Both d - And l -Glucose Polyphosphates Mimic d - myo-Inositol 1,4,5-Trisphosphate: New Synthetic Agonists and Partial Agonists at the Ins(1,4,5)P3Receptor

Article abstract of DOI:10.1021/acs.jmedchem.0c00215

Chiral sugar derivatives are potential cyclitol surrogates of the Ca2+-mobilizing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Six novel polyphosphorylated analogues derived from both d- and l-glucose were synthesized. Binding to Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R] and the ability to release Ca2+ from intracellular stores via type 1 Ins(1,4,5)P3Rs were investigated. β-d-Glucopyranosyl 1,3,4-tris-phosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3, and α-d-glucopyranosyl 1,3,4-tris-phosphate are full agonists, being equipotent and 23-fold less potent than Ins(1,4,5)P3, respectively, in Ca2+-release assays and similar to Ins(1,4,5)P3 and 15-fold weaker in binding assays. They can be viewed as truncated analogues of adenophostin A and refine understanding of structure-activity relationships for this Ins(1,4,5)P3R agonist. l-Glucose-derived ligands, methyl α-l-glucopyranoside 2,3,6-trisphosphate and methyl α-l-glucopyranoside 2,4,6-trisphosphate, are also active, while their corresponding d-enantiomers, methyl α-d-glucopyranoside 2,3,6-trisphosphate and methyl α-d-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both l-glucose-derived ligands are partial agonists: they are among the least efficacious agonists of Ins(1,4,5)P3R yet identified, providing new leads for antagonist development.

Full text of DOI:10.1021/acs.jmedchem.0c00215

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Article
Both D- and L-glucose polyphosphates mimic D-myo-
inositol 1,4,5-trisphosphate: new synthetic agonists
and partial agonists at the Ins(1,4,5)P3 receptor
Megan Shipton, Andrew M. Riley, Ana M Rossi, Charles A. Brearley, Colin W. Taylor, and Barry V. L. Potter
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00215 • Publication Date (Web): 14 Apr 2020
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Both D- and L-glucose polyphosphates mimic D-myo-inositol 1,4,5-trisphosphate:
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new synthetic agonists and partial agonists at the Ins(1,4,5)P receptor
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Megan L. Shipton, Andrew M. Riley, Ana M. Rossi, Charles A. Brearley, Colin W.
‡
†
Taylor and Barry V. L. Potter*
†
Drug Discovery & Medicinal Chemistry, Department of Pharmacology, University of
Oxford, Mansfield Road, OX1 3QT, United Kingdom
‡Department of Pharmacology, Tennis Court Road, University of Cambridge, CB2 1PD,
United Kingdom.
§
School of Biological Sciences, University of East Anglia, Norwich Research Park,
Norwich NR4 7TJ, UK.
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ABSTRACT: Chiral sugar derivatives are potential cyclitol surrogates in the Ca -
mobilizing intracellular messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P ]. Six
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novel polyphosphorylated analogues derived from both D- and L-glucose were synthesized.
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Binding to Ins(1,4,5)P receptors [Ins(1,4,5)P R] and abilities to release Ca from
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intracellular stores via type 1 Ins(1,4,5)P Rs were investigated. β-D-Glucopyranosyl 1,3,4-
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trisphosphate, with similar phosphate regiochemistry and stereochemistry to Ins(1,4,5)P3
and α-D-glucopyranosyl 1,3,4-trisphosphate are full agonists, being equipotent and 23-fold
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less potent than Ins(1,4,5)P respectively in Ca -release assays, and similar to Ins(1,4,5)P
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and 15-fold weaker in binding assays. They can be viewed as truncated analogues of
adenophostin A and refine structure-activity relationship understanding for this
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Ins(1,4,5)P R agonist. L-Glucose-derived ligands, methyl α-L-glucopyranoside 2,3,6-
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trisphosphate and methyl α-L-glucopyranoside 2,4,6-trisphosphate are also active, while
their corresponding D-enantiomers, methyl α-D-glucopyranoside 2,3,6-trisphosphate and
methyl α-D-glucopyranoside 2,4,6-trisphosphate, are inactive. Interestingly, both L-
glucose-derived ligands are partial agonists: they are amongst the least efficacious agonists
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of Ins(1,4,5)P R yet identified, providing new leads for antagonist development.
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INTRODUCTION
D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P , 1] is a second messenger that binds
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to tetrameric D-myo-inositol 1,4,5-trisphosphate receptors [Ins(1,4,5)P Rs] on the
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endoplasmic reticulum. Ins(1,4,5)P Rs are Ca channels that open to release Ca to
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,2
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the cytosol. The resulting local or global increases in cytosolic Ca concentration
regulate diverse cellular processes, including mitochondrial metabolism, cell
proliferation, differentiation, smooth muscle contraction, secretion, exocytosis and
ion channel opening.3
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Ins(1,4,5)P (Figure 1a) binds to the Ins(1,4,5)P -binding core (IBC; residues 224-
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04) close to the N-terminus of each of the four Ins(1,4,5)P R subunits (Figure 1b,
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c). The IBC consists of an α-helical domain and a β-trefoil domain, between which
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there is a cleft rich in basic amino acid residues. Ins(1,4,5)P binding within the
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cleft allows phosphates at positions 1 and 5 to interact with the α-domain, while the
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4-phosphate interacts with the β-domain (Figure 1c). As Ins(1,4,5)P interacts with
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both domains, it pulls the two sides of the clam-like IBC together. The clam
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closure leads to channel opening, possibly by re-arranging Ca -binding sites such
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that Ca can bind to the Ins(1,4,5)P R and trigger conformational changes that lead
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
-domain
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Arg568
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P = phosphate
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Figure 1. Structures of Ins(1,4,5)P R and its agonists (a) Structure of Ins(1,4,5)P
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(1). (b) Binding mode of 1 to the IBC of Ins(1,4,5)P R with key amino acid residues
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involved in binding labelled. (c) Crystal structure of the IBC of type 1 InsP R with
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Ins(1,4,5)P (1) bound (PDB: 1N4K). The α-domain is shown in green and the β-
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domain in yellow. (d) Structure of the Ins(1,4,5)P R agonist adenophostin A (AdA).
