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Document Type and Number:
WIPO Patent Application WO/1980/000338
Kind Code:
New fluorine-substituted vitamin D compounds, methods for preparing such compounds and fluorinated intermediate compounds used in such methods. The fluorine-substituted vitamin D compounds of this invention are characterized by vitamin D-like activity in their ability to stimulate intestinal calcium transport and bone mobilization and in promoting the calcification of rachitic bone. These compounds would find ready application as a substitute for the vitamin D compounds in the treatment of disease states evincing calcium-phosphorus unbalance and which may be nonresponsive to normal vitamin D therapy.

Application Number:
Publication Date:
March 06, 1980
Filing Date:
July 24, 1979
Export Citation:
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International Classes:
A61K31/59; A61K31/593; A61P3/02; C07C67/00; C07C401/00; G06F11/14; G06F11/18; G06F11/20; G06F15/16; G06F15/177; (IPC1-7): C07C45/00; C07C35/22; C07C69/02; C07C69/76; C07C17/00; C07B1/00
Foreign References:
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1. A method for the preparation of fluorovitamin D compounds and fluoro5,6transvitamin D compounds of the formulae where R is a steroid side chain of the configuration and where, R, is hydroxy, 0acyl or 0lower alkyl, and R, ^ and Rg are each selected froirf the group consisting of hydrogen, hydroxy, 0lower alkyl, 0acyl and fluoro, except that at least one of Rg, ^ and Rr must be fluoro, and R, is hydrogen or lower alkyl, which. comprises treating 3,5cyclovitamin D compounds of the formula where Z is lower alkyl, and chain and where R2, Rg, R4, and & are each selected from the group consisting of hydrogen, hydroxy, 0acyl, 0lower aklyl and fluoro, except that at least one of Rg, R^, and Rg must be hydroxy, and Rg is hydrogen or lower alkyl, with a fluor ating agent, recovering the fluorinated product, subjecting the recovered product to acid catalyzed solvolysis whereby a product containing in a mixture fluorovitamin D and fluoro5,6transvitamin D products is obtained, chromatographically separating said products and recovering each of them.
2. The process of claim 1 wherein one or more hydroxy groups present in the 3,5cyclovitamin D starting material are protected against fluorination by acylation prior to treatment with the fluormating reagent.
3. The process of Claims 1 or 2 wherein one or more protective acyl groups, or any acyl groups intro¬ duced during the solvolysis are removed by hydrolysis under basic conditions, either prior to or sub¬ sequent to chromatographic separation of the solvolysis products.
4. The process of Claim 1 wherein the fluorinating reagent is a dialkylaminosulfur trifluoride.
5. The process of Claim 4 wherein the fluorinating reagent is diethylaminosulfur trifluoride.
6. Compounds having the formula where R is a steroid side chain of the structure and where Rj^ is hydroxy, 0acyl or 0lower alkyl R2 is hydrogen, hydroxy or 0acyl, Rg, R^ and Rg are each selected from the group consisting of hydrogen, hydroxy, 0acyl, 0lower alkyl and fluoro, except that at least one of Rg, R^ and Rg must be fluoro, and O PI Rg is hydrogen or lower alkyl.
7. The compounds of Claim 6 wherein R^ is fluoro.
8. The compounds of Claim 6 wherein R is fluoro.
9. The compounds of Claim 7 or 8 wherein R, is hydroxy.
10. The compounds of Claim 9 wherein R2 is hydrogen or hydroxy.
11. The compounds of Claim 6 wherein Rg is methyl having the side chain stereochemical configuration of ergosterol.
12. Compounds having the formula where R is a steroid side chain of the structures w here R, is hydroxy, 0acyl or 0lower alkyl, and ^2 is hydrogen, hydroxy or 0acyl, and R3, R^ and Rg are each selected from the group con¬ sisting of hydrogen, hydroxy, 0acyl, 0lower alkyl and fluoro, except that at least one of Ro, j, and Rg must be fluoro, and R_ is hydrogen or lower alkyl.
13. Compounds of Claim 12 where R is fluoro.
14. Compounds of Claim 12 where g is fluoro.
15. The compounds of Claims 13 or 14 where R, is hydroxy.
16. The compounds of Claim 15 where R2 is hydrogen or hydroxy.
17. The compounds ofClaim 12 wherein Rg is methyl having the side chain stereochemical configuration of ergosterol.
18. 25fluorovitamin D„.
19. lαhydroxy25fluorovitamin D„.
20. 25fluoro5,6transvitamin D2.
21. 25fluoro5,6transvitamin D3.
22. lαhydroxy25fluoro5,6transvitamin D2.
23. 24hydroxy25fluoro5,6transvitamin D .
24. lαhydroxy25fluoro5,6trans.vitamin Dg.
25. lα,2 (R)dihydroxy25fluoro5,6transvitamin Dg. OMPI.

Process for Preparing Side-Chain Fluorinat ' ed Vitamin D Compounds

Technical Field This invention relates to compounds having vitamin D-like activity. More specifically, this invention relates to fluoro-derivatives of vitamin D.

