Fragment-based development of triazole-substituted O-galactosyl aldoximes with fragment-induced affinity and selectivity for galectin-3

Article abstract of DOI:10.1039/b909091f

A fragment-based development of 3C-triazol-1-yl-O-galactopyranosyl aldoximes led to the discovery of highly selective and high affinity (K _d down to 11 μM) small monosaccharide based inhibitors of galectin-3. Galectin-7, 8 N-terminal CRD, and 9

Full text of DOI:10.1039/b909091f

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Fragment-based development of triazole-substituted O-galactosyl aldoximes
with fragment-induced affinity and selectivity for galectin-3†
Johan Tejler,a Bader Salameh,b Hakon Lefflerc and Ulf J. Nilsson*a
Received 8th May 2009, Accepted 19th June 2009
First published as an Advance Article on the web 20th July 2009
DOI: 10.1039/b909091f
A fragment-based development of 3C-triazol-1-yl-O-galactopyranosyl aldoximes led to the discovery of
highly selective and high affinity (Kd down to 11 mM) small monosaccharide based inhibitors of
galectin-3. Galectin-7, 8 N-terminal CRD, and 9 N-terminal CRD bound the inhibitors only weakly.
The galectin-3 selectivity was hypothesized to stem from interaction of the aldoxime moiety with a site
not present in the other galectins.
charides bind, subsite D is occupied by another pyranoside that
is glycosylated by the subsite C-binding galactose. Typically, this
Introduction
Galectins were defined in 1994 as proteins with “affinity for
subsite D-binding pyranoside is (1→4)-linked Glc or GlcNAc or
b-galactosides and a significant sequence similarity in the carbohy-
(1→3)-linked GlcNAc or GalNAc.
drate binding site. . .”.1 They are widely spread throughout nature
In order to investigate the proposed biological role of galectins,
from plants to invertebrates and now includes 15 mammalian
potent and selective inhibitors are essential. However, the use
proteins.2 The galectins are suggested to control several impor-
of natural ligands as inhibitors is hampered by their difficult
tant biological events, including intracellular trafficking,3 cell
synthesis, sensitivity to hydrolysis, and their high polarity. In
signalling,4–7 apoptosis,8 and cell adhesion.2 Observations of these
attempts to circumvent these disadvantages, we have previously
events on a cellular or organismal level related to inflammation,9,10
presented the replacement of the saccharide in subsite D with
immunity,11,12 and cancer progression13 have been reported. A
coherent picture on the molecular mechanisms of galectins
simpler organic fragments by introducing aldoxime ethers at
b-galactose C1 via reaction of O-b-D-galactopyranosyl hydroxy-
lamine with aldehydes.25 We herein demonstrate a high selectivity
of O-galactosyl aldoximes towards galectin-3 as a result of further
evaluation of oxime ethers against galectin-1, 7, 8 N (N-terminal
domain), and 9 N (N-terminal domain). Furthermore, trans-
formations of the O-galactosyl aldoximes through a reduction–
acylation protocol are demonstrated to lead to decreased inhibi-
tion, which emphasizes the importance of the oxime functionality
for selectivity and affinity for galectin-3. Finally, we show that
both selectivity and affinity for galectin-3 can be greatly improved
by combining structural fragments earlier optimized as subsite
B-binding entities (aromatic amides and 1,2,3-triazoles17,18,23) with
the best subsite D-binding aldoxime structures in a fragment-
based manner.
underlying the cellular and organism level observations has begun
to emerge during recent years. Central are the discoveries that
galectin-1 regulates selective T helper cell apoptosis via selective
recognition of glycans,4 that intracellular targeting of proteins is
related to galectin-8 activities,14,15 and that cell-surface glycopro-
tein localization and residence time is controlled by lattices formed
via N-glycan and galectin-3 cross-linking.5–7 Recent important
additions to knowledge about galectin molecular mechanisms are
that glycan-mediated intracellular clustering with galectin-3 was
shown to influence apical sorting of proteins3 and that galectin-3-
mediated lattice formation with T cell receptors prevented their co-
localization with CD8 thus conferring anergy in tumor-infiltrating
CD8+ lymphocytes.16 Clearly, galectins are indeed emerging as
attractive targets for new anti-cancer and anti-inflammatory drugs
and development discovery of high-affinity and selective small-
molecule inhibitors17–31 is hence critical.32–34
Results and discussion
The carbohydrate recognition domain (CRD) of galectins is a
beta-sandwich of about 135 aa which forms a groove that binds
up to a tetrasaccharide and can schematically be described as four
subsites (A–D). Subsite C is built from most of the conserved
sequence elements and binds galactose. When natural oligosac-
Evaluation of oxime ethers against galectin-1, 3, 7, 8 N, and 9N
The first overall important observation when analyzing the
dissociation constants for the panel of oxime ethers (Fig. 1) for
galectin-1, 3, 7, 8 N, and 9 N was that the structure of the
aldehyde component greatly influenced the affinity for the different
galectins. When comparing the three best inhibitors for each of the
five tested galectins (Table 1), structure preferences of the galectins
were seen. Oxime ethers with selectivity for galectin-1, 3, 7, and
8 N were found, while the best inhibitors for galectin-9N displayed
higher affinity for other galectins.
aOrganic Chemistry, Lund University, POB 124, SE-22100, Lund, Sweden.
