An improved synthetic route to the potent angiogenesis inhibitor benzyl manα(1→3)-Manα(1→3)-Manα(1→3) -Manα(1→2)-Man Hexadecasulfate

Article abstract of DOI:10.1071/CH09015

An improved synthetic route to α(1→3)/α(1→2)-linked mannooligosaccharides has been developed and applied to a more efficient preparation of the potent anti-angiogenic sulfated pentasaccharide, benzyl Manα(1→3)-Manα(1→3)-Manα(1→3) -Manα(1→2)-Man hexadecasu

Full text of DOI:10.1071/CH09015

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2009, 62, 546–552
www.publish.csiro.au/journals/ajc
An Improved Synthetic Route to the Potent Angiogenesis
Inhibitor Benzyl Manα(1→3)-Manα(1→3)-Manα(1→3)-
Manα(1→2)-Man Hexadecasulfate∗
Ligong Liu,A,B Ken D. Johnstone,A Jon K. Fairweather,A,C Keith Dredge,A
and Vito FerroA,D,E
ADrug Design Group, Progen Pharmaceuticals Limited, Darra, Qld 4076, Australia.
BCurrent address: Centre for Drug Design and Development, Institute for
Molecular Bioscience, University of Queensland, Brisbane,
Qld 4072, Australia.
CCurrent address: Chemical Analysis Pty Ltd, 41 Greenaway Street, Bulleen,
Vic. 3105, Australia.
DCurrent address: CRC for Polymers, School of Physical and Chemical Sciences,
Queensland University of Technology, GPO Box 2434, Brisbane,
Qld 4001, Australia.
ECorresponding author. Email: vito.ferro@qut.edu.au
An improved synthetic route to α(1→3)/α(1→2)-linked mannooligosaccharides has been developed and applied to a
more efficient preparation of the potent anti-angiogenic sulfated pentasaccharide, benzyl Manα(1→3)-Manα(1→3)-
Manα(1→3)-Manα(1→2)-Man hexadecasulfate, using only two monosaccharide building blocks. Of particular note are
improvements in the preparation of both building blocks and a simpler, final deprotection strategy. The route also provides
common intermediates for the introduction of aglycones other than benzyl, either at the building block stage or after
oligosaccharide assembly. The anti-angiogenic activity of the synthesized target compound was confirmed via the rat
aortic assay.
Manuscript received: 8 January 2009.
Final version: 5 February 2009.
Introduction
synthesis. Herein we describe improvements to the synthesis
of α(1→3)/α(1→2)-linked mannooligosaccharides and their
application to the preparation of 3.
Recently we described the synthesis[1] of α(1→3)/α(1→2)-
linked mannooligosaccharides such as 1 and 2 (Fig. 1), which
were subsequently sulfonated to provide a series of heparan sul-
fatemimeticsrelatedtotheangiogenesisinhibitorPI-88.[2–4] The
synthetic strategy involved the use of only two or three building
blocks in a reiterative ‘1 + 1’strategy that provides ready access
to all homologues in the series compared with the alternative
‘3 + 2’ block approaches to the parent pentasaccharide.[5,6] The
building blocks used were the starting block 4[7] and an ortho-
gonally protected elongating block 6.[8,9] The strategy allowed
for a final glycosylation, if desired, with a simple capping
block depending on the desired length of the target oligosac-
charide. Pentasaccharide 2 is also a starting material for the
preparation of various polysulfated glycosides,[10] such as 3,
which have shown potent anti-angiogenic activity in various
in vitro and in vivo models of angiogenesis.[11] However, as
a route to 3 the previous synthesis is somewhat inefficient as
it requires complete deprotection to the polyol 2 followed by
acetylation and reintroduction of the benzyl group by glyco-
sylation. As part of our ongoing studies into anti-angiogenic
heparan sulfate mimetics we sought improved, more direct routes
to compounds such as 3 that are amenable to multigram scale
Results and Discussion
The alcohol 5 was selected as the glycosyl acceptor for the prepa-
ration of 3. It was considered that 5 would be a superior starting
block to 4 because the protecting groups are the same as in
the elongating block 6, thus eliminating one deprotection step
before sulfonation. More importantly, later in the synthesis the
anomeric benzyl group can be selectively removed and activated
(e.g., by hydrogenolysis followed by trichloroacetimidate forma-
tion) to provide access to various different glycosides, if desired.
Alternatively, the required aglycone can be introduced into the
building block itself by substituting the appropriate alcohol for
benzyl alcohol (Scheme 1). For the synthesis of 5 (Scheme 1) the
readily available[12] orthoester 7 was deacetylated with saturated
ammonia in methanol and then treated with benzoyl chloride in
pyridine at room temperature[13] to give the tribenzoate 9 in good
overall yield following crystallization from ethyl acetate/hexane.
The orthoester was then hydrolyzed quantitatively by treatment
with 90% trifluoroacetic acid (TFA) in water at room tempera-
ture for 45 min, and the resulting hemiacetal 10 was acetylated
∗Dedicated to Professor Bob Stick on the occasion of his retirement.
© CSIRO 2009
10.1071/CH09015
0004-9425/09/060546

RESEARCH FRONT
Routes to Potent Angiogenesis Inhibitors
547
to give the diacetate 11 as an anomeric mixture (α:β = 5:1) in
94% yield.[14] Without further purification, diacetate 11 was
treated with benzyl alcohol and BF3 etherate in dichloromethane
at room temperature for 16 h to give the glycoside 12 in 73%
yield after flash chromatography. Deacetylation was smoothly
carried out in quantitative yield by treatment with 0.5% acetyl
chloride in methanol. The resulting building block 5, obtained
as an oil, was pure enough for use in glycosylations without fur-
ther purification.This sequence represents a more efficient route
to 5 than our previously published method via the correspond-
ing benzyl orthoester[15] (overall yield from the orthoester 38%
versus 23%).
