Phosphatidylinositol mannosides: Synthesis and suppression of allergic airway disease

Article abstract of DOI:10.1016/j.bmc.2006.04.037

Phosphatidylinositol mannoside (PIM) extracts from mycobacteria have been shown previously to suppress allergic airway inflammation in mice. To help determine the structural requirements for activity, PIM1₂ (1), PIM1₆ (2) and PIM2 (3

Full text of DOI:10.1016/j.bmc.2006.04.037

Bioorganic & Medicinal Chemistry 14 (2006) 5632–5642
Phosphatidylinositol mannosides: Synthesis and suppression
of allergic airway disease
Gary D. Ainge,a Jennifer Hudson,b David S. Larsen,c,* Gavin F. Painter,a
Gurmit Singh Gillc and Jacquie L. Harperb
aIndustrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand
bThe Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
cUniversity of Otago, PO Box 56, Dunedin, New Zealand
Received 1 March 2006; revised 9 April 2006; accepted 13 April 2006
Available online 11 May 2006
Abstract—Phosphatidylinositol mannoside (PIM) extracts from mycobacteria have been shown previously to suppress allergic
airway inflammation in mice. To help determine the structural requirements for activity, PIM12 (1), PIM16 (2) and PIM2 (3) were
synthesized and tested for their ability to suppress cellular inflammation in a mouse model of allergic asthma. The synthetic PIMs
were all effective in suppressing airway eosinophilia in the asthma model, with PIM16 being the most effective. Suppression of all
inflammatory cells monitored was observed, indicating a general blockade of cellular inflammation. Non-mannosylated phospha-
tidylinositol (PI) had no suppressive effect, indicating that at least one a-D-mannopyranosyl residue is necessary for activity. The
suppressive effect of the three PIM compounds indicates that other members of this set may be of value in treatment of a range
of diseases driven by infiltration of inflammatory cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
in the treatment of immune-mediated diseases ranging
from allergies to tuberculosis and cancer.12–14
Allergic asthma is increasing in prevalence and severity
worldwide.1 The specific reasons for these increases are
unclear, however a variety of contributing factors have
been proposed, including a lower incidence of exposure
to infectious agents in industrialised nations.2 It has
been postulated that exposure to various bacteria early
in childhood moderates the body’s response to allergens
and lowers the likelihood of the development of allergies
later in life.3–5 Mycobacteria have been identified as
organisms that act in this way6–8 by inducing complex
immune responses. These responses have been attribut-
ed to the diverse chemical features of the organism that
include unmethylated copies of DNA9 that are released,
component proteins such as tuberculin purified protein
derivative (PPD)10 and cell wall constituents.11 Many
studies have been undertaken to determine the potential
for exploiting mycobacteria and mycobacterial products
Members of our team have demonstrated that lipoarabi-
nomannan (LAM) and phosphatidylinositol mannan
(PIM), glycolipids isolated from the cell wall of
Mycobacterium bovis, can suppress the infiltration of
inflammatory cells into the lung in a mouse model
of allergic airway disease.15 The suppression by the
PIM-containing extracts was shown to be associated
with the production of the regulatory cytokine IL-10,
indicating that the extracts trigger an ‘anti-inflammato-
ry’ response in vivo. The suppressive effect was shown to
be dependent on the presence of the acyl chains and, in
the case of LAM, mannose capping on the glycolipids,
identifying these moieties as suitable targets for
structure–activity relationship studies.
The compositions of the LAM and PIM components
of the cell walls vary between different species of
Mycobacteria,16–20 the variability being observed in
both the structures of the fatty acid chains and the
number of mannose substituents in the PIM com-
pounds. Separation of the different PIM compounds
present in cell wall extracts can be technically chal-
lenging, however their relatively small molecular sizes
Keywords:
Phosphatidylinositol
mannans;
PIMs;
Asthma;
Inflammation.
*
Corresponding author. Tel.: +64 3 479 7816; fax: +64 4 4797906;
e-mail: dlarsen@alkali.otago.ac.nz
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.037

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
5633
HO
OH
O
HO
OCOC17H35
OH
O
OH
HO
R =
O
HO
P
HO
HO
O
O
OCOC17H35
O
HO
HO
HO
OH
O
HO
OR
HO
OR
HO
HO
O
OH
HO
OR
HO
OH
OH
O
OH
OH
OH
OH
O
OH
OH
PIM12 1
PIM16 2
PIM2 3
make them amenable to syntheses. To help determine
the structural requirements of PIM compounds for
anti-inflammatory responses in vivo, we have under-
taken the chemical syntheses of PIM compounds
PIM12 (1), PIM16 (2) and PIM2 (3) and tested them
for inhibitory properties in a mouse model of allergic
airway inflammation.
2. Results and discussion
2.1. Synthesis of PIM12 (1)
The known 4-methoxybenzyl ether 4 was made by
selective monoetherification of the previously described
tetrabenzyldiol by the procedures of Martin and
Wagman.29 Mannosylation of the C-2 hydroxyl group
of product 4 was achieved by reaction with mannosyl
phosphite 5,26,27 promoted by trimethylsilyl trifluorome-
thanesulfonate (TMSOTf), to give an inseparable
mixture of a-mannoside (6) and its b-anomer in 83%
combined yield. After removal of the p-methoxyben-
zyl-protecting group with ceric ammonium nitrate
(CAN), 2-O-mannosyl-myo-inositols a-7 and its b-ano-
mer were isolated in 51% and 15% yields, respectively,
and a mixed fraction containing these two glycosides
(21%, in similar proportions) was also obtained
The key difference between previously synthesized
PIM12 derivatives and the PIM12 now described lies in
the composition of the acyl group of the phosphatidyl
unit. Previous PIM12 derivatives have incorporated di-
O-palmitoyl21 or myristoyl22–24 acyl groups, whereas
we have synthesized a stearoyl PIM12 derivative, as
the stearoyl acyl group has been identified in active
PIM extracts.15 We also describe a versatile synthesis
of PIM16 and PIM2 via a chemoselective mannosylation
and dimannosylation of inositol acceptor 11, respective-
ly. Although various acylated-PIM2 compounds, man-
nosylated at O-2 and O-6, have been prepared by the
groups of Van Boom,25 Watanabe26,27 and Fraser-
Reid,28 to our knowledge this is the first report on the
synthesis of a PIM16 entity.
(Scheme 1). The specific optical rotation of product a-
18
D
7 {½aꢀ +15.3 (c 1.5, CH2Cl2)} compared well with that
reported by Elie et al. {½aꢀ +16 (c 1, CHCl3)},22 and 1H
D
and 13C NMR data were consistent with those reported
in the literature.22
BnO
OBn
O
OH
BnO
BnO
BnO
BnO
BnO
OBn
O
BnO
(i)
+
BnO
OPMB
BnO
O
OBn
BnO
OP(OMe)2
BnO
OR
BnO
OBn
4
5
6 R = PMB
RO
(ii)
OR
O
7 R = H
RO
RO
Et3NH+
+
-
R1
OCOC17H35
O
O
(iii)
RO
7
+
-
OCOC17H35
H
O
O
P
P
RO
O
O
RO
OCOC17H35
OR
O
O
OCOC17H35
8
9
R = Bn, R1 = Et3NH
(iv)
10 R = H, R1 = Et3NH
(salt of compound 1)
t
Scheme 1. Reagents: (i) TMSOTf, MS-4A, Et2O (83%); (ii) CAN, MeCN–H2O (9:1), (51%); (iii) BuCOCl, py, then I2 in py-H2O (88%); (iv) H2,
Pd/C, EtOAc–THF–EtOH–H2O (86%).

5634
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
The installation of the phospholipid moiety was
achieved using the H-phosphonate method30 which
involved coupling of alcohol a-7 with phosphonate 8
in the presence of pivaloyl chloride as an activator.
The disubstituted hydrogen phosphonate produced
was converted, by in situ oxidation with iodine in wet
pyridine, to the corresponding O-protected phosphate
diester of PIM12 which was isolated as the triethylam-
monium salt (9) in 88% yield (Scheme 1). Hydrogenoly-
sis of the benzyl-protecting groups over palladium gave
the triethylammonium salt (10) of PIM12 (1) as a white
solid after lyophilisation in 86% yield (Scheme 1). High
resolution mass spectrometry established the molecular
formula of the anion as C51H96O18P. The 1H NMR
spectrum showed all the relevant resonances with the
appropriate intensities. In particular, the sn-2 proton
was evident as a broad multiplet at d 5.13–5.06 ppm.