E-mail: ulf.nilsson@organic.lu.se; Fax: +46 46 2228209; Tel: +46 46
2228218
bChemistry Department, The Hashemite University, PO Box 150459, Zarka
13115, Jordan
cSection MIG, Department of Laboratory Medicine, Lund University,
So¨lvegatan 23, SE-223 62, Lund, Sweden
The oxime ether with highest affinity for galectin-1 (the aromatic
26, Kd = 910 mM) showed 10 times affinity enhancement as
compared to the reference methyl b-D-galactoside 51, while
† Electronic supplementary information (ESI) available: Experimental
procedures and 1H NMR data for compounds 1–50. 1H NMR for
compounds 54–66, 68–71, and 73–77. See DOI: 10.1039/b909091f
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Table 1 Galectin dissociation constants (mM) of oxime inhibitors and the
three reference compounds methyl b-D-galactoside 51, methyl b-lactoside
52, and methyl b-LacNAc 53. The three best inhibitors for each galectin
are in bold-face
galectin-7 over galectin-1 and no, or very small, affinity could
be seen for the other galectins. Bicyclic ring systems were not
preferred by galectin-7 and 9N. A 3-hydroxy-2-naphthyl oxime
50 turned out to be the second best inhibitor for galectin-8N,
although not selective for this galectin. It is noteworthy that
the corresponding unsubstituted naphthyl analogue 41 showed
no binding to galectin-8N. Interestingly, the 2,5-dihydroxylated
phenyl oxime 25, which also had a hydroxyl group at the ortho
position, had about three times higher affinity for galectin-8N
than for galectin-3 and 7 and seven times higher than for galectin-
9N. However, neither ortho nor meta hydroxylated phenyl oximes
(i.e. 2 or 10) showed affinity for galectin-8N. Galectin-9N prefers
large substituents, but no selectivity was observed for this galectin.
A closer analysis of the structure–activity relationships reveals
that aliphatic moieties had no effect on binding to any of the
galectins. It is also apparent that an unsubstituted phenyl had
no or only minor effects upon binding no matter if the oxime
linker was one, two or three carbons long. However, hydroxyl
substituents on various positions on the phenyl ring increase the
affinity for all galectins. As natural ligands position a sugar moiety
to form hydrogen bonds in subsite D, it is reasonable to assume
that the affinity-enhancing effects are due to hydrogen bonding to
subsite D.
Galectin
1
3
7
8N
5200
9N
3400
23
490
N.I.
> 2000
N.I.
5119
5220
5320
10
26
34
35
41
49
8
10000
190
65
1000
910
4400
160
59
2800
6300
650
360
370
330
4600
4800
110
550
62
1000
1900
N.I.a
N.I.
N.I.
1200
8400
> 2000
> 2000
> 2000
> 5000
340
> 2000
> 2000
N.I.
> 5000
780
N.I.
160
370
> 2000
> 2000
N.I.
> 5000
> 5000
N.I.
N.I
950
19
25
50
39
1200
2200
> 5000
550
390
N.I.
510
1200
> 5000
1500
N.D.b
N.I.
480
820
840
1000
a Not inhibitory. b Not determined.
both methyl b-lactoside 52 and methyl b-LacNAc 53 showed
much stronger affinity for galectin-1. Larger ring systems seem
to be disadvantageous for galectin-1 (i.e. naphthyl 41, indol
49, anthracene 8, and naphthyl 50), while bicyclic ring systems
were preferred by galectin-3 (i.e. 41 and 49). The anthracene
moiety in 8 appeared to be too large for the CRD in galectin-
3, but not for galectin-7, 8 N, and 9N. Galectin-7 also showed
good affinity for other large ring systems, such as the tricyclic
16 or 39 (Kd = 780 mM, and 1000 mM, respectively). Two
oxime ethers, the acetamidophenyl 19 and anthracene 8, were
as good as LacNAc as inhibitors for galectin-7 although not
as good as lactose. Compound 19 showed 3-fold selectivity for
Synthesis and evaluation of galactosyl hydroxylamines and
N-acetylated hydroxylamines
Reducing the oxime ethers to hydroxylamines would lead to
structural variations, as well as a secondary amino groups
amenable for further derivatizations (e.g. acylations), which in turn
was expected to provide potential novel galectin inhibitors with
altered biological properties. Various ways to reduce the oxime
ether were investigated. Pyridine–borane reduction35 initially
looked promising, but significant byproduct formations, including
Fig. 1 Aldoximes 1–50.25
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Table 2 Dissociation constants (mM) for hydroxylamines 54–62
O–N bond cleavage, were observed after work-up. The use of
NaCNBH3 in glacial acetic acid finally proved to be the best
method, although varying amounts of unreacted starting material
typically were recovered thus compromising isolated yields. Rea-
sonable yields of the hydroxylamine could nevertheless be obtained
with large excess of and subsequent additions of NaCNBH3
(Scheme 1).36,37 A panel of nine structurally diverse oxime ethers
2, 4, 10, 12, 27, 38, 41, 44, and 48 were selected for reduction to
give the hydroxylamines 54–62 of which four (55, 57, 60, and 62)
underwent quantitative acylation to give 63–66 (Scheme 2).
Galectin
1
3
7
8N
9N
54
55
56
57
58
59
60
61
62
1900
N.I.a
N.I.a
N.I.a
N.I.a
N.I.a
N.I.a
> 5000
N.I.a
820
490
2500
> 5000
2400
> 5000
2100
> 5000
1200
> 5000
N.D.b
N.D.b
3200
4600
3700
2200
4100
4600
4100
4600
> 5000
> 5000
> 5000
> 5000
> 5000
> 5000
> 5000
N.I.a
> 5000
> 5000
> 5000
> 5000
N.I.a
N.I.a
> 5000
> 5000
a Not inhibitory. b Not determined.
Galactosyl oxime with an aromatic subsite B-binding amide at C3
Aromatic amides at C3¢ of LacNAc have been shown to be efficient
galectin-3 subsite-B binding fragments and detailed structure–
activity relationships are available.17,18 One of the optimized
LacNAc C3¢ amides, 3,5-dimethoxybenzamide 72, was selected as
the subsite B-binding fragment to combine with the best galectin-
3-binding O-galactosyl oxime fragment, the indol derivative 49.
