Novel 1α,25-dihydroxyvitamin D3 analogues with the side chain at C12

Article abstract of DOI:10.1021/jm049016g

The plethora of actions of 1α,25(OH)₂D₃ in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmu

Full text of DOI:10.1021/jm049016g

J. Med. Chem. 2006, 49, 1509-1516
1509
Articles
Novel 1r,25-Dihydroxyvitamin D3 Analogues with the Side Chain at C12
Xose´ C. Gonza´lez-Avio´n,† Antonio Mourin˜o,† Natacha Rochel,*,‡ and Dino Moras‡
Departamento de Qu´ımica Orga´nica y Unidad Asociada al Consejo Superior de InVestigaciones Cient´ıficas, UniVersidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain, and De´partement de Biologie et de Ge´nomique Structurales, Institut de Ge´ne´tique et de
Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Sante´ et de la Recherche Me´dicale/
UniVersite´ Louis Pasteur, Parc d’InnoVation BP10142, 67404 Illkirch Cedex, France
ReceiVed December 2, 2004
The plethora of actions of 1R,25(OH)2D3 in various systems suggested wide clinical applications of vitamin
D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis,
cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to
reduce the calcemic side effects while maintaining the transactivation potency of the natural ligand. In light
of the crystal structures of the vitamin D nuclear receptor (VDR), novel analogues of the hormone 1R,25-
(OH)2D3 with side chains attached to C-12 were synthesized via the convergent Wittig-Horner approach.
Among the compounds studied, the analogue 2b showed the highest binding affinity for VDR and was the
most potent at inducing VDR transcriptional activity in a transient transfection assay (20% of the
transactivation activity of the natural ligand).
Introduction
The biologically active form of vitamin D3 (cholecalciferol;
1a in Figure 1) is its dihydroxy derivative, the steroid hormone
1R,25-dihydroxyvitamin D3 [1R,25(OH)2D3,a calcitriol; 1b]. The
first action of this hormone to be discovered was its contribution
to bone maintenance through control of calcium and phosphate
metabolism.1 Subsequently it has been found to participate in
the regulation of the cell cycle, affecting cell proliferation,
differentiation, and apoptosis, and to have immunosuppressive
effects.2 These roles have suggested that calcitriol could be used
not only for treatment of bone disorders such as osteoporosis
or renal osteodystrophy, but also to treat leukemia, cancer of
the breast, colon, and prostate gland, psoriasis, autoimmune
diseases, and graft rejection.3,4 However, the calcemic effects
of excess calcitriol (bone resorption, hypercalcemia, and
calcification of soft tissue) limit its use for these therapeutic
purposes. As a result, analogues have been sought that have
Figure 1. Chemical formulas of vitamin D3 (1a, cholecalciferol), the
steroid hormone 1R,25-dihydroxyvitamin D3 [1b, 1R,25(OH)2D3,
calcitriol], and the new analogues 2a-c.
less intense calcemic effects without appreciable loss of other
activities. To date, more than 3000 calcitriol analogues have
been synthesized, but few are of clinical interest.5,6
of several complexes in which calcitriol or agonist analogues
are bound to a modified human VDR ligand binding domain
(hVDR∆ LBD) that has the same ligand-binding, RXR-
associating, and transactivation capacities as those of wild-type
hVDR.11-14 We report here the synthesis of three new calcitriol
analogues designed in the light of these crystal structures, 2a-
c, together with the results of preliminary evaluation of their
molecular biological properties. The hope that the new ligands
may display novel interaction patterns of pharmacological
interest rests on their side chains being located at C12 rather
than at C17, the position of the side chain in the natural hormone
and in most existing analogues.
Rational design of new pharmacologically useful calcitriol
analogues must be based on precise understanding of the
mechanisms of action of the native hormone. It is known that
most of its actions are mediated by a specific nuclear receptor,7
the vitamin D receptor (VDR), which upon binding calcitriol
undergoes a series of events leading to the activation of the
transcription of target genes through the association with the
retinoid X nuclear receptor (RXR), coactivators of the p160
family (SRC-1),8 cointegrators that remodel chromatin (CBP),9
and mediator complexes that recruit RNA polymerase (DRIP/
TRAP).10 In previous work we determined the crystal structures
* Corresponding author: tel (33) 3 88 65 33 50; fax (33) 3 88 65 32 76;
e-mail rochel@igbmc.u-strasbg.fr.
Results and Discussion
† Universidad de Santiago de Compostela.
Design. The C25-hydroxyl group of calcitriol is essential for
its high-affinity binding to hVDR, since it forms hydrogen bonds
with hVDR histidines 305 and 397.11 Inspection of the cal-
‡ CNRS/INSERM/Universite´ Louis Pasteur.
a Abbreviations: NR, nuclear receptor; VDR, vitamin D NR; LBD, ligand
binding domain; 1R,25(OH)2-D3, 1alpha,25-dihydroxyvitamin D3.
10.1021/jm049016g CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

1510 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Gonza´lez-AVio´n et al.
Scheme 1. Retrosynthetic Analysis of Analogues 2a-c
Scheme 2. Synthesis of Ketone 5 from the Known Alcohol 6a
Figure 2. Docking of 2b into the LBP of hVDR∆. (A) Possible
conformation for the side chain of 2b (gray) superimposed with the
active conformation of 1R,25(OH)2D3 (blue). (B) VDR-2b complex.
The view is restricted to the region of the protein (H3, H6, H11, and
H12) contacting the aliphatic side chain. Only residues closer than 4.0
Å are shown. The ligand is shown in stick representation with carbon
and oxygen atoms in gray and red, respectively. The hydrogen bonds
formed by the 25-OH group of 2b are shown as green dashed lines.
citriol-hVDR∆ LBD complex mentioned above showed that
only side chains attached to the “northern” positions of the
calcitriol CD system (C17, C18, and C12) would be able to
interact with these histidines via a terminal hydroxyl group.