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Modulators of Ins(1,4,5)P R activity are highly sought after, and many studies have
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examined structure-activity-relationship (SARs) of ligands binding to Ins(1,4,5)P R
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attempting to identify partial agonists or antagonists.8–10 Synthetic analogues have
played a key role in this process, including the potent glyconucleotide
adenophostins5,11 (Figure 1d), with a recent synthetic study reporting the effects of
replacing the glucose moiety of adenophostin A (AdA) with a chiro-inositol core.12
This highlights developing interest in examining less explored isomers of inositol
than the myo-form.13
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The phosphates attached to the 4- and 5-positions of Ins(1,4,5)P (Figure 1a) are
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thought to be essential to agonist activity as each interacts with a different domain
of the IBC. 4,14 The 1-phosphate increases affinity, but it is not essential for receptor
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activation. The hydroxyl group attached to the 6-position of Ins(1,4,5)P appears to
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be important for optimal activity but it is not essential, while hydroxyl groups
attached to the 2- and 3-positions are less involved in ligand binding.16
A few Ins(1,4,5)P R antagonists have been identified, but these suffer major
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drawbacks including poor target selectivity, cell impermeablility (heparin),17
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inconsistent effectiveness in assays (2-aminoethoxydipheylborane, 2-APB), low
potency (caffeine),17,18 and disputed activity (xestospongins).17 For decades,
analogues of Ins(1,4,5)P have been synthesized and investigated in attempts to
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discover a selective antagonist for Ins(1,4,5)P R that could be made, at least
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temporarily, cell-permeable with enzymatic- or photo-labile protecting groups.19,20
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Very recently, there has also been work carried out by Li et al. that describes the
delivery of inositol phosphates into cells via lysosomes rendering phosphate
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protecting groups unecessary. To date, however, only a small number of analogues
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of Ins(1,4,5)P with minor structural modifications have been identified as partial
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agonists or antagonists at Ins(1,4,5)P R. Most of these compounds demonstrate that
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conservative modifications to the phosphates attached to positions 4 and 5 and the
hydroxyl group attached to position 3 can lead to degrees of antagonist activity. 22–
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However, many analogues of Ins(1,4,5)P with modifications to the same regions
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are inactive. Attaching a bulky substituent to the axial 2-position hydroxyl can also
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lead to partial agonist activity and interestingly even using a simple benzene ring
as a surrogate for inositol in a benzene polyphosphate approach, as a dimer or
biphenyl, can provide low-affinity antagonists.28,29
Stimulated in part by the discovery of the adenophostins,5,10,30 there have been a
number of studies to investigate polyphosphates of D-glucose and of other sugars,30–
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as inositol phosphate analogues (see below). By using such carbohydrates, the
need for optical resolution of protected cyclitol precursors or resulting phosphate
regioisomers is bypassed, as chiral starting materials are readily available. Also, the
structural features of carbohydrates offer additional opportunities for synthetic
versatility. In this study, we return to and expand upon, the use of D-glucose to try
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to identify novel Ins(1,4,5)P R ligands. We also investigate, perhaps counter-
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intuitively, the use of L-glucose as a starting material.
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Although several studies have generated D-glucose-based ligands of
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Ins(1,4,5)P R,
no ligands based on the L-glucose enantiomer have been
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synthesized and nor is it known whether ligands with this scaffold would bind to
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Ins(1,4,5)P R. In a previous study, Ins(4,5)P [albeit a low-affinity Ins(1,4,5)P R
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ligand] was effectively mimicked by a D-Gluc(3,4)P surrogate. We noted that both
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D- and L-glucose offer three hydroxyl groups of the requisite relative configuration
that could in principle be used to mimic the 4,5,6-hydroxyl groups in myo-inositol.
Thus, we anticipated that we could use this similarity (Figure 2, highlighted in red),
alongside intrinsic structural differences of L- and D-glucose to prepare diverse chiral
ligands with appropriately located phosphates These would present different
structural motifs to the Ins(1,4,5)P R and allow further investigation of the binding
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site and perhaps identify novel activity. With this in mind we designed and
synthesized six novel ligands based on L-glucose and D-glucose (Figure 3) and
evaluated their activity at Ins(1,4,5)P R.
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Figure 2: Structures of methyl -D-glucopyranoside and methyl -L-
glucopyranoside relative to myo-inositol with the shared stereochemistry of the
hydroxyl groups highlighted in red.
D-glucose-based
L-glucose based
OH
O
OH
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OH
OH
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HO OH
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O
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HO
OH
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OH
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OH
O
OH
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R =H, R =P(O)(OH)
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4 R =H, R =P(O)(OH)
2
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R =P(O)(OH) , R =H 5 R =P(O)(OH) , R =H
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2
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Figure 3. Structures of Ins(1,4,5)P (1) analogues: methyl -L-glucopyranoside 2,3,6-
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trisphosphate (2) and methyl -L-glucopyranoside 2,4,6-trisphosphate (3), methyl -D-
glucopyranoside 2,3,6-trisphosphate (4) and methyl -D-glucopyranoside 2,4,6-
trisphosphate (5), -D-glucopyranosyl 1,3,4-trisphosphate (6), -D-glucopyranosyl 1,3,4-
trisphosphate (7).