An improved preparation of the elongating block 6 starting
from methyl α-d-mannopyranoside (13) was also developed, as
illustrated in Scheme 2. The key step for the synthesis of 6
is the dibutyltin oxide-mediated selective allylation of 13 to
give the 3-O-allyl ether 14. At the completion of the reaction
the solvent was evaporated and the residue was perbenzoylated
with benzoyl chloride in pyridine to give the tribenzoate 15 in
51% yield after tedious flash chromatography. The major by-
product from this reaction was the tetrabenzoate 16 formed
from perbenzoylation of unreacted starting material 13. On a
large scale, it was possible to remove significant quantities of
16 by crystallization from methanol/ethyl acetate before chro-
matography. Acetolysis of compound 15 was accomplished by
stirring in acetic anhydride at room temperature for 1 h with
a catalytic amount (1% v/v) of conc. sulfuric acid. The result-
ing product was not purified but was treated directly with 5%
acetyl chloride in methanol to give the hemiacetal 18 in excel-
lent overall yield after chromatography. This procedure avoided
the use of toxic amines (e.g., benzylamine, hydrazine) typi-
cally employed in large excess for similar transformations. The
trichloroacetimidate 6 was then prepared under catalysis with
diazobicycloundecane (DBU, 5 mol-%).
RO
OR
O
HO
OH
O
RO
RO
HO
HO
OH
OH
OR
OR
O
O
HO
RO
O
O
n
3
O
O
HO
HO
HO
RO
RO
RO
O
O
OH
OBn
1
2
n ꢀ 2
n ꢀ 3
3 R ꢀ SO3Na
OH
O
OBz
O
RO
RO
RO
BzO
BzO
O
With the building blocks in hand, 5 was glycosylated with 6
in 1,2-dichloroethane at 0◦C under catalysis by trimethylsilyltri-
flate (TMSOTf, 0.2 equiv.) to give the disaccharide 19 in good
yield (84%). The allyl group was removed by treatment with
PdCl2 to give the alcohol 20 in 80% yield after flash chromato-
graphy, ready for further glycosylation with 6. Two subsequent
OBn
R ꢀ Bn
R ꢀ Bz
O
NH
CCI3
4
5
6
Fig. 1.
OMe
OMe
OMe
O
O
O
AcO
AcO
AcO
HO
HO
HO
BzO
BzO
BzO
(a)
(b)
(c)
O
O
O
O
O
O
7
8
9
OAc
O
OAc
O
OAc
O
(d)
(e)
(f)
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
5
OH
OAc
OBn
10
11
12
Scheme 1. Reagents and conditions: (a) NH3/MeOH, 4◦C, 16 h (or r.t., 4 h), 92%. (b) BzCl, DMAP, pyridine, r.t.,
1–16 h, 60%. (c) ∼90% TFA/water, r.t., 45 min, 100%. (d) Ac2O, DMAP, pyridine, r.t., o/n, 94%. (e) BnOH, BF3
etherate, CH2Cl2, r.t., o/n, 73%. (f) 0.5% AcCl in MeOH, r.t., o/n, 100%.
OH
O
OH
O
OBz
O
HO
HO
HO
HO
BzO
BzO
RO
(a)
(b)
(c)
HO
O
OMe
OMe
OMe
13
14
15 R ꢀ allyl
16 R ꢀ Bz
OBz
O
OBz
O
BzO
BzO
(d)
(e)
BzO
BzO
6
O
O
OAc
OH
17
18
Scheme 2. Reagents and conditions: (a) (i) Bu2SnO, MeOH, 3 h, 80◦C, (ii) allyl-Br, Bu4NBr, PhMe, 60◦C, 2 days.
(b) BzCl, pyridine, r.t., o/n, 27–51% (two steps). (c) Conc. H2SO4 in Ac2O (1% v/v), r.t., 1 h. (d) 5% AcCl in MeOH,
r.t., 5 h, 72% (two steps). (e) Cl3CCN (2 equiv.), DBU (5 mol-%), CH2Cl2, 0◦C, 5 h, 98%.

RESEARCH FRONT
548
L. Liu et al.
rounds of glycosylation/deallylation followed in similar yields
(Scheme 3), which led to the pentasaccharide 26. Removal of
the benzoates was accomplished by treatment with NaOMe in
MeOH, although the limited solubility of compound 26 resulted
in incomplete deprotection, which necessitated purification of
the polyol by reverse phase HPLC in this case. Sulfonation with
SO3·pyridine complex in N,N-dimethylformamide (DMF) then
gave the desired sulfated pentasaccharide 3 in 72% yield fol-
lowing purification by dialysis, identical in all respects to a
previously prepared sample.[10]
anti-angiogenic sulfated pentasaccharide 3. Of particular note
are the use of a benzoyl protected starting block 5, which results
in a simpler final deprotection strategy, and improvements in
both the preparation of 5 and of the elongating block 6. The
route also provides common intermediates for the introduc-
tion of any desired aglycone, either at the building block stage
or after oligosaccharide assembly, without requiring complete
deprotection and re-acetylation/activation/glycosylation.
Experimental
Compound 3 has displayed potent anti-angiogenic activity
in several cell-based assays indicative of angiogenesis such as
growth factor-induced endothelial cell proliferation assays and
the endothelial cell tube formation (Matrigel) assay.[11] In addi-
tion, compound 3 has shown potent anti-angiogenic activity
in vivo in two murine models.[11] In this study, compound 3
was evaluated in a modified version of the rat aortic assay[16,17]
(Fig. 2), which confirmed its potent anti-angiogenic activity.This
assay is considered to come closest to simulating the in vivo
situation, not only because it includes the surrounding non-
endothelial cells but also because the endothelial cells have not
been preselected by passaging and thus are not in a proliferative
state at the time of explantation.[18] The data presented supports
further investigation of 3 as an anti-angiogenic agent.
