WO1996030338A1 - 1-substituted vitamin d3 analogues - Google Patents

Abstract

Vitamine D3 analogues which include a hydroxyalkyl substituent in the 1-position and a modified D-ring side chain are described. These novel compounds are potent anti-proliferative substances with activity comparable to that of calcitriol but with a vitamin D3 receptor binding rating of approximately 10-3 compared to that of calcitriol.

Description

1-SUBSTITUTED VITAMIN D3 ANALOGUES

BACKGROUND OF THE INVENTION

The present invention relates to novel biologically active vitamin D₃ analogues which include at least one hydroxyalkyl substituent on the A-ring (e.g. in the 1-position). More

specifically, the present invention relates to hybrid compounds YB, JK 276-1, JK 276-2, JK 277-1 and JK 277-2 which are vitamin D₃ analogues each of which includes both a hydroxyalkyl substituent in the 1-position and a modified D-ring side chain. These compounds are potent anti-proliferative vitamin D₃ analogues but have a very low (~10^-3 relative to calcitriol) binding affinity to the calf thymus vitamin D₃ receptor (VDR).

Field of the Invention

Vitamin D₃ analogues have been recognized as having important biological activities. It is known, for example, that vitamin D₃ analogues can be used to control calcium and phosphate

metabolism.

It is also known that such analogues are useful for inducing cell differentiation and for inhibiting undesired cell proliferation. For example, it is well recognized that during normal metabolism vitamin D₃ produces 1α,25-dihydroxy-vitamin D₃ (calcitriol) which is a potent regulator of cell differentiation and proliferation as well as intestinal calcium and phosphorus absorption and bone calcium mobilization. Calcitriol is also known to affect the immune system and this compound, as well as a variety of synthetic vitamin D₃ derivatives have been used in practical, clinical chemotherapy of such diverse human illnesses as osteoporosis, cancer, immunodeficiency syndromes and skin disorders such as dermatitis and psoriasis. However, major research efforts are underway in an effort to prepare vitamin D₃ analogues as drugs in which calcitropic activity is effectively separated from cell growth regulation.

Calcitriol may be structurally represented as follows:

The upper and lower ring portions of calcitriol may be called, for ease of reference, the C/D-ring and A-ring, respectively.

Description of the Related Art

Numerous references can be cited as showing prior work with respect to vitamin D₃ analogues, calcitriol or the like. See, for example:

Vitamin D. Chemical, Biochemical and

Clinical Update, Proceedings of the Sixth Workshop on Vitamin D, Merano, Italy, March 1985; Norman, A.W., Schaefer, K., Grigoleit, H . G . , Herrath, D.V.

Eds.; W. de Gruyter; New York, 1985; Brommage, R.,

DeLucca, H.F., Endocrine Rev., 1985, 6, 491;

Dickson, I., Nature, 1987, 325, 18; Cancela, L.,

Theofon, G., Norman, A.W., in Hormones and Their Actions. Part I; Cooke, B.A., King, R.J.B., Van der Molen, H.J. Eds.; Elsevier, Holland, 1988;

Tsoukas, D.C., Provvedini, D.M., Manolagas, S.C.,

Science, (Washington, D.C.) 1984, 224, 1438;

Provvedini, D.M., Tsoukas, C.D., Deftoe, L. J., Manolagas, S.C., Science, (Washington, D.C.) 1983,

221, 1181; Vitamin D. Chemical Biochemical, and

Clinical Endocrinology of Calcium Metabolism.

Proceedings of the Fifth Workshop on Vitamin D,

Williamsburg, VA, Feb. 1982, Norman, A.W.,

Schaefer, K., Herrath, D.V., Grigoleit, H.G.,

Eds., W. de Gruyter, New York, 1982, pp. 901-940;

Calverley, M.J. in Vitamin D: Molecular,

Cellular, and Clinical Endocrinology, Norman,

A.W., Ed., de Gruyter; Berlin, 1988, p. 51;

Calverley, M. J., Tetrahedron, 1987, 43, 4609.

The entire contents of each reference are hereby incorporated by reference.

Many analogues of calcitriol have been synthesized and evaluated. Among these, all the leading candidates include the 1α-hydroxyl A-ring substituent characteristic of calcitriol, i.e. they differ in the side chain attached to the

D-ring of the steroid framework.

Some calcitriol analogues lacking the

1α-hydroxyl group have also been prepared, e.g. the 1 β-hydroxyl , 1α-fluoro and the 1- -unfunctionalized (i.e. 25-hydroxyvitamin D₃).

However, these have been found to be much less biologically active than calcitriol and other synthesized 1α-hydroxy analogues.

Accordingly, it appears to be axiomatic among workers in the field that the 1α-hydroxy group is essential for desirable biological activity. See, for example, Biochem. Biophys. Res. Commun.. 97:

1031 (1980); Chem. Pharm. Bull., 32:3525 (1984) and Bull. Soc. Chim. France, II:98 (1985), the entire contents of each are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that the A-ring portion of vitamin D₃ analogues can be modified without negatively affecting the biological activity of the resulting compounds. In its broadest aspects, the invention provides vitamin D₃ analogues which include at least one hydroxyalkyl substituent on the ring-A.

It is contemplated that this hydroxyalkyl substituent may be placed on the 1, 2, 3- and/or 4- positions of the A-ring. However, the preferred embodiment of the invention is the vitamin D₃ analogue wherein the 1α-hydroxy group is replaced by a hydroxyalkyl group of, for example, 1-6 carbon atoms.

Structurally, the preferred D₃ analogues of the invention may be shown as follows:

including the stereoisomers thereof, wherein R is

3

-R³OH, R being straight or branched alkyl of 1 to

6 carbons; R¹ is hydrogen and R² represents the substituents completing a vitamin D₃ analogue.

However, also contemplated are the corresponding analogues which include one or more hydroxyalkyl substituents in the 2,3- and/or 4- position in lieu of, or in addition to, the hydroxyalkyl in the 1-position of the ring-A.

It will be appreciated that the D-ring may include the conventional D₃ substitution or any other known modification thereof. See, for

example, the variations shown in Cancer Research, 50:6857-6864 (November 1, 1990), the entire contents of which are incorporated herein by reference.

The preferred compound according to the invention is 1-hydroxymethyl-25-hydroxyvitamin D₃ represented by the formula:

or the formula:

A particularly preferred compound of the present invention is the compound YB represented by the formula:

Compound YB includes a hydroxyalkyl substituent in the 1-position and a modified D- ring side chain. As a result, compound YB demonstrates potent anti-proliferative activity comparable to that of calcitriol but has a VDR binding affinity of ~10^-3 relative to that of calcitriol.

Other particularly preferred compounds according to the invention are

These compounds also demonstrate potent anti-proliferative activity comparable to

calcitriol but have VDR binding affinities of less than 10^-3 relative to calcitriol.

However, as noted, the invention is not to be viewed as limited to these compounds as other hybrid analogues involving the attachment of one or more additional hydroxyalkyl groups on the ring-A, with various other modifications as substituents in the ring-D, are contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the growth inhibition of keratinocyte cell line PE by 1,25-dihydroxy vitamin D₃ and 1-hydroxymethyl homologues at 3μM.

Figure 2A shows the inhibition of TPA-induced ornithine decarboxylase activity by pretreatment with 1,25-dihydroxy vitamin D₃ and 1- hydroxymethyl homologues.

Figure 2B shows a dose-response curve for the inhibition of TPA-induced ODC activity with the 1-hydroxymethyl vitamin D₃ diastereomers (-)-2 and (+)-3.

Figure 3 shows a first example of the dose-response effects of calcitriol, compound YA and compound YB on keratinocyte proliferation. N₀ represents the number of cells at zero hours and N₁ represents the number of cells at 96 hours.

Figure 4 shows a second example of the dose-response effects of calcitriol, compound YA and compound YB on keratinocyte proliferation.

Figure 5 shows a comparison of the effects of 1,25(OH)₂D₃ (calcitriol), compound YA and compound YB on the proliferation of RWLeu-4 human chronic myelogenous leukemic cells as a function of dose. MCW-II5-y-A is compound YA and MCW-II5-y-B is compound YB.

Figure 6 shows a comparison of the effects of 1,25(OH)₂D₃ (filled squares), YA (filled circles) and YB (filled triangles) on thymidine incorporation by human breast cancer cell lines MDA 468 (Figure 6A) and SKBr 3 (Figure 6B).

Figure 7 shows a comparison of the effects of calcitriol (filled circles), JK 276-1 (open triangles), JK 276-2 (filled triangles), JK 277-1 (open squares), and JK 277-2 (filled

squares) on proliferation of keratinocyte cell line PE as a function of dose. Open circles are control cells.

Figure 8 shows a comparison of the inhibitory effects of YA, YB, JK 276-1, JK 276-2 JK 277-1 and JK 277-2 on growth of HL-60 cells. YA and YB were at concentrations of 10^-7 M; the other compounds were at concentrations of 10^-6 M. DETAILED DESCRIPTION OF THE INVENTION

Preferred procedures for preparing the 1-hydroxyalkyl analogues of the invention are shown hereinafter although it will be appreciated that other procedures or modifications thereof can be used and will be evident to those in the art. Thus, the preparation of the two diastereomeric forms of 1-hydroxymethyl-25-hydroxyvitamin D₃, is illustrated, but not limited, by the following reaction Schemes I-III in conjunction with the examples which follow:

The reaction scheme illustrated in

Scheme III hereinafter utilizes methodology described earlier (J. Org. Chem., 56:4339 (1991); Ibid 57:7012 (1992); Tetrahedron Lett., 32:5295 (1991); J. Org. Chem., 55:3967 (1990) and Accts. Chem. Res. , 20 : 72 (1987)), the entire contents of which are hereby incorporated by reference, to prepare ring-A phosphine oxide 11 for

Horner-Wittig coupling with C, D-ring ketone 12 in a convergent approach to the vitamin D₃ family that was pioneered by Lythgoe et al. (J. Chem. Soc., Perkin I, 2608 (1977)), the entire contents of which are hereby incorporated by reference.

The preparation process begins, as shown in Scheme I, using ambiphilic (chameleon-like) 3-bromo-2-pyrone (4) to undergo regiospecific, and stereoselective Diels-Alder cycloaddition with acrolein under sufficiently mild thermal

conditions (70-90°C) to allow isolation on gram scale of the desired, unsaturated, bridged, bicyclic lactone adduct. Because this bicyclic aldehyde was unstable to chromatography, it was immediately reduced and then O-silylated to give chromatographically stable, crystalline, bicyclic, primary alcohol derivative 5 in 46% overall yield. Reductive cleavage of the bridgehead

carbon-bromine bond was achieved in high yield under neutral radical conditions using

tributyltinhydride and azobisisobutyronitrile (AIBN). The halogen-free bicyclic lactone product is the synthetic equivalent of the product derived from 2-pyrone itself cycloadding to acrolein, a Diels-Alder reaction that requires high pressures and that cannot be accomplished simply by heating because of loss of CO₂ from the lactone bridge.

Basic methanolysis of the lactone bridge and in situ conjugation of the carbon-carbon double bond gives the conjugated cyclohexene ester alcohol 6. Resolution of this alcohol 6 is achieved via formation and separation by preparative HPLC and preparative tic of diastereomeric-esters 7a and 7b, derived from enantiomerically pure α- methoxyphenylacetic acid. Analytical HPLC

indicated purified diastereomer 7a to have a diastereomeric excess (d.e.) of 98.8% and 7b of 96.5%. Methanolysis of diastereomeric esters 7a and 7b separately gave back the original alcohol 6 as a pair of enantiomers, 6a and 6b; each

enantiomer was carried on separately.

The absolute stereochemistry of

enantiomer 6a (and therefore also 6b) has been assigned by chemical correlation with a closely related compound of established absolute

configuration (J. Chem. Soc., (C), 2352 (1971), the entire contents of which are hereby

incorporated by reference), as outlined in Scheme II.

Referring back to Scheme I, O-silylation of alcohols 6 gave bis-silyl ethers 8, and then reduction of the conjugated methyl ester

functionality produced allylic alcohol 9. A [3,3] sigmatropic rearrangement using sulfinyl

orthoester allowed efficient, one-flask,

regiospecific formation of 2-carbon-extended conjugated dienoate esters 10 (J. Org. Chem.,

56:6981 (1991), the entire contents of which are hereby incorporated by reference). This mixture of geometric isomers was photochemically

isomerized into the desired Z-10. Based on literature precedent (J. Org. Chem., 51: 3098

(1986), the entire contents of which are hereby incorporated by reference), dienoate esters 10 were reduced, chlorinated, converted into the corresponding phosphines, and finally oxidized to give ring-A phosphine oxides 11 as two enantiomers (11a and 11b) having almost equal but opposite specific rotations of approximately 54°.

Lythgoe-type coupling (J. Chem. Soc., Perkin I, 2608 (1977), the entire contents of which are hereby incorporated by reference) of 60-100 mg of ring-A phosphine oxides 11a and 11b with enantiomerically pure ring-C,D chiron 12 was followed immediately by fluoride-promoted

desilylation to form (-)-1αhydroxymethyl-25-hydroxyvitamin D₃ [(-)-2] and (+)1β-hydroxymethyl-3α,25-hydroxy analogue (+)-3 in good yields

(Scheme III). Two aspects of this coupling should be noted in particular. First, a systematic study of bases used to deprotonate phosphine oxides like 11 (e.g., MeLi, MeLi•TMEDA, n-BuLi, PhLi, LDA) showed PhLi to be best as determined by the yield of the coupled triene product. Second, the scale of the coupling reaction was critical to its success. Thus, while coupling using 60-100 mg of ring-A phosphine oxide proceeded routinely in good yields, coupling on 10-20 mg scale proceeded poorly even if such special precautions were taken such as scrupulous drying of the gaseous nitrogen or argon gas used as the atmosphere above the reaction mixture, scrupulous drying of solvents and reagents, use of molecular sieves, and

azeotroping off any adventitious water by adding and removing benzene from the A and the C,D-ring units repeatedly.

