Isolation of steroids containing a 5,7-diene functionality from a sterol mixture

A method is provided for isolating 5,7-diene-containing steroids, particularly 3.beta.-ols and esters of 3.beta.-ols, from a sterol mixture. The method involves (1) treating the mixture with a dienophile or with an oxidizable dienophile precursor in combination with an oxidizing agent so as to provide a Diels-Alder adduct of the 5,7-diene to be isolated, followed by (2) removal of the adduct from the mixture and (3) regeneration of the 5,7-diene with a suitable reducing agent. The invention also encompasses subsequent purification steps and intermediate modification of the Diels-Alder intermediate, e.g., wherein chemical conversion of the Diels-Alder adduct is effected prior to regeneration of the 5,7-diene. Novel compounds which are Diels-Alder adducts of 5,7-diene-containing steroids are provided as well.

TECHNICAL FIELD 
This invention relates generally to synthetic methods involving steroids, 
and more particularly relates to a novel method for isolating steroids 
containing a 5,7-diene functionality from a mixture of sterols. The method 
is especially useful for isolating 5,7-diene-containing sterols from a 
mixture containing yeast sterol metabolites. 
BACKGROUND 
The present invention derives from the development of a biotechnological 
fermentation process that produces a yeast sterol mixture enriched in 
cholesta-5,7,24-triene-3.beta.-ol and accompanied by other di-olefinic 
yeast sterol metabolites. The aforementioned trienol, having the chemical 
structure (I), 
##STR1## 
is a valuable compound useful, inter alia, as an intermediate in the 
synthesis of a variety of compounds related to vitamin D.sub.3 
derivatives, e.g., cholesta-5,7-diene-3.beta.-25-diol and other 
25-substituted vitamin D.sub.3 precursors. Accordingly, it is necessary to 
provide a feasible process for isolating and purifying the trienol (I) 
from a fermentation mixture containing yeast sterol metabolites, including 
lanosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, zymosterol and 
cholesta-7,24-diene-3.beta.-ol. The mixture may also include squalene. As 
may be seen from the following structures, a number of these compounds 
containing two or more degrees of unsaturation. Thus, the process of 
isolation and separation must be specific. 
##STR2## 
S.C. Eyley et al., J. C. S. Perkins Trans. I, pp. 731-735 (1976), describe 
a method for synthesizing 25-hydroxyprovitamin D.sub.3 and 
25.xi.,26-dihydroxyprovitamin D.sub.3. The method involves initial 
reaction of the C-22 aldehyde derived by degradation of ergosterol with a 
Grignard reagent derived from 4-chloro-2-methylbut-1-ene, followed by 
reductive elimination of the mesylate of the resulting C-22 alcohol. 
J. P. Moreau et al., J. Org. Chem. 39(14):2018-2023 (1974), is a background 
reference which describes the synthesis of 
5.alpha.-cholesta-7,24-dien-3.beta.-ol and 
cholesta-5,7,24-trien-3.beta.-ol. 
J. W. Blunt et al., Biochemistry 8(2):671-675 (February 1969), describe 
methods of synthesizing cholesta-5,7-diene-3.beta.,25-diol, followed by 
conversion to 25-hydroxycholecaliferol. 
S. S. Yang et al., Tetrahedron Letters 27:2315-2316 (1977), is a background 
reference describing a method for synthesizing 25-fluorovitamin D.sub.3. 
D. R. Crump et al., J. C. S. Perkins Trans. I, pp. 2731-2733 (1973), 
describes a method for synthesizing (22S)-hydroxyvitamin D.sub.4 using 
ergosterol acetate as a starting material. The synthesis involves 
selective epoxidation of the 22,23-double bond of ergosterol acetate, 
followed by a Grignard reaction on the hexanor-22-aldehyde, and 
irradiation. 
SUMMARY OF THE INVENTION 
Accordingly, it is a primary object of the invention to provide a method 
for isolating and removing a 5,7-diene-containing steroids, particularly 
3.beta.-ols and esters of 3.beta.-ols, from a mixture of sterols. 
