NO945001L - Steroid glycosides for the treatment of hypercholestrolemia - Google Patents

Abstract

Certain steroid glycosides are useful as hypocholesterolemic agents and anti-atherosclerotic agents.

Description

Background for the invention

This invention relates to steroidal glycosides and methods for using the same, particularly as hypo-cholesterolemia agents and antiatherosclerosis agents in mammals.

Many known products that have hypocholesterolemia activity are cross-linked synthetic polymer derivatives, e.g. of polystyrene. E.g. have cross-linked, water-insoluble, bile acid-binding polystyrene-based resins, e.g. Cholestyramine® agents, a sandy "mouth feel", and thus have poor palatability. Moreover, these resin beads typically have a low in vivo efficiency. The effective hypocholesterolemidose of these materials is thus very large, typically 18-24 g of formulated product per day. Other known polymers that have hypocholesterolemia activity include the natural product chitosan and chitosan derivatives as described in European application pub. No. 0212145. However, the effective hypocholesterolemic dose of these materials is also high.

Other known hypercholesterolemia controlling agents include plant extracts such as "alfalfa saponins". However, these plant extracts are of variable composition, and contain significant amounts of useless chemical substances. Because of. the variations in composition make it difficult to set a standard dosage or predict the contaminants that are present. Such extracts are thus not well suited for human use. Purification of these extracts would also be expensive. Alternatively, certain synthetically produced pure "sapogenin-derived" compounds e.g. substances composed of spirostane, spirostene or sterol-derived compounds reduce cholesterol absorption more effectively than alfalfa extracts on a weight basis, and thus can be administered in reasonably sized doses. Because the chemical composition of these substances is known and because they can be synthesized with a high degree of purity, they are suitable for use by any warm-blooded animal, including humans.

However, if they are not administered in massive amounts, pure sapongenins will not significantly inhibit the absorption of cholesterol. It is only when they are compounded with another group that the sapogenins have the desired effect. Examples of such sapogenin compounds are compounds of tigogenin and diosgenin, especially glycosides thereof. P.K. Kintia, hi. K. Vasilenko, G.M. Gorianu, V.A. Bobeiko, I.V. Suetina, N.E. Mashchenko, Kim. Pharm. Zh., 1981, 15(9), 55 describes 3-O-(/3-D-galactopyranosyl)hecogenin and its use as a hypocholesterolemic agent. U.S. patents Nos. 4,602,003 and 4,602,005 disclose certain steroidal glycosides, particularly 3-O-(/J-D-glucopyranosyl)tigogenin and 3-O-(/3-D-cellobiosyl)tigogenin and their use in the control of hypercholesterolemia. 3-0-(/3-D-cellobiosyl) tigogenin has superior hypocholesterolaemic activity compared to e.g. cholestyramine.

In addition, certain other steroidal glycosides described below have been published, however, these publications do not relate to hypocholesterolemia activity. "Structural Features of the Antioxidant and fungicidal Activity of Steroid Glycosides", Dimoglo, A. S.; Choban I. N.; Bersuker, I.B.; Kintya, P.K.; Balashova, N.N.; Bioorg. Khim, 11(3), 408-413, 1985 describes rockogenin /?-D-galactopyranoside and tigogenin D-lactoside. "Preparation and Properties of Some New Steroid/3-D-Glucopyranosides,/9-D-Glucopyranosiduronic Acids, and Derivatives", Schneider, J.J.; Carb. Research, 17, 199-207, 1971 describes tigogenin /3-D-glucopyranuronoside. "Sterol Glycoside with Activity as Prostaglandin Synthetase Inhibitor", Pegel, K.H.; Walker, H.; US Patent 4,260,603,

April 7, 1981 discloses hecogenin /3-D-glucopyranoside. "Hemolytic Properties of Synthetic Glycosides", Segal, R.; Shud, F.; Milo-Goldzweig, i.; J. Pharm. Sei., 67 (11) 1589-1592, 1978 describes tigogenin /3-D-maltoside, tigogenin /9-L-fucopyranoside, smilagenin /3-maltoside and tigogenin α-L-rhamno-sid. "Steroid Glycosides from the Roots of Capsicum Annuum II: The Structure of the Capsicosides", Gutsu, E.V.; Kintya, P. K.; Lazurevskii, G.V.; Kim. Price. Soedin., (2), 242-246, 1987 describes tigogenin α-D-arabanopyranoside and tigogenin D-galactopyranoside. "Molluscicidal Saponins from Cornus Florida L.", Hostettmann, K.; Hostettmann-Kaldas, M. ; Nakanishi, K.; Hooray. Chim. Acta, 61, 1990-1995, 1978 describes smilagenin /?-D-galactopyranoside. "Steroidal Saponins from Several Species of Lilliflorae Plants", Yang, C.; Li, K.; Ding, Y.; Yunnan Zhiwu Yanjiu Zengkan, Suppl. 3, 13-23, 1990 describes (25S)-hecogenin cellobioside. "Determination of the Absolute Configuration of a Secondary Hydroxy Group in a Chiral Secondary Alcohol Using Glycosidation shifts in Carbon-13 NMR Spectroscopy", Seo, S.: Tomita, Y.; Tori; K.; Yoshimura, Y.; J. Am. Chem. Soc., 100(11), 3331-3339, 1978 describes smilagenin β-glucoside and smilagenin β-glucoside. "Steroid Glycosides from Asparagus Officinalis", Lazurevskii, G. V.; Goryanu, G.M.; Kintya, P. K.; Doc. Acad. Nauk. SSSR, 231(6), 1479-81, 1976 describes sarsasapogenin/3-glucoside.

Although the hypocholesterolemia compounds described above constitute a significant contribution to the technology, there is continuous research in this area to find improved hypocholesterolemia pharmaceuticals.

Summary of the invention

This invention relates to steroidal glycosides, particularly spirostanyl glycosides, which are useful as hypocholesterolemic agents and antiatherosclerosis agents. The compounds of this invention have the formula

where either (A): Q<1>is carbonyl Q<2>is carbonyl, methylene, R<1>0-alkylene(C2-C3;

R^-O-alkylene (C2-C3)

or

Q<*> and Q<5> are both methylene;

and where

R<1>'s

Ø-D-glucopyranosyl, jØ-D-glucopyranuronosyl, 0/3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-galactopyranosyl, Ø-D-fucopyranosyl, p- L- fucopyranosyl, /3- D-xylopyranosyl, Ø-L-xylopyranosyl α-D-arabanopyranosyl, α-L-arabanopyranosyl, α-D-cellobiosyl,

jS-D-cellobiosyl,

/3-D-lactosyl,

Ø-D-maltosyl,

/?-D-gentiobiosyl, 3-O-/?-D-galactopyranosyl-α-D-arabanopyranosyl or Δ-D-maltotriosyl;

or (B):

Q<1>, Q<*> and Q<5> are all methylene;

orR<1>0-alkylene(C2-<C>3)

and where

R<1>'s

/3-D-glucopyranosyl, Ø-D-glucopyranuronosyl, /?-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-fucopyranosyl, ( 3- L- f ukopyranosyl, /3-D-xylopyranosyl, /3-L-xylopyranosyl a-D-arabanopyranosyl, a-L-arabanopyranosyl,/3-D-eellobiosyl,/9-D-lactosyl,/3-D-maltosyl, /3-D-gentiobiosyl, 3-o-/?-D -galactopyranosyl-α-D-arabanopyranosyl or β-D-maltotriosyl;

or (C):

Q<1>, Q 1* and Q<5> are all methylene; Q2 is carbonyl;

or

R<1>0-alkylene(C2-<C>3)

C25er (R) ;

and where

R<1>'s

/3-D-glucopyranuronosyl,

/3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-fucopyranosyl,

/3-L-f ukopyranosyl,

/3-D-xylopyranosyl,

/3-L-xylopyranosyl

α-D-arabanopyranosyl,

α-L-arabanopyranosyl,

/3-D-cellobiosyl,

/3-D-lactosyl,

/3-D-maltosyl,

/3-D-gentiobiosyl,

3-O-β-D-galactopyranosyl-α-D-arabanopyranosyl or β-D-maltotriosyl;

or (D):

Q<1>, Q<2>, Q'' and Q<5> are all methylene; R<1>0-alkylene(C2-C3)

orR<1>0-alkylene(C2-<C>3)

and where

R<1>'s

/3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-fucopyranosyl,

/3-D-xylopyranosyl,

/3-L-xylopyranosyl

α-L-arabanopyranosyl,

/3-D-cellobiosyl,

/3-D-gentiobiosyl,

3-O-Ø-D-galactopyranosyl-α-D-arabanopyranosyl or β-D-maltotriosyl;

or (E):Q<1>, Q<2> and Q<5> are all methylene;

Q<4>is carbonyl or

C5 is alpha;

C25er (R) ; and where

R<1>'s

/3-D-galactopyranosyl, /3-D-cellobiosyl, Ø-D-lactosyl,

/3-D-maltosyl or /3-D-maltotriosyl;

or (F)<:>c Q<1>, Q<2> and Q<*>are all methylene;

Q<5>is carbonyl or

C5 is alpha;

C25er (R) ; and where

R<1>'s

/3-D-galactopyranosyl,

/3-D-cellobiosyl,

/3-D-lactosyl,

/3-D-maltosyl or /3-D-maltotriosyl;

provided that (3/3,5a,25R)-3-[(/3-D-cellobiosyl)oxy]spirostane is not included.

A first group of preferred compounds of formula IA consists of those compounds wherein Q<1> is carbonyl,

or Q<2>is the methylene, Q<3>is , Q<*>is the methylene, Q<5>is the methylene, the C5 hydrogen is alpha and C25 has the R configuration. Particularly preferred within this group are compounds where Q<1>is carbonyl and R<1>is Ø-D-cellobiosyl, α-D-cellobiosyl, /3-D-glucopyranosyl, /3-D-galactopyranosyl, /3-D-lactosyl , /3-D-maltosyl or /3-D-maltotriosyl. Particularly preferred within this group is also a compound where Q<1> is and R<1> is /3-D-cellobiosyl. Another particularly preferred compound within this group is a compound where Q-1 is -

and

R<1> is Ø-D-cellobiosyl.

A second group of preferred compounds of formula IA

are compounds where Q<1> is methylene, Q<2> is

or Q<4> is the methylene, Q<5> is the methylene, the C5 hydrogen is alpha and the C25 is (R). Particularly preferred within this second group is a compound where Q<2> is and -R* is /3-D-cellobiosyl. A third group of preferred compounds of formula IA are compounds wherein Q<1>is carbonyl, is carbonyl,

, Q<4>'s

is the methylene, Q<5> is the methylene, the C5 hydrogen is alpha and C25 is (R). Particularly preferred within this group is a compound

where Q1 is carbonyl, Q<2> is carbonyl and R<1> is /3-D-cellobiosyl. Another particularly preferred compound within this group

is a compound where Q<1> is carbonyl, Q<2>, is

and R<1> is /3-D-cellobiosyl. Another particularly preferred compound within this group is a compound where Q<1> is carbonyl, l Q<2> is and r! is /3-D-lactosyl. Another particularly preferred compound within this group is a compound where Q<1> is Q<2> is carbonyl and R-<*> is /3-D-cellobiosyl. Another particularly preferred compound within this group is a compound where Q<1> is

Q<2> is carbonyl and R<1> is /3-D-cellobiosyl.

A fourth group of preferred compounds of formula IA are compounds wherein Q<1>is methylene, Q<2>is carbonyl, Q<3>is

Q<4> is the methylene, Q<5> is the methylene, the C5 hydrogen is alpha and

C25er (R). Particularly preferred within this fourth group are compounds where R<1> is /3-D-lactosyl or /3-D-cellobiosyl.

A fifth group of preferred compounds of formula IA are compounds where Q<1> and Q<2> are both methylene, Q<3> is

Q<4>and Q<5> are both methylene and C25 (R). Especially

preferred within this fifth group is a compound where the C5 hydrogen is alpha and R<1> is /3-D-gentiobiosyl. Another particularly preferred compound within this group is a compound where the C5 hydrogen is beta and R<1>er/3-D-cellobiosyl.

A sixth group of preferred compounds of formula IA are compounds wherein Q<1>, Q<2> and Q5 are all methylene, Q<3> is

Q<4> is carbonyl, the C5 hydrogen is alpha and C25 is (R).

Particularly preferred within this group is a compound where R<1> is/3-D-cellobiosyl.

A seventh group of preferred compounds of formula IA are compounds wherein Q<1>, Q<2> and Q<4> are all methylene, Q<3> is

Q<5>is carbonyl, C5h<y>drogen is alpha and C25 is (R).

Particularly preferred within this group is a compound where R<1> is /3-D-cellobiosyl.

Still another aspect of this invention relates to a method of controlling hypercholesterolemia or atherosclerosis in a mammal by administering to a mammal suffering from hypercholesterolemia or atherosclerosis a hypercholesterolemia or atherosclerosis controlling amount of a spirostanyl glycoside of formula I

where either (A):Q<1>is methylene, carbonyl, Q<2>is carbonyl, methylene

or Q<3>is R<1>0-alkylene(C2-C3)

orR<1>0-alkylene(C2-<C>3)

Q'1 and Q<5> are both methylene;

and where

R<1>'s

/3-D-glucopyranosyl, /3-D-glucopyranuronosyl, /3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-galactopyranosyl, /3-D-f ukopyranosyl, ,-. /3-L-f ukopyranosyl,

/3-D-xylopyranosyl,

/3-L-xylopyranosyl

α-D-arabanopyranosyl, α-L-arabanopyranosyl, α-D-cellobiosyl,

/3-D-cellobiosyl,

/3-D-lactosyl,

/3-D-maltosyl,

/3-D-gentiobiosyl,

3-O-β-D-galactopyranosyl-α-D-arabanopyranosyl or β-maltotriosyl;

or (B):

Q<1>, Q<2> and Q<5> are all methylene;

Q<4>is carbonyl or

C5 is alpha;

C25er (R);

and where

R<1>'s

/3-D-galactopyranosyl,

/3-D-cellobiosyl,

/3-D-lactosyl,

/3-D-maltosyl or

/3-D-maltotriosyl;

or (C):

Q<1>, Q2 and Q<4> are all methylene; Q<3>'s

or Q<5>is carbonyl or

C5 is alpha; c

C25er (R);

and where;

R<1>'s

/3-D-galactopyranosyl,

/3-D-cellobiosyl,

/3-D-lactosyl,

/3-D-maltosyl or

/3-D-maltotriosyl ;

under the condition that

(3/3, 5a, 25R) -3- [ (α-D-cellobiosyl) oxy ] spirostane, (3/3, 5a, 25R) -3-[ (/3-D-glucopyranosyl) oxy] spirostane, (3/ 3, 5a, 25R)-3-[(/3-D-cellobiosyl)oxy]spirostane or

(3/3, 5a, 25R)-3-[(jS-D-galactopyranosyl)oxy]spirostan-12-one is not included.

