Note: Descriptions are shown in the official language in which they were submitted.
"" ~21~S~
--1--
Description
vitamin D Glycosides
Technical Field
The present inven-tion relates to water-soluble
synthetic glycosides of vitamin D, and their use in the
regulation of calcium metabolism.
Background Art
vitamin D3 deficiency, or disturbances in the
metabolism of vitamin D3 cause such diseases as ricketts,
renal osteodystrophy and related bone diseases, as well
as, generally, hypo- and hyper-calcemic states. vitamin
D3 and its metabolites are therefore crucial in
maintaining normal development of bone structure by
regulating blood calcium levels.
vitamin D3 is rapidly converted to 25-OH-D3 in the
liver. In response to hypocalcemia, 25-OH-D3, the major
circulating metabolite of the vitamin, undergoes further
metabolism in the kidney to 1~, 25-(OH)2D3. 1~, 25-
(OH)2D3 acts more rapidly than either D3 or 25-OH-D3.
Additionally, the dihydroxy form of the vitamin is 5-10
times more potent than D3 and about 2-5 times more potent
- than the monohydroxy form of the vitamin, in vivo,
provided it is dosed parenteraly and daily (Napoli, J.L.
and Deluca, H.F., "Blood Calcium Regulators" and
references cited therein in: Burger's Medicinal
Chemistry, 4th Ed., part II, edited by Manfred Wolf,
Wiley-Interscience, 1979, pp-725-739.)
vitamin D2, vitamin D3 or their metabolites which
are hydroxylated at positions 1; 1, 25; 1,24, 25; 24,25;
25,26; or 1,25,26 are water-insoluble compounds. When a
5~
--2--
drug is relatively insoluble in a aqueous environmént or
in the gastrointestinal lumen, post-administration
dissolution may become the rate-limiting step in drug
absorption. On the other hand, with water-soluble drugs,
dissolution occurs rapidly and thus facilitates transport
through the blood and to the site of activity. It would
therefore be desirable to provide a form of vitamin D (D3
or D2) which is hydrophilic and/or water-soluble, yet
preserves the normal biological properties of the water-
insoluble drug.
The extracts from the leaves of a South American
plant, Solanum malacoxylon (hereinafter "S.m."), have
been demonstrated to contain a water-soluble principle
which is different than l,25(OH)2D3 and which, upon
treatment with glycosidase enzymes yields 1,25(OH)2D3,
plus a water-soluble unidentified fragment. (See, for
example, Haussler, M.R., et al, Life Sciences, Volume
18: 1049~1056 (1976); Wasserman R.H. et al, Science
19~: 853-855 (1976); Napoli, J.L. et al, The Journal of
Biological Chemistry, 252: 2580-2583 (1977)).
A very similar water-soluble principle, which upon
treatment with glycosidases also yields 1,25 dihydroxy
vitamin D3, is found in the plant Cestrum diurnum
(hereinafter "C.d."; Hughes, M.R., et al, Nature, 268:
3~7-3~9 (1977)). The water soluble extracts from S.m. or
C.d. have biological activity which is similar to that of
1~, 25-dihydroxy vitamin D3.
The only evidence existing to date concerning the
structure of the water-soluble fragment released during
glycosidase treatment of the water-soluble principles
from these plants is indefinite. The authors of the
aforementioned publications have concluded that the
structure is probably a glycoside, on the basis of
enzymatic evidence, the water-solubility, and the use of
~L2:1~S~
--3--
chemical detectlon reagents ~Peterlik~ N. and Wasserman,
R.H. FEBS Lett. 56: 16-l9, (1973)). Humphreys (Nature
(London) New Biology 246: 155 (1973)), however, has cast
some doubt on this conclusion since he demonstrated that
the Molisch carbohydrate test was negative Eor the
principle.
Since it is known that the molecular weight of the
water-soluble vitamin D3-containing principle, prior to
enzymatic release, is considerably greater than 1000
(Humphreys, D.J., Nature (London) New Biology 246: 155
(1973)), the molecular weight of the water-soluble
conjugated fragment released by enzymatic hydrolysis can
be calculated to be considerably greater 584, the
molecular weight of dihydroxy vitamin D3 being 416. Thus
if the water-soluble fragment released by enzymatic
hydrolysis were in fact a glycoside, it would contain
more than 3 glycosidic (glycopyranosyl or glycofuranosyl)
units.