It is another object of the invention to provide such a method wherein the 
mixture of sterols comprises a plurality of yeast sterol metabolites. 
It is still another object of the invention to provide such a method 
wherein the plurality of yeast sterol metabolites includes squalene, 
lanosterol, 4,4-dimethylzymosterol, 4-methylzymosterol, zymosterol and 
cholesta-7,24-diene-3.beta.-ol. 
It is yet another object of the invention to provide such a method wherein 
the 5,7-diene-containing steroid is cholesta-5,7,24-triene-3.beta.-ol. 
It is a further object of the invention to provide such a method which 
involves treating the mixture with a dienophile as will be described in 
detail herein. 
It is still a further object of the invention to provide such a method 
which involves treating the mixture with a oxidizable dienophile 
precursor, and with an oxidizing agent capable of oxidizing the dienophile 
precursor to a dienophile. 
It is yet a further object of the invention to provide such a method which 
enables preparation of the isolated 5,7-diene-containing steroids in 
purified form. 
It is still a further object of the invention to provide novel compounds 
which are selected Diels-Alder adducts of 5,7-diene-containing steroids as 
will be described in detail herein. 
Additional objects, advantages and novel features of the invention will be 
set forth in part in the description which follows, and in part will 
become apparent to those skilled in the art upon examination of the 
following, or may be learned by practice of the invention. 
In one aspect, the invention is a method of isolating asteroid, typically a 
3.beta.-ol or an ester of a 3.beta.-ol, containing a 5,7-diene 
functionality from a mixture of sterols, wherein the method involves: (a) 
treating the mixture with a dienophile having the structural formula 
X--R.dbd.R--Y, in which the R's are both N or both C--Q where the Q's are 
H or together form a third bond, and wherein X and Y are 
electron-withdrawing groups which may or may not be linked together; (b) 
removing the Diels-Alder adduct so formed from the mixture; and (c) 
treating the Diels-Alder adduct with a reducing agent which is effective 
to cleave the adduct and regenerate the 5,7-diene-containing steroid. 
In another aspect, the invention is a method of isolating asteroid 
containing a 5,7-diene functionality from a mixture of sterols, wherein 
the method involves: (a) treating the mixture with (i) an oxidizable 
dienophile precursor having the structural formula X--NH--NH--Y where X 
and Y are as above, and (ii) an oxidizing agent effective to oxidize the 
precursor; (b) removing the Diels-Alder adduct so formed from the mixture; 
and (c) treating the Diels-Alder adduct with a reducing agent which is 
effective to cleave the adduct and regenerate the 5,7-diene-containing 
steroid. 
In either case, the isolated 5,7-diene-containing steroid can then be 
purified via crystallization, chromatography, precipitation, or the like. 
In still another aspect of the invention, modification of the Diels-Alder 
adduct is carried out between steps (a) and (b), typically a modification 
which could not be made with the reactive 5,7-diene functionality present. 
In still another aspect of the invention, novel compounds are provided in 
the form of Diels-Alder adducts of certain 5,7-diene-containing steroids.

DETAILED DESCRIPTION OF THE INVENTION 
Before the present methods are disclosed and described, it is to be 
understood that this invention is not limited to the isolation and 
purification of specific 5,7-diene-containing steroids, to the treatment 
of specific sterol mixtures, or to the use of specific synthetic reagents, 
i.e., dienophiles, oxidizing agents, reducing agents, or the like, as such 
may, of course, vary. It is also to be understood that the terminology 
used herein is for the purpose of describing particular embodiments only 
and is not intended to be limiting. 
It must be noted that, as used in the specification and the appended 
claims, the singular forms "a", "an" and "the" include plural referents 
unless the context clearly dictates otherwise. Thus, for example, 
reference to "a sterol" includes mixtures of sterols, reference to 
"asteroid" includes mixtures of two or more steroids, and the like. 
