Synthesis and Preliminary Biological Evaluation of 1,3,5-Triazine Amino Acid Derivatives to Study Their MAO Inhibitors

Three series of 4,6-dimethoxy-, 4,6-dipiperidino- and 4,6-dimorpholino-1,3,5-triazin-2-yl) amino acid derivatives were synthesized and characterized. A preliminary study for their monoamine oxidase inhibitory activity showed that compounds 7, 18, and 25 had MAO-A inhibition activity comparable to that of the standard clorgyline, with apparently more selective inhibitory activity toward MAO-A than MAO-B and no significant acute toxicity.


Introduction
Human monoamine oxidases A and B (MAO-A and B) are the most widely studied flavin-dependent amine oxidases. They are located in the mitochondrial outer membranes of neuronal, glial, and other cells particularly abundant in the liver and brain [1,2]. These FAD-dependent enzymes catalyze the oxidative deamination of several endogenous and exogenous monoamines and are responsible for the regulation and metabolism of major monoamine neurotransmitters, such as serotonin (5-OH tryptamine), noradrenaline, and dopamine [1][2][3]. The two mammalian isoforms of these enzymes are characterized by their distinct sensitivity to inhibitors and specificity to substrates. Thus, MAO-A is selectively inhibited by clorgyline and preferentially metabolizes serotonin: whereas MAO-B is inhibited by L-deprenyl and preferentially metabolizes benzylamine and phenylethylamine as substrates [4]. Among selective MAO inhibitors, those against MAO-A are used as anti-depressant and anti-anxiety drugs and have been claimed to protect neuronal cells against apoptosis [5,6]. In contrast, MAO-B inhibitors have been found to be beneficial in the treatment of Parkinson's disease and Alzheimer's disease. Early MAO-inhibitors introduced into clinical practice for the treatment of depression were abandoned due to adverse side effects, such as the "cheese effect", which is characterized by hypertensive crises [4], and because the mechanism of interaction of several new drugs with MAOs has not been yet fully characterized. For these reasons, research has been aimed at the synthesis of new potential agents with clinical applications.
Here, we prepared three small libraries of molecules based on amino acid-substituted 1,3,5-triazine and evaluated their capacity to inhibit MOAs.
The aim of the present study was to tailor MAO-A inhibitors by designing a hybrid from different possible active sites of previously known MAO-A inhibitors, based on the following considerations: (i) the presence of electron-rich aromatic moieties (e.g., moclobemide [34],bazinaprine [35], quinoxaline derivatives [7,8]); (ii) the presence of morpholine moiety (e.g., moclobemide [34], bazinaprine [35]); and (iii) the presence of amino acid moiety [9,10]). The target compounds were designed to study the effect of molecular variation on MAO inhibitory activity, Figure 1.
The 1 H-NMR spectrum of 7 in DMSO-d6 (Supplementary Data Figure S9) showed two doublet of doublet (dd) peaks at δ 3.00 ppm and 3.14 ppm, corresponding to the two diastereotopic methylene protons Ha and Hb, respectively, as shown in the staggered conformation using Newman projection ( Figure 2).
Compounds 29 or 30 were prepared by reaction of cyanuric chloride with equiv. of piperidine/morpholine in the presence of Et3N (Scheme 2) [36]. In contrast, the preparation of N-(4,6-dipiperidino/dimorpholino-1,3,5-triazin-2-yl) amino acid derivatives through the reaction of free α-amino acids with 29/30 was not successful (Scheme 2). The difficulty to displace the third chlorine by the rather weak nucleophilicity of the amino group of α-amino acids can be attributed to the presence of two electron-donating piperidine/ morpholine groups, which decreases the positivity of the third chlorine-bearing carbon and prevents the

Preliminary Biology
The newly synthesized compounds 3-9 and 15-28 were tested to determine selectivity for MAO-A and MAO-B in the presence of the specific substrate serotonin or benzylamine, respectively. Compounds 7, 18, and 25 showed MAO-A inhibition activity comparable to that of the standard clorgyline with apparently more selective inhibitory activity toward MAO-A than MAO-B, and without no significant acute toxicity. More formal studies to confirm these preliminary results will be carried out and published elsewhere.

Conclusions
The synthesis and a preliminary biochemical evaluation of the newly synthesized N-(2,4-disubstituted-1,3,5-triazin-2-yl) amino acid derivatives as MAO inhibitors were described. Compounds 7, 18 and 25 showed the highest activity within the test compounds comparable to that of the standard clorgyline. These preliminary tests have also shown remarkable selectivity within the test compounds as MAO-A inhibitors. Therefore, such compounds would represent a fruitful matrix for the development of a new class of MAO-A inhibitors that would deserve further investigation and derivatization.