Moreover, the results of enzymatic release are fully
consistent with a wide variety of structures. For
example, Haussler, M.R., et al, Life Sciences 18: 1049-
1056 (1976) disclose the use of mixed glycosidases
derived from Charonia lampus to hydrolyze the water-
soluble principle. This enzyme is really a mixture of
enzymes as follows (Miles Laboratories, 1977
Catalog): ~- glucosidase (11 units), - mannosidase (33
units), ~- mannosidase (5.2 units), ~- glucosidase,
~- glucosidase (4.8 units), ~- galactosidase ~44
units), - galactosidase~(26 units), a- fucosidase (24
units), ~- xylosidase (8.2 units), ~- N-
acetylglucosaminidase (210 units), a- N-
acetylgalactosaminidase (41 units), and ~- N-
acetylgalactosaminidase (25 units) Peterlik, M., et al
(Biochemical and Biophysical Research Communications, 70:
797-804 (1976)) in their study of S.m. extract
s~
with ~-glucosidase (almond) from Sigma Chemical Company
utilized an enzyme that also contained ~- D-
galactosidase, and ~- D-mannosidase activities (Sigma
Chemical Company, February 19~1 Catalog; see also
Schwartz, J., et al, Archives of Biochemistry and
Biophysics, 137: 122-127 (1370)).
In sum, the results observed by these authors are
consistent with a wide range of structures, none of which
have been well characterized but which, even if proven to
be glycosides, contain at least more than 3 glycosidic
units per vitamin D unit.
A need, therefore, continues to exist for a well-
defined, well-characterized water-soluble form of vitamin
D, which will be hypocalcemically active and maintain
calcium and phosphorus homeostasis.
Disclosure of the Invention
It is therefore an object of the invention to
provide well-characteri~ed, well-defined synthetic, water
soluble forms of vitamin D3, vitamin D2, and hydroxylated
metabolites thereof.
It is another object of the invention to provide
water-soluble forms of the aforementioned vitamins D
which are hypocalcemically active, and which are active
in maintaining calcium and phosphorous homeostasis in the
animal bodyO
~ till another object of the invention is to provide
a pharmaceutical composition containing the
aforementioned vitamins.
Yet another object of the invention is to provide a
method for the treatment of hypocalcemia and calcium and
phosphorous metabolic disorders in animals, by using the
aforementioned water-soluble forms of vitamin D.
s~
--5--
These and olher objects of the invention, as will
hereinafter become more readily apparent, have been
attained by providing:
A synthetic compound which is biologically active in
maintaining calcium and phosphorous homeostasis in
animals, ot the formula (I)
~ X
(1)
~J
4 ~ ~
R10 J~x
wherein the bond between carbons C-22 and C-23 is single
or double; R2 is hydrogen, -CH3 or -CH2CH3;
wherein X is selected from the group consisting of
hydrogen and -ORl, wherein Rl is hydrogen or a straight
or branched chain glycosidic residue containing 1-20
glycosidic units per residue; with the proviso that at
least one of said Rl is a glycosidic residue.
~ u~ vi~_Out the Invention
The present invention provides for the first time
well-defined and substantially pure characteri~ed
synthetic, water-soluble forms of vitamins D3 and D2, as
well as hydroxylated derivatives of these vitamins. The
compounds of the present invention may in many instances
~ be crystalline. They represent a distinct advance over
- the partially purified, poorly characterized presumed
"glycoside" of lal 25-dihydroxy vitamin D3 of the prior
~9S~
--6--
art.
The compounds of the invelltion are those having
the formula (I): X
~ X
~ (I)
~
R 0 ~ X
wherein the bond between carbons C-22 and C-23 is single
of double; R is hydrogen, -CH3 or -CH2CH3,
wherein X is selected from the group consisting of
hydrogen and -ORl, where ~1 is hydrogen or a straight or
branched chain glycosidic residue containing 1-20
glycoside units per residue,
with the proviso that at least one of said Rl is
a glycosidic residue.