Since analogues with side chains at C17 have already been
extensively investigated and the effects of moving the side chain
to C18 have also been examined,15,16 we decided to explore
the possibilities of placing the side chain at C12â. According
to our calculations, a C12-borne hydroxy-terminated side chain
must contain seven carbon atoms between C12 and terminal
25-OH groups (standard C-C distances equal to 1.5-1.6 Å
for sp3 carbons) in order to fit into the VDR ligand-binding
pocket, considered in our design that both C12 and the terminal
25-OH occupy the same locations, relative to the VDR, as in
the VDR bound to the natural hormone. Imposing the additional
requirement that the side chain should terminate in the same
dimethylhydroxymethyl group as calcitriol, we therefore set out
to prepare compound 2b (Figures 1 and 2), in which the side
chain is seven carbons long, and compounds 2a and 2c, in which
it has one less and one more carbon, respectively. Interactions
of the 2b side chain with the protein (Figure 2) are similar to
those of 1R,25(OH)2D3 except with Leu309 and decreased
interactions with H305 and H397.
a Reagents and conditions: (a) PDC, CH2Cl2 (97%). (b) LDA, THF;
TMSCl. (c) Pd(OAc)2, CH3CN (89%, two steps). (d) DIBAL-H, THF (91%).
(e) TBSCl, imidazole, DMF (99%). (f) m-CPBA, CH2Cl2 (93%). (g) LiNEt2,
HMPA, Et2O (97%). (h) m-CPBA, CH2Cl2 (98%). (i) MsCl, Et3N, CH2Cl2
(98%). (j) Na/naphthalene, THF (84%). (k) H2, 10% Pd/C, EtOAc (94%).
(l) PDC, CH2Cl2 (91%).
the known alcohol 6 (itself obtained from commercially
available vitamin D2)16,18 by methods recently developed in our
laboratory.19
The preparation of compound 5 (Scheme 2) began with
oxidation of 6 followed by R,â-unsaturation of the resulting
ketone 7 by Saegusa’s methodology.20 Reduction of the resulting
enone 8 provided the alcohol 9, which was protected as the
silyl ether derivative 10. Epoxidation of 10 from the less-
hindered face afforded 11, which was opened with freshly
prepared lithium diethylamine,21 to afford the allylic alcohol
12 in excellent yield. Epoxidation of 12 followed by mesylation
afforded the epoxymesylate 14, which was treated with soldium
naphthalenide in dry THF22 to afford allylic alcohol 15 (81%
yield from its isomer 12). Finally, standard catalytic hydrogena-
tion of 15 to 16, followed by oxidation of the C12 hydroxyl
group, afforded 5 in 49% overall yield from 6 (12 steps).
Ketones 4 were prepared by palladium-catalyzed coupling
between the appropriate side chains, in terminal alkyne form
(18), and compound 17, the enol triflate of 5 (Scheme 3). The
required alkynes were prepared straightforwardly by metalation
of 1-pentyne, 1-hexyne, and 1-heptyne with n-hexyllithium in
dry THF, followed by trapping of the resulting alkynyl carban-
Synthesis. Our strategy for the synthesis of analogues 2a-c
was based on the convergent Wittig-Horner approach,17 which
is based on the coupling of the anion of compound 3 (the
phosphine oxide of ring A) with ketones 4a-c, which comprise
the CD ring system and a C12-borne side chain (Scheme 1).
Ketones 4a-c would be obtained by attaching the appropriate
side chain to the C12-ketone 5, which would be prepared from

NoVel 1R,25-DihydroxyVitamin D3 Analogues
Scheme 3. Retrosynthesis of Ketones 4a-c
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1511
Scheme 6. Synthesis of Analogues 2a-ca
Scheme 4. Synthesis of Side-Chain Building Blocks 18a-ca
a Reagents and conditions: (a) nBuLi, THF, -78 °C; 4a-c. (b) TBAF,
THF.
17 and alkynes 18. Best yields of the desired products 19
(>96%) were obtained when catalytic amounts of copper iodide
and bis(triphenylphosphine)palladium(II) chloride were sequen-
tially added to a mixture of 17 and the appropriate alkyne 18
in dry diethylamine at 0 °C. Because of their lability, enynes
19 were immediately hydrogenated (10% Pd/C, EtOAc) to
compounds 22, which after desilylation and standard oxidation
of the resulting alcohols 23 afforded ketones 4a-c.
a Reagents and conditions: (a) nHexLi, THF; acetone (67-90%). (b)
KH, 1,3-diaminopropane (77-86%).
Scheme 5. Synthesis of Ketones 4a-ca
Finally, Wittig-Horner coupling of the anion of phosphine
oxide 3 with ketones 4a-c, followed by desilylation of the
protected analogues 24a-c, afforded the target compounds 2a,
2b, and 2c in overall yields of 19%, 16%, and 19%, respectively,
from known alcohol 6 (Schemes 6 and 1).
