Synthesis of a Heparan Sulfate Mimetic Library Targeting FGF and VEGF via Click Chemistry on a Monosaccharide Template

Article abstract of DOI:10.1002/cmdc.201200151

A disulfated methyl 6-azido-6-deoxy-α-D-mannopyranoside template was used as a core structure for binding to the angiogenic growth factors FGF-1, FGF-2, and VEGF. The core structure was diversified in a rapid, parallel manner by employing the Cu^I-catalyzed Huisgen azide-alkyne cycloaddition ("click") reaction. The diversity was further extended by incorporating a Swern oxidation-Wittig reaction sequence on a click adduct of propargyl alcohol. Thus, the sulfated core was linked by various spacers to selected hydrophobic or polar motifs, which were designed to probe the protein surface surrounding the cationic heparan sulfate binding sites of the growth factors in order to improve affinity and selectivity. The affinities of the compounds for the growth factors were measured by surface plasmon resonance solution affinity assays. A lead compound was identified with micromolar binding affinity toward both FGF-1 and VEGF (K_d=84 and 49μM, respectively) and good selectivity over FGF-2 (29- and 51-fold, respectively).

Full text of DOI:10.1002/cmdc.201200151

MED
DOI: 10.1002/cmdc.201200151
Synthesis of a Heparan Sulfate Mimetic Library Targeting
FGF and VEGF via Click Chemistry on a Monosaccharide
Template
Ligong Liu,*[a, b] Caiping Li,[a] Siska Cochran,[a] Shane Jimmink,[a] and Vito Ferro*[a, c]
A
disulfated methyl 6-azido-6-deoxy-a-d-mannopyranoside
motifs, which were designed to probe the protein surface sur-
rounding the cationic heparan sulfate binding sites of the
growth factors in order to improve affinity and selectivity. The
affinities of the compounds for the growth factors were mea-
sured by surface plasmon resonance solution affinity assays. A
lead compound was identified with micromolar binding affinity
toward both FGF-1 and VEGF (Kd =84 and 49 mm, respectively)
and good selectivity over FGF-2 (29- and 51-fold, respectively).
template was used as a core structure for binding to the an-
giogenic growth factors FGF-1, FGF-2, and VEGF. The core
structure was diversified in a rapid, parallel manner by employ-
ing the CuI-catalyzed Huisgen azide–alkyne cycloaddition
(“click”) reaction. The diversity was further extended by incor-
porating a Swern oxidation–Wittig reaction sequence on
a click adduct of propargyl alcohol. Thus, the sulfated core was
linked by various spacers to selected hydrophobic or polar
Introduction
Heparan sulfate (HS) is a member of the glycosaminoglycan
family and is ubiquitously present on mammalian cell surfaces
and in the extracellular matrix. Structurally, HS is a linear poly-
anionic polymer that consists of repeating disaccharide se-
quences of 1!4 linked uronic acid and d-glucosamine. Much
structural diversity is introduced into HS during its biosynthe-
sis, e.g., by epimerization at C5 of the uronic acid, N-acetyla-
tion or N-sulfation of glucosamine, and O-sulfation at O2 of
the uronic acid and/or O6 and O3 of glucosamine.[1] Such di-
verse structures facilitate HS binding to a variety of proteins
and lead to its important role in numerous physiological and
pathological processes, including angiogenesis, cancer, inflam-
mation, and infectious diseases.[2] However, such a broad pro-
tein binding spectrum also hinders the clinical application of
HS and the related heparin as drugs because of unwanted side
effects. Therefore it is of interest to develop selective HS mim-
etics that can differentiate specific structural motifs of target
HS binding proteins.
Fibroblast growth factors 1 and 2 (FGF-1 and -2)[3] and vas-
cular endothelial growth factor (VEGF)[4] are intimately involved
in angiogenesis associated with tumor growth and metastasis.
Their interactions with their corresponding receptors are medi-
ated by HS to initiate subsequent intracellular responses. Inhib-
ition of HS binding to FGFs or VEGF could therefore be an ef-
fective strategy for the prevention or mitigation of tumor-in-
duced angiogenesis.[5] A recent study indicated that one possi-
ble mechanism of tumor escape from anti-VEGF therapy is the
up-regulation of FGF-2. Thus an anti-FGF-2 approach could be
used as a strategy to compensate for resistance to anti-VEGF
therapy.[6] This further suggests the importance of developing
selective ligands or cocktails of selective ligands that target
these HS binding proteins.
Many classes of structurally diverse HS mimetics have been
shown to bind FGF-(1,2) and/or VEGF. Apart from polyanionic
polymers such as sulfated polysaccharides,[7] poly(N-acrylamino
acids),[8] sulfonic acid polymers,[9] hydroxylated and carboxylat-
ed polyaromatic compounds,[10] and oligoribonucleic acids,[11]
small-molecule candidates derived from dipeptides,[12] glyco-
conjugates,[13] suramin and its analogues,[14] sulfated pep-
tides,[15] and heparin-like oligosaccharides[1c,16] also compete
with HS to block cell signaling and proliferation in vitro. The
above studies have provided some knowledge of the location,
number, and pattern of sulfates required for binding, but have
also pointed to the importance of hydrogen bonding and hy-
drophobic interactions.[17] To probe such structural variables,
the most popular approach has been to use heparin-like oligo-
saccharides, which have inherent challenges due to their com-
plex syntheses. Therefore, there is significant interest in the de-
velopment of simpler approaches to HS mimetics that can de-
liver advantages such as A) robust chemistry, ideally tolerant to
various functional groups and with easy incorporation of di-
verse motifs into the core structure; B) simple, scalable, and
[a] Dr. L. Liu, Dr. C. Li, Dr. S. Cochran, S. Jimmink, Dr. V. Ferro
Drug Design Group, Progen Pharmaceuticals Limited
2806 Ipswich Road, Darra, QLD 4076 (Australia)
[b] Dr. L. Liu
Institute for Molecular Bioscience
The University of Queensland, Brisbane, QLD 4072 (Australia)
E-mail: l.liu@imb.uq.edu.au
[c] Dr. V. Ferro
School of Chemistry and Molecular Biosciences
The University of Queensland, Brisbane, QLD 4072 (Australia)
E-mail: v.ferro@uq.edu.au
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200151: detailed characterization
data for all intermediates and sulfated final products.
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L. Liu, V. Ferro, et al.
