Antagonists of myo-inositol 3,4,5,6-tetrakisphosphate allow repeated epithelial chloride secretion

Bioorganic & Medicinal Chemistry 11 (2003) 3315–3329

Antagonists of myo-Inositol 3,4,5,6-Tetrakisphosphate Allow

Repeated Epithelial Chloride Secretion

Marco T. Rudolf,^aCarlo Dinkel,^bAlexis E. Traynor-Kaplan^cand Carsten Schultz^a,b,

*

^aInstitut fur Organische Chemie, Universitat Bremen, UFT, 28359 Bremen, Germany

¨

^bEuropean Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany

^cInologic, Inc., 101 Elliot Ave. W, Suite 400, Seattle, WA 98119, USA

Received 11 November 2002; accepted 18 March 2003

Abstract—Cystic ﬁbrosis (CF) patients suﬀer from a defect in hydration of mucosal membranes due to mutations in the cystic

ﬁbrosis transmembrane regulator (CFTR), an apical chloride channel in mucosal epithelia. Disease expression in CF knockout mice

is organ speciﬁc, varying with the level of expression of calcium activated Cl^ꢀchannels (CLCA). Therefore, restoring transepithelial

Cl^ꢀsecretion by augmenting alternate Cl^ꢀchannels, such as CLCA, could be beneﬁcial. However, CLCA-mediated Cl^ꢀsecretion is

transient, due in part to the inhibitory eﬀects of myo-inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P₄]. This suggests that antago-

nists of Ins(3,4,5,6)P₄could be useful in treatment of CF. We have, therefore, synthesized a series of membrane-permeant

Ins(3,4,5,6)P₄derivatives, carrying alkyl substituents on the hydroxyl groups and screened them for eﬀects on Cl^ꢀsecretion in a

human colonic epithelial cell line, T₈₄. While membrane-permeant Ins(3,4,5,6)P₄derivatives had no direct eﬀects on carbachol-

stimulated Cl^ꢀsecretion, Ins(3,4,5,6)P₄derivatives, but not enantiomeric Ins(1,4,5,6)P₄derivatives, reversed the inhibitory eﬀect of

Ins(3,4,5,6)P₄on subsequent thapsigargin activation of Cl^ꢀsecretion. The extent of the antagonistic eﬀect of the Ins(3,4,5,6)P₄

derivatives varied with the position of the alkyl substituents. Derivatives with a cyclohexylidene ketal or a butyl-chain at the 1-

position reversed the Ins(3,4,5,6)P₄-mediated inhibition of Cl^ꢀsecretion by up to 96 and 85%, respectively, whereas butylation of

the 1- and 2-position generated a reversal eﬀect of only 65%. Derivatives carrying the butyl chain only at the 2-position showed no

antagonistic eﬀect. These data: (1) Support the hypothesis that Ins(3,4,5,6)P₄stereospeciﬁcally inhibits Ca²⁺activated Cl^ꢀsecretion

and that Ins(3,4,5,6)P₄mediates most, if not all of the cholinergic-mediated inhibition of chloride secretion in T₈₄cells; (2)

Demonstrate Ins(3,4,5,6)P₄-mediated inhibition can be completely reversed with rationally designed membrane-permeant

Ins(3,4,5,6)P₄antagonists; (3) Demonstrate that a SAR for membrane-permeant Ins(3,4,5,6) P₄antagonists can be generated and

screened in a physiologically relevant cell-based assay; (4) Indicate that Ins(3,4,5,6)P₄derivatives could serve as a starting point for

the development of therapeutics to treat cystic ﬁbrosis.

Introduction

ﬁbrosis transmembrane regulator (CFTR) can be trig-

gered by elevating cAMP levels, and consequently

PKA-dependent phosphorylation.⁵The vast majority of

CF patients suﬀer from a defect in hydration of mucosal

membranes due to mutations in the gene coding for the

CFTR. Mucosal epithelia also express Cl^ꢀchannels

other than the CFTR such as the outwardly rectifying

Cl^ꢀchannel (ORCC), calcium-activated Cl^ꢀchannels

(CLCA) and volume-regulated Cl^ꢀchannels (ClC).

While the ORCC appears to be controlled by the CFTR

and therefore is dysfunctional in CF,^6ꢀ9active CLCA

Transepithelial chloride ﬂux by polarized epithelia controls

a variety of physiological processes, such as intestinal and

pancreatic secretion and renal functions.¹For example, Cl^ꢀ

secretion regulatesthe ﬂuid balance of the intestinallumen,²

and is a driving force for the hydration of airway epithelia.³

Given this key role in physiological functions, defects in Cl^ꢀ

transport can result in life-threatening diseases, such as

diarrhea and cystic ﬁbrosis (CF).⁴

Transepithelial Cl^ꢀsecretion is gated through multiple

apical Cl^ꢀchannels. One of these channels, the cystic

are reportedly more abundant in CF tissue.^10,11

A

number of studies indicate that phenotypes with

increased activity of alternate Cl^ꢀchannels such as the

CLCA correlate to milder clinical manifestations.^9,12ꢀ15

Therefore, in CF, cAMP-mediated Cl^ꢀtransport is

defective,^16,17leaving Cl^ꢀsecretion via the Ca²⁺

*Corresponding author. Tel.: +49-6221-387-210; fax: +49-6221-387-

206; e-mail: schultz@embl.de

doi:10.1016/S0968-0896(03)00188-3

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M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

dependent pathway intact and even enhanced. Thus, it

has been hypothesized that alternate ion channels may

compensate for defects in CFTR function and could be

therapeutically useful. There are compelling arguments

for pursuing artiﬁcial activation of alternate Cl^ꢀchan-

nels to counteract CF pathophysiology. This has lead to

tests with Ca²⁺-elevating agents such as purinergic

agonists in the treatment of CF.¹⁸Currently, two com-

pounds are in clinical development that elevate intra-

cellular calcium [(Ca²⁺)_i] and thereby modulate Cl^ꢀ

secretion; INS365, a PY2Y receptor agonist and dur-

amycin, an antibiotic that triggers an increase in intra-

cellular Ca²⁺levels. However, an increase in [(Ca²⁺)_i]

does not always lead to Cl^ꢀsecretion. Activation of

phospholipase C (PLC) can lead to a transient activa-

tion of Cl^ꢀsecretion through the myo-inositol 1,4,5-tris-

demonstrated to elevate, for instance, intracellular

Ins(3,4,5,6)P₄levels.²⁰Preferred masking groups were

acyloxymethyl groups. Here we demonstrate that some

of these Ins(3,4,5,6)P₄derivatives are potent antagonists

of this inhibition following carbachol stimulation. Fur-

thermore, the selection of modiﬁed Ins(3,4,5,6)P₄deri-

vatives enables us to elucidate some of the structural

requirements for the putative Ins(3,4,5,6)P₄interactions

to its targets.

Results

Design and synthesis

We have previously shown that modiﬁcations of the

hydroxyl groups of Ins(3,4,5,6)P₄selectively inﬂuence

the ability of membrane-permeant Ins(3,4,5,6)P₄deri-

vatives to inhibit carbachol-induced Cl^ꢀ-secretion. In

that study we demonstrated that a 2-deoxy derivative

was a partial agonist for Ins(3,4,5,6)P₄, inhibiting Cl–

secretion, while the corresponding 1-deoxy derivative

showed no eﬀect on Cl^ꢀ-secretion.³⁶Therefore, the

complete deletion of a hydroxyl group eliminated both

the hydrogen bonding donor and acceptor potential for

interaction with targets. In the present study, we sought

derivatives that would provide the acceptor potential

but are void of the hydrogen donor properties. Apart

from the compounds mentioned above, some deoxy-

ﬂuoro-Ins(3,4,5,6)P₄derivatives were previously pre-

pared.³⁷Some of these compounds are suspected to

have eﬀects on protein phosphatases.²²

phosphate [Ins(1,4,5)P₃]/[Ca²⁺

]

pathway, but also

i

promotes a long-term inhibitory feedback, preventing

Cl^ꢀsecretion from being sustained.¹⁹We have demon-

strated that the intracellular signaling molecule,

Ins(3,4,5,6)P₄, becomes elevated after prolonged PLC

activation and ‘uncouples’ Cl^ꢀsecretion from the rise in

intracellular Ca²⁺in mucosal epithelia.²⁰This reg-

ulatory role for Ins(3,4,5,6)P₄has been conﬁrmed by

several investigators.^21,22

The observations that cholinergic but not histaminergic

stimulation uncoupled the Ca²⁺-mediated Cl^ꢀsecretion

(CaMCS), and the inhibitory action of membrane-per-

meant derivative of Ins(3,4,5,6)P₄, have led to the con-

clusion that Ins(3,4,5,6)P₄is the endogenous negative

regulator of this conductance.^20,23Studies by Xie et al.

employing whole cell patch clamp technique with intra-

cellular perfusion of inositol tetrakisphosphates indi-

cated that Ins(3,4,5,6)P₄modulated an apically located

Ca²⁺/calmodulin kinase regulated Cl^ꢀchannel in T₈₄

cells.^22,24Furthermore, Ca²⁺-dependent Cl^ꢀchannels

reconstituted in planar lipid bilayers could also be

regulated by low levels of Ins(3,4,5,6)P₄in the absence

of phosphatase activities.²⁵Electrophysiological analy-

sis of the eﬀects of Ins(3,4,5,6)P₄in the CFPAC-1 cell

line, originally derived from a CF patient suﬀering from

the ꢀF508 defect,²⁶provided further evidence for an

important and widespread intracellular function of

Ins(3,4,5,6)P₄as a negative regulator of Cl^ꢀsecre-

tion.^21,27,28

We prepared a series of membrane-permeant myo-ino-

sitol 3,4,5,6-tetrakisphosphate derivatives alkylated at

the hydroxyl groups, namely 1-O-butyl-2-O-butyryl-

myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetox-

ymethyl) ester [1-O-Bu-2-O-Bt-Ins(3,4,5,6)P₄/AM] (1),

2-O-butyl-1-O-butyryl-myo-inositol 3,4,5,6-tetrakispho-

sphate octakis(acetoxymethyl) ester [2-O-Bu-1-O-Bt-

Ins(3,4,5,6)P₄/AM] (2), 1,2-di-O-butyl-myo-inositol

3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester

[1,2-di-O-Bu-Ins(3,4,5,6)P₄/AM] (3), and the corre-

sponding enantiomeric compounds 3-O-Bu-2-O-Bt-

Ins(1,4,5,6)P₄/AM

(ent-1),

2-O-Bu-3-O-Bt-

Ins(1,4,5,6)P₄/AM (ent-2) and 2,3-O-Bu-Ins(1,4,5,6)P₄/

AM (ent-3), respectively³⁸(Fig. 1).

The pharmaceutical development of activators of Ca²⁺

-

mediated Cl^ꢀsecretion in CF epithelia could be limited

by the inhibitory action of Ins(3,4,5,6)P₄on Cl^ꢀtrans-

port. Therefore, there is an urgent need to understand

the mechanism by which Ins(3,4,5,6)P₄inhibits CaMCS,

speciﬁcally identifying the downstream targets of

Ins(3,4,5,6)P₄. Accordingly, we have synthesized a set of

membrane-permeant Ins(3,4,5,6)P₄derivatives to study

the interaction of Ins(3,4,5,6)P₄with its targets, and—

more importantly—to ﬁnd antagonists of the inhibitory

action of Ins(3,4,5,6)P₄(Fig. 1). Prodrug approaches, in

which the highly polar phosphate groups are masked,

are commonly used to help deliver biologically active

molecules to the cytosol.²⁹In fact, membrane-permeant

derivatives of inositol polyphosphates have been suc-

cessfully employed in the past,^20,30ꢀ36and have been

Synthesis of the Ins(3,4,5,6)P₄derivative rac-1,2-O-cyclo-

hexylidene-myo-inositol 3,4,5,6-tetrakisphosphate octaki-

s(acetoxymethyl) ester (rac-4) started from the common

precursor rac-1,2-O-cyclohexylidene-myo-inositol (rac-5)

(Scheme 1). The phosphorylation of rac-5 proceeded

smoothly using the method of Yu and Frazer-Reid³⁹to

give the fully protected tetrakisphosphate rac-6 after

puriﬁcation by preparative HPLC. Deprotection of the

phosphoric acid triester was achieved by catalytic

hydrogenolysis with Pd/C in ethanol. Equal amounts of

ethyl-N,N-diisopropylamine (DIEA) with respect to the

number of deprotected phosphates produced during the

reaction prevented the otherwise common hydrolysis of

the acid labile cyclohexylidene group. After ﬁltration

and drying rac-7 was obtained as its DIEA salt. rac-7 was

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3317

Figure 1. Structures of myo-inositol 3,4,5,6-tetrakisphosphate derivatives. Bioactivatable protecting groups are shown in red: 1-O-butyl-2-O-butyryl-

myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (1) and the enantiomer 3-O-butyl-2-O-butyryl-myo-inositol 1,4,5,6-tetrakispho-

sphate octakis(acetoxymethyl) ester (ent-1), as well as 2-O-butyl-1-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (2),

1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (3), and the racemic rac-1,2-O-cyclohexylidene-myo-inositol

3,4,5,6-tetrakisphosphate octakis(acetoxymethyl) ester (rac-4).

