Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5′-Adenosine Monophosphate-Activated Protein Kinase (AMPK)

Abstract

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK)

“…Initial pharmacological evaluation was performed with the human α1β1γ1 AMPK isoform that is predominantly expressed in the kidney, 27,28 and the same assay format was also used to assess isoform selectivity (human α1β2γ1, human α2β1γ1, human α2β2γ1, and human α2β2γ3). Consistent with our previous reports, 21,29 parent carboxylic acids 1−3 potently activate human AMPK isoforms that contained the β1 subunit (including α1β1γ1) (Table 2 and Figure 9). Under these experimental conditions, acyl glucuronide standards of M1, M2, and M3 also selectively activated human β1 isoforms, albeit with 9−19-fold loss in potency (Table 2).…”

“…The present article describes our efforts in the biochemical synthesis, purification, and structural characterization of the glucuronide conjugate of 1 and related analogues PF-06885249 (2) and PF-06679142 (3) (Figure 1) from the indole-3carboxylic acid series of AMPK activators, which have been recently described as potential back-up agents with extended half-lives arising from a net reduction in glucuronidation rates and renal excretion, relative to 1. 29 The biotransformation profile of 2 and 3 in human hepatocytes demonstrated that the predominant route of metabolism also involved glucuronidation (similar to the metabolic fate of 1 in human hepatocytes). Considering the similarities in log D values for 1, 2, and 3, we reasoned that the increased resistance of 2 and 3 toward glucuronidation occurred from a reduction in metabolic rate caused by a diminished affinity for the UGT binding site due to unfavorable active site interactions of the 5-aryl (compound 2) or 4-fluoro (compound 3) substituents rather than a straightforward decrease in lipophilicity.…”

“…Considering the similarities in log D values for 1, 2, and 3, we reasoned that the increased resistance of 2 and 3 toward glucuronidation occurred from a reduction in metabolic rate caused by a diminished affinity for the UGT binding site due to unfavorable active site interactions of the 5-aryl (compound 2) or 4-fluoro (compound 3) substituents rather than a straightforward decrease in lipophilicity. 29 During the course of this investigation, available analytical (mass and NMR spectra) data and chemical derivatization studies suggested that the glucuronide conjugates obtained upon incubation of the parent compounds 1−3 in UDPGAsupplemented human liver microsomes were acyl glucuronides. In vitro pharmacological evaluation revealed that the acyl glucuronide metabolites were selective activators of the human β1-containing AMPK isoforms, which was consistent with their ability to inhibit de novo lipogenesis in primary human hepatocytes.…”

Design, synthesis, and biological evaluation of 1,2,4‐oxadiazole‐containing pyrazolo[3,4‐b]pyridinones as a new series of AMPKɑ1β1γ1 activators

“…[ 53 ] It further reported PF‐06685249 (EC 50 [AMPKα1γ1β1] = 12.0 nM) ( 10 ), which exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption. [ 54 ] We previously reported a structure‐based virtual screening based on our “in‐house” compounds library that led to the discovery of compound 17f ( 11 ), which potently activated AMPKα1γ1β1 (EC 50 = 420 nM). [ 55 ] However, compound 17f has insufficient activation capacity as compared with PF‐06409577, which may be related to their different binding modes with AMPK.…”

Evolution of the Synthesis of AMPK Activators for the Treatment of Diabetic Nephropathy: From Three Preclinical Candidates to the Investigational New Drug PF-06409577

“…5′-Adenosine monophosphate-activated protein kinase (AMPK) is a heterotrimeric serine-threonine kinase (consisting of α-, β-, and γ-subunits) that plays a key role in energy homeostasis. Indole acids 1 , 2 , and 3 (Figure ) were recently identified as potent activators of β1-containing isoforms of AMPK and were evaluated in safety and efficacy studies. − In addition, compound 3 was advanced to first-in-human clinical trials as a potential treatment for diabetic nephropathy. The development of improved synthetic routes for producing multigram quantities of 1 and 2 and kilogram quantities of 3 is described herein.…”