General
NMR spectra were recorded at 400 MHz for 1H and 100 MHz for
13C, either in deuteriochloroform (CDCl3) with residual CHCl3
(1H, δ 7.26) or deuterium oxide (D2O), employing residual HOD
(1H, δ 4.78) as an internal standard, at ambient temperatures
(298 K). Where appropriate, analyses of 1H NMR spectra were
aided by gradient-selected correlation spectroscopy (gCOSY)
experiments. Flash chromatography was performed on silica gel
(40–63 µm) under a positive pressure with specified eluants. All
solvents used were of analytical grade. The progress of reac-
tions was monitored by TLC using commercially prepared silica
gel 60 F254 aluminium-backed plates. Compounds were visual-
ized by charring with 5% sulfuric acid in methanol and/or by
visualization under ultraviolet light. HPLC was performed on
a Gilson preparative HPLC system, controlled using Trilution
software, running a gradient of 5 to 95% acetonitrile in water
In conclusion, we have developed an improved synthetic
route to α(1→3)/α(1→2)-linked mannooligosaccharides and
have applied this to a more efficient preparation of the
OBz
O
OBz
OBz
BzO
BzO
RO
OBz
OBz
(a)
(a)
O
O
BzO
RO
5 ꢁ 6
BzO
(a)
6
O
6
O
O
BzO
BzO
BzO
BzO
BzO
BzO
O
O
OBn
(b)
OBn
19 R ꢀ allyl
20 R ꢀ H
21 R ꢀ allyl
22 R ꢀ H
(b)
OBz
OBz
OBz
O
BzO
BzO
RO
OBz
OBz
O
OBz
OBz
O
BzO
RO
O
OBz
OBz
BzO
O
(a)
BzO
O
BzO
O
6
O
3
O
O
BzO
BzO
BzO
23 R ꢀ allyl
24 R ꢀ H
O
(b)
BzO
BzO
BzO
O
OBn
OBn
OH
OH
O
25 R ꢀ allyl
26 R ꢀ H
(b)
HO
(c)
(d)
OH
OH
HO
3
O
HO
O
3
O
HO
HO
HO
O
27
OBn
Scheme 3. Reagents and conditions: (a) TMSOTf (0.2–1 equiv.), DCE (0.1 M), 3 Å MS, Ar, 0◦C, 84–94%.
(b) PdCl2 (40 mg mmol−1), MeOH/DCE (1/1, v/v, 0.05 M), 70◦C, 40 min, 64–91%. (c) NaOMe, MeOH, r.t., o/n, 67%.
(d) (i) SO3·pyridine (3 equiv./OH), DMF (0.04 M), 60◦C, o/n, (ii) NaOH, 72% (two steps).

RESEARCH FRONT
Routes to Potent Angiogenesis Inhibitors
549
(a) 6
washed sequentially with NaHCO3 (sat.) (3 × 100 mL), ice-cold
0.5 M HCl (1 × 100 mL), and NaHCO3 (sat.) (1 × 100 mL),
dried (Na2SO4), filtered, and the solvent evaporated to yield
an orange syrup. Recrystallization from EtOAc/hexane gave
5
4
3
2
1
0
1
the tribenzoate 9 as needles (7.6 g, 70% from triol). H NMR
(CDCl3) data were in accord with the literature.[13]
3,4,6-Tri-O-benzoyl-1,2-di-O-acetyl-D-mannopyranose 11
**
The tribenzoate 9 (6.5 g, 12 mmol) was treated with 90% aque-
ous TFA (20 mL) at 0◦C and was stirred at room temperature
for 45 min. The solvent was evaporated and co-evaporated
with toluene (3 × 50 mL) and dried for 16 h under vacuum to
give the hemiacetal 10 as a yellow foam (6.87 g, 100%), used
directly in the next step. 1H NMR (400 MHz) data were in
accord with the literature.[14] The product and DMAP (cat.)
were dissolved in dry pyridine (30 mL) and acetic anhydride
(0.033 mol, 3.1 mL) in dry pyridine (6 mL) was added drop-
wise over 15 min at 0◦C and stirring continued for 2 h at room
temperature. The solution was quenched by adding dry MeOH
(10 mL) at 0◦C and stirred for 15 min. The solution was concen-
trated under vacuum, and co-evaporated with toluene (50 mL)
and dichloromethane (5 mL) to give the diacetate 11 as a yellow
foam (7.05 g, 94%, α:β = 5:1), used in the next step without fur-
ther purification. 1H NMR (CDCl3) data were in accord with the
literature.[14]
Control
3 (10 µM)
3 (50 µM)
(b)
(c)
(d)
Fig. 2. Pentasaccharide 3 significantly inhibits the sprouting of new
microvessels in the rat aortic assay for angiogenesis. Aortic explants were
embedded in Matrigel on day 0. Treatment with 3 commenced on day 1
and the extent of angiogenesis in each group was determined on day 8 (a).
The assay was scored from 0 (least positive) to 5 (most positive) and the
data presented as mean (n = 6); bars + s.e.m.[16] ∗∗P < 0.01 versus control.
Statistical analysis was conducted using a oneway ANOVA followed by a
post hoc Dunnett’s test (Graphpad Instat v3.0). The photographs taken on
day 4 illustrate the inhibitory effects of 3 at 10 µM (c) and 50 µM (d) in
comparison to control (b).
Benzyl 3,4,6-Tri-O-benzoyl-2-O-acetyl-
α-D-mannopyranoside 12
The diacetate 11 (101 mg, 0.175 mmol) and dry benzyl alco-
hol (0.21 mmol, 1.2 equiv., 0.022 mL) was dissolved in
dry dichloromethane (1 mL) and boron trifluoride etherate
(0.875 mmol, 5 equiv., 0.124 mL) was added dropwise within
15 min at 0◦C under Ar and stirring continued for 16 h at
room temperature. The solution was quenched by adding tri-
ethylamine (0.175 mL) at 0◦C (pH 8). The mixture was taken
up in dichloromethane (50 mL), washed with NaHCO3 (sat.)
