Development of inositol-based antagonists for the d-myo-inositol 1,4,5-trisphosphate receptor

Article abstract of DOI:10.1039/c0cc03003a

The syntheses of four d-myo-inositol 1,4,5-trisphosphate (InsP₃) derivatives, incorporating phosphate bioisosteres at the 5-position, are reported. The methyl phosphate ester and sulfate derivatives retain InsP ₃ receptor (InsP₃

Full text of DOI:10.1039/c0cc03003a

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COMMUNICATION
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Development of inositol-based antagonists for the D-myo-inositol
1,4,5-trisphosphate receptorwz
Neil S. Keddie,ya Yulin Ye,yb Tashfeen Aslam,a Tomas Luyten,c Davide Bello,a
Clive Garnham,d Geert Bultynck,c Antony Galioned and Stuart J. Conway*b
Received 3rd August 2010, Accepted 24th September 2010
DOI: 10.1039/c0cc03003a
The syntheses of four D-myo-inositol 1,4,5-trisphosphate (InsP3)
derivatives, incorporating phosphate bioisosteres at the 5-position,
are reported. The methyl phosphate ester and sulfate derivatives
retain InsP3 receptor (InsP3R) agonist activity; the compounds
that possess a methylphosphonate or a carboxymethyl moiety
are InsP3R antagonists.
borate (2-APB), the most commonly used InsP3R antagonist,9
displays a number of biological actions, including inhibition of
SERCA pumps10 and inhibition of store operated Ca2+
entry.11,12
Most existing InsP3R antagonists are not structurally
related to InsP3; indeed, only one InsP3 derivative (3) has
been reported to act as an InsP3R antagonist and no biological
data were provided for this compound.7,8 However, it seems
probable that InsP3-based antagonists are most likely to be
selective for InsP3Rs over other cellular targets. The elucida-
tion of the X-ray crystal structure of the mouse type 1 InsP3R
in complex with InsP3 enables the rational design of InsP3R
ligands.13 This structure shows that InsP3 binds between two
flexible lobes, the a-domain and the b-domain, leading to
proposals of a ‘‘clam-shell-like’’ mode of receptor activation.14
The 1- and 5-position phosphates of InsP3 bind to the a-domain,
whereas the 4-position phosphate binds predominantly to the
b-domain. The structure confirms the findings of previous
structure–activity studies that the 4- and 5-position phosphate
groups are the most important for InsP3-receptor interactions.3
We previously hypothesised that reduction of charge at the
4-position, by introducing a phosphate isostere, would lead to
compounds that can bind to the InsP3Rs. The reduced charge
of these compounds would lead to them being unable to evoke
the conformational change required for receptor activation,
hence they would acts as competitive antagonists.15 None of
the 4-position-modified compounds synthesised exhibited
agonist or antagonist activity at InsP3Rs, leading to the
conclusion that the 4-position phosphate group is essential
for InsP3R binding.15 Furthermore, we hypothesised that the
conformational change undergone by InsP3Rs upon InsP3
binding is modest, and mediated by interaction of the 5-position
phosphate of InsP3 with its receptor. To test this hypothesis,
our attention turned to the development of InsP3 derivatives
that contain phosphate bioisosteres at the 5-position (Fig. 1).
Again, phosphate isosteres that possess reduced charge,
compared to a phosphate group, were utilised. However, more
polar isosteres than used previously have been employed, to
maximise the likelihood of these compounds binding to the
polar InsP3 binding pocket of the InsP3Rs.
D-myo-Inositol 1,4,5-trisphosphate (InsP3, 1, Fig. 1) is a
ubiquitous intracellular second messenger that mediates a
plethora of cellular functions via interaction with defined
receptors (InsP3Rs).1,2 These receptors are ligand-gated
Ca2+-releasing channels that are located predominantly on
the endo- or sarcoplasmic reticulum (ER/SR). The central
signalling role of InsP3 has promoted many chemical syntheses
of the natural compound and numerous unnatural derivatives.3,4
With only a few exceptions,5–8 these compounds have all
displayed full agonist activity at InsP3Rs. However, to enable
chemical dissection of the InsP3 signalling pathway, the
development of selective InsP3R antagonists is an essential
aim. Although some antagonists of this receptor do exist, these
compounds are not selective for the InsP3Rs over other Ca2+
channels and pumps. For example, 2-aminoethoxydiphenyl
Fig. 1 The structures of InsP3 (1) and its four 5-position derivatives,
2–5.
a School of Chemistry and Centre for Biomolecular Sciences,
University of St Andrews, North Haugh, St Andrews, Fife,
KY16 9ST, UK
b Department of Chemistry, Chemistry Research Laboratory,
University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.
E-mail: stuart.conway@chem.ox.ac.uk; Fax: +44 (0)1865 285002;
Tel: +44 (0)1865 285109
c Laboratory of Molecular Signalling, Campus Gasthuisberg O/N1,
K.U. Leuven, Herestraat 49, Bus 802, B-3000 Leuven, Belgium
d Department of Pharmacology, University of Oxford,
Mansfield Road, Oxford, OX1 3QT, UK
Herein, we report the synthesis of four 5-position-modified
InsP3 derivatives (Fig. 1). It is shown that, while compounds
with a methyl phosphate ester (2) or a sulfate group (5) at the
5-position retain InsP3R agonist activity, compounds with a