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environmentally friendly reactions; C) simple and efficient pu-
rification steps; and D) suitability for parallel operations.
mine complex (3 equiv per hydroxy group) gave the parent tri-
sulfate 19 in moderate yield (47%, Scheme 1). Both FGF-1 and
-2 possess a primary ionic HS binding site enriched with basic
amino acids such as Lys and Arg. In previous docking studies,
particularly with the cyclitol analogues 1, this site is preferen-
tially occupied by the vicinal cis-disulfates in the six-membered
ring.[18b,23] Decreasing the number of sulfates to one proved to
be detrimental for binding affinity.[24] To investigate the influ-
ence of the sulfation pattern on binding, we maintained two
sulfates in the ring, but capped the third hydroxy group with
a hydrophobic group. The benzyl ether group was initially se-
lected (compounds 20, 29, and 30) for this purpose,
Over the last decade, we have explored HS mimetics based
on cyclitol[18] or a-d-mannopyranose building blocks[19] as
probes for HS binding proteins and confirmed that some selec-
tivity can be obtained either by varying the spacer between
two polyanionic motifs or introducing hydrophobic groups
onto a central polyanionic core. The above two cyclic scaffolds
share a common pattern of presenting sulfates around the
ring, including a pair of cis-oriented vicinal sulfo groups. This is
illustrated in Figure 1, which shows the cyclitol 1 and manno-
based mainly on the ease of access to all three ana-
logues as well as its aromatic and hydrophobic prop-
erties desirable for probing the surrounding nonionic
surface. The 4-O-benzyl analogue 20 was prepared
by benzylation of 2,3-O-isopropylidene 6[25] followed
by hydrolysis and sulfation. Another common ap-
proach to monobenzylated carbohydrates is through
regioselective reductive ring opening of the corre-
sponding benzylidene acetals.[26] The 2,3-O-benzyli-
dene 26 was thus prepared[27] and subjected to re-
duction with sodium cyanoborohydride. The reaction
was very sluggish and offered only a moderate yield
Figure 1. Comparison of the cyclitol 1 and methyl 6-azido-6-deoxy-a-d-mannopyranoside
templates 2–4: varied presentation of sulfate patterns (OX), hydrophobic substituent
(OR) and linkage point for derivatization.
pyranose 2–4 (where X=SO3Na) core structures and the spatial
relationship between their respective sulfo groups. Therefore,
to further expand the search for VEGF and FGF inhibitors, we
selected methyl 6-azido-6-deoxy-a-d-mannopyranoside (5) as
a building block for the synthesis of a library of HS mimetics
targeted against these growth factors with the specific aims to
A) minimize the number of sulfates in the monosaccharide ring
to help identify critical sulfate groups and decrease overall
charge, and B) to explore any nonionic binding surfaces
around the primary cationic binding site. This resulted in the
selection of three template structures in order to identify the
optimal placement of the two sulfates, i.e., 2 (3,4-), 3 (2,4-),
and 4 (2,3-di-O-sulfo) (Figure 1).
of the corresponding monobenzyl ether products with low se-
lectivity. The slow and incomplete reaction could be due to
the presence of the unprotected 3-hydroxy group. With tri-
fluoroacetic acid (TFA) as electrophile, at room temperature,
only a small amount of 2-O-benzylated product 27 was ob-
served and separated (2.7%), with the majority being recov-
ered starting material (44%). At elevated temperature (708C),
the reaction afforded two separable regioisomers in equal
amounts (26% each) plus 26% of recovered starting material.
Replacement of TFA with hydrochloric acid (1m solution in di-
ethyl ether) offered a slightly improved selectivity for 2-O-
benzyl isomer 27 (24 versus 15%) at room temperature; how-
The azido group provides a link-
ing point for rapid derivatization
of the scaffold via the CuI-cata-
lyzed Huisgen [3+2] azide–
alkyne cycloaddition or “click” re-
action, one of the most powerful
synthetic methods for building
diverse libraries,[20] including in
the glycomics area.[21]
Results and Discussion
Selection of sulfation pattern
Methyl
6-azido-6-deoxy-a-d-
mannopyranoside
5
was pre- Scheme 1. Reagents and conditions: a) 2,2-dimethoxypropane (0.3m), CSA (5 mol%), RT, 3 h, 100%; b) 1. NaH
(3 equiv), DMF, RT, 1 h; 2. RBr or RCl or RI (3 equiv), RT, 6 h, 48–97%; c) p-TsOH (20 mol%), MeCN/MeOH/H2O
pared from methyl a-d-manno-
pyranoside by following a pub-
lished procedure.[22] Sulfation
(10:10:1), 408C, 3 h, 97–100%; d) 1. SO3·Me3N complex (3 equiv per OH), DMF, 608C, 6–18 h; 2. 08C, pH 10 with
5m NaOH and sat. aq. Na2CO3; 3. silica column chromatography, SPE with Strata-NH2 or Strata-X, 3–89%; e) PhCH-
(OMe)2 (2 equiv), CSA (0.2 equiv), DMF/MeCN (1:1), 608C, 18 h, 38%; f) NaBH3CN (12 equiv), MS (3 ꢁ), TFA (6 equiv),
with sulfur trioxide trimethyla- DMF, 708C, 6 h, 27 (26%), 28 (26%), and recovered SM 26 (26%).
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at C6 with a spacer of varying structure and
length.
Table 1. Dissociation constants for sulfated products binding to FGF-(1,2) and VEGF as
measured by SPR solution affinity assays.
Compd Yield[a]
tm [min][b] Purity [%][b]
Kd [mm][c]
FGF-1
FGF-2
VEGF
Click library
19
24
29
30
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
47[d]
89[d]
59[d]
66[d]
100[f]
71
7.8
8.9
ND
ND
7.1
100
99
343ꢀ19
210ꢀ15
630ꢀ100
470ꢀ60
The alkynes used for click coupling to the mono-
saccharide azide were either commercially avail-
able or were easily prepared from acylation of
propargylamine or alkylation of various alcohols
with propargyl bromide to provide a diverse li-
brary (Scheme 2). The rationale behind the choice
of each alkyne derivative was based on A) availa-
bility of the reagents; B) structural diversity in
order to target diverse features surrounding the
primary ionic binding site, i.e., size and location of
hydrophobic, charge/dipole, or hydrogen bonding
groups; C) linker groups such as amides, ethers, or
alkenes in order to control rigidity; and D) feed-
back from binding assays.
