Note: Descriptions are shown in the official language in which they were submitted.
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INOSITOL POLYPHOSPHATE DERIVATIVES AND METHODS OF USING
SAME
This invention was made with government support
under grant number R01 DK47240-01A1 awarded by National
Institutes of Health. The government has certain rights
- in the invention.
BACKGRQUND OF THE INVENTION
FAD OF THE IN_VENTIC~N_
The present invention relates generally to
compounds and methods for modulating chloride ion
transport, and, more specifically, to antagonists and
agonists of inositol polyphosphates.
BACKGROUND INFORMATION
A11 living organisms are made of cells. The
boundary of a cell is determined by the plasma membrane
which serves to segregate materials on the inside from
those on the outside. The cell membrane functions by
regulating the passage of materials such as nutrients
into or out of the cell. The transport of ions across
cell membranes serves many important functions in a cell,
including the regulation of oncotic pressure of the cell.
The regulation of ion transport provides cells with the
appropriate concentration of ions to maintain their
integrity and function. Under inappropriate conditions,
for example, if the ion concentration inside the cell is
too high, water moves into the cell and causes it to
swell or burst. If the ion concentration in the cell is
too low, the cell shrinks.
Organisms exploit the cell membrane ion
transport properties to regulate fluids in particular
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tissue types. For example, cells lining the intestine
regulate water uptake using ion transport. One
particularly important ion transported by cells is
chloride ion. Chloride ion transport plays a key role in
a variety of cellular functions such as osmoregulation,
intracellular pH regulation, and salt and fluid
secretion.
In the intestine, high levels of chloride ion
secretion are associated with diseases such as secretory
diarrhea. Secretory diarrhea has many causes, including
infection by microorganisms such as Salmonella, Shigella,
and certain strains of Escherichia col.i. Other
conditions associated with the regulation of fluid levels
in tissues and chloride ion secretion include tissue
swelling associated with inflammation, infection or
trauma. Thus, methods for inhibiting undesirable levels
of chloride ion secretion could be used to alleviate
symptoms of these pathologies.
Cystic fibrosis is the most common lethal
genetic disease in Caucasians, affecting approximately
one in 2,000 births among Americans of European descent.
It is characterized by abnormally viscous mucous
secretions, which lead to chronic pulmonary disease,
pancreatic insufficiency and intestinal obstructions. In
the United States, approximately 30,000 people have
cystic fibrosis and about 1,000 new cases are diagnosed
every year. Although, in the past, afflicted children
often died as infants, individuals can now survive into
their twenties and thirties. Nevertheless, there is no
cure for cystic fibrosis and current therapies do not
correct the underlying cellular defect but only manage
the symptoms of the disease.
Current treatments for cystic fibrosis are
largely confined to symptom management or control of
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opportunistic infections. For example, the use of
vaccinations for viral pathogens and culture-specific
antibiotics for bacterial infections can be used to
prevent infection. Corticosteroids are sometimes useful
in the treatment of the inflammatory response to
infections but have a variety of undesirable long term
side effects. Other treatments focus on the symptoms of
chronic pulmonary disease associated with abnormally
viscous mucous secretions in the lung. For example,
physical assistance such as chest percussion and postural
drainage aids in clearing the secretions from the lungs.
Bronchodilators have been beneficial in some patients,
but in others have resulted in decreased gas exchange.
Other symptoms associated with abnormally viscous mucous
secretions include intestinal obstructions and pancreatic
insufficiency. Intestinal obstructions occur in 20% of
adults and are treated with enemas, or, where enemas are
not effective, by surgery.
Pancreatic insufficiency occurs in 80 to 90% of
the patients and is caused by decreased secretion of
bicarbonate ions and fluid due to impaired recycling of
chloride ions out of the cell. The corresponding
pancreatic fluid becomes hyperconcentrated in protein and
becames inspissated, causing obstruction of the
pancreatic ducts and subsequent atrophy of pancreatic
acini. Pancreatic insufficiency is treated with enzyme
supplementation, which usually restores adequate
digestive and absorptive functions, thereby correcting
symptoms of malnourishment. However, the underlying
disease persists, often causing recurrent bouts of
pancreatitis that can lead to atrophy of the pancreas.
A treatment that restores normal pulmonary
function, normal secretory function to the colon or
normal pancreatic function would provide a great
advantage over currently available therapies. Gene
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therapy has been used on cystic fibrosis patients.
However, attempts at gene therapy have been unsuccessful
for a variety of reasons, including the extraordinary
size of the gene, immune reactions to adenovirus vectors
used to transfer the gene and the rapid turnover of
epithelial tissue.
Thus, there exists a need for a means to alter
chloride ion secretion so as to ameliorate the symptoms
of pathologies such as cystic fibrosis and secretory
diarrhea which are associated with abnormal chloride
transport. The present invention satisfies this need and
provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides compositions
that are cell permeable antagonists of inositol
polyphosphates. For example, the invention provides
antagonists of myo-inositol 3,4,5,6-tetrakisphosphate and
phosphatidylinositol trisphosphate. In addition, the
invention provides methods for enhancing chloride ion
secretion from a cell by contacting the cells with cell
permeable antagonists of inositol polyphosphates. The
invention also provides methods for enhancing chloride
ion secretion in an individual by administering cell
permeable antagonists of inositol polyphosphates to the
individual. The invention additionally provides methods
for alleviating a sign or symptom associated with cystic
fibrosis in an individual by administering a cell
permeable antagonist of inositol polyphosphates to the
individual.
' The invention also provides compositions that
are cell permeable agonists of inositol polyphosphates.
For example, the invention provides agonists of
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myo-inositol 3,4,5,6-tetrakisphosphate and
phosphatidylinositol trisphosphate. In addition, the
invention provides methods for decreasing chloride ion
secretion from a cell by contacting the cell with cell
5 permeable agonists of inositol polyphosphates. The
invention also provides methods for decreasing chloride
- ion secretion in an individual by administering cell
permeable agonists of inositol polyphosphates to the
individual. The invention additionally provides methods
for alleviating a sign or symptom associated with
secretory diarrhea in an individual by administering cell
permeable agonists of inositol polyphosphates to the
individual.
BRIEF DESCRIPTION O~~' THE DRAWINGS
Figure 1 shows the structures of myo-inositol,
scyllo-inositol and a representative cell permeable
inositol polyphosphate derivative,
2-Bt-1-Me-Ins (3, 4, 5, 6) P~/AM.
Figure 2 shows Bt,-Ins(3,4,5,6)PQ/AM inhibition
of chloride ion secretion in rabbit colon.
Figure 3 shows enhanced chloride ion secretion
in colonic epithelial T8q cells treated with 200 ~M
2-Bt-1-Me-Ins(3,4,5,6)PQ/AM due to the reversal of
Btu-Ins(3,4,5,6)Pq/AM-mediated inhibition of histamine-
stimulated chloride ion secretion.
Figure 4 shows that Bt~- Ins ( 3 , 4 , 5 , 6 ) P4/AM does
not increase intracellular calcium and does not affect
calcium response to thapsigargin.
Figure 5 shows enhanced chloride ion secretion
in colonic epithelial Tg4 cells treated with
D,L-1,2-cyclohexylidene-Ins(3,4,5,6)P4/AM (Figure 5A);
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1-Bu-2-Bt-Ins (3, 4, 5, 6) PQ/AM (Figure 5B) ;
BuZ-Ins (3, 4, 5, 6) Pq/AM (Figure 5C) ;
3-Bt-2-Bu-Ins(1,4,5,6)Pq/AM (Figure 5D);
Bu2-Ins (l, 4, 5, 6) PQ/AM (Figure 5E) ;
S 1-Bt-2-Bu-Ins{3,4,5,6)P9/AM (Figure 5F); and
3-Bu-2-Bt-Ins(1,4,5,6)P4/AM (Figure 5G).
Figure 6 shows enhanced chloride ion secretion
in colonic epithelial T84 cells treated with
Bt2-Ins(1,4,5,6)PQ/AM due to reversal of EGF-mediated
inhibition of chloride ion secretion.
Figure 7 shows decreased chloride ion secretion
in colonic epithelial T84 cells treated with EGF or
treated with cell permeable derivatives of PtdInsP:.
Btu-Ins(1,4,5,6)P4/AM enhances chloride ion secretion due
to reversal of EGF- and PtdInsP3-mediated inhibition of
chloride ion secretion.
Figure 8 shows decreased chloride ion secretion
in colonic epithelial TBQ cells treated with increasing
concentrations of PtdInsP3/AM.
Figure 9 shows the structure of cell permeable
PtdInsP3 and diCl6-Bt-PtdInsP/AM.
Figure 10 shows a schematic of the synthesis of
cell permeable PtdInsP3/AM derivatives.
Figure 11 shows a schematic of the synthesis of
1,2-cyclohexylidene-Ins(3,4,5,6)P4//AM.
Figure 12 shows structures of myo-inositol
1,3,4-trisphosphate and 2,5,6-Btu-Ins(1,3,4)P3/AM. Only
the D-configuration of the compounds is shown.
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Figure 13 shows a schematic for synthesis of
cell permeable inositol polyphosphate derivatives.
The invention provides compositions and methods
for altering chloride ion secretion by contacting cells
with cell permeable compounds that are antagonists or
agonists of inositol polyphosphates. As used herein, the
term "altering" refers to enhancing or decreasing
chlaride ion secretion such that the level of chloride
ion transport into or out of a cell is different than the
level of secretion in cells not treated with an agonist
or antagonist of the invention. As used herein, the term
"antagonist" means that a compound has the function of
reducing the physiological activity of another compound.
For example, an antagonist of a compound that inhibits
chloride ion secretion reverses that inhibition.
Similarly, the term "agonist" means that a compound has
the function of mimicking the physiological activity of
another compound.
An antagonist of the invention enhances
chloride ion secretion from cells. As used herein, the
term "enhancing," when used in reference to increasing
chloride ion secretion, means that the level of chloride
ion transport out of a cell is higher than the level of
secretion in corresponding cells not treated with an
antagonist. Conversely, the term "decreasing," when used
in reference to inhibiting chloride ion secretion, means
that the level of chloride ion transport out of a cell is
lower than the level of secretion in corresponding cells
not treated with an agonist. As used herein, the term
"contacting" refers to incubating or exposing a cell to a
cell permeable compound such that the compound can pass
through the cell membrane.
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Abnormal chloride ion secretion is associated
with various pathological conditions. Accordingly, the
compounds of the invention are useful for enhancing or
decreasing chloride ion secretion, thereby alleviating
symptoms of such diseases. In certain pathological
conditions, such as cystic fibrosis, enhancement of
chloride ion secretion can be useful for alleviating
symptoms of the disease, whereas, in other pathological
conditions, such as secretory diarrhea, decreasing
chloride secretion can alleviate the symptoms. As used
herein, the term "alleviating" refers to relieving or
lessening a symptom of a disease. Thus, depending on the
chloride ion secretion abnormality, the invention
provides compositions and methods to compensate for
abnormal chloride ion secretion associated with a
particular disease.
The invention provides compounds that are
agonists or antagonists of inositol polyphosphates.
Inositol polyphosphate derivatives can function as
inositol polyphosphate agonists and antagonists. The
effects of compounds of the invention on Ins(3,4,5,6)Pq-
and PtdInsP3-mediated inhibition of calcium-mediated
chloride ion secretion are described in the Examples and
summarized in Table I.
The invention provides cell permeable compounds
that regulate chloride ion secretion through calcium-
dependent chloride ion channels by mimicking or
antagonizing the activity of the endogenous inhibitors of
calcium-mediated chloride ion secretion, Ins(3,4,5,6)PQ or
PtdInsP3. As disclosed herein, compounds that increase
the intracellular concentration of Ins(3,4,5,6)PQ or
PtdInsP3 are useful for inhibiting chloride ion secretion.
In contrast, compounds that antagonize the effect of
Ins(3,4,5,6)Pq or PtdInsP3 are useful for enhancing
chloride ion secretion.
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Table I. The Effects of Inositol Polyphosphate
Derivatives on Chloride Ion Secretion
TABLE I
Derivative' Abbrev- Inhib- Ability Mechanism
to and
iation= ition Block Inositol
of
C1- Inhibition Polyphosphate
Secret- Pathway
ion Affected
1,2-di-O- Bt,- Agonist of
butyryl- Ins(3,4,5,6) Ins(3,4,5,6)
myo-inositol P,/AM + P4
3,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
2-O-butyryl-1-O-2-Bt-1-Me- Antagonist
of
methyl- Ins(3,4,5,6) Ins(3,4,5,6)
myo-inositol P,/AM - + PQ
3,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
1,2-di-O-methyl-Men- Antagonist
of
myo-inositol Ins(3,4,5,6) Ins(3,4,5,6)P
3,4,5,6_ P4/AM _ + a
TKphosphate oct
(acetoxymethyl)
ester
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Derivative) Abbrev- Inhib- Ability Mechanism
to and
iation~ ition Block Inositol
of
C1- Inhibition Polyphosphate
Secret- Pathway
ion Affected
D,L-1,2-di- D,L-1,2- Antagonist
of
cyclohexylidene-cyclohexy- Ins(3,4,5,6)
myo-inositol lidene- + P,
3,4,5,6- Ins(3,4,5,6)
5 TKphosphate oct P,/AM
(acetoxymethyl)
ester
1-O-butyl-2-O- 1-Bu-2-Bt- Antagonist
of
butyryl-myo- Ins(3,4,5,6) Ins(3,4,5,6)
10 inositol PQ/AM + P,
3,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
1,2-di-O-butyl- BuZ- Antagonist
of
myo-inositol Ins(3,4,5,6) Ins
3,4,5,6- PQ/AM + (3,4,5,6)P4
TKphosphate oct
(acetoxymethyl)
2 ester
0
1-O-butyryl-2-O-1-Bt-2-Bu-
butyl-myo- Ins(3,4,5,6)
inositol P4/AM -
3,4,5,6-
2 TKphosphate oct
5
(acetoxymethyl)
ester
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Derivative' Abbrev- Inhib- Ability Mechanism
to and
iationz ition Block Inositol
of
C1- Inhibition Polyphosphate
Secret- Pathway
ion Affected
2, 3-di-O-methyl-Mez-
myo-inositol Ins(1,4,5,6)
3, 4, 5, 6- P,/AM - -
TKphosphate oct
(acetoxymethyl)
ester
2-O-butyryl-3-O-2-Bt-3-Me-
methyl- Ins(1,4,5,6)
myo-inositol Pq/AM - -
3,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
D,L-1,2-O- D,L-Bt~- Agonist of
butyryl-scyZZo- scyZlo-Ins Ins(3,4,5,6)
inositol (3,4,5,6) + P4
3,4,5,6- P4/AM
TKphosphate oct
(acetoxymethyl)
ester
D,L-1-0-butyryl-D,L-1-Bt-2- Agonist of
2-U-methyl-myo- Me- Ins(3,4,5,6)
inositol Ins(3,4,5,6)+ P,
3,4,5,6- P4/AM
TKphosphate oct
(acetoxymethyl)
ester
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Derivative) Abbrev- Inhib- Ability Mechanism
to and
iation~ ition Block Inositol
of
C1- Inhibition Polyphosphate
Secret- Pathway
ion Affected
D,L-1,2- D,L-1,2 C1~-
dichloro-1,2- 2,2 dideoxy-
dideoxy-myo- Ins(3,4,5,6)-
inositol P4/AM
3,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
3-O-butyryl-2-O-3-Bt-2-Bu-
butyl-myo- Ins(1,4,5,6)
inositol P4/AM -
1,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
2,3-di-O-butyl- Bu,-
myo-inositol Ins(1,4,5,6)
1,4,5,6- PQ/AM -
TKphosphate oct
(acetoxymethyl)
ester
3-O-butyl-2-O- 3-Bu-2-Bt-
butyryl-myo- Ins(1,4,5,6)
inositol P4/AM -
1,4,5,6-
TKphosphate oct
(acetoxymethyl)
ester
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Derivative' Abbrev- Inhib- Ability Mechanism
to and
iation~ ition Block Inositol
of
C1- InhibitionPolyphosphate
Secret- Pathway
ion Affected
D,L-2,5,6-tri-O-D,L-Bt3- Agonist of
butyryl-myo- Ins(1,3,4) Ins(3,4,5,6)
inositol P3/AM + PQ
1,3,4-
trisphosphate
hex
(acetoxymethyl)
ester
2,3-di-O Bt~_ Antagonist
of
butyryl- Ins(1,4,5,6) PtdInsP,
myo- -
inos itol Pa /AM +
1,4,5,6-
TKphosphate oct
(acetoxymethyl
)
ester
sn-di-O- diC,b-Bt- Agonist of
palmitoyl-D,L-6-PtdIns PtdInsP:
O-butyryl- P3/AM +
phosphatidyl-
inositol
3,4,5-
trisphosphate
hepta
(acetoxymethyl)
ester
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Derivative) Abbrev- Inhib- Ability Mechanism
iation= ition to and
of Block Inositol
C1- Inhibition Polyphosphate
Secret- Pathway
ion Affected
sn-di-O- dice-Bt- Agonist of
octanoyl-D,L-6- PtdIns PtdInsP,
O-butyryl- (3,4,5) +
phosphatidyl- P,/AM
inositol
3,4,5-
trisphosphate
hepta
(acetoxymethyl)
ester
D,L-I-0-butyryl-D,L-1-Bt-2- Agonist of
2-0-deoxy- deoxy-Ins Ins(3,4,5,6)
inositol (3,4,5,6) P,
3,4,5,6- P4/AM
TKphosphate oct
(acetoxymethyl)
ester
1 Derivatives listed use the following abbreviations: TK,
tetrakisphosphate; oct, octakis, hex, hexakis. Unless
otherwise indicated, all compounds are the D form.
z Abbreviations used in the table and throughout the
application: Ins, inositol; P, phosphate; P~, n designates
the number of phosphates; AM, acetoxymethyl ester; Bt,
butyryl; Me, methyl; Bu, butyl; C16, palmitoyl; C~,
octanoyl; PtdIns, phosphatidylinositol.
Unless otherwise indicated, all derivatives are myo-
inositol derivatives.
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The cell permeable compounds of the invention,
which readily pass through the plasma membrane, can be
useful for directly increasing the intracellular
concentration of Ins(3,4,5,6)P9 or PtdInsP3 or for
5 mimicking the effects of Ins(3,4,5,6)PQ or PtdInsP~ due to
structural similarities. Alternatively, the cell
permeable compounds of the invention are useful for
antagonizing the effects of Ins(3,4,5,6)Pq or PtdInsP~.
As disclosed herein, inositol polyphosphate derivatives
10 can function as cell permeable agonists or antagonists of
Ins ( 3 , 4 , 5 ( 6 ) PQ or PtdInsP~ .
Cell permeable inositol polyphosphate
derivatives were synthesized as described in Example XI.
These compounds are cell permeable because the charged
15 phosphate groups are masked by acetoxymethyl ester groups
and the hydroxyl groups are masked by hydrophobic
chemical groups (see Figure 1). A hydrophobic chemical
group useful for masking a hydrophilic group can be, for
example, an acyl group, an alkyl group, an alkylidene
group, or any hydrophobic chemical group that can mask
hydrophilic groups on inositol polyphosphates. The alkyl
groups include methyl, ethyl, propyl, iso-propyl, butyl,
iso-butyl, tert-butyl, sec-butyl, 1-methylbutyl,
2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl,
pentyl and hexyl as well as alkylene groups, cyclic
chains of carbon atoms such cyclohexyl and cyclopentyl
groups, as well as combinations of linear or branched
chains and cyclic chains of carbon atoms such as a
methyl-cyclohexyl: or cyclopropyl-methylene group. With
respect to methyl derivatives, however,
2-Bt-1-Me-Ins (3, 4, 5, 6) PQ/AM, Men-Ins (3, 4, 5, 6) P4/AM,
Me~-Ins (1, 4, 5, 6) PQ/AM, 2-Bt-3-Me-Ins (1, 4, 5, 6) Pq/AM and
D,L-1-Bt-2-Me-Ins(3,4,5,6)PQ/AM are not within the claimed
compositions. In addition, it should be recognized that
an alkyl as defined herein can be substituted with a
substituent. The alkylidene groups, which are acetal
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groups, include, for example, methylidene, ethylidene,
propylidene, butylidene, pentylidene, hexylidene,
cyclobutylidene, cyclopentylidene, cyclohexylidene and
cycloheptylidene (Kemp and Vellaccio, Organic Chemistry,
Worth Publishers, New York (1980)), which is incorporated
herein by reference. The acyl groups include, for
example, acetyl, propanyl, butyryl, hexanoyl and valeryl.
With respect to acyl derivatives, however,
Btz-Ins (3, 4, 5, 6) Pq/AM, D, L-Bt~-scyllo-Ins (3, 4, 5, 6) Pq/AM and
Bt,-Ins(1,4,5,s)Pq/AM are not within the claimed
compositions. Once inside the cell, butyryl and
acetoxymethyl ester groups are cleaved by intracellular
esterases. Thus, modification with these hydrophobic
functional groups allows compounds to cross the cell
membrane where they are hydrolyzed to expose hydrophilic
groups of the non-derivatized compound. The alkyl and
alkylidene groups, in contrast, are non-hydrolyzable and
thus are retained on the derivative after crossing the
cell membrane.
Additional modifications of inositol
polyphosphates provide cell permeable derivatives. For
example, Ins(3,4,5,6)PQ derivatives, including
1, 2-dideoxy-Ins (3, 4, 5, 6) Pq/AM,
1-Bt-2-deoxy-Ins(3,4,5,6)PQ/AM,
2-Bt-1-deoxy-Ins(3,4,5,6)PQ/AM and
1,2-dichloro-1,2-dideoxy-myo-Ins(3,4,5,6)PQ/AM, also are
cell permeable derivatives (see Table I). In addition,
cell permeable PtdInsP3 derivatives include, for example,
diCl6-6-O-Bt-PtdIns (3,4, 5) P3/AM and
dice-s-O-Bt-PtdIns (3, 4, 5) P3/AM, as well as
diC,6-2-O-Bt-PtdIns (3, 4 ( 5) P3/AM,
dice-2-.O-Bt-PtdIns (3, 4, 5) P3/AM,
diC_6-2, 6-O-Bt~-PtdIns (3, 4, 5) P3/AM and
dice-2, 6-O-Bt~-PtdIns (3 , 4, 5) P3/AM. Methods for
synthesizing the acylated and acetoxymethyl ester
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derivatives are described by Roemer et al. (J. Chem.
~oc,~ Chem. Commun. N4:411 (fat 1995); J. Chem. Soc..
PPrkins Trans. 1 N14:1683 (fat 1996)), each of which is
incorporated herein by reference.
The invention provides cell permeable compounds
that are antagonists of inositol polyphosphates. In one
embodiment, the cell permeable compounds are
Ins(3,4,5,6)P4 antagonists. As disclosed herein,
Ins ( 3 , 4 , 5 , 6 ) Pq derivatives function as Ins ( 3 , 4 , 5 , 6 ) Pq
antagonists. For example, Ins(3,4,5,6)PQ derivatives with
alkyl groups attached to the hydroxyl group at position
1, position 2 or both function as antagonists of
Ins(3,4,5,6)P~ (see Table I and Examples III and IV).
Particularly useful alkyl group derivatives are methyl
(Me) and butyl (Bu) derivatives. For example,
2-Bt-1-Me-Ins (3, 4, 5, 6) P4/AM, Men-Ins- (3, 4, 5, 6) PQ/AM,
1-Bu-2-Bt-Ins (3, 4, 5, 6) P4/AM and Buy-Ins (3, 4, 5, 6) P4/AM each
reversed Ins(3,4,5,6)PQ-mediated inhibition of chloride
ion secretion and, therefore, are Ins(3,4,5,6)Pq
antagonists. Other useful Ins(3,4,5,6)PQ derivatives are
those containing an alkylidene group on position 1 and
position 2. For example, D,L-1,2-cyclohexylidene-
Ins(3,4,5,6)Pq/AM reversed carbachol-mediated inhibition
of chloride ion secretion and, therefore, is an
Ins(3,4,5,6)PQ antagonist. In addition, Ins(1,4,5,6)PQ/AM
derivatives with an alkylidene group on position 2 and
position 3 also can be used as an Ins(1,4,5,6)P4
derivative.
