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Description  |
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This invention relates to the tigogenin cellobioside, tigogenin
cellobioside heptaacetate and to the use of these compounds for treatment
of hypercholesterolemia and atherosclerosis.
RELATED DISCLOSURES
French Pat. No. 2,425,859 and U.S. Pat. No. 4,260,603 describe the
medicaments having activity as prostaglandin synthetase inhibitors which
medicaments comprise certain sterol glycosides, a fatty acid ester
thereof, or a spiroketal-steroid glucosides.
Hemolytic properties of tigogenin .alpha.-L-rhampyranoside and tigogenyl
-maltoside are described in J. Pharm. Sci., 67(11):1589 (1978).
SUMMARY
One aspect of this invention is a compound of formula
##STR1##
wherein R is hydrogen, namely tigogenin cellobioside, as an individual
.alpha.- or .beta.-anomer or a mixture thereof, which compound is useful
in treatment of hypercholesterolemia and atherosclerosis.
Another aspect of this invention is a compound of the formula
##STR2##
wherein R is --C(O)CH.sub.3, namely tigogenin cellobioside heptaacetate,
as an individual .alpha.- or .beta.-anomer or a mixture thereof, which is
an intermediate in the synthesis of tigogenin cellobioside.
Yet another aspect of this invention is the method of treating
hypercholesterolemia and atherosclerosis by administering the compound of
this invention to a subject in need of such treatment.
Still another aspect of this invention is a pharmaceutical composition
comprising a therapeutically effective amount of a compound of this
invention in admixture with suitable pharmaceutically acceptable
excipient.
The last aspect of this invention is a process of making the compound of
this invention, particularly the process of preparation of .alpha.- and
.beta.-anomers.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used hereinafter:
"Tigogenin" means a compound of 5.alpha.,20.alpha.,22.alpha.,
25D-spirostan-3-ol, represented by the formula
##STR3##
"Tigogenin cellobioside" means a non-separated mixture of .alpha. and
.beta.-anomers and an individual .alpha.- and .beta.-anomer. It is
depicted by the formula
##STR4##
wherein R is hydrogen.
The wavy line illustrates the possibility that the steroid is attached to
the cellobioside either above or below of the plane. "Tigogenin
cellobioside heptaacetate" means a non-separated mixture of .alpha.- and
.beta.-anomers and an individual .alpha.- and .beta.-anomer. It is
depicted by the formula
##STR5##
wherein R is --C(O)CH.sub.3.
The wavy line illustrates the possibility that the steroid is attached to
the cellobioside heptaacetate either above or below of the plane.
".alpha.-Anomer" means the compound wherein the steroid is attached below
the plane of the cellobioside.
".alpha.-Tigogenin cellobioside" or ".alpha.-Tigogenin cellobioside
heptaacetate" is depicted by the formula
##STR6##
wherein R is hydrogen or --C(O)CH.sub.3, respectively.
".beta.-Anomer" means the compound wherein the steroid is attached above
the plane of the cellobioside.
".beta.-Tigogenin cellobioside" or ".beta.-Tigogenin cellobioside
heptaacetate" is depicted by the formula
##STR7##
wherein R hydrogen or --C(O)CH.sub.3, respectively.
"Mammals" means a class of warm-blooded vertebrates characterized by
mammary glands, including but not limited to humans, laboratory or
domestic animals such as dogs, cats, mice, rats or rabbits, and livestock.
"Treatment" covers any treatment of the disease in a mammal, particularly
human, and includes:
(i) preventing the disease from occurring in a subject which may be
predisposed to the disease but has not yet been diagnosed as having it;
(ii) inhibiting the disease, i.e. arresting the development of said
disease; or
(iii) relieving the disease, i.e. causing regression of the disease.
"Hypercholesterolemia", also known as hypercholesteremia or
hypercholesterinemia, means the presence of an abnormally large amount of
cholesterol in the cells and plasma of the circulating blood.
"Arteriosclerosis" as used herein means a degenerative arterial sclerosis
marked by hardening and thickening of the vessel walls.
The types of arteriosclerosis generally recognized are atherosclerosis,
Monckerberg's arteriosclerosis, hypertensive arteriosclerosis,
arteriolosclerosis or senile arteriosclerosis.
"Atherosclerosis" as used herein means:
(i) deposition of lipid with proliferation of fibrous connective tissue
cells in the inner walls of arteries;
(ii) modular sclerosis; arteriosclerosis characterized by irregularly
distributed lipid deposits in the intima of large and medium-sized
arteries. Such deposits are associated with fibrosis and calcification,
and are almost always present in some degree in the middle-aged and
elderly.
"Hypertensive arteriosclerosis" means progressive increase in muscle and
elastic tissue of arterial walls resulting from hypertension. In
longstanding hypertension elastic tissue forms numerous concentric layers
in the intima and there is replacement of muscle collagen fibers and
hyaline thickening of the intima of arterioles. Such changes can develop
with increasing age even in the absence of hypertension and may then be
referred to as senile arteriosclerosis.
"Monckeberg's arteriosclerosis" means
(i) degeneration,
(ii) sclerosis, or
(iii) calcification.
