A disaccharide polyphosphate mimic of 1D-myo-inositol 1,4,5-trisphosphate

Article abstract of DOI:10.1039/a608208d

A concise route from D-glucose and D-ribose to a potent sugar polyphosphate second messenger mimic related to adenophostin A is described; a role for the adenine base of the adenophostins is suggested.

Full text of DOI:10.1039/a608208d

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A disaccharide polyphosphate mimic of 1d-myo-inositol 1,4,5-trisphosphate
David J. Jenkins, Rachel D. Marwood and Barry V. L. Potter*†
Department of Medicinal Chemistry, School of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath,
UK BA2 7AY
A concise route from D-glucose and D-ribose to a potent sugar
polyphosphate second messenger mimic related to adeno-
phostin A is described; a role for the adenine base of the
adenophostins is suggested.
which should clarify the relative importance of conformational
restriction of this phosphate for the potency of 2a.
2,6-Di-O-benzyl-3,4-di-O-(p-methoxybenzyl)-d-glucopyra-
nose 5 was prepared in five steps from d-glucose.8b Reaction of
10
5 with Cl3CCN in the presence of K2CO3 gave the
1d-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3, 1] is a sec-
ond messenger responsible for increasing the intracellular Ca2+
concentration in stimulated cells.1 Structure–activity studies
now allow a good understanding of the relationship between 1
and its receptor.2 A specific vicinal d-threo bisphosphate is
essential for activity, while the third phosphate and 6-hydroxy
group help to enhance binding affinity.
a-trichloroacetimidate 6 ([a]D +13.6) and the crystalline
b-anomer 7 (mp 80–81 °C; [a]D +16.5), separated by flash
chromatography, in the ratio of 1:2.5, based on isolated
products.
With a suitable glycosyl donor in hand, we required 8 as an
acceptor. d-Ribose was converted into known11 9. Reaction of
9 with 1.05 equiv. of p-methoxybenzaldehyde dimethyl
Most active inositol polyphosphates and related compounds
tested at the Ins(1,4,5)P3 receptor exhibit potencies comparable
to or less than that of 1.2 However, in 1993 a Japanese group
reported the isolation of two potently agonistic trisphosphate
glyconucleotides from culture broths of Penicillium brevi-
compactum,3 named adenophostins A and B and identified as 2a
and 2b, respectively.4 The structure of 2a has now been
confirmed by total synthesis.5 Both adenophostins are effective
at concentrations 10–100-fold lower than for 1; these relative
potencies are consistent with binding data.6,7 Thus, 2a and 2b
are by far the most potent agonists yet described at the
Ins(1,4,5)P3 receptor, despite their structural dissimilarity to 1,
and are attractive leads to carbohydrate-based Ins(1,4,5)P3
mimics and receptor modulators.
To elucidate the structural motifs of the adenophostins
responsible for their activity we8 and others9 prepared the
minimal structure 3, which exhibited a potency 10-fold lower
than 1, demonstrating that while the glucose bisphosphate
contained the pharmacophore responsible for Ca2+ release, at
least part of the adenosine moiety was responsible for the high
activity of the adenophostins. Molecular modelling studies on 3
established9 that the staggered conformation of its bimethylene
chain did not allow the 2A-phosphate to mimic the corresponding
phosphate of either 1 or 2a. We report here the synthesis of
methyl 3-O-(a-d-glucopyranosyl)-b-d-ribofuranoside 2,3A,4A-
trisphosphate 4, in which the adenine ring of 2a has effectively
been deleted, but the third phosphate is held similarly to 2a, and
D-Glucose
5 steps
D-Ribose
ii
BnO
OMe
OBn
O
O
PMBO
PMBO
BnO
5
R2O OR1
9 R1 = R2 = R3 = H
10 R1, R2 = p-methoxybenzylidene;
R3 = H
11 R1, R2 = p-methoxybenzylidene;
R3 = Bn
12 R1 = H; R2 = PMB; R3 = Bn (33%)
13 R1 = PMB; R2 = Ac; R3 = Bn
14 R1 = Ac; R2 = PM B; R3 = Bn
15 R1 = H; R2 = allyl; R3 = Bn
16 R1 = allyl; R2 = H; R3 = Bn
17 R1 = R2 = H; R3 = Bn
OH
iii
iv
i
v
vi
OBn
O
PMBO
PMBO
BnO
O
CCl3
NH
+
6
+
BnO
OMe
O
OBn
O
PMBO
PMBO
O
CCl3
NH
BnO
HO OPMB
8 (43%)
7
vii
BnO
OMe
O
BnO
BnO
OBn
OMe
O
+
O
O
RO
RO
RO
RO
O
OR
BnO
BnO
O
OR
18 R = PMB
20 R = H
22 R = P(O)(OBn)2
19 R = PMB
21 R = H
viii
ix
viii
NH2
N
x
N
OH
HO
O
HO
O
OR
OH
OH
OMe
N
N
HO
O
O
2–O3PO
2–O3PO
2–O3PO
2–O3PO
2–O3PO
2–O3PO
2–
OPO3
OH
2–
O
OPO3
HO
2–
4
O
OPO3
PMB = p-MeOC6H4CH2
Ins(1,4,5)P3 1
2a R = H
b R = COMe
Scheme 1 Reagents and conditions: i, Cl3CCN, K2CO3, CH2Cl2, N2, room
temp. 2 h, 65%; ii, MeOH, H2SO4, room temp., 20 h (b-anomer obtained by
crystallisation); iii, p-MeOC6H4CH(OMe)2, PTSA, DMF, 70 °C, 2MeOH,
4 h, 93%; iv, NaH, BnBr, DMF, 3 h, 82%; v, DIBAL-H, CH2Cl2, room
temp., 3 h, 76%; vi, DDQ, CH2Cl2, 3 Å sieves, room temp., 3 h, 71%; vii,
Me3SiOSO2CF3, Et2O, 3 Å sieves, room temp., 10 min; viii, DDQ, CH2Cl2–
H2O (10:1), room temp., 1 h, 56%; ix, (BnO)2PNPri2, 1H-tetrazole, room
temp., 30 min, then MCPBA, 278 °C to room temp., 10 min, 82%; x, H2,
Pd–C, 40 psi, MeOH–H2O 4:1, room temp., 18 h, 70%
OH
O
HO
O
OH
OMe
O
2–O3PO
2–O3PO
2–O3PO
2–O3PO
HO
O
HO
2–
O
OPO3
2–
OPO3
3
4
Chem. Commun., 1997
449

