Indole-Containing Amidinohydrazones as Nonpeptide, Dual RXFP3/4 Agonists: Synthesis, Structure–Activity Relationship, and Molecular Modeling Studies

Abstract

The central relaxin-3/RXFP3 system plays important roles in stress responses, feeding, and motivation for reward. However, exploration of its therapeutic applications has been hampered by the lack of small molecule ligands and the cross-activation of RXFP1 in the brain and RXFP4 in the periphery. Herein, we report the first structure–activity relationship studies of a series of novel nonpeptide amidinohydrazone-based agonists, which were characterized by RXFP3 functional and radioligand binding assays. Several potent and efficacious RXFP3 agonists (e.g., 10d) were identified with EC50 values <10 nM. These compounds also had high potency at RXFP4 but no agonist activity at RXFP1, demonstrating > 100-fold selectivity for RXFP3/4 over RXFP1. In vitro ADME and pharmacokinetic assessments revealed that the amidinohydrazone derivatives may have limited brain permeability. Collectively, our findings provide the basis for further optimization of lead compounds to develop a suitable agonist to probe RXFP3 functions in the brain.

Discovery and Characterization of the First Nonpeptide Antagonists for the Relaxin-3/RXFP3 System

“…In cell-based systems, relaxin-3 can also bind and activate RXFP1 and RXFP4, in addition to RXFP3. ,, RXFP1 and RXFP3 have an overlapping expression profile in the brain, while RXFP4 is mainly located in the periphery. ,, Because of these cross-activations, we next assessed the receptor selectivity against RXFP1 and RXFP4. RXFP1 is a Gα s -coupled receptor, and therefore, the endogenous agonist peptide relaxin-2 potently stimulated cAMP production with an EC 50 = 0.02 nM . Compound 33 had no detectable agonist activity (Figure A) or antagonist activity (Figure B) at RXFP1 at all concentrations tested up to 30 μM.…”

“…To expedite the development of RXFP3 small-molecule ligands, we have previously developed a CHO-hRXFP3 cell-based cAMP assay using a time-resolved fluorescence energy transfer (TR-FRET)-based immunoassay (PerkinElmer) . Because the RXFP3 cAMP assay was originally developed in the 96-well format, we automated and miniaturized the assay to a 384-well format for HTS.…”

“…The BBB permeability of 33 was evaluated in the MDCK-MDR1 bidirectional transport assay . The compound had an apparent permeability P app (A → B) of 1.3 × 10 –6 cm/s, indicating modest permeability .…”

“…Despite the emerging pharmacological implications of the relaxin-3/RXFP3 system, small-molecule antagonists of RXFP3 have not been previously disclosed. Relaxin-3 binds and activates RXFP3, a class A Gα i/o -coupled GPCR, leading to inhibition of adenylyl cyclase and stimulation of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. , Relaxin-3 also binds and activates RXFP1 and RXFP4, two members of the relaxin/insulin superfamily, and these cross-activations make a selective RXFP3 ligand critical for precise elucidation of RXFP3 functions. − Although several potent and selective RXFP3 peptide ligands have been developed and extensively used in pharmacological studies, these peptides have limitations as potential therapeutics as they are metabolically unstable and require administration via central injection. ,− Recently, small-molecule RXFP3 agonists have been reported, but they often have dual agonism at RXFP3/4. − On the other hand, efforts from both academic laboratories and pharmaceutical companies to develop small-molecule antagonists have not been successful . To identify small-molecule RXFP3 antagonists, we performed a high-throughput screen (HTS) of a 19,000-member chemical library with a focus on GPCR pharmacophore features using a functional cAMP accumulation assay.…”

“…To expedite the development of RXFP3 small-molecule ligands, we have previously developed a CHO-hRXFP3 cell-based cAMP assay using a time-resolved fluorescence energy transfer (TR-FRET)-based immunoassay (PerkinElmer). 24 Because the RXFP3 cAMP assay was originally developed in the 96-well format, we automated and miniaturized the assay to a 384-well format for HTS. The assay was optimized for cell density, incubation times, and dimethyl sulfoxide (DMSO) sensitivity to give relaxin-3 potency (EC 50 = 0.12 nM) and efficacy consistent with the 96-well assay.…”