Benzimidazole–galactosides bind selectively to the Galectin-8 N-Terminal domain: Structure-based design and optimisation

Article abstract of DOI:10.1016/j.ejmech.2021.113664

We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline–galactoside ligand at a resolution of 1.6 ?. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)–galactoside with a K_d of 1.8 μM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole–galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Full text of DOI:10.1016/j.ejmech.2021.113664

European Journal of Medicinal Chemistry 223 (2021) 113664
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Benzimidazoleegalactosides bind selectively to the Galectin-8 N-
Terminal domain: Structure-based design and optimisation
,
b
,
, b,
Mujtaba Hassan a 1, Sjors van Klaveren a 1, Maria Håkansson c, Carl Diehl c,
a
a
b
c
ꢀ ꢀ c
ꢁ
ꢀ
€
Rebeka Kovacic , Floriane Baussiere , Anders P. Sundin , Jaka Dernovsek , Bjorn Walse ,
d
e
b
, **
ꢀ ꢀ b
Fredrik Zetterberg , Hakon Leffler , Marko Anderluh , Tihomir Tomasic
,
Ziga Jakopin ***, Ulf J. Nilsson a
b,
, *
ꢀ
a Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Box 124, SE-221 00, Lund, Sweden
b
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva cesta 7, 1000 Ljubljana, Slovenia
c SARomics Biostructures AB, Medicon Village, SE-223 81, Lund, Sweden
d Galecto Biotech AB, Sahlgrenska Science Park, Medicinaregatan 8 A, SE-413 46, Gothenburg, Sweden
e Department of Laboratory Medicine, Section MIG, Lund University BMC-C1228b, Klinikgatan 28, 221 84, Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a
previously reported quinolineegalactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a
collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole
Received 4 February 2021
Received in revised form
13 June 2021
Accepted 19 June 2021
Available online 25 June 2021
moieties were designed and synthesised. This led to the discovery of
a 3-O-(N-methyl-
benzimidazolylmethyl)egalactoside with a Kd of 1.8 M for galectin-8N, the most potent selective syn-
m
thetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole
egalactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selec-
tivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological
lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-
derivatised galactosides represent promising compounds for studies of the pathological implications
of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory
therapeutics targeting galectin-8.
Keywords:
Benzimidazole
Galectin-8N
Quinoline
Selectivity
Molecular dynamics
X-ray crystallography
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
contains a single CRD that can aggregate with its glycine and
proline-rich N-terminal domain [3]. The third subclass comprises
Galectins are a family of carbohydrate-binding proteins found in
most organisms [1]. While all lectins bind (oligo-) saccharides,
the tandem repeat galectins with two different CRDs, at the N- and
C-terminals, respectively [1]. Galectin-8 belongs to the subclass of
the tandem repeat lectins.
galectins have a specific affinity for
b-galactosides due to the
structural and sequence similarity in their carbohydrate recogni-
tion domains (CRD) [2]. The family of galectins is divided into three
subclasses according to their CRD distribution. The first subclass
contains the monomeric galectins, with one CRD that dimerises
depending on the concentration and ligand density. The second
subclass consists of only the chimera-type galectin-3, which
The two distinct CRDs of galectin-8 and the connecting hinge
domain are all required for the protein to function [4]. Therefore,
blocking the recognition in one CRD is sufficient to hinder the
overall functionality of the protein. Although both CRDs bind b-
galactosides as found in natural glycans, binding of native oligo-
saccharides and glycosphingolipids to the CRD on the N-terminal
domain of galectin-8 occurs with a higher affinity [5,6]. For this
reason, we aimed to design selective ligands for the galectin-8 N-
terminal domain.
* Corresponding author.
** Corresponding author.
*** Corresponding author.
Galectins participate in a wide variety of cellular processes such
as cell adhesion, migration, proliferation, survival, and differentia-
tion, and are involved in a broad range of pathologies [3,7].
E-mail address: ulf.nilsson@chem.lu.se (U.J. Nilsson).
These authors contributed equally.
1
https://doi.org/10.1016/j.ejmech.2021.113664
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
Galectin-8 promotes VEGF-C mediated lymphangiogenesis, which
is involved in many pathological conditions, including tumour
metastasis, organ graft rejection, corneal inflammation, and type 2
diabetes [8]. Galectin-8 is upregulated in several cancers, including
lung, kidney, bladder, and breast cancer [9]. By modulating the
innate and adaptive immune system through the regulation of T-
cell homeostasis, galectin-8 is involved in several inflammatory and
autoimmune disorders [10,11]. A few reports link the unique
functions of galectin-8 to the specificity of the N-terminal CRD [12].
Due to its ability to induce selective autophagy upon recognising
damaged intracellular vesicles, galectin-8 also has antibacterial
activity [13]. Because of this wide range of functions, selective and
high-affinity galectin-8N ligands would be valuable research tools,
as well as potential anti-tumour and anti-inflammatory drug can-
didates. The literature reports a few synthetic galactose-based in-
hibitors of galectin-8N, including a methyl 3-O-[1-carboxyethyl]-
-galactopyranoside [14], galactomalonyl phenyl esters [15], and
fused tricyclic galactoseebenzene hybrids [16]. The
quinolineegalactoside 1 (Fig. 1) is the only galactose-based ligand
reported to bind galectin-8N (Kd 110 M) with good selectivity over
b-
D
m
most other galectins [17]. The quinolineegalactoside 2, lacking the
carboxylic acid functionality, had about a 7-fold lower affinity for
galectin-8N with a Kd value of 700
quinolineegalactoside 3, bearing
aglycon, binds galectin-3 and galectin-8N with Kd values of
1.2 M and 1.5 M Kd, respectively [17]. The improved affinity
caused by the -3,4-dichlorophenyl aglycon likely results from a
halogen bonding interaction between the 3-chloro substituent and
the Gly87 backbone carbonyl, as well as an Se interaction be-
mM. On the other hand, the
a-3,4-dichlorothiophenyl
m
m
a
p
tween the anomeric sulfur and Trp86 [18]. Using
quinolineegalactoside 1 as a vantage point, our immediate focus
aimed at further improving galectin-8N selectivity over galectin-3.
Herein we report the X-ray crystallography of compound 1 with
galectin-8N and the use of this structure in the design, synthesis,
and evaluation of galectin-8N inhibitors with improved selectivity
over other mammalian galectins.
2. Results and discussion
2.1. Structural analysis
Fig. 2. (A) The electron density map (green mesh) 2|F0| ꢀ |Fc|
ac contoured at 1s, for
The protein used for crystallisation was produced from an N-
terminal 6-His-tagged construct where the His-tag was removed
using TEV cleavage before final purification. The purified protein
starts with Ser4 and ends with Gln158. The three first and the two
last amino acids of the construct are not visible in the electron
density.
compound 1 in complex with galectin-8N (PDB ID: 7AEN; grey cartoon representa-
tion). (B) The green dashed lines in the figure reveal the interactions of the carboxy
quinoline moiety compound 1 (in sticks: carbon in green, and oxygen in red) with
Arg45 and Gln47 (in sticks: carbon in grey, oxygen in red, and nitrogen in blue), ac-
counting for galectin-8N selectivity.
The galectin-8Nelactose complex was obtained by co-
crystallisation using 10 mM lactose added to the protein prior to
crystallisation, whereas the galectin-8Ne1 complex was obtained
by soaking the ligand into galectin-8N-lactose crystals. The crystal
structures were determined by X-ray diffraction at resolutions of
1.35 Å for the galectin-8Nelactose complex and 1.6 Å for the
corresponding galactose in the galectin-8Nelactose complex. The
HO4 of the methyl b-D-galactopyranoside is involved in a hydrogen-
bonding network with Arg45, Arg69, and His65. The HO6 engages
in hydrogen bonding interactions with Asn79 and Glu89. The ni-
trogen atom of the quinoline ring is placed in a favourable position
for the electron-rich ring nitrogen to establish an ionedipole
interaction with the guanidinium of Arg45. Furthermore, the
galectin-8Ne1 complex (Fig. 2). The binding mode of the methyl
b-
D
-galactopyranoside in compound is identical to the
1
Fig. 1. The structure of the previously reported galectin-8N quinolineegalactoside inhibitors 1e3 [17].
2

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
carboxylate moiety of the quinoline is placed in a favourable
orientation to establish a water-mediated hydrogen bond with
Arg45 (Fig. 2). The ionedipole interaction and the water-mediated
hydrogen bond with Arg45 may account for the higher affinity and
selectivity of the quinolineecarboxylate to galectin-8N. The pre-
dicted binding mode of compound 1, previously obtained from
molecular dynamics simulations [17], is nearly identical to the
binding mode in our galectin-8Ne1 crystal structure.
5ae5c (Scheme 1). Esterification of the latter in methanol yielded
the methyl azidobenzoates 6ae6c. In parallel, b-D-galactopyrano-
sides 7a and 7b were used in a protecting group-free, dibutyl-
stannylene-mediated, 3-O-alkylation to give alkynes 8a and 8b
(Scheme 2) [21]. In a copper-catalysed alkyneeazide click reaction,
azides 6ae6c were reacted with alkynes 8a and 8b to give the
methyl benzoates 9ae9f. Alkaline hydrolysis of esters 9ae9e pro-
duced the respective benzoic acids 10ae10e. Using the click reac-
tion, benzoic acid 10f was synthesised directly from azide 5c and
alkyne 8b. The binding affinities of compounds 9ae9f and 10ae10f
were determined in a competitive protein-binding fluorescence
polarisation assay (Table 1) [22e24].
