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Document Type and Number:
WIPO Patent Application WO/1980/000339
Kind Code:
New fluorine-substituted vitamin D compounds, methods for preparing such compounds and new 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; C07C17/00; C07J9/00; C07C35/22; C07C69/02; C07C69/76
Foreign References:
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1. A process for preparing 1fluorovitamin D compounds or 1fluoro5,6transvitamin D compounds or 1fluor 3,5cyclovitamin D compounds which comprises treatin the corresponding 1hydroxylate'd vitamin D compounds or 1hydroxylated 5,6transvitamin D compounds or 1hydroxylated 3,5cyclovitamin D compounds with a fluorinating reagent, thereby replacing the hydroxy group present in such treated compounds with fluorin and recovering the respective 1fluorinated product.
2. The process of Claim 1 wherein a 1fluoro3,5cyclo vitamin D compound is subjected to acid catalyzed solvolysis thereby obtaining both 1fluorovitamin D and 1fluoro5,6trans_vitamin D products and recovering each of them.
3. The process of Claims 1 or 2 wherein one or more hydroxyl goups present in the 1hydroxylated vitamin D, 5,6trans amd 3.5cyclovitamin D com¬ pounds are protected against fluorination by acylati prior to treatment with the fluorinating reagent.
4. The process of Claim 3 where, with 1hydroxyvitamin compounds or lhydroxy5,6transvitamin D compounds as starting material, only the C3 hydroxy function is acylated prior to treatment with the fluorinating reagent.
5. The process according to Claims 3 or 4 wherein the protective acyl groups, after the fluorination has been completed, are reconverted to hydroxyl groups by hydrolysis under basic conditions.
6. Compounds having the formulae where R has the structures and where R, is hydrogen, hydroxy, 0acyl or 0lower alkyl and where R2 is fluoro, R3, R^ and Rr are each selected from the group consisting of hydrogen, hydroxy, 0acyl, 0lower alkyl and fluoro, and where Rg is hydrogen or lower alkyl. The compounds of Claim 8 where R3 is hydrogen, hydroxy, or fluoro. 0, The compounds of Claim 8 where R^ is hydrogen, hydroxy, or fluoro.
7. 11 The compounds of Claims 9 or 10 where Rg is methyl having the stereochemical configuration of the ergosterol side chain.
8. 12 Compounds having the formula where R is a steroid side chain of the structures and where Z is lower alkyl, R2 is fluoro, 3 R^ and R5 are each selected from the group con¬ sisting of hydrogen, hydroxy, 0lower alkyl, 0acyl and fluoro, and Rg is hydrogen or lower alkyl.
9. 13 The compounds of Claim 12 wherein R^ is hydrogen, hydroxy or fluoro.. 14, The compounds of Claim 13 wherein Rg is hydrogen, hydroxy, or fluoro.15 The compounds of Claims 13 or 14 wherein Rg is hydrogen and Rg is methyl, having the side chain stereochemical configuration of ergosterol.
10. 16 1fluorovitamin D3 17 1fluoro5,6tra'nsvitamin D3 18 lfluoro25hydroxyvitamin Dg.
11. 19 1fluoro25hydroxy5,6transvitamin Dg,.
12. 1,25difluorovitamin Dg.
13. 1,25difluoro5,6transvitamin' Dg.
14. 1fluorovitamin D2.
15. 1fluoro5,6transvitamin D2«.
16. 1fluoro25hydroxyvitamin D2.
17. 1fluoro25hydroxy5,6transvitamin D2 "BUREA OMPI μ. W1PO N \O.

Process for Preparing 1-Fluorinated 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 3 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 mobilization and are effective in preventing rickets. Research during the past decade has shown, however, that vitamins D~ and Ω. must be metabolized to their hydroxylated forms before biological activity is expressed. Current evidence indicates, for example, that 1,25-dihydroxyvitamin D_, the dihydroxylated metabolite of vitamin D 3 is the com¬ pound responsible for the biological effects mentioned earlier. Similarly, 1,25-dihydroxyvitamin D j is the active form of vitamin D„. 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 lα- hydroxyergocalciferol; 3,715,374 directed to 24,25- dihydroxycholecalciferol; 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 tc 3-deoxy-lα-hydroxycholecalciferol; 4,069,321 directed

to the preparation of various side chain fluorinated vitamin D g derivatives and side chain fluorinated dihydrotachysterolg analogs. Disclosure of Invention

It has now 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- malacia, osteodystrophy, and hyperparathyroidism.

