Synthesis and biological activity of 2-(3′-hydroxypropylidene)- 1α-hydroxy-19-norvitamin D analogues with shortened alkyl side chains

Article abstract of DOI:10.1021/jm200743p

As a continuation of our efforts directed to vitamin D compounds of promising biological properties, 19-norvitamins 9-13, possessing a 3′-hydroxypropylidene fragment attached to C-2 and shortened 17β-alkyl chains, were synthesized. A new synthetic pathway

Full text of DOI:10.1021/jm200743p

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Activity of 2-(30-Hydroxypropylidene)-
1α-hydroxy-19-norvitamin D Analogues with Shortened Alkyl Side Chains
Agnieszka Glebocka,†,‡ Rafal R. Sicinski,†,‡ Lori A. Plum,‡ and Hector F. DeLuca*,‡
†Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
‡Department of Biochemistry, University of Wisconsin—Madison, 433 Babcock Drive, Madison, Wisconsin 53706, United States
S Supporting Information
b
ABSTRACT: As a continuation of our efforts directed to vitamin D compounds
of promising biological properties, 19-norvitamins 9ꢀ13, possessing a 30-hydroxy-
propylidene fragment attached to C-2 and shortened 17β-alkyl chains, were
synthesized. A new synthetic pathway providing the CD-ring ketones 20ꢀ24 is
described starting from the epimeric aldehydes 25 and 26. The hydrindanones
20ꢀ24 were subjected to the WittigꢀHorner reaction with the phosphine oxide
14, and the vitamin D compounds 9ꢀ13 were obtained after hydroxyl deprotec-
tion. In comparison to 1α,25-(OH)2D3 (1), the prepared analogues, except for
the 20R-compound 12, were only ca. 3 times less potent in binding to VDR.
Compounds 9ꢀ11 and 13 exhibited HL-60 cellular activity 5ꢀ20 times lower and
transcriptional activity ca. 10 times decreased related to those for the hormone 1.
When tested in vivo, all the analogues showed no ability to mobilize calcium from
bone, and intestinal calcium transport activity was observed only at high doses of
the vitamins 10, 12, and 13.
’ INTRODUCTION
exhibiting the unique ability to induce bone formation in vitro
and in vivo.5,6 During the following years, several vitamin D
analogues characterized by such A-ring modification were
synthesized, and many of them showed promising biological
activities.7ꢀ17
Since the isolation and structural characterization of the
most active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3
(1α,25-(OH)2D3, calcitriol, 1; Figure 1) representing its hormo-
nal form,1 the biological functions of this compound have been
extensively studied.2 It has been established that calcitriol, in addition
to its classical action as the regulator of calcium and phosphorus
homeostasis, also plays a pivotal role in cellular differentiation, inhibi-
tion of cell proliferation, and immunomodulation.3 Such a broad
array of biological activities of the natural hormone 1 encouraged
many research groups to undertake the synthesis of vitamin
D analogues characterized by a selective biological profile. This
great synthetic effort resulted in the preparation of more than
3000 calcitriol analogues.4 There were two main goals of intro-
ducing structural modifications: to search for the vitamin D
compounds with increased calcemic potency (superagonists)
or to search for noncalcemic compounds, which could be useful
in treating specific diseases (cancer, psoriasis, multiple
sclerosis, etc.) without causing undesired side effects related
to hypercalcemia.
In 1998, we discovered an important modification of the
vitamin D carbon skeleton, which led to the compounds with
interesting biological properties. This structural change consists
of the removal of the exomethylene group from C-10 and its shift
to C-2.5 Thus, the 2-methylene-1α,25-dihydroxy-19-norvitamin
D3 analogue 2 bound to the vitamin D receptor (VDR) equally
well as the natural hormone 1 but showed an enhanced ability
to mobilize calcium from bone. The isomeric (20S)-com-
pound (3, 2MD) exerted even stronger activity in bone,
Seven years ago, a set of noncalcemic 2-methylene-19-nor-
1α-hydroxyvitamin D analogues 4ꢀ6 with shortened side
chains were described.18 Interestingly, it was established that
these compounds, substituted at C-17 with a branched (or
even unbranched) alkyl group, retained some valuable geno-
mic functions related to those of 2MD but lacked its calcemic
activity. Therefore, these analogues show promise of being
used as anticancer agents or, if they have an ability to decrease
the PTH level, for the treatment of secondary hyperthyroid-
ism. Continuing our structureꢀactivity studies in the vitamin
D area, we also prepared 19-nor-2-(30-hydroxypropylidene)
vitamin D compounds and found that their E-geometrical
isomers 7 and 8 were more potent in the in vitro and in vivo
tests.19 Taking into account these findings, we decided to
synthesize different hybrid compounds combining both struc-
tural modifications of the vitamin D molecule: shortened alkyl
side chains and the presence of the ring A of 19-norvitamins
substituted at C-2 with a 30-hydroxypropylidene group.20 This
article describes the preparation of the vitamin D analogues
9ꢀ13 and the results of their biological testing.
Received: June 9, 2011
Published: September 08, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), its analogues, and the building blocks for the synthesis of vitamins 9ꢀ13.
’ RESULTS AND DISCUSSION
It turned out that alkylation of the nitrile 28 proceeded quite
efficiently. Thus, treatment of its anion, generated using LDA,
with ethyl or isobutyl bromide resulted in the formation of single
substitution products 33 and 34 (Scheme 2) in 80% and 74%
yield, respectively. A lower yield was obtained when silylated
2-bromoethanol was used as an alkylating agent for the prepara-
tion of 35. Interestingly, in all cases, the nucleophilic substitution
turned out to be a highly diastereoselective process, resulting in
the products having the S-configuration at the newly formed
stereogenic center. Therefore, it might be suggested that alkyl
bromide reacts with a rotamer of the nitrile 28 anion possessing
the α-methyl substituent directed to C-7 and the CN group
facing C-2. The approach of the alkylating agent from the si-face
of this anion is incomparably easier than from the opposite side,
significantly hindered by an angular methyl group. Such a stereo-
chemical outcome of this alkylation reaction was supported by
X-ray crystal structure analysis of the compound obtained by
alkaline hydrolysis of the benzyloxy group in 34. The ORTEP
drawing of the hydroxy nitrile 37 is shown in Figure 2.
