Removal of the 26-methyl group from 19-nor-1α,25-dihydroxyvitamin D3 markedly reduces in vivo calcemic activity without altering in vitro VDR binding, HL-60 cell differentiation, and transcription

Article abstract of DOI:10.1021/jm1010447

Twelve new analogues of 19-nor-1α,25-dihydroxyvitamin D₃ (5-16) were prepared by convergent syntheses, employing the Wittig Horner reaction. The necessary Grundmann type ketones (45-48), possessing fixed configurations of the hydroxyl group at

Full text of DOI:10.1021/jm1010447

8642 J. Med. Chem. 2010, 53, 8642–8649
DOI: 10.1021/jm1010447
Removal of the 26-Methyl Group from 19-nor-1r,25-Dihydroxyvitamin D3
Markedly Reduces in Vivo Calcemic Activity without Altering in Vitro VDR Binding, HL-60 Cell
Differentiation, and Transcription
Pawel Grzywacz, Grazia Chiellini, Lori A. Plum, Margaret Clagett-Dame, and Hector F. DeLuca*
Department of Biochemistry, University of Wisconsin;Madison, 433 Babcock Drive, Madison, Wisconsin 53706, United States
Received August 11, 2010
Twelve new analogues of 19-nor-1R,25-dihydroxyvitamin D3 (5-16) were prepared by convergent
syntheses, employing the Wittig-Horner reaction. The necessary Grundmann type ketones (45-48),
possessing fixed configurations of the hydroxyl group at C-25, were obtained by a multistep procedure
from commercial vitamin D2 and enantiomers of 1,3-butanediol (23 and 24). We have examined the
influence of removal of one of the methyl groups located at C-25 on the biological in vitro and in vivo
activity. The in vivo tests showed that the synthesized vitamin D compounds (5-16) exhibit reduced
calcemic activity both in bone and in the intestine. However, in vitro potency of 2-methylene and 2R-
methyl compounds (5-10, 13, and 14) remained similar or enhanced as compared to that of
1R,25-(OH)2D3.
Introduction
approach required using new Grundmann type ketones
(45-48) (Scheme 1), which we intended to prepare from the
known alcohols (17 and 18) and commercially available en-
antiomers of 1,3-butanediols (23 and 24) (Scheme 2). To obtain
C,D ring ketones (45-48), we employed the Wittig reaction16 to
attach new side chains to the C,D ring aldehydes (19 and 20).
Commercial (S)- and (R)-1,3-butanediols (23 and 24), respec-
tively, appeared to be suitable starting materials for construction
of the side chains, possessing fixed configurations of the hydro-
xyl group at C-25 (steroidal numbering). Selective tosylation of
primary hydroxy groups of diols (23 and 24), followed by a
triethylsilyl protection of secondary hydroxy groups, provided
corresponding tosylates 25 and 26 (Scheme 2). These com-
pounds (25 and 26) were converted into primary iodides 27 and
28, which were treated with triphenylphosphine in acetonitrile
under reflux. The triethylsilyl ethers appeared to be unstable
under these reaction conditions, which led to the generation of
hydroxyphosphonium iodides 21 and 22. Despite the loss of the
protective group, we used the salts 21 and 22, based on the
efficient application of the analogous salt in the synthesis of the
Windaus-Grundmann ketone as described by Mourino.17
The C,D ring aldehydes 19 and 20 were obtained by the
oxidation of the corresponding alcohols 17 and 18, which
were previously prepared in our laboratory from commercial
vitamin D2.12 The Wittig reaction between compounds 19
and 20 and ylides, generated from the hydroxyphosphonium
iodides 21 and 22 by n-butyllithium, provided only olefinic
products (29-32) with the E-geometry of the introduced
double bond. The catalytic hydrogenation of the compounds
(29-32) furnished the tertiary alcohols 33-36, which were
protected as tert-butyldimethylsilyl ethers (37-40). The
removal of the benzoyl group under basic conditions gave the
secondary alcohols 41-44, which were subjected to catalytic
oxidation with tetrapropylammonium perruthenate18 in the
presence of 4-methylmorpholine N-oxide and afforded the
expected ketones 45-48. The Horner-Wittig reaction between
1R,25-Dihydroxyvitamin D3 [1R,25-(OH)2D3, calcitriol, 1;
Figure 1], the active form of vitamin D3, is one of the primary
biological regulators of calcium and phosphorus homeostasis
in humans and animals.1-4 Its important biological effects
include the inhibition of proliferation and increased differ-
entiation of various malignant cells,5-8 as well as suppression
of the autoimmune disease.9 Such a broad range of diverse
functions suggests enormous therapeutic potential of vitamin
D compounds.10 However, the application of these com-
pounds has been limited by the danger of hypercalcemia.