Specifically, this invention relates to fluoro¬ vitamin D compounds of general structure I below, and 5,6-trans-fluorovitamin D compounds of general structure

where R represents a steroid side chain of the configur¬ atio


- -

where R-^ is selected from the group consisting of hydrogen, hydroxyl, 0-lower alkyl or 0-acyl, where R 2 is fluoro and where each of R~', R^ and Rg is selected from the group consisting of hydrogen, hydroxyl, 0- lower alkyl, 0-acyl, and 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 a hydroxyvitamin D compound or hydroxyvitamin D analog with a fluorinating agent and obtaining directly the corresponding fluoro- vitamin D compound or fluorovitamin D analog in which fluorine is located at the carbon originally occupied by the hydroxy function(s) of the starting material.

Suitable starting materials for this fluorination process include hydroxyvitamin D compounds of general structure III below, or hydroxy-5,6-trans-vitamin D compounds of general structure IV, below, or hydroxy- 3,5-cyclo-vitamin D compounds of general structure V below,


> - OMPI •fr. W1PO £$NAT.O

where R represents a steroid side chain of the con¬ figuration

, W

and where R, is sleeted from the group consisting of hydrogen, hydroxy, 0-lower alkyl, and 0-acyl, and where R„ is hydroxy and each of R 3 , R^ and Rg is selected from hydrogen, hydroxyl, 0-lower alkyl, and 0-acyl, and where R- represents hydrogen or lower alkyl, and where 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) and the word "acyl" denotes an aliphatic acyl group containing 1 to about 5 carbon atoms (e v g. acetyl, propionyl) or an aromatic or substituted aromatic acyl group (e.g. benzoyl or nitro-benzoyl).

Fluorovitamin D compounds of general structure I are prepared from starting materials of general structure III whereas fluoro-5,6-trans-vitamin D compounds of structure II are prepared from compounds of general structure IV, and fluorination of cyclovitamin D starting materials of general structure V, followed by subsequent conversions as described hereinafter leads to both fluorinated products I and II. The starting materials for fluorination, e.g. com¬ pounds of general structure III-V can be prepared by known methods. Hydroxyvitamin D compounds of general structure III 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, NY 1978). The 5,6-trans-vitamin D compounds of general structure IV can be prepared from the corresponding 5,6-cis com- pounds (general structure III) by the well-known isomeriza- tion reaction using iodine catalyst (Verloop et ^ al_. Rec. Trav. Chim. Pays-Bas. 78, 1004 (1969)).

3,5-Cyclovitamin D compounds of general structure V can be prepared by the procedures of Sheves and Mazur, J_. Am. Chem. Soc. 97, 6249 (1975), and Paaren et al. , Proc. Nat! Acad. Sci. USA, 75_, 2080 (1978).

Jones e aα. (U.S. Patent No. 4,069,321) have claimed the preparation of various side chain fluorinated vitamin D„ derivatives and side chain fluorinated dihydro-. tahysterol 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 fluorovitamin D compound. It has now been found, however, that side chain , t 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 intact hydroxyvitamin D compounds and analogs of general structures III-V above, by means of flourinating reagents such as a dialkylaminosulfur trifluoride, e.g. diethylaminosulfur trifluoride. With such reagents, one or more 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 hdyroxy 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-

- 6 -

duction of fluorine into vitamin D compounds without alteration of the sensitive triene chromophore. It is this chemical reactivity of the vitamin D chromo¬ phore that has led other investigators skilled in the art to adopt indirect and laborious routes when attempt the synthesis of fluorovitamin D compounds. For exampl Jones e al. (U.S. Patent 4,069,321) in suggesting methods for the preparation of several fluorovitamin D analogs, use diethylaminosulfur trifluoride only for th introduction of fluorine into precursor steroids which are subsequently converted to the fluorovitamin D analo There is indeed only one previous application of diethylaminosulfur trifluoride for the direct synthesis of 25-fluorovitamin D, from 25-hydroxyvitamin D 3 (see Onisko, Schnoes, and DeLuca, Tetrahedron Letters (No. 13 1107 (1977)). •

It has now been found that the aforesaid diethyl¬ aminosulfur trifluoride reagent can be used not only for the introduction of fluorine at various positions - (e.g. carbon-24,25,26) of the side chain of a vitamin D molecule or analog but also for the introduction of fluorine at carbon 1, and thus affords an efficient and direct route to the desired 1-fluoro products as well as C-l and side chain fluorinated products. These C-l fluorovitamin D and C-l-fluoro-5,6-trans-vitamin D products are novel compounds.

