WO1996001811A1

WO1996001811A1 - 2-substituted 1,25-dihydroxyvitamin d3 derivatives

Info

Publication number: WO1996001811A1
Application number: PCT/US1995/007595
Authority: WO
Inventors: Gary H. Posner
Original assignee: The Johns-Hopkins University
Priority date: 1994-07-11
Filing date: 1995-06-22
Publication date: 1996-01-25

Abstract

Vitamin D3 analogues which include a 2-substituted alcohol or fluoride are described. The preferred alcohol substituent is exemplified by the structural formula -(CH2)4OH and the preferred fluoride substituents by the structural formulas -(CH2)3F and -(CH2)4F. Methods for the preparation of a vitamin D3 analogue which includes a 2-substituted alcohol or fluoride starting with 2+4-cycloaddition of commercially available methyl 2-pyrone-3-carboxylate are also described.

Description

2-SUBSTITUTED 1,25-DIHYDROXYVITAMIN D₃ DERIVATIVES

BACKGROUND OF THE INVENTION

The present invention relates to novel biologically active vitamin D₃ analogues which include an alcohol or fluoride substituent in the 2-position and methods for their preparation.

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 vitamin D₃ produces 1α,25-dihydroxyvitamin D₃ (calcitriol) during normal metabolism. Calcitriol 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

Especially for post-menopausal women,

osteoporosis is a very serious illness that causes physical deformity and high susceptibility to bone (e.g., hip) fractures. In the general population aged above 65 years, osteoporosis ranks third to heart disease and cancer in terms of prevalence. It is estimated that 30% of women at 75 years and 40% of women at 85 years have abnormal bone loss. Calcitriol is being used, especially in Japan where dietary intake of calcium is low, for treatment of osteoporosis . The Chugai

Pharmaceutical Company has developed ED-71 (1) as a synthetic derivative of calcitriol, having a batter therapeutic index than calcitriol⁴. This

2ß-(3'-hydroxypropyloxy)-calcitriol has a two-fold stronger binding affinity to the rat plasma vitamin D-binding protein (DBP) than does

calcitriol, suggesting that it circulates in the plasma with a longer half-life than calcitriol⁴. Furthermore, in animal models with osteoporosis, ED-71(1) is more effective than calcitriol⁴.

To probe structure-medicinal activity

relationships in the hope of preparing a new osteoporosis drug with an even better therapeutic index than ED-71(1), we targeted the 2-carbaanalogues 2 and 3, structurally represented below. These analogues were chosen because replacing one oxygen atom by a methylene group, a relatively small change in a large steroid molecule, was anticipated, based on the current working model for receptor binding of ED-71(1)⁴, not to interfere with such critical receptor binding, also, these analogues were chosen for chemical reasons, probing whether recently developed Diels-Alder methodology using 2-pyrones and monosubstituted alkenes could be extended to 1,2-disubstituted alkene dienophiles with reliable and faithful transfer of olefin geometry ultimately into the stereochemical relationships at the 1- and 2- positions of the steroid targets. We record here the results of these chemical explorations and biological evaluations.

SUMMARY OF THE INVENTION

The present invention is for a vitamin D₃ analogue which includes a 2-substituted alcohol or fluoride. The preferred alcohol substituent is exemplified by the structural formula -(CH₂)₄OH and the preferred fluoride substituents by the

structural formulas -(CH₂)₃F and -(CH₂)₄F.

The preferred diastereomers of the 2- substituted -(CH₂)₄OH are represented by the following structural formulas:

The present invention is also for the related method of preparation of a vitamin D₃ analogue which includes a 2-substituted alcohol or fluoride starting with 2+4-cycloaddition of commercially available methyl 2-pyrone-3-carboxylate. Diastereomeric 2-substituted calcitriol analogues were prepared in only eleven chemical operations, starting with 2+4-cycloaddition of commercially available methyl

2-pyrone-3-carboxylate. Highlights of this convergent and stereo controlled synthetic

approach are as follows: (1) retention of reactant dienophile geometry in the product bicyclic lactone, characteristic of a concerted

inverse-electron-demand Diels-Alder cycloaddition; (2) an improved decarboxylation procedure

involving chemospecific allyloxide opening of a lactone ring in the presence of a methyl ester and then non-high pressure palladium-promoted allylic ester decarboxylation; and (3) use of the

enantiomerically pure C,D-ring chiron 14 to resolve racemic A-ring phosphine oxide 13.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EXEMPLARY EMBODIMENTS Uncatalyzed Diels-Alder cycloadditions between highly polarized dienes and dienophiles can occur step-wise rather than in the usual concerted fashion. To probe this issue within the context of inverse-electron-demand Diels-Alder cycloadditions using 2-pyrones substituted at the 3-position with highly electron-withdrawing (e.g. 3-sulfonyl, 3-acyl) substituents⁵, 3-p- toluenesulfonyl-2-pyrone and 1,2-disubstituted alkenes 4E and separately 4Z were placed under high pressure. In both of these electronically matched cases, the electron-poor 2-pyrone diene and the electron-rich vinylic ether cycloadded to produce isolable bicyclic lactone adducts without undesirable and often-encountered extrusion of CO₂ (Scheme I)

The most important aspect of these Diels- Alder reactions from a mechanistic viewpoint is that the cycloadducts retained the stereochemical information in the reactant vinylic ethers:

dienophile 4E led exclusively to trans-4,5- oriented products 5a and 5b, whereas dienophile 4Z led exclusively to cis-4, 5-oriented products 6a and 6b. Therefore, these polarized 2+4- cycloadditions must occur in a concerted rather than in a step-wise fashion. The assignments of the 4,5-positional relationships were based on extensive precedent 5, and the assignments of the 4,5-stereochemical relationships were based on the match of the 400 MHz ¹H NMR J_4,5 coupling constants with those calculated using the Karplus eguation for energy-minimized structures generated using