Vitamins D^ and D 2 are well-known agents for the control of calcium and phosphorus homeostasis. These compounds in the normal animal or human stimulate intestinal calcium transport and bone calcium mobiliza¬ tion and are effective in preventing rickets. Research during the past decade has shown, however, that vitamins D 2 and D 3 must be metabolized to their hydroxylated forms before biological activity is expressed. Current evidence indicates, for example, that 1,25-dihydroxy- vitamin Dg, the dihydroxylated metabolite of vitamin Dg is the compound responsible for the biological effects mentioned earlier. Similarly, 1,25-dihydroxy- vitamin D 2 is the active form of vitamin D 2 - Background Art

References to various vitamin D derivatives are extant in the patent and other literature. See, for example, U.S. Patent Numbers: 3,565,924 directed to 25- hydroxycholecalciferol; 3,697,559 directed to 1,25- dihydroxycholecalciferol; 3,741,996 directed to lα- hydroxycholecalciferol; 3,743,661 directed to 5,6-trans- 25-hydroxycholecalciferol; 3,907,843 directed to l - hydroxyergocalciferol; 3,715,374 directed to 24,25- d ihydroxycholecalciferol; 3,739,001 directed to 25,26- dihydroxycholecalciferol; 3,786,062 directed to 22- dehydro-25-hydroxycholecalciferol; 3,847,955 directed to

1,24,25-trihydroxycholecalciferol; 3,906,014 directed to 3-deoxy-lα-hydroxycholecalciferol; 4,069,321 directed to the preparation of various side chain fluorinated vitamin D 3 derivatives and side chain fluorinated dihydrotachysterolg analogs. Disclosure of Invention

It has also been found that fluorinated vitamin D compounds also possess vitamin D-like activity. Such fluoro-analogs, therefore, represent useful compounds for the treatment of various diseases such as osteo- alacia, osteodystrophy, and hyperparathyroidism.

Specifically, this invention relates to fluorovitami D compounds of general structure I below, and 5,6-trans- fluorovitamin D compounds of general structure II below,


where R represents a steroid side chain of the configuration

where R, is selected from the group consisting of hydroxyl, 0-lower alkyl or 0-acyl, where R 2 , R g , R^, and R 5 are selected from the group consisting of hydrogen, hydroxyl, 0-lower alkyl, 0-acyl, or fluoro except that at least one of R 3 , R^ and R 5 must be fluoro, and where R g represents hydrogen or lower alkyl. Best Mode for Carrying Out the Invention

These fluoro compounds are prepared by a process which involves the treatment of hydroxylated 3,5- cyclovitamin D compounds with a fluorinating agent and obtaining directly the corresponding fluoro- 3,5-cyclovitamin D compound in which fluorine is located at the carbon originally occupied bythe hydroxy functionCs) of the starting material. These fluoro cyclovitamin D intermediates are then solvolyzed in an aqueous*or alcoholic solvent containing acid catalyst or in a solvent consisting of a low-molecular weight organic carboxylic acid to obtain both the 5,6-cis and 5,6-trans- fluorovitamin D compounds of general structure I and II respectively, from which, optionally, any acyl functions if present in the starting material or introduced during solvolysis, can be removed by hydrolysis (or reduction) to obtain the respective free hydroxy compounds.

"gJ ET,

_ O PI_

Suitable starting materials for this fluorination process include hydroxy-3,5-cyclo-vitamin D compounds of general structure III below,

( .

HE where R represents a steroid side chain of the configuration


and where R 2 , 3 , R^ and Rr are selected from hydrogen, hydroxyl, 0-lower alkyl, and 0-acyl, except that at leas one of R 3 , R^ and Rr must be hydroxy, and where R g repres hydrogen or lower alkyl, and were Z represents lower alkyl.

In this specification and in the claims the words "lower alkyl" denotes a hydrocarbon radical containing from 1 to about 5 carbon atoms which may be of normal or branched chain configuration (e.g. methyl, ethyl-,

isopropyl, butyl, isobutyl) and the word "acyl" denotes an aliphatic acyl group containing 1 to about 5 carbon atoms (e.g. acetyl, propiony], butyryl) or an aromatic or substituted aromatic acyl group (e.g. benzoyl, nitro-benzoyl, or chloro-benzoyl) .

The starting materials for fluorination, i.e. the 3,5-cyclovitamin D compounds of general structure III can be prepared by known methods from 3-hydroxyvitamin D compounds which in turn are available by synthesis or as isolated natural products (see for example, Schnoes and DeLuca, in Bioorganic Chemistry, E. E. Van Tamelen-, ed. , Vol. 2., Chap. 12, pp. 299-335, Academic Press, N.Y., 1978).

3,5-Cyclovitamin D compounds of general structure III can be prepared by the procedures of Sheves and

Mazur, J. Am. Che . Soc. 9_7_, 6249 (1975), and Paaren et_ al. Proc. Nat. Acad. Sci. USA 7_5, 2080 (1978). The procedures given by these authors are general and starting materials of structure III with any of the side chains indicated above are, therefore readily acces¬ sible from the available 3-hydroxyvitamin D compoμnds.

