2988 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Liu et al.
ceuticals Ltd. TLC was performed on precoated plates (Merck
TLC aluminum sheets silica 60F254). Spots were visualized by
spraying of plates with phosphomolybdic acid in methanol
followed by heating. Flash chromatography was carried out
using Sorbsil C60 silica gel. NMR spectra were recorded with
a J EOL GX-270 or EX-400 spectrometer. 1H chemical shifts
were measured in ppm relative to tetramethylsilane (TMS),
and signals are expressed as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), or br (broad).31P chemical shifts were
measured in ppm relative to external 85% H3PO4 and are
positive when downfield from this reference. Melting points
(uncorrected) were determined using a Reichert-J ung Thermo
Galen Kofler Block. Microanalysis was carried out by the
University of Bath microanalysis service. Optical rotations
were measured using an Optical Activity Ltd. AA-10 polarim-
eter, and [R]D values are given in 10-1 deg cm2 g-1. Mass
spectra were recorded at the Mass Spectrometry Service of the
University of Bath. Ion-exchange chromatography was per-
formed on a LKB-Pharmacia medium-pressure ion-exchange
chromatograph using Q Sepharose Fast Flow with gradients
of triethylammonium hydrogen carbonate (TEAB) as eluent.
Column fractions containing inositol polyphosphate analogues
were assayed for total phosphate content by a modification of
the Briggs test.27
then H2O. The organic phase was dried (MgSO4) and concen-
trated, followed by flash chromatography to give the fully
protected di-O-benzyl ether 6 (1.13 g, 96%); mp: 120-124 °C
(from methanol/water); [R]D ) -160 (CHCl3, c ) 0.2) δH (400
MHz, CDCl3) 1.27, 1.28 (12 H, 2 s, 4 × CH3), 3.18, 3.27 (12 H,
2 s, 4 × OCH3), 3.66-3.67 (2 H, m, H-1 and H-6), 3.90-3.93
(2 H, m, 2 × CH), 4.02-4.05 (2 H, m, 2 × CH), 4.46 and 4.83
(4 H, AB, J AB)12.2 Hz, 2 × CH2), 7.21-7.51 (10 H, m, ArH);
m/z (+ve ion FAB) 587 [(M+ - H•) 100%], 557 (7), 101.0 (40),
91.0 (100); Anal. (C32H44O10) C, H.
L-(2′S,3′S)-1,6-Di-O-ben zyl-bis-O-2,3;4,5-(2′,3′-dim eth oxy-
bu ta n e-2′3′-d iyl)-ch ir o-in ositol (en t-6) was obtained in a
fashion identical to that described for 6; mp 117-120 °C (from
methanol/water); [R]D ) +152 (CHCl3, c ) 5); spectroscopic
data were identical to those obtained for 6; Anal. (C32H44O10
)
C, H.
D-1,6-Di-O-ben zyl-ch ir o-in ositol (7). To 6 (1.4 g, 2.4
mmol) was added a mixture of trifluoroacetic acid (20 mL) and
water (20 mL). The mixture was stirred at room temperature
for 20 min and then evaporated to dryness in vacuo to give a
white solid, which was recrystallized from ethyl acetate to give
tetraol 7 (0.73 g, 84%); mp: 174-175 °C (from ethyl acetate);
[R]D ) -24 (MeOH, c ) 0.4); δH (400 MHz, DMSO d6) 3.34-
3.36 (2 H, m, 2 × CH), 3.53-3.56 (2 H, m, 2 × CH), 3.71-3.72
(2 H, m, H-1 and H-6), 4.56 and 4.69 (4 H, AB, J AB ) 12.2 Hz,
2 × CH2), 4.55-4.65 (4 H, br, partially buried, D2O exch., 4 ×
OH), 7.25-7.35 (10 H, m, ArH); m/z (+ve ion FAB) 359 [(M+
-H•) 20% ], 721 (75), 383 (28), 181 (28), 91 (100); Acc. Mass
(C20H23O6).
