New 2-alkylidene 1α,25-dihydroxy-19-norvitamin D3 analogues of high intestinal activity: Synthesis and biological evaluation of 2-(3′-alkoxypropylidene) and 2-(3′-hydroxypropylidene) derivatives

Article abstract of DOI:10.1021/jm051082a

In a search for novel vitamin D compounds of potential therapeutic value, E- and Z-isomers of 1α,25-dihydroxy-2-(3′-hydroxypropylidene)-19- norvitamin D₃, as well as a derivative of the former compound possessing a 3′-(methoxymethoxy)propyliden

Full text of DOI:10.1021/jm051082a

J. Med. Chem. 2006, 49, 2909-2920
2909
New 2-Alkylidene 1r,25-Dihydroxy-19-norvitamin D3 Analogues of High Intestinal Activity:
Synthesis and Biological Evaluation of 2-(3′-Alkoxypropylidene) and 2-(3′-Hydroxypropylidene)
Derivatives
Agnieszka Glebocka,†,‡ Rafal R. Sicinski,†,‡ Lori A. Plum,† Margaret Clagett-Dame,† 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 October 25, 2005
In a search for novel vitamin D compounds of potential therapeutic value, E- and Z-isomers of 1R,25-
dihydroxy-2-(3′-hydroxypropylidene)-19-norvitamin D3, as well as a derivative of the former compound
possessing a 3′-(methoxymethoxy)propylidene substituent at C-2, were efficiently prepared. All vitamins
were obtained in convergent syntheses, starting with (-)-quinic acid and the protected 25-hydroxy Grundmann
ketones. Quinic acid was converted into keto lactone 11, and a substituted hydroxypropylidene group was
attached by Wittig reaction yielding pairs of isomeric compounds 12, 13 and 14, 15. These olefinic products
were then transformed into phosphine oxides 32-34 which were subjected to Lythgoe type Wittig-Horner
coupling with C,D-fragments 35a and 35b. An alternative route was also elaborated that comprised Julia
coupling of sulfones 39a and 39b with the cyclohexanone derivative 23. The binding of all synthesized
vitamins to the full-length rat recombinant vitamin D receptor (VDR) is either similar to or within one log
of 1R,25(OH)2D3. The in vivo tests have revealed that the calcemic activity of all analogues in the E-series
(5a, 6a, 6b) is considerably higher than that of the native hormone.
Introduction
It is well-known that 1R,25-dihydroxyvitamin D3 (1R,25-
(OH)2D3, calcitriol, 1; Figure 1), the hormonally active form
of vitamin D3, exhibits a multitude of biological activities.1,2 It
has been established that the hormone 1R,25-(OH)2D3 controls
the expression of numerous genes whose products are involved
in calcium and phosphorus homeostasis as well as in cellular
differentiation.3 The vitamin D receptor (VDR),4 which belongs
to the nuclear receptor superfamily, plays a crucial role in these
genomic biological actions.5 Upon binding to vitamin D, VDR
undergoes heterodimerization with retinoic X receptor (RXR)
and coactivators, and then the resulting complex binds to a
vitamin D response element (VDRE) inducing transcription.6
In view of such an important role of the vitamin D-VDR
complex we became interested in designing a vitamin D
analogue that could not only bind effectively to VDR, but also
introduce additional interactions that would alter the complex.
In 1998 we reported the synthesis of vitamin D analogues
2a,b which had the A-ring exocyclic methylene unit attached
to C-2, instead of C-10 as in the natural hormone 1.7 These
analogues are characterized by VDR binding affinity comparable
to 1R,25-(OH)2D3 but exhibit a significantly higher (and
selective) ability to mobilize calcium from bone, in particular
20S-isomer 2b (2MD). Recently, 2-ethylidene analogues have
been prepared in our laboratory, and it has been determined
that E-isomers 3a,b are two times more active than the hormone
Figure 1. Chemical structures of 1R,25-dihydroxyvitamin D3 (calcitriol,
1 in binding to VDR.8 Moreover, in vivo studies have shown
1) and analogues.
that these analogues have higher intestinal activity than 1 and
they exhibit similar (3a) or significantly increased (3b) ability
to mobilize calcium from bone. The isomeric Z compounds 4a,b
are less potent, showing no activity in bone. Conformational
analysis and spectral data of 2-ethylidene-19-norvitamin D
analogues prove that their A-ring conformational equilibrium
is significantly shifted, due to a strong A(1,3)-strain interaction,9,10
to the â-chair (E-isomers, Figure 2a) or R-chair form (Z-isomers,
Figure 2b).
Previously we noted very high biological activity of the 2R-
(3′-hydroxypropyl) and 2R-(ω-hydroxyalkoxy) derivatives of the
hormone 111 as well as crystallographic data indicating that the
VDR binding pocket can accommodate rather bulky fragments
* 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.
10.1021/jm051082a CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/25/2006

2910 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Glebocka et al.
Figure 2. Preferred A-ring conformations of 2-alkylidene-1R-hydroxy-19-norvitamin D analogues: E-isomers (a) and Z-isomers (b).
Scheme 1
attached to C-2.12 These facts encouraged us to synthesize 2-(3′-
hydroxypropylidene) derivatives of 19-nor-1R,25-(OH)2D3 (struc-
tures 6 and 7) as well as compounds possessing 2-(3′-
alkoxypropylidene) substituents terminated with an alkoxyl
group, containing an additional oxygen atom capable of forming
hydrogen bonds with the VDR, i.e., the analogues substituted
at C-2 with dCHCH2CH2O(CH2)nOR moiety, where n ) 1, 2,
3, etc. Our choice fell on a relatively simple vitamin D structure
5 with the (methoxy)methoxyl unit attached to the terminal C′-3
atom of the propylidene moiety at C-2. In view of preliminary
results of molecular modeling of the free ligand 5a and its
complex with rVDR,13 such an analogue seemed to be an
interesting synthetic target. The strategy of our synthesis was
based on the Wittig-Horner coupling14 of the readily available
protected 25-hydroxy Grundmann ketones 35a15 and 35b7 with
the corresponding phosphine oxides of structures 32-34.
816 (Scheme 1) easily prepared from the commercially available
(1R,3R,4S,5R)-quinic acid.17 The next step was selective protec-
tion of the equatorial hydroxyl group at C-3 as the tert-
butyldimethylsilyl (TBDMS) ether. Vandewalle described the
reaction of 8 with TBDMSCl and separation of the isomeric
silyl ethers by preparative HPLC.18 We established that the lower
temperature of the reaction and small excess (1.15 molar) of
the silylating agent resulted in considerably better chemoselec-
tivity of the process and higher yields of the desired 3-OTBDMS
derivative 9 that can be isolated by crystallization and column
chromatography. The axial 4-hydroxyl was subsequently oxi-
dized with Dess-Martin periodinane reagent, and the remaining
tertiary hydroxyl in the formed 10 was acetylated. Esterification
of the hydroxyl at C-1 was advantageous, because the presence
of a free 1-hydroxyl group resulted in significantly diminished
yields of the following Wittig reactions. According to our
expectations, reaction of ketone 11 with the ylide generated from
phosphonium bromide A and n-butyllithium proceeded smoothly,
providing two isomeric olefinic products 12 and 13 (5:1 ratio)
in 77% yield. Configuration of the propylidene unit at C-4 in
Results and Discussion
Chemistry. As a starting compound for the synthesis of the
A-ring fragments we chose a known bicyclic trihydroxy lactone

1R,25-Dihydroxy-19-norVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2911
Scheme 2
1
compounds 12 and 13 was unambiguously determined by H
NOE difference spectroscopy experiments (see Supporting
Information). Wittig reaction of 11 with the ylide generated from
phosphonium salt B proved to be even more efficient giving
the olefinic products 14 and 15 (3:2 ratio) in 87% yield.
