New 1α,25-dihydroxy vitamin D3 analogues with side chains attached to C-18: Synthesis and biological activity

Article abstract of DOI:10.1016/S0968-0896(98)00161-8

A new class of analogues of 1α,25-dihydroxy vitamin D₃ has been synthesised, in which the side chain (C-23 to C-27) has been removed and where new hydroxylated side chains have been attached to the C-18 methyl group. These analogues show antipr

Full text of DOI:10.1016/S0968-0896(98)00161-8

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 2029±2039
New 1ꢀ,25-dihydroxy Vitamin D3 Analogues with Side Chains
Attached to C-18: Synthesis and Biological Activity
Gunnar Grue-Sùrensena* and Christina Mùrk Hansenb
aDepartment of Chemical Research, Leo Pharmaceutical Products, DK-2750 Ballerup, Denmark
bDepartment of Biochemistry, Leo Pharmaceutical Products, DK-2750 Ballerup, Denmark
Received 1 May 1998; accepted 12 June 1998
AbstractÐA new class of analogues of 1a,25-dihydroxy vitamin D3 has been synthesised, in which the side chain (C-23
to C-27) has been removed and where new hydroxylated side chains have been attached to the C-18 methyl group.
These analogues show antiproliferative activity in U937 and HaCaT cells comparable to that of 1a,25-dihydroxy
vitamin D3. Lack of calcemic activity makes these analogues potentially useful in the treatment of proliferative dis-
eases. # 1998 Published by Elsevier Science Ltd. All rights reserved.
Introduction
Numerous analogues of 1a,25-dihydroxy vitamin D3
(1,25(OH)2 D3) have been investigated in biological tests
in order to explain structure±activity relationships.
1,25(OH)2 D3 and its analogues exhibit a multitude of
biological activities,1 including antiproliferative e€ects
and the ability to stimulate cell di€erentiation, to induce
apoptosis, to regulate the immune system and to mobi-
lise calcium. Vitamin D3 compounds are therefore
obvious candidates as therapeutic agents in the treatment
of immune diseases, bone disorders and hyperprolifera-
tive diseases, such as cancer and psoriasis. However, the
fact that vitamin D3 compounds a€ect the calcium
homeostasis often leads to induction of hypercalcemia
and thus limits the usefulness of such compounds in the
clinic. Therefore, the current objective of synthesising
analogues of 1,25(OH)2 D3 is to develop compounds
with potent cellular e€ects but with reduced e€ects on
the calcium metabolism.2
with the calculated preferred orientation of the side
chain in these analogues to the `left'4 prompted us to
test the biological activity of analogues in which the side
chain (carbon atoms C-23 to C-27) of 1,25(OH)2 D3 has
been removed and a variety of new hydroxylated side
chains have been attached to C-18. In these compounds,
the reach of the side chain hydroxy group would be
restricted to a space in or above the plane of the CD-
ring system. Furthermore, due to the steric hindrance
of the residual 2-propyl group in position 17, the side
chain hydroxy group in the new 18-analogues would
not be able to reach as far to the `right' of C-22 as the
25-hydroxy group of 1,25(OH)2 D3 is able to. We
describe the synthesis and biological activity of these
compounds.
The present work describes our e€ort to shed some light
on the structure±activity relationship of a new class of
analogues of 1,25(OH)2 D3.3 The high anti-proliferative
2
activity of 20-epi analogues of 1,25(OH)2 D3 together
Key words: Vitamins; antiproliferative agents.
*Corresponding author. Tel.: 0045 44 94 58 88 ext. 2237; fax:
+45 44 94 55 10; e-mail: gunnar.soerensen@leo.dk
0968-0896/98/$ - see front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00161-8

2030
G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
ꢀ
.
Scheme 1. (a) LiAlH4, THF, re¯ux, 21 h, 93%; (b) Pb(OAc)4, benzene, pyridine, hn, 15 C, 3.5 h, 88%; (c) BF3 Et2O, Ac2O, 25 to
25 ꢀC, 1 h, 39%; (d) KOH, MeOH, H2O, re¯ux, 2.5 h, 65%; (e) tert-butyldimethylsilyl chloride, imidazole, DMF, 25 ꢀC, 4 h, 94%.
Results and discussion
7i gave elimination of HBr to the conjugated enyne
side chain fragment. Thus, the side chain of compound
7i was attached to 6 in a 5-step sequence (Scheme 3).
Similarly, attempts to alkylate 6 with a preformed
alkyl bromide to give a compound with the side chain
of compound 8h were unsuccessful. In this case, the
Synthesis
The synthesis of the new analogues with side chains
attached to C-18 is divided into three parts: (1) synthesis
of the key CD-ring intermediate 6 (Scheme 1), (2)
attachment of the new side chains at C-18 (Schemes
2±4), and (3) attachment of the A-ring followed by
removal of protecting groups to give the new analogues
12a±i (Scheme 2). Chemical yields for compounds 7 to
12 are given in Table 1.
side chain was attached in
(Scheme 4).
a two-step sequence
In order to successfully join the CD-ring moiety with the
A-ring phosphine oxide 19, the deprotected alcohol at
C-8 in compounds 8a±i was oxidised to the corre-
sponding ketone with pyridinium dichromate and any
unprotected tertiary alcohol in the side chain was pro-
tected as its TMS ether (compounds 10a±c and 10h).
The tertiary hydroxy group of compounds 9d±g and 9i
was protected as THP ether. The coupling of the A-
with the CD-ring fragments and deprotection of
hydroxy groups followed known methods.13
The mono-tosylate 15 was reduced with LiAlH4 to the
alcohol 2, in which the hydroxy group and the 18-
methyl group share a 1,3 trans diaxial relationship
(Scheme 1). Oxidation mediated by lead tetraacetate
and UV-light6,7 produces 3, in which an ether bridge
between C-8 and C-18 has been established. The ether
was cleaved with a Lewis acid (boron tri¯uoride ethe-
rate) in acetic anhydride6 to give the diacetate 4, where
the con®guration at C-8 was inverted. Hydrolysis to the
diol 5, followed by mono-silylation completes the
synthesis of the key CD-ring intermediate 6 in an over-
all yield of 20% from 1.