The binding data presented in Table 1 show that none of the
triazole compounds had a higher affinity than the previously
discovered quinoline 1. Nevertheless, the presented data did give us
some insight and steered our further design. Among the function-
alised triazoles, the most promising structure for binding to
2.2. Design, synthesis, and binding affinities
For a systematic investigation of the galectin-8N binding pocket,
we synthesised a collection of galactosides derivatised at O3 with
triazoles carrying substituted benzoic acids (Fig. 3 and Scheme 2).
Triazoles are easily accessible through reliable ‘click’ chemistry and
are found in several other scaffolds targeting different galectins
with high affinity [19].
The triazole scaffold loosely mimics the general scaffold of
quinoline 1, with a benzoic acid attached to a heteroaromatic ring,
but with an additional measure of rotational freedom between
these two rings (Fig. 3, panel A). A prospective docking approach in
the crystal structure of galectin-8e1 revealed that the triazole ni-
trogen of compounds 10ae10f is positioned in proximity to engage
in ionedipole interactions with the guanidinium side chains (Arg45
or Arg69) on either side of the galectin-8N binding site, analogous
to the nitrogen in quinoline 1 (Fig. 3, panel B and C). Additionally,
the carboxylic acids of 10d and 10e are suggested to plausibly
overlap that of quinoline 1, where it engages in binding with the
structural water. At the anomeric position of the conserved galac-
galectin-8N was meta-benzoic acid 10e with an affinity of 640 mM.
The meta-substituted congeners 9e and 10b also had a higher af-
finity than their ortho- and para-counterparts. Although the range
of binding affinities of compounds 9ae9f and 10ae10f on galectin-
8N is relatively narrow, the SAR extracted from this series suggests
that aromatic structures with meta-substituted carboxylic acids are
optimal for galectin-8N ligands with a diaromatic scaffold. Our
subsequently investigated compound class corroborates this trend.
tose, both
b-S-tolyl and b-O-methyl aglycons have been widely
applied in synthetic galactose-based inhibitors of galectins [17]. In
this series, both these groups are included as a control for the
structureeactivity relationship (SAR) of the differently substituted
benzoic acid moieties.
The convergent synthesis of triazoles started from a tert-butyl-
nitrite-promoted azidotrimethylsilane substitution of amino-
benzoic acid derivatives 4ae4c [20], which yielded aryl azides
Scheme 1. Reagents and conditions: (a) TMSN3, t-BuONO, MeCN,
(81e99%); (b) H2SO4, MeOH, 70 ꢁC, overnight (70e92%).
0
ꢁCert, 1 h,
Fig. 3. (A) Visualised rationale for the diaromatic scaffold. (B), (C) Overlay of the docking poses of triazole-benzoic acids 10e (magenta sticks) and 10d (cyan sticks) with the
experimental binding mode of quinoline 1 (green sticks) in complex with galectin-8N (PDB ID: 7AEN; grey cartoon representation). Hydrogen bonding of 10e is shown in magenta
dashed lines. The hydrogen bonding of the carboxylic acid of quinoline 1 (in green dashed lines) overlaps with the predicted binding of triazole-benzoic acid 10d (in cyan dashed
lines).
3

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
Scheme 2. Reagents and conditions: (a) propargyl bromide, K2CO3, n-Bu2SnCl2, n-Bu4NBr, MeCN, 80 ꢁC, 3 h, (35e86%); (b) Azides 6ae6c, CuI, DIPEA, MeCN, rte50 ꢁC, overnight,
(59e96%); (c) NaOH, H2O, MeCN, 50 ꢁC, 30 min, (44e99%).
The stannylene-mediated 3-O-alkylation of methyl
b-D-gal-
Table 1
d values (m
M)a of compounds 9ae9f and 10ae10f.
K
actopyranoside 7b with alkyl halides 11ae11d afforded alkylated
galactosides 12ae12d (Scheme 3). It is worth mentioning that the
synthesis of compound 12a did not proceed without the Boc pro-
Compound
Galectin-8N
Galectin-3
7b [24]
Lactose [5,25]
1 [17]
2 [17]
9a
6300
91
110
4400
231
380 11
620 50
>2000
tection of benzimidazole 11a. The alkylation of methyl
b-D-gal-
actopyranoside 7b with benzimidazole 11a resulted in the
spontaneous deprotection of the Boc group to give benzimidazole
12a.
6
700 41
1100 250
1200 280
940 40
9b
9c
490
8
Compounds 12ae12d were evaluated for their binding affinities
against galectin-8N and galectin-3 (Table 1). The benzimidazole
12a and benzothiazole 12c had binding affinities for galectin-8N
comparable to that of the non-functionalised quinoline 2, while
the N-methylbenzimidazole 12b and benzoxazole 12d had slightly
weaker affinities. Benzimidazoles 12a and 12b showed selectivity
for galectin-8N over galectin-3 and were chosen to investigate the
effect of the added carboxylic acid moiety similar to quinoline 2.
The benzimidazole chlorides 14ae14c were synthesised by 2-
chloro-1,1,1-trimethoxyethane condensation from the corre-
sponding 1,2-diamines 13ae13c [29]. The Boc protection of 14c
afforded the two regioisomers of the benzimidazole 14d. Alkylation
>2000
9d
9e
1400 240
810 86
330 75
510 79
700 94
690 140
250 40
230 49
470 70
500 63
190 18
9f
>2000
10a
1200 180
10b
10c
10d
10e
10f
12a
12b
12c
12d
15a
15b
15c
16a
760
8
1500 200
N.B.b
640 38
1300 200
860 91
1200 72
870 37
1600 51
1100 120
1600 310
1200 210
190 24
>2000
1500
7
1600 180
510 71
620 148
N.B.b
2000 56
1600 110
1400 84
1100 140
590 16
of methyl
b-D-galactopyranoside 7b with the benzimidazoles
14ae14d via stannylene acetal-mediated 3-O-alkylation afforded
the methyl esters 15ae15c (Scheme 4). The subsequent alkaline
hydrolysis produced carboxylic acids 16ae16c.
Next, the affinities of esters 15ae15c and their corresponding
carboxylic acids 16ae16c for galectin-8N and galectin-3 were
determined (Table 1). The esters 15ae15c had a similar affinity to
the non-functionalised benzimidazole 12b, with negligible selec-
tivity over galectin-3. In contrast, the carboxylic acids 16a and 16c
16b
16c
310 26
a
Results represent the mean SEM of n ¼ 4 to 8.
b
Non-binding up to the highest tested concentration of 1500 mM.
Benzimidazoles, benzothiazoles, and benzoxazoles are privi-
leged scaffolds in drug discovery [26e28] and were investigated
because they fit the diaromatic scaffold presented in Fig. 3, panel A.
had galectin-8N affinities of 190
mM and 310 mM, respectively;
about four-fold higher than their congeners 12a and 12b. The car-
boxylic acid 16b had a lower binding affinity for galectin-8N and a
similar binding affinity for galectin-3 when compared to those of
12a and 12b. The benzimidazole 16a is the most selective mono-
saccharide ligand for human galectin-8N reported to date with a 7-
fold selectivity over galectin-3.
The heterocyclic rings were hypothesised to engage in a catione
p
stacking with Arg45, or form ionedipole interactions with Arg45 or
Arg69, similar to the nitrogen in quinoline 1. With synthetic
accessibility in mind, we initially synthesised the galactosides
lacking the carboxylic acid moiety for comparison to the previously
reported quinoline 2.
Based on the previously published SAR (Fig. 1), we hypothesised
that combining the carboxy-benzimidazole with
a-3,4-
dichlorothiophenyl aglycon might significantly increase the bind-
ing affinity for galectin-8N [17]. A microwave-assisted tin-mediated
alkylation of galactosides was adopted to alkylate 3,4-
dichlorophenyl
a-thiogalactoside 17 with the alkyl chloride 14a
and 14d to afford compounds 18a and 18b [30]. The following
alkaline hydrolysis of the methyl esters yielded the carboxy-
benzimidazoles 19a and 19b (Scheme 5).
The affinities of compounds 19a and 19b for galectin-1, -3, -4C
(C-terminal), -4N (N-terminal), ꢀ7, -8C, -8N, -9C, and -9N are given
in Table 2. Compounds 19a and 19b achieved over 100-fold gain in
binding affinity for galectin-8N compared to the parent compounds
16a and 16c with Kd values of 1.8
Furthermore, N-methylbenzimidazole 19a displayed some
mM and 2.7 mM, respectively.
Scheme 3. Reagents and conditions: (a) i. 7b, n-Bu2SnO, MeOH, 70 ꢁC, 2 h; ii. n-
Bu4NBr, 1,4-dioxane, 85 ꢁC, overnight, (47e64%).
4

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
Scheme 4. Reagents and conditions: (a) 2-chloro-1,1,1-trimethoxyethane, BF3.Et2O, DCM, rt, (92e94%); (b) Boc2O, DMAP, DMF, rt, (82%); (c) i. 7b, Bu2SnO, MeOH, 70 ꢁC, 2h; ii. n-
Bu4NBr, 1,4-dioxane, 85 ꢁC, overnight, (65e77%); (d) KOH, EtOH, H2O, 80 ꢁC, 4e6 h (37e56%).
Scheme 5. Reagents and conditions: (a) 14a or 14d, Bu2SnO, n-Bu4NI, PhMe, MeCN, MW, 120 ꢁC, 1 h (31e34%); (b) 18a, KOH, EtOH, H2O, 80 ꢁC, 6 h (31%). (c) 18b, LiOH, EtOH, H2O,
80 ꢁC, overnight (41%).
