# Patent

## Title
Process for the preparation of pure aqueous betaine solutions

## Abstract

Biologically active 1.alpha.-hydroxy-22-iodinated vitamin D.sub.3 compounds and a process for the preparation of 1.alpha.-hydroxy-22-iodinated vitamin D.sub.3 compounds are disclosed. The 22-iodovitamin D.sub.3 compounds show relatively high binding affinity for the vitamin D receptor demonstrating their potential for high in vivo biological activity. Further, these compounds induce relatively high differentiation of malignant cells. Also, these 22-iodo compounds show high in vivo calcium transport activity with little or no bone calcium mobilization activity. These compounds thus show promise in the treatment of osteoporosis.

## Background
This invention relates to biologically active vitamin D.sub.3 compounds. More specifically, the invention relates to 1.alpha.-hydroxylated-22-iodinated vitamin D.sub.3 compounds and a process for the preparation thereof.

The 1.alpha.-hydroxylated metabolites of vitamin D--most importantly 1.alpha.,25-dihydroxyvitamin D.sub.3 and 1.alpha.,25-dihydroxyvitamin D.sub.2 - are known as highly potent regulators of calcium homeostasis in animals and humans, and more recently their activity in cellular differentiation has also been established. V. Ostrem et al, Proc. Natl. Acad. Sci. USA. (1987), 84, 2610. As a consequence, many structural analogs of these metabolites, such as compounds with different side chain structures, different hydroxylation patterns, or different stereochemistry, have been prepared and tested. Important examples of such analogs are 1.alpha.-hydroxyvitamin D.sub.3, 1.alpha.-hydroxyvitamin D.sub.2, various side chain fluorinated derivatives of 1.alpha.,25-dihydroxyvitamin D.sub.3, and side chain homologated analogs. Several of these known compounds exhibit highly potent activity in vivo or in vitro, and some of these have been found to exhibit an interesting separation of activities in cell differentiation and calcium regulation. This difference in activity provides these compounds with advantageous therapeutic activity profiles and thus numerous of these compounds are in use, or have been proposed for use, in the treatment of a variety of diseases such as renal osteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

In a continuing effort to explore pharmacologically important vitamin D analogs, several new 22-iodinated vitamin D.sub.3 compounds have now been synthesized. The 22-iodinated compounds show relatively high binding affinity for the vitamin D receptor thus demonstrating their potential for high in vivo biological activity. Further, these compounds induced relatively high differentiation of malignant cells. Also, these 22-iodo compounds showed high in vivo calcium transport activity with little or no bone calcium mobilization activity. Accordingly, the 22-iodo compounds show promise in the treatment of osteoporosis, particularly senile osteoporosis and postmenopausal osteoporosis. The iodo compounds can also be labeled with radio iodine to be used for 1.alpha.,25-(OH).sub.2 -D.sub.3 assays.

FIG. 1 is a graph of the percent differentiation of HL-60 cells versus concentration for a prior art vitamin D.sub.3 compound and two of the new 22-iodo-vitamin D.sub.3 compounds; and

FIG. 2 is a graph of the competitive binding ability versus concentration for the same three compounds as in FIG. 1.

The present invention is directed to biologically active 1.alpha.-hydroxy-22-iodinated vitamin D.sub.3 compounds and to a process for the preparation of biologically active 1.alpha.-hydroxy-22-iodinated vitamin D.sub.3 compounds having the general formulas I and II shown below: ##STR1## where Y.sup.1 and Y.sup.2, which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, and where R is hydrogen, an aryl, alkyl, hydroxyalkyl or fluoroalkyl group, or R may represent the following side chain fragment: ##STR2## wherein R.sup.1 represents hydrogen, hydroxy or protected hydroxy, R.sup.2 and R.sup.3 are each selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, or, when taken together represent the group--(CH.sub.2).sub.m --where m is an integer having a value of from 2 to 5, R.sup.4 is selected from the group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R.sup.5 is selected from the group consisting of hydrogen, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, and wherein n is an integer having a value of from 1 to 5.

Specific important examples of side chains are the structures represented by formulas (a), (b), (c) and (d) below, i.e. the side chain as it occurs in (22S)-iodo-25-hydroxyvitamin D.sub.3 (a); (22S)-iodovitamin D.sub.3 (b); (22R)-iodo-25-hydroxyvitamin D.sub.3 (c); and (22R)-iodovitamin D.sub.3 (d). ##STR3##