In another embodiment, the cell permeable
compounds are PtdInsP3 antagonists. As disclosed herein,
cell permeable Ins(1,4,5,6)P4 derivatives are PtdInsP:
antagonists. For example, Ins(1,4,5,6)PQ derivatives with
butyryl groups attached at positions 2 and 3 reversed
EGF-mediated and PtdInsP,-mediated inhibition of calcium-
mediated chloride ion secretion and, therefore, are
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PtdInsP3 antagonists (see Table I and Examples VII
and VIII).
The invention also provides cell permeable
compounds that are agonists of inositol polyphosphates.
In one embodiment, the cell permeable compounds are
Ins(3,4,5,6)PQ agonists. As disclosed herein,
Ins(3,4,5,6)PQ derivatives are Ins(3,4,5,6)P4 agonists.
For example, Bt~-Ins (3, 4, 5, 6) P4/AM,
Bt2-scyllo-Ins (3, 4, 5, 6) Pq/AM and
1-Bt-2-deoxy-Ins(3,4,5,6)PQ/AM (36o inhibition) inhibit
calcium-mediated chloride secretion by uncoupling
chloride ion secretion from intracellular calcium
concentrations and, therefore, are Ins(3,4,5,6)Pq agonists
(see Table I and Examples I to III). In addition,
Ins(1,3,4)P3 derivatives are Ins(3,4,5,6)Pq agonists. For
example, Btj-Ins(1,3,4)P3/AM inhibits calcium-mediated
chloride ion secretion by increasing intracellular
concentrations of Ins(3,4,5,6)P4 and, therefore, are
Ins (3, 4, 5, 6) PQ agonists (see Example V) .
In another embodiment, the cell permeable
compounds are PtdInsP3 agonists. As disclosed herein,
PtdInsP3 derivatives are PtdInsP~ agonists. For example,
diCl6-Bt-PtdInsP3/AM and diCS-Bt-PtdInsP3/AM inhibited
calcium-mediated chloride ion secretion and, therefore,
are PtdInsP3 agonists (see Table I and Example VIII).
Molecular modeling of the structure of inositol
polyphosphate derivatives can~be used to design synthetic
compounds with structural features that mimic the desired
activity of the inositol polyphosphate derivative.
Alternatively, cell permeable synthetic compounds that
act as agonists or antagonists of inosi.tol polyphosphates
can be identified, for example, by screening a
combinatorial chemistry library. Using the methods
described herein, synthetic compounds can be screened for
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19
the desired activity of mimicking or antagonizing the
effect of a given inositol polyphosphate on calcium-
mediated chloride ion secretion. Methods for making and
screening combinatorial chemistry libraries are well
known to those skilled in the art (Combinatorial Peptide
and NonpP~r~de Libraries: A Handbook, Jung, ed., VCH, New
York (1996)), which is incorporated herein by reference.
The invention additionally provides methods for
altering chloride ion secretion by contacting cells with
cell permeable compounds that are antagonists or agonists
of inositol polyphosphates. Movement of chloride ions is
used by cells to regulate water flow within the body of
an organism. Cells have specific chloride ion channels
that only allow chloride ions to pass through the
membrane when the channels are open. These channels are
especially important in cells in mucous membranes, which
secrete mucins and regulate water flow. When water flow
into and out of cells is deficient, mucin secretion
results in viscous plugs, which obstruct airways in the
lungs and the mucosal lining of the intestine in diseases
such as cystic fibrosis. Not a11 chloride ion channels
are the same, however, and different channels are
regulated in different ways.
A specific group of chloride ion channels, the
calcium-dependent chloride ion channels, are linked to
calcium ion flux. When the intracellular concentration
of calcium ions increases, chloride ion secretion
increases. As used herein, the term "chloride ion
secretion" refers to the transport of chloride ions
across the plasma membrane of a cell. Treatment of cells
with certain compounds such as histamine, a
physiologically relevant agonist that does not have a
measurable long term effect on inositol polyphosphate
metabolism, causes an increase in intracellular calcium
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ion concentration that results in increased chloride ion
secretion.
Carbachol, a muscarinic cholinergic compound,
also increases intracellular calcium ion concentration
5 and stimulates chloride ion secretion with short term
treatment. However, following long-term treatment of
colonic epithelial T84 cells with carbachol, increased
concentrations of intracellular calcium ions no longer
stimulate chloride ion secretion (Kachintorn et al., Am.
10 ~. PhSr_siol. Cell 264:C671 (1993)), which is incorporated
herein by reference. Long-term treatment with carbachol
leads to a slow and prolonged rise of intracellular
Ins(3,4,5,6)P4 levels (Vajanaphanich, et al., Nature
371:711 (1994)), which is incorporated herein by
15 reference. Treatment of T84 cells with a mixture of
Btu-Ins (3, 4, 5, 6) PQ/AM and Bt~-Ins (1, 4, 5, 6) Pq/AM inhibited
chloride ion secretion in response to increased
concentrations of intracellular calcium induced by
thapsigargin whereas Bt~--Ins(1,4,5,6)Pq/AM had no effect.
20 As exemplified herein, enantiomerically pure
Bt2-Ins(3,4,5,6)Pq/AM inhibited chloride ion secretion in
response to increased concentrations of intracellular
calcium (see Example I). Thus, Ins(3,4,5,6)PQ is an
endogenous negative regulator of calcium-mediated
chloride ion secretion because it uncouples chloride ion
secretion from calcium ion concentration.
Another mechanism for inhibiting calcium-
mediated chloride ion secretion distinct from that
mediated by Ins(3,4,5,6)PQ is by increasing levels of
PtdInsP3. EGF inhibits calcium-mediated chloride ion
secretion induced by carbachol and thapsigargin in Te4
cells (Uribe et al., Am. J. Ph~rsiol. 271:C914 (1996a)).
EGF triggers a 1.7-fold increase in Ins(3,4,5,6)Pq levels,
compared to carbachol, which triggers up to a 20-fold
increase in Ins(3,4,5,6)P4. These results suggest that
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21
EGF modulates calcium-mediated chloride ion secretion
through a different mechanism than Ins(3,4,5,6)P4.
EGF activates phosphatidylinositol 3-kinase
(PI 3-kinase) and the inhibitory effect of EGF on
calcium-mediated chloride ion secretion is mediated by
PI 3-kinase (Uribe et al., J. Biol. Chem. 271:26588
(1996b)). EGF stimulated an increase in levels of the
PI 3-kinase products, phosphatidyl-inositol
3,4-bisphosphate (PtdInsP2) and PtdInsP3. These PI 3-
kinase products increased with a time course that
paralleled EGF inhibition of chloride ion secretion.
Wortmannin, which is a highly specific PI 3-kinase
inhibitor, blocked formation of PtdInsP, and PtdInsP, and
reversed the EGF induced inhibition of chloride ion
secretion. Thus, a mechanism involving PtdInsP3 was
responsible for EGF induced inhibition of calcium-
mediated chloride ion secretion.
Since the compounds of the invention are
agonists and antagonists of inositol polyphosphates,
which regulate chloride ion secretion, these compounds
are useful for altering chloride ion secretion in cells.
The cell permeability of the compounds allows delivery of
the compounds to the cytoplasm, the site of inositol
polyphosphate action, by contacting cells with the
compounds.
The invention provides a method for enhancing
chloride ion secretion across a cell membrane by
contacting a cell with a cell permeable antagonist of an
inositol polyphosphate. In one embodiment,
Ins(3,4,5,6)Pn antagonists are used to enhance chloride
ion secretion. As disclosed herein, Ins(3,4,5,6)P4
derivatives can be used as Ins(3,4,5,6)P4 antagonists.
In another embodiment, PtdInsP3 antagonists are used to
enhance chloride ion secretion. As disclosed herein,
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22
Ins(1,4,5,6)P4 derivatives can be used as PtdInsP~
antagonists.
The invention also provides a method for
decreasing chloride ion secretion across a cell membrane
by contacting a cell with a cell permeable agonist of an
inositol polyphosphate. In one embodiment, Ins(3,4,5,6)PQ
agonists are used to decrease chloride ion secretion. As
disclosed herein, Ins(3,4,5,6)Pq derivatives can be used
as Ins ( 3 , 4 , 5 ( 6 ) P9 agonists . In addition, Ins ( 1, 3 , 4 ) P3
derivatives can be used as Ins(3,4,5,6)PQ agonists. In
another embodiment, PtdInsP: agonists are used to decrease
chloride ion secretion. As disclosed herein, PtdInsP3
derivatives can be used as PtdInsP, agonists.
The invention provides a method for enhancing
chloride ion secretion in an individual by administering
to the individual an antagonist of Ins(3,4,5,6)PQ or
PtdInsP3. As used herein, the term "individual" refers to
an organism, which generally is a mammal, particularly a
human, to be treated with agonistic or antagonistic
compounds of the invention that enhance or decrease
chloride ion secretion. The chloride ion secretion
enhancement or decrease occurs in the cells comprising
one or more tissue organs in the individual. As used
herein, the term "administering" or "administration"
refers to introducing the antagonist or agonist to an
individual in a manner and form such that the antagonist
or agonist is delivered to the appropriate target tissue
or tissues, where it can contact the target cells,
thereby altering chloride ion secretion of the cells.
The invention additionally provides a method for
alleviating a sign or symptom associated with cystic
fibrosis by administering an antagonist of Ins(3,4,5,6)PQ
or PtdInsP~ or both to an individual exhibiting the sign
or symptom.
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23
The invention also provides a method for
decreasing chloride ion secretion in an individual by
administering to the individual an agonist of
Ins(3,4,5,6)Pg or PtdInsP3. In addition, the invention
provides a method for alleviating a sign or symptom
associated with secretory diarrhea by administering an
agonist of Ins(3,4,5,6)P4 or PtdInsP3 to an individual
exhibiting the sign or symptom.
An antagonist or agonist generally is
administered to an individual as a pharmaceutical
composition. Such a pharmaceutical composition contains
the antagonist or agonist and a pharmaceutically
acceptable vehicle. Pharmaceutically acceptable vehicles
are well known in the art and include aqueous solutions
such as physiologically buffered saline or other solvents
or vehicles such as glycols, glycerol, oils such as olive
oil or injectable organic esters.
A pharmaceutically acceptable vehicle can
contain physiologically acceptable compounds that act,
for example, to stabilize the antagonist or agonist or
increase the absorption of the agent. Such
physiologically acceptable compounds include, for
example, carbohydrates such as glucose, sucrose or
dextrans, antioxidants such as ascorbic acid or
glutathione, chelating agents, low molecular weight
proteins or other stabilizers or excipients. One skilled
in the art would know that the choice of a
pharmaceutically acceptable vehicle, including a
physiologically acceptable compound, depends, for
example, on the route of administration of the antagonist
or agonist of the invention and the target tissue or
tissues.
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Several routes of administration can be used
depending on the individual to be treated and the target
tissue or tissues. The pharmaceutical composition can be
administered by various routes, including, for example,
S orally, rectally, or parenterally, such as intravenously,
intramuscularly, subcutaneously, intraorbitally,
intracapsularly, intraperitoneally, intracisternally or
by passive or facilitated absorption through the skin
using, for example, a skin patch or transdermal
iontophoresis, respectively. Furthermore, the
composition can be administered by injection, intubation
or topically, the latter of which can be passive, for
example, by direct application of an ointment or powder,
or active, for example, using a nasal spray or inhalant.
The total effective dose can be administered to
a subject as a single dose, either as a bolus or by
infusion over a relatively short period of time, or can
be administered using a fractionated treatment protocol,
in which the multiple doses are administered over a more
prolonged period of time. One skilled in the art would
know that the concentration of the agent required to
obtain an effective dose in a subject depends on many
factors including the age and general health of the
subject as well as the route of administration and the
number of treatments to be administered. In view of
these factors, the skilled artisan would adjust the
particular dose so as to obtain an effective dose.
The dosage of a particular compound is
determined by the intracellular concentration required to
achieve the desired effect, for example, enhancing or
decreasing chloride ion secretion. The effective
intracellular concentration can be determined using
methods as disclosed herein or otherwise known in the
art. For example, the effective intracellular
concentration of the agonist Btu-Ins(3,4,5,6)P4/AM was
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determined to be about 4 ~cM using an in vitro
Ins(1,3,4)P3-5/6-kinase assay (Vajanaphanich et al. supra,
1994) . Thus, for Bt~-Ins (3, 4, 5, 6) PQ/AM, the dosage is
chosen so as to provide an intracellular derivative
5 concentration of about 4-20 uM, preferably 4 uM. Initial
studies are conducted in a model system to determine the
effectiveness of particular compounds. Determination of
the effective concentration necessary to treat a
pathological condition in an individual is determined in
10 Phase I and Phase II clinical trials.
The compounds of the invention can be used
alone, or in combination with each other or with other
therapeutic agents to treat a pathological condition
associated with abnormal chloride ion secretion. For
15 example, the treatment protocol can begin with one
compound and additional compounds can be administered
with the first compound as needed if the relief due to
the first compound is insufficient. The treatment can
also be combined with other modes of treatment, such as
20 administration of uridine 5'-triphosphate plus amiloride
(see Bennett et al., Am, J, Resx~ir. Crit. Care
Med. 153:1796 (1996)), which is incorporated herein by
reference.
The compositions and methods of the invention
25 are useful for treating various pathologies, such as
cystic fibrosis or secretory diarrhea, which are
characterized, in part, by abnormal chloride ion
secretion. Defective chloride ion secretion occurs in
cystic fibrosis due to mutations in an epithelial cell
chloride ion channel, the cystic fibrosis transmembrane
regulator (CFTR). Chloride ion channels associated with
the apical surface of epithelial cells are the principal
control point regulating salt and fluid secretion. Most
cases of cystic fibrosis are the result of a single point
mutation, known as the F508 mutation, which results in
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26
the loss of the amino acid phenylalanine at amino acid
position 508. This mutation interferes with transfer of
the CFTR to the cell membrane, thereby reducing chloride
ion secretion by the cell.
One approach to treatment of cystic fibrosis is
to artificially activate other chloride ion channels.
For example, in epithelial cells, the outwardly
rectifying chloride channel (ORCC) is a chloride ion
channel that is regulated by cyclic AMP. However, the
ORCC is also believed to be regulated by the CFTR and,
therefore, is less active in cystic fibrosis (Schwiebert
et al., Cell 81:1063 (1995); Egan, et al., Nature 358:581
(1992); and Gabriel, et al., Nature 363:263 1993J. In
contrast, calcium-dependent chloride ion channels are
more abundant in cells of a mouse model of cystic
fibrosis (Grubb et al., Am. J. Phvsiol. 266:C1478
(1994)). Because calcium-dependent chloride ion channels
are distinct from CFTR and are likely more abundant in
cells of patients with cystic fibrosis, it should be
possible to augment flux through these channels to
compensate for the lack of flux through the CFTR. As
disclosed herein, one or more antagonists of
Ins(3,4,5,6)PQ or PtdInsP3 can be used to restore chloride
ion secretion in epithelial cells affected by defects in
the CFTR.
Several model systems are useful for assessing
the ability of inositol polyphosphate derivatives of the
invention to enhance chloride ion secretion. One such
model is the human colonic epithelial T84 cell line, which
has been studied extensively as a model secretory
epithelium (Dharmsathaphorn et al. Am. J. Ply iol.
246:G204 (1984)). Tgq cells exhibit a relatively
differentiated phenotype when grown to confluence on
permeable supports, forming polarized monolayers with
tight junctions and vectorial transport. T~4 monolayers
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27
retain many receptor-mediated chloride ion secretory
mechanisms including those involving changes in free
cytosolic calcium and the CFTR (Cohn et al., Proc. Natl.
Acad. Sci. L1~ 89:2340 (1992)). Agents such as
histamine, carbachol, calcium ionophores and thapsigargin
elevate intracellular calcium ions and stimulate varying
degrees of chloride ion secretion across T84 monolayers
(Dharmsathaphorn et al., J. Clin. Invest. 84:945 (1989);
Kachintorn, et al., supra, 1993). Cyclic AMP also
stimulates chloride ion secretion through the CFTR in T84
cells (Anderson and Welsh, Proc. Natl. Acad. Sci. USA
88:6003 (1991)). Thus, T84 cells provide an in vitro
model for the regulation of chloride ion secretion in
normal and pathological conditions that involve defective
chloride ion transport, such as cystic fibrosis.
Additionally, epithelial cells of rabbit colon also
provide a model of colonic epithelial cells.
Three of the more debilitating symptoms of
cystic fibrosis are chronic pulmonary disease, pancreatic
insufficiency and intestinal obstructions. Several model
systems are available for characterizing the therapeutic
effectiveness of inositol polyphosphate derivatives of
the invention for treatment of these symptoms. For
example, knockout of the CFTR gene in T8q cells (CFTR- T84
cells) provides a model for colonic epithelial cells that
have a genetic background similar to that of colonic
cells of cystic fibrosis patients (see Example X). The
CFTR- T84 cell line is useful for assessing the
effectiveness of compounds of the invention, which are
antagonists of Ins(3,4,5,6)P4 and PtdInsP3, at increasing
chloride ion secretion.
Primary human nasal epithelial cells are a
model of respiratory epithelium for testing the effect of
various compounds on chloride ion secretion. Primary
human nasal epithelial cells are useful for testing the
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28
function of compounds of the invention, which are
antagonists of Ins(3,4,5,6)PQ and PtdInsP3, on chloride
ion secretion in these cells (see Example IX).
A suitable model system for studying pancreatic
function uses the pancreatic duct epithelial cell CFTR-
cell line, CFPAC, which is a human cell line derived from
a pancreatic adenocarcinoma of a patient homozygous for
the F508 mutation. CFPAC cells lack cAMP-stimulated
channel function but exhibit calcium-activated chloride
ion channel activity (Shoemacher et al., Proc. Natl.
Acad. Sci. USA 87:4012 (1990)). Dog pancreatic duct
epithelial cells also are useful for testing the function
of compounds of the invention, which are antagonists of
Ins(3,4,5,6)PQ and PtdInsP3, on chloride ion secretion in
these cells (Nguyen et al., Am. J. PhSrsiol. 272:G172
(1997)), which is incorporated herein by reference.
Animal models of cystic fibrosis also are
available for assessing the activity of Ins(3,4,5,6)P~ and
PtdInsP3 antagonists. For example, CFTR knockout mice
have been generated that provide models of cystic
fibrosis {Rozmahel et al. Nature Gen. l2:280 (1996);
Snouwaert et al., Science 257:1083 (1992); Ratiff et al.,
Nature Gen. 4:35 (1992); O'Neal et al., Hum. Molec.
Genet. 2:1561 (1993); and Dorin et al., Nature 359:211
{1992)), each of which is incorporated herein by
reference. Increased levels of expression of calcium-
dependent chloride ion channels correspond to improved
viability in CFTR knockout mice (Clarke et al., Proc.
Natl. Acad. Sci USA_ 91:479 (1994); and Rozmahel, et al.,
supra, 1996). Animal models are used to test the
efficacy of compounds of the invention, which are
antagonists of Ins(3,4,5,6)PQ and PtdInsP3, on chloride
ion secretion in these animals. Animal models also are
useful for testing the effectiveness of compounds of the
invention on particular tissues in the animal.
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29
Since cystic fibrosis symptoms arise in several
organ systems, one skilled in the art would select a
particular route and method of administration of the
compound based on the symptom to be treated. The
compounds are somewhat amphipathic and are soluble in
aqueous solution. Alternatively, the compounds can be
dissolved in dimethyl sulfoxide containing a surfactant.
The compounds also can be dissolved in a solution
containing FREON (1,1,2 trichlorotrifluoromethane), which
has an advantage of low viscosity, allowing access to
more diseased tissue and, thus, is useful for treatment
during acute inflammation or during early stages of
disease. For example, in a subject suffering from
respiratory pathology, an antagonist of the invention can
be suspended or dissolved in the appropriate
pharmaceutically acceptable carrier and administered
directly into the lungs using an inhalation device such
as an inhaler nebulizer or turboinhaler. Thus, to
alleviate mucous accumulation in the lung, the compounds
can be administered in aerosol form directly to the
target lung tissue. Alternatively, the compounds can be
dissolved in FREON and can be delivered by lung
perfusion. Such treatment can be maintained through
periodic breathing of an atomized aqueous solution.
The clinical manifestations of cystic fibrosis,
such as mucous viscosity, are well known and one in the
art would know how to determine if a treatment protocol
is effective in alleviating or preventing these known
respiratory signs and symptoms of cystic fibrosis. For
example, measurement of mucociliary clearance using
(99MTc)iron oxide particles can be used to determine the
effectiveness of treatment with compounds of the
invention (Bennett, supra, 1996).
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Other clinical manifestations of cystic
fibrosis also can be treated with antagonists of
Ins(3,4,5,6)PQ or PtdInsP3 using appropriate modes of
administration. For example, sinus complications can be
5 treated by administering pharmaceutical compositions in a
nasal spray. This treatment is advantageous over current
methods for treating sinus complications which often
require multiple endoscopic surgeries that can result in
facial deformities in patients with cystic fibrosis. For
10 alleviation of intestinal disorders in cystic fibrosis,
the agent can be administered by suppository or enema in
an appropriate pharmaceutical vehicle. Alternatively,
enteric coated tablets can be administered orally. In a
subject suffering from pancreatitis, a pharmaceutical
15 composition can be administered intravenously, orally or
by any other method that distributes the compound
systemically, including to the pancreas.
In addition to cystic fibrosis, other
pathological conditions related to fluid buildup in
20 tissues are amenable to treatment by altering chloride
ion secretion. For example, swelling associated with
inflammation, infection or trauma, such as brain
swelling, can be alleviated by decreasing chloride ion
secretion (Berger et al., Acta Neurochir. SuRpl. (alien)
25 60:534 (1994); and Staub et al., J. Neurotrauma 11:679
(1994) ) .
In contrast to pathological conditions such as
cystic fibrosis, where chloride ion secretion is
abnormally low, other pathological conditions are
30 characterized, in part, by abnormally high levels of
chloride ion secretion. For example, high levels of
chloride ion secretion occur in secretory diarrhea. The
invasive enteric bacteria Salmonella, which causes
secretory diarrhea, is one of the most important causes
of childhood death in the developing world and of health
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problems in the developed world. More than 1.3 billion
cases of gastroenteritis and approximately 3 million
deaths occur each year due to Salmonella infections.
Because secretory diarrhea is associated with increased
chloride ion flux across the gut epithelia, decreasing
chloride ion secretion can alleviate symptoms of
secretory diarrhea. The skilled artisan would recognize
that methods of treating individuals with agonists of
inositol polyphosphates to alleviate symptoms of
secretory diarrhea are similar to those described above.
The invention provides compositions and methods
for altering chloride ion secretion across a cell
membrane by contacting a cell with a cell permeable
agonist or antagonist of inositol polyphosphates. The
invention also provides methods for altering chloride ion
secretion in an individual by administering to the
individual an agonist or antagonist of inositol
polyphosphates. The invention further provides a method
for alleviating a symptom associated with pathological
conditions associated with abnormal chloride ion
secretion by administering a pharmaceutical composition
containing an agonist or antagonist of inositol
polyphosphates. Thus, the compounds of the invention are
useful as medicaments for alleviating the signs and
symptoms of a pathological condition characterized, in
part, by abnormal chloride ion secretion.
The following examples are intended to
illustrate but not limit the present invention.
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EXAMPLE I
nP~rPa~P~ Calcium-mediated Chloride Ion Secretion by a
('P11 permeable Derivative of Ins (3. 4, 5. 6) PQ in e4 Cells
This example demonstrates that a cell permeable
derivative of Ins(3,4,5,6)Pq decreases calcium-mediated
chloride ion secretion in the colonic epithelial T99 cell
line by increasing the intracellular concentration of
Ins (3, 4, 5, 6) P4 .