Chemistry. Availability of the phosphine oxide 14, synthesized
previously in our laboratory,19 allowed us to focus on the prepara-
tion of the corresponding CD-ring ketones 20ꢀ24, which can be
condensed with an anion of 14 in a WittigꢀHorner process.
Considering the hydrindanols 15ꢀ19 as the immediate precur-
sors of the desired Grundmann ketone analogues, we have
decided to explore the usefulness of the 20-cyano compound as
a crucialsyntheticprecursor. Its α-alkylation processseemed to be
a convenient method of introducing the desired alkyl fragment at
C-20, and the subsequent alkali metal reduction should be
suitable for the removal of the cyano group.
Preparation of the nitrile 28 (Scheme 1) was easily accom-
plished from the known bicyclic aldehyde 25 obtained,21 to-
gether with its epimer 26,22 by the degradation of vitamin D2.
Thus, conversion of 25 into the oxime 27 followed by acetolysis
provided the nitrile compound 28 in the overall 81% yield. Both
aldehydes 25 and 26 proved also to be suitable substrates for the
preparation of hydrindanols 15 and 16. The Wittig methylena-
tion of the aldehydes (carried out separately for each isomer) and
the following hydrogenation of the formed olefins 29 and 30 gave
the benzoates 31 and 32, which after hydrolysis provided
alcohols 15 and 16 in the overall yield ca. 50%.
The benzoyloxy nitrile 33 proved to be a convenient precursor
of 17. Its hydrolysis to the hydroxy nitrile 36 and the following
DIBALH reduction gave the hydroxy aldehyde 38 (64%; direct
reduction 33 f 38 proved to be less efficient). After conversion
of the formyl substituent in 38 to the p-tosylhydrazone derivative
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Scheme 1a
a (a) NH2OH ꢁ HCl, py, 89%; (b) Ac2O, 91%; (c) Ph3P+ CH3 Brꢀ, n-BuLi, THF; (d) H2/Pd, AcOEt; (e) KOH, MeOH.
Scheme 2a
a (a) RCH2Br, LDA, THF; (b) KOH, MeOH; (c) DIBALH, CH2Cl2; (d) p-TsNHNH2, MeOH, 67%; (e) NaBH3CN, DMF, 65%; (f) K, HMPA, t-
BuOH, Et2O, 84%; (g) hν.
and its subsequent reduction with sodium cyanoborohydride to
methyl, the desired hydridanol 17 was obtained in 44% overall yield.
For subsequent removal of the cyano group in the prepared
tertiary nitriles, we decided to test two synthetic pathways: the
reduction with dissolving metal and the less frequently used
strategy, involving conversion of the nitrile to the formyl group
and the photolysis of the formed aldehyde. The first procedure
rules out the presence of the benzoyloxy group in the nitrile molecule
because it could be reduced by alkali metal to the hydrocarbon.23
Thus, the reduction of the hydroxy nitrile 37 with a branched side
chain was performed by its treatment with potassium in ether and
hexamethylphosphoric triamide.24 The reaction resulted in the
efficient formation of two epimeric products 18 and 19 (in 84%
yield) possessing alkyl side chains. However, no stereoselectivity
was found in the examined nitrile removal process; both epimers
were formed in virtually identical quantities.
Pursuing the second synthetic strategy, aldehyde 39, prepared
by DIBALH reduction of hydroxy nitrile 37, was subjected to
photolysis in hexane solution under an oxygen-free atmosphere
at 0 °C, and the obtained complex mixture of products was
separated by column chromatography and HPLC. According to
our expectations, the UV irradiation resulted mainly in the forma-
tion of the desired compounds 18 and 19. However, the photo-
chemical removal of carbon oxide from aldehyde 39 occurred
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nonstereoselectively, and the yield of these photoproducts (20%,
ca. 1:1) was disappointing. Similar decarbonylation processes of
10β-formyl steroids (19-aldehydes) in 5α- and 5β-series, as
reported in the literature, were much more efficient.25 The
mechanism of such a photochemical process is well-known and
involves radical cleavage of the bond between the carbonyl group
and α-carbon atom (Norrish type I reaction)26 in the excited
aldehyde or ketone, with the following extrusion of carbon mon-
oxide and recombination of the radicals (or hydrogen incorpora-
tion from the solvent). Rather to our surprise, among the isolated
and characterized products of the examined photolysis of 39, the
olefinic compound 40 (12%) was also present, resulting from the
extrusion of formaldehyde molecule. Most likely, in this case, an
alternative chain scission process operates (Norrish type III
reaction),26,27 involving abstraction of the β-hydrogen by the
oxygen atom of the excited aldehyde 39 and cleavage of the
resulting diradical to give the olefin 40 and hydroxycarbene,28
which further photochemically rearranges to formaldehyde.29
Although theoretically possible,30 such photodissociation pro-
cesses were very rarely observed during the photolysis of aliphatic
aldehydes.31,32 In our case, the observed preferential formation of
the least substituted olefin 40 can be explained by conformational
analysis of the aldehyde 39, possessing a formyl group attached to
the tertiary carbon. Assuming that the preferred ground-state
conformations of the aldehyde can be correlated with an out-
come of its intramolecular photoreactions, a preference for
hydrogen abstraction from the 2-methyl group emerges from
molecular modeling (HyperChem release 7.0) indicating that a
side-chain rotamer of 39, characterized by the synperiplanar
orientation of the carbonyl group with respect to C(2)-CH3, is
energetically favored.
These results prompted us to examine also the stereochemical
course of the photochemically induced decarbonylation reaction
occurring with the β,γ-unsaturated aldehyde 41, and this model
compound was easily synthesized from 35 (unpublished results).
