2-Oxoesters: A Novel Class of Potent and Selective Inhibitors of Cytosolic Group IVA Phospholipase A2.

Cytosolic phospholipase A2 (GIVA cPLA2) is the only PLA2 that exhibits a marked preference for hydrolysis of arachidonic acid containing phospholipid substrates releasing free arachidonic acid and lysophospholipids and giving rise to the generation of diverse lipid mediators involved in inflammatory conditions. Thus, the development of potent and selective GIVA cPLA2 inhibitors is of great importance. We have developed a novel class of such inhibitors based on the 2-oxoester functionality. This functionality in combination with a long aliphatic chain or a chain carrying an appropriate aromatic system, such as the biphenyl system, and a free carboxyl group leads to highly potent and selective GIVA cPLA2 inhibitors (X I(50) values 0.00007-0.00008) and docking studies aid in understanding this selectivity. A methyl 2-oxoester, with a short chain carrying a naphthalene ring, was found to preferentially inhibit the other major intracellular PLA2, the calcium-independent PLA2. In RAW264.7 macrophages, treatment with the most potent 2-oxoester GIVA cPLA2 inhibitor resulted in over 50% decrease in KLA-elicited prostaglandin D2 production. The novel, highly potent and selective GIVA cPLA2 inhibitors provide excellent tools for the study of the role of the enzyme and could contribute to the development of novel therapeutic agents for the treatment of inflammatory diseases

Synthesis of new phospholipase A2 inhibitors and study of their antiinflammatory activity

