Synthesis and biological activity of 25-hydroxy-2-methylene-vitamin D3 analogues monohydroxylated in the A-ring

Article abstract of DOI:10.1021/jm500750b

The 20R- and 20S-isomers of 25-hydroxy-2-methylene-vitamin D₃ and 3-desoxy-1α,25-dihydroxy-2-methylene-vitamin D₃ have been synthesized. Two alternative synthetic routes were devised for preparation of the required A-ring synthons, s

Full text of DOI:10.1021/jm500750b

Article
pubs.acs.org/jmc
Synthesis and Biological Activity of 25-Hydroxy-2-methylene-vitamin
D3 Analogues Monohydroxylated in the A‑ring
Izabela K. Sibilska,†,‡ Rafal R. Sicinski,†,‡ Justin T. Ochalek,† Lori A. Plum,† and Hector F. DeLuca*,†
†Department of Biochemistry, University of WisconsinMadison, 433 Babcock Drive, Madison, Wisconsin 53706, United States
‡Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
S
* Supporting Information
ABSTRACT: The 20R- and 20S-isomers of 25-hydroxy-2-methylene-
vitamin D3 and 3-desoxy-1α,25-dihydroxy-2-methylene-vitamin D3
have been synthesized. Two alternative synthetic routes were devised
for preparation of the required A-ring synthons, starting from the chiral
compound derived from the (−)-quinic acid and, alternatively, from
the commercially available achiral precursor, monoprotected 1,4-
cyclohexanedione. The A-ring dienynes were coupled by the
Sonogashira process with the respective C,D-ring fragments, the enol
triflates derived from the protected (20R)- or (20S)-25-hydroxy
Grundmann ketones. All four compounds possessed significant in vivo
activity on bone calcium mobilization and intestinal calcium transport. The presence of a 2-methylene group increased intestinal
calcium transport activity of all four analogues above that of the native hormone, 1α,25-dihydroxyvitamin D3. In contrast, bone
calcium mobilization was equal to that produced by 1α,25-dihydroxyvitamin D3 in compounds having a (20S)-configuration or
diminished to one-tenth that of 1α,25-dihydroxyvitamin D3 in compounds with a (20R)-configuration.
and 3β-hydroxyl groups, it was of interest to investigate
whether 2-methylene-19-norvitamins can be enzymatically 1α-
hydroxylated in kidney by cytochrome P450 (CYP27B1),
analogously to 25-hydroxyvitamin D3. Therefore, we synthe-
sized such compounds (analogues 4 and 5) and examined their
activity.11,12 It has been established that 1-desoxy-2MD (5)
binds to the VDR 1 log less strongly than the natural hormone
and 2MD. Further, in comparison with 2MD, the examined
compound is significantly less active in vivo, which might
indicate its reduced tendency to undergo enzymatic 1α-
hydroxylation. Also, other A-ring monohydroxylated 19-
norvitamin D compounds were obtained in our laboratory,
namely, 3-desoxy-2MD (7) and its (20R)-epimer 6, and it
turned out that they are characterized by significant calcemic
potency.13 High biological activity of compounds discussed
above, as well as an intriguing problem of enzymatic oxidation
at C-1, encouraged us to perform synthesis of the related series
of vitamin D analogues possessing the “natural” 10-exo-
methylene group, which can play a crucial role in the 1-
hydroxylation process. Thus, to elucidate further the effects of
an A-ring possessing two exocyclic methylene groups at C-2
and C-10 on biological in vitro and in vivo activities, we
designed 25-hydroxy-2-methylene-vitamin D3 analogues 8−11
(Figure 2). As a convenient method for their synthesis, we
chose the Sonogashira coupling14 of the A-ring dienynes (12−
14) and CD-ring vinyl triflates (17 and 18), derived from the
INTRODUCTION
■
25-Hydroxyvitamin D3 (25-OH-D3, 1, Figure 1) is the major
metabolite of vitamin D3 circulating in the blood, and
undergoing hydroxylation at C-1 in the kidneys to biologically
active form, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3,
calcitriol, 2].1 This natural hormone is not only responsible
for regulation of mineral homeostasis, but it also has been
recognized for numerous antiproliferative, pro-differentiative,
and immunomodulatory activities.2 Calcitriol exerts its major
actions through the classical genomic pathway, which involves
binding to the vitamin D receptor (VDR)−retinoid X receptor
(RXR) complex and subsequent modulation of gene
expression;3,4 rapid actions of 1α,25-(OH)2D3 have been also
established.5 The unusually broad range of these activities
explains increasing interest in the preparation of its analogues
characterized by more selective biological profiles and
considered as promising therapeutic agents.6 As a consequence
of the efforts of many research groups, directed to the syntheses
of calcitriol analogues with interesting structural modifications,
some of the discovered compounds have been already clinically
used in the treatment of bone diseases, secondary hyper-
parathyroidism, and psoriasis.1,6−8
One of the most interesting structural alterations of the
calcitriol molecule, discovered in our laboratory,9 was the “shift”
of its 10-exomethylene substituent from C-10 to C-2. Such
modification, combined with an inversion of the natural
configuration at C-20, resulted in the analogue, named 2MD
(3), that strongly acts on bone and leads to dramatic increase in
bone density in ovariectomized rats.10 Taking into account the
structure of 2MD, characterized by the presence of allylic 1α-
Received: May 14, 2014
Published: September 15, 2014
© 2014 American Chemical Society
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Figure 1. Chemical structure of 25-hydroxyvitamin D3 (1), 1α,25-dihydroxyvitamin D3 (calcitriol, 2), 2MD (3), and its desoxy analogues 4−7.
Figure 2. Chemical structures of the obtained 25-hydroxy-2-methylene-vitamin D3 analogues 8−11 and the building blocks for their synthesis.
a
Scheme 1
a
(a) Martin sulfurane, CCl4, 91%; (b) CH2N2, Et2O, 99%; (c) DMSO, 125 °C, 98%; (d) DIBALH, CH2Cl2, toluene, 96%; (e) PDC, CH2Cl2, 44%;
(f) n-BuLi, TMSCHN2, 52%.