Biological Activity. The strength of the interaction of 2a,
2b, and 2c for hVDR∆ LBD, was evaluated by titration and
competition experiments with electrospray ionization mass
spectrometry (ESI-MS), a technique that allows characterization
of noncovalent interactions24 and is accordingly increasingly
being used to study ligand binding.25-30 This methodology is
used to evaluate electrostatic and H-bond specific contacts
involved in the binding of a ligand to a receptor. Changes in
electrostatic and H-bond contacts upon chemical modification
of the ligand are directly reflected in the ESI-MS data, which
allows a qualitative classification of ligands. This qualitative
classification obtained by ESI-MS is similar to that obtained
by classical competition experiments.14,26-30 ESI-MS titration
experiments were performed for hVDR∆ LBD binding with 2a,
2b, 2c, 1R,25(OH)2D3, and as negative control 9-cis-retinoic
acid, ligand for the retinoid NRs with no known affinity for
VDR. The dominant species were found to be [M + 12H]12+
and [M + 11H]11+ charged states of the monomeric hVDR∆
LBD. All analogues, 2a, 2b, and 2c, were shown by ESI-MS
to bind to VDR LBD. Figure 3 shows the ESI mass spectra
obtained after addition of a 3-fold molar excess of 2b to
hVDR∆. ESI mass spectra for 2a, 2c, 1R,25(OH)2D3, and 9-cis-
retinoic acid are shown in Figure S3 in the Supporting
Information. The ligand with the highest affinity is shown as
the species with the highest relative abundance. The relative
abundances of liganded hVDR∆ are the following: 95%, 69%,
71%, and 60% of complexes in the presence of 1R,25(OH)2D3,
2a, 2b, and 2c, respectively. Increasing the incubation time from
15 min to 24 h does not change the relative abundance values.
The relative affinity of hVDR∆ for the ligand is in the order 2c
< 2a < 2b , 1R,25(OH)2D3. Competition experiments (Sup-
porting Information for 2b) confirmed the titration experiments
results. Together these results suggest a gradual increase in the
affinity of hVDR∆ for 2c, 2a, and 2b.
a Reagents and conditions: (a) LDA, THF; 21 (72%). (b) 18a-c, CuI,
(Ph3P)2PdCl2, Et2NH. (c) H2, 10% Pd/C, EtOAc. (d) TBAF, THF. (e) PDC,
CH2Cl2.
ions with acetone (Scheme 4). Treatment of the resulting internal
alkynes 20a-c with KAPA base23 (potassium 3-aminopropyl-
amide) caused triple-bond migration to the terminal positions
they have in the desired products 18. Enol triflate 17 was
prepared by treating ketone 5 with freshly prepared LDA and
reaction of the resulting lithium enolate with triflimide 21
(Scheme 5). Various sets of reaction conditions were then
evaluated for the key coupling reaction between the enoltriflate

1512 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Gonza´lez-AVio´n et al.
Figure 3. ESI mass spectra of hVDR LBD. The mass spectra were acquired at 20 V. (A) After addition of 3 molar equiv of 2b (MM ) 431 Da).
71% of the detected species correspond to the hVDR∆-2b complex and 29% are related to unliganded VDR. (B) Before any ligand.
The functional activity of compounds 2a-c was evaluated
in a transient transfection assay in COS-7 cells transfected with
luciferase reporter plasmid and an expression vector for a Gal4
DBD-hVDR∆ LBD fusion protein. All ligands have low to
very low transactivation properties. At a concentration of 10-7
M, the transactivation activities of 2a-c were only 8%, 20%,
and 6%, respectively, compared with that of calcitriol (Figure
4A). This low response may reflect the difficulty for the C12-
side chain analogues to reach the H305 and H397 contact points
due to the influence of C18. Figure 4B shows the dose-response
curve of the 2b analogue with 1R,25(OH)2D3 as reference. At
10-5 M 2b had the same transactivation activity as the natural
ligand. The agonist behavior of 2b was confirmed by obtaining,
under standard conditions, small crystals of hVDR∆ LBD-2b
complex that were isomorphous to hVDR∆ LBD-1R,25-
(OH)2D3.10-12 The finding that increasing the concentration of
2b from 10-7 to 10-5 M increases its relative transactivation
activity from 17% to 100% might be due either to a partial
stabilization of an inactive VDR conformation or to a faster
catabolic degradation in vivo than that of calcitriol (sufficiently
faster to reduce its activity at 10-7 M but not so fast as to achieve
any significant reduction at 10-5 M). Both mechanisms might
allow 2b to be of pharmacological utility.
Conclusion. Analysis of the crystal structure of hVDR∆
LBD-calcitriol complex led to the design and synthesis of novel
calcitriol analogues bearing a side chain at C12 instead of C17
(compounds 2a-c). The affinities of these analogues for
hVDR∆ LBD were 60-71% that of calcitriol, and at a
concentration of 10-7 M their abilities to activate a Gal4 DBD-
Figure 4. (A) Transactivation of a luciferase reporter gene induced
hVDR∆ LBD construct ranged from 6% to 20% that of
by 10-7 M concentrations of 2a-c, as percentages of that induced by
calcitriol, with the 2b analogue being the most active.
calcitriol. (B) Dose-response curves for 2a (O), 1R,25(OH)2D3 (2),
2c (×), and 2b (9). COS cells were transiently transfected with a Gal4-
Experimental Section
VDR expression plasmid and 17m5-TATA-Luc reporter. Cells were
des-A,B-Androstan-8-one (7). Pyridinium dichromate (15.48 g,
41.19 mmol) was added to a solution of 6 (1.58 g, 10.2 mmol) in
dry CH2Cl2 (40 mL). The mixture was stirred for 8 h at room
temperature (RT) and filtered through a layer of silica gel. The
solids were washed with Et2O and the solution was concentrated.
The residue was purified by flash chromatography on silica gel
treated with either vehicle or the indicated concentration of the various
compounds.
(4% EtOAc/hexanes) to afford 7 [1.55 g, 39.95 mmol, 97%, Rf )
0.4 (15% EtOAc/hexanes), colorless oil].

