Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-04-22
2002-07-23
Padmanabhan, Sreeni (Department: 1621)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C424S450000, C552S542000
Reexamination Certificate
active
06423702
ABSTRACT:
In particular the present invention relates to a process for synthesizing steroid-type compounds hydroxylated in a position a to a double bond.
It also relates to the use of molecules thus synthesized as cyclotoxic agents active against cells which have a high proliferative potential.
The steroids are molecules which comprise a cyclopentanophenanthrene skeleton, widely distributed in the living world, and which have great physiological significance. This structure is thus found in bile acids, sex or cortical hormones, certain vitamins, and cardenolides of plant origin.
A great number of them have a therapeutic potential or properties, and it is thus particularly useful to have available a simple and inexpensive process for synthesizing them. A key stage of this synthesis consists of the selective hydroxylation of certain positions of the skeleton.
Among the steroids which are of significance, the cardenolides constitute a group of steroids having 23 carbons which exist in the form of glucosides in various plants, mainly in the foxglove, lily and corn crowfoot families. Many of these glucosides have a considerable cardiotonic activity and some are used in therapeutics, especially digitalin, digitoxin and digoxin. Schematically, the aglucon part of these molecules is characterized by the presence of a “butenolide”-type (unsaturated lactone) side chain, a 14-hydroxyl in the cis position with respect to the 18 methyl and a 3-hydoxyl [sic]. For example, this structure is found in the digitoxigenin molecule of formula:
The sugars which form a glucoside with the steroid via a bond established with the C-3 hydroxyl comprise glucose and rhamnose, as well a certain number of unusual sugars, such as 2,6-dideoxyhexose, often methylated in the C-3 position.
The way to construct an &agr;,&bgr;, lactone [sic] on C17 [lacuna] cyclic structure necessary for the pharmacological activity of this type of compound has been described, especially by Marini-Bettolo et al., (Can. J. Chem. 1981, 59, 1403-1404). Conventionally, 17-keto-&agr;,&bgr;-ene derivatives are used as starting compound and lithium ethoxyacetalide [sic], lithium &bgr;-furyl and lithium 2-methoxyfuryl are used as reactants. These processes of synthesis cannot be used on the industrial scale because they have a certain number of disadvantages. The time required to obtain the product is relatively long and, moreover, the synthesis is carried out under severe conditions (organic solvents, high temperature). Moreover, the yield of the reaction is low (of the order of 10%) and the purification of the product is difficult, in view of the many additional products which appear during the reaction. Finally, the starting material is both very expensive and very difficult to obtain.
Another group of steroid compounds which has a potential significance in therapeutics consists of the oxysterols, the structure of which derives directly from that of cholesterol via the presence of one or more oxygenated functional groups carried either by the rings or by the side chain of cholesterol. A role in the inhibition of cholesterol synthesis was first attributed to the oxysterols.
The oxysterols were then studied as antiproliferative agents.
In a recent review, Smith & Johnson (Free Radical Biology and Medicine, 1989, 7, 285-332) itemize the mammary cells attacked in vitro by these compounds. In a completely different approach, Cheng et al., (J. Chem. Res., 1977, 217, 2501-2521) have isolated and identified antitumoral active principles from drugs used in the traditional Chinese pharmacopoeia.
Thus the drug Bombix Cum Botryte [sic] contains 7&bgr;-hydroxycholesterol which has a cytotoxic activity against cultured cells showing significant multiplication, such as lymphoma (mouse RDM4 and YAC-1) (Cancer Biochem. Biophys., 1986, 9, 75-83), HTC cells (rat liver tumoral cells) (Biochem. Biophys. Res. Commun., 1984, 120, 192-198), rat fibroblasts originating from the heart (C.R. Acad. Sci. Paris, 1984, 299, 221-225) and rat liver epithelial cells (Cell Biol. Toxicol., 1989, 5, 261-270). On the other hand, cells which have a weaker potential for division and which achieve a certain degree of differentiation when cultured, such as cultured heart cells and hepatocytes from newborn rats, are not detrimentally affected by this molecule. The cytotoxicity of 7-hydroxycholesterol [sic] is shown by a halt in cell growth, followed by morphological modifications which precede the detachment and the rapid lysis of the cells.
Rong et al., (C.R. Acad. Sci. Paris, 1985, 300, 89-94) have shown that the sodium salts of 7&bgr;-hydroxycholesterol 3,7-bis(hemisuccinate) have an antitumoral activity, injected intraperitoneally into mice carrying ascitic tumors, [lacuna] Krebs-II transplantable carcinoma. It [sic] also shown, in female Sprague-Dawley rats, that (22 R)-cholest-5-ene-3&bgr;,7&bgr;22-triol effectively reduced the development of tumors induced by 7,12-di-methylbenz(&agr;)anthracene [sic] (Iversen et al., Virchows Archiv B, 1986, 51, 313-320).
It is generally assumed that the cytotoxic effect of the oxysterols results from the superimposition of several phenomena, the most important of which are the inhibition of the synthesis of cholesterol by inhibition of a key enzyme (HMGR) and a destabilization of the plasmic membranes. In this context, and with a long-term therapeutic prospective, the inventors have studied the effects and the mechanism of action of 7&bgr;-hydroxycholesterol on spontaneously transformed lines obtained from primary cultures of astrocytes from newborn rats (normal cells). In the central nervous system (CNS), the astrocytes (glial cells) have retained the potentiality of multiplying, which phenomenon is manifested in certain pathological states, such as reactional gliosis (inflammation) and formation of glioblastomas (neoplasia).
Now, a recent publication shows that 7&bgr;-hydroxycholesterol is metabolized, in transformed astrocytes, to esters of fatty acids.
The conventional route of chemical synthesis used for obtaining 7&bgr;-hydroxycholesteryl ester resorts to 7-ketocholesterol as starting material.
After esterification of the OH in the 3-position, the functional group is reduced with NaBH
4
. The two a and &bgr;, isomers are obtained with a yield of 70% for the &bgr; isomer. This process is difficult to transpose to the industrial level, given its duration.
This is why the present invention relates to a gentle process for the hydroxylation of an acyclic or cyclic methylene radical in the allylic position, characterized in that a peroxidase, iodide ions and hydrogen peroxide (H
2
O
2
) are reacted with this methylene radical to produce a compound hydroxylated in a position a to a double bond.
The peroxidase used is preferably lactoperoxidase (LPO), the iodide ions generally arise from KI and the hydrogen peroxide can be generated in situ.
In contrast to the conventional processes for hydroxylation, this process does not involve a powerful reducing agent.
The OH radical is provided by the decomposition of H
2
O
2
, catalyzed by lactoperoxidase, an enzyme which is commercially available at reasonable cost.
The reaction can be carried out at temperatures of between 20° C. and 40° C., and will not affect functional groups present on the molecule.
The present invention thus relates to a process for the hydroxylation of an acyclic or cyclic methylene in the allylic position, characterized in that the starting compound contains at least one unprotected ester functional group, and in that this ester functional group is recovered intact in the compound after hydroxylation.
The present invention also relates to a process for the hydroxylation of an acyclic or cyclic methylene in the allylic position, in which the starting compound contains an ether functional group.
The allylic methylene radical is preferably:
a secondary carbon (1) arranged on [sic] the following Scheme Ia:
in which the carbon (2) is preferably connected to a hydrogen or to other carbo
Behr Patrick
Kupferberg Alexandre
Mersel Marcel
Privat Alain
Centre National de la Recherche Scientifique "CNRS"
Jacobsoh Holman, PLLC
Padmanabhan Sreeni
LandOfFree
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