Enantiomerically pure 1,2-Btu-Ins(3,4,5,6)PQ/AM
was synthesized to determine if 1,2-Btz-Ins(3,4,5,6)PQ/AM
mediates the uncoupling of chloride ion secretion from
intracellular calcium concentrations. Te4 cells (passages
18-48) were maintained as described previously (Weymer,
et al., -?_ Clin. Invest. 76:1828 (1985)), which is
incorporated herein by reference. After 7-10 days of
incubation on SNAP-WELLS (Corning Costar; Cambridge, MA),
monolayers had formed tight junctions. Cell monolayers
were preincubated with 100, 200 or 400 ~.M
Bt~-Ins (3, 4, 5, 6) Pq/AM for 30 min prior to mounting. After
an additional 30 min, 0.1 mM histamine was added and
chloride ion secretion was monitored. Chloride ion
secretion was measured as short circuit current (IS~)
across T89 monolayers grown to confluence and mounted in
Ussing chambers equipped with voltage clamps (Physiologic
Instruments; San Diego, CA). Data was acquired and
analyzed using "Acquire and Analyze" software
(Physiologic Instruments).
Maximum decreases of histamine-stimulated
chloride ion secretion was attained with 200 ~cM
1,2-Bt~-Ins(3,4,5,6)PQ/AM with no further decrease with
400 ~cM (Table II). This result is consistent with
previous studies showing that intracellular levels of
Ins ( 3 , 4 , 5 , 6 ) Pq near 4 ,uM, equivalent to 2 0 0 ~.~.M
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33
extracellular concentration, correspond to the onset of
maximum inhibition (Vajanaphanich, et al., supra, 1994).
Table II. Decreased Calcium-dependent Chloride Ion
Secretion by a Cell Permeable Ins(3,4,5,6)P4 Derivative.
TABLE II
BtZ=na(3,4,5,6)Peak 4lsc (% Control)n vs control (l00%)
P,/AM after histamine
(concentration)(100uM)
MAan t- SEM
100~tM 75.1 7.4 8 p<0.012
200M S7.2 15.1 6 p<0.037
400~tM 69.3 8.7 4 p<0.041
In contrast to the decrease in chloride ion
secretion by cell permeable derivatives of Ins(3,4,5,6)P4,
no inhibition of dibutyryl cyclic AMP acetoxymethyl ester
stimulated chloride ion secretion was observed (peak DIS
- 96.3 ~ 9.1% or control; mean ~ SEM; n=4). This result
is consistent with the observation that cyclic AMP-
mediated chloride ion secretion occurs through a chloride
ion channel distinct from the channel involved in
calcium-mediated chloride ion secretion (McRoberts et
al., J. Biol. Chem. 260:14163 (1985)).
These results demonstrate that increasing the
intracellular concentration of Ins(3,4,5,6)PQ by exposing
cells to cell permeable derivatives of Ins(3,4,5,6)P~
decreases calcium-mediated chloride ion secretion by
uncoupling chloride ion secretion from intracellular
calcium concentrations and that cell permeable
Ins(3,4,5,6)P4 derivatives are agonists of Ins(3,4,5,6)P4.
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34
EXAMPLE II
Decreased Calcium-mediated Chloride Ion
Secretion by a Cell Permeable Ins(3.4,5.6)P,; Derivative
in Rabbit Colon
This example demonstrates that increasing the
intracellular concentration of Ins(3,4,5,6)P4 using a cell
permeable derivative of Ins(3,4,5,6)P4 causes decreased
calcium-mediated chloride ion secretion in rabbit colonic
tissue.
The results obtained above using T:4 colonic
epithelial cells in culture were confirmed using a rabbit
colon model system (Frizzell et al., J. Membrane Biol.
27:297 (1976); and Frizzell and Schultz, Int. Rev.
Physiol. 19:205 (1979)), each of which is incorporated
herein by reference. Segments of rabbit colon were
excised and used as a source of rabbit colonic epithelia.
Briefly, segments of rabbit colon were washed with
Ringers solution containing 141 mM Na', 5 mM K',
1.2 mM Caz', 1.2 mM Mg'', 122 mM CI-, 25 mM HC03,
1.6 mM HPO4, 0.4 mM H~POq and 10 mM glucose. At 37oC, this
solution is pH 7.4 when gassed with 5o CO~ and 95o O,.
Epithelium was stripped of the underlying muscle layer
and mounted in modified SNAP WELLS (Frizzell et al.,
supra, 1976). Epithelia were preincubated with 200 ~M
1,2-Bt2-Ins(3,4,5,6)PQ/AM for 30 min at 37C and mounted
in Ussing chambers. Short circuit current, conductance
and resistance were measured at 4 sec intervals as
described in Example I. Peak IS~ was 50.9% ~ 10.4 of
controls which were incubated with vehicle (Figure 2;
mean ~ SEM, n=3; p<0.0S by Student's two tailed t-test).
The o control shown in Figure 2 represents ~IS~ over peak
I5~ in control monolayers stimulated with histamine.
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These results demonstrate that the cell
permeable derivative Btz-Ins (3, 4, 5, 6) PQ/AM acts as an
agonist of Ins(3,4,5,6)PQ by increasing the intracellular
concentration of Ins(3,4,5,6)PQ and decreases chloride ion
5 secretion in rabbit colon. These results also confirm
than the elevation of intracellular Ins(3,4,5,6)Pa
uncouples chloride ion secretion from intracellular
calcium concentration in colon epithelial tissue.
EXAMPLE III
10 Rey,~rsal of Ins(3,4.5.6)Pq Inhibition of Histamine-
stimulated chloride Ion Secreti~z Using Ins(3f4,5.6)PQ
Ant,~aonists
This example demonstrates that the inhibitory
effect of Ins(3,4,5,6)P' on histamine-stimulated chloride
15 ion secretion can be reversed by cell permeable
derivatives of Ins(3,4,5,6)Pa that function as antagonists
of Ins (3,4,5,6)P4.
The results shown in Examples I and II indicate
that increasing concentrations of Ins(3,4,5,6)PQ decreases
20 calcium-mediated chloride ion secretion. Therefore,
compounds that antagonize the function of Ins(3,4,5,6)PQ
in cells should reverse this decrease.
Initial studies were directed towards
understanding structural determinants of Ins(3,4,5,6)P4
25 responsible for the inhibitory effect on calcium-mediated
chloride ion secretion. A series of cell permeable
inositol polyphosphate derivatives were synthesized.
These derivatives contained either one or two non-
hydrolyzable chemical groups on the 1, 2 or 3 position.
30 T84 monolayers were preincubated for 30 minutes with
200 ~,M of 2-Bt-1-Me-Ins (3, 4, 5, 6) Pq/AM,
Me2-Ins(3,4,5,6)PQ/AM, Mez-Ins(1,4,5,6)Pq/AM or
2-Bt-3-Me-Ins(1,4,5,6)PQ/AM.
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Following the incubation, cells were mounted in
Ussing chambers. Calcium-mediated chloride ion secretion
was stimulated with histamine (10-' M). Control values
were the response to histamine in coincubated monolayers.
Data are represented as mean peak DIS~ ~ SEM, expressed as
% control for 8 to 10 experiments. Significant
differences were identified using the Student's two-
tailed t test.
As shown in Table III, no inhibitory effect
occurred in cells treated with any of the derivatives.
These results indicate that the hydrogen-bonding donor
potential at the 1 and 2 position participates in
mediating the inhibitory effect of Ins(3,4,5,6)P4 on
calcium-mediated chloride ion secretion. The scyllo
derivative of the cell permeable Btu-Ins(3,4,5,6)Pq/AM was
at least as effective an inhibitor as the myo derivative,
indicating that the directionality of the 2-hydroxyl
position is irrelevant for inhibitory activity.
T94 cells were preincubated with cell permeant
inositol polyphosphates as described above to determine
if any of the inositol polyphosphate derivatives function
as antagonists of Ins(3,4,5,6)Pq inhibition of calcium-
mediated chloride ion secretion. Following histamine
stimulation, short circuit current was measured. As
shown in Figure 3, incubation with
Bt2-Ins(3,4,5,6)P4/AM decreased histamine-stimulated
chloride ion secretion relative to control cells. The %
control shown in Figure 3 represents ~IS~ over peak I5~ in
control monolayers stimulated with histamine.
However,preincubation with 2-Bt-1-Me-Ins(3,4,5,6)P9/AM
reversed the inhibitory effect of Bt~-Ins(3,4,5,6)PQ/AM.
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Table III. Reversal of Ins(3,4,5,6)P4-mediated Decrease
in Chloride Ion Secretion by Derivatives of Tns(3,4,5,6)P,
TABLE III
BtzIns(3,4,5,6)Antagonists Peak ~Isc n vs control
p~/p,M (~ Control) (100$)
after
(concentration) histamine
( 100uht)
Maan t SEM
100~tM 75.1 7.4 8 p<0.012
200M 57.2 15.1 6 p<0.037
400~tM 69.3 8.7 4 p<0.041
- 2-Ht-1-Me- 87.3 6.6 8 ns
Ins(3,4,5,6)
P,/AM (200~tM)
- Me~-Ins(3,4,5,6)103.9 11.1 8 ns
PQ/AM (200~.tM)
- Me2- 120. B 10 . 8 ns
6
Ins{1,4,5,6)
PQ/AM (2001M)
- 2-Bt-3-Me- 105.5 3.7 10 ns
Ins(1,4,5,6)
P4/AM (200~tM)
200M 2-Bt-1-Me- 117.7 10.6 8 ns
Ins(3,4,5,6)
P,/AM (200~tM)
200M Me,- 107.13 10.2 8 ns
Ins(3,4,5,6)
P,/AM (200M)
200.M 2-Bt-3-Me- 69.7 5.04 B p<0.0005
Ins(1,4,5,6)
P4/AM (200~tM)
200uM Me2- 68.4 7.3 8 p<0.003
Ins(1,4,5,6)
P4/AM (200~tM)
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Bt2Ins(3,4,5,6)Antagonists Peak ~Isc n vs control
P,/AM (% Control) (100%)
after
(concentration) histamine
(100uM)
Mean SEM
- Bt,AMP/AM 96.3 9.1 4 ns
(2~tM}
- Bts-Ins(2,4,5,6)137.9 20.7 4 ns
P, /AM
(400.M)
- D,L-1-Bt-2-Me- 114.2 23.7 8 ns
Ins(3,4,5,6)
P4/AM
(800M)
200~tM D,L-1-Bt-2-Me- 96.7 14.8 3 ns
Ins(3,4,5,6)
P4 /AM
(400~CM)
5 - D,L-1,2-C1_- 89.39 7.8 8 ns
dideoxy-
Ins(3,4,5,6)
P 4 SAM
(400 /~M)
The effect of various inositol polyphosphate derivatives
on histamine-stimulated chloride ion secretion are shown
in Table III. The Ins(3,4,5,6)PQ derivatives
2-Bt-1-Me-Ins (3, 4, 5, 6) PQ/AM and Me~-Ins (3, 4, 5, 6) PQ/AM,
reversed the inhibitory effect of Bt~-Ins(3,4,5,6)P4/AM on
histamine-stimulated chloride ion secretion. In
contrast, derivatives of Ins(1,4,5,6)Pq,
Mez-Ins (1, 4, 5, 6) Pa/AM and 2-Bt-3-Me-Ins (1, 4, 5, 6) Pq/AM, had
essentially no effect. These results indicate that the
reversal of the Bt,-Ins(3,4,5,6)PQ/AM decrease on chloride
ion secretion is stereospecific and demonstrate that
these alkyl derivatives of Ins(3,4,5,6)P9 function as
antagonists of Ins(3,4,5,6)P4.
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Other derivatives were also tested.
Preincubation with Btz-Ins(1,4,5,6)PQ/AM did not inhibit
chlaride ion secretion and in fact led to a slight
increase in chloride ion secretion. The Ins(3,4,5,6)PQ
derviative, D,L-1,2-C12-dideoxy-Ins(3,4,5,6)Pq/AM had a
slight inhibitory effect on chloride ion secretion.
D,L-1-Bt-2-Me-Ins(3,4,5,6)P4/AM did not inhibit chloride
ion secretion and did not reverse the inhibition of
Bt2-Ins (1, 4, 5, 6) PQ/AM.
Elevation in intracellular calcium
concentration leads to increased chloride ion secretion.
To ensure that none of the effects of the inositol
polyphosphate derivatives on chloride ion secretion were
due to changes in calcium levels, the concentration of
intracellular calcium was measured in cells treated with
the inositol polyphosphate derivatives. As shown in
Figure 4, Bt~-Ins (1, 4, 5, 6) P4/AM did not elevate
intracellular calcium or the calcium response to
thapsigargin. None of the compounds tested altered
intracellular calcium levels by themselves or modified
levels of intracellular calcium stimulated with carbachol
or thapsigargin. Therefore, the effects of inositol
polyphosphate derivatives on chloride ion secretion are
not due to any effect on intracellular calcium
concentrations.
These results demonstrate that cell permeable
derivatives of Ins(3,4,5,6)P4 reverse the decrease in
calcium-mediated chloride ion secretion resulting from
increased intracellular concentrations of Ins(3,4,5,6)P9
due to treatment of cells with 1,2-Btu-Ins(3,4,5,6)PQ/AM,
thereby enhancing calcium-mediated chloride ion secretion
from these cells. In contrast, derivatives of the
stereoisomer Ins(1,4,5,6)PQ do not affect inhibition of
chloride ion secretion mediated by Ins(3,4,5,6)P4. Thus,
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cell permeable derivatives of Ins(3,4,5,6)P9 function as
Ins (3, 4, 5, 6) Pq antagonists.
EXAMPLE IV
Reversa7~ of Carbachol-mediated Decreases in Chloride Ion
5 Secretion Using Ins(3.4.5.6)P4 Antagonists
This example demonstrates that carbachol-
mediated decreases in chloride ion secretion can be
reversed by cell permeable Ins(3,4,5,6)Pq derivatives that
function as antagonists of Ins(3,4,5,6)P4.
10 In Example III above, intracellular
Ins(3,4,5,6)PQ was increased by the addition of the cell
permeable derivative, Bt,-Ins (3, 4, 5, 6) Pq/AM, which is
hydrolyzed by endogenous esterases to produce
Ins(3,4,5,6)P4. In this example, intracellular
15 Ins(3,4,5,6)PQ is increased by prolonged treatment of
cells with carbachol.
T84 colonic epithelial cells were treated with
D,L-1,2-cyclohexylidene-Ins(3,4,5,6)P4/AM,
1-Bu-2-Bt-Ins (3, 4, 5, 6) P4/AM, Buz-Ins (3, 4, 5, 6) P9/AM,
20 3-Bt-2-Bu-Ins (1, 4, 5, 6) Pq/AM, Bu~-Ins (1, 4, 5, 6) P~/AM,
1-Bt-2-Bu-Ins(3,4,5,6)Pq/AM or 3-Bu-2-Bt-Ins(1,4,5,6)Pq/AM
and the short circuit current was measured.
One set of cells was preincubated for 30 min
with an inositol polyphosphate derivative. The other two
25 sets of cells were preincubated with vehicle, alone.
Measurement of IS~ was initiated at time 0. At 5 min,
carbachol (10-4 M) was added to one set of cells
preincubated with vehicle alone (designated "Carbachol"
in Figure 5) and to the set of cells preincubated with
30 inositol polyphosphate derivatives (designated
"Carbachol + D,L-1,2-Cyclohexylidene-Ins(3,4,5,6)PQ/AM" in
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Figure 5A). At 30 min, 1 ~M thapsigargin was added.
Data were collected for n = 4 to 6 experiments.
Figure 5A shows an experiment using
D,L-1,2-cyclohexylidene-Ins(3,4,5,6)PQ/AM. The addition
of carbachol caused a transient increase in short circuit
current in cells preincubated with the Ins(3,4,5,6)Pq
derivative and in cells treated with carbachol alone. At
30 min, following the return of ~IS~ to control levels,
1 ~M thapsigargin was added to stimulate increased
intracellular calcium. The expected increase in ~IS~ was
observed with thapsigargin treated control cells, whereas
~IS~; was attenuated in cells receiving prolonged treatment
with carbachol. Pretreatment with
D,L-1,2-cyclohexylidene-Ins(3,4,5,6)P4/AM reversed the
inhibitory effect of carbachol on calcium-mediated
chloride ion secretion. Thus,
D,L-1,2-cyclohexylidene-Ins(3,4,5,6)PQ/AM functions as an
antagonist of Ins(3,4,5,6)P4.
As shown in Figure 5, the Ins(3,4,5,6)PQ
derivatives, D,L-1,2-cyclohexylidene-Ins(3,4,5,6)PQ/AM,
1-Bu-2-Bt-Ins(3,4,5,6)Pq/AM and Bu--Ins(3,4,5,6)P4/AM,
reversed the carbachol-mediated inhibition of chloride
ion secretion stimulated by thapsigargin, indicating that
these Ins(3,4,5,6)PQ derivatives function as antagonists
of Ins (3, 4, 5, 6) P9 (Figure 5A to 5C, respectively) . In
contrast, the 1-Bt-2-Bu-Ins(3,4,5,6)PQ/AM as well as
3-Bt-2-Bu-Ins (1, 4, 5, 6) Pq/AM, 2, 3-Bu~-Ins (1, 4, 5, 6) P~/AM and
3-Bu-2-Bt-Ins(1,4I,5,6)PQ/AM did not reverse carbachol-
mediated inhibition of chloride ion secretion stimulated
by thapsigargin (Figure 5D to 5G, respectively).
These results demonstrate that cell permeable
derivatives of Ins(3,4,5,6)Pq reverse the inhibitory
effect of prolonged treatment with carbachol on calcium-
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mediated chloride ion secretion and, therefore, function
as antagonists of Ins(3,4,5,6)P4.
EXAMPLE V
Elevation of Intracellular Ins(1,3.4)P3 Levels Increases
Ins(3.4.5.6)PQ and Decreases Calcium-mediated
Chloride Ion Secretion
This example demonstrates that treatment of
cells with a cell permeable derivative of Ins(1,3,4)P~
increases Ins(3,4,5,6)P4, thereby decreasing calcium-
mediated chloride ion secretion.
Almost 30 inositol polyphosphates have been
identified in cells. Ins(1,3,4,5)PQ is converted to
Ins(1,3,4)P3 by a 5' inositol polyphosphate phosphatase.
In an in vitro assay system, Ins(1,3,4)P3 inhibited
inositol 1 kinase, which catalyzes the conversion of
Ins(3,4,5,6)PQ to Ins(1,3,4,5,6)PS (Tan et al., J. Biol.
Chem. 272:2285 (1997)), which is incorporated herein by
reference.
The cell permeable derivative
D,L-Bt3-Ins(1,3,4)P3/AM was synthesized. Colonic
epithelial T~4 cells were preincubated for 30 min with 400
~M D,L-Btu-Ins(1,3,4)P3/AM prior to mounting in Ussing
chambers and measuring ~IS~ . At 10 min, carbachol ( 10-'' M)
was added to transiently stimulate calcium-mediated
chloride ion secretion. Data were the average of 8
experiments with p<0.04, unpaired, two-sided student's t-
test.
Incubation of cells with 400 ~.M
D,L-Bt3-Ins(1,3,4)P3/AM caused a three-fold increase in
Ins(3,4,5,6)PQ measured in cells labeled with
(3H)-inositol, indicating that Ins(1,3,4)P3 increased
Ins ( 3 , 4 , 5 , 6 ) P4 in vivo. Control cells , which were
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43
preincubated with vehicle had a peak ~IS~ of I5.2 ~ 2.8.
Cells preincubated with D,L-Bt3-Ins(1,3,4)P3/AM had a peak
DIs~ of 7.9 ~ 1.6. Thus, calcium-mediated chloride ion
secretion was decreased approximately 50% by treating
cells with the cell permeable D,L-Btj-Ins(1,3,4)P3/AM
derivative.
These results demonstrate that cell permeable
Ins(1,3,4)P3 derivatives can increase the intracellular
concentration of Ins(3,4,5,6)P4, resulting in decreased
calcium-mediated chloride ion secretion and, therefore,
function as agonists of Ins(3,4,5,6)P4.
EXAMPLE VI
Salmonella Invasion of Human Colonic Epithelial Cells
Tnr~reases the Intracellular Concentration of
Ins(1,4,~,6)P4
This example demonstrates that invasion of
human colonic epithelial cells by Salmonella increases
concentrations of intracellular Ins(1,4,5,6)P4.
Invasive enteric bacteria enter and penetrate
the intestinal epithelium to gain access to the
underlying mucosa and initiate systemic infection.
Previous studies showing that Salmonella invasion of
epithelial cells caused an increase in inositol
polyphosphate turnover indicated that inositol
polyphosphates could play a role in the response of
epithelial cells to bacterial invasion (Ruschkowski et
al., FEMS Microbiol. Lett. 74:l21 (1992)). To
characterize changes in inositol polyphosphates induced
by bacterial invasion, the effect of invading bacteria on
colonic epithelial cells was assessed.
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Confluent colonic epithelial T84 cells in 6 well
plates were incubated for 4 days with 50 ~.Ci/well of
(3H)-inositol in inositol-free 50% Dulbecco's modified
Eagle's (DME) medium and 50% Ham's F12 media supplemented
with 5% dialyzed newborn calf serum. Cultures were
washed three times with prewarmed medium (50% DME,
50o Ham's F12 and 1 mg/ml bovine serum albumin) and
infected with 5x108 bacteria/well in the same media for
various periods of time. Cultures were washed twice with
ice-cold phosphate buffered saline (PBS) and lysed for 5
min on ice in 10% trichloroacetic acid and 10 mM phytic
acid. Extracts were neutralized using FREON/alamine and
resolved on an Adsorbosphere SAX column to separate the
('H)-inositol polyphosphates. Radioactive peaks were
quantitated as described previously using an HPLC
equipped with an on-line radioactivity detector
(Kachintorn et al., supra, 1993). To quantitate
enantiomers unresolved on HPLC, the peak corresponding to
(3H) -Ins (3, 4, 5, 6) PQ and Ins (1, 4, 5, 6) Pq was separated from
a11 other (3H)-InsP4 isomers by HPLC, desalted, then
incubated with partially purified Ins(1,4,5,6)P4 3-kinase
with an internal standard of (32P)-labeled Ins(1,4,5,6)PQ
as described previously (Vajanaphanich et al., supra,
1994). By comparing the relative amounts of ('H)- and
(3zP) -labeled Ins (1) 3, 4, 5, 6) PS formed, the ratio of
(3H) -Ins (3, 4, 5, 6) PQ to (3H) -Ins (1, 4, 5, 6) Pq in the original
peak was determined.
Monolayers of T8q cells were labeled with
(3H)-inositol and, infected for varying periods of time
with Salmonella dublin. During the 60 min period
immediately following infection, there were no
significant changes in the levels of
myo-inositol hexakisphosphate (InsP6) or other inositol
polyphosphates such as Ins(1,4,5)P3 that typically
accumulate when phospholipase C is activated. In
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contrast, levels of unresolved enantiomers
( 3H ) - Ins ( 3 , 4 , 5 , 6 ) PQ and ( jH ) - Ins ( 1 , 4 , 5 , 6 ) PQ
increased
within 10 minutes after infection with S. dublin.
Approximately 85% of this peak corresponded to
5 (3H)-Ins(1,4,5,6)P4. A maximum 14-fold increase over
control uninfected cells was reached 30-40 minutes after
infection (see Table IV) . Levels of (3H) -Ins (1, 4, 5, 6) P9
decreased slowly after 40 min and returned to near
baseline by 3 h after infection (see Table IV). Similar
10 observations were made using another human intestinal
epithelial cell line, LS174T. (3H)-Ins(1,4,5,6)PQ was
found to increase 11.3-fold after S. dublin infection,
indicating that the changes in cellular inositol
polyphosphates after infection represent a general
15 response of epithelial cells.
Infection of T89 cells with another invasive
Salmonella strain, Salmonella typhi BRD691, also
increased ( 3H) -Ins ( 1, 4 , 5 , 6 ) PQ levels ( see Table IV) . In
contrast, a mutant strain of S. dublin, SB133, that
20 attaches normally to epithelial cells but does not invade
them, increased (3H)-Ins(1,4,5,6)PQ levels only minimally,
indicating that invasion of host cells by Salmonella was
required for this response. However, bacterial invasion
alone was not sufficient to increase Ins(1,4,5,6)PQ
25 levels, since infection of T84 cells with several other
invasive gram negative bacteria, including Shigella
flexner.i, Shigella dysenteriae, Yersinia enterocolitica
and enteroinvasive Escherichia coli (serotype 029:NM),
caused only small increases in (3H)-Ins(1,4,5,6)Pq levels.
