CA2079543A1 - Process for making cholesterol lowering saponins - Google Patents

Abstract

PROCESS FOR MAKING CHOLESTEROL LOWERING SAPONINS
ABSTRACT

Disclosed is a process for preparing novel compounds containing 5-C-hydroxymethylhexose and a sterol or triterpene. These materi-als, when consumed by humans and animals, lower the cholesterol level in the blood. The compositions are prepared by preparing and then reacting a fluoro or bromo hexaacylated 5-C-hydroxymethylhexose with a sterol. In a second method the hexaacylated 5-C-hydroxymethylhexose is reacted with a sterol in the presence of trimethylsilyl trifluoromethane sulfonate.

Description

~ L ~

2079~3 PROCESS FOR MAKING CHOLESTEROL LOWERING SAPONINS

Stanislaw Pikul Adam W. Mazur TECHNICAL FIELD
This invention relates to a process for m~king novel 5-C-hydroxymethyl hexose- derivatives of sterols. These compounds are used to lower both serum cholesterol and serum triglyceride levels in animals and humans.
BACKG~OUND OF THE INVENTION
Saponins are a type of glycoside found in nature. A saponin is composed of a sapogenin and a sugar. The sugar can be a monosaccha-ride or an oligosaccharide. The sapogenin is a steroid or a triterpene.
The saponins found in soybeans, alfalfa, and ginseng have been studied extensively for their effect of lowering cholesterol. Both the animal's ability to absorb cholesterol as well as its level of serum cholesterol are reduced.
U.S. 4,602,003 issued to Malinow (1986) describes synthetic sapogenin and sterol compounds which inhibit the absorption of cholesterol and are used to treat hypercholesterolemia. These compounds are synthetic glycosides of tigogenin, diosgenin, smilagenin, and the like. Cellobiose-tigogenin and cellobiose-diosgenin were also made as were the ester derivatives.
U.S. 4,602,005 issued to Malinow (1986) is related to the 4,602,003 patent. Tigogenin cellobioside is described as being particularly effective for treating hypercholesterolemia and atherosclerosis.
While it is well known that saponins have a cholesterol lower-ing benefit, it is also well known that these materials hydrolyze in the digestive system. When the sugar moiety is removed, i.e., the glycosidic linkage is cleaved, the cholesterol is no longer removed.
Therefore, a saponin derivative which does not hydrolyze in the

-2- 2079~3 stomach or intestine would be highly des;rable. Such a compound could even be derived from cholesterol.
Surprisingly it has been found that derivatizing a sterol with a 5-C-hydroxymethyl substituted sugar provides a saponin derivative which is not hydrolyzed in the stomach or intestine, but still functions to lower cholesterol and serum triglycerides. The 5-C-hydroxymethyl sugars are the subject of U.S. 5,041,541 (1991).
It is an object of this invention to provide a process for making these novel saponins. It is a further object of this inven-tion to provide these novel compounds in an economical and efficienc process. These and other objects will be evident from the discus-sion herein.
All percentages are by weight unless otherwise indicated.
SUMMARY OF THE INVENTION
The novel saponins of the present invention encompass 5-C-hydroxymethylhexose mono-, di- or trisaccharides which are bonded to sterols through a glycosidic linkage. These 5-C-hydroxymethyl saponins are prepared by the following a process comprising the steps of:
(1) acylating a 5-C-hydroxymethyl hexose in a two step acylation comprising:
(a) reacting an acid anhydride with said hexose in the presence of a base;
(b) reacting the product of step (a) with an acid anhydride and a catalytic amount of a strong acid; and (2) reacting the acylated hexose with a fluoride in the pres-ence of boron trifluoride etherate, reacting the acylated hexose with a bromide in the presence of mercuric cyanide and then reacting the fluoro or bromo derivative with the sterol.
Alternatively, step (2) involves a reaction of the acylated hydroxymethyl hexose with the sterol under anhydrous conditions in the presence of trimethylsilyl trifluoromethane sulfonate.
The acyl groups are hydrolyzed in the presences of an alkali metal alcoholate catalyst to yield the sterol-5-C-hydroxymethyl arabinohexopyranoside.

