CA1219581A - Vitamin d glycosides and a method for use - Google Patents

Abstract

VITAMIN D GLYCOSIDES
Abstract A synthetic compound which is biologically active in maintaining calcium and phosphorous metabolism in animals, of the formula I

Description

"" ~21~S~

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~

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~

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~

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~

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-~

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;

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~:~

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

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~

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~

"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~

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~

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~

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~

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.

Claims (24)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Process for preparing a synthetic compound of the formula (I):

(I) wherein the double bond between positions C-22 and C-23 is single or double;
R2 is hydrogen, methyl or ethyl;
X is selected from the group consisting of hydrogen and -OR1, where R1 is hydrogen or a straight or branched chain glycosidic residue:
with the proviso that at least one said R1 is a glycosidic residue, and that the total number of glycosidic units per compound is not larger than 3, said glycosidic residue excluding residues wherein the glycoside is glucuronic acid or derivatives thereof, which comprises condensing a hydroxylated vitamin D of the formula:

with a fully acylated glycoside or fully acylated straight or branched chain glycosidic polymer, followed by deacylation of the glycosidic residues to give a syn-thetic compound of formula (I).

2. Process according to claim 1, which comprises providing a beta bond at position C-3.

3. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound I wherein X at position C-1 is -OR1, and the bond at C-1 is beta.

4. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound I wherein the bond between C-22 and C-23 is single, and R2 = H.

5. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

6. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

7. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosiaic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

8. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

9. Process according to claim 1, which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

10. Process according to claim 1 which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

11. Process according to claim 1 which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

12. Process according to claim 1 which comprises selecting said hydroxylated vitamin D and said glycoside or glycosidic polymer to give a compound of the formula:

wherein R1 is as defined in claim 1.

13. A synthetic compound which is biologically active in maintaining calcium and phosphorous metabolism in animals, of the formula I:

(I) wherein the double bond between positions C-22 and C-23 is single or double, R2 is hydrogen, methyl or ethyl, X is selected from the group consisting of hydrogen and -OR1, where R1 is hydrogen or a straight or branched chain glycosidic residue, with the proviso that at least one of said is a glycosidic residue, and that the total number of glycosidic units per compound is not larger than 3, said glycosidic residue excluding residues wherein the glycoside is glucuronic acid or derivatives thereof, whenever prepared by the process of claim 1 or its obvious chemical equivalents.

14. The compound of claim 13 wherein the bond at position C-3 is beta, whenever prepared by the process-of claim 2 or its obvious chemical equivalents.

15. The compound of claim 13 wherein, when X at position C-1 is -OR1, the bond at C-1 is beta, whenever prepared by the process of claim 3 or its obvious chemical equivalents.

16. The compound of claim 13 wherein the bond between C-22 and C-23 is single, and R2 = H, whenever prepared by the process of claim 4 or its obvious chemical equivalents.

17. The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 5 or its obvious chemical equivalents.

18. The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 6 or its obvious chemical equivalents.

19. The compound of claim 13 which is wherein R1 is as defined in claim 1, whever prepared by the process of claim 7 or its obvious chemical equivalents.

20, The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 8 or its obvious chemical equivalents.

21. The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 9 or its obvious chemical equivalents.

22. The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 10 or its obvious chemical equiv-alents.

23. The compound of claim 13 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 11 or its obvious chemical equiv-alents.

24. The compound of claim 1 which is wherein R1 is as defined in claim 1, whenever prepared by the process of claim 12 or its obvious chemical equivalents.