Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
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This invention relates tO a novel pro_ess for the preparation of
1~,25-dihydroxycholeclaciferol, a compound exhibiting greater activity
than vitamins D2 and D3 as measured by its antirachitic properties, and
which has value as a nutritional adjuvant, and to intermediates used for
preparing the same.
Processes for preparing 1~,25-dihydroxycholecalciferol have
heretofore been known (see DeLuca et al, Tetrahedron Letters,
page 4147 (1972) and Barton et al, J. Chem. Soo. Chem. Comm.,
page 203 (1974)). Each of these Icnown processes passes through a
triacyl derivative of 1c~,25-dihydroxyprovitamin D3 as an intermediate,
and in order ~o form the 5, 7-diene system in this intermediate,
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such severe reactions as introduction of a bromine atom into the
7-position and dehydrobromination must be conducted. Accordingly,
in these known processes, hydroxy groups at the 1Q-~ 3,~- and 25-
positions should be protected with an acyl group or the like, and the
protective groups should naturally be removed in subsequent steps.
A series of such severe reactions as introduction of a bromine atom
into the 7-position and dehydrobromination and removal of acyl groups
causes formation of various by-products. Consequently, complicated
purification means must be adopted and the yield is inevitably lowered.
This is particularly true with the process of Barton et al, since the
process passes through 1~, 2cy-epoxycholesta-4, 6-dien-3-one-25-ol
as an intermediate, requiring a very dangerous Birch reduction (using
liquid ammonia-metallic lithium) and such a cumbersome purification
process as preparative TLC, several times in the process steps.
Accordingly, each of these known processes cannot be considered tc~
be an industrially practical process. ~ -
The process of this invention provides a method for making t~
intendedcompound, 1~,25-dihydroxycholecalciferol, underverymild
conditions, whereby such side reactions as elimination of hydroxyl groups
and rearrangement of double bonds can be prevented. In addition, hydroxyl
groups need not particularly be protected and the intended compound can
be obtained very efficiently without adopting complicated reaction or
purification means.
In accordance with this invention, 1~,25-dihydroxycholecalciferol (I)
is prepared by isomerizing cholesta-l, 4,6-trien-3-on-25-ol (IX) in the
presence of a basic catalyst to form cholesta-l, 5, 7-trien-3-on-25-ol
(VIIl), reducing the so formed compound with a metal hydride to form
cholesta-1,5,7-trien-3,B~25-diol (VII), reacting the so formed compound
with a 1, 2, 4-triazoline-dione derivative represented by the following
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general formula (Vl)
¦¦ N-R
N /
O
wherein R represents an organic residue, to form a 1, 4-cyclic adduct
represented by the following general formula (V)
.
.
~\/\~ OH
':`' ' ~ ' ., '
HO
N )=O
LN
\R
wherein R is as defiried above, reacting the so formed compound with
a peroxide to obtain a la, 2a-epoxide compound represented by the
following general formula (IV)
1~7~7028
~ OH
O~ ~
HCi~ / N
~N ~-O
N
\R
whèrein R is as defined above, reducing the so formed compound with
a metal hydride to form cholesta-5,7-diene-1~,3,B,25-triol (IIl), irrad-
iating the so forme~ compound with ultraviolet rays to form 1c~,25-
dihydroxyprevitamin D3 (II), isomerizing the so formed previtamin
D3, and recovering la, 25-hydroxycholecalciferol.
DETAILED DESCRIPllON OF ~HE
PREFERRED EMBODIMEN~S
'rhe starting material for the process of this invention, namely,
cholesta-l, 4, 6-trien-3-on-25-ol, was obtained from cholesterol as
follows. Cholesterol was converted to cholesterol acetate by known
procedures after which the cholesterol acetate was converted to 25-
hydroxycholesterol acetate utilizing the procedures of A. Rotman and
Y. Mazur a. Chem. Soc., Chem. Comm., 1974, 15). ~he recovered
25-hydroxycholesterol acetate was hydrolyzed quantitatively with
aqueous 5% KH/MeOH to 25-hydroxycholesterol.
