Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) selectively eliminates bone calcium mobilization activity

Article abstract of DOI:10.1016/j.bmc.2009.09.047

The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D₃ (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled by the Wittig-Horner process with the correspo

Full text of DOI:10.1016/j.bmc.2009.09.047

Bioorganic & Medicinal Chemistry 17 (2009) 7658–7669
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1
dihydroxyvitamin D3 (2MD) selectively eliminates bone calcium
mobilization activity
a,25-
Rafal Barycki a, Rafal R. Sicinski a,b, Lori A. Plum a, Pawel Grzywacz a, Margaret Clagett-Dame a,
Hector F. DeLuca a,
*
a Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
b Department of Chemistry, University of Warsaw, ul. Pasteura 1, 02-093 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2009
Revised 22 September 2009
Accepted 24 September 2009
Available online 29 September 2009
The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1a,25-dihydroxy-2-methylene-19-norvi-
tamin D3 (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled
by the Wittig–Horner process with the corresponding C,D-fragments (13–15), obtained by a multi-step
procedure from commercial vitamin D2. The goal of our studies was to examine the influence of removal
of the methyl groups located at carbons 13 and 20 on the biological potency of 2MD in the hope of finding
analogs with improved therapeutic profiles.
Keywords:
Replacement of the 20-methyl with hydrogen in 2-methylene-19-nor-(20S)-1a,25-dihydroxyvitamin
Vitamin D analogs
19-Norvitamin D
Vitamin D receptor
Calcemic activity
D3 (2MD) did not affect binding to the rat vitamin D receptor and had little effect on transcription activity
and on HL-60 differentiation. However, the mobilization of calcium from bone was largely eliminated
while intestinal calcium transport remained strong. Curiously, removal of both the C-13-methyl and
20-methyl restored slightly the bone calcium mobilizing activity. Thus, the 21-nor analog of 2MD may
provide a potent analog with a greater margin of safety than 2MD.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
exerted a more pronounced ability to elevate serum calcium from
bone than 1a,25-(OH)2D3, and such calcemic in vivo activity was
1
a
,25-Dihydroxyvitamin D3 (1
a
,25-(OH)2D3, calcitriol, 1; Fig. 1)
significantly enhanced in its epimer 6 with an ‘unnatural’ configu-
ration at C-20. Further biological tests of this analog (2MD) showed
its unique ability to induce bone formation.16,17 2MD (6) has pro-
ven anabolic for bone in in vivo studies but has a tendency to cause
hypercalcemia because of its bone calcium mobilizing activity.15
Because of docking calculations, the idea that removal of the 20-
methyl might reduce bone calcium mobilization occurred to us.18
We, therefore, prepared the 21-nor derivative of 2MD (8). As a
control, we also removed the 13-methyl (7) or both the 20- and
13-methyl (9). The results are consistent with the idea that the
20-methyl of 2MD is central to its activity on bone calcium mobi-
lization. The synthetic strategy consisted of the Wittig–Horner
coupling of the known A-ring fragment, the phosphine oxide
10,14 with the corresponding bicyclic ketones 13–15 which, in turn,
were prepared from the hydrindanols 11 and 12 (Fig. 2).
is the active hormonal form of vitamin D3.1–3 It not only regulates
calcium and phosphorus homeostasis but promotes differentiation
ofvariousmalignantcells,4–7 andsuppresses autoimmunediseases.8
Such a broad range of diverse functions of the natural hormone 1 is
mediated by its binding to the intracellular vitamin D receptor
(VDR), belonging to the nuclear receptor superfamily.9 Therefore, a
strong affinity of the vitamin D compound to VDR is of considerable
importancetothefunctionsof vitaminD. Formationof acomplexbe-
tween VDR and the ligand changes the conformation of the protein10
and influences its subsequent hetero-dimerization with retinoid X
receptor (RXR) and recruitment of several coactivators allowing
the transactivation process to begin.10,11 Many analogs of calcitriol
have been prepared; most of them are described.12
In 1990, we reported the synthesis of the analog 3 of the natural
hormone in which the exomethylene group at C-19 was re-
moved.13 During the subsequent years, similar 19-norvitamins
were obtained in our and other laboratories.14 Another very prom-
ising structural change of the ring A consisted in a ‘shift’ of the exo-
methylene group from carbon 10 to carbon 2.15 Thus, the analog 5
2. Results
2.1. Synthesis
Since the hydroxy compound 11 was previously obtained in our
laboratory, by degradation of the commercial vitamin D2 16
* Corresponding author. Tel.: +1 608 262 1620; fax: +1 608 262 7122.
E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.047

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
7659
21
20
R
R
18
25
H
H
H
OH
OH
17
OH
13
14
9
8
H
H
H
7
6
19
5
4
3
10
1
2
HO
OH
HO
OH
HO
OH
1: R = CH3 (calcitriol)
2: R = H
3: R = CH3
4: R = H
5
R
H
R
H
OH
H
OH
H
HO
OH
HO
OH
6: R = CH3 (2MD)
7: R = H
8: R = CH3
9: R = H
Figure 1. Chemical structures of 1a,25-dihydroxyvitamin D3 (calcitriol, 1) and its analogs.
followed by reconstruction of the side chain,19 we focused our
efforts on the preparation of its 21-nor analog 12 (steroidal number-
ing). As a starting compound, we have chosen methylketone 17
(Scheme 1) prepared from vitamin D2 16 as described by Posner.20
For the removal of the acetyl substituent, we used the synthetic se-
quence elaborated by Mourino for the analogous compound.21 Thus,
the Bayer–Villiger oxidation of 17 provided the acetate 18 which
was hydrolyzed and the resulting alcohol 19 oxidized to the 17-ke-
tone 20. Its subsequent Wittig reaction with the ylide, generated
from the phosphonium salt 21,22 yielded a mixture of olefinic com-
pounds. As expected,23 the isomer 22 with Z-geometry of the intro-
duced double bond was a major product. Hydrogenation of the
resulted olefinic mixture furnished the protected alcohol 23 which,
following removal of the silyl group, gave the desired hydrindanol
12. This compound was a convenient precursor of both Grundmann
ketones 14 and 15. Thus, the 8b-alcohol 12 was subjected to the cat-
alytic oxidation with RuO4 which provided the expected hydroxy
ketone 24.24 Protection of its tertiary hydroxyl as TES ether was
the last step of the hydrindanone 14 synthesis.
For the conversion of 12 to the 18-nor ketone 15, the removal of
13b-methyl group was necessary. The synthetic sequence leading
to the 18-nor derivative was initially performed for the homolo-
gous compound 11 by applying the method we used satisfactorily
in the synthesis of the analog 2.25 Thus, hydrindanol 11 was treated
with tert-butyl nitrite and the formed ester 25 (Scheme 2) was irra-
diated with a UV lamp yielding, as a result of the Barton reaction,26
the E-oxime 27. The unstable primary photolysis product, 18-ni-
trite, was not isolated. Dehydration of 27 was achieved by its heat-
ing in acetic anhydride, resulting in acetylation and the subsequent
elimination of the residues of acetic acid. The formed acetoxy ni-
trile 29 was hydrolyzed and the cyano group in the hydroxy nitrile
31 was removed by reduction with metallic potassium.27 The
obtained hydrindanol 33 was subjected to oxidation with RuO4
and the resulted hydroxy ketone 35 was silylated to provide the
18-nor Grundmann ketone 13. By application of the analogous
reaction sequence, as indicated in Scheme 2, the 21-nor compound
12 was converted into 18,21-dinor Grundmann ketone 15, thus
accomplishing the synthesis of the ‘upper’ building blocks required
P(O)Ph2
R2
R1
R
H
H
OTES
H
H
TBSO
OTBS
OH
O
13: R1 = H, R2 = CH3
14: R1 = CH3, R2 = H
15: R1 = R2 = H
11: R = CH3
12: R = H
10
Figure 2. The building blocks for the synthesis of 7, 8 and 9.