Direct successful fluorination at carbon 1 of .the vitamin D molecule is particularly noteworthy and sur¬ prising because available information suggested that the vitamin triene chromophore, which is highly prone to rearrangement, would undergo undesirable and irreversibl alteration attendant upon fluorine introduction at carbon 1. Indeed the seemingly analogous reaction, namely, introduction of fluorine at carbon 3 by treating a C-3-hydroxyvitamin D 3 compound or analog with diethyl¬ aminosulfur trifluoride is not successful precisely

because of chromophore rearrangement. Displacement of the hydroxy function at that position with diethylamino¬ sulfur trifluoride leads to an undesired product in which the chromophore is altered. The facile preparation of 5 l-fluorovitamin D compounds and vitamin D analogs is, therefore, a novel and unexpected result. It has also been observed that more than one fluorine can be intro¬ duced simultaneiously simply by subjecting multiply hydroxylated (e.g. di-, trihydroxy-)vitamin D starting

-■- materials to fluorination. Since the fluorination process entails the replacement of free hydroxy function(s) it is 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

15 benzoylation. In particular, a C-3-hydr.oxy function if present in the starting material, needs to be protected by acylation since, as already mentioned above, treatment of C-3-hydroxyvitamin D compounds or analogs with diethyl¬ aminosulfur trifluoride leads to undesired chromophore

20 rearrangement. Protection of hydroxy groups can be accomplished readily by known methods and after fluorina¬ tion the acyl groups can, of course, be readily removed if desired, by hydrolysis under basic conditions.

The scope and versatility of the direct fluorina- 25 tion process by which fluorovitamin D compounds of general structure I above, and fluoro-5,6-trans-vitamin D com¬ pounds of general structure II above can be prepared from starting materials of general structures III TV, and V, is more specifically illustrated by the following

30 typical conversions .




( 1 ) lα,25-dihydroxyvitamin D 3 3-0-Acyl >- 1,25- difluorovitamin g 3-0-Acyl

(2) lα,-hydroxyvi amin D g 3-0-Acyl —J- 1-fluorovitamin D 3 3-0-Acyl (3) lα,25-dihydroxyvitamin D g 3,25-di-O-Acyl -→ 1-fluo 25-hydroxyvitamin D g 3,25-di-O-Acyl (4) 1,24,25-trihydroxyvitamin D g 3,24,25-tri-O-Acyl→ l-fluoro-24,25-dihydroxyvitamin Dg 3,24,25-tri- O-Acyl (5) 3-deoxy-lα-hydroxyvitamin D g — 3-deoxy- 1-fluorovitamin D 3

(6) 3-deoxy-lα,25-dihydroxyvitamin D g — > 3-deoxy-l,25 difluorovitamin Do

(7) 1,25-dihydroxyvitamin Do 3-0-Acyl — > 1,25-difluo vitamin D 2 3-0-Acyl

(8) lα,25-dihydroxy-5,6-trans-vitamin D 3 3-0-Acyl — > l,25-difluoro-5,6-trans-vitamin D 3 3-0-Acyl

(9) 1,25-dihydroxy-5,6-trans-vitamin D ? 3,25-di-O- Acyl —>> 1-fluoro-25-hydroxy-5,6-trans-vitamin Do 3,25-di-O-Acyl

(10) lα,25-dihydroxy-6-methoxy-3,5-cyclcwitamin D 3

1 ,25-difluoro-6-methoxy-3,5-cyclo-

vitamin D 3 1, '25-difluorovitamin D3 +

1,2.5-difluoro 5,6-trans-vitamin Do (11) lα-hydroxy-6-methoxy-3,5-cyclovitamin Do

