Note: Descriptions are shown in the official language in which they were submitted.
lOB~61~7
R~N UN 4212/11
The present invention relates to 3-deoxy-1~ -
hydroxycholecalciferol 13,
CH3 4~,~,,~
~ H
d' -
C~CH2 ' ' "
OH
and 3-deoxy-~, 25-dihydroxycholecalciferol 13a
.~
CH~ ~
H ~ OH
11 ' '
~f CH2 ~ `
~ OE~ ' `,,:
... .
13a
- 2 - ~ ~ :
.
826~37
novel analogs of vitamin D bearing the necessary 1~-
hydroxyl group and possessing the uni~ue characteristic
of stimulating intestinal calcium transport without
significantly mobilizing bone calcium.
The present invention also relates to novel
processes for the preparation of 3-deoxy-ld-hydroxy-
cholecalciferol 13 starting from cholesterol 2. More
particularly, the present invention relates to processes
for the preparation of 3-deoxy-1~-hydroxycholecalciferol
13 com~rising the steps of converting cholesterol 2
to 1~ ,2~ -oxido-4,6-cholestadien-3-one 1, reductively
cleaving 1~ ,2~.-oxido-4,6-cholestadien-3-one 1 to
4,6-cholestadien-1~ ,.3 B -diol _, selectively hydro-
genolyzing 4,6-cholestadien-1~,3~ -diol 4 to ~
l~hydroxy-5-cholestene:S..... and transforming 1~- :
hydroxy-5-cholestene 5 to 3-deoxy-1~hydroxycholeCal-
ciferol 13, or alternatively, reductively cleaving 1~,
2C~-oxido-4,6-cholestadien-3-one 1 to 1~ -hydroxy-
cholesterol 6, selectively sulfonating 1~ -hydroxy-
cholesterol 6 to 1~ -hydroxycholesteryl sulfonate 7
and reducing lc~-hydroxycholesteryl sulfonate 7 to 1~-
hydroxy-5-cholestene 5.
In addition the present invention also relates
to a novel process for the preparation of 3-deoxy-1~~
25,dihydroxycholecalciferol 13a starting from cholesterol 2.
More particularly, the present invention relates to a
process for the preparation of 3-deoxy-1~,25-
dihydroxycholecalciferol 13a comprising the steps of
, .
~ - 3 - ;.
- .- , ~ - . : . ~ . .. ; :
~08;~87 ` `
selectively sulfonating 1~ ,25-dihydroxycholesterol
14 to 1~ ,25-dihydroxycholesteryl sulfonate 16,
reducing 1~ ,25-dihydroxycholesteryl sulfonate 16
to 1~ ,25-dihydroxy-5-cholestene 17 and transforming
1~ r25-dihydroxy-5-cholestene 17 to 3-deoxy-1~ ,
25-dihydroxycholecalciferol 13a.
The invention described herein was made in the
performance of work under research grants from the United
States Public l~ealth Service.
In the formulas presented herein, the various
subs~ituents are illustrated as joined to the steroid
nucleus by one of three notations: a solid line (
indicating a substituent which is in the beta-orientation
(i.e., above the plane of the molecule), a dotted line
(------) indicating a substituent which is in the `
alpha-orientation (i.e., below the plane of the m~lecule),
or a wiggly line (____A__~_) indicating a substituent which
may be in the alpha- or beta-orientation or may be a "
mixture of both forms. The formulas have all been drawn ;
to show the compounds in their absolute stereochemical
cvnfigurations. Since the starting materials are derived
rom naturally occurring materials, the final products
exist in the single absolute configuration depicted herein.
However, the processes of the present invention are
intended to apply as well to the synthesis of steroids
of the racemic series. Thus, one may begin the synthesis
utilizing racemic starting materials to prepare racemic
products. Optically active products can then be prepared
by resolution of the racemic products utilized in the
preparation thereof, as hereinafter described, by standard
resolution techniques well-known in the art.
~L~826~
As used throughout the specification and appended
claims, the term "alkyl" denotes a straight or branched chain
saturated hydrocarbon radical having 1 to 8 carbon atoms,
such as, for example, methyl, 2-propyl, 2-methylpropyl,
3-methylpentyl, octyl and the like; the term "alkylphenyl"
denotes a group mono- or polysubstituted by alkyl, such as, :
for example, tolyl, xylyl, mesityl and the like; the term
"alkanoyl" denotes a radical derived by abstraction of the
hydroxyl group from an alkylcarbo~xylic acid having 2 to 8
carbon atoms, such as acety~, 2-methylpropionyl, 2-methyl-
pentanoyl, octanoyl and the like; the term "alkanol" denotes
~n alcohol derived by combination of alkyl and hydroxyl
radicals, such as, for example, methanol, 2-propanol, 2-methyl-
propanol, 3-methylpentanol, octanol and the like; the term
"alkoxy" denotes a radical derived by abstraction of the
hydroxyl proton from an alkanol, such as, for example,
methoxy, 2-propoxy, 2-methylpropoxy, 3-methylpentoxy, octoxy
and the like; and the term "halide" denotes chloride and
bromide. The term "lower" refers to the numerical range 1 to 8.
In the first step of the process of the present
invention for the preparation of 3-deoxy-1~ -hydroxychole-
calciferol 13, 1~ ,2C~-oxido-4~6-cholestadien-3-one 1,
CH3
H
~Q CH3 ~H I ¦
~ '' '
- 5 -
:: ,
~'
l~Z~
prepared by dehydrogenation of cholesterol 2
CH3"" ~
~ H :
H ~
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to 1,4,6- ~ `
ch~lestatrien-3-one 3
CH3~
H ¦ '
0~ ~
3 .
according to the procedùre described by A. B. Turner (J.
Chem. Soc. C, 2568 (19`68)) followed by selective epoxidation
of the 1,2-double bond with alkaline hydrogen peroxide
according to the known procedure of B. Pelc and E. Kodicek
(J. Chem. Soc. C, 1568 (1971)), is reduced to 4,6-cholestadien-
1~ ,3~ -diol 4.
~.
< - 6 - :~-, : :
'~,
~26~7
3'~",-~~
~ H
H
The reduction of 1~ ,2~ -oxido-4,6-cholestadien-3-
one 1 is accomplished using a suitable aluminum hydride
reducing agent suspended or dissolved in an inert organic
solvent at a reaction temperature of up to about 50C.
