Note: Descriptions are shown in the official language in which they were submitted.
lZS4$25
This invention relates to a method for preparing
compounds having vitamin D-like activity and to compounds
which are key intermediates in such method.
More specifically, this invention relates to a
method for preparing compounds having vitamin D-like activity
which contain an oxygen function at carbon 1 in the molecule.
Still more specifically, this invention relates to
a method for preparing l~-hydroxylated compounds which are
characterized by vitamin D-like activity via a cyclovitamin
D intermediate.
It is well known that the D vitamins exhibit certain
biological effects, such as stimulation of intestinal calcium
absorption, stimulation of bone mineral resorption and the
prevention of rickets. It is also well known that such
20 '
--1--
~254225
biological ac-tivity is dependent upon th~se vitamins being
, altered in vivo, i.e. metabolized, to hydroxylated derivatives.
¦ For example, current evidence indicates that la~25-dihydroxy-
! vitamin D3 is the in vivo active form of vitamin D~ and is
the compound responsible ~or the aforementioned biological
effects.
The synthetic l~-hydroxyvitamin D analogs, such as 1~-
hydroxyvitamin D3, and l~-hydroxyvitamin D2 also exhibit
pronounced biological potency and such compounds as well as
~ the natural metabolites show great promise as agents for the
l treatment of a variety of calcium metabolism and bone disorders,
i such as osteodystrophy, osteomalacia and osteoporosis.
I Since l~-hydroxylation is an essential element in
¦~imparting biological activity to the vitamin D compounds and
¦,their derivatives there has been increasing interest in
methods for chemically accomplishing such hydroxylation.
Except for one suggested method for the total synthesis of
l~-hydroxyvitamin D3 (Lythgoe et al, J. Chem. Soc., Perkin
¦~Trans I, p. 2654 ~1974)), all syntheses of l~-hydroxylated
I vitamin D compounds prior to the conception of the present
iinvention involved the preparation of a l~-hydroxylated
~steroid, from whi~h, after conversion to the corresponding
-hydroxy-5,7-diene sterol derivative, the desired vitamin
~D compound is obtained by well known photochemical methods.
,Thus available syntheses are multistep processes and in most
~cases are inefficient and laborious.
I A new method for introducing a hydroxyl group at the
,carbon 1 (C-l) position in the vitamin D or vitamin D derivative
~`molecule has now been found which in concept and execution
! differs radically from existing syntheses. This method,
~ - 2 -
¦! l
Il I
1Z~422S
which will be more fully described hereinafter, provides for
~the direct introduction of an oxygen function at C-l by
allylic oxidation.
¦ In general, the method of this invention comprises
~preparing 1~-hydroxylated ~ s having the formula
-...~ I
lO ,! - Ho~. OH
¦1by subjecting compounds (hereinafter referred to by the
general term "cyc~ovitam ~ in8 the formula
1' Z O ~ 7 - `
;Ito allylic oxLdation, rscovering the re5u1ting 1~-hydroxy~ated
: cyclovitamin D compound from the allylic oxidation reaction
mixture, acylating the recovered compound and recovering the
. . resulting la-0-acyl derivative, subjecting said derivative
: to acid catalyzed solvolysis, recovering the desired la~
: O-acyl vitamin D compound and hydrolyzing (or reducing with
! hydride reagents) the la-0-acylated product to obtain la-
j~hydroxyvitamin D compounds.
¦' In the above described process, R in the formulae
represents a steroid side c~ain; most commonly a substituted
¦l or unsubstituted, or saturated or unsaturated, or substituted
j. and unsaturated cholesterol side chain group and Z represents
, hydrogen or a Iower alkyl or lower acyl group or aromatic acyl
~254~ S
I
¦ ~roup. Preferably R will be a cholesterol or ergosterol
, side chain group characterized by the presence of a hydrogen
¦ or hydroxy group at what will be the 25-carbon (C-25)
position in the desired product molecule.
~ Wherever herein and in the claims the word "lower" is
'used as a modifier for alkyl or acyl, it is intended to
identify a hydrocarbon chain having from 1 to about 4 carbon
jatoms and can be either a straight chain or branched chain
configuration. An aromatic acyl group is a group such as
,benzoyl or substituted benzoyl. Also, in the various formulae
depicted, a wavy line to any substituent is indicative of
that particular substituent being in either the or S
stereoisomeric form.
More specifically, in the practice of the process of
this invention, R in the formulae set forth above and those
to follow, and in the claims, is preferably a cholesterol
side chain group characteri~ed by the formula
,
o ~ R~
wherein each of Rl, R2 and ~3 are selected from the group
consisting of hydrogen, hydroxy, lower alkyl, substituted
lower alkyl, O-lower alkyl, substituted O-lower alkyl, and
¦ fluorine. The most preferred side chain group having the
above configuration is one where Rl and R3 are hydrogen and
, R2 is hydroxyl. Other preferred side chain groups are those
where Rl, R2 and R3 are hydrogen, or where Rl is hydroxyl
1 and R2 and R3 are hydrogen, or where Rl and R2 are hydroxyl
i and R3 is hydrogren.
I~ ' ' '_ ~ _ '
~1 .
~5422~i 1
I Another preferred side chain group represented by R is
the ergosterol side chain group characterized by the formula
~ ~ 3
'wherein each of Rl, R2 and R3 are selected from the group
~consisting of hydrogen, hydroxyl, lower alkyl, substituted
ilower alkyl, O-lower alkyl, substituted O-lower alkyl, and
Ifluorine, and R4 is selected from the group consisting of
hydrogen and lower alkyl. The most preferred side chain
I groups having the designated ergosterol side chain configuration
are where Rl and R3 are hydrogen, R2 is hydroxyl and R4 is
~ methyl or where Rl, R2 and R3 are hydrogen and R4 is methyl
¦ and where the stereochemistry of R~ is that of ergosterol~
It is understood that wherever hydroxy groups occur in
'the side chain group R o~ the cyclovitamin D starting material,
such groups may ~e acylated, e.g. lower acyl such as acet~l
¦ or substituted lower acyl, benzoyl or substituted benzoyl,
~ I although-such acylation is not required for success of the
¦~ process.
It is to be noted further that the side chain group R
! need not be limited to the types enumerated above. The
¦ process described in this invention is-a general one that is
¦ applicable to cyclovitamin D compounds possessing many of
the common steroid side chains, e.g. the side chain of
I pregnenolone, desmosterol, cholenic acid~ or homocholenic
¦ acid. In addition -to the side chain groups defined above,
¦ cyclovitamin D compounds wherein the side chain R group is
' represented for ~xample by the following structures
~CCOA~
` or ~\/~ rl ~ 5 ~/~~~ .
i.
.
12~;42~
are conveniently prepared and are suitable starting materials
for the process of this invention.
, The cyclovitamin starting material for the oxidation
process is conveniently prepared from a vitamin D compound
i by a two-step procedure which comprises converting a vitamin
D compound carryin~ a 3~-hydroxy group to the corresponding
3~-tosylate derivative and then solvolyzing this tosylate in
a suitable buffered solvent mixture, such as methanol/acetone
~ containing sodium acetate, to yield the cyclovitamin product.
Sheves and Mazur ~J. Am. Chem. Soc. 97, 6249 (1975~) applied
this sequence to vitamin D3, and obtained as major product a
cyclovitamin D3 to which they assigned the structure shown
below, i.e. 6R-methoxy-3,5-cyclovitamin D3. A minor cyclo-
vitamin formed in this process was identifed as the corresponding
compound with the methoxy in the 6S configuration.
i It has now been found that if the solvolysis reaction is
carried out in methanol using Na~C03 buffer, a better yield of
cyclovitamin product than that reported by Sheves and Maz~r
can be obtained.
C H30
I It has now been found that vitamin D compounds carrying
other chemically reactive substituents (e.g., side chain hydroxy
1, groups) can be converted efficiently to their cyclovitamin D
derivatives. ~or example, with 25-hydroxyvitamin D3 as the
1, starting material in the above described process 25~hydroxy-6-
l~ methox~-3~s-c~clovitemin D3 is observed. The st~ucture of Ibis
j,l - 6 _
. .
' .
1, ~ ~ 4~ 25
!
¦ compound is shown below, where R represents the 25-hydroxy-
¦ cholesterol side chain. Similarly with 24,25-dihydroxyvitamin
¦'D3 as starting material, the above described process leads to
¦24,25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 represented by
the structure shown below where R represents the 24,25-dihydroxy-
¦cholesterol side chain. With vitamin D2 as the starting materialthe same process sequence leads to cyclovitamin D2, also
¦represented by the structure below but where R signifies the
ergosterol side chain. These cyclovitamin D compounds are new
compounds.
i In analogy with the results of Sheves and Mazur cited
,earlier, the 6R-methoxy sterochemistry can be assigned to the
major cyclovitamin D product obtained in these reactions, and
~to the minor constituent ~5-10%~ of the cyclovitamin product
mixture the 6S-methoxy configuration. The process of this
invention does not require separation of these stereoisomers, it
being understood, however~ that, if desired, such separation
can be accomplished by known methods, and that either C~ -epimer
ican be used although not necessarily with the same process
jefficiency. For these reasons stereochemical configuration
~at C-6 of the cyclovitamin D compounds is not designated in
¦ the structures of the speciPication and the claims.