known Grundmann ketones (15 and 16). Part of this work has
diazomethane. Such 1,3-dipolar cycloaddition proved to be a
regio- and stereospecific process and provided the single
bicyclic product in very high yield. The structure of the adduct
22, resulting from the attack of the diazomethane from the less
hindered side of the molecule, was unequivocally established by
consideration of literature data concerning similar pro-
cesses,17,18 molecular modeling, and inspection of the 1H
NMR spectrum of the product. The most informative were
proton resonances derived from the NN−CH2 fragment of
the formed dihydropyrazole ring: multiplicity of these signals
(doublets of doublets) proves that this methylene group is
attached to a hydrogen-bearing carbon, whereas the magnitudes
been previously communicated.15
RESULTS AND DISCUSSION
■
Chemistry. First, we focused our efforts on the preparation
of the A-ring fragment 12. The synthesis started from the
hydroxy ester 20 (Scheme 1), obtained from the commercially
available (−)-(1S,3R,4S,5R)-quinic acid (19) by the method
elaborated by Sibilska et al.12 The synthetic strategy was based
on the work of Desmaele and Tanier.16 Thus, the tertiary
alcohol was dehydrated using Martin sulfurane reagent, and the
formed α,β-unsaturated ester 21 was subjected to reaction with
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Figure 3. Preferred energy-minimized (HyperChem) conformations of bicyclic diazomethane adduct 22 (a), three hydroxy ketones 29 (b), 30 (c),
1
and 31 (d), and three propargylic alcohols 36 (e), 42 (f), and 48 (g). The most informative H−1H constants are listed; values of calculated
couplings (PC MODEL) are enclosed in parentheses.
of the respective vicinal couplings confirm the ascribed
structure (Figure 3a).
position of the silyl-protected hydroxyl group. A racemic
mixture of trans-3-methyl-4-pivaloyloxy-cyclohexanones 27 and
28 (Scheme 2) was easily obtained in four steps from
commercially available 1,4-cyclohexanedione monoethylene
acetal 26 in 55% overall yield.19 These compounds were
subjected to enantioselective α-aminoxylation using the
procedure described by Hayashi et al.20 Thus, enantiomers
27 and 28 were subjected to the reaction with nitrosobenzene
carried out in the presence of a catalytic amount of L-proline,
and three main products, 29, 30, and 31, were isolated by
column chromatography in 23%, 19%, and 22% yield,
respectively. The synthetically advantageous outcome of this
The subsequent thermolysis of the bicyclic compound 22 led
to efficient extrusion of nitrogen and formation of unsaturated
ester 23. Its reduction with DIBALH furnished the allylic
alcohol 24, which was oxidized with PDC to the α,β-
unsaturated aldehyde 25. Treatment of this product with
(trimethylsilyl)diazomethane provided the desired A-ring
building block 12.
Another synthetic path to the A-ring precursors of 2-
methylene-vitamin D analogues was also elaborated, involving
total synthesis of both fragments 13 and 14, differing in the
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a
Scheme 2
a
(a) PhNO, L-Pro, CHCl3, 29/30/31 = 1.14:1:1.17, 64%; (b) TBDPSCl, AgNO3, DMF; 32 97%, 38 95%, 44 73%; (c) Ph3P+CH3Br−, n-BuLi, THF,
33 97%, 39 96%, 45 79%; (d) DIBALH, CH2Cl2, toluene; 34 96%, 40 96%, 46 97%; (e) Dess−Martin periodinane, CH2Cl2; 35 95%, 41 96%, 47
95%; (f) TMSCCH, n-BuLi, THF; 36 96%, 42 94%, 48 99%; (g) MsCl, TEA, CH2Cl2; 37 38%, 43 97%; (h) Martin sulfurane, CCl4; 43 98%, 49/
50 = 2:1, 81%; (i) K2CO3, THF, MeOH; 13 96%, 14 60%.
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a
Scheme 3
a
(a) LDA, PhNTf2, THF; 18 82%; (b) (PPh3)2Pd(OAc)2, CuI, Et2NH, DMF; 51 87%, 52 93%, 53 74%, 54 58%; (c) H2, Lindlar cat., quinoline,
hexane; 55 25%, 56 80%, 57 80%, 58 80%; (d) 65 °C, hexane; 59 76%, 60 87%, 61 96%, 62 64%; (e) TBAF, THF; 8 29%, 9 23%, 10 33%, 11 56%.
process, directly providing the required α-hydroxylated
products, was rather unexpected. Absence of the usual α-
aminoxylated products is obviously a consequence of the
different reaction conditions. The procedures described in the
literature20 used an excess of carbonyl compound to nitro-
sobenzene (from 1.2 to as much as 10 equiv) whereas we
employed 1.75 equiv of nitrosobenzene and higher catalyst
loading. The structural assignments of the obtained hydroxy
ketones was based on molecular modeling and careful analysis
of their 1H NMR spectra as well as assumption that an
introduced secondary hydroxyl has the R-configuration.20 Thus,
for example, multiplicity (doublet of dublets) of the signal
derived from the proton attached to the carbinol C-2 in
hydroxy ketones 29 and 30 indicates that the newly introduced
hydroxyl and the methyl substituent are located in these
products on the opposite site of the cyclohexanone ring,
whereas a single splitting of 2-H signal in the spectrum of
isomeric compound 31 confirms that hydroxyl is located
between carbonyl and methyl group. Configurational assign-
ment of methyl and pivaloyloxy substituents is facilitated by the
fact that they remain in the trans-relationship. Molecular
mechanics calculations have shown that, for all three products,
their conformations possessing equatorially oriented hydroxyl
(Figure 3b−d) are strongly preferred (steric energies lower by
at least 1.3 kcal/mol). These results were supported by
comparison of selected vicinal coupling constants found in the
1H NMR spectra of the hydroxy ketones with the data reported
by Anet for cyclohexanol analogues (Jax,ax = 11.1 Hz, Jeq,eq = 2.7
Hz).21
Three hydroxy ketones 29−31 were then separately
transformed to the respective cyclohexanones 35, 41, and 47,
employing the following four-step reaction sequence: silylation
with tert-butyldiphenyl silyl chloride, Wittig methylenation with
an ylide generated from methyltriphenylphosphonium bromide,
reductive removal of the pivalate through treatment with
DIBALH, and finally oxidation of cyclohexanol derivatives with
Dess−Martin periodinane. The obtained cyclohexanones were
then subjected to reaction with lithium (trimethylsilyl)acetylide,
which provided in each case the single product. The structures
of the formed propargylic alcohols 36 and 48 were proposed by
assuming an anion addition at the less hindered face of carbonyl
and in the case of 42 by an attack of nucleophile from the side
opposite to the bulky OTPS group. Although the analysis of the
1H NMR spectra (Figure 3e−g) of products provided some
support for these structural assignments, the important proof
for the ascribed configurations of the tertiary hydroxyls was
obtained from the results of the subsequent dehydration
processes. Thus, a facile although not very efficient conversion
of tertiary alcohol 36 to the desired dienyne product 37 was
achieved by its treatment with methanesulfonyl chloride-
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a
b
c
Table 1. Relative VDR Binding Activities, HL-60 Differentiating Activities, and Transcriptional Activities of the 25-OH-D3
(1), Vitamin D Hormone (2), 2MD (3), and the Vitamin D Analogues 4−11
a
Competitive binding of 1α,25-(OH)2D3 (2) 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 (refs 11−13; Figures
12 and 13 in Supporting Information) and represent the inhibition constant when radiolabeled 1α,25-(OH)2D3 is present at 1 nM and a Kd of 0.2
b
nM is used. The numbers shown in the table are expressed as the average ratio of the 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 (2) 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 (refs 11−13; Figures 14 and 15 in Supporting Information) and represent the analogue
concentration capable of inducing 50% maturation. The numbers shown in the table are expressed as the average ratio of the 1α,25-(OH)2D3 ED50
c
to the ED50 for 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 (refs 11−13; Figures 16 and 17 in Supporting Information) and represent the analogue
concentration capable of increasing the luciferase activity by 50%. The numbers shown in the table are expressed as the average ratio of the 1α,25-
(OH)2D3 ED50 to the ED50 for the analogue.