Jones e__ al. (U.S. Patent No. 4,069,321) have claimed the preparation of various side chain fluorinated vitamin D derivatives and side chain fluorinated dihydro- tachysterol 3 analogs. The method of preparation sug¬ gested by these investigators involves fluorination of a precursor steroid and subsequent conversion of the fluoro-steroid to the desired fluoroyitamin D compound. It has now been found, however, that side chain and/or ring A-fluorinated vitamin D compounds or analogs of general structures I and II above can be much more conveniently prepared by direct introduction of fluorine into hydroxy-cyclovitamin D compounds of general struc¬ ture III above, by means of fluorinating reagents such as dialkylaminosulfur trifluoride, e.g. diethyla ino- sulfur trifluoride. With such reagents, one or more

- υ R

fluorine substituents can be introduced into a vitamin D molecule, by direct replacement of hydroxy function(s) in the starting material.

Middleton (J. Org. Chem. 40, 574, (1975)) has described the use of diethylaminosulfur trifluoride for the displacement of hydroxy functions by fluorine in organic compounds, but his applications are limited to simple and stable molecules, which provide no basis for judging the efficacy of the reagent for direct intro- duction of fluorine into vitamin D compounds without alteration of the sensitive double bond system. It is this chemical reactivity of the vitamin D chromophore that has lead other investigators skilled in the art to adopt indirect and laborious routes when attempting the synthesis of fluorovitamin D compounds. For example, Jones et_ aJL. (U.S. Patent 4,069,321) in suggesting methods for the preparation of several fluorvitamin D analogs, used diethylaminosulfur trifluoride only for the introduction of fluorine into precursor steroids which are subsequently converted to fluorovitamin D analogs. There is indeed only one previous application of diethylaminosulfur trifluoride for the direct synthe¬ sis of 25-fluorovitamin D 3 from 25-hydroxy itamin D 3 (see Onisko, Schnoes, and DeLuca, Tetrahedron Letters (No. 13) 1107 (1977)). This one example however gives no indication- of the general utility of the fluorinating agent or of practical procedures for preparing other fluorovitamin D compounds.

It has now been found that the aforesaid diethyl- aminosulfur trifluoride reagent can be used for the introduction of fluorine at various positions (e.g. carbon-24,25,26) of the side chain of a cyclovitamin D molecule, as well as for the introduction of fluorine at carbon 1, and readily permits the efficient prepara- tion of a variety of fluorovitamin D compounds

- U E »

(structures I and II above) inclduing those of Jones et al. patent (referenced above).

A further advantage of the present method is the production of both 5,6-cis and 5,6-trans fluorovitamin D compounds from the same cyclovitamin starting material and in the same process thereby obviating the need of separate synthesis of both these desired products. In addition, the use of 3,5-cyclovitamin D starting materials provides a highly convenient temporary protection of the C-3 position and the triene system during fluorination. In the example of Onisko et l ^ . cited above the C-3- hydroxy group is protected by formation of a 3-0-acyl function, to avoid both fluorination at C-3 and the rearrangement of the ' triene chromophore. We can confirm that fluorination of a vitamin D compound bearing an unprotected C-3-hydroxy group does indeed lead to undesired products in which the triene chromophore is altered. The use of the stable 3,5-cyclovitamin D compounds of general structure III as substrates for fluorination not only avoids these difficulties, but also permits the convenient regeneration of the desired C-3 substituent (C-3-hydroxy, 0-acyl, 0-alkyl) by subsequent acid catalyzed solvolysis.

Finally, it has also been observed that more than one fluorine can be introduced simultaneously simply by subjecting multiple hydroxylated (e.g. di-, trihydroxy-) cyclovitamin D starting materials to fluorination. Since the fluorination process entails the replacement of free hydroxy function(s) it is therefore also essential that any such functions in the starting material that are not to be replaced by fluorine be suitably protected, e.g. by acylation such as acetylation or benzoylation Protection of hydroxy groups can be accomplished readily by known methods and after fluorination the acyl groups can, of course, be readily removed, if desired, by hydrolysis under basic conditions.

Inherent protection of the C-3 position and the triene system and the simultaneous production of both cis and trans fluorovitamin products are the most notable aspects of the process of this invention.. The general nature, the scope and versatility of the direct fluorination process by which fluorovitamin D compounds of general structure I above, and fluoro-5,6- transa-vitamin D compounds of general structure II above can be prepared from starting materials of general structure III, is more specifically illustrated by the following typical conversions.

(1) 25-hydroxy-6-methoxy-3,5-cyclovitamin D 3

(fluori tion? 25-fluoro-6-methoxy-3,5-cyclovitamin

D 3- 7 (solvo e Plys 2 i.s/ 25-fluorovitamin D-3 + 25-fluoro-5,'6- trans-vitamin D 3

(2) lα-acetoxy-25-hydroxy-6-methoxy-3,5-cyclovitamin D 3 step 1 (fluorination) lα-acetoxy-25-fluoro-6-methoxy-3,5-cyclovitamin D 3

I step 2 (solvolysis) lα-acetoxy-25-fluorovitamin D 3

Istep 3 (hydrolysis) lα-hydroxy-25-fluorovitamin D 3

+ lα-acetoxy-25-fluoro-5,6-trans-vitamin D 3 ^ step 3 (hydrolysis) lα-hydroxy-25-fluoro-5,6-trans-vitamin D g (3) lα,25-dihydroxy-6-methoxy-3,5-cyclovitamin D 3

(fluorinatio f >25-difluoro-6-methoxy-3,5-cyclo- vitamin D- ■ Bteτ> 2 1,25-difluorovitamin D + 3 (solvolysis/ 1,25-difluoro-5,6-trans-vitamin D 3'