3 2~79~43 The sterols are selected from the group of spirostanols such as diosgenin and tigogenin, as well as others, including, cholesterol, ~-sitosterol, ~-sitosterol, stigmasterol, sitostanol, ergosterol and campesterol.
DETAILED DESCRIPTION OF THE INVENTION
Definitions The term "comprising" as used herein encompasses the terms "consisting of" and "consisting essentially of".
The terms "novel sa)onins" and "hydroxymethyl saponins" as used herein refer to the 5-C-hydroxymethyl derivatives of the hexoses afiU
their stereoisomers which are bonded to a sterol to provide a saponin. The bond is through a glycosidic linkage.
Monosaccharides, di- and trisaccharide derivatives of the hexoses may be used to derivatize the sterol.
The term "sterol" as used herein refers to natural or synthetic plant or animal sterols or triterpenes. This includes the phytosterols and the mycosterols as well as cholesterol for a more detailed discussion of sterols see for example, Nes, W.D., Parish, E.J. Ed., "Analysis of Sterols and Other Biologically Significznt Steroids", Academic Press, Inc. (1989). Preferred sterols are selected from the group of diosgenin, stigmastanol, tigogenin, cholesterol, ~-sitosterol, ~-sitosterol, stigmasterol, ergosterol, campesterol, oleanoic acids, soyasapogenols, protoascigenin, togenols, protoparaxadiols, and protopanaxadiols.
The term "galactose oxidase" as used herein refers to D-galactose:oxygen 6-oxidoreductase which is identified as E.C.
1.1.3.9 or as Chemical Abstracts Registry Number 9028-79-9.
The term "catalase", as used herein, refers to H202:H202 oxidoreductase which is identified as E.C. 1.11.1.6. Catalase is an enzyme which decomposes hydrogen peroxide. These enzymes occur in both plant and animal cells.
The term "hexose" means a carbohydrate containing six carbons.
This term encompasses both aldehyde-containing hexoses (aldohexoses) and ketone-containing hexoses (ketohexoses).

4- 2079~3 The term "aldohexoses" refers to the group of sugars whose molecule contains six carbon atoms, one aldehyde group and five alcohol groups. The sixteen stereoisomers of the aldohexose series are D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, L-allose, L-altrose, L glucose, L-mannose, L-gulose, L-idose, L-galactose and L-talose. These sugars exist in solution as an equilibrium mixture of several "tautomeric forms": a pyran-ring formi a furan-ring form; or a straight-chain aldehyde form. Aldohexoses may also occur in an ~ or ~ anomeric configura-tion, depending on the position of the C-l hydroxyl group.
The term "D-ketohexose" refers to the group of sugars which contain six carbon atoms, one ketone group and five alcohol groups.
The eight stereoisomers are D- and L- isomers of psicose, fructose, sorbose and tagatose. L;ke the aldohexoses, these ketohexoses can exist in solution as an equilibrium mixture of several "tautomeric forms": pyran-ring; a furan ring and a straight chain ketone form.
DescriDtion of the Novel ComDounds

5-C-hydroxymethyl aldohexose mono-, di- or trisaccharides can be used to make the sterols of the present invention. Preferred 5-C-hydroxymethylaldohexose derivatives are those of galactose, glucose, and mannose. Due to the relative ease of synthesizing galactose-based compounds, derivatives of D-galactose are the most preferred compounds. These can be in the form of aldohexopyranoses or aldohexofuranoses.
Preferred embodiments are 5-C-hydroxymethyl derivatives of fructose and sorbose, due to the availability of natural sugars.
Preferred disaccharides comprise at least one S-C-hydroxymethyl-aldohexose or 5-C-hydroxymethylketohexose.
The sterol derivatives of this invention contain at least one 5-C-hydroxymethyl sugar group (i.e., monosaccharides, monosaccharide derivatives) from monosaccharides covalently bound through glycoside linkages. The sterols contain an alcohol group which reacts with the sugar to form a glycosidic bond.
The synthesis of the sterol glycosides involves the reaction of an acetylated or other protected 5-C-hydroxymethyl hexose with a 5 2079~3 sterol in the presence of a catalyst and an inert solvent. The protecting group is removed and the sterol derivative is prepared.
The corresponding fluoro or bromo derivative of the sugar can also be reacted with the sterol.
The method of converting a hexose to a 5-hydroxymethyl-D-aldohexose-based compound is accomplished by employing the following steps.
1. Enzymic Oxidation of D-aldohexose-based ComDound with D-aldohexose:oxyqen 6-oxidoreductase The reaction is conducted in a clean vessel under agitation.
mixer with a tip speed of about 100-400 feet/min is preferred.
Sterile conditions prevent enzyme deactivation by microbial contami-nation.
An aqueous solution having a concentration of from about 1% to about 50%, preferably from about 10% to about 20% of D-aldohexose-based compound is prepared. The pH of the solution is adjusted to enhance reaction kinetics. A solution pH of from about 6 to about 8 is desired when using galactose oxidase as the enzyme. Galactose oxidase enzymic conversion requires a temperature of from about 1C
to about 50C. The reaction can be run at temperatures up to the inactivation temperature of the enzyme. However, at higher tempera-tures microbial growth can be an issue. A temperature of from about 3'C to about 25~C provides good enzyme stability, good oxygen saturation values at standard pressure, and reasonable reaction kinetics for galactose oxidase. Typical reaction times are in the range of from about 1 to about 24 hours.
From about 1,000 to about 1,000,000 unit activity of enzyme per mole of D-aldohexose or D-aldohexose based compound is typically added to the solution. Preferably from about 100,000 to 300,000 unit activity is used.
The level of available oxygen in solution also affects the oxidation step; a solution saturated with oxygen is preferred. Air and/or oxygen may be continuously bubbled through the solution to maintain oxygen saturation. Continùously pumping 2 to 3 volumes of air per volume of solution per minute using sparge rings having a