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Figure 4. Bone calcium mobilization of 1α,25-(OH)2D3 (2) and the synthesized 25-hydroxy-2-methylene-vitamin D3 analogues 8 and 10. Asterisks
indicate p < 0.05.
Figure 5. Bone calcium mobilization of 1α,25-(OH)2D3 (2) and the synthesized 25-hydroxy-2-methylene-vitamin D3 analogues 9 and 11. Asterisks
indicate p < 0.05.
triethylamine. In contrast, the same procedure applied to the
epimeric propargylic alcohol 42 resulted in its smooth and
quantitative conversion into the anti-Zaitsev elimination
product 43. An almost identical result of the process was
observed with Martin sulfurane as a dehydrating agent, whereas
very poor yield of 43 was obtained with Burgess reagent (syn-
elimination). Also in the case of alcohol 48, its dehydration
with Martin sulfurane reagent proved to be an efficient process;
however, the prevailing product was the undesired dienyne 49
with the newly formed trisubstituted double bond, whereas the
Zaitsev product 50 represented the minor component of the
reaction mixture. Attempted dehydration of the alcohol 48,
performed according to the procedure used for 36, yielded a
complex mixture of products with the dienyne 49 as the main
component. Removal of the trimethylsilyl group in the dienyne
37 provided in quantitative yield the A-ring building block 13
suitable for synthesis of the target 25-hydroxy-2-methylene-
vitamin D3 compounds 8 and 9. Desilylation of the mixture of
49 and 50 allowed us to separate the isomeric mixture of
dienynes; the obtained compound 14 also constituted an useful
building fragment that could be used for a construction of 3-
desoxy-1α,25-dihydroxy-2-methylene-vitamin D3 (10) and its
(20S)-epimer 11.
The enol triflate 18, representing a C,D-ring/side chain
fragment, was obtained from the protected (20S)-25-hydroxy
Grundmann ketone 16,9 employing the method used by De
Clercq for the preparation of the epimeric enol triflate 17 from
the hydrindanone 15.22 Thus, treatment of the kinetic enolate,
generated from the ketone 16 by addition of the LDA at −78
°C, with N-phenyltriflimide afforded 18 in a good yield
(Scheme 3). Then, the enol triflates 17 and 18 were subjected
to Sonogashira coupling13 with the dienynes 13 and 12,
respectively. The coupling reactions were carried out in the
presence of bis(triphenylphosphine)palladium(II) acetate−
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Figure 6. Intestinal calcium transport of 1α,25-(OH)2D3 (2) and the synthesized 25-hydroxy-2-methylene-vitamin D3 analogues 8 and 10. Asterisks
indicate p < 0.05.
Figure 7. Intestinal calcium transport of 1α,25-(OH)2D3 (2) and the synthesized 25-hydroxy-2-methylene-vitamin D3 analogues 9 and 11. Asterisks
indicate p < 0.05.
copper(I) iodide catalyst and diethylamine, and they yielded
the expected trienynes 51 and 52, which were further
hydrogenated in the presence of Lindlar catalyst and quinoline
to the respective previtamin D analogues 55 and 56. After
purification by preparative TLC they were subjected to the
thermal reaction in hexane, and the protected vitamin D
compounds 59 and 60 were isolated by HPLC. Finally,
hydroxyl deprotection with tetrabutylammonium fluoride
provided the target 25-hydroxy-2-methylene-vitamin D3 (8)
and its (20S)-epimer 9. In their proton NMR spectra the
corresponding signals, derived from A-ring and olefinic protons,
were characterized by virtually identical chemical shifts. These
data exclude the possibility that vitamins 8 and 9 can have
different configuration at C-3 and, consequently, prove that
their precursors, dienynes 12 and 13, must have had the same
configuration of the hydroxyl group.
Using as substrates the dienyne 14 and both vinyl triflates 17
and 18, (20R)- and (20S)-3-desoxy-2-methylene-1α,25-dihy-
droxyvitamins (10 and 11), respectively, were obtained via a
four-step reaction sequence, applying analogous synthetic
transformations as described above for the synthesis of 8 and 9.
Biological Activity. We evaluated in vitro and in vivo
biological activities of the previously (compounds 4−7) and
newly (compounds 8−11) synthesized vitamin D analogues. In
comparison with 2MD, in vitro activities of all tested analogues
were significantly lower (Table 1). The same was true when the
activities were compared with those of the natural hormone 1,
with the exception of 1α-hydroxylated compounds. As
expected, analogues missing the 1α-hydroxyl showed lower
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binding activity then those hydroxylated at the C-1 position.
This was reflected also in the HL-60 differentiation activity and
in the induction of the 24-hydroxylase. Largely the addition of
the 10-exomethylene group improved in vitro activity of (20S)-
1-desoxy compounds, whereas in the (20R)-series only VDR
binding ability was increased. No clear effect of the presence of
an additional exomethylene group was found for 3-desoxy
analogues. In the case of in vivo assays (Figures 4−7), all
analogues possessed significant activity. The presence of the 2-
exomethylene group improved intestinal calcium transport
above that of the native hormone, 1α,25-(OH)2D3 but
decreased by a factor of 10 bone calcium mobilization of the
(20R) series. A similar reduction might also be true for the
(20S) series. However, this is based on historical data.9,11−13,23
Enzymatic Hydroxylation. Since 1α-desoxy compounds
with two methylene groups showed higher in vivo activities
compared with their monosubstituted counterparts, we could
assume that the 10-exomethylene group facilitates the
enzymatic 1α-hydroxylation. Examination of this process
using compounds 4, 8, and 9 showed that CYP27B1 was able
to catalyze 1α-hydroxylation of all three vitamins, but the
reaction rate was much lower than that of 25-OH-D3 as
substrate (Table 2). This may be due to the presence of the 2-
Taking into account these results, the ability of 1α-desoxy
analogues to act as a prodrug is plausible since its 1α-
hydroxylation could occur in a regulated manner and the half-
life of the compound might be extended. However, double
substitution significantly reduced the rate of the process, and by
that, the usefulness of these compounds might be limited.