Among the suitable aluminum hydride reducing agents .
are alkali metal aluminum hydrides, such as lithium aluminum :~
hydride and the like, alkali metal aluminum alkoxy hydrides,
such as lithium tri-(tert.-butoxy)aluminum hydride, lithium ~;
diethoxy-aluminum hydride and the like, and alkyl aluminum
hydrides, such as diisobutylaluminum hydride and the like.
Alkali metal aluminum hydrides are preferred; lithium .~ .
aluminum hydride is mos~ preferred.
Among the inert organic solvents are ethereal
solvents, such as diethyl ether, diisopropyl ether, tetra- ~ :
hydrofuran, dioxane, 1,2-dimethoxyethane and the like, when
alkali metal aluminum hydrides and alkall metal~ alkoxyaluminum . .
hydrides are used as the reducing agents, and aromatic -- :
, : .
hydrocarbons, such as benzene, toluene, xylene and~the llke, .;
- 7 ~
, .
-- . ,, - ,,: . : : . ~. - . ..
1~82~;~7
when alkylaluminum hydrides are used as the reducing agents.
Diethyl ether and tetrahydrofuran are the preferred ethereal
solvents; diethyl ether is most preferred. Benzene and toluene
are the preferred aromatic hydrocarbon solvents; toluene
is most preferred.
While reaction temperatures below about 50C are
not critical, reaction temperatures above about 50C should
be avoided to minimize possi~ble hydrogenolysis of the 3~ -
hydroxyl group of the diendiol 4.
Similarly, the molar ratio of the aluminum hydride
reducing agent to the diendiol 4 is not critical as long
as the ratio is greater than 0.5. Molar ratios of about 1
to about 10 are preferred. A molar ratio of about 5 is most
preferred.
In the second step of the process, the diendiol 4
is subjected to the conditions of the Birch reduction where-
upon the 3~ -hydroxyl group is hydrogenolyzed and the ~4,6_
diene system is conjugatively reduced to afford 1~ -hydroxy-5-
cholestene 5.
CH3""
H ¦
~--~ ''
H
1~8Z~
The reductive-hydrogenolysis of the diendiol 4
is effected by a solution of an alkali metal in a suitable
ammoniacal solvent containing an appropriate inert organic
cosolvent under an inert atmosphere at a temperature of
about -33 to about 25C.
Included among the alkali metals are sodium, potassium,
lithium and the like. Sodium and lithium are preferred;
lithium is most preferred.
Suitable ammoniacal solvents include ammonia and
primar~ and secondary amines, such as methylamine, ethylamine,
dimethylamine and the like. Ammonia and methylamine are
preerred; ammonia is most preferred.
Among inert organic cosolvents are ethereal `
cosolvents, such as diethyl ether, tetrahydrofuran, dioxane
and the like. Diethyl ether and tetrahydrofuran are pre-
ferred; tetrahydrofuran is most preferred.
Appropriate inert atmospheres include nitrogen,
argon, helium and the like. Nitrogen and helium are
preferred; nitrogen is most preferred.
While reaction temperatures within the range of
about -33 to about 25C are no~ critical, the preferred ;
reaction tempexatures for reactions utilizing ammoniacal
solvents boiling below about 25C are the boiling points of
the solvents, and the preferred reaction temperatures for
reactions utilizing ammoniacal solvents boiling above about
25C are about 25C. The most preferred reaction temperature
is the boiling point of ammonia, -33C.
`.- 9
1~)13Z687
As in most Birch-type reductions, the molar ratio
of the dissolving alkali metal to the diendiol 4 is not
crucial. For the reduction of diendiol 4 to the enol 5,
molar ratios within the range of about 25 to about 100 are
preferred; molar ratios of about 50 are most preferred.
Alternatively, 1~ -hydroxy-S-cholestene 5 may be
prepared by reductive-cleavage of 1~ ~2C~-oxido-4~6-cholestadien-
3-one 1 to lc~-hydroxycholesterol 6
CH
CH
HO ~ H
~ . '
HO
according to the procedure of Barton, et al. (J. Am. Chem.
Soc., 95, 2748 ~1973)) followed by selective sulfonation of ;
the 3~ -hydroxyl group of the endiol 6 to the sulfonate
of formula 7
':
CH3
H
^C~ " ~,
R'-S02-0 ~ H
~.
-- 10 -- ~ ' '
~OB;~68~
wherein R' is lower alkyl, phenyl or lower-alkylphenyl,
and reduction of 7.
The selective sulfonation of the 3 -hydroxyl group
of the endiol 6 is performed by treatment with about 1 to
about 5 molar-equivalents of a sulfonyl halide of formula 8
R SO2 X
.
wherein R' is lower alkyl, phenyl or lower-alkylphenyl and
X is chloro or bromo,
in the presence of a basic solvent at a reaction temperature
of about 0C to afford the sulfonate 7.
Among the basic solvents which have been found to ;
be useful in the sulfonation step are trialkylamines, such
as triethylamine, tripropylamine and the like, N,N-dialkylani-
lines, such as N,N-dimethylaniline and the like, and hetero-
aromatic amines, such as pyridine, and alkylpyridines, such
as picolines, lutidines and collidines and the like.
Preferred basic solvents are triethylamine and pyridine;
pyridine is the most preferred basic solvent.
While the molar ratio of the sulfonyl halide 8 to
the endiol 6 within the range of about 1 to about 5 is not
crucial, a molar ratio of about 2 is preferred.
While reaction temperatures above about 0C are
to be avoided to suppress disulfonate formation, i.e., sulfona-
tion of the 1~ - as well as the 3~ -hydroxyl groups, a
reaction temperature of about -10C is preferred.
.
, ~ ..
- 11 - ~ .
, ~,., : .
1~'82~'ff~3f'7'
Included among the preferred 3ffB -sulfonates of
formula 7 are those compounds of formula 7 wherein R' is
methyl, phenyl or 4-tolyl. The most preferred 3~ -sulfonate
of formula 7 is the compound of formula 7 wherein R' is 4-tolyl.
The last step of the alternative process for the
preparation of lf~ -hydroxy-5-cholestene 5, the reduction of
a 3~ -sulfonate of formula 7, is accomplished by dissolving
a compound of formula 7 in an inert ethereal solvent, such
as ether, dimethoxyethane, tetrahydrofuran, dioxane and the
like, ether and tetrahydrofuran being preferred; ether
being most preferred, and treating the solution with an
alkali metal aluminum hydride, such as sodium aluminum
hydride, lithium aluminum hydride and the like, lithium
aluminum hydride being the preferred alkali metal aluminum
hydride, at a temperature range from about 25C to the boiling
point of the ethereal solvent, the boiling point of the inert
ethreal solvent being thè preferred reaction temperature.