Compounds 7a±g were prepared by alkylation of the
alcohol at C-18 in compound 6 with a series of hydroxy
group protected side chain alkyl bromides (20a,8 20b,9
20c,9 20d,10 20e,10 20f,11 20g,12 see Fig. 1), and potas-
sium hydride/18-crown-6 in THF (Scheme 2). Attempts
to alkylate 6 with a preformed alkyl bromide to give

G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
2031
Scheme 2. (a) KH, 18-Crown-6, Y-O-CR2-Q-Br (20a±g), THF, rt, 20 min; (b) 7a±c: HF, EtOAc, MeCN, H2, rt, 40 min or 7d±g and 7i:
TBA ¯uoride trihydrate, THF, 60 ꢀC, 3 h; (c) pyridinium dichromate, CH2Cl2, rt, 18 h; (d) 9a±c and 9h: trimethylsilyl chloride, ethyl-
diisopropylamine, CH2Cl2, rt, 140 min; (e) Ref. 13; (f) HF, EtOAc, MeCN, H2O, rt, 40 min.
In vitro and in vivo activity
adding increasing concentrations of the test compounds.
Displacement of 50% of the bound of [3H]-1,25(OH)2
D3 was obtained with 1,25(OH)2 D3 at (10.0‹4.5)Â
10 11 M (n=11). As shown in Table 2, the test com-
pounds bound only weakly to the VDR. The com-
pounds with the most ¯exible side chains (12a±c)
displayed the highest binding anities, although these
were still reduced by a factor 3-5 when compared to the
parent compound, 1,25(OH)2 D3.
The in vitro potencies of the compounds 12a±i were
investigated by determining their antiproliferative e€ects
in two human cell lines, the U937 lymphoma cell line
and the HaCaT keratinocyte cell line. In addition, the
binding anity of the compounds for the vitamin D
receptor (VDR) was assessed using isolated VDR's from
the intestine of rachitic chickens. Table 2 summarises
the results obtained from these in vitro studies. The in
vivo calcemic activity of the compounds 12a±i was
determined after 7 days of oral administration to rats
(Table 3).
The antiproliferative activity of the test compounds in
the HaCaT cell line was assessed by [3H]-thymidine
uptake, whereas the activity of the compounds in the
U937 cell line was determined by counting the cells.
50% inhibition of the cell growth (IC50) was obtained
with 1,25(OH)2 D3 at (4.8‹1.5)Â10 8 M (n=13) and
The receptor binding studies were performed by dis-
placement of [3H]-1,25(OH)2 D3 from the receptor by

2032
G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
Scheme 3. (a) KH, 18-Crown-6,3-bromoprop-1-ene, THF, rt, 80 min, 87%; (b) ozone, CH2Cl2, MeOH, 70 ꢀC, 30 min, 100%; (c)
NaBH4, THF, MeOH, 0 ꢀC, 35 min, 76%; (d) TsCl, pyridine, 0 ꢀC, 4 h, 59%; (e) H-CꢁC-CMe2OTHP, n-BuLi, dioxane, 90 ꢀC, 48 h,
55%.
.
Scheme 4. (a) tert-BuOK, 18-Crown-6, THF, 3-bromoprop-1-yne, rt, 19 h, 50%; (b) n-BuLi, THF, BF3 Et2O, 2,2-dimethyloxirane,
78 ꢀC to rt, 1 h, 45%; (c) HF, EtOAc, MeCN, H2O, rt, 40 min, 85%.
(3.4‹2.3)Â10 8 M (n=12) in the HaCaT and in the
Table 3 shows the mean urinary excretion of calcium
and the serum calcium levels after 7 days of oral
administration of the test compounds to rats. The data
clearly show that all the compounds 12a±i exerted only
weak e€ects on the calcium metabolism in vivo and were
at least 20 times less calcemic than 1,25(OH)2 D3.
U937 cell line, respectively.
Interestingly, the compounds with the highest binding
anity for the VDR (12a±c) also appeared to be the
most potent analogues with respect to their e€ects on
cell proliferation, but the di€erence in antiproliferative
activity between the nine analogues (12a±i) is quite small
(within one order of magnitude). This comparable anti-
proliferative activity of these analogues of 1,25(OH)2 D3
together with the limited range of the C-18 side chain
point to a binding site for the side chain hydroxy group
onto the VDR on the same side of the CD ring system
as the C-18 methyl group but not far to the right of the
2-propyl group at C-17 (cf. Ref. 14).
Conclusion
The synthesis of 1a,25-dihydroxy vitamin D3 analogues,
in which the side chain from C-23 to C-27 was removed
and in which new side chains were attached to the C-18
methyl group have succesfully been carried out. These
analogues show a weak binding to the VDR and an

G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
2033
Table 1. Chemical yields
Compound
Y/Y0
Chemical yield (%)
R
Q
7a
8a
H
9a 10a 11a 12a Me
(CH2)3
(CH2)3
TMS
72
7b
H
TMS
80
9b 10b 11b 12b Et
55
8b
H
78
80
66
TMS
66
7c
H
TMS
59
9c 10c 11c 12c Me
73
8c
H
81
65
75
(CH2)4
TMS
64
7d
H
80
9d
TMS
59
11d 12d Me
79
8d
61
67
m-C6H4CH2
m-C6H4CH2
C ˆ CCH2
C ˆ CCH2
THP THP THP
THP
61
95
7e
87
8e
79
9e
54
11e 12e Et
THP
THP THP THP
80
75
74
43
11f
60
12f Me
7f
THP THP THP
8f
9f
THP
41
65
7g
77
8g
67
9g
48
11g 12g Et
THP
THP THP THP
73
86
8h
H
74
13
69
9h 10h 11h 12h Me CH2C ˆ CCH2
H
81
9i
TMS
70
11i
85
8i
34
50
12i Me
7i
C ˆ C(CH2)2
H
55
THP THP
62 60
THP
40
57
antiproliferative activity comparable to that of 1a,
25-dihydroxy vitamin D3. This activity indicates a
binding site for the side chain hydroxy group to the
vitamin D receptor in or above the plane of the CD-ring
system. Lack of calcemic activity makes these analo-
gues potentially useful in the treatment of proliferative
diseases.
Figure 1. Hydroxy group protected side chain alkyl bromides
20a±g. TMS=trimethylsilyl and THP-tetrahydropyran-2-yl.
Ozone was produced on a Fisher ozone generator model
500.