CH30 [~
1
..
Il .
i2542:i:5
, By appropriate choice of suit~ble reagents or conditions
the process of this invention will yield cyc~ovitamin D
' I
where Z represents hydrogen, alkyl or acyl, and R can represent
any of the side chain structure types defined earlier. For
example, if ethanol instead of methanol is used in the
solvolyzing medium, a cyclovitamin of the structure shown
above is ob-tained, where Z represents ethyl. It is evident
that other 0-alkylated cyclovitamin D products can be
obtained by the use of the appropriate alcohol in the reaction
medium.
Similarly a solvolysis reaction medium composed of
solvents containing H20, such as acetonelH20, or dioxanelH20,
in the presence of an acetate salt or other buffering agent
yields the corresponding cyclovitamin D compound of the
formula shown above where Z is hydrogen. Sheves and Mazur
[Tetrahedron Letters tNo. 34) pp. 2987-2990 (19762] have in
fact prepared 6-hydroxycyclovitamin D3; i.e. the compound
represented by the structure above where`Z is hydrogen and
R represents the cholesterol sidechain, by treating vitamin
D3 tosylate with aqueous acetone buffered with KHC03.
It has now been found that a 6-hydroxy cyclovitamin, if
desired, can be converted to the correspond;ng acyl derivative
¦!
_ I . ".
- B -
~Z~4225
~i.e. Z = acyl, such as acetyl or benzoyl) by acylation
using standard conditions (e.g. acetic anhydride/pyridine~.
The acylated cyclovitamin D of the structure shown above,
¦ with Z representing acetyl, can also be obtained as a minor
llproduct, when the solvolysis reaction is carried out in a
¦ medium of dry methanol containing sodium acetate. The
cyclovitamin D compound where Z represents methyl is a
preferred starting material for subsequent reactions.
In the process of this invention the allylic oxidation
'is normally carried out in a suitable solvent, such as, for
example, CH2C12, CHC13, dioxane or tetrahydrofuran, utilizing
¦ selenium dioxide as the oxidizing agent. Because of the
nature of this oxidation reaction, it is preferable that it
be carried out at room temperature or lower temperatures.
-The oxidation reaction is also most advantageously conducted
i ¦ in the presence of a hydroperoxide, for example, hydrogen
,peroxide or an alkyl hydroperoxide such as tert-butyl hydro-
lperoxide. The oxidation product, i.e. the la-hydroxycyclovitamin
IID compound~ is readily recovered from the reaction mixture
¦,by solvent extraction (e.g. ether), and is conveniently
further purified by chromatography.- Other allylic oxidants
jcan be used if desired, it being understood that with such
~other oxidants variation in product yield may be encountered
¦'and that adjustment of the conditions under which the oxidation
¦reaction is carried out may have to be made, as will be
'evident to those skilled in the art. The products resulting
from allylic oxidation of cyclovitamin D compounds of the
~,structure shown abDve where Z represents lower alkyl te.g.
methyl) are readily illustrated by the following formula
l' ' ..................................................... .
_ g _
!1l
~ ~5~225
.1 . 1,
1~ ~~ l
where R represents any of the side chain structures defined
~ earlier, and Z represents lower alkyl te.g. methyl).
; Oxidation of the cyclovitamins by the process of this
, invention results in the formation of l-hydroxycyclovitamins
; possessing the l~-stereochemistry which ;s desired, i.e.,
; the stereochemistry of biologically active l-hydroxyiated
¦' vitamin D metabolites. The positional and stereochemical
selectivity and the remarkable efficiency of the oxidation
process is both novel and unexpected and all l~-hydroxy-
cyclovitamins disclosed are new compounds.
Minor products resulting from selenium dioxide oxidation
, of cyclovitamin D compounds are l-oxocyclovitamin D derivatives
I of the following structurè
. R .
I,. . Z~ ~ . .
where Z represen-ts lower alkyl and R represents any of the
side chain groups defined ëarlier. These l-oxocyclovitamin
- 10 -
2s4æs
1. .. .
D derivatives are readily reduced by hydride reagents (e.g.
LiAlH4 or NaBH4 or equivalent reagents) to form predominantly
l~-hydroxycyclovitamin D derivatives of the formula illustrated
i~previously. The facile reduction of l-oxocyclovitamin D
¦'compounds and especially the predominant form~tion of l-hydroxy-
cyclovitamin D compounds possessing the l~-stereochemistry
! is an unexpected finding, since mechanistic arguments-would
have predicted approach of the hydride reducing agent ~rom
¦the l~ss hindered side of the l-oxocyclovitamin D molecule
which would lead to the predominant function'of the l~-hydroxy-
cyclovitamin epimer.
The acylation of the recovered l~-hydroxycyclovitamin D
compound is conveniently accomplished by standard methods
with well-known acylating reagents, acetic anhydride beir.g
. one example, in a suitable solvent, e.g. pyridine, and is
normally conducted at room temperature over a period of
several hours, e.g. overnight. The product of acylation is
the corresponding l-O-acylcyclovitam~n D compound, which is
I conveniently recovered in a purity sufficient for further
reactions by solvent (e.g. ether)'extraction from the medium
; ! with subsequent evaporation of solvents.
Any primary or secondary hydroxyl groups present in the
side chain (R) of t~e'l~-hydroxycyclovitamin D compound can
be expected to be acylated also under these conditions~ If
complete acylation of tertiary hydroxy groups (e.g. the 25-
hydroxy-group) is desired, more vigorous acylating conditions
are normally required, e.g. elevated temperatures (75-
100C). It is advisable in such cases to conduct the
I reaction under a n:itrogen atmosphere to avoid decomposition
I of labile compounds. Products of such acyla-tions can be
llustrated ~y the formula ¦
-11- 1~
1. ` ~ I
. . I
%~ l
1 1
i ~
'where Y represents a lower acyl group or aromatic acyl group
'and Z represents lower alkyl and lwhere R can represent any
¦ of the steroid side chains defined earlier in this specification,
¦ it being understood that secondary or primary hydroxyl
groups originally present, will now occur as the correspond-
ing 0-acyl substituent, and any tertiary hydroxy group
originally present, may be hydroxy or 0-acyl depending on
the condition chosen.
; ¦ Conversion of the l~-0-acyl cyclovitamin to the 1~-0-
acyl vitamin D derivative is accomplished by acid-catalyzed
solvolysis of the cyclovitamin. Thus, warming la-0-acyl-
' cyclovitamin D with p-toluenesulfonic acid, in a suitable
i solvent mixture (e.g. dioxane/H20) yields l~-0-acyl vitamin
D compound. Sheves and Mazur used this reaction for the
`I conversion of cyclovitamin D3 to vitamin D3`~J. Am. Chem.
i l Soc. 97, 6249 ~1975)].
¦ A novel and unexpected surprising finding, not evident
from the prior art, was that la-0-acyl cyclovitamin D
¦, compounds are cleanly converted and in good yield to the
' corresponding l~-0-acyl vitamin by acid solvolysis. This
,, result was completed unpredictable since the allylic la-
il oxygen Punction of an la-hydroxycyclovitamin D compound
¦~ would be expected to be labile to the solvolysis conditions.
I - 12 - `
~ .
i
12~422S
Direct solvolysis of the l~-hydroxycyclovitamin D
can be accomplished in the presence of organic carboxylic
acids, e.g., acetic, formic, with subsequent recavery of
the corresponding 3-O-acyl la-hydroxyvitamin D derivative
and conversion of such derivative to the corresponding
hydroxy compound.
It is also important that any tertiary or allylic
alcohol functions that may occur in the side chain be pro-
tected as the corresponding acylates or other suitable,
acid-stable protecting group. The product l~-O-acyl vitamin
D is readily recovered from the solvolysis mixture by solvent
extraction and is further purified by chromatography. The
solvolysis reaction yi~lds both la-O-acyl vitamin D possessing
; the naturalS,6-cis double bond geometry, and the corresponding
l~-O-acyl vitamin D with a 5,6-trans geometry, in a ratio of
ca. 5:1. These products are readily separated by solvent
extraction and chromatography to yield in pure form l~-O-acyl
vitamin D product of the general formula illustrated below
~as well as, if desired, the corresponding 5,6-trans-isomer~,
R
., ~
' . Il
HO ~ OY
where Y represents a lower acyl group (e.g. acetyl) or aromatic
acyl group (e.g. benzoyl) and where R represents any of the
steroid side chains defined earlier, it being understood that
all hydroxy functions are present as their corresponding O-acyl
derivatives.
