26-Desmethyl-2-methylene-22-ene-19-nor-1α,25-dihydroxyvitamin D 3 compounds selectively active on intestine

Article abstract of DOI:10.1016/j.steroids.2014.01.012

Six new analogs of 2-methylene-19-nor-1α,25-dihydroxyvitamin D ₃, 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

Full text of DOI:10.1016/j.steroids.2014.01.012

Steroids 83 (2014) 27–38
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Steroids
journal homepage: www.elsevier.com/locate/steroids
26-Desmethyl-2-methylene-22-ene-19-nor-1
compounds selectively active on intestine
a,25-dihydroxyvitamin D3
⇑
Grazia Chiellini, Pawel Grzywacz, Lori A. Plum, Margaret Clagett-Dame, Hector F. DeLuca
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new analogs of 2-methylene-19-nor-1
a
,25-dihydroxyvitamin D3, 6–7 and 8a,b–9a,b, have been
Received 14 November 2013
Received in revised form 10 January 2014
Accepted 22 January 2014
Available online 7 February 2014
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
D
22E-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.
Keywords:
Vitamin D
Calcemic activity
Transcription activity
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.
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
derivatives with various side chain modifications. This endeavor
has yielded several tissue-selective compounds with therapeutic
1
a
,25-Dihydroxyvitamin D3, [1
central regulator of calcium homeostasis [1,2]. In addition,
,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 1 ,25(OH)2D3 are
a
,25(OH)2D3] (1) is perhaps the
potential [3,11], among them 2MD (4) (Fig. 1), one of the most
promising analogs [12]. This analog is at least 30-fold more effec-
tive than 1a,25(OH)2D3 in stimulating osteoblast-mediated bone
calcium mobilization while being approximately equally potent
1a
a
in supporting intestinal calcium transport [13].
mediated by the vitamin D receptor (VDR), which is a member of
the nuclear receptor superfamily. It acts as a ligand-dependent
A very recent addition to our ongoing structure–activity rela-
tionship studies has been the development of 2-methylene-
gene transcription factor [1]. 1a,25(OH)2D3 and its analogs have
19,26-dinor-1a,25-dihydroxyvitamin D3 analogs [14]. Indeed, the
significant therapeutic potential in the treatment of osteoporosis,
vitamin D-resistant rickets, secondary hyperparathyroidism, psori-
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,
asis, and renal osteodystrophy [1]. However, use of 1
itself is limited because it induces significant hypercalcemia. A
number of 1 ,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-1 ,25-(OH)2D2 (paricalc-
itol, Zemplar) (2) and 1 -(OH)D2 (doxercalciferol, Hectorol) (3)
a,25(OH)2D3
a
with the (20S,25R)-2-methylene-19,26-dinor-1
5 (Fig. 1), being the most potent of the new series [14]. We have
now prepared two new 2-methylene- ,25(OH)2D3
22E-19-nor-1
a,25(OH)2D3 analog
a
a
D
a
have been developed and used to treat secondary hyperpathyroid-
ism (SH) [3,10].
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
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-1a,25-dihydroxyvitamin D3 compound 5
(Fig. 1), might improve tissue selectivity while reducing the
⇑
Corresponding author. Tel.: +1 608 262 1620; fax: +1 608 262 7122.
E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).
calcemic activity, we also synthesized four new 2-methylene-D22
http://dx.doi.org/10.1016/j.steroids.2014.01.012
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

28
G. Chiellini et al. / Steroids 83 (2014) 27–38
Fig. 1. Chemical structures of 1a,25-dihydroxyvitamin D3 (calcitriol, 1) and its analogs.
E-19,26-dinor-1
a
,25(OH)2D3 compounds (8a, 8b, 9a, 9b; Fig. 1).
Lambda 3B UV–Vis spectrophotometer in ethanol. 1H nuclear mag-
netic 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 (d)
in parts per million are quoted relative to internal Me4Si (d 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 addition-
ally confirmed by inspection of their 1H NMR, 13C NMR, UV absorp-
tion, and high-resolution mass spectra.
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 (D
22E).
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 re-
placed 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-
D
22E-19,26-dinor-1
a,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