Chem-3D (Scheme I)⁷. Bicyclic lactone 6a, the very major cycloadduct, differed in a characteristic way from bicyclic lactone 6b in terms of the

chemical shift of the bridgehead hydrogen atom ( δ 4.98 vs. 5.04) and the chemical shift of the C₄ hydrogen atom ( δ 4.55 vs. 3.83). Also,

irradiation of the C₅ hydrogen atom of lactone 6a caused an 11 % nOe on the C₄ hydrogen atom. The large discrepancy between the observed and the calculated J_4.5 coupling constant for cis-4.5- disubstituted bicyclic adduct 6b was of

considerable concern. Therefore, a series of similar cis-4.5-disubstituted bicyclic lactones was prepared. Examination of their J_4.5 coupling constants (Table I) showed a very subtle effect of the nature of the substituents on this magnitude of the vicinal coupling constant. For example, the cycloadduct derived from the 6-membered cyclic vinyl ether showed J_4.5=0 Hz (entry 2), whereas that derived from the 5-membered cyclic vinyl ether showed J_4.5=8.5 Hz (entry 3). Finally, based on literature analogies in which E-alkenes

underwent 2+4-cycloadditions more easily than Z- alkenes^6e-9, we were surprised to find that vinylic ether geometric isomer 4Z reacted considerably faster than isomer 4E with 3-tolysufonyl-2-pyrone. Whereas vinylic ether isomer 4Z yielded almost exclusively the endo-cycloadduct 6a, as expected from previous results, vinylic ether isomer 4E gave a 1:2 mixture of cycloadducts 5a:5b.

From a synthesis viewpoint, cycloadducts 5 and 6 turned out to be disappointing. Despite close structural similarity with other bridgehead- substituted toluenesulfonyl cycloadducts and related cyclohexene systems that underwent smooth reductive-desulfonylation, we were unsuccessful using a variety of conditions (e.g., Al-Hg, Na-Hg, Raney nickel, Li/NH₃) to effect high-yielding reductive-desulfonylation. Also, it was

anticipated that ultimate conversion of the methyl ether functionality into the desired alcohol group would be more difficult than deprotection of a silyl ether. Therefore, silylated vinylic ether 7Z, prepared according to literature precedent as illustrated in Scheme II⁹, and commercially available methyl 2-pyrone-3-carboxylate were subjected to high pressure cycloaddition (eq. 1).

Bicycloadduct 8 was the major product, isolated on gram scale in 60% yield, with the oxygen substituted at position-4, as expected based on the polar nature of the Diels-Alder cycloaddition and also on literature precedent, with a cis-4,5-stereochemical relationship. This stereochemical outcome was expected based on the results in Scheme I with the 3-sulfonyl-2-pyrone and was confirmed by the observed large ¹H NMR J_4.5 coupling constant (8.6 Hz) and by the

characteristic chemical shifts of the bridgehead

hydrogen atom at δ 5.1 and the C₄ hydrogen atom at δ 4.7. Also, irradiation of the C₅ hydrogen atom caused a 14% nOe on the C₄ hydrogen atom,

confirming their cis-4,5-relationship. We have not succeeded in preparing cleanly the isomeric silylated vinylic ether 7E for cycloaddition with methyl 2-pyrone-3-carboxylate.

Having served its chemical function of activating the pyrone diene (the unsubstituted parent 2-pyrone is unreactive)^5d'¹⁰ for

cycloaddition with the electron-rich vinylic ether 7Z, the bridgehead carboxylate ester group in bicyclic lactone adduct 8 had to be removed. To complement our two-step lactone methanolysis and high-pressure procedure for this type of

decarboxylation¹¹, we report now a new and more convenience (i.e., not high pressure) two-step protocol (Scheme III). Although bicyclic lactone methyl ester 8 has two ester carbonyl groups, it was gratifying to find that the lactone ring was chemospecifically attacked by lithium allyloxide to produce mixed methyl allyl malonate 9 in 75% yield. In accord with literature precedent¹², allyl ester 9 was smoothly decarboxylated using palladium acetate; an unexpected but desired benefit of this procedure was conjugation of the cyclohexene double bond, giving the contiguously

tetrasubstituted cyclohexene 10 in 92% yield

(Scheme III).

Without any surprises, highly functionalized cyclohexene alcohol ester 10 was O-silylated and then reduced to form allylic alcohol 11 (Scheme

IV). One flask Claisen rearrangement followed by spontaneous thermal sulfoxide elimination was achieved using our sulfinylated ortho ester protocol giving an unusually favorable >10:1 Z:E ratio of dienoate esters from which the desired Z- dienoate 12 was isolated by chromatography in 80% yield.

Established reactions as outlined in Scheme IV below provided the crucial, fully O-protected, racemic, A-ring phosphine oxide 13. Lythgoe-type coupling of the conjugate base of phosphine oxide 13, generated using phenyllithium , with C,D-ring ketone 14 of natural absolute configuration produced O-silylated derivatives of diastereomeric 2-(4'-hydroxylated) calcitriol analogues 3 and 3', isolated in 50% yield.

Fluoride-induced cleavage of the silyl protecting groups proceeded easily at three of the four silylated alcohol groups; desilylation at the C, secondary alcohol position, however, was unexpectedly slow, requiring considerably more vigorous reaction conditions. In another context¹⁶, we have observed that the C₁ secondary alcohol unit in calcitriol is chemically less reactive than the C₃ secondary alcohol toward esterifying reagents.

Nevertheless, fluoride-assisted quadruple

desilylation under more vigorous conditions eventually yielded the desired calcitriol

analogues 3 and 3'.

Easy HPLC separation gave each diastereomer in enantiomerically pure form. Tentative

assignments of stereochemistry to diastereomers 3 and 3' were made by H NMR in analogy with closely related calcitriol analogues; in diastereomeric pairs differing only by inversions of

stereochemistry at positions 1-3 but not in the

C,D-ring or in the side chain, the lα-substituted diastereomer characteristically showed a lower field absorption for the C₁₈-methyl group and for one proton of the C₁₉-methylene group (Table II).

