Novel 1.alpha.-Hydroxyvitamin D.sub.2 Epimer and Derivatives

This invention was made in the course of work supported by a grant or award from the Department of Health and Human Services. The Government has certain rights in this invention.

This invention relates to vitamin D.sub.2 compounds, and more specifically to the preparation of the novel (24S)-epimer of 1.alpha.-hydroxyvitamin D.sub.2, and certain derivatives thereof.

The natural vitamin D-derived hormone, 1.alpha.,25-dihydroxy-vitamin D.sub.3, and its 25-deoxy analog, 1.alpha.-hydroxyvitamin D.sub.3, both exhibit high activity in vivo, being known as potent stimulators of the intestinal absorption of calcium and the mobilization of calcium from bone and as effective promoters of bone calcification. A very similar activity pattern is shown by 1.alpha.,25-dihydroxyvitamin D.sub.2 (U.S. Pat. No. 3,880,894) and its 25-deoxy analog, 1.alpha.-hydroxyvitamin D.sub.2 (U.S. Pat. No. 3,907,843). These compounds likewise elicit the full spectrum of vitamin D-type responses such as intestinal calcium transport, bone mineral mobilization and bone calcification response in the 1.alpha.-hydroxyvitamin D.sub.2 are characterized by having a C-24 stereochemistry as it occurs in the side chain of ergosterol, i.e. these compounds are defined by the structures shown below, where R represents side chains (a) and (b), respectively: ##STR1## More recently the C-24-epimer of 1.alpha.,25-dihydroxyvitamin D.sub.2 has been prepared and tested (U.S. Pat. Nos. 4,588,716 and 4,769,181). This compound is characterized by the structure shown above, where R represents side chain (c). Remarkably, this C-24-epimeric vitamin D derivative exhibits a distinctly different biological activity profile, in that it is active in stimulating intestinal calcium absorption and promoting the calcification of bone, but does not elicit a bone calcium mobilization response.

This invention provides a new vitamin D analogue, namely 1.alpha.-hydroxy-24-epi-vitamin D.sub.2, which may be represented by the structure below, as well as the acyl and alkylsilyl derivatives of that compound. ##STR2##

This compound, therefore, is distinguished from the known 1.alpha.-hydroxyvitamin D.sub.2, by having the opposite methyl stereochemistry at C-24 (i.e. the 24S configuration), and it is further distinguished by exhibiting a strikingly different pattern of biological activity than the known vitamin D.sub.2 derivative, as more fully described below.

The synthesis of 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 requires the construction of an appropriate side chain unit having the desired (S) stereochemistry at the carbon center that is to become carbon-24 in the target compound, and the condensation of that side chain unit with a suitable 1.alpha.-hydroxylated vitamin D nucleus so as to generate the desired final product.

The synthesis of the optically active side chain unit comprised the conversion of commercially available, racemic 2,3-dimethylbutanol to the corresponding bromide and then to the magnesium bromide derivative (1) according to published procedures (see T. Suda et al., Chem. Soc. 82, 3396, 1960; Martinez et al., Gazz. Chim. Ital. 97, 96, 1967; Organic Synthesis, Collective volume 2, p. 358, Wiley & Sons, N.Y., 1943).

The magnesium bromide derivative (1) shown below was then reacted with (R)-(+)-p-toluenesulfinic acid (-)-menthyl ester, compound (2) below, under Grignard reaction conditions. This reaction is the key step for it provides a mixture of diastereomeric sulfoxides, namely compounds (3) and (4), which may be readily separated by column chromatography or by high pressure liquid chromatography (hplc) to give both the 2R (compound (3) and the (compound 4) stereoisomers. ##STR3##

Subsequent oxidation of p-tolyl-2,3-dimethylbutylsulfoxides 3 and 4 then affords the corresponding optically active sulfones. Thus, as shown in the scheme above, oxidation of sulfoxide (3) with chloroperbenzoic acid gives the (2R)-2,3-dimethylbutyl-p-tolysulfone (5), whereas analogous treatment of sulfoxide (4) gives (2S)-2,3-dimethylbutyl-p-tolysulfone (6).

The preceding reaction sequence provides a novel and efficient method for the preparation of optically active side chain units as their sulfonyl derivatives, which then may be used according to known procedures for the construction of a variety of steroid or vitamin D side chains having a chiral center at C-24. The tolylsulfones (5) and (6) above are new compounds; the corresponding enantiomeric phenylsulfones have been obtained previously by lengthy and elaborate synthesss [Mori et al., Tetrahedron Letters 38, 2099 (1982); Sakakibara et al., Heterocycles 17, 301 (1982); Ferraboschi and Santaniello, Synth. Commun. 14, 1199 (1984); Kocienski et al., J. Chem. Soc. Perkin Trans. 1, 834 (1978)].