G. Chiellini et al. / Steroids 83 (2014) 27–38
29
2.2. Synthesis of compounds
the phosphonium iodide 15b (310 mg, 0.67 mmol) and n-butyllith-
ium (1.6 M, 840
l
L, 1.34 mmol). ½a D24
ꢁ
= +98.7° (c 1.75, CHCl3); 1H
2.2.1. General procedure for the synthesis of compounds 13, 14, 16a,
16b, 17a, 17b
NMR (500 MHz, acetone-d6) d 8.05 (2H, m, o-HBz), 7.63 (1H, m, p-
HBz), 7.52 (2H, m, m-HBz), 5.42 (1H, dt, J = 15.2, 7.0 Hz, 23-H) 5.38
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.
(1H, d, J = 2.5 Hz, 8 -H), 5.32 (1H, dd, J = 15.2, 8.5 Hz, 22-H), 3.72
a
(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) d 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-[(40R)-hydroxy-pent-(10E)-
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-butyllith-
2.2.2. (8S,20R)-Des-A,B-8-benzoyloxy-20-[40-hydroxy-40-methyl-
pent-(10E)-en-yl]-pregnane (13)
ium (1.6 M, 720
l
L, 1.15 mmol). ½a D24
ꢁ
= ꢀ28.8° (c 0.8, CHCl3); 1H
NMR (500 MHz, acetone-d6) d 8.05 (2H, m, o-HBz), 7.63 (1H, m, p-
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)
H
Bz), 7.52 (2H, m, m-HBz), 5.46 (1H, dt, J = 15.4, 6.9 Hz, 23-H) 5.38
(1H, s, 8 -H), 5.36 (1H, dd, J = 15.4, 8.5 Hz, 22-H), 3.76 (1H, m,
a
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) d 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
and n-butyllithium (1.95 M, 1.14 mL, 2.22 mmol). ½a D24
ꢁ
= +87.8° (c
2.75, CHCl3); 1H NMR (400 MHz, CDCl3) d 8.06 (2H, m, o-HBz),
7.55 (1H, m, p-HBz), 7.44 (2H, m, m-HBz), 5.41 (1H, s, 8a-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) d 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.
2.2.7. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(40S)-hydroxy-pent-(10E)-
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-butyllith-
2.2.3. (8S,20S)-Des-A,B-8-benzoyloxy-20-[40-hydroxy-40-methyl-pent-
(10E)-en-yl]-pregnane (14)
ium (1.6 M, 560
NMR (500 MHz, acetone-d6) d 8.04 (2H, m, o-HBz), 7.63 (1H, m, p-
l
L, 0.9 mmol). ½a 2D4
ꢁ
= ꢀ11.4° (c 1.4, CHCl3); 1H
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-butyllith-
HBz), 7.52 (2H, m, m-HBz), 5.46 (1H, dt, J = 15.4, 6.8 Hz, 23-H) 5.39
(1H, s, 8 -H), 5.35 (1H, dd, J = 15.4, 6.3 Hz, 22-H), 3.78 (1H, m,
a
ium (1.61 M, 1.38 mL, 2.22 mmol). ½a D24
ꢁ
= ꢀ25.1° (c 2.5, CHCl3); 1H
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) d 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.
NMR (500 MHz, CDCl3) d 8.04 (2H, m, o-HBz), 7.55 (1H, m, p-HBz),
7.44 (2H, m, m-HBz), 5.42 (3H, m, 8a-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) d 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.
2.2.8. General procedure for the synthesis of compounds 18, 19, 22a,
22b, 23a, 23b
2.2.4. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(40R)-hydroxy-pent-(10E)-
en-yl]-pregnane (16a)
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 anhydrous methylene chloride
(3 mL), tert-butyldimethylsilyl trifluoromethane-sulfonate (1.8 equiv)
was added at ꢀ20 °C. The reaction mixture was stirred at 0 °C for 1 h. It
was quenched with water and extracted with methylene chloride.
Combined organic phases were washed with brine, dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (3% ethyl ace-
tate/hexane) to give the product 18, 19, 22a, 22b, 23a, 23b.
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-butyllith-
ium (1.6 M, 980
NMR (500 MHz, acetone-d6) d 8.05 (2H, m, o-HBz), 7.62 (1H, m, p-
l
L, 1.56 mmol). ½a D24
ꢁ
= +69.6° (c 1.3, CHCl3); 1H
HBz), 7.52 (2H, m, m-HBz), 5.41 (1H, dt, J = 15.4, 7.0 Hz, 23-H) 5.38
(1H, d, J = 1.8 Hz, 8 -H), 5.31 (1H, dd, J = 15.4, 8.4 Hz, 22-H), 3.72
a
(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) d 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.
2.2.9. (8S,20R)-Des-A,B-8-benzoyloxy-20-[40-(tert-
butyldimethylsilyloxy)-40-methyl-pent-(10E)-en-yl]-pregnane (18)
According to a general procedure the pure product 18 (67 mg,
96% yield) was obtained from the alcohol 13. [
CHCl3); 1H NMR (400 MHz, CDCl3) d 8.06 (2H, m, o-HBz), 7.55
(1H, m, p-HBz), 7.44 (2H, m, m-HBz), 5.41 (1H, d, J = 2.3 Hz, 8 -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
a] +62.9 (c 3.35,
D
a
2.2.5. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(40S)-hydroxy-pent-(10E)-
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),
(100 MHz)
d 166.44, 139.08, 132.65, 130.90, 129.53, 128.32,