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We ensured that the designed ligands retained structures equivalent to the critical 4,5-
bisphosphate motif of Ins(1,4,5)P and had no major structural modifications in regions
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believed to be necessary for ligand binding; modifications in regions equivalent to the 2-
O-position were permitted, as these were expected to remain outside the binding pocket.
We hypothesized that the L-glucose-based ligands [methyl -L-glucopyranoside 2,3,6-
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trisphosphate (2) and methyl -L-glucopyranoside 2,4,6-trisphosphate (3)] might have
sufficiently conservative structural changes relative to Ins(1,4,5)P that they could still
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bind to Ins(1,4,5)P R, possibly with novel activity. The D-glucose-based ligands [methyl
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-D-glucopyranoside 2,3,6-trisphosphate (4) and methyl -D-glucopyranoside 2,4,6-
trisphosphate (5)] were not expected to adopt orientations that position the phosphate
groups in appropriate regions of the IBC (see Supporting Information for details of all
possible predicted binding modes). Their bioassay was designed to enable confirmation of
enantioselectivity. From a practical standpoint, the synthesis of ligands 4 and 5 was
optimized first with D-glucose before the commercially available, but considerably more
costly, L-glucose was used to make the respective L-enantiomers, 2 and 3. We anticipated
that the two ligands with phosphates at the anomeric carbon, -D-glucopyranosyl 1,3,4-
trisphosphate (6) and -D-glucopyranosyl 1,3,4-trisphosphate (7), would help to elucidate
the basis for the high affinity of AdA.5,12,36 We expected both novel truncated analogues to
be agonists due to their structural similarity to Ins(1,4,5)P , but were interested to compare
3
their activities with AdA and the previously analysed truncated analogue, Glu(3,4)P2.5
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS
Chemistry
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Methyl -L-glucopyranoside 2,3,6-trisphosphate (2) was synthesized in a five-step route
from readily available L-glucose (Scheme 1). Refluxing L-glucose in an acidic methanol
solution resulted in protection of the anomeric hydroxyl with a methyl group. Multiple
recrystallizations from ethanol afforded methyl -L-glucopyranoside 8 in 45% yield. The
4
- and 6-position hydroxyls were protected with a benzylidene group to form 9 in 91%
yield. This benzylidene group was reduced regioselectively, opening to form methyl 4-O-
benzyl--D-glucopyranoside (10). The benzylidene reduction was first attempted with
borane-THF and AlCl , but this was found to be insufficiently regioselective and produced
3
inseparable regioisomers. The reaction was successfully carried out with borane-THF and
37
La(Tf) following the method of Shie et al. to yield 10 in 31% yield following
3
purification. The hydroxyl groups in triol 10 were then phosphitylated with dibenzyl
diisopropylphosphoramidite and subsequent oxidation with mCPBA formed 11 in 86%
yield. The benzyl protecting groups on phosphates and O-4 were then removed by stirring
a solution of 11 with Pearlman’s catalyst under hydrogen overnight. After filtration to
remove catalyst and evaporation of the solvent, 2 was collected as the triethylammonium
salt in 53% yield.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of L-glucose-derived ligands: methyl -L-glucopyranoside 2,3,6-
trisphosphate (2) and methyl -L-glucopyranoside 2,4,6-trisphosphate (3)a
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OH
OH
OH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
Ph
O
a
O
b
O
c
O
O
OH
OH
OH
OBn
OH
OH
OBn
OH
OH
OH
OH
O
O
O
OH
13
L-glucose
8
12
d
f
OH
O
g
O
Ph
O
OBn
O P OBn
OBn
OH
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O P OH
OH
OH
O
OH
O
O
O
O
O
OBn
OBn
O
P
10
OH
P
O
9
e
O
O
O
OH
OBn
O
d
O
O
HO
O
P O
P O
BnO
BnO
O
OH
OBn
O
O
3
P
P
O
14
OBn
O
O
O
O
O
O
OH
O
OBn
e
P
P
O
O
OH
OBn
OBn
O
O
O
P O
O
O
P O
OBn
HO
BnO
11
2
aReagents and conditions: (a) MeOH, reflux, 5d (b) (1) TMSCl, py, 22h (2) DCM,
.
benzaldehyde, FeCl 6H O, MeCN, triethylsilane, 0°C - room temperature, 1.5h (c)
2
2
i
MeOH, H O, 1M HCl , reflux, 3h (d) (1) DCM, 5-phenyl-1H-tetrazole, (BnO) PN( Pr) ,
2
(aq)
2
2
2
0h (2) mCPBA, -78°C - room temperature (e) MeOH:H O (10:1 v/v), cat. Pd(OH) /C,
2
2
H , 24h (f) MeCN, benzaldehyde dimethyl acetal, cat. CSA, 24h (g) BH -THF, La(Tf) ,
2
3
3
7d.