30 Furthermore, addition of non-invasive gram negative
bacteria such as enterohemorrhagic E. coli (serotype
O157) or a non-pathogenic E. coli (DHSa) to T54 monolayers
had little effect on (3H)-Ins(1,4,5,6)Pq levels. Addition
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of 10 ~g/ml bacterial lipopolysaccharide (LPS) had no
effect on (3H) -Ins (1, 4, 5, 6) Palevels.
Table IV. Increase in Ins(1,4,5,6)P4 Levels after
Salmonella Infection of Epithelial Cells
Bacteria Added Time after('H)-(1,9,5,6)P) n
levels
(Ratio Infected/Control)
Salmonella dublin lane 30 13.9 0.8 4
Salmonella dublin lane 60 10.3 1.3 5
Salmonella dublin lane 120 3.6 2
Salmonella dublin lane 180 1.9 2
Salmonella typhi BRD691 30 10.5 2
Salmonella dublin SB133 30 1.9 0.1 3
( invA)
Shigella flexneri 60 1.9 2
Shigella dysenteriae 60 2.3 2
Yersinia enterocolitica 60 2.6 0.1 3
Escherichia coli 029:NM 60 2.0 0.1 5
Escherichia coli O157 60 2.3 0.2 4
Escherichia coli DHSa 60 2.1 0.2 4
LPS 60 1.0 2
These results demonstrate that invasion of
colonic epithelial cells with Salmonella increases the
intracellular concentration of Ins(1,4,5,6)P4. Increased
chloride ion secretion occurs in secretory diarrhea, a
pathological manifestation of Salmonella infection,
suggesting a correlation with Ins(1,4,5,6)P9
concentrations.
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EXAMPLE VII
Ins(1.4,5,6)PQ Reverses EGF-Induced Inhibition of Chloride
Ion Secretion
This example demonstrates that increasing the
intracellular concentration of Ins(1,4,5,6)Pq by
incubating cells with a cell permeable Ins(3,4,5,6)Pq
derivative reverses inhibition of chloride ion secretion
induced by epidermal growth factor (EGF).
In Examples III and IV above, Ins(1,4,5,6)P4
derivatives had no effect on the Ins(3,4,5,6)P4-mediated
decrease in chloride ion secretion. To test whether
Ins(1,4,5,6)P9 affects EGF induced inhibition of chloride
ion secretion, T84 cell monolayers were preincubated for
30 min with Bt~Ins (1, 4, 5, 6) P4/AM. Measurement of ~IS~ was
initiated (see Example I) and, after 5 min of
measurements, 16.3 nM EGF was added to the basolateral
surface of the cells. After an additional 15 min
incubation, 100 ~M carbachol was added to acutely elevate
intracellular calcium concentrations. Controls also were
stimulated with carbachol but were not pretreated with
EGF. Data are the means of duplicate measurements from a
representative experiment (total of three experiments).
As shown in Figure 6 and Figure 7 (panels a and
b), EGF inhibited carbachol stimulated chloride ion
secretion. Preincubation with BtZIns(1,4,5,6)Pq/AM, a
cell permeable derivative that is hydrolyzed to
Ins(1,4,5,6)PQ by endogenous cellular esterases,
significantly reversed EGF induced inhibition of chloride
ion secretion. In contrast, the inhibitory function of
EGF was not attenuated by addition of a cell permeable
derivative of Ins(1,3,4,5,6)PS (Figure 7, panel C).
Addition of a cell permeable Ins(3,4,5,6)Pq derivative,
the enantiomer of Ins(1,4,5,6)P4, also did not attenuate
EGF inhibitory function on chloride ion secretion.
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Addition of Bt~Ins(1,4,5,6)P4/AM did not reverse EGF
inhibition of cyclic AMP-mediated chloride ion secretion,
indicating that the action of Ins(1,4,5,6)PQ was specific
for calcium-mediated chloride ion secretion.
These results demonstrate that increased levels
of Ins(1,4,5,6)P4, delivered to cells as a cell permeable
derivative, enhances calcium-mediated chloride ion
secretion by reversing EGF-mediated inhibition of
chloride ion secretion.
EXAMPLE VIII
Ins ( 1 , 4, 5 , 6 ) P4 Reverses PtdInsP3 Decreases in Calcium
mediated Chloride Ion Secretion
This example demonstrates that a cell permeable
derivative of PtdInsP3 decreases calcium-mediated chloride
ion secretion. A cell permeable Ins(1,4,5,6)Pq derivative
reverses the PtdInsP3-mediated decrease in chloride ion
secretion.
T84 cell monolayers were preincubated for 30 min
with PtdInsP3/AM and DIS~ was measured. As shown in
Figure 8, increasing concentrations of PtdInsP,/AM
inhibited carbachol-stimulated chloride ion secretion,
indicating that an inositol polyphosphate distinct from
Ins(3,4,5,6)PQ also inhibits calcium-mediated chloride ion
secretion.
Additional cell permeable derivatives of the PI
3-kinase product PtdInsP3 were synthesized and tested for
the effect on chloride ion secretion. Cells were
preincubated with diCib-Bt-PtdInsP3/AM or
diCs-Bt-PtdInsP3/AM and ~IS~ was measured as described in
Example VII.
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As shown in Figure 7, pretreatment of T84 cells
with diCl6-Bt-PtdInsP3/AM (Figure 7d) inhibited calcium-
mediated chloride ion secretion by up to 740. Similarly,
dice-BtPtdInsP3/AM inhibited chloride ion secretion by
790. No inhibition of calcium-mediated chloride ion
secretion was observed when cells were pretreated with
PtdInsP3, which is not expected to enter cells. These
cell permeable derivatives of PtdInsP3 had no effect on
calcium levels after carbachol stimulation. The level of
decrease observed with these derivatives of PtdInsP, was
comparable to that observed with EGF treatment of cells.
Furthermore, addition of EGF to monolayers preincubated
with cell permeable PtdInsP: derivatives did not result in
additional inhibition of calcium-mediated chloride ion
secretion, indicating that PtdInsP~ and EGF function
through the same mechanism.
Teq cells also were preincubated with cell
permeable Ins(1,4,5,6)PQ derivatives to determine whether
Ins(1,4,5,6)P4 can reverse PtdInsP,-mediated decreases in
chloride ion secretion. As shown in Figure 7d,
preincubation of T84 monolayers with membrane-permeable
Ins(1,4,5,6)P4 derivatives partially reversed PtdInsP~-
mediated decreases in chloride ion secretion.
These results demonstrate that PtdInsP~
decreases calcium-mediated chloride ion secretion. In
addition, Ins(1,4,5,6)P4 reverses PtdInsP, decreases in
ca'~cium-mediated chloride ion secretion.
Example IX
Chloride Ion Transport in Human Nasal Epithelia
This example provides a primary human nasal
epithelial cell system useful as a model for examining
the effectiveness of compounds to alter chloride ion
secretion in respiratory epithelia.
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Primary human nasal epithelia are obtained by
biopsy. For bioelectric and calcium measurement in
monolayers, primary human nasal epithelial cells are
plated on porous Transwell Coll filters (pore diameter,
5 0.4 /.~.M; Costar) affixed to O-rings and studied 5-7 days
after seeding, a time coincident with the development of
maximal transepithelial potential difference. Cells are
maintained in serum-free Hams' F12 medium supplemented
with insulin ( 10 ,ug/ml ) , transferrin ( 5 E.cg/ml ) ,
10 endothelial cell growth supplement (3.75 ,ug/ml),
hydrocortisone (5 nM)~ endothelial cell growth supplement
(3.75 ,ug/ml), triiodothyronine (30 nM) and 1 mM CaCl~.
Human nasal epithelial monolayers are mounted in
miniature Ussing chambers, 5-7 days after plating.
15 Transepithelial potential difference is
monitored by a Voltage-Clamp/Pulse Generator (Physiologic
Instruments, San Diego, California) and the bioelectrics
recorded on a two channel recorder. Monolayers are
exposed to sodium free Ringers to calculate changes in
20 transepithelial chloride resistance, a measure of the
change from its native sodium absorptive state to a
chloride ion secretory state. For intracellular calcium
measurements, monolayers are loaded with Fura-2 and
mounted over an objective of a microscope coupled to a
25 microfluorimeter. The fluorescence intensity ratio is
collected either from a field of 30-40 cells or from a
single cell. DIsc to intracellular calcium levels are
normalized in each preparation. To ensure that the
results reflect response from true monolayers, monolayers
30 are routinely fixed and cross-sectional segments
examined.
Primary human nasal epithelial cells are useful
for treating with antagonists of Ins(3,4,5,6)PQ and
PtdInsP3, such as those described in Examples III, IV, VII
35 and VIII. Compounds effective at enhancing chloride ion
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secretion in primary human nasal epithelia are good
candidates for alleviating symptoms of cystic fibrosis
such as pulmonary insufficiency.
EXAMPLE X
Generation of CFTR- Ta4_Cells
This example provides a method for preparing
CFTR- T8q cells, which are useful as a model of cystic
fibrosis.
To screen Ins(3,4,5,6)P4 derivatives and to
ultimately determine if the CFTR regulates the expression
of elements involved in Ins(3,4,5,6)Pq-mediated inhibition
of chloride ion secretion, CFTR- T84 cells are generated.
The CFTR genes) in T~4 cells are inactivated
using a double selection approach, originally developed
to generate gene knockout mutations in mouse embryonic
stems cells (ES cells) (Mansour et al., a re 336:348
(1988)), which is incorporated herein by reference.
Mutagenic CFTR gene targeting constructs are generated in
pSSC-9 vector (Chauhan and Gottesman, Gene 120:281
(1992)), which is incorporated herein by reference. This
vector carries the neomycin resistance gene, driven by a
thymidine kinase promoter, flanked on both sides by the
hsv-tk genes. In addition, pSSC-9 carries convenient
cloning sites on both sides of the gene, which can be
used to insert gene targeting segments, and two Sfil
restriction sites, which can be used to excise the
mutagenic cassette in a linear form. Selection for the
expression of the neomycin gene, by resistance to G418,
allows screening for stably transfected clones.
Subsequent selection against hsv-tk expression, using
cyclovir, expands the clones in which the construct
integrated via homologous recombination into the targeted
site (Mansour et al., supra, 1988). An 8.5 kb fragment
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52
of the CFTR gene (TE 2611E8.5, containing exon 21) is
used as a source of CFTR gene sequences (Rommens et al.,
Science 245:1059 (1989); and Rommens et al., Am. J. Hum.
Genet. 45:932 (1989)), each of which is incorporated
herein by reference. Alternatively, the polymerase chain
reaction (PCR) can be used to directly clone appropriate
sequences from T89 cells. Several PCR primers for
amplification of CFTR gene segments have been described
(Zielinski et al., Genomics 10:214 (1991)), which is
incorporated herein by reference. Fragments at least 2-4
kb long should be amplified to ensure sufficient sequence
homology for homologous recombination. PCR primers carry
restriction site extensions compatible with pSSC-9
cloning sites.
Mutagenic constructs are transfected into T8q
cells, using DNA transfection methods, for example,
calcium phosphate or electroporation-based protocols
(Molecular Biology, A Laboratory Manual, 2nd ed.,
Sambrook et al., Eds., Cold Spring Harbor Laboratory
Press, Plainview, NY (1989)), which is incorporated
herein by reference. Stable transfectants are selected
using G418, expanded and subjected to the second round of
selection using cyclovir. Resistant clones, presumably
derived by homologous recombination of the vector into
the CFTR locus, are subjected to an additional round of
selection, using increasing amounts of G418. This
approach, developed to facilitate generation of double
knockout mutants in mice using ES cells, can be also
applied for selection of homozygous mutations in cell
lines (Chen et al., Proc. Natl. Acad. Sci. USA 90:4528
(1993); and Mortenson, Hy~ertens~,on 22:646 (1993)), each
of which is incorporated herein by reference. Selected
clcznes are analyzed by Southern blot and PCR analyses to
verify that the neo cassette was inserted into the CFTR
3 5 gene ( s ) .
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A "double construct method" can alternatively
be used in which the mutagenesis of target genes is
performed sequentially, using two different mutagenic
constructs (Mortenson, supra, 1993; Feldhaus et al.,
EMBO J. 12:2763 (l993); and Porter and Itzhaki, Eur. J.
Biochem. 218:273 (1993)), each of which is incorporated
herein by reference. These constructs carry a neo gene
or a gpt gene cassette, allowing for sequential
transfection and selection of the neo gene using G418 and
selection of the gpt gene using mycophenolic acid plus
xanthine. Double knockout CFTR mutants of T~Q cells are
used to assess the efficacy of Ins(3,4,5,6)P~ and PtdInsP~
antagonists.
These results demonstrate the generation of a
CFTR- mutant of Te4 cells, which can be used as a model of
cystic fibrosis. These cells provide a genetic
background similar to that found in epithelial cells of
cystic fibrosis patients. Antagonists of Ins(3,4,5,6)Pq
and PtdInsP3, which effectively enhance chloride ion
secretion in CFTR- T84 cells, are good candidates for
enhancing chloride ion secretion,in cystic fibrosis
patients.
EXAMPLE XI
Preparation of Cell Permeable Inositol Polyphosphate
Derivatives
This example describes the synthesis of cell
permeable inositol polyphosphate derivatives.
The synthetic reactions for some cell permeable
inositol polyphosphate derivatives were described
previously (Roemer et al., supra, 1996; and Roemer et
al., supra, l995).
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The synthetic reactions of additional inositol
polyphosphate derivatives are described below. Figure 9
shows the structure of cell permeable PtdInsP~ and
diCl6-Bt-PtdInsP3/AM. Figure 10 shows a schematic of the
synthesis of PtdInsP3/AM derivatives. Figure 11 shows a
schematic of the synthesis of 1,2-cyclohexylidene-
Ins(3,4,5,6)Pq//AM. Figure 12 shows structures of myo-
inositol 1,3,4-trisphosphate and 2,5,6-Bt3-
Ins(1,3,4)P3/AM. Only the D-configuration of the
compounds is shown. Figure 13 shows a schematic for
synthesis of cell permeable inositol polyphosphate
derivatives. In Figure 13, the following conditions were
used: (i) Bt~O, DMAP, pyr. ; (ii) TFA, CH3CN / HzO; (iii) a
Bu~SnO, toluene, refl., b BnBr, CsF, DMF; (iv) Bt~O, DMAP,
pyr. , v Pd / C (10%) , AcOH; (vi) a (Bn0) ~PNiPr~,
tetrazole, CH3CN, b AcO00H, -40~C; (vii) Pd / C (l00),
AcOH; and (viii) AMBr, DIEA, CH3CN.
A11 chemical reagents were obtained in the
highest purity available. Where necessary, solvents were
dried and/or distilled before 'use. Acetonitrile was
0
distilled from phosphorus(V) oxide and stored over 3 A
molecular sieves, as was dimethylformamide (DMF).
0
Pyridine and toluene were stored over 4 A molecular
sieves Ethyl-diisopropylamine (DIEA) was dried over
sodium wire. Palladium on charcoal (l00) and
trifluoroacetic acid were from Acros Chemie. Dibenzyl
~l-diisopropylphosphoramidite, peracetic acid (32% v/w),
tetrazole, sodium hydride and acetoxymethyl bromide were
from Aldrich. Milwaukee, Wisconsin Butyric anhydride,
DIEA and tris(triphenylphosphin)-rhodium (I)-chloride
were from Merck Benzyl bromide, allyl bromide, butyl
iodide', cesium fluoride and 4-dimethylamino pyridine
(DMAP) were from Fluka. The ion-exchange resin Dowex 50
WX 8, H+-form, was from Serva, Heidelberg Germany. A11
other reagents were from Riedel-de Haen.
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High performance liquid chromatography (HPLC)
was performed on a LDC/Milton Roy Consta Metric III pump
with a LDC/Milton Roy UV Monitor D (254 nm) or a Knaur
refractive index detector. The analytical column was a
5 Merck Hibar steel tube (250 mm x 4 mm) filled with RP 18
material (Merck, LiChrosorb:l0 E,cm). Preparative HPLC was
performed using a Shimadzu LC 8A pump with a preparative
LDC UV III Monitor (254 nm) or a Knaur refractive index
detector and a Merck Prepbar steel column (25~ mm x 50
10 mm) filled with RP 18 material (Merck, LiChrospher 100,
10 ~cm). The eluents were methanol-water mixtures;
composition are given in % methanol (MeOH).
1H-NMR and 31P-NMR spectra were recorded on a
Brucker AM 360 AM spectrometer Chemical shifts were
15 measured in ppm relative to tetramethylsilane for -H NMR
spectra and external 85% H3P0, for '1P NMR spectra. J-
values are given in Hz. Mass spectra were recorded using
a Finnigan Mat 8222 mass spectrometer with fast atom
bombardment (FAB) ionization. High resolution masses
20 were determined relative to known compounds with a mass
not differing more than 10%. Melting points (MP)
(uncorrected) were determined using a Buchi B-540
apparatus. Optical rotations were measured at the sodium
D-line in a 10 cm cell with a Perkin-Elmer 1231
25 polarimeter. Ultrafiltration of the palladium/charcoal
catalyst was performed with a Sartorius filtration
apparatus SM 162 O1 using filters from regenerated
cellulose (Sartorius, SM 116 04)Sartorius Edgewood, New
York. Element analysis were performed by
30 Mikroanalytisches (Labor Beller, Gottingen, Germany).
D-1,4,5,6-tetra-Q-benzyl-mvo-inositol (ent-9)
and D-3,4,5,6-tetra-O_-benzyl-mvo-inositol (~) were
synthesized as described before (Roemer et al., supra,
1996). The compound rac-1,2-Di-Q-cyclohexylidene-m_vo-
35 inositol was synthesized by the method of Angyal and Tate
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56
(Angyal and Tate, J. Chem. Soc. 1965:6949 (1965)), which
is incorporated herein by reference.
C",~neral procedure for phosphor~rlation. The
selectively protected r~,vo-inositol derivative and
tetrazole were dissolved under argon in dry acetonitrile
before dibenzyl N,N-diisopropylphosphoramidite was added.
After stirring at room temperature for the indicated
time, the reaction mixture was cooled to -40~C and
peracetic acid (32% v/w; 1 mol equivalent for each mol
equivalent of phosphoramidite) was added. After the
mixture has reached room temperature the solvent was
removed under reduced pressure and the residual oil was
purified by preparative HPLC to give the desired inositol
tetrakisphosphate derivative.
General procedure for removina the benz~l
groups by hydrogenolysis. The fully protected m_vo-
inositol tetrakisphosphate or the tetrabenzyl-inositol,
respectively, in acetic acid were vigorously stirred with
palladium on carbon (10%; 0.1 mol palladium for each mol
of benzyl groups) under an hydrogen atmosphere in a self
built hydrogenation apparatus for the indicated time.
The catalyst was removed by ultrafiltration and the
filtrate was freeze dried to give the respective product.
General x~rocedure for the introduction of
a.cetox et 1 esters. The thoroughly dried inositol
tetrakisphosphate derivative (free acid) was suspended in
dry acetonitrile under argon~before dry DIEA (2.25 mol
DIEA for each mol of hydroxy groups) and acetoxymethyl
bromide (1 mol equivalent for each mol equivalent of
DIEA) were added. After stirring of the mixture at room
temperature in the dark for 4 days a11 volatile
components were evaporated off under reduced pressure and
the crude residue was purified by preparative HPLC with
the solvent specified to give the inositol
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57
tetrakisphosphate octakis (acetoxymethyl) ester as a
syrup.
D-3 4 5 6-Tetra-O benz~l-1-O-butvl-myo-inositol
l10) Dry ~ (250 mg, 463 ~.cmol) and dry dibutyltin oxide
(116 mg, 167 ~mo1) were heated under reflux in dry
toluene (100 ml) in a Soxhlet apparatus with activated
molecular sieve (3 A) for 18 h. The reaction mixture was
cooled to room temperature and evaporated to dryness
under diminished pressure. CsF (140 mg, 926 ,umol) was
added to the residual oil, and the mixture was kept under
high vacuum for 2 h. The residual syrup was dissolved in
dry DMF (10 ml) under argon and 1-butyl iodide (300 ,ul,
2.62 mmol) was added. After stirring the solution for 48
h, HPLC analysis (90% MeOH; 1.5 ml/min; tR=7.43 min)
showed no further reaction. Excess of 1-butyl iodide and
DMF were removed in high vacuum. The crude product was
chromatographed by preparative HPLC (93%MeOH; 40 ml/min;
tR=22.30 min) to give compound ~Q (175 mg, 74%) as a
solid. Mp: 75.4~-75.9~C (from methanol) . [~] z4o: + 8.4~
(c=0.98 in CHC13) . 1H-NMR (CDC13, 360 MHZ) : b0.91 (3 H,
t, J =7.25 Hz, CH3) , 1.34-1.44 (2 H, m, CHz) , 1.57-1.65 (2
H, m, CH~) , 2 .46 (1 H, s (br) , OH) , 3 .23 (1 H, dd, 7 =
9.31, 2.33 Hz, H-1), 3.43 (1 H, dd, _J = 9.78, 2.80 Hz, H-
3)) 3.45 (1 H, dd, J =9.78, 9.31 Hz, H 4), 3.57 (1 H, dt,
~= 6.99, 6.52 Hz, OCCH_2CH~CH3) , 3,57 (1 H, dt, ~T = 6.99
Hz, OCCH~CH~CH3) , 3.91 (1H, dd, J = 9.31, 9.31 Hz, H-5) ,
3.99 (1 H, dd, ~ = 9.78, 9.31 Hz, H-6), 4.27 (1 H, dd, J
- 2.80, 2.33 Hz, H-2), 4.71-1.91 (8 H, m, C~ZPh), 7.25-
7.39 (20 H, m, CHZpl1) MS: ~ (+ve ion FAB) 597 [(M + H)',
1] , 91 [Bn , 100] .
D-1.4~~.6-Tetra-0-benzvl-3-O-butvl-mvo-inositol
lent-10). A similar reaction and work-up of the diol
ent-9 gave compound ent-~.Q. [a] zq" - 8. 7~ (c = 1 . O1 in
CHC13). Spectral data were in accordance with those
obtained for enantiomer ~Q.
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58
D-3 , 4 . 5 . 6-Tetra-O-benzyl-1-O-butyl-2-O-butyr~rl-
myo-inositol (I1). A solution of ~Q (178 mg, 298 ,umol),
butyric anhydride (210 /,cl, 596 ,ccmol) and DMAP (38 mg, 30
~mol) in dry pyridine (3 ml) was stirred at room
temperature for 18 h. The solvents were evaporated under
high vacuum to give an oil. Residual pyridine was
removed by evaporating three times with octane. The
residue was dissolved in tert-butyl methyl ether (10 ml)
and was washed once with phosphate buffer (10 ml),once
with sodium hydrogen carbonate (10 ml), once with sodium
hydrogen sulfate (10 ml), once again with phosphate
buffer (10 ml) and then with brine (10 ml). The organic
layer was dried over NaZS04 and filtered. Evaporation of
the solvent gave pure ~7 (194 mg, 98 0 ) as an oil, [a] 'QO:
- 10.4~ (c - 1.97 in CHClj). 'H-NMR (CHC13, 360 MHZ). S
0,91 (3 H, t, ~,T = 7.25 Hz, CH3}, 0.98 (3 H, t, ~ = 7.46
Hz, CH3) , 1.32-1.42 (2 H, m, CHZ) , 1.50-1.60 (2 H, m,
CHZ) , 1 . 69 (2 H, tq, ~= 7.46, 7 .46 Hz, O (O) CCH~CH_ZCH3) ,
2 . 39 (2H, t, ~T = 7.46 Hz, 0 (O) CCI~-z, CHZCHj, ) , 3 . 31 (1 H,
dd, ~ = 9.66, 2.63 Hz, H-1), 3.44 (1 H, dt, T = 9.07,
6.81 Hz, OCH~CH~CH~CH3) , 3.48 (1 H, dd, _J = 9.66, 3.07 Hz,
H 3), 3.49 (1 H, dd, ~ = 9.66, 9.22 Hz, H-5), 3.67 (1 H
dt, T = 8.78, 6.81 Hz, OCH,CH~CH~CH3) , 3.81 (1 H, dd, T~=
9.66, 9.66 Hz, H-4), 3.86 ( 1 H, dd ~T = 9.66, 9.22 Hz,
H-6 ) , 4 . 51-4 . 93 ( SH, m, CH,Ph) , 5 . 83 ( 1 H, dd, J = 3 . 07,
2.63 Hz, H-2}, 7.26-7.36 (20 H, m, CHZPh). MS: m/z (+ve
ion FAB) 665 [ (M + H) + , 21] , 91 [Bn', 100] .