Structural modificationsofthe vitaminD molecule have led to
a search for new analogues that exhibit reduced calcemic
potency and selective actions.11 In 2007, we reported the
biological properties of two 2-methylene-19-nor analogues
(2 and 3) in which the side chains were modified at C-25.12,13
Removal of the 25-hydroxy group from compound 414 led to
reduced activity in vitro, but in vivo calcemic potency re-
mained unchanged likely because of in vivo 25-hydroxylation.
Methyl replacement of the 25-hydroxy group in 4 reduced the
overall potency but retained bone selectivity (compound 3).
These results suggest that a possible approach to reducing
calcemic activity might be the elimination of the methyl
groups surrounding C-25. We now report several new ana-
logues produced by removing the 26-methyl group from
the 2-carbon-substituted 19-nor-1R,25-dihydroxyvitamin
D3 and its 20S isomer.
Results and Discussion
Chemistry. The synthetic strategy for new analogues (5-16)
was based on the Lythgoe type Horner-Wittig olefination
reaction,15 which was successfully utilized by us earlier
for preparation of the vitamin D compounds (2 and 3).12 This
*To whom correspondence should be addressed. Tel: 608-262-1620.
Fax: 608-262-7122. E-mail: deluca@biochem.wisc.edu.
r
pubs.acs.org/jmc
Published on Web 11/24/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8643
Figure 1. Chemical structures of 1R,25-dihydroxyvitamin D3 (calcitriol, 1) and 19-nor analogues (2-16).
Scheme 1. Synthesis of the Grundmann Ketones 45-48a
a Reagents: (i) SO3 pyridine, Et3N, DMSO, CH2Cl2. (ii) (1) 21 or 22, n-BuLi, THF; (2) 19 or 20, THF. (iii) H2, Pd/C, MeOH. (iv) TBSOTf, 2,
3
6-lutidine, CH2Cl2. (v) (1) KOH, EtOH; (2) aqueous HCl. (vi) TPAP, NMO, molecular sieves 4 A, CH2Cl2.
˚

8644 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Scheme 2. Synthesis of the Phosphonium Iodides 21 and 22a
Grzywacz et al.
a Reagents: (i) (1) TsCl, DMAP, Et3N, CH2Cl2; (2) TESOTf, 2,6-lutidine, CH2Cl2. (ii) KI, acetone. (iii) Ph3P, MeCN.
Scheme 3. Syntheses of the Vitamin D Analogues 5-16a
a Reagents: (i) (1) 49, PhLi, THF; (2) 45, 46, 47, or 48, THF. (ii) aqueous HF, THF, MeCN. (iii) H2, (Ph3P)3RhCl, PhH.
the corresponding C,D fragments (45-48) and the anion,
generated from the phosphine oxide 49 by phenyllithium,
produced the protected vitamin D compounds 50-53
(Scheme 3). The silyl-protective groups were cleaved in the
presence of hydrofluoric acid, and after the final purification
by high-performance liquid chromatography (HPLC), the
target vitamin D analogues 5-8 were obtained. The X-ray
analysis of a single-crystal of the compound 5 (Figure 2)
confirmed the stereochemistry at C-25 as (25S). The homo-
geneous catalytic hydrogenation of 2-methylene compounds
5-8, in the presence of tris(triphenylphosphine)rhodium(I)
chloride, provided approximately an equimolar mixture of
2-methyl-19-norvitamins, which were easily separated by HPLC.
