The First Locked Side-Chain Analogues of Calcitriol (1α,25- Dihydroxyvitamin D3) Induce Vitamin D Receptor Transcriptional Activity

Article abstract of DOI:10.1021/ol0351246

(Equation presented) We describe the synthesis of the first locked side-chain analogues of the natural hormone 1α,25-(OH)₂-D ₃ and their effects on gene transcription in human colon cancer cells. Analogue 2 was more potent than 1α.25

Full text of DOI:10.1021/ol0351246

ORGANIC
LETTERS
2003
Vol. 5, No. 22
4033-4036
The First Locked Side-Chain Analogues
of Calcitriol (1r,25-Dihydroxyvitamin D )
3
Induce Vitamin D Receptor
Transcriptional Activity
Xenxo Pe´rez-Garc´ıa,† Antonio Rumbo,† Mar´ıa Jesu´s Larriba,‡ Paloma Ordo´n˜ez,‡
,†
Alberto Mun˜oz,‡ and Antonio Mourin˜o*
Departamento de Qu´ımica Orga´nica y Unidad Asociada al CSIC, UniVersidad de
Santiago de Compostela, 15706 Santiago de Compostela, and Instituto de
InVestigaciones Biome´dicas (CSIC-UAM), Arturo Duperier, 4, 28029 Madrid, Spain
qomourin@usc.es
Received June 18, 2003
ABSTRACT
We describe the synthesis of the first locked side-chain analogues of the natural hormone 1r,25-(OH)2-D and their effects on gene transcription
3
in human colon cancer cells. Analogue 2 was more potent than 1r,25-(OH)2-D at inducing vitamin D receptor (VDR) transcriptional activity.
3
Analogues 3a and 3b show potency similar to that of 1r,25-(OH)2-D , whereas 3c was less active. The novel analogues efficiently bind VDR
3
in vivo to induce transcription from a consensus vitamin D responsive element (VDRE).
It is now known that 1R,25-dihydroxyvitamin D3 [1a, 1R,-
25-(OH)2-D3, calcitriol], the hormonally active form of
vitamin D3 (1b, cholecalciferol), exerts a wide range of
genomic biological actions through a multistep mechanism
that includes binding to the nuclear vitamin D receptor
(VDR),1 heterodimerization of the VDR with retinoid X
receptor (RXR), and binding of the resulting complex to
specific DNA sequences, named vitamin D responsive
element (VDRE), to induce transcription.2,3 The hormone
1R,25-(OH)2-D3, besides its important role in calcium
† Universidad de Santiago de Compostela.
‡ Instituto de Investigaciones Biome´dicas. E-mail (A. Mun˜oz): amunoz@
iib.uam.es.
(1) Chawla, A.; Repa, J. J.; Evans, R. M.; Mangelsdorf, D. J. Science
2001, 294, 1866-1870.
(2) Vitamin D; Feldman, D., Glorieux, F. H., Pike, J. W., Eds.; Academic
Press: New York, 1997.
homeostasis, also promotes cell differentiation and inhibits
cell proliferation of various tumor cells, a fact that suggests
its possible use in the treatment of cancer. Unfortunately,
the therapeutic value of 1R,25-(OH)2-D3 as an antitumor
agent finds serious limitations due to its potent calcemic side
effects.2,4 Recent interest in the development of an analogue
of 1R,25-(OH)2-D3 with selective properties for treatment
of cancer and dermatological diseases has led to an increased
activity in the vitamin D field.5,6
Prior to Moras’ X-ray study of a mutant VDR complexed
to 1R,25-(OH)2-D3, incomplete understanding of the con-
(4) Vitamin D Endocrine System: Structural, Biological, Genetic, and
Clinical Aspects; Norman, A. W., Bouillon, R., Thomasset, M., Eds.;
Vitamin D Workshop, Inc.: University of California-Riverside, Riverside,
CA, 2000.
(5) Kabat, M. M.; Radinov, R. Curr. Opin. Drug DiscoV. DeV. 2001, 4,
808-833.
(3) Calberg, C. J. Cell. Biochem. 2003, 88, 274.
10.1021/ol0351246 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/04/2003