Its irradiation with UV light resulted in smooth decarbonylation
leading to the hydrindanol 42 (59%) with an unsaturated side
chain. Even careful examination of the reaction mixture by HPLC
did not indicate the presence of the epimeric product. These results
are in agreement with the data reported in the literature.25,33 The
results of the photolysis of 41 presented here could also be
explained by molecular modeling. As in the case of 39, the least-
energy side-chain rotamer of 41 has a synperiplanar orientation
of the carbonyl group with respect to C(2)-CH3. Assuming that
such an excited rotamer then undergoes an α-cleavage (Norrish
type I) process, the aldehyde hydrogen is suitably oriented in
this biradical intermediate for a selective incorporation at the
α-carbon upon the extrusion of carbon monoxide. Although such
a stereoselective photodecarbonylation process occurring in a
conformationally labile system is of interest, its application to the
construction of the steroidal alkylidene side chains would be
limited because of the alternative use of the Wittig reactions of
20-aldehydes.
Successful preparation of hydrindanols 15ꢀ19 allowed us to
accomplish the last steps of the synthesis of the desired vitamin D
analogues. Oxidation of these alcohols with tetrapropylammonium
perruthenate furnished the respective Grundmann ketones 20ꢀ24
(Scheme 3), and the obtained CD-ring fragments were coupled
with the lithium phosphinoxy carbanion generated from the
phosphine oxide 14, to give the protected vitamin D compounds
43ꢀ47. These, in turn, after treatment with tetrabutylammonium
Figure 2. ORTEP derived from the single-crystal X-ray analysis of hydroxy
nitrile 37 (one of the two independent structures, slightly differing in their
side chain conformation, which were found in the asymmetric part of the
unit cell).
Scheme 3a
a (a) NMO, TPAP, CH2Cl2; (b) 14, PhLi, THF; (c) n-Bu4NF, THF.
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Table 1. VDR Binding Properties,a HL-60 Differentiating Activities,b and Transcriptional Activitiesc of the Vitamin D Analogues
9ꢀ13
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 doseꢀresponse 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 1α,25-(OH)2D3
Ki to the Ki for the analogue. b 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 doseꢀresponse 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 the analogue. c Transcriptional assay in
rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The ED50 values are derived from doseꢀresponse curves and
represent the analogue concentration capable of increasing the luciferase activity 50%. The luciferase activity ratio is the average ratio of the
1α,25-(OH)2D3 ED50 to the ED50 for the analogue.
fluoride, afforded the final 2-(30-hydroxypropylidene)-1α-hydroxy-
19-norvitamin D compounds 9ꢀ13.
vitro and in vivo methods. Deletion of 2 or even 4 carbons from
the vitamin D3 side chain and an absence of 25-hydroxyl, commonly
considered as crucial for the VDR binding, had little effect on the
affinity for the receptor. Thus, all tested vitamins, with a notable
exception for analogue 12, bound equally well to the full length
Biological Evaluation. Biological activity of the side chain
modifications in the synthesized 2-(30-hydroxypropylidene)-19-
norvitamin D analogues 9ꢀ13 were determined using both in
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Figure 3. Bone calcium mobilization activity of calcitriol (1) and the synthesized analogues 9ꢀ13.
recombinant rat vitamin D receptor, being only ca. 3 times less
active than 1α,25-(OH)2D3 (Table 1). It was established that
compounds 9, 10, and 11 were also approximately equal in their
ability to elicit cellular differentiation of human promyelocytic
HL-60 cells into monocytes showing only 5ꢀ8 times lower
activity than the natural hormone 1. Not surprisingly, these
vitamins tested in the transcription assay showed a similar activity
in inducing transcription of a vitamin D-responsive gene, i.e.,
the 24-hydroxylase (CYP-24) promoter driving the luciferase
reporter gene system. The 20R-compound 12, possessing an
iso-butyl fragment attached to C-20, showed the reduction of
receptor affinity by 20-fold. Moreover, its cell differentiation
and transcriptional activities were decreased 600- and 500-fold,
respectively. Except for this analogue, all analogues prepared had
significant in vitro activity. On the basis of their in vitro activity,
it was expected that some in vivo activity would be found. Sur-
prisingly, none of the synthesized compounds 9ꢀ13 showed an
ability to mobilize calcium from bone, even at high doses
(Figure 3). Similarly, low doses of the tested compounds failed
to support intestinal calcium transport (Figure 4). Of consider-
able importance was finding that compound 9 and its homologue
11, substituted at C-20 with two methyl groups, showed little or
no intestinal calcium transport activity. However, compounds
10 and 12 did have some intestinal calcium transport activity
at high doses. Vitamins differing in their configurations at C-20
(9 versus 10 and 12 versus 13) revealed that 20S-epimers are
characterized by higher intestinal activity. Also, the elongation
of 17β-alkyl substituent seems to enhance the biological effect
in vivo. We previously prepared 2-(30-hydroxypropylidene)-
1α,25-dihydroxyvitamin D3 and showed it to be more potent
than 1α,25-(OH)2D3 on both intestine and bone.19 The
absence of a full chain in this series clearly eliminates intestinal
and bone activities in vivo, despite their similarity in in-vitro
measurements.
Discussion. The presented results show that an α-alkylation
process of the 22-nitrile can provide the desired extension of the
steroidal 17β-substituent by the attachment of the different
(even branched) alkyl fragments. Such a side-chain construction
strategy can be used if both series of epimeric compounds,
differing in configuration at C-20, are of interest. Although
isomer separation is necessary, this synthetic path is comparable
with other methods, for instance, those involving equilibration of
22-aldehydes and their reduction and separation of epimeric 22-
alcohols.
All vitamin D analogues described in this work represent a new
class of hybrid compounds with structural modifications combin-
ing the presence of a long 30-hydroxypropylidene substituent at
C-2 and an abbreviated 17β-side chain. The synthesized com-
pounds have significantly diminished calcemic activity, but, at the
same time, they are characterized by very good binding ability to
VDR and possess high cell differentiation and transcriptional
activities. These properties suggest that they might be useful in
suppressing cancer growth or some other disease where calcemic
activity is not desired.
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Figure 4. Intestinal calcium transport activity of calcitriol (1) and the synthesized analogues 9ꢀ13.