The anomeric proton signal resonated as a doublet
Route A. Selective mannosylation of diol 11, which
is readily accessible and has been used previously for
the synthesis of PIM derivatives,28 with the a-directing
2-O-acetyl mannosyl donor 1231,32 (molar ratio
1:1.35), gave 6-mannoside a-13 in 50% yield, along
with 17% of the dimannoside 20 (see Route B). The
positioning of the mannosylation in the major product
13 was assigned by O-benzoylation of the free hydrox-
yl to give compound 17 (65%). The expected down-
field shift (5.99 ppm) of the characteristic (br t,
J = 2.7 Hz) inositol H-2 proton was evident. Conse-
quently glycosylation of diol 11 occurred preferentially
at the equatorial C-6 hydroxyl group. The a-anomeric
configuration of glycoside 13 was established from the
C-1 heteronuclear one-bond 13C–1H coupling constant
of 175.5 Hz.33,34
Compound 13 was then transformed into alcohol 16 by
standard deacetylation, O-benzylation and deallylation
via compounds 14 and 15. Introduction of the glycero-
phosphate moiety and debenzylation, as illustrated in
Scheme 1, then led in sequence to the O-protected salt
18 and then the triethylammonium salt (19) of the
required product (2). High-resolution mass spectrome-
try established the molecular formula of the anion as
C51H96O18P and the NMR data were consistent with
the structure of 19. The sn-2 proton signal was observed
as a broad multiplet at d 5.10–5.02 and the anomeric
(J1 ,2 = 1.5 Hz) at d 4.96 ppm. The 13C NMR spectrum
was also consistent with the structure of salt 10, with the
main features being the carbonyl signals at d 173.7 and
174.0 ppm, the anomeric carbon signal at d 101.4 and
the three oxygenated methylene carbon resonances evi-
dent at d 63.5, 62.8 and 61.3 ppm. A singlet in the 31P
NMR spectrum was observed at d 0.08 ppm.
0
0
2.2. Synthesis of PIM16 (2)
0
0
The 6-substituted inositol derivative PIM16 (2) was
synthesized using the strategy shown in Scheme 2,
proton signal resonated as a doublet (J1 ,2 = 2.0 Hz) at
d 4.91.
+
R1
OR
RO
OR
BnO
-
OCOC17H35
OCOC17H35
O
P
RO
BnO
Route A
O
OR1
O
RO
BnO
O
O
(i)
O
BnO
BnO
OAc
O
OR
OR
OBn
OBn
O
O
BnO
OBn
OR2
OR
OR
OC(NH)CCl3
12 (1.35 equiv.)
13
14
15
16
17
R = Bn, R1 = Et3NH 18
R = H, R1 = Allyl, R2 = Ac
R = R2 = H, R1 = Allyl,
R = R2 = Bn, R1 = Allyl,
R = R2 = Bn, R1 = H
(ii)
(iii)
(iv)
(vii)
19
R = H, R1 = Et3NH
(vi)
+
(v)
(salt of acid 2)
OH
BnO
BnO
R = Bz, R = Allyl, R = Ac,
OAll
1
2
BnO
OH
11
BnO
RO
+
OR
O
OR
O
BnO
BnO
BnO
BnO
RO
OAc
O
RO
(i)
+
BnO
R1
O
O
BnO
RO
-
OCOC17H35
OC(NH)CCl3
O
P
BnO
RO
OR1
O
O
BnO
RO
12 (2.71 equiv.)
O
O
O
OCOC17H35
OBn
OBn
OR
OR
O
O
Route B
OBn
OR
OR
OR
(vi)
R = Ac, R1 = All 20
24
25
(ii)
(iii)
(iv)
R = Bn, R1 = NH4
R = H, R1 = Na
(salt of acid 3)
(viii)
21
22
23
R = H, R1 = All
R = Bn, R1 =All
R = Bn, R1 = H
Scheme 2. Reagents: (i) TMSOTf, MS-4A, DCM (50% for 13 and 57% for 20); (ii) NaOMe, MeOH (95% for 14 and 72% for 21); (iii) BnBr, NaH,
DMF (100% for 15 and 79% for 22); (iv) (Ph2MeP)2(COD)Ir+ PF6ꢁ (77% for 16 and 76% for 22); (v) Py, BzCl (65%); (vi) 8, tBuCOCl, py, then I2 in
t
py-H2O (84% for 18 and 74% for 24); (vii) H2, Pd/C, EtOAc–THF–EtOH–H2O (72%); (viii) H2, Pd(OH)2/C, BuOH (37%).

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
5635
2.3. Synthesis of PIM2 (3)
asthma mouse model.15 Eosinophils are a key inflamma-
tory cell associated with inflammatory diseases and
tissue damage including airways remodelling in asthmat-
ics. In the current study, intranasal administration of the
triethylammonium salts of PIM12 (10) and PIM16 (19)
and the sodium salt of PIM2 (25) resulted in a decrease
in the number of eosinophils per unit volume in the
bronchoalveolar lavage (BAL) fluid compared with the
phosphate-buffered saline (PBS) controls (Fig. 1A–C),
with PIM16 appearing to cause the greatest suppressive
effect.
The inositol acceptor 11 was also used for the synthesis
of PIM2 (25) by dimannosylation with excess donor 12
(2.71 mol equiv) (Scheme 2, Route B). By this method
the main product from the glycosylation reaction was
the dimannoside 20 (57%). The a-anomeric configura-
tions for compound 20 were established from the C-1
one-bond 13C–1H coupling constants. Dimannoside 20
was readily converted to the known25 alcohol 23. The
diacyl glycerol moiety was installed as before by use of
compound 8, and the final deprotection was effected
by hydrogenolysis over Pd(OH)2 on carbon at 150 psi
to afford the sodium salt of PIM2 (25). High-resolution
mass spectrometry established the molecular formula of
the anion C57H106O23P. The 1H NMR spectrum showed
the appropriate intensities with all the expected signals
present. In particular, the sn-2 proton was evident at d
5.3 ppm as were the two anomeric protons at d 5.14
and 5.11 ppm. The 13C NMR was also consistent, with
the main observations being: the carbonyl signals at d
174.8 and 174.5 ppm, the two anomeric signals at d
101.92 and 101.89 ppm and the four oxygenated methy-
lene carbons evident at d 63.9, 63.3, 61.7 and 61.5 ppm.
The 31P NMR displayed a single peak at d ꢁ3.8 ppm.
Interestingly, there was also a general decrease in both
macrophage and lymphocyte cell numbers in the BAL
fluid. In the case of all three cell types, eosinophils, mac-
rophages and lymphocytes, the percentage of each cell
type in the BAL fluid did not change significantly
(Fig. 2A–C). It therefore appears that, like the PIM
extracts from mycobacteria,15 the suppression of lung
eosinophilia by the PIM compounds results from overall
suppression of cell infiltration rather than a blockade of
a specific cell type.
Previous work by Sayers et al.15 showed that the pres-
ence of the acyl chains and mannose capping was
important for suppressive activity of the larger lipogly-
can extract lipoarabinomannan (LAM). LAM isolated
from Mycobacterium smegmatis, which lacks mannose
caps, failed to suppress airway eosinophilia. Because
PIM extracted from M. smegmatis contains the
D-mannopyranosyl residues, we chose to test
2.4. Suppression of airway eosinophilia
In earlier research, members of our group have shown
that PIMs isolated from the cell wall of M. bovis signif-
icantly suppress the recruitment of eosinophils in an
Figure 1. Effects of intranasal administration of PIMs on cell infiltration in a mouse model of allergic airways disease (AAD). Cells per mL in the
BAL of ovalbumin-(OVA) challenged mice treated with (A) PIM12, (B) PIM16, (C) PIM2 or (D), PI. The bar-graphs each represent results of 2–4
experiments each comprising five mice.

5636
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
Figure 2. Percentages of different cell types in the BAL fluid of OVA-challenged mice following intranasal administration of (A) PIM12, (B), PIM16,
(C) PIM2 or (D) PI. Graphs are representative of 2–4 experiments comprising five mice per group.
1,2-diacyl-sn-glycero-3-phospho-(1-D-myo-inositol) (PI,
Aldrich P-5954) in the ovalbumin (OVA) model to
determine if the activity of PIMs was dependent on
the presence of mannose. As shown in Figures 1D
and 2D, PI had no effect on either cell infiltration
or cell percentages, respectively, indicating a key role
for the mannose sugars in the activity of the synthetic
PIM compounds.
4. Experimental procedures for synthesis of PIM
analogues
4.1. General
Mps were measured on a Gallenkamp capillary melting
point apparatus and are uncorrected. Specific optical
rotations, given in 10ꢁ1 deg cm2 gꢁ1, were measured at
ambient temperature using either a Jasco DIP-370 or
DIP-1000 polarimeter with a cell of path length
1
3. Conclusions
1.0 dm. H NMR spectra were obtained at either 300
or 500 MHz and referenced to the residual solvent peak.
Chemical shifts are reported using the d scale, and cou-
pling constants (J) and separations are reported in Hz.
13C NMR spectra were recorded at 75 or 125 MHz
and chemical shifts are reported using the d scale.
FAB mass spectra were recorded on a Kratos MSORF
or a VG70-250S double focusing, magnetic sector mass
spectrometer under chemical ionization conditions using
isobutene, ammonia or xenon as the ionizing gas, or un-
der high-resolution FAB conditions in a glycerol or
nitrobenzyl alcohol matrix. Electro-spray ionization
(ESI) mass spectra were recorded on a PerSeptive
Biosystems Mariner time-of-flight mass spectrometer.
