Synthesis, structure, and biological activity of des-side Chain analogues of 1α,25-Dihydroxyvitamin D3 with substituents at C18

Article abstract of DOI:10.1002/cmdc.201100021

An improved synthetic route to 1α,25-dihydroxyvitamin D₃ des-side chain analogues 2a and 2b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF-7 breast cancer cells and prodifferentiation activity toward SW480-ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitaminD receptor (hVDR) complexed to one of these analogues, 20(17→18)-abeo-1α,25-dihydroxy-22-homo-21-norvitamin D₃ (2a) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25-dihydroxyvitamin D₃. Vitamin D₃ supplements: The synthesis and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D₃ with substituents at C18 are described. Crystallographic analysis revealed that the new side chain introduced at C18 adopts the same orientation as the natural side chain at C17 in the parent molecule 1α,25-dihydroxyvitamin D₃.

Full text of DOI:10.1002/cmdc.201100021

MED
DOI: 10.1002/cmdc.201100021
Synthesis, Structure, and Biological Activity of des-Side
Chain Analogues of 1a,25-Dihydroxyvitamin D3 with
Substituents at C18
Lieve Verlinden,[b] Annemieke Verstuyf,[b] Guy Eelen,[b] Roger Bouillon,[b] Paloma OrdꢀÇez-
Morꢁn,[c] Marꢂa Jesffls Larriba,[c] Alberto MuÇoz,[c] Natacha Rochel,[d] Yoshiteru Sato,[d]
Dino Moras,[d] Miguel Maestro,[e] Samuel Seoane,[f] Fernando Dominguez,[f] Silvina Eduardo-
Canosa,[a] Daniel Nicoletti,[a] Edelmiro Moman,[a] and Antonio MouriÇo*[a]
Dedicated to Argentinean Professors Rita H. Rossi, Julio C. Podestꢀ, Manuel Gonzꢀlez Sierra, and Oscar S. Giordano for their
commitment and contribution to organic chemistry.
An improved synthetic route to 1a,25-dihydroxyvitamin D3
des-side chain analogues 2a and 2b with substituents at C18
is reported, along with their biological activity. These ana-
logues display significant antiproliferative effects toward MCF-
7 breast cancer cells and prodifferentiation activity toward
SW480-ADH colon cancer cells; they are also characterized by
a greatly decreased calcemic profile. The crystal structure of
the human vitamin D receptor (hVDR) complexed to one of
these analogues, 20(17!18)-abeo-1a,25-dihydroxy-22-homo-
21-norvitamin D3 (2a) reveals that the side chain introduced at
position C18 adopts the same orientation in the ligand binding
pocket as the side chain of 1a,25-dihydroxyvitamin D3.
Introduction
The growth inhibition, prodifferentiating, and immunomodula-
tory activity of 1a,25-dihydroxyvitamin D3 (1a), the hormonally
active form of vitamin D3 (1b), opens new avenues for the use
of this compound in cancer, psoriasis, and immune-related dis-
orders such as multiple sclerosis and inflammatory bowel dis-
ease. However, the therapeutic applicability of compound 1a
is limited by hypercalcemia, a condition in which serum calci-
um levels are elevated, which occurs when supraphysiological
doses of 1a are administered.[1] Therefore, it remains a major
challenge to design and synthesize analogues of 1a that have
the best possible balance between the advantageous antiproli-
ferative and prodifferentiating effects on the one hand and the
adverse calcemic activity on the other hand.
