Design, synthesis, and biological evaluation of a 1α,25-dihydroxy-19- norvitamin D3 analogue with a frozen A-ring conformation

Article abstract of DOI:10.1021/jm070635+

To establish the conformation of vitamin D compounds responsible for biological activity, a 1α,25-dihydroxy-19-norvitamin D analogue 4 possessing a 1α-hydroxy group fixed in the axial orientation (β-chair form) was synthesized. The starting compounds were

Full text of DOI:10.1021/jm070635+

6154
J. Med. Chem. 2007, 50, 6154-6164
Design, Synthesis, and Biological Evaluation of a 1r,25-Dihydroxy-19-norvitamin D3 Analogue
with a Frozen A-Ring Conformation
Rafal R. Sicinski,†,‡ Agnieszka Glebocka,†,‡ Lori A. Plum,† and Hector F. DeLuca*,†
Department of Biochemistry, UniVersity of WisconsinsMadison, 433 Babcock DriVe, Madison, Wisconsin 53706, and Department of Chemistry,
UniVersity of Warsaw, ul. Pasteura 1, 02-093 Warsaw, Poland
ReceiVed June 4, 2007
To establish the conformation of vitamin D compounds responsible for biological activity, a 1R,25-dihydroxy-
19-norvitamin D analogue 4 possessing a 1R-hydroxy group fixed in the axial orientation (â-chair form)
was synthesized. The starting compounds were bicyclic lactones 6, 7a, and 7b, derived from the quinic acid
lactone, which were converted to the bicyclic ketone 13. Julia coupling of this compound with sulfone 15
produced the 19-norvitamin D analogue 4, possessing an additional ring connecting the 3â-oxygen and
C-2, and the isomeric 3â-hydroxy compound 5. In Vitro, both analogues 4 and 5 exhibit reduced activity
compared to the natural hormone 1, but the binding, differentiation, and transcriptional activities of analogue
5 are markedly higher than that of 4 constrained in the R-chair conformation. Surprisingly, in ViVo tests in
mice showed that the analogue 4 significantly increases serum calcium at dose levels similar to 1R,25-
(OH)2D3. These seemingly discordant results are discussed.
Introduction
It has been established that 1R,25-dihydroxyvitamin D3a (1R,-
25-(OH)2D3, calcitriol, 1; Figure 1), the functional metabolite
of vitamin D, exerts its control over a multitude of biological
processes related to calcium and phosphorus homeostasis, cell
proliferation and differentiation, and immune regulation.1,2
Numerous studies have indicated that these biological functions
of the natural hormone 1R,25-(OH)2D3 are mediated through
the vitamin D receptor (VDR),3 a nuclear protein which belongs
to the nuclear receptor superfamily.4 A specific interaction
between 1R,25-(OH)2D3 and the VDR promotes some confor-
mational changes in the protein and allows its subsequent
binding to the retinoid X receptor (RXR) and several coacti-
vators. The formed complex binds to a vitamin D response
element (VDRE) inducing transcription.5 It is well-known that
the presence of a 1R-hydroxyl group in the vitamin D molecule
is crucial for the receptor binding.6,7 Accumulated crystal-
lographic data indicate that vitamin D compounds are bound to
the VDR with the ring A existing in a â-chair conformation
(Figure 2a) and possessing an equatorially oriented 1R-hydroxyl.
However, in all vitamins which formed these crystalline
complexes with the human, rat, or zebra fish VDR, i.e., the
natural hormone 1R,25-OH)2D38-10 and its analogues with the
modified side chains10-14 or 2R-substituents15 as well as 19-
Figure 1. Chemical structure of 1R,25-dihydroxyvitamin D3 (calcitriol,
nor-1R-hydroxylated D vitamins with 2-methyl9 or 2-methyl-
1) and its analogues.
ene9,16 substituents, equilibration between the ligand’s A-ring
We have recently described the preparation of 2-(3′-hydrox-
ypropylidene)-19-nor-1R,25-(OH)2D3 (2) and its alkoxy deriva-
tive 3.17,18 Since both of these analogues were characterized by
high binding ability to the nuclear vitamin D receptor (VDR),
it encouraged us to attempt the synthesis of a closely related
19-norvitamin D compound 4 in which the propylidene sub-
stituent at C-2 is connected to a 3â-oxygen atom.19 The
structural features of this molecule (presence of an additional
ring and a “flattening bond” system)20 should restrain the A-ring
from interconversion to the alternative chair conformation and
“freeze” it in the R-form (Figure 2b).
chair forms was possible. As a consequence of this fact, the
actual A-ring conformation of the complexed ligand, dissolved
in the physiological milieu, could be still considered as
uncertain. Therefore, a vitamin D analogue existing only in the
opposite R-chair A-ring conformation and possessing a 1R-
hydroxyl group “frozen” in the axial orientation seemed to be
an interesting synthetic target.
* To whom correspondence should be addressed. Telephone: 608-262-
1620, fax: 608-262-7122, e-mail: deluca@biochem.wisc.edu.
† University of WisconsinsMadison.