-6- 20795~3 high contact area works well. Suitable anti-foam agents include dimethyl silîcone, other organosilicone compounds, and FG-10 silicone (Dow Chemical). The level of anti-foaming agent is from 10 to 100 ppm.
It is also advantageous to reduce or eliminate the amount of free peroxide in the reaction vessel. Adding from about 10,000 to about 2,000,000 unit activity of catalase per mole of D-aldohexose-based compound can be used. Other procedures for the removal of peroxide can be employed. Copper catio,.s in the oxidation solution have been found to enhance enzyme stability. From about 0.1 mM to about 2 mM of CuS04 is used. Serum albumen is also a good en7yme stabili~er.
Finally, the catalase and D-aldohexose:oxygen 6-oxidoreductase are removed from the product solution. This can be done using conventional methods. The preferred separation technique is ultrafiltration through a membrane with from about 1,000 to about 30,000 molPcular weight cut-off (MWCO).
2. Condensation of Oxidation Product With Formaldehyde to the 5-C-hydroxymethyl derivatives of D-qalactose-based Compound From about 4 to about 40 molar equivalents of formaldehyde (most preferably from about 4 to about 8 molar equivalents) and from about 1 to about 13 molar equivalents of sodium hydroxide (most preferably from about 1 to about 3 molar equivalents) are added to the filtrate solution from step 1 (substrate). A resulting concen-tration of from about 10% to about 30% substrate is preferred. A pH
between about 12 and about 13 is preferred. The reaction solution is maintained at a temperature of from about 15C to about 40'C
until completion of the reaction. Cooling may be required until the exothermic reaction has ceased (typically about 1 hour). The solution is agitated until the condensation reaction has achieved the desired degree of completion (about 1 to about 24 hours, typi-cally 16 hours).
In order to control temperature and pH during the condensation re~ction (thereby preventing aldehyde destruction)~ it is preferred

-7- ~7~3 to prereact the formaldehyde and sodium hydroxide in a separate operation. Aqueous formaldehyde and sodium hydroxide solut;ons are combined and agitated at from about 15~C to about 35~C until the exothermic reaction ceases (typically about 30 min.). The solution is then warmed to a temperature of from about 15C to about 40C and quickly added to the filtrate solution of step 1 while maintaining a temperature of from about 15C to about 40C. The solution is agitated until the condensation reaction has achieved the desired degree of completion.
Other bases (e.g., Ca(OH)2, KOH and mixtures of them) can be used in place of all or part of the sodium hydroxide.
Another method of conducting the condensation reaction with formaldehyde is through the reaction of the sugar aldehyde which is produced via the galactose oxidase oxidation reaction and formalde-hyde on a strongly basic resin. The oxidation product and the formaldehyde are contacted with a resin which has a pH of at least 11.5 at a temperature of from about 20 C to about 50C for from 0.5 to 24 hours. A ratio of formaldehyde to sugar aldehyde of 4:1 to ~:1 is used. Preferably the ratio is about 4:1 to about 5:1.
Sufficient salts and buffers are present in the oxidation reaction mixture to form and to maintain the highly basic conditions necessary to conduct the condensation reaction. As the reaction progresses, additional salts are generated from the formic acid formed and these are adsorbed by the resins. Use of methanol-free formaldehyde and cupric oxide as a catalyst facilitates the reac-tion.
The product of this reaction can be purified using fractional distillation to remove the excess formaldehyde and/or adsorption techniques in a manner similar to the other condensation reaction.
3. _rification Unwanted ions (e.g., Na+,OH~,H+) and residual formaldehyde should be removed from the resulting reaction solution. Purifica-tion can be accomplished by conventional means, such as by utilizing adsorption resins, dialysis, precipitation, or a combination of 207~