differentiation, and gene transcription activity. Table 1 shows
the results of the competitive VDR binding assays as com-
pared to the natural hormone 1 and to 2-methylene-19-
nor-(20S)-1R,25-dihydroxyvitamin D3 (4). No remarkable
differences in binding to the receptor were noted except with
(20R)-2β-methyl compounds 11 and 15. The VDR binding
affinities of these analogues are approximately one log less
than those of the native hormone 1 or analogues possessing
2-methylene (4-8) or 2R-methyl (9, 10, 13, and 14) groups.
The results of the HL-60 cell differentiation assays are
depicted in Table 1. In these assays, cells were given one dose
of the indicated amounts, and after 4 days, the extent of
differentiation was assessed by incubating the cells with a
substrate (nitro blue tetrazolium), which is reduced bythe cells
if they have differentiated into monocytes. Although (25R)
analogues demonstrate slightly more activity than (25S)
compounds, the configuration of the 25-hydroxy group exerts
Biological Evaluation
A standard set of in vitro assays was conducted with all
12 compounds (5-16) to ascertain receptor binding, cell

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8645
Figure 2. X-ray crystal structure of (20R,25S)-2-methylene-19,26-dinor-1R,25-dihydroxyvitamin D3 (5).
Table 1. VDR Binding Properties,a HL-60 Differentiating Activities,b and Transcriptional Activitiesc of the Vitamin D Analogues (5-16)
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 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 and the synthesized vitamin D analogues. The differentiation state was determined by measuring the percentage of cells
reducing nitro blue tetrazolium (NBT). The EC50 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 EC50 to the EC50 for 1R,25-(OH)2D3. c Transcriptional
assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene reporter plasmid. The EC50 values are derived from dose-response curves
and represent the analogue concentration capable of increasing the luciferase activity by 50%. The luciferase activity ratio is the average ratio of the EC50
for the analogue to the EC50 for 1R,25-(OH)2D3. All of the experiments were carried out in duplicate on at least two different occasions.
a small impact on the overall potency in cell differentiation.
Two (20S,25R) compounds, 8 and 14, possessing 2-methylene
or 2R-methyl group, respectively, have differentiation activity
20 times higher than 1,25(OH)2D3 1 but approximately 0.8

8646 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Grzywacz et al.
Figure 3. Serum calcium levels of vitamin D-deficient rats on a 0.02% calcium diet and given vehicle or vehicle plus the indicated compound
each day for 4 days as an intraperitoneal injection. Serum was harvested 24 h after the last dose. Note: Dose ranging studies were performed
with these compounds, but only the highest dose levels tested are shown to highlight the differences and make it easier for the reader.
times lower than parent compound 4. Four (20R) compounds
(5, 7, 9, and 13) and two (20S,25S)-analogues (6 and 10)
exhibit the next highest activity in this assay, which is approxi-
mately similar to that observed with the native hormone 1.
Not surprisingly, the derivatives 11, 12, 15, and 16, containing
a 2β-methyl substituent, have the lowest activities, 3-35 times
lower than 1,25(OH)2D3 1.
The pattern of potencies in in vitro transcription assays is
shown in Table 1. Similar to that observed in the HL-60 cell
differentiation assays, (25R) compounds demonstrate higher
transcriptional potency than in the case of (25S) analogues.
This difference is more noticeable in the case of the (20S)
compounds. In this cell-based assay, two (20S,25R) com-
pounds 8 and 14 express the highest transcriptional potency.
The activity of the other 2-methylene and 2R-methyl ana-
logues (5-7, 9-13) is similar to that observed with natural
hormone 1. Consistent with the other two in vitro assays, the
derivatives possessing a 2β-methyl group 11, 12, 15, and 16
possess the lowest transcriptional activities.
In vivo biological activities are shown in Figure 3 and Table 2.
