In order to study the

In order to study the role of haspin’s kinase activity in mitosis (and other cellular processes) and its potential role in cancer, we sought to identify and optimize inhibitors. Utilizing a recently developed time-resolved fluorescence resonance energy transfer (TR-FRET) high throughput screening (HTS) assay with histone H3 peptide as substrate and a europium-labeled phosphospecific monoclonal antibody for detecting phosphorylated substrate (H3T3ph), the acridine derivative was discovered as a potent inhibitor (; IC=0.010μM). Kinase profiling of revealed potent DYRK2 inhibitory activity as well. Herein, we describe the structure–activity relationship (SAR) of the acridine series for both haspin and DYRK2 inhibition. The synthesis of many of the acridine analogs was accomplished using the procedure outlined in . 2-Bromobenzoic acids were coupled to anilines using a copper-mediated procedure to give . Cyclization of to 9-chloroacridines was accomplished using phosphorus oxychloride. Treatment of with PS in the presence of DMPU gave . Alternatively, Sephin1 was cyclized to ketone in the presence of polyphosphoric acid (PPA), which was subsequently treated with Lawesson’s reagent with microwave (MW) heating at 110°C to produce . The thioketone could be alkylated with various amino-protected alkylbromides (BrCH(CH)Y; Y=NHBoc, NMeBoc, or NPhthalimide) in the presence of base (KOH) and the phase-transfer catalyst tetrabutylammonium iodide (TBAI) in a mixture of toluene and water to give . Boc-protected analogs of (Y=NHBoc or NMeBoc) upon treatment of 4N HCl in a mixture of 1,4-dioxane and methanol gave (Z=NH or NHMe). Alternatively for analogs of with Z=NH, they could also be prepared directly from via alkylation. Acridine analogs where the alkylamine groups were connected through an O or NH were prepared according to the procedure illustrated in . Ketone was converted to as previously described. Then a nucleophilic aromatic substitution with a mono-N-protected diamine followed by removal of the protecting group gave . Ketone was also alkylated with -Boc-protected 1-amino-3-bromopropane in the presence of KOH and phase-transfer catalyst (i.e., TBAI) followed by de-protection to give . Acridine analogs where the alkylamine group was connected through a methylene were prepared using the method described in . Diphenylamine derivative was condensed with acetic acid to give acridine analog . The methyl substituent in the 9-position was oxidized with selenium dioxide to give aldehyde . Addition of the anion of an -Boc-protected alkyne gave alcohol . Exposure of to reducing conditions (Pd/C and EtSiH) resulted in concomitant reduction of the alkyne and alcohol. Finally, removal of the protecting group on the amine yielded . The synthesis of a tetrahydroacridine analog is outlined in . β-Ketoester was allowed to react with 4-anisidine, , to produce . Cyclization of in sulfuric acid gave ketone . This material was converted to the corresponding thioketone . Alkylation with 1-amino-3-bromopropane hydrobromide gave . Finally, a 2-phenylquinoline analog was synthesized accord to the procedure outlined in . The acetophenone derivative was coupled with benzamide in the presence of a catalytic amount of CuI to give . Base-mediated cyclization of gave . Conversion of this material to the thiol analog was accomplished with Lawesson’s reagent. Then alkylation and de-protection as previously described for other analogs yielded . Compound was initially profiled for functional inhibitory activity against a panel of two hundred and seventy kinases at 10μM. The results demonstrated that this compound was quite selective and only inhibited ten of the other kinases by ⩾90% (Supplementary Table S1 and Figure S1)., IC values were determined for these kinases (), with only five being potently inhibited (IC <1μM). DYRK2 was the most sensitive of these kinases (IC=2nM).,