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The 25-hydroxycholesterol was dissolved in dioxane and boiled
with 2, 3-dichloro-5, 6-dicyanobenzo-quinone (DDQ) to obtain 25-hydroxy-
cholesta-1,4-diene-3-one. This latter compound was subjected to
dehydrogenation with chloroanil in the presence of the reduced derivative
of DDQ-hydroquinone (2HD~Q) to obtain 25-hydroxy-cholesta- 1, 4, 6-
triene-3-one. Alternatively, 25-hydroxy-cholesta- 1, 4, 6-trien-3-one
can be obtained by DDQ oxidation of 25-hydroxycholesterol a. J.
Partridge et al, Helv. Chim. Acta 57, 764 (1974))which was converted
from 6,~-methoxy-25-hydroxy-3~, 5-cyclo-5~-cholestane.
The cholesta l, 4, 6-trien-3-on-25-ol (IX) is subjected to isomeri-
zation in the presence of a basic catalyst whereby the two double bonds
located at ~ 4' 5 and a6' 7 positions are isomerized to ,5 ' and ~ 7~ 8
positions respectively. Although any of the well known basic catalysts
which promote such isomerization can be used the preferred catalysts
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are the alcoholates, such as methylates, ethylates and particularly
potassium t-butoxide.
The isomerization is preferably carried out in a solvent which
has a high dissol~,ing power for the starting compound including, in
general, organic solvents such as ethers, benzene type solvents and
hydrocarbon solvents. Solvents such as ether (ethyl), isopropyl ether,
tetrahydrofuran, dimethylsulfoxide, and tertiary butyl alcohol find ready
application. Excellent results are obtained when, for example, dimethyl-
sulfoxide as the solvent and potassium t-butoxide as the catalyst are used
in combination for the isomerization reaction. In addition, it is desirable
that the reaction be carried out in an atmosphere of inert gas such as
argon or nitrogen.
The isomerization reaction can be carried out over a reasonably
-broad temperature range although the preferred range is from about
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0 tO about 200 C. Higher or lower temperatures may be used but find
practical limits in their inlluence upon time of reaction, stability, or
instability of reactants to certain temperatures,
The compound (VllI) is isolated from the reaction mixture
according to a customary procedure, for example, by neutralizing
the resulting reaction mixture liquid, extracting it, concentrating the
extract and subjecting the concentrate to column chromatography or
the like. Because of yield considerations, however, it is preferred
that the reaction mixture be forwarded to the next step directly without
isolation of the compound (VIII).
The compound (VII) is obtained by reducing compound (VIII)
with a metal hydride. Metal hydrides suitable for such reduction, are,
for example, lithium aluminum hydride, metal boron hydrides such as
sodium boron hydride, potassium boron hydride or lithium boron
hydrides, so~ium borohydride and calcium borohydride. It is preferred
that the reduction reaction be carried out in a solvent, and use of an
ether type solvent such as ether, tetrahydrofuran, 1,2-dimethoxyethane,
diglyme (diethylene glycol dimethyl ether3 and the like~ When sodium
borohydride or calcium borohydride is used as the metal hydride, a
hydroxyl group-containing solvent such as water, methanol and ethanol
can be used. The reaction temperature is suitably chosen within a range
of from lowered temperatures to elevated temperatures. The preferred
temperatur,e range for the reaction is from about 0 C. to about 200 C.
Isolation of the compound (VIl) from the reaction mixture can
be accomplished according to customary methods, for example, by
neutralizing and extracting the reaction mixture liquid, concentrating
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the extract and subjecting the concentrate to column chromatography
or the like. lt is possible to forward the reaction mixture directly
to the subsequent step without isolation of the compound (VlI).
The compound (V) is prepared by reacting compound (VII)
with comp~und (Vl). It is preferred that the reaction be carried out
in a solvent that does not participate in the reaction.
In general, use of organic solvents such as ethers, benzene-type
solvents and hydrocarbon solvents is preferred. Specific examples of
preferred solvents are benzene, tetrahydrofuran and methylene chloride.
The reaction temperature is suitably chosen within a range of from
lower temperatures to elevated reflux temperatures.
ln the 1, 2, 4-triazoline-3, 5-dione derivative represented by the
general formula (VI), any of organic residues inactive to the reaction
can be used as R. With the preferred solvents for the reaction it is
generally preferred that R be a monocyclic aromatic residue or a
lower alkyl group. Specific examples of such preferred organic residues
are phenyl, mono-sub3tituted phenyl, methyl and ethyl grQupS.
