Development of carbohydrate-derived inhibitors of acid sphingomyelinase

Article abstract of DOI:10.1016/j.bmc.2009.11.030

The acid sphingomyelinase is an emerging drug target, especially for inflammatory lung diseases. Presently, there are no directly-acting potent inhibitors available for cell-based studies. The potent inhibitor phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2) is not only unsuited for cell culture studies, but also does not provide hints for further structural improvements. In the SAR study described here, we replaced the inositolphosphate moiety by a carbohydrate derivative and the phosphatidic acid residue by an alkylsulfone ester. The resulting compound is more active than its parent compound and offers new means for further structural modification.

Full text of DOI:10.1016/j.bmc.2009.11.030

Bioorganic & Medicinal Chemistry 18 (2010) 939–944
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of carbohydrate-derived inhibitors of acid sphingomyelinase
*
Anke G. Roth, S. Redmer, Christoph Arenz
Institut für Chemie, Humboldt Universität zu Berlin, 12489 Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The acid sphingomyelinase is an emerging drug target, especially for inflammatory lung diseases. Pres-
ently, there are no directly-acting potent inhibitors available for cell-based studies. The potent inhibitor
phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2) is not only unsuited for cell culture studies, but also
does not provide hints for further structural improvements. In the SAR study described here, we replaced
the inositolphosphate moiety by a carbohydrate derivative and the phosphatidic acid residue by an alkyl-
sulfone ester. The resulting compound is more active than its parent compound and offers new means for
further structural modification.
Received 17 August 2009
Revised 30 October 2009
Accepted 13 November 2009
Available online 11 December 2009
Keywords:
Sphingolipids
Enzyme inhibitors
SAR studies
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Dawson and co-workers14 found out that the inositol ring (number
of phosphate groups, stereochemistry, and the existence of hydro-
The acid sphingomyelinase (aSMase) is an emerging drug tar-
get in a variety of diseases.1–3 The enzyme is essential for infec-
tion of non-phagocytotic cells with Neisseria gonorrhoeae4 and
inhibition of aSMase prevents bacterial infections in a rat model
of cystic fibrosis.5 Formation of acute lung injury (ALI) elicited
by endotoxin, acid instillation or platelet-activating factor (PAF)6
and the formation of pulmonary emphysema,7 is regulated by
aSMase. Pharmacological or genetic inhibition of aSMase prevents
apoptosis and degeneration of liver cells in a mouse model for
Wilson’s disease.8 In addition, there are several reports that aSM-
ase significantly contributes to the formation of atherosclerotic
plugs.9 The rapid advancement in dissecting the biological and
patho-physiological roles of aSMase however, stands in sharp con-
trast to the lack of potent and selective inhibitors of this enzyme.
For animal and cell culture studies, the tricyclic antidepressants
desipramine and imipramine are commonly used, despite of their
known indirect way of action10 and despite their known lack of
specificity towards other lysosomal sphingolipid hydrolases like
acid ceramidase.11
xyl groups) is more or less invariant. In contrast, the phosphatidic
acid residue could be replaced by a simple alkylsulfonyl residue,
leading to inhibitors of the same potency, when compared to
PtdIns3,5P2. These inhibitors have improved biophysical properties
and a marked cellular activity.13 However, since PtdIns3,5P2 is a
naturally occurring lipid, involved in signal transduction, we envi-
sion the development of aSMase inhibitors that are less likely to
interfere with phosphoinositide signalling and that may provide
further possibilities for introducing structural modifications lead-
ing to more potent aSMase inhibitors.
2. Results and discussion
Preceding the identification of PtdIns3,5P2, Sandhoff and col-
leagues found out that various nucleoside-3,5-bisphosphates inhi-
bit the aSMase with IC50 values of ꢀ5
l
M, respectively.15 In the
light of our newer observations, clearly showing that the inhibitory
potency of phosphoinositide analogs dramatically increases with
lipophilicity, we were amazed by such a potent inhibition, despite
of the relatively moderate lipophilicity of the nucleoside bisphos-
phates. Thus we wondered if carbohydrate analogues of PtdIns
3,5P2 may be potent inhibitors of aSMase, too. While in the inositol
moiety, the 1,3,5-trisphosphate motif is essentially in a cis, cis
arrangement, the 3,5-bisphosphate motif at the ribose has a trans
orientation. We believe that the trans orientation is possible only
because of the flexibility at the primary 50-carbon atom of the ri-
Some time ago, we were able to identify phosphatidylinositol-
3,5-bisphosphate (PtdIns3,5P2) as the most potent inhibitor of
aSMase (KM = 0.53 l
M).12 Yet, this naturally occurring phospho-
lipid has unfavourable biophysical characteristics like its fivefold
negative charge and its two long fatty acid chains that would
firmly fix it within cellular membranes. Moreover, the compound
is a substrate for phospholipases A1, A2, C and D and phosphoino-
sitide phosphatases, making it ill-suited for cell culture studies. In
first structure–activity-relationship (SAR) studies, we12,13 and
bose scaffold. Because D-glucose is structurally very similar and
is a very low-cost starting material and in addition the arsenal of
established regio- and chemo-selective procedures at the glucose
scaffold is nearly unlimited, we decided to synthesize PtdIns3,5P2
analogs derived from glucose.
* Corresponding author. Tel.: +49 30 2093 8393; fax: +49 30 2093 6947.
E-mail address: christoph.arenz@chemie.hu-berlin.de (C. Arenz).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.030

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A. G. Roth et al. / Bioorg. Med. Chem. 18 (2010) 939–944
Table 1
In order to mimic the 1,3,5-trisphosphate and the 3,5-bisphos-
Inhibition of aSMase from rat brain
phate patterns of PtdIns3,5P2 and of the nucleoside inhibitors
diisopropylidene glucofuranose 1 was chosen as a starting mate-
rial, allowing for modification at the 3-position. The starting mate-
rial was benzylated in quantitative yield to give 2, which then was
a
Compds
Inhibition IC50 (lM)
7a
7b
7c
7d
39.6 ( 0.68)
4.16 ( 0.07)
2.34 ( 0.07)
0.44 ( 0.01)
converted into 3-O-benzyl-D-glucose 3, upon treatment with acetic
acid (Fig. 1).16 Benzyl-protection at the anomeric position gave 4 in
moderate yield. In the next step, alkyl sulfonyl groups were regio-
selectively attached to the 6-O-position to form 5a–d. Each of the
compounds then was treated with an excess of phosphoramidate
and the P(III) intermediates were converted into the protected
phosphates 6a–d by oxidation, using hydroperoxide. The final
deprotection by catalytic hydrogenation afforded the phosphoino-
sitide-analogs 7a–d.
a
Values are means of three experiments, standard deviation is given in
parentheses.