In this specification and in the claims which follow reference will be made 
to a number of terms which shall be defined to have the following 
meanings: 
"Alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 
to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 
isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, 
tetracosyl and the like. Preferred "alkyl" groups herein contain 1 to 12 
carbon atoms. "Lower alkyl" refers to an alkyl group of 1 to 6, more 
preferably 1 to 4, carbon atoms. 
"Alkylene" refers to a difunctional saturated branched or unbranched 
hydrocarbon chain containing from 1 to 24 carbon atoms, and includes, for 
example, methylene (--CH.sub.2 --), ethylene (--CH.sub.2 --CH.sub.2 --), 
propylene (--CH.sub.2 --CH.sub.2 --CH.sub.2 --), 2-methylpropylene 
[--CH.sub.2 --CH(CH.sub.3)--CH.sub.2 --], hexylene [--(CH.sub.2).sub.6 --] 
and the like. "Lower alkylene" refers to an alkylene group of 1 to 6, 
more preferably 1 to 4, carbon atoms. 
"Alkenylene" refers to a difunctional branched or unbranched hydrocarbon 
chain containing from 2 to 24 carbon atoms and at least one double bond. 
"Lower alkenylene" refers to an alkenylene group of 2 to 6, more 
preferably 2 to 5, carbon atoms. 
"Alkynyl" refers to a branched or unbranched acetylenically unsaturated 
hydrocarbon group of 2 to 24 carbon atoms such as ethynyl, 1-propynyl, 
2-propynyl, 1-butynyl, 2-butynyl, octynyl, decynyl, tetradecenyl, 
hexadecynyl, and the like. "Lower alkynyl" refers to an alkynyl group of 2 
to 6, more preferably 2 to 4, carbon atoms. 
"Acyl" refers to a group of the structure --(C.dbd.O)--R', where R' is as 
described herein. Acyl, therefore, includes such groups as, for example, 
acetyl, propanoyl (or propionyl), isopropanoyl, n-butanoyl (or n-butyryl), 
benzoyl, phenylacetyl, and the like. "Lower acyl" refers to an acyl group 
wherein R' is lower alkyl as defined above. 
"Aryl" refers to a phenyl or 1- or 2-naphthyl group. "Monocyclic aryl" 
refers to a phenyl group. Optionally, these groups are substituted with up 
to five ring substituents selected from the group consisting of 
--(CH.sub.2).sub.n --NH.sub.2, --(CH.sub.2).sub.n --COOH, --NO.sub.2, 
halogen and lower alkyl, where n is an integer in the range of 0 to 6 
inclusive. 
"Arylene" refers to a difunctional aromatic moiety; "monocyclic arylene" 
refers to a phenylene group. These groups may be substituted with up to 
four ring substituents selected from the group consisting of 
--(CH.sub.2).sub.n --NH.sub.2, --(CH.sub.2).sub.n --COOH, --NO.sub.2, 
halogen and lower alkyl, where n is an integer in the range of 0 to 6 
inclusive. 
"Halo" or "halogen" refers to fluoro, chloro, bromo or iodo, usually 
regarding halo substitution for a hydrogen atom in an organic compound. Of 
the halos, chloro and bromo are generally preferred with chloro generally 
being the more preferred. 
"Optional" or "optionally" means that the subsequently described event or 
circumstance may or may not occur, and that the description includes 
instances where said event or circumstance occurs and instances in which 
it does not. For example, "optionally substituted phenyl" means that the 
phenyl may or may not be substituted and that the description includes 
both unsubstituted phenyl and phenyl wherein there is substitution. 
In describing the location of groups and substituents, the following 
numbering system will be employed. 
##STR3## 
This system is intended to conform the numbering of the 
cyclopentanophenanthrene nucleus to the convention used by the IU or 
Chemical Abstracts Service. The term "steroid" as used herein is intended 
to mean compounds having the aforementioned cyclopentanophenanthrene 
nucleus. 