I ~2s4225
.~, I
! I
la-O-acyl vitamin D derivatives are readily converted
j to the desired l-hydroxyvitamin D compounds by hydrolytic
¦ or reductive removal of the acyl protecting group. The
¦, specific method chosen would depend on the nature of the
compound, in particular also the nature of the side chain R
group and its substituents. It is understood for example
'that hydride reduction would not be employed, if simultaneous
~reduction of another function susceptible to reduction, e.g.
1 ketone or ester, is to be avoided, or else such functions
¦ would be suitably modified prior to reductive removal of
acyl groups. Thus, treatment of the acylated compound with
a suitable hydride reducing agent te.g. lithium aluminum
hydride) yields the corresponding la-hydroxyvitamin D
compound. Similarly mild basic hydrolysis (e.g. KOH/MeOH~
~converts the acylated compound to the desired la-hydroxy
¦,derivative, it being understood that in cases where the side
~chain carries sterically hindered (~.g. tertiary~ O-acyl
l~groups, more vigorous conditions (elevated temperatures, ~
¦~prolonged reaction times) may be required. The la-hydroxyvitamin
j,D compound prepared by either method, is readily recovered
j~in pure form by solvent extraction (e.g. ether~ and chroma-
tography and/or crystallization from a suitable solvent.
¦~ An alternative and novel method for converting the la-
¦ O-acyl cyclovitamin D compounds to corresponding vitamin D
¦Iderivatives cons1sts of acid-catalyzed solvolysis of the
cyclovitamin compound in a medium consisting of an organic
~'acid te.g. acetic acid, formic acid) or of an organic acid
with a co-solvenl, such as acetone, or dioxane, if required
!Ifor solubilizing the cyclovitamin. It is a particular
! advantage of this method that if the side chain group R
¦'contains any tertiary hydroxy groups (e.g. the 25-hydroxy
I, .
- 14 -
~Z~ii42~S
j group) protection of such functionalities, e.g. as their
acyl derivatives, is not necessary. Thus, by way of example,
solvolysis of l~-O-acetoxyvitamin D3 in glacial acetic acid
yields la-acetoxy vitamin D3 3B-acetate, as well as some of
the corresponding 5,6-trans-compound tproduct ratio ca.
¦~ 3:1). These products can be separated by chromatography or
¦ the mixture can be hydrolyzed uncler basic conditions ~such
as KOHtMeOH) to yield l~-hydroxyvitamin D3 and the corresponding
l-hydroxy-5,6-trans-vitamin D3, which can then be separated
~ by chromatography. This method can be applied to any 1~-0-
acyl cyclovitamin D compound possessing any of the side
I chain groups R defined earlier in this specification.
Even more advantageously, solvolysis of l-O-acyl
¦~cyclovitamins can be carried out in formic acid or formic
! acid plus a suitabie co-solvent such as dioxane. This
¦ process leads to the formation of l~-O-acyl-vitamin D 3~-
formate dcrivatives, illustraee tbe Folloving formula
~where Y is a lower acyl group (preferably not formyl) or
¦~aromatic acyl group and R represents any of the side chain
groups defined earlier. Again the corresponding 5~6-trans
~compound is formed also as a minor product. Since the 3~-0-
j ~ormyl group is very readily hydrolyzed under conditions
I where the l~-O-acyl group is not affected (e.g. by treatment
¦! - 15 -
:l2542:~5
1.
:
¦ with potassium carbonate in a few minutes, as shown by the
specific Examples), the above miY.ture of 3-0-formyl products
are readily converted to la-0-acyl vitamin D and its corresponding
i 5,6-trans isomer. This mixture can be conveniently separated
at this stage by chromatographic methods to yield pure 1-
0-acyl vitamin D and the corresponding 5,6-trans-1~ 0-acyl
i vitamin D which can now separately be subjected to bas~c
hydrolysis, or to reductive cleavage of the acyl group to
j yield l~-hydroxyvitamin D compound, and 5,6-t'r'ans-1-
- ' 10 I hydroxyvitamin D compound.
¦ Another novel procedure for the conversion of la-0-acyl
' cyclovitamin derivatives to la-0-acyl-3~-formyl vitamin D
compounds of the formula illustrated above involves use of "crown
'ether" catalysts. For example, a two-phase system
consisting of formic acid and a hydrocarbon (e.g. hexane/benzene~
solution of l-0-acyl cyclovitamin D containing a suitable
crown ether (e.g. 15-crown-5~ Aldrich Chemical Co., Milwaukee)
and formate ion, converts the l~-0-acyl cyclovitamin to the
' l~-O-acyl-3~-0-formyl vitamin D derivative in good yield.
¦ The corresponding 5,6-trans isomer is formed as a minor
, product and is conveniently separated by chromatography.
Ii A further variation of the methods just described
¦I consists of converting a l~-hydroxycyclovitamin D compound
¦ to the corresponding l~-0-formyl derivative (e.g. by means
I of acetic-formic anhydride, in pyridine) reprecented by the
following formula ~ '
30 1' zo~l
¦' ~ C H
li - 16 -
11 . I
l! ¦
1 ........................................................... I
li ~2542~
I` ,
where R represents any of the side chain groups defined herein
before and Z represents lower alkyl, and subjecting this
intermediate to solvolysis in glacial acetic acid, as previously
described, to obtain, l-formyloxy vitamin D ~-acetate and
as a minor product the corresponding 5,6-trans isomer.
jRemoval of the formyl group, as described above, yields la-
Ihydroxyvitamin D 3-acetate and its 5,6-trans isomer which
lare conveniently separated at this stage by chromatography
¦and then separately subjected to hydrolysis or reductive
,cleavage of the acetates to yield a pure la-hydroxyvitamin D
compound and its 5,6-trans isomer.
Ii The allylic oxidation process of this invention can also
¦ be applied to cyclovitamin D compounds bearing 6-hydroxy or
¦l6-0-acyl groups. Thus, cyclovitamin D compounds of the
followin~ structuDe
~
! where ~ represents hydrogen and R represents any of the
I sidechain groups defined herein before can-be oxidized at
!~ carbon 1 by the ally~ic oxidation process of this invention
¦ to yield la-hydroxy-6-hydroxycyclovitamin D compounds and 1-
oxo-6-hydroxycyclovitamin cyclovitamin D compounds. Under
the oxidation conditions previously described, some cycloreversion
.l of the la-hydroxy-6-hydroxycyclovitamin D compound to a
! mixture of 5~6-cis and 5~6-trans-la-hydroxyvitamin D compounds
1 also occurs. All products are readily recovered from the
oxidation mixture by chromatography. The la-hydroxy-6-
hydroxycycyclov;tamin D compounds obtained by allylic oxidation
- 17 -
1254225
`;
j can ~e acylated (e.g. acetylated) by the standard process
, described previously and the resulting 1,6-diacyl cyclovitamin
! D intermediates are readily converted by ac;d solvolysis as
¦ discussed above to 5,6-cis and 5,6-trans-1~-0-acyl vitamin D
compounds which are easily separ~ted by chromatography.
Hydrolysis (by known methods) of the l-O-acyl derivatives
leads to the desired l~-hydroxyvitamin D products and their
~5,6-trans isomers respectively. The l-oxo-6-hydroxycyclovitamin
~D products are readily reduced by hydride reagents the la-
hydroxycyclovitamin derivatives.
Similarly, cyclovitamin D compounds of the structureshown above where Z represents acyl (e.g. acetyl, benzoyl~
'and R represents any of the sidechain groups previously
¦defined, can be converted by the sequence of allylic oxidation,
acylation, acid solvolysis, and finally hydrolysis of the
¦~acyl groups as described for the case of the 6-hydroxy analogues
¦to la-hydroxyvitamin D products and their corresponding 5,6-
,trans isomers.
I A further noteworthy and unexpeeted finding made i~ the
jcourse of this invention is the discovery that la-hydroxyvitamin
tD compounds are readily and efficiently converted to la-hydroxy-
~cyclovitamin D compounds by solvolysis of the 3~-tosylates (or
mesylates~ of la-hydroxy- or la-O-acyl vitamin D derivatives.
I~For example, la-acetoxyvitamin D3 3-tosylate, upon solvolysis
¦tusing conditions described herein before, e.g., heating in
methanol solvent containing NaHC03, yields la-hydroxy-6-methoxy-
3,5-cyclovitamin D3. Oxidation of this product te.g. with
MnO2 in CH2C12 solvent) yields the corresponding 1-oxo-6-methoxy-
1~3,5-cyclovitamin D3 analog as described in the specific examples.
~ In the following examples, which are intended to be
illustrative only, the numbers identifying particular products,
e.~. 3a for la-hydroxycyclovitemin D3, correspond to the
i - 18 -
225
! numbers designating the various structures for such products
as set forth below.
o D~" CH., J Cl13 ;/
I, 1 2 3
lC ' L
OAc ~o - OA. HO "- Olt
I a: R =
I b: R = ~H
c: R = ~ .-
ll d: R = ~OH
30 1' :
I - 19 _
.,
I!
