Phosphatidyl myo-inositol mannosides mimics built on an acyclic or heterocyclic core: Synthesis and anti-inflammatory properties

Article abstract of DOI:10.1002/cmdc.201100291

Phosphatidyl myo-inositol mannosides (PIMs) are constituents of the mycobacterial cell wall and possess immunomodulatory activities. Certain PIM derivatives have immunoprotective activity and are of interest as anti-inflammatory agents. In order to identi

Full text of DOI:10.1002/cmdc.201100291

DOI: 10.1002/cmdc.201100291
Phosphatidyl myo-Inositol Mannosides Mimics Built on an
Acyclic or Heterocyclic Core: Synthesis and Anti-
inflammatory Properties
Sophie Front,[a, b] Nathalie Court,[b] Marie-Laure Bourigault,[b] Stꢀphanie Rose,[b]
Bernhard Ryffel,[b] FranÅois Erard,[b] Valꢀrie F. J. Quesniaux,[b] and Olivier R. Martin*[a]
Phosphatidyl myo-inositol mannosides (PIMs) are constituents
of the mycobacterial cell wall and possess immunomodulatory
activities. Certain PIM derivatives have immunoprotective activ-
ity and are of interest as anti-inflammatory agents. In order to
identify simplified analogues of PIMs that retain this interesting
activity, we have prepared a series of new analogues based
either on an acyclic or on a heterocyclic scaffold that replaces
the inositol moiety, and evaluated these compounds for their
inhibition of LPS-induced release of NO and pro-inflammatory
cytokines by macrophages. It was found that the inositol
moiety can be favourably replaced by an aza-cyclitol (trihy-
droxy-piperidine) or an oxa-cyclitol (trihydroxy-tetrahydropyr-
an) unit, and that the configuration of the OH-carrying carbons
does not play a significant role. The biological activity is re-
duced if the nitrogen atom is free in the aza-cyclitol unit.
Introduction
Phosphatidyl-myo-inositol mannosides (PIMs) are biosynthetic
precursors of the mycobacterial cell-wall glycolipids lipomann-
an (LM) and lipoarabinomannan (LAM).[1,2] Mycobacterial LAM,
LM and PIM are recognised by macrophages and dendritic
cells through various pattern recognition receptors, including
Toll-like receptors (TLRs)[3–5] and C-type lectins such as the man-
nose receptor and dendritic cell-specific intercellular adhesion
molecule-3 grabbing nonintegrin (DC-SIGN).[6–10] PIMs have
slight pro-inflammatory activities,[11] and PIM dimannoside
(PIM2) and hexamannoside (PIM6), the two most abundant
classes of PIM found in M. bovis BCG and M. tuberculosis H37Rv,
activate macrophages to secrete low levels of TNFa through
TLR2 and MyD88 receptors.[11,12]
Thus, not only complex mycobacterial lipoglycans like
ManLAM and LM, but also small-molecular-weight PIMs are
potent inhibitors of host inflammatory responses and could
represent interesting lead compounds for new anti-inflamma-
tory agents.[24]
However, the large-scale production of PIM-related mole-
cules is thwarted in the main by the complexity of the synthe-
sis of selectively protected myo-inositol precursors. Preliminary
investigations on acyclic analogues in which the cyclitol is re-
placed by a 3-C glycerol scaffold (type I analogues) have al-
The purification of mycobacterial PIMs and their chemical
characterisation revealed four major acyl forms, mono- to
tetra-acylated, for both PIM2 and PIM6.[12–15] Higher-order PIMs
with mannose-cap-like structures were found to preferentially
associate with the human mannose receptor and to contribute
to phagosome–lysosome fusion,[6] whereas PIM2s were recog-
nised by DC-SIGN independently of their acylation degree.[10]
Several complex mycobacterial glycolipids exhibit anti-in-
flammatory activities. For example, ManLAM inhibition of lipo-
polysaccharide (LPS)-induced IL-12 production in dendritic cells
was attributed to DC-SIGN.[16,17] In addition, we have shown
that di-acylated LM, as well as PIM2 and PIM6, inhibited the
LPS/TLR4-induced cytokine response, independently of TLR2,
SIGN-R1 and the mannose receptor.[18,19] Recently, we demon-
strated that synthetic PIM1 and a PIM2 mimetic repeated these
in vitro activities and inhibited endotoxin-induced airway in-
flammation, TNFa and KC secretion and neutrophil recruitment
in vivo.[19] Suppression of ovalbumin-induced allergic airway
eosinophilia, a model dependent on LPS response,[20] by natu-
ral or synthetic PIMs was also reported.[21–23]
[a] S. Front, Prof. O. R. Martin
ICOA, UMR 6005, Universitꢀ d’Orlꢀans and CNRS
rue de Chartres, B.P. 6759
4067 Orlꢀans (France)
E-mail: olivier.martin@univ-orleans.fr
[b] S. Front, N. Court, M.-L. Bourigault, S. Rose, Dr. B. Ryffel, Prof. F. Erard,
Dr. V. F. J. Quesniaux
IEM, UMR 6218, Universitꢀ d’Orlꢀans and CNRS
3B rue de la Fꢀrollerie, 45071 Orlꢀans Cedex 2 (France)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100291.
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ready shown that the inositol moiety is dispensable, and that
the key structural elements of PIMs are the phosphodiester,
the fatty acid residues and at least one mannosyl group.[19,23]
In order to identify novel structures that mimic PIMs and
retain or improve upon their biological activity, we designed a
series of analogues of PIM2 in which the core inositol scaffold
was replaced by a more readily accessible saturated N- or O-
heterocycle carrying the HO functionalities of myo-inositol at
C-6, C-1 and C-2 (type II). In addition, the structure of acyclic
analogues of type I was further abbreviated by removing one
of the mannosyl residues in order to generate highly simplified
PIM1 analogues. Specifically this article reports the preparation
of PIM1 mimic 2 and of three types of heterocyclic analogues
of PIM2, namely aza-cyclitol analogues having the xylo configu-
ration (compounds 6a and 6b, and two oxa-cyclitol analogues
having the xylo and arabino configurations (compounds 7 and
8, respectively), and the comparison of their anti-inflammatory
activity (inhibition of LPS-promoted NO, TNFa and IL-12 pro-
duction) with that of the reference compounds PIM1 (1), PIM2
mimic 4 and PIM1 isomer 5.[24]
age without affecting the glycerolipid esters is facilitated at
the end of the synthesis by the higher reactivity of the me-
thoxyacetyl groups[32] compared to normal acetyl groups.
Synthesis of mannosyl donor 11: d-Mannose was converted
into tetra-O-methoxyacetyl-a-d-mannopyranosyl trichloroacet-
imidate (11) by peracylation, selective cleavage of the anome-
ric methoxyacetate with hydrazine, and treatment with
trichloroacetonitrile (Scheme 1).
Synthesis of phosphoramidite 15: Phosphorylating agent 15
was prepared according largely to known procedures. Mono-
acylation of 3-O-benzyl-sn-glycerol with a limited amount of
stearic acid in the presence of a coupling agent, followed by
acylation with palmitic acid provided the selectively acylated
1-O-stearoyl-2-O-palmitoyl-sn-gycerol derivative 13 in 60%
overall yield (Scheme 2).[33] Debenzylation of 13 (H2/Pd) was
performed immediately before phosphitylation with freshly
prepared
benzyloxy-bis-(N,N-diisopropylamino)phosphine,[34]
which gave reagent 15 (48%); this reagent was purified by
rapid flash chromatography on silica gel. The success of the
phosphodiester synthesis strongly relies on the quality and the
purity of this reagent (see ref. [35] for an improved procedure
for the preparation of benzyloxy-bis(N,N-diisopropylamino)-
phosphine).
Results and Discussion
Synthesis of PIM1m: As PIM1 conserves a major part of the
activity of natural PIM2 and of more highly mannosylated
PIMs, and in view of the significant activities[19,23] of the acyclic
PIM2 mimic 4, we thought that it would be of interest to pre-
pare the equivalent, simple PIM1 mimic 2, which is built on a
glycerol scaffold and has only one mannosyl residue. In order
to simplify the synthesis to the greatest extent, compound 2
was prepared from racemic 1,2-O-isopropylidene glycerol (sol-
ketal).1 Glycosylation of this compound with trichloroacetimi-
Synthetic procedures
The general strategy for the synthesis of PIM analogues follows
synthetic sequences similar to those developed by other
groups for the preparation of PIM1 and PIM2 structures,[25–31]
namely the preparation of a (di)mannosylated scaffold and of a
glycerolipid carrying two fatty acid residues, followed by the
connection of these entities by a phosphodiester linkage in
the last stage of the synthesis. In our synthesis, a notable im-
provement is the use of a methoxyacetylated mannopyranosyl
donor in the glycosylation step: such esters provide efficient
stereocontrol of the glycosylation, while their selective cleav-
1
Note: parallel studies have shown that the configuration in the glycerol unit
has little effect on the biological activity (unpublished data).
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Phosphatidyl myo-Inositol Mannosides Mimics
idation of the resulting phosphite. The methoxyacetyl groups
were cleaved by using tert-butylamine in methanol, and final
debenzylation afforded PIM1 mimic 2.
Synthesis of PIM2 analogues incorporating an aza-inositol:
The synthesis of PIM2 analogues built on a 3,4,5-trihydroxy-pi-
peridine scaffold was achieved by way of the symmetrical 3-O-
benzyl-iminoxylitol derivative 21, which is readily available
from diacetone-d-glucose in five simple steps and in very high
yield.[36,37] This approach leads to a core structure that has the
non-natural xylo configuration. Thus compound, 21, was N-de-
benzylated with Pearlman’s catalyst and then N-trifluoroacety-
lated with trifluoroacetic anhydride (TFAA) to give piperidine
diol 23 (Scheme 4). This compound underwent double glycosy-
lation with donor 11 to afford 24 in 47% yield. The 3-OH
group in 24 was deprotected by hydrogenolysis and then con-
verted into the corresponding phosphoester by treatment with
phosphorimidate 15, under the same conditions as before. The
resulting compound, 26, exhibited very complex NMR spectra
because of the existence of P* diastereoisomers, of slowly ro-
tating amide linkage conformers, and of the nonequivalence of
the two sugar moieties (which are diastereotopic in this struc-
ture); this leads, for example, to the observation of eight sig-
nals for anomeric carbons, all of equal intensities, and to four
signals in the 19F NMR spectrum! Hydrogenolysis of the benzyl
phosphate gave 27, which was O-deacylated by using tert-
butylamine in methanol; this treatment promoted the partial
cleavage of the N-COCF3 group, to give compounds 6a and
6b in an approximately 2:3 ratio. These two compounds could
be efficiently separated by flash chromatography and charac-
terised independently.
Scheme 1. a) MAcCl, Py, 87%; b) hydrazine acetate, DMF, À178C, 93%; c) tri-
chloroacetonitrile, DBU, CH2Cl2, 30 min, 73%.
