Synthesis and Biological Evaluation of Polysulfated Oligosaccharide Glycosides as Inhibitors of Angiogenesis and Tumor Growth

Article abstract of DOI:10.1021/jm901449m

A series of polysulfated penta- and tetrasaccharide glycosides containing α(1→3)/α(1→2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid, tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

Full text of DOI:10.1021/jm901449m

1686 J. Med. Chem. 2010, 53, 1686–1699
DOI: 10.1021/jm901449m
Synthesis and Biological Evaluation of Polysulfated Oligosaccharide Glycosides as Inhibitors of
Angiogenesis and Tumor Growth
Ken D. Johnstone,†,@ Tomislav Karoli,†,# Ligong Liu,†,# Keith Dredge,† Elizabeth Copeman,† Cai Ping Li,† Kat Davis,†
Edward Hammond,† Ian Bytheway,†,1 Edmund Kostewicz,‡,r Francis C. K. Chiu,‡ David M. Shackleford,‡
Susan A. Charman,‡ William N. Charman,‡ Job Harenberg,§ Thomas J. Gonda, and Vito Ferro*,†,^,@
†Drug Design Group, Progen Pharmaceuticals Limited, Toowong, Queensland 4066, Australia, ‡Centre for Drug Candidate Optimisation, Monash
Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia, §Clinical Pharmacology, Faculty of Medicine
Mannheim, Ruprecht-Karls University of Heidelberg, Mannheim, Germany, Molecular Oncogenesis Group, Diamantina Institute for Cancer,
Immunology and Metabolic Medicine, The University of Queensland, Brisbane, Queensland, Australia, and ^School of Physical and Chemical
Sciences, Queensland University of Technology, Brisbane, Queensland 4001, Australia. @ Current address: School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia. # Current address: Centre for Drug Design and Development,
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. r Current address: Institute of
Pharmaceutical Technology, Goethe University, 60438 Frankfurt am Main, Germany. 1 Please address any compound inquiries to Progen
Pharmaceuticals. E-mail: IanB@progen-pharma.com.
Received September 30, 2009
A series of polysulfated penta- and tetrasaccharide glycosides containing R(1f3)/R(1f2)-linked
mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability
to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2,
and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent
activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting
activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor
activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1
(muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoa-
gulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic
profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further
investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.
Introduction
Chart 1
To grow and metastasize, tumors are critically dependent on
angiogenesis,1,2 that is, the growth of new blood vessels from
preexisting ones surrounding a tumor, and inhibition of
angiogenesis is now well established as an important thera-
peutic strategy for cancer patients.3 Cell surface and extra-
cellular matrix (ECM)a heparan sulfate (HS) glycosa-
minoglycans are complex polysaccharides that are ubiquitous
in nature and play important roles in the regulation of several
aspects of cancer biology, including angiogenesis, tumor pro-
gression, and metastasis.4,5 The use of HS mimetics to mod-
ulate these processes is a promising approach for new cancer
therapeutics.4-7 This is well-illustrated by 1 [muparfostat (PI-
88) (Chart 1)],8,9 a complex mixture of highly sulfated oligo-
saccharides that recently progressed to phase III clinical trials
in postresection hepatocellular carcinoma.10
HS mimetics such as 1 are promising as anticancer ther-
apeutics because they simultaneously inhibit both angiogenesis
and metastasis.8 1 inhibits angiogenesis directly by antagoniz-
ing the interactions of angiogenic growth factors such as FGF-
2 and VEGF with HS, thus blocking the formation of the
ternary HS-growth factor receptor complexes11 that initiate
the angiogenic cell signaling cascade. Compound 1 blocks
metastasis by potently inhibiting heparanase,12,13 an endo-β-
glucuronidase that cleaves HS side chains of proteoglycans
*To whom correspondence should be addressed. Current address:
School of Chemistry and Molecular Biosciences, The University of
Queensland, Brisbane, Queensland 4072, Australia. Phone: 61-7-3346
9598. Fax: 61-7-3365 4299. E-mail: v.ferro@uq.edu.au.
a Abbreviations: HS, heparan sulfate; FGF, fibroblast growth factor;
VEGF, vascular endothelial growth factor; ECM, extracellular matrix;
CMC, chemistry, manufacturing, and controls; APTT, activated partial
thromboplastin time; HUVEC, human umbilical vein endothelial cell;
TGI, tumor growth inhibition.
r
pubs.acs.org/jmc
Published on Web 02/03/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1687
Chart 2
that are a major component of the ECM and basement
membranes, thus allowing metastatic tumor cells to escape
into the bloodstream. Heparanase also plays a key role in
angiogenesis;14 thus, its inhibition by 1 contributes indirectly
to the antiangiogenic activity of 1.
Compound 1 is still being assessed in a phase II trial for
melanoma. Its administration is associated with side effects
such as anticoagulant activity15 and immune-mediated
thrombocytopenia.16 The pharmacokinetic profile17 supports
daily dosing, but because it is a complex mixture, its develop-
ment has been challenging compared with that of typical small
molecules that are single chemical entities. To improve upon
this technology, HS mimetics such as 2 and 4 (Chart 1) were
prepared and evaluated for their ability to inhibit heparanase
and tobind toangiogenicgrowthfactors.18 The compounds in
this small initial series were mostly glycosides of the major
pentasaccharide found in 1. Compounds such as 2 and 4
exhibited biological activity similar to that of 1, but com-
pound 4 in particular, with its more lipophilic octyl group,
showed improved pharmacokinetics in a rat model (cleared
from the plasma approximately 3 times more slowly than 1).
Both 2 and 4 were subsequently shown to be potent inhibitors
of in vivo angiogenesis in two separate murine models when
administered as subcutaneous injections at a dose of 30 mg/
kg/day (b.i.d.).19 To build on these promising early results,
herein we describe the synthesis of analogues of compounds 2
and 4 and their biological evaluation as potential antiangio-
genic agents.
Results and Discussion
Analogues of 2 and 4 in which the length and nature of the
aglycone were altered for the purpose of examining the effects
of such changes on the activity of the compounds were
prepared [3 and 5-17 (Charts 1 and 2)]. In some cases, the
aglycones contained various chromophores in the anticipa-
tion that they would render the compounds easier to detect in
biological fluids (Chart 2). In the earlier, preliminary series,18
the length of the oligosaccharide portion was limited to
pentasaccharide because other studies had shown that the
lower homologues were less potent. For example, highly
sulfated di- and trisaccharides are only partial competitive
inhibitors of heparanase and do not completely inhibit the
enzyme, even at very high concentrations.20 However, in this
study, tetrasaccharide glycosides were also prepared because
sulfated tetrasaccharides have exhibited antiangiogenic8 and
anti-heparanase18,20 activity comparable to that of pentasac-
charides but lower anticoagulant activity.15,21 It was also
desirable to test whether the greater lipophilicity of the
tetrasaccharide glycosides would have any significant effect
on the pharmacokinetic profile of these compounds. In addi-
tion, from a drug development perspective, such compounds
would be prepared by stepwise total synthesis20,22 from
monosaccharide building blocks, and thus, tetrasaccharides
would be cheaper to manufacture, requiring one fewer round
of glycosylation.
were conducted via treatment of a mixture of the peracetate and
the alcohol with BF3 etherate at 60 ꢀC for 2-6 h, which resulted
in the desired glycoside in 48-75% yield after chromatography.
The expected exclusive formation of the R-glycoside, due to
blocking of the β-face by the tri- or tetrasaccharide substituent at
the 2-position, was confirmed by NMR spectroscopy (e.g., a
1
measured JC-1,H-1 value of 170 Hz for compound 39 is char-
acteristic24 for the R-linkage). Glycosylation of 4-butylbenzyl
alcohol, dodecanol, and 12-azido-1-dodecanol required the
more reactive trichloroacetimidates 32 and 33 as the glycosyl
donors.18 These donors were readily prepared from their corre-
sponding peracetates 30 and 31, respectively, by anomeric
deacetylation with benzylamine followed by reaction with tri-
chloroacetonitrile and base; however, the products contained
traces of the byproduct benzylacetamide which could not be
completely removed by chromatography. The benzylacetamide
was carried through with the glycoside but was easily removed
at the polyol stage by washing with EtOAc. The glycosylation
reactions were conducted in dichloromethane at -20 ꢀC with
catalysis by TMSOTf and once again gave exclusively the
R-anomer as the sole product in moderate yield. The dansyl
derivatives 22 and 25 were prepared by Staudinger reaction of
the corresponding azides 38 and 39 with triphenylphosphine in
aqueous THF followed by acylation with dansyl chloride in the
presence of triethylamine.
The compounds were prepared from the penta- and tetra-
saccharides 28 and 29, respectively (Scheme 1), which were
available as byproducts23 from the manufacture of 1, or by total
synthesis using readily prepared monosaccharide building
blocks.20,22 The starting materials were converted into their
peracetates 30 and 31, respectively, by treatment with excess
acetic anhydride in pyridine. Glycosylations with the more
reactive alcohols benzyl alcohol, octanol, and 8-azido-1-octanol
Compounds 18-21, 23, 24, 26, and 27 contain a phenyl- or
naphthyltriazole group at the end of the alkyl chain introduced
via a Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reac-
tion25,26 between the azidoalkyl glycoside and phenyl- or
naphthylacetylene. In the first instance, the cycloaddition

1688 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Scheme 1. Synthesis of Polysulfated Glycosides 3 and 5-17a
Johnstone et al.
a (a) Ac2O, Py; (b) (i) BnNH2, (ii) Cl3CCN, Cs2CO3 (10 mol %), K2CO3 (2 equiv); (c) alcohol (3 equiv), BF3 Et2O, 1,2-DCE, 60 ꢀC, 48-75%; (d)
3
alcohol (2 equiv), TMSOTf, DCM, -20 ꢀC; (e) (i) NaOMe/MeOH, (ii) SO3 Me3N (or pyridine) complex (3 equiv/OH), DMF, 60 ꢀC, (iii) NaOH
3
(aqueous); (f) (i) Ph3P, THF/H2O, (ii) dansyl chloride, Et3N; (g) phenyl- or naphthylacetylene (2 equiv), t-BuOH, CuSO4 (10 mol %), sodium ascorbate
3
(30 mol %).
