A practical synthesis of nitrone-derived C5a-functionalized isofagomines as protein stabilizers to treat Gaucher disease

Abstract

Isofagomine (IFG) and its analogues possess promising glycosidase inhibitory activities. However, a flexible synthetic strategy toward both C5a-functionalized IFGs remains to be explored. Here we show a practical synthesis of C5a-S and R aminomethyl IFG-based derivatives via the diastereoselective addition of cyanide to cyclic nitrone 1. Nitrone 1 was conveniently prepared on a gram scale and in high yield from inexpensive (-)-diethyl D-tartrate via a straightforward method, with a stereoselective Michael addition of a nitroolefin and a Nef reaction as key steps. A 268-membered library (134 × 2) of the C5a-functionalized derivatives was submitted to enzyme- or cell-based bio-evaluations, which resulted in the identification of a promising β-glucocerebrosidase (GCase) stabilizer demonstrating a 2.7-fold enhancement at 25 nM in p.Asn370Ser GCase activity and a 13-fold increase at 1 μM in recombinant human GCase activity in Gaucher cell lines.

Fig. 1. Design and general strategy to discover new GCase stabilizers for potential treatment of Gaucher disease.

a Structures of fagomine, isofagomine (IFG), and their derivatives with unique IFG skeleton and promising bioactivities. b Mechanism of pharmacological chaperones. c Schematic diagram of interactions between GCase and IFG-based molecules to design (d) C5a-aminomethyl IFG-based scaffolds using cyclic nitrone 1 as the key intermediate followed by combinatorial parallel synthesis and in-situ biological evaluation to develop GCase stabilizers for chemical chaperones and (e) co-administration.

Fig. 2. Retrosynthetic analysis of C5a-aminomethyl IFG-based scaffolds and cyclic nitrone 1.

The retrosynthetic plan of desired C5a-aminomethyl IFG scaffolds from (−)-diethyl d-tartrate.

Fig. 3. Preparation of cyclic nitrone 1 from (−)-diethyl d-tartrate.

a Synthetic pathway of IFG-type cyclic nitrone 1. b Optimization of Grignard Michael addition of 4.

Fig. 4. Synthesis of compound libraries and evaluation of selected compounds.

a Scaffold 11 and scaffold 14 were synthetically prepared to generate library A and library B for in-situ cell-based chaperone screening in Gaucher fibroblasts (GM00372), respectively. Several selected hits were found, resynthesized, and further characterized as potent GCase chemical chaperones. b The β-glucosidase activity enhancement effects of IFG, 15, 16, and 17 in Gaucher fibroblasts (GM00372) and the stabilization of rh-GCase with these small molecules at 100 µM. The fold change in enzyme activity is compared to untreated cells (normalized value = 1). The maximal fold increase was observed at a given compound concentration. Data are the mean of three determinations.ΔTm (^oC) refers to melting temperature change compared to Tm of rh-GCase in the absence of small molecules. c The influence of 16 on α- and β-glucosidase activities in GM00372 fibroblasts. Enzyme activity is normalized to untreated cells and assigned a relative activity of 1.

Fig. 5. Structures of resynthesized IFG derivatives and bioevaluation results.

a Chemical structure of the resynthesized IFG derivatives 18–27 and their GCase inhibitory activities. b The influence of 15–27, S1, and IFG (100 nM) toward cellular β-glucosidase activity in p.Asn370Ser/p.Leu29Alafs*18 Gaucher fibroblasts (GM00372). Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. Mean values ± SD are shown for triplicate experiments. NT refers to cells without treatment of molecules.

Fig. 6. Enhancement effects of compound 16 on β-glucosidase in Gaucher fibroblasts.

Co-administration of rh-GCase (0.17 μM) with small molecule 16 (1 μM) significantly increased β-glucosidase activity in Gaucher fibroblasts (GM00372 and GM00877). Enzyme activity is normalized to cells treated only with rh-GCase, assigned a relative activity of 1. Mean values ± SD are shown for triplicate experiments. ***p < 0.005 (One-way ANOVA with Tukey’s post-hoc).