:L~54225
. CH3~ Z~IJ ~o~l
H
~' 7 8 Z = H 10
10 ¦ `- 9 Z = Ac
C~O~ C~
C110 AeO''~OCHo
- ! 11 12
- 1I b: R - ~----~ H
c: R = = ~
d: R = ~OH
ii ' " , . . .
_ 20-
0
12542~5
Ex~ple 1
lu~Hydroxycyclovita~in D3 r3a) and l-oxo-cyclovita~in_ 3 (7a):
' To a stirred suspension of 1.4 mg (1.2 x 10 5 moles) of SeO2 in 1.0 ml
of dr~- CH2C12 is added 7 ~1 ~5.1 x 10 5 moles~ of a 70% solution of tert.
utyl hydrop~roxid2 (t-BuOOH). After stirrin~ for 25 ~in a solution of 9 mg
(2.3 x 10 5 moles) of 3,5-cyclovita~in D3 (compound 2a, prepared from vitamin
¦D3 ~la) by the method of Sheves ~ Mazur, J. Am. Chem. Soc. 97, 6249 (1975)~
in 0.5 ml of CH2C12 is added drop~ise. The mixture is stirred at room temper-
¦,ature for an addi~ional 25 ~in. Then 2.0 ~1 of 10% NaOH iR added, and this
¦-resulting mixture is diluted with 15 ml of diethylether. The organ-fc phase
; ¦is fieparated and washed successively with 10~ NaOH (2 x 10 ml), H2O (2 x 10
Iml), sat. FeS04 t3 x 10 ~1), and sat. NaCl (15 ml); and then dried over
¦MgSO4. Removal of solvent in vacuo yields a crude oily product that after
! chromatograph7 on a silica gel thin layer plate (10 x 20 cm, 750 ~m) developed
¦in 30% ethylacetate: Skellysolve B yields 4.5 mg (43% yield) of l~-hydroxy-
- !3,5-cyclovitamin D3 (3a): mass spectrum: (m/e) 414(30), 382~70), 341(35),
269(20), 247(45), 174(25), 165(30), 135(65); NMR, ~, ~.53 (3~, s, 18-~3),
'0.61 (2H, m, 4-H2), 0.87 (6H, d, 26-H3 and 27-H3~, 0.92 (3H, d, 21-H3), 3-26
(3E, s, 6-OCE3), 4.18 (lH, d9 J=9.0 ~z, 6-H), 4.22 ~lH, m, l-H), 4.95~(1H, d,
IJ=9 Hz, 7-H), 5.17 (lH, d, ~=2.2 ~z, l9(Z)-H), 5.25 (lH, d, J=2.2 Hz, l9(E~-
.H),
~ s a minor component 2.0 mg (19Z yield) of l-oxo-cyclovitamin D3 (7a)
was isolated from the reaction ~ixture: ~ass spectrum: ~m/e) 412 t40),
~380 (50), 267 (15), 247 (23), 135 (50)9 133 (100); NMR, ~, 0.49 (3H, s, 18-H3),
IQ-58 (2H, m, 4-H2), 0.87 (6H, d, 26-E3 and 27-~ ), 0.93 (3H, d, 21-E3), 3.30
¦~3~, s, 6-OCH3), 4.07 (lE, d, J=9.0 Hz, 6-H), 5.02 (lE, d, ~=9.0 Hz, 7-H),
!5.62 (lH, s, l9(Z)-~), 6.04 (lH, s, 19(E)-H); UV 248 (4,000).
l - - .
Example 2
~l~-Acetoxy-cyclovitamin D3 (4a):
Compound 3a (1.5 m,g) is dissolved in 200 ~1 of dry pyridine and 50 ~1 of
acetic anhydride. The reaction is ~ept at room temperature overnight, then
* Trade Mark 21
! diluted with 5 ml of sat. NaHC03 solution. This solution is extracted with
.three 5 ml portions of ether and the organic extracts are washed with H20 (2
x 10 ml), dried over MgS04, and the solvent is removed in vacuo to give
compound 4a: NMR, ~, 0.53 (3H, s, 18-H3), 0.69 (2H, m, 4-H2), 0.87 (6H, d,
26-H3 and 27-H3)~ 0.92 (3H~ d~ 21-H3)~ 2.10 (3H~ s~ l-OAc), 3.26 (3H~ 8~ 6-
iOCH3), 4.18 (lH, d, J=9.2 Hz~ 6-H), 4.98 (lH~ d, J=9.2 Hz~ 7-H~, 4.98 (lH~ d,
¦J=2.1 Hz, l9(Z)-H)~ 5.23 (lH, m~ l-H), 5.25 (lH, d~ J=2.1 Hz, l9(E)-H).
¦ Example 3
~ Hydroxyvitamin D3 (6a):
A solution of 1.3 mg of (4a) in 0.5 ml of a 3:1 mixture of 1,4-dioxane
and H20 is heated to 55~, 0.2 mg of p-toluenesulfonic acid in 4 ~1 of H20 iS
,added and heating is continued for 0.5 hr. The reaction is then quenched
with 2 ml of sat. NaHC03 and extracted with two 10 ml portions of ether. The
,organic extracts are dried over MgS04 and the solvent removed in vacuo. The
crude product is then applied to a 10 x 20 cm silica gel plate developed in
~i30% EtOAc: Skellysolve B to yield 400 ~g of product 5a: UY, ~maX 264 nm;
,mass spectrum, m/e 442 (M , 75)~ 382 a 0~, 269(15~ 134(100?; NMR~ ~ 0.52
¦(3H, s~ 18-H3)~ 0.86 (6H~ d~ J=5.5 HZ~ 26-H3 and 27-H3)~ 0.91 (3H~ d~ J=5.9
IH~, 21-H3), 2.03 (3H, s, l-OCOCH3), 4.19 (lH, m, 3-H), 5.04 ~lH, d~ J=1.5 Hz~
jl9(Z)-H)~ 5.31 (lH, m(sharp)~ l9(E)-H)~ 5.49 (lH~ m, l-H~ 5.93 (lH~ d~
IJ=11. 4 HZ~ 7-H), 6 . 37 ~lH~ d, J=11. 4 Hz, 6-H).
i Product 5a is taken up in 0.5 ml of ether and treated with excess LiAlH4.
The reaction is quenched with sat. NaCl solution and product is isolated by
~filtration and evaporation of the solvent in vacuo. The slngle product (6a~
,co-chromatographs with a standard sample of la-hydroxyvitamin D3 in 97:3
,CHC13:CH30H (l~-hyclroxyvitamin D3 Rf = 0.10, l~-hydroxyvitamin D3 Rf = 0.15,
,reaction product (6a), Rf ~ 0.10). This product possesses ~max = 264 nm and
a mass spectrum and nmr speCtrum identical to that of authentic l~-hydroxy-
vitamin D3-
~ -
Il - 22 -
~L2542ZS
Example 4
25-Hydroxycyclovitamin D3 (2b):
A solution of 100 mg of 25-hydroxyvitamin D3 (lb) and 150 mg of p-
, toluene-solfonyl chloride in 0.5 ml of dry pyridine is allowed to react for
24 hr at 3, and is then quenched with 5 ml of sat. NaHCO3. The aqueous
l.phase is extracted with ether (2 x 10 ml~ and the ether extract is washed
¦t~ith sat- NaHC03 (3 x 10 ml), 3% HCl (2 x 10 ml), and H2O (2 x 10 ml) and
then dried over MgSO4. The solvent is removed in vacuo and the crude
!,residue t25-hydroxyvitamin D3 3-tosyla~e) is taken up in 1.5 ml of anhydrous
¦methanol and 0.3 ml of anhydrous acetone~ 170 mg ~8 eq.~ of NaOAc is added
¦'and the solution is warmed to 55 for 20 hr. The mixture is cooled, diluted
~ith 10 ml of H2O and extracted with 3 x 10 ml of ether. The organic extracts
~are washed with three 10 ml portions of H20, dried over MgS04, and the
solvent is removed in vacuo. This crude residue is applied to a 20 cm x 20
cm silica gel TLC plate (750~m thick) which is developed once in a Skellysolve
B:ethyl acetate (8:2) system to yield 48 mg (45Z overall yield from lb~ of
(2b): mass spectrum, m/e: 414 (M , 40), 399(10), 382(80), 253(50), 59(100);
iN~ , 0.53 (3H, s, 18-~3), 0.74 (2H, m, 4-H2), 0.94 (3~s d, J=6.2 H2~ 21-
¦~H3), 1.21 ~6H, s, 26-H3 and 27-H3), 3.25 (3H, s, 6-OC~), 4.16 (1~, d, J=9.2
IHz, 6-H), 4.~9 (lH, m(sharp), l9(Z)-H), 4.99 (lH, d, J-9.3 Rz, 7-H)s 5.04
i~lH, m(sharp), l9(E)-H).