As used in the description, and in the claims, the term "hydroxy-protecting group" refers to any group commonly used for the protection of hydroxy functions during subsequent reactions, including, for example, acyl or alkylsilyl groups such as trimethylsilyl, triethylsilyl, t-butyldimethylsilyl and analogous alkyl or arylsilyl radicals, or alkoxyalkyl groups such as methoxymethyl, ethoxymethyl, methoxyethoxyethyl, tetrahydrofuranyl or tetrahydropyranyl. A "protected-hydroxy" is a hydroxy function derivatized by one of the above hydroxy-protecting groupings. "Alkyl" represents a straight-chain or branched hydrocarbon radical of 1 to 10 carbons in all its isomeric forms, such as methyl, ethyl, propyl, isopropyi, butyl, isobutyl, pentyl, etc., and the terms "hydroxyalkyl," "fluoroalkyl" and "arylalkyl" refer to such an alkyl radical substituted by one or more hydroxy, fluoro or aryl groups respectively. An "acyl" group is an alkanoyl group of 1 to 6 carbons in all its isomeric forms, or an aroyl group, such as benzoyl, or halo-, nitro- or alkyl-substituted benzoyl groups, or an alkoxycarbonyl group of the type alkyl--O--CO--, such as methoxycarbonyl, ethoxycarbonyl, propyloxycarbonyl, etc., or a dicarboxylic acyl group such as oxalyl, malonyl, succinoyl, glutaroyl, or adipoyl. The term "aryl" signifies a phenyl-, or an alkyl-, nitro- or halo-substituted phenyl group. The term alkoxy signifies the group alkyl--O--.

The wavy lines to the substituents at C-20 (methyl) and C-22 (iodine) indicate that these substituents may have either the R or S configuration.

The preparation of 1.alpha.-hydroxy-22-iodovitamin D compounds having the basic structures shown above can be accomplished starting from the diene-protected derivative of structure III or vitamin D compounds of the general structures IV and V ##STR4## where Y.sup.1 and Y.sup.2 are as defined above and R.sup.6 represents hydrogen, O--alkyl or O--aryl. Reaction of the C-22 aldehydes III. IV or V (R.sup.6 =hydrogen) with an alkylmagnesium halide having the structure RMgX (R as defined above, X=halogen) or alkyllithium reagent having the structure RLi (R as defined above) in the appropriate inert solvent, provides 22-hydroxy compounds of the general formulas VI, VII and VIII, respectively (Y.sup.3 =H). Alternatively, the same compounds VI-VIII can be obtained by reaction of the above mentioned organometallic reagents with esters III, IV and V (R.sup.6 =O--alkyl or O--aryl) under carefully controlled reaction conditions providing 22-ketones of the general formulas III, IV and V (R.sup.6 =R as defined above) and by subsequent reduction of the 22-oxo group in these compounds by the appropriate reducing agents (e.g. hydride reduction). ##STR5## Adduct VI (Y.sup.3 =H) subjected to basic conditions can be then converted to 5,7-diene steroid which in turn via the well known process consisting of irradiation with UV light and thermal isomerization gives VII (Y.sup.3 =H).

The 22-hydroxy intermediates VII and VIII (Y.sup.3 =H) having, preferably, all remaining hydroxy groups protected are then treated with an alkyl- or arylsulfonylhalide (e.g. methanesulfonylchloride, p-toluenesulfonylchloride) in a suitable solvent (e.g. pyridine) to obtain the corresponding 22--O--alkyl-- or --arylsulfonyl derivatives (the compounds having the structures shown VII and VIII above, where Y.sup.3 is alkyl--SO.sub.2 -- or aryl--SO.sub.2 --). These sulfonates are then subjected to nucleophilic substitution reaction with a reagent which can serve as an iodine ion source (inorganic iodides e.g. alkali metal iodides, ammonium iodide etc. and organic iodides e.g. tetraalkylammonium iodides etc.) in an appropriate solvent (preferably acetone, 2-butanone, 2-propanol, acetonitrile etc.), at a temperature ranging from 0.degree. C. to the boiling temperature of the solvent, thereby displacing the sulfonate group and obtaining the 22-iodo compounds, represented by the structures I and II above. To avoid undesired isomerization of the 5,6-double bond which can occur due to evolution of small amounts of iodine (radical dissociation of C-I bond) in the reaction medium it is required to perform such substitution reaction in the presence of mercury and in the dark. In order to neutralize strong acids which can be formed in the side processes (concurrent elimination reactions) it is advantageous to add calcium carbonate to the reaction medium.

It is also evident that the reaction of the 22-sulfonates with isotopically labeled iodides (e.g. NH.sub.4.sup.123 I, Na.sup.125 I, Na.sup.129 I, Na.sup.131 I, etc.) provides a convenient means for preparing 22-iodo compounds I and II in isotopically-labeled form. Due to known susceptibility of aliphatic iododerivatives to nucleophilic substitution such radioiodinated compounds can also be prepared by reaction of the unlabeled derivatives I or II with the isotopically labeled iodides (i.e. isotope exchange process). Vitamins of high specific activity (up to 2200 Ci/mmol) labeled with carrier-free radioiodine can be conveniently produced in that manner. These radiolabeled vitamin D derivatives find ready application as tracers in various known binding assays and for other experimental research studies.

