A 20 S combined with a 22 r configuration markedly increases both in vivo and in vitro biological activity of 1α,25-Dihydroxy-22-methyl-2-methylene- 19-norvitamin D3

Article abstract of DOI:10.1021/jm300187x

Six new analogues of 1α25-dihydroxy-19-norvitamin D₃ (3a-4b, 5, and 6) were prepared by a convergent synthesis applying the Wittig-Horner reaction as a key step. The influence of methyl groups at C-22 on their biological activity was examined.

Full text of DOI:10.1021/jm300187x

Article
pubs.acs.org/jmc
A 20S Combined with a 22R Configuration Markedly Increases both
in Vivo and in Vitro Biological Activity of 1α,25-Dihydroxy-22-
methyl-2-methylene-19-norvitamin D3
Agnieszka Flores,† Rafal R. Sicinski,‡ Pawel Grzywacz,† James B. Thoden,† Lori A. Plum,†
Margaret Clagett-Dame,† and Hector F. DeLuca*,†
†Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison, 433 Babcock Drive,
Madison, Wisconsin 53706-1544, United States
‡Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
S
* Supporting Information
ABSTRACT: Six new analogues of 1α,25-dihydroxy-19-norvitamin D3
(3a−4b, 5, and 6) were prepared by a convergent synthesis applying the
Wittig−Horner reaction as a key step. The influence of methyl groups at
C-22 on their biological activity was examined. It was established that
both in vitro and in vivo activity is strongly dependent on the
configuration of the stereogenic centers at C-20 and C-22. Introduction
of the second methyl group at C-22 (analogues 5 and 6) generates the
compounds that are slightly more potent than 1α,25-(OH)2D3 in the in
vitro tests but much less potent in vivo. The greatest in vitro and in vivo
biological activity was achieved when the C-20 is in the S configuration
and the C-22 is in the R configuration. The building blocks for the synthesis, the respective (20R,22R)-, (20R,22S)-, (20S,22R)-,
and (20S,22S)-diols, were obtained by fractional crystallization of mixtures of the corresponding diastereomers. Structures and
absolute configurations of the diols 21a, 21b, and 22a as well as analogues 3a, 5, and 6 were confirmed by the X-ray
crystallography.
min D3 (2, 2MD) showing a very strong activity in bone and,
moreover, inducing bone formation in vitro and in vivo.7,8
Recently, Yamamoto et al. described 2-methylene-19-norvita-
min D compounds bearing a 22S-butyl and 22S-ethyl group and
discovered that they can act as agonists or antagonists for the
VDR.9
In the present study we examined all possible conformers
3a−4b resulting from C-20 epimerization and introduction of a
methyl group at the two available positions on C-22 in the
1α,25-dihydroxy-2-methylene-19-norvitamin D3. We also used
the attachment of two methyl groups on C-22 (analogues 5 and
6) to help understand the biological impact of the double
substitution of this side chain carbon. Our results show that
superior biological activity both in vitro and in vivo is obtained
in the case of 20S,22R-configuration. How this occurs is
discussed in relation to the ligand interaction with the receptor.
INTRODUCTION
■
Vitamin D3 must be metabolized to its active form, 1α,25-
dihydroxyvitamin D3 [calcitriol, 1α,25-(OH)2D3 (1); Figure 1]
before it is functionally active.1 This hormonal form of vitamin
D3 is responsible for calcium and phosphorus homeostasis, but
it also plays a role in other biological systems that are still under
investigation.2 A number of analogues of 1α,25-dihydroxyvita-
min D3 have been successfully developed as pharmaceuticals
used in the treatment of renal osteodystrophy, osteoporosis,
and vitamin D-resistant rickets.3 Although these compounds are
an improvement over previous therapies, further improvements
are clearly possible.
Modification of the configuration of vitamin D compounds
definitely affects biological activity. Inverting the configuration
at C-20 usually results in increased activity both in vivo and in
vitro.4 Yamada et al. designed conformationally restricted
vitamin D analogues, postulating that introduction of a methyl
group at C-22 hinders rotation around the C(20)−C(22)
bond.5 Moreover, restricting the side chain rotation by the
introduction of a C(17)−C(20) double bond has a marked
impact on biological activity, with the Z-configuration strongly
favoring calcemic potency.6
RESULTS
■
Synthesis. The first approach to prepare 22-methyl-1α,25-
(OH)2D3 analogues was based on the stereoselective conjugate
addition of organocuprate to the steroidal (E)- and (Z)-22-ene-
24-ketones.10 We describe herein a convergent synthesis of all
the four diastereomers of 22-methyl-1α,25-(OH)2D3 where the
In 1998 we discovered A-ring modification of vitamin D
compounds consisting in “shift” of the exomethylene unit at C-
10 to carbon 2.7 One of the most potent analogues of this series
proved to be (20S)-1α,25-dihydroxy-2-methylene-19-norvita-
Received: February 10, 2012
Published: April 10, 2012
© 2012 American Chemical Society
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Figure 1. Structures of the 1α,25-(OH)2D3 (1), 2MD (2), and vitamin D analogues (3a−6).
key precursors are nitriles 9 and 10, readily obtainable from the
Inhoffen−Lythgoe diol.11 In contrast to similar synthetic
approaches, independently proposed by Fujishima12 and
21b was oxidized with PDC, and after hydroxy protection, the
hydrindanone 23b was obtained in 53% yield (over two steps).
Coupling of a phosphine oxide A-ring with a C,D-ketone is
one of the most useful methods for the synthesis of vitamin D
analogues.15 In this approach, first developed by Lythgoe,16 an
anion of an allylic phosphine oxide reacts with the Grundmann
ketone via the Wittig−Horner reaction. In our case, this
method proved to be very useful. The anion, generated from
the known phosphine oxide 257 with phenyllithium, was
coupled with the ketones 23a and 23b to give the
corresponding protected 19-norvitamin D analogues 26a and
26b in 91% and 90% yield, respectively. The silyl protecting
groups were removed with hydrofluoric acid to give the final
compounds 3a and 3b in 78% and 79% yield, respectively. The
structure and absolute configuration of the vitamin 3a were
confirmed by X-ray crystallography (Figure 3).
The synthesis of the diastereomeric vitamins 4a and 4b
started from the 20R-tosylate 811 and followed the analogous
path. Also in this case, fractional crystallization from ethyl
acetate of the epimeric pair of 20,22-diols 22a and 22b,
obtained in a ratio of 2:1, played a crucial role. The absolute
(20S,22R)-configuration of the prevailing diol 22a, which
crystallized first, was established by X-ray analysis (Figure 2).
The pure (20S,22S)-diol 22b was obtained in the crystalline
form from the mother liquors. Both diols were then separately
converted into the respective protected 25-hydroxy Grund-
mann ketones 24a,b by the same consecutive processes as
described above. Finally, they were subjected to the Wittig−
Horner coupling with the anion generated from 25, and after
removal of the silyl protecting groups in the products 27a,b, the
corresponding vitamin D3 analogues 4a and 4b were obtained
in 77% and 50% yield, respectively.