OR
P
OH
O
OR
O
O
RO
RO
O
When the analogs 7a–d were tested as inhibitors of aSMase, we
noticed a strong correlation between inhibition and increasing al-
kyl chain length. Fortunately, the carbohydrate analogue 7d
O
P
i
HO
OR
O
OR
OR
OR
(IC50 = 0.44 lM) is approximately twice as potent when compared
to PtdIns3,5P2, itself (Table 1). In analogy to PtdIns3,5P2, the IC50
values for neutral sphingomyelinase (nSMase) inhibition by com-
RO
OH
RO
O
O
P
4
8
R = Bn
pounds 7a-d are all >100 lM, respectively (data not shown).
ii
R = H 9a
In order to learn more about the SAR of the carbohydrate deriv-
atives, we furthermore envisioned the synthesis of the unlipidated
Figure 2. Reagents and conditions: (i) (BnO)2PN(iPr)2, tetrazole, CH3CN, rt, 24 h,
then tBuOOH, 0 °C to rt, 30 min; (ii) H2, Pd/C, MeOH/H2O (4:1), rt, 16 h.
scaffold
log 9b (see Figs. 2 and 3). Previous studies in our group had shown
that myo-inositol 1,3,5-trisphosphate (IC50 = 27 M), but not cyclo-
hexane-cis, cis-1,3,5-trisphosphate (IC50 >100 M), shows moder-
D-glucose-2,4,6-trisphosphate 9a and its 1,3-dideoxy-ana-
l
l
ate inhibition of aSMase.13 We wondered whether in contrast to
the inositol ring, the hydroxyl groups of the glucose ring would
not play a role for aSMase inhibition. The synthesis of 9a started
from the intermediate 4, which was exhaustively phosphorylated
under the conditions described above (Fig. 2). The yield for this
one-pot reaction leading to 8 was only 32%. In the final step, the
benzyl protecting groups were removed in a single quantitative
step using hydrogen on palladium-charcoal catalyst, to afford 9a.
whether the inhibitor 7d is active in cell culture, we stimulated
apoptosis in cultured HEK-293 cells using 10ꢂ8 M dexamethasone.
When 1 lM or 2 lM of the inhibitor together with the dexameth-
asone were added to the culture media, the apoptosis was gradu-
ally suppressed down to the level of untreated cells (Fig. 4).
3. Conclusions
Despite the fact that aSMase is emerging as an important drug
target, there are no potent and selective inhibitors that can be used
in cell culture. We have synthesized the first selective aSMase
inhibitor based on a carbohydrate scaffold. The inhibitor signifi-
cantly differs from the structure of PtdIns3,5P2, is more active than
its parent compound and provides a new starting point for SAR
studies leading to more potent inhibitors that may replace the tri-
cyclic antidepressants in cell culture and model animal studies. In-
deed, we were able to show that the carbohydrate-based inhibitor
is active in cell culture and inhibits dexamethasone-induced apop-
tosis. However, whether the inhibitor affects the lysosomal aSMase
or the secreted aSMase only will be a matter of future investiga-
tions (Table 1).
The synthesis of the dideoxy-analog 9b started from
D-glucose
diacetonide 1, which was de-hydroxylated using the Barton–
McCombie procedure.17 After acidic deprotection the resulting 3-
deoxy-D-glucopyranose 12 was peracetylated and transformed into
the glycosyl bromide (Fig. 3). The latter was de-functionalized to
yield the tetrahydropyrane derivative 14.18 This compound was
deprotected using cation exchange resin. The triol 15 then was
phosphorylated according to the known procedure and was quan-
titatively deprotected to give the final product 9b. To our surprise,
9a as well as 9b only marginally inhibited aSMase, with IC50 values
ꢁ50
lM, respectively (data not shown).
Inhibitors of aSMase are potentially worthwhile as suppressors
of apoptosis in a variety of inflammatory diseases. In order to test
O
S
O
O
O
S
O
O
O
OH
O
R''
O
R''
OR
O
R'O
R'O
O
v
ii
iv
O
O
P
HO
BnO
OR
O
OR'
OR'
HO
R'O
OR'
O
O
OH
R'O
O P
OH
5a-d
R' = Bn
OR'
O
1
3
6a-d
(R' = Bn)
(R = H)
i
(R = H)
iii
a R'' = C2H5
vi
b R'' = C6H13
c R'' = C8H17
d R'' C12H25
R = Bn
2
R = Bn
4
R' = H 7a-d
Figure 1. Reagents and conditions: (i) NaH, BnBr, DMF, 0 °C–rt, 3 h; (ii) AcOH (90%), 105 °C, 45 min; (iii) BnOH, AcCl, rt, 24 h; (iv) R0SO2Cl, NEt3, CH2Cl2, rt, 16 h; (v)
(BnO)2PN(iPr)2, tetrazole, CH3CN, rt, 24 h, then tBuOOH, 0 °C to rt, 30 min; (vi) H2, Pd/C, MeOH/H2O (4:1), rt, 16 h.