D-1 4 5 6-Tetra-0-benzyl-3-O-butyl-2-O-butyryl-
myo-inositol (ent-11l. Compound ent-10 was butyrylated
as described above for the other enantiomer to give
compound ent-11 . [a] 2"D: + l0.7~ (c- 2 . 05 in CHC13) .
Spectral data were in accordance with those of enantiomer
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D-1-O-Butyl-2-O-butyryl-myo-inositol (12).
Compound ~,,~ (178 mg, 267 ,umol) was hydrogenated palladium
(10%) on carbon under hydrogen as described in the
general procedure to give tetrol ~ (81 mg, 99%) as a
solid after freeze-drying. [a]z'D: + 26.5~ (c = 2.0 in
MeOH) . 1H NMR ( [D6]DMSO, 360 MHZ) : b 0.84 (3 H, t, 7 =
7.38 Hz, CH3), 0.90(3 H, t, ~ = 7.38 Hz, CH3), l.21-1.31
2 H, m, CH2) , l.36-1.44 (2 H, m, CHz) , 1.54 (2 H, tq, s
7. 38, 700 Hz, O (O) CCHZ, CH~CH3) , 2 . 24 (2 H, t, ~ = 7. 00 Hz,
O(O)CCH~,CH~CH3), 2.97 (1 H) dd, ~ - 8.94, 8.55 Hz, H-5},
3.10 (1 H, dd, s~ = 9.72, 2.72 Hz, H- 1), 3.25-3.35 (4 H,
m, H-3 , H-4 , H- 6 , OC~-IZ CH~CH,CH3 ) , 3 . 50 ( 1 H, dt , 7~~= 8 . 94 ,
6.60 Hz, OC~ZCH~ CH~CH3) , 4.85 (4 H, s (br) , OH) , 5.36 (1 H
dd, J_ = 2.72, 2.33 Hz, H-2). MS: m z (+ve ion FAB) 307
[ (M + H)+, 21] , 71 [Bt+, 100] . MS: ~ (-ve ion FAB) 305
[ (M-H') -, 27] , 87 [B + O-, 100] . Anal . ( ) C: calculated,
54.89; found 54 45; H: calculated, 8.55, found 8.56.
D-3-0-Butyl-2-O-butyl-2-O-Butyrx~yo-inositol
(ent-12). A similar reaction and work-up of the fully
protected compound ent-11 afforded tetrol ent-~ [a] '~~
26.6~ (c = 0.76 in MeOH}. Spectral data were in
accordance with those obtained for enantiomer ~2..
D-1-O-butyryl-myo-inositol 3 4 5 6-tetrakis
S ;hPn~y1_1 phosphate (20). A solution of tetrol ~ (63
mg 206 /.cmol) and tetrazole (174 mg, 2.47 mmol) in
acetonitrile (2 ml) was treated with dibenzyl Ice, ~-
diisopropylphosphoramidite (834 /,~1, 2.47 mmol) for 18 h.,
oxidized with peracetic acid, and worked up as described.
Purification by preparative HPLC (93% MeOH; 40 ml/min; tR
- 26.45 min) gave compound ~0, (165 mg, 60%) as an oil.
[a] z'~: -4 . 9~ (c =1 . 08 in CHC13) . 1H-NMR (CDC13, 360 MHZ)
b 0.77 (3 H, t, J = 7.27 Hz, CH3), 0.93 (3 H, t, ~ = 7.27
Hz, CH3) , 1.12-1.21 (2 H, m, CHz) , 1.31 -1.46 (2 H, m,
CHZ) , 1.62 (2 H tq, J= 7.26, 7.26 Hz, O (O) CCI~zCH~CH;) , 2 .24
(2 H, t ~=7.26 Hz, O (O) CC$zCH~CH3) , 3 .26 (1, H, dt,
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8.23, 5.81 Hz, OCH_zCH~CH~CH3) , 3 .37 (1 H, dd, J = 9, 20,
2.90 Hz, H-1), 3.44 (1 H, dt, _J = 8.23, 7.26 Hz,
OC$ZCHZCH~CH3) , 4.35 (1 H, ddd, ~ = 9.69, 9.69, 2.42 Hz,
H-3), 4.53 (1 H, ddd, J = 9.69, 9.69, 9.69 Hz, H-5), 4.68
5 (1 H, ddd, ~= 9.69, 9.69, 9.20 Hz, H-6), 4.91 (1 H, ddd
~T = 9.84, 9.69, 9.69 Hz, H-4) , 4.92-5.02 (16 H, m, CHzPh) ,
5.91 (1 H, dd, ~T = 2.90, 2.42 Hz, H-2), 7.11-7.30 (40 H,
m, CHZp~) , 31P NMR (CDC13, 1H decoupled, 145.8 MHZ)
b-1.81 (1 P, s), -1.18 (1 P, s), -0.72 (1 P, s), -0.66 (1
10 P, s) . MS. n (+ve ion FAB) 1347 [ (M + H)', 8] ; 91 [Bn',
100] .
D-3-O-But~rl-2-O-but~~yl-myo-inositol 1 . 4 . 5 . 6-
tetrakis (dibenzyl) phosphate (ent-20). Tetrol ent-12
was phosphitylated and oxidized as described above for
15 compound ?Q to give the fully protected phosphate ent-
~. [a] Zqp : + 4 . 8 ~ ( c = 2 . 09 in CHC13 ) . Spectral data were
in accordance with those obtained for enantiomer 20.
D-1-O-Butyl-2-O-butvryl-myo-inositol 3.4.5.6-tetrakis
phosphate ( 21 ) . Compound ,~Q ( 16 0 mg, 118 ,umol ) was
20 hydrogenated with palladium (10%) on carbon as described
in the general procedure to give title compound 21 (73
mg, 99 0 ) as a solid after freeze-drying. [a] '''J 4 . 1~ (c =
1.04 in HzO, pH 1.6) . 1H NMR (DzO, 360 MHZ) . b 0.81 (3
H, t, ~ = 7.30 Hz, CH3) . 0.91 (3 H, t, ~7 -7.30 Hz, CH3) ,
25 1.22-1.30 (2 H, m, CHZ), 1.42-1.49 (2 H, m CHZ), 1.62 (2
H, tq, J = 7. 30, 7.30 Hz, O (O) CC~?CH~CH3) , 2.36, 2 .54 (2
H, m, O (O) CC~zCH~CH3) , 3 . 56 (1 H, dt, ,1 = 9 .49, 6.46 Hz,
OCH_2CH~CHZCH3) , 3.63 (1 H, dt, J= 9.36, 6.74 Hz,
OC~-jzCH~CHZCH3) , 3.70 (1 H, dd, J=9.74, 2.62 Hz , H-1) , 4.27
30 (1 H, ddd, J = 9.36, 9.36, 9.36 Hz, H-5), 4.31 (1 H, ddd,
~T = 9.74, 9,74, 2.25 Hz, H-3), 4.39 (I H, ddd, J= 9.74,
9,36, 9.36 Hz, H-6), 4.49 (1 H, ddd, J_-9.74, 9.36, 9.00
Hz, H-4), 5.75 (1 H, dd, J = 2.62, 2.25 Hz, H-2), 3'P NMR
(DzO, 1H decoupled, 145.8 MHZ): b-0.10 (2 P, s), 0.50(1
35 P. s). 0.80 ( 1 P, s).
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61
MS: rte, (+ve ion FAB) 627 [ (M + H)', 4] , 71 [Bt+, 100J .
MS: ~. (-ve ion FAB) 625 [ (M-H') 100] .
D-3-O-Bui-'yl-2-O-butyryl-myo-inositol 1~ 4 5 6-
tPrrak;s~hosphate (ent-21) A similar reaction with the
fully protected substrate ent-2020 afforded the free acid
ent-2121 after freeze drying [a] z4p:=4.1~ (c=0.78 in HBO, pH
1.6). Spectral data were in accordance with those
obtained for enantiomer 21.
D-1--O-Butyl-mvo-inositol 3 4 5 6-tetrakisphosphate (5).
Compound _2~ (17 rng, 27 ~.mol) was treated with 1 M KOH
(26U ~.1) to adjust the pH value to 12.8. The solution
was stirred at room temperature for 2 days. The reaction
mixture was directly poured onto an ion-exchange column
(Dowex 50 WX 8, Ht) for purification. Lyophilization
gave compound 5_ (14 mg, 94%) - [a]29~: + 4.9~ (c=0.53 in
HZO, pH 1.6) . 'H NMR (DSO, 360 MHZ) : b 0.78 (3 H t,
J=7.44 Hz, CH3) , 1.26 (2 H, tq, ~=7.44, 7.44 Hz, CH~) ,
1.43-l.52 (2H, m, CH,), 3.42 (1 H, dd, ~T=9.60, 2.78 Hz, H-
1) , 3.52 (1 H, dt, ~=9.71, 6.74 Hz, OC-~izCH~CH~CH3) 3.59 (1
H, dt , ~7=9 . 81, 6 . 74 Hz , OC~ZCH,CH~CH; ) 4 . 16 ( 1 H, ddd, _J =
9.60, 9.60, 2.78 Hz, H-3) 4.21 (1 H, ddd, J=9.37, 9.37,
9.37 Hz, H-5), 4.35 (1H, dd, J=2.78, 2.78 Hz, H-2), 4.39
(1H, ddd, J=9.60, 9.60, 9.37 Hz, H-6), 4.60 (1 H, ddd, J-
9.60, 9.60, 9.37 Hz, H-4), 31P NMR (DzO, 1H decoupled,
145.8 MHZ): b 0.10 (2P, s), 0.70 (1P, s), 1.05 (1P, s).
MS: r~ (-ve ion FAB) 555 [ (M-H') , 100J .
D-3-O-Butvl-mvo-illo~i~.Ql 1,4.5,6-tetrakisnhosnhate (ent-
The butyryl groups of substrate ent-2121 were
hydrolyzed by the same method described above to give the
tetrakisphosphate ent-55 - [a] z4~: + 4.6~ (c = 0.35 in H,O,
pH 1.6). Spectral data were in accordance with those
obtained for enantiomer ~..
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62
D-1-O-Butyl-2-O-buty~vl-mxo-inositol 3 4 5 6-
h h a x DIEA
( 131 ~.1, 768 ~,mol ) and acetoxymethyl bromide ( 77 ~1, 768
umol) were added to a suspension of compound ~ (30 mg,
47 ~,mol) in acetonitrile (2 ml) as described in the
general procedure. Purification by preparative HPLC (73%
MeOH, 37.5 ml/min, tR = 19.30) gave compound 1 (31 mg,
55 0 ) as a syrup. - [a] Zqp: + 2. 0~ (c=1 . 00 in toluene) . 1H
NMR ([D]etoluene, 360 MHZ): ~ 0.85 (3 H, t, J-7.48 Hz,
CH3) 0.97 (3H, t, J = 7.48 Hz, CH3) ( 1.39 (2H, tq, J =
7.48, 7.28 Hz, CHI) , 1.51-1.67 (4H, m, 2x CHI) 1.75-1.75
(24 H, 8 s, 8x OAc) , 2.08-2.13 (2 H, m, CHz) , 3.07 (1 H,
dd, J-9.45, 2.76 Hz, H-1), 3.46 (1H, dt, J = 7.68, 5.91
Hz, OCH~CHzCH~CH3) , 3.62 (1 H, dt, J = 7.74, 7.68 Hz,
OCH,CHzCH~CH3) , 4 . 62 ( 1H, ddd, J = 9 . 84 , 9 . 84 , 2 . 76 Hz, H-
3), 4.67 (1 H, ddd, J = 9.84, 9.84, 9.45 Hz) H-5), 4.80
(1H, ddd, J = 9.45, 9.45, 9.16 Hz, H-6) S.04 (1H, ddd, J-
9.84, 9.84, 9.45 Hz, H-4), 5.63-5.96 (16 H, m, CH~OAc),
6.00 (1 H, dd, J=2.76, 2.76 Hz, H-2) . 31P NMR ( [D] 8-
toluene, 1H decoupled, 145.8 MHZ): b-5.14 (1 P, s), -4.49
(1P, s), -4.06 (1P, s), -3.99 (1 P, s), MS:n z (+ve ion
FAB) 1131 [ (M-CHZOAc' + 2H)', 58] , 98.7 [ ( (M-3 CH~OAc' +
4H) , 100] , MS:~ (-ve ion FAB) 1129 [ (M-CH,OAc')-, 38] ,
241 [OP (OCH~OAc) -2, 100] .
D-3-O-Butyl-2-O-butyryl-myn-inositol 1 4 5 6-
~etrakisphos~hate octakis(acetox~~l)ester (ent-1).
Alkylation of the phosphate ent-2121 as described above
afforded the octakis(acetoxymethyl) ester ent-11. [a]24D:
+ 1.9~ (c = 1.46 in toluene). Special data were in
accordance with those obtained for enantiomer ,~,.
D-1-O-Aryl-3.4,5,6-tetra-0-benzyl-myo-inositol(13). Dry
9 (690 mg, 1.28 mmol) and dry dibutyltin oxide (324 mg,
1.3 mmol) were heated under reflux in dry toluene (150
ml) in a Soxhlet apparatus with activated molecular sieve
(3 /~) for 20 h. The reaction mixture was cooled to room
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63
temperature and evaporated to dryness under diminished
pressure. CsF (388 mg, 2.56 mol) was added to the
residual oil, and the mixture was kept under high vacuum
for 2 h. The residual syrup was dissolved in dry DMF (10
ml) under argon and 1-allyl iodide (329 ~.1, 3.58 mmol)
was added. After stirring the solution for 20 h, HPLC
analysis (95% MeOH; 1.5 ml/min; tR=3.20 min) showed no
further reaction. Excess of 1-allyl iodide and DMF were
remaved in high vacuum. The crude product was
chromatographed by preparative HPLC (90% MeOH; 40 ml/min;
tR=26.15 min) to give compound ~,~, (448 mg, 60%) as a
solid. Mp: 71-72~C (from methanol) . - [a] z'~: + 4.6~
(c=0.98 in CHC13) . 1H NMR (CDC13, 360 MHZ) : S 3.30 (1H,
dd, J-973, 3.10 Hz, H-1), 3.4l (1H, dd, J=9.73, 2.65, H-
3), 3.44 (1H, dd, J=9.51, 9.51, H-5), 3.95 (1H, dd, J-
9.73, 9.51 Hz, H-6), 3.99 (1H, dd, J=9.73, 2.51 Hz, H-4),
4.18 (1H, dd, J=1.65, 1.33 Hz, OCH_ZCHCH~) , 4.19 (1H, dd,
J=2.65, 1.33 Hz, OCI-~zCHCH~) , 4.20 (1H, dd, J-3.10, 2.65
Hz, H-2), 4.71-4.92 (8H, m, CH_zPh), 5.19 (1H, ddt,
J=11.50, 1.33, 1.33 Hz, OCHzCHC~iz), 5.29 (1H, ddt, J-
16.91, 2.65, 1.33 Hz, OCH~CHCI-~2) , 5.94 (1H, dddd, J=16. 91,
11.50, 1.33, 1.33 Hz, OCHZC~ICHZ) , 7.26-7.37 (20 H, m,
CHZPh) . MS:rr (+ve ion FAB) 581 [ (M+H)', 1] , 91 [Bn',
100] . MS:~ (ve ion FAB) 580 [M-H')'6] 489 [M-Bn')~,
100] .
n-3-O-Al7vl-1.4.5.6-tetra-O-benzvl-mvo-inositQ~ (ent-13).
A similar reaction and work-up of the diol ent-~ gave
compound ent-,~. - [a] z4p: + 3 . 9~ (c=0 . 82 in CHC13) .
Spectral data were in accordance with those obtained for
enantiomer
D-
(l41. .Sodium hydride (46 mg, 1.92 mmol) was added to a
stirred solution of ~ (445 mg, 767 ~mol) in dry DMF (5
ml) at room temperature in the dark. The mixture was
stirred for 5 h, after which 1-butyl iodide (306 ~.1, 2.68
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64
mmol) was added. The suspension was stirred for 18 h at
80~C, after HPLC (95o MeOH; 1.5 ml/min; tR= 7.35) showed
a product. Excess of 1-butyl iodide and DMF were
evaporated off under reduced pressure. The mixture was
then dissolved in tert-butyl methyl ether f40 ml) and
washed once with phosphate buffer (20 ml). aqueous.
sodium dithionate (20 ml) and brine (20 ml) successively.
The organic layer was dried over NaZS04, filtered, and the
ether was evaporated off to give an oil. The crude oil
was purified by preparative HPLC (93% MeOH; 40 ml/min;
tR=37.10 min) to give the title compound ~4 (4S8 mg, 940)
as an oil . - [a] 29p: + 1 .5~ (c=2.29 in CHC13) . 1H NMR
(CDC13, 360 MHZ); b 0.95 (3 H, t, J=7.27 H2, CH3}, 1.39-
1.49 (2H, m, CHI) , 1.58-1.66 (2H, m, CH2) , 3.24 (1H, dd,
J=9.69, 2.42 Hz, H-1), 3.36 (1H, dd, J=9.93, 2.42, H-3),
3.46 (1H, dd, J=9.45, 9.45, H-5), 3.78 (2H, t, J=6.54 Hz,
OC~IZCH~CHZCH3) , 3.89 (1H, dd, J=2.42, 2.42 Hz, H-2) , 3.98
(1H, dd, J-9.4S, 9.45 Hz, H-6), 4.03 (1H, dd, J-9.45,
9.45 Hz, H-4), 4.15 (1H, dd, J-5.81, 1.00 Hz, OC~2CHCH,),
4.16 (1H, dd, J-6.06, 1.00 Hz, OC~-iZCHCH2) , 4.72, 4. 95 (8H,
m, C~ZPh) , 5.19 (1H, ddt, J-10.66, 1.45, 1.45 Hz,
OCH~CHCI~2) , 5.32 (1H, ddt, J-16.95, 1.45, 1.45 Hz,
OCH~CHCj~2), 5.95 (1H, dddd, J-l6.95, 10.66, 1.45, 1.45 Hz,
OCH,CI-~CHZ) , 7.26-7.40 (20 H, m, CH2P~) . MS:m z (+ve ion
FAB) 637 [ (M+H} +, 1] , 91 (Bn', 100] .
D-3-O-Allyl-1,4.5.6-tetra-O-benzyl-2-O-butyl-myo-inositol
Pent-14). A similar reaction and work-up of compound
ent-1_~ gave compound ent-~. [a] zqp: - 1.6~ (c=1.87 in
CHC13}. Spectral data were in accordance with those
obtained for enantiomer ~,.
D-3,4,5,6-Tetra-0-benzvl-2-O-butyl-mvo-inositol (15).
Tris(triphenylphosphin)-rhodium(I}-chloride (140 mg, 150
~,mol) and DIEA (25 ~,1, 140 kmol) were added to a
suspension of ~ (458 mg, 720 ~,mol) in 50% ethanol (90
ml), before the suspension was heated under reflux for
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7 h. The reaction mixture was cooled to room temperature
before trifluoroacetic acid (7 ml) was added and the
solution was stirred for an additional 24 h, after HPLC
(90% MeOH; 1.5 ml/min; tR=4.03 min) showed no starting
5 material. After neutralization with aqueous. 2N NHQOH the
ethanol was evaporated off under reduced pressure to give
a syrup. The syrup was dissolved in pert-butyl methyl
ether (40 ml) and washed once with phosphate buffer (20
ml) and brine (20 ml) successively. The organic layer
10 was dried over Na~S04, filtered, and the ether was
evaporated off to give an oil. The crude oil was
purified by preparative HPLC (92% MeOH; 40 ml/min; tR-
27.40 min) to give ~ (275 mg, 74%) as a solid. - [a] ~so:
+ 24.6~ (c=0.97 in CHC13). -H NMR (CDC13, 360 MHZ): ~ 0.92
15 (3H, t, J-7.36 Hz, CH3) , 1.32-1.43 (2H, m, CHz) , 1.53-1.61
(2H, m, CHI), 3.41 (1H, dd, J=9.96, 2.60 Hz, H-1), 3.45
(1H, dd, J-9.52, 2.60 Hz, H-3), 3.46 (1H, dd, J-9.31;
9.31 Hz, H-5), 3.61 (1H, dt, J-9.09, 6.49 Hz,
OCCfj2CHZCH3) , 3.74 (1H, dd, J-9.52, 9.31 Hz, H-4) , 3.86
20 (1H, dd, J=2.60, 2.60 Hz, H-2), 3.95 (1H, dt, J-8.95,
6.49 Hz, OCC~ZCH,CH3) , 3.98 (1H, dd, J-9.96, 9.31, Hz, H-
6) , 4.79-4.94 (8H, m, CI~zPh) ( 7.26-7.36 (20 H, m, CH~P~) .
MS;~ (+ve ion FAB) 597 [ (M+H) ~, 1] , 91 [BnT, 100] . MS:~/~
(-ve ion FAB) 595 [ (M-H'}-, 100] , [ (M-Bn')-, 20] . C:
25 calculated, 76.48; found 76.52; H: calculated, 7.43,
found 7.40.
D-1 4 5 6-Tetra-O-benzyl-2-O-butyl-myo-inositol (ent-15).
A similar reaction and work-up of the diol ent-15 gave
compound ent-15. ~ [a] ZQO: + 24.9~ (c=1. 00 in CHC13) .
30 Spectral data were in accordance with those obtained for
enantiomer ~.
D-:~ 4~ 5~, 6-Tetra-O-benz5rl-2-O-but~rl-1-O-butyrvl m~ro
i~,ositol (16). A solution of alcohol 15 (178 mg, 298
~.mol) in dry pyridine (4 ml) were treated with butyric
35 anhydride (158 ul, 447 ~.mol) and DMAP (38 mg, 29 ~.mol)
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66
and stirred at room temperature. When HPLC analysis (90%
MeOH; 1.5 ml/min, tA=6.40 min) showed no more starting
material (18 h), the reaction mixture was evaporated
under reduced pressure to give a crude oil. To remove
residual pyridine the oil was dissolved in octane and
evaporated three times. The residue was dissolved in
tert-butyl methyl ether (20 ml) and was washed once with
phosphate buffer (10 ml), once with sodium hydrogen
carbonate (10 ml), once with sodium hydrogen sulfate (10
ml), once again with phosphate buffer (l0 ml) and then
with brine (10 ml). The organic layer was dried over
Na~S04 and filtered. Evaporation of the solvent gave pure
,7~ (176 mg, 89%) as an oil. - [a] '4,,: - 15.4~ (c=1.00 in
CHC13) . iH NMR (CHC13, 360 MHZ) ; b 0.91 (3H, t, J = 7.21
Hz ) CH3) , 0. 93 (3H, t, J = 7.2l Hz, CH3) , 1 .35-1 . 56 (4 H,
m, 2 x CHI) , 1.58-1.72 (2H, m, CHz) , 2.21-2.26 (2H, m,
CHI), 3.48 (1H, dd, J-9.61, 2.40 Hz, H-3), 3.51 (1H, dd,
J-9.66, 9.37, Hz, H-5), 3.52 (2H) tq, J-8.97, 6.25, Hz,
O (O) CC~2CH~CHz) , 3 . 76 (2H, t, J-9 . 13, 6 .25 Hz,
O(O)CCHzCHZCH,) , 3.94 (1H, dd, J-2.40, 2.40 Hz, H-2) , 4.00
(1H, dd, J=9.61, 9.6l Hz, H-4), 4.02 (1H, dd, J = 9.85,
9.31 Hz, H-6), 4.73 (1H, dd, J=9.85, 2.40 Hz, H-1), 4.65-
4.93 (8H, m, CH~Ph) , 7.25-7.34 (20 H, m, CHZP~I ) . MS:m z
(+ve ion FAB) 66S [ (M+H) +, 1] , 91 [Bn+, 100] .