Further studies on the natural hormone analogues with A-
ring exomethylene substituents are underway and will be
reported in due course.
EXPERIMENTAL SECTION
■
Chemistry. Optical rotations were measured in chloroform using a
PerkinElmer models 241 and 343 polarimeters at 22 °C. Ultraviolet
(UV) absorption spectra were obtained on a Shimadzu UV-1800 UV
spectrophotometer in 100% EtOH. All nuclear magnetic resonance
spectra were recorded in deuteriochloroform using Varian Unity plus
(200 MHz), Bruker DMX-400 (400 MHz), and Bruker DMX-500
(500 MHz). COSY spectra and spin decoupling, as well as NOE,
DEPT 90, and DEPT 135, experiments were used to assign particular
signals in the 1H and 13C NMR spectra. 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), and multiplet (m). High resolution mass spectra were
registered on LCT (TOF) or Mass Quattro LC spectrometers. High-
performance liquid chromatography (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 detectors. Solvents were dried and
distilled following standard procedures.
Table 2. Enzymatic 1α-Hydroxylation of Vitamin D
Compounds 1, 4, and 8 with CYP27B1
enzymatic rate constant,
kcat (min−1
cat. efficiency, kcat/KM
(M−1 s−1
compd
)
KM (μM)
)
The purity of final compounds was determined by HPLC, and they
were judged at least 99% pure. Two HPLC columns (9.4 mm × 25 cm
Zorbax-Sil and 9.4 mm × 25 cm Zorbax Eclipse XDB-C18) were used
as indicated in Table 3 (Supporting Information). The purity and
identity of the synthesized vitamins were additionally confirmed by
1
4
8
9
2.47 0.05
0.72 0.14
0.43 0.02
0.53 0.05
0.48 0.03
0.44 0.07
0.42 0.02
0.45 0.04
8.5 × 104
2.7 × 104
1.7 × 104
1.9 × 104
1
inspection of their H NMR and high-resolution mass spectra.
The known vinyl triflate 1724 was obtained according to the
procedure of De Clercq et al.;22 an analogous method was used for the
preparation of the isomeric (20S)-triflate 18 (described below) from
the protected (20S)-25-hydroxy Grundmann ketone 16.9 The starting
hydroxy ester 20 was prepared from (−)-quinic acid (19),11,12 whereas
cyclohexanone compounds 27 and 28 were obtained from commercial
1,4-cyclohexanedione monoethylene ketal (26) according to the
published procedures.19,24
(20S)-25-[(Triethylsilyl)oxy]-8-trifluoromethanesulfonyloxy-des-
A,B-cholest-8-ene (18). A solution of the ketone 16 (28.5 mg, 72.19
μmol) in anhydrous THF (350 μL) was slowly added to the solution
of LDA (2.0 M in THF/heptane/ethylbenzene; 40 μL, 80 μmol) in
dry THF (100 μL) at −78 °C under argon. Then a solution of N-
phenyltriflimide (28.3 mg, 79.27 μmol) in dry THF (100 μL) was
added. After 2 h, the cooling bath was removed, and the reaction
mixture was allowed to warm to room temperature. Stirring was
continued for 30 min, and water was added. The mixture was extracted
with hexane, dried over MgSO4, and concentrated. The residue was
applied on a silica Sep-Pak cartridge and eluted with hexane to afford
the enol triflate 18 (17.2 mg, 82% considering recovered substrate)
and unreacted ketone 16 (12 mg).
(5R)-5-[(tert-Butyldimethylsilyl)oxy]-4-methylene-cyclohex-1-ene-
carboxylic Acid Methyl Ester (21). To a solution of hydroxy ester 20
(162.5 mg, 541 μmol) in anhydrous carbon tetrachloride (5.1 mL) at
room temperature under argon was added solution of bis[α,α-
bis(trifluoromethyl)benzenemethanolato]diphenylsulfur (546 mg, 812
μmol) in anhydrous carbon tetrachloride (1.8 mL). Reaction was
stirred for 2 h, water was added, and the mixture was extracted with
methylene chloride, dried (Na2SO4), and concentrated. The resulting
product was applied on a silica Sep-Pak cartridge and eluted with
hexane/diethyl ether (97:3) to give the desired product contaminated
with dehydrating agent. Further purification was performed on
preparative TLC plates (Silica Gel 60F254, 20 cm × 20 cm, 250
methylene group, which could shift the juxtaposition of the A-
ring relative to the enzyme active site, making it less than ideal
to form the necessary bonding interactions for catalysis. The
side chain, on the other hand, probably played a minor role
judged by the similar kcat values of 8 and 9. Removal of the 10-
methylene moiety seemed to have offset the impact of the 2-
exomethylene group, slightly increasing the turnover rate of the
analogue 4. All the structural variations did not change the KMs
of CYP27B1 for these compounds.
CONLUSIONS
■
We report here an extension of our structure−activity studies
on the A-ring modified vitamin D analogues. 25-Hydroxyvita-
min D3 compounds possessing a 2-exomethylene group and a
single hydroxyl substituent located at C-1 or C-3 were designed
and synthesized by Sonogashira coupling of the vinyl triflates,
derived from the Grundmann ketones, with the respective A-
ring fragments prepared by different synthetic pathways.
The in vitro potencies and in vivo activities of the synthesized
2-methylene-25-hydroxyvitamins D3 8−11 not only proved the
importance of the 1α-hydroxyl but also showed that the 10-
exomethylene moiety is responsible for sustaining some of the
biological activity of the compounds. Also earlier indication of
easier hydroxylation of 2,10-dimethylene vitamins 8 and 9 was
not proven by the enzymatic CYP27B1 oxidation experiments.
The rate of this process was significantly lower and the
hydroxylation proceeded even more slowly for disubstituted
vitamins.
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nm), developed in hexane/diethyl ether (92:8), to give an unsaturated
ester 21 (139 mg, 91%) as a colorless oil.
mL) under argon at room temperature; white precipitate formed
immediately. The reaction was stirred for 17 h and then quenched
with water. The mixture was extracted with hexane, dried (MgSO4),
and concentrated. Purification by column chromatography on silica
(1% → 4% diethyl ether/hexane) gave protected α-hydroxy ketone 32
(1.2 g, 97%).