Therefore, we have also decided to test an alternative way to
the final compounds 6 and 7 using intermediates possessing a
TBDMS group as a protection of the terminal hydroxyl in the
A-ring hydroxypropylidene moiety.
executed with the prevailing E-geometrical isomer, providing
the desired A-ring fragment, the phosphine oxide 32. A synthetic
sequence analogous to the conversion of 12 to 32 was used for
the transformation of isomeric compounds 14 and 15 into the
corresponding phosphine oxides 33 and 34. Since a separation
of the isomeric E- and Z-compounds was rather difficult, all
synthetic steps were performed with the mixture of isomers.
The anion generated from 32 and phenyllithium reacted with
the protected 25-hydroxy Grundmann ketone 35a to give the
19-norvitamin D compound 36a in 48% yield. Silyl protecting
groups were removed by treatment with tetrabutylammonium
fluoride and the final 2-[3′-(methoxymethoxy)propylidene]
derivative of 19-nor-1R,25-(OH)2D3 (5a) was obtained in 71%
Both compounds 12 and 13 can be easily converted to the
cyclohexanone derivative 22 (Scheme 2). However, since
chromatographical separation of these geometrical isomers was
relatively easy, we only used compound 12 for this purpose,
whereas the isomer 13 served as a starting compound for the
synthesis of a different vitamin D analogue (unpublished results).
Thus, sodium borohydride reduction of 12 followed by periodate
oxidation of the resulting triol 16 gave hydroxy ketone 19 that
was subsequently silylated to the desired protected ketone 22.
Peterson olefination of 22 resulted in the mixture of R,â-
unsaturated esters 24 and 25 (7:1 ratio) in 86% yield. 1H NOE
difference spectroscopy and analysis of proton couplings allowed
us to unequivocally establish the configurations of the obtained
products (see Supporting Information), which could be separated
by HPLC. However, it was more convenient to first perform
DIBALH reduction and then column chromatography separation
of the allylic alcohols 28 and 29. The next three steps were
1
yield. Comparison of its H NMR spectrum with that of the
respective 2-ethylidene-substituted analogue 3a8 indicated their
close similarity. Half-widths of the multiplets derived from the
methine protons at C-1 (δ 4.44, w/2 ) 20 Hz) and C-3 (δ 4.84,
w/2 ) 10 Hz), found in the spectrum of 5a, indicated that its
1R- and 3â-hydroxy groups occupy, respectively, equatorial and
axial orientation. Conformational equilibrium of the A ring is
in this case significantly shifted to the â-chair form (Figure 2a)
because in the alternative R-chair conformation strong interac-
tion exists between the propylidene group and 3â-hydroxyl.
Obviously, it was expected that ketal derivative 5a could be
hydrolyzed in appropriate conditions to yield the parent 3′-
hydroxypropylidene compound 6a. Unfortunately, all attempts

2912 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Glebocka et al.
Scheme 3
(MgBr2 and n-BuSH, LiBF4, AlI3, camphor-10-sulfonic acid in
MeOH) to deprotect the terminal 3′-hydroxyl of the propylidene
unit in 5a failed. Wittig-Horner reaction between the mixture
of phosphine oxides 33 and 34 and the protected Grundmann
ketones 35a and 35b provided 19-norvitamin D compounds 37a
and 37b in 41% and 47% yield, respectively. Only traces of
the corresponding Z-isomers were detected in the reaction
mixtures. Such reaction outcome could be expected because our
previous studies on the synthesis of 2-ethylidene vitamin D
analogues 3 and 4 indicated low reactivity of the phosphine
oxides in the Z-series.8 Deprotection of the four hydroxy goups
in the obtained products 37a,b gave the desired 2-(3′-hydroxy-
propylidene)-substituted 19-norvitamin D3 analogues 6a,b.
Similarly as in the case of 5a, their A-rings exist preferentially
in the â-chair conformation.
norvitamin D3 compounds 7a,b their A-ring conformational
equilibrium is strongly biased toward the R-chair form (Figure
2b).
Biological Evaluation. The synthesized derivative of 19-nor-
1R,25-(OH)2D3 with a 3′-(methoxymethoxy)propylidene moiety
at C-2 (5a) was tested for its ability to bind the full-length
recombinant rat vitamin D receptor. The competitive binding
analysis shows (Table 1) that 5a exhibits ca. 6 times lower
affinity for the receptor than 1R,25-(OH)2D3 (1). Thus, despite
the fact that an alkoxyalkylidene chain consisting of seven atoms
is attached to C-2, this analogue still retains a significant binding
ability that is most likely due to the presence of two additional
oxygen atoms. In the case of the closely related E-isomer 6a,
and its analogue 6b with the unnatural configuration at C-20,
the presence of a free terminal hydroxy group in the 2-propyl-
idene substituent results in increasing affinity to receptor,
essentially identical to that of the hormone 1. Compounds in
the Z-series (7a,b) are ca. 3-8 times less active than 1R,25-
(OH)2D3 in binding to VDR.
High binding potency of the synthesized E-isomers is not
surprising when the results of the docking experiments, per-
formed for analogue 6a and full length rVDR-LBD,8 are taken
into account. It turned out that this analogue, regardless of its
initial positioning in the binding pocket, is finally oriented
(Figure 3) quite similar as the natural hormone 1 in its crystalline
complex with the VDRmt.12 Thus, the plane of the intercyclic
diene moiety of 6a is approximately parallel to the Trp 282
rings, whereas the 1R- and 3â- and 25-hydroxy groups create
strong hydrogen bonds with Ser 274, Arg 270, and His 301,
respectively. The terminal hydroxyl from the ligand’s 2-pro-
pylidene fragment contacts Arg 270 and, additionally, Asp 144.
Studies on the cellular activity of the synthesized compounds
to induce the differentiation of human promyelocyte HL-60 cells
into monocytes have indicated that they are almost equivalent
to hormone 1, with the exception of the analogue 6b being 10
times more active (Table 1).
Promising results of preliminary biological tests performed
with E-geometrical isomers 5a and 6a,b encouraged us to also
synthesize their counterparts in the Z-series. To achieve this
goal an alternative synthesis was chosen, employing the
modified Julia olefination19 as a crucial step for providing the
19-norvitamin D skeleton. Such an attempt, based on the Kittaka
approach,20 involved preparation of the isomeric sulfones 39a,b
(Scheme 3), as the C,D-fragments, and their coupling with the
A-ring cyclohexanone derivative 23. Thus, both Grundmann
ketones 35a,b were coupled with the anion of triethyl phospho-
noacetate and the prepared esters reduced with DIBALH to give
the allylic alcohols 38a,b. They were, in turn, subjected to
Mitsunobu reaction with 2-mercaptobenzothiazole and the
formed sulfides easily converted to the allylic sulfones 39a,b.
According to our expectations, Julia coupling of the anions,
generated from these compounds with LiHMDS, and the
protected ketone 23 provided the corresponding pairs of E- and
Z-isomeric products: 37a, 40a and 37b, 40b. Each pair of the
vitamin D compounds was initially purified by column chro-
matography, then treated with tetrabutylammonium fluoride, and
the final isomeric vitamins 6a, 7a and 6b, 7b were separated
1
by reversed-phase HPLC. Analysis of H NMR spectra con-
The ability of the vitamin D analogues in the E-series to
induce transcription of vitamin D-responsive genes was exam-
firmed that in the 2-(3′-hydroxypropylidene)-substituted 19-

1R,25-Dihydroxy-19-norVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2913
Table 1. VDR Binding Properties,a HL-60 Differentiating Activities,b and Transcriptional Activities of the Vitamin D Compounds 5a, 6a, 6b, 7a, and
7b
VDR binding
HL-60 differentiation
ED50 (M) activity ratio
24-OHase transcription
compound
compd no.