several techniques. Fract;onal distillation can also be use to effectively remove the formaldehyde.
The resulting solution from the above-mentioned purification step will generally contain from about 1% to 50% 5-C-hydroxy-methylated product. The purified aqueous product solution may beused directly, or it can be concentrated to higher levels (e.g., from about 90% to about 95% sugar).
It is desirable to concentrate the solution at low temperatures to prevent thermal breakdown of the 5-C-hydroxymethylated compound.
Reverse osmosis employing a membrane with about a 100 MWC0 and a 99~O
~aCl rejection at from about 10C to about 387C is preferred.
Examples of these membranes include HR-98 or HR-99 polysulfone/polyamide thin film composite membranes, manufactured by Niro Corporation.
The most straightforward method of crystallizing 5-C-hydroxymethyl-aldohexose-based compounds is by saturating an aqueous solution at an an elevated temperature and cooling it to precipitate out the product crystals. However, this technique can be hindered by impurities and by-products in the solutions. The following technique is the most effective for precipitating the product and reducing the level of impurities and by-products.
A 90-95% solution of the product compound is prepared. Water is removed using ethanol (1:1) additions/evaporations (usually 1 or 2 such procedures are sufficient).
The solid residue resulting from the final ethanol evaporation is dissolved in methanol under reflux; a ratio of 1:1 to about 3:1 of methanol to solid is used. This is followed by the cooling of the solution to from about -10'C to about 20'C, for from about 1 to about 12 hours. The crystals are then filtered out and washed with cool methanol (about O'C).
Residual methanol may be removed by drying and/or by recrystallization from water. The crystals can be washed with acetone to further remove impurities.
The crystallization solvents can be removed by vacuum, fluid-ized bed drying and other techniques known in the art.

-9- 20795~3 A detailed description of the synthesis of these compounds is found in the application of Mazur et al., EP0 341,063 (1989), incorporated herein by reference.
4. Acvlation of 5-C-HYdroxvmethvl Hexoses First the 5-C-hydroxymethyl hexose is converted to an acylated 5-C-hydroxymethyl hexose to protect the sugar during subsequent reactions and to control the reaction products when the sugar is reacted with the sterol.
The 5-C-hydroxymethyl hexose is converted to the acylated derivative by a two-step esterification reaction. Any carboxylic acid anhydride can be used to make the esters. Preferably the anhydrides of acids having from 2 to 6 carbon atoms are used. Most preferably, acetic anhydride is used to protect the hydroxyl groups.
The 5-C-hydroxymethyl hexose is reacted with an acid anhydride in the presence of a base. Pyridine, used as the solvent works well in this reaction. The reaction is usually carried out at ambient temperatures.
In this initial reaction, 4 of the hydroxyls are esterified in a monosaccharide and up to 6 or 7 hydroxyls of a disaccharide are acylated. The anhydro derivative of the sugar forms during this reaction.
The partially acylated sugar is then reacted with additional carboxylic acid anhydride and a catalytic amount of concentrated or anhydrous sulfuric acid or other strong acid. This opens the anhydro bridge and adds 2 more acyl groups. The 5-C-hydroxymethyl hexose is now fully acylated. The acylated derivatives are usually crystalline and are formed in good yield.
The acylation can be done in one step using an excess of acid anhydride and anhydrous or very concentrated acids, preferably sulfuric acid, as a catalyst. However, this process is not pre-ferred.
8y way of example, the following reaction conditions work best:
Temperature: Ambient.
Solvent: Pyridine.
3~ Time: 1 to 24 hours.

-lO- 20795~3 Acid Anhydride:Carbohydrate Substrate Ratio: 1 to 2 equiva-lents for each hydroxyl group.
The partially acetylated sugar derivative or the 5-C-hydroxymethyl hexose can be esterified using acid catalyst under these conditions:
remperature: 0 to 20~C.
Solvent: Acetic Anhydride.
Time: 1 to 24 hours Catalyst: Concentrated sulfuric acid, phosphoric acid, or trifluoromethanesulfonic acid.
5. Condensation with Sterol The hexaacylated hexose is coupled with the sterol by one of three pathways:
1. Reaction with sterol using trimethylsilyl trifluoromethanesulfonate.
2. Conversion to 1-bromo- derivative and then reacted with the sterol.
3. Conversion to 1-fluoro derivative and reacted with sterol.
In option 1, the sterol is mixed with the hexaacyl 5-C-hydroxymethyl hexose and trimethylsilyl trifluoromethanesulfonate in an inert solvent. Preferably chlorinated hydrocarbons are used as the solvent. A ratio of from 1:1 to 2:1 hexose to sterol is used.
This reaction provides the pentaacyl 5-C-hydroxymethyl hexose sterol in yields of up to about 20%.
In option 2, an unstable 1-bromo pentaacyl 5-C-hydroxymethyl hexose is formed by react;ng hydrogen bromide in acetic acid with the acylated sugar derivative. The bromo derivative is usually unstable and must be reacted immediately with the sterol and a catalyst. The reaction is run in smaller batches using chlorinated hydrocarbons as the solvent. It results in a 30% or less yield.
Mercury cyanide works well as a catalyst in this reaction.
In option 3, a stable 1-fluoro-pentaacyl 5-C-hydroxymethyl hexose is formed by reacting the acylated hexose with pyridine and hydrogen fluoride. A possible side reaction involves cleavage of the acetyl group from the hexose 2-position. This hydroxyl group is -11- 2079~3 easily reacylated by reacting the 1-fluoro compound with an acyl anhydride as described above.
The l-fluoro derivatives prepared by this process are crystal-line and stable. They can be crystallized from ethanol or other alkyl alcohols and stored. These materials make good sources of 5-C-hydroxymethyl hexose for derivatization of a number of materi-als. They are very reactive in coupling reactions with sterols.
The reaction of these stable l-fluoro pentaacyl 5-C-hydroxymethyl hexoses with the sterol proceeds best when the sterol has been converted to the trimethylsilyl (TMS) derivative. The reaction with the TMS-sterol is carried out in chlorinated hydrocar-bon solvents using boron trifluoride etherates as catalysts. The resultant product is a pentaacyl-5-C-hydroxymethyl hexose derivative of the sterol.
The pentaacyl derivatives of hydroxymethyl saponins are crys-talline and easily purified by conventional techniques. The acyl groups are now hydrolyzed to make steryl 5-C-hydroxymethyl hexoside.
To hydrolyze the pentaacyl compounds, they are suspended in methanol or other lower alkyl alcohol and an alkali metal alcoholate (e.g., sodium methoxide) is added. This mixture is stirred for about 4-24 hours at ambient or reflux temperature.
The product is filtered and washed with alcohol. The product is a sol;d. The alcohol reactant and wash contain additional 5-C-hydroxymethyl hexose sterol derivatives which can be recovered by passing the alcohol through an amberlyst R120 acid (from Aldrich) form resin column until the alcohol (methanol) is neutral. The saponin is recovered from the alcohol eluent.
The sterol derivative can be dried under vacuum to remove any traces of solvent.
It has been found that the above reaction conditions provide good yields of hydroxymethyl saponins.
The preparation of the 5-C-hydroxymethyl hexose sterols is described in the following examples. The examples are illustrative of the invention and are not intended to be limiting of it.