Elemental analyses were carried out at the Campbell
Microanalytical Laboratory, University of Otago, Dun-
edin, New Zealand. Thin-layer chromatography (TLC)
was performed on Merck silica gel DC Alurolle Kiesel-
gel 60F254 plates and were visualized under UV light
and/or with a spray (5% w/v dodecamolybdophosphoric
acid in EtOH) with subsequent heating. Flash column
We have prepared samples of PIM12 (10), PIM16 (19)
and PIM2 (25), and evaluated their ability to suppress
eosinophil recruitment in the lung in an asthma mouse
model. The profile of cell type in the BAL fluid sug-
gests that this activity results from an overall suppres-
sion of cell infiltration. All three PIMs, 10, 19 and 25,
possessed this suppressive activity with PIM16 (19)
showing the greatest effect. These results suggest that
only one a-D-mannopyranosyl residue is required for
the observed activity and that the mannosylation site
can be at either O-2 or O-6 of the inositol moiety.
In addition we have shown that the activity of PIM
appears to be dependent on the presence of mannose,
however, at this point it is unclear as to whether the
type of sugar group is important. We are currently
preparing analogues of PIM structures to ascertain
the minimal structural requirements for the suppres-
sion of recruitment of eosinophils by PIM-based
compounds.

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
5637
18
D
20
D
chromatography was carried out using Merck Kieselgel
60 (230–400 mesh). All chromatography solvents were
of reagent grade. THF was distilled from sodium-benzo-
phenone ketyl under nitrogen and dichloromethane was
distilled from phosphorus pentoxide. All other solvents
and reagents were purified using the methods described
by Perrin et al.35 Organic solutions were dried over
MgSO4.
Data for a-7 ½aꢀ +15.3 (c 1.5, CH2Cl2), lit.22 ½aꢀ +16 (c
1, CHCl3). 1H NMR (500 MHz, CDCl3) d 7.40–7.16 (m,
40H), 5.37 (br s, 1H), 4.95 (d, J = 11.0 Hz, 2H), 4.93 (d,
J = 11.0 Hz, 1H), 4.87 (d, J = 11.0 Hz, 1H), 4.79 (d,
J = 11.0 Hz, 2H), 4.78 (d, J = 12.5 Hz, 1H), 4.71–4.47
(m, 8H), 4.36 (d, J = 12.0 Hz, 1H), 4.33 (br t,
J = 2.0 Hz, 1H), 4.11–4.09 (m, 2H), 3.88–3.85 (m, 1H),
3.79 (t, J = 9.5 Hz, 1H), 3.66–3.64 (m, 1H), 3.57 (dd,
J = 11, 2.5 Hz, 1H), 3.53 (t, J = 9.5 Hz, 1H), 3.44–3.34
(m, 4H). 13C NMR (125 MHz, CDCl3) d 138.8,
138.68, 138.65, 138.55, 138.52, 138.45, 138.2, 128.8,
128.53, 128.50, 128.4, 128.36, 128.32, 128.25, 128.23,
128.19, 128.16, 128.08, 128.07, 128.05, 128.03, 127.99,
127.96, 127.8, 127.64, 127.60, 127.57, 127.48, 127.42,
127.41, 127.3, 98.9, 83.7, 81.8, 80.9, 79.2, 78.9, 75.9,
75.8, 75.4, 75.2, 73.50, 73.47, 72.37, 72.26, 71.96,
71.90, 71.7, 68.9. HRMS-ESI (M+H)+ calcd for
C68H71O11: 1063.4996. Found: 1063.5017.
4.2. 3,4,5,6-Tetra-O-benzyl-2-O-(2,3,4,6-tetra-O-benzyl-
D-mannopyranosyl)-1-O-p-methoxybenzyl-D-myo-inositol
(6)
3,4,5,6-Tetra-O-benzyl-1-O-p-methoxybenzyl-D-myo-
inositol (4) (200 mg, 0.30 mmol) and mannosyl phos-
phite (5)26,27 (200 mg, 0.32 mmol) were dissolved in dry
˚
ether (10 mL) and molecular sieves 4 A (100 mg) were
added. The mixture was stirred at room temperature
under nitrogen for 15 min, TMSOTf (10 lL,
0.06 mmol) was added and the stirring was continued
for a further 30 min. Et3N (1.0 mL) was added, the
mixture was diluted with more ether, washed with
water and dried. Removal of the solvent and purifica-
tion of the residue by chromatography over silica (hex-
ane/ether, 8:2) gave the title compound 6 (296 mg, 83%,
undetermined mixture of anomers) as a colourless syr-
19
D
Data for b-7: ½aꢀ ꢁ2.1 (c 1.0, CH2Cl2). 1H NMR
(500 MHz, CDCl3) d 7.47–7.18 (m, 40H), 4.94–4.82
(m, 9H), 4.78 (d, J = 11.5 Hz, 1H), 4.67 (d,
J = 11.0 Hz, 1H), 4.64–4.51 (m, 5H), 4.45 (d,
J = 11.5 Hz, 1H), 4.41 (s, 1H), 4.11 (t, J = 2.5 Hz, 1H),
4.04 (d, J = 3.0 Hz, 1H), 3.91–3.85 (m, 2H), 3.79 (dd,
J = 10.5, 2.0 Hz, 1H), 3.78–3.68 (m, 2H), 3.53 (dd,
J = 10.0, 2.0 Hz, 1H), 3.50–3.42 (m, 3H). 13C NMR
(125 MHz, CDCl3) d 139.1, 139.0, 138.8, 138.6, 138.3,
138.22, 138.19, 138.12, 128.6, 128.5, 128.45, 128.43,
128.39, 128.29, 128.19, 128.17, 128.07, 128.04, 127.87,
127.71, 127.66, 127.60, 102.6, 83.4, 83.1, 82.4, 81.6,
80.9, 80.1, 75.9, 75.8, 75.7, 75.3, 74.6, 74.3, 74.1, 73.6,
73.2, 72.0, 71.6, 69.4. MS-ESI (M+H)+ calcd for
C68H71O11: 1063.4996. Found: 1063.4983.
1
up. H NMR (500 MHz, CDCl3) d 7.36–7.13 (m, 42H),
6.77 (d, J = 8.5 Hz, 2H), 5.43 (d, J = 1.5 Hz, 1H), 4.92–
4.76 (m, 8H), 4.62–4.40 (m, 9H), 4.36 (d, J = 12.5 Hz,
1H), 4.13 (br d, J = 10.0 Hz, 1H), 4.07 (t, J = 9.5 Hz,
1H), 3.84 (dd, J = 10.0, 2.5 Hz, 1H), 3.78 (t,
J = 9.5 Hz, 1H), 3.74–3.69 (m, 3H), 3.71 (s, 3H), 3.54
(dd, J = 11.5, 3.5 Hz, 1H), 3.42 (t, J = 9.5 Hz, 1H),
3.35 (t, J = 9.5 Hz, 1H), 3.30 (dd, J = 9.0, 2.5 Hz,
1H). 13C NMR (125 MHz, CDCl3) d 159.4, 138.9,
138.8, 138.6, 138.5, 138.4, 138.3, 130.0, 129.8, 128.47,
128.45, 128.42, 128.39, 128.35, 128.28, 128.26, 128.16,
128.12, 128.09, 128.04, 127.98, 127.94, 127.68, 127.57,
127.50, 127.44, 127.38, 127.28, 113.9, 98.4, 83.5, 81.5,
81.2, 80.9, 79.2, 78.9, 76.2, 75.8, 75.1, 74.8, 74.4,
73.5, 73.2, 72.1, 72.0, 71.8, 71.7, 68.9, 55.3. MS-ESI
(M+H)+ calcd for C76H79O12: 1183.5572. Found:
1183.5564.
4.4. Triethylammonium 3,4,5,6-tetra-O-benzyl-2-O-
(2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl)-1-O-(1,2-
di-O-stearoyl-sn-glycero-3-phosphoryl)-D-myo-inositol (9)
Mannopyranosyl-inositol a-(7) (40 mg, 0.038 mmol) and
H-phosphonate 830 (56 mg, 0.76 mmol) were mixed,
dried by co-evaporation from dry pyridine (2· 10 mL)
and dissolved in dry pyridine (4 mL) under nitrogen.
Pivaloyl chloride (50 lL, 0.041 mmol) was added and
the mixture was stirred for 30 min at 20 ꢁC. A freshly
prepared solution of iodine (33 mg) in pyridine–H2O
(9:1, 20 mL) was added and the mixture was stirred
for a further 30 min. The solution was diluted with
CHCl3 (20 mL), stirred for 15 min and then washed with
Na2S2O3 solution (10%, 20 mL) and H2O. The solvents
were removed at 50 ꢁC and the residue was dissolved in
CH2Cl2 (20 mL), washed with triethylammonium bicar-
bonate buffer (1 M, 2· 20 mL) and with H2O (100 mL),
and dried. The solvent was removed and the residue was
purified by chromatography over silica gel (CH2Cl2/
4.3. 3,4,5,6-Tetra-O-benzyl-1-O-(2,3,4,6-tetra-O-benzyl-
a-D-mannopyranosyl)-D-myo-inositol a-(7) and the
anomer b-(7)
Ceric ammonium nitrate (200 mg, 0.37 mmol) was
added to
a solution of compound 6 (200 mg,
0.17 mmol) in MeCN–H2O (9:1, 5 mL) at 0 ꢁC. The
mixture was stirred at 0 ꢁC for 5 min and then at
20 ꢁC for 45 min. NaHCO3 solution (satd 10 mL)
was added and the mixture was diluted with CH2Cl2.