Monckeberg's arteriosclerosis generally means arterial sclerosis involving
the peripheral arteries, especially of the legs of older people, with
deposition of calcium in the medial coat (pipe-stem arteries) but with
little or no encroachment on the vessel lumen.
"Arteriolosclerosis" means arteriolar sclerosis. Arteriolosclerosis affects
mainly the small vessels called arterioles. Arteriolosclerosis can be seen
especially in patients with chronic hypertension.
PREFERRED EMBODIMENTS
This invention concerns a compound of the formula
##STR8##
wherein R is either hydrogen or --C(O)CH.sub.3, namely tigogenin
cellobioside or tigogenin cellobioside heptaacetate, as an individual
.alpha.-anomer, .beta.-anomer or mixture thereof.
One preferred group of compounds are those wherein R is --C(O)CH.sub.3,
namely:
.alpha.-tigogenin cellobioside heptaacetate;
.beta.-tigogenin cellobioside heptaacetate; or
the mixture of .alpha.- or .beta.-tigogenin cellobioside heptaacetate.
The second and most preferred group of compounds are those wherein R is
hydrogen, namely:
.alpha.-tigogenin cellobioside;
.beta.-tigogenin cellobioside; and
the mixture of .alpha.- and .beta.-tigogenin cellobioside.
PREPARATION PROCEDURES
Reaction Scheme 1 illustrates the preparation of tigogenin cellobioside
(A). In the formula (A) and in the following text the term tigogenin
cellobioside is meant to include the mixture of .alpha.- and
.beta.-anomers of tigogenin cellobioside or individual .alpha.-anomer, or
.beta.-anomer.
The R in formula (III) is --C(O)CH.sub.3 and the R in formula (A) is
hydrogen.
##STR9##
Reaction Scheme 1 illustrates processes of preparation of an individual
.alpha.-anomer, or an individual .beta.-anomer or a nonseparated mixture
of both. All three processes use the same starting compounds, cellobiose
octaacetate (I) and tigogenin (II), have the same intermediate, tigogenin
cellobioside heptaacetate (III), and result in the same final compound
(A).
Three procedures differ only in reaction conditions to give either
individual .alpha.-anomer (Procedure 1) or individual .beta.-anomer
(Procedure 2) or the mixture of both (Procedure 3).
Procedures 1 and 2
Reaction conditions in Procedures 1 and 2 are essentially the same, except
that a stereochemical modification is obtained using the different
reaction solvent. Tetrahedron Letters, 28:1379 (1984) describe such
solvent induced stereochemical modification. In this case, the use of
acetonitrile gives the .alpha.-anomer. The use of the methylene chloride,
on the other hand, gives .beta.-anomer.
Step 1: Step 1 illustrates the conversion of tigogenin and cellobiose
octaacetate to tigogenin cellobioside heptaacetate (III).
Both starting materials, cellobiose octaacetate and tigogenin, are
commercially available from Aldrich and from Research Plus.
Cellobiose octaacetate (I) is reacted with tigogenin (II) in an organic
solvent, preferably acetonitrile for preparation of .alpha.-anomer and
methylene chloride for preparation of .beta.-anomer, in approximate
amounts of 65:200:5000; wt:wt:vol. Metal chloride, preferably stannic
chloride, is added over a period of 1-5 minutes, preferably 2 minutes. The
reaction mixture is warmed to a temperature between 50.degree.-75.degree.
C., preferably to 65.degree. C. or to the temperature when the mixture
becomes homogeneous. That temperature is maintained for 10-60 minutes,
preferably for 20 minutes, then cooled to 20.degree.-40.degree. C.,
preferably 30.degree. C. The mixture is then submitted to purification
procedures by methods known in the art to give, depending on the solvent
which is used, the .alpha.- or .beta.-tigogenin cellobioside heptaacetate
(III).
Step 2: Step 2 illustrates the preparation of tigogenin cellobioside (A) by
hydrolysis of compound (III).
Compound (III) is reacted with water and a mixture of organic solvents,
preferably methylene chloride, triethylamine and methanol, in approximate
amounts of 4:10:6:12:24; wt:v:v:v:v, at temperature of
20.degree.-80.degree., preferably at reflux, for 2-10 hours, preferably
for 6 hours. Then, the reaction mixture is stirred overnight at
temperature of 15.degree.-30.degree. C., preferably at room temperature.
The resulting material is evaporated, purified and separated by methods
known in the art to give .alpha.- or .beta.-tigogenin cellobioside (A),
depending on which isomer of tigogenin cellobioside heptaacetate was used
in the Step 2.
Procedure 3
Procedure 3 illustrates the preparation of the mixture of predominantly
.beta.-tigogenin cellobioside.
Step 1: Both tigogenin (I) and cellobiose octaacetate (II) are dissolved in
an organic solvent, preferably in methylene chloride, in approximate
amounts 4:80; w/v and 14:80; w/v, respectively. Metal salt, preferably
stannic tetrachloride, is added to cellobiose octaacetate in an amount
approximately equal to that of cellobiose octaacetate. Cellobiose solution
is then added to tigogenin and reacted to 1-8 hours, preferably for 3
hours, at a temperature of 15.degree.-30.degree. C., preferably at room
temperature. The mixture is washed with buffer, preferably with
bicarbonate and the gas which develops during the reaction is removed. The
mixture is submitted to purification and separation by techniques known in
the art to yield the mixture of .alpha.- and .beta.-tigogenin cellobioside
heptaacetate (III).
