CA1079718A - 1.alpha.,24-DIHYDROXYCHOLECALCIFEROLS, NOVEL PRECURSORS THEREOF, AND PROCESSES FOR PREPARING THEM - Google Patents

Abstract

ABSTRACT
This invention relates to novel 1.alpha.,3.beta.,24-trihydroxycholesta-5,7-diene, derivatives thereof and a process for preparing them. The novel 1.alpha.,3.beta.,24-trihydroxycholesta-5,7-diene and derivatives thereof can be converted to novel 1.alpha.,24-dihydroxycholecalciferol expressed by the following formula 5 - a) which is useful for controlling the calcium metabolism of warm-blooded animals. The novel 1.alpha.,3.beta.,24-trihydroxycholesta-5,7-diene and derivatives thereof are expressed by the following formula

Description

~C~7~3~

This invention relates to novel 1~,3~,24-trihydroxycholesta-5,7-diene, derivatives thereof and a process for preparing theln.
m e novel 1~,3~,24-trihydroxycholesta-5,7-diene and derivatives thereof can be converted to novel 1~,24-dihydroxycholecalciferol expressed by the following formula OH
~'l~/
I ¦ (5-a) ,y 0 (~1 [
. which is useEul for controlling the calc:iun metabolism of wc~nm-blooded . animals.
The novel 1~,3~,24-trihydroxycholesta-5,7-diene and derivatives thereof are expressed by the following formula ~ OR3 ,~ 20 ~\,`,`~
'.',. R10 wherein Rl, R2 and R3 are identical or different and represent a hydrogen ' ato~n or a protective group convertible to a hydrogen at~n.
According to the present invention there is provided a process for preparing 1~,3~,24-trihydroxycholesta-5,7-diene or its derivative :,', ~ ~

~I r3 ~ 1 :It, ~ :
.~ , . . .
.,.

,. .

,. .

~7~

expressed by the follcwing formula OR2 ~ ~ (3) J

wherein Rl, ~ and R3 are identical or different and represent a hydrogen . atcm or a protective group c~nvert.ible to a hydrogen atc~ without changing the structure of the above form~la, which comprises ~ reacting a 1~,24-dihydroxycholesterol derivative expressed by the formula s~ OR6 ~:: OR5 ~ ~ (2) RL~O~W
wherein R4, ~ and R6 are identical or different, and represent a protective . group convertible to a hydrogen atc~ without changing the struc-ture of ;~ 20 the resulting formula (3), ,:~
.; with an allylic brc~mnating agen-t in an inert organic medium, then conkacting the resulting reaction m1xture with a clehydrobrc ~ nating agent to form a 1~,3~,24-trihydroxycholesta-5,7-diene derivative expressed ~:.
. by the follc~ ng formula :, s .,.
, ~ ~ -la-, ~, .
,,~ - , .

797~8 OR~

. ~ ~ (3') ~
~~ .
wherein R4, R5 and R6 are as defined above, and, ~here required, splitting . off the protective grcups to form the corresponding la,3~,24-trihydroxycholesta-. 10 5,7-diene-The above formLla (3) generically represents la,3~,24(S)-trihydroxy-cholesta-5,7-diene and i~s derivative (to be referred to as la,3~,24(S)-(OH)3 diene and its derivatives of the follcwin~ for~ula, ... .

.~ .

.,. --Ll~
:.

~,. .

:~' ..,

2 ~ ~ ~ (3-1) ~b~

wherein Rl, R2 and R3 are the same as defined above, 1~,3~,2~R)-trihydroxy-cholesta-5,7-diene and its derivatives ~to be referred to as 1~,3~,24(R)-(OH)3 diene and its derivatives) of the ~ollowing formula : OR3 C ~ t3-2 .~ .

wherein Rl, R2 and R3 are the same as defined above and a mixture of a lal3~,24(S)-epimer of formula (3-1) and a la,3~,24(R)-epimer ~., of formula (3-2).
i~ In the present specification, the above m.ixture oE 1,3~,24(S)-opimer of formula (3-1) and a la,3R,2~(R)-ep.imor of formula ~3-2) in optional ratio is expresse~ by tho followi.n~ formula ~,, \ ~/ ' ' OR2 (3-3) .

: .

~ : :

1~7~8 In other ~orcLs, -OR3 at the 24-position is expressed by `\~

The same representation as in the above formulae (3), (3-1) and (3-2) is used to express precursors and final products of these compounds. Of the la,24-dihydroxycholecalciferols and derivatives thereof, which can be derived from the la,3~,24-trihydroxycholesta-5,7-diene and derivatives thereof of formula ~3) ha~e most useful pharmacological effects, as has been ascer-tained by the work of the inventors of the present applications.
The lct,2~-dihyclroxycholecalciEorols oE Eorrnula (5-a) also represont (i) la,24(S)-clillydroxycholoc~llc:i:f():rol [la,2(1(S)-L)ll('CI, t:i:i) 10~.,2/l(R)-cl.illydroxy-cholocalciferol [la,24(R)~DLICC], ancl ~iii) a mixturc of la,Z~S)-DllCC alld ~, la,24~R)-D~ICC [la,24-D~lCC epimeric mixture].
As wi~l be shown hereinbelow by detailed animal tests, the la,24~S)- .
DHCC alone, the la,24~R)-DHCC alone, and a mixture of these all show a very , useful pharmacological activity as a controlling agent for the calcium metabolism of warm-blooded animals, and have a far lower toxicity ~LD50) than la-hydroxycholecalcierol ~la-LlCC) which is a known analog o-E active forms of vitamin D3.
The la-hydroxycholecalc~erol~la-~lCC) is oxpresscd by -the follow-i 20 ing formula ,:
: ,~
~ - 3 -,~
:;'.~. '' ~5 , . .

97~

B-l) t~O` ~ O~l To the best of our knowledge, there has been no report on the above ld,24-dihydroxycholecalciferols, and the only exception is the report of the present inventors in The Journal of Steroid Biochemistry, Vol. 5, No. 4, June 1974 - Fourth International Congrc~ss on tlormonal Steroicls - Mexico City, 2-7 Soptembor 197~, ~bstracts Oe Papors Prosolltocl (actually pub.l:ishccl on L6th ~ugust, :L97~). Ihc ropor-t stntos tll.lt 24-hyclroxycholosto:ro:L ~24-llC) of tho following formula OH

C~ ~ (B-2) $ 1-10 2~J25-d:ihydroxycholesterol t24,25-l)tlC) o:E tlle follow:ing formula v 0~
OH

n (B-3~

;'.
:
~ . .
.~ - 4 -) :', , : :

i; ~

~L~7~

and their lq-hydroxy derivatives can be converted to vitamin n derivatives by conventional establishcd procedures. I-lowever, it was only the compounds of formula (B-2~ and (B-3) which tlle inventors could then convert to vitamin D
deriva~ives. Accordingly, in the above-cited Congress in Mexico City, they only stated that the conversion of the l~-hydroxy derivatives of (B-2) to vita-min D derivatives were still under investigation, and did not give any report on the conversion of the l~-hydroxy derivatives of (B-3) to vitamin D
derivatives. According to the works of the inventors made then and their sub-sequent investigations, the procedures known prior to the filing of the present invention could not aford vitamin D derivatives cholecalciferol-type from these l~-hydroxy derivatives, as will be mentioned below. Now, however, the inventors succeeded in synthesi~ing a group of novel 1~,3~,24-trihydroxy-cholosta-5,7-di.ono ~mcl lts dorivativos oE Eormula (3) an~ a gro~lp oe novcl L~,2~-~llhy(lroxycllolocalcieoroL an~ its ~lorivat;ives, preEorahly oe eormula ~5-a~
by tho procoss ot this lnvontion to bo ~cscrlbo~ horoillbelow.
The process of this invention will be described below in detail.
[1] First reaction step:-According to this invention, 1~,24-dihydroxycholesterol (to be referred to as 1~,24-~HC) of the following formula (2-a) 0~1 `~~Y
~l ~ ~ 1 t2-a) 1~0~
is prepared by reacting 1~-2~-epoxy-24-ketocholesta-4,6-dien-3-one (to be referred to as 1~-2~-epoxy) of the following formula :

~0~7~1~

~' -`T' "~ , L~
o with an alkali metal and a proton donor in the presence of liquid ammonia or a liquid amine.
la,24-DIIC expressed by formula (2-a) means a mixture o:E la,24~S~-dihydroxycholesterol [la,24(S)-DHC] and la,24(R)-dihydroxycholesterol Ela/24(R)-DHC] in optional ratios, and according to the method of the above first step, la,24-DHC is obtained as a mixture of (S)-epimer and (R)-cpimer.
To the best of our knowlodgu, thc la,2~-~poxy of formula (l) ancl la,2~-DIIC oE ~ormula (2-a) aro hoth novel compouIlds not doscribod in tho priorLitcratur~, but synthcs;izcd tor thu first time by the inventors of tlle prescntapplication.
The above la,24-~HC can be converted to a group of la,24-DHCC and its derivatives, especially of formula (5-a), by the process described below.
In addition, these compounds are useful intermediates for other steroids hav-ing physiological activatives, for example, intermediates for the synthesis of la,2~,25-trihydroxycholecalciferol which is an active form of vitamin D3.
Exampl~s of tho liquid amines used in the ~irst st~p are primary, secondary or tertiary alkylamines such as methylamine, cthylamine, diethyl-amine, or triethylamine. The use of liquid ammonia is preferred, however.
Although there is no particular restriction on the liquid ammonia, it is pre-ferably treated, for example, by distillation, to remove water from it as much as possible. The suitable amount of the liquid ammonia or the liquid amine is 5 to 500 times, especially 10 to 200 times, the weight of the la,2a-epoxy-24-,, '' ' ' ,' ~:

::: ',:,' ' ketocholesta-4,6-dien-3-one.
Examples of preferred alkali metals are lithium, sodium, and potassium. Lithium is especially suitable. Ihe amount of the alkali metal is excessive with regard to the 1~,2~-epoxy of formula (1), for example, 5 to 250 times (atomic equivalent), especially 10 to 200 times, the amount of the lc~,2~-epoxy of formula (1).
Preferably, an inert organic solvent for the 1~,2~-epoxy of formula (1) is used in order to have the reaction proceed smoothly. Examples of pre-ferred solvents are ethers such as ethyl ether, tetrahydrofuran, dioxane or 1,2-dimethoxyethane, aliphatic hydrocarbons such as ligroine, pentane, hexane, cyclohexane or methyl cyclohexane, and mixtures of two or more of these.
The amount of the orcganic solvent is at least equal to the volume of thc liquid ammonia uscd, pro~orahLy I to 2 timos ttlO voLumo ot thc liquid no~
Suitlhlo proton donors aro ~Immoniulll s~alts ~ucl~ ag alllmorliulll salts ot organic acids and ammonium salts of inorganic acids. Specific examples of the ammonium salts are ammonium chloride, ammonium bromide, ammonium carbonate, ammonium sulfate, ammonium phosphate, ammonium hydrogcnp hosphate, ammonium acetate, ammonium formate, ammonium benzoate, ammonium benzenesulfonate, and ammonium p-toluenesulfonate. Of these, the ammonium salts of inorganic acids, such as ammonium chloride, are especially preferred. The amount of the alnlnonium salt is at least equimolar to the alkaLi mctal uscd, preEerably up to 10 molar times the latter.
Lower alcohols such as methanol, ethanol or tert-butyl alcohol can also be used as the proton donor The method of adding the reaction reagents is very important in performing the first step of the process of this invention, and advantageously, any one of the following three adding methods is chosen.

9~3L8 (A) The 4~,2~ epoxy is added to a mixture containing liquid a~nonia and the alkali metal, and then the a~nonium salt is added. At this time, it is more preferred to add the ammoniwn salt successively in two or more small portions.
(B) A minor proportion (desirably less than 0.7 molar time the amount of the alkali metal used) of the ammonium salt is previously added to a mixture containing the liquid anmonia and the alkali metal. The 1~,2~-epoxy is added to this system, and then the remainder of the ammonium salt is addecl.
(C) The la,2a-epoxy and the ammonium salt are added simultaneously in small portions to a mixture containing the liquid ammonia and the alkali metal.
Of the above methods, the method (A) is especially preferred.
The reaction temperature is usually from -70 to the refluxing temperaturo oE li(luid amlllonia ln tho roact;Loll systom.
It is advmtagoous tllat a~tor a(ldillg all Oe tho proton donor, tho reaction is carried out until tho alkali metal used in excess is compl~tely decomposed under reflux of the liquid ammonia. This can afford the final la,24-DHC in a higher yield.
A process has previously been known which comprises reacting l~,a2 -epoxy-cholesta-4,6-dien-3-one of the formula ~/y 0 1 ~ ~ ~ (b-d) O
with metallic lithium and ammonium chloride in the presence of liquid ammonia to orm la-hydroxycholesterol (la-HC) of the formula .. ' ,' ,: ':, :
.
' .

1~7~7~LB

o~
~ 5) H0~J
for the purpose of obtaining l~-hydroxycholecalciferol (b-l) (D.H.R. Barton et al., J. Am. Chem. Soc., 95, 2748, 1973).
It has been found that the 1~,2~-epoxy used in this invention is substituted by an oxo group (-0) at the 24-position, and according to this invention, the four-stage reducing reaction of such a 1~,2~-epoxy, that is, ~i) the reduction of the carbonyl group at the 24-position, (ii) the reduction of the 1~, 2~-cpoxy group, (iii) tho ro~ ction Oe tho ~,6-clior~o to a 5-ono, antl L0 (iv) tho rotluctioll oE tho carbollyl group ,at tho 3-l)ositit)ll, procoeds smoothly Ln a singLo stop, and thclt when preterrotl condltions aro employed, 1~,2~-dihydroxycholesterol (1~,2~-DIIC) can be synthesized in a high yield of, say, 60 to 75%. The fact that such a four-stage reducing reaction ~stages (i) to (iv)] proceeds smoothly in a single step is neither disclosed in any prior publication, and this is believed to be a novel reaction dis-covered for the first time by the inventors of the present application.
[2] Preparation of the lt~,2t~-epoxy:-Tht) 1~,2 -epoxy Oe tormula tl) use(l as a starting ~nator:ial in the first step of the process of this invention can be easily prepared, for example, by the following process using as a starting material fucosterol of following formula - 9 _ ~o~

``~\r'"
~ I (A-l) H~,~J
present in great quantities iTI brown marine algae. One example of this pro-cess is as follows:
Fucosterol is oxidized with ozone at low temperatures, and the resulting ozonide is reduced with an acetic acid/zinc system to form 24-ketocholesterol of the following formula o ``1--`''"1~1' ~ A-2) 110~
in a yield of about 60 to 75%. The resulting 24-ketocholesterol is oxidized, for example, with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) under reflux of, for example, dioxane to form cholesta-1,4,6-triene-3,24-dione of the :Eollowing .eormula o ~/' ) ~ ~ (A-3) ~ .
.' o - : ' . .
. .
: , ' ' ' ' ., ' ' ' ' ; ~ :
.
,~' . ' ,, , , ~ , ~t'971~

and then this oxidation product is epoxidized at room temperature under alkaline conditions ~pll about 7.5-9) using hydrogen peroxide, for example.
'~lis method makes it possible to synthesize the la,2~-epoxy of formula (1) in a yield of about 40 to 55% from the 24-ketocholesterol of formula (A-2).
The cholesta-1,4,6-triene-3,24-dione of formula (A-3) is also a novel compound synthesized for the first time by the inventors of the present application.
When a lower alcohol such as methanol is used as a solvent for the above epoxidation reaction, the resulting 1~,2~-epoxy precipitates from the lower alcohol solvent. The precipitated epoxy can be separated from the reaction mixture by filtration, ancl clirect'Ly used as a start-ing material in tho first stop of tho procoss of this invorltion.
[31 Soparat;Lon o~ (S)- an~l (R)-oyimors of L~,24~
As ~llro~l~ly St1tO~, tllo Ll~,24-~ ( o~ or~ lLI (2) ol)t~lino~ l tllo first step of the process of this invention is obtained as a mixture of 1~, 24~S)-DHC and la,24~R)-DHC.
It has been found that according to this invention the (S)-epimer and the (R)-epimer can be separated smoothly by substituted- or unsubstituted-benzoylation of the hydroxyl groups at the 3- and 24-positions of the 1~,24-DHC. In other words, the l-position may be a hydrvxyl group itself or protect-o~l by a protoctivo group convortible thcrcto Ln order to perform the scparation of the (S)- and (R)-epimers.
Substituted-or unsubstituted-benzoylation of -the 3- and 24-positions of the 1~,24-DHC can be performed by reacting the 1~,24-DHC with substituted or unsubstituted benzoyl chloride for example in an inert organic solvent in ~' the presence of an organic base as an acid acceptor. This reaction is a conventional reaction kno~n as a Schotten-Baumann reaction. An organic base . , , :
.'' ' , , such as pyridine can be used in the above reaction as the inert organic solvent, and in this case, thc use of an acid acceptor is not particularly required.
Substituted- or unsubstituted-benzoylation of the 3- and 24-positions of the 1~,24-DHC can be selectively achieved by reacting it with substituted or unsubstituted benzoyl chloride for example in a pyridine solution at a low temperature ~e.g., -5 to 10C.) for a suitable period of time (e.g.~ 10 to 24 hours). The hydroxyl group at the l-position can be acylated by, for example, reacting a sterol whose hydroxyl groups at the 3- and 24-positions are pro-tected, with an acyl chloride in a pyridine solution at 0 to 50C. The hydroxyl group at the l-position can be trimethyl-silylated, for example, by reacting a sterol whose hydroxyl groups at the 3- and 24-positions are pro-tectod, with N-trimothylsiIyl imidLIæoIo in n pyridlIlo solution at a higl t~mpor~tur~ (o.~., 50 to 110C).
By perEorming tho su~stitutcd- or unsubst:ituted-bQnzoylatioIl oE
1~,24-DHC at higher temperatures, for example, 15 to 100C., preferably 30 to 80C., the 1-, 3- and 24-positions o~ 1~,24-DHC can be benzoylated in one step.
Examples of the substituted or unsubstituted benzoyl groups are benzoyl, p-bromobenzoyl, 2,4-dibromobenzoyl, p-chlorobenzoyl, and p-nitro-benzoyl groups. OE tllese, benzoyl and p-bromobenzoyl groups are particularly pro~orrocI.
The l-position of 1~,24-DIIC may be a hycIroxyl group itself. Ex-- amples of protective groups for it are the above substituted- or unsubstituted benzoyl groups, acyl groups (organic carboxylic acid residues) or groups cap-able of forming an ether linkage. Examples of such acyl groups are acetyl, propanoyl, butanoyl, pentanoyl, caproyl, cyclohexanoyl, chloroacetyl, and bromoacetyl groups. Examples of protective groups capable of forming ether . - . . . .
.
': '; ' ' ' :, .

~ .~ ' ' , ' .

97~L8 linkages with thc hydroxyl group at the l-position are a t-butyl group, a benzyl group, a triaryl group such as a triphenylmethyl group, a tetrahydro-pyranyl group, a methoxymethyl group, or an alkyl-substituted silyl group such as a trimcthylsilyl group.
The above acyl groups including the substituted or unsubstituted benzoyl groups are preferred as protective groups for the hydroxyl group at the l-position.
In order to separate the 1~,24-DIIC into the (S)-epimer and the (R)-epimer, the la,24-D~lC of formula (2-a) is first converted to a benzoyl derivative of the 1~,24--D~IC expressed by the :Eollowing formula OR~4 '`~

~ 2-t~) V ~''~ ''''''' R"40 wherein R"4 is a substituted or unsubstituted benzoyl group, and R5 is a hydro-gen atom or a protective group convertible to a hydrogen atom.
This benzoyl derivative is then subjected to chromatography using a carrier at least containing silicon dioxido (SiO2) to separate it into a 1,24(S)-epimer and a 1,24~R~cpimor.
rhcso opimors aro also novol compoullds synthos:izocl and soparcltod successful.ly for the first timo by the lnventors of the present application.
The carrier containing silicon dioxide may, for example, be silica gel, silicic acid, silica-alumina gel, diatomaceous earth, or kaolin. The silica gel, silicic acid and silica-alumina gel consisting mainly of silica are especially suitable.
Chromatography can be carried out by any desired type of procedure, -~ - 13 -~'797~&1 such as high pressure liquid chromatography, thin-layer chromatography, or column chromatography. tIowever, for separation on a large scale, colwnn chromatography is preferably used. The colwnn chromatography is carried out~
for example, by using silica gel as a carrier and n-hexane, benzene, ethyl acetate, methylene chloride, or ether as a developing solvent to obtain the epimers in a pure form. Impure fractions may be repeatedly subjected to column chromatography, or fractionally crystallized to remove small amounts of impurities.
The developing speed of the benzoyl derivative of 1,24tR)-D~IC is high, whereas the developing speed of the benzoyl derivative of 1~24(S)-DIIC
is low.
[~] Second reaction stcp (synthosis of 5,7-diene de-rivativo):-~ccording to this inventLon, -tho 1~,24-I)I-IC ot formula (2-a~ obtain-od by thc Elrst-stcI~ roaction is convertocl to a protcctod derivativo ot 1~,3~,24-trihydroxycholesta-5,7-diene after protecting the hydrogen atoms of the hydroxyl groups at the 1-, 3- and 24-positions with a protective group convertible to a hydrogen atom, or after separating the 1~,24-DHC benzoyl derivative of formula (2-b) into the 1~,24(S)-epimer and the 1,24(R)-epimer, or after converting the benzoyl protective groups in the 1-, 3- and 24-pGsitions of formula (2-a~ to other protective groups convertible to hydroxyl groups.
IhUS, according to the prcsent invention, at least onc of protected I~3~24(s)-trihydroxychol0sta-5~l-d:iollo dcrivative an~ protectod 1~ ,24(R)-trihydroxycholesta-5,7-diene derivative (these will be referred to as 5,7-diene derivatives for simplicity)of the formula .. - 1~ -.. . O.' ~
.' ., .

!
':
,, ,` . . .

'9~i8 OR'6 OR'5 ~ ~ I (3~) , 1/~ -R' O ~ ~\ ~
can be prepared by reacting at leas~ one of a protec:ted derivative of 1~,24(S)~dihydroxycholesterol and a protected derivative of 1~,24(R)-dihydroxycholesterolof the following formula OR'6 5 ~ \ ~ ~ I (2) L
~,,,1 o wherein R'4, R'5 and R'6 are identical or different, and represent a protective group convertible to a hydrogen atom without changing the structure of the following formula ~3'), with an allylic brominating agent in an inert organic medium, and then con-tacting the reaction mixture with a dehydrobrominating agent.
It is important that in the second step o:E forming the above 5,7-diene derivativcs, the hydroxyl groul)s at tho 1-, 3- and 24-po~:itions oE tlle 1~,24-D~lC and its epimers of ormula (2) are protected. I:E the second-step reaction is carried out without protecting these three hydroxyl groups of the 1~,24-DHC, the oxidation and decomposition of the hydroxyl groups cannot be prevented, and the yield o the intended 5,7-diene derivatives becomes very low.
The protective group may be any protective group capable of being __ - 15 -.

' ~ ~ . . . .

; ' :

7~

converted to a hydroxyl group at its 1-~ 3- and 24-positions without breaking the cholesta-5,7-diene skeleton after conversion to the 5,7-diene derivative.
Examples of such protective groups are shown below.
(1) Acyl groups:
Cl-C12 aliphatic or aromatic carboxylic acid residues or their nitro-, halogen- and alkoxy-substituted derivatives, for example, acetyl, propanoyl, butanoyl, pentanoyl, capronyl, cyclohexanoyl, chloroacatyl, bromo-acetyl, benzoyl, p-bromobenzoyl, p-nitrobenzoyl, ethylbenzoyl, and toluyl groups. Of these, acetyl, benæoyl and propanoyl groups are especially pre-ferred.
(2) Groups which form ether linkages with hydroxyl groups:
A tert.-butyl group, a benzyl group, a triarylmethyl group such as a triphonylmethyl group~ a totrahydropyr~myl grou~), a Illothoxymothyl group and an alkyL-substitutod silyl grouL) such as a trimothylsilyl group. Ot thc abovc protoctivo groups, the acyl groups (l~ aro ospccialLy I7roeorrod, but tho invention is in no way limited to them.
The allyl-position brominating agent may be any compounds which are usually employed for the bromination of an allyl position, and for example, N-bromosuccinimide, 1,3-dibromo-5,5-dimethylhydantoin and N-bromocaprolac~am are preferably used. Usually, the amount of the allylic brominating agent is 1 to 2 equivalent times the amount of the 5-ene of formula ~2).
Iho reaction in tho prosont invention is first botweon the l~r,2~-D~IC of formula t2), preferably a protccted 1~,2~-U~IC or its protccted epimer, and the allylic brominating agent in an inert organic medium. Suitably, this reaction is carried out at room temperature to 1~0C.
The inert organic medium is one which does not react with the brominating agent. Advantageously, it is, for example, a hydrocarbon or halo-genated hydrocarbon, such as hexane, heptane, cyclohexane, ligroine, benzene, ~ .~

~97~8 toluene, xylene, bromobenzene, chlorobenzene, nitrobenzene, carbon tetra-chloride, 1,2-dichloroethane, or 1,2-dibromoethane. An ether solven~, such as Ce //~so/~ e ~ /v5~ /~e ethyl ether, tetrahydrofuran, dioxane, metllyl e~ w~ or phenyl er~es~}~e, can also be used. These inert organic solvents can be used either alone or in admixture.
The reaction proceeds at the above temperature. If desired, the reaction can be carried out under the irradiation of actinic light having a wavelength corresponding to infrared to ultraviolet rays. In this case, the reaction proceeds at a temperature lower than room temperature. A radical initiator, such as azobisisobutyronitrile, benzoyl peroxide or cyclohexyl hydroperoxide, can also be added in a small amount.
The dehydrobrominat:ing agent is preferably trimethyl phosphite, s-collidille, or diethyl aniline, ancl thoy may bc usod in combincLtion. Theoreti-cally, tho rcactioll proccods tully when tho anlount oE tl-o dohydrobrorninating agont is o~luimol-lr to tllo bromill.ltocl product obtained by tllo provious brominat-ing reaction, but in order to secure clesirable rates of reaction and yields, the dehydrobrominating agent is used preferably in an amount of at least about 2 molar times the amount of the brominated product. Since the dehydrobrominat-ing agent also acts as a reaction medium, there is no particular upper limit to its amount.
UsuallyJ the reaction proceeds in good condition at a temperature o~ 80 to 250C., preferably 120 to 150C. 'I'he reaction time varios according to the type of the dehydrobrominating agent, the type of tlle reaction solvent, etc. For example, in a preferred embodiment wherein the reaction is carried out under reflux in a xylene-s-collidine system, the reaction is completed ~ithin a short time of about 20 minutes.
After the end of the reaction, the solvent is distilled off at reduced pressure to afford an oily crude 5,7-diene derivative.

1~qdemar k ., [5] Third Reaction Step (Purification of Crude 5,7-Diene Derivative):-As stated above, a method for preparing l~-hydroxycholesterol (1~-IIC) of the formula ~ ",~ ~

01l ~ ~ (B-5) ~10~
has already been known (D.ll.R. Barton et al., J. Am. Chem. Soc., 95, 2748, 1973).
It has al.so been known that the above l~-hydroxycholesterol (l~-HC) is converted tol~,3~-diacetoxycholesta-5,7-diene of the formula ~\C ~ ~ I (U-6) CO ;~
wherein Ac is CH3CO-, by the same reaction as in the second step of the present invention described above, and this product is then thermally rearranged by irradiation of ultraviolet rays to convert it to 1~,3~-diacetoxycholecalci-ferol (vitamin D derivative) of the formula ~/\,/y ~ ~ ~B-l) ~~- .

~ .
Ac ~ Ac : - 18 -.

.

- ' ~

~ 9~

The inventors, therefore, attempted to thermally rearrange the 5,7-diene derivative of the formula OR5 / ~ 1 ~3~
1~'"~

obtained in the second step by direct irradiation of ultraviolet rays by the method known in regard to the synthesis of the 1~,3~-diacetoxycholecalciferol of formula (B-l), but the desired 1~,24-dihydroxycholecalciferol derivative of the formula l1~6 `~'~' `r" `

~ ~ ~5) C~
4 OR5 .
: tYherein R4, R5 and R6 are the same as defined in formula (2), could not be :LO lso:lated.
'I'he inventors presumed that th:is is becausc the 5,7-dione dcriva-tive of formula ~3') is impure and probably contains a 4,6-diene derivative , of the formula ~' ~ .u, ~,,, ~,:
, .
', - . ~ ' , ' .' ' ' ' ~' ' .

~ ~97~3 lR6 R O ` (6) wherein R4, R5 and R6 are the same as defined above. On this presumption, ~he inventors attempted to purify the crude 5,7-diene derivative formed and separated in the second step described above, by a known adsorption chromato-graphy using silica gel as an adsorbent, that is, thin-layer chromatography or column chromatography, but none of these chromatographic methods could separate the 4,6-diene derivativo of formula (6) from the cruclc 5,7-diene dorivativo oE Eormula (3').
~n oxanlplo is known in which cllromato~raplly using silver nitrato was applied to tho separltLon ancl purificatioll of acetoxycholosta-5,7-dienos (Tetrahedron Letters, No. 40, 4147, 1972, and German OLS 2,400,931).
Thus, the inventors hit upon an idea of applying the above silver nitrate chromatography to the above crude 5,7-diene derivative of formula (3') and attempted to purify the crude 5,7-diene derivative by column chromatography using 2~ silver nitrate-silica gel. But the attempt failed, and the 4,6-diene dcrivative could not be separated.
Various subscqucnt invostlgat:ions about the mcthod oE purifying the crude 5,7-diene derivative finally led to the discovery tllat the free 5,7-diene can be separated very well from the free 4,6-diene and other impurities by a chromatographic method comprising splitting off the protective group from the crude 5,7-diene derivative of formula ~3') to convert it to 1~,3~,24-trihydroxycholesta-5,7-diene (the free 5,7-diene) of the formula ,. _ : .
. .

~'797~8 ~1 0~l ~ ~ (3-a) ~ 10~/
and bringing it into contact with a carrier containing silicon dioxide and hav-ing adsorbed thereto non-metallic silver.
This purifying process of the invention can be applied not only to the crude protected 5J7-diene derivative obtained in the second step, but also to the 5,7-diene derivative in 2~tS)-epimer form or the 5,7-diene derivative in 24(R)-epimer form. In any case, it is essential that such a crude compound is subjected to the purifying process of this invention after splitting off the protective groups at the 1-, 3- and 2~-positions to convcrt it to the froe 5,7-dlene o~ ormula (3-a) Suitable carriers containing sLlicon dioxicle and having adsorbed thereto non metallic silver are silica gel and silica-alumina gel containing or having adhered thereto silver nitrate. The silica gel is particularly advan-tageous.
Such silica gel is prepared, for example, by dissolving silver nitrate in water or a suitable organic solvent such as acetonitrile or alcohols, mixing the solution with silica gol, and then evaporating the solvent ~rom the mixture. When it is used ~or coLumn chromatography, it is activated>
if desired, and filled in a glass column. When it is used for thin-layer chromatography, a suitable fixing agent, such as gips or starch is incorporated in the silica gel prepared in the above manner, and if desired, a fluorescent agent is also added to spread the mixture on a suitable plate ~glass or a , ~:

~ , :, ". .
.. .
. ..

~0797~8 polyester film) in a thickness of 0.2 mm to 2mm, fi~ed, and activated.
In order to impr0gnate silver nitrate in a thin-layer chromato-graphic plate made only of silica gel, the plate is immersed in a solution of silver nitrate in an organic solvent ~for example, acetonitrile or an alcohol) in a suitable concentration, dried, and then activated.
The amount of silver nitrate used in the process of this invention is suitably 0.1 to 20% by weight based on silica gel, especially preferably 0.5 to 10% by weight. When the amount o silver nitrate is less than 0.1% by weight or larger than 20% by weight, the separating ability becomes poor. In the latter case, the silver nitrate is wastefully used.
Examples of a developing solvent or an eluting solvent used for thin-layer chromatography or column chromatography are organic solvents such as cyclohexane, n-hexane, benzeneJ toluone, chloroform, 1,2-dichloromethane, 1,2-dlchloroothano, acoton~, diethyl othor, tctrahycl~ouran, ~thyl acetate, methanol, ethanol, and propanol, and mixtures o~ these. OE these, mixtures of low-boiling solvents such as n-hexane, benzene, chloroform, acetone, ethyl acetate or methanol are particularly suitable. Suitable mixing ratios can be easily determined experimentally.
The methods o development, elution, and detection can be satis-factorily performed in accordance with conventional chromatographic techniques.
The inventors found as a result of applying this purifying method, tho crude 5,7-dien~ derivative obtain~d in th0 second step contains the 4,6-diene in a weight ratio o about 1/2 to 1/4 based on the puri~ied 5,7-diene.
Such a great quantity o the 4,6-diene can be separated and removed rom the 5,7-diene substantially completely by the purifying ~ethod of ~his invention.
It has been found that this puriying method makes it possible to obtain the above free 5,7-diene easily in high purity and yield, and that as will be described hereinbelow, this 5,7-diene, either as such or after protect-' '' ' . ' .
. , : " ' ' '. : . . :, ' , . .
.

~0797~L8 ing at least one of the hydro~yl groups at its 1-, 3- and 24 positions with the same protective group as described hereinabove, can be converted to 1~,24-dihydroxycholecalciferol or its protected derivatives by irradiating ultra-violet rays and then rearranging it ~isomerization~.
It is belie~ed therefore that the purifying~ method in the third step of the present invention is of very high commercial value.
[6] Synthesis of la,24-cholecalciferol or its protected derivative and its pharmacological activities:
la,24-Cholecalciferol or its derivatives resulting from the protec-tion of at least one hydroxyl group thereof with a protective group as repre-sented by the following formula OR~

(5) R '` ~ R2 wherein Rl, R2 and R3 are the same as deined in formula (3-b), can be obtain-ed byirradiating ultraviolet rays on at least one of the ree 5,7-diene purified and separated in the ~hird step or its derivative result:ing from the protection of at least one of the hydroxyl groups at the 1-, 3- and 24-positions of the free 5,7-diene expressed by the formula .~. .. ~ .

~ 1~3797~ :

O-R2 ~ ~ (3-b) Rl O ~
wherein Rl, R2 and R3 are identical or different and represent a hydrogen atom or a protective group convertible to a hydrogen atom without changing the structure of formula C5), in an inert organic solvent, and then i.somerizing the resulting product.
The free 5,7-diene or its protected derivatives of formula ~3-b) may be laJ3~,24(S)-trihydroxycholesta-5,7-diene or its protected derivatives, or lu,3~,2~R)-trihydroxychole~ta-5,7~iene or its protected derivat:lvos, or mixturos of those :in arbitrary rE~tios, and they can bo convorto~ to 1~,24 (S)-type, la,24(R)-type or mixod typo dlllydroxycholocalciCerol or :Its protoctecl derivatives by the process.
When Rl, R2 and/or R3 in formulae ~3-b) and ~5) represents a pro-tective group, it may be the same protective group as represented by R'~, R'5 andjor R'6 in formula ~2). The purified la,3~,24-trihydroxycholesta-5,7-diene of formula (3-a) can be converted to its protected derivative by the same method as described hereinabove with respect to the separatlon of the la,24-WIC into the ~S)- and ~R)-opimers in paragraph ~3~ abovo.
In the process, however, the use of a purified Eree 5,7-diene in which all of Rl, R2 and R3 in formula (3-b) are hydrogen atoms is more advan-tageous than its protected derivatives. Since 1~,24-dihydroxycholecalciferol of the following formula __ ., -.

. . . ~ . . . : .

: ~ ~ ' ' ' . ' ' ' . ': ' ; ' - . . . ' , . .

1~797~8 o~l \, ~ (5-a) ~1 ., .

HO~ ~ OH
is a so-called analog of active forms of vitamin D3, the use of a purified free 5,7-diene of formula (3-b) permits the direct preparation of the analog of active form of vitamin D3 of formula ~5-a). On the other hand, when the protected derivative of ormula (3-b) is used, two extra steps Oe protecting the hydroxyl groups of thc purified froe 5,7-dicne and splLtting off the pro-toctlvo groups o-f tho resultinK protocted 1~,2~-dihydroxycholccalciEorol. ~r particular, the splitting off of the protective groups involves a partial decomposition or degeneration of 1~,24-dihydroxycholecalciferol.
For this reason, too, the purifying process in the third step of this invention is a very advantageous purifying method.
The purified 5,7-diene or its protected derivatives of the formula (3-b), preferably purified free 5,7-diene, is converted to the 1~,24-dihydroxy-cholecalciferol or its protected derivative of formula (5) or (5-a) by first irradiating ultraviolet rays having a wavel~ngth of about 200 to 360 nm to the free purified 5,7-diene or its protected derivative in an inert organic solvent such as hydrocarbonsand halogenated hydrocarbons and then isomerizing so formed 1~,24-dihydroxycholecalciferol derivative at a temperature of about 10 to 120~C and finally separating and purifying so formed 1~,24-dihydroxy- !
cholecalciferol or its derivative.

., , , . - , ~ , .~- . ~ , . ~ .
, : ' ' ,, : , . ,: .
. , - :

7~L8 The 1~,2~-dihydroxycholecalciferol tl~,24-DllCC~ of ~rmula (5-a~
obtained by the metho~ described above, rnore specifically ~i~ lu,24~S~-di-hydroxycholecalciferol, (ii~ la,24(R)-dihydroxycholecalciEerol, or (iii) a mixture of tlle dihydroxycholecalciferols (i) and (ii) in optlonal ratios, is a novel compound successfully synthesi~ed and isolated by the inventors of the present application. These compounds are vitamin D3 analogs having super-ior activities, and their toxicity is low.
Thè inventors of the present application made a comparatiYe study on the activities of the novel la,24-DHCC with regard to the known la-IICC, and found that these novel compounds as will be shown in the follow:ing refer-ential examples, have at least an equivalent effect to la-HCC in intestinal calcium transport, their efect on bone resorption is less than about one-third of the effoct of the la-llCC, ancl tha~ their I,115() val~Ics arc ~ess than about ono-tcntIl o~ tllat Oe tho la-tI(:C. It has boon ascert,Iinod in particular thIt la,24(S)-DllCC has a slightly weakor action of promoting intest:inaL calciu transport than la,24(R)-DHCC, but exhibits almost no bone resorption.
Accordingly, la,24-DHCC of this invention can be used as a unique medicine having reduced side-effects and high safety as compared with the known analogs of active forms of vitamin D3 such as la-HCC and 1~,25-dihydroxy-cholecalciferol when applied, for example, to diseases induced by the abnormal metabolization of calcium. Pharmacological tests conducted by the inventors have made :it clear that various optimum pharmaceutical preparations can be formed according to different diseased cond:itions.
The following examples illustrate the present invention more spe-cifically.
Characteristics of the novel intermediates and final compounds are shown in the accompanying drawings, in which:
Figure 1 shows the mass spectrum of 1~,24-DHC;

' -`` 10~

Figure 2 shows the W spectrum of the free 5,7-d:iene;
Figure 3showsthe mass spectrum of the free 5,7-diene;
Figure 4 shows the UV spectrum of 1~,24-DHCC;
Figure 5 shows the mass spectrum of 1~,24-DHCC;
Figure 6 shows the effect on bone resorption in rats of the compounds l~-IICC and 1~,24-DHCC: and Figure 7 shows the effect on body weight change in rats of the compounds la-HCC; lu,24-DHCC; 1~,24(R)-DHCC and 1~,24~S)-DIICC.
The test methods used in these examples for the determination of the characteristics of the final products were as follows:
Unless otherwise specified, NMR spectra were determined by Varian - 26a -- ..~.
...... . .. . .

. . . . : . , .
~' ' ' :' '' ... .
. .

97~

EM 360 or ~EOL PS/PFT-100 (Nippon Electronics Co., Ltd.) in deuterochloroform (CDC13) using tetramethylsilane as internal standard.
Mass spectra and high resolution mass spectra were determined by using Shimad~u LKB-9000 (trademark for a product of Shimazu Seisakusho Co., Ltd.).
UV spectra were determined by tlitachi EPS-3T (trademark for a product of Hitachi Limited) using an ethanol solution.
The melting point was measured by means of a hot stage microscope, and the resulting values were not corrected.
The absolute configuration of 1~,24-dihydroxycholesterol was deter-mined as follows:
An epimeric mixture of 2~,2~-cpoxycholestorol 3-bonzoate, that is, a mixturo of "~

C~
BzO

and ~ .

B O ~

:~0797~8 wherein ~ represents a benzoyl group, is chromato~raphed using silica gel as a carrier to separate and recover thc two epimers. Each of the epimers is methanolyzed to form a 24-OII derivative and a 25-OCI13 derivative. The method for determining the absolute configuration of the two epimers by applying the modified Horean's Method, which is disclosed in Tetra'hedron Letters 1 J 15 ~
1975, was utilized. By reducing the two epimers whose absolute configuration has thus been determined with an AlC13-LiA11~4 system, a 24(R~-hydroxy derivative is obtained from the 24(R),25-epoxy derivative, and a 24(S)-hydroxy derivative is obtained from the 24(S),25-epoxy derivative. Since it is known that the latter S-derivative is more polar than the former R-derivative, it is assumed that this fact is applicable also to la,24-dihydroxycholesterol. Thus, this la,24-dihydroxycholesterol is converted to its tribcnzoatc- or dibenzoate-~erivatlvc which is thcn chromatographcd through a silica gel column. 'I'hus, a nloro po~nr opimor ls convortecl to a la,24tS)-clorivativo ancl a loss polar epimor, to a la,24-(R)-derivative.
Where in the present application, there is no specific reference to an R-derivative or an S-derivative, for example, when reference is merely to la, 3~,24-~OH)3 diene, it represents an equimolar mixture of la,3~,24(R)-(OH)3 diene and la,3~,24(S)-(OH)3 diene.
Referential Example 1. Synthesis of Starting Material:
la,2~-dihydroxycholesterol was prepared by the following four stcps (1) to t~.
(1) Synthesis of 24-ketocholesterol from fucosterol:-Fucosterol (4.1 g) was dissolved in 100 ml. of methylene chloride~
and while cooling the solution with dry ice-acetone to about -20C., the fucosterol was oxidized for 30 minutes with ozone at a rate of generation of 0.86 g/hr and in a concentration of 17.2 g/m3 (2)' After the reaction,8 g of zinc powder and 200 ml. of acetic acid were added, and the resulting , ~ . ' '~' . ~"', : . , `. ' : -. .
~' ' ' ~ ' . ... ..

107~7~
ozonide was reductively decomposed at room temperature for 24 hours. The resulting ~inc acetate was separated by filtration, and washed with an aqueous solution of sodiwn carbonate. The methylene chloride phase separated was thoroughly washed with water, and dried with anhydrous sodium sulfate. The methylene chloride was evaporated off at reduced pressure, and the resulting white crystals were column-chromatographed using silica gel as a carrier (eluted with a benzene-n-hexane mixed solvent) to afEord 2.8 g of 24-keto-cholesterol in a yield of 70.3%.
(2) Synthesis of 24-ketocholesta-1,4,6-trien-3-one from 24-keto-cholesterol:-4.0 g of 24-ketocholesterol and 7.0 g of 2,3-dichloro-5,6-dicyano-benzoquinone were dissolved in 140 ml. of dioxane, and the solution was stirred under reflux oE dioxanc for 27 hours. After the roaction, the reaction mLxture was coolecl to room temperaturc. Thc roslllting hydroquinono ~lorLvatlvo was separated by filtration, ancl washed with 30 ml. of dioxane~ I`he filtrate and the wash liquid were combined, and dioxane was e~aporated off at reduced pressure to afford a black-brown oi~y substance.
The oily substance was separated and purified by column chromato-graphy using alumina as a carrier (eluted with a methylene chloride-acetone mixed solvent) to afford 2.76 g of 24-ketocholesta-1,~,6-trien-3-one as cry-stals. Analysis of the product showed the following results.
NMR spectrum:
0.86 (3H, s, C-18-CH3), 1.20 ~3H, s, C-l9-CH3).
1.15 (6H, d, J=10 Hz, C-26,27-CH3), 5.95 - 6.10 ~3H, m, C-4, 6, 7-H), 6.29 ~lH, dd, J=ll, 15Hz, C-2-H), 7.10 ~lH, J=ll Hz, C-l-H) , . . .
.
. .

lQ7971~

Molecular weight ~by gas mass spectrum):
394 ~M ) ~3) Synthesis of 1~,2q-epoxy-24-ketocholesta-4,6-dien-3-one from 24-ke~ocholesta-1,4,6-trien-3-one-~-

3.53 g of 24-ketocholesta-1,4,6-trien-3-one was dissolved in a mixture of 100 ml. of methanol, 20 ml. of tetrahydrofuran and 50 ml. o~
dioxane, and 1.6 ml. of a 5% by weight methanol solution of sodium hydroxide and 5 ml. of 30% aqueous hydrogen peroxide were added. The reaction was car-ried out at room temperature for 24 hours. After the reaction, a small amount of acetic acid was added to neutralize the solution to a pH of 7. The reaction mixture was then extracted by adding water and ether. The ethereal phase was thoroughly washed with water, and dried with anhydrous sodium sulfate. lhe ether was evaporated oef at reduced pressure to aford 4.04 g Oe a light yellow solid. Tllo solid product was column-chromatographod usLng silica ~el (oluted with a bonzeno-cthor mixcd solvollt~ to aeEord 2.52 g of 1~,2~-epoxy-2~-koto-cholesta-4,6-dien-3-one which showe~ the following characteristics.
Melting point: 150 to 151.5C.

UV spectrum ~ethanol 291 nm max High resolution mass spectrum:
Found=410.2793 Req~lire (M ~=410.2821 ~C27H3803) NMR spoctrum:
0.80 (3~l, s, C-18-~ls), 1.15 ~3~1, s, C-19-lls), 3,43 (1~l, dd, J=4Hz, J=1.5Hz, C-2-H) 3.60 ~lH, d, J=4Hz, C-l-H), 5.68 ~lH, d, J=1.5Hz, C-4-H) 6.10 (2H, s, C=6, C-7 Hs) , .;., . . ; ,: . . . :
..
' ' ' '' - ~ ' ' ~ ' ' : :
. ' ~ ' ' ~, , .
,: . . . ~

~L~797~8

(4) Synthesis of 1~,24-dihydroxycholesterol (the first step) Liquid ammonia (10 ml.) dried with metallic sodium was trapped in a three-necked flask equipped with a cIropping funnel and a clry ice cooler while cooling the flask with a cooling medium consis1;ing of acetone and dry ice. Metallic lithium ~150 mg) was added to the liquid ammonia, and they were stirred for 10 minutes. The mixture was dissolved in 15 ml. oE tetrahydro-furan, and 100 mg of 1~,2~-epoxy-24-ketocholesta-4,6-dien-3-one was added dropwise in the course of 10 minutes.
After the addition, the flask was separated from the cooling medium~
and ammonia was refluxed for 20 minutes. Again, the flask was immersed in the cooling medium, and 1.5 g of a fully dried powder of ammonium chLoride was added slowly over the course of 2 hours. The flask was withdrawn Erom the cooling medium, and the reac-tion was contlnuecI wLth st;Lr~ing.
Stirring was stopped whon the bluo color o~ the soLutiQn completely cIisappoarod. Ihe cooler was removecI from the flc~sk, ancI nitrog~rI gas w.as introduced to remove the ammonia.
Ethyl acetate t60 ml.) and 60 ml. of lN-hydrochloric acid were added to the residue to subject it to distributive extraction. Ihe ethyl acetate phase separated was thoroughly washed with water, and dried, followed by evaporating off the ethyl acetate at reduced pressure. The residue was dissolved in 3 ml. o ethyl acetate, and chromatographed through a column containing silica gel as a carrier using an eluting soLvent consisting o~ a mixture of benzene and acetate. There was obtained 61 mg of a purified pro-duct having the following characteristics.
NMR spectrum (in C5D5N), ~ ~ppm):
0.70 (3H, s, 18-CIE3), 0.99 ~3H, s, l9-CH3), 3.28 (4H, m, 24-H and hydroxy H), .

': ,.

.
. , .

~0797~8 3.82 ~1~l, m, 1~
4.00 (lH, m, 3~-ll),

5.50 (1ll, m, 6-ll) Mass spectrum (see Figure 1):
418 (M ), 400, 382 High resolution n-ass spectrum:
Found=418.3428 Require, M ~C27H46O3) 418-3449 From the above characteristics, ~he resulting product was identi-fied as 1,24-dihydroxycholesterol.
Example 1 (A) Synthesis of la,3~,Z4-triacetoxycholest-5-ene:-300 mg of 1~,24-dihydroxycholesterol was reacted with 40 ml. of acetic anhyclrido and 125 ml. oE pyridine for 3 hours at 95C. Water was added to tho roaction mi.xture, an~l it was extracted with ~iethyl ethor. 'I'he ethereal phase was washed with an acid and then with an alkali, drie~, and evaporated to remov0 the ether to afford crude 1~,3~,24-triacetoxycholest-5-ene. The crude product was chromatographed through a column containing silica gel as a carrier using an eluting solvent consisting of benzene to afford 325 mg of purified 1~,3~,24-triacetoxycholest-5-ene as an oily purified pro-duct having the following NMR spectrum and mass spectrum.
NMR spectrum:
0.67 (3~1, s, C-18), 2.Q2 ~9tl, s, 3-acetyl), 4.8 ~2H, m~3~ and 24-H2), 5.05 ~lH, m~l~-H), 5.65 ~lH, m, 6-H) . ,: ., . . .
. ;.. , . , . .. , ~ ,:
.. ;, .. : . ,. , :
.
' . , . . ~ . . ., ~, , "

1~17~3718 Mass spectrum:
484 (~ C~13C00ll), ~24, 364 ~B) Synthesis of 1~,3~,24-triacetoxycholesta-5,7-diene (second step):~
300 mg of 1,3~,24-triacetoxycholest-s-ene was reacted with 90 mg of 1,3-dibromo-5,5-dimethylhydantoin in ~..5 ml. of n~hexane under reflux for 15 minutes. The reaction mixture was cooled, and the resul~ing 5,5-dimethyl-hydantoin were and the excessive 1,3-dibromo-515-dimethylhydratoin were removed by filtration. The filtrate was concentratecl at reduced pressure to afford 349 mg of a yellow oily substance. Xylene (2.5 ml.) was added to this substance to form a solution. The resulting solution was added dropwise over ~he course of 15 minutes to a solution held at 165C. of 0.85 ml. of s-collidine in 1.9 ml. of xylene, and the reaction was carried o~lt for an addi-tional 10 mLnutos. After tho reaction, tho hydrobromido oE s-collidine was romovod by Elltration, and the s-colLidine and xylene wore evaporate(l oEE nt reduced pressure. The residue was dissolved in diethyl ether. The ethereal phase was washed with lN-hydrochloric acid and a 5% aqueous solution of sodium bicarbonate, and repeatedly with water. It was then treated with activated carbon, and the ether was evaporated off at reduced pressure to afford 310 mg of a yellow oily substance. This oily substance was carefully separated twice by column chromatography ~elution with benzene) using silicic acid to aEford 69 mg (yield 23%) of a non-crystalline puriEied product. ~`rom tho Eollowing spectrum data, this product was identiEied as 1~,3~,2~-tri-acetoxycholesta-5,7-diene.
W spectrum, ~max (nm) 262, 271, 282, 294 NMR spectrum:
2.02 and 2.04 (9H, s, CH3C00-), -- ~,75 ~2H, b, 3~- and 24-H), ... .. .

. - , :'., . ' ~
''"', ' .......... ., : .

~9718 5.01 ClH, b, l~-H), 5.35 (lH, d, J=5.5~1z, 6 or 7-H), 5.69 (lH, d, J=5.5Hz, 6 or 7-H) Mass spectrum:
542 (M ), 514~ 482, 422, 362, 249, 204 Example 2 Synthesis of 1~,3~,24-triacetoxycholesta-5,7-diene ~second step):-150 mg of 1~,3B,24-triacetoxycholest-5-ene was reacted with 45 mg of 1,3-dibromo-5,5-dimethylhydantoin in 3 ml. of n-hexane for 15 minutes under reflux. The reaction mixture was cooled, and the resulting crystals were removed by filtration. The filtrate was concentrated at reducecl pres-sure to afford a yellow oily substance. To this substance was added 1.3 ml.
of xylene. The resulting solution was added dropwise over the course of about 15 minutes to a solution of 0.25 ml. oE trimcthylphosphitc in 4 ml. of xylcno unclor roflux, ~nd thc roaction was performed for an additiollal 90 minutes. Iho reaction mixturc was tllen concentratod at a temporature o~ 10ss than 75C. to afford 148 mg of an oily substance. This substance was treated in the same way as in Example 1, ~B). The final product showed the same W , mass, and MMR spectrum data as the 1~,3~,24-triacetoxycholesta-5,7-diene obtained in Example 1, ~B).
Example 3 Synthesis of la,3~,24-tribenzoyloxy-cholesta-5,7-diene ~second step):-200 mg of 1,3~,24-tribenzoyloxy-cholest-5-ene prepared in the same way as in Eixample 1, ~A) was heated for 15 minutes under reflux together with 55 mg of N-bromosuccinimide in 15 m~ of carbon tetrachloride. The reaction mixture was cooled, and the resulting crystals were removed by fil-tration. The filt~ate was concentrated, and 5 ml. of xylene was added to the :, :: , -... . . . .. . .

-~07~'718 residue to form a solution.
The resulting solution was added dropwise over the course of 15 minutes to a solution of 0.2 ml. of trimethyl phosphite in 4 ml. of xylene under reflux~ and the mixture was further re1uxed for 90 minutes.
The reaction mix~u~e was then concentrated at a temperature of less than 75C., and the residue was carefully chromatographed twice through a column containing silicic acid using benzene as an eluting solvent to aford 50 mg ~yield 25%) of a purified non-crystalline product. This product showed the following spectra~ and was identified as la,3~,24-tribenzoyloxycholesta-5,7-diene.
W spectrum, yetahxanol Cnm):

231, 262, 271, 282, 294 NMR spectrum:
.95 t2~l, b, 3~-~l and 2~-~1), 5.32 ~2}l, b, 1~-ll and 6 or 7-}l~, 5.72 tl~l, d, J=6~1z, 6 or 7-H), 7.49 and 8.02 ~15H, m. aromatic H) Mass spectrum:

728 ~), 638~ 620, 606, 484, 362.
Example 4 CA) Synthesis of la-hydroxy-3~,24CS)-dibenzoyloxyGholest-5-en:
2.5 g o~ la,24tS)-dihydroxycholesterol was mixed with 2.10 g o~
benzoyl chloride and 40 ml. o~ pyridine, and the mixture was allowed to stand for one day at 20 G.
The reaction mixture was treated in the same way as in Example lCA) to obtain crude l~-hydroxy-3~,24cS)-dibenzo~loxycholest-5-ene.
CB) Synthesis of 1~,3~,24CS)-triacetoxycholesta-5,7-diene (second step):-. . .
la-Hydroxy-3~,24~S~-dibenzoyloxy-cholest-5-ene was reduced with .... . ..

.

~079718 LiA1114 in dry diethyl ether to form 1~,3~,24~S)-trihydroxy-cholest-5-ene.
This product (180 mg) was then reacted with 24 ml. of acetic anhydride and 75 ml. of pyridine at 95C. for 3.5 hours. The product was treated in the same way as in Example 1, (A~, and chromatographed through a column containing silica gel as a carrier to afford 195 mg of 1~,3~,24(S)-triacetoxy-cholest-5-ene.
150 mg of this product was purified in the same way as in Example 1 to afford 43.5 mg (yield 29%) of a purified product having the following spectrum data. This purified product was identified as la,3~,24(S)-triacetoxy-10 cholesta-5,7-diene.
W spectrum, ~ ethanol (nm 262, 271, 282, 294 NMR spectrum:
2.02 and 2.04 ~9~1, s, Cl13C00-), ~.75 (2l-l, b, c-3-, C-2~-ll), 5.01 (lH, b, c-l-H), 5.35 (lH, d, J=5-6Hz, C-6 or C-7-H), 5.69 ~lH, d, J=5-6Hz, C-6 C-7-H) Mass spectrum:
542 (M ), 514, 482, 422, 362, 249, 209 Example 5 (A) Separation o~ 24(R~-der:ivative and 24(S)-derivative o la,3~, 24-tribenzoyloxycholest-5-ene:-An n-hexane solution o 1.58 g of the crude laJ3~24-triben cholest-5-ene was chromatographed through a column containing 100 g of silica gel as a carrier to divide it into fractions each having a volume of 50 ml.
The purity of each of the fractions was ascertained by high pressure liquid chromatography, and the corresponding fractions were combined and the solvent ~_ 36 : ` ' ' , :
' '. : ,. ' . '''' ' .
` . ~ , . . .
.
.

~o~,g7~8 was evaporated of. T~o epimers having the following NMR spectra were obtain-ed. A less polar epimer ~500 mg) eluted at an early stage was the 24(R)- ~`
derivative, and a more polar epimer (490 mg) eluted at a stage toward the end was the 24(S)-derivative.
NMR spectra of 1~,3~,24(R)-tribenzoyloxycholest-5-ene:
0.65 (3}l, s, C 18)~ L
0.92 (3~l, s, C-21), 1.01 ~6H, b, s, C-25, 26), 1.20 (3H, s, C-19), 5.00 (lH, m, C-24), 5.20 (lH, m, C-3), 5.44 Cl~l, m, C~
5.70 (1~1, m, C-6), 7.5 t9~l, m, benzoyl), 8.1 (6~1, m, bellzoyL) NMR spectrum of 1~,3~,24(S)-tribenzoyloxycholest-5-ene:
0.63 (3H, s, C-18) Other spectrum data are the same as those of the 24~R)-derivative.
(B) Synthesis of 1~,3~,24(S)-tribenzoyloxy-cholesta-5,7-diene ~second step):-10Q mg o~ 1~,3~,24CS)-tribenzoyloxy-cholest-5-ene was heatecl under reflux for 15 minutes together with 23 mg o~ 1,3-dibromo-5,5-dimethylhydantoine in 16 ml. of carbon tetrachloride. The reaction mixture was cooled, and the resulting crystals were removed by filtration. The filtrate was concentrated and 2.5 ml. of xylene was added to the residue. The solution was added drop-~ise over the course of 15 minutes to a solution under reflux of 0.3 ml. of s-collidine in 2 ml. of xylene, and further refluxed for 10 minutes. ~ -After the reaction, the hydrobromide of s-collidine was removed by ` - . ~, . .
': . ' . ' ' , ' ' ' ' , , ~L0797~8 filtration, and s-collidine and xylene ~cre evaporated off at reduced pressure.
The residue was dissolved in diethyl ether. The ethereal phase was washed with lN-hydrochloric acid and a 5% aqueous solution of sodium bicarbonate, and then repeatedly ~ith water. The ethereal phase was then dried, and ~he ether was evaporated off at reduced pressure to afford a ye:Llow oily substance.
The oily substance was carefully chromatographed twice through a column containing silicic acid using benæene as an eluting solution to afford 28 mg ~yield 28%) of a purified non-crystalline product. This product had the following spectra, and was identified as la,3~,24(S)-tribenzoyloxycholesta-5,7-diene.
W spectrum, ~ ~tahanl (nm):
231, 262, 271, 282, 29 NMR spcctrum:
4.~5 ~21l, b, C-3 ancl C-24-11s) J
5.32 ~2ll, b, C-L and C-6 or C-7-tl), 5.72 ~lH, d, J=6Hz, C-6 or C-7-tl), 7.49 and 8.02 (15H, m, aromatic Hs), Mass spectrum:
728 (M ), 638, 620, 606, 484, 362 Example 6 ~A) Separation of 24~R)-derivative and 24~S)-derivative of l~-hydroxy-38,24-dibenzoyloxycholest-5-eno:-2.1 g of the crude la-hydroxy-3~,24-dibenzoyloxycholest-5-ene was chromatographed through a column containing 30 g o silica gel using an elut-ing solvent consisting of a 200:1 mixture of benzene and ethyl acetate to divide it into fractions each having a volume of 50 ml. Each of the fractions was subjected to high pressure liquid chromatography to ascertain their purity.
The corresponding fractions were combined, and the solvent was evaporated off . ~ .
':' ...... . ' . ' . ' ',. ' ~ : ~:
' ., . , ~ .. . . .
. :, . . , ,: .