NoVel 1R,25-DihydroxyVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1513
des-A,B-Androst-9(11)-en-8-one (8). A lithium diisopropyl-
amide solution was prepared by the addition of BuLi (8.69 mL,
and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (7% EtOAc/hexanes) to give 12 [2.45
g, 8.71 mmol, 97%, Rf ) 0.55 (20% EtOAc/hexanes), white solid
(mp 34 °C)].
n
21.29 mmol, 2.20 M solution in hexanes) to dry diisopropylamine
(3.20 mL, 22.9 mmol) at -78 °C. The stirred mixture was allowed
to warm until the formation of LDA as semisolid slurry. After
cooling at -78 °C, the LDA slurry was dissolved with dry THF
(60 mL) and a solution of 7 (2.49 g, 16.38 mmol) in dry THF (50
mL) was then added dropwise. The mixture was allowed to warm
to RT and was stirred for 1 h. The enolate was trapped by the
addition of dry chlorotrimethylsilane (3.11 mL, 24.57 mmol) at
-78 °C. The mixture was allowed to warm to 0 °C for 2.5 h and
the reaction was quenched with aqueous NaHCO3 (60 mL). The
aqueous layer was extracted with Et2O (2 × 50 mL) and the
combined organic solution was dried, filtered, and concentrated in
a Vacuum. The residue was dissolved in dry CH3CN (100 mL),
and palladium(II) acetate (3.86 g, 17.2 mmol) was added. After
the mixture was stirred overnight at RT, the formation of a
palladium mirror was observed. The solids were removed by
filtration through a layer of silica gel and washed with Et2O (50
mL). Aqueous NaHCO3 (100 mL) was added to the organic
solution, and the aqueous layer was extracted with Et2O (3 × 80
mL). The combined organic solution was dried, filtered, and
concentrated in vacuo. The residue was purified by flash chroma-
tography on silica gel (8% EtOAc/hexanes) to give 8 [2.19 g, 14.58
mmol, 89% in two steps, Rf ) 0.35 (15% EtOAc/hexanes), colorless
oil].
des-A,B-Androst-9(11)-en-8â-ol (9). Diisobutylaluminum hy-
dride (21.1 mL, 21.1 mmol, 1 M solution in hexanes) was added
dropwise to a stirred solution of 8 (2.11 g, 14.1 mmol) in dry THF
(80 mL) at -78 °C. The reaction mixture was stirred for 15 min
and quenched by the successive addition of water (50 mL) and
HCl (200 mL, 5% aqueous solution). The aqueous layer was
extracted with Et2O (3 × 100 mL) and the combined organic
solution was washed with aqueous NaHCO3 (100 mL), dried,
filtered, and concentrated in vacuo. The residue was purified by
flash chromatography on silica gel (9% EtOAc/hexanes) to give 9
[1.95 g, 12.8 mmol, 91%, Rf ) 0.4 (20% EtOAc/hexanes), colorless
oil]. Due to its volatility, high-vacuum drying is not recommended
for 9.
8â-tert-Butyldimethylsilyloxy-des-A,B-androst-9(11)-ene (10).
Imidazole (3.42 g, 50.3 mmol) and tert-butyldimethylsilyl chloride
(6.06 g, 40.23 mmol) were successively added to a solution of 9
(1.53 g, 10.1 mmol) in dry DMF. The mixture was stirred overnight
at room temperature. Brine (100 mL) was added and the aqueous
layer was extracted with hexanes (3 × 50 mL). The combined
organic solution was dried, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography on silica gel (hexanes)
to yield 10 [2.66 g, 10.0 mmol, 99%, Rf ) 0.8 (hexanes), colorless
oil].
8â-tert-Butyldimethylsilyloxy-des-A,B-9r,11r-epoxyandros-
tane (11). 3-Chloroperoxybenzoic acid (4.87 g, 27.7 mmol) was
added in portions to a stirred solution of 10 (2.61 g, 9.81 mmol) in
dry CH2Cl2 (100 mL) at 0 °C. The mixture was stirred in the dark
for 2 h at 0 °C and for 3 h at room temperature. The reaction was
quenched by the addition of Na2S2O4 (80 mL, saturated aqueous
solution). The resulting mixture was vigorously shaken and the
aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic solution was dried, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography on silica
gel (3% Et2O/hexanes) to afford 11 [2.58 g, 9.12 mmol, 93%, Rf
) 0.3 (hexanes), colorless oil].
8â-tert-Butyldimethylsilyloxy-des-A,B-11r,12r-epoxyandrostan-
9r-ol (13). 3-Chloroperoxybenzoic acid (3.05 g, 17.6 mmol) was
added in portions to a solution of 12 (1.66 g, 5.88 mmol) in dry
CH2Cl2 (40 mL) at 0 °C. The mixture was stirred in the dark for 2
h at RT. The reaction was quenched by the addition of aqueous
Na2S2O4 (40 mL). The resulting mixture was vigorously shaken
and the aqueous layer was extracted with CH2Cl2 (2 × 30 mL).
The combined organic solution was washed with NaHCO3 (50 mL,
saturated aqueous solution), dried, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography on silica
gel (6% EtOAc/hexanes) to give 13 [1.71 g, 5.76 mmol, 98%, Rf
) 0.5 (20% EtOAc/hexanes), white solid (mp 64 °C)].
8â-tert-Butyldimethylsilyloxy-des-A,B-11r,12r-epoxyandrostan-
9r-ylmethanesulfonate (14). Dry Et3N (1.01 mL, 7.27 mmol) and
dry methanesulfonyl chloride (0.52 mL, 6.7 mmol) were succes-
sively added to a solution of 13 (1.67 g, 5.59 mmol) in dry CH2Cl2
(15 mL) at -10 °C. After the mixture was stirred for 1 h, the
reaction was quenched with water (25 mL). The aqueous layer was
extracted with CH2Cl2 (2 × 25 mL) and the combined organic
solution was dried, filtered, and concentrated in vacuo to give crude
14 [2.10 g, 5.55 mmol, 99%, Rf ) 0.5 (20% EtOAc/hexanes), same
Rf as 13, colorless oil]. Crude 14 was submitted to the next reaction
without further purification.