Step 2: An organic solvents/water mixture, preferably
triethylamine/methanol/methylene chloride/water and the solution of
heptaacetate (III), obtained above, is reacted under constant stirring for
6-24 hours, preferably overnight at a temperature of 15.degree.-30.degree.
C., preferably at room temperature. The solvent is removed and the residue
is submitted to purification and separation by techniques known in the
art. The purified mixture is dialyzed in a tilting dialyzer (described in
Example 3) for 1-4 days, preferably for 3 days. The mixture is submitted
to another purification procedure and extracted with an organic solvent,
preferably heptane, in a soxhlet. The resulting residue is dried at
15.degree. to 30.degree. C., preferably at room temperature, for 1-4 days,
preferably for 3 days, to yield a predominantly .beta.-tigogenin
cellobioside.
The mixture of .alpha.- and .beta.-tigogenin cellobioside is obtained by
mixing the proportional amounts of individual .alpha.- and .beta.-anomers.
Isolation and purification of the compounds and intermediates described
herein can be effected, if desired, by any suitable separation or
purification procedure such as, for example, filtration, extraction,
crystallization, column chromatography, thin-layer chromatography or
thick-layer chromatography, or a combination of these procedures. Specific
illustrations of suitable separation and isolation procedures can be had
by reference to the examples hereinbelow. However, other equivalent
separation or isolation procedures could, of course, also be used.
UTILITY AND ADMINISTRATION
Utility
This invention relates to certain glycosides which are potent inhibitors of
cholesterol absorption and are therefore primarily useful for treatment of
hypercholesterolemia. Since the hypercholesterolemia is closely related to
the development of generalized cardiovascular, cerebral vascular or
peripheral vascular disorders, secondarily these compounds prevent the
development of atherosclerosis, particularly arteriosclerosis.
Cholesterol, which belongs to the body major plasma lipids, is highly
soluble in fat but only slightly soluble in water. It is capable of
forming esters with fatty acid and approximately 70% of the cholesterol
present in plasma is in the form of cholesterol esters.
Cholesterol present in the body is either of endogenous or exogenous
origin. Exogenous cholesterol is present in the diet and is absorbed
slowly from the gastrointestinal tract into the intestinal lymph.
Endogenous cholesterol, in a rather large quantity, is formed in the cells
of the body. Essentially, all the endogenous cholesterol that circulates
in the lipoproteins of the plasma is formed by the liver, but all other
cells of the body can and do form at least some cholesterol.
The major plasma lipids, including cholesterol, do not circulate free in
the plasma, but are bound to proteins and transported as macromolecular
complexes called lipoproteins.
The plasma lipoproteins can be separated into four major classes:
chylomicrons;
very low density lipoproteins (VLDL);
low density lipoproteins (LDL); and
high density lipoprotein (HDL).
Because of a varying ratio of lipid to protein, the densities of
lipoproteins differ. The lipoproteins can be succefully separated by
ultracentrifugation or by electrophoresis. The pathological
hyperlipoproteinemias, which will be treated by the method of this
invention, are classified on the basis of the pattern of lipoprotein
abnormalities.
Chylomicrons: The largest lipoprotein particles, the chylomicrons, contain
the most lipids and are thus of the least density. They have high
molecular weights (10.sup.9 to 10.sup.10) and consist of a core of
nonpolar lipids (mostly triglycerides) surrounded by a coat of protein,
phospholipid, and free cholesterol. Chylomicrons are secreted into the
intestinal lymphatics by the intestinal mucosa following the absorption of
a lipid-containing meal, and their triglycerides are eventually stored in
adipose tissue.
Very-Low-Density-Lipoproteins: The (VLDL) are also triglyceride-rich. Their
molecular weights are approximatey 5.times.10.sup.6. These molecules are
secreted by the liver, and their triglyceride is in part derived from
dietary carbohydrates. Similar to the chylomicrons, VLDL triglycerides are
mostly destined for storage in adipose tissue. On conventional
electrophoresis, the VLDL migrate between the .beta.- or low-density
lipoproteins (LDL) and the .alpha.- or high-density lipoproteins (HDL). In
this electrophoretic scheme the VLDL are thus termed
pre-.beta.-lipoproteins. Because of the high triglyceride content of the
chylomicrons and the VLDL, an increase in their concentration is
accompanied by elevation in the concentration of the plasma triglycerides.
The fraction of VLDL which is rich in cholesterol is called .beta.-VLDL,
which term is derived from the mobility of these lipoproteins. Like
chemically altered LDL, .beta.-VLDL are transported by scavenger cells
into the blood vessel wall thus resulting in formation of atheromatous
foam cells, the initiator of atheromatous plaques.