30
G. Chiellini et al. / Steroids 83 (2014) 27–38
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.
SiMe2); 13C NMR (125 MHz) d 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.
2.2.10. (8S,20S)-Des-A,B-8-benzoyloxy-20-[40-(tert-
butyldimethylsilyloxy)-40-methyl-pent-(10E)-en-yl]-pregnane (19)
According to a general procedure the pure product 19 (65 mg,
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, neutral-
ized with 5% aqueous solution of HCl and extracted with methy-
lene chloride. Combined organic phases were washed with a
saturated aqueous NaHCO3 solution, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (5–10% ethyl ace-
tate/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.
93% yield) was obtained from the alcohol 14. [
CHCl3); 1H NMR (500 MHz, CDCl3) d 8.05 (2H, m, o-HBz), 7.54
(1H, m, p-HBz), 7.43 (2H, m, m-HBz), 5.41 (2H, m, 8 -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) d 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.
a
]
ꢀ21.2 (c 4.95,
D
a
2.2.11. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(40R)-(tert-
butyldimethylsilyl)oxy-pent-(10E)-en-yl]-pregnane (22a)
According to a general procedure the pure product 22a (30 mg,
78% yield) was obtained from the alcohol 16a. [
CHCl3); 1H NMR (600 MHz, CDCl3) d 8.05 (2H, m, o-HBz), 7.55
(1H, m, p-HBz), 7.44 (2H, m, m-HBz), 5.41 (1H, s, 8 -H), 5.35–5.26
a] +53.8 (c 1.1,
D
a
(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)
d 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.
2.2.16. (20R)-Des-A,B-20-[40-(tert-butyldimethylsilyloxy)-40-methyl-
pent-(10E)-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. [a]
D
ꢀ5.8 (c 1.1, CHCl3); 1H NMR (400 MHz, CDCl3) d 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) d 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
2.2.12. (8S,20R)-Des-A,B-8-benzoyloxy-20-[(40S)-(tert-
butyldimethylsilyl)oxy-pent-(10E)-en-yl]-pregnane (22b)
According to a general procedure the pure product 22b (37 mg,
95% yield) was obtained from the alcohol 16b. [
CHCl3); 1H NMR (600 MHz, CDCl3) d 8.05 (2H, m, o-HBz), 7.55
(1H, m, p-HBz), 7.44 (2H, m, m-HBz), 5.41 (1H, s, 8 -H), 5.35–5.26
a] +54.1 (c 1.2,
D
mass calculated for
415.3022.
C
24H44O2SiNa (MNa+) 415.3008, found
a
(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)
d 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.
2.2.17. (20S)-Des-A,B-20-[40-(tert-butyldimethylsilyloxy)-40-methyl-
pent-(10E)-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. [a]
D
ꢀ39.3 (c 1.35, CHCl3); 1H NMR (400 MHz, CDCl3) d 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) d
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.
2.2.13. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(40R)-(tert-
butyldimethylsilyl)oxy-pent-(10E)-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) d 8.05 (2H, m, o-HBz), 7.54 (1H, m, p-HBz), 7.42 (2H, m, m-
HBz), 5.41 (1H, s, 8a-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) d 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.
2.2.18. (20R)-Des-A,B-20-[(40R)-(tert-butyldimethylsilyl)oxy-pent-
(10E)-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) d 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) d 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.
2.2.14. (8S,20S)-Des-A,B-8-benzoyloxy-20-[(40S)-(tert-
butyldimethylsilyl)oxy-pent-(10E)-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) d 8.05 (2H, m, o-HBz), 7.56 (1H, m, p-HBz), 7.45 (2H, m, m-
HBz), 5.45 (1H, s, 8a-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,