The symbols ".alpha." and ".beta." indicate the specific stereochemical 
configuration of a substituent at an asymmetric carbon atom in a chemical 
structure as drawn. Thus ".alpha.", denoted by a broken line, indicates 
that the group at the position in question is below the general plane of 
the molecule as drawn, and ".beta.", denoted by a bold line, indicates 
that the group at the position in question is above the general plane of 
the molecule as drawn. In the present disclosure, if bonds are not 
indicated explicitly as ".alpha." or ".beta.", it should be assumed that 
the structural formula encompasses both types of compounds, with the 
stereochemical configuration of the naturally occurring steroid molecule 
preferred. 
In addition, the five- or six-membered rings of the steroid molecule are 
often designated A, B, C and D as shown. 
The term "sterol" as used herein is intended to mean asteroid molecule 
having the backbone structure illustrated above, and containing at least 
one hydroxyl group. Generally, the sterols of the present invention have a 
single hydroxyl group at the 3-position. 
The term "purified compound" as used herein intends a composition which 
contains at least about 80 wt. % of that compound, preferably at least 
about 90 wt. %, and most preferably at least about 99 wt. %. 
The 5,7-diene-containing steroids which may be isolated and purified using 
the present technique have the general structural formula 
##STR4## 
wherein R.sup.3 is selected from the group consisting of H and R'CO-- 
where R' is lower alkyl or monocyclic aryl of 5 to 7 carbon atoms, and 
R.sup.4, R.sup.5 and R.sup.6 are independently selected from the group 
consisting of H, hydroxyl and lower alkyl. The R.sup.3 moiety, if other 
than H, is thus a hydroxyl-protecting group; typical R.sup.3 moieties are 
H, CH.sub.3 CO-- and C.sub.6 H.sub.5 CO--. If R.sup.4, R.sup.5 and R.sup.6 
are other than H and OH, they will generally be methyl or ethyl, more 
typically methyl. 
As noted earlier, the method of the present invention is particularly 
useful for isolating and purifying cholesta-5,7,24-triene-3.beta.-ol from 
a mixture of sterols, e.g., a mixture of yeast sterol metabolites 
(squalene, lanosterol, 4,4-dimethylzymosterol, and the like). Virtually 
any sterol may be present in the mixture so long as the compounds do not 
contain a conjugated diene functionality. As illustrated by the yeast 
sterol metabolite mixture shown above, the compounds present in the 
composition from which the 5,7-diene-containing sterol is to be isolated 
may contain two or more degrees of unsaturation. 
In a first embodiment of the invention, the mixture of sterols is treated 
with a dienophile having the structural formula X--R.dbd.R--Y wherein the 
R's are both N or both C--Q where the Q's are H or together form a third 
bond. Thus, the dienophile in this embodiment will have the structure 
X--N.dbd.N--Y, X--(CQ).dbd.(CQ)--Y, or X--C.tbd.C--Y. This type of 
reaction will sometimes be referred to herein as reaction type (1). The 
substituents X and Y are electron-withdrawing groups which are 
independently selected from the group consisting of --COOH, --CHO, 
--NO.sub.2, --CN, --COOR.sup.1 and --COR.sup.1 where R.sup.1 is lower 
alkyl, or X and Y may be linked together to form a --(CO)--Z--(CO)-- 
bridge. In the latter case, i.e., when X and Y are linked together, the 
"Z" linkage is lower alkylene, lower alkenylene, monocyclic arylene of 5 
to 7 carbon atoms with up to 4 ring substituents, --S--, or --NR.sup.2 -- 
wherein R.sup.2 is H, lower alkyl or monocyclic aryl of 5 to 7 carbon 
atoms with up to 5 ring substituents. Ring substituents are selected from 
the group consisting of --(CO.sub.2).sub.2 --NH.sub.2, --(CH.sub.2).sub.n 
--COOH, --NO.sub.2, halogen and lower alkyl, wherein n is an integer in 
the range of 0 to 6 inclusive. Dienophiles within the aforementioned group 
may be available commercially or may be readily synthesized using starting 
materials and techniques known to those skilled in the art of synthetic 
organic chemistry. Examples of particular dienophiles useful herein 
include the following: 
##STR5## 
These dienophiles are available commercially from a number of sources, 
e.g., from the Aldrich Chemical Company, Milwaukee, Wis. As will be 
appreciated by those skilled in the art, such dienophiles may also be 
readily synthesized using conventional techniques (see, e.g., S. W. Moje 
and P. Beak, J. Org. Chem. 39(20):2951 (1974), and K. Rufenacht, Helv. 