8â-tert-Butyldimethylsilyloxy-des-A,B-androst-9(11)-en-12r-
ol (15). A 3 M sodium naphthalenide solution was prepared by the
addition of a solution of naphthalene (7.89 g, 60 mmol) in dry THF
(20 mL) to Na (1.38 g, 60 mmol, small pieces) at RT. After being
stirred overnight, the resulting deep blue solution (18.2 mL, 54.5
mmol) was added to a solution of crude 14 (2.05 g, 5.45 mmol) in
dry THF (25 mL) at -10 °C. The mixture was stirred for 20 min
and the reaction was quenched by the careful addition of H2O (50
mL). The aqueous layer was extracted with EtOAc (3 × 30 mL)
and the combined organic solution was dried, filtered, and
concentrated in vacuo. The residue was purified by flash chroma-
tography on silica gel (7% EtOAc/hexanes) to afford 15 [1.30 g,
4.58 mmol, 84%, Rf ) 0.4 (20% EtOAc/hexanes), white solid (mp
113 °C)].
8â-tert-Butyldimethylsilyloxy-des-A,B-androstan-12r-ol (16).
A catalytic amount of 10% palladium on active carbon (0.050 g)
was suspended in a solution of 15 (1.244 g, 4.41 mmol) in EtOAc
(60 mL). The mixture was stirred under hydrogen atmosphere
(balloon pressure) for 2 h at RT. The solids were removed by
filtration through a short layer of silica gel, and the product was
eluted with Et2O. After concentration in vacuo, the residue was
purified by flash chromatography on silica gel (7% EtOAc/hexanes)
to afford 16 [1.18 g, 4.15 mmol, 94%, Rf ) 0.5 (20% EtOAc/
hexanes), white solid (mp 73 °C)].
8â-tert-Butyldimethylsilyloxy-des-A,B-androstan-12-one (5).
Pyridinium dichromate (5.90 g, 15.7 mmol) was added to a solution
of 16 (1.11 g, 3.91 mmol) in CH2Cl2 (60 mL). After the mixture
was stirred for 36 h at RT, the solids were removed by filtration
through a layer of silica gel and washed with Et2O. The solution
was concentrated in vacuo and the resulting residue was purified
by flash chromatography on silica gel (5% EtOAc/hexanes) to afford
5 [1.00 g, 3.56 mmol, 91%, Rf ) 0.6 (20% EtOAc/hexanes), white
solid (mp 32 °C)].
n
2-Methyl-3-heptyn-2-ol (20a). Hexyllithium (35.7 mL, 80.0
8â-tert-Butyldimethylsilyloxy-des-A,B-androst-11-en-9r-ol (12).
A lithium diethylamide solution was prepared by the addition of
nBuLi (17.9 mL, 44.7 mmol, 2.5 M solution) to a stirred solution
of Et2NH (5.87 mL, 53.9 mmol) in dry Et2O (20 mL) at -40 °C.
After 20 min, a solution of 11 (2.53 g, 8.98 mmol) in dry Et2O (25
mL) and dry HMPA (12.0 mL, 69.0 mmol) were sequentially added.
The mixture was stirred for 13 h at RT, and the reaction was
quenched with a few drops of aqueous NH4Cl and HCl (100 mL,
2% aqueous solution). The aqueous layer was extracted with Et2O
(3 × 50 mL) and the combined organic solution was dried, filtered,
mmol, 2.24 M solution in hexanes) was added to a stirred solution
of 1-pentyne (7.88 mL, 80.0 mmol) in dry THF (100 mL) at -78
°C. After the mixture was stirred for 1 h at RT, acetone (6.17 mL,
84.0 mmol) was added at -78 °C. The mixture was allowed to
warm to RT for 3 h and the reaction was quenched with aqueous
NH4Cl (100 mL). The aqueous layer was extracted with Et2O (3 ×
70 mL) and the combined organic solution was dried, filtered, and
concentrated in vacuo. The residue was purified by flash chroma-
tography on silica gel (9% EtOAc/hexanes) to give 20a [8.51 g,
67.2 mmol, 84%, Rf ) 0.65 (25% EtOAc/hexanes), colorless oil].

1514 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Gonza´lez-AVio´n et al.
2-Methyl-3-octyn-2-ol (20b). See 20a for reaction procedure.
Reagents: nhexyllithium (35.7 mL, 80.0 mmol, 2.24 M solution in
hexanes), 1-hexyne (9.00 mL, 80.0 mmol), and dry THF (100 mL).
Product: 20b [10.1 g, 72.0 mmol, 90%, Rf ) 0.65 (25% EtOAc/
hexanes), colorless oil].
2-Methyl-3-nonyn-2-ol. (20c). See 20a for reaction procedure.
Reagents: nhexyllithium (35.7 mL, 80.0 mmol, 2.24 M solution in
hexanes), 1-heptyne (10.5 mL, 80.0 mmol), and dry THF (100 mL).
Product: 20c [8.30 g, 53.6 mmol, 67%, Rf ) 0.65 (25% EtOAc/
hexanes), colorless oil].
2-Methyl-6-heptyn-2-ol (18a). A suspension of KH in mineral
oil was washed with dry hexane (3 × 40 mL) under an argon
atmosphere and dried under high vacuum to give KH (9.06 g, 225
mmol) as a gray powder. Dry 1,3-diaminopropane (120 mL) was
added at 0 °C and the mixture was stirred for 3 h at this temperature
and then overnight at room temperature. Caution must be taken
due to hydrogen evolution during the first 3 h. The above solution
(40 mL, 75 mmol) was added to neat 20a (3.15 g, 25.0 mmol) at
room temperature. After being stirred for 20 h, the mixture was
cautiously poured over crushed ice/NaCl. NH4Cl (150 mL, saturated
aqueous solution) was added and the aqueous layer was extracted
with Et2O (6 × 40 mL). The combined organic solution was dried,
filtered, and concentrated in vacuo. The residue was purified by
flash chromatography on silica gel (8-12% EtOAc/hexanes) to give
18a [2.44 g, 19.25 mmol, 77%, Rf ) 0.55 (25% EtOAc/hexanes),
colorless oil].