(4) 25-hydroxy-6-methoxy-3,5-cyclovitamin D 2

(fluorination ' 25-fluoro-6-methoxy-3,5-cyclovitamin

D 2 (solvolysis ) * 25-fluorovitamin D + 25-fluoro- 5,6-trans_-vitamin D 2 (5) 25,26-dihydroxy-6-methoxy-3,5—cyclovitamin D- (fluorinlfcion ) > 25,26-difluoro-6-methoxy-3,5-

cyclovitamin D 3 ( so voi ys i s ) 25,26-difluorovitamin Dg + 25,26-difluoro-5,6-trans-vitamin D 3

The cyclovitamin starting materials shown in reaction 1-5 above are prepared as described by Paaren et_ al. and Mazur et_ al_. in the above cited references. In any of the illustrated examples, step 1 represents the fluorina ¬ tion reaction using diethylaminosulfur trifluoride, and step 2 represents acid catalyzed solvolysis using any of the conditions of Paaren et_ al_. , in the above cited reference and step 3 represents hydrolysis of acyl pro¬ tecting groups or of acyl groups introduced by solvolysis (if present) .

It is to be appreciated that the foregoing reactions are meant to be illustrative only. The examples are presented to show that the process provides for the convenient introduction of fluorine into both ring A and the side chain of the steroid molecule, and, specifically that vitamin D compounds and analogs having fluorine substituents at any one or more of carbons 1,

24, 25, or 26 can be readily made from the correspondingly hydroxylated cyclo-vitamin starting materials.

A preferred reagent for fluorination is diethylamino- sulfur trifluoride (Middleton, J. Org. ' Chem. 40, 574 ( 1975 )) . The reaction is conveniently conducted in a halo-carbon solvent, such as methylene chloride, carbon

tetrachloride or trichlorofluoromethane, at low tempera¬ ture, e.g. -78°C. For the displacement of hydroxy groups by fluorine, short reaction times, e.g. 15-45 min, are adequate. Although the reagent also attacks keto groups in the molecule, it does so at a much slower rate, and therefore, protection of any keto function present is not normally required under conditions where displacement of hydroxy groups is desired.

As mentioned previously, and as illustrated by the reactions above, any hydroxy groups in the starting material that are not to be replaced by fluorine in this process must be protected, e.g. acylated (acetylated, benzoylated) . Acylation of hydroxy groups is, of course, a well-known procedure, and is normally accomplished by treating the hydroxy compound with an acylating agent

(e.g. acetic anhydride, benzoyl chloride) in a suitable solvent (e.g. pyridine). Primary and secondary hydroxy groups in vitamin D compounds or their analogs are readily acylated using such reagents and solvents, at roo temperature (or slightly elevated temperature, e.g. 50°C) over a period of 2-6 hr. Acylation of tertiary hydroxy groups requires, of course, more vigorous conditions, e.g elevated temperatures (e.g. 75-100°C), and appropriate reaction times, e.g. 4-24 hr. It is preferable to conduc the reaction under a nitrogen atmosphere to avoid decomposition of material. Selective acylation of specif hydroxyl groups is also readily accomplished. Thus, by way of example 1,25-dihydroxy-6-methoxy-3,5-cyclovitamin D g 1-ace ate or l,24,25-trihydroxy-6-methoxy-3,5-cyclo- vitamin D~ 1,24-diacetate can be obtained by acetylation of 1,25-dihydroxy-6-methoxy-3,5-cyclovitamin D 3 , and 1,24,25-trihydrdxy-6-methoxy-3,5-cyclovitamin D~, respectively at room temperature, since under such conditions the tertiary hydroxy group does not react. Where selective acylation of chemically similar hydroxy groups is required, chromatographic separation of product may be necessary. Thus, the C-l and C-24-monoacetates of

l,24,25-trihydroxy-6-methoxy-3,5-cyclovitamin Dg can be prepared by conducting an acetylation at room tempera¬ ture and stopping the reaction before complete acetylation has occurred. Under such circumstances, a mixture of four compounds is obtained. The unacetylated starting material, the 1,24-diacetylated product, and the 1-acetoxy and 2 -acetoxy-monoacetylated compounds from which the desired mono-acetates (as well as the 1,24-diacetate) are obtained by chromatography. Other partially acylated products that are required as starting materials for subsequent fluorination are obtained in the analogous fashion. Alternatively, partially acylated compounds can be obtained by first acylating all hydroxy groups and then removing one or more of the acyl functions by hydrolysis. Thus the 24,25-dihydroxy-6-methoxy-3,5- cyclovitamin D g 25-0-acyl compound can be obtained by partial hydrolysis under basic conditions of the 24,25- di-0-acyl derivative. These examples are cited, to show that many different methods are known and available "to produce O-protected vitamin D compounds as may be required for the subsequent fluorination reaction.