Table 1. Inhibition of Heparanase,a Growth Factor Binding,b and Anticoagulant Activity (at 0.1 mg/mL)c of Test Compoundsd
compd
heparanase Ki (nM)
Kd for FGF-1 (pM)
Kd for FGF-2 (nM)
Kd for VEGF (nM)
APTT (s)
heptest (s)
1
240 ( 30
370 ( 120
280 ( 60
350 ( 90
330 ( 100
310 ( 70
280 ( 90
420 ( 110
310 ( 60
360 ( 80
270 ( 50
330 ( 70
350 ( 110
610 ( 120
570 ( 140
270 ( 90
270 ( 70
240 ( 40
160 ( 60
240 ( 50
161 ( 22
118 ( 9
210 ( 160
6000 ( 3000
160 ( 80
280 ( 60
120 ( 50
190 ( 120
200 ( 70
100 ( 40
360 ( 130
790 ( 100
2300 ( 1300
190 ( 100
61 ( 9
95 ( 22
129 ( 12
82 ( 21
160 ( 40
160 ( 40
480 ( 70
43 ( 10
140 ( 21
35 ( 9
110 ( 40
130 ( 60
83 ( 14
550 ( 80
86 ( 11
253 ( 25
73 ( 23
2.2 ( 2.3
1.8 ( 0.5
21 ( 4
1.5 ( 0.4
18 ( 5
>500
>500
417.9
250.8
96.7
73.1
125.9
96.9
53.0
89.0
59.5
nde
>500
>500
281.1
124.4
79.5
49.8
64.2
38.4
24.4
20.0
28.8
nde
2
3
4
5
6
12.0 ( 1.1
12 ( 3
7
8
0.62 ( 0.24
10.4 ( 2.2
0.70 ( 30
7.1 ( 2.0
8.9 ( 2.0
1.4 ( 0.8
25 ( 17
12.2 ( 0.4
5.1 ( 1.8
0.79 ( 0.24
9
10
11
12
13
14
15
16
17
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
a Ki values ( the standard error for inhibition of recombinant human heparanase as determined from inhibition response curves as previously
described.20 b Binding affinities for growth factors FGF-1, FGF-2, and VEGF were determined using a surface plasmon resonance-based solution
affinity assay, as previously described.28,29 The Kd values represent the average of at least duplicate measurements ( the standard deviation. c The normal
ranges for coagulation values in pooled human plasma are 26-36 s for APTT and 13-20 s for heptest. APTT and heptest values are the means of two
determinations. d See the Experimental Section for assay details. e Not determined.
reaction was performed on the aglycone in a convenient one-
pot procedure27 whereby the azido alcohol, initially formed by
displacement of the corresponding bromide by azide ion,
reacted in situ with the alkyne in the presence of a copper
catalyst. This simple procedure provided ready access to
the triazole-functionalized alcohols for glycosylation with
the trichloroacetimidate donor 32 or 33. Unfortunately,
these alcohols proved to be completely unreactive under our
glycosylation conditions, even at elevated temperatures.
The compounds were therefore prepared via the correspond-
ing azidoalkyl glycosides 38-42 which underwent a Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition with phenyl-
or naphthylacetylene in aqueous tert-butanol. The cycloaddi-
tions generally proceeded satisfactorily and gave a moderate
yield (43-75%) of product after overnight reaction; however,
it is noteworthy that some reactions with naphthylacetylene
were rather sluggish and required prolonged reaction
times and an excess of reagents to produce a reasonable
yield of product, e.g., 3 days for 21 (50%) and 11 days for 27
(26%).
The peracetylated glycosides were then converted into the
polysulfated final products 3 and 5-17 by deprotection with
sodium methoxide in methanol followed by sulfonation with
excess sulfur trioxide pyridine (or dimethylamine) complex in
DMF at 60 ꢀC for 16 h. The polysulfates were obtained in
good yield following purification by size exclusion chroma-
tography or dialysis to remove salts and low-molecular weight
impurities. Product purity was determined by a combination
of CE21 and size exclusion HPLC and was generally >95%.
Mass spectrometric analysis (ESMS) was conducted for some
compounds and, in accord with previous observations,28 gave
spectra that consistedofclusters of peaks correspondingtothe
loss of sulfo groups. However, it was evident from the 1H and
13C NMR spectra that the compounds generally were fully
sulfated (single sets of peaks with resonances shifted down-
field). This is in contrast to polysulfated oligosaccharides that
are sulfated at the reducing end such as 1, which contain a
small subpopulation of slightly undersulfated species. Such
compounds are difficult to obtain fully sulfated because they
are susceptible to anomeric hydrolysis and are poorly soluble

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1689
in DMF, typically forming insoluble gums at the completion
of the sulfonation reaction,21 resulting in incomplete sulfona-
tion. The improved level of sulfation may thus be due to the
greater solubility conferred on the compounds by the lipo-
philic aglycones as well as the increased stability to hydrolysis.
The compounds were initially evaluated for their ability to
3
inhibit the hydrolysis of H-labeled HS by recombinant hu-
man heparanase using an ultrafiltration assay20 (Table 1). The
results indicate that the compounds are potent inhibitors of
heparanase (Ki = 270-610 nM), with tetrasaccharides dis-
playing activity comparable to that of pentasaccharides. With
the exception of 14 and 15, which were approximately 2.5-fold
weaker inhibitors than 1, the compounds had Ki values similar
to those of 1, indicating that the presence of a lipophilic
aglycone does not greatly impair the compounds’ ability to
inhibit heparanase.
The affinity of the compounds for angiogenic growth
factors FGF-1, FGF-2, and VEGF was then determined via
a surface plasmon resonance-based solution affinity assay.28,29
All compounds bound tightly to the growth factors (Kd values
in the nanomolar to picomolar range), in some cases with
slightly better affinities than 1, with tetrasaccharides displaying
an affinity comparable to that of pentasaccharides (Table 1).
With the exception of 8 and 10, which showed improved
affinity for all three growth factors compared with 1, the
aglycone modifications had variable effects on affinity depend-
ing on the growth factor, with FGF-1 seemingly more sensitive
(10 of 16 compounds exhibited improved affinity).
The compounds were subsequently subjected to a suite of
cell-based assays indicative of angiogenesis in which the
improved antiangiogenic activity of many compounds com-
pared with that of 1 became apparent. The first of these
focuses on the ability of the compounds to inhibit the pro-
liferation of endothelial cells in response to angiogenic growth
factors. In human umbilical vein endothelial cell (HUVEC)
proliferation assays, many compounds inhibited FGF-1-,
FGF-2-, and VEGF-induced endothelial cell proliferation
with IC50 values similar to or lower than that of 1 (Figure 1a).
These data and the Kd data from Table 1 were also expressed
as a ratio to provide a measure of the efficiency with which
growth factor affinity is translated into antiproliferative
activity against HUVECs whose division is being promoted
by the same growth factor (Figure 1b). The ratios are
normalized with respect to 1, allowing for easier comparison
with this compound, and the data indicate that compounds
3, 5, 7, and 16 show significant improvement in the transla-
tion of growth factor affinity to antiproliferative activity.
This suggests that the aglycone groups used in these com-
pounds facilitate significantly higher bioavailability in these
cell experiments which, if translated to increased bioavail-
ability in organisms, could significantly improve the efficacy
of this potent class of HS mimetics.
Endothelialcell tubeformation onanECM-derivedprotein
matrix using HUVECs was also strongly inhibited by several
compounds at concentrations of 10, 50, and 100 μM
(Figure 2a), with many compounds achieving 100% inhibi-
tion at concentrations of 50 μM (Figure 2b and data not
shown). In contrast, 1 has only modest activity in this assay
and does not reach 100% inhibition, even at very high
concentrations (e.g., only 58% inhibition at 100 μM).19 It is
important to note that the compounds do not induce cyto-
toxicity at the concentrations tested in the models described
above with the LC50 values (lethal concentration required
to kill 50% of the endothelial cells) for all the compounds
Figure 1. Inhibition of growth factor-induced HUVEC prolifera-
tion by test compounds. (a) The IC50 data represent the mean and
standard deviation to indicate interexperimental error where se-
lected compounds were tested in two to eight independent experi-
ments. Data without error bars are from single experiments. An
asterisk indicates IC50 > 50 μM (50 μM was typically the highest
concentration tested). (b) These data and those in Table 1 have been
used to calculate the ratio of growth factor affinity (Kd) to cell
proliferation efficacy (IC50). The ratio, which has been normalized
with respect to compound 1 to ease comparison, indicates the
efficiency with which affinity for a growth factor carries over to
the cell proliferation experiment specific for that same growth
factor. A ratio of >1 indicates that that compound has a higher
efficiency than 1.
being >100 μM (data not shown). The LC50 for compound 5,
for example, is more than 10-fold higher than its IC50 con-
centration in the endothelial cell proliferation assays.
Selected compounds were also examined in the ex vivo rat
aortic angiogenesis assay. In this model, rat aortic endothe-
lium exposed to a three-dimensional matrix of ECM-derived
proteins switches to a microvascular phenotype, generating
branching networks of microvessels. Angiogenesis is triggered
by the injury caused by the dissection procedure and does not
require stimulation by exogenous growth factors. With the
exception of 2 and 3, the tested compounds significantly
inhibited angiogenesis at a concentration of 10 μM, with
compound 4 showing the most potent inhibition at 50 μM
of all compounds tested (see Figure 3). To examine the
viability of the aortic tissue following treatment with the test
compounds, withdrawal of these compounds on day 6 or 7
(depending on the individual experiment) was followed by

1690 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Johnstone et al.
Figure 2. (a) Inhibition of HUVEC tube formation on a Matrigel by selected test compounds at 10, 50, and 100 μM taken from a representative
experiment (control n = 9, treatment groups n = 3). An asterisk indicates P < 0.05 vs vehicle control. Statistics were determined using a one-
way ANOVA followed by a Dunnett’s post hoc test (Graphpad Instat, version 3.05). (b) Percentage inhibition of selected compounds in the
tube formation assay. For compounds tested on multiple occasions, standard deviations are shown to indicate interexperimental error. (c)
Some representative images of control and compound 5 at 10 and 100 μM.
compounds exert their inhibitory effects via an antiangiogenic
mechanism as opposed to the induction of a toxic effect on the
tissue (data not shown).
The anticoagulant activity of selected test compounds was
determined by measuring the effect of a solution of a com-
pound at a concentration of 0.1 mg/mL in PBS on the
elevation of the activated partial thromboplastin time
(APTT) of pooled normal human plasma. The compounds
were also evaluated in the heptest, a clot-based assay which
measures both anti-Xa and anti-IIa activity but reflects pre-
dominantly anti-Xa activity at low concentrations. The data
are presented in Table 1 and clearly show that the presence of
the aglycone attenuates the anticoagulant activity. The com-
Figure 3. Inhibition of angiogenesis by selected compounds at con-
pounds with the smallest aglycones, such as 2 and 3 which
centrations of 10 and 50 μM in the rat aortic assay. An asterisk
have only a benzyl group, still have some significant anti-
indicates P < 0.05 vs vehicle control. Statistics were determined using
coagulant activity, but in general, the remaining compounds
a one-way ANOVA followed by a Dunnett’s post hoc test (Graphpad
Instat, version 3.05). The data are representative of an individual
angiogenesis experiment with each concentration assessed in triplicate.
could be described as possessing only modest anticoagulant
activity. The APTT data were independently confirmed by
testing in another laboratory.
treatment with VEGF for up to an additional 7 days. The
appearance of microvessel sprouts demonstrated that the
To test the effectiveness of these compounds in a tumor
model, the B16 melanoma model was chosen. B16 melanoma

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1691
Figure 4. Antitumor activity in the B16 mouse melanoma model.
Compounds 4 and 5 were administered for 7 days at 30 mg/kg/day
(b.i.d.) to C57 mice (n = 10), 3 days after tumor challenge at day 0.
Both 4 and 5 significantly reduced the tumor size up to day 10.
P < 0.05 is considered significant.
is a commonly used cell line for the induction of tumors in
syngeneic C57/BL6 mice. B16 melanoma is a nonmetastatic,
fast-growing tumor unresponsive to most anticancer agents,
including 1. The tetrasaccharide 5 and its pentasaccharide
homologue, the previously prepared 4, were tested in this
model because they exhibited good activity across all the
assays and are among the simplest to synthesize in this series.