Scheme 1. (i) (a) (BnO)₂PNiPr₂, 1H-tetrazole, MeCN; (b) ꢀ40 ^ꢁC, AcOOH; (ii) H₂, Pd/C (10%), ethanol, DIEA, 6d; (iii) AMBr, DIEA, MeCN.

alkylated with acetoxymethyl bromide (AMBr) in the pre-

sence of DIEA to give the uncharged, bioactivatable octa-

kis(acetoxymethyl) ester rac-4.

above to ﬁnally give the octakis(acetoxymethyl) esters 2

and ent-2, respectively. The 1,2-di-O-butyl-myo-inositol

(24) and 2,3-di-O-butyl-myo-inositol (ent-24) precursors

were synthesized by alkylation of 8 and ent-8, respec-

tively, with butyl iodide in the presence of NaH in

DMF, followed by hydrogenolysis. The reaction

sequence to the octakis(acetoxymethyl) esters 3 and ent-

3, respectively, was performed as described above for

the other tetrakisphosphates.

The synthesis strategy (Scheme 2) for mono- and dibu-

tyl ether derivatives followed those previously reported

for similar compounds.⁴⁰In brief, enantiomerically pure

3,4,5,6-tetra-O-benzyl-myo-inositol (8) and 1,4,5,6-tetra-

O-benzyl-myo-inositol (ent-8), respectively, were regio-

selectively alkylated with 1-butyl iodide at the 1/3-OH

group via a cyclic dibutylstannyl intermediate. The

remaining hydroxyl group was esteriﬁed with butyric

anhydride. After removal of the benzyl groups, stan-

dard phosphorylation/deprotection procedures gave the

tetrakisphosphates, which were alkylated with acetoxy-

methyl bromide, to yield the uncharged octakis(ace-

toxymethyl) esters 1 and ent-1, respectively. In case of 2-

OH alkylated compounds, the initial alkylation of the 1/

3-OH group was performed with allyl iodide, followed

by the alkylation of the 2-OH group with 1-butyl iodide

in the presence of sodium hydride. Selective removal of

the allyl ether with tris(triphenyl)phosphin rhodium(I)

chloride, followed by treatment with triﬂuoroacetic

acid, gave alcohols 9 and (ent-9), respectively. Butyr-

ylation and subsequent hydrogenolysis resulted in

tetrols 11 and (ent-11), respectively. Phosphorylation

and deprotection steps were performed as described

Finally, the monobutyrates 13, ent-13, 19, and ent-19

were hydrolyzed with KOH to give the potassium salts

of 1-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate (14)

and 3-O-butyl-myo-inositol 1,4,5,6-tetrakisphosphate

(ent-14) as well as 2-O-butyl-myo-inositol 3,4,5,6-tetra-

kisphosphate (22) and 2-O-butyl-myo-inositol 1,4,5,6-

tetrakisphosphate (ent-22), respectively. Combined with

the 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakispho-

sphate (26) and its enantiomer ent-26 a set of six ortho-

gonally alkylated tetrakisphosphates is now available

for in vitro studies.

Eﬀect of inositol 3,4,5,6-tetrakisphosphate derivatives on

calcium-mediated chloride secretion

The colonic epthelia cell line T₈₄, an established model

for the study of Cl^ꢀsecretion, was grown to conﬂuence

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M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

Scheme 2. (i) (a) Bu₂SnO, toluene, reﬂux, soxhlet with molecular sieves, 3 A; (b) butyl iodide, CsF, DMF; (ii) Bt₂O, pyridine, DMAP; (iii) H₂, Pd/C

(10%), AcOOH; (iv) (a) (BnO)₂PNiPr₂, tetrazole, MeCN; (b) ꢀ40 ^ꢁC, AcOOH; (v) AMBr, DIEA, MeCN; (vi) (a) Bu₂SnO, toluene, reﬂux, soxhlet

with molecular sieves, 3 A; (b) allyl iodide, CsF, DMF; (vii) butyl iodide, NaH, DMF; (viii) (a) tris(triphenylphosphine)rhodium(I) chloride, EtOH/

H₂O; (b) TFA. For details on intermediates, see text and Experimental. Only the enantiomers of the Ins(3,4,5,6)P₄series are shown.

on polycarbonate membranes in Snapwells (Costar).

The monolayers were mounted into modiﬁed Ussing

chambers and Cl^ꢀsecretion was measured as short

circuit current (I_sc) as described before.³⁶We have

previously reported that enantiomerically pure 1,2-

di-O-butyryl-Ins(3,4,5,6)P₄/AM was capable of inhi-

biting charbachol-stimulated CaMCS by about 50%

and that of thapsigargin by about 60%.³⁰To com-

pare the inhibitory eﬀects of membrane-permeant

derivatives of Ins(3,4,5,6)P₄and Ins(1,4,5,6)P₄on car-

bachol-stimulated CaMCS, cells were pre-incubated

with 600 mM of the membrane-permeant derivatives 1–

3, respectively, for 30 min prior to mounting, or with

800 mM of the racemic compound rac-4. After an addi-

tional 10 min, 100 mM carbachol was added to the solu-

tion bathing the basolateral surface and the I_scwas

monitored. We choose to stimulate CaMCS with car-

bachol and not with histamine due to the higher mag-

nitude of the eﬀect.²³However, no signiﬁcant change in

I_scwas observed although some of the derivatives led to

a slighty (10–15%) higher I_sccompared to control.

These results indicate that the Ins(3,4,5,6)P₄-derivatives

1–3 and derivative rac-4 did not inhibit CaMCS in T₈₄

cells.

using the membrane-permeant Ins(3,4,5,6)P₄derivative,

Bt₂Ins(3,4,5,6)P₄/AM, which generated intracellular

Ins(3,4,5,6)P₄levels of about 4 mM.²⁰In this way, we

tested whether the membrane-permeant derivatives 1–4

could reverse carbachol-induced inhibition of Cl^ꢀ

secretion.

This protocol could potentially verify whether carba-

chol-mediated inhibition of Cl^ꢀsecretion is due in its

entirety to the elevation of Ins(3,4,5,6)P₄or whether

other processes initiated through carbachol also con-

tribute to the inhibition. To test this possibility, T₈₄

monolayers were preincubated with 600 mM extra-

cellular 1-O-Bu-2-O-Bt-Ins(3,4,5,6)P₄/AM (1) for

30 min prior to mounting. 25 min after stimulation with

carbachol, 1 mM thapsigargin was added to both halves

of the Ussing chamber in order to elevate [Ca²⁺]_iand

subsequently stimulate Cl^ꢀsecretion (Fig. 2). As is

shown in Figure 3, 1-O-Bu-2-O-Bt-Ins(3,4,5,6)P₄/AM

(1) abolished the inhibitory eﬀect of carbachol on thap-

sigargin-stimulated Cl^ꢀsecretion by about 88%, while

the enantiomeric Ins(1,4,5,6)P₄/AM derivative (ent-1)

was ineﬀective (Fig. 3). Lower concentrations (200 mM)

showed no signiﬁcant inhibition (n=4, data not shown).

To investigate whether the other butylated compounds

were equally eﬀective, cells were preincubated with

similar amounts of 2-O-Bu-1-O-Bt-Ins(3,4,5,6)P₄/AM

(2) (Fig. 2) or 1,2-di-O-Bu-Ins(3,4,5,6)P₄/AM (3) and I_sc

was monitored under the same conditions. As shown in

Figure 3, 1,2-di-O-Bu-Ins(3,4,5,6)P₄/AM (3) reversed

the carbachol-mediated inhibition of CaMCS by about

65% compared to control, whereas 2-O-Bu-1-O-Bt-

Ins(3,4,5,6)P₄/AM (2) was ineﬀective (Figs 2 and 3). The

enantiomeric Ins(1,4,5,6)P₄derivatives ent-2 and ent-3

Eﬀect of membrane-permeant Ins(3,4,5,6)P₄-derivatives

on carbachol-induced inhibition of chloride secretion

The response of T₈₄cells to carbachol is followed by a

prolonged period in which subsequent CaMCS is sup-

pressed.²³This inhibition of the Cl^ꢀsecretory response

is due, at least in part, to carbachol-induced elevation in

intracellular Ins(3,4,5,6)P₄levels, uncoupling Cl^ꢀsecre-

tion from [Ca²⁺]_i. This eﬀect could be simulated by

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3319

Figure 2. Time course of the reversed Ins(3,4,5,6)P₄inhibition of thapsigargin-stimulated Cl^ꢀ-secretion by membrane-permeant Ins(3,4,5,6)P₄deri-

vatives. T₈₄monolayers were incubated for 30 min with (A) 1, (B) 2, (C) 3 where indicated, or vehicle (DMSO/5% Pluronic) prior to mounting into

Ussing chambers. Cl^ꢀsecretion was measured as short circuit current (ꢀI_sc). Carbachol (10^ꢀ4M) was added to the basolateral side to induce ele-

vated Ins(3,4,5,6)P₄levels and a transient onset of Cl^ꢀ-secretion.²⁰After 25 min, thapsigargin (10^ꢀ6M) was added to the basolateral and the apical

side to stimulate Cl^ꢀ-secretion a second time. Data reﬂect monitoring every 4 s and are the average of six experiments.

were also unable to reverse the inhibition of Cl^ꢀsecre-

tion (Fig. 3). The racemic compound 1,2-O-cyclohex-

ment with 600 mM extracellular for the compounds 1–3

or 800 mM for the derivative rac-4, respectively (n=2,

data not shown). Furthermore, the rise in [Ca²⁺]_iin

response to thapsigargin was not altered by any of the

derivatives (n=4, data not shown).

ylidene-Ins(3,4,5,6)P₄/AM

(rac-4)

reversed

the

carbachol-induced inhibition of CaMCS by about 95%

compared to control, at an extracellular concentration

of 800 mM (Figs 2 and 3), while lower doses (400 mM)

were not eﬀective (n=4, data not shown). These data

suggest that the Ins(3,4,5,6)P₄derivatives 1, 3, and rac-

4, but not 2, are able to bind to the putative

Ins(3,4,5,6)P₄-binding site, preventing Ins(3,4,5,6)P₄

from inhibiting CaMCS.

Discussion

The recent discovery of Ins(3,4,5,6)P₄as an endogenous

negative regulator of CaMCS in T₈₄cells may assist in

the design of therapeutic agents which modulate ﬂuid

secretion in diseased epithelia of patients suﬀering from

hypersecretory or hyposecretory disorders such as

secretory diarrhea or CF.

Eﬀect of membrane-permeant Ins(3,4,5,6)P₄derivatives

on intracellular calcium levels

We have previously demonstrated that Ins(3,4,5,6)P₄

inhibits Ca²⁺-dependent Cl^ꢀsecretion without altering

intracellular Ca²⁺mobilization.²⁰Therefore, it was

unlikely that the reversal of the inhibition could be due

to an eﬀect on intracellular calcium levels ([Ca²⁺]_i). As

expected, ratio imaging experiments with Fura-2-loaded

T₈₄cells showed unchanged [Ca²⁺]_ilevels after treat-

However, the downstream eﬀector in the Ins(3,4,5,6)P₄-

signaling pathway, the Ins(3,4,5,6)P₄target(s), has not

yet been identiﬁed. Photoaﬃnity labeled derivatives or

aﬃnity columns may help to characterize these proteins

in the future. Knowledge of the canonical ligand/protein

interaction properties of Ins(3,4,5,6)P₄with its putative

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M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

Bt-2-O-Bu-Ins(3,4,5,6)P₄-derivative (2) on Cl^ꢀsecretion

resulted from steric hindrance of the butyl group at the

2-position rather than from loss of hydrogen bonding

donor potential.

Of course, the lack of eﬀect on Cl^ꢀsecretion does not

necessarily reﬂect the binding properties to protein(s).

