The initial beta carboline derivatives used in

The initial beta-carboline derivatives used in the in silico docking experiments were prepared using the procedure outlined in . Harmol, , was -alkylated with -Boc-protected alkylamines in the presence of cesium carbonate to give and . The carbamate protecting group was removed under acidic conditions to give and . Harmine, , could be -alkylated. However, phthalimide-protected alkylamines were required and a stronger base (i.e. NaH) was also needed to give and . Removal of the protecting group was accomplished using hydrazine hydrate to yield and . Haspin kinase inhibitory activity for the various compounds was assessed using the same assay utilized for the HTS, except in the presence of varying concentrations of test compounds., As anticipated and were inactive haspin inhibitors at 10μM. However, and were both active demonstrating IC values of 0.46 and 0.17μM, respectively (). Based on these results additional beta-carboline analogs were prepared using the general procedure outlined in . Indoles were either directly converted to derivative using MeNCHCHNO (generated from MeNO and MeNCH(OMe) at 85°C for 0.5h) or in a two step procedure (i.e. Vilsmeier–Haak followed by Henry reactions). The alkene and nitro groups were both reduced in the presence of lithium aluminum hydride (LAH) to give . A Pictet–Spengler reaction between and various aldehydes generated . In the case where R=CF, the hemiacetal CFCHOH(OEt) was used. Oxidation of the tetrahydro-beta-carboline with manganese dioxide gave . Finally as previous outlined, alkylation generated , which was de-protected to give . Several other beta-carboline derivatives were also prepared using the procedures outlined in . Intermediate was de-methylated using HBr in acetic dihydrofolate reductase to give phenol . This material was converted to triflate and then a methylsulfonamide was introduced utilizing a Pd-catalyzed reaction to yield . Removal of the protecting group gave amine . Likewise, intermediate was converted to the -butyl ether in the presence of MeNCH(O--Bu), which was again de-protected to liberate amine . Intermediate was also de-protected to give , which was converted to the tertiary amine through reductive amination. Finally, intermediate was converted to the secondary amine via the formamide . The additional compounds prepared were used to further explore the structure–activity relationship of for haspin inhibition (). Removal of the methyl at the 1-position () or replacement with an isopropyl () were detrimental, where as replacement with an ethyl ) was equivalent. Replacing the methoxy in the 7-position with a hydroxyl () was tolerated, but a fluorine (), methyl sulfonamide () or -butoxy () were detrimental. Transposition of the methoxy to the 5- or 6-positions ( and ) resulted in loss of activity, while introduction of the methoxy to the 8-position () was more tolerated. The 6,7- and 7,8-disubstituted analogs and demonstrated less activity. The primary amine also appeared to be optimal, where the secondary () and tertiary () amines were less active. Finally, in order to potentially improve metabolic stability, analogs and ) that replace the methyl group in the 1-position with a trifluoromethyl were evaluated. Gratifyingly, displayed an IC value of 100nM for inhibiting haspin. This compound was also assessed for in vitro metabolic stability in pooled mouse liver microsomes and demonstrated excellent stability with a of 341min and a CL of 3.8μL/min/mg protein suggesting that this compound may be useful as an in vivo probe., The beta-carboline series was also evaluated for inhibition of human DYRK2 using a previously described assay. Harmine and harmol gave IC values for DYRK2 inhibition of 0.69 and 1.5μM, consistent with previous studies. Introduction of the tethered amine onto the N-position substantially reduced the potency for DYRK2 inhibition in all cases examined (). For example, had an IC value for DYRK2 of 15μM and demonstrated 150-fold selectivity for haspin over DYRK2.