Ki (M)
binding ratio
1
ED50 (M)
activity ratio
1
1R,25-(OH)2D3
2-(3′-MOMO-propylidene)-
1
1.1 × 10-10
5.0 × 10-10
1.3 × 10-10
6.2 × 10-11
6.1 × 10-10
1.5 × 10-10
2.4 × 10-9
4.7 × 10-9
1.3 × 10-9
4.0 × 10-10
3.0 × 10-9
1.4 × 10-9
1
2.6 × 10-10
19-nor-1R,25-(OH)2D3
5a
6a
6b
7a
7b
6.6
1.3
0.9
7.7
2.8
1.5
0.6
0.1
1.7
0.8
3.8 × 10-9
4.2 × 10-11
6.5 × 10-12
15
2-(3′-hydroxypropylidene)-19-nor-
1R,25-(OH)2D3 (E-isomer)
0.2
0.02
2-(3′-hydroxypropylidene)-19-nor-
(20S)-1R,25-(OH)2D3 (E-isomer)
2-(3′-hydroxypropylidene)-19-nor-
1R,25-(OH)2D3 (Z-isomer)
2-(3′-hydroxypropylidene)-19-nor-
(20S)-1R,25-(OH)2D3 (Z-isomer)
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 to three 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 two to three
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 3. View of the three-dimensional structure of ligand binding cavity of the rat VDR with the docked vitamin D analogue 6a (FlexiDock,
TRIPOS). The four amino acids (Asp 144, Ser 274, Arg 270, and His 301) forming the shortest hydrogen bonds (the Å distances are marked in
green) with the ligand are depicted. Also the Trp 282 residue is shown (pink).
ined using the 24-hydroxylase (CYP-24) luciferase reporter gene
system and it was established that compound 5a has 15 times
lower activity with respect to 1R,25-(OH)2D3 (Table 1).
However, analogues 6a and 6b are characterized by significantly
higher transcriptional potencies, 5 and 50 times higher, respec-
tively, in comparison with 1.
Considering the results of the in vitro tests, a relatively low
calcemic activity might be expected for the analogue 5a.
However, tests in vivo in mice indicate that the potency of
compound 5a in raising serum calcium is higher than that of
the parent hormone 1 (Figure 4). Taking into account a relatively
weak transcriptional activity of 5a and its significant potency
in vivo, it can be suggested that the analogue must be first
metabolized in the living organism, most likely to the parent
3′-hydroxypropylidene derivative 6a. This idea is supported by
studies done in the mouse and the rat. A single oral dose of 5a
raises serum calcium 3 days later to the same level as 6a (Figure
5). In addition, 5a causes similar bone calcium mobilization
and intestinal calcium transport activity as 6a in D-deficient
rats (Figure 6). The distinct activity differences observed in vitro
between 6a and 6b were not observed in vivo (Figures 5 and
6). However, the Z-isomers are at least 10 times less active than
their E-counterparts which is consistent with the results obtained
in vitro (Figure 5). Finally, it is important to note that all three
analogues 5a, 6a, and 6b, while more potent than the native
hormone in both bone calcium mobilization as well as intestinal
calcium transport, have selective intestinal activity. Note that
100 pmol/day of compound 2b (Figure 7) gave an increase in
intestinal calcium transport equivalent to that induced by 5.7
pmol of 6a or 6b. On the other hand, compound 2b was shown
previously to have approximately equal activity on intestine as
1 but was found 30 times more active on bone.21
Conclusions
The structures of all synthesized compounds were mainly
established by different NMR methods. NOE difference spec-
troscopy and heteronuclear correlation NMR spectra (HMQC,
HMBC) were used for the complete assignments of 1H and 13
C
signal groups. Analysis of the observed vicinal proton coupling
constants, confirmed by spin decoupling experiments, and their

2914 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Glebocka et al.
in shedding light on the design of new compounds with intestine
or bone only activity. In addition, these compounds might have
some utility in the therapeutic treatment of intestinal diseases.
The results of the tests presented above support our previous
findings concerning 2-ethylidene 19-norvitamins 3a,b and 4a,b.8
It has been found that introduction of alkylidene, hydroxyalkyl-
idene, or alkoxyalkylidene substituents at position 2 in the 19-
nor-1R,25-(OH)2D3 molecule results in the creation of vitamin
D analogues characterized by strong affinities to VDR and
highly elevated in vivo potencies. Such high potent in vivo
activity is limited to E-geometric isomers possessing â-chair
conformation of the cyclohexane ring A.
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
UV-Vis spectrophotometer in ethanol. 1H nuclear magnetic
resonance (NMR) spectra were recorded at 400 and 500 MHz with
a Bruker Instruments DMX-400 and DMX-500 Avance console
spectrometers in deuteriochloroform. 13C nuclear magnetic reso-
nance (NMR) spectra were recorded at 125 MHz with a Bruker
Instruments DMX-500 Avance console in deuteriochloroform.
Chemical 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 bicyclic lactone 8 was obtained from commercial
(-)-quinic acid (90% yield) according to the published procedure.17
(1R,3R,4S,5R)-3-[(tert-Butyldimethylsilyl)oxy]-1,4-dihydroxy-
6-oxa-bicyclo[3.2.1]octan-7-one (9). To a stirred solution of lactone
8 (1.80 g, 10.34 mmol) and imidazole (2.63 g, 38.2 mmol) in
anhydrous DMF (14 mL) was added tert-butyldimethylsilyl chloride
(1.80 g, 11.9 mmol) at 0 °C. The mixture was stirred at 0 °C for
30 min and 1 h at room temperature, poured into water and extracted
with ethyl acetate and ether. The organic layer was washed several
times with water, dried (MgSO4), and evaporated to give a colorless
crystalline residue that was crystallized from hexane/ethyl acetate
to give 2.12 g of pure ether 9. The mother liquors were evaporated
and purified by flash chromatography. Elution with hexane/ethyl
acetate (8:2) gave additional quantity of monosilylated (3-OTB-
DMS) derivative 9 (0.14 g, overall yield 76%) and some quantity
of crystalline isomeric (4-OTBDMS) ether (0.10 g, 3%).
(1R,3R,5R)-3-[(tert-butyldimethylsilyl)oxy]-1-hydroxy-6-oxa-
bicyclo[3.2.1]octane-4,7-dione (10). To a stirred suspension of
Dess-Martin periodinane reagent (6.60 g, 15.5 mmol) in anhydrous
CH2Cl2 (100 mL) was added 3-silyloxy compound 9 (3.86 g, 13.4
mmol). The mixture was stirred at room temperature for 18 h,
poured into water and extracted with ethyl acetate. The organic
layer was washed several times with water, dried (MgSO4), and
evaporated to give an oily residue which slowly crystallized on
cooling (3.67 g, 95%). TLC indicated high purity of the obtained
ketone 10 which could be used in the next step without further
purification. The analytical sample was obtained by recrystallization
from hexane.
(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-6-oxa-
bicyclo[3.2.1]octane-4,7-dione (11). A solution of a crude hydroxy
ketone 10 (1.64 g, 5.8 mmol) in anhydrous pyridine (12 mL) and
acetic anhydride (5.5 mL) was stirred for 3 h at room temperature.
It was poured into water and extracted with ethyl acetate. The
organic layer was washed with saturated NaHCO3, saturated CuSO4,
and water, dried (MgSO4), and evaporated to give an oily residue
which was dissolved in hexane/ethyl acetate (8:2) and filtered
through short path of silica gel. Evaporation of solvents gave pure
crystalline acetate 11 (1.51 g, 81%). An analytical sample was
obtained by recrystallization from hexane/ethyl acetate.
Figure 4. Serum calcium changes in response to a single oral dose of
the analogue 5a or native hormone in CD-1 mice. Six to seven week
old female mice were given one oral dose of 5a or 1R,25-(OH)2D3 (1),
and blood was collected at several timepoints following dose admin-
istration for calcium concentration determination in the serum.
correlation with the couplings obtained by force field calcula-
tions allowed us to successfully assign both stereochemistries
of the compounds and their preferred solution conformations.