formation that the side chain of 1R,25-(OH)2-D3 adopts in
the binding pocket of the vitamin D receptor (VDR) led to
the synthesis of numerous side-chain-modified analogues,
of which only a few have been identified as promising for
the treatment of certain cancers and psoriasis.7,8 To study
indirectly the location of the C25 hydroxyl group of 1R,25-
(OH)2-D3 in its bioactive conformation or conformations, we
recently reported the synthesis and conformational analysis
of side-chain analogues of 1R,25-(OH)2-D3 that incorporate
conformationally locked units in the form of a double bond,
a cyclopropane ring, an aromatic ring, or an additional five-
membered ring.9,10 The fact that some of these analogues
improved the biological profile of the natural hormone for
potential therapeutic applications has now led us to synthesize
novel analogues of 1R,25-(OH)2-D3 with side chains with
higher degrees of rotational restriction in order to define the
topography of the side-chain hydroxyl group that is required
to induce gene transcription. Conformational and docking
studies of a series of analogues using Moras’ X-ray crystal
structure11 led us to design the locked side-chain analogues
2 and 3, which incorporate two triple bonds or a triple bond
and an aromatic unit in their respective side chains. Here
we describe the synthesis of these four novel analogues of
1R,25-(OH)2-D3 and report preliminary data on their biologi-
cal behavior.
The upper fragments 5 and 6 are prepared from alkyne 7
under metal-catalyzed couplings. Methyl ketone 8, which is
readily prepared by degradation of Inhoffen-Lythgoe diol
(9) as shown in earlier work,13 serves for the preparation of
the key alkyne 7.
The preparation of the upper fragments 5 and 6 required
for the convergent synthesis of the target compounds 2 and
3 is shown in Scheme 2. Treatment of ketone 8 with LDA
Scheme 2 a
The synthesis of analogues 2 and 3 follows the mild
convergent Wittig-Horner approach originally developed by
Lythgoe and later improved by the Hoffmann La Roche
group (Scheme 1).5,12 In this route, 5 or 6 is coupled with
Scheme 1. Retrosynthetic Analysis of Analogues 2 and 3
a Reagents and reaction conditions: (a) LDA; N-(5-chloro-2-
n
pyridyl)-triflimide, THF -78 °C. (b) LDA, THF, rt. (c) HexLi;
I2, THF, -78 °C. (d) 12 (1.3 equiv), CuI (10%), pyrrolidine. (e)
13 (2 equiv), Et3N (4 equiv), (PPh3)2PdCl2 (10%), DMF, 80 °C.
13a (X ) m-Br, R ) OTBS), 13b (X ) m-OSO2CF3, R ) CO2Me),
13c (X ) p-OSO2CF3, R ) CO2Me).
and reaction of the resulting kinetic enolate with N-(5-chloro-
2-pyridyl)-triflimide14 gave vinyl triflate 10 (64%), which
(10) (a) For leading references on conformational analysis of nonlocked
side-chain analogues of 1R,25-(OH)2-D3, see: Yamada S.; Shimizu M.;
Yamamoto, K. Med. Res. ReV. 2003, 23, 89-115. (b) For analogues of
1R,25-(OH)2-D3 with partially locked side chains, see: Okamura, W. H.;
Graig, A. S.; Curtin, M. L.; De Lera, A. R.; Figade´re, B.; Muralidharan, K.
R.; Palenzuela, A. In: Vitamin D: Gene Regulation, Structure-Function
Analysis and Clinical Application; Norman, A. W., Bouillon, R., Thomasset,
M., Eds.; Walter de Gruyter: Berlin, 1991, pp 169-177.
the anion of phosphine oxide 4 to provide, after deprotection,
the desired 1R,25-dihhydroxyvitamin D3 analogues 2 or 3.
(6) Carsten, C.; Mourin˜o, A. Expert Opin. Ther. Patents 2003, 13, 761-
772.
(7) Brown, A. J. Am. J. Kidney Dis. 2001, 38, S3-S19.
(8) Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. ReV. 1995,
16, 200-257.
(9) (a) Mart´ınez-Pe´rez, J. A.; Sarandeses, L.; Granja, J.; Palenzuela J.
P.; Mourin˜o, A. Tetrahedron Lett. 1998, 39, 4725. (b) Ferrna´ndez-Gacio,
A.; Vitale, C.; Mourin˜o, A. J. Org. Chem. 2000, 65, 6978. (c) Varela, C.;
Nilsson, K.; Torneiro, M.; Mourin˜o, A. HelV. Chim. Acta 2002, 85, 3251.
(d) Riveiros, R. Synthesis of Analogues of Calcitriol (1R,25-Dihydroxy-
vitamin D3) with a Semi-rigid Side Chain. Ph.D. Thesis, 2001.
(11) Rochel, H.; Wurtz, J. M.; Mitscher, A.; Klaholz, B.; Moras, D. Mol.
Cel 2000, 5, 173.
(12) For synthesis of vitamin D metabolites and analogues, see: (a) Zhu,
G.-D.; Okamura, W. H. Chem. ReV. 1995, 95, 1877. (b) Krause, S.; Schmalz,
H.-G. In Organic Synthesis Highliths IV; Schmalz, H.-G., Ed.; Wiley-
VCH: Weinheim, Germany, 2000; pp 212-217.
(13) (a) Sardina, F. J.; Mourin˜o, A.; Castedo, L. J. Org. Chem. 1986,
51, 1264 (b) Ferna´ndez, B.; Mart´ınez-Pe´rez, J.; Granja, J.; Castedo, L.;
Mourin˜o, A. J. Org. Chem. 1992, 57, 3173.
4034
Org. Lett., Vol. 5, No. 22, 2003