-12- 20795~3 EXAMPLE I
PreDaration of methYl 5-C-hvdroxYmethYl-~-L-arabino-hexoDvranoside from methyl B-D-galactoside.
1. Oxidation of MethYl B-D-GalactoPYranoside with Galactose Oxidase ~ OH H ~ O

HO Lo OMe gala~ose HO ~ O OMe I /\ I oxidase ¦ / \

OH OH

Reagents MW Moles Amount methyl ~-D- 194.18 0.103 20.0 9 galactopyranoside Sigma Chemical Co., (No. M-6757) Phosphate Buffer, 100 mM --- --- 412.0 ml Catalase, 16900 units/mg --- --- 7.5 mg Sigma Chemical Co., (No. C-40) Galactose Oxidase --- ---9000 units The reaction is conducted in a one-liter vessel equipped with an aerator and a gentle stirrer. Sterile conditions are used to prevent enzyme deactivation by microbial contamination. The reac-tion is run at 4~C to minimize deactivation of galactose oxidase.

13 207~3 Methyl ~-D-galactopyranoside (1) is dissolved in the aerated phosphate buffer. The volume flow of air discharged by the aerator is regulated to produce an oxygen-saturated solution while prevent-ing foaming of the solution. At 4~C, the galactose oxidase and catalase are added and this solution is aerated for 20 hours.
The enzymes are removed from the product solution by ultrafiltration using a 10,000 MWCO membrane (Diaflo 13242, manu-factured by Amicon). The resulting filtrate contains the oxidation product, methyl ~-D-galacto-hexodialdo-1,5-pyranoside (2).
2. Condensation of Oxidation Product With FormaldehYde to Methvl 5-C-HYdroxvmethYl-~-L-arablno-hexoDvranoside (3 H ,~0 OH

~ CH20,NaOH

OH OH

Reagents Amount filtrate solution containing the oxidation product methyl ~ X~ D-galacto-hexodialdo-1,5-pyranoside from step 1. 400 ml 37% formaldehyde solution (aqueous) 400 ml 50% sodium hydroxide solution (aqueous) 144 ml The filtrate solution from step 1 and the formaldehyde solution are combined in a one-liter vessel. The sodium hydroxide solution is added to the filtrate/formaldehyde solution over a period ~

_14~ 2079~3 hour while the solution temperature is maintained between 20-C and 25~C with an ice-water bath. After the exothermic reaction has ceased, the ice-water bath is removed and the reaction mixture is stirred at room temperature for 16 hours. The reaction mixture is heated to 55C and deionized using ion exchange columns: first Amberlite IR-120(H+), then Amberlite IRA-400(0H-), both packings manufactured by Rohm & Haas. Finally, the deionized solution of the product is eluted through an Amberlite IRA-400 (HS03-) ion exchange column to remove remaining formaldehyde. Slow room-temperature evaporation to dryness, followed by drying of the residue at room temperature under vacuum overnight produces 18.5 9 (80%) of (3).
EXAMPLE II
Preparation of 1~6-anhvdro-5-C-hYdroxYmethYl-~-L-altro~ranose (4) OH ~ OH
/
HO O OMe Ho _ o 1~ ~ H2SO4 1( ~