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acetal,12 with continuous removal of the liberated MeOH via an
air condenser,13 gave the 2,3-O-(p-methoxybenzylidene) deri-
vative 10 ([a]D 243.0) as a ca. 3:2 diastereoisomeric mixture,
as judged by NMR. Benzylation of 10 gave 11 ([a]D 227.2).
Cleavage of the acetal with LiAlH4–AlCl3 in refluxing THF,14
Footnotes
† E-mail: B.V.L.Potter@bath.ac.uk
‡ Spectroscopic data for compound 20: dH (400 MHz; CDCl3; J/Hz) 1.69
(1 H, br s, exch. D2O, OH), 2.74, 2.87 (2 H, 2 br s, exch. D2O, 2 3 OH), 3.32
(3 H, s, OCH3), 3.38 (1 H, dd, J 3.4, 9.8, 2A-H), 3.45–3.57 (5 H, m, 4A-H,
5-Ha, 5-Hb, 6A-Ha, 6A-Hb), 3.74 (1 H, dt, J 3.9, 9.8, 5A-H), 3.92 (1 H, t, J 9.3,
3A-H), 4.01 (1 H, m, 3-H), 4.22 (2 H m, 2-H, 4-H), 4.44, 4.52 (2 H, AB, JAB
12.2, CH2Ph), 4.51 (2 H, s, CH2Ph), 4.69, 4.74 (2 H, AB, JAB 11.7, CH2Ph),
4.69 (1 H, d, J 3.4, 1A-H), 4.88 (1 H, s, 1-H), 7.23–7.36 (15 H, m, 3 3 Ph);
dC(100.4 MHz; CDCl3) 55.03 (q, OCH3), 69.00 (t, C-5 or C-6A), 70.74 (d,
C-4A), 70.84 (d, C-5A), 71.69 (t, C-5 or C-6A), 73.24 (d, C-3A), 73.28 (d, C-3),
73.32, 73.55, 74.14 (3 t, CH2Ph), 78.35 (d, C-2A), 79.11 (d, C-2 or C-4),
80.27 (d, C-2 or C-4), 97.79 (d, C-1A), 108.34 (d, C-1), 127.57, 127.63,
127.69, 127.74, 128.33, 128.42, 128.51, 128.75 (8 d, Ph), 137.18, 137.86,
138.06 (3 s, 3 3 C-1 of phenyl ring); m/z (FAB+) 597 [(M + 1)+, 12%], 565
[(M 2 OCH3)+, 48], 343 [(M 2 C13H18O5)+, 3], 255 [(M 2 C20H24O5)+, 2];
m/z (FAB2) 595 [(M 2 1)2, 28%], 505 [(M 2 C7H7)2, 15], 253 [(M 2
C20H24O5)2, 25].
§ Spectroscopic data for compound 4 (triethylammonium salt): dH(400
MHz; CD3OD; J/Hz) 3.35 (3 H, s, OCH3), 3.54 (1 H, ABX, 2JAB 11.9, 3J
6.4, 5-Ha), 3.62 (1 H, dd, J 3.8, 9.6, 2A-H). 3.66 (1 H, br s, OH), 3.69–3.73
(3 H, m, 5-Hb, 5A-H, 6A-Ha), 3.93 (1 H, ABX, JAB 13.0, 3J 3.5, 6A-Hb),
4.04–4.11 (2 H, m, 4-H, 4A-H), 4.44 (1 H, dd, J 4.3, 7.3, 3-H), 4.45 (1 H, q,
J3-H, 2-H = J3-H, 4-H = JHP = 9, 3A-H), 4.58 (1 H, dd, J 4.3, JHP 9.5, 2-H),
4.94 (1 H, s, 1-H), 5.03 (2 H, br s, 2 3 OH), 5.13 (1 H, d, J 3.7, 1A-H);
dC(100.4 MHz; CD3OD) 55.18 (q, OCH3), 61.98, 64.87 (2 t, C-5, C-6A),
73.32, 73.52, 73.78, 76.29, 76.46 (with C–P coupling), 78.83 (6 d, C-2, C-3,
15
NaCNBH3–Me3SiCl in MeCN12 or DIBAL-H in CH2Cl2 all
gave the required 8 (mp 42–43 °C; [a]D +34.6), and the more
polar 12 ([a]D 228.8), in approximately equal proportions, the
latter reagent giving by far the best yield. The structures of 8 and
12 were confirmed by preparation of acetates 13 ([a]D +17.5)
and 14 ([a]D +21.5), the 1H NMR spectra of which respectively
revealed a triplet at d 5.13 (J 5.3 Hz) corresponding to H-3, and
a doublet at d 5.18 (J 4.4 Hz; H-1 presented as a singlet)
corresponding to H-2. Although the regioselectivity of acetal
cleavage was disappointing, isomer 12 was easily reoxidised to
11 (as a 92:8 diastereoisomeric mixture, [a]D 226.4; diaster-
eoisomers not assigned) using DDQ in dry CH2Cl2.16
A preparation of the related allyl-protected ribosides 15 and