Mourino,13 we used crystallization, avoiding the expensive
̃
separation of the C,D-ring isomers by flash chromatography or
HPLC. Since only a few 22-alkyl-19-nor-1α,25-dihydroxyvita-
min D3 analogues have been synthesized to date,9 we have also
decided to prepare and test the analogues double-substituted at
C-22 (Scheme 1).
As shown in Scheme 1, the vitamin D analogues 3a and 3b
were prepared from the 20S-tosylate 7.11 Its substitution with
cyanide provided the nitrile 9 (in 97% yield) that was alkylated
at the α-position with bromide A using LDA as a base to give
the compounds 11a,b (93% yield) being a mixture of epimers
at C-22. The two consecutive reductions, first with DIBALH,
then with NaBH4, afforded the epimeric mixture of the alcohols
15a,b in 56% yield (over two steps). The hydroxy group in
these compounds was removed in a two-step reaction
sequence: conversion into the tosylates 17a,b followed by
reduction with LiAlH4 (80% overall yield). Removal of both
silyl protecting groups in the formed products 19a,b afforded a
mixture of the epimeric diols 21a and 21b in a ratio of 1:2,
1
respectively (as established by H NMR). The stereoisomers
were separated by crystallization from ethyl acetate, and their
absolute configurations were determined by X-ray crystallog-
raphy (Figure 2). This is one of the rare examples of a process
known as fractional crystallization used in chemical engineering
for purification or analysis. In this method, based on differences
in solubility, two or more substances dissolved in a solvent
crystallize from the solution at different rates.14 In our case, the
prevailing (20R,22S)-diol 21b crystallized first. Then pure
crystals of the (20R,22R)-diol 21a were obtained from the
mother liquors. The diol 21a was subsequently oxidized with
tetrapropylammonium perruthenate in the presence of 4-
methylmorpholine N-oxide, and in the formed product the 25-
hydroxy group was protected as a TES ether to give the
Grundmann ketone 23a in 85% overall yield. The epimeric diol
The synthesis of 22,22-dimethyl vitamin D analogues 5 and
6, performed for the compounds in the 20R- and 20S-series
separately, is shown in Scheme 2. Thus, α-alkylation of the 20R-
nitrile 9 with methyl iodide was achieved using LDA as a base,
and the product 28 double-substituted at C-20 was obtained in
56% yield. The conversion of the 20S-nitrile 10 into 29 was
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a
Scheme 1
a
(a) NaCN, DMSO; (b) LDA, THF; (c) DIBAl-H, CH2Cl2; (d) NaBH4, MeOH; (e) p-TsCl, pyridine; (f) LiAlH4, Et2O; (g) TBAF, THF; (h)
crystallization; (i) (1) PDC, CH2Cl2 or NMO, TPAP, 4 Å molecular sieves, CH2Cl2; (2) TESOTf, 2,6-lutidine, CH2Cl2; (j) PhLi, THF; (k) 48% aq
HF, MeCN, THF.
analogous but significantly more efficient. The reduction of the
obtained nitriles with DIBALH afforded the respective
aldehydes 30 and 31 in 75% and 95% yield, respectively.
These were next subjected to the Wittig−Horner−Emmons
reaction with the triethylphosphonoacetate anion to give the
α,β-unsaturated esters 32 and 33 (55% and 91% yield,
respectively), which were subsequently hydrogenated using a
palladium catalyst. The esters 34 and 35, obtained in 68% and
95% yield, were treated with methylmagnesium bromide to give
the diols 36 and 37 in 100% and 84% yield, respectively. These
diols were in turn oxidized to the ketones with tetrapropy-
lammonium perruthenate in the presence of 4-methylmorpho-
line N-oxide. The terminal 25-hydroxy group in the formed
products was protected as a triethylsilyl ether. The obtained
Grundmann ketones 38 and 39 (86% and 78% overall yield)
were coupled with a lithium phosphinoxy carbanion derived
from the phosphine oxide 25 to give the protected 19-
norvitamins 40 and 41 in 78% and 91% yield, respectively.
Acidic cleavage of the silyl groups furnished the final 20R- and
20S-vitamins 5 and 6 in 79% and 75% yield, respectively. The
structures and absolute configurations of these compounds
were confirmed by X-ray crystallography (Figure 3).
Biological Evaluation. Biological activities in vitro are
summarized in Table 1. Single 22-methylation of the (20R)-25-
hydroxylated side chain did not significantly change the affinity
of the analogues 3a and 3b to the nuclear receptor, compared
to their parent compound, 2-methylene-19-nor-1α,25-
(OH)2D3.7 However, addition of a 22-methyl group to the
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Figure 2. ORTEP drawings derived from the single-crystal X-ray analysis of the (20R,22R)-diol 21a, (20R,22S)-diol 21b, and (20S,22R)-diol 22a.
Figure 3. ORTEP drawings derived from the single-crystal X-ray analysis of the vitamins 3a, 5, and 6.
analogue 2 (2MD), characterized by an “unnatural” 20S side
chain, caused a much stronger effect; the 22R-compound 4a
bound 2.5 times more strongly than 2, and it proved to be 250-
fold more potent than its 22-epimer 4b. Also, clear distinctions
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a
Scheme 2
a
(a) LDA, THF, CH3I; (b) DIBAl-H, CH2Cl2; (c) LDA, THF, (EtO)2P(O)CH2COOEt; (d) 10% Pd/C, H2, MeOH; (e) CH3MgBr, Et2O; (f) (1)
NMO, TPAP, 4 Å molecular sieves, CH2Cl2; (2) TESOTf, 2,6-lutidine, CH2Cl2; (g) PhLi, THF; (h) 48% aq HF, MeCN, THF.
in the biological activity appeared in the cell differentiation
assay, where the analogue 3b with an additional 22S-methyl
group in the “natural” side chain was 10-fold more active than
its 22-epimer 3a, whereas in the case of 20-epi compounds, the
22R-isomer 4a was found to be 4-fold more potent than 2MD
and, compared to its 22S-counterpart 4b, showed higher
potency by 3 orders of magnitude. A similar pattern of relative
activities of the four diastereomeric at C-20 and C-22, 22-
methyl vitamins 3a,b−4a,b, was found in the transcriptional
assay. The transactivation potencies of the analogues 3b and 4a
were significantly higher compared to those of compounds 3a
and 4b, being their 22-epimers.
Noteworthy, the introduction of two methyl groups at the 22
position, regardless of the configuration at the 20-carbon,
created the vitamins 5 and 6 having higher binding affinities to
VDR compared to their parent vitamins. This was especially
notable in the case of the latter compound.7 However, in the
two remaining in vitro tests, double-substitution at C-22 of the
“natural” side chain resulted in a marked increase of potency of
the analogue (6),7 but for the corresponding 20-epi compound
(2 versus 5), this modification caused a reversed effect. Thus,
the 22,22-dimethyl analogue 5 showed cell differentiating
potency and transcriptional activity lower by 1 and 2 orders of
magnitude, respectively, than the structurally related analogue,
2MD.
substantially more potent than 1α,25-(OH)2D3. Despite the
fact that 3b, 5, and 6 appear to have the VDR binding affinity
and the cell differentiation activity similar to or slightly higher
than that of 1α,25-(OH)2D3, their ability to cause the release of
bone calcium in vitamin D deficient rats was at least 50 times
lower. In the intestine, the order of potency of the analogues
was the same as that observed for the bone.