Example 5
i1,25-Dihydroxycyclovitamin D3 (3b) and:l-oxo-25-hydroxycyclovitamin D~ (7b):
j A mixture of 2.45 mg (0.5 eq.) of SeO2, 14 ~1 (2 eq.) of t-BuOOH and 1.2
`ml of dry CH2C12 is allowed to react at room temperature for 30 min. A
solution of the cyclovitamin (2b) in 0.5 ml of C~2C12 is added dropwise to
¦this oxidizing medium, and the reaction is continued for 15 min. The reaction
; I;is then quenched with 2.0 ml of 10X NaOH and diluted with 20 ml of diethyl
~., 'Il, ' ' , .
ll
1 - - 23 -
Il. ' ,
~1
254~22S
ether. The organic phase is separated and washed successively wlth 10% NaOH,
, H2O, sat. FeS04 solution, sat. NaHC03, and again with H~O, and then dried
I over MgSO4. The solven~ is removed in vacuo and the crude residue is applied
to a silica gel thin layer plate (20 cm x 20 cm, 750~mthick), which is
~developed in a Skellysolve B:ethyl acetate (6:4) system to yield 11 mg (53%
Iyield) of (3b): mass spectrum: m/e 430(M+, 15), 412(12). 380(35~, 269(10),
¦59~100); NMR, ~, 0.53 (3H, s, 18-H3), 0.61 (2H, m, 4-H2), 0.93 (3H, d, J=6.2
jHz, 21-H3), 1.21 (6H, 5, 26-H3 and 27-H3), 3.25 (3H, s, 6-OCH3?, 4-17 (lH, d,
J=9.2 Hz, 6-H), 4.20 (lH, m, l-H), 4.95 (lH, d, J=9.2 Hz, 7-H~, 5.19 (lH, d,
~J=l.9 Hz, l9tZ)-H), 5.22 (lH, d, J=l.9 Hz, l9(E)-H). As a minor component 1-
oxo-25-hydroxycyclovitamin D3 (7b) was isolated (15~) from the reaction
.mixture. Mass spectrum: m/e 428 (M ).
- Example 6
'1~,25-Dihydroxycyclovitamin D3-1,25-diacetate (4b-25-OAc):
A solution of 7 mg of (3b) in 200 ~1 of-dry pyridine is treated with
,10 ~1 of acetic anhydride. The syseem is flushed with N2 and heated to 97
¦for 16.0 hr. After cooling, the mixture is diluted with 5 ml of sat. NaHC03.
The aqueous mixture is extracted with two 10 ml portions of ether and the
¦lorganic phase is washed successively with two 10 ml portions o~ sat. ~aHC03,
liand then with 10 ml of H20. After drying over MgSO4, the solvent and residual
pyridine are removed by azeotropic distillation with benzene in vacuo. The
crude product is then applied to a silica gel thin layer piate (10 cm x 20
jcm, 750~m thick) daveloped in Skellysolve B:ethyl acetate (8:2) to yield 6 mg
(72Z) of the diacetate (4b,25-OAc) and 1.2 mg of the corresponding 3-acetoxy-
25-hydroxy derivative.
~', ' .
.ji ' . ' .
!l . . .
!i~
, ' .
- ~4 -
I!
Il
`~25~225
Example 7
jl,25-Dihydroxyvitamin D3-1,25-diacetate (5b,25-OAc):
! To 3.8 mg of t4b,25-OAc), dissolved in 400 ~1 of dioxane:H2O (3:1~ and
,warmed to 55~, is added 8 ~1 of a solutiQn of p-tvluene sulfonic acid in }l2O
'and heating is continued for 10 min. The reaction is quenched with sat.
NaHC03 and extracted with two 10 ml portions of ether. The ether solution is
washed with two 10 ml portions of H~O and dried over MgS04. The solvent is
,removed in vacuo, and the residue is applied to a silica gel thin layer plate
!~5 x 20 cm, 250~m thick) which is developed in Skellysolve B:ethyl acetate
¦!(8:2) to yield 1.8 mg (45Z) of (5b,25-OAc): W; 1 ax 265 nm; mass spectrum:
m/e 500(M+~ 25), 440(55), 422(15), 398(10), 380~45), 134(100); NMR, ~, 0.52
'(3H, s, 18-H33, 0.92 (3H, d, J=6.2 Hz, 21-H3), 1.42 (6H, s, 26-H3 and 27-H3),
1-97 (3H, s, 25-OCoCH3), 2.03 (3H, s, l-OCOCH3), 4-18 (lH, m, 3-H2, 5-03 (lH9
d, J=l.l Hz, l9(Z)-H), 5.31 (lH, m(sharp), l9(E)-H), 5.49 (lH, m, l-H), 5.93
(lH, d, J=11.4 Hz, 7-H), 6.37 (lH, d, J=11.4 Hz, 6-H).
' Example 8
~1~,25-Dihydroxyvitamin D3 (6b).
To a stirred solution of 1.0 mg of the diacetate, (5b,25-QAc~ ill 1.5 ~1
lof ether is added 0.5 ml of an ether solution saturated with LiAlH4. After
1lO miu at room temperature, the reaction is quenched with sat. NaCl solution
and the salts are dissolved by addition of 3Z ~Cl. The aqueous phase is
~extracted with ether and the ether extracts are washed with H2O and dried
¦over MgSO4. Thin layer chromatography (5 x 20 cm silica gel plates, 250 ~m
'thick) using 5~ MeOH: CnC13 yields 0.6 mg (70~? of 1~,25-dihydroxyvitamin D3
~(6b), exhibiting a W-spectrum with ~ 265 nm. The identity of-6b as
¦1~,25_dihydroxyvitamin D3 is established by direct COmpariSOD of mass and nmr
spectra with those of authentic material, as well as by co-chromatography of
6b with authentic 1~,25-dihydroxyvitamin D3.
j, . .
I' ., . ' .
j, - 25 -
~i ' .
iæ54225
~xample 9
;Cyclovitamin D2 (2c):
A solution of 100 mg of vitamin D2 (lc) and 100 mg of p-toluenesulfonyl
chloride in 0.3 ml of pyridine is allowed to react for 24 hr at 3, and is
then quenched with 10 ml of sat. NaHC03. The aqueous mixture is extracted
with two 10 ml portions of ether and the ether extract is washed successively
with sat. NaHC03 (3 x 10 ml), 3~ HCl (2 x 10 ml), and H20 (2 x 10 ml), and iS
then dried over MgS04. The solvent is rlemoved in vacuo and the crude vitamin
D2-3-tosylate is taken up in 1.5 ml of anhydrous methanol and 0.3 ml of
anhydrous acetone. After addition of 170 mg of sodium acetate, the solutlon
is warmed to 55~ for 20 hr. After cooling, the solution is diluted with
10 ml of H20 and extracted with three 10 ml portions of ether. The organic
extracts are washed with three 10 ml portions of H20, dried with MgSO4, and
the solvent is removed in vacuo. The residue is chromatographed on a silica
,gel thin layer plate (20 x 20 cm, 750~m) in Skellysolve B:ethyl acetate (8:2)
to yield 60 mg (59%) of (2c): mass spectrum: m/e 410 ~ , 152, 378(403,
253C40~, 119(60); N~R~ ~ 0.55 (3H~ s, 18-H3)~ 0.74 (2~ m~ 4-~22~ 0-82 and
'o.84 (6H, dd, J=4.1 ~z, 26-H3 and 27-~3), 0.91 (3H, d, J=7.0 Hz, 21
l1.02 (3H, d~ J=6.6 HZ~ 28-H3), 3.26 (3~ s~ 6-OCH3)~ 4.13 ~lH~ d~ J=9.6 ~z~
l6-H), 4.89 (lH, m, l9(Z)-H), 5.00 (lH, d, J=9.4 ~z, 7-H), 5.04 ~ m(sharp~,
~l9(E)-H), 5.20 (2H, m, 22-H and 23-H).