Once fluorine has been introduced into the molecule all acyl protecting groups can be removed at this stage if desired or convenient by basic hydrolysis, e.g. treat- ment of the acylated fluoro cyclovitamin analog with 5% KOH in MeOH, at a temperature of 25-80°C for 1-4 hr. Alternatively deacylation can be performed after solvolysis of the cyclovitamin intermediate to the cis and trans vitamin products as described below. After fluorine introduction, the fluoro cyclovitamin derivatives are subjected to acid catalyzed solvolysis using, for example, the conditions described by Sheves and Mazur and Paaren et_ a^L. in the above cited references. As shown by the above illustrative reactions, solvolysis yields both the 5,6-cis-fluoro-vitamin D


products and the corresponding 5,6-trans-fluorovitamin D analogs. These fluorinated cis and trans reaction products can be conveniently separated by chrσmatography at this stage (as described by Paaren et _ύ_. in the above cited reference) and then can be separately sub¬ jected to hydrolysis (if acyl protecting, groups are to be removed) using standard conditions, e.g. 0.1 M KOH in methanol, 60°C, 1-4 hr. Alternatively, removal of protecting groups can also be accomplished, of course, prior to separation of the cis and trans isomers.

Depending upon the exact solvolyzing conditions, 5,6-cis and trans products with different C-3 substituents are obtained. Thus, conducting the solvolysis in a medium - consisting of aqueous dioxane and a catalytic amount of acid (e.g. p_-toluene sulfonic acid or methane sulfonic acid) yields fluorinated 5,6-cis and trans products beari a C-3-hydroxy substituent. Conducting the solvolysis in an anhydrous alcoholic solvent (e.g. methanol, ethanol, isopropanol,. etc.) containing the acid catalysts mentione leads to 5,6-cis and trans products bearing a C-3 O-alkyl substituent where the alkyl group corresponds to the alkyl portion of the alcohol solvent used. Solvolysis • of fluorocyclovitamin D compounds in warm acetic acid (e.g. 50-60°C) yields 5,6-cis and trans fluorovitamin D compounds bearing a C-3-acetoxy substituent, and if the solvolysis of fluorocyclovitamins is conducted in dioxane/formic acid solvents, the corresponding 3-0- formyl 5,6-cis and trans products are obtained. The use of other organic acids (e.g. trifluoro acetic acid) yields, of course, the analogous 3-0-acyl products. If desired these C-3 O-acylated products can be readily converted to the ' corresponding C-3-hydroxy compounds by mild base hydrolysis. From cyclovitamin D compounds of general structure III the production of 5,6-cis and trans-fluorovitamin D compounds according to the process of this invention thus comprises an essential two-step

sequence, namely, (1) direct fluorine introduction using a dialkylaminosulfur trifluoride reagent and (2) sub¬ sequent solvolysis under acid catalysis. Preliminary hydroxy protection (e.g. by O-acylation) is required only in those cases where any one of several available hydroxy groups in the starting material is to be replaced by fluorine. These 0-acyl protecting groups are readily removed, if desired, arid as indicated in the foregoing, this deprotection step can be performed at any time after fluorine introduction. For example, removal of 0-protect- ing group is possible immediately after fluorination (prior to solvolysis), or immediately after solvolysis (prior to separation of cis and trans products) or finally after separation of the cis and trans isomers resulting from solvolysis. A choice between these possibilities would be based on convenience and/or the specific product desired. Example 1 lα,25-Dihydroxy-6-methoxy-3,5-cylovitamin D g 1- acetate: 1,25-Dihydroxy-6-methoxy-3,5-cyclovitamin D 3 (5 mg) prepared by the method of Paaren et_ al. (Proc. Nat. Acad. Sci. USA, 75_, 2080 (1978)) is acetylated with acetic anhydride (0.5 ml) and pyridine (2 ml) at 50°C under argon for 2 hr. After the usual work-up, the 1-acetate product (5.5 mg) is isolated by high- pressure liquid chromatography over a silica gel column eluted with 2% 2-propanol/hexane. Example 2

25-Fluoro-lα-hydroxy-6-methoxy-3,5-cyclovitamin Dp 1-acetate: l,25-Dihydroxy-6-methoxy-3,5-cyclovitamin Do 1-acetate (3 mg) in CH 2 C1 2 at -78°C under argon is treated with diethylaminosulfur trifluoride (5 mg) . After several minutes, the reaction mixture is allowed to warm to room temperature and quenched with 5% K 2 C0g. Additional CH 2 C1 2 is added, the organic phase is separated and the



25-fluorinated product is isolated by thin-layer chromatography using a silica gel plate and 20% ethyl acetate in hexane as solvent, to yield 2 mg of pure product. Example 3

25-Fluoro-lα-hydroxy-6-methoxy-3,5-cyclovitamin Dp: A sample of 25-fluoro-cyclovitamin derivative as obtained in Example2 is dissolved in 0.1 M KOH/methanol solvent an warmed to 50°C for 2 hr. Water is then added and the hydrolyzed product is extracted into ether. Purification by thin-layer chromatography on silica gel plates developed with 30% ethyl acetate in Skellysolve B yields pure 25-fluoro-l-hydroxy-6-methoxy-3,5-cyclovitamin D g . Example 4 25-Fluoro-lα-hydroxyvitamin D. and 25-fluoro-lα- hydroxy-5,6-trans-vitamin D.: A solution of 3 mg of 25-fluoro-lα-acetoxy-6-methoxy-3,5-cyclovitamin D (the product of Example 2) in a. 3:1 mixture of dioxane and water (1.5 ml) containing 0.2 mg of p-toluenesulfonic acid is warmed to 55°C for 15 min. Saturated NaHC0 3