G. Chiellini et al. / Steroids 83 (2014) 27–38
31
2.2.19. (20R)-Des-A,B-20-[(40S)-(tert-butyldimethylsilyl)oxy-pent-
(10E)-en-yl]-pregnan-8-one (24b)
3
a
-H), 2.83 (1H, dm, J = 12.5 Hz, 9b-H), 2.51 (1H, dd, J = 13.2,
5.9 Hz, 10 -H), 2.46 (1H, dd, J = 12.6, 4.3 Hz, 4 -H), 2.33 (1H, dd,
a
a
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) d 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)
d 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.
J = 13.2, 2.7 Hz, 10b-H), 2.18 (1H, dd, J = 12.6, 8.4 Hz, 4b-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, 2ꢂ 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 calcu-
lated for C45H84O3Si3Na (MNa+) 779.5626, found 779.5652.
2.2.20. (20S)-Des-A,B-20-[(40R)-(tert-butyldimethylsilyl)oxy-pent-
(10E)-en-yl]-pregnan-8-one (25a)
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
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) d 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)
d 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.
(67 mg, 115
l
mol), PhLi (1.8 M in di-n-butylether, 75
l
L,
137
l
mol) and the ketone 21 (26 mg, 66 lmol). UV (in hexane)
kmax 262.5, 253.0, 245.0 nm; 1H NMR (400 MHz, CDCl3) d 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, 1b- and 3
H), 2.81 (1H, dm, J = 13.1 Hz, 9b-H), 2.52 (1H, dd, J = 13.2, 5.8 Hz,
10 -H), 2.46 (1H, dd, J = 12.7, 4.3 Hz, -H), 2.33 (1H, dd,
a-
2.2.21. (20S)-Des-A,B-20-[(40S)-(tert-butyldimethylsilyl)oxy-pent-
(10E)-en-yl]-pregnan-8-one (25b)
a
4a
J = 13.2, 2.4 Hz, 10b-H), 2.17 (1H, dd, J = 12.7, 8.4 Hz, 4b-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, 3ꢂ SiMe), 0.047 (3H, s, SiMe),
0.025 (3H, s, SiMe); 13C NMR (100 MHz) d 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.
According to a general procedure the pure product 25b (10 mg,
67% yield) was obtained from the benzoate 23b. 1H NMR
(400 MHz, CDCl3) d 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) d
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
2.2.25. (20R,25R)-1a-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
To a stirred solution of the phosphine oxide 26 (3.7 equiv) [15]
[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D3
in anhydrous THF (500 lL), a solution of phenyllithium (1.8 M in
tert-butyldimethylsilyl ether (29a)
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 pre-
cooled solution of the Grundmann0s type ketone 20, 21, 24a, 24b,
According to a general procedure the pure protected analog 29a
(8 mg, 48% yield) was obtained from the phosphine oxide 26
(41 mg, 70
l
mol), PhLi (1.8 M in di-n-butylether, 48
lL, 86 lmol)
25a or 26b (1 equiv) in anhydrous THF (200 + 100
l
L) was added
and the ketone 24a (9 mg, 23 lmol). For analytical purpose a sam-
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 re-
duced 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.
ple of the protected vitamin 29a was further purified by HPLC
(9.4 ꢂ 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol
(99.9:0.1) solvent system, Rt = 3.70 min); UV (in hexane) kmax
263.1, 253.2, 244.3 nm; 1H NMR (400 MHz, CDCl3) d 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, 1b- and 3
9b-H), 2.52 (1H, dd, J = 13.5, 6.1 Hz, 10
4.5 Hz, 4 -H), 2.34 (1H, dd, J = 13.5, 6.3 Hz, 10b-H), 2.18 (1H, dd,
a-H), 3.78 (1H, m, 25-H), 2.78 (1H, dm, J = 12.1 Hz,
a-H), 2.48 (1H, dd, J = 12.7,
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
a
J = 12.7, 8.6 Hz, 4b-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) d 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.
(62 mg, 107
l
mol), PhLi (1.8 M in di-n-butylether, 60
lL,
114 mol) and the ketone 20 (22 mg, 56
l
lmol). UV (in hexane)
kmax 262.5, 253.0, 245.0 nm; 1H NMR (500 MHz, CDCl3) d 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, 1b- and