[0.222 g, 0.547 mmol, 96%, Rf ) 0.6 (25% EtOAc/hexanes),
colorless oil].
20(17f12)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-tri-
homo-21-norcholest-11-en-20(22)-yn-25-ol (19c). See 19a for
experimental procedure. Reagents: CuI (5 mg), bis(triphenylphos-
phine)palladium(II) chloride (5 mg), 17 (0.194 g, 0.462 mmol),
18c (0.216 g, 1.41 mmol), and dry Et2NH (12 mL). Product: 19c
[0.193 g, 0.457 mmol, 99%, Rf ) 0.6 (25% EtOAc/hexanes),
colorless oil].
(17f12â)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-homo-
21-norcholestan-25-ol (22a). A catalytic amount of 10% palladium
on active carbon (5 mg) was suspended in a solution of 19a (0.060
g, 0.144 mmol) in EtOAc (5 mL). The mixture was stirred under
hydrogen atmosphere (balloon pressure) for 16 h at RT. The solids
were removed by filtration through a layer of silica gel, and the
product was eluted with Et2O. The solution was concentrated in
vacuo and the residue was purified by flash chromatography on
silica gel (10% EtOAc/hexanes) to afford 22a [0.055 g, 0.131 mmol,
of 91%, Rf ) 0.5 (20% EtOAc/hexanes), colorless oil].
(17f12â)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-di-
homo-21-norcholestan-25-ol.(22b). See 22a for experimental
procedure. Reagents: 10% palladium on active carbon (10 mg),
19b (0.204 g, 0.504 mmol), and EtOAc (10 mL). Product: 22b
[0.193 g, 0.469 mmol, 93%, Rf ) 0.5 (20% EtOAc/hexanes),
colorless oil].
(17f12â)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-tri-
homo-21-norcholestan-25-ol (22c). See 22a for experimental
procedure. Reagents: 10% palladium on active carbon (10 mg),
19c (0.177 g, 0.504 mmol), and EtOAc (10 mL). Product 22c [0.165
g, 0.463 mmol, 92%, Rf ) 0.5 (20% EtOAc/hexanes), colorless
oil].
20(17f12â)-abeo-des-A,B-24-Homo-21-norcholestan-8â,25-
diol (23a). Tetrabutylammonium fluoride trihydrate (0.644 g, 2.04
mmol) was added to a solution of 22a (0.054 g, 0.136 mmol) in
dry THF (5 mL). The stirred mixture was refluxed for 2 days. Water
(15 mL) was added and the aqueous layer was extracted with Et2O
(3 × 10 mL). The combined organic solution was dried, filtered,
and concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (22% EtOAc/hexanes) to give 23a
[0.032 g, 0.116 mmol, 85%, Rf ) 0.35 (30% EtOAc/hexanes),
colorless oil] and recovered 22a (0.008 g, 15%).
20(17f12â)-abeo-des-A,B-24-Dihomo-21-norcholestan-8â,25-
diol (23b). See 23a for experimental procedure. Reagents: Tetra-
butylammonium fluoride trihydrate (1.14 g, 3.63 mmol), 22b (0.149
g, 0.363 mmol), and dry THF (3 mL). Product: 23b [0.101 g, 0.341
mmol, 94%, Rf ) 0.35 (30% EtOAc/hexanes), colorless oil].
20(17f12â)-abeo-des-A,B-24-Trihomo-21-norcholestan-8â,-
25-diol (23c). See 23a for experimental procedure. Reagents:
Tetrabutylammonium fluoride trihydrate (0.921 g, 2.92 mmol), 22c
(0.124 g, 0.292 mmol), and dry THF (3 mL). Product: 23c [0.088
g, 0.283 mmol, 97%, Rf ) 0.35 (30% EtOAc/hexanes), colorless
oil].
20(17f12â)-abeo-25-Hydroxy-des-A,B-24-homo-21-nor-
cholestan-8-one (4a). Pyridinium dichromate (0.140 g, 0.374 mmol)
was added to a solution of 23a (0.035 g, 0.125 mmol) in dry CH2-
Cl2 (4 mL). The mixture was stirred in the dark for 5 h at RT. The
reaction was monitored by TLC (double elution with 3:3:7 EtOAc/
CH2Cl2/hexanes). The solids were removed by filtration through a
layer of silica gel and the product was eluted with Et2O. The
solution was concentrated in vacuo and the residue was purified
by flash chromatography on silica gel (20% EtOAc/hexanes) to
afford 4a [0.034 g, 0.123 mmol, >95%, Rf ) 0.35 (30% EtOAc/
hexanes), colorless oil].
2-Methyl-7-octyn-2-ol (18b). See 18a for experimental proce-
dure. Reagents: 40 mL (75 mmol approximately) of KH solution
and 20b (3.51 g, 25.0 mmol). Product: 18b [2.91 g, 20.8 mmol,
83%, Rf ) 0.55 (25% EtOAc/hexanes), colorless oil].
2-Methyl-8-nonyn-2-ol. (18c). See 18a for experimental pro-
cedure. Reagents: 40 mL (75 mmol approximately) of KH solution
and 20c (3.73 g, 24.2 mmol). Product: 18c [3.22 g, 20.9 mmol,
86%, Rf ) 0.55 (25% EtOAc/hexanes), colorless oil].