The 3,5-dimethoxybenzamide 68 was prepared in 91% yield by
catalytic hydrogenation in ethanol/HCl over Pd/C of the azido
group of the known 6738 (a/b 1/1) followed by acylation with
3,5-dimethoxybenzoyl chloride (Scheme 3). Conversion of 68
into the corresponding bromide, followed by substitution with
N-hydroxyphthalimide under phase-transfer conditions gave 69
Scheme 1 Synthesis of hydroxylamines 54–62 (yield in %).
Scheme 2 Synthesis of acylated hydroxylamines 63–66. Yields were near
quantitative.
Dissociation constants for hydroxylamines 54–62 and the
acylated products 63–66 were determined for galectin-1, 3, 7, 8
N, and 9 N (Table 2), but in most cases the activity was lost
as compared to the parent oxime ether. The only exception was
galectin-7 towards which the naphthyl 54 was much more potent
than the parent oxime ether (48, Kd > 2 mM).
As the biological evaluations of the hydroxyl amines 54–62
and their acetylated analogs 63–66 were disappointing and the
oxime reduction–acylation protocol showed modest efficiency,
this approach was not further pursued. Instead, project focus
turned towards exploiting possibilities of combining selected
galectin-3 subsite D-binding oximes with earlier reported subsite
B-binding structural elements in order to develop monosaccha-
ride derivatives with selectivity and high affinity for galectin-3.
Scheme
3 Synthesis of a galactosyl oxime 71 with an aromatic
subsite B-binding amide at C3. (a) i. H2, Pd/C, HCl, EtOH. ii.
3,5-dimethoxybenzoyl chloride, pyridine, CH2Cl2, 91%. (b) i. HBr,
CH2Cl2. ii. N-hydroxyphthalimide, tetrabutylammonium hydrogensulfate,
CH2Cl2, Na2CO3, 55%. (c) Hydrazine hydrate, MeOH, 72%. (d) In-
dole-3-carboxaldehyde, H2O/THF, HCl, 53%, E/Z 5:1.
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Table 3 Dissociation constants (mM) for galactosyl oximes having a
C3-amide (71) or C3-triazole (76) fragments
Galectin
3
7
8N
9N
71
76
77
46
11
17
390
410
870
>> 3000
370
920
>> 3000
N.D.a
> 600
a Not determined.
in 55% yield (no a-isomer was detected). Deprotection of the
phthalimide and acetyl groups in 69 was accomplished using
hydrazine hydrate in methanol to give the hydroxylamine 70 in 72%
yield. The oxime ether 71 was formed in 53% yield after treating 70
with indole-3-carboxaldehyde in an acidified H2O/THF mixture.
Compound 71 (Kd = 46 mM, Table 3) showed an affin-
ity enhancement of 7 times for galectin-3 as compared to
the oxime ether analogue 49 (Fig. 2), but the corresponding
3,5-dimethoxybenzamido LacNAc derivative 72 had 67 times
higher affinity (Kd = 1 mM) for galectin-3 than the parent LacNAc
structure, suggesting that the 3,5-dimethoxybenzamido moiety is
not an optimal subsite B-binding fragment to combine with the
subsite D-binding indole oxime. Further analysis showed that 71
showed some selectivity for galectin-3 with about 8 times higher
affinity for galectin-3 as compared to galectin-7, 8 N, and 9 N
(Kd = 390 mM, 410 mM and 370 mM).
Scheme 4 Synthesis of galactosyl oximes 76 and 77 with subsite B-bind-
ing 1,2,3-triazoles at C3. (a) N-hydroxyphthalimide, boron trifluoride
diethyl etherate, CH2Cl2, 33%. (b) 2.3 M MeNH2 in MeOH, 88%.
(c) Indole-3-carboxaldehyde, HCl (0.1 eq), H2O/THF, 67%. (d) i. Cu(I),
methyl propiolate, propanol. ii. 40% MeNH2 in H2O, 88%. (e) Cu(I), phenyl
acetylene, propanol, 45%.
Galactosyl oximes with subsite B-binding 1,2,3-triazoles at C3
Although the C3 amides described above indeed corroborated
our hypothesis that a subsite B-binding aromatic amide fragment
combined with a subsite D-binding oxime fragment could lead to
efficient monosaccharide inhibitors of galectin-3, the absence of
a synergistic affinity enhancement and thus lower than expected
affinity of the derivative 71 was disappointing. The effects of the
aromatic amide and the oxime ether were not even fully additive.
Hence, we decided to investigate 1,2,3-triazoles as alternative
subsite B-binding C3-fragments,23 as they have been demonstrated
to be about as efficient subsite B-binding fragments as aromatic
amides (Scheme 4).
the moderate yield. Deprotection with methylamine in methanol
gave hydroxylamine 74 in 88%. The oxime ether 75 was formed
in 67% yield after reacting 74 with indole-3-carboxaldehyde in an
acidified H2O/THF mixture. Two triazoles were then prepared
from the indole oxime ether 75. First, Cu(I)-catalyzed 1,3-dipolar
cycloaddition39–42 with methyl propiolate, followed by treatment
with methylamine furnished 76 in 88% yield. The second triazole
was prepared in 45% by cycloaddition between phenyl acetylene
and 75 to give 77.
Compound 73 was obtained in 33% yield (a/b 2/98) from
glycosylation of 67 (a/b 1/1) using N-hydroxyphthalimide and
boron trifluoride-diethyl etherate in CH2Cl2. The less reactive
a-anomer of thestartingmaterialwas recovered, which can explain
Compounds 76 (Kd = 11 mM) and 77 (Kd = 17 mM) were
significantly better than the amide 71 as inhibitors of galectin-3
(Table 3). A comparison with the previously published results for
galectin-3 for the methyl amide and the phenyl analogs of the
Fig. 2 Combination of a subsite B-binding aromatic amide with a subsite D-binding indole-3-carbaldoxime for selective galectin-3 inhibition.