DISCUSSION
■
The above-presented results of biological tests, performed for
the synthesized vitamins monosubstituted at C-22, indicated
that the order of their in vitro activity was 4a ≫ 3b > 3a > 4b.
Such an activity pattern could be quite successfully predicted
taking into account the published data on the closely related
diastereomers of 22-methyl-1α,25-(OH)2D3 differing in con-
figurations at C-20 and C-22. On the basis of conformational
analyses of 25-hydroxylated side chains present in these
compounds and other side chain restricted vitamin D
analogues, Yamada and collaborators developed an “active
space region” concept.5,17 They created three-dimensional “dot
maps” showing the spatial regions that can be potentially
occupied by the 25-oxygen present in the energetically
preferred conformations of the vitamin D compounds
possessing different side chains. It was established that the
25-hydroxy group occupied only one spatial region (A, G, EA,
and EG) in each of the four C-20 and C-22 diastereomers of
the 22-methylated hormone. Interestingly, further conforma-
tional studies allowed the Japanese scientists to correlate these
defined regions with the biological activities of vitamin D
In vivo biological activities are shown in Figures 5 and 6. The
activity profiles in bone calcium mobilization partially paralleled
the results observed in the in vitro assays, where 3a and 4b
exhibited lower potency than the native hormone, and 4a was
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a
b
c
Table 1. VDR Binding Properties, HL-60 Differentiating Activities, and Transcriptional Activities of the Vitamin D
Hormone (1), 2MD (2) and the Vitamin D Analogues 3a−6
a
Competitive binding of 1α,25-(OH)2D3 (1) and the synthesized vitamin D analogues to the full-length recombinant rat vitamin D receptor. The
experiments were carried out in duplicate on two different occasions. The Ki values are derived from the dose−response curves and represent the
inhibition constant when radiolabeled 1α,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. The binding ratio is the average ratio of the
b
1α,25-(OH)2D3 Ki to the Ki for the analogue. Induction of differentiation of HL-60 promyelocytes to monocytes by 1α,25-(OH)2D3 (1) and the
synthesized vitamin D analogues. Differentiation state was determined by measuring the percentage of cells reducing nitro blue tetrazolium (NBT).
The experiment was repeated in duplicate two times. The ED50 values are derived from the dose−response curves and represent the analogue
concentration capable of inducing 50% maturation. The differentiation activity ratio is the average ratio of the 1α,25-(OH)2D3 ED50 to the ED50 for
c
the analogue. Transcriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The ED50 values are
derived from dose−response curves and represent the analogue concentration capable of increasing the luciferase activity by 50%. The luciferase
d
activity ratio is the average ratio of the 1α,25-(OH)2D3 ED50 to the ED50 for the analogue. Active side chain spatial regions occupied by 25-hydroxy
group, as defined by Yamada.5
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Figure 4. Side chain conformational analysis of the model 8-methylene compounds 42a−c possessing side chains identical to those of the vitamin D
analogues 4a, 5, and 6, respectively. Energy-minimized conformations of these compounds (energy window, 1 kcal/mol) were overlaid. The circles
show the location of 25-oxygen atoms in the corresponding conformers. Side chain carbons and hydrogen atoms are omitted for clarity. There are
indicated spatial regions EG, G, EA, and A as defined by Yamada.5
compounds such as the receptor (VDR) affinity, cell differ-
entiation, and transactivation potency.5,17 Thus, in terms of the
space region, the orders of activity were established as follows:
EA > A > G > EG (affinity for the VDR), and EA > A > EG ≥
G (cell differentiation and gene transactivation). Inspired by the
above-described findings, we performed a conformational
analysis of the model 8-methylene des-A,B-compounds 42a−c
possessing 25-hydroxy-22-methyl-substituted side chains of the
vitamins, whose synthesis is presented in this study. Compound
42a was chosen to prove that our calculations provide the same
spatial region that was ascribed by Yamada for the side chain of
the analogous (20S,22R)-isomer of 22-methyl-1α,25-(OH)2D3.
We carried out the MM+ force field calculations of the model
8-methylene compounds by using the HyperChem molecular
modeling program. The conformational searches were
performed according to protocol described by us previously
in detail.18 For the lowest energy conformers of (20S,22R)-
compound 42a, only the gauche(+) conformation in relation to
the dihedral angle C(16−17−20−22) was found. On the
contrary, in the case of 22,22-dimethylated compounds 42b
and 42c, only the anticlinal and antiperiplanar, respectively,
conformation was present with respect to this torsion angle.
Figure 4 shows the results of superimposition of the most stable
side chain conformers of 42a−c, falling into the 1 kcal/mol
energy window. As a consequence of the different torsion
angles adopted by low-energy side chain conformers of the
examined compounds, the 25-oxygen tends to reside in the
following regions: EA (42a), EG and G (42b), A and G (42c).
The first result confirms the correctness of our calculations
because the most active front region EA was also found by
Yamada for the 22-methylated vitamin having analogous
additional alkyl substituents at C-22. The 25-oxygen in 5 is
distributed over the inactive rear regions of EG−G, whereas in
6 it also tends to locate in the active front region A.
In summary, the vitamin D analogues presented in this work
are interesting candidates in the structure−function studies.
Moreover, they have shown a promising therapeutic value that
is under investigation now.
EXPERIMENTAL SECTION
■
Chemistry. Melting points (uncorrected) were determined on a
Thomas-Hoover capillary melting point apparatus. Optical rotations
were measured in chloroform using a Perkin-Elmer model 343
polarimeter at 22 °C. Ultraviolet (UV) absorption spectra were
recorded with a Perkin-Elmer Lambda 3B UV−vis spectrophotometer
1
in ethanol or hexane. H nuclear magnetic resonance (NMR) spectra
were recorded in deuteriochloroform at 400 and 500 MHz with Bruker
DMX-400 and DMX-500 Avance console spectrometers. In the case of
diastereomeric mixtures of compounds, proton signals belonging to
the major isomer are listed; selected signals of the minor isomer are
marked in italic. 13C NMR spectra were recorded in deuteriochloro-
form at 100 and 125 MHz with the same Bruker instruments.
Chemical shifts (δ) are reported in parts per million relative to CH3Si
(δ 0.00) as an internal standard. Abbreviations used are singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m). Numbers in
parentheses following the chemical shifts in the 13C NMR spectra refer
to the number of attached hydrogens as revealed by DEPT
experiments. 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. Solvents were
dried and distilled following standard procedures.