The organic layer was washed with water and dried
(MgSO4). Removal of the solvent and purification of
the residue by chromatography on silica gel (hex-
anes/ether 8:2 to 6:4) gave the title compound a-7
(91 mg, 51%) as a colourless syrup. A mixed fraction
of a-7 and b-7 (37 mg, 21%) and a further fraction
containing 3,4,5,6-tetra-O-benzyl-2-O-(tetra-O-benzyl-
b-D-mannopyranosyl)-D-myo-inositol b-(7) (27 mg,
15%) were obtained as colourless syrups.
MeOH/Et3N, 98:1:1) to give the title compound 9
18
(61 mg, 88%) as colourless glass. ½aꢀ +17.0 (c 1.0,
D
CH2Cl2). 1H NMR (500 MHz, CDCl3) d 7.53 (dd,
J = 6.5 Hz, 2H), 7.42 (dd, J = 6.5 Hz, 2H), 7.33–7.17
(m, 36H), 5.74 (d, J = 3.0 Hz, 1H), 5.18–5.08 (m, 1H),
4.99 (dd, J = 11.5, 4.0 Hz, 1H), 4.92–4.79 (m, 10H),
4.73 (d, J = 12.0 Hz, 1H), 4.60–4.52 (m, 4H), 4.39 (dd,
J = 12.0, 5.0 Hz, 1H), 4.27 (d, J = 12.0 Hz, 1H), 4.20

5638
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
(br d, J = 10.5 Hz, 1H), 4.15–4.04 (m, 3H), 4.03–3.86
(m, 5H), 3.80 (t, J = 9.5 Hz, 1H), 3.75 (t, J = 9.5 Hz,
1H), 3.50 (dd, J = 9.5, 3.0 Hz, 1H), 3.48–3.44 (m, 2H),
3.16 (dd, J = 9.5, 5.5 Hz, 1H), 2.97 and 2.96 (2 · q,
J = 7.5 Hz, 6H), 2.20 and 2.17 (2 · t, J = 8.0 Hz, 4H),
1.59–1.47 (m, 4H), 1.32–1.19 (m, 65H), 0.88 (t,
J = 7.0 Hz, 6H). 13C NMR (125 MHz, CDCl3) d 173.3,
172.91, 172.89, 139.12, 139.10, 139.0, 138.8, 138.7,
138.61, 138.57,128.39, 128.36, 128.35, 128.26, 128.25,
128.22, 128.17, 128.11, 128.09, 128.08, 128.01, 127.67,
127.64, 127.59, 127.55, 127.54, 127.52, 127.47, 127.38,
127.34, 127.17, 127.0, 98.0, 83.3, 81.1, 80.9, 80.8, 79.4,
79.3, 76.2, 75.7, 75.5, 75.1, 74.7, 74.0, 73.2, 72.1, 71.5,
71.4, 70.6, 70.5, 70.4, 69.1, 63.8, 63.64, 63.60, 62.81,
62.78, 45.7, 34.32, 34.1, 32.0, 29.8, 29.73, 29.71, 29.59,
29.58, 29.41, 29.38, 29.2, 27.4, 27.3, 24.94, 24.91, 22.7,
14.2, 8.5. 31P NMR (202 MHz, CDCl3) d 0.0, +0.01.
MS-ESI (M-NHEt3)ꢁ calcd for C107H144O18P:
1748.0090. Found: 1748.0118.
inositol (11) (226 mg, 0.461 mmol), 2-O-acetyl-3,4,6-tri-
O-benzyl-a-D-mannopyranosyl trichloroacetimidate
˚
(12) (399 mg, 0.626 mmol) and 4 A molecular sieves
(120 mg) in CH2Cl2 (15 mL) at ꢁ40 ꢁC. The reaction
mixture was allowed to warm to 0 ꢁC over 70 min when
Et3N (2.5 mL) was added. The mixture was filtered
through Celite and the filter cake washed with further
CH2Cl2 (2· 100 mL). The solvent was removed and
the residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:9 to 3:7)
afforded the dimannosylated inositol derivative 20
(110 mg, 0.076 mmol, 17%) followed by the title com-
20
D
pound 13 (222 mg, 0.230 mmol, 50%) as an oil. ½aꢀ
1
+11.0 (c 5.48, CHCl3). H NMR (300 MHz, CDCl3) d
7.36–7.01 (m, 30H), 5.97–5.84 (m, 1H), 5.45–5.40 (m,
2H), 5.25–5.15 (m, 2H), 4.90–4.52 (m, 10H), 4.41 (d,
J = 11.0 Hz, 1H), 4.25 (d, J = 12.0 Hz, 1H), 4.18–3.90
(m, 8H), 3.40–3.20 (m, 5H), 2.39 (s, 1H), 2.10 (s, 3H).
13C NMR (75 MHz, CDCl3) d 170.8, 139.4, 139.0,
138.9, 138.6, 138.5, 138.3, 134.7, 128.9, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 118.5, 98.6, 81.8, 81.6, 81.0, 80.2, 78.5, 76.3,
76.1, 75.7, 75.3, 74.7, 73.7, 73.1, 72.1, 71.7, 71.6, 69.3,
4.5. Triethylammonium 2-O-(a-D-mannopyranosyl)-1-O-
(1,2-di-O-stearoyl-sn-glycero-3-phosphoryl)-D-myo-inosi-
tol (10)
68.7, 67.1, 21.5. Gated decoupled 13C NMR (75 MHz,
CDCl3) selected data, d 98.6, JC1 -H1 175.5 Hz. MS-
ESI (M+NH4)+ calcd for C59H68NO12: 982.4736.
Found: 982.4726. Anal. (C59H64O12) C, H.
1
0
0
A mixture of protected PIM12 9 (55 mg, 0.03 mmol) and
Pd/C (10%, 100 mg) in a mixture of EtOAc/THF/EtOH/
H2O (2:1:1:1, 20 mL) was stirred at 20 ꢁC under hydrogen
for 16 h. The mixture was filtered through a pad of Celite,
the pad was washed with MeOH–H2O (1:1, 10 mL), THF
(5 mL) and CH2Cl2 (10 mL). The solvent was removed
from the combined filtrate and washings, toluene was
addedandremoved. The residue was applied toa prepara-
tive silica gel plate (20 cm · 20 cm) and eluted with
CH2Cl2/MeOH/Et3N (90:8:2). The silica near the baseline
was removed and washed with warm MeOH (30 mL) and
then CH2Cl2 (30 mL). The solvent was removed and the
product was lyophilised from dioxane–water (2:3) to give
the title compound 10 (29 mg, 86%) as a white solid. 1H
NMR (500 MHz, CDCl3/CD3OD/D2O, 35:20:3) d 5.13–
5.06 (m, 1H), 4.96 (d, J = 1.5 Hz, 1H), 4.29–4.23 (m,
1H), 4.20–3.82 (m, 6H), 4.82–3.78 (m, 1H), 3.72 (br t,
J = 8.5 Hz, 1H), 3.65 (dd, J = 10.0, 2.5 Hz, 1H), 3.61–
3.53 (m, 2H), 3.48–3.42 (m, 2H), 3.31 (dd, J = 10.0,
2.5 Hz, 1H), 3.10 (t, J = 9.5 Hz, 1H), 2.95 (q,
J = 7.0 Hz, 6H), 2.17 and 2.14 (2t, J = 8.0 Hz, 6H),
1.48–1.38 (m, 4H), 1.75–1.07 (m, 65H), 0.73 (t, J = 8 Hz,
6H). 13C NMR (125 MHz, CDCl3/CD3OD/D2O
4.7. 1-O-Allyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-2-O-benzoyl-3,4,5-tri-O-benzyl-D-myo-
inositol (17)
Benzoyl chloride (100 lL, 0.861 mmol) was added drop-
wise to a stirred solution of alcohol 13 (37 mg,
0.040 mmol) in pyridine (10 mL). After being stirred for
8 h, the mixture was diluted with H2O (50 mL) and
extracted with ether (2· 50 mL). The combined extracts
were washed with HCl (0.5 M, 70 mL) and NaHCO3 solu-
tion (satd, 50 mL). After drying and filtration, the solvent
was removed and the residue purified by column chroma-
tography on silica gel. Elution with EtOAc/light petro-
leum (3:7 to 2:3) afforded the title compound 17 as an
oil (28 mg, 0.026 mmol, 65%). ½aꢀ22 +34 (c 0.48, CH2Cl2).