A first group of preferred compounds of formula I are compounds where Q<1>, Q<2>, Q<4> and Q<5> are methylene, C25 is (R)

and Q<3>'s

Particularly preferred within this group are compounds where the C5 hydrogen is alpha and RI is /3-D-<g>luco-

pyranuronosyl, /3-D-maltosyl, /3-D-lactosyl, /3-D-gentiobiosyl or /3-D-galactopyranosyl. Another particularly preferred compound within this group is a compound where the C5 hydrogen is beta and R1 is /3-D-cellobiosyl.

A second group of preferred compounds of formula I are

compounds where Q<1>is carbonyl,

or , Q<2> is methylene, Q<3> is Q<4>and Q<5> are both methylene, C25 is (R) and the C5 hydrogen is alpha. Particularly preferred within this second group are compounds where Q<1> is carbonyl and R<1> is /3-D-cellobiosyl, Q-D-cellobiosyl, /3-D-glucopyranosyl, /3-D-galactopyranosyl, /3-D -lactosyl, /3-D-maltosyl or /3-D-maltotriosyl. Another particularly preferred compound within this second group is a compound where Q 1 is and R 1 is 3-D-cellobiosyl. Another particularly preferred compound within this second group is a compound where Q<1> is and R<1> is/3-D-cellobiosyl. A third group of preferred compounds of formula I are compounds where Q<1> is methylene, Q<2> is carbonyl, or Q<3> is Q<4> and Q<5> are both methylene, C25 is (R) and The C5 hydrogen is alpha. Particularly preferred within this third group are compounds where Q<2> is carbonyl and R<1> is /3-D-cellobiosyl or /3-D-lactosyl. Other particularly preferred compounds within this third group are compounds where Q<2> is carbonyl,

and R<1>er/3-D-cellobiosyl or

/3-D-galactopyranosyl.

A fourth group of preferred compounds of formula I

are compounds where Q<1>is carbonyl,

or Q<2> is carbonyl, or , Q<3> is , Q<4> and Q<5> are both methylene, the C5 hydrogen is alpha and C25 is (R) . Particularly preferred within this fourth group is a compound where Q<1> is carbonyl, Q<2> is carbonyl and R<1> is/3-D-cellobiosyl. Particularly preferred within this fourth group are compounds where Q<1> is carbonyl, Q<2> is and R<1> is /3-D-cellobiosyl or /3-lactosyl. Another particularly preferred compound within this group is a compound in which Q<1> is Q<2> is carbonyl, and R<1> is /3-D-cellobiosyl. Another particularly preferred compound within this group is a compound where Q<1> is

, Q<2> is carbonyl and R<1> is

/3-D-cellobiosyl.

A fifth group of preferred compounds of formula I are compounds wherein Q<1>, Q<2> and Q<5> are all methylene, Q<3> is

Q<4> is carbonyl, the C5 hydrogen is alpha and C25 is (R).

Particularly preferred within this group is a compound where R<1> is/3-D-cellobiosyl.

A sixth group of preferred compounds of formula I are compounds wherein Q<1>, Q<2> and Q<4> are all methylene, Q<3> is

Q<5> is carbonyl, the C5 hydrogen is alpha and C25 is (R).

Particularly preferred within this group is a compound where R<1> is/3-D-cellobiosyl.

This invention also relates to pharmaceutical preparations for the control of hypercholesterolemia or atherosclerosis in mammals, and which comprise a compound of formula IA and a pharmaceutically acceptable carrier.

A further feature according to this invention relates to a preparation comprising a hydrate of a compound of formula

IA.

The compounds with the formulas IA and I are defined here as the one enantiomer which has the absolute stereochemistry given in the formulas respectively IA and I.

Other features and advantages will be obvious from this description and the claims describing the invention.

Detailed description of the invention

The Formula IA compounds are a subset of the Formula I compounds. In the following detailed descriptions of the invention (e.g. how to carry out the invention, how to use the invention) reference to the formula I group of compounds in itself shall thus include the formula Ia compounds.

The following description of reaction cores I, II & III describes the preparation of formula I compounds where Q<4> and Q<5> are both methylene.

According to reaction scheme I, the desired compounds of formula I, in which Q<1>, Q<2> and Q<3> are as defined above, can be prepared by deacetylation of the appropriate alpha-per-acetylated formula III compound or beta-peracetylated formula IV compound where Q ^- and Q<2> are as defined above and X is either a bond or alkylene-O-.

Typically, the deacetylation is carried out by combining the formula III or IV compound with a nucleophilic base such as sodium methoxide or potassium cyanide in a solvent such as methanol, tetrahydrofuran, n-propanol or mixtures thereof at an elevated temperature of approx. 40"C to approx. 100"C (typically with reflux) and pressure of 3 kPa to approx. 345 kPa (typical ambient) for approx. 0.25 hours to approx. 2 hours. For formula I compounds, when the sugar is glucopyranuronosyl, the resulting deacetylated compound is furthermore further hydrolysed by e.g. exposure to sodium hydroxide. If desired, these compounds where either Q<*> or Q<2> is carbonyl can also be reduced to form the corresponding alcohols in a method alternative to carrying out the reduction before the coupling (described in reaction scheme IV and the accompanying text). In a similar way, where appropriate, these compounds where either Q<1> or Q<2> is hydroxy can be oxidized to form the corresponding carbonyl in a method alternative to carrying out the oxidation before coupling.

The desired formula III compound where Q1 and Q<2> are as defined above can be prepared by anomerization of the appropriate formula IV compound where Q<1> and Q<2> are as defined above. The stereochemical terms alpha and beta refer to the configuration of the bond carbon in the sugar.

Typically, the anomerization is carried out by treatment with a mineral acid such as hydrobromic acid in an anhydrous aprotic solvent such as methylene chloride at temperatures of 20°C to about 40°C (typically ambient) for at least 24 hours, typically for several days. For arabanopyranosyl derivatives, however, the alpha anomer is obtained directly from the saccharide-steroid linkage described below and the beta anomer from the procedure above (ie, the nomenclature reverses).

According to reaction scheme II, the desired formula IV compounds where Q<1> and Q<2> are as defined above can be prepared by coupling the suitable acetylated sugar halide (e.g. bromide) and steroid. Specifically, for those formula IV compounds where the sugar is different from /3-D-maltosyl, /3-D-gentiobiosyl or /3-D-2-acetamido-2-deoxy-glucopyranosyl, a zinc fluoride promoted coupling is used of the appropriate formula V compound (where Q<1> and Q<2> are as defined above and X is either a bond or alkylene-O-) and peracetylated sugar halide, and for those formula IV compounds where the sugar is/3-D -maltosyl, /3-D-gentiobiosyl or /3-D-2-acetamido-2-deoxy-glucopyranosyl, a mercury (II) bromide and mercury (II) cyanide-promoted coupling of the appropriate formula VI is used compound (e.g. trimethylsilyl ether of the formula V compound where Q<1> and Q<2> are as defined above, and X is either a bond or alkylene-O-) and peracetylated sugar halide.

In general, the zinc fluoride-promoted coupling of the formula V compound and the peracetylated sugar bromide takes place in a non-protic, anhydrous reaction-inert solvent (e.g. acetonitrile) at a temperature of approx. 20°C to approx. 100°C for approx.0.5 to approx. 12 hours. Typically, approx. 0.5 to approx. 4 equivalents (based on the formula V compound) of zinc fluoride and about 0.5 to about 3 equivalents of acetylated sugar bromide. Preferably, the coupling is acid-catalyzed, and it is particularly preferred that hydrohalic acid formed during the reaction is used as acid catalyst. The desired compounds can be prepared at a pressure of 3 to 345 kPa, although ambient pressure is typically used. In a preferred isolation technique, the glycosides can be precipitated from the crude filtered reaction mixture (e.g. the acetonitrile product solution) by adding approx. 25% to 75% water and the rest alcohol (eg methanol). Precipitation of the product from aqueous methanol/acetonitrile requires less processing than an extractive isolation, and gives a product of higher purity.

In general, the mercury (II) bromide and mercury (II) cyanide-promoted coupling of the formula VI compound and the acetylated sugar bromide is carried out in an aprotic, anhydrous solvent such as methylene chloride at a temperature of approx. 20°C to approx. 100"C for about 0.5 to 6 hours. Typically about 0.5 to about 4 equivalents (based on formula IV compound) of mercury (II) bromide and mercury (II) cyanide are used and about 0.5 to about 3 equivalents of peracetylated sugar bromide (e.g. /3-D-maltosyl, /3-D-gentiobiosyl or /3-D-2-acetamido-2-deoxy-glucopyranosyl).The desired compounds can be prepared by pressing 3 to 345 kPa, although ambient pressure is typically used.Preferably, they are isolated as described for the zinc fluoride promoted coupling of the formula V compound above.

The desired formula VI compounds where Q<1> and Q<2> are as defined above and X is either a bond or alkylene-O-, can be prepared by silylation of the appropriate formula V compound, where Q<1> and Q< z> is as defined above and X is either a bond or alkylene-O-.

In general, the formula V compound, a base such as triethylamine and an activated trialkylsilyl compound (eg, trimethylsilyltrifluoromethanesulfonate or trimethylsilyl chloride) are reacted in an aprotic, anhydrous solvent such as methylene chloride at a temperature of less than about 10°C to about 0.5 hours to approx. 2 hours.

According to reaction scheme III, the desired formula V compounds where Q<1> and Q<2> are as defined above and X is alkylene-O-, can be prepared by reduction of the suitable formula VII compound where Q<1> and Q<2> is as defined above.

In general, the reduction is carried out by reacting the formula VII compound with lithium aluminum hydride in an anhydrous solvent such as tetrahydrofuran at temperatures of less than about 10"C for about 0.5 hours to about 3 hours.

The desired formula VII compounds where Q<1> and Q<2> are as defined above can be prepared by coupling the appropriate formula VIII compound where Q<1> and Q<2> are as defined above with ethyl diazoacetate in the presence of rhodium acetate dimer. The formula VIII compound and the ethyl diazoacetate are thus reacted in an aprotic solvent such as methylene chloride in the presence of rhodium acetate dimer at ambient temperature for about 0.5 hours to about 3 hours.

The starting materials for the above described reaction schemes (eg ethyl diazoacetate, peracetylated sugar halides) are readily available, or can be readily synthesized by those skilled in the art using conventional methods in organic synthesis.

As an aid to the preparation of the above steroids, the following section will also describe the preparation of the various formula VIII compounds. Literature references for the preparation of formula VIII steroid compounds (where Q<1> is the methylene and Q<2> and the stereochemistry of the C5 hydrogen and C25 carbon are as defined below) are described in Table I.

The following sections describe and/or provide literature references for the preparation of the various steroids used as starting materials (ie the alternative stereochemistry at the C3 position and the oxygenation and various epimers at Cu and C12) from the formula VIII compounds above described in Table I .Generally, the preparation of the various oxygenated steroids is independent of the stereochemistry at the C3, C5 and C25 positions. When the appropriate stereochemistry at the C3, C5 and C25 positions is achieved where Q<1> and Q<2> are both methylene or where Q<1> is methylene and Q<2> is carbonyl, the various oxygenated compounds can in Q<1>and Q<2> are thus produced from there.

Some of the manufacturing methods described herein will

require protection of remote functionality (eg Q<1>, Q<2>andQ<3>). The need for these protecting groups will vary depending on the nature of the remote functionality and the conditions of the manufacturing processes. This need can easily be determined

mes by professionals in this area. For a general description of protecting groups and their application, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 191.

The formula VIII compounds where Q<1> is methylene, Q<2> is either methylene or carbonyl and C3-hydroxy is beta can be converted into the corresponding formula VIII compounds where C3-hydroxy is alpha by the following two methods. These manufacturing methods can be used regardless of the C25 stereochemistry.

If Q<2> is carbonyl, the carbonyl is protected as a ketal (eg, ethylene ketal) by reacting the steroid with ethylene glycol and an acid catalyst according to the method of Engel and Rakhit, Can. J. Chem. 40, 2153, 1962. When the C5 hydrogen is alpha, the C3 hydroxy group is oxidized to the ketone using pyridinium chlorochromate (PCC) in methylene chloride at ambient conditions. The C3 ketone is then reduced with a sterically hindered reducing agent such as K-Selectride® reducing agent at low temperature in tetrahydrofuran to form the C3 alpha alcohol according to Gondos and Orr, J. Chem. Soc. Chem. Commun. 21, 1239, 1982. If desired, the Q<2-> protecting group is removed with an acid, such as hydrochloric acid, in a suitable solvent such as e.g. acetone.

For the compounds where the C5 hydrogen is beta, the same methods are used as were used when the C5 hydrogen is alpha, except that the C3 ketone is reduced by using sodium borohydride in ethanol to form the C3 alpha alcohol.

Reaction scheme IV illustrates the reaction mechanism to obtain the formula VIII compounds where Q<1> and Q<2> are defined above starting from the formula VIII compound, where Q<1> is methylene and Q<2> is carbonyl.

In general, the preparation methods for these compounds can be found in L.F. Fieser and M. Fieser, Steroids, Reinhold Pub. Corp i, New York, 1959 and references therein, with the following descriptive text (which is the key to reaction scheme IV) provides specific guidelines.

Briefly, according to reaction scheme IV method 1, the starting material is acetylated and brominated according to the method described in J. Chem. Soc, 1956, 4344. This intermediate is then reduced with lithium aluminum hydride and treated with silver oxide by a method similar to that described in Heiv. Act. Chim., 1953, 36, 1241. The resulting 0-11,12-epoxide is opened with trichloroacetic acid, saponified and reduced with zinc and acetic acid using the procedure described in J. Chem. Soc, 1956, 4330 to form the product shown for Method 1.

In method 2, the starting material is selectively acetylated using the method described in J. Chem. Soc. 1956, 430. When applying the method described in Org. Syn., 1976, 55, 84, the resulting product is oxidized with chromium trioxide and pyridine. Using the method described in Synthesis, 1973, 790, the resulting product is saponified with potassium cyanide in water, methanol and THF to form the product shown for method 2.