OH OH

Methyl 5-C-hydroxymethyl-~-L-arabino-hexopyranose (3) (59.0g, 0.263 moles) is dissolved in 0.70M sulfuric acid (260 ml), and stirred at 100C for 90 minutes. The solution is cooled to room temperature and neutralized using an ion exchange resin (Amberlite IRA-400 (OH-). The resin is filtered off, and the filtrate is refluxed for 15 minutes with activated carbon (4.0 9). Carbon is removed with a glass fiber filter, and the filtrate is evaporated to dryness with ethanol. The white waxy residue is refluxed for 15 minutes with methanol (50 ml). The solution is stored overnight at 0C. The product is filtered to yield 20.09 (39.6%) of 1 9 6-anhydro-5-C-hydroxymethyl-~-L-altropyranose (4).
M.P. = 166.5-C - 168.5C. [~]23 = +145.1 (C 7.2 in water) D

2079~3 EXAMPLE III
PreDaration of S-C-acetoxYmethYl-1~2.3.4.6-penta-O-acetYl-L-arabino-hexoPyranose ~6) OH OAc HO~ O, pyndine OH OAc OAc OAc AcO AcO - -O
I / \ Ac20,H2SO4 I/

~ - ~ ~ ~ OAC
OAc OAc A solution of crude 1,6-anhydro-5-C-hydroxymethyl-~-L-altropyranose (4) (10.0 g, 52 mmol) in a mixture of acetic anhydride (100 ml) and pyridine (lOOml) is stirred at room temperature for h. The reaction mixture is poured into ice water (300 ml), the product is extracted with methylene chloride (300 ml). The organic phase is washed with 1 M HCl (3 x 400 ml), sodium bicarbonate (300 ml) and water (300 ml).
Evaporation of solvent produces crude 19.0 9 of 5-C-acetoxy-methyl-1,6-anhydro-2,3,4-tri-0-acetyl-~-L-altropyranose (5) which, without further purification, is dissolved in acetic anhydride (30"
ml) and the solution is cooled to 0 -5'C. While maintaining this -16- 2079~3 temperature, sulfuric acid (30.0 9) is slowly added. When the addition is complete, the ice bath is removed and the solution is stirred at ambient temperature for 2 h. At that time, TLC
(Analtech GF plates, toluene:acetone 2:1) shows a single major product with a small amount of more polar impurities. An excess of acetic anhydride is destroyed by slow addition of water (45 ml) with cooling at temperatures below 30 C. The resulting solution is partitioned between methylene chloride (300 ml) and aqueous sodium bicarbonate (300 ml), the organic phase is washed repeatedly with sodium bicarbonate (3 x 300 ml) and water (300 ml). Evaporation of the solvent gives 17.0 9 (70% yield) of (6).
[~]26.2 = +39.5O (C 8.3 in CHCl3) D

Anal. ~alc. for ClgH26013: C, 49.35; H, 5.67. Found: C, 49.16; H, 5.60.
EXAMPLE IY
Svnthesis of 5-C-acetoxvmethYl-1-fluoro-2,3,4,6,-tetra-0-acetYl-L-arabino-hexoPYranose (7) ~ OAc ~OAc AcO ~ o 1.HF-pyridine AcO ~ - O
/ \ 2. Ac2O, E~3N, DMAP 1 /
OAc ~ ~ F

OAc OAc 1,2,3,4,6,6'-Hexa-0-acetyl-5-C-hydroxymethyl-~/~-L-arabino-hexopyranose (749) is dissolved in cold (0 C) HF-pyridine complex (2009). A cooling bath is removed and stirring is continued until thin layer chromatographic analysis shows complete disappearance of the starting material (-3-4 h). The reaction mixture is diluted -17- 207~3 with methylene chloride (1.5 l) and the solution is washed with a saturated aqueous NaHC03 solution (2x300 ml) and brine ~300 ml).
The solution is dried over Na2S0l, filtered and solvents are concen-trated to about 500 ml. Triethylamine (22 ml) and acetic anhydride (15 ml) are then added followed by 4-dimethylaminopyridine (50 mg) and the mixture is stirred at ambient temperature for about 1 h.
The mixture is then diluted with methylene chloride (1.0 1), washed with NaHC03 aq. sat. solution (2x300 ml) and brine (300 ml), dried over Na2SOi and filtered. The solvents are removed in vacuo and the crude product is crystallized (ethanol) to give 5-C-acetoxymethyl -l-fluoro-2,3,4,6,-tetraacetyl-L-arabino-hexopyranoside (46.5 9).
m.p. 107-110C
EXAMPLE V
Diosqenin-5-C-acetoxYmethYl-2.3~4~6~-tetra-0-acetvl-B-D-arabino-hex-oDvranoside (9) AcO~

