Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythm

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of known enzyme inhibitors. Until now, the development of photocontrolled protein kinases inhibitors proves to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity by photoswitching ligands, together with the accompanying modulation of cellular circadian rhythms in which by CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a hit reduced azopurine with a circadian period lengthening effect more pronounced than its parent molecule, longdaysin. Altogether, the results presented here provide challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.