The molar ratio of the alkali metal aluminum
hydride to the sulfonate 7 is not critical. The reduction is `
conveniently carried out with a molar ratio of the reducing
agent to the sulfonate 7 of about 1 to about 25, a molar
ratio of about 10 being preferred.
In the next step of the process for the preparation
of 3-deoxy-lf~ -hydroxycholecalciferol 13, 1~ -hydroxy-5-
cholestene 5 is converted to the acylate of formula 9
- 12 -
~,
f . , ~
,
~8~7
CH3
H
~ , :
wherein R is lower alkanoyl,
by means of acylating agents derived from straight or branched
chain saturated alkanecarboxylic acids having 2 to 8 carbon
atoms, such as alkanoyl halides and symmetrical alkanoic
anhydrides, in the presence of a tertiary heteroaromatic amine,
such as, for example, pyridine, picoline, lutidine, collidine :
and the like as the solvent system and acid-acce~tor and N,N-
dimethyl-4-amino-pyridine as the catalyst at from about 15C ~
to about the boiling point of the solvent system using from . ~ .
about 5 to about 20 moles of the acylating agent for each
molar equivalent of 1~ -hydroxy-5-cholestene 5. The :~
acylation is preferably performed at about room temperature
with about 10 moles of acylating agent for each mole of ~:`
alcohol 9. :~
Suitable alkanoyl halides include acetyl halides, . :
propionyl halides, 2-methylpropionyl halides, trimethylacetyl ; .
halides, hexanoyl halides, dimethylpentanoyl halides, : ~:
octanoyl halides and the like; acetyl chloride, hexanoyl
. t ~
__
~ 13 - -
" ~ .
~Zl6~7
chloride and octanoyl chloride are preferred; acetyl chloride
and octanoyl chloride are most preferred. Suitable symmetrical
alkanoic anhydrides include acetic anhydride, proprionic
anhydride, 2-methylpropionic anhydride, trimethylace~ic
anhydride, hexanoic anhydride, dimethyl-pentanoic anhydride,
octanoic anhydride and the like; acetic anhydride, hexanoic
anhydride and octanoic anhydride are preferred; acetic
anhydride and hexanoic anhydride are most preferred.
The subsequent steps of the process for the prepara- `
tion o 3-deoxy-lc~ -hydroxycholecalciferol 13 are performed
by utilizin~ procedures well-known in the art. Thus 1~ -
acyloxy-5-cholestene 9 is allylically brominated by means of
1,3-dibromo-5,5-dimethyl-hydantoin in a suitable aromatic-
aliphatic hydrocarbon solvent system, such as l:l-benzene-
hexane to a mixture of 7~ - and 7~ -bromo-l~ -acyloxy-5-
cholestenes of formula 10
.:' `
CH3", ~
H
~ .
`
Br
,~ -,
wherein R is as hereinbefore defined,
which without further purification is dehydrobrominated by
means of trimethylphosphite in an aromatic hydrocarbon
~ .
~. - 14 -
~08~ 7
solvent, such as xylene, to 1~ -acyloxy-5,7-cholestadienes
of formula 11
~ CH3"~
wherein R is as hereinbefore defined
by procedures essentially the same as those employed by Barton,
et al., supra for the synthesis of 1~ ,25-diacetoxy-7,8-
dehydrocholesteryl acetate, and hydrolyzed to 1~ -hydroxy-5, ;
7-cholestadiene of formula 11 (wherein R is hydrogen) by
means of an alkali metal hydroxide, such as sodium or
potassium hydroxide and the like, dissolved in a suitable lower
alkanol, such as methanol, ethanol and the like, at about
room temperature under an inert atmosphere, such as nitrogen,
helium and the like. ~he saponification of 1~ -acyloxycholestane
derivatives, such as compounds of formula 11 is well-known in
~ . .... .
the art (see for example, J. Rubio-~ightbourn, et al, Chem.
Pharm. Bull., 21, 1854 (1973)).
-- 1 5
' '
.
.,. . . ~ . . , .. .. . .. . . .. .. .. . , , .. , . ., .. . ... ; . . .... . . . . .. ..
1~382~B7
In the final steps of the process for the pre~ara~
tion of 3-deoxy-1~ -hydroxycholecalciferol 13, 1~ -hydroxy-
5,7-cholestadiene tll, R is hydrogen) dissolved in a suitable
saturated aliphatic hydrocarbon, such as pentane, hexane and
the like, or an ethereal solvent, such as ether, tetrahyd`ro-
~uran and the like, is irradiated by means of a medium pressure
mer~ury lamp equipped with a Corex ~lass filter under an inert
atmQsphere, such as nitrogen, helium and the like, at a
temperature from about -40 to about 25C for about 8 minutes
to afford 3-deoxy-lC~-hydroxyprecholecalciferoI 12
CH3r~
,~H ~ EI ¦ ,
` ~ ~
12
) which is then isomerized t~ 3-deoxy-1C~-hydroxycholecalciferol
CH3
H
' ` ~ ''` ' " " '
~ 2
~ OH .~:
* Trade Mark
- 16 -
. :'
~ .. . . .. . . .
.. .. .. . . : : . . : .
32~;87
by heating the previtamin 12 in an inert or~anic solvent,
such as dioxane, tetrahydrofuran and the like, under an inert
atmosphere, such as nitrogen, helium and the like at about .``
75C for about 2 hours. The irradiation and isomerization
steps follow paths well-trodden in the art (see for example,
Barton, et al., supra).
In the first step of the process o the present
invention for the preparation of 3-deoxy-1~ ,25-dihydroxy-
cholecalciferol 13a, 1~ ,25-dihydroxycholesterol 14,
3~"~ ~ ~
CH ~ ~ I OH `
H ~
14
prepared from cholesterol 2 vla 25-hydroxycholesterol 15
3~" ~
¦ OH
H .
~, . ' .
- 17 - :~
:
1~3Z687
according to ,he procedures of Narwid, et al. (Helv. Chim.
Acta., 57, 781 (1974) and sarton~ et al. (J. Chem. Soc. Chem.
Commun., 203 (1974), is selectively sulfonated to a compound
of formula 16
C~3"~
R'-SO2-O
16
~ .
wherein ~ is lower alkyl, phenyl or lower-alkylphenyl
by the method ~or the conversion of lo~-hydroxycholesterol
6 to the sulfonate of formula 7.