The activities of 4 and 5 in cell-based assays were similar, so
the tumor study would be useful for the examination of
whether any differences between a tetra- and a pentasacchar-
ide became apparent in vivo. The compounds were adminis-
tered as a subcutaneous injection in PBS for 7 days at 30 mg/
kg/day (b.i.d.) to C57 mice (n = 10), 3 days after tumor
challenge at day 0. Treatment with both 4 and 5 significantly
reduced the tumor size [percentage of tumor growth in-
hibition (TGI) values of 94 and 86%, respectively] up to day
10 (Figure 4). Both compounds significantly inhibited tumor
progression in this model, indicating that HS mimetics have
the potential to inhibit tumor growth. The experiments were
repeated at a daily dose of 15 mg/kg, i.e., half the previous
dose, and both compounds once again significantly inhibited
tumor growth, producing TGI values of 42% for compound 4
and 64% for compound 5. No appreciable differences were
noted between the two compounds in this model, indicating
that tetra- and pentasaccharides have similar activity in vivo.
The pharmacokinetics of 5 were investigated in male
Sprague-Dawley rats following intravenous (iv) and subcuta-
neous (sc) administration at a nominal dose of 10 mg/kg
(Figure 5). Measurable concentrations of 5 were detected in
plasma up to 6-8 h postdose, and the apparent half-life was
approximately 1.7 h for both routes of administration. The
apparent volume of distribution was very low (26 mL/kg) and
comparable to the total plasma volume in the rat (∼31 mL/kg),30
suggesting that 5 was not extensively distributed out of the
vasculature into peripheral tissue compartments. The total
plasma clearance was low (12 mL h-1 kg-1), and renal
elimination of intact 5 was not a significant clearance path-
way, as 5 was not detected in urine after either sc or iv
administration. These data suggest that 5 may have improved
pharmacokinetics versus those of 4, which was previously
found to exhibit a slightly shorter half-life (1.1 h) and a higher
plasma clearance (∼84 mL h-1 kg-1).18 In addition, 5 was well
absorbed following subcutaneous dosing, with a bioavailabil-
ity of approximately 67%.
Figure 5. Plasma concentrations of 5 following iv and sc adminis-
tration to male Sprague-Dawley rats at a nominal dose of 10 mg/kg
(n = 2 rats per dosing route). Concentrations observed in individual
rats are presented (empty symbols) together with the mean for each
dosing route (filled symbols).
Conclusions
A series of polysulfated glycosides of R(1f3)/R(1f2)-
linked mannopenta- and -tetrasaccharides were readily
synthesized in four or five steps from the parent oligosacchar-
ides. These HS mimetics were evaluated for their antiangio-
genic activity in a series of in vitro and ex vivo assays. In
general, the compounds exhibited superior activity to the
phase III HS mimetic 1 in cell-based assays indicative of
angiogenesis, and importantly, tetrasaccharides, which are
likely to be cheaper to manufacture than pentasaccharides,
were shown to have activity similar to that of pentasacchar-
ides. Moreover, compounds 4 and 5 showed potent antitumor
activity in vivo in a mouse melanoma model which is resistant
to 1. The lipophilic modifications also resulted in significant
attenuation of anticoagulant activity (a common side effect
of HS mimetics) and improved in vivo pharmacokinetics,
with compound 5 exhibiting a good pharmacokinetic profile
in rats. Taken together, the data support the continued deve-
lopment of HS mimetics of this type as antiangiogenic, anti-
cancer agents.
Experimental Section
General Information. Unless otherwise stated, nuclear mag-
netic resonance (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 in deuterium oxide (D2O),
employing residual HOD (1H, δ 4.78) as the internal standard, at

1692 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Johnstone et al.
ambient temperatures (298 K). Where appropriate, analyses of
1H NMR spectra were aided by gCOSY experiments. Flash
chromatography was performed on silica gel (40-63 μm) under
a positive pressure with specified eluants. All solvents used were
analytical grade. The progress of reactions was monitored by
thin layer chromatography (TLC) using commercially prepared
silica gel 60 F254 aluminum-backed plates. Compounds were
visualized by charring with 5% sulfuric acid in methanol and/or
under ultraviolet light. Analytical HPLC was performed on a
Waters Alliance 2690 Separations Module equipped with a
Waters 2410 refractive index detector, using a Phenomenex
PolySep-GFC-P2000 (300 mm ꢀ 7.8 mm) column and 0.1 M
NaNO3 as the mobile phase. The flow rate was 0.8 mL/min, and
the column temperature was set to 35 ꢀC. Capillary electrophor-
esis (CE) was performed in reverse polarity mode on an Agilent
CE System using 10 mM 5-sulfosalicylic acid (pH 3) as the
background electrolyte and detection by indirect UV absor-
bance at 214 nm, as previously described.21 Compound homo-
(3.5 mL) was stirred at 60 ꢀC under argon for 5 h. The solution was
cooled to room temperature, and Et3N (250 μL) was added before
the mixture was poured into brine and extracted with DCM. The
organic layer was washed with brine, dried (Na2SO4), and evapo-
rated and the residue purified by flash chromatography (1:1 f 7:3
EtOAc:hexane) to give the glycoside 34 (102 mg, 64%) as a colorless
1
oil: H NMR (CDCl3) δ 7.36-7.28 (m, 5 H, Ph), 5.33-5.14 (m,
7 H), 5.02 (dd, 1 H, J = 3.1, 1.9 Hz), 4.99-4.96 (m, 2 H), 4.95 (d,
1H, J= 1.5 Hz), 4.94 (d, 1 H, J= 1.6 Hz), 4.89 (d, 1 H, J=1.5Hz),
4.70, 4.52 (ABq, JAB = 11.9 Hz, PhCH2), 4.30-3.83 (m, 12 H), 2.17,
2.15, 2.12, 2.11, 2.10, 2.09, 2.08, 2.04, 2.03, 2.02, 1.99, 1.96 (12 s, 39 H,
13 ꢀ Ac); 13C NMR (CDCl3) δ 170.7, 170.6, 170.5, 170.4, 170.2,
170.1, 169.9, 169.7(1), 169.6(9), 169.6, 169.5, 169.3, 136.2, 128.6,
128.3, 127.9, 99.4, 99.0, 98.9, 97.4, 76.7, 75.3, 75.2, 71.0, 70.9, 70.2,
69.7, 69.6(5), 69.5(8), 69.4, 69.2, 68.7, 68.3, 67.1, 66.7, 66.2, 65.6, 62.5,
62.2, 62.0, 61.8, 20.9, 20.8(2), 20.7(9), 20.6(3), 20.6(0), 20.5(7), 20.5.
Benzyl 2,3,4,6-Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-
tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-man-
nopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside, Tride-
casodium Salt (3). Glycoside 34 (102 mg, 78 μmol) was sequentially
deacetylated and sulfonated according to the general procedures
and then purified by SEC to give the polysulfate 3 (104 mg, 64%)
as a white amorphous solid: 1HNMR(D2O, solvent suppressed on
HOD) δ 7.37-7.24 (m, 5 H, Ph), 5.36 (br s, 1 H), 5.28 (d, 1 H, J =
1.4 Hz), 5.26 (d, 1 H, J = 1.1 Hz), 5.06 (t, 1 H, J = 2.0 Hz), 5.01 (d,
1 H, J = 1.8 Hz), 4.87 (br s, 1 H), 4.69-4.65 (m, 2 H affected by
suppression), 4.59-3.68 (m, 22 H affected by suppression); 13C
NMR (125 MHz, D2O) δ 136.6, 129.0, 128.9, 128.6, 99.6, 97.4,
76.7, 76.5, 76.1, 75.0, 74.9, 74.0, 72.8, 72.5, 72.2, 71.4, 70.2, 69.9,
69.4, 69.2, 67.5, 67.4, 66.1, 66.0.
1
geneity was determined by H and/or 13C NMR spectroscopy
and for the final products by CE and/or HPLC. Unless indicated
otherwise, the purity of the final products was >95%.
General Procedure for Deacetylation. A solution of the per-
acetate in anhydrous MeOH (0.1 M) (or MeOH-THF) was
treated with a solution of NaOMe in MeOH (1.35 M, 0.2-0.6
equiv). The mixture was stirred at room temperature for 1-3 h
(monitored by TLC) and then neutralized with ion-exchange
resin AG-50W-X8 (Hþ form). The mixture was filtered, and the
resin was rinsed with MeOH. The combined filtrate and wash-
ings were concentrated in vacuo and thoroughly dried to give the
polyol product.
General Procedure for Sulfonation. A mixture of the polyol
and SO3 pyridine (or trimethylamine) complex (2 equiv per
Octyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-
tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-
mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (35).
3
hydroxyl group) in DMF (0.04 M) was heated (60 ꢀC, over-
night). The ice-cooled reaction mixture was treated with MeOH
(3 volumes) and then made basic (to pH >10) by the addition of
aqueous Na2CO3 (10%, w/w). The mixture was filtered and the
filtrate evaporated and coevaporated (H2O). The crude poly-
sulfated product was dissolved in H2O and purified by size
exclusion chromatography (see below). When required, after
lyophilization the product was passed through an ion-exchange
resin column (AG-50W-X8, Naþ form, 1 cm ꢀ 4 cm, deionized
H2O, 15 mL) to transfer the product uniformly into the sodium
salt form. The solution collected was evaporated and lyophilized
to give the final product.
Size Exclusion Chromatography. Size exclusion chromatog-
raphy (SEC) was performed on Bio-Gel P-2 in a 5 cm ꢀ 100 cm
column at a flow rate of 2.8 mL/min with 0.1 M ammonium
bicarbonate as the eluant, collecting 2.8 min (7.8 mL) fractions.
Fractions were analyzed for carbohydrate content by being
spotted onto silica gel plates and visualization by charring,
and/or analyzed for polyanionic species by the dimethyl methy-
lene blue (DMB) test.31 Finally, fractions were checked for
purity by CE, and those deemed to be free of salt were pooled
and lyophilized.
2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-man-
nopyranosyl-(1f2)-1,3,4,6-tetra-O-acetyl-D-mannopyranose (30).
The polyol 2823 (2.3 g, 3.45 mmol) was acetylated [excess Ac2O
(9.4 mL, 99.4 mmol, 2.06 equiv per hydroxyl group), pyridine
(15 mL), and DMAP (10 mg) at room temperature for 4 days] and
purified by flash chromatography (3:2 f 19:1 EtOAc:hexane) to
give the peracetate 30 as an oil (4.11 g, 95%): ESMS m/z 1277.19
[M þ Na]þ; 1H NMR (CDCl3) δ 6.20 (d, 1 H, J1,2 = 1.8 Hz, H-1I),
5.35-5.15 (m, 7 H), 5.05-4.92 (m, 5 H), 4.30-3.85 (m, 15 H), 2.18,
2.14, 2.12, 2.10, 2.08, 2.06, 2.03, 2.02, 1.97 (9 s, 42 H, 14 ꢀ Ac).
Benzyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-
r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside
(34). A solution of peracetate 30 (150 mg, 120 μmol), benzyl alcohol
˚
1-Octanol (47 mg, 360 μmol, dried over 3 A sieves) was glycosy-
lated with peracetate 30 (150 mg, 120 μmol) as described above for
compound 34 to give, following flash chromatography (2:3 f 3:2
EtOAc:hexane), glycoside 35 (100 mg, 63%) as a colorless oil: 1H
NMR (CDCl3) δ 5.32-5.13 (m, 7 H), 5.01 (dd, 1 H, J = 2.9, 1.9
Hz), 4.99-4.96 (m, 2 H), 4.93 (d, 1 H, J = 1.4 Hz), 4.89 (d, 1 H,
J = 1.2 Hz), 4.86 (d, 1 H, J = 1.1 Hz), 4.28-3.95 (m, 12 H),
3.92-3.82 (m, 3 H), 3.63 (dt, 1 H, J = 9.5, 6.8 Hz, OCH2CH2 A),
3.39 (dt, 1 H, J = 9.4, 6.6 Hz, OCH2CH2 B), 2.15(3), 2.14(6), 2.11,
2.09, 2.08, 2.06, 2.04, 2.03, 2.00, 1.98, 1.95 (11 s, 39 H, 13 ꢀ Ac),
1.60-1.52 (m, 2 H, OCH2CH2), 1.31-1.21 (m, 10 H), 0.85 (t, 3 H,
J = 6.8 Hz, CH3); 13C NMR (CDCl3) δ 170.6, 170.5(4), 170.4(5),
170.3, 170.2, 170.1, 170.0, 169.9, 169.7(4), 169.6(9), 169.6, 169.5,
169.4, 99.4, 99.0, 98.8, 98.1, 77.0, 75.2, 71.0, 70.8, 70.2, 69.6, 69.5,
69.4, 69.3, 68.5, 68.3, 67.3, 66.7, 66.3, 65.6, 62.5(4), 62.4(7), 62.0,
61.8, 31.7, 29.2(4), 29.2(0), 29.1, 26.0, 22.5, 20.9, 20.7(9), 20.7(6),
20.6(1), 20.5(8), 20.5(5), 20.5, 14.0.
Octyl 2,3,4,6-Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-
tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-man-
nopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside, Tride-
casodium Salt (5). Glycoside 35 (97 mg, 73 μmol) was sequentially
deacetylated and sulfonated according to the general procedures
and then purified by SEC to give polysulfate 5 (100 mg, 65%) as a
1
white amorphous solid: H NMR (D2O, solvent suppressed on
HOD) δ 5.38 (br s, 1 H), 5.28 (m, 2 H), 5.07 (br t, 1 H, J = 2.3 Hz),
4.91 (d, 1 H, J = 1.7 Hz), 4.88 (br s, 1 H), 4.70 (br s, 1 H), 4.66 (dd,
1H affected by suppression, J = 8.0, 2.7 Hz), 4.58-4.19 (m, ∼16 H
affected by suppression), 4.04-3.95 (m, 4 H), 3.62 (dt, 1 H, J =
9.4, 6.9 Hz, OCH2 A), 3.48 (dt, 1 H, J = 9.5, 6.1 Hz, OCH2 B),
1.53-1.42 (m, 2 H), 1.25-1.10 (m, 10 H), 0.71 (t, J = 6.7 Hz,
CH3); 13C NMR (125 MHz, D2O) δ 99.6, 98.0, 76.8, 76.5, 76.1,
75.0, 74.9, 73.9, 72.8, 72.6, 72.2, 71.4, 70.0, 69.9, 69.4, 69.2, 68.7,
67.6, 66.1, 65.9, 31.1, 25.6, 28.4(3), 28.4(1), 25.4, 22.0, 13.5.
2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-man-
nopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranosyl
Trichloroacetimidate (32). Peracetate 30 (500 mg, 398 μmol) in
(38 mg, 360 μmol), and BF3 Et2O (34 mg, 240 μmol) in dry DCE
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1693
diethyl ether (3.0 mL) and THF (750 μL) was treated with
benzylamine (0.137 g, 1.3 mmol, 139 μL) at 0 ꢀC. The mixture
was allowed to warm slowly to room temperature and react
overnight. The solvent was evaporated and the residue dissolved
in DCM and washed with cold 0.5 M HCl (three times), followed
by brine, and the organic solution was dried (Na2SO4), filtered,
and evaporated. The residue was dissolved in dry DCM (10 mL),
and sulfonated according to the general procedures and then
purified by SEC to give the polysulfate 7 (64%) as a white powder
(77mg):1H NMR (D2O, internal DOH at 4.60 ppm) δ5.19 (s, 1 H),
5.15 (d, 1 H, J=1.9Hz), 5.10(d, 1H, J= 1.9 Hz), 5.07 (d, 1 H, J=
1.9 Hz), 4.89 (m, 1 H), 3.77-3.64 (m, 30 H), 3.48-3.41 (m, 1 H,
OCH2), 3.33-3.27 (m, 1 H, OCH2), 1.30 (m, 2 H, CH2), 1.10-0.90
(m, 18 H, 9 ꢀ CH2), 0.54 (t, 3 H, J = 6.7 Hz, CH3).
˚
8-Azidooctyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside (38). 8-Azidooctanol32 (171 mg, 1.2 mmol, 3 equiv)
was glycosylated with peracetate 30 (500 mg, 0.398 mmol) as
described for compound 34 to give, following flash chromato-
graphy (1:4 f 3:1 EtOAc:hexane), glycoside 38 (331 mg, 61%) as
a glass: 1H NMR (CDCl3) δ 5.34-5.16 (7 H, m), 5.03-4.87 (6 H,
m), 4.30-3.84 (15 H, m), 3.64 (1 H, m, CH2), 3.39 (1 H, m, CH2),
3.24 (2 H, t, CH2N3) 2.17, 2.16, 2.13, 2.11 (ꢀ2), 2.10 (ꢀ2), 2.07,
2.06, 2.05, 2.02, 2.00, 1.96 (39 H, 11s, OAc), 1.60-1.56 (4 H, m,
CH2), 1.39-1.29 (8 H, m, CH2).
and molecular sieves (3 A, 30 mg), anhydrous cesium carbonate
(12.9 mg, 39.8 μmol), and potassium carbonate (110 mg, 796 μmol)
were added. The mixture was stirred at 0 ꢀC before trichloroace-
tonitrile (115 mg, 80 μL, 796 μmol) was added. The mixture was
stirred for 5 h at room temperature until complete conversion
by TLC. The mixture was filtered, and the solvent was evaporated
to give the crude product which was subjected to flash chroma-
tography (1:6 f 3:1 EtOAc:hexane) to give the trichloroacetimi-
date 32 (307.5 mg, 57%) as a clear oil which solidified on standing
in the refrigerator. Note that the product contains benzy-
lacetamide which is best removed with an EtOAc wash after
1
polyol formation: H NMR (CDCl3) δ 6.40 (d, 0.7 H, J1,2
=
1.5 Hz, H-1IR), 6.22 (d, 0.3 H, J1,2 =1.5Hz, H-1Iβ), 5.40-5.14 (m,
7 H), 5.05-4.89 (m, 5 H), 4.31-3.84 (m, 15 H), 2.19-1.98 (m,
39 H, Ac).
8-(4-Phenyl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-acetyl-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-
acetyl-r-D-mannopyranoside (19). Glycoside 38 (74.5 mg, 54.5 μmol),
phenylacetylene (16.7 mg, 164 μmol), tert-butanol (600 μL),
CuSO4 (20 μL of a 0.3 M solution), and sodium ascorbate (25
μL of a 1 M solution) were shaken at room temperature overnight.
The solvent was evaporated and the crude mixture subjected to
flash chromatography (1:4 f 3:1 EtOAc:hexane) to give the
phenyltriazole 19 as a glass (34.4 mg, 43%): Rf = 0.36 (3:1
4-Butylbenzyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-
r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside
(36). To a solution of trichloroacetimidate 32 (200 mg, 147 μmol)
in DCM (5 mL) was added 4-butylbenzylalcohol (48.3 mg,
50.2 μL, 294 μmol). The mixture was stirred at -20 ꢀC for
20 min. TMSOTf (10 μL, 56 μmol) was added. The mixture
was stirred for a further 20 min at -20 ꢀC. Triethylamine (17 μL,
122 μmol) was added, before the mixture was warmed to room
temperature and filtered. The solution was evaporated and the
crude product purified by flash chromatography (1:4 f 5:1
EtOAc:hexane) to give glycoside 36 (106 mg, 53%) as a clear
1
EtOAc:hexane); H NMR (CDCl3) δ 7.82 (2 H, d, Ph-H), 7.76
(1 H, s, triazole-H), 7.42 (2 H, t, Ph-H), 7.32 (1 H, t, Ph-H),
5.35-5.14 (7 H, m), 5.04-4.88 (6 H, m), 4.39 (2 H, t, CH2N),
4.30-3.86 (15H, m), 3.65 (1 H, m, CH2), 3.39 (1 H, m, CH2), 2.18,
2.17, 2.14, 2.11(9), 2.11(5), 2.10(6), 2.10(3), 2.07, 2.06 (ꢀ2), 2.03,
2.00, 1.97 (39 H, 12s, OAc), 1.95 (2 H, m, CH2), 1.57 (2 H, m,
CH2), 1.38-1.30 (8 H, m, CH2).
1
oil containing traces of benzyl acetamide: H NMR (CDCl3) δ
7.20 (2 H, d, Ar), 7.14 (2 H, d, Ar), 5.32-5.14 (7 H, m), 5.02-4.87
(6 H, m), 4.65, 4.46 (2 H, AB q, PhCH2), 4.28-3.83 (15 H, m),
2.58 (2 H, t, CH2), 2.16, 2.15, 2.12, 2.10, 2.09, 2.08 (ꢀ2), 2.03, 2.01
(ꢀ2), 2.00(6), 1.98, 1.95 (39H, 11s, OAc), 1.56(2 H, m, CH2), 1.32
(2 H, m, CH2), 0.89 (3 H, t, CH3).
4-Butylbenzyl 2,3,4,6-Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-
mannopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside, Tri-
decasodium Salt (6). Glycoside 36 (106 mg, 78.3 μmol) was
sequentially deacetylated and sulfonated according to the general
procedures and then purified by SEC to give polysulfate 6 (64%)
as an amorphous solid: 1H NMR (D2O) δ 7.30 (2 H, d, Ar), 7.21
(2 H, d, Ar), 5.39 (1 H, m), 5.33 (2 H, m), 5.13 (1 H, m), 5.06 (1 H,
m), 4.91 (1 H, m), 4.73-3.93 (22 H, m), 2.53 (2 H, t, CH2), 1.48
(2 H, m, CH2), 1.20 (2 H, m, CH2), 0.78 (3 H, t, CH3).
8-(4-Phenyl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-sulfo-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-
sulfo-r-D-mannopyranoside, Tridecasodium Salt (9). Phenyltria-
zole 19 (34.4 mg, 23.4 μmol) was sequentially deacetylated and
sulfonated according to the general procedures and then purified
by SEC to give the polysulfate 9 (43.7 mg, 85%) as a white
powder: 1H NMR (D2O) δ 8.30 (1 H, s, triazole-H), 7.70 (2 H, m,
phenyl-H), 7.45-7.34 (3 H, m, phenyl-H), 5.39 (1 H, m), 5.32 (2
H, m), 5.12 (1 H, m), 4.89 (2 H, m), 4.74-3.95 (22 H, m), 4.38 (2
H, m, CH2N), 3.59 (1 H, m, CH2O), 3.44 (1 H, m, CH2O), 1.84 (2
H, m, CH2), 1.45 (2 H, t, CH2), 1.19 (8 H, m); 13C NMR (125
MHz, D2O) δ 147.4, 129.7, 129.3, 128.8, 125.7, 122.4, 99.6, 98.8,
97.9, 76.7, 76.5, 76.1, 75.0, 74.9, 74.0, 72.8, 72.6, 72.2, 71.4, 70.0,
69.4, 69.2, 68.6, 67.6, 66.1, 66.0, 50.8, 29.1, 28.3, 27.9, 27.7, 25.3,
25.1.
8-Azidooctyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-man-
nopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (39).