Methyl -L-glucopyranoside 2,4,6-trisphosphate (3) was also synthesized in five steps
from L-glucose, diverging from the synthesis of 2 after the initial methylation step. Methyl
α-L-glucopyranoside (8) was protected in a regioselective one-pot reaction that involved
persilylation followed by FeCl -catalyzed benzylidene protection as described by
2
3
8
Bourdreux et al. to yield 12 in 79% yield. The acid-labile benzylidene group was removed
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Journal of Medicinal Chemistry
through reflux with HCl(aq) to form 13 in 93% yield. Phosphorylation using standard
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
9
phosphoramidite methodology was then employed to give 14 in 40% yield. After
debenzylation with hydrogen and Pearlman’s catalyst and filtration and evaporation of the
solvent, the final product, 3, was collected as its triethylammonium salt in 90% yield.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of ligands with phosphate groups at the anomeric carbon: -D-
glucopyranosyl 1,3,4-trisphosphate (6) and -D-glucopyranosyl 1,3,4-trisphosphate (7)a
O
O
O
HO
BnO
BnO
O
BnO
P
HO
P
O
O
O
O
c
O
O
O
P
O
HO
P
O
O
BnO
OBn
HO
O
P
O
BnO
P
BnO
O
BnO
HO
OBn
BnO
17
a
HO
HO
O
b
6
HO
HO
O
BnO
OH
BnO
15
O
16
BnO
O
BnO
O
O
P
HO
BnO
O
P
O
O
O
O
HO
c
O
O
O
P
O
P
O
O
O
P
BnO
OBn
OBn
BnO
P
HO
BnO
OH
O
O
OH
18
7
a
Reagents and conditions: (a) MeOH, cat. PdCl , 6h (b) (1) DCM, 5-phenyl-1H-tetrazole,
2
i
(
BnO) PN( Pr) , 20h (2) mCPBA, -78°C - room temperature (c) MeOH:H O (10:1 v/v),
2
2
2
cat. Pd(OH) /C, H , NaHCO , 24h.
2
2
3
Both -D-glucopyranosyl 1,3,4-trisphosphate (6) and -D-glucopyranosyl 1,3,4-
trisphosphate (7) were synthesized via a divergent route, starting with allyl 2,6-di-O-Bn-
-D-glucopyranoside (15).34 Palladium chloride-catalyzed deallylation yielded 16,
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Journal of Medicinal Chemistry
Page 14 of 70
although purification of this compound was found to be very difficult at this step and
purification after phosphorylation proved to be much more effective. Thus, slightly impure
16, was phosphorylated to yield a pure, partially separable mixture of the epimeric
phosphates 17 and 18 (56% yield total: 14% 17, 19% 18, 23% mixed epimers). It was
unclear how stable 17 and especially the phosphorylated -epimer 18 would be, as there
are reports of compounds with phosphate groups at the anomeric carbon atom being unable
to survive purification by silica column chromatography in some cases and not in others.40–
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
We found in this case that both compounds survived silica gel chromatography, although
3
1
1
8 showed slight degradation by PNMR over time (approx. 30%. after three weeks at
C). Catalytic hydrogenolysis of both compounds 18 and 19 was carried out in the
4
presence of sodium bicarbonate to prevent acidic hydrolysis of the potentially labile C-1
phosphates. The final products 6 and 7 were purified by reverse phase ion pair
chromatography and collected as their triethylammonium salts.
Scheme 3: Synthesis of D-glucose-derived ligands: methyl -D-glucopyranoside 2,3,6-
trisphosphate (5) and methyl -D-glucopyranoside 2,4,6-trisphosphate (4)a
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Journal of Medicinal Chemistry
HO
HO
HO
1
2
a
Ph
BnO
O
c
HO
HO
O
b
O
HO
BnO
O
HO
HO
O
O
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HO
HO
HO
HO
O
O
O
OH
19
D-glucose
23
24
f
d
HO
g
Ph
O
BnO
HO
O
O
O
HO
OH
OBn
P OBn
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
HO
HO
O
P
O
O
BnO
2
1
OBn
20
O
OH
P
P
O
O
d
O
O
e
O
O
OH
P
HO
BnO
O
OBn
O
O
BnO
O
O
O
O
O
P
P
P
OH
OBn
OBn
25
O
O
e
HO
O
O
O
HO
O
O
BnO
O
O
BnO
BnO
P
O
5
O
P
O
O
P
O
P
O
OBn
OBn
OH
O
O
O
4
22
aReagents and conditions: (a) MeOH, reflux, 5d (b) (1) TMSCl, py, 22h (2) DCM,
.
benzaldehyde, Fe(II)Cl 6H O, MeCN, triethylsilane, 0°C - room temperature, 1.5h (c)
2
2
i
MeOH, H O, 1M HCl , reflux, 3h (d) (1) DCM, 5-phenyl-1H-tetrazole, (BnO) PN( Pr) ,
2
(aq)
2
2
20h (2) mCPBA, -78°C - room temperature (e) MeOH:H O (10:1 v/v), cat. Pd(OH) /C,
2
2
H , 24h (f) MeCN, benzaldehyde dimethyl acetal, cat. CSA, 24h (g) BH -THF, La(Tf) ,
2
3
3
7
d.
Methyl -D-glucopyranoside 2,3,6-trisphosphate (4) and methyl -D-glucopyranoside
,4,6-trisphosphate (5) were synthesized using the same methods as described for their
2
enantiomers (1 and 2) starting the route with D-glucose (Scheme 3).
The relative stabilities of α-D-glucopyranosyl 1,3,4-trisphosphate (6) and β-D-
glucopyranosyl 1,3,4-trisphosphate (7) were first investigated by allowing each compound
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Page 16 of 70
(
as triethylammonium salts) to remain in a solution of D O at room temperature at pH 7.
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Over the course of two months, neither isomer showed any sign of degradation. Following
this, a mixture of the compounds in a known starting ratio was exposed to increasingly
1
harsh conditions and relative isomer degradation was monitored through H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
spectroscopy (see Supporting Information Figs. S5 and 6). From the results of the
hydrolysis study, we determined that the β-epimer (7) degraded more readily than the
corresponding α-epimer (6). Both compounds were however surprisingly durable and
required strongly acidic conditions to be fully hydrolyzed (ie at pH1 for 1d), while strongly
basic conditions (ie pH 14 for 1d) only produced limited degradation (and at pH 10 for a
week there was no change). We are therefore confident that both compounds remained
intact during the near-neutral conditions of the biological assays. All final compounds were
assessed by HPLC for purity (Fig. S6) and products from acid hydrolysis were examined
5
by HPLC using a synthetic standard of D-glucose 3,4-bisphosphate to confirm the
expected hydrolysis product of both compounds (see Supporting Information Fig. S7).