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R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
O
OR
O
H
H
i
iii
Vitamin D2
16
H
OTES
H
OTES
H
OTES
18: R = Ac
19: R = H
ii
17
20
Br-
+
Ph3P
H
H
OR
21
v
vii
iv
H
H
O
OR
H
OTES
24: R = H
14: R = TES
23: R = TES
12: R = H
viii
vi
22
Scheme 1. Synthesis of the Grundmann ketone 14. Reagents: (i) m-CPBA, cyclohexane; (ii) MeONa, MeOH; (iii) PDC, PPTS, CH2Cl2; (iv) tert-BuOK, THF; (v) H2, Pd/C, AcOEt; (vi)
CSA, n-BuOH; (vii) RuCl3 ꢀ H2O, NaIO4, CCl4, MeCN, H2O; (viii) TESOTf, 2,6-lutidine, CH2Cl2.
for the Wittig–Horner coupling with the A-ring fragment. Reten-
tion of configuration of the stereogenic center at C-13 in the syn-
thesized hydrindanols and hydrindanones was confirmed by
circular dichroism (CD) data. Thus, circular dichroism curve of
the hydroxy ketone 36, displaying two negative Cotton effects
around 290 and 190 nm, showed close similarity to the CD curves
recorded for the Grundmann ketone with a ‘natural’ 20R-configu-
ration and its 18-nor analog.25
C,D-fragments 13–15 provided the protected vitamin D com-
pounds 37–39 (Scheme 3) which, after removal of the silyl groups
and purification by HPLC, afforded the target vitamin D analogs 7, 8
and 9, respectively.
2.2. Biological evaluation
The synthesized vitamin D analogs 7, 8 and 9 were tested for their
ability to bind the full-length recombinant rat vitamin D receptor
(rVDR). Inagreementwithourexpectations, thecompetitivebinding
The final reaction between the anion, generated from the
phosphine oxide 10 by phenyllithium, and the corresponding
OH
R2
R
R2
N
CN
H
H
H
ii
iii
H
H
H
OR1
OH
OR1
11: R1 = H, R2 = CH3
29: R1 = Ac, R2 = CH3
12: R1 = R2 = H
30: R1 = Ac, R2 = H
i
iv
25: R1 = NO, R2 = CH3
26: R1 = NO, R2 = H
27: R = CH3
28: R = H
31: R1 = H, R2 = CH3
32: R1 = R2 = H
i
iv
v
R
R
R
H
H
H
H
H
H
OTES
OH
vii
vi
H
H
H
O
O
OH
13: R = CH3
15: R = H
35: R = CH3
36: R = H
33: R = CH3
34: R = H
Scheme 2. Synthesis of the Grundmann ketones 13 and 15. Reagents: (i) tert-BuONO, CHCl3; (ii) h
RuCl3 ꢀ H2O, NaIO4, CCl4, MeCN, H2O; (vii) TESOTf, 2,6-lutidine, CH2Cl2.
m, PhH; IPA; (iii) Ac2O; (iv) MeONa, MeOH; (v) K, HMPA, tert-BuOH, Et2O; (vi)

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
7661
P(O)Ph2
R2
R1
R2
R1
H
H
OTES
OH
R2
R1
TBSO
OTBS
H
H
H
OTES
10
ii
i
H
O
TBSO
OTBS
HO
OH
13: R1 = H, R2 = CH3
14: R1 = CH3, R2 = H
15: R1 = R2 = H
37: R1 = H, R2 = CH3
38: R1 = CH3, R2 = H
39: R1 = R2 = H
7: R1 = H, R2 = CH3
8: R1 = CH3, R2 = H
9: R1 = R2 = H
Scheme 3. Synthesis of the vitamin D analogs 7, 8 and 9. Reagents: (i) PhLi, THF; (ii) TBAF, molecular sieves 4 Å, THF for 37?7; CSA, n-BuOH for 38?8, and 39?9.
analysis shows (Table 1) that all compounds bind rVDR very
effectively.
homeostasis and influence the ability of the analogs to mobilize
calcium from bone.
Studies on the cellular activity of the prepared compounds to
induce the differentiation of human promyelocyte HL-60 cells into
monocytes show that all vitamins are more potent (3–7 times)
than the vitamin D hormone (Table 1).
The results of the transcriptional assay indicate that the analog
7 exhibits a significantly enhanced ability to induce transcription
of vitamin D-responsive genes. When the 24-hydroxylase (CY-24)
luciferase reporter gene system was used, 18,19-dinorvitamin 7
An experimental confirmation of these predictions, based on the
docking simulations, was obtained soon afterwards when we
determined the X-ray crystallographic data of a ternary complex
of 2MD with the ligand binding domain (LBD) of rVDR and a
LXXLL-containing coactivator peptide.28 Inspection of this complex
reveals that the angular methyl group, located at C-13 in the 2MD
molecule, as well as its 20-methyl substituent, are surrounded in
the LBD by the most hydrophobic amino acids. Thus, strong hydro-
phobic contacts (distances between hydrogens shorter than 3.5 Å)
were found29 between the angular methyl at C-13 and two amino
acids (V230 and I267) whereas, for the methyl attached to C-20,
four residues (I264, M268, L305 and L309) were involved in such
interactions. That all these amino acids influence the transactiva-
tion activity of 2MD (6) was nicely demonstrated by the results
of alanine scanning mutational analysis (ASMA). Yamada and
Yamamoto established that Ala mutations of I267 and L305 (rVDR
numbering) in the ligand binding pocket of the human vitamin D
receptor (hVDR) nearly abolished the mutant transactivation po-
tency (less than 20% of the activity of the wild-type VDR) induced
by 2MD, mutation of V230 and I264 had also significant effect,
whereas the remaining residues M268 and L309 seemed to play
less important role (more than 90% of the wild-type receptor
activity).30
proved to be 15 times more active than 1
a
,25-(OH)2D3 (Table 1).
have
The 19,21-dinorvitamin and 18,19,21-trinorvitamin
8
9
slightly higher (ca. 1.5 and 5 times, respectively) transcriptional
potencies with respect to the native hormone 1.
For in vivo comparison, we have included Figure 3, a summary
of the activity of 2MD. While all compounds showed excellent
intestinal calcium transport activity, the change in bone calcium
mobilization, by removing the 20-methyl group, was dramatic.
The 21-nor (8) is essentially devoid of bone calcium mobilization
activity (Fig. 4). Removal of C-13 methyl (7) reduced bone calcium
mobilizing activity of 2MD (Fig. 5) to a level that is very similar to
that of the native hormone. Removal of both the 18- and 21-methyl
(9) resulted in a compound with activity on bone in between that
observed for the 21-nor compound (8) and the 18-nor analog (7)
(Fig. 6).
Thus, docking calculations suggest that removal of these methyl
constituents, especially C-21, from 2MD may result in a reduction
of bone calcium mobilizing activity. Clearly, removal of the 20-
methyl abolishes bone calcium mobilizing activity. Removal of
the 13-methyl reduces but does not eliminate bone calcium mobi-
lizing activity. When both are removed, some restoration of bone
calcium mobilizing activity occurs.
3. Discussion
A few years ago, we reported results of our docking experiments
performed with 2-substituted vitamin D analogs and aimed at
establishing how the binding pocket of the full-length ligand bind-
ing domain (LBD) of rVDR could accommodate these ligands.18 It
was found that, in complexes of ligands possessing elevated BCM
activity (such as the 2-methylene compounds 5 and 6), short
hydrophobic contacts existed between vitamin D methyl group
at C-20 and the conserved amino acids L305 and L309. Moreover,
the detailed analysis of the VDR binding sites, capable of creating
contacts with ligand methyl groups attached to C-13 and C-20,
showed the existence of two hydrophobic patches on the receptor
surface which could be responsible for specific methyl–methyl
interactions. We concluded that the hydrophobic interactions of
the VDR with the ligand methyl substituents might modulate its
interaction with coactivators and corepressors involved in calcium
4. Conclusions
Removal of the 20-methyl from 2MD essentially eliminates its
bone calcium mobilizing activity specifically while not affecting
all other measured activities including intestinal calcium trans-
port. Removal of 13-methyl also reduces somewhat bone calcium
mobilizing activity while removal of both methyls is not additive
in lowering bone calcium activity. These results suggest that the
20-methyl is an essential group for 2MD’s strong activity on
bone.