8-Azido-1-octanol (51 mg, 0.356 mmol) was glycosylated with
peracetate 3118 (200 mg, 0.13 mmol) as described for compound
34 to give, after flash chromatography (1:2 f 3:1 EtOAc:hexane),
glycoside 39 (103 mg, 48%) as a glass: 1H NMR (CDCl3) δ
5.28-5.14 (8 H, m), 5.02-4.86 (8 H, m), 4.27-3.75 (19 H, m), 3.63
(1 H, m, CH2), 3.38 (1 H, m, CH2), 3.23 (2 H, t, CH2N3), 2.15,
2.14(5), 2.14(3), 2.11, 2.10, 2.09, 2.08(8), 2.08(3), 2.07(9), 2.07(0),
2.06, 2.04, 2.03(5), 2.00, 1.99, 1.95 (48 H, 16s, OAc), 1.60-1.54 (4
H, m, CH2), 1.35-1.28 (8 H, m, CH2); 13C NMR (150 MHz,
CDCl3) δ 170.6(4), 170.6(2), 170.6(1), 170.5, 170.4, 170.3, 170.2,
170.1, 170.0(2), 169.9(8), 169.8, 169.7, 169.6, 169.5, 169.4, 99.3,
99.0, 98.9, 98.2, 77.2, 75.2, 75.1, 75.0, 71.1, 71.0, 70.9, 70.3, 69.7,
69.6, 69.5, 69.4, 69.3, 68.4(5), 68.3(8), 68.3(5), 67.3, 66.8, 66.7,
66.3, 65.6, 62.6, 62.5, 62.0, 61.7, 51.4, 29.6, 29.2, 29.1, 29.0, 28.7,
Dodecyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-
D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (37). 1-Dodecanol
(0.849 mmol, 3 equiv) was glycosylated with trichloroacetimidate
3318 (0.469 g, 0.285 mmol) as described for compound 36 to give,
following flash chromatography (99:1 f 98:2 CHCl3:MeOH),
glycoside 37 as a colorless gum. Two fractions were obtained each
containing the byproduct BnNHAc (76 mg, 5:3 37:BnNHAc
ratio; 179 mg, 5:11 37:BnNHAc ratio): 1H NMR (CDCl3) δ
5.30-5.16 (m, 8 H), 4.99-4.87 (m, 8 H), 4.31-3.77 (m, 19 H),
3.68-3.61 (m, 1 H, OCH2), 3.44-3.36 (m, 1 H, OCH2), 2.18, 2.17,
2.15, 2.11, 2.10, 2.09, 2.07, 2.06, 2.05, 2.02, 2.01, 1.97, 1.95 (13 s, 48
H, 16 ꢀ Ac), 1.58 (quintet, 2 H, J = 6.7 Hz, CH2), 1.33-1.22 (m,
18 H, 9 ꢀ CH2), 0.86 (t, 3 H, J = 6.7 Hz, CH3).
Dodecyl 2,3,4,6-Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside, Hexadecasodium Salt
(7). Glycoside 37 (72 mg, 0.043 mmol) was sequentially deacetylated

1694 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Johnstone et al.
26.6, 26.0, 20.9(2), 20.8(9), 20.8(7), 20.8, 20.7, 20.6(2), 20.5(8),
20.5.
flash chromatography (1:1 f 3:1 EtOAc:hexane), naphthyltria-
zole 21 (42.5 mg, 50%) as a glass: Rf = 0.37 (3:1 EtOAc:hexane);
1H NMR (CDCl3) δ 8.35 (1 H, m, naphth-H), 7.90-7.87 (2 H, m,
naphth-H), 7.82 (1 H, s, triazole-H), 7.73 (1 H, d, naphth-H),
7.54-7.50 (3 H, m, naphth-H), 5.30-5.15 (7 H, m), 5.04-4.88 (6
H, m), 4.48 (2 H, t, CH2N), 4.31-3.85 (15 H, m), 3.65 (1 H, m,
CH2), 3.40 (1 H, m, CH2), 2.18, 2.17, 2.13, 2.12, 2.11, 2.10(4),
2.10(2), 2.07, 2.06, 2.05, 2.03, 2.00, 1.97 (39 H, 13s, OAc), 2.03
(2 H, m, CH2), 1.59 (2 H, m, CH2), 1.39 (2 H, m, CH2), 1.36-1.30
(6 H, m, CH2).
8-(4-Phenyl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-acetyl-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside (18). Azide 39 (25 mg, 0.015 mmol) was reacted with
phenylacetylene (5 mg, 0.045 mmol) as described for compound
19 to give, after flash chromatography (2:1 EtOAc:hexane, 1:2 f
4:1), phenyltriazole 18 (20 mg, 75%) as a glass: Rf = 0.30 (3:1
1
8-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-
sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-manno-
pyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-
3,4,6-tri-O-sulfo-r-D-mannopyranoside, Tridecasodium Salt (11).
Naphthyltriazole 30 (42.5 mg, 28 μmol) was sequentially deacety-
lated and sulfonated according to the general procedures and then
purified by SEC to give polysulfate 11 (48.8 mg, 83%) as a white
powder: 1H NMR (D2O) δ 8.20 (1 H, s, triazole-H), 7.93 (3 H, m,
naphth-H), 7.57-7.48 (4 H, m, naphth-H), 5.39 (1 H, m), 5.34 (2
H, m), 5.13 (1 H, m), 4.91 (1 H, m), 4.85 (1 H, m), 4.74-3.93 (22 H,
m), 4.42 (2 H, m, CH2N), 3.52 (1 H, m, CH2O), 3.40 (1 H, m,
CH2O), 1.85 (2 H, m, CH2), 1.41 (2 H, t, CH2), 1.18 (8 H, m).
8-(5-Dimethylaminonaphthalene-1-sulfonamido)octyl 2,3,4,6-
Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-
r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (22). (a) Azide 38
(161 mg, 118 μmol) and triphenylphosphine (92.7 mg, 353 μmol,
3 equiv) were dissolved in THF (3 mL). Water (63.7 mg, 64 μL,
3.54 mmol, 30 equiv) was added and the mixture stirred at 50 ꢀC for
2 h. The solvent was evaporated to give the crude amine which was
used without further purification or characterization. (b) The
product from part a (70 mg, 52.2 μmol) was dissolved in DMF
(1.3 mL). Dansyl chloride (42.2 mg, 156.6 μmol) was added,
followed by triethylamine (11 μL). The solution was stirred for 3 h
at room temperature. The solvent was evaporated and the crude
product purified by flash chromatography (1:2 f 3:1 EtOAc:
hexane) to give sulfonamide 22 (24.3 mg, 25%) as a pale yellow
oil: 1H NMR (CDCl3) [first set of rotamer aromatic peaks
(integration of 0.4 per proton)] δ 8.61 (1 H, m-broad, Ar), 8.32
(1 H, m-broad, Ar), 7.58-7.51 (2 H, m-broad, Ar), 7.45 (1 H, m-
broad, Ar), 7.24 (1 H, m-broad, Ar); 1H NMR (CDCl3) [second set
of rotamer aromatic peaks (integration of 0.6 per proton)] δ 8.23
(1 H, d, Ar), 7.66 (1 H, m, Ar), 7.64(1 H, m, Ar), 7.58-7.51 (2 H, m,
Ar), 7.44 (1 H, m, Ar); 1H NMR (CDCl3) (nonaromatic peaks) δ
5.34-5.15 (7 H, m), 5.03-4.86 (6 H, m), 4.79 (1 H, t, NH),
4.30-3.85 (15 H, m), 3.64 (1 H, m, CH2), 3.37 (1 H, m, CH2),
2.93 [6 H, s, (CH3)2N], 2.86 (2 H, q, CH2NH) 2.17, 2.16, 2.13, 2.11
(ꢀ2), 2.10 (ꢀ2), 2.07, 2.05, 2.04(7), 2.02, 2.00, 1.96 (39 H, 11s, OAc),
1.52 (2 H, m, CH2), 1.37 (2 H, m, CH2), 1.24 (2 H, m, CH2),
1.20-1.08 (6 H, m, CH2).
EtOAc:hexane); H NMR (CDCl3) δ 7.82 (2 H, d, Ph-H), 7.74
(1 H, s, triazole-H), 7.41 (2 H, t, Ph-H), 7.31 (1 H, t, Ph-H),
5.30-5.15 (8 H, m), 5.02-4.87 (8 H, m), 4.39 (2 H, t, CH2N),
4.29-3.76(19H, m), 3.63(1 H, m, CH2), 3.39(1H, m, CH2), 2.17,
2.16, 2.15(6), 2.13, 2.11, 2.10(6), 2.10(3), 2.09(5) (ꢀ2), 2.08, 2.06,
2.05, 2.03, 2.02, 2.00, 1.96 (48 H, 15s, OAc), 1.94 (2 H, m, CH2),
1.57 (2 H, m, CH2), 1.35-1.30 (8 H, m, CH2).
8-(4-Phenyl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-sulfo-r-
D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyr-
anoside, Hexadecasodium Salt (8). Phenyltriazole 18 (57.5 mg,
0.033 mmol) was sequentially deacetylated and sulfonated accord-
ing to the general procedures and then purified by SEC to give
polysulfate 8 (58.6 mg, 66%) as a white powder: 93% pure by CE;
1H NMR (D2O) δ 8.43 (s, dCH), 7.90-7.88 (m, 2 H, Ph),
7.62-7.57, 7.32-7.49 (2m, 3 H, Ph), 5.55 (s, 2 H), 5.50 (s, 2 H),
5.29 (s, 1 H), 5.08 (s, 2 H), 5.06 (s, 1 H), 4.91-4.35 (m, 25 H),
4.25-4.10 (m, 4 H), 3.76, 3.61 (2m, OCH2), 2.01 (m, 2 H, CH2),
1.61 (m, 2 H, CH2), 1.36 (m, 8 H, CH2).
8-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-man-
nopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-
r-D-mannopyranoside (20). Azide 39 (27 mg, 0.016 mmol),
1-ethynylnaphthalene (8 mg, 0.05 mmol), tert-butanol (200 μL),
CuSO4 (5 μL of a 0.3 M solution), and sodium ascorbate (6.25 μL
of a 1 M solution) was shaken at room temperature overnight. The
solvent was evaporated and the crude mixture subjected to flash
chromatography (1:1 f 4:1 EtOAc:hexane) to give naphthyltria-
zole 20 (18 mg, 64%) as a glass: Rf = 0.20 (3:1 EtOAc:hexane); 1H
NMR (CDCl3) δ 8.35 (1 H, m, naphth-H), 7.87-7.90 (2 H, m,
naphth-H), 7.82 (1 H, s, triazole-H), 7.73 (1 H, d, naphth-H),
7.54-7.50 (3 H, m, naphth-H), 5.30-5.15 (8 H, m), 5.02-4.87 (8
H, m), 4.48 (2 H, t, CH2N), 4.29-3.76 (19 H, m), 3.65 (1 H, m,
CH2), 3.39 (1 H, m, CH2), 2.17, 2.16, 2.15, 2.13, 2.11, 2.10(6),
2.10(3), 2.09(5) (ꢀ2), 2.08, 2.06, 2.05, 2.04, 2.02, 1.99, 1.96 (48 H,
15s, OAc), 2.03 (2 H, m, CH2), 1.61 (2 H, m, CH2), 1.39 (2 H, m,
CH2), 1.37-1.29 (6 H, m, CH2).