20-Methyl-de-A,B-pregnan-(8S)-ol (2). A solution of
20S-(4-methylphenylsulfonyloxymethyl)-de-A,B-pregnan-
(8S)-ol (1)5 (44.0 g, 120 mmol) in dry THF (450 mL) was
re¯uxed under argon with LiAlH4 (8.5 g, 223 mmol) for
21 h. After cooling the mixture was quenched with drops
of water and ®ltered through ®lter aid. The ®ltrate was
mixed with water (1.0 L) and ether (0.5 L). The organic
phase was washed with water (0.5 L) and brine (0.5 L),
and removal of solvent gave compound 2 as a colourless
oil (22.0 g, 93%). An analytical sample was obtained by
Experimental
General
1H and 13C NMR spectra were recorded in CDCl3 on a
Bruker AM300 spectrometer and chemical shifts are
reported in ppm down®eld from tetramethylsilane (s, d,
t, m and b for singlet, doublet, triplet, multiplet and
broad, respectively). Mass spectra were recorded on an
Autospec Micromass spectrometer (EI spectra) and a
Quattro II Micromass spectrometer (ES spectra). For
column chromatography Merck silica gel 60 (40±63 mm)
was used. Organic extracts were dried with magnesium
sulfate before evaporation in vacuo on a rotary evaporator.
1
chromatography with EtOAc/petroleum ether (1/2). H
NMR d 0.84 (d, 3H), 0.91 (d, 3H), 0.93 (s, 3H), 0.90±
1.92 (m, 13H), 1.98 (m, 1H), 4.07 (m, 1H). 13C NMR d
69.4, 58.8, 52.7, 41.9, 40.3, 33.6, 30.6, 27.4, 23.1, 22.6,
22.4, 17.5, 13.7. MS (EI+) m/z 196.2 (M+).

2034
G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
Table 2. In vitro biological activities
Compound
Side chain
VDR bindinga Inhibition of proliferation HaCaTb
Inhibition of proliferation U937c
d
12a
0.2Â
0.5Â
2Â
e
12b
0.3Â
2Â
1Â
12c
12d
0.2Â
2Â
3Â
<0.02Â
0.5Â
0.4Â
<0.5Â
12e
<0.04Â
0.3Â
12f
0.02Â
0.07Â
<0.4Â
0.8Â
0.3Â
0.4Â
12g
12h
12i
0.02Â
0.02Â
0.9Â
0.8Â
0.4Â
0.4Â
aThe binding anity for the vitamin D receptor (VDR) of each compound was measured (n=3) relative to 1,25(OH)2 D3:
IC50=1.00Â10 10 M (coecient of variation (CV)=45%, n=11).
bThe inhibitory e€ect of each compound was measured (n=2) relative to 1,25(OH)2 D3: IC50=4.8Â10 8 M (CV=31%, n=13).
cThe inhibitory e€ect of each compound was measured (n=3) relative to 1,25(OH)2 D3: IC50=3.4Â10 8 M (CV=68%, n=12).
dn=1.
en=3.
8S,18-Epoxy-20-methyl-de-A,B-pregnane (3). (cf. Ref. 6).
A mixture of compound 2 (1.40 g, 7.1 mmol), pyridine
(3.5 mL) and lead tetraacetate (15.9 g, 35.8 mmol) in
benzene (375 mL) in an atmosphere of argon was irra-
diated with UV-light from a Hanau TG 700 W mercury
lamp at 15 ꢀC for 3.5 h. The reaction mixture was ®l-
tered, the precipitate was washed with ether (3Â100 mL)
and the solvent was removed from the combined ®l-
trates. The residue was mixed with EtOAc/petroleum
ether (1/3), ®ltered and chromatographed with EtOAc/
petroleum ether 1/3 to give compound 3 (1.22 g, 88%). 1H
NMR d 0.85 (d, 3H), 0.91 (d, 3H), 0.90±2.10 (m, 13H),
3.70 (m, 2H), 4.14 (d, 1H). 13C NMR d 78.9, 70.9, 57.9,
54.2, 53.4, 37.1, 32.4, 32.4, 28.5, 24.9, 22.9, 22.5, 18.9.
8R,18-Diacetoxy-20-methyl-de-A,B-pregnane (4). Boron
tri¯uoride etherate (90 mL) was added dropwise under
argon and at 25 to 20 ꢀC to a solution of compound

G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
Table 3. Calcemic activity in normal ratsa
2035
Compound
Dose mg/kg/day
oral 7x
Calcium in urine
mmol/day mean‹SD
p-valueb
Calcium in serum
mmol/L mean‹SD
p-valueb
12a
1.0
10
24.8‹9.9
37.8‹14.6
ns
p<0.01
2.93‹0.04
2.96‹0.15
ns
ns
1,25(OH)2 D3
Control
12b
0.5
Ð
111.9‹49.3
25.9‹4.1
17.5‹6.0
20.7‹7.1
p<0.001
Ð
ns
3.11‹0.08
2.91‹0.03
2.76‹0.17
2.78‹0.10
p<0.02
Ð
ns
ns
1.0
10
p<0.001
1,25(OH)2 D3
Control
12c
0.5
Ð
57.2‹37.4
16.5‹6.7
20.5‹7.0
34.1‹13.6
p<0.001
Ð
ns
2.87‹0.08
2.70‹0.10
2.91‹0.20
2.71‹0.06
ns
Ð
ns
ns
1.0
10
p<0.01
1,25(OH)2 D3
Control
12d
0.5
Ð
152.7‹39.8
20.2‹6.0
33.5‹12.8
24.5‹8.9
p<0.001
Ð
3.03‹0.14
2.69‹0.12
2.73‹0.15
3.00‹0.04
p<0.05
Ð
ns
10
100
p<0.05
ns
p<0.01
1,25(OH)2 D3
Control
12e
1.0
Ð
155.7‹58.6
24.2‹8.4
p<0.001
Ð
2.88‹0.22
2.61‹0.12
2.87‹0.08
2.71‹0.13
ns
Ð
10
100
31.4‹10.0
31.8‹20.7
p<0.001
ns
p<0.05
ns
1,25(OH)2 D3
Control
12f
1.0
Ð
155.7‹58.6
24.2‹8.4
25.9‹13.0
17.9‹8.0
p<0.001
Ð
ns
2.88‹0.22
2.61‹0.12
2.79‹0.02
2.75‹0.13
ns
Ð
ns
ns
10
100
p<0.01
1,25(OH)2 D3
Control
12g
0.5
Ð
93.3‹37.1
29.4‹9.6
24.1‹10.2
27.4‹15.8
p<0.001
3.04‹0.05
2.81‹0.17
2.88‹0.13
2.87‹0.14
ns
Ð
ns
ns
Ð
ns
ns
1.0
10
1,25(OH)2 D3
Control
12h
0.5
Ð
152.7‹39.8
20.2‹6.0
25.4‹6.7
17.3‹6.3
p<0.01
Ð
3.03‹0.14
2.69‹0.12
3.07‹0.22
3.06‹0.06
p<0.05
Ð
ns
1.0
10
p<0.02
ns
p<0.02
1,25(OH)2 D3
Control
12i
0.5
Ð
80.3‹32.6
19.7‹4.5
22.7‹6.6
17.5‹3.7
p<0.001
3.04‹0.02
2.85‹0.06
1.98‹0.02
1.71‹0.06
p<0.01
Ð
Ð
ns
ns
10
100
p<0.01
p<0.001
1,25(OH)2 D3
Control
0.5
Ð
112.7‹29.7
19.6‹5.2
p<0.001
Ð
2.37‹0.45
2.23‹0.06
ns
Ð
aFor experimental details, see text.
bns: not signi®cant.
