A Simple and Effective Synthesis of 3- and 4-((Phenylcarbamoyl)oxy)benzoic Acids

Phenserine, posiphen, tolserine and cymserine and its derivatives are experimental Alzheimer’s disease drugs that contain a phenyl phenylcarbamate moiety that is responsible for their anti-Alzheimer activities. We have developed a simple (3 steps) and effective (overall yields 76–90%) method for preparing 3and 4-((phenylcarbamoyl)oxy)benzoic acids which can be reacted with amines to produce phenyl phenylcarbamate moiety containing amides as new potential anti-Alzheimer disease drugs. The synthesized carboxylic acids are thus important building blocks with potential use in medicinal chemistry and drug discovery.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder. 1 The synaptic dysfunction and neurodegeneration in AD most severely affects the cholinergic system. 2 This decreases the levels of the neurotransmitter acetylcholine (ACh), 3 which then produces cognitive impairment and memory loss, 4 characteristic for patients with AD. Several compounds are currently being evaluated in preclinical and clinical trials for efficacy in AD, including cholinesterase (ChE) inhibitors which increase the levels of ACh in the brain: phenserine, posiphene, tolserine and cymserine and its derivatives ( Figure  1). 5 These experimental Alzheimer's disease drugs all contain the phenyl phenylcarbamate moiety or its derivative. Phenserine 6 and posiphen 7 contain a phenyl phenylcarbamate moiety, tolserine 8 contains a phenyl ortho-tolylcarbamate moiety and cymserine and its derivatives 9,10 contain a phenyl (4-isopropylphenyl)carbamate moiety ( Figure 1).
Phenserine, posiphen, tolserine and cymserine and its derivatives are pseudo-irreversible carbamate inhibitors of ChEs where the phenyl phenylcarbamate moiety is responsible for their biological activity. Their mechanism of inhibition involves a rapid initial covalent reaction between their carbamate carbonyl group and the catalytic serine in the active site of ChEs (carbamoylation). The inhibited (carbamoylated) ChE is then reactivated by a slow hydrolysis (decarbamylation) of the active enzyme serine (Scheme 1). 11,12 As part of our development of new ChE inhibitors as potential anti-Alzheimer disease drugs, we designed compounds with the general formula 1 that contain the phenyl phenylcarbamate moiety (Scheme 2A). These compounds were designed based on the structures of our previously reported ChE inhibitors. [13][14][15] We planned to synthesize compounds with the general formula 1 by utilizing one of several methods for the synthesis of carbamates, 16,17 i.e. reacting phenols with the general formula 2 with various phenyl isocyanates (3) in the presence of a catalytic amount of 4-dimethylamino pyridine (4-DMAP) in CH 2 Cl 2 or DMF (Scheme 2A). 18,19 However, this reaction did not produce the desired carbamates as no reaction was observed. Therefore, we had to plan an alternative synthetic route. We decided to use 3-and 4-((phenylcarbamoyl)oxy) benzoic acids (4) and react them with various amines (5) which we have previously used to synthesize amide 13,14 and sulfonamide 14,15 ChE inhibitors, in the presence of coupling reagent TBTU and N,N-diisopropylethylamine (DIPEA) in CH 2 Cl 2 20 to produce the designed amides (Scheme 2B).
The problem was that 3-and 4-((phenylcarbamoyl) oxy)benzoic acids (4; Scheme 2B) are not commercially available and procedures for their preparation have also not been reported yet. Herein we describe how we solved this problem by developing a simple procedure to produce these building blocks in high overall yields.

1. General Chemistry Methods
1 H NMR and 13 C NMR were recorded at 400.130 MHz and 100.613 MHz, respectively, on an NMR spectrophotometer (Bruker Avance III). The chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the deuterated solvent used. The coupling constants (J) are reported in Hz, and the splitting patterns are indicated as: s, singlet; br. s, broad singlet; d, doublet; dd, doublet of doublets; td, triplet of doublets; h, hextet; m, multiplet; t, triplet; br. t, broad triplet; dt, doublet of triplets; tt, triplet of triplets; q, quartet; qd, quartet of doublets. Infrared (IR) spectra were recorded on a FT-IR spectrometer (System Spectrum BX; Perkin-Elmer). ATR IR spectra were recorded on a FT-IR spectrometer (Thermo Nicolet Nexus 470 ESP). Micro-analyses were performed on a Perkin-Elmer C, H, N Analyzer 240 C. The analyses are indicated by the symbols of the elements and they were within ±0.4% of the theoretical values. Mass spectra were recorded on a LC-MS/MS system (Q Executive Plus; Thermo Scientific, MA, USA). Melting points were determined on a Leica hot-stage microscope and are uncorrected. Evaporation of the solvents was performed under reduced pressure. Reagents and solvents were purchased from Acros Organics, Alfa Aesar, Euriso-Top, Fluka, Merck, Sigma-Aldrich, and TCI Europe, and were used without further purification, unless otherwise stated. Flash column chromatography was performed on silica gel 60 for column chromatography (particle size, 230-400 mesh). Analytical thin-layer chromatography was performed on silica gel aluminum sheets (0.20 mm; 60 F254; Merck), with visualization using ultraviolet light and/or visualization reagents. Analytical reversed-phase UPLC method A was performed on an LC system (Dionex Ultimate 3000 Binary Rapid Separation; Thermo Scientific) equipped with an autosampler, a binary pump system, a photodiode array detector, a thermostated column compartment, and the Chromeleon Chromatography Data System. The detector on UPLC system was set to 210 nm and 254 nm. The column used for method A was a C18 analytical column (50 × 2.1 mm, 1.8 µm; Acquity UPLC HSS C18SB). The column was thermostated at 40 °C.