Biology
Permeabilized HEK-Ins(1,4,5)P R1 cells were used to determine the ability of compounds
3
2
+
2
-7 (with Ins(1,4,5)P and AdA as controls) to evoke Ca release from intracellular stores
3
(Figure 4 and Table). Maximally effective concentrations of Ins(1,4,5)P , -D-glucopyran-
3
osyl 1,3,4-trisphosphate (6) or -D-glucopyranosyl 1,3,4-trisphosphate (7) released the
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Page 17 of 70
Journal of Medicinal Chemistry
2
+
same fraction (ca. 80%) of the intracellular Ca stores, suggesting that these two epimeric
compounds are both full agonists (Figure 4). Compound 7 was equipotent with Ins(1,4,5)P3
and 6 was ca. 20-times less potent than Ins(1,4,5)P3.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The L-glucose-based ligands methyl -L-glucopyranoside 2,3,6-trisphosphate (2) and
methyl -L-glucopyranoside 2,4,6-trisphosphate (3) were much less potent than
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ins(1,4,5)P (Figure 4), while their enantiomers methyl -D-glucopyranoside 2,3,6-
3
trisphosphate (4) and methyl -D-glucopyranoside 2,4,6-trisphosphate (5) were, as
2
+
predicted, inactive. The Ca release evoked by maximally effective concentrations of 2 or
was only ca. 70% of that evoked by Ins(1,4,5)P , suggesting that 2 and 3 are partial
3
3
agonists. Since partial agonists bind to Ins(1,4,5)P Rs, but activate them less effectively
3
than full agonists, a partial agonist must bind to more Ins(1,4,5)P Rs than a full agonist to
3
2
+
evoke comparable Ca release. We performed equilibrium competition binding assays
3
using [ H]-Ins(1,4,5)P and the active ligands to examine relationships between ligand
3
binding and functional responses. The affinities of 6 and 7 for Ins(1,4,5)P R aligned with
3
their potencies in functional assays, with 7 having an affinity indistinguishable from that
of Ins(1,4,5)P , while 6 had ca. 15-fold lower affinity (Figure 5, Table). The EC /K values
3
50
d
for Ins(1,4,5)P , AdA, 6 and 7 were similar, consistent with each being a full agonist
3
(
Table). Comparison of the concentrations of 2 and 3 required to occupy 50% of binding
2
+
2+
sites (K ) and to evoke release of 39% of the Ca stores (EC , i.e. the Ca release evoked
d
39
by a half-maximally effective Ins(1,4,5)P concentration) confirmed that 2 and 3 are weak
3
partial agonists: their EC /K values (132 and 462, respectively) were much greater than
3
9
d
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Journal of Medicinal Chemistry
Page 18 of 70
that of Ins(1,4,5)P (17). HPLC was used to confirm the purity of the compounds used in
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the biological assays (details in the Supporting Information).
Table 1. Receptor binding and Ca2+ Release by Ins(1,4,5)P R mediated by
3
a
Ins(1,4,5)P , AdA and compounds 2-7
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2+
Ca release
Binding
pKd
^
pEC50 Releas
h
h
EC /K
EC /K
50
d
39
d
e
EC50
K (nM)
d
(%)
6
.90 ± 78.8 ±
0.12 1.3
26nM
8.06 ±
0.03
14
Ins(1,4,5)P3
0.7 ±
0.1
1.1 ±
0.2
(5 - 46)
17
1
8.7
(5 - 63)
2
4.06 ± 56.2 ± 1.4 ±
5.91 ±
0.03
0.9 ±
0.1
71
132
*
*
*
0
.09
7.7µM
3.98 ± 53.1 ± 1.3 ±
2.6
0.2
(34 –
148)
191*
(49 –
355)
462*
8
1230
3
6.26 ±
0.9 ±
0.1
*
*
*
0
.04
5.0
0.2
ND
ND
0.07
(
123 –
295)
(128 –
1667)
104µM
549
4
5
6
ND
4.3 ±
48 ± 12$
ND
ND
ND
ND
23
#
2
.1
ND
5.9 ±
53 ± 3$
#
2
.1
5.53 ± 78.8 ± 0.9 ±
6.89 ±
0.7 ±
0.1
*
*
0
.20
.96µM
7.09 ± 75.2 ±
.18 1.4
0nM
3.0
0.2
0.09
(5 - 105)
2
129
7.95 ±
0.05
7
0
7
1.0 ±
0.1
1.1 ±
0.2
(2 - 29)
8
11.2
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Journal of Medicinal Chemistry
AdA
7.62 ± 77.8 ± 0.8 ±
8.86 ±
0.14
1.2 ±
0.2
17
1
2
3
4
5
6
7
*
*
0.12
4.5
0.1
(
5 – 51)
2
4nM
1.4
8
a
2+
Effects of ligands on Ca release from the intracellular stores of permeabilized HEK-
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Ins(1,4,5)P R1 cells and on [ H]-Ins(1,4,5)P binding to cerebellar membranes are
3
3
summarized. Results from functional assays are means ± SEM (pEC (-log of the half-
5
0
2
+
maximally effective concentration), Ca release (%) and Hill coefficient, h) and means
EC ) from 5-11 independent experiments, each performed in duplicate. Results from
(
5
0
binding experiments are means ± SEM (pK (-log of the equilibrium dissociation
d
constant) and h) and means (K ) from 3 independent experiments. The pK values for
d
d
4
3
Ins(1,4,5)P and AdA have been published (Mills et al.) and are reproduced with
3
permission. Final columns show EC /K or (for partial agonists and Ins(1,4,5)P )
5
0
d
3
*
EC /K (mean and 95% CI). ND, not determined. P < 0.05 relative to Ins(1,4,5)P .