We investigated the competition of our derivatives with

naturally produced Ins(3,4,5,6)P₄after carbachol sti-

mulation. Since carbachol-mediated inhibition of thap-

sigargin-induced Cl^ꢀsecretion was essentially abolished

by pretreatment with 1-O-Bu-2-O-Bt-Ins(3,4,5,6)P₄/AM

(1) as well as rac-1,2-O-cyclohexylidene-Ins(3,4,5,6)P₄/

AM (rac-4), compounds modiﬁed at the 1-hydroxyl

position seemed to retain the ability to bind to the

Ins(3,4,5,6)P₄target protein thereby antagonizing the

inhibitory eﬀect of Ins(3,4,5,6)P₄. 1,2-Di-O-Bu-

Ins(3,4,5,6)P₄/AM (3) had a slightly weaker eﬀect and 2-

O-Bu-1-O-Bt-Ins(3,4,5,6)P₄/AM (2) was inactive. Thus,

a ﬂexible bulky substituent at the 2-position appeared to

abolish the binding properties of the Ins(3,4,5,6)P₄-

derivative probably due to steric hindrance (Fig. 4). On

the other hand, the more rigid positioning of the cyclo-

hexylidene ketal in rac-4 did not restrict binding, sug-

gesting steric discrimination factors of the

Ins(3,4,5,6)P₄-binding site. The binding ability of the 1-

O-butyl compound (1) to the Ins(3,4,5,6)P₄-binding site

points to the role of the 1-hydroxyl group as a hydrogen

bonding acceptor rather than a donor group (Fig. 4). In

addition, the lipophilicity of the butyl group (or the

cyclohexylidene ketal) seems to promote binding as the

sterically hindered 1,2-di-O-Bu-Ins(3,4,5,6)P₄derivative

(3) is also an antagonist, albeit with lower potency (Fig.

3). All enantiomeric Ins(1,4,5,6)P₄/AM-derivatives (ent-

1 through ent-3) did not prevent Ins(3,4,5,6)P₄from

inhibiting Cl^ꢀsecretion (Fig. 3), again indicating the

importance of the proper orientation of the phosphates.

In addition to the above ﬁndings, the complete reversal

of the carbachol/Ins(3,4,5,6)P₄-mediated inhibition of

the thapsigargin-induced Cl^ꢀsecretion suggests that

carbachol inhibits subsequent CaMCS exclusively via

Ins(3,4,5,6)P₄, with negligible contribution from other

negative regulators.

Figure 3. Ins(3,4,5,6)P₄/AM derivatives, but not Ins(1,4,5,6)P₄/AM

derivatives reversed Ins(3,4,5,6)P₄inhibition of thapsigargin-stimu-

lated Cl^ꢀ-secretion in T₈₄cells. Monolayers were preincubated with

the indicated membrane-permeant tetrakisphosphate acetoxymethyl

ester for 30 min prior to mounting into the Ussing chamber. CaMCS

was ﬁrst stimulated with carbachol (10^ꢀ4M) and after 25 min for a

second time with thapsigargin (10^ꢀ6M). Data are mean peak

ꢀI_scꢂSEM expressed as% control for six experiments. Control values

represent the response to thapsigargin in co-incubated monolayers.

Signiﬁcant diﬀerences are denoted by probability values derived by

Students’ two-tailed t-test (* p <0.03; ** p <0.04; *** p<0.0009).

Concentration of the InsP₄derivatives were 1-O-Bu-2-O-Bt-

Ins(3,4,5,6)P₄/AM (1, 600 mM); 2-O-Bu-1-O-Bt-Ins(3,4,5,6)P₄/AM (2,

600 mM); 1,2-di-O-Bu-Ins(3,4,5,6)P₄/AM (3, 600 mM); rac-1,2-cyclo-

Ins(3,4,5,6)P₄/AM (rac-4, 800 mM), 3-O-Bu-2-O-Bt-Ins(1,4,5,6)P₄/AM

(ent-1, 600 mM); 2-O-Bu-3-O-Bt-Ins(1,4,5,6)P₄/AM (ent-2, 600 mM);

2,3-di-O-Bu-Ins(1,4,5,6)P₄/AM (ent-3, 600 mM).

binding sites will enable us to identify more potent

ligands.

The

modiﬁed

membrane-permeant

Ins(3,4,5,6)P₄-derivatives presented here now enable us

to map the unidentiﬁed binding-sites in the natural

environment of the living cell with transepithelial Cl^ꢀ-

secretion as a read-out.

Derivatives for mapping the Ins(3,4,5,6)P₄-binding sites

require the systematic modiﬁcation of each of the func-

tional groups, namely the hydroxyl and phosphate

moieties to alter particular sites of interaction with the

target. In this study, we modiﬁed the interaction poten-

tial of the hydroxyl groups by simple alkylation since

previous studies have shown that the proper orientation

of the phosphate moieties is essential to exhibit physi-

ological function.^20,24Alkylation of the respective

hydroxyl group yielded Ins(3,4,5,6)P₄derivatives, which

exhibit the same stereochemistry, and an unchanged

acceptor potential for hydrogen bonding. All alkylated

Ins(3,4,5,6)P₄-derivatives (1–4) were unable to inhibit

CaMCS. Therefore, it appears that the hydrogen bond-

ing donor potential of the hydroxyl groups is essential

for the ability to inhibit CaMCS. Furthermore, we pre-

viously reported that a 1-deoxy-derivative, where the

donor and acceptor potential is missing, was incapable

of inhibiting Cl^ꢀsecretion, indicating that the hydrogen

bonding acceptor potential of the 1-hydoxyl group is

critical. In contrast, the 2-hydroxyl group was shown to

play a relatively minor role (Fig. 4), because the 2-deoxy

Ins(3,4,5,6)P₄-derivative was partially active. We can

therefore conclude that the lack of an eﬀect of the 1-O-

For the development of future tools such as tethered

compounds, the 1-hydroxyl group appears to be the

most obvious choice to link a (hydrophobic) spacer to

Ins(3,4,5,6)P₄. Furthermore, membrane-permeant deri-

Figure 4. Relative importance of the hydroxyl groups of Ins(3,4,5,6)P₄

with respect to binding and inhibitory properties.

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3321

vatives of ﬂuorescently labeled Ins(3,4,5,6)P₄-derivatives

may help to locate the Ins(3,4,5,6)P₄targets in living

cells.

¹H NMR and ³¹P NMR spectra were recorded on a

Bruker DPX 200 or a Bruker AM 360 spectrometer.

Chemical shifts were measured in ppm relative to tetra-

methylsilane for ¹H NMR spectra and external 85%

H₃PO₄for ³¹P NMR spectra. Coupling constants (J) are

given in Hz. Mass spectra were recorded using a Finni-

gan Mat 8222 mass spectrometer with fast atom bom-

bardment (FAB) ionization. Peak intensities are given

on a scale from 0 to 100. High resolution masses

(HRMS) were determined relative to known com-

pounds with a mass not diﬀering more than 10%.

Where indicated, high resolution masses were deter-

mined with direct chemical ionisation (DCI) and mat-

ched with peaks from perﬂuorinated kerosins (PFK)

giving a maximum diﬀerence of 5 ppm. Meltings points

(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

polarimeter. Ultraﬁltration of the palladium/carbon

catalyst was performed with a Sartorius ﬁltration appa-

ratus SM 162 01 using ﬁlters from regenerated cellulose

(Sartorius, SM 116 04). Element analyses were per-

formed by Mikroanalytisches Labor Beller, Gottingen,

Germany.

The antagonists identiﬁed in this study can serve as lead

compounds for the development of pharmaceuticals,

which can augment CaMCS by inactivating a feedback

inhibitory mechanism. Such agents may overcome the

chronically reduced electrolyte and water ﬂux of epi-

thelia from CF-patients. An important step for future

optimization and drug development would be the iden-

tiﬁcation of the putative Ins(3,4,5,6)P₄-binding proteins.

In summary, we have demonstrated that modiﬁed

membrane-permeant derivatives are useful tools for

probing inositol phosphate/protein interaction in intact

living cells. This methodology enabled us to identify the

critical nature of the hydrogen bonding acceptor poten-

tial of the 1-hydroxyl group for binding to the

Ins(3,4,5,6)P₄binding site. Furthermore, we were able

to identify speciﬁc antagonists of Ins(3,4,5,6)P₄prior to

the identiﬁcation of the Ins(3,4,5,6)P₄targets. The pre-

paration of additional derivatives in their membrane-

permeant form might both increase our knowledge of

the important messenger Ins(3,4,5,6)P₄and open new

possibilities for developing treatments for CF.

Cell culture

All studies were performed using monolayers of the T₈₄

cell line, and cells from passage numbers 20–29 only.

Methods for the maintenance of T₈₄cells for use in

transepthelial electrolyte transports studies have been

described previously.⁴¹In brief, T₈₄cells were grown in

Dulbecco’s modiﬁed Eagle’s/F12 media (JRH, Lenexa,

KS, USA) supplemented with 5% newborn calf serum

(Hyclone, Logan, UT, USA) and 50 U/mL each of

penicillin/streptomycin (Core Cell Culture Facility,

UCSD). Medium was replaced every third day. For the

measurement of chloride secretion, 10⁶cells were seeded

onto microporous ﬁlter inserts (see above) and main-

tained for 8–12 days prior to experiments in order to

develop conﬂuent monolayers with stable transepithelial

resistance.

Experimental

All chemical reagents were obtained in the highest pur-

ity 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). Ethyl-N,N-diisopropylamine (DIEA, from

Merck) was dried over sodium wire. Palladium on

charcoal (10%) was from Acros Organics. Dibenzyl

N,N-diisopropylphosphoramidite, tetrazole, peracetic

acid (32%), dibutyltin oxide, and acetoxymethyl bro-

mide were from Aldrich. 4-(Dimethylamino)pyridine

(DMAP) and cesium ﬂuoride (CsF) were from Fluka.

Thapsigargin was purchased from Alexis Biochemicals,

La Jolla, CA, USA. Carbachol was obtained from ICN,

Irvine, CA, USA. Fura-2 acetoxymethyl ester (fura-2/

AM) was purchased from Calbiochem, La Jolla, CA,

USA. Cell culture membrane inserts (Snapwell, 0.4 mm

pore size polycarbonate) were obtained from Corning

Costar Corporation (Cambridge, MA, USA). All other

reagents were at least reagent grade and were obtained

commercially.

Chloride secretion

Chloride secretion was measured as short circuit current

(I_sc) across monolayers of T₈₄cells, mounted in mod-

iﬁed Ussing chambers (Physiologic Instruments, San

Diego, CA, USA), bathed with Ringer solution warmed

to 37 ^ꢁC and gassed continuously with 95% O₂, 5% CO₂

at a rate of 30–35 mL/min, as described before.³⁶The

spontaneous potential diﬀerence across the monolayer

was short-circuited with a voltage clamp (Model VCC

MC6, Physiologic Instruments, San Diego, CA, USA).

Short circuit current (I_sc) and conductance were recor-

ded at 4-s intervals using Acquire and Analyze Software

1.2 (Physiologic Instruments, San Diego, CA, USA).

Increased I_scstimulated through cholinergic pathways

or through calcium-mobilizing agonists in T₈₄cells are

fully reﬂective of Cl^ꢀsecretion.⁴²The Ringer’s solution

HPLC was performed on a LDC/Milton Roy Consta-

metric III pump with a LDC/Milton Roy UV Monitor

D (254 nm) or a Knauer refractive index detector. The

analytical column was a Merck Hibar steel tube (250 ꢃ

4 mm) ﬁlled with RP 18 material (Merck, LiChrosorb;

10 mm). Preparative HPLC was performed using a Shi-

madzu LC 8A pump with a preparative LDC UV III

Monitor (254 nm) or a Knauer refractive index detector

and a Merck Prepbar steel column (250 ꢃ 50 mm) ﬁlled

with RP 18 material (Merck, LiChrospher 100, 10 mm).

The eluents were methanol–water mixtures; composi-

tions are given in % methanol (MeOH).

contained (in mM) 140 Na⁺_ꢀ, 5.2 K⁺, 1.2 Ca²⁺

,

0.8 Mg²⁺, 119.8 Cl^ꢀ, 25 HCO₃, 2.4 H₂PO₄^ꢀ, 0.4 HPO^ꢀ₄,

and 10 glucose and was adjusted to pH 7.4.