Thus, in only 11 steps from commercially available methyl 2-pyrone-3-carboxylate, two new vitamin D₃ analogues have been prepared, and the C,D-ring chiron 14 has been used to resolve racemic A-ring phosphine oxide 13.

Preliminary biological evaluation of

synthetic diastereomers 3 and 3' involved

measuring their relative binding affinities to rat vitamin D binding protein (DBP) and also to the vitamin D receptor of bovine thymus (Table III) . Diastereomer 3 had significantly higher affinity than ED-71 (1) for the vitamin D binding protein, but it had extremely low affinity for the vitamin D receptor; whether this separation of binding affinities has important mechanistic and/or medicinal value remains to be established.

Diastereomer 3', on the other hand, had lower affinity than ED-71 (1) for the vitamin D binding protein, but it had higher affinity than ED-71 (1) for the vitamin D receptor. Determining the impact of these differences on possible use of these new analogues for chemotherapy of

osteoporosis requires further biological testing.

Relative binding affinities of diastereomers 3 and 3' to the vitamin D binding protein and to the vitamin D receptor showed some unusual trends. Diastereomer 3' surprisingly bound much more effectively to the vitamin D receptor than did the established osteoporosis drug candidate ED-71 (1).

EXAMPLES

General

Tetrahydrofuran and diethyl ether were distilled from benzophenone ketyl prior to use. Methylene chloride and triethylamine were

distilled from calcium hydride immediately prior to use. Commercially available anhydrous solvents were used in other instances. All reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) and unless otherwise specified were used as received without further purification. All reactions were run in flame-dried flasks under nitrogen unless otherwise specified. FT-IR spectra were determined using a Perkin-Elmer Model 1600 FT-IR spectrophotometer. The ¹H NMR were recorded on a Varian XL-400 spectrometer and

Bruker AMX-300 spectrometer operating at 400 and 300 MHz, respectively. The ¹³C spectra were recorded on the same instruments operating at 100 and 75 MHz, respectively. High resolution mass spectra were obtained on a two sector-high- resolution VG-70S mass spectrometer run at 70 eV. A Leco Corp. Model No. PG-200-HPC 13 Kbar

apparatus was used for the high-pressure studies.

Silylated Vinylic Ether 7Z

To a 100 mL round-bottomed flask containing 2.1 g (17.5 mmol) of 1,6-hexanediol, 2.7mL (19.2 mmol) of triethylamine, and 10 mg of N,N- dimethylaminopyridine in 35 mL CH₂Cl₂ was added 5 mL (19.2 mmol) of t-butylchlorodiphenylsilane. The reaction was stirred at room temperature for 12 h, or until complete by TLC. The reaction was quenched with 10 mL water, the organic layer was separated, and the aqueous layer was washed with CH₂Cl₂. The combined organic layers were dried over MgSO₄, filtered, and concentrated. The crude product was purified by column chromatography (10- 20% EtOAc/Hexane) to afford 3.73 g (10.45 mmol, 60% yield) of the monosilated product as a clear oil.

To a 250 mL round-bottomed flask containing 10.14 mL (125.4 mmol) of pyridine in 50 mL of CH₂Cl₂ was slowly added 6.27 g (62.7 mmol) of chromium trioxide. The resultant deep burgundy solution was stirred for 15 minutes at room temperature. At the end of this period, a

solution of the above monosilated diol in 50 mL CH₂Cl₂ was added via cannula. A tarry, black deposit separated immediately. This mixture was stirred for lh at which time the CH₂Cl₂ was removed on a rotary evaporator and the tar was diluted with Et₂O. This heterogeneous mixture was filtered through silica gel to give a yellow liquid which was concentrated. The crude product was purified by column chromatography (5% EtOAc/Hexane) to give 3.17 g (8.9 mmol, 85% yield) of the aldehyde as a clear oil. ¹H NMR (300 MHz, CDCl₃) 5 9.74 (s, 1H), ,.675 (m, 4H), 7.40 (m, 6H), 3.66 (t, J=6.2 Hz, 2H), 2.34 (m, 2H), 1.58 (m, 4H), 1.41 (m, 2H),

1.04 (s, 9H), IR (CHCl₃) 3013 cm^-1, 2931 cm^-1, 1713 cm^-1.

The above aldehyde was then diluted with 20 mL of benzene and 1.64 mL (11.75 mmol) of

triethylamine. The resulting mixture was cooled to 0°C and 2.45 ml (10.68 mmol) of t- butyldimethylsilyl triflate was added. The reaction was allowed to warm to room temperature and stirred for 1 h. The reaction was quenched with brine and diluted with Et₂O. The organic layer was separated, and the aqueous layer was washed with Et₂O. The combined organic layers were dried over MgSO₄, filtered, and concentrated. The crude product was purified by column

chromatography (Hexane) or silica gel that was slurry-packed with 1% triethylamine/hexane. The silylated vinylic ether 7Z (1.67 g, 3.56 mmol, 40% yield) was a clear oil; R_f=0.8 (25% EtOAc/Hexane); ¹H NMR (300 MHz, CHCl₃) δ 7.65 (m, 4H), 7.41 (m, 6H), 6.17 (dt, J=5.9, 1.5 HZ, 1H), 4.43 (dd,

J=7.4, 5.9 HZ, 1H), 3.61 (t, J=6.6 Hz, 2H), 2.09 (ddt, J=14.7, 7.4, 1.5 Hz, 2H), 1.54 (m, 2H), 1.37

(m, 2H), 0.92 (s, 9H), 0.12 (s, 6H) ; ¹³C NMR (75 MHz, CHCl₃) δ 138.50, 135.54 (4), 134.15 (2), 129.50, 129.43, 127.55 (2), 127.53, 110.54, 63.93, 32.31, 26.87 (3) , 25.99 (3), 25.80, 25.66, 23.36, 19.22, -2.92, -5.36; IR (CHCl₃) 3013, 2931, 1654,

1108 cm^-1; HRMS m/e calcd. for C₂₄H₃₅O₂Si₂ 411.2176, found 411.2179.