Low-Density-Lipoproteins: The low density lipoproteins have the
electrophoretic mobility of .beta.-globulins and are therefore known as
.beta.-lipoproteins. These lipoproteins contain the major portion of the
total plasma cholesterol. When LDL are present in increased concentration,
plasma cholesterol concentration is increased, while the triglyceride
concentration is relatively normal.
High-Density Lipoproteins: The high density lipoproteins are considerably
smaller particles. These lipoproteins contain about 50% of protein and
have the electrophoretic mobility of .alpha.-globulins and are therefore
termed .alpha.-lipoproteins. Of their lipids, phospholipids predominate.
Plasma levels of HDL are inversely correlated with risk of
atherosclerosis.
Depending on the plasma lipoprotein pattern, it is possible to classify
patients with three types of hyperlipemia abnormalities:
hypercholesterolemia, combined hyperlipemia and hypertriglyceridemia.
Hypercholesterolemia, combined hyperlipemia and hypertriglyceridemia occur
commonly and involve two classes of lipoproteins (VLDL and LDL) for which
there is a positive correlation between plasma concentration and the
incidence of atherosclerosis.
1. Hypercholesterolemia is characterized by the presence of the LDL
.beta.-lipoproteins. It may be genetic, sporadic, or secondary to various
defined causes such as hypothyroidism, nephrotic syndrome, myeloma, and
excess dietary cholesterol. If the hypercholesterolemia is of genetic
origin, clinical manifestations of the disorder are usually evident before
the age of 30. Until that age the risk of vascular disease seems to be
greatly increased. The studies have shown that about 50% of individuals
suffering from the genetic (familial) hypercholesterolemia have myocardial
infarction before the age of 50.
2. Combined hyperlipemia is characterized by the presence of both LDL
.beta.-lipoproteins and VLDL pre-.beta.-lipoproteins. In combined
hyperlipemia, both plasma cholesterol and triglyceride concentrations are
elevated. In a study of 500 survivors of myocardial infarction, one third
had hyperlipemia. Familial combined hyperlipemia, often associated with a
.beta.- and pre-.beta.-lipoprotein pattern, is the most common genetic
cause and accounts for 30% of the hyperlipemic group.
3. Hypertriglyceridemia is characterized by the VLDL
pre-.beta.-lipoproteins. Hypertriglyceridemia is frequently encountered
and is likewise associated with an increased risk of atherosclerosis.
Patients with hypertriglyceridemia exhibit sensitivity to carbohydrates;
that is, to a diet high in carbohydrates. Such diet results in elevated
plasma concentrations of VLDL, the triglyceride of which is in part
synthesized by the liver from carbohydrates. In this disorder, glucose
tolerance is commonly abnormal, and diabetes mellitus is frequently
associated with such an excess of VLDL.
For reference to the above, see Guyton, Medical Biology, 5th Ed.,
pp.926-927 (1976); The Merck Index, 13th Ed., pp.381-383 (1977) and
Goodman and Gilman, The Pharmacological Basis of Therapeutics, 5th Ed., pp
744-747 (1975).
Inhibition of cholesterol absorption was studied in monkeys and rats using
the commonly recognized screening tests.
The screening test which was used for determination of inhibition of
cholesterol absorption in monkeys is described in J. Clin. Invest., 67:156
(1981). An inhibitory activity of the compounds of this invention were
compared to that of cholestyramine, known antilipoproteinemic drug, and
were found to be five times or more potent. The same results as those
obtained with 2% cholestyramine were obtained with the 0.4% tigogenin
cellobioside. Since it is well known that the current treatment of
hypercholesterolemia requires enormous daily dosages, this finding is of
great importance. For example, the recommended adult dose of
cholestyramine is 4 g three to four times daily which represents the total
dose of 12-16 g of cholestyramine in admixture with 15-20 g of excipient.
Thus, the total volume of the drug administered daily is between 27-36 g.
On the other hand, the same effect has been obtained with the daily dose
of only 0.8 g three to four times daily in a total dose of 2.4-3.2 g in
admixture with 3-4 g of excipient, i.e. the total volume of the drug which
is expected to be administered daily is between 5.4-7.2 g per day.
The percentage of cholesterol absorption in rat was studied by using the
test described in Am. J. Clin. Nutr., 30:2061 (1977). When compared to
other synthetic glycosides such as structurally related diosgenin
cellobioside, tigogenin glucoside, diosgenin glucoside or alfalfa
saponins, compounds of the current invention had lower percentage of
cholesterol absorption and thus were more effective in removing the
cholesterol from the plasma.
Administration
Administration of tigogenin-cellobioside can be via any of the accepted
modes of administration suitable for treatment of the hypercholesterolemia
or atherosclerosis. These methods include oral routes, parenteral routes
such as intravenous, subcutaneous, intradermal, or intramuscular and other
systemic routes of administration such as, for example, by suppositories.
The amount of tigogenin cellobioside administered will, of course, be
dependent on the subject being treated, on the severity of the affliction,
on the manner of administration and on the judgment of the prescribing
physician. However, an effective dosage is in the range of 7 to 115
mg/kg/day, preferably 28 to 57 mg/kg/day. For an average 70 kg human, this
would amount to 0.5 to 8 g/day, preferably 2-4 g/day, most preferably
2.4-3.2 g/day.