Isolation of the compound (V) from the reaction mixture is
performed by concentrating the reaction mixture, extracting the con-
centrate with a solvent such as benzene or methylene chloride, and
subjecting the extract to column chromatography.
The compound (IV) is prepared by react ing compound (V) with
a peroxide. An organic peracid is preferably used as the peroxide.
As the peracid, there can be employed, aromatic peracids such as
perbenzoic acid and m-chloroperbenzoic acid and aliphatic peracids
such as permaleic acid, peracetic acid and pertrifluoroacetic acid.
lt is preferred that the reaction be carried out in a solvent, for example,
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ethers, such as ethyl ether and tetrahydrofuran chlorinated hydrocarbon
solvents, such as chloroform and methylene chloride, alkyl esters of organic
acids such as ethyl acetate, and fatty acids such as acetic acid the
peroxide being present in an amount of about 1 to 5 moles per mole
of compound V. In view of the inactivity toward the peroxide used
and the dissolving power toward the reactants, use of hydrocarbon
solvents such as methylene chloride and chloroform is especially
preferred. The reaction temperature is chosen within a rarlge of from
lowered temperatures to elevated temperatures depending on the kind
of the peroxide used.
The compound (lIl) is obtained by reducing compound (IV) ~
with a metal hydride. The metal hydrides which are preferably employed
are lithium aluminum hydride and lithium borohydride. The reaction
is normally carried out in a solvent, such as the ether type solvents,
for example, ethyl ether, tetrahydrofuran, 1,2-dimethoxyethane and
diglyme, which are especially preferred. The reaction temperature .
is chosen suitably within a range of from lowered tempexatures to
elevated temperatures, for example from about 50 to about 150~ C.
Isolation of the compound (lIT) from the reaction mixture is
performed according to customary methods, e. g. by decomposing the
excessive metal hydride, extractlng the reactl;on mixture, concentrating
the extract and subjecting the concentrate to column chromatography
or the like.
Compound (III) is then irradiated with ultraviolet rays to form
compound (II) in which the ring B of the steroid skeleton is cleaved.
The irradiation IS generally conducted in a solvent. Hydrocarbon-type
solvents, ether-type solvents and alcohols can be readily employed.
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Examples of preferable hydro_arbon-type solvents are saturated
hydrocarbon solvents, especially those having a low boiling point,
such as hexane and octane. Ether type solvents which are preferably
employed are the low-boiling-point saturated ethers, such as ethyl
ether and tetrahydrofuran and the preferable alcohols are the lower
alcohols such as methanol and ethanol. It is preferred that the reaction
be carried out at a temperature approximating room temperature and
in an atmosphere of an inert gas such as argon. The time for irradiation
of ultraviolet rays varies depending upon the intensity of the ultraviolet
source lamp and the reaction scale. In general, the irradiation time
is within a range of from scores of seconds to several hours.
Compound (II) is isolated from the reaction mixture according
to customary methods, for example; by concentrating the reaction mixture
and subjecting the concentrate to chromatography or the like. It is possible
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to forward the reaction mixture to the subsequent step directly without
isolation of the compound (II).
The compound (I) is formed by isomerizing the compound (II)
with thermal equilibrium being established between the compound (II)
and the desired end compound (I). The isomerization reaction is carried
out in accordance with known and customary methods, for example, by
allowing the compound (II) to stand for a period in the dark at room
temperature, or by heating it in a solvent. Any solvents which are
inactive to the reaction e. g., hydrocarbon-type s~lvents, ether-type solvents
and alcohols can be used. Preferred solvents are those having a low
boiling point and a high dissolving power for the compound (II), such as
hexane, isoctane, toluene, ether and te~rahydrofuran. Depending upon
the reaction conditions the reaction tlme may range from several hours
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tO several weeks. \7\1hen cornpound (II) is allowed to stand in the
dar};, it is preferred that the reaction continue for several weeks, When
~he reaction is carried out under heating and refluxing, it is preferred
thal the reaction continue only for several hours.
Isolation of the end product, 1~, 25-dihydroxycholecalciferol from
the reaction mixture is accomplished by known and custornary purification
means such as extraction, recrystallization, column chromatography
and partition chromatography. Column chromatography using a column
packed with Sephadex (manufactured by Pharmacia Pine Chemicals) is
especially effective. I~nisomerized compound (II) is also obtained in
the separation procedure and this unisomerized compound (II) can be
recycled for further isomerization after recovery.