novel analogues of vitamin D3 in which side chains homolo-
gous to that of the natural hormone are linked to C18 through
a CꢀC bond and that have no substituents on C17.[3] However,
the structural analysis and biological activity of these com-
pounds was not yet reported. In this paper we present an im-
proved synthetic route to two of these analogues, 20(17!18)-
abeo-1a,25-dihydroxy-22-homo-21-norvitamin D3 (2a) and
20(17!18)-abeo-1a,25-dihydroxy-22,23-dihomo-21-norvitamin
[a] Dr. S. Eduardo-Canosa, Dr. D. Nicoletti, Prof. E. Moman, Prof. A. MouriÇo
Departamento de Quꢁmica Orgꢀnica y Unidad Asociada al CSIC
Universidad de Santiago, 15782 Santiago de Compostela (Spain)
Fax: (+34)981-595-012
E-mail: antonio.mourino@usc.es
[b] Dr. L. Verlinden, Prof. A. Verstuyf, Dr. G. Eelen, Prof. R. Bouillon
Laboratorium voor Experimentele Geneeskunde en Endocrinologie
Katholieke Universiteit Leuven, 3000 Leuven (Belgium)
[c] Dr. P. OrdꢂÇez-Morꢀn, Dr. M. J. Larriba, Prof. A. MuÇoz
Instituto de Investigaciones Biomꢃdicas, “Alberto Sols”
Consejo Superior de Investigaciones
Cientꢁficas-Universidad Autꢂnoma de Madrid, 28029 Madrid (Spain)
[d] Dr. N. Rochel, Dr. Y. Sato, Dr. D. Moras
Institut de Gꢃnꢃtique et de Biologie Molꢃculaire et Cellulaire (IGBMC), Insti-
tut National de Santꢃ et de Recherche Mꢃdicale (INSERM) U964/Centre Na-
tional de Recherche Scientifique (CNRS) UMR 7104/Universitꢃ de Strasbourg,
67404 Illkirch (France)
[e] Dr. M. Maestro
Departamento de Quꢁmica Fundamental
Universidad de A CoruÇa, 15071 A CoruÇa (Spain)
One of our research strategies is focused on the synthesis of
vitamin D3 analogues with side chains attached to the angular
C18 methyl group. Most of the reported C18 substituted ana-
logues are linked to C18 through an oxygen atom and carry re-
sidual groups at C17.[2] We recently reported the synthesis of
[f] Dr. S. Seoane, Dr. F. Dominguez
Departamento de Fisiologꢁa, Facultad de Medicina
Universidad de Santiago, 15782 Santiago de Compostela (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100021.
788
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 788 – 793

Vitamin D3 Analogues
D3 (2b). A structural study of the seven-membered C18 side
chain analogue 2a complexed to hVDR was performed to in-
vestigate the binding mode of this newly introduced side
chain at C18. Furthermore, the biological activity profile of
these analogues was determined in several cancer cell lines.
dation, and methylation of the resulting ketone. Desilylation of
6 followed by hydrogenation of the double bond and oxida-
tion of the secondary hydroxy group provided ketone 7 in
82% yield. Coupling of ketone 7 with the lithium anion of
phosphine oxide 3 afforded, after desilylation, the desired
eight-membered vitamin D3 side-chain analogue 2b in 78%
yield over the three steps (12 steps from 5, 15.7% overall
yield).
Results and Discussion
Chemistry
Structural analysis
The target compounds 2a and 2b were synthesized by modi-
fying our previous strategy as outlined in Scheme 1.[3] The re-
placement of the tert-butyldimethylsilyl protecting group by
A structural study of the hVDR–2a complex was performed to
gain more insight into the interaction of the 2a analogue with
the VDR. In this complex, the protein adopted the canonical
conformation of all previously reported structures of VDR
bound to agonist and superagonist ligands with helix H12
folded in the agonistic position.[5] The seven-membered side
chain introduced at position C18 adopted the same orientation
in the pocket. An adaptation of its conformation was observed
to maintain the hydrogen bonds forming the anchoring
points. Compared with the structure of the hVDR–1a complex,
the atomic coordinates of hVDR bound to compound 2a
showed a root-mean-square deviation of 0.25 Š of all Ca
3
atoms. The sizes of the ligands were 396 and 391 Š for com-
pound 1a and 2a, respectively. The volume of the ligand bind-
3
ing cavity was 673 and 638 Š and the ligand occupied 61 and
59% of the pocket for 1a and 2a, respectively.
