Ligand Design toward Multifunctional Substrate Reductive Transformations.

The synthesis of bis(N1-phenyl-5-hydroxypyrazol-3-yl)pyridines ("L") is described, and these are silylated to achieve analogues ("Si2L") without the variable of the hydroxyl proton mobility. One hydroxyl example is characterized in its bis-pincer iron(II) complex, which shows every OH proton involved in hydrogen bonding. The steric bulk of the silylated N-phenyl-substituted ligands allows the synthesis and characterization of paramagnetic (Si2L)FeCl2 complexes, and one of these is reduced, under CO, to give the diamagnetic (Si2L)Fe(CO)2 species. Structural comparison and density functional theory calculations of the dichloride and dicarbonyl species show that much, but not all, of the reduction occurs at both the ligand pyridine and pyrazole rings, and thus this ligand type is more resistant to reduction than the simpler bis(iminopyridines). The OSiR3 substituent offers a useful diagnostic of reduction at pyrazole via the degree of π-donation to pyrazole by the oxygen lone pairs, and the stereoelectronic features of the NPh moiety are analyzed. The X-ray photoelectron spectroscopy binding energies of both iron and nitrogen are analyzed to show details of the locus of reduction.

Noncompetitive immunoassay optimized for pharmacokinetic assessments of biologically active efruxifermin.

Efruxifermin (EFX) is a homodimeric human IgG1 Fc-FGF21 fusion protein undergoing investigation for treatment of liver fibrosis due to nonalcoholic steatohepatitis (NASH), a prevalent and serious metabolic disease for which there is no approved treatment. Biological activity of FGF21 requires its intact C-terminus, which enables binding to its obligate co-receptor ß-Klotho on the surface of target cells. This interaction is a prerequisite for FGF21 signal transduction through its canonical FGF receptors: FGFR1c, 2c, and 3c. Therefore, the C-terminus of each FGF21 polypeptide chain must be intact, with no proteolytic truncation, for EFX to exert its pharmacological activity in patients. A sensitive immunoassay for quantification of biologically active EFX in human serum was therefore needed to support pharmacokinetic assessments in patients with NASH. We present a validated noncompetitive electrochemiluminescent immunoassay (ECLIA) that employs a rat monoclonal antibody for specific capture of EFX via its intact C-terminus. Bound EFX is detected by a SULFO-TAG™-conjugated, affinity purified chicken anti-EFX antiserum. The ECLIA reported herein for quantification of EFX demonstrated suitable analytical performance, with a sensitivity (LLOQ) of 20.0 ng/mL, to support reliable pharmacokinetic assessments of EFX. The validated assay was used to quantify serum EFX concentrations in a phase 2a study of NASH patients (BALANCED) with either moderate-to-advanced fibrosis or compensated cirrhosis. The pharmacokinetic profile of EFX was dose-proportional and did not differ between patients with moderate-to-advanced fibrosis and those with compensated cirrhosis. This report presents the first example of a validated pharmacokinetic assay specific for a biologically active Fc-FGF21 fusion protein, as well as the first demonstration of use of a chicken antibody conjugate as a detection reagent specific for an FGF21 analog.