Structure‐Activity Relationship and Crystallographic Studies on 4‐Hydroxypyrimidine HIF Prolyl Hydroxylase Domain Inhibitors

Abstract The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.


Preparation of tPHD2 (residues 181 -426)
In brief, cDNA encoding for the catalytic domain of tPHD2 (181-426 residues) was cloned into the pET28a(+)/ pET24a(+) vectors (Novagen), as reported, [8] to enable production of tPHD2 (residues 181-426) protein with/without an N-terminal His6-tag. The tPHD2 (residues 181-426) encoding construct was transformed into the Escherichia coli BL21 DE3 cell line; protein production was induced with 0.5 mM isopropyl-b-D-thiogalactosidase (3-5 hr at 28℃). Cells were harvested and lysed by sonication in 20 mM Tris-HCl (pH 7.0) and 0.3 M NaCl; soluble protein (about 5% total soluble extract) was purified by immobilized Ni ion affinity chromatography using pentadentate tris-carboxymethyl ethylene diamine resin followed by cleavage of the His6-tag by thrombin (or alternately by cation exchange chromatography) with a final purification by gel filtration chromatography. [8] The protein was exchanged into 50 mM Tris-HCl buffer (pH 7.5) and concentrated to % 40 mg/ml. The protein was of > 95% purity, as determined by SDS-PAGE analysis and electrospray ionization mass spectrometric analysis.

X-ray Crystallography
Crystallisation procedures were as reported. [1][2]9] Crystals of truncated PHD2 in complex with 8 were grown in 0.2 M ammonium formate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 30% PEG 4000 and were cryoprotected by transferring to a solution of mother liquor supplemented with 25% (v/v) glycerol. Data were collected at 100K using synchrotron radiation at the Diamond Light Source (DLS) beamline I03 and were processed by using MOSFLM and SCALA. [10], [11] The structure was solved by molecular replacement using PHASER (search model PDB ID 4BQX) [12] and refined by PHENIX using the maximum-likelihood function. [13], [14] Iterative cycles of model building in COOT and refinement proceeded until the Rcryst/Rfree values converged. [15] Studies on the optimization of PHD-ligand crystallization conditions will be reported in due course.
RapidFire-MS PHD2 hydroxylation assays [1] Inhibition of PHD2 activity was assessed by mass spectrometry. % conversion = 100 x hydroxylated / (hydroxylated + non-hydroxylated peptide) IC50 data were determined from non-linear regression plots using GraphPad prism 6.0. The level of +16 (methionine residue oxidation) as observed in the no enzyme control was around 4 -5%. All data was normalized to no enzyme control. 2

RapidFire-MS FIH hydroxylation assays
Tris(hydroxymethyl)aminomethane was from Fisher; all other reagents were from Sigma Aldrich and of the highest available purity. Ferrous ammonium sulphate (FAS) was prepared freshly each day as a 400 mM stock solution in 20 mM HCl, this was then diluted to 1 mM in deionized water. [16] L-ascorbic acid (50 mM) and 2OG stock solutions (10 mM) were prepared freshly every day in deionized water, as for the PHD2 assays. The synthetic consensus ankyrin peptide (HLEVVKLLLEAGADVNAQDK-CONH 2 ) was synthesized by GL Biochem (Shanghai) Ltd and dissolved to 1 mM in deionized water. [16][17] FIH was prepared as reported. [2,18] 20 μL FIH (100 nM) in the assay buffer (50 mM Tris-HCl pH 7.8, 50 mM NaCl) was pre-incubated for 15 minutes in the presence of the inhibitors and the enzyme reaction initiated by addition of 20 μL substrate (200 μM L-ascorbic acid, 20 μM Fe(II) sulfate, 10 μM synthetic ankyrin peptide and 20 μM 2OG). [16] After 15 minutes the reaction was quenched by addition of 4 μL 10% formic acid and the reaction transferred to a RapidFire RF360 high throughput sampling robot. Samples were aspirated under vacuum onto a C4 SPE cartridge. After an aqueous washing step (0.1% (v/v) aqueous formic acid), to remove non-volatile buffer components from the C4 SPE cartridge , peptide was eluted in an organic washing step (85% acetonitrile in water, 0.1% formic acid) and injected into an Agilent peptide substrate and the hydroxylated peptide product; the peak area data for extracted ion chromatograms were integrated using RapidFire Integrator software. % conversion of substrate to product was calculated in Microsoft excel and IC 50 curves generated using Graph pad Prism.