Scheme 2. a) stearic acid, DCC, DMAP, CH2Cl2, 63%; b) palmitic acid, EDCI,
DMAP, CH2Cl2, 95%; c) H2, Pd/C 10%, EtOH/CH2Cl2, 99%; d) benzyloxy-
bis(N,N-diisopropylamino)phosphine, 1H-tetrazole, CH2Cl2, 30 min, 48%.
date 11 afforded a-mannoside 16 (56%), which was then de-
protected in the glycerol unit to give diol 17 (Scheme 3). Selec-
tive benzylation at the primary position of 17 to give 18, was
achieved in modest yield (29%) by using benzyl trichloroaceti-
midate. The phosphodiester linkage was then created by treat-
ing 18 with 15 in the presence of 1H-tetrazole, followed by ox-
Synthesis of PIM analogues incorporating a xylo-oxa-inositol:
The core 3-O-benzyl 1,5-anhydroxylitol (32) was obtained in
high yield from 3-O-benzyl-d-xylofuranose derivative 28 (three
steps; Scheme 5). Hydrolysis of the isopropylidene group in 28
followed by peracetylation of the resulting d-xylopyranose 29
gave glycosyl acetate 30.[38]
The anomeric substituent of 30 was reductively cleaved by
using Et3SiH in the presence of BF3·Et2O[39] to give 31 in 50%
yield. Further elaboration of this compound as described
above for diol 23 gave, after deprotection, the oxa analogue of
PIM2, compound 7, in 12% overall yield. It is interesting to
note that the xylo core tetrahydropyran in 7 exists as an equi-
librium of two chair conformations with a significant contribu-
1
tion (~40%) from the C4-type conformation (all substituents in
an axial disposition), as indicated by the rather small coupling
constants between trans-related protons (3JH,H ~7 Hz). This be-
haviour can be explained by the large size of the three sub-
stituents, which partially shifts the conformational equilibrium
toward the all-axial conformation.
Synthesis of PIM analogues incorporating an arabino-oxa-ino-
sitol: By building a PIM2 mimic on a 1,5-anhydro-d-arabinitol
scaffold, the mimic’s structure matches the configurations of
the natural myo-inositol derivatives at the positions C-6, C-1
and C-2, and therefore the spatial arrangements of the sub-
stituents are essentially identical to those of natural PIMs; the
structure lacks only the inositol 3-, 4- and 5-OH groups. The
synthesis of such an oxa-heterocyclic analogue of PIMs was
Scheme 3. a) 11, TMSOTf, CH2Cl2, MS 4 ꢂ, 56%; b) AcOH, H2O, 18 h, qt;
c) benzyl trichloroacetimidate, TMSOTf, CH2Cl2, 4 ꢂ molecular sieves, 2 h,
29%; d) 15, 1H-tetrazole, CH2Cl2, 3 ꢂ molecular sieves, 2 h; À208C to RT,
then mCPBA, 1 h, 27%; e) tBuNH2, MeOH/CHCl3, 45%; f) Pd/C 10%, H2, EtOH/
CH2Cl2, 83%.
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Scheme 4. a) Pd(OH)2, H2, EtOH, 24 h; b) TFAA, CH2Cl2, 18 h, 45%; c) 11, TMSOTf, CH2Cl2, 4 ꢂ molecular sieves, 2 h, 47%; d) H2, Pd/C 10%, EtOH/CH2Cl2, 97%;
e) 15, 1H-tetrazole, CH2Cl2, 3 ꢂ molecular sieves, 2 h, then mCPBA, 1 h, 37%; f) Pd/C 10%, H2, EtOH/CH2Cl2, 3 h, 95%; g) tBuNH2, MeOH/CHCl3, 1 h; 32% for 6a
and 48% for 6b.
Scheme 5. a) DOWEX 50WX8, dioxane/H2O, 758C, 18 h; b) Ac2O, Py, 98%;
c) BF3·Et2O, Et3SiH, CH3CN, 24 h, 50%; d) Na, MeOH, 1 h, quant.; e) 11,
TMSOTf, CH2Cl2, 4 ꢂ molecular sieves, 2 h, 67%; f) H2, Pd/C 10%, EtOH/
CH2Cl2, 97%; g) 15, 1H-tetrazole, CH2Cl2, 3 ꢂ molecular sieves, 2 h, À208C to
RT, then mCPBA, 47%; h) tBuNH2, MeOH/CHCl3, 43%; i) Pd/C 10%, H2, EtOH/
Scheme 6. a) HCl, MeOH, 1.5 h, 688C, quant.; b) Bu2SnO, CsF, toluene then
CH2Cl2, 92%.
DMF, BnBr, 33%; c) trifluoroacetic acid, dioxane/H2O, 45%; d) Ac2O, Py, 35%;
e) BF3·Et2O, Et3SiH, CH3CN, 6 h, 94%; f) Na, MeOH, 1 h, 96%; g) 11, TMSOTf,
CH2Cl2, 4 ꢂ molecular sieves, 2 h, 70%; h) H2, Pd/C 10%, EtOH/CH2Cl2, 99%;
achieved via 3-O-benzyl-1,5-anhydro-d-arabinitol (41). This
compound was prepared from methyl d-lyxopyranosides 37:
formation of a stannylidene derivative of 37 provided a means
of selectively benzylating the 3-OH group to give 38,[40] albeit
in modest yield and regioselectivity (3-OBn/2-OBn ratio: 2:1),
acetylation to give 39, conversion of the methyl glycoside into
i) 15, 1H-tetrazole, CH2Cl2, 3 ꢂ molecular sieves, 2 h, then mCPBA, 1 h, 55%;
j) tBuNH2, MeOH/CHCl3, 45%; k) Pd/C 10%, H2, EtOH/CH2Cl2, 79%.
glycosyl acetate 40 and reductive cleavage of this group under
the same conditions as for xylopyranose derivative 30 to afford
tetrahydropyran derivative 41 (Scheme 6). This compound was
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Phosphatidyl myo-Inositol Mannosides Mimics
then deacylated and submitted to the same sequence of reac-
tions as described above for 23 to provide the oxa-inositol
PIM2 analogue 8.
pounds in a concentration-dependent manner, reaching a pla-
teau at 10–20 mm, with a slightly higher activity for PIM2 than
PIM1 or PIM2 mimetic (Figure 1a). The inhibition of TNFa and
IL-12p40 was stronger, with IC50 values of 2.5–5 and 0.4–
0.7 mm, respectively, and a slightly higher activity for PIM2 than
for PIM1 or PIM2 mimetic (Figure 1b, c).
Biological investigations
A mannosyl residue was removed from the acyclic PIM2
mimetic analogue to generate a simplified PIM1 mimetic 2.
PIM1 mimetic 2 was clearly less active than native PIM1 or the
PIM1 isomer 5 in inhibiting the release of NO and TNFa, and
to a lesser extent in inhibiting IL-12p40 release (Figure 2).
Therefore, the acyclic mimetic of PIM2 retained the capacity to
inhibit NO or pro-inflammatory cytokine release by LPS-activat-
ed macrophages, while a simplified, acyclic PIM1 mimetic
Comparison of the acyclic structures with reference PIM1 and
PIM2 compounds: We first confirmed that an acyclic analogue
of PIM2 in which the cyclitol is replaced by a 3-C glycerol scaf-
fold (type I analogues), PIM2 mimetic (PIM2m), has a biological
activity similar to those of the synthetic, native PIM2[29] and
PIM1 (Figure 1). Murine bone-marrow-derived macrophages
(BMDM) were stimulated with LPS in the absence or in the
presence of the PIMs. NO release was inhibited by the com-
Figure 2. Reduced activity of a simplified PIM1 analogue (2) for inhibiting
NO, TNFa and IL-12p40 release by LPS-stimulated macrophages. Bone-
marrow-derived macrophages were incubated for 24 h with LPS in the pres-
Figure 1. PIM1, PIM2 and a PIM2 mimetic inhibit NO, TNFa and IL-12p40 re-
lease by LPS-stimulated macrophages. Bone-marrow-derived macrophages
~
*
*
*
were incubated for 24 h with LPS in the presence of PIM1 (1, ), synthetic
ence of PIM1 (1, ), PIM1 isomer (5, ), or the simplified PIM1m (2, ) in
which a mannosyl residue was removed from the acyclic PIM2 mimetic ana-
logue. a) NO, b) TNFa and c) IL-12p40 were measured in the supernatants.
Results are expressed as a percentage of inhibition of the control stimulation
performed with LPS plus vehicle.
&
^
PIM2 (3, ), or PIM2m (4, ), an acyclic analogue of PIM2 in which the cy-
clitol is replaced by a 3-C glycerol scaffold. a) NO, b) TNFa and c) IL-12p40
were measured in the supernatants. Results are expressed as a percentage
of inhibition of the control stimulation performed with LPS plus vehicle.
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structure bearing only one mannosyl residue was essentially in-
active.
Biological activities of the heterocyclic structures: Heterocyclic
analogues of PIM2 in which the core inositol scaffold is re-
placed by a more readily accessible saturated N- or O-hetero-
cycle carrying the HO functionalities of myo-inositol at C-6, C-1
and C-2 (type II) were then tested, namely aza-cyclitol ana-
logues with the xylo configuration (compounds 6a and 6b)
and two oxa-cyclitol analogues with the xylo and arabino con-
figurations (compounds 7 and 8, respectively).
All four compounds retained significant anti-inflammatory
activity, although the xylo-aza-cyclitol NH 6a was less active in
inhibiting LPS-induced NO and TNFa release; no difference
was observed, however, on the release of IL-12p40 by 6a com-
pared to the other compounds (Figure 3). This compound car-
ries a free secondary amine function, which, in protonated
form, induces a positive charge at this position that does not
exists in the parent structures, and might explain the lower ac-
tivity in the first two tests. On the other hand, the activities of
the two oxa isomers are very similar; this indicates, surprisingly,
that the configurations present in the natural myo-inositol scaf-
fold do not play an important role. This is further supported
by the fact that the PIM1 isomer 5 has an inhibitory activity in
these tests comparable to that of PIM1 (Figure 2). This PIM1
isomer can indeed be considered as a doubly inverted epimer
(at C-1 and C-2) of PIM1.
Figure 3. Heterocyclic analogues of PIM2 inhibit NO, TNFa and IL-12p40 re-
lease by LPS-stimulated macrophages. Bone-marrow-derived macrophages
were incubated for 24 h with LPS in the presence of heterocyclic PIM2 aza-
*
cyclitol analogues that have the xylo configuration ( : xylo-aza-cyclitol
^
NCOCF3, 6a; : xylo-aza-cyclitol NH, 6b) and two oxa-cyclitol analogues that
~
&
have the xylo and arabino configurations ( : xylo-oxa-cyclitol, 7; : arabino-
oxa-cyclitol, 8). a) NO, b) TNFa and c) IL-12p40 were measured in the super-
natants. Results are expressed as a percentage of inhibition of the control
stimulation performed with LPS plus vehicle.
structure (2) was essentially inactive, thus suggesting a require-
ment for both mannosyl residues to conserve an active acyclic
structure.
Conclusions
A new acyclic analogue of PIM1 was prepared, as well as a
series of PIM2 mimics based on a heterocyclic scaffold in place
of the native myo-inositol moiety. The anti-inflammatory prop-
erties of the new compounds were evaluated by their capacity
to inhibit the release of nitric oxide and pro-inflammatory cyto-
kines (TNFa, IL12) induced by LPS in macrophages. We showed
here that, although an acyclic mimetic of PIM2 carrying two
mannosyl residues retained the activity of the parent PIM2
compound, the corresponding simplified, acyclic PIM1 mimetic
Parallel biological assays of the heterocyclic analogues of
PIM2, namely compounds 6a, 6b, 7 and 8, led to the remark-
able result that the inositol moiety can be replaced by an oxa-
or aza-cyclitol; this indicates that the OH groups at C-3, C-4
and C-5 of the inositol are not essential, although the slightly
lower activity observed indicates that they might contribute to
the activity. It appears that the ring nitrogen of the aza-cyclitol
structure should be deactivated (as an amide) for the best ac-
tivity, although the inhibition of LPS-induced release of IL-
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Phosphatidyl myo-Inositol Mannosides Mimics
silyl triflate (TMSOTf; 20% mol equiv vs donor) was added, and the
mixture was stirred for another hour at RT. The reaction was
stopped by the addition of excess triethylamine, and the solids
were removed by filtration through a pad of celite. The solvent
was removed under vacuum, and the residue was purified by silica
gel column chromatography to give the desired glycoside.