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R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
Table 1
VDR binding properties,a HL-60 differentiating activities,b and transcriptional activitiesc of the vitamin D compounds 7, 8 and 9
Compound
Compd no.
VDR binding
Ki (M)
HL-60 differentiation
EC50 (M) Ratio
24-OHase transcription
EC50 (M) Ratio
Ratio
H
H
H
OH
1 ꢀ 10ꢁ10
2 ꢀ 10ꢁ11
2 ꢀ 10ꢁ10
1 ꢀ 10ꢁ10
1
2 ꢀ 10ꢁ9
4 ꢀ 10ꢁ10
7 ꢀ 10ꢁ10
3 ꢀ 10ꢁ10
1
3 ꢀ 10ꢁ10
2 ꢀ 10ꢁ11
2 ꢀ 10ꢁ10
6 ꢀ 10ꢁ11
1
H
1
HO
OH
H
OH
H
7
8
9
0.2
0.2
0.35
0.15
0.07
0.67
0.2
HO
OH
OH
H
2
HO
OH
H
H
OH
H
1
HO
OH
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 EC50 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 EC50 to the EC50 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 24-hydroxylase gene reporter plasmid. The EC50 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 EC50 for the analog to the
EC50 for 1a,25-(OH)2D3. All the experiments were carried out in duplicate on at least two different occasions.
solutions. 1H nuclear magnetic resonance (NMR) spectra were re-
corded at 400 and 500 MHz with Bruker Instruments DMX-400
and DMX-500 Avance console spectrometers in deuteriochloro-
form. 13C nuclear magnetic resonance (NMR) spectra were
recorded at 100 and 125 MHz with the same Bruker Instruments
in deuteriochloroform. Chemical shifts (d) are reported downfield
from internal Me4Si (d 0.00). Electron impact (EI) mass spectra
were obtained with a Micromass AutoSpec (Beverly, MA) instru-
ment. High-performance liquid chromatography (HPLC) was
performed on a Waters Associates liquid chromatograph equipped
with a Waters 600 Controller, a Rheodyne 7725i injector and
5. Experimental
5.1. Chemistry
Melting points (uncorrected) were determined on a Thomas–
Hoover capillary melting-point apparatus. Infrared (IR) spectra
were recorded with a Perkin–Elmer FT-IR Spectrum 2000. Ultravi-
olet (UV) absorption spectra were recorded with a Perkin–Elmer
Lambda 3B UV–vis spectrophotometer in ethanol. Circular dichro-
ism (CD) spectra were recorded between 185 and 300 nm at room
temperature with a JASCO J-715 spectropolarimeter in hexane

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
7663
Figure 3. Bone calcium mobilization and intestinal calcium transport activity of 2MD (6).
Figure 4. Bone calcium mobilization and intestinal calcium transport activity of calcitriol (1) and the synthesized analog 8. Letters above the bars indicate statistically
significant differences between treatment groups (p <0.05).
Waters 2487 Dual k Absorbance Detector. THF was freshly distilled
before use from sodium benzophenone ketyl under argon.
The starting compounds 11 and 17 were obtained from com-
mercial vitamin D2 (16) according to the published procedures.19,20
was added at 0 °C. Then the reaction mixture was allowed to warm
up to room temperature and left stirred for five days. Next portions
of meta-chloroperbenzoic acid (1.0 g, 0.8 g, and 0.6 g) were added
after one day, two days, and four days, respectively. The suspen-
sion was filtered off and the filtrate was washed with a saturated
aqueous solution of NaHCO3 (20 mL). The organic phase was dried
over anhydrous MgSO4, concentrated under reduced pressure and
the residue was purified by column chromatography on silica
(1?3% ethyl acetate/hexane) to give the acetate 18 (0.89 g, 58%);
5.2. des-A,B-8b-[(Triethylsilyl)oxy]-testosterone acetate (18)
To a stirred solution of the methylketone 17 (1.13 g; 3.65 mmol)
in cyclohexane (50 mL), meta-chloroperbenzoic acid (77%, 1.50 g)

7664
R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
Figure 5. Bone calcium mobilization and intestinal calcium transport activity of calcitriol (1) and the synthesized analog 7.
½
a 2D4
ꢂ
+18.7 (c 0.90, CHCl3); 1H NMR (400 MHz, CDCl3) d 0.56 (6H, q,
dichloride (90 mL), pyridinium dichromate (2.25 g, 5.98 mmol)
J = 7.9 Hz), 0.95 (9H, t, J = 7.9 Hz), 1.11 (3H, s), 2.03 (3H, s), 4.05
(1H, narrow m); 13C NMR (100 MHz, CDCl3) d 4.9, 6.9, 13.6, 17.2,
21.2, 22.2, 26.7, 34.5, 37.8, 42.0, 47.8, 69.0, 82.9, 171.3; MS (EI)
m/z (relative intensity) 326 (M+, 3), 297 (18), 283 (8), 145 (70),
135 (100); exact mass calculated for C18H34O3Si 326.2277, mea-
sured 326.2269.
was added at 0 °C. The cooling bath was removed and the reaction
mixture was stirred for 9 h. Then the solvent was removed under
reduced pressure and the residue was purified by column chroma-
tography on silica (5?10% ethyl acetate/hexane) to give the ketone
20 (642 mg, 85%); ½a D24
ꢂ
+82.9 (c 0.90, CHCl3); 1H NMR (500 MHz,
CDCl3) d 0.60 (6H, q, J = 7.9 Hz), 0.97 (9H, t, J = 7.9 Hz), 1.11 (3H,
s), 2.43–2.47 (1H, m), 4.19 (1H, narrow m); 13C NMR (125 MHz,
CDCl3) d 4.9, 6.9, 16.3, 17.0, 21.3, 32.3, 34.5, 35.3, 47.5, 48.8, 69.9,
221.2; MS (EI) m/z (relative intensity) 282 (M+, 10), 252 (100),
133 (17), 103 (39); exact mass calculated for C16H30O2Si
282.2015, measured 282.2013.
5.3. des-A,B-8b-[(Triethylsilyl)oxy]-testosterone (19)
The acetate 18 (972 mg, 2.98 mmol) was dissolved in methanol
(25 mL) and treated with 10% solution of sodium methoxide in
methanol (5 mL) for 2.5 h. A saturated aqueous solutions of NH4Cl
(10 mL) and water (15 mL) were added and the product was ex-
tracted with methylene chloride (5 ꢀ 75 mL). The organic phase
was dried over anhydrous MgSO4, concentrated under reduced
pressure and the residue was purified by column chromatography
on silica (5?15% ethyl acetate/hexane) to give the semi-crystalline
5.5. des-A,B-8b-[(Triethylsilyl)oxy]-21-norcholest-17-ene (22,
mixture of Z and E isomers)
To a stirred suspension of 5-methylhexyltriphenylphosphoni-
um bromide23 (21; 4.10 g, 9.35 mmol) in anhydrous THF
(13.5 mL), a solution of potassium tert-butoxide (1 M in THF,
8.9 mL, 8.9 mmol) was added dropwise at ꢁ10 °C. The suspension
was stirred and allowed to warm up to 0 °C over 30 min. Then a
solution of the ketone 20 (716 mg, 2.53 mmol) in anhydrous THF
(3.0 mL) was added via cannula and the resulting mixture was stir-
red at 45 °C for four days. The saturated aqueous solution of NH4Cl
(20 mL) and water (30 mL) were added and the mixture was ex-
tracted with diethyl ether (3 ꢀ 100 mL). The organic phase was
dried over anhydrous MgSO4, concentrated under reduced pres-
sure, and the residue was purified by column chromatography on
silica (0?5% ethyl acetate/hexane) to give a mixture of isomeric
olefins 22 (572 mg, 63%; Z/E ratio of 5:1); 1H NMR (400 MHz,
alcohol 19 (764 mg, 90%); ½a D24
ꢂ
+39.6 (c 0.95, CHCl3); mp 93–95 °C;
1H NMR (400 MHz, CDCl3) d 0.56 (6H, q, J = 7.9 Hz), 0.95 (9H, t,
J = 7.9 Hz), 0.96 (3H, s), 3.56 (1H, m), 4.02 (1H, narrow m); 13C
NMR (100 MHz, CDCl3) d 5.0, 6.9, 12.4, 17.3, 22.2, 29.9, 34.6, 37.5,
42.2, 48.1, 69.1, 82.2; MS (EI) m/z (relative intensity) 284 (M+, 9),
255 (100), 237 (40), 135 (42), 103 (66); exact mass calculated for
C14H27O2Si (M+ꢁC2H5) 255.1780, measured 255.1770.
5.4. des-A,B-8b-[(Triethylsilyl)oxy]-androstane-17-one (20)
To a stirred solution of the alcohol 19 (760 mg, 2.68 mmol) and
pyridinium p-toluenesulfonate (30 mg, 0.12 mmol) in methylene