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Fig. 3 Combination of subsite B-binding triazoles fragments with a subsite D-binding indole-3-carbaldoxime fragment for efficient and selective
galectin-3 inhibition.
3-deoxy-3-(1H-1,2,3-triazol-1-yl)-1-thio-galactoside (i.e. 78 and
79, Kd = 230 and 150 mM, respectively)23 and oxime ether 49 (Kd =
330 mM)25 clearly shows an additive effect between the substituents
at galactose C1 and C3 (Fig. 3). Indeed, the 30-fold affinity-
enhancement conferred by the triazole moiety of 76 (compare
76 with 49) surpasses the 12-fold enhancement observed when
the same triazole group was attached to a LacNAc disaccharide
(i.e. 80). This suggests a synergistic effect by the galactose C1 and
C3 substituents in 76 and 77. Furthermore, both monosaccharide
derivatives 76 and 77 are superior to LacNAc (Kd = 67 mM) and
lactose (Kd = 220 mM) methyl glycosides. Importantly, 76 and 77
have high selectivity for galectin-3 and show virtually no or very
weak binding to galectin-7, 8 N, or 9N. Such high selectivity is
very rarely observed for natural galectin ligands and the unnatural
triazole and oxime fragments of 76 and 77 thus not only confer
affinity but also high selectivity for galectin-3.
Fig. 4 Calculated energy minima of 76 in complex with galectin-3.
Molecular modeling
Conclusions
In an attempt to explain the improved affinity and selectivity of
the triazoles 76 and 77 towards galectin-3, these two inhibitors
were modeled into the binding sites of galectin-1, 3, 7, 8 N, and
9N. Energy minima of 76 and 77 in complex with the galectins
did support the hypothesis that the substituted triazole fragments
occupied the extended binding pocket near galactose C3 (subsite
B). However, the indole oxime fragment of 76 and 77 adopted a
bent conformation and consequently did not fully occupy subsite
D extending from galactose C1 (Fig. 4). In the case of galectin-3,
the indole aldoxime fragment was instead positioned in a cavity
extending above the b-face of the galactose moiety, which resulted
in arg144 being pinched by the two fragments. This cavity is not
present in the other galectins investigated, which possibly explains
the high galectin-3 selectivity conferred by the indole aldoxime
fragment.
In conclusion, we have from a panel of structurally simple
galactosyl oxime ethers identified potent and selective ligands for
galectin-3 and 7. Furthermore, ligands were synthesized using
a fragment based approach where amide and triazole moieties
optimized for subsite B in galectin-3 were combined with a
subsite D-binding oxime ether. This approach proved particularly
successful for galactose C3-triazoles as two compounds, 76 and
77, displayed low-micromolar affinity and indeed high selectivity
for galectin-3. Hence, the results herein represent a significant
progress towards potentially important research tools and towards
a lead for the development of galectin-3-targeting drugs as the
compounds are small, selective, show promising affinities, and are
less polar than natural saccharide ligands (e.g. 77 has Kd = 17 mM,
MW 449, and clogP 2.3).
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Table 4 Yields (%) and 1H NMR for hydroxylamines 54–62. Chemical shifts for substituents and H-1
Yield
22
1H NMR d:
HRMS calcd/found
336.1447/336.1441a
336.1447/336.1445a
302.1240/302.1237a
318.1189/318.1189a
386.0215/386.0211b
304.1196/304.1198a
302.1240/302.1246a
286.1291/286.1293a
342.1917/342.1920a
54
55
5f
7.87 (br s, 1H, Ar-H), 7.84–7.81 (m, 3H, Ar-H), 7.54 (dd, 1H, J 8.5 Hz, 1.7 Hz, Ar-H), 7.47–7.44 (m, 2H,
Ar-H), 4.54 (d, 1H, J 7.9 Hz, H-1), 4.30 (s, 2H, NCH2).
8.26 (d, 1H, J 8.3 Hz, Ar-H), 7.87–7.79 (m, 2H, Ar-H), 7.57–7.48 (m, 3H, Ar-H), 7.42 (dd, 1H, J 8.2 Hz, J
7.1 Hz, Ar-H), 4.63 (d, 2H, J 5.8 Hz, NCH2), 4.58 (d, 1H, J 7.9 Hz, H-1).
7.12 (t, 1H, J 8.0 Hz, Ar-H), 6.85–6.83 (m, 2H, Ar-H), 6.69 (ddd, 1H, J 8.1 Hz, J 2.4 Hz, J 1.1 Hz, Ar-H),
4.51 (d, 1H, J 7.7 Hz, H-1), 4.06 (S, 2H, NCH2).
6.99 (d, 1H, J 8.2 Hz, Ar-H), 6.28 (d, 1H, J 2.3 Hz, Ar.H), 6.24 (dd, 1H, J 8.1 Hz, J 2.3 Hz, Ar-H), 4.51 (d,
1H, J 7.6 Hz, H-1), 4.11 (d, 1H, J 12.0 Hz, NCH2), 4.04 (d, 1H, J 11.3 Hz, NCH2).
7.46 (br d, 2H, J 8.3 Hz, Ar-H), 7.32 (br d, 2H, J 8.3 Hz, Ar-H), 4.48 (d, 1H, J 7.6 Hz, H-1), 4.09 (d, 2H, J
1.1 Hz, NCH2).
7.34–7.28 (m, 1H, Ar-H), 7.21–7.17 (m, 2H, Ar-H), 7.01–6.96 (m, 1H, Ar-H), 4.50 (d, 1H, J 7.7 Hz, H-1),
4.13 (s, 2H, NCH2).
