Design , Synthesis and Pharmacological Evaluation of New Coumarin Derivatives as Monoamine Oxidase A and B Inhibitors

“Abstract.” With the aim to find out the structural features for the MAO inhibitory activity and selectivity, in the present communication we report the design, synthesis and pharmacological evaluation of a new series of coumarin derivatives with 4-methyl or cycloalkene or benzene ring condensed in the 3,4 position. The substituents in this new scaffold were introduced in the 5, 7 and/or 8 positions of the coumarin moiety. The synthesized compounds 1-13 were evaluated as MAO A and B inhibitors using clorgyline and selegiline, respectively, as reference inhibitors, showing, most of them, activities in the nanomolar range. Compounds 6 (IC50 = 1.18 nM) and 10 (IC50 = 1.48 nM), show higher activity than selegiline (IC50 = 19.60 nM), and high MAO-B selectivity with 100-fold and 1600-fold inhibition levels, with respect to the MAO-A isoform.


Introduction
Mono amine oxidases (MAOs) are flavoenzymes bound to the outer mitochondrial membrane and are responsible for the oxidative deamination of neurotransmitters and dietary amines. 1,2Two isoforms, namely MAO-A and MAO-B, have been identified on the basis of their amino acid sequences, threedimensional structure, substrate preference and inhibitor selectivity. 3,4MAO-A has a higher affinity for serotonin and noradrenaline whereas MAO-B preferentially deaminates phenylethylamine and benzylamine.These properties determine the clinical importance of MAO inhibitors.Selective MAO-A inhibitors such as clorgyline (irreversible) and moclobemide (reversible) are used in the treatment of neurological disorders such as depression, 5,6 whereas the selective and irreversible MAO-B inhibitors such as selegiline and rasagiline are useful in the treatment of Parkinson's 7,8 and Alzheimer's diseases. 9,10l of these aspects have led to an intensive search for novel MAO inhibitors (MAOIs) and this effort has increased considerably in recent years.However, earlier MAOIs introduced into clinical practice were abandoned due to adverse effects, such as hepatotoxicity, orthostatic hypotension and the so-called "cheese effect", which was characterized by hypertensive crisis. 11,124][15] The major breakthrough has been brought about by the crystallization of hMAO-B with different inhibitors. 16This fact explains the subsequent elucidation and determination of the 3D structure of the active site of hMAO-A 3 and opened new possibilities for the design of more selective and reversible drugs and facilitated the computer-assisted development of more selective inhibitors.Molecular docking calculations, 17,18 comparative molecular field analysis (CoMFA) 19,20 as well as QSAR methods are among the computational methods that have been used to predict MAOIs.Respect of this, in previous works our group has developed a new QSAR methodology called MARCH-INSIDE 21,22 methodology to seek theoretical models for the prediction of different biological activities.Applying this method is possible to predict the in vitro MAO A/B inhibitory activity of new series of coumarin derivatives.The coumarin analogs are a family of natural and/or synthetic compounds with different pharmacological activities, one of which is MAO inhibitory activity. 23,24In many cases, it is known that activity and selectivity are determined by the nature of the substituents at the 7-and the 4/3-positions. 25- 27 n the other hand, in a previous investigation 28 we developed QSAR studies for the MAO-A inhibitory activity in a series of coumarin derivatives and found that the most active compounds were the 7-acetonyloxy-substituted compounds, which showed higher activity than their cyclic analogs and were also more active than the 7-hydroxy precursors.

Results and discussion
The theoretical QSAR model was created using a very large data base of heterogeneous compounds in which activities were measured in cellular lines from different organs and species.The model classified correctly 3222 out of 3408 inputs (94.5%) and was then used to predict the activity of 13 compounds in our designed coumarin library.All of the active compounds were correctly predicted by the model, and a total of 81.82% and 72.73% compounds were correctly predicted as MAO-A and MAO-B inhibitors, respectively.
On the basis of the QSAR model information -and with the aim of exploring the structure-affinity and MAO-A/B selectivity relationships -in the present work we designed a series of 13 coumarin derivatives (Table 1) that include the aforementioned chemical diversity with particular attention paid to the most interesting 7-(β-ketoether)coumarin derivatives.The compounds were synthesized according to Scheme 1 and details are given in the Experimental section.
5][26] The 7-hydroxy derivatives have been shown to be inactive, even with the introduction of bulky substituents in any other positions of the coumarin (compounds 1 and 2).The introduction of acetonyl/bromoallyloxy groups in the 7-position of the coumarin yielded compounds (3 to 10) with greater MAO-A and MAO-B inhibitory activity.The substitution in the 5 position (compounds 11 and 12) of the coumarin ring leads to the activity loss either in the case of the ketoether substituent or the cyclation to the corresponding furocoumarin 13.Finally, the most relevant findings are the introduction of bulky groups such as cyclohexyl or phenyl in the 3,4-positions of the 7-acetonyl derivatives increased both MAO-A and MAO-B inhibitory activities with concomitant loss of selectivity, whereas replacement of the acetonyl substituent at position 7 by the bromoallyloxy group resulted in compounds 6 and 10, which had very high MAO-B inhibitory activity (IC 50 of about 1.2 nM and 1.5 nM) and the highest MAO-B selectivity (approximately 100-fold and 1600-fold) with respect to the MAO-A isoform.The production of H 2 O 2 catalyzed by MAO isoforms can be detected using the previously mentioned reagent, a non-fluorescent, highly sensitive and stable probe that reacts with H 2 O 2 in the presence of horseradish peroxidase to produce a fluorescent product: resorufin.In this study hMAO activity was evaluated using the above method following the general procedure described previously by us. 29e tested drugs (new compounds and reference inhibitors) inhibited the control enzymatic MAO activities and the inhibition was concentration dependent.

