Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reissue Patent
1992-02-03
2003-02-18
Jordan, Kimberly (Department: 1205)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
C514S824000, C552S546000
Reissue Patent
active
RE037999
ABSTRACT:
FIELD OF INVENTION
The field of the invention is medicine agents and methods for combatting atherogenesis.
BACKGROUND OF INVENTION
The role of cholesterol oxidation products in atherogenesis has long been a controversial topic. Peng and Taylor (1983), for example, hypothesized that cholesterol oxidation products may be responsible for an initial arterial cell injury that eventually results in atherosclerosis. On the other hand, Higley et al. (1986) disputed that hypothesis, claiming instead that oxidized cholesterol is substantially less atherogenic than purified cholesterol. The analysis of the oxidized cholesterol shown in Table 1 of Higley et al. indicated the presence of diols and epoxides (78%) as the major oxidized compounds with a minor amount of hydroperoxide (18%) identified as 7&agr;-hydroperoxide.
Hypercholesteroloemia is widely considered to be a major risk factor for the development of atherosclerosis. The lowering of blood cholesterol levels has therefore been an important goal in the search for ways to prevent or treat atherosclerosis. Medicinal agents reported as reducing development of athersclerotic lesions typically result in the lowering of blood cholesterol levels. Beneficial agents which do not affect blood cholesterol are rare. But Bell and Schaub (1986) have reported that chlorpromazine reduced the development of such lesions in rabbits fed an atherogenic diet without lowering of blood cholesterol. Chlorpromazine may function as an inhibitor of calmodulin (Gietzen, 1986) but it is classified pharmacologically as a tranquilizer and sedative (Merck Index, 1976), and therefore is not likely to be useful as a treatment for atherosclerosis.
Tipton, et al. (1987), have reported that a mixture of cholesterol auto-oxidation products prepared from an aged sample of cholesterol inhibits calmodulin irreversibly in a Ca
2
+-dependent reaction. The reactive material was destroyed by chemical reduction. It was concluded that the calmodulin inhibition was due to one or more cholesterol hydroperoxides. The mixture of cholesterol hydroperoxides was further purified by chromatography and used in feeding experiments with rabbits. Three diets were compared, one with added cholesterol, one with added cholesterol hydroperoxides, and the third with both the cholesterol and cholesterol hydroperoxides. The diet containing cholesterol alone was found to have caused extensive atheroma formation, while the addition of the cholesterol hydroperoxides to the cholesterol diet markedly reduced atheroma formation. The cholesterol hydroperoxides were not found to lower cholesterol concentration in blood plasma, liver or heart. In a related experiment, it was found that the chemically reduced hydroperoxides were not effective in reducing atheroma formation.
Cholesterol oxidizes readily in contact with air, and the oxidation proceeds at ambient room temperature. The oxidation products as initially formed are largely cholesterol hydroperoxides. See Smith, “Cholesterol Autooxidation” (1981, Plenum Press, New York). This reference lists the initial auto-oxidation products in Table 11, pages 238 to 239. These included 3&bgr;-hydroxycholest-5-ene-7&agr;-hydroperoxide; 3&bgr;-hydroxycholest-5-ene-7&bgr;-hydroperoxide; and 3&bgr;-hydroxycholest-5-ene-25-hydroperoxide, which is presently preferred for use in this invention. Other hydroperoxides included 3&bgr;-hydroxycholest-5-ene-17-hydroperoxides; 3&bgr;-hydroxycholest-5-ene-20-hydroperoxides; 3&bgr;-hydroxycholest-5-ene-22-hydroperoxides; and 3&bgr;-hydroxycholest-5-ene-24-hydroperoxides.
When it is desired to accelerate the oxidation of cholesterol to hydroperoxides, a photochemical oxidation procedure can be employed as described by Schenk et al. (1958). This reference describes the preparation and isolation of 3&bgr;-hydroxycholest-5-ene-7&agr;-hydroperoxide. The 7-&bgr; isomer of this compound can be synthesized and isolated as described by Teng et al. (1973). Other cholesterol hydroperoxides can be prepared as an oxidized mixture, and separated by fractionation. See van Lier et al. (1970).
