In this study we present the synthesis

In this study, we present the synthesis and structure–activity studies of a structurally distinct series possessing high potency and selectivity in HEK cell lines together with a potentially promising overall biopharmacological profile. Focusing on creating sufficient structural dissimilarity with glutamate and improvement of pharmacological characteristics, we constructed variable lipophilic structural fragments with the terminal carboxy- and amino-groups of aspartic and 2,3-diaminopropionic hippo signaling correspondingly. In comparison with TBOA series, these inhibitors possess only one chiral center and a free carboxylic acid group, potentially simplifying the syntheses and improving physico-chemical properties. The general synthetic routes, leading to aspartate and 2,3-diaminopropionate analogs, are shown in . As outlined in , aspartic acid analogs – were synthesized through standard carbodiimide-mediated coupling between an amine and an appropriately protected aspartic acid. It is worth mentioning that step (ii) in proved to be challenging in the synthesis of aspartamides. For example, basic hydrolysis or even exposure of the coupled protected material to elevated temperatures led to the contamination of the target materials with the product of intramolecular cyclization (1) and its subsequent non-selective opening (2) (). Hydrogenolysis with vigorous stirring at elevated pressure (50psi) or Cu-mediated debenzylation (step (ii) in , and steps (iv) and (viii) in ) allowed us to overcome these difficulties. Diaminopropionic acid analogs were prepared via in situ formation of hydroxysuccinimide ester with the appropriate aryl carboxylic acid, followed by coupling with diaminopropionic acid. To simplify isolation and purification of the intermediate, the crude material was subjected to esterification with trimethylsilyl diazomethane. Final hydrolysis of a methyl ester with diluted (1N) sodium hydroxide and removal of Boc-group with TFA afforded analogs –. Examples of the synthesis of the targets with highly elaborated lipophilic cores are shown in . Yields usually ranged from good to excellent (40–90%). The structural integrity of the analogs was confirmed by traditional analytical techniques (NMR, MS, CHN, and IR). It is noteworthy that among the plethora of nitro-group reduction methods, only SnCl in ethanol (step (ii) in ) showed excellent regioselectivity preserving the bromine atom intact. A potentially problematic Pd-catalyzed formation of dibenzofuran (v) and polyphosphoric acid mediated cyclization of biaryl methanols into corresponding fluorenes (xi), both requiring strenuous conditions (110–120°C), took place without major complications, routinely providing products with satisfactory yield (40–70%). The EAAT inhibitory properties of compounds, prepared in the course of the study, are shown in . The compounds under study were tested in a HEK cell line expressing each of the human transporter subtypes EAAT-1–3. Compound was also tested for its EAAT-2 uptake inhibitory effect on MDCK cells in an effort to benchmark its potency with TBOA series. It showed potent inhibition of EAAT-2 uptake with an IC<10nM, which is comparable with the best TBOA analogs reported to date. Overall, both aspartamide and diaminopropionamide series routinely elicited high EAAT-2 inhibitory potency in HEK cells with an IC<100nM (, , , and ). Linear arrangement of amino acid fragment with respect to distal aromatic ring is a prerequisite for potency in diaminopropionamide series (weakly active ). The selectivity proved to be an elusive and complex issue. As exemplified by compounds , , and , breaking of aryl–aryl bond with concomitant loss of rigidity did not deteriorate EAAT-2 inhibition but gave rise to some selectivity (10- to 20-fold) versus EAAT-1 and EAAT-3. On the other hand, fluorenone , not only lost EAAT-2 potency, but also reversed the sense of inhibitory preference. The best results achieved in diaminopropionamide series are represented by analogs and . Possessing significant EAAT-2 potency, they also showed excellent separation for EAAT-1 (60- to 300-fold) and a moderate one for EAAT-3 (∼10-fold). Linear, in respect to amino acid residue, arrangement in biaryl analogs, while lacking selectivity, revealed a uniformly high blocking potency against EAAT-2 transporter in both series (aspartamides- and diaminopropionamides). Planarity derived from the conversion of biaryls into fluorenes (–, ) did not affect the properties, leaving them potent non-selective inhibitors (with the exception of ) of EAAT-2 transporter. On the other hand, perturbations in the proximity of aryl–aryl linkage in some cases (, –) produced the desired selectivity trend. Our attempts to exploit these results led to the synthesis and evaluation of the ethers –. While potent EAAT-2 blockers dibenzofuran and phenoxazine showed no selectivity, less rigid ethers and, especially, showed both increased potency and desired inhibitory preference. In addition to being the most potent compound, bromo-ether (EAAT-2 IC∼85nM) was the most selective with 59- and 45-fold selectivity over EAAT-1 and EAAT-3, respectively. Because of superior combination of potency and selectivity, the compound was fully characterized pharmacologically. It showed high potency in rat cortical synaptosomes and EAAT-2 expressing oocytes (). In addition, the compound did not show cross-receptor reactivity (failed to activate both ionotropic and metabotropic glutamate receptors) and proved to be a competitive non-substrate inhibitor of EAAT-2 (by failure to activate transporter-like current when applied to oocytes expressing EAAT-1–3 transporters).