Both intestinal calcium transport and bone calcium mobiliza-
tion were measured in the same rats made vitamin D-deficient
prior to administering any of the experimental compounds. A
rise in blood calcium levels reflects the ability of an analogue
to stimuate the mobilization of bone calcium because the
animals are fed a diet essentially devoid of calcium. Analogue
activity in the intestine was assessed ex vivo by analyzing
calcium transport in the everted gut sac preparation. Figure 3
highlights the fact that all of the analogues have low bone
mobilizing activity. The most potent compound 8 is at least 25
times (21060 pmol/780 pmol) less active than the native hormone
1 in vivo; yet, analogue 8 is much more potent than 1,25-
(OH)2D3 1 in causing cellular differentiation and stimulating
in vitro 24-OHase transcription. The order of bone calcium
mobilizing activity of the 19,26-dinor compounds (5-16)
parallels that observed in vitro, among them 2-methylene
and 2R-methyl analogues of (20S) configuration 6, 8, 10,
and 14 being the most potent. It is worth noting that (25R)
diastereoisomers are more potent on bone.
Table 2 summarizes the results obtained from the intestinal
calcium transport assays. In contrast to the bone, the (20S,25R)
compound8 has similaractivity tothenativehormone1 orthe
parent analogue 4 in the intestine. The remainder of the
compounds has reduced intestinal calcium transport activity,
among them 2β-methyl compounds 11, 15, and 16 have the
lowest in particular when coupled with a (20R) configuration.
Conclusions
The present study clearly shows that removal of one methyl
from C-25 of the 19-norvitamin D3 analogues (5-16) mark-
edly reduces the calcemic activity derived from either bone or
intestine. On the other hand, the lack of the same methyl
group has little impact on receptor binding, HL-60 differen-
tiation, and in vitro transcription. The exceptions are the 2β-
methyl analogues that are virtually devoid of any activity. In
the case ofthe 20S compounds analogues(6, 8, 10, and14), the
in vitro activity is increased by removal of the methyl at C-25
accompanied by a 25R configuration. Thus, the most potent
of the 19,26-dinorvitamin D series are (20S,25R)-2-methylene
and 2R-methyl analogues 8 and 14, respectively. In general,
25R-hydroxy analogues exhibit higher potency, measured
both in vitro and in vivo, than 25S diastereoisomers. The
greatly reduced calcemic activities of the presented com-
pounds (5-10, 13, and 14) coupled with their ability to
promote differentiation make them drug candidates for treat-
ment of secondary hyperparathyroidism of renal failure and,
possibly, certain cancers.
Experimental Section
Chemistry. Melting points (uncorrected) were determined on
a Thomas-Hoover capillary melting point apparatus. Optical
rotations were measured in chloroform or methanol using a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8647
Table 2. Summary of Intestinal Ca Transport
a S/M ratio = the amount of calcium found in the serosal compartment divided by the amount of calcium present in the mucosal compartment.
Perkin-Elmer model 343 polarimeter at 22 °C. Ultraviolet (UV)
absorption spectra were recorded with a Perkin-Elmer Lambda
3B UV-vis spectrophotometer in ethanol or hexane. 1H nuclear
magnetic resonance (NMR) spectra were recorded in deuterio-
chloroform, acetone-d6, or methanol-d4 at 400 and 500 MHz
with Bruker Instruments DMX-400 and DMX-500 Avance
console spectrometers. 13C NMR spectra were recorded in
deuteriochloroform, acetone-d6, or methanol-d4 at 100 and
125 MHz with the same Bruker Instruments. Chemical shifts (δ)
in parts per million are quoted relative to internal Me4Si
(δ 0.00). Numbers in parentheses following the chemical shifts in
the 13C NMR spectra refer to the number of attached hydrogens
as revealed by DEPT experiments. 31P NMR spectra were
recorded at 162 MHz with Bruker Instruments DMX-400
Avance console spectrometer in methanol-d4. Chemical shifts
(δ) in parts per million are quoted relative to external H3PO4
(δ 0.00). Electron impact (EI) mass spectra were obtained with a
Micromass AutoSpec (Beverly, MA) instrument. HPLC was
performed on a Waters Associates liquid chromatograph
equipped with a model 6000A solvent delivery system, model
U6K Universal injector, and model 486 tunable absorbance
detector. The known alcohols 17 and 18 were prepared accord-
ing to the published procedure.12 THF was freshly distilled
before use from sodium benzophenone ketyl under argon. A
designation “(volume þ volume)”, which appears in general
procedures, refers to an original volume plus a rinse volume.
