Substituted benzyl N -phenylcarbamates – their solvolysis and inhibition activity to acetylcholinesterase and butyrylcholinesterase

Kinetic evidence has indicated that methanolysis of synthesized 4-acetoxybenzyl carbamates proceeds via a one-step (concerted) mechanism. Concerted 1,6-elimination produces the very reactive 1,4-quinonemethide, which was trapped in the form of 4-methoxymethylphenol. The inhibition activity of benzyl N -phenylcarbamates to acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) was also tested. The found IC 50 values varied within the limits of 199-535 µmol·l –1 for ACHE and 21-177 µmol·l –1 for BCHE. The found values of partition coefficient (P ow ) in the range of 1.5-11.5 represent a prerequisite of good transport of benzyl N-phenylcarbamates through haemato-encephalic barrier.


Introduction
Carbamates 1 are a class of organic compounds possessing a number of suitable properties which can be utilized in industry, agriculture or medicine.For instance, in textile industry they have found applications as auxiliary textile agents. 2Eradication of dicotyledonous weeds 3 is achieved by means of the carbamates Desmedipham and Fenmedipham.However, in recent years the greatest attention has been focused on research into new medical drugs.1a,b Priority is given to treatment of Alzheimer's disease, 4 which affects a continuously increasing part of human population all over the world.An example of carbamate drug 5 used for treatment of Alzheimer's disease is Rivastigmin ((S)-N-ethyl-3-[(1-dimethylamino)ethyl] N-methylphenylcarbamate, Exelon ® ) (Fig. 1) having the effect of cholinesterase inhibitors (CHEIs).Rivastigmin (Exelon ® ) -a drug for treatment of Alzheimer's disease. 5 Alzheimer's disease, there is a severe loss of cholinergic cells in the brain that leads to diminished levels of the neurotransmitter acetylcholine (ACH). 6The pharmacology of CHEIs has been recently reviewed. 5nother medical application of carbamates consists in utilization of the so-called labile carbamate bond as connecting link between a polymeric carrier and medical drug. 7For example 7a , in the antimycotic conjugate of amphotericin B with star poly(ethylene glycol), amphotericin B was linked through a phenyl-β-D-glucopyranoside fragment by the labile carbamate linkage.Enzymatic hydrolysis of the β-glucosidic bond is followed by 1,6-elimination reaction and decomposition of carbamate linkage whereby amphotericin B is released from its polymeric carrier.This system was suggested for targeting therapy of systemic mycoses (Scheme 1).Although the 1,4-and 1,6-elimination reactions of benzyl carbamates are well known for a long time 7b and often used 7a,c,d for controlled drug release from polymeric carriers, the literature lacks a more detailed kinetic study of mechanism of this reaction.The aim of the present work is to study hydrolysis and methanolysis of benzyl N-phenylcarbamates 1a-k (Scheme 2).