i Example l0
~l-Hydroxycyclovitamin D2 (3c) and l-oxo-cyclovitamin D2 ~7C):
l A miXture of 2.7 mg of SeO2 and 13.4 ~1 of 70% t-BuOOH, in 1.5 ml of dry
CH2C12~ is allowed to react for 30 min~ Compound 2c (20 mg) in 0.5 ml of
¦CH2C12 is then added dropwise~ the reaCtion is continued for 15 min~ and then
quenched with 2.0 ml of 10Z NaOH. The solution is dilutèd with 15 ml of
¦ether, the ether phase is separated and washed successively with 10% NaOH~
H20, sat . FeS04 solution, sat. ~aHCO3~ and again with H2O. After drying over
¦,MgSO4~ the solvent is removed in Vacuo, and the residue is appliFd to a
1~
! - 26 -
S4225
I silica gel thin layer plate (20 x 20 cm, 750l~m) which is developed once in
I Skellysolve B:ethyl acetate (8 2) system to yield 9.5 mg (45%) of (3c):
mass spectrum: m/e 426(M+ SS)~ 394(75) 353(30)~ 269(40~ 135(95); N~
1 0 53 (3H S 18-H3) 0 63 (2H, m, 4-H2), 0 8~ and 0 84 (6H dd, 26-H3 and
! 27-H33~ 0 92 (3H d, J=6 0 HZ 21-H3) 1 02 (3H d, J=6 4 HZ 28-H3) 3 26
¦I(3H S 6-OCH3) 4 18 (1H d, J=9 6 HZ 6-H), 4 21 (1H m, 1-H~ 4.94 (1H d,
liJ=9.6 HZ, 7-H), 5.17 (lH, m(sharp), 19(Z)-H3, 5.19 (2H, m, 22-H and 23-H),
1l5 24 (1H m~sharp~ 19(E)-H) A second minor component isolated from the
¦lreaction mixture proved to be l-oxo-cyclovitamin D2 (7c): mass spectrum,
~10 Iim/e 424 (M~)
Example 11
Hydroxycyclovitamin D2-1-acetate (4C3
TO 6 5 mg of (3C) in 300 yl of dry pyridine is added 150 yl of acetic
anhydride. This solution is heated to 55~ for 1.5 hr, then diluted with 5 ml
jof sat. NaHC03 and extracted with two 10 ml portions of ether. The organic
¦!extracts are washed with sat. NaHCO3, and H20, dried over MgS04 and the
ilresidual pyridine and solvent is removed by azeotropic distillation with
¦-benzene in vacuo, to yield compound 4c: mass spectrum: m/e 468(M~, 40~,
408(~0), 376(65), 251(603, 135(100).
li Example 12
¦l-Hydroxyvitamin D~-l-acetate (5c):
!~ A solution of 5.0 mg of (4c) in 400 yl of dioxane: H20 (3 1) is heated
to 55; 12 yl of an aqueous solution of p-toluenesulfonic acid (50 yg/yl) is
added and heating is continued for 10 min. The reaction is then quenched
with sat. NaHC03 and extracted with two 10 ml portions of ether. The separated
,ether phase is washed with 10 ml of sat. NaHC03 and two 10 ml portions of
'B20, dried over MgS04, and the solvent is removed in vacuo. Preparative thin
1. ' .
i, .
. ,1-' ' ' ' .
~ 7 -
Il
1;Z542~:5
layer chromato~raphy on silica gel (Skellysolve B:ethyl acetate, 8:2) gives
' 1.6 mg of 5c (32% yield); UV; ~ 265 nm; mass spectrum: m/e 454(M , 80),
! 394(80), 376(20), 269(40), 135(100); NMR, ~, 0.53 (3H, s, 18-H3~, 0.81
and 0.84 (6H, d, J=4.4 Hz, 26-H3 and 27-H3), 0.91 (3H, d, J=7.0 Hz, 21-H3),
1.01 (3H, d, J=6.7 Hz, 28-H3), 2.03 (3H, s, 3-OCOCH3), 4.18 (lH, m, 3-H),
5 03 (lH, d, J=1.5 Hz, l9(Z)-H), 5.19 (2H, m, 22-H and 23-H), 5.3 (lH,
m(sharp), l9(E)-H), 5.48 (lH, m, l-H), 5.92 (lH, d, J=11.0 Hz, 7-H), 6.37
¦' (lH, d, J=11.0 Hz, 6-H).
- I Example 13
l-Hydroxyvitamin D2 (6c):
i A solution of 1.1 mg of (5c) in 1.5 ml of ether is treated with 0.5 ml
of an ether solution ~aturated with LiAlH4. After 10 min at room temperature
-the reaction is quenched with sat. NaCl and the salts dissolved in 3~ HCl.
This aqueous solution is extracted with ether and the organic extracts are
washed with water and dried over MgS04. TLC on 250 ~ thick, 5 x 20 cm,
plates in 5% methanol:chloroform yields 0.8 mg (75% yield~ of l~-hydroxy-
vitamin D2: W: ~max 265 nm; mass spectrum: m~e 412 ~ ), 394, 376, 287,
,269, 251, 152, 134 (base peak); NMR: ~, 0.56 (3H, s, 18-H3), 0.82 and 0.84
i(6~, dd, J=4.4 ~z, 26-H3 and 27-H3), 0.92 (3H, d, J=6.6 Hz, 21-H3), 1.02 (3H,
j'd, J=6.6 Hz, 28-H3~, 4.23 (lH, m, 3-H~, 4.42 (lH, m, l-H), 5.00 (lH, m(sharp),
¦19(Z)-H), 5.20 C2H, m, 22-H and 23-H), 5.32 (lH, dd, J=1.4 Hz, l9(E~-H), 6.02
, d, J=l~ z, 7-H~, 6.38 (lH, d, J=11.6 Hz, 6-~). These spectral data
iare in full accord with data obtained for l-hydroxyvitamin D2, prepared by
an entirely different method lLam et al. Science, 186, 1038-1040 (1974~].
Example 14
Solvolysis of l-Acetoxycyclovitamin D in Acetic Acid:
; A solution of 3.0 mg of l~-hydroxycyclovitamin D3-1-acetate ( ~ in 200
~1 of glacial acetic acid is warmed to 55 for 15 min and subsequently
~30
- 28 - .
i
I
~2S422~
.
! quenched with ice-cold sat. NaHC03. The aqueous mixture is ex~racted with
diethylether and the organic phase is washed with sat. NaHC03 and water,
dried over MgS04, and filtered to yield a solution of 5,6-cis and 5,6-trans-
l~-acetoxyvitamin D3 3-acetates (UV: ~ 267-269 nm). The dried ether
solution is treated with a small amount (1.0 mg) of lithium aluminum
hydride, quenched with sat. NaCl, f~ltered and the ~olvent is removed in
vacuo. The crude oil is applied to a 5 x 20 cm silica gel tlc plate
¦I(250 ~mthick) which is developed in 5~ methanol:chloroform to yield 1.6 mg
,of a mixture (W , ~ 267-269 nm~ of la-hydroxyvitamin D3 (6a) and the
,corresponding 5,6-trans isomer (5,6-trans-la-hydroxyvita~in D3) in a ratio of
',3:1 as determined by NMR analysis: Characteristic resonances for the cis
isomer (6a): ~, 6.38 and 6.01 (d, J=11.4 Hz, 6-H and 7-H), 5.33 (dd,
J=1.5 Hz, l9(E)-H), 5.01 (sharp m, l9(Z)-H), 0.54 (s, 18-H3); for the
5,6-trans isomer: 6.58 and 5.88 (d, J=11.4 Hz, 6-H and 7-~), 5.13 (d, J-1.4
¦.HZ, l9(E)-H), 4.98 (sharp m, l9(Z)-H), 0.56 (s, 18-~
j. The same procedure may be used to effect the cleavage of the cycloprane
¦~ring (cycloreversion) of other cyclovitamins or their C-l-oxygenated analogs.
¦,Thus heating l~-acetoxy-25-hydroxyvitamin D3 (compound 4b, no protecting
group required for 25-OH function) in glacial acetic acid as described above,
Iyields l-acetoxy-25-hydroxyvitamin D3 3-acetate as the major product
; ¦(plus some of the corresponding 5,6-trans isomer~ as mi~or product~ and this
, 'mixture may be directly hydrolyzed (MeOH/KOH~ or subjected to hydride
reduction as described above, to yield la,25-dihydroxyvitamin D3 as the
ma~or product and 5,6-trans-1~,25-dihydroxyvitamin D3 as a minor product.
Example 15
.Formic acid catalyzed solvolysis of la-acetoxycyclovitamin D3:
A solution of the la-acetoxycyclovitamin D3 (4a) in dry dioxane is
¦ warmed to 55 and treated with a 1:1 solution of 98% formic acid:dioxane
j (50 ~l/mg cyclovita~in) for 15 min. The reaction is then quenched with
'
, .
I .
~ 42:~5
ice-water and extracted with ether. The ether extracts are washed with
water, sat. NaHC03, sat. NaCl, dried over ~IgS04, and the solvent removed in
vacuo. The crude product (l~-acetoxy-3~-formylvitamin V3 and its 5,6 trans
isomer) is dissolved in a 1:1 solution of dioxane:methanol and an equivalent
amount of aqueous ~2C03 (10 mg/100 ~ Ls added. After 5 min at room tempera-
ture, the solution is diluted with water and extracted repeatedly with
ether. The ether extracts are washed with water, dried over MgS04, and the
~solvent is removed in vacuo. The crude cis and trans mixture of l-acetoxy-3-
Ihydroxyvitamins is then chromatographed on a 10 x 20 cm, 750~im thick silica
'gel plate in 1:3 ethyl acetate:Skellysolve B to yield the pure cis-l~-~cetoxy-
vitamin D3. Basic hydrolysis, (NaOH in methanol) yields a product which is
chromatographically and spectrally identical to an authentic sample of 1~-
.hydroxyvitamin D3.
