Based on compound RC-1 we synthesized additional compounds (Fig. ), RC-2 with a CH2 group replacing the oxygen nearest the b-carbon, their analogous acrylamides IR-1 and IR-2, and the non-covalent analog NC-1. We also synthesized the cyanoacrylamide RC-3 (Fig. ), replacing the Ca hydrogens with methyl groups. Similar dimethylated cyanoacrylamides were reported to have improved cellular permeability . We evaluated the ability of the compounds to induce degradation of BTK in human cell lines. We incubated Mino cells with the compounds and followed BTK levels after 24 hours by western blot (Fig. , Supp. Fig. ). The non-covalent PROTAC NC-1 showed the highest degradation potency with DC50 = 2.2 nM (Maximal degradation -Dmax = 97%). The irreversible acrylamides IR-1 and IR-2, and the cyanoacrylamide RC-3 followed closely with DC50's under 10 nM and Dmax near 90%. The cyanoacrylamides RC-1 and RC-2 were less potent. Similar trends were observed in Ramos cells (Supp. Fig. ). We conducted metabolomics studies to estimate if cellular penetration and stability may contribute to the relative potencies (Supp. Table ). NC-1 and RC-3 reached an effective concentration which was ~2 times higher than IR-2 and ~10 times higher than RC-2. Therefore, the lower potency of RC-2 can at least in part be explained by lower permeability or stability. We followed the rate of BTK degradation facilitated by this compound series via a time course experiment in Ramos and Mino cells (Supp. Fig. ). The rates of degradation correlated well with the DC50 observed after 24 hours, with NC-1, IR-1, IR-2 and RC-3 degrading BTK within 2-4 hours, while RC-2 and RC-1 required 6-24 hours to reach maximum degradation.