The A and seco B rings of the C18 side chain analogue 2a
presented conformations similar to those of the natural ligand
(Figure 1a and b). As a consequence of the modified side
chain, the C and D rings were shifted by 0.4 Š. The distance
between the 1-hydroxy and the 25-hydroxy groups varied
from 13.1 Š for 1a to 12.8 Š for analogue 2a complex. All the
residues of VDR forming the binding pocket adopted the same
conformation as those of the VDR–1a complex except for the
side chain of Ile271 (H5). The interactions between the ligand
and the receptor involved hydrophobic contacts and electro-
static interactions. The hydroxy groups made the same hydro-
gen bonds as in the VDR–1a complex, 1-OH with Ser237 (H3)
and Arg274 (H5), 3-OH with Tyr143 (H1) and Ser278 (b0), and
the 25-OH with His305 (loop H6–H7) and His397 (H11). As a
result of the modification of the ligand, the side chain of com-
pound 2a took another pathway in the pocket and made addi-
tional contacts with the CB atom of Leu230 (H3) at 3.9 Š of
C20 and 3.6 Š of C22 atom, respectively. Its side chain, howev-
er, lost a contact with the CD2 atom of Leu309 (H7) at 3.8 Š of
C21 for 1a (Figure 1c). The elongated side chain of 2a in-
duced contacts between C26 and Leu404 (H11), C26 and
Leu414 (loop H11–H12), and C27 and Phe422 (H12) at the end
of the ligand. A Ca atom of His305 was shifted by 0.4 Š to
maintain the hydrogen bonds with the hydroxy group.
Scheme 1. Synthesis of 2a and 2b: a) nBuLi, THF, ꢀ788C, then 4, 3 h; b) HF–
Py, CH2Cl2, RT, 1 h; c) 1. LiAlH4, Et2O, D, 12 h, 2. p-TsCl, DMAP, Py, CH2Cl2, 08C,
48 h, 3. NaCN, DMSO, 908C, 2 h, 4. iBu2AlH, CH2Cl2, ꢀ108C, 2 h, 5. MeLi, Et2O,
ꢀ788C, 1 h, 6. PDC, CH2Cl2, RT, 24 h, 7. MeLi, Et2O, ꢀ788C, 2 h; d) 1. 48% HF–
Py, CH3CN, CH2Cl2, Et3N, RT, 48 h, 2. H2, 5% Pd/C, EtOAc, RT, 16 h, 3. PDC,
CH2Cl2, RT, 24 h; e) 1. 3–Li anion, THF, ꢀ788C, 6 h, 2. HF–Py, CH2Cl2, RT, 2 h
(12 steps from 5: 15.7% yield).
the triethylsilyl group was thought to have benefits at the final
deprotection-purification stage. To set the vitamin D triene
system corresponding to 2a, we coupled hydroxyketone 4
with the lithium anion of phosphine oxide 3 in THF at ꢀ788C
to obtain the protected vitamin D3 analogue 2a-TES in 85%
yield. Desilylation of 2a-TES took place cleanly using pyridini-
um fluoride to give the desired vitamin D3 analogue 2a in high
yield. This result validates the use of phosphine oxide 3 in the
syntheses of future vitamin D3 analogues following Lythgoe’s
Wittig–Horner approach.[4] The synthesis of 2b commenced
with known carboxylic acid 5,[3] which was converted into alco-
hol 6 in 24% yield through a seven-step sequence involving
reduction of the carboxylic group, tosylation, displacement of
the resulting tosylate with sodium cyanide, reduction of the re-
sulting nitrile to the homologated aldehyde, methylation, oxi-
To confirm the crystallographic data, several VDR mutants
were used to investigate the capacity of compound 2a to
transactivate a vitamin D response element (VDRE)-containing
reporter construct. Compound 1a and the seven-membered
C18 side chain analogue 2a were unable to transactivate the
reporter construct when cells were transfected with VDRs car-
ChemMedChem 2011, 6, 788 – 793
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
789