D
1H NMR (300 MHz, CDCl3) d 8.06–8.00 (m, 2H), 7.59–
7.52 (m, 1H), 7.48–7.40 (m, 2H), 7.35–7.01 (m, 30H), 5.99
(t, J = 2.7 Hz, 1H), 5.97–5.81 (m, 1H), 5.50–5.41 (m, 2H),
5.25–5.13 (m, 2H), 4.95–4.55 (m, 10H), 4.42 (d,
J = 10.9 Hz, 1H), 4.25 (d, J = 12.0 Hz, 1H), 4.19–3.90
(m, 7H), 3.58 (dd, J = 12.5, 2.8 Hz, 1H), 3.48–3.31 (m,
4H), 2.07 (s, 3H). 13C NMR (75 MHz, CDCl3) d 170.6,
166.3, 139.3, 138.9, 138.5, 138.2, 138.0, 134.6, 133.5,
130.3, 128.8, 128.7, 128.6, 128.4, 128.2, 128.1, 128.0,
127.9(2), 127.8(6), 127.7(5), 127.6(9), 118.4, 98.7, 82.2,
81.5, 79.0, 78.8, 78.4, 76.7, 76.3, 76.2, 75.4, 74.7, 73.6,
72.5, 72.0, 71.7, 71.3, 69.1, 68.8, 67.1, 21.4. MS-ESI
(M+NH4)+ calcd for C66H72NO13: 1086.4998. Found:
1086.4952.
6
35:20:3) d 174.0 (CO, d, JP–C 2.5 Hz), 173.7 (CO, d,
5JP–C 5 Hz) 101.4, 78.5, 76.34, 76.29, 74.5, 72.7, 72.3,
71.9, 70.5, 70.44, 70.37, 70.33, 70.1, 67.0, 63.5 (d,
2JC,P = 0.5 Hz), 62.8, 61.3, 49.0, 48.8, 48.7, 48.6, 48.4,
48.2, 48.1, 47.9, 46.1, 34.04, 34.02, 33.9, 31.7, 29.5, 29.4,
29.4, 29.3, 29.2, 29.1, 28.94, 28.92, 24.7, 24.61, 22.4,
13.7, 8.3. 31P NMR (202 MHz, CDCl3/CD3OD/D2O
35:20:3)
d
0.08. MS-ESI (M-NHEt3)ꢁ calcd for
C51H96O18P: 1027.6334. Found: 1027.6330.
4.6. 1-O-Allyl-6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-3,4,5-tri-O-benzyl-6-O-D-myo-inositol
(13)
4.8. 1-O-Allyl-3,4,5-tri-O-benzyl-6-O-(3,4,6-tri-O-benzyl-
a-D-mannopyranosyl)-D-myo-inositol (14)
TMSOTf (25 lL, 0.14 mmol) was added dropwise to a
stirred mixture of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-
Sodium methoxide in MeOH (30%, ca 0.05 mL) was
added dropwise to a stirred solution of acetate 13

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
5639
(255 mg, 0.264 mmol) in MeOH (12 mL). After being
stirred for 3 h, the reaction mixture was diluted with
NH4Cl solution (satd 50 mL). The aqueous phase was
extracted with CHCl3 (3· 40 mL) and the combined
organic extracts were washed with H2O (100 ml). After
drying and filtration the solvent was removed to afford
the title compound 14 as an oil (232 mg, 0.251 mmol,
(200 mg, 2.38 mmol) was added. The mixture was stirred
for an additional 5 min when H2O (50 mL) was added.
This mixture was extracted with CHCl3 (2· 60 mL)
and after drying and filtration the solvent was removed
and the residue purified by column chromatography on
silica gel. Elution with EtOAc/light petroleum (1:4 to
3:7) afforded the title compound 16 as an oil (192 mg,
20
1
1
95%). ½aꢀ +30.5 (c 4.64, CHCl3). H NMR(300 MHz,
0.181 mmol, 77%). ½aꢀ +25.6 (c 3.84, CHCl3); 1H
D
D
CDCl3) d 7.37–7.04 (m, 30H), 5.96–5.82 (m, 1H), 5.46
(s, 1H), 5.30–5.18 (m, 2H), 4.96–4.61 (m, 9H), 4.55 (d,
J = 12.1 Hz, 1H), 4.43 (d, J = 11.1 Hz, 1H), 4.25 (d,
J = 12.1 Hz, 1H), 4.19–3.81 (m, 9H), 3.41–3.21 (m,
5H), 2.42 (br s, 2H). 13C NMR (75 MHz, CDCl3) d
139.3, 139.0, 138.9, 138.6, 138.3, 134.5, 128.9, 128.8,
128.7, 128.5, 128.3, 128.2, 128.0, 127.7, 118.4, 100.3,
81.8, 81.7, 81.0, 80.5, 80.2, 76.3, 76.1, 76.0, 75.2, 74.7,
73.7, 73.1, 72.3, 71.4, 69.1, 68.8, 67.1, 21.5. MS-ESI
(M+NH4)+ calcd for C57H66NO11: 940.4630. Found:
940.4637.
NMR (500 MHz, CDCl3) d 7.38–7.06 (m, 40H), 5.56
(d, J = 2.0 Hz, 1H), 5.07 (d, J = 11.5 Hz, 1H), 4.90 (d,
J = 11.0 Hz, 1H), 4.85 (d, J = 11.0 Hz, 1H), 4.80 (d,
J = 10.5 Hz, 1H), 4.77 (d, J = 10.0 Hz, 1H), 4.74–4.58
(m, 6H), 4.55 (s, 2H), 4.45 (d, J = 11.0 Hz, 1H), 4.30
(d, J = 11.0 Hz, 1H), 4.05–3.80 (m, 7H), 3.49 (dd,
J = 11.5, 4.5 Hz, 1H), 3.46–3.42 (m, 3H), 3.27 (t,
J = 9.5 Hz, 1H), 2.34 (d, J = 10.1 Hz, 1H). 13C NMR
(125 MHz, CDCl3) d 139.0, 138.69, 138.65, 138.53,
138.51, 138.4, 138.3, 138.2, 128.7, 128.6, 128.55, 128.4,
128.30, 128.28, 128.23, 128.20, 128.16, 128.03, 128.00,
127.99, 127.90, 127.85, 127.75, 127.70, 127.6, 127.51,
127.46, 127.39, 127.35, 127.31, 97.3, 81.6, 81.3, 81.2,
79.6, 78.4, 78.1, 75.9, 75.8, 74.90, 74.87, 74.81, 74.6,
73.4, 73.2, 73.1, 72.1, 71.79, 71.76, 68.9. MS-ESI
(M+NH4)+ calcd for C68H74NO11: 1080.5256. Found:
1080.5197.
4.9. 1-O-Allyl-2,3,4,5-tetra-O-benzyl-6-O-(2,3,4,6-tetra-
O-benzyl-a-D-mannopyranosyl)-D-myo-inositol (15)
Sodium hydride (60% dispersion in mineral oil, 40.0 mg,
1.00 mmol) was added to a stirred solution of diol 14
(232 mg, 0.251 mmol) and benzyl bromide (100 lL,
0.840 mmol) in DMF (8 mL) cooled to 0 ꢁC. After
20 min, the ice bath was removed and the reaction mix-
ture stirred at 20 ꢁC for 3 h after which NH4Cl solution
(50 mL, satd) was added. The mixture was extracted
with ether (2· 50 mL) and the combined ethereal ex-
tracts were washed with H2O (2 · 50 mL). After drying
and filtration, the solvent was removed and the residue
purified by column chromatography on silica gel. Elu-
tion with EtOAc/light petroleum (1:9 to 1:4) afforded
4.11. Triethylammonium 3,4,5,6-tetra-O-benzyl-6-O-
(2,3,4,6-tetra-O-benzyl-a-D-mannopyranosyl)-1-O-(1,2-
di-O-stearoyl-sn-glycero-3-phosphoryl)-D-myo-inositol
(18)
A mixture of protected inositol 16 (30 mg, 0.03 mmol)
and phosphonate salt 8 (50 mg, 0.06 mmol) was dried
by co-evaporation from dry pyridine (2· 10 mL) and
dissolved in the same solvent (4 mL) under nitrogen.