7.21 (dd, 1H, J 7.7 Hz, J 1.5 Hz, Ar-H), 7.11 (dd, 1H, J 7.8 Hz, J 1.6 Hz, Ar-H), 6.81–6.76 (m, 2H, Ar-H),
4.53 (d, 1H, J 7.52 Hz, H-1), 4.20 (d, 1H, J 12.4 Hz, NCH2), 4.13 (d, 1H, J 12.6, NCH2).
7.39 (br dd, 2H, J 8.3 Hz, J 1.6 Hz, Ar-H), 7.33–7.24 (m, 3H, Ar-H), 4.51 (d, 1H, J 7.7 Hz, H-1), 4.13 (s,
2H, NCH2).
44
20
57
58
59
60
61
62
33
15
15
34
37
32
7.36 (br d, 2H, J 8.54 Hz, Ar-H), 7.31 (br d, 2H, J 8.5 Hz, Ar-H), 4.51 (d, 1H, J 7.7 Hz, H-1), 4.10 (s, 2H,
NCH2), 1.30 (s, 9H, CH3).
a [M + H]+. b [M + Na]+.
9.6 Hz, J 3.2 Hz, H-3); ESIMS m/z calcd. for [C13H19NO6 + H]+:
286.1291. Found: 286.1293.
Experimental
General methods
Typical procedure for acylation of hydroxylamines
All commercial chemicals were used without further purification.
Thin layer chromatography (TLC) was carried out on 60F254 silica
(Merck) and visualization was made by UV light followed by
heating with aqueous sulfuric acid. Column chromatography (CC)
Compound 55 was dissolved in a freshly prepared mixture of
methanol, acetic acid anhydride and water (85:10:5, 2 mL) and
the reaction mixture was stirred overnight. Lyophilization gave
1
˚
was performed on silica (Amicon 35–70 mm, 60 A). Reversed
63 in quantitative yield: H NMR (300 MHz, MeOD): d 8.18–
phase chromatography was performed on Waters Sep-Pack Vac
35 cc C18–5 g columns. NMR experiments were recorded with
Bruker ARX 300 MHz or Bruker DRX 400 MHz spectrometers
at ambient temperature. 1H-NMR assignments were derived from
COSY experiments. Chemical shifts are given in ppm relative
to TMS, using the solvent residual peaks of CD2HOD at 3.31,
HDO at 4.79 and CHCl3 at 7.26. The optical rotations were
measured with a Perkin-Elmer 341 or a 241 polarimeter. HRMS
(FAB) were recorded with a JEOL SX-120 instrument or (ESI)
with a Micromass Q-TOF micro spectrometer. MALDI TOF MS
was recorded with a Bruker Biflex III. Fluorescence polarization
experiments and calculations were performed as described19,43 and
performed at 4 ◦C except for galectin-3 and -8N, which were
done at ambient temperature. Concentrations and probes used
for galectins-1, -3, -7, -8N and -9N were as described.19,43 The
probes were used at 0.1 mM.
8.16 (m, 1H, Ar-H), 7.89–7.79 (m, 2H, Ar-H), 7.55–7.41 (m, 4H,
Ar-H), 5.61 (d, 1H, J 16.2 Hz, NCH2), 5.35 (d, 1H, J 16.1 Hz,
NCH2), 4.72 (d, 1H, J 7.9 Hz, H-1), 3.84 (d, 1H, J 3.0 Hz, H-4),
3.72–3.54 (m, 3H), 3.49–3.43 (m, 2H), 2.31 (s, 3H, NAc); ESIMS
m/z calcd. for [C19H24NO7 + H]+: 378.1553. Found: 378.1555.
1,2,4,6-Tetra-O-acetyl-3-(3,5-dimethoxybenzamido)-3-deoxy-D-
galactopyranose 68
To a stirred solution of 3-azido-1,2,4,5-tetra-O-acetyl-3-deoxy-
D-galactopyranose 6738 (102 mg, 0.27 mmol) in EtOH (15 mL)
was added 1 M HCl (2.7 mL) and Pd/C (10%, 107 mg).
The mixture was hydrogenated (H2, 1 atm) for 70 minutes,
filtered through Celite, and concentrated under reduced pressure
to give the intermediate 3-amino-1,2,4,5-tetra-O-acetyl-3-deoxy-
D-galactopyranoside, which was immediately dissolved in dry
CH2Cl2 (10 mL). To the solution was added 3,5-dimethoxybenzoyl
chloride (546 mg, 2.7 mmol) dissolved in CH2Cl2 (10 mL) followed
by pyridine (1.64 mL). The reaction mixture was stirred under a ni-
trogen atmosphere overnight, concentrated, and co-concentrated
with toluene. Flash chromatography (EtOAc/heptane, 5:4) gave
68 (126 mg, 91%) as a mixture of anomers: [a]2D4 76 (c 0.5, MeOH);
1H NMR (400 MHz, CDCl3): d 6.77 (t, 4H, J 2.8 Hz, Ar-H), 6.56
(t, 2H J 2.3 Hz, Ar-H), 6.46 (t, 2H, J 7.4 Hz, a and b NH), 6.34
(d, 1H, J 3.6 Hz, aH-1), 5.84 (d, 1H, J 8.2 Hz, bH-1), 5.61 (br d,
1H, J 2.1 Hz, aH-4), 5.54 (d, 1H, J 3.2 Hz, bH-4), 5.36 (dd, 1H, J
11.5 Hz, J 3.6 Hz, aH-2), 5.17 (dd, 1H, J 11.2 Hz, J 8.3 Hz, bH-2),
4.81 (ddd, 1H, J 11.2 Hz, J 7.9 Hz, J 3.0 Hz, aH-3), 4.53 (ddd,
1H, J 11.1 Hz, J 7.9 Hz, J 3.3 Hz, bH-3), 4.41 (br t, 1H, aH-5),
Typical procedure for reduction of oxime ethers
Compound 44 (25 mg, 88 mmol) was dissolved in glacial acetic
acid (2 mL) and stirred under a nitrogen atmosphere. NaCNBH3
(28 mg, 446 mmol) was added, the reaction mixture was stirred
for 75 minutes, and then lyophilized. Flash chromatography gave
1
61 (9.3 mg, 37%): H NMR (400 MHz, MeOD): d 7.39 (br dd,
2H, J 8.3 Hz, J 1.6 Hz, Ar-H), 7.33–7.24 (m, 3H, Ar-H), 4.51 (d,
1H, J 7.7 Hz, H-1), 4.13 (s, 2H, NCH2), 3.81 (dd, 1H, J 1.0 Hz,
J 3.2 Hz, H-4), 3.77 (dd, 1H, J 11.4 Hz, J 7.1 Hz, H-6), 3.69 (dd,
1H, J 11.3 Hz, J 5.1 Hz, H-6¢), 3.59–3.49 (m, 2H), 3.46 (dd, 1H, J
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Table 5 1H NMR for acetylated hydroxylamines 63–66. Chemical shifts for substituents and H-1
1H NMR d:
HRMS calcd/found
378.1553/378.1555a
382.1114/382.1116b
366.1165/366.1155b
384.2022/384.2023a
63
64
65
66
8.18–8.16 (m, 1H, Ar-H), 7.89–7.79 (m, 2H, Ar-H), 7.55–7.41 (m, 4H, Ar-H), 5.61 (d, 1H, J 16.2 Hz, NCH2), 5.35 (d,
1H, J 16.1 Hz, NCH2), 4.72 (d, 1H, J 7.9 Hz, H-1), 2.31 (s, 3H, NAc).