12p40 appears not to be perturbed by the positive charge in
6a. Furthermore, our results show that, unexpectedly, the con-
figuration of the inositol moiety at C-1–C-2–C-6 does not
appear to play a significant role: the activity of the xylo epi-
mers 6b and 7 is essentially the same as that of the arabino
epimer 8, the latter reproducing the configurations of the
native structure. This is also supported by the fact that the
double epimer of PIM1 at C-1 and C-2 (compound 5) retains
the activity of the parent PIM1.
General procedure B (debenzylation): Protected alcohol was dis-
solved in CH2Cl2/MeOH (1:1, ~10 mL per 150 mg of protected alco-
hol). 10% Palladium on charcoal (50 mg per 100 mg of protected
alcohol) was added, and the reaction mixture was stirred at room
temperature under H2 for 1–4 h. The catalyst was removed by fil-
tration through a Millipore membrane and washed with CH2Cl2/
MeOH (1:1, 2ꢃ10 mL). The residual solvents were evaporated to
give the expected compound, which was used in the next step
without purification.
Thus, while there is a strict requirement for the phosphate
group, fatty acid residues and at least one mannosyl unit, the
myo-inositol moiety appears to behave only as a scaffold that
can be replaced by a heterocyclic core or by an acyclic tether.
This information on the structural elements of PIMs should
provide a basis for the design of more readily available com-
pounds that have therapeutic interest as anti-inflammatory
agents.
General procedure C (phosphorylation): A solution of phosphora-
midite 15 (1.5 equiv) was added to a solution of the deprotected
alcohol in dry CH2Cl2. Molecular sieves (3 ꢂ) were added, and the
suspension was stirred for 30 min at room temperature. A commer-
cial solution of 1H-tetrazole (~0.45m in acetonitrile; 3 or 5 equiv)
previously dried over 3 ꢂ molecular sieves was then added at 08C.
The resulting mixture was stirred for 2 h at RT and was then cooled
to À408C before the dropwise addition of a dried (MgSO4) solution
of meta-chloroperoxybenzoic acid (mCPBA; purity ~50%; 3 equiv)
in CH2Cl2 (2 mL). After the mixture had been stirred for 10 min at
À408C and 2 h at RT, the reaction was quenched by the addition
of sat. aq. Na2S2O3 (20 mL), and the combined mixture was extract-
ed with Et2O (80 mL). The organic phase was washed with sat. aq.
Na2S2O3 (3ꢃ15 mL), NaHCO3 (15 mL) and NaCl (15 mL) and dried
(MgSO4). The solvents were removed in vacuo, and the residue was
purified by silica gel column chromatography to give the phospho-
diester.
Experimental Section
General methods: Unless otherwise stated, all reactions requiring
anhydrous conditions were carried out under dry Ar. Dichlorome-
thane and toluene were distilled over calcium hydride. Tetrahydro-
furan was distilled over sodium/benzophenone ketyl. Diethyl ether
was distilled over CaH2 and stored at 48C, on 4 ꢂ molecular sieves,
under Ar. All reagent-grade chemicals were obtained from com-
mercial suppliers (Sigma–Aldrich, Acros) and were used as received.
1H, 13C, 19F and 31P NMR spectra were recorded on a Bruker DPX250
or a Bruker Avance 400 spectrometer. Proton chemical shifts are
reported relative to Me4Si (d=0.000 ppm) as internal standard.
Carbon chemical shifts are referenced to the central line of the
CDCl3 triplet (d=77.16 ppm), unless otherwise stated. 31P and 19F
chemical shifts are reported downfield from the external standard,
phosphoric acid and boron trifluoride diethyl etherate, respectively.
General procedure D (removal of methoxyacetyl (MAc) groups):
The starting material (0.03 mmol) was dissolved in CHCl3/MeOH
(1:4, 1 mL). The mixture was cooled to 08C, and tert-butylamine
(164 mL) was added. After the mixture had been stirred at 08C for
10 min and at about 108C for an additional hour, the solvents
were evaporated under high vacuum, without heating, and the
residue was purified by flash chromatography to give the desired
product.
1
Carbon multiplicities were assigned by DEPT experiments. H and
13C NMR signals were assigned with the help of H,H and H,C corre-
lations (COSY and HSQC spectra). Flash chromatography was per-
formed according to the established protocol on silica gel 60 (40–
63 mm). Reactions were monitored by thin-layer chromatography
(silica gel 60F254 precoated aluminium plates), and the compounds
were detected under UV light and by staining with phosphomolyb-
dic acid solution. ESI-MS was performed on a Perkin–Elmer SCIEX
API 300 spectrometer. High-resolution mass spectra were recorded
on a Waters Q-TOF micro spectrometer at the Centre Rꢀgional de
Mesures Physiques in Clermont–Ferrand (France) or on a Bruker
maXis UHR-Q-TOF spectrometer in Orlꢀans.
Penta-O-methoxyacetyl-d-mannopyranose (9): Methoxyacetyl
chloride (33 mL, 0.361 mol, 6.5 equiv) was added dropwise to a so-
lution of d-mannose (10 g, 0.056 mol) in pyridine (100 mL) at 08C,
and the mixture was stirred overnight at RT. The solvent was
evaporated under vacuum, and the residue was diluted with ethyl
acetate (200 mL); the resulting solution was washed with aq. HCl
(1n, 80 mL) and brine (80 mL), and dried (MgSO4). Removal of the
solvent and purification of the residue by silica gel column chro-
matography (light petroleum/ethyl acetate 1:2) gave compound 9
(a-anomer) as a yellow syrup (26 g, 87%). Rf =0.40 (toluene/ace-
NMR numbering scheme: Mannose numbering: C1’–C6’ (although
the two sugar units are not equivalent in most dimannosyl com-
pounds, no attempt has been made to assign signals to one or the
other sugar unit)
1
tone 2:1); H NMR (250 MHz, CDCl3): d=3.41 (s, 6H), 3.45 (s, 3H),
3.49 (s, 6H), 3.85–4.26 (m, 12H; 5CH2O, H6A, H5’), 4.40 (dd, J6’A,6’B
=
Glycerol numbering: C1a–C3a
Heterocyclic moiety: C1–C5
12.3, J6’B,5’ =4.3 Hz, 1H; H6’B), 5.25–5.53 (m, 3H; H2’, H3’, H4’),
6.21 ppm (s, 1H; H1’); 13C NMR (62.9 MHz, CDCl3): d=59.34, 59.42,
59.50, 61.83, 65.47, 68.35, 68.82, 69.14, 69.26, 69.37, 70.44, 90.49,
167.61, 169.08, 169.22, 169.33, 169.90 ppm; MS (ESI) m/z 558.5
[M+NH4]+, 563.5 [M+Na]+, 579.5 [M+K]+.
General procedure A (glycosylation): The glycosyl donor (11;
2.5 equiv) and the acceptor were previously dried over P2O5 over-
night under high vacuum; the acceptor was then dissolved in
CH2Cl2 (1 mL per 100 mg of donor) under argon. Molecular sieves
(4 ꢂ) were added, the mixture was stirred for 30 min, and then a
solution of donor in CH2Cl2 (0.5 mL per 100 mg donor) was added
under argon. After the mixture had been stirred for 1 h, trimethyl-
2,3,4,6-Tetra-O-methoxyacetyl-d-mannopyranose (10): Freshly
prepared hydrazine acetate (1.36 g, 14.8 mmol, 1.6 equiv) was
added to a stirred solution of compound 9 (5 g, 9.25 mmol) in
DMF (30 mL) cooled to À178C (ice–salt bath). After having been
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2087

O. R. Martin et al.
MED
stirred at À158C for 2 h, the reaction mixture was allowed to warm
up to 108C over 1 h, then diluted with ethyl acetate (250 mL). The
solution was washed with aq. HCl (1n, 100 mL), then with brine
(100 mL), dried (MgSO4) and concentrated in vacuo to give com-
pound 10 as a yellow syrup (4.021 g, 93%). Rf =0.33 (toluene/ace-
CH2Ph), 5.24 (m, 1H; H2a), 7.25–7.36 ppm (m, 5H; CHar); 13C NMR
(100 MHz, CDCl3): d=14.23 (CH3), 22.82, 25.01, 25.08, 29.22–29.83
(8 lines), 32.06, 34.23, 34.44 (all CH2’s), 62.76 (C3a), 68.38 (C1a),
70.12 (C2a), 73.42 (CH2Ph), 127.72, 127.87, 128.51, 137.83, 173.16,
173.45 ppm; MS (ESI): m/z 687.5 [M+H]+, 704.5 [M+NH4]+, 709.5
[M+Na]+.
1
tone 2:1); H NMR (400 MHz, CDCl3; anomer ration a/b ~9:1): d=
3.34 (s, 3H), 3.35 (s, 3H), 3.39 (s, 3H), 3.41 (s, 3H), 3.87 (AB, J=
16.8 Hz, 2H; CH2O), 3.95 (s, 2H; CH2O), 4.02 (s, 2H; CH2O), 4.08 (AB,
J=16.4 Hz, 2H; CH2O), 4.13–4.20 (m, 2H; H6A, OH), 4.22–4.32 (m,
O-Benzyl
O-(2R)-2-hexadecanoyloxy-3-octadecanoyloxypropyl
N,N-diisopropyl phosphoramidite (15): Compound 13 (250 mg,
0.36 mmol) was dissolved in CH2Cl2/EtOH (2:5, 14 mL). An excess of
10% palladium on charcoal (10 mg) was added, and the reaction
mixture was stirred for 4 h at RT under H2. The mixture was heated
to 408C to completely dissolve the final product before being fil-
tered through a Millipore membrane to remove the catalyst. The
Pd/C was washed with CH2Cl2/EtOH (1:1, 3ꢃ20 mL) previously
heated to 408C. The combined organic phases were concentrated
to give compound 14 (215 mg, 99%) as a white solid. 1H-Tetrazole
(11 mg, 0.151 mmol, 0.6 equiv) and compound 14 (150 mg,
0.251 mmol) were dried separately over P2O5 for 1 h under high
vacuum. After both compounds had been combined, dry CH2Cl2
(2 mL) was added, and then a solution of freshly prepared benzyl-
oxy-bis(N,N-diisopropylamino)phosphine (0.84m in CH2Cl2; 358 mL,
0.301 mmol, 1.2 equiv). After 30 min at RT, the reaction mixture
was diluted with CH2Cl2 (50 mL), cooled to 08C and washed with
cooled sat. aq. NaHCO3 (10 mL). The organic phase was separated
and dried (MgSO4), and the solvent was removed in vacuo below
308C. Fast purification by flash silica gel chromatography (light pe-
troleum/ethyl acetate 6:1 containing 1% NEt3) gave compound 15
as a colourless oil (100 mg, 48%). 1H NMR (400 MHz, CDCl3): d=
0.88 (t, J=6.8 Hz, 6H; 2CH3), 1.17–1.19 (2d, 12H; isopropyl-CH3),
1.20–1.35 (m, 52H; 26CH2), 1.57–1.65 (m, 4H; 2CH2), 2.29 (2t, 4H;
2CH2CO), 3.56–3.92 (several m, 4H; 2 isopropyl-CH, 2H3a), 4.17
(ddd, J1aA,1aB =12.0, J1aA,2, J1aA,P =4.8, 6.4 Hz, 1H; H1aA), 4.34 (ddd,
2H; H5’, H6’B), 5.19 (d, J1’,2’ =1.8 Hz, 1H; H1’), 5.27 (t, J4’,3’ =J4’,5’
=
10 Hz, 1H; H4’), 5.30 (dd, J2’,3’ =3.2 Hz, 1H; H2’), 5.49 ppm (dd, 1H;
H3’); 13C NMR (100 MHz, CDCl3): d=59.45 (CH3), 59.48 (CH3), 59.50
(CH3), 62.62 (C6’), 66.53 (C4’), 68.00 (C5’), 69.29 (C3’), 69.34, 69.41,
69.49, 69.52 (4CH2O), 70.50 (C2’), 91.98 (C1’), 169.48 (CO), 169.71
(CO), 170.20 ppm (CO); HRMS (ESI): calcd for C18H28O14Na: m/z
491.13713 [M+Na]+, found: 491.13740.