‡ University of Warsaw.
a Abbreviations: 25-OH-D3, 25-hydroxyvitamin D3; 1R,25-(OH)2D3,
1R,25-dihydroxyvitamin D3; VDR, vitamin D receptor; hVDR, human
vitamin D receptor; LBD, ligand binding domain; MOM, methoxymethyl;
SEM, 2-(trimethylsilyl)ethoxymethyl.
The strategy of our synthesis was based on the Julia
coupling21,22 of the bicylic ketone of structure 13 (Scheme 1)
with the protected sulfone 15 serving as the C,D-ring fragment.
10.1021/jm070635+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/26/2007

1R,25-Dihydroxy-19-norVitamin D3 Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6155
Figure 2. (a) A-Ring conformational equilibrium of 1R-hydroxyvitamin D analogues. The A-ring conformations of the synthesized vitamin D
analogues 4 (b) and 5 (c).
We have recently described a conversion of the commercially
available (1R,3R,4S,5R)-(-)-quinic acid into the bicyclic lac-
tones substituted with alkoxyalkylidene groups (6 and 7a,b).17
We anticipated that these compounds, especially the Z-isomer
6, might be useful for the construction of the desired A-ring
unit 13. Sulfone 15, in turn, could be easily prepared22 from
the protected 25-hydroxy Grundmann ketone.
“below” (R) the plane of C(4)-C(3)dC(2)-C(7) fragment
(Figure 3a), is energetically preferred (steric energy lower by
2.44 kcal/mol) to an alternative conformation in which five-
atom moiety C(4)-C(3)dC(2)-C(7)-O is almost planar
(Figure 3b). Also, the comparison of the respective values of
dihydropyran proton couplings observed in the 1H NMR
spectrum of 10 with those predicted by the molecular modeling
program fully supported the half-chair form (Figure 3a,b).
Sodium borohydride reduction of 10 furnished a bicyclic triol
11. Periodate cleavage of the vicinal diol and subsequent
silylation of the formed hydroxy ketone 12 provided the desired
Results and Discussion
Chemistry. As a first starting compound for the synthesis
of the ketone 13, we chose bicyclic lactone 6 (Scheme 1)
prepared from the quinic acid as described previously.17 A wide
array of different reagents has been tried for deprotection of
the terminal primary hydroxy group in 6. Unsuccessful attempts
to remove the MOM group included HCl in 2-propanol, AG
50W-X4 in methanol, CF3COOH in methylene chloride, LiBF4
in acetonitrile, Me3SiBr in methylene chloride, and Ph3CBF4
in methylene chloride, whereas low yields of deprotected
derivative were achieved with BuSH and MgBr2 in ethyl ether
and B-chlorocatecholborane in methylene chloride. Treatment
of 6 with aluminum iodide in acetonitrile provided the best yield
(71%) of the desired 3′-hydroxypropylidene compound 8a that
was subsequently tosylated under standard conditions. Consistent
with our expectations,23,24 reaction of the tosylate 9a with
tetrabutylammonium fluoride gave an excellent yield (89%) of
the cyclized product 10. The 1H NMR spectrum of 10 supported
its tricyclic structure. The signals of the olefinic proton at C-3
and a methine proton at C-7 appeared as a narrow (w/2 ) 11
Hz) and a broad (w/2 ) 22 Hz) multiplet, respectively. Spin
decoupling experiments confirmed that broadening of the latter
signal was due to additional homoallylic couplings with the
protons at C-4; such effects are often observed in cyclic systems.
Detailed analysis of proton-proton coupling data allowed us
to establish that an additional 5,6-dihydro-2H-pyran ring exists
in the half-chair conformation. Molecular modeling calculations
proved that a form of 10, which possess an oxygen atom situated
1
A-ring fragment 13. Interestingly, analysis of H NMR data
seem to indicate that half-chair conformation of the dihydro-
pyran moiety is also favored in all compounds subsequently
synthesized from 10. Magnitudes of the vicinal couplings of
5-H confirmed the axial orientation of the oxygen substituent
at C-5 in both hexahydrochromenone derivatives 12 (Figure 3c)
and 13 (Figure 3d).
We also explored an alternative pathway to 13 starting from
the mixture of isomeric compounds 7a,b prepared by us
previously.17 Selective deprotection of the primary hydroxyl was
achieved without problems by acetic acid-catalyzed hydrolysis,
and the formed alcohols 8a,b were separated by column
chromatography. The main E-isomer 8b was tosylated and the
tosylate 9b subjected to reduction with sodium borohydride.
Spectral data of the obtained product 14 revealed that reduction
of the lactone moiety was followed by nucleophilic substitution
of the tosyl group resulting in the closure of the dihydropyran
ring. Periodate oxidation of the diol 14 furnished the ketone
13, identical with the previously obtained. Thus, it was proved
that both isomeric hydroxypropylidene lactones 8a,b can be
efficiently converted to the bicyclic ketone 13. This hexahy-
drochromenone derivative was in the next step subjected to
modified Julia olefination.
Through preparation of the C,D-ring fragment, sulfone 15
was achieved by the synthetic path depicted in the Scheme 2.

6156 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sicinski et al.
