Discovery, synthesis, and biological evaluation of novel SMN protein modulators

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure-property relationships including microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models and a starting point for clinical development.

Figure 1

SMN2-luciferase reporter assay.

Figure 2

Overview of screening and hits selection process.

Figure 3

A. Chemical structures of 1 and 2 from qHTS. B. Activity of compounds 1 and 2 in SMN1 (cyan) and SMN2 (blue) reporter assays, indicating the rate of SMN induction (ROI) at different concentrations, and luciferase inhibition assay (black). C. Quantification by western blot of SMN levels after treatment with compounds 1 and 2 at different concentrations, as indicated.

Figure 4

SAR strategy.

Figure 5

Quantification by western blot of SMN levels after treatment with drug compounds at different concentrations, as indicated.

Figure 6

Number of gems per 100 nuclei after treatment with drug compounds at different concentrations, as indicated.

Scheme 1

Synthesis of the hit compound 2 and 4-arylthiazolyl piperidine analogues.a ^a Conditions and reagents: (i) triethylamine, µW, 100 °C; (ii) arylboronic acids or pinacol esters, 2.0 N Na₂CO₃, 5% Pd(PPh₃)₄, µW, 100 °C; (iii) a. 4-bromophenylboronic acid, 2.0 N Na₂CO₃, 5% Pd(PPh₃)₄, µW, 100 °C; b. MCPBA; (iv) triethylamine, µW, 100 °C.

Scheme 2

Modifications at the thiazole core template.a ^a Conditions and reagents: (i) piperidine-4-carboxamide, triethylamine, µW, 100 or 120 °C; (ii) arylboronic acids or pinacol esters, 2.0 N Na₂CO₃, 5% Pd(PPh₃)₄, µW, 100 °C; (iii) piperidine-4-carboxamide, N,N-diisopropylethylamine, 0 °C.

Scheme 3

Synthesis of the piperidine modification analogues.a ^a Conditions and reagents: (i) triethylamine, µW; (ii) arylboronic acids or pinacol esters, 2.0 N Na₂CO₃, 5% Pd(PPh₃)₄, µW, 100 °C; (iii) LiOH, THF, H₂O; (iv) EDC, DMAP, µW, 100 °C; (v) KOCN, H₂O, or R₅COCl, triethylamine, or R₆SO₂Cl, triethylamine; (vi) trifluoroacetic acid; (vii) acetyl chloride, triethylamine; (viii) NaN₃, ZnBr₂, NaOH, dioxane, H₂O, 120 °C.