Synthesis of 1R,25-dihydroxy-19-norvitamin D3 analogues
substituted at C-2 with (3′-methoxymethoxy)propylidene (5a),
and 3′-hydroxypropylidene moiety (6a,b and 7a,b) allowed
detailed biological testing of these compounds. Biological
evaluation of the analogue 5a revealed that it is highly potent
in vivo. This result was unexpected because of its relatively
low transcriptional activity observed in vitro compared to the
native hormone. One explanation for the differences observed
in vitro compared to in vivo, is that 5a is metabolized in the
living organism to a more potent derivative and the most likely
metabolite is generated by cleavage of the terminal meth-
oxymethoxy group in the 2-alkylidene substituent resulting in
2-(3′-hydroxypropylidene) compound 6a. This is further sup-
ported by the delay of serum calcium response noted in Figure
4 for 5a. This explanation is also supported by the results
obtained with analogue 6a and other literature reports indicating
that ether moieties are readily cleaved in the body.22 Both
analogues 6a and 6b showed increased potency in vitro
compared to 1R,25(OH)2D3, but in vivo, these compounds were
no more potent than 5a. In vitro the analogue with the 20 carbon
in the unnatural configuration (6b) exhibited higher potency than
6a which has the natural 20R-configuration. This result was
expected as several other compounds have been shown to exhibit
enhanced potency when the 20-carbon is switched to the
unnatural configuration.23 Another structure/activity pattern that
has been reported before is that Z-isomers of 2-alkylidene
vitamin D analogues have less activity than their counterparts
in the E-configuration.8 This pattern was repeated with this series
of compounds as well in that 7a and 7b were less active than
6a and 6b both in vitro and in vivo. Besides the unique in vitro/
in vivo activity associated with 5a, this series of compounds
show an interesting biological profile in that compounds 5a,
6a and 6b have intestinal selective activity which could be useful

1R,25-Dihydroxy-19-norVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2915
Figure 5. Serum calcium changes in response to a single oral dose of 5a, 6a, 6b, 7a and 7b in CD-1 mice. Six to seven week old female mice were
given one oral dose of the designated compounds, and blood was collected for serum calcium analysis 3 days following dose administration.
Figure 6. Bone calcium mobilization and intestinal calcium transport activity of 5a, 6a, and 6b. Rats were made vitamin D-deficient and placed
on a diet nearly devoid of calcium. Blood and duodenum were collected 24 h following the last of 4 consecutive daily ip doses.
HCl (150 mL), the organic phase was separated and the water phase
extracted with CH2Cl2. The combined organic phases were washed
with water and diluted in NaHCO3, dried (MgSO4), and evaporated
to give a yellowish oil. The residue was purified by flash
chromatography. Elution with hexane/ethyl acetate (95:5) afforded
pure oily 1-bromo-3-(methoxymethoxy)propane (1.12 g, 55%).
To a solution of 1-bromo-3-(methoxymethoxy)propane (0.46 g,
2.5 mmol) in anhydrous toluene (1.5 mL) was added triphenyl-
phoshine (0.71 g, 2.7 mmol) under argon with stirring. The mixture
was heated at 100 °C for 20 h and cooled to room temperature.
The liquid was decanted and the solid residue was ground with a
spatula, filtered, and washed several times with ether. After drying
overnight in a vacuum desiccator, colorless crystals of phosphonium
salt A (0.98 g, 88%) could be used in the Wittig reaction without
further purification.
Figure 7. Dose-dependent intestinal calcium transport activity of 2b.
Rats were made vitamin D-deficient and placed on a diet nearly devoid
of calcium. Duodena were collected for gut sac preparation 24 h
following the last of four consecutive daily ip doses.
[3-[(tert-Butyldimethylsilyl)oxy]propyl]triphenylphospho-
nium Bromide (B). To a solution of 1-bromo-3-[(tert-butyldi-
methylsilyl)oxy]propane (Aldrich, 2.18 g, 8.56 mmol) in anhydrous
benzene (1.6 mL) was added triphenylphoshine (2.64 g, 10.2 mmol)
under argon with stirring. The mixture was heated at 85 °C for 18
h and cooled to room temperature. The liquid was decanted and
the solid residue was ground with a spatula, filtered, and washed
several times with ether. Colorless crystals of phosphonium salt B
[3-(Methoxymethoxy)propyl]triphenylphosphonium Bromide
(A). To a solution of bromomethyl methyl ether (1.3 mL, 16 mmol)
and N,N-diisopropylethylamine (4.5 mL, 27.7 mmol) in anhydrous
CH2Cl2 (50 mL) at 0 °C was added 3-bromo-1-propanol (1.0 mL,
11 mmol), and the mixture was stirred at 0 °C for 1 h and at room
temperature for 20 h. The reaction mixture was poured into 1 N

2916 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Glebocka et al.
(3.7 g) were purified by silica column chromatography. Pure salt
B (3.04 g, 69%) was eluted with chloroform/methanol (96:4).
[(4E)- and (4Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimeth-
ylsilyl)oxy]-6-oxa-4-[3′-(methoxymethoxy)propylidene]bicyclo-
[3.2.1]octan-7-one (12 and 13). To the phoshonium bromide A
(420 mg, 0.94 mmol) in anhydrous THF (5 mL) at 0 °C was added
dropwise n-BuLi (1.6 M in hexanes, 1.12 mL, 1.8 mmol) under
argon with stirring. After 5 min another portion of A was added
(420 mg, 0.94 mmol) 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 in 2 equal portions
(30 min interval) to a solution of keto lactone 11 (300 mg, 0.91
mmol) in anhydrous THF (8 mL). The reaction mixture was stirred
at -78 °C and stopped by addition of brine cont. 1% HCl (3 h
after addition of the first portion of the Wittig reagent). Ethyl acetate
(9 mL), benzene (6 mL), ether (3 mL), sat. NaHCO3 (3 mL), and
water (3 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
(consisting mainly of isomeric 12 and 13 in the ratio of ca. 5:1)
was separated by flash chromatography on silica. Elution with
hexane/ethyl acetate (85:15) resulted in partial separation of
products: 29 mg of 13, mixture of 12 and 13 (ca. 3.5:1, 85 mg),
and pure 12 (176 mg). Rechromatography of the mixed fractions
resulted in almost complete separation of the products (total yield
of both isomers 77%).
[(4E)- and (Z)-(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethyl-
silyl)oxy]-6-oxa-4-[3′-((tert-butyldimethylsilyl)oxy)propylidene]-
bicyclo[3.2.1]octan-7-one (14 and 15). To the phosphonium
bromide B (1.55 g, 3.04 mmol) in anhydrous THF (42 mL) at -
20 °C was added dropwise n-BuLi (2.0 M in cyclohexane, 1.50
mL, 3.00 mmol) under argon with stirring. The solution was stirred
at - 20 °C for 15 min. The orange-red mixture was cooled to -45
°C and siphoned during 15 min to a solution of keto lactone 11
(700 mg, 2.13 mmol) in anhydrous THF (24 mL). The reaction
mixture was stirred at -40 °C for 2 h and stopped by addition of
brine cont. 1% HCl. Ethyl acetate (30 mL), benzene (20 mL), ether
(10 mL), saturated NaHCO3 (10 mL), and water (10 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 residue (consisting mainly of
isomeric 14 and 15 in the ratio of ca. 3:2) was purified by flash
chromatography on silica. Elution with hexane/ethyl acetate (9:1)
gave the mixture of products 14 and 15 (905 mg, 87%). Analytical
samples of both isomers were obtained after HPLC (10 mm × 25
cm Zorbax-Sil column, 4 mL/min) separation using a hexane/ethyl
acetate (9:1) solvent system. Pure oily compounds 14 and 15 were
eluted at RV 28 mL and 29 mL, respectively.