~y glycosidic units are meant glycopyranosyl or
glycofuranosyl, as well as their amino sugar derivatives
bu~ do not include glucuronic acid or derivatives thereof.
The residues may be homopolymers, random, or alternating
or block copolymers thereof. The glycosidic units have
free hydroxy groups, or hydroxy groups acylated with a
group R3-C-, wherein R3 is hydrogen, lower alkyl, aryl or
o
aralkyl. Preferably R3 is Cl-C6 alkyl, most preferably
acetyl or propionyl, phenyl,
s'~
nitrophenyl, halophenyl, lower al]~yl-substituted phenyl,
lower alkoxy-substituted phenyl and the like; or benzyl,
nitrobenzyl, halobenzyl, lower-alkyl-substituted benzyl,
lower alkoxy-substituted benzyl, and the like.
When the compouncls of Eormula (I) have a double bond
at position C-22, they are derivatives of vitamin D2,
whereas if the bond at that position is single, and there
is a lack of C24 alkyl they are derivatives of vitamin
D3. The latter are preferred.
The compounds of the invention contain at least one
glycosidic residue at positions 1, 3, 24, 25 or 26. They
may, however contain more than one, and up to five such
glycosidic residues simultaneously.
Preferred are those compounds derived from vitamins
15 D3 or D2; l-hydroxy-vitamins D3 or D2; 1,25-dihydroxy-
vitamins D3 or D2; 25-dihydroxy-vitamins D3 or D2; 25,26-
dihydroxy-vitamins D3 or D2; 1,24,25-trihydroxy-vitamins
D3 or D2 and 1,25,26-trihydroxy-vitamins D3 or D2. Most
preferred among these are vitamins D3 or D2; l-hydroxy-
20 vitamins D3 or D2; and 1-25-dihydroxy-vitamins D3 or D2.
In the case of multihydroxylated forms of the
vitamins (e.g., lr25-dihydroxy-vitamin D3 has three
hydroxy groups, at positions 1, 3, and 25) r the preferred
compounds of the invention are those wherein less than
25 all of the multiple hydroxy groups are glycosilated, most
preferably those where only one of the multiple hydroxy
groups is glycosilated.
The glycosides can comprise up to 20 glycosidic
units. Preferred, however, are those having less than
30 10, mose preferred, those having 3 or less than 3
glycosidic units. Specific examples are those containing
1 or 2 glycosidic units in the glycoside residue.
The glycopyranose or glycofuranose rings or amino
``` ~L~lq;~'S'~
derivatives thereof may be fully or partially acylated or
completely deacylated. The completely or partially
acylated glycosides are useful as deined intermediates
for the synthesis of the deacylated materials.
Among the possible glycopyranosyl structures are
glucose, mannose, galactose, gulose, allose, altrose,
idose, or talose. Among the furanosyl structures, the
preferred ones are those derived from fructose,
arabinose, cellobiose, maltose, lactose, trehalose,
10 gentiobiose, and melibiose. Among the triglycosides, the
preferred ones are those derived from fructose, arabinose
or xylose. Among preferred diglycosides are sucrose,
cellobiose, maltose, lactose, trehalose, gentiobiose, and
melibiose. Among the triglycosides, the preferred ones
15 may be raffinose or gentianose. Among the amino
derivatives are N-acetyl-D-galactosamine, N-acetyl-D-
glucosamine, N-acetyl-D-mannosamine, N-acetylneuraminic
acid, D-glucosamine, lyxosylamine, D-galactosamine, and
the like.
When more than one glycosidic unit is present on a
single hydroxy group (i.e., di or polyglycosidic
residues), the individual glycosidic rings may be bonded
by 1-1, 1-2, 1-3, 1-4, 1-5, or 1-6 bonds, most preferably
1-2, 1-4, and 1-6. The linkages between individual
25 glycosidic rings may be a or ~.
