5-Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji-C Domain-Containing Protein 5

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.

(a) Overview of the JMJD5:20i complex structure (2.13 Å resolution); (b) active site view of the JMJD5:20i complex structure reveals that the pyridine heterocycle of 20i is positioned to interact with the indole side chain of W310 via π-stacking (distance from the centers of the pyridine and W310 phenyl rings: 3.7 Å), that the C4 carboxylate of the 2,4-PDCA core is positioned to interact with the side chain amino group of K336 (2.5 Å) and the side chain hydroxyl group of Y272 (2.6 Å), and that the C2 carboxylate is positioned to interact with the side chain amide group of N327 (2.7 and 2.9 Å) and the indole NH group of W414 (3.5 Å). In addition, the C2 carboxylate is positioned to chelate the Mn ion (2.3 Å) together with the pyridine N atom (2.3 Å). The Mn ion is also complexed by the side chain carboxylate of D323 (2.2 Å) and the side chain imidazole Nτ atoms of H321 (2.2 Å) and H400 (2.4 Å); a water molecule ligates the Mn ion (2.4 Å). The 2-methoxyphenyl group of 20i occupies a conformation in which its para C-H is positioned to interact with the indole side chain of W248 via a σ-π interaction (distance Cpara to the center of the W248 phenyl ring: 3.7 Å).
(a) Overview of the JMJD5:20j complex structure (2.27 Å resolution); (b) active site view of the JMJD5:20j complex structure reveals that the pyridine heterocycle of 20j is positioned to interact with the indole side chain of W310 via π-stacking (distance from the centers of the pyridine and W310 phenyl rings: 3.6 Å), that the C4 carboxylate of the 2,4-PDCA core is positioned to interact with the side chain amino group of K336 (2.5 Å) and the side chain hydroxyl group of Y272 (2.7 Å), and that the C2 carboxylate is positioned to interact with the side chain amide group of N327 (2.7 and 2.8 Å) and the indole NH group of W414 (3.5 Å). In addition, the C2 carboxylate is positioned to chelate the Mn ion (2.2 Å) together with the pyridine N atom (2.3 Å). The Mn ion is also complexed by the side chain carboxylate of D323 (2.2 Å) and the side chain imidazole Nτ atoms of H321 (2.2 Å) and H400 (2.2 Å); a water molecule ligates the Mn ion (1.8 Å). The 2-methoxyphenyl group of 20j occupies a conformation in which its para C-H is positioned to interact with the indole side chain of W248 via a σ-π interaction (distance Cpara to the center of the W248 phenyl ring: 3.4 Å).
Purifications were performed using an automated Biotage Isolera One purification machine ( Infrared (IR) spectroscopy was performed using a Bruker Tensor-27 Fourier transform infrared (FT-IR) spectrometer. High-resolution mass spectrometry (HRMS) was performed using electro-spray ionization (ESI) mass spectrometry (MS) in the positive or negative ionization modes employing a Thermo Scientific Exactive mass spectrometer (ThermoFisher Scientific); data are presented as a mass-to-charge ratio (m/z).
Single crystal X-ray diffraction data were collected using an Oxford Diffraction SuperNova diffractometer (Rigaku). Structures were solved using SUPERFLIP software 20 and refined using the CRYSTALS software suite [21][22]

General Procedure B
To dimethyl 26 and an N-alkylamine (3.0 equiv.) at ambient temperature. Nitrogen gas was bubbled through the reaction mixture for 15 min, the reaction mixture was then placed into a preheated sand bath (170 °C) and stirred for 24 h under a nitrogen atmosphere. The reaction mixture was cooled to ambient temperature, concentrated, and purified by column chromatography to afford the desired purified dimethyl 5aminoalkyl pyridine-2,4-dicarboxylate which was used in the next reaction according to General Procedure C.

General Procedure C
A procedure reported for the synthesis of C3 substituted pyridine-2,4-dicarboxylate derivatives was followed: [18][19] To a solution of a 5-substituted dimethyl pyridine-2,4-dicarboxylate (1.0 equiv.) in methanol (0.2 M, HPLC grade) was added an aqueous solution of lithium hydroxide (0.4 M, 2.8 equiv.) under an ambient atmosphere at 0 °C. The reaction mixture was slowly warmed to ambient temperature overnight (14 -18 h). The methanol was then removed under reduced pressure and the remaining aqueous reaction mixture was extracted three times with dichloromethane (the organic extracts were discarded). The aqueous phase was acidified (pH ≈ 7.0 to 7.7) using Dowex ® 50XW8 (H + -form, mesh 200-400), filtered, and lyophilized to afford the solid C5 substituted pyridine-2,4-dicarboxylate. The crude product was sufficiently pure as judged by 1 H and 13 C NMR and used without further purification in the biological assays. pKa-values for the 2,4-PDCA derivatives were not determined, thus, some might have been isolated as the corresponding mono-or dilithium salts; note that some 2,4-PDCA derivatives contain trace amounts of acetic acid, as confirmed by 1 H and 13 C NMR analysis, which has likely been introduced into the samples during lyophilization.