[(4E)-(1′R,3′R,5′R)-3′-[(tert-Butyldimethylsilyl)oxy]-1′,5′-dihy-
droxy-4′-[3′′-(methoxymethoxy)propylidene]cyclohexyl]meth-
anol (16). To a stirred solution of compound 12 (165 mg, 0.40
mmol) in anhydrous ethanol (5 mL) at 0 °C was added NaBH4
(151 mg, 4.0 mmol), and the mixture was stirred at 0 °C for 1 h,
then for 10 h at 6 °C, and for 2 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 flash chromatography.
Elution with hexane/ethyl acetate (2:8) gave pure triol 16 as a
colorless oil (115 mg, 79%).
[(E)- and (Z)-(1′R,3′R,5′R)-3-[(tert-Butyldimethylsilyl)oxy]-
1′,5-dihydroxy-4′-[3′′-[((tert-butyldimethylsilyl)oxy)propylidene]-
cyclohexyl]methanol (17 and 18). Triols 17 and 18 were obtained
by NaBH4 reduction of compounds 14 and 15, performed analo-
gously to the process described above for 12 except the reaction
was carried out for 21 h at room temperature. Products were purified
by column chromatography. Elution with hexane/ethyl acetate
(4:6) gave a semicrystalline mixture of triols 17 and 18 in 98%
yield.
iodate-saturated water (1.2 mL) was added to a solution of the
triol 16 (79 mg, 0.21 mmol) in methanol (5 mL) at 0 °C. The
solution was stirred at 0 °C for 1 h, poured into brine, and extracted
with ethyl acetate and ether. The extract was washed with brine,
dried (MgSO4), and evaporated. An oily residue was redissolved
in hexane/CH2Cl2 and applied on a Sep-Pak cartridge. Pure hydroxy
ketone 19 (64 mg, 88%) was eluted with hexane/ethyl acetate
(7:3) as an oil slowly crystallizing in the refrigerator.
[(4E)- and (4Z)-(3R,5R)-3-[(tert-Butyldimethylsilyl)oxy]-5-hy-
droxy-4-[3′-[((tert-butyldimethylsilyl)oxy)propylidene]]cyclohex-
anone (20 and 21). Sodium periodate cleavage of the triols 17
and 18 was performed analogously to the process described above
for 16. The mixture of hydroxy ketones 20 and 21 (88% yield)
was eluted from a Sep-Pak cartridge with hexane/ethyl acetate
(8:2) as an oil slowly crystallizing in the refrigerator.
[(3R,5R)-3,5-Bis[(tert-Butyldimethylsilyl)oxy]-4-[3′-(methoxy-
methoxy)propylidene]cyclohexanone (22). To a solution of hy-
droxy ketone 19 (40 mg, 117 µmol) in anhydrous CH2Cl2 (0.4 mL)
at -50 °C was added 2,6-lutidine (32 µL, 274 µmol) and tert-
butyldimethylsilyl triflate (56 µL, 240 µmol). The mixture was
stirred for 5 min at -50 °C, then it was allowed to warm to -15
°C and stirred at this temperature for an additional 30 min. Benzene
and water were added, and the mixture was poured into water and
extracted with benzene. The extract was washed with saturated
CuSO4 and water, dried (MgSO4), and evaporated. The oily residue
was redissolved in hexane and purified by flash chromatography
on silica. Elution with hexane/ethyl acetate (95:5) gave pure
protected ketone 22 as a colorless oil (30 mg, 57%; 66% based on
recovered substrate) and unreacted 19 (6 mg).
[(3R,5R)-3,5-Bis[(tert-Butyldimethylsilyl)oxy]-4-[3′-[((tert-bu-
tyldimethylsilyl)oxy)propylidene]cyclohexanone (23). Silyla-
tion of hydroxy ketones 20 and 21 was performed analogously
to the process described above for 19 except the reaction was
carried out for 50 min at -50 °C. The product was purified by
flash chromatography on silica. Elution with hexane/ethyl acetate
(95:5) gave pure protected ketone 23 as a colorless oil (18 mg,
64%; 74% based on recovered substrates) and a mixture of
unreacted 20 and 21 (3 mg).
[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-(meth-
oxymethoxy)propylidene]cyclohexylidene]acetic Acid Methyl
Esters (24 and 25). To a solution of diisopropylamine (25 µL,
0.18 mmol) in anhydrous THF (0.15 mL) was added n-BuLi (2.5
M in hexanes, 72 µL, 0.18 mmol) under argon at -78 °C with
stirring. Methyl(trimethylsilyl)acetate (30 µL, 0.18 mmol) was then
added. After 15 min, the ketone 22 (38.4 mg, 84 µmol) in anhydrous
THF (0.2 mL) was added. The solution was stirred at -78 °C for
an additional 2 h, and the reaction mixture was quenched with wet
ether, poured into brine and extracted with ether and benzene. The
combined extracts were washed with brine, dried (MgSO4), and
evaporated. An oily residue was redissolved in hexane and applied
on a Sep-Pak cartridge. Pure allylic esters 24 and 25 (37.2 mg,
86%; isomer ratio of 24:25 ) ca. 7:1) were eluted with hexane/
ethyl acetate (97:3). Separation of the products was achieved by
HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/min) using the
hexane/ethyl acetate (95:5) solvent system. Pure compounds 24 and
25 were eluted at RV 41 mL and 44 mL, respectively, as colorless
oils.
[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-[((tert-
butyldimethylsilyl)oxy)propylidene]cyclohexylidene]acetic Acid
Methyl Esters (26 and 27). Peterson olefination of protected ketone
23 was performed analogously to the process described above for
22. The mixture of allylic esters 26 and 27 (60 mg, 68%; isomer
ratio of 26:27 ) ca. 6:1) was eluted from a Sep-Pak cartridge with
hexane/ethyl acetate (98.5:1.5).
2-[(3′R,5′R)-3′,5′-bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-(meth-
oxymethoxy)propylidene]cyclohexylidene]ethanol (28 and 29).
Diisobutylaluminum hydride (1.0 M in toluene, 0.35 mL, 0.35
mmol) was slowly added to a stirred solution of the allylic esters
24 and 25 (7:1, 37.2 mg, 74 µmol) in toluene/methylene chloride
(2:1, 1.5 mL) at -78 °C under argon. Stirring was continued at
-78 °C for 1 h, the mixture was quenched by addition of potassium
(4E)-(3R,5R)-3-[(tert-Butyldimethylsilyl)oxy]-5-hydroxy-4-[3′-
(methoxymethoxy)propylidene]cyclohexanone (19). Sodium per-

1R,25-Dihydroxy-19-norVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2917
sodium tartrate (2 N, 2 mL), aq. HCl (2 N, 2 mL) and H2O (24
mL), and then diluted with ether and benzene. The organic layer
was washed with diluted NaHCO3 and brine, dried (MgSO4), and
evaporated. The residue was purified by flash chromatography.
Elution with hexane/ethyl acetate (95:5) resulted in partial separation
of allylic alcohols: pure major product 28 (16 mg), mixture of both
isomers (15 mg) and pure minor Z-isomer 29 (3 mg; total yield
97%). Rechromatography of the mixed fractions resulted in almost
complete separation of the products.
2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-[((tert-
butyldimethylsilyl)oxy)propylidene]cyclohexylidene]ethanol (30
and 31). DIBALH reduction of allylic esters 26 and 27 was
performed analogously to the process described above for 24 and
25. The products were purified by flash chromatography. Elution
with hexane/ethyl acetate (95:5) yielded a mixture of allylic alcohols
30 and 31 (86% yield). Analytical samples of both isomers were
obtained after HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/
min) using a hexane/ethyl acetate (9:1) solvent system. Pure oily
compounds 30 and 31 were eluted at RV 28 mL and 29 mL,
respectively.