While both 1-hydroxymethyl-25- hydroxyvitamin D₃ diastereomers (-)-2 and (+)-3 demonstrate useful biological activity, it is surprising to find that there are considerable physical difficulties between these diastereomers. For example, whereas 1α-hydroxymethyl diastereomer (-)-2 is easily crystallized, 1β-hydroxymethyl diastereomer (+)-3 is very difficult to

crystallize. This difference in crystallinity offers a significant advantage since a mixture of diastereomers (-)-2 and (+)-3, produced from racemic ring-A phosphine oxide 11 and

enantiomerically pure ring-C,D chiron 12, could be induced to yield crystals of only diastereomer (-)-2. Also, 1α-hydroxymethyl diastereomer (-)-2 demonstrates unexpectedly poor solubility in such organic solvents as methylene chloride, chloroform and methanol. Nevertheless, both hydroxymethyl diastereomers (-)-2 and (+)-3 have extremely similar W and high field ¹H and ¹³C NMR spectra as well as extremely similar chromatographic

properties.

EXAMPLE 1

Bromobicyclic Lactone 5

A 25 mL hydrolysis tube was charged with 1.43 g (8.2 mmol, 1.0 eq.) of 3-bromo-2-pyrone 4, 3.69 g (65.7 mmol, 8.0 eq.) of acrolein, 23.0 mg of barium carbonate and 10 mL of methylene

chloride. This was sealed under nitrogen and warmed to 70-90°C for 91 hours with constant stirring. Examination of an aliquot of the reaction mixture by 400 MHz ¹H NMR indicated that complete formation of a single bicycloadduct had occurred. A stream of nitrogen was then blown over the reaction mixture so as to remove the acrolein.

After holding this under high vacuum, the crude product was diluted with methylene

chloride/diethyl ether (ca. 1:1) and passed through a plug of celite. The solvent was

evaporated to give 3.32 g of a yellow oil which was dissolved in 50 mL ethanol and 20 mL of diglyme and cooled to -78°C (dry ice/acetone) under argon. To this, a solution of 476 mg (12.6 mmol, 1.5 eq.) of NaBH₄ in 8 mL of ethanol was added. After stirring for 30 minutes, the mixture was diluted with methylene chloride. and then 4 mL of saturated aqueous ammonium chloride was added.

After warming to room temperature, this mixture was dried over anhydrous magnesium

sulfate, filtered through a plug of celite, and purified by column chromatography (silica gel, 20% to 50% ethyl acetate/hexane) to afford 1.42 g of a yellow oil which was immediately dissolved in 20 mL of anhydrous methylene chloride under argon and cooled to 0°C. To this 0.75 mL (6.4 mmol, 1.05 eq.) of 2,6-lutidine was added followed by the addition of 1.5 mL (6.5 mmol, 1.07 eq.) of tert-butyldimethylsilyl trif luoromethanesulfonate.

This was stirred for 30 minutes, warmed to room temperature, diluted with methylene chloride, washed with water, the organic portion dried over magnesium sulfate, and the solvent evaporated.

Purification by silica gel column

chromatography (10 to 20% ethyl acetate/hexane) afforded 1.32 g (3.8 mmol, 46%) of the silyloxy bromo bicycloadduct 5 as a white solid (Rf=0.7, 50% ethyl acetate/hexane), mp 100.5-102°C. ¹H NMR (CDCl₃) δ 6.37-6.40 (m, 1H), 6.33 (dd, 8, 5 Hz, 1H), 5.18-5.22 (m, 1H), 3.96 (dd, J=10.1, 3.5 Hz, 1H), 3.65 (dd, J=10.1, 7.1 Hz, 1H), 2.43-2.49 (m, 1H), 2.31-2.37 (m, 1H), 1.91 (ddd, J=13.2, 3.9,

1.3 Hz, 1H), 0.86 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H) ; ¹³C NMR (CDCl₃) 5 169.0, 136.4, 1-30.4, 73.5, 64.3, 62.1, 41.1, 31.2, 25.7 (3C), 18.1, -5.4, -5.5; FT-IR (CHCl₃) 1763 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₁₄H₂₃O₃SiBr 288.9896, found 288.9901. EXAMPLE 2

Hydroxy α, β-Unsaturated Ester 6 (from 5)

To a 25 mL flame-dried round-bottomed flask 179.6 mg (0.52 mmol, 1.0 eq.) of silyloxy bromo bicycloadduct 5, and a total of 0.20 mL of tri-n-butyltin hydride, 15 mg of

azobisisobutyronitrile (AIBN), and 4.0 mL of an hydrous benzene was added and refluxed (placed in a preheated oil bath) for a total of 75 minutes. This was cooled to room temperature and then diluted with wet ether. A few drops of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were added and the mixture stirred for 5 minutes at which time the white precipitate was removed by

filtration through a plug of silica gel with ether. The solvent was evaporated and the

resulting oil placed in a 50 mL f lame-dried round-bottomed flask under argon. The oil was dissolved in 3 mL of anhydrous tetrahydrofuran (THF) and cooled to -45°C. To this, 0.6 mL of a freshly prepared sodium methoxide solution (20 mg of sodium in 4.0 mL of anhydrous methanol) was added and stirred at -45°C for 2.5 hours and then at 25°C for 1 hour. The reaction mixture was diluted with methylene chloride, quenched with saturated aqueous ammonium chloride, dried over anhydrous magnesium sulfate, filtered, and the solvent evaporated. Purification by silica gel chromatography afforded 119.2 mg (0.40 mmol, 77%) of hydroxy ester 6 as a colorless oil (Rf=0.2, 25% ethyl acetate/hexane). ¹H NMR (CDCl₃) δ 6.94 (ddd, J=5, 3, 1 Hz, 1H), 4.20-4.12 (m, 1H), 3.72 (s, 3H), 3.74-3.71 (m, 1H), 3.50 (dd, J=10.0, 8.0 Hz, 1H), 2.90 (bs, 1H), 2.60 (dtdd, J=19.2, 6, 1.6, 1 Hz, 1H), 2.23 (dddd, J=12.4, 4, 2.8, 1.6 Hz, 1H), 2.09 (dddd, J=19.2, 8.8, 3.0, 2.0 Hz, 1H), 1.65 (bs, 1-OH, this signal disappears upon D₂O quench), 1.57 (ddd, J=12.4, 11.2, 6 Hz, 1H), 0.87 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H) ; ¹³C NMR (CD₂Cl₂) δ

167.4, 139.9, 130.5, 65.1, 63.6, 51.8, 38.1, 35.6,

33.8, 26.1 (3C), 18.5, -5.3, -5.4; FT-IR (thin film) 3412, 1716 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₁₅H₂₈O₄Si 243.1053, found 243.1059.

EXAMPLE 3 Hydroxy α, β-Unsaturated Ester 6 (from 7)

A round-bottomed flask was charged with 0.632 g (1.41 mmol) of the diester 7b which was dissolved in 10 mL of tetrahydrofuran and 10 mL of methanol and then cooled to 0°C. To this, 0.20 mL of a freshly prepared sodium methoxide stock solution (32.1 mg of sodium in 5.0 mL of methanol) was added and rapidly stirred for 1 hour and then warmed to room temperature. Rapid stirring was maintained and the progress of the reaction was monitored by TLC. Periodic addition of sodium methoxide stock solution was made until the reaction was complete (ca. 8 hours). Most of the solvent was evaporated and the mixture was diluted with diethyl ether and passed through a two-inch plug of silica gel. Purification by silica gel column chromatography (25% to 75% ethyl

acetate/hexane) gave 0.386 g (1.28 mmol, 91%) of the hydroxy ester (+)- 6a as a colorless oil:

[α]_D ^23°C+59.7° (C = 0.082, CH₂Cl₂, d.e. 98.8%) The same procedure was used for the conversion of 0.900 g (2.01 mmol) of the diester 7b into 0.548 g (1.82 mmol, 91%) of the hydroxy ester (-)- 6b as a colorless oil:

[α]_D ^23°C+59.4º (C = 0.085, CH₂Cl₂, d.e. 98.8%) EXAMPLE 4 α-Methoxyphenylacetic Esters 7a and 7b

To flame-dried 250 mL round-bottomed flask 3.11 g (10.4 mmol of hydroxy ester 6, 2.06 g (12.4 mmol, 1.2 eq.) of (R)-(-)-α-methoxyphenyl acetic acid, 2.45 g (11.9 mmol, 1.15 eq.) of 1,3-dicyclohexylcarbodiimide, and 0.15 g (1.2 mmol, 0.1 eq.) of 4-dimethylaminopyridine were dissolved in 150 mL of anhydrous Et₂O under argon. This reaction mixture was stirred at room temperature for 12 h. The white precipitate was then removed by filtration, the organic layer was washed twice with water, dried over MgSO₄, and the solvent removed by rotary evaporation to leave a very light yellow oil. All impurities were removed from the diastereomeric ester 7a and 7b by silica gel column chromatography (0-20% EtOAc/hexane). The diastereomers were then separated by

preparative normal phase HPLC (4.5%

EtOAc/hexane, 30 mL/min) and by preparative thick layer chromatography (PTLC, multiple elutions with 15% EtOAc/hexane, 1500 μ plates). On a

preparative scale, the diastereomers overlapped on both HPLC and PTLC; therefore, fractions were cut and repurified by numerous injections (ca. 8) and applications, respectively. The diastereomeric excess (d.e.) of fractions was deduced by

analytical normal phase HPLC (7a: RT = 13.4; 7b: RT = 15.1, 1.0 mL/min, 10% EtOAc/hexane). A 1.09 g (2.43 mmol, 23%) sample of 7a (d.e. 98.5%) and a 0.90 g (2.01 mmol, 19%) sample of 7b (d.e. 96.5%) were obtained. A 1.22 g (2.72 mmol, 26%) mixture of 7a and 7b was not adequately separated so as to be used in the subsequent synthetic

transformations. 7a: ¹H NMR (CDCl₃) δ 7.44-7.32 (m, 5H), 6.80 (ddd, J = 4.7, 3.35, 1.1 Hz, 1H), 5.34-5.24 (m, 1H), 4.75 (s, 1H), 3.72 (s, 3H), 3.69 (d, J = 3.4 Hz, 1H), 3.57 (dd, J = 10, 7.2 HZ, 1H), 3.41 (s, 3H), 2.90 (bs, 1H), 2.57-2.51 (m, 1H), 2.20-2.15 (m, 1H), 1.95 (dddd, J = 19.1,

8.1, 3.35, 1.9 Hz, 1H), 1.72 (ddd, J = 12.8, 11.2, 6.0 Hz, 1H), 0.85 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H); ¹³C NMR (CDCl₃) δ 169.9, 166.5, 137.9, 136.1,

130.0, 128.4, 128.3 (2C), 126.9 (2C), 82.4, 67.6, 64.3, 57.1, 51.3, 36.7, 30.9, 29.7, 25.7 (3C),

18.0, -5.7, - 5.8; FT-IR (thin film) 1749, 1716 cm-¹; HRMS, m/z (M+ - t-Bu) calcd for C₂₄H₃₆O₆Si

391.1577, found 391.1580. 7b: ¹H NMR (CDCl₃) δ 7.43-7.31 (m, 5H), 6.88 (ddd, J = 4.75, 3.3 1 Hz, 1H), 5.29-5.21 (m, 1H), 4.73 (s, 1H), 3.71 (s,

3H), 3.64 (dd, J = 9.9, 3.5 Hz, 1H), 3.52 (dd, J = 9.9, 7.1 HZ, 1H), 3.40 (s, 3H), 2.77 (bs, 1H), 2.67 (dddd, J = 19, 6, ≈4.75, 1Hz, 1H), 2.16 (ddd, J = 19, 8, 3.3, 2 Hz, 1H), 2.00 (m, 1H), 1.59

(12.8, 11.0, 6, 1H), 0.81 (s, 9H), -0.03 (s, 3H),

-0.07 (s, 3H); ¹³C NMR (CDCl₃) δ 170.1, 166.7,

138.1, 136.2, 130.3, 128.6, 128.5 (2C), 127.0

(2C), 82.5, 67.8, 64.3, 57.2, 51.5, 36.7, 31.4,

29.6, 25.7 (3C), 18.1, -5.6, -5.7; FT-IR (thin film) 1749, 1716 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₂₄H₃₆O₆Si 391.1577, found 391.1576.

EXAMPLE 5

Bis Silyloxy α,β-Unsaturated Ester 8

In a 50 mL flame-dried round bottomed flask 202.5 mg (0.67 mmol, 1.0 eq.) of hydroxy ester 6 was dissolved in 15 mL of anhydrous

methylene chloride under argon. To this 0.100 mL (0.84 mmol, 1.25 eq.) of 2,6-lutidine was added and stirred for 3 minutes followed by the addition of 0.195 mL (0.84 mmol, 1.25 eq.) of

tert-butyldimethylsilyl trifluoromethanesulfonate. After 30 minutes, the solvent was evaporated and purification by silica gel column chromatography (5 to 10% ethyl acetate/hexane) gave 240.4 (0.58 mmol, 86%) of the silyloxy ester 8 as a colorless oil (Rf=0.6, 10% ethyl acetate/hexane). ¹H NMR (CDCl₃) δ 6.92 (ddd, J=5.2, 2.8, 1 Hz, 1H), 4.15 (m, 1H), 3.72-3.69 (m, 1H), 3.71 (s, 3H), 3.52 (dd, J=9, 8 Hz, 1H), 2.76 (bs, 1H), 2.47 (dtd, J=19.2, ca. 5.2, 1 Hz, 1H), 2.17-2.12 (m, 1H), 2.13-2.05 (dddd, J=19.2, 9, 2.8, 2.0 Hz, 1H), 1.58-1.51 (ddd, J=12.8, 11.2, 2.0, Hz, 1H), 0.88 (s, 9H), 0.87 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H); ¹³C NMR (CD₂Cl₂) δ

167.4, 140.3, 130.4, 65.3, 64.6, 51.7, 38.4, 36.5, 34.6, 26.1 (6C), 18.6, 18.5, -4.4 to -5.3 (4C); FT-IR (thin film) 1716 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₂₁H₄₂O₄Si₂ 357.1917, found 357.1922.