8-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-
sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-manno-
pyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-
2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-sulfo-
r-D-mannopyranoside (10). Naphthyltriazole 20 (22.6 mg,
0.0125 mmol) was sequentially deacetylated and sulfonated
according to the general procedures and then purified by SEC
to give polysulfate 10 (86.9 mg, 80%) as an off-white powder:
1H NMR (D2O) δ 8.24 (1 H, s, triazole-H), 8.02 (1 H, m,
naphth-H), 7.93 (2 H, m, naphth-H), 7.73 (1 H, d, naphth-H),
7.55-7.48 (3 H, m, naphth-H), 5.37 (2 H, m), 5.32 (2 H, m), 5.12
(1 H, m), 4.91 (2 H, m), 4.82 (1 H, m), 4.72-3.89 (27 H, m), 4.42
(2 H, m, CH2N), 3.53 (1 H, m, CH2O), 3.38 (1 H, m, CH2O),
1.88 (2 H, m, CH2), 1.42 (2 H, t, CH2), 1.19 (8 H, m).
8-(5-Dimethylaminonaphthalene-1-sulfonamido)octyl 2,3,4,6-
Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside, Tridecasodium Salt
(12). Sulfonamide 22 (24.3 mg, 15.4 μmol) was sequentially
deacetylated and sulfonated according to the general procedures
and then purified by SEC to give polysulfate 12 (29.4 mg, 82%) as
an off-white powder: 1H NMR (D2O) δ 8.71 (1 H, d, naphth-H),
8.33 (2 H, m, naphth-H), 8.05 (1 H, d, naphth-H), 7.85 (2 H, m,
naphth-H), 5.36-5.29 (3 H, m), 5.12 (1 H, m), 4.88 (2 H, m),
4.74-3.90 (22 H, m), 3.50 (1 H, m, CH2O), 3.45 [6 H, s, (CH3)2N],
3.34 (1 H, m, CH2O), 2.87 (2 H, m, CH2N), 1.22 (2 H, m, CH2),
0.97 (2 H, t, CH2), 0.81 (2 H, m), 0.66-0.40 (6 H, m).
8-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)octyl 2,3,4,6-Tetra-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-manno-
pyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f2)-
3,4,6-tri-O-acetyl-r-D-mannopyranoside (21). Azide 38 (76.4 mg,
55.9 μmol) was reacted with 1-ethynylnaphthalene (25.5 mg, 168
μmol) as described for compound 20 for 3 days (extra 25 μL
portions of sodium ascorbate were added each day) to give, after
3-Azidopropyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-man-
nopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (41).
3-Azidopropanol33,34 (98 mg, 972 μmol) was glycosylated with
peracetate 31 (500 mg, 324 μmol) as described for compound 34
to give, following flash chromatography (3:2 f 3:1 EtOAc:

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1695
hexane), glycoside 41 (387 mg, 75%) as an oil: ESMS m/z 1601.81
[M þ NH4]þ; 1H NMR (CDCl3) δ 5.30-5.13 (m, 8 H), 5.02-4.88
(m, 8 H), 4.28-3.75 (m, 20 H), 3.49 (dt, 1 H, J = 9.8, 6.1 Hz,
OCH2CH2B), 3.42-3.35 (m, 2 H, CH2N3), 2.16, 2.14(9), 2.14(7),
2.11, 2.10, 2.09(2), 2.08(8), 2.08(7), 2.08, 2.07, 2.06, 2.05, 2.04,
2.00, 1.99, 1.95 (16s, 16 ꢀ 3 H, 16 ꢀ AcO), 1.89-1.84 (m, 2 H,
CCH2C); 13C NMR (CDCl3) δ 170.4, 170.3, 170.2, 170.1, 169.9,
169.8(2), 169.7(7), 169.6, 169.5, 169.4, 169.3, 169.2, 99.1(2),
99.1(0), 98.8(4), 98.7(7), 98.1, 76.7, 75.0, 74.9, 74.7, 71.0, 70.8,
70.7, 70.0, 69.5, 69.3, 69.2, 68.5, 68.2, 67.2, 66.7, 66.6, 66.0, 65.4,
64.6, 62.4, 62.3, 61.9, 61.5, 47.9, 28.5, 20.7(4), 20.7(2), 20.6(9),
20.6, 20.4(9), 20.4(6), 20.4.
3-(4-Phenyl[1,2,3]triazol-1-yl)propyl 2,3,4,6-Tetra-O-acetyl-
r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside (23). Azide 41 (59 mg, 37 μmol) was reacted with
phenylacetylene (13.5 mg, 132 μmol) as described for compound
19 to give, after flash chromatography (4:1 f 1:0 EtOAc:hexane),
phenyltriazole 23 (47 mg, 75%) as an oil: ESMS m/z 1686.94
[M þ H]þ; 1H NMR (CDCl3) δ 7.83-7.78 (m, 3 H, triazole-H þ
ArH), 7.43-7.30 (m, 3 H, ArH), 5.31-5.14 (m, 8 H), 5.03-4.90
(m, 8 H), 4.52 (t, 2 H, J = 6.6 Hz, triazole-CH2), 4.30-3.73 (m, 19
H), 3.46 (dt, 1 H, J = 10.1, 5.8 Hz, OCH2B), 2.30-2.24 (m, 2H,
CH2CH2CH2), 2.18, 2.17, 2.16, 2.13, 2.12, 2.11(1), 2.10(8), 2.10(2),
2.10(0), 2.09, 2.06, 2.03, 2.02(3), 2.01(8), 2.00, 1.97 (16s, 16 ꢀ 3 H,
16 ꢀ Ac).
6%) as an off-white solid: 95% pure by CE; 1H NMR (D2O) δ
8.22-8.26 (1 H, m), 7.69-7.71 (2 H, d), 7.29-7.40 (3 H, m),
3.52-5.33 (30 H, m), 2.14-2.17 (2 H, m).
3-(5-Dimethylaminonaphthalene-1-sulfonamido)propyl 2,3,4,6-
Tetra-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-
D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-
acetyl-r-D-mannopyranoside (25). (a) A mixture of polymer-
bound triphenylphosphine (124 mg, 194 μmol, Fluka catalog
no. 93094) and azide 41 (105 mg, 66 μmol) in anhydrous THF
(2 mL) was stirred for 2 h; then water (36 μL, 2.0 mmol) was
added, and the mixture was stirred at 50 ꢀC for 4 h. The mixture
was cooled to room temperature, filtered, and evaporated in
vacuo to give the amine, used without further purification: APCI-
MS m/z 1558.25 [M þ Hþ]. (b) A solution of the amine (47 mg,
30.2 μmol), dansyl chloride (16.3 mg, 60.3 μmol), Et3N (15.3 mg,
21 μL, 150 μmol), and DMAP (12 μL of a 135 mM solution in
CHCl3, 1.5 μmol) in CHCl3 (900 μL) was stirred for 4 h.
Phenomenex StrataNH2 medium (30 mg) was added, and the
mixture was stirred for 30 min then applied directly to a prepared
flash chromatography column (4:1 f 9:1 EtOAc:hexane) to give
sulfonamide 25 (18 mg, 30%) as an oil: 1H NMR (CDCl3) δ 8.68
(br s, 1 H), 8.36 (br s, 1 H), 8.23 (dd, 1 H, J = 7.3, 1.1 Hz),
7.61-7.53 (m, 2 H), 7.29 (br s, 1 H), 5.31-5.15 (m, 8 H), 5.03 (dd,
1 H, J = 3.2, 1.9 Hz), 5.00-4.93 (m, 5 H), 4.91 (d, 1 H, J = 1.6
Hz), 4.88 (d, 1 H, J = 1.7 Hz), 4.30-3.87 (m, 19 H), 3.80 (ddd, 1
H, J = 2.3, 4.7, 10.1 Hz), 3.74 (dt, 1 H, J = 10.0, 5.5 Hz), 3.48 (dt,
1 H, J = 10.0, 5.8 Hz), 3.00-2.97 (br s, 8 H), 2.18, 2.17(1),
2.16(6), 2.14, 2.12(1), 2.11(7), 2.11(2), 2.11(1), 2.10(5), 2.10, 2.09,
2.06, 2.05, 2.04, 2.03, 2.01, 1.97 (17s, 17 ꢀ 3 H), 1.77-1.71 (m, 2
H); APCI-MS m/z 1791.35 [M þ Hþ].
3-(4-Phenyl[1,2,3]triazol-1-yl)propyl 2,3,4,6-Tetra-O-sulfo-r-
D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside, Hexadecasodium Salt (13). Phenyltriazole 23 (45 mg,
27 μmol) was sequentially deacetylated (ESMS of polyol m/z
1014.59 [M þ H]þ) and sulfonated according to the general
procedures and then purified by SEC to give polysulfate 13
(31 mg, 43%) as a white powder: 1H NMR (D2O) δ 8.25-8.19
(m, 1 H), 7.72-7.67 (m, 2 H), 7.41-7.34 (m, 2 H), 7.32-7.27 (m,
1 H), 5.43-4.64 (m, 7 H), 4.55-3.59 (m, 23 H), 3.55-3.49 (m,
1 H), 2.17-2.10 (br s, 2 H).
3-Azidopropyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside (40). 3-Azidopropanol (67 mg, 660 μmol) was glyco-
sylated with peracetate 30 (276 mg, 220 μmol) as described for
compound 34 to give, following flash chromatography (3:2 f 4:1
EtOAc:hexane), glycoside 40 (176 mg, 61%) as an oil: ESMS m/z
1319.69 ([M þ Na]þ); 1H NMR (CDCl3) δ 5.31-5.12 (m, 7 H),
5.00-4.87 (m, 6 H), 4.26-3.94 (m, 11 H), 3.90-3.81 (m, 2 H),
3.77 (dt, 1 H, J = 9.9, 6.0 Hz), 3.47 (dt, 1 H, J = 9.9, 6.0 Hz),
3.42-3.32 (m, 2 H), 2.14(1), 2.13(5), 2.10, 2.08 (ꢀ2), 2.06 (ꢀ2),
2.05, 2.03(2), 2.02(5), 1.99, 1.98, 1.93 (13s, 13 ꢀ 3 H, 13 ꢀ OAc),
1.84 (pentet, 1 H, J = 6.2 Hz); 13C NMR (CDCl3) δ 170.6, 170.5,
170.4, 170.3, 170.1(4), 170.0(9), 169.9(2), 169.8(8), 169.7, 169.6,
169.5(4), 169.4(5), 169.3, 99.4, 98.9, 98.8, 98.1, 76.8, 75.1(5),
75.1(1), 70.9, 70.8, 70.1, 69.6, 69.5, 69.4, 69.2, 68.5, 68.3, 67.3,
66.6, 66.1, 65.5, 64.7, 62.5, 61.9, 61.7, 48.0, 28.5, 20.8(4), 20.7(6),
20.7, 20.6, 20.5(4), 20.5(2), 20.4(9), 20.4(7).
3-(4-Phenyl[1,2,3]triazol-1-yl)propyl 2,3,4,6-Tetra-O-sulfo-r-
D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-
sulfo-r-D-mannopyranoside, Tridecasodium Salt (14). Azide 40
(174 mg, 134 μmol) was reacted with phenylacetylene as
described for compound 19 to give, following flash chromatog-
raphy (4:1 f 17:3 EtOAc:hexane), the phenyltriazole (109 mg,
58%) as an oil, used directly in the next step without further
characterization. This material (109 mg, 78 μmol) was sequen-
tially deacetylated and sulfonated according to the general
procedures and then purified by dialysis (Pierce Slide-A-Lyzer
2000 MW cutoff cartridge, 10 ꢀ 2 L HPLC grade water, pH
adjusted to 8 with 3 M NaHCO3) to give polysulfate 14 (10.5 mg,
3-(5-Dimethylaminonaphthalene-1-sulfonamido)propyl 2,3,4,6-
Tetra-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-
mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-
sulfo-r-D-mannopyranoside (15). Sulfonamide 25 (18 mg, 10.1 μmol)
was sequentially deacetylated and sulfonated according to the
general procedures and then purified by dialysis against purified
water (Pierce Slide-A-Lyzer, 2000 MWCO, 4 ꢀ 5 L) to yield 15 (3
mg, 11%) as a white amorphoussolid:95%purebyCE; 1H NMR
(D2O) δ 8.75-8.69 (m, 1 H), 8.40-8.32 (m, 2 H), 8.03-7.82 (m, 3
H), 5.45-4.67 (m, 7 H affected by irradiation), 4.60-3.52 (m, 15
H affected by irradiation), 3.46 (s, 6 H), 3.07-2.87 (m, 4 H),
1.45-1.30 (m, 2 H).