L-1,6-Di-O-ben zyl-ch ir o-in ositol (en t-7) was obtained in
a fashion identical to that described for 13; mp 172-173 °C
(from ethyl acetate); [R]D ) +26 (MeOH, c ) 0.3); spectroscopic
data were identical to those obtained for 7; Acc. Mass (C20H23O6).
D-1,6-Di-O-b en zyl-ch ir o-in osit ol 2,3,4,5-t et r a k is(O,O-
d iben zyl p h osp h a te) (8). The tetraol 7 (0.18 g, 0.50 mmol)
and 1H-tetrazole (0.35 g, 5.0 mmol) in dry dichloromethane
(10 mL) were stirred at room temperature for 10 min, and then
bis(benzyloxy)(diisopropylamino)phosphine (0.83 g, 2.4 mmol)
was added. The mixture was stirred for 1 h and cooled to 0
°C, followed by addition of m-chloroperoxybenzoic acid (60%,
1.0 g, 3.5 mmol). After further stirring for 30 min, the mixture
was diluted with ethyl acetate (50 mL) and washed with 10%
sodium metabisulfite (50 mL), aqueous NaHCO3, brine, and
water (50 mL of each). The organic phase was dried over
magnesium sulfate and evaporated to dryness. The residue
was chromatographed on silica gel to give compound 8 (0.59
g, 84%) as an oil; [R]D ) + 4.7 (CHCl3, c ) 0.6); δH (400 MHz,
CDCl3) 4.13 (2 H, br s, H-1 and H-6), 4.35 and 4.44 (4 H, AB,
J AB ) 11.7 Hz, 2 × CH2), 4.55-4.65 (2 H, br m, 2 × CH), 4.86-
5.01 (18 H, m, 8 × CH2 and 2 × CH), 6.92-7.31 (50 H, m,
ArH); δP (160 MHz, CDCl3), 1H-decoupled) -2.23 (2 P, s), -1.36
(2 P, s); m/z (+ve ion FAB) 1401 [(M + H)+ 10%], 271 (8), 181
(12), 91 (100); Acc. Mass (C76H77O18P4).
Biology. The activity of highly purified Ins(3,4,5,6)P4
1-kinase from bovine aorta was assayed as described previ-
ously.8 Aliquots of the purified enzyme (0.7 µg) were incubated
at 37 °C, with approximately 6000 dpm of 0.3 µM [3H]Ins-
(3,4,5,6)P4, plus the concentration of D- or L-chiro-Ins(2,3,4,5)-
P4 indicated in the figure, in a final volume of 100 µL buffer
containing 20 mM HEPES (pH 7.2), 6 mM MgSO4, 0.4 mg/mL
saponin, 100 mM KCl, 0.3 mg/mL bovine serum albumin, 5
mM ATP, 10 mM phosphocreatine, and 2.5 Sigma units of
phosphocreatine kinase. The reaction was stopped after 30 min
by adding 1 mL of ice-cold quenching buffer, which contained
1 mg/mL InsP6, 0.2 M ammonium formate, and 0.1 M formic
acid. The amount of Ins(1,3,4,5,6)P5 formed was analyzed by
chromatography on gravity-fed anion exchange columns. The
experiments were repeated three times for the analogues (3
and en t-3); data for a single inhibition curve for Ins(1,3,4)P3
for comparison with previous data only are presented.