3 (8.0 g, 41.2 mmol) in acetic anhydride (500 mL). Stir-
ring was continued for 45 min at 20 ꢀC and for 1 h at
rt. The mixture was poured into ice cold saturated aqu-
eous sodium hydrogencarbonate (1 L). After stirring for
1 h the aqueous phase was extracted with ether
(3Â300 mL). The combined ether phases were washed
with saturated aqueous sodium hydrogencarbonate
(6Â200 mL), water (500 mL) and brine (250 mL), and
were dried and evaporated. Chromatography with
EtOAc/petroleum ether (1/10 to 1/4) gave compound 4
(4.7 g, 39%). 1H NMR d 0.85 (d, 3H), 1.02 (d, 3H), 2.01
(s, 3H), 2.06 (s, 3H), 0.90±2.35 (m, 13H), 3.97 (d, 1H),
4.16 (d, 1H), 4.90 (m, 1H). 13C NMR d 171.0, 170.6,
73.0, 62.2, 58.8, 53.9, 47.8, 34.5, 32.0, 31.2, 27.7, 23.4,
23.4, 22.8, 21.5, 21.2, 21.0. MS (ES+) m/z 297 ([M+H]+).
20-Methyl-de-A,B-pregnane-8R,18-diol (5). Compound
4 (4.7 g, 15.9 mmol) was dissolved in methanol (50 mL)
and a 10% (w/v) solution of potassium hydroxide in
methanol (30 mL) was added. After re¯ux for 2.5 h the
reaction mixture was ®ltered through silica gel. The ®l-
trate was evaporated and chromatographed with
EtOAc/petroleum ether (2/1) to give compound 5
(2.2 g, 65%). Melting point 100±101 ꢀC (from chloroform).

2036
G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
1H NMR d 0.88 (d, 3H), 1.04 (d, 3H), 0.90±2.10 (m,
14H), 2.33 (m, 1H), 3.62 (m, 2H), 3.67 (m, 1H). 13C
NMR d 70.6, 60.5, 58.9, 56.9, 49.2, 35.8, 34.1, 31.4, 28.0,
23.4, 23.4, 23.2, 21.9. MS (EI+) m/z 212.1 (M+).
brine (40 mL) and concentrated. Chromatography with
ether/petroleum ether (1/20) as eluant gave compound
13 (0.25 g, 50%). 1H NMR d 4.06 (d, 2H), 3.66 (dt, 1H),
3.42 (d, 1H), 3.29 (d, 1H), 2.39 (t, 1H), 2.24 (m, 1H),
1.95±0.85 (m, 12H), 0.96 (d, 3H), 0.86 (s, 9H), 0.82 (d,
3H), 0.029 (s, 3H), 0.024 (s, 3H). 13C NMR d 80.0, 73.8,
71.1, 68.2, 58.9, 58.2, 56.9, 48.2, 36.3, 34.8, 31.0, 27.8,
25.7, 24.2, 23.2, 22.8, 21.7, 18.0, 4.3, 4.8.
8R-tert-Butyldimethylsilyloxy-20-methyl-de-A,B-pregnan-
18-ol (6). Compound 5 (2.2 g, 10.4 mmol), imidazole
(1.8 g, 26.4 mmol) and tert-butyldimethylsilyl chloride
(1.7 g, 11.3 mmol) was stirred in dry DMF (95 mL) at rt
for 4 h. The reaction mixture was partitioned between
water (200 mL) and ether (200 mL). The organic phase
was washed with water (100 mL) and brine (100 mL)
and evaporated. The residue was chromatographed with
EtOAc/petroleum ether (1/3) to give compound 6 (3.2 g,
94%). 1H NMR d 0.01 (s, 3H), 0.02 (s, 3H), 0.85 (s, 9H),
0.85 (d, 3H), 1.02 (d, 3H), 0.90±1.98 (m, 13H), 2.29 (m,
1H), 3.52±3.70 (m, 3H). 13C NMR d 71.0, 60.5, 58.7,
56.7, 48.9, 36.2, 33.8, 31.3, 27.6, 25.7, 24.1, 23.2, 23.0,
21.8, 4.4, 4.9. MS (EI+) m/z 326.2 (M+).