The purity of final compounds was determined by HPLC, and they
were judged to be at least 99% pure. Two HPLC columns (9.4 mm ×
25 cm Zorbax-Sil and Zorbax RX-C18) were used as indicated in
Table 1 (Supporting Information). The purity and identity of the
synthesized vitamins were additionally confirmed by inspection of
(20S,22R)-configurations of the side chain substituents.5
A
comparison of the in vitro activities of the 22-monomethylated
vitamins 3a,b−4a,b with the spatial regions occupied by their
side chain hydroxy group (Table 1) indicates that their order of
biological potencies is in agreement with the correlations found
by Yamada, despite the fact that different A-ring fragments are
present in these two series of compounds. When considering
the biological in vitro activity of the analogues 5 and 6,
conformational analysis also explains the effect of two
1
their H NMR and high-resolution mass spectra.
(8S,20R)-des-A,B-20-(Cyanomethyl)-8β-[(triethylsilyl)oxy]-
pregnane (9). Sodium cyanide (2 g, 41 mmol) was added to a
solution of the tosylate 7 (0.84 g, 1.75 mmol) in dry DMSO (8 mL).
The resulting mixture was stirred at 90 °C for 3 h. Then it was cooled,
diluted with water, and extracted with ethyl acetate. The combined
organic phases were dried (Na2SO4) and concentrated. The residue
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Figure 5. continued
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Figure 5. Total serum calcium levels reflecting the ability of each analogue to release bone calcium stores. The values shown are the difference from
the vehicle controls. Each value represents the mean SEM of six values.
was purified by column chromatography on silica gel using 10% ethyl
acetate/hexane to give the nitrile 9 (0.57 g, 97%).
nitrile 12a,b was performed as described for 11a,b. A mixture of the
aldehydes 14a,b was obtained in 76% yield.
(8S,20S)-des-A,B-20-(Cyanomethyl)-8β-[(triethylsilyl)oxy]-
pregnane (10). The substitution reaction of the tosylate 8 with
cyanide, performed as described for 9, gave the nitrile 10 (85%).
(8S,20R,22R)- and (8S,20R,22S)-des-A,B-22-Cyano-8β,25-bis-
[(triethylsilyl)oxy]cholestane (11a,b). n-Butyllithium (1.6 M in
hexane, 2.7 mL, 4.32 mmol) was added to a solution of diisopropyl-
amine (0.6 mL, 0.43 g, 4.25 mmol) in THF (4 mL) at 0 °C. The
resulting mixture was stirred at 0 °C for 30 min. Then it was cooled to
−78 °C, and a solution of the nitrile 9 (0.57 g, 1.70 mmol) in THF (5
mL) was added. The mixture was stirred at −78 °C for 30 min and a
solution of the bromide A (0.96 g, 3.42 mmol) was added. The
reaction mixture was stirred at −78 °C for 1 h and at 0 °C for 1 h.
Then it was quenched with a saturated aqueous NH4Cl solution and
extracted with ethyl acetate. The combined organic phases were
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by column chromatography on silica gel (elution with
1.5%, 3%, and 10% ethyl acetate/hexane) to give a mixture of nitriles
11a,b (0.85 g, 93%).
(8S,20R,22R)- and (8S,20R,22S)-des-A,B-22-(Hydroxymethyl)-
8β,25-[(triethylsilyl)oxy]cholestane (15a,b). Sodium borohydride
(0.44 g, 11.63 mmol) was added to a solution of the aldehydes 13a,b
(0.64 g, 1.19 mmol) in methanol (10 mL) at 0 °C. The reaction
mixture was warmed and stirred at room temperature for 2 h, then
quenched with water and extracted with ethyl acetate. The combined
organic layers were washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography
on silica gel (elution with 3% and 10% ethyl acetate/hexane) to give a
mixture of the alcohols 15a,b (0.46 g, 71%).
(8S,20S,22R)- and (8S,20S,22S)-des-A,B-22-(Hydroxymethyl)-
8β,25-[(triethylsilyl)oxy]cholestane (16a,b). Sodium borohydride
reduction of the aldehydes 14a,b was performed as described for
13a,b. A mixture of the alcohols 16a,b was obtained in 70% yield.
(8S,20R,22R)- and (8S,20R,22S)-des-A,B-22-Methyl-8β,25-
[(triethylsilyl)oxy]cholestane (19a,b). A solution of tosyl chloride
(0.66 g, 3.46 mmol) in pyridine (2 mL) was added to a solution of the
alcohols 15a,b (0.46 g, 0.85 mmol) in dry pyridine (4 mL) at −20 °C.
The reaction mixture was stirred at −20 °C for 1 h and at +4 °C for 18
h. Then it was poured into a saturated aqueous CuSO4 solution and
extracted with dichloromethane. The combined organic phases were
dried (Na2SO4) and concentrated. The residue was purified by column
chromatography on silica gel (elution with 3% ethyl acetate/hexane)
to give a mixture of the tosylates 17a,b (0.54 g, 92%).
LiAlH4 (0.4 g, 10.53 mmol) was added to a solution of the tosylates
17a,b (0.53 g, 0.76 mmol) in dry diethyl ether (10 mL) at 0 °C. The
reaction mixture was stirred at +4 °C for 20 h. The excess of LiAlH4
was decomposed with water. The reaction mixture was diluted with
diethyl ether and subsequently filtered through Celite. The filtrate was
extracted with ethyl acetate, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography on silica gel (3% ethyl
acetate/hexane) to give a mixture of the products 19a,b (0.32 g, 80%).
(8S,20S,22R)- and (8S,20S,22S)-des-A,B-22-Methyl-8β,25-
[(triethylsilyl)oxy]cholestane (20a,b). Tosylation of the alcohols
16a,b and reduction of the formed tosylates with LiAlH4, performed as
(8S,20S,22R)- and (8S,20S,22S)-des-A,B-22-Cyano-8b,25-bis-
[(triethylsilyl)oxy]cholestane (12a,b). Reaction of the nitrile 10
anion with the bromide A, carried out as described for 9, gave a
mixture of nitriles 12a,b (79%).
(8S,20R,22R)- and (8S,20R,22S)-des-A,B-22-Formyl-8b,25-
bis[(triethylsilyl)oxy]cholestane (13a,b). Diisobutylaluminum hy-
dride (1.5 M in toluene, 1.4 mL, 2.1 mmol) was added to a solution of
the nitriles 11a,b (0.81 g, 1.51 mmol) in dichloromethane (10 mL) at
−10 °C. The reaction mixture was stirred at −10 °C for 1 h, and it was
quenched with a saturated aqueous potassium sodium tartrate solution
(5 mL). The water phase was extracted with dichloromethane, and the
combined organic layers were washed with brine, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography on
silica gel (elution with 3% ethyl acetate/hexane) to give a mixture of
the aldehydes 13a,b (0.64 g, 79%).