32
G. Chiellini et al. / Steroids 83 (2014) 27–38
2.2.26. (20R,25S)-1
[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D3
tert-butyldimethylsilyl ether (29b)
a
-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
m, 1b- and 3
9b-H), 2.52 (1H, dd, J = 13.2, 6.0 Hz, 10
4.5 Hz, 4 -H), 2.33 (1H, dd, J = 13.2, 2.9 Hz, 10b-H), 2.18 (1H, dd,
a
-H), 3.78 (1H, m, 25-H), 2.85 (1H, dm, J = 12.6 Hz,
a-H), 2.47 (1H, dd, J = 12.6,
a
According to a general procedure the pure protected analog 29b
(15 mg, 48% yield) was obtained from the phosphine oxide 26
J = 12.6, 8.5 Hz, 4b-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, 3ꢂ SiMe), 0.027 (3H, s, SiMe);
13C NMR (125 MHz) d 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
(73 mg, 127
l
mol), PhLi (1.8 M in di-n-butylether, 85
lL,
180 mol) and the ketone 24b (16 mg, 42
l
lmol). For analytical
purpose a sample of the protected vitamin 29b was further purified
by HPLC (9.4ꢂ250 mm Zorbax Sil column, 4 mL/min, hexane/2-
propanol (99.9:0.1) solvent system, Rt = 3.80 min); UV (in hexane)
kmax 263.1, 253.2, 244.3 nm; 1H NMR (400 MHz, CDCl3) d 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,
C
44H82O3Si3Na (MNa)+ 765.5468, found 765.5461.
m, 1b- and 3
9b-H), 2.52 (1H, dd, J = 13.1, 5.9 Hz, 10
4.3 Hz, 4 -H), 2.33 (1H, dd, J = 13.1, 2.3 Hz, 10b-H), 2.17 (1H, dd,
a
-H), 3.78 (1H, m, 25-H), 2.82 (1H, dm, J = 11.8 Hz,
a-H), 2.47 (1H, dd, J = 12.6,
2.2.29. General procedure for the synthesis of compounds 6, 7, 8a, 8b,
9a, 9b
a
J = 12.6, 8.7 Hz, 4b-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, 3ꢂ SiMe), 0.027 (3H, s, SiMe);
13C NMR (100 MHz) d 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.
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 un-
der 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 per-
formed by straight phase HPLC (15% 2-propanol/hexane; 4 mL/min;
9.4 mm ꢂ 25 cm Zorbax Sil column), and/or by reversed-
phase HPLC (15% water/methanol; 3 mL/min; 9.4 mm ꢂ 25 cm
Zorbax Eclipse XDB-C18 column) to give the analytically pure
19,26-dinorvitamin D analogs 6, 7, 8a, 8b, 9a or 9b.
2.2.27. (20S,25R)-1a-[(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
lmol), PhLi (1.8 M in di-n-butylether, 34
l
L, 61
lmol) and the
2.2.30. 2-Methylene-19-nor-22(E)-ene-1a,25-dihydroxyvitamin D3
ketone 25a (6.5 mg, 17
lmol). For analytical purpose a sample of
(6)
the protected vitamin 30a was further purified by HPLC
(9.4 ꢂ 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol
(99.9:0.1) solvent system, Rt = 3.70 min): UV (in hexane) kmax
262.6, 253.0, 244.8 nm; 1H NMR (600 MHz, CDCl3) d 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, 1b-
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 lmol). The vitamin 6 was further purified by
straight phase HPLC [Rt = 6.66 min.] and then by reverse phase
HPLC [Rt = 11.53 min.], as it’s described in a general procedure.
UV (in EtOH) kmax 261.5, 252.5, 244.5 nm; 1H NMR (500 MHz,
CDCl3) d 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,
and 3
2.52 (1H, dd, J = 13.2, 6.0 Hz, 10
-H), 2.33 (1H, dd, J = 13.2, 2.9 Hz, 10b-H), 2.18 (1H, dd, J = 12.6,
a-H), 3.77 (1H, m, 25-H), 2.83 (1H, dm, J = 12.6 Hz, 9b-H),
a-H), 2.46 (1H, dd, J = 12.6, 4.5 Hz,
4a
@CH2), 4.48 (2H, m, 1b- and 3
10b-H), 2.82 (1H, br d, J = 11.9 Hz, 9b-H), 2.57 (1H, dd, J = 13.3,
3.2 Hz, 4 -H), 2.33 (1H, dd, J = 13.3, 6.0 Hz, 4b-H), 2.29 (1H, dd,
J = 12.8, 8.6 Hz, 10 -H), 1.20 (6H, s, 26,27-H6), 1.04 (3H, d,
a-H), 2.85 (1H, dd, J = 12.8, 4.3 Hz,
8.3 Hz, 4b-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, 3ꢂ SiMe), 0.027 (3H, s, SiMe); 13C NMR (125 MHz) d
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 calcu-
lated for C44H82O3Si3Na (MNa)+ 765.5468, found 765.5461.
a
a
J = 6.6 Hz, 21-H3), 0.570 (3H, s, 18-H3); 13C NMR (125 MHz) d
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-1a,25-
2.2.28. (20S,25S)-1a-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
dihydroxyvitamin D3 (7)
[(tert-butyldimethyl-silyl)oxy]-19,26-dinor-22-(E)-ene-vitamin D3
According to a general procedure the pure 2-methylene analog
tert-butyldimethylsilyl (30b)
7 (12.74 mg, 59% yield) was obtained from the protected vitamin
According to a general procedure the pure product 30b (10 mg,
46% yield) was obtained from the phosphine oxide 26 (52 mg,
28 (39.44 mg, 52 lmol). The vitamin 7 was further purified by
straight phase HPLC [Rt = 6.46 min] and then by reverse phase
HPLC [Rt = 10.19 min], as it’s described in a general procedure.
UV (in EtOH) kmax 261.0, 252.0, 244.5 nm; 1H NMR (400 MHz,
CDCl3) d 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,
89
lmol), PhLi (1.8 M in di-n-butylether, 61
lL, 110 lmol) and
the ketone 25b (9 mg, 24
lmol). For analytical purpose a sample
of the protected vitamin 30b was further purified by HPLC
(9.4 ꢂ 250 mm Zorbax Sil column, 4 mL/min, hexane/2-propanol
(99.9:0.1) solvent system, Rt = 3.51 min): UV (in hexane) kmax
263.1, 253.2, 244.3 nm; 1H NMR (500 MHz, CDCl3) d 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,
@CH2), 4.48 (2H, m, 1b- and 3
10b-H), 2.80 (1H, br d, J = 14.2 Hz, 9b-H), 2.56 (1H, dd, J = 13.4,
3.6 Hz, 4 -H), 2.32 (1H, dd, J = 13.4, 6.0 Hz, 4b-H), 2.28 (1H, dd,
J = 13.3, 8.4 Hz, 10 -H), 1.20 (6H, d, J = 1.2 Hz, 26,27-H6), 0.95
a-H), 2.84 (1H, dd, J = 13.3, 4.4 Hz,
a
a