The configuration of the oxygen linkage of a hydroxy
group, or glycosidic residue attached to the vitamin D3
or D2 molecule may be either a (out of the plane of the
paper) or ~ (into the plane of the paper). It is
30 preferred if the configuration of the 3-hydroxy or
glycosidoxy group at C-3 be ~ , and that, independently
or simultaneously the configuration of the hydroxy or
glycosidoxy at C-l be a . It is also preferred that the
configuration around C-24 be R. When, at C-24, X=H and
lZl~P-~
- 9 -
R2=-CH3 or -CH2CH3, the configuratlon at C-2~ is
preferably S.
Specific Examples of compounds o:E the invention are:
vitamin D3, 3~ - D-glucopyranoside);
vitamin D3, 3~ D-fructofuranoside);
vitamin D3, 3~ - cellobioside);
vitamin D3, 3~ - maltoside);
vitamin D3, 3~ - lactoside);
vitamin D3, 3~ - trehaloside);
vitamin D3, 3~- raffinoside;
vitamin D3, 3~- gentiobioside;
la- hydroxy-vitamin D3, 3~ D-
glucopyranoside);
la- hydroxy-vitamin D3, 3
D-fructofuranoside);
la- hydroxy-vitamin D3, 3~ - cellobioside);
la- hydroxy- 3~ - maltosyl) vitamin D3;
1- hydroxy- 3~- raffinosyl-vitamin D3;
la- hydroxy- 3~- gentiobiosyl-vitamin D3;
la-(~- D-glucopyranosyl)-vitamin D3;
la-(~- D-fructofuranosyl)-vitamin D3;
la-(~- cellobiosyl)-vitamin D3;
1-(~- maltosyl)~vitamin D3;
la-(~- lactosyl)-vitamin D3
la-(~- trehalosyl)-vitamin D3;
1- raffinosyl-vitamin D3;
1- gentiobiosyl-vitamin D3;
la, 25-dihydroxy-vitamin D3, 3~ - D-
: fructofuranoside)
la, 25-dihydroxy-vitamin D3, 3~ - D-
glucopyranoside);
la-(~- D-glycopyranosyl)-25-hydroxy-vitamin D3;
la-(~- D-fructofuranosyl)~25-hydroxy-vitamin D3;
la- hydroxy-25- (~- D-fructofuranosyl)-vitamin
35. D3;
--10--
1- hydroxy, 25- (~- cellobiosyl)-vitamin D3;
1~- hydroxy, 25~ maltosyl)-vitamin D3;
1~- hydroxy, 25- (~- lactosyl) vitamin D3;
1~- hydroxy, 25- (~- trehalosyl)-vitamin D3;
1~- hydroxy, 25-raffinosyl-vitamin D3;
1~- hydroxy, 25-gentiobisyl-vitamin D3.
All of the aforementioned derivatives can also be
prepared with vitamin D2.
The glycosidic derivatives of vitamins D of the
10 present invention can be prepared by standard synthetic
methods well known to those skilled in the art. These
methods depend on whether the starting vitamin D3 or
vitamin D2 contains one or more hydroxy groups. When the
vitamin contains only one hydroxy group, the syntheses
15 are straightforward, since the monohydroxylated vitamin
(hydroxylated at position 3) is treated wi-th silver
carbonate in a refluxing solution of an inert nonpolar
solvent such as benzene or toluene, to which is added
fully acylated glycoside or fully acylated straight or
20 branched chain glycosidic polymer, either of these
containing an appropriate leaving group (L.G.) at
position C~l' of the terminal ring (or on the single
ring, as called for). Condensation occurs according to
the following reaction, indicated here for a single
25 glycoside for purpose of illustration only:
~LZ19~B:l
o
o o ~ o ~
o
o o o l ~
~,~ 7`~ ~
o I
~o ~g
o ~ o
o
~ ~ o
o~
`
o
P~ P; ~,
o o ~ o ~
C~ W P; , o
0~
~ ~ ~. o
~O
I m
~ o
o, I
~4
.~ ~
'o o
~ a)
~Q
....