(fluorinationft 1-f uoro-6-methoxy-3,5-cyclovitam

Ω - lsolvolysisft 1-fluorovitamin D 2 + 1-fluoro-5,6 trans-vitamin D 2

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 ring A and if desired 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 starting materials. As indicated by the above illustrative reactions, fluorination of starting materials of type III or IV yields the corresponding: 1-fluoro products directly but depending on the starting material used and the specific product desired, a preliminary hydroxy protection step (e.g. by acylation) and a final deprotection step may be required. With cyclovita in D starting materials of. general structure V, the overall process', and specifically the fluorination step, is the same, except that sub¬ sequent to fluorine introduction, a solvolysis step is used to regenerate both cis and trans forms of the fluorovitamin products. The solvolysis step, however, is in a formal sense an alternative deprotection step in which the "protected" (i.e. temporarily absent) C-3 substituent as well as both the cis and trans triene chromophore are regenerated.

A perferred 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 a methylene.chloride, carbon tetrachloride or trichlorofluoromethane, at low temperature 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 the fluorine in this process . must be protected, e.g. acylated (acetylat benzoylated). In particular, a hydroxy function at carbon 3 (as is commonly present in vitamin D compounds or analogs) needs to be protected by acylation, since, as mentioned above, fluorination in the presence of a C-3 hydroxy group can lead to undesired products. 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 acylat using such reagents and solvents,- at room 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 tie s, e.g. 4-24 hr. It is preferable to con¬ duct the reaction under a nitrogen atmosphere to avoid decomposition of material. Selective acylation of specific, chemically different hydroxyl groups is there¬ fore readily accomplished. Where selective acylation of chemically similar hydroxy groups is required, chromato- graphic separation of products may be necessary. Thus, lα-hydroxyvitamin D_ 3-acetate can be prepared by con¬ ducting an acetylation at room temperature and stopping the reaction before complete acetylation has occurred. Under such circumstances, a mixture of four compounds is obtained: lα-hydroxyvitamin D 3 , lα-hydroxyvitamin D 3 1-acetate, lα-hydroxyvitamin D g 3-acetate and lα-hydroxy vitamin D 3 1,3-diacetate, from which the desired product

e.g. lα-hydroxyvitamin D 3 3-acetate (see reaction ( 2 ) ) is obtained by chromatography. Other partially acylated products that are required as starting materials for subsequent fluorination (e.g. as in reactions 1, 7, 8 ) 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 1,25-dihydroxyvitamin D g 3,25-di-0-acyl compound (reaction 3) can be obtained by partial hydrolysis under basic conditions of the 1,25-dihydroxyvitamin D 3 1,3,25- tri-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 groups, if present, can be removed by basic hydrolysis, e.g. treatment of the acylated fluoro analog with 5% KOH in MeOH, at a temperature of 25-80°C for 1-4 hr. The resulting deacylated fluorinated products are then conveniently further purified by chromatography. The use of cyclovitamin D compounds of structure V pro¬ vides, " of course, starting materials with inherent protection of the C-3-position and triene chromophore and for purposes of this fluorination process the 3,5- cyclopropano-"protecting group" may be considered operationally equivalent to the 3-0-acyl protection in the case of cis and trans vitamin starting materials III and IV, except that, of course, the eventual "deprotection" of these cyclopropano-intermediates requires acid catalyzed solvolysis. Whenever a cyclovitamin starting material carries hydroxy functions that are not to be fluorinated (e.g. in the side chain) these hydroxy functions can be protected by acylation using methods entirely analogous to those discussed above.

BUREj . -_QMPf

Fluorination of cyclovitamin D compounds of structure V represents a general and convenient method for the production of both fluorovitamin D compounds and fluoro-5,6-trans-vitamin D compounds represented respec- tively by general structures I and II above in which R. is selected from the group consisting of hydroxyl, 0-acyl or 0-lower alkyl and where R, 2 , Rg, R^, R,- and represent substituents as previously defined.

These required cyclovitamin D starting materials ar conveniently prepared from C-3-hydroxyvitamin D compound by the methods published by Sheves and Mazur J_. Am. Chem. Soc. 97, 6249 (1975)) and Paaren et al. (Proc. Nat. Acad. Sci. USA 7_5_, 2080 (1978)). Using the process of the present invention these compounds are readily fluorinated at carbon 1 and/or any of the side chain positions. After fluorine introduction, the fluoro- cyclovita in derivatives can be subjected to acid catalyzed solvolysis as described by Sheves and Mazur and Paaren et_ al_ in the above cited references, to yield both fluorovitamin D compounds and 5,6-trans-fluoro- vitamin D compounds.