3322

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

Preincubation of T₈₄cells with membrane-permeant

InsP₄-derivatives

rac-1,2-O-Cyclohexylidene-myo-inositol 3,4,5,6-tetrakis

(dibenzyl)phosphate (rac-6). A solution of rac-1,2-O-

cyclohexylidene-myo-inositol (rac-5) (130 mg, 500 mmol)

and tetrazole (350 mg, 5.00 mmol) in acetonitrile (6 mL)

was treated with dibenzyl N,N-diisopropylphos-

phoramidite³⁹(1.68 mL, 5.00 mmol) for 26 h and subse-

quently oxidized with peracetic acid at ꢀ40 ^ꢁC. After the

mixture warmed to room temperature the solvent was

removed under reduced pressure and the residual oil

was puriﬁed by preparative HPLC (93% MeOH;

40 mL/min; t_R=22.35 min) to give the fully protected

Conﬂuent T₈₄cells on Snapwell inserts were rinsed with

1 mL of Ringer’s solution on each side of the Snapwell.

100 mL of Ringer’s solution containing inositol tetraki-

sphosphate octakis(acetoxymethyl) ester and 2 mL of

DMSO/5% Pluronic 127 was added to the basolateral

reservoir. The cells were incubated at 37 ^ꢁC for 30 min.

The incubation mixture was discarded and the cells were

washed with Ringer’s solution (1 mL) and were ready

for mounting. Cells for control experiments were incu-

bated only with 100 mL of Ringer’s solution with 2 mL of

DMSO/5% Pluronic 127.

1

compound rac-6 (306 mg, 47%) as a colorless gum. H

NMR (CDCl₃, 360 MHz): d 1.20–1.80 (10H, m, CH₂

[C₆H₁₀]), 4.26 (1H, dd, J=6.01, 6.01 Hz, H-1), 4.66 (1H,

dd, J=6.01, 3.43 Hz, H-2), 4.76–4.81 (2H, m, H-3, H-5),

4.92–5.17 (18H, m, CH₂Ph, H-4, H-6), 7.20–7.40 (40H,

m, CH₂Ph). ³¹P NMR (CDCl₃, ¹H decoupled,

145.8 MHz): d ꢀ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) 1209

[(MꢀC₇H⁺₇)^ꢀ, 1], 277 [OPO(OBn)₂, 100]. HRMS

(DCI): m/z 1209.312ꢂ10 ppm (MꢀC₇H⁺₇)^ꢀ(calcd for

C₆₁H₆₅O₁₈P₄, 1209.312).

Synthesis

Enantiomerically pure 3,4,5,6-tetra-O-benzyl-myo-ino-

sitol (8) and 1,4,5,6-tetra-O-benzyl-myo-inositol (ent-8)

were synthesized as described before.⁴⁰rac-1,2-O-

Cyclohexylidene-myo-inositol (rac-5) was synthesized by

the method of Angyal and Tate.⁴³

rac-1,2-O-Cyclohexylidene-myo-inositol 3,4,5,6-tetrakis-

phosphate (rac-7). rac-6 (91 mg, 70 mmol) was dissolved

in dry ethanol (4 mL) before dry ethyl-N,N-diisopropyl-

amine (95 mL, 560 mmol) was added, followed by palla-

dium (10%) on charcoal (84 mg, 800 mmol). After

stirring the solution at room temperature for 6 days

under a hydrogen atmosphere the catalyst was removed

by ﬁltration and the ﬁltrate was dried to give the ethyl-

N,N-diisopropylammonium salt of tetrakisphosphate rac-

7 (108mg, 96%). ¹H NMR (D₂O, 360 MHz): d 1.30–1.90

(10H, m, CH₂[C₆H₁₀]), 4.01(1H, ddd, J=9.08, 9.08,

8.23Hz, H-5), 4.18–4.26 (2H, m, H-1, H-3), 4.32–4.40

(2H, m, H-4, H-6), 4.55 (1H, dd, J=4.26, 3.69Hz, H-2).

³¹P NMR (D₂O, ¹H decoupled, 145.8 MHz): d ꢀ0.24 (1 P,

s), 0.10 (1 P, s), 0.85 (1 P, s), 0.90 (1 P, s). MS: m/z (+ve

ion FAB) 581 [(M+H)⁺, 80], 81 [PO(OH)⁺₂, 100]. MS:

m/z (ꢀve ion FAB) 579 [(MꢀH⁺)^ꢀ, 100].

General procedure for phosphorylation

The selectively protected myo-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 indi-

cated time, the reaction mixture was cooled to ꢀ40 ^ꢁC

and peracetic acid (32% v/w; 1 mol equiv for each mol

equiv of phosphoramidite) was added. After the mixture

reached room temperature the solvent was removed

under reduced pressure and the residual oil was puriﬁed

by preparative HPLC to give the desired inositol tetra-

kisphosphate derivative.

General procedure for removing the benzyl groups by

hydrogenolysis

Either the fully protected myo-inositol tetrakisphos-

phate or the tetra-O-benzyl-myo-inositol derivative,

respectively, was vigorously stirred with palladium on

charcoal (10%; at least 0.1 mol palladium for each mol

of benzyl groups) suspended in acetic acid under a

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.

rac-1,2-O-Cyclohexylidene-myo-inositol 3,4,5,6-tetrakis-

phosphate octakis(acetoxymethyl) ester (rac-4). DIEA

(205 mL, 1.20 mmol) and acetoxymethyl bromide

(120 mL, 1.20 mmol) were added under argon to a sus-

pension of compound rac-7 (108 mg, 66 mmol) in aceto-

nitrile (2 mL). After stirring the reaction mixture in the

dark for 4 days all volatile compounds were evaporated

oﬀ under reduced pressure and the crude residue was

puriﬁed by preparative HPLC (68% MeOH, 40 mL/

min, t_R=19.35) to give compound rac-4 (50 mg, 65%)

as a syrup. ¹H NMR (toluene-d₈, 360 MHz): d 1.20–1.75

(10H, m, CH₂[C₆H₁₀]), 1.81–1.92 (24H, 8 s, 8 ꢃ OAc),

4.28 (1H, dd, J=5.51, 5.51 Hz, H-1). 4.77 (1H, dd,

J=5.51, 3.54 Hz, H-2), 4.91–4.99 (2H, m, H-5, H-6),

5.07 (1H, ddd, J=8.66, 8.27, 3.54 Hz, H-3), 5.17 (1H,

ddd, J=9.05, 8.66, 7.08 Hz, H-4), 5.65–5.94 (16H, m,

CH₂OAc). ³¹P NMR (toluene-d₈, ¹H decoupled,

145.8 MHz): d ꢀ4.56 (1 P, s), ꢀ4.07 (1 P, s), ꢀ3.67 (1 P,

s), ꢀ3.55 (1 P, s). MS: m/z (ꢀve ion FAB) 1083

[(MꢀCH₂OAc⁺)^ꢀ, 35], 241 [OPO(OCH₂OAc)^ꢀ₂, 100].

HRMS: m/z 1157.168 [(M+H)⁺] (calcd for

C₃₆H₅₇O₃₄P₄, 1157.167).

General procedure for the introduction of acetoxymethyl

esters

The thoroughly dried inositol tetrakisphosphate deriva-

tive 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 equiv for each

mol equiv of DIEA) were added. After stirring the mix-

ture at room temperature in the dark for 4 days, all vola-

tile components were evaporated under reduced pressure

and the crude residue was puriﬁed by preparative HPLC,

with the solvent speciﬁed to give the inositol tetrakispho-

sphate octakis(acetoxymethyl) ester as a clear gum.

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3323

D-3,4,5,6-Tetra-O-benzyl-1-O-butyl-myo-inositol

(9).

2.63 Hz, H-2), 7.26–7.36 (20H, m, CH₂Ph). MS: m/z

(+ve ion FAB) 667 [(M+H)⁺, 21], 91 [C₇H₇⁺, 100].

HRMS: m/z 667.364 [(M+H)⁺] (calcd for C₄₂H₅₁O₇,

667.363).

Dry 8 (250 mg, 463 mmol) and dry dibutyltin oxide

(116 mg, 467 mmol) were heated under reﬂux in dry tol-

uene (100 mL) in a Soxhlet apparatus with activated

molecular sieves (3 A) for 18 h. The reaction mixture

was cooled to room temperature and evaporated to

dryness under diminished pressure. CsF (140 mg,

926 mmol) 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 mL, 2.62 mmol) was added. After stir-

ring the solution for 48 h, HPLC analysis (90% MeOH;

1.5 mL/min; t_R=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;

t_R=22.30 min) to give compound 9 (175 mg, 74%) as a

D-1,4,5,6-Tetra-O-benzyl-3-O-butyl-2-O-butyryl-myo-in-

ositol (ent-10). Compound ent-9 was butyrylated as

described above for the other enantiomer to give com-

24

pound ent-10. ½aꢄ_D+10.7^ꢁ(c 2.05 in CHCl₃). HRMS:

m/z 667.367 [(M+H)⁺] (calcd for C₄₂H₅₁O₇, 667.363).

¹H NMR and MS data were in accordance with those of

enantiomer 10.

D-1-O-Butyl-2-O-butyryl-myo-inositol (11). Compound

10 (178 mg, 267 mmol) was hydrogenated with palladium

(10%) on charcoal under hydrogen as described in the

general procedure to give tetrol 11 (81 mg, 99%) as a

solid after freeze-drying. Mp 125–125.9 ^ꢁC (from

24

colorless solid. Mp: 75.4–75.9 ^ꢁC (from methanol). ½aꢄ_D

24

ꢁ

+8.4 (c 0.98 in CHCl₃). H NMR (CDCl₃, 360 MHz):

d 0.91 (3H, t, J=7.25 Hz, CH₃), 1.34–1.44 (2H, m,

CH₂), 1.57–1.65 (2H, m, CH₂), 2.46 (1H, s (br), OH),

3.23 (1H, dd, J=9.31, 2.33 Hz, H-1), 3.43 (1H, dd,

J=9.78, 2.80 Hz, H-3), 3.45 (1H, dd, J=9.78, 9.31 Hz,

MeOH). ½aꢄ_D+26.5^ꢁ(c 2.0 in MeOH). ¹H NMR

1

(DMSO-d₆, 360 MHz): d 0.84 (3H, t, J=7.38 Hz, CH₃),

0.90 (3H, t, J=7.38 Hz, CH₃), 1.21–1.31 (2H, m, CH₂),

1.36–1.44 (2H, m, CH₂), 1.54 (2H, tq, J=7.38, 7.00 Hz,

O(O)CCH₂CH₂CH₃), 2.24 (2H, t, J=7.00 Hz,

O(O)CCH₂CH₂CH₃), 2.97 (1H, dd, J=8.94, 8.55 Hz,

H-5), 3.10 (1H, dd, J=9.72, 2.72 Hz, H-1), 3.25–3.35

(4H, m, H-3, H-4, H-6, OCH₂CH₂CH₂CH₃), 3.50 (1H,

dt, J=8.94, 6.60 Hz, OCH₂CH₂CH₂CH₃), 4.85 (4H, s

(br), OH), 5.36 (1H, dd, J=2.72, 2.33 Hz, H-2). MS: m/

z (+ve ion FAB) 307 [(M+H)⁺, 21], 71 [Bt⁺, 100]; m/z

(ꢀve ion FAB) 305 [(MꢀH⁺)^ꢀ, 27], 87 [BtO^ꢀ, 100].

Anal. (C₁₄H₂₆O₇) C: calcd 54.89; found 54.45; H: calcd

8.55, found 8.56.

H-4),

3.57

(1H,

dt,

J=6.99,

6.52 Hz,

OCH₂CH₂CH₂CH₃), 3.57 (1H, dt, J=6.99 Hz,

OCH₂CH₂CH₂CH₃), 3.91 (1H, dd, J=9.31, 9.31 Hz, H-

5), 3.99 (1H, dd, J=9.78, 9.31 Hz, H-6), 4.27 (1H, dd,

J=2.80, 2.33 Hz, H-2), 4.71–4.94 (8H, m, CH₂Ph),

7.25–7.39 (20H, m, CH₂Ph). MS: m/z (+ve ion FAB)

597 [(M+H)⁺, 1], 91 [C₇H₇⁺, 100]. Anal. (C₃₈H₄₄O₆) C:

calcd 76.48; found 76.87; H: calcd 7.43, found 7.50.

D-1,4,5,6-Tetra-O-benzyl-3-O-butyl-myo-inositol (ent-9).