'5~:~
-12-
In this reaction sequence, R3 is as deEined
previously, LG is a cornmon leaviny group such as bromine,
chlorine, iodine, p-toluenesulfonyl, and the like,
capable of being replaced in a bimolecular nucleophilic
substitution reaction.
When the vitamin D3 or D2 is reacted with a
glycosldic polymer, one or more of the oCOR3 groups in
the glycopyranoside or glycofuranoside rings is replaced
by a fully acylated glycosidic unit, with the proviso
10 that the total number of glycosidic units not exeed 20.
The reaction is carried out at from room temperature
to refluxing conditions for a period of 1-lO hours, and
is thereafter cooled and filtered to remove the silver
salt. The filtrate is dried and the inert solvent is
15 evaporated. The resulting product can be purified by any
of the standard modern purification methods such as high
performance liquid chromatography, silicic acid
chromatography, thin layer preparative chromatography,
and the like. A mixture of two products is normally
20 obtained, being the ~ and ~ glycofuranosyl or
glycopyranosyl derivatives at the point of ring
attachment. These can normally be separated by the
aforementioned chromatographic methods.
After separation of the individual products, the
25 glycosidic residues are deacylated in base, such as
sodium methoxide in methanol, of ammonia in methanol.
Further purification by high performance chromatography
is usually indicated to obtain the highly purified
product.
When the starting vitamin D (D3 or D2) carries two
hydroxy groups (such as l-hydroxy vitamin D3, or 25-
hydroxy vitamin D3) one of these needs to be selectively
protected with a protecting group which can be ultimately
removed after the condensation , and before, during or
~z~
after the deacylation of the glycosidic residues. The
same is true if three or more hydroxy groups are present
in the vitamin starting materials, and less than all of
these require to be glycosylated.
The selective protection of hydroxy groups in the
starting materials can be carried out by using standard
protection and deprotection reactions, well known to
those skilled on Organic Chemistry.
Because each of the hydroxyl groups on the vitamin D
10 molecule have different reactivities either due to the
fact that they are either primary (e.g. 26-OH), secondary
(eg. 24-OH, 3~-OH, etc.) or tertiary (eg. 25-OH)
hydroxyl functions, selectivity can be achieved.
Furthermore, because of steric considerations the
3~-OH has different reactivity than the la-OH which is
both a vicinyl hydroxyl function as well as sterically
hindered by the exocylic Clg methylene function on C10.
A good example of these reactivities is illustrated in
Holick et al, Biochemistry: 10, 2799, 1971, where it is
20 shown that the trimethylsilyl ether derivative of 1,25-
(OH)2-D3 can be hydroxyled in HCl-MeOH under mild
conditions to yield 3,25-disilyl ether, and 25-monosilyl
ether derivatives of l,25-(OH)2-D3. Furthermore, to
obtain a 1,25-(OH)2-D3 whereby the 3 and 1 hydroxyls are
25 protected, the 25-monosilyl ether derivative of 1,25-
(OH)2-D3 can be acetylated to form the 1,25--tOH)2-D3-1,3-
diacetyl-25-trimethyl silyl ether. Because the acetates
are quite stable to acid hydrolyis, this derivative can
be acid hydrolyzed to yield l,3-diacetoxy-25-
30 hydroxyvitamin D3. An alternative approach would simplybe to acetylate 1,25-(OH)2-D3 in acetic anhydride in
pyridine at room temperature for 24 to 48 to yield 1,3-
diacetoxy-25 hydroxyvitamin D3.
For protecting the 25-hydroxyl group for 25-
~L~5~L
-14-
hydroxyvitamin D3 the following can be done: 25-OH-D3
can be completely acetylated in acetic anhydride and
pyridine under refluxing conditions for 24 h. The 3-Ac
can be selectively removed by saponification (KOH in 95
MeOH-water) at room temperature for 12 h.