A. G. Roth et al. / Bioorg. Med. Chem. 18 (2010) 939–944
941
O
O
R''
OR
R
O
iii
vi
O
O
R''
R'
RO
O
O
R''
OR
1
12
15
(R = H)
(R = OH)
(R = OH)
iv
vii
i
13 R'= R'' = OAc
16 R = PO(OBn)2
9b R = PO(OH)2
10 R = SC(S)Me
11 R = H
v
viii
ii
14 R'= H, R'' = OAc
Figure 3. Reagents and conditions: (i) MeI, NaH, CS2; (ii) Bu3SnH; (iii) AcOH (90%), 105 °C, 45 min; (iv) Ac2O, pyridine; (v) HBr, then Bu3SnH; (vi) Na, MeOH; (vii)
(BnO)2PN(iPr)2, tetrazole, CH3CN, rt, 24 h, then tBuOOH, 0 °C to rt, 30 min; (viii) H2, Pd/C, MeOH/H2O (4:1), rt, 16 h.
five column volumes (CV) of equilibration buffer (nSMase:
20 mM Tris/HCl, 1 mM EDTA, 1 mM EGTA, protease inhibitor mix,
pH 7.4; aSMase: 50 mM NaOAc, 1 mM EDTA, 1 mM EGTA, protease
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
inhibitor mix, pH 5.0). After sample loading, the column was
washed with 10CV of equilibration buffer, followed by a linear gra-
dient (3CV) of washing buffer (0–100%, equilibration buffer, con-
taining 1 M sodium chloride) and maintained for 8CV at washing
buffer (100%). The final elution was carried out with a 10CV gradi-
ent Triton X-100 (0–1.1%, v/v) in washing buffer.
4.1.2. Sphingomyelinase assays
The micellar nSMase assays using 14C-labeled sphingomyelin as a
substrate were performed as described before.19 The fluorescent
aSMase assay was performed in a 384-well-plate using the 6-hexa-
decanoylamino-4-methylumbelliferyl-phosphorylcholine (HMU-PC)
substrate.13,20 Reaction mixtures consisted of HMU-PC-
substrate (13.3
100 mM NaOAc, pH 5.2, 0.2% (w/v) Na-TC, 0.02% (w/v), NaN3 0.2%
(v/v) Triton X-100) and enzyme preparation (13.3 L). The inhibi-
lL), reaction-buffer (13.3 lL total, containing
Figure 4. The aSMase inhibitor 7d inhibits dexamethasone-induced apoptosis in
HEK-293 cells. Absorption from
a DNA fragmentation ELISA. Apoptosis was
l
stimulated using 10ꢂ8 M dexamethasone for 12 h. Control: untreated cells.
tors, which are well-soluble, were taken from 1 mM stock solutions
(100 mM NaOAc, pH 4.5). Inhibitors were added in various concen-
trations and the reactions were incubated for 3 h at 37 °C in a plate
reader (FLUOstar OPTIMA, BMG labtech, Offenburg, Germany). The
fluorescence of 6-hexadecanoylamino-4-methylumbelliferone
(HMU) was measured (kex = 380 nm, kem = 460 nm) in real time.
Michaelis–Menten analysis was done using radio-labelled sphingo-
myelin. The assays using either the HMU-PC or the 14C-sphingomy-
elin as a substrate gave the same results.
4. Experimental
4.1. Enzyme preparation and activity assays
4.1.1. Partial purification of aSMase and nSMase
The isolation and partial purification of aSMase has been
slightly modified from a method described previously.19 Briefly,
aSMase was obtained as follows: One rat brain (stripped rat brains,
Pel-Freez Biologicals, Rogers, AR, USA) was homogenized in NaOAc
buffer (10 mL, 100 mM, pH 5.0), containing Triton X-100 (0.1%, v/v)
and protease inhibitor mix. The brain tissues were homogenized
for three rounds of 10 passes each. Afterwards, the crude homoge-
nate was centrifuged to remove debris (1000g, 15 min). The super-
natant was centrifuged at 35,000g for 45 min and was filtered
4.2. Apoptosis assay
DNA fragmentation ELISA: First, the kinetics of DNA fragmenta-
tion after dexamethasone donation were measured in the lysate
and in the supernatant, respectively. Between 6 h and 8 h, there
was a steep increase in absorbance in the probes from the superna-
tant; this is typical for apoptosis (data not shown). The apoptosis
assay was performed according to the manufacturer’s protocol
(Roche Cat. No. 11 585 045). Briefly, cells were harvested and sus-
pended in culture medium (2 ꢃ 105 cells per mL) containing BrdU
trough a sterile filter (£ = 0.45 lm). The enzyme preparation was
stored at ꢂ80 °C or loaded onto a strong anion exchange column
(Mono Q 5/50 GL, GE Healthcare). For nSMase containing micro-
somes, one rat brain was homogenized with a Teflon pestle in
Tris/HCl buffer (10 mL, 50 mM, pH7.4) containing EDTA (2 mM),
EGTA (5 mM), DTT (5 mM), b-mercaptoethanol (5 mM) and a pro-
tease inhibitor mix (Roche) after washing with isotonic sodium
chloride solution. The crude homogenate was centrifuged (1000g,
15 min) to remove debris. The supernatant was centrifuged at
100,000g, 90 min, and the pellet was solubilized in homogenisation
buffer containing Triton X-100 (1.7%, v/v). After rocking at 4 °C for
2 h, the microsome preparation was stored at ꢂ80 °C or loaded
onto a strong anion exchange column (Mono Q 5/50 GL, GE Health-
care). For both sphingomyelinases, the column was flushed with
labelling solution (10 lM final concentration) and plated in a 96-
well cell culture dish (ꢀ0.5 ꢃ 103 cells per well). After 16 h, cells
were washed and new media was added. Then, cells were treated
with dexamethasone (10ꢂ8 M) and 7d (2
lM). After 7 h of incuba-
tion, the supernatant (100
lL) was collected and added to a 96-
well plate containing immobilized anti-BrdU antibody. After incu-
bation, removal of the supernatant and extensive washing, the sec-
ondary antibody and the TMB substrate were added and
absorbance was measured at 370 nm (FLUOstar OPTIMA, BMG Lab-
tech). The experiment was performed in quintuplicate.

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A. G. Roth et al. / Bioorg. Med. Chem. 18 (2010) 939–944
4.3. Synthesis
4.3.3. General procedure for preparation of 1,3-di-O-benzyl-6-O
-alkylsulfonyl- -glucose (5a–d)
To a stirred solution of 1,3-di-O-benzyl-
D
The syntheses of 416 and 1518 have been described previously.
Synthetic protocols can be found in the Supplementary data.
