26-Desmethyl-2-methylene-22-ene-19-nor-1α,25-dihydroxyvitamin D₃ compounds selectively active on intestine

Abstract

Six new analogs of 2-methylene-19-nor-1α,25-dihydroxyvitamin D₃, 6–7 and 8a,b–9a,b, have been synthesized. All compounds are characterized by a trans double bond located in the side chain between C-22 and C-23. While compounds 6 and 7 possess C-26 and C-27 methyls, compounds 8a,b and 9a,b lack one of these groups. A Lythgoe-based synthesis, employing the Wittig–Horner reaction was used for these preparations. Two different types of Δ²²E-25-hydroxy Grundmann’s ketone, having either only one stereogenic center located at position C-20 (20 and 21), or two stereogenic centers located at 20- and 25-positions (24a,b–25a,b) were obtained by a multi-step procedure from commercial vitamin D₂. The introduction of a double bond at C-22 appeared to lower biological activity in vitro and in vivo. Further removal of a 26-methyl in these analogs had little effect on receptor binding, HL-60 differentiation and CYP24A expression but markedly diminished or eliminated in vivo activity on bone calcium mobilization while retaining activity on intestinal calcium transport.

1. Introduction

1α,25-Dihydroxyvitamin D₃, [1α,25(OH)₂D₃] (1) is perhaps the central regulator of calcium homeostasis [1,2]. In addition, 1α,25(OH)₂D₃ plays a role in controlling differentiation and growth of a variety of cells and may play a significant role in the activity of B and T cells [3–8]. The biological responses to 1α,25(OH)₂D₃ are mediated by the vitamin D receptor (VDR), which is a member of the nuclear receptor superfamily. It acts as a ligand-dependent gene transcription factor [1]. 1α,25(OH)₂D₃ and its analogs have significant therapeutic potential in the treatment of osteoporosis, vitamin D-resistant rickets, secondary hyperparathyroidism, psoriasis, and renal osteodystrophy [1]. However, use of 1α,25(OH)₂D₃ itself is limited because it induces significant hypercalcemia. A number of 1α,25(OH)₂D₃ analogs have therefore been synthesized, and some of them have been shown to have low calcemic activity [9] (Fig. 1). Two of these analogs, 19-nor-1α,25-(OH)₂D₂ (paricalcitol, Zemplar) (2) and 1α-(OH)D₂ (doxercalciferol, Hectorol) (3) have been developed and used to treat secondary hyperpathyroidism (SH) [3,10].

Fig. 1.

Chemical structures of 1α,25-dihydroxyvitamin D₃ (calcitriol, 1) and its analogs.

In our continuing effort to identify vitamin D₃ hormone analogs with selective biological activity, we have recently given focus to the synthesis and characterization of 2-substituted 19-nor derivatives with various side chain modifications. This endeavor has yielded several tissue-selective compounds with therapeutic potential [3,11], among them 2MD (4) (Fig. 1), one of the most promising analogs [12]. This analog is at least 30-fold more effective than 1α,25(OH)₂D₃ in stimulating osteoblast-mediated bone calcium mobilization while being approximately equally potent in supporting intestinal calcium transport [13].

A very recent addition to our ongoing structure–activity relationship studies has been the development of 2-methylene-19,26-dinor-1α,25-dihydroxyvitamin D₃ analogs [14]. Indeed, the results of our biological studies revealed that removing only one of the two methyl groups at C-25 and maintaining the 25-hydroxy group is an effective method of weakening calcemic activity [14]. In general, (25R)-hydroxy analogs exhibit more efficacy, measured both in vitro and in vivo, than (25S) diastereoisomers, with the (20S,25R)-2-methylene-19,26-dinor-1α,25(OH)₂D₃ analog 5 (Fig. 1), being the most potent of the new series [14]. We have now prepared two new 2-methylene-Δ²²E-19-nor-1α,25(OH)₂D₃ compounds 6 (20R) and 7 (20S) (Fig. 1), which are characterized by the presence of a double bond between C-22 and C-23 in the side chain, as in vitamin D2 analogs 2 and 3 (Fig. 1). Then, to probe whether combining the introduction of a double bond at C-22 with the absence of one of the two methyl groups at C-25 (as in 2-methylene-19,26-dinor-1α,25-dihydroxyvitamin D₃ compound 5 (Fig. 1), might improve tissue selectivity while reducing the calcemic activity, we also synthesized four new 2-methylene-Δ²²E-19,26-dinor-1α,25(OH)₂D₃ compounds (8a, 8b, 9a, 9b; Fig. 1). Structurally all these six new 2-methylene-19-nor-vitamin D ana-logs have a hydroxyl substituent attached to C-25 in the side chain, and a trans double bond located between C-22 and C-23 in the side chain (Δ²²E).

In addition, in compounds 8a,b–9a,b one of the two methyl groups normally located at C-25 in the side chain has been replaced with a hydrogen atom (26-nor). Therefore, the side chains of these last four compounds have two stereogenic centers located at the 20- and 25-positions, and all the four possible 2-methylene-Δ²²E-19,26-dinor-1α,25(OH)₂D₃ stereoisomers 8a (20R,25R), 8b (20R,25S), 9a (20S,25R), and 9b (20S,25S) (Fig. 1) are described.

2. Experimental methods

2.1. General

Optical rotations were measured in chloroform or methanol using a Perkin–Elmer model 343 polarimeter at 22 °C. Ultraviolet (UV) absorption spectra were recorded with a Perkin–Elmer Lambda 3B UV–Vis spectrophotometer in ethanol. ¹H nuclear magnetic resonance (NMR) spectra were recorded in deuteriochloroform, or acetone-d₆, at 400 and 500 MHz with Bruker Instruments DMX-400 and DMX-500 Avance console spectrometers. ¹³C NMR spectra were recorded in deuteriochloroform, at 100 and 125 MHz with the same Bruker Instruments. Chemical shifts (δ) in parts per million are quoted relative to internal Me₄Si (δ 0.00). Electron impact (EI) mass spectra were obtained with a Micromass AutoSpec (Beverly, MA) instrument. HPLC was performed on a Waters Associates liquid chromatograph equipped with a model 6000A solvent delivery system, model U6K Universal injector, and model 486 tunable absorbance detector. THF was freshly distilled before use from sodium benzophenone ketyl under argon. A designation “(volume + volume)”, which appears in general procedures, refers to an original volume plus a rinse volume.

Both final vitamin D analogues synthesized by us gave single sharp peaks on HPLC, and they were judged at least 99.5% pure. The purity and identity of the synthesized vitamins were additionally confirmed by inspection of their ¹H NMR, ¹³C NMR, UV absorption, and high-resolution mass spectra.

2.2. Synthesis of compounds

2.2.1. General procedure for the synthesis of compounds 13, 14, 16a, 16b, 17a, 17b

To a stirred suspension of the phosphonium salt 12 or 15a–b (3.0 equiv) [14] in anhydrous THF (5 mL), n-butyllithium (6.0 equiv) was added at −20 °C. The solution was stirred at −20 °C for 1 h and it turned deep orange. A pre-cooled solution of aldehyde 10 or 11 (1 equiv) [14] in anhydrous THF (1 + 1 mL) was added and the reaction mixture was stirred at −20 °C for 4 h and at room temperature for 18 h. The reaction was quenched with water and the mixture was extracted with ethyl acetate. Combined organic phases were washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica (5–10% ethyl acetate/hexane) to give the product 13, 14, 16a, 16b, 17a, 17b.

2.2.2. (8S,20R)-Des-A,B-8-benzoyloxy-20-[4′-hydroxy-4′-methylpent-(1′E)-en-yl]-pregnane (13)

According to a general procedure the pure product 13 (67 mg, 47% yield) was obtained from the aldehyde 10 (117 mg, 0.37 mmol), the phosphonium iodide 12 (476 mg, 1.11 mmol) and n-butyllithium (1.95 M, 1.14 mL, 2.22 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=+87.8°$ (c 2.75, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.06 (2H, m, o-H_Bz), 7.55 (1H, m, p-H_Bz), 7.44 (2H, m, m-H_Bz), 5.41 (1H, s, 8α-H), 5.39 (2H, m, 22-H and 23-H), 1.19 (6H, s, 26,27-H₆), 1.07 (3H, s, 18-H₃), 1.06 (3H, d, J = 6.7 Hz, 21-H₃); ¹³C NMR (100 MHz) δ 166.40, 141.29, 132.64, 130.80, 129.48, 128.29, 122.80, 72.11, 70.46, 55.93, 51.60, 46.79, 41.79, 40.00, 39.77, 30.45, 28.97, 27.69, 22.61, 20.55, 17.96, 13.70; exact mass calculated for C₂₅H₃₆O₃Na (MNa⁺) 407.2562, found 407.2548.