[2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-(meth-
oxymethoxy)propylidene]cyclohexylidene]ethyl]diphenylphos-
phine Oxide (32). To the allylic alcohol 28 (16 mg, 33 µmol) in
anhydrous THF (0.4 mL) was added n-BuLi (2.5 M in hexanes, 13
µL, 33 µmol) under argon at 0 °C with stirring. Freshly recrystal-
lized tosyl chloride (6.7 mg, 35 µmol) was dissolved in anhydrous
THF (90 µL) and added to the allylic alcohol-BuLi solution. The
mixture was stirred at 0 °C for 5 min and set aside at 0 °C. In
another dry flask with air replaced by argon, n-BuLi (2.5 M in
hexanes, 64 µL, 0.16 mmol) was added to Ph2PH (30 µL, 0.16
mmol) in anhydrous THF (200 µL) at 0 °C with stirring. The red
solution was siphoned under argon pressure to the solution of
tosylate until the orange color persisted (ca. 1/4 of the solution
was added). The resulting mixture was stirred an additional 40 min
at 0 °C and quenched by addition of H2O (20 µL). Solvents were
evaporated under reduced pressure, and the residue was redissolved
in methylene chloride (0.5 mL) and stirred with 10% H2O2 (0.25
mL) at 0 °C for 1 h. The organic layer was separated, washed with
cold aq. sodium sulfite and H2O, dried (MgSO4), and evaporated.
The residue was subjected to flash chromatography. Elution with
hexane/ethyl acetate (85:15) gave an unchanged allylic alcohol (2
mg). Subsequent elution with benzene/ethyl acetate (7:3) provided
pure phosphine oxide 32 (15.5 mg, 68%; 78% based on recovered
starting material) as a colorless oil. An analytical sample was
obtained after HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/
min) purification using a hexane/2-propanol (9:1) solvent system
(RV 41 mL).
[2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-[3′′-[((tert-
butyldimethylsilyl)oxy)propylidene]cyclohexylidene]ethyl]-
diphenylphosphine Oxides (33 and 34). The phosphine oxides
33 and 34 were obtained from the mixture of allylic alcohols 30
and 31 analogously as described above for compound 32. The crude
reaction products were subjected to flash chromatography. Elu-
tion with hexane/ethyl acetate (95:5) gave unchanged allylic
alcohols. Subsequent elution with hexane/ethyl acetate (7:3) resulted
in a mixture of isomeric phosphine oxides 33 and 34 (49% yield;
81% yield based on recovered substrates 30 and 31).
1R-[(tert-Butyldimethylsilyl)oxy]-2-[3′-(methoxymethoxy)pro-
pylidene]-25-[(triethylsilyl)oxy]-19-norvitamin D3 tert-Butyldi-
methylsilyl Ether (36a). To a solution of phosphine oxide 32 (15.5
mg, 23 µmol) in anhydrous THF (0.25 mL) at -78 °C was slowly
added phenyllithium (1.8 M in cyclohexane/ether, 13 µL, 23 µmol)
under argon with stirring. The solution turned deep orange. The
mixture was stirred at -78 °C for 20 min and a precooled (-78
°C) solution of protected hydroxy ketone 35a (19 mg, 48 µmol;
prepared according to a published procedure)15 in anhydrous THF
(0.25 mL) was slowly added. The mixture was stirred under argon
at -78 °C for 3 h and at 6 °C for 16 h. Ethyl acetate and water
were added, and the organic phase was washed with brine, dried
(MgSO4), and evaporated. The residue was dissolved in hexane,
applied on a silica Sep-Pak cartridge, and washed with hexane/
ethyl acetate (98:2, 10 mL) to give 19-norvitamin derivative 36a
(9.5 mg, 48%). The Sep-Pak was then washed with hexane/ethyl
acetate (96:4, 10 mL) to recover some unchanged C,D-ring ketone
35a (10 mg), and with ethyl acetate (10 mL) to recover diphenyl-
phosphine oxide 32 (1 mg).
1R-[(tert-Butyldimethylsilyl)oxy]-2-[3′-[((tert-butyldimethyl-
silyl)oxy)propylidene]-25-[(triethylsilyl)oxy]-19-norvitamin D3
tert-Butyldimethylsilyl Ether (37a). Protected vitamin 37a was
obtained by the Wittig-Horner coupling of the corresponding
phosphine oxides 33 and 34 (ca. 6:1) and protected hydroxy ketone
35a, performed analogously to the process described above for
coupling of 32 and 35a. The crude mixture resulting from the
reaction was separated by column chromatography on silica. Elution
with hexane/ethyl acetate (99.5:0.5) yielded 19-norvitamin deriva-
tive 37a (41%, 47% based on recovered substrates). The column
was then washed with hexane/ethyl acetate (96:4) to recover some
unchanged C,D-ring ketone 35a, and with ethyl acetate to recover
unreacted diphenylphosphine oxides. An analytical sample of the
product was obtained by HPLC (10 mm × 25 cm Zorbax-Sil
column, 4 mL/min) purification using a hexane/ethyl acetate
(99.8:0.2) solvent system. Pure protected vitamin 37a was eluted
at RV 28 mL as a colorless oil.
(20S)-1R-[(tert-Butyldimethylsilyl)oxy]-2-[3′-[((tert-butyldi-
methylsilyl)oxy)propylidene]-25-[(triethylsilyl)oxy]-19-norvita-
min D3 tert-Butyldimethylsilyl Ether (37b). Silylated 19-norvi-
tamin D3 compound 37b was obtained by Wittig-Horner coupling
of protected 25-hydroxy Grundmann ketone 35b (prepared accord-
ing to a published procedure)7 with the phosphine oxides 33 and
34 performed analogously to the process described above for the
preparation of (20R)-isomer 37a. The protected vitamin was purified
on a silica column, using a hexane/ethyl acetate (99.5:0.5) solvent
system, and it was obtained in ca. 47% yield. An analytical sample
of the protected vitamin 37b was obtained by HPLC (10 mm × 25
cm Zorbax-Sil column, 4 mL/min) purification using a hexane/
ethyl acetate (99.7:0.3) solvent system. Pure compound 37b was
eluted at RV 25 mL as a colorless oil.
1R,25-Dihydroxy-2-[3′-(methoxymethoxy)propylidene]-19-
norvitamin D3 (5a). To a solution of the protected 19-norvitamin
D3 36a (3.0 mg, 3.5 µmol) in anhydrous THF (200 µL) was added
tetrabutylammonium fluoride (1.0 M in THF, 210 µL, 210 µmol).
The mixture was stirred under argon at room temperature for 18 h,
poured into brine and extracted with ethyl acetate. Organic extracts
were washed with brine, dried (MgSO4), and evaporated. The
residue was purified by HPLC (10 mm × 25 cm Zorbax-Sil column,
4 mL/min) using a hexane/2-propanol (75:25) solvent system.
Analytically pure 19-norvitamin 5a (1.27 mg, 71%) was collected
at RV 26 mL. The compound gave also a single peak on reversed-
phase HPLC (6.2 mm × 25 cm Zorbax-ODS column, 2 mL/min)
using a methanol/water (8:2) solvent system: it was collected at
RV 35 mL.
1R,25-Dihydroxy-2-[3′-hydroxypropylidene]-19-norvitamin D3
(6a). Treatment of the protected vitamin 37a with tetrabutylam-
monium fluoride, performed as described for 36a, gave a vitamin
D compound that was purified by HPLC (10 mm × 25 cm Zorbax-
Sil column, 4 mL/min) using a hexane/2-propanol (8:2) solvent
system. Analytically pure E-isomer 6a (97%) was collected at RV
37.5 mL. The compound gave also a single peak on reversed-phase
HPLC (6.2 mm × 25 cm Zorbax-ODS column, 2 mL/min) using
a methanol/water (8:2) solvent system: it was collected at RV 23
mL.
