Novel dual inhibitors of calpain and lipid peroxidation

Abstract

A series of molecules with dual inhibitory activities on calpain and lipid peroxidation were synthesized. These hybrid compounds were built on the calpain pharmacophore 2-hydroxytetrahydrofuran linked to a set of antioxidants via a l-leucine linker. Compound 7, the most potent in cellular calpain and lipid peroxidation inhibitions, provided effective protection against glial cell death induced by maitotoxin.

Introduction

Calpains, members of the thiol protease superfamily, are implicated in a number of patho-physiological processes. In particular, the role of calpain 1 in central nervous system conditions such as stroke, Parkinson's and Alzheimer's diseases, subarachnoid hemorrhage or head trauma, as well as in peripheral pathologies like muscular dystrophy, cataract, cardiac ischemia, restenosis or arthritis has been extensively studied.¹ Most of these pathologies are associated with inflammatory processes and the production of free radical species such as reactive oxygen species (ROS).

In pioneering experiments, we demonstrated that the combination of a calpain inhibitor (Z-LL-H)² and an antioxidant (BHT)³ led synergistically to protection against glial cell death.⁴ Consequently, we set up a chemical program aimed at designing molecules possessing both activities.

The free aldehyde group of Z-LL-H was regarded as a chemically reactive function incompatible with in vivo administration. Therefore the 2-hydroxy-tetrahydrofuran group, a masked aldehyde, was selected as a calpain pharmacophore^5a suitable for our purposes. In this context, the biologically active species was hypothesized to be the hydroxyalkyl-aldehyde (the opened semi-acetal),^5b which is in equilibrium with the cyclic semi-acetal. Thus, the available hydroxyalkyl-aldehyde forms a hemithioacetal with the cysteine residue of the calpain active site.

On the other hand, the selection of the antioxidant part of these hybrid compounds was facilitated by our previous work in the field of dual NOS-ROS inhibitors.⁶

Furthermore, we reasoned that these hybrid compounds should have balanced calpain inhibitory and free radical scavenging properties. Attention was therefore focused on a series of potent antioxidants (1a–d) as illustrated in Scheme 1.

As a first approach to the design of these dual inhibitors, the connection between the calpain pharmacophore and the antioxidant was inspired by the peptidic backbone of classical calpain inhibitors. Thus, the optimization of the molecules focused on the nature of the antioxidant and its impact on the calpain and lipid peroxidation inhibitory activities. A 4-step synthetic pathway (Scheme 1) was used to convert commercial (S)-leucine methyl ester·HCl, 2, into compounds 6a–d. Compound 2 was either condensed with trolox^® 1a–CO₂H, phenothiazines 1b–CO₂H⁷ or 1c–CO₂H⁸ to afford carboxamides 3a–c, or converted to the urea 3d by reaction with 1d (NH₂) in the presence of triphosgene and DIEA. The methyl esters of 3a–d were saponified and the resulting carboxylic acids 4a–d were condensed with commercial (S)-α-amino-γ-butyrolactone using EDC/HOBT to yield the lactones 5a–d which, on reduction with DIBAL at −60 °C, afforded the hybrid molecules 6a–d. It should be noted that each compound of the 6a–d series exists as an equilibrated two-diastereomeric mixture. This is attributed to the reversible and nonstereoselective closing and opening of the semi-acetal ring, with intermediate formation of a transient hydroxyalkyl aldehyde, with traces of water.

The aminoindoline 1d (NH₂)⁹ was accessible (Scheme 2) through a short straightforward strategy from commercial 5-nitroindoline. Alkylation with benzyl bromide and reduction of 8 by a mixture of Raney Ni and hydrazine hydrate in EtOH readily gave 1d (NH₂).