(8S,20S,22R)- and (8S,20S,22S)-des-A,B-22-Formyl-8β,25-bis-
[(triethylsilyl)oxy]cholestane (14a,b). DIBALH reduction of the
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Figure 6. continued
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Figure 6. In vivo intestinal calcium transport compared to the native hormone. The values shown are the difference from the vehicle controls and
represent the average SEM of six measurements.
described for 15a,b, gave a mixture of the products 20a,b (96 mg,
75%).
that was further washed with ethyl acetate. After removal of the
solvent, the 25-hydroxy-8-ketone (14.4 mg, 89%) was obtained as a
colorless oil.
(8S,20R,22R)- and (8S,20R,22S)-des-A,B-22-Methylcholes-
tane-8β,25-diol (21a and 21b). Tetrabutylammonium fluoride
(1.0 M in THF, 3.4 mL, 3.4 mmol) was added to a solution of the
compounds 19a,b (0.31 g, 0.59 mmol) in THF (3 mL) at 0 °C. The
reaction mixture was stirred at +4 °C for 20 h. Then it was diluted with
water and extracted with ethyl acetate. The combined organic extracts
were dried (Na2SO4) and concentrated. The residue was purified by
column chromatography on silica gel (elution with 10% and 50% ethyl
acetate/hexane) to give a mixture of diols 21a and 21b (0.17 g, 99%)
Triethylsilyl trifluoromethanesulfonate (20 μL, 23 mg, 88 μmol)
was added dropwise to a solution of the obtained 25-hydroxy-8-ketone
(14.4 mg, 49 μmol) and 2,6-lutidine (20 μL, 18 mg, 0.17 mmol) in
dichloromethane (2 mL) at −40 °C. The reaction mixture was stirred
at −40 °C for 15 min. Then it was diluted with dichloromethane and
washed with water. The organic layer was dried (Na2SO4) and
concentrated. The residue was applied on a Waters silica Sep-Pak
cartridge (5 g). Elution with ethyl acetate/hexane (1:99, then 2:98)
gave the protected ketone 23a (19 mg, 95%).
(20R,22S)-des-A,B-22-Methyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (23b). Pyridinium dichromate (0.18 g, 0.48 mmol)
and pyridinium p-toluenesulfonate (24 mg, 95 μmol) were added in
one portion to a solution of diol 21b (24.9 mg, 84 μmol) in dry
dichloromethane (5 mL). The reaction mixture was stirred at room
temperature for 75 min. Then it was quenched with water and
extracted with dichloromethane. The combined organic layers were
dried (Na2SO4) and concentrated. The residue was applied on a
Waters silica Sep-Pak cartridge (2 g). Elution with dichloromethane
gave the pure 25-hydroxy-8-ketone (23.6 mg).
Silylation of the 8-ketone with triethylsilyl trifluoromethanesulfo-
nate was performed according to procedure described above. The
crude product was applied to a Waters silica Sep-Pak cartridge (10 g).
Elution with ethyl acetate/hexane (2:98, then 5:95) gave the protected
ketone 23b (18.2 mg, 53% yield in two steps).
(8S,20S,22R)-des-A,B-22-Methyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (24a). Oxidation of the diol 22a with tetrapropy-
lammonium perruthenate and 4-methylmorpholine oxide and the
subsequent silylation of the resulting 25-hydroxy-8-ketone were
performed as described for conversion of 21a into 23a. The protected
ketone 24a was obtained in 68% yield.
1
in a 1:2 ratio, respectively (based on H NMR). The isomers were
separated by crystallization from ethyl acetate, and the absolute
configuration was established by X-ray analysis. Pure crystals (96 mg)
of the 22S-isomer 21b were obtained after first crystallization. Then
pure crystals (44.6 mg) of the other 22R-isomer 21a were obtained
from the concentrated mother liquors. Additionally, a second crop of
pure crystals (16 mg) of the diol 21b was obtained from the filtrate
after second crystallization.
(8S,20S,22R)- and (8S,20S,22S)-des-A,B-22-Methylcholes-
tane-8β,25-diol (22a and 22b). Deprotection of the silyl groups
in the compounds 20a,b with tetrabutylammonium fluoride was
performed analogously as described for 19a,b. The products were
purified by column chromatography on silica gel (30% ethyl acetate/
hexane) to give a mixture of the diols 22a and 22b (99%) in a 2:1
1
ratio, respectively (as established by H NMR). The isomers were
separated by crystallization from ethyl acetate, and the absolute
configuration was established by X-ray analysis. Pure crystals of the
22R-isomer 22a were obtained after two crystallizations, and the
isomeric 22S-diol 22b was obtained from the mother liquors.
(20R,22R)-des-A,B-22-Methyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (23a). Molecular sieves (4 Å, 60 mg) were added to
a solution of 4-methylmorpholine oxide (33 mg, 0.282 mmol) in
dichloromethane (0.25 mL). The mixture was stirred at room
temperature for 15 min, and tetrapropylammonium perruthenate (2
mg, 5.7 μmol) was added, followed by a solution of the diol 21a (16
mg, 54 μmol) in dichloromethane (300 + 250 μL). The resulting
suspension was stirred at room temperature for 1 h. The reaction
mixture was filtered through a Waters silica Sep-Pak cartridge (2 g)
(8S,20S,22S)-des-A,B-22-Methyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (24b). Oxidation of the diol 22b and subsequent
silylation of the 25-hydroxy-8-ketone were carried out according to the
procedure described above for conversion of 21a into 23a to give the
protected ketone 24b in 73% yield.
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(20R,22R)-1α,25-Dihydroxy-22-methyl-2-methylene-19-nor-
vitamin D3 (3a). Phenyllithium (1.8 M in di-n-butyl ether, 60 μL, 108
μmol) was added to a stirred solution of the phosphine oxide 25 (54
mg, 93 μmol) in anhydrous THF (500 μL) at −30 °C. After 30 min,
the mixture was cooled to −78 °C, and a precooled solution of the
ketone 23a (19 mg, 47 μmol) in anhydrous THF (300 + 200 μL) was
added. The reaction mixture was stirred under argon at −78 °C for 4 h
and then at +4 °C for 19 h. Ethyl acetate was added, and the organic
phase was washed with brine, dried (Na2SO4), and concentrated. The
residue was applied on a Waters silica Sep-Pak cartridge (5 g). The
cartridge was eluted with hexane and ethyl acetate/hexane (1:99) to
give the protected vitamin 26a (32.64 mg, 91%).
The protected vitamin 26a (32.64 mg, 42 mmol) was dissolved in
THF (4 mL) and acetonitrile (3 mL). A solution of aqueous 48% HF
in acetonitrile (1:9 ratio, 4 mL) was added at 0 °C, and the resulting
mixture was stirred at room temperature for 2 h. Saturated aqueous
NaHCO3 solution was added, and the reaction mixture was extracted
with dichloromethane. The combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. The residue was
diluted with 2 mL of hexane/ethyl acetate (7:3) and applied on a
Waters silica Sep-Pak cartridge (5 g). An elution with hexane/ethyl
acetate (7:3, then 1:1) gave the crude product 3a. The vitamin 3a was
further purified by a normal-phase HPLC [9.4 mm × 250 mm Zorbax
silica column, 5 mL/min, hexane/2-propanol (85:15) solvent system,
tR = 6.5 min] and a reversed-phase HPLC [9.4 mm × 250 mm Zorbax
RX-C18 column, 3 mL/min, methanol/water (85:15) solvent system,
tR = 13.2 min] to give the pure compound 3a (15.3 mg, 78%). Pure
crystals of the analogue 3a were obtained after crystallization from
hexane/2-propanol, and they were characterized by an X-ray analysis.