2.2.3. (8S,20S)-Des-A,B-8-benzoyloxy-20-[4′-hydroxy-4′-methyl-pent-(1′E)-en-yl]-pregnane (14)

According to a general procedure the pure product 14 (52 mg, 45% yield) was obtained from the aldehyde 11 (93 mg, 0.30 mmol), the phosphonium iodide 12 (476 mg, 1.11 mmol) and n-butyllithium (1.61 M, 1.38 mL, 2.22 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=-25.1°$ (c 2.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (2H, m, o-H_Bz), 7.55 (1H, m, p-H_Bz), 7.44 (2H, m, m-H_Bz), 5.42 (3H, m, 8α-H, 22-H, 23-H), 1.22 (6H, s, 26,27-H₆), 1.04 (3H, s, 18-H₃), 0.94 (3H, d, J = 6.6 Hz, 21-H₃); ¹³C NMR (125 MHz) δ 166.41, 141.34, 132.64, 130.83, 129.50, 128.29, 122.86, 72.06, 70.68, 56.30, 51.46, 46.92, 41.91, 40.23, 39.33, 30.57, 29.12, 29.11, 26.83, 22.49, 21.57, 17.78, 13.80; exact mass calculated for C₂₅H₃₆O₃Na (MNa⁺) 407.2562, found 407.2561.

2.2.4. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(4′R)-hydroxy-pent-(1′E)-en-yl]-pregnane (16a)

According to a general procedure the pure product 16a (47 mg, 49% yield) was obtained from the aldehyde 10 (81 mg, 0.26 mmol), the phosphonium iodide 15a (361 mg, 0.78 mmol) and n-butyllithium (1.6 M, 980 µL, 1.56 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=+69.6°$ (c 1.3, CHCl₃); ¹H NMR (500 MHz, acetone-d₆) δ 8.05 (2H, m, o-H_Bz), 7.62 (1H, m, p-H_Bz), 7.52 (2H, m, m-H_Bz), 5.41 (1H, dt, J = 15.4, 7.0 Hz, 23-H) 5.38 (1H, d, J = 1.8 Hz, 8α-H), 5.31 (1H, dd, J = 15.4, 8.4 Hz, 22-H), 3.72 (1H, m, 25-H), 3.37 (1H, d, J = 4.0 Hz, OH) 1.102 (3H, d, J = 6.4 Hz, 27-H₃), 1.096 (3H, s, 18-H₃), 1.05 (3H, d, J = 6.6 Hz, 21-H₃); ¹³C NMR (100 MHz) δ 166.44, 140.80, 132.66, 130.84, 129.51, 128.32, 123.25, 72.14, 67.20, 55.97, 51.64, 42.37, 41.84, 39.91, 39.80, 30.49, 27.58, 22.57, 22.57, 20.59, 17.99, 13.72; exact mass calcd for C₂₄H₃₄O₃ (M⁺) 370.2508, found 370.2503.

2.2.5. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(4′S)-hydroxy-pent-(1′E)-en-yl]-pregnane (16b)

According to a general procedure the pure product 16b (42 mg, 52% yield) was obtained from the aldehyde 10 (70 mg, 0.22 mmol), the phosphonium iodide 15b (310 mg, 0.67 mmol) and n-butyllithium (1.6 M, 840 µL, 1.34 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=+98.7°$ (c 1.75, CHCl₃); ¹H NMR (500 MHz, acetone-d₆) δ 8.05 (2H, m, o-H_Bz), 7.63 (1H, m, p-H_Bz), 7.52 (2H, m, m-H_Bz), 5.42 (1H, dt, J = 15.2, 7.0 Hz, 23-H) 5.38 (1H, d, J = 2.5 Hz, 8α-H), 5.32 (1H, dd, J = 15.2, 8.5 Hz, 22-H), 3.72 (1H, m, 25-H), 3.32 (1H, d, J = 4.4 Hz, OH) 1.102 (3H, d, J = 6.1 Hz, 27-H₃), 1.096 (3H, s, 18-H₃), 1.05 (3H, d, J = 6.6 Hz, 21-H₃); ¹³C NMR (100 MHz) δ 166.43, 140.86, 132.66, 130.82, 129.50, 128.32, 123.42, 72.12, 67.15, 55.87, 51.63, 42.48, 41.81, 39.93, 39.79, 30.47, 27.65, 22.59, 22.48, 20.47, 17.98, 13.72; exact mass calcd for C₂₄H₃₄O₃ (M⁺) 370.2508, found 370.2491.

2.2.6. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(4′R)-hydroxy-pent-(1′E)-en-yl]-pregnane (17a)

According to a general procedure the pure product 17a (39 mg, 48% yield) was obtained from the aldehyde 11 (70 mg, 0.22 mmol), the phosphonium iodide 15a (221 mg, 0.66 mmol) and n-butyllithium (1.6 M, 720 µL, 1.15 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=-28.8°$ (c 0.8, CHCl₃); ¹H NMR (500 MHz, acetone-d₆) δ 8.05 (2H, m, o-H_Bz), 7.63 (1H, m, p-H_Bz), 7.52 (2H, m, m-H_Bz), 5.46 (1H, dt, J = 15.4, 6.9 Hz, 23-H) 5.38 (1H, s, 8α-H), 5.36 (1H, dd, J = 15.4, 8.5 Hz, 22-H), 3.76 (1H, m, 25-H), 3.49 (1H, d, J = 4.0 Hz, OH) 1.13 (3H, d, J = 6.2 Hz, 27-H₃), 1.07 (3H, s, 18-H₃), 0.92 (3H, d, J = 6.7 Hz, 21-H₃); ¹³C NMR (100 MHz) δ 166.45, 140.74, 132.67, 130.86, 129.53, 128.32, 123.33, 72.08, 67.70, 56.33, 51.48, 42.46, 41.94, 40.16, 39.48, 30.60, 26.86, 22.74, 22.50, 21.46, 17.81, 13.89; exact mass calcd for C₂₄H₃₄O₃Na (MNa⁺) 393.2406, found 393.2407

2.2.7. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(4′S)-hydroxy-pent-(1′E)-en-yl]-pregnane (17b)

According to a general procedure the pure product 17b (37 mg, 50% yield) was obtained from the aldehyde 11 (65 mg, 0.2 mmol), the phosphonium iodide 15b (201 mg, 0.6 mmol) and n-butyllithium (1.6 M, 560 µL, 0.9 mmol). ${[\alpha ]}_{\mathrm{D}}^{24}=-11.4°$ (c 1.4, CHCl₃); ¹H NMR (500 MHz, acetone-d₆) δ 8.04 (2H, m, o-H_Bz), 7.63 (1H, m, p-H_Bz), 7.52 (2H, m, m-H_Bz), 5.46 (1H, dt, J = 15.4, 6.8 Hz, 23-H) 5.39 (1H, s, 8α-H), 5.35 (1H, dd, J = 15.4, 6.3 Hz, 22-H), 3.78 (1H, m, 25-H), 3.40 (1H, d, J = 4.2 Hz, OH) 1.13 (3H, d, J = 6.2 Hz, 27-H₃), 1.07 (3H, s, 18-H₃), 0.93 (3H, d, J = 6.7 Hz, 21-H₃); ¹³C NMR (100 MHz) δ 166.45, 141.11, 132.66, 130.87, 129.53, 128.32, 123.41, 72.09, 67.23, 56.34, 51.47, 42.56, 41.95, 40.15, 39.37, 30.59, 26.80, 22.73, 22.49, 21.56, 17.83, 13.85; exact mass calcd for C₂₄H₃₄O₃Na (MNa⁺) 393.2406, found 393.2410.

2.2.8. General procedure for the synthesis of compounds 18, 19, 22a, 22b, 23a, 23b

To a stirred solution of the alcohol 13, 14, 16a, 16b, 17a or 17b (1.0 equiv) and 2,6-lutidine (3.5 eq.) in anhydrousmethylene chloride (3 mL), tert-butyldimethylsilyl trifluoromethane-sulfonate (1.8 equiv) was added at −20 °C. The reaction mixture was stirred at 0 °C for1 h. It was quenched with water and extracted with methylene chloride. Combined organic phases were washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (3% ethyl acetate/hexane) to give the product 18, 19, 22a, 22b, 23a, 23b.