For oral administration, which is preferred, a pharmaceutical composition
takes the form of solutions, suspensions, tablets, pills, capsules,
powders, sustained release formulations and the like.
Parenteral route of administration is the administration of drugs to a
patient by injection under or through one or more layers of the skin or
mucous membrane. Parenteral administration would preferably be reserved
for crisis situations, wherein the subject is unable to swallow or
administer the medication to himself.
Systemic administration via suppository is the administration of the drug
in a solid but readily meltable cone or cylinder made of a tigogenin
cellobioside and a suitable pharmaceutical excipient. Suppository must be
suitable for insertion into a bodily passage or cavity, and is usually
inserted into the rectum. This way of administration would be preferred in
the patient with severe ingestion disturbance such as repeated vomiting.
Pharmaceutical Composition
Depending on the intended mode of administration, the pharmaceutical
compositions may be in the form of solid, semi-solid or liquid dosage
forms, such as, for example, tablets, pills, capsules, powders, liquids,
suspensions, or the like, preferably in unit dosage forms suitable for
single administration of precise dosages. The pharmaceutical compositions
will include a conventional pharmaceutical carrier or excipient and,
tigogenin cellobioside as an active ingredient. In addition, it may
include other medicinal or pharmaceutical agents, carriers, adjuvants,
etc.
A pharmaceutical composition may contain 0.1%-95% of tigogenin
cellobioside, preferably 1%-70%. In any event, the composition or
formulation to be administered will contain a quantity of tigogenin
cellobioside in an amount effective to alleviate the signs of the subject
being treated, e.i. hypercholesterolemia or atherosclerosis.
For solid pharmaceutical compositions, conventional non-toxic solid
carriers include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like.
Liquid pharmaceutically administerable compositions can be prepared by
dissolving or dispersing, or otherwise preparing tigogenin cellobioside,
and mixing it optionally with a pharmaceutical adjuvant in a carrier, such
as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and
the like, to thereby form a solution or suspension.
For parenteral administration, such as, for example, intravenous
injections, the tigogenin cellobioside is dissolved in a vehicle. Vehicle
may be, for example, aqueous vehicle, such as sodium chloride injection,
Ringer's injection, dextrose injection and others, water miscible vehicle,
such as ethyl alcohol, polyethylene glycol of the liquid series or
propylene glycol, or nonaqueous vehicles such a corn oil, peanut oil or
sesame oil. Vehicle will be buffered to the proper pH in order to
stabilize a solution against chemical degradation and formed in such a way
as to control isotonicity of injection. Other substances may also be added
as antimicrobial or antioxidant agents.
A more recently devised approach for parenteral administration employs the
implantation of a slow-release or sustained-release system, such that a
constant level of dosage is maintained. See, e.g., U.S. Pat. No.
3,710,795.
For systemic administration via suppository, the tigogenin-cellobioside may
be formulated as suppositories using as the carrier traditional binders
and carriers such as, for example, polyalkylene glycols, or triglycerides.
Such suppositories may be formed from mixtures containing tigogenin
cellobioside in the range of 0.5%-10%; preferably 1-2%.
Methods of preparing various pharmaceutical compositions with a certain
amount of active ingredient are known, or will be apparent, to those
skilled in this art. For examples, see Remington's Pharmaceutical
Sciences, Mack Publishing Company, Easton, Pa., 15th Edition (1975).
EXAMPLE 1
Preparation of .alpha.-Tigogenin Cellobioside
1. Preparation of .alpha.-Tigogenin Cellobioside Heptaacetate
A 12 l three neck round bottom flask fitted with a mechanical stirrer and a
500 ml dropping funnel was flushed with nitrogen and charged with 650 g
(0.96 moles) of cellobiose octaacetate, 200 g (0.48 moles) of tigogenin,
and 5 l of acetonitrile. A total of 250 g (0.96 moles) of stannic chloride
was then added over two minutes through the dropping funnel. The reaction
mixture was warmed on a steam bath, until it became homogeneous
(65.degree. C.). The 65.degree. C. temperature was maintained for 20 min,
then the mixture was cooled to 30.degree. C. 4 liters of saturated aqueous
sodium bicarbonate solution was added carefully and the mixture was
vigorously stirred for 90 minutes. The layers were separated and the
aqueous phase reextracted with 4 liters of methylene chloride. The
combined organic phases were dried over magnesium sulphate and evaporated
under reduced pressure to a gum. The gum was passed through a silica gel
column (1.5 kg) eluted with 25% methylene chloride/hexane (v/v). The
fraction which was obtained was evaporated under vacuum. The residue was
crystallized from ethanol to give 120 g (24% of theory yield) of
.alpha.-tigogenin cellobioside heptaacetate.
2. Preparation of .alpha.-Tigogenin Cellobioside
A mixture of 38.2 g of .alpha.-tigogenin cellobioside heptaacetate in 60 ml
of methylene chloride, 240 ml of methanol, 120 ml of triethylamine and 100
ml of water was refluxed for 6 hours and then stirred overnight at room
temperature. The reaction mixture was evaporated under reduced pressure to
a thick paste which was washed with water and hexane and dried. The
resulting material was chromatographed on a column of 3 kg of silica gel
using 10% methanol/methylene chloride (v/v) to elute desired material.