Cycloadduct 8

A 12 cm piece of 3/8" heat shrinkable teflon tubing (Ace Glass cat. #12685-40) was sealed on one end with a glass dowel plug by using a heat gun. To this 500.0 mg (3.24 mmol) of methyl 2- pyrone-3-carboxylate (Aldrich), 2g (4.26 mmol) of silylated vinylic ether 7Z, 10 mg of barium carbonate, and 2 mL of dry CH₂Cl₂ was added. The open end of tubing was then sealed in a similar fashion with a second glass dowel plug. This 'sealed tube' was the pressurized at 10-11 Kbar at room temperature for 4 days. The reaction mixture was concentrated on a rotary evaporator and the residue was purified by column chromatography (5% EtOAc/Hexane) to give 1.21 g (1.94 mmol, 60%) of the cycloadduct 8 as a clear oil; R_f=0.58 (25% EtOAc/Hexane); ¹H NMR (300 MHz, CHCl₃) δ 7.65 (m, 4H), 7.41 (m, 6H), 6.78 (dd, J=7.8, 2.8 Hz , 1H), 6.46 (dd, J=7.8, 5.1 Hz, 1H), 5.09 (ddd, J=5.1, 3.8, 1.0 HZ, 1H), 4.71 (dd, J=7.6, 1.00 Hz, 1H),

3.89 (s, 1H), 3.66 (t, J=6.1 Hz , 3H), 2.35 (ddd, J=7.6, 3.8, 2.8 Hz, 1H), 1.56 (m, 2H), 1.26 (m, 2H), 1.06 (S, 9H), 0.91 (m, 2H), 0.78 (s, 9H), 0.012 (s, 3H), 0.006 (s, 3H) ; ¹³C NMR (75 MHz, CHCl₃) δ 168.93, 167.51, 135.51 (4), 133.70 (2),

130.46, 129.53 (2), 128.64, 127.54 (4), 76.12, 69.36, 63.12, 52.7, 44.72, 32.37, 27.09, 26.75 (3), 25.64 (3), 25.56, 25.31, 23.34, 19.09, 18.04, -3.90, -5.03, IR (CHCl₃) 1760, 1743 cm^-1; HRMS m/e calcd. for C₃₁H_A1O₆Si₂ 565.2442, found 565.2450.

Mixed Malonate 9

To a 25 mL round-bottomed flask with 500 mg (0.80 mmol) of cycloadduct 8 and 2 mL of CH₂Cl₂ at 0°C was added dropwise, via syringe, 962 μL of a freshly made 1.0 M solution of lithium allyloxide in allyl alcohol. The reaction mixture was allowed to warm to room temperature after the addition. Reaction was complete by TLC after 2 hours. The mixture was quenched with 2 mL aq.

NH₄Cl and extracted with CH₂Cl₂. The organic layer was dried with MgSO₄, filtered, and concentrated on a rotary evaporator. The residue was purified by column chromatography (0-20% EtOAc/Hexane) to give 410 mg (0.60 mmol, 75%) of the desired malonate as a clear oil; R_f=0.42 (25% EtOAc/Hexane); ¹H NMR (300 MHZ, CHCl₃) δ 7.68 (m, 4H), 7.41 (m, 6H), 5.96 (S, 2H), 5.83 (ddt. J=17.2, 10.5, 5.6 Hz, 1H),

5.36 (dd, J=17.2, 1.5 Hz, 1H), 5.22 (dd, J=10.5, 1.5 HZ, 1H), 4.76 (s, 1H), 4.63 (ddt, J=13.3, 5.6, 1.4 Hz, 1H), 4.51 (ddt, J=13.3, 5.6, 1.4 Hz, 1H), 4.02 (t, J=8.6 HZ, 1H), 3.74 (s, 3H), 3.69 (t, J=6.1 Hz, 2H), 1.71 (m, 2H), 1.64 (m, 2H), 1.57

(S, 1H), 1.28 (m, 2H), 1.05 (s, 9H), 0.81 (s, 9H), 0.07 (s, 3H), -0.06 (s, 3H) ; ¹³C NMR (75 MHz,

CHCl₃) δ 169.11, 167.95, 135.41 (4), 134.07 (2), 133.89, 131.17, 129.34 (2), 127.44 (4), 122.47, 118.43, 71.70, 68.64, 65.72, 63.89, 61.38, 52.57, 46.04, 32.45, 26.76 (3), 25.94 (3), 23.25, 19.04, 18.27, 14.02, -3.79, -4.44; IR (CHCl₃) 1737 cm^-1; HRMS m/e calcd. for C₃₄H₄₇O₇Si₂ 623.2860, found

623.2865.

To a 25 mL round-bottomed flask with 380 mg (0.56 mmol) of the allyl ester alcohol, 78 μL (0.67 mmol) of 2,6-lutidine, and 1.5 mL of CH₂Cl₂ at 0°C was added 154 μL (0.67 mmol) of t- butyldimethylsilyl trifluoroethanesulfonate dropwise via syringe. The reaction was complete by TLC after 10 minutes. The reaction was

quenched at 0°C with 1 mL water and allowed to warm to room temperature. Extraction with CH₂Cl₂ followed by drying with MgSO₄, filtration, and concentration afforded a viscous oil which was purified by column chromatography (5%

EtOAc/Hexane) giving 390 mg (0.49 mmol, 87%) of O- silylated mixed malonate 9 as a clear oil; R_f=0.6 (25% EtOAc/Hexane); ¹H NMR (300 MHz, CHCl₃) δ 7.68 (m, 4H), 7.41 (m, 6H), 5.87 (d, J=2.68 Hz, 2H), 5.83 (ddt, J=17.2, 10.5, 5.6 Hz, 1H), 5.36 (dd, J=17.2, 1.5 Hz, 1H), 5.22 (dd, J=10.5, 1.5 Hz, 1H), 4.76 (s, 1H), 4.63 (ddt, J=13.3, 5.6, 1.4 Hz, 1H), 4.51 (ddt, J=13.3, 5.6, 1.4 Hz, 1H), 4.13 (d, J=9.2 Hz, 1H), 3.74 (s, 3H), 3.69 (t, J=6.1 Hz, 2H), 1.76 (m, 2H), 1.61 (m, 2H), 1.55 (s, 1H), 1.28 (m, 2H), 1.06 (s, 9H), 0.88 (s, 9H), 0.83 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H), 0.04 (s, 3H), -