’ CONCLUSIONS
AutoSpec (Beverly, MA) instrument. High-performance liquid chroma-
tography (HPLC) was performed on a Waters Associates liquid
chromatograph equipped with a Model 6000A solvent delivery system,
a Model U6K Universal injector, and a Model 486 tunable absorbance
detector. THF was freshly distilled prior to use from sodium benzophe-
none ketyl under argon.
Preparation of the phosphine oxide 14 from commercial (ꢀ)-quinic
acid was reported by us previously.19 The starting 22-aldehydes 25 and 26
were obtained from vitamin D221,22 according to the published procedures.
The aldehyde 41 was prepared from 35 (Supporting Information).
The purity of final compounds was determined by HPLC, and they
were judged to be at least 99% pure. Two HPLC systems (straight phase,
hexane/2-propanol, and reversed-phase, water/methanol) were em-
ployed as indicated in Table 1 (Supporting Information). The purity
and identity of the synthesized vitamins were additionally confirmed by
inspection of their 1H NMR and high-resolution mass spectra.
Compound 41: 1H NMR (400 MHz, CDCl3) δ, 0.989 (3H, s, 7a0-
CH3), 1.240 (3H, s, 2-CH3), 4.10 (1H, narr m, 40α-H), 5.04 (1H, dd, J =
17.6, 0.9 Hz, 4-HZ), 5.22 (1H, dd, J = 10.9, 0.9 Hz, 4-HE), 6.07 (1H, dd,
J = 17.6, 10.9 Hz, 3-H), 9.51 (1H, s, CHO).
The vitamin D side-chain modification consisting in signifi-
cant shortening to the small branched alkyl fragments contain-
ing no hydroxyls can be very beneficial for the biological proper-
ties of the analogues, making them possible candidates for
therapeutic use. Such truncation of the normal side chain present,
for example, in higly calcemic compounds as 2MD or 2-(30-hydro-
xypropylidene)-19-norvitamins 7 and 8, can result in noncalcemic
compounds still retaining other genomic activities.
A significant potency of the synthesized vitamin D analogues 9
and 11, possessing the shortened 17β-side chain, in displacement
of the radiolabeled 1α,25-(OH)2D3 from the receptor protein, as
well as their high cellular and transcriptional potency combined
with lack of ability to raise serum calcium concentration by either
a bone mobilization or intestinal transport, suggests that both
compounds could be effective in psoriasis or as anticancer agents.
’ EXPERIMENTAL SECTION
Chemistry. Melting points (uncorrected) were determined on a
Thomas-Hoover capillary melting-point apparatus. Ultraviolet (UV)
absorption spectra were recorded with a Perkin-Elmer Lambda 3B UVꢀvis
(E)- and (Z)-Benzoic Acid (1R,3aR,4S,7aR)-1-[(20-Hydroxyi-
mino-10-methyl)ethyl]-7a-methyl-octahydro-inden-4-yl Es-
ters (27a,b). To a solution of aldehyde 25 (284 mg, 0.90 mmol) in
anhydrous pyridine (5 mL) was added NH2OH ꢁ HCl (210 mg). The
mixture was stirred at room temperature for 20 h, poured into water, and
extracted using ethyl acetate. The combined organic phases were
separated, washed with saturated NaHCO3, water, and saturated CuSO4,
dried (MgSO4), and evaporated. The oily residue was purified using
1
spectrophotometer in ethanol. H nuclear magnetic resonance (NMR)
spectra were recorded at 400 and 500 MHz with Bruker Instruments DMX-
400 and DMX-500 Avance console spectrometers in deuteriochloroform.
Chemical shifts (δ) are reported downfield to internal Me4Si (δ 0.00).
Electron impact (EI) mass spectra were obtained with a Micromass
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column chromatography on silica. Elution with hexane/ethyl acetate
(9:1) gave pure isomeric oximes, the less polar 27a (167 mg), and the
more polar 27b (105 mg; total yield 89%).
(2 mL). The solution was stirred at 0 °C for 20 min, cooled to ꢀ78 °C,
and siphoned to the solution of 28 (430 mg, 1.31 mmol) in THF
(1.5 mL). The resulting yellow mixture was stirred for 30 min, then
HMPA(600μL) was added, and stirring was continued for another 15 min.
Then, CH3CH2Br (310 μL, 4.08 mmol) was added, and the solution was
stirred at ꢀ78 °C for 40 min. Saturated NH4Cl was added, and the
mixture was extracted with ethyl acetate. The combined organic phases
were washed with water, dried (MgSO4), and evaporated. The residue
was subjected to column chromatography on silica. Elution with hexane/
ethyl acetate (95:5) gave the pure compound 33 (280 mg, 60%; 80%
based on recovered substrate). Further elution with hexane/ethyl acetate
(95:5) gave unreacted 28 (107 mg).
Benzoic Acid (1R,3aR,4S,7aR)-1-[(R)-10-Cyano-ethyl]-7a-
methyl-octahydro-inden-4-yl Ester (28). The solution of oximes
27a,b (1.6:1; 248 mg, 0.75 mmol) in acetic anhydride (8 mL) was
refluxed for 1.5 h. The reaction mixture was cooled, poured carefully on
ice, and extracted with toluene. The extracts were combined, washed
with water, saturated NaHCO3, and brine, dried (MgSO4), and evapo-
rated. The residue was applied on a silica Sep-Pak (5 g). Elution with
hexane/ethyl acetate (95:5) gave the pure, semicrystalline nitrile 28
(212 mg, 91%).
Benzoic Acid (1S,3aR,4S,7aR)-1-[(S)-10-Cyano-10,30-dimethyl-
butyl]-7a-methyl-octahydro-inden-4-yl Ester (34). Alkylation
of the nitrile 28 with (CH3)2CHCH2Br was performed analogously as
described above for the preparation of 33, except that after the addition
of iso-butyl bromide, the reaction mixture was allowed to warm up to
ꢀ40 °C during 1 h. The crude product was applied to a silica Sep-Pak
(2 g) and eluted with hexane/ethyl acetate (98:2) to give a pure
semicrystalline compound 34 (60 mg, 66%; 74% based on recovered
substrate); further elution with hexane/ethyl acetate (97:3) gave
unreacted 28 (8.5 mg).
