Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-03-16
2001-01-23
Badio, Barbara (Department: 1616)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C514S177000, C514S179000, C514S172000, C514S173000, C514S824000, C540S007000, C540S046000, C540S076000, C540S114000, C552S508000, C552S510000, C552S530000
Reexamination Certificate
active
06177418
ABSTRACT:
This invention is directed to a class of tetracyclic triterpenes, in particular protostane and fusidane (29-desmethylprotostane) derivatives, that are useful as cholesterol-lowering and anti-atherosclerosis agents.
More particularly, the invention relates to a hitherto unknown metabolite of the fungus
Fusidium coccineum
represented by the formula (I)
and pharmaceutically acceptable salts and in vivo hydrolysable esters thereof, and to derivatives of said compound having the general formula (II)
wherein
R
1
stands for hydrogen or methyl;
R
2
is hydrogen, methyl, CH
2
OH, CH
2
OR
3
, CHO, CH═CH
2
, COOH or COOR
4
;
R
3
stands for straight or branched (C
1
-C
6
) alkyl, aralkyl or aryl, optionally substituted with halogen, hydroxy or carboxy; alkanesulfonyl or arenesulfonyl; (C
1
-C
4
)alkanoyl or aroyl, optionally substituted with halogen, hydroxy or carboxy;
R
4
stands for straight or branched (C
1
-C
6
)alkyl, (C
2
-C
6
)alkenyl, (C
2
-C
6
)alkynyl, aralkyl, aryl, alkanoyloxyalkyl or dialkylaminoethyl;
Q
1
and Q
2
are each independently hydrogen, hydroxy or a group OR
3
; or, taken together, Q
1
and Q
2
stand for oxygen; or Q
1
(Q
2
) and R
1
(R
2
), when taken together, constitute a double bond that connects carbon atoms 3 and 4; or Q
2
and R
2
, when taken together with carbon atoms 3 and 4, may form an oxetane ring.
X is hydrogen; or X and Q
1
(Q
2
), when taken together, form a double bond connecting carbon atoms 2 and 3;
the C24,25-bond is a double bond or a single bond;
and, additionally, one or more of the double bonds connecting carbon atoms 2 and 3, 3 and 4, 17 and 20, and/or 24 and 25 may optionally be epoxidized with formation of an oxirane ring or hydrated to give a carbon-carbon single bond where one of the carbon atoms is substituted with hydroxy;
with the proviso that when, at the same time, the C24,25-bond is a double bond, Q
1
is hydrogen, Q
2
is hydroxy, and R
1
is methyl, then R
2
cannot be methyl or hydroxymethyl;
and pharmaceutically acceptable salts and in vivo hydrolysable esters thereof.
The compounds of the invention derived from the compounds of formula (II) by epoxidation or hydration can comprise several diastereomeric forms (e.cg. R and S configuration at the carbon atoms which are part of the oxirane ring or at the carbon atom bearing the hydroxy group). The invention covers all these diastereoisomers in pure form as well as mixtures thereof.
Atherosclerosis, a chronic disease related to the vascular system, is one of the most common causes of death in the Western world, and a high cholesterol level in the blood is a key risk factor in its development.
The inhibition of the biosynthesis of cholesterol constitutes an important approach to lowering serum cholesterol, and several therapeutic agents based on this principle are already available.
These agents (e.g. lovastatin, simvastatin, pravastatin and fluvastatin) interfere with an early step in the cholesterol biosynthesis—namely the conversion of hydroxymethylglutaryl-CoA (HMG-COA) to mevalonate (cf.
Scheme A).
Scheme A is a schematic presentation of multivalent feedback regulation of HMG-CoA-reductase. The dashed lines indicate probable nonsterol regulators and the dotted lines indicate regulation by cholesterol which is derived from LDL uptake. This cholesterol suppresses HMG-CoA reductase and to a limited extent squalene synthetase (Brown & Goldstein, 1980, J. Lipid Research 21, 505-517).
However, mevalonate is also the obligate precursor of a number of non-steroidal isoprenoids such as dolichol, ubiquinone and isopentenyl t-RNA and the formation of these essential compounds will therefore also be inhibited by inhibitors of HMG-CoA reductase. This is an undesired effect and efforts have therefore been concentrated on the finding of cholesterol lowering compounds that interfere with a later step in the biosynthesis of cholesterol.
