Discovery and In Vivo Proof of Concept of a Highly Potent Dual Inhibitor of Soluble Epoxide Hydrolase and Acetylcholinesterase for the Treatment of Alzheimer’s Disease

With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer’s disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)–TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, human microsomal stability, lack of neurotoxicity, and it rescued memory, synaptic plasticity, and neuroinflammation in an AD mouse model, after low dose chronic oral administration.


TABLE OF CONTENTS
Synthesis of intermediates 5, 6, 8a,b, 9a,b, 10b,c, and 11b S4 In vitro and in vivo biological methods S8 Figure S1. Best scoring poses for 12c in each of the AChE models evaluated S17 Figure S2. Interactions of the 6-chlorotacrine moiety of 12c in the CAS of AChE S17 Figure S3. RMSD values of the 6-chlorotacrine moiety of 12c over the last 100 ns of each simulation S18 Figure S4. RMSD values of the TPPU moiety of 12c over the last 100 ns of each simulation S18 Figure S5. Binding mode of donepezil in control simulations S19 Figure S6. In vivo treatment and experimental timeline S19 Figure S7. Familiarization phase of the NORT test S19 Table S1. Summary of the AChE structures available in the PDB that were analyzed to identify prior knowledge of the cryptic pocket in the PAS S20 Table S2. Reported and experimental PAMPA-BBB permeability of the commercial drugs used for assay validation S21 Table S3. Quantities of reagents, microsomes, and test compounds used in the microsomal stability assays S22 Table S4. Gradient used in the UPLC-MS/MS analysis for microsomal stability studies S22 Table S5. Antibodies used in Western Blot (WB) S23 Table S6. Primers and probes used in qPCR studies S23 Table S7. Reference c1 and c2 values for Trp286 rotamers in AChE S24 (5). 1 To a solution of 4-(trifluoromethoxy)phenyl isocyanate, 4 (800 mg, 3.94 mmol), in CH2Cl2 (4 mL), 1benzylpiperidin-4-amine (900 mg, 4.73 mmol) was added. The reaction mixture was stirred at room temperature overnight. Then, the solvent was evaporated under vacuum to obtain a yellowish residue (2.52 g). After column chromatography purification (35-70 μm silica gel, CH2Cl2 / MeOH mixtures), urea 5 was isolated as a white solid (1.50 g, 97%): 1

1-(Piperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (6). 2 To a solution of N-benzyl
piperidine 5 (1.50 g, 3.81 mmol) in MeOH (60 mL), 10% Pd/C (150 mg) and concd. HCl (2 mL) were added, and the mixture was hydrogenated at atmospheric pressure for 5 days. The suspension was filtered and the resulting colorless solution was evaporated at reduced pressure. The residue was dissolved in CH2Cl2 (20 mL) and treated with 2 N NaOH until basic pH. The organic phase was separated and dried over anhydrous Na2SO4 and evaporated under vacuum, to afford piperidine 6 (953 mg, 82% yield) as a white solid: 1   Propidium Displacement Studies. The affinity of 12c for the PAS of Electrophorus electricus AChE (EeAChE) (type VI-S, Sigma-Aldrich) was determined using the PASspecific ligand propidium iodide (P) (Sigma-Aldrich), following a described procedure. 9 Complexation of propidium iodide and AChE leads to a shift in the excitation wavelength. 9

S10
Fluorescence intensity was monitored with a Jasco 6200 spectrofluorometer (Jasco Europe, Italy) using a 0.5 mL quartz cuvette at rt. EeAChE (2 μM PAMPA-BBB assay for brain permeability. The brain permeability (Pe) of the target compounds was assessed by an in vitro parallel artificial membrane permeability assay for blood-brain barrier penetration (PAMPA-BBB assay), 12 which uses a lipid extract of porcine brain membrane as a BBB model. The assay was validated by comparing experimental and reported Pe values of a set of fourteen commercial drugs (Table S2 of  U mL -1 glucose-6-phosphate dehydrogenase, using the volumes indicated in Table S3. exhibits cognitive and emotional abnormalities from young ages. 13,14 Moreover, inflammatory and oxidative stress markers are present at early ages and during adultness. 15 In this work, 5month-old male SAMP8 mice were used to perform behavioral and molecular analyses. Animals were randomly divided into SAMP8 control (n=8) and SAMP8 treated with 12c S13 (n=8, 2 mg kg -1 day -1 ). The animals had free access to food and water and were kept under standard temperature conditions (22 ± 2 ºC) and 12-h/12-h light/dark cycles (300 lux/0 lux).
Compound 12c was dissolved in 1.8% (2-hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, St. Louis, MO) and administered through drinking water for 4 weeks ( Figure S6  Catalunya, Spain (10291). All studies and procedures for the behavioral tests, brain dissection and extractions followed the ARRIVE. Every effort was made to minimize animal suffering and to reduce the number of animals.

Behavioural tests. Novel Object Recognition Test (NORT). The NORT protocol was
performed. 16 Briefly, mice were placed in a 90º two-arm (25 × 20 × 5 cm) black maze, with removable walls for easy cleaning and light intensity in mid-field was 30 lux. Before the memory trials, mice were habituated to the apparatus for 10 min for 3 days. On day 4, the animals were subjected to a 10 min acquisition trial, in which they were allowed to freely explore two identical objects located at the end of each arm (First trial-Familiarization). After 2 h (short-term memory determination) and 24 h (long-term memory determination) from the first trial, mice were subjected to a 10 min retention trial, in which one of the two old objects S14 had been replaced by a novel one. The behavior was recorded, and the time that the mice spent exploring the new object (TN) and the old one (TO) were measured manually.
Exploration was defined as sniffing or touching the objects with the nose and/or forepaws.