Carborane-based design of a potent Vitamin D receptor agonist

Article abstract of DOI:10.1039/c5sc03084f

The vitamin D nuclear receptor (VDR) is a potential target for cancer therapy. It is expressed in many tumors and its ligand shows anticancer actions. To combine these properties with the application of boron neutron capture therapy (BNCT), we design and

Full text of DOI:10.1039/c5sc03084f

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Carborane-Based Design of a Potent Vitamin D Receptor Agonist
Rocio Otero,a,§ Samuel Seoane,b,§ Rita Sigüeiro,a,c Anna Y. Belorusova,c Miguel A. Maestro,d Roman
Pérez-Fernández,b Natacha Rochel c,* and Antonio Mouriñoa,*
Received 00th January 20xx,
Accepted 00th January 20xx
The Vitamin D nuclear receptor (VDR) is a potential target for cancer therapy. It is expressed in many tumors and its ligand
shows anticancer actions. To combine these properties with the application of boron neutron capture therapy (BNCT), we
design and synthetize a potent VDR agonist based on the skeleton of the hormone 1α,25-dihydroxyvitamin D3 (1,25D) and
an o-carborane (dicarba-o-closo-1,2-dodecaborane) at the end of its side chain. The present ligand is the first secosteroidal
analog with the carborane unit that efficiently binds to VDR and functions as an agonist with 1,25D-like potency in
transcriptional assay on vitamin D target genes. Moreover it exhibits similar antiproliferative and pro-differentiating
activities but is significantly less hypercalcemic than 1,25D. The crystal structure of its complex with VDR ligand binding
domain reveals its binding mechanism involving boron-mediated dihydrogen bonds that mimic vitamin D hydroxyl
interactions. In addition to the therapeutic interest, this study establishes the basis for the design of new unconventional
vitamin D analogs containing carborane moieties for specific molecular recognition, and drug research and development.
DOI: 10.1039/x0xx00000x
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shown to have broader applications in medicinal chemistry as active
biomolecules such as carborane compounds that have anticancer
activity or act as HIV replication inhibitors.3,4
Introduction
Icosahedral carboranes (dicarba-closo-dodecaboranes) are
carbon-boron clusters with spherical geometry and a hydrophobic
surface. They are thermally and metabolically stable, with high
lipophilicity and ability to form an unusual proton-proton
(dihydrogen) bond B-H···H-X (X = N, O, C).1,2 Carboranes have been
used as unique pharmacophores in biologically active
Among the receptors expressed in tumors that can be targeted by
carborane ligands for cancer therapy, the vitamin D receptor (VDR),
a member of the nuclear receptor superfamily, is a potential
candidate.11 Vitamin D3, before eliciting its physiological functions,
undergoes two hydroxylations, first in the liver and then in the
compounds.3,4 Initial development of the medicinal chemistry kidney, to produce its active form, 1α,25-dihydroxyvitamin D3
(1,25D, Figure 1). The hormone 1,25D through binding to VDR
application of boron was applied to boron neutron capture therapy
(BNCT) of cancer with the objective to selectively destroy cancer regulates a number of physiological and pathological processes,
cells as an alternative to conventional radiation therapy. BNCT is including cell proliferation and differentiation, which has led to the
based on the nuclear capture of neutrons by 10B to produce high study of its effects in cancer. Significant antitumor activity (i.e. anti-
energy particles that selectively destroy tumors without affecting proliferative effects, induction of apoptosis, stimulation of
healthy tissue.5-8 The development of this technique depends on
diffrentiation, and inhibition of invasion and metastasis) has been
the availability of boron compounds which bind receptors in the demonstrated for 1,25D in different types of cancers including
breast, prostate, colon, and acute myeloid leukemia.12,13 In fact,
tumors. To target tumors more specifically, derivatives of natural
ligands that target receptors but also bear boron atoms have been recent studies have established that VDR expression should be
taken into account for breast cancer treatment.14
synthesized.4,9,10 More recently, boron compounds have been
Results and discussion
In this study, we describe the design, the synthesis and the
functional and structural characterization of a novel vitamin D
analog bearing an o-carborane group at the side chain (1, Scheme
1). To date, only a few simple carborane mimics of vitamin D have
been developed. These compounds, which show reduced activity,
lack the 1,25D A-ring moiety and possess a p-carborane unit that
replaces the natural CD-rings.15-18 The p-carborane unit of theses
vitamin D mimics interacts with the VDR only through van der
Waals interactions.15 More recently19 the same authors synthetized
structure-related compounds which exhibit similar pro-
differentiation activities in HL-60 cells as the natural 1,25D
hormone.
§ R. Otero and S. Seoane contributed equally.