Once the desired protected vitamin D derivative is
prepared, the same is reacted with silver carbonate or
other methods for coupling (as described e.g. by
Igarashi, K., in "Advances in Carbohydrate Chemistry and
10 Biochemistry," Vol 34, 243-283, or Warren, C.D. et al,
Carbohydrate Research, 82: 71-83 (1980)), and the
glycosidic or polyglycosidic residue as in Scheme I
above, followed by deacylation, deprotection and
purification. Among the starting vitamin D derivatives
15 which are readily available, are, for example:
Vitamin D3;
Vitamin D2;
l-hydroxy-Vitamin D3;
l-hydroxy-Vitamin D2;
25-OH-Vitamin D3;
25-OH-Vitamin D2;
1,24-(OEI)2-Vitamin D3;
1,25-dihydroxy-Vitamin D3;
1,25-dihydroxy-Vitamin D2;
24,25-dihydroxy-Vitamin D3;
25,26~dihydroxy-Vitamin D3;
24,25-dihydroxy-Vitamin D2;
1,24,25-trihydroxy-Vitamin D3;
1,25,26-trihydroxy-Vitamin D3;
Some material.s, such as 25,26-Vitamin D2, 1,24,25-
trihydroxy Vitamin D2 or 1,25,26-trihydroxy Vitamin D2
have not yet been fully identified in the art, but can
nevertheless be used if synthetically prepared.
The acylated glycoside containing a leaving group at
35 position C-l' of the first (or only) glycosidic ring can
" ~Zl~S~
-15-
be prepared, for example, by the methods of Fletcher,
H.G., Jr., "Methods in Carbohydrate Chemistry" 2: 228
(1963), or Bonner, W.A., Journal of Organic Chemistry
26: 908-911 (1961), or Lemieux, R.U., "Methods in
Carbohydrate Chemistry", Vol. II, 221,222.
Oligosacchacide intermediates can be prepared, for
example by the methods of Lemieux, R.U., J. of Amer.
Chem. Soc. 97: 4063-4069 (1975); of Frechet, J.M.J.,
"Polymer-Supported Reactions in Organic Synthesis" (1980)
10 407-434, or Kennedy, J.F., "Carbohydrate Chemistry" 7:
496-585 (1975).
Commercially available sugars include (Pfanstiehl
Laboratories, Inc.): Pentoses, such as: D-arabinose, L-
arabinose, D-Lyxose, L-Lyxose, D-Ribose, D-Xylose, L-
15 Xylose; Hexoses, such as: Dextroses, D-Fructose, D-
Galactose, a-D-GluCOse, ~-D-Glucose, L-Glucose,
Levulose, D-mannose, L-Mannose, L-Sorbose; Heptoses, such
as: D-Glucoheptose, D-Mannoheptulose, Sedoheptulosan;
Disaccharides, such as: Cellobiose,
3-O-~-D- Galactopyranosyl-D-arabinose, Gentiobiose,
Lactoses, ~-Lactulose, Maltose, ~-Melibiose, Sucrose,
Trehalose, Turanose; Trisaccharides, such as :
Melezitose, Raffinose; Tetrasaccharides, such as:
Stachyose; Polysaccharides and derivatives, such as:
25 Arabic Acid, Chitin, Chitosan, Dextrin, Cyclo-Dextrins,
Glycogen, Inulin.
Alternatively, the whole synthetic sequence
, (protection, condensation and deprotection) can be
carried out starting with a ~5,7 steroidal diene which
30 is a provitamin D. After glycosylation, the provitamin
; is ring-opened photochemically, and the resulting
previtamin is thermally rearranged to yield glycosilated
vitamin.
It is known (Napoli, J.L. and DeLuca, H.F., in
S~
-16-
"Burger's Medicinal Chemistry" ~th Ed., part II, page 728
ff) that the active form of vitamin D is 1,25-
dihydroxyvitamin D3. When 1,25-dihydroxy-vitamin D3
glycoside is used in the treatment of hypocalcemic
states, or the regulation of phosphorus and calcium
metabolism in an animal, especially in a human, the
endogenous glycosidase enzymes of the animal directly
release the active form of the vitamin. On the other
hand, when non-hydroxylated derivatives of the vitamin
10 are used (such as, e.g., vitamin D3 glycoside), enzymatic
release of the hydroxylated vitamin is followed by
hydroxylation in the liver and then in the kidney in
order to form the active 1,25-dihydroxy vitamin.
