28–7.34 (m, 1H, Harom), 7.41–7.47 (m, 1H, Harom),7.52–7.59 (m, 1H, Harom), 7.92–7.99 (m, 2H, Harom), 8.06–8.11 (m, 1H, H-1), 8.44 (s, 1H, H-6); EI-MS m/z: 362 (M+, 100 %); Anal. calcd. for C21H22N4S: C, 69.58; H, 6.12; N, 15.46; S, 8.84. Found: C, 69.54; H, 6.07; N, 15.40; S, 8.82. 12-(3-(N,N-dimethylamino)propyl)-12(H)-pyrido[2,4-e]quino[3,4-b][1,4]thiazine (7e) Yield 58 %; an oil;
1H NMR (CDCl3, 500 MHz) δ (ppm): 1.63–1.78 (m, 2H, CH2 CH 2CH2), 1,98 (s, 6H, N(CH3)2), 2.18–2.24 (t, J = 7.2 Hz, 2H, (CH3)2NCH 2), 4.01–4.12 Selleck AZD6244 (t, J = 7.3 Hz, 2H, NCH2), 7.04–7.11 (m, 1H, H-11), 7.28–7.36 (m, 1H, Harom),7.41–7.48 (m, 1H, Harom), 7.53–7.61 (m, 1H, Harom), 7.98-8.01 (m, 2H, Harom), 8.08–8.14 (m, 1H, H-1), 8.46 (s, 1H, H-6); EI-MS m/z: 336 (M+, 100 %); Anal. calcd. for C19H20N4S: C, 67.83; H, 5.99; N, 16.65; S, 9.53. Found: C, 67.74; H, 5.93; N, 16.61; S, 9.50. Antiproliferative assay in vitro Cell culture The synthesized compounds were evaluated for their anticancer activity using two cultured cell lines: SNB-19 (human glioblastoma, DSMZ – German Collection of Microorganisms and Cell Cultures, Selleck Fosbretabulin Braunschweig, Germany) and C 32 (human amelanotic melanoma, ATCC—American Type Culture Collection,
Rockville, MD, USA). The cultured cells were kept at 37 °C and 5 % CO2. The cells were seeded (1 × 104 cells/well/100 μl D-MEM supplemented with 12 % FCS and streptomycin and penicillin) using 96-well plates (Corning). WST-1 assay Antiproliferative effect of compounds 4 and 7 was determined using
the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). This colorimetric assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells, Protein kinase N1 leading to formazan formation. After exposure to tested compounds (at concentrations between 0 and 100 μg/ml) for 72 h, cells were incubated with WST-1 (10 μl) for 2 h, and the absorbance of the samples against a background control was read at 450 nm using a microplate reader. Results are expressed as means of at least two independent experiments performed in triplicate. Acknowledgments The study is supported by the Medical University of Silesia (Grant KNW-1-073/P/1/0). Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Amaral L, Kristiansen JE (2000) Phenothiazines: an alternative to conventional therapy for the initial management of suspected multi-drug resistant tuberculosis. Int J Antimicrob Agents 14:173–CCI-779 cost 176PubMedCrossRef Bansode TN, Shelke JV, Dongre VG (2009) Synthesis and antimicrobial activity of some new N-acyl substituted phenothiazines. Eur J Med Chem 44:5094–5098PubMedCrossRef Clarke FH, Silverman GB, Wotnick CM, Sperber N (1961) 3-Azaphenothiazine and dialkylaminoalkyl derivatives.