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Carborane 1 is the first vitamin D analog with the carborane unit characterization. Construction of the carborane-containing side was
that efficiently binds to the VDR ligand binding domain (LBD) and is envisaged to arise by reaction between alkyne 2 and decaborane
as transcriptionally active as 1,25D on vitamin D target genes, but is (B10H14).4 It was not clear at this point if the labile vitamin D triene
significantly less hypercalcemic than 1,25D. In addition to the system would withstand the reaction conditions. The vitamin D
therapeutic interest, our study helps clarify the functional behavior triene system would arise by a Pd-catalyzed cascade process21
of this molecule.
involving ring-closure of enol-triflate 4 and coupling with boronate
3 (Scheme 1).
25
OH
TMS
H
H
C
D
H
H
=Boron
=Carbon
H
A
H
H
1
HO
OH
HO
OH
1,25D
1
H
H
H
O
B
3
Fig. 1 Vitamin D hormone (1,25D) and the target carborane 1.
Design
O
TfO
SiO
OSi
SiO
OSi
HO
OH
1
2
4
On the basis of the crystal structure of an engineered active ligand-
binding domain of the human vitamin D receptor (hVDR LBD)
bound to 1,25D,20 which reveals an additional hydrophobic space
around the side chain, and inspired by the biological profile of
carboranes,3,4 we designed a new analog bearing an o-carborane
moiety at the end of the side chain (1, Fig. 1). Docking results show
that the novel vitamin D analog fits reasonably well in the ligand
binding pocket. The ligand adopts the canonical active
conformation in which the rings and the triene system of the new
ligand have similar positions as the natural hormone 1,25D. The
A-ring hydroxyl groups form hydrogen bonds with the same amino
acid residues as 1,25D. Remarkably, the side-chain carborane
moiety binds by unusual dihydrogen bonding with the His305 and
His397 residues (Fig. 2).
Scheme 1 Retrosynthesis of target carborane 1. Si = protecting group, TMS =
SiMe3.
The synthesis of carborane 1 is illustrated in Scheme 2 and started
with known Inhoffen-Lythgoe diol (5a),22 which was converted to
known tosylate 5b. Exposure of tosylate 5b to magnesium reagent
623 in the presence of catalytic Li2CuCl4 provided alcohol 7a (96%),
which was oxidized with pyridinium dichromate to the
corresponding ketone 7b (98%). Wittig chemistry on 7b using ylide
RO
TMS
MgBr
6
H
b
TMS
H
c, d
96%
77%
H
H
HO
HO
5a, R = H
7a
a (80%)
5b
, R = pTs
TfO
f
TBSO
OTBS
TMS
H
TMS
H
e
4
88%
98%
H
H
pinB
Br
3
8
H
TMS
H
H
g
h
i
93%
H
H
H
1
92%
43%
TBSO
OTBS
TBSO
OTBS
TBSO
OTBS
9
2
10
Scheme 2 Synthesis of target analog 1. Reagents and conditions: a) pTsCl
(1.5 equiv), py, 0 °C, 12 h; b) 6 (4 equiv), Et2O/THF (1:1); Li2CuCl4 (0.01
equiv), THF, -78 °C → 23 °C, 12 h; c) Oxidation of 7a to the corresponding
ketone 7b: PDC (3 equiv), CH2Cl2, 23 °C, 5 h; d) (Ph3PCH2Br)Br (8 equiv), tol,
ultrasound, 15 min; KOtBu in THF (7.9 equiv, 1 M), -15 °C → 0 °C, 2.5 h, then
7b in tol, -15 °C → 23 °C, 3 h; e) Tol/THF (3:1), -78 °C, tBuLi (2 equiv), 1 h;
B(OiPr)3 (1.5 equiv), 1.5 h; pinacol (1.3 equiv), -78 °C → 23 °C, 4 h; f) 4
(1 equiv), K3PO4 (27 equiv, aqueous sol, 2 M), THF, PdCl2(PPh3)2 (0.05 equiv),
2 h; g) K2CO3, MeOH, 23 °C, 14 h; h) PhNMe2 (6.5 equiv), tol, decaborane
(B10H14, 3 equiv), 110 °C, 1 h; i) HF (48%, 2.5 equiv), CH2Cl2/CH3CN (2:1), 23
Fig 2. A) Structure of 1,25D (yellow) in complex with hVDR LBD
(PDB ID: 1DB1). B) Docked structure of analog 1 (green) into hVDR LBD.
Distances are shown in Å.