After evaporation of the fraction which contained .alpha.-tigogenin
cellobioside, the residue was stirred in boiling isopropanol, cooled, and
collected to give 13.9 g of .alpha.-tigogenin cellobioside.
NMR data for .alpha.- and .beta.-tigogenin cellobioside are given in Table
I which follows Example 3.
EXAMPLE 2
Preparation of .beta.-Tigogenin Cellobioside
1. Preparation of .beta.-Tigogenin Cellobioside Heptaacetate
A 12 liters three neck round bottom flask fitted with a mechanical stirrer
and a 40 ml dropping funnel is flushed with nitrogen and charged with 650
g (0.96 moles) of cellobiose octaacetate, 200 g (0.48 moles) of tigogenin,
and 500 ml of methylene chloride. A total of 250 g (0.96 moles) of stannic
chloride is then added over two minutes through the dropping funnel. The
reaction is warmed on a steam bath, and refluxed for 20 min, then cooled
to 30.degree. C. 4 liters of saturated aqueous sodium bicarbonate solution
is added carefully and the mixture is vigorously stirred for 90 minutes.
The layers are separated and the aqueous phase reextracted with 4 liters
of methylene chloride. The combined organic phases are dried over
magnesium sulphate and evaporated under reduced pressure to a gum. The gum
is passed through a silica gel column (1.5 kg) eluting with 25% methylene
chloride/hexane (v/v). The fraction containing .beta.-tigogenin
cellobioside is evaporated under vacuum. The residue is crystallized from
ethanol to give .beta.-tigogenin cellobioside heptaacetate.
2. Preparation of .beta.-Tigogenin Cellobioside
A mixture of 38.2 of .beta.-tigogenin cellobioside heptaacetate in 60 ml of
methylene chloride, 240 ml of methanol, 120 ml of triethylamine and 100 ml
of water, is refluxed for 6 hours, then stirred overnight at room
temperature. The reaction mixture is evaporated under reduced pressure to
a thick paste which is washed with water and hexane and dried. The
resulting material is chromatographed on a silica gel column of 3 kg of
silica gel using 10% methanol/methylene chloride (v/v) to elute desired
material. After evaporation of the appropriate fractions, the residue is
stirred in boiling isopropanol, cooled, and evaporated to give
.beta.-tigogenin cellobioside.
EXAMPLE 3
Preparation of .beta.-Tigogenin Cellobioside
General Information and Procedures
Tigogenin was obtained from Research Plus.
Cellobiose octaacetate was obtained from Aldrich.
Tilting dialyzer: A tilting dialyzer was built using a large container
provided with a siphon in such a way that the water level was continuously
rising and falling. The dialysis bags were tied to a vertical metal rod
midway between the upper and lower water levels. The tilting bags moved
the powdered material and increased its contact with water.
Experimental Procedure
Tigogenin: 41.9 g of tigogenin (100 mmoles) was dissolved in 800 ml of
water-free methylene chloride.
Water-free methylene chloride: Water-free methylene chloride was prepared
by adding the MgCl.sub.2 to a commercially obtained methylene chloride.
The solution was stirred for 30 min and filtered.
Cellobiose octaacetate: 135 g of cellobiose octaacetate (200 mmoles) is
dissolved in 800 ml of water-free methylene chloride and 25 ml of stannic
tetrachloride (200 mmoles) was added. The mixture was stirred for 10
minutes.
The cellobiose octaacetate mixture, obtained above, was introduced into a
separatory funnel and added dropwise to a solution of tigogenin, obtained
above, at an approximate rate of 150 ml/min. The mixture was stirred for 3
hours. The resulting solution was poured into 4 liter separatory funnel
containing 500 ml of saturated bicarbonate solution saturated with
methylene chloride. 500 ml of water saturated with methylene chloride was
added and the gas was allowed to escape. The solution was mixed by
inversion and separated into an organic and water phases. Lower phases
were transferred to a separatory funnel, washed twice with water saturated
with methylene chloride and, after separation, the upper phase was
aspirated and discarded. The remaining phase was washed twice with 250 ml
of water, separated, and again the upper phase was discarded. The lower
phase was washed twice with water, separated and again the lower phases
were collected and evaporated to 100-200 ml at approximately 50.degree. C.
under vigorous stirring and nitrogen atmosphere to give predominantly
.beta.-tigogenin cellobioside heptaacetate.
1400 ml of a mixture containing triethylenamine/methanol/water (1:2:1) was
added slowly and under constant stirring to the solution of the above
tigogenin cellobioside heptaacetate. The mixture was stirred overnight.