A. MouriÇo et al.
MED
Figure 2. Transactivating potency of various VDR point mutants in COS-1
cells. Cells were stimulated with compound 1a (black bars) or the analogue
2a (open bars), each applied at their EC50 concentrations (6 nm for com-
pound 1a and 3 nm for analogue 2a). Luciferase activity was normalized for
b-galactosidase activity (Luc/b-gal) and expressed relative to transfected ve-
hicle-stimulated cells. Bars represent the mean ꢁSD of three independent
experiments (*p<0.01, Student’s t-test).
Heterodimerization with RXR
Figure 1. Conformation of the VDR-bound 2a analogue. a) Compound 2a is
shown in its FOꢀFC electron density omit map contoured at 3.0 s; the ligand
is shown in stick representation. b) A stereo view of the ligand conforma-
tions of 1a (dark gray) and 2a (light gray) in their ligand binding pockets.
c) Superposition of the VDR–2a (gray) and VDR–1a complexes. The view is
restricted to the region of the protein (H3, H7, H11, and H12) that contains
the side chain of the ligand. Only residues closer than 4.0 Š are shown.
Leu309 and Tyr401 for the 2a complex and Leu230, Leu404, Leu414, and
Phe422 for the 1a complex are shown for comparison. The ligands are
shown in stick representation in light gray for analogue 2a and dark gray
for compound 1a. Hydrogen bonds formed by the 25-OH groups are shown
as dashed lines.
To assess whether VDR bound to 2a was able to heterodimer-
ize with RXR, we monitored its interaction by electrospray ioni-
zation mass spectrometry (ESI-MS) under nondenaturing condi-
tions. Addition of fivefold molar excess of 2a in the heterodi-
mer VDR–RXR LBDs resulted in the appearance of a novel
series of mass/charge (m/z) ions corresponding to a fully
bound VDR–2a–RXR (see Supporting Information for details).
The different conformation of the VDR-bound 2a analogue
does not affect the VDR–RXR heterodimer stability. We then
monitored by ESI-MS the recruitment of the SRC-1 NR2 co-acti-
vator peptide to VDR–2a–RXR complex (data not shown) and
found it to be similar to VDR–1a–RXR.
rying either the Leu233Ala, Ile271Ala, Arg274Ala, Trp286Ala,
His397Ala, or Tyr401Ala mutation (Figure 2). The transactivation
potency of compound 1a was moderately decreased when
cells were transfected with the VDR mutants Val234Ala,
Ile268Ala, Val300Ala, and His305Ala (50% of the activity of
wild-type VDR), whereas transfection with the mutants
Ser275Ala and Ser278Ala had little effect on the transactivation
capacity of compound 1a. Introduction of a seven-membered
side chain at C18 rendered analogue 2a less potent than 1a in
transactivating a VDRE-containing reporter construct in cells
transfected with the VDR mutants Ile268Ala, Ser275Ala,
Val300Ala, and His305Ala. These findings are in agreement
with the crystal structure and suggested that interactions with
these amino acids became more critical when a side chain is
introduced at C18 instead of at position C17. The transactivat-
ing capacity of the VDR mutant Val234Ala became more
potent in cells that were incubated with compound 2a, which
confirmed crystallographic data that showed a weaker interac-
tion between this amino acid and the side chain of the 2a ana-
logue.
