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note
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R = 11.69 min. Compound 2 (285 mg, 0.13 mmol) was dissolved in TFA/thioanisole/water/DCM (16:2:1:1, 10 mL) and stirred at room temperature for 2 h. The solvent was removed in vacuo and the resulting residue partitioned between water (20 mL) and Et...
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0032190770
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The solid phase synthesis of a 2-ALA derivative has been reported-see
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note
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4 cells per well. After 48 h, the culture medium was removed, and the wells were washed with PBS. The cells were incubated with freshly prepared solutions of 3 at 0.005, 0.01, 0.05, 0.01, and 0.5 mM. Serum-free medium (100 μL) containing varying prodrug concentrations was added to a designated series of wells. Each plate contained control wells with cells, but without added drug for determination of the background reading, and reference wells containing cells incubated with the same ALA concentrations. Fluorescence readings were taken at 4, 5, 7, 24, and 48 h after addition of 3, and the plates were incubated at 37 °C between measurements. The fluorescence signal from each well was measured using a Perkin-Elmer LS-50B spectrofluorimeter, with excitation at 405 nm and 635 nm emission, with slit widths set to 10 nm and an internal 530 longpass filter used on the emission side. The mean fluorescence was calculated after subtraction of the control values (i.e., without addition of ALA). For temperature studies, PAM212 were seeded in two 96-well plates, as before, and treated with 3 (0.3mM in clear media, 100 μL/well). The plates were wrapped in foil and incubated at 37 and 4 °C, respectively, for 12 h, whereupon the prodrug solution was removed. The cells were rinsed in PBS and clear media (100 μL) were added to each well. Generation of PpIX fluorescence was then monitored as before.
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