All final vitamin D analogues synthesized by us gave single
sharp peaks on HPLC, and they were judged at least 99.5%
pure. Two HPLC systems (straight- and reversed-phase) were
employed as indicated in Table 3 in the Supporting Information.
General Procedure for the Synthesis of Compounds 19 and 20.
To a solution of the alcohol 17 or 18 (1 equiv), triethylamine (5 equiv)
in anhydrous methylene chloride, and anhydrous DMSO, the
sulfur trioxide pyridine complex (6 equiv) was added at 0 °C.
The reaction mixture was stirred under argon at 0 °C for 1 h, and
then, it was concentrated under reduced pressure. The crude
mixture was diluted with ethyl acetate, washed with brine, dried
over Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel
(2 f 5% ethyl acetate/hexane) to give the aldehyde 19 or 20.
General Procedure for the Synthesis of Compounds 25 and 26.
To a stirred solution of 1,3-butanediol 23 or 24 (1 equiv), DMAP
(0.02 equiv), and triethylamine (3 equiv) in anhydrous methy-
lene chloride, p-toluenesulfonyl chloride (1.2 equiv) was added
at 0 °C. The reaction mixture was stirred at 4 °C for 22 h. It was
diluted with methylene chloride, washed with water, dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica
(20 f 50% ethyl acetate/hexane) to give a tosylate.
To a stirred solution of tosylate (1 equiv) and 2,6-lutidine (1.1
equiv) in anhydrous methylene chloride, triethylsilyl trifluoro-
methanesulfonate (1 equiv) was added at -50 °C. The reaction
mixture was allowed to warm to room temperature (4 h), and
stirring was continued for an additional 20 h. It was diluted with
methylene chloride, washed with water, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue
was purified by column chromatography on silica (3% ethyl
acetate/hexane) to give the product 25 or 26.
General Procedure for the Synthesis of Compounds 27 and 28.
To a stirred solution of the tosylate 25 or 26 (1 equiv) in
anhydrous acetone (50 mL), potassium iodide (5.5-6.3 equiv)
was added. The reaction mixture was refluxed for 10 h. Water
(30 mL) was added to dissolve salts, and the mixture was
extracted with ethyl acetate. Combined organic phases were
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was purified by column chromatography
on silica (3% ethyl acetate/hexane) to give the iodide 27 or 28.
General Procedure for the Synthesis of Compounds 21 and 22.
To a stirred solution of the iodide 27 or 28 (1 equiv) in anhydrous
acetonitrile (50 mL), triphenylphosphine (3 equiv) was added.
The reaction mixture was refluxed for 2 days. The solvent was
evaporated under reduced pressure to give the solid. Ethyl
acetate (50 mL) was added, the mixture was stirred at room
temperature for 4 h, and the solvent was removed by filtration.
The solid was stirred again with ethyl acetate for 1 h, and the
solvent was removed. After it was dried, the pure phosphonium
iodide 21 or 22 was obtained. An alternative to this procedure is
to dissolve the solid mixture of the phosphonium iodide and
triphenylphosphine in methylene chloride and reprecipitate the
salt by adding diethyl ether.

8648 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Grzywacz et al.
General Procedure for the Synthesis of Compounds 29-32. To a
stirred suspension of the phosphonium salt 21 or 22 (3 equiv) in
anhydrous THF (5 mL), n-butyllithium (4.5-6 equiv) was added
at -20 °C. The solution was stirred at -20 °C for 1 h, and it turned
deep orange. A precooled solution of aldehyde 19 or 20 (1 equiv) in
anhydrous THF (1 þ 1 mL) was added, and the reaction mixture
was stirred at -20 °C for 4 h and at room temperature for 18 h. The
reaction was quenched with water, and the mixture was extracted
with ethyl acetate. Combined organic phases were washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica (5 f 10% ethyl acetate/hexane) to give the product
29, 30, 31, or 32.