For the preparation of the desired 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 analogue, the (2S)-2,3-dimethyl-p-tolylsulfone (6) as obtained by the above procedure is the appropriate side chain unit. Accordingly, compound (6) is reacted with the known 1.alpha.-hydroxyvitamin D-22-aldehyde derivative (structure 7, below, where X.sup.1 and X.sup.2 are hydroxy-protecting groups, e.g. an alkylsilyl group, such as t-butyldimethylsilyl), using the general procedures of Kutner et al., J. Org. Chem. 53, 3450 (1988). This condensation yields the side chain aduct represented by structure (8) below (X.sup.1 and X.sup.2 =hydroxy-protecting group), which is then reduced with a metal amalgam to provide the hydroxy-protected 24-epi-vitamin D.sub.2 derivative, structure (9, X.sup.1 and X.sup.2 -hydroxy-protecting groups). Upon removal of the hydroxy-protecting groups according to standard procedures there is obtained the desired 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 (compound 10, X.sup.1 =X.sup.2 =H). ##STR4##

As shown by the above structures, the process of this invention yields both the free hydroxy compound (10), where X.sup.1 and X.sup.2 are hydrogen, as well as hydroxy-protected derivatives, such as compound (9), where X.sup.1 and X.sup.2 represent an alkylsilyl group. Furthermore, the hydroxy compound (10) can be converted to other derivatives, by the corresponding 1- and/or 3-acyl derivatives, by standard acylation procedures, to provide the compounds of structure (9), where X.sup.1 and X.sup.2 represent acyl groups. Alkylsilyl and acyl derivatives of compound (10) find use in applications where enhanced lipid-solubility is desired.

In this specification and the claims, the term `alkylsilyl` means a trialkylsilicon radical, where each of the alkyl groups may have from 1 to 5 carbons in all isomeric forms. Common examples include trimethylsilyl, triethylsilyl and t-butyldimethylsilyl. The term `acyl` signifies an aliphatic acyl group (alkanosyl group) from 1 to 5 carbons in all isomeric forms (e.g. formyl, acetyl, propionyl, etc.), or an aromatic acyl group, such as benzoyl, or nitro, halo or methyl substituted benzoyl groups.

The process of this invention is more particularly described by the following illustrative examples. In these examples, designation of products or intermediates by Arabic numerals, e.g. 1, 2, 3, . . . etc. refers to the structures so numbered in the preceding description.

Magnesium turnings (0.24 g, 10 mmol) and a crystal of I.sub.2 were placed in a dry flask and covered with 5.0 mL of anhydrous tetrahydrofuran. 1-Bromo-2,3-dimethylbutane (1.54 g, 8 mmol) was added slowly with stirring under nitrogen atmosphere and occasional cooling. The mixture was stirred at room temperature for 1.5 h or until most of the magnesium was consumed. This mixture (containing compound 1) was cooled and 2.35 g (R)-(+)-p-toluenesulfinic acid (-)-menthyl ester (compound 2) (10 mmol) in 10.0 mL of anhydrous tetrahydrofuran was added. The mixture was stirred under nitrogen atmosphere at room temperature for 16 h, cooled and decomposed with saturated NH.sub.4 Cl solution. The organic layer was separated and the squeous phase extracted several times with ether. The combined organic phase was washed with water and brine, dried with MgSO.sub.4, filtered and evaporated. The residue was chromatographed on a 70-270 mesh silica gel column to give 1.26 g of diastereomeric sulfoxide mixture. This was separated by flash chromatography on a 230-400 mesh silica gel column with ethyl acetate and hexane mixtures or by semipreparative HPLC (Zorbax Sil, 9.4.times.25 cm column) using ethyl acetate-hexane mixtures. The first compound to elute was the (S)-(-)-p-tolyl-(2R)-2,3-dimethylbutylsulfoxide (3) and the second compound was the (S)-(-)-p-tolyl-(2S)-2,3-dimethylbutyl sulfoxide (4) MS m/z (relative intensity 224 (M.sup.+, 6), 208 (14), 140 (100), 139 (8), 124 (30), 92 (22), 91 (21), 44 (10), 43 (71), 28 (34), 27 (25); .sup.1 H NMR (CDCl.sub.3) 6 0.80 (3H, d, J=7.0 Hz), 0.89 (3H, d, J=7.0 Hz), 0.98 (3H, d, J=6.5 Hz), 1.6-1.82 (2H, m), 2.42 (3H, s, CH.sub.3 --Ar), 2.71 (2H, m), 7.34 (2H, d, J=15 Hz) (H-aryl ortho), 7.54 (2H, d, J= 15 Hz, H-aryl ortho). (2S) sulfoxide 4 [.alpha.].sub.D.sup.20 =153.5 (c=4 in CHCl.sub.3); (2R) sulfoxide 3sulfoxide [.alpha.].sub.D.sup.20 =-444.8 (c=4 in CHCl.sub.3).