D-1.4.5,6-Tetra-O-benzyl-3-O-butyl-2-O-butyryl-myo-
inositol (ent-16). Compound ent-1515 was butyrylated as
described above for the other enantiomer to give compound
ent-16 . - [a] ZQp: + 15. 9~ (c=1 . 10 in CHC13) . Spectral
data were in accordance with those of enantiomer
D-2-O-Butyl-1-O-butyryl-myo-inositol t17) . Compound ~
(170 mg, 255 ~.mol) was hydrogenated with palladium (10%)
on carbon under hydrogen as described in the general
procedure to give tetrol 17 (75 mg, 97%) as a solid after
freeze drying. [a] 2~~: + 41. 9~ (c=1 . 10 in MeOH) . 'H NMR
( [D6] DMSO, 360 MHZ) . b 0 .87 (3H, t, J=7.16 Hz, CH3) , 0 . 89
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(3H, t, J = 7.33 Hz, CH3) , 1.28-1.48 (4H, m, 2 x CHI) ,
l.52-1.62 (2H, m, CHI) , 2.24-2.36 (2H, m, CHI) , 2.96 (1H,
dd, J=9.21, 9.21 Hz, H-5), 3.26 (1H, dd, J-9.55, 2.39 Hz,
H-3), 3.35 (1H, dd, J=9.55, 9.21 Hz, H-4), 3.44 (1H, dt,
J = 9.43, 6.39 Hz, OC~2CH~CH~CH3) , 3.52 (1 H, dd, J =
10.23, 9.21 Hz, H-6), 3.57 (1H, dd, J = 2.39, 2.39 Hz, H-
2), 4.47 (2 H, dt, J = 9.21, 6.39 Hz, OCH2CH2CH3), 4.47
(1H, dd, J = 10.23, 2.39 Hz, H-1), 4.71 (2H, s (br), OH),
4.82 (1H, s (br) , OH) , 4.86 (1H, s (br) , OH) , MS:,pl/~ (+ve
ion FAB) 307 [(M+H)+, 100], MS:~ (-ve ion FAB) 305
[(M+H)~, 34], 87 (Bt0-, 100]. C: calculated, 54.89; found
54.90; H: calculated, 8.55, found 8.51.
D-2-O-Butyl-3-O-butyryl-m~Q-inositol (ent-17). A similar
reaction and work-up of the fully protected compound ent-
~ afforded tetrol ent-17. - [a]''D: -40.5~ (c=1.00 in
MeOH). Spectral data were in accordance with those
obtained for enantiomer
D-2-O-Butyl-1-O-butyryl-mho-inositol 3 4 5 6-
tetrakis(dibenzyl)phosx~hate (22). A solution of compound
~ (55 mg, 178 ~.mol) and tetrazole (152 mg, 2.1S mmol) in
acetonitrile (2 ml) was treated with dibenzyl I~,~T-
diisopropylphosphoramidite (726 ~.1, 2.15 mmol) for 22 h,
oxidized with peracetic acid, and worked up as described.
Purification by preparative HPLC (92% MeOH; 40 ml/min;
tR=29.00 min) gave compound 2,~, (192 mg, 80%) as an oil. -
[a] ''qp: -3 .4~ (c=1. 02 in CHC13) . 1H NMR (CDC1;, 360 MHZ) : b
0.79 (3H, t, J=7.30 Hz, CHI) , 0.86 (3 H, t, J=7.30 Hz,
CH3) , 1.23-1.38 (2H, m, CHZ) , 1.41-1.57 (4H, m, 2 x CHZ) ,
2.01-2.17 (2 H, m, CHZ), 3.54 (1H, dt, J=8.52, 6.63 Hz,
OC~,CH~CHZCH3) , 3.60 (1H, dt, J=8.52, 7.74 Hz,
OC~i>CH~CH~CH3) , 4.13 (1H, dd, J=2.52, 2.52 Hz, H-2) , 4.22
(1H, ddd, J=9.73, 9.73, 2.52 Hz, H-3), 4.43 (1H, ddd,
J=9.51, 9.51 Hz, H-5), 4.44 (1H, ddd, J=9.51, 9.51, 9.28
Hz, H-6), 4.89 (1H, ddd, J-9.73, 9.73, 9.51 Hz, H-4),
4.91-5.08 (17 H, m, Cj~2Ph, H-1) , 7.13-7.27 (40 H, m,
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68
CHz~) . 31P NMR (CDC13, 'H decoupled, 145.8 MHZ) : b -1.64
(1P, s), -1.42 (1P, s), -0.76 (1P) s), -0.53 (1P, s).
MS:m z (+ve ion FAB) 1347 [ (M+H)', 33] , 71 [Bt', 100] .
MS:m z (-ve ion FAB) 1255 [ (M-Bn+)-, 16] , 277 [OPO(OBn)2-,
100] .
D-2-O-Butyl-3-O-bu~rryl-myo-inositol 1.4,5.6-
tetrakis (dibenzyll~hosx~hate (ent-22) . Compound ent-12
was phosphitylated and oxidized as described above for
compound 22 to give the fully protected phosphate en -2
[a] 2"p : + 3 . 1 ~ ( c=1 . 10 in CHC13 ) . Spectral data were in
accordance with those obtained for enantiomer ~2.
D-2 -O-But~rl -1-O-butyryl -myo- inositol 3 . 4 . 5 . 6 -
tetrakisnhos~hate (23). Compound ~2 {190 mg, l41 ~.mol)
was hydrogenated with palladium (10%) on carbon as
described in the general procedure to give title compound
(87 mg, 99%) as a solid after freeze drying. - [a]'9D:
+ 9.9~ (c=l.10 in H~O, pH 1.6) . 1H NMR (DZO, 360 MHZ) : b
0.75 (3H, t, J-7.15 Hz, CH3), 0.76 {3H, t, J=7.31 Hz,
CH3) , 1.19-l.29 (2H, m, CHZ) , 1.38-1.53 (4H, m, 2 x CH~) ,
2.30 (2H, t, J-7.47, O(0)CC$2CH~CH3) , 3.58 (1H, dt, J-
9.64, 6.44 Hz, OCH_ZCH~CH~CH3) , 3.67 (1H, dt, J-9.64, 6.36
Hz , OCHzCH,CH~CH3 ) , 4 . O 1 ( 1H, dd, J=2 . 54 , 2 . 23 Hz , H-2 ) ,
4.22 (1H, ddd, J-9.22, 9.22, 8.90 Hz, H-5), 4.23 (1H,
ddd, J=9.54, 9.54, 2.54 Hz, H-3), 4.43 (1H, ddd, J=9.90,
9.22, 9.22 Hz, H-6), 4.48 (1H, ddd, J-9.54, 9.54, 9.54
Hz, H-4), 4.94 (1H, dd, J=9.90, 2.23 Hz, H-1). 31P NMR
(D20, 'H decoupled, 145.8 Hz), S -0.20 (2P, s), 0.40 (1P,
s), 0.50 (1P, s).~ MS:m/z (+ve ion FAB) 627 [(M+H)', 8],
71 [Bt+, 100l. MS/~ (-ve ion FAB) 625 [(M-H')+, 35], 79
[OP (O) 2, 100] .
D-2-O-But~l-3-O-butvryl-myo-inositol 1.4.5.6-
tetrakisphosphate (ent-23). A similar reaction with the
fully protected substrate ent2323 afforded the free acid
ent-23 after freeze drying. [cx] ZQO: -9.7~ (c=1. 00 in H,O,
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69
pH 1.6). Spectral data were in accordance with those
obtained for enantiomer
D 2 O-Butyl-mvo-inositol 3~ 4 5 6-tetrakisx~host~hate (6) .
Compound ~ (33 mg, 52 umol) was treated with 1 M KOH
(453 ~,l) to adjust the pH value to 12.8. The solution
was stirred at room temperature for 2 days. The reaction
mixture was directly poured onto an ion-exchange column
(Dowex 50 WX 8, H') for purification. Lyophilization gave
compound ~ (27 mg, 93%) . -- [a] ''';,: -1.1~ (c=0.89 in HBO,
pH 1.6) . 1H NMR (D20, 360 MHZ) : b 0.77 (3H, t, J-7.37 Hz,
CH3) _, 1.21-1.31 (2H, m, CH2) , 1.39-1.52 (2H, m, CH,) , 3 .63
(1H, dt, J=9.30, 6.18 Hz, OCH~CH~CH~CH3) , 3.67 (1H, dd,
J=10.00, 2.63 Hz, H-1), 3.75 (1H, dt, J-9.30, 6.56 Hz,
OC~I2CH,CH~CH~) , 3.94 (1H, dd, J-2.63, 2.37 Hz, H-2) , 4. 13
(1H, ddd, J-9.21, 9.21, 9.21 Hz, H-5), 4.17 (1H, ddd, J-
9.47, 9.47, 2.37 Hz, H-3), 4.30 (1H, ddd, J-9.47, 9.47,
9.21 Hz, H-4), 4.43 (1H, ddd, J-10.00, 9.73, 9.21 Hz, H-
6) . ''P NMR (D20, 1H decoupled, 145.8 MHZ) : b -0.18 (1P,
s), 0.55 (2P, s), 0.95 (1P, s). MS:~ (+ve ion FAB)
557 [ (M+H')-, 100] . MS:~ (ve ion FAB) 555 [ (M-H)-, l00] .
D-2-O-Butyl-myo-inositol 1y4 5 6-tetrakisphosphate (ent-
~ The butyryl groups of substrate a t- were
hydrolyzed by the same method described above to give the
tetrakisphosphate ent-6. - [a]'4" + 1.6~ (c=0.60 in H~O,
pH 1.6). Spectral data were in accordance with those
obtained for enantiomer
a D-2-O-Butyl-1-O-butyrvl-mho-ino~itol 3 4 5 6-
tetrak~ sx~hosphate octakis lacetox~rmeth~l) ester
DIEA (182 ~1, 1.06 mmol) and acetoxymethyl bromide (107
~1, 1.06 mmol) were added to a suspension of compound
(37 mg, 59 /.~.mol) in acetonitrile (2 ml) as described in
the general procedure. Purification by preparative HPLC
(72o MeOH, 40 ml/min, tR =21.12) gave compound ~ (33 mg,
46%) as a syrup [a]24p + 1.1~ (c= 1.07 in toluene). 'H NMR
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([D)etoluene, 360 MHZ): b 0.93 (3 H, t, J =7.28 Hz, CH3),
0.97 (3 H, t, ~ - 7.48 Hz, CH3), 1.39 - 1.49 (2 H, m,
CHz), 1.51-1.60 (2 H, m, CH~), 1.77-1.86 (24 H, 8 s, 8 x
OAc), 2.39 (1 H, dt, _J - 16.67, 7.38 Hz, CHz), 2.63 ( 1 H,
5 dt, J =16.67, 7.68, CH3), 3.78 (1, H, dt, s,7 = 9.4S, 6.30
Hz, OC~-IzCH~CH~CH3) , 3.87 ( 1 H, dt, _J = 9.06, 6.30 Hz,
OC~,izCH~CH2CH3) , 4.40 ( 1 H, dd, ~7 = 2.36, 2.36 Hz, H-2) ,
4.68 (1 H, ddd, _J = 9.55, 9.55, 2.36 Hz, H-3), 4.79 ( 1
H, ddd, J = 9.55, 9.45, 9.45 Hz, H-5) , 5.03 (1 H, ddd,
10 ~=9.55, 9.55, 9.S5 Hz, H-4), 5.09 (1 H, ddd, 7 - 10.04,
9.55, 9.55 Hz, H-6), 5.21 ( 1 H, dd, J - 10.04, 2.36 Hz,
H-1) . 5.58-S.93 (16 H, m, CH~OAc) . 31P NMR ( [D] e-toluene,
'H decoupled, 145.8 MHZ): b-4.42 (1 P, s), -3.98 (1 P, s),
-3.48 (2 P, s) . MS: m z (+ ve ion FAB) l131 [ (M-CH,OAci +
15 2 H) , 44] , 987 [ (M-3 CH~OAc~ + 4 H) ~, 100) , MS: ~ (-ve
ion FAB) 1129 [ (M-CH~OAc') , 18) , 241 [OP (OCH~OAc) ~, 100] .
p-2-O-But,~yl-3-O-Butyr~rl-m~ro-inositol 1. 4 ) 5, 6-
t-Prrak;s~hosphate octakis (acetox et yl) ester (ent-2)
Alkylation of the phosphate ent 23 as described above
20 afforded the octakis (acetoxymethyl) ester ent-2. [a]z4p:
1.3~ (c = 0.60 in toluene). Spectral data were in
accordance with those obtained for enantiomer 2_.
D-3 4~ 5 6-Tetra-O-benzyl-1 2-di-O-buty_1-mho-inositol
(18). Sodium hydride (13 mg, 522 ,umol) was added to a
25 stirred solution of ~ (94 mg, 174 /Cmol) in dry DMF (3 ml)
at room temperature in the dark. The mixture was stirred
for 5 h, after which 1-butyl iodide (120 /cl, 1.04 mmol)
was added. The suspension was stirred for 36 h at 80~C,
after HPLC (95% MeOH; 1.5 ml/min; tR = 7.26) showed a
30 product. Excess of 1-butyl iodide and DMF were
evaporated off under reduced pressure. The mixture was
then dissolved in tert-butyl methyl ether (30 ml) and
washed once with phosphate buffer (10 ml), aqueous sodium
dithionate (10 ml) and brine (10 ml) successively. The
35 organic layer was dried over Na~S04, filtered, and the
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ether was evaporated off to give an oil. The crude oil
was purified by preparative HPLC (95% MeOH, 40 ml/min; tR
- 27.24 min) to give the title compound 1$. (100 mg, 88%)
as an oil . [a] z"o: + 1.3~ (c=1.00 in CHC13) . -H NMR
(CDC13, 360 MHZ} : S 0.092 (3 H, t, T = 7.52 Hz, CH3) ,
1.36- 1.47 (4 H, m, 2 x CHz} , 1.55-1.63 (4 H, m, 2 x CHI) ,
3.14 (1 H, dd, T - 9.57, 2.28 Hz, H-3), 3.34 (1 H, dd, J-
10.02, 2.28, H-1), 3.43 (1 H, dd, J = 9.34, 9.34, H-5),
3.52 (1 H, dt, J=9.1I, 6.60 Hz, OCCHZCH~CH3) , 3 .60 (1 H,
dt, ,~ = 9.11, 6.60 Hz, OCC$ZCHZCH3 ) , 3.75 (2 H, t,
6.60 Hz, OCC~i~CH~CH3) , 3 .89 ( 1 H, dd, _J = 2.28, 2.28 Hz,
H-2), 3.92 (1 H, dd, s~ - 9.57, 9.34 Hz, H-4), 4.00 (1 H,
dd, ~T = 10.02, 9.34 Hz, H-6), 4.71 - 4.93 (8 H, m, C-~.I3Ph),
7.24-7.39 (20 H, m, CH~Ph) . MS: Ir {+ve ion FAB) 653 [ (M
+ H) ', 1] , 91 [Bn', 100] . MS : c~ 653 . 383 (M + H)
(calculated for C4~H53O6 653.384) .
D-1~ 4 . 5 , 6-Tetra-O-benz5rl-2 , 3-di-O-butyl-m~ro-inositol
(ent-18). A similar reaction and work-up of the compound
ent-99 gave compound ent-18. [a]z4p: 1.2~ (c=2.2o in
CHC13). Spectral data were in accordance with those
obtained for enantiomer ~$.
D-1.2-Di-O-butyl-rr~ro-inositol (1~). Compound 18 (100 mg,
153 ,umol) was hydrogenated with palladium (10%) on carbon
under hydrogen as described in the general procedure to
give tetrol .1~ (40 mg, 92%) as a solid after freeze
drying. (from ethanol) [a]24 + 18.7~ (c = 0.80 in MeOH).
1H NMR ( [D6] DMSO, 360 MHZ) : b 0. 89 {3 H, t, ~=7. 52 Hz,
CH3) , 0.91 (3 H, t, J = 7.38 Hz, CH3) , 1.24-1.39 (4 H, m,
CHZ) , 1.41 1.53 (4 H, m, CH2) , 2.88 (1 H, dd, J_ = 9,55,
2.63 Hz, H-3), 2.93 (1 H, dd,~,T = 9.93, 2.63 Hz, H-1),
3 . 14 ( 1 H ( dt , 7 = 9 . 51, 6 . 4 2 Hz , OCC~-IZCH~CH~CHi ) , 3 . 31 ( 1
H, dd, ~ = 9.52, 9.52 Hz, H-5), 3.39-3.67 (6 H, m, 3x
OCC-LizCH~CH~CH,, H-2, H-4, H-6) , 4.43 (1 H, s, OH) , 4.52 (1
H, S, OH), 4.59 (2 H, s (br), OH), MS: ~z. (+ve ion FAB)
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293 [ (M + H) ', 100] . MS : m/z (-ve ion FAB) 291 [ (M-H" ) ,
100] .
D-2. 3-Di-O-butyl-myo-inositol (ent-19). A similar
reaction and work-up of the fully protected compound ent-
.~$ afforded tetrol ent-19. [a] z4p: - 18 . 9~ (c=1 . 36 in
MeOH). Spectral data were in accordance with those
obtained for enantiomer ~9.
D-1.2-Di-O butyl-myo-inositol 3.4.5.6-
~etrakis(dibenzvl)phosmhate (24). A solution of compound
1~ (40 mg, 137 ~mol) and tetrazole (115 mg, 1.64 mmol) in
acetonitrile (2 ml) was treated with dibenzyl ~N-
diisopropylphosphoramidite (552 ~1, 1.64 mmol) for 18 h,
oxidized with peracetic acid, and worked up as described.
Purification by preparative HPLC (93o MeOH; 40 ml/min; tR
- 26.05 min) gave compound 24 (133 mg, 73%) as an oil.
[a] z~D: - 2.3~ (c- 1. 00 in CHC13) . -H NMR (CDClj, 360 MHZ)
b 0.79 (3 H, t, s~ =7.31 Hz, CH3) . 0.86 (3 H, t, _J = 7.31
Hz, CH3) , 1.13-1.38 (4 H,- m, 2 x CHI) ( 1.41-1.56 (4 H, m,
2 x CHZ), 3.24 (1 H, dd, ~ = 9.89, 2.15 Hz, H-1), 3.36 (1
H, dt, J = 8.31, 7.41 Hz, OCH_~CH~CH~CH3) , 3.41 (1 H, dt,
- 8.31, 5.87 Hz, OC,#~ZCH~CH=CH3) , 3 .60 (1 H, dt, 7 = 8.88,
6.55 Hz, OC~zCH~CH~CH3) , 3.71 (1 H, dt, _J = 8.88, 6.55 Hz,
OCH_~CH~Ch~Ch3) , 4.16 (1 H, ddd, ~T = 9.89, 9.89, 2.47 Hz, H-
3), 4.21 (1 H, dd, J = 2.47, 2.15 Hz, H-2), 4.48 (1 H,
ddd, ~7 = 9.67, 9.67, 9.67 Hz, H-5), 4.79 (1 H, ddd, J =
9.89, 9.67, 9.67 Hz, H-6), 4.95 (1 H, ddd, ~7 = 9.89,
9.67, 9.67 Hz, H-4), 4.96-5.09 (16 H, m, C$ZPh), 7.12-7.32
(40 H, m, CH2~) . 31P NMR (CDC13, 1H de coupled, 145.8
MHZ): ~ - 1.82 (1 P, s), -1.59 (1 P, s), -0.81 (1 P, s),
-0.68 (1 P, s) . MS: n z (+ve ion FAB) 1333 [ (M + H)', 2] ,
91 [Bn', 100] . MS: ~(-ve ion FAB) 1241 [ (M-Bn-)-, 8] ,
277 [OPO (OBn) -z, 100] .
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D-2 3-Di-O-butyl-m~o~ nositol 1 4,, 5 6-tetrak~ s
(dibenzyl)~~ate (ent-24). Compound ent-19 was
phosphitylated and oxidized as described above for
compound ,?.~ to give the fully protected phosphate ent-24.
[a] 29p: + 2.5~ (c=1.15 in CHC1~) . Spectral data were in
accordance with those obtained far enantiomer 24.
D- 1- 'n i h
(7). Compound ~ (134 mg, 100 ~cmol) was hydrogenated
with palladium (10%) on carbon as described in the
general procedure to give title compound 7 (52 mg, 870)
as solid after freeze drying. (c =1.10 in H~O, pH 1.6).
1H NMR (D20, 360 MHZ) : b 0.70 (3 H, t, ~T = 7.37 Hz, CH~) ,
0.71 (3 H, t, ~ =7.37 Hz, CH3) , 1.13-1.24 (4 H, m, 2 x
CH~) , 1.32-1.46 (4 H, m, 2 x CH,) , 3.39 (1 H, dd, _J =
9.47, 2.33 Hz, H-1), 3.45 (1 H, dt, ~ = 8.59, 8.16 Hz
OC$;~CH~CH,CH~) , 3.5I (1 H, dt, ~ = 8.59, S.79 Hz,
OCR;>CH~CHZCH3) , 3.56 (1 H, dt, J_ = 9.47, 6.31 Hz,
OCH_>CH~CH~CHz), 3.65 {1 H, dt, J = 9.47, 6.84 Hz,
OCH_,CH~CH~CH~) , 4.02-4.08 (2 H, m, H-2, H-3) , 4.11 (1 H,
ddd, ~T = 9.47, 9.47, 9.47 Hz, H-5), 4.27 (1 H, ddd, J_
=9.47, 9.47 9.47 Hz, H-4), 4.39 (1 H, d J = 9.47 ,
9.21, 9.21 Hz, H-6) . '1P NMR (D~O, 'H decoupled, 145.8
MHZ): b -0.69 (1 P, s), -0.41 (2 P, s), -0.12 (1 P, s).
MS: ~ (+ve ion FAB) 613 [ (M + H)', 15] , 81 [PO (OH) ~=,
100] . MS: r ~ (-ve ion FAB) 611 [(M-H')-, 90] 79 [OP (O)-~,
100] .
D-2,3-Di-O-butyl mvoinositol 1.4.5,6-tetrakisphosnha
(ent 7). A similar reaction with the fully protected
substrate ent-24 afforded the free acid ent-7 after
freeze drying. (c = 1.00 in H20, pH 1.6). Spectral data
were in accordance with those obtained for enantiomer 2.
D-1.2-Di-O-butyl-myo-inositol 3.4.5.6-tetrakisphosr~hate
octakis (acetoxymeth~t~) ester (3) . DIEA (l87 ~,1, 1.00
mmol) and acetoxymethyl bromide (111 ~l, 1.00 mmol) were
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added to a suspension of compound 7 (34 mg, 55 ,umol) in
acetonitrile (2 ml.) as described in the general
procedure. Purification by preparative HPLC (73% MeOH,
40 ml/min, tR = 20.40) gave compound 3_ (42 mg, 650) as a
syrup . [a] 2'" - 2 . 3 ~ ( c=1 . 03 in toluene ) . 1H NMR ( [D] a
toluene, 360 MHZ): ~ 0.92 (3 H, t, ~7 = 7.28 Hz, CH3),
0.99 (3 H, t, ~ = 7.48 Hz, CH3), l.33-1.47 (4 H, m, 2 x
CH2) , 1.48 - l.54 (2 H, m, CHZ) , 1.61-1.69 (2 H, m, CHI) ,
1.77-l.87 (24 H, 8 s, 8 x OAc), 3.11 (1 H, dd, T = 9.84,
2.36 Hz, H-1). 3.46 (1 H, dt, ~ =8.79, 7.28 Hz,
OC~ZCHzCH2CH3) , 3.56 (1 H, dt, ~ = 8.79, 7.28 Hz
OC~2CH~CH~CH3) , 3.84 (1 H) t, T = 6.50 Hz, 2 x
OCHzCH~CH~CH3) . 4.34 (1 H, dd, _J = 2.36, 2.36 Hz, H-2) ,
4.49 (1 H, ddd, _J = 9.45, 9.45, 2.36 Hz, H-3), 4.68 (1 H,
ddd, ~ = 9.65, 9.65, 9.65 Hz, H-5), 4.90 (1 H, ddd, _J =
9.65, 9.45, 9.45 Hz, H-4), 5.06 (1 H, ddd, ~T = 9.84,
9.65, 9.65 Hz, H-6) , 5.68-5.96 (16 H, m, C~-zOAc) . 'iP NMR
([D]e-toluene, 1H decoupled, 14S.8 MHZ) b - 5.12 (1 P, s),
-3.98 (1 P, s), -3.69 (1 P, s), -3.42 (1 P, s) MS: ~r z
(+ve ion FAB) 1189 [ (M + ~H)', 3] , 1045 [ (M-2x CHzOAc' + 3
H)', 100] , MS: ~z_ (-ve ion FAB) 1116 [ (M-CH~OAc')-, 30] ,
241 [OP (OCH~OAc) -z, 100] .