1. General Procedure for Synthesis of Benzyl Esters 6 and 8 (General Procedure 1)
To a 100-mL round-bottom flask equipped with a stirring bar, hydroxybenzoic acid (5.000 g, 36.177 mmol, 1.0 mol. equiv.) and DMF (50 mL) were added. The resulting solution was stirred and Na 2 CO 3 (3.837 g, 36.177 mmol, 1.0 mol. equiv.) was added. Benzyl bromide (4.297 mL, 36.177 mmol, 1.0 mol. equiv) was added dropwise to the suspension and the reaction mixture was stirred for 24 hours at room temperature, then poured into a 500-mL separating funnel. Water (100 mL) was added and the mixture was extracted with Et 2 O (3 × 150 mL). The combined organic phases where transferred into a 1-L separating funnel, washed with water (3 × 450 mL) followed by sat. brine solution (450 mL), dried over anhyd. Na 2 SO 4 , and evaporated to produce the benzyl hydroxybenzoate as a colourless oil which solidified into a white solid after cooling. This product was used in the next step without further purification.

General Procedure for Synthesis of Carbamates 10-15 (General Procedure 2)
To a round-bottom flask equipped with a stirring bar, benzyl hydroxybenzoate (1.0 mol. equiv.) and CH 2 Cl 2 (c = 0.3 M) were added. The resulting solution was stirred and 4-DMAP (0.01 mol. equiv.) was added. Phenyl isocyanate, 2-methylphenyl isocyanate or 4-isopropylphenyl isocyanate (1.0 mol. equiv.) was added dropwise and the reaction mixture was stirred for 24 hours at room temperature, then evaporated to produce the carbamates. These products were used in the next step without further purification.

3. General Procedure for Debenzylation of Benzyl Esters Yielding 16-21 (General Procedure 3)
To a round-bottom flask equipped with a stirring bar, benzyl ester (1.0 mol. equiv.) and inhibitor-free THF (c = 0.02 g/mL) were added. The resulting solution was stirred and agitated with a stream of argon for 30 min. 10% Pd/C (5% mass of benzyl ester) was added and the resulting suspension was agitated with a stream of hydrogen for 30 min. The reaction mixture was stirred under an atmosphere of hydrogen for 24 hours then agitated with a stream of argon for 30 min, filtered with suction through a pad of Celite and evaporated to produce the carboxylic acid.

3. 15. Synthesis of 3-((1-(2,3-Dihydro-1Hinden-2-yl)piperidin-3-yl)carbamoyl) phenyl Phenylcarbamate (22)
To a 50-mL round-bottom flask equipped with a stirring bar, compound 16 (0.100 g, 0.389 mmol, 1.0 equiv) was added followed by CH 2 Cl 2 (10 mL). The resulting suspension was stirred and cooled to 0 °C. N,N-Diisopropylethylamine (0.135 mL, 0.778 mmol, 2.0 equiv) was added dropwise and the suspension transformed into a solution. TBTU was added and 30 min later solution A (see below) was added dropwise. The reaction mixture was allowed to warm to room temperature and then stirred for 24 hours. During this time a white precipitate formed. The suspension was filtered with suction to produce 0.133 g of compound 22 as a white solid (75% yield).
Preparation of solution A: To 25-mL round-bottom flask equipped with a stirring bar, compound 23 (0.112 g, 0.389 mmol, 1.0 equiv) was added followed by CH 2 Cl 2 (11 mL). The resulting suspension was stirred and cooled to 0 °C. N,N-Diisopropylethylamine (0.135 mL, 0.778 mmol, 2.0 equiv) was added dropwise and the suspension transformed into a solution.

Conclusions
In summary, we have developed method for the synthesis of previously unreported 3-and 4-((phenylcarbam-oyl)oxy)benzoic acids from commercially available 3-and 4-hydroxybenzoic acids, respectively. The main advantages of our method are the simplicity, as no purification of intermediates or final acids is required, and effectiveness, as the overall yields are very good to excellent (76-90%). As we have shown, the synthesized carboxylic acids can be converted further, e.g. reacted with amines to produce amides with potential application in drug discovery.