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    • For the SAR study human haspin kinase inhibitory activity

    2022-05-20

    For the SAR study, human haspin kinase inhibitory activity of the various compounds was evaluated using the same assay utilized for the HTS, except in the presence of varying test compound concentrations. DYRK2 kinase inhibitory activity was measured by P-incorporation into Woodtide peptide substrate in the presence of human DYRK2 containing an N-terminal GST-fusion protein and γP-ATP. Only one of the methoxys in appears necessary for potent haspin inhibition. When both of the methoxys were removed () inhibitory activity was dramatically reduced (). However, when only one of the methoxys was removed () or replaced with a methyl () or chlorine () potent activity (IC <100nM) was retained. The methoxy substituents of could also be replaced with hydroxyls (). Interestingly, transposition of the 7-methoxy to the 3-position () resulted in loss of activity. However, the 2-methoxy-3-chloro analog () was still quite active. The three aromatic rings that comprise the acridine also appeared necessary. Both and lacked haspin inhibitory activity. Next, the tether length between the thioether at the 9-position of the acridine and the primary amine was examined. Truncation () resulted in reduced activity, while addition of another methylene unit () had only a minimal impact on potency. Next, the contribution of the amine was examined. A secondary amine () was equally potent and a tertiary amine () only resulted in a slight decrease in activity. However, incorporation of the nitrogen into a phthalimide ( and ) resulted in a significant loss of activity. Finally, the thioether was examined (). Replacement of the thioether with an amine () or ether () was detrimental. But replacement of the sulfur with a methylene () retained potent inhibitory activity. The SAR for DYRK2 inhibition had many similarities to that observed for haspin inhibition with some notable exceptions. Both methoxy groups appear to be necessary for DYRK2 inhibitory activity. For example, removal of both methoxy groups () was very detrimental, while removal of one methoxy () still resulted in a significant erosion of potency. Likewise, replacement of one methoxy with a methyl () or a chlorine nae inhibitor () was not as tolerated compared to the results observed for haspin inhibition. Transposition of the 7-methoxy to the 3-position () also lead to loss in activity. Similarly, and unlike in the haspin SAR, removal of the 7-methoxy and addition of a 3-chloro group () was not tolerated for DYRK2 inhibition. The three aromatic rings that comprise the acridine also appear necessary for DYRK2 inhibition, with both and lacking activity. The effect of the tether length between the thioether at the 9-position of the acridine and the primary amine ( and ) was the same as previously observed for haspin inhibition. For DYRK2 inhibition the primary amine was better than secondary () or tertiary () amines. Interestingly, incorporation of the nitrogen into a phthalimide resulted in a less dramatic impact on DYRK2 inhibition compared to haspin and provided a moderately potent analog () that was 5.4-fold selective for DYRK2 verses haspin. Similar to the observations made with haspin inhibition, replacement of the thioether with an amine () or ether () was detrimental to DYRK2 inhibition, while replacement of the sulfur with a methylene () was tolerated, albeit with a fivefold reduction in potency. In conclusion, a SAR study of the acridine derivative , identified utilizing a recently developed HTS assay, was conducted for both haspin and DYRK2 inhibition. The study revealed that several structural features of , such as the three acridine aromatic rings, the presence of one or both methoxy groups, a three or four methylene tether between the thioether and the acridine, and a thioether or CH (but not an amine or ether) link to the acridine were necessary for both haspin and DYRK2 inhibition. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor LDN-209929 (, IC <60nM) with 180-fold selectivity verses DYRK2. In addition, a moderately potent DYRK2 inhibitor LDN-211848 (, IC <400nM) with a 5.4-fold selectivity verses haspin was also identified. Additional optimization of the acridine series, potentially utilizing co-crystallization of these inhibitors with haspin and DYRK2, might provide more potent and selective inhibitors for both kinases. In addition, these inhibitors will serve as valuable molecular probes to study the cellular functions of these kinases and their potential roles in various diseases.