In method 3, the starting material is converted to the corresponding toluenesulfonylhydrazone which in turn is treated with sodium methoxide using a method similar to that described in J. Am. Chem. Soc, 1954, 76, 4013. The resulting 11-ene product is oxidized with osmium tetroxide and N-methylmorpholine-N-oxide according to the procedure described in Tetrahedron Letters, 1976, 1973 to form the product shown for method 3.

In method 4, the starting material is monobrominated using a method described in US patent no. 3,178,418. Hydrolysis of this intermediate using the method described in J. Chem. Soc, 1956, 4330 gives the product shown for Method 4.

In methods 5 and 6, the starting material is reduced with lithium aluminum hydride according to the method described in J. Am. Chem. Soc, 1954, 76, 4013. The two products shown in methods 5 and 6 are separated chromatographically.

In method 7, the starting material is reduced with lithium aluminum hydride according to the method described in J. Am. Chem. Soc, 1951, 73, 1777 to form the product shown.

In method 8, the starting material is reduced with lithium and ammonia according to the method described in J. Am. Soc, 1953, 75, 1282 to form the product shown.

In method 9, the starting material is acetylated according to the method described in J. Am. Chem. Soc, 1955, 77, 1632, forming a mixture of acetates, from which the 3,11-diacetate can be isolated. The unprotected 12-alcohol is then oxidized with chromium trioxide and pyridine according to the method described in Org. Syn., 1976, 55, 84. Saponification of the acetates gives the product shown for method 9.

In method 10, the starting material is diacetylated using the method described in J. Chem. Soc, 1956, 4330. The diacetate is reduced with calcium and ammonia using the method described in J. Chem. Soc 1956, 4334

forming the product shown for Method 10.

In method 11, the starting material is reduced with lithium and ammonia according to the method described in J. Am. Chem. Soc, 1953, 75, 1282 to form the product shown.

In method 12, the starting material is reduced with lithium aluminum hydride according to the method described in J. Am. Chem. Soc, 1951, 73, 1777 to form the product shown.

In method 13, the starting material is selectively protected on the 3-alcohol with t-butyldimethylchlorosilane and imidazole using the method described in J. Am. Chem. Soc, 1972, 94, 6190. Using the method described in Org. Sight. 1976, 55, 84, the product is oxidized with chromium trioxide and pyridine. The 3-alcohol is then desilylated with hydrofluoric acid in acetonitrile using the method described in J. Am. Chem. Soc, 1972, 94, 6190 to form the product shown for Method 13.

In Method 14, the starting material is selectively protected on the 3-alcohol with t-butyldimethylchlorosilane and imidazole using the method described in J. Am. Chem. Soc, 1972, 94, 6190. The resulting intermediate is reduced with lithium aluminum hydride using the method described in J. Am. Chem. Soc, 1951, 73, 1777. The resulting intermediate is selectively acetylated on the 12-alcohol, silylated on the 11-alcohol with trimethylsilyl triflate and 2,6-lutidine using the procedure described in Tetrahedron Letters, 1981, 22, 3455, and then deacetylated on the 12-alcohol with lithium aluminum hydride and an aqueous ammonium chloride quench. The 12-alcohol is oxidized with chromium trioxide and pyridine in methylene chloride using the method described in Org. Syn., 1976, 55, 84, and then desilylated with hydrofluoric acid in acetonitrile using the method described in J. Am. Chem. Soc, 1972, 94, 6190 to form the product shown in Method 14.

According to reaction scheme V, the desired formula IA compounds where Q<1>, Q<2>, Q<3>, Q<*>, Q<5>, C5 and C25 are as described in (E) above (i.e. oxygenated in Q <*->position) is produced by the following methods.

The desired formula X compound can be prepared by oxidation of tigogenin IX. In general, the oxidation is carried out by reacting tigogenin with pyridinium chlorochromate in a reaction-inert solvent such as methylene chloride at CC to ambient temperature for approx. 2 hours to approx. 10 hours.

The desired formula XI compound can be prepared by bromination of the formula X compound followed by an elimination reaction. Typically, the bromination is carried out by reacting the compound of formula X with bromine in tetrahydrofuran at a temperature of approx. -78°C, followed by heating to ambient temperature for approx. 1 hour to approx. 3 hours. The elimination reaction is carried out by reacting the brominated product prepared above with lithium bromide and lithium carbonate in a polar aprotic solvent such as dimethylformamide at a temperature of approx. 100°C to approx. 140°C for approx. 1 hour to approx. 4 hours.

The desired formula XII compound can be prepared by epoxidation of the appropriate formula XI compound followed by reduction with lithium aluminum hydride. In general, the epoxidation is carried out by reacting the compound of formula XI with hydrogen peroxide and sodium hydroxide in a polar, protic solvent such as methanol at ambient temperature for approx. 2 hours to approx. 6 hours. The reduction is carried out on the 11-alcohol with trimethylsilyl triflate and 2,6-lutidine using the method described in Tetrahedron Letters, 1981, 22, 3455, and then deacetylated on the 12-alcohol with lithium aluminum hydride and an aqueous ammonium chloride suppression. The 12-alcohol is oxidized with chromium trioxide and pyridine in methylene chloride using the method described in Org. Syn., 1976, 55, 84, and then desilylated with hydrofluoric acid in acetonitrile using the method described in J. Am. Chem. Soc, 1972, 94, 6190 to form the product shown in Method 14.

According to reaction scheme V, the desired formula IA compounds where Q<1>, Q<2>, Q<3>, Q", Q<5, <C>5 and C25 are as described in (E) above (i.e. oxygenated in Q<4->position) is produced by the following methods.

The desired formula X compound can be prepared by oxidation of tigogenin IX. In general, the oxidation is carried out by reacting tigogenin with pyridinium chlorochromate in a reaction-inert solvent such as methylene chloride at 0°C to ambient temperature for about 2 hours to about 10 hours.

The desired formula XI compound can be prepared by bromination of the formula X compound followed by an elimination reaction. Typically, the bromination is carried out by reacting the compound of formula X with bromine in tetrahydrofuran at a temperature of approx. -78"C, followed by heating to ambient temperature for about 1 hour to about 3 hours. The elimination reaction is carried out by reacting the brominated product prepared above with lithium bromide and lithium carbonate in a polar aprotic solvent such as dimethylformamide at a temperature of about 100°C to about 140°C for about 1 hour to about 4 hours.

The desired formula XII compound can be prepared by epoxidation of the appropriate formula XI compound followed by reduction with lithium aluminum hydride. In general, the epoxidation is carried out by reacting the compound of formula XI with hydrogen peroxide and sodium hydroxide in a polar, protic solvent such as methanol at ambient temperature for approx. 2 hours to approx. 6 hours. The reduction is carried out by reacting the epoxide prepared above with lithium aluminum hydride in a reaction-inert solvent such as tetrahydrofuran at ambient temperature for 2 hours to approx. 6 hours.

The desired formula IA compound as described in (E) above where Q<4> contains a hydroxy group, can then be prepared from the appropriate formula XII compound by a zinc fluoride-catalyzed coupling followed by deacetylation with sodium methoxide as previously described. The compounds where Q<*> is carbonyl can be prepared in a similar way, with the addition of oxidation with pyridinium chlorochromate (as described for formula X compounds) before the deacetylation.

According to reaction scheme VI, the desired formula IA compounds where Q<1>, Q<2>, Q<3>, Q<4>, Q<5>, C5 and C25 are as described in (F) above (i.e. oxygenated in Q <5->position) is produced by the following methods.

The desired compound of formula (XIV) can be obtained by protection (as denoted by P) of the alcohol function in diosgenin (XIII) followed by hydroboration of the olefin. Typically, the alcohol is protected as an ethoxymethyl ether by reacting diosgenin with ethoxymethyl chloride and diisopropylethylamine in an anhydrous solvent such as methylene chloride at ambient temperature for approx. 2 hours to 6 hours. The hydroboration is carried out by reacting the compound prepared above with a borane-tetrahydrofuran complex in a reaction-inert solvent such as tetrahydrofuran at ambient temperature for approx.

1 hour to approx. 6 hours.

The desired formula XV compound can be prepared by oxidation of the appropriate formula XIV compound followed by removal of the alcohol protecting group. In general, the oxidation is carried out by reacting the formula XIV compound with pyridinium chlorochromate in an anhydrous solvent such as methylene chloride at ambient temperature for approx.

2 hours to approx. 8 hours. The removal of the alcohol protecting group can be carried out by reacting the oxidized product prepared above with concentrated hydrochloric acid in a mixed solvent containing methanol and tetrahydrofuran at a

temperature of approx. 40"C to approx. 65°C for approx. 5 min. to approx.

1 hour.

The desired formula IA compound as described in (F) above, where Q<5> is carbonyl, can then be prepared from the appropriate formula XV compound by a zinc fluoride-catalyzed coupling reaction followed by deacetylation using sodium methoxide as previously described. The compounds where Q<5> contains a hydroxy group can be prepared in a similar way with the addition of reduction before deacetylation. Typically, the reduction is carried out by reaction with sodium borohydride in a mixed solvent of ethanol and dichloromethane at ambient temperature for approx. 1 hour to approx. 6 hours.

The compounds of formula I which have been obtained and have asymmetric carbon atoms can be separated into their diastereomers on the basis of their physicochemical differences by methods known per se, e.g. by chromatography and/or fractional crystallization.

The compounds according to this invention where the sugar is /3-D-glucopyranuronosyl are acidic and form base salts. All such base salts are within the scope of this invention, and they can be prepared by conventional methods. E.g. they can be prepared simply by bringing the acidic and basic units into contact with each other, usually in stoichiometric ratio, in either an aqueous, non-aqueous or semi-aqueous medium, as appropriate. The salts are recovered either by filtration, by precipitation with a non-solvent followed by filtration, by evaporation of the solvent, or in the case of aqueous solutions, by lyophilization, as appropriate.

Moreover, many of the compounds of this invention can be isolated as hydrates.

The compounds according to this invention are potent inhibitors of cholesterol absorption and are thus all suitable for therapeutic use as hypercholesterolemia controlling agents in mammals, especially humans. Since hypercholesterolemia is closely related to the development of generalized cardiovascular, cerebral vascular or peripheral vascular disorders, these compounds can prevent the development of atherosclerosis, particularly arteriosclerosis.

The hypercholesterolemia controlling activity of these compounds can be demonstrated by methods based on standard procedures. E.g. the in vivo activity of these compounds to inhibit absorption of cholesterol in the intestines can be determined by the method of Melchoir and Harwell (J. Lipid Res., 1985, 26, 306-315).

Activity can be determined by the amount of hypo-cholesterolemic agent that reduces cholesterol absorption, relative to control, in male golden Syrian hamsters. Male golden Syrian hamsters are administered either a cholesterol-free diet (the control animals) or a diet supplemented with 1% cholesterol and 0.5% carbonic acid for 4 days. On the following day, the animals are fasted for 18 hours, and are then administered a 1.5 ml oral bolus of water containing 0.25% methylcellulose, 0.6% Tween 80 and 10% ethanol (the control animals) or an oral bolus also containing the desired concentration of the compound to be tested. Immediately after the bolus administration, the animals receive a second 1.5 ml oral bolus of liquid hamster diet containing 1% [<3>H] cholesterol (2.0 juCi/animal; 210 dpm/nmol) and 0.5% carbonic acid, and are fast for another 24 hours. At the end of this second fasting period, the animals are killed, the liver excised, saponified and aliquots are decolorized by the addition of hydrogen peroxide, and determined for radioactivity. Total radioactivity in the liver is calculated based on measured liver weight. The degree of cholesterol absorption is expressed as a percentage of the total radioactivity administered as an oral bolus that is present in the liver 24 hours after bolus administration.

The anti-atherosclerosis effects of the compounds can be determined by the amount of agent that reduces lipid deposition in the rabbit aorta. Male New Zealand White rabbits are fed a diet containing 0.4% cholesterol and 5% peanut oil for 1 week (meal provided once a day). After 1 week, the rabbits receive a daily dose with the desired concentration of the compound to be tested. After 8.5 weeks, the drug treatment is terminated, and the animals are kept on the cholesterol-containing diet for another 2 weeks and then switched to a cholesterol-free diet for 5 weeks. The animals are euthanized and the aorta removed from the thoracic arch to the branch of the iliac crest. The major life arteries are cleaned of adventitia, opened longitudinally and then stained with Sudan IV as described by Holman et al. (Lab. Invet. 1958, 7, 42-47). The percentage of surface area stained is quantified by densitometry using an Optimas Image Analyzing System (Image Processing Systems). Reduced lipid deposition is indicated by a reduction in the percent surface area stained in the drug-treated group, compared to the control rabbits.

Administration of the compounds according to this invention can be via any method that delivers the compounds to the intestinal spaces. These methods include oral routes, intraduodenal routes, etc.

The amount of steroid glycoside administered will naturally depend on the individual being treated, on the degree of the ailment, on the method of administration and the judgment of the attending physician. However, an effective dosage is in the range of 0.71 to 200 mg/kg/day, preferably 2 to 50 m9/fcq/day, most preferably 2 to 7 mg/kg/day. For a human of average weight 70 kg, this would be 0.05 to 14 g/day, preferably 0.14 to 3.5 g/day, most preferably 0.14 to 0.5 g/day.

For oral administration, which is preferred, a pharmaceutical preparation may take the form of solutions, suspensions,

tablets, pills, capsules, powders, formulations with extended release, etc.

Depending on the intended method of administration, the pharmaceutical preparations can be in the form of solid, semi-solid or liquid dosage forms, such as e.g. tablets, pills, capsules, powders, liquids, suspensions etc., -preferably in unit dosage form, suitable for single administration of precise dosages. The pharmaceutical preparations will include a conventional pharmaceutical carrier or excipient and a compound according to this invention as active ingredient. Moreover, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc.

Pharmaceutical preparations according to this invention may contain 0.1% to 95% of the compound, preferably 1% to 70%. In any case, the preparation or formulation to be administered will contain a quantity of a compound of this invention in an amount which will effectively alleviate the symptoms of the individual being treated, i.e. hypercholesterolemia or atherosclerosis.

For solid pharmaceutical preparations, conventional non-toxic solid carriers will include e.g. pharmaceutical qualities of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate etc.

Liquid pharmaceutically administrable preparations can be prepared by dissolving or dispersing, or otherwise preparing a compound according to this invention, and optionally mixing it with a pharmaceutical adjuvant in a carrier, such as e.g. water, saline, aqueous dextrose, glycerol, ethanol, etc., thereby forming a solution or suspension.