(3 × 20 mL), dried (Na2SO4), filtered, and the solvent was co-
evaporated with toluene (2 × 20 mL) to yield an orange syrup.
The compound was purified by flash chromatography (hexane/
EtOAc 3/1 → EtOAc) to give the glycoside 12 as a colourless
over 30 min through an Atlantis Prep dC18 5 µm 19 × 150 mm
column. HPLC detection was performed with a Gilson Pre-
pELSdetectorandaWatersdualwavelengthabsorbancedetector
measuring the absorbance at 254 and 214 nm. Capillary elec-
trophoresis (CE) was performed on an Agilent CE System with
the use of 10 µm M 5-sulfosalicylic acid at pH 3 as the back-
ground electrolyte and detection by indirect UV absorbance at
214 nm, as previously described.[19] Compound homogeneity
was determined by 1H and/or 13C NMR spectroscopy and, where
appropriate, by CE.
1
syrup (80 mg, 73%). H and 13C NMR (CDCl3) data were in
accord with the literature.[15]
Benzyl 3,4,6-Tri-O-benzoyl-α-D-mannopyranoside 5
3,4,6-Tri-O-benzoyl-1,2-O-(methoxyethylidene)-
β-D-mannopyranose 9
A solution of the glycoside 12 (100 mg, 0.16 mmol) in dry
MeOH/1,2-dichloroethane (DCE) (2/1, 3 mL) was treated with
AcCl (0.3 mL, 10% v/v). The mixture was stirred at 0◦C
overnight and then at room temperature for 3 h. The solution
was poured into a mixture of ice/water and chloroform (1/1,
100 mL). The organic phase was separated, washed with sat.
NaHCO3 (20 mL) and brine (20 mL), was dried (Na2SO4), fil-
tered, and evaporated to give the alcohol 5 as an oil (97 mg,
100%). 1H and 13C NMR (CDCl3) data were in accord with the
literature.[15]
3,4,6-Tri-O-acetyl-1,2-O-(methoxyethylidene)-β-d-manno-
pyranose[12] 7 (8.0 g, 0.022 mol) was dissolved in dry MeOH
(70 mL). Saturated NH3 in MeOH (25 mL) was added dropwise
over 15 min at 0◦C and stirring continued for 16 h at 4◦C. The
solvent was evaporated and the residue purified on a column
of silica gel (MeOH/EtOAc 1/10 → 1/1 → EtOAc → MeOH)
and co-evaporated with toluene (3 × 50 mL) to give the triol
8 as a yellow syrup (4.8 g, 92%), used directly in the next
step. The triol and DMAP (cat.) were dissolved in dry pyri-
dine (20 mL) and benzoyl chloride (6 equiv., 0.12 mol, 14 mL)
was added at 0◦C and stirring was continued for 16 h. The
mixture was poured onto ice-water (200 mL) and stirred with
MeOH/water (1/1, 200 mL). The precipitate was washed with
MeOH/water (1/5, 3 × 100 mL) and water (3 × 100 mL) and
then dissolved in dichloromethane (200 mL). The solution was
Methyl 3-O-Allyl-2,4,6-tri-O-benzoyl-
α-D-mannopyranoside 15
A mixture of methyl α-d-mannopyranoside (2.0 g, 10.5 mmol)
and Bu2SnO (2.9 g, 11.5 mmol) in MeOH (50 mL) was heated
(80◦C, overnight) with azeotropic removal of H2O throughout.

RESEARCH FRONT
550
L. Liu et al.
The solvent was then evaporated and co-evaporated (CH3CN,
2 × 50 mL) to yield a yellow powder. Bu4NBr (3.7 g, 11.5 mmol)
and allyl bromide (4.5 mL, 50 mmol) in toluene (100 mL) were
added to the above powder and the combined mixture was heated
(60◦C, 2 days). The solvent was evaporated and co-evaporated
(CH3CN, 2 × 50 mL) to yield a white powder. The powder
was dissolved in pyridine (15 mL) and DCE (50 mL), and ben-
zoyl chloride (2.4 mL, 20.9 mmol) was added. The mixture was
stirred at room temperature overnight, cooled (0◦C), and treated
with MeOH (5 mL) with continued stirring (0◦C → room tem-
perature, 5 min) before evaporation of the solvent. The residue
was subjected to workup (EtOAc) and flash column chromato-
graphy (EtOAc/hexane, 1/19 to 3/17) to give the tribenzoate 15
as a colourless oil (3.2 g, 51%, 3 steps). 1H NMR (CDCl3) data
were in accord with the literature.[8] δC (CDCl3) 55.59 (OMe),
63.46 (C-6), 68.68, 68.97, 69.34, 70.98, 74.63 (C-2, -3, -4, -5,
OCH2), 99.16 (C-1), 117.74 (=CH2), 128.58–134.41 (16C,
=CH, Ar), 165.59, 165.95, 166.44 (3C, C=O).
12.0, J5,6a 2.6, H-6a), 4.49 (ddd, 1H, J5,6b 3.5, H-5), 4.37 (dd, 1H,
H-6b), 4.23 (dd, 1H, H-3), 4.16–4.09 (m, 1H, allyl-1ꢀ), 4.02–3.97
(m, 1H, allyl-1ꢀ), 1.65 (br s, 1H, OH).
3-O-Allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyl
Trichloroacetimidate 6
The hemiacetal 18 (9.4 g, 13.9 mmol) was dissolved in anhy-
drous dichloromethane (28 mL, 0.5 M) and trichloroacetonitrile
(2.8 mL, 27.7 mmol, 2equiv.)wasadded.Themixturewasstirred
at 0◦C while DBU (104 µL, 0.69 mmol, 5 mol-%) was added.