880ꢀ100 >4300
>95[e]
>95[e]
100
>95[e]
99
5400ꢀ1300
>2500
2500ꢀ300 12900ꢀ2700
18000ꢀ4000 10600ꢀ1100
270ꢀ70
710ꢀ40
320ꢀ40
>1000
ND
>700
>1000
ND
>500
ND
260ꢀ50
84
12.4
23.8
11.8
13.9
264ꢀ24
64[f]
22
99
96
95
89
99
95
99
94
99
96
100
100
98
>500
ND
4200ꢀ500
107ꢀ26
>500
850ꢀ200
>500
77
>3200
78, 99[g] 14.7
1370ꢀ300 >1000
1500ꢀ300 >2900
45
63
69
27
69
15
40[g]
39
67
46
23
17.6
16.3
13.8
19.7
13.4
21.8
8.1
13.1
14.0
13.2
13.8
750ꢀ60
610ꢀ70
260ꢀ50
170ꢀ30
280ꢀ60
49ꢀ9
1100ꢀ90
340ꢀ30
1200ꢀ70
340ꢀ90
84ꢀ22
>2800
>3100
>2900
1800ꢀ600
>2500
>1000
>3200
>500
>500
The click library could be assembled via three
alternative routes with similar overall yields as
highlighted in Scheme 3 by starting from three
different azide building blocks. Route A from disul-
fate 24 gave direct and rapid access to the final
products. Purification proved challenging due pri-
marily to the very polar nature of starting materi-
als, products, and reagents (such as ascorbate
used to generate CuI in situ). However, an effective
protocol using solid-phase extraction (SPE: C18 or
Strata-NH2 cartridge) was developed and adapted for parallel
purification of the library. Route B from diol 17 gave quantita-
tive yields and easy purification of intermediates by conven-
tional flash chromatography. A final sulfation to give the de-
sired product was straightforward. Route C from acetal 11 pro-
vided protected diol intermediates, which could be used for
further structural modifications (e.g., via a Swern–Wittig ap-
550ꢀ50
>5000
3000ꢀ600
>4000
380ꢀ60
1200ꢀ200 >3100
3000ꢀ400 >3100
97
99
129ꢀ25
>2500
[a] Yield of click coupling step via Route A unless noted otherwise. [b] Determined by
CE. [c] Values represent the mean ꢀSD of at least two measurements. ND: no data avail-
able; ’>’ signs indicate Kd value is above the maximum tested concentration. [d] Yield
of sulfation step. [e] Purity by 1H NMR spectroscopy. [f] Route B. [g] Route C.
ever, TFA remained the electrophile of choice because it is
easier to handle. Standard sulfation gave the corresponding di-
sulfates 29 and 30 in good yield (59 and 66%, respectively).
Initial binding studies with FGF-(1,2) and VEGF (Table 1) indi-
cated the trisulfate 19 had moderate selectivity and binding af-
finity to all three proteins (Kd: 210–630 mm). Both 3,4- and 2,4-
disulfates 29 and 30 were totally inactive, but 2,3-disulfate 20
showed activity similar to that of
trisulfate 19 toward VEGF and
FGF-1, but bound weakly to
FGF-2 (Kd =3.30 mm). Therefore,
the template with the 2,3-disul-
fate was selected for further in-
vestigation. A small series of an-
alogues with aromatic or alkyl
substitution at the 4-position
were assembled (21–25) using
a similar synthetic protocol for
20. Preliminary SAR analysis indi-
cated that the allyl group (com-
pound 24) further improved the
affinity and selectivity for VEGF.
Based on the above observa-
Scheme 2. Diverse alkyne blocks: A) commercially available blocks; B) acylation of propargylamine; C) O-alkylation
tions, a click library based on of alcohol with propargyl bromide; D) synthesis of acid starting materials. Reagents and conditions: a) RCOCl
(1 equiv) or toluenesulfonyl chloride (1 equiv), propargylamine (1 equiv), Et3N (1.2 equiv), CH2Cl2, RT, 18 h; or
scaffold 24 was quickly assem-
RCO2H (1 equiv), propargylamine (1 equiv), EDCI (1.2 equiv), Et3N (2 equiv), DMAP (cat.), CH2Cl2, RT, 18 h; or propar-
bled by incorporating either hy-
gylamine (1 equiv), phthalic anhydride (1 equiv), THF, RT, 18 h, 31–85%; b) NaH (1.5 equiv), propargyl bromide
drophobic (substituted aromatic-
(1.5 equiv), DMF, RT, 18 h, or K2CO3 (1.1 equiv), propargyl bromide (1.1 equiv), RT, 3 days, 50–88%; c) 1. NaH (60%,
or alkyl-type) or polar groups 2.1 equiv), RBr (2 equiv), DMF, RT, 18 h; 2. NaOH (1m), RT, 18 h; 3. conc. HCl, 49 (31%) and 50 (3.7%).
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Click–Swern–Wittig library
To further diversify the click li-
brary, particularly to introduce
some rigidity within the linker
between the disulfate core and
the remote functional motif,
a strategy was explored involv-
ing conversion of the primary al-
cohol 69 to the corresponding
aldehyde 70 followed by Wittig
olefination (Scheme 4). It is well
known that the selectivity of the
Wittig olefination[28] can be con-
trolled to obtain either of two
geometric isomers by choosing
the phosphine auxiliaries. At this
stage, only the most common
triphenylphosphine was investi-
gated.
During the initial investiga-
tions, the commercially available
and stable Wittig reagent methyl
(triphenylphosphoranylidene)a-
cetate was used to trap the in-
situ-generated aldehyde 70. The
Wittig reaction was carried out
at room temperature for 30 min
to give exclusively (E)-72 in 74%
isolated yield. Following this en-
couraging result, a small set of
compounds was designed to
mainly include aromatic motifs.
Scheme 3. The assembly of a compound library by click reaction: A) Synthetic routes; B) structures of the 2,3-dis-
ulfated mannosides. Reagents and conditions: a) CuSO4 (aq. 0.3m, 5 mol%), sodium ascorbate (aq. 1m, 20 mol%),
tBuOH/H2O (1:1, 1m), RT, 18 h, Route A: 15–84%, Route B,C: 86–100%; b) SO3·Me3N complex (3 equiv per OH),
DMF, 608C, 6–18 h, 40–87%; c) p-TsOH (20 mol%), MeCN/MeOH/H2O (10:10:1), 408C, 3 h, 97–100%.
proach; see below). The modest yields of the disulfated prod-
ucts derived from Routes B and C were generally due to in-
complete sulfation. In some cases, monosulfated products
were isolated. In our hands the critical factor concerning the
success of the click reaction was the concentration, although
the reaction can also be influenced by the pH. In general, the
optimum concentration based on the azide was 0.15–0.35m.