8â-tert-Butyldimethylsilyloxy-des-A,B-androst-11-en-12-yl-tri-
fluoromethanesulfonate (17). A lithium diisopropylamide solution
was prepared by the addition of nhexyllithium (1.03 mL, 2.46 mmol,
2.38 M solution in hexanes) to neat iPr2NH (0.367 mL, 2.65 mmol)
at -78 °C. The cooling bath was removed and the temperature
was allowed to warm until the formation of LDA as semisolid
slurry. After cooling again at -78 °C the solid was dissovled in
THF (8 mL). A solution of 5 (0.534 g, 1.89 mmol) in dry THF (8
mL) was added by cannula. The mixture was stirred for 4 h at RT
and the enolate was trapped with a solution of the triflimide 21
(1.49 g, 3.78 mmol) in dry THF (8 mL) at -30 °C. The mixture
was allowed to warm to RT and then stirred for 2 days. Water (50
mL) was added and the aqueous layer was extracted with Et2O (3
× 40 mL). The combined organic solution was dried, filtered, and
concentrated in vacuo. The residue was purified by flash chroma-
tography on silica gel (20% CH2Cl2/hexanes and 8% EtOAc/
hexanes) to give 17 [0.564 g, 1.36 mmol, 72%, Rf ) 0.6 (2%
EtOAc/hexanes), white solid (mp 41 °C)] and 0.139 g of recovered
starting material (26%).
20(17f12)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-
homo-21-norcholest-11-en-20(22)-yn-25-ol (19a). A catalytic
amount of CuI (2 mg) and of (Ph3P)2PdCl2 (2 mg) were sequentially
added to a mixture of 17 (0.065 g, 0.157 mmol) and 18a (0.059 g,
0.471 mmol) in dry Et2NH (4 mL) at 0 °C. The mixture was stirred
at 0 °C for 1 h and at RT for 1 h. NH4Cl (10 mL, saturated aqueous
solution) was added and the aqueous layer was extracted with Et2O
(3 × 10 mL). The combined organic solution was dried, filtered,
and concentrated in vacuo. The residue was purified by flash
chromatography (8% EtOAc/hexanes) to give 19a [0.060 g, 0.154
mmol, 98%, Rf ) 0.6 (25% EtOAc/hexanes), colorless oil].
Compound 19a decomposes in contact with air at RT. It must be
stored at -20 °C under argon.
20(17f12â)-abeo-25-Hydroxy-des-A,B-24-dihomo-21-nor-
cholestan-8-one (4b). See 4a for experimental procedure. Re-
agents: Pyridinium dichromate (0.208 g, 0.553 mmol), 23b (0.082
g, 0.277 mmol), and dry CH2Cl2 (6 mL). Product: 4b [0.074 g,
0.252 mmol, 91%, Rf ) 0.45 (40% EtOAc/hexanes), colorless oil].
20(17f12â)-abeo-25-Hydroxy-des-A,B-24-trihomo-21-nor-
cholestan-8-one (4c). See 4a for experimental procedure. Re-
agents: Pyridinium dichromate (0.169 g, 0.450 mmol), 23c (0.070
20(17f12)-abeo-8â-tert-Butyldimethylsilyloxy-des-A,B-24-di-
homo-21-norcholest-11-en-20(22)-yn-25-ol (19b). See 19a for
experimental procedure. Reagents: CuI (5 mg), bis(triphenylphos-
phine)palladium(II) chloride (5 mg), 17 (0.236 g, 0.570 mmol),
18b (0.239 g, 1.71 mmol), and dry Et2NH (12 mL). Product: 19b

NoVel 1R,25-DihydroxyVitamin D3 Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1515
g, 0.225 mmol), and dry CH2Cl2 (5 mL). Product: 4c [0.066 g,
0.214 mmol, 95%, Rf ) 0.45 (40% EtOAc/hexanes), colorless oil].
20(17f12â)-abeo-1r-Triisopropylsilyloxy-25-hydroxy-24-homo-
21-norvitamin D3 Triisopropylsilyl Ether (24a). nBuLi (0.312 mL,
0.449 mmol, 1.44 M solution in hexanes) was added dropwise to
a solution of dry 3 (0.333, 0.499 mmol) in THF (3 mL) at -78 °C.
The resulting deep red solution was stirred at -78 °C for 1 h
followed by the slow addition of a solution of the ketone 4a (0.035
g, 0.125 mmol) in dry THF (3 mL). The mixture was stirred in the
dark for 3 h at -78 °C and for 4 h at -40 °C. The reaction was
quenched with H2O (15 mL) and the aqueous layer was extracted
with Et2O (3 × 10 mL). The combined organic solution was dried,
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (15% EtOAc/hexanes)
to afford 24a [0.065 g, 0.089 mmol, 71%, Rf ) 0.7 (30% EtOAc/
hexanes), colorless oil].
20(17f12â)-abeo-1r-Triisopropylsilyloxy-25-hydroxy-24-di-
homo-21-norvitamin D3 Triisopropylsilyl Ether (24b). See 24a
for experimental procedure. Reagents: nBuLi (0.46 mL, 0.66 mmol,
1.44 M solution in hexanes), 3 (0.490, 0.735 mmol), dry THF (4.5
mL), and 4b (0.054 g, 0.184 mmol) in dry THF (4 mL). Product:
24b [0.090 g, 0.121 mmol, 66%, Rf ) 0.65 (30% EtOAc/hexanes),
colorless oil].
20(17f12â)-abeo-1r-Triisopropylsilyloxy-25-hydroxy-24-tri-
homo-21-norvitamin D3 Triisopropylsilyl Ether (24c). See 24a
for experimental procedure. Reagents: nBuLi (0.41 mL, 0.59 mmol,
1.44 M solution in hexanes), 3 (0.441, 0.661 mmol), THF (4 mL),
and 4c (0.054 g, 0.184 mmol) in dry THF (4 mL). Product: 24c
[0.080 g, 0.118 mmol, 64%, Rf ) 0.65 (30% EtOAc/hexanes),
colorless oil].
