Application of the four-component Ugi condensation for the preparation of sulfated glycoconjugate libraries

Article abstract of DOI:10.1016/j.bmcl.2004.02.017

A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic or mon

Full text of DOI:10.1016/j.bmcl.2004.02.017

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2221–2226
Application of the four-component Ugi condensation for the
preparation of sulfated glycoconjugate libraries
Ligong Liu, Cai Ping Li, Siska Cochran and Vito Ferro*
Drug Design Group, Progen Industries Ltd, 2806 Ipswich Rd, Darra, Qld 4076, Australia
Received 17 December 2003; revised 4 February 2004; accepted 4 February 2004
Abstract—A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-
component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic
or monosulfated moiety, or a second sulfated monosaccharide. The affinities of these heparan sulfate (HS) mimetics for the HS-
binding fibroblast growth factors FGF-1 and FGF-2 were measured via a surface plasmon resonance solution affinity assay.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Heparin and heparan sulfate (HS) are two important
members of the glycosaminoglycan (GAG) family of
linear, polyanionic polysaccharides that consists of
chemistry. In this communication we report the first
examples of the synthesis of sulfated glycoconjugates
based on the Ugi reaction and their affinities for the HS-
binding fibroblast growth factors 1 and 2 [FGF-(1,2)].
repeating disaccharide units of uronic acid-(1 fi 4)-
D-
glucosamine.1;2 Variable patterns of substitution of each
disaccharide unit with N-sulfo, O-sulfo and N-acetyl
groups provide a large number of complex sequences
that encode a high density of biochemical information.
While such diverse structures contribute to their wide
range of biological activities,3;4 they also limit their
clinical usefulness as drugs because of unwanted side
effects. It is therefore desirable to develop HS mimetics
that could specifically target HS-binding proteins.
The choice of FGF-(1,2) as targets is based on their
important roles in a wide variety of physiological pro-
cesses related to cell proliferation, differentiation and
migration, as well as disease processes such as tumour
angiogenesis.15–17 These FGFs are also among the most-
studied HS-binding proteins with a number of X-ray
crystal structures published, including FGF in complex
with heparin-derived oligosaccharides,18;19 and FGF in
ternary complex with its receptor (FGFR) and a hepa-
rin-derived oligosaccharide.20;21 FGF-(1,2) are attractive
targets for antiangiogenic and antitumour drug design22
and a number of small molecules have been shown to
inhibit FGF-mediated mitogenic activity by binding to
FGF-(1,2) and blocking the formation of the hepa-
rin:FGF:FGFR complex.23–31
During the previous decade, multicomponent reactions
such as the four-component Ugi condensation5 (Scheme
1) have attracted much attention in drug discovery and
lead optimization,6–8 including the glycomics area,9–14
because of their synthetic potential for the generation of
molecular diversity and applications in combinatorial
A focused library of HS mimetics was designed with
at least one component derived from a persulfated
monosaccharide (Fig. 1) to target the positively charged
Scheme 1. Generic representation of the four-component Ugi con-
densation.
Figure 1. Design of the library of sulfated glycoconjugates. R ¼ an
aromatic, an aliphatic or a negatively charged group (e.g., sulfo
group).
* Corresponding author. Tel.: +61-7-3273-9150; fax: +61-7-3375-6746;
e-mail: vito.ferro@progen.com.au
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.017