Benzoic Acid (1S,3aR,4S,7aR)-1-[(S)-30-[(tert-Butyldimethyl-
silyl)oxy]-10-cyano-10-methyl-propyl]-7a-methyl-octahydro-
inden-4-yl Ester (35). Alkylation of the nitrile 28 with BrCH2-
CH2OTBDMS was performed analogously as described above for
the preparation of 33. The crude product was applied to a silica Sep-
Pak (5 g) and eluted with hexane/ethyl acetate (96:4) to give the
compound 35 as an oil (15 mg, 45%).
Benzoic Acid (1R,3aR,4S,7aR)-7a-Methyl-1-[(R)-10-methyl-
prop-20-enyl]-octahydro-inden-4-yl Ester (29). To the methyl-
triphenylphoshonium bromide (31 mg, 87 μmol) in anhydrous THF
(0.5 mL) at 0 °C was added dropwise n-BuLi (2.65 M in hexanes, 64 μL,
0.170 mmol) under argon with stirring. After 5 min, another portion of
Ph3P+CH3 Brꢀ was added (31 mg, 87 μmol), and the solution was
stirred at 0 °C for 10 min and then at room temperature for 20 min. The
orange-red mixture was cooled to ꢀ78 °C and siphoned to a solution of
aldehyde 25 (33 mg, 0.109 mmol) in anhydrous THF (0.1 mL). The
reaction mixture was stirred at ꢀ78 °C and stopped by the addition of
brine containing 1% HCl 3 h after the addition of the first portion of the
Wittig reagent. Ethyl acetate (3 mL), benzene (2 mL), ether (1 mL),
saturated NaHCO3 (1 mL), and water (1 mL) were added, and the
mixture was vigorously stirred at room temperature for 18 h. Then, the
organic phase was separated, washed with brine, dried (MgSO4), and
evaporated. The oily residue was filtered through a silica Sep-Pak (2 g).
Elution with hexane/ethyl acetate (99:1) resulted in the pure olefinic
product 29 (19 mg, 68%).
(S)-2-[(10S,3a0R,40S,7a0R)-40-Hydroxy-7a0-methyl-octahydro-
inden-10-yl]-2-methyl-butylnitrile (36). Alkaline hydrolysis of the
benzoyloxy nitrile 33, performed as described for 31, gave the crude
product that was purified on a silica Sep-Pak (2 g) and eluted with
hexane/ethyl acetate (8:2) to furnish the pure hydroxy nitrile 36
(179 mg, 99%).
Benzoic Acid (1R,3aR,4S,7aR)-7a-Methyl-1-[(S)-10-methyl-
prop-20-enyl]-octahydro-inden-4-yl Ester (30). Wittig reaction
of aldehyde 26, performed as described above for 25, gave an olefinic
product that was purified on a silica Sep-Pak (2 g). Elution with hexane/
ethyl acetate (98:2) provided pure compound 30 (73%).
Benzoic Acid (1R,3aR,4S,7aR)-1-[(R)-10-Methyl-propyl]-7a-
methyl-octahydro-inden-4-yl Ester (31). To a solution of olefin
29 (45 mg, 0.146 mmol) in ethyl acetate (5.5 mL) was added Pd/C
(10%, 27 mg), and the resultant suspension was stirred under constant
flow of hydrogen at room temperature for 19 h. Then, the suspension
was filtered, the filtrate was evaporated and applied to a silica Sep-Pak
cartridge (2 g). Elution with hexane/ethyl acetate (96:4) gave the pure,
oily ester 31 (40 mg, 87%).
(S)-2-[(10S,3a0R,40S,7a0R)-40-Hydroxy-7a0-methyl-octahydro-
inden-10-yl]-2,4-dimethyl-pentanenitrile (37). The benzoyloxy
nitrile 34 was treated with 10% methanolic KOH as described above for
33. The crude product was purified on a silica Sep-Pak (2 g) and eluted
with hexane/ethyl acetate (95:5) to afford the pure hydroxy nitrile 37
(92%).
(S)-2-[(10S,3a0R,40S,7a0R)-40-Hydroxy-7a0-methyl-octahydro-
inden-10-yl]-2-methyl-butyraldehyde (38). To the solution of
nitrile 36 (172 mg, 0.773 mmol) in anhydrous methylene chloride
(3.3 mL) was slowly added a solution of diisobutylaluminum hydride
(1.5 M in toluene; 1.66 mL, 2.3 mmol) at ꢀ78 °C. The solution was
stirred for 1 h, then it was allowed to warm up to ꢀ30 °C during 1 h 30
min and was cooled to ꢀ78 °C again. The reaction was quenched by the
addition of brine containing 5% HCl, and it was extracted with ethyl
acetate. The combined organic layers were washed with NaHCO3 and
brine, dried (MgSO4), and evaporated. The remaining residue was
purified on a silica Sep-Pak (2 g). Elution with hexane/ethyl acetate
(8:2) gave the pure hydroxy aldehyde 38 (112 mg, 65%).
(S)-2-[(10S,3a0R,40S,7a0R)-40-Hydroxy-7a0-methyl-octahydro-
inden-10-yl]-2,4-dimethyl-pentanal (39). Reduction of nitrile 37
performed as described for 36 gave the crude product that was purified
on a silica Sep-Pak (2 g) and eluted with hexane/ethyl acetate (95:5) to
afford the pure hydroxy aldehyde 39 (60%).
(1R,3aR,4S,7aR)-1-(10,10-Dimethyl-propyl)-7a-methyl-octahy-
dro-inden-4-ol (17). A solution of aldehyde 38 (10 mg, 0.42 μmol)
and p-toluenesulfonyl hydrazide (31 mg, 0.168 mmol) in dry methanol
(0.5 mL) was stirred with molecular sieves of 4 Å at 55 °C for 19 h. Then,
the reaction mixture was cooled, poured into water, and extracted with
toluene. The combined organic phases were washed with water, dried
Benzoic Acid (1R,3aR,4S,7aR)-1-[(S)-10-Methyl-propyl]-7a-
methyl-octahydro-inden-4-yl Ester (32). Hydrogenation of ole-
fin 30 was performed as described for 29. The crude product was
purified on a silica Sep-Pak (2 g). Elution with hexane/ethyl acetate
(97:3) yielded the pure, oily ester 32 (86%).
