The identification of novel kinase inhibitor
The identification of novel kinase inhibitor scaffolds is highly desirable in order to develop selective kinase inhibitors. Small-molecule inhibitors of Interleukin-2-inducible T-cell kinase (Itk) that are based on the 3-aminopyridin-2-one fragment 1 have been reported. Despite derivatisation of 1 yielding potent Itk inhibitors, this 3-aminopyridin-2-one fragment is poorly represented in small-molecule protein kinase modulators. This fragment is capable of forming multiple hydrogen bonds to the backbone of the hinge region, possesses good physicochemical properties and contains several suitable points for derivatisation (Fig. 1), representing a good starting point for the synthesis of a kinase-targeted fragment library. Herein, we report the synthesis and kinase profiling of a 3-aminopyridin-2-one based fragment library against a panel of 26 protein kinases. This led to the rapid identification of aminopyridin-2-one derivatives as a novel class of inhibitors of monopolar spindle 1 (MPS1) and Aurora kinases, two kinases intimately involved in mitosis.
Results and discussion A fragment library based on 3-aminopyridin-2-one was synthesised from 5-bromo-2-methoxypyridin-3-amine by first introducing a variety of aromatic and heteroaromatic groups at the 3-aminopyridin-2-one C5-position under standard Suzuki cross-coupling conditions, followed by deprotection of the 3-amino-2-methoxy-pyridine intermediates by generating TMS-I in situ (Figure 2). 3-Amino-6-methyl-5-(pyridin-4-yl)pyridin-2-one (15) was synthesised in two steps from the phosphodiesterase inhibitor Milrinone (13). Hydrolysis of the nitrile 13 to the carboxamide intermediate 14 by treatment with concentrated sulfuric 5-Methyl-UTP mg at 120 °C, was followed by a Hofmann rearrangement to give compound 15 (Fig. 2). In order to test the biochemical activity of our fragment library, a panel of 26 structurally diverse protein kinases was used. The panel represented members of the TK, CMGC, CAMK, CK1 and AGC families (Supplementary data Table 1). By choosing this panel, we recognised that the exact composition of a kinase panel could affect selectivity scores in particular for small assay panels, as suggested by Karaman et al. Compounds 1–3, 5–8 and 15 were initially screened at a single concentration of 100 µM using a mobility shift-based biochemical assay from Caliper Life Sciences. The 3-aminopyridin-2-one fragment 1 showed very little activity against our kinase panel with the exception of Aurora A and AKT2, 58% and 77% inhibition at 100 μM, respectively (Fig. 3, and Supplementary data Table 2). This may be expected from screening such a small fragment at a relatively low concentration, as even weak activity such as a Ki of 1 mM would give a ligand efficiency of 0.52. Introduction of an aromatic ring at the C5-position generally resulted in an increase in biochemical activity. Introduction of a N-methylpyrazole (compound 2) gave high percentage inhibition against a large number of kinases in the panel, resulting in a S(50%) of 0.77 (Supplementary data Table 2) showing good potency but poor selectivity. The phosphodiesterase inhibitor Amrinone (3), which contains a 4-pyridyl substitution, showed reasonable activity against four members of the panel (MPS1, CHK1, PKCζ and PKA), resulting in a S(50%) score of 0.08. Introduction of a methyl group at the C6-position of the aminopyridinone 3 gave compound 15 which showed low potency against the 26-kinase panel, only one kinase, Aurora B, was inhibited by>50% (77%) (Fig. 3, and Supplementary data Table 2). This may suggest the methyl group in compound 15 clashes with the protein-binding site, or induces an unfavourable conformation of the biaryl system. Retention of the exocyclic 3-NH2 hydrogen bond donor in 3, accompanying by the removal of the hydrogen bond donor of the pyridone ring by conversion to the 3-amino-2-methoxy-pyridine analogue 5 resulted in a similar inhibitory profile against MPS1 and Aurora B compared to compound 3 (Table 1). An increase in activity against Aurora A, MPS1, ABL and CHK2 was observed when the 4-pyridyl ring in compound 3 was substituted with a quinoline moiety, giving compound 6 (Table 1, Fig. 3). This analogue displayed a selectivity score S(50%) of 0.19 (Supplementary data Table 2). A significant increase in activity across the entire panel was observed by the introduction of a substituted 2-aminopyrimidine moiety. 2-Methylaminopyrimidine analogue 7 gave>50% inhibition against 23 members of our 26-kinase panel (Fig. 3) which resulted in a selectivity score S(50%) of 0.92 (Supplementary data Table 2). Substitution of 2-methylaminopyrimidine in 7 with 2-cyclopropylaminopyrimidine (compound 8) maintained activity against some members of the panel (AurA, MPS1, GSK3b, RSK1 PKD2) resulting in a selectivity score S(50%) of 0.35 (Supplementary data Table 2). The poor selectivity of the aminopyrimidine analogues 7 and 8 is possibly due to the aminopyrimidine acting as a hinge binding motif as previously reported.18, 35, 36, 37, 38 In order to validate these single point assay results, the dose-response curves for the compound libraries were obtained against Aurora A, Aurora B and MPS1 kinases. These kinases were selected because of their high Skinase(50%) against this compound library (Supplementary data Table 4) and their biological relevance in cancer cell proliferation and survival. The Aurora kinases are well recognised as attractive targets for cancer therapeutics and have extensively been investigated.39, 40 The inhibition of MPS1 has been also proposed as an effective method for treatment of human cancers, with a relatively limited number of small-molecule MPS1 modulators being reported.41, 42, 43, 44, 45, 46, 47