Thus the amount of solvent was kept to a minimum as long as
practical for operation and solubility of the reagents.
The Wittig reagents were obtained either from commercial
sources or were synthesized by reaction of the corresponding
alkyl bromide with triphenylphosphine followed by base-in-
duced (sodium hydride or n-butyllithium) ylide formation. The
crude mixtures of Wittig reagents showed characteristic color
changes after addition of the base, i.e., colorless solution or
white suspension to instant bright-yellow to red. The Wittig
coupling was carried out in parallel with freshly prepared alde-
hyde solution (70 in CH2Cl2), split and transferred into the
above freshly prepared Wittig reagent vessels (all operations
Scheme 4. Assembly of Swern–Wittig library. Reagents and conditions: a) 1. (COCl)2 (3.1 equiv), DMSO (6.5 equiv), CH2Cl2, ꢁ788C, 1 h; 2. 69 in CH2Cl2, ꢁ788C,
1.5 h; 3. Et3N (6.5 equiv), ꢁ788C, 1 h then warm to RT; b) Ph3P=CHR (2–13 equiv), PhMe or DMF or NaH, THF, RT, 1–18 h; c) 1. standard hydrolysis of acetonide;
2. standard sulfation.
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were carried out at room temperature). The reaction mixture
was purified by flash chromatography. However, in the cases of
the presence of two geometric isomers, they were generally
difficult to separate. Thus, various conditions (solvent and
base) were investigated to improve the selectivity of Z/E olefin
formation, and the best results are summarized in Table 2. The
geometry of the products was assigned based on characteristic
Growth factor binding
The affinities of the test compounds for FGF-(1,2) and VEGF
were measured by a surface plasmon resonance (SPR)-based
solution affinity assay in which free ligand competes with im-
mobilized heparin for protein.[29] An important aspect of this
assay is its specificity, as protein–ligand binding is only detect-
ed if the HS binding site is involved. Thus, nonspecific binding
to other sites of the protein is not evaluated. The first-round
18-member click library based on 24 was assembled, with
functional groups protruding from the triazole ring covering
amine, carboxylate, cyclic, and acyclic alkyl and aromatic
groups (51–68). In general, all ligands showed weak binding to
FGF-2; thus the improved binding to other proteins translated
to significantly improved selectivity against FGF-2. The extra
sulfate in 51 did not improve the binding at all, whilst the
amine 52 showed slightly higher affinity for VEGF. A phenyl
group, either directly linked (53) or slightly removed (56, 60)
from the triazole ring improved the binding to FGF-1 and/or
VEGF, particularly the selectivity of 56 for FGF-1 and 61 for
VEGF. The addition of an extra anionic motif such as carboxyl-
ate (compound 61) or sulfonamide (compound 62) also slight-
ly improved binding. Electron-donating groups on the phenyl
ring (57–59) or an ether linkage (65–68) did not improve bind-
ing. To deliver an electron-rich polar but non-anionic group to
the distal secondary cationic groove as observed in FGF-1,[30]
compound 63 was designed by incorporating a trifluoromethyl
group at the end of the enlongated hydrophobic aromatic
spacer. Indeed, this compound showed the best binding to
FGF-1 (Kd =84 mm) and VEGF (Kd =49 mm) with significantly im-
proved selectivity against FGF-2 (29- and 51-fold, respectively).
An attempt with compound 64 to target this secondary bind-
ing site with a remote sulfate group delivered by a flexible
methylene chain, however, failed to improve the binding to
both FGF-1 and VEGF. Introduction of rigidity between the tria-
zole ring to the extended functional motifs, such as the olefin
double bond in the Swern–Wittig library (76–81), proved to be
detrimental, with no binding observed at concentrations up to
1 mm.
1
coupling constants of olefinic protons in the H NMR spectra,
that is, Jcisffi11 Hz and Jtransffi16 Hz.
The triphenylphosphoranylidene acetate always gave the
E isomer (72) under conditions such as in toluene at 908C or
room temperature or in DMF at room temperature. The nitrile
analogue 71 was obtained as an inseparable mixture of Z/E
isomers in a molar ratio of 2:1, which were separated at the
diol stage after hydrolysis. The benzyl reagent gave only
a small amount of unidentified products with the majority
(84%) of intermediate aldehyde 70 recovered when sodium
hydride in DMF was used as the base. However, the use of n-
butyllithium in THF gave an inseparable isomeric mixture of 73
in good yield (Z/E=7:3, 78%). The naphthyl analogue gave
predominantly the Z olefin with NaH/DMF, and both isomers
were separable by flash chromatography. The 4-trifluorome-
thylbenzyl reagent gave only the Z isomer with NaH/DMF in
contrast to a Z/E mixture with nBuLi/THF, which could be sepa-
rated by flash chromatography. The alkyl Wittig reagents such
as cyclopropylmethylene-, cyclohexylmethylene-, or allylidene-
triphenylphosphorane all failed to give the desired products
with either NaH or nBuLi. In these cases, the intermediate alde-
hyde 70 was recovered in 48–90% yield.
The corresponding olefins were deprotected to release the
diol, which was separated where achievable to give enriched
or sole isomers before being subjected to the standard sulfa-
tion procedure. The product was purified using the SPE Strata-
NH2 protocol. It was also found that in some cases the diols
partially isomerized during the sulfation step and reverted
back to Z/E mixtures (78, 79). Fortunately, such sulfated Z/E
mixtures could be separated or enriched by the SPE Strata-NH2
protocol. The purity of the final sulfated products was deter-
mined by NMR and capillary electrophoresis (CE) analysis, and
is summarized in Table 2.
Conclusions
By mapping the sulfation pattern around methyl 6-azido-6-
deoxy-a-d-mannopyranoside 5, an interesting template bear-
Table 2. Results of the Swern–Wittig library.