Conclusions
In summary, we have developed theoretical models to predict inhibitory MAO activity.On the basis of this model we have found new coumarin derivatives with inhibitory activity comparable to those of clorgyline and selegiline, respectively, which are used as reference inhibitors and have a very high MAO-A and MAO-B selectivity.These findings have encouraged us to continue our investigations into the design of more potent and selective analogs by introducing appropriate substituents into the coumarin scaffold.

Experimental section
Chemistry.Melting points were determined using a Reichert Kofler thermopan or in capillary tubes on a Büchi 510 apparatus and are uncorrected.IR spectra were recorded on a Perkin-Elmer 1640FT spectrophotometer. 1 H and 13 C NMR spectra were recorded on a Bruker AMX spectrometer at 300 and 75.47 MHz, respectively, using TMS as internal standard (chemical shifts in δ values, J in Hz).Mass spectra were obtained using a Hewlett Packard 5988A spectrometer.Elemental analyses were performed using a Perkin-Elmer 240B microanalyser and were within ±0.4% of calculated values in all cases.Silica gel (Merck 60, 230-00 mesh) was used for flash chromatography (FC).Analytical thin layer chromatography (TLC) was performed on plates precoated with silica gel (Merck 60 F254, 0.25 mm).
General procedure for the preparation of General procedure for the preparation of oxoether derivatives 3, 4, 5, 6, 7 and 12.To a solution of the substituted 7-hydroxycoumarin derivatives 1, 2 or 11 (62 mmol) in dry acetone (300 mL) were added K 2 CO 3 (4 g) and the corresponding 2-chloroketones or 2,3-dibromopropene (120 mmol).The mixture was heated under reflux for 24 h.The mixture was cooled and the solid residue was filtered off.
The solvent was removed under reduced pressure and the crude product was purified by FC and/or crystallization to give the desired compound.General procedure for the preparation of 3,4-benzocoumarins 8-10.To a solution of the acyclic ether 4, 5 30 or 7 (0.30 mmol) in toluene (15 mL) was added DDQ (0.60 mmol).The solution was heated under reflux for 5 h.The mixture was cooled, the precipitate filtered off and the solvent evaporated under reduced pressure.The resulting residue was purified by FC to give the desired compound.Control experiments were carried out simultaneously by replacing the test drugs (new compounds and reference inhibitors) with appropriate dilutions of the vehicles.In addition, the possible capacity of the above test drugs to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition was determined by adding these drugs to solutions containing only the Amplex Red reagent in a sodium phosphate buffer.
To determine the kinetic parameters of hMAO-A and hMAO-B (K m and V max ), the corresponding enzymatic activity of both isoforms was evaluated (under the experimental conditions described above) in the presence of a number of p-tyramine concentrations.
The specific fluorescence emission (used to obtain the final results) was calculated after subtraction of the background activity, which was determined from vials containing all components except the MAO isoforms, which were replaced by a sodium phosphate buffer solution.

A
specific way to new lead discovery involves several general common steps when used in terms of a QSAR: (a) construction of a suitable molecular database of compounds that either show the property in question or do not; (b) calculation of the molecular descriptors; (c) construction of the model; (d) estimation of the biological activity using QSAR; (e) synthesis and characterization of selected compounds; (f) assay of the candidate compounds in order to corroborate the predicted biological activity.

Scheme 1 .
Scheme 1. Synthetic strategy for prepared compounds recombinant hMAO-A or hMAO-B and adjusted to obtain in our experimental conditions the same reaction velocity in the presence of both isoforms were incubated for 15 min at 37 ºC in a flat-blackbottom 96-well microtest plate placed in the dark fluorimeter chamber.After this incubation period, the reaction was started by adding (final concentrations) 200 μM of 10-acetyl-3,7-dihydroxyphenoxazine reagent (Amplex Red assay kit), 1 U/ml horseradish peroxidase and 1 mM p-tyramine.The production of H 2 O 2 and, consequently, of resorufin was quantified at 37 °C in a Multi-Detection microplate fluorescence reader (FLX800) based on the fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15 min period, during which the fluorescence increased linearly.

Table 2 .
The corresponding IC 50 values and MAO-B selectivity ratios [IC 50 (MAO-A)]/[IC 50 (MAO-B)] are shown in Table 2.In vitro evaluation of MAO inhibitory activities of compounds 1-13 and reference inhibitors Each IC 50 value is the mean ± S.E.M. from five experiments (n = 5).Values obtained under the assumption that the corresponding IC 50 against MAO-A/B is the highest concentration tested.