3
9
d
3
#
$
^
2+
Ca release evoked by 300 µM of the ligand
3
Specific binding of [ H]-Ins(1,4,5)P in presence of 30µM of competing ligand
3
2+
EC reports the concentration of ligand required to evoke the same Ca release (39% of
3
9
the intracellular stores) as evoked by a half- maximally effective concentration of
Ins(1,4,5)P3.
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Page 20 of 70
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
00
Ins(1,4,5)P3
75
2
3
4
5
6
7
AdA
5
0
25
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
10
-8
-6
-4
log [ligand] (/M)
Figure 4. Concentration-dependent effects of Ins(1,4,5)P and related ligands on
3
2
+
Ca release from intracellular stores of permeabilized HEK-Ins(1,4,5)P R1 cells. Results
3
are mean ± SEM from 5-11 independent experiments, each with duplicate determinations.
1
00
Ins(1,4,5)P3
2
3
6
7
7
5
0
5
25
0
AdA
-12
-10
-8
-6
-4
log [ligand] (/M)
3
Figure 5. Equilibrium-competition binding to cerebellar membranes using [ H]-
Ins(1,4,5)P (1.5nM) and the indicated concentrations of competing ligands. Results are
3
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Journal of Medicinal Chemistry
means ± SEM from 3 independent experiments. The results for Ins(1,4,5)P and AdA have
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
3
been published (Mills et al.) .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DISCUSSION
Of the glucose polyphosphates considered in this study, the two that bound to Ins(1,4,5)P R
3
with highest affinity were -D-glucopyranosyl 1,3,4-trisphosphate (6) and -D-
glucopyranosyl 1,3,4-trisphosphate (7). Both compounds, which can be considered as
44
truncated analogues of adenophostin A (AdA, Figure 6), were found to be full agonists
of Ins(1,4,5)P R, and the -epimer (7) was equipotent with Ins(1,4,5)P .
3
3
Compounds containing a phosphate group attached to the anomeric carbon atom, as
featured in 6 and 7, have not been investigated as Ins(1,4,5)P R ligands, presumably due
3
3
1
to concerns over their stability, at least in the case of the β-epimer. Nevertheless, we
found that both α-D-glucopyranosyl 1,3,4-trisphosphate (6) and -D-glucopyranosyl 1,3,4-
trisphosphate (7) were surprisingly durable as their triethylammonium salts and neither
compound showed signs of degradation after two months in neutral aqueous solution at
room temperature (see Supporting Information for details). Both 6 and 7 were eventually
degraded under strongly acidic conditions, and HPLC traces were taken to confirm their
hydrolysis to glucose 3,4-bisphosphate (details in the Supporting Information).
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OH
OH
OH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
O
HO
P
O
P
O
O
P
O
O
O
P
O
O
P
P
5 HO 1
O
P
HO
P
OH
O
P
1
Ins(1,4,5)P
6
3
7
NH2
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
OH
HO
O
N
OH
OH
OH
4
''
P
O
O
P O
O
P
O
O
P
O
O
(CH )
O
P
O
P
2 n
O
2'
P
HO
(H C)
3''
OH
HO
P
OH
OH
O
P
O P
O
n
2
O P
4
Sureshan et al.
n= 1, 2, 3
Terauchi et al.31
n= 1, 2, 3
Terauchi et al.
31
AdA
Figure 6. Structural comparison of Ins(1,4,5)P (1), AdA and some of its truncated
3
analogues including -D-glucopyranosyl 1,3,4-trisphosphate (6) and -D-glucopyranosyl
1
,3,4-trisphosphate (7). The conserved regions of the structures involved in binding are
drawn in blue while the differing auxiliary phosphate is shown in red.
Previous studies using synthetic analogues of AdA30–34,45 demonstrated that the adenine
moiety significantly increases potency of the agonist, the vicinal phosphates are crucial to
activity, and minor adjustments to the placement of the auxiliary phosphate can be
tolerated.5,34 The general consensus for AdA binding has been that the ligand interacts with
the binding site of Ins(1,4,5)P R with the 3″, 4″ and 2′ phosphates mimicking the 4, 5 and
3
2
7,46
1
phosphates of Ins(1,4,5)P respectively.
However, these previous studies employed
3
analogues which differed from Ins(1,4,5)P in several ways and it has therefore been
3
difficult to isolate the specific impact of replacing the myo-inositol ring with D-
glucopyranose.
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Journal of Medicinal Chemistry
This has implications for the possible mode of action of AdA and related compounds.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
7
Indeed, a cryo-EM study of tetrameric Ins(1,4,5)P R1 has recently proposed that AdA
3
interacts with the IBC in a completely different way to Ins(1,4,5)P , with the two domains
3
of the IBC being pulled together by the 3″- and 4″-phosphate groups of AdA interacting
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
47
with one domain and the adenine moiety interacting with the other. In this model of AdA
binding to Ins(1,4,5)P R, the glucose bisphosphate structure of AdA only coincidentally
3
resembles the myo-inositol 4,5-bisphosphate of Ins(1,4,5)P and there is no structural
3
correspondence between the glucose ring of AdA and the inositol ring of Ins(1,4,5)P3.