Methods for the production of various pharmaceutical preparations with a certain amount of active ingredient are known, or will be obvious, to those skilled in the art. See e.g. Remington's Pharmaceutical Sciences., Mack Publishing Company, Easter, Pa., 15th ed. (1975).

Example 1

(30.5a.120.25R)-3-(/3-D-galactocyl)oxy}-12-hydroxyspirostane

REDUCTION OF KETONES

To a room temperature solution of ( 30 , 5a , 25R )-3-(β-D-galactocyl)oxy]-spirostan-12-one (1.33 g, 2.24 mmol; obtained via deacetylation of ( 30 , 5a , 25R)-3-[(tetraacetyl-/3-D-galactosyl)-oxy]spirostan-12-one (preparation B2) according to the procedure described in example 3), ethanol (130 ml) and chloroform (260 ml) were added a solution of sodium borohydride (0.51 g, 13.4 mmol) and ethanol (50 mL). After stirring for 4 hours, methanol (200 mL) was added and stirring was resumed for 2 hours. The reaction mixture was concentrated in vacuo to give 3.38 g of crude product. Recrystallization from a mixture of methanol (80 mL) and water (8 mL), followed by washing with cold methanol (20 mL) and drying gave 1.33 g (quantitative yield) of the title compounds. MS: 595 (M+H).

Melting point: > 200°C. High resolution FAB (m/e): calcd for C 33 H 55 O 9 : 595.3846, found : 595.3861.

The title compound was prepared from the appropriate starting material in a similar manner using the method in example 1.

Example 2

( 30 , 5a . 120 . 2 5R)- 3-( fl- D- cellobiocvl) oxvl- 12- hydroxyspirostane

Example 3

f 30 . 5a, 120 . 25R)-3-(fl-D-cellobiocyl)oxy1spirostan-ll-one

DEACETYLATION

A mixture of (30,5a,25R)-3-[(heptaacetyl-/?-D-cellobiosyl)oxy]spirostan-ll-one (6.57 g, 6.26 mmol), sodium methoxide (68 mg, 1, 25 mmol), methanol (35 mL) and tetrahydrofuran (75 mL) were heated to reflux for 1 h, followed by stirring at room temperature for 12 h. A white precipitate formed within 30 min. The final suspension was concentrated in vacuo to give 6.0 g of crude product. This material was purified by flash chromatography (eluent: chloroform followed by 8:2 chloroform:methanol) to give 2.71 g (57% yield) of the title compound.

<X>H NMR (DMSO-d6) <S: 5.22 (d, dJ=5Hz, 1H); 5.00 (m, 3H);

4.64 (s, 1H); 4.58 (t, J=5Hz, 1H); 4.54 (t, J=6Hz, 1H) ;

4.34 (q, J=8 Hz, 1H); 4.27 (d, J=8Hz, 1H); 4.23 (d, J=8HZ, 1H); 3.68-2.94 (m, 15 H); 2.34 (itl, 2H); 2.08-0.81 (m, 23H); 0.92 (s, 3H); 0.86 (d, J=7 Hz, 3H); 0.72 (d, J= 6 Hz, 3H); 0.59 (s, 3H) . DEPT 13 C NMR (DMSO-d 6 ) 6 : 210.4

(s), 108.8 (s), 103.6 (d), 100.6 (d), 81.1 (d), 80.6 (d), 77.2 (d), 76.9 ( d), 76.5 (d), 75.5 (d), 75.1 (d), 73.7 (d), 73.6 (d), 70.5 (d), 66.4 (t ), 63.5 (d), 61.5 (t), 60.9 (t), 50.5 (d), 57.1 (t), 54.7 (d), 44.3 (s) , 44.1 (d), 41.7 (d), 36.8 (d), 35.6 (t), 35.2 (s), 34.0 (t), 32.6 (t), 31.3 (S), 30.2 (d), 29.2 (t), 28.9 (t), 28.2 (t), 17.5 (q), 17.3 (q), 14 .8 (q) , 12.3 (q). IR (KBr): 3407 (s) , 1700 (m) cm"<1>. High resolution FAB MS (m/e) : calculated for C39H620nNa 777.4037, found 777.4108. Analysis: calculated for C39H6201A»2H20, C 59.22 , H 8.41; found

C 59.48, H 8.48. Melting point: >300°C.

A monohydrate crystalline form of the above title product was prepared as follows: A mixture of 20 g of the crude product prepared according to the above procedure, 600 ml of n-propanol, and 400 ml of water was stirred and heated to reflux. To the resulting solution was added 2.0 g of diatomaceous earth. Still under reflux, the insoluble components were removed by filtration. The filtrate was atmospherically distilled to a total volume of 600 ml and cooled to ambient temperature. The resulting suspension was granulated for 1 hour and the product collected by filtration. The undried recrystallized cake from the above recrystallization was slurried in 500 mL of methanol. This suspension was heated to reflux for 16 hours, cooled to ambient temperature, granulated for 48 hours, and isolated by filtration. Vacuum drying gave 16.1 g (81% recovery) of the crystalline monohydrate of the title compound in Example 3.

Examples 4-47

The following compounds were prepared from the appropriate starting materials in a similar manner using the above procedures.

Example 48

( 3B . 5a . llfl. 25PJ- 3- r ( fl- D- cellobiosvH oxvl- 11- hvdroxv-spirostan- 12- one

DEACETYLATION

Based on the procedure described in Synthesis, 1973, 790, (/3,5α,11/3,25R)-3-[(heptaacetyl-/9-D-cellobiosyl)oxy]-spirostan-11-ol-12-one (240 mg, 0.225 mmol) dissolved in methanol (20 mL) and tetrahydrofuran (10 mL). To this solution was added a solution of potassium cyanide (146 mL, 2.25 mmol) in water (0.1 mL) and methanol (5 mL). The resulting mixture was heated to 80°C for four hours. After cooling, the mixture was concentrated to dryness and purified by flash chromatography (9:1 chloroform:methanol eluent) to give the title compound. Temp. 245-247°C. MS (m/e): 771 (P+1), 793 (P+Na). Analysis: calculated for C39H62015»3H20, C 56.70, H 8.31.

found, C 56.97, H 7.80.

Preparation Al

( 36 , 5a . 2 5R) - 3- f ( heptaacetyl- a- D- cellobiosvl) oxy1spirostan- 11-one

ANOMERIZATION

Hydrobromic acid (30% in acetic acid, 1.2 mL) was added to a room temperature solution of (3(3,5a,25R)-3-[(heptaacetyl-/3-D-cellobiosyl)-oxy]spirostan-ll-one ( 2.0 g) in methylene chloride

(35 mL) and the resulting mixture was stirred at room temperature for 94 hours. The reaction was quenched by slow addition of saturated aqueous sodium bicarbonate (20 mL). The organic layer was separated, dried over magnesium sulfate and dried in vacuo to give 1.637 g of a black solid. Purification by repeated flash chromatography (1:1 hexane:ethyl acetate) afforded 651 mg (33% yield) of the title compound.

<X>H NMR (CDCl 3 ) 5:5.41 (t, J=10Hz, 1H); 509 (complex, 3H); 4.91 (t, J=8 Hz, 1H); 4.69 (dd, J=4 & 10 Hz, 1H) ;

4.49 (complex, 3H); 4.36 (dd, J=4 & 13 Hz, 1H); 3.99 (m, 3H); 3.67 (m, 2H); 3.40 (m, 3H); 2.45 (m, 1H); 2.22 (s, 2H); 2.11 (s, 3H); 2.07 (s, 3H); 2.03 (s, 6H); 2.00 (s, 3H); 1.99 (s, 3H); 1.97 (s, 3H); 2.00 - 0.80 (m, 22H);

1.02 (s, 3H); 0.92 (d, J=7 Hz, 3H); 0.77 (d, J=7 Hz, 3H);

0.69 (S, 3H) . DEPT 13 C NMR (CDCl 3 ) 5:210.0 (s) , 170.5

(s) , 170.3 (S) , 170.2 (S) , 169.6 (s) , 169.3 (s) , 169.1 (s), 109.2 (s) , 100.9 ( d) , 94.3 (d) , 80.6 (d) , 78.0 (d) , 77.0 (d), 73.1 (d), 71.9 (d), 71.8 (d), 71.2 (d), 69.6 (d), 68.1, (d), 67.8 (d), 66.9 (d), 64.4 (d), 62.0 (t), 61.5 (t), 60.7 (d), 57.6 (t), 55.7 (d), 45.0 (d), 44.3 (s), 41.8 (d), 36 .9 (d), 35.5 (t), 35.4 (t), 35.1 (s), 32.7 (t), 31.3 (t), 31.2 (t), 30, 2 (d), 28.7 (t), 28.0 (t), 27.4 (t), 20.9 (q), 20.7 (q), 20.6 (q), 20.5 (q), 17.1 (q), 17.0 (q), 14.2 (q), 12.1 (q). IR (Kbr): 1751 (s) , 1706 (m) cm"<1>. MS (m/e): 1049 (M+H) , 1071 (M + Na). Analysis: calculated for C53H76021»H20, C 59.65 H 7.37; found C 59.66 H 7.00.Mp: 248-249°C.

Preparation Bl

(36.5a.25R)-3-f(heptaacetyl-g-D-cellobiosyl)oxvlspirostan-11-one

ZINC FLUORIDE PROMOTED COUPLING OF FREE SPIROSTANE

A suspension of (30,5α,25R)-3-hydroxyspirostan-11-one (3.0 g, 6.97 mmol) and anhydrous zinc fluoride (2.88 g,

27.9 mmol) in dry acetonitrile (175 mL) was dried by removing.

75 ml of acetonitrile by distillation. The suspension was allowed to cool, heptaacetyl-/3-D-cellobiosyl bromide (9.75 g, 13.9 mmol) was added, and the resulting suspension was heated to 65°C

for 3 hours. After cooling to room temperature, methylene chloride (150 ml) was added, the suspension was stirred for 10 min. and filtered. The filtrate was concentrated in vacuo to give 10 g of crude product. This material was dissolved in 8:2 chloroform:methanol, preadsorbed on silica gel and purified by flash chromatography (eluent: 1:1 ethyl acetate:hexane followed by neat ethyl acetate) to give 6.81 g (93% yield) of the title material.

<X>H NMR (CDCl 3 ) 6: 5:11 (complex, 2 H); 5.04 (t, J=9 Hz, 1H); 4.90 (t, J=9 Hz, 1H); 4.83 (t, J=8 Hz, 1H); 4.49 (complex, 4H); 4.34 (dd, J=4.5 & 12.5 Hz, 1H); 4.04 (t, J=13 Hz, 1H), 4.03 (t, J=11 Hz, 1H); 3.72 (t, J=9.5 Hz,

1H); 3.65 (m, 1H); 3.56 (m,1H); 3.45 (m, 1H); 2.47 (m,

1H); 2.22 (s, 2H); 2.08 (s, 3H); 2.06 (s, 3H); 2.00 (p, 6H); 1.99 (p, 6H); 1.96 (s, 3H); 2.00 - 1.00 (m, 22H);

0.98 (s, 3H); 0.92 (d, J=7 Hz, 3H); 0.77 (d, J=7 Hz, 3H);

0.68 (s, 3H) . DEPT<13>C NMR (CDC13) S: 209.9 (s), 170.5

(s), 170.3 (s), 170.2 (s), 169.9 (s), 169.8 (s), 169.5

(s), 169.3 (s) , 169.0 (s), 109.2 (s), 100.8 (d), 99.4

(d), 90.0 (s), 80.6 (d), 79.4 (d), 76.6 (d), 75.3 (s), 72.9 (d), 72.6 ( d), 72.5 (d), 71.9 (d), 71.8 (d), 71.6 (d), 67.8 (s), 66.9 (t), 64.4 (d ), 62.1 (t), 61.5 (t),

60.8 (s), 60.7 (d), 57.6 (t), 55.7 (d), 44.8 (d), 44.3

(s), 41.8 (d), 36.9 (d), 35.6 (t), 35.2 (s), 34.1 (t),

32.7 (h), 31.3 (h), 31.2 (h), 30.2 (d), 29.0 (h), 28.7

(t), 28.0 (t), 20.9 (q), 20.7 (q), 20.6 (q), 20.5 (q),

20.5 (q), 17.1 (q), 17.0 (q), 14.2 (q), 12.0 (q). IR (KBr) : 1756 (s) , 1706 (m) cm"<1>. MS (m/e) : 1049 (M+H) . Analysis: calculated for C53H76021»H20, C 59.65, H 7, 37; found

C 59.86, H 7.25. Melting point: 210-212°C.

In a similar manner, the following compounds, preparations B2-B41, were prepared from the appropriate starting materials using the general method above.

Preparation B2

(30.5a,25R)-3-[(tetraacetvl-Ø-D-qalactosyl)oxy]spirostan-12-one

Preparation B3

(30.5a.25R)-3-\(heptaacetyl-Ø-D-cellobiosyl)oxy]spirostane

Preparation B4

(30.50.25R)-3-r(heptaacetyl-Ø-D-cellobiosyl)oxvlspirostane

Preparation B5

( 30 , 5a . 25R)-3-\(triacetyl-Ø-D-qlucuronosyl)oxy1spirostane methyl ester

Preparation B6

( 30 . 5a . 2 5R) - 3- [ ( tetraacetyl- Ø- D- qlucupvranosyl) oxy") spirostan-12- one

Preparation B7

(.