General methods and material: All solvents employed were of
reagent grade and were dried by refluxing over appropriate dry-
ing agents. All reactions were performed under argon unless sta-
ted otherwise. All reactions were monitored by thin-layer
chromatography (TLC) by using Silica Gel 60 F254 and the com-
pounds were detected with UV light (254 nm) or by spraying
the plates with Seebach solution (25 g MoO3 ꢃ H3PO4 ꢃ H2O,
10 g Ce(IV)SO4 ꢃ 4H2O, 60 ml sulfuric acid and 905 ml of water)
or by sulfuric acid solution (5% sulfuric acid in ethanol) followed
by heating at 250 °C. Flash chromatography was performed on
silica gel MERCK 60 (0.040–0.063 mm). Organic phases were
dried over MgSO4 or Na2SO4 and solvents were evaporated under
reduced pressure while maintaining the water bath temperature
between 40 and 45 °C. NMR spectra (1H, 13C, APT, and 31P) were
recorded in CDCl3, CD3OD, CD3CN or D2O on Bruker DPX-300
NMR spectrometer at 300 MHz (1H), 75 MHz (13C, APT), and
121 MHz (31P), temperature 25 °C. Chemical shifts are given in
ppm with TMS as internal standard (d = 0.00); 31P, 85% H3PO4
(d = 0.00) as external standard. Mass spectra were recorded with
a Hewlett–Packard GCMS 5995-A spectrometer.
D-glucose 4 (1.0 equiv)
and triethylamine (3.0 equiv) in dichloromethane (5 ml) was
added the corresponding sulfonyl chloride (1.1 equiv) at 0 °C.
The reaction mixture was warmed up from 0 °C to room temper-
ature overnight and diluted with diethyl ether. The mixture was
washed successively with 100 ml each of water, 1 M HCl, water,
saturated sodium bicarbonate solution and brine. The solution
dried over anhydrous sodium sulfate. Filtration and concentra-
tion of the organic layer gave an oily residue, which was
purified by silica gel chromatography (cyclohexane/ethyl acetate
4:1 (v/v)).
4.3.4. 1,3-Di-O-benzyl-6-O-ethylsulfonyl-a/b-D-glucose (5a)
The desired product was obtained as a colourless oil (92 mg,
73%). 1H NMR (300 MHz, CDCl3): d = 1.43 (dt, J = 2.2, 7.4 Hz, 3H);
2.52 (s, 2H); 3.17 (q, J = 7.4 Hz, 2H); 3.50–3.74 (m, 3H); 4.30–4.47
(m, 2H); 4.76 (td, J = 2.7, 11.4 Hz, 2H); 4.89–5.05 (m, 2H); 7.30–
7.40 (m. 10H) ppm. 13C NMR (75 MHz, CDCl3): d = 8.1; 44.9; 68.1;
68.2; 69.1; 69.7; 70.2; 71.2; 72.6; 73.5; 74.3; 74.8; 75.1; 82.5;
83.2; 98.0; 101.7; 127.9; 128.0; 128.1; 128.2; 128.2; 128.6;
136.7; 136.7; 138.3; 138.4 ppm. HRMS for: C22H28O8SNa [M+Na]+
calcd: 475.1397, found: 475.1407.
4.3.1. 1,5-Anhydro-3-deoxy-2,4,6-dibenzylphosphoryl-
glucitol (16)
D-
4.3.5. 1,3-Di-O-benzyl-6-O-hexylsulfonyl-D-glucose (5b)
The desired product was obtained as a colourless oil (70 mg,
31%). 1H NMR (300 MHz, CDCl3): d = 0.89 (t, J = 6.8 Hz, 3H); 1.27–
1.34 (m, 4H); 1.41 (tt, J = 4.9, 9.6 Hz, 2H); 1.86 (ddd, J = 5.0, 7.64,
18.2 Hz, 2H); 2.14–2.55 (br s, 2H); 3.08–3.17 (m, 2H); 3.49–3.58
(m, 1H); 3.59–3.72 (m, 2H); 3.83 (ddd, J = 1.8, 4.5, 9.6 Hz, 1H);
4.31 (dd, J = 1.9, 11.3 Hz, 1H); 4.41 (dd, J = 4.6, 11.3 Hz, 1H); 4.58
(d, J = 11.7 Hz, 1H); 4.75 (dd, J = 5.5, 11.6 Hz, 2H); 4.99 (dd,
J = 5.5, 7.4 Hz, 2H); 7.30–7.40 (m, 10H) ppm. 13C NMR (75 MHz,
CDCl3): d = 13.9; 22.2; 23.3; 27.8; 31.1; 50.4; 67.9; 69.1; 69.7;
70.2; 72.6; 75.1; 82.5; 98.0; 127.9; 128.2; 128.3; 128.6; 136.6;
138.4 ppm. HRMS for: C26H35O8S [MꢂH]ꢂ calcd: 507.2058, found:
507.2064.
To a stirred solution of 1,5-anhydro-3-deoxy-D-glucitole 15
(0.17 g, 0.72 mmol) in acetonitrile and 1H-tetrazole (0.45 M,
8.00 ml, 3.61 mmol) was added N,N-Diisopropyl-O,O-dibenzyl
phosphoramidite (1.25 g, 3.61 mmol) in an argon atmosphere.
The reaction mixture was stirred at room temperature for 24 h.
The solution was cooled to 0 °C and tBuOOH (670 ll) was added
and stirred for 30 min at room temperature. It was diluted with
chloroform (150 ml), washed with saturated sodium bicarbonate,
water and brine, dried over anhydrous magnesium sulfate and
concentrated in vacuum. The crude product was purified with a
short column chromatography on SiO2 gel (cyclohexane/ethyl
acetate 1:1 (v/v)). Evaporation of the solvent gave the product
in 10% (0.07 g) yield. 1H NMR (300 MHz, CD3OD): d = 1.65 (q,
J = 11.3 Hz, 1H); 2.53–2.64 (m, 1H); 3.13 (t, J = 10.5 Hz, 1H);
3.84 (ddd, J = 1.5, 5.2, 10.7 Hz, 1H); 3.96–4.25 (m, 5H); 4.95–
5.07 (m, 12H); 7.25–7.38 (m, 30H) ppm. 13C NMR (75 MHz,
CD3OD): d = 38.5 (d, J = 4.7 Hz); 67.3 (d, J = 5.6 Hz); 70.2 (d,
J = 5.7 Hz); 70.8; 70.9; 71.1; 71.2; 71.2; 71.3; 79.0 (dd, J = 3.3,
6.5 Hz); 129.1; 129.2; 129.4; 129.4; 129.7; 129.8; 129.9;
130.0; 136.9; 136.9; 137.0; 137.1; 137.1; 137.2 ppm. 31P
NMR (121 MHz, CD3OD): d = ꢂ1.19 (1P); ꢂ0.91 (1P); ꢂ0.17 (1P)
ppm. HRMS for: C48H52O13P3 [M+H]+ calcd: 929.2615, found:
929.2622.