The mixture was stirred at 0◦C. After 4 h, TLC (hexane/EtOAc,
4/1) indicated the presence of a small amount of starting mate-
rial. Another portion of trichloroacetonitrile (1 mL) was added.
After stirring at 0◦C for another 1 h (TLC: no further change),
the mixture was concentrated, treated with Et3N (6 mL), and
evaporated onto silica gel. Purification by flash chromatography
(hexane/EtOAc/Et3N, 450/50/1 → 360/120/1) gave the imidate
6 as a white foam (9.2 g, 98%). 1H NMR (CDCl3) data were in
accord with the literature.[8,9]
3-O-Allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranose 18
To a solution of the methyl glycoside 15 (6.70 g, 12.26 mmol) in
dichloromethane (40 mL, 0.3 M) and acetic anhydride (2.31 mL,
24.52 mmol, 2 equiv.) was added concentrated H2SO4 (0.4 mL,
1% v/v, ∼7.52 mmol). The mixture was stirred at room tem-
perature while the reaction was monitored by TLC (a 20 µL
aliquot was treated with 20 mg of Na2CO3. Dichloromethane
(0.2 mL) was added and the mixture was shaken well and the
dichloromethane solution was used for TLC in hexane/EtOAc,
3/1).After completion (1 h), the mixture was treated with NaOAc
(2.1 g, 25 mmol). After stirring at room temperature for 1 h, the
mixture was filtered through a plug of celite. The solid was
washed with dichloromethane (2 × 10 mL). The combined fil-
trate and washings were evaporated to dryness, co-evaporated
with MeOH (2 × 10 mL) under vacuum to give the acetate 17 as
a pale-yellow gum/foam (7.98 g), and used directly in the next
step without purification. δH (CDCl3) 8.08–8.04 (m, 6H, Bz),
7.60–7.53 (m, 3H, Bz), 7.46–7.36 (m, 6H, Bz), 6.30 (d, 1H, J1,2
2.4, H-1), 5.90 (dd, 1H, J3,4 = J4,5 9.8, H-4), 5.76–5.65 (m, 1H,
allyl-2ꢀ), 5.60 (dd, 1H, J2,3 3.4, H-2), 5.17 (dm, 1H, J 17.1, allyl-
3ꢀ), 5.06 (dm, 1H, J 10.3, allyl-3ꢀ), 4.66 (dd, 1H, J6a,6b 12.2,
J5,6a 2.9, H-6a), 4.40 (dd, 1H, J5,6b 3.9, H-6b), 4.33 (ddd, 1H,
H-5), 4.18–4.12 (m, 1H, allyl-1ꢀ), 4.16 (dd, 1H, H-3), 4.01 (ddm,
1H, J 12.7, 5.9, allyl-1ꢀ), 2.22 (s, 3H, Ac). The above product
was suspended in MeOH (24.5 mL, 0.5 M) and acetyl chloride
(1.23 mL, 5% v/v, 17.3 mmol) was added at room temperature.
(Note: the solubility was improved significantly after the addi-
tion of AcCl.) The resulting slightly pink suspension was stirred
at room temperature for 5 h. The mixture was cooled to 0◦C and
Et3N (4.8 mL, 34.6 mmol, 2 equiv. based on AcCl) was added.
The mixture was concentrated to a small volume (∼14 mL). The
yellow suspension was stirred at 0◦C while water (42 mL) was
added dropwise to result in the formation of a thick gummy pre-
cipitate. After standing at 4◦C overnight, the top clear solution
was decanted. The gum was dissolved in MeOH/EtOAc (2/1,
30 mL) and loaded onto silica gel. Purification by flash chro-
matography (hexane/EtOAc, 8/1 → 3/1) gave the hemiacetal 18
as a white foam (4.70 g, 72%, 2 steps). 1H NMR (CDCl3) data
were in accord with the literature:[8,9] δH 8.12–8.01 (m, 6H,
Bz), 7.60–7.52 (m, 3H, Bz), 7.47–7.31 (m, 6H, Bz), 5.87 (dd,
1H, J3,4 = J4–5 9.7, H-4), 5.76–5.64 (m, 1H, allyl-2ꢀ), 5.60 (dd,
1H, J2,3 3.5, J1,2 1.8, H-2), 5.44 (d, 1H, H-1), 5.16 (dm, 1H, J
17.5, allyl-3ꢀ), 5.04 (dm, 1H, J 10.5, allyl-3ꢀ), 4.72 (dd, 1H, J6a,6b
Benzyl 2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-
3,4,6-tri-O-benzoyl-α-D-mannopyranoside 20
A mixture of the imidate 6 (1.00 g, 1.36 mmol, 1.1 equiv.) and the
alcohol 5 (717 mg, 1.23 mmol) in DCE (10 mL) in the presence
of molecular sieves (500 mg of 3 Å powder) was treated with
TMSOTf (246 µL, 1.36 mmol) at 0◦C. The combined mixture
was stirred at 0◦C for 1.5 h at which point TLC (EtOAc/hexane,
3/7) indicated complete conversion. Et3N (100 µL) was intro-
duced, the mixture was filtered, and the solvent was evaporated.
The residue was subjected to flash column chromatography
(EtOAc/hexane, 1/9 to 1/1) to yield the disaccharide 19 as a
colourless foam (1.20 g, 84%). PdCl2 (50 mg) was added to a
solution of the above disaccharide 19 (1.13 g, 0.97 mmol) in
MeOH (10 mL) and DCE (10 mL), and the combined mixture
was heated (70◦C, 40 min). The mixture was cooled and filtered.