Phospholipases A2 comprise a family of lipolytic enzymes that catalyze the hydrolysis of the sn-2 ester bond of phospholipids, to release free fatty acids, including arachidonic acid, and lysophospholipids. The products of this enzymatic hydrolysis are converted to eicosanoids, prostaglandins and leukotrienes, which are considered to be potent mediators of inflammation. Therefore, reducing the formation of arachidonic acid, by inhibition of phospholipases A2, would be valuable for prevention and treatment of various inflammatory conditions, such as brain injury, allergic symptoms and other diseases and is an attractive target for the design of new anti-inflammatory drugs. In the first chapter of this thesis, we present the structure, function, and catalytic mechanism of cPLA2 and iPLA2. Both of these cytosolic phospholipases selectively cleave the sn-2 ester bond of phospholipids. Moreover, cPLA2 is an 85 kDa protein, and shows selectivity for arachidonic acid. iPLA2 is an 80-88 kDa protein, shows no selectivity for arachidonic acid and is calcium-independent. Most of the already known inhibitors of cPLA2 and iPLA2 and some representative methods of their synthesis are summarized in the second chapter. Among these inhibitors, several fattty acid derivatives, pyrrole and indole derivatives, 2-propanone derivatives, pyrrolidine derivatives, trifluoromethylketones, natural products and enol lactones are included. In the third chapter, we discuss the enzymatic removal of carboxyl-, amino- and hydroxyl-protecting groups. The synthesis of complex, polyfunctional organic molecules, such as oligonucleotides, oligosaccharides and in general natural products, requires the proper introduction and removal of protecting groups. For this purpose a large number of protecting groups as well as chemical methods for protection and deprotection of various functionalities have been developed and are widely used in synthetic organic chemistry. A major problem for the synthesis of polyfunctional molecules is that a given functional group has to be protected or deprotected selectively in the presence of functionalities of similar reactivity as well as functionalities sensitive to acids, bases, oxidation or reduction. The application of biocatalysts in the protecting group chemistry may offer excellent alternatives to chemical methods, because enzymes: (i) carry out highly chemo- and regioselective transformations, (ii) usually operate at neutral pH values, and (iii) combine a high selectivity for the reactions they catalyze and the structure they recognize with a broad substrate tolerance. The aim of this thesis was the synthesis and study of new bioactive molecules that could show inhibitory activity against cPLA2 and iPLA2. Furthermore, we were interested in finding an enzyme that could be used in organic synthesis for the selective removal of ester protecting groups, and its applicability in the synthesis of 2-oxoamide inhibitors. It has been demonstrated that the 2-oxoamide inhibitor based on δ-amino acid has shown the best in vitro inhibitory activity against cPLA2. Dipeptides are considered to be δ-amino acid analogues, because of the distance between N- and C-terminal groups. L-Valine and L-norleucine were used as starting materials to synthesize a series of derivatives that contain the activated 2-oxoamide group and an amide group as replacement of two methylenes of the δ-amino acid derivative. The 2-oxoamide group can be synthesized by coupling the amino group of the protected dipeptide with 2-hydroxy-acid, followed by oxidation. Dipeptides can be synthesized by coupling of suitably N-protected valine or norleucine with several hydrochloric salts of glycine esters. The final derivatives contain a free or a protected carboxyl group. In a similar manner, pseudodipeptides based on ethers, can be considered as δ-amino acid analogues. They can be formed by reduction of Boc- and Z-L-norleucine and reaction with bromoacetic acid esters. Removal of Boc- and Z-protecting group, followed by coupling with 2-hydroxy acid and oxidation, yielded 2-oxoamide derivatives, containing an ether bond instead of one methylene. .............................Οι φωσφολιπάσες Α2 είναι ένζυμα που χαρακτηρίζονται από την ικανότητά τους να καταλύουν την υδρόλυση του μεσαίου εστερικού δεσμού (sn-2) φωσφολιπιδικών υποστρωμάτων, παράγοντας ελεύθερα λιπαρά οξέα και λυσοφωσφολιπίδια. Τα προϊόντα αυτής της ενζυμικής υδρόλυσης αποτελούν πρόδρομες ενώσεις των εικοσανοειδών, τα οποία θεωρείται ότι εμπλέκονται ως διαμεσολαβητές σε πολλές παθολογικές καταστάσεις, όπως η φλεγμονή και ο πόνος, Επομένως, η σύνθεση ισχυρών και εκλεκτικών αναστολέων των φωσφολιπασών Α2 μπορεί να οδηγήσει σε νέα φάρμακα για την καταπολέμηση πολλών ασθενειών, όπως φλεγμονή, αλλεργικά συμπτώματα, βλάβες του εγκεφάλου, καρδιαγγειακές διαταραχές και καρκίνος. Στο πρώτο κεφάλαιο της παρούσας διατριβής, παρουσιάζονται η δομή, η λειτουργία, η τοπολογία και ο μηχανισμός δράσης των φωσφολιπασών cPLA2 και iPLA2. Πρόκειται για δύο κυτοσολικές φωσφολιπάσες που εξειδικεύονται στην υδρόλυση του μεσαίου εστερικού δεσμού (sn-2) των φωσφολιπιδίων. Η cPLA2 είναι ένα ένζυμο με μοριακό βάρος 85 kDa, εμφανίζει εκλεκτικότητα για το αραχιδονικό οξύ και η κρυσταλλική της δομή έγινε γνωστή μόλις το 1999. Η iPLA2 έχει μοροακό βάρος 85-88 kDa, δεν εμφανίζει εκλεκτικότητα για συγκεκριμένο λιπαρό οξύ και είναι ανεξάρτητη ιόντων ασβεστίου. Στο δεύτερο κεφάλαιο, συνοψίζονται διάφορες κατηγορίες ενώσεων που δρουν ως αναστολείς των δύο παραπάνω ενζύμων και έχουν ανακοινωθεί στη βιβλιογραφία μέχρι σήμερα. Ανάμεσα σε αυτούς συμπεριλαμβάνονται παράγωγα λιπαρών οξέων, πυρρολίου και ινδολίου, πυρρολιδινών, 2-προπανόνης, τριφθορομεθυλοκετονών, ενολολακτονών, καθώς και φυσικά προϊόντα. Για κάποιες από αυτές τις ενώσεις αναφέρονται αντιπροσωπευτικές μέθοδοι σύνθεσης. Στο τρίτο κεφάλαιο αυτής της εργασίας, γίνεται λόγος για ενζυμική απομάκρυνση προστατευτικών ομάδων της αμινομάδας, του καρβοξυλίου και του υδροξυλίου. Στη συνθετική Οργανική Χημεία, μία από τις πιο σημαντικές διαδικασίες είναι η κατάλληλη εισαγωγή ή απομάκρυνση προστατευτικών ομάδων. Παρ’όλο που έχουν αναπτυχθεί πολυάριθμες χημικές μέθοδοι για το χειρισμό προστατευτικών ομάδων, υπάρχουν προβλήματα, ιδίως σε περιπτώσεις όπου μία συγκεκριμένη δραστική ομάδα πρέπει να προστατευτεί ή να αποπροστετευτεί εκλεκτικά κάτω από πολύ ήπιες συνθήκες και παρουσία άλλων ομάδων με παρόμοια δραστικότητα, καθώς επίσης και παρουσία ομάδων που είναι ευαίσθητες σε όξινο, αλκαλικό, οξειδωτικό ή αναγωγικό περιβάλλον. Εναλλακτικά, για το σκοπό αυτό, μπορούν να χρησιμοποιηθούν βιοκαταλύτες, προσφέροντας διάφορα πλεονεκτήματα, όπως υψηλή εκλεκτικότητα ως προς τα υποστρώματα και πολύ ήπιες συνθήκες αντίδρασης. Σκοπός της διατριβής ήταν η σύνθεση και η μελέτη νέων μορίων που εμφανίζουν ανασταλτική δράση έναντι της cPLA2 και της iPLA2. Επιπλέον, μας απασχόλησε η εύρεση ενός ενζύμου που να μπορεί να χρησιμοποιηθεί στη συνθετική Οργανική Χημεία για την εκλεκτική απομάκρυνση εστέρων, καθώς και η εφαρμογή του στις αντιδράσεις σύνθεσης των αναστολέων. Έχει βρεθεί ότι το 2-οξοαμίδιο που βασίζεται στο δ-αμινοξύ εμφανίζει την ισχυρότερη in vitro ανασταλτική δράση. Τα διπεπτίδια, λόγω της απόστασης της Ν- και C-τελικών ομάδων, μπορεί να θεωρηθούν ανάλογα δ-αμινοξέων. Ξεκινώντας από πρώτη ύλη L-βαλίνη, L-νορλευκίνη και γλυκίνη, παρασκευάστηκε μία σειρά ενώσεων που περιέχουν τη δραστική 2-οξοαμιδική ομάδα και φέρουν αμιδικό δεσμό σε αντικατάσταση δύο μεθυλενίων που παραγώγου δ-αμινοξέος. Η 2-οξοαμιδική ομάδα μπορεί να προκύψει από σύζευξη της αμινομάδας κατάλληλα προστατευμένου διπεπτιδίου, με 2-υδροξυ-δεκαεξανοϊκό οξύ ακολουθούμενη από οξείδωση. Τα διπεπτίδια προέρχονται από τη σύζευξη κατάλληλα Ν-προστατευμένης L-βαλίνης και L-νορλευκίνης με διάφορους εστέρες υδροχλωρικών αλάτων της γλυκίνης. Το καρβοξύλιο της τελικής ένωσης μπορεί να βρίσκεται ελεύθερο ή με τη μορφή εστέρα. Με παρόμοια λογική, και τα αιθερικού τύπου ψευδοδιπεπτίδια μπορούν να θεωρηθούν ανάλογα δ-αμινοξέων. ........................