The sulfonate of formula 16 is then reductively
cleaved to lC~,25-dihydroxy-S-cholestene 17
3~", ~
OH
H `~ `
,.
17
- - 18 -
~(~8~6~3~
by the method described for the conversion of the 3~ -
sulfonate of formula 7 to 1~ -hydroxy-5-cholestene 5.
The subsequent steps of the process for the prepara-
tion of 3-deoxy-1C~25-dihydroxycholecalciferol 13a follow
those employed for the transformation of lC~-hydroxy-5-
cholestene 5 to 3-deoxy-lc~-hydroxycholecalciferol 13.
Thus lc~,25-dihydroxy-5-cholestene 17 is acylated to the
diacylate of formula 18
3/ ~ `
H ~ OR ~
~ .
~ ~ H
wherein R is lower alkanoyl "
by the method described for the acylation of 5 to compounds
of formula 9 followed by bromination to a mixture of 7~ - .
and 7~ -bromo-10~,25-diacyloxy-5-cholestenes of formula 19
'. ~
3~//
H ~ OR
H
Br :
9 ',.
, ` .
- 19 - , . . .
6B7
by the proce~ure described for the formation of the
previtamin 12 from the 5,7-cholestadiene 11 and isomerized to
3-deoxy-lCX,25-dihydroxycholecalciferol 13a following the
method used for the conversion of the previtamin 12 to 3-deoxy-
lo~-hydroxycholecalciferol _. .
The processes of the present invention are useful
for the preparation of the potent selective intestinal
calcium transport stimulators, 3-deoxy-lG~-hydroxycholecal-
ciferol 13 and 3-deoxy-1 ~,25-dihydroxycholecalciferol 13a.
4,6-Cholestadien-lc~,3~ -diol 4, lCX -hydroxy-5-
cholestene 5 and its lower alkanoyI derivatives of formula 9,
~ hydroxy-5,7-cholestadiene of formula 11 (wherein R is
hydrogen) and its lower al]canoyl derivatives of formula 11
(wherein R is lower alkanoyl) and the lower alkyl-, phenyl
and lower-alkylphenylsulfonates of l~-hydroxycholesterol
of formula 7 (wherein R' is lower alkyl, phenyl or lower-
alkylphenyl) are useful intermediates for the preparation
of 3-deoxy-1CC-hydroxycholecalciferol.
lOC,25-Dihydroxycholesteryl sulfonates of formula
16 (wherein R' is lower alkyl, phenyl or lower-alkylphenyl),
lo~,25-dihydroxy-5-cholestene 17 and its lower alkanoyl
derivatives of formula 18 (wherein R is lower alkanoyl) are `
.
useful intermediates for the preparation of 3-deoxy-loC,25-
dihydroxycholecalciferol 13a and dehydrobromination and
hydrolysis to 1~ ,25-dihydroxycholesta-5,7-diene 20 ~.
.
~ ;
- 20 -
~8Z6~37
3"" ~ ~
HO ~ ~ ~OH
by the procedure described for the conversion of acylates of
formula 9 to lo~-hydroxy-5~7-cholestadiene of formula 11
by means of the intermediate mixture o 7O~- and 7~ -bromo- `:
1~-acyloxy-5-cholestenes of formula 10.
The provitamin, ~ ,25-dihydroxycholesta-5,7-diene
20 is then irradiated to the previtamin 21
" ' .
.
CH3 . -.
HO
- 21 -
~L~82SO~7
3-Deoxy~ hydroxycholecalciferol 13 and 3-deoxy-
1~ ,25-dihydroxycholecalciferol 13a stimulate intestinal
calcium transport without significant concomitant mobilization
of bone calcium release and thus are useful not only for the
~reatment of vitamin D deficiency and metabolic disorders
in mammals where release of bone calcium is not detrimental,
but also for the treatment of those disorders when release
of bone calcium is undesirable. Bearing the lc~-hydroxyl group
required for biological activity and introduced metabolically
into vitamin D and its analogs in healthy subjects by the
kidney, 3-deoxy-lC~-hydroxycholecalciferol and 3-deoxy-
lC~,25-dihydroxycholecalciferol 13a are also useful for the
treatment of vitamin D diseases and metabolic disorders
associated with renal malfunction and uremic conditions.
In vitamin D deficient chicks, 3-deoxy-1C-hydroxy-
cholecalciferol 13 stimulates calcium transport to a maximum
level of about 7 times greater than that of rachitic control
birds and elicits a response about 1.5 times greater than the
res~onse elicited by cholecalciferol or its potent, rapid-
acting natural metabolite, l~ ,25-dihydroxycholecalciferol.
Maximum biological response is obtained at about 12 hours
af~er administration of 3-deoxy-lC~-hydroxycholecalciferol.
Due to the presence of the lc~-hydroxyl group, the character-
istic time lag of the 3-deoxy derivative 13 is about l/3-
1~2 that of the parent vitamin, cholecalciferol, and about
the same as those of 16~25-dihydroxycholecalciferol and
lo~-hydroxycholecalciferol. ~ ~
''
- 22 - ~
1082~
. . - .
In vitamin D deficient chicks, 3-deoxy-lc~25-
dihydroxycholecalciferol 13a stimulates calcium transport to
a maximum level of about 5-7 times greater than that of
rachitic control birds and elicits a response about equivalent
to the response elicited by cholecalciferol or its potent,
rapid-acting natural metabolite, 1~,25-dihydroxycholecalci-
ferol. Maximum biological response is obtained at about 12
hours after administration of 3-deoxy-lc~,25-dihydroxychole-
calciferol. Due to the presence of the lC~-hydroxyl group,
the characteristic time lag of the 3-deoxy derivative 13a
is about 1/3 that of the parent vitamin, cholecalciferol,
and about the same as those of lcx~25-dihydroxycholecalcifer
ana lC~-hydroxycholecalciferol.
Compared to 1~-hydroxycholecalciferol and lC~, `
25-dihydroxycholecalciferol, 3-deoxy-1~ -hydroxycholecalciferol
is virtually devoid of bone calcium mobilization activity
in the in vivo system described in Example 13. While lcC,
.
25-dihydroxycholecalciferol and 1~-hydroxycholecalciferol
xelease significant amounts of calcium at a dose of 5x10-5
ug/ml, 3-deoxy-lo~-hydroxycholecalciferol does not significantly
mobilize bone calcium at 5xlO-1 ug/ml, i.e., 3-deoxy-~ -
hydroxycholecalciferol is about 10,000 times less active than
metabolites. 3-Deoxy-1C~,25-dihydroxycholecalciferol is about
1/50 as active as 1 0~,25-dihydroxycholecalciferol.