A similar reaction with the diol ent-8 gave compound

D-3-O-Butyl-2-O-butyryl-myo-inositol (ent-11). A similar

reaction of the fully protected compound ent-10 aﬀor-

24

ent-9. ½aꢄ_Dꢀ8.7^ꢁ(c 1.01 in CHCl₃). Spectral data were

24

ded tetrol ent-11. ½aꢄ_Dꢀ26.6^ꢁ(c 0.76 in MeOH). H

NMR and MS data were in accordance with those

obtained for enantiomer 11.

1

in accordance with those obtained for enantiomer 9.

D-3,4,5,6-Tetra-O-benzyl-1-O-butyl-2-O-butyryl-myo-ino-

sitol (10). A solution of 9 (178 mg, 298 mmol), butyric

anhydride (210 mL, 596 mmol), and DMAP (38 mg,

30 mmol) 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 Na₂SO₄and ﬁltered.

Evaporation of the solvent gave pure 10 (194 mg, 98%)

D-1-O-Butyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakis(di-

benzyl)phosphate (12). A solution of tetrol 11 (63 mg,

206 mmol) and tetrazole (174 mg, 2.47 mmol) in acetoni-

trile (2 mL) was treated with dibenzyl N,N-diisopropyl-

phosphoramidite (834 mL, 2.47 mmol) for 18 h, oxidized

with peracetic acid, and worked up as described above.

Puriﬁcation by preparative HPLC (93% MeOH; 40 mL/

min; t_R=26.45 min) gave compound 12 (165 mg, 60%)

24

as a colorless gum. ½aꢄ_Dꢀ4.9^ꢁ(c 1.08 in CHCl₃). H

NMR (CDCl₃, 360 MHz): d 0.77 (3H, t, J=7.27 Hz,

CH₃), 0.93 (3H, t, J=7.27 Hz, CH₃), 1.12–1.21 (2H, m,

CH₂), 1.31–1.46 (2H, m, CH₂), 1.62 (2H, tq, J=7.26,

7.26 Hz, O(O)CCH₂CH₂CH₃), 2.24 (2H, t, J=7.26 Hz,

O(O)CCH₂CH₂CH₃), 3.26 (1H, dt, J=8.23, 5.81 Hz,

OCH₂CH₂CH₂CH₃), 3.37 (1H, dd, J=9.20, 2.90 Hz, H-

1), 3.44 (1H, dt, J=8.23, 7.26 Hz, OCH₂CH₂CH₂CH₃),

4.35 (1H, ddd, J=9.69, 9.69, 2.42 Hz, H-3), 4.53 (1H,

ddd, J=9.69, 9.69, 9.69 Hz, H-5), 4.68 (1H, ddd,

J=9.69, 9.69, 9.20 Hz, H-6), 4.91 (1H, ddd,

J=9.84,9.69, 9.69 Hz, H-4), 4.92–5.02 (16H, m,

CH₂Ph), 5.91 (1H, dd, J=2.90, 2.42 Hz, H-2), 7.11–7.30

(40H, m, CH₂Ph). ³¹P NMR (CDCl₃, ¹H decoupled,

145.8 MHz): d ꢀ1.81 (1 P, s), ꢀ1.18 (1 P, s), ꢀ0.72 (1 P,

s), ꢀ0.66 (1 P, s). MS: m/z (+ve ion FAB) 1347

1

24

ꢁ

1

as a colorless oil. ½aꢄ_Dꢀ10.4 (c 1.97 in CHCl₃). H

NMR (CDCl₃, 360 MHz): d 0.91 (3H, t, J=7.25 Hz,

CH₃), 0.98 (3H, t, J=7.46 Hz, CH₃), 1.32–1.42 (2H, m,

CH₂), 1.50–1.60 (2H, m, CH₂), 1.69 (2H, tq, J=7.46,

7.46 Hz, O(O)CCH₂CH₂CH₃), 2.39 (2H, t, J=7.46 Hz,

O(O)CCH₂CH₂CH₃), 3.31 (1H, dd, J=9.66, 2.63 Hz,

H-1),

3.44

(1H,

dt,

J=9.07,

6.81 Hz,

OCH₂CH₂CH₂CH₃), 3.48 (1H, dd, J=9.66, 3.07 Hz, H-

3), 3.49 (1H, dd, J=9.66, 9.22 Hz, H-5), 3.67 (1H, dt,

J=8.78, 6.81 Hz, OCH₂CH₂CH₂CH₃), 3.81 (1H, dd,

J=9.66, 9.66 Hz, H-4), 3.86 (1H, dd, J=9.66, 9.22 Hz,

H-6), 4.51–4.93 (8H, m, CH₂Ph), 5.83 (1H, dd, J=3.07,

3324

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

[(M+H)⁺, 8], 91 [C₇H⁺₇, 100]. HRMS: m/z 1347.416

[(M+H)⁺] (calcd for C₇₀H₇₉O₁₉P₄, 1347.417).

HRMS: m/z 554.979 [(MꢀH⁺)^ꢀ] (calcd for

C₁₀H₂₃O₁₈P₄, 554.984).

D-3-O-Butyl-2-O-butyryl-myo-inositol 1,4,5,6-tetrakis

(dibenzyl)phosphate (ent-12). Tetrol ent-11 was phosphi-

tylated and oxidized as described above for compound

D-3-O-Butyl-myo-inositol 1,4,5,6-tetrakisphosphate (ent-

14). The butyryl group of ent-13 was hydrolyzed by the

same method described above to give the tetrakispho-

24

12 to give the fully protected phosphate ent-12. ½aꢄ_D

sphate ent-14. ½aꢄ_Dꢀ4.6^ꢁ(c 0.35 in H₂O, pH 1.6).

+4.8^ꢁ(c 2.09 in CHCl₃). HRMS (DCI): m/z

HRMS: m/z 554.982 [(MꢀH⁺)^ꢀ] (calcd for

[(MꢀC₇H⁺₇)^ꢀ]

(calcd

for

C₁₀H₂₃O₁₈P₄, 554.984). H NMR, ³¹P NMR, and MS

data were in accordance with those obtained for enan-

tiomer 14.

1

1255.345ꢂ10 ppm

1

C₆₃H₇₉O₁₉P₄, 1255.345). H NMR, ³¹P NMR, and MS

data were in accordance with those obtained for enan-

tiomer 12.

D-1-O-Butyl-2-O-butyryl-myo-inositol

3,4,5,6-tetraki-

D-1-O-Butyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakis-

phosphate (13). Compound 12 (160 mg, 118 mmol) was

hydrogenated with palladium (10%) on carbon as

described in the general procedure to give compound 13

sphosphate octakis(acetoxymethyl) ester (1). DIEA

(131 mL, 768 mmol) and acetoxymethyl bromide (77 mL,

768 mmol) were added to a suspension of compound 13

(30 mg, 47 mmol) in acetonitrile (2 mL) as described in

the general procedure. Puriﬁcation by preparative

HPLC (73% MeOH, 37.5 mL/min, t_R=19.30) gave

24

(73 mg, 99%) as a solid after freeze-drying. ½aꢄ_D+4.1^ꢁ

1

(c 1.04 in H₂O, pH 1.6). H NMR (D₂O, 360 MHz): d

24

0.81 (3H, t, J=7.30 Hz, CH₃), 0.91 (3H, t, J=7.30 Hz,

CH₃), 1.22–1.30 (2H, m, CH₂), 1.42–1.49 (2H, m, CH₂),

1.62 (2H, tq, J=7.30, 7.30 Hz, O(O)CCH₂CH₂CH₃),

2.36–2.54 (2H, m, O(O)CCH₂CH₂CH₃), 3.56 (1H, dt,

J=9.49, 6.46 Hz, OCH₂CH₂CH₂CH₃), 3.63 (1H, dt,

J=9.36, 6.74 Hz, OCH₂CH₂CH₂CH₃), 3.70 (1H, dd,

J=9.74, 2.62 Hz, H-1), 4.27 (1H, ddd, J=9.36, 9.36,

9.36 Hz, H-5), 4.31 (1H, ddd, J=9.74, 9.74, 2.25 Hz, H-

3), 4.39 (1H, ddd, J=9.74, 9.36, 9.36 Hz, H-6), 4.49

(1H, ddd, J=9.74, 9.36, 9.00 Hz, H-4), 5.75 (1H, dd,

compound 1 (31 mg, 55%) as a colorless syrup. ½aꢄ_D

ꢀ2.0^ꢁ(c 1.00 in toluene). ¹H NMR (toluene-d₈,

360 MHz): d 0.85 (3H, 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, CH₂),

1.51–1.67 (4H, m, 2 ꢃ CH₂), 1.75–1.75 (24H, 8 s, 8 ꢃ

OAc), 2.08–2.13 (2H, m, CH₂), 3.07 (1H, dd, J=9.45,

2.76 Hz, H-1), 3.46 (1H, dt, J=7.68, 5.91 Hz,

OCH₂CH₂CH₂CH₃), 3.62 (1H, dt, J=7.74, 7.68 Hz,

OCH₂CH₂CH₂CH₃), 4.62 (1H, ddd, J=9.84, 9.84,

2.76 Hz, H-3), 4.67 (1H, 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), 5.04

(1H, ddd, J=9.84, 9.84, 9.45 Hz, H-4), 5.63–5.96 (16H,

m, CH₂OAc), 6.00 (1H, dd, J=2.76, 2.76 Hz, H-2). ³¹P

1

J=2.62, 2.25 Hz, H-2), ³¹P NMR (D₂O, H decoupled,

145.8 MHz): d ꢀ0.10 (2 P, s), 0.50 (1 P, s), 0.80 (1 P, s).

MS: m/z (+ve ion FAB) 627 [(M+H)⁺, 4], 71 [Bt⁺,

100]. MS: m/z (ꢀve ion FAB) 625 [(MꢀH⁺)^ꢀ, 100].

HRMS: m/z 625.025 [(MꢀH⁺)^ꢀ] (calcd for

C₁₄H₂₉O₁₉P₄, 625.025).

1

NMR (toluene-d₈, H decoupled, 145.8 MHz): d ꢀ5.14

(1 P, s), ꢀ4.49 (1 P, s), ꢀ4.06 (1 P, s), ꢀ3.99 (1 P, s).

MS: m/z (+ve ion FAB) 1131 [(MꢀCH₂OAc⁺+2H)⁺,

58], 987 [(Mꢀ3 CH₂OAc⁺+4H)⁺, 100], m/z (ꢀve ion

D-3-O-Butyl-2-O-butyryl-myo-inositol 1,4,5,6-tetrakis-

phosphate (ent-13). A similar reaction with the fully

protected compound ent-12 aﬀorded the free acid ent-13

FAB)

1129

[(MꢀCH₂OAc⁺)^ꢀ,

38],

241

[OPO(OCH₂OAc)^ꢀ₂, 100]. HRMS: m/z 1131.189

[(MꢀCH₂OAc⁺+2H)⁺] (calcd for C₃₅H₅₉O₃₃P₄,

1131.189).

24

after freeze-drying. ½aꢄ_Dꢀ4.1^ꢁ(c 0.78 in H₂O, pH 1.6).

HRMS: m/z 625.023 [(MꢀH⁺)^ꢀ] (calcd for

1

C₁₄H₂₉O₁₉P₄, 625.025). H NMR and MS data were in

accordance with those obtained for enantiomer 13.

D-3-O-Butyl-2-O-butyryl-myo-inositol

1,4,5,6-tetraki-

sphosphate octakis(acetoxymethyl) ester (ent-1). Alkyl-

ation of the phosphate ent-21 as described above

24

D-1-O-Butyl-myo-inositol 3,4,5,6-tetrakisphosphate (14).

An aqueous solution of compound 13 (17 mg, 27 mmol)

was treated with 1 M KOH (260 mL) 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 puri-

ﬁcation. Lyophilization gave compound 14 (14 mg,

aﬀorded the octakis(acetoxymethyl) ester ent-1. ½aꢄ_D

+1.9^ꢁ(c 1.46 in toluene). HRMS: m/z 1129.171

[(MꢀCH₂OAc⁺)^ꢀ] (calcd for C₃₅H₅₇O₃₃P₄, 1129.173).

¹H NMR, ³¹P NMR, and MS data were in accordance

with those obtained for enantiomer 1.

D-1-O-Allyl-3,4,5,6-tetra-O-benzyl-myo-inositol

(15).