was subjected to elimination (LDA) to deliver the key alkyne
7 in good yield (98%). With the common intermediate 7 in
hand, we first accomplished the synthesis of the upper
fragment 5 precursor of analogue 2. Metalation of 7
(HexLi)15 and subsequent reaction of the resulting lithium
acetylide with iodine provided the iodo alkyne 11 (85%).
Coupling between 11 and propargylic ether 12 in the
presence of copper(I) iodide16 and pyrrolidine gave the
desired diyne 5 in good yield (88%) (five steps from 8, 47%).
The aromatic upper fragments 6 required for the synthesis
of analogues 3 were prepared from alkyne 7 using Pd-
catalyzed alkynylation under the Sonogashira reaction.17
Thus, heating of a mixture of 7 and 1-bromo-3-tert-
butyldimethylsilyloxybenzene (13a) in DMF at 80 °C in the
presence of Et3N and (PPh3)2PdCl2 provided 6a (53%).
Alkynes 6b (69%) and 6c (67%) were prepared in a similar
manner using methyl 3-[[(trifluoromethyl)sulfonyl)]oxy]-
benzoate (13b) and methyl 4-[[(trifluoromethyl)sulfonyl)]-
oxy]benzoate (13c) as the aromatic partners (Scheme 2).
Ketone 14 required for the synthesis of analogue 2 was
prepared from diyne 5 by sequential desilylation (HF) and
oxidation with pyridinium dichromate (two steps, 87%)
(Scheme 3). Ketone 14 was coupled at -78 °C with the anion
Scheme 4 a
a Reagents and reaction conditions: (a) HF (45%), CH3CN, rt;
PDC (2 equiv), CH2Cl2, rt. (b) Anion of 4, -78 °C. (c) 17a: Bu4NF
(3 equiv), THF, rt. 17b-c: MeLi (3 equiv), THF, 0°C; Bu4NF.
n
n
Desilylation and subsequent oxidation of 6b and 6c delivered
ketones 16b and 16c, which were coupled as above to
provide esters 17b and 17c, respectively.
Scheme 3 a
Treatment of 17b and 17c with methyllithium followed
by desilylation (nBu4NF) led to the respective desired
analogues 3b and 3c (29 and 54% overall yields from 6b
and 6c, respectively).
Finally, the biological activity of the four novel locked
vitamin D analogues was assayed in the human SW480-ADH
colon cancer cell line. These cells express endogenous VDR
and respond to 1R,25-(OH)2-D3 addition by inhibition of
proliferation and differentiation to an epithelial phenotype.19
a Reagents and conditions: (a) HF (45%), CH3CN, rt; PDC
n
(2 equiv), CH2Cl2, rt. (b) Anion of 4, THF, -78 °C. (c) Bu4NF
(3 equiv), THF, rt; AG50 WX4, MeOH, rt.
of phosphine oxide 418 to form stereoselectively the corre-
sponding protected analogue 15. Sequential deprotection of
15 (nBu4NF; AG50 WX4) provided the requisite analogue 2
(80% from 14).
Analogues 3 were prepared in a similar way from the upper
fragments 6 (Scheme 4). Desilylation and oxidation of 6
provided ketones 16. Wittig-Horner coupling of 16a with
the anion of phosphine oxide 4 led, after desilylation (nBu4-
NF), the desired analogue 3a (four steps from 6a, 36%).
Figure 1. SW480-ADH colon cancer cells were transfected with
the 4xVDRE-DR3.tk-luc construct containing the luciferase gene
under the control of four copies of direct repeat 3 (DR3) VDRE.
A â-galactosidase expression vector (pRSV-LacZ) was also trans-
fected as an internal control. After 72 h of incubation in the presence
or absence of the indicated concentrations of 1R,25-(OH)2-D3,
luciferase and â-galactosidase activities in total cell extracts were
measured. Mean values and standard deviations of the mean
obtained in three experiments using triplicates are shown.
(14) Comings, D. L.; Dehghani, A. Tetrahedron Lett. 1992, 33, 6299.
n
(15) nHexLi is a cheaper, more stable, and stronger base than BuLi.
(16) Alami, M.; Ferri, F. Tetrahedron Lett. 1996, 37, 2763.
(17) (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
4467. (b) Sonogashira, K. Metal-catalyzed Cross-coupling Reactions;
Diederich, F., Stang, P. J., Eds.; Wiley-VCH: Weinheim, Germany, 1998,
pp 203-229.
(18) Mourin˜o, A.; Torneiro, M.; Vitale, C.; Ferna´ndez, S.; Pe´rez-Sestelo,
J.; Anne, A.; Gregorio, C. Tetrahedron Lett. 1997, 38, 4713.
Org. Lett., Vol. 5, No. 22, 2003
4035