Compd
Wittig reagent
Condition[a]
A
Geometry[b]
Yield [%]
100
Sulfated product
Geometry[b]
Yield [%]
tm [min][c]
Purity [%][c]
71
Ph3P=CHCN
Z/E=2:1
76
77
78
79
80
81
Z/E=9:1
Z/E=1:9
29
20
9
58
40
21
11.2
10.7
7.6
8.9
9.6
95
99
95
100
100
100
72
73
74
75
Ph3P=CHCO2Me
Ph3P=CHPh[d]
Ph3P=CHPh(4-CF3)[d]
Ph3P=CH(2-naphthyl)[d]
B
C
D
D
E only
Z/E=7:3
Z/E=10:1[e]
Z/E=7:1
74–100
78
82
E
Z
Z
Z
89
12.1
[a] Conditions for Wittig olefination: A) PhMe; B) DMF; C) nBuLi, THF; D) NaH, THF. All at 188C, 18 h. [b] Determined by 1H NMR spectroscopy. [c] Deter-
mined by CE. [d] Wittig reagents prepared from the corresponding alkyl bromide and Ph3P followed by base-promoted elimination. [e] Under condition C:
Z/E=2:1 (53% yield).
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methyl 4-O-allyl-6-azido-6-deoxy-2,3-di-O-isopropylidene-a-d-man-
nopyranoside (11) as a colorless gum (281 mg, 66%). 1H NMR
ing 4-O-allyl-2,3-disulfate was selected for further optimization
for the targeted growth factors. A small library was assembled
in a parallel fashion by employing click chemistry. SPE proto-
cols were developed to streamline efficient purification of such
polar compounds. The method was applied to produce an 18-
membered library incorporating various functional motifs via
several linker groups. The diversity of the library was further
extended by employing a Swern oxidation–Wittig olefination
sequence to deliver several functional motifs at varied geomet-
rical presentation. Binding studies indicated that both the se-
lectivity and affinity can be improved by incorporating non-
anionic motifs, designed to target the hydrophobic surface sur-
rounding the primary cationic HS binding site of the proteins.
A clear pattern of structural requirements for selectivity against
FGF-2 was observed. Aromatic groups presented at varied lo-
cations from the disulfate core were found to contribute to
the binding either by hydrophobic or p-stacking interactions.
An aromatic spacer with the appropriate rigidity and length to
deliver a polar group (CF3) for interaction with the distal polar
groove did lead to the discovery of a more potent and selec-
tive ligand (compound 63) for FGF-1 and VEGF. Compounds
were also identified with selectivity for only a single growth
factor, e.g., 65 for FGF-1 and 61 for VEGF. The strategy pro-
vides a rapid, parallel approach to explore chemical space
around a core structure, which can be employed as a chemical
tool to differentiate a family of similar proteins or to map bind-
ing space around other privileged drug templates.
(CDCl3, 400 MHz): d=5.85 (m, 1H, allyl-2’), 5.23 (ddd, 1H, J2-3’trans
=
17.2, J3’-gem =3.6, J1’-3’ =1.6, H3’trans), 5.17–5.13 (m, 1H, H3’cis), 4.89 (s,
1H, H1), 4.35 (dddd, 1H, J1’gem =12.4, J1’-2’ =5.2, J=1.6, H1’), 4.16
(dd, 1H, J2-3 =5.6, J3-4 =7.2, H3), 4.09 (d, 1H, H2), 4.05 (dddd, 1H,
H1’), 3.67 (ddd, 1H, J4-5 =10.4, J5-6a =6.8, J5-6b =2.4, H5), 3.48 (dd,
1H, J6a-6b =13.2, H6b), 3.40 (dd, 1H, H6a), 3.39 (s, 3H, OMe), 3.33
(dd, 1H, H4), 1.50 (s, 3H, Me), 1.30 ppm (s, 3H, Me); 13C NMR
(CDCl3, 100 MHz): d=134.4, 117.1, 109.2, 98.0, 78.2, 76.2, 75.6, 71.5,
68.0, 54.9, 51.6, 27.8, 26.1 ppm.
For library production, 460 mL of the stock solution of
6
(0.185 mmol, 0.4m) was transferred into each reaction vial and al-
kylated as above. The crude residue was hydrolyzed directly with-
out intermediate purification.
General procedure for hydrolysis of isopropylidene acetal: Iso-
propylidene acetals 7–12 (0.185 mmol) were dissolved in MeCN/
MeOH/H2O (3, 3, and 0.2 mL, respectively) and treated with para-
toluenesulfonic acid monohydrate (7 mg, 0.037 mmol, 20 mol%).
The mixture was stirred at 408C for 3 h and cooled. After addition
of Et3N (0.4 mL), the mixture was evaporated, and the residue was
purified by flash chromatography (EtOAc/hexane 1:6!2:1) to give
the diol products 13–18.
Methyl 4-O-allyl-6-azido-6-deoxy-a-d-mannopyranoside (17):
Colorless, waxy solid (48 mg, 100%, Rf =0.69, EtOAc/MeOH/H2O
1
50:2:1); H NMR (CDCl3, 400 MHz): d=5.91 (m, 1H, allyl-H2’), 5.28
(ddd, 1H, J2’-3’trans =16.8, J3’trans-3’cis =3.0, J1’-3’ =1.6, allyl-H3’trans), 5.20
(ddd, 1H, J2’-3’cis =10.0, J3’trans-3’cis =3.0, J1’-3’ =1.6, allyl-H3’cis), 4.72 (d,
1H, J1-2 =2.0, H1), 4.28 (dddd, 1H, Jgem =12.4, J1’-2’ =5.6, J=1.6,
allyl-1’), 4.13 (dddd, 1H, allyl-1’), 3.92 (dd, 1H, J2-3 =3.6, H2), 3.88
(dd, 1H, J3-4 =9.6, H3), 3.70 (ddd, 1H, J4-5 =9.6, J5-6a =5.6, J5-6b =2.8,
H5), 3.54–3.44 (m, 3H, H4, H6a, H6b), 3.39 (s, 3H, MeO), 2.62 ppm
(br s, 2H, OH); 13C NMR (CDCl3, 100 MHz): d=134.4, 117.3, 100.5,
76.2, 73.6, 71.4, 70.8, 70.5, 54.9, 51.3 ppm.