2.2.9. (8S,20R)-Des-A,B-8-benzoyloxy-20-[4′-(tertbutyldimethylsilyloxy)-4′-methyl-pent-(1′E)-en-yl]-pregnane (18)

According to a general procedure the pure product 18 (67 mg, 96% yield) was obtained from the alcohol 13. [α]_D +62.9 (c 3.35, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.06 (2H, m, o-H_Bz), 7.55 (1H, m, p-H_Bz), 7.44 (2H, m, m-H_Bz), 5.41 (1H, d, J = 2.3 Hz, 8α-H), 5.38 (1H, m, 23-H), 5.24 (1H, dd, J = 15.4, 8.4 Hz, 22-H), 1.15 (6H, d, J = 2.0 Hz, 26,27-H₆), 1.07 (3H, s, 18-H₃), 1.04 (3H, d, J = 6.6 Hz, 21-H₃), 0.86 (9H, s, Si-t-Bu), 0.06 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 166.44, 139.08, 132.65, 130.90, 129.53, 128.32, 124.31, 73.67, 72.20, 56.26, 51.69, 48.33, 41.82, 39.97, 39.84, 30.54, 29.74, 29.40, 27.66, 25.81, 22.66, 20.57, 18.03, 18.03, 13.72, −2.05; exact mass calculated for C₃₁H₅₀O₃SiNa (MNa⁺) 521.3427, found 521.3422.

2.2.10. (8S,20S)-Des-A,B-8-benzoyloxy-20-[4′-(tert-butyldimethylsilyloxy)-4′-methyl-pent-(1′E)-en-yl]-pregnane (19)

According to a general procedure the pure product 19 (65 mg, 93% yield) was obtained from the alcohol 14. [α]_D −21.2 (c 4.95, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.05 (2H, m, o-H_Bz), 7.54 (1H, m, p-H_Bz), 7.43 (2H, m, m-H_Bz), 5.41 (2H, m, 8α-H and 23-H), 5.29 (1H, dd, J = 15.4, 9.1 Hz, 22-H), 1.18 (6H, d, J = 4.5 Hz, 26,27-H₆), 1.04 (3H, s, 18-H₃), 0.93 (3H, d, J = 6.6 Hz, 21-H₃), 0.87 (9H, s, Si-t-Bu), 0.08 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 166.43, 139.10, 132.62, 130.91, 129.53, 128.30, 124.39, 73.72, 72.14, 56.44, 51.52, 48.29, 41.94, 40.30, 39.28, 30.63, 29.76, 29.65, 26.88, 25.83, 22.56, 21.53, 18.04, 17.82, 13.68, −2.04; exact mass calculated for C₃₁H₅₀O₃SiNa (MNa⁺) 521.3427, found 521.3450.

2.2.11. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(4′R)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnane (22a)

According to a general procedure the pure product 22a (30 mg, 78% yield) was obtained from the alcohol 16a. [α]_D +53.8 (c 1.1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 8.05 (2H, m, o-H_Bz), 7.55 (1H, m, p-H_Bz), 7.44 (2H, m, m-H_Bz), 5.41 (1H, s, 8α-H), 5.35–5.26 (2H, m, 22-H and 23-H), 3.81 (1H, m, 25-H), 1.12 (3H, d, J = 6.0 Hz, 27-H₃), 1.06 (3H, s, 18-H₃), 1.03 (3H, d, J = 6.6 Hz, 21-H₃), 0.88 (9H, s, Si-t-Bu), 0.06 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 166.86, 138.78, 132.56, 131.11, 129.77, 128.50, 124.63, 72.62, 69.13, 56.51, 51.96, 43.16, 42.15, 39.87, 30.53, 27.54, 26.08, 23.53, 22.88, 22.60, 18.45, 18.36, 18.05, 13.98, −4.32, −4.45.

2.2.12. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(4′S)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnane (22b)

According to a general procedure the pure product 22b (37 mg, 95% yield) was obtained from the alcohol 16b. [α]_D +54.1 (c 1.2, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 8.05 (2H, m, o-H_Bz), 7.55 (1H, m, p-H_Bz), 7.44 (2H, m, m-H_Bz), 5.41 (1H, s, 8α-H), 5.35–5.26 (2H, m, 22-H and 23-H), 3.78 (1H, m, 25-H), 1.10 (3H, d, J = 6.0 Hz, 27-H₃), 1.07 (3H, s, 18-H₃), 1.03 (3H, d, J = 6.6 Hz, 21-H₃), 0.89 (9H, s, Si-t-Bu), 0.05 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 166.70, 138.93, 132.88, 131.13, 129.77, 128.55, 124.44, 72.44, 69.23, 56.47, 51.92, 43.15, 42.05, 39.99, 30.77, 27.74, 26.12, 23.45, 22.85, 22.63, 18.40, 18.26, 18.04, 13.96, −4.32, −4.45.

2.2.13. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(4′R)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnane (23a)

According to a general procedure the pure product 23a (24 mg, 84% yield) was obtained from the alcohol 17a. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (2H, m, o-H_Bz), 7.54 (1H, m, p-H_Bz), 7.42 (2H, m, m-H_Bz), 5.41 (1H, s, 8α-H), 5.40–5.20 (2H, m, 22-H and 23-H), 3.78 (1H, m, 25-H), 1.11 (3H, d, J = 6.0 Hz, 27-H₃), 1.02 (3H, s, 18-H₃) 0.88 (9H, s, Si-t-Bu), 0.82 (3H, d, J = 6.5 Hz, 21-H₃), 0.04 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 166.52, 138.87, 132.66, 130.90, 129.55, 128.33, 124.17, 72.15, 68.74, 56.38, 52.18, 42.89, 41.88, 40.08, 34.86, 30.61, 26.98, 25.80, 23.67, 22.68, 18.61, 18.48, 18.03, 13.78, −4.47, −4.75.

2.2.14. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(4′S)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnane (23b)

According to a general procedure the pure product 23b (35 mg, 89% yield) was obtained from the alcohol 17b. ¹H NMR (600 MHz, CDCl₃) δ 8.05 (2H, m, o-H_Bz), 7.56 (1H, m, p-H_Bz), 7.45 (2H, m, m-H_Bz), 5.45 (1H, s, 8α-H), 5.33–5.24 (2H, m, 22-H and 23-H), 3.80 (1H, m, 25-H), 1.18 (3H, d, J = 6.0 Hz, 27-H₃), 1.05 (3H, s, 18-H₃), 0.95 (3H, d, J = 6.6 Hz, 21-H₃), 0.88 (9H, s, Si-t-Bu), 0.051 (6H, s, SiMe₂); ¹³C NMR (125 MHz) δ 166.47, 139.55, 132.85, 131.11, 129.73, 128.52, 124.42, 72.40, 69.17, 56.67, 51.26, 43.25, 42.15, 40.22, 39.40, 30.77, 26.81, 26.12, 23.45, 22.85, 19.42, 18.26, 18.04, 13.96, −4.32, −4.45.

2.2.15. General procedure for the synthesis of compounds 20, 21, 24a, 24b, 25a, 25b

To a stirred solution of the benzoate 18, 19, 22a, 22b, 23a or 23b in anhydrous ethanol (10 mL), a solution of sodium hydroxide in anhydrous ethanol (2.5 M, 2 mL) was added. The reaction mixture was refluxed for 18 h. It was cooled to room temperature, neutralized with 5% aqueous solution of HCl and extracted with methylene chloride. Combined organic phases were washed with a saturated aqueous NaHCO₃ solution, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (5–10% ethyl acetate/hexane) to give the alcohol. Pyridinium dichromate (5 equiv) was added to a solution of the alcohol (1 equiv) and pyridinium p-toluenesulfonate (0.3 equiv) in anhydrous methylene chloride (5 mL). The resulting suspension was stirred at room temperature for 3 h. The reaction mixture was filtered through a Waters silica Sep-Pak cartridge (5 g) that was further washed with hexane/ethyl acetate (8:2). After removal of solvents the ketone 20, 21, 24a, 24b, 25a, or 25b was obtained.

2.2.16. (20R)-Des-A,B-20-[4′-(tert-butyldimethylsilyloxy)-4′-methylpent-(1′E)-en-yl]-pregnan-8-one (20)

According to a general procedure the pure product 20 (22 mg, 93% yield) was obtained from the benzoate 18 in two steps. [α]_D −5.8 (c 1.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.40 (1H, ddd, J = 15.3, 7.8, 7.1 Hz, 23-H), 5.25 (1H, dd, J = 15.3, 8.4 Hz, 22-H), 2.45 (1H, dd, J = 11.2, 7.6 Hz), 1.16 (6H, s, 26,27-H₆), 1.05 (3H, d, J = 6.6 Hz, 21-H₃), 0.86 (9H, s, Si-t-Bu), 0.66 (3H, s, 18-H₃), 0.06 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 212.01, 138.45, 124.87, 73.63, 62.03, 56.48, 49.77, 48.30, 40.96, 39.86, 38.84, 29.76, 29.42, 27.87, 25.80, 24.06, 20.76, 19.06, 18.04, 12.66, −2.05; exact mass calculated for C₂₄H₄₄O₂SiNa (MNa⁺) 415.3008, found 415.3022.