(20R,22S)-1α,25-Dihydroxy-22-methyl-2-methylene-19-nor-
vitamin D3 (3b). The protected vitamin 26b was prepared in 90%
yield by the Wittig−Horner reaction of ketone 23b and the phosphine
oxide 25, performed analogously to the process described above for
the preparation of 26a. The protected vitamin 26b was hydrolyzed as
described for 26a, and the product 3b was further purified by a
normal-phase HPLC [9.4 mm × 25 cm Zorbax silica column, 5 mL/
min, hexane/2-propanol (85:15) solvent system, tR = 8.5 min] and a
reversed-phase HPLC [9.4 mm × 25 cm Zorbax RX-C18 column, 3
mL/min, methanol/water (85:15) solvent system, tR = 15.2 min] to
give the pure compound 3b (13.5 mg, 79%).
(20S,22R)-1α,25-Dihydroxy-22-methyl-2-methylene-19-nor-
vitamin D3 (4a). The protected vitamin 27a was prepared from the
ketone 24a in 90% yield analogously to the isomeric vitamin 26a.
Hydrolysis of silyl protecting groups in 27a was performed as
described for 26a, and the obtained vitamin 4a was purified by a
normal-phase HPLC [9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min,
hexane/2-propanol (85:15) solvent system, tR = 7.9 min] and a
reversed-phase HPLC [9.4 mm × 25 cm Zorbax RX-C18 column, 3
mL/min, methanol/water (85:15) solvent system, tR = 14.7 min] to
give the pure compound 4a (10.3 mg, 77%).
(20S,22S)-1α,25-Dihydroxy-22-methyl-2-methylene-19-nor-
vitamin D3 (4b). The protected vitamin 27b was prepared from the
ketone 24b in 87% yield analogously to 26a. The silyl protecting
groups in 27b were hydrolyzed as described for 26a, and the obtained
vitamin 4b was purified by a normal-phase HPLC [9.4 mm × 25 cm
Zorbax-Sil column, 4 mL/min, hexane/2-propanol (85:15) solvent
system, tR = 7.3 min] and a reversed-phase HPLC [9.4 mm × 25 cm
Zorbax RX-C18 column, 3 mL/min, methanol/water (85:15) solvent
system, tR = 11.7 min] to give the vitamin 4b (6.6 mg, 50%).
(8S,20R)-des-A,B-20-(Cyanodimethylmethyl)-8β-
[(triethylsilyl)oxy]pregnane (28). n-Butyllithium (1.6 M in hexane,
2.4 mL, 3.8 mmol) was added to a solution of diisopropylamine (0.54
mL, 0.384 g, 3.8 mmol) in THF (2 mL) at 0 °C. The resulting mixture
was stirred at 0 °C for 30 min. Then it was cooled to −78 °C and a
solution of the nitrile 9 (0.326 g, 0.973 mmol) in THF (2 mL) was
added. The mixture was stirred at −78 °C for 30 min, and then
iodomethane (1.2 mL, 2.73 g, 19.2 mmol) was added. The reaction
mixture was stirred at −78 °C for 1 h and at room temperature for 1 h,
then quenched with saturated aqueous NH4Cl solution and extracted
with ethyl acetate. The combined organic phases were washed with
brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (elution with 5%, then 10% ethyl
acetate/hexane) to give the product 28 (0.197 g, 56%).
(8S,20S)-des-A,B-20-(Cyanodimethylmethyl)-8β-
[(triethylsilyl)oxy]pregnane (29). Alkylation of the nitrile 10,
performed as described for 9, gave the product that was purified by
column chromatography on silica gel (elution with 10%, then 20%
ethyl acetate/hexane) to furnish the compound 29 in 99% yield.
(8S,20R)-des-A,B-20-(1′,1′-Dimethyl-2′-oxoethyl)-8-
[(triethylsilyl)oxy]pregnane (30). Diisobutylaluminum hydride (1.0
M in dichloromethane, 3.1 mL, 3.1 mmol) was added to a solution of
the nitrile 28 (0.197 g, 0.543 mmol) in dichloromethane (4 mL) at
−10 °C. The reaction mixture was stirred at −10 °C for 1 h. Then it
was quenched with a saturated aqueous potassium sodium tartrate
solution (5 mL). The water phase was extracted with dichloro-
methane. The combined organic layers were washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by column
chromatography on silica gel (elution with 10% ethyl acetate/hexane)
to give the aldehyde 30 (0.15 g, 75%).
(8S,20S)-des-A,B-20-(1′,1′-Dimethyl-2′-oxoethyl)-8-
[(triethylsilyl)oxy]pregnane (31). DIBALH reduction of the nitrile
29, performed as described for 28, gave the aldehyde 31 in 95% yield
as colorless crystals.
(8S,20R)-des-A,B-20-(1′,1′-Dimethyl-3′-ethoxycarbonylallyl)-
8β-[(triethylsilyl)oxy]pregnane (32). n-Butyllithium (1.6 M in
hexane, 5.2 mL, 8.3 mmol) was added to a solution of diisopropyl-
amine (1.2 mL, 0.84 g, 8.3 mmol) in dry THF (2 mL) at 0 °C. After
30 min the mixture was cooled to −10 °C and triethylphosphonoa-
cetate (1.9 mL, 2.13 g, 9.5 mmol) was added. The reaction mixture
was stirred at −10 °C for 30 min, and then a solution of the aldehyde
30 (0.15 g, 0.41 mmol) in anhydrous THF (5 mL + 3 mL) was added
via cannula. The mixture was stirred under argon at −10 °C for 1 h.
Then it was warmed at 37 °C for 2.5 h and then stirred at room
temperature overnight. Dichloromethane was added, and the organic
phase was washed with water, dried (Na2SO4), and concentrated. The
product was purified on a Sep-Pak cartridge (5 g). Elution with ethyl
acetate/hexane (2%) provided 32 (99 mg, 55%).
(8S,20S)-des-A,B-20-(1′,1′-Dimethyl-3′-ethoxycarbonyl-
allyl)-8β-[(triethylsilyl)oxy]pregnane (33). The olefination reac-
tion of the aldehyde 31 gave the product that was purified on a Sep-
Pak cartridge (5 g). Elution with ethyl acetate/hexane (2%, then 3%
and 5%) furnished 33 in 91% yield.
( 8 S , 2 0 R ) - d e s - A , B - 2 0 - ( 1 ′ , 1 ′ - D i m e t h y l - 3 ′ -
ethoxycarbonylpropyl)pregnan-8β-ol (34). A solution of the ester
32 (99 mg, 0.23 mmol) in methanol (5 mL) was hydrogenated in the
presence of 10% palladium on powdered charcoal (10 mg) at room
temperature for 20 h. Filtration of the reaction mixture through a
Waters silica Sep-Pak cartridge (2 g) provided the ester 34 (50.4 mg,
68%).