Pivolyl chloride (50 lL, 0.04 mmol) was added and the
mixture stirred at 20 ꢁC for 30 min. A freshly prepared
solution of iodine (30 mg, 0.12 mmol) in pyridine–H2O
(9:1, 20 mL) was added and stirring was continued for
30 min. The solution was diluted with CHCl3 (20 mL)
and stirred for 15 min then washed with Na2S2O3 solu-
tion (10%, 20 mL) and H2O. After drying, the solvent
was removed and the residue was dissolved in CH2Cl2
(20 mL), washed with triethylammonium bicarbonate
(1 M, 2· 20 mL) and with H2O (100 mL). The solvent
was removed and the residue was purified by chroma-
tography over silica gel (CH2Cl2/MeOH/Et3N, 98:1:1)
the title compound 15 as an oil (278 mg, 0.252 mmol,
+17.6 (c 5.56, CHCl3). 1H NMR
22
D
100%). ½aꢀ
(300 MHz, CDCl3) d 7.42–7.03 (m, 40H), 5.82–5.69
(m, 1H), 5.57 (s, 1H), 5.27–5.10 (m, 2H), 4.93–4.57 (m,
14H), 4.45 (d, J = 11.0 Hz, 1H), 4.27–3.77 (m, 10H),
3.41–3.12 (m, 5H). 13C NMR d 139.6, 139.3, 139.2,
139.1, 138.9, 138.7, 134.9, 128.8, 128.7, 128.6(3),
128.5(9) 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9(2), 127.8(5), 127.7(7), 127.6(7), 127.5(8), 117.5,
98.8, 82.4, 82.3, 81.8, 81.4, 80.5, 76.3, 76.2, 76.0, 75.8,
75.2, 74.4, 73.6, 73.3, 73.2, 72.5, 72.4, 72.2, 71.0, 69.1,
68.8, 67.1, 21.5. MS-ESI (M+NH4)+ calcd for
C71H78NO11: 1120.5569. Found: 1120.5628.
to give the title phosphate 18 (44 mg, 84%) as a colour-
20
D
less glass. ½aꢀ +30.6 (c 1, CH2Cl2). 1H NMR (500 MHz,
4.10. 2,3,4,5-Tetra-O-benzyl-6-O-(2,3,4,6-tetra-O-ben-
zyl-a-D-mannopyranosyl)-D-myo-inositol (16)
CDCl3) d 7.50–6.98 (m, 40H), 5.78 (d, J = 2.0 Hz, 1H),
5.28-5.19 (m, 1H), 5.02 (d, J = 11.5 Hz, 1H), 4.91–4.54
(m, 16H), 4.48 (d, J = 11.0 Hz, 1H), 4.54–4.39 (m,
1H), 4.30–4.23 (m, 1H), 4.20–3.94 (m, 8H), 3.55–3.49
(m, 2H), 3.41 (dd, J = 11.5, 1.5 Hz, 1H), 3.38–3.33 (m,
1H), 2.72 (q, J = 7.5 Hz, 6H), 2.25–2.17 (m, 4H), 1.57–
1.50 (m, 4H), 1.29–1.20 (m, 56H), 1.09 (t, J = 7.5 Hz,
9H), 0.88 (t, J = 7.5 Hz, 6H). 13C NMR (125 MHz,
CDCl3) d 173.4, 173.0, 139.8, 139.7, 139.58, 139.55,
139.3, 138.9, 138.8, 138.6, 138.3, 128.33, 128.30,
128.23, 128.18, 128.15, 128.12, 128.11, 128.09, 128.07,
127.98, 127.85, 127.7, 127.64, 127.62, 127.60, 127.55,
127.54, 127.45, 127.30, 127.26, 127.18, 127.0, 97.3,
81.8, 81.1, 79.7, 77.3, 77.1, 76.8, 76.1, 75.8, 75.4, 74.9,
(1,5-Cyclooctadiene)bis(methyldiphenylphosphine)iridi-
um(I) hexafluorophosphate (40 mg, 0.047 mmol) was
added to a stirred solution of allyl ether 15 (259 mg,
0.235 mmol) in THF (10 mL) under argon. This atmo-
sphere was replaced with hydrogen for ca 1 min and
the hydrogen was replaced with argon. The mixture
was stirred at 20 ꢁC for 70 min, the solvent was removed
in vacuo and the residue dissolved with stirring in
CH2Cl2/MeOH (2:1, 9 mL). Acetyl chloride (0.1 mL,
1.29 mmol) was added to this solution and stirring was
continued for a further 90 min when solid NaHCO3

5640
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
74.8, 74.6, 73.0, 72.5, 72.1, 71.5, 71.4, 70.7, 69.1, 63.7,
62.9, 45.6, 34.4, 34.1, 32.0, 29.8, 29.7, 29.6, 29.43,
29.40, 29.2, 27.9, 25.0, 24.9, 22.8, 14.2. 31P NMR
(202 MHz, CDCl3) d ꢁ0.6, ꢁ0.7. MS-ESI (M-NHEt3)ꢁ
calcd for C107H144O18P: 1748.0090. Found: 1748.0052.
NMR (75 MHz) d 170.7, 170.2, 139.4, 139.2, 138.8,
138.6, 138.4, 134.4, 129.0, 128.8, 128.7, 128.6, 128.5,
128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
118.0, 99.6, 98.9, 81.8, 81.3, 79.2, 78.6, 77.7, 76.6, 76.4,
76.1, 75.4 (2· CH2), 74.6, 74.5, 73.8, 73.7, 72.9, 72.6,
72.1, 71.9, 71.7, 69.1, 69.0, 68.9, 68.6, 21.5. Gated decou-
pled 13C NMR (75 MHz, CDCl3) selected data, d 99.6,
4.12. Triethylammonium 6-O-(a-D-mannopyranosyl)-1-
O-(1,2-di-O-stearoyl-sn-glycero-3-phosphoryl)-D-myo-
inositol (19)
1
1
0
0
00
00
JC1 –H1 173 Hz, d 98.9, JC1 –H1 174 Hz. MS-ESI
(M+NH4)+ calcd for C88H98NO18: 1456.6778. Found:
1456.6777.
A mixture of protected PIM16 18 (25 mg, 0.014 mmol)
and Pd/C (10%, 50 mg) was stirred at 20 ꢁC under
hydrogen for 16 h in EtOAc/THF/EtOH/H2O (2:1:1:1,
15 mL). The mixture was filtered through a pad of Cel-
ite. The filter pad was washed successively with MeOH/
H2O (1:1, 2· 10 mL), THF (5 mL) and CH2Cl2 (10 mL).
The solvents were removed from the combined filtrate
and washings and the residue was dried by co-evapora-
tion from toluene. The residue was purified by prepara-
tive thin-layer chromatography on silica gel (CH2Cl2/
MeOH/Et3N, 90:8:2 as eluent). The baseline region of
silica was cut and washed with warm MeOH (30 mL)
and CH2Cl2 (30 mL). The solvent was removed and
the white residue was lyophilised from dioxane–H2O
water (2:3) to give the deprotected phosphate 19
(11 mg, 72%) as a white fluffy powder. 1H NMR
(500 MHz, CDCl3/CD3OD/D2O 35:20:3) d 5.10–5.02
(m, 1H), 4.91 (d, J = 2.0 Hz, 1H), 4.20–3.75 (m, 4H),
3.54 (dd, J = 12.5, 5.5 Hz, 1H), 3.46 (t, J = 9.5 Hz,
1H), 3.22 (dd, J = 10.0, 2.0 Hz, 1H), 3.08 (t,
J = 9.5 Hz, 1H), 2.93 (q, J = 7.0 Hz, 6H), 2.16 and
2.12 (t, J = 7.0 Hz, 2H), 1.46–1.35 (m, 4H), 1.13 (t,
J = 7.0 Hz, 9H), 0.70 (t, J = 7.5 Hz, 6H). 13C NMR
(125 MHz, CDCl3) d 174.0, 173.7, 101.3, 73.1, 72.6,
70.9, 70.4, 69.9, 67.0, 62.6, 61.3, 48.9, 48.7, 48.5, 48.3,
48.2, 48.0, 47.8, 46.0, 33.9, 33.8, 31.6, 29.40, 29.35,
29.29, 29.25, 29.11, 29.05, 28.9, 28.8, 24.64, 24.56,
22.4, 13.7, 8.2. 31P NMR (202 MHz, CDCl3) d ꢁ0.11,
4.14. 1-O-Allyl-3,4,5-tri-O-benzyl-2,6-di-O-(3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-D-myo-inositol (21)
Sodium methoxide in MeOH (30%, ca 0.05 mL) was
added dropwise to a stirred solution of diacetate 20
(603 mg, 0.419 mmol) in MeOH/DCM (2:1, 12 mL).
After being stirred for 24 h, the reaction mixture was
diluted with CH2Cl2 (50 mL) and washed with NH4Cl
(satd, 50 mL). The aqueous phase was re-extracted with
CH2Cl2 (2· 50 mL) and the combined organic extracts
were washed with H2O (100 ml). After drying (MgSO4)
and filtration, the solvent was removed and the residue
purified by column chromatography on silica gel. Elu-
tion with EtOAc/light petroleum (3:7 to 13:7) afforded
20
D
the diol 21 as an oil (411 mg, 0.303 mmol, 72%). ½aꢀ
1
+63 (c 0.82, CHCl3). H NMR (300 MHz, CDCl3) d
7.40–7.02 (m, 45H), 5.92–5.80 (m, 1H), 5.42 (br s, 1H),
5.29–5.17 (m, 3H), 4.93–3.82 (m, 31H), 3.48 (dd,
J = 10.7, 3.6 Hz, 1H), 3.33–3.16 (m, 6H), 2.42 (br s,
2H). 13C NMR (75 MHz, CDCl3) d 139.2, 139.1,
138.9, 138.6, 138.4, 138.3, 134.2, 129.0, 128.8, 128.7,
128.6, 128.5, 128.3, 128.2, 127.9, 127.7, 118.3, 100.8,
100.6, 82.0, 81.9, 81.8, 80.5, 79.8, 79.3, 77.1, 76.3, 76.1,
75.4, 75.3, 74.6, 74.5, 73.8, 73.7, 72.6, 72.3, 72.2, 71.7,
71.5, 69.1, 69.1, 68.7, 68.6. MS-ESI (M+NH4)+ calcd
for C84H94NO16: 1372.6573. Found: 1372.6565.