2.2.17. (20S)-Des-A,B-20-[4′-(tert-butyldimethylsilyloxy)-4′-methylpent-(1′E)-en-yl]-pregnan-8-one (21)

According to a general procedure the pure product 21 (27 mg, 92% yield) was obtained from the benzoate 19 in two steps. [α]_D −39.3 (c 1.35, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 5.42 (1H, ddd, J = 15.4, 8.2, 7.0 Hz, 23-H), 5.28 (1H, dd, J = 15.4, 9.1 Hz, 22-H), 2.42 (1H, dd, J = 11.4, 7.6 Hz), 1.17 (6H, s, 26,27-H₆), 0.94 (3H, d, J = 6.6 Hz, 21-H₃), 0.86 (9H, s, Si-t-Bu), 0.61 (3H, s, 18-H₃), 0.069 and 0.065 (each 3H, each s, each SiMe₂); ¹³C NMR (100 MHz) δ 212.10, 138.69, 124.97, 73.65, 61.91, 56.57, 50.03, 48.23, 41.03, 40.44, 38.28, 29.79, 29.62, 27.21 (t), 25.79, 23.93, 21.52, 18.97, 18.03, 12.49, −2.06; exact mass calculated for C₂₄H₄₄O₂SiNa (MNa⁺) 415.3008, found 415.3018.

2.2.18. (20R)-Des-A,B-20-[(4′R)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnan-8-one (24a)

According to a general procedure the pure product 24a (9 mg, 61% yield) was obtained from the benzoate 20a in two steps. ¹H NMR (400 MHz, CDCl₃) δ 5.38–5.23 (2H, m, 22-H and 23-H), 3.79 (1H, m, 25-H), 2.44 (1H, m), 1.07 (3H, d, J = 6.6 Hz, 27-H₃), 0.95 (3H, d, J = 6.6 Hz, 21-H₃), 0.89 (9H, s, Si-t-Bu), 0.67 (3H, s, 18-H₃), 0.046 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 211.97, 138.20, 124.71, 68.97, 62.08, 56.51, 49.78, 42.90, 40.87, 39.67, 38.85, 27.73, 25.84, 24.09, 23.17, 20.70, 19.07, 18.11, 12.68, −4.52, −4.68; exact mass calcd for C₂₃H₄₂O₂Si Na (MNa)⁺401.2852, found 401.2847.

2.2.19. (20R)-Des-A,B-20-[(4′S)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnan-8-one (24b)

According to a general procedure the pure product 24b (16 mg, 81% yield) was obtained from the benzoate 22b in two steps. ¹H NMR (400 MHz, CDCl₃) δ 5.34–5.20 (2H, m, 22-H and 23-H), 3.74 (1H, m, 25-H), 2.41 (1H, dd, J = 11.5, 7.6 Hz), 1.05 (3H, d, J = 6.1 Hz, 27-H₃), 0.99 (3H, d, J = 6.6 Hz, 21-H₃), 0.84 (9H, s, Si-t-Bu), 0.61 (3H, s, 18-H₃), 0.043 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 211.97, 138.10, 124.74, 68.92, 62.02, 56.46, 49.76, 42.89, 40.95, 39.69, 38.85, 27.73, 25.88, 24.05, 23.23, 20.61, 19.03, 18.17, 12.68, −4.54, −4.69; exact mass calcd for C₂₃H₄₂O₂Si Na (MNa)⁺ 401.2852, found 401.2845.

2.2.20. (20S)-Des-A,B-20-[(4′R)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnan-8-one (25a)

According to a general procedure the pure product 25a (7 mg, 67% yield) was obtained from the benzoate 23a in two steps. ¹H NMR (400 MHz, CDCl₃) δ 5.35–5.22 (2H, m, 22-H and 23-H), 3.74 (1H, m, 25-H), 2.41 (1H, dd, J = 11.5, 7.6 Hz), 1.13 (3H, d, J = 6.1 Hz, 27-H₃), 0.89 (9H, s, Si-t-Bu), 0.84 (3H, d, J = 5.9 Hz, 21-H₃), 0.63 (3H, s, 18-H₃), 0.053 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 212.13, 139.12, 124.44, 68.66, 62.22, 56.49, 50.04, 42.66, 41.05, 40.18, 33.85, 27.13, 25.89, 24.03, 23.78, 21.61, 18.93, 18.16, 12.70, −4.38, −4.70; exact mass calculated for C₂₃H₄₂O₂SiNa (MNa)⁺ 401.2852, found 401.2848.

2.2.21. (20S)-Des-A,B-20-[(4′S)-(tert-butyldimethylsilyl)oxy-pent-(1′E)-en-yl]-pregnan-8-one (25b)

According to a general procedure the pure product 25b (10 mg, 67% yield) was obtained from the benzoate 23b. ¹H NMR (400 MHz, CDCl₃) δ 5.35–5.22 (2H, m, 22-H and 23-H), 3.76 (1H, m, 25-H), 2.41 (1H, dd, J = 11.5, 7.6 Hz), 1.15 (3H, d, J = 6.1 Hz, 27-H₃), 1.01 (3H, s, 18-H₃), 0.88 (3H, d, J = 6.6 Hz, 21-H₃), 0.84 (9H, s, Si-t-Bu), 0.052 (6H, s, SiMe₂); ¹³C NMR (100 MHz) δ 211.97, 139.10, 124.44, 68.72, 62.32, 56.46, 51.76, 42.89, 41.15, 40.19, 33.85, 27.73, 25.88, 24.05, 23.60, 20.61, 19.03 18.27, 12.68, −4.54, −4.69; exact mass calculated for C₂₃H₄₂O₂Si Na (MNa)⁺ 401.2852, found 401.2848.

2.2.22. General procedure for the synthesis of compounds 27, 28, 29a, 29b, 30a, 30b

To a stirred solution of the phosphine oxide 26 (3.7 equiv) [15] in anhydrous THF (500 µL), a solution of phenyllithium (1.8 M in di-n-butylether, 1.2 equiv) was added at −20 °C under argon. The mixture was stirred for 30 min and then cooled to −78 °C. A precooled solution of the Grundmann′s type ketone 20, 21, 24a, 24b, 25a or 26b (1 equiv) in anhydrous THF (200 + 100 µL) was added via cannula and the reaction mixture was stirred for 4 h at −78 °C. Then the reaction mixture was stirred at 4 °C for 19 h. Ethyl acetate (20 mL) was added and the organic phase was washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified on a Waters silica Sep-Pak cartridge (0–2% ethyl acetate/hexane) to give the protected vitamin D compound 27, 28, 29a, 29b, 30a or 30b.

2.2.23. (20R)-1a-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19-nor-22(E)-ene-vitamin D₃ tert-butyldimethylsilyl ether (27)

According to a general procedure the pure protected analog 27 (35.06 mg, 83% yield) was obtained from the phosphine oxide 26 (62 mg, 107 µmol), PhLi (1.8 M in di-n-butylether, 60 µL, 114 µmol) and the ketone 20 (22 mg, 56 µmol). UV (in hexane) λ_max 262.5, 253.0, 245.0 nm; ¹H NMR (500 MHz, CDCl₃) δ 6.22 and 5.84 (each 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.38 (1H, ddd, J = 15.3, 7.7, 7.2 Hz, 23-H), 5.26 (1H, dd, J = 15.3, 8.5 Hz, 22-H), 4.97 and 4.92 (each 1H, each s, ═CH₂), 4.43 (2H, m, 1β- and 3α-H), 2.83 (1H, dm, J = 12.5 Hz, 9b-H), 2.51 (1H, dd, J = 13.2, 5.9 Hz, 10α-H), 2.46 (1H, dd, J = 12.6, 4.3 Hz, 4α-H), 2.33 (1H, dd, J = 13.2, 2.7 Hz, 10β-H), 2.18 (1H, dd, J = 12.6, 8.4 Hz, 4β-H), 1.165 (6H, s, 26,27-H₆), 1.02 (3H, d, J = 6.6 Hz, 21-H₃), 0.897 (9H, s, Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.863 (9H, s, Si-t-Bu), 0.562 (3H, s, 18-H₃), 0.081 (3H, s, SiMe), 0.071 (6H, s, 2× SiMe), 0.068 (3H, s, SiMe), 0.050 (3H, s, SiMe), 0.027 (3H, s, SiMe); ¹³C NMR (125 MHz) d 152.97, 141.15, 139.34, 132.76, 124.25, 122.40, 116.14, 106.26, 73.75, 72.51, 71.64, 56.36, 48.37, 47.60, 45.60, 40.49, 38.56, 29.79, 29.42, 28.74, 28.05, 25.84, 25.78, 23.43, 22.23, 20.88, 18.25, 18.17, 18.06, 12.28, −2.02, −4.86, −5.10; exact mass calculated for C₄₅H₈₄O₃Si₃Na (MNa⁺) 779.5626, found 779.5652.