Scheme 1
Thus, the tertiary hydroxyl in the starting 25-hydroxy Grund-
mann ketone 1625 was protected as an SEM ether, and the
product was transformed into an R,â-unsaturated aldehyde 17
by treatment with the deprotonated N-tert-butyl-2-(trimethyl-
silyl)acetaldimine26 followed by oxalic acid hydrolysis. It was
established that only the product with E-stereochemistry was
formed in such reaction sequence. The aldehyde 17 was reduced
with DIBALH, and the resulting allylic alcohol 18 was subjected
to Mitsunobu reaction with 2-mercaptobenzothiazole followed
by oxidation to form the desired C,D-ring fragment 15. Coupling
of the ketone 13 with an anion, generated from the thiazoline
sulfone 15 and lithium bis(trimethylsilyl)amide, followed by
removal of the silyl protecting groups gave the expected mixture
of two 19-norvitamin D analogues 4 and 5 which were purified
and separated by straight- and reversed-phase HPLC. The
structures of both products were unequivocally established by
analysis of their 1H NMR, UV, and mass spectra. Spin
1R,25-dihydroxy-19-norvitamin D3,27 characterized by a sig-
nificant bias toward one chair form of the ring A, proved
especially useful. Chemical shifts of the corresponding olefinic
and A-ring protons as well as their multiplet patterns show close
similarity in the respective vitamin D pairs: (a) analogue 4 and
(Z)-2-ethylidene-19-nor-1R,25-(OH)2D3 (Figure 4a), (b) ana-
logue 5 and (E)-2-ethylidene-19-nor-1R,25-(OH)2D3 (Figure 4b).
It has, therefore, been confirmed that the secondary A-ring
hydroxyl in vitamin D analogues 4 (Figure 2b) and 5 (Figure
2c) has an axial orientation. Ring A in these compounds, because
of the presence of an exocyclic double bond being a part of an
additional six-membered ring, is prevented from interconversion
to the alternative chair conformer. Thus, the compound 4
represents a unique analogue of the vitamin D hormone, with a
“frozen” A-ring conformation and can be used for evaluation
of the role of 1R-OH orientation in binding with VDR and
subsequent biological activity.
1
decoupling and H NOE difference spectroscopy experiments
Biological Evaluation. Among the synthesized vitamin D
compounds 4 and 5, possessing an additional dihydropyran ring,
only the latter has significant binding affinity to the vitamin D
receptor (Table 1) albeit decreased more than 500 times
compared to 1R,25-(OH)2D3 (1). Studies on the ability of the
involving olefinic protons at C-6 and C-7 were helpful in
establishing the A-ring conformations of the synthesized 19-
norvitamins. Comparison of their proton NMR spectra with
those of the previously synthesized 2-ethylidene analogues of

1R,25-Dihydroxy-19-norVitamin D3 Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6157
Figure 3. Preferred, energy-minimized conformations of the synthesized compounds 10 (a), 12 (c), and 13 (d); a higher energy conformer of 10
1
(b) is also shown. The most informative H-1H coupling constants are given. Values of the calculated couplings are given in parentheses.
vitamins 4 and 5 to induce differentiation of human promyelo-
cyte HL-60 cells into monocytes show that their potency is
decreased by 3 and 2 orders of magnitude, respectively, in
comparison with the natural hormone 1. Analogue 5 also has
weak transcriptional activity, indicated in the 24-hydroxylase
(CYP-24) promoter driving luciferase reporter gene system,
whereas 4 has been found completely inactive in this regard.
Taking into account the above-described in Vitro test results,
very low calcemic activity or even complete lack of in ViVo
activity might be expected for the synthesized analogues 4 and
5. However, studies conducted with these compounds in ViVo
in vitamin D-deficient mice in doses exceeding 30 times that
of the natural hormone, showed that these compounds have a
similar calcemic response after 24 h, as 1 and isomer 4 was
markedly more active 48 h after the dose was administered
(Figure 5). A second study conducted in vitamin D-sufficient
CD-1 mice again showed the remarkable in ViVo activity of
analogue 4 to raise blood calcium levels (Figure 6). However,
no significant increase in serum calcium was detected with
analogue 5, which is consistent with the vitamin D-deficient
mouse study in that the apparent increase in serum calcium after
administration of analogue 5 did not turn out to be statistically
significant given the variation and the few animals per group
(n ) 3). Interestingly, the analogue that had the least amount
of activity in Vitro (analogue 4 in the R-chair form) has the
most in ViVo activity, and furthermore its potency in ViVo is
similar to that of the native hormone. The last experiment
conducted with analogue 4 is shown in Figure 7. This study
was again performed in vitamin D-sufficient CD-1 mice. Two
different routes of administration (intraperitoneal injection vs
oral gavage) were studied. Both routes of administration are
effective at causing an increase in serum calcium; however,
intraperitoneal injection is more effective than oral gavage. This
observation is similar to that of the native hormone. It should
also be noted that in the vitamin D-deficient animals, there is a
significant delay compared to 1R,25-(OH)2D3 for an observed
increase in serum calcium to occur when the animals are given
analogue 4. It can be suggested that in ViVo both analogues 4

6158 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sicinski et al.