- Each of intermediate compounds (VlII), (VII), (V), (IV), (III) and
(II) formed in the process of this invention are novel and form another
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aspect of this i~vention.
A more complete understandlng of the invention can be obtained
from reference to the following sp~cific examples which are included
for purposes of illustration only and are not intended to belimiting.
Example 1
1. Preparation of cholesta- 1, 5, 7-trien-3-on-25-ol.
A reaction vessel was charged with 125 ml of
dry dimethylsulfoxide and 2. 5 g of choles$a-1, 4, 6-trien-
- 3-on-25-ol was suspended in the dimethylsulfoxide. The
suspension was heated, and 10 ml of dry ether was added
to the resulting milky suspension tO obtain a transparent
solution. ~he reaction vessel was filled wilh dry argon
gas. Powdery potassium t-buloxide prepared from 1. 25 g
potassium` and 50 ml of butanol was added al one time to
the solution under ice cooling and wilh violenl agilalion.
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The reaction mixture liquid was violently agitated for
12 minutes and was then poured into ice water saturated
with carbon dioxide gas, which was prepared by adding
dry ice to water. The mixture was then extracted with
600 ml of ether cooled to 0 to S C. The extract was
promptly washed with cooled water saturated with a large
quantity of carbon dioxide until the washing liquid became
neutral, and the ether was then distilled off to obtain crude
cholesta- 1, 5, 7-trien-3-on-25-ol.
2. Preparation of cholesta-l, 5, 7-triene-3~, 25-diol.
100 ml of an ethanol solution containing 2. 5 g
of sodium borohydride was added dropwise under agitation - -
to 110 ml of methanol containing 4. 9 g of calcium chloride,
which was cooled below - 10 C. The mixture was agitated
for 20 minutes while maintaining the temperature at about
-10 C. Then, 100 ml of the ether solution containing crude
cholesta- 1, 5, 7-trien-3-on-25-ol, which was prepared in 1.
above, was added dropwise to the above mixture. The reaction
mixture was further agitated at -10 C. for 1 hour and at
0 C. for 1 hour, and was then neutralized with 10% acetic
acid after which the solvent was distilled off under reduced
pressure. The residue was extracted with methylene chloride
and the extract was washed with water and dried with
magnesium sulfate. Methylene chloride was distilled off
to obtain crude cholesta- 1, 5, 7-triene-3~, 25-diol (about 1 g).
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3. Preparation of 1,4-cyclized adduct of cholesta-1,5,7-
triene-3~B, 25-diol and 4-phenyl- 1, 2, 4-triæoline-
3, S-dione.
About S00 mg of 4-phenyl-1,2,4-triæoline-3,5-
dione was added little by little under agitation to 50 ml
of a methylene chloride solution containing the crude
cholesta-1,5,7-triene-3~,25-diol ~about 1 g)obtained
in 2. above, until the methylene chloride solution became
red. 'rhe solution was agitated at room temperature for
1 hour, and methylene chloride was distilled off and the
residue was subjected to column chromatography using
a column packed with 200 g of alumina. A chloroform
effluent fraction was collected and recrystallized from
methand to obtain 1. 4 g (the overall yield being 38.1%)
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of a cyclic adduct of cholesta-1,5,7-triene-3,~,25-diol
and 4-phenyl-1,2,4-triazoline-3,5-dione having a meltin~
point of 159 to 160 C.
Elementary Analysis Values as C35H4704N3- 1/2H2O:
Calculated: C = 72. 13%, H = 8. 30~57c~ N = 7. 21%
Found: C - 72. 06%, H = 8. 15%, N = 7. 45%
Example 2
197. 3 mg of the 1, 4-cyclic adduct obtained in
Exarnple 1 was dissolved in 5 ml of chloroform and 237
mg of m-chloroperbenzoic acid was added to the solution
after which the mixture was agitated for 40 hours at room
temperature. The reaction mixture liquid was diluted
with chloroform, washed with a lO~YC aqueous solution of
potassium carbonate, dried with anydrous magnesium
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sulfate, and the solvenL was distilled off under reduced
pressure. The residue was subjected to silica gel
column chromatography. A 1~, 2,~-epoxide of the 1, 4-cyclic
adduct melting at 159 to 160. 5 C. was recovered from a
fraction primarily eluted with methanol-containing ether,
and from the subsequent fraction was obtained 68. 3 mg
of a 1~,2~r-epoxide of the 1,4-cyclic adduct having a melting
point of 162 to 163. S C. (as measured with respect to a
product recrystallized from methanol).