7.09 (d, 1H, J 7.8 Hz, Ar-H), 6.27–6.24 (m, 2H, Ar-H), 4.74 (d, 1H, J 8.0 Hz, H-1), 4.85 (d, 1H, NCH2, partly
obscured by solvent peak), 4.81 (d, 1H, J 15.8 Hz, NAc), 2.26 (s, 3H, CH3).
7.22 (dd, 1H, J 7.9 Hz, J 1.1 Hz, Ar-H), 7.10 (dt, 1H, J 7.7 Hz, J 1.7 Hz, Ar-H), 6.79 (br t, 2H, J 8.6 Hz, Ar.H), 4.93
(d, 1H, J 16.2 Hz, NCH2), 4.92 (d, 1H, J 16.4 Hz, NCH2), 4.74 (d, 1H, J 8.0 Hz, H-1), 2.29 (s, 3H, NAc).
7.34 (d, 2H, J 8.4 Hz, Ar-H), 7.27 (d, 2H, J 8.47 Hz, ArH), 4.98 (d, 1H, J 15.8 Hz, NCH2), 4.88 (d, 1H, NCH2, partly
obscured by solvent peak), 4.72 (d, 1H, J 8.0 Hz, H-1), 2.25 (s, 3H, NAc), 1.30 (s, 9H, CCH3).
a [M + H]+. b [M + Na]+.
4.17–3.97 (m, 5H), 3.80 (s, 12H, OMe), 2.20 (s, 3H, OAc), 2.14 (s,
6H, OAc), 2.13 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.04 (s, 9H, OAc);
FAB-HRMS m/z calcd. for [C23H29NO12Na]+: 534.1587. Found:
534.1591.
O-[3-Deoxy-3-(3,5-dimethoxybenzamido)-b-D-galactopyranosyl]-
indole-3-carbaldoxime 71
To 70 (11 mg, 31 mmol) and indole-3-carboxaldehyde (6.2 mg,
43 mmol) dissolved in H2O (3 mL) and THF (1 mL) was added
0.1 M HCl (100 mL). The reaction mixture was stirred overnight,
neutralized with 0.1 M NaHCO3, and concentrated under reduced
pressure. The residue was dissolved in H2O and purified with
C-18 RP HPLC. Elution with a gradient of acetonitrile in H2O
and lyophilization gave 71 (8 mg, 53%) as a E/Z (4:1) mixture:
[a]2D2 36 (c 0.3, MeOH; 1H NMR (400 MHz, MeOD) for E isomer:
d 8.48 (s, 1H, NCH), 8.09 (br d, 1H, J 7.7 Hz, Ar-H), 7.56 (s, 1H,
Ar-H), 7.40 (br d, 1H, J 8.1 Hz, Ar-H), 7.22–7.11 (m, 2H, Ar-H),
7.08 (d, 2H, J 2.3 Hz, Ar-H), 6.65 (br t, 1H, J 2.3 Hz, Ar-H),
5.19 (d, 1H, J 8.2, H-1), 4.23 (dd, 1H, J 10.9 Hz, J 3.1 Hz, H-3),
4.06 (d, 1H, J 3.0 Hz, H-4), 3.99 (dd, 1H, J 10.9 Hz, J 8.2 Hz,
H-2), 3.84 (s, 6H), 3.84–3.72 (m, 3H). For Z isomer: 8.35 (s, 1 J,
NCH), 7.91 (s, 1H, Ar-H), 7.82 (br d, 1H, J 7.1 Hz, Ar-H), 5.19
(d, 1H, J 8.1, H-1): FAB-HRMS m/z calcd. for [C24H27N3O8Na]+:
508.1696. Found: 508.1700.
O-(2,4,6-Tri-O-acetyl-3-(3,5-dimethoxybenzamido-3-deoxy-b-D-
galactopyranosyl)-N-hydroxyphthalimide 69
To a stirred solution of 68 (92 mg, 0.18 mmol) in dry CH2Cl2 (2 mL)
was added Ac2O (34 mL) and after 10 minutes 33% HBr in glacial
acetic acid (1.2 mL). After 2 h, more CH2Cl2 (15 mL) was added,
the solution washed using H2O (15 mL), followed by saturated
NaHCO3 (20 mL), dried over Na2SO4, filtered, and concentrated at
ambient temperature. The intermediate was immediately dissolved
in 20 mL CH2Cl2 and tetrabutylammonium hydrogen sulfate
(61 mg, 0.18 mmol), N-hydroxyphthalimide (146 mg, 0.90 mmol)
and finally 1 M Na2CO3 (1.8 mL) were added. After 2 h, 20 mL
CH2Cl2 were added, the organic layer washed with 15 mL H2O,
15 mL saturated NaCl, and 15 mL H2O. The organic layer was
dried over Na2SO4 and concentrated under reduced pressure.