Experimental Section
1
General methods: Unless otherwise stated, H and 13C NMR spec-
tra were recorded on a Varian 400 instrument (at Griffith University,
Brisbane, Australia). All signals were referenced to solvent peaks
(CDCl3: d=7.26 ppm for 1H or d=77.0 ppm for 13C; D2O: d=
4.60 ppm for 1H). Where appropriate, analyses of 1H NMR spectra
were aided by gCOSY experiments. Flash chromatography was per-
formed on silica gel (40–63 mm) under positive pressure with speci-
fied eluents. All solvents used were analytical grade. The progress
of reactions was monitored by thin-layer chromatography (TLC)
using aluminum-backed Merck silica gel 60 F254 sheets. Compounds
were visualized by charring with 5% H2SO4 in MeOH and/or under
UV light. Capillary electrophoresis (CE) was performed in reverse
polarity mode on an Agilent CE System using 10 mm 5-sulfosalicyl-
ic acid (pH 3) as the background electrolyte and detection by indi-
rect UV absorbance at l 214 nm, as previously described.[31] Com-
General procedure for sulfation: The alcohol (0.163 mmol) was
dissolved in anhydrous DMF (4.1 mL, 0.04m) and treated with
sulfur trioxide trimethylamine complex (3 equiv per OH group).
The mixture was stirred at 608C for 6–18 h, and cooled in an ice–
water bath. The mixture was brought to pH 10 by careful addition
of 5m aqueous NaOH followed by saturated aqueous Na2CO3. The
mixture was evaporated to dryness and purified by the following
protocols.
Flash chromatography: A silica gel column (1ꢂ18 cm) was pre-
washed with EtOAc/MeOH/H2O (10:2:1, then 50:2:1). A solution of
the crude product in a minimal amount of H2O was loaded. The
column was eluted with EtOAc/MeOH/H2O (50:2:1 to 20:2:1 to
10:2:1, up to 7:2:1 and 5:2:1 if necessary). The fractions were
pooled and evaporated. The residue (a white powder) was re-dis-
solved in H2O and lyophilized to give the product as an amor-
phous powder.
1
pound homogeneity was determined by H and/or 13C NMR spec-
troscopy and for the sulfated products by CE. Compounds 5 and 6
were prepared according to published procedures.[22,25] Only repre-
sentative examples for library production are listed in this section.
Detailed experimental data for all compounds and intermediates
can be found in the Supporting Information.
SPE with Strata-X cartridge: A Strata-X cartridge (Phenomenex,
500 mg) was preconditioned by washing with MeCN (10 mL) and
H2O (5 mL). The sample was loaded in H2O (2ꢂ0.5 mL) at a flow
rate of ~1 dropsꢁ1. The cartridge was eluted with H2O (5–15 mL)
and MeCN/H2O (1%, 10%, then 50%; 3 mL each) and collection
volume was 0.5 mL per vial. The carbohydrate-containing fractions
were identified by charring with 5% H2SO4 in H2O after spotting
onto a TLC plate. An aliquot of 20 mL of each identified fraction
was diluted with H2O (200 mL) and used directly for CE analysis for
purity. Pure fractions were pooled and lyophilized. This method
General procedure for alkylation of the 4-hydroxy group: A
stock solution of isopropylidene acetal 6 in anhydrous DMF
(3.5 mL, 0.4m) was treated with NaH (60% dispersion in mineral
oil, 163 mg, 4.26 mmol, 3 equiv). The mixture was stirred at RT for
1 h. Allyl bromide (4.26 mmol, 3 equiv) was added. The mixture
was stirred at RT for another 6 h, treated with MeOH (1 mL) and
evaporated to dryness. The residue was purified by flash chroma-
tography (EtOAc/hexane 1:10!1:6) to give the desired product,
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Heparan Sulfate Mimetic Library
MED
was generally used in combination with silica column chromatog-
raphy to give completely desalted pure product.
1H, allyl-3’trans), 5.10 (dm, 1H, Jallyl-2’-3’cis =10.3, allyl-3’cis), 4.87 (d,
1H, J1-2 =2.0, H1), 4.66–4.58 (overlapped with DOH, 2H, H2 and
H6b), 4.54 (dd, 1H, J6a-6b =14.3, J5-6a =7.6, H6a), 4.50 (dd, 1H, J3-4
=
SPE with Strata-NH2 cartridge: The crude residue was extracted
with MeOH (3ꢂ1 mL). The mixture was centrifuged and the above
clear solution was transferred using a Pasteur pipette. Generally,
the second extraction was already very weak and the third did not
show any product. This step removed most inorganic salts. The
combined MeOH extracts were checked for pH, acidified by the ad-
dition of 10–20 mL acetic acid (adjusted to pH 6). The solution was
evaporated to dryness and re-dissolved in H2O (1 mL) for loading.
A Strata-NH2 cartridge (Phenomenex, 500 mg) was primed by elu-
tion with MeOH (9 mL), H2O (9 mL), 5% aq. acetic acid (6 mL) and
H2O (9 mL). The sample solution was loaded and the cartridge was
eluted with H2O, then aqueous NH4HCO3 (0.01m, 0.02m, 0.05m,
0.07m, 0.1m and 0.2m; 12 mL each gradient for 4ꢂ3 mL collec-
tions). Disulfates were generally eluted with 0.02m gradient, and
clearly separated from the related trisulfate (if applicable) and un-
dersulfated by-products. Fractions were checked by TLC and CE,
pooled and lyophilized.
9.5, J2-3 =3.2, H3), 4.24 (ddm, 1H, Jgem =11.9, J=6.0, allyl-1’), 3.98
(ddm, 1H, J=6.8, allyl-1’), 3.88 (ddd, 1H, J5ꢁ6b =2.8, J4ꢁ5 =9.5, H5),
3.41 (dd, 1H, H4), 3.00 ppm (s, 3H, MeO).