The filtrate was evaporated and the residue subjected to flash
column chromatography (EtOAc/hexane, 1/9 to 1/1) to yield the
disaccharide alcohol 20 as a colourless glass (873 mg, 80%). δH
(CDCl3) 8.09–7.89, 7.57–7.28 (2 m, 35H, ArH), 5.98 (dd, 1H,
J3I,4I–4I,5I 9.9, H-4I), 5.87 (dd, 1H, J2I,3I 3, 7, H-3I), 5.64 (dd,
1H, J3II,4II–4II,5II 9.7, H-4II), 5.60 (dd, 1H, J1II,2II 1.8, J2II,3II 3.3,
H-2II), 5.18 (d, 1H, H-1II), 5.14 (d, 1H, J1I,2I 1.8, H-1I), 4.71 (d,
1H, A of ABq, JA,B 11.9, CH2Ph), 4.59 (dd, 1H, J5I,6I 2.7, J6I,6I
12.2, H-6Ia), 4.48–4.42, 4.35–4.30 (2 m, 8H, H-2I, -3II, -5I,II
,
-6Ib, -6aII, -6bII, CH2Ph). δC (CDCl3) 166.97, 166.40, 166.23,
165.90, 165.61, 165.58 (6C, C=O), 136.58 (ArCCH2), 133.78,
133.64, 133.53, 133.35, 133.30 (6C, ArCCO), 130.20–128.14
(35C, ArCH), 99.79, 97.98 (C1I,II), 77.43, 72.61, 70.98, 70.27,
69.97, 69.58, 69.46, 69.02, 67.49 (C-2I,II, -3I,II, -4I,II, -5I,II
,
CH2Ph), 63.39, 63.13 (C-6I,II). m/z (ESMS) 1015.2 [M + H]+,
1037.2 [M + Na]+.
Benzyl 2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-
2,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-
3,4,6-tri-O-benzoyl-α-D-mannopyranoside 22
A mixture of the imidate 6 (900 mg, 1.2 mmol, 1.1 equiv.) and the
disaccharide alcohol (20, 1.24 g, 1.1 mmol) in DCE (8 mL) in the
presence of molecular sieves (500 mg of 3 Å powder) was treated
withTMSOTf (50 µL, 0.27 mmol, 0.25 equiv.) at 0◦C. The com-
bined mixture was stirred at 0◦C for 4 h. TLC (EtOAc/hexane,

RESEARCH FRONT
Routes to Potent Angiogenesis Inhibitors
551
2/3) indicated complete conversion. Et3N was then introduced.
The mixture was filtered and the solvent was evaporated.
The residue was subjected to flash column chromatography
(EtOAc/hexane, 1/9 to 1/1) to yield the trisaccharide 21 as a
colourlessoil, whichwasuseddirectlyforde-O-allylation. PdCl2
(50 mg) was added to a solution of the above trisaccharide 21
in MeOH (10 mL) and DCE (10 mL) and the combined mixture
was heated (80◦C, 2 h). The mixture was cooled and filtered.
The filtrate was evaporated and the residue subjected to flash
column chromatography (EtOAc/hexane, 1/9 to 1/1) to yield
the trisaccharide alcohol 22 as a colourless oil (1.14 g, 71%,
2 steps). δH (CDCl3) 8.07–7.78, 7.62–7.13 (2 m, 50H, ArH),
5.96 (dd, 1H, J3I,4I–4I,5I 9.9, H-4I), 5.93 (dd, 1H, J3III,4III–4III,5III
9.7, H-4III), 5.82 (dd, 1H, J2I,3I 3.3, H-3I), 5.76 (dd, 1H,
J1III,2III 2.0, J2III,3III 3.1, H-2III), 5.65 (dd, 1H, J3II,4II–4II,5II 9.9,
H-4II), 5.32 (d, 1H, J1II,2II 1.5, H-1II), 5.21 (d, 1H, H-1III),
5.16 (d, 1H, J1I,2I 1.5, H-1I), 5.08 (dd, 1H, J2II,3II 3.1, H-2II),
4.69 (d, 1H, A of ABq, JA,B 11.8, CH2Ph), 4.66 (dd, 1H, H-
3III), 4.60–4.30 (m, 11H, H-2I, -5I–III, -6aI–III, -6bI–III, CH2Ph),
4.17 (dd, 1H, H-3II). δC (CDCl3) 166.64–165.22 (9C, C=O),
136.53–132.99 (10C,ArC), 130.24–128.48 (50C,ArCH), 99.78,
99.56, 97.94 (C-1I–III), 77.64, 75.91, 72.49, 71.63, 71.17, 69.93,
Benzyl 2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-
2,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-
tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-
tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-
tri-O-benzoyl-α-D-mannopyranoside 26
A mixture of the imidate (6) (161 mg, 0.223 mmol, 2 equiv.) and
the tetrasaccharide alcohol 24 (229 mg, 0.112 mmol) in DCE
(4 mL) in the presence of molecular sieves (100 mg of 3 Å pow-
der) was treated with TMSOTf (2 µL, 0.012 mmol, 0.11 equiv.)
at 0◦C. The combined mixture was stirred at 0◦C for 80 min.