4.3.6. 1,3-Di-O-benzyl-6-O-octylsulfonyl-D-glucose (5c)
The desired product was obtained as a colourless oil (46 mg,
62%). 1H NMR (300 MHz, CDCl3): d = 0.88 (t, J = 6.8 Hz, 3H); 1.24–
1.33 (m, 8H); 1.37–1.45 (m, 2H); 1.79–1.93 (m, 2H); 2.44 (br s,
2H); 3.13 (ddd, J = 3.3, 6.3, 8.1 Hz, 2H); 3.49–3.63 (m, 2H); 3.64–
3.72 (m, 1H); 3.83 (ddd, J = 1.9, 4.6, 9.7 Hz, 1H); 4.28–4.47 (m,
2H); 4.54 (td, J = 2.0, 11.3 Hz, 1H); 4.70–4.78 (m, 2H); 4.87–5.04
(m, 2H); 7.30–7.38 (m, 10H) ppm. 13C NMR (75 MHz, CDCl3):
d = 14.0; 22.5; 23.4; 28.1; 28.9; 31.6; 50.4; 67.9; 69.1; 69.8; 70.2;
72.6; 75.1; 82.5; 98.0; 127.9; 128.0; 128.2; 128.2; 128.3; 128.6;
136.6; 138.4 ppm. HRMS for: C28H41O8S [M+H]+ calcd: 537.2517,
found: 537.2523.
4.3.2. 1,5-Anhydro-3-deoxy-
D
-glucitole-2,4,6-triphosphate (9b)
-gluco-
A mixture of the 3-deoxy-2,4,6-dibenzylphosphoryl-
D
4.3.7. 1,3-Di-O-benzyl-6-O-dodecylsulfonyl-D-glucose (5d)
pyranositole 16 and 10% Pd on carbon in 25 ml methanol and
water (MeOH/H2O 4:1 (v/v)) was stirred under H2 (1 atm) over-
night. The solution was filtered and concentrated in vacuum. The
residue was diluted with water and lyophilized twice. 1H NMR
(300 MHz, D2O): d = 1.63 (q, J = 11.4 Hz, 1H); 2.56–2.69 (m, 1H);
3.21 (t, J = 10.6 Hz, 1H); 3.39 (dd, J = 3.4, 9.5 Hz, 1H); 3.85–4.20
(m, 5H) ppm. 13C NMR (75 MHz, D2O): d = 37.6 (d, J = 3.6 Hz);
64.2 (d, J = 5.1 Hz); 68.0 (t, J = 5.9 Hz); 69.4 (d, J = 5.5 Hz); 78.3 (t,
J = 8.6 Hz) ppm. 31P NMR (121 MHz, D2O): d = ꢂ0.51 (1P); 0.03
(1P); 1.03 (1P) ppm. HRMS for: C6H16O13P3 [M+H]+ calcd:
388.9798, found: 388.9801.
The desired product was obtained as a colourless oil (107 mg,
40%). 1H NMR (300 MHz, CDCl3): d = 0.91 (t, J = 6.7 Hz, 3H); 1.24–
1.35 (m, 16H); 1.42 (dd, J = 7.1, 13.5 Hz, 2H); 1.81–1.94 (m, 2H);
2.15–2.78 (m, 2H); 3.07–3.24 (m, 2H); 3.49–3.67 (m, 2H); 3.67–
3.75 (m, 1H); 3.85 (ddd, J = 1.9, 4.6, 9.7 Hz, 1H); 4.27–4.48 (m,
2H); 4.60 (dd, J = 2.0, 12.3 Hz, 1H); 4.72–4.80 (m, 2H); 4.91–5.11
(m, 2H); 7.35–7.42 (m, 10H) ppm. 13C NMR (75 MHz, CDCl3):
d = 14.1; 22.6; 23.4; 28.1; 29.0; 29.2; 29.3; 29.4; 29.5; 31.8; 50.4;
67.9; 69.1; 69.8; 70.2; 72.6; 75.1; 82.5; 98.0; 127.9; 128.2;
128.3; 128. 6; 136.7; 138.4 ppm. HRMS for: C32H47O8S [MꢂH]ꢂ
calcd: 591.2997, found: 591.3003.

A. G. Roth et al. / Bioorg. Med. Chem. 18 (2010) 939–944
943
4.3.8. General procedure for preparation 1,3-di-O-benzyl-2,4-
dibenzylphosphoryl-6-O-alkylsulfonyl- -glucose (6a–d)
To a stirred solution of the corresponding 1,3-di-O-benzyl-6-O-
sulfonyl- -glucose derivatives (5a–d) (1.0 equiv) in acetonitrile
(121 MHz, CDCl3): d = ꢂ1.23 (1P); ꢂ1.09 (1P) ppm. HRMS for:
D
C56H67O14P2S [M+H]+ calcd: 1057.3721, found: 1057.3733.
D
4.3.12. 1,3-Di-O-benzyl-2,4-dibenzylphosphoryl-6-O-dodecyl-
and 1H-tetrazole (0.45 M, 3.5 equiv) was added N,N-diisopropyl-
O,O-dibenzylphosphoramidite (3.5 equiv) in an argon atmosphere.