Biological evaluation
Despite the fact that the analogue 2a fitted well in the VDR
binding pocket and that the hydrogen bonds, which form the
anchoring points, were maintained, the affinity of compound
2a for pig VDR was greatly diminished (10% of the affinity of
compound 1a; Table 1). Moreover, binding to the transport
protein vitamin D binding protein (hDBP) was significantly de-
creased (2.5% of the affinity of compound 1a). Homologation
of the side chain in compound 2b further decreased the bind-
ing affinity to DBP and VDR (1.3 and 3.3% of the affinity of 1a,
respectively). The low affinity for hDBP may be explained by at-
tachment of the 2a and 2b side chain fragments to C18 that
orientates the side chain hydroxy groups toward the b-face of
the CD rings. Notwithstanding this low affinity for VDR, but in
accordance with the transactivation results, the seven-mem-
bered C18 side chain analogue 2a was as potent as compound
1a in decreasing the proliferation of human breast adenocarci-
790
www.chemmedchem.org
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 788 – 793

Vitamin D3 Analogues
Table 1. In vitro binding affinities, antiproliferative activities, and in vivo calcemic effects of compound 1a and its 20(17!18)-abeo1a,25-dihydroxy-21-nor-
vitamin D3 analogues 2a and 2b.
Compd
Binding EC50 [nm][a]
Growth Inhib. EC50 [nm][b]
Calcemic Act.[c]
[mgkgꢀ1 dayꢀ1
]
DBP
VDR
MCF-7
1a
2a
2b
50
2000
4000
0.2
2
6
60
50
200
0.1
>60
>100
[a] Binding of compounds 1a, 2a, and 2b to human DBP and pig VDR is expressed as the concentration needed to displace 50% of [3H]1a,25(OH)2D3
from DBP or VDR. [b] The antiproliferative effects of 1a,25(OH)2D3 and its analogues on MCF-7 cells are expressed as the concentrations required for half-
maximal inhibition of [3H]thymidine incorporation. [c] The calcemic activity of compounds 1a, 2a, and 2b was determined in mice (n=6 per group) by in-
traperitoneal injections for seven consecutive days. This activity is expressed as the maximal dose that can be administered without exceeding the serum
calcium concentration observed when mice were treated with compound 1a at 0.1 mgkgꢀ1 dayꢀ1. Serum calcium in mice treated with vehicle was 11.5ꢁ
0.4 mgdLꢀ1, 14.5ꢁ0.7 mgdLꢀ1 in mice treated with compound 1a at 0.1 mgkgꢀ1 dayꢀ1, 11.9ꢁ0.7 mgdLꢀ1 upon treatment with compound 2a at
60 mgkgꢀ1 dayꢀ1, and 11.4ꢁ0.4 mgdLꢀ1 in mice treated with compound 2b at 100 mgkgꢀ1 dayꢀ1. Serum calcium levels in mice treated with compounds 2a
or 2b were not different from control mice, but were significantly lower than in mice treated with compound 1a at 0.1 mgkgꢀ1 dayꢀ1 (p<0.01, one-way
ANOVA followed by Fisher LSD test).
noma MCF-7 cells (Table 1). Addition of an extra carbon atom
Conclusions
in the side chain in compound 2b decreased the growth-inhib-
itory potential of this analogue because threefold higher con-
centrations of this compound were required to obtain a 50%
decrease in proliferation. The ability to induce cell differentia-
tion was investigated in human colon cancer SW480-ADH cells,
which upon incubation with compound 1a undergo epithelial
differentiation with increased adhesiveness as a result of a
drastic change in their pattern of gene expression that in-
cludes the induction of E-cadherin, occludin, and several other
adhesion proteins.[6] Both C18 side chain analogues induced a
similar morphological change as compound 1a, promoting the
formation of compact epithelioid cell islands, but again the an-
alogue with the longer side chain (2b) was less efficient (Fig-
ure 3a). Likewise, compounds 2a and 1a were equipotent in
increasing the cellular content of E-cadherin, a hallmark of the
differentiated phenotype, whereas the 2b analogue was less
potent (Figure 3b). In agreement with these observations, ana-
logue 2a and the parent molecule 1a, but not analogue 2b,
increased VDR expression (Figure 3b). The mechanistic basis
for the discrepancy between receptor binding and biological
activity may be attributed to changes in VDR conformation
and a modified interaction of liganded VDR with co-activator
molecules, which alters the structural and functional properties
of the entire DNA-binding complex.[7]
We describe improved syntheses of two des-side chain ana-
logues of vitamin D3 with substituents at C18. In the VDR crys-
tallographic study we demonstrated that the seven-membered
side chain introduced at position C18 in compound 2a adopts
the same orientation in the ligand binding pocket as the side
chain of compound 1a with maintenance of the hydrogen
bonds that form the anchoring points of the ligand. Mutational
analysis confirmed the interactions of compound 2a with the
amino acids lining the VDR ligand binding pocket as deter-
mined in the crystal structure. Despite the fact that analogue
2a fitted well in the VDR binding pocket, it displayed a weak
binding to VDR and homologation of the side chain further de-
creased the affinity. Nevertheless both des-side chain ana-
logues of vitamin D3 possessed significant antiproliferative and
prodifferentiating properties with greatly decreased calcemic
effects. This biological profile makes these analogues, and es-
pecially compound 2a, potential candidates for the treatment
of hyperproliferative disorders such as breast or colon cancer.