These C-1-fluorinated cis and trans reaction pro¬ ducts can be conveniently separated by chromatography at this stage (as described by Paaren et_ aJ in the abov cited reference) and then can be separately subjected to hydrolysis (if acyl protecting groups are to be removed) using the standard conditions described earlier e.g. 0.1 M KOH in methanol, 60°C, 1-4 hr. It should be noted that, in principle, the removal of the acyl groups (using the conditions cited) can be accomplished at any stage after fluorine introduction. Thus acyl groups could be removed also prior to solvolysis, or prior to separation of the cis and trans solvolysis products. A choice between these options would depend on practical convenience and/or the eventual product desired. In general, deacylation as the final step is the preferred mode of operation.

Depending upon the exact solvolyzing conditions, 5,6-cis and -trans products with different C-3 sub- stituents are obtained. Thus, conducting the solvolysis in a medium consisting of aqueous dioxane and a catalytic amount of p_-toluene sulfonic acid yields fluorinated 5,6- cis and trans products bearing a C-3-hydroxy substituent. Solvolysis of fluorocyclovitamin D intermediates in acidified alocholic solvents (e.g. methanol, ethanol, isopropanol) leads to 3-0-alkyl-5,6-cis and trans products where the alkyl group corresponds to the alkyl portion of the alcoholic solvent used. Solvolysis of fluoro¬ cyclovitamin 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. If desired these C-3-O-acylated products can be readily converted to the corresponding C-3-hydroxy compounds by mild base hydrolysis. The production of both 5,6-cis and 5,6-trans-l- fluorovitamin D compounds from 3,5-cyclovitamin D starting materials (which are readily separable by chromatography) is an advantageous feature of this fluorination process, and the C-1-fluorinated 3,5-cyclovitamin D derivatives are new and highly useful intermediates.

- 14 -

Example 1 lα-Hydroxyvitamin Do 3-acetate: lα-hydroxyvitamin D o (25 g, 0.063 mmole) and acetic anhydride (50 μl) in pyridine-benzene (2 ml, 1:1) are heated at 50°C under argon for 4 hr. The reaction mixture is cooled, water and ether are added, and the phases are separated. The ether layer is washed with water, 1 N HC1 (2 times), dilute NaHC0 3 , saturated NaCl and dried (Na^O^). The solvent is evaporated and the residue is chromatographed on a silica gel plate (20 x 20 cm, 0.75 cm thick) developed with ethyl acetate/Skellysolve B (1:1). Four bands of R f 0.20, 0.38, 0.49 and 0.64 are apparent. The band with R~ 0.49 consists of product, lα-hydroxyvitamin D 3 3-acetate, 6.0 mg (22%). The bands with R f 0.64, 0.38, and 0.20 consists of lα-hydroxyvitamin D, 1,3-diacetate, lα-hydroxyvitamin D 3 1-acetate, and starting material (7.3 mg, 29%), respectively. The undesired 1-acetate and the diacetate are combined (6.8 mg, 23-25%), hydrolyzed (1 ml 0.1 M OH/MeOH;-l ml ether, 1.25 hr. , room temperature), and pooled with starting material to give 14 mg lα-hydroxy¬ vitamin D 3 . This process is repeated twice more to give a total of 12 mg (43%) lα-hydroxyvitamin Do 3-acetate: uv (95% EtOH) λjiicL v x 265, λmm. . 228; n r (270 MHz, CDCl3 o ) δ 0.54 (s, 18-CH 3 ), 0.87 (d, J = 6.6 Hz, 26,27-(CH 3 ) 2 ), 0.92 (d, J = 6.1 Hz, 21-CH 3 ), 2.03 (s, 3 AcO-), 4.41 ( , lβ-H), 5.02, 5.34 (19-H's), 5.34 (3α-H), 6.02, 6.34 (AB quartet, J = 11.4 Hz, 6 and 7 H'.s). Example 2 1-Fluorovitamin Dp-. To 2 mg lα-hydroxyvitamin D g