Elementary Analysis Values as C35H47OSN3- 1/2H2O:
Calculated: C = 70.21~C, H - 8. 03~c, N - 7.02%
Found: C = 70. 02%, H - 7. 83%, N = 7. 03%
Example 3
Preparation of cholesta-5, 7-diene- 1~, 3~, 25-triol.
. ~
140. 3 g of the 1~, 2~-epoxide of the 1, 4-cyclic adduct of
cholesta- 1, 5, 7-triene-3,8, 25-diol and 4-phenyl- 1-2, 4-
triazoline-3,5-dione obtained in Example 2 was dissolved
in 15 ml of tetrahydrofuran, and 142 mg of lithium aluminum
hydride was added little by little to the solution under
agitation. Then, the mixture was mildly refluxed and
boiled for 1 hour and cooled, and 20 ml of tetrahydrofuran
was added to the reaction mixture. A saturated aqueous
solution of Glauber salt was added to the reaction mixture
until generation of bubbles was stopped, whereby excessive
lithium aluminum hydride was decomposed. The reaction
mixture was then filtered, and the residue was washed 3
times with 10 ml of tetrahydrofuran. The organic solvent
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layer was dried and the solvent was distilled off, The
residue was purified by using a column packed with 20 g
of Sephadex LH_20, and a fraction eluted by chloroform-
hexane (65: 35 V/V ratio) was collected and recrystallized
from ether-hexane to obtain 49. 9 mg of cholesta-5, 7-
diene- lc, 3~, 25-t~iol in the form of needles melting at
149 to 152 C.
Mass Spectrum: mk 416(M+), 398, 380, 362
UV Spectrum: il. 2 263, 272, 282, 294 m,u
max
~xaniple 4
Preparation of 1~,25-dihydroxyprevitamin D3.
A soluti~n of 31. 5 mg of cholesta-5, 7-d~ene-1~, 3~,
25-triol in 380 ml of ether was exposed for 1 minute to
ultraviolet rays through;a Vycor filter in an argon gas
atmosphere by using a 400-W high pressure mercury
lamp (manufactured by Toshiba). The solvent was distilled
off at room ternperature under reduced pressure, and
the residue was subjected to colurnn chromatography
sing a column packed with 20 g of ~ephadex LH-2~. From
a fraction primarily eluted by chloroform-hexane (65: 35
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V/V Tatio) was obtained 10. 5 mg of oily- 1~, 25-dihydroxy-
previtamin D3 ~having a maximum ultraviolet absorption
at 260 m,u in an ether solution), and from the subsequent
fraction was recovered 14. 3 mg of cholesta-5, 7-diene~
3~, 25-triol. When Ihis compound was exposed IO ultraviolet
rays in the same manner as above, 4. 5 mg of lc~, 25-dihydroxy-;
previtamin D3 was obtained.
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Example 5
Preparation of l ~, 25- dih~droxycholecalci~erol.
The 1~,25-dihydroxyprevitamin D3 obtained in
Example 4 above was dissolved in 100 ml of ether, and the
solution was allowed to stand at room temperature in
the dark in a vessel filled with argon gas for 2 weeks,
during which the position of the maxirnum ultraviolet
absorp;ion was shifted from 260 m}l to 264 m,~ and the
absorption intensity was increased to l. 6 times the initial
intensity. The solvent was distilled off under reduced
pressure and the residue was subjected to chromatography
using a column packed with 10 g of $ephadex LH-20. From
a fraction eluted by chloroform-hexane (65: 35 v!v ratio)
was obtained 9. 3 mg of oily 1~, 25-dihydrox$~cholecalciferol
~ ..... .
having a melting point of 9i to 99~ C. (as measured with
respect to a needle crystal recrystallized from chloroform).
Mass Spectrum: m/e416(M~), 398, 380, 362, 269, 251,
152, 134
UV Spectrum: ~ 264 m,u
x
lt will be apparent to one of ordinary skill in the art that in the
foregoing Examples other solvents and reactants, as well as reaction
conditions cornp~tible with such solvents and reactants, such as those
set forth in the foregoing specification, can be employed with comparable
results.
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