Example 16
¦ ~ ln D3-tosylate:
To a suspension of 170 mg of vitamin D3-tosylate ir. 6.0 ml of anhydrous
,methanol is added 213 mg (8.0 eq.) of NaHC03. The system is flushed with
; 'nitrogen and heated to 58 for 20 hr. The reaction is then diluted w~th sat.
i~aCl solution, transferred to a separatory funnel and extracted with 2 x 10 ml
portions of Et20. The organic extracts are washed with 1 x 10 ml portion
¦ of sat. NaCl and dried over MgS04. Aft'er removal of the solvent in vacuo,
the oily residue is chromatographea on a 750~m, 20 x 20 cm silica gel prep
plate inethyl acetate:Skellysolve B 2:8 to yield 94 mg (75%) of cyclo-
¦ vitamin D3 ( ~
! Example 17
¦~6-Hydroxy-cyclovitamin D3 (8a):
A'solution of 100 mg of vitamin D3, 100 mg of TsCl and 500 ~il of dry
,pyridine is kept at 5 for 24 ~r then diluted ~th ether and washed several
Ii
~ ' ' ' ' ' '
i ' - 30 -
.
~2S422~
times with sat. NaHCO3. The organic layer is dried over MgS04 and the solvent
~ is removed in vacuo. The crude D3-tosylate is suspended in 4 . O ml of
¦ acetone:H20 9:1 along with 175 mg (8 eq.) of NaHC03. The resulting mixture
liis heated at 55~ overnight, diluted with sat. NaCl and extracted twice
¦with ether. The ether extract is washed once ~ith water, dried over MgS04,
¦:and the solvent removed in vacuo. Preparative TLC (20 x 20 cm, 750~m,8:2
acetates
¦Skellysolve B: ethyl /yields 55 mg of the 6-hydroxy-3,5-cyclovitamin D3 ~8a);
¦~mass spectrum, m/e 384 (M ), 366, 253, 247.
i~
I Example 18
'6-Acetoxycyclovitamin D3 (9a):
To a solution of 300 ~1 of dry pyridine and 200 ~1 of Ac2O is added 6 mg
;of 6-hydroxy-cyclovitamin D3 (8a) in 200 ~1 of pyridine. The reaction is
warmed at 55 for 2.0 hr under ~2 then diluted with a large excess of
~toluene. The solution is evaporated to dryness at 40 in vacuo to yield the
crude 6-acetoxycyclovitamin D3 (9a); mass spectrum, m/e 426 (M ~.
! Example 19
1 .
~,~ydride reduction of l-oxo-cvclovitamin D (7a) to 3a:
i~ -~~ - 3 --
A solution of 2.0 mg of l-oxo-cyclovieamin D3 in 500 ~1 of ether'~s
~treated with 300 ~1 of ether saturated with LiAln4. After 30 min the
treaction is carefully quenched by the dropwise addition of sat. NaCl. The
¦insoluble salts are removed by filtration and the filtrate is dried over
MgS04. The solvent is removed in vacuo to yield 1.7 mg of a 95:5 mixture of
l~-hydroxycyclovitamin D3 (3a) and the corresponding l~-hydroxycyclovitamin D3
isomer, which are separated by chromatography. Similar treatment of l-oxo-
~cyclovitamin D3 with 300 ~i of lOOZ ethanol saturated with NaBH4 yields an
8:2 mixture of l~-hydroxy and l~-hydroxycyclovitamin D3 compounds (3a
and its l~-epimer).
~1 .
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~254~225
i
I Example 20
¦SeO~/t-BuOOH oxidation of 6-hydroxy cyclovitamin D3 (8a~:
To a stirring suspenSion of 2.0 mg of SeO2 in 1.5 ml dry CH2C12 i5
added 10 ~1 of 70~ t-BuOOH. When homogeneous~ a solution of 14 mg of
6-hydroxy 1 i of dry CH~Cl~
iis continued for 1.5 hr at room temperature. The reaction is quenched with
jlO% NaOH, diluted with ether, washed with lO~ NaOH and water, dried over
~MgSO4, and the solvent removed in vacuo. The crude oily residue is
.chromatographed tlO X 20 cm, 750~m,1:1 ethyl acetate.Skellysolve B) to
jyield 1.5 mg (10%) 1-oxo-6-hydroxy-cyclovitamin D3: mass spectrum~ (m/e~
398 (35)~ 380 (25), 247 (25), 135 (40)~ 133 (lOQ~; 2.0 mg ~15%~ of la,6-
¦laihydroxy cyclovitamin D3 (10a): maSs spectrum; (m/e~, 400 (50~ 382 (80)9
269 (20)~ 247 (40)~ 135 (80), 133 (40); and 2.0 mg (15Z) of la-hydro~y-
jvitamin D3 (6a)~ and the corresponding la-hydroxy-5,6-trans isomer.
Example 21
¦Conversion-of la,6-dihydroxy-cyclovitamin_~ (lOa~ to ~ roxyvitamin D3 ~6a):
. A solution of 400 ~1 dry pyridine, 200 yi acetic anhydride, and ~2.0 mg-of
jla,6-dihydroxy-cyclovitamin D3 (10a) is warmed to 55~ for 2.0 hr. The reaCtion
lis then diluted with toluene and stripped to dryness. The resulting oil
¦(la,6-diacetoxy-cyclovitamin D3) is taken up in 100 ~1 of THF and treated
,I ith 200 ~1 of 97% HC02~ for 15 min at 55'9. Dilution with sat. NaCl,
extraction with ether, washing with sat. NaHCO3, drying over MgSO4, and
¦removal of the ether in vacuo gives the crude l-acetoxy-3-formate cis- and
¦ tranS- Vitamin derivatives. Selective formate hydrolysis with R2CO3 followed
l by chromatography yields pure la-acetoxyvitamin D3 ~5a) ~hiCh iS converted
¦ to la-hydroxyvitamin D3 (6a) by simple ~OH/MeOH hydrolysis.
Il . , - , .
Il - 32 - -
54225
, Example 22
24(R)~25-Dihydrox~-cycl-ovitamin D3 ~2d):
To 150 ~1 of dry pyridine is added 10.4 mg of 24R,25-(OH)2D3 and 7.13 mg
~(1.5 eq.) of TsCl. The reaction is maintained at 0~ for 72 hr then diluted
with sat. NaHC03 and extracted with ether. After washing the ether
extract with sat. NaHCO3, drying over MgSO4, and removing the solvent in
vacuo, the crude tosylate (~70X by TLC) is suspended in 2 ml of anhydrous
~eOH along with 25 mg of NaHCO3 and heated under N2 at 58~ for 20 hr. The
l~eaction is then diluted with sat. NaCl and extracted with ether. The
ether extracts are washed with water, dried over MgS04 and the solvent removed
in vacuo. Preparative TLC (10 x 20 cm, 750 ~msilica gel, 6:4 Skellysolve
B:ethyl aCetate)yields 2.5 mg of recovered 24R,25-(OH~2D3 and 4.4 mg of
~24R,25-dihydroxy-cyclovitamin D (2d). mass spectrum, (m1e~, 430 (15),
398 (65), 253 (40), 159 (45), 119 (55), 59 (100), NMR, ~, 0.55 (3H~ s, 18-H3),
0.74 (2H, m, 4-H2), 0.94 (3H, d, J=6.2 Hz, 21-H3), 1.17 (3H, s, 26-H3),
1.22 (3H, s~ 27-H3), 3.26 (3H, s, 6-OCH3), 3.34 ~lH, m, 24-H), 4.17 (lH, d,
;~J=9.0 Hz, 6-H), 4.88 (lH, m(sharp), l9(Z)-H), 5.00 ~l-H, d, J=9.O Hz, 7-H),
,5 4 ClH, m(sharp), l9(E)-H).
~ - .
I Example 23
ZO 11~,24(R),25-Trihydroxy-cyclovitamiD _3 (3d):
To a previously prepared solutioD of 1.12 mg SeO2 and 12 ~1 of 70%
It BuOOH in 1.0 ml of dry CH2C12 is added 4.2 mg of 24R,25-dihydroxy-cyclo-
vitami~ D3 in 500 ~1 of CH2C12. After 30 min an additional portion of 1.12 mg
e2 and 12 ~1 70X t-BuOOH, in 500 ~1 of CH2C12 is added and the reaction
continued for an hour longer. The reaction is quenched with 10% NaOH,
~iluted with ether, and washea twice with 10% NaOH followed by a water wash.
The organic solution is dried over MgSO4, the solvent removed in vacuo, and
the resulting oll is chro=ato3r phed on a 5 x 20 c= 250P= sllica gel plate
_ 33 _
Il .
