Εξαγωγή και μελέτη ποσοτικών σχέσεων δομής-δράσης (QSAR) ενώσεων με δράση κατά των σερινοπρωτεασών. Σύνθεση και φαρμακοχημική μελέτη κουμαρινικών παραγώγων με πιθανή βιολογική δράση κατά των σερινοπρωτεασών και της φλεγμονής

Abstract

Inflammation is a complex phenomenon involving interrelationships between humoral and cellular reactions through a number of inflammatory mediators. It is an usual symptom covering different pathologies and there are still many questions to be answered in order to understand the inflammatory process, as well as a need for better-tolerated and more efficient anti-inflammatory drugs. Recently it has been clarified that serine proteases are implicated in various inflammatory disorders. Some of the most well known members of serine proteases are trypsin, chymotrypsin, thrombin and Factor Xa. Coumarin derivatives are mostly active compounds. Many of them have been isolated from natural sources and especially from plants. In previous studies many coumarin derivatives have shown significant anti-inflammatory activity as well as antioxidant. Coumarins were found to highly inhibit various enzymes implicated in inflammation. Among them lipoxygenase and serine proteases, especially chymotrypsin and thrombin. In this study a number of new synthesized coumarin Mannich bases is presented. Among them: compound 2 [9-methyl-9,10-dihydro-2H,8H-hromen[8,7-e][1,3]-oxazin-2-one] and compounds 3-15 with the general structure of 7-hydroxy-8-methylene-amine-substituted coumarins. Another group of new coumarin derivatives has also been synthesized. These compounds contain an azomethine group at position 7 of the coumarin ring. The structure of all the new compounds (bases, salts, azomethine derivatives) are confirmed spectrophotometrically and elemental analysis have been performed. Lipophilicity of the compounds was determined experimentally as RM values by RPTLC and theoretically by computational methods (C-QSAR for Clog P and McSpartan). The compounds are novel and in relation to their pharmacological properties are of high interest, since they present major differences to the classical anti-inflammatory agents. All the compounds have been conducted to a preliminary in vitro and in vivo study. The new compounds were studied for their interaction with the stable free radical DPPH and most of them shown high ability to scavenge free radicals. Moreover many of them were found to have a good competition with DMSO for scavenging free hydroxy radicals, which were found to be involved in inflammatory process. A number of enzymes is well known that they are implicated in inflammation. Some of them are lipoxygenase, cyclooxygenase and #-glucuronidase. Most of the new tested coumarin derivatives have shown high inhibitory activity on these enzymes in two different concentrations (0.01 mM and 0.1 mM). Compounds were tested in vitro against various serine proteases. In the case of trypsin and chymotrypsin two different experiments were performed examining their proteolytic activity and their ability to function as esterases. Almost all the compounds presented high inhibitory activity against both enzymic activities Two different experiments were performed to delineate the inhibitory activity of the tested coumarins against thrombin. Some of the compounds presented significant inhibition. All the azomethine - derivatives were studied in vitro for the nitric oxide release in the presence of thiol cofactors. It was found that ##2 - release was dependent on the nature of thiol. Some of them were found to highly release ##2 . For the anti-inflammatory activity of all the new compounds the carraggenin induced rat paw edema has been used. All the tested compounds highly inhibit the carrageen induced rat paw edema at 0.01 mmol/ kg b.w. For some of the synthesized compounds the adjuvant induced arthritis (AID) model has been used. AID is considered to be one of the best experimental models for investigation of anti-inflammatory drugs and quite close to the human rheumatoid arthritis. The examined compounds suppressed very effectively the AID manifestations (inflammation – arthritic score – cachexia impairment of the in vivo drug metabolism). In continuation, we tried to linearly correlate the different biological responses in order to delineate the mechanism of action of these derivatives: in vivo anti-inflammatory and in vitro antioxidant activity, free radical scavenging activity and inhibitory activity of enzymes induced by the tested compounds. The relationships between structure, physicochemical properties and biological activities for several classes of serine proteases (trypsin, thrombin and Factor Xa) inhibitors reported in literature, have been evaluated. An attempt has been also made to correlate all the biological data of this research with the physicochemical properties of the corresponding compounds. The QSAR analysis presented here is an attempt to organize the knowledge available from the compounds able to inhibit trypsin, thrombin and Factor Xa. The C-QSAR methodology of Biobyte can greatly help in this effort being focused on the physicochemical and statistical bases as to extract useful results that can be used in the further design of serine proteases inhibitors.