3-Deoxy-lC~-hydroxycholecalciferol and 3-deoxy-lo~,
25-dihydroxycholecalciferol may be formulated with various `
conventional inert organic and inorganic pharmaceutical
carriers suitable for parenteral or enteral administration `~
such as, for example, water, gelatin, lactose, starch,
,
~ - 23 -
. : . .: - ., .. : . ,
~L~8i:~87
magnesium sterate, talc, vegetable and fish liver oil,
gums and the like. 3-Deoxy-l~ -hydroxycholecalciferol can
be administered in conventional pharmaceutical forms such as
solid forms, for example, tablets, dragees, capsules,
suppositories or the like, or in liquid forms such as solutions,
suspension, suppositories or the like. The pharmaceutical
co~positions containing 3-deoxy-loC-hydroxycholecalciferol
and 3-deoxy-lG~,25-dihydroxycholecalciferol can be subjected
to conventional pharmaceutical processes such as steriliza-
tion, and can contain conventional pharmaceutical excipients
such as preservatives, stabilizing agents, emulsifying
agents, salts for ad~usting osmotic pressure or buffers.
The pharmaceutical compositions can also contain other
therapeutically valuable substances.
A suitable pharmaceutical dosage unit might contain
about 10 to 1000 ug of 3-deoxy-l~C-hydroxycholecalciferol and
1-100 ug of 3-deoxy-lC~,25-dihydroxycholecalciferol.
Suitable parenteral dosage regimens in mammals
comprise from about 1 ug/kg to about 25 ug/kg per day. For
any particular subject, the specific dosage regimen should be
adjusted according to the disorder being treated, the
individual needs of the patient and the professional judgments
of those administering or supervising the administration of ;` ` .:
3-deoxy-lo<-hydroxycholecalciferol or 3-deoxy-10C1,25-
dihydroxycholecalciferol. The dosages set forth herein are
exemplary. They do not to any extent limit the scope or
practice of this invention.
.:'
.
,' ~
.. .. . . . . . ..
~82~;8~ `
EX~PLES
The following examples are illustrative only of
the invention and are not to be construed as limiting the
invention in any manner.
Example 1
.
1,4,6-Cholestatrien-3-one (3). 1,4,6-Cholestatrien-
3-one was prepared in 56% yield from cholesterol, 2,3-dichloro-
5,~-dicyano-1,4-benzoquinone and dioxane by the procedure of
A. B. Turner, J. Chem. Soc. C, 2568 (1963).
'~"'' " '. .
Example 2
`' ..
1 ~,2~ -Oxido-4,6-cholestadien-3-one (1).
1O~,2'X-oxido-4,6-cholestadien-3-one was prepared in 72%
yield from 1,4,6-cholestatrien-3-ones, aqueous 30% hydrogen
peroxide, 15% sodium hydroxide solution and methanol by the
procedure of B. Pelc and E. Kodicek, J. Chem. Soc. C, 1568
(1971).
Example 3
4,6-Cholestadien-1O~,3 ~-diol (4). A solution of
lC~2Oc-oxido-4~6-cholestadien-3-one (1, 5.0 g, 0.013 mole)
in anhydrous ether (200 ml) was heated under reflux with
lithium aluminum hydride (2.5 gt 0.066 mole) under anhydrous
~.
:
- 25 -
~ ~ .
, .: '
~8Z6i87
conditions for 5 hours. The reaction mixture was cooled
in an ice-bath and water (2.5 ml), 15% sodium hydroxide
solution, and water (7.5 ml) were added successively to the
well-stirred reaction mixture. The precipitate was collected
on a filter and the filter cake was washed with e~her. The
filtrate was evaporated under vacuum and the residue was
chromatographed on Woelm neutral alumina, grade III (150 g).
The material (3.8 g) eluted with benzene-ether (2:1) was
recrystallized from acetone-methanol to give the diol 4
as needles (3.25 g, 62% yield), mp 120-121C.
Anal. Calc'd for C~7H44O2: C, 80.94; H, 11.07.
~ound: C, 80.81; H, 11.21.
Example 4
lCC-Hydroxy-5-cholestene (5). A three-necked standard
.
taper round-bottom flask equipped with a mechanical stirrer, ;
dry ice condenser, a nitrogen inlet and an ammonia inlet
was thoroughly dried, flushed with nitrogen, cooled in a dry
ice-acetone bath and charged with ammonia (60 ml). Lithium
(0.4 g, 0.06 mole) was added portionwise under an atmosphere
of nitrogen, with stirring. A solution of ~,6-cholestadien-
1c~,3~ -diol (4, 0.518 g, 1.29 mmoles) in freshly distilled ~ ;
tetrahydrofuran (60 ml) was added and, after removal of the
cooling bath, the reaction mixture was stirred for 3 hours.
Ammonium chloride (ca 0.5 g) was added and after stirring
for 1 hour, saturated ammonium chloride solution was added.
'
'..' ~ .': '
- 26 ~
,..,,,. ~
'' ' '
` 10~26137
The ammonia was allowed to evaporate. Water was added to the
residue and the solution was extracted with ether. The
combined organic extracts were washed with water, dried over
anhydrous sodium sulfate, filtered and the filtrate was
evaporated under vacuum. Filtration of the residual solid
(0.509 g) dissolved in low boiling petroleum ether through a
column of silica gel gave 0.3 g (60%) of carbinol 5.
For analysis, a sample was purified by preparative
thin-layer chromatography on silica gel followed by recrystal-
lization from 95~ ethanol. The carbinol 5 had mp
n2 0-l03 0c. `
Anal. Calc'd for C27H46O C, 83-87; H~ 11.99.
Found: C, 83.71; H, 12.34.
Example 5
'
1~-Hydroxycholesterol (6). lC~,2C-Oxido-4,
,
6-cholestadien-3-one (1, 3~0 g, 7.6 mmoles) in tetrahydrofuran
(100 ml) was treated with lithium (4.0 g, 0.58 mole) for
3 hours by the procedure described above for the reduction of
4. Ammonium chloride (25 g) was added to the reaction mixture,
with stirring, and after one hour, saturated ammonium chloride
solution was added cautiously with vigorous stirring. The
ammonia was allowed to evaporate. Water was added and the
solution was extracted with ether. The combined ethereal
extracts were washed with water, dried over anhydrous sodium
~'
. .
- 27 - .`~
- ~ .
'
~ . . ; -
~C~8Z687
sulfate, filtered and the filtrate was evaporated under
vacuum. Chromatography of the residue (3.05 g) over alumina
(Woelm neutral, grade III, 50~ ethyl acetate-ethanol)
followed by recrystalli~ation from acetone gave 1.5 g
(49~) of the carbinol 5, mp 156-157C.
Example 6
lo~-~ydroxycholesteryl Tosylate (7, R' is 4-methyl-
phenyl). A solution of lc~-hydroxycholesterol (6, 1.0 g,
2.5 mmoles), p-toluenesulfonyl chloride (1.0 g, 5.2 mmoles)
and anhydrous pyridine (5 ml) was allowed to ctand overnight
in a freezer having a temp2rature less than 0C. Cold water
and ether were added to the reaction mixture. The phases
were separated and the ethereal phase was washed with cold
water, dried over anhydrous sodium sulfate, filtered and
the filtrate was evaporated. The crystalline residue
(1.35 g) was suitable without further purification for use -~
in the subsequent steps.
For analysis, a sample was recrystallized from
acetone-low boiling petroleum ether. The analytical sample
had mp 147 C (dec.).
~ nal. Calc'd for C35H5204S: C, 73-33; H, 9.41.
Found: C, 7 3 . 0 2; H, 9.48.
- 2~ - :
." .:
,5~
' '
.. . , - : . . .: .. . . -. . . : .
~082~87
Example 7
lCc-Hydroxy-5-cholestene (5). ~ solution of lc~-
hydroxycholesteryl tosylate (7, R' is 4-methylphenyl, 1.35 g)
in anhydrous ether (80 ml) was heated under reflux with lithium
aluminu~ hydride (0.908 g, 23.9 mmoles) under anhydrous
conditions for 5 hours. Work-up of the reaction mixture as
described in the above procedure for the preparation of 4,6-
cholestadien-lc~,3 ~-diol 4 followed by chromatography of
the crude carbinol on silica gel (25 g) using low boiling
petroleum ether-benzene as the eluent gave 705 mg (72%
yield based on 1~ -hydroxycholesterol 6) of 5.
Example 8
1~-Acetoxy-5-cholestene (9, R is acetyl). A
. .
solution of 1~ -hydroxy-5-cholestene (5, 1.5g, 3~9 mmoles),
acetic anhydride (5 ml), pyridine (5 ml) and 4-dimethylamino-
pyridine (.10 g) was allowed to stand at room temperature
overnight. Cold water and ether were added to the reaction `
mixture. The phases were separated and the ethereal extract ;
was washed with cold dilute hydrochloric acid, water and
sodium bicarbonate solution. The organic extract was dried `
over anhydrous sodium sulfate, filtered and the filtrate
was evaporated under reduced pressure. The residue was
dissolved in benzene and filtered through a column of silica
gel. Evaporation of the eluent followed by recrys-talliza-
tion of the residue from benzene gave 1.40 g (85~ yield)
- 29 -
,. . : ,,
~o~3z687
of the aceto~y derivative (9, R is acetyl) as needles, mp
69-70C.
Anal. Calc'd for C29H48O2: C, 81-25; H, 11-29-
Found: C, ~1.51; H, 11.20.
Example 9 -~
lC~-Acetoxy-5,7-cholestadiene (11, R is acetyl).
To a magnetically stirred solution of 1~ -acetoxy-5-cholestene
(9, R is acetyl, 0.415 g, 0.97 mmole) in 1:1 benzene-hexane
(90 ml) heated under reflux under anhydrous conditions was
added 1,3-dibromo-5, 5-dimethylhydantoin (0.145 g, 0.51
mmole) in one portion. The reaction mixture was heated
under reflux for 15 minutes and then cooled in an ice-bath.
The precipitate was collected on a filter and washed with
cold low boiling petroleum ether. The combined filtrates
were evaporated to dryness at room temperature under va~cuum
to give a yellow residual syrup. ;~
The yellow residual syrup was dissolved in xylene
~(50 ml) and the solution was added dropwise, with stirring,
to a refluxing solution of trimethylphosphite tl.5 ml)
in xylene (25 ml). After the addition was complete (ca 1/2
hour), the reaction mixture was heated under reflux for one
hour, and after cooling, the mixture was evaporated to
dryness at water pump vacuum and then under high vacuum.
The residue was dissolved in a small volume of low
boiling petroleum ether and was chromatographed on 10~ -
silver nitrate impregnated silica gel (15 g) using ether-low ; `
'~',~ ' `'
- 3~ - ;
, ';', .
`''`
~L~82~87
boiling petroleum ether (0~, 200 ml; 2%, 350 ml; 4%, 500 ml;
10%, 100 ml) as the eluents. Fourteen milliliter fractions
were collected. Fractions 52-75 contained mainly lcx-acet
5,7-cholestadiene as determined by ultraviolet spectroscopy.
These fractions were pooled and evaporated under vacuum.
The residue (55 mg) was chromatographed on silica gel (8 g)
using ether-low boiling petroleum ether (0%, 60 ml; 2%,
90 ml; 6%, 30 ml) as the eluents. Thirteen milliliter
fractions were collected and evaporation of fraction 4 under
reduced pressure gave 45 mg (11~ yield) of 1~ -acetoxy-5,
7-cholestadiene, mp 105-106C.
In a subsequent experiment, further purification by
preparative thin-layer chromatography followed by
recrystallization from ethanol afforded the acetate as
colorless needles, mp 108-109C.
Example 10 "
lC~-Hydroxy-5,7-cholestadiene (11, R is hydrogen).
A solution of lCx-acetoxy-s~7-cholestadiene (11, R is acetyl,
8.3 mg) and 5% methanolic potassium hydroxide (10 ml) was
allowed to stand at room temperature under an atmosphere of
nitrogen overnight. Work-up of the reaction mixtùre in the
usual way gave 1--hydroxy-5,7-cholestadiene.
11~8Z6~7
Example 11
3-Deoxy-l~ -hydroxycholecalciferol (13). ~X -
Hydroxy-5,7-cholestadiene (11, R is hydrogen) from the above
experiment was dissolved in ether and irradiated with a 100
~att medium pressure mercury lamp equipped with a Corex glass
filter under an atmosphere of nitrogen for 8 minutes by the
method of Barton, et al., J. Am. Chem. Soc., 95, 2748 (1973).
