Optimisation of a triazolopyridine based histone demethylase inhibitor yields a potent and selective KDM2A (FBXL11) inhibitor

A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC50 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity.

triethylamine (87.1 mL, 130 mmol) in tetrahydrofuran (43.5 mL) was evacuated and refilled with nitrogen (x 5), trimethylsilylacetylene (7.6 mL, 55.0 mmol) was then added and the resulting solution stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo to give a black gum that was taken up in dichloromethane (40 mL) and filtered. The filtrate was concentrated in vacuo and purified by flash column chromatography (9:1 heptane:ethyl acetate) to give 12 as an orange oil (4.94 g, 85%). R f 0.20 (9:1 heptane:ethyl acetate); δ H (400 MHz, CDCl 3  Compound s1 was synthesized according to a literature procedure and its physical and spectroscopic properties are in agreement to those reported. 2
The organic layers were washed with brine (2 x 15 mL) then combined, dried over sodium sulfate and concentrated in vacuo to give a brown oil that was purified by flash column chromatography (10% à 30% ethyl acetate in cyclohexane) to give 13 as a white solid (41 mg, 53%).

Representative Syntheses of Amide Derivatives
Compound 31 was synthesized according to a literature procedure and its physical and spectroscopic properties are in agreement to those reported. 3
The reaction mixture was concentrated in vacuo then purified on a 10 g strong cation exchange column, washing with methanol (100 mL) then eluting with methanolic ammonia (100 mL of a 2M solution) to give s3 as an orange gum (789 mg, 97%

Lysine Demethylase (KDM) Catalytic Turnover Assay using Alphascreen
All reagents were from Sigma Aldrich unless otherwise stated and were of the highest purity. was dispensed into each well using a Multidrop (Thermo Scientific) and allowed to incubate with the compounds for 15 min at room temperature. After transfer of substrate (5 µL) the enzyme reaction was progressed for the indicated time period (see Table S1 for final concentrations and assay parameters). The enzyme reaction was stopped after the indicated time by addition of 5 µL of Stop Solution (30 mM EDTA, 800 mM NaCl in assay buffer).
Streptavidin Donor beads (0.08 mg/ml) and Protein-A conjugated acceptor beads (0.08 mg/ml) were pre-incubated for 1 hour with an antibody to the product methyl mark (Table   S1) and the presence of biotin-H3-product was detected by addition of 5 µL of the preincubated alphascreen beads (final concentrations of 0.02 mg/ml with respect to acceptor and donor beads). Detection was allowed to proceed for 1 hour at room temperature and the assay plates read in a BMG Labtech Pherastar FS plate reader. Data were normalized to the no enzyme control and the IC 50 determined from the nonlinear regression curve fit using GraphPad Prism 5.

Crystallography
Crystals of JMJD2A were grown as described. 4 The crystals were soaked with compound 14a by mixing 0.5 µL of 10mM compound 14a (in ethylene glycol) with 1.5 µL reservoir solution (0.1M BIS-TRIS pH 5.9, 0.15M ammonium sulfate, 11% PEG3350) and adding it to the crystals. The crystals were directly flash frozen in liquid nitrogen after incubating them for 12 hours. A dataset was collected at the Diamond Light Source, beamline I04-1 with a Pilatus 2M CCD detector at 0.92Å. Data were integrated with XDS 5 and scaled with AIMLESS. 6 Ligand dictionaries for compound 14a where generated with GRADE. 7 Refinement was done with PHENIX 8 and after several cycles of manual rebuilding with COOT, 9 the model converged to a Rfactor/ Rfree of 20.7 and 25.9%, respectively. The quality of the model was validated with MOLPROBITY. 10 For data collection and refinement statistics see Table S2. Numbers in parentheses refer to the highest resolution shell.