G. Chiellini et al. / Steroids 83 (2014) 27–38
33
(3H, d, J = 6.6 Hz, 21-H3), 0.528 (3H, s, 18-H3); 13C NMR (100 MHz)
d 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.
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-1a,25-
dihydroxyvitamin D3 (8b)
According to a general procedure the pure 2-methylene analog
2.2.32. (20R,25R)-2-Methylene-19,26-dinor-22-(E)-ene-1
a
,25-
8b (4 mg, 54% yield) was obtained from the protected vitamin 29b
dihydroxyvitamin D3 (8a)
(15 mg, 34 lmol). The final compound 8b was purified by straight-
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 lmol). The final compound 8a was purified by re-
verse-phase HPLC (Rt = 13.7 min) as it’s described in a general pro-
cedure. UV (in EtOH) kmax 261.4, 252.4, 244.4 nm; 1H NMR
(900 MHz, CDCl3) d 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),
phase HPLC (Rt = 9.3 min) and then by reverse-phase HPLC
(Rt = 12.9 min) as it’s described in a general procedure. UV (in
EtOH) kmax 262.1, 252.6, 244.1 nm; 1H NMR (800 MHz, CDCl3) d
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, 1b- and 3
dd, J = 13.3, 4.5 Hz, 10b-H), 2.81 (1H, br d, J = 13.2 Hz, 9b-H), 2.57
(1H, dd, J = 13.4, 3.7 Hz, 4 -H), 2.33 (1H, dd, J = 13.4, 6.1 Hz, 4b-
H), 2.29 (1H, dd, J = 13.3, 8.3 Hz, 10 -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
a-H), 3.75 (1H, m, 25-H), 2.83 (1H,
5.10 and 5.08 (each 1H, each s, @CH2), 4.47 (2H, m, 1b- and 3
H), 3.78 (1H, m, 25-H), 2.84 (1H, dd, J = 13.3, 5.4 Hz, 10b-H), 2.81
(1H, br d, J = 13.5 Hz, 9b-H), 2.56 (1H, dd, J = 13.5, 3.6 Hz, 4 -H),
2.32 (1H, dd, J = 13.5, 5.4 Hz, 4b-H), 2.28 (1H, dd, J = 13.3, 8.1 Hz,
10 -H), 1.17 (3H, d, J = 6.3 Hz, 27-H3), 1.03 (3H, d, J = 6.3 Hz, 21-
a
-
a
a
a
mass calculated for
423.2874.
C
26H40O3Na+ (MNa+) 423.2875, found
a
Scheme 1. Reagents: (i) n-BuLi, THF; (ii) TBSOTf,2,6-lutine, CH2CL2; (iii) (1) KOH, EtOH; (2) PDC CH2CL2.

34
G. Chiellini et al. / Steroids 83 (2014) 27–38
2.2.34. (20S,25R)-2-Methylene-19,26-dinor-22-(E)-ene-1
a,25-
2.2.35. (20S,25S)-2-Methylene-19,26-dinor-22-(E)-ene-1a,25-
dihydroxyvitamin D3 (9a)
dihydroxyvitamin D3 (9b)
According to a general procedure the pure 2-methylene analog
According to a general procedure the pure 2-methylene analog
9a (1 mg, 43% yield) was obtained from the protected vitamin 30a
9b (1.3 mg, 36% yield) was obtained from the protected vitamin
(4.5 mg, 6
lmol). The final compound 9a was purified by reverse-
30b (7 mg, 9 lmol). The final compound 9b was purified by
phase HPLC (Rt = 11.8 min) as it’s described in a general procedure.
UV (in EtOH) kmax 262.1, 252.6, 244.1 nm; 1H NMR (900 MHz,
CDCl3) d 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
straight-phase HPLC (Rt = 9.3 min) and then by reverse-phase HPLC
(Rt = 11.1 min) as it’s described in a general procedure. UV (in EtOH)
kmax 262.1, 252.6, 244.1 nm; 1H NMR (800 MHz, CDCl3) d 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,
s, @CH2), 4.40 (2H, m, 1b- and 3
(1H, dd, J = 13.1, 4.5 Hz, 10b-H), 2.73 (1H, br d, J = 13.5 Hz, 9b-H),
2.51 (1H, dd, J = 13.5, 4.5 Hz, 4 -H), 2.27 (1H, dd, J = 13.5, 6.3 Hz,
4b-H), 2.22 (1H, dd, J = 13.1, 8.1 Hz, 10 -H), 1.11 (3H, d,
a-H), 3.76 (1H, m, 25-H), 2.78
@CH2), 4.48 (2H, m, 1b- and 3
dd, J = 12.8, 4.8 Hz, 10b-H), 2.80 (1H, br d, J = 12.8 Hz, 9b-H), 2.57
(1H, dd, J = 12.8, 2.5 Hz, 4 -H), 2.32 (1H, dd, J = 12.8, 6.4 Hz, 4b-
H), 2.29 (1H, dd, J = 12.8, 8.0 Hz, 10 -H), 1.19 (3H, d, J = 6.4 Hz,
a-H), 3.78 (1H, m, 25-H), 2.84 (1H,
a
a
a
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.
a
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.
Scheme 2. Reagents: (i) PhLi, (ii) aq. HF, THF, MeCN.

G. Chiellini et al. / Steroids 83 (2014) 27–38
35
Table 1
VDR binding properties,a HL-60 differentiating activities,b and transcriptional activitiesc of the vitamin D analogs 6–7; 8a,b–9a,b.
VDR bindinga
HL-60 differentiationb
CYP24A1 transcriptionc
Compound
,25-(OH)2D3
19-Nor-1 ,25-dihydroxyvitaminD2 (Zemplar)
(20S)-2-Methylene-19-nor-1 ,25-(OH)2D3 (2MD)
(20S,25R)-2-Methylene-19,26-dinor-1 ,25-(OH)2D3 (SR1)
Compd no.
Ki (M)
Ratio
EC50 (M)
Ratio
EC50 (M)
Ratio
1a
1
2
4
5
6
7
8a
8b
9a
9b
1 ꢂ 10ꢀ10
1 ꢂ 10ꢀ10
1 ꢂ 10ꢀ10
9 ꢂ 10ꢀ11
6 ꢂ 10ꢀ11
6 ꢂ 10ꢀ11
9 ꢂ 10ꢀ11
3 ꢂ 10ꢀ10
8 ꢂ 10ꢀ11
2 ꢂ 10ꢀ10
1
1
1
0.9
0.6
0.6
0.9
3
3 ꢂ 10ꢀ9
4 ꢂ 10ꢀ9
8 ꢂ 10ꢀ11
9 ꢂ 10ꢀ11
2 ꢂ 10ꢀ10
2 ꢂ 10ꢀ10
2 ꢂ 10ꢀ10
1 ꢂ 10ꢀ9
8 ꢂ 10ꢀ10
1 ꢂ 10ꢀ9
1
1.3
2 ꢂ 10ꢀ10
3 ꢂ 10ꢀ10
7 ꢂ 10ꢀ12
1 ꢂ 10ꢀ11
2 ꢂ 10ꢀ11
2 ꢂ 10ꢀ11
2 ꢂ 10ꢀ11
1 ꢂ 10ꢀ10
1 ꢂ 10ꢀ10
1 ꢂ 10ꢀ10
1
a
1.5
0.035
0.05
0.1
0.1
0.1
0.5
0.5
0.5
a
0.027
0.03
0.07
0.07
0.07
0.33
0.27
0.33
a
(20R)-2-Methylene-
(20S)-2-methylene-
(20R,25R)-2-Methylene-
D
D
22E-19-nor-1
22E-19-nor-1
a
,25-(OH)2D3 (AT3)
a
,25-(OH)2D3 (N23)
D
22E-19,26-dinor-1
a
,25-dihydroxyvitamin D3
(20R,25S)-2-Methylene-
(20S,25R)-2-Methylene-
D
D
22E-19,26-dinor-1
a,25-(OH)2D3
22E-19,26-Dinor-1
a,25-(OH)2D3
0.8
2
(20S,25S)-2-Methylene-
D
22E-19,26-dinor-1
a,25-(OH)2D3
a
Competitive binding of 1
dose–response curves and represent the inhibition constant when radiolabeled 1
ratio of the analog Ki to the Ki for 1 ,25-(OH)2D3.
Induction of differentiation of HL-60 promyelocytes to monocytes by 1
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 1 ,25-(OH)2D3.
a
,25-(OH)2D3 (1) and the synthesized vitamin D analogs to the full-length recombinant rat vitamin D receptor. The Ki values are derived from
a,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. The binding ratio is the average
a
b
a
,25-(OH)2D3 and the synthesized vitamin D analogs. Differentiation state was determined by
a
c
Transcriptional 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 1a,25-(OH)2D3. All the experiments were carried out in duplicate on at least two different occasions.
Bone Calcium Mobilization
10
8.9
9
8.2
7.8
8
7.0
6.5
7
6
5
4
3
2
1
0
6.0
5.8
5.3
5.2
4.9
4.7
4.3
4.0
Intestinal Calcium Transport
14
12
10
8
10.9
8.1
8.1
7.3
7.0
6.9
6.6
5.7
6
4.3
4
2
0
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).