SUMMARY OF INVENTION
This invention is based in part on the discovery of a novel class of cholesterol hydroperoxide compounds in which the hydroperoxide group is in the 20-position and the compounds also contain other added oxygenation. As far as is known, no oxidized derivatives of cholesterol have heretofore been reported containing
a 20-hydroperoxy group together with a 25-hydroxyl group. An example of such novel compounds is 20-hydroperoxy-25-hydroxycholesterol which has two isomeric forms (R and S). Specific compounds are 20(R)-hydroperoxy-25-hydroxycholesterol and 20(S)-hydroperoxy-25-hydrocholesterol. These compounds can also be named as: 20-(R)-hydroperoxy-cholest-5-ene-3&bgr;,25-diol, and 20(S)-hydroperoxy-cholest-5-ene-3&bgr;,25-diol.
hydroperoxy groups at two positions. An example of such novel compounds is
20
,
25
-
dihydroperoxy cholesterol which has two isomeric forms
(
20
R and
20
S
).
Specific compounds are
20
-(
R
)-
20
,
25
-
dihydroperoxycholesterol and
20
-(
S
)-
20
,
25
-
dihydroperoxycholesterol. These compounds can also be named as:
20
-(
R
)-
20
,
25
-
dihydroperoxy
-
3
&bgr;
-
hydrocholest
-
5
-
ene and
20
(
S
)-
20
,
25
-
dihyroperoxy
-
3
&bgr;
-
hydroxycholest
-
5
-
ene.
The 20-hydroperoxy cholesterol compounds of this invention are highly active as calmodulin inhibitors, and also display a high level of activity as inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT). ACAT is an index for anticholesterol therapeutic agents (Suckling et al., 1985). The novel compounds of this invention are therefore believed to be useful as therapeutic agents for suppressing atherogenesis in human patients.
EXPERIMENTAL BASIS OF INVENTION
Isolation
The starting material is cholesterol that has been “aged” in air at room temperature for a long time (in this case 20 years) or cholesterol that has been heated at 55-60 C. in the dark in a thin layer for 14 days.
The starting material is dissolved in boiling methanol, 30 ml per gram of starting material; the solution is cooled slowly to 4 C. to allow crystallization of unchanged cholesterol. The cholesterol is removed by vacuum filtration and the filtrate is reduced to one eighth the original volume by distillation on a rotary evaporator under reduced pressure. After cooling the concentrated filtrate to 4 C., a second crop of cholesterol is removed by vacuum filtration. The filtrate from this step is extracted three times with hexane and the methanol-rich (lower) layer is evaporated to dryness under vacuum with a rotary evaporator to yield 2.5 g of residue from 50 g of starting material.
This mixture is fractionated by flash chromatography on silicic acid (60-230 mesh, 200 g) eluted with hexane, 2-propanol mixtures according to the following protocol:
hexane-2-propanol ratio,
volume,
v/v
ml.
12:1
260
11:1
240
10:1
440
9:1
500
6:1
280
Elution was at the rate of 50 mL/min and 50 mL fractions were collected. The desired product elutes in fractions 16-21, with hexane:2-propanol 10:1 v/v. The combined fractions 16-21 are evaporated to dryness to yield about 0.3 g of material which is then rechromatographed on silicic acid (200-400 mesh, 75 g) with hexane and 2-propanol mixtures according to the following protocol:
hexane-2-propanol ratio,
volume,
v/v
ml.
10:1
440
9:1
250
8:1
270
7:1
240
5:1
240
The flow rate is 20-25 mL/min. After the first 100 mL of solvent, one-minute fractions are collected. Elution of the hydroperoxides is monitored by thin-layer chromatography on silica gel, with benzene and ethyl acetate 3:2 (v.v) as the developing solvent. Hydroperoxides are detected by spraying the plates with 1% N,N-dimethyl-p-phenylenediamine in methanol and water 1:1 (v/v) containing 1% acetic acid. The desired product elutes in fractions 25-31, with hexane:2-propanol 8:1 v/v and the yield is 40 mg.
This mixture is then fractionated by reverse-phase high-performance liquid chromatography on an Ultrasphere (Beckman Instrument Co.) octadecylsilane-treated silica column,
Shih Meiling
Tipton Carl L.
Iowa State University & Research Foundation, Inc.
Jordan Kimberly
Tilton, Fallon, Lungmus & Chestnut
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