The next step of the process comprises the removal of the hydroxy protecting groups to produce the free hydroxy compounds represented by 1.alpha.-hydroxyvitamin structures I and II above (where Y.sup.1, Y.sup.2 and R.sup.1 are hydrogens) or 1.alpha.,25-dihydroxyvitamin D structures (where Y.sup.1, Y.sup.2 are hydrogens and R.sup.1 is hydroxyl). Alternatively, the deprotection of hydroxyl groups may be performed on 22-sulfonates to obtain compounds of structures VII and VIII (where Y.sup.3 is alkyl--SO.sub.2 -- or aryl--SO.sub.2 -- and Y.sup.1, Y.sup.2 and R.sup.1 are hydrogens) which can be then subjected to substitution reaction giving rise to 22-iodo compounds I and II (where Y.sup.1, Y.sup.2 and R.sup.1 are hydrogens). If obtained as a mixture of the two C22 epimers, 22-alcohols, sulfonates and/or iodides can be separated by chromatographic methods. If desired, the 5,6-cis compounds I, IV and VII can be easily converted to the corresponding 5,6-trans counterparts (and vice versa) II, V and VIII, respectively by the known iodidecatalyzed isomerization process.

This invention is more specifically described by the following illustrative examples. In these examples specific products identified by Arabic numerals (e.g. 1, 2, 3, etc.) refer to the specific structures so identified in the preceding description and in the Schemes. Bromocompound A (Scheme 1)

A solution of known 4-bromo-2-methyl-2(triethylsilyloxy) butane (A) (94 mg, 0.33 mmol) in anhydrous ether (0.3 mL) was added dropwise to a stirred mixture of magnesium powder (9.7 mg, 0.4 mmol; .about.50 mesh, Aldrich) in anhydrous ether (0.2 mL) under argon at room temperature with occasional warming it up to 35.degree. C. After addition was complete the mixture was stirred for 15 min at room temperature and 30 min at 40.degree. C. Then it was cooled to 0.degree. C. and a solution of known C-22 aldehyde (1) [32 mg, 0.056 mmol; see A. Kutner et al., J. Org. Chem. 53, 3450 (1988)] in anhydrous ether (0.3 mL, cooled to 0.degree. C.) was added dropwise. After the reaction mixture was stirred for 20 min at 0.degree. C. and 75 min at room temperature it was quenched with aqueous solution of NH.sub. 4 Cl (2 mL) and diluted with 4:1 (v/v) benzene/ether (20 mL). The organic layer was separated, washed with water and diluted NaHCO.sub.3, dried, and evaporated. TLC and HPLC control indicated formation of only one (22S) of the two possible isomers. Pure (22S)-hydroxyvitamin D derivative (2) was obtained as a colorless oil (31 mg, 72% yield) by preparative HPLC (Zorbax-Silica column 6.2 mm.times.25 cm) using 3.5% ethyl acetate in hexane as an eluent; peak at 30 mL was collected. UV (EtOH) .lambda..sub.max 264 nm, .lambda..sub.min 225 nm, A.sub.264 /A.sub.225 =1.6; .sup.1 H-NMR (CDCl.sub.3, 500 MHz): .delta. 0.062 (12H, br s, 4.times.SiMe), 0.546 (3H, s, 18-H.sub.3), 0.583 (6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.877 (18H, s, 2.times.Si-t-Bu), 0.920 (3H, d, J=6.4 Hz, 21-H.sub.3), 0.947 (9H, t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 1.221 and 1.225 (3H and 3H, each s, 26- and 27-H.sub.3), 2.83 (1H, br d, J=12.5 Hz, 9.beta.-H), 3.63 (1H, m, 22-H), 4.19 (1H, m, 3.alpha.-H), 4.37 (1H, m, 1.beta.-H), 4.87 and 5.18 (1H, and 1H, each s, 19-H.sub.2), 6.02 (1H, d, J=11.2 Hz, 7-H), 6.24 (1H, d, J=11.2 Hz, 6-H); MS, m/z (rel intensity) 774 (M.sup.+, 15), 642 (43), 75 (100); exact mass calcd for C.sub.45 H.sub.86 O.sub.4 Si.sub.3 774.5834, found 774.5850. Chloride

To a solution of alcohol (2) (28 mg, 0.036 mmol) in dry pyridide (100 .mu.L) was added freshly recrystallized p-toluenesulfonyl chloride and the reaction was allowed to proceed for 64 h at 4.degree. C. The reaction mixture was poured into ice/saturated NaHCO.sub.3 with stirring. After 40 min of stirring the aqueous suspension was extracted with 4:1 (v/v) benzene/ether (3.times.10 mL). The combined organic extracts were washed with saturated NaHCO.sub.3, water, saturated CuSO.sub.4, again water, dried (Na.sub.2 SO.sub.4) and evaporated. The oily yellowish residue was purified by preparative HPLC (Zorbax-Silica column 6.2 mm.times.25 cm) using 2% ethyl acetate in hexane as an eluent. Pure tosylate (3) (26 mg, 78%; collected at 20 mL) was obtained as a colorless oil: UV (hexane) .lambda..sub.max 264 and 223 nm, .lambda..sub.min 238 nm; .sup.1 H-NMR (CDCl.sub.3, 500 MHz): .delta. 0.059 and  0.067 (6H and 6H, each s, 2.times.SiMe.sub.2), 0.479 (3H, s, 18-H.sub.3), 0.528 (6H,q, J=8 Hz, 3.times.SiCH.sub.2), 0.877 (18H, s, 2.times.Si-t-Bu), 0.915 (9H, t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 0.929 (3H, d, J=6.0 Hz, 21-H.sub.3), 1.103 and 1.141 (3H and 3H, each s, 26- and 27- H.sub.3), 2.43 (3H, s, Ar-Me), 2.80 (1H, br d, J=12.3 Hz, 9.beta.-H), 4.19 (1H, m, 3.alpha.-H), 4.38 (1H, m, 1.beta.-H), 4.58 (1H, t, J=7.1 Hz, 22-H), 4.86 and 5.19 (1H and 1H, each s, 19 -H.sub.2), 5.99 (1H, d, J=11.2 Hz, 7-H), 6.22 (1H, d, J=11.2 Hz, 6-H), 7.32 (2H, d, J=8 Hz, Ar-H), 7.80 (2H, d, J=8 Hz, Ar-H); MS, m/z (rel intensity) 928 (M.sup.+, 1), 796 (2), 756 (3), 664 (3), 624 (47), 492 (27), 173 (100); exact mass calcd for C.sub.52 H.sub.92 O.sub.6 Si.sub.3 S 928.5922, found 928.5894.