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
7665
Figure 6. Bone calcium mobilization and intestinal calcium transport activity of calcitriol (1) and the synthesized analog 9. Letters above the bars indicate statistically
significant differences between treatment groups (p <0.05).
CDCl3) d 0.56 (6H, q, J = 7.9 Hz), 0.86 (6H, d, J = 6.7 Hz), 0.95 (9H, t,
J = 7.9 Hz), 1.10 (3H, s), 4.11 (1H, narrow m), 4.94 (0.83H, t,
J = 7.3 Hz), 5.36 (0.17H, t, J = 4.7 Hz); 13C NMR (100 MHz, CDCl3) d
5.0, 7.0, 18.0, 19.9, 22.6, 22.7, 23.8, 27.5, 27.6, 27.7, 28.0, 28.7,
30.6, 34.6, 38.3, 38.7, 38.9, 44.2, 52.8, 69.7, 119.3, 130.0, 149.9;
MS (EI) m/z (relative intensity) 364 (M+, 6), 335, (23), 321 (10),
279 (37), 232 (54), 205 (43), 171 (51), 147 (100); exact mass calcu-
lated for C23H44OSi 364.3161, measured 364.3175.
(330 mg, 1.42 mmol). After one day, the solvent was removed un-
der reduced pressure, and the residue was purified by column
chromatography on silica (5?15% ethyl acetate/hexane) to give
the 8b-alcohol 12 (328 mg, 98%); ½a D24
ꢂ
+34.1 (c 1.25, CHCl3); 1H
NMR (400 MHz, CDCl3) d 0.84 (3H, s), 0.86 (6H, d, J = 6.6 Hz), 4.10
(1H, narrow m); 13C NMR (100 MHz, CDCl3) d 14.0, 17.4, 2 ꢀ 22.7,
22.9, 27.6, 2 ꢀ 28.0, 29.0, 29.9, 33.9, 38.5, 39.1, 41.3, 51.5, 52.3,
69.3; MS (EI) m/z (relative intensity) 252 (M+, 43), 237 (44), 219
(22), 209 (19), 125 (49), 111 (100); exact mass calculated for
C17H32O 252.2453, measured 252.2446.
5.6. des-A,B-8b-[(Triethylsilyl)oxy]-21-norcholestane (23)
To a solution of the olefins 22 (552 mg, 1.52 mmol) in ethyl ace-
tate (20 mL), 5% palladium on activated carbon (160 mg) was
added and the mixture was hydrogenated overnight. The catalyst
was filtered off and the filtrate was concentrated under reduced
pressure. The residue was purified on a Waters silica Sep-Pak car-
tridge (washed with hexane) to give the saturated silyl ether 23
5.8. des-A,B-25-Hydroxy-21-norcholestane-8-one (24)
A solution of hydrindanol 12 (74 mg, 0.29 mmol) in acetonitrile/
carbon tetrachloride (1:1; 1.5 mL) was added to a vigorously stir-
red solution of RuCl3 ꢀ H2O (10 mg, 0.05 mmol) and NaIO4
(227 mg, 1.06 mmol) in water (1 mL). After 2 h, the next portion
of RuCl3 ꢀ H2O (8 mg, 0.04 mmol) was added and the reaction mix-
ture was stirred for three days. Then a few drops of 2-propanol
were added, followed by water (5 mL) and the mixture was ex-
tracted with diethyl ether (3 ꢀ 15 mL). The organic phase was
dried over anhydrous MgSO4, concentrated under reduced pres-
sure and the residue was purified on a Waters silica Sep-Pak car-
tridge (3?25% ethyl acetate/hexane) to give the hydroxy ketone
(515 mg, 93%); ½a D24
ꢂ
+41.8 (c 1.15, CHCl3); 1H NMR (400 MHz,
CDCl3) d 0.55 (6H, q, J = 7.9 Hz), 0.81 (3H, s), 0.86 (6H, d,
J = 6.6 Hz), 0.95 (9H, t, J = 7.9 Hz), 4.05 (1H, narrow m); 13C NMR
(100 MHz, CDCl3) d 5.0, 7.0, 14.1, 17.6, 2 x 22.7, 23.4, 27.8, 28.0,
29.1, 30.0, 35.0, 38.9, 39.1, 41.5, 51.7, 52.8, 69.2; MS (EI) m/z (rel-
ative intensity) 337 (100), 323 (59), 271 (67), 233 (77); exact mass
calculated for C21H41OSi (M+ꢁC2H5) 337.2927, measured 337.2911.
24 (24 mg, 31%); ½a D24
ꢂ
ꢁ14.0 (c 1.25, CHCl3); 1H NMR (400 MHz,
5.7. des-A,B-21-Norcholestane-8b-ol (12)
CDCl3) d 0.55 (3H, s), 1.21 (6H, s), 2.42 (1H, dd, J = 11.2, 7.7 Hz);
13C NMR (100 MHz, CDCl3) d 12.9, 19.6, 23.9, 24.8, 27.8, 29.2,
2 ꢀ 29.3, 29.9, 30.2, 37.1, 41.0, 44.0, 49.7, 51.5, 61.4, 71.0, 211.9;
MS (EI) m/z (relative intensity) 266 (M+, 18), 248 (87), 233 (84),
To a stirred solution of the silyl ether 23 (485 mg, 1.33 mmol) in
n-butanol (25 mL) was added (+)-10-camphorsulfonic acid