Flash chromatography (EtOAc/heptane, 5:4) gave 69 (60 mg, yield
55%): [a]2D2 47 (c 0.5, CH2Cl2); 1H NMR (300 MHz, CDCl3):
d 7.88–7.78 (m, 4H, H-Ar), 6.78 (d, 2H, J 2.3 Hz, Ar-H), 6.59
(d, 1H, J 7.8 Hz, NH), 6.55 (t, 1H, J 2.3 Hz, H-Ar), 5.57 (br d,
1H, J 2.4 Hz, H-4), 5.35 (dd, 1H, J 10.9 Hz, J 8.1 Hz, H-2), 5.25 (d,
1H, J 8.1 Hz, H-1), 4.59 (ddd, 1H, J 10.9 Hz, J 7.8 Hz, J 3.3 Hz,
H-3), 4.19–4.07 (m, 3H), 3.81 (s, 6H, OMe), 2.26 (s, 3H, OAc),
2.17 (s, 3H, OAc), 2.01 (s, 3H, OAc); FAB-HRMS m/z calcd. for
[C29H30N2O13Na]+: 637.1646. Found: 637.1660.
O-(3-Azido-3-deoxy-2,4,6-tri-O-acetyl-b-D-galactopyranosyl)-N-
hydroxyphthalimide 73
To 67 (0.31 g, 0.84 mmol) and N-hydroxyphthalimide (0.68 g,
4.18 mmol) was added dry CH2Cl2 (15 mL). The mixture was
cooled on an ice bath under a nitrogen atmosphere. A mixture of
boron trifluoridediethyl etherate (2.1 mL, 16.7 mmol) and CH2Cl2
(5 mL) was slowly added and the reaction mixture was left at 4 ◦C
for 48 hours. After filtration through Celite, the filtrate was washed
with saturated NaHCO3 and H2O. The combined organic layer
was dried (Na2SO4) and concentrated. Flash chromatography
(heptane/EtOAc, 1:1) gave 73 (130 mg, 33%): [a]2D2 1.6 (c 0.5,
CHCl3); 1H NMR (300 MHz, CDCl3): d 7.88–7.77 (m, 4H, Ar-H),
5.45–5.42 (m, 2H, J 3.5 Hz, J 1.1 Hz, J 2.3 Hz, J 8.0 Hz, H-2,
H-4), 4.99 (d, 1H, J 8.2 Hz, H-1), 4.17 (br dd, 2H, J 7.0 Hz, J
0.7 Hz, H-6, H-6¢), 3.90 (dt, 1H, J 6.7 Hz, J 1.2 Hz, H-5), 3.74 (dd,
1H, J 10.5 Hz, J 3.4 Hz, H-3), 2.28 (s, 3H, OAc), 2.21 (s, 3H, OAc),
2.00 (s, 3H, OAc); FAB-HRMS m/z calcd. for [C20H20N24O10Na]+:
499.1077. Found: 499.1072.
O-[3-Deoxy-3-(3,5-dimethoxybenzamido)-b-D-galactopyranosyl]-
hydroxylamine 70
To 69 (26 mg, 43 mmol) dissolved in MeOH (2 mL) was added
hydrazine hydrate (0.5 mL), and the reaction mixture was stirred
overnight, and then concentrated. The residue was dissolved in
H2O and applied on to C-18 silica (5 g). Elution with a gradient
1
of MeOH in H2O and lyophilization gave 70 (15 mg, 72%): H
NMR (300 MHz, MeOD): d 7.05 (d, 2H, J 2.3 Hz, Ar-H), 6.94 (d,
0.4H, J 2.3 Hz, NH partly exchanged with solvent), 6.64 (t, 1H, J
2.3 Hz, Ar-H), 4.53 (d, 1H, J 8.0 Hz, H-1), 4.10 (dd, 1H, J 10.8 Hz,
J 3.1 Hz, H-3), 3.97 (br d, 1H, J 2.7 Hz, H-4), 3.81 (s, 6H, OMe),
3.75–3.69 (m, 3H); FAB-HRMS m/z calcd. for [C15H22N2O8Na]+:
381.1274. Found: 381.1283.
O-(3-Azido-3-deoxy-b-D-galactopyranosyl)-hydroxylamine 74
Compound 73 (0.19 g, 0.41 mmol) dissolved in 2.3 M MeNH2
in MeOH was stirred overnight and concentrated under reduced
pressure. Flash chromatography (CH2Cl2/MeOH, 5:1–4:1) gave
1
74 (80 mg, 88%): [a]2D2 -5.3 (c 1, MeOH); H NMR (300 MHz,
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D2O): d 4.58 (d, 1H, J 8.0 Hz, H-1), 4.07 (d, 1H, J 2.9 Hz, H-4),
3.76–3.72 (m, 3H), 3.67 (dd, 1H, J 10.5 Hz, J 8.0 Hz, H-2), 3.56
(dd, 1H, J 10.5 Hz, J 3.2 Hz, H-3); FAB-HRMS m/z calcd. for
[C6H12N4O5Na]+: 243.0705. Found: 243.0706.