To a stirred solution of tosylate (3) (4.6 mg, 5 .mu.mol) in 1:1 (v/v) acetone/2-butanone (200 .mu.L) was added a drop of mercury (220 mg) and anhydrous calcium carbonate (1 mg, 10 .mu.mol) followed by sodium iodide (3.7 mg, 25 .mu.mol). The resultant mixture was stirred and heated for 100 h at 45.degree. C. in the dark under argon, by which time almost no starting material remained. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (2.times.10 mL). The combined organic layers were washed with 1% Na.sub.2 S.sub.2 O.sub.3 and water, dried (Na.sub.2 SO.sub.4) and evaporated. The work up of the reaction mixture and following chromatographic separations were done using subdued light in the laboratory and exposure of products to any light, particularly of wavelength close to the carbon-iodine bond absorption (259 nm) was avoided as much as possible. The mixture of products was then repeatedly chromatographed by HPLC (6.2 mm.times.25 cm Zorbax-Silica column) using 0.1% ethyl acetate in hexane as an eluent. The peaks of 22S-iodocompound (4) and 22R-isomer (5) partially overlapped (R.sub.v 's 48 mL and 53 mL, respectively) but rechromatography (or recycling of both peaks) afforded pure material. (22S)-iodocompound (4) (0.8 mg, 18%): UV (hexane) .lambda..sub.max 264 nm, .lambda..sub.min 227 nm; A.sub.264 /A.sub.227 =1.6; .sup.1 H-NMR (CDCl.sub.3, 500 MHz) .delta. 0.058 and 0.064 (6H and 6H, each s, 2.times.SiMe.sub.2), 0.569 (6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.597 (3H, s, 18-H.sub.3), 0.875 (18H, br s, 2.times.Si-t-Bu), 0.944 (9H, t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 0.964 (3H, d, J=6 Hz, 21-H.sub.3), 1.194 and 1.211 (3H and 3H, each s, 26- and 27-H.sub.3), 2.82 (1H, br d, J.about.13 Hz, 9.beta. -H), 4.19 (2H, m, 3.alpha.- and 22-H), 4.37 (1H, m, 1.beta.-H), 4.86 and 5.18 (1H and 1H, each s, 19-H.sub.2), 6.02 (1H, d, J=11.2 Hz, 7-H), 6.23 (1H, d, J=11.2 Hz, 6-H); MS, m/z (rel intensity) 884 (M.sup.+, 21), 752 (62), 624 (22), 248 (100); exact mass calcd for C.sub.45 H.sub.85 O.sub.3 Si.sub.3 I 884.4851, found 884.4875. (22R)-iodocompound (5) (0.4 mg, 9%): UV (hexane) .lambda.max 264 nm, .lambda..sub.min 227 nm; A.sub.264 /A.sub.227 =1.8; .sup.1 H-NMR .delta. 0.064 (12H, br s, 4.times.SiMe), 0.517 (3H, s, 18-H.sub.3), 0.568 (6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.875 (18H, br s, 2.times.Si-t-Bu), 0.950 (9H, t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 1.158 (3H, d, J=6.8 Hz, 21-H.sub.3), 1.179 (6H, br s, 26- and 27-H.sub.3), 2.83 (1H, br d, J.about.13 Hz, 9.beta.-H), 4.19 (1H, m, 3.alpha.-H), 4.37 (1H, m, 1.beta.-H), 4.45 (1H, d, J=11 Hz, 22-H), 4.86 and 5.18 (1H and 1H, each s, 19-H.sub.2), 6.02 (1H, d, J=11.2 Hz, 7-H), 6.23 (1H, d, J=11.2 Hz, 6-H); MS, m/z (rel intensity) 884 (M.sup.+, 20), 752 (54), 624 (24), 248 (100); exact mass calcd for C.sub.45 H.sub.85 O.sub.3 Si.sub.3 I 884.9851, found 884.4899. Both iodovitamins (4) and (5), when protected from light can be stored for a prolonged time in a freezer with neither 5,6-cis/trans isomerization nor epimerization at C-22. If desired, however, both processes can be easily performed. In order to achieve the interconversion between both C(22)-epimers each compound (4) and (5) can be subjected to an analogous nucleophilic substitution reaction with NaI as described above for the tosylate (3). In both cases the similar equilibrium mixture can be obtained consisting of epimeric (22S)- and (22R)-iodocompounds (4) and (5) in a ratio of about 3-4:1. Transformation of 5,6-cis isomers to their 5,6-trans analogs is described in the following Example.