(1R,3aR,4S,7aR)-1-[(R)-10-Methyl-propyl]-7a-methyl-octa-
hydro-inden-4-ol (15). Solution of the ester 31 (40 mg, 0.129 mmol)
in 10% methanolic KOH (2 mL) was heated at 50 °C for 24 h, poured
into water, and extracted with ethyl acetate. The organic phase was
washed with water and then dried (MgSO4) and evaporated. The oily
residue was applied on a silica Sep-Pak (2 g) and eluted with hexane/
ethyl acetate (96:4) to give the pure product 15 (22 mg, 81%).
(1R,3aR,4S,7aR)-1-[(S)-10-Methyl-propyl]-7a-methyl-octa-
hydro-inden-4-ol (16). Alkaline hydrolysis of the ester 32, per-
formed as described for 31, gave the crude hydrindanol that was purified
on a silica Sep-Pak (2 g). Elution with hexane/ethyl acetate (97:3)
furnished the pure alcohol 16 (80%).
Benzoic Acid (1S,3aR,4S,7aR)-1-[(S)-10-Cyano-10-methyl-
propyl]-7a-methyl-octahydro-inden-4-yl Ester (33). n-Butyl-
lithium (1.6 M in hexanes, 1.0 mL, 1.6 mmol) was added at 0 °C to
the flask containing diisopropylamine (262 μL, 1.54 mmol) and THF
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Journal of Medicinal Chemistry
ARTICLE
(MgSO4), evaporated, and applied on a silica Sep-Pak (2 g). Elution with
hexane/ethyl acetate (85:15) gave p-tosylhydrazone (12 mg, 67%)
slightly contaminated with p-TsNHNH2. This crude tosylhydrazone
was dissolved in DMF (0.15 mL), and p-TsOH (2 mg, evaporated twice
with benzene) was added followed by NaBH3CN (8 mg, 0.126 mmol).
The mixture was stirred at 100 °C for 19 h, then it was cooled, poured
into water, and extracted with hexane and ethyl acetate. The combined
organic phases were washed with water, dried (MgSO4), and evaporated.
The remaining oily residue was applied on a silica Sep-Pak (2 g). Elution
with hexane/ethyl acetate (98:2) gave pure hydrindanol 17 (4 mg, 65%).
(1R,3aR,4S,7aR)-1-[(R)-10,30-Dimethyl-butyl]- and (1R,3aR,
4S,7aR)-1-[(S)-10,30-Dimethyl-butyl]-7a-methyl-octahydro-
inden-4-ol (18 and 19). (a) A solution of the hydroxy nitrile 37
(49 mg, 0.186 mmol) in t-BuOH (50 μL) and ether (200 μL) was added
dropwise to a blue mixture of potassium (55 mg, 1.4 mmol) in hexa-
methylphosphoric triamide (HMPA; 170 μL) and ether (420 μL) at
0 °C under argon with stirring. A cooling bath was removed and stirring
continued for 4 h at room temperature. The reaction mixture was diluted
using benzene, unreacted potassium was removed, and a few drops of
2-propanol were added. The organic phase was washed using water,
dried (MgSO4), and evaporated. The residue was applied to a silica Sep-
Pak (2 g) and eluted with hexane/ethyl acetate (95:5) to give a 1:1
mixture of epimeric alcohols 18 and 19 (37 mg, 84%).
(b) A solution of the hydroxy aldehyde 39 (20 mg, 0.084 mmol) in
hexane (350 mL) was irradiated with a 350 W Hanau S 81 mercury arc
lamp in an apparatus consisting of a Pyrex vessel with a water-cooled
Vycor immersion well. A slow stream of argon was passed into the vessel,
and the temperature of the solution was maintained at 0 °C. The
solution was irradiated for 70 min and allowed to warm up to room
temperature. Then, the solution was concentrated under vacuum and
the residue applied on a silica column. Elution with hexane/ethyl acetate
(95:5) gave a mixture of three compounds (5 mg). Separation of the
products was achieved by HPLC (10 mm ꢁ 25 cm, Zorbax-Sil column,
4 mL/min) using a hexane/ethyl acetate (88:12) solvent system. A 1:1
mixture of the epimeric alcohols 18 and 19 (2.8 mg, 15%) was collected
at Rv 30 mL, and the olefinic product 40 (1.7 mg, 9%) was collected at
Rv 34 mL.
(1R,3aR,4S,7aR)-7a-Methyl-1-[(R)-10-methyl-allyl)-octahy-
dro-inden-4-ol (42). A solution of hydroxy aldehyde 41 in hexane
was irradiated for 25 min with a 350 W Hanau S 81 mercury arc lamp in
an analogous manner as was described above for 38. The solution was
concentrated under vacuum, and the residue was applied to a silica Sep-
Pak (2 g) and eluted with hexane/ethyl acetate (97:3) to afford
unsaturated alcohol 42 (60%).
(1R,3aR,4S,7aR)-1-[(R)-10-Methyl-propyl]-7a-methyl-octahy-
dro-inden-4-one (20). To a solution of NMO (23 mg) in methylene
chloride (0.9 mL) were added molecular sieves of 4 Å (120 mg), and the
mixture was stirred at room temperature for 15 min. Then, a solution of
hydrindanol 15 (21 mg, 0.10 mmol) in methylene chloride (0.15 mL)
was added followed by tetrapropylammonium perruthenate (TPAP;
2.5 mg). The resulted dark mixture was stirred for 30 min and applied on
a silica Sep-Pak (2 g). Elution using hexane/ethyl acetate (95:5) gave
pure ketone 20 (18.5 mg, 88%).