(-)-8 from (-)-6b: [α]_D ^23ºC-46.7° (c = 0.094, CH₂CI₂, d.e. 96.5%)

(+)-8 from (+)-6a: [α]₀ ^23°C-47.1o (c = 0.100, CH₂CI₂, d.e. 98.8%)

EXAMPLE 6 Dienoates E-10 and Z-10

A flame-dried 50 mL round-bottomed flask was charged with 240.4 mg (0.58 mmol, 1.0 eg.) of the silyloxy ester 8, dissolved in 4.0 mL of anhydrous toluene, and cooled to -78°C under argon. To this 1.3 mL (1.2 mmol, 2.2 eq.) of diisobutylaluminum hydride DIBAL-H (1.0 M in hexane) was added and stirred at -78°C for 30 minutes and then at 25°C for 90 minutes. This was quenched with 5 drops of 2 N sodium potassium tartrate, 1.5 mL of water, and diluted with methylene chloride. This was separated, the organic portion dried over anhydrous magnesium sulfate. Purification by silica gel column

chromatography (10 to 25% ethyl acetate/hexane) gave 194.2 mg (0.050 mmol, 87%) of the allylic alcohol 9 as a colorless oil (Rf=0.5, 25% ethyl acetate/hexane) which was immediately used in the preparation of E-10 and Z-10. A 25 mL hydrolysis tube was charged with 184.7 mg (0.48 mmol, 1.0 eq.) of the allylic alcohol 9, a total of 427 mg (1.5 mmol, 3.1 eq.) of 1-phenylsulfinyl-2,2,2-triethoxyethane, 3 mg of 2,4,6-trimethylbenzoic acid, and 9 mL of anhydrous methylene chloride. This was sealed under nitrogen and warmed to 135-145°C for a total of 12.5 hours. After cooling the reaction mixture, the solvent was evaporated and purification by PTLC (3 × 1000 μ, 3% ethyl acetate/hexane) gave 141. 6 mg (0.31 mmol, 65%) of E-10 and 19.9 mg (0.04 mmol, 9%) of Z-10 as oils. Shorter reaction times lead to increased Z/E ratios. E-10: ¹H NMR (CDCl₃) δ 5.84 (t, J=1.4 HZ, 1H), 511 (s, 1H), 4.81 (t, J=1.4 Hz , 1H); ¹³C NMR (CDCl₃) δ 1.664, 158.0, 149.6, 115.4, 111.4, 66.7, 65.2, 59.6, 42.2, 38.4, 36.5, 25.8 (3C), 25.7 (3C), 18.1, 18.0, 14.3, -4.89, -4.94, -5.48, -5.53; FT-IR (thin film) 1716 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₂₄H₄₆O₄Si₂ 397.2230, found 397.2235. Z-10: ¹H NMR (CDCl₃) δ 5.58 (t, 1 Hz, 1H), 4.96- 4.93 (m, 2H), 4.15-4.04 (m, 3H), 3.71 (dd, J=10, 5.0 Hz, 1H), 3.52 (t, 10 Hz, 1H),

2.75-2.68 (m, 1H), 2.44 (ddt, 12.4, 4.0, 1 Hz, 1H), 2.26 (dddd, 12.4, 8.0, 1.6 Hz, 1H), 2.03 (dddd, J=13, 5.6, 4.0, 1.6 Hz, 1H), 1.7 (ddd, 13, 4, 1 Hz, 1H), 1.23 (t, 7.2 Hz, 3H), 0.089 (s, 9H),

0.087 (s, 9H), 0.06 (s, 6H), 0.043 (s, 3H), 0.040 (s, 3H) ; ¹³C NMR (CDCl₃) δ 166.3, 154.2, 145.6, 116.4, 112.3, 67.5, 64.2, 60.0, 47.2, 44.0, 36.9, 25.84 (3C), 25.75 (3C), 18.2, 18.0, 14.0, -4.73, -4.80, -5.42, -5.50; FT-IR (CDCl₃) 1718 cm^-1; HRMS, m/z (M⁺ - t-Bu) calcd for C₂₄H₄₆O₄Si₂ 397.2230, found 397.2231. (-)-E-10 from ( + )-8: [α]_D ^23°C -38.0°

(c=0.094, CHCI₃, d.e. 98.5%)

( + )-E-10 from (-)-8: [α]_D ^23°C -37.2° (c=0.051, CHCI₃, d.e. 96.5%)

EXAMPLE 7 Photoisomerization to dienoate Z-10

A borosilicate test tube was charged with 141.1 mg (0.31 mmol) of dienoate E-10, 9.3 mg of 9-fluorenone, and 9.0 mL of tert-butyl methyl ether. The tube was sealed with a rubber septum, placed in a solution of 2 M sodium orthovanadate and irradiated with a medium pressure mercury arc lamp for 16 hours. This was purified by PTLC (1 × 1000 μ, 1 × 1500 μ, 3% ethyl acetate/hexane) to give 132.3 mg of an inseparable mixture of Z-10 and 9-fluorenone (therefore, the yield of Z-10 would be 123.0 mg (0.27 mmol, 87%); that is, 132.3 mg of starting material minus 9.3 mg of

fluorenone).

EXAMPLE 8 Phosphine oxide 11

A flame-dried round-bottomed flask was charged with 123.0 mg (0.27 mmol, 1.0 eq.

containing 9.3 mg of 9-fluorenone) of Z-10 and 1.5 mL of anhydrous toluene under argon and then cooled to 0°C. To this 0.60 mL (0.60 mmol, 2.2 eq.) of diisobutylaluminum hydride DIBAL-H (1 M in hexane) was added and stirred at 0°C for 35 minutes and then warmed to 25°C. An additional 0.06 ml (0.06 mmol, 0.2 eq.) of DIBAL-H was added and stirred for 2 hours. The reaction mixture was quenched with 0.5 mL of 2 N sodium potassium tartrate, diluted with methylene chloride, separated, and the organic portion dried over an hydrous magnesium sulfate. Purification by PTLC (2 × 1000 μ), (2 elutions) 10% ethyl

acetate/hexane and then 15% ethyl acetate/hexane gave 56.8 mg (0.14 mmol, 51%) of the allylic alcohol as an oil.

A flame-dried 25 mL round-bottomed flask was charged with 90 mg (0.67 mmol, 4.8 eq.) of

N-chlorosuccinimide and dissolved in 1.5 mL of anhydrous methylene chloride and then cooled to 0°C under argon. To this 0.052 mL (0.71 mmol, 5.1 eq.) of dimethyl sulfide was added. The white precipitate that immediately formed was stirred at 0°C for 10 minutes and then at -20°C (dry

ice/ethylene glycol) for 10 minutes. To this a solution of the freshly prepared allylic alcohol in 1.5 mL of anhydrous methylene chloride was added via cannula (the flask containing the alcohol solution was rinsed with 0.5 mL of

anhydrous methylene chloride and this also

transferred to the reaction mixture via cannula). This was stirred at -20°C for 15 minutes and then at 25°C for 50 minutes. The reaction mixture was quenched with water, diluted with methylene chloride, separated, the organic portion dried over anhydrous magnesium sulfate, filtered, and the solvent evaporated. This was passed through a column of florisil with 10% ethyl acetate/hexane to give 46.7 mg (0.11 mmol, 79%) of the allylic chloride. This was then dissolved in 2.0 mL of anhydrous tetrahydrofuran in a flame dried 50 mL round-bottomed flask under argon and to this a freshly prepared tetrahydrofuran solution of lithium diphenylphosphide (Ph₂PLi, this deep orange reactant was prepared by the equimolar addition of n-butyllithium to diphenylphosphine) was added slowly until a yellow color persisted. This was then quenched with 0.5 mL of water, the

tetrahydrofuran evaporated, diluted with 10 mL of methylene chloride, 6 drops of 30% hydrogen peroxide were added, and then rapidly stirred for 10 minutes. This was diluted with methylene chloride, dried over anhydrous magnesium sulfate, filtered, and the solvent evaporated.

Purification by silica gel column

chromatography (5 to 50% ethyl acetate/hexane) afforded 29.3 mg (0.049 mmol, 45%) (18% from Z-10) of the phosphine oxide 11 as a white solid after removal from benzene, (Rf=0.3, 50% ethyl

acetate/hexane), mp 118-122°C. ¹H NMR (C₆D₆) δ

7.83-7.78 (m, 4H), 7.05-7.03 (m, 6H), 5.46 (ddt, J=14.0, 7.6, 1.2 Hz, 1H), 5.42 (d, J=2 Hz 1H), 4.99 (dd, J=2, 1.2 Hz, 1H), 3.95-3.90 (m, 1H), 3.69 (dd, J=10.0, 6.4 Hz, 1H), 3.55 (dd, J=10.0, 8.8 Hz, 1H), 3.32-3.12 (m, 2H), 2.70-2.63 (m, 1H), 2.40-2.33 (m, 1H), 2.26-2.19 (m, 1H), 1.94-1.87

(m, 1H), 1.83 (ddd, J=13, 7.6, 4.8 Hz, 1H), 0.98 (s, 9H), 0.95 (s, 9H), 0.071 (s, 3H), 0.065 (s, 3H), 0.049 (s, 3H), 0.014 (s, 3H) ; ¹³C NMR (C₆D₆) δ 145.4 (d, J = 2.5 Hz), 142.0 (d, J = 12.2 Hz), 132.8 (d, J = 98.0 Hz) 132.7 (d, J = 98.2 Hz),

131.62 (d, J = 2.5 HZ), 131.58 (d, J = 2.6),

130.93 (d, J = 9.2 Hz), 130.88 (d, J = 9.2 Hz), 128.42 (d, J = 11.7 Hz), 128.40 (d, J = 11.6), 114.0 (d, J = 7.8 Hz), 112.6, 67.32, 67.30, 64.1, 46.7, 44.1, 37.4, 31.2 (d, J = 70.9 Hz), 25.8

(3C), 25.7 (3C), 18.0 (2C), -4.8, -4.9, -5.4 (2C); IR (CHCl₃) 3020, 2956, 2930, 2857, 1680, 1472, 1463, 1438, 1255, 1100 cm^-1; MS, m/z (El) 596 (M⁺, 3), 540 (43), 539 (100), 407 (58), 332 (22), 202 (27), 201 (25), 75 (30), 73 (86); HRMS, m/z (M⁺) calcd for C₃₄H₅₃O₃Si₂P 596.3271, found 596.3277.

(-)-11a from (-)-Z-10: [α]_D ^23.5°C-54.0° (c=0.061,

CH₂CI₂, d.e. 98.5%)

(+)-11b from (+)-Z-10: [α]_D ^23.5°C-54.4°(c=0.096, CH₂CI₂, d.e. 96.5%)

EXAMPLE 9 1α-hydroxymethyl-25-hydroxyvitamin D₃ [(-)-2]

A flame-dried 10 mL round-bottomed flask was charged with 79.7 mg (0.13 mmol, 1.9 eq.) of the phosphine oxide (-)-11a and dissolved in 1.0 mL of freshly distilled anhydrous tetrahydrofuran and cooled to -78°C under argon. Phosphine oxide (-)-11a was azeotropically dried with benzene and held under high vacuum for 24 hours immediately prior to use. To this 0.091 mL (0.138 mmol, 2.0 eq.) of PhLi (1.52 M in diethyl ether) was added drop wise over a 5 minute period. A deep

orange-red color persisted after the second drop of the PhLi solution was added. This was allowed to stir an additional 8 minutes at -78°C at which time a precooled (-78°C) solution consisting of 24.3 mg (0.069 mmol, 1.0 eq.) of the CD ring ketone in 0.5 mL of freshly distilled anhydrous tetrahydrofuran was added drop wise via cannula.

The C,D ring ketone 12 was also

azeotropically dried with benzene and held under high vacuum immediately prior to use. The flask containing the C,D ring ketone 12 was rinsed with 0.4 mL of tetrahydrofuran and this was also slowly added to the reaction mixture via cannula. This deep orange-red solution was stirred in the dark at -78°C for 2.5 hours and then warmed to -65°C over 30 minutes. At this temperature, the reaction mixture turned to a light yellow. This was immediately quenched with 0.3 mL of 2 N sodium potassium tartrate followed by the addition of dilute aqueous potassium carbonate. After warming to room temperature, the reaction was diluted with methylene chloride, separated, the organic portion dried over anhydrous magnesium sulfate, and filtered.

Purification by silica gel column

chromatography (5% to 10% ethyl acetate/hexane) afforded 37.9 mg (0.049 mmol, 69%) of the crude coupled product. This was immediately placed in a flame-dried 10 mL round-bottomed flask and

dissolved in 3.0 mL of freshly distilled anhydrous tetrahydrofuran under argon. To this 0.17 mL (0.17 mmol, 3.5 eq.) of tetrabutylammonium

fluoride (1 M in tetrahydrofuran) was added and stirred at 25°C in the dark for 14 hours.