OAc Mc M~ ~--M~

M~
~ ~

AcO ~_o J~J

OAc -18- 20795~3 To a cold (O~C) solution of 5-C-acetoxy-methyl 2,3,4,6-tetraacetoxy-1-fluoro-L-arabino-hexopyranoside (12.7 9) and trimethylsilyldiosgenin (15 9) in dried 1,2-dichloroethane (300 ml) is added BF3 Et20 complex (19.5 ml). The cooling bath is removed and the mixture is stirred for 1 h when TLC analysis shows complete disappearance of the fluoride. The reaction mixture is diluted with methylene chloride (300 ml), transferred to a separatory funnel and washed with NaHC03 aq. sat. solution (2xlO0 ml) and brine (100 ml).
The solution is dried over Na2S0~, filtered and solvents are concen-trated to about 100 ml. Triethylamine (4 ml) and acetic anhydride(2.5 ml) are then added, followed by 4-dimethylaminopyridine (10 mg) and the mixture is stirred at ambient temperature for about 1 h.
The mixture is then diluted with methylene chloride (200 ml), washed with NaHC03 aq. sat. solution (2xlO0 ml) and brine (100 ml), dried over Na2S0~ and filtered. The solvents are then removed in vacuo and the crude product is purified by silica gel chromatography to give diosgenin 5-C-acetoxymethyl-2,3,4,6,-tetra-0-acetyl-B-D-arabino-hexopyranoside (11.8 9) as a white solid.
m.p. l~0-185C
HYdrolYsis to diosqenin 5-C-hYdroxvmethvl-B-D-arabino-hexopyranoside ~b O~Me Me ~

M~
~ ~
L~o c ,J~, ~J M~ONa. MeOH

30 ~Jc 9 ~ t r ue HO~O

OH

-19- ~0795~

This material is suspended in methanol (qO0 ml) and sodium methoxide (2 mL of 25% solution in methanol) is added. After stirring for 20 h at room temperature the mixture is filtered and thoroughly washed with rnethanol. The combined methanol washings are neutralized with Amberlite ~ IR-120 and solvent is evaporated. The solids are combined with the filtrant and dried in vacuo to give

8.22 g of diosgenin 5-C-hydroxymethyl-~-D-arabino-hexopyranoside.

EXAMPLE VI
Synthesis of d osqenin 5-C-hydroxYmethvl-L-arabino-hexopyranoside OAc AcO _ 1. diosgenin. Hg(CN)2 I / ~ 2. MeONa. MeOH
V ~ OAc~
~Br OAc Me -- Me ~~ Me ~C I .
Me \~
~OH
~7 OH

2,3,4,6-tetraacetyl-5-C-acetoxymethyl-1-bromo-L-arabinohexopy-ranoside prepared from per-0-acetyl-5-C-hydroxymethyl-L-arabinohexo-pyranose (18 g) according to the standard method, was mixed with diosgenin (12.3 9), mercuric cyanide (14.5 9) in dry 1,2-dichloroethane (250 ml) and stirred at 50~C for 20 h. The reaction mixture was washed successively with water (1000 ml), 10%
aqueous sodium iodide (2 x 200 ml), aqueous saturated solution of sodium bicarbonate (200 ml), and water (200 ml). The organic layer was next dried and evaporated. The crude residue was purified and hydrolyzed as described in Example V.

Claims (35)

1. A process for preparing 5-C-hydroxymethyl hexose deriva-tives of sterols comprising:
(1) acylating a 5-C-hydroxymethyl hexose in a two step acylation comprising;
(a) reacting an acid anhydride with said hexose in the presence of a base;
(b) reacting the product of step (a) with an acid anhydride and a catalytic amount of a strong acid to form a hexaacylated hexose derivative; and (2) preparing the 1-fluoro derivative of the hexaacylated 5-C-hydroxymethyl hexose by reacting the acylated hexose with fluoride ion in the presence of boron trifluoride etherate; and (3) reacting the product of step (2) with a sterol.

2. A process according to Claim 1 wherein the acyl groups are hydrolyzed using an alkali metal alcoholate catalyst.

3. A process according to Claim 1 wherein said acid anhydride is a carboxylic acid anhydride having from 2 to 6 carbon atoms.

4. A process according to Claim 3 wherein said base is pyridine.

5. A process according to Claim 4 wherein said acid anhydride is acetic anhydride.

6. A process according to Claim 3 wherein said acid is selected from the group of sulfuric acid, phosphoric acid and trifluoromethanesulfonic acid.

7. A process according to Claim 1 wherein the sterol is first converted to the trimethylsilyl derivative.

8 A process according to Claim 5 wherein the 5-C-hydroxy-methyl hexose is selected from the group consisting of 5-C-hydroxymethyl derivatives of galactose, glucose, mannose, fructose, sorbose and tagatose and wherein said sterol is selected from the group consisting of cholesterol, diosgenin, sitosterol, ergosterol, campesterol, stigmastanol and tigogenin.