2,3,4,6-Tetra-O-methoxyacetyl-a-d-mannopyranosyl trichloroace-
timidate (11): Trichloroacetonitrile (1.28 mL, 12.80 mmol, 12 equiv)
was added to a mixture of compound 10 (500 mg, 1.067 mmol)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 45 mL, 0.299 mmol,
0.28 equiv) in dry CH2Cl2 (10 mL) at RT under Ar. After the mixture
had been stirred for 10 min, the solvent was removed to give a
residual volume of 2–3 mL. The concentrated solution of 11 was
rapidly purified by silica gel column chromatography (light petrole-
um/ethyl acetate 1:2 containing 1% of Et3N) to afford compound
11 (479 mg, 73%) as yellow syrup: C20H28O14NCl3 (M=
614.81 gmolÀ1). Rf =0.39 (light petroleum/ethyl acetate 1:3 contain-
1
ing 1% Et3N); H NMR (250 MHz, CDCl3): d=3.41 (s, 3H; CH3), 3.42
(s, 3H; CH3), 3.45 (s, 3H; CH3), 3.50 (s, 3H; CH3), 3.95 (AB, J=
16.8 Hz, 2H; CH2O), 4.03 (s, 2H; CH2O), 4.08 (s, 2H; CH2O), 4.19 (AB,
J=15.6 Hz, 2H; CH2O), 4.22–4.33 (m, 2H; H6A, H5’), 4.40 (dd,
J
6’B,5’ =5.3, J6’B,6’A =13.0 Hz, 1H; H6’B), 5.45 (t, J4’,5’ =J4’,3’ =9.8 Hz, 1H;
H4’), 5.54 (dd, J3’,2’ =3.2 Hz, 1H; H3’), 5.59 (dd, 1H; H2’), 6.32 (d,
1’,2’ =1.5 Hz, 1H; H1’), 8.84 ppm (s, 1H; NH).
J
J
1aB,2 =4.0, J1aB,P =8.0 Hz, 1H; H1aB), 4.65 [2dd (Dd=0.01 ppm),
A,B =12.4, A,P =8.4 Hz, 1H; HA of CH2Ph], 4.73 [2dd (Dd=
J
J
0.003 ppm), JB,P =8.4 Hz, 1H; HB of CH2Ph], 5.19 ppm (m, 1H; H2a),
7.3–7.4 (m, 5H; 5CHar); 31P NMR (162 MHz, CDCl3): d=148.70,
148.80 ppm.
3-O-Benzyl-2-O-hexadecanoyl-1-O-octadecanoyl-sn-glycerol (13):
4-(N,N-Dimethylamino)pyridine (DMAP; 124 mg, 1.02 mmol,
0.1 equiv) and dicyclohexylcarbodiimide (DCC; 4.19 g, 20 mmol,
2.4 equiv) were added to a solution of stearic acid (2.41 g,
8.46 mol) and 3-O-benzyl-sn-glycerol (1.85 g, 10 mmol, 1.2 equiv) in
dry CH2Cl2 (50 mL) under argon at 08C. The mixture was stirred at
08C for 1 h, then warmed up to RT and stirred for an additional
18 h. The solids were removed by filtration through a cotton plug,
the filtrate was concentrated in vacuo, and the residue was puri-
fied by flash silica gel chromatography (light petroleum/ethyl ace-
tate 6:1) to give compound 12 (2.5 g, 66%) as a white solid (m.p.
41–438C) (ref. [33], no m.p. reported).
1-O-(2,3,4,6-Tetra-O-methoxyacetyl-a-d-mannopyranosyl)-2,3-O-
isopropylidene-dl-glycerol (16): By using general glycosylation
procedure A with solketal (282 mL, 2.26 mmol, 1.3 equiv) and com-
pound 11 (1.07 g, 1.74 mmol), pure compound 16 (569 mg, 56%)
was obtained as a 1:1 mixture of diastereoisomers in the glycerol
unit after purification by silica gel chromatography (toluene/ace-
1
tone 6:1). Rf =0.57 (CH2Cl2/acetone 4:1); H NMR (400 MHz, CDCl3):
d=1.37 (s, 3H; CH3), 1.43, 1.44 (2s, 3H; CH3), 3.40, 3.41, 3.46, 3.48
(4s, 4ꢃ3H; 4OCH3), 3.53–3.62 (m, 1H), 3.70–3.83 (m, 3H), 3.87–
4.33 (several m, 15H), 4.35–4.45 (2dd, 1H), 4.90 (d, J1’,2’ =1.2 Hz,
0.5H; H1’), 4.93 (d, J1’,2’ =1.6 Hz, 0.5H; H1’), 5.33 (t, J=10.0 Hz,
1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide
hydrochloride
(EDCI; 1.78 g, 9.30 mmol, 2.5 equiv) and DMAP (91 mg,
0.744 mmol, 0.2 equiv) were added to a solution of compound 12
(1.67 g, 3.72 mmol) and palmitic acid (1.91 g, 7.44 mmol, 2 equiv)
in dry CH2Cl2 (30 mL) at RT. The reaction mixture was stirred over-
night, then diluted with CH2Cl2 (150 mL), washed successively with
aq. HCl (1n, 60 mL), water (60 mL) and brine (60 mL), and then
dried (MgSO4). The organic phase was concentrated, and the resid-
ual product was purified by flash chromatography (light petrole-
um/ethyl acetate 25:1 to 20:1) to give compound 13 as a white
solid (2.45 g, 96%). m.p.=44–458C; Rf =0.40 (light petroleum/ethyl
acetate 6:1); [a]D =+5.5 (c=1.63, CHCl3); 1H NMR (400 MHz,
CDCl3): d=0.88 (t, J=6.6 Hz, 3H; CH3), 1.22–1.40 (m, 52H; 26CH2),
1.54–1.67 (m, 4H; 2CH2), 2.24–2.34 (2t, 4H; 2CH2CO), 3.59 (~d, 2H;
2H1a=CH2OBn), 4.19 (dd, J3aA,3aB =12.0, J3aA,2a =6.4 Hz, 1H; H3aA),
4.35 (dd, J3aB,2a =3.2 Hz, 1H; H3aB), 4.53 (AB, J=12.0 Hz, 2H;
0.5H; H4’), 5.34 (t, J=10.0 Hz, 0.5H; H4’), 5.39 (dd, J1,2 =1.8, J2,3
3.4 Hz, 0.5H; H2’), 5.41 (dd, J1,2 =1.8, J2,3 =3.4 Hz, 0.5H; H2’), 5.484
(dd, J2,3 =3.6, J3,4 =10 Hz, 0.5H; H3’), 5.488 ppm (dd, J2,3 =3.6, J3,4
=
=
10 Hz, 0.5H); 13C NMR (101 MHz, CDCl3): d=25.40 (CH3), 25.49
(CH3), 26.77 (CH3), 26.85 (CH3), 59.48 (OCH3), 59.54 (OCH3), 62.31,
62.38, 63.10, 65.80, 66.24 (all CH2’s), 66.31, 66.37 (C4’), 66.44 (CH2),
68.40 (CH), 68.47 (CH), 68.56 (CH2), 69.03 (CH2), 69.28 (CH), 69.31
(CH), 69.36, 69.42, 69.44, 69.50, 69.57, 69.58 (all CH2), 69.65 (CH),
69.68 (CH), 74.16 (CH), 74.29 (CH), 97.54 (C1’), 97.77 (C1’), 109.81
(Cq), 109.86 (Cq), 169.33, 169.41, 169.53, 170.07 ppm; MS (ESI): m/z
600.5 [M+NH4]+, 605.5 [M+Na]+, 621.0 [M+K]+.
1-O-(2,3,4,6-Tetra-O-methoxyacetyl-a-d-mannopyranosyl)-dl-
glycerol (17): Compound 16 (472 mg, 0.810 mmol) was dissolved
in a mixture of acetic acid (10 mL) and H2O (4.5 mL). After the mix-
2088
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ChemMedChem 2011, 6, 2081 – 2093

Phosphatidyl myo-Inositol Mannosides Mimics
ture had been stirred for 18 h, the solvents were evaporated, and
the residue was dried by coevaporation with toluene (10 mL) to
give compound 17 as a colourless syrup, as a 1:1 mixture of diaste-
reoisomers (439 mg, quant.). 13C NMR (101 MHz, CDCl3): d=59.34
(CH3), 59.38 (CH3), 62.35 (C6’), 62.38 (C6’), 63.37 (C1a), 63.40 (C1a),
66.22 (C4’), 68.25 (C5’), 69.22, 69.29, 69.35, 69.41, 69.43 (5 CH2),
69.54, 69.58, 69.64 (3 lines for C2’ and C3’), 70.47 (C2a), 70.72 (C2a),
97.44 (C1’), 97.78 (C1’), 169.33, 169.53, 169.60, 169.61, 170.07,
170.10 ppm; HRMS (ESI) calcd for C21H34O16Na: m/z 565.1745
[M+Na]+, found m/z 565.1758.
under H2 for 1.5 h. The solids were then removed by filtration
through a Millipore membrane. The catalyst was washed with
CH2Cl2/EtOH (1:1, 3ꢃ20 mL). The organic phases were combined,
and the solvents were evaporated to give compound 2 as a white
solid (6 mg, 83%). 1H NMR (400 MHz, CD3OD 0.35 mL/CDCl3
0.15 mL): d=0.87 (t, J=6.8 Hz, 6H; 2CH3), 1.20–1.36 (m, 52H),
1.54–1.65 (m, 4H), 2.31 and 2.34 (2t, 4H; 2CH2CO), 3.51–3.93 (m,
10H), 4.05–4.22 (m, 3H), 4.39–4.45 (m, 2H), 4.78 (brs, 1H), 4.80
(brs, 1H), 5.25 ppm (m, 1H; H2a); 31P NMR (162 MHz, CDCl3): d=
À2.56 ppm; HRMS (ESIÀ) calcd for C46H89O15P: m/z 911.5861
[MÀH]À, found: 911.5878.
3-O-Benzyl-1-O-(2,3,4,6-tetra-O-methoxyacetyl-a-d-mannopyra-
nosyl)-dl-glycerol (18): Compound 17 (168 mg, 0.310 mmol) was
dissolved in CH2Cl2 (5 mL), and benzyl trichloroacetimidate (58 mL,
0.310 mmol, 1 equiv) was added. Molecular sieves (4 ꢂ) were
added, and the mixture was stirred for 30 min and then cooled at
08C. TMSOTf (12 mL, 0.062 mmol, 0.2 equiv) was added, and the
mixture was stirred for 1.5 h at 08C. The reaction was stopped by
the addition of excess triethylamine, and the solids were removed
by filtration through a pad of celite. The solvent was removed
under vacuum, and the product was purified by silica gel column
chromatography (petroleum ether/ethyl acetate 1:2) to give pure
N-Trifluoroacetamido-3-O-benzyl-1,5-dideoxy-1,5-imino-xylitol
(23): Compound 21 (300 mg, 0.957 mmol) was dissolved in EtOH
(10 mL). Excess palladium hydroxide was added, and the reaction
mixture was stirred at RT under H2 for 20 h. More catalyst was
added, and the mixture was stirred for another 18 h. The solids
were then removed by filtration through a Millipore membrane.