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R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
151 (97), 111 (94), 97 (95), 81 (100); exact mass calculated for
11. Photolysis of the crude nitrite with the mercury arc lamp fol-
lowed by rearrangement of the intermediate nitroso compound
was carried out as described above for 25. After evaporation of
the solvents, the residue was purified on a Waters silica Sep-Pak
cartridge (15?25% ethyl acetate/hexane) to give the oxime 28
C17H28O (M+ꢁH2O) 248.2140, measured 248.2131.
5.9. des-A,B-25-[(Triethylsilyl)oxy]-21-norcholestane-8-one
(14)
(40% yield with respect to 12); ½a D24
ꢂ
+35.5 (c 0.90, CHCl3); 1H
To a stirred solution of the hydroxy ketone 24 (21 mg, 79
and 2,6-lutidine (20 L, 18 mg, 152 mol) in anhydrous methylene
chloride (500 L), dropwise triethylsilyl trifluoromethanesulfonate
(32 L, 37 mg, 132
mol) was added at ꢁ50 °C. After 20 min, a few
l
mol)
NMR (400 MHz, CDCl3) d 0.86 (6H, d, J = 6.6 Hz), 4.06 (1H, narrow
m), 6.81 (1H, br s), 7.23 (1H, s), 10.91 (1H, s); 13C NMR
(100 MHz, CDCl3) d 17.3, 22.0, 2 ꢀ 22.6, 27.7, 27.9, 28.1, 29.1,
34.4, 34.6, 38.9, 49.1, 51.8, 52.0, 67.4, 152.4; MS (EI) m/z (relative
intensity) 281 (M+, 11), 264 (72), 246 (57), 205 (49), 183 (100); ex-
act mass calculated for C17H30NO (M+ꢁOH) 264.2327, measured
264.2326.
l
l
l
l
l
drops of wet methylene chloride were added, followed by water
(5 mL) and the mixture was extracted with methylene chloride
(3 ꢀ 15 mL). The combined organic phases were washed with a
saturated aqueous solution of CuSO4 (5 mL), dried over anhydrous
MgSO4 and concentrated under reduced pressure. The residue was
purified on a Waters silica Sep-Pak cartridge (0?5% ethyl acetate/
hexane) to give the protected hydroxy ketone 14 (20 mg, 67%);
5.12. (20S)-8b-(Acetoxy)-des-A,B-cholestan-18-nitrile (29)
A solution of 27 (100 mg, 0.34 mmol) in acetic anhydride (5 mL)
was refluxed for 1.5 h. The reaction mixture was cooled, poured
carefully into ice water (ca. 25 mL) and extracted with benzene
(3 ꢀ 40 mL). The combined organic phases were washed with a
saturated aqueous solution of NaHCO3 (2 ꢀ 40 mL), water
(30 mL), dried over anhydrous Na2SO4 and evaporated. The residue
was purified on a Waters silica Sep-Pak cartridge (5% ethyl acetate/
½
a 2D4
ꢂ
ꢁ7.3 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d 0.55 (3H, s),
0.56 (6H, q, J = 7.9 Hz), 0.95 (9H, t, J = 7.9 Hz), 1.19 (6H, s), 2.42
(1H, dd, J = 11.1, 7.7 Hz); 13C NMR (100 MHz, CDCl3) d 6.8, 7.1,
24.0, 24.8, 27.7, 29.2, 29.9, 30.2, 37.1, 41.1, 45.1, 49.7, 51.5, 61.4,
73.4, 212.1; MS (EI) m/z (relative intensity) 365 (32), 351 (54),
322 (45), 231 (66), 173 (92), 135 (86), 103 (100).
hexane) to give the nitrile 29 (91 mg, 84%); ½a D24
ꢂ
ꢁ26.4 (c 0.75,
5.10. (18E)-(20S)-18-(Hydroxyimino)-des-A,B-cholestan-8b-ol
CHCl3); IR (CHCl3) mmax 2228, 1741, 1241 cmꢁ1 1H NMR
;
(27)
(500 MHz, CDCl3) d 0.87 (6H, d, J = 6.6 Hz), 0.91 (3H, d, J = 6.6 Hz),
2.15 (3H, s), 2.46 (1H, br d, J = 3.2 Hz), 5.20 (1H, narrow m); 13C
NMR (125 MHz, CDCl3) d 17.9, 18.8, 22.6, 22.7, 23.3, 23.8, 27.1,
28.0, 29.9, 35.6, 36.2, 36.3, 39.1, 45.6, 51.9, 54.1, 68.7, 121.2,
171.0; MS (EI) m/z (relative intensity) 319 (M+, 18), 304 (10), 290
(3), 277 (84), 259 (100), 244 (54), 234 (27), 216 (40), 202 (33),
188 (60), 174 (47), 147 (39), 134 (34), 121 (95); exact mass (ESI)
calculated for C20H33NO2Na (M++Na) 342.2409, measured
342.2413.
A solution of the hydrindanol 11 (185 mg, 0.69 mmol) in chloro-
form (5 mL) was treated with tert-butyl nitrite (1 mL) for 1 h in
darkness. Then anhydrous benzene (10 mL) was added and the sol-
vents were removed under reduced pressure. The crude oily (20S)-
des-A,B-cholestan-8b-yl nitrite (25) was sufficiently pure to be
used in the next synthetic step without purification.
Compound 25: 1H NMR (500 MHz, CDCl3) d 0.76 (3H, s), 0.81
(3H, d, J = 6.5 Hz), 0.87 (6H, d, J = 6.6 Hz), 5.78 (1H, narrow m);
13C NMR (125 MHz, CDCl3) d 13.1, 17.9, 18.5, 22.2, 22.6, 22.7,
23.9, 27.1, 28.0, 31.5, 34.9, 35.3, 39.3, 39.7, 41.9, 51.9, 56.0.
A solution of the crude nitrite 25 in anhydrous benzene
(150 mL) was irradiated in an apparatus consisting of a Pyrex ves-
sel with a water-cooled immersion well and a Hanovia high-pres-
sure mercury arc lamp equipped with a Pyrex filter. A slow stream
of argon was passed through the solution and the temperature was
maintained at about 10 °C. Monitoring of the reaction progress by
TLC indicated that the reaction was completed within 30 min. Ben-
zene was removed under reduced pressure, the residue was dis-
solved in 2-propanol (5 mL) and refluxed for 2 h. Then the
mixture was allowed to cool and it was left overnight to accom-
plish the isomerization of the initially formed nitroso compound
to the desired oxime form. The solvent was evaporated and the res-
idue was purified on a Waters silica Sep-Pak cartridge (25% ethyl
acetate/hexane) to give the oxime 27 (102 mg, 51% yield with re-
5.13. 8b-(Acetoxy)-des-A,B-21-norcholestane-18-nitrile (30)
Transformation of the oxime 28 to the corresponding nitrile by
heating in acetic anhydride was accomplished as described above
for 27. The product was purified on a Waters silica Sep-Pak car-
tridge (3?5% ethyl acetate/hexane) to give the nitrile 30 in 92%
yield; ½a 2D4
ꢂ
+5.2 (c 1.30, CHCl3); 1H NMR (400 MHz, CDCl3) d 0.86
(6H, d, J = 6.6 Hz), 2.13 (3H, s), 2.25–2.28 (1H, m), 5.21 (1H, narrow
m); 13C NMR (100 MHz, CDCl3) d 18.5, 20.9, 22.6, 23.3, 27.2, 27.6,
27.9, 28.2, 30.1, 30.8, 34.0, 38.9, 46.5, 49.1, 51.2, 68.4, 121.4,
170.9; MS (EI) m/z (relative intensity) 305 (M+, 7), 288 (3), 263
(83), 245 (44), 220 (79), 205 (100); exact mass calculated for
C19H31NO2Na 328.2252, measured 328.2249.
5.14. (20S)-des-A,B-Cholestan-18-nitrile-8b-ol (31)
spect to 11); ½a 2D4
ꢂ
+8.2 (c 0.80, CHCl3); 1H NMR (400 MHz, CDCl3)
A solution of the acetoxy nitrile 29 (90 mg, 0.28 mmol) in meth-
anol (3 mL) was treated with 5% solution of sodium methoxide in
methanol (3 mL) for 2 h. A saturated aqueous solution of NH4Cl
(5 mL) was added, followed by water (10 mL), and the mixture
was extracted with methylene chloride (5 ꢀ 40 mL), dried over
anhydrous Na2SO4 and evaporated. The residue was purified on a
Waters silica Sep-Pak cartridge (20% ethyl acetate/hexane) to give
d 0.84 (3H, d, J = 6.3 Hz), 0.87 (6H, d, J = 6.6 Hz), 2.20 (1H, br d,
J = 13.1 Hz), 4.04 (1H, narrow m), 7.33 (1H, s), 10.8 (1H, br s); 13C
NMR (100 MHz, CDCl3) d 17.5, 18.6, 21.8, 22.6, 22.7, 24.1, 27.2,
28.0, 34.3, 35.0, 35.6, 39.3, 49.5, 52.6, 56.7, 67.6, 152.2; MS (EI)
m/z (relative intensity) 295 (M+, 2), 278 (28), 260 (20), 245 (8),
206 (19), 183 (38), 165 (13), 148 (15), 121 (100); exact mass calcu-
lated for C18H33NO2Na (M++Na) 318.2409, measured 318.2412.
the hydroxy nitrile 31 (73 mg, 94%); ½a D24
ꢁ6.1 (c 0.75, CHCl3); IR
ꢂ
(CHCl3) 3486, 2228; 1H NMR (500 MHz, CDCl3) d 0.87 (6H, d,
J = 6.6 Hz), 0.92 (3H, d, J = 6.7 Hz), 2.43 (1H, br d, J = 3.1 Hz), 4.10
(1H, narrow m); 13C NMR (125 MHz, CDCl3) d 17.9, 22.6, 22.7,
22.9, 23.9, 27.1, 28.0, 32.8, 35.7, 36.2, 36.3, 44.7, 53.4, 54.2,
122.5; MS (EI) m/z (relative intensity) 277 (M+, 28), 262 (34), 259
(18), 248 (16), 244 (24), 220 (30), 216 (18), 206 (100); exact mass
calculated for C18H31NO 277.2496, measured 277.2395.
5.11. (18E)-18-(Hydroxyimino)-des-A,B-21-norcholestane-8b-ol
(28)
The des-A,B-21-norcholestane-8b-yl nitrite (26) was obtained
from the alcohol 12 by the trans-esterification with tert-butyl
nitrite, performed analogously to the process described above for