D-2.3-Di-O-butyl-rr~ro-inositol 1,4.5.6-tetrakisphosphate
octakis (acetoxymethyl) ester (ent-3). Alkylation of the
phosphate ~t-7 as described above afforded the
octakis(acetoxymethyl) ester ent-33. [a]Z'p: + 2.5~ (c=
1.10 in toluene). Spectral data were in accordance with
those obtained for enantiomer ~.
rac-1.2-Di-O-c~clohexvlidene-myo-inositol 3.4,5.6
tetrakis (dibenzy~ ~hosnhate (rac-26). A solution of
compound rac-25 (130 mg, 500 ~cmol) and tetrazole (350 mg,
5.00 mmol) in acetonitrile (6 ml) was treated with
dibenzyl ~ diisopropylphosphoramidite (1.68 ml, 5.00
mmol) for 26 h, oxidized with peracetic acid, and worked
up as described. Purification by preparative HPLC (93%
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MeOH; 40 ml/min, tR = 22.35 min) gave compound r~c-26 (306
mg, 47%) as an oil. 1H NMR (CDC13, 360 MHZ) ~ 1.20-1.80
(10 H, m, CH~ (cyclohex..)), 4.26 (1 H, dd, 7 = 6.01, 6.01
Hz, H-1}, 4.66 ( 1 H, dd, ~ = 6.01, 3.43 Hz, H-2), 4.76-
5 4.81 (2 H, m, H-3, H-5) 4.92-5.17 (18 H, m, CI-_I,Ph, H-4, H-
6) , 7.20-7.40 (40 H, m, CHZp~) . 31P NMR (CDC1:, 'H
decoupled, 145.8 MHZ): ~-1.69 (1 P, s), -1.59 (1 P, s), -
1.52 (1 P, s), -1.00 (1 P, s). MS: m z (-ve ion FAB)
l209 ( (M-Bn') -, 1] , 277 [OPO (OBn) -2, 100] .
10 rac-1 2-Di-O-cyclohexylidene-myo-inositol 3.4.5.6-
c- la o
rac-26 (91 mg, 70 ~cmol) was dissolved in dry ethanol (4
ml), before dry ethyl-diisopropylamine (95 ~cl, 560 ~cmol)
was added, followed by palladium (100) on carbon (84 mg,
15 840 umol). After stirring the solution at room
temperature for 6 days under hydrogen atmosphere the
catalyst was removed by ultrafiltration and the filtrate
was freeze dried to give title compound rac-88 (108 mg,
96%) . 1H NMR, (DSO, 360 MHZ) : b 1.30-l.90 (10 H, m, CHI
20 (cyclohex.)), (1 H, ddd, ~ = 9.08, 9.08, 8.23 Hz, H-5),
4.18-4.26 (2 H, m, H-1, H-3), 4.32-4.40 (2 H, m, H-4, H-
6) _, 4.55 (1 H, dd, _J = 1.26, 3.69 Hz, H-2) . "P NMR (D~O,
1H decoupled, 145.8 MHZ): b - 0.24 (1 P, s), 0.10 (1 P,
s) , 0. 85 (1 P, s) , 0. 90 (1 P, s) .
25 rac-1 2-Di-0-cyclohexylidene-myo-inositol 3.4.5.6-
DIEA (205 ~.1, 1.20 mmol) and acetoxymethyl bromide
(120A , 1.20 mmol) were added to suspension of compound
rac-88 (108 mg, 66 E.cmol) in acetonitrile (2 ml) as
30 described in the general procedure. Purification by
preparative HPLC (68% MeOH, 40 ml/min, tR = 19.35) gave
compound rac-44 (50 mg, 65%) as a syrup. 'H NMR
([D]etoluene, 360 MHZ): ~ 1.20-1.75 (10 H, m, CH2
(cyclohex.)), 1.81l.92 (24 H, 8 s, 8 x OAc), 4.28 (1 H,
35 dd, ~ = 5.51, 5.51 Hz, H-1). 4.77 (1 H, dd, T = 5.51,
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3.54 Hz, H-2), 4.91-4.99 (2 H, m, H-5, H-6), 5.07 (1 H,
ddd, ~ = 8.66, 8.27, 3.54 Hz, H 3 ), 5.17 ( 1 H, ddd, J =
9.05, 8.66, 7.08 Hz, H-4), 5.65-5.94 (16 H, m, CH_~OAc).
31P NMR ( [D] e-toluene, 1H decoupled, 145. 8 MHZ) : ~ -4 .56 (1
P, s) , -4.07 (1 P, s) ( -3.67 (1 P, s) , -3.55 (1 P, s) .
MS: ~ (-ve ion FAB) 1083 ( (M-CHZOAc') , 35] , 241
[OP (OCH~OAc) -z, 100] .
rac-3.4.5,6-Tetra-O-benz~rl-2-deoxy-2-iodo-1-O-(4-
r~ethoxybenzvl)-myo-inositol (rac-56). A mixture of dry
rac=55 (rac-3,4,5,6-tetra-0-benzyl-1-O-(4-methoxybenzyl)-
myo-inositol) (1.32 g, 2 mmol), triphenylphosphine (2.15
g, 8.2 mmol), imidazole (549 mg, 8.2 mmol), and iodine
(1.52 g, 6 mmol) were stirred under reflux in dry toluene
(100 ml) for 41 h. The reaction mixture was cooled to
room temperature, saturated aqueous sodium
hydrogencarbonate (100 ml) was added, and the mixture was
stirred for 10 min. Iodine was added in portions. When
the toluene phase remained iodine-colored it was stirred
for an additional 15 min: Excess of iodine was removed
by the addition of aqueous sodium dithionite solution.
The organic and the aqueous phase were separated in a
separating funnel and the organic phase was washed
(twice) with brine. The toluene phase was dried over
Na~S04 and filtered. Evaporation of the mixture and
crystallization gave pure ra - (1.01 g, 73%). Mp..
I31.5~-132.4~C (from methanol) . 1H NMR (CDC1:) 360 MHZ)
3.49 (1 H, dd, _J = 9.27, 9.27 Hz), 3.50 (1 H, dd, ~ _
9.27, 9.27 Hz), 3.80 (3 H, s, OMe), (1 H, dd, J = 9.78,
9.31 Hz, H-4), 4.06 (1H, dd, ~ = 11.08, 11.08 Hz), 4.82-
4.92 (10 H, m, 4x C~ZPh and CI-~z in PMB) , 6.87 (2 H, d, PMB
ArH), 7.23-7.37 (22 H, m, 4 x CHz~ and PMB ArH). MS:
(+ve ion FAB) 924 [ (M + NBA + H)+, 1] , 121 [PMB', 100] .
rac-3,4.5,6-Tetra-O-benzyl-2-deoxy-1-O-(4-methoxybenzyl)-
myo-inositol (rac-57). Compound rac-5656 was dissolved in
dry toluene (150 ml) and AIBN (63 mg, 355 ~Cmol) and n-
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BujSnH (l.63 ml, 6.3 mmol) were added. The solution was
heated under an argon atmosphere for 2 h. The reaction
mixture was cooled and was washed once with phosphate
buffer (30 ml) and once with brine (30 ml). The organic
layer was dried over Na~S04 and filtered. Evaporation of
the mixture and crystallization gave ~7 (692 mg, 76%).
Mp. . 76.2~-76.8~C (from methanol) . ~~H NMR (CDC13, 360
MHZ): b 1.64 (1 H, ddd, ~7 = 12.73, 12.73, 12.73 Hz, H-2
(ax)), 2.39 (1 H, ddd, T = 12.73, 4.39, 4.39 Hz, H-2
(eq)), 3.39-3.46 (2 H, m, H-1, H-3), 3.46 (1 H, dd, T =
9.22, 8.87 Hz, H-5), 3.55 (1 H, dd, 7 = 9.22, 9.22 Hz, H-
4), 3.56 (1 H, dd, ~ = 9.22, 9.22 Hz, H-6), 3.81 (3 H, s,
OMe), 4.60-4.94 (10 H, m, 4 x CH_~Ph and C~~ in PMB), 6.86
( 2 H , d , PMB ArH ) , 7 . 2 4 - 7 . 3 3 ( 2 2 H , m , 4 x CH=~ and PMB
ArH) . MS: ~ (-ve ion FAB) 643 [ (M-H') , 1] , 523 [M-
PMB') -, 100] .
rac-3 4 5 6-Tetra-0-benzyl-2-deoxy-myo-inositol (58).
DDQ (367 mg, 1.62 mmol) was added to a solution of rac-5757
(600 mg, 931 ~.mol) in CHzCl~ (10 ml) containing small
amounts of water (5%). After the suspension had been
stirred at room temperature for 28 min HPLC analysis (90%
MeOH; 1.5 ml/min; tP = 4.27 min) showed the reaction to be
complete. The reaction mixture was evaporated under
reduced pressure and purified by preparative HPLC (900
MeOH; 40 ml/min; 20.00 min) to give rac-5858 (232 mg, 480)
as a. solid. Mp.. I26.7~C (from methanol). -H NMR (CDC13,
360 MHZ): b 1.64 (1 H, ddd, ~T = 12.73, 12.73, 12.73,
12.73 Hz, H-2 (ax)), 2.39 (1 H, ddd, _J = 12.73, 4.39,
4.39 Hz, H-2 (eq))~, 3.39-3.46 (2 H, m, H-1, H-3), 3.46 (1
H, dd, ~ = 9.22, 8.87 Hz, H-5), 3.55 (1 H, dd, J_ = 9.22,
9.22 Hz, H-4), 3.56 (1 H, dd, ~7 = 9.22, 9.22 Hz, H-6),
3.81. (3 H, s, OMe), 4.60-4.94 (10 H, m, 4 x CH_zPh and CH_z
in PMB) , 6.86 (2 H, d, PMB ArH) , 7.24-7.33 (22 H, m, 4 x
CHzPh and PMB ArH) . MS: m~ (+~Je ion FAB) 525 [ (M + H)+,
1] , 91 [Bn') , l00] .
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rar-~ a 5 6-Tetra-O-benzyl-1-O-bu~yrvl-2-deoxy-mvo-
inositol (rac-59). A solution of rac-5858 (186 mg, 355
~.mol) butyric anhydride (70 ~Cl, 426 ~.mol) and DMAP (12
mg, 10 ~.mol) in dry pyridine (5 ml) was stirred at room
temperature for 18 h. The solvents were evaporated under
high vacuum to give an oil. Residual pyridine was
removed by evaporating three times with octane. The
residue was dissolved in tert-butyl methyl ether (30 ml)
and was washed once with phosphate buffer (20 ml), once
with sodium hydrogen carbonate (20 ml), once with sodium
hydrogen sulfate (20 ml), once again with phosphate
buffer (20 ml) and then with brine (20 ml). The organic
layer was dried over NaZS09 and filtered. Evaporation of
the solvent gave pure rac-59 (196 mg, 94%) as a solid.
Mp.. 110.1~-110.3~C (from methanol). 1H NMR (CHC13, 360
MHZ} : b 0.91 (3 H, t, ,~ = 7.25 Hz, CH3) , 0.98 (3 H, t, J =
7.46 Hz, CH3) , 1.32-1.42 (2 H, m, CHI) , 1.50-1.60 (2 H, m,
CHz) , 1 . 69 (2 H, tq, ~ = 7.46, 7.46 Hz, O (O) CCHZCH_ZCH3)
2 .39 (2 H, t, ~ = 7.46 Hz, O (0) CC~-zCHZCH3) , 3 .31 (1 H, dd,
~ = 9.66, 2.63 Hz, H-1), 3.44 (1 H, dt, ~ = 9.07, 6.8Z
Hz, OCH_ZCHZCH~CH3) , 3.48 (1 H, dd, J_ = 9.66, 3.07 Hz, H-3) ,
3.49 (1 H, dd, 7 = 9.66, 9.22 Hz, H-5), 3.67 (1 H, dt,
- 8.78, 6.81 Hz, OCHZCH.,CHZCH3) , 3.81 (1 H, dd, ~ = 9.66,
9.66 Hz, H-4), 3.86 (1 H, dd, ~ = 9.66, 9.22 Hz, H-6),
4.51-4.93 (8H, m, CH_ZPh), 5.83 (1 H, dd, T = 3.07, 2.63
Hz, H-2), 7.26-7.36 (20 H, m, CHzp~). MS: ~ (+ve ion
FAB} 595 [ (M + H)+, 21] , 91 [Bn', 100] .
sac-1-O-butyryl-2-deoxy-myo-inositol (rac-60). Compound
(177 mg, 297 umol) was hydrogenated with palladium
(10%) on carbon under hydrogen as described in the
general procedure to give tetrol rte- 0 (68 mg, 98%) as a
solid after freeze drying. Mp: 174.9~-175.9~C (from
ethanol) . ~H NMR ( [D6] DMSO, 360 MHZ) : b 0.84 (3 H, t,
7.38 Hz, CH3), 0.90 (3 H, t, ~ = 7.38 Hz, CH3), 1.21-1.31
(2 H, m, CHz) , 1.36-1.44 (2 H) m, CH2) , 1.54 (2 H, tq, ~7 =
7 . 38 , 7. 00 Hz, 0 (O) CCH2C~IzCH3) , 2 .24 (2 H, t, ~ = 7. 00
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Hz, O (O) CC~izCH~CH3) , 2.97 (1 H, dd, ~ = 8.94, 8 .55 Hz, H-
5), 3.10 (1 H, dd, ~7 = 9.72, 2.72 Hz, H-1), 3.25-3.35 (4
H, m, H-3, H-4, H-6, OCI32CH~CHzCH3) , 3.50 (1 H, dt, J_ _
8.94, 6.60 Hz, OC~zCH2CHZCH3) , 4.8S (4 H, s (br) , OH) , 5.36
(1 H, dd, ~ = 2.72, 2.33 Hz, H-2). MS: m~ (+ve ion FAB)
235 [ (M + H)', 100] . MS: ~z, (-ve ion FAB) 233 [ (M - H')
27] , 87 [Bt0-, 100] .
sac -1-O-buty~r,~~l - 2 -deoxy-myo- inos itol 3 . 4 . 5 . 6 -
rPrrak;~(dibenzv,~~hosnhate (rac-61). A solution of
tetrol rac-6060 (44 mg, 190 ~.mol) and tetrazole (160 mg,
2.28 mmol) in acetonitrile (2 ml) was treated with
dibenzyl ~-diisopropylphosphoramidite (767 ~,1, 2.28
mmol) for 36 h, oxidized with peracetic acid, and worked
up as described. Purification by preparative HPLC (92%
MeOH; 40 ml/min; tR = 21.55 min) gave compound rac-6161 (172
mg, 71%) as an oil. 1H NMR (CDC13, 360 MHZ) : b 0.79 (3 H,
t, ~ = 7.57 Hz, CH3), 1.45 (2 H, tqu, ~ = 7.57, 7.57 Hz (3-
CHZ), 1.46 (1 H, ddd, ~7 = 12.92, l1.14, 11.14 Hz, H-2
(ax)), 2.08 (2 H, t, ~ = 7.57 Hz, a-CHz), 2.58 (1 H, ddd,
~ = 12.92, 4.90, 4.90 Hz, H-2 (eq)}, 4.28-4.37 (1 H, m,
H-3), 4.44 (1 H, dd, ,T = 9.24, 9.24, 9.24 Hz, H-5), 4.53
(1 H, ddd, J = 9.24, 9.02, 9.02 Hz, H-4), 4.56 (1 H, ddd,
~7 = 9.24, 9.24, 9.24 Hz, H-6), 4.83-5.08 (17 H, m, 8 x
C~,ZPh, H-1) , 7.12-7.32 (40 H, m, 8 x CHz~) . 31P NMR
(CDC13, 1H decoupled, 145.8 MHZ): b -1.67 (1 P, s), -1.33
(1 P, s), -0.85 (1 P, s), -0.64 (1 P, s). MS: ~ (+ve
ion FAB) 1275 [ (M + H)+, 4] 91 [Bn+, 100] . MS: ~, (-ve
ion FAB) 1183 j (M - Bn+) -, 16] , 277 jOPO (OBn) -, 100] .
_rac-1-O-butrrvl-2-deoxy-myo-inositol 3.4.5.6-
tetrakis,~phate (rac-62). Compound _r~c-61 (165 mg, 130
~mol) was hydrogenated with palladium (10%) on carbon as
described in the general procedure to give title compound
_ac~-62_ (65 mg, 90%) as a solid after freeze drying. 1H
NMR (Dz0) , 360 MHZ) : b 0.67 (3 H, t, ,1 = 7.41 Hz, CH,} ,
1.33-1.43 (2 H, m, (3-CHZ) , 1.62 (1 H, ddd, sI = 11.72,
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11.71, 11.38 Hz, H-2 (ax) ) , 2.17 (2 H, m, a-CHz) , 2.25 (1
H, ddd, ,~ = 11.38, 2.24, 2.24 Hz, H-2 (eq)), 4.07-4.23 (4
H, m, H-3, H-4, H-5, H-6), 4.85 (1 H, m, H-1), 3'P NMR
(D20, 1H decoupled. 145.8 MHz): b -0.39 (2 P, s), -0.13 (1
5 P, s) , 0.41 (1 P, s) . MS: cue. (+ve ion FAB) 555 [ (M +
H)', 100] , 485 [M - Bt' + 2H'] , 40] . MS: ,~z (-ve ion
FAB) 553 [ (M - H+) -, 100] .
sac-~-O-butyrx,l-2-deoxy-myo-inositol 3 4 5.6-
ki r
10 DIEA (201 ~.1, 1.19 mmol) and acetoxymethyl bromide (119
~.1, 1.19 mmol) were added to a suspension of compound
rac-6262 (37 mg, 66 ~,mol) in acetonitrile (2 ml) as
described in the general procedure. Purification by
preparative HPLC (69% MeOH, 40.00 ml/min, tR = l2.10) gave
15 compound rac5151 ( 31 mg, 50% ) as a syrup . 'H NMR ( [D] 8
toluene, 360 MHZ): b 0.96 (3 H, t, ~7 = 7.48 Hz, CHI), 1.54
(1 H, ddd, ~ = 12.70, 12.70, 12.3l Hz, H-2 (ax)), 1.63-
1.74 (2 H, m, (3-CHZ), 1.8l-1.87 (24 H, 8 s, 8 x OAc), 2.30
(1 H, dt, ~ = l6.28, 7.68 Hz, a-CH2), 2.52 (1 H, dt,
20 16.28, 7.88 Hz, a-CHz), 2.64 (1 H, ddd, 7 = 12.31, 5.31,
5.31 Hz, H-2 (eq)), 4.40-4.59 (4 H, m, H-3, H-4, H-5, H-
6), 5.03 (1 H, ddd, T = 12.20, 9.53, S.31 Hz, H-1), 5.65-
5.95 (16 H, m, 8 x CHzOAc) . 31P NMR ( [D] 8-toluene, 'H
decoupled, 145.8 MHZ): b -4.68 (1 P, s), -4.32 (1 P, s),
25 -3.77 (1 P, s), -3.65 (1 P, s). MS:~ (+ve ion FAB) 1131
[ (M + H)', 12] , 987 [ (M - 2 CHZOAc+ + 3 H)', 100] , MS:
(-ve ion FAB) 1059 [ (M - Bt+) -, 16] , 241 [OP (OCHZOAc) z,
100] .
rac-2-deoxy-mtro-inositol 3.4.S.6-tetrakisphosphate lrac-
30 ~1. Compound ~ was treated with 1 M KOH (260 ~.1) to
adjust the pH to 12.8. The solution was stirred at room
temperature for 2 days. The reaction mixture was
directly poured onto an ion-exchange column (Dowex 50 WX
8, H') for purification. Lyophilization gave compound
35 rac-5353. 1H NMR (D20, 360 MHZ) : ~ 1.55 (1 H, ddd, ~,T =
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12.09, 12.09, 12.09 Hz, H-2 (ax)), 2.24 (1 H, ddd, 7 =
12.09, 4.31, 4.31 Hz, H-2 (eq)), 4.79 (1 H, ddd,
12.09, 8.77, 4.31 Hz, H-1), 5.14 (1 H, ddd, ~ = 9.25,
9.25, 8.83 Hz, H-5), 5.18-5.73 (3 H, m, H-3, H-4, H-6).
31P NMR (DzO, 'H decoupled, 145.8 MHZ) : ~ -0.08 (1 P, s) ,
0.2B (1 P, s), 0.34 (1 P, s), 0.90 (1 P, s). MS:
(+ve ion FAB) 485 [(M + H)+, 100] MS: m/z (-ve ion FAB)
483 [ (M-H+) , 100] .
rar--1-O-benzyl-6-O-butyryl-2 3-4 5-di-O-~yclohexylidene-
rc~,vo-inositol (rac-100). A solution of ~-1-Q-benzyl-
2,3-4,5-di-Q-cyclohexylidene-mvo-inositol (200 mg, 465
~,mol ) , butyric anhydride ( 144 ~,1, 571 ~,mol ) and DMAP ( 6
mg, 50 ~,mol) in dry pyridine (3 ml) was stirred at room
temperature for 36 h. The solvents were evaporated under
high vacuum to give an oil. Residual pyridine was
removed by evaporating three times with octane. The
residue was dissolved in tent-butyl methyl ether (40 ml)
and was washed once with phosphate buffer (20 ml), once
with sodium hydrogen carbonate (20 ml), once with sodium
hydrogen sulfate (20 ml), once again with phosphate
buffer (20 ml) and then with brine (20 ml). The organic
layer was dried over Na~S04 and filtered. Evaporation of
the solvent gave pure rac-100 (186 mg, 80%) as an oil. iH
NMR (CHC13, 360 MHZ) : b 0.94 (3 H, t, ~ = 7.40 Hz, CH3) ,
1.39-1 . 79 (12 H, m, CHz (cyclohex. ) , CHZ ((3-CHz) ) , 2.23-
2.37 (2 H, m, a-CHZ), 3.60 (1 H, dd, s~ = 2.85, 2.08 Hz, H-
1), 3.63 (1 H, dd, ~ = 10.86, 8.09 Hz, H-5), 4.3l (1 H,
dd, ~ = 7.17, 2.85 Hz, H-2), 4.34 (1 H, dd, ~ = 10.86,
8.56 Hz, H-4), 4.38 (1 H, dd, ,~ = 8.56, 7.17 Hz, H-3),
4.78 (2 H, AB, T = 10.01 Hz, ,~2-Ph, 5.24 (1 H, dd, ~ _
8.09, 2.08 Hz, H-6), 7.24-7.39 (S H, m, CHzp~). MS: rr
(+ve ion FAB) 50l [ (M + H)+, 2] , 91 [Bn', 100] .
rac-1-O-benzyl-6-O-butyryl-myo-inositol (rac-101) . A
solution of rac-100 (186 mg, 372 ~.mol) in CH3CN/H20
(100:Z, 8 ml) was stirred with trifluoroacetic acid (4
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82
ml) at room temperature for 2 h. The solvent was
evaporated in high vacuum to give the title compound rac-
1~1 (I25 mg, 98%) as an oil. 1H NMR (CHC13, 360 MHZ) : b
0.86 (3 H, t, ~ = 7.35 Hz, CH3) , 1.44-1.56 (2 H, m, (3-
CHI) , 2. 13-2 .31 (2 H, m, a-CHZ) , 3 .12 (1 H, dd, ~T = 9.13 )
9.13 Hz, H-5), 3.15 (1 H, dd, J = 9.58, 2.45 Hz, H-3),
3.32 (1 H, dd, sl = 10.05, 2.45 Hz, H-1), 3.44 (1 H, dd,
- 9.36, 9.36 Hz, H-4), 4.01 {1 H, dd, ~ = 2.45, 2.45 Hz,
H-2) , 4.44 {2 H, AB, s~ = 9.93 Hz, ~I 2Ph, ) , 5.Z2 (1 H, dd,
~ = 10.05, 9.13 Hz, H-6) , 7.24-7.34 (5 H, m, CHz,~) . MS:
r~ (+ve ion FAB)341 [(M + H)+, 10] 91 [Bn+, 100] . MS:
tt~Z (-ve ion FAB) 339 [ (M - H+)-, 14] , 87 [Bt0-, 100] .
rac-1-O-benzxl-6-O-butyryl-myo-inositol 2~, 3. 4. 5-
tetrakis (dibenzyl) pho~~hate (rac-102). A solution of
compound rac-101 (145 mg, 426 ~.mol) and tetrazole (358
mg, 5.I1 mmol) in acetonitrile (3 ml) was treated with
dibenzyl N-N-diisopropylphosphoramidite (1.72 ml, 5.I1
mmol} for 22 h, oxidized with peracetic acid, and worked
up as described. Purification by preparative HPLC (94%
MeOH; 40 ml/min; tR=22.40 min) gave compound rac-102 (297
mg, SO%) as an oil. 'H NMR (CDClj, 360 MHZ); S 0.78 (3H,
t, ~T=7.38 Hz, CH3) , 1.21-1.52 (2H, m, [3-CH2) , 2.06-2.20 (2
H, m, a-CHZ), 3.41 (1H, ddd, s~, =10.01, 2.03, 2.03 Hz, H-
3), 4.28-4.34 (2 H, m, H-1, H-5), 4.49 ( 1 H, ddd, ~T,
2S =9.41, 9.41, 9.41 Hz, H-4), 4.78-5.18 (18 H, m, Cj~2Ph),
5.50 (1 H, ddd, s~, =9.15, 2.03, 2.03 Hz, H-2), 5.64 (1H,
dd, ~7, =10.14, 10.14 Hz, H-6) , 7.07-7.32 (45 H, m, CHz~) .