Table 2
K
d values (
m
M)a of compound 19a and 19b towards galectins.
Galectin (N: N-terminal domain, C: C-terminal domain)
1
2
3
4 N
4C
7
8N
8C
9N
9C
45
19a
19b
130 11 a
87 6.1
220 19
100
5.0 1.1
4.1 0.4
130 12
78 14
120 37
160 12
190 33
43 5.5
1.8 0.1
2.8 0.1
760 160
450 23
16 1.5
13 1.2
2
9
18 0.7
a
Results represent the mean SEM of n ¼ 4 to 8.
selectivity for galectin-8N over galectin-3 and galectin-9N together
with about 25e400-fold selectivity over the remaining assayed
galectins. The unsubstituted benzimidazole 19b binds galectin-8N
with lower selectivity over galectin-3 and with about 5- and 9-
fold selectivity over galectin-9N and galectin-9C, respectively.
Hence, the benzimidazole N-methyl group in 19a apparently
induced a small selectivity for galectin-8N over galectin-3.
Taken together, the previously published quinolineegalactoside
ligands [17], the structural analysis of 1 in complex with galectin-
8N (Fig. 2), and the affinity data presented here show that galac-
tosides derivatised at O3 with fused bicyclic heterocycles carrying a
carboxylic acid moiety enhance galectin-8N affinity. A further
comparison of the benzimidazoles 16a and 16c with 19a and 19b
2.3. Molecular modelling
Molecular dynamics simulations were performed to understand
the selectivity of the benzimidazoleegalactosides for galectin-8N.
In all simulations, the N-methyl benzimidazole ring stacked with
Arg45 in galectin-8N or the corresponding Arg144 in galectin-3
(Fig. 4). The main difference between the binding modes in the
two galectin CRDs is the placement of the benzimidazole. In
galectin-8N, both benzimidazoles 16a and 19a point the N-methyl
group towards the galactose O2, whereas in galectin-3 the N-
methyl group, and consequently the carboxylic acid, point away
from the O2. As a result, the basic nitrogen of the benzimidazole can
form a hydrogen bond with Gln47 of galectin-8N, which is not
possible with the corresponding Ala146 or Asp148 in galectin-3.
The N-methyl group of a bound benzimidazole-based inhibitor
covers and thus shields the arginine side chain from bulk water,
which may partly explain the binding affinity of compound 16b. As
for compound 19a, a halogen bond was indicated by the close
confirms earlier observations that an
a-anomeric 3,4-
dichlorothiophenyl aglycon improves the binding affinity for
galectin-8N by two orders of magnitude. However, the 3,4-
dichlorothiophenyl aglycon also improves affinity for galectin-3
and thus reduces the selectivity.
5

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
Fig. 4. (A), (B) Molecular dynamics simulation snapshots of representative low-energy conformations of 16a (in yellow sticks) in complex with galectin-8N and galectin-3,
respectively. (C), (D) Molecular dynamics simulation snapshots of representative low-energy conformations of 19a (in teal sticks) in complex with galectin-8N and galectin-3,
respectively. Hydrogen bonding is shown in corresponding dashed lines.
distance and straight angle between the 3-chloro substituent and
the backbone carbonyl oxygen of Gly87 in galectin-8N, and Gly182
in galectin-3, respectively (Fig. 4, panels C and D). This halogen
bonding is consistent with previous findings [18]. The carboxylate
moiety of the benzimidazole was placed in a favourable position to
establish a water-mediated hydrogen bond with Gly142 in galectin-
8N (Fig. 4, panel A). The binding of the nitrogen lone pair of the
benzimidazole to the non-conserved Gln47 likely contributes the
higher selectivity of the benzimidazoleegalactosides compared to
the quinolineegalactosides.
benzimidazoleegalactosides. The benzimidazole 19a represents a
promising tool to investigate the biomedical implications of
galectin-8 and a steppingstone towards highly selective galectin-
8N ligands with potential therapeutic applications.
4. Experimental section
4.1. Competitive fluorescence polarisation experiments
Human galectins-1, -3, -4N, -4C, -7, -8N, -9N, and -9C were
expressed and purified as previously described [5,23,31]. Fluores-
cence polarisation experiments were performed using the PHER-
Astar FS plate reader (software version 2.10 R3), and the
fluorescence anisotropy of fluorescein tagged probes was measured
by excitation at 485 nm and emission at 520 nm. The specific
conditions for galectins-1, 3, -4N, -4C, -7, -8N, -9N, and -9C were
kept as reported [22,31,32]. The synthesised compounds were
dissolved in pure DMSO at 20 mM concentration and diluted in PBS
to 3e6 different concentrations, and each concentration was tested
in duplicate. The highest inhibitor concentrations tested were
1.5 mM. The average values of Kd and SEM were calculated from 4 to
8 duplicate measurements, showing 10e90% inhibition.
3. Conclusion
We have crystallised the quinolineegalactoside 1 with galectin-
8N and unravelled the interactions accounting for the selectivity for
galectin-8N. This crystal structure was used to design heterocycles
as quinoline surrogates. The N-phenyltriazoles, benzoxazole, and
benzothiazoleegalactosides showed no direct improvement over
the quinolineegalactoside. The benzimidazole and N-methyl
benzimidazole, however, showed higher selectivity for galectin-8N
compared to the original quinolineegalactoside. N-methyl benz-
imidazole 16a is the most selective monosaccharide galectin-8N
ligand to date with 7-fold selectivity over galectin-3. The position
of the carboxylic acid proved crucial for both affinity and selectivity
for galectin-8N, as evidenced by the comparison of the binding
affinities of 16a and 16b. The binding affinity of 16a was greatly
4.2. Protein expression and purification
The clone for galectin-8N was ordered from GenScript (Leiden,
the Netherlands). The DNA sequence encoding amino acids 5e158
from human galectin-8 (UniProt entry O00214 isoform 1) was
cloned into pET28a with an N-terminal His-tag and a TEV cleavage
site. Ser4 in galectin-8N originates from the remaining Ser in the
TEV site after cleavage. The plasmid was transformed into E. coli
BL21 (DE3) R3 and the expression was started by inoculating LB
enhanced by the introduction of the
moiety, resulting in compound 19a with a Kd of 1.8
a
-3,4-dichlorothiophenyl
M for
m
galectin-8N, while maintaining selectivity over the other galectins.
Thus, compound 19a represents the most potent selective galectin-
8N ligand reported to date. Molecular dynamics simulations sug-
gested that benzimidazole galactosides bind the non-conserved
amino acid Gln47 in galectin-8N, accounting for the selectivity of
media containing Kanamycin (50 mg/mL) with an overnight culture.
6

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
Cells were grown to OD ~0.6 at 37 ꢁC, induced with 0.5 mM IPTG,
and incubated overnight at 18 ꢁC. Cells were harvested by centri-
fugation, resuspended in lysis/IMAC A buffer (10 mM Tris pH 8,
150 mM NaCl, 0.5 mM TCEP) supplemented with Triton-X, lyso-
zyme, benzonase, and PMSF, and lysed using sonication. Lysed cells
were clarified using centrifugation and the supernatant was added
mesh, 40e63
1260 Infinity system with
m
m). Preparative HPLC was performed on an Agilent
a
SymmetryPrep C18,
5
mM,
19 mm ꢂ 100 mm column using a gradient (water with 0.1% formic
acid and acetonitrile). Monitoring and collection were based on
UVevis absorbance at 210 and 254 nm. NMR spectra 1H, 13C, 2D
COSY, and HMQC were recorded with a Bruker Avance II 400 MHz
spectrometer (400 Hz for 1H, 100 Hz for 13C) or a Bruker Avance III
500 MHz spectrometer (500 Hz for 1H, 125 Hz for 13C) at ambient
to a drop column containing 5 mL
a-Lactose Agarose (Sigma
Aldrich) and incubated under rotation for 1 h at 4 ꢁC. The column
was washed with lysis buffer, and galectin-8N was eluted using
200 mM lactose. Eluted fractions were cleaved using TEV protease
overnight at 4 ꢁC and loaded on a 5 mL Histrap column equilibrated
with lysis buffer. The column was washed with 2% IMAC B (10 mM
Tris pH 8, 150 mM NaCl, 0.5 mM TCEP, 500 mM imidazole) and
uncleaved protein and cleaved His-tag eluted using a linear
gradient of 2e50% IMAC B. Flowthrough and wash fractions con-
taining galectin-8N were concentrated and injected on a Superdex
S75 16/60 equilibrated with SEC buffer (10 mM Tris pH 8, 150 mM
NaCl, 0.5 mM TCEP). The purity was analysed using SDS-PAGE and
fractions containing galectin-8N were concentrated to 20 mg/mL.
temperature. Chemical shifts are reported in
(ppm), with multiplicity (b ¼ broad, s ¼ singlet, d ¼ doublet,
triplet, quartet, quin quintet, hept heptet,
d parts per million
t
¼
q
¼
¼
¼
m ¼ multiplet, app ¼ apparent), coupling constants (in Hz) and
integration. High-resolution mass analyses were performed using a
Micromass Q-TOF mass spectrometer (ESI). Purities of final com-
pounds were determined by UPLC (Waters Acquity UPLC system,
column Waters Acquity CSHC18, 0.5 mL minꢀ1H2OeMeCN gradient
5e95% 10 min with 0.1% formic acid). Analytical data are given if the
compound is novel or not fully characterised in the literature. All
tested compounds were ꢃ95% pure according to analytical HPLC
analysis.