The crude irradiation product was chromatographed on silica
gel (12 g) using low boiling petroleum ether (100 ml),
2~ ether-low boiling petroleum ether (100 ml), 5% ether-low
boiling petroleum ether (100 ml), 8% ether-low boiling
petroleum ether (200 ml) and 10% ether-low boiling petroleum
ether (100 ml) as the eluents. Ten milliliter fractions were
collected. Fractions 36-39, the ultraviolet spectra of which `
showed maxima at 260 nm and minima at 230 nm, were combined ~ `
and evaporated to afford 3-deoxy-1~ -hydroxyprecholecalciferol
( _ ) ~ ` `
The 3-deoxy-1~-hydro~yprecholecalciferol from the
preceding experiment was dissolved in the required volume of
iso-octane and the solution was heated at 75C for 2.15 hours ;
under a nitrogen atmosphere,according to the procedure of
Barton, et al., supra. The solvent was evaporated and the `~ "
resi~ue was chromatographed on silica gel (10 g) using low `
boiling petroleum ether (150 ml) and ether-low boiling
petroleum ether (3%, 150 ml; 5%, 150 ml; 8%, 100 ml) as the
eluents. Ten milliliter fractions were collected. Fractions
37-40, the ultraviolet spectra of which showed maxima at ~
262 nm and minima at 227 nm, were combined and evaporated ;
to give 0.63 mg of 3-deoxy-1~ -hydroxycholecalciferol~ The
~` "
thin-layer chromatography of the vitamin showed one spot. ~ ~ `
- 32 -
.~ ~, .... .
1~8Z~B7
The vitamin exhibited the expected mass spectrum having
the calculated molecular ion.
Example 12
Determination of sone Calcium Mobilization in vitro.
.= .~ . _ . . .
45Calcium chloride (200 uCi) was administered to
17-day pregnant rats, and after 48 hours, the rats were
sacrificed and the fetuses were separated. Fetal radii
and ulnae were isolated and cultured in Biggers-Gwatkin-
~leyner medium. Paired radii and ulnae were employed. One
bone was treated with a solution of the vitamin D3 derivative
in 95% ethanol and its pair was used as the control. The
bones were cultured in a carbon dioxide incubator for 72 hours.
At the end of the culture period, aliquots of the media were
collected and the released 45calcium was counted.
The effectiveness of the vitamin D3 derivative in
prompting bone calcium release is expressed as the ratio of
the number of counts per minute (T) of released 45calcium from
the treated bone to the number of counts per minute (C) of
released 45calcium from its paired control. A T/C ratio
~reater than 1 indicates a significant release of bone calcium
in response to the vitamin D3 derivative.
- 33 -
~ .
.. . .
82~8~
Mobilization of Bone Calcium
Compound Dose (ug/ml) T/C - S.E.
1cC,25-(OH)2-D3 2xl0 5 1.62 - O.17
5x10 5 1.93 + 0.12
1~-OH-D3 5x10 3 1.75 + 0.20
3-D-lc~-OH-D3 0.5 0.83 + 0.05
1.0 1.~9 + 0.20
3-D-lo~,25-(OH)~-D3 ~ 0.5x10 3 1.66 + O.04
1.0x10 3 1.9~ + 0~13
5.0xl0 3 2.21 + 0.27
Four bone pairs were used for each determination
lCX,25-Dihydroxycholecalciferol (1O~,25-(OH)2-D3)
1~-Hydroxycholecalciferol (lc~-oH-D3)
3-Deoxy-1~-hydroxycholecalciferol (3-D-lC~-OH-D3)
3-Deoxy-lo~ r 25-dihydroxycholecalciferol (3-D-1O~,25-(OH)2D
Standard error of the mean (S.E.)
. .
Example 13
'" `',
Determination of Intestinal Calclum Transport ln vivo.a
Chicks were maintained on a rachitogenic diet for
3 weeks. The vitamin D3 derivative aissolved in 0.2 ml of
1:1-1,2-propandiol and ethanol was administered interperi-
~.- . . .
toneally. After 24 hours, the duodenal loop was lifted out,
' :
~- 34 -' ,
~3826~
0.2 ml of a solution of 45calcium chloride (2 uCi) in 95~
ethanol was placed in the loop and ~he loop was returned to
the cavity. Thirty minutes thereafter, the chicks were
sacrificed by decapitation, the blood was collectea, the
serum was separated and the amount of 45calcium absorbed
~rom the duodenal loop was determined radiographically.
Stimulation of Intestinal Calcium Transporta
Intestinal
Compound Administered Time of Assay Calciumb Relative
D~se After Absorption E~tancement
~osing(plasma 45Ca2+~ Control
__
(~noles) (hours)(cpm/0.20 ml+SEM)
Cbntrol None - 430 + 15 1.0
D3 1.3 10 620 + 18 1.4
D3 1.3 24 1360 + 40* 3.2
D3 2.6 24 2060 + 65* 4.8
D3 26.0 24 1730 _ 72* 4.0
___ _ __ ____ ________ __ _ __ __ _____ _ __ __ ___ __ __ __ __ ___ ___ _ _ _ __ _ __ __ __ _ _ _ _ _ _ _ _ _ _ _
10~,25-(OH)2-D3 0.6 10 1950 + 68* 4.5
loc~25-(oH)2-D3 0.6 24 780 + 21 1.8
_____ ______________________________________ __________ ________________________
aThe steroids were administered intraperitoneally in 0.20 ml of 1,2-
prop~utediol: ethanol, 1:1. At the indicated time an assay of
intestinal calcium transport was carried out exactly as described by i~ `
Hibberd and Herman (19). For this assay 4.0 mg f 40Ca2+ + 45Ca2+
(2 uCi) are placed in a duodenal loop, in vivo. Thirty minutes later
the appearance of 45Ca2+ is measured in~~te blood. Each nL~ber is the
average + SEM for gxoups of 6-8 birds.
lues indicated by * are significantly different from ~te control (-D)
~t P ~ 0.01.
Cholecalciferol (D3)
1CY,25-Dihydroxycholecalciferol (1~,25-OH)2-D3)
lC~-Hydroxycholecalciferol (lo~-oH-D3)
3-Deoxy-lC~-hydroxycholecalciferol (3-D-lo~-oH-D3)
- 35 -
~ .
~ . . . . ..
~2~i87
~~ Stimulation of Intestinal Calcium Transporta
Intestinal
Ccm~ound A~ministeredTime of AssayCalciumb Relative
D~se After Absorption Enhancement
Dosing (plasma 45Ca2+~ Overl
.