Results and Discussion
From the structural formula (Fig. 2) of the carbamates (1a-c) possessing an acetoxy group at 4position of benzylic moiety it can be generally deduced that there will be four sites of attack by hydroxide or methoxide anion.The first possible site of attack is the carbonyl carbon atom in the acetoxy group in position 4-i.e.hydroxide-or methoxide-ion-catalyzed solvolysis of the ester bond (B Ac 2) with subsequent 1,6-elimination.The second reaction can be an S N 1 reaction at the benzylic carbon atom.This possibility is less probable due to the low dielectric constant of the methanol.The third site of attack is the carbon atom of carbamate bond, where the B Ac 2 solvolysis is possible too.In the case of strong electron-withdrawing groups (R 2 ), such as a nitro group, it is also possible to consider the E1 CB mechanism.First, we verified the possibility of spectrophotometric monitoring of the hydrolysis kinetics of carbamates 1a-c in sodium hydroxide solutions in the concentration range of 0.01-0.10mol•l -1 at the temperature of 25 °C.It was found that in all the cases the starting carbamates are decomposed to give 4-hydroxybenzyl alcohol and sodium salts of substituted N-phenylcarbamic acids.However it was impossible to precisely evaluate kinetic records due to turbidity or opalescence which was always formed at the substrate concentrations above 1 × 10 -5 mol•l -1 .Therefore, we studied methanolysis in sodium methoxide solutions in the concentration range of 0.01-0.10mol•l -1 at the temperature of 25 °C.In these cases the reactions proceeded in homogeneous medium, and the Lambert-Beer law was also fulfilled.The methanolyses were measured under pseudo-first order conditions when the system kinetically behaved as a single reaction (A → B). Figure 3 presents the dependences of the observed rate constants k obs (s -1 ) on the sodium methoxide concentration for the methanolyses of carbamates 1a-c and 2a-c.Figure 3. Dependence of the observed rate constants k obs (s -1 ) on the sodium methoxide concentration (mol•l -1 ) for methanolyses of derivatives: The graph shows that all the dependences are linear with relatively small differences in slopes between the individual carbamates 1a-c.The reaction order in methoxide ion is one.The reaction rate ν (mol•l -1 •s -1 ) and the observed rate constant k obs (s -1 ) could be expressed by the following equations, where c s stands for concentration of substrate (mol•l -1 ), k (mol -1 •l•s -1 ) is the catalytic constant of decomposition of ester, [CH 3 O -] is actual concentration of methoxide ion: ) against the Hammett substituent constants σ gave a linear dependence with slope practically equal to zero, which indicates that the attack by methoxide ion must take place far from the substituent, hence on the carbonyl carbon atom of acetoxy group.This means that either the initial methanolysis of ester linkage (B Ac 2) is followed by 1,6elimination giving the reactive 1,4-quinonemethide (Scheme 3); or alternatively a concerted mechanism combines the attack by methoxide ion with simultaneous cleavage of the bond between carbon atom and phenoxide oxygen atom (Scheme 4).
The presence of 1,4-quinonemethide formed was proved by analysis of the reaction products.In these cases mass spectrometry proved the presence of 4-methoxymethylphenol, i.e. product of addition of methanol to 1,4-quinonemethide.The same conditions as those used in the cases of carbamates 1a-c were also adopted in the study of methanolysis of 4-methylphenyl acetate (2a), 4-hydroxymethylphenyl acetate (2b), and 4-acetoxymethylphenyl acetate (2c).In the cases of methanolyses of esters 2a and 2b, methyl acetate is produced along with sodium phenoxide and sodium 4-hydroxymethylphenoxide, respectively.In the case of 4acetoxymethylphenyl acetate (2c), methyl acetate is produced along with sodium 4methoxymethylphenoxide.The kinetic dependences presented in Fig. 3 show that the methanolysis of 4-methylphenyl acetate (2a) is the slowest: k = 1.96 mol -1 •l•s -1 .Also the methanolysis of 4-hydroxymethylphenyl acetate (2b) is slower ( k = 2.93 mol -1 •l•s -1 ) than that of 4-acetoxymethylphenyl acetate (2c) ( k = 4.56 mol -1 •l•s -1 ), where the dependence of the observed rate constant on the methoxide concentration lies at a virtually identical straight line as that for carbamates 1a-c (Fig. 3).The formation of 1,4-quinonemethide and practically the same kinetic dependence indicate that the methanolysis of 4-acetoxymethylphenyl acetate (2c) will proceed by a mechanism similar to that operating in the cases of carbamates 1a-c.From the literature 8 it is known that the methanolysis of phenyl acetates proceeds by the B Ac 2 mechanism in which the rate-limiting (the slowest) reaction step involves addition of methoxide ion with formation of a tetrahedral intermediate, which is rapidly decomposed in the second reaction step to give phenoxide anion.The acceleration of methanolysis of 4-acetoxymethylphenyl acetate (2c), as compared with 4-methylphenyl acetate (2a) and 4-hydroxymethylphenyl acetate (2b), can be explained with high probability by operation of concerted mechanism.This means that the attack by methoxide ion and the cleavage of the bond between carbon atom and phenoxide oxygen atom are simultaneous processes.This process involves delocalization of negative charge with concomitant lowering of the transition state energy and acceleration of methanolysis in comparison with the other acetates.
Moreover, we studied the methanolysis of carbamates 1d-f i.e. carbamates having 4-Cl substituent in the benzyl moiety; the Hammett σ constant 9 of this substituent (0.22) is close to that of acetoxy group (0.31), but the methanolysis of these carbamates cannot involve 1,6elimination; it can proceed only by the B Ac 2 or E1 CB mechanism on the carbamate group.However, in all the cases it was found that these carbamates are relatively stable in this medium, and the methanolysis under given conditions does not take place.From this observation it is clear that the B Ac 2 or E1 CB mechanisms on the carbamate group will not operate in the case of carbamates 1a-c either.
As already stated, the methanolysis of carbamates 1a-c produces a very reactive intermediate 1,4-quinonemethide which reacts with nucleophiles present: hydroxide, methoxide or others.This fact can be useful in the case of potential ACHE inhibitors, because one of the principles of ACHE inhibition lies in alkylation of nucleophilic functional groups in active sites of ACHE.
Acetylcholine is hydrolytically destroyed in the brain by cholinesterases. 10In vertebrates, there are two types of cholinesterases, which are distinguished on the basis of their substrate specificities, distribution in various tissues and sensitivity toward various inhibitors.There exist acetylcholinesterase (true cholinesterase, specific cholinesterase, ACHE; E.C. 3.1.1.7)and butyrylcholinesterase (pseudocholinesterase, non-specific cholinesterase, BCHE; E.C. 3.1.1.8). 11he effectiveness of the inhibitor could be described by the 50% inhibitory concentration IC 50 .Table 1 summarizes values of the 50% inhibitory concentration IC 50 and partition coefficients P ow of tested carbamates 1a-k.From the results obtained it follows that all the tested carbamates 1a-k inhibit ACHE as well as BCHE.Generally it is possible to conclude that inhibition of BCHE by the tested compounds is stronger than that of ACHE.The effectiveness of tested carbamates 1a-j for inhibition of ACHE is higher than that of Exelon ® (ACHE: IC 50 = 501 µmol•l -1 ).12a On the other hand, the effectiveness of all the tested carbamates for inhibition of BCHE is lower than that of Exelon ® (BCHE: IC 50 = 20 µmol•l -1 ).12a This could be caused by several structural differences between the hydrophobic gorge of active centre in ACHE and that in BCHE.At the base of the gorge in ACHE, the binding of the substrate is represented by two phenylalanine molecules whose aromatic residues protrude into the gorge. 13In BCHE, these molecules are replaced by two smaller amino acid molecules, such as valine and leucine.This conformational change creates a larger space within the deepest area of the gorge of BCHE to allow the fit of larger-size substrates and inhibitors of BCHE. 14The most effective inhibitor of ACHE and BCHE is compound 1a.The worst inhibitor is compound 1k.The values of partition coefficient showed that the tested carbamates 1a-k are much better soluble in n-octanol than in water and that is why the potential for crossing the blood-brain barrier should be good.

Experimental Section
General Procedures.The starting chemicals were purchased from Sigma-Aldrich.Methanol (UV-Vis grade) was refluxed under argon to remove traces of carbon dioxide and stored under argon atmosphere.Its quality was checked by means of UV VIS spectroscopy.Absorbance of the pure solvent against empty cell was lower than 0.08 in the wavelength range above λ = 250 nm, l = 1 cm.The given melting points were not corrected.Structures of the products prepared were verified by means of 1 H and 13 C NMR.The 1 H and 13 C NMR spectra were recorded on a Bruker Avance 500 instrument.The chemical shifts δ are referenced to the solvent residual peaks δ(DMSO-d 6 ) = 2.55 ppm ( 1 H) and 39.6 ppm ( 13 C).The coupling constants J are given in Hz.The mass spectra were recorded on an Agilent Technologies Comp.gas chromatograph 6890N with a mass detector 5973 Network for samples dissolved in either ether or acetone.The microanalyses were performed on an apparatus of FISONS Instruments, EA 1108 CHN.The kinetic measurements were carried out using HP UV/VIS 8453 Diode Array apparatus in 1cm quartz closable cell at 25 °C.At this temperature, 10 µl methanolic solution of substrate was injected into 2 ml aqueous sodium hydroxide or methanolic sodium methoxide, so that the final concentration of substrate was ca 5 × 10 -4 mol•l -1 .The measured absorbance-time dependences at appropriate wavelength (1a,b and 2b,c at 245 nm; 1c: 292 nm; 2a: 240 nm) were used to calculate the observed rate constant k obs using an optimizing program.The IC 50 values were determined by the method of initial reaction rates. 12The determination of partition coefficient (P ow ) in n-octanol/water system was carried out by a known method. 15

General method of preparation of substituted N-phenyl-O-benzylcarbamates (1a-1k)
A solution of corresponding substituted benzyl alcohol (2.5 mmol) with 2 drops of triethylamine in chloroform (5 ml) was treated with a solution of corresponding substituted phenyl isocyanate (2.5 mmol) in chloroform (5 ml).The separated solid was collected by filtration, dried and recrystallized from the below-given solvent.

Figure 2 .
Figure 2. Possible sites of attack by hydroxide or methoxide anion in carbamates 1a-c.

Table 1 .
The IC 50 values and partition coefficient P ow of carbamates 1a-k