24

ꢁ

1

94%). ½aꢄ_D+4.9 (c 0.53 in H₂O, pH 1.6). H NMR

(D₂O, 360 MHz): d 0.78 (3H, t, J=7.44 Hz, CH₃), 1.26

(2H, tq, J=7.44, 7.44 Hz, CH₂), 1.43–1.52 (2H, m,

CH₂), 3.42 (1H, dd, J=9.60, 2.78 Hz, H-1), 3.52 (1H,

dt, J=9.71, 6.74 Hz, OCH₂CH₂CH₂CH₃), 3.59 (1H, dt,

J=9.81, 6.74 Hz, OCH₂CH₂CH₂CH₃), 4.16 (1H, ddd,

J=9.60, 9.60, 2.78 Hz, H-3), 4.21 (1H, 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), 4.46

(1H, ddd, J=9.60, 9.60, 9.37 Hz, H-4). ³¹P NMR (D₂O,

¹H decoupled, 145.8 MHz): d 0.10 (2 P, s), 0.70 (1 P, s),

1.05 (1 P, s); m/z (ꢀve ion FAB) 555 [(MꢀH⁺)^ꢀ, 100].

Dry 8 (690 mg, 1.28 mmol) and dry dibutyltin oxide

(324 mg, 1.3 mmol) were heated to reﬂux in dry toluene

(150 mL) in a Soxhlet apparatus with activated mole-

cular sieves (3 A) for 20 h. The reaction mixture was

cooled to room 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 dis-

solved in dry DMF (10 mL) under argon and 1-allyl

iodide (329 mL, 3.58 mmol) was added. After stirring the

solution for 20 h, HPLC analysis (95% MeOH; 1.5 mL/

min; t_R=3.20 min) showed no further reaction. Excess

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3325

24

of 1-allyl iodide and DMF were removed in high

vacuum. The crude product was chromatographed by

preparative HPLC (90% MeOH; 40 mL/min;

t_R=26.15 min) to give compound 15 (448 mg, 60%) as a

pound ent-15 gave compound ent-16. ½aꢄ_Dꢀ1.6^ꢁ(c 1.87

in CHCl₃). HRMS: m/z 637.357 [(M+H)⁺] (calcd

C₄₁H₄₉O₆, 637.353). ¹H NMR and MS data were in

accordance with those obtained for enantiomer 16.

24

solid. Mp 71–72 ^ꢁC (from methanol). ½aꢄ_D+4.6^ꢁ(c 0.98

1

in CHCl₃). H NMR (CDCl₃, 360 MHz): d 3.30 (1H,

D-3,4,5,6-Tetra-O-benzyl-2-O-butyl-myo-inositol

(17).

dd, J=9.73, 3.10 Hz, H-1), 3.41 (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, OCH₂CHCH₂),

4.19 (1H, dd, J=2.65, 1.33 Hz, OCH₂CHCH₂), 4.20

(1H, dd, J=3.10, 2.65 Hz, H-2), 4.71–4.92 (8H, m,

CH₂Ph), 5.19 (1H, ddt, J=11.50, 1.33, 1.33 Hz,

OCH₂CHCH₂), 5.29 (1H, ddt, J=16.91, 2.65, 1.33 Hz,

OCH₂CHCH₂), 5.94 (1H, dddd, J=16.91, 11.50, 1.33,

1.33 Hz, OCH₂CHCH₂), 7.26–7.37 (20H, m, CH₂Ph).

MS: m/z (+ve ion FAB) 581 [(M+H)⁺, 1], 91 [C₇H₇⁺,

100], m/z (ꢀve ion FAB) 580 [(MꢀH⁺)^ꢀ, 6], 489

[(MꢀC₇H⁺₇)^ꢀ, 100].

Tris(triphenylphosphine)-rhodium(I) chloride (140 mg,

150 mmol) and DIEA (25 mL, 140 mmol) were added to a

suspension of 16 (458 mg, 720 mmol) in 50% ethanol

(90 mL). The suspension was heated to reﬂux for 7 h.

The mixture was cooled to room temperature before

triﬂuoroacetic acid (7 mL) was added and the solution

was stirred for additional 24 h, when HPLC analysis

(90% MeOH; 1.5 mL/min; t_R=4.03 min) showed no

more starting material. After neutralization with aq

NH₃(2 N) the solvents were evaporated under reduced

pressure to give a syrup. The syrup was dissolved in tert-

butyl methyl ether (40 mL) and washed once with

phosphate buﬀer (20 mL) and brine (20 mL), succes-

sively. The organic layer was dried over Na₂SO₄, ﬁltered

and the ether was evaporated oﬀ to give an oil. The

crude oil was puriﬁed by preparative HPLC (92%

MeOH; 40 mL/min; t_R=27.40 min) to give 17 (275 mg,

74%) as a solid. Mp 112.3–113.1 ^ꢁC (from MeOH).

D-3-O-Allyl-1,4,5,6-tetra-O-benzyl-myo-inositol (ent-15).

A similar reaction and workup of the diol ent-8 gave

24

compound ent-15. ½aꢄ_Dꢀ3.9^ꢁ(c 0.82 in CHCl₃). ¹H

NMR and MS data were in accordance with those

obtained for enantiomer 15.

24

½aꢄ_D+24.6^ꢁ(c 0.97 in CHCl₃). ¹H NMR (CDCl₃,

360 MHz): d 0.92 (3H, t, J=7.36 Hz, CH₃), 1.32–1.43

(2H, m, CH₂), 1.53–1.61 (2H, m, CH₂), 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, OCH₂CH₂CH₂), 3.74 (1H, dd,

J=9.52, 9.31 Hz, H-4), 3.86 (1H, dd, J=2.60, 2.60 Hz,

H-2), 3.95 (1H, dt, J=8.95, 6.49 Hz, OCH₂CH₂CH₂),

3.98 (1H, dd, J=9.96, 9.31 Hz, H-6), 4.79–4.94 (8H, m,

CH₂Ph), 7.26–7.36 (20H, m, CH₂Ph). MS: m/z (+ve ion

FAB) 597 [(M+H)⁺, 1], 91 [C₇H⁺₇, 100]. MS: m/z (ꢀve

ion FAB) 595 [(MꢀH⁺)^ꢀ, 100], [(MꢀC₇H⁺₇)^ꢀ, 20].

Anal. (C₃₈H₄₄O₆) C: calcd 76.48; found 76.52; H: calcd

7.43, found 7.40.

D-1-O-Allyl-3,4,5,6-tetra-O-benzyl-2-O-butyl-myo-inosi-

tol (16). Sodium hydride (46 mg, 1.92 mmol) was added

to a stirred solution of 15 (445 mg, 767 mmol) in dry

DMF (5 mL) at room temperature in the dark. The

mixture was stirred for 5 h, after which 1-butyl iodide

(306 mL, 2.68 mmol) was added. The suspension was

stirred for 18 h at 80 ^ꢁC when HPLC (95% MeOH;

1.5 mL/min; t_R=7.35) showed a complete reaction.

Excess of 1-butyl iodide and DMF were evaporated oﬀ

under reduced pressure. The mixture was then dissolved

in tert-butyl methyl ether (40 mL) and washed once with

phosphate buﬀer (20 mL), aq sodium dithionate (20 mL)

and brine (20 mL), successively. The organic layer was

dried over Na₂SO₄, ﬁltered, and the ether was evapo-

rated oﬀ to give an oil. The crude oil was puriﬁed by

preparative HPLC (93% MeOH; 40 mL/min;

t_R=37.10 min) to give the title compound 16 (458 mg,

94%) as an oil. ½aꢄ_D+1.5^ꢁ(c 2.29 in CHCl₃). H NMR

(CDCl₃, 360 MHz): d 0.95 (3H, t, J=7.27 Hz, CH₃),

1.39–1.49 (2H, m, CH₂), 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, OCH₂CH₂CH₂CH₃), 3.89 (1H, dd,

J=2.42, 2.42 Hz, H-2), 3.98 (1H, dd, J=9.45, 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, OCH₂CHCH₂), 4.16 (1H, dd, J=6.06,

1.00 Hz, OCH₂CHCH₂), 4.72–4.95 (8H, m, CH₂Ph),

5.19 (1H, ddt, J=10.66, 1.45, 1.45 Hz, OCH₂CHCH₂),

5.32 (1H, ddt, J=16.95, 1.45, 1.45 Hz, OCH₂CHCH₂),

5.95 (1H, dddd, J=16.95, 10.66, 1.45, 1.45 Hz,

OCH₂CHCH₂), 7.26–7.40 (20H, m, CH₂Ph). MS: m/z

(+ve ion FAB) 637 [(M+H)⁺, 1], 91 [C₇H₇⁺, 100].

HRMS: m/z 637.373 [(M+H)⁺] (calcd C₄₁H₄₉O₆,

637.353).

D-1,4,5,6-Tetra-O-benzyl-2-O-butyl-myo-inositol (ent-

17). A similar reaction of compound ent-16 gave

24

ꢁ

1

ent-17. ½aꢄ_Dꢀ24.9 (c 1.00 in CHCl₃). H NMR and

MS data were in accordance with those obtained for

enantiomer 17.

24

1

D-3,4,5,6-Tetra-O-benzyl-2-O-butyl-1-O-butyryl-myo-in-

ositol (18). A solution of alcohol 17 (178 mg, 298 mmol)

in dry pyridine (4 mL) was treated with butyric anhy-

dride (158 mL, 447 mmol) and DMAP (38 mg, 29 mmol)

and stirred at room temperature. After 18 h, HPLC

analysis (90% MeOH; 1.5 mL/min, t_R=6.40 min)

showed no more starting material. 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 (10 mL), and then with brine (10 mL). The

organic layer was dried over Na₂SO₄and ﬁltered. Evap-

oration of the solvent gave pure 18 (176 mg, 89%) as a

D-3-O-Allyl-1,4,5,6-tetra-O-benzyl-2-O-butyl-myo-inositol

(ent-16). A similar reaction and workup of the com-

24

colorless oil. ½aꢄ_Dꢀ15.4^ꢁ(c 1.00 in CHCl₃). H NMR

1

3326

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

(CHCl₃, 360 MHz): d 0.91 (3H, t, J=7.21 Hz, CH₃),

0.93 (3H, t, J=7.21 Hz, CH₃), 1.35–1.56 (4H, m, 2 ꢃ

CH₂), 1.58–1.72 (2H, m, CH₂), 2.21–2.26 (2H, m, CH₂),

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)CCH₂CH₂CH₃), 3.76 (2H, t, J=9.13, 6.25 Hz,

O(O)CCH₂CH2CH₃), 3.94 (1H, dd, J=2.40, 2.40 Hz,

H-2), 4.00 (1H, dd, J=9.61, 9.61 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, CH₂Ph), 7.25–7.34 (20H, m,

CH₂Ph). MS: m/z (+ve ion FAB) 667 [(M+H)⁺, 1], 91

[C₇H⁺₇, 100]. HRMS: m/z 667.368 [(M+H)⁺] (calcd for

C₄₂H₅₁O₇, 667.363).

OCH₂CH₂CH₂CH₃), 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, 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 (17H, m, CH₂Ph,

H-1), 7.13–7.27 (40H, m, CH₂Ph). ³¹P NMR (CDCl₃,

¹H decoupled, 145.8 MHz): d ꢀ1.64 (1 P, s), ꢀ1.42 (1 P,

s), ꢀ0.76 (1 P, s), ꢀ0.53 (1 P, s). MS: m/z (+ve ion

FAB) 1347 [(M+H)⁺, 33], 71 [Bt⁺, 100], m/z (ꢀve ion

FAB) 1255 [(MꢀC₇H₇⁺)^ꢀ, 16], 277 [OPO(OBn)^ꢀ₂, 100].

HRMS: m/z 1347.418 [(M+H)⁺] (calcd for

C₇₀H₇₉O₁₉P₄, 1347.417).

D-2-O-Butyl-3-O-butyryl-myo-inositol 1,4,5,6-tetrakis(di-

benzyl)phosphate (ent-20). Compound ent-19 was phos-

phitylated and oxidized as described above for

compound 19 to give the fully protected phosphate ent-

D-1,4,5,6-Tetra-O-benzyl-2-O-butyl-3-O-butyryl-myo-ino-

sitol (ent-18). Compound ent-17 was butyrylated as

described above for the other enantiomer to give com-

24

pound ent-18. ½aꢄ_D+15.9^ꢁ(c 1.10 in CHCl₃). HRMS:

20. ½aꢄ_D+3.1^ꢁ(c 1.10 in CHCl₃). HRMS (DCI): m/z

m/z 667.362 [(M+H)⁺] (calcd for C₄₂H₅₁O₇, 667.363).

¹H NMR and MS data were in accordance with those of

enantiomer 18.