3 -acetate in CH 2 C1 2 (0.4 ml) at -78°C is added diethyl¬ aminosulfur trifluoride (12 μl) with good stirring. The cooling bath is removed and 5 in later the reaction is quenched with 5% K 2 C0 3 - Ether is added and the phases are separated. The organic phase is washed with water, and brine, and concentrated to 0.5 ml. To the organic

phase 0.1 M KOH/MeOH (1 ml) is added. After 1.5 hr at room temperature the solvent is removed, ether and water are added, the phases are separated. The ether phase is washed with water and brine and filtered through Na 9 S0^. The residue obtained after evaporation of the ether is chromatographed over a microparticulate silica gel column (5 μparticles, 0.7 x 25 cm) eluted with 0.5% isopropanol/hexane. The desired product elutes in 78 ml (0.85 mg & ): uv λm__,a_- v x 265,' λm„m. 226 nm,' λ_max/λmm.„ 2.1; nmr (benzene-dg) δ 0.63 (s, C-18 methyl), 0.98 (d, J = 6.1 Hz, C-26,27 methyls), 1.01 (d, J - 6.5 Hz, C-21 methyl), 3.52 (m, 1/2 24 Hz, 3α-proton), 4.71 (doublet of ultiplets, J = 50 Hz, w 1/2 20 Hz), 5.23, 5.55 (two s, 19-protons), 6.38, 6.52 (ABq, J = 11 Hz, 6- and 7- protons); mass spectrum m/e (relative intensity) 402.3320 •(M + , 0.10, calcd. for C^H^FO, 402.3298), 382 (M + -HF, 0.40), 364 (M + -HF-H 2 0, 0.28), 349 (M + -HF-H 2 0, 0.28), 349 (M + -HF-H 2 0-CH 3 , 0.04), 289 (M + -side chain, 0.05), 269 (M + -side chain-HF, 0.08), 251 (M + -side chain-HF- H 2 0, 0.10), 135 (1.00). Example 3,

• lα,25-Dihydroxyvitamin Do 3-acetate: lα,25-Dihydroxy- vitamin D 3 (25 mg) and acetic anhydride (50 μl) in pyridine-benzene (12 ml, 1:1) are heated at 50°C under argon for 4 hr. The usual work-up gives a mixture of partially acetylated products (including the 1-acetate, the 3-acetate and the 1,3-diacetate)- from which the desired 3-acetate product- is separated by preparative-layer chromatography on silica gel developed with 40% ethyl acetate in Skellysolve B. Recycling of the unreacted starting material and hydrolysis of undesired 1,3-diacetate and 1-acetate improves the overall yield.

Example 4

1,25-Difluorovitamin D,: lα,25-Dihydroxy itamin D g 3-acetate (3 mg) in CHoCl 2 at -78°C under argon is treated with diethylaminosulfur trifluoride (20 μl). Th solution is allowed to warm to room temperature and quenched with 5% K 2 C0 3 - Work-up of'the reaction is done as described in Example 2,. Chromatography of the recovered material over a silica gel plate developed with 25% ethyl acetate in Skellysolve B yields purified 1,25-difluorovitamin D 3 3-acetate. Hydrolysis (1 ml of 0.1 M KOH/MeOH, 1 ml ether, room temperature, 2 hr) and rechromatography over silica gel (ethyl acetate/ Skellysolve B) gives 1,25-difluorovitamin D~. Example 5 1,25-Dihydroxyvitamin D 25-acetate: A solution of 10 mg of 1,25-dihydroxyvitamin D g is heated at 90°C for 16 hr under argon with 0.5 ml of acetic anhydride and ' 2 ml of yridine. The usual work-up gives a mixture from which 1,25-dihydroxyvitamin D 3 1,3,25-triacetate ( 9.5 mg) is separated by chromatography over a prepara¬ tive layer of silica gel developed with 25% ethyl acetate/Skellysolve B or by high-pressure liquid chroma¬ tography with a silica gel column eluted with mixtures of 0.5% 2-propanol in hexane. Selective hydrolysis ( 0.1 M KOH/MeOH, ether, 1.5 hr, 25°C) of the 1 and 3 acetates gives 7 mg of 1,25-dihydroxyvitamin D 3 25- acetate, which is purified by chromatography on silica gel thin layer plates using 50% ethyl acetate in Skelly ¬ solve B as solvent. Example 6

