Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-05-09
2002-01-15
Gerstl, Robert (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S119000, C548S414000
Reexamination Certificate
active
06339160
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATION
The present application is the national stage under 35 U.S.C. 371 of PCT/IT98/00202, filed Jul. 17, 1998.
DESCRIPTION
The present invention has as its object new compounds usable in the therapy of a series of human pathologies such as tumoral growth and metastatization, atherosclerosis, multiple sclerosis, Alzheimer's disease, osteoporosis, rheumatoid arthritis and other inflammatory diseases. Said compounds in fact, following in vitro experiments extensively described in the following chapters, showed a remarkable inhibitory capability on certain human enzymes, the zinc-dependent metalloproteinases, which have been related with such pathologies (see for example: “Inhibition of matrix metalloproteinases. Therapeutic potential” - Annuals N.Y. Acad.Sci. 732 (1994)). Thus although an integration of experimental data with adequate evidence in vitro is naturally necessary, the results collected already allow to expect their usability in specific therapies. Moreover such inhibitory capacity was originally demonstrated also in a series of zinc-dependent metalloproteinases extracted from snake venoms, also denominated “hemorrhagines” for their capacity of inducing extensive internal herorrhagies in victims of snake bites, and constitute the most damaging agent in the venomous mixtures elaborated by Crotalidae and Viperidae. Thus their usability also in preparing specific antidotes against venom of Crotalidae and Viperidae seems evident.
The design and synthesis of such compounds in fact constitutes the last step in a long course of research, based on the study of the structure and action mechanism of certain particular zinc-dependent metalloproteinases called hemorhagines.
The article IL FARMACO (1993), 48 (9), 1271-7 shows that in the study of peptidic inhibitors a conformationally restricted model of compound tested on proteinase II from Crotalus Adamanteus snake venom has a sensibly lower inhibitory activity than that of related substrates. This result indicates that the structure of the inhibitor compound has direct influence on the fitting and bind at the enzyme active site and is not foreseeable a priori.
The so-called Hemorrhagic Factors of Hemorrhagines constitute a very important class of enzymes detected in the venom of snakes belonging to the Crotalidae family. They are structurally of use to the snake as they rapidly induce extended internal hemorrhagies in victims, causing circulatory collapse and preventing the victim from escaping its fate. The mechanism of the hemorrhagic action is due to the particular ease with which the enzymes are capable of degrading a large number of filiform proteins which bind the various vasal endothelid cells, allowing the elements of the blood to escape from the vessels. Although their molecular weights differ greatly, the hemorrhagines maintain however some fixed characteristics on the catalytic site, in the way that Zinc bonds with certain amino acids of the proteic chain, and in the way in which they attack the proteins of the basal membrane of blood vessels. They also seem to have in common the mechanism which ensures the protection of the snake's organism from the toxic effects of its own metalloproteinases, which seems based on the production of tripeptides capable of functioning as competitive inhibitors, interacting with the active site of the enzyme containing Zinc (Biomed.Biochim.Acta 50, 769-773, 1991).
Now the presence of Zinc in the active site of the enzyme constitutes one of the most interesting aspects of the study of Hemorrhagines. In fact this characteristic is not exclusively theirs, but characterizes a wide number of proteolytic enzymes which perform a series of important and diversified physiological and pathological functions in other animal organisms, evolutively also quite distant from each other. In relation to this a comparative study was carried out to determine possible similarities and differences in structure.
By studying the sequences of residues of the proteic chains and the amino acids involved in Zinc bonding it has been possible to obtain a sort of “family tree” for this family of proteinases (see for example FEBS Letters 312, 110-114, 1992 and Developmental Biology 180, 389-401, 1996): it has thus been seen that the active site of enzymes belonging to living beings quite distant from each other, such as Astatin (extracted from a river crustacean), Seratin (obtained from a microorganism), Matrixines (present in the organism of mammals) and those of Hemorrhage Factors of snake venom, in reality differ only in one of the four amino acids binding Zinc. Thus in spite of the fact that there are strong differences in the rest of the proteic structure, they can be considered to be in some way evolutively correlated. This is particularly interesting when considering the fact that the functions performed by these enzymes are not in any way analogous. In fact it has been ascertained that proteolytic enzymes of snake venom if on one hand are very similar and have thus allowed the definition of a proper new family of proteinases: snake venoms metalloproteinases (see for example Biol.Chem. Hoppe-Seyler 373, 381-385, 1992), on the other hand, they do not show any functional similarity with any other protein of the plant or animal world. In particular an extremely relevant fact is the difference between the functionality of hemmorhagines and those of Human Matrixines, which exercise important effects on cell migration and on the reconstruction of damaged tissues. Moreover, while Matrixines are released in the form of “zimogens” (that is, inactive enzymes which must be made functional through other proteinases intervention), and can be inhibited by particular proteins (TIMP), hemorrhagines are immediately active at the moment of dilution in the blood flow. In spite of such structural and functional differences the Applicant has determined the existence of a close correspondence between the inhibition of snake hemorrhagines and the pharmacological results obtained on animal models in which the patogenous agent is presumed to be a zinc-dependent metalloproteinase produced by the tissues of the mammal. Such correspondence seems the consequence of a structural resemblance existing albeit only in the active site between two different types of metalloproteinases and, based on this, the Applicant has developed a method for the selection of compounds for potential therapeutic use in man (Italian patent application RM95A000557; European patent application EP0758021).
Many mammal zinc-dependent metalloproteinases in fact have been related with pathological situations, some of which have been mentioned above. For example gelatinases seem involved in tumoral metastatization, while collagenases have a pathogenic role in arthritic phenomena.
EP-A-0 401 963 describes phosphonopeptides showing an inhibitory activity in regard to enzymes of the collagenase family and as such the compounds are considered useful in the treatment of arthritic and other diseases.
Certain compounds which inhibit matrixines have begun the phases of clinical development in patients suffering from tumour or arthritis: however they are usually scarcely absorbed when administered orally, and are constituted by hydroxamates, compounds which can present toxicity problems in chronic administration.
Finally, a new family of zinc-dependent metalloproteinases was recently identified, localized on the cell membrane, which possess the same proteic domains of hemorrhagines, and are thus considered their closest relatives (see Developmental Biology 180, 389-401, 1996). These proteinases, called ADAM (A Disintegrin and A Metalloproteinase Domain), are correlated with the functionality of the reproductive apparatus, but are also responsible for releasing TNF-a (Tumor Necrosis Factor alfa) and ACE in the circulation and seem correlated to SNC diseases, including Multiple Sclerosis.
SUMMARY OF THE INVENTION
The aim of the present invention is to supply compounds with pharmacological activity towards human pathologies, which have b
Cirilli Maurizio
D'Alessio Silvana
Di Stazio Giovanni
Gallina Carlo
Gavuzzo Enrico
Browdy and Neimark
Gerstl Robert
Polifarma S.p.A.
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