Figure 4. Comparison of preferred, energy-minimized A-ring conformations of (a) the synthesized analogue 4 and (Z)-2-ethylidene-19-nor-1R,-
25-(OH)2D3, (b) the synthesized analogue 5 and (E)-2-ethylidene-19-nor-1R,25-(OH)2D3. The most informative proton NMR data (chemical shifts
and multiplet patterns) and NOEs are given.
Scheme 2
and 5 might undergo some metabolic change (cleavage of
dihydropyran rings at the ether bond?)28 resulting in one
compound with very little biological activity and the other with
pronounced in ViVo activity. If ring cleavage is the metabolic
conversion taking place, it is understandable why analogue 4
is much more active than analogue 5 in ViVo. Unlike analogue
4, cleavage of the dihydropyran ring in analogue 5 would not
produce a 1R-hydroxyl group which is required for VDR

1R,25-Dihydroxy-19-norVitamin D3 Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6159
Table 1. VDR Binding Properties,a HL-60 Differentiating Activities,b and Transcriptional Activitiesc of the Vitamin D Analogues 4 and 5
a Competitive binding of 1R,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 dose-response curves and represent the inhibition constant when
radiolabeled 1R,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 analogue Ki to the Ki for 1R,25-
(OH)2D3. b Induction of differentiation of HL-60 promyelocytes to monocytes by 1R,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 dose-response curves and represent the analogue concentration capable of inducing 50% maturation. The differentiation
activity ratio is the average ratio of the analogue ED50 to the ED50 for 1R,25-(OH)2D3. c Transcriptional assay in rat osteosarcoma cells stably transfected
with a 24-hydroxylase gene reporter plasmid. The ED50 values are derived from dose-response curves and represent the analogue concentration capable of
increasing the luciferase activity 50%. The lucerifase activity ratio is the average ratio of the analogue ED50 to the ED50 for 1R,25-(OH)2D3.
Figure 5. Serum calcium change in response to a single, intraperitoneal injection in D-deficient CD-1 mice. Statistical significance (p < 0.05)
compared to the vehicle group is indicated by an asterisk.
binding.6,7 Further studies will be required to provide an
understanding for the in ViVo biological activity.
nor counterparts obtained in our laboratory34 prompted us to
suggest that an axial orientation of 1R-OH might be of crucial
importance for exertion of calcemic activity. It was found that
2R-alkylated vitamins, characterized by strong bias (above 90%)
toward conformers with the axial hydroxyl at C-1, are much
more biologically potent then the respective 2â-isomers existing
in solution primarily in the opposite â-chair form.34 Afterward,
however, the Moras group reported the crystal structure of the
hVDR ligand binding domain (LBD) complexed with hormone
18 and several other ligands characterized by an unnatural
configuration at C-20.11 All these results clearly indicated that
the receptor binds (at least in the crystalline state) vitamin D
compounds having their A-rings in the â-chair conformation.
Even more convincing was the recent report from our laboratory,
Discussion
Conformational equilibrium of the cyclohexane ring A of
vitamin D compounds and its influence on biological activity
has been studied for more than three decades.29,30 In 1974
Okamura hypothesized that the â-chair form, which possesses
an equatorial 1R-hydroxyl, is responsible for the biological
activities of vitamin D analogues.31 Our early studies on 1R,-
25-dihydroxy-10,19-dihydrovitamin D3 seemed to contradict this
suggestion.32 Then, the results of biological testing and con-
formational analysis of 2-methyl-substituted analogues of the
hormone 1, synthesized by Japanese scientists,33 and their 19-

6160 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sicinski et al.
Figure 6. Serum calcium change in response to a single, intraperitoneal injection in D-sufficient CD-1 mice. Statistical significance (p < 0.05)
compared to the vehicle group is indicated by an asterisk.
Figure 7. Serum calcium change in response to different routes of administration in D-sufficient CD-1 mice. Statistical significance (p < 0.05)
compared to the vehicle group is indicated by an asterisk.
in which Vanhooke described the crystal structure of the rat
VDR LBD complexed with a 1R,25-dihydroxy-2R-methyl-19-
norvitamin D3,9 possessing highly elevated biopotency in both
intestine and bone.34 The study shows that this vitamin D
compound also adopts the â-chair A-ring conformation in its
crystalline complex with VDR. However, in all these cases
interconversion between the ligand’s A-ring chair conformers
was not prohibited, and therefore some doubts could arise which
form of the ligand-VDR complex actually exists in the real
physiological environment. Thus, it was tempting to synthesize
and biologically evaluate a vitamin D compound possessing an
axially oriented hydroxyl group at C-1 and unable to change
its A-ring conformation. The 1R,25-dihydroxyvitamin D3 ana-
logue 4, described in the present work, fulfills these require-
ments. It was established that such vitamin D does not bind to
the VDR and lacks activity in cellular differentiation and in
inducing transcription of a vitamin D-responsive gene. Notably,
its isomer 5 possessing free 3â- and 25-hydroxyl groups, but
characterized by a “frozen” A-ring â-chair conformation, was
found to be ca. 560 times less potent in binding to the receptor
than the hormone 1. Such binding ability could be expected for
a 25-OH-D3 derivative in which the 1R-oxygen function cannot
act as a hydrogen donor to create the hydrogen bonds with the
amino acids from the LBD.6 Considering the structure of the
vitamins 4 and 5, possessing a trans-conformation of the
intercyclic 5,7-diene fragment and the 25-hydroxycholestane
side chain, it would be expected that only genomic VDR ligand
binding pocket35 can accommodate both these compounds.