0.06 (s, 3H) ; ¹³C NMR (75 MHz, CHCl₃) 5 169.27, 167.96, 135.45 (4), 133.99 (2), 133.97, 131.33, 129.38 (2), 127.48 (4), 121.83, 118,43, 71.70, 69.16, 65.69, 64.00, 61.47, 52.52, 46.10, 32.67, 26.81 (3) , 25.99 (3), 25.80 (3), 23.25, 19.10,

18.35, 17.99, 14.11, -3.83, -4.20, -4.34, -4.70; IR (CHCl₃) 1737 cm^-1; HRMS m/e calcd. for C₄₀H₆₁O₇Si₃ 737.3725, found 737.33730.

Cyclohexene Ester 10

A mixture of 380 mg (0.48 mmol) of malonate

9, 23 μL (0.60 mmol) formic acid, 87 μL (0.62 mmol) triethylamine, 10 mg (0.04 mmol) triphenylphosphine, and 2 mg (0.01 mmol) palladium acetate in 1.5 mL dioxane was sealed in a 5 mL hydrolysis tube and heated at 100°C for 12 h.

After evaporation of dioxane, 1 N HCl (lmL) was added, and the mixture was extracted with CH₂Cl₂ (2 mL x 2). The organic solution was washed with saturated NaHC0₃ and dried over MgSo₄, filtered, and concentrated. The oily residue was purified by column chromatography (0-10% EtOAc/Hexane) to give 310 mg (0.44 mmol, 92%) of cyclohexene ester 10 as a clear oil: R_f=0.50 (25% EtOAc/Hexane); ¹H NMR (300 MHz, CHCl₃) δ 7.68 (m, 4H), 7.41 (m, 6H), 6.87 (t, J=1.5 Hz, 1H), 5.76 (s, 1H), 4.72 (s, 1H), 4.16 (m, 1H), 3.99 (m, 1H), 3.74 (s, 3H), 3.69 (t, J=6.1 HZ, 2H), 2.60 (dt, J=14.3, 5.6 Hz, 1H), 2.10 (ddd, J=16.9, 8.4, 1.5 Hz, 1H), 1.58 (m, 2H), 1.55 (s, 1H), 1.38 (m, 2H), 1.26 (m, 2H), 1.05 (s, 9H), 0.89 (S, 9H), 0.83 (s, 9H), 0.05 (s, 3H), 0.04 (S, 3H), -0.07 (s, 3H), -0.09 (s, 3H) ; ¹³C NMR (75 MHZ , CHCl₃) δ 167.01, 135.51 (4),

134.05 (2), 133.16, 132.35, 129.45 (2), 127.55 (4), 66.89, 64.16, 51.50, 47.43, 33.1, 26.87, 25.95, 25.92, 25.89, 25.84, 25.68, 23.31, 19.17, 18.42, 18.06, -4.09, -4.37, -4.74, -5.39; IR

(CHCl₃) 1713 cm^-1; HRMS m/e calcd. for C₃₆H₅₇O₅Si₃ 653.3514, found 653.3516. Allylic Alcohol 11

To a 25 mL round-bottomed flask containing 695 mg (0.98 mmol) of cyclohexene ester 10 in 8 mL toluene at -78°C was added 2.15 ml (2.15 mmol) of 1 M diisobutylaluminum hydride in toluene dropwise via syringe. The reaction was complete by TLC after lh. The reaction was quenched by addition at -78°C of 5 mL of 2 M potassium sodium tartrate followed by dilution with 10 mL EtOAc. After the mixture was allowed to warm to room temperature and stirred for 0.5 h, two layers were visible. These were separated and the aqueous layer was washed with EtOAc. The combined organic fractions were washed with water, and brine, and dried over MgSO₄, filtered, and concentrated. The residue was purified by column chromatography (0-25%

EtOAc/Hexane) to give 518 mg (0.76 mmol, 77%) of allylic alcohol 11 as a clear oil: R_f=0.35 (25% EtOAc/Hexane); ¹H NMR (300 MHz, CHCl₃) δ 7.68 (m, 4H), 7.41 (m, 6H), 5.51 (t, J=1.5 Hz, 1H), 4.80 (s, 1H), 4.22 (d, J=12.0 Hz, 1H), 4.16 (m, 1H), 3.99 (m, 1H), 3.74 (s, 3H), 3.69 (t, J=6.1 Hz, 2H), 2.60 (dt, J=14.3, 5.6 Hz, 1H), 2.10 (ddd, J=16.9, 8.4, 1.5 HZ, 1H), 1.58 (m, 2H), 1.55 (s, 1H), 1.38 (m, 2H), 1.26 (m, 2H), 1.05 (s, 9H),

0.89 (ε, 9H), 0.83 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H), -0.07 (s, 3H), -0.09 (s, 3H) ; ¹³C NMR (100 MHz, CHCl₃) δ 137.81, 135.51 (4) , 134.04 (2), 129.47 (2), 127.55 (4), 122.65, 70.03, 68.32, 65.68, 63.77, 46.36, 33.11, 31.28, 26.85 (3), 25.90 (3), 25.82 (3), 25.51, 25.43, 24.52, 24.03, 19.21, 18.06, -4.20, -4.82 (2), -4.92; IR (CHCl₃) 3401, 1713 cm^-1; HRMS m/e calcd. for C₃₄H₅₇O₄Si₃ 625.3565, found 625.3570.