Synthesis
Our plan for the synthesis of the target analog 1 entailed formation
of the o-carborane unit late in the synthesis to circumvent
anticipated problems related to carborane chemistry and
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°C, 12 h. pTsCl = p-toluenesulfonyl chloride, PDC = pyridinium dichromate, We next analyzed the biological properties of carborane 1 in
py = pyridine, tol = toluene, TBS = SitBuMe2.
multiple cell lines. The biological transcriptional activity of 1, as
compared to 1,25D, was evaluated in human MCF-7 breast cancer
Ph3P=CHBr (prepared by treatment of Ph3PCH2Br2 with KOtBu)21,24
provided vinyl bromide 8 (77%), which was converted to boronate 3
(88%) by metallation with tBuLi, reaction of the organolithium with
triisopropyl boronate, and transesterification with pinacol. The
triene system of 2 was constructed in 90% yield by Pd-catalyzed
assembling21 of boronated 3 with the Pd-intermediate resulting
from ring-closure of enol-triflate 425 in the presence of PdCl2(PPh3)2
and K3PO4, followed by desilylation of the side chain (HF). In the
crucial step, the carborane unit of 1 was introduced in the side
chain in 43% yield by reaction of alkyne 2 with decaborane in the
presence of N,N-dimethylaniline. Finally, deprotection of 10
furnished the desired carborane analog 1 in 93% yield (19% overall
yield, 9 steps).
cells. Both compounds at 10-8 and 10-7
M concentrations
significantly decreased cell proliferation with respect to control cells
(Fig. 3a) although they are 3-fold less effective than calcipotriol.26
Carborane 1 also showed similar differentiation-inducing activity
compared to 1,25D in HaCaT keratinocytes (Fig. 3b). Competitive
binding assay demonstrated that VDR binding affinity of 1 was two
times higher than that of 1,25D (IC50: 2.9 x 10-9 M vs. 6.8 x 10-9 M,
respectively) (Fig. 3c). Ligand-induced transcriptional activities were
determined in HEK 293 cells transiently transfected with expression
vectors encoding the full-length hVDR and a luciferase reporter
plasmid encompassing the promoter region of hCYP24A1. We found
that VDR transcriptional activity is induced by lower doses of ligand
1 as compared with 1,25D, with EC50 of 1.4 x 10-10 M (Fig. 3d). In
addition, using MCF-7 cells, we evaluated the effects of carborane 1
administration on a vitamin D target gene by real-time PCR. 1,25D
and carborane 1 showed similar biological potency to induce
CYP24A1 mRNA expression (Fig. 3e). Finally, serum calcium levels
were evaluated in mice 21 days after intraperitoneal administration
of 1,25D or the carborane 1. Mice treated with carborane 1 did not
develop hypercalcemia and they had significantly lower calcium
levels than 1,25D-injected mice (P=0.03, Fig. 3f). Thus, carborane 1
strongly binds to VDR, induces similar biological activities and
displays reduced calcemic effects.
Crystal structure
To decipher the mode of recognition of carborane 1 by the VDR, we
co-crystallized the zebrafish wild-type VDR LBD in complex with the
ligand and the coactivator peptide corresponding to the second
LXXLL motif of SRC-2 and solved the structure with a resolution up
to 2.4 Å. Although numerous carborane ligands for nuclear
receptors have been synthesized 27-29 very few of them have been
crystallized as protein-ligand complexes.16 The data collection and
refinement statistics of the structure are summarized in
Supplementary Table 1. The overall structure (Fig. 4a) is highly
homologous to the VDR-1,25D structure with the insertion region
between helices H2 and H3 not visible in the electron density map
indicating local disorder of this region.30 Despite the bulky
carborane moiety at the end of the side chain, the ligand-VDR
complex adopts the canonical active conformation of the 1,25-VDR
complex. The position and conformation of the activated helix H12
in the zVDR complex is strictly maintained in its agonistic position
(Fig. 4a).30 When compared to the zVDR LBD-1,25D structure, the
Cα atoms of the zVDR LBD-carborane complex have a root mean
square deviation of 0.3 Å over 238 residues. However, small shifts in
secondary structural elements are observed for the loop H6-H7 and
the last turn of helix H11, shifted by 0.6 Å. The ligand is buried in
the predominantly hydrophobic pocket and adopts the same
orientation as 1,25D (Fig. 4b-c). Regarding the interface
VDR/coactivator peptide, we observed that contacts between
protein residues and the coactivator peptide are conserved. The
C1-OH and C3-OH groups of the ligand form similar H-bonds as
1,25D.
Fig. 3 Biological characterization of the carborane 1. a) Cell proliferation in
human MCF-7 breast adenocarcinoma cells. P < 0.01 vs. control cells. b)
Differentiation activity in human HaCaT keratinocytes cells. c) Vitamin D
receptor binding. Competitive binding of 1,25D and carborane 1 to the full-
length human VDR. IC50 values are derived from dose-response curves. d)
Transcriptional responses of hVDR in HEK293 EBNA cells. e) Real-time PCR of
CYP24A1 mRNA levels in human MCF-7 breast cancer cells. f) Calcium levels
in sera of mice treated with 1,25D and carborane 1. Error bars represent
standard deviation (SD).