The biological results obtained in Vitro on the synthesized
analogues 4 and 5 clearly confirm that the A-ring â-chair
conformation and, consequently, equatorial orientation of the
1R-OH are necessary for the vitamin D compound to ensure its
binding with VDR and exertion of biological activity. Biological
evaluation of the tested vitamins in ViVo does not generate results
consistent with those obtained in Vitro, most likely due to
metabolic transformation of both compounds occurring in the
animal.
Conclusions
The conformations of the A-rings of vitamins 4 and 5, as
well as the structures of intermediate compounds used for the
construction of their A-ring parts, were established by NMR
spectroscopic methods. Analysis of the observed vicinal proton
coupling constants proved that in the synthesized vitamin D
analogues 4 and 5, possessing an additional dihydropyran ring,
their A rings could only exist in a single conformation, R- and
â-chair, respectively. Biological in Vitro testing of the analogues
4 and 5 allowed us to conclude that the presence of an
equatorially oriented free hydroxyl at C-1 is necessary for
binding to the vitamin D receptor. Thus, only a vitamin D
analogue that can assume a â-chair A-ring conformation can
be accepted by the VDR, further inducing conformational
changes crucial for the ligand-receptor activation.
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
1
UV-vis spectrophotometer in ethanol. H nuclear magnetic reso-
nance (NMR) spectra were recorded at 400 and 500 MHz with a
Bruker Instruments DMX-400 and DMX-500 Avance console
spectrometers in deteriochloroform. 13C nuclear magnetic resonance
(NMR) spectra were recorded at 125 MHz with a Bruker Instru-
ments DMX-500 Avance console in deuteriochloroform. Chemical

1R,25-Dihydroxy-19-norVitamin D3 Analogue
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6161
shifts (δ) are reported downfield from internal Me4Si (δ 0.00).
Electron impact (EI) mass spectra were obtained with a Micromass
AutoSpec (Beverly, MA) instrument. High-performance liquid
chromatography (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 before
use from sodium benzophenone ketyl under argon.
The starting compounds 6 and 7a,b were obtained from com-
mercial (-)-quinic acid according to the published procedures.17
(4Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-4-
[3′-hydroxypropylidene]-6-oxabicyclo[3.2.1]octan-7-one (8a). To
a solution of 6 (26 mg, 63 µmol) in anhydrous CH3CN (0.6 mL)
was added AlI3 (170 mg, 0.42 mmol) at 0 °C under argon. The
mixture was stirred at 0 °C for 50 min, poured into aq Na2S2O3,
and extracted with ethyl acetate. The extract was washed with brine,
dried (Na2SO4), and evaporated. The residue was purified by flash
chromatography. Elution with hexane/ethyl acetate (6:4) afforded
pure alcohol 8a (16.5 mg, 71%) as a colorless oil.
extracts were combined and evaporated to dryness with benzene.
The solid residue was then washed a few times with warm
chloroform. The combined chloroform extracts were concentrated
to a small volume and applied on a silica Sep-Pak cartridge. Elution
with hexane/ethyl acetate (1:9, 20 mL) yielded a pure semisolid
triol 11 (6 mg, 75%).
(5R,8aR)-5-Hydroxy-2,3,5,6,8,8a-hexahydrochromen-7-one (12).
Sodium periodate-saturated water (50 µL) was added to a solution
of the triol 11 (5 mg, 2.6 µmol) in methanol (200 µL) at 0 °C. The
mixture was stirred at 0 °C for 1 h, thioanisole (3 µL) was added,
and stirring was continued for 10 min. The mixture was diluted
with benzene/ethyl acetate (1:1, 1 mL) and filtered through a silica
Sep-Pak. The Sep-Pak was washed with an additional 5 mL of the
same solvent system, the combined solutions were evaporated, and
the residue was redissolved in hexane/ethyl acetate (7:3) and applied
on a silica gel Sep-Pak. Elution with the same solvent system (10
mL) provided aromatic compounds whereas a pure hydroxy ketone
12 (4.1 mg, 98%) was eluted with hexane/ethyl acetate (1:1, 10
mL) as a colorless oil.
