Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors

Abstract

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Figure 1

Overview of the ceramide metabolism and some related enzymes.

Figure 2

Representative known and structurally diversified AC inhibitors.

Figure 3

Rational design of the novel class of AC inhibitors and hit identification.

Scheme 1. Synthesis of 8a–d

Reagents and conditions: (a) KNCO, AcOH, and i-PrOH, 70 °C, 3 h (36–53%) and (b) RNCO and DMAP, CH₃CN, rt, 3–16 h (15–82%).

Scheme 2. Synthesis of 11a–q

Reagents and conditions: (a) TZD, K₂CO₃, and DMF, rt, 1–2 h. (b) LiOH and THF, rt, 30 min–1 h (6%- quant. over two steps for 11a, 11c–p) or t-BuOK and THF, rt, 30 min (for 11b and 11q: 10 and 38% over two steps; respectively).

Scheme 3. Synthesis of 12a–y and 13a–c

Reagents and conditions: (a) RNCO and DMAP (10–83% for 12a–b, h–w) or Et₃N (78% for 12g) and CH₃CN, rt, 3–16 h or RNH₂, Boc₂O, DMAP and CH₃CN, rt, 1–3 h (48–80% for 12c–d) or RNH₂, triphosgene, and DIPEA (76% for 12e) or Et₃N (24% for 12f) and DCM, rt, 3–12 h; (b) 4 M HCl, 1,4-dioxane, rt, 2 h; (c) HCHO, AcOH, NaBH(OAc)₃, and CH₃CN, rt, 1 h (34% over two steps); (d) for 13a: N-methyl-4-phenylbutylamine, triphosgene, DIPEA, and DCM, 0 °C to rt, 3 h (98%); for 13b: 4-phenyl-1-butanol, triphosgene, DIPEA, and DCM, 0 °C to rt, 3 h (53%); for 13c: 6-phenylhexanoic acid, SOCl₂, and DCM, rt, 6 h; then 11a, Et₃N and THF, 0 °C to rt, 16 h (55%).

Scheme 4. Synthesis of 15a–b and 18a–b

Reagents and conditions: for the synthesis of 15a–b: (a) 4-phenylbutyl isocyanate, DMAP, DMF, 50 °C, 2 h, (47–81%); for the synthesis of 18a–b: (a′) CDI, imidazole, DCM, rt, 16 h (86–92%); (b′) 4-phenylbutyl isocyanate, DMAP, DMF, 50 °C, 4 h (68–79%).

Scheme 5. Synthesis of 25c–f

Reagents and conditions: (a) Pd(PPh₃)₄, 2 M Na₂CO₃, and 1,4-dioxane, reflux, 16 h (77–87%); (b) 10% Pd/C, cyclohexene, and EtOH, 60 °C, 24 h (65%); (c) TMSOTf, Et₃N, and THF, −78 to −50 °C, 4 h; then, NBS and THF, −40 to −20 °C, 30 min; (d) TZD, K₂CO₃, and DMF, rt, 1–2 h (93% over three steps for 23b); (e) t-BuOK and THF, rt, 1 h (29% over 4 steps for 24a) or LiOH and THF, rt, 30 min (72% for 24b); (f) 4-phenylbutyl isocyanate, DMAP, and pyridine, rt, 16 h (69%); (g) 4 M HCl and 1,4-dioxane, rt, 2 h (46%); (h) HCHO, AcOH, NaBH(OAc)₃, and CH₃CN, rt, 1 h (60%) (i) 4 M HCl and 1,4-dioxane, rt, 2 h; (j) HCHO, AcOH, NaBH(OAc)₃, and CH₃CN, rt, 1 h (71% over two steps); and (k) RNCO, DMAP, and pyridine, rt, 16 h (25–36% for 25d–e) or i-BuNH₂, triphosgene, Et₃N, and DCM, rt, 3 h (82% for 25f).

Scheme 6. Synthesis of 32a–c

Reagents and conditions: (a) Pd(PPh₃)₄, 2 M Na₂CO₃, and 1,4-dioxane, reflux, 16 h (85%); (b) HCO₂NH₄, 20% Pd(OH)₂, and MeOH, 60 °C, 4 h (69%); (c) TMSOTf, Et₃N, and THF, −78 to −50 °C, 4 h; then, NBS and THF, −40 to −20 °C, 30 min; (d) TZD, K₂CO₃, and DMF, rt, 2 h (66% over three steps); (e) t-BuOK and THF, rt, 30 min (67%); (f) 4 M HCl and 1,4-dioxane, rt, 2 h; (g) HCHO, AcOH, NaBH(OAc)₃, and CH₃CN, rt, 1 h and (h) RNCO, DMAP, and pyridine, rt, 16 h (62% for 32a; 64% for 32b) or i-BuNH₂, triphosgene, Et₃N, and DCM, rt, 3 h (20% for 32c).

Figure 4

Planned SAR exploration.

Figure 5

(A) Concentration–response curve for the inhibition of hAC activity by 11a and 12a; (B) Michaelis–Menten analysis of the reaction of hAC in the presence of vehicle (DMSO 1%, ●) or 12a (25 nM, ▲; 100 nM, ■). Rbm14-12: fluorogenic substrate of hAC. The graph is representative of two independent experiments, each performed in three technical replicates.

Figure 6

Concentration dependence of the effects of 32b in SH-SY5Y cells on hAC activity after a 3 h incubation (A) and SL levels (B–F). GraphPad Prism software (GraphPad Software, Inc., USA) was used for statistical analysis. Data were analyzed using the Student t-test or 1-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons. Differences between groups were considered statistically significant at values of p < 0.05. Values are expressed as means SEM of at least six determinations. Experiments were repeated twice with similar results.

Figure 7

Time course of the effects of 32b (10 μM) in SH-SY5Y cells on hAC activity (A) and SL levels (B–F). GraphPad Prism software (GraphPad Software, Inc., USA) was used for statistical analysis. Data were analyzed using the Student t-test or 1-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons. Differences between groups were considered statistically significant at values of p < 0.05. Values are expressed as means SEM of at least six determinations. Experiments were repeated twice with similar results.

Figure 8

Concentration–response curve for the inhibition of hAC activity by 31c and 32b (A) and putative docking pose of compound 32b in hAC (PDB code: 6MHM) (B).

Figure 9

Putative docking poses of compound 32b in hAC (PDB code: 6MHM) (A) and in hNAAA (PDB code: 6DXX) (B).