''P NMR (CDCL3, 1H decoupled, 145.8 MHZ) ; b -1.94 (1 P,
s),-1.03 (1 P, s), -0.79 (1 P, s), -0.32 (1 P, s). MS:
cr (+ve ion FAB) 138Z [ (M+H} +, 1] , 71 [Bt+, 100] . MS;
m/z (-ve ion FAB) 1279 [ (M-Bn+}-, 16] ( 277 [OPO {OBn) Z,
100] .
z~c-6-O-butyr~~myo-inositol 2. 3. 4J 5 ~etrakis hos hn ate
lrac-IO,~), Compound rac-102 {290 mg, 2I0 ~.mol) was
hydrogenated with palladium (10%) on carbon as described
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in the general procedure to give title compound
(119 mg, 99%) as a solid after freeze drying. 1H NMR
(D20, 360 MHZ): b 0.87(3 H, t, ~ =7.38 Hz, CH3), l.51-1.6I
(2 H, m, (3-CHZ) , 2.39 (2 H, d, ~=7.52 Hz, a-CHZ) 2.96 ( 1
H, ddd, 7, =9.36, 7.45, 2.03 Hz, H-1), 3.88 (1 H, ddd, T
=9.S1, 2.03, 2.03 Hz, H-3), 4.29 (1 H, ddd, 7, =9.94,
9.51, 9.5l Hz, H-5), 4.30 (1 H, dddd, ~ =9.36, 9.36,
2.03, 2.03 Hz, H-2), 4.52 (1 H, ddd, .~, =9.51, 9.5l, 9.51
Hz, H-4) , 5.22 (1H, dd, ~Z =9.94, 9.94 Hz, H-6) . 31P NMR
(DZO, 1H decoupled, l45.8 MHZ); b 0.19 (2P, s), 0.42 (1 P,
s) , 0.89 (1 P, s) . MS:~ (+ve ion FAB) 57l [ (M+H)', 10] ,
71 [Bt+, 100] MS: ~ (-ve ion FAB) 569 [M-H')-, 35] , 79
[OP (O) ~, 100] .
rac 1 O f1 2 dip~lmitovl-sn-glycerolel-6-O-butyryl-m~ro-
1
(rac-104). DIEA (190 ~1, 1.12 mmol) and acetoxymethyl
bromide (126 ~.1, 1.26 mmol) were added to a suspension of
compound rac-103 (40 mg, 70 ~mol) in dry acetonitrile
(1.5 ml). After stirring of the mixture at room
temperature in the dark for 3 h, 1,2-dipalmitoyl-sn-
glycerole (20 mg, 35 ~.mol) was added and the solution was
stirred at 4~C in the dark for 4 days. The solvent was
evaporated under reduced pressure and the crude residue
was extracted with toluene, to give the title compound
rac-104 as a syrup.
rac-1-O-fl 2-dioctanoy_ -sn-glvcerolel-6-O-butyryl-m3ro-
~nos~to~ 3 4 5-trisphos~hate hexakis (acetoxymethy~
ester (rac-105) DIEA (136 ~.1, 800 ~Cmol) and acetoxymethyl
bromide (90 ~,1, 900 ~,mo1) were added to a suspension of
compound rac-103 (28 mg, 50 ~.mol) in dry acetonitrile
(1.5 ml.) After stirring of the mixture at room
temperature in the dark for 3h, 1, 2-dipalmitoyl-sn-
glycerole (23 mg, 70 ~.mol) was added and the solution was
stirred at 4~C in the dark for 4 days. The solvent was
evaporated under reduced pressure and the crude residue
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84
was extracted with toluene, to give the title compound
rac-105 as a syrup.
Materials - A11 chemical reagents were obtained in the
highest purity available. Where necessary, solvents were
dried and/or distilled before use. Acetonitrile was
distilled from phosphorus(V) oxide and stored over
molecular sieves 3 A, as was dimethylformamide (DMF).
Pyridine and toluene were stored over molecular sieves 4
P.. Ethyl-diisopropylamine (DIEA) was dried over sodium
wire. Palladium on charcoal (10%) and trifluoroacetic
acid were from Acros Chemie. Dibenzyl ~T,I~-
diisopropylphosphoramidite, peracetic acid (32o v/w),
tetrazole and acetoxymethyl bromide were from Aldrich.
Butyric anhydride, DIEA were from Merck. Benzyl bromide,
cesium fluoride and 4-dimethylamino pyridine (DMAP) were
from Fluka. A11 other reagents were from Riedel-de Haen.
rac-3,4-Di-O-benzyl-1,2-cyclohexylidene-mvo-inositol (1)
was synthesized in the 3 steps from mvo-inositol as
described previously (Angyal et al., J. Che~m. Soc. 4116
(1961); Jiang and Baker Carbohydr. Chem. 5:615 (1986)),
each of which is incorporated herein by reference. All
compounds were racemic.
4-Di-O-benzyl-5 6-di-O-butyryl-1 2-cvclohex5rlidene-myo-
inositol (152) - A solution of 151 (130 mg, 295 ~mol),
butyric anhydride (193 ~,1, 1.18 mmol) and DMAP (40 mg, 33
~.mol) in dry pyridine (2 ml) was stirred at room
temperature for 2 days. The solvents were evaporated
under high vacuum to give an oil. Residual pyridine was
removed by evaporating three times with octane. The
residue was dissolved in tert-butyl methyl ether (10 ml)
and was washed once with phosphate buffer (10 ml), once
with sodium hydrogen carbonate (10 ml), once with sodium
hydrogen sulfate (10 ml), once again with phosphate
buffer (10 ml) and then with brine (10 ml). The organic
layer was dried over NazS04 and filtered. Evaporation of
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the solvent gave pure ~ (171 mg, 98%) as an oil. 1H NMR
(CHC13, _360 MHz) : b 0.87 (3 H, t, ~ = 7.30 Hz, CH,) , 0.91
(3 _H, t, ~ = 7.30 Hz, CH3) , 1.26-1.87 (14 H, m, CH,
(cyclohex.), 2 x (3-CHZ (Bt)), 2.l2-2.17 (2 H, m, a-CHz),
5 2.22-2.31 (2 H, m, a-CHz) , 3.76 (1H, dd, J_ = 9.29, 4.05
Hz, H-3), 3.97 (1 H, dd, ,~ = 9.29, 8.85 Hz, H-4), 4.01 (1
H, dd, ~T = 7.74, 5.09 Hz, H-1), 4.27 (1 H, dd, ,~, = 5.09,
4.05 Hz, H-2), 4.65-4.99 (4 H, m, 2 x C~2Ph), 4.96 (1 H,
dd, sl = l0.51, 8.85 Hz, H-5), 5.29 (1 H, dd) ~ = 10.51,
(+ve
10 7.74 Hz, H-6), 7.26-7.38 (10 H, m, CHZp,~). MS:
ion FAB) 581 [ (M + H)', 1] , 91 [Bn', 100] . MS: ~ 581.31l
(M + H)+ (calcd for C34H450a 581.311) .
3 , 4-,DiO-~enzyl-5 6-c,~ -O-buty,~~-mvo-inositol (~ 53 ) - A
solution of ~ (166 mg, 286 ~.mol) in 5 ml CH3CN/H~O (100
15 . 1) was stirred with trifluoroacetic acid (2 ml) at room
temperature for 2 h. The raection mixture was evaporated
under diminshed pressure and the product was extracted
with tert.-butyl methyl ether. The organic layer was
washed with brine, dried over Na2S04, filtered and
20 evaporated to give as an oil (140 mg, 99%). -H NMR
(CHC13, 360 MHz) : b 0. 88 (3 H, t, ,l = 7.40 Hz, CH,) , 0.92
(3 H, t, ~ = 7.40 Hz, CH3), 1.50-1.66 (4 H, m, 2 x a-CHZ),
2.11-2.13 (2 H, m, a-CH2), 2.21-2.36 (2 H, m, a-CHI), 3.05
(2 H, s (br), 2 x OH), 3.54 (1H, dd, J = 9.48, 2.77 Hz,
25 H-3), 3.58 (1 H, dd, ~T = 9.86, 2.77 Hz, H-1), 3.95 (1 H,
dd, ~ = 9.71, 9.48 Hz, H-4), 4.17 (1 H, dd, ~ = 2.77,
2.77 Hz, H-2), 4.62-4.87 (4 H, m, 2 x C-~i2Ph), 5.01 (1 H,
dd, ~ = 9.71, 9.71 Hz, H-5), 5.29 (1 H, dd, ~T = 9.86,
9.7Z Hz, H-6) , 7.23-7.34 (10 H, m, CH2P~,) . MS: ~ (+ve
30 ion FAB) 523 ( (M + Na)+, 22] , 91 (Bn+, 100] . MS: r~Lz (-ve
ion FAB) 499 [ (M + H+)-, 2] , 87 [Bt-, 100] . MS: tr
539.207 (M + K)' (calcd for C28H3608K 539.205) .
1 3 4-Tri-O-benz3rl-5~, 6-di-O-butyr5rl-mvo-inositol (154) -
Dry 153 (143 mg, 286 ~,mol) and dry dibutyltin oxide (71
35 mg, 300 ~mol) were heated under reflux in dry toluene
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86
(100 ml) in a Soxhlet apparatus with activated molecular
sieve (3 A) for 4 h. The reaction mixture was cooled to
room temperature and evaporated to dryness under
diminshed pressure. CsF (92 mg, 572 ~,mol) was added to
the residual oil, and the mixture was kept under high
vacuum for 2 h. The residual syrup was dissolved in dry
DMF (5 ml) under argon and benzyl bromide (270 ~,1, 2.28
mmol) was added. After stirring the solution for 18 h,
HPLC (Merck Hibar steel tube (250 mm x 4 mm) filled with
RP 18 material (Merck, Lichrosorb; 10 Vim)) analysis (95%
MeOH; 1.5 ml/min; tR = 3.20 min) showed no further
reaction. Excess of benzyl bromide and DMF were removed
in high vacuum. The crude product was chromatographed by
preparative HPLC (Merck Prepbar steel column (250 mm x 50
mm) filled with RP 18 material (Merck, LiChrospher; 10
~Cm)) (88% MeOH; 40 ml/min; tR = 17.20 min) to give
compound 154 (448 mg, 75%) as a solid. 1H NMR (CHClj, 360
MHz) : b 0.87 (3 H, t, _J = 7.46 Hz, CH3) , 0.90 (3 H, t, ~ _
7.25 Hz, CH3), 1.48-1.68 (4 H, m, 2 x a-CHz), 2.09-2.22 (4
H, m, 2 x a-CHZ) , 2.52 (1 H, s (br) , OH) , 3 .40 (1H, dd,
- 9.99, 2.63 Hz, H-1), 3.44 (1 H, dd, ~ = 9.66, 2.63 Hz,
H-3), 4.03 (1 H, dd, J = 9.88, 9.66 Hz, H-4), 4.19 (1 H,
dd, s~ = 2.63, 2.63 Hz, H-2), 4.51-4.88 (6 H, m, 3 x
C~zPh) , 5.04 (1 H, dd, s~ = 10.09, 9.88 Hz, H-5) , 5.48 (1
H, dd, ~ = 10.09, 9.99 Hz, H-6), 7.23-7.34 (15 H, m,
CH2~) . MS: ~ (+ve ion FAB) 613 [ (M + Na)', 6j , 91 [Bn+,
100) . MS: ~ 6I3.277 (M + Na)+ (calcd for C35HQZO8Na
613.278).
3 4-Tri-O-benz5rl-2 5 6-tri-O-bu~yrvl-myo-inositol (155)
- A solution of ~ (98 mg, 166 ~mol), butyric anhydride
( 68 ~.l , 415 ~,mol ) and DMAP ( 5 mg, 4 ~.mol ) in dry pyridine
(2 ml) was stirred at room temperature fox 2 h. A similar
work-up as described above for compound 2_ gave pure 1~
(106 mg, 99%) as an oil. 1H NMR (CHC13, 360 MHz): S 0.87
(3 H, t, ~ = 7.47 Hz, CH3), 0.89 (3 H, t, ~ = 7.96 Hz,
CH3) , 0.96 (3 H, t, ~T = 7.47 Hz, CHj) , 1.23-1.41 (2 H, m,
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CH2) , 1.51-1,60 {2 H, m, CHZ} , l.65-l.73 (2 H, m, CHZ) ,
2.13 (2 H, t, ~7 = 7.47 Hz, a-CHz) , 2.20 (2 H, t, 7, = 7.47
Hz, a-CHz), 2.43 (2 H, t, T = 7.47 Hz, a-CHZ), 3.46 (1H,
dd, ~T = 10.03, 2.77 Hz, H-1), 3.53 (1 H, dd, _J = 9.B2,
2.99 Hz, H-3), 3.86 (1 H, dd, ~T = 9.82, 9.82 Hz, H-4),
4.40-4.83 (6 H, m, 3 x C$ZPh) , 5.06 (1 H, dd, 7 = 10.03,
9.82 Hz, H-5), 5.37 (1 H, dd, ~ = 10.03, 10.03 Hz, H-6),
5.86 (1 H, dd, ~T = 2.99, 2.77 Hz, H-2), 7.19-7.34 (15 H,
m, CHz,p~) . MS: ~ (+ve ion FAB) 661 [ (M + H) *, 1] , 91
[Bn~, 100] . MS: ~ 661.338 (M + H)* (calcd for CzgHqgOg
661.334).
5-6-Tri-O-buty~,yl-myo-inc~sitol (156) - (105 mg, 159
~,mol) was dissolved in acetic acid (3m1), before
palladium (l00) on charcoal (80 mg, 795 ~.mol) was added.
After stirring the solution at room temperature for 6 h
under hydrogen atmosphere the catalyst was removed by
ultrafiltration and the filtrate was freeze dried to give
title compound 1~5.~ (53 mg, 86%). Mp: 113.4~-114.4~C {from
MeOH) . 1H NMR ( [D] 6-DMSO) : S 0. 94 (3 H, t, ~ = 7. S3 Hz,
CH3) , 0.95 (3 H, t, ~ = 7.53 Hz, CH3) , 0.99 (3 H, t, T =
7.53 Hz, CH3) , 1.59-1.76 (6 H, m, 3 x [3-CHZ} , 2.25-2.36 (4
H, m, 2 x a-CHZ}, 2.43 (2 H, t, ~7 = 7.32 Hz, a-CHI), 3.72
(1H, dd, ~ = 9.77, 2.84 Hz, H-3), 3.83 (1 H, dd, _J =
10.17, 2.84 Hz, H-1), 3.85 (1 H, dd, ~ = 9.77, 9.77 Hz,
H-4), S.02 (1 H, dd, ~T = 9.77, 9.77 Hz, H-5), 5.25 (1 H,
dd, ~ = 10.17, 9.77 Hz, H-6), 5.53 (1 H, dd, ~T = 2.84,
2.84 Hz, H-2) , MS: ~, (+ve ion FAB) 391 [ (M + H}+, 5] , 71
[Bt', 100] . MS: m(,~ (-ve ion FAB) 389 [ (M - H*)-, 11] , 87
[Bt0-, 100] . Anal. Calcd for C18H3oO9: C, 55.37; H, 7.74.
Found: C, 55.43; H, 7.84.
~,,~,. 6-Tri-O-butyry~~-myQ-inositol 1, 3 , ~-
~,r_,'-~ (d,'_~P~7,~r1 )phosphate (157) - A solution of triol
(48 mg, 123 ~.mol) and tetrazole (104 mg, 1.48 mmol) in
acetonitrile (2 ml) was treated with dibenzyl ~,~1-
diisopropylphosphoramidite (500 ~,1, I.48 mmol) for 18 h,
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cooled to -40~C and oxidized with peracetic acid (32%
v/w, 340 ~.1, 1.48 mmol) under vigorous stirring (Yu and
Fraiser-Reid, Tetrahedron Lett. 29:979 (1988)), which is
incorporated herein by reference. The mixture was allowed
to warm to room temperature. The solvent was removed
under reduced pressure and the residual oil was purified
by preparative HPLC (92o MeOH; 40 ml/min; tR = 17.50 min)
to give compound 157 (104 mg, 72%) as an oil. 1H NMR
(CHC13, 360 MHz) : b 0.76 (3 H, t, ~ = 7.32 Hz, CH3) , 0.78
(3 H, t, ~ = 7.32 Hz, CH3), 0.93 (3 H, t, _J = 7.32 Hz,
CH3) , 1.32-1.50 (4 H, m, 2 x (3-CHz) , 1.57-1.67 (2 H, m, (3-
CH~) , 2.04 (2 H, t, ~ = 7.57 Hz, a-CHz) , 2.13 (2 H, t, T =
7.57 Hz, a-CHI), 2.35 (2 H, t, ~ = 7.57 Hz, a-CH2), 4.38
(1 H, ddd, sI = 9.28, 9.28, 2.69 Hz, H-3), 4.47 (1H, ddd,
_J = 9.99, 9.99, 2.69 Hz, H-1), 4.82-5.04 (13 H, m, 3 x
C~-zPh, H-4), 5.14 (1 H, dd, ~T = 9.77, 9.77 Hz, H-5), 5.45
(1 H, dd, 7 = 9.99, 9.77 Hz, H-6), 6.04 (1 H, dd, _J =
2.69, 2.69 Hz, H-2) , 7.20-7.34 (30 H, m, CHzP~) . 3'P-NMR
(CHC13, 'H-decoupled, 145.8 MHz) : b -l.25 (1 P, s) , -1.18
(1 P, s), -1.15 (1 P, s). MS: ~ (+ve ion FAB) 1171 [(M
+ H)+, 4] , 91 [Bn', 100] . MS: m z (-ve ion FAB) 1079 [ (M -
Bn~) , 45] , 277 [OPO (OBn) 2 , 100] .
2 5,6-Tri-O-butvryl-myo-inositol 1f3 4-trisphospha a
(1S8) - The free acid 157 (100 mg, 85 ~.mol) was
hydrogenated as described above to give title compound
$ (47 mg, 88%) after freeze drying. 1H NMR (D20, 360
MHz) : b 0.83 (3 H, t, ~7 = 7.40 Hz, CH3) , 0.84 (3 H, t,
7.40 Hz, CH3) , 0. 92 (3 H, t, ,T = 7.40 Hz, CH3) , 1.46-1.56
(4 H, m, 2 x (3-CHZ) , 1.61-1.71 (2 H, m, (3-CHZ) , 2.32 (2 H,
t, ~T = 7.22 Hz, a-CHZ) , 2.34 (2 H, t, sl = 7.22 Hz, a-CHZ) ,
2.47 (2 H, t, s~ = 7.22 Hz, a-CHz), 4.44-4.S5 (2H, m, H-1,
H-3), 4.6l (1 H, ddd, ~7 = 9.57, 9.39, 9.39 Hz, H-4), 5.26
(1 H, dd, J_ = 9.57, 9.57 Hz, H-5), S.35 (1 H, dd, T =
9.75, 9.57 Hz, H-6) , 5.77 (1 H, s (br) , H-2) . 3'P-NMR
(D20, 1H-decoupied, 14S.8 MHz) : b -0.94 (1 P, s) , -0.09 (1
P, s), -0.06 (1 P, s). MS: ~ (+ve ion FAB) 669 [(M +
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K)', 20] , 71 [Bt', 100] . MS: ~ (-ve ion FAB) 629 [ (M -
H') ~, 30] , 97 [OP (OH) 2-, 100] .
~ ~-Tri-O-butvryl-myo-inositol 1,3.4-trisphos~hate
~Pxa] ; s (acetoxh_yl ) ester (1 59) - DIEA (209 ~1, 1.23
mmol) and acetoxymethyl bromide {123 ~,1, l.23 mmol) were
added to a suspension of compound 158 (39 mg, 61 ~,mol) in
dry acetonitrile (2 ml). After stirring of the mixture at
room temperature in the dark for 4 days a11 volatile
components were evaporated off under reduced pressure and
the crude residue was purified by preparative HPLC (73%
MeOH, 40 ml/min, tR = 20.25) to give compound 1~ (44 mg,
67%) as a syrup. 1H NMR ( [D] etoluene, 360 MHz) : S 0 . 83 (3
H, t, s~ = 7.28 Hz, CH3) , 0.92 (3 H, t, ~ = 7.28 Hz, CH3) ,
0.97 (3 H, t, ~T = 7.48 Hz, CH3) , 1.50-1.60 (2 H, m, (3-
CHz) , 1.67-1.76 (4 H, m, 2 x (3-CHz) , 1.79-1. 96 (18 H, 6 x
s, 6 x OAc) , 2, 08-2.13 (2 H, m, a-CHz) , 2.33-2.47 (2 H, m,
a-CHz) , 2.52-2.61 (2 H, m, a-CHz) , 5.05 (1H, ddd, ~T =
9.64, 9.64, 2.17 Hz, H-3), 5.09 {1 H, ddd, J = 9.64,
9.06, 9.06 Hz, H-4), 5.11 (1 H, ddd, ~ = 9.84, 9.84, 2.17
Hz, H-1), 5.50-5.S3 (14 H, m, 6 x C~zOAc, H-5, H-6), 6.21
(1 H, dd, J = 2.17, 2.17 Hz, H-2) . 31P-NMR (DzO, 'H-
decoupled, l45.8 MHz): S -4.17 {1 P, s), -4.05 (1 P, s),
-3.97 (1 P, s) . MS: ~ (+ve ion FAB) [ (M + K)', 20] , 71
[Bt', 100J . MS: ~ (-ve ion FAB) [ {M - H') ~, 30] , 97 [,
100] .
Although the invention has been described with
reference to the examples provided above, it should be
understood that various modifications can be made without
departing from the spirit of the invention. Accordingly,
the invention is limited only by the claims.