4.3. X-ray data collection and atomic structure determination
4.4.1. General method for the preparation of azides 5ae5c
Co-crystals with lactose were formed at 20 ꢁC using 12 mg/mL
human galectin-8N in buffer (10 mM Tris/HCl pH 8.0, 150 mM NaCl,
10 mm lactose and 1 mM TCEP) mixed with reservoir 25% (w/v) PEG
Aminobenzoic acids 4ae4c (1 equiv.) were dissolved in aceto-
nitrile (5 mL). At 0 ꢁC, t-butyl nitrite (1.5 equiv.) was added, fol-
lowed by the dropwise addition of trimethylsilyl azide (1.2 equiv.).
The reaction mixture was allowed to cool to room temperature and
stirred for 1 h. The solvent was evaporated in vacuo. The crude
material was loaded on silica for flash chromatography purification
to give azides 5ae5c. Because of the expected instability of the
aromatic azides, these products were stored in the dark at 4 ꢁC. All
of the 1H NMR spectra match the literature [40].
2000 monomethylethyer (MME) in a 1:1 ratio in a 2 mL hanging
drop over 1 mL reservoir in a NEXTAL plate. Crystals were formed in
a few days. Before data collection crystals were transferred to a cryo
solution (10 mM Tris/HCl pH 8.0, 50 mM NaCl, 10 mM lactose, 25%
(w/v) PEG 2000 MME, 20% ethylene glycol (EG) and 1 mM TCEP)
and flash-frozen in liquid nitrogen. A co-crystal with lactose grown
from a seeded drop with 24% (w/v) PEG 2000 MME in the reservoir
was used to soak in compound 1 by transferring crystals in three
4.4.1.1. 2-Azidobenzoic acid (5a). Following the general method,
the reaction was performed with 4a (250 mg, 1.46 mmol), t-butyl
nitrite (327 mL, 2.73 mmol, 1.5 equiv.), trimethylsilyl azide (286 mL,
steps to different soaking drops. First to a 2
(20% (v/v) glycerol, 25% (w/v PEG 2000 MME, 10 mM Tris/HCl pH
8.0, 50 mM NaCl and 1 mM TCEP), and secondly to a 2 L drop with
mL drop with glycerol
m
2.19 mmol 1.2 equiv.). Purification by flash chromatography (1:3,
EtOAc/hexane) gave compound 5a as a solid in 81% yield (240 mg).
5 mM compound 1 (10 mM Tris/HCl pH 8.0, 50 mM NaCl, 5 mM
compound 1, 25% (w/v) PEG 2000 MME and 1 mM TCEP), and
thirdly to a second drop with 5 mM compound 1 (same composi-
tion as before). The incubation times in the first drops were about
15 min and in the third drop about 24 h. Then the crystal was
transferred to a cryo solution (10 mM Tris/HCl pH 8.0, 50 mM NaCl,
5 mM compound 1, 25% (w/v) PEG 2000 MME, 20% ethylene glycol
(EG) and 1 mM TCEP) and flash-frozen in liquid nitrogen. 1.35 Å was
collected for the galectin-8N - lactose complex at Diamond Light
Source, beamline I04. 1.6 Å data was collected for the galectin-8N -
compound 1 complex at Swiss Light Source, beamline PXIII. The
lactose data set was processed using the Xia2 pipeline [33], and the
compound 1 data set using XDS [34] and Aimless [35] programs.
Both structures were determined with two molecules in the
asymmetric unit in space group P212121. The structures have been
determined using molecular replacement and the Phaser software
[36], and the galectin-8N structure with lactose PDB id: 5GZD as a
template. The refinement was made using Refmac5 [37], the model
building was made in Coot [38], and the models have been analysed
using Molprobity [39], (Table S1 in the supporting information).
1H NMR (400 MHz, CDCl3)
J ¼ 8.1, 7.4, 1.6 Hz, 1H), 7.31e7.24 (m, 2H).
d
8.14 (dd, J ¼ 7.8, 1.4 Hz, 1H), 7.62 (ddd,
4.4.1.2. 3-Azidobenzoic acid (5b). Following the general method,
the reaction was performed with 4b (200 mg, 1.46 mmol), t-butyl
nitrite (260 mL, 2.19 mmol, 1.5 equiv.), trimethylsilyl azide (230 mL,
1.75 mmol 1.2 equiv.). Purification by flash chromatography (1:20,
MeOH/DCM) gave compound 5b as a solid in 90% yield (215 mg). 1H
NMR (400 MHz, CDCl3) d 7.93e7.86 (m, 1H), 7.81e7.75 (m, 1H), 7.47
(t, J ¼ 7.9 Hz, 1H), 7.29e7.24 (m, 1H).
4.4.1.3. 4-Azidobenzoic acid (5c). Following the general method,
the reaction was performed with 4c (250 mg, 1.46 mmol), t-butyl
nitrite (327
mL, 2.73 mmol, 1.5 equiv.), and trimethylsilyl azide
(286 L, 2.19 mmol 1.2 equiv.). Purification by flash chromatography
m
(1:3, EtOAc/hexane) gave compound 5c as a solid in 99% yield
(296 mg). 1H NMR (400 MHz, CDCl3)
J ¼ 8.8 Hz, 1H).
d
8.11 (d, J ¼ 8.8 Hz,1H), 7.11 (d,
4.4.2. General method for the preparation of methyl benzoates
6ae6c
4.4. Chemistry
Azides 4ae4c (1 equiv.) were dissolved in dry methanol (2 mL).
A catalytic amount of sulfuric acid was added and the mixture
heated to 60 ꢁC overnight. After cooling, 5% sodium bicarbonate
solution was added to quench the reaction. This mixture was
extracted with ethyl acetate. The organic layers were combined,
washed with water, dried over Na2SO4, and concentrated in vacuo
to give methyl benzoates 6ae6c. The reported 1H NMR matches the
All reagents and solvents were dried before use according to
standard methods. Commercial reagents were used without further
purification. TLC analysis was performed on precoated Merck silica
gel 60 F254 plates using UV light and charring solution (H2SO4:
EtOH 1: 9). Flash column chromatography was performed on SiO2
purchased from Aldrich (technical grade, 60 Å pore size, 230e400
7

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
literature [41]. Because of the expected instability of the aromatic
azides, these products were stored in the dark at 4 ꢁC.
4.4.5.1. Tolyl 3-O-((2-methylcarboxyphenyl)-1,2,3-triazol-4-
ylmethyl)-1-thio-b-D-galactopyranoside (9a). Following the gen-
eral method, the reaction was performed with 6a (150 mg,
0.85 mmol), 8a (250 mg, 0.77 mmol), CuI (15 mg, 0.08 mmol), and
4.4.2.1. Methyl 2-azidobenzoate (6a). Following the general
method, the reaction with 5a (250 mg, 1.53 mmol) gave compound
6a as a solid in a 78% yield (214 mg). 1H NMR (400 MHz, CDCl3)
DIPEA (148
(1:30, MeOH/DCM) gave compound 9a as a white solid in 61% yield
(237 mg). 1H NMR (400 MHz, CDCl3)
mL, 0.85 mmol). Purification by flash chromatography
d
7.86 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.53 (ddd, J ¼ 8.1, 7.4, 1.6 Hz, 1H),
d
8.03 (dd, J ¼ 7.8, 1.5 Hz, 1H),
7.26e7.23 (m, 1H), 7.19 (ddd, J ¼ 8.5, 7.7, 1.1 Hz, 1H), 3.91 (s, 3H).
7.88 (s, 1H), 7.69 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.62 (td, J ¼ 7.6, 1.3 Hz, 1H),
7.51e7.43 (m, 3H), 7.12 (d, J ¼ 7.9 Hz, 2H), 5.01 (d, J ¼ 13.1 Hz, 1H),
4.92 (d, J ¼ 13.1 Hz, 1H), 4.52 (d, J ¼ 9.7 Hz, 1H), 4.17e4.12 (m, 1H),
3.97 (ddd, J ¼ 10.8, 6.3, 4.2 Hz, 1H), 3.87e3.77 (m, 2H), 3.74 (s, 3H),
3.62e3.55 (m, 2H), 3.13 (s, 1H), 2.92 (d, J ¼ 2.2 Hz, 1H), 2.38 (dd,
J ¼ 8.4, 4.4 Hz, 1H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
4.4.2.2. Methyl 3-azidobenzoate (6b). Following the general
method, the reaction with 5b (270 mg, 1.66 mmol) gave compound
6b as a solid in a 95% yield (280 mg). 1H NMR (400 MHz, CDCl3)
d
7.83e7.79 (m, 1H), 7.72e7.68 (m, 1H), 7.42 (t, J ¼ 7.9 Hz, 1H), 7.20
(ddd, J ¼ 8.0, 2.4, 1.0 Hz, 1H), 3.93 (s, 3H).
d 166.22, 145.64, 136.41, 135.70, 133.44, 131.64, 130.91, 130.82,
130.38, 129.91, 127.57, 126.47, 125.52, 88.64, 82.90, 79.47, 68.68,
65.46, 62.70, 60.90, 52.91, 21.06. HRMS calcd for C24H28O7N3S þ Hþ
(M þ H)þ: 502.1642, found: 502.1635.
4.4.2.3. Methyl 4-azidobenzoate (6c). Following the general
method, the reaction with 5c (200 mg, 1.23 mmol) gave compound
6c as a solid in a 74% yield (160 mg). 1H NMR (400 MHz, CDCl3)
d
8.08e7.99 (m, 2H), 7.11e7.03 (m, 2H), 3.91 (s, 3H).