(1R,3aR,7aR)-1-[(R)-10,30-Dimethyl-butyl]- and (1R,3aR,7aR)-
1-[(S)-10,30-Dimethyl-butyl]-7a-methyl-octahydro-inden-4-
one (23 and 24). The mixture of alcohols 18 and 19 (1:1) was
oxidized with TPAP as described above for 15. The crude products were
purified on a silica Sep-Pak (2 g). Elution with methylene chloride gave a
1:1 mixture of epimeric ketones 23 and 24 (91%). Separation of isomers
was achievedby HPLC(9.4 mm ꢁ 25 cm Zorbax-Sil column, 4 mL/min)
using a hexane/ethyl acetate (95:5) solvent system. The (20S)-ketone
24 was collected at RV 39 mL and the R-isomer 23 at RV 40 mL.
1α-[(tert-Butyldimethylsilyl)oxy]-2-[30-[((tert-butyldimethyl-
silyl)oxy)propylidene]-19,24,25,26,27-pentanorvitamin D3 tert-
Butyldimethylsilyl Ether (43). To a solution of phosphine oxide 14
(11.5 mg, 15.6 μmol) in anhydrous THF (0.30 mL) at ꢀ78 °C,
phenyllithium (1.8 M in butyl ether, 9 μL, 16 μmol) was slowly added
under argon with stirring. The solution turned deep orange. The mixture
was stirred at ꢀ78 °C for 20 min. A precooled (ꢀ78 °C) solution of
ketone 20 (19 mg, 91 μmol) in anhydrous THF (0.10 mL) was slowly
added. The mixture was stirred under argon at ꢀ78 °C for 2 h and at
6 °C for 16 h. Ethyl acetate and water were added, and the organic phase
was washed with brine, dried with MgSO4, and evaporated. The residue
was dissolved in hexane, applied on a silica column, and washed with
hexane/ethyl acetate (98.5:1.5) to produce silylated vitamin 43 (1.44 mg,
13%; 20% based on recovered 14). The column was then washed with
hexane/ethyl acetate (95:5) to recover a portion of unchanged C,D-ring
ketone 20 (7 mg), and hexane/ethyl acetate (6:4) was used to recover
diphenylphosphine oxide 14 (4.2 mg).
(20S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-[30-[((tert-butyldi-
methylsilyl)oxy)propylidene]-19,24,25,26,27-pentanorvi-
tamin D3 tert-Butyldimethylsilyl Ether (44). Silylated 19-norvi-
tamin D compound 44 was obtained by WittigꢀHorner coupling of
hydrindanone 21 with the phosphine oxide 14 performed analogously to
the process described above for the preparation of 43. After separation of
the reaction mixture by column chromatography on silica, using hexane/
ethyl acetate (98.5:1.5) as an eluent, the protected vitamin 44 was
obtained (19%; 30% based on recovered 14).
1α-[(tert-Butyldimethylsilyl)oxy]-2-[30-[((tert-butyldimethyl-
silyl)oxy)propylidene]-20-methyl-19,24,25,26,27-pentanor-
vitamin D3 tert-Butyldimethylsilyl Ether (45). Silylated 19-
norvitamin D compound 45 was obtained by WittigꢀHorner coupling
of hydrindanone 22 with the phosphine oxide 14 performed analogously
to the process described above for the preparation of 43. After separation
of the reaction mixture by column chromatography on silica, using
hexane/ethyl acetate (99.5:0.5) as an eluent, the protected vitamin 45
was obtained (48%).
1α-[(tert-Butyldimethylsilyl)oxy]-2-[30-[((tert-butyldimethyl-
silyl)oxy)propylidene]-19,23,24-trinorvitamin D3 tert-Butyl-
dimethylsilyl Ether (46). Silylated 19-norvitamin D compound 46
was obtained by WittigꢀHorner coupling of hydrindanone 23 with the
phosphine oxide 14 performed analogously to the process described
above for the preparation of 43. After separation of the reaction mixture
by column chromatography on silica, using hexane/ethyl acetate
(98.5:1.5) as an eluent, the protected vitamin 46 was obtained (50%;
83% based on recovered 23).
(20S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-[30-[((tert-butyldi-
methylsilyl)oxy)propylidene]-19,23,24-trinorvitamin D3 tert-
Butyldimethylsilyl Ether (47). Silylated 19-norvitamin D com-
pound 47 was obtained by WittigꢀHorner coupling of hydrindanone
24 with the phosphine oxide 14 performed analogously to the process
described above for the preparation of 43. After separation of the
reaction mixture by column chromatography on silica, using hexane/
ethyl acetate (98.5:1.5) as an eluent, the protected vitamin 47 was
obtained (53%; 62% based on recovered 14).
(1R,3aR,4S,7aR)-1-[(S)-10-methyl-propyl]-7a-methyl-octahy-
dro-inden-4-one (21). Oxidation of hydrindanol 16 with TPAP was
performed in a manner analogous to that for the isomeric 15. The crude
hydrindanone was purified on silica Sep-Pak (2 g). Elution with hexane/
ethyl acetate (96:4) gave pure ketone 21 (82%).
(1R,3aR,7aR)-1-(10,10-Dimethyl-propyl)-7a-methyl-octahy-
dro-inden-4-one (22). Oxidation of hydrindanol 17 with TPAP was
performed in a manner analogous to that for the isomeric 15. The crude
hydrindanone was purified on silica Sep-Pak (2 g). Elution with hexane/
ethyl acetate (96:4) gave pure ketone 22 (59%).
1α-Hydroxy-2-(30-hydroxypropylidene)-19,24,25,26,27-
pentanorvitamin D3 (9). To a solution of the protected vitamin 43
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Journal of Medicinal Chemistry
ARTICLE
(1.4 mg, 1.91 μmol) in anhydrous THF (1.3 mL), tetrabutylammonium
fluoride (1.0 M in THF, 86 μL, 86 μmol) and triethylamine (16 μL)
were added. The mixture was stirred under argon at room temperature
for 18 h, poured into brine, and extracted using ethyl acetate and diethyl
ether. The organic extracts were washed with brine, dried using MgSO4,
and evaporated. The residue was purified by HPLC (9.4 mm ꢁ 25 cm
Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol (8:2) solvent
system. Pure 19-norvitamin 9 (0.56 mg, 72%) was collected at RV
25.5 mL. On a reversed-phase HPLC (9.4 mm ꢁ 25 cm Eclipse XDB-
C18 column, 4 mL/min) using a methanol/water (95:5) solvent system,
compound 9 was collected at RV 42 mL.