9. A process according to Claim 8 wherein the sterol is diosgenin or cholesterol.

10. A process according to Claim 9 wherein the aldohexoside is 5-C-hydroxymethyl-L-arabino-hexopyranoside.

11. A process for preparing 5-C-hydroxymethyl hexose deriva-tives of sterols comprising:
(1) acylating a 5-C-hydroxymethyl hexose in a two step acylation comprising;
(a) reacting an acid anhydride with said hexose in the presence of a base;
(b) reacting the product of step (a) with an acid anhydride and a catalytic amount of a strong acid to form a hexaacylated hexose derivative; and (2) preparing the 1-bromo derivative of the hexaacylated 5-C-hydroxymethyl hexose by reacting the acylated hexose with bromide ion in the presence of acetic acid; and (3) reacting the product of step (2) with a sterol in the presence of mercury cyanide.

12. A process according to Claim 11 wherein the acyl groups are hydrolyzed using an alkali metal alcoholate catalyst.

13. A process according to Claim 11 wherein said acid anhydride is a carboxylic acid anhydride having from 2 to 6 carbon atoms.

14. A process according to Claim 13 wherein said base is pyridine.

15. A process according to Claim 14 wherein said acid anhydride is acetic anhydride.

16. A process according to Claim 13 wherein said acid is selected from the group of sulfuric acid, phosphoric acid and trifluoromethanesulfonic acid.

17. A process according to Claim 15 wherein the 5-C-hydroxy-methyl hexose is selected from the group consisting of 5-C-hydroxymethyl derivatives of galactose, glucose, mannose, fructose, sorbose and tagatose and wherein said sterol is selected from the group consisting of cholesterol, diosgenin, sitosterol, ergosterol, campesterol, stigmastanol and tigogenin.

18. A process for making 5-C-hydroxymethyl hexose derivatives of sterols comprising the steps of:
(1) acylating a 5-C-hydroxymethyl hexose in a two step acylation comprising;
(a) reacting an acid anhydride with said hexose in the presence of a base;
(b) reacting the product of step (a) with an acid anhydride and a catalytic amount of a strong acid; and (2) reacting the acylated hexose with a sterol under anhydrous conditions in the presence of trimethylsilyl trifluoromethane sulfonate.

19. A process according to Claim 18 wherein the acyl groups are hydrolyzed using an alkali metal alcoholate catalyst.

A process according to Claim 18 wherein said acid anhydride is a carboxylic acid anhydride having from 2 to 6 carbon atoms.

21. A process according to Claim 20 wherein said base is pyridine and wherein said acid anhydride is acetic anhydride.

22. A process according to Claim 21 wherein the hexose group is selected from the group consisting of the D or L isomers of galactose, glucose, mannose, fructose, sorbose and tagatose and wherein said sterol is selected from the group consisting of spirostanols, diosgenin, cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, tigogenin, oleanoic acid, soya sapogenols, protoascigenin, protoparaxadiols and protopanaxadiols.

23. Sterol 5-C-acyloxymethyl-2,3,4,6-tetraacyl arabino-hexopyranosides.

24. A compound according to Claim 23 where the hexose group is selected from the group consisting of the D or L isomers of galactose, glucose, mannose, fructose, sorbose and tagatose and wherein said sterol is selected from the group consisting of spirostanols, diosgenin, cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, tigogenin, oleanoic acid, soya sapogenols, protoasciyenin, protoparaxadiols and protopanaxadiols.

25. A compound according to Claim 24 where the galactose group is qalacto-pyranose and qalacto-furanose.

26. A compound according to Claim 25 where the aldohexo-pyranose group is selected from the group consisting of altro-hexopyranose, ido-hexopyranose and qulo-hexopyranose.

27. A compound according to Claim 23 wherein said hexose is 5-C-hydroxymethyl-L-arabino-hexopyranoside and said sterol is diosgenin, tigogenin or cholesterol.

28. A compound according to Claim 23 which is diosgenin-5-C-hydroxymethylarabino-hexopyranoside.

29. A compound according to Claim 23 which is tigogenin-5-C-hydroxymethylarabino-hexopyranoside.

30. A compound according to Claim 23 which is cholester-ol-5-C-hydroxymethylarabino-hexopyranoside.

31. 1-Halo-5-C-acyloxymethyl-2,3,4,6-tetraacyl arabinn-hexopyranosides.

32. A compound according to Claim 31 where the hexose group is selected from the group consisting of the D or L isomers of galactose, glucose, mannose, fructose, sorbose and tagatose.

33. A compounds according to Claim 32 wherein the halogen is bromine or fluorine.

34. A compound according to Claim 33 where the galactose group is qalacto-pyranose and qalacto-furanose.

35. A compound according to Claim 34 where the aldohexo-pyranose group is selected from the group consisting of altro-hexopyranose, ido-hexopyranose and qulo-hexopyranose.

APP/4492/rad