D-(2′R,3′R)-2,3;4,5-Bis-O-(2′,3′-d im et h oxyb u t a n e-2′3′-
d iyl)-ch ir o-in ositol (5). To a stirred suspension of D-chiro-
inositol (4) (5.00 g, 27.7 mmol) in dry methanol (90 mL) were
added trimethyl orthoformate (24 mL, 220 mmol), butanedione
(5.8 mL, 66 mmol), and Et2O‚BF3 (0.4 mL, 3 mmol). The
mixture was stirred at room temperature for 30 h. The solvent
was evaporated, and the residue was partitioned between CH2-
Cl2 (100 mL) and H2O (100 mL). The organic phase was dried
over MgSO4 and evaporated to dryness. Purification of the
residue by flash chromatography on silica gel gave the bisac-
etal 5 (11.0 g, 97%); mp 103-120 °C (from diisopropyl ether);
[R]D ) -151 (CH2Cl2, c ) 1); δH (400 MHz, DMSO d6) 1.16,
1.20 (12 H, 2 s, 4 × CH3), 3.12, 3.14 (12 H, 2 s, 4 × OCH3),
3.64-3.75 (6 H, m, 6 × CH), 5.13 (2 H, d, J ) 3.4 Hz, D2O
exch., 2 × OH); δC (100 MHz, DMSO d6) 17.63 (2 × CH3), 17.68
(2 × CH3), 47.09 (2 × OCH3), 47.13 (2 × OCH3), 66.33 (2 ×
CH), 67.55 (2 × CH), 70.15 (2 × CH), 97.99 (2 × Cq of BDA),
99.21 (2 × Cq of BDA); m/z (+ve ion FAB) 407 (M, 100%), 377
(40), 101 (100), 73 (28); Anal. (C18H32O10) C, H, N.
L-1,6-Di-O-b en zyl-ch ir o-in osit ol 2,3,4,5-t et r a k is(O,O-
d iben zyl p h osp h a te) (en t-8) was obtained in a fashion
identical to that described for 8; [R]D ) -3.7 (CHCl3, c ) 0.8);
spectroscopic data were identical to those obtained for 7; Acc.
Mass (C76H77O18P4).
D-ch ir o-In ositol 2,3,4,5-tetr a k isp h osp h a te (3). A solu-
tion of 8 (0.30 g, 0.21 mmol) in methanol (20 mL) and a few
drops of water was stirred vigorously with 10% Pd/C (1.2 g)
under atmosphere of H2 at room temperature overnight. The
catalyst was removed by filtration, and the solvents were
evaporated in vacuo. The residue was purified by ion-exchange
chromatography on Pharmacia Q Sepharose Fast Flow resin
eluting with a gradient of TEAB buffer (0.1-1 M) gave the
pure triethylammonium salt of compound 3 (0.17 mmol, 81%),
which eluted at 750-850 mM buffer; [R]D ) +8.9 (MeOH, c )
1); δH (400 MHz, CD3OD) 4.10 (2 H, br s, H-1 and H-6), 4.49-
4.53 (2 H, m, 2 × CH), 4.53-4.57 (2 H, m, 2 × CH); δP (160
L-(2′S,3′S)-2,3;4,5-Bis-O-(2′,3′-dim eth oxybu tan e-2′3′-diyl)-
ch ir o-in ositol (en t-5) was obtained in a fashion identical to
that described for 5; mp 105-120 °C (from diisopropyl ether);
[R]D ) +145 (CH2Cl2, c ) 0.8); spectroscopic data were identical
to those obtained for 5; Acc. Mass (C18H32O10).
D-(2′R,3′R)-1,6-Di-O-ben zyl-bis-O-2,3;4,5-(2′,3′-dim eth oxy-
bu ta n e-2′,3′-d iyl)-ch ir o-in ositol (6). A mixture of bisacetal
(5) (0.82 g, 2.0 mmol), benzyl bromide (1.0 g, 5.8 mmol) and
NaH (0.24 g of a 60% dispersion in oil, 6.0 mmol) in dry DMF
was stirred at room temperature for 20 h, and the excess of
NaH was destroyed by adding methanol. The solvents were
evaporated to dryness in vacuo, and the residue was taken up
in ethyl acetate and washed with saturated aqueous NaHCO3,
1
MHz, CDCl3), H-decoupled) -0.14 (2 P, s), 0.38 (2 P, s); Acc.
Mass (C6H15O18P4).
L-ch ir o-In ositol 2,3,4,5-tetr a k isp h osp h a te (en t-3) was
obtained from en t-8 in a fashion identical to that described