The reaction mixture was stirred at room temperature for 24 h. The
solution was cooled to 0 °C and tBuOOH (3.5 equiv) was added and
stirred for 30 min at room temperature. It was diluted with chloro-
form (50 ml), washed with saturated sodium bicarbonate, water
and brine, dried over anhydrous magnesium sulfate and concen-
trated in vacuum. The crude product was purified with a short col-
umn chromatography on SiO2 gel (cyclohexane/ethyl acetate 3:1
(v/v)).
sulfonyl-D-glucose (6d)
The desired product was obtained as a colourless oil (86 mg,
43%). 1H NMR (300 MHz, CDCl3): d = 0.91 (t, J = 6.7 Hz, 3H); 1.25–
1.35 (m, 16H); 1.41 (dd, J = 10.4, 11.5 Hz, 2H); 1.85 (td, J = 7.5,
10.4 Hz, 2H); 3.02–3.22 (m, 2H); 3.46–4.25 (m, 3H); 4.25–4.42
(m, 2H); 4.48–4.55 (m, 1H); 4.67–5.08 (m, 12H); 5.26 (d,
J = 3.6 Hz, 1H), 7.25–7.42 (m, 30H) ppm. 13C NMR (75 MHz, CDCl3):
d = 14.1; 22.6; 23.3; 28.1; 29.0; 29.3; 29.5; 29.6; 31.8; 50.5; 67.5;
68.5 (d, J = 4.5 Hz); 69.2 (d, J = 5.6 Hz); 69.4 (d, J = 5.6 Hz); 69.7 (t,
J = 5.0 Hz); 70.4; 74.9 (d, J = 4.4 Hz); 75.1; 76.3 (d, J = 5.9 Hz);
78.1 (dd, J = 3.4, 8.0 Hz); 96.0; 126.9; 127.6; 127.7; 127.9; 127.9;
128.1; 128.2; 128.4; 128.5; 135.3; 135.4; 135.4; 135.5; 135.5;
135.5; 136.5; 137.8 ppm. 31P NMR (121 MHz, CDCl3): d = ꢂ1.16
(1P); ꢂ1.02 (1P) ppm. HRMS for: C60H74O14P2SNa [M+Na]+ calcd:
1135.4172, found: 1135.4167.
4.3.9. 1,3-Di-O-benzyl-2,4-dibenzylphosphoryl-6-O-ethylsulfon-
yl-a/b-D-glucose (6a)
The desired product was obtained as a colourless oil (106 mg,
54%). 1H NMR (300 MHz, CDCl3): d = 1.38 (dt, J = 4.1, 7.4, 7.4 Hz,
3H); 3.11 (dq, J = 4.2, 7.4 Hz, 2H); 3.77 (dd, J = 8.5, 17.4 Hz, 1H);
3.99–4.12 (m, 1H); 4.28–4.56 (m, 3.5H); 4.59–4.72 (m, 2H);
4.73–5.02 (m, 11H); 5.27 (d, J = 3.6 Hz, 0.5H); 7.20–7.35 (m, 30H)
ppm. 13C NMR (75 MHz, CDCl3): d = 8.0; 44.9; 67.5; 67.8; 68.5 (d,
J = 4.3 Hz); 69.2; 69.3; 69.3; 69.4; 69.5; 69.5; 69.7; 69.7–69.9
(m); 70.4; 70.9; 72.7 (d, J = 4.0 Hz); 74.0; 74.6 (d, J = 6.3 Hz); 74.9
(d, J = 6.3 Hz); 75.0; 76.3 (d, J = 6.0 Hz); 78.0 (d, J = 6.1 Hz); 81.0
(t, J = 3.8 Hz); 96.1; 99.4 (d, J = 3.5 Hz); 127.5; 127.7; 128.0;
128.1; 128.2; 128.3; 128.4; 128.4; 128.5; 135.2; 135.5; 135.5;
135.6; 135.7; 135.8; 136.2; 136.5; 137.5; 137.7 ppm. 31P NMR
(121 MHz, CDCl3): d = ꢂ1.35 (1P); ꢂ1.31 (1P); ꢂ1.28 (1P); ꢂ1.10
(1P) ppm. HRMS for: C50H55O14P2S [M+H]+ calcd: 973.2782, found:
973.2788.
4.3.13. 1,3-Di-O-benzyl-2,4,6-dibenzylphosphoryl-D-glucose (8)
The desired product was obtained as a colourless oil (56 mg,
32%). 1H NMR (300 MHz, CD3OD): d = 3.88–3.94 (m, 1H); 3.96–
4.04 (m, 1H); 4.05–4.12 (m, 1H); 4.18–4.22 (m, 1H); 4.24–4.30
(m, 1H); 4.34–4.44 (m, 1H); 4.60–4.94 (m, 12H); 5.01 (s, 2H);
5.03 (s, 2H); 5.10 (d, J = 3.6 Hz, 1H); 7.08–7.36 (m, 40H) ppm. 13C
NMR (75 MHz, CD3OD): d = 28.0; 71.2; 76.1; 76.2; 76.3; 78.0;
79.5; 97.3; 127.5; 127.6; 127.7; 127.8; 127.9; 128.0; 128.3;
128.4; 128.6; 135.3; 135.4; 135.5; 135.6; 135.7; 136.6; 137.6;
137.8 ppm. 31P NMR (121 MHz, CD3OD): d = ꢂ1.30 (1P); ꢂ0.95
(1P); ꢂ0.18 (1P) ppm. HRMS for: C62H64O15P3 [M+H]+ calcd:
1141.3453, found: 1141.3458.