12-Azidododecanol. To a mixture of 12-bromo-1-dodecanol
(246 mg, 0.927 mmol) in DMF (1.8 mL, 0.5 M) were added
sodium azide (121 mg, 1.855 mmol, 2 equiv) and tetrabutylam-
monium iodide (17 mg, 0.0464 mmol, 0.05 equiv), and the
mixture was stirred at 80 ꢀC for 3 h. The mixture was filtered
through a plug of Celite and the cake rinsed with EtOAc (20
mL). The combined filtrate and washings were evaporated onto
silica gel and purified by flash chromatography (6:1 f 2:1
hexane:EtOAc) to give 12-azido-1-dodecanol as a colorless oil
(193 mg, 92%). The 1H NMR (CDCl3) spectrum was in accord
with the literature.35
12-Azidododecyl 2,3,4,6-Tetra-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-manno-
pyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-mannopyranoside (42).
12-Azidododecanol (1.5 equiv) was glycosylated with trichloro-
acetimidate3318 (0.325 g, 0.197 mmol) as described for compound
36 to give, following flash chromatography (CHCl3 f CHCl3:
MeOH, 99:1 f 98:2), glycoside 42 as a colorless gum containing
1
the byproduct BnNHAc (71.1 mg, 1:1 42:BnNHAc ratio): H
NMR (CDCl3) δ 5.29-5.14 (m, 8 H), 5.01-4.86 (m, 8 H),
4.28-3.75 (m, 19 H), 3.64 (dt, 1 H, J = 9.5, 7.0 Hz, OCH2),
3.39 (dt, 1 H, J = 9.5, 7.0 Hz, OCH2), 3.22 (t, 2 H, J = 7.0 Hz,
NCH2), 2.16, 2.15, 2.11, 2.10, 2.09, 2.09, 2.09, 2.09, 2.07, 2.06,
2.04, 2.04, 2.01, 1.95 (14s, 48 H, 16 ꢀ Ac), 1.57 (m, 4 H, 2 ꢀ CH2),
1.36-1.22 (m, 16 H, 8 ꢀ CH2).

1696 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Johnstone et al.
12-(4-Phenyl[1,2,3]triazol-1-yl)dodecyl 2,3,4,6-Tetra-O-acetyl-
r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-
acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-manno-
pyranoside (26). Azide 42 (71 mg, 41.5 μmol) was reacted with
phenylacetylene (83 μmol, 2 equiv) as described for compound 19
to give, following flash chromatography (1:6 f 3:1 EtOAc:
hexane), phenyltriazole 26 as a colorless gum (46.3 mg, 62%):
1H NMR (CDCl3) δ 7.82 (d, 2 H, J = 7.2 Hz, Ph), 7.75 (s, 1 H,
triazole-CH), 7.41 (t, 2 H, J = 7.2 Hz, Ph), 7.31 (t, 1 H, J = 7.2
Hz, Ph), 5.30-5.15 (m, 8 H), 5.03-4.87 (m, 8 H), 4.38 (t, 2 H, J =
7.2 Hz, NCH2), 4.29-3.77 (m, 19 H), 3.64 (dt, 1 H, J = 9.6, 6.8
Hz, OCH2), 3.40 (dt, 1 H, J = 9.6, 6.8 Hz, OCH2), 2.17, 2.16,
2.16, 2.13, 2.11, 2.11, 2.11, 2.10, 2.09, 2.07, 2.05, 2.05, 2.02, 2.00,
1.96 (15s, 48 H, 16 ꢀ Ac), 1.93 (m, 2 H, CH2), 1.57 (m, 2 H, CH2),
1.34-1.24 (m, 16 H, 8 ꢀ CH2).
affinity assay, as previously described.28,29 Heparin-coated
CM4 sensor chips were prepared via reductive amination of
the reducing end aldehyde with 1,4-diaminobutane, as pre-
viously described.29 Ligands binding to FGF-1 and VEGF were
assessed in HBS-EP buffer [10 mM HEPES (pH 7.4), 150 mM
NaCl, 3.0 mM EDTA, and 0.005% (v/v) polysorbate 20], while
binding to FGF-2 was assessed in HBS-EP buffer containing
300 mM NaCl. Sensorgram data were analyzed using BIAeva-
luation (BIAcore) and Kd values determined as previously
described.29 The Kd values represent the average of at least
duplicate measurements ( the standard deviation. The results
are presented in Table 1.
Growth Factor-Induced Endothelial Cell Proliferation Assay.
(a) Endothelial Cell Culture. HUVECs were maintained and
subcultured according to standard cell culture protocols essen-
tially as described by Lonza (Basel, Switzerland). Briefly, cells
were maintained in Lonza endothelial growth medium (EGM)
with recommended supplements and growth factors [VEGF,
FGF-2, EFG, IGF, hydrocortisone, fetal bovine serum (FBS),
ascorbic acid, heparin, and gentamicin]. Cells were subcultured
when they reached 70-80% confluence by trypsinization and
reseeding in fresh growth medium in new culture vessels at
densities of 2500-5000 cells/cm2 of vessel surface area. Cell
counts were performed using a hemocytometer, and viable cells
were visualized with trypan blue. Medium for the proliferation
studies was prepared using EGM with 2% FBS and gentamicin
only. Test compounds were weighed out from powder stocks
and diluted in PBS to 10 mM stock solutions and stored at
-80 ꢀC. For experiments, compounds were subsequently di-
luted in EBM-2 medium (supplemented with 2% FBS and
gentamicin) to various working concentrations as required. (b)
Proliferation Assay. Proliferation was induced in HUVECs
using various concentrations of growth factor VEGF, FGF-1,
or FGF-2 over a period of 72 h. In the first of a series of
experiments, the assay was further optimized by examination of
the cell density and growth factor concentration required to
induce maximal proliferation by growth factors. Briefly, 100 μL
of cells was added to each well at concentrations between 1 and 3
ꢀ 103 per well. Growth factors and test compounds were then
added in 50 μL volumes at specified concentrations (typically
seven ranging from 1 to 50 μM in triplicate) to yield a final
volume of 200 μL. Following incubation for 70 h, 20 μL of the
CellTiter 96 Aqueous One Solution Cell Proliferation Assay
(Promega) was added for 2 h prior to the absorbance at 490 nm
being read, yielding OD values. Once these values were sub-
tracted from the blanks (medium only), the data were imported
into BIAevaluation to determine IC50 values for each curve. The
data are presented in Figure 1.
Matrigel Microtubule Formation Assay. The tube formation
assay was performed essentially as described by Malinda et al.,37
with modifications. HUVECs in the fourth or fifth passage at
70-80% confluence were harvested and resuspended in Lonza
endothelial growth medium (EGM2) containing all supple-
ments as directed by the manufacturer, except heparin, at a cell
density of 4 ꢀ 105 cells/mL. For each set of triplicate wells,
200 μL of cells (4 ꢀ 105 cells/mL) was treated with an equal
volume of compound to produce final concentrations of 10, 50,
and 100 μM (thus ensuring 1 ꢀ 300 μL is available for each
condition). A 100 μL aliquot of cells was then plated onto 96-
well plates precoated with growth factor-reduced Matrigel
(50 μL for 30 min followed by a further 30 μL for 1 h) and
incubated for 18 h. Tube formation was examined by phase-
contrast microscopy, and images were collected using an Olym-
pus C5050 digital camera. Tube formation inhibition was
quantified manually from images by recording the total number
of branching nodes connecting three or more tubules. Results
are expressed as the number of branching nodes for individual
experiments (Figure 2a) or percentage inhibition compared to
control to facilitate interexperiment comparison (Figure 2b).
Untreated HUVECs were used as a control for normal cell
12-(4-Phenyl[1,2,3]triazol-1-yl)dodecyl 2,3,4,6-Tetra-O-sulfo-
r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-
(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-
r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-mannopyranoside,
Hexadecasodium Salt (16). Phenyltriazole 26 (46mg, 25.4μmol) was
sequentially deacetylated and sulfonated according to the general
procedures and then purified by SEC to give polysulfate 16 as a
1
white powder (45 mg, 64%): 94% pure by CE; H NMR (D2O,
internal DOH at 4.60 ppm) δ 7.88 (s, 1 H, triazole-CH), 7.47-7.44
(m, 2 H), 7.21-7.10 (m, 3 H), 5.23 (br s, 1 H), 5.19 (d, 1 H, J =
1.5Hz), 5.12 (d, 1 H, J = 1.8 Hz), 5.10 (d, 1 H, J =1.5Hz), 4.91 (m,
1 H), 4.76-3.72 (m, 32 H), 3.37-3.30 (m, 1 H, OCH2), 3.23-3.17
(m, 1 H, OCH2), 1.51 (m, 2 H, CH2), 1.12 (m, 2 H, CH2), 0.90-0.63
(m, 16 H, 8 ꢀ CH2).
12-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)dodecyl 2,3,4,6-Tetra-
O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-manno-
pyranosyl-(1f3)-2,4,6-tri-O-acetyl-r-D-mannopyranosyl-(1f3)-2,4,
6-tri-O-acetyl-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-acetyl-r-D-
mannopyranoside (27). Azide 42 (86 mg, 50.3 μmol) was reacted
with 1-ethynylnaphthalene (83 μmol, 2 equiv) as described for
compound 20 (11 days). The mixture was then evaporated onto
silica gel and purified by flashchromatography (1:6 f 3:1 EtOAc:
hexane) to give naphthyltriazole 27 as a colorless gum (24.2 mg,
26%): 1H NMR (CDCl3) δ 8.38-8.33 (m, 1 H), 7.92-7.86 (m, 2
H), 7.80 (s, 1 H, triazole-CH), 7.72 (dd, 1 H, J = 7.3, 1.5 Hz),
7.54-7.48 (m, 3 H), 5.31-5.15 (m, 8 H), 5.03-4.87 (m, 8 H), 4.47
(t, 2 H, J = 7.3 Hz, N-CH2), 4.30-3.77 (m, 19 H), 3.64 (dt, 1 H,
J = 9.7, 6.8 Hz, OCH2), 3.40 (dt, 1 H, J = 9.7, 6.8 Hz, OCH2),
2.17, 2.16, 2.16, 2.13, 2.12, 2.11, 2.11, 2.10, 2.09, 2.07, 2.06, 2.05,
2.02, 2.00, 1.97, 1.57 (16s, 48 H, 16 ꢀ Ac), 2.07-1.95 (m, over-
lapped with Ac singlets, 2 H, CH2), 1.57 (m, 1 H, CH2), 1.42-1.23
(m, 16 H, 8 ꢀ CH2).