methylphosphonate (3) and a carboxymethyl (4) derivative act
as InsP3R antagonists.
w This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
z Electronic supplementary information (ESI) available: Additional
biological data, compound characterisation data and NMR spectra
for compounds 2–5. See DOI: 10.1039/c0cc03003a
y These authors contributed equally to this work.
The synthesis of compounds 3–5 commenced from
myo-inositol, and employed a well-established sequence of
c
242 Chem. Commun., 2011, 47, 242–244
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Scheme 3 The synthesis of the 5-position-modified InsP3 derivative 2.
Reagents and conditions: a. i. TBAF, THF, 89%; ii. 1. (BnO)2PNiPr2,
1H-tetrazole, CH2Cl2; 2. mCPBA, CH2Cl2, 89%; b. i. CAN,
MeCN, H2O, 75%; ii. 1. (BnO)(OCH3)PNiPr2, 1H-tetrazole, CH2Cl2;
Scheme 1 The synthesis of the key intermediate 9. Reagents and
conditions: a. i. AcCl, MeOH, CH2Cl2, 83%; ii. Bu2SnO, BnBr,
TBABr, 3 A molecular sieves, MeCN, 79%; b. i. 1. (BnO)2PNiPr2,
1H-tetrazole, CH2Cl2; 2. mCPBA, CH2Cl2, 81%; ii. PdCl2, MeOH,
80–84%.
t
2. mCPBA, CH2Cl2, 92%; c. Pd black, H2, BuOH, H2O, NaHCO3,
51%.
the TIPS group, using TBAF, furnished a 1,4-diol. This com-
pound was phosphitylated and oxidised to give the protected
bisphosphate 12 (Scheme 3). Oxidative cleavage of the PMB ether,
followed by phosphitylation and oxidation, gave the fully
protected methyl phosphate ester 13. Hydrogenolysis, as above,
furnished 2 as the presumed pentakis sodium salt.
transformations to afford the chiral, non-racemic, camphor
derivative 7 (Scheme 1).16,17 Removal of the chiral auxiliary
under acidic conditions followed by treatment with dibutyltin
oxide and benzyl bromide furnished the desired 3-O-benzyl
regioisomer (8) in high yield. Phosphitylation and oxidation of
the 1- and 4-position hydroxyl groups gave the protected
bisphosphate. The allyl group was removed by treatment with
PdCl2 in methanol, yielding the key intermediate 9. The
methyl phosphonate was installed using conditions reported
by Dreef et al. (Scheme 2).7,8 In our hands, this reaction was
capricious and the greatest success was achieved when using
freshly prepared reagents and rigorously dry conditions.
Hydrogenolysis in the presence of sodium bicarbonate
afforded the methyl phosphonate 3 as its presumed pentakis
sodium salt. The carboxymethyl derivative 4 was synthesised
by cleavage and oxidation of the allyl group,18,19 followed
by hydrogenolysis as before. The sulfate 5 was afforded by
reaction of 9 with sulfur trioxide in pyridine, followed
by hydrogenolysis.
The ability of 2–5 to mobilise Ca2+ or inhibit InsP3-induced
Ca2+ release (IICR) was investigated using either a unidirectional
45Ca2+ flux assay in permeabilised cells or a Ca2+ fluorescence
assay in sea urchin egg homogenate. In the 45Ca2+ assay, either
MEF cells or L15 cells were employed.z Using the 45Ca2+ assay
in MEF cells, the methyl phosphate ester (2) acts as an InsP3R
agonist, with an EC50 = 9.7 mM (InsP3 EC50 = 5.2 mM in the
same system, see ESIz). The sulfate 5 also acts as a weak InsP3R
agonist, evoking InsP3R-mediated Ca2+ release when applied at
100 and 300 mM in permeabilised L15 cells (see ESIz). The
methylphosphonate 3 showed little activity when applied to
permeabilised L15 cells alone. However, application of 3
(300 mM) before and during application of InsP3 (0.25 mM)
caused approximately 40% reduction in the amount of IICR
(Fig. 2).
The methyl phosphate ester 2 was synthesised from intermediate
11, which was prepared as described previously.20 Deprotection of
The biological activity of 3 and 4 was also investigated using
the sea urchin egg homogenate-based assay.21–238 In this assay
both 3 and 4 behaved as InsP3R antagonists (Fig. 3). The
methylphosphonate (3) gave 37% inhibition of IICR when
applied at a concentration of 1.67 mM. The carboxymethyl
derivative 4 demonstrated 58% inhibition of IICR when
applied at a concentration of 5.00 mM. In both cases, the
compounds evoked no Ca2+ release when applied in the
absence of InsP3. Additionally, preliminary data (not shown)
indicate that 4 has no significant effect on cyclic adenosine
diphosphate ribose-induced Ca2+ release, which occurs via
ryanodine receptors. These data indicate that 4 is acting at
InsP3Rs and not non-selectively at other Ca2+ channels or
pumps. Taken together, our data show that 3 and 4 are acting
as InsP3R antagonists. These data represent the first conclusive
proof that InsP3 derivatives can act as antagonists at InsP3Rs.
The structural similarity of the methyl phosphate ester 2 and
the methylphosphonate 3, which differ by one oxygen atom yet
possess opposing activity at InsP3Rs, potentially provides
unique insight into the mechanism of InsP3R activation and
inhibition. Our current hypothesis is that all compounds bind
Scheme 2 The synthesis of the 5-position-modified InsP3 derivatives
3–5. Reagents and conditions: a. i. 1. Bis[6-(trifluoromethyl)benzo-
triazol-1-yl] methylphosphonate, pyridine; 2. BnOH, 36–60%; ii. Pd
t
black, H2, BuOH, H2O, NaHCO3, 93%. b. i. RuCl3ꢀH2O, NaIO4,
t
CCl4, MeCN, H2O, 44%; ii. Pd black, H2, BuOH, H2O, NaHCO3,
t
98%. c. i. SO3, pyridine, 52%; ii. BuOH, H2O, NaHCO3, 94%.
c
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Chem. Commun., 2011, 47, 242–244 243