ꢁ0.15 ppm.
MS-ESI
(M-NHEt3)ꢁ
calcd
for
4.15. 1-O-Allyl-3,4,5-tri-O-benzyl-2,6-di-O-(2,3,4,6-tet-
ra-O-benzyl-a-D-mannopyranosyl)-D-myo-inositol (22)
C51H96O18P: 1027.6334. Found: 1027.6316.
4.13. 1-O-Allyl-2,6-di-O-(2-O-acetyl-3,4,6-tri-O-benzyl-
a-D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol
(20)
Sodium hydride (60% dispersion in mineral oil, 50.0 mg,
1.25 mmol) was added to a stirred solution of diol 21
(411 mg, 0.303 mmol) and benzyl bromide (150 lL,
1.26 mmol) in DMF (15 mL) cooled to 0 ꢁC. After
20 min the ice bath was removed and the reaction mix-
ture stirred at 20 ꢁC for 3 h and NH4Cl (satd, 50 mL)
was added. The mixture was extracted with ether
(2· 100 mL) and the combined ethereal extracts were
washed with H2O (2· 70 mL). After drying and filtra-
tion the solvent was removed and the residue purified
by column chromatography on silica gel. Elution with
EtOAc/light petroleum (1:4 to 3:7) afforded the title
compound 22 as an oil (367 mg, 0.239 mmol, 79%).
TMSOTf (100 lL, 0.552 mmol) was added dropwise to a
stirred mixture of 1-O-allyl-3,4,5-tri-O-benzyl-D-myo-
inositol (11) (400 mg, 0.815 mmol), 2-O-acetyl-3,4,6-tri-
O-benzyl-a-D-mannopyranosyl
trichloroacetimidate
˚
(12) (1.41 g, 2.21 mmol) and 4 A molecular sieves
(270 mg) in DCM (50 mL) cooled to ꢁ40 ꢁC. The reac-
tion mixture was allowed to warm to 0 ꢁC over 90 min
when Et3N (2.5 mL) was added. The mixture was fil-
tered through Celite and the filter cake washed with fur-
ther CH2Cl2 (2· 100 mL). The solvent was removed and
the residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:9 to 3:7)
22
1
½aꢀ +31 (c 0.88, CHCl3). H NMR (300 MHz, CDCl3)
D
d 7.38–7.04 (m, 55H), 5.80–5.67 (m, 1H), 5.52 (br s, 1H),
5.25 (br s, 1H), 5.25–5.07 (m, 2H), 4.92–3.78 (m, 35H),
3.56–3.14 (m, 7H). 13C NMR (75 MHz) d 139.5, 139.3,
139.2, 139.1, 139.0, 138.9, 138.8, 138.5, 134.4, 128.8,
128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8,
127.7, 127.6, 118.2, 99.1, 82.2, 81.9, 81.8, 80.6, 79.4,
79.2, 76.4, 76.4, 76.1, 75.4, 75.3, 75.2, 75.1, 75.0, 73.8,
73.6, 72.8, 72.6, 72.5, 72.4, 72.3, 72.0, 71.5, 71.2, 69.4,
afforded the diglycoside 20 (665 mg, 0.462 mmol, 57%)
20
D
as an oil. ½aꢀ +27.5 (c 4.75, CHCl3). 1H NMR
(300 MHz, CDCl3) d 7.38–7.00 (m, 45H), 5.96–5.83
(m, 1H), 5.47–5.43 (m, 3H), 5.23–5.12 (m, 3H), 4.95–
4.65 (m, 8H), 4.60–3.79 (m, 21H), 3.55–3.50 (m, 1H),
3.34–3.20 (m, 6H), 2.12 (s, 3H), 2.11 (s, 3H). 13C

G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
5641
69.1. MS-ESI (M+NH4)+ calcd for C98H106NO16:
1552.7506. Found: 1552.7566.
4.18. 2,6-(Di-O-a-D-mannopyranosyl)-1-O-(1,2-di-O-ste-
aroyl-sn-glycero-3-phosphoryl)-D-myo-inositol (25)
4.16. 3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-ben-
zyl-a-D-mannopyranosyl)-D-myo-inositol25 (23)
Compound 24 was converted to the sodium salt by ion-
exchange chromatography (Dowex 50W X8—100 Na+
form, eluting with MeOH/CHCl3). A sample (42 mg,
0.019 mmol) was dissolved in tBuOH (10 mL) and
hydrogenated in the presence of Pd(OH)2/C (40 mg,
H2 150 psi, 40 ꢁC) over 72 h during which TLC (5:95
MeOH/CH2Cl2) showed disappearance of the starting
material (Rf 0.3) to give a baseline product. The mixture
was filtered through Celite and purified on silica gel elut-
ing with CHCl3 and CHCl3/MeOH/H2O (7:4:1) to afford
(1,5-Cyclooctadiene)bis(methyldiphenylphosphine)iridi-
um(I) hexafluorophosphate (40 mg, 0.047 mmol) was
added to
a stirred solution of allyl ether 22
(313 mmol, 0.204 mmol) in THF (12 mL) under argon.
The argon was replaced with hydrogen for ca 1 min
and the hydrogen atmosphere was, in turn, replaced
with argon. After the mixture was stirred at 20 ꢁC
for 90 min, the solvent was removed and the residue
dissolved with stirring in CH2Cl2/MeOH (2:1, 9 mL).
Acetyl chloride (0.1 mL) was added to this solution
and stirring was continued for 4 h and solid NaHCO3
was added. The mixture was stirred for an additional
5 min when H2O (50 mL) was added. The mixture was
extracted with CHCl3 (2· 80 mL). After drying
(MgSO4) and filtration, the solvent was removed in
vacuo and the residue purified by column chromatog-
raphy on silica gel. Elution with EtOAc/light petro-
leum (1:4 to 3:7) afforded the title compound 23
(234 mg, 0.156 mmol, 76%) as an oil. Spectroscopic
data are in agreement with literature25 values; MS-
FAB (+ve) 1626 [(M+Cs)+, 10%], 286 (10), 181 (30),
133 (25), 91 (100). HRMS-FAB (M+H)+ calc for
C95H99O16: 1495.6933. Found: 1495.6992.
phosphate 3 (8 mg, 37%) as a white powder after lyo-
20
philisation from H2O. ½aꢀ +23.0 (c, 0.20, CDCl3/
D
CD3OD/D2O 70:40:6). 1H NMR (500 MHZ, CDCl3/
CD3OD/D2O, 70:40:6), d 5.30–5.25 (br s, 1H), 5.14 (br
s, 1H), 5.11 (br s, 1H), 4.43 (m, 1H), 4.31 (br s, 1H),
4.08–3.95 (m, 7H), 3.85–3.72 (m, 7H), 3.69–3.59 (m,
3H), 3.47 (m, 1H), 3.30 (t, J = 9.1 Hz, 1H), 2.37 (t,
J = 7.6 Hz, 2H), 2.33 (t, J = 7.7 Hz, 2H), 1.67–1.55 (m,
4H), 1.35–1.21 (m, 56H), 0.89 (t, J = 6.9 Hz, 6H). 13C
NMR (75 MHz, CDCl3/CD3OD/D2O 70:40:6),
d
174.8, 174.5, 101.92, 101.89, 78.9, 73.7, 73.3, 73.2,
71.0, 70.9, 70.7, 70.5, 67.5, 67.4, 63.9, 63.3, 61.7, 61.5,
34.6, 34.5, 32.2, 30.1, 30.0, 29.9, 29.8, 29.7, 29.5(3),
29.4(9), 25.3, 25.2, 23.0, 14.2. 31P NMR (CDCl3/
CD3OD/D2O, 70:40:6, 121.5 MHz) d ꢁ3.8; MS-FAB
(ꢁve) 1190 [(MꢁNa)ꢁ 20%] 338 (15), 283 (15), 97 (25),
79 (100). HSMS-FAB (MꢁNa+)ꢁ calcd for
C57H106O23P: 1189.6863. Found: 1189.6879.
4.17. 3,4,5-Tri-O-benzyl-2,6-di-O-(2,3,4,6-tetra-O-ben-
zyl-a-D-mannopyranosyl-1-O-(1,2-di-O-stearoyl-sn-glyce-
ro)-D-myo-inositol (24)
5. Experimental procedures for the biological tests
5.1. Airway inflammation model
A mixture of alcohol 23 (44 mg, 0.029 mmol) and the
H-phosphonate triethylammonium salt
8
(46 mg,
0.058 mmol) was dried by evaporation with pyridine
(2· 5 mL) and re-dissolved in pyridine (5 mL) under ar-
gon. Pivaloyl chloride (43.0 lL, 0.348 mmol) was added
and the reaction stirred at room temperature for 1 h un-
der argon. TLC analysis (2:3 EtOAc/light petroleum)
showed conversion of 23 (Rf 0.35) into a mixture of
the diastereomeric H-phosphonates (Rf 0.60 and 0.65).