The solvent was evaporated and the crude product passed through a column of silica gel with 5% to 10% methanol/diethyl ether and then purified by PTLC (3 × 1000 μ, 8% methanol/diethyl ether) to afford 17.2 mg (0.039 mmol, 83%) (58% from

(-)-11a) of 1α-hydroxymethyl-25-hydroxyvitamin D₃ ((-)-2). This compound was only sparingly soluble in organic solvents (e.g. MeOH, CHCl₃, CH₂Cl₂). ¹H NMR (CDCl₃) δ 6.32 (d, J=11.2 Hz, 1H), 5.95 (d, J=11.2 Hz, 1H), 5.18 (d, J=2 Hz, 1H), 5.02 (d, J=2 Hz, 1H), 0.93 (d, J=6.4 Hz, 3H), 0.54 (s, 3H). ¹³C NMR (CD₃OD) δ 147.7, 142.6, 136.7, 124.0 119.0, 114.1, 71.5, 67.4, 64.7, 58.0, 57.6, 47.4, 47.0, 46.5, 45.3, 41.9, 37.8, 37.6, 37.5, 30.0, 29.3, 29.1, 28.7, 24.7, 23.3, 22.0, 19.4, 12.3; UV

(EtOH) Λ Max 264 nm; mp 181-184°C. EXAMPLE 10

1β-hydroxymethyl-3β-norhydroxy-3α,25-dihydroxyvitamin D₃ ((+)-3)

This procedure was similar to the one used for the preparation of vitamin (-)-2. The amounts of reagents utilized were as follows:

phosphine oxide (+)-11b: 101.3 mg (0.17 mmol, 2.7 eq.), PhLi (1.52 M in Et₂O) : 0.135 mL(0.21 mmol, 3.3 eq.), C,D ring 12: 22.3 mg (0.063 mmol, 1.0 eq.). This afforded 21.1 mg (0.049 mmol, 76%) of the vitamin (+)-3 as an off white solid. ¹H NMR

(CDCl₃) δ 6,31 (d, J = 11.3 Hz, 1H), 5.94 (d, J = 11.3 Hz, 1H), 5.15 (dd, J = 2.1, 1.0 Hz, 1H), 4.99 (d, J = 2 Hz, 1H), 4.03-3.97 (m, 1H), 3.63-3.55 (m, 2H), 2.832.78 (m, 1H), 2.65-2.57 (m, 1H),

2.30-2.24 (m, 1H), 0.93 (d, J = 9.8 Hz, 3H), 0.5 (s, 3H); ¹³C NMR (CDCl₃) δ 145.4, 143.3, 134.1, 123.7 117.0, 113.9, 71.1, 67.2, 64.4, 56.5, 56.3, 46.3 45.9, 44.5, 40.5, 37.5, 36.4, 36.1, 29.4, 29.2, 29.1, 27.7, 23.6, 22.3, 20.8. 18.8, 11.9; UV (EtOH) Amax 264 nm; mp 118-123°C.

The 1-hydroxymethyl derivatives of the invention have been compared with calcitriol for biological activity. The compounds were tested for growth inhibition of murine keratinocyte cells (cell line PE) and for the inhibition of

TPA-induced ornithine decarboxylase (ODC)

activity.

The cell line PE was derived from a papilloma induced in female SENCAR mice by a standard skin initiation/promotion protocol

(Carcinogenesis, 7=949-958 (1986), the entire contents of which are hereby incorporated by reference) and was chosen for its particular sensitivity to the induction of ornithine decarboxylase (ODC) activity by the extensively characterized tumor promoter TPA. The PE cell line culture medium used in the tests consisted of Eagle's minimal essential medium without calcium chloride supplemented with 8% chelexed fetal calf serum and 1% antibiotic-antimycotic and the addition of CaCl₂ to 0.05 mM Ca⁺⁺.

EXAMPLE 11

Compound YB Four of the currently known analogues of

1,25D3 (calcitriol) that are among the most active inducers of leukemic cell differentiation are shown below.

The potencies, relative to 1,25D3 and determined using HL-60 cells, are shown underneath each compound. Compound KH 1060 was 133 times as effective as 1,25D3 in the induction of leukemic cell (HL-60) differentiation.

Table 1 shows the results of further investigations into the effect of the D-ring side chains on the inhibition of proliferation (Anzano et al., Cancer Research, 54: 1653-1656, 1994;

Vitamin D, Proceedings of the Eighth Workshop on Vitamin D, Paris, France, July 8-10, 1991, Norman et al., eds., W. deGruyter, New York, 1991, the entire contents of which are hereby incorporated by reference); induction of differentiation

(Ostrem et al., J. Biol. Chem., 262: 14164-14171, 1987); and calcemic effects of calcitriol

analogues.

These results led to the synthesis of compound YB having a hydroxymethyl group in the 1- position and the side chain of compound KH 1060 attached to the D ring. The structure of compound YB is represented by the formula:

Preparation of Compound YB

Compound YA and YB were prepared according to the following procedures and as outlined in

Schemes IV, V and VI below. Lythgoe-Inhoffen Diol 3.

A flame-dried 500 mL, three-necked round bottomed flask was charged sequentially with the following: 42 mg (0.5 mmol, 0.07 equiv.) of

NaHCO₃, 22.5 mL of anhydrous MeOH, 100.5 mL of anhydrous CH₂Cl₂, and 3.0 g (7.0 mmol) of

ergocalciferol 1 (Vitamin D₂, [α]²⁵+100°, c=1.5, EtOH). While vigorously stirring, the solution was cooled to -78°C and treated with O₃(O₂ pressure = 7.5 psi) until a deep, blue color developed and persisted (approximately 45-50 min.). The

solution was subsequently flushed with O₂ (7.5 psi) for 10-15 min. until the blue color faded. Upon addition of triphenylphosphine (Ph₃P, 2.6 g, 0.01 mol, 1.4 equiv.), the reaction mixture was allowed to warm to room temperature and stirred for 3 hrs.

Concentration of the solution by rotary evaporator, followed by purification via silica gel chromatography (15% EtOAc/hexane), afforded 851 mg of impure (slowly decomposing, evidenced by TLC and ¹H NMR) "Grundmann Ketone" 2 in 50% yield as a yellow oil (immediately used in preparation of "Lythgoe-Inhoffen Diol" 3 (Rf=0.7, 50%

EtOAc/hexane); ¹H NMR (CDCl₃) δ 9.54 (d, J=2.9 Hz, 1H), 2.5-1.2 (m, 13H), 1.1 (d, J = 6.9 Hz, 3H), 0.62 (3H, s).

A flame-dried 50 mL round-bottomed flask was charged with 851 mg (4.0 mmol) of ketone aldehyde 2, dissolved in 20 mL of anhydrous MeOH, and cooled to 0°C. Solid sodium borohydride was added in a portionwise manner over a period of 30 min. until complete disappearance of starting material was observed by TLC. After stirring for an additional 30 min. at room temperature, the mixture was quenched with water, extracted three times with Et₂O, dried over MgSO₄, filtered, and concentrated by rotary evaporation.

Purification by silica gel chromatography (50% EtOAc/hexane) afforded 498.5 mg (2.4 mmol) of diol 3 in 59% yield as a white solid (R_f = 0.5, 50% EtOAc/hexane); m.p. 108-110°C (Inhoffen et al., Chem . Ber . , 91: 781, 1958, the entire contents of which are hereby incorporated by reference, m.p. 109-110°C); ¹H NMR (CDCl₃) δ 4.05 (m, 1H), 3.62 and 3.59 (2d, J = 3.2 and 3.6 Hz, 1H), 3.34 (dd, J = 6.8, 10.4 Hz, 1H), 1.97 (m, 1H), 1.86-1.77 (m, 3H), 1.59-1.13 (m, 11H), 1.0 (d, J = 6.8 Hz, 3H), 0.93 (s, 3H); ¹³C NMR (CDCl₃) δ 69.1, 67.7, 52.9, 52.3, 41.8, 40.2, 38.2, 33.5, 26.6, 22.5, 17.4, 16 . 6 , 13 . 5 ; FT-IR 4212 , 3621 , 3464 , 3017 , 2943 ,

2871 , 2400 , 1473 , 1458 , 1446 , 1372 cm^-1 . HRMS , (M⁺ ) calcd for C₁₃H₂₄O₂ 2 12 . 1776 , found 212 . 1779 . O-Silylated Aldehyde 5

A flame-dried 50mL round bottomed flask was charged with 344 mg (0.36 mmol, 0.8 equiv.) of RuCl₂(PPh₃)₃ and 10 mL of benzene. Diol 3 (95.2 mg, 0.45 mmol) was taken up in 20 mL of benzene and added portionwise to the stirring RuCl₂(PPh₃)₃ solution. The reaction mixture was stirred at room temperature for approximately 12 h. The benzene was removed by rotary evaporation and the resulting dark green solid was washed with Et₂O (7 × 10 mL). The Et₂O washings were collected and quickly passed through a plug of silica gel. Due to its instability, hydroxy aldehyde 4 was

typically carried on without further purification.

Removal of all traces of ruthenium related compound(s) (i.e., hydridochlorotris- (triphenylphosphine)ruthenium), visually

identifiable as insoluble dark green solids, required proper purification by silica gel column chromatography (10% EtOAc/hexane) and afforded

66.9 mg (0.32 mmol) of hydroxy aldehyde 4 in 71% yield as a yellov; oil (slowly decomposing,

evidenced by TLC and ¹H NMR) Rf=0.45, 50%

EtOAc/hexane), ¹H NMR (CDCl₃) δ 9.55 (d, J =3.2 Hz, 1H), 4.08 (m, 1H), 2.34 (m, 1H), 1.92-1.1 (m,

13H), 1.08 (d, J = 6.8 Hz, 3H), 0.96 (s, 3H); ¹³C NMR (CDCl₃) δ 205.06, 68.80, 51.88, 51.44, 49.06, 42.31, 40.10, 33.55, 26.05, 22.78, 17.32, 13.83, 13.23, FT-IR 4214, 3617, 3020, 2940, 2873, 2715, 1720, 1474, 1458, 1446, 1374 cm^-1.

Hydroxy aldehyde 4 obtained from 520 mg of diol 3 (as described above) and contaminated with trace amounts of unidentified ruthenium

compound(s), was dissolved in 12 mL of

dimethylformamide (DMF) and cooled to 0°C. To this solution was added 0.21 mL (1.8 mmol) of 2,6-lutidine followed by 0.40 mL (1.7 mmol) of tert -butyldimethylsilyl trifluoromethanesulfonate (TBDMS-OTf=Z-OTf). The progress of the reaction was monitored closely by TLC. Further addition of 2,6-lutidine (0.21 mL) and TBDMS-OTf (0.40 mL) was made until the reaction was complete (ca. 2h, 0°C).

The reaction mixture was quenched with 70 mL of water, extracted with Et₂O (3 × 25 mL), the organic portion was dried over MgSO₄, filtered, concentrated by rotary evaporation and immediately purified by silica gel chromatography (100% hexane) to afford 462.1 mg of O-silylated aldehyde 5 as a light yellow oil in 57% yield from diol 3 (Rf = 0.48, 15% EtOAc/hexane). ¹H NMR (CDCl₃) δ 9.57 (d, J = 3.2 Hz, 1H), 4.01 (m, 1H), 2.34 (m, 1H), 1.9-1.1 (m, 12H), 1.08 (d, J = 6.8 Hz, 3H), 0.95 (s, 3H), 0.88 (s, 9H, t-BuSi), 0.004 and - 0.012 (2s, 6H, Me₂Si); ¹³C NMR (CDCl₃) δ 205.34, 69.05, 52.34, 51.66, 49.16, 42.62, 40.40, 34.33,

26.19, 25.78, 23.31, (18.0 - questionable), 17.55, 14.08, 13.33, -4.82, -5.18, FT-IR 4201, 3679, 3014, 2919, 2848, 2705, 2385, 1712cm^-1.

Spectroscopic data corresponds to that previously reported (Fernandez et al., J. Org .

Chem . , 57: 3173-3178, 1992, the entire contents of which are hereby incorporated by reference). Due to the instability of O-silylated aldehyde 5 (as evidenced by TLC and ¹H NMR), storage at -20°C did not exceed a 12h period.

O-Silylated Ketone 6

O₂ was bubbled through a solution of KO-t-Bu (0.43 mL, 0.43 mmol) in dry t-BuOH (0.942 mL, freshly distilled from CaH₂) for 10-15 min. A solution of O-silylated aldehyde 5 (28.5 mg, 0.088 mmol) in 0.54 mL of t-BuOH was added and O₂ was bubbled through the solution for an additional 10 min. followed by N₂ for 15 min.

The solution was quenched with 20 mL of H₂O, extracted with Et₂O (3 × 15 mL), dried over MgSO₄, filtered, concentrated, and chromatographed on a silica gel column (100% hexane) to afford 22.1 mg of O-silylated ketone 6 in 80% yield as a white solid (R_f = 0.8, 20% EtOAc/hexane); m.p. 33-34°C (Fernandez et al., J. Org. Chem . , 57: 3173-3178,

1992, m.p. 34-35°C); ¹H NMR (CDCl₃) δ 4.04 (m, 1H), 2.47 (t, J=8.7 Hz, 1H), 2.09 (s, 3H), 0.87 (s, 9H, t-BuSi), 0.85 (s, 3H), 0.01 and 0.005 (s, 6H, Me₂Si); FT-IR 1700 cm^-1, with physical and

spectroscopic properties corresponding to those reported (Fernandez et al., J . Org . Chem . , 57:

3173-3178, 1992).

Alcohol (20R)-7

A flame dried 25 mL round bottomed flask was charged with 41.8 mg (0.13 mmol) of O-silylated ketone 6, dissolved in 10 mL of anhydrous MeOH, and cooled to 0°C. Solid sodium borohydride (24.6 mg, 0.65 mmol, 5 equiv.) was added portionwise to the solution until the disappearance of all starting material was observed by TLC.

The reaction mixture was quenched with water, extracted with Et₂O (3 × 25 mL), dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography (10% EtOAc/hexane) to afford 26.0 mg of the desired (20R)-7 alcohol epimer and 10.4 mg of the (20S)-7 alcohol epimer (2.5:1) both as light yellow oils in 86% total yield (Rf = 0.8 (20R)-7, 0.7 (20S)-7, 50% EtOAc/hexane); (20R)-7 alcohol: ¹H NMR (CDCl₃) δ 4.01 (m, 1H), 3.74 (m, 1H), 2.02-1.16 (m, 13H), 1.12 (d, J=6.4 Hz, 3H), 1.0 (bs, 3H), 0.88 (s, 9H), 0.01 and -0.007 (2s, 6H); ¹³C NMR (CDCl₃) δ 70.19, 69.16, 59.15, 52.60, 41.95, 40.93, 34.42, 25.82, 24.75, 23.35, 23.25, (18.04 - questionable), 17.54, 14.36, -4.78,

-5.17; FT-IR 3475, 2925, 2975, 2852, 1763, 1487, 1375 cm^-1; (20S)-7 alcohol; ¹H NMR (CDCl₃) δ 4.02 (m, 1H), 3.68 (m, 1H), 2.02-1.16 (m, 13H), 1.25 (S, 3H), 1.20 (d, J = 6.0 Hz, 3H), 0.88 (s, 9H) 0.012 and 0.003 (2s, 6H).