The catalyst was washed with EtOH (2ꢃ10 mL). The organic phases
were combined, and the solvent was evaporated under reduced
pressure to give compound 22 (172 mg, containing about 35% of
triol). This sample of 22 (172 mg) was dissolved in CH2Cl2 (5 mL).
Pyridine (100 mL, 1.31 mmol, 1.7 equiv) and trifluoroacetic anhy-
dride (160 mL, 1.16 mmol, 1.5 equiv) were added, and the mixture
was stirred for 18 h at RT. CH2Cl2 (30 mL) was added, the organic
phase was washed with aq. HCl (1n, 2ꢃ15 mL), then with water
(15 mL), dried over MgSO4 and concentrated under high vacuum.
The residue was purified by silica gel column chromatography (pe-
troleum ether/ethyl acetate 3:2) to give compound 23 (110 mg,
45% from 21) as colourless syrup. Rf =0.40 (petroleum ether/ethyl
1
18 (57 mg, 29%). Rf =0.41 (CH2Cl2/acetone 4:1); H NMR (400 MHz,
CDCl3): d=3.33, 3.34, 3.36, 3.40 (4s, 4ꢃ3H; 4CH3), 3.43–3.58 (m,
3H), 3.67 (dd, J=6.1, 10.4 Hz, 0.5H), 3.71 (dd, J=4.1, 10.5 Hz,
0.5H), 3.86 (AB, J=16.7 Hz, 2H; CH2O), 3.91–4.15 (m, 12H), 4.27
(dd, J 6’B,6’A =12.2, J6’B,5’ =5.0 Hz, 1H; H6’B), 4.49 (s, 2H; CH2Ph), 4.80
(d, J1’,2’ =1.4 Hz, 0.5H; H1’), 4.81 (d, J1’,2’ =1.1 Hz, 0.5H; H1’), 5.24 (t,
J
4’,5’ =J4’,3’ =10.0 Hz, 1H; H4’), 5.30 (dd, J2’,3’ =3.4 Hz, 0.5H; H2’), 5.31
(dd, J2’,3’ =3.4 Hz, 0.5H; H2’), 5.39 [2dd (Dd=0.0018 ppm), 1H;
H3’], 7.20–7.31 ppm (m, 5H; CHar); 13C NMR (101 MHz, CDCl3): d=
59.47 (CH3), 59.53 (CH3), 62.38 (C6’), 62.42 (C6’), 66.35 (C4’), 68.38
(C5’), 69.27 (C3’), 69.31 (C3’), 69.35 (CH2), 69.39 (CH), 69.42 (CH2),
69.49 (CH2), 69.56 (CH2), 69.58 (CH2), 69.70 (C2’), 69.76 (CH2), 69.83
(CH2), 70.90 (CH2), 70.95 (CH2), 73.58 (CH2Ph), 73.61 (CH2Ph), 97.65
(C1’), 97.88 (C1’), 127.83, 127.85, 128.0, 128.58, 137.75 (Cq), 137.76
(Cq), 169.39, 169.56, 170.03, 170.05 ppm; MS (ESI): m/z 655.5
[M+Na]+; HRMS (ESI) calcd for C28H40O16Na: m/z 655.22086
[M+Na]+, found m/z 655.22134.
1
acetate 3:2). H NMR (400 MHz, CDCl3; the molecule is desymmetr-
ised by rotamers at the NÀCO bond): d=3.51 (d, J=13.5 Hz, H5A/
1A), 3.61 (brs, 1H; H3), 3.65–3.79 (brAB, 2H; 2H1/2H5), 3.82 (brs,
2H; OH, H2/4), 3.96 (brs, 1H; H4/2), 4.11 (dd, J=4.0, 13.6 Hz, 1H;
H5B/1B), 4.66 (~s, 2H; OCH2Ph), 7.25–7.40 ppm (m, 5H; CHar);
13C NMR (101 MHz, CDCl3): d=45.77 (C1/5), 48.26 (C5/1), 67.80
(C2/4), 67.97 (C4/2), 73.04 (CH2Ph), 76.52 (C3), 116.51 (q, CF3, J=
289 Hz), 127.78, 128.23, 128.72, 137.71 (Cq), 157.72 ppm (q, COCF3,
J=36 Hz); 19F NMR (376 MHz, CDCl3): d=À67.72 ppm; HRMS (ESI)
calcd for C14H16NO4F3Na: m/z 342.0929 [M+Na]+, found: m/z
342.0927.
1-O-(2,3,4,6-Tetra-O-methoxyacetyl-a-d-mannopyranosyl)-3-O-
benzyl-2-O-((S)-2-O-hexadecanoyloxy-3-O-octadecanoyloxy-pro-
pyl)(benzyl)phosphoryl)-dl-glycerol (19): Compound 18 (49 mg,
0.077 mmol) was phosphorylated by the general procedure C
using a solution of compound 15 (85 mg, 0.102 mmol, 1.3 equiv)
in CH2Cl2 (5 mL) and 1H-tetrazole (3 equiv). Pure compound 19
(29 mg, 27%) was obtained after purification of the crude product
by silica gel column chromatography (petroleum ether/ethyl ace-
tate 1:1) as a mixture of P* and glycerol diastereoisomers. Rf =0.25
(petroleum ether/ethyl acetate 1:1); 13C NMR (101 MHz, CDCl3): d=
14.26, 22.82, 24.97, 29.2–29.8 (several CH2’s), 32.06, 34.12, 34.25,
59.5, 59.58, 61.89, 62.18, 65.7–69.9 (several signals), 73.61 (OCH2Ph),
75.44, 75.53, 97.45, 97.86, 127.8–128.8 (CHar), 135.6–135.8 (Carq),
137.56 (Carq), 169.4, 169.5, 172.92, 173.3 ppm; 31P NMR (162 MHz,
CDCl3): d=À1.57 ppm; MS (ESI): m/z 1382.0 [M+H]+, 1398.0
[M+NH4]+, 1403.0 [M+Na]+.
N-Trifluoroacetamido-3-O-benzyl-2,4-bis-O-(2,3,4,6-tetra-O-meth-
oxyacetyl-a-d-mannopyranosyl)-1,5-dideoxy-1,5-imino-xylitol
(24): By using the general procedure for glycosylation A with 23
(105 mg, 0.330 mmol) and 11 (500 mg, 0.813 mmol, 2.5 equiv),
pure compound 24 (188 mg, 47%) was obtained as a syrup after
purification by silica gel column chromatography (CH2Cl2/acetone
1
6:1). Rf =0.21 (CH2Cl2/acetone 6:1); H NMR (400 MHz, CDCl3; com-
plex because of amide rotamers and nonequivalence of the man-
nosyl residues), selected signals: d=2.92 (dd, J=9.9, 12.9 Hz, 1H),
3.06 (dd, J=10.1, 12.9 Hz, 1H), 3.18 (dd, J=9.8, 13.9 Hz, 1H), 3.26
(dd, J=10.3, 13.9 Hz, 1H; H1A/5A, 4 signals), 3.36, 3.38, 3.38, 3.40
3.41, 3.44, 3.45, 3.48 (8CH3), 4.94 (s, 1H), 5.05 (s, 1H), 5.14 (s, 1H),
5.17 ppm (s, 1H; H1’, 4 signals); 13C NMR (101 MHz, CDCl3): d=
43.44, 45.54, 45.85, 47.91 (C1/5), 59.32–59.53 (OCH3, 7 signals),
61.75, 62.23, 62.55 (C6’, 3 signals), 65.43, 65.56, 65.97, 66.07, 68.49,
68.62, 68.68, 68.94, 69.35, 69.49 (10CH), 69.14, 69.22, 69.26, 69.29,
69.39, 69.43, 69.46 (CH2OMe), 71.17, 72.20, 76.56, 77.08, 81.88,
82.00 (6CH), 75.48, 75.57 (CH2Ph), 94.41, 94.75, 98.82, 99.07 (C1’, 4
signals), 116.14 and 116.21 (2q, J=289 Hz, CF3), 125.32–129.06
(CHar), 137.09 (Cq), 169.12–170.18 ppm (14 lines); 19F NMR
(376 MHz, CDCl3): d=À68.79, À68.55 ppm; HRMS (ESI) calcd for
C50H68F3NO30Na2: m/z 632.6787 [M+2Na]2+, found: m/z 632.6782.
1-O-(a-d-Mannopyranosyl)-2-O-((S)-2-O-hexadecanoyloxy-3-O-oc-
tadecanoyloxypropyl)phosphoryl)-dl-glycerol (2): Compound 20
(10 mg, 45%) was obtained from compound 19 (28 mg,
0.02 mmol) by using general method D to cleave the methoxyace-
tyl esters and purification by flash chromatography (CH2Cl2/MeOH
12:1). Compound 20 (9 mg, 0.008 mmol) was then dissolved in
CH2Cl2/EtOH (1:2, 6 mL). An excess of 5% palladium on charcoal
(20 mg) was added, and the reaction mixture was stirred at RT
ChemMedChem 2011, 6, 2081 – 2093
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2089

O. R. Martin et al.
MED
N-Trifluoroacetamido-2,4-bis-O-(2,3,4,6-tetra-O-methoxyacetyl-a-
d-mannopyranosyl)-(3-O-((S)-2-O-hexadecanoyloxy-3-O-octa-
decanoyloxypropyl)(benzyl)phosphoryl)-1,5-dideoxy-1,5-imino-
xylitol (26): Compound 24 (188 mg, 0.154 mmol) was first depro-
tected by general procedure B to give compound 25 (168 mg,
97%). 13C NMR (101 MHz, CDCl3): d=44.37, 45.69, 46.24, 47.74 (4
signals for C1 and C5), 59.36, 59.45, 59.49, 59.55, 59.56, 62.39 (C6’),
62.64 (C6’), 66.03 and 66.17 (2CHsugar), 68.75 (C5’), 68.80 (C5’),
69.12, 69.20, 69.27 and 69.65 (4CHsugar), 69.31, 69.34, 69.43, 69.45
and 69.59 (5 signals of CH2 for MAc), 74.32, 75.16, 75.80, 75.96,
76.61 and 77.35 (6 signals of CH for C2, C3, C4), 95.94 and 95.98
(C1’), 99.03 and 99.04 (C1’), 169.27, 169.37, 169.42, 169.44, 169.47,
169.52, 169.65, 169.66, 169.69, 170.19, 170.24, 170.28 ppm; 19F NMR
(376 MHz, CDCl3): d=À68.86, À68.69 ppm; Compound 25
(168 mg, 0.149 mmol) was phosphorylated by general procedure C
using a solution of compound 15 (0.26m in CH2Cl2; 861 mL,
0.223 mmol, 1.5 equiv) in CH2Cl2 (8 mL) and 1H-tetrazole (5 equiv).
Pure compound 26 (104 mg, 37%) was obtained after purification
of the crude product by silica gel column chromatography (tolu-
ene/acetone 4:1). Rf =0.22 (toluene/acetone 4:1); 1H NMR
(400 MHz, CDCl3): very complex because of amide rotamers, P* iso-
mers and nonequivalence of the mannosyl residues; 13C NMR
(101 MHz, CDCl3), most significant data: d=94.47, 94.50, 94.91,
95.00, 98.69, 98.76, 99.18 ppm (2C1’, 8 signals! A copy of the spec-
trum is shown in the Supporting Information); 19F NMR (376 MHz,
CDCl3): d=À68.68, À68.67, À68.47, À68.44 ppm; 31P NMR
(162 MHz, CDCl3): d=À0.92 ppm; HRMS (ESI) calcd for
C87H139F3NO37PNa2: m/z 961.9256 [M+2Na]2+, found: m/z 961.9268.
data: d=97.26, 97.88, 99.43, 100.45 ppm (2C1’); 19F NMR
(376 MHz, CDCl3/CD3OD 0.4:0.2 mL): d=À68.34, À67.83 ppm;
31P NMR (162 MHz, CDCl3/CD3OD 0.4:0.2 mL): d=0.161 ppm; IS-
MS: m/z 1210.5 [MÀH]À; HRMS (ESI) calcd for C56H101F3NO21PNa:
m/z 1234.6454 [M+Na]+, found: m/z 1234.6442. A fluorinated im-
purity (d19F =À76.26) is observed in both samples.