Methylene chloride was removed at 30.degree. C. under vacuum. Three liters
of water was added and the mixture was left in the refrigerator for 1 to 2
hours. Afterwards, the mixture was centrifuged at 4000 RPM for 20 min,
precipitate dried at room temperature, crushed, dissolved in small amount
of methanol and water, and dialized for 3 days in tilting dialyzer against
running tap water. The resulting mixture was filtered, dried at room
temperature, crushed into fine powder and extracted in Soxhlet (jacketed
at 40.degree.) with heptane for 3 days. .beta.-tigogenin cellobioside
which remained in the thimble was removed and dried at room temperature
for 2 to 3 days to evaporate heptane. This procedure yielded 30 to 40% of
.beta.-tigogenin celobioside.
The 'H NMR spectra of 1'.alpha.- and 1'.beta.-tigogenin cellobioside were
measured on the Bruker WM300 Fourier transform NMR spectrometer in d.sub.6
DMSO solution with reference to internal tetramethylsilane.
TABLE I
______________________________________
300 MHz 'H NMR Data in d.sub.6 DMSO
Chemical Shifts in ppm
Cellobioside
Assignments
.alpha.-tigogenin
.beta.-togoenin
No. of Protons
______________________________________
16.alpha. 4.28 m 4.25 m 1
18 0.72 s 0.72 s 3
19 0.78 s 0.78 s 3
21 0.89 d, 0.89 3
d,J=6.8 Hz d,J=6.8 Hz
26 0.74 0.73 3
d,J=6.3 Hz d, J=6.3 Hz
1' 4.78 4.29 1
d,J=3.5 Hz d,J=7.9 Hz
1" 4.23 4.23 1
d,J=7.6 Hz d,J=7.6 Hz
______________________________________
J = coupling constant
s = singlet
d =doublet
m =multiplet
EXAMPLE 4
Effect of Tigogenin Cellobioside on Plasma Cholesterol
This example illustrates the effect of tigogenin cellobioside on plasma
cholesterol in monkeys Macaca fascicularis. The procedure is described in
J. Clin. Invest., 67:156 (1981).
Experimental procedure
Experimental Regimen
The animals were divided into groups I and II. Group I was treated with 2%
cholestyramine, Group II was treated with 0.4% tigogenin cellobioside.
Each group was divided into two subgroups. Animals in the first subgroup
served as cholesterol controls, i.e. they received no drug treatment but
were fed with a cholesterol diet. Animals in the second group received a
drug treatment and were also fed with a cholesterol diet.
Subgroup 1: Before the beginning of the experimental regimen, the control
blood was obtained from all animals in both groups and a total cholesterol
and a high density lipoprotein cholesterol were determined. Then the
animals were fed the cholesterol diet. After three weeks on the diet
without treatment, the blood from treated monkeys was collected and a
total cholesterol and a high density lipoprotein cholesterol were
determined.
Subgroup 2: Before the beginning of the regimen of subgroup 2, the animals
from subgroup 1 were fed cholesterol-free semipurified diet for five
weeks. After five weeks on the cholesterol-free diet, the control blood
was again obtained from all animals and a total cholesterol and a high
density lipoprotein cholesterol were determined. Then, the animals were
put on a cholesterol diet combined with drug treatment. After three weeks
of the cholesterol diet and of the appropriate treatment, the blood from
all monkeys was collected and a total cholesterol and a high density
lipoprotein cholesterol were determined.
Animals and diet
Twelve adult female cynomolgus (Macaca fascicularis) were fed for 3 weeks
the cholesterol-free semipurified diet (SPD) of the following content:
______________________________________
Ingredient g/100 g of diet
______________________________________
casein 18
sugar 30
honey 10
Alphacel 12
butter 3
coconut oil 8.5
safflower oil 2.5
vitamins (OWP) 2
salts (Hegsted IV) 4
vitamin D-3 (2,000 IU/ml
0.2 ml
banana (wet weight) 10
proteins (% calories) 20.6
fat (% of calories) 33.5
carbohydrate (% of calories)
45.9
______________________________________
This diet can optionally contain 0.118 g/100 g of diet or 0.35 mg/kCal of
cholesterol.
The experimental protocol is outlined and the results are summarized in
Table A.
Results (summarized in Table A)
The monkeys were assigned to two groups (I and II) according to their serum
cholesterol response; they were stratified and assigned randomly, thus
resulting in groups with similar elevations of cholesterolemia.
Control 1, Sample 1
Control venous blood was obtained for analysis and the mean values of the
serum cholesterol were determined to be 240 mg/dl for Group I and 228
mg/ml for Group II.
Then, the monkeys were offered semipurified diet containing 0.1%
cholesterol for 3 weeks. At the end of this period, blood sample 2 was
obtained.
Cholesterol Diet, Sample 2
After three weeks on the cholesterol diet, the mean value of cholesterol
was 324 mg/dl in Group I and 316 mg/dl in Group II. The level of
cholesterol was significantly higher in samples 2, with degree of
significance .ltoreq.0.01.
The monkeys were again given semipurified diet without cholesterol for 5
weeks and bled at the end of this period.
Control 2, Sample 3
After five weeks on the cholesterol-free diet, the level of cholesterol in
both groups decreased to 221 mg/dl in Group I and to 189 mg/dl in Group
II.
