Synthesis of compounds with activated carbonyl groups as lipolytic enzyme inhibitors

Phospholipases A2 constitute of super family of hydrolytic enzymes that catalyse the hydrolysis of the ester bond at the sn-2 position of phospholipids, releasing fatty acids, including arachidonic acid and lysophospholipids. Arachidonic acid is metabolized by enzymes to various eicosanoids, as prostaglandins and leukotrienes. On the other hand, lysophospholipids are also bioactive molecules but also precursors of other of bioactive mediators, as the platelets activation factor (PAF). However all these compounds are involved in inflammation and pain. Consequently, phospholipases A2 inhibitors constitute new agents for the treatment of inflammation and pain. The development of new potent but also selective inhibitors of these enzymes constitutes an interesting approach for the synthesis of medicines with particular interest (new anti-inflammatory drugs, drugs for the treatment multiple sclerosis etc.). The importance of fluorine in modern medicinal chemistry is very crucial because, mainly in the past few years, has been proved that the properties of fluorine can alter drastically pKa but also logD of molecules that acting as inhibitors. Moreover, have been recorded selective interactions between proteins and fluorine substitutents. Based on this fact, until today there have been composed enough inhibitors that have fluorine atoms. These inhibitors are mainly based on the trifluoromethyl ketone group and the pentafluoroethyl ketone group, but d difluoromethyl-ketones are also known. Inhibitors of such type have been studied for many enzymes such as phospholipase, zinc metalloproteinases, fatty acid amide hydrolase, pepsin, a-chymotrypsin, pancreatic elastase etc. However, in the literature (included patents), the synthesis of inhibitors of phospholipase A2 is limited in compounds that contain the trifluoromethyl group and in one only inhibitor that contains the pentafluoroethyl ketone group. The need of synthesis of specific inhibitors for phospholipases GIVA cPLA2, GVIA iPLA2 and GV sPLA2 led to the idea for developing inhibitors that they contain activated carbonyl groups, as heptafluoropropyl ketones, tetrafluoro ketones, 1,1,1,3,3-[pentafluoro] ketones and β-fluoro-oxoamides except the already known groups (trifluoromethyl ketones, pentafluoroethyl ketones and oxoamides). ..........................................Οι φωσφολιπάσες Α2 αποτελούν μια πολυμελή οικογένεια υδρολυτικών ενζύμων που καταλύουν την υδρόλυση του εστερικού δεσμού στην μεσαία θέση (sn-2) των φωσφολιπιδίων απελευθερώνοντας λιπαρά οξέα, συμπεριλαμβανομένου του αραχιδονικού οξέος, και λυσοφωσφολιπίδια. Το αραχιδονικό οξύ με τη δράση διαφόρων ενζύμων μετατρέπεται σε πλήθος εικοσανοειδών, όπως οι προσταγλανδίνες και τα λευκοτριένια. Από την άλλη πλευρά, τα λυσοφωσφολιπίδια είναι επίσης βιοδραστικά μόρια, αλλά και πρόδρομα μόρια άλλων βιοδραστικών μεσολαβητών, όπως ο παράγοντας ενεργοποίησης αιμοπεταλίων (PAF). Όλες όμως αυτές οι ενώσεις εμπλέκονται στην φλεγμονή και τον πόνο. Συνεπώς, οι αναστολείς της φωσφολιπάσης Α2 μπορεί να αποτελέσουν νέου τύπου μέσα για την καταπολέμηση της φλεγμονής και του πόνου. Η ανάπτυξη νέων ισχυρών αλλά και εκλεκτικών αναστολέων αυτών των ενζύμων αποτελεί ενδιαφέρουσα ερευνητική πρόταση, καθώς θα μπορούσε να οδηγήσει στην παρασκευή-σύνθεση φαρμάκων με ιδιαίτερο ενδιαφέρον (όπως νέου τύπου αντιφλεγμονώδη, ή φάρμακα για την καταπολέμηση της σκλήρυνσης κατά πλάκας κ.ά.). Η σημασία του φθορίου στη σύγχρονη Φαρμακοχημεία είναι πολύ μεγάλη καθώς, κυρίως τα τελευταία χρόνια, έχει αποδειχτεί ότι οι ιδιότητες του φθορίου μπορούν να μεταβάλλουν δραστικά το pKa αλλά και το logD των μορίων που δρουν ως αναστολείς. Επιπλέον, έχουν καταγραφεί αξιόλογες επιλεκτικές αλληλεπιδράσεις μεταξύ πρωτεΐνης και υποκαταστάτη φθορίου. Με βάση τις παραπάνω διαπιστώσεις έχουν συντεθεί έως σήμερα αρκετοί αναστολείς που φέρουν άτομα φθορίου. Οι αναστολείς αυτοί βασίζονται τόσο στην ομάδα της τριφθορομέθυλο κετόνης, όσο και της πενταφθοροαίθυλο κετόνης, αλλά έχουν συντεθεί ως ενζυμικοί αναστολείς και διφθορομεθυλενοκετόνες. Αναστολείς τέτοιου τύπου έχουν δοκιμαστεί σε πλήθος ενζύμων πέραν των φωσφολιπασών (π.χ. i εταλλοπρωτεάσες ψευδαργύρου, αμιδοϋδρολάσες λιπαρού οξέος, πεψίνη, α-χυμοθρυψίνη, παγκρεατική ελαστάση κ.ά.). Ωστόσο, ανατρέχοντας στην βιβλιογραφία (συμπεριλαμβανομένων των διπλωμάτων ευρεσιτεχνίας), η σύνθεση αναστολέων της φωσφολιπάσης Α2 περιορίζεται σε ενώσεις που φέρουν την τριφθορομέθυλο κετονική ομάδα και σε ένα μόνο αναστολέα που να φέρει την πενταφθοροαίθυλο κετονική ομάδα. Η ανάγκη σύνθεσης εκλεκτικών αναστολέων για τις φωσφολιπάσες GIVA cPLA2, GVIA iPLA2 και GV sPLA2 οδήγησε στην ιδέα σχεδιασμού αναστολέων που φέρουν ενεργοποιημένο καρβονύλιο, όπως οι επταφθοροπρόπυλο κετόνες, οι τετράφθορο κετόνες, οι 1,1,1,3,3-πεντάφθορο κετόνες, και τα β-φθορο-2-οξοαμίδια πέραν των ήδη γνωστών ομάδων (τριφθορομέθυλο κετόνες, πενταφθοροαίθυλο κετόνες και οξοαμίδια). .........................................................................

Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective

Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

The development of selective inhibitors for individual PLA(2) enzymes is necessary in order to target PLA(2)-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA(2) inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA(2), a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the alpha' position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA(2) inhibitors

Different extraction methodologies and their influence on the bioactivity of the wild edible mushroom Laetiporus sulphureus (Bull.) Murrill

Laetiporus sulphureus is an edible wood-rooting basidiomycete. The nutritional and medicinal properties of this mushroom have long been known by traditional practitioners. The aim of this study was to determine the proximate composition, total phenol antioxidant capacity and antimicrobial activities of different extracts of L. sulphureus. Different extraction methodologies, including high energy techniques, were employed and their effect was examined on the activity of the extracts. Optimum extraction methodologies (classical and ultrasound-assisted) provided one fraction containing neutral and polar lipids and the other fraction containing fungal carotenoids and pigments. Fatty acid analysis indicated a predominant level of polyunsaturated fatty acids followed by saturated and mono-unsaturated fatty acids. Both the aqueous methanolic and water extracts contained higher TPC and showed better antioxidant capacity than the ethanolic extract. Irrespective of the type of extraction applied, L. sulphureus showed good antimicrobial activity against all the tested bacteria and fungi, being in some cases stronger than the used antibiotics and mycotics. Therefore, this edible mushroom could be considered as a positive candidate to be utilised by the food industry, not only for obtaining bioactive compounds to be used as natural antioxidants/antimicrobial agents, but possibly also for its nutritional value and health benefits.Serbian Ministry of Education, Science and Technological Development {[}173032]; FP7 Regpot ``ARCADE{''} {[}245866

Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective

Intracellular phospholipase A(2) group IVA and group VIA play important roles in Wallerian degeneration and axon regeneration after peripheral nerve injury.

We provide evidence that two members of the intracellular phospholipase A(2) family, namely calcium-dependent group IVA (cPLA(2) GIVA) and calcium-independent group VIA (iPLA(2) GVIA) may play important roles in Wallerian degeneration in the mouse sciatic nerve. We assessed the roles of these PLA(2)s in cPLA(2) GIVA(-/-) mice, and mice treated with a selective inhibitor of iPLA(2) GVIA (FKGK11). Additionally, the effects of both these PLA(2)s were assessed by treating cPLA(2) GIVA(-/-) mice with the iPLA(2) inhibitor. Our data suggest that iPLA(2) GVIA may play more of a role in the early stages of myelin breakdown, while cPLA(2) GIVA may play a greater role in myelin clearance by macrophages. Our results also show that the delayed myelin clearance and Wallerian degeneration after sciatic nerve crush injury in mice lacking cPLA(2) and iPLA(2) activities is accompanied by a delay in axon regeneration, target re-innervation and functional recovery. These results indicate that the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) contribute significantly to various aspects of Wallerian degeneration in injured peripheral nerves, which is then essential for successful axon regeneration. This work has implications for injury responses and recovery after peripheral nerve injuries in humans, as well as for understanding the slow clearance of myelin after CNS injury and its potential consequences for axon regeneration

Differing roles for members of the phospholipase A2 superfamily in experimental autoimmune encephalomyelitis.

The phospholipase A(2) (PLA(2)) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) and two of the secreted PLA(2)s (sPLA(2) GIIA and sPLA(2) GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA(2) GIVA plays a role in the onset, and iPLA(2) GVIA in the onset and progression of EAE. We also show a potential role for sPLA(2) in the later remission phase. These studies demonstrate that selective inhibition of iPLA(2) can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA(2) is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA(2) might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis