Synthesis, proapoptotic screening, and structure-activity relationships of novel aza-lupane triterpenoids

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 18, 2010, Pages 5389-5393

  Mar, Aye Aye ^a   Szotek, Erika L ^a   Koohang, Ali ^a   Flavin, William P ^a   Eiznhamer, David A ^a   Flavin, Michael T ^a   Xu, Ze Qi ^a  

^a Advanced Life Sciences Inc   (United States)

Author keywords

Apoptosis; Aza lupane triterpenoid; Betulinic acid; Cytotoxicity

Indexed keywords

BETULIC ACID; CYTOCHROME C;

ANTINEOPLASTIC ACTIVITY; APOPTOSIS; ARTICLE; CELL DEATH; CONTROLLED STUDY; CYTOTOXICITY TEST; DRUG DESIGN; DRUG SCREENING; DRUG STRUCTURE; DRUG SYNTHESIS; HUMAN; HUMAN CELL; IC 50; STRUCTURE ACTIVITY RELATION;

ANTINEOPLASTIC AGENTS; APOPTOSIS; CELL LINE, TUMOR; CYTOTOXINS; DRUG SCREENING ASSAYS, ANTITUMOR; HUMANS; NEOPLASMS; STRUCTURE-ACTIVITY RELATIONSHIP; TRITERPENES;

EID: 77956174639     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.07.120     Document Type: Article

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20. 1H NMR δ 0.66 (s, 3H), 0.76 (s, 3H), 0.68 (d, J = 11.0 Hz, 1H), 0.78-1.03 (m, 4H), 0.89 (s, 3H), 0.92 (s, 3H), 0.98 (s, 3H), 1.02-1.27 (m, 5H), 1.31-1.50 (m, 8H), 1.57 (d, J = 10.0 Hz, 3H), 1.67 (t, J = 11.5 Hz, 1H), 1.81-1.98 (m, 3H), 2.32 (m, 3H), 2.58 (q, J = 6.5 Hz, 2H), 2.64 (q, J = 6.5 Hz, 2H), 3.08 (m, 3H), 3.51 (d, J = 10.5 Hz, 1H), 4.19 (br s, 1H), 4.74 (s, 2H), 6.65 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 9.12 (br s, 1H). MS (ESI+) m/e 577 (M+H).
21. 4 per well) were plated onto a 96-well plate the evening before treatment in triplicate. Test compounds were initially dissolved in DMSO at 10 mM concentration and serially diluted in growth media to various concentrations. Each compound was tested at five different concentrations, 2, 10, 25, 50 and 75 μM, and each concentration was replicated in four wells. The negative control (0 μM of test compound) contained the same amount of DMSO as that of the highest test concentration. Upon treatment with the test compound solutions in growth media, they were incubated for 72 h for all the cell lines with exception for OVCAR which was treated for 120 h. Next, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium, inner salt) reagent was added and incubation was continued for 1.5-4 h at 37 °C. During the incubation, MTS is bioreduced to formazan by viable cells. The inhibitory effects were obtained by measuring the absorbance of the formazan at 490 nm on a Wallac Victor II plate reader and calculated by subtracting the absorbance measured at the same wavelength from DMSO-treated cells.
22. 5 per well) were plated in a 60 mm dish the evening before treatment. Upon treatment with 7.5 μM test compound solutions in growth media without FBS, the cells were incubated at 37 °C for 16 h. Next 10% FCS was added to prevent cell damage and improve the efficiency of cell pelleting during subsequent centrifugation steps. After trypsinization and cell pelleting, the Nexin staining solution (including Annexin V and 7-ADD) was added and incubation was continued on ice for 20 min. Apoptosis was measured on a Guava instrument by setting the gates as described in the manufacturer's protocol to establish quadrants and to measure the population of viable, mid apoptotic and late apoptotic/necrotic cells in each quadrant.