General Procedure for the Synthesis of Compounds 33-36. To
a solution of the olefin 29, 30, 31, or 32 in methanol (6 mL), 10%
palladium on activated carbon (7 mg) was added, and the
mixture was hydrogenated overnight. The catalyst was filtered
off, and the filtrate was concentrated under reduced pressure. The
residue was purified by column chromatography on silica (5 f
10% ethyl acetate/hexane) to give the product 33, 34, 35, or 36.
General Procedure for the Synthesis of Compounds 37-40. To
a stirred solution of the alcohol 33, 34, 35, or 36 (1 equiv) and
2,6-lutidine (3.6-4 equiv) in anhydrous methylene chloride
(3 mL), tert-butyldimethylsilyl trifluoromethanesulfonate
(1.8-2 equiv) was added at -20 °C. The reaction mixture was
stirred at 0 °C for 1 h. It was quenched with water and extracted
with methylene chloride. Combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel (3% ethyl acetate/hexane) to give
the product 37, 38, 39, or 40.
General Procedure for the Synthesis of Compounds 41-44. To
a stirred solution of the benzoate 37, 38, 39, or 40 in anhydrous
ethanol (10 mL), a solution of sodium hydroxide in anhydrous
ethanol (2.5 M, 2 mL) was added. The reaction mixture was
refluxed for 18 h. It was cooled to room temperature, neutralized
with 5% aqueous solution of HCl, and extracted with methylene
chloride. Combined organic phases were washed with a satu-
rated aqueous NaHCO3 solution, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (5 f 10% ethyl
acetate/hexane) to give the alcohol 41, 42, 43, or 44.
General Procedure for the Synthesis of Compounds 45-48. To
a stirred solution of 4-methylmorpholine N-oxide (3.2-3.6 equiv)
in anhydrous methylene chloride (0.5 mL), pulverized molecular
sieves A4 (ca. 60 mg) were added, and the mixture was stirred for
15 min. Then tetrapropylammonium perruthenate (0.13-0.22
equiv) was added, followed by a solution of the alcohol 41, 42, 43,
or 44 (1 equiv) in anhydrous methylene chloride (300 þ 300 μL).
The resulting dark mixture was stirred at room temperature for
1 h, and then, it was filtered through a silica Sep-Pak, which was
further washed with methylene chloride. After evaporation of
the solvent, the ketone 45, 46, 47, or 48 was obtained.
acetonitrile (1:9 ratio, 2 mL) was added at 0 °C, and the resulting
mixture was stirred at room temperature for 6 h. The reaction
was quenched with a saturated aqueous NaHCO3 solution and
extracted with ethyl acetate. Combined organic phases were
washed with brine, dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The residue was purified on a Waters
silica Sep-Pak cartridge (10 f 30% ethyl acetate/hexane) to give
the crude products. Final purifications of the vitamin D com-
pounds were performed by straight phase HPLC (15% 2-pro-
panol/hexane; 4 mL/min; 9.4 mm ꢀ 25 cm Zorbax Sil column),
followed by reversed-phase HPLC (15% water/methanol; 3 mL/
min; 9.4 mm ꢀ 25 cm Zorbax Eclipse XDB-C18 column) to give
the analytically pure 19,26-dinorvitamin D analogues 5, 6, 7, or 8.
General Procedure for the Synthesis of Compounds 9-16.