11.1 Hz, J 8.0 Hz, H-2), 4.16 (d, 1H, J 3.0 Hz, H-4). 3.99–3.95 (m,
1H, H-5), 3.84 (dd, 1H, 11.4 Hz, H-6), 3.76 (dd, 1H, J 11.4 Hz, J
5.8 Hz, H-6¢). For Z isomer: 8.38 (s, 1H, NCH), 7.93 (s, 1H, Ar-H),
7.82–7.80 (m, 2H, Ar-H), 5.30 (d, 1H, J 7.8 Hz, H-1), 4.59 (dd, 1H,
J 11.0 Hz, J 8.0 Hz, H-2); ESIMS m/z calcd. for [C23H24N5O5]+:
450.1777. Found: 450.1754.
O-(3-Azido-3-deoxy-b-D-galactopyranosyl)-indole-3-
carbaldoxime 75
Computational methods
To 74 (27 mg, 123 mmol) and indole-3-carboxaldehyde (23 mg,
159 mmol) dissolved in H2O (2 mL) and THF (2 mL) was added
0.1 M HCl (123 mL). The reaction mixture was stirred overnight,
neutralized with 0.1 M NaHCO3, and concentrated under reduced
pressure. Flash chromatography (CH2Cl2/MeOH, 4:1) gave 75
(13 mg, 67%) as a E/Z (10:1) mixture: [a]2D2 -27 (c 0.5, MeOH); 1H
NMR (400 MHz, MeOD) for E isomer: d 8.48 (s, 1H, NCH), 8.06
(d, 1H, J 7.9 Hz, Ar-H), 7.57 (s, 1H, Ar-H), 7.41 (d, 1H, J 8.1 Hz,
Ar-H), 7.20 (br dt, 1H, J 7.6 Hz, J 1.1 Hz, Ar-H), 7.12 (dt, 1H, J
1.0 Hz, J 7.5 Hz, Ar-H), 5.11 (d, 1H, J 8.2 Hz, H-1), 3.99 (d, 1H,
J 3.0 Hz, H-4), 3.95 (dd, 1H, J 10.3 Hz, J 8.2 Hz, H-2), 3.81–3.69
(m, 2H), 3.45 (dd, 1H, J 10.4 Hz, J 3.1 Hz, H-3). For Z isomer:
8.35 (s, 1H, NCH), 7.90 (s, 1H, Ar-H), 7.80 (br d, 1H, J 7.8 Hz,
Ar-H), 7.45 (br d, 1H, J 7.3 Hz, Ar-H), 5.10 (d, 1H, J 8.1 Hz, H-1);
FAB-HRMS m/z calcd. for [C15H17N5O5Na]+: 370.1127. Found:
370.1137.
Molecular modeling was performed with the MMFFs force field
withwater implementedinMacroModel (version9.1, Schro¨dinger,
LLC, New York, 2005). All triazole and oxime torsions were
systematically varied, minimized, and finally minimized in com-
plex with galectins. Starting conformations were built from the
published crystal structures of galectin-1, 7, and 9 N in complex
with lactose,44–46 of galectin-3 in complex with a C3¢-amido-
derivatised LacNAc-based inhibitor,17 and a homology model of
galectin-8N in complex with LacNAc.14 Starting conformations
of the amides were positioned in the two possible orientations.
Acknowledgements
The authors would like to thank Barbro Kahl-Knutson for help
with fluorescence polarization analysis and galectin production
and Susanne Carlsson for providing galectins 8 N and 9 N. Support
from the Lund University Research School of Medicinal Sciences,
the Swedish Research Council, and the programs “Glycoconju-
gates in Biological Systems” and “Chemistry for Life Sciences”
sponsored by the Swedish Strategic Research Foundation is
acknowledged.
O-[3-Deoxy-3-(4-methylaminocarbonyl-1H-1,2,3-triazol-1-yl)-b-
D-galactopyranosyl]-indole-3-carbaldoxime 76
To 75 (0.9 mg, 2.6 mmol) dissolved in propanol (0.5 mL) was added
Cu wire, followed by methyl propiolate (0.5 mL). The reaction
mixture was stirred for 24 hours then concentrated. The residue
was dissolved in 40% MeNH2 in H2O, stirred overnight, and
concentrated. Flash chromatography (CH2Cl2/MeOH, 10:1) gave
76 (0.72 mg, 65%) as a E/Z (10:1) mixture: 1H NMR (400 MHz,
MeOD) for E isomer: d 8.48 (s, 1H, NCH), 8.48 (s, 1H, triazole-H),
8.20 (d, 1H, J 7.8 Hz, Ar-H), 7.57 (s, 1H, Ar-H), 7.40 (d, 1H, J
8.1 Hz, Ar-H), 7.21–7.18 (m, 1H, Ar-H), 7.16–7.11 (m, 1H, Ar-H),
5.21 (d, 1H, J 8.0 Hz, H-1), 4.98 (dd, 1H, J 11.1 Hz, J 3.1, H-3),
4.35 (dd, 1H, J 11.1 Hz, J 8.0 Hz, H-2), 4.11 (d, 1H, J 2.8 Hz, H-4),
3.82 (dd, 1H, J 11.4 Hz, J 6.4 Hz, H-6), 3.74 (dd, 1H J 11.5 Hz, J
5.8 Hz, H-6¢), 3.84–3.72 (m, 2H), 2.94 (s, 3H, CH3). For Z isomer:
8.52 (s, 1H, triazole-H), 8.37 (s, 1H, NCH), 7.92 (s, 1H, Ar-H),
7.81 (br d, 1H, J 7.1 Hz, Ar-H), 5.28 (d, 1H, J 7.9 Hz, H-1);
MALDI TOF m/z calcd. for [C19H22N6O6Na]+: 454.15. Found:
454.67; ESIMS m/z calcd. for [C19H23N6O6]+: 431.1679. Found:
431.1678.
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