The water-soluble glycosilated vitimin D conjugates
15 of the present invention include hydrophilic derivatives
of good water solubility to derivatives of excellent
water-solubility. They can be used generally in any
application where the use of vitamin D3, vitamin D2 or
hydroxylated derivatives thereof has been called for in
20 the prior art. The advantage of the conjuyates of the
invention resides in their water-solubility and thus
their ease of administration in aqueous media such as,
for example, saline or aqueous buffers. This allows the
utilization of these conjugates in such devices as
25 vitamin D releasing in-line pumps, intravenous
dispensation and the like. Other advantages include
treatment of fat malabsorption syndromes, as well as
release of the biologically active form of Vitamin D3 in
the gut, e.g. 1,25-(OH)2-D3 glycoside -~ gut ~ 1,25(0H)2-
30 D3 ~ biological action.
The conjugates of the inven-tion can be administered
by any means that eEfect the regulation of calcium and
phosphorus homeostasis and metabolism in animals,
especially humans. For example, administration can be
35 topical, parenteral, subcutaneous, intradermal,
S~3~
-17-
intravenous , intramuscular, or interperitoneal.
Alteratively or concurrently, administration can be by
the oral route. The dosage administered will be
dependent upon the age, health and weight of the
recipient, kind of concurrent treatment if any, frequency
of treatment, and the nature of the effect desired.
Generally, a dosage of active ingredient compounds will
be from about 0.1 ~g to 1 mg per kg of body weight.
Normally, from 0.1 ~g to 10 ~g per kg per application,
10 in one or more applications per therapy, is effective to
obtain the desired result.
An additional unexpected property of the co~pounds
of the invention is that some of them may demonstrate
promotion of calcium absorption through the intestine
15 without effecting calcium mobilization brought about by
calcium release from bones. Calcium mobilization by bone
release is a common feature of 1,25 dihydroxy-vitamin
D3. Its selective absence in some of the compounds of
the invention has a beneficial therapeutic consequence by
20 promoting an increase in serum calcium levels by
~ stimulating intestinal calcium transport. It is
i disadvantageous for patients with severe bone disease to
maintain serum calcium levels at the expense of
mobilizing calcium from their already wasting bones.
The compounds can be employed in dosage Eor~s such
as tablets, capsules, powder packets or liquid solutions,
suspensions or elixirs for oral administration, or
sterile liquids for formulations such as solutions or
suspensions Eor parenteral use. In such compositions,
30 the active ingredient will ordinarily always be present
in an amount of at least lx10-6% by wt. based upon the
total weight of a composition, and not more than 90% by
wt. An inert pharmaceutically acceptable carrier is
preferably used. Among such carriers are 95% ethanol,
35 vegetable oils, propylene glycols, saline buffers, etc.
5 i3~
-18-
Having now generally described this invention, a
more complete understanding can be obtained by reEerence
to certain examples, which are included herein for
purposes of illustration only and are not intended to be
limiting unless otherwise specified.
Example 1
Preparation of Vitamin D3, 3~ -~lucoside.
In a 3-neck 100-ml round-bottom flask, equipped with
dropping funnel and distillation head, was suspended 1.00
10 g (3.63 mmole) of dry silver carbonate, freshly prepared
according to the procedure of Becker, Biochem. Biophys.
Act: 100: 574-581 (1965), in 5 ml of dry benzene, in
which was dissolved 147 mg (0.382 mmole) of vitamin D3.