2,4-Bis-O-(2,3,4,6-tetra-O-methoxyacetyl-a-d-mannopyranosyl)-
1,5-anhydroxylitol (34): Diglycosyl xylitol 33 (709 mg, 67%) was
obtained by using diol 32 as glycosyl acceptor (212 mg,
0.945 mmol), compound 11 as glycosyl donor (1.69 g, 2.74 mmol,
2.9 equiv) and TMSOTf (248 mL, 50% mol equiv vs donor) in gener-
al procedure A, followed by purification by silica gel column chro-
matography (petroleum ether/ethyl acetate 1:3). Rf =0.25 (petrole-
um ether/ethyl acetate 1:3). This compound (333 mg, 0.296 mmol)
was O-debenzylated according to general procedure B to give
1
pure compound 34 (297 mg, 97%). H NMR (CDCl3, 400 MHz): d=
3.15–3.25 (m, 2H; H1A,5A), 3.41, 3.42, 3.45, 3.46, 3.47, 3.49 (6s,
24H; 8OMe), 3.58–3.68 (m, 3H; H2,3,4), 3.88–4.20 (several AB and
s, 8CH2OMe), ~3.98 (hidden m, 2H; H1B,5B), ~4.06 (hidden m, 1H;
H5’), 4.21 (dd, J=2.2, 12.2 Hz, 1H; H6A), 4.28 (dd, J=2.3, 12.2 Hz,
1H; H6A), 4.33 (dd, J=5.2, 12.3 Hz, 1H; H6’B), 4.36 (dd, J=5.6,
12.3 Hz, 1H; H6’B), 4.48 (ddd, J=2.4, 5.1, 10 Hz, 1H; H5’), 4.93
(narrow d, J1’,2’ =1.4 Hz, 1H; H1’), 5.18 (narrow d, J1’2’ =1.2 Hz, 1H;
H1’), 5.30 (t, J=10 Hz, 2H; 2H4’), 5.32, (narrow dd, 1H; H2’), 5.40–
5.47 ppm (m, 3H; H2’, 2H3’); 13C NMR (CDCl3, 101 MHz): d=59.45,
59.46, 59.47, 59.50, 59.51, 59.54 (MeO), 62.47, 62.58 (2CH2; C6’),
66.25 (CH; C4’), 67.62, 68.74 (2CH2; C1,5), 68.69, 68.86, 69.14, 69.26,
69.87 (5CH; C2’,C3’,C5’), 69.3–69.6 (CH2, 5 lines, CH2OMe), 75.75,
76.57, 78.49 (3CH; C2,C3,C4), 96.42, 99.03 (2CH; C1’), 169.33,
169.40, 169.45, 169.46, 169.57, 169.62, 170.0, 170.16 ppm (MAc);
HRMS (ESI) calcd for C41H63O30: m/z 1035.33987 [M+H]+, found:
m/z 1035.33857; calcd for C41H62O30Na: m/z 1057.32181 [M+Na]+,
found: m/z 1057.32010.
2,4-Bis-O-(a-d-mannopyranosyl)-(3-O-((S)-2-O-hexadecanoyloxy-
3-O-octadecanoyloxy-propyl)phosphoryl)-1,5-dideoxy-1,5-imino-
xylitol (6a) and N-trifluoroacetamido-2,4-bis-O-(a-d-mannopyra-
nosyl)-(3-O-((S)-2-O-hexadecanoyloxy-3-O-octadecanoyloxypro-
pyl)phosphoryl)-1,5-dideoxy-1,5-iminoxylitol (6b): Compound 26
(90 mg, 0.048 mmol) was dissolved in CH2Cl2/EtOH (2:3, 10 mL).
Excess 10% palladium on charcoal (100 mg) was added, and the
reaction mixture was stirred at RT under H2 for 3 h. The solids were
removed by filtration through a Millipore membrane. The catalyst
was washed with CH2Cl2/EtOH (1:1, 3ꢃ20 mL). The residual sol-
vents were evaporated to give compound 27 (82 mg, 95%) as a
syrup. 13C NMR (101 MHz, CDCl3), significant signals: d=94.54,
94.98, 98.65, 99.17 ppm (C1’, 4 signals; a copy of the spectrum in
given in the Supporting Information). Compound 27 (80 mg,
0.045 mmol) was then deprotected according to general method D
in CHCl3/MeOH (1 mL) and, after separation and purification by
two successive flash chromatographies (CHCl3/MeOH/H2O 70:40:1),
two compounds were obtained as white solids: the first is com-
pound 6a, with a free amino group (16 mg, 32%), and the second
is compound 6b, which retained the COCF3 group (21 mg, 48%).
2,4-Bis-O-(2,3,4,6-tetra-O-methoxyacetyl-a-d-mannopyranosyl)-
3-O-((S)-2-O-hexadecanoyloxy-3-O-octadecanoyloxypropyl)(ben-
zyl)phosphoryl-1,5-anhydroxylitol (35): Alcohol 34 (297 mg,
0.287 mmol) was phosphorylated according to general procedure C
with compound 15 (850 mg, 1.02 mmol, 3.5 equiv), 1H-tetrazole
solution (3 mL, 1.204 mmol, 4.2 equiv) and mCPBA (416 mg,
1.204 mmol, 4.2 equiv) for the oxidation step to give compound
35 (243 mg, 47%) after purification by silica gel column chroma-
tography (toluene/acetone 3:1). Rf =0.27 (toluene/acetone 2:1);
1H NMR (400 MHz, CDCl3; complex spectrum due to P* diastereo-
isomers and nonequivalence of mannosyl units): signals of H1’ d=
4.91 (s, 1H), 5.00 (s, 0.5H), 5.03 ppm (s, 0.5H); signals of H2’: d=
5.54 (t, 0.5H), 5.56 (t, 0.5H), 5.59 (dd, 0.5H), 5.61 ppm (dd, 0.5H);
13C NMR (CDCl3, 101 MHz): d=14.23 (CH3), 22.81, 24.95, 24.99,
29.25–29.83, 32.04, 34.09, 34.21, 34.25 (CH2), 59.42–59.61 (OMe),
61.93, 62.11, 62.69 (CH2), 65.56 (CH), 66.12–66.26 (CH2), 67.39, 67.44
(CH2), 68.33, 68.38 (CH2), 68.82, 69.03 (CH), 69.3–69.64 (CH2OMe),
69.96 (CH), 70.43–70.55 (CH2), 71.44–71.55 (CH), 77.19, 77.35 (CH),
78.42–78.50 (CH), 94.88 (C1’), 99.41 and 99.36 (C1’), 128.14–129.00
(CHar), 135.88, 135.93 (Car), 169.25–169.66 (11 lines, MAc), 170.02,
170.07 (MAc), 172.98, 173.04, 173.35, 173.38 ppm (Acyl-CO);
31P NMR (CDCl3, 162 MHz): d=À1.21, À0.98 ppm; HRMS (ESI) calcd
for C85H139O37PNa2: m/z 914.4264 [M+2Na]2+, found m/z 914.4267.
Compound 6a: Rf =0.06 (CHCl3/MeOH/H2O 70:40:1); 1H NMR
(250 MHz, CDCl3/CD3OD/D2O 0.35:0.3:0.05 mL, poor solubility): d=
0.89 (t, J=6.8 Hz, 6H; 2CH3), 1.20–1.50 (m, 52H), 1.50–1.70 (m,
4H), 2.26–2.40 (2t, 4H; 2CH2CO), 3.01–4.50 (several m), 4.93 (brs,
1H; H1’), 5.08 (brs, 1H; H1’), 5.16–5.30 ppm (m, 1H; H2a); 31P NMR
(162 MHz, CDCl3/CD3OD/D2O, poor solubility): d=À0.67 ppm;
HRMS (ESI) calcd for C54H102NO20PNa: m/z 1138.6631 [M+Na]+,
found: m/z 1138.6617.
Compound 6b: Rf =0.28 (CHCl3/MeOH/H2O 70:40:1); 1H NMR
(400 MHz, CDCl3/CD3OD 0.4:0.2 mL): d=0.89 (t, J=6.7 Hz, 6H;
2CH3), 1.20–1.50 (m, 52H), 1.51–1.71 (m, 4H), 2.30–2.36 (2t, 4H;
2CH2CO), 3.20–3.80 (several m), 4.40 (H3, hidden by HOD signal),
4.91 (s, 0.5H), 5.03 (s, 0.5H), 5.12 (s, 0.5H), 5.17 (s, 0.5H; 2H1’),
5.25 ppm (m, 1H; H2a); 13C NMR (101 MHz, CDCl3): significant
2,4-Bis-O-(a-d-mannopyranosyl)-3-O-((S)-2-O-hexadecanoyloxy-3-
O-octadecanoyloxypropyl)(benzyl)phosphoryl-1,5-anhydroxylitol
(36): Compound 36 (58 mg, 43%) was obtained from 35 (200 mg,
0.112 mmol) by following general method D (2 mL of solvent mix-
ture) and after purification by flash chromatography (CHCl3/MeOH
70:15). 1H NMR (400 MHz, CD3OD): signals of H1’: 5.18 (brs, 1H),
2090
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ChemMedChem 2011, 6, 2081 – 2093

Phosphatidyl myo-Inositol Mannosides Mimics
5.20 (brs, 1H); signals of H2’: 3.99 (dd, J=1.6, 3.2 Hz, 1H), 4.01 (dd,
J=1.6, 3.2 Hz, 1H); signals of H2a: 5.22 (m, 1H), 5.27 ppm (m, 1H);
13C NMR (101 MHz, JMod, CD3OD/CDCl3 3:1; presence of P* diaste-
reoisomers): d=14.43 (CH3), 23.45, 25.69, 29.9–30.5 (CH2), 32.77,
34.72, 34.86, 62.53, 62.82, 62.89 (C6’, 3 signals), 66.84 (br), 67.02 (d,
CH2, C1a, C3a), 67.39 and 69.64 (CH2, C1, C5), 68.0 (CH), 70.70 (d,
tion by silica gel column chromatography (petroleum ether/ethyl
acetate 1:4). Rf =0.16 (petroleum ether/ethyl acetate 1:3); [a]D =
+21 (c=1.07, CDCl3); 1H NMR (400 MHz, CDCl3): d=3.41, 3.45,
3.46, 3.48, (4s, 24H; 8OCH3), 3.25–3.5 (m, 2H), 3.54–3.61 (m, 1H;
H3), 3.87–4.19 (several m and AB systems, 22H), 4.19–4.38 (2dd,
4H; 2H6B’), 4.67 (AB, J=11.9 Hz, 2H; CH2Ph), 4.94 (narrow d, J=
1.0 Hz, 1H; H1’), 5.14 (s, 1H; H1’), 5.30 (t, J4’,3’ =J4’,5’ =9.9 Hz, 2H;
J
C,P =7.0 Hz, CH, C2a), 71.06 and 71.20 (2d, JC,P =6.0 Hz, CH2,
OCH2Ph), 71.32 (CH), 71.64, 71.76, 71.96, 74.38 (d), 74.82, 77.08,
77.26, 81.3 (very br, C3; all CH), 98.32 (d, CH, C1’), 103.0 (d, CH,
C1’), 129.19, 129.26, 129.51, 129.53, 129.65 (CHar), 136.5–136.62
(2d, Car), 174.22 (2 signals, CO), 174.61 ppm (2 signals, CO);
31P NMR (162 MHz, CD3OD/CDCl3 3:1): d=À3.98 ppm; HRMS (ESI)
calcd for C61H108O21P: m/z 1207.71152 [M+H]+„ found: m/z
1207.71066; calcd for C61H107NaO21P: m/z 1229.69347 [M+Na]+,
found: m/z 1229.69174.