1255.354ꢂ10 ppm

[(MꢀC₇H⁺₇)^ꢀ]

(calcd

for

C₆₃H₇₁O₁₉P₄, 1255.354). ¹H NMR and MS data were in

accordance with those obtained for enantiomer 20.

D-2-O-Butyl-1-O-butyryl-myo-inositol (19). Compound

18 (170 mg, 255 mmol) was hydrogenated with palladium

(10%) on carbon under hydrogen as described in the

general procedure to give tetrol 19 (75 mg, 97%) as a

solid after freeze-drying. Mp 131.2–131.8 ^ꢁC (from eth-

D-2-O-Butyl-1-O-butyryl-myo-inositol 3,4,5,6-tetrakis-

phosphate (21). Compound 20 (190 mg, 141 mmol) was

hydrogenated with palladium (10%) on carbon as

described in the general procedure to give title com-

pound 21 (87 mg, 99%) as a solid after freeze-drying.

24

anol). ½aꢄ_D+41.9^ꢁ(c 1.10 in MeOH). ¹H NMR

½aꢄ_D+9.9^ꢁ(c 1.10 in H₂O, pH 1.6). H NMR (D₂O,

360 MHz): d 0.75 (3H, t, J=7.15 Hz, CH₃), 0.76 (3H, t,

J=7.31 Hz, CH₃), 1.19–1.29 (2H, m, CH₂), 1.38–

1.53 (4H, m, 2 ꢃ CH₂), 2.30 (2H, t, J=7.47,

O(O)CCH₂CH₂CH₃), 3.58 (1H, dt, J=9.64, 6.44 Hz,

OCH₂CH₂CH₂CH₃), 3.67 (1H, dt, J=9.64, 6.36 Hz,

OCH₂CH₂CH₂CH₃), 4.01 (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 (D₂O, ¹H decoupled, 145.8 MHz): d ꢀ0.20

(2 P, s), 0.40 (1 P, s), 0.50 (1 P, s). MS: m/z (+ve ion

FAB) 627 [(M+H)⁺, 8], 71 [Bt⁺, 100], m/z (ꢀve ion

FAB) 625 [(MꢀH⁺)^ꢀ, 35], 79 [OP(O)₂^ꢀ, 100]. HRMS:

m/z 625.030 [(MꢀH⁺)^ꢀ] (calcd for C₁₄H₂₉O₁₉P₄,

625.025).

1

(DMSO-d₆, 360 MHz): d 0.87 (3H, t, J=7.16 Hz, CH₃),

0.89 (3H, t, J=7.33 Hz, CH₃), 1.28–1.48 (4H, m, 2 ꢃ

CH₂), 1.52–1.62 (2H, m, CH₂), 2.24–2.36 (2H, m, CH₂),

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,

OCH₂CH₂CH₂CH₃), 3.52 (1H, dd, J=10.23, 9.21 Hz,

H-6), 3.57 (1H, dd, J=2.39, 2.39 Hz, H-2), 4.47 (1H, dt,

J=9.21, 6.39 Hz, OCH₂CH₂CH₂CH₃), 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: m/z (+ve ion

FAB) 307 [(M+H)⁺, 100]. MS: m/z (ꢀve ion FAB) 305

[(MꢀH⁺)^ꢀ, 34], 87 [BtO^ꢀ, 100]. Anal. (C₁₄H₄₄O₆) C:

calcd 54.89; found 54.90; H: calcd 8.55, found 8.51.

D-2-O-Butyl-3-O-butyryl-myo-inositol (ent-19). A similar

reaction of the fully protected compound ent-18 aﬀor-

24

ded tetrol ent-19. ½aꢄ_Dꢀ40.5^ꢁ(c 1.00 in MeOH). H

NMR and MS data were in accordance with those

obtained for enantiomer 19.

D-2-O-Butyl-3-O-butyryl-myo-inositol 1,4,5,6-tetrakis-

phosphate (ent-21). A similar reaction with the fully

protected substrate ent-20 aﬀorded the free acid ent-21

1

24

after freeze-drying. ½aꢄ_Dꢀ9.7^ꢁ(c 1.00 in H₂O, pH 1.6).

D-2-O-Butyl-1-O-butyryl-myo-inositol 3,4,5,6-tetrakis(di-

benzyl)phosphate (20). A solution of compound 19

(55 mg, 178 mmol) and tetrazole (152 mg, 2.15 mmol) in

acetonitrile (2 mL) was treated with dibenzyl N,N-diiso-

propylphosphoramidite (726 mL, 2.15 mmol) for 22 h,

then oxidized with peracetic acid, and worked up as

described. Puriﬁcation by preparative HPLC (92%

MeOH; 40 mL/min; t_R=29.00 min) gave compound 20

HRMS: m/z 625.027 [(MꢀH⁺)^ꢀ] (calcd for

1

C₁₄H₂₉O₁₉P₄, 625.025). H NMR and MS data were in

accordance with those obtained for enantiomer 21.

D-2-O-Butyl-myo-inositol 3,4,5,6-tetrakisphosphate (22).

Compound 21 (33 mg, 52 mmol) was treated with 1 M

KOH (453 mL) 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 puriﬁca-

tion. Lyophilization gave compound 22 (27 mg, 93%).

24

(192 mg, 80%) as a colorless oil. ½aꢄ_Dꢀ3.4^ꢁ(c 1.02 in

1

CHCl₃). H NMR (CDCl₃, 360 MHz): d 0.79 (3H, t,

J=7.30 Hz, CH₃), 0.86 (3H, t, J=7.30 Hz, CH₃), 1.23–

1.38 (2H, m, CH₂), 1.41–1.57 (4H, m, 2 ꢃ CH₂), 2.01–

2.17(2H, m, CH₂), 3.54 (1H, dt, J=8.52, 6.63 Hz,

OCH₂CH₂CH₂CH₃), 3.60 (1H, dt, J=8.52, 7.74 Hz,

24

½aꢄ_Dꢀ1.1^ꢁ(c 0.89 in H₂O, pH 1.6). H NMR (D₂O,

360 MHz): d 0.77 (3H, t, J=7.37 Hz, CH₃), 1.21–1.31

(2H, m, CH₂), 1.39–1.52 (2H, m, CH₂), 3.63 (1H, dt,

1

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

3327

J=9.30, 6.18 Hz, OCH₂CH₂CH₂CH₃), 3.67 (1H, dd,

J=10.00, 2.63 Hz, H-1), 3.75 (1H, dt, J=9.30, 6.56 Hz,

OCH₂CH₂CH₂CH₃), 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). ³¹P NMR (D₂O, ¹H decoupled,

145.8 MHz): d ꢀ0.18 (1 P, s), 0.55 (2 P, s), 0.95 (1 P, s).

MS: m/z (+ve ion FAB) 557 [(M+H⁺)⁺, 100], m/z

(ꢀve ion FAB) 555 [(MꢀH⁺)^ꢀ, 100]. HRMS: m/z

554.983 [(MꢀH⁺)^ꢀ] (calcd for C₁₀H₂₃O₁₈P₄, 554.984).

1.04 mmol) was added. The suspension was stirred for

36 h at 80 ^ꢁC, until HPLC (95% MeOH; 1.5 mL/min;

t_R=7.26) showed predominantly one product. Excess of

1-butyl iodide and DMF were evaporated under

reduced pressure. The mixture was then dissolved in

tert-butyl methyl ether (30 mL) and washed once with

phosphate buﬀer (10 mL), aq sodium dithionate (10 mL)

and brine (10 mL), successively. The organic layer was

dried over Na₂SO₄, ﬁltered, and the ether was evapo-

rated oﬀ to give an oil. The crude oil was puriﬁed by

preparative HPLC (95% MeOH, 40 mL/min,

t_R=27.24 min) to give the title compound 23 (100 mg,

24

D-2-O-Butyl-myo-inositol 1,4,5,6-tetrakisphosphate (ent-

22). The butyryl group of substrate ent-21 was hydro-

lyzed by the method described above to give the tetra-

88%) as an oil. ½aꢄ_D+1.3^ꢁ(c 1.00 in CHCl₃). ¹H NMR

(CDCl₃, 360 MHz): d 0.92 (6H, t, J=7.52 Hz, CH₃),

1.36–1.47 (4H, m, 2 ꢃ CH₂), 1.55–1.63 (4H, m, 2 ꢃ

CH₂), 3.14 (1H, dd, J=9.57, 2.28 Hz, H-3), 3.34 (1H,

dd, J=10.02, 2.28, H-1), 3.43 (1H, dd, J=9.34, 9.34, H-

5), 3.52 (1H, dt, J=9.11, 6.60 Hz, OCCH₂CH₂CH₃),

3.60 (1H, dt, J=9.11, 6.60Hz, OCCH₂CH₂CH₃), 3.75

(2H, t, J=6.60 Hz, OCH₂CH₂CH₂CH₃), 3.89 (1H, dd,

J=2.28, 2.28Hz, H-2), 3.92 (1H, dd, J=9.57, 9.34Hz, H-

4), 4.00 (1H, dd, J=10.02, 9.34Hz, H-6), 4.71–4.93 (8H,

m, CH₂Ph), 7.24–7.39 (20H, m, CH₂Ph). MS: m/z (+ve

ion FAB) 653 [(M+H)⁺, 1], 91 [C₇H⁺₇, 100]. HRMS: m/z

653.383 (M+H)⁺(calcd for C₄₂H₅₃O₆653.384).

24

kisphosphate ent-22. ½aꢄ_D+1.6^ꢁ(c 0.60 in H₂O, pH

1.6). HRMS: m/z 554.985 [(MꢀH⁺)^ꢀ] (calcd for

1

C₁₀H₂₃O₁₈P₄, 554.984). H NMR and MS data were in

accordance with those obtained for enantiomer 22.

D-2-O-Butyl-1-O-butyryl-myo-inositol 3,4,5,6-tetrakis-

phosphate octakis(acetoxymethyl) ester (2). DIEA

(182 mL, 1.06 mmol) and acetoxymethyl bromide

(107 mL, 1.06 mmol) were added to a suspension of

compound 21 (37 mg, 59 mmol) in acetonitrile (2 mL) as

described in the general procedure. Puriﬁcation by pre-

parative

t_R=21.12 min) gave compound 2 (33 mg, 46%) as a

HPLC

(72%

MeOH,

40 mL/min,

D-1,4,5,6-Tetra-O-benzyl-2,3-di-O-butyl-myo-inositol (ent-

23). A similar reaction of the compound ent-8 gave

24

ꢁ

syrup. ½aꢄ_D+1.1 (c 1.07 in toluene). H NMR (tolu-

ene-d₈, 360 MHz): d 0.93 (3H, t, J=7.28 Hz, CH₃), 0.97

(3H, t, J=7.48 Hz, CH₃), 1.39–1.49 (2H, m, CH₂), 1.51–

1.60 (2H, m, CH₂), 1.77–1.86 (24H, 8 s, 8 ꢃ OAc), 2.39

(1H, dt, J=16.67, 7.38 Hz, CH₂), 2.63 (1H, dt,

J=16.67, 7.68, CH₂), 3.78 (1H, dt, J=9.45, 6.30 Hz,

OCH₂CH₂CH₂CH₃), 3.87 (1H, dt, J=9.06, 6.30 Hz,

OCH₂CH₂CH₂CH₃), 4.40 (1H, dd, J=2.36, 2.36 Hz, H-

2), 4.68 (1H, ddd, J=9.55, 9.55, 2.36 Hz, H-3), 4.79

(1H, ddd, J=9.55, 9.45, 9.45 Hz, H-5), 5.03 (1H, ddd,

J=9.55, 9.55, 9.55 Hz, H-4), 5.09 (1H, ddd, J=10.04,

9.55, 9.55 Hz, H-6), 5.21 (1H, dd, J=10.04, 2.36 Hz, H-

1), 5.58–5.93 (16H, m, CH₂OAc). ³¹P NMR (toluene-d₈,

¹H decoupled, 145.8 MHz): d ꢀ4.42 (1 P, s), ꢀ3.98

(1 P, s), ꢀ3.48 (2 P, s). MS: m/z (+ve ion FAB)

1131 [(MꢀCH₂OAc⁺+2H)⁺, 44], 987 [(Mꢀ3

CH₂OAc⁺+4H)⁺, 100], m/z (ꢀve ion FAB) 1129

[(MꢀCH₂OAc⁺)^ꢀ, 18], 241 [OPO(OCH₂OAc)^ꢀ₂, 100].