The solution was brought to boiling. At that point, 647
15 mg (1.57 mmole) of tetra-O-acetyl -~- D-glucopyranosyl
bromide, prepared according to the procedure of Lemieux,
supra, dissolved in 25 ml of benzene, was added
drop-by-drop. Benzene continued to distill and about 1/2
hour later more silver carbonate (approximately 1 g) was
20 added to the reaction mixture. The reaction was followed
by thin layer chromatography (20:80 v/v ethyl
acetate/hexane). The minor product had an Rf of 0.24,
and the major product had an Rf of 0.20. After two
hours, the reaction mixture was cooled and then filtered
25 through glass wool to remove the silver salt. The
filtrate was dried over anhydrous sodium sulfate, and the
benzene was evaporated under nitrogen. The resulting
yellow oil was applied to a preparative ~-Porasil high-
pressure liquid chromatographic column (dimensions, 8
30 mmx30cm; flow rate: 2 ml/min; solvent 15/85 v/v ethyl
acetate/hexane) The major product, 9,10-secocholesta-
5,7,10(19)-trien -3~- yl-2',3',4',6'-tetra-O-
acetyl -~- D-glucopyranoside, with a retention time of 58
minutes, exhibited an absorbance maximum o~ 265 nm, and
- ~LZ~5~
--19--
an absorbance minimum of 228 nm, characteristic of the
triene chromophore in vitamin D. Its mass spectrum
contains a peak Eor the parent molecular ion and at m/e
714, 2.5% (M+); peaks at 383, 5~ (M-pyronium ion); 366,
28% (M-pyronium ion~water)+; 351, 18%; 331, 15~ (pyronium
ion)+; 271, 2.5%; 253, 14%; 169 100%; (C8HgO~)+; 109,63
(C6H5O2)+; and 60, 20% (metnyl Eormate or acetic acid).
A minor product 9,10-secocholesta-5,7,10(19)-
triene -3~- yl-2',3',4',6',-tetra-O-acetyl -~- D-
10 glucopyranoside with a retention time of 45 minutes, alsoexhibited an absorbance maximum at 265 nm and an
absorbance minimum at 228 nm. Its mass spectrum
exhibited a molecular ion of m/e 714.
The major product, having the retention time of 58
15 minutes, was then deacylated with sodium methoxide and
methanol. A small piece of sodium metal was added to the
compound dissolved in anhydrous methanol. After 1/2 hour
the solution was neutralized with dilute acetic acid.
The solution was dried under nitrogen and then applied to
20 a reverse-phase high-pressure liquid chromatographic
column (Radial Pak A column Waters Associates, dimensions
0.8 ~ 10 cm; flow rate 1 ml/min; solvent 98/2 v/v
methanol/water). The product, 9,10-secocholesta-
5,7,10(19)triene -3~- yl -~- D-glucopyranoside, had a
25 retention time of 12.5 minutes and exhibited the UV
spectrum, ~max 265 nm, ~min 228 nm , typical of the
vitamin D chromophore.
The vitamin D3, 3~-glucoside vitamin D3,
3-glucoside and the vitamin D3, 3~ glucoside acetate
30 were tested for biological activity. Male weanling rats
from Holtzmann Company, Madison, Wisconsin, U.S.A., were
fed a vitamin D deficient diet that was adequate in
phosphorus and low in calcium (0.02~) for 3-1/2 weeks.
Groups of five animals received orally either
4 ~g, 1 ~g, 0.5 ~g, 0.25 ~g of Vitamin
`` ~%~5S~
-20-
D3 -3~- glucoside, 1 ~g Vitamin D3 -3~- glucoside or
2 ~g Vitamin D3 -3~- glucoside acetate in 50 ~1 of 95%
ethanol or vehicle alone. 24 hours later the animals
were sacrificed and the small intestine and blood were
collected Intestinal calcium transport studies were
performed by the everted gut sac technique, and blood was
used for serum calcium determinations. The results are
shown in the following table:
1d~1~ 5 ~
I/O (inside Ca45 Serum
/outside Ca45) Calcium
Compound ~ S.D. + S.D.
95% ethanol1.6 i 0.34.3 i 0.2
4~g Vitamin D3
3~- glucoside 3.9 i 0.2 5.3 ~ 0.2
1 ~g do.3.6 ~ 0.3
0.5 ~g do.2.5 i 0.2
0.25 ~g do.2.0 i 0.2
l~g Vitamin D3
3a- glucoside 2.0 i 0.4
2~g Vitamin D3
3~- glucoside
acetate 1.7 i 0.2
The data show that the vitamin D3, 3~ -glucoside is
capable of stimulating intestinal calcium absorption, and
bone calcium mobilization. The 3a glucoside is somewhat
less active, while the 3~- acetate appears inactive.