Stereochemical assignments were made by comparing ¹H NMR chemical shift and coupling constant data reported in the literature for similar compounds (see Murayama et al., Chem .

Pharm . Bull . , 34: 4410-4413, 1986, the entire contents of which are hereby incorporated by reference, for assignment of (20S)-7 alcohol; see Wilson et al., Bioorganic & Medicinal Chemistry Letters , 3: 341-344, 1993, the entire contents of which are hereby incorporated by reference, for assignment of (20R)-7 alcohol).

Williamson Ether Coupling Reaction of Alcohol (20R)-7 to Side Chain Bromide Synthon 13

An oven-dried 35 mL round bottomed flask equipped with a magnetic stirring bar was charged with 611 mg of KH (35% suspension in mineral oil). The KH was washed with anhydrous THF (3 × 6 mL), dried in vacuo, reweighed (dry weight = 205 mg,

5.1 mmol, 14.2 equiv.), suspended in 10 mL of anhydrous THF and maintained under an argon atmosphere. A solution of 111.4 mg (0.36 mmol) of alcohol (20R)-7 dissolved in 5 mL of anhydrous THF was added via syringe. After 15 min. upon

addition of alcohol (20R)-7, the solution turned yellow indicating formation of the alkoxide anion; however, the mixture was stirred for 1 h to ensure formation was complete. A solution of side chain bromide synthon 13 (508 mg, 1.8 mmol, 5 equiv.) dissolved in 5 mL of anhydrous THF was added via syringe and the progress of the reaction was monitored closely by TLC.

Upon disappearance of alcohol (20R)-7(1h), the reaction was quenched with H₂O, extracted with Et₂O (3 × 25 mL), dried over MgSO₄, filtered and purified by silica gel column chromatography (0010% EtOAc/hexane) to afford 176 mg (0.34 mmol) of O-silyl protected ether 8 as a light brown oil in 96% yield from (20R)-7 (Rf = 0.75, 10%

EtOAc/hexane); ¹H NMR (CDCl₃) δ 3.99 (m, 1H), 3.54 (m, 2h), 3.24 (dt, J = 6.0, 15.6 Hz, 2H),

3.14-3.08 (m, 2H), 2.11 (dt, J = 2.4, 12.8 Hz,

2H), 1.8-1.1 (m, 14H), 1.03 (q, J = 5.6 Hz, 4H), 0.92 (bs, 3H), 0.87 (s, 9H), 0.79 (dt, J = 2.0, 9.6 Hz, 6H), 0.07 (s, 9H), -0.008 and -0.026 (2s, 6H); ¹³C NMR (CDCl₃) δ 78.67, 77.72, 69.33, 68.92, 57.09, 52.63, 42.02, 40.53, 35.38, 34.63, 31.38, 31.30, 25.81, 24.97, 24.71, 23.24, 18.25, 18.03, 17.6, 14.44, 8.25, 8.12, 2.69, -4.77, -5.17, FT-IR 2954, 2931, 2884, 2860, 1461, 1372cm^-1.

O-Silylated Ketone Ether 11 A flame dried 25 mL round bottomed flask equipped with a magnetic stirring bar was charged with 143 mg (0.28 mmol) of O-silylated ether 8, 7 mL THF, 20 mg of 4 angstrom powdered molecular sieves (oven-dried), and 510 mg (1.9 mmol, 7 equiv.) of tetrabutylammonium fluoride hydrate (TBAF). TBAF was added portionwise at room temperature and the progress of the reaction was monitored closely by TLC. After 4.5 h, two lower runnings spots A and B were observed (in addition to starting material) (Rf = 0.7 (8), 0.4(A), 0.3(B), 10% EtOAc/hexane). The most polar spot (B) corresponded to desired product diol ether 9, whereas A was thought to correspond to the single deprotected tertiary alcohol compound. After stirring at room temperature for 8h, the mixture was refluxed at 70°C for 20 h and full conversion to B (9) was observed.

The reaction mixture was cooled to room temperature, concentrated by rotary evaporation, and purified by silica gel column chromatography (10% EtOAc/hexane) to afford 81.5 mg (0.25 mmol) of diol ether 9 contaminated with a small amount of impurities (as evidenced by TLC and ¹H NMR) as a colorless oil in 89% yield (Rf=0.45, 20%

EtOAc/hexane); ¹H NMR (CDCl₃) δ 4.06 (m, 1H), 3.59 (2t (overlapping), J = 6.4 Hz, 2H), 3.33-3.19 (m, 4H), 246-1.20 (m, 17H), 1.1 (d, J = 6.0 Hz, 3H), 0.85 (t, J = 7.5 Hz, 6H), 0.65 (s, 3H).

A flame dried 25 mL round bottomed flask was charged with 600 mg (2.8 mmol, 11 equiv.) of pyridinium chlorochromate (PCC) and 400 mg of NaAc (4.9 mmol, 20 equiv.). The flask was flushed with Ar and maintained under an Ar atmosphere.

Approximately 40 mL of anhydrous CH₂Cl₂ was added via syringe and the mixture was allowed to stir 5-10 min.

Diol ether 9 (81.5 mg, 0.25 mmol) was

dissolved in 10 mL anhydrous CH₂Cl₂ and added dropwise via cannula; upon its addition the reaction mixture turned a darker shade of orange (orange/brown). Progress of the reaction as followed by TLC showed the reaction was complete after 2h stirring at room temperature.

The solution was filtered through a plug of silica gel, concentrated, and purified by silica gel chromatography (50% EtOAc/hexane) to afford 52.3 mg (0.16 mmol) of hydroxy ketone ether 10 (KH-1060) as an oil in 64% yield (Rf = 0.5, 50% EtOAc/hexane); ¹H NMR (CDCl₃) δ 3.58 and 3.56 (2t (overlapping), J = 6.4 Hz, 2H), 3.33-3.19 (m, 4H), 2.46-1.2 (m, 13H), 1.47 (q, J = 7.6 Hz, 4H), 1.1 (d, J = 6.0 Hz, 3H), 0.85 (t, J = 7.5 Hz, 6H),

0.65 (s, 3H), FT-IR 2968, 2938, 2878, 1706, 1458, 1382 cm^-1. Spectroscopic properties correspond to those reported (Wilson et al., Bioorganic &

Medicinal Chemistry Letters , 35: 3280-3287, 1993).

A flame dried 25 mL round bottomed flask was charged with 50.3 mg (0.15 mmol) of hydroxy ketone ether 10, dissolved in 7 mL of anhydrous CH₂Cl₂ and maintained under an Ar atmosphere,

1-(trimethylsilyl)imidazole (TMS-imidazole, 0.32 mol, 2.2 mmol, 15 eguiv.) was added dropwise via syringe over 5-10 min.

The mixture was stirred at room temperature overnight, quenched with 10 mL H₂O, extracted with EtOAc (3 × 25 mL), dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography (20% EtOAc/hexane) to afford 57.8 mg (0.15 mmol) of O-silylated ketone ether 11 as a light brown oil in quantitative yield (Rf=0.25, 10% EtOAc/hexane); [α]²⁸D-31° (c=2.8 × 10^-3 g/ml, EtOAc); ¹H NMR (CDCl₃) δ 3.57 (dt, J = 6.4, 12.4

Hz, 2H), 3.29-3.10 (m, 4H), 2.5-1.2 (m, 17H), 1.08 (d, J = 6.0 HZ, 3H), 0.81 (dt, J = 3.2, 10.8 Hz, 6H), 0.65 (s, 3H), 0.082 (s, 9H); ¹³C NMR (CDCl₃) δ 212.15, 78.59, 77.49, 68.91, 61.51, 56.84, 49.92, 41.19, 38.91, 35.31, 31.33, 25.07, 24.59, 24.15,

19.39, 18.24, 12.94, 8.27, 8.14, 2.69; FT-IR 2966, 2884, 1702, 1455, 1373, 1067 cm^-1.

The synthesis of O-silylated ketone ether 11 (compound (-)-11 (KH-1060)) is outlined

diagrammatically in Scheme IV below:

Side Chain Bromide Synthon 13

A flame dried 100 mL round bottomed flask was charged with 1.0 g (5.0 mmol) of ethyl

4-bromobutyrate dissolved in 20 mL of anhydrous Et₂O. The solution was cooled down to -78 °C under an Ar atmosphere and 10 mL (20 mmol, 5 equiv.) of ethylmagnesium chloride was added. The reaction mixture was warmed to room temperature, stirred 4 h, quenched with 50 mL of H₂O, extracted with Et₂O (3 × 50 mL), dried over MgSO₄, filtered,

concentrated, and purified by silica gel column chromatography (10-15% EtOAc/hexane) to afford 728.0 (3.0 mmol) of hydroxy bromide 12 in 60% yield. A flame-dried 100 mL round bottomed flask was charged with 728 mg (3.0 mmol) of hydroxy bromide 12 dissolved in 50 mL of anhydrous CH₂Cl₂. To this solution was added 1.6 mL (10.9 mmol, 3.6 equiv.) of 1-(trimethylsilyl)imidazole. The reaction mixture was stirred at room temperature overnight under an Ar atmosphere, quenched with 20 mL H₂O, extracted with CH₂Cl₂ (2 × 25 mL), dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography (100% hexane) to afford 777.2 mg (2.8 mmol) of O-silylated bromide 13 in 92% yield as an oil ¹H NMR (CDCl₃) δ 3.39 (t, J = 6.8 Hz, 2H), 1.84 (m, 2H), 1.47 (m, 6H) 0.81 (t, J = 7.4 Hz, 6H), 0.084 (s, 9H); ¹³C NMR (CDCl₃) δ 78.39, 37.04, 34.77, 31.41, 27.37, 8.25, 2.68.

The synthesis of O-silylated bromide 13 is outlined diagrammatically in Scheme V below:

22-oxa-1-(hydroxymethyl)-26-(hydroxydiethyl) vitamin D3 compounds [(-)-16ya] and [(+)-16yb]

Racemic phosphine oxide 14 (60.4 mg, 0.1 mmol, 1.4 equiv.) was dissolved in 1 mL freshly distilled anhydrous THF and cooled to -78°C under an Ar atmosphere. To this was added 0.062 ml (0.112 mmol, 1.1 equiv.) of PhLi (1.8 M in Et₂O) dropwise over 5 min. during which time a deep red-orange color developed and persisted. The mixture was allowed to stir an addition 7-8 min. at -78°C at which time a precooled (-78°C) solution of C-D ring (-)-11 (26.4 mg, 0.07 mmol, 1.0 equiv.) dissolved in 0.5 mL freshly distilled anhydrous THF was added dropwise via cannula. The flask containing C-D ring(-)-11 was rinsed with an additional 0.5 mL of THF and slowly added to the reaction mixture via cannula. The deep red-orange solution was stirred in the dark for 3.0 h during which time (periodically checked visually) it was observed turning progressively lighter in color until it reached a light yellow color. Upon observation of the light yellow color, the

reaction mixture was immediately quenched at -78°C with 0.3 mL of 2N sodium potassium tartrate followed by addition of dilute aqueous potassium carbonate. After warming to room temperature, the reaction was extracted with EtOAc (3 × 20 mL), the organic portion was dried over MgSO₄, filtered, concentrated, and purified by silica gel column chromatography (7% EtOAc/hexane) to afford 49.1 mg (0.063 mmol) of the crude coupled product in 90% yield from C-D ring (-)-11. This was immediately placed in a flame-dried 25 mL round bottomed flask and dissolved in 10 ml of freshly distilled anhydrous THF under argon. To this was added 70 mg (0.27 mmol, 4.5 equiv.) of solid TBAF and it was stirred at room temperature for approximately 12h in the dark. The solvent was evaporated and the mixture was roughly purified by silica gel column chromatography (100% EtOAc) to afford 5.0 mg (0.01 mmol, 15% from precursor) of a mixture of two diastereomers [(-)-16ya] and [(+)-16yb] slightly contaminated with impurities (as

evidenced by TLC and ¹H NMR). The mixture of diastereomers was subject to HPLC separation (40% MeOH/ Acetonitrile; reverse phase; C₁₈ column, semi-prep, flow rate 1 ml/min., retention times: [(-)-16ya] 21.62 min., [(+)-16yb] 22.83 min.) to give pure diastereomers. Both diastereomers are sparingly soluble in organic solvents (MeOH, CHCl₃, EtOAc, Acetone) and readily "stick" to glass thus often strongly resisting removal with solvents (both organic and inorganic). [(-)-16ya]

[α]^28.8D-81°(c = 0.9 × 10^-3 g/ml, MeOH); ¹H NMR

(CDCl₃) δ 6.32 (d, J = 11.2 Hz, 1H), 5.93 (d, J = 11.2 Hz, 1H), 5.17 (d, J = 2Hz, 1H) 5.01 (d, J = 2.0 Hz, 1H), 4.0-3.88 (m, 1H), 3.6-3.5 (m, 2H), 3.30-3.18 (m, 2H), 2.85-2.77 (m, 1H), 2.67-2.56 (m, 1H), 2.29-2.22 (m, 1H), 2.18-2.12 (m, III), 1.08 (d, J = 6.0 Hz, 3H), 0.84 (dt, J = 2.0, 9.6 Hz, 6H), 0.55 (s, 3H); MS m/e M⁺474; UV (MeOH) λ _max 265 nm. [(+)-16yb] [α]^32.9D+12.5 ° (c = 0.4 × 10-3 g/ml, MeOH); ¹H NMR (CDCl₃) δ 6.32 (d, J = 11.2 Hz, 1H), 5.92 (d, J = 11.6 Hz, 1H), 5.15 (dd, J = 2.1, 1.0 Hz, 1H), 4.99 (d, J = 2.0 Hz, 1H), 4.03-3.97 (m, 1H), 3.63-3.52 (m, 2H), 3.30-3.17 (m, 2H), 2.83-2.78 (m, 1H), 2.65-2.56 (m, 1H), 2.30-2.22 (m, 1H), 2.15 (m, 1H), 1.08 (d, J = 6.0 Hz, 3H), 0.84 (dt, J = 2.0, 9.6 Hz, 6H), 0.52 (s, 3H); MS m/e M⁺ 474; UV(MeOH) λ_max 265 nm.