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L. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2221–2226
ꢀ
Scheme 2. Reagents and conditions: (a) ethylene glycol, MS 3 A, CH2Cl2, BF3 Æ Et2O, )20 °C, 1.5 h; (b) (i) MsCl, Et3N, CH2Cl2, 0–25 °C, 3h, (ii)
NaN3, DMF, N2, 50 °C, 2 h, 45% (three steps); (c) (i) NaOMe, MeOH, 25 °C, 1.5 h, (ii) NaH, BnBr, DMF, 25 °C, 2 h, 58% (two steps); (d) PPh3,
THF–H2O (7:1), 25 °C, o/n, 96%, or NaBH4, NiCl2, MeOH–THF (3:2), 0–25 ° C, 1 h, 89%; (e) (i) Ac2O, HCO2H, 25 °C, 3h, 92%, (ii) POCl 3, Et3N,
CH2Cl2, 0 °C, 1 h, 92%; (f) NaH, DMF, BrCH2CO2But, 25 °C, 4 h; (g) 5% TFA in CH2Cl2, 25 °C, 0.5 h, 90% (two steps); (h) NaH, DMF,
BrCH2CO2Et, 25 °C, 19 h, 51%; (i) NaOH, THF–MeOH (1:1), 70 °C, 3h, 70%.
residues on the surface of the HS binding site of FGF-
(1,2).18;19 The monosugar series of compounds contains
a single, persulfated monosaccharide linked via a spacer
(formed by the Ugi reaction) to either an aromatic, an
aliphatic or a negatively charged functional group.
These structures are similar in size to a disaccharide and
are useful for studying the contributions of hydrophobic
or additional ionic motifs within the HS binding site.
The bis-sugar series of compounds contains two per-
sulfated monosaccharide units linked by a spacer. These
structures are designed to mimic sulfated oligosaccha-
rides with the central sugar units replaced by a spacer,
and be suitable for investigating the spatial relationship
between the two negatively charged monosaccharide
units. In addition, due to restricted rotation about the
amide bonds, the conformational flexibility of these
types of structures is greatly reduced and may offer a
restricted presentation of sulfo groups.13;14 Recently a
large, random combinatorial library was prepared via
the Ugi reaction and screened for inhibition of heparin
binding to FGF-2.25 As expected, the identified hits all
contained some negatively charged functional groups.
trichloroacetimidate32 1 with ethylene glycol followed by
standard functional group transformations and pro-
tecting group manipulation provided the amino block 5
in 25% overall yield. This was converted into the iso-
cyanide block 6 in high yield via formylation and
dehydration. The acid blocks 9 and 12 were prepared via
esters (tert-butyl or ethyl) by alkylation of the corre-
sponding alcohols 733 and 10,34 followed by hydrolysis.ꢁ
The commercially available
used as an acid block.
D-glucuronic acid was also
The components used for the Ugi reaction are outlined
in Table 1. The monosugar series (Table 1, entries 1–6
and 10) was constructed by using a carbohydrate acid
block (D-glucuronic acid or 12) with commercially
available amine, isocyanide and carbonyl components.35
One example using the carbohydrate isocyanide block 6
(entry 10) was also prepared. The bis-sugar series was
prepared by Ugi reaction incorporating two carbohy-
drate blocks (entries 7 and 9), or by one incorporating a
single carbohydrate block and a bis-amine (trans-1,4-
diaminocyclohexane, entry 8) or bis-acid (3,3-dimethyl-
glutaric acid, entry 11). The products, isolated in good
to moderate yield (36–72%), were deprotected (NaOMe/
MeOH, entries 1–4; or H2/Pd/C, entries 5–11) and sul-
fonated36 to provide compounds 14–24 (Fig. 2).37 The
partially sulfated product 13 was separable from its fully
sulfated counterpart 14 by flash chromatography
(MeCN–Et3N–H2O, 110:2:11). Interestingly, the sul-
To illustrate the potential diversity offered by this type
of library, monosaccharide building blocks were pre-
pared containing three of the four types of functional
groups required for the Ugi condensation (amine, iso-
cyanide and carboxylic acid) at various positions around
the hexopyranose ring. To simplify the characterization
of the products, a symmetrical carbonyl component,
formaldehyde,ꢀ was generally used in library produc-
tion. The preparation of the carbohydrate building
blocks is outlined in Scheme 2. Glycosylation of the
fated products in the D-glucuronic acid series (13–17)
were isolated exclusively as a-anomers, as determined by
1H NMR spectroscopy (typically J1;2 ꢀ 3:5 Hz).
ꢀ
Formaldehyde was added from a stock solution in MeOH (2 M)
ꢁ
made from the corresponding commercially available aqueous
solution (36.5–38.0% w/w, aqueous solution stabilized with 10%
MeOH).
The corresponding tert-butyl ester of 10 was susceptible to polymer-
ization upon acid catalyzed deprotection. The ethyl ester 11 was
therefore used in this case.

L. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2221–2226
2223
Table 1. Components used for the Ugi reaction, sulfated products (shown in Fig. 2), and dissociation constants measured for sulfated products
binding to FGF-(1,2)
Components for Ugi reaction
Yield
(%)a
Sulfated
product
Kd (lM)b
Entry
Acid
Amine
Aldehyde
Isocyanide
FGF-1
FGF-2
1
D
-GlcA
BnNH2
HCHO
66
13c
14
587 246
1160 190
122 13254 12
62.5 1.3>800
d
2
3
D
D
-GlcA
-GlcA
BnNH2
BnNH2
PhCHO
HCHO
46
36
15
16
104 52.3206 11
4
5
D
-GlcA
HCHO
HCHO
47
72
17
18
2340 690
>800d
12
BnNH2
BnNH2
260
296
4
4
200 36
6
7
12
12
HCHO
HCHO
66
64
19
20
551 96
6
4.84 0.16
16.0 3.0
8
9
12
9
HCHO
HCHO
43
36
21
22
32.6 23.6
6.36 1.2
27.9 1.5
BnNH2
BnNH2
5
6
6
85.0 13.0
10
11
CH3CO2H
HCHO
HCHO
67
67
23
24
34.3 0.2
10.0 4.0
287 27
34.0 13.0
a Unoptimized percentage yields for initial Ugi reactions (including peracetylation, if required).
b The average and standard deviation of at least two independent measurements.
c Undersulfated product isolated by chromatography.
d No binding observed up to this concentration.
The binding affinities of the sulfated glycoconjugates
13–24 for FGF-(1,2) were measured using a surface
plasmon resonance (SPR) solution affinity assay38
(Table 1). The principle of the SPR assay is that a
solution, at equilibrium, of the growth factor and a
ligand is passed over a sensor chip containing immobi-
lized heparin. As the unbound growth factor binds to
the heparin, an increase in the SPR response is detected
from which the concentration can be determined. The
dissociation constant, Kd, can be calculated from the
decrease in the free growth factor concentration as a
function of ligand concentration.
group leads to a significant decrease in affinity for the
D
-glucuronic acid series (14 vs 17) and no change for the
-mannose series (18 vs 19), indicating that the spatial
D
arrangement of the sulfo groups is also important for
good binding.
The potential for synthesizing larger, more diverse
combinatorial libraries of this type is enormous given
that (i) carbohydrate blocks can be used for any of the
four Ugi components,10 (ii) a large number of different
carbohydrates are available and (iii) there is the ability
to introduce the required functional groups at any
position(s) around the hexopyranose ring via standard
carbohydrate protecting group manipulations. In addi-
tion, it should be possible to limit both the number of
sulfo groups and their placement in specific positions
around the hexopyranose ring by using either orthogo-
nally protected, or differentially sulfated carbohydrate
Ugi blocks. These latter possibilities are currently under
investigation.
The measured Kd values range over three orders of
magnitude from 10À3 to 10À6 M. Although the affinities
are not as strong as for heparin/HS or the phospho-
sulfomannan PI-88,38 some of the compounds show
promising affinities, particularly the monosugar com-
pounds, which have relatively few sulfo groups. The
small size of the library does not allow a complete SAR
analysis; however, some trends are evident. The number
of sulfo groups is clearly important, as indicated by the
decrease in affinity upon loss of a single sulfo group (13
vs 14), and the generally higher affinity seen in the bis-
sugar series (20–22, 24). The introduction of additional
charge by replacement of an aromatic group with a sulfo
In conclusion, a library of sulfated glycoconjugates as
heparin/HS mimetics has been synthesized via the four-
component Ugi condensation utilizing one or two
monosaccharide-derived building blocks. These com-
pounds show promising affinities for the HS binding,