Ii
li l
I'
22S
I in cthyl acetate:Skellysolve B 1:1 to yield 1.6 mg of 1~,24(R),25-trihydroxy-
, cyclovitamin D3 (3d): mass spectrum, (m/e), 446 (30), 414 (50), 396 (40),
269 (30), 135 (80), 59 (100); NMR, ~, 0.55 (3H, s, 18-H3~, 0.65 (2H, m, 4-H2),
,0.96 (3H, d, J=6.0 H~, 21-H3), 1.19 (3H, s. 26-H3), 1.24 (3H, s, 27-H3~, 3.28
(3~, s, 6-OCH3), 3.35 (lH, m, 24-H), 4.20 (la, d, J=9.0 Hz, b-H), 4.Z2 (lH,
m, l-H), 4.97 (lH, d, J=9.0 Hz, 7-H), 5.18 (lH, m(sharp~, l9(Z)-H), 5.26
(lH, d, J=2.2 Hz, l9(E)-H). l-oxo-24(R),25-dihydroxy-cyclovitamin D3 (7d)
is also isolated as a minor component (<20%).
¦~ Example 24
,1~,24(R),25-Trihydroxyvitamin D3 (6d):
To 200 ~1 or dry pyridine and 150 ~1 of Ac2O is added 1.4 mg of lct,24R,25-
.trihydroxy-cyclovitamin D3 (3d). The system is flushed with N2 and heated to
95~ for 20 hr. The reaction is then diluted with dry toluene and azeo-
¦tropically distilled to dryness~ The oily product, 1,24CRj,25-triacetoxy-
cyclovitamin D3 (4d-24,25-diacetate), is dissolved in 200 ~1 of TEF and
added to 500 ~1 of a 1:1 solution o~ 97% HCQ2H:l~ and heated to 55 for 15
¦.min. The cooled reaction is diluted with ether, washed with H20, sat. NaHC03,
¦~sat. NaCl, and dried over MgS04. After removal of the solvent in vacuo the
¦icrude 1,24R,25-triacetoxy-3~-formate vitamin D intermediate is dissolved in
1~200 ~1 of THF and treated with 1.0 mg K2C03 in 10 ~1 H20 and 90 ~1 MeOH for
¦~5 min at room temperature. Dilution with sat. NaCl, extraction with ether, and
j~chromatography on a 5 x 20 cm, 250~m, silica gel plate in ethyl acetate:
¦~Skellysolve B 4 6 yields 1,24R,25-triacetoxy-vitamin D3. Treatment of this
triacetate with LiA1~4 gives 1,24R,25-trihydroxyvitamin D3 (6d) which
¦lis identical in all respects to an authentic sample.
Example 25
'Conversion of l-hydroxycyclovitamin D3 (3a) to l-hydroxyvitamin D3 (6a)
via the l-formyl intermediate (lla): ~
, A 200 ~1 portiOn of acetic anhydride is cooled to 0 and 100 ~1 of 97Z
Iformic acid is addecl slowly. The solution is brielly (15 min) heated to 50
Il - 34 -
I
1i,
225
then cooled to 0. A 100 ~1 portion of the acetic-formic anhydride is then
added to a solution of 5 mg of 1~-hydroxy-cyclovitamin D3 (3a) in
¦ pyridine at 0~. After 2.0 hr the reaction is diluted with sat. NaC1, extracted
j with ether, washed wlth H20, and dried over MgS04. The crude la-formyl-
cyclovitamin D3 (lla) obtained after removing the solvent in vacuo is
dissolved in glacial acetic acid and heated to 55~ for 15 min. Dilution with
sat. NaCl, extraction with ether, and isolation of the organic products
¦igive the crude product consisting of l-formyloxyvitamin D3 3-acetate (12a)
land the corresponding 5J6-trans -isomer. Treatment of the crude mixture with
K2C03 in H20/MeOH followed by chromatography (5 x 20 cm, 250~m, silica gel,
3:7 ethyl acetate:Skellysolve B) yields the pure l-hydroxyvitamin D3 3-acetate
¦;and 5,6-trans l~-hydroxyvitamin D3 3-acetate, which are hydrolytically
¦ converted (KOH/MeO~) to the corresponding la-hydroxy-vitamin D3 (6a) and its
5,6-trans isomer respectively.
' Example 26
! Cro~n e~her catalyzed cycloreversion of l~-acetoxy-cyclovitamin D3:
, A 0.5 M hexane:benzene Cl:lj solution of 15-cro~n-5 (Aldrich Chemical
Co., Milwaukee) is saturated with finely divided anhydrous sodium acetate.
To 300 ~1 of this so~ution is added 11.0/ of l-acetoxy-cyclovitamin D3 ~4a)
~in 600 ~1 of dry hexanes followed by 200 ~1 of 97~ formic acid. The two-
phase mixture is vortexed occasionally over 30 mln, then diluted with hexanes
jand the acid layer removed. The organic phase is washed with sat. NaHC03,
~sat. ~aCl, dried over MgS04 and the solvent removed in vacuo. The crude oil
llis taken up in 300 ~1 of THF and 300 ~1 of methanol and treated with 10 mg
¦ of K2C03 in 100 ~1 of H20. After 5 min at ambient temperature the reaction
¦'is diluted with sat. ~aCl and extracted with two portions of ether. The
¦ organic/is washed with H20, dried over MgS04, and the solvent removed in vacuo.
, .
!
!i. . .
- 35 -
22s
The resulting mixture is subjected to preparative Tl,C (750 ~m, 10 x 20 cm,
75:25 Skellysolve B:ethyl acetate) to yield 5.7 mg. (54~) of la-acetoxy-
vitamin D3 (5a) and 2.1 mg (20%) of 5,6-trans-1~-acetoxy-vitamin D3.
Example 27
Conversion of la-hydroxyvitamin D3 (6a? to l~-hyaroxycyclovitamin D3 (3a):
To 0.2 ml of pyridine is added 3.0 mg of la-acetoxyvitamin D3 (5a),
obtained by either selective acetylation of l~-hydroxyvitamin D3 (3a)(2 molar
excess acetic anhydride in pyridine, 4 hours, room temperature, followed by
separation of the desired la-acetoxyvitamin D3 derivative on preparative
silica gel tlc, using Skellysolve B:ethyl acetate, 3:13 or as the product from
Example 2, and 6.0 my of tosylchloride. After 18 hr. at 3 the reaction is
quenched with saturated NaCl solution, extracted with ether, ana the ether
extracts washed repeatedly with a saturated NaHCO3 solution. After drying over
MgSO4, and removal of the solvent in vacuo the crude la-acetoxyvitamin D3
3-tosylate is taken up in 3.0 ml of anhydrous MeOH bufferea with 12.0 mg of
NaHCO3. The reaction mixture is heatea to 55D overnight, quenched with
saturated solution of NaCl, extracted with ether and the solvent in removed
in vacuo. The crude product is subjected to preparative tlc ~5.X 20 cm, 250 ~m
silica gel, Skellysolve B:ethyl acetate, 3:1) to yield 2.2 mg of la-hydroxy-
cyclovitamin D3 t3a) which is-identical in all respects to-the product obtained
in Example 1.
Example 28
MnO2 oxidation of la-hydroxycyclovitamin D3 ~3a) to l-oxo-cyclovitamin D3 ~7a)
To 1.0 ml of dry CH2C12 is added 3.0 mg of la-hyaroxycyclovitamin D3
(3a) and 35 mg of finely divided MnO2. ~See for example, Paaren et al J. Chem.
Soc., Chem. Comm. 890 (1977)~. After 2.0 hr. the reaction mixture is filtered
through Celite to yield, after preparative tlc (5 x 20 cm. 250 ~m, silica gel,
Skellysolve B:ethyl acetate), 2.6 mg of l-oxo-cyclovitamin D3 ~7a) identical in
all respects to the product described in Example 1.
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* Trade Mark
~4æs ~'
Example_29
Direct solvolysis of la-Hydroxycyclovi-tamin D compounds
3.8 ml of glacial acetic acid is added to 380 mg of 1~-
hydroxycyclovitamin D3 and the solution warmed for 10 min. at 60.
After cooling the mixture is added to a stirring solution of
ice/NaHCO3. The neutralized aqueous solution is extracted with
diethyl ether, the combined organic extracts washed once with
water and dried over MgSO4. The crude product after solvent re-
moval is chromatagraphed on a 1.5 x 60 cm column, of 50 g of
neutral silica gel eluted with 100 ml of 4%, 100 ml of 8%, 100 ml
of 12%, and 400 ml of 16~ EtOAc/Skellysolve B. The desired 1~-
hydroxyvitamin D3 3-acetate isomer elutes before la-hydroxy-5,6-
trans-vitamin D3 3-acetate; 175 mg of la-hydroxyvitamin D3 3-acetate
is obtained; UV: ~maX264 nm; MS(m/e)442(M ,8), 382(70), 364(15),
269(20), 134(100).
Hydrolysis of la-hydroxyvitamin D3 3-acetate (10% NaOH/
MeOH, 2hr. RT)yields l~-hydroxyvitamin D3.
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