4.3.10. 1,3-Di-O-benzyl-2,4-dibenzylphosphoryl-6-O-hexylsul-
4.3.14. General procedure for deprotection of benzylethers, syn-
fonyl-
D
-glucose (6b)
thesis of 1,3-di-O-benzyl-2,4-dibenzylphosphoryl-6-O-dodecyl-
The desired product was obtained as a colourless oil (104 mg,
76%). 1H NMR (300 MHz, CDCl3): d = 0.87 (t, J = 6.8 Hz, 3H); 1.23–
1.30 (m, 4H); 1.33–1.41 (m, 2H); 1.81 (td, J = 7.6, 12.7 Hz, 2H);
2.98–3.11 (m, 2H); 4.01 (dd, J = 8.7, 17.5 Hz, 2H); 4.23–4.35 (m,
3H); 4.45–4.51 (m, 2H); 4.69 (d, J = 11.7 Hz, 1H); 4.76–4.83 (m,
3H); 4.85–4.89 (m, 2H); 4.89–4.95 (m, 4H); 4.98–5.11 (m, 1H);
5.23 (d, J = 3.6 Hz, 1H); 7.25–7.40 (m, 30H) ppm. 13C NMR
(75 MHz, CDCl3): d = 13.9; 22.3; 23.3; 27.8; 31.1; 50.4; 67.5; 68.5
(d, J = 4.2 Hz); 69.2 (d, J = 5.6 Hz); 69.4 (d, J = 5.5 Hz); 69.7 (dd,
J = 5.4, 7.0 Hz); 70.4; 76.3 (d, J = 5.6 Hz), 78.1 (dd, J = 3.4, 8.1 Hz);
96.1; 127.4; 127.5; 127.7; 127.9; 127.9; 128.1; 128.1; 128.2;
128.4; 128.5; 135.4; 135.4; 135.4; 135.4; 135.5; 135.5; 136.5;
137.8 ppm. 31P NMR (121 MHz, CDCl3): d = ꢂ1.78 (1P); ꢂ1.64
(1P) ppm. HRMS for: C54H63O14P2S [M+H]+ calcd: 1029.3408,
found: 1029.3420.
sulfonyl-
A mixture of the 1,3-di-O-benzyl-2,4-dibenzylphosphoryl-6-O-
alkylsulfonyl- -glucose derivatives (6a–d) and 10% Pd on carbon
in 50 ml methanol and water (MeOH/H2O 4:1 (v/v)) was stirred
under H2 (1 atm) overnight. The solution was filtered and concen-
trated in vacuum. The residue was diluted with water and lyophi-
lized twice.
D-glucose (7a–d)
D
4.3.15. 6-O-Ethylsulfonyl-D-glucose-2,4-bisphosphate (7a)
The desired product was obtained as a colourless solid (42 mg,
89%). 1H NMR (300 MHz, D2O): d = 1.26 (t, J = 7.4 Hz, 3H); 3.28 (dq,
J = 2.4, 7.4 Hz, 2H); 3.72–4.02 (m, 3H); 4.10 (ddd, J = 1.8, 4.5, 9.4 Hz,
1H); 4.34 (ddt, J = 2.8, 7.1, 8.4 Hz, 1H); 4.45–4.56 (m, 1H); 5.30 (d,
J = 3.3 Hz, 1H) ppm. 13C NMR (75 MHz, D2O): d = 7.2; 44.6; 67.8 (d,
J = 6.4 Hz); 68.8 (d, J = 5.0 Hz); 70.6 (d, J = 6.1 Hz); 72.2 (d,
J = 6.1 Hz); 73.5 (dd, J = 5.9, 11.8 Hz); 74.0; 74.9 (d, J = 5.6 Hz);
78.4 (d, J = 6.3 Hz); 90.5; 94.8 (d, J = 4.4 Hz) ppm. 31P NMR
(121 MHz, D2O): d = ꢂ0.09 (1P); ꢂ0.04 (1P); 0.18 (1P); 0.23 (1P)
ppm. HRMS for: C8H17O14P2S [MꢂH]ꢂ calcd: 430.9820, found:
430.9818.
4.3.11. 1,3-Di-O-benzyl-2,4-dibenzylphosphoryl-6-O-octylsul-
fonyl-D-glucose (6c)
The desired product was obtained as a colourless oil (110 mg,
72%). 1H NMR (300 MHz, CDCl3): d = 0.92 (t, J = 6.7 Hz, 3H); 1.24–
1.36 (m, 8H); 1.38–1.48 (m, 2H); 1.79–1.94 (m, 2H); 3.03–3.17
(m, 2H); 4.01–4.13 (m, 2H); 4.27–4.43 (m, 3H); 4.53 (dd, J = 2.1,
11.7 Hz, 2H); 4.74 (d, J = 11.7 Hz, 1H); 4.79–4.86 (m, 3H); 4.89–
5.00 (m, 6H); 5.09 (dd, J = 6.5, 9.6 Hz, 1H); 5.28 (d, J = 3.6 Hz,
1H); 7.25–7.40 (m, 30H) ppm. 13C NMR (75 MHz, CDCl3):
d = 14.0; 22.5; 23.3; 28.1; 28.9 (d, J = 1.8 Hz); 31.6; 50.4; 67.5;
68.5 (d, J = 4.3 Hz); 69.2 (d, J = 5.6 Hz); 69.5 (d, J = 5.7 Hz); 69.7
(dd, J = 4.6, 5.3 Hz); 70.4; 74.9 (d, J = 4.4 Hz); 75.0; 76.3 (d,
J = 5.7 Hz); 78.0 (dd, J = 3.2, 8.0 Hz); 96.0; 127.4; 127.5; 127.7;
127.9; 127.9; 128.1; 128.1; 128.4; 128.6; 135.3; 135.3; 135.3;
135.4; 135.4; 135.4; 135.5; 136.5; 137.7 ppm. 31P NMR
4.3.16. 6-O-Hexylsulfonyl-D-glucose-2,4-bisphosphate (7b)
The desired product was obtained as a colourless solid (45 mg,
85%). 1H NMR (300 MHz, D2O): d = 0.74 (t, J = 7.1 Hz, 3H); 1.12–
1.24 (m, 4H); 1.27–1.39 (m, 2H); 1.62–1.79 (m, 2H); 3.24–3.34
(m, 2H); 3.70–3.81 (m, 1H); 3.92 (td, J = 8.5, 12.7 Hz, 2H); 4.09
(ddd, J = 1.9, 4.7, 9.6 Hz, 1H); 4.32 (ddd, J = 5.3, 11.5, 14.6 Hz,
1H); 4.44–4.56 (m, 1H); 5.30 (d, J = 3.3 Hz, 1H) ppm. 13C NMR
(75 MHz, D2O): d = 13.2; 21.6; 22.6; 26.8; 30.2; 49.6; 67.8 (d,
J = 6.3 Hz); 68.8; 70.7 (d, J = 5.5 Hz); 72.3 (d, J = 6.3 Hz)M; 73.5
(dd, J = 6.0, 11.1 Hz); 74.0; 74.9 (d, J = 5.5 Hz); 78.4 (d, J = 6.2 Hz);

944
A. G. Roth et al. / Bioorg. Med. Chem. 18 (2010) 939–944
90.5; 94.9 (d, J = 4.5 Hz) ppm. 31P NMR (121 MHz, D2O): d = ꢂ0.06
(1P); 0.25 (1P) ppm. HRMS for: C12H25O14P2S [MꢂH]ꢂ calcd:
487.0446, found: 487.0446.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.030.