4.4.5.2. Tolyl 3-O-((3-methylcarboxyphenyl)-1,2,3-triazol-4-
ylmethyl)-1-thio-
method, the reaction was performed with 6b (60 mg, 0.34 mmol),
8a (100 mg, 0.31 mmol), CuI (6 mg, 0.04 mmol), and DIPEA (59 L,
b-D-galactopyranoside (9b). Following the general
4.4.3. Tolyl 3-O-(prop-2-ynyl)-1-thio-
b-D-galactopyranoside (8a)
Tolyl 1-thio- -galactopyranoside 7a (1.00 g, 3.49 mmol), n-
b
-D
m
Bu4NBr (113 mg, 0.35 mmol, 0.1 equiv.), K2CO3 (724 mg, 5.24 mmol,
1.5 equiv.) and n-Bu2SnCl2 (106 mg, 0.35 mmol, 0.1 equiv.), were
loaded to a round-bottom flask and put under inert atmosphere.
Acetonitrile (10 mL) and DMF (1 mL) were added to give a white
suspension. Propargyl bromide solution (0.97 mL, 80 wt% in
toluene, 8.73 mmol, 2.5 equiv.) was added and the reaction mixture
heated to 80 ꢁC for 3 h. Solvents evaporated in vacuo. The mixture
was taken up in DCM, washed with HClaq (0.5 M), purified by flash
chromatography (1:20, MeOH/DCM) to give compound 8a as a
white solid in a 49% yield (550 mg). 1H NMR (400 MHz, CDCl3)
0.34 mmol). Purification by flash chromatography (1:30, MeOH/
DCM) gave compound 9b as a white solid in 90% yield (140 mg). 1H
NMR (400 MHz, CDCl3)
d
8.26 (s, 1H), 8.14 (s, 1H), 8.05 (d, J ¼ 7.5 Hz,
1H), 7.94 (d, J ¼ 7.8 Hz, 1H), 7.55 (t, J ¼ 7.8 Hz, 1H), 7.41 (d, J ¼ 7.7 Hz,
2H), 7.06 (d, J ¼ 7.5 Hz, 2H), 4.92 (s, 2H), 4.53 (d, J ¼ 9.7 Hz, 1H), 4.25
(s, 1H), 3.94 (s, 4H), 3.85 (t, J ¼ 9.2 Hz, 2H), 3.77e3.51 (m, 4H), 3.00
(s, 1H), 2.28 (s, 3H), 2.02 (s, 1H). 13C NMR (101 MHz, DMSO‑d6)
d
165.27, 146.31, 136.88, 135.91, 131.26, 131.10, 130.66, 130.34, 129.41,
128.97, 124.37, 122.23, 120.03, 88.03, 82.52, 78.91, 68.17, 64.88,
62.22, 60.36, 52.58, 30.67, 20.55. HRMS calcd for C24H28O7N3S þ Hþ
(M þ H)þ: 502.1642, found: 502.1639.
d
7.51e7.44 (m, 2H), 7.19e7.11 (m, 2H), 4.52 (d, J ¼ 9.7 Hz, 1H), 4.43
(d, J ¼ 2.4 Hz, 2H), 4.20e4.14 (m, 1H), 4.05e3.95 (m, 1H), 3.83 (ddd,
J ¼ 12.1, 8.4, 4.4 Hz, 1H), 3.77 (td, J ¼ 9.4, 2.1 Hz, 1H), 3.65e3.56 (m,
2H), 2.58e2.53 (m, 2H), 2.51 (t, J ¼ 2.4 Hz, 1H), 2.36 (s, 3H). 13C NMR
4.4.5.3. Tolyl 3-O-((4-methylcarboxyphenyl)-1,2,3-triazol-4-
ylmethyl)-1-thio-b-D-galactopyranoside (9c). Following the general
method, the reaction was performed with 6c (150 mg, 0.85 mmol),
8a (250 mg, 0.77 mmol), CuI (15 mg, 0.08 mmol), and DIPEA
(101 MHz, CDCl3)
d
138.50, 133.26, 129.86, 127.85, 88.76, 81.09,
79.52, 78.28, 75.31, 68.69, 67.70, 62.81, 57.71, 21.16. HRMS calcd
C
16H20O5S þ Naþ (M þ Na)þ: 347.0929, found: 347.0922.
(148
MeOH/DCM) gave compound 9c as a white solid in 61% yield
(235 mg). 1H NMR (400 MHz, CDCl3)
8.24e8.18 (m, 2H), 8.11 (s,
mL, 0.85 mmol). Purification by flash chromatography (1:30,
4.4.4. Methyl 3-O-(prop-2-ynyl)-
b-
D-galactopyranoside (8b)
d
Methyl -galactopyranoside 7b (205 mg, 1.06 mmol), n-Bu4NBr
b
-
D
1H), 7.86e7.80 (m, 2H), 7.49e7.43 (m, 2H), 7.13 (d, J ¼ 7.9 Hz, 2H),
4.96 (q, J ¼ 13.3 Hz, 2H), 4.50 (d, J ¼ 9.7 Hz, 1H), 4.21 (s, 1H),
4.04e3.95 (m, 4H), 3.89e3.78 (m, 2H), 3.59 (dd, J ¼ 9.0, 3.2 Hz, 2H),
3.02 (s, 1H), 2.86 (d, J ¼ 1.7 Hz, 1H), 2.33 (s, 3H), 2.24 (dd, J ¼ 8.4,
(34 mg, 0.11 mmol, 0.1 equiv.), K2CO3 (219 mg, 1.58 mmol, 1.5 equiv.)
and n-Bu2SnCl2 (32 mg, 0.11 mmol, 0.1 equiv.), were loaded to a
round-bottom flask and put under inert atmosphere. Acetonitrile
(2 mL) and DMF (0.2 mL) were added to give a white suspension.
4.3 Hz, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 165.82, 146.92, 140.30,
Propargyl bromide solution (235
m
L, 80 wt% in toluene, 2.11 mmol, 2
136.41, 131.60, 131.52, 130.83, 129.91, 129.75, 122.70, 120.25, 88.54,
82.99, 79.41, 68.67, 65.38, 62.66, 60.85, 52.91, 52.90, 21.06, 21.05.
HRMS calcd for C24H28O7N3S þ Hþ (M þ H)þ: 502.1642, found:
502.1638.
equiv.) was added and the reaction mixture heated to 80 ꢁC for 3 h.
Solvents were evaporated in vacuo.The mixture was taken up in
DCM, washed with HClaq (0.5 M), purified by flash chromatography
(1:10, MeOH/DCM) to give compound 8a as a white solid in a 77%
yield (190 mg). The 1H NMR matches the reported literature data
4.4.5.4. Methyl 3-O-((2-methylcarboxyphenyl)-1,2,3-triazol-4-
[42]. 1H NMR (400 MHz, CD3OD)
d
4.39 (dd, J ¼ 3.4, 2.4 Hz), 4.20 (d,
ylmethyl)-
method, the reaction was performed with 6a (60 mg, 0.34 mmol),
8b (63 mg, 0.27 mmol), CuI (5 mg, 0,03 mmol), and DIPEA (47 L,
b-D-galactopyranoside (9d). Following the general
J ¼ 7.7 Hz), 4.10 (dd, J ¼ 3.2, 0.7 Hz), 3.77 (dd, J ¼ 6.1, 2.5 Hz), 3.61 (dd,
J ¼ 9.7, 7.6 Hz), 3.55 (s), 3.54e3.49 (m), 2.91 (t, J ¼ 2.4 Hz).
m
0.27 mmol). Purification by flash chromatography (1:5, MeOH/
DCM) gave compound 9d as a off-white solid in 77% yield
4.4.5. General method for the preparation of methyl
benzoatedtriazoles 9ae9f, and 10f
(86 mg).1H NMR (400 MHz, CDCl3)
d
8.04 (dd, J ¼ 7.8, 1.5 Hz, 1H),
Propynyl 8a or 8b (1 equiv.), azide 5c, 6a, 6b, or 6c (1.1 equiv.),
and CuI (0.1 euiv.) were charged to a round-bottom flask and taken
up in acetonitrile (10 mL). N,N-Diisopropylethylamine (DIPEA, 1.1
equiv.) was added, and the reaction mixture stirred overnight at
50 ꢁC. The solvent was removed from the reaction mixture and the
crude material loaded on silica for purification by flash chroma-
tography to give triazoles 9ae9f and 10f.
7.90 (s, 1H), 7.70 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.63 (td, J ¼ 7.6, 1.3 Hz, 1H),
7.49 (dd, J ¼ 7.8, 1.2 Hz, 1H), 5.03 (d, J ¼ 13.1 Hz, 1H), 4.92 (d,
J ¼ 13.1 Hz, 1H), 4.25 (d, J ¼ 7.7 Hz, 1H), 4.14 (s, 1H), 4.03e3.96 (m,
1H), 3.92e3.79 (m, 2H), 3.76 (s, 3H), 3.63e3.54 (m, 5H), 3.49 (d,
J ¼ 5.2 Hz, 2H), 3.25 (d, J ¼ 1.7 Hz, 1H), 2.78 (d, J ¼ 2.2 Hz, 1H), 2.41
(dd, J ¼ 8.3, 4.6 Hz, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 166.22,
145.64,135.71,133.44, 130.90, 130.38, 127.59, 126.50, 125.50, 104.84,
8

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
81.63, 75.49, 69.99, 65.20, 62.56, 60.81, 56.35, 52.91. HRMS calcd for
C
J ¼ 5.1 Hz, 1H), 4.80 (dd, J ¼ 27.5, 12.4 Hz, 2H), 4.63 (d, J ¼ 4.8 Hz,
1H), 4.55 (d, J ¼ 9.7 Hz, 1H), 4.01e3.95 (m, 1H), 3.64e3.37 (m, 6H),
18H24O7N3S þ Hþ (M þ H)þ: 410.1558, found: 410.1551.