(2R,4S,5S)-2-[(tert-Butyldiphenylsilyl)oxy]-5-methyl-4-pivaloy-
loxy-cyclohexanone (38). Silylation of the hydroxy ketone 30 was
performed according to procedure described above. The crude
product was purified by column chromatography on silica. Elution
with hexane/diethyl ether (98:2) gave an oily protected compound 38
in 95% yield.
(3aR,6R,7aR)-6-[(tert-Butyldimethylsilyl)oxy]-5-methylene-
3,3a,4,5,6,7-hexahydro-indazole-7a-carboxylic Acid Methyl Ester
(22). Solution of diazomethane in diethyl ether [5.5 mL; prepared
according to the procedure of Arndt]25 was added to the solution of
unsaturated ester 21 (375 mg, 1.33 mmol) in anhydrous diethyl ether
(2 mL) at room temperature. Reaction mixture was protected from
light and stirred overnight. Solvent was evaporated; the residue was
dissolved in hexane, applied on a silica Sep-Pak cartridge, and eluted
with hexane/ethyl acetate (96:4) to give bicyclic compound 22 (423
mg, 99%) as a colorless oil.
(5R)-5-[(tert-Butyldimethylsilyl)oxy]-2-methyl-4-methylene-cyclo-
hex-1-enecarboxylic Acid Methyl Ester (23). A solution of compound
22 (310 mg, 955 μmol) in freshly distilled anhydrous DMSO (16 mL)
was stirred at 125 °C for 5 h under argon. Heating bath was removed,
water added and the mixture was extracted with hexane, dried
(Na2SO4), and concentrated. Separation by column chromatography
on silica using hexane/diethyl ether (97:3) gave unsaturated ester 23
(239 mg, 84%; 98% considering recovered substrate) and unchanged
adduct 22 (43 mg).
(5′R)-5′-[(tert-Butyldimethylsilyl)oxy]-2′-methyl-4′-methylene-cy-
clohex-1′-enyl)-methanol (24). Diisobutylaluminum hydride (1.0 M
in toluene; 1.33 mL, 1.33 mmol) was slowly added to a stirred solution
of the ester 23 (89 mg, 300 μmol) in toluene/methylene chloride (2:1;
8 mL) at −78 °C under argon. Stirring was continued at −78 °C for 1
h. The mixture was quenched with potassium−sodium tartrate (2 N, 4
mL), aqueous HCl (2 N, 4 mL), and H2O and extracted with ethyl
acetate. The combined organic layers were washed with brine, dried
(MgSO4), and concentrated. The residue was applied on a silica Sep-
Pak cartridge and eluted with hexane/ethyl acetate (95:5) to afford the
alcohol 24 (78 mg, 96%).
(2R,3R,4S)-2-[(tert-Butyldiphenylsilyl)oxy]-3-methyl-4-pivaloy-
loxy-cyclohexanone (44). Silylation of the hydroxy ketone 31 with t-
BDPSCl was performed as described for 29, and it was completed after
30 h. Purification by column chromatography on silica gave protected
compound 44 in 73% yield.
(2R,4R,5R)-2-[(tert-Butyldiphenylsilyl)oxy]-5-methyl-1-methylene-
4-pivaloyloxy-cyclohexane (33). n-Buthyllithium (1.6 M in hexanes;
430 μL, 688 μmol) was added dropwise to methyltriphenylphospho-
nium bromide (122 mg, 342 μmol) in anhydrous THF (1.4 mL) at 0
°C. After 15 min, another portion of phosphonium salt (122 mg, 342
μmol) was added, and the solution was stirred at 0 °C for 10 min and
at room temperature for 20 min. The orange-red mixture was then
cooled to −78 °C and siphoned to the precooled (−78 °C) solution of
the ketone 32 (160 mg, 344 μmol) in anhydrous THF (350 μL). The
reaction mixture was stirred at −78 °C for 4 h and then at room
temperature for 1 h. The mixture was poured into brine and extracted
with hexane. The organic layer was dried (MgSO4) and evaporated to
give an orange oily residue, which was applied on a Waters silica Sep-
Pak cartridge. Elution with hexane/diethyl ether (97:3) gave pure
olefinic compound 33 (153 mg, 97%) as a colorless oil.
(5R)-5-[(tert-Butyldimethylsilyl)oxy]-2-methyl-4-methylene-cyclo-
hex-1-enecarbaldehyde (25). The mixture of alcohol 24 (77 mg, 287
μmol), and pyridinium dichromate (347 mg, 1.61 mmol) in anhydrous
methylene chloride (4.6 mL) was stirred vigorously at room
temperature for 16 h under argon. Then the reaction mixture was
filtered through a pad of Celite (washed with methylene chloride), and
solvent was removed under reduced pressure. The residue was applied
on a silica Sep-Pak cartridge and eluted with hexane/ethyl acetate
(98:2) to yield the aldehyde 25 (33.5 mg, 44%) as a colorless oil.
(5R)-5-[(tert-Butyldimethylsilyl)oxy]-1-ethynyl-2-methyl-4-meth-
ylene-cyclohexene (12). n-Butyllithium (1.6 M in hexanes; 101 μL,
161.71 μmol) was added to a solution of (trimethylsilyl)diazomethane
(2.0 M in hexane, 76 μL, 151.5 μmol) in anhydrous THF (150 μL) at
−78 °C under argon, and a solution of aldehyde 25 (33.5 mg, 125.7
μmol) in dry THF (100 μL +100 μL) was added via cannula. The
cooling bath was removed after 1 h, and stirring was continued at
room temperature overnight. Water was added, and the mixture was
extracted with hexane, dried (Na2SO4), and concentrated. The residue
was applied on a silica Sep-Pak cartridge and eluted with hexane to
afford the dienyne 12 (16 mg, 52%) and the recovered substrate (1.5
mg).
(2R,4R,5R)- and (2R,3R,4S)-2-Hydroxy-5-methyl-4-pivaloyloxy-
cyclohexanone (29, 31) and (2R,4S,5S)-2-Hydroxy-5-methyl-4-
pivaloyloxy-cyclohexanone (30). To a stirred solution of racemic
mixture of 27 and 28 (551 mg, 2.59 mmol) and L-proline (143.6 mg,
1.25 mmol) in chloroform (5 mL), a solution of nitrosobenzene (485
mg, 4.53 mmol) in chloroform (10 mL) was slowly added by a syringe
pump at 4 °C over 24 h. Then the mixture was stirred at room
temperature for additional 2 h. Reaction was quenched with brine and
extracted with ethyl acetate, dried (MgSO4), and concentrated. The
residue was separated by column chromatography on silica using
hexane/ethyl acetate (9:1) to give the isomeric α-hydroxy ketones (in
the elution order) 29, 30, and 31 (ratio of 1.14:1:1.17; 380 mg, 64%).