(20S)-1α-Hydroxy-2-(30-hydroxypropylidene)-19,24,25,
26,27-pentanorvitamin D3 (10). Treatment of the protected vita-
min 44 with tetrabutylammonium fluoride, performed as described for
43, gave a vitamin D compound that was purified by HPLC (9.4 mm ꢁ
25 cm Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol (8:2)
solvent system. Pure 19-norvitamin 10 (53%) was collected at RV
25.5 mL. On a reversed-phase HPLC (9.4 mm ꢁ 25 cm Eclipse XDB-
C18 column, 4 mL/min) using a methanol/water (95:5) solvent system,
vitamin 10 was collected at RV 42 mL.
1α-Hydroxy-2-(30-hydroxypropylidene)-20-methyl-19,24,
25,26,27-pentanorvitamin D3 (11). Treatment of the protected
vitamin 45 with tetrabutylammonium fluoride, performed as described
for 43, gave a vitamin D compound that was purified by HPLC (9.4 mm
ꢁ 25 cm Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol
(7:3) solvent system. Pure 19-norvitamin 11 (75%) was collected at RV
24.5 mL. On a reversed-phase HPLC (9.4 mm ꢁ 25 cm Eclipse XDB-
C18 column, 4 mL/min) using a methanol/water (95:5) solvent system,
vitamin 11 was collected at RV 27 mL.
1α-Hydroxy-2-(30-hydroxypropylidene)-19,23,24-trinorvita-
min D3 (12). Treatment of the protected vitamin 46 with tetrabuty-
lammonium fluoride, performed as described for 43, gave a vitamin D
compound that was purified by HPLC (9.4 mm ꢁ 25 cm Zorbax-Sil
column, 4 mL/min) using a hexane/2-propanol (7:3) solvent system.
Pure 19-norvitamin 12 (98%) was collected at RV 24 mL. On a reversed-
phase HPLC (9.4 mm ꢁ 25 cm Eclipse XDB-C18 column, 4 mL/min),
using a methanol/water (95:5) solvent system, vitamin 12 was collected
at RV 31.5 mL.
Crystallographic Studies. Crystal Data (For Compound 37).
C17H29NO, M = 263.41, T = 100(2) K, orthorhombic, space group
P212121, Z = 8, a = 6.2985(5), b = 21.9702(16), c = 22.7730(14) Å, αβγ =
90°, V = 3151.3(4) Å3, Dx = 1.110 g cmꢀ3
.
3
Structure Determination. The data were collected using the Kuma
KM4CCD k-axis diffractometer with graphite-monochromated MoKα
radiation. The crystal was positioned at 65 mm from the KM4CCD
camera. Frames (1204) were measured at 1.0° intervals with a counting
time of 20 s. The data were corrected for Lorentz and polarization effects.
No absorption correction was applied. Data collection, cell refinement,
and data reduction were carried out with the Kuma Diffraction programs
CrysAlis CCD and CrysAlis RED.35
The structure was solved by direct methods36 and refined using
SHELXL.37 The refinement was based on F2 for all reflections except
those with very negative intensities. Weighted R factors wR and all
goodness-of-fit S values were based on F2. Conventional R factors were
2
based on F with F set to zero for negative F2. The F02 > 2s(F0 ) criterion
was applied only for the R factors calculation and was not relevant to the
choice of reflections for the refinement. The R factors based on F2 are
about twice as large as those based on F. All hydrogen atoms were located
from a differential map and refined isotropically. Scattering factors were
taken from Tables 6.1.1.4 and 4.2.4.2 from the International Tables for
Crystallography.38
Crystallographic data for the structure reported in this paper has been
deposited at the Cambridge Crystallographic Data Centre with deposi-
tion number CCDC-838768.
’ ASSOCIATED CONTENT
S
Supporting Information. Purity criteria of the vitamin D
b
analogues 9ꢀ13, their 1H NMR spectra and spectral data of the
all synthesized compounds, experimental procedures for the chemi-
cal synthesis of compound 41, and figures with doseꢀresponse
curves derived from cellular differentiation assay of the vitamin D
analogues 9ꢀ13. CCDC-838768 contains the supplementary
crystallographic data for this article. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif. This material is available
free of charge via the Internet at http://pubs.acs.org.
(20S)-1α-Hydroxy-2-(30-hydroxypropylidene)-19,23,24-
trinorvitamin D3 (13). Treatment of the protected vitamin 47 with
tetrabutylammonium fluoride, performed as described for 43, gave a
vitamin D compound that was purified by HPLC (9.4 mm ꢁ 25 cm
Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol (7:3) solvent
system. Pure 19-norvitamin 13 (98%) was collected at RV 24 mL. On a
reversed-phase HPLC (9.4 mm ꢁ 25 cm Eclipse XDB-C18 column,
4 mL/min) using a methanol/water (95:5) solvent system, vitamin 13
was collected at RV 30 mL.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 608-262-1620. Fax: 608-262-7122. E-mail: deluca@
biochem.wisc.edu.
’ ACKNOWLEDGMENT
Biological Studies. In Vitro Studies. VDR binding, HL-60 differ-
entiation, and 24-hydroxylase transcription assays were performed as
previously described.19
The work was supported by a fund from the Wisconsin Alumni
Research Foundation. A special thanks to Jean Prahl and Xiao-
hong Ma for carrying out the in vitro studies and to Shinobu
Miyazaki and Xiaohong Ma for conducting the in vivo studies.
In Vivo Studies. Bone Calcium Mobilization and Intestinal Cal-
cium Transport. Male weanling SpragueꢀDawley rats were purchased
from Harlan (Indianapolis, IN). The animals were group housed and
placed on Diet 11 (0.47% Ca) + AEK oil for one week followed by Diet
11 (0.02% Ca) + AEK oil for 3 weeks. The rats were then switched to a
diet containing 0.47% Ca34 for one week followed by two weeks on a diet
containing 0.02% Ca. Dose administration began during the last week on
the 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 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.19
’ ABBREVIATIONS USED
1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; VDR, vitamin D
receptor
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