The synthetic process described in the Example 3 above ensures the preparation of pure 5,6-cis isomers (4) and (5) uncontaminated by 5,6-trans counterparts. Such geometrical isomers are easily distinguishable by both chromatographical and spectral properties. If required, iodine-catalyzed isomerization of 5,6-cis compounds (4) and (5) to the respective 5,6-trans products (6) and (7) can be accomplished according to a known procedure [see A. Verloop et al., Rec. Trav. Chim. Pays-Bas 78, 1004 (1959)]. Thus, treatment of compound (4) in ether with a catalytic amount of iodine [2% of the amount of (4)], while keeping the solution under diffuse daylight for 1 h, results in cis to trans isomerization and after HPLC separation (Zorbax-Silica column 6.2 mm.times.25 cm, 0.1% ethyl acetate in hexane), the 5,6-trans isomer (6) is obtained (R.sub.v 60 mL). Similarly, compound (5), upon treatment with iodine under the above conditions, is isomerized to (7), which can be obtained in pure form after HPLC (conditions as above) separation (R.sub.v 70 mL). (6). .sup.1 H-NMR (CDCl.sub.3, 500 MHz) .delta. 0.609 (3H, s, 18-H.sub.3), 0.967 (3H, d, J=6 Hz, 21-H.sub.3), 1.201 and 1.216 (3H and 3H, each s, 26- and 27-H.sub.3), 2.88 (1H, br d, J.about.13 Hz, 9.beta.-H), ca 4.2 (2H, m, 3.alpha.- and 22-H), 4.57 (1H, m, 1.beta.-H), 4.95 and 4.99 (1H and 1H, each s, 19-H.sub.2), 5.83 and 6.47 (1H and 1H, each d, J=11 Hz, 7- and 6-H). (7). .sup.1 H-NMR (CDCl.sub.3, 500 MHz) .delta. 0.532 (3H, s, 18-H.sub.3), 1.167 (3 H, d, J=6  Hz, 21-H.sub.3), 1.185 (6H, br s, 26- and 27-H.sub.3), 2.88 (1H, br d, J.about.13 Hz, 9.beta.-H), 4.22 (1H, m, 3.alpha.-H), 4.46 (1H, d, J=11 Hz, 22-H), 4.54 (1H, m, 1.beta.-H), 4.95 and 4.99 (1H and 1H, each s, 19-H.sub.2), 5.83 and 6.46 (1H and 1H, each d, J=11 Hz, 7- and 6-H).

Each of 22-iodovitamins was separately hydrolyzed using the same procedure. To a solution of protected triol (4) (1 mg) in anhydrous benzene (50 .mu.L) was added AG 50W-X4 ion exchange resin (20 mg, prewashed with methanol) as a slurry in an anhydrous methanol (200 .mu.L). A drop of mercury (400 mg) was added and the resultant mixture was vigorously stirred at room temperature for 10 h in the dark under argon. The reaction was diluted with 1:1 (v/v) ether/ethyl acetate (1 mL), the solution was decanted and transferred to a separatory funnel and the resin was washed with 1:1 ether/ethyl acetate (2.times.2 mL). The combined organic phase was washed with 5 mL portions of brine, 1% Na.sub.2 S.sub.2 O.sub.3, saturated NaHCO.sub.3, and brine again, dried (Na.sub.2 SO.sub.4) and evaporated (temp. below 35.degree. C.). The work up of the reaction mixture and following chromatographic separations were done using subdued light in the laboratory and exposure of iodinated compounds to any light, particularly of wavelength close to the carbon-iodine absorption (259 nm), was avoided as much as possible. Preparative HPLC [6.2 mm.times.25 cm Zorbax-Silica column, 1:1 (v/v) hexane/ethyl acetate as an eluent] provided small amount of (22R,25)-epoxy-1.alpha.-hydroxyvitamin D.sub.3 (8) (eluted at R.sub.v 34 mL). Further elution gave (22S)-iodo-1.alpha.25-dihydroxyvitamin D.sub.3 (9): R.sub.v 79 mL; UV (EtOH) .lambda..sub.max 264 nm, .lambda..sub.min 227 nm, A.sub.264 /A.sub.227 =2.0; .sup.1 H-NMR (CDCl.sub.3, 500 MHz) .delta. 0.607 (3H, s, 18-H.sub.3), 0.975 (3H, d, J=5.9 Hz, 21-H.sub.3), 1.23 and 1.24 (3H and 3H, each s, 26- and 27-H.sub.3), 2.83 (1H, br d, J.about.13 Hz, 9.beta.-H), 4.21 (2H, m, 3.alpha.- and 22-H), 4.44 (1H, m, 1.beta.-H), 5.00 and 5.33 (1H and 1H, each s, 19-H.sub.2), 6.02 (1H, d, J=11.3 Hz, 7-H), 6.38 (1H, d, J=11.3 Hz, 6-H); MS, m/z (rel intensity)  542 (M.sup.+, 2), 524 (M.sup.+  - H.sub.2 O, 31), 506 (M.sup.+  - 2H.sub.2 O, 17), 414 (M.sup.+  - HI, 8), 396 (M.sup.+  - HI - H.sub.2 O, 54) 378 (M.sup.+  - HI - 2H.sub.2 O, (21), 99 (100); exact mass calcd for C.sub.27 H.sub.41 O.sub.2 I (M.sup.+  - H.sub.2 O) 524.2151, found 524.2145.