2H4’), 5.41 (s, 2H; 2H2’), 5.40–5.44 (m, 1H; H3’), 5.48 (dd, J2’,3’ =
3.3 Hz, 1H; H3’), 7.27–7.36 ppm (m, 5H; CHar); 13C NMR (101 MHz,
CDCl3): d=59.36, 59.41 (CH3), 62.35, 62.43 (2C6’), 66.21, 66.34
(2C4’), 68.30, (very br, C1,C5), 68.67, 68.68, 69.15, 69.75 (CH, 2C2’,
2C3’, 2C5’), 69.24, 69.31, 69.33, 69.38, 69.44 (8CH2OMe), 72.67
(CH2Ph), 75.12, 75.40 (br, C2, C4), 79–80 (very br, C3), 98.75 (br, C1’),
98.90 (C1’), 127.82, 127.99, 128.59, 137.53, 169.20, 169.21, 169.27,
169.31 (2C), 169.35, 169.88, 170.01 ppm (8CO MAc); HRMS (ESI)
calcd for C48H68O30Na: m/z 1147.3693 [M+Na]+, found: m/z
1147.3715.
2,4-Bis-O-(a-d-mannopyranosyl)-3-O-((S)-2-O-hexadecanoyloxy-3-
O-octadecanoyloxypropyl)phosphoryl)-1,5-anhydroxylitol
(7):
Compound 36 (58 mg, 0.048 mmol) was dissolved in CH2Cl2/EtOH
(2:3, 10 mL). Excess 10% palladium on charcoal (40 mg) was
added, and the reaction mixture was stirred at RT under H2 for 2 h.
The catalyst was removed by filtration through a Millipore mem-
brane, then washed with CH2Cl2/EtOH (1:1, 3ꢃ20 mL). The organic
phases were combined, and the solvents were evaporated to give
2,4-Bis-O-(2,3,4,6-tetra-O-methoxyacetyl-a-d-mannopyranosyl)-
3-O-((S)-2-O-hexadecanoyloxy-3-O-octadecanoyloxy-propyl)(ben-
zyl)phosphoryl-1,5-anhydro-d-arabinitol (45): Compound 43
(194 mg, 0.172 mmol) was deprotected by general method B to
give compound 44 as colourless syrup (176 mg, 99%; NMR data
are given in the Supporting Information). Alcohol 44 (170 mg,
0.164 mmol) was phosphorylated according to general method C
with compound 15 (200 mg, 0.217 mmol, 1.3 equiv), 1H-tetrazole
solution (966 mL, 0.435 mmol, 2.7 equiv) and mCPBA (170 mg,
0.435 mmol, 2.7 equiv) for the oxidation to give compound 45
(160 mg, 55%) after purification by silica gel chromatography (tolu-
ene/acetone 3:1). Rf =0.51 (toluene/acetone 2:1); 13C NMR
(101 MHz, CDCl3; complex because of the presence of P* diaste-
reoisomers): d=14.08 (CH3), 22.65, 24.79, 29.07–29.67, 31.88, 33.93,
33.95, 34.05 (CH2), 59.3–59.4 (OMe’s), 61.72 and 61.74 (CH2), 62.37
and 62.42 (CH2; 2C6’), 65.78 (d) and 65.95 (d, JC,P =5.4 Hz, CH2,
C1a), 66.16 (CH), 66.22 (CH), 67.86 (very br), 68.86–69.45 (several
lines, CH and CH2’s), 70.02 (d), 70.16 (d, JC,P =5.9 Hz, CH2’s, 2CH2Ph),
75.22 (br, CH), 98.80 (very br, C1’), 99.12 (C1’), 127.89–128.98
(CHar), 135.46 (d, JC,P =6.4 Hz), 135.62 (d, JC,P =5.2 Hz; Cq ar),
169.21–169.36, 169.89, 169.94 (CO’s MAc groups), 172.77, 172.84,
173.10, 173.17 ppm (CO’s acyl chains); 31P NMR (162 MHz, CDCl3):
d=À1.83, À1.18 ppm; HRMS (ESI) calcd for C85H139O37PNa2: m/z
914.4264 [M+2Na]2+, found: m/z 914.4274.
1
compound 7 as an amorphous white solid (50 mg, 92%). H NMR
(400 MHz, CD3OD/CDCl3 0.3/0.15 mL): d=0.86 (t, J=6.8 Hz, 6H;
2CH3), 1.20–1.33 (m, 52H), 1.53–1.65 (m, 4H), 2.31–2.34 (2t, 4H;
2CH2CO), 3.35 (dd, J=7.6, 12.0 Hz, 1H; H1A/5A), 3.42 (dd, J=6.8,
12.8 Hz, 1H; H1A/5A), 3.52–3.58 (m, 1H), 3.60–3.85 (several m,
12H), 3.93–4.0 (m, 2H; H2’, H1B/5B), 4.03 (dd, J=4.4, 11.6 Hz, 1H;
H3aA), 4.10–4.16 (m, 2H; 2H1a), 4.18 (dd, J=6.8 Hz, 1H; H3aB),
4.30 (brq, J2,3 =J3,4 ~7, J3,P ~7 Hz, 1H; H3), 4.39 (dd, J=2.8, 12.0 Hz,
1H; H1B/5B), 4.88 (s, 1H; H1’), 4.95, (s, 1H; H1’), 5.33 ppm (m, 1H;
H2a); 13C NMR (101 MHz, JMod, CD3OD/CDCl3 0.3:0.15 mL): d=
14.34 (CH3), 23.21, 25.43, 25.47, 29.69, 29.72, 29.92, 29.96, 30.11,
30.14, 30.26, 32.50, 34.58, 34.72, 62.13 (C6’), 62.21 (C6’), 62.90 (C3a),
65.53 (d, 2JCÀP =4.8 Hz, C1a), 66.94 (C1/5), 67.58 (CHsugar), 67.87
(CHsugar), 69.08 (C1/5), 70.60 (d, 3JCÀP =7.3 Hz, C2a), 70.99 (C2’),
71.39 (2CHsugar), 71.61 (CHsugar), 71.73 (d, 3JCÀP =3.9 Hz, C2/4),
73.78 (C2/4), 74.22 (CHsugar), 76.18 (CHsugar), 77.88 (br, hidden by
CDCl3 signal, C3), 98.28 (C1’), 102.18 (C1’), 174.22 (CO), 174.58 ppm
(CO); 31P NMR (162 MHz, CD3OD/CDCl3 3:2): d=À2.1 ppm; HRMS
(ESI) calcd for C54H101O21P: m/z 1115.6495 [MÀH]À, found:
1115.6504.
2,4-Bis-O-(a-d-mannopyranosyl)-3-O-((S)-2-O-hexadecanoyloxy-3-
O-octadecanoyloxy-propyl)(benzyl)phosphoryl-1,5-anhydro-d-
arabinitol (46): Compound 46 (47 mg, 45%) was obtained from
compound 45 (155 mg, 0.087 mmol) by using general method D in
CHCl3/MeOH (2 mL) and purification by flash chromatography
(CHCl3/MeOH 70:15). 1H NMR (400 MHz, CD3OD): d=0.90 (t, 6H;
2CH3), 1.29 (m, 52H), 1.60 (m, 4H), 2.28–2.35 (m, 4H), 3.35–3.45 (m,
1H), 3.50–4.27 (several m, 21H), 4.36 (dd, 1H; J=3.3, 12.1 Hz),
4.43–4.54 (brm, 1H), 4.92 (narrow d, J=1.4 Hz, 0.5H; H1’), 4.96
(narrow d, J=1.5 Hz, 0.5H; H1’), 4.993 and 4.999 (2d, appt, 1H;
2H1’), 5.11–5.22 (AB coupled to 31P, JH,P =5.4 Hz, 2H; CH2Ph), 5.28
(m, 1H; H2a), 7.33–7.50 ppm (m, 5H; CHar); 13C NMR (101 MHz,
CD3OD): d=14.49 (CH3), 23.76, 26.01, 30.19–30.86 (several CH2),
33.10, 34.88, 35.05, 62.90 (C6’), 62.96, 63.01 (C1a or 3a), 67.29–
67.37 (C1a or 3a), 68.50, 68.58 (CH sugar), 68.78–69.45 (very br, CH2
arabinitol according to CÀH correlation), 70.99–71.11 (C2a), 71.38,
71.40 ppm (2d, Dd=0.016, JC,P =6 Hz, CH2, CH2Ph), 71.98 (CH),
72.00 (CH), 72.05 (CH), 72.37 (CH), 72.39 (CH), 72.42 (CH), 75.19
(CH), 75.22 (CH), 75.25 (CH), 75.36 (CH), 102.8–102.85 (br, C1’),
103.27 (C1’), 129.3–129.95 (CHar), 136.92, 136.98, 137.03 (2d, Car),
174.44, 174.79, 174.81 ppm (a copy of spectrum is given in the
3-O-Benzyl-2,4-bis-O-(2,3,4,6-tetra-O-methoxyacetyl-a-d-manno-
pyranosyl)-1,5-anhydro-d-arabinitol (43): A catalytic amount of
sodium was added to a solution of compound 41 (92 mg,
0.298 mmol) in MeOH (6 mL). The mixture was stirred for 2 h at RT
under Ar, then neutralised by the addition of IR120 ion-exchange
resin (H+ form). The resin was removed by filtration, and the sol-
vent was evaporated under vacuum to give compound 42 as a
yellow syrup (64 mg, 96%). [a]D =À82 (c=1.0, CHCl3); 1H NMR
(CDCl3, 400 MHz): d=3.21 (dd, J=7.7, 11.2 Hz, 1H; H5A), 3.44 (dd,
J=3.2, 7.8 Hz, 1H; H3), 3.47 (dd, J=2.4, 12 Hz, 1H; H1A), 3.75 (dd,
J=5.0, 11.8 Hz, 1H; H1B), 3.82 (dd, J=4.2, 11.3 Hz, 1H; H5B), 3.90
(td, J=4.2, 7.6, 7.6 Hz, 1H; H4), 4.01 (dt, J=2.8, 2.8, 5 Hz, 1H; H2),
4.66 (d, JAB =11.8 Hz, AB, 1H) and 4.76 (d, AB, 1H; CH2Ph), 7.26–
7.44 ppm (m, 5H; Ph); 13C NMR (CDCl3, 101 MHz): d=67.07 (C2),
67.87 (C4), 70.64 (2C, C1, C5), 72.81 (CH2Ph), 81.90 (C3), 128.58,
128.97, 129.27 (CHar), 139.97 ppm (Car). By using compound 42
(62 mg, 0.276 mmol) as the glycosyl acceptor and compound 11
(425 mg, 0.691 mmol, 2.5 equiv) as glycosyl donor in general meth-
od A, compound 43 (219 mg, 70%) was obtained after a purifica-
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O. R. Martin et al.
MED
Supporting Information); 31P NMR (162 MHz, CDCl3): d=À2.35,
À2.22 ppm; HRMS (ESI) m/z calcd for C61H107O21P: 1207.7121
[M+H]+, found: 1207.7112.
[3] H. D. Brightbill, D. H. Libraty, S. R. Krutzik, R. B. Yang, J. T. Belisle, J. R. Ble-
harski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, P. J. Brennan,
B. R. Bloom, P. J. Godowski, R. L. Modlin, Science 1999, 285, 732–736.
[4] K. A. Heldwein, M. J. Fenton, Microbes Infect. 2002, 4, 937–944.
