1816 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 Table 2. Inhibition Data for JMJD2A with Selected Compounds by the MALDI-TOF Assaya compd JMJD2E IC50 by FDH assay (μM) JMJD2E IC50 by MALDI-TOF (μM) JMJD2A IC50 by MALDI-TOF (μM) 7f 5.4 21 12 7c 8.6 61 13 7e 11.7 38 20 10k 14.7 47 14 7d 14.7 43 17 8m 14.8 59 13 10e 16.6 25 10 10a 37.1 76 33 7i 82.9 ND ND 8b 185.1 ND ND 9h 326.5 ND ND a JMJD2A was used at 2 μM, 2OG at 10 μM and ARKme3STGGK peptide at 10 μM. JMJD2E IC50s were also determined by MALDI-TOF MS for comparison. ND = not determined. Figure 9. Binding of 10a to JMJD2A likely interferes with both 2OG and histone substrate binding. Superimposition of H3K9me3 substrate (orange and pink sticks) with JMJD2A 3 Ni(II) 3 Zn(II).10a structure (PDB ID 2OQ6) is shown. The surface of JMJD2A (blue) illustrates separate K9me3 (pink) and cosubstrate (NOG, cyan, replaces 2OG in this structure) subpockets. Compound 10a (yellow sticks) occupies a large hydrophobic pocket adjacent to the sub- strate binding cleft. It may interfere with binding of the Thr11 side chain of the H3K9 substrate. of the binding pockets. Binding within the identified JMJD2 subpocket may be a way of achieving more potent inhibition with other series of “template” inhibitors that bind to the active site iron. These compounds may be useful as chemical tools to probe the in vivo roles of the JMJD2 demethylases. However, those inhibitors screened against FIH did inhibit HIF asparaginyl hydroxylation, demonstrating that further expansion of this, or related series of compounds that chelate the active site iron, is required to achieve higher selectivity within the JmjC subfamily. In this regard, it is notable that although both FIH and the JMJD2 demethylases contain a relatively large subpocket, there are significant differences in the residues that form these subpockets. Experimental Section All reagents and solvents were purchased from Aldrich or Alfa Aesar and used without further purification. Reactions were monitored by TLC, which was performed on precoated Rose et al. aluminum-backed plates (Merck, silica 60 F254). Melting points were determined using a Leica Galen III hot-stage melting point apparatus and microscope. Infrared spectra were recorded from Nujol mulls between sodium chloride discs on a Bruker Tensor 27 FT-IR spectrometer. NMR spectra were acquired using a Bruker DPX500 NMR spectrometer. Chemical shifts (δ) are given in ppm, and the multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Coupling constants J are given in Hz to the nearest 0.1 Hz. High resolution mass spectra (HRMS) were recorded using Bruker MicroTOF. The purity of all compound synthesized were >95% as deter- mined by analytical reverse-phase HPLC (Ultimate 3000). The chemical synthesis and purity of 3a and 3b are described in the Supporting Information. The 73 N-oxalyl-D-tyrosine deriva- tives were purchased from ChemOvation (Horsham, UK). The identity and purity of the lead compounds were confirmed by 1H NMR, HRMS, and HPLC (for all compounds), and these data (for compounds 6d, 7c, 7d, 7e, 7f, 7i, 8b, 8m, 9h, 10a, 10e, and 10k) are given in the Supporting Information. The catalytic domain of human JMJD2E (residues 1-337) was produced as N-terminally His6-tagged protein in Escherichia coli and purified by Ni-affinity chromatography as reported.18 JMJD2E inhibition was assessed using a FDH coupled assay, as reported.9 All compounds were initially tested at 100 μM, and the initial rates of demethylation measured by measuring NADH production using an Envision multilabel reader (Perkin-Elmer, Waltham, MA). For JMJD2A, a MALDI-TOF MS based assay was used becuase JMJD2A was not optimized for analysis in our current FDH assay. JMJD2A 2 μM, Fe(II) 10 μM, and ascorbate 100 μM in 50 mM HEPES pH 7.5 with inhibitor stock DMSO solutions where final inhibitor concentrations varied but final DMSO concentration was always 5% of assay mix were incubated for 15 min at 25 C, after which time reactions were initiated by addition of 2OG (10 μM) and peptide (10 μM), followed by 30 min incubation at 37 C. Reactions were quenched with methanol 1:1 (v/v) followed by addition of four volumes of 20 mM triammonium citrate. The diluted assay mixture (1 μL) was then mixed with 1 μL of R-cyano-4-hydroxycinnamic acid (the MAL- DI-TOF-MS matrix) and spotted onto a MALDI-TOF-MS plate.18 The relative intensities of different methylation states observed in the mass spectra were then used to calculate percen- tage demethylation. IC50s were calculated from the variation in percentage demethylation at different inhibitor concentrations. FIH and PHD2 assays were carried out as reported.27,34 The binding of compounds to JMJD2E was evaluated by nondena- turing ESI-MS as described.9 His-tagged JMJD2E was desalted using a Bio-Spin 6 Column (Bio-Rad, Hemel Hempstead, UK) in 15 mM ammonium acetate pH 7.5. The stock solution was diluted with the same buffer to a final concentration of 100 μM. FeSO4 3 7H2O was dissolved in 20 mM HCl at a concentration of 100 mM. This was then diluted with Milli-Q water to give final working concentrations of 100 μM. The protein (15 μM) was mixed with 1 equiv of Fe(II) and 1 equiv of inhibitor and incubated for 30 min at 37 C prior to nondenaturing ESI-MS analysis. For competition experiments, the protein was mixed with equimolar amounts of Fe(II) and two inhibitors each at concentration of 15 μM and incubated for 30 min at 37 C prior to nondenaturing ESI-MS analysis. Data were acquired on a Q-TOF mass spectrometer (Q-TOF micro, Micromass, Altrinc- ham, UK) interfaced with a Nanomate (Advion Biosciences, Ithaca, NY) with a chip voltage of 1.70 kV and a delivery pressure 0.25 psi (1 psi = 6.81 kPa). The sample cone voltage was typically 80 V, with a source temperature of 40 C and with an acquisition/ scan time of 10 s/1 s. Calibration and sample acquisition were performed in the positive ion mode in the range of 500-5000 m/z. The pressure at the interface between the atmospheric source and the high vacuum region was fixed at 6.60 mbar. External instru- ment calibration was achieved by using sodium iodide. Data were processed with MASSLYNX 4.0 (Waters). The limit of detection