RapidFire-MS KDM4A hydroxylation assays
The potency of 4-hydroxypyrimidines for KDM4A was assessed by RapidFire mass spectrometry (RF-MS). Truncated KDM4A was prepared as reported. [19] The . [20] LAA was prepared freshly each day to a concentration of 50 mM in deionized water, ferrous ammonium sulphate was prepared freshly each day by dissolving to 400 mM in 10 mM HCl and then dilution to 1 mM in deionized water, 2OG was prepared freshly each day by dissolving in 10 mM in deionized water. The KDM4A RF-MS assay was performed in 384-well plate format in assay buffer (50 mM MES pH 7.0). All reagent dispenses were performed using a multidrop reagent dispenser (Thermo). Dilutions of compounds were prepared using an ECHO 550 acoustic dispenser (Labcyte) and dry dispensed (250 nL) in duplicate into 384-well polypropylene plates (Greiner Bio-One); DMSO (250 nL) was dispensed into columns 12 and 2, 4-pyridinedicarboxylic acid (a known JmjC KDM inhibitor) [21] (250 nL of 10 mM) was dispensed into column 24 as a no enzyme control. KDM4A (300 nM) was IC50 data were determined from non-linear regression plots using GraphPad prism 6.0

MALDI-TOF MS vCPH Hydroxylation Assays
Recombinant viral collagen prolyl hydroxylase (vCPH) was prepared and the assay was conducted as reported . [24]

NMR Experiments
Nuclear Magnetic Resonance (NMR) spectra were recorded using a Bruker AVIII 700 MHz NMR spectrometer equipped with a 5-mm inverse cryoprobe using 3 mm MATCH NMR tubes (Cortectnet). The relaxation delay was 2 s and the 1 JCH was set to 160 Hz. A 6.8 ms Q3.1000 180degree pulse was used and selective irradiation was applied at the selective chemical shift.

General Considerations For Synthesis
All reactions involving moisture-sensitive reagents were carried out under a nitrogen atmosphere using standard vacuum line techniques. Glassware was oven dried and cooled under nitrogen before use. Commercial anhydrous solvents used in reactions and HPLC grade solvents were employed for work-up and chromatography. Water was purified using an Elix UV-10 system. Aqueous solutions were made using de-ionized water. Thin layer chromatography (TLC) was carried out using Merck (Darmstadt, Germany) silica gel 60 F254 TLC plates. TLC visualization was carried out under UV light and stained with one of three stains; ninhydrin, potassium permanganate, or anisaldehyde.
Chromatographic purifications were carried out using a Biotage ® (Uppsala, Sweden) Isolera One or Biotage ® SP4 flash purification system, using Biotage ® pre-packed SNAP columns. Reactions were monitored using an Agilent (Cheshire, UK) 1200 series, 6120 quadrupole LC-MS system using a Merck Chromolith ® Performance RP-18 HPLC column. Deuterated solvents were obtained from Sigma-Aldrich, and 1 H NMR spectra were obtained using Bruker AVANCE AVIII HD 400 nanobay (400 MHz) machine or a machine Bruker AV500 (500M Hz) with a 13 C cryoprobe. All signals are described in δ ppm with multiplets being denoted as singlet, doublet, triplet, quartet, and multiplet using the abbreviations s, d, t, q, and m, respectively. Chemical shifts in presented NMR spectra were referenced using residual solvent peaks with coupling constants, J, reported in hertz Elmer 241 Polarimeter were used to obtain optical rotations. All compounds were highly purified (est. >95% by 1 H and 13 C NMR).
All chemicals, reagents, and solvents were obtained from Sigma-Aldrich (Dorset, UK) and used without further purification. HPLC grade solvents were used for reactions, chromatography, and work-ups.
General Procedure for ethyl ester amide coupling 6 (1 equiv.), the relevant amine (1 equiv.) and DABACO-(AlMe3)2 (0.8 equivs) were added to a microwave vial, which was then was flushed with N2 and evacuated (3 times) before the addition of anhydrous THF (2 ml). The reaction mixture was then heated at 130 o C for 8 minutes with microwave radiation. The reaction mixture was diluted with CH2Cl2 (15 ml), followed by the addition of KNaC4H4O6·4H2Oaq (50 ml), then stirred overnight. The phases were then separated and the organic phase was washed with water, brine and dried over Na2SO4. The solvent was removed in vacuo. The crude compound was purified by flash column chromatography using (0% -10 % MeOH, CH2Cl2, 1 % NH3) over 20 column volumes to give the desired compound.