Preparation for coupling: Two 10 mL round

bottomed flasks were equipped with magnetic stir bars, oven dried for 12 h, cooled in a desiccator, rinsed with benzene and evaporated on a rotary evaporator (3X), and held under high vacuum for 5- 6 h. A 10 mL round bottomed flask was charged with 60.4 mg (0.1 mmol) of racemic phosphine oxide 14 (synthesized as previously reported⁵) which was azeotropically dried with benzene (3 X), sealed with a rubber septum, kept under high vacuum for 5-6h, re-azeotroped with benzene added via syringe through the septum, and kept under high vacuum (0.05 mm Hg) overnight (approximately 12 h). A 10 mL round bottomed flask was charged with 26.4 mg (0.07 mmol) of C-D ring (-)11 which was dried by the procedure described for racemic phosphine oxide 14.

The synthesis of 22-oxa-1-(hydroxymethyl)-26- (hydroxydiethyl)vitamin D3 [(-)-16ya] (compound YA) and [(+)-16yb] (compound YB) are outlined diagrammatically in Scheme VI below:

EXAMPLE 12

Compounds JK 276-1 and JK 276-2

Another currently known analogue of

1,25(OH)₂D₃ which has been proposed as a possible candidate for development as an anticancer agent and/or a drug for the treatment of immune related diseases is 20-epi-23-oxa-24a,24b-dihomo-1α,25-dihydroxyvitamin D3, also called MC 1357:

However, pharmacokinetic studies in rat indicate that this compound has a very short serum half-life, a characteristic that may limit its

effectiveness in therapeutic use (Calverley and Binderup, Bioorganic & Medicinal Chemistry

Letters , 3:1845-1848, 1993, the entire contents of which are hereby incorporated by reference).

These results led to the synthesis of hybrid compounds JK 276-1 and JK 276-2 having a

hydroxymethyl group in the 1-position and the side chain of compound MC 1357 attached to the D ring. The structures of compound JK 276-1 and JK 276-2 are represented by the formulas:

Preparation of Compounds JK 276-1 and JK 276-2

Compounds JK 276-1 and JK 276-2 were prepared according to the following procedures and as outlined in Schemes VII and VIII below. General

Tetrahydrofuran (THF) and diethyl ether (Et₂O) were distilled from benzophenone ketyl prior to use. Methylene chloride (CH₂Cl₂) and triethylamine (NEt₃) were distilled from calcium hydride prior to use. Commercially available anhydrous solvents were used in other instances. All reagents were purchased from Aldrich Chemical Co (Milwaukee, WI) and were used as received without further

purification. FT-IR spectra were recorded using a Perkin-Elmer Model 1600 FT-IR spectrophotometer. The ¹H and ¹³C NMR spectra were recorded on a

Varian XL-400 spectrometer operating at 400 MHz and 100 MHz respectively. Chemical shifts are expressed in parts per million downfield from tetramethylsilane. High resolution mass spectral data were obtained using a VG-70S mass

spectrometer run at 70 eV. Concentrations of optical rotation were given in grams per 100 mL.

20(R)-epimer alcohol (+)-19. The mixture of aldehyde 17 (400 mg, 1.23 mmol) (prepared by following the general procedure of Posner, G.H.; White, M.C.; Dolan, P.; Kensler, T.W.; Yukihiro, S.; Guggino, S.E. Bioorg . & Med . Chem . Lett . 1994, 3 , 2919, incorporated herein by reference), and 40% Bu₄NOH aqueous solution (0.40 mL, 0.62 mmol) in CH₂Cl₂ (6 mL) was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, and

chromatographed on silica gel (1% EtOAc/hexane) with anhydrous NaSO₄ (2 g) on top of the column. This gave 260 mg (0.82 mmol, 65%) of a 2:1 mixture of aldehyde 18 and 17. This mixture was dissolved in THF (5 mL), NaBH₄ (30 mg, 0.79 mmol) was added, followed by dropwise addition of EtOH (4 mL). The resulting reaction mixture was stirred at room temperature for 30 min., quenched with saturated NH₄Cl solution (10 mL), and

extracted with ether. The combined organic phase was washed with brine solution (saturated NaCl), dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was

chromatographed on silica gel (5% EtOAc/hexane) to afford 147 mg (0.45 mmol, 37% overall from 17) of the desirable 20(R)-epimer as an oil: ¹H NMR

(CDCl₃) δ 4.03-3.97 (m, 1 H), 3.71 (dd, J = 10.6 and 3.6 Hz, 1 H), 3.45 (dd, J = 10.6 and 7.2 Hz, 1H), 1.90-1.07 (m, 13 H), 0.94 (d, J = 6.8 Hz, 3 H), 0.93 (s, 3 H), 0.88 (s, 9 H), 0.006 (s, 3 H), -0.007 (s, 3 H); ¹³C NMR (CDCl₃) δ 69.29, 66.83, 53.01, 52.96, 41.91, 40.12, 37.48, 34.39, 26.73, 25.80, 22.86, 18.03, 17.66, 16.60, 14.09, -4.79, -5.16; [α]²³ _D +40.6° (c = 2.80, CH₂Cl₂); IR (CHCl₃, cm^-1), 3628, 2931, 2857, 2360, 1253, 1023, 903, 837, 746, 652; HRMS m/z (M⁺-t-Bu) calcd. for

C₁₉H₃₈O₂Si: 269.1937; Found: 269.1938.

Tosylate-(+)-20.

To a stirred solution of alcohol-(+)-19 (50 mg, 0.15 mmol) in THF (5 mL) at 0°C, was added 1.0 M NaHMDS solution (0.23 mL, 0.23 mmol) in THF, the resulting reaction mixture was stirred at room temperature for 30 min. before TsCl (44 mg, 0.23 mmol) in THF (2 mL) was added via cannula. The resulting reaction mixture was stirred at room temperature for 2 h, quenched with saturated NaHCO₃ solution, and extracted with ether. The combined organic phase was washed with brine solution, dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was

chromatographed on silica gel (10% EtOAc/hexane) to give 58 mg (0.12 mmol, 79%) of (+)-20 as an oil: ¹H NMR (CDCl₃) δ 7.77 (d, J = 8.4 Hz, 2 H), 7.34 (d, J = 8.4 Hz, 2 H), 4.11 (dd, J = 9.4 and 3.2 Hz, 1 H), 3.99-3.93 (m, 1 H), 3.77 (dd, J = 9.4 and 7.2 Hz, 1 H), 1.79-0.94 (m, 16 H), 0.89-0.83 (m, 12 H), 0.81 (s, 3 H), 0.011 (s, 3 H), -0.028 (s, 3 H); ¹³C NMR (CDCl₃) δ 144.49, 133.13, 129.66, 127.88, 74.25, 69.12, 52.68, 52.55, 41.74, 39.85, 34.66, 34.18, 26.58, 25.76, 22.67, 21.59, 17.97, 12.97, 17.50, 16.64, 14.00, -4.83, -5.20; [α]²³ _D +16.7° (c = 2.90, CH₂Cl₂); IR (CHCl₃, cm^-1) 2931, 2857, 1359, 1189, 1176, 922, 902, 838, 744, 652; HRMS m/z (M⁺-t-Bu) calcd. for C₂₆H₄₄O₄SSi:

423.2025; Found: 423.2027.

CD ring diol-(+)-21

To stirred solution of alcohol-(+)-19 (50 mg, 0.15 mmol) and 18-crown-6 (122 mg, 0.46 mmol) in THF ( 4 mL) at room temperature, was added 1.0 M KO-t-Bu solution (0.40 mL, 0.40 mmol) in t-BuOH, the resulting reaction mixture was stirred at room temperature for 2 h before bromo side-chain (156 mg, 0.62 mmol) (Calverley, M.J.; Binderup, L.

Bioorg . & Med . Chem . Lett . 1993, 3 , 845,

incorporated herein by reference) in THF (3 mL) was added via cannula. The resulting reaction mixture was stirred for 3 h at room temperature, quenched with saturated NH₄Cl solution, and

extracted with ether. The combined organic phase was washed with brine solution, dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (1% EtOAc/hexane) to give 65 mg (0.13 mmol, 85%) of the O-alkylated intermediate. This was dissolved in THF (4 mL), NEt₃ (0.5 mL) was added, followed by 1.0 M TBAF solution (1.2 mL, 1.2 mmol) in THF. The resulting reaction mixture was refluxed for 3 days, cooled to room temperature, and chromatographed on silica gel (30%

EtOAc/hexane) to afford 34 mg (0.11 mmol, 71% overall) of the desired diol as an oil: ¹H NMR (CDCl₃) δ 4.38-4.02 (m, 1H), 3.48 (dd, J = 9.2 and 4.0 Hz, 1H), 3.43-3.35 (m, 2 H), 3.12 (dd, J = 9.2 and 7.6 Hz, 1 H), 2.85-2.68 (br s, 1 H); 1.85-1.05 (m, 23 H), 0.93-0.85 (m, 6 H); ¹³C NMR (CDCl₃) δ 76.68, 74.98, 71.42, 70.03, 69.06, 53.50, 52.43, 41.60, 41.03, 39.67, 35.32, 33.48, 29.35, 29.14, 26.52, 24.70, 22.28, 17.39, 17.18, 13.81; [α]²³ _D +9.67° (c = 1.50, CH₂Cl₂); IR (CHCl₃, cm^-1) 3616, 2937, 2871, 2244, 1455, 1375, 1099, 926, 899, 757, 727, 708; HRMS m/z (M⁺) calcd. for C₁₉H₃₆O₃:

312.2664; Found: 312.2670.

CD ring-(-)-22.

The mixture of alcohol-(+)-21 (50 mg, 0.16 mmol), PCC (69 mg, 0.32 mmol) and dry celite (70 mg) in CH₂Cl₂ (2 mL) was stirred for 2 h at room temperature. The resulting mixture was passed through silica gel (4 g) eluting with 1:1 mixture of hexane/ether (20 mL). Evaporation of solvents under reduced pressure afforded the crude ketone intermediate. This was dissolved in CH₂Cl₂ (0.5 mL), TMS-imidazole (112 mg, 0.80 mmol) was added. The resulting reaction mixture was stirred

overnight at room temperature, quenched with water, and extracted with ether. The combined organic phase was washed with brine solution, dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was

chromatographed on silica gel (10% EtOAc/hexane) to provide 58 mg (0.15 mmol, 94%) of the O-silylated CD ring ketone as an oil: ¹H NMR (CDCl₃) δ 3.44-3.29 (m, 3 H), 3.21 (dd, J = 9.2 and 6.4 Hz, 1 H), 2.44 (dd, J = 11.6 and 7.6 Hz, 1 H), 2.30-2.15 (m, 2 H), 2.04-1.30 (m, 14 H), 1.20 (s, 6 H), 0.94 (d, J = 6.4 Hz, 3 H), 0.64 (s, 3 H), 0.087 (s, 9 H); ¹³C NMR (CDCl₃) δ 211.91, 74.85, 73.68, 71.46, 61.84, 53.54, 49.69, 41.25, 40.88,

38.08, 35.54, 29.84, 29.81, 26.67, 24.76, 23.94

18.91, 17.24, 12.84, 2.58; [α]²³ _D -40.0 (c = 1.60, CH₂Cl₂); IR (CHCl₃, cm^-1) 3154, 2967, 2876, 2284, 2239, 1790, 1698, 1250, 1036, 840; HRMS m/z (M⁺) calcd. for C₂₅H₄₀O₂SSi: 432.2518; Found: 432.2515.

CD ring diol-(-)-23.

To a stirred solution of m-(1'.1'-dimethylhydroxy-methyl)thiophenol (105 mg, 0.62 mmol) (see Grue-Sorensen, G.G.; Binderup, E.;

Binderup, L.; in Vitamin D, A Pluripotent Steroid Hormone : Structural Studies, Molecular

Endocrinology and Clinical Applications, eds.

Norman, Boullion and Thomasset, 1994, Walter de Gruyter, New York, 1993, pp. 75-76, incorporated herein by reference) in DMF (6 mL), was added 1.0 M KO-t-Bu solution (0.62 mL, 0.62 mmol) in THF, the resulting solution was stirred for 2 h at rt before tosylate-(+)-20 (150 mg, 0.31 mmol) in THF (4 mL) was added via cannula. The resulting reaction mixture was stirred at room temperature overnight, quenched with saturated NH₄Cl solution, and extracted with ether. The combined organic phase was washed with brine solution, dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was chromotagraphed on silica gel (5% EtOAc/hexane) to afford the S-alkylated intermediate. This was dissolved in THF (4 mL), NEt₃ (0.5 mL) was added, followed by 1.0 M TBAF solution (2.0 mL, 2.0 mmol) in THF. The resulting reaction mixture was refluxed for 3 days, cooled to room temperature, and

chromatographed on silica gel (50% EtOAc/hexane) to provide 87 mg (0.22 mmol, 75% ) of (-)-23 as an oil: ¹H NMR (CDCl₃) δ 7.41-7.37 (m, 1 H), 7.21-7.08 (m, 3 H), 4.00-3.94(m, 1 H), 3.54 (dd, J = 12.4 and 3.6 Hz, 1 H), 2.61 (dd, J = 12.4 and 8.8 Hz, 1 H), 2.09-2.04 (br s, 2 H), 1.86-1.12 (m, 19 H), 0.94 (d, J = 6.4 Hz, 3 H), 0.80 (s, 3 H); ¹³C NMR (CDCl₃) δ 149.76, 137.34, 128.53, 127.15, 125.20, 121.83, 72.88, 69.05, 55.74, 52.32, 41.75,

40.48, 40.19, 34.66, 33.40, 31.61, 26..68, 22.23,

18.80, 17.40, 13.93; [α]^{2 3} _D -22.3 (c = 2.00, CH₂Cl₂); IR (CHCl₃, cm^-1) 3614, 2933, 2872, 2248,

1471, 1175, 909, 894, 746, 712, 649; HRMS m/z (M⁺) calcd. for C₂₂H₃₄O₂S: 362.2280; Found: 362.2276.

