Based on these results we investigated the cytotoxicity of prodrug 12 and its bioorthogonal activation by reaction with Tz in HT1080 fibrosarcoma cancer cells. In comparison to the parent drug CA4, a 700-fold higher concentration of cTCO-caged 12 (910 nM) was needed to reduce the cell count by 50% (IC50) over the course of a 72 h treatment (Fig. ), also demonstrating the high stability of the bis(carbamate)-linked cTCO-DMEDA-prodrug under physiologically relevant conditions. In situ reaction of 12 with DMT (7) and PymK ( ) at a non-toxic Tz concentration of 5 µM restored drug cytotoxicity via Tz-triggered bioorthogonal release of CA4, shifting the IC50 from 910 nM to 4.2 nM (220-fold) and 6.2 nM (150-fold), respectively (Fig. ). As a point of reference, the cTCO-caged prodrug SQP33, currently being tested in phase 1 clinical trials, was reported to be 83-fold less cytotoxic than its parent drug doxorubicin.