The animals were then given semipurified diet with 0.1% cholesterol and
either 2% cholestyramine (Group I) or 0.4% cellobiose tigogenin (Group II)
for 3 weeks. At the end of this period, sample 4 was obtained.
Cholesterol Diet and Drug Treatment, Sample 4
After three weeks on the cholesterol diet combined with the drug treatment,
the values of cholesterol in Group I was 226 mg/dl and 202 mg/dl in Group
II.
Results shown in Table A and FIG. 1 demonstrate that both drugs prevented
the expected rise in serum cholesterol. There were small, nonsignificant
elevations in high-density lipoprotein-cholesterol associated with drug
intake. See Samples 1, 2, 3 and 4 in HDL cholesterol section of the Table
A. Although the changes in the triglyceride levels would be a natural
response to the ingestion of a high-cholesterol diet, no changes were
found. This indicates that both drugs prevented the increase in
low-density lipoprotein cholesterol.
TABLE A
__________________________________________________________________________
Serum cholesterol (mg/dl; mean .+-. SE).sup.a
Total Cholesterol HDL Cholesterol
Choles-
Choles- Choles-
Cholesterol terol diet
terol diet terol diet
Period Control 1
diet Control 2
+ drug
Control 1
diet Control 2
+ drug
Sample 1 2 3 4 1 2 3 4
__________________________________________________________________________
GROUP I N = 6
240 .+-. 19
324 .+-. 24
221 .+-. 19
226 .+-. 16
126 .+-. 12
148 .+-. 17
155 .+-. 9
166 .+-. 8
Drug: 2% cholestyramine
paired t test
P: vs column 2
0.01 -- 0.001
0.01 NS -- NS NS
vs column 1
-- 0.01 NS NS -- NS 0.02 0.05
vs column 3 NS NS
GROUP II N = 6
228 .+-. 19
316 .+-. 21
189 .+-. 10
202 .+-. 8
104 .+-. 12
139 .+-. 14
148 .+-. 10
163 .+-. 7
Drug: 0.4% cellobiosetigogenin
paired t test
p: vs column 2
0.01 -- 0.01 0.01 0.05 -- NS NS
vs column 1
-- 0.01 0.05 0.05 -- 0.05 0.05 0.01
vs column 3 NS NS
__________________________________________________________________________
.sup.a Abbreviations:
HDLcholesterol, high density lipoprotein cholesterol;
N, number of animals;
NS, not significant.
EXAMPLE 5
Effect of Synthetic Glycosides on Intestinal Absorption of Cholesterol in
Rats
This example illustrates the effect of synthetic glycosides on intestinal
absorption of cholesterol in rats. The method used for this experiment is
described in detail by Malinow et al., Am. J. Clin. Nutr., 30:2061 (1977).
Experimental Protocol
2-mg bolus of 4[.sup.14 C]cholesterol was given intragastrically to rats.
Feces were collected for 72 hours, and the fecal excretion of labeled
neutral steroids was determined.
Rats were divided into three groups I-III.
In Groups I and II the animals were divided into subgroups of 6 rats. The
first subgroup served as control and never received any treatment. The
second and third subgroups were treated with either a compound of this
invention or with one of the others closely related compounds. Thus, in
Groups I and II, the rats were treated with tigogenin cellobioside,
diosgenin cellobioside, tigogenin glucoside and diosgenin glucoside.
Results summarized in Table B confirm that the tigogenin cellobioside
decreased an intestinal absorption of cholesterol more markedly than
structurally similar compounds diosgenin cellobioside, tigogening
glucoside and diosgenin glucoside. Tigogenin cellobioside was
significantly more effective than closest related diosgenin cellobioside
(see Group I, subgroups 2 and 3). When compared to control group,
tigogenin cellobioside decreased the cholesterol absorption by almost 50%
(with significance of .ltoreq.0.001). The tigogenin cellobioside was also
more effective than tigogenin glucoside and diosgenin glucoside (see Group
II, subgroups 2 and 3, and compare Group I and II).
Group III has two subgroups with 12 animals in each. An inhibition of the
cholesterol absorption in rats treated with tigogenin cellobioside was
compared to the inhibition of the cholesterol absorption in nontreated
controls.
Similarly to Group I, wherein treatment with tigogenin cellobioside
inhibited absorption of cholesterol by 47% (No=6), in Group III the
treatment with tigogenin cellobioside inhibited absorption of cholesterol
by 41.5% (No=12). And, the absorption of cholesterol in experimental rats
in Group I was only 53% and the absorption of cholesterol in Group III was
only 58.5% of the absorption of cholesterol in control group.
TABLE B
______________________________________
Effects of Synthetic Saponins of
Cholesterol Absorption in Rats
Rela-
P (Stu-
tive
Number Cholesterol
dent's t
Ab-
Treat- of Dose absorption
test vs
sorp-
Group ment animals (mg) (% I.D.)
control)
tion
______________________________________
I none 6 0 74.8 .+-. 1.6 100
C-T 6 14 39.6 .+-. 1.8
<0.001 53
C-D 6 14 53.7 .+-. 1.3
<0.001 72
II none 6 0 74.6 .+-. 2.3 100
G-T 6 14 46.2 .+-. 1.8
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