CD ring-(-)-24.

The mixture of alcohol-(-)-23 (50 mg, 0.13 mmol), PCC (39 mg, 0.18 mmol), NaOAc (30 mg) and dry celite in CH₂Cl₂ (5 mL) was stirred at 0°C for 40 min. The reaction mixture was passed through silica gel (5 g) eluting with 1:1 mixture of hexane/ether (30 mL), evaporation of solvents under reduced pressure gave the ketone

intermediate. The ketone intermediate was

dissolved in CH₂Cl₂ (0.5 mL) and TMS-imidazole (109 mg, 0.78 mmol) was added. The resulting reaction mixture was stirred overnight at room temperature, quenched with water, and extracted with ether. The combined organic phase was washed with brine solution, dried over MgSO₄, and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel (10% EtOAc/hexane) to provide 40 mg (0.09 mmol, 67%) of (-)-24 as an Oil: ¹H NMR (CDCl₃) δ 7.47-7.43 (m, 1 H), 7.25-7.16 (m, 3 H), 3.18 (dd, J = 12.0 and 3.6 Hz, 1 H), 2.79 (dd, J = 12.0 and 8.0 Hz, 1 H), 2.46 (dd, J = 11.6 and 7.6 Hz, 1 H), 2.30-1.31 (m, 18 H), 1.05 (d, J = 6.8 HZ, 3 H), 0.58 (s, 3 H), 0.094 (s, 9 H); ¹³C NMR (CDCl₃) δ 211.48, 150.80,

136.50, 128.32, 127.31, 125.97, 122.44, 74.99, 61.57, 55.28, 49.65, 40.94, 40.76, 38.60, 34.66, 32.42, 32.24, 26.58, 23.91, 18.80, 18.76, 12.87, 2.36; [α]²³ _D -53.4° (c = 1.05, CH₂Cl₂); IR (CHCl₃, cm^-1) 2965, 2254, 1706, 1382, 1252, 1219, 1040, 910, 842, 781, 774, 651. HRMS m/z (M⁺-CH₃) calcd. for C₂₂H₄₂O₂SSi: 367.2668; Found: 367.2676.

Synthesis of calcitriol analogs JK276-1 (26-1) and JK276-2 (26-2)

Referring to Scheme VIII, a solution of 53 mg (0.11 mmol, 1.5 equiv.) of phosphine oxide (±)-25 (Posner, G. H.; Nelson, T. D.; Guyton, K. Z.; Kensler, T. W. J. Med . Chem . 1992, 35, 3280- 3287) in 1 mL of anhydrous THF was cooled to - 78° C and treated dropwise under argon with 110 μL (0.11 mmol, 1.5 equiv.) of 1 M solution of phenyllithium in THF. The resulting orange solution was stirred for 30 min at -78° C. To the solution, was added a solution of 25.3 mg (0.066 mmol, 1 equiv.) of C,D-ring 22 in 0.5 mL of anhydrous THF dropwise. After being stirred for 1 h at the same temperature, the reaction mixture was allowed to warm up to room temperature for 10 h, quenched with 2 mL of a 1:1 mixture of 2 N sodium potassium tartrate and 2 N K₂CO₃, extracted with EtOAc (30 mL × 2) and washed with brine (15 mL × 2). The combined organic portion was dried with anhydrous MgSO₄, concentrated in vacuo and then purified by silica gel column chromatography (3 %

EtOAc/hexane) to afford 44.4 mg (0.058 mmol, 88 %) of the coupled product as a colorless oil. The silyl ethers were dissolved in 2 mL of anhydrous THF. To the solution were added 0.35 mL (0.35 mmol, 6 equiv.) 1 M tetrabutylammonium fluoride solution in THF, and 50 μL (0.35 mmol, 6 equiv.) of triethylamine. After 12 h at room temperature, the mixture was extracted with EtOAc (30 mL × 2) and washed with brine (15 mL × 2). The combined organic portion was dried with anhydrous MgSO₄, concentrated in vacuo and then purified by silica gel column chromatography (EtOAc / MeOH / NEt₃) to afford 25.5 mg (0.055 mmol, 95 %) of mixture of two diastereomers as a viscous colorless oil. The diastereomers were separated by reverse phase HPLC (C-18 semipreparative column, 50 % MeCN / H₂O, 3 ml / min) to afford 8.5 mg (26.8 %) of 26-1 ( 1α, 3β, RT=38.9 min ) as a white solid, and 11.5 mg (42.8 %) of 26-2 (1β, 3α, RT=46.0 min) as a colorless oil. Rf=0.39 (3 % MeOH / EtOAc). (-)-26-1; [α ]²⁸ _D -131° (c=2 mg/ mL, CHCl₃); mp 129° C; ¹H NMR (400 MHz, CDCl₃) δ 6.32 (d, J =11.2 Hz, 1H), 5.95 (d, J =11.2 Hz, 1H), 5.18 (dd, J =1.6, 0.8 Hz, 1H), 5.02 (d, J =2 Hz, 1H), 3.99-3.93 (m, 1H), 3.54-3.52 (m, 1H), 3.50 (dd, J =5.2, 4 Hz, 1H), 3.47-3.36 (m, 2H), 3.20 (dd, J =9.2, 7.6 Hz, 1H), 2.83 (dd, J =12.4, 4 Hz, 1H), 2.67-2.59 (m, 2H), 2.56(br s,

OH), 2.26 (dd, J =12.0, 9.6 Hz, 1H), 2.00-1.21 (m, 21H), 1.22 (s, 6H), 0.95 (d, J =6.4, 3H) 0.55 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 145.11, 142.79, 134.01, 123.71, 117.14, 114.56, 75.34, 71.58, 70.14, 67.15, 64.32, 56.14, 53.56, 46.36, 45.69, 45.07, 41.19, 39.73, 37.43, 36.14, 29.48, 29.26, 28.98, 26.77, 24.81, 23.54, 22.12, 17.29, 12.37; IR (CHCl₃, cm^-1) 3607, 3398, 3008, 2934, 2874, 1644, 1453, 1377, 1247, 1100, 1036; UV (MeOH) λ_max 266 nm (e=66,000); MS m/z (70 eV, El) 460 (10.3 %, M⁺), 148(100 %); HRMS m/z (M⁺ ) Calcd. for C₂₉H₄₈O₄ 460.3553, found 460.3556. (+)-26-2 (1β, 3α); [α ]²⁸ _D + 45° (c=1 mg/ mL, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 6.30 (d, J =11.2 Hz, 1H), 5.94 (d, J =11.2 Hz, 1H), 5.14 (d, J =1.2 Hz, 1H), 4.98 (d, J =2 Hz, 1H), 4.03-3.97 (m, 1H), 3.64-3.54 (m, 1H), 3.50 (dd, J =5.2, 4 Hz, 1H), 3.46-3.36 (m, 2H),

3.50 (dd, J =9.2, 7.6 Hz, 1H), 2.82 (dd, J =12.4, 4.0 Hz, 1H), 2.65-2.57 (m, 2H), 2.56(br s, OH), 2.27 (dd, J =12.8, 6.8 Hz, 1H), 2.00-1.25 (m, 21H), 1.22 (s, 6H), 0.94 (d, J =6.8, 3H) 0.53 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 145.33, 142.84,

134.28, 123.57, 117.07, 113.84. 75.33, 71.55, 70.15, 67.09, 64.29, 58.09, 53.47, 46.23, 45.63, 44.46, 41.16, 39.73, 37.36, 36.07, 29.45, 29.24, 28.97, 26.81, 24.79, 23.43, 22.05, 17.28, 12.32; IR (CHCl₃, cm^-1) 3613, 3402, 3014, 2943, 2873,

1602, 1467, 1350, 1238, 1114; UV (MeOH) λmax 262 nm (∈=16,000); MS m/z (70 eV, EI) 460 (11.43 %, M⁺), 148(100 %); HRMS m/z (M^*) Calcd. for C₂₉H₄₈O₄

460.3553, found 460.3552.

Example 13

Compounds JK 277-1 and JK 277-2

Another currently known analogue of 1,25(OH)₂D₃ which shows promise of high

antiproliferative and low calcemic activity, designated GS 1500 (see Grue-Sorensen et al., in Vitamin D, A Pluripotent Steroid Hormone:

Structural Studies, Molecular Endocrinology and Clinical Applications, ed. Norman Boullion and Thomasset, 1994, Walter de Gruyter, New York, pp 75-76, which is hereby incorporated by reference) is represented by the formula:

In this compound the configuration of 1,25(OH)₂D₃ is reversed at C-20, the 23 methylene group is replaced by sulfur, and the 24 methylene group is replaced by a meta-substituted phenyl ring.

Compounds JK 277-1 and JK 277-2 are further structural modifications of 1,25(OH)₂D₃, having a hydroxymethyl group in the 1-position and the side chain of compound GS 1500 attached to the D ring. The structures of compound JK 277-1 and JK 277-2 are represented by the formulas:

Preparation of Compounds JK 277-1 and JK 277-2 Compounds JK 277-1 and JK 277-2 were prepared according to the following procedures and as outlined in Schemes VII and IX. Referring to Scheme IX, a solution of 30 mg (0.06 mmol, 1.5 equiv.) of phosphine oxide (±)-25 in 0.7 mL of anhydrous THF was cooled to - 78° C and treated dropwise under argon with 63 μL (0.06 mmol, 1.5 equiv.) of 1 M solution of phenyllithium in THF. The resulting orange solution was stirred for 30 min at -78° C. To the solution, was added a solution of 19.0 mg (0.044 mmol, 1 equiv.) of C,D- ring 24 in 0.5 mL of anhydrous THF dropwise.

After being stirred for 1 h at the same

temperature, the reaction mixture was allowed to warm up to room temperature for 10 h, quenched with 2 mL of a 1:1 mixture of 2 N sodium potassium tartrate and 2 N K₂CO₃, extracted with EtOAc (30 mL × 2) and washed with brine (15 mL × 2). The combined organic portion was dried with anhydrous MgSO₄, concentrated in vacuo and then purified by silica gel column chromatography (3 % EtOAc / hexane) to afford 27.8 mg (0.058 mmol, 78 %) of the coupled product as a colorless oil. The silyl ethers (60.0 mg, 0.074 mmol) were dissolved in 3 mL of anhydrous THF. To the solution were added 0.44 mL (0.44 mmol, 6 equiv) 1 M

tetrabutylammonium fluoride solution in THF, and 65 μL (0.35 mmol, 5 equiv.) of triethylamine.

After 12 h, the mixture was extracted with EtOAc (30 mL × 2) and washed with brine (15 mL × 2). The combined organic portion was dried with anhydrous MgSO₄, concentrated in vacuo and then purified by silica gel column chromatography (EtOAc / MeOH /NEt₃) to afford 37.0 mg (0.073 mmol, 98 %) of mixture of two diastereomers as a viscous

colorless oil. The diastereomers were separated by reverse phase HPLC (C-18 semipreparative column, 60 % MeCN / H₂O, 3 ml / min) to afford 9.5 mg (31.7 %) of 27-1 ( 1α, 3β, RT=28.5 min ) as a white solid, and 14.9 mg (42.8 %) of 27-2 (1β, 3α,

RT=35.5 min) as a colorless oil. Rf=0.40 (3 % MeOH / EtOAc). (-)-27-1 (1α, 3β); [α ]²⁸ _D -127° (c=1.4 mg/ mL, CHCl₃); mp 148 C; ¹H NMR (400 MHz, CDCl₃) δ 7.48-9-7.49 (m, 1H), 7.27-7.23 (m, 3H), 6.31 (d, J =11.6 Hz, 1H), 5.95 (d, J =11.6 Hz, 1H), 5.17

(dd, J =2.0, 0.8 Hz, 1H), 5.01 (d, J =1.2 Hz, 1H), 3.99-3.92 (m, 1H), 3.57-3.54 (m, 2H), 3.26 (dd, J =12.4, 3.6 Hz, 1H), 2.83-2.79 (m, 1H), 2.75 (dd, J =12.0, 8.4 Hz, 1H), 2.66-2.58 (m, 2H), 2.25 (dd, J =12.0, 9.6 HZ, 1H), 2.03-1.28 (m, 20H), 1.04 (d, J

= 6.8, 3H) 0.52 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 149.77, 145.14, 142.50, 137.49, 134.17, 128.64, 127.24, 125.21, 123.68, 117.27, 114.52, 72.45, 67.13, 64.31, 56.03, 55.74, 46.34, 45.80, 45.01, 40.82, 40.27, 37.41, 35.41, 31.71, 28.95, 26.84,

23.57, 22.07, 18.89, 12.46; IR (CHCl₃, cm^-1) 3605,

2934, 1642, 1379, 1100, 1036, 914; UV (MeOH) λ_max

258 nm (∈=23,000). MS m/z (70 eV, EI) 510 (45.2 %, M), 148 (100 %). (-)-27-2 (1β,3α); [α ]²⁸ _D -16 (c=11.9 mg/ mL, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ

7.48-9-7.49 (m, 1H), 7.27-7.18 (m, 3H), 6.30 (d, J =11.2 Hz, 1H), 5.94 (d, J =11.2 Hz, 1H), 5.15 (dd, J =1.2, 1H), 4.98 (d, J =2.0 Hz, 1H), 4.03-3.97 (m, 1H), 3.61-3.58 (m, 2H), 3.25 (dd, J =12.4, 3.6 Hz, 1H), 2.84-2.79 (m, 1H), 2.74 (dd, J =12.4, 8.8 HZ, 1H), 2.66-2.57 (m, 2H), 2.27 (dd, J =12.6, 8.0 HZ, 1H), 2.03-1.33 (m, 20H), 1.04 (d, J =6.8, 3H)

0.49 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 149.77, 145.33, 142.58, 137.49, 134.41, 128.63, 127.23, 125.22, 123.52, 117.18, 113.86, 72.43, 67.09, 64.29, 56.00, 55.68, 46.22, 45.74, 44.47, 40.82, 40.29, 37.36, 35.37, 31.71, 28.95, 26.89, 23.46,

22.00, 18.88, 12.42; IR (CHCl₃, cm^-1) 3604, 3015, 2932, 1590, 1452, 1381, 1036; UV (MeOH) λ_max 258 nm (∈=53,000); MS m/z (70 eV, EI) 510 (68.8 %, M⁺), 135 (100 %); HMRS m/z (M⁺) Calcd. for C₃₂H₄₆O₃S 510.3168, found 510.3163.