2.2.24. (20S)-1a-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19-nor-22(E)-ene-vitamin D₃ tert-butyldimethylsilyl ether (28)

According to a general procedure the pure protected analog 28 (39.54 mg, 79% yield) was obtained from the phosphine oxide 26 (67 mg, 115 µmol), PhLi (1.8 M in di-n-butylether, 75 µL, 137 µmol) and the ketone 21 (26 mg, 66 µmol). UV (in hexane) λ_max 262.5, 253.0, 245.0 nm; ¹H NMR (400 MHz, CDCl₃) δ 6.21 and 5.83 (each 1H, each d, J = 11.1 Hz, 6- and 7-H), 5.38 (1H, ddd, J = 15.4, 8.4, 6.8 Hz, 23-H), 5.29 (1H, dd, J = 15.4, 8.7 Hz, 22-H), 4.97 and 4.92 (each 1H, each s, ═CH₂), 4.42 (2H, m, 1β- and 3α-H), 2.81 (1H, dm, J = 13.1 Hz, 9β-H), 2.52 (1H, dd, J = 13.2, 5.8 Hz, 10α-H), 2.46 (1H, dd, J = 12.7, 4.3 Hz, 4α-H), 2.33 (1H, dd, J = 13.2, 2.4 Hz, 10β-H), 2.17 (1H, dd, J = 12.7, 8.4 Hz, 4β-H), 1.16 (6H, s, 26,27-H₆), 0.93 (3H, d, J = 6.4 Hz, 21-H₃), 0.895 (9H, s, Si-t-Bu), 0.865 (9H, s, Si-t-Bu), 0.855 (9H, s, Si-t-Bu), 0.518 (3H, s, 18-H₃), 0.077 (3H, s, SiMe), 0.066 (9H, s, 3× SiMe), 0.047 (3H, s, SiMe), 0.025 (3H, s, SiMe); ¹³C NMR (100 MHz) δ 152.99, 141.32, 139.40, 132.63, 124.25, 122.42, 116.00, 106.24, 73.78, 72.53, 71.63, 56.67, 56.20, 48.29, 47.61, 45.76, 40.91, 39.86, 38.55, 29.83, 29.62, 28.77, 27.41, 25.84, 25.78, 23.23, 22.11, 21.57, 18.25, 18.16, 18.07, 12.11, −2.04, −4.86, −4.90, −5.10; exact mass calculated for C₄₅H₈₄O₃Si₃Na (MNa⁺) 779.5626, found 779.5651.

2.2.25. (20R,25R)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D₃ tert-butyldimethylsilyl ether (29a)

According to a general procedure the pure protected analog 29a (8 mg, 48% yield) was obtained from the phosphine oxide 26 (41 mg, 70 µmol), PhLi (1.8 M in di-n-butylether, 48 µL, 86 µmol) and the ketone 24a (9 mg, 23 µmol). For analytical purpose a sample of the protected vitamin 29a was further purified by HPLC (9.4 × 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol (99.9:0.1) solvent system, R_t = 3.70 min); UV (in hexane) λ_max 263.1, 253.2, 244.3 nm; ¹H NMR (400 MHz, CDCl₃) δ 6.23 and 5.83 (each 1H, each d, J = 11.7 Hz, 6- and 7-H), 5.40–5.24 (2H, m, 22-H and 23-H), 4.97 and 4.93 (each 1H, each s, ═CH₂), 4.40 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H), 2.78 (1H, dm, J = 12.1 Hz, 9β-H), 2.52 (1H, dd, J = 13.5, 6.1 Hz, 10α-H), 2.48 (1H, dd, J = 12.7, 4.5 Hz, 4α-H), 2.34 (1H, dd, J = 13.5, 6.3 Hz, 10β-H), 2.18 (1H, dd, J = 12.7, 8.6 Hz, 4β-H), 1.11 (3H, d, J = 6.3 Hz, 27-H₃), 0.96 (3H, d, J = 6.3 Hz, 21-H₃), 0.897 (9H, s, Si-t-Bu), 0.895 (9H, s, Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.56 (3H, s, 18-H₃), 0.081 (3H, s, SiMe), 0.067 (3H, s, SiMe), 0.055 (9H, s, 3xSiMe), 0.027 (3H, s, SiMe); ¹³C NMR (100 MHz) δ 152.96, 141.13, 138.98, 132.76, 124.74, 122.40, 116.09, 106.25, 72.54, 71.63, 68.73, 56.68, 56.33, 47.59, 45.57, 38.55, 36.13, 35.98, 28.76, 27.73, 26.11, 25.85, 25.72, 23.62, 23.46, 22.18, 20.70, 18.77, 18.25, 18.17, 12.24, −4.34, −4.62, −4.85, −4.87, −5.10; exact mass calculated for C₄₄H₈₂O₃Si₃₋ Na (MNa)⁺ 765.5470, found 765.5456.

2.2.26. (20R,25S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D₃ tert-butyldimethylsilyl ether (29b)

According to a general procedure the pure protected analog 29b (15 mg, 48% yield) was obtained from the phosphine oxide 26 (73 mg, 127 µmol), PhLi (1.8 M in di-n-butylether, 85 µL, 180 µmol) and the ketone 24b (16 mg, 42 µmol). For analytical purpose a sample of the protected vitamin 29b was further purified by HPLC (9.4×250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol (99.9:0.1) solvent system, R_t = 3.80 min); UV (in hexane) λ_max 263.1, 253.2, 244.3 nm; ¹H NMR (400 MHz, CDCl₃) δ 6.22 and 5.83 (each 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.38–5.27 (2H, m, 22-H and 23-H), 4.96 and 4.90 (each 1H, each s, ═CH₂), 4.43 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H), 2.82 (1H, dm, J = 11.8 Hz, 9β-H), 2.52 (1H, dd, J = 13.1, 5.9 Hz, 10α-H), 2.47 (1H, dd, J = 12.6, 4.3 Hz, 4α-H), 2.33 (1H, dd, J = 13.1, 2.3 Hz, 10β-H), 2.17 (1H, dd, J = 12.6, 8.7 Hz, 4β-H), 1.11 (3H, d, J = 6.0 Hz, 27-H₃), 1.02 (3H, d, J = 6.5 Hz, 21-H₃), 0.897 (9H, s, Si-t-Bu), 0.895 (9H, s, Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.56 (3H, s, 18-H₃), 0.081 (3H, s, SiMe), 0.067 (3H, s, SiMe), 0.055 (9H, s, 3× SiMe), 0.027 (3H, s, SiMe); ¹³C NMR (100 MHz) δ 152.96, 141.13, 138.98, 132.76, 124.74, 122.40, 116.09, 106.25, 72.54, 71.63, 68.73, 56.33, 47.59, 45.57, 42.97, 40.47, 38.55, 36.13, 35.98, 28.76, 27.73, 26.11, 25.85, 25.22, 23.42, 23.30, 22.18, 20.70, 18.25, 18.17, 12.24, −4.34, −4.62, −4.87, −5.10; exact mass calcd for C₄₄H₈₂O₃Si₃Na (MNa)⁺ 765.5470, found 765.5439.

2.2.27. (20S,25R)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D₃ tert-butyldimethylsilyl ether (30a)

According to a general procedure the pure product 30a (6 mg, 48% yield) was obtained from the phosphine oxide 26 (25 mg, 43 µmol), PhLi (1.8 M in di-n-butylether, 34 µL, 61 µmol) and the ketone 25a (6.5 mg, 17 µmol). For analytical purpose a sample of the protected vitamin 30a was further purified by HPLC (9.4 × 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol (99.9:0.1) solvent system, R_t = 3.70 min): UV (in hexane) λ_max 262.6, 253.0, 244.8 nm; ¹H NMR (600 MHz, CDCl₃) δ 6.22 and 5.84 (each 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.38–5.27 (2H, m, 22-H and 23-H), 4.97 and 4.91 (each 1H, each s, ═CH₂), 4.43 (2H, m, 1β and 3α-H), 3.77 (1H, m, 25-H), 2.83 (1H, dm, J = 12.6 Hz, 9β-H), 2.52 (1H, dd, J = 13.2, 6.0 Hz, 10α-H), 2.46 (1H, dd, J = 12.6, 4.5 Hz, 4α-H), 2.33 (1H, dd, J = 13.2, 2.9 Hz, 10β-H), 2.18 (1H, dd, J = 12.6, 8.3 Hz, 4β-H), 1.12 (3H, d, J = 6.0 Hz, 27-H₃), 0.898 (9H, s, Si-t-Bu), 0.892 (9H, s, Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.84 (3H, d, J = 6.5 Hz, 21-H₃), 0.54 (3H, s, 18-H₃), 0.082 (3H, s, SiMe), 0.067 (3H, s, SiMe), 0.052 (9H, s, 3× SiMe), 0.027 (3H, s, SiMe); ¹³C NMR (125 MHz) δ 152.98, 141.22, 138.98, 132.74, 124.74, 122.40, 116.11, 106.25, 72.53, 71.65, 68.74, 56.62, 56.19, 47.61, 45.67, 38.57, 36.13, 35.92, 28.76, 27.37, 26.13, 25.84, 25.78, 23.67, 23.45, 22.32, 20.80, 18.76, 18.25, 18.17, 12.23, −4.38, −4.71, −4.87, −5.09; exact mass calculated for C₄₄H₈₂O₃Si₃Na (MNa)⁺ 765.5468, found 765.5461.