2.27 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 167.17, 145.42, 136.41,
4.4.5.5. Methyl 3-O-((3-methylcarboxyphenyl)-1,2,3-triazol-4-
ylmethyl)- -galactopyranoside (9e). Following the general
method, the reaction was performed with 6b (77 mg, 0.47 mmol),
8b (100 mg, 0.43 mmol), CuI (14 mg, 0.04 mmol), and DIPEA (66 L,
135.73, 132.82, 131.64, 130.84, 130.32, 129.91, 126.76, 125.67, 88.60,
83.03, 79.43, 68.66, 65.42, 62.83, 60.90, 21.06. HRMS calcd for
C
b-D
23H26O7N3S þ Hþ (M þ H)þ: 488.1486, found: 488.1485.
m
0.47 mmol). Purification by flash chromatography (1:30, MeOH/
4.4.6.2. Tolyl 3-O-((3-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-1-
thio- -galactopyranoside (10b). Following the general method,
DCM) gave compound 9e as a white solid in 40% yield (68 mg). 1H
b-D
NMR (400 MHz, CD3OD)
d
8.69 (s, 1H), 8.50 (t, J ¼ 1.8 Hz, 1H),
the reaction was performed with 9b (50 mg, 0.10 mmol) to give
8.17e8.12 (m, 2H), 7.74 (t, J ¼ 8.0 Hz, 1H), 4.96 (d, J ¼ 12.6 Hz, 1H),
4.88 (d, J ¼ 12.5 Hz, 1H), 4.20 (d, J ¼ 7.7 Hz, 1H), 4.15 (d, J ¼ 2.5 Hz,
230H), 3.99 (s, 3H), 3.80 (dd, J ¼ 6.1, 3.7 Hz, 2H), 3.68 (dd, J ¼ 9.6,
7.8 Hz, 1H), 3.58e3.52 (m, 4H), 3.49 (dd, J ¼ 9.6, 3.2 Hz, 1H). 13C
compound 10b as a white solid in a 97% yield (47 mg). 1H NMR
(400 MHz, DMSO‑d6) d 8.92 (s,1H), 8.42e8.35 (m,1H), 8.18e8.11 (m,
1H), 8.06e8.02 (m, 1H), 7.74 (t, J ¼ 7.9 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz,
2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 5.31 (d, J ¼ 6.0 Hz, 1H), 4.85 (d,
J ¼ 12.6 Hz, 1H), 4.77 (d, J ¼ 12.6 Hz, 1H), 4.69 (s, 1H), 4.61 (d,
J ¼ 4.9 Hz,1H), 4.55 (d, J ¼ 9.7 Hz,1H), 4.03e3.98 (m,1H), 3.62e3.39
NMR (101 MHz, DMSO‑d6)
d 164.92, 145.36, 136.41, 131.04, 129.20,
128.47, 123.52, 121.10, 119.88, 103.66, 80.79, 74.25, 69.42, 64.65,
61.33, 60.25, 55.03, 50.85. HRMS calcd for C18H24O7N3S þ Hþ
(M þ H)þ: 410.1558, found: 410,1548.
(m, 7H), 2.27 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 166.81, 146.77,
137.29, 136.40, 133.11, 131.59, 130.93, 130.84, 129.91, 129.61, 124.46,
122.71, 88.49, 82.98, 79.39, 68.66, 65.32, 62.70, 60.82, 21.06. HRMS
calcd for C23H26O7N3S þ Hþ (M þ H)þ: 488.1486, found: 488.1481.
4.4.5.6. Methyl 3-O-((4-methylcarboxyphenyl)-1,2,3-triazol-4-
ylmethyl)-
method, the reaction was performed with 6c (47 mg, 0.27 mmol),
8b (51 mg, 0.22 mmol), CuI (4 mg, 0.02 mmol), and DIPEA (38 L,
b-D-galactopyranoside (9f). Following the general
4.4.6.3. Tolyl 3-O-((4-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-1-
thio-b-D-galactopyranoside (10c). Following the general method,
m
0.22 mmol). Purification by flash chromatography (1:4, MeOH/
the reaction was performed with 9c (50 mg, 0.10 mmol) to give
DCM) gave compound 9f as a white solid in 59% yield (53 mg). 1H
compound 10c as a white solid in a 76% yield (37 mg). 1H NMR
NMR (400 MHz, DMSO‑d6)
d
8.93 (s, 1H), 8.22e8.14 (m, 2H), 8.08 (d,
(400 MHz, DMSO‑d6)
d
8.91 (s, 1H), 8.15 (d, J ¼ 8.8 Hz, 2H), 8.04 (d,
J ¼ 8.8 Hz, 2H), 5.07 (d, J ¼ 4.9 Hz, 1H), 4.84 (d, J ¼ 12.6 Hz, 1H), 4.76
(d, J ¼ 12.6 Hz, 1H), 4.65 (t, J ¼ 5.6 Hz, 1H), 4.52 (d, J ¼ 5.2 Hz, 1H),
4.05 (d, J ¼ 7.6 Hz, 1H), 3.96e3.92 (m, 1H), 3.91 (d, J ¼ 5.1 Hz, 3H),
3.61e3.42 (m, 3H), 3.39 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
J ¼ 8.8 Hz, 2H), 7.37 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 5.31
(d, J ¼ 5.9 Hz, 1H), 4.81 (dd, J ¼ 30.1, 12.6 Hz, 2H), 4.68 (s, 1H), 4.61
(d, J ¼ 4.8 Hz,1H), 4.55 (d, J ¼ 9.6 Hz,1H), 4.00 (s, 1H), 3.63e3.39 (m,
6H), 2.27 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 146.86, 136.41,
d
165.81, 146.92, 140.31, 131.51, 129.74, 122.68, 120.25, 104.80, 81.77,
131.61, 130.83, 129.91, 122.68, 120.11, 88.54, 82.99, 79.41, 68.67,
65.38, 62.68, 60.85, 21.05. HRMS calcd for C23H26O7N3S þ Hþ
(M þ H)þ: 488.1486, found: 488.1483.
75.44, 70.02, 65.18, 62.58, 60.77, 56.35, 52.89. HRMS calcd for
C
18H24O7N3S þ Hþ (M þ H)þ: 410.1558, found: 410,1544.
4.4.5.7. Methyl 3-O-((4-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-
-galactopyranoside (10f). Following the general method, the re-
action was performed with 5c (71 mg, 0.44 mmol), 8b (101 mg,
0.43 mmol), CuI (6 mg, 0.04 mmol), and DIPEA (75 L, 0.43 mmol).
b
-
4.4.6.4. Methyl 3-O-((2-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-
b-
D
D
-galactopyranoside (10d). Following the general method, the re-
action was performed with 9d (63 mg, 0.15 mmol) to give com-
pound 10d as a yellow solid in 97% yield (59 mg).1H NMR (400 MHz,
m
Purification by flash chromatography (1:4, MeOH/DCM) gave
DMSO‑d6)
d
13.16 (s), 8.49 (s, 1H), 7.92 (d, J ¼ 6.7 Hz, 1H), 7.77 (td,
compound 9f as a white solid in 44% yield (75 mg). 1H NMR
J ¼ 7.6,1.1 Hz,1H), 7.68 (t, J ¼ 7.2 Hz,1H), 7.60 (d, J ¼ 7.7 Hz,1H), 5.08
(s, 1H), 4.82 (d, J ¼ 12.4 Hz, 1H), 4.74 (d, J ¼ 12.4 Hz, 1H), 4.53 (d,
J ¼ 4.5 Hz, 1H), 4.05 (d, J ¼ 7.6 Hz, 1H), 3.93 (s, 1H), 3.63e3.43 (m,
(400 MHz, CD3OD)
d
8.69 (s, 1H), 8.24 (d, J ¼ 8.7 Hz, 2H), 8.01 (d,
J ¼ 8.7 Hz, 2H), 4.98e4.94 (m, 1H), 4.88 (d, J ¼ 12.9 Hz, 1H), 4.20 (d,
J ¼ 7.7 Hz, 1H), 4.15 (dd, J ¼ 3.2, 0.9 Hz, 1H), 3.85e3.72 (m, 2H), 3.68
(dd, J ¼ 9.6, 7.7 Hz, 1H), 3.56 (d, J ¼ 2.8 Hz, 3H), 3.55e3.52 (m, 1H),
3H), 3.37e3.29 (m, 4H). 13C NMR (101 MHz, DMSO‑d6)
d 167.12,
145.43,135.76,132.86, 130.86, 130.32,129.19, 126.79, 125.65, 104.82,
81.79, 75.46, 70.00, 65.19, 62.73, 60.82, 56.34. HRMS calcd for
3.51e3.46 (m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 146.83, 139.87,
131.61, 122.66, 120.13, 104.79, 81.76, 75.44, 70.02, 65.16, 62.58,
60.77, 56.35. HRMS calcd for C17H22O8N3 þ Hþ (M þ H)þ: 396.1401,
found: 396.1399.
C
17H22O8N3 þ Hþ (M þ H)þ: 396.1401, found: 396.1399.