TLC (EtOAc/hexane, 40/60) indicated complete conversion and
Et3N was introduced. The mixture was filtered and the sol-
vent was evaporated. The residue was subjected to flash column
chromatography (EtOAc/hexane, 1/9 to 1/1) to yield the pen-
tasaccharide 25 as a colourless oil, which was used directly for
de-O-allylation. PdCl2 (20 mg) was added to a solution of the
above pentasaccharide in MeOH (5 mL) and DCE (5 mL) and
the combined mixture was heated (70◦C, 2 h). The mixture was
cooled and filtered. The filtrate was evaporated and the residue
subjected to flash column chromatography (EtOAc/hexane, 1/9
to 1/1) to yield the pentasaccharide alcohol 26 as a colour-
less glass (206 mg, 71%, 2 steps). δH (CDCl3) 8.12–7.08 (m,
80H, Ph), 6.01–5.88 (m, 3H, H-4I–III), 5.84 (dd, J2I,3I 3.2, J3I,4I
9.6, H-3I), 5.80 (dd, J1III,2III 2.0, J2III,3III 3.6, H-2III), 5.78 (dd,
J3IV,4IV∼J4IV,5IV 9.6, H-4IV), 5.44 (dd, J3V,4V∼J4V,5V 10, H-
4V), 5.33 (d, J1II,2II 1.6, H-1II), 5.28 (dd, J2II,3II 3.2, H-2II), 5.23
(d, H-1III), 5.15 (d, J1I,2I 2.0, H-1I), 5.10 (dd, J1IV,2IV 2.0, J2IV,3IV
2.8, H-2IV), 4.88 (d, H-1IV), 4.87 (dd, J1V,2V 1.6, J2V,3V 3.2, H-
2V), 4.84 (d, H-1V), 4.72 (d, 1H,A ofABq, JAB 12, PhCH2), 4.64
(dd, J3III,4III 9.6, H-3III), 4.59(dd, J5,6a 2.8, J6a,6b 12, H-6a), 4.54–
4.24 (m, 10H), 4.39 (dd, H-2I), 4.24 (dd, H-3IV), 4.01 (dd, H-3V),
3.99–3.82 (m, 6H). δC (CDCl3) 166.46–165.10 (15C, C=O),
136.51–132.92 (16C,ArC), 130.26–128.23 (80C,ArCH), 99.71,
99.29, 99.03, 97.89 (C-1I–V), 76.98, 76.61, 76.29, 72.46, 71.66,
71.41, 71.10, 69.97, 69.85, 69.73, 69.46, 69.29, 68.91, 68.52,
68.15, 67.80, 67.51, 67.28 (C-2I–V, -3I–V, -4I–V, -5I–V, CH2Ph),
63.86, 63.01, 62.42, 62.23 (C-6I–V).
69.91, 69.76, 69.57, 69.25, 68.70, 68.33, 67.70 (C-2I–III, -3I–III
,
-4I–III, -5I–III, CH2Ph), 63.82, 63.07, 62.34 (C-6I–III). m/z
(ESMS) 1511.4 [M + Na]+.
Benzyl 2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-
2,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-
tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-
tri-O-benzoyl-α-D-mannopyranoside 24
A mixture of the imidate 6 (121 mg, 0.167 mmol, 1.2 equiv.)
and the trisaccharide alcohol 22 (201 mg, 0.139 mmol) in DCE
(5 mL) in the presence of molecular sieves (50 mg of 3 Å pow-
der) was treated with TMSOTf (10 µL, 0.055 mmol, 0.4 equiv.)
at 0◦C. The combined mixture was stirred at 0◦C for 40 min.
TLC (EtOAc/hexane, 40/60) indicated the complete conversion
and Et3N was introduced. The mixture was filtered and the
solvent was evaporated. The residue was subjected to flash col-
umn chromatography (EtOAc/hexane, 1/9 to 1/1) to yield the
tetrasaccharide 23 as a colourless oil, which was used directly
for de-O-allylation. PdCl2 (50 mg) was added to a solution
of the above tetrasaccharide 23 in MeOH (10 mL) and DCE
(10 mL) and the combined mixture was heated (80◦C, 2 h).
The mixture was cooled and filtered. The filtrate was evapo-
rated and the residue subjected to flash column chromatography
(EtOAc/hexane, 1/9 to 1/1) to yield the tetrasaccharide alco-
hol 24 as colourless oil (206 mg, 77%, 2 steps). δH (CDCl3)
8.12–7.08 (m, 65H, Ph), 6.00–5.90 (m, 3H, H-4I–III), 5.85 (dd,
J2I,3I 3.2, J3I,4I 10, H-3I), 5.80 (dd, J1III,2III 2.0, J2III,3III 3.2,
H-2III), 5.48 (dd, J3IV,4IV∼J4IV,5IV 9.6, H-4IV), 5.34 (d, J1II,2II
1.6, H-1II), 5.28 (dd, J2II,3II 2.8, H-2II), 5.22 (d, H-1III), 5.16
(d, J1I,2I 1.6, H-1I), 4.92 (dd, J1IV,2IV 1.6, J2IV,3IV 3.2, H-2IV),
4.88 (d, H-1IV), 4.72 (d, 1H, A of ABq, JAB 11.6, PhCH2),
4.65 (dd, J3III,4III 9.6, H3III), 4.59 (dd, J5,6a 3.2, J6a,6b 12.4,
H6a), 4.55–4.20, 3.97–3.94 (2m, 13H), 4.06 (dd, H3IV), 3.98 (m,
H5IV). δC (CDCl3) 166.50–165.15 (12C, C=O), 136.51–132.93
(13C, ArC), 130.34–128.23 (65C, ArCH), 99.68, 99.31, 99.28,
97.89 (C-1I–IV), 77.30, 76.48, 76.24, 72.49, 71.58, 71.43, 71.08,
69.99, 69.86, 69.72, 69.29, 69.00, 68.54, 68.17, 67.82, 67.54 (C-
Benzyl 2,3,4,6-Tetra-O-sulfo-α-D-mannopyranosyl-(1→3)-
2,4,6-tri-O-sulfo-α-D-mannopyranosyl-(1→3)-2,4,6-tri-O-
sulfo-α-D-mannopyranosyl-(1→3)-2,4,6-tri-O-sulfo-α-D-
mannopyranosyl-3,4,6-tri-O-sulfo-α-D-mannopyranoside,
Hexadecasodium Salt 3
The perbenzoate 26 (172 mg, 69.3 µmol) was dissolved in dry
MeOH (2.0 mL). NaOMe (11 M) in MeOH (20 µL) was added
and the mixture was stirred at room temperature for 6 h, before
the solution was neutralized with AG50WX8 resin (H+ form).