1,25-Dihydroxyvitamm D,. 3,25-diacetate: 1,25- Dihydroxyvitamin D 3 25-acetate (7 mg) is acetylated using the conditions described in Example 1. From the product mixture 1,25-dihydroxyvitamin D 3 3,25-diacetate (4 g) is isolated by silica gel thin layer chromatography using

ethyl acetate/Skellysolve B (1:1) as solvent system. Example 7

1-Fluo o-25-hydroxyvitamih D g : 1,25-dihydroxyvitamm D 3 3,25-diacetate ( mg) in CH 2 C1 2 at -78°C under argon is treated with diethylaminosulfur trifluoride (4 mg). The reaction mixture is allowed to warm.to room temperature and quenched with 5% K 2 C0 3 . Chromatography o the recovered material over silica gel high-pressure liquid chromatography eluted with 1% 2-propanol in hexane gives 0.7 mg of l-fluoro-25-hydroxyvitamin D 3 3,25-diacetate. Hydrolysis (0.1 M KOH/MeOH, ether) and rechromatography over silica gel high-pressure liquid chromatography developed with 5% 2-propanol/hexane yields 1-fluoro- 25-hydroxy-vitamin D 3 in pure form. Example 8 l-Fluoro-25-hydroxy-5,6-trans-vitamin D 3 : A solution of 2 mg of lα,25-dihydroxyvitamin D p 3,25-diacetate in 2 ml of ether containing a drop of pyridine is treated with 0.1 ml of a solution of iodine in Skellysolve B (0.5 mg/ml) and stirred for 15 min. After addition of 1 ml of an aqueous solution of sodium thiosulfate, the organic " phase is separated, and solvent is evaporated. The desired product, lα,25-dihdyroxy-5,6-trans-vitamin D 3,25-diacetate is isolated by thin layer chromatography on silica gel using 20% ethyl acetate in Skellysolve B as solvent system, (yield 0.8 mg). This material is directly fluorinated using the conditions described in Example 2 (CH 2 C1 2 solution, 10 μl diethylaminosulfur trifluori.de, -78°C) and after work-up as described in Example 2 the recovered product is hydrolyzed (0.1 M KOH, MeOH/ether, 60°C, 3 hr.). Purification of the hydrolysis product by high-pressure liquid chromatography on silica gel columns using 3% 2-propanol in Skellysolve B as solvent yields 200 μg of l-fluoro-25-hydroxy-5,6-trans- vitamin D .

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Example 9

1,25-Difluoro-6-methoxy-3,5-cyclovitamin D_: A solution of 1 mg of l,25-dihydroxy-6-methoxy-3,5-cyclo- vitamin D 3 (prepared by the method of Paaren et al_. Proc Nat. Acad. Sci. USA, 5_, 2080 (1978)) in CH 2 C1 2 at -78°C under argon is treated with diethylaminosulfur tri¬ fluoride (2 mg). After 10 min. , the reaction mixture is allowed to warm to room temperature and quenched with 5% K 2 C0 3 - After the usual work-up (E.G. see Example 2) 1,25- difluoro-6-methoxy-3,5-cyclovitamin D g (250 μg) is isolated in pure form by high-pressure liquid chromatog¬ raphy using a silica gel column eluted with 10% tetra- hydrofuran/hexane. Alternatively, the product can be purified by thin-layer chromatography on silica gel usin 20% ethyl acetate in Skellysolve B as developing solvent Example 10 l-Fluoro-6-methoxy-3,5-cyclovitamin p: A methylen chloride solution of 10 mg lα-hydroxy-6-methoxy-3,5- cyclovitamin D„ (prepared as described by Paaren et_ al. Proc. Nat. Acad. Sci. USA 7j>_, 2080 (1978)) is cooled to -78°C and treated with 20 mg of diethylaminosύlfur"'tri- fluoride. After 15 min. the solution is warmed to room temperature, aqueous NaHC0 3 (5 ml) and CH 2 C1 2 (10 ml) ar added. The organic phase is separated, solvent is evaporated and the product is purified by preparative thin-layer chromatography (silica gel plates), 10% ethyl acetate in Skellysolve B as developing solvent, to yield 1-fluoro-6-methoxy-3,5-cyclovitamin ' Do. Example 11