Methyl
4-O-allyl-6-deoxy-6-{4-[4-(4-trifluoromethylbenzyloxy)-
benzamidyl]methyl-[1,2,3]triazol-1-yl}-2,3-di-O-sulfo-a-d-manno-
pyranoside, disodium salt (63): White powder, 4.3 mg, 15% (Rf =
1
0.23, EtOAc/MeOH/H2O 10:2:1); H NMR (D2O, 400 MHz): d=7.85 (s,
1H, triazole), 7.55 (dm, 2H, J=9.0, Ar), 7.54 (d, 2H, J=8.6, Ar), 7.44
(d, 2H, J=8.6, Ar), 6.86 (dm, 2H, J=9.0, Ar), 5.79 (dddd, 1H, Jallyl-2’-
3’trans =17.3, Jallyl-2’-3’cis =10.2, J=6.3, J=6.0, allyl-2’), 5.16 (dm, 1H,
allyl-3’trans), 5.09 (s, 2H, OCH2), 5.05 (dm, 1H, Jallyl-2’-3’cis =10.2, allyl-
3’cis), 4.79 (d, 1H, J1-2 =1.8, H1), 4.64 (dd, 1H, J6a-6b =14.4, J5-6b =2.8,
H6b), 4.58 (dd, 1H, J2-3 =3.2, H2), 4.52 (dd, 1H, J5-6a =7.4, H6a), 4.50,
4.44 (AB q, 2H, Jgem =15.8, NCH2-triazole), 4.45 (dd, 1H, J3-4 =9.5,
H3), 4.22 (ddm, 1H, Jgem =12.0, J=6.0, allyl-1’), 3.96 (ddm, 1H, J=
6.7, allyl-1’), 3.78 (ddd, 1H, J4-5 =9.5, H5), 3.35 (dd, 1H, H4),
2.84 ppm (s, 3H, MeO).
Methyl 4-O-allyl-6-azido-6-deoxy-2,3-di-O-sulfo-a-d-mannopyra-
noside, disodium salt (24): White amorphous powder (55 mg,
89%; CE: tm =8.9 min, 99% purity; Rf =0.20, EtOAc/MeOH/H2O
General procedure for Swern–Wittig reactions: A dry round-bot-
tomed flask was loaded with anhydrous CH2Cl2 (4.5 mL) and DMSO
(1.12 mmol, 6.5 equiv), and the resulting solution was stirred at
ꢁ788C while oxalyl chloride (0.533 mmol, 3.1 equiv) was added
dropwise. After stirring at ꢁ788C for 10 min, a solution of triazole
alcohol 69 (0.172 mmol) in anhydrous CH2Cl2 (0.7 mL) was added.
After another 10 min, Et3N (1.12 mmol, 6.5 equiv) was added. The
mixture was stirred and allowed to warm to RT after removal of
the dry-ice–acetone bath. The resulting aldehyde stock solution
was used directly for Wittig reactions. (During library production,
the stock solution was split accordingly before addition of the
Wittig reagent.) Wittig reagent (2 equiv, 0.344 mmol) was added. In
some cases, CH2Cl2 was removed by evaporation, and the mixture
was treated with an alternative solvent such as toluene, THF, or
DMF. The mixture was stirred at room or elevated temperature for
a few hours or overnight. The mixture was evaporated under
vacuum and purified by flash chromatography (hexane/EtOAc
7:1!1:1). The product fractions were pooled according to TLC,
evaporated, and dried under high vacuum.
1
10:2:1); H NMR (D2O, 400 MHz): d=5.88–5.76 (m, 1H, allyl-2’), 5.20
(d, 1H, J2’-3’trans =17.2, allyl-3’trans), 5.12 (d, 1H, J2’-3’cis =10.0, allyl-
3’cis), 4.91 (s, 1H, H1), 4.65 (br s, 1H, J2-3 =3.2, H2), 4.48 (dd, 1H, J2-
=3.2, J3-4 =9.2, H3), 4.21 (dd, 1H, Jgem =11.6, J1’-2’ =5.6, allyl-1’), 4.00
(dd, 1H, J1’-2’ =6.4, allyl-1’), 3.71–3.67 (m, 1H, H5), 3.58 (dd, 1H, J6a-
6b =13.6, J5-6b =2.0, H6b), 3.58 (dd, 1H, J4-5 =9.6, H4), 3.46 (dd, 1H,
J
5-6a =5.6, H6a), 3.30 ppm (s, 3H, MeO); 13C NMR (D2O, 100 MHz, in-
ternal MeOH at 49.1 ppm): d=133.9, 119.2, 98.4, 76.1, 75.3, 74.2,
73.1, 70.7, 55.4, 50.8 ppm.
General procedure for click chemistry (Route A, unless otherwise
specified): To a 2 mL sample vial was loaded a solution of the sulfat-
ed sugar azide (0.67m in H2O, 52 mL, 35 mmol) and a solution of
the acetylene (0.25–0.83m in tert-butanol, 42 mmol, 1.2 equiv). To
this mixture was added a solution of copper(II) sulfate (0.3m in
H2O, 5.8 mL, 1.75 mmol, 5 mol%) and a solution of sodium ascor-
bate (1m in H2O, 7.0 mL, 6.99 mmol, 20 mol%). Note: in the case of
propargylamine, 5ꢂ7.0 mL of 1m stock solution of sodium ascor-
bate was added at intervals of 10 min. The color of the reaction
mixture changed from blue to yellow after each addition of
sodium ascorbate, but quickly back to blue, possibly due to facile
air oxidation of amine-coordinated CuII/CuI complexes. Thus several
portions of reducing agent were added to maintain the presence
of CuI. The mixture was shaken on a Minishaker at 600 rpm, RT,
overnight, and purified by flash chromatography (EtOAc/MeOH/
H2O 50:2:1!20:2:1!10:2:1). In most cases, the above product
was purified again by using Strata-X cartridge to give the corre-
sponding triazole product.
A sample of intermediate aldehyde (70) was purified by flash chro-
matography (hexane/EtOAc 7:1!2:1) to give a colorless gum (Rf =
1
0.57, hexane/EtOAc 1:1); H NMR (CDCl3, 400 MHz): d=10.12 (s, 1H,
CHO), 8.26 (s, 1H, triazole), 5.91 (dddd, 1H, Jallyl-2’ꢁ3’trans =17.4, Jallyl-2’-
3’cis =10.3, J=6.2, J=5.7, allyl-2’), 5.29 (dm, 1H, allyl-3’trans), 5.20
(dm, 1H, Jallyl-2’-3’cis =10.3, allyl-3’cis), 4.88 (dd, 1H, J6a-6b =14.1, J5-6b
2.7, H6b), 4.84 (s, 1H, H1), 4.52 (dd, 1H, J5-6a =7.9, H6a), 4.36 (dddd,
1H, Jgem =12.5, J=5.4, J=1.4, J=1.4, allyl-1’), 4.21 (dd, 1H, J2-3
5.7, J3-4 =6.8, H3), 4.08 (dddd, 1H, J=6.5, J=1.4, J=1.1, allyl-1’),
4.07 (dd, 1H, J1-2 =0.8, H2), 3.89 (ddd, 1H, J4-5 =10.0, H5), 3.18 (dd,
1H, H4), 3.02 (s, 3H, MeO), 1.43 (s, 3H, Me), 1.31 ppm (s, 3H, Me).