C57BL/6 male mice were bred and housed in a conven-
tional animal facility at the Wellington School of Med-
icine. All animals used for the experiments were aged
between 8 and 12 weeks. Experimental procedures were
approved by the Animal Ethics Committee in accor-
dance with University of Otago (Dunedin, New Zea-
land) guidelines for the care of animals.
A
freshly prepared solution of iodine (30.0 mg,
0.116 mmol) in pyridine/H2O (95:5, 10 mL) was added
and reaction stirred for 30 min. CHCl3 (20 mL) was
added and the mixture stirred for a further 15 min.
The organic layer was washed with Na2S2O3 (10% aq,
20 mL) and aqueous triethylammonium bicarbonate
(1 M, 3· 20 mL), dried (MgSO4) and concentrated in
vacuo to give the crude product which was purified by
semi-preparative reverse phase HPLC (acetonitrile/iso-
propanol/0.05% ammonia, 40:40:20 to 45:45:10) to af-
5.2. Immunisation and airway challenge
Mice were injected intraperitoneally (ip) with 2 lg of
OVA (Sigma–Aldrich, St Louis, MO) in 200 ll of alum
adjuvant (Serva, Heidelberg, Germany) on day 0 and 14
(n = 5 mice/group). On day 28 mice were anaesthetised
by an ip injection of a mixture of Ketamine and Xyla-
zine (Sigma–Aldrich) and intranasally (in) challenged
with 50 ll PBS containing 100 lg OVA.
1
ford 24 (48 mg, 74%) as an oil. H NMR (300 MHz,
CDCl3) d 7.40–7.00 (m, 55H), 5.73 (br s, 1H), 5.67 (br
s, 1H), 5.12 (br s, 1H), 4.83–3.70 (m, 38H), 3.40–3.02
(m, 6H), 2.16–2.07 (m, 4H), 1.55–1.40 (m, 4H), 1.32–
1.10 (m, 56H), 0.89–0.80 (m, 6H). 13C NMR (75 MHz,
CDCl3), selected data, d 173.8, 173.4, 98.6, 97.9. 31P
NMR (CDCl3, 121.5 MHz) d 0.01. Compound 24 was
converted to the sodium salt, see below, HRMS-FAB
(M+H)+ calcd for C134H173O23PNa: 2204.2003. Found:
2204.1904.
5.2.1. Treatment with PIM analogues. One week (day 21)
prior to OVA challenge mice were anaesthetised and the
PIM analogues administered intranasally (50 ll, 20 lg/
ml, in). Control mice were given 50 lL PBS.
5.2.2. Detection of cell types in the bronchoalveolar lavage
fluid. Four days (day 32) post-OVA challenge (day 28)
the mice were sacrificed and bronchoalveolar lavage

5642
G. D. Ainge et al. / Bioorg. Med. Chem. 14 (2006) 5632–5642
14. Walker, C.; Sawicka, E.; Rook Graham, A. W. Curr.
Opin. Allergy Clin. Immunol. 2003, 3, 481–486.
15. Sayers, I.; Severn, W.; Scanga, C. B.; Hudson, J.; le Gros,
G.; Harper, J. L. J. Allergy Clin. Immunol. 2004, 114,
302–309.
16. Gilleron, M.; Ronet, C.; Mempel, M.; Monsarrat, B.;
Gachelin, G.; Puzo, G. J. Biol. Chem. 2001, 276, 34896–
34904.
(BAL) was performed (3 washes with 1 ml PBS). Total
BAL cell numbers were counted, cells were fixed onto
slides using a cytospin and stained with the Diff-Quick
Staining Kit (Dade Behring, Newark, USA). The per-
centages of different cell types were determined micro-
scopically using standard histological criteria.
17. Nigou, J.; Gilleron, M.; Puzo, G. Biochimie 2003, 85,
153–166.
Acknowledgments
18. Gilleron, M.; Quesniaux, V. F. J.; Puzo, G. J. Biol. Chem.
2003, 278, 29880–29889.
19. Mempel, M.; Ronet, C.; Suarez, F.; Gilleron, M.; Puzo,
G.; Van Kaer, L.; Lehuen, A.; Kourilsky, P.; Gachelin, G.
J. Immunol. 2002, 168, 365–371.
20. Severn, W. B.; Furneaux, R. H.; Falshaw, R.; Atkinson, P.
H. Carbohydr. Res. 1998, 308, 397–408.
21. Stepanov, A. E.; Shvets, V. I.; Evstigneeva, R. P. Zh.
Obshch. Khim. 1977, 47, 1653–1656.
The authors thank ComOne, the Foundation of
Research Science and Technology and the Marsden
Fund (Royal Society of New Zealand, IRL0401 to
GFP) for financial support. We also thank Dr Wayne
Severn (AgResearch) for helpful discussions. Professor
Robin Ferrier assisted in the preparation of the
manuscript.
22. Elie, C. J. J.; Dreef, C. E.; Verduyn, R.; Van der Marel,
G. A.; Van Boom, J. H. Tetrahedron 1989, 45, 3477–
3486.
References and notes
23. Stadelmaier, A.; Schmidt, R. R. Carbohydr. Res. 2003,
338, 2557–2569.
24. Rademacher, T. W.; Schmidt, R.; Stadelmaier, A.; (Lasc-
aux Pharmaceuticals Limited, UK). Application: WO,
2003; p. 87.
1. Zhang, J.; Smith, K. R. Br. Med. Bull. 2003, 68, 209–225.
2. Ramsey Clare, D.; Celedon Juan, C. Curr. Opin. Pulm.
Med. 2005, 11, 14–20.
3. von Mutius, E. Thorax 2001, 56, 153–157.
25. Elie, C. J. J.; Verduyn, R.; Dreef, C. E.; Van der Marel, G.
A.; Van Boom, J. H. J. Carbohydr. Chem. 1992, 11,
715–739.
4. Farooqi, I. S.; Hopkin, J. M. Thorax 1998, 53, 927–932.
5. Bjorksten, B.; Sepp, E.; Julge, K.; Voor, T.; Mikelsaar, M.
J. Allergy Clin. Immunol. 2001, 108, 516–520.
26. Watanabe, Y.; Yamamoto, T.; Okazaki, T. Tetrahedron
1997, 53, 903–918.
6. Smit Joost, J.; Folkerts, G.; Nijkamp Frans, P. Curr. Opin.
Allergy Clin. Immunol. 2004, 4, 57–62.
27. Watanabe, Y.; Yamamoto, T.; Ozaki, S. J. Org. Chem.
1996, 61, 14–15.
28. Jayaprakash, K. N.; Lu, J.; Fraser-Reid, B. Bioorg. Med.
Chem. Lett. 2004, 14, 3815–3819.
29. Martin, S. F.; Wagman, A. S. J. Org. Chem. 1996, 61,
8016–8023.
30. Crossman, A., Jr.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1997, 2769–2774.
31. Yamazaki, F.; Sato, S.; Nukada, T.; Ito, Y.; Ogawa, T.
Carbohydr. Res. 1990, 201, 31–50.
32. Mayer, T. G.; Schmidt, R. R. Eur. J. Org. Chem. 1999,
1153–1165.
33. Podlasek, C. A.; Wu, J.; Stripe, W. A.; Bondo, P. B.;
Serianni, A. S. J. Am. Chem. Soc. 1995, 117, 8635–8644.
34. Bock, K.; Lundt, I.; Pedersen, C. Tetrahedron Lett. 1973,
1037–1040.
7. Shirakawa, T.; Enomoto, T.; Shimazu, S.-i.; Hopkin, J.
M. Science (Washington, DC) 1997, 275, 77–79.
8. Hopkin, J. M. Thorax 2000, 55, 443–445.
9. Yamamoto, S.; Kuramoto, E.; Shimada, S.; Tokunaga, T.
Jpn. J. Cancer Res. 1988, 79, 866–873.
10. O’Brien, R. L.; Happ, M. P.; Dallas, A.; Palmer, E.;
Kubo, R.; Born, W. K. Cell (Cambridge, MA, United
States) 1989, 57, 667–674.
11. Barnes, P. F.; Chatterjee, D.; Abrams, J. S.; Lu, S.; Wang,
E.; Yamamura, M.; Brennan, P. J.; Modlin, R. L. J.
Immunol. 1992, 149, 541–547.
12. Young, S. L.; Murphy, M.; Zhu, X. W.; Harnden, P.;
O’Donnell, M. A.; James, K.; Patel, P. M.; Selby, P. J.;
Jackson, A. M. Int. J. Cancer 2004, 112, 653–660.
13. Juffermans, N. P.; Leemans, J. C.; Florquin, S.;
Verbon, A.; Kolk, A. H.; Speelman, P.; Van Deventer,
S. J. H.; Van der Poll, T. Infect. Immun. 2002, 70, 147–
152.
35. Perrin, D. D.; Armarego, W. L. F. Purification of
Laboratory Chemicals. 3rd ed., 1988.