The compounds were sufficiently pure to be used for the next
synthetic steps without further purification.
(2R,4S,5S)-2-[(tert-Butylodiphenylsilyl)oxy]-5-methyl-1-methyl-
ene-4-pivaloyloxy-cyclohexane (39). Wittig reaction of the ketone
38, performed according to procedure described above, gave an oily
olefinic compound 39 in 96% yield.
(2R,3R,4S)-2-[(tert-Butyldiphenylsilyl)oxy]-3-methyl-1-methylene-
4-pivaloyloxy-cyclohexane (45). Wittig reaction of the ketone 44 was
performed as described for 32. The product was purified on a silica
Sep-Pak cartridge. Elution with hexane/diethyl ether (98:2) gave an
oily olefinic compound 45 in 79% yield.
(1R,2R,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
cyclohexanol (34). Diisobutylaluminum hydride (1.0 M in toluene;
6.12 mL, 6.12 mmol) was slowly added to a stirred solution of the
ester 33 (710 mg, 1.53 mmol) in toluene/methylene chloride (2:1, 45
mL) at −78 °C under argon. Stirring was continued at −78 °C for 1 h
and at −40 °C for 30 min. The mixture was quenched with
potassium−sodium tartrate (2 N, 4 mL), aqueous HCl (2 N, 4 mL),
and H2O (14 mL) and extracted with ethyl acetate. The organic phase
was washed with brine, dried (MgSO4), and evaporated. The residue
was purified by column chromatography on silica using hexane/ethyl
acetate (9:1) to give alcohol 34 (558 mg, 96%).
(1S,2S,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
cyclohexanol (40). Reduction of the ester 39 with diisobutylaluminum
hydride, performed according to the procedure described above, gave
the alcohol 40 in 96% yield.
(1S,2R,3R)-3-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
cyclohexanol (46). Reduction of the ester 45 with diisobutylaluminum
hydride, performed as described for 33, gave the alcohol 46 in 97%
yield.
(2R,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-cy-
clohexanone (35). Dess−Martin periodinane (529 mg, 1.25 mmol)
was added to a stirred solution of alcohol 34 (396 mg, 1.04 mmol) in
anhydrous methylene chloride (20 mL) at room temperature under
argon. Stirring was continued for 1 h and saturated NaHCO3 was
slowly added. The mixture was extracted with methylene chloride,
dried (MgSO4), and concentrated. The residue was applied on a silica
Sep-Pak cartridge and eluted with hexane/diethyl ether (98:2) to
afford ketone 35 (389 mg, 95%) as a colorless oil.
(2R,4R,5R)-2-[(tert-Butyldiphenylsilyl)oxy]-5-methyl-4-pivaloy-
loxy-cyclohexanone (32). t-Butylchlorodiphenylsilane (1.16 mL, 4.53
mmol) was added to a solution of α-hydroxy ketone 29 (765 mg, 3.6
mmol) and silver nitrate (1.72 g, 10.14 mmol) in anhydrous DMF (16
(2S,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-cy-
klohexanone (41). Oxidation of the alcohol 40 with Dess−Martin
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Journal of Medicinal Chemistry
Article
periodinane, performed according to procedure described above, gave
the oily ketone 41 in 96% yield.
(2S,3R)-3-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-cy-
clohexanone (47). Oxidation of the alcohol 46 with Dess−Martin
periodinane, performed as described for 34, gave the oily ketone 47 in
95% yield.
(5R)-5-[(tert-Butyldiphenylsilyl)oxy]-1-ethynyl-2-methyl-4-meth-
ylene-cyclohexane (13). Anhydrous potassium carbonate (34.5 mg,
250 μmol) was added to a stirred solution of protected acetylene 37
(58 mg, 126 μmol) in anhydrous THF/MeOH (1:1, 6 mL) at room
temperature under argon. The stirring was continued for 19 h, and
then water and saturated NH4Cl were added; the mixture was
extracted with hexane, dried (MgSO4), and concentrated. The residue
was applied on a silica Sep-Pak cartridge and eluted with hexane to
afford compound 13 (47 mg, 96%).
(3S)-3-[(tert-Butyldiphenylsilyl)oxy]-1-ethynyl-2-methyl-4-methyl-
ene-cyclohexane (14). Treatment of a mixture of protected enynes 49
and 50 (ratio of 1:2) with potassium carbonate in anhydrous THF/
MeOH, performed according to the procedure described above, gave
products, which were separated by HPLC (9.4 mm × 25 cm Zorbax-
Sil column, 4 mL/min) using hexane as an eluent. Pure dienyne 14
(60% yield from 50) was collected at RV = 22 mL.
3β-[(tert-Butyldiphenylsilyl)oxy]-2-methylene-25-[(triethylsilyl)-
oxy]-9,10-secocholesta-5(10),8-dien-6-yne (51). To a solution of the
dienyne 13 (47 mg, 121.6 μmol) and vinyl triflate 17 (50 mg, 95
μmol) in anhydrous DMF (0.95 mL) were added CuI (2.7 mg, 14.2
μmol), (PPh3)2Pd(OAc)2 (2.0 mg, 2.7 μmol), and Et2NH (945 μL) at
room temperature under argon. After 20 min, the reaction mixture
turned deep reddish-brown. Water was added, and the mixture was
extracted with hexane, dried (MgSO4), and concentrated. The residue
was applied on a silica Sep-Pak cartridge and eluted with hexane to
afford compound 51 (63 mg, 87%).
(20S)-3β-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne (52). Sono-
gashira coupling of the dienyne 12 and vinyl triflate 18, performed
according to the procedure described above, gave the trienyne 52 in
93% yield.
3-Desoxy-1α-[(tert-butyldiphenylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne (53). Sono-
gashira coupling of the dienyne 14 and vinyl triflate 17, performed as
described for preparation of 51, gave the trienyne 53 in 74% yield.
(20S)-3-Desoxy-1α-[(tert-butyldiphenylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne (54). Sono-
gashira coupling of the dienyne 14 and vinyl triflate 18, performed as
described for preparation of 51, gave the trienyne 54 in 58% yield.