2.2.28. (20S,25S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D₃ tert-butyldimethylsilyl (30b)

According to a general procedure the pure product 30b (10 mg, 46% yield) was obtained from the phosphine oxide 26 (52 mg, 89 µmol), PhLi (1.8 M in di-n-butylether, 61 µL, 110 µmol) and the ketone 25b (9 mg, 24 µmol). For analytical purpose a sample of the protected vitamin 30b was further purified by HPLC (9.4 × 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol (99.9:0.1) solvent system, R_t = 3.51 min): UV (in hexane) λ_max 263.1, 253.2, 244.3 nm; ¹H NMR (500 MHz, CDCl₃) δ 6.22 and 5.83 (each 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.38–5.27 (2H, m, 22-H and 23-H), 4.96 and 4.90 (each 1H, each s, ═CH₂), 4.43 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H), 2.85 (1H, dm, J = 12.6 Hz, 9β-H), 2.52 (1H, dd, J = 13.2, 6.0 Hz, 10α-H), 2.47 (1H, dd, J = 12.6, 4.5 Hz, 4α-H), 2.33 (1H, dd, J = 13.2, 2.9 Hz, 10β-H), 2.18 (1H, dd, J = 12.6, 8.5 Hz, 4β-H)1.11 (3H, d, J = 6.0 Hz, 27-H₃), 1.02 (3H, d, J = 6.5 Hz, 21-H₃), 0.898 (9H, s, Si-t-Bu), 0.895 (9H, s, Si-t-Bu), 0.867 (9H, s, Si-t-Bu), 0.52 (3H, s, 18-H₃), 0.082 (3H, s, SiMe), 0.067 (3H, s, SiMe), 0.055 (9H, s, 3× SiMe), 0.027 (3H, s, SiMe); ¹³C NMR (125 MHz) δ 152.96, 141.13, 138.98, 132.76, 124.74, 122.40, 116.09, 106.25, 72.54, 71.63, 68.73, 56.63, 56.29, 47.61, 45.67, 40.61, 40.24, 38.55, 36.13, 35.98, 28.76, 27.73, 25.93, 25.85, 25.78, 23.89, 23.45, 22.33, 22.22, 18.77, 18.25, 18.17, 12.06, −4.37, −4.66, −4.86, −5.09; exact mass calculated for C₄₄H₈₂O₃Si₃Na (MNa)⁺ 765.5468, found 765.5461.

2.2.29. General procedure for the synthesis of compounds 6, 7, 8a, 8b, 9a, 9b

To a solution of the protected vitamin 27, 28, 29a, 29b, 30a or 30b in THF (2 mL) and acetonitrile (2 mL), a solution of aqueous 48% HF in acetonitrile (1:9 ratio, 2 mL) was added at 0 °C and the resulting mixture was stirred at room temperature for 6 h. The reaction was quenched with a saturated aqueous NaHCO₃ solution and extracted with ethyl acetate. Combined organic phases were washed with brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure. The residue was purified on a Waters silica Sep-Pak cartridge (10–30% ethyl acetate/hexane) to give the crude products. Final purification of the vitamin D compounds was performed by straight phase HPLC (15% 2-propanol/hexane; 4 mL/min; 9.4 mm × 25 cm Zorbax Sil column), and/or by reversed-phase HPLC (15% water/methanol; 3 mL/min; 9.4 mm × 25 cm Zorbax Eclipse XDB-C18 column) to give the analytically pure 19,26-dinorvitamin D analogs 6, 7, 8a, 8b, 9a or 9b.

2.2.30. 2-Methylene-19-nor-22(E)-ene-1α,25-dihydroxyvitamin D₃ (6)

According to a general procedure the pure 2-methylene analog 6 (12.24 mg, 64% yield) was obtained from the protected vitamin 27 (35.05 mg, 46 µmol). The vitamin 6 was further purified by straight phase HPLC [R_t = 6.66 min.] and then by reverse phase HPLC [R_t = 11.53 min.], as it’s described in a general procedure. UV (in EtOH) λ_max 261.5, 252.5, 244.5 nm; ¹H NMR (500 MHz, CDCl₃) δ 6.36 and 5.88 (1H and 1H, each d, J = 11.2 Hz, 6-H and 7-H), 5.39 (2H, m, 22,23-H₂), 5.11 and 5.09 (each 1H, each s, ═CH₂), 4.48 (2H, m, 1β- and 3α-H), 2.85 (1H, dd, J = 12.8, 4.3 Hz, 10β-H), 2.82 (1H, br d, J = 11.9 Hz, 9β-H), 2.57 (1H, dd, J = 13.3, 3.2 Hz, 4α-H), 2.33 (1H, dd, J = 13.3, 6.0 Hz, 4β-H), 2.29 (1H, dd, J = 12.8, 8.6 Hz, 10α-H), 1.20 (6H, s, 26,27-H₆), 1.04 (3H, d, J = 6.6 Hz, 21-H₃), 0.570 (3H, s, 18-H₃); ¹³C NMR (125 MHz) δ 151.95, 143.13, 141.60, 130.53, 124.15, 122.74, 115.37, 107.72, 71.78, 70.62, 70.55, 56.33, 56.06, 46.84, 45.75, 45.69, 40.54, 40.30, 38.13, 29.02, 28.90, 28.04, 23.44, 22.28, 20.86, 12.30; exact mass calculated for C₂₇H₄₂O₃ (M⁺) 414.3134, found 414.3135.

2.2.31. (20S,22E)-2-Methylene-19-nor-22-ene-1α,25-dihydroxyvitamin D₃ (7)

According to a general procedure the pure 2-methylene analog 7 (12.74 mg, 59% yield) was obtained from the protected vitamin 28 (39.44 mg, 52 µmol). The vitamin 7 was further purified by straight phase HPLC [R_t = 6.46 min] and then by reverse phase HPLC [R_t = 10.19 min], as it’s described in a general procedure. UV (in EtOH) λ_max 261.0, 252.0, 244.5 nm; ¹H NMR (400 MHz, CDCl₃) δ 6.35 and 5.88 (1H and 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.44 (2H, m, 22-H and 23-H), 5.11 and 5.09 (each 1H, each s, ═CH₂), 4.48 (2H, m, 1β- and 3α-H), 2.84 (1H, dd, J = 13.3, 4.4 Hz, 10β-H), 2.80 (1H, br d, J = 14.2 Hz, 9β-H), 2.56 (1H, dd, J = 13.4, 3.6 Hz, 4α-H), 2.32 (1H, dd, J = 13.4, 6.0 Hz, 4β-H), 2.28 (1H, dd, J = 13.3, 8.4 Hz, 10α-H), 1.20 (6H, d, J = 1.2 Hz, 26,27-H₆), 0.95 (3H, d, J = 6.6 Hz, 21-H₃), 0.528 (3H, s, 18-H₃); ¹³C NMR (100 MHz) δ 151.96, 143.27, 141.71, 130.45, 124.14, 122.64, 115.26, 107.69, 71.76, 70.75, 70.60, 56.54, 56.13, 46.90, 45.80, 45.74, 40.72, 39.80, 38.11, 29.11, 29.05, 28.91, 27.26, 23.24, 22.09, 21.61, 12.28; exact mass calculated for C₂₇H₄₂O₃ (M⁺) 414.3134, found 414.3142.