Preparation B8

( 30, 5a. 25R) - 3- f ( heptaacetvl-/ 3- D- cellobiosyl) oxy" | spirostane

Preparation B9

(30, 5a. 25R)-3-r(heptaacetyl-Ø-D-cellobiosyl)oxy1ethoxy)-spirostane

Preparation B10

(30.5a.25R)-3-(f(tetraacetyl-Ø-D-galactopyranosyl)oxvletoxy)-spirostane

Preparation Car

(30.5a.25R)-3-[(heptaacetvl-Ø-D-lactosyl)oxy]spirostane

Preparation B12

( 3fl. 5a. 25R)- 3- r( heptaacetvl- g- D- lactosyl) oxy1spirostan- 12- one

Preparation B13

(30.5a.25R)-3-r(triacetyl-a-L-arabanopyranosyl) oxylspirostane

Preparation B14

(30.5a.25R)-3-[(triacetvl-a-D-arabanopuranosyl) oxylspirostane

Preparation B15

( 30 , 5a. 25R) - 3- r ( triacetvl-/ 3- L- xylopvranosyl) oxvl spirostane

Preparation B16

( 30 . 5a, 25R)- 3- r ftriacetyl- g- L- fucopyranosyl) oxvlspirostane

Preparation B17

f 30 . 5a. 25R)-3-r(triacetvl-fl-D-xvlopyranosyl)oxyl spirostane

Preparation B18

(30.5a.25R)-3-r (triacetyl-fi-D-fucopyranosyl) oxvl spirostane

Preparation B19

( 30 . 5a . 25R )- 3- f ( tetraacetyl- if- D- qalactopyranosvl) oxylspirostane

Preparation B20

(30.5a.25R)-3- f

Preparation B21

( 30 . 5a . 25 S )- 3-[ ftetraacetvl- if- D- qalactopyranosyl) oxy]-spirostane

Preparation B22

( 30 , 5a . 120 . 25R )- 3- f ( heptaacetyl- g- D- cellobiosvl) oxvl- 12-hydroxyspirostan- ll-one

Preparation B23

( 3/ 3. 5a. lla. 25R) - 3- T ( heptaacetvl-fi- D- cellobiosvl) oxvl- 11-hydroxyspirostane

Preparation B24

( 36 . 5a. llfl . 25PJ - 3- r ( heptaacetvl-/ 3- D- cellobiosvl) oxvl - 11-hydroxyvspirostane

Preparation B25

( 36 . 5a . 25R) - 3- r ( tetraacetyl- <3- D- qlukopvranosvl) oxvl- 11- one

Preparation B26

( 36 . 5a . 11/ 3. 25R) - 3- r ( heptaacetvl- fl- D- cellobiosvl) oxvl - 11,12-di( hydroxysv) spirostane

Preparation B27

( 36 . 5a . Ila . 126 . 25R )- 3- f( heptaacetvl- fl- D- cellobiosvl)- 11. 12-di( hydroxy) spirostane

Preparation B28

(36.5a.12a.25R)-3-f(heptaacetvl-fl-D-cellobiosyl)oxy]-12-hydroxyspirostane

Preparation B29

( 36 , 5a . 25R)-3-f(heptaacetyl-fl-D-lactosyl)oxy1spirostan-ll-one

Preparation B30

(36.5a.25R)-3-r(heptaacetyl-fl-D-cellobiosyl)oxy 1 spirostan-12-one

Preparation B31

( 36 . 5a . Ila . 2 5R) - 3- f ( heptaacetyl-/ 3- D- cellobiosyl) oxy] 11. 12-dihydroxyspirostane

Preparation B32

( 36 , 5a . Ila . 2 5R) - 3 - f ( heptaacetvl-/ 3- D- cellobiosyl) oxvl- 11-hydroxyspirostan- 12- one

Preparation B3 3

( 30 , 5a. 25R) - 3- r ( heptaacetvl-/ 3- D- cellobiosyl) oxvl spirostane-11. 12-dione

Preparation B34

( 30 . 5a . 11/ 3 . 12a . 25R) - 3- r ( heptaacetvl- fl- D- cellobiosvl) oxy)-11. 12-di (hydroxy) spirostane

Preparation B35

( 36 . 5a . 12a . 2 5R)- 3- f( fl- D- cellobiosvl) oxvl- 12- hvdroxv-spirostan- ll- one

Preparation B36

( 36 . 5a . 126 . 25R )- 3- r fheptaacetvl- fl- D- lactosvl) oxvl- 12-hvdroxyspirostan- ll- one

Preparation B37

f 36 . 5a. 25R )- 3- f( dodecaacetvl- g- D- maltotriosevlVoxyl spirostan-11- one

Preparation B38

(30.5a.25R)-3-fheptaacetyl-/3-D-maltosyl)oxy]spirostan-ll-one

Preparation B39

( la. 30 . 5a . 25R )-3-f(heptaacetyl-g-D-cellobiosvl)oxy1-1-hydroxyspirostane

Preparation B40

(3i3.5a.25R)-3-f (heptaacetyl-fi-D-cellobiosvl)oxylspirostan-6-one

Preparation B41

( 30 . 5a . 110 , 2 5R)- 3- f( heptaacetvl- fl- D- cellobiosyl) oxy] 11-hydroxyspirostan- 12-one

Preparation Cl

(30.5a.25R)-3-r (heptaacetvl-lf-D-lactosyl)oxvlspirostane

MERCURY( II) BROMIDE/ MERCURY( II) CYANIDE PROMOTED COUPLING OF SYLYLATED SPIROSTANE

Powdered 4Å molecular sieves (1 g) was added to a solution of trimethylsilyltigogenin (1.17 g, 2.4 mmol) and acetobromolactose (3.36 g, 4.8 mmol) in CH 2 Cl 2 (15 mL) and CH 3 CN (5 ml) at room temperature. After stirring for 15 min. was added Hg(CN) 2 (2.4 g, 9.6 mmol) and HgBr 2 (3.4 g, 9.6 mmol), and the mixture stirred at room temperature for 3 h. The mixture was diluted with ethyl acetate (50 mL) and filtered. The filtrate was washed with IN (HCl (3 x 30 mL) and brine (1 x 30 mL), dried (Na 2 SO 2 ) filtered and concentrated in vacuo. The product was purified by flash chromatography (10-20% EtOAc/CH 2 Cl 2 ) to give 400 mg product as a colorless solid MS 489 (M+H)<+>.

<*>H NMR (250 MHZ, CDCl 3 ) S 5.35 (d, 1H, J = 1.0 Hz); 5.2 (dd, 1H, J=4.5, 4.5 Hz); 5.15 (dd, 1H, J=6.0, 5.0 Hz);

4.95 (dd, 1H, J=4.5, 1.0 Hz); 4.85 (dd, 1H, J=5.0, 4.5 Hz); 4.55 (d, 1H, J=6.0 Hz); 4.4 (m, 3H); 4.1 (m, 3H);

3.85 (t, 1H, J=3.0Hz); 3.8 (t, 1H, J=4.5Hz); 3.5 (m, 3H); 3.35 (t, 1H, J=5.0 Hz); 2.15 (s, 3H), 2.12 (s, 3H);

2.07 (s, 12H); 2.0 (s, 3H); 2.0 - 0.5 (m, 27H); 0.98 (d, 3H; J=4.0Hz); 0.82 (s, 3H); 0.8 (d, 3H, J=4.0Hz); 0.73 (s, 3H).

In a similar manner, the following compounds, the preparations C2-C4, were prepared from the suitable starting materials using the general method above.

Preparation C2

( 30 . 5a . 2 5R) - 3- f ( heptaacetyl- <3- D- maltosyl) oxy 1 - spirostane

Preparation C3

( 30 , 5a . 25R) - 3- F ( triacetyl- fl- D- 2- acetamido- 2- deoxygluco-pyranosvl) oxvl- spirostane

Preparation C4

( 36 , 5a . 2 5R)-3-f( heptaacetyl- g- D- genthiobiosvl) oxy]- spirostane

Preparation Dl

(36.5a.25R)-3-trimethylsilvloxvspirostane

SYLYLATION OF SPIROSTANES

Trimethylsilyl trifluoromethanesulfonate (4 mL, 22.1 mmol) was added dropwise to a solution of tigogenin (6 g, 14.4 mmol) and triethylamine (6 mL, 45 mmol) in CH 2 Cl 2 (50 mL) at 0°C. After 1 h the mixture was diluted with ether (100 mL) and washed with saturated NaHCO 3 solution (2 x 50 mL) and brine (1 x

50 ml), dried (Na2SOJ filtered and concentrated in vacuo.

After addition of methanol, a precipitate formed which was filtered off and washed with methanol and dried to give 6.2 g of product as a colorless solid.

S.mp. 197-198°C. MS 489 (M + H)<+>.<*>H NMR (250 MHZ, CDCl 3 ) S 4.35 (q, 1H, J=3.0 Hz); 3.5 (m, 2H); 3.4 (t, 1H, J=5.5Hz); 2.0-0.5 (m, 27H); 1.0 (d, 3H, J=4.0 Hz); 0.85 (s, 3H); 0.8 (d, 3H, J=4.0Hz); 0.75 (s, 3H); 0.1 (s, 9H).

Preparation El

( 36 . 5a . 25R)- 3-( 2- hydroxyethoxy)- spirostane

LAH- REDUCTIONS

Lithium aluminum hydride (0.285 g, 7.5 mmol) was added to a solution of tigogenin-O-acetic acid ethyl ester (2.5 g,

4.98 mmol) in THF (50 mL) at 0 °C. After 1 hour, the reaction was quenched by sequential addition of H 2 O (0.285 mL), 15% NaOH (0.285 mL) and H 2 O (0.85 mL). The mixture was diluted with ether (25 mL) and dried with MgSO 4 , filtered and concentrated in vacuo to give 2.1 g of product as a colorless solid. S.mp. 207-208°C. MS 461 (M+H)<+>.

<X>H NMR (250 MHz, CDCl 3 ) S 4.4 (q, 1H, J=3.0 Hz); 3.7 (m, 2H); 3.6 (m, 2H); 3.5 (m, 1H); 3.4 (t, 1H, J=5.5Hz); 3.3 (m, 1H); 2.0-0.5 (m, 28H), 1.0 (d, 3H, J=4.0 Hz); 0.85

(p, 3H); 0.8 (d, 3H, J=4.0 Hz); 0.75 (s, 3H).

Preparation Fl

( 13B . 5a . 25PJ - spirostan- 3-yl) O- acetic acid ethyl ester rRhfOAcKl-, CATALYZED COUPLINGS

Ethyl diazoacetate (5.5 mL, 0.048 mol) dissolved in 30 mL CH 2 Cl 2 was added dropwise over 1 hour to a solution of tigogenin (10 g, 0.024 mol) and rhodium acetate dimer (250 mg) in CH 2 Cl 2 (250 mL) at room temperature. Gas evolved throughout the addition, and when the addition was complete, the mixture was stirred for an additional 1 hour. The mixture was diluted with hexanes (100 mL) and filtered through a plug of silica gel. The filtrate was concentrated in vacuo and after addition of methanol to the residue, a precipitate formed which was filtered off and washed with methanol and dried to give 6.0 g of product as a colorless solid. S.mp. 119-120°C. MS 503 (M+H)<+>.

<*>H NMR (250 MHz, CDCl 3 ) S 4.35 (q, 1H, J=3.0 Hz); 4.2 (m, 2H); 4.1 (s, 2H); 3.4 (m, 3H); 2.0 - 0.5 (m, 30H); 0.95 (d, 3H, J=4.0 Hz); 0.8 (s, 3H); 0.75 (d, 3H, J=4.0Hz);

0.72 (s, 3H).

Preparation Gl

(( 3g. 5a. lllf. 12a, 25R)- spirostane- 3. 11. 12- triol

(3fl. 5a. Ila. 12a. 25R)- 11. 23-dibromo-3-acetoxyspirostan-12-one: The title compound was synthesized from (3fl,5a,25R)-3-acetoxyspirostan-12-one according to the procedure described in J .Chem♦ Soc.. 1956, 4344.

( 3fl. 5a. Ila. 12fl. 2 5R)- 11. 2 3- dibromospirostan- 3. 12- diol: (3fl, 5a, lia, 25R)-11,23-dibromo-3-acetoxyspirostan-12-one ( 20.00 g, azeotropically dried with toluene) was dissolved in THF

(600 ml) and cooled to -78°C. Lithium aluminum hydride (96.0 mL of 1.0 M THF solution) was slowly added and the resulting mixture stirred at -7 8°C for 2 hours and 0°C for 0.5 hours. Using a cannula, the mixture was carefully transferred to stirred 3 M aqueous ammonium chloride (200 mL).

The organic phase was separated, combined with THF washings of the solid residues, and concentrated to give the title compound.

(( 3/ 3. 5a . 11/ 3 . 12/ 3. 25R) - 23- bromo- ll. 12- epoxyvspirostan- 3-ol: The following method is a variation of the one described in Heiv. Act. Chim.. 1953, 36, 1241.

((3/3, 5a, 11a, 12/3, 25R)-11, 23-dibromospirostane-3,12-diol (18.08 g) was dissolved in pyridine (500 mL) at room temperature and treated with silver oxide (70 .0 g). The resulting mixture was stirred in the dark for 71 hours. The mixture was filtered and the solid washed with ether and then chloroform. These washings were combined with the filtrate and concentrated. The resulting solid was purified by flash chromatography (1 :1 hexane:ethyl acetate) to give 12.2 g of a 1:1 mixture of the title compound and (3/3,5a,25R)-23-bromo-spirostan-3-ol-12-one. Further chromatography (7: 3 hexane:ethyl acetate) gave the pure title compound.

( 3/ 3, 5a. 11/ 3. 12a. 25R) - 23- bromo- 12- ( trichloroacetoxy) spirostane-3. 11-diol: Using the method described in J. Chem. Soc. 1956, 4330, (3/3,5a,11/3,12/3,25R)-23-bromo-11,12-epoxyspirostan-3-ol was treated with trichloroacetic acid in toluene at room temperature for 3 days to give the title compound .

( 3/ 3 . 5a. 11/ 3. 12a. 25R) - 23- bromo- spirostane- 3 . 11. 12. triol: Using the method described in J. Chem. Soc, 1956, 4330, (3/3,5a,11/3,12a,25R)-23-bromo-12-(trichloroacetoxy)-spirostane-3,11-diol was saponified with sodium hydroxide in water and ethanol to give the title compound.

(3/ 3. 5a, 11/ 3. 12a. 25R) - spirostane-3, 11. 12-triol: Using the method described in J. Chem. Soc, 1956, 4330, (3/3, 5a, 11/3,12a, 25R)-23-bromo-12-(trichloroacetoxy)-spirostane-3,11-diol was reduced with zinc and acetic acid to give the title compound.

Preparation G2

( 30. 5a. 12a. 25R) spirostan- 3, 12- diol- ll-one

( 3/ 3. 5a. 11) 3, 12a. 25R) - 3 . 12-di (acetoxy) spirostan-ll-ol: Using the method described in J. Chem. Soc, 1956, 4330, (3/3, 5a, 110,12a, 25R)-spirostane-3,11,12-triol (preparation G1) was selectively acetylated with acetic anhydride and pyridine to give the title compound.

(3/ 3. 5a. 12a. 25R) - 3. 12-di (acetoxy) spirostan-ll-one: When using the method described in Org. Syn., 1976, 55, 84, (3/3, 5a, 11/S, 12a, 25R)-3,12-di(acetoxy)-spirostan-11-ol was oxidized with chromium trioxide and pyridine in methylene chloride to give the title connection.

