Synthesis of Novel Pyridine‐Carboxylates as Small‐Molecule Inhibitors of Human Aspartate/Asparagine‐β‐Hydroxylase

Abstract

The human 2‐oxoglutarate (2OG)‐dependent oxygenase aspartate/asparagine‐β‐hydroxylase (AspH) is a potential medicinal chemistry target for anticancer therapy. AspH is present on the cell surface of invasive cancer cells and accepts epidermal growth factor‐like domain (EGFD) substrates with a noncanonical (i. e., Cys 1–2, 3–4, 5–6) disulfide pattern. We report a concise synthesis of C‐3‐substituted derivatives of pyridine‐2,4‐dicarboxylic acid (2,4‐PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed by using a mass spectrometry‐based assay with a stable thioether analogue of a natural EGFD AspH substrate. Certain C‐3‐substituted 2,4‐PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine‐carboxylate‐related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid in the development of AspH inhibitors suitable for in vivo use.

Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-β-hydroxylase

“…2OG-competition for binding the AspH active site ( e.g. N -oxalylglycine, 28 , 36 , 86 pyridine-2,3-dicarboxylic acid 21. , 87 ) and Fe(II)-chelation ( e.g.…”

“…11 , 12 Studies on naturally occurring mutations of human AspH presumably resulting in inactive AspH (Traboulsi syndrome) 13 , 14 , 15 , 16 and animal models 8 , 17 , 18 suggest that AspH might affect cell motility through the notch signalling pathway. Despite the combined evidence suggesting that AspH is a promising medicinal chemistry target for the development of small-molecule-based cancer therapeutics, comparatively few AspH inhibition studies using small-molecules are reported, with most of these relying either on the use of likely non-selective 19 , 20 or partially selective 21 small-molecules or on the use of l -ascorbic acid (LAA)-derived small-molecules. 18 , 22 , 23 , 24 This likely reflects the historic lack of simple and reliable assays to monitor recombinant human AspH activity in vitro .…”

“… 30 High-throughput solid phase extraction coupled to mass spectrometry (SPE-MS) AspH inhibition assays using a stable substrate analogue were developed and employed to identify potent AspH inhibitor templates. 21 …”

Aspartate β-hydroxylase as a target for cancer therapy

“…Nortrilobolide and related compounds are reported to be potent cytotoxic agents with subnanomolar sarco/endoplasmic reticulum calcium ATPase (SERCA) inhibition [93]. Recently, a family of potent pyridine dicarboxylates have also been published [65] utilizing a mass spectrometry-based inhibition assay [94]. These compounds are related to known iron-dependent dioxygenase inhibitors 2,3-pyridine dicarboxylate, 2,4-pyridine dicarboxylate and 2.6-pyridine dicarboxylate.…”

“…The synthesized pyridine dicarboxylates were assayed for activity against a range of other enzymes, to include PHD2, FIH, and lysine-specific demethylase 4E (KDM4E) in addition to ASPH, with varying degrees of selectivity. However, while cell-based activities have not been evaluated, the dicarboxylate nature of the compounds may be useful for cell surface ASPH inhibitors that may not have cell penetrating activity [94].…”