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. 2011 Jan 7;9(1):127-35.
doi: 10.1039/c0ob00592d. Epub 2010 Nov 15.

Inhibition of the Histone Demethylase JMJD2E by 3-substituted Pyridine 2,4-dicarboxylates

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Free PMC article

Inhibition of the Histone Demethylase JMJD2E by 3-substituted Pyridine 2,4-dicarboxylates

Armin Thalhammer et al. Org Biomol Chem. .
Free PMC article

Abstract

Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone N(ε)-methyl lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that structure-based inhibitor design can enable selective inhibition of histone demethylases over related human 2OG oxygenases.

Figures

Fig. 1
Fig. 1
Crystallographic analyses on JMJD2A and PHD2. A. View from a crystal structure of JMJD2A in complex with NiII (substituting for FeII) and the inhibitor 2,4-PDCA (derived from PDB ID 2VD7). B. Active site of JMJD2A showing chelation of the active site metal (green) by the conserved iron-binding Hx[D/E] … H motif (His188, Glu190 and His276). Selected active site residues are shown. C. View from a crystal structure of the prolyl hydroxylase PHD2 in complex with MnII, a fragment of the hypoxia inducible factor 1α peptide substrate (not shown), and the 2OG cosubstrate analogue N-oxalyl glycine (NOG), a relatively non-specific 2OG oxygenase inhibitor (PDB ID 3HQR). D. Closeup view of the PHD2 active site showing chelation of MnII (green) by the conserved iron-binding Hx[D/E] … H motif (His313, Asp315 and His374).
Scheme 1
Scheme 1
A. The Nε-methyl lysine demethylation reaction catalysed by the 2OG-dependent JMJD2 histone demethylases, which preferentially act on tri- and di-Nε-methylated lysines. Demethylation of Nε-trimethyllysyl residues (1) proceeds via hydroxylation to give an unstable hemiaminal intermediate, which collapses spontaneously to release the demethylation product 2. The formation of the by-product formaldehyde can be measured spectrophotometrically using formaldehyde dehydrogenase and NAD+ in a coupled enzymatic assay. B. Structures of the 2OG cosubstrate (3), its analogues NOG (4) and 2,4-PDCA (5), and the targeted C-3 substituted 2,4-PDCA derivatives. The ligand atoms involved in chelation of the ferrous iron are shown in red and blue.
Scheme 2
Scheme 2
Synthesis of 3-bromopyridine 14. Reagents and conditions: (i) Br2 (0.9 eq.), 20% oleum, 165 °C, 24 h, 11 (12%), 12 (22%), 13 (35%); (ii) KMnO4 (5 eq.), NaOH (0.7 eq.), H2O, 110 °C, 3 h; (iii) cat. H2SO4, MeOH, reflux, 24 h, 53%; (iv) n-BuLi (1.1 eq.), THF, −78 °C, 1 h, or i-PrMgCl (1.1 eq.), THF, RT; then MeOH workup; (v) tetrahydropyran-2-ol (1.1 eq.), PPh3 (1.1 eq.), diisopropyl azodicarboxylate (1.1 eq.), RT, 2 h, 46%; (vi) methoxymethyl chloride (1.05 eq.), KOtBu (1.1 eq.), DMF–CH3CN, RT, 1 h; (vii) base (1.5 eq.), then excess CO2(s); base (1.5 eq.), then excess CO2(s); then HCl/dioxane; *Ratios as determined by 1H NMR.
Scheme 3
Scheme 3
Synthesis of pyridine dicarboxylate derivatives 34–36. Reagents and conditions: (i) Pd(OAc)2 (10 mol%), PPh3 (0.2 eq.), Cs2CO3 (1.1 eq.), DMF, 70 °C, 3–5 h, 60–75%; (ii) CuI (0.1 eq.), N-(n-butyl)imidazole (0.5 eq.), Cs2CO3 (2 eq.), toluene, MW, 140 °C, 1 h, 44%; (iii) NaOH, MeOH, H2O, 6 h-overnight, 49–75%.
Scheme 4
Scheme 4
Synthesis of pyridine dicarboxylate derivatives 37–50. Reagents and conditions: (i) Pd2dba3 (2 mol%), ligand L (6 mol%), base (1.4 eq.), toluene, MW, 110–150 °C; L = PPh3 or BINAP or 30: <5% conversion; L = DPEPhos: 11% yield; L = XantPhos: 77% yield; (ii) NaOH, MeOH–H2O, RT; (iii) Pd2dba3 (2 mol%), XantPhos (6 mol%), Cs2CO3 (1.4 eq.), toluene, MW, 110 °C, 12 h, 69%; (iv) CF3CO2H, CH2Cl2, 0 °C to RT, 89%; (v) NaOH, MeOH–H2O, 2 h, RT, 67%.

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