107'~71~

to form two epimers having the following NMR spectrum data.
A less polar epimer (500 mg, melting point 168 - 169 C.) was the i.
24(R)-derivative, and a more polar epimer ~G00 mg, melting point 139.5 -140.5C.) was the 24~S)-derivative.
NMR spectrum of l~,hydroxy-3~,24~R)-dibenzoyloxycholest-5-ene:
0.67 ~3~l, s, C-18) 0.96 (611, b, s, C-19,21), 1.00 (6~l, d, J=6Hz, C-25,26), 3.96 (1~1, m, C-l), 5.06 (1l1, r,l, C-24), 5.36 ~1~1, m, C-3), 5.71 (1ll, m, C-6), 7.52 (611, m, benzoyl), 8.12 (411, m~ benzoyl) NMR spectr~ll of la-hydroxy-3~,2~S)-dLbenzoyloxycholost-5-ono:
0.70 (31-l, s, C-18) Other spectrum data are the same as those of the 24~S)-derivative.
C~) Synthesis of l~-acetoxy-3~,24~S)-dibenzoyloxy-cholesta-5,7-diene ~second step):-1~-Hydroxy-3~,24(S)-dibenzoyloxy-cholest-5-ene was repeated with acetic anhydride and pyridine in the same way as in Example 1, (A). The reaction product was purLfied in the same way as in ExampLe 1 to af~ord 1~-acetoxy-3~,2~(S)-dibenzoyloxy-cholest-5-ene.
462 mg o 1~-acetoxy-3~,24(S)-dibenzoyloxy-cholest-5-ene was reacted with 188.6 mg of N-bromosuccinimide in 16 ml. of carbon tetrachloride under reflux for 30 minutes. The reaction mixture was cooled, and the resulting crystals were removed by filtration. The filtrate was concentrated at reduced pressure to afford a yellow oily substance. To the substance was added 6.8 _ 39 -~,; .. - - :

~ .
.. ,''. ,,' '~ ', , ' ' .' '' , -~79'718 ml. of xylene to form a sol~ltion. The solution ~Yas addecl dropwise to a solution under reflux of 0.4 ml. of trimethyl phosphite in 8 ml. of xylene, and the reaction was carried out for an additional 90 minutes. After the reaction, the reaction mixture was concentrated at reduced pressure to afford an oily product. This product showed the following spectra, and was identi-fied as l-acetoxy-3~,24(S)-dibenzoyloxy-cholesta-5,7-diene.
W spectrum, ~, ethanol ~llm):
231, 262, 2~1, 282, 294 NMR spectrum:
2.04 ~3~l, s, C~13COO-), 4.7-5.4 (3H, m, 1,B,3~ and 24-113), 5.33 (lH, d, J-6Hz, 6 or 7-H), 5.70 (lH, d, J=611z, 6 or 7~
7.4 - 8.2 (101-1, m, aromatic lls) 13xamplo 7 Synthesis o~ la,3B,24~R)-trlacetoxy-cholesta-5,7-~l:iene tsecond step):-la-Hydroxy-3B,24(R)-dibenzoyloxy-cholest-5-ene prepared and separ-ated in the same way as in Example 6, (A) was subjected to the same procedure as in Example 4. A purified product having quite the same UV, NMR and mass 2û spectra as the corresponding S-epimer obtained in Example 4 was formed in a yield of 28%. This product was identified as la,3~B,24~R)-triacetoxy-cholesta~
5,7-diene.
Example 8 Synthesis of la,3~,24(R)-tribenzoyloxy-cholesta-5,7-diene (second step) :-1,3~,24(R)-tribenzoyloxy-cholest-5-ene prepared and separated in the same way as in Example 5, (A) was subjected to the same procedure as in Example 5, (B).

... .. :. :

.
': : . , ', ,, ~L~797~8 A purified product having quite the same W , NMR, and mass spectra as the corresponding S-epimer obtained in Example 5 was obtained in a yield of 27%. This product was identified as la,3~,24(R)-tribenzoyloxy-cholestra-5,7-diene.
Example 9 Synthesis of la-acetoxy-3~,24tR)-dibenzoyloxy-cholesta-5,7-diene (second step):-la-Hydroxy-3~,24~R)-dibenzoyloxy-cholest-5-ene prepa~ed and separ-ated in the same way as in Example 6, (A) was subjected to the same procedure as in Example b, ~B).
A purified product having quite the same W and mass spectra as the corresponding S-epimer obtained in Example 6 was formed. This product was identifie~l as la-acetoxy-3~,24(R)-diben~oyloxy-cholesta-5,7-diene.
~xamplo 10 ~ Synthcsis ol: 1~,3~,24-triacotoxy-cholesta-5,7-di~rle iErom la,3~,24-triacetoxy-cholest-5-ene:-In accordance with the procedure of Example 1, ~B), 1~,3~,24-tri-acetoxy-cholest-5-ene was reacted with 1,3-dibromo-5,5-dimethylhydantoin and s-collidine in n-hexane. The reaction product was washed with acid and alkali in ether, and treated with activated carbon to aEord a crude reaction mixture containing la,3~,24-triacetoxy-cholesta-5,7-diene.
(B~ ~Iydrolysis of la,3~,24-triacetoxy-cholesta-5,7-diene:-The crude product obtained in (h) above ~as hydrolyzed in a 5%
methanol solution of potassium hydroxide to afford a crude reaction mixture containing la,3~,24-trihydroxycholesta-5,7-diene.
~C) Determination of the purity of la,3~,24-trihydroxycholesta-5,7-diene:-The W spectrum of 100% pure la,3~,24-trihydroxy-cholesta-5,7-,,~....

.

797~8 diene is ~ethanol =282 nm~ and the UV spectrum of 100% p~lre 1~,3~24-tri-hydroxy-4,6-diene is ~neltax =240 nm. Our investigations have shown that the mixing ratio between these two compounds can be determined by comparing their ~max values-The ratio of the 5,7-diene derivative and the 4,6-diene derivative in the crude reaction mixture obtained in (B) above was determined by this procedure, and it was found that the molar ratio of 1~,3~,24-trihydroxy-cholesta-5,7-diene to 1~,3~,24-trihydroxycholesta-4~6-diene was about 3:1.
(D) Separation of 1~,3~,24-trihydroxycholesta-5,7-diene and 1~,3~, 24-trihydroxycholesta-4,6-diene (third step):-(D-l) Separation by preparative thin-layer chromatography:-A commercially available plate for preparativc thin-layer chromato-graphy (sllica gel, a product oE Merck Company, 20 cm x 20 cm x 0.5 mm) was immorsed Ln a solution Oe silvor nitrate Ln acotonLtrilo to impregnate it :iTl an amount of about 1.5% by weight as silver nitrate, and heat-treated at 70C.
for 2 hours. The chromatographic plate so obtained was used for the subse-quent separating operation.
The crude reaction mixture (30 mg) obtained in (B) above was ad-sorbed to the plate and developed through about 20 cm with a 6% methanol-chloroorm mixed solvent. After air drying the plate, the reaction mixture was again developed with the same solvent. It showed two bands at an RP of about 0.23 ancl about 0.33. These ractions wero scraped ofE, and extractecl with ethyl acetate rom the silica gel. There were obtained 17.1 mg (57%) of a white solid substance (Rf=0.23), and 6.0 mg ~20%) of a white solid sub-stance ~Rf=0.33).
A part of the plate was colored using sulfuric acid, and a band o Rf=about 0.38 was confirmed. This fraction was scraped off, and extracted in the same way as above to aford about 1 mg of a solid substance.
.

1~9~1~

These products had the follo~ing spectra. The product with an Rf of 0.23 was identified as la,3~,24-trihydroxycholesta-5,7-diene; the product with an Rf of 0.33, as la,3~,24-trihydroxychoLesta-4,6-diene; and the product with an Rf of 0.38, as la,24-dihydroxycholes~erol.
(1) Product with Rf=0.23 (la,3~,24-trihydroxycholesta-5,7-diene) W spectrum (see Figu-re 2) ~ ethanol (nm) 262, 271 (F=ll,000), 282 (~=12,000), 294 (~=7000 NMR spectrum ~in C3D6O):
0.63 ~3~1, s, 18-C~13), 3.30 (111, m, 24-H), 3,70 ~1~1, m, l~-H), 4.08 (lH, m, 3a-11), 5.30 ~111, d, J=6tlz, 6 or 7-1l), 5.60 tl~l, cl, J=611~, 6 or 7-ll) Mass spectrum (see Figure 3):
4.16 (M ), 398, 380, 357, 251, 227, 197, 157 Melting point (C.):
102 to 103C.
(2) Product with Rf=0.33 (la,3~,24-trihydroxycholesta-4,6-diene):-UV spectrum, ~ ethanol ~nm 233, 240, 2~8 Mass spectrum:
416 (M ), 398, 380 ~3) Product with Rf~0.38 ~la,24-dihydroxycholesterol):-This corresponded with a standard sample with Rf=0.38.
~D-2) Separation by column chromatography:-Silica gel commercially available for use in column chromatography ~C-200, trademark for a product of Wako Jyunyaku Kogyo Kabushiki Kaisha} was 1~797~8 impregnated with about 2% by weight of silver nitrate while using it as a solution, and heat-treated at 70C. for 2 hours. The silica gel was then packed in a glass column with a diameter of 1 cm and a length of 30 cm. ~0 mg of ~he crude reaction mixture obtained in (B) above was poured into the column, and eluted with a mixed solvent consisting of ben~ene and ethyl acetate. It was divided into fractions each with a volume of about 20 ml. These fractions were separated while monitoring by thin-layer chromatography and UV spectrum.
When the volume ratio of benzene to ethyl acetate was 2:1 to 1:1, 1.6 mg (yield 19%) of a product corresponding to Rf=0.33 in ~D-I) was ob~ained.
When this ratio was 1:1, 19.6 mg ~49%) of a product corresponding to Rf=0.23 was obtained.
These products were identified by various spectrum data.
_omparative Example I
'rhis ['xamplo illustrates thc soparation oE L~,3~,2~-trihyclroxy-cholosta-5,7-diono and l~,3~,24-trihydroxycllolesta-~,6-dLene by a carrior containing silicon dioxide but not containing non-metallic silver (comparison with regard to the third step).
30 mg of a mixture of 1~,3~,24-trihydroxycholesta-5,7-diene and 1~,3~,24-trihydroxycholesta-4,6-diene in a molar ratio of 3:1, which had been prepared in the same way as in Example 1~, (A) and (B) and identified in the same way as in Example 14, (C), was chromatographed through a column with a dlamoter of 1 cm and a length of 30 cm using silica gel as a carrier in an attempt to separate it into the constituents.
A mixed solvent of benzene and ethyl acetate was used as a develop-ing solvent in varying mixing volume ratios from lO:l to 1:1, and the starting mixture was divided into fractions each with a volume of about 20 ml. The separation was attempted while monitoring by thin-layer chromatography and W
spectrum. When the mixing ratio of benzene to ethyl acetate was about 2:1, ~7971~

the main component began to flow out. Analysis of each of these fractions by UV spectrum showed that in all of the fractions analyzed, an absorption was observed at 283, 240, 248, 262, 271, 282, and 294 nm. As a result, i~ was found that when silica gel was used as a carrier, the 1~,3~,24-trihydroxy-cholesta-5,7-diene could not be separated from the 1~,3~,24-trihydroxycholesta-4,6-diene. Furthermore, it was found that there was hardly any difference in composition among these fractions, and the molar ratio of the 5,7-diene to the 4,6-diene remained at about 3:1. (See Comparative Example 3 below.) Comparative Example 2 This Example illustrates the separation of 1~,3~,24-triacetoxy-cholesta-5,7-diene and 1~,3~,24-triacetoxycholesta-4,6-diene using a carrier containing silicon dioxide but not containing non-metallic silver ~comparison wlth respect to the third step).
A mixture o~ La,3~,2~-triacetoxycholesta-5l7-dieno and 1~,3~,2~-triacotoxycholesta-~,6-dione in a moLar rntio oE 3:1, which had been ~repared in the same way as in Example lO, (A), and identified in the same way as in Example 10, ~C), was subjec~ed to preparative thin-layer chromatography using the same plate as used in Example 10, ~D-l) and a silica gel carrier treated with silver nitrate (developing solvent, 0.4% methanol-chloroform). Only one spot was detected by W spectrum at Rf=about 0.4.
This fraction was isolated to obtain an oily substance whose UV
spectrum showed an absorption at 233, 2~0, 2~8, 262, 271, 282, and 294 nm.
Thus, it was found that the separation of 1~,3~,24-triacetoxy-cholesta-5,7-diene from 1~,3~,24-triacetoxycholesta-4,6-diene failed even when silica gel containing non-metallic silver was used as a carrier ~see Comparative Example 4).
Exam~le 11 Separation of 1~,3~,24~R)-trihydroxycholesta-5,7-diene and 1~,3~, _ -45 -~79'718 24(R)-trihydroxychol~sta-4,6-diene ~third st~p):-la-~lydroxy-3B,24(R)-dibenzoyloxycholest-5-ene prepared and separated in the same way as in Example 6, (A) was treated in the same way as in Example 1, (A) to form la-acetoxy-3~,24(R)-dibenzoyloxycholest-5-ene. This product was treated in the same way as in Example 6 to afford a crude reaction mixt~re containing l~-acetoxy-3~,24(R)-dibenzoyloxy-cholesta-5,7-diene. The crude mix~ure was hydrolyzed in the same way as in Fxample 10, (B), and separated by preparative thin-layer chromatography in the same way as in ~D-l).
As a result, the reaction mixture containing la,3~,24(R)-tri-hydroxycholesta-5,7-diene and la,3~,24(R)-trihydroxycholesta-4,6-diene in a molar ratio of about 3:1 was distinctly separated into these two constituents.
The product separated from the spot basecl on 1,3~,24(R)-trihydroxy-cholesta-5,7-cliono had tho Eollowing chnracteristics.
UV spoctrum, A ~tkhanol tnm) 262, 271 ~ ~11,000), 282 (11,800), 294 ~7,000).
MMR spectrum tC~D60):
3.25 ~lH, m, C-24-}1), 3.68 tlH, m, 1~-~1), 4.10 (ltl, m, 3a-H), 5.30 (lH, d, J=6Hz, 6 or 7-H), 5.60 (11l, cl, J=6~1z, 6 or 7-H), Mass spectrum:
416 (M ), 398, 380, 357, 251, 227, 197, 157 Melting point t C.):
96 - 99C.
Example 12 Separation of 1~,3~,24(S)-trihydroxycholesta-5,7-diene and la,3~,24 , 1~797~3 (S)-trihydroxycholesta-4,6-diene (third step):-l~-llydroxy-3~,24~S)-dibenzoyloxycholest-5~ene prepared an~ separat-ed in the same way as in Example 6, (A) was treated in the same way as in Example 1, (A) to aff~rd la-acetoxy-3~,24(S)-dibenzoyloxycholest-5-ene. This product was treated in the same way as in Example 6 to afford a crude reaction mixture containing la-acetoxy-3~,24(S)-dibenzoylcholesta-5,7-diene. This reaction mixture was hydrolyzed in the same way as in Example 10, (B), and separated by preparative thin-layer chromatography in the same way as in (D-l) above.
As a resul~, the above crude reaction mixture containing la,3~,24 (S)-trihydroxycholesta-5,7-diene and la,3~,24~S)-trihydroxycholesta-4,6-diene in a molar ratio of about 3:1 could be separated distinctly into these constituents.
Tho procluct scpuratclcL erom th~ s~ot bas~d on La,3~,2~$~-trihydroxy-cholostu-5,7-(lienQ hacl tho EolLowLng charactoristics.