(nmoles) (hours)(c~m/0.20 ml+SEM)
1~-OH-D3 1.6 10 2010 52* 4.7
1~ -D3 0.8 24 1920 + 64* 4.5
____________________________________________________________ ____________________ .
3-D-16X-OH-D3 26.0 9 1047 + 67* 2.4
3-D-~X -D3 26.0 12 3000 + 220* 7.0
3-D-1~-QH-D3 26.0 24 1930 + 95* 4 5
3-D-lo~-OH-D3 5.2 24 1880 + 96* 4.4
_
~ontrol (-D) none -- 310 + 15 1.0
_____________ _____________________________ _ ___________________________________ :
loC~25-(oH)2-D3 6.5 8 1100 + 30* 3.5
l~x~25-(oH)2-D3 6.5 12 1200 + 60* 3.9
1~ ,25-(QH)2-D3 6.5 16 1230 + 40* 4.0
C~,25-(OH)2-D3 6.5 36 580 + 15 1.9
o~,25-(OH)2-D3 0.26 12 1000 + 40* 3.2
la ,25-(QH)2-D3 1.30 12 1010 + 25* 3.2
______________________________________________ _______________________________ .
3D-lC~,25-(QH)2-~3 6.5 8 750 + 30* 2.4
3D-la ,25-(OH)2-D3 6.5 12 800 + 20* 2.4
3D-lc~,25-(OH)2-D3 6.5 16 1060 + 40* 3.4
3D-~ ,25-(QH)2-D3 6.5 36 400 + 12 1.3
3D-lCx,25-(OH)2-D3 0.26 16 370 + 20 1.2
_______________ -- . .
aThe steroids were administered intraperitoneally in 0.20 ml of 1,2-
propanediol: ethanol, 1:1. At the indicated time an assay of intestinal
ç~lcium tr~nsport was carried out (13). For this assay 4.0 ~q of
~UCa~+ + 4~Ca~+ (2 uCi) are placed in duodenal loop, in vivo. l'hirty
munutes later the appearance of 45Ca2+ is measured in the bload. Each
number is the average _ SEM of groups of 8-10 birds.
~alues indicated by a * are significantly different from the control ~`
(-D) at P~C û.01. ;
- 36 -
~0~32~
Example 14
lC~,25-Dihydroxycholesteryl Tosylate (16, R' is 4-
methylphenyl). A solution of 1 ~ ,25-dihydroxycholesterol
(15, 0.5 g, 1.19 mole), p-toluenesulfonyl chloride (0.575 g,
3 mmole) and anhydrous pyridine (5 ml) was allowed to stand in a
freezer having a temperature less than 0C for 30 hours. Wor~-
up of the reaction mixture as described in Example 6 followed
by recrystallization from acetone-petroleum ether afforded
0.552 ~ (81%) of 'he tosylat~e (16, R' is 4-methylphenyl),
mp 13~-139~C.
Example 15
lCX,25-Dihydroxy-5-cholestene (17). A solution of
lCC,25-dihydroxycholesteryl tosylate tl6, R' is 4-methylphenyl,
0.570 g, 1.00 mmole), lithium aluminum hydride (1.033 g, 27 mmole)
and anhydrous ether (150 ml) was heated under reflux for 20 hours.
Work-up of the reaction mixture by the procedure described in
Example 3 followed by chromatography of the crude reaction `
mixture on silica gel using low boiling petroleum ether-
benzene as the eluent gave 0.282 g (70%) of the diol 17, ;
mp 127-128C and 135-136C.
- 37 -
. '': ~
. .
, :
~8'~687
. ~
Example 16
10~,25-Diacetoxy-5-cholestene (19, R is acetyl).
A solution of lC~,25-dihydroxy-5-cholestene (17, 0.195 g,
0.48~ mmole), acetic anhydride (4 ml) and anhydrous pyridine
~4 ml) was heated at 90C for 24 hours. Work-up by the
procedure described in Example 8 followed by filtration of a
solution of the residue in 2% acetone-benzene through
silica gel and recrystallization from methanol gave the
diacetate 20, mp 106-107C.
Example 17
1 ~,25-Dihydroxy-5,7-cholestadiene (20). 1O~,25-
Diacetoxy-5-cholestene (19, R is acetyl, 0.223 g, 0.46 mmole)
in 1:1 benzene-hexane was treated with 1,3-dibromo-5,5-dimethyl-
hydantoin (0.675 g, 0.23 mmole) as described in Example 9.
The crude bromo compound in xylene (10 ml) was
added dropwise to boiling s-collidine (14 ml) under an atmos-
phere of nitrogen. After the addition was complete, the
reaction mixture was heated under reflux ~or 30 minutes,
allowed to cool, worked up in the usual manner and chromato-
graphed on 10% silver nitrate impregnated silica gel (linear
gradient between equal volumes of petroleum ether and 1:1
ether-petroleum ether). Fractions showing ultra-violet
absorption maxima at 280 nm and 312 nm were pooled and
concentrated.
': . .
- 38 -
: ' . " ',
,,; , :
,
A solution of the residue, 5% methanolic potassium
hydroxide (45 ml) was allowed to stand at 25C for 12 hours.
T~ork-up of the reaction mixture in the usual way ~ollowed by
chromatography of the residue on silica gel (linear gradient
between equal volumes of petroleum ether and ether) gave 17 mg
of the dihydroxy diene 20, pure by thin-layer chromatography
as detected by ultraviolet irradiation.
The product had mp 151-152~C after recrystalliza-
tion from methanol-water.
Example 18
3-Deoxy-1O~,25-dihydroxycholecalciferol (13a).
1C,25-Dihydroxy-5,7-cholestadiene (12 mg) in ether was
irradiated with a 100 watt medium pressure mercury lamp equipped
with a Corex glass filter under an atmosphere of nitrogen with
ice cooling for 8 minutes by the method described in Example 11
to ~ive 3-deoxy-lC~,25-dihydroxyprecholecalciferol (21).
The 3-deoxy-16~,25-dihydroxyprecholecalciferol (21)
from the above was isomerized by heating in iso-octane at
75C for 2.25 hours according to the procedure of Example 11.
Cl~romatography of the residue twice on silver nitrate impreg-
nated with silica gel as in Example 17 gave 1.4 mg (12%) of
3-deoxy-1O~,25-dihydroxycholecalciferol (13a), homogeneous
by thin-layer chromatography. 3-Deoxy-1c~,25-dihydroxychole-
calciferol showed the expected ultraviolet absorption maximum
at 263 nm and minimum at 288 nm. It also exhibited a mass
spectrum having the calculated molecular ion. -~
''
- 39 -
~ -. , : :. . . . . . :