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
7667
5.15. des-A,B-21-Norcholestane-18-nitrile-8b-ol (32)
(100 MHz, CDCl3) d 14.3, 21.3, 22.2, 22.6, 27.8, 29.3, 29.7, 33.0,
36.5, 41.6, 44.1, 49.6, 51.0, 58.0, 71.0, 212.0; MS (EI) m/z (relative
intensity) 266 (M+, <1), 248 (57), 233 (19), 215 (4), 208 (15), 163
(29), 137 (100); exact mass (ESI) calculated for C17H30O2Na
(M++Na) 289.2144, measured 289.2136.
Hydrolysis of the acetoxy nitrile 30 with sodium methoxide in
methanol was performed analogously as it was described above
for 29. The crude product was purified on a Waters silica gel
Sep-Pack cartridge (15?25% ethyl acetate/hexane) to give the hy-
droxy nitrile 32 in 89% yield; ½a D24
ꢂ
+23.0 (c 1.10, CHCl3); 1H NMR
d 0.86 (6H, d, J = 6.6 Hz), 2.24 (1H, d,
5.19. des-A,B-25-Hydroxy-18,21-dinorcholestane-8-one (36)
(400 MHz, CDCl3)
J = 13.0 Hz), 4.12 (1H, narrow m); 13C NMR (100 MHz, CDCl3) d
17.8, 22.6, 22.9, 27.3, 27.6, 27.9, 28.3, 30.7, 33.0, 34.2, 38.9, 45.7,
49.0, 52.6, 67.2, 122.5; MS (EI) m/z (relative intensity) 263 (M+,
64), 246 (39), 236 (77), 220 (92), 206 (84), 193 (95), 134 (100); ex-
act mass calculated for C17H29NO 263.2249, measured 263.2256.
The ruthenium tetroxide oxidation of the alcohol 34 was per-
formed analogously to the process described above for 33. The
products were separated on a Waters silica Sep-Pak cartridge
(5?30% ethyl acetate/hexane) to give the hydroxy ketone 36 in
31% yield; ½a 2D4
ꢂ
+9.0 (c 0.95, CHCl3); CD
D
e
(kmax) ꢁ1.70 (312),
ꢁ2.86 (300), ꢁ2.93 (293), ꢁ3.10 (191); 1H NMR (400 MHz, CDCl3)
d 1.21 (6H, s), 13C NMR (100 MHz, CDCl3) d 21.1, 24.9, 27.7, 28.7,
29.1, 29.2, 29.6, 34.1, 41.5, 43.9, 45.7, 54.8, 56.8, 58.1, 211.79; MS
(EI) m/z (relative intensity) 252 (M+, 47), 234 (68), 219 (63), 194
(75), 67 (100); exact mass calculated for C16H28O2 252.2089, mea-
sured 252.2080.
5.16. (20S)-des-A,B-18-Norcholestan-8b-ol (33)
To a stirred mixture of potassium (110 mg, 2.82 mmol) in hex-
amethylphosphoramide (280
drous diethyl ether (700 L), a solution of the hydroxy nitrile 31
(70 mg, 0.25 mmol) in anhydrous tert-butyl alcohol (65 L) and
anhydrous diethyl ether (250 L) was added dropwise at 0 °C un-
lL, 288 mg, 1.62 mmol) and anhy-
l
l
l
5.20. (20S)-25-[(Triethylsilyl)oxy]-des-A,B-18-norcholestane-8-
der argon. The mixture was allowed to warm to the room temper-
ature and then stirred for 5 h. The remaining potassium was
removed, and a few drops of 2-propanol were added followed by
benzene (20 mL). The organic phase was washed with water
(10 mL), dried over anhydrous Na2SO4 and concentrated under re-
duced pressure. The residue was purified on a Waters silica Sep-
Pak cartridge (10% ethyl acetate/hexane) to give the alcohol 33
one (13)
To a stirred solution of 35 (12 mg, 45
(13 L, 12 mg; 111 mol) in anhydrous methylene chloride
(250 L), triethylsilyl trifluoromethanesulfonate was added drop-
lmol) and 2,6-lutidine
l
l
l
wise at ꢁ50 °C under argon. After 20 min, a few drops of wet meth-
ylene chloride were added, followed by water (7 mL). The reaction
mixture was extracted with methylene chloride (3 ꢀ 7 mL), the
combined organic phases were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified
on a Waters silica Sep-Pak cartridge (3% ethyl acetate/hexane). Fi-
nal purification was performed by HPLC (5% ethyl acetate/hexane,
4 mL/min, 10 mm ꢀ 25 cm Zorbax-Sil column; Rt = 5.42 min) to
give the TES-protected hydroxy ketone 13 (13 mg, 76%); H NMR
(500 MHz, CDCl3) d 0.56 (6H, q, J = 7.9 Hz), 0.77 (3H, d, J = 6.8 Hz),
0.94 (9H, t, J = 7.9 Hz), 1.19 (6H, s), 13C NMR (125 MHz, CDCl3) d
6.8, 7.1, 14.3, 21.4, 22.2, 22.7, 27.8, 29.7, 29.8, 29.9, 32.9, 36.4,
41.6, 45.2, 49.6, 51.1, 58.0, 73.4, 212.1; MS (EI) m/z (relative inten-
sity) 365 (M+ꢁMe, 8), 351 (100), 322 (6), 239 (2), 231 (25), 220 (4),
205 (15), 189 (4), 173 (92); exact mass (ESI) calculated for
C23H44O2SiNa (M++Na) 403.3008, measured 403.2995.
(54 mg, 85%); ½a 2D4
ꢂ
+32.6 (c 0.90, CHCl3); 1H NMR (500 MHz, CDCl3)
d 0.78 (3H, d, J = 6.8 Hz), 0.87 (6H, d, J = 6.6 Hz), 4.06 (1H, narrow
m); 13C NMR (125 MHz, CDCl3) d 14.7, 20.2, 22.7, 22.9, 24.7, 25.3,
28.0, 30.8, 33.1, 33.5, 36.3, 39.3, 39.7, 48.6, 50.3, 67.9; MS (EI) m/
z (relative intensity) 252 (M+, 6), 234 (21), 219 (23), 209 (26),
191 (8), 179 (4), 167 (13), 149 (89), 139 (47), 122 (90), 107 (35),
95 (80), 79 (87), 67 (88), 58 (100); exact mass calculated for
C17H32O 252.2453, measured 252.2448.
5.17. des-A,B-18,21-Dinorcholestane-8b-ol (34)
Decyanation of the hydroxy nitrile 32 to the hydrindanol 34 was
performed analogously to the process described above for 31. The
crude product was purified on a Waters silica Sep-Pak cartridge
(5?15% ethyl acetate/hexane) to give the alcohol 34 in 80% yield;
½
a 2D4
ꢂ
+66.8 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3) d 0.86 (6H, d,
5.21. des-A,B-25-[(Triethylsilyl)oxy]-18,21-dinorcholestane-8-
one (15)
J = 6.6 Hz), 4.04 (1H, narrow m); 13C NMR (100 MHz, CDCl3) d 20.2,
2 ꢀ 22.6, 24.4, 27.7, 27.9, 28.6, 29.2, 30.6, 33.5, 34.4, 39.0, 43.6,
44.6, 50.3, 67.8; MS (EI) m/z (relative intensity) 238 (M+, 50), 220
(76), 205 (41), 195 (79), 135 (82), 122 (86), 93 (100); exact mass
calculated for C16H30O 238.2297, measured 238.2323.
Silylation of the hydroxy ketone 36 was performed analogously
to the process described above for 35. The crude product was puri-
fied on a Waters silica Sep-Pak cartridge (2?5% ethyl acetate/hex-
ane) to give the silylated compound 15 (15 mg, 58%); 1H NMR
(400 MHz, CDCl3) d 0.56 (6H, q, J = 7.9 Hz), 0.94 (9H, t, J = 7.9 Hz),
1.18 (6H, s), 13C NMR (100 MHz, CDCl3) d 6.8, 7.1, 21.4, 24.7,
27.8, 28.7, 29.1, 29.6, 29.9, 34.2, 41.5, 45.1, 45.8, 54.8, 58.1, 73.4,
211.7; MS (EI) m/z (relative intensity) 366 (M+, 1), 351 (33), 337
(76), 308 (13), 217 (64), 173 (100); exact mass (ESI) calculated
for C22H42O2SiNa 389.2852, measured 389.2840.
5.18. (20S)-des-A,B-25-Hydroxy-18-norcholestane-8-one (35)
To a stirred solution of RuCl3 ꢀ H2O (10 mg, 0.05 mmol) and
NaIO4 (227 mg, 1.06 mmol) in water (1 mL), a solution of the 8b-
alcohol 33 (74 mg, 0.29 mmol) in tetrachloromethane (0.75 mL)
and acetonitrile (0.75 mL) was added. The reaction mixture was
stirred vigorously for three days. Then a few drops of 2-propanol
were added followed by water (10 mL). The reaction products were
extracted with methylene chloride (3 ꢀ 20 mL). The combined or-
ganic phases were dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The mixture of products was separated on
a Waters silica Sep-Pak cartridge (10?60% ethyl acetate/hexane)
to give the desired hydroxy ketone 35 (13 mg, 17%) and (20S)-
des-A,B-18-norcholestane-8-one (27 mg, 37%).
5.22. (20S)-1a,25-Dihydroxy-2-methylene-18,19-dinorvitamin
D3 (7)
To a stirred solution of the phosphine oxide 10 (46 mg;
mol) in anhydrous THF (600 L), a solution of phenyllithium
(1.5 M in THF, 63 L, 95
mol) was added at ꢁ20 °C under argon.
The mixture was stirred for 20 min and then cooled to ꢁ78 °C. A
pre-cooled solution of the Grundmann ketone 13 (13 mg; 34 mol)
in anhydrous THF (300 L) was added via cannula and the reaction
79
l
l
l
l
Compound 35: 1H NMR (400 MHz, CDCl3) d 0.78 (3H, d,
J = 6.7 Hz), 1.22 (6H, s), 2.01 (1H, br d, J = 12.3 Hz); 13C NMR
l
l