(2S)-2,3-Dimethylbutyl-p-tolylsulfoxide (4) (52 mg, 0.2 mmol) was dissolved in 1.0 mL of anhydrous dichloromethane and 60 mg (0.3 mmol) of 3-chloroperoxybenzoic acid (80-85%, Sigma) added with stirring. The reaction mixture was stirred for 2 h and quenched with 10% sodium bicarbonate. More dichloromethane was added and the combined organic extracts were washed with aqueous sodium sulfite and brine and dried with MgSO.sub.4. The solvent was removed in vacuo and the crude sulfone was purified by silica gel flash chromatography using hexane ethyl acetate mixtures to afford sulfone (6) as a colorless oil. For analytical purposes this was also purified by HPLC (Zorbax Sil 9.4.times.25 cm column) using 10% ethyl acetate in hexane to give 42 mg of pure (2S)-sulfone (6): [.alpha.].sub.D.sup.20 =+17 (c=3.5 in CHCl.sub.3); MS m/z (relative intensity) 240 (M.sup.+, 3), 197 (5), 157 (100), 92 (19), 91 (27), 85 (25), 84 (31), 43 (72); .sup.1 H NMR .delta. 0.77 (3H, d, J=7 Hz), 0.82 (3H, d, J=7.0 Hz), 1.00 (3H, d, J=7.0 Hz), 1.66-1.98 (2H, m), 2.45 (3H, s, CH.sub.3 -Aryl), 2.86 (1H, dd, J=8, 11 Hz), 3.06 (1H, dd, j=4, 12 Hz), 7.35 (2H, d, J=7.0 Hz, H-aryl ortho), 7.75 (2H, d, J=8, H-aryl ortho).

The (2R)-sulfone (5) was prepared by oxidation of sulfoxide 3, using the experimental procedure as described in Example 2 above. The resulting (2R) sulfone (5) showed an optical rotation of [.alpha.].sub.D.sup.20 =-19 (c-1.4, CHCl.sub.3).

1.alpha.-Hydroxy-24-epi-vitamin D.sub.2 (10)

To a stirred solution of 30 mg (125 .mu.mol) of (2S)-2,3-dimethylbutyl-p-tolylsulfone (6) in 300 .mu.L anhydrous tetrahydrofurane (containing 1.10-phenanthroline as an indicator) was added under argon at -78.degree. C. 18 .mu.L (130 .mu.mol) of diisopropylamine followed by 86 .mu.L of a solution of n-BuLi in hexane (1.50M, 130 .mu.mol). The solution was stirred at -78.degree. C. for 15 min (dark brown color), and 4 mg (7 .mu.mol) of the protected aldehyde (7, X.sup.1 =X.sup.2 =t-BuMe.sub.2 Si) in 0.3 mL of anhydrous tetrahydrofurane was added and the mixture stirred under argon at -78.degree. C. for 1 h. The reaction mixture was quenched with 1 mL of saturated NH.sub.4 Cl solution, warmed to 0.degree. C. and extracted with ethyl acetate, and the organic phase was washed with saturated NaCl. The organic phase was dried with MgSO.sub.4, filtered and evaporated. The residue was redissolved in ethyl acetate, passed through a Sep Pak column in ethylacetate and evaporated. The residue was purified by HPLC (Zorbax Sil 9.4.times.25 cm column) using 10% ethylacetate in hexane to give 3.3 mg (58%) of the hydroxysulfones (8, X.sup.1 =X.sup.2 =t-BuMe Si). MS m/z (relative intensity) 8.2 (M.sup.+, 20), 680 (34), 440 (52), 248 (64), 157 (65), 75 (100).

