DESIGN, SYNTHESIS AND PRELIMINARY BIOLOGICAL EVALUATION OF GLUTAMATE IONOTROPIC RECEPTOR LIGANDS

AMPA receptors, like NMDA and kainate receptors, belong to the glutamate ion channel receptor family. As ligand-gated ion channels, AMPA receptors are composed of domains that span the membrane to form a pore or channel, and the channel is gated by glutamate. Excessive AMPA receptor activity has been implicated in various neurological diseases, such as amyotrophic lateral sclerosis (ALS), ischemia and epilepsy , by a pathogenic mechanism known as excitotoxicity. Several studies demonstrate that excessive activity of Ca2+-permeable AMPA receptors is involved in a wide range of neurological diseases. Thus, blocking excessive AMPA receptor activity would be a promising therapeutic approach for the treatment of these diseases. AMPA receptor antagonists show a broad range of neuroprotection and are better tolerated with lesser side effects as compared with NMDA receptor antagonists. Various AMPA receptor antagonists have been synthesized; for example, NBQX (6- nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione) is a potent, competitive antagonist of AMPA channels, but it also blocks kainate receptors. Mechanistically, noncompetitive antagonists are considered better suited for a more selective blockade of AMPA receptors, because they bind to a regulatory site(s) distinct to the agonist site and their actions should not depend on the oncentration of an agonist. Noncompetitive antagonists have also the theoretical advantage to counteract excitotoxicity even at high concentration of glutamate and to show less side-effects than competitive antagonists. It should be pointed out that all of these small-molecules are typically drug-like and amenable to chemical optimization for oral bioavailability and favourable pharmacokinetic properties. The most promising group of noncompetitive antagonists of AMPA receptors are 2,3-benzodiazepine derivatives, whose prototype GYKI 52466, 1-(4-aminophenyl)-4- methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine, demonstrated significant anticonvulsant and neuroprotective action. The research group with whom I worked during my PhD has been involved by many years in the synthesis of new 1-(4-aminophenyl)-3,5-dihydro-7,8- ethylenedioxy-4H-2,3- benzodiazepin-4-ones and in the characterization of their mechanism of action. It has been recently reported that the introduction of a thiadiazole moiety at the N-3 position of the 2,3-benzodiazepine scaffold yields an enhancement in potency and selectivity on AMPA receptors. The two 2,3-benzodiazepines GYKI 47409 and GYKI 47654 were found to be far more potent inhibitors of both the closed and open conformations of all four homomeric AMPA receptor channels than the unsubstituted 2,3-benzodiazepine GYKI 52466 as well as N-3 substituted derivatives Talampanel and GYKI 53784. The Authors proposed that the heterocycle at the N-3 position is able to well accommodate into a “side pocket”, generating a strong interaction with residues surrounding this binding site, as predicted by a four-point pharmacophore model that suggests a role of the heteroatom of the thiadiazole moiety as H-bond acceptor. To better define the structure–activity relationship (SAR) of this class of compounds, we planned to synthesize two groups of analogues of 2,3-benzodiazepines, The first group of these compounds bears a methyl group on 7,8- ethylenedioxy moiety in order to create additional hydrophobic interactions between the 7,8-ethylenedioxy portion and the receptor site. The second group of 2,3-benzodiazepines has been designed starting from the abovementioned thiadiazole derivatives GYKI 47409 and GYKI 47654. We decided to insert at the N-3 position a more flexible heterocycle that could better fit into the binding pocket and presumably maintain the same capability to interact with the receptor site via hydrogen bond as the thiadiazole nucleus does. In particular, I synthesized derivatives 4a-4c in which a 3-bromoisoxazolin-5-yl has been linked to N-3 and derivatives 5a-5c in which the same heterocycle was linked to N-3 by means of a methylene spacer. The choice of the 3- romoisoxazolin-5-yl is in agreement with literature data , in which the substituent at N-3 position could be represented by a bstituted or unsubstituted 5-or 6-membered, aromatic, saturated or partially saturated heterocyclic ring containing at least two heteroatoms, with one of them being a nitrogen atom. Over the past years several lines of evidences implicated glutamate in the development and proliferation of different types of cancers inside and outside of the central nervous system. Beside to its excitatory role in the CNS, glutamate is involved in others cellular and biochemical functions such as proliferation, differentiation and survival of the neural cells. A number of findings revealed that the inhibition of AMPA receptor activity was able to inhibit migration and to induce apoptosis in human glioblastoma cells , and to decrease cell growth in different non-neuronal cancer cell lines. With the aim to elucidate the potential mechanism in cell cycle regulation elicited by 2,3-benzodiazepine derivatives, compound 2c, based on primary screening on six different tumor cell lines, has been selected as the most active 2,3-benzodiazepine-4-one derivatives. The ability of compound 2c to modulate the cell cycle distribution and the molecular determinants involved in the cell cycle check points, together with its potential ability to modulated apoptotic pathways in human Jurkat T cell line has been evaluated.The NMDA receptors are heterologous complexes consisting of several subunits assembled to form tetrameric arrangements. So far, seven different subunits have been identified, each with a specific modulatory influence on the receptor: one GluN1subunit,four GluN2 subunit (GluN2A-D) and two subunits GluN3 (GluN3A-B) . During the past several decades, a great number of iGluR ligands have been developed, but few of them are specific for a single subtype. For the glycine site in the GluN1 subunit of NMDA receptors, a large number of antagonists exists, but relatively few full and partial agonists have been reported. In order to further evaluate the ability of L-Cys derivatives to differentiate between the glycine binding site of GluN1 in a GluN2 subunit-dependent manner, and to assess the effect of substituents with different electronic characteristics on the agonist properties of the lead compound 6a, I designed and synthesized new L-cysteine derivatives S-substituted 6b-6g in which a chlorine atom or a methoxy group has been introduced in different positions of the benzyl moiety and compound 6h in which the aromatic ring has been replaced by a 4-pyridyl nucleus.