Polymerizations of n-butyl acrylate under stable free radical polymerization (SFRP) conditions, using TEMPO as the nitroxide mediator, are reported at reaction temperatures between 133 and 135 °C. The polymerizations proceed in a living manner under both bulk and miniemulsion conditions with polydispersities of around 1.3. Plots of number-average molecular weight vs conversion and ln([M]0/[M]) vs time are linear, indicating a controlled polymerization. It is argued that the ability of a particular nitroxide to control the polymerization of n-butyl acrylate is not only due to a favorable equilibrium constant (K) between the dormant and active polymeric species in the system but also due to the amount of excess nitroxide that is present. In these experiments excess TEMPO is controlled by the continuous addition of a dilute solution of ascorbic acid or an ascorbic acid derivative, leading to successful n-butyl acrylate polymerizations. Chain extension of a homopoly(n-butyl acrylate) prepared under miniemulsion conditions is demonstrated.

MLN4924 is a selective inhibitor of the NEDD8-activating enzyme (NAE) and has advanced into clinical trials for the treatment of both solid and hematological malignancies. In contrast, the structurally similar compound 1 (developed by Millennium: The Takeda Oncology Company) is a pan inhibitor of the E1 enzymes NAE, ubiquitin activating enzyme (UAE), and SUMO-activating enzyme (SAE) and is currently viewed as unsuitable for clinical use given its broad spectrum of E1 inhibition. Here, we sought to understand the determinants of NAE selectivity. A series of compound 1 analogues were synthesized through iterative functionalization of the purine C6 position and evaluated for NAE specificity. Optimal NAE specificity was achieved through substitution with primary N-alkyl groups, while bulky or secondary N-alkyl substituents were poorly tolerated. When assessed in vitro, inhibitors reduced the growth and viability of malignant K562 leukemia cells. Through this study, we have successfully identified a series of sub-10 nM NAE-specific inhibitors and thereby highlighted the functionalities that promote NAE selectivity.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562MLN, R-U937MLN) were selected. R-K562MLN and R-U937MLN cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562MLN cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.