( 3/ 3. 5a. 12a. 25R) - spirostane- 3 . 12-diol-ll-one: Using the procedure described in Syn., 1973, 790, (3/3,5a, 12/3, 25R)-3,12-di(acetoxy)spirostan-ll-one was saponified with potassium cyanide in water, methanol and THF to form the title compound.

Preparation G3

( 30. 5a. 110. 25R) spirostane- 3. 11- diol

(30.5a.110.25R)spirostan-3.11-diol: (30.5a.25R)spirostan-3-ol-11-one (Aldrich Chemical Companyl, Milwaukee, WI or Steraloids Inc., Wilton, N.H., or see preparation G13) was converted to the title compound via reduction with lithium aluminum hydride in THF at room temperature according to the method described in J. Am. Chem. Soc, 1951, 73, 1777.

Preparation G4

( 30. 5a. Ila. 25R) spirostane- 3. 11- diol

f 30. 5a. Ila. 25R) spirostan- 3 . 11-diol: (30,5a,.25R)spirostan-3-ol-11-one (Aldrich Chemical Companyl, Milwaukee, WI or Steraloids Inc, Wilton, N.H., or see preparation G13) was

converted to the title compound via reduction with lithium and ammonia according to the method described in J. Am. Chem. Soc, 1953, 75, 1282.

Preparation G5

( 38 . 5a . 11B . 12 B . 25R ) spirostane-3, 11. 12-triol

( 3B . 5a . 11B . 12B . 25R ) spirostan- 3. 11. 12- triol: (30,5a,120,25R)-3,12-di(acetoxy)spirostan-ll-one (purchased from Steraloids, Inc ., or see preparation G13) was converted to the title compound via reduction with lithium aluminum hydride in THF at room temperature according to the method described in J. Am. Chem. Soc, 1951, 73, 1777.

Preparation G6

( 30. 5a. Ila. 120. 25R) spirostane- 3. 11. 12- triol

(30.5a.120.25R) spirostan-3.12-diol-ll-one; (30,5a,120,25R)-3,12-di(acetoxy)spirostan-11-one (purchased from Steraloids, Inc, or see preparation G13) was saponified with potassium carbonate in water, methanol and THF to give the title compound.

(3B.5a.Ila.120.25R)spirostane-3.11.12-triol: (30.5a.120.25R)-spirostane-3,12-diol-11-one was converted to the title compound via reduction with lithium and ammonia according to the method described in J. Am. Chem. Soc, 1953, 75, 1282.

Preparation G7

( 30. 5a. 12a. 25R) spirostane- 3. 12- diol

(30.5a.12a.25R) spirostane-3.12-diol; Using the method described in J. Am. Chem. Soc, 1954, 76, 4013, (30,5a,25R)-spirostan-3-ol-12-one was reduced with lithium aluminum hydride in ether to give a mixture of C-12 alcohols, from which the title compound was isolated.

Preparation G8

( 3/ 9. 5a. 25R) - spirostane- 3- ol- ll. 12-dione

( 30 . 5a . 120 . 25R )-3-( t -butyldimethylsilyloxy) spirostan-12-ol-11-one: Using the method described in J. Am. Chem. Soc, 1972, 94, 6190, (3/9, 5a, 120, 25R)-spirostan-3,12-diol-11-one (see preparation G6) was silylated with t-butyldimethylchlorosilane and imidazole in DMF to give the title compound .

(30.5a.25R)-3-(t-butvldimethylsilvloxv)spirostane-11.12-dione: When using the method described in Org. Syn., 1976, 55, 84, (30,5a,120,25R)-3-(t-butyldimethylsilyloxy)-spirostan-12-ol-11-one was oxidized with chromium trioxide and pyridine in methylene chloride to give the title compound.

( 30, 5a. 25R)- spirostane- 3- ol- ll. 12-dione: Using the method described in J. Am. Chem. Soc, 1972, 94, 6190, (30,5a,25R)-3-(t-butyldimethylsilyloxy)spirostane-11,12-dione was desilylated with hydrofluoric acid in acetonitrile to give the title compound.

Preparation G9

( 30. 5a. 110. 25R) spirostan- 3. ll- diol- 12- one

(30.5a.110.120.25R)-3-(t-butyldimethylsilyloxy)spirostane-11. 12-diol: (30,5a,120,25R-3-(t-butyldimethylsilyloxy)-spirostan-12-ol-11-one (see method G8) was converted to the title compound via reduction with lithium aluminum hydride in THF at room temperature according to the method described in J. Am. Chem. Soc, 1951, 73, 1777.

(30.5a.110.120.25R)-3-(t-butyldimethylsilyloxy)-12-acetoxyspirostan-ll-ol: (30.5a,110,120,25R-3-(t-butyldimethylsilyloxy)spirostan-11,12- diol was selectively acetylated with acetic anhydride, pyridine and dimethylaminopyridine in methylene chloride to give the title compound.

(36.5a.110.120.25R)-3-(t-butyldimethylsilyloxy)-11-(trimethylsilyloxy)-12-acetoxyspirostane: (30.5a,110.120.25R)-3-(t-butyldimethylsilyloxy)- 12-acetoxyspirostan-11-ol was silylated with trimethylsilyl triflate and 2,6-lutidine in methylene chloride according to the procedure described in Tetrahedron Letters, 1981, 22, 3455.

(36.5a.116.126.25R)-3-(t-butyldimethylsilyloxy)-11-(trimethylsilyloxy)spirostan-12-ol: (30,5a,110,120,25R)-3-(t-butyldimethylsilyloxy) -11-(trimethylsilyloxy)-12-acetoxyspirostane was deacetylated by treatment with lithium aluminum hydride in THF followed by careful addition of aqueous ammonium chloride. The resulting title compound was subjected to 11 to 12 silyl group migration on silica gel, and thus had to be used crude.

(30,5a.110.25R)-3-(t-butyldimethylsilyloxy)-11-(trimethylsilyloxy)spirostan-12-one r (30.5a,110,120,25R)-3-(t-butyldimethylsilyloxy)-11 -(trimethylsilyloxy)spirostan-12-ol was oxidized with chromium trioxide and pyridine in methylene chloride according to the method described in Org. Syn., 1976, 55, 84 during formation of the title compound.

(30.5a.110.25R)-spirostane-3.ll-diol-12-one: The title compound synthesized from (30.5a.110.25R)-3-(t-butyldimethylsilyloxy)-11-(trimethylsilyloxy)spirostane -12-one was desilylated with hydrofluoric acid in acetonitrile according to the procedure described in J. Am. Chem. Soc, 1972, 94, 6190. The title compound must be handled carefully because it will rearrange to (30,5α,120,25R)-spirostan-3,12-diol-11-one if exposed to base.

Preparation G10

( 30. 5a. Ila. 2 5R) spirostan- 3. ll- diol- 12- one

(30.5a.Ila.120.25R) 3.11-di(acetoxy)spirostan-12-ol:

(30,5a,11a,120,25R)-spirostane-3,11,12-triol (see preparation G6)

was acetylated according to the method described in J. Am. Chem. Soc, 1955, 77, 1632 to form a mixture of acetates from which the title compound could be isolated.

( 30 , 5a . Ila . 25R) 3. 11-di(acetoxy)spirostan-12-one: (3/3, 5a, lia, 120, 25R) 3,11-di(acetoxy)spirostan-12-ol was oxidized with chromium trioxide and pyridine in methylene chloride according to the method described in Org. Syn., 1976, 55, 84 during formation of the title compound.

( 30. 5a. Ila. 25R) spirostan- 3. 11- diol- 12- one; (30,5a,11a,25R)-3,11-di(acetoxy)spirostan-12-one was saponified with sodium methoxide in methanol and THF to give the title compound.

Preparation Gil

( 30, 5a, lia. 12a. 25R) spirostane- 3. 11. 12- triol

( 36 , 5a . 25R ) spirostan-3-ol-12-tosylhydrazone: (30,5a,25R)-spirostan-3-ol-12-one (8.00 g) was dissolved in glacial acetic acid (200 mL) and heated to 50°C. Paratoluenesulfonylhydrazide (6.928 g) was added and the solution stirred at 50°C for 30 min. After a further 2 hours of stirring at room temperature, water (200 ml) was added. The resulting solid was collected, washed with water (100 mL), dried, triturated with refluxing acetone (300 mL), filtered hot and dried to give 3.903 g of the title compound.

(30,5a,25R)spirostan-ll-en-3-ol: A mixture of (30,5a,25R)-spirostan-3-ol-12-tosylhydrazone (9.100 g) and sodium methoxide (8.379 g) in DMF ( 200 ml) was heated to 150°C for 35 min., then cooled to room temperature. The mixture was then poured into ice water (1200 ml) and the resulting suspension filtered. The collected solid was washed with water (100 mL), air dried, and dissolved in methylene chloride (700 mL). This solution was washed with water (2 x 200 mL), dried with MgSO 4 , and concentrated to give a white solid. After

flash chromatography, 2.384 g of the title compound were isolated (m.p. 179-181°C, lit. 188-192°C - J. Am. Chem. Soc., 1954, 76 4013).

( 3/ 3. 5a . Ila. 12a, 25R) spirostan- 3 . 11. 12-triol: (3/3,5a,25R)-spirostan-11-en-3-ol was oxidized to the title compound with osmium tetroxide and N-methylmorpholine-N-oxide in water, t-butanol and acetone according to the procedure described in Tetrahedron Letters.1976, 1973.

Preparation G12

( 3/ 3. 5a. 12/ 3, 25R) spirostan- 3 . 12- diol- ll- one ( 3/ 3. 5a, 11/ 3. 25R) - ll- bromospirostan- 3- ol- 12- one: A glass-lined reactor was filled with 190 1 of methanol and then blown in below the surface with hydrochloric acid gas until 7.7 kg (5.0 eq.) was filled. After completion of this blow-in, the reactor was charged with 18.8 kg (42.2 mol) of (3/3, 5a, 25R) spirostan-3-ol-12-one, 190 L of methanol and 38 L of methylene chloride. This mixture was

cooled to 10°C, and a solution of 8.4 kg of bromine (52.7 mol, 1.25 eq.) in 38 L of methylene chloride was added over 2 hours while maintaining a vessel temperature of ca. 10°C. After the addition was complete, the reaction was allowed to warm to room temperature and stirred for 2 hours. TLC at this point indicated complete turnover.

The reaction was diluted with 190 L of water and stirred in

10 min. After separation of the layers, the aqueous layer was extracted twice with 114 1 methylene chloride. The three combined organic extracts were washed twice with 114 L of water, once with 114 L of saturated saline, then dried using 7.0 kg of magnesium sulfate. The desiccant was removed by filtration on a 30 inch Lapp followed by two 11 L methylene chloride washes. The combined filtrates and washings were atmospherically distilled to a total volume of 26 L. Twice 38 L of methanol were added, followed by continued distillation. When a final volume of <38 L was achieved, the mixture was cooled to room temperature. The resulting suspension was granulated for 2 hours, filtered on a 30-in

Patch, and the filter cake washed twice with 11 1 methanol. Vacuum drying of the filter cake at 45-50°C gave 12.6 kg (58.6% yield) of the title compound.

( 30 , 5a. 120 . 25R) spirostan- 3 . 12-diol-ll-one: A glass-lined reactor was charged with 12.4 kg of (30.5a,110.25R)-11-bromospirostan-3-ol-12-one (24.34 mol), 125 1 of t-butanol, 125 L of water and 7.5 kg (189 mol, 7.75 eq.) of sodium hydroxide pellets. The reaction was heated to reflux for 1.5 hours, held at reflux for 4.5 hours (vessel temperature was 83°C), then cooled to room temperature. TLC at this point indicated complete turnover.

The reaction was distilled to remove the t-butanol. This was achieved both by vacuum and atmospheric distillation. During the concentration, two 123 1 portions of water were added. Once the t-butanol was removed, the aqueous suspension was cooled to room temperature and granulated for two hours. The suspension was filtered on a 30 inch pad, washed twice with 11 L of water, and the filter cake was air dried at 60°C. This gave 11.1 kg of the title compound.

Preparation G13

( 30. 5a. 25R) spirostan- 3- ol- ll- one

(30.5a,120.25R)-3.12-diacetoxspirostan-ll-one: A glass-lined reactor was filled with 98 L of pyridine, 98 L of acetic anhydride and 11.0 kg of (30.5a,120.25R)spirostan- 3,12-diol-11-one (preparation G12). This mixture was refluxed for 2 hours (vessel temperature 128°C) and allowed to cool to room temperature. The reaction was vacuum distilled to a total volume of 57 1 (container temperature approx. 45°C during distillation). The suspension was diluted with 95 1 acetic acid and further vacuum distilled to a total volume of 57 1 (container temperature at the end approx. 80°C). The mixture was diluted with 330 L of water and cooled to room temperature. After 5 hours of granulation, the title compound was isolated by filtration on a 30 inch Lapp followed by two washings with 11 L of water.

The filter cake was dried at 60°C under vacuum with a yield of 12.2 kg (93.3%).

( 3/ 3, 5Q. 25R) spirostan-3-ol-11-one: A stainless steel reactor was cooled to -80°C by passing liquid nitrogen through the inner coils. Ammonia was added to the reactor until 54.5 kg (80 L, 3,200 mol, 170 eq.) was filled.

At the same time as the ammonia charge was begun, a glass-lined reactor was charged with 10.0 kg of (3/3, 5a, 125, 25R)-3,12-diacetoxyspirostan-11-one (18.84 mol) and 151 L of THF. This solution was atmospherically distilled until a total volume of 98 L had been obtained.

At the end of the ammonia charge, 2.8 kg of calcium shavings (69.0 g atoms, 3.7 eq.) were added over 30 min. while the reactor temperature was maintained at -50°C. At the end of this addition, the THF solution of (3/3,5a,12/3,25R)-3,12-di-acetoxyspirostan-11-one was added over 20 min. (reactor temperature at the end of the addition was -35°C) followed by a rinse with 3.8 L of THF. The reaction mixture was stirred for 30 min. at -35"C to -40"C. While the reaction was at -35°C to -40°C, 3.33 L of bromobenzene (4.98 kg, 31.7 mol, 1.68 eq.) was added followed by 3.33 L of water.