Chim. Acta 51:518 (1968)). 
In a second embodiment, a dienophile precursor is used which may be 
converted to a dienophile with a suitable oxidizing agent. This reaction 
will sometimes be referred to herein as reaction (2). Here, the sterol 
mixture is simultaneously treated with the dienophile precursor and with 
an oxidizing agent effective to oxidize the precursor to an active 
dienophile. The dienophile precursor has the structural formula 
X--NH--NH--Y wherein X and Y are as defined above. Exemplary dienophile 
precursors are wherein X and Y are linked together to form a 
--(CO)--Z--(CO)-- bridge, with Z as defined above. Preferably, in this 
embodiment, Z is monocyclic arylene of 5 to 7 carbon atoms substituted 
with up to 2 substituents selected from the group consisting of 
--(CH.sub.2).sub.n --NH.sub.2 and --(CH.sub.2).sub.n --COOH, wherein n is 
an integer in the range of 0 to 6 inclusive. Dienophile precursors within 
the aforementioned group may be available commercially or may be readily 
synthesized using starting materials and techniques known to those skilled 
in the art of synthetic organic chemistry (see, e.g., H. D. K. Drew and H. 
H. Hatt, J. Chem. Soc. 16 (1937)). Examples of particular dienophile 
precursors useful herein (again, such compounds are available 
commercially, or may be readily synthesized) include the following: 
##STR6## 
Any oxidizing agent capable of oxidizing the dienophile precursor to an 
active dienophile may be used, with the exception of oxidizing agents 
which could interfere with the formation of the Diels-Alder adduct or 
which could interact detrimentally in some other way with any of the 
sterols in the sterol mixture. Exemplary oxidizing agents include 
potassium peroxymonosulfate, lead tetraacetate, iodosobenzene diacetate, 
N-bromosuccinimide and t-butyl hypochlorite. 
Either of the aforementioned reactions, i.e., treatment of the sterol 
mixture with a dienophile having the structure X--R.dbd.R--Y, or with a 
dienophile precursor of the structure X--NH--NH--Y and an oxidizing agent, 
results in the formation of a Diels-Alder adduct. These reactions are 
illustrated in the following schemes: 
##STR7## 
Both types of reactions are carried out in an inert atmosphere, in a 
non-reactive, preferably polar organic solvent effective to dissolve the 
reactants. With reaction (2), it is preferred that the oxidizing agent be 
added gradually to a solution of the steroid and the dienophile in the 
selected solvent, and that the procedure be carried out at a relatively 
low temperature, i.e., 10.degree. C. or lower (0.degree. C. to 5.degree. 
C., as may be obtained by an ice/water bath, is optimal). At least about 
15 minutes, preferably at least 1 hour, should be allowed for the reaction 
to occur. 
After preparation of the Diels-Alder adduct using either reaction (1) or 
reaction (2), the adduct is removed from the reaction mixture and 
regenerated to give the 5,7-diene-containing steroid in isolated form. 
Removal of the adduct from the reaction mixture is preferably done 
chromatographically, using, for example, a silica gel column which will 
preferentially bind the Diels-Alder adduct. The chemical and physical 
properties of the Diels-Alder adduct can be varied by manipulating the 
substituents present on the dienophile as well as by varying R.sup.3. For 
example, basic properties can be imparted to the Diels-Alder adduct by the 
use of a dienophile containing a basic substituent, e.g., --NH.sub.2, 
--(CH.sub.2).sub.n --NH.sub.2, or the like. The adduct is then a basic 
molecule and separable from the sterol mixture using acid extraction. 
Similarly, acidic properties can be imparted to the Diels-Alder adduct by 
the use of a dienophile containing an acid substituent, e.g., --COOH, 
--(CH.sub.2).sub.n --COOH, or the like. The adduct will then be an acidic 
molecule and separable from the sterol mixture using basic extraction. 