Experimental Section
Chemistry
Interestingly, introduction of a side chain at position C18 led
to a decrease in calcemic activity. Indeed, in comparison with
compound 1a, at least 600-fold higher doses of the seven- or
eight-membered C18 side chain analogues could be adminis-
tered on a daily basis in NMRI vitamin D-replete mice (Table 1).
The lower calcemic activity may be due, in part, to the low
binding affinity of these compounds for DBP, which results in
an increased clearance. However, other mechanisms such as in-
creased metabolic degradation, tissue-specific differences in
the binding affinities of compound 1a and its analogues for
the VDR, and different signaling pathways have been suggest-
ed to contribute.[7,8] The low calcemic activity of compound 2a
together with its potent antiproliferative and prodifferentiating
activity make this compound appealing for therapeutic appli-
cation.
In addition to NMR, HPLC analysis was used to determine the
purity (>95%) of vitamin D3 analogues.
20(17!18)-abeo-3-(Triethylsilyl)-1a-[(triethylsilyl)oxy]-25-hy-
droxy-22-homo-21-norvitamin D3 (2a-TES): A solution of nBuLi
(0.45 mL, 1.14 mmol, 2.5m in hexanes) was added to a solution of
phosphine oxide 3 (0.702 g, 1.204 mmol) in dry THF (8 mL) at
ꢀ788C. The deep-red solution was stirred for 1 h. A solution of
ketone 4 (0.063 g, 0.277 mmol) in dry THF (8 mL) was added drop-
wise. The reaction mixture was stirred in the dark at ꢀ788C for 3 h
and at ꢀ308C for 4 h. H2O (0.5 mL) was added and the resulting
mixture was concentrated to give a residue which was dissolved in
Et2O (100 mL). The combined organic phase was washed with satu-
rated NaHCO3 (20 mL), brine (3”20 mL), H2O (60 mL), dried, fil-
tered, and concentrated. The residue was purified by flash chroma-
tography (SiO2, 2”15 cm, 6–12% Et2O/hexanes) to give the pro-
ChemMedChem 2011, 6, 788 – 793
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
791

A. MouriÇo et al.
MED
at ꢀ788C. The deep-red solution was stirred for 1 h. A solution of
ketone 7 (0.050 g, 0.17 mmol) in dry THF (5 mL) was added drop-
wise. The reaction mixture was stirred in the dark at ꢀ788C for 2 h
and at ꢀ558C for 4 h. H2O (0.5 mL) was added. Concentration of
the reaction mixture gave a residue which was dissolved in Et2O
(100 mL). The combined organic phase was washed with saturated
NaHCO3 (20 mL), saturated NaCl (3”20 mL), H2O (60 mL), dried, fil-
tered, and concentrated. The residue was purified by flash chroma-
tography (SiO2, 2”15 cm, 6–12% Et2O/hexanes) to give the pro-
tected analogue 2b-TES [0.091 g, 82%, Rf =0.8 (30% EtOAc/hex-
anes), colorless oil].