Growth Inhibition Test

The growth inhibition test was carried out as follows:

Growth curves for PE cells treated with calcitriol and its 1-hydroxymethyl homologues were generated by assay for the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) according to the method described by Carmichael et al., Cancer Res., 47:936-942 (1987), the entire contents of which are hereby

incorporated by reference. A mitochondrial dehydrogenase reduces MTT to a blue formazan product with an absorbance maximum of 505 nm in DMSO; the number of viable cells can thus be determined spectrophotometrically.

PE cells were seeded at a density of 5,000 cells/well in 50 μl medium into 96-well microtiter plates. Twelve hours later, the medium was removed, and cells were treated with 100 μl fresh medium into which the appropriate amount of vitamin D₃ or derivative dissolved in dimethyl sulfoxide (DMSO) had been added, with the

concentration of DMSO held constant at 0.1%. The plates were fed once at 48 hours, with the

readdition of the vitamin D₃ analogues at this time. At 24 hour intervals following the initial treatment of the cells with compounds, 0.1 mg (50 μg of a 2 mg/ml solution) of MTT was added to the plates. After 4 hours, the MTT was removed and DMSO added to dissolve the blue formazan dye.

Using a microtiter plate reader, the A₅₀₅ was then determined and cell number calculated from

blank-subtracted absorbance values. Results from the MTT assay for the inhibition of cell growth were independently confirmed by treating 100 cm² dishes of cells in an analogous manner for 96 hours, whereupon the cells were harvested by trypsinization and counted. Further, the

viability of the cells treated with vitamin D₃ or derivatives was determined to be identical to control cells at 96 hours by try pan blue

exclusion.

Inhibition of TPA-induced ODC Activity

100 cm² dishes of PE cells were treated with vitamin D₃ or analogues dissolved in DMSO by direct addition into the culture medium. Fifteen minutes later, the plates were treated with 100 ng/ml TPA dissolved in ethanol. For both

additions, the solvent concentration was held constant at 0.1%, and control values represent the results from plates treated with these solvents. Three plates were used for each experimental group.

Following incubation for 4 hours after

addition of TPA, the medium was removed and the dishes washed with ice cold phosphate-buffered saline (PBS). The excess PBS was then removed and the dishes rinsed with an ice cold solution of pyridoxal phosphate in PBS (50 μg/ml). The excess liquid was removed, and the dishes were frozen at -80°C. The dishes were scraped into Eppendorf tubes while still partially frozen, and the cells further lysed by freeze-thawing for generation of the 12,000 x g cytosol.

Cytosolic ODC activity was determined in triplicate by measuring the release of ¹⁴CO₂ from

L-[¹⁴C]ornithine using an Eppendorf microvessel assay as previously described (Cancer Res.,

43 :2555-2559 (1983), the entire contents of which are hereby incorporated by reference). Results

Results of the foregoing tests are illustrated by the accompanying drawings wherein:

Figure 1 graphically shows the growth inhibition of keratinocyte cell line PE by vitamin D₃ and 1-hydroxymethyl homologues at 3 μM. The values shown represent the mean from 12 wells + S.D. Arrows indicate administration of fresh medium into which the compounds dissolved in DMSO had been added. Control cells were treated with DMSO alone (0.1% in culture medium). The-treated values are significantly different from the solvent control at 72 and 96 hours (p< 0.001, Student's t-test).

Figures 2A and 2B illustrate the inhibition of TPA-induced ornithine decarboxylase activity by pretreatment with vitamin D₃ and

1-hydroxymethyl homologues. Fig. 2A shows

inhibition of TPA stimulated response by

pretreatment of cells for 15 minutes with 1 μM of the compounds. Values represent the mean ± S.D. for 3 measurements. Pretreatment with calcitriol or its synthetic derivatives resulted in a

statistically significant reduction in TPA-induced ODC activity (p < 0.001, Student's t-test). Fig. 2B shows a dose-response curve for the inhibition of TPA-induced ODC activity with the

1-hydroxymethyl vitamin D₃ diastereomers (-)-2 and (+)-3.

As shown, calcitriol and its 1-hydroxymethyl derivatives were equipotent at inhibiting growth of PE cells. The anti-proliferative effects of the three compounds as demonstrated by reduction in cell number over time as compared to control plates is shown in Figure 1. While the control cells continued in the exponential phase of cell growth from 24 hours onward, this rapid rate of cell proliferation was significantly blunted by treatment with calcitriol or its 1-hydroxymethyl derivatives. Further, the treated cell populations had reached a plateau by 72 hours, days before the control cells would become confluent and senescent. Thus, all three vitamin D₃ compounds were active in inhibiting cell growth and division. The activity of these compounds was due to cytostatic rather than cytotoxic effects, as cell viability was unchanged in the treatment groups as determined by dye exclusion assay.

Calcitriol and the 1-hydroxymethyl diastereomers also significantly inhibited the effects of TPA (12-O-tetradecanoylphorbol-13-acetate) on the activity of ornithine

decarboxylase (ODC). ODC catalyzes the initial and rate-limiting step in the polyamine

biosynthetic pathway; while the function of polyamines is not fully understood, they are essential for growth, differentiation and

replication. This enzyme can be induced rapidly and dramatically by many growth stimuli, including the tumor promoter TPA (Annu. Rev. Biochem.,

53:749-790 (1984); the entire contents of which are hereby incorporated by reference).

The ability of TPA to induce ODC is associated with its proliferative and tumor promoting

properties (Cancer Res., 35:2426-2433 (1975);

Biochem. Biophys. Res. Commun., 105:969-976 (1982) and Proc. Natl. Acad. Sci. USA, 70: 6028-6032

(1982); the entire contents of which are hereby incorporated by reference).

A variety of agents have been shown to inhibit TPA effects on ODC induction as well as TPA -stimulated tumor promotion, including calcitriol (Cancer Res., 45: 5426-5430 (1985); Biochem.

Biophys. Res. Commun., 116:605-611 (1983); the entire contents of which are hereby incorporated by reference), anti-inflammatory steroids and vitamin A analogues (Biochem. Biophys. Res.

Commun., 91:1488-1496 (1979); the entire contents of which are hereby incorporated by reference), as well as free radical scavenging compounds (Adv. Free Radical Biol. and Med., 2:347-387 (1986); the entire contents of which are hereby incorporated by reference).

Similarly, Figure 2A shows the effects of vitamin D₃ and its 1-hydroxymethyl derivatives on the TPA-stimulated ODC activity in vi tro . The potency of the three compounds as inhibitors of the effects of TPA on this enzyme were not

significantly different from each other. Fig. 2B illustrates the similar dose-response

characteristics of the 1-hydroxymethyl vitamin D₃ diastereomers.

Taken together, these results indicate that replacing the 1α-hydroxyl group in calcitriol does not diminish biological activities

characteristic of vitamin D₃. Further, the results demonstrate that changing the stereochemistry of a 1-substituent does not necessarily change anti-proliferative activity.

The foregoing shows unexpectedly high anti-proliferative and cell growth inhibitory activities for the 1-hydroxyalkyl analogues of the invention, the expectation from the prior art being that replacement of the lα-hydroxyl group of calcitriol would be damaging to such activities. It is also surprising that changing the

stereochemistry of the 1-hydroxyalkyl (1α to 1β, compound 2 to compound 3) did not change the antiproliferative or cell growth inhibitory activity. Both (-)-2 and (+)-3 showed less than, or equal to, 2% of calcitriol's binding to the

1,25(OH)₂-vitamin D₃ receptor (VDR).

The VDR binding assay was performed according to the procedure of Reinhardt, T.A., Horst, R.L., Orf, J.W., Hollis, B.W., J . Clin . Endocrin .

Metab . , 58 : 91-98 (1984), the entire contents of which are hereby incorporated within by reference.

Results with Compound YB Compound YB has been studied in the above-described assays. As seen in Table 2, compound YB has a rating of 1 for inhibition of proliferation, a rating of 1 for induction of differentiation, and a rating of approximately 10^-3 for VDR binding.

Thus, there is an extremely wide spread between the ratings for inhibition of

proliferation and induction of differentiation compared to VDR binding.

Further support for the extremely low VDR binding is seen in Table 3.

Compound YB is designated MCW-II-5yb in Table 3. These results show the amount of each compound that results in 50% displacement of [³H]- 1,25(OH)₂D₃ from the calf thymus VDR. Compound YB bound about 1300 times less strongly than

1,25(OH)₂D₃.

Figures 3 and 4 are separate examples showing the effects of various concentrations of the compound YB and calcitriol on cell

proliferation. The calcium channel opening assay was performed according to the procedure of

Caffrey, J.M., Farach-Carson, M.C., J. Biol .

Chem . , 264 : 20265-20274 (1989), the entire

contents of which are hereby incorporated within by reference. Results from these separate

examples confirm that compound YB is a potent anti-proliferative and differentiation inducing analogue of vitamin D₃ with both anti-proliferative and differentiation inducing activity comparable to that of calcitriol. Additional confirmation of the anti- proliferative activity of compound YB is shown in Figure 5. Treatment of RWLeu-4 human CML cell line with compound YB resulted in an anti- proliferative effect comparable to or slightly greater than that of calcitriol, even at 50 nM.

Compound YB is designated as MCW-II5-Y-B and calcitriol as 1,25-(OH)₂-D₃ in Table 4. These results show that compound YB is similar to calcitriol in its ability to open calcium channels in an instantaneous non-genomic fashion.

Figures 6A and 6B provide data on the ability of compounds YA and YB to inhibit growth in human breast cancer cells, as evidenced by suppression of thymidine incorporation. It can be seen in this regard that YB is very comparable to

calcitriol in this regard, while YA appears to have no effect on MDA 468 cells.

Additional data has been obtained in U 937 cancer cells as shown in Table 5.

These data indicate that YB is about one third as active in inhibiting cell proliferation as

1,25(OH)₂D₃ in contrast to YA, which was

ineffective at concentrations up to 10^-7 M. In addition, YB, like 1,25(OH)₂D₃, is effective in inducing cell differentiation at a concentration of 10^-8 M, whereas YA is inactive at 10^-7 M.

In summary, compound YB is a vitamin D₃ analogue having a hydroxyalkyl substituent in the 1-position and a modified D-ring side chain. This novel compound exhibits a wide spread between the ratings for proliferation inhibition plus

differentiation induction compared to VDR binding. RESULTS WITH COMPOUNDS JK 276-1, JK 276-2,

JK 277-1 AND JK 277-2

As shown in Figure 7, all four of analogues JK 276-1, JK 276-2, JK 277-1 and JK 277-2 inhibited proliferation of PE cells. In HL-60 cells, compounds JK 276-2 and JK 277-2 were even more effective than YB in inhibiting growth, while JK 276-1 and JK 277-1 showed a relatively modest effect.

All four compounds show VDR binding affinities of less than 10^-3 compared to calcitriol. Table 6 shows the amount of each compound that results in 50% displacement of [³H]-1,25(OH)₂D₃ from calf thymus VDR. Compounds JK 276-2 and JK 277-2 bound, respectively, about 1500 and 3750 times less strongly than 1,25(OH)₂D₃.

Thus, there is a useful therapeutic window for these compounds in terms of separation of antiproliferative activity from calcemic activity. The results with the HL-60 cells indicate that YB, JK 276-2 and JK 277-2 will be especially useful in this regard. Therapeutic Potential

Because of their ability to inhibit cell proliferation and stimulate differentiation, and their low affinity for calcemic vitamin D3

receptors, compositions of this invention, in particular YB, JK 276-2 and JK 277-2, should prove valuable as therapeutic agents in diseases such as psoriasis and cancer wherein regulation of cell proliferation is an important aspect of treatment.

It will be appreciated that various modifications may be made in the foregoing without departing from the spirit and scope of the

invention-as defined in the following claims wherein:

Claims

WHAT IS CLAIMED IS:

1. A vitamin D₃ analogue of the formula:

and stereoisomers thereof, wherein R is -R¹OH, where R¹ is straight or branched alkyl of 1 to 6 carbons.

2. A vitamin D₃ analogue of the formula:

3. A vitamin D₃ analogue of the formula:

and stereoisomers thereof, wherein R is -R¹OH, where R¹ is straight or branched alkyl of 1 to 6 carbons.

4. A vitamin D₃ analogue of the formula:

5. A method of inhibiting cell proliferation by administering a vitamin D₃ analogue of the formula:

and stereoisomers thereof, wherein R is -R¹OH, where R¹ is straight or branched alkyl of 1 to 6 carbons.

6. The method of claim 5 wherein said vitamin D₃ analogue has the formula:

7. A method of inhibiting cell proliferation by administering a vitamin D₃ analogue of the formula:

and stereoisomers thereof, wherein R is -R¹OH, where R¹ is straight or branched alkyl of 1 to 6 carbons.

8. The method of claim 7 wherein said vitamin D₃ analogue has the formula: