Synthesis and biological evaluation of 3-alkyloxazolidin-2-ones as reversible MAO inhibitors

The discovery of the two forms of MAO had led to significant advances in the understanding of the physiological and biochemical roles that these enzymes play in normal processes and in disease states. The recent development of a new generation of highly selective reversible MAO inhibitors (MAOIs) have led to a renewed interest in the therapeutic potential of these new compounds with respect to early irreversible MAOIs. In fact, reversible MAOIs offer new hopes for generating superior anti-depressant and anti-parkinsonian agents by virtue of the selective inhibition of MAO-A and MAO-B, respectively. Aryloxazolidinones are one of the relatively new classes of MAOIs. Their “lead compound” is Toloxatone (Humoryl ® ), the first reversible and selective MAO-A inhibitor introduced in clinical practice as an anti-depressant. In order to investigate the importance and hence the biological role played by the anisyl ( p -methoxyphenyl) group linked to N 3 atom of Toloxatone oxazolidinone ring or more in general of an aromatic group, some new N 3 -alkyloxazolidinones were synthesized and their ability to inhibit MAO-A and MAO-B enzymes was evaluated by a fluorimetric method that uses the kynuramine as substrate. Also modifications at C 5 of the 2-oxazolidinone ring were considered. The set of N 3 - alkyl substituted and at C 5 modified compounds showed ability to inhibit MAO-A and MAO-B but with lower extent than Toloxatone used as reference drug. Such biological results provide insights into structure-activity relationships, confirming that is necessary the presence of N 3 -aryl moiety to act as a potent reversible MAO inhibitor, not possible in the same extent when the aryl is replaced by an alkyl group (Ki ≈ 10 -7 M versus 10 -3 –10 -4 M).


Introduction
Similar to other neurotransmitting systems, the catecholamines are massively liberated in the synaptic space after a depolarization of the presynaptic membrane.They, then, act at the receptor level and are subsequently eliminated rapidly by two main mechanisms: catabolism and reuptake.
Inactivation of the effects of catecholamines at the sympathetic neuroeffector junction can take place by one or more mechanisms: a) uptake or reuptake, b) O-methylation, c) oxidative deamination.
The two main degradation pathways of these endogen amines are represented both by oxidative deamination, due to the presence of MAO enzymes (monoamino oxidase) and Omethylation, catalyzed by COMT enzymes (Catechol-O-methyl transferase). 1euronal MAO is a flavine containing enzyme and is mainly located on the outer membrane of the mitochondria at the catecholaminergic terminals. 2AO is important in regulating the levels of catecholamines in tissues (particularly intraneuronally), but can also act on the 3-O-methyl metabolites of catecholamines (i.e., COMT then MAO).Thus, the major metabolite of norepinephrine and epinephrine that appears in urine is 3-methoxy-4-hydroxymandelic acid also called vanillylmandelic acid (VMA).So far, at least two isoforms of neuronal MAO have been recognized: MAO-A and MAO-B, deriving from two different genes that are identified on the basis of their specificity for inhibitor and substrate.MAO-A enzyme preferably metabolizes serotonine and noradrenaline, and is inhibited by clorgyline; MAO-B enzyme preferencially metabolizes dopamine and is inhibited by deprenyl.2 In the actual pharmacological therapy, several drugs with anti-MAO properties are commonly used.These compounds, block degradation of the endogen amines, determine an increment of their concentration and are thus useful for those pathologies where the neuronal transmission is faulting.3 However, these drugs are almost always not selective.In particular, selective inhibitors of MAO-A seem to be useful in the treatment of the depression, while MAO-B inhibitors, by determining an increased synaptic availability for dopamine, are used as agents for the treatment of the Parkinson's disease.4 Recent studies showed that derivatives such as 3-aryloxazolidin-2-one (oxazolidinones, Figure 1) are regularly used to treat infections caused by those Gram positive agents, 5,6 that are resistant to traditional antibiotic agents, here represent a new class of reversible as well irreversible inhibitors for MAO-A and MAO-B enzymes.Hence, these antibacterial agents are able to inhibit protein synthesis 7,8 and are also used for neurodegenerative type pathologies.The 3-phenyloxazolidin-2-one derivatives substituted in position 5 with alcoholic or ethereal group (Toloxatone, Cimoxatone, Figure 1) are proved to be anti-depressant agents, while the 5-amino-3-phenyloxazolidin-2-one derivatives are effective as anti-Parkinson agents (e.g., Almoxatone, MD 780236, Figure 1).8 The inhibition produced by these molecules is stereoselective due to the presence of a stereogenic center.The most powerful and selective MAO-A inhibitors have (R)-configuration (e.g.Cimoxatone, Tolaxotane, etc.).In particular, (R, S)-Toloxatone has Ki= 1.8 µM (MAO-A) and Ki= 44 µM (MAO-B); whereas (R)-Toloxatone has Ki= 0.8 µM (MAO-A) and Ki= 38.9 µM (MAO-B); (S)-Toloxatone has Ki= 12.5 µM (MAO-A) and Ki= 78.6 µM (MAO-B).On the contrary, the most powerful and selective MAO-B inhibitors have (S)-configuration [e.g.(R)-Almoxatone has Ki= 1.77 µM (MAO-A) and Ki= 0.28 µM (MAO-B), (S)-Almoxatone has Ki= 0.38 µM (MAO-A) and Ki= 0.17 µM (MAO-B)].9,10 Oxazolidinones derivatives are particularly versatile drugs whose study is extremely relevant from a pharmacological point of view.[11][12][13][14][15] Continuing our studies aimed at developing new potent and more selective β-adrenoceptor antagonists, 16 some oxazolidinones were prepared as synthetic suitable precursors of aryloxypropanolamines.In this paper, we report the synthesis of a set of racemic and enantiomerically pure N 3 -alkyl substituted oxazolidinones as well as their pharmacological evaluation as inhibitors of the two MAO-isomeric forms.This set of compounds is structurally different from the already known reversible MAOIs because an alkyl group is bonded to the N 3 atom of the 2-oxazolidinone ring, instead of an aromatic one such as in Toloxatone, Cimoxatone, Almoxatone, etc. (Figure 1).Among these molecules there are also some modification at the methylene-C 5 of the oxazolidin-2-one.The results of such an investigation are reported below.