( 8 S , 2 0 S ) - d e s - A , B - 2 0 - ( 1 ′ , 1 ′ - D i m e t h y l - 3 ′ -
ethoxycarbonylpropyl)pregnan-8β-ol (35). Hydrogenation of the
ester 33, carried out as described above for 32, afforded compound 35
in 95% yield as a colorless oil.
(8S,20R)-des-A,B-22,22-Dimethylcholestane-8β,25-diol (36).
Methylmagnesium bromide (3.0 M solution in diethyl ether, 130
μL, 0.39 mmol) was added to a solution of the ester 34 (50 mg, 0.154
mmol) in anhydrous diethyl ether (3 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 2 h and then at room temperature for
18 h. It was quenched with saturated aqueous NH4Cl solution,
extracted with ethyl acetate, dried (Na2SO4), and concentrated. The
residue was applied on a Waters silica Sep-Pak cartridge (5 g). Elution
with ethyl acetate/hexane (1:1) gave the diol 36 (48 mg, 100%) as
colorless crystals.
(8S,20S)-des-A,B-22,22-Dimethylcholestane-8β,25-diol (37).
Reaction of the ester 35 (24 mg, 0.074 mmol) with the Grignard
reagent, performed analogously to that described above for 34, yielded
the diol 37 (19.2 mg, 84%).
(20R)-des-A,B-22,22-Dimethyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (38). Oxidation of the diol 36 with tetrapropy-
lammonium perruthenate and 4-methylmorpholine oxide and the
subsequent silylation of the formed 25-hydroxy-8-ketone were carried
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out analogously as described for the conversion of 21a into 23a. The
protected ketone 38 was obtained in 86% yield.
and finding the global minimum structures was analogous to that
described by us previously18 and involved the Conformational Search
module.
Crystallographic Studies. Crystal Data for the Diol 21a.
C19H36O2, M = 296.48, T = 100 (1) K, monoclinic, P2(1), a =
11.394 (2) Å, b = 16.535 (3) Å, c = 11.450 (2) Å, α = γ = 90°, β
= 119.26 (3)°, V = 1881.9 (7) Å3, Z = 4, Dx = 1.046 Mg/m3, μ
= 0.497 mm−1, F(000) = 664. Two molecules of the diol 21a
were present in the asymmetric unit.
Crystal Data for the Diol 21b. C19H36O2, M = 296.48, T = 100 (1)
K, monoclinic, C2, a = 26.391 (5) Å, b = 6.0830 (12) Å, c = 12.688 (3)
Å, α = γ = 90°, β = 118.38 (3)°, V = 1792.1 (6) Å3, Z = 4, Dx = 1.099
Mg/m3, μ = 0.522 mm−1, F(000) = 664.
Crystal Data for the Diol 22a. C19H36O2, M = 296.48, T = 100 (1)
K, monoclinic, P2(1), a = 9.6840 (19) Å, b = 19.156 (4) Å, c = 9.6870
(19) Å, α = γ = 90°, β = 91.27 (3)°, V = 1796.6 (6) Å3, Z = 4, Dx =
1.096 Mg/m3, μ = 0.521 mm−1, F(000) = 664.
(20S)-des-A,B-22,22-Dimethyl-25-[(triethylsilyl)oxy]-
cholestan-8-one (39). Oxidation of the diol 37 with tetrapropy-
lammonium perruthenate and 4-methylmorpholine oxide and the
subsequent silylation of the resulting 25-hydroxy-8-ketone were
performed as described for the conversion of 21a into 23a. The
protected ketone 39 was obtained in 78% yield.
(20R)-1α,25-Dihydroxy-22,22-dimethyl-2-methylene-19-
norvitamin D3 (5). The protected vitamin 40 was obtained by the
Wittig−Horner reaction of the ketone 38 and the phosphine oxide 25,
performed analogously to the process described above for preparation
of 26a. The vitamin was purified by a normal-phase HPLC [9.4 mm ×
25 cm Zorbax-Sil column, 4 mL/min, 2-propanol/hexane (0.1:99.9)
solvent system, tR = 3.2 min] to give the pure protected compound 40
(78%).
The protected vitamin 40 was hydrolyzed as described for 26a, and
the product 5 was purified by a normal-phase HPLC [9.4 mm × 25 cm
Zorbax-Sil column, 5 mL/min, hexane/2-propanol (85:15) solvent
system, tR = 7.4 min] and a reversed-phase HPLC [9.4 mm × 25 cm
Zorbax RX-C18 column, 3 mL/min, methanol/water (85:15) solvent
system, tR = 13.3 min] to give the pure compound 5 (79%), mp 154
°C (from 2-propanol/hexane).
Crystal Data for the Vitamin 3a. C31H54O4, M = 490.74, T = 100
(1) K, monoclinic, C2(1), a = 27.039 (5) Å, b = 6.4790 (13) Å, c =
17.412 (4) Å, α = γ = 90°, β = 103.35 (3)°, V = 2967.9 (10) Å3, Z = 4,
Dx = 1.098 Mg/m3, μ = 0.544 mm−1, F(000) = 1088. In addition to
one molecule of the vitamin 3a, there was also one molecule of 2-
propanol in the asymmetric unit.
Crystal Data for the Vitamin 5. C32H56O4, M = 504.77, T = 298
(1) K, monoclinic, C2, a = 27.3382 (16) Å, b = 6.6860 (13) Å, c =
19.221 (10) Å, α = γ = 90°, β = 113.57 (3)°, V = 3320.3 (11) Å3, Z =
4, Dx = 1.041 Mg/m3, μ = 0.513 mm−1, F(000) = 1120. In addition to
one molecule of the vitamin 5, there was also one molecule of 2-
propanol in the asymmetric unit.
Crystal Data for the Vitamin 6. C33H58O4, M = 518.79, T = 100
(1) K, monoclinic, P2(1), a = 7.5780 (15) Å, b = 14.792 (3) Å, c =
14.481 (3) Å, α = γ = 90°, β = 102.22 (3)°, V = 1586.5 (6) Å3, Z = 2,
Dx = 1.086 Mg/m3, μ = 0.532 mm−1, F(000) = 576. In addition to one
molecule of the vitamin 6, there was also one molecule of diethyl ether
in the asymmetric unit.
Structure Determination. The data were collected using a Bruker
AXS Platinum 135 CCD detector controlled with the PROTEUM
software suite (Bruker AXS Inc., Madison, WI). The X-ray source was
Cu K radiation (1.541 78 Å) from a Rigaku RU200 X-ray generator
equipped with Montel optics, operated at 50 kV and 90 mA. The X-ray
data were processed with SAINT, version 7.06A (Bruker AXS Inc.),
and internally scaled with SADABS, version 2005/1 (Bruker AXS
Inc.). The sample was mounted on a glass fiber using vacuum grease
and cooled to 100 K. The intensity data were measured as a series of φ
and ω oscillation frames each of 1° for 5−20 s/frame. The detector
was operated in 512 × 512 mode and was positioned 4.5 cm from the
sample. Cell parameters were determined from a nonlinear least-
squares fit in the range 4.0° < θ < 55°.