After this addition, the distillation of ammonia from the reactor was started. This distillation was directed to a water scrubber. When all the ammonia was removed, the reaction (now at 24°C) was transferred to a glass-lined reactor followed by a rinse with 15 L of THF. The combined solution and rinse was vacuum distilled to a thick oil. To this was added 132 1 of methanol and 3.3 kg (59 mol) of potassium hydroxide pellets. This mixture was heated at reflux for 1 hour, cooled, then 10 L of acetic acid and 167 L of water were added. This suspension was further cooled to room temperature and granulated for 1 hour. The title compound was isolated by filtration on a 30 inch Lapp followed by washing with 19 L of 3:1 water:methanol mixture. Vacuum drying at 55°C gave 7.05 kg (86.9%).

Preparation G: Physical data

Satisfactory MS and IR data were obtained on all of the (30,5a,25R)spirostan-3-ol compounds described in Preparation G (see Table 1). The different diol and triol products could be separated from each other by proton NMR (see Table 2.)

1 - IR data suggest that this compound readily tautomerizes enol ketone forms in CHC13. 2 - All samples run in CDC13 except for 11/3-ol-12-one which was run in DMSO-d6. Peaks for H16, H3f H26eq and H26axer also observed at >2ppm. In CDC13 these are observed at 4.37 (ddd, J = 9.9 & 7 Hz, 1H), 3.56 (heptet, J = 4 Hz, 1H), 3.45 (ddd, J = 10.6 & 2 Hz , 1H), 3.35 (t, J = 11 Hz, 1H) .

Preparation Hl

( 5a. 25R)- spirostan- 3-one

Pyridinium chlorochromate (PCC) was added to a mixture of tigogenin (50.00 g, 120.0 mmol), celite (160 g), in CH 2 Cl 2

(1000 ml) at 0°C. The reaction was allowed to come to ambient temperature and was stirred for 5 hours. The reaction was diluted with 4000 mL of Et 2 O and filtered through a plug of silica gel. The plug was washed with an additional 6000 mL of Et 2 O. The filtrate was concentrated in vacuo to give 45.00 g of the title compound (90.4%).

<J>HNMR (250 MHz, CDCl 3 ) d 4.38 (q, J = 7Hz, 1H); 3.40 (m, 2H); 2.20-2.45 (m, 3H); 0.70-2.14 (m, 36H); 1.02 (s, 3H);

0.96 (d, J = 7 Hz, 3H); 0.76 (s, 3H); 0.76 (d, J = 7Hz, 3H). MS: 415 (M + H)<+>; S.mp. 209-211°C.

Preparation H2

( 2a. 5a. 25R)- 2- bromospirostan- 3- one

A mixture of (5α,25R)-spirostan-3-one (1.00 g,

2.41 mmol) and tetrahydrofuran (10 mL) were cooled to -78°C under a nitrogen atmosphere. Bromine (0.39 g, 2.41 mmol) was added and the reaction mixture gradually warmed to room temperature. After 3 hours the reaction was suppressed by the addition of

saturated sodium hydrogen sulphite solution. The mixture was diluted with ethyl acetate, washed with saturated sodium hydrogensulfite solution (1x), saturated sodium hydrogencarbonate (1x), brine (1x), dried (sodium sulfate), filtered, and concentrated in vacuo. After addition of ether, a precipitate formed which was filtered off and washed with hexanes to give 1.20 g (85%) of the title compound.

<1>HNMR (250 MHZ, CDCl 3 ) d 4.75 (q, J = 7Hz, 1H); 4.40 (q,

J = 7Hz, 1H) ; 3.40 (m, 2H); 2.64 (q, J=6, 1H); 2.40 (m, 2H); 0.70 - 2.55 (m, 34H); 1.10 (s, 3H); 0.96 (d, J = 7Hz, 3H); 0.80 (s, 3H); 0.80 (d, J = 7, 3H). MS 493 (M+

H)<+>.

Preparation H3

( 5a. 25R)- spirost- 1- en- 3- one

A mixture of lithium bromide (0.700 g, 8.06 mmol), lithium carbonate (1.20 g, 16.24 mmol) and anhydrous N,N-dimethylformamide (30 mL) was heated under a nitrogen atmosphere to 95°C. To this mixture was added (2a,5a,25R)-2-bromo-spirostan-3-one (4.00 g, 8.11 mmol). The reaction mixture was stirred at 130°C for 3 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water (3x), brine (1x), dried (sodium sulfate), filtered and concentrated in vacuo to give 3.31 g, (98%) of the title compound.

^NMR (250 MHz, CDCl 3 ) d 7.10 (d, J = 10Hz, 1H), 5.85 (d, J = 10Hz, 1H); 4.40 (q(J = 7Hz, 1H); 3.40 (m, 2H); 2.30 (m, 2H); 0.70-2.05 (m, 33H); 1.02 (s, 3H); 0.96 (d, J =

7Hz, 3H); 0.80 (s, 3H); 0.78 (d, J=7Hz, 3H). MS 413 (M

+ H)<+>.

Preparation H4

( la . 2a . 5a , 25R )- 1. 2- epoxy- spirostan- 3- one

A mixture of (5α,25R)-spirost-1-en-3-one (2.87 g,

6.96 mmol), tetrahydrofuran (30 mL), methanol (50 mL) and 15% sodium hydroxide (1 mL) were stirred under a nitrogen atmosphere. The mixture was cooled to 0°C and 30% hydrogen peroxide (5 mL) was added. The reaction mixture was gradually warmed to room temperature and stirred for 4 hours. The reaction was diluted with ethyl acetate, cooled to 0°C, and then quenched with saturated sodium hydrogen sulfite solution. The mixture was washed with saturated sodium bisulfite (2x), brine (1x), (dried sodium sulfate), filtered and concentrated in vacuo to give 2.64 g (88%) of the title compound.

<1>HNMR (250 MHz, CDCl 3 ) d 4.40 (q, J = 7Hz, 1H); 3.40 (m, 3H), 3.24 (d, J = 6Hz, 1H); 2.25 (dd, J = 18.4 Hz, 1H);

0.70-2.28 (m, 34H); 0.98 (d, J = 7Hz, 3H); 0.92 (s, 3H);

0.80 (s, 3H); 0.78 (d, J = 7Hz, 3H). MS 429 (M + H)<+>.

Preparation H5

(la,. 36 f 5a. 25R)- 1. 3- di(hvdroxy) spirostane

Lithium aluminum hydride (0.43 g, 15.38 mmol) was added to a solution of (1α,2α,5α,25R)-1,2-epoxy-spirostan-3-one in THF (20 mL) at 0 °C . The reaction was gradually warmed to room temperature and after 3 hours additional lithium aluminum hydride (0.10 g, 3.58 mmol) was added. After 1 hour, the reaction was cooled to 0°C and quenched by sequential addition of H 2 O (0.75 mL), 15% NaOH (0.75 mL), and H 2 O (1.50 mL). The mixture was dried with MgSO 4 , filtered, and concentrated in vacuo. The product was purified by flash chromatography (50% EtOAc/50% hexane to 95% EtOAc/5% MeOH) to give 0.460 g, (34%) of the title compound.

<:>HNMR (250 MHZ, CDCl 3 ) d 4.48 (q, J = 7Hz, 1H); 4.04 (m, 1H); 3.80 (m, 1H); 3.40 (m, 2H); 0.75-2.05 (m, 37H); 0.96 (d, J = 6Hz, 4H); 0.84 (s, 3H); 0.78 (d, J=6Hz, 3H); 0.76 (p, 3H). MS 433 (M+H)<+>.

Preparation II

(30, 5a. 25R)-3-f(heptaacetvl-Ø-D-cellobiosyl)oxy]spirostan-1-one

Pyridinium chlorochromate (0.123 g, 0.57 mmol) was added to a mixture of (1a,30,5a,25R)-3-[(heptaacetyl-Ø-D-cellobiosyl)oxy]-1-hydroxyspirostane (0, 2 g, 0.19 mmol, see preparation B39), celite (0.2 g), in CH 2 Cl 2 (5 mL) at 0°C. The reaction was allowed to come to ambient temperature and was stirred for 2 hours. The reaction was diluted with 15 mL of Et 2 O and filtered through a plug of silica gel. The plug was washed with an additional 500 mL of Et 2 O. The filtrate was concentrated in vacuo to give 0.18 g of the title compound (90%).

<1>HNMR (250 MHZ, CDCl 3 ) d 5.15 (m, 3H); 4.90 (m, 2H); 4.50 (m, 2H); 4.35 (m, 2H); 4.05 (m, 2H); 3.65 (m, 3H); 3.40 (m, 2H); 2.35 (t, J = 12.5 Hz, 1H); 2.60 (q, J = 6 Hz, 1H); 1.95-2.20 (m, 21H); 0.70-1.90 (m, 37H); 1.15 (s, 3H); 0.95 (d, J = 7 Hz, 3H); 0.80 (d, J = 6 Hz, 3H); 0.76 (p, 3H). MS: 1049 (M + H)<+>.

Preparation Jl

( 30, 25R)- 3- ethoxymethoxy- 5- spirostene

A mixture of diosgenin (2.5 g, 6.0 mmol), chloromethyl ethyl ether (1.14 g, 12.0 mmol), diisopropylethylamine (3.90 g, 30.0 mmol) and 1,2-dichloroethane ( 75 ml) was stirred under a nitrogen atmosphere at ambient temperature for 4 hours. Methanol (<1 mL) was added to suppress the reaction. The mixture was diluted with ethyl acetate and washed with water (2x), brine (1x), dried over sodium sulfate, filtered and concentrated in vacuo to give 2.17 g (76.5%) of the title compound as a colorless solid.

<X>H NMR (250 M Hz, CDCl 3 ) d 5.35 (d, 2H, 3=1.0 Hz); 4.75 (s, 2H); 4.4 (m, 1H); 3.6 (q, 2H, J=7.0Hz); 3.4 (m, 2H);

3.35 (t, 1H, J=11 HZ); 2.4-0.7 (m, 38H); 1.2 (s, 3H);

0.95 (d, 3H, J=7Hz); 0.8 (d, 3H, 3=1Hz); 0.75 (s, 3H). MS: 777 (M+Na)<+>; m.p. 125-127°C.

Production 32

(3) 3, 5a. 6a. 25R) - 3- ethoxymethoxys- 6- hydroxyspirostane

Borane-tetrahydrofuran complex (0.68 mL, 0.68 mmol) was added to a solution of (30,25R)-3-ethoxymethoxy-5-spirostene (0.10 g, 0.21 mmol) in tetrahydrofuran (8 mL) . The mixture was stirred under a nitrogen atmosphere at ambient temperature for 3.5 hours. The reaction mixture was cooled to 0°C and methanol (1.5 mL), 15% sodium hydroxide solution (1.5 mL) and 30% hydrogen peroxide (1.5 mL) were added. The reaction mixture was then gradually warmed to ambient temperature and stirred overnight. The reaction was quenched at 0'C by addition of saturated sodium hydrogen sulphite solution. The mixture was diluted with ethyl acetate and washed with ammonium chloride solution (1x), brine (1x), dried (sodium sulfate), filtered and concentrated in vacuo to give 0.11 g of a mixture of the 6a-alcohol and the 6/3-alcohol. The two products were separated by flash chromatography on silica gel (6:4 hexane:ethyl acetate). The major product (Rf=0.40) was identified to be the title compound.

<*>H NMR (250 MHz; CDCl 3 ) d 4.7 (s, 2H); 4.4 (m, 1H); 3.6 (q, 2H, J=11.0Hz); 3.45 (m, 3H); 3.35 (t, 1H, J=11.0Hz); 2.3-0.6 (m, 41H); 1.8 (d, 3H, 3=1, 0 Hz); 0.82 (s, 3H), 0.78 (d, 3H, J=7.0 Hz); 0.75 (s, 3H). MS: 491 (M+H)<+>; m.p. 171°C.

Preparation J3

( 30 , 5a . 25R )- 3- ethoxymethoxv- spirostan- 6- one

Pyridinium chlorochromate (1.98 g, 9.20 mmol) was added to a mixture of (30,5α,6α,25R)-3-ethoxymethoxy-6-hydroxyspiro stane (0.90 g, 1.8 mmol) and celite ( 8.0 g) in anhydrous dichloromethane at 0°C. The reaction mixture was gradually warmed to ambient temperature over 1 hour and was stirred for an additional 5 hours. The reaction mixture was then filtered through a plug of silica gel using ether as eluent. The combined ether fractions were concentrated in vacuo to give 0.80 g (91%) of the title compound as a colorless solid.

<X>HNMR (250 MHz; CDCl 3 ) d 4.75 (m, 2H); 4.4 (m, 1H); 3.6 (q, 2H; J=7.0 Hz); 3.45 (m, 2H); 3.35 (t, 1H; J=11.0Hz) ;

2.4-0.6 (m, 40H); 0.9 (d, 3H, J=7.0 Hz); 0.8 (d, 3H, J=7.0 Hz); 0.78 (p, 6H). MS: 489.0 (MH)<+>; m.p. 191-193°C.

Preparation J4

( 3/ 3, 5a. 25R) - spirostan- 6- one

Concentrated hydrochloric acid (2 drops) was added to a solution of (3(3,5a,25R)-3-ethoxymethoxy-spirostan-6-one (0.70 g, 1.43 mmol) in methanol (10 mL) and tetrahydrofuran (10 mL). The mixture was stirred under a nitrogen atmosphere and heated to 62° C. After 15 min, the reaction was cooled to 0° C. and neutralized with 15% sodium hydroxide solution. The mixture was concentrated in vacuo and then diluted with ethyl acetate. The organic layer was washed with water (2x), brine (1x), dried (sodium sulfate), concentrated in vacuo and purified by flash chromatography (1:1 hexane:ethyl acetate) to give 0.55 g (89.4%) of the title compound as a colorless solid.

<*>H NMR (250 MHz; CDCl 3 ) d 4.4 (m, 1H); 3.45 (m, 2H); 3.35 (t, 1H, J=11.0 (Hz); 2.35-0.6 (m, 38H); 0.95 (d, 3H, J=7.0Hz); 0.75 (d , 3H, J=7.0); 0.71 (s, 6H), MS: 431 (M+H) + ; mp 210-212°C.

Preparation At

(30.5a.6a.25R)-3-r(heptaacetvl-Ø-D-cellobiosyl)oxy 1-6-hydroxyspirostane

Sodium borohydride (0.11 g, 2.86 mmol) was added to a solution of (3/3, 5a, 25R)-3-[(heptaacetyl-Ø-D-cellobiosyl)oxy]-spirostan-6-one (1 .5 g, 1.43 mmol, see preparation B41) in ethanol (20 ml) and dichloromethane (5 ml), and the mixture was stirred under a nitrogen atmosphere at room temperature for 4 hours. The reaction mixture was then cooled to 0°C and neutralized with 1N hydrochloric acid. The mixture was partially concentrated in vacuo and then diluted with ethyl acetate, washed with 1N hydrochloric acid (lx), brine (lx), dried (sodium sulfate), filtered and concentrated in vacuo to give 1.00 g (66%) of the title compound as a colorless solid.