(5R,8aR)-5-[tert-Butyldimethylsilyl)oxy]-2,3,5,6,8,8a-hexahy-
drochromen-7 -one (13). (a) To a solution of hydroxy ketone 12
(4 mg, 24 µmol) in anhydrous methylene chloride (90 µL) were
added 2,6-lutidine (7 µL, 60 mmol) and tert-butyldimethylsilyl
trifluoromethanesulfonate (12 µL, 51 mmol) at -50 °C. The
reaction mixture was stirred at -50 °C for 50 min, diluted with
cold methylene chloride, and poured into water. The organic phase
was washed with saturated CuSO4 and water, dried (Na2SO4), and
evaporated. The residue was redissolved in hexane and applied on
a silica gel Sep-Pak. Elution with hexane/ethyl acetate (95:5, 10
mL) provided a less polar compound (2.1 mg), the TBDMS
derivative of the enol ether derived from 13. The desired protected
hydroxy ketone 13 (3.3 mg, 49%) was eluted with hexane/ethyl
acetate (9:1, 10 mL) as a colorless oil. Further elution with the
same solvent system afforded 2,3,8,8a-tetrahydrochromen-7-one
(0.6 mg).
(b) To a solution of compound 9b (20 mg, 38 µmol) in anhydrous
ethanol (0.5 mL) at 0 °C was added NaBH4 (14 mg, 0.38 mmol),
and the mixture was stirred at 5 °C for 18 h and then for 20 h at
room temperature. The saturated NH4Cl was added, and the mixture
was poured into brine and extracted several times with ether and
methylene chloride. The extracts were washed with brine, combined,
dried (MgSO4), and evaporated. The oily residue was purified by
silica Sep-Pak cartridge. Elution with hexane/ethyl acetate (7:3)
gave pure product 14 as semicrystals (115 mg, 79%).
(4E)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-4-
[3′-hydroxypropylidene]-6-oxabicyclo[3.2.1]octan-7-one (8b). To
a solution of a mixture of isomeric compounds 7a and 7b (2:3 ratio;
700 mg, 1.49 mmol) in THF (6 mL) were added acetic acid (18
mL) and water (6 mL). The solution was stirred at room temperature
for 5 h, cooled to 0 °C, poured into saturated NaHCO3, and extracted
with ethyl acetate. The organic layer was dried (MgSO4) and
evaporated. The oily residue was purified by column chromatog-
raphy on silica. Elution with hexane/ethyl acetate (75:25) afforded
a less polar Z-isomer 8a (84 mg), a mixture of 8a and 8b (147
mg), and a pure more polar E-isomer 8b (180 mg); overall yield
of both products 77%.
(4Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-6-
oxa-4-[3′-(p-toluenesulfonyloxy)propylidene]bicyclo[3.2.1]octan-
7-one (9a). To a solution of hydroxy compound 8a (16 mg, 43
µmol) in anhydrous pyridine (140 µL) were added at 0 °C
p-toluenesulfonyl chloride (24 mg, 127 µmol) and a catalytic
quantity of 4-(dimethylamino)pyridine. The mixture was stirred at
0 °C for 1 h and at 6 °C for 18 h. It was then poured into ice/
saturated NaHCO3, shaken for 15 min, and extracted with ethyl
acetate and benzene. The combined extracts were washed with
saturated NaHCO3, water, saturated CuSO4, and water again, dried
(Na2SO4), and evaporated. The residue (ca. 16 mg) was dissolved
in benzene/hexane, applied on a silica Sep-Pak cartridge, and
washed with hexane/ethyl acetate (95:5, 10 mL) to remove polar
impurities. Elution with hexane/ethyl acetate (85:15, 20 mL)
provided a pure oily tosylate 9a (19 mg, 84%).
(4E)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-6-
oxa-4-[3′-(p-toluenesulfonyloxy)propylidene]bicyclo[3.2.1]octan-
7-one (9b). Tosylate 9b was obtained from hydroxy compound 8b
by tosylation performed analogously to the process described above
for 8a except the product was purified by column chromatography.
Apolar impurities were removed with hexane/ethyl acetate (95:5).
Further elution with hexane/ethyl acetate (75:25) provided a pure
oily tosylate 9b in 90% yield.
(1R,7R,9S)-(9-Acetoxy-6,11-dioxa-tricyclo[7.2.1.0*2,7*]dodec-
2-en-10-one (10). Solution of tosylate 9a (18.8 mg, 36 µmol) in
dry THF (8 mL) was treated with tetrabutylammonium fluoride
(1.0 M in THF, 180 µL, 180 µmol). The mixture was stirred under
argon at room temperature for 18 h, poured into brine, and extracted
with ethyl acetate and benzene. Organic extracts were washed with
brine, dried (Na2SO4), and evaporated. The oily residue was
dissolved in hexane, applied on a silica Sep-Pak cartridge, and
washed with hexane/ethyl acetate (85:15, 20 mL) to give a pure
tricyclic product 10 (7.6 mg, 89%) as an oil.
Sodium periodate cleavage of the diol 14 was performed
analogously to the process described above for 11. The semicrys-
talline ketone 13 (97% yield) was eluted from a Sep-Pak cartridge
with hexane/ethyl acetate (9:1).