20(17f12â)-abeo-1r,25-Dihydroxy-24-homo-21-norvitamin
D3 (2a). Tetrabutylammonium fluoride trihydrate (0.052 g, 0.165
mmol) was added to a solution of 24a (0.036 g, 0.049 mmol) in
dry THF (1.5 mL). The mixture was stirred in the dark for 20 h at
room temperature. Aqueous NH4Cl (10 mL) was added and the
aqueous layer was extracted with Et2O (3 × 8 mL). The combined
organic solution was dried, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(60-65% EtOAc/hexanes) and by reverse-phase chromatography
(RP 18, 18% H2O/MeOH) to give 2a [0.020 g, 0.048 mmol, >95%,
Rf ) 0.5 (EtOAc), white solid].
10 concentrators (Amicon), and diluted at a final concentration of
10-5 M. Experiments were performed on an electrospray ionization
time-of-flight mass spectrometer (LCT, Micromass) with continu-
ously infusion of the sample into the ion source at a flow rate of 4
µL/min by a Harward Model 11 syringe pump (Harward Ap-
paratus). All ligands were dissolved in ethanol at 10-2 M. Ligand
(0.6 µL) was added to 40 µL of protein solution (2.4 nmol) to
maintain an ethanol level below 1.5%. Titration experiments were
done by adding 3 molar equiv of ligands to protein solution,
followed by 15 min of incubation at 4 °C. Competition experiments
were performed by adding an equimolar mixture of two ligands
(each in 3-fold molar excess) to the protein, followed by 15 min
of incubation. To prevent dissociation in the gas phase during the
ionization and desorption process, the cone voltage was optimized
to 10-20 V. Mass data were acquired in the positive ion mode on
a mass range of 1000-5000 m/z. Calibration of the instrument was
performed with the multiply charged ions produced by a separate
injection of horse heart myoglobin diluted to 2 µM in 1:1 water/
acetonitrile (v/v) acidified with 1% (v/v) formic acid. The relative
abundance of the different species present on ESI mass spectra was
measured from their respective peak intensities. All experiments
were reproduced at least three times.
Cell Culture, Transfection, and Transactivation Assays. COS
cells (SV40-transformed African Green monkey kidney) were
maintained in Dulbecco’s modified Eagle’s medium supplemented
with 5% dextran-coated charcoal-stripped foetal bovine serum and
gentamycin. Transient transfection assays were carried out in 24-
well plates (105 cells/well) by a standard calcium phosphate
coprecipitation technique as described previously.11 Cells were
cotransfected with precipitates containing 250 ng of receptor
expression vectors PXJ440 VDR∆ (118-427 ∆166-216), 2 µg
of reporter gene 5 × 17m-TATA-Luc(luciferase), 2 µg of an internal
control recombinant expressing â-galatosidase pCH110lacZ (Phar-
macia), and completed to 20 µg with carrier DNA ppSK+. Cells
were treated with 10-11 to 10-5 M 1R,25(OH)2D3, analogues, or
ethanol vehicle. Thirty-six hours following transfection, cells
extracts were assayed for luciferase and â-galactosidase activity.
Luciferase values were normalized to â-galactosidase activity. The
data were expressed as luciferase x-fold induction values, where
the value 1 is assigned to the normalized luciferase activity of blank
cultures.
20(17f12â)-abeo-1r,25-Dihydroxy-24-dihomo-21-norvita-
min D3 (2b). See 2a for experimental procedure. Reagents:
Tetrabutylammonium fluoride trihydrate (0.068 g, 0.215 mmol),
24b (0.040 g, 0.054 mmol), and dry THF (1.5 mL). Product: 2b
[0.022 g, 0.050 mmol, 92%, Rf ) 0.5 (EtOAc), white solid].
20(17f12â)-abeo-1r,25-Dihydroxy-24-trihomo-21-norvita-
min D3 (2c). See 2a for experimental procedure. Reagents:
Tetrabutylammonium fluoride trihydrate (0.095 g, 0.301 mmol),
24c (0.057 g, 0.075 mmol), and dry THF (2 mL). Product: 2c
[0.032 g, 0.074 mmol, >95%, Rf ) 0.5 (EtOAc), colorless oil].
Expression, Purification, and Crystallization of hVDR∆. The
LBD of the human VDR (residues 118-427 ∆166-216) was
cloned in pET28b expression vector, to obtain an N-terminal
hexahistidine-tagged fusion protein, and overproduced in Escheri-
chia coli BL21 (DE3) strain. Cells were grown in LB medium and
subsequently induced for 6 h at 20 °C with 1 mM isopropyl thio-
â-D-galactoside. The purification included a metal affinity chro-
matography step on a cobalt-chelating resin. After tag removal by
thrombin digestion, the protein was further purified by gel filtration.
The final protein buffer was 10 mM Tris, pH 7.5, 100 mM NaCl,
and 5 mM dithiothreitol. Purity and homogeneity were assessed
by sodium dodecyl sulfate (SDS) and native polyacrylamide gel
electrophoresis (PAGE) and denaturant and native electrospray
ionization mass spectrometry. Crystallization trials of the different
complexes were performed at 4 °C by vapor diffusion in hanging
drops with 0.1 M 2-(N-morpholino)ethanesulfonic acid (Mes), pH
6.0, and 1.4 M ammonium sulfate as precipitant agent.
Acknowledgment. We thank H. Nierengarten for mass
spectrometry analysis. We are grateful to the DIGICYT (Spain,
projects SAF 2001-3187 and 2004-01885) and CNRS, INSERM,
Hoˆpital Universitaire de Strasbourg, and Ministe`re de la
Recherche et de la Technologie for financial support. This work
benefits from the technical platform of structural genomics
supported by the Genopole and SPINE programs. We thank J.P.
van de Velde, J. Zorgdrager, and M. Sno (Solvay Pharmaceu-
ticals BV, Weesp, The Netherlands) for the gift of starting
materials. X.C.G.-A. thanks the Spanish Ministry of Science
and Technology for an FPI fellowship.
Supporting Information Available: Experimental data, NMR
and analytical data, and ESI MS data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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