UV trum A ethanol ~nm) 262, 271 ~10,800), 282 (11,500), 294 ~6,900) NMR spectrum Cln C3D60):
3.27 (lH, m, C-24-tl), 3.67 ~lH, m, l~-H), 4.10 ~1~l, m, 3a-~l), 5.30 ~1ll, d, J~611z, 6 or 7-ll), 5.60 tlH, d, J=6Hz, 6 or 7-~l) Mass spectrum:
416 (M ), 398, 380, 357, 251, 227, 197, 157 Melting point (C):
120 to 124C.

.

~L~179~
Referential Example l.
Synthesis of la,24-dihydroxycholecalciferol:
16 mg of 1~,3~,24-trihydroxycholesta-5,7-diene prepared and separated in the same way as in Example 10, (D-l) was dissolved in S00 ml. of diethyl ether, and this solution was irradiated with ultraviolet rays for 2.5 minutes at 5C. in an atmosphere of argon using a 200 W high pressure mercury lamp (654A-36, trademark for a product of Hanovia Company). A part of the solution was taken out, and its W spectrum was dete~nined. There was an increase in absorption at 262 to 263 nm which was considered to be due to 1~,24-dihydroxy-pre-cholcalciferol. After the reaction, ether was evaporated off at room temperature under reduced pressure. Benzene ~50 ml.) was added to the residue, and isomerization was carried out for 2 hours under reflux of benzene.
A~tor th~ reaction, tho bonzeno was cvaporntod oeE at rcducod pros-suro to afford 16 mg o a whito solid. 'lllis product was carceully separatc(l by preparati~e thin-layer chromatography using a silica gel carrier containing about 1.5% of silver nitrate ~obtained in the same way as in Example 10, (D-l)) Ideveloped twice with 6% methanol-chloroform). It showed three bands that could be confirmed by ultraviolet rays. From the least polar band, 2.8 mg of a white solid was obtained. This product had the following properties, and was identified as 1~,24-dihydroxycholecalciferol.

UV spectr~ml t~igure 4) ~ethanol (nm) = 265 ~ethanol (nm) = 228 NMR spectrum ~C3D6O): ; -0.57 ~6H, d, 18-CH3), ~ ;
0.87 (6H, d, J=7Hz, 26- ~ 27-CH3), 0.96 (3H, d, J=5Hz, 21-CH3)~

, ,: -~797:~8 3.19 (lH, m, 24-tl), 4.15 (ltl, m, 1B-II), 4.36 (lH, m, 3a-tl), 4.85 (ltl, b, s, 19-tl), 5.30 ~1H, b, s, 19-H),

6.05 ~ltl, d, JAB = 11tlz, 6 or 7-tl) J
6- 26 ~1~1, d, JAE~ = lltlz, 6 or 7-H), Mass spectrum (Figure S):
416 ~M ), 398, 380, 269, 251, 134, High resolution mass spectrum:
Found = 416.32768 Requir~, M ~C27~14~03) = 416.32927 Me1~ ing po int ~C. ):
8(1 to 85.
1~1,24~R)-clihyclroxycholocaLci~erol havin~ the fo11owlng propcrties.
UV spectrum: ),ethanol ~nm) = 265 ~.mthanol (nm) - 228 NMR spectrum (C3D6O):
0.59 (3H, s, 18-Ctl3), 0.87 ~6tl, d, J=7tlz, 26- ~27-Ctl3) 3.20 ~ltl, m, 29-tl) 4.14 ~ltl, m. 1B-tl), 4.42 ~lH, m, 3a-H), 4.87 ~lH, b, s, l9-H), 5.32 (lH, b, s, 19-H), 6.08 ~lH, d, JAB = llHz, 6- or 7-H), 6.30 ~lH, d, JAB = lltlz, 6- or 7-H), . - . . . .
:: :
, , .. .. . . . .
'' ~" , ' ' ~ , ' ' , ' ' '. ' ' .~ " ' ' ', ' ' ' Mass spectrum:
416 ~+), 398, 380, 269, 251, Hi~h resolution mass spectrum:
Found = 416.33084 Require ~M ) - 416.32927 (C27l-14~O3) and 1~,24(S)-dihydroxycholecalciferol having the following proper~ies.
W spectrum: ~ e~hanol (nm)=265 A mtihanol ~nm)=228 NMR spectrum (in C3D6O):
0.58 (3~l, s, 18-C~13}, 0.87 ~6H, d, J=7Hz, 26- and 27-C~I3), 3.20 ~ltl, m, 24-ll), ~.14 ~Ill, m, I~-ll), 4.42 ~11l, m, 3a-ll), 4.87 ~lH, b, s, l9-H), 5.32 (ltl, b, s, l9-H), 6-08 ~1~1, d, JAB = llHz, 6- or 7-H), 6.30 ~lH, d, JAB = llHz, 6- or 7 H), Mass spectrum:
416 ~M ), 398, 380, 269, 251, 134, lligh resolution mass spectrum:
Found = 416.33095 Require (M ) = 416.32927 (C27H4403) were also prepared in the same way as above using 1~,3~,24~R) and 1~,3~,24 ~S)-trihydroxycholesta-5,7-diene as starting material respectively.
Comparative Example 3 Synthesis of 1~,24-dihydroxycholecalciferol from 1~,3~,24-tri-, : .' , . .:...... '.- " : ' ' , . ',. ' ' ' ' ~0'797~8 hydroxycholesta-5,7-diene containing 1~,3~,24-trihydroxycholesta-4~6-diene:-lO mg of a mixture of 1~,3~,24-trihydroxycholesta-5,7-diene and 1~,3~,24-trihydroxycholesta-4,6-diene in a molar ratio of about 3:1 obtained in the same way as in Comparative Example 1 was dissolved in S00 ml. of di-ethyl ether, and the solution was irradiated with ultraviolet rays at 5C. for 2 minutes. After the reaction, the diethyl ether was carefully evaporated off at reduced pressurc. To the residue was added 50 ml. of benzene, and isomerization was carried out in an atmosphere of argon for 2 hours under reflux of benzene. After the reaction, the benzene was evaporated of to afford 10 mg of a brown oily substance.
The W spectrum of this product was complicated. In particular, it was devoid of a specific absorption of 1~,24-dihydroxycholecalciferol which exhibits an absorption maximum at 265 nm, and the formation of la,24-dihyclroxy-cholocalcifcrol could not be con~irmod.
rhis was coneirmed by subjecting the above substance to preparative thin-layer chromatography using a silica gel carrier having adsorbed thereto silver nitrate, and comparing the chromatogram wi~h that of a standard sample of 1~,24-dihydroxycholecalciferol. The product did not show any spot which corresponded to the standard sample.
It can be seen therefore that when the purity of 1~,3~,24-tri-hydroxycholesta-5,7-diene is low, 1~,3~,24-trihydroxycholecalciferol is not formed at all by isomerization using ultraviolet irrad:iation.
Comparative ~xample 4 Synthesis of la,24-diacetoxycholecalciferol-3~-acetate from 1~3~J
24-triacetoxycholesta-5,7-diene containing 1~,3~,24-triacetoxycholesta-4,6-diene 10 mg of a mixture consisting of 1~,3~,24-triacetoxycholesta-5,7-diene and 1~,3~,24-triacetoxy-4,6-diene in a molar ratio o about 3:1 obtained , - ' ,. .
, ' ':: , ' .

797:18 in the same ~ay as in Comparative Example 2 was dissolved i.n 500 ml. of diethyl ether, and the solution was irradiated with ultraviolet rays at 5C.
for 2 minutes. The reaction mixture tur~ed yellowish brown. After the reaction, the diethyl ether was carefully evaporated off at reduced pressure.
Benzene ~50 ml.) was added to the residue, and isomerization was carried out for 2 hours in an atmosphere of argon under reflux of benzene. After the reackion, most of the benzene was evaporated off at reduce~ pressure. To the residue was added 1 ml. of 5% potassium hydroxide-methanolJ and the mixture was allowed to stand at room temperature for 24 hours in an atmosphere o argon to perform hydrolysis. After the reaction, the benzene was evaporated off, and its UV spectrum was determined. The product was subjected to preparative thin-layer chromatography. The results were ~uite the samo as in Comparative Example 3, and no formation oE 1~,24-dihydroxycholecalciferol was obsorve~I.
Referential Bxample 2.
Effect of promoting intestinal calcium transport by 1~,24-dihydroxy-cholecalciferol ~la,24-DHCC):-~a) Comparison with ~y-hydroxycholecalciferol ~la-HCC):-Male Wistar rats with a body weight of about 200 g were fasted overnight, and then orally administered with 625 p moles of a solution of each of la,24-DHCC and l~-~ICC in corn oil. After a predetermined period of time, ~ solution of radioactivo calcium chloride (45Ca C12, 30 ~Ci!mQ) was admlnis-tered orally. The radioactivity level in blood was measured over the course of 10 to 60 minutes. The maximum value was made an index of intestinal calcium absorption.
A control group of rats was administered with corn oil alone in the same amount. The dosages of the corn oil solution of la,24-DHCC or l~-HCC and the corn oil were 0.0125 ml per 100 g of the body weight of each , : . . . . .
' ' : : ' ' : . ,: , . . :

i~'79'7~3 rat, and the dosage of the radioactive calci~ chloride aqueous solution ~p~l

7.0) was 0.1 ml per 100 g of the body weight o each rat.
The radioactivity level was measured by placing 0.2 ml. of serum in a Vial bot~le, adding 12 ml. o a cocktail containing a scintillator (containing 1200 ml of toluene, 800 ml of ethyl cellosolve, 8 g of 2,5-diphenyloxazole, and 300 mg of 2,2'-p-phenylenebis~5-phenyloxa~ole)), and determining the radioactivity by means of a liquid scintillation counter.
The results are shown in Table 1.
Table 1 Time after administration Radioactivity in serum ~cpm) ~hours) la,24-DllCCla-HCC
Control 275 ~ 95 (4)* 275 -~ 95 ~4) 4 360 ~ l50 (4) 697 :~. 221 (~)

8 996 ~ 80 (4) 1035 ~ 150 (4) 12 924 ~ 130 t~) 6~ 210 ~4~
24 426 -~ 61 ~4) 332 ~ 28 ~4) *The numbers in the parentheses show the number of rats in a particular group.
The above results demonstrate that la,24-D~lCC has nearly an equivalent effect to la-llCC with rogard to the promotion o~ intestinal calcium absorption.
~b) Comparison between la,2~R)-D~ICC and la,24(S)-D~lCC:-Male Wistar rats were fasted ovcrni~ht, and then orally administer-ed with 625 p moles of a solution of each of la,24(R)-D~lCC and la,24~S)-DHCC in corn oil. Eight hours after the administration, the rats were killed, and intestinal calcium absorption was dete~mined by the everted gut sac method LMartin, D.L. and H.F. DeLuca, Amer. J. Physiol. 216, 1351 ~1969~]. The results are shown in Table 2.