(20S)-1R,25-Dihydroxy-2-[3′-hydroxypropylidene]-19-norvi-
tamin D3 (6b). Vitamin 6b was obtained by hydrolysis of the silyl
protecting groups in the 19-norvitamin derivative 37b performed
analogously to the process described above for the preparation of
(20R)-isomers 5a and 6a. The product was purified by HPLC (10
mm × 25 cm Zorbax-Sil column, 4 mL/min) using a hexane/
2-propanol (8:2) solvent system. Pure 19-norvitamin 6b (95% yield)
was collected at RV 36.5 mL. The compound gave also a single
peak on reversed-phase HPLC (6.2 mm × 25 cm Zorbax-ODS

2918 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10
Glebocka et al.
column, 2 mL/min) using a methanol/water (8:2) solvent system:
it was collected at RV 18 mL.
1R,25-Dihydroxy-2-[3′-hydroxypropylidene]-19-norvitamin D3
(6a and 7a). To a solution of sulfone 39a (26.6 mg, 44 µmol) in
dry THF (150 µL) was added LiHMDS (1 M in THF, 41 µL, 41
µmol) at -78 °C under argon. The solution turned deep red. The
mixture was stirred at -78 °C for 1 h, and a solution of the ketone
23 (17.0 mg, 32.1 µmol) in THF (150 µL) was added. The stirring
was continued at -78 °C for 2 h, and the reaction mixture was
allowed to warm slowly to -50 °C. After stirring for an additional
50 min at -50 °C, it was poured into saturated NH4Cl and extracted
with ethyl acetate and hexane. The extract was washed with brine,
dried (Na2SO4) and evaporated. The yellow oily residue was
chromatographed on silica gel column. Elution with hexane
provided a mixture of protected vitamins 37a and 40a (1.3:1, 16
mg, 40%, 77% based on recovered starting material). Further elution
with hexane/ethyl acetate (97:3) gave unreacted sulfone 39a (13
mg).
Treatment of the obtained mixture of protected vitamin D
compounds 37a and 40a with tetrabutylammonium fluoride,
performed as described for 36a, gave a mixture of vitamin D
analogues 6a and 7a which was purified by HPLC (10 mm × 25
cm Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol
(8:2) solvent system. A pure mixture of 19-norvitamins 6a and 7a
(1.3:1, 95% yield) was collected at RV 37.5 mL. Separation of both
isomers was easily achieved by reversed-phase HPLC (6.2 mm ×
25 cm Zorbax-ODS column, 2 mL/min) using a methanol/water
(8:2) solvent system. Analytically pure E-isomer 6a was collected
at RV 23 mL and Z-isomer 7a at RV 29 mL.
(20S)-1R,25-Dihydroxy-2-[3′-hydroxypropylidene]-19-norvi-
tamin D3 (6b and 7b). Julia coupling between sulfone 39b and
ketone 23 followed by chromatographical separation of the products
was carried out as described above for the 20R-isomer 39a. Elution
with hexane provided a mixture of protected vitamins 37b and 40b
(1.3:1, 42%, 78% based on recovered starting material).
Treatment of the obtained mixture of protected vitamin D
compounds 37b and 40b with tetrabutylammonium fluoride,
performed as described for 36a, gave a mixture of vitamin D
analogues 6b and 7b which was purified by HPLC (10 mm × 25
cm Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol
(8:2) solvent system. A pure mixture of 19-norvitamins 6b and 7b
(1.3:1, 96% yield) was collected at RV 36.5 mL. Separation of both
isomers was easily achieved by reversed-phase HPLC (6.2 mm ×
25 cm Zorbax-ODS column, 2 mL/min) using a methanol/water
(8:2) solvent system. Analytically pure E-isomer 6b was collected
at RV 18 mL and Z-isomer 7b at RV 28 mL.
Biological Studies. 1. Measurement of Intestinal Calcium
Transport and Bone Calcium Mobilization. Male, weanling
Sprague-Dawley rats were purchased from Harlan (Indianapolis,
IN), housed in D-deficient lighting conditions and fed a D-deficient
diet.24 The first week the animals were fed Diet 11 (0.47% Ca) +
AEK oil. During the next 3 weeks, the rats were fed the same diet
with the calcium carbonate removed (0.02% Ca). The rats were
then switched back to a diet containing 0.47% Ca for one week
before returning to a diet containing 0.02% Ca for two more weeks.
Dose administration began during the last week on the 0.02%
calcium diet. Four consecutive intraperitoneal doses in 0.1 mL of
(95:5) 1,2-propanediol/ethanol were given approximately 24 h apart.
Twenty-four hours after the last dose, blood was collected from
the severed neck and the concentration of serum calcium determined
as a measure of bone calcium mobilization. The first 10 cm of the
intestine was also collected for intestinal calcium transport analysis
using the everted gut sac method as previously described except
radioactive tracer was not used.25
2-[(1R,3aS,7aR)-7a-Methyl-[(R)-6-[(triethylsilyl)oxy]-6-meth-
ylheptan-2-yl]-octahydro-inden-(4E)-ylidene]ethanol (38a). To
a suspension of NaH (49 mg, 2.04 mmol) in anhydrous THF (1.2
mL) was added (EtO)2P(O)CH2COOEt (500 µL, 2.53 mmol) at 0
°C. The mixture was stirred at room temperature for 10 min and
lithium chloride (13 mg, 0.30 mmol) was then added. The stirring
was continued for 1 h and cooled to 0 °C, and a solution of the
protected hydroxy ketone 35a (100 mg, 0.25 mmol) in THF (0.6
mL) was added. After stirring at room temperature for 32 h, the
reaction mixture was diluted with ethyl acetate and poured into
saturated ammonium chloride. The organic phase was separated,
washed with brine, dried (Na2SO4) and evaporated. The residue
was separated by flash chromatography. Elution with hexane/ethyl
acetate (99:1) afforded pure oily [(1R,3aS,7aR)-7a-methyl-1-[(R)-
6-[(triethylsilyl)oxy]-6-methylheptan-2-yl]-octahydro-inden-(4E)-
ylidene]acetic acid ethyl ester (82 mg, 70%).
Diisobutylaluminum hydride (1 M in toluene, 200 µL, 0.2 mmol)
was added to a stirred solution of allylic ester (29 mg, 62 µmol) in
anhydrous toluene (0.5 mL) at -78 °C under argon. The mixture
was stirred at -78 °C for 1 h, and the reaction was quenched by
addition of potassium sodium tartrate (2 N, 1 mL), aq HCl (2 N, 1
mL), and water (12 mL). The mixture was poured into brine and
extracted with ethyl acetate and ether. The combined extracts were
washed with diluted NaHCO3 and brine, 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, 20
mL, and 9:1, 10 mL) gave allylic alcohol 38a (23 mg, 87%) as a
colorless oil.
2-[(1R,3aS,7aR)-7a-Methyl-[(S)-6-[(triethylsilyl)oxy]-6-meth-
ylheptan-2-yl]-octahydro-inden-(4E)-ylidene]ethanol (38b). Trans-
formation of the protected Grundmann ketone 35b into allylic
alcohol 38b was achieved using an analogous two-step reaction
sequence as described above for 20R-isomer 35a. Thus, the allylic
ester, prepared in 68% yield by Wittig reaction of the ketone 35b
and triethyl phosphonoacetate, was reduced with DIBALH to give
the alcohol 38b in 98% yield.
(1R,3aS,7aR)-4-[2-(Benzothiazole-2-sulfonyl)-(4E)-ethylidene]-
7a-methyl-1-[(R)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl]-oc-
tahydro-indene (39a). To a solution of 2-mercaptobenzotriazole
(12.5 mg, 75 µmol) and Ph3P (19.5 mg, 75 µmol) in dry methylene
chloride (150 µL) at 0 °C was added a solution of allylic alcohol
38a (21 mg, 50 µmoL) in methylene chloride (150 µL) followed
by DIAD (14 µL, 50 µmol). The mixture was stirred at 0 °C for 1
h, and the solvents were evaporated in vacuo. The residue was
dissolved in ethanol (300 mL) and cooled to 0 °C, and 30% H2O2
(30 µL) was added, followed by (NH4)6Mo7O24 ¨I 4H2O (12.3 mg,
10 µmol). The mixture was stirred at room temperature for 3 h,
poured into cold saturated Na2SO3, and extracted with ethyl acetate.