Functional activity
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As hydrogen atoms at CH of carborane are far more acidic than
those at BH, interaction between carborane CH and Histidine would
also be possible.31 Unconventional dihydrogen bonds with amino
acids residues have been already reported.32 Overall, the numerous
contacts among the atoms of the carborane and hydrophobic
residues of H3, H11 and H12 significantly stabilize the agonist
conformation of VDR in agreement with the agonist potency of this
novel compound. A recently described non-steroidal vitamin D
mimics30 with a p-carborane moiety replacing the CD-rings of 1,25D
form only hydrophobic interactions with VDR and exhibit weaker
pro-differentiationg activities in HL-60. The crystal structures of
some of these p-carborane VD mimics revealed weaker H-bonds
compared to 1,25D and lack the crucial CH-π interaction with Trp.18
Remarkably, our carborane 1 that occupies the crucial anchoring
points of the natural ligand to VDR, binds tighter (2.4x) to VDR and
Fig. 4. a) Overall structure of the zVDR LBD bound to the carborane 1 and
the coactivator peptide. The stars indicate the insertion region not visible in
the electron density map. b) Overlay of 1,25D (carbon atoms in yellow and
oxygen atoms in red) with 1 (violet) within zVDR LBD complexes. c) The
carborane 1 shown in the 2Fo-Fc electron density map contoured at 1σ.
is slightly more potent in transcriptional activation than 1,25D
.
Conclusions
An unique secosteroidal vitamin D analog bearing an o-carborane
unit at the side chain has been designed, synthesized and
biologically tested. In comparison with the natural hormone 1,25D,
the novel analog, which lacks the 25-hydroxy group, strongly binds
to VDR, induces similar biological activities and displays reduced
calcemic effects. The carborane unit mimics the interaction of the
25-hydroxyl group of the natural hormone with the VDR and shows
additional hydrophobic contacts between the boron atoms and
important residues of helix H12. The crystal structure of the analog
in complex with the zebrafish wild-type VDR-LBD, reveals that the
carborane cage interacts efficiently through extensive van der
Waals interactions and dihydrogen bonds with the two histidines
His333 and His423. These results establish the basis for the rational
The secosteroidal part of the ligand forms similar interactions with
the zVDR ligand binding pocket compared to 1,25D. The side-chain
adopts a conformation slightly different than that observed for
1,25D as a consequence of the carborane moiety and the longer
length of the side chain (8.9 Å instead of 6.5 Å). The carborane
group forms extensive interactions with Leu255, Leu258, Ala259,
Val262, Ala331, His333, His423, Tyr427, Leu430, and C-terminal
residues: Leu440, Val444 and Phe448. Stronger contacts are
observed with Ala259, Ala331 and Leu440 compared to 1,25D. The
two histidines His333 and His423 interacts with boron atoms
(Fig. 5). Although the resolution of the crystal structure (2.4 Å) is
not sufficient to resolve the position of the H atoms, on the basis of
the hydride-like hydrogens at the boron atoms, we considered that
in the VDR-carborane complex the two histidines interact with
boron atoms through unconventional (BH····HN) dihydrogen bonds
(Fig. 5), replacing the H-bonds formed between the C25-OH group
of the natural ligand and both histidines.
design of new unconventional vitamin
D analogs containing
carborane moieties for specific molecular recognition, and drug
research and development.
Acknowledgements
We thank the Spanish Ministry of Economy and Innovation
(MEI, SAF2010-15291 and SAF2012-38240), Xunta de Galicia
(project GPC2014/001), Agence Nationale de la Recherche
(ANR-13-BSV8-0024-01), French Infrastructure for Integrated
Structural Biology (FRISBI) (ANR-10-INSB-05-01), and INSTRUCT
as part of the European Strategy Forum on Research Infra-
structures (ESFRI), for financial support and CESGA for
computing time. R. O. thanks the Spanish MEI for an FPI
fellowship. S. S. thanks the Asociación Española Contra el
Cáncer for a fellowship. R. S. thanks the Xunta de Galicia for a
postdoctoral fellowship. We thank Oliver Smart (Global
Phasing Ltd), Alastair McEwen (IGBMC) and the staff of PX1
(SOLEIL synchrotron) for their collaborations.
Figure 5 Details of the interactions mediated by the carborane moiety of
ligand 1 with residues of the zVDR LBD at a 3.8 Å distance cutoff. Dihydrogen
bonds between boron atoms and His333 and His423 are shown by red
dashed lines. Distances are shown in Å.
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