7668
R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
mixture was stirred for 3 h at ꢁ78 °C. Then the reaction mixture
was stirred at 4 °C overnight, ethyl acetate (20 mL) was added
and the organic phase was washed with brine (3 mL), 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). Final purification was performed by HPLC
(0.5% 2-propanol/hexane; 4 mL/min, 10 mm ꢀ 25 cm Zorbax-Sil
column; Rt = 6.59 min) to give the protected vitamin D compound
37 (13.5 mg, 53%); UV (hexane) kmax = 242, 251, 261 nm; 1H NMR
(500 MHz, CDCl3) d 0.06 (3H, s), 0.11 (3H, s), 0.17 (3H, s), 0.19
(3H, s), 0.56 (6H, q, J = 8.0 Hz), 0.76 (3H, d, J = 6.7 Hz), 0.86 (9H,
s), 0.89 (9H, s), 0.94 (9H, t, J = 8.0 Hz), 1.19 (6H, s), 2.18 (1H, dd,
J = 12.5, 8.1 Hz), 2.86 (1H, br d, J = 13.8 Hz), 4.93 (1H, s), 4.96 (1H,
s), 5.92 (1H, d, J = 11.1 Hz), 6.19 (1H, d, J = 11.1 Hz); 13C NMR
(125 MHz, CDCl3) d ꢁ5.1, ꢁ4.9, ꢁ4.9, ꢁ4.8, 6.8, 7.1, 18.2, 18.2,
22.3, 23.1, 25.8, 25.8, 27.8, 29.0, 29.7, 29.8, 29.9, 31.3, 33.6, 36.5,
38.7, 45.3, 47.5, 49.0, 50.2, 52.3, 71.9, 72.3, 73.4, 106.3, 113.7,
122.4, 132.9, 143.8, 152.9; MS (EI) m/z (relative intensity) 687
(M+ꢁt-Bu, 6), 628 (2), 612 (100), 583 (6), 555 (4), 480 (29), 366
(44); exact mass calculated for C40H75O3Si3 (M+ꢁt-Bu) 687.5024,
measured 687.5028.
residue was purified on a Waters silica Sep-Pak cartridge
(20?50% ethyl acetate/hexane) to give 17 mg of the crude com-
pound 8. The vitamin was further purified by HPLC (13% 2-propa-
nol/hexane, 4 mL/min, 10 mm ꢀ 25 cm Zorbax-Sil column;
Rt = 7.66 min) to give the analytically pure 19,21-dinorvitamin D
analog 8 (11.9 mg, 74%); ½a D24
ꢂ
+8.0 (c 0.80, CHCl3); UV (EtOH)
kmax = 243, 251, 261 nm; 1H NMR (400 MHz, CDCl3) d 0.45 (3H,
s), 1.21 (6H, s), 2.26–2.35 (2H, m), 2.57 (1H, dd, J = 13.4, 3.5 Hz),
2.84 (2H, m), 4.48 (2H, m), 5.09 (1H, s), 5.11 (1H, s), 5.89 (1H, d,
J = 11.2 Hz), 6.38 (1H, d, J = 11.2 Hz); 13C NMR (100 MHz, CDCl3) d
12.6, 22.6, 23.3, 27.8, 29.1, 29.2, 2 ꢀ 29.3, 30.5, 38.2, 38.5, 44.0,
45.4, 45.8, 51.3, 55.8, 70.7, 71.1, 71.8, 107.7, 115.2, 124.3, 130.4,
143.5, 152.0; MS (EI) m/z (relative intensity) 402 (M+, 36), 299
(15), 231 (17), 107 (50), 91 (64), 49 (100); exact mass calculated
for C26H42O3 402.3134, measured 402.3145.
5.24. 1a,25-Dihydroxy-2-methylene-18,19,21-trinorvitamin D3
(9)
The protected vitamin D compound 39 was prepared in the
same way as 37, except starting from the corresponding Grund-
mann ketone 15 and using 1.8 M solution of phenyl lithium in
di-n-butyl ether. The crude product was purified on a Waters silica
Sep-Pak cartridge (0?2% ethyl acetate/hexane) to give the vitamin
To a solution of the protected vitamin 37 (13 mg, 17
anhydrous THF (5 mL) tetra-n-butylammonium fluoride (1 M in
THF, 260 L, 260 mol) was added dropwise, followed by addition
lmol) in
l
l
of activated molecular sieves 4 Å (200 mg). The reaction mixture
was stirred under argon for 2 h. Then the solvent was removed un-
der reduced pressure, and the residue was purified on a Waters sil-
ica Sep-Pak cartridge (40?50% ethyl acetate/hexane). Final
purification of the vitamin D compound 7 was performed by HPLC
(20% 2-propanol/hexane; 4 mL/min; 10 mm ꢀ 25 cm Zorbax-Sil
column; Rt = 5.58 min) to give the analytically pure 18,19-dinorvi-
tamin D analog 7 (3.8 mg, 56%); UV (EtOH) kmax = 242, 250,
260 nm; 1H NMR (500 MHz, CDCl3) d 0.77 (3H, d, J = 6.6 Hz), 1.21
(6H, s), 2.58 (1H, dd, J = 13.2 Hz, 3.9 Hz), 2.81 (1H, dd, J = 13.3,
4.4 Hz), 2.87 (1H, br d, J =13.9 Hz), 5.10 (1H, s), 5.11 (1H, s), 5.97
(1H, d, J = 11.3 Hz), 6.35 (1H, d, J = 11.3 Hz); MS (EI) m/z (relative
intensity) 402 (M+, 39), 384 (41), 366 (14), 351 (11), 299 (58),
231 (36), 142 (58), 69 (100); exact mass calculated for C26H42O3
402.3134, measured 402.3121.
39 in 72% yield; ½a D24
ꢂ
+4.5 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3) d 0.03 (3H, s), 0.04 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.56
(6H, q, J = 7.9 Hz), 0.87 (9H, s), 0.90 (9H, s), 0.94 (9H, q,
J = 7.9 Hz), 1.18 (6H, s), 2.18 (1H, dd, J = 12.5, 7.9 Hz), 2.42 (3H,
m), 2.86 (1H, d, J = 13.6 Hz), 4.42 (2H, m), 4.93 (1H, s), 4.96 (1H,
s), 5.92 (1H, d, J = 11.1 Hz), 6.19 (1H, d, J = 11.1 Hz); 13C NMR
(100 MHz, CDCl3) d ꢁ5.1, ꢁ4.9, ꢁ4.9, 6.8, 7.1, 18.2, 24.8, 2 ꢀ 25.8,
25.8, 27.7, 28.9, 29.1, 29.9, 31.0, 34.9, 38.7, 45.1, 45.2, 47.5, 52.3,
53.9, 71.9, 72.3, 73.4, 106.3, 113.8, 122.5, 132.9, 143.6, 153.0; MS
(EI) m/z (relative intensity) 468 (50), 366 (61), 337 (35), 219 (54),
173 (64), 73 (100); exact mass (ESI) calculated for C43H82O3Si3Na
753.5470, measured 753.5450.
Removal of the silyl protecting groups in the vitamin 39 was
performed analogously to the process described above for 38.
The crude product was purified on a Waters silica Sep-Pak car-
tridge (30?55% ethyl acetate/hexane) to give 10 mg of the crude
compound 9. The vitamin was further purified by HPLC (acetoni-
trile/water/methanol 7:18:75, 4 mL/min, 9.4 mm ꢀ 25 cm Zorbax
Eclipse XDB-C18 column; Rt = 10.35 min) to give 18,19,21-trinorvi-
tamin D analog 9 in 77% yield; UV (EtOH) kmax = 243, 251, 260 nm;
1H NMR (500 MHz, CDCl3) d 1.09 (6H, s), 2.30–2.36 (2H, m), 2.58
(1H, dd, J = 13.2, 3.8 Hz), 2.81 (1H, dd, J = 13.2, 4.4 Hz), 2.86 (1H,
d, J = 13.8 Hz), 4.48 (2H, m), 5.09 (1H, s), 5.11 (1H, s), 5.97 (1H, d,
J = 11.2 Hz), 6.35 (1H, d, J = 11.2 Hz); 13C NMR (125 MHz, CDCl3) d
24.7, 25.3, 27.7, 28.8, 29.0, 29.2, 30.9, 34.7, 38.0, 44.0, 45.0, 45.9,
52.2, 53.8, 70.8, 71.1, 71.7, 107.7, 113.0, 124.2, 130.7, 145.8,
152.0; MS (EI) m/z (relative intensity) 388 (M+, 61), 370 (42), 352
(20), 337 (20), 285 (100); exact mass calculated for C25H40O3
388.2977, measured 388.2962.
5.23. 1a,25-Dihydroxy-2-methylene-19,21-dinorvitamin D3 (8)
The protected vitamin D compound 38 was prepared in the
same way as 37, except starting from the corresponding Grund-
mann ketone 14. The crude product was purified on a Waters silica
Sep-Pak cartridge (0?2% ethyl acetate/hexane) to give the vitamin
38 in 90% yield; ½a D24
ꢂ
+20.4 (c 1.05, CHCl3); 1H NMR (500 MHz,
CDCl3) d 0.02 (3H, s), 0.05 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.45
(3H, s), 0.56 (6H, q, J = 7.9 Hz), 0.86 (9H, s), 0.90 (9H, s), 0.94 (9H,
q, J = 7.9 Hz), 1.18 (6H, s), 2.18 (1H, dd, J = 12.2, 8.3 Hz), 2.33 (1H,
m), 2.50 (2H, m), 2.86 (1H, m), 4.42 (2H, m), 4.92 (1H, s), 4.97
(1H, s), 5.84 (1H, d, J = 11.0 Hz), 6.22 (1H, d, J = 11.0 Hz); 13C
NMR (125 MHz, CDCl3) d ꢁ5.1, ꢁ4.9, 6.8, 7.1, 12.4, 18.2, 18.3,
22.6, 23.2, 24.9, 25.8, 25.9, 27.9, 28.9, 29.4, 29.9, 30.6, 38.6, 45.2,
45.3, 47.6, 51.3, 55.7, 71.7, 72.5, 73.5, 106.3, 115.9, 122.4, 132.7,
141.4, 153.0; MS (EI) m/z (relative intensity) 687 (M+ꢁt-Bu, 34),
628 (12), 612 (100), 583 (38), 480 (49), 366 (86); exact mass calcu-
lated for C40H75O3Si3 (M+ꢁt-Bu) 687.5024, measured 687.5052.
To a stirred solution of the protected vitamin 38 (30 mg,
5.25. Biological in vitro studies
VDR binding, HL-60 differentiation and 24-hydroxylase tran-
scription assays were performed as previously described.31
40
l
mol) in n-butanol (3 mL), (+)-10-camphorsulfonic acid
5.26. Biological in vivo studies: bone calcium mobilization and
intestinal calcium transport
(21 mg, 90
l
mol) was added at 0 °C. Then the cooling bath was re-
moved and the reaction mixture was stirred for 1 week. A few
drops of a saturated aqueous solution of NaHCO3 were added, fol-
lowed by water (5 mL), and the mixture was extracted with ethyl
acetate (5 ꢀ 10 mL). The combined organic phases were dried over
anhydrous MgSO4, concentrated under reduced pressure, and the
Male, weanling Sprague-Dawley rats were purchased from Har-
lan (Indianapolis, IN). The animals were group housed and placed
on Diet 11 (0.47% Ca) + AEK oil for one week followed by Diet 11
(0.02% Ca) + AEK oil for three weeks. The rats were then switched