8R-tert-Butyldimethylsilyloxy-20-methyl-18-(5-hydroxy-
5-methyl-hex-2-yn-1-yloxy)-de-A,B-pregnane (14). n-
Butyl lithium (0.47 mL, 1.5 M in hexane) was slowly
added under argon to a solution of compound 13
(0.25 g, 0.69 mmol) in dry THF at 78 ꢀC. The dark
solution was stirred at this temperature for 40 min and
boron tri¯uoride diethyletherate (0.100 mL, 0.79 mmol)
was added. After stirring for 10 min 2,2-dimethyloxirane
(0.060 g, 0.83 mmol) in dry THF (2 mL) was added and
the mixture was stirred for 1 h at rt. Saturated aq
ammonium chloride (10 mL) was added and the mixture
was extracted with EtOAc (2Â30 mL). The organic
phase was washed with saturated sodium hydro-
gencarbonate (30 mL), water (30 mL) and brine (30 mL)
and concentrated. Chromatography with ether/petroleum
The alkylation of compound 6 to compound 7g is
representative for the series of reactions leading from
6 to 7a±g
1
Alkylation of compound 6 to compound 7g. Compound 6
(0.234 g, 0.72 mmol) and 1-bromo-4-ethyl-4-(tetra-
hydropyran-2-yloxy)-hex-2-yne (20g) (0.42 g, 1.45 mmol)
were dissolved in dry THF (5 mL) under argon and
potassium hydride (0.22 mL of a 20% oil emulsion,
1.1 mmol) was added. After 5 min a solution of 18-
crown-6 (0.19 g, 0.72 mmol) in dry THF (1.5 mL) was
added. After stirring for 20 min at rt the reaction mix-
ture was partitioned between water (50 mL) and ether
(50 mL). The ether phase was washed with brine (25 mL)
and the residue after evaporation of the solvent was
chromatographed with ether/petroleum ether (1/10) to
give the desired compound 7g (1/1 mixture of two THP
ether (1/2) gave 14 (0.137 g, 45%). H NMR d 4.08 (t,
2H), 3.64 (dt, 1H), 3.46 (d, 1H), 3.27 (d, 1H), 2.40 (t,
2H), 2.25 (m, 1H), 1.95±0.85 (m, 13H), 1.31 (s, 6H), 0.96
(d, 3H), 0.85 (s, 9H), 0.81 (d, 3H), 0.02 (s, 6H). 13C
NMR d 82.6, 79.4, 71.2, 69.8, 67.8, 58.9, 58.5, 56.8, 48.2,
36.2, 34.7, 34.3, 31.0, 28.4, 27.7, 25.7, 24.2, 23.1, 22.9,
21.7, 18.0, 4.3, 4.8.
8R-tert-Butyldimethylsilyloxy-20-methyl-18-(prop-2-en-1-
yloxy)-de-A,B-pregnane (15). Potassium hydride
(3.14 mL, 20% emulsion in oil) was added to a solution
of compound 6 (0.945 g, 2.9 mmol) and 18-Crown-6
(0.767 g, 2.9 mmol) in dry THF (9 mL) at 78 ꢀC under
argon. Allylbromide (2.7 g, 22 mmol) in THF (3 mL)
was added and the mixture was stirred for at rt for
80 min. Ether (150 mL) was added and the mixture was
quenched and washed with water (100 mL). The organic
phase was washed with water (100 mL) and brine
(100 mL) and concentrated. Chromatography with ether/
1
isomers). Yield: 0.28 g (73%). H NMR d 0.01 (s, 3H),
0.02 (s, 3H), 0.81 (d, 3H), 0.86 (s, 9H), 0.90±1.95 (m,
31H), 2.27 (m, 1H), 3.26 (dd, 1H), 3.46 (m, 2H), 3.64 (m
1H), 3.93 (m, 1H), 4.13 (m, 2H), 5.03 (m, 1H). 13C
NMR d 95.7, 95.7, 86.5, 86.4, 82.4, 82.4, 78.3, 71.2, 67.5,
67.5, 63.1, 63.1, 58.9, 58.3, 56.8, 48.1, 36.2, 34.5, 32.5,
31.9, 31.8, 31.7, 31.7, 31.0, 27.7, 25.7, 25.2, 24.2, 23.1,
22.9, 21.6, 20.2, 20.2, 17.9, 8.6, 8.6, 8.3, 8.3, 4.4, 4.9.
1
petroleum ether (2/98) gave 15. Yield: 0.92 g (87%). H
NMR (CDCl3) d 5.86 (m, 1H), 5.24 (m, 1H), 5.12 (m,
1H), 3.86 (m, 2H), 3.69 (dt, 1H), 3.34 (d, 1H), 3.21 (d,
1H), 2.28 (m, 1H), 1.95±0.90 (m, 12H), 0.98 (d, 3H),
0.86 (s, 9H), 0.82 (d, 3H), 0.024 (s, 3H), 0.018 (s, 3H).
13C NMR (CDCl3) d 135.1, 115.5, 72.1, 71.2, 68.7, 58.9,
56.9, 48.3, 36.3, 35.1, 31.0, 27.8, 25.7, 24.2, 23.2, 22.9,
22.0, 18.0, 4.3, 4.8.
8R-tert-Butyldimethylsilyloxy-20-methyl-18-(prop-2-yn-1-
yloxy)-de-A,B-pregnane (13). A solution of compound 6
(0.446 g, 1.37 mmol) , 18-crown-6 (0.36 g, 1.36 mmol)
and potassium tert-butoxide (0.31 g, 2.8 mmol) in dry
THF (12 mL) was stirred for 10 min and 3-bromoprop-
1-yne (0.32 g, 2.7 mmol) was added, and after stirring
for 1 h 3-bromoprop-1-yne (0.64 g, 5.4 mmol) and
potassium tert-butoxide (0.62 g, 5.5 mmol) were ®nally
added. After stirring for 19 h ether was added and the
reaction mixture was washed with water (40 mL) and
8R-tert-Butyldimethylsilyloxy-20-methyl-18-(carbonyl-
methoxy)-de-A,B-pregnane (16). Ozone was passed
through a solution of compound 15 (0.90 g, 2.45 mmol)
in a mixture of dichloromethane (60 mL) and methanol

G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
2037
(20 mL) at 70 ꢀC for 30 min until no more starting
compound 15 could be detected (TLC, ether/petroleum
ether, 1/10). Triphenylphosphine (0.8 g, 3.0 mmol) was
added and the mixture was stirred at 70 ꢀC for 30 min.
The solvent was removed and the residue was chroma-
tographed with ether/petroleum ether (1/4) followed by
and the combined organic phases were washed with
brine and concentrated. The residue was chromato-
graphed with ether/petroleum ether (1/10) to give the
desired compound 7i: 0.152 g (55%). This compound
was not pure and was used in the next step without fur-
ther puri®cation.
1
ether as eluant to give compound 16 (0.90 g, 100%). H
NMR d 9.72 (t, 1H), 3.95 (t, 2H), 3.63 (dt, 1H), 3.47 (d,
1H), 3.31 (d, 1H), 2.33 (m, 1H), 1.95±0.90 (m, 12H),
1.00 (d, 3H), 0.87 (s, 9H), 0.83 (d, 3H), 0.024 (s, 3H),
0.014 (s, 3H). 13C NMR d 201.2, 76.7, 71.0, 70.3, 58.8,
56.7, 48.5, 36.2, 34.6, 31.3, 27.7, 25.7, 24.2, 23.2, 22.8,
21.8, 17.9, 4.3, 4.8.