[(1R,3aS,7aR)-7a-Methyl-1-[(R)-6-[(2-trimethylsilyl)-
ethoxymethoxy]-6-methylheptan-2-yl]-octahydroinden-(4E)-ylide-
ne]acetaldehyde (17). To a solution of 25-hydroxy Grundmann
ketone 1625a (186 mg, 0.663mmol) in anhydrous methylene chloride
(0.3 mL) and N,N-diisopropylethylamine (448 mg, 3.46 mmol) was
added 2-(trimethylsilyl)ethoxymethyl chloride (166 mg, 0.995
mmol). The mixture was strirred at room temperature for 1.5 h
and then poured into 2% aq HCl solution and extracted with
methylene chloride. The extracts were washed with diluted NaH-
CO3, dried (MgSO4), and evaporated. An oily residue was purified
by column chromatography. Elution with hexane/ethyl acetate (95:
5) gave pure protected Grundmann ketone, (1R,3aR,7aR)-7a-methyl-
1-[(R)-6-[(2-trimethylsilyl)ethoxymethoxy]-6-methylheptan-2-yl]-
octahydroinden-4-one (220 mg, 81%) as a colorless oil.
To a solution of diisopropylamine (70 µL, 0.507 mmol) in
anhydrous THF (0.58 mL) was added n-BuLi (2.0 M in cyclohex-
ane, 254 µL, 0.507 mmol) under argon at -78 °C. After being
stirred for 5 min, the mixture was warmed to 0 °C and a solution
of N-tert-butyl-2-(trimethylsilyl)acetaldimine26c (504 µmol, 86 mg)
in anhydrous THF (0.5 mL) was added. The mixture was stirred
for 20 min and cooled to -78 °C, and a solution of protected
Grundmann ketone (86 mg, 0.205 mmol) in anhydrous THF (1.1
mL) was added. The resulting mixture was allowed to warm to
(5R,7R,8aR)-7-Hydroxymethyl-3,5,6,7,8,8a-hexahydro-2H-
chromene-5,7-diol (11). To a solution of tricyclic compound 10
(9.5 mg, 40 µmol) in anhydrous ethanol (1.0 mL) at 0 °C was added
NaBH4 (15 mg, 0.4 mmol). The resultant mixture was then stirred
at room temperature for 18 h, a small volume of brine/saturated
NH4Cl was added, and the solvents were removed in vacuum. The
residue was washed several times with warm ethanol. The ethanol

6162 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Sicinski et al.
-20 °C during 30 min. After the mixture was stirred for additional
1.5 h at this temperature, 5% aq solution of oxalic acid solution
(1.4 mL) was added and the mixture was stirred for 1.5 h at room
temperature. Then it was poured into water and extracted with ether.
The organic phase was washed with diluted NaHCO3 and water,
dried (MgSO4), and evaporated. An oily residue was purified by
column chromatography. Elution with hexane/ethyl acetate (95:5)
gave pure oily aldehyde 17 (64 mg, 70%).
2-[(1R,3aS,7aR)-7a-Methyl-1-[(R)-6-[(2-trimethylsilyl)-
ethoxymethoxy]-6-methylheptan-2-yl]octahydroinden-(4E)-ylide-
ne]ethanol (18). Diisobutylaluminum hydride (1.0 M in toulene,
357 µL, 0.357 mmol) was added to a solution of the aldehyde 17
(57 mg, 0.131 mmol) in anhydrous toluene (1 mL) at -78 °C under
argon. The mixture was stirred for 1 h at -78 °C, quenched by
addition of 2 N potassium sodium tartrate, poured into the water,
and extracted with ethyl acetate. The organic extract was washed
with brine, dried (MgSO4), and evaporated. The residue was purified
by column chromatography. Pure allylic alcohol 18 (50 mg, 87%)
was eluted with hexane/ethyl acetate (9:1).
(1R,3aS,7aR)-4-[2-(Benzothiazole-2-sulfonyl)-(4E)-ethylidene]-
7a-methyl-1-[(R)-6-[(2-trimethylsilyl)ethoxymethoxy]-6-methyl-
heptan-2-yl]-octahydroindene (15). To a stirred solution of
2-mercaptobenzotriazole (52.9 mg, 317 µmol) and triphenylphos-
phine (82.4 mg, 317 µmol) in anhydrous methylene chloride (750
µL) were added a solution of the allylic alcohol 18 (91.5 mg, 210
µmoL) in anhydrous methylene chloride (750 µL) and DIAD
(61 µL, 210 µmol) at 0 °C. After being stirred for 1 h at this
temperature, the solvents were evaporated in vacuo. The residue
was redissolved in ethanol and cooled to 0 °C, and 30% H2O2 (130
µL) followed by (NH4)6Mo7O24·4H2O (51.6 mg, 41.9 µmol) was
added. After being stirred for 3 h at room temperature, the mixture
was poured into cold saturated Na2SO3 and extracted with ethyl
acetate. The organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was redissolved in a small volume of
benzene/hexane (1:1) and applied on a silica column. Pure oily
allylic sulfone 15 (105 mg, 81%) was eluted with hexane/ethyl
acetate (98:2).
1R,25-Dihydroxy- and 25-Hydroxy-19-norvitamin D3 Ana-
logues (4 and 5). To a solution of sulfone 15 (38.0 mg, 61 µmol)
in dry THF (200 µL) was added LiHMDS (1 M in THF, 60 µL, 60
µmol) at -78 °C under argon. The solution turned deep red. The
mixture was stirred at -78 °C for 20 min, and a solution of the
ketone 13 (7.5 mg, 26.6 µmol) in THF (100 + 80 µL) was added.
