Design and synthesis of novel methoxypyridine-derived gamma-secretase modulators

Abstract

The evolution of gamma-secretase modulators (GSMs) through the introduction of novel heterocycles with the goal of aligning activity for reducing the levels of Aβ42 and properties consistent with a drug-like molecule are described. The insertion of a methoxypyridine motif within the tetracyclic scaffold provided compounds with improved activity for arresting Aβ42 production as well as improved properties, including solubility. In vivo pharmacokinetic analysis demonstrated that several compounds within the novel series were capable of crossing the BBB and accessing the therapeutic target. Treatment with methoxypyridine-derived compound 64 reduced Aβ42 levels in the plasma of J20 mice, in addition to reducing Aβ42 levels in the plasma and brain of Tg2576 mice.

Keywords: Alzheimer’s disease; Amyloid beta; Aβ42 reduction; Gamma secretase modulator; Methoxypyridines; Structure activity relationship.

Figure 1.

Representative methyl imidazole-derived GSM scaffolds. E2012 was developed by Eisai. PF-06442609 was developed by Pfizer and illustrates that the general GSM tetracyclic system may be fused.^–

Figure 2.

Fluorophenyl-derived GSMs selected for focused B-ring modification in order to broaden SAR.

Figure 3.

Stable H4 human neuroglioma cells over-expressing human APP751 (H4-APP751 cells) were transfected with the NΔE construct, and then treated with different concentrations GSM compound 22d (lane 3 = 10 μM; lane 4 = 1 μM) or GSI (DAPT, lane 5 = 300 nM, lane 6 = 100 nM) for another 24hrs. Vehicle control treatment shown in lanes 1–2. Cells were harvested 48hrs post transfection and applied to Western blotting analysis. Myc antibody was utilized to assess the NΔED and NICD tagged with Myc on their N-termini. β-Actin was utilized as the loading control. Compound 22d did not inhibit Notching processing; however, DAPT significantly inhibits Notching processing.

Figure 4.

Concentration response curves of compound 22d using orthogonal medium throughput SHSY5Y-APP cell-based screening assays. Aβ38, Aβ40, Aβ42, and total Aβ peptide levels were determined using Meso Scale Sector 6000 Multiplex assays.

Figure 5.

Levels of Aβ38, Aβ40, and Aβ42 in plasma following daily oral administration of either vehicle, compound 46 (A), or compound 64 (B) to female 5–6 month-old J20 mice (n = 5/dose) were measured following a 3-day treatment course. Percent reduction in amyloid is indicated for the study. Aβ38, Aβ40, and Aβ42 peptide levels were determined using Meso Scale Sector 6000 Multiplex assays. Statistical analysis was performed using Graphpad Prism software and results are expressed as mean ± SEM, and Anova was used to detect a significant effect. *: p < 0.05. **: p < 0.005. ***: p < 0.0005.

Figure 6.

Concentration response curves of compound 46 (A) and compound 64 (B) using orthogonal medium throughput SHSY5Y-APP cell-based screening assays. Aβ38, Aβ40, Aβ42, and total Aβ peptide levels were determined using Meso Scale Sector 6000 Multiplex assay.

Figure 7.

Levels of Aβ38, Aβ40, and Aβ42 in plasma and brain following daily oral administration of either vehicle or compound 64 to female 5–6 month-old Tg2576 mice (n = 12/dose) were measured following a 14-day treatment course. Percent reduction is indicated for the study. Aβ38, Aβ40, and Aβ42 peptide levels were determined using Meso Scale Sector 6000 Multiplex assays. Statistical analysis was performed using Graphpad Prism software and results are expressed as mean ± SEM, and ANOVA was used to detect a significant effect. *: p < 0.05. **: p < 0.005. ***: p < 0.0005.

Scheme 1.

GSM B-ring optimization. Aminothiazole 3 (NGP-555) represents the initial lead compound for efforts to discover a potent GSM with improved ADMET properties. Tetrahydroindazole D-ring analog 4 was developed with the aim of improving critical properties of compound 3, including solubility. The rings are labeled A-D for clarity.

Scheme 2.

Synthesis of phenyl-, methoxyphenyl-, and pyridyl B-ring-containing analogs. Reagents and conditions: a) 4-methylimidazole, K₂CO₃, DMSO, 55 °C, 16 h, 44–67%; b) Br₂, 33% HBr in AcOH, DCM, r.t., 1.5 h, 75–80%; c) NaOMe, DMF, 0 °C to 50 °C, 1 h, 27%; d) Br₂, 33% HBr in AcOH, r.t., 0.75 h, 100%.

Scheme 3.

Synthesis of methoxypyridine and methoxypyrazine B-ring-containing analogs. Reagents and conditions: a) NaOMe, 1,4-dioxane, reflux, 18 h, 98%; b) Ac₂O, formic acid, THF, 0 °C to r.t., 2 h, 95–100%; c) CH₃COCH₂Cl, K₂CO₃, KI, DMF, r.t., 4–18 h, 91–93%; d) NH₄OAc, acetic acid, 120 °C, 10 h, 85%; e) X=C n-ethylvinylether, Pd(dppf)Cl₂, TEA, ethylene glycol, 100 °C, 4 h, 73% or X=N tributyl(1-ethoxyvinyl) tin, Pd(PPh₃)₂Cl₂, dioxane, 100 °C, 18 h, 85%; f) 2N HCl, acetone, r.t., 0.5–18h, 89–96%; g) Br₂, 33% HBr in acetic acid, EtOAc, CHCl₃, r.t., 0.5–5 h, 85–90%.

Scheme 4.

Reagents and conditions: a) ethylhydrazine oxalate, EtOH, reflux, 18 h, 88%; b) benzoyl isothiocyanate, 60 °C, 3.5 h, 91%; c) K₂CO₃, MeOH, THF, r.t., 18 h, 92%.

Scheme 5.

Reagents and conditions for substrates 8a, 8c, and 10: a) EtOH, DIPEA, 55 °C, 24 h, 18–33%. Reagents and conditions for substrates 17a and 17b: a) EtOH, reflux, 18 h, 40–42%.

Scheme 6.

Reagents and conditions: a) 33% HBr in AcOH, 50 °C, 1.5 h, 92%.

Scheme 7.

Reagents and conditions: a) ethylhydrazine oxalate or methylhydrazine, EtOH, reflux, 18 h, 61–66%; b) benzoyl isothiocyanate, acetone, reflux, 3.5 h, 88–96%; c) K₂CO₃, MeOH, THF, r.t., 18 h, 81–92%.

Scheme 8.

Reagents and conditions: a) EtOH, reflux, 18 h, 33–51%.