HRMS: m/z 1131.187 [(MꢀCH₂OAc⁺+2H)⁺] (calcd

for C₃₅H₅₉O₃₃P₄, 1131.189).

compound ent-23. ½aꢄ_Dꢀ1.2^ꢁ(c 2.20 in CHCl₃). Spec-

1

tral data were in accordance with those obtained for

enantiomer 23.

D-1,2-Di-O-butyl-myo-inositol (24). Compound 23

(100 mg, 153 mmol) was hydrogenated with palladium

(10%) on carbon under hydrogen as described in the

general procedure to give tetrol 19 (40 mg, 92%) as a

solid after freeze-drying. Mp 143.7–144.5 ^ꢁC (from eth-

24

anol). ½aꢄ_D+18.7^ꢁ(c 0.80 in MeOH). ¹H NMR

(DMSO-d₆, 360 MHz): d 0.89 (3H, t, J=7.52 Hz, CH₃),

0.91 (3H, t, J=7.38 Hz, CH₃), 1.24–1.39 (4H, m, CH₂),

1.41–1.53 (4H, m, CH₂), 2.88 (1H, dd, J=9.55, 2.63 Hz,

H-3), 2.93 (1H, dd, J=9.93, 2.63 Hz, H-1), 3.14 (1H, dt,

J=9.51, 6.42 Hz, OCH₂CH₂CH₂CH₃), 3.31 (1H, dd,

J=9.52, 9.52 Hz, H-5), 3.39–3.67 (6H, m,

3

ꢃ

OCH₂CH₂CH₂CH₃, H-2, H-4, H-6), 4.43 (1H, s, OH),

4.52 (1H, s, OH), 4.59 (2H, s (br), OH), MS: m/z (+ve

ion FAB) 293 [(M+H)⁺, 100]. MS: m/z (ꢀve ion FAB)

291 [(MꢀH⁺)^ꢀ, 100], Anal. (C₁₄H₂₈O₆) C: calcd 57.51;

found 56.32; H: calcd 9.65, found 9.91.

D-2-O-Butyl-3-O-butyryl-myo-inositol 1,4,5,6-tetrakis-

phosphate octakis(acetoxymethyl) ester (ent-2). Alkyl-

ation of the phosphate ent-23 as described above

D-2,3-Di-O-butyl-myo-inositol (ent-24). A similar reac-

tion and workup of the fully protected compound ent-23

24

aﬀorded the octakis(acetoxymethyl) ester ent-2. ½aꢄ_D

aﬀorded tetrol ent-24. ½aꢄ_Dꢀ18.9^ꢁ(c 1.36 in MeOH). ¹H

ꢀ1.3^ꢁ(c 0.60 in toluene). HRMS: m/z 1129.177

[(MꢀCH₂OAc⁺)^ꢀ] (calcd for C₃₅H₅₇O₃₃P₄, 1129.173).

¹H NMR and MS data were in accordance with those

obtained for enantiomer 2.

NMR and MS data were in accordance with those

obtained for enantiomer 24.

D-1,2-Di-O-butyl-myo-inositol 3,4,5,6-Tetrakis(dibenzyl)-

phosphate (25). A solution of compound 24 (40 mg,

137 mmol) and tetrazole (115 mg, 1.64 mmol) in acetoni-

trile (2 mL) was treated with dibenzyl N,N-diisopropyl-

phosphoramidite (552 mL, 1.64 mmol) for 18 h, oxidized

with peracetic acid, and worked up as described in the

general procedures. Puriﬁcation by preparative HPLC

D-3,4,5,6-Tetra-O-benzyl-1,2-di-O-butyl-myo-inositol (23).

Sodium hydride (13 mg, 522 mmol) was added to a stir-

red solution of 8 (94 mg, 174 mmol) in dry DMF (3 mL)

at room temperature in the dark. The mixture was stir-

red for 5 h, after which 1-butyl iodide (120 mL,

3328

M. T. Rudolf et al. / Bioorg. Med. Chem. 11 (2003) 3315–3329

(93% MeOH; 40 mL/min; t_R=26.05 min) gave com-

1.00mmol) and acetoxymethyl bromide (111mL,

1.00mmol) were added to a suspension of compound 26

(34 mg, 55mmol) in acetonitrile (2 mL) as described in the

general procedure. Puriﬁcation by preparative HPLC

(73% MeOH, 40mL/min, t_R=20.40min) gave com-

24

pound 25 (133 mg, 73%) as an oil. ½aꢄ_Dꢀ2.3^ꢁ(c 1.00 in

1

CHCl₃). H NMR (CDCl₃, 360 MHz): d 0.79 (3H, t,

J=7.31 Hz, CH₃), 0.86 (3H, t, J=7.31 Hz, CH₃), 1.13–

1.38 (4H, m, 2 ꢃ CH₂), 1.41–1.56 (4H, m, 2 ꢃ CH₂),

3.24 (1H, dd, J=9.89, 2.15 Hz, H-1), 3.36 (1H, dt,

J=8.31, 7.41 Hz, OCH₂CH₂CH₂CH₃), 3.41 (1H, dt,

J=8.31, 5.87 Hz, OCH₂CH₂CH₂CH₃), 3.60 (1H, dt,

J=8.88, 6.55 Hz, OCH₂CH₂CH₂CH₃), 3.71 (1H, dt,

J=8.88, 6.55 Hz, OCH₂CH₂CH₂CH₃), 4.16 (1H, ddd,

J=9.89, 9.89, 2.47 Hz, H-3), 4.21 (1H, dd, J=2.47,

2.15 Hz, H-2), 4.48 (1H, ddd, J=9.67, 9.67, 9.67 Hz, H-

5), 4.79 (1H, ddd, J=9.89, 9.67, 9.67 Hz, H-6), 4.95

(1H, ddd, J=9.89, 9.67, 9.67 Hz, H-4), 4.96–5.09 (16H,

m, CH₂Ph), 7.12–7.32 (40H, m, CH₂Ph). ³¹P NMR

24

pound 3 (42 mg, 65%) as a syrup. ½aꢄ_Dꢀ2.3^ꢁ(c 1.03 in

1

toluene). H NMR (toluene-d₈, 360 MHz): d 0.92 (3H, t,

J=7.28 Hz, CH₃), 0.99 (3H, t, J=7.48 Hz, CH₃), 1.33–

1.47 (4H, m, 2 ꢃ CH₂), 1.48–1.54 (2H, m, CH₂), 1.61–1.69

(2H, m, CH₂), 1.77–1.87 (24H, 8 s, 8 ꢃ OAc), 3.11 (1H,

dd, J=9.84, 2.36Hz, H-1). 3.46 (1H, dt, J=8.79, 7.28Hz,

OCH₂CH₂CH₂CH₃), 3.56 (1H, dt, J=8.79, 7.28Hz,

OCH₂CH₂CH₂CH₃), 3.84 (1H, t, J=6.50 Hz, 2 ꢃ

OCH₂CH₂CH₂CH₃), 4.34 (1H, dd, J=2.36, 2.36 Hz, H-

2), 4.49 (1H, ddd, J=9.45, 9.45, 2.36 Hz, H-3), 4.68

(1H, ddd, J=9.65, 9.65, 9.65 Hz, H-5), 4.90 (1H, ddd,

J=9.65, 9.45, 9.45 Hz, H-4), 5.06 (1H, ddd, J=9.84,

9.65, 9.65 Hz, H-6), 5.68–5.96 (16H, m, CH₂OAc). ³¹P

1

(CDCl₃, H decoupled, 145.8 MHz): d ꢀ1.82 (1 P, s),

ꢀ1.59 (1 P, s), ꢀ0.81 (1 P, s), ꢀ0.68 (1 P, s). MS: m/z

(+ve ion FAB) 1333 [(M+H)⁺, 2], 91 [C₇H₇⁺, 100], m/z

(ꢀve ion _ꢀFAB) 1241 [(MꢀC₇H⁺₇)^ꢀ, 8], 277

[OPO(OBn)₂, 100]. HRMS: m/z 1333.416 [(M+H⁺)⁺]

(calcd for C₇₀H₈₁O₁₉P₄, 1333.417).

1

NMR (toluene-d₈, H decoupled, 145.8 MHz): d ꢀ5.12

(1 P, s), ꢀ3.98 (1 P, s), ꢀ3.69 (1 P, s), ꢀ3.42 (1 P, s).

MS: m/z (+ve ion FAB) 1189 [(M+H)⁺, 3], 1045

[(Mꢀ2 ꢃ CH₂OAc⁺+3H)⁺, 100], m/z (ꢀve ion FAB)

1115 [(MꢀCH₂OAc⁺)^ꢀ, 30], 241 [OPO(OCH₂OAc)^ꢀ₂,

100]. HRMS: m/z 1117.210 [(MꢀCH₂OAc⁺+2H)⁺]

(calcd for C₃₅H₆₁O₃₂P₄, 1117.210).

D-2,3-Di-O-butyl-myo-inositol 1,4,5,6-tetrakis(dibenzyl)-

phosphate (ent-25). Compound ent-24 was phosphity-

lated and oxidized as described above for compound 25

24

to give the fully protected phosphate ent-25. ½aꢄ_D

+2.5^ꢁ(c 1.15 in CHCl₃). HRMS (DCI): m/z

D-2,3-Di-O-butyl-myo-inositol 1,4,5,6-tetrakisphosphate

octakis(acetoxymethyl) ester (ent-3). Alkylation of the

phosphate ent-26 as described above aﬀorded the octa-

1241.3748ꢂ10 ppm

[(MꢀC₇H⁺₇)^ꢀ]

(calcd

C₆₃H₇₃O₁₈P₄, 1241.3748). H NMR and MS data were

for

1

24

in accordance with those obtained for enantiomer 25.

kis(acetoxymethyl) ester ent-3. ½aꢄ_D+2.5^ꢁ(c 1.10 in

toluene). HRMS: m/z 1115.192 [(MꢀCH₂OAc⁺)^ꢀ]

1

D-1,2-Di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate

(26). Compound 25 (134 mg, 100 mmol) was hydro-

genated with palladium (10%) on carbon as described in

the general procedure to give title compound 26 (52 mg,

(calcd for C₃₅H₅₉O₃₂P₄, 1115.194). H NMR and MS

data were in accordance with those obtained for enan-

tiomer 3.

24

87%) as a solid after freeze-drying. ½aꢄ_D+4.1^ꢁ(c 1.10 in

H₂O, pH 1.6). ¹H NMR (D₂O, 360 MHz): d 0.70 (3H, t,

J=7.37 Hz, CH₃), 0.71 (3H, t, J=7.37 Hz, CH₃), 1.13–

1.24 (4H, m, 2 ꢃ CH₂), 1.32–1.46 (4H, m, 2 ꢃ CH₂),

3.39 (1H, dd, J=9.47, 2.33 Hz, H-1), 3.45 (1H, dt,

J=8.59, 8.16 Hz, OCH₂CH₂CH₂CH₃), 3.51 (1H, dt,

J=8.59, 5.79 Hz, OCH₂CH₂CH₂CH₃), 3.56 (1H, dt,

J=9.47, 6.31 Hz, OCH₂CH₂CH₂CH₃), 3.65 (1H, dt,

J=9.47, 6.84 Hz, OCH₂CH₂CH₂CH₃), 4.02–4.08 (2H,

m, H-2, H-3), 4.11 (1H, ddd, J=9.47, 9.47, 9.47 Hz, H-

5), 4.27 (1H, ddd, J=9.47, 9.47, 9.47 Hz, H-4), 4.39

(1H, ddd, J=9.47, 9.21, 9.21 Hz, H-6). ³¹P NMR (D₂O,

¹H decoupled, 145.8 MHz): d ꢀ0.69 (1 P, s), ꢀ0.41 (2 P,

s), ꢀ0.12 (1 P, s). MS: m/z (+ve ion FAB) 613

[(M+H)⁺, 15], 81 [PO(OH)⁺₂, 100], m/z (ꢀve ion FAB)

611 [(MꢀH⁺)^ꢀ, 90]. 79 [OP(O)^ꢀ₂, 100]. HRMS: m/z

611.043 [(MꢀH⁺)^ꢀ] (calcd for C₁₄H₃₁O₁₈P₄, 611.046).

Acknowledgements

We thank Dr. Peter Schulze for MS spectra and Ms.

Nicole Heath for revising the manuscript. This work

was

supported

by

the

Deutsche

For-

schungsgemeinschaft and the Fonds der Chemischen

Industrie.

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Article Doi

Antagonists of myo-inositol 3,4,5,6-tetrakisphosphate allow repeated epithelial chloride secretion

DOI: 10.1016/S0968-0896(03)00188-3

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