4.4.6.5. Methyl 3-O-((3-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-
b-
D
-galactopyranoside (10e). Following the general method, the re-
4.4.6. General method for the preparation of benzoic acids 10ae10e
Methyl benzoates 9ae9e were taken up in acetonitrile (5 mL),
NaOHaq (1 mL, 1 M) was added and the reaction mixture heated to
50 ꢁC for 30 min. The reaction mixture was concentrated, water
(5 mL) and HCl (1.5 mL, 1 M) was added, and the mixture was
extracted with EtOAc (3 ꢂ 5 mL). Organic layers were combined,
washed with water, and concentrated in vacuo to give benzoic acids
10ae10e.
action was performed with 9e (38 mg, 0.08 mmol) to give com-
pound 10e as a white solid in a quantitative yield (37 mg). 1H NMR
(400 MHz, DMSO‑d6)
d 13.47 (s, 1H), 13.47 (s, 1H), 8.96 (s, 1H),
8.44e8.37 (m, 1H), 8.17 (dd, J ¼ 8.1, 1.3 Hz, 1H), 8.07e8.01 (m, 1H),
7.75 (t, J ¼ 7.9 Hz, 1H), 5.08 (d, J ¼ 5.0 Hz, 1H), 4.90e4.66 (m, 3H),
4.57 (d, J ¼ 5.1 Hz, 1H), 4.05 (d, J ¼ 7.6 Hz, 1H), 4.00e3.89 (m, 1H),
3.63e3.41 (m, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 166.75, 146.77,
137.30, 133.04, 130.93, 129.58, 124.50, 122.77, 120.70, 104.76, 81.73,
75.39, 70.01, 64.93, 62.57, 60.56, 56.33. HRMS calcd for
4.4.6.1. Tolyl 3-O-((2-carboxyphenyl)-1,2,3-triazol-4-ylmethyl)-1-
thio-b-D-galactopyranoside (10a). Following the general method,
C
17H22O8N3 þ Hþ (M þ H)þ: 396.1401, found: 396.1397.
the reaction was performed with 9a (50 mg, 0.10 mmol) to give
4.4.7. General method for the preparation of compounds 12a-12d
Methyl -galactopyranoside 1 (1 equiv.) and Bu2SnO (1.1e1.2
compound 10a as a white solid in a 84% yield (41 mg). 1H NMR
b-D
(400 MHz, DMSO‑d6)
d
8.48 (s, 1H), 7.91 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.76
equiv.) were dissolved in dry MeOH. The mixture was stirred under
reflux conditions for 2 h under N2 atmosphere. The solvent was
evaporated in vacuo. The crude was co-evaporated with toluene to
(td, J ¼ 7.7, 1.5 Hz, 1H), 7.67 (td, J ¼ 7.6, 1.2 Hz, 1H), 7.59 (dd, J ¼ 7.8,
1.0 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 5.33 (d,
9

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
remove any remaining MeOH. The residue was dried under vacuum
to give the stannylene acetal intermediate as an amorphous white
solid. The dry crude was dissolved in 1,4-dioxane and Bu4NBr (1.5
equiv.) and the halide 11a-11d (1.5 equiv.) was added. The reaction
mixture was stirred under N2 atmosphere overnight at 85 ꢁC. The
solvent was evaporated in vacuo. The crude material was purified
by flash chromatography. The compounds were further purified
with preparative HPLC prior to galectin binding evaluations.
1H, ArH), 7.66e7.63 (m, 1H, ArH), 7.51e7.29 (m, 2H, ArH), 5.06 (q,
J ¼ 14.9, 2.9 Hz, 2H, CH2C7H4NO), 4.20 (d, J ¼ 7.7 Hz, 1H, H-1), 4.13
(d, J ¼ 2.1 Hz, 1H, H-4), 3.85e3.69 (m, 3H, H2, H-6a, H-6b),
3.60e3.50 (m, 6H, H-3, H-5, OCH3). 13C NMR (101 MHz, CD3OD)
d
150.78, 139.93, 125.44, 124.59, 119.21, 110.58, 104.41, 82.97, 74.97,
70.34, 65.90, 63.92, 60.97, 55.90, 48.25, 48.04, 47.82, 47.61, 47.40,
47.19, 46.97. HRMS calcd for C15H19NO7 þ Hþ (M þ H)þ: 326.1234,
found: 326.1229.
4.4.7.1. Methyl
3-O-(1H-benzo[d]imidazole-2-ylmethyl)-
b
-
D
-gal-
4.4.8. General method for the preparation of compounds 14a-14c
To a 0 ꢁC solution of 2-chloro-1,1,1-trimethoxyethane (1.5 equiv.)
in anhydrous DCM, BF3$Et2O (1.1e2 equiv.) was added dropwise
followed by the addition of the diamines 13a-13c (1 equiv.). The
reaction mixture was stirred at room temperature overnight. The
reaction was then quenched by the addition of a saturated aqueous
solution of NaHCO3. The product was extracted by DCM 3 times,
and the combined organic phases were washed with brine, dried
over anhydrous Na2SO4, and the solvent was evaporated in vacuo to
afford the desired product which was used without further
purification.
actopyranoside (12a). Following the general method, the reaction
was performed with 7b (50 mg, 0.25 mmol), dry MeOH (3 mL),
Bu2SnO (76.9 mg, 0.32 mmol, 1.2 equiv.), anhydrous 1,4-dioxane
(3 mL), Bu4NBr (124.5 mg, 0.38 mmol, 1.5 equiv.), 11a [43]
(70.9 mg, 0.38 mmol, 1.5 equiv.). Compound 12a was obtained as a
white amorphous solid in 60% yield (52.8 mg). 1H NMR (400 MHz,
CD3OD)
d
8.01 (dd, J ¼ 8.0, 1.2 Hz, 1H, ArH), 7.96 (dd, J ¼ 8.3, 1.0 Hz,
1H, ArH), 7.53 (td, J ¼ 8.1, 7.7, 1.3 Hz, 1H, ArH), 7.45 (td, J ¼ 7.7, 1.2 Hz,
1H, ArH), 5.17 (ABq, J ¼ 14.1, 13.5 Hz, 2H, CH2C7H4N2), 4.20 (d,
J ¼ 7.7 Hz, 1H, H-1), 4.16 (dd, J ¼ 3.3, 1.0 Hz, 1H, H-4), 3.84e3.74 (m,
3H, H-2, H-6a, H-6b), 3.56 (s, 3H, OCH3), 3.55e3.51 (m, 2H, H-3, H-
5). 13C NMR (101 MHz, CD3OD)
d
152.23, 135.37, 123.62, 114.12,
4.4.8.1. Methyl 2-(chloromethyl)-3-methyl-benzimidazole-5-
carboxylate (14a). Following the general procedure, the reaction
was performed with 2-chloro-1,1,1-trimethoxyethane (516 mg,
3.34 mmol, 1.5 equiv.), 12 mL anhydrous DCM, 13a (400.2 mg,
2.22 mmol, 1 equiv.) and BF3$Et2O (727.4 mg, 2.46 mmol, 1.1 equiv.).
Compound 14a was obtained as a pale-yellow solid in 94% yield
104.43, 82.60, 74.92, 70.42, 65.77, 63.94, 60.94, 55.96, 48.24, 48.03,
47.81, 47.60, 47.39, 47.18, 46.96. HRMS calcd for C15H20N2O6 þ Hþ
(M þ H)þ: 325.1400, found: 325.1399.
4.4.7.2. Methyl 3-O-(1-methyl-1H-benzo[d]imidazole-2-ylmethyl)-
b-
D
-galactopyranoside (12b). Following the general method, the re-
(496.4 mg). The reported 1H NMR matches the literature [44]. 1
NMR (400 MHz, CD3OD)
8.19 (dd, J ¼ 1.7, 0.7 Hz,1H, ArH), 7.94 (dd,
H
action was performed with 1 (50.6 mg, 0.26 mmol), dry MeOH
(3 mL), Bu2SnO (70.8 mg, 0.28 mmol, 1.1 equiv.), anhydrous 1,4-
dioxane (3 mL), Bu4NBr (125.9 mg, 0.39 mmol, 1.5 eq), 11b
(71.4 mg, 0.39 mmol, 1.5 eq). Compound 12b was obtained as a
white amorphous solid in 64% yield (54.4 mg). 1H NMR (500 MHz,
d
J ¼ 8.5, 1.6 Hz, 1H, ArH), 7.67 (dd, J ¼ 8.5, 0.8 Hz, 1H, ArH), 4.98 (s,
2H), 3.95 (d, J ¼ 0.6 Hz, 6H). 13C NMR (101 MHz, CD3OD)
d 167.18,
152.82, 144.50, 135.48, 125.29, 123.67, 118.43, 112.18, 51.33, 35.37,
29.45. HRMS calcd for C11H11N2O2Cl þ Hþ (M þ H)þ: 239.0587,
found: 239.0589.
CD3OD)
d
7.71 (q, J ¼ 7.4 Hz 2H, ArH), 7.51e7.42 (m, 2H, ArH), 5.17 (q,
J ¼ 14.1 Hz, 2H, CH2C8H7N2), 4.19 (d, J ¼ 7.8 Hz, 1H, H-1), 4.17 (d,
J ¼ 2.4 Hz, 1H, H-4), 3.98 (s, 3H, NCH3), 3.83e3.70 (m, 3H, H2, H-6a,
H-6b), 3.57e3.52 (m, 6H, H-3, H-5, OCH3). 13C NMR (126 MHz,
4.4.8.2. Ethyl 2-(chloromethyl)-3-methyl-benzimidazole-5-
carboxylate (14b). Following the general procedure, the reaction
was performed with 2-chloro-1,1,1-trimethoxyethane (656 mg,
4.24 mmol, 1.5 equiv.), 7 mL anhydrous DCM, 13b (551.3 mg,
2.84 mmol, 1 equiv.) and BF3$Et2O (916.2 mg, 3.1 mmol, 1.1 equiv.).