16 by a different route has recently been reported.17 Anomerisa-
tion of methyl 5-O-benzyl-2,3-O-isopropylidene-b-d-ribofur-
anoside on acidic hydrolysis, to give a ca. 1:4 a:b-anomeric
mixture of products was described. We found that the more
labile p-methoxybenzylidene acetal of 11 could be removed
without anomerisation by treatment with 80% (v/v) aqueous
acetic acid at 60 °C for 25 min, to give 17 ([a]D 242.0; lit.17
247.7). Therefore, 10 may be a more suitable intermediate than
methyl 2,3-O-isopropylidene-b-d-ribofuranoside to prepare
derivatives of methyl b-d-ribofuranoside substituted at position
5.
2
3
C-2A–C-5A), 82.71 (d, C-4), 98.93 (d, C-1A), 108.96 (dd, JCP 3.7, C-1);
dP(161.7 MHz; CD3OD) 20.38, 1.05, 1.10 (3s); m/z (FAB2) 565 [M2,
100%] (Found: M2, 565.012. C12H24O19P3 requires, 565.012).
Coupling of 7 and 8 was achieved using Me3SiOSO2CF3 as
promoter.10 The 1H NMR spectrum indicated that although the
a-glucopyranosyl compound 18 was the major product (H-1A, d
5.09, J 3.4 Hz), the anomer 19 was present as a ca. 20%
contaminant which could not be removed at this stage.
However, on treatment with DDQ, the required triol 20‡ (mp
103–105 °C; [a]D +28.3) could be separated from 21 by column
chromatography. Phosphitylation of 20 followed by oxidation
gave the trisphosphate 22 ([a]D +34.7). Compound 22 was
hydrogenolysed to give the required trisphosphate 4§ which was
purified on Q Sepharose resin eluting with a 0–1 mol dm23
gradient of triethylammonium hydrogen carbonate, pH 7.5. The
triethylammonium salt of 4 eluted at ca. 800–850 mmol dm23
buffer. As this form was surprisingly poorly soluble in water, it
was converted to the freely soluble hexapotassium salt ([a]D
+67.3 calc. for free acid, H2O, pH 8.5) before quantification by
total phosphate assay18 and biological evaluation.
Preliminary biological evaluation of ‘ribophostin’ 4 using
permeabilised hepatocytes19 revealed a Ca2+-mobilising po-
tency 10-fold better than 3 and very close to that of Ins(1,4,5)P3.
Full biological characterisation will be reported elsewhere.
Noting that 2A-dephosphorylation of 2a reduces its binding
affinity 1000-fold,4 this suggests that conformational restriction
of the 2A-phosphate alone can engender Ins(1,4,5)P3-like, but
not adenophostin-like, potency. Therefore the adenine base of
adenophostin plays an important, but as yet undefined, role in
enhancing activity. We believe that it probably contributes to
the positioning of the ribose phosphate such that it can mimic
the 1-phosphate of Ins(1,4,5)P3 in a unique way. Further
clarification must now await the synthesis of suitably conforma-
tionally restricted compounds for accurate positioning of the
2A-phosphate group.
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We thank Dr H. Dietrich for discussions, Dr C. Taylor for
preliminary biological evaluations, the Wellcome Trust for a
Prize Studentship (R.D.M.) and the BBSRC (Intracellular
Signalling Programme) for financial support.
Received, 5th December 1996; Com. 6/08208D
450
Chem. Commun., 1997