200 MHz, CDCl3) δ 4.73 (s, 4H), 6.58 (dd, J = 5.0, 1.6 Hz, 2H), 7.36–7.59 (m, 8H), 8.26 (dd, J = 5.0, 1.6 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 53.1, 107.1, 123.3 (q, J = 3.7 Hz), 123.8 (q, J = 272.3 Hz), 124.5 (q, J = 3.8 Hz), 129.7, 131.4 (q, J = 32.4 Hz), 137.7, 150.4, 153.4.
5.6. Biological Assays
5.6.1. Antifungal Assay
Organisms. For the antifungal evaluation, strains obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), the German Collection of Microorganisms (DSMZ, Braunschweig, Germany), and the Pharmaceutical Microbiology Culture Collection (PMC, Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy) were tested. The strains were Candida albicans (ATCC 10231, ATCC 10261, ATCC 24433, ATCC 90028, 3153, PMC 1002, PMC 1011, and PMC 1030), C. parapsilosis ATCC22019, C. parapsilosis DSM 11224, C. tropicalis DSM 11953, C. tropicalis PMC 0908, C. tropicalis PMC 0910, C. glabrata PMC 0805, C. krusei DSM 6128, and C. krusei PMC 0613, Cryptococcus neoformans (DSM 11959, PMC 2102, PMC 2107, PMC 2111, and PMC 2136), dermatophytes (Trichophyton mentagrophytes DSM 4870, T. mentagrophytes PMC6509, Microsporum gypseum DSM 7303, and M. gypseum PMC 7331). All of the strains were stored and grown in accordance with the procedures of the Clinical and Laboratory Standards Institute (CLSI) [26, 27].
Antifungal Susceptibility Assays. In vitro antifungal susceptibility was evaluated using the CLSI broth microdilution methods [26, 27]. Fluconazole and Amphotericin B were used as reference drugs. The final concentration ranged from 0.125 to 64 μg/mL. The compounds were dissolved previously in DMSO at concentrations 100 times higher than the highest desired test concentration and successively diluted in test medium in accordance with the procedures of the CLSI [28]. Microdilution trays containing 100 μL of serial twofold dilutions of compounds in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) were inoculated with an organism suspension adjusted to attain a final inoculum concentration of 1.0×103–1.5×103 cells/mL for yeasts and 0.4×104–5×104 CFU/mL for dermatophytes. The panels were incubated at 35°C and observed for the presence of growth at 48 h (Candida spp.) and 72 h (C. neoformans and dermatophytes).
The minimal inhibitory concentration (MIC) was, for yeasts, the lowest concentration that showed ≥ 50% growth inhibition compared with the growth control and, for dermatophytes, the lowest concentration that showed ≥ 80% growth inhibition compared with the growth control. The MIC100 was the lowest drug concentration that prevented 100% of growth with respect to the untreated control. According to CSI protocols, the fluconazole MIC50 and the amphotericin B MIC100 were calculated (22,23). The results were expressed as the geometric mean (G M) of the MIC values.
5.6.2. Antiprotozoal Assay
For the evaluation of antiprotozoal and cytotoxic activity an integrated panel of microbial screens and standard screening methodologies were adopted as previously described [29] on the following organisms: chloroquine-resistant P. falciparum K 1-strain; L. infantum MHOM/MA (BE)/67 amastigote stage; suramin-sensitive Trypanosoma brucei Squib-427 strain; Trypanosoma cruzi Tulahuen CL2 (benznidazole-sensitive) strain; human fetal lung fibroblast cells (MRC-5 SV2).
All assays were performed in triplicate. Compounds were tested at 5 concentrations (64, 16, 4, 1, and 0.25 μg/mL) to establish a full dose titration and determine the IC50 (inhibitory concentration 50%). The final in-test concentration of DMSO did not exceed 0.5%, which is known not to interfere with the different assays [29].