Analogously performed hydrolysis of 22-iodocompound (5) gave after HPLC separation (conditions as above) a trace of (22S,25)-epoxy-1.alpha.-hydroxyvitamin D.sub.3 (8) (eluted at R.sub.v 28 mL) and (22R)-iodo-1.alpha.,25-dihydroxyvitamin D.sub.3 (11): R.sub.v 63 mL; UV (EtOH) .lambda..sub.max 264 nm, .lambda..sub.min 228 nm, A.sub.264 /A.sub.228 =1.7; .sup.1 H-NMR (CDCl.sub.3, 500 MHz) .delta. 0.535 (3H, s, 18-H.sub.3), 1.172 (3H, d, J=6.5 Hz, 21-H.sub.3), 1.224 (6H, br s, 26- and 27-H.sub.3), 2.82 (1H, br d, J.about.13 Hz, 9.beta.-H), 4.24 (1H, m, 3.alpha.-H), 4.45 (1H, m, 1.beta.-H), 4.47 (1H, d, J=12 Hz, 22-H), 5.00 and 5.33 (1H and 1H, each s, 19-H.sub.2), 6.02 (1H, d, J=11.2 Hz, 7-H), 6.37 (1H, d, J=11.2 Hz, 6-H); MS m/z (rel intensity 542 (M.sup.+, 8), 524 (M.sup.+  - H.sub.2 O, 64), 506 (M.sup.+  - 2H.sub.2 O, 19), 414(M.sup.+ -HI, 10), 396 (M.sup.+  - HI - H.sub.2 O, 62), 378 (M.sup.+  - HI - 2H.sub.2 O, 9), 99 (100); exact mass calcd for C.sub.27 H.sub.43 O.sub.3 I 542.2257, found 542.2248; calcd for C.sub.27 H.sub.41 O.sub.2 I (M.sup.+ - H.sub.2 O) 524.2151, found 524.2136. Elongation of the hydrolysis time results in the formation of increasing proportions of cyclic ethers (8) or (10). These conversions are useful for correlating the respective C(22)-epimeric iodocompounds with starting tosylates. Thus, for example, (22S)-tosylate (3) subjected to the analogous, (mercury is not necessary) hydrolysis conditions as described above formed (22R,25)-epoxy-1.alpha.-OH-D.sub.3 8 (63% yield) as the only isolable product. ##STR6##

The novel compounds of this invention exhibit an unexpected pattern of biological activity. All of the 22-iodo compounds exhibited high potency in promoting the differentiation of malignant cells. Competitive binding tests also showed that these compounds have the potential for high in vivo biological activity. In addition, the 22-iodo compounds showed high in vivo calcium transport activity with little or no bone calcium mobilization activity. This is illustrated by the biological assay results obtained for the claimed 22-iodo-vitamin D.sub.3 compounds, which are summarized in FIGS. 1 and 2, and Table 1. FIG. 1 shows a comparison of the activity of the known active metabolite 1.alpha.,25-dihydroxyvitamin D.sub.3 and two of the 22-iodo analogs in inducing the differentiation of human leukemia cells HL-60 cells) in culture to normal cells (monocytes). Differentiation activity was assessed by a standard differentiation assay, abbreviated in FIG. 1 as NBT (nitrobluc tetrazolium reduction). The assay was conducted according to known procedures, as given, for example, by DeLuca et al U.S. Pat. No. 4,717,721 and Ostrem et al, J. Biol. Chem. 262, 14164, 1987.  For the assay, the differentiation activity of the test compounds is expressed in terms of the percent of HL-60 cells having differentiated to monocytes in response to a given concentration of test compound.

The results summarized in FIG. 1 clearly show that the new 22-iodo-vitamin D.sub.3 analogs and specifically (22S)-iodo-1.alpha.,25-dihydroxyvitamin D.sub.3 and (22R)-iodo-1.alpha.,25-dihydroxyvitamin D.sub.3, are as potent as 1.alpha.,25-dihydroxyvitamin D.sub.3 in promoting the differentiation of leukemia cells. Thus in NBT assay close to 90% of the cells are induced to differentiation by 1.alpha.,25-dihydroxyvitamin D.sub.3 at a concentration of 1.times.10.sup.-7 molar, and the same degree of differentiation is achieved by the two 22-iodo analogs.