The solution was filtered before the solvent was evaporated and
the residue taken up in water. The solution was passed through
a solid phase extraction cartridge (Waters Sep-Pak Vac 6cc C18)
to remove methyl benzoate before purification by reverse phase
HPLC. After purification the sample was lyophilized to give the
polyol 27 as a white solid (43 mg, 67%, 97% pure by HPLC),
identical in all respects to an authentic sample.[10] (HRMS found
919.3279. Calc. for C37H59O26: 919.3296 [M + H]+.)
The above polyol (43 mg, 46.8 µmol) was dissolved in DMF
(1.17 mL, 0.04 M). SO3·py (2.25 mmol, 358 mg) was added in
one portion and the mixture was stirred overnight at 60◦C.
The mixture was cooled to 0◦C before 5 M NaOH (5.4 mmol,
1.08 mL) was added in one portion. The solvent was evaporated
and the residue dissolved in water and dialyzed (Slide-A-Lyzer
2
I–IV, -3I–IV, -4I–IV, -5I–IV, CH2Ph), 63.88, 63.02, 62.42, 62.31
(C-6I–IV).

RESEARCH FRONT
552
L. Liu et al.
2K Dialysis Cassette) over 1 week with daily water changes
(final water change done with HPLC-grade water). The sample
was lyophilized to yield the persulfate 3 as an off-white solid
(85.7 mg, 72%, 92% pure by CE). 1H NMR and CE data were
in accord with the literature.[10]
[2] M. Basche, D. L. Gustafson, S. N. Holden, C. L. O’Bryant, L.
Gore, S. Witta, M. K. Schultz, M. Morrow, A. Levin, B. R. Creese,
M. Kangas, K. Roberts, T. Nguyen, K. Davis, R. S. Addison,
J. C. Moore, S. G. Eckhardt, Clin. Cancer Res. 2006, 12, 5471.
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[3] K. D. Lewis, W. A. Robinson, M. J. Millward, A. Powell, T. J. Price,
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K. L. Roberts, J. R. Zalcberg, R. Gonzalez, Invest. New Drugs 2008,
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Rat Aortic Assay for Angiogenesis
RataortaswereharvestedfromSpragueDawleyrats, 6–10weeks
of age by Tetra-Q, Brisbane, Australia and trimmed of remain-
ing fat and connective tissue before cutting 1 mm ring sections,
which were subsequently bisected and transferred to complete
EBM-2 media (Lonza, Basel, Switzerland) that contained 2%
fetal calf serum and all singlequot reagents except for heparin.
Matrigel (BD Biosciences, San Jose, USA) was allowed to cool
oniceandonceinaliquidform, 180 µLwaspipettedinto48-well
tissue culture plates (Nunc, Rochester, USA). The plates were
incubated at 37◦C for 30 min to allow the Matrigel to solid-
ify. Aortic segments were then carefully placed on top of the
Matrigel in the centre of each well and 60 µL of Matrigel was
pipetted to cover the aortic segment. The plate was then returned
to the incubator for a further 20 min before each well was supple-
mented with 1.0 mL of media with or without 10 µM or 50 µM
of compound 3 and replenished every 24 h with fresh media 3.
The stock concentration (10 mM) of 3 was prepared using sterile
phosphate buffered saline, pH 7.2. As a control, medium alone
as assayed was used. On day 8, the microvessels were scored
from 0 (least) to 5 (most positive) by two investigators in an
independent fashion. The results are presented in Fig. 2.
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[6] S. Valerio, A. Pastore, M. Adinolfi, A. Iadonisi, J. Org. Chem. 2008,
73, 4496. doi:10.1021/JO8003953
[7] T. Ogawa, H. Yamamoto, Carbohydr. Res. 1982, 104, 271.
doi:10.1016/S0008-6215(00)82588-4
[8] L. Chen, F. Kong, J. Carbohydr. Chem. 2002, 21, 341.
doi:10.1081/CAR-120014899
[9] L. Chen, Y. Zhu, F. Kong, Carbohydr. Res. 2002, 337, 383.
doi:10.1016/S0008-6215(02)00011-3
[10] T. Karoli, L. Liu, J. K. Fairweather, E. Hammond, C. P. Li, S. Cochran,
K. Bergefall, E. Trybala, R. S. Addison, V. Ferro, J. Med. Chem. 2005,
48, 8229. doi:10.1021/JM050618P
[11] V. Ferro, K. Dredge, L. Liu, E. Hammond, I. Bytheway, C. Li,
K. Johnstone, T. Karoli, K. Davis, E. Copeman, A. Gautam, Semin.
Thromb. Hemost. 2007, 33, 557. doi:10.1055/S-2007-982088
[12] Z. J. Li, H. Li, M. S. Cai, Carbohydr. Res. 1999, 320, 1.
doi:10.1016/S0008-6215(99)00107-X
[13] A. K. Pathak, V. Pathak, J. M. Riordan, S. S. Gurcha,
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[14] L. Heng, J. Ning, F. Kong, J. Carbohydr. Chem. 2001, 20, 285.
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[15] J. K. Fairweather, T. Karoli, V. Ferro, Bioorg. Med. Chem. 2004, 12,
6063. doi:10.1016/J.BMC.2004.09.005
Accessory Publication
Copies of 1H and 13C NMR spectra for compounds 20, 22, 24,
and 26 are available from the Journal’s website.
[16] J. K. Min, K. Y. Han, E. C. Kim, Y. M. Kim, S. W. Lee, O. H. Kim,
K. W. Kim, Y. S. Gho, Y. G. Kwon, Cancer Res. 2004, 64, 644.
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Acknowledgements
The authors thank Drs Richard Furneaux, Derek Watt, and Colin Hayman
(Glycosyn, NZ) for useful discussions and suggestions. Ms Kat Davis is
gratefully acknowledged for technical assistance with the rat aortic assay.
[19] G. Yu, N. S. Gunay, R. J. Linhardt, T. Toida, J. Fareed,
D. A. Hoppensteadt, H. Shadid, V. Ferro, C. Li, K. Fewings,
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