Recently, the isolation and characterisation of two new families of compounds, called squalestatins and zaragozic acids, respectively, have been reported. These compounds are potent inhibitors of the enzyme squalene synthetase (cf. Scheme A) and therefore lower the formation of the cholesterol-precursor squalene without interfering with the production of non-steroidal isoprenoids.
The conversion of 2,3-oxidosqualene into lanosterol—another intermediate in the biosynthesis of cholesterol—is another target for inhibition. This conversion, which is catalyzed by the enzyme oxidosqualene cyclase, is believed to take place as outlined in scheme B.
The 2,3-oxidosqualene, formed by enzymatic epoxidation of squalene, is folded in a pre-chair-boat-chair-boat conformation and the proton initiated cyclization proceeds through a series of rigidly-held carbocationic intermediates. The intermediate C-20 protostercl cation then undergoes backbone rearrangement to yield lanosterol.
Because of the similarity between the conformation of the protosterol cation and that of a protosterol (e.g. compound 6, Scheme 1) we hypothesized that certain compounds containing the protostane ring system might act as inhibitors of oxidosqualene cyclase and thereby inhibit the formation of cholesterol in a very specific way.
The effect of the compounds of the invention on cholesterol synthesis (
14
C-acetate incorporation into cholesterol, separated by TLC) in human Hep G2 cells can be tested in vitro according to the method described by A. Boogards et al, (Biochem. J., 1987, 241, 345-351).
The effect of the compounds of the invention on cholesterol biosynthesis from [
14
C]acetate or [
3
H]mevalonate by isolated rat heptocytes and by rat or mouse liver in vivo can be tested according to the method described by Y. Tsujita et al. (Biochem. Biophys. Acta, 1986, 877, 50-60).
Two of the compounds represented by the general formula (II), i.e. those in which the C24,25-bond is a double bond, Q
1
is hydrogen, Q
2
is hydroxy, R
1
is methyl and R
2
stands for either methyl or hydroxymethyl have been described previously. (S. Okuda et al., Tetrahedron Letters 1968, 4769-4772; T. Hattori et al., Tetrahedron Letters 1969, 1023-1026; G. Visconti, Ph.D. Thesis No. 4156, ETH Zurich, 1968). Both compounds have been isolated in small amounts from the mycelium of the helvolic acid-producing fungus
Cephalosporium caerulens
and, independently, from the mycelium of
Fusidium coccineum
, the fungus known to produce fusidic acid, but an investigation of their biological activities has never been reported.
However, the discovery and recent isolation in substantial amounts of the compound of formula (I) offered the possibility to prepare larger amounts of said two compounds of formula (II) by chemical means and to study their biological activities.
It has now been found that said two compounds of formula (II) and other compounds of the present invention show activity as inhibitors of hepatic cholesterol synthesis in vitro and in vivo.
The invention also relates to methods of preparing the compounds of the formulae (I) and (II) as defined above.
The compound of formula (I) is a hitherto unknown metabolite of the fungus
Fusidium coccineum
, formed during the fermentation process in addition to fusidic acid, and can be isolated in substantial amounts by fractionation of mother liquors from which fusidic acid has been recovered.
It is noteworthy in this context that the production of fusidic acid by fermentation of
Fusidium coccineum
has been described in detail (see Biotechnology of Industrial Antibiotics, E. J. Vandamme, ed.; Marcel Dekker, Inc., New York, 1984, 427-449, and references cited therein).
The new compound is a tetracyclic triterpenoid acid C
30
H
48
O
3
, containing a secondary hydroxyl group and two isolated double bonds, one trisubstituted, the other tetra-substituted. Chemical and spectral data obtained for this compound were in agreement with the structure shown in formula (I). The compound can be used as such or in the form of salts or in vivo hydrolysable esters.
The compounds of formula (II) may conveniently be prepared from the compound of formula (I) by
Daehne Welf von
Godtfredsen Wagn Ole
Badio Barbara
Leo Pharmaceutical Products Ltd. A/S
Pillsbury Madison & Sutro LLP
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