2.2.32. (20R,25R)-2-Methylene-19,26-dinor-22-(E)-ene-1α,25-dihydroxyvitamin D₃ (8a)

According to a general procedure the pure 2-methylene analog 8a (1.6 mg, 44% yield) was obtained from the protected vitamin 29a (7 mg, 9 µmol). The final compound 8a was purified by reverse-phase HPLC (R_t = 13.7 min) as it’s described in a general procedure. UV (in EtOH) λ_max 261.4, 252.4, 244.4 nm; ¹H NMR (900 MHz, CDCl₃) δ 6.35 and 5.87 (1H and 1H, each d, J = 10.8 Hz, 6- and 7-H), 5.40–5.38 (1H, m, 22-H), 5.34–5.31 (1H, m, 23-H), 5.10 and 5.08 (each 1H, each s, ═CH₂), 4.47 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H), 2.84 (1H, dd, J = 13.3, 5.4 Hz, 10β-H), 2.81 (1H, br d, J = 13.5 Hz, 9β-H), 2.56 (1H, dd, J = 13.5, 3.6 Hz, 4α-H), 2.32 (1H, dd, J = 13.5, 5.4 Hz, 4β-H), 2.28 (1H, dd, J = 13.3, 8.1 Hz, 10α-H), 1.17 (3H, d, J = 6.3 Hz, 27-H₃), 1.03 (3H, d, J = 6.3 Hz, 21-H₃), 0.55 (3H, s, 18-H₃); exact mass calcd for C₂₆H₄₀ONa (MNa⁺) 423.2875, found 423.2873.

2.2.33. (20R,25S)-2-Methylene-19,26-dinor-22-(E)-ene-1α,25-dihydroxyvitamin D₃ (8b)

According to a general procedure the pure 2-methylene analog 8b (4 mg, 54% yield) was obtained from the protected vitamin 29b (15 mg, 34 µmol). The final compound 8b was purified by straight-phase HPLC (R_t = 9.3 min) and then by reverse-phase HPLC (R_t = 12.9 min) as it’s described in a general procedure. UV (in EtOH) λ_max 262.1, 252.6, 244.1 nm; ¹H NMR (800 MHz, CDCl₃) δ 6.35 and 5.88 (1H and 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.41–5.32 (2H, m, 22-H and 23-H), 5.11 and 5.09 (each 1H, each s, ═CH₂), 4.47 (2H, m, 1β- and 3α-H), 3.75 (1H, m, 25-H), 2.83 (1H, dd, J = 13.3, 4.5 Hz, 10β-H), 2.81 (1H, br d, J = 13.2 Hz, 9β-H), 2.57 (1H, dd, J = 13.4, 3.7 Hz, 4α-H), 2.33 (1H, dd, J = 13.4, 6.1 Hz, 4β-H), 2.29 (1H, dd, J = 13.3, 8.3 Hz, 10α-H),1.19 (3H, d, J = 6.2 Hz, 27-H₃), 1.03 (3H, d, J = 6.4 Hz, 21-H₃), 0.55(3H, s, 18-H₃); exact mass calculated for C₂₆H₄₀O₃Na⁺ (MNa⁺) 423.2875, found 423.2874.

2.2.34. (20S,25R)-2-Methylene-19,26-dinor-22-(E)-ene-1α,25-dihydroxyvitamin D₃ (9a)

According to a general procedure the pure 2-methylene analog 9a (1 mg, 43% yield) was obtained from the protected vitamin 30a (4.5 mg, 6 µmol). The final compound 9a was purified by reverse-phase HPLC (R_t = 11.8 min) as it’s described in a general procedure. UV (in EtOH) λ_max 262.1, 252.6, 244.1 nm; ¹H NMR (900 MHz, CDCl₃) δ 6.28 and 5.81 (each 1H, each d, J = 11.7 Hz, 6- and 7-H), 5.38–5.25 (2H, m, 22-H and 23-H), 5.04 and 5.02 (each 1H, each s, ═CH₂), 4.40 (2H, m, 1β- and 3α-H), 3.76 (1H, m, 25-H), 2.78 (1H, dd, J = 13.1, 4.5 Hz, 10β-H), 2.73 (1H, br d, J = 13.5 Hz, 9β-H), 2.51 (1H, dd, J = 13.5, 4.5 Hz, 4α-H), 2.27 (1H, dd, J = 13.5, 6.3 Hz, 4β-H), 2.22 (1H, dd, J = 13.1, 8.1 Hz, 10α-H), 1.11 (3H, d, J = 6.3 Hz, 27-H₃), 0.87 (3H, d, J = 6.3 Hz, 21-H₃), 0.45 (3H, s, 18-H₃); exact mass calculated for C₂₆H₄₀O₃Na⁺ (MNa⁺) 423.2875, found 423.2881.

2.2.35. (20S,25S)-2-Methylene-19,26-dinor-22-(E)-ene-1α,25-dihydroxyvitamin D₃ (9b)

According to a general procedure the pure 2-methylene analog 9b (1.3 mg, 36% yield) was obtained from the protected vitamin 30b (7 mg, 9 µmol). The final compound 9b was purified by straight-phase HPLC (R_t = 9.3 min) and then by reverse-phase HPLC (R_t = 11.1 min) as it’s described in a general procedure. UV (in EtOH) λ_max 262.1, 252.6, 244.1 nm; ¹H NMR (800 MHz, CDCl₃) δ 6.35 and 5.87 (1H and 1H, each d, J = 11.2 Hz, 6- and 7-H), 5.45–5.42 (1H, m, 22-H), 5.34–5.30 (1H, m, 23-H), 5.11 and 5.09 (each 1H, each s, ═CH₂), 4.48 (2H, m, 1β- and 3α-H), 3.78 (1H, m, 25-H), 2.84 (1H, dd, J = 12.8, 4.8 Hz, 10β-H), 2.80 (1H, br d, J = 12.8 Hz, 9β-H), 2.57 (1H, dd, J = 12.8, 2.5 Hz, 4α-H), 2.32 (1H, dd, J = 12.8, 6.4 Hz, 4β-H), 2.29 (1H, dd, J = 12.8, 8.0 Hz, 10α-H), 1.19 (3H, d, J = 6.4 Hz, 27-H₃), 0.95 (3H, d, J = 6.4 Hz, 21-H₃), 0.52 (3H, s, 18-H₃); exact mass calcd for C₂₆H₄₀O₃Na (MNa)⁺ 423.2875, found 423.2870.

2.3. Biological studies

2.3.1. In vitro studies

VDR binding, HL-60 differentiation and 24-hydroxylase transcription assays were performed as previously described [16,17].

2.3.2. In vivo studies

Bone calcium mobilization and intestinal calcium transport were performed as previously described [16,17]. Briefly, weanling rats were made vitamin D-deficient by housing under lighting conditions that block vitamin D production in the skin. In addition, the animals were fed a diet devoid of vitamin D. Experimental compounds were administered intraperitoneally once per day for four consecutive days. Twenty-four hours after the last dose was given, the blood was collected, and everted gut sacs were prepared. Calcium transport was measured ex vivo and bone calcium mobilization was carried out as previously described [16,17]. There were 5–6 animals in each group. The control animals received vehicle only, while positive control animals received the indicated dose of 1,25-(OH)₂D₃ in the vehicle.

3. Results and discussion

3.1. Chemistry

The synthesis strategy of the new 2-methylene-Δ²²E-19-nor-1α,25(OH)₂D₃ compounds 6 (20R) and 7 (20S) and 2-methylene-Δ²²E-19,26-dinor-1α,25(OH)₂D₃ compounds 8a,b–9a,b was based on the Wittig–Horner olefination reaction [18] between Grundmann-type ketones (20–21; 24a,b–25a,b) and the phosphine oxide 26 (Scheme 2). The A-ring fragment 26 was prepared according to the published procedure [15] whereas the syntheses of the necessary Δ²²E-25-hydroxy C,D-ring ketones (20–21; 24a,b–25a,b) are presented in Scheme 1. As recently reported by us [14], the Wittig reaction between the C,D-ring aldehydes 10 and 11, previously prepared in our laboratory from commercial vitamin D₂ [19], and either the hydroxyphosphonium bromide 12 [20] or the hydroxyphosphonium iodides 15a and 15b, easily prepared in our laboratory from commercially available (S)- and (R)-1,3-butanediols [14,20], efficiently provided only the olefinic products with the E-geometry of the introduced double bond 13–14 and 16a,b–17a,b, respectively [14,20]. Then, after protection of the tertiary hydroxyl groups as tert-butyldimethylsilyl ethers 18–19 and 22a,b–23a,b, the removal of the benzoyl group under basic conditions gave the secondary alcohols, which were immediately subjected to oxidation with PDC affording the Grundmann ketones 20–21 and 24a,b–25a,b in very good yields. As outlined in Scheme 2, each of the six Δ²²E-25-hydroxy Grundmann ketones (20–21, 17a,b–18a,b) was coupled with the anion, generated from phosphine oxide 26 and phenyllithium, affording the corresponding six protected 19-norvitamin D analogs (27–28, 29a,b–30a,b). Then, after silyl groups removal using hydrofluoric acid the final 2-methylene-Δ²²E-19-nor-1α,25(OH)₂D₃ (6–7) and 2-methylene-Δ²²E-19,26-dinor-1α,25(OH)₂D₃ (8a,b–9a,b) compounds were complete.