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' :. . , ,: , ~079t718 Table 2 _ Ca ( / ) Control 1.29 ~ 0.16 (4)*
1~,24tR)-DHCC 5.86 ~ 1.98 ~4) la,24(S)-DHCC 2.68 t 0.80 (4) *The numbers in the parenthesss show the number of rats in a particular group.
The experimental conditions were as follows:
Intestinal tract used: duodenum, 7 cm Amount of the medium poured in the everted duodenum: 0.7 ml Composition of the medium:
~aCl 125 mM
Fructose 10 mM
Tris-Cl buffer 30 mM (ptl 7.4) CaCI2 0.25 mM
45CaC12 10 ~ICi/~ ' Incubation conditions:
37C., 90 minutes a gaseous mixture of 9S% 2 and 5% C02 was passed Amount of the liquid used for radioactivity measurement: 0.1 ml Other conditions were the same as in ~a) above.
Ihe above results demonstrate tha~ L~,24tR)-D~ICC cxhibited a greater effect of promoting intestinal calcium absorption than 1~,24(S)-DHCC.
Referential exam~le 3 Comparison of l~-HCC, 1~,24-DHCC, 1~,24(R~-DHCC and 1~,24(S)-DHCC ~`
with regard to the effect of promoting intestinal calcium absorption:-The experimental procedure was quite the same as in Referential ,.-- . , . , : :
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1~:)797~8 example 2 ~b). The results obtainecl are shown in Table 3.

Table 3 .~ . I _ _ ~ ~ 125 p mole 625 p mole 3~125 p mole _ _ Control 3.31 + 0.12 (15~*
la-HCC 3-59 + 0.23 (4) 4.10~ 0.2~ (4) 5.1û + 0.27 (4) la,24-DHCC 4.12 + 0.25 (4) 4.84 +0.32 (4) 5.47 + 0-33 (4) la,24~S)-DHCC 3.97 ~ 0.28 (4) 4.17 + 0.31 (4) 5.18 + 0.20 (4) la,24(R)-DHCC 4.42 + 0.41 (4) 5.93 + 0.33 (4) 5.84 + 0.39 (4) The numbers in the parentheses show the number o:E rats in a particular group.
The results shown in Table 3 led to the reconfirmation of the experimental results in Referential Example 2. In other words, with regard to intestinal calcium absorption, la,24-D~lCC has at least an cquivalent acti-vity to la-llCC, and among lu,2~-D~ICC, lc~,24tR)-I)tlCC and 1~,24~S)-D~ICC, the Eollowing rolation holds good with regard to the degrce of activity oE pro-moting intestinal calcium absorption.
la,24(R)-DHCC > la,24-DllCC ~ la,24(S)-DHCC
Referential example 4 Comparison of bone absorption effect between la,24-D~ICC and la-HCC:-Male Wistar rats (each with a body weight of about 150 g) were sub-cutaneously administered with an aqueous solution of 45CaC12 in a dose of 50 ~Ci per rat, and kept for 6 weeks. 45Ca so administered was rapidly absorbed, and a greater portion of it gathered at th~ bones in several hours. rhree weeks later, the 45Ca level in blood became constant, and only the bones were in the specifically labelled state. This was confirmed by a macro-autoradiographic analysis of the whole body. After keeping the rats for 6 weeks, they were orally administered continuously once a day with a solution of 2,125 P moles each of la-HCC and la,24-D~lCC. Blood was taken out with - ^ -- 55 --, - , , ' ; ' ' ' ' ' , l~g~

the passage of time, cmd the serum radioactivity level was measured by the method described in Referential example 2 ~a). An increase in the serum radioactivity level was made an index of the effect of bone absorption. The control group was administered with corn oil alone. The number of rats tested was 4 in each group. The results obtained are shown :Ln Figure 6. In this figure, the symbol * indicates a significant difference in serum radioactivity level from the control.
As can be seen from the results shown in Figure 6, with la-HCCJ
bone absorption increased significantly two days after the start of adminis-tration, and this tendency became greater with time. On the other handJ with l~J24-DHCCJ bone absorption increased significantly only after a lapse of 4 daysJ but after that, the degree of increase was far lower than with la-HCC
(about 1/3 on the eighthday). This appoars to sug~est that la,24-DtlCC ex-hlbits a weakor bono absorbing oEEoct than l~-IICC, an(l :is lowor in toxicity.
RoEorontlal oxamplo 5 Comparison of bone absorbing effect and body weight change among la-HCC, la,24-DHCC, 1,24(R)-DHCC and la,24~S)-DHCC:-A similar test to Referential example 4 was performed on the above four compounds. Unlike Referential example 4, the experiment was begun three weeks after the administration of 45CaC12. Both the serum radi~activity level and the total calcium concentration in the serum were measured. For the moasuremont o~ the calciwn concontration, a calcium measuring kit ~made by Iatron Company) was used. 'I'he results obtained are shown in 'rables 4 and 5.

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Table 4 Serum 45Ca level 1~ _ \ Time Serum radioac~ivity ~cpm) \ ~days) _ -5 - 7 9 _ Control 699 + 53.7 621 + 48.6 597 ~ 33.9 608 + 49.3 1-~ICC 690 + 39.4 1056 ~ 66.8 1184 ~ 51.1 904 + 57.1 lc~,24-DHCC 615 + 54.9 948 + 64.6 7ûO 1- 32.8 661 -~ 78.8 1,24~R)-DtlCC648 ~ 44.3 874 + 30.4 778 + 26.9 681 -~ 63.7 la,24(S)-DHCC780 ~ 43.5 539 + 45.2 520 + 53.8 609 + 91.5 Table 5 Serum Ca level ~ _ _ ~ .
~ ~Y~ O Ser Im ca ~ L_ , _ _ Control 11.4 ~ 0.32 10.0 ~ 0.29 11.1 ~ 0.22 10.6 ~ 0.15 la-HCC 10.4 + 0.21 14.1 + 0.29 14.7 + 0.33 13.8 + 0.41 la,24-DHCC 11.7 + 0.26 12.9 + 0.18 11.5 + 0.23 12.1 + 0.19 la,24tR)-DHCC 11.3 -~ 0.35 12.6 ~ 0.23 13.3 + 0.25 12.8 + 0.35 la,24(S)-DHCC 11.2 + 0.16 11.5 + 0.33 11.2 -~ 0.29 11.5 + 0.24 From the results shown in Tables 4 and 5, the experimental results in Reerential example 4 were ro-conEirmed. In other words~ these results show that la,24-DHCC is far weaker in bone absorbing effect than la-~lCC, and among la,24-DHCC, la,24(R)-DHCC and la,24~S)-DHCC, the following relation holds good with regard to the bone absorbing eff.ect.
1,24CR)-DHCC > 1,24-DHCC > 1~,24(S)-DHCC.
What is particularly noteworthy is that scarcely any bone absorbing effect is observed with la,24~S)-DHCC, and its effect is almost equi~ralent to . .

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~79~8 the control. Accordingly, this indicates that 1~,24 (S) -DHCC has an action of specifically promoting intestinal calcium absorption, and this considered to be of utmost importance for clinical applications.
Changes in body weight as determined by the present experiment are shown in Figure 7. It is interesting to note that la,24-DHCC causes a lesser degree of change in body weight than la-I-lCC, and in paLrticular~ la,24(S)-DHCC
shows little difference from the control group. This also suggests that similarly to the case of bone absorption, the toxicity of 1~,24-DHCC is lower than that oi la-HCC.
Referential example 6 Comparison of toxicity between la,24-DHCC and la-HCC:-The toxicity of each of the above two compounds was tested by awell known method. The results are shown :in Tablc 6.
Table 6 _ _ _ _ LD50 (~gtkg) Method of Species Sex administration 1~ -HCC1~,24-DHCC

Mouse ~Peros ______ = 2500_____________ __ ~_____ Intravenous 167 ¦ 33,000 - 3,300 ~ 100 3,300 - 1,000 _ . _ .
nat ~ Poros --~ L --------------------It is clear from the above table that the toxicity of 1~,24-DHCC is one-tenth or less of that of la-HCC. This result can also be suggested by the results of Referential example 4 and 5. -On the basis of the results obtained in Referential example 1 to 6, the characteristics of la,24-DHCC can be compared with those of la-HCC as ., ~ ' .'' ' .
,~ , ' - ~ o797~8 shown in Table 7. In these data, the activity of l~ lCC is set at 1.
Table 7 la,24-DHCC la-}lCC

Effecto of pTomoting intestinal calci~ 1 1 absorption Bone absoTbing effect less than Acute ~oxicity less than 1/1~
Since it has been demonstrated that 1~i-24-DHCC in accordance with this invention selectively promotes intestinal calcium absorption and exhibits a weak bone absorbing effect as compared with the known analogs of active form of vitamin D3, it is considered to be very useful as medicines that can bo applied with litt:Le s:ide-offects to the treatment o:E cliseascs inducecl by abnormal mctabolism of calcium. In particular, la,24(S~-DHCC is a very desirable substance in this regard, and is expected to come into wide use.

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Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing 1.alpha.,3.beta.,24-trihydroxycholesta-5,7-diene or its derivative expressed by the following formula (3) wherein R1, R2 and R3 are identical or different and represent a hydrogen atom or a protective group convertible to a hydrogen atom without changing the structure of the above formula, which comprises reacting a 1.alpha.,24-dihydroxycholesterol derivative expressed by the formula (2) wherein R4, R5 and R6 are identical or different, and represent a protective group convertible to a hydrogen atom without changing the structure of the resulting formula (3), with an allylic brominating agent in an inert organic medium, then contacting the resulting reaction mixture with a dehydrobrominating agent to form a 1.alpha.,3.beta.,24-trihydroxycholesta-5,7-diene derivative expressed by the following formula (3') wherein R4, R5 and R6 are as defined above, and, where required, splitting off the protective groups to form the corresponding 1.alpha.,3.beta.,24-trihydroxycholesta 5,7-diene.

2. The mixture in arbitrary ratios of 1.alpha.,3.beta.,24(S)-trihydroxy-cholesta-5,7-diene or its derivative and 1.alpha.,3.beta.,24(R)-trihydroxycholesta-5,7-diene or its derivative, expressed by the following formula (3) wherein R1, R2 and R3 are identical or different and represent a hydrogen atom or a protective group convertible to a hydrogen atom, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A process as claimed in claim 1 which includes the subsequent step of separating and recovering at least one of 1.alpha.,3.beta.,24(S)- and 1.alpha.,3.beta.,24(R)-trihydroxycholesta-5,7-diene or derivatives thereof from trihydroxycholesta-4,6-diene chromatographically by contacting a mixture containing at least one of 1.alpha.,3.beta.,24(S)- and 1.alpha.,3.beta.,24(R)-trihydroxycholesta-5,7-diene of the following formula (3-a) and trihydroxycholesta-4,6-diene of the following formula (4) as a solution in an inert organic solvent with a carrier containing silicon dioxide and having adsorbed thereto non-metallic silver, and where required, protecting at least one of the hydroxy groups at the 1-, 3- and 24- positions with a protective group convertible to a hydrogen atom without changing the structure of the resulting formula (3).

4. 1.alpha.,3.beta.,24(S)-trihydroxycholesta-5,7-diene or a derivative thereof resulting from the protection of at least one hydroxyl group thereof, expressed by the following formula (3-1) wherein R1, R2 and R3 are identical or different and represent a hydrogen atom or a protective group convertible to a hydrogen atom without changing the structure of the above formula, when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

5. 1.alpha.,3.beta.,24(R)-trihydroxycholesta-5,7-diene or a derivative thereof resulting from the protection of at least one hydroxyl group thereof, expressed by the following formula (3-2) wherein R1, R2 and R3 are identical or different and represent a hydrogen atom or a protective group convertible to a hydrogen atom without changing the structure of the above formula, when prepared by the process of claim 3 or by an obvious chemical equivalent thereof.