20(17!18)-abeo-1a,25-Dihydroxy-22,23-dihomo-21-norvitamin
D3 (2b): HF–pyridine complex (10 drops) was slowly added to a so-
lution of 2b-TES (0.085 g, 0.136 mmol) in dry CH3CN (3 mL), dry
CH2Cl2 (1 mL) and dry Et3N (1 mL). The reaction mixture was stirred
in the dark at RT for 1.5 h. Saturated NaHCO3 (30 mL) was added
slowly and the aqueous layer was extracted with Et2O (4”20 mL).
The combined organic layer was washed with brine (2”25 mL),
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 1.5”15 cm, 10–12% iPrOH/hexanes) to give
2b [0.05 g, 95%, Rf =0.5 (30% iPrOH/hexanes), white solid].
Details on the syntheses of compounds 3, 6a, 6b, 6c, 6d, 6e, 6 f,
6g, 6, 7a, and 7 are described in the Supporting Information.
Structural analysis of hDVR complexed to compound 2a: Details
on expression, purification, crystallization, and subsequent X-ray
crystallography, data collection, and processing can be found in
the Supporting Information. The accession number for the coordi-
nates of the complex reported herein is PDB ID: 3P8X. The meth-
odology applied for the analysis of heterodimerization with RXR is
described in the Supporting Information. The transactivating assay
was performed as described by Verlinden et al.[7]
Biological activity
Figure 3. Analogues 2a and 2b induce an adhesive epithelial phenotype in
SW480-ADH cells. a) Differentiation of SW480-ADH cells demonstrated by
phase-contrast micrographs of cells treated with each compound at 100 nm
or vehicle for 48 h. A representative experiment is shown. b) Western blot
analysis of the expression of E-cadherin, VDR, and b-actin (loading control)
at 8 and 48 h treatment with each compound at 100 nm. Numbers show the
quantification of E-cadherin induction (ratio of levels in treated versus un-
treated cells) after normalization to b-actin. A representative experiment is
shown.
Affinity of the analogues to hDBP and pVDR and their antiprolifera-
tive activity were determined as described previously.[8] The proto-
col for in vivo evaluation of calcemic activity was approved by the
ethical committee for animal research at the Katholieke Universiteit
Leuven and was in accordance with the NIH Guide for the Care
and Use of Laboratory Animals. The ability of the compounds to
induce differentiation of SW480-ADH colon cancer cells was mea-
sured as described by Palmer et al.[6]
tected analogue 2a-TES [0.125 g, 85%, Rf =0.8 (30% EtOAc/hex-
anes), colorless oil].
Acknowledgements
20(17!18)-abeo-1a,25-Dihydroxy-22,23-dihomo-21-norvitamin
D3 (2a): HF–pyridine complex (10 drops) was slowly added to a so-
lution of 2a-TES (0.125 g, 0.194 mmol) in dry CH3CN (6 mL), dry
CH2CL2 (4 mL), and dry Et3N (3 mL). The reaction mixture was
stirred in the dark at RT for 1 h. Saturated NaHCO3 (30 mL) was
added slowly and the aqueous layer was extracted with Et2O (4”
20 mL). The combined organic layer was washed with brine (2”
25 mL), dried, filtered, and concentrated. The residue was purified
by flash chromatography (SiO2, 1.5”15 cm, 10–12% iPrOH/hex-
anes) to give 2a [0.079 g, 98%, Rf =0.4 (20% iPrOH/hexanes),
white solid].