2224
L. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2221–2226
Figure 2. Structures of compounds evaluated for binding to FGF-(1,2) in the SPR solution affinity assay (dissociation constants given in Table 1).
X ¼ SO3Na.
angiogenic growth factors FGF-(1,2). This strategy
provides useful biological tools to study heparin/HS-
binding proteins as well as information for refinement of
ligands to achieve improved activity and selectivity. The
use of unprotected components in the Ugi reaction such
NMR experiments. We also thank Drs. Robert Don,
Jon Fairweather, Tomislav Karoli and Ian Bytheway
(Progen Drug Design Group) for useful discussions
and critical reading of this manuscript. This work was
partially funded by an AusIndustry START grant to
Progen Industries Ltd.
as
D-glucuronic acid, greatly simplifies the synthetic
procedures and represents a platform for the rapid
assembly of such products by using inexpensive, readily
available starting materials.
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13: 1H NMR (D2O, 400 MHz, 25 °C, internal ref.: acetone,
d 2.05): two rotamers in a molar ratio of 56:44. Major
rotamer: d 7.36–7.11 (m, 5H, Ph), 5.95 (d, J1;2 ¼ 3:4 Hz,
H1), 4.89 (d, J4;5 ¼ 9:6 Hz, H5), 4.75, 4.69 (AB,
JA;B ¼ 16 Hz, a-CH2), 4.50 (dd, J2;3 ¼ J3;4 ¼ 9:6 Hz, H3),
4.31 (dd, H2), 4.00 (dd, H4), 3.87 (s, b-CH2), 3.42–3.32 (m,
1H, cyclohexyl CH), 1.64–1.36, 1.20–0.92 (2m, 5H each,
cyclohexyl CH2); minor rotamer: d 7.36–7.11 (m, 5H, Ph),
5.90 (d, J1;2 ¼ 3:2 Hz, H1), 4.58 (d, J4;5 ¼ 9:8 Hz, H5), 4.52
(s, 2H, c-CH2), 4.48 (dd, J2;3 ¼ 10, J3;4 ¼ 9:4 Hz, H3), 4.32,
3.90 (AB, JA;B ¼ 17:6 Hz, d-CH2), 4.28 (dd, H2), 4.04 (dd,
H4), 3.42–3.32 (m, 1H, cyclohexyl CH), 1.64–1.36, 1.20–
0.92 (2m, 5H each, cyclohexyl CH2); 13C NMR (D2O,
100 MHz, internal ref.: acetone, d 30.37): doubling-up of
signals due to two rotamers: d 170.0, 169.8, 168.9, 168.8
(2 · C ¼ O), 135.4, 135.2, 129.2, 129.2, 128.5, 128.3, 128.2,
128.1 (Ph), 95.6(7), 95.6(5) (C1), 77.8, 77.7 (C3), 73.8, 73.7
(C2), 70.7, 70.2 (C4), 69.2, 68.7 (C5), 52.9 (a-CH2), 51.1
(c-CH2), 50.3(d-CH 2), 50.0 (b-CH2), 49.2, 49.0 (cyclo-
hexyl CH), 31.8(9), 31.8(6), 25.1, 25.0, 24.5, 24.4 (cyclo-
hexyl CH2); HRMS: m=z calcd for C21H28N2Na3O16S3:
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1
729.0294; found: 729.0242 [M + H]þ. 14: H NMR: two
rotamers in a molar ratio of 70:30. Major rotamer: d 7.34–
7.16 (m, 5H, Ph), 5.95 (d, J1;2 ¼ 3:5 Hz, H1), 5.24 (d,
J4;5 ¼ 9:6 Hz, H5), 4.90, 4.47 (AB, JA;B ¼ 15:6 Hz, a-CH2),
4.67–4.57 (overlapped with water, 1H, H3), 4.54 (dd,
J3;4 ¼ 8:8 Hz, H4), 4.39 (dd, J2;3 ¼ 9:8 Hz, H2), 3.93, 3.75
(ABq, JA;B ¼ 16:8 Hz, b-CH2), 3.30–3.20 (m, 1H, cyclo-
hexyl CH), 1.65–1.35, 1.18–0.92 (2m, 5H each, cyclohexyl
CH2); minor rotamer: d 7.34–7.16 (m, 5H, Ph), 5.91 (d, 1H,
J1;2 ¼ 3:4 Hz, H1), 4.77–4.72 (m, 2H, H5 and H3or H4),
4.69, 4.22 (AB, JA;B ¼ 15:2 Hz, c-CH2), 4.67–4.56 (over-
lapped with water, 1H, H3or H4), 4.37 (dd, J2;3 ¼ 9:8 Hz,
H2), 4.26, 3.94 (AB, JA;B ¼ 18:4 Hz, d-CH2), 3.36–3.26 (m,
1H, cyclohexyl CH), 1.65–1.35, 1.18–0.92 (2m, 5H each,
cyclohexyl CH2); 13C NMR: major rotamer: d 172.4, 171.7
(2 · C ¼ O), 137.2, 131.7, 131.6, 131.1 (Ph), 97.7 (C1), 78.1
(C4), 77.6 (C3), 76.4 (C2), 70.1 (C5), 55.8 (a-CH2), 53.2
(b-CH2), 51.8 (cyclohexyl CH), 34.2, 27.6, 27.1 (cylcohexyl
CH2); minor rotamer: 97.6 (C1), 78.2 (C4), 77.8 (C3), 76.3
(C2), 71.0 (C5), 53.3 (d-CH2), 53.8 (c-CH2), 52.1 (cyclo-
hexyl CH), 34.3, 27.5, 27.1 (cylcohexyl-CH2); HRMS: m=z
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35. General procedure for the Ugi reaction: Solutions in MeOH
of the acid (1 equiv), amine (1 equiv), aldehyde (1 equiv)
and isocyanide (1 equiv) were transferred sequentially, in
that order, into a reaction vial (final concentration: 0.1–
0.5 M). When
D-glucuronic acid was the acid component,
it was added as a solid. When a bis-acid or bis-amine was
used, only 0.5 equiv of this component was added. The
mixture was shaken at rt for 24 h and the reaction was
monitored by TLC. The mixture was then evaporated in
vacuo and the residue either directly purified by flash
chromatography, or, for entries 1–4 in Table 1, subjected

2226
L. Liu et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2221–2226
calcd for C21H27N2Na4O19S4: 830.9682; found: 830.9635
[M+H]þ.
38. Cochran, S.; Li, C.; Fairweather, J. K.; Kett, W. C.;
39. Yu, G.; Gunay, N. S.; Linhardt, R. J.; Toida, T.; Fareed,
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