Z-Dienoate 12

To 510 mg (0.75 mmol) of allylic alcohol 11 in a sealable hydrolysis tube was added 642 mg (2.2 mmol) triethylphenyl sulfinyl orthoacetate and 1.0 mg 2,4,6-trimethylbenzoic acid and 2 mL CH₂Cl₂. The tube was purged with argon and sealed, then heated at 100°C for 12h. The reaction mixture was concentrated. Crude H NMR showed a >10:1 mixture of Z:E isomers. The crude product was purified via PTLC (10%EtOAc/Hexane) to give 450 mg (0.60 mmol, 80%) of Z dienoate 12 as a clear oil; R_f=0.61 (25% EtOAc/Hexane); ¹H NMR (400 MHZ, CHCl₃) δ 7.68 (m, 4H), 7.41 (m, 6H), 5.61 (s, 1H), 5.17 (t, J=1.8 Hz , 1H), 5.09 (t, J=1.3 Hz, 1H) 4.55 (dd, J=5.5, 1.3 Hz, 2H), 4.12 (q, J=7.2 HZ, 2H), 3.97 (t, J=3.8 Hz, 1H), 3.66 (t, J=6.1 HZ, 2H), 2.48 (dd, J=5.5, 1.4 Hz, 1H), 2.15 (dd, J=3.8, 1.7 HZ, 1H), 1.70 (m, 1H), 1.58 (m, 2H), 1.55 (S, 1H) , 1.38 (m, 2H), 1.26 (m, 2H), 1.04 (S,

9H), 0.87 (s, 18H), 0.05 (s, 6H), 0.04 (s, 6H) ; ¹³C NMR (75 MHz, CHCl₃) δ 165.92, 157.09, 153.29, 145.39, 135.52 (4), 134.09 (2), 129.47 (2), 127.55 (4), 117.17, 70.23, 63.91, 59.67, 50.54, 32.98, 26.86 (3), 25.80 (3), 25.74 (3), 19.20, 18.18, 17.98, 14.30, 14.13, -4.66, -4.83, -4.87, -5.17; IR (CHCl₃) 3025, 2931, 1713 cm^-1; HRMS m/e calcd. for C₄₃H₇₀O₅Si₃ 750.4531, found 750.4538.

Phosphine Oxide 13

To a 25 mL round-bottomed flask containing 180 mg (0.24 mmol) of dienoate 12 in 2 mL toluene at -78°C was added dropwise 530 μL (0.53 mmol) of a 1 M solution of diisobutylaluminum hydride via syringe. After lh the reaction was complete by TLC. The reaction was quenched at -78°C by addition of 2 mL of 2 N potassium sodium tartrate and dilution with 5 mL EtOAc. The mixture was allowed to warm to room temperature and stirred 0.5 h until two distinct phases appeared. The organic phase was separated, and the aqueous phase was washed with EtOAc. The combined organic extracts were washed with water and brine, dried over MgSO₄, filtered, and concentrated. The crude mixture was quickly purified by column

chromatography (10-20% EtOAc/Hexane) to give 167 mg (0.24 mmol) of the desired allylic alcohol.

To a 10 mL round-bottomed flask containing 152 mg (1.14 mmol) N-chlorosuccinimide in 3 mL CH₂Cl₂ at 0°C was added 90 mL (1.22 mmol) of dimethyl sulfide via syringe. A white precipitate formed immediately upon addition. This mixture was cooled to -20°C and stirred for 20 minutes. The above allylic alcohol in 2 mL CH₂Cl₂ was then added to the heterogeneous mixture via cannula. The reaction was stirred for 0.5 h at -20°C and then allowed to warm to room temperature and stirred for an additional 1 h. The organic layer was washed with water, brine, dried with MgSO₄, filtered, and concentrated. The crude product was passed through florisil (5% EtOAc/Hexane) to afford 154 mg (0.21 mmol) of the desired allylic chloride as a yellow oil.

To a 10 mL round-bottomed flask containing the above allylic chloride in 2 mL THF at -78°C was added dropwise via cannula a 0.5 M solution of potassium diphenylphosphide in THF. The addition was stopped once the red color persisted. After 1 h the reaction was allowed to slowly warm to 0°C at which point it was complete by TLC. The reaction was quenched with 2 drops of water and the THF was removed. The residue was diluted with 2 mL CH₂Cl₂ and 10 drops of 30% H₂O₂ was added.

After 1 h the reaction was diluted with CH₂Cl₂ and water and the layers were separated. The organic phase was concentrated. The crude product was purified by column chromatography (25-50%)

EtOAc/Hexane) to give 121 mg (0.13 mmol, 54% from Z-dienoate 12) of the phosphine oxide 13 as a clear oil: R_f=0.56 (75% EtOAc/Hexane); ¹H NMR (400 MHz, CHCl₃) δ 7.74-7.36 (m, 20H), 5.3 (dd, J=15.1 HZ, 6.6H), 5.11 (S, 1H), 4.75 (s, 1H), 4.42 (d, J=3.2 HZ, 1H), 3.87 (dd, J=8.9, 4.9 Hz, 1H), 3.65 (t, J=6.6 HZ, 3H), 3.40 (dt, J=15.1, 8.9 Hz, 1H), 3.14 (dt, J=16.0, 6.7 HZ, 1H), 2.4 (dd, J=13.3, 3.2 HZ, 1H), 2.06 (dd, J=14.2, 2.9 Hz, 1H), 1.56 (m, 1H), 1.53 (m, 2H), 1.43 (m, 2H), 1.35 (m, 2H), 1.04 (s, 9H), 0.88 (s, 9H), 0.82 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H), -0.01 (s, 3H), -0.03 (s, 3H); ¹³C NMR (75 MHz, CHCl₃) δ 146.20 (2), 141.17,

135.50 (4), 134.06 (2), 131.71, 131.04, 130.92 (4), 129.45 (2), 128.63 (4), 128.47 (2), 127.53 (4), 114.39, 70.15, 63.90, 50.72, 32.97, 30.09,