Also, as alluded to above, after preparation of the Diels-Alder adduct, the 
moiety present at R.sup.3 may be converted to a functionality which 
imparts desirable crystallization and/or precipitation parameters. For 
example, a hydroxyl group present at C-3 may be readily converted to a 
benzoate species, which in turn will make the adduct more crystalline and 
more readily separable from the sterol mixture. 
Regeneration of the 5,7-diene-containing steroid is then accomplished by 
treatment of the adduct with a reducing agent such as lithium aluminum 
hydride ("LAH"), diisobutyl aluminum hydride ("DiBAL"), Red-Al.RTM. (a 
solution of sodium bis(2-methoxy-ethoxy)aluminum hydride in toluene, 
available from the Aldrich Chemical Company, Inc., Milwaukee, Wis.), or 
the like. Lithium aluminum hydride is particularly preferred. The reaction 
proceeds initially at a low temperature, i.e., 10.degree. C. or lower 
(again, as may be obtained by an ice/water bath), followed by, after at 
least about 30 minutes, warming to at least about 50.degree. C. for at 
least several minutes. Excess reducing agent and any salts or derivatives 
thereof are then removed, e.g., by filtration through celite or the like. 
Evaporation of the reaction mixture will then give rise to the 
5,7-diene-containing steroid. 
Purification of the regenerated 5,7-diene-containing steroid may then be 
carried out using any of a number of techniques which will be readily 
appreciated by those of ordinary skill in the art. For example, 
purification can be effected via recrystallization e.g., using methanol, 
ethanol, or the like, or using precipitation or chromatographic 
techniques. 
Where the 5,7-diene-containing steroid is 
cholesta-5,7,24-triene-3.beta.-ol, the isolated, purified material may be 
used to prepare cholesta-5,7-diene-3.beta.,25-diol as described in 
commonly assigned U.S. patent application Ser. No. 07/869,328, now 
abandoned entitled "Method of Preparing Cholesta-5,7-Diene-3,25-Diol and 
Analogs Thereof", inventors J. Johansson et al., filed on even date 
herewith. The disclosure of that patent application is hereby incorporated 
by reference in its entirety. Cholesta-5,7-diene-3.beta.,25-diol is a 
biologically important hydroxylated pro-vitamin D.sub.3 metabolite which 
may be converted by sunlight or other well-established photochemical 
methods to 25-hydroxy vitamin D.sub.3. Such vitamin D.sub.3 derivatives 
are useful in a number of contexts, e.g., in topical pharmaceutical 
formulations (for the treatment of skin disorders or the like), in oral 
vitamin compositions, and as livestock feed additives. 
In a variation on the above-described reactions, chemical conversion of one 
or more sites on the 5,7-diene-containing steroid may be effected while 
the molecule is protected in the form of the Diels-Alder adduct. For 
example, the .DELTA..sup.24 double bond may be converted to a 
24-amino-25-hydroxyl species, a 24,25-dihydroxyl species, or the like. 
Also, the "A" ring of the steroid may be oxidized when the compound is in 
adduct form. Examples of chemical conversions which may be carried out on 
the Diels-Alder adduct are described in co-pending patent application Ser. 
No. 07/869,328 incorporated by reference above. 
It should also be pointed out that a number of the Diels-Alder adducts 
described hereinabove generically represented by structural formula (XIX) 
above and include the following specific adducts: 
##STR8## 
The advantages of the invention include the following: selectivity of 
reaction of the 5,7-diene-containing steroid with a reactive dienophile; 
crystallinity of the Diels-Alder adduct, which readily enables removal 
from the initial sterol mixture; very high yield of the final purified 
product, on the order of or higher; simplicity of "scaling up" to a 
manufacturing context; and the ability to modify the 5,7-diene in 
Diels-Alder adduct form. 
It is to be understood that while the invention has been described in 
conjunction with preferred specific embodiments thereof, the foregoing 
description, as well as the examples which follow, are intended to 
illustrate and not limit the scope of the invention. Other aspects, 
advantages and modifications within the scope of the invention will be 
apparent to those skilled in the art to which the invention pertains. 