Inhibitors of secreted phospholipase A2 suppress the release of PGE2 in renal mesangial cells

The upregulation of PGE2 by mesangial cells has been observed under chronic inflammation condition. In the present work, renal mesangial cells were stimulated to trigger a huge increase of PGE2 synthesis and were treated in the absence or presence of known PLA2 inhibitors. A variety of synthetic inhibitors, mainly developed in our labs, which are known to selectively inhibit each of GIVA cPLA2, GVIA iPLA2, and GIIA/GV sPLA2, were used as tools in this study. Synthetic sPLA2 inhibitors, such as GK115 (an amide derivative based on the non-natural amino acid (R)-γ-norleucine) as well as GK126 and GK241 (2-oxoamides based on the natural (S)-α-amino acid leucine and valine, respectively) presented an interesting effect on the suppression of PGE2 formation

Structure-activity relationships of natural and non-natural amino acid-based amide and 2-oxoamide inhibitors of human phospholipase A(2) enzymes.

A variety of 2-oxoamides and related amides based on natural and non-natural amino acids were synthesized. Their activity on two human intracellular phospholipases (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase (GV sPLA(2)) was evaluated. We show that an amide based on (R)-gamma-norleucine is a highly selective inhibitor of GV sPLA(2)

Development of Potent and Selective Inhibitors for Group VIA Calcium-Independent Phospholipase A2 Guided by Molecular Dynamics and Structure-Activity Relationships.

The development of inhibitors for phospholipase A2 (PLA2) is important in elucidating the enzymes implication in various biological pathways. PLA2 enzymes are an important pharmacological target implicated in various inflammatory diseases. Computational chemistry, organic synthesis, and in vitro assays were employed to develop potent and selective inhibitors for group VIA calcium-independent PLA2. A set of fluoroketone inhibitors was studied for their binding mode with two human cytosolic PLA2 enzymes: group IVA cPLA2 and group VIA iPLA2. New compounds were synthesized and assayed toward three major PLA2s. This study led to the development of four potent and selective thioether fluoroketone inhibitors as well as a thioether keto-1,2,4-oxadiazole inhibitor for GVIA iPLA2, which will serve as lead compounds for future development and studies. The keto-1,2,4-oxadiazole functionality with a thioether is a novel structure, and it will be used as a lead to develop inhibitors with higher potency and selectivity toward GVIA iPLA2

2-Oxoamide inhibitors of cytosolic group IVA phospholipase A2 with reduced lipophilicity.

Cytosolic GIVA phospholipase A2 (GIVA cPLA2) initiates the eicosanoid pathway of inflammation and thus inhibitors of this enzyme constitute novel potential agents for the treatment of inflammatory diseases. Traditionally, GIVA cPLA2 inhibitors have suffered systemically from high lipophilicity. We have developed a variety of long chain 2-oxoamides as inhibitors of GIVA PLA2. Among them, AX048 was found to produce a potent analgesic effect. We have now reduced the lipophilicity of AX048 by replacing the long aliphatic chain with a chain containing an ether linked aromatic ring with in vitro inhibitory activities similar to AX048

2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2 inhibitors.

Calcium-independent phospholipase A2 (GVIA iPLA2) has recently attracted interest as a medicinal target. The number of known GVIA iPLA2 inhibitors is limited to a handful of synthetic compounds (bromoenol lactone and polyfluoroketones). To expand the chemical diversity, a variety of 2-oxoamides based on dipeptides and ether dipeptides were synthesized and studied for their in vitro inhibitory activity on human GVIA iPLA2 and their selectivity over the other major intracellular GIVA cPLA2 and the secreted GV sPLA2. Structure-activity relationship studies revealed the first 2-oxoamide derivative (GK317), which presents potent inhibition of GVIA iPLA2 (XI(50) value of 0.007) and at the same time significant selectivity over GIVA cPLA2 and GV sPLA2