Tris(triphenylphosphine)rhodium(I) chloride (1.0-1.5 equiv)
was added to dry benzene (5 mL) presaturated with hydrogen
(15 min). The mixture was stirred at room temperature until a
homogeneous solution was formed (ca. 25 min). A solution of
the analogue 5, 6, 7, or 8 (1 equiv) in dry benzene (3 þ 1 mL) was
added, and the reaction was allowed to proceed under a
continuous stream of hydrogen for 4 h. The solvent was
removed under reduced pressure, and the residue was redis-
solved in hexane/ethyl acetate (1:1) and applied on a Waters
silica Sep-Pak cartridge (2 g). A mixture of 2-methyl vitamins
was eluted with the same solvent system. A mixture of com-
pounds was further purified by HPLC (9.4 mm ꢀ 250 mm
Zorbax-Sil column, 4 mL/min) using a hexane/2-propanol
(85:15) solvent system. The separation of 2R-methyl analogues
9, 10, 13, or 14, from the 2β-methyl ones 11, 12, 15, or 16, was
achieved by reversed-phase HPLC (9.4 mm ꢀ 250 mm Zorbax
RX-C18 column, 3 mL/min) using a methanol/water (85:15)
solvent system.
Biological Studies. In Vitro Studies. VDR binding, HL-60
differentiation, and 24-hydroxylase transcription assays were
performed as previously described.19
In Vivo Studies. Bone calcium mobilization and intestinal
calcium transport were performed as previously described.19
Briefly, weanling rats were made vitamin D-deficient by housing
under lighting conditions that block vitamin D production in the
skin. In addition, the animals were fed a diet devoid of vitamin D
and alternating levels of calcium. Experimental compounds
were administered intraperitoneally once per day for four con-
secutive days. Twenty-four hours after the last dose was given,
the blood was collected, and everted gut sacs were prepared.
Calcium was measured in the blood and two different intestinal
compartments using atomic absorption spectrometry. Each
study was comprised of at least 5-6 animals/experimental
group and was controlled with a vehicle group (5% ethanol:95%
propylene glycol) and one or more positive control groups
[1,25(OH)2D3].
Acknowledgment. The work was supported in part by
funds from the Wisconsin Alumni Research Foundation.
We gratefully acknowledge Jean Prahl, Julia Zella, and
Jennifer Vaughan for carrying out the in vitro studies and
Heather Neils, Shinobu Miyazaki, and Xiaohong Ma for
conducting the in vivo studies. We thank Dr. Mark Anderson
for his assistance in recording NMR spectra. This study made
use of the National Magnetic Resonance Facility at Madison,
which was supported by the NIH Grants P41RR02301
(BRTP/NCRR) and P41GM66326 (NIGMS). Additional
equipment was purchased with funds from the University of
Wisconsin, the NIH (RR02781 and RR08438), the NSF
(DMB-8415048, OIA-9977486, and BIR-9214394), and the
U.S. Department of Agriculture.
General Procedure for the Synthesis of Compounds 50-53. To
a stirred solution of the phosphine oxide 49 (2.5-3.8 equiv) in
anhydrous THF (500 μL), a solution of phenyllithium (3.3-4.6
equiv) was added at -20 °C under argon. The mixture was stirred
for 30 min and then cooled to -78 °C. A precooled solution of
the Grundmann’s type ketone 45, 46, 47, or 48 (1 equiv) in
anhydrous THF (200 þ 100 μL) was added via cannula, and the
reaction mixture was stirred for 4 h at -78 °C. Then, the reaction
mixture was stirred at 4 °C for 19 h. Ethyl acetate (20 mL) was
added, and the organic phase was washed with brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The
residue was purified on a Waters silica Sep-Pak cartridge (0 f
2% ethyl acetate/hexane) to give the protected vitamin D
compound 50, 51, 52, or 53.
General Procedure for the Synthesis of Compounds 5-8. To a
solution of the protected vitamin 50, 51, 52, or 53 in THF (5 mL)
and acetonitrile (4 mL), a solution of aqueous 48% HF in
Supporting Information Available: Purity criteria, spectral
and X-ray data of the synthesized compounds; representative

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8649
figures with either the competitive binding curves or the dose-
response curves derived from the binding, cellular differentia-
tion, and transcriptional assays of the vitamin D analogues
(5-16). This material is available free of charge via the Internet
at http://pubs.acs.org.
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