Chiral Cyclohexane 1,3-Diones as Inhibitors of Mutant SOD1-Dependent Protein Aggregation for the Treatment of ALS

Abstract

Cyclohexane 1,3-diones were identified as a class of molecules exhibiting a protective effect against mutant SOD1 induced toxicity in PC-12 cells, but an optimized analogue had little or no effect on life extension in the G93A SOD1 mouse model for amyotrophic lateral sclerosis (ALS). Additional testing showed that these compounds were inactive in neurons, and further analogue synthesis was carried out to identify compounds with neuronal activity. Starting from two racemic derivatives that were active in cortical neurons, two potent analogues (1b and 2b) were resolved, which were protective against mutant SOD1 induced toxicity in PC-12 cells. Both compounds were found to be active in cortical neurons and presented good ADME profiles in vitro. On the basis of these results, an ALS mouse trial with 1b was carried out, which showed slightly greater life extension than the FDA-approved ALS drug riluzole, thereby validating cyclohexane 1,3-diones as a novel therapeutic class for the treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a rare and fatal neurodegenerative disease characterized by progressive motor neuron loss in the central and peripheral neuron systems, leading to clinical muscle atrophy, paralysis, and final death from respiratory failure, generally, in 3–5 years.¹ It is estimated that the incidence of ALS is 1–2 cases per 100,000 people, with an increased risk for military personnel.^2,3 Although there has been progress in the identification of potential targets for the disease, and many new therapeutics have been tested in animals and in clinical trials over the last two decades,⁴ no effective treatment is currently available; the only FDA-approved drug, riluzole, a presumptive antiglutamatergic drug, extends survival by only 2–3 months.⁵

Although ALS is principally a sporadic disease, approximately 10% of all cases are familial (FALS), and over 100 genes are potentially responsible for FALS.⁶ Mutations in Cu/Zn superoxide dismutase (SOD1) are the most common cause of FALS.⁷ Although mutations in SOD1 account for only 2% of ALS patients, it has recently been shown that astrocytes from both FALS and sporadic ALS (SALS) patients are similarly toxic to motor neurons and that knockdown of SOD1 significantly attenuates astrocyte-mediated toxicity of motor neurons, indicating that SOD1 is a viable target for SALS.⁸ Also, because mutant SOD1 leads to oxidative stress, protein misfolding, and aggregation, all of which are associated with ALS pathogenesis,⁹ it is reasonable to include inhibitors of mutant SOD1-induced protein aggregation as a viable strategy to identify novel ALS therapeutics.

Three different scaffolds, arylsulfanylpyrazolones (ASP), pyrimidine-2,4,6-triones (PYT), and cyclohexane-1,3-diones (CHD), were identified by a high-throughput cell-based screen¹⁰ based on cell lines developed by Morimoto and co-workers.¹¹ Extensive modification of the ASP^12,13 and PYT¹⁴ leads afforded excellent therapeutic candidates, with favorable potency, pharmacokinetics, toxicity, and life extension in the ALS mouse model. However, the most potent of the CHD derivatives did not show any significant extension of life in the ALS mouse model, despite having comparable potency in the PC-12 cell assay and favorable pharmacokinetic properties.¹⁵ Aggregation of mutant G93A SOD1 is induced in the PC-12 assay, which produces a concomitant loss in cell viability. Cell viability is restored through treatment with compounds that reduce protein aggregation. The proposed explanation was the lack of in vitro activity in cortical neurons. Two racemic analogues (1 and 2) were identified with enhanced activity in cortical neurons that retained their activity in the PC-12 assay. Here we have synthesized the enantiomers of the active compounds and show that both enantiomers of each scaffold penetrate cortical neurons, that the pharmacokinetics of the eutomers are favorable, and that one of the isomers produces a slightly greater extension of life in the ALS mouse model than riluzole, the only FDA-approved drug for ALS.

Glossary

Abbreviations

ADME

absorption, distribution, metabolism, excretion

ALS

amyotrophic lateral sclerosis

CHD

cyclohexane 1,3-dione

CNS

central nervous system

FALS

familial ALS

PBS

phosphate buffered saline

PK

pharmacokinetics

SALS

sporadic ALS

SOD1

Cu/Zn superoxide dismutase

Supporting Information Available

Detailed description of experimental procedures, biological evaluations, and characterization of 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

We thank the National Institutes of Health (Grant 1R43NS057849), the ALS Association (TREAT program), and the Department of Defense (AL093052), for their generous support of this research.

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States