The stirring was continued at -78 °C for 1.5 h, and the reaction
mixture was allowed to warm to -10 °C during ca. 1.5 h. Then
it was poured into saturated NH4Cl and extracted with ether. The
extract was washed with brine, dried (Na2SO4), and evaporated.
The yellow oily residue was purified by column chromatography.
Elution with hexane/ethyl acetate (97:3) afforded the silylated
19-norvitamins (7 mg, 44%). Further elution with hexane/ethyl
acetate (95:5) gave unreacted sulfone 15 (27 mg) whereas un-
changed ketone 13 (1 mg) was eluted with hexane/ethyl acetate
(94:6).
2. In ViWo Studies. First Study. Female, CD-1 mice (6 weeks
old) were purchased from Harlan Sprague-Dawley Co. (India-
napolis, IN). Upon arrival, the animals were placed in a room with
filtered lighting and fed a purified, vitamin D-deficient diet36 for
17 weeks. The mice were then assigned to four groups (n )
3/group) and given a single intraperitoneal injection of vehicle (95:5
propylene glycol/ethanol), 1R,25-(OH)2D3, analogue 4, or analogue
5. Blood was collected prior to dose administration and multiple
time points thereafter. Serum calcium was measured by diluting
with 0.1% lanthanum chloride and reading the absorbance using
an atomic absorption spectrometer. The change in serum calcium
from predose values (baseline) is reported.
Second Study. Female CD-1 mice (6-7 week old) were
purchased from Harlan (Indianapolis, IN). The animals were group
housed and fed a purified diet containing 0.47% calcium.36 After a
5-7 day acclimation period, the animals were assigned to treatment
groups (n ) 5-6/group) and given a single dose of the designated
analogues by intraperitoneal injection. Blood was collected for
serum calcium concentration analyses immediately prior to dose
administration and 72 h following dose delivery. Serum calcium
was analyzed as described above.
Third Study. Female, CD-1 mice (6-7 week old) were
purchased from Harlan (Indianapolis, IN). The animals were group
housed and fed a purified diet containing 0.47% calcium.36 After a
5-7 day acclimation period, the animals were assigned to treatment
groups (n ) 5/group) and given a single dose of the designated
analogues by intraperitoneal injection or oral gavage. Blood was
collected for serum calcium concentration analyses at various
timepoints following dose delivery. Serum calcium was analyzed
as described above.
Statistical Analysis. In vivo data were analyzed by one-way
ANOVA followed by pairwise comparisons when significant overall
differences were detected. Post-hoc analyses included Tukey’s,
Scheffe’s, and Fisher’s LSD tests. Only differences (p < 0.05) that
were present in two out of the three post-hoc tests were considered
significant.
Molecular Modeling. Molecular mechanism studies were used
to establish the energy-minimized structures of the most important
synthetic intermediates. The calculation of optimized geometries
and steric energies was carried out using the algorithm from the
MM+ HyperChem (release 7.0) software package (Autodesk, Inc.).
MM+ is an all-atom force field based on the MM2 functional form.
The couplings observed in the 1H NMR spectra of the synthesized
compounds were compared to those calculated using PC MODEL
(release 9.0) molecular modeling software (Serena Software);
molecular modeling was performed in the MMX mode. The force
field MMX is an enhanced version of MM2, with the pi-VESCF
routines taken from MMP1.
Acknowledgment. The work was supported in part by funds
from the Wisconsin Alumni Research Foundation and the
National Foundation for Cancer Research. A special thanks to
Jean Prahl and Xiaohong Ma for carrying out the in Vitro studies
and to Aaron Hirsch and Jennifer Vaughan for conducting the
in ViVo studies.
The obtained mixture of protected 19-norvitamins (3.5 mg, 5.1
µmol) was dissolved in anhydrous methanol (0.6 mL) and treated
with (+)-10-camphorosulfonic acid (10 mg, 43 µmol). The solution
was stirred under argon at room temperature for 19 h, poured into
brine, and extracted with ethyl acetate. The extract was washed
with diluted NaHCO3 and brine, dried (Na2SO4), and evaporated.
The residue was purified by HPLC (9.4 mm × 25 cm Zorbax-Sil
column, 4 mL/min) using a hexane/2-propanol (9:1) solvent system.
Isomeric 19-norvitamins 4 (0.25 mg, 11%) and 5 (0.87 mg, 39%)
were collected at RV 31 mL and RV 35 mL, respectively. Final
purification and separation of both isomers was achieved by
reversed-phase HPLC (9.4 mm × 25 cm Zorbax-ODS column, 4
mL/min) using methanol/water (97:3) solvent system. 1R,25-
Dihydroxy-19-norvitamin D analogue 4 was collected at RV 31.5
mL and its isomer 5 at RV 18 mL.
Supporting Information Available: Purity criteria and spectral
data of the synthesized compounds; figures with either the competi-
tive binding curves or dose-response curves derived from the
binding and cellular differentiation and transcriptional assays of
the vitamin D analogues 4 and 5. This material is available free of
charge via the Internet at http://pubs.acs.org.
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