The ability of the four derivatives to active VDR was
examined in transactivation experiments using cells that were
transfected with a plasmid encoding the luciferase reporter
gene under the control of a vitamin D response element
(VDRE). Treatment with 10-7 M or 10-9 M 1R,25-(OH)2-
D3 for 48 h led to 39- and 7-fold increases, respectively, in
luciferase expression over vehicle-treated cells (Figure 1).
Remarkably, analogue 2 was more potent that calcitriol,
causing higher increases in VDR transactivating activity (50-
and 12.5-fold at 10-7 or 10-9 M, respectively). Analogues
3a and 3b showed potency similar to that of calcitriol at
10-7 M (around 40-fold activation) but lower potency at 10-9
M, whereas analogue 3c was less active (18-fold activation
at 10-7 M). These results show that our compounds ef-
ficiently bind VDR in vivo, inducing its ability to activate
transcription from a consensus VDRE.
structural information with regard to the bioactive conforma-
tion of the natural hormone. Further biological results, the
clinical potential of the novel analogues, and the development
of new analogues in this series will be reported in due course.
Acknowledgment. Generous financial support for this
research was provided by the DIGICYT (Spain, Projects
SAF2001-3187 and SAF2001-2291). We acknowledge J. P.
van de Velde and J. Zorgdrager (Solvay Pharmaceuticals BV,
Weesp, The Netherlands) and L. Binderup (Leo Pharma-
ceuticals, Ballerup, Denmark) for the gift of starting materi-
als. X.P.-G. thanks the Xunta de Galicia for a Predoctoral
fellowship. M.J.L. thanks the Spanish Ministry of Education,
Culture and Sports for a fellowship (MECD). P.O. thanks
the Spanish Ministry of Science and Technology for a
fellowship (MCYT). A.R. thanks the Spanish Ministry of
Education and Technology for a Ramon y Cajal grant.
In summary, we have developed the first locked side-chain
analogues of 1R,25-(OH)2D3. It is noteworthy that the novel
analogues 2 and 3a-c lead to significant transcription
activation in comparison to 1R,25-(OH)2-D3, giving important
Supporting Information Available: Experimental pro-
cedures and spectral data (1H and 13C NMR). This material
is available free of charge via the Internet at http://pubs.acs.org.
(19) Pa´lmer, H. G.; Gonzale´z-Sancho, J. M.; Espada, J.; Berciano, M.
T.; Puig, I.; Baulida, J.; Quintanilla, M.; Cano, A.; Garc´ıa de Herreros, A.;
Lafarga, M.; Mun˜oz, A. J. Cell. Biol. 2001, 154, 369.
OL0351246
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Org. Lett., Vol. 5, No. 22, 2003