R. Barycki et al. / Bioorg. Med. Chem. 17 (2009) 7658–7669
6. Ostrem, V. K.; DeLuca, H. F. Steroids 1987, 49, 73.
7669
to a diet containing 0.47% Ca for one week followed by two weeks
on a diet containing 0.02% Ca. Dose administration began during
the last week on 0.02% Ca diet. Four consecutive intraperitoneal
doses were given approximately 24 h apart. Twenty-four hours
after the last dose, blood was collected from the severed neck,
and the serum calcium level was determined as a measure of bone
calcium mobilization. The first 10 cm of the intestine was also col-
lected for the intestinal calcium transport analysis using the evert-
ed gut sac method.32 Statistical analyses were conducted in all
in vivo bone calcium mobilization studies. ANOVA followed by
pairwise comparisons using Tukey’s test was done in vehicle;
260 pmol and 780 pmol dose groups for each of the studies.
7. Abe, E.; Miyaura, C.; Sakagami, H.; Takeda, M.; Konno, K.; Yamazaki, T.; Yoshiki,
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Acknowledgments
17. Plum, L. A.; Fitzpatrick, L. A.; Ma, X.; Binkley, N. C.; Zella, J. B.; Clagett-Dame,
The work was supported in part by funds from the Wisconsin
Alumni Research Foundation. We would like to express our grati-
tude to Dr. Wanda Sicinska for her valuable comments concerning
the X-ray crystallographic data of a 2MD complex with the rVDR
and a coactivator peptide. A special thanks to Jean Prahl and Jenni-
fer Vaughan for carrying out the in vitro studies and to Heather
Neils and Xiaohong Ma for conducting the in vivo studies.
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 Wis-
consin, the NIH (RR02781, RR08438), the NSF(DMB-8415048, OIA-
9977486, BIR-9214394), and the USDA.
M.; DeLuca, H. F. Osteoporosis Int. 2006, 17, 704.
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