EXAMPLE 1 
Example 1(a) 
##STR9## 
To a stirred solution under argon of a crude sterol mixture (obtained from 
Amoco; 50 g) containing squalene, lanosterol, 4,4-dimethylzymosterol, 
4-methylzymosterol, zymosterol, cholesta-7,24-diene-3.beta.-ol and 
cholesta-5,7,24-triene-3.beta.-ol (11.0 g, pure trienol, 28.8 mmol) 
dissolved in dichloromethane (600 ml) was added phthalhydrazide (obtained 
from Aldrich; 15.0 g, 92.5 mmol). This solution was cooled in an ice/water 
bath (0.degree.-5.degree. C.). To the cooled solution was added dropwise a 
solution of lead tetraacetate (15.0 g, 33.9 mmol) and acetic acid (1.95 
ml) in dichloromethane (215 ml). Addition time 30 min. After stirring at 
0.degree.-5.degree. C. for 1.5 hr, the reaction mixture was stirred at 
room temperature for a total of 4 hr. TLC on silica gel impregnated with 
silver nitrate showed no trienol present. The reaction mixture was 
filtered through celite and the combined dichloromethane solution was 
washed with water and sodium bicarbonate, and again with water. 
Evaporation of the solvent gave a yellow crude product that was purified 
in the following way. The crude product was dissolved in a mixture of 
ethyl acetate and 20% hexane, and filtered through silica gel (125 g). 
After all the non-reacted sterols had been washed off the column, the 
adduct was eluted with 50% ethyl acetate in hexane. The yield of pure 
adduct was 14.1 g, or 90.4%. NMR, IR and mass. spec. were in agreement 
with the proposed structure. 
EXAMPLE 1(b) 
##STR10## 
To a solution of the Diels-Alder adduct prepared in the previous section 
(13.9 g, 25.6 mmol) dissolved in THF (300 ml) and cooled to 
0.degree.-5.degree. C. was added dropwise 1M lithium aluminum hydride 
(LAH) in THF (80 ml, 0 mmol). After stirring at (0.degree.-5.degree. C.) 
for 1 h, the reaction mixture was warmed to 60.degree. C. for 20 min. The 
mixture was then cooled to 0.degree.-5.degree. C. and a saturated ammonium 
chloride solution was added dropwise until excess LAH was destroyed 
(.about.20 ml). The reaction mixture was stirred for 30 min and 
subsequently was left to rest for 30 min. The reaction mixture was then 
filtered through celite to remove aluminum and lithium salts. The clear 
THF solution was evaporated to give the crude trienol as a thick yellow 
syrup. The crude material was recrystallized from methanol to give a total 
of 7.3 g (74.5%) of pure cholesta-5,7,24-triene-3.beta.-ol. MP 
114.degree.-117.degree. C. NMR, IR, and Mass spectra were in accordance 
with the proposed structure. 
EXAMPLE 2 
The procedure of Examples 1(a) and 1(b) may be repeated, except that the 
phthalhydrazide contains an --NH.sub.2 substituent at the 3-position of 
the phenyl ring (as may be obtained from Aldrich Chemical Co.). The 
Diels-Alder adduct so provided may be easily separated using acid 
extraction from the remaining neutral sterols. 
EXAMPLE 3 
The procedure of Examples 1(a) and 1(b) may be repeated, except that the 
phthalhydrazide contains an --NH.sub.2 substituent at the 4-position of 
the phenyl ring (as may be obtained from Aldrich Chemical Co.). The 
Diels-Alder adduct so provided may be easily separated using basic 
extraction from the remaining neutral sterols. 
EXAMPLE 4 
The procedure of Examples 1(a) and 1(b) may be repeated, except that 
4-phenyl-1,2,4-triazoline-3,5-dione (e.g., as may be obtained from Aldrich 
Chemical Co.) is substituted for phthalhydrazide and no oxidizing agent is 
needed or used.