[5] V. Quesniaux, C. Fremond, M. Jacobs, S. Parida, D. Nicolle, V. Yeremeev,
F. Bihl, F. Erard, T. Botha, M. Drennan, M. N. Soler, M. Le Bert, B. Schnyd-
er, B. Ryffel, Microbes Infect. 2004, 6, 946–959.
[6] P. B. Kang, A. K. Azad, J. B. Torrelles, T. M. Kaufman, A. Beharka, E. Tibesar,
L. E. DesJardin, L. S. Schlesinger, J. Exp. Med. 2005, 202, 987–999.
[7] L. Tailleux, O. Schwartz, J.-L. Herrmann, E. Pivert, M. Jackson, A. Amara,
L. Legres, D. Dreher, L. P. Nicod, J. C. Gluckman, P. H. Lagrange, B. Gic-
quel, O. Neyrolles, J. Exp. Med. 2003, 197, 121–127.
[8] N. Maeda, J. Nigou, J. L. Herrmann, M. Jackson, A. Amara, P. H. Lagrange,
G. Puzo, B. Gicquel, O. Neyrolles, J. Biol. Chem. 2003, 278, 5513–5516.
[9] S. Pitarque, J. L. Herrmann, J. L. Duteyrat, M. Jackson, G. R. Stewart, F.
Lecointe, B. Payre, O. Schwartz, D. B. Young, G. Marchal, P. H. Lagrange,
G. Puzo, B. Gicquel, J. Nigou, O. Neyrolles, Biochem. J. 2005, 392, 615–
624.
2,4-Bis-O-(a-d-mannopyranosyl)-3-O-((S)-2-O-hexadecanoyloxy-3-
O-octadecanoyloxy-propyl)phosphoryl-1,5-anhydro-d-arabinitol
(8): Compound 46 (45 mg, 0.037 mmol) was dissolved in CH2Cl2/
EtOH (1:1, 8 mL). An excess of 5% palladium on charcoal (50 mg)
was added, and the reaction mixture was stirred at RT under H2 for
2 h. The catalyst was then removed by filtration through a Milli-
pore membrane. The solid was washed with CH2Cl2/EtOH (1:1, 3ꢃ
20 mL). The combined organic phases were evaporated to give
1
compound 8 (33 mg, 79%) as an amorphous white solid. H NMR
(400 MHz, CD3OD/CDCl3 0.35:0.17 mL): d=0.86 (t, 6H; 2CH3), 1.2–
1.3 (m, 52H), 1.52–1.65 (m, 4H), 2.28–2.38 (2t, 4H; 2CH2CO), 3.34–
3.45 (m, 1H), 3.45–3.58 (m, 2H), 3.58–3.69 (m, 4H), 3.69–4.03 (m,
10H), 4.07–4.23 (m, 4H), 4.33–4.43 (m, 2H), 4.96 (s, 1H; H1’), 5.01
(s, 1H; H1’), 5.26 ppm (narrow m, 1H; H2a); 31P NMR (162 MHz,
CD3OD/CDCl3 0.35:0.17 mL): d=À1.82 ppm; HRMS (ESI) m/z calcd
for C54H100O21P: 1115.65002 [M+H]+, found: 1115.64938.
[10] J. B. Torrelles, A. K. Azad, L. S. Schlesinger, J. Immunol. 2006, 177, 1805–
1816.
[11] B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle,
M. J. Fenton, J. Leukoc. Biol. 2001, 69, 1036–1044.
Biological assays
[12] M. Gilleron, V. F. Quesniaux, G. Puzo, J. Biol. Chem. 2003, 278, 29880–
29889.
[13] M. Gilleron, C. Ronet, M. Mempel, B. Monsarrat, G. Gachelin, G. Puzo, J.
Biol. Chem. 2001, 276, 34896–34904.
[14] F. F. Hsu, J. Turk, R. M. Owens, E. R. Rhoades, D. G. Russell, J. Am. Soc.
Mass Spectrom. 2007, 18, 466–478.
[15] F. F. Hsu, J. Turk, R. M. Owens, E. R. Rhoades, D. G. Russell, J. Am. Soc.
Mass Spectrom. 2007, 18, 479–492.
[16] T. B. H. Geijtenbeek, S. J. van Vliet, E. A. Koppel, M. Sanchez-Hernandez,
C. M. J. E. Vandenbroucke-Grauls, B. Appelmelk, Y. van Kooyk, J. Exp.
Med. 2003, 197, 7–17.
[17] S. I. Gringhuis, J. den Dunnen, M. Litjens, B. van Het Hof, Y. van Kooyk,
T. B. Geijtenbeek, Immunity 2007, 26, 605–616.
[18] E. Doz, S. Rose, J. Nigou, M. Gilleron, G. Puzo, F. Erard, B. Ryffel, V. F.
Quesniaux, J. Biol. Chem. 2007, 282, 26014–26025.
[19] E. Doz, S. Rose, N. Court, S. Front, V. Vasseur, S. Charron, M. Gilleron, G.
Puzo, I. Fremaux, Y. Delneste, F. Erard, B. Ryffel, O. R. Martin, V. F. Ques-
niaux, J. Biol. Chem. 2009, 284, 23187–23196.
Primary macrophage cultures: Murine bone-marrow cells were iso-
lated from femurs and cultivated (106 mLÀ1) for 7 days in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with l-gluta-
mine (2 mm), 20% horse serum and 30% L929 cell-conditioned
medium as source of M-CSF. After further three days in fresh
medium, the bone-marrow-derived macrophages (BMDMs; 105
cells per well) in DMEM supplemented with l-glutamine (2 mm), b-
mercaptoethanol (2ꢃ10À5 m) and 0.1% FCS were stimulated with
LPS (100 ngmLÀ1; E. coli, serotype O111:B4, Sigma) in the presence
of IFN-g (500 UmLÀ1). The PIM preparations or DMSO vehicle con-
trols were added 30 min prior to the stimuli. Lyophilised PIM prep-
arations were solubilised in DMSO and used at a non-cytotoxic,
maximum 1% DMSO final concentration. The lack of cytotoxicity
was controlled by incorporating MTT.
Cytokine determination: Cell culture supernatants were harvested
after 24 h, and the contents of TNFa and IL-12p40 cytokine were
measured by using commercially available ELISA reagents (Duoset
R&D Systems, Abingdon, UK).
[20] S. C. Eisenbarth, D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, K.
Bottomly, J. Exp. Med. 2002, 196, 1645–1651.
[21] I. Sayers, W. Severn, C. B. Scanga, J. Hudson, G. Le Gros, J. L. Harper, J.
Allergy Clin. Immunol. 2004, 114, 302–309.
[22] G. D. Ainge, J. Hudson, D. S. Larsen, G. F. Painter, G. Singh-Gill, J. L.
Harper, Bioorg. Med. Chem. 2006, 14, 5632–5642.
[23] J. L. Harper, C. M. Hayman, D. S. Larsen, G. F. Painter, G. Singh-Gill,
Bioorg. Med. Chem. 2011, 19, 917–925.
[24] V. Quesniaux, O. R. Martin, S. Front (CNRS & University of Orleans), PCT
Patent Application WO 2009/153434A2.
Nitrite measurements: Nitrite concentrations were determined by
using the Griess reaction (3% phosphoric acid, 1% p-aminoben-
zene-sulfonamide, 1% N-(1-naphtyl)ethylenediamine. Results are
expressed as percent of inhibition compared with full release in
the presence of DMSO but in the absence of PIM.
[25] C. J. J. Elie, C. E. Dreef, R. Verduyn, G. A. van der Marel, J. H. van Boom,
Tetrahedron 1989, 45, 3477–3486.
[26] A. Stadelmaier, R. R. Schmidt, Carbohydr. Res. 2003, 338, 2557–2569.
[27] a) Y. Watanabe, T. Yamamoto, S. Ozaki, J. Org. Chem. 1996, 61, 14–15;
b) Y. Watanabe, T. Yamamoto, T. Okazaki, Tetrahedron 1997, 53, 903–
918.
[28] K. N. Jayaprakash, J. Lu, B. Fraser-Reid, Bioorg. Med. Chem. Lett. 2004, 14,
3815–3819.
[29] G. D. Ainge, N. A. Parlane, M. Denis, C. M. Hayman, D. S. Larsen, G. F.
Painter, Bioorg. Med. Chem. 2006, 14, 7615–7624.
[30] B. S. Dyer, J. D. Jones, G. D. Ainge, M. Denis, D. S. Larsen, G. F. Painter, J.
Org. Chem. 2007, 72, 3282–3288.
Acknowledgements
Financial support of this study by grants from the CNRS, Universi-
ty of Orlꢀans, and European Union FEDER N8 1649–32264 is
gratefully acknowledged. We are also very grateful to Dr. Gavin
Painter (Industrial Research Limited, Lower Hutt, New Zealand)
for providing us with a sample of synthetic PIM2.
[31] G. D. Ainge, N. A. Parlane, M. Denis, B. S. Dyer, A. Harer, C. M. Hayman,
D. S. Larsen, G. F. Painter, J. Org. Chem. 2007, 72, 5291–5296.
[32] R. Rosseto, N. Bibak, R. DeOcampo, T. Shah, A. Gabrielian, J. Hajdu, J.
Org. Chem. 2007, 72, 1691–1698.
Keywords: anti-inflammatory agents
walls · phosphatidyl inositol mannosides · PIM mimics
· mycobacterial cell
[33] N. Morisaki, K. Morita, A. Nishikawa, N. Nakatsu, Y. Fukui, Y. Hashimoto,
R. Shirai, Tetrahedron 2000, 56, 2603–2614.
[1] G. S. Besra, C. B. Morehouse, C. M. Rittner, C. J. Waechter, P. J. Brennan, J.
Biol. Chem. 1997, 272, 18460–18466.
[34] a) W. Bannwarth, A. Trzeciak, Helv. Chim. Acta 1987, 70, 175–186;
b) R. B. King, P. M. Sundaram, J. Org. Chem. 1984, 49, 1784–1789.
[2] M. Gilleron, J. Nigou, B. Cahuzac, G. Puzo, J. Mol. Biol. 1999, 285, 2147–
2160.
2092
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2081 – 2093

Phosphatidyl myo-Inositol Mannosides Mimics
[35] G. Guanti, L. Banfi, A. Basso, L. Bondanza, G. Guglieri, K. Powles, R. Riva,
Amino Acids 2010, 39, 367–373.
[39] a) J. A. Bennek, G. R. Gray, J. Org. Chem. 1987, 52, 892–897; b) A. Jeffery,
V. Nair, Tetrahedron Lett. 1995, 36, 3627–3630.
[36] a) B. B. Shankar, M. P. Kirkup, S. W. McCombie, A. K. Ganguly, Tetrahedron
Lett. 1993, 34, 7171–7174; b) T. Tite, M.-C. Lallemand, E. Poupon, N. Ku-
nesch, F. Tillequin, C. Gravier-Pelletier, Y. Le Merrer, H.-P. Husson, Bioorg.
Med. Chem. 2004, 12, 5091–5097.
[40] For related structures and technique, see: M. E. Haque, T. Kikuchi, K.
Yoshimoto, Y. Tsuda, Chem. Pharm. Bull. 1985, 33, 2243–2255.
[37] F. Oula¨ıdi, S. Front-Deschamps, E. Gallienne, E. Lesellier, K. Ikeda, N.
Asano, P. Compain, O. R. Martin, ChemMedChem 2011, 6, 353–361.
[38] S. Mandal, N. Sharma, B. Mukhopadhyay, Tetrahedron: Asymmetry 2010,
21, 2172–2176. See also J. N. Johnston, L. A. Paquette, Tetrahedron Lett.
1995, 36, 4341–4344.
Received: June 13, 2011
Published online on September 7, 2011
ChemMedChem 2011, 6, 2081 – 2093
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
2093