Results and Discussion
The compounds reported in Table 1 have been synthesized in enantiomerically pure and/or racemic form through the synthetic route depicted in Scheme 1 and 2. This synthetic strategy proved to be particularly advantageous since the use of easy-to-find and low cost starting materials is foreseen and a few reaction steps will easily led to the compounds to be pharmacologically evaluated.These molecules were biologically tested by bovine brain mitochondria as enzyme source.Activities of MAO-A and MAO-B were determined by a fluorimetric method with the kynuramine as substrate.The structural modification present in all the compounds, here below described, with respect to well-known MAOIs would provide useful information on the electronic and steric requirements of the active site of the two enzymes.In particular, to the nitrogen atom of the oxazolidinone ring are bonded alkyl groups of different shape and size and on methylene-C 5 directly bonded to the ring there is an aromatic moiety such as the p-toluenesulfonyl or benzothiazolyl-2-thiol.On the contrary, in the Toloxatone molecule the nitrogen atom bears an aryl such as the anisyl (pmethoxyphenyl) and to the methylene is bonded only a hydroxy group.Hence, the structural modifications introduced would provide insights on the stereoelectronic demand of the two enzyme catalytic site.Biological data of oxazolidinones bearing on C 5 amine, amide and ester functionality other than the hydroxy group and always on N 3 an aromatic group have also been reported.Instead, the compounds here described bear on the N 3 an alkyl, cycloalkyl and benzyl residue, and on C 5 -methylene bearing a hydroxy, benzothiazolyl-2-thiol and p-toluenesulfonyl group.The results obtained are summarized in the Table 1.All the compounds have shown an inhibitory activity (Ki around 10 -3 M) less than Toloxatone (Ki ≈ 10 -7 M) and than many other reported compounds in which the C 5 bonds an aromatic or heteroaromatic group.
Finally, among all the tested compounds resulted ten-fold more active those ones bearing an aromatic group either bonded to the ring (phenyl of the N-benzyl derivative 4e) or to the side chain.
In conclusion, the results reported and discussed above, definitely prove that N 3 -aryl moiety is an indispensable structural requirement of the oxazolidinone class compounds to be potent MAOIs.

Biological evaluation procedure: preparation of brain mitochondria and amine oxidase assay
Bovine brain mitochondria, containing MAO-A and B, were isolated according to Basford. 17nzyme activity was determined by fluorimetric method with kynuramine as substrate. 18ompounds dissolved in dimethylsulfoxide (DMSO) were added to the reaction in the presence of kynuramine and then incubated 30 min at 37°C in a thermostated bath for enzyme activity determination.
Spectroscopic and analytical data were identical to those ones reported above for the racemic compound.
Spectroscopic and analytical data were identical to those ones reported above for the racemic compound.

3 a
Data represent mean values of at least three separate experiments.b N.T.= not tested.

Issue in Honor of Prof. Vincenzo Tortorella ARKIVOC 2004 (v) 118-130 ISSN 1424-6376 Page 124
1elting points were taken on Electrothermal apparatus and are uncorrected.1HNMR spectra were recorded in CDCl 3 and in DMSO-d 6 on a Varian EM 390 or XL200 spectrometer and chemical shifts are reported in parts per million (δ) from internal Me 4 Si.Absolute values of the coupling constant (J) are reported.IR spectra were recorded on a Perkin-Elmer 681 spectrometer.GC analyses were performed by using a HP1 column (methyl silicone gum; 5 m × 0.53 mm × 2.65 µm film thickness) on a HP 5890 model, Series II.Optical rotations measurements were obtained using a Perkin-Elmer digital polarimeter, model 241 MC.Thin layer chromatography (TLC) was performed on silica gel sheets with fluorescent indicator (Statocrom SIF, Carlo Erba), the spots on the TLC were observed under ultraviolet light or were visualised with I 2 vapour.Flash chromatography was conducted by using silica gel with an average particle size of 60 µm, a particle size distribution of 40-63 µm and 230-400 ASTM.GC-MS analyses were performed on a HP 5995C model and microanalyses on an Elemental Analyser 1106-Carlo Erba -instrument.Chemicals and solvents were of the highest quality grade available and purchased from the Aldrich Chemical Co. or the Sigma Chemical Co.
© ARKAT USA, Inc Experimental Section General Procedures.