( 2 ml ) is then added and the products are extracted into ether. The ether solvent is dried and evaporated and the residue is chromatographed on silica gel plates to separate the 5,6-cis and 5,6-trans 25-fluorovitamin D products. Development with 25% ethyl acetate in

Skellysolve B gives 1 mg of pure 25-fluoro-lα-acetoxy- vitamin Dg and 0.3 mg of 25-fluoro-l -acetoxy-5,6-trans vitamin Dg. Hydrolysis of 25-fluorp-lα-acetoxy vitamin D_ in 0.1 M KOH/MeOH, for 2 hr. at 50°C gives a single product, 25-fluoro-l -hydroxyvitamin D 3 : uv 265 nm (ε = 18,200); n r (270 MHz) 0.55 (s, 18-CHg), 0.94 (d, J = 6 Hz, 21-CHg ) , 1.34 (d, J = 22 Hz, 26 9 27-(CHg) 2 ), 4.23 . (m, 3α-H), 4.43 (m, 1B-H) , 5.33, 5.01 (2 , 19-CHg), 5.85, 6.38 (AB quartet, J = 11 Hz , 6 and 7-H's); mass spectrum m/e_ 418.3197 (M + , 0.07, C^H^O^ calcd.

418.3222), 400.3141 (M + -H 0), 0.22, calcd. 400.3143), 382.3027 (M + -2 x H 2 0) , 0.23, calcd. 382.3031), 380.3059 (M + -H 2 0)-HF, 0.18, calcd. 380.3069), 362.2974 (M + -2 x H 2 0-HF, 0.18, calcd. 362.2997), 152.0842 (0.42, calcd. 152.0840), 134.0736 (1.00 calcd. 134.0734). Similar hydrolysis of 25-fluoro-lα-acetoxy-5,6-trans.vitamin g yields 25-fluoro-lct-hydroxy-5,6-trans vitamin Dg in pure form (λmax 270 run); mass spectrum, m—/:—e 418.

Example 5. let,25-Difluoro-6-methoxy-3,5-cyclovitamin Dg: A solution of 1 g of 1,25-dihydroxy-6-methoxy-3,5-cyclo- vitamin D 3 (prepared by the method of Paaren et_ al_, (Proc. Nat. Acad. Sci. USA, 7j5_, 2080 (1978)) in CH 2 C1 2 at -78°C under argon is treated with diethylaminosulfur trifluoride (2 mg) . After 10 min. , the reaction mixture is allowed to warm to room temperature and quenched with 5% K 2 C0 g . After the usual work-up (e.g. see Example 2) 1,25-difluoro- 6-methoxy-3,5-cyclovitamin Dg (250 μg) is isolated in pure form.by high-pressure liquid chromatography using a silica gel column eluted with 10% tetrahydrofuran/hexane. Alternatively, the product can be purified by thin-layer chromatography on silica gel using 20% ethyl acetate in Skellysolve B as developing solvent. Example 6 l ,24 ( R)25-trihydroxy-6-methoxy-3,5-cyclovitamin Dg 1,24-diacetate: A pyridine solution (0.2 ml) of 10 mg of 24(R)25-dihydroxyvitamin Dg is treated with 1.5 eq. of p-toluene sulfonyl chloride at 0°C for 48 hr. Dilution with saturated NaHCOg solution, and extraction with ether yields crude 3-tosyl product. This product in 2 ml MeOH is treated with 25 mg of NaHCOg and heated under N at 50°C for 20 hr. Dilution -with water and extraction with ether gives the cyclovitamin product. Chromatography of the product on silica gel plates (40% ethyl acetate in Skellysolve B) gives 4 mg of 24(R),25- dihydroxy-6-methoxy-3,5-cyclovitamin D g in pure form.


' °

This product is oxidized by treatment with Se0 2 according to the procedures of Paaren et al_. (Proc. Nat. Acad. Sci. USA, 75_, 2080 (1978)): 4 mg of the cyclovitami in CH 2 C1 2 solution (0.5 ml) is treated with Se0 2 ( 1 mg) and 10 yl of 70% t-butylhydroperoxide for 30 min. at 25°C. After addition of NaOH solution (10%), the 1-hydroxy- cyclovita in product is extracted into ether.. Evaporatio of solvent gives an oil which is chromatographed on silic gel plates using 50% ethyl acetate in Skellysolve B, to yield 1.5 mg of lα,24(R) ,25-trihydroxy-6-methoxy-3,5- cyclovitamin D g . This product is acetylated under the usual conditions (0.2 ml of acetic anhydride in 1 ml pyridine, 50°c,2 hr). Dilution of the reaction mixture with water, extraction with ether, and evaporation of the ether yields ca. 2 mg of lα,24(R),25-trihydroxy-6- methoxy-3,5-cyclovitamin D 3 1,24-diacetate, sufficiently pure for subsequent fluorination. Example 7

