A solution of BL660 (17.3 mg, 0.052 mmol, 1 equiv.) and pyridine (4.23 mL, 0.052 mmol, 1 equiv.) in anhydrous CH2Cl2 (1.5 mL) was cooled to -20 °C under the N2 atmosphere. Cyanuric fluoride (4.85 mL, 0.056 mmol, 1.08 equiv.) was slowly added to the reaction and stirred at the same temp. for ~40 min. A solution of compound 6 (28.6 mg, 0.146 mmol, 2.8 equiv.) in anhydrous CH2Cl2 (0.5 mL) was added dropwise at the same temperature. The reaction was slowly brought to room temperature and was stirred for an additional 1.5 h at room temperature. After quenching with crushed ice, the organic layer was removed, and the aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, concentrated, and purified using silica gel column chromatography (eluent: 1:4 v/v EtOAc:Hexanes) to afford BL660-NTR in 10% yield (2.7 mg, 0.005 mmol). MTT Assay using MTX-Me2 and MTX-Ph i Pr2 48-well plates were seeded with 30,000 A549 cells per well (500 µL of 60,000 cells/mL) and incubated at 37 °C with 5% CO2 for 24 hours. Media was removed and fresh serum-free RPMI 1640 containing 0, 0.001, 0.01, 0.1, 1, 10 μL of MTX-Me2 or MTX-(Pr i Pr)2 were added (0.1% DMSO final v/v). The media was removed after 24 hours incubation and replaced with 500 µL of a 20∶1 mixture of serum-free RPMI 1640 and (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL stock in PBS). The cells were incubated for 2 hours under the same condition and then the medium was removed and replaced with DMSO (500 µL/well). The absorbance of each well was recorded at 555 nm on a microplate reader. Viability was calculated by the absorbance relative to the vehicle control.