However, this conclusion does not support the observed activities of the compounds in this
study and other AdA analogues; it should therefore be viewed with caution.12,47
In the present study, we found that the closest possible glucose-containing analogue of
Ins(1,4,5)P , namely compound 7, is effectively indistinguishable from Ins(1,4,5)P in our
3
3
2
+
assays of Ins(1,4,5)P R binding and Ca release. This is entirely consistent with the idea
3
that, in the Ins(1,4,5)P -binding site, the glucopyranoside ring of 7 is closely analogous to
3
the myo-inositol ring of Ins(1,4,5)P . In turn, this establishes that the flexible Ins(1,4,5)P -
3
3
binding site can accommodate the equatorial glucopyranoside hydroxymethyl (CH OH)
2
group and pyranoside ring oxygen in place of the myo-inositol 3-OH group and C-2,
respectively, with no impact on activity. The -epimer 6 is approximately 27-fold less
2
+
potent than -epimer 7 in Ca release. This shows that the axial phosphate group in 6 can
still contribute to binding [Gluc(3,4)P is much less potent] and is also consistent with an
2
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Journal of Medicinal Chemistry
Page 24 of 70
earlier report that D-chiro-Ins(1,3,4)P , the C-1 epimer of Ins(1,4,5)P having an axial 1-
3
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
phosphate group, had 25-fold lower potency than Ins(1,4,5)P3.35,48
Thus, the effects of trisphosphates 6 and 7 provide strong support for the argument that
compounds containing the D-glucopyranosyl 3,4-bisphosphate structure mimic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ins(1,4,5)P due to the direct structural analogy between glucopyranosyl and myo-inositol
3
rings depicted in Figures 2 and 6. In such compounds, it is highly likely that the glucose
3
,4-bisphosphate structure simply pulls together the two domains of the IBC in the manner
proposed for the inositol 4,5-bisphosphate motif of Ins(1,4,5)P . We recently reported
3
1
2
43
studies in which the glucose ring of AdA and ribophostin was replaced by D-chiro-
inositol, leading to both modest and significant increases in biological activity respectively.
It therefore remains to be conclusively established whether the additional components
present in the AdA molecule can, counter-intuitively, induce a completely unrelated role
for the glucose bisphosphate component of AdA itself, as suggested in the cryo-EM
study.47
Previous studies of C-glycosidic truncated analogues of AdA with different chain lengths
tethering the third, auxiliary phosphate group (Figure 6) have demonstrated that
positioning of this phosphate has a significant effect on the affinity of the agonist.30–34 It
2
+
has been observed that the Ca -releasing potency of these analogues at Ins(1,4,5)P R
3
decreases as the length and flexibility of the linkage to the auxiliary phosphate increases.34
The orientation of the linkage also plays a role, with axial linkages usually resulting in
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Journal of Medicinal Chemistry
more potent compounds. However, even the most potent of these compounds [Figure 6,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
Terauchi et al. axial linkage, n = 2] is still weaker than Ins(1,4,5)P and compound 7.
3
OH
HO
P
P
O
O
O
O
P
O
O
O
O
P
P
P
O
P
O
O
O
HO
HO O
P
P
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
1
2
Ins(1,4,5)P3
3
Figure 7. Ins(1,4,5)P (1) and analogues methyl α-L-glucopyranoside 2,3,6-trisphosphate
3
(2) and methyl α-L-glucopyranoside 2,4,6-trisphosphate (3) with their structural
differences contribute to their Ins(1,4,5)P R partial agonist activity in dark red.
3
Methyl -L-glucopyranoside 2,3,6-trisphosphate (2) was found to be a partial agonist of
Ins(1,4,5)P R1, while its D-glucose-based enantiomer, compound 4, was inactive. This
3
supports the structural alignment of trisphosphate 2 with Ins(1,4,5)P shown in Figure 7
3
and in our molecular modeling in Figure S3. No such alignment is possible for compound
4
because it does not possess a vicinal bisphosphate motif whose stereochemistry matches
that of Ins(1,4,5)P3.
In the predicted binding conformation of methyl α-L-glucopyranoside 2,3,6-trisphosphate
(2) (Figure S3), the axial methyl group is positioned in a region of the binding site normally
occupied by the 3-hydroxyl of Ins(1,4,5)P . In the design of 2, we further anticipated that
3
the phosphate group at C-6 of L-glucose would mimic the auxiliary 1-phosphate of
Ins(1,4,5)P to some extent as there is evidence from previous studies showing that the
3
Ins(1,4,5)P R can accommodate more sterically demanding groups in this region of the
3
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Journal of Medicinal Chemistry
Page 26 of 70
binding site.5,49–51 A very recent example of this is that replacement of the Ins(1,4,5)P 1-
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
phosphate by a pyrophosphate, which increases both charge and steric bulk, does not affect
3
7
activity. In addition, trisphosphate 2 contains an hydroxyl group appropriately placed to
mimic the important 6-OH group of Ins(1,4,5)P3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In studies of Ins(1,4,5)P analogues as partial agonists, it has been shown that perturbations
3
5
2
in the equivalent of the 3-hydroxyl of Ins(1,4,5)P can result in partial agonist activity,
3
especially when this disruption (often by means of stereochemical inversion) occurs in
conjunction with a modification to the vicinal phosphate pair or other region of the
ligand.18,22,25,52 It has been observed in multiple studies that limited, equatorial extension
of substituents from the 3 position equivalent can be tolerated,53–55 but larger groups hinder
binding56,57 and inversion of the 3-hydroxyl to axial results in a slight decrease in ligand
activity.22,58–60 Figure 7 therefore suggests that the axial O-methyl group is the most likely
component of 2 that causes it to display partial agonist activity, perhaps by interfering with
the ligand binding to the β-domain of the IBC or by reducing the extent of domain closure.