25-Fluoro-l,24(R)-dihydroxy-6-methoxy-3,5-cyclo- vit-amin D- 1,24-diacetate: The diacetoxy-cyclovitamin product obtained in Example 6 is fluorinated under the usual conditions (CH 2 C1 2 solvent, 3 mg of diethylamino- sulfur trifluoride, -78°, 15 min.). The reaction mixture is then- allowed to warm to room temperature, quenched with 5% K 2 C0g solution and additional CH 2 C1 2 is added. The organic phase is separated and the 25-fluorinated product is isolated and purified by thin-layer chroma¬ tography on silica gel plates using- 25% ethyl acetate in Skellysolve B as solvent system to yield 1 mg of 25- fluoro-lα,24(10-dihydroxy-5-methoxy-3,5-cyclovitamin D_ 1,24-diacetate. Example 8

25-Fluoro-lα,24(R)-dihydroxyvitamin D anc *ι 25- fluoro-lα,24(R)-dihdyroxy-5,6-trans-vitamin D„: A dioxane solution of the 25-fluoro cyclovitamin product as obtained


in Example 7 is warmed to 55 * ^; and treated with a 1:1 mixture of dioxane: 98% formic acid (200 yl). After 15 min. ice-water is added and the products are extracted with ether. After evaporation of solvent, the residue is taken up in dioxane/methanol (1:1, 1 ml) and treated with 0.1 ml of an aqueous COg solution for 5 min. (to hydrolyze the C-3-formate groups). Extraction of the products into ether, and chromatography on silica gel plates (using 40% ethyl acetate in Skellysolve B) yields 0.3 mg of 25-fluoro-lα,24(R)-dihydroxyvitamin Dg 1,24-diacetate and 0.1 mg of 25-fluoro-lα,24(R)-dihydroxy- 5,6-trans-vitamin D g 1,24-diacetate. The former product is hydrolyzed (0.1 M KOH/MeOH, 50°C,3 hr.) to 25-fluoro- lα,24(R)-dihydroxyvitamin Dg. Similar hydrolysis of the 5,6-trans-diacetate yields 25-fluoro-lα,24(R)-dihydroxy- 5,6-trans-vitamin D 3 . Example 9

25-Fluorovitamin D g and 25-fluoro-5,6-trans-vitamin Dg: A solution of 15 mg of 25-hydroxy-6-methoxy-3,5- cyclovitamin Dg [prepared by the method of Paaren __ al. in Proc. Nat. Acad. Sci. USA, 75_, 2080 (1978)3 in 0.5 ml of dichloromethane is added dropwise to a cooled (-78°c mixture of diethylaminosulfur trifluoride (30 mg) in 0.5 ml of CHoClo. After stirring for 5 min. the cooling bath is removed and the reaction mixture is warmed to room temperature. Aqueous NaHC0 3 (4%, 5 ml) and 10 ml of CH 2 C1 2 are added. After separation of the organic phase, washing with H 2 0, drying (N S0 4 ) and evaporation of solvent, the product, 25-fluoro-6-methoxy- 3,5-cyclovitamin D g is purified on silica gel thin-layer plates, developed with 10% ethyl acetate in Skellysolve B to give 10 mg of product. This intermediate is directly solvolyzed using the conditions of Paaren et aL. (reference cited above) (2.5 ml of a 3:1 mixture of dioxane/water containing 0.2 mg of p-toluene sulfonic acid, warmed to

55°C for 15 min., followed by addition of NaJCOg (3 ml) and extraction of the product into ether) to give a

3:1 mixture (50% yield) of 25-fluorovitamin D 3 and 25- fluoro-5,6-transvitamin Dg. Separation on silica gel tic using 15% ethyl acetate in Skellysolve B as solvent gives

25-fluorovitamin Dg as a colorless oil: uv (EtOH) ~ maχ

265 n ; ir (CCl^) 3620 (hydroxyl), 3080 cm " (exocyclic methylene); nmr (CDClg) δ 6.24 (d, J = 11 Hz, 1H, C-6),

6.03 (d, J = 11 Hz, 1H, C-7), 5.05 (m, 1H, C-19), 4.82

(d, J = 2.6 Hz, 1H, C-19), 3.95 (t of t, J - 7.1 Hz,

J 2 = 3.6 Hz, 1H, C-3), 1.34 (d, J Hp = 21.3 Hz, 6H, C-26,27

0.93 (d, J = 6 Hz, 3H, C-21), 0.54 (s, 3H, C-18); mass spectrum m/e (relative intensity) 402.3284 (13, M ,

402.3298 calcd, for C^H^gOF), 360 (4), 271 (4), 253 (5), 136 (100), 118 (88), 61 (12), 59 (1); homogeneous on glc

{ _- is.. - 3.6 and 3.9 min for the pyro and isopyro derivatives

2 mm x 6' 3% OV-101, 260°C oven isothermal). Example 10

25-Fluorovitamin Dp and 25-fluoro-5,6-trans-vitamin D A methylene chloride solution of 25-hydroxy-6- methoxy-3,5-cyclovitamin D 2 [prepared from 25-hydroxyvita- min D 2 according to the procedures of Paaren et al_. Proc. Nat. Acad. Sci. 2ϋ 2080 (1978)] is fluorinated and the product, 25-fluoro-6-methoxy-3,5-cyclovitamin D ? is isolated as described in Example __. Upon solvolysis of this intermediate exactly as described in Example 9 a ( 4:1 ) mixture of 25-fluorovitamin D 2 and 25-fluoro-5,6- trans-vitamin D 2 is obtained, which is separated on a silica gel thin layer plates (12% ethyl acetate/Skelly- solve B as solvent) to yield pure 25-fluorovitamin D.