2-Methylene-25-[(triethylsilyl)oxy]-vitamin D3 tert-Butyldiphenyl-
silyl Ether (59). To a solution of the trienyne 51 (63 mg, 82.5 μmol) in
hexane (7.6 mL) and quinoline (13.5 μL) was added Lindlar catalyst
(189 mg), and the mixture was stirred at room temperature under a
positive pressure of hydrogen. Lindlar catalyst was added four times
during 8 h (in 30 mg portions), and then the mixture was applied on a
silica Sep-Pak cartridge and eluted with hexane/ether (99.7:0.3) to
yield a mixture of previtamin D product and unreacted substrate.
Further purification by preparative TLC (Silica Gel 60F254, 20 cm × 20
cm, 250 μm layer) with hexane/ether (98:2) gave the previtamin 55
(12.7 mg, 20%; 25% based on recovered substrate) and 12.1 mg of the
unchanged trienyne. Previtamin compound was then dissolved in
anhydrous hexane (6 mL) and stirred at 65 °C for 5 h and at 40 °C
overnight under argon. Solvent was evaporated, and the residue was
purified by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min)
using hexane/ethyl acetate (99:1) solvent system. Pure protected
vitamin 59 (9.7 mg, 76%) was eluted at RV 15.5 mL.
(1S,2R,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
1-[(trimethylsilanyl) ethynyl]-cyclohexanol (36). A solution of n-BuLi
(1.6 M in hexanes, 325 μL, 520 μmol) was added dropwise to a
solution of trimethylsilylacetylene (76 μL, 537 μmol) in anhydrous
THF (2 mL) under argon at 0 °C. The solution was stirred for 30 min
and cooled to −78 °C; then a precooled (−78 °C) solution of the
ketone 35 (158 mg, 417.3 μmol) in dry THF (2 mL) was slowly
added. After 15 min, the mixture was warmed to 0 °C and stirred for
additional 30 min. Then it was quenched with water, extracted with
ether, dried (MgSO4), and concentrated. The resulting product was
applied on a silica Sep-Pak cartridge and eluted with hexane/ethyl
acetate (98:2) to afford alcohol 36 (190 mg, 96%) as a colorless oil.
(1S,2S,5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
1-[(trimethylsilanyl) ethynyl]-cyclohexanol (42). Reaction of the
ketone 41 with an anion of trimethylsilylacetylene, performed
according to procedure described above, gave an oily alcohol 42 in
94% yield.
(1R,2S,3R)-3-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-
1-[(trimethylsilanyl) ethynyl]-cyclohexanol (48). Reaction of the
ketone 47 with an anion of trimethylsilylacetylene, performed as
described for 35, gave an oily alcohol 48 in 99% yield.
(5R)-5-[(tert-Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-1-
[(trimethylsilanyl)ethynyl]-cyclohexene (37). Mesyl chloride (170 μL,
2.19 mmol) was slowly added to a stirred solution of alcohol 36 (174
mg, 364 μmol) and TEA (310 μL, 2.21 mmol) in dry methylene
chloride (6 mL) at room temperature under argon. The reaction was
quenched after 1 h with 5% HCl and extracted with methylene
chloride. The organic phase was washed with saturated NaHCO3,
dried (MgSO4), and concentrated. The resulting product was applied
on a silica Sep-Pak cartridge and eluted with hexane/diethyl ether
(99:1) to afford dienyne 37 (64 mg, 38%) as a colorless oil.
(3R,6S)-3-[(tert-Butyldiphenylsilyl)oxy]-6-methyl-4-methylene-1-
[(trimethylsilanyl)ethynyl]-cyklohexene (43). For method a, dehy-
dration of the alcohol 42, performed according to procedure described
above, gave an oily dienyne 43 in 97% yield.
For method b, to a solution of the alcohol 42 (21 mg, 44 μmol) in
anhydrous carbon tetrachloride (0.5 mL) at room temperature under
argon was added solution of bis[α,α-bis(trifluoromethyl)-
benzenemethanolato]diphenylsulfur (44 mg, 65.4 μmol) in anhydrous
carbon tetrachloride (2 mL). Reaction was stirred for 6 h, and during
this time dehydrating reagent was added twice (in ca. 15 mg portions).
Water was added, and the mixture was extracted with methylene
chloride, dried (Na2SO4), and concentrated. The resulting product was
applied on a silica Sep-Pak cartridge and eluted with hexane/diethyl
ether (98:2) to afford the product contaminated with dehydrating
agent. Further purification was performed on preparative TLC plate
(Silica Gel 60F254, 20 cm × 20 cm, 250 μm), developed in hexane/
diethyl ether (92:8), to give the dienyne 43 (19.8 mg, 98%).
For method c, a solution of alcohol 42 (30 mg, 62.9 μmol) and
methyl N-(triethylammoniumsulfonyl)carbamate (45 mg, 188 μmol)
in anhydrous toluene (2.2 mL) was stirred at room temperature for 5 h
and at 40 °C for 14 h under argon. The reaction mixture was cooled to
room temperature; then water and brine were added. The mixture was
extracted with diethyl ether, dried (Na2SO4), and concentrated. The
residue was applied on a silica Sep-Pak cartridge. Elution with hexane/
diethyl ether (98:2) afforded the dienyne 43 (6 mg, 21%).
(20S)-2-Methylene-25-[(triethylsilyl)oxy]-vitamin D3 tert-Butyldi-
methylsilyl Ether (60). Hydrogenation of the trienyne 52, performed
according to procedure described above, gave after purification on a
silica Sep-Pak cartridge the previtamin 56 in 80% yield. The previtamin
compound was then subjected to thermal isomerization to give
protected vitamin 60 in 87% yield.
3-Desoxy-1α-[(tert-butyldiphenylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-vitamin D3 (61). Hydrogenation of the trienyne 53,
performed as described for 51, gave after purification on a silica Sep-
Pak cartridge the previtamin 57 in 80% yield. The previtamin
compound was then subjected to thermal isomerization to give the
product that was purified by HPLC (9.4 mm × 25 cm Zorbax-Sil
column, 4 mL/min) using hexane/ethyl acetate (99:1) solvent system.
Pure protected vitamin 61 (96%) was eluted at RV 16.3 mL.
(5R,6R)-5-[(tert-Butyldiphenylsilyl)oxy]-6-methyl-4-methylene-1-
[(trimethylsilyl)ethynyl]-cyclohexene (49) and (3S)-3-[(tert-
Butyldiphenylsilyl)oxy]-2-methyl-4-methylene-1-[(trimethylsilyl)-
ethynyl]-cyclohexene (50). Reaction of the alcohol 48 with the Martin
sulfurane dehydrating agent, performed as described for 42, gave
products, which were purified on a silica Sep-Pak cartridge. Elution
with hexane/diethyl ether (98:2) afforded the oily isomeric dienynes
49 and 50 (62 mg, 81%; isomer ratio of 2:1).