Scheme 2.

Reagents: (i) PhLi, (ii) aq. HF, THF, MeCN.

Scheme 1.

Reagents: (i) n-BuLi, THF; (ii) TBSOTf,2,6-lutine, CH₂CL₂; (iii) (1) KOH, EtOH; (2) PDC CH₂CL₂.

3.2. Biological activity

All compounds bound the receptor with very similar affinities. Only one of the analogs, compound 8b (20R, 25S), had a slightly lower affinity (Table 1). The two 2-methylene-Δ²²E-19-nor-1α,25(OH)₂D₃ compounds 6 and 7 exhibited approximately 10 times higher HL-60 differentiation activity as compared to the natural hormone 1 and 20 times higher than19-nor-1α,25-dihydroxyvitaminD₂ 2. The 25R isomers 8a and 9a displayed higher cell differentiation activity as compared to the corresponding 25S isomers 8b and 9b, with isomer 8a being the most potent of this series, having about 10 times more HL-60 differentiation potency as compared to the natural hormone 1. As shown in Table 1, the 25S isomers 8b and 9b are equally potent in inducing cell differentiation and their efficacy is 3 times higher than that of the natural hormone 1 and 4 times higher than that of 19-nor-1α,25-dihydroxyvitaminD₂ 2. Compound 8a (20R, 25R) is the most potent of the 2-methylene-Δ²²E-19,26-dinor-1α,25(OH)₂D₃ analogs, and its potency is comparable to that of 5, 6 and 7. The pattern of potencies in in vitro transcription assays are shown (Table 1). Similar to that observed in the HL60 cell differentiation assays, compounds 6–7, and the (20R,25R) compound 8a, express the highest transcriptional potency. They are more potent than both 2 and 1α,25(OH)₂D₃ (1), but less active than 2MD (4) and 5. As shown in Table 1, the transcriptional activity of the 20R,25R isomer 8a is about 20 times that of the corresponding 20S,25R isomer 9a. Usually the 20-epimerization increases the transcriptional activity, and this result constitutes an exception to that pattern. On the other hand, the 20-epimerization does not affect the transcriptional activity of the 25S isomers 8b and 9b, whose activity is comparable to that of the natural hormone (1).

Table 1.

VDR binding properties,^a HL-60 differentiating activities,^b and transcriptional activities^c of the vitamin D analogs 6–7; 8a,b–9a,b.

		VDR bindinga		HL-60 differentiationb		CYP24A1 transcriptionc
Compound	Compd no.	Ki(M)	Ratio	EC50(M)	Ratio	EC50(M)	Ratio
1α,25-(OH)2D3	1	1 × 10−10	1	3 × 10−9	1	2 × 10−10	1
19-Nor-1α,25-dihydroxyvitaminD2 (Zemplar)	2	1 × 10−10	1	4 × 10−9	1.3	3 × 10−10	1.5
(20S)-2-Methylene-19-nor-1α,25-(OH)2D3 (2MD)	4	1 × 10−10	1	8 × 10−11	0.027	7 × 10−12	0.035
(20S,25R)-2-Methylene-19,26-dinor-1α,25-(OH)2D3 (SR1)	5	9 × 10−11	0.9	9 × 10−11	0.03	1 × 10−11	0.05
(20R)-2-Methylene-Δ22E-19-nor-1α,25-(OH)2D3 (AT3)	6	6 × 10−11	0.6	2 × 10−10	0.07	2 × 10−11	0.1
(20S)-2-methylene-Δ22E-19-nor-1α,25-(OH)2D3 (N23)	7	6 × 10−11	0.6	2 × 10−10	0.07	2 × 10−11	0.1
(20R,25R)-2-Methylene-Δ22E-19,26-dinor-1α,25-dihydroxyvitamin D3	8a	9 × 10−11	0.9	2 × 10−10	0.07	2 × 10−11	0.1
(20R,25S)-2-Methylene-Δ22E-19,26-dinor-1α,25-(OH)2D3	8b	3 × 10−10	3	1 × 10−9	0.33	1 × 10−10	0.5
(20S,25R)-2-Methylene-Δ22E-19,26-Dinor-1α,25-(OH)2D3	9a	8 × 10−11	0.8	8 × 10−10	0.27	1 × 10−10	0.5
(20S,25S)-2-Methylene-Δ22E-19,26-dinor-1α,25-(OH)2D3	9b	2 × 10−10	2	1× 10−9	0.33	1 × 10−10	0.5

^aCompetitive binding of 1α,25-(OH)₂D₃ (1) and the synthesized vitamin D analogs to the full-length recombinant rat vitamin D receptor. The K_i values are derived from dose–response curves and represent the inhibition constant when radiolabeled 1α,25-(OH)₂D₃ is present at 1 nM and a K_d of 0.2 nM is used. The binding ratio is the average ratio of the analog K_i to the K_i for 1α,25-(OH)₂D₃.

^bInduction of differentiation of HL-60 promyelocytes to monocytes by 1α,25-(OH)₂D₃ and the synthesized vitamin D analogs. Differentiation state was determined by measuring the percentage of cells reducing nitro blue tetrazolium (NBT). The ED₅₀ values are derived from dose–response curves and represent the analog concentration capable of inducing 50% maturation. The differentiation activity ratio is the average ratio of the analog ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃.

^CTranscriptional assay in rat osteosarcoma cells stably transfected with a CYP24A1 gene reporter plasmid. The ED₅₀ values are derived from dose–response curves and represent the analog concentration capable of increasing the luciferase activity by 50%. The luciferase activity ratio is the average ratio of the ED₅₀ for the analog to the ED₅₀ for 1α,25-(OH)₂D₃. All the experiments were carried out in duplicate on at least two different occasions.

It is unclear why compounds 4 and 5 have more than 10× the activity of 1α,25(OH)₂D₃ in causing differentiation of HL-60 cells and CYP24A1 transcription while having similar activity in binding to the VDR. The former assays involve a cell culture assay in which 5% serum is present. The vitamin D binding protein (DBP) in serum binds 1α,25(OH)₂D₃ reducing the free 1α,25(OH)₂D₃. The compounds with a 20S configuration are bound poorly by the DBP. Thus, compounds 4, 5, 7, 9a, and 9b would have a much higher free concentration resulting in high activity. Because this unexpected high activity did not occur in in vivo assays (Figs. 2–4), its significance is unlikely. Nevertheless, activity in HL-60 may represent anti-cancer activity, which is not assessed by the in vivo assays used in the present study.

Fig. 2.

Effects of 1,25(OH)₂D₃ (1) and compounds 6–7 on bone calcium mobilization and intestinal calcium transport (structures are shown in Figure 1).

Fig. 4.

In vivo intestinal calcium transport compared to the native hormone (1). Compounds 1, 5, 8a, 8b, 9a and 9b are shown in Figure 1.

In the present series¹⁴, removal of the 26-methyl group has little impact on receptor binding (compounds 4 and 5) and slightly reduces HL-60 differentiation (compounds 4 and 7). A combination of a double bond at carbon 22 with removal of the 26-methyl results in a compound with one log less in vitro potency (compare compounds 4 and 9a).

In vivo biological activities of compounds 6 and 7 are shown in Fig. 2. Consistent with the in vitro results, these two analogs show increased potency in bone calcium mobilization compared to the native hormone. However, introduction of a trans double bond between C-22 and C-23 resulted in significantly decreased activity in bone compared to 2MD (260 pmol 2MD will raise serum calcium by 5.3 mg/dL [12] compared to 2.5 mg/dL for compound 6 and 2.0 mg/dL for compound 7). As shown in Figs. 3 and 4, removal of the 26-methyl group from 2-methylene-Δ²²E-19-nor-1α,25(OH)₂D₃ compounds 6 and 7 selectively reduced in vivo activity. In fact, while all the new 26-nor analogs 8a,b–9a,b have virtually no bone calcium mobilization activity in vivo (Fig. 3) but retain calcium transport activity in the intestine (1) (Fig. 4). As shown in Fig. 3 the 25S isomers 8b and 9b are 200× times less potent on bone than 1α,25(OH)₂D₃ (1), and the 25R isomers 8a and 9a are at least 30 times less active in mobilizing calcium from bone. Thus, the in vivo results reaffirm the potency profile observed in vitro: coupling a double bond at C-22 with 26-methyl removal results in analogs with significantly lower potencies.

Fig. 3.

Total serum calcium levels reflecting the ability of each analog to release bone calcium stores. Compounds 1, 5, 8a, 8b, 9a and 9b are shown in Figure 1.

4. Conclusion

Removing the 26-methyl group from 2-methylene-22-ene-19-nor-1α,25-dihydroxyvitamin D₃ results in compounds that are selectively active on intestinal calcium transport. Additionally this activity is increased by a 20S configuration.