This study was supported by the Spanish Ministry of Education
and Science (Grants SAF2004-01885, SAF2007-60341, and
SAF2007-67205), the European Union (MRTN-CT-2005-019496,
NucSys), the Xunta de Galicia (Projets INCITE08PXIB-209130PR
and ACEUIC-2006/XA050), Consolider, Nanobiomed, Fondo de In-
vestigaciones Sanitarias, Fonds voor Wetenschappelijk Onderzoek
(FWO) – Vlaanderen (G.553.06 and G.0587.09), CNRS, INSERM,
Universitꢃ de Strasbourg, ANR, and the European Commission
Structural Proteomics in Europe SPINE2-Complexes (LSHG-CT-
2006-031220). CESGA (Santiago, Spain) is acknowledged for com-
puting time granted to the research group of A.M. For structural
analysis, we thank the staff of the beamlines at ESRF for experi-
mental assistance during data collection. We thank IGBMC mass
spectrometry common service for MS analysis. D.N. thanks the
20(17!18)-abeo-3-(Triethylsilyl)-1a-[(triethylsilyl)oxy]-25-hy-
droxy-22,23-dihomo-21-norvitamin D3 (2b-TES): A solution of
nBuLi (0.41 mL, 1.02 mmol, 2.5m in hexanes) was added to a solu-
tion of phosphine oxide 3 (0.627 g, 1.076 mmol) in dry THF (5 mL)
792
www.chemmedchem.org
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 788 – 793

Vitamin D3 Analogues
[4] E. G. Baggiolini, J. A. Iacobelli, B. M. Hennessy, A. D. Batcho, J. F. Sereno,
M. R. Uskokovic, J. Org. Chem. 1986, 51, 3098–3108, and references
therein.
[5] N. Rochel, D. Moras, Curr. Top. Med. Chem. 2006, 6, 1229–1241.
[6] H. G. Palmer, J. M. Gonzalez-Sancho, J. Espada, M. T. Berciano, I. Puig, J.
Baulida, M. Quintanilla, A. Cano, A. G. de Herreros, M. Lafarga, A. Munoz,
J. Cell Biol. 2001, 154, 369–387.
Argentinian National Council for Scientific research (CONICET) for
a postdoctoral fellowship. S.E.-C. thanks the Spanish MEC for an
FPU fellowship. G.E. is a postdoctoral researcher for FWO. We
thank Dishman Netherlands B.V. (Weesp, the Netherlands) for the
gift of vitamin D2.
[7] L. Verlinden, A. Verstuyf, C. Verboven, G. Eelen, C. De Ranter, L. J. Gao,
Y. J. Chen, I. Murad, M. Choi, K. Yamamoto, S. Yamada, D. Van Haver, M.
Vandewalle, P. J. De Clercq, R. Bouillon, J. Biol. Chem. 2003, 278, 35476–
35482.
Keywords: vitamin D analogues · biological activity · cancer ·
steroids · vitamins
[8] A. Verstuyf, L. Verlinden, E. van Etten, L. Shi, Y. Wu, C. D’Halleweyn, D.
Van Haver, G. D. Zhu, Y. J. Chen, X. Zhou, M. R. Haussler, P. J. De Clercq, M.
Vandewalle, H. Van Baelen, C. Mathieu, R. Bouillon, J. Bone Miner. Res.
2000, 15, 237–252.
[1] G. Eelen, C. Gysemans, L. Verlinden, E. Vanoirbeek, P. J. De Clercq, D.
Van Haver, C. Mathieu, R. Bouillon, A. Verstuyf, Curr. Med. Chem. 2007, 14,
1893–1910.
[2] a) G. Grue-Sørensen, C. M. Hansen, Bioorg. Med. Chem. 1998, 6, 2029–
2039; b) I. Cornella, S. J. Perez, A. Mourino, L. A. Sarandeses, J. Org. Chem.
2002, 67, 4707–4714; c) K. Nilsson, M. J. Valles, L. Castedo, A. Mourino,
Bioorg. Med. Chem. Lett. 1993, 3, 1855–1858.
Received: January 21, 2011
Revised: February 24, 2011
[3] E. Momꢁn, D. Nicoletti, A. MouriÇo, J. Org. Chem. 2004, 69, 4615–4625.
Published online on March 29, 2011
ChemMedChem 2011, 6, 788 – 793
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
793