Structure-guided design of purine-based probes for selective Nek2 inhibition

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.


N-Methyl-3-nitrobenzamide (S1)
To a stirred solution of 3-nitrobenzoyl chloride (2.0 g, 11 mmol) in DCM (15 mL) was added methylamine (33% in EtOH, 6.7 mL, 54 mmol) slowly over 15 min. After stirring at room temperature for 2 h the solvent was removed. The white residue was extracted into EtOAc (2 × 100 mL). The combined extracts were washed with aq. sodium bicarbonate (1 × 100 mL) and water (1 × 100 mL). The organic layer was dried (Na 2 SO 4 ) and concentrated to give the title compound as a white solid (1.93

Reduction of aryl-nitro derivatives to corresponding anilines (compounds S3 and S4)
To a stirred solution of compound S1 or S2 in anhydrous solvent as indicated was added 10% palladium on activated carbon. The resulting mixture was stirred under an atmosphere of H 2 at room temperature for 24 h. Alternatively, as indicated, hydrogen was replaced by ammonium formate (5 mol. equiv.), which was added to the suspension. The reaction mixture was filtered through a bed of Celite eluting with MeOH-DCM (1:9) to afford the title compound following removal of the solvent in vacuo.

Reduction of nitro-aryl thioamides (compounds S7 and S8)
To a stirred solution of the nitroaryl thioamide S5 or S6 in glacial acetic acid (25 mL/g) was added zinc powder. The resulting mixture was stirred under nitrogen at room temperature for 18 h. The suspension was filtered through Celite eluting with MeOH. After removal of the MeOH, the residual acetate salt was disolved in water (20 mL) and neutralised by dropwise addition of conc. ammonia solution until pH 8. After cooling for 1 h, the precipitated aniline was isolated by filtration and washed with water (30 mL).

N,N-Dimethyl-3-nitrobenzenesulfonamide (S9)
To a stirred solution of dimethylamine hydrochloride (1.1 g, 14 mmol) and DIPEA (3.6 mL, 20 mmol) in DCM (10 mL) was added 3-nitrobenzenesulfonyl chloride (1.0 g, 4.5 mmol) over 15 min, and the reaction mixture was stirred at room temperature for 18 h. The mixture was concentrated to give a viscous oil that was extracted into EtOAc (100 mL) and washed with aqueous sodium bicarbonate solution (100 mL). The aqueous layer was re-extracted with EtOAc (100 mL) and the organic layers were combined, dried (Na 2 SO 4 ) and concentrated to give the title compound as an off-white solid (1.0

Precursor to homocarboxamide 15 2-(3-Nitrophenyl)-N-methylacetamide (S11)
To a solution of 3-nitrophenylacetic acid (0.50 g, 2.8 mmol) in THF (5 mL) was added SOCl 2 (0.22 mL, 3.1 mmol) and the reaction mixture was stirred at room temperature for 2 h. Solvents were removed in vacuo and the brown residue was re-dissolved in THF (4 mL), which was added dropwise to a solution of methylamine (40% aq., 0.70 mL, 12.6 mmol) over 15 min, and the reaction mixture was stirred for 18 h at room temperature. Solvents were removed, and the resulting brown residue was extracted into DCM (50 mL). The extract was washed with aq. NaHCO 3 (50 mL) and water (50 mL). The organic layer was dried (Na 2 SO 4 ) and concentrated to give the title compound as a brown solid (

Precursors to secondary ureas 16-18; and further examples
Preparation of nitrophenyl-ureas (S13-S17) To a stirred solution of the required 3-or 4-nitrophenyl isocyanate (1.00 g, 6.1 mmol) in anhydrous THF (50 mL) was added the required amine (7.3 mmol) and the resulting mixture was stirred at ambient temperature under an atmosphere of nitrogen for 3 h. The mixture was concentrated in vacuo and purified as indicated below.

Reduction of aryl-nitro derivatives (compounds S18-S22)
To a stirred solution of compound S13-S17 in MeOH (10 mL) was added 10% palladium on activated carbon. The resulting mixture was stirred under an atmosphere of H 2 at room temperature for 24 h. Alternatively, as indicated, hydrogen was replaced by ammonium formate (5 mol. equiv.), which was added to the suspension. The product was purified by filtration through a bed of Celite eluting with 10% MeOH in DCM, to afford the title compound following removal of the solvent in vacuo.

2-(3-Nitrophenyl)-N-(3-dimethylaminopropyl) acetamide (S48)
To a stirred solution of 3-nitrophenylacetic acid (2.0 g, 11 mmol) in THF (15 mL) was added SOCl 2 (0.88 mL, 12 mmol) and the reaction mixture was stirred at room temperature for 2 h. Solvents were removed in vacuo and the residual oil was redissolved in THF (15 mL). N,N-dimethyl-1,3-propanediamine (6.9 mL, 55 mmol) was added dropwise over 15 min and the reaction mixture was stirred for an additional 18 h. The mixture was concentrated to give a viscous oil that was extracted