The organic layer was washed with brine, dried (Na2SO4), and
evaporated. The residue was dissolved in a small volume of
benzene/hexane (1:1) and applied on a silica Sep-Pak. Elution with
hexane/ethyl acetate (9:1, 20 mL and 85:15, 20 mL) and removal
of the solvents gave an oily product (33 mg) that was dissolved in
anhydrous DMF (300 µL). Imidazole (18 mg, 0.26 mmol) was
added followed by triethylsilyl chloride (50 µL, 0.29 mmol), and
the mixture was stirred at room temperature for 3 h. Ethyl acetate
was added and water, and the organic layer was separated, washed
with brine, dried (Na2SO4), and evaporated. The residue was
purified by HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/
min) using a hexane/ethyl acetate (9:1) solvent system. Analytically
pure sulfone 39a (22.8 mg, 76%) was collected at RV 24 mL.
(1R,3aS,7aR)-4-[2-(Benzothiazole-2-sulfonyl)-(4E)-ethylidene]-
7a-methyl-1-[(S)-6-[(triethylsilyl)oxy]-6-methylheptan-2-yl]-oc-
tahydro-indene (39b). Conversion of the allylic alcohol 38b into
sulfone 39b involved an analogous reaction sequence as that
described above for 20R-isomers 38a and 39a. The final product
was purified by HPLC (10 mm × 25 cm Zorbax-Sil column, 4
mL/min) using a hexane/ethyl acetate (9:1) solvent system. Analyti-
cally pure sulfone 39b (80%) was collected at RV 23 mL.
Calcium was measured in the presence of 0.1% lanthanum
chloride by means of a Perkin-Elmer atomic absorption spectrometer
Model 3110. Intestinal calcium transport is expressed as the serosal:
mucosal ratio of calcium in the sac to the calcium in the final
incubation medium. Bone calcium mobilization represents the rise
in serum calcium of the rats maintained on a very low calcium
diet.
2. Mouse Studies. 6-7 week old female CD-1 mice were
purchased from Harlan (Indianapolis, IN). The animals were group

1R,25-Dihydroxy-19-norVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 10 2919
housed and fed a purified diet containing 0.47% calcium.24 After a
5-7 day acclimation period, the animals were given a single dose
of the designated analogues by gastric gavage. The compounds were
formulated in Neobee oil and ethanol (5%) so that the dose could
be delivered in a volume of 4 mL/kg body weight. Blood was
collected for serum calcium concentration analyses at various
timepoints following dose delivery. Serum calcium was analyzed
as described above.
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 5.0) software package (Autodesk, Inc.).
MM+ is an all-atom force field based on the MM2 functional form.
The procedure used for generation of the respective conformers of
the alkoxypropylidene substituent and finding the global minimum
structures was analogous to that described previously by us for the
vitamin D side chain conformers 28 and involved the Conformational
Search module of the ChemPlus (release 1.5) program (Hypercube,
Inc.). The couplings observed in the 1H NMR spectra of the
synthesized compounds were compared to those calculated using
the PC MODEL (release 6.0) 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.
3. Measurement of Binding to the Full-Length Rat Recom-
binant Vitamin D Receptor (VDR). Purified full-length rat
recombinant receptor was prepared as described earlier with a few
modifications.25 The entire coding region for the rat VDR was
inserted into the p29 plasmid including the flexible insertion region
(residues 165-211). During the purification of the full-length
receptor, the eluate from the metal affinity column was dialyzed
against the same buffer but at a pH of 8.0 instead of 7.0 and 50
mM sodium phosphate was used instead of 20 mM. The size of
the SP-Sepharose Fast Flow column was slightly different, 1.5 ×
17 cm, and the salt gradient used for elution of the VDR from this
column linearly increased from 0 to 0.8 M phosphate buffer over
a total volume of 300 mL. Fractions judged pure by SDS-PAGE
were combined and dialyzed against 25 mM EPPS at pH 8.5,
containing 50 mM NaCl and 0.02% NaN3. Following dialysis, the
protein was concentrated by ultracentrifugation to approximately
1.4 mg/mL. Aliquots of the purified protein were flash-frozen in
liquid nitrogen and stored at -80 °C until use. On the day of each
binding assay, the protein was diluted in TEDK50 (50 mM Tris,
1.5 mM EDTA, pH 7.4, 5 mM DTT, 150 mM KCl) with 0.1%
Chaps detergent. The receptor protein and ligand concentration were
optimized such that no more than 20% of the added radiolabeled
ligand was bound to the receptor. Unlabeled ligands were dissolved
in ethanol and the concentrations determined using UV spectro-
photometry (1R,25(OH)2D3: molar extinction coefficient ꢀ )
18 200 and λmax ) 265 nm; the tested 19-norvitamin D com-
pounds: ꢀ ) 42 000 and λmax ) 252 nm). Radiolabeled ligand (3H-
1R,25(OH)2D3, ∼159 Ci/mmol) was added in ethanol at a final
concentration of 1 nM. Radiolabeled and unlabeled ligands were
added to 100 µL of the diluted protein at a final ethanol
concentration of e10%, mixed, and incubated overnight on ice to
reach binding equilibrium. The following day, 100 µL of hydroxyl-
apatite slurry (50%) was added to each tube and mixed at 10-min
intervals for 30 min. The hydroxylapatite was collected by
centrifugation and then washed three times with Tris-EDTA buffer
(50 mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron
X-100. After the final wash, the pellets were transferred to
scintillation vials containing 4 mL of Biosafe II scintillation cocktail,
mixed, and placed in a scintillation counter. Total binding was
determined from the tubes containing only radiolabeled ligand. The
displacement experiments were carried out in duplicate on two to
three different occasions.
4. Measurement of Cellular Differentiation. Human promy-
elocytic leukemia (HL-60) cells were grown in RPMI-1640 medium
containing 10% fetal bovine serum at 37 °C in the presence of 5%
CO2. HL-60 cells were plated at 1.2 × 105 cells/plate. Eighteen
hours after plating, cells in duplicate were treated with the
compound tested so that the final concentration of ethanol was less
than 0.2%. Four days later, the cells were harvested and a nitro
blue tetrazolium (NBT) reduction assay was performed. The
percentage of differentiated cells was determined by counting a
total of 200 cells and recording the number that contained
intracellular black-blue formazan deposits.26 The experiment was
repeated 2 to 3 times, and the results are reported as the mean.
Verification of differentiation to monocytic cells was determined
by measuring phagocytic activity (data not shown).
5. Transcriptional Assay. Transcription activity was measured
in ROS 17/2.8 (bone) cells that were stably transfected with a 24-
hydroxylase (24OHase) gene promoter upstream of a luciferase
reporter gene.27 Cells were given a range of doses. Sixteen hours
after dosing, the cells were harvested and luciferase activities were
measured using a luminometer. Each experiment was performed
in duplicate two to three separate times.
Acknowledgment. The work was supported by funds from
the Wisconsin Alumni Research Foundation. We gratefully
acknowledge Jean Prahl for carrying out the binding studies
and the HL-60 differentiation measurements, Julia Zella for
carrying the transcriptional studies, and to Xiaohong Ma for
the testing of these compounds in the animals.
Supporting Information Available: Purity criteria for the
synthesized compounds; spectral data of the synthesized com-
pounds; figures with preferred, energy-minimized conformations
of the synthetic intermediates 12, 13, 19, 22, 24, 25; figures with
either the competitive binding curves or dose-response curves
derived from the binding, cellular differentiation, and transcriptional
assays of the vitamin D analogues 5a, 6a,b, 7a,b. This material is
available free of charge via the Internet at http://pubs.acs.org.
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