1-Fluorovitamin D 2 and 1-Fluoro-5,6-trans-vitamin D A solution of 3 mg of l-fluoro-6-methoxy-3,5-cyclovitami D 2 (see Example 10) in dry dioxane (2 ml) is warmed to 55°C and treated with a 1:1 mixture of 98% formic acid: dioxane ( 150 μl) ' . After 15 min., ice-water is added and the products are extracted with ether. The solvent is

evaporated and the product mixture (consisting of 1- fluorovitamin D 2 3-formate and 1-fluoro-5,6-trans- vitamin D 2 3-formate) is directly hydrolyzed by dissolution in dioxane:methanol (1:1) and treatment with aqueous 2 C0 3 solution (10 mg/0.1 ml). Hydrolysis is complete after 5 min. at room temperature, and the solution is diluted with water and the products extracted, into ether. Chromatography on silica gel plates (750 μ thick) using 1:3 ethyl acetate:Skellysolve B as eluting solvent, separates the 5,6-cis and 5,6-trans products and yields pure 1-fluorovitamin 2 (1 mg) and 1-fluoro-5,6-trans- vitamin D 2 (0.3 mg). Biological Activity of fluorovitamin D compounds

The novel fluorovitamin D 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 these fluorovitamin D compounds include the stimulation of intestinal calcium transport, the stimulation of calcium mobilization from bone and the calcification of bone.

For the demonstration of these effects a, desirable test animals is the male weanling rat maintained on a vitamin D-deficient low calcium diet, or a vitamin D-deficient, low-phosphorus diet, as described by Suda et_ al. J. Nutr. 100- 1049 (1970). With animals maintained on a low calcium diet, intestina'l calcium absorption can be assayed by the everted gut sac technique of Martin and DeLuca (Am. J. Physiol. 216, 1351 (1969)) and bone calcium mobiliza¬ tion can be determined by the rise of serum calcium as described " for example by Blunt et al. Proc. Nat. Acad. Sci. USA 61_, 1503 (1968); degree of endochondral calcification can be assayed by the "line-test" method described in the U.S. Pharmacopeia (15th revision, p. 889, Mack Publ. Eas on, PA (1955)) using animals maintained on


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the low phosphorus diet. Using such methods the biological activity of the 1-fluorovitamin D or 1- luoro-5,6-trans-vitamin D products of this invention is readily demonstrated. Thus 1-fluorovitamin D 3 administered intraperitoneally to rats maintained on a vitamin D-deficient low-phosphorus diet, causes significant calcification of bone as illustrated in Table 1.

TABLE 1 Antirachitic Properties of 1-Fluorovitamin D 3 a

Serum phosphorus Daily does (ng) (mg/100 ml) Line-test Sco

1,2-propanediol 3.1 +_ 0.3 0

Vitamin D 3 (20) 4.5 +_ 0.3 3.6 + 0.7

1-Fluorovitamin D 3 (270) 4.0 +_ 0.2 3.2 + 0.4

Data are expressed as mean +_ SEM from 3-5 rats. Test compounds given intraperitoneally in 1,2- propanediol solvent for 7 days.

Similarly, administration of 1-fluo'rovitamin D p to vitamin D-deficient animals maintained on a low calcium diet, produces significant elevations of serum calcium levels and will effectively stimulate intestinal calcium transport, as demonstrated by the typical dose response data given in Table 2.

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TABLE 2 Dose Response Data for 1-Fluorovitamin D 3

Bone Calcium Mobilization

Serum calcium Intestinal Calcium Dose (ng) (mg/100 ml) transport

Ethano1 (contro1) 4.6 +_ 0.1 1.7 + 0.2

Vitamin D 3 (24) 5.2 +_ 0.2 3.1 + 0.3

1-Fluorovitamin D, , (280) 5.1 +_ 0.02 2.0 +_ 0.2

II (700) 5.5 +_ 0.2 2.7 f 0.2

It (1260) 5.8 + 0.1 3.8 + 0.4

It (2450) 6.2 +_ 0.3 2.8 +_ 0.2

Data given as mean +_ SEM for 5-6 rats. C Coommppoouunnddss aaddmmiinniisstteerreedd bbyy intrajugular injections in 50 μl of ethanol solvent.

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