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Journal of Medicinal Chemistry
Article
(20S)-3-Desoxy-1α-[(tert-butyldiphenylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-vitamin D3 (62). Hydrogenation of the trienyne 54,
performed as described for 51, gave after purification on a silica Sep-
Pak cartridge the previtamin 58 in 80% yield. The previtamin
compound was then subjected to thermal isomerization to give the
product that was purified on a silica Sep-Pak cartridge. Elution with
hexane gave pure protected vitamin 62 in 64% yield.
25-Hydroxy-2-methylene-vitamin D3 (8). To a solution of
protected vitamin 59 (8.7 mg) in THF (0.7 mL) was added
tetrabutylammonium fluoride (1.0 M in THF; 546 μL, 546 μmol) at
room temperature under argon. The stirring was continued for 18 h,
brine was added, and the mixture was extracted with ethyl acetate. The
organic extracts were dried (MgSO4) and evaporated. The residue was
purified by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min)
using hexane/2-propanol (97:3) solvent system; vitamin 8 (1.357 mg,
29%) was collected at RV 35 mL. Analytical sample of the vitamin was
obtained after HPLC (9.4 mm × 25 cm Zorbax Eclipse XDB-C18
column, 4 mL/min) using methanol/water (93:7) solvent system (RV
28 mL).
(20S)-25-Hydroxy-2-methylene-vitamin D3 (9). Hydroxyl depro-
tection in the silylated vitamin 60, performed according to procedure
described above, gave product that was purified by HPLC (9.4 mm ×
25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (96:4)
solvent system; vitamin 9 (23% yield) was collected at RV 39 mL. An
analytical sample of the vitamin was obtained after HPLC (9.4 mm ×
25 cm Zorbax Eclipse XDB-C18 column, 4 mL/min) using methanol/
water (93:7) solvent system (RV 35 mL).
3-Desoxy-1α,25-dihydroxy-2-methylene-vitamin D3 (10). Hydrox-
yl deprotection in the silylated vitamin 61, performed as described
above for 59, gave product that was purified by HPLC (9.4 mm × 25
cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (97:3)
solvent system; vitamin 10 (33% yield) was collected at RV 25 mL. An
analytical sample of the vitamin was obtained after HPLC (9.4 mm ×
25 cm Zorbax Eclipse XDB-C18 column, 4 mL/min) using methanol/
water (93:7) solvent system (RV 42 mL).
followed by Diet 11 (0.02% Ca) + AEK oil for 3 weeks. The rats were
then switched to a diet containing 0.47% Ca29 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 intra-
peritoneal 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 was determined as a measure
of bone calcium mobilization. The first 10 cm of the intestine was also
collected for the intestinal calcium transport analysis using the everted
gut sac method.26
Molecular Modeling. The molecular mechanics studies were used
to establish the energy-minimized structures of the synthesized
compounds shown in Figure 3. The calculation of optimized
geometries and steric energies was initially 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 finding the global minimum
structures involved the Conformational Search module; these
structures were further energy-minimized using the semiempirical
AM1 method. Finally, the respective vicinal proton−proton coupling
constants in the preferred energy-minimized conformations were
calculated using PCMODEL (release 9.0) software package (Serena
Software).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Purity criteria of the vitamin D analogues 8−11, their H and
13C NMR spectra, competitive binding curves or dose−
response curves derived from cellular differentiation and
transcriptional assays, and spectral data of the all synthesized
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
(20S)-3-Desoxy-1α,25-dihydroxy-2-methylene-vitamin D3 (11).
Hydroxyl deprotection in the silylated vitamin 62, performed as
described above for 59, gave product that was purified by HPLC (9.4
mm × 25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol
(97:3) solvent system; vitamin 11 (56% yield) was collected at RV 36
mL. An analytical sample of the vitamin was obtained after HPLC (9.4
mm × 25 cm Zorbax Eclipse XDB-C18 column, 4 mL/min) using
methanol/water (91:9) solvent system (RV 56 mL).
■
Corresponding Author
*Dr. Hector F. DeLuca. Telephone: 608-262-1620. Fax: 608-
262-7122. E-mail: deluca@biochem.wisc.edu.
Notes
The authors declare no competing financial interest.
Biological Studies. In Vitro Studies. VDR binding, HL-60
differentiation, and 24-hydroxylase transcription assays were per-
formed as previously described.26,27
ACKNOWLEDGMENTS
■
The work was supported in part by funds from the Wisconsin
Alumni Research Foundation. We thank Jean Prahl for
assistance on tissue culture work and conducting VDR binding
and HL-60 differentiation studies and Xiaohong Ma for
conducting the in vivo studies.
CYP27B1 Activity Assay. 25-Hydroxyvitamin D3 was purchased
from SAFC-Pharma (Madison, WI). Mouse CYP27B1, bovine
adrenodoxin (Adx), and adrenodoxin reductase (AdR) were purified
as described previously.28 The reconstituted CYP27B1 enzymatic assay
was carried out in a buffer containing 20 mM Tris (pH 7.7), 125 mM
NaCl, 0.1% CHAPS, 2 μM Adx, 0.2 μM AdR, 0.05 μM CYP27B1, and
0−5 μM vitamin D substrate. All the vitamin D analogues were
dissolved in ethanol, and the concentrations were determined based on
UV absorption at appropriate wavelengths (ε254 = 42 mM−1 cm−1 for
4; ε265 = 18.2 mM−1 cm−1 for 25-OH-D3; ε270 = 18.0 mM−1 cm−1 for 8
and 9). The reactions were initiated by the addition of NADPH at a
final concentration of 1 mM and incubated at 37 °C for 5−20 min to
keep substrate conversion ≤20%. Then the reactions were quenched
and extracted with dichloromethane, and the organic layer was
collected and dried under ambient conditions. After being redissolved
in hexane/2-propanol (93:7) solvent system, the mixture was analyzed
by HPLC (Zorbax Rx-Sil, 9.4 mm × 25 cm, 4 mL/min) at 265 nm.
The initial rates calculated from product formation were fitted to the
Michaelis−Menten equation V = kcat[E]0[S]/(KM + [S]) using
GraphPad Prism 5 to obtain the kcat and KM values.
ABBREVIATIONS USED
■
1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; 2MD, (20S)-
1α,25-dihydroxy-2-methylene-19-norvitamin D3; VDR, vitamin
D receptor
REFERENCES
■
(1) Feldman, D., Pike, J. W., Adams, J. S., Eds. Vitamin D, 3rd ed.;
Elsevier Academic Press: San Diego, CA, 2011.
(2) Jones, G.; Strugnell, S. A.; DeLuca, H. F. Current understanding
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