The space group was determined by systematic absences and
statistical tests and verified by subsequent refinement. The structure
was solved by direct methods22 and refined by the full-matrix least-
squares methods on F2. The hydrogen atom positions were
determined from difference peaks and ultimately refined by a riding
model with idealized geometry. Non-hydrogen atoms were refined
with anisotropic displacement parameters. The absolute structure was
determined by refinement of the Flack parameter.23
(20S)-1α,25-Dihydroxy-22,22-dimethyl-2-methylene-19-nor-
vitamin D3 (6). The protected vitamin 41 was prepared by the
Wittig−Horner reaction of the ketone 39 and the phosphine oxide 25,
performed analogously to the process described above for the
preparation of 26a. The vitamin was purified by a normal-phase
HPLC [9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min, 2-propanol/
hexane (0.1:99.9) solvent system, tR = 3.4 min] to give the pure
protected compound 41 (91%).
The protected vitamin 41 was hydrolyzed as described for 26a, and
the product 6 was purified by a normal-phase HPLC [9.4 mm × 25 cm
Zorbax-Sil column, 4 mL/min, hexane/2-propanol (85:15) solvent
system, tR = 7.8 min] and a reversed-phase HPLC [9.4 mm × 25 cm
Zorbax RX-C18 column, 3 mL/min, methanol/water (85:15) solvent
system, tR = 15.7 min] to give the pure compound 6 (75%).
Biological Studies. 1. In Vitro Studies. VDR binding, HL-60
differentiation, and 24-hydroxylase transcription assays were
performed as previously described and are shown in the
footnote of Table 1.19,20
2. In Vivo Studies. 2.1. Bone Calcium Mobilization and
Intestinal Calcium Transport. Male, weanling Sprague−Dawley
rats were purchased from Harlan (Indianapolis, IN). The
animals were group housed and placed on diet 11 (0.47% Ca as
CaCO3) + AEK oil for 1 week followed by diet 11 (0.02% Ca)
+ AEK oil for 3 weeks. The rats were then switched to a diet
containing 0.47% Ca21 for 1 week followed by 2 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 concentration of serum calcium determined as a measure of
bone calcium mobilization. Because there is essentially no
calcium in the intestine (the diet contains 0.02% calcium), the
rise in serum calcium is derived from mobilization of calcium
from bone. Further, no calcium is found in urine when a low-
calcium diet is fed; thus, the rise in serum calcium cannot be the
result of increased renal reabsorption. The first 10 cm of the
intestine was also collected for the intestinal calcium transport
analysis using the everted gut sac method.20 Statistical
significance was ascertained by application of the Student’s
“t” test.
Crystallographic data for the structures reported in this paper have
been deposited at the Cambridge Crystallographic Data Centre with
the deposition numbers CCDC 851675 (21a), CCDC 851674 (21b),
CCDC 851677 (22a), CCDC 851676 (3a), CCDC 851679 (5), and
CCDC 851678 (6). These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.
2.2. Molecular Modeling. The molecular mechanism studies were
used to establish the energy-minimized structures of the model
compounds 42a−c. The calculation of optimized geometries and steric
energies was carried out using the algorithm from the MM+
HyperChem (release 8.0) software package (Autodesk, Inc.). MM+
is an all-atom force field based on the MM2 functional form. The
procedure used for generation of the respective side chain conformers
ASSOCIATED CONTENT
■
S
* Supporting Information
Purity criteria of the vitamin D analogues 3a−4b, 5, and 6; their
1H and 13C NMR spectra; figures with dose−response curves
derived from cellular differentiation assay; and spectral data of
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Article
(7) Sicinski, R.; Prahl, J.; Smith, C.; DeLuca, H. F. New 1α,25-
dihydroxy-19-norvitamin D3 compounds of high biological activity:
synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and
2-methylene analogues. J. Med. Chem. 1998, 41, 4662−4674.
(8) Shevde, N. K.; Plum, L. A.; Clagett-Dame, M.; Yamamoto, H.;
Pike, J. W.; DeLuca, H. F. A potent analog of 1α,25-dihydroxyvitamin
D3 selectively induces bone formation. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 13487−13491.
the all synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 608-262-1620. Fax: 608-262-7122. E-mail:
deluca@biochem.wisc.edu.
(9) Sakamaki, Y.; Inaba, Y.; Yoshimoto, N.; Yamamoto, K. Potent
antagonist for the vitamin D receptor: vitamin D analogues with
simple side chain structure. J. Med. Chem. 2010, 53, 5813−5826.
(10) (a) Yamamoto, K.; Yamada, S. Highly diastereoselective
conjugate addition of organocuprate to acyclic E- and Z-enones:
reversal stereoselectivity under kinetic and thermodynamic conditions.
Tetrahedron Lett. 1992, 33, 7521−7524. (b) Yamamoto, K.; Takahashi,
J.; Hamano, K.; Yamada, S. Stereoselective syntheses of (22R)- and
(22S)-22-methyl-1α,25-dihydroxyvitamin D3: active vitamin D3
analogs with restricted side chain conformation. J. Org. Chem. 1993,
58, 2530−2537.
(11) (a) Posner, G. H.; Lee, J. K.; White, C.; Hutchings, R. H.; Dai,
H.; Kachinski, J. L.; Dolan, P.; Kensler, T. W. Antiproliferative hybrid
analogs of the hormone 1α,25-dihydroxyvitamin D3: design, synthesis,
and preliminary biological evaluation. J. Org. Chem. 1997, 62, 3299−
3314. (b) DeLuca, H. F.; Flores, A.; Grzywacz, P.; Plum, L. A.; Clagett-
Dame, M.; Thoden, J. B.; Holden, H. M. Diastereomers of 2-
Methylene-19-nor-22-methyl-1α,25-dihydroxyvitamin D3. U.S. Patent
0,237,557, September 29, 2011.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge Jennifer Vaughan, Jean Prahl, and
Erin Gudmundson for their excellent technical assistance. We
also thank Dr. Mark Anderson for his assistance in recording
NMR spectra. This study made use of the National Magnetic
Resonance Facility 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 Wisconsin, the NIH (Grants RR02781,
RR08438), the NSF (Grants DMB-8415048, OIA-9977486,
BIR-9214394), and the USDA.
ABBREVIATIONS USED
■
1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; VDR, vitamin D
receptor
(12) Fujishima, T.; Zhaopeng, L.; Konno, K.; Nkagawa, K.; Okano,
T.; Yamaguchi, K.; Takayama, H. Highly potent cell differentiation-
including analogues of 1α,25-dihydroxyvitamin D3: synthesis and
biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-
chain modifications. Bioorg. Med. Chem. 2001, 9, 525−535.
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