4.3.17. 6-O-Octylsulfonyl-D-glucose-2,4-bisphosphate (7c)
The desired product was obtained as a colourless solid (38 mg,
67%). 1H NMR (300 MHz, D2O): d = 0.74 (t, J = 6.6 Hz, 3H); 1.12–
1.26 (m, 8H); 1.31 (dd, J = 6.7, 12.0 Hz, 2H); 1.64–1.78 (m, 2H);
3.12–3.32 (m, 2H), 3.69–3.82 (m, 1H); 3.92 (dd, J = 6.5, 13.6 Hz,
2H); 4.05–4.16 (m, 1H); 4.34 (dd, J = 4.5, 11.4 Hz, 1H), 4.42–4.54
(m, 1H), 5.31 (s, 1H) ppm. 13C NMR (75 MHz, D2O): d = 13.5;
22.1; 22.7; 27.3; 28.2 (d, J = 4.1 Hz); 31.1; 49.7 (d, J = 3.7 Hz);
67.8 (d, J = 6.4 Hz); 68.8; 70.7; 72.3 (d, J = 5.7 Hz); 73.7 (dd,
J = 5.4, 9.4 Hz); 75.0 (d, J = 4.9 Hz); 90.5; 94.9 (d, J = 2.1 Hz) ppm.
31P NMR (121 MHz, D2O): d = ꢂ0.04 (1P); 0.23 (1P) ppm. HRMS
for: C14H29O14P2S [MꢂH]ꢂ calcd: 515.0759, found: 515.0758.
References and notes
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F.; Meyer, T. F. Cell 1997, 91, 605.
5. Teichgraber, V.; Ulrich, M.; Endlich, N.; Riethmuller, J.; Wilker, B.; De Oliveira-
Munding, C. C.; van Heeckeren, A. M.; Barr, M. L.; von Kurthy, G.; Schmid, K. W.;
Weller, M.; Tummler, B.; Lang, F.; Grassme, H.; Doring, G.; Gulbins, E. Nat. Med.
2008, 14, 382.
6. Goggel, R.; Winoto-Morbach, S.; Vielhaber, G.; Imai, Y.; Lindner, K.; Brade, L.;
Brade, H.; Ehlers, S.; Slutsky, A. S.; Schutze, S.; Gulbins, E.; Uhlig, S. Nat. Med.
2004, 10, 155.
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Hubbard, W. C.; Berdyshev, E. V.; Tuder, R. M. Nat. Med. 2005, 11, 491.
8. Lang, P. A.; Schenck, M.; Nicolay, J. P.; Becker, J. U.; Kempe, D. S.; Lupescu, A.;
Koka, S.; Eisele, K.; Klarl, B. A.; Rubben, H.; Schmid, K. W.; Mann, K.;
Hildenbrand, S.; Hefter, H.; Huber, S. M.; Wieder, T.; Erhardt, A.; Haussinger,
D.; Gulbins, E.; Lang, F. Nat. Med. 2007, 13, 164.
4.3.18. 6-O-Dodecylsulfonyl-D-glucose-2,4-bisphosphate (7d)
The desired product was obtained as a colourless solid (31 mg,
70%). 1H NMR (300 MHz, D2O): d = 0.77 (t, J = 5.6 Hz, 3H); 1.13–
1.25 (m, 16H); 1.29–1.40 (m, 2H); 1.71 (s, 2H); 3.19 (s, 2H);
3.63–3.83 (m, 1H); 3.87- 4.06 (m, 2H); 4.08–4.21 (m, 1H); 4.27–
4.40 (m, 1H); 4.41–4.54 (m, 1H); 5.35 (m, 1H) ppm. 13C NMR
(75 MHz, D2O): d = 13.8; 22.6; 23.1; 28.0; 29.3; 29.5; 29.9; 32.0;
49.9 (d, J = 4.6 Hz); 67.9 (d, J = 6.1 Hz); 69.0 (d, J = 4.0 Hz); 70.7
(d, J = 7.3 Hz); 74.0; 75.1; 90.5 ppm. 31P NMR (121 MHz, D2O):
d = 0.77 (1P); 2.26 (1P) ppm. HRMS for: C18H37O14P2S [MꢂH]ꢂ
calcd: 571.1385, found: 571.1384.
9. Tabas, I. Circulation 2004, 110, 3400.
10. Kölzer, M.; Werth, N.; Sandhoff, K. FEBS Lett. 2004, 559, 96.
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Mahdy, A.; Zeidan, Y.; Bielwaska, A.; Hannun, Y. A.; Norris, J. S. FEBS Lett. 2006,
580, 4751.
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Sandhoff, K. Biol. Chem. 2003, 384, 1293.
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2004, 89, 636.
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Biochim. Biophys. Acta 1987, 922, 323.
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Carbohydr. Res. 2004, 339, 1511.
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4.3.19. D-Glucose-2,4,6-trisphosphate (9)
The desired product was obtained as a colourless oil (31 mg,
70%). 1H NMR (300 MHz, D2O): d = 4.00 (dd, J = 4.8, 11.2 Hz, 2H);
4.05–4.16 (m, 4H); 5.39 (d, J = 2.5 Hz, 1H) ppm. 13C NMR
(75 MHz, D2O): d = 63.9 (d, J = 4.3 Hz); 68.7–69.1 (m); 73.5 (d,
J = 6.3 Hz); 74.8 (d, J = 5.2 Hz); 90.6 ppm. 31P NMR (121 MHz,
D2O): d = ꢂ0.21 (1P); 0.81 (1P); 1.39 (1P) ppm. HRMS for:
C6H14O5P3 [MꢂH]ꢂ calcd: 418.9551, found: 418.9545.
Acknowledgements
The authors gratefully acknowledge funding by the DFG
(AR376/2-1) and the Volkswagen Stiftung.