Discovery of Orally Active Hydroxyethylamine Based SPPL2a Inhibitors

Abstract

SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely described in the literature. Recently, we have disclosed a selective, small molecular weight agent SPL-707 which confirmed that pharmacological inhibition of SPPL2a leads to the accumulation of its substrate CD74/p8 and as a consequence to a reduction in the number of B-cells as well as myeloid dendritic cells in mice. In this paper we describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit, several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410) which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.

Figure 1

Structures of the known SPPL2a inhibitors LY-411,575 and SPL-707, HTS hit 1, and HIV protease inhibitor darunavir.

Figure 2

(A) Pharmacokinetic profile of compound 15 determined as a mean plasma concentration of three animals (Balb\c mice) after dosing the compound using a PEG300/D5W (3:1) formulation for i.v. and p.o. application. (B) Inhibition of CD74/p8 NTF processing in mice after oral treatment with 15. Mice received single oral doses of 100 mg/kg LY-411,575 or 10, 30, and 100 mg/kg 15 using PEG300/D5W (3:1) formulation. Four h after dosing, inhibition of SPPL2a in vivo was assessed by measuring CD74/p8 NTF accumulation in splenocyte lysates by Western blot analysis with an antimouse CD74 antibody. Relative quantification of the p8 bands is shown, and the reference sample with LY-411,575 was set arbitrarily as 100%. (C) Total plasma and spleen concentrations (means ± SEM) of 15 at termination of the study 4 h after dosing. Statistics are one-way ANOVA followed by Dunnett’s test compared to matched vehicle group: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Scheme 1. Synthesis of Compound 15 and Its Analogs

Reagents and conditions: (a) DIPEA, CH₂Cl₂, 23 °C, 16 h (85%); (b) DBU, i-PrOH, 85 °C, 16 h (69%); (c) Me₃P, THF/H₂O, 23 °C, 3 h (71%).