Methyl α-L-glucopyranoside 2,4,6-trisphosphate (3) was designed to bind to the
Ins(1,4,5)P R in a manner that would potentially satisfy the essential binding requirements
3
by positioning the pyranoside ring oxygen in place of the non-essential 3-OH group of
Ins(1,4,5)P while the axial 1-methoxy group occupied the place of the unimportant 2-OH
3
group of Ins(1,4,5)P (Figure 7 and S4). In this binding mode, the L-glucose 6-phosphate
3
group would enter the region of the binding site usually occupied by the 4-phosphate of
Ins(1,4,5)P3.
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Journal of Medicinal Chemistry
In previous studies, it has been shown that conservative modifications to the phosphates
attached to the 4 and 5 equivalent positions (and sometimes in conjunction with a
modification to the 3 position equivalent) can produce partial agonists and even low-
affinity antagonists.22–25,52,56,61 Bello et al. hypothesized that if a ligand could bind to only
one side of the IBC (through disruption of the interactions of either the 4 or 5 equivalent
phosphates), it would be unable to pull the clam-like structure of the binding site closed
and would therefore be unable to activate the receptor. Thus, a suitable modification to the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
26
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-phosphate of Ins(1,4,5)P might weaken the important interaction with the β-domain of
3
the clam-shell structure and induce sub-optimal activation of Ins(1,4,5)P R. However
3
previous studies attempting to generate partial agonists with modifications solely to the 4-
26
phosphate have failed to identify any active Ins(1,4,5)P analogues.
3
Pleasingly, our assays show that L-glucose trisphosphate 3 also behaves as a partial agonist
of Ins(1,4,5)P R1, with improved binding affinity and higher EC /K ratio than partial
3
50
d
agonist 2. The fact that the D-enantiomer 5 is inactive supports the structural alignment of
3 with Ins(1,4,5)P depicted in Figure 7. The "extended" 4-phosphate group equivalent in
3
3
may thus disrupt the interaction of the ligand with the -domain of the IBC as theorized.26
It is likely that the absence in 3 of an equivalent to the 3-OH group in Ins(1,4,5)P also
3
contributes to a decreased interaction between the -domain of the IBC and the ligand.
Indeed, activity for 3-deoxy-Ins(1,4,5)P at Ins(1,4,5)P R has been reported to drop to up
3
3
to 40 fold.10
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The two partial agonists, -L-glucopyranoside 2,3,6-trisphosphate (2) and -L-
glucopyranoside 2,4,6-trisphosphate (3), indicate that perturbations of the ring structure of
the ligand are sufficient to induce partial agonism. Both ligands suffer from low affinity
and as a result, structurally related compounds are currently being developed that will
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
incorporate similar structural differences to Ins(1,4,5)P and hopefully maintain the desired
3
decreased efficacy while increasing affinity. The most promising avenue seems to be
adapting the structure of 3 by generating other ligands with extended 4-position phosphate
equivalents. This could hypothetically be continued with L-glucose, but inositol could also
prove to be a useful starting material as ligands could be synthesized with a similar
extension of the 4-position hydroxyl without loss of an equivalent hydroxyl to position 3
in Ins(1,4,5)P , perhaps thereby improving ligand affinity while retaining partial agonist
3
activity. Such work is in progress.
CONCLUSIONS
We have synthesized four novel active ligands for the Ins(1,4,5)P R based on both D-
3
glucose and L-glucose templates as inositol surrogates. The two ligands based on L-
glucose, namely methyl α-L-glucopyranoside 2,3,6-trisphosphate (2) and methyl α-L-
glucopyranoside 2,4,6-trisphosphate (3), are low-affinity, low-efficacy partial agonists of
Ins(1,4,5)P R, while their respective D-glucose-based enantiomers 4 and 5 are inactive.
3
Two further synthetic D-glucose-based trisphosphates, -D-glucopyranosyl 1,3,4-
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Page 29 of 70
Journal of Medicinal Chemistry
trisphosphate (6) and -D-glucopyranosyl 1,3,4-trisphosphate (7) can be regarded as close
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
analogues of Ins(1,4,5)P but they are also related structurally to the naturally-occurring
3
glyconucleotide Ins(1,4,5)P R agonist adenophostin A (AdA). They can therefore further
3
our understanding of how AdA binds to Ins(1,4,5)P R. Both 6 and 7 were found to be full
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
agonists of Ins(1,4,5)P R, with the perhaps surprisingly stable -epimer 7 being equipotent
3
to Ins(1,4,5)P itself and potentially useful as a chemical biology tool under physiological
3
conditions (with degradation induced under extremes of pH). The potency of 7
demonstrates that the structural differences between myo-inositol and D-glucose need not
result in any decrease in ligand activity. This is consistent with the D-glucopyranosyl 3,4-
bisphosphate moiety of AdA directly mimicking the D-myo-inositol 4,5-bisphosphosphate
structure of Ins(1,4,5)P at the binding site of Ins(1,4,5)P Rs.
3
3
Partial agonists 2 and 3 are the first L-glucose-derived ligands that have been synthesized
for Ins(1,4,5)P R. Both compounds provide evidence for the viability of generating partial
3
agonists and potential antagonists of Ins(1,4,5)P R by deliberately disrupting the crucial
3
moieties involved in binding to the IBC clam shell and pulling the domains together upon
ligand binding. We hypothesize that the axial O-methyl group of compound 2 and the
extended phosphate in the equivalent of the 4-position phosphate in Ins(1,4,5)P of
3
compound 3 cause the partial agonist activity of these compounds by either disrupting the
interactions of the ligand with the β domain of the IBC or by preventing complete closure
of the IBC upon binding. These partial agonists could prove to be interesting starting points
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