26.83 (3), 25.79 (3), 25.05, 24.06, 19.18, 18.04, -4.58, -4.69, -4.89, -5.07; IR (CHCl₃) 3025, 2954, 1472, 1255 cm^-1; HRMS m/e calcd. for C₄₉H₆₈O₄Si₃ 835.4163, found 835.4169. Calcitriol Analogues 3 and 3'

To a 10 mL round-bottomed flask containing 95 mg (0.103 mmol) of phosphine oxide 13 in 1.4 mL of THF at -78°C was added dropwise 67 μL (0.103 mmol) of a 1.54 M solution of phenyllithium in THF. The resultant red solution was allowed to stir for 10 minutes. A solution of 20 mg (0.056 mmol) of C-D ring 14 in 1 mL of THF was added via cannula. The reaction was complete after 1 h by TLC following disappearance of C-D ring. The reaction was quenched by addition of 1 mL 1:1 KHC0₃/2 N

potassium sodium tartrate. The layers were separated and the organic phase was washed with brine, dried over MgSO₄, filtered and concentrated. The crude product was rapidly purified by

filtration through silica gel using 50%

EtOAc/Hexane as solvent to give 47 mg (0.052 mmol, 50% yield) of the coupled product. The silyl ethers were cleaved by redissolving the product in 1 mL of THF and treating with 220 μL (0.22 mmol) of tetrabutylammonium fluoride. After 24 h the reaction was diluted with water and the layers were separated, dried over MgSO₄, filtered and concentrated. The diastereomers were separated and purified by reverse phase HPLC (30-20%

H₂O/CH₃CN on a C-18 semi-prep, column) to afford 1.1 mg (0.002 mmol, 4% yield) of analogue 3, and 4.4 mg (0.008 mmol, 16% yield) of analogue 3', both as white solids. ¹H NMR of 3 (300 MHz, CHCl₃) δ 6.41 (d, J=11.6 HZ, 1H) , 6.02 (d, J=11.6 Hz, 1H) , 5.29 (S, 1H) , 5.02 (s, 1H) , 4.35 (s, 1H) , 3.82 (m, 1H) , 3.68 (t, J=6.1 Hz, 2H) , 2.78 (d, J=11.5 Hz, 1H) , 2.62 (dd, J=11.5, 3.5 Hz, 1H) ,

2.20 (dd, J=10.2, 3.5 HZ, 1H) , 1.98 (m, 2H) , 1.68- 1.20 (m) , 0.92 (s, 3H) , 0.90 (s, 3H) , 0.54 (s, 3H). ¹H NMR of 3' (300 MHz, CHCl₃) δ 6.40 (d, J=11.6 Hz, 1H), 5.98 (d, J=11.6 Hz ,1H) , 5.27 (s, 1H), 4.98 (s,1H) , 4.38 (s,1H) , 3.87 (m,1H) , 3.68 (t, J=6.1 HZ, 2H), 2.82 (d, J=11.5 Hz ,1H) , 2.65 (dd, J=11.5, 3.5 Hz ,1H) , 2.24 (dd, J=10.2, 3.5 Hz, 1H) , 1.68-1.20 (m), 0.92 (s, 3H), 0.90 (s,

3H), 0.52 (s, 3H) ; UV-Vis (MeOH) λ_max 268 nm (e 15,600); HRMS m/e calcd. for C₃₁H₅₀O₃ 470.3760, found 470.3771.

2-Fluorides

The above-described methods for producing the vitamin D₃ analogues with alcohols substituted in the 2-position can be readily modified to produce comparable analogues with fluorides substituted in the two position (18-21). Table IV lists the fluoride analogues of the present invention.

The following scientific articles and

references have been cited throughout this

application and the entire contents of each article or reference is hereby incorporated by reference.

Scientific Articles

1. Yamaguchi, K.; Matsumura, G.; Kagechika, H.; Azumaya, I.; Ito, Y; Itai, A.; Shudo, K.; J. Am . Chem . Soc. , 1991, 113 , 5474 and references therein.

2. Tilyard, M.W. in "Vitamin D: Gene

Regulation, Structure-Function Analysis, and

Clinical Applications", A.W. Norman, R. Bouillon, M. Thomasset, Eds., W. de Gruyter, NY, 1991, p. 779.

3. Nordin, B.E.C.; Need, A.G.; Morris, H.A. ; Horowitz, M. in "Vitamin D: Gene Regulation, Structure-Function Analysis, and Clinical

Applications", A.W. Norman, R. Bouillon, M.

Thomasset, Eds., W. de Gruyter, NY, 1991, p. 787.

4. a) Nishii, Y.; Abe, S.; Kobayashi, T.; Okano, T.; Tsugawa, N.; Slatopolsky, E.; Brown, A.J.; Dusso, A.; Raisz, L.G. in "Vitamin D: Gene Regulation, Structure-Function Analysis, and

Clinical Applications", A.W. Norman, R. Bouillon, R. Thomasset, Eds., W. de Gruyter, NY, 1991, p. 294; b) Okano, T.; Tsugawa, N.; Masuda, S.;

Takeuchi, A.; Kobayashi, T.; Takita, Y.; Nishii, Y.; Biochem . Biophys . Res . Commun . , 1989, 163 , 1444; c) Miyamoto, K.; Murayama, E.; Ochi, K.;

Watanabe, H.; Kubodera, N.; Chem . Pharm . Bull . , 1993, 41 , 1111.

5. a) Posner, G.H.; Nelson, T.D.; Kinter, CM.; Johnson, N.; J. Org. Chem . , 1992, 57 , 4083; b) Posner, G.H.; Nelson, T.D.; Kinter, CM.,

Afarinkia, K.; Tetrahedron Lett . , 1991, 32 , 5295; c) Afarinkia, K.; Posner, G.H.; Ibid, 1992, 33 , 7839; d) for a review, see Afarinkia, K.; Vinader, V.; Nelson, T.D.; Posner, G.H.; Tetrahedron , 1992, 48 , 9111. 6. a) Gupta, R.B.; Frank, R.W.; J. Am.