The desilylation of compound 7g to compound 8g is
representative for the series of reactions leading from
7d±g and 7i to 8d±g and 8i, respectively
Desilylation of compound 7g with TBA ¯uoride. Com-
pound 7g (0.250 g, 0.47 mmol) and tetra-n-butylammo-
nium ¯uoride trihydrate (0.445 g, 1.4 mmol) in THF
(5 mL) was stirred at 60 ꢀC for 3 h. Saturated aq sodium
hydrogen carbonate was (25 mL) added and the mixture
was extracted with EtOAc (50 mL). The organic phase
was washed with water (25 mL) and brine (25 mL) and
concentrated. Chromatography with EtOAc/petroleum
ether (1/1) as eluant gave the desired compound 8g (1/1
mixture of two isomers). Yield: 0.170 g (86%). 1H NMR
d 0.84 (d, 3H), 0.90±2.10 (m, 32H), 3.29 (m, 1H), 3.34
(d, 1H), 3.48 (m, 2H), 3.73 (m, 1H), 3.94 (m, 1H), 4.13
(m, 2H), 5.06 (m, 1H). 13C NMR d 95.8, 86.7, 82.7, 82.6,
78.5, 77.2, 70.8, 70.7, 67.9, 67.8, 63.2, 59.0, 58.6, 57.2,
48.5, 35.8, 34.9, 32.7, 32.1, 31.9, 31.1, 28.2, 28.2, 25.5,
23.5, 23.5, 23.4, 23.0, 21.9, 20.3, 20.3, 8.8, 8.5.
8R-tert-Butyldimethylsilyloxy-20-methyl-18-(2-hydroxy-
ethoxy)-de-A,B-pregnane (17). Sodium borohydride
(0.43 g, 11.4 mmol) was added to an ice cold solution of
compound 16 (0.90 g, 2.45 mmol) in a mixture of THF
(20 mL) and methanol (40 mL). After stirring for 35 min
the mixture was evaporated and the residue was chro-
matographed with ether/petroleum ether (1/7) followed
by ether to give compound 17 (0.69 g, 76%). 1H NMR d
3.68 (m, 2H), 3.65 (dt, 1H), 3.45 (m, 2H), 3.39 (d, 1H),
3.25 (d, 1H), 2.28 (m, 1H), 1.95±0.80 (m, 13H), 0.99 (d,
3H), 0.86 (s, 9H), 0.82 (d, 3H), 0.022 (s, 3H), 0.014 (s,
3H). 13C NMR d 72.1, 71.1, 69.2, 61.8, 58.8, 56.7, 48.4,
36.2, 34.6, 31.3, 27.8, 25.7, 24.2, 23.1, 22.8, 21.8, 17.9,
4.3, 4.8.
The desilylation of compound 7c to compound 8c is
representative for the series of reactions leading from
7a±c and 14 to 8a±c and 8h, respectively
8R-tert-Butyldimethylsilyloxy-20-methyl-18-[2-(4-toluene-
sulfonyloxy)-ethoxy]-de-A,B-pregnane (18). 4-Toluene-
sulfonyl chloride (0.70 g, 3.7 mmol) was added to an ice
cold solution of 17 (0.69 g, 1.86mmol) in pyridine
(7.5 mL). After stirring for 4 h at 0 ꢀC ether (40 mL) was
added and the mixture was washed with water (2Â40 mL)
and brine (40 mL) and concentrated. Chromatography
with ether/petroleum ether (1/4) to give compound 18
(0.57 g, 59%). 1H NMR d 7.77 (d, 2H), 7.32 (d, 2H), 4.11
(m, 2H), 3.58 (dt, 1H), 3.52 (m, 2H), 3.31 (d, 1H), 3.16 (d,
1H), 2.42 (s, 3H), 2.15 (m, 1H), 1.95-0.90 (m, 14H), 0.89
(d, 3H), 0.84 (s, 9H), 0.77 (d, 3H), 0.007 (s, 3H), 0.003 (s,
3H). 13C NMR d 144.5, 132.9, 129.6, 127.7, 71.1, 69.4,
69.0, 68.4, 58.7, 56.6, 48.3, 36.2, 34.5, 31.0, 27.7, 25.7,
24.2, 23.1, 22.7, 21.7, 21.4, 17.9, 4.4, 4.8.
Desilylation of compound 7c with hydrogen ¯uoride. A
mixture of compound 7c (0.380 g, 0.73 mmol) in EtOAc
(2.5 mL), acetonitrile (8.5 mL) and a 5% (w/v) solution
of hydro¯uoric acid in acetonitrile/water (7/1, v/v,
7.5 mL) was stirred for 40 min at rt EtOAc (50 mL) was
added and the mixture was washed with saturated aq.
sodium hydrogen carbonate (50 mL), water (50 mL),
brine (25 mL) and concentrated. Chromatography with
EtOAc as eluant gave the desired compound 8c. Yield:
0.189 g (79%). 1H NMR d 0.84 (d, 3H), 1.00 (d, 3H),
1.21 (s, 6H), 0.85±2.10 (m, 20H), 3.31 (m, 1H), 3.20 (d,
1H), 3.31 (m, 3H), 3.72 (m, 1H). 13C NMR d 71.2, 71.0,
70.8, 69.0, 59.0, 57.1, 48.7, 43.7, 35.9, 35.2, 31.2, 30.3,
29.2, 29.2, 28.3, 23.5, 23.4, 23.1, 22.1, 21.2.
8R-tert-Butyldimethylsilyloxy-20-methyl-18-[5-hydroxy-
5-methylhex-3-yn-1-yloxy]-de-A,B-pregnane (7i). n-Butyl
lithium (1.4 mL, 1.5 M in hexane) was added to a solu-
tion of 3-(tetrahydropyral-2-yloxy)-3-methylbut-1-yne
(0.37 g, 2.2 mmol) in dry dioxane (6 mL) at 5 ꢀC. After
stirring for 30 min at 5 ꢀC and 1 h at rt a solution of 18
(0.28 g, 0.53 mmol) in dry dioxane (3 mL) was added.
The mixture was stirred at 90 ꢀC for 48 h. The reaction
mixture was partitioned between saturated aq. sodium
hydrogencarbonate (25 mL) and ethyl acetate (25 mL).
The aq phase was extracted with ethyl acetate (25 mL)
The oxidation of compound 8c to compound 9c is
representative for the series of reactions leading from
8a±h to 9a±h
Oxidation of compound 8c with PDC
A solution of compound 8c (0.180 g, 0.55 mmol) and
pyridinium dichromate (0.621 g, 1.65 mmol) in dichloro-
methane (15 mL) was stirred at rt for 18 h. Dichloro-

2038
G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
methane (100 mL) was added and the mixture was
washed with saturated aq. sodium hydrogencarbonate
(100 mL), water (100 mL) and brine (50 mL) and con-
centrated. The residue was chromatographed with
EtOAc (compounds 9d±g use EtOAc/petroleum ether,
1/3) as eluant to give the desired ketone 9c. Yield:
0.142 g (80%). 1H NMR d 0.86 (d, 3H), 1.03 (d, 3H),
1.20 (s, 3H), 1.21 (s, 3H), 1.10±2.40 (m, 19H), 2.45 (m,
1H), 3.12 (d, 1H), 3.28 (m, 3H). 13C NMR d 211.5, 71.3,
70.8, 70.0, 60.4, 58.6, 52.7, 43.4, 40.5, 36.2, 30.9, 30.0,
29.3, 29.2, 27.8, 23.9, 23.2, 23.0, 20.8, 19.5.
45.4, 43.7, 42.9, 35.8, 31.5, 30.4, 29.2, 29.1, 29.0, 27.9,
23.5, 23.3, 23.3, 22.4, 21.2. MS (EI+) m/z 460.3 (M+).
1
12c: H NMR d 0.85 (m, 9H), 1.03 (d, 3H), 1.00±2.20
(m, 24H), 2.30 (dd, 1H), 2.38 (m, 1H), 2.61 (dd, 1H),
2.86 (m, 1H), 3.12 (s, 2H), 3.29 (m, 2H), 4.24 (m, 1H),
4.42 (m, 1H), 4.98 (m, 1H), 5.30 (m, 1H), 6.03 (d, 1H),
6.38 (d, 1H). 13C NMR d 147.6, 142.8, 133.3, 124.8,
117.3, 112.1, 74.4, 72.0, 71.2, 69.3, 66.8, 58.9, 55.7, 49.5,
45.5, 42.8, 36.1, 35.4, 31.4, 30.9, 30.9, 29.0, 28.1, 24.1,
23.6, 23.3, 23.3, 22.4, 7.9, 7.8. MS (EI+) m/z 474.3 (M+).
12d: 1H NMR d 0.85 (d, 3H), 1.07 (d, 3H), 1.57 (s, 6H),
0.80±2.15 (m, 16H), 2.30 (dd, 1H), 2.52 (m, 1H), 2.60
(dd, 1H), 2.85 (m, 1H), 3.18 (m, 2H), 4.23 (m, 1H), 4.40
(m, 3H), 4.94 (m, 1H), 5.28 (m, 1H), 5.98 (d, 1H), 6.36
(d, 1H), 7.16 (d, 1H), 7.29 (t, 1H), 7.38 (d, 1H), 7.42 (s,
1H). 13C NMR d 149.1, 147.5, 143.0, 139.0, 133.2, 128.1,
125.5, 124.9, 123.2, 123.2, 117.3, 112.1, 77.2, 73.6,
71.1,68.5, 66.8, 58.9, 55.7, 49.5, 45.4, 42.8, 35.7, 31.7,
31.6, 31.5, 29.1, 27.8, 23.6, 23.4, 23.3, 22.3. MS (EI+)
m/z 494.3 (M+).
The silylation of compound 9c to compound 10c is
representative for the series of reactions leading from
9a±c and 9h to 10a±c and 10h, respectively
Silylation of side chain hydroxy group in compound 9c.
A
solution of compound 9c (0.132 g, 0.41 mmol),
N-ethyldiisopropylamine (0.106 g, 0.82 mmol) and
trimethylsilyl chloride (0.089 g, 0.82 mmol) in dichloro-
methane (4 mL) was stirred at rt for 140 min. Dichloro-
methane (30 mL) was added and the mixture was diluted
with phosphate bu€er (30 mL, pH 6.5). The organic
phase was washed with brine (30 mL) and concentrated.
Chromatography with EtOAc/petroleum ether (1/4) as
eluant gave the desired TMS-ether 10c. Yield: 0.100 g
1
12e: H NMR d 0.74 (t, 3H), 0.75 (t, 3H), 0.84 (d, 3H),
1.06 (d, 3H), 1.00±2.15 (m, 20H), 2.31 (dd, 1H), 2.52 (m,
1H), 2.60 (dd, 1H), 2.84 (m, 1H), 3.17 (m, 2H), 4.24 (m,
1H), 4.40 (m, 2H), 4.42 (m, 1H), 4.95 (m, 1H), 5.30 (m,
1H), 5.99 (d, 1H), 6.36 (d, 1H), 7.10±7.35 (m, 4H). 13C
NMR d 147.6, 145.7, 143.0, 138.7, 133.2, 127.8, 127.3,
124.9, 124.4, 124.4, 117.3, 112.0, 77.2, 73.7, 71.0, 68.4,
66.8, 58.9, 55.7, 49.4, 45.4, 42.9, 35.7, 34.9, 34.9, 31.5,
29.0, 27.8, 23.5, 23.4, 23.3, 22.3, 7.8. MS (EI+) m/z
522.3 (M+).
1
(61%). H NMR d 0.08 (s, 9H), 0.84 (d, 3H), 1.02 (d,
3H), 1.18 (s, 6H), 1.10±2.40 (m, 18H), 2.47 (m, 1H),
3.06 (d, 1H), 3.24 (m, 3H). 13C NMR d 211.3, 73.8,
71.4, 69.2, 60.3, 58.4, 52.6, 44.5, 40.4, 35.6, 30.8, 29.9,
29.6, 29.6, 27.5, 23.7, 23.0, 22.9, 20.9, 19.2, 2.4.
Coupling of the phosphine oxide 19 with CD-ring ketone
9d±g, 9i, 10a±c and 10h. The method described in Ref.
13 was used. Yields for compounds 11a±i are given in
Table 1.
12f: 1H NMR d 6.40 (d, 1H), 6.05 (d, 1H), 5.31 (bs, 1H),
4.99 (bs, 1H), 4.43 (m, 1H), 4.21 (m, 1H), 4.11 (d, 1H),
3.92 (d, 1H), 3.32 (d, 1H), 3.06 (d, 1H), 2.87 (bd, 1H),
2.65 (bs, 1H), 2.60 (dd, 1H), 2.42 (m, 1H), 2.30 (dd, 1H),
2.15±1.05 (m,15H), 1.49 (s, 3H), 1.47 (s, 3H), 1.01 (d,
3H), 0.85 (d, 3H). 13C NMR d 147.3, 143.1, 133.5, 124.7,
117.1, 112.6, 91.1, 78.4, 71.7, 67.3, 66.6, 65.0, 58.7, 58.4,
55.8, 49.2, 45.5, 42.7, 35.4, 31.6, 31.4, 31.1, 29.1, 27.8,
23.5, 23.2, 22.1. MS (EI+) m/z 442.2 (M+).
Deprotection of compounds 11a-i with hydrogen ¯uoride.
The procedure for deprotection of compounds 7a±c was
used. Yields for compounds 12a±i are given in Table 1.
12a: 1H NMR d 6.38 (d, 1H), 6.04 (d, 1H), 5.30 (bs, 1H),
4.39 (m, 1H), 4.21 (m, 1H), 3.30 (m, 2H), 3.14 (s, 2H),
2.84 (bd, 1H), 2.55 (dd, 1H), 2.45±1.05 (m, 22H), 1.21 (s,
3H), 1.20 (s, 3H), 1.01 (s, 3H), 0.86 (s, 3H). 13C NMR d
147.3,142.4, 133.2, 124.4, 117.1, 111.9, 71.8, 71.0, 70.4,
69.2, 66.5, 58.6, 55.5, 49.2, 45.3, 42.6, 40.8, 35.9, 31.2,
29.1, 28.8, 28.8, 27.9, 24.8, 23.3, 23.1, 22.2. MS (EI+) m/
z 446.2 (M+).
1
12g: H NMR d 0.85 (d, 3H), 1.00 (t, 6H), 1.00 (d, 3H),
1.00±2.25 (m, 20H), 2.30 (dd, 1H), 2.43 (m, 1H), 2.61
(dd, 1H), 2.87 (m, 1H), 3.10 (d, 1H), 3.30 (d, 1H), 4.09
(m, 2H), 4.23 (m, 1H), 4.43 (m, 1H), 5.00 (m, 1H), 5.32
(m, 1H), 6.03 (d, 1H), 6.39 (d, 1H). 13C NMR d 147.4,
143.0, 133.4, 124.8, 117.2, 112.3, 88.8, 80.7, 72.1, 71.4,
67.4, 66.7, 58.8, 58.4, 55.7, 49.1, 45.5, 42.9, 35.3, 34.4,
34.1, 31.4, 29.0, 27.7, 23.4, 23.2, 23.2, 22.2, 8.6, 8.6. MS
(EI+) m/z 470.3 (M+).
12b: 1H NMR d 0.85 (d, 3H), 1.03 (d, 3H), 1.20 (s, 6H),
1.00±2.15 (m, 22H), 2.31 (dd, 1H), 2.44 (m, 1H), 2.61
(dd, 1H), 2.85 (m, 1H), 3.08 (s, 2H), 3.27 (t, 2H), 4.23
(m, 1H), 4.43 (m, 1H), 4.99 (m, 1H), 5.32 (m, 1H), 6.00
(d, 1H), 6.39 (d, 1H). 13C NMR d 147.6, 143.2, 133.1,
125.0, 117.3, 112.0, 71.1, 71.0, 68.7, 66.8, 58.9, 55.6, 49.4,
12h: 1H NMR d 6.38 (d, 1H), 6.02 (d, 1H), 5.31 (bs, 1H),
4.98 (bs, 1H), 4.41 (m, 1H), 4.23 (m, 1H), 4.06 (m, 2H),

G. Grue-Sùrensen, C. Mùrk Hansen/Bioorg. Med. Chem. 6 (1998) 2029±2039
2039
3.27 (d, 1H), 3.10 (d, 1H), 2.77 (bd, 1H), 2.60 (dd, 1H),
2.43 (bd, 1H), 2.37 (bt, 2H), 2.30 (dd, 1H), 2.15±1.05 (m,
16H), 1.29 (s, 6H), 1.01 (d, 3H), 0.84 (d, 3H). 13C NMR
d 147.4, 142.9, 133.2, 124.9, 117.3, 112.4, 82.8, 79.6,
71.4, 70.0, 67.6, 66.7, 58.8, 58.7, 55.7, 49.1, 45.5, 42.9,
35.3, 34.4, 31.5, 29.0, 28.6, 27.7, 23.4, 23.3, 23.2, 22.2.
MS (EI+) m/z 456.2 (M+).
calcium was calculated from days 3±7 (steady state
conditions) and expressed in mmol/day (mean‹SD).
Serum calcium was measured on day 7 and expressed in
mmol/L (mean‹SD). Statistical analysis was carried
out using Student's t-test. Results are shown in Table 3.
Calculations. In all in vitro experiments 1,25(OH)2 D3
served as reference. The results were calculated using
the actual value of 1,25(OH)2 D3 in each separate
experiment.
12i: 1H NMR d 6.38 (d, 1H), 6.01 (d, 1H), 5.32 (bs, 1H),
4.99 (bs, 1H), 4.43 (m, 1H), 4.23 (m, 1H), 3.37 (bt, 2H),
3.14 (ABq, 2H), 2.85 (bd, 1H), 2.59 (dd, 1H), 2.53 (m,
1H), 2.38 (t, 2H), 2.30 (dd, 1H), 2.05±1.00 (m, 16H),
1.47 (s, 6H), 1.02 (d,3H), 0.85 (d.3H). 13C NMR d 147.5,
143.0, 133.3, 124.8, 117.3, 112.3, 85.7, 79.9, 71.1, 69.7,
69.0, 66.8, 65.2, 58.8, 55.6, 49.4, 45.4, 42.8, 35.7, 31.6,
31.5, 29.0, 28.0, 23.5, 23.3, 23.3, 22.4, 20.1. MS (EI+)
m/z 456.2 (M+).
Acknowledgements
The authors wish to thank Ms. Helle Selvig, Ms. Hanne
Olsen, Mr. Lars Borup, Ms. Dorthe Hansen for skilful
technical assistance, Niels Rastrup Andersen and
Grethe Aagaard for recording and discussion of NMR
spectra, Lene Ho€meyer for mass spectral data and Ms.
Adriana Boye for help with the manuscript.
Vitamin D receptor binding anity.15 The binding a-
nity of the compounds for the vitamin D receptor was
assessed by displacement of bound 3H-labelled 1,25-
(OH)2 D3 from isolated receptors obtained from intest-
inal epithelium of rachitic chickens (Amersham Den-
mark Aps, Birkerd, DK). For each compound, three
separate experiments were performed. Results are
shown in Table 2.
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