A saturated solution of Na.sub.2 HPO.sub.4 in methanol (1.0 mL) was added to a stirred solution of the 3.3 mg sulfone (8) in 1.0 mL of anhydrous tetrahydrofuran followed by 160 mg of powdered anhydrous Na.sub.2 HPO.sub.4. The mixture was stirred under argon for 15 min, cooled to 0.degree. C. and fresh 5% sodium amalgam (ca. 400 mg) added. The mixture was stirred at 5.degree. C. for 20 h; 5 mL of hexane added and the hexane layer decanted. The solid material was then extracted with 10% ethyl acetate in hexane (3.times.5 mL). The combined organic phase was washed with saturated NaCl and filtered through a Sep Pak cartridge and evaporated. Final purification on HPLC (Zorbax Sil 9.4.times.25 cm column) (10% ethyl acetate in hexane as solvent) gave 1.05 mg (40%) of vitamin D.sub.2 derivative (9, X.sup.1 =X.sup.2 =t-BuMe.sub.2 Si). (As a byproduct, 0.47 mg of the 22-hydroxylated derivative was also obtained.) MS m/z (relative intensity) 640 (M.sup.+, 24), 508 (65), 248 (67), 147 (13), 73 (100), 69 (58); .sup.1 H NMR .delta.0.54 (3H, s, 18-CH.sub.3), 4.19 (1H, m, 3-H), 4.35 (1H, m, 1-H), 4.86 (1H, S, 19Z-H), 5.17 (3H, m, 19E-H and 22-23-H-S), 6.00 (1H, d, J=9.6 Hz, 7-H), 6.23 (1H, d, J=8.8 Hz, 6-H). The hydroxy-protected diol (9, 800 .mu.g) was dissolved in 0.5 mL of anhydrous tetrahydrofuran, and to this solution was added 90 .mu.L 1M solution of tetrabutylammonium fluoride in tetrahydrofuran. The mixture was stirred under argon at 55.degree. C. for 1 h. The mixture was cooled and 5 mL of ether added. The organic phase was washed with saturated NaCl solution and dried over anhydrous MgSO.sub.4, evaporated and redissolved in 20% 2-propanol in hexane and filtered through Sep-Pak. Preparative HPLC (Zorbax-Sil 9.4 mm x 25 cm column) in 20% 2-propanol in hexane gave 308 pg 1.mu.-hydroxy-24-epi-vitamin D.sub.2 (10, X.sup.1 =X.sup.2 =H) 1.alpha.-Hydroxy-24-epi-vitamin D.sub.2 exhibited the following spectral properties: UV (EtOH) .lambda..sub.max : 264 nm, .lambda..sub.min 228; MS m/z (relative intensity) 412 (M.sup.+, 13), 394 (21), 376 (7), 287 (4), 269 (7), 251 (6), 252 (31), 251 (6), 152 (35), 151 (15), 134 (100), 69 (50), 55 (73); .delta.0.49 (3-H, S, 18-CH.sub.3), 0.77 (3-H, d, J=7.1 26 or 27-CH.sub.3), 0.85 (3H, d, J=6.8, 28-CH3), 0.94 (3H, d, J=6.5, 21-CH.sub.3), 4.94 (1H, S, 19Z-H), 5.13 (2H, m, 22 and 23 H) (5.11, 5.13, 5.14), 5.26 (1H, S, 19E-H), 5.99 (1H, d, J=11.2 Hz, 7-H), 6.35 (1H, d, J= 11.2 Hz, 6-H), 4.21 (1H, m, 3-H), 4.41 (1H, m, 1-H). 1.alpha.-Hydroxy-24-epi-vitamin D.sub.2 can be distinguished from the previously known 1.alpha.-hydroxyvitamin D.sub.2 by reverse phase HPLC (4.6 mm.times.25 cm, ODS-Zorbax column) with 15% water in acetonitrile. The first compound to elute in this system was 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 and the second, the known

The new analogue was tested in the vitamin D-deficient rat. These tests indicate that lo-hydroxy-24-epi-vitamin D.sub.2 has a biological activity spectrum that is distinctly different from that of the previously known 1.alpha.-hydroxyvitamin D.sub.2. In Table 1 below, representative assay results are given. These include tests of intestinal calcium transport activity ("S/M ratios"), and of bone mineral mobilization as reflected by serum calcium levels. These assays were conducted according to standard procedures (see e.g., U.S. Pat. No. 4,588,716). The rats used in these assays were made vitamin D-deficient by maintenance on a vitamin D-free, low calcium diet (0.02% Ca, 0 37% P) for 31/2 weeks. They received the test compounds (or vehicle alone; -D control group) 20 h prior to sacrifice.