=
=
Other routes: similar to Route A except the diol 17 (Route B) or
isopropylidene acetal 11 (Route C) was used as azide source, both
of which were added as a tert-butanol stock solution (0.67m). The
crude product was purified by flash chromatography (hexane/
EtOAc 4:1!1:4!EtOAc, or extra elution with EtOAc/MeOH 95:5
for diols).
Methyl
4-O-allyl-6-deoxy-6-{4-[(2-(E)-methoxycarbonyl)vinyl]-
[1,2,3]triazol-1-yl}-2,3-di-O-isopropylidene-a-d-mannopyranoside
[(E)-72]: White powder (52.0 mg, 100%, Rf =0.46, hexane/EtOAc=
1:1); 1H NMR (CDCl3, 400 MHz): d=7.84 (s, 1H, triazole), 7.64 (d,
1H, J=16.1, trans-vinyl), 6.66 (d, 1H, J=16.1, trans-vinyl), 5.90
(dddd, 1H, Jallyl-2’-3’trans =16.8, Jallyl-2’-3’cis =10.3, J=5.9, J=5.1, allyl-2’),
Methyl 4-O-allyl-6-deoxy-6-(4-phenyl-[1,2,3]triazol-1-yl)-2,3-di-O-
sulfo-a-d-mannopyranoside, disodium salt (53): Pale-yellow
powder, 18.0 mg, 84% (Rf =0.19, EtOAc/MeOH/H2O 10:2:1);
1H NMR (D2O, 400 MHz): d=8.18 (s, 1H, triazole), 7.64–7.61 (m, 2H,
Ph), 7.37–7.32 (m, 2H, Ph), 7.30–7.25 (m, 1H, Ph), 5.82 (dddd, 1H,
5.28 (d, 1H, allyl-3’trans), 5.18 (d, 1H, Jallyl-2’
=3’cis =10.3, allyl-3’cis),
4.84 (s, 1H, H1), 4.81 (dd, 1H, J6a-6b =13.9, J5-6b =2.2, H6b), 4.47 (dd,
1H, J5-6a =7.3, H6a), 4.35 (dd, 1H, Jgem =12.5, J=5.9, allyl-1’), 4.20
(dd, 1H, J2-3 =5.9, J3-4 =6.6, H3), 4.10–4.05 (m, 2H, H2 and allyl-1’),
J
allyl-2’-3’trans =17.1, Jallyl-2’-3’cis =10.3, J=6.8, J=6.0, allyl-2’), 5.20 (dm,
ChemMedChem 2012, 7, 1267 – 1275
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1273

L. Liu, V. Ferro, et al.
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19, 1321–1338; b) T. Kessler, F. Fehrmann, R. Bieker, W. E. Berdel, R. M.
Mesters, Curr. Drug Targets 2007, 8, 257–268; c) L. Sepp-Lorenzino, K. A.
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tokine Network 2009, 20, 225–234.
[7] a) B. Casu, M. Guerrini, A. Naggi, M. Perez, G. Torri, D. Ribatti, P. Carmi-
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3.87 (ddd, 1H, J4-5 =10.3, H5), 3.78 (s, 3H, MeO), 3.17 (dd, 1H, H4),
3.04 (s, 3H, MeO), 1.42 (s, 3H, Me), 1.31 ppm (s, 3H, Me).
4-O-allyl-6-{4-[2-(E)-(carbonato)vinyl]-[1,2,3]triazol-1-yl}-6-deoxy-
2,3-di-O-sulfo-a-D-mannopyranoside, trisodium salt [(E)-78]: Fol-
lowing the general hydrolysis procedure, isopropylidene acetal (E)-
72 (51 mg, 0.125 mmol) afforded the corresponding diol as white
1
powder (31 mg, 67%, Rf =0.35, EtOAc); H NMR (CDCl3, 400 MHz):
d=7.93 (s, 1H, triazole), 7.65 (d, 1H, J=16.0, vinyl), 6.68 (d, 1H, J=
16.0, vinyl), 5.95 (dddd, 1H, Jallyl-2’-3’trans =16.9, Jallyl-2’-3’cis =10.1, J=5.8,
J=5.3, allyl-2’), 5.31 (dm, 1H, allyl-3’trans), 5.20 (dm, 1H, Jallyl-2’-3’cis
=
10.1, allyl-3’cis), 4.75 (dd, 1H, J6a-6b =14.0, J5-6b =2.4, H6b), 4.68 (d,
1H, J1-2 =1.0, H1), 4.61 (dd, 1H, J5-6a =6.8, H6a), 4.32 (ddt, 1H,
J
gem =12.5, J=5.8, J=1.4, J=1.4, allyl-1’), 4.22 (ddt, 1H, J=5.8, J=
1.4, J=1.4, allyl-1’), 3.95–3.86 (m, 3H, H5, H3 and H2), 3.79 (s, 3H,
MeO), 3.28 (dd, 1H, J3-4 =8.7, J4-5 =10.1, H4), 3.13 (s, 3H, MeO),
2.55 ppm (br s, 2H, OH). The above pure E-configured ester diol
(30 mg, 0.0812 mmol) was sulfated following the standard proce-
dure to give (E)-78 as white powder (4.4 mg, 9%). 1H NMR (D2O,
400 MHz): d=8.14 (s, 1H, triazole), 7.17 (d, 1H, J=16.1, trans-
olefin), 6.44 (d, 1H, J=16.1, trans-olefin), 5.83 (dddd, 1H, Jallyl-2’-
3’trans =17.2, Jallyl-2’-3’cis =10.3, J=6.6, J=5.9, allyl-2’), 5.21 (dm, 1H,
allyl-3’trans), 5.11 (dm, 1H, Jallyl-2’-3’cis =10.3, allyl-3’cis), 4.85 (d, 1H,
[8] A. Bentolila, I. Vlodavsky, R. Ishai-Michaeli, O. Kovalchuk, C. Haloun, A. J.
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We thank Drs. Tommy Karoli, Jon Fairweather, Robert Don, Ian
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Received: March 20, 2012
Revised: April 26, 2012
Published online on May 21, 2012
ChemMedChem 2012, 7, 1267 – 1275
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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