Chem. Soc, 1987, 109, 5393; b) Jensen, F.; Foote, C.S.; Ibid, 1987, 109, 6376; c) Baran, J.; Mayr, H.; Ruster, V.; Klarner, F.G.; J. Org. Chem., 1989, 54, 5016; d) Gassman, P.G.; Gorman, D.B.; J. Am. Chem. Soc, 1990, 112, 8624; e) Reissig, H-U.; Hippeli, C; Arnold, T.; Chem. Ber., 1990, 123, 2403; f) Van Mele, B.; Huybrechts, G; Int. J.

Chem. Kinetics, 1989, 21, 967; g) Sauer, J.; Lang, D.; Wiest, H.; Chem. Ber., 1964, 97, 3208; g) cf. Li, Y.; Houk, K.N.; J. Am. Chem. Soc, 1993, 115, 7478 and references therein.

7. a) Karplus, M.; J. Chem. Phys., 1959, 30, 11; b) Karplus, J.; J. Am. Chem. Soc, 1963, 85, 2870; c) Jackman, L.M.; Sternhell, S.;

Applications of NMR Spectroscopy in Organic

Chemistry, 2nd Ed.; Pergamon, NY, 1969.

8. a) Posner, G.H., Kinter, CM.; J. Org. Chem., 1990, 55, 3967; b) Posner, G.H.; Nelson, T.D.; Tetrahedron, 1990, 46, 4573; c) Posner,

G.H.; Wettlaufer, D.G.; J. Jim. Chem. Soc, 1986, 108, 7373.

9. Moeller, K.D.; Tinao, L.V.; J. Am. Chem. Soc, 1992, 114, 1033 and references therein.

10. 2-Pyrone, prone to rapid polymerization under thermal conditions, undergoes non- regioselective cycloadditions under very high pressure conditions (e.g. 18.5 Kbar): Markό, I.E.; Seres, P.; Swarbrick, T.M.; Staton, I.; Adams, H.; Tetrahedron Lett., 1992, 33, 5649.

11. Posner, G.H.; Carry, J.-C; Anjeh,

T.E.N.; French, A.N.; J. Org. Chem., 1992, 57, 7012.

12. Mandi, T.; Imaji, M. ; Takada, H.;

Kawata, M. ; Nokami, J. ; Tsuji, J.; J. Org. Chem., 1989, 54, 5395.

13. a) Posner, G.H.; Crouch, R.D.; Kinter, CM.; Carry, J.-C; J. Org. Chem., 1991, 56, 6981; b) Posner, G.H.; Dai, H. ; BioMed. Chem. Lett., 1993, 3, 1829.

14. Lythgoe, B. ; Moran, T.A.; Nambudiry, M.E.N.; Tideswell, J. Wright, P.W.; J. Chem. Soc Perkin Trans. 1, 1978, 590. 15. Posner, G.H.; Nelson, T.C; Guyton, K.Z.; Kensler, T.W.;; J. Med . Chem. , 1992, 35 , 3280.

16. Unpublished results of G.H. Posner and H. Liu.

17. Unpublished results of G.H. Posner, T. D. Nelson, and C.H. Oh.

18. Filler, R. et al. (Eds.); Organo fluorine Compounds in Medicinal Chemistry and Biomedical Applications , Elsevier Science Publishers, 1993.

19. Kobayashi, Y; Nakazawa, M. ; Kumadaki, I., Taguchi, T., Ohshima, E.; Ikekawa, N.; Tanaka, Y.; DeLuca, H.F.; Chem. Pharm . Bull . , 1986, 34 , 1568-1572.

20. Ohshima, E.; Sai, H.; Takatsuto, S.;

Ikekawa, N.; Kobayashi, Y.; Tanaka, Y.; DeLuca, H.F.; Chem . Pharm . Bull . , 1984, 32 , 3525-3531.

21. Oshida, L-i; Morisaki, M. ; Ikekawa, N.; Tetrahedron Lett . , 1980, 21 , 1755-1756. While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not to be limited to the disclosed embodiments, but on the contrary is intended to cover various

modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A vitamin D₃ analogue which includes a substituent in the 2-position selected from the group consisting of alcohols and fluorides.

2. The analogue of claim 1 wherein said substituent is -(CH₂)₄OH.

3. The analogue of claim 2 having the following structural formula:

4. The analogue of claim 2 having the following structural formula:

5. The analogue of claim 1 wherein said substituent is -(CH₂)₃F.

6. The analogue of claim 1 wherein said substituent is -(CH₂)₄F.

7. A method for the preparation of a vitamin D₃ analogue which includes an alcohol substituent in the 2-position, comprising the steps of:

a) subjecting methyl 2-pyrone-3-carboxylate and a silylated vinylic ether to high pressure cycloaddition to form a bicycloadduct;

b) reacting the bicycloadduct with lithium allyloxide to produce a mixed methyl allyl malonate; c) decarboxylating the mixed methyl allyl malonate with palladium acetate to yield a

cyclohexene ester;

d) silylating and reducing the cyclohexene ester to form an alcohol;

e) subjecting the allylic alcohol to a Claisen rearrangement followed by spontaneous thermal sulfoxide elimination to form a

Z-dienoate;

f) reacting the Z-dienoate with a hydride to form an allylic alcohol;

g) reacting the allylic alcohol with N- chlorosuccinimide and dimethyl sulfide to form an allylic chloride;

h) reacting the allylic chloride with diphenylphosphide and then with hydrogen peroxide to form a phosphine oxide;

i) reacting the phosphine oxide with

phenyllithium to produce a conjugate base of the phosphine oxide;

j) coupling the conjugate base of phosphine oxide with a C,D-ring ketone to produce O- silylated derivatives of diastereomeric 2-(4'- hydroxylated) calcitriol analogues; and

k) separating and isolating the analogues.