The data of Table 1 show that the new analogue, 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 exhibits high activity in stimulating intestinal calcium transport being essentially equivalent in this activity to the known 1.alpha.-hydroxyvitamin D.sub.2. In contrast, the new compound exhibits no activity in mobilizing calcium from bone. Thus the new compound, although structurally closely related to the known 1.alpha.-hydroxyvitamin D.sub.2, exhibits a remarkably different activity profile. In stimulating the absorption of calcium, but not its liberation from home, the new analogue is highly suitable as a therapeutic agent for the prevention or treatment of physiological conditions characterized by the loss of bone mass.

The results of Table 1 demonstrate that, in terms of its calcemic action, the novel 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 exhibits a biological activity spectrum similar to that of the known 1.alpha.,25-dihydroxy-24-epi-vitamin D.sub.2. However, further tests showed that the new compound is quite different from the known 24-epi-D.sub.2 derivative in its activity in inducing the differentiation of malignant cells to normal monocytemacrophages. Differentiation activity was assayed using human leukemia cells (HL-60 cells), according to two standard tests, namely the nitroblue tetrazolium reduction (NBT-reduction) and the phagocytosis assays, and as shown in Table 2, the new compound was compared against 1.alpha.,25-dihydroxyvitamin D.sub.3 (a highly potent differentiation agent) and 1.alpha.,25-dihydroxy-24-epi-vitamin D.sub.2.

The assays were conducted as described by Ostrem et al. (J. Biol. Chem. 262, 14164-14171, 1987), and by DeLuca et al. (U.S. Pat. No. 4,717,721). The results given in Table 2 demonstrate that 1.alpha.,25-dihydroxyvitamin D.sub.3 standard has, as expected, remarkable HL-60 cell differentiation activity. Even at doses as low as 10.sup.-8 M, this compound produced approximately 64-67% differentiation in the 4-day trial period in both the NBT-reduction and the phagocytosis assay. 1.alpha.,25-Dihydroxy-24-epi-vitamin D.sub.2 is somewhat less active (about 5 times less active than 1.alpha.,25-dihydroxyvitamin D.sub.3 standard), but also shows very potent activity in this system, e.g. better than 60% differentiation at 5.times.10.sup.-8 M and 80% differentiation at a concentration of 10.sup.-7 M. In contrast, 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 possesses little or no cell differentiation activity. At best, only 16-20% differentiation was observed at a concentration of 10.sup.-7 M, and at a concentration of 1-2.times.10.sup.-8 M, where the 1.alpha.,25-dihydroxy-24-epi-vitamin D.sub.2 compound shows 40-50% differentiation, the new analogue does not elicit a significant differentiation response. Thus, 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 has little or no activity in promoting differentiation of promyelocytes to monocytes. These results show a marked biological difference between the present compound and the previously produced 1,25-dihydroxy-24-epi-vitamin D.sub.2.

Thus the preceding assays demonstrates that the new 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 exhibits a distinct and unique spectrum of activities--namely high potency in stimulating calcium transport, no activity in mobilizing calcium from bone, and little, if any, differentiation activity--which clearly distinguishes the compound from those of the prior art.

The new compound, therefore, represents a valuable addition to the repertoire of useful therapeutic agents, and may be applied advantageously in situations where the specific stimulation of intestinal calcium transport is desired, e.g. diseases such as osteodystrophy or osteoporosis characterized by loss of bone mass.

For treatment purposes, the novel compound of this invention may be formulated as a solution in innocuous solvents, or as an emulsion, suspension or dispersion in suitable solvents or carriers, or as pills, tablets or capsules, together with solid carriers, according to conventional methods known in the art. The compound is advantageously administered by injection, or by intravenous infusion of suitable sterile solutions, or in form of liquid or solid doses via the alimentary canal. Doses of from 1 .mu.g to 50 .mu.g per day of 1.alpha.-hydroxy-24-epi-vitamin D.sub.2 are appropriate for treatment purposes, such doses being adjusted according to the disease to be treated and the response of the subject as is well understood in the art. Since the new compound exhibits specificity of action, it is suitably administered alone, in situations where only calcium transport stimulation is desired, or together with graded doses of another active vitamin D compound--e.g. 1.alpha.-hydroxyvitamin D.sub.2 or D.sub.3, or 1.alpha.,25-dihydroxyvitamin D.sub.3 --in situations where some degree of bone mineral mobilization (together with calcium transport stimulation) is found to be advantageous.