(uv, λm_a_x 265 nm; mol. wgt. = 414) and 25-fluoro-5,'6- trans-vitamin D (uv, \ ^ 270 nm; mol. wgt. = 414).


Example 11

25-Fluoro-24(R)-hydroxyvitamin Dg and 25-fluoro- 24(R)-hydroxy-5,6-trans-vitamin D^: From 24(R),25- dihydroxyvrtamin Dg, the required 3,5-cyclovitamin D intermediate, 24(R),25-hydroxy-6-methoxy-3,5-cyclovitamin Dg, is prepared as described in Example _ . This compound (5 mg) is acetylated (0.3 ml acetic anhydride, in 1.5 ml of pyridine, at 50°Cfor 2 hr) to yield the corresponding 2 -mono-acetate derivative (5 mg) . This product is directly fluorinated and isolated as described in Example 7_ and the desired 25-fluoro-product is then purified by thin layer chromatography (15% ethylacetate in Skelly¬ solve b ) . Solvolysis of this material exactly as described in Example 8_ gives the expected mixture 3-formyloxy- 24-acetoxy-25-fluorovitamin Dg and the corresponding 5,6- trans isomer, which is subjected to hydrolysis (0.1 M KOH/methanol, 1.5 hr. , room temperature) to remove for yl and acetyl groups. Evaporation of solvent, addition of water and extraction with the ether yields 2.5 mg of crude products. High pressure liquid chromatography on a * 0.7. x 25 cm 'Column of 5 micron silica gel, eluted with 2% isopropanol/hexane, yields 25-fluoro-24(R)- hydroxyvitamin D« (1 mg) : uv \ m^ __ 265 nm; nmr δ 0.55 (singlet, 18-methyl) , 0.94 (doublet J = 5.9 Hz, 21- methyl), 1.34, 1.33 (two doublets, J = 22 Hz each, 26- and 27-methyl), 3.52 (multiplet, 24S-proton), 3.95 ( multiplet, 3α-proton) , 4.82, 5.06 (multiplets, 19-protons) 6.04, 6.24 ( AB quartet, J = 12 Hz, . 6,7-protons); mass spectrum m/e ( composition, m/e calcd.) 418.3264 (C 2y H 43 0 F, 418.3247 ) , 398.3132 (C^H^O^ 398.3185) ,385.2904 ( C 26 H 38 OF, 385.2906), 271.2050 (C lg H 27 0, 271.2061), 253.1962 ( C lg H 25 , 253.1956), 136.0891 (C g H 12 0, 136.0888), il8.0783 ( C g H 10 , 118.0782); and 25-fluoro-24(R)-hydroxy- 5,6-trans-vitamin Dg: uv λ 272 nm; subjecting the 5-nitial solvolysis product mixture to hydrolysis with


< 0

0.1 M aqueous potassium carbonate as described in Example 8_ removes the 3-formyl group and chromatography of the resulting mixture (silica gel plates, 20% ethyl acetate Skellysolve B as solvent) yields pure 2 (R)- acetoxy-25-fluorovitamin Dg and 24(R)-acetoxy-25-fluoro- 5,6-trans-vitamin Dg. Biological activity of fluorovitamin D compounds:

The novel fluorovitaminD compounds prepared as described above exhibit significant vitamin D-like biological activity when tested in vitamin D-deficient animals. The vitamin D-like activities exhibited by the fluoro-vitamin D compounds include the stimulation of intestinal calcium transport, the stimulation of calcium mobilization from bone and the calcification of bone. F the demonstration of these effects a desirable test animal is the male weanling rat maintained on a vitamin D-deficient low calcium diet, or a vitamin D-deficient, low-phorphorus diet, as described by Suda et_ al_. J. Nutr. 100, 1049 (1970). With animals maintained on a low calcium diet, intestinal calcium absorption can be assayed by the everted gut sac technique of Martin and DeLuca (Am. J. Physiol. 216, 1351 (1969)) and bone calci mobilization can be determined by the rise of serum calcium as described for.example by Blunt et_ al^ Proc. Nat. Acad. Sci. USA 6^, 1503 (1968); degree of endo- chondral calcification can be assayed by the "line-test" method described in the U.S. Pharmacopeia (15th revision, p. 889, Mack Publ. Easton, Pa. (1955)) using animals maintained on the low phosphorus diet. Using such methods the biological efficacy of the novel fluoro-vitamin D compounds of this invention is readily demonstrated. Thus, a 5..μg dose of 25-fluoro- v i tam i n D administered to vitamin D-deficient, calcium- depleted animals will produce a significant stimulation of intestinal calcium transport and elevation of serum clacium levels within 24 hrs after administration.

Similarly a 1 yg dose of lα-hydroxy-25-fluoro- vitamin D 2 will be highly effective in stimulating intestinal calcium transport and bone calcium mobiliza¬ tion (as measured by the rise • of serum calcium levels), and the compound also promotes the calcification of rachitic bone as measured by the "line-test" score.

Fluoro-5,6-trans-vitamin D compounds also exhibit pronounced vitamin D-like activity. For example 25- fluoro-5,6-trans-vitamin Dg is effective in stimulating intestinal calcium transport, bone calcium mobilization and the healing of rickets in these test animals.