36
G. Chiellini et al. / Steroids 83 (2014) 27–38
Bone Calcium Mobilization
25R, 20R or 20S
8
7
6
5
4
3
2
1
0
5.8
5.4
4.8
4.4
4.3
4.2
4.1
4.0
3.9
Bone Calcium Mobilization
25S, 20R or 20S
8
7
6
5
4
3
2
1
0
5.8
4.3
4.0
3.8
3.7
3.7
3.5
3.5
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.
2.3. Biological studies
D a,25(OH)2D3 compounds 8a,b–9a,b was based
22E-19,26-dinor-1
on the Wittig–Horner olefination reaction [18] between Grund-
mann-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 neces-
2.3.1. In vitro studies
VDR binding, HL-60 differentiation and 24-hydroxylase tran-
scription assays were performed as previously described [16,17].
sary
D
22E-25-hydroxy C,D-ring ketones (20–21; 24a,b–25a,b) are
2.3.2. In vivo studies
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-butan-
ediols [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
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 admin-
istered intraperitoneally once per day for four consecutive days.
Twenty-four hours after the last dose was given, the blood was col-
lected, 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 ani-
mals received the indicated dose of 1,25-(OH)2D3 in the vehicle.
3. Results and discussion
Scheme 2, each of the six D
22E-25-hydroxy Grundmann ketones
(20–21, 17a,b–18a,b) was coupled with the anion, generated from
phosphine oxide 26 and phenyllithium, affording the correspond-
ing six protected 19-norvitamin D analogs (27–28, 29a,b–30a,b).
Then, after silyl groups removal using hydrofluoric acid the final
3.1. Chemistry
The synthesis strategy of the new 2-methylene-
,25(OH)2D3 compounds 6 (20R) and 7 (20S) and 2-methylene-
D
22E-19-nor-
1a
2-methylene-D a,25(OH)2D3 (6–7) and 2-methylene-
22E-19-nor-1

G. Chiellini et al. / Steroids 83 (2014) 27–38
37
D
22E-19,26-dinor-1
complete.
a
,25(OH)2D3 (8a,b–9a,b) compounds were
cies 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 tran-
scriptional potency. They are more potent than both 2 and
3.2. Biological activity
1a,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. Usu-
ally 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).
It is unclear why compounds 4 and 5 have more than 10X the
activity of 1a,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
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-
,25(OH)2D3 compounds 6 and 7 exhibited approximately 10
times higher HL-60 differentiation activity as compared to the nat-
ural hormone 1 and 20 times higher than19-nor-1 ,25-dihydroxy-
D
22E-19-nor-
1a
a
vitaminD2 2. The 25R isomers 8a and 9a displayed higher cell
differentiation activity as compared to the corresponding 25S iso-
mers 8b and 9b, with isomer 8a being the most potent of this ser-
ies, 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 differen-
tiation and their efficacy is 3 times higher than that of the natural
binds 1a,25(OH)2D3 reducing the free 1a,25(OH)2D3. The com-
pounds 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
hormone 1 and 4 times higher than that of 19-nor-1
oxyvitaminD2 2. Compound 8a (20R, 25R) is the most potent of
the 2-methylene- ,25(OH)2D3 analogs, and its
22E-19,26-dinor-1
potency is comparable to that of 5, 6 and 7. The pattern of poten-
a,25-dihydr-
D
a
Intestinal Calcium Transport
25R, 20R or 20S
16
14
12
10
8
11.0
10.9
9.3
8.5
8.3
8.2
7.2
5.0
6
4.4
4
2
0
Intestinal Calcium Transport
25S, 20R or 20S
16
14
12
10
8
11.4
10.4
9.6
8.6
7.4
6.7
6.3
5.3
6
4
2
0
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.

38
G. Chiellini et al. / Steroids 83 (2014) 27–38
significance is unlikely. Nevertheless, activity in HL-60 may repre-
sent anti-cancer activity, which is not assessed by the in vivo assays
used in the present study.
Wisconsin, the NIH (RR02781, RR08438), the NSF (DMB-8415048,
OIA-9977486, BIR-9214394), and the USDA.
In the present series14, removal of the 26-methyl group has lit-
tle 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 re-
sults in a compound with one log less in vitro potency (compare
compounds 4 and 9a).
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Acknowledgments
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[17] Chiellini G, Grzywacz P, Plum LA, Barycki R, Clagett-Dame M, DeLuca HF.
The work was supported in part by funds from the Wisconsin
Alumni Research Foundation. We gratefully acknowledge Jean
Prahl, Julia Zella and Jennifer Vaughan for carrying out the
in vitro studies, and Heather Neils, Shinobu Miyazaki and Xiaohong
Ma for conducting the in vivo studies. We thank Dr. Mark Anderson
for his assistance in recording NMR spectra.
Synthesis and biological properties of 2-methylene-19-nor-25-dehydro-1a-
hydroxyvitamin D3-26,23-lactones
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This study made use of the National Magnetic Resonance Facil-
ity at Madison, which was supported by the NIH Grants
P41RR02301 (BRTP/NCRR) and P41GM66326 (NIGMS). Additional
equipment was purchased with funds from the University of
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