FIG. 2 shows a comparison of the sarnc three compounds as in FIG. 1 illustrating their relative activity with regard to competitive binding to the vitamin D receptor. The competitive receptor binding was done with pig nuclear extract (PNE) as described in Perlman et al. Biochemistry 29, 190-196 (1990) using the porcine extract prepared as described by Dame et al PNAS 82, 7825-7829 (1985). These data are used to demonstrate that the 22-iodo compounds, and in particular the 22S (compound 9) and 22R (compound 11) analogs tested, have potential for high in vivo biological activity.

In regard to the biological data on the calcemic activity of these compounds reported in Table 1, the data in Table 1 illustrate that (22S)-iodo-1.alpha.,25-dihydroxyvitamin D.sub.3 has biological activity in intestinal calcium transport much higher, i.e. about double, to that of 1,25-(OH).sub.2 D.sub.3. The data also illustrates that this compound possesses little or no bone calcium mobilizing activity even when given at 1 .mu.g per day for six days.

The 22-iodo compounds would thus find utility as a treatment for osteoporosis, particularly postmenopausal osteoporosis and senile osteoporosis. The utility for these compounds results from their low bone calcium mobilizing activity with normal differentiative activity, normal binding to the receptor, and high calcium transport activity. ______________________________________ INTESTINAL CALCIUM TRANSPORT AND BONE CALCIUM MOBILIZATION ACTIVITY OF 22S-IODO-1,25-(OH).sub.2 D.sub.3 Intestinal Calcium Bone Calcium Dose      Transport    Mobilization (.mu.g/d/6 (Serosal/Mucosal (Serum Calcium) Compound days)     Ratio)       (mg/100 ml) ______________________________________ None (-D) 0          4.0 .+-. 0.2 4.1 .+-. 0.1 1,25-(OH).sub.2 D.sub.3 0.1       11.2 .+-. 1.2 5.9 .+-. 0.3 22S-Iodo- 1.0       22.8 .+-. 1.4 4.9 .+-. 0.1 1,25-(OH).sub.2 D.sub.3 ______________________________________

Rats were fed the vitamin D-deficient, low calcium (0.02%) diet for 3 weeks and then given the indicated dose in 0.1 ml propylene glycol:ethanol (95:5) intraperitoneally each day for 6 days. On day 7, serum was analyzed for calcium and the duodenum used to measure intestinal calcium transport as described by Martin et al. Am. J. Physiol. 216, 1351-1359, 1969, as modified by Periman et al. Biochemistry, 29, 190-196, 1990.

For treatment purposes, the novel compounds of this invention can be formulated as solutions in innocuous solvents, or as emulsions, suspensions or dispersions in suitable innocuous solvents or carriers, or as pills, tablets or capsules, containing solid carriers according to conventional methods known in the art. For topical applications the compounds are advantageously formulated as creams or ointments or similar vehicle suitable for topical applications. Any such formulations may also contain other pharmaceutically-acceptable and nontoxic excipients such as stabilizers, anti-oxidants, binders, coloring agents or emulsifying or taste-modifying agents.

The compounds are advantageously administered by injection, or by intravenous infusion of suitable sterile solutions, or in the form of oral doses via the alimentary canal, or topically in the form of ointments, lotions, or in suitable transdermal patches. For the treatment of malignant diseases, the 22-iodovitamin D compounds of this invention are administered to subjects in dosages sufficient to inhibit the proliferation of malignant cells and induce their differentiation into normal monocyte-macrophages. Suitable dosage amounts are from 0.5 to 500 .mu.g of compound per day, such dosages being adjusted, depending on the disease to be treated, its severity and the response or condition of the subject as is well-understood in the art.

## Claims

1. A method for preparing 22-iodo-vitamin D.sub.3 compounds, comprising the steps of: compounds VII or VIII where the structures of compounds VII and VIII comprise: ##STR7## where Y.sup.1 and Y.sup.2, which may be the same or different, are hydroxy-protecting groups, Y.sup.3 is hydrogen, and where R is hydrogen, an aryl, alkyl, hydroxyalkyl or fluoroalkyl group, or R may represent the following side chain fragment: ##STR8## wherein R.sup.1 represents hydrogen, hydroxy or protected hydroxy, R.sup.2 and R.sup.3 are each selected from the group consisting of alkyl, hydroxylalkyl and fluoroalkyl, or, when taken together represent the group--(CH.sub.2).sub.m --where m is an integer having a value of from 2 to 5, R.sup.4 is selected from the group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R.sup.5 is selected from the group consisting of hydrogen, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, and wherein n is an integer having a value of from 1 to 5; alkylsulfonylhalide or an arylsulfonylhalide in a suitable solvent to obtain the corresponding 22--O--alkyl-- or 22--O--arylsulfonyl derivative having the structure VII or VIII as follows: ##STR9## where Y.sup.3 is --SO.sub.2 --alkyl or --SO.sub.2 --aryl; reacting the sulfonyl derivative with a reagent providing an iodide ion source in an appropriate solvent at a temperature ranging from 0.degree. C. to the boiling temperature of the solvent in the presence of mercury and in the absence of light of a wavelength close to the carbon-iodine bond absorption wavelength to obtain 22-iodo compounds I and II where the structure of compounds I and II comprise ##STR10## where Y.sup.1 and Y.sup.2 are hydroxy-protecting groups and R is as defined above; and derivatives VII or VIII and before reacting with the reagent providing an iodide ion source or after obtaining the 22-iodo compounds I and II above to provide 22-iodo-1.alpha.-hydroxylated vitamin D.sub.3 compounds of structure I or II where Y.sup.1 and Y.sup.2 are both hydrogen.

2. The method of claim 1 wherein the step of preparing the 22-hydroxy-intermediate comprises compounds IV and V where the structures of compounds IV and V comprise ##STR11## where Y.sup.1 and Y.sup.2 are hydroxy-protecting groups and R.sup.6 represents hydrogen, and an inert solvent.

3. The method of claim 2 wherein the organometallic reagent is selected from the group consisting of an alkylmagnesium halide having the structure RMgX where R is as defined in claim 1 and X is halogen and an alkyllithium having the structure RLi where R is as defined in claim 1.

4. The method of claim 1 wherein the step of preparing the 22-hydroxy intermediate comprises IV and V where the structures of compounds IV and V comprise ##STR12## where Y.sup.1 and Y.sup.2, which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, and R.sup.6 represents O--alkyl or O--aryl, provide a 22-ketone compound of the structure IV or V where R.sup.6 represents R as defined in claim 1: reducing agent.

5. The method of claim 4 wherein the organometallic reagent is selected from the group consisting of an alkylmagnesium halide having the structure RMgX where R is as defined in claim 1 and X is halogen and an alkyllithium having the structure RLi where R is as defined in claim 1.

6. The method of claim 1 wherein the step of preparing the 22-hydroxy intermediate comprises derivative III comprises ##STR13## where Y.sup.1 and Y.sup.2 are hydroxy-protecting groups and R.sup.6 represents hydrogen, with an organometallic reagent in an inert solvent, to produce an adduct VI having the structure ##STR14## where R is as defined in claim 1, and Y.sup.3 is hydrogen, and converting adduct VI to the 22-hydroxy-intermediate VII.

7. The method of claim 6 wherein the step of converting comprises removal of the triazoline group to form a 5,7-diene steroid intermediate, subjecting said 5,7-diene steroid intermediate to irradiation with ultraviolet light to obtain the corresponding previtamin D compound, and isomerizing said previtamin D compound in a solvent at a temperature from about 50.degree. C. to about 90.degree. C. for a time sufficient to obtain intermediate VII.

8. The method of claim 6 wherein the organometallic reagent is selected from the group consisting of an alkylmagnesium halide having the structure RMgX where R is as defined in claim 1 and X is halogen and an alkyllithium having the structure RLi where R is as defined in claim 1.

9. The method of claim 1 wherein the step of preparing the 22-hydroxy intermediate comprises derivative III comprises ##STR15## where Y.sup.1 and Y.sup.2 are as defined in claim 4 and R.sup.6 represents O--alkyl or O--aryl, with an organometallic reagent, to produce a 22-ketone compound of structure III where R.sup.6 represents R as defined in claim 1; reducing agent to produce an adduct VI having the structure ##STR16## where Y.sup.3 is hydrogen and converting adduct VI to the 22-hydroxy-intermediate VII.

10. The method of claim 9 wherein the step of converting comprises removal of the triazoline group to form a 5,7-diene steroid intermediate, subjecting said 5,7-diene steroid intermediate to irradiation with ultraviolet light to obtain the corresponding previtamin D compound, and isomerizing said previtamin D compound in a solvent at a temperature from about 50.degree. C. to about 90.degree. C. for a time sufficient to obtain intermediate VII.

11. The method of claim 9 wherein the organometallic reagent is selected from the group consisting of an alkylmagnesium halide having the structure RMgX where R is as defined in claim 1 and X is halogen and an alkyllithium having the structure RLi where R is as defined in claim 1.

12. The method of claim 1 wherein the iodide ion is selected from the group consisting of tetraalkylammonium iodides, and mixtures thereof.

13. The method of claim 1 wherein the iodide ion is selected from the group consisting of alkali metal iodides, ammonium iodide, and mixtures thereof.

14. The method of claim 1 wherein the step of reacting the sulfonyl derivative with a reagent providing an iodide ion source occurs in the absence of light having a wavelength of about 259 nm.

15. The method of claim 1 further comprising the step of adding calcium carbonate to the reaction medium during nucleophilic substitution of sulfonyloxy derivatives by iodide ion.

16. Vitamin D compounds having the structure I and II shown below: ##STR17## where X is radioiodine, Y.sup.1 and Y.sup.2, which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, and where R is hydrogen, an aryl, alkyl, hydroxyalkyl or fluoroalkyl group, or represents the following side chain fragment: ##STR18## wherein R.sup.1 represents hydrogen, hydroxy or protected hydroxy, R.sup.2 and R.sup.3 are each selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, or, when taken together represent the group--(CH.sub.2).sub.m --where m is an integer having a value of from 2 to 5, R.sup.4 is selected from the group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R.sup.5 is selected from the group consisting of hydrogen, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, and wherein n is an integer having a value of from 1 to 5.

17. The vitamin D compounds of claim 16 wherein said radioiodine is selected from the group consisting of .sup.123 I, .sup.125 I, .sup.129 I and .sup.131 I.