12-(4-Naphthalen-1-yl[1,2,3]triazol-1-yl)dodecyl 2,3,4,6-Tetra-
O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-manno-
pyranosyl-(1f3)-2,4,6-tri-O-sulfo-r-D-mannopyranosyl-(1f3)-2,4,
6-tri-O-sulfo-r-D-mannopyranosyl-(1f2)-3,4,6-tri-O-sulfo-r-D-
mannopyranoside, Hexadecasodium Salt (17). Naphthyltriazole
27 (39.6 mg, 0.0213 mmol) was sequentially deacetylated and
sulfonated according to the general procedures and then pur-
ified by SEC to give polysulfate 17 as a white powder (40 mg,
1
68%): H NMR (D2O, internal DOH at 4.60 ppm) δ 8.05 (s,
1 H, triazole-CH), 7.97 (d, 1 H, J = 8.3 Hz), 7.90 (d, 2 H, J =
7.8 Hz), 7.55-7.40 (m, 4 H), 5.44-5.22 (m, 4 H), 5.09-3.82 (m,
33 H), 3.41-3.33 (m, 1 H, OCH2), 3.25-3.16 (m, 1 H, OCH2),
1.78 (quintet, 2 H, CH2), 1.14-0.79 (m, 18 H, 9 ꢀ CH2).
Heparanase Inhibition Assay. The inhibitory potencies of the
compounds toward recombinant human heparanase36 were de-
termined using a Microcon ultrafiltration assay, which utilizes
3H-labeled HS as the substrate (Km = 1.64 μM), as previously
described.20 The results are presented in Table 1.
Growth Factor Binding Assays. The binding affinities of the
compounds for growth factors FGF-1, FGF-2, and VEGF were
determined on a BIAcore 3000 instrument (BIAcore, Uppsala,
Sweden) using a surface plasmon resonance-based solution

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1697
growth and tube formation in Matrigel. Representative images
are shown in Figure 2c.
artery cannulations were performed on the day prior to dosing
to allow iv dosing and blood sampling, respectively. Compound
5, formulated as a 2.8 mg/mL solution in PBS, was infused
intravenously over a period of 5 min (1.0 mL/rat; n = 2 rats) or
injected subcutaneously between the scapulae (1.0 mL/rat; n =
2 rats) to achieve a nominal dose of 10 mg/kg. Samples of blood
[collected from the arterial cannula using a Culex autosampler
(BASi)] and total urine were collected periodically for up to 8
and 24 h postdose, respectively. To minimize the potential for
ex vivo degradation of 5 in blood/plasma samples, blood samples
were collected directly into heparinized borosilicate vials (at 4 ꢀC)
containing Complete (a protease inhibitor cocktail, 80 mg/mL,
Roche, Mannheim, Germany), potassium fluoride (4 mg/mL),
and EDTA (0.1 M). Once collected, blood samples were centri-
fuged, and the plasma supernatant was removed and stored at
-20 ꢀC for subsequent analysis by LC-MS.
Ex Vivo Rat Aortic Angiogenesis Assay. Explants from rat
aortas were prepared by a modification of protocols previously
described.38-41 Briefly, thoracic aortas were excised from
2-4-month-old Sprague-Dawley rats and trimmed of remaining
fat and connective tissue. Great care was taken at every stage to
reduce physical damage of the aorta. Tissue was transferred to
complete EBM-2 medium (Cambrex) containing 2% FCS and
all singlequots (Cambrex) reagents except for heparin. Mean-
while, Matrigel (BD Biosciences) was allowed to cool on ice, and
once in a liquid form, 180 μL was pipetted into 48-well tissue
culture plates (Nunc). The plates were incubated at 37 ꢀC for
30 min to allow Matrigel to solidify.
We prepared aortas by cutting 1 mm ring sections and then
bisecting them. Aortic segments were then carefully placed on top
on the Matrigel in the center of each well, and once they were
orientated as required, 60 μL of extra Matrigel was placed on top
and the plate returned to the incubator for a further 20 min. Each
well was then supplemented with 1.0 mL of medium in the
absence (control) or presence of test compounds usually at two
concentrations within the range of 1-50 μM, depending on the
particular compound or experiment. Cultures were replenished as
appropriate every 48 h, and scoring of microvessels was con-
ducted at various time points up to 8 days by two assessors. The
extent of microvessel sprouting was determined as previously
described41 and expressed as a semiquantitative score. Sprouting
vessels were photographed using the 4ꢀ objective with an Olym-
pus C-7070 camera and an adaptor for the eyepiece.
The terminal elimination half-life, total plasma clearance, and
apparent volume of distribution were estimated by standard
noncompartmental analysis using WinNonlin (version 4.0,
Pharsight Corp., Mountain View, CA).
Analysis of Compound 5 in Rat Plasma. LC-MS analysis of 5
was performed on a Waters Micromass Quattro Ultima Pt triple
quadrupole instrument coupled with a Waters 2795 HPLC
system. Mass spectrometry was performed with negative mode
electrospray ionization with a capillary voltage of 2.7 kV, a
detector multiplier gain of 650 V, and source block and desolva-
tion temperatures of 90 and 350 ꢀC, respectively. Elution of the
analyte was monitored using selected ion recording (SIR) using
the combined signals of the seven strongest doubly charged
fragment ions (m/z 708.5, 668.5, 628.5, 588.5, 548.5, 508.5, and
468.5) corresponding to the loss of 5-11 sulfate units from the
molecular ion of 5. The molecular ion was not observed due to
extensive in-source fragmentation. Chromatographic separa-
tion was performed on a Shodex Ohpak 6 μm SB-802.5 HQ 80
In some instances, to determine the potential toxicity of
compounds in this assay, we assessed the viability of the tissue
by withdrawing the compound or medium from the culture on
day 6 or 7 and adding complete medium with VEGF (typically
10 ng/mL) for up to an additional 7 days. In the absence of
toxicity, the viable tissue should sprout microvessels in response
to the exogenous growth factor.
˚
A 250 mm ꢀ 2 mm column equipped with a 0.22 μm particle
filter. The mobile phase consisted of 20% acetonitrile, 50%
ammonium formate (0.1 M), and 30% water delivered at a rate
of 0.6 mL/min. Leucine enkaphalin was used as the internal
standard.
Anticoagulant Activity. The anticoagulant activity of the test
compounds was determined by measuring the effect of various
concentrations of the compound (0-100 μg/mL in PBS) on the
elevation of the APTT of pooled normal human plasma. APTT
measurements were performed on a STAGO STA-Compact
Coagulation Analyzer using standard protocols according to
the manufacturer’s instructions. Unfractionated heparin (UFH)
was used as the control. The normal range of APTT for pooled
normal human plasma is 26-36 s. The results are presented in
Table 1. The coefficient of variation for APTT coagulation
values of 36 s is 2% and for coagulation values of g90 s is 5-7%.
The compounds were also evaluated in the heptest42 as
follows. Venous blood was collected by clean venipuncture
using 3.8% sodium citrate as an anticoagulant. One volume of
anticoagulant was gently mixed with 9 volumes of blood in
plastic tubes. Plasma was prepared by centrifugation at 1800g
for 10 min. Plasma samples were analyzed within 30 min; 50 μL
of plasma was incubated with 10 μL of each anticoagulant
thereafter for 2 min at 37 ꢀC, and 50 μL of factor Xa
(Haemachem, St. Louis, MO) was added. After incubation for
an additional 2 min at 37 ꢀC, coagulation was induced by
addition of 50 μL of Recalmix (Haemachem). Clotting assays
were performed with a ball coagulameter KC10 (Amelung,
Lemgo, Germany). Heptest values in normal plasma samples
ranged from 13 to 20 s (mean of 17.1 ( 2.1 s). The coefficient of
variation for heptest coagulation values is 1.8% for values of
20-90 s and 3-4% for values of >90 s.
Plasma samples were prepared for analysis by protein pre-
cipitation using acetonitrile under alkaline conditions. Solution
standards were diluted from a stock solution (prepared in a 10%
methanol/water mixture) with a 10% methanol/water mixture.
Plasma standards were prepared by spiking blank plasma
(100 μL) with solution standards (20 μL in a 10% methanol/
water mixture) and the internal standard (IS) leucine enkephalin
(20 μL in a 50% acetonitrile/water mixture) to give a final IS
concentration of 0.2 μg/mL. Protein precipitation for plasma
samples was conducted via the addition of sodium hydroxide (40
μL, 0.1 M) and acetonitrile (180 μL) followed by vortexing (60 s)
and centrifugation (10000 rpm) in a microcentrifuge for 3 min.
The supernatant was transferred to a clean Eppendorf tube and
freeze-dried. Subsequently, the residue was reconstituted in
10% aqueous methanol (100 μL) and 50 μL injected onto the
column for LC-MS analysis. Calibration standards were pre-
pared in the range of 20-400 μg/mL. Plasma samples from the
dosing studies were prepared similarly except the solution
standard was replaced with a blank solvent. Extraction recovery
from rat plasma was confirmed by comparison of the peak area
of the spiked plasma samples to the peak area of the control
sample where the spike was added to a blank plasma extract.
In Vivo Mouse Melanoma Model. B16F1 cells were cultured in
complete DMEM medium containing 10% FCS, penicillin/
streptomycin, L-glutamine, sodium pyruvate, and 2-mercap-
toethanol. Cells were harvested for tumor inoculation, B16F1
cells by disruption with trypsin/EDTA, washed with HBSS, and
centrifuged for 5 min at 1500 rpm. Cells were then resuspended
in PBS to ensure 5 ꢀ 105 cells were injected in a volume of 50 μL.
The tumor was implanted between the shoulder blades. Three
days following tumor inoculation, each treatment group was
Pharmacokinetic Studies. Studies were conducted in accor-
dance with the Australian and New Zealand Council for the
Care of Animals in Research and Training Guidelines and were
approved by the Monash University animal experimentation
ethics committee. Adult male Sprague-Dawley rats (weighing
279-303 g) were supplied by Monash University Animal Ser-
vices (Clayton, Victoria, Australia). Jugular vein and carotid

1698 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Johnstone et al.
injected subcutaneously at different sites each day and at a dose
of 30 mg/kg twice daily for 7 days in a volume of injection of
100 μL. Injections continued until day 15, providing a 12-day
treatment period. Mice were monitored daily from the start of
injections, and palpable tumors were measured daily. Tumor
size was determined from the measurement in two dimensions,
l ꢀ w, where l is the longest dimension and w the shortest
dimension. To estimate tumor volume, the formula 0.5l(w2)
was employed. Data are presented as the median tumor growth
(Figure 4) and were analyzed by a one-way analysis of variance
(ANOVA) followed by a post hoc Newman-Keuls multiple-
comparison test (Graphpad Prism, version 3). Data are pre-
sented as means ( the standard error of the mean. Differences
with a p value of less than 0.05 were considered statistically
significant. The percentage of tumor growth inhibition (%TGI)
was also calculated to correct for interexperimental differences.
TGI values were calculated using the formula TGI = (1 - ΔT/
ΔC) ꢀ 100, where ΔT and ΔC represent the change in mean
tumor mass between the last day of therapy and the first day of
therapy in the sample compound-treated (T) and vehicle control
(C) groups, respectively.
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Acknowledgment. We thank Shane Jimmink and Danielle
Wilson for technical assistance and Anand Gautam for discus-
sions on the tumor models. We also thank Peter Gambell (Peter
MacCallum Cancer Institute, Melbourne, Australia) for carry-
ing out repeat APTT measurements and David Hume, Keith
Watson, and Ron Dickenson for useful discussions.
Supporting Information Available: Copies of 1H and 13C
NMR spectra for selected new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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