<*>H NMR (250 MHz; CDCl 3 ) d 5.2-4.4 (m, 14H), 4.3 (m, 1H);

3.75-3.35 (m, 3H); 3.3 (t, 1H, J=11.0Hz); 2.15-0.5 (m, 59H); 0.95 (s, 3H); 0.90 (d, 3H, J=7, 0 Hz); 0.75 (s, 3H);

0.70 (d, 3H, J=7.0 Hz). MS: 1051 (M+H)<+>.

It is to be understood that the invention is not limited to the particular embodiments shown and described herein, but that various changes and modifications can be made without deviating from the spirit and scope of this new concept as defined by the following claims.

Claims (63)

1. Spirostanyl glycoside, characterized by formula IA where either (A): Q<1> is carbonyl Q<2> is carbonyl, methylene, Q <3> is R ^-al k <y>l en (C2 - <C>3 ) or R<1>0 -alkylene(C2 - <C>3 ; Q <*> and Q <5> are both methylene; and where R <1> is / 3-D-glucopyranosyl, Æ-D-glucopyranuronosyl,/ 3-D-2-acetamido-2-deoxy-glucopyranosyl,/ 3-D-galactopyranosyl, /3-D-fucopyranosyl,/ 3-L-fucopyranosyl, /3-D-xylopyranosyl, /3-L-xylopyranosyl α-D-arabanopyranosyl, α-L-arabanopyranosyl, α-D-cellobiosyl, /3-D-cellobiosyl, /3-D-lactosyl,/ 3-D-maltosyl, /3-D-gentiobiosyl, 3-0-/ 3-D-galactopyranosyl-α-D-arabanopyranosyl or Æ-D-maltotriosyl; or (B): Q1 , Q<A> and Q<5> are all methylene; R ^-al k <y>l one (C2 -C3 ) or R<1>0 -alkylene(C2 - <C>3 ) and where R <1> is Ø-D-glucopyranosyl, /3-D-glucopyranuronosyl,/ 3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-f ukopyranosyl, 0-L-fucopyranosyl, / 3-D-xylopyranosyl, /3-L-xylopyranosyl α-D-arabanopyranosyl, α-L-arabanopyranosyl, /3-D-cellobiosyl, /3-D-lactosyl, /3-D-maltosyl, /3-D-gentiobiosyl, 3-O-/3-D-galactopyranosyl-α-D-arabanopyranosyl or α-D-maltotriosyl; or (C): Q1 , Q<4> and Q <5> are all methylene; Q 2 is carbonyl; Q <3> is R<1>0 -alkylene(C2 -C3 ) or R<1>0 -alkylene(C2 -C3 ) C 25 is (R); and where R <1> is /3-D-glucopyranuronosyl,/ 3-D-2-acetamido-2-deoxy-glucopyranosyl, Æ-D-fucopyranosyl, /9-L-fucopyranosyl, /3-D-xylopyranosyl, /3-L-xylopyranosyl a-D- arabanopyranosyl, α-L-arabanopyranosyl, Æ-D-cellobiosyl, /3-D-lactosyl, /3-D-maltosyl, /3-D-gentiobiosyl, 3-O-/3-D-galactopyranosyl-α-D-arabanopyranosyl or /3-D-maltotriosyl; or (D): Q <1> , Q <2> / Q u and Q <5> are all methylene; andQ<3> R<1>0 -alkylene(C2 - <C>3 ) or R<1> 0-alkylene(C2 - <C>3 ) and where R<1> is /3-D-2-acetamido-2-deoxy-glucopyranosyl, Æ-D-xylopyranosyl, / 3-L-xylopyranosyl α-L-arabanopyranosyl, Æ-D-cellobiosyl, 3-O-β-D-galactopyranosyl-α-D-arabanopyranosyl or β-D-maltotriosyl; or (E): Q<1> , Q<2> and Q<5> are all methylene; Q<4> is carbonyl or Q<3> C5 is alpha; C 25 is (R); and where R <1> is Ø-D-galactopyranosyl, /3-D-cellobiosyl,/ 9-D-lactosyl, /3-D-maltosyl or/ 3-D-maltotriosyl; or (F): Q <1> , Q <2> and Q 4 are all methylene; Q<5> is carbonyl or C5 is alpha; C 25 is (R); and where R <1> is /3-D-galactopyranosyl, /3-D-cellobiosyl, / 3-D-lactosyl, /3-D-maltosyl or /3-D-maltotriosyl; provided that (3/3,5a,25R)-3-[(/3-D-cellobiosyl)oxy]spirostane is not included. 2. Connection according to claim 1, characterized in that Q <1> is carbonyl, Q<2> ,Q<4> andQ<5> are all methylene, Q <3> is The Cg hydrogen is alpha and C25 is (R). 3. Connection according to claim 2, characterized in that Q<1> is carbonyl and R<1> is /3-D-cellobiosyl. 4. Connection according to claim 2, characterized in that Q <1> is carbonyl and R <1> is /3-D-galactopyranosyl. 5. Connection according to claim 2, characterized in that Q<1> is carbonyl and R<1> is α-D-cellobiosyl. 6. Connection according to claim 2, characterized in that Q <1> is carbonyl and R <1> is Æ-D-glucopyranosyl. 7. Connection according to claim 2, characterized in that Q <1> is carbonyl and R <1> is /3-D-lactosyl. 8. Connection according to claim 2, characterized in that Q <1> is carbonyl and R <1> is/ 3-D-maltosyl. 9. Connection according to claim 2, characterized in that Q <1> is carbonyl and R <1> is/ 3-D-maltotriosyl. 10. Connection according to claim 2, characterized in that 0.1 is and R<1> is /3-D-cellobiosyl. 11. Connection according to claim 2, characterized in that Q <1> is and R<1> is/ 3-D-cellobiosyl. 12. Compound according to claim 1, characterized in that C1/Q4°gQ5 are all the methylene, q <2> is , the C5 hydrogen is alpha and the C25 is (R). 13. Connection according to claim 12, characterized by thatQ<2> is and r! is /3-D- <c>ellobiosyl. 14. Connection according to claim 1, characterized in that Q <1> is carbonyl, q2 is carbonyl, Q <3> is Q<4> and Q<5> are all methylene, C25 is (R), and the C5 hydrogen is alpha. 15. Connection according to claim 14, characterized in that Q <1> is carbonyl, Q <2> is carbonyl and R <1> is/ 3-D-cellobiosyl. 16. Connection according to claim 14, characterized in that Q1 is carbonyl, Q<2> is and R<1> is/ 3-D-cellobiosyl. 17. Connection according to claim 14, characterized in that Q <1> is carbonyl, Q <2> is and R<1> is/ 3-D-lactosyl. 18. Connection according to claim 14, characterized in that Q <1> is Q <2> is carbonyl and R <1> is /9-D-cellobiosyl. 19. Connection according to claim 14, characterized in that Q <1> is , Q<2> is carbonyl and R<1> is /3-D-cellobiosyl. 20. Compound according to claim 1, characterized in that Q <1> , Q <4> and Q<5> are all methylene, Q <2> is carbonyl, Q <3> is The c5 hydrogen is alpha and C25 is (R). 21. Compound according to claim 20, characterized in that R <1> is/ Ø-D-lactosyl. 22. Compound according to claim 20, characterized in that R<1> is/ 3-D-cellobiosyl. 23. Connection according to claim 1, characterized in that Q <1> and Q <2> ,Q<4> and Q <5> all is the methylene, Q <3> is and C25 is (R). 24. Connection according to claim 23, characterized in that the C5 hydrogen is beta and R<1> is /3-D-cellobiosyl. 25. Compound according to claim 1, characterized in that Q <1,> Q <2> and Q <5> all is the methylene, Q <3> is Q <4> is carbonyl, the C5 hydrogen is alpha and C25 is (R). 26. Compound according to claim 25, characterized in that R<1> is/ 3-D-cellobiosyl. 27. Connection according to claim 1, characterized in that Q <1> , Q <2> and Q <4> all is the methylene, Q<3> is Q 5 is carbonyl, the C5 hydrogen is alpha and C25 is (R). 28. Connection according to claim 27, characterized in that R<1> is Æ-D-cellobiosyl. 29. Method for controlling hypercholesterolemia or atherosclerosis in a mammal, characterized by administering to a mammal suffering from hypercholesterolemia or atherosclerosis a hypercholesterolemia or atherosclerosis controlling amount of a spirostanyl glycoside of formula where either (A): Q<1> is methylene, carbonyl Q<2> is carbonyl, methylene, Q <3> is R<1>0 -alkylene(C2 - <C>3 ) or <R1> 0-alkylene(C2 -C3 ) Q<4> and Q<5> are both methylene; and where R <1> is /3-D-glucopyranosyl, /3-D-glucopyranuronosyl, /3-D-2-acetamido-2-deoxy-glucopyranosyl, /3-D-galactopyranosyl, /3-D-fucopyranosyl, 0- L-fucopyranosyl, /3-D-xylopyranosyl, /3-L-xylopyranosyl a-D-arabanopyranosyl, a-L-arabanopyranosyl, a-D-cellobiosyl,/ 3-D-cellobiosyl,/ 3-D-lactosyl, /3-D- maltosyl, /3-D-gentiobiosyl, 3-O-/3-D-galactopyranosyl-α-D-arabanopyranosyl or /3-D-maltotriosyl; or (B): Q <1> , Q <z> and Q <5> are all methylene; Q <3> is Q<4> is carbonyl or C5 is alpha; C 25 is (R); and where R<1> is /3-D-galactopyranosyl,/ 3-D-cellobiosyl,/ 3-D-lactosyl, /3-D-maltosyl or /3-D-maltotriosyl; or (C): Q<1> , Q<2> and Q<4> are all methylene; Q <3> is Q<5> is carbonyl or C5 is alpha; C 25 is (R); and where; R <1> is / 3-D-galactopyranosyl, /3-D-cellobiosyl, /3-D-lactosyl, / 3-D-maltosyl or / 3-D-maltotriosyl; under the condition that (3/3, 5a, 2 5R) -3- [ (α-D-cellobiosyl) oxy] spirostane, (3/3, 5a, 25R) -3- [ (/3-D-glucopyranosyl) oxy] spirostane, (3 /3, 5a, 25R)-3-[(/3-D-cellobiosyl)oxy]spirostane or (3/3, 5a, 25R)-3-[(/3-D-galactopyranosyl)oxy]spirostan-12-one is not included. 30. Method according to claim 29, wherein Q1, Q<2>, Q<*> and Q<5> all are methylene, C25 is (R) and Q<3> is 31. Method according to claim 30, in which the C5 hydrogen is beta and R<1> is /3-D-cellobiosyl. 32. Method according to claim 30, wherein the C5 hydrogen is alpha and R<1> is /3-D-glucopyranuronosyl. 33. Method according to claim 30, in which the C5 hydrogen is alpha and R<1> is /3-D-maltosyl. 34. Method according to claim 30, wherein the C5 hydrogen is alpha and R<1> is /3-D-lactosyl. 35. Method according to claim 30, wherein the C5 hydrogen is alpha and R<1> is /3-D-gentiobiosyl. 36. Method according to claim 30, in which the C5 hydrogen is alpha and R<1> is/3-D-galactopyranosyl. 37. Method according to claim 29, wherein Q <1> is carbonyl, Q<2> ,Q<4> and Q <5> are all methylene, Q <3> is C25 is (R) and the C5 hydrogen is alpha. 38. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-cellobiosyl. 39. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-galactopyranosyl. 40. The method of claim 37, wherein Q<1> is carbonyl and R<1> is Qf-D-cellobiosyl. 41. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-glucopyranosyl. 42. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-maltosyl. 43. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-maltotriosyl. 44. Method according to claim 37, wherein Q<1> is carbonyl and R<1> is/3-D-lactosyl. 45. Method according to claim 37, wherein Q is <1> and R<1> is/ 3-D-cellobiosyl. 46. Method according to claim 37, wherein Q is <1> and R<1> is /3-D-cellobiosyl. 47. Method according to claim 29, wherein Q <1> , Q <*> and Q <5> is methylene, Q<2> is carbonyl, or Q <3> is c25 is (R) °9 The C5 hydrogen is alpha. 48. Method according to claim 47, wherein Q <2> is carbonyl and R <1> is/3-D-cellobiosyl. 49. Method according to claim 47, wherein Q<2> is carbonyl and R<1> is/3-D-lactosyl. 50. Method according to claim 47, wherein Q is <2> and R<1> is /3-D-cellobiosyl. 51. Method according to claim 47, wherein Q2 is and R<1> is/ 3-D-galactopyranosyl. 52. The method according to claim 29, wherein Q is <1> carbonyl, Q<2> is carbonyl, or Q<3> is Q<4> and Q<5> are both methylene, <C>25 is (R), and the C5 hydrogen is alpha. 53. The method of claim 52, wherein Q<1> is carbonyl, Q<2> is carbonyl and R<1> is /3-D-cellobiosyl. 54. Method according to claim 52, wherein Q is <1> carbonyl, Q <2> is and R<1> is /3-D-cellobiosyl. 55. Method according to claim 52, wherein Q is <1> carbonyl,Q<2> is and R<1> is/ 3-D-lactosyl. 56. Method according to claim 52, wherein Q is <1> °-2 is carbonyl and R<1> is /3-D-cellobiosyl. 57. Method according to claim 52, wherein Q is <1> Q<2> is carbonyl and R<1> is/3-D-cellobiosyl. 58. Method according to claim 29, wherein Q <1> , Q <z> and Q <5> all are methylene, Q <3> is ten ,Q<4> is carbonyl, the C5 hydrogen is alpha and C25 is (R). 59 . The method of claim 58, wherein R<1> is /3-D-cellobiosyl. 60. Method according to claim 29, wherein Q <1> , Q <2> and Q <A> all are methylene, Q3 is Q<5> is carbonyl, the C5 hydrogen is alpha and C25 is (R ). 61. Method according to claim 60, wherein R<1> is Æ-D-cellobiosyl. 62. Pharmaceutical preparation for the control of hypercholesterolemia or atherosclerosis in mammals, characterized by a compound according to claim 1 and a pharmaceutically acceptable carrier. 63. Preparation, characterized by a hydrate of a compound according to claim 1.