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In conclusion, four InsP3 derivatives with phosphate bio-
isosteres at the 5-position have been synthesised. Biological
evaluation of these compounds has revealed the methyl
phosphate ester (2) and sulfate (5) derivatives retain InsP3R
agonist activity. However, the methylphosphonate (3) and
carboxymethyl (4) derivatives behave as InsP3R antagonists.
These studies provide the first proof that InsP3 derivatives can
act as antagonists at InsP3Rs. The compounds developed
herein will likely prove useful tools for the study of InsP3Rs
and provide new insight into the mechanism of InsP3R
activation and inhibition.
The authors thank the University of St Andrews (DB) and
the BBSRC (NSK, TA) for funding. SJC thanks St Hugh’s
College, Oxford, for research support.
Fig. 2 Fractional loss plots of 45Ca2+ in L15 cells when treated with
the methylphosphonate analogue 3. The cells were permeabilised,
leading to an initial loss of 45Ca2+ (0–3 min). Application of InsP3
evokes a large loss of 45Ca2+ as a result of InsP3R activation. This loss
of 45Ca2+ is reduced by B40% in the presence of the methyl-
phosphonate 3 (300 mM), indicating that this compound is behaving
as an InsP3R antagonist.
Notes and references
z MEF cells are immortalised wild-type mouse embryonic fibroblast
cells expressing normal levels of InsP3R1 and InsP3R3. L15 cells are
mouse L-fibroblast cells stably over-expressing InsP3R1 by B8–10 fold.
8 Sea urchin eggs possess only one subtype of InsP3R.
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Fig. 3 Compounds 3 and 4 inhibit IICR. (A) A representative trace
obtained from sea urchin egg homogenate loaded with the fluorescent
Ca2+ dye Fluo-3. Application of InsP3 (400 nM) evokes an increase in
fluorescence due to Ca2+ release from InsP3Rs (black trace). Applica-
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however, application of InsP3 (400 nM) in the presence of 4 evokes
significantly reduced Ca2+ release, demonstrating that 4 is an InsP3R
antagonist (grey trace). (B) A comparison of the Ca2+ release evoked
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compounds unable to evoke the conformation change required
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