Compound 14b was obtained as a pale-yellow solid in 98% yield
CD3OD)
d 151.16, 135.93, 134.51, 124.30, 123.99, 116.17, 110.69,
104.42, 82.24, 74.88, 70.22, 65.69, 62.55, 60.87, 55.86, 48.03, 47.86,
47.69, 47.52, 47.35, 47.18, 47.01, 29.69. HRMS calcd for
C
16H22N2O6 þ Hþ (M þ H)þ: 339.1551, found: 339.1556.
(705 mg). 1H NMR (400 MHz, CD3OD)
d 8.32 (s, 1H), 8.03 (d,
4.4.7.3. Methyl
3-O-(benzo[d]thiazol-2-ylmethyl)-
b
-
D
-galactopyr-
J ¼ 8.6 Hz,1H), 7.60 (d, J ¼ 8.6 Hz,1H), 4.99 (s, 2H), 4.40 (q, J ¼ 7.1 Hz,
anoside (12c). Following the general method, the reaction was
performed with 1 (50 mg, 0.25 mmol), dry MeOH (3 mL), Bu2SnO
(76.9 mg, 0.32 mmol, 1.2 equiv.), anhydrous 1,4-dioxane (3 mL),
Bu4NBr (124.5 mg, 0.38 mmol, 1.5 equiv.), 11c (70.9 mg, 0.38 mmol,
1.5 equiv.). Compound 12c was obtained as a white amorphous
2H), 3.95 (s, 3H), 1.43 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, CD3OD)
d
166.86, 152.08, 140.69, 139.03, 125.09, 124.59, 120.78, 109.92,
60.79, 35.46, 29.50, 13.27. HRMS calcd for C12H13ClN2O2 þ Hþ
(M þ H)þ: 253.0744, found: 253.0746.
solid in 60% yield (52.8 mg). 1H NMR (400 MHz, CD3OD)
d
8.01 (dd,
4.4.8.3. Methyl 2-(chloromethyl)-1H-benzo[d]imidazole-6-
carboxylate (14c). Following the general procedure, the reaction
was performed with 2-chloro-1,1,1-trimethoxyethane (1.4 g,
9.03 mmol, 1.5 equiv.), 30 mL anhydrous DCM, 13c (1 g, 6.02 mmol,
1 equiv.) and BF3$Et2O (3.56 mg, 12.04 mmol, 2 equiv.). Compound
14c was obtained as a pale-yellow solid in 77% yield (1.04 g). 1H
J ¼ 8.0, 1.2 Hz, 1H, ArH), 7.96 (dd, J ¼ 8.3, 1.0 Hz, 1H, ArH), 7.53 (td,
J ¼ 8.1, 7.7, 1.3 Hz, 1H, ArH), 7.45 (td, J ¼ 7.7, 1.2 Hz, 1H, ArH), 5.18 (q,
J ¼ 14.5 Hz, 2H, CH2C7H4NS), 4.20 (d, J ¼ 7.7 Hz, 1H, H-1), 4.16 (dd,
J ¼ 3.3, 1.0 Hz, 1H, H-4), 3.84e3.74 (m, 3H, H-2, H-6a, H-6b), 3.56 (s,
3H, OCH3), 3.55e3.51 (m, 2H, H-3, H-5). 13C NMR (101 MHz, CD3OD)
d
171.62, 152.31, 134.71, 126.01, 125.07, 121.95, 121.70, 104.52, 82.61,
NMR (400 MHz, CD3OD)
d
8.32e8.23 (m, 1H), 7.96 (dd, J ¼ 8.6,
75.03, 70.44, 68.63, 66.01, 61.00, 55.88. HRMS calcd for
C
1.6 Hz, 1H), 7.62 (dd, J ¼ 8.5, 0.6 Hz,1H), 4.89 (s, 2H), 3.94 (s, 3H). 13
C
15H19NO6S þ Hþ (M þ H)þ: 342.1011, found: 342.1019.
NMR (101 MHz, CD3OD) d 167.42, 152.66, 124.78, 124.10, 117.54,
114.16, 51.26, 36.66. HRMS calcd for C10H8N2O2Cl þ Hþ (M þ H)þ:
4.4.7.4. Methyl
3-O-(benzo[d]oxazol-2-ylmethyl)-
b
-D
-galactopyr-
225.0431, found: 225.0435.
anoside (12d). Following the general method, the reaction was
performed with 1 (50.2 mg, 0.25 mmol), dry MeOH (3 mL), Bu2SnO
(77.4 mg, 0.31 mmol, 1.2 equiv.), anhydrous 1,4-dioxane (3 mL),
Bu4NBr (125.2 mg, 0.39 mmol, 1.5 equiv.), 11d (88.2 mg, 0.56 mmol,
2 equiv.). Compound 12c was obtained as a white amorphous solid
4.4.8.4. 1-(tert-butyl) 5-methyl 2-(chloromethyl)-1H-benzo[d]imid-
azole-1,5-dicarboxylate and 1-(tert-butyl) 6-methyl 2-(chlor-
omethyl)-1H-benzo[d]imidazole-1,6-dicarboxylate
(14d).
Compound 14c (500.8 mg, 2.23 mmol), di-tert-butyl dicarbonate
(550 g, 2.52 mmol, 1.2 equiv.) and anhydrous DMF (8 mL) were
in 47% yield (39.1 mg). 1H NMR (400 MHz, CD3OD)
d
7.75e7.70 (m,
10

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
added to an oven dried round flask, then the temperature was
adjusted at 5e10 ꢁC. DMAP (20 mg, 0.16 mmol, 0.06 equiv.) was
then added and the reaction was stirred at the same temperature
for 2 h. After the reaction was stopped, the reaction mixture was
diluted with EtOAc (70 mL), washed with aq. NaHCO3, brine, dried
over anhydrous sodium sulfate and EtOAc was then removed in
vacuo. Purification with flash column chromatography (EtOAC:
Heptane 1:2) afforded the desired product 14c as a pale yellow oil
that solidified at room temperature overnigt (591 mg, 82% yield). 1H
120.44, 109.61, 104.56, 82.03, 75.04, 70.18, 65.51, 63.27, 61.04, 60.75,
55.91, 29.53, 13.30. HRMS calcd for C19H26N2O8 þ Hþ (M þ H)þ:
411.1767, found: 411.1762.
4.4.9.3. Methyl 3-O-((5-methoxycarbonyl)1H-benzo[d]imidazole-2-
ylmethyl)-methoxy)-b-D-galactopyranoside (15c). Following the
general method, the reaction was performed with 7b (61.3 mg,
0.31 mmol), dry MeOH (3 mL), Bu2SnO (86.5 mg, 0.34 mmol, 1.1
equiv.), anhydrous 1,4-dioxane (3 mL), Bu4NBr (152.2 mg,
0.47 mmol, 1.5 equiv.), 14d (113.5 mg, 0.35 mmol, 1.1 equiv.).
Compound 15c was obtained as a white amorphous solid in 69%
NMR (400 MHz, CDCl3)
d
8.74 (d, J ¼ 1.0 Hz, 1H), 8.46 (d, J ¼ 1.0 Hz,
1H), 8.16e8.03 (m, 3H), 7.80 (d, J ¼ 8.4 Hz, 1H), 5.09 (d, J ¼ 5.9 Hz,
4H), 3.98 (s, 6H), 1.78 (d, J ¼ 6.9 Hz, 18H). 13C NMR (101 MHz, CDCl3)
yield (82.9 mg). 1H NMR (400 MHz, CD3OD)
d
8.27 (d, J ¼ 1.5 Hz, 1H,
d
166.96,166.83,152.87, 151.63, 147.67, 144.93,141.47, 136.57,133.09,
ArH), 7.97 (dd, J ¼ 8.5, 1.5 Hz, 1H, ArH), 7.63 (d, J ¼ 8.5 Hz, 1H, ArH),
5.05 (q, J ¼ 14.2 Hz, 2H, CH2C9H7N2O2), 4.23 (d, J ¼ 7.7 Hz, 1H. H-1),
4.17 (dd, J ¼ 3.3, 1.1 Hz, 1H, H-4), 3.95 (s, 4H, CO2CH3), 3.85e3.74 (m,
3H, H2, H-6a, H-6b), 3.57 (s, 3H, OCH3), 3.57e3.54 (m,1H, H-5), 3.54
127.44, 127.04, 126.87, 126.02, 122.43, 120.16, 117.35, 114.92, 87.23,
52.32, 52.27, 39.40, 39.31, 27.90. HRMS calcd for C15H17N2O8Cl þ Hþ
(M þ H)þ: 325.0955, found: 325.0953.
(d, J ¼ 3.3 Hz, 1H, H-3). 13C NMR (126 MHz, CD3OD)
d 167.47, 155.27,
4.4.9. General procedure for compounds 15a-15c
123.59, 117.87, 115.78, 114.81e113.39 (m), 104.36, 82.48, 74.89,
70.30, 65.54, 64.56, 60.92, 55.83, 51.12, 48.02, 47.85, 47.68, 47.51,
47.34, 47.17, 47.00. HRMS calcd for C17H22N2O8 þ Hþ (M þ H)þ:
383.1454, found: 383.1444.
Methyl
b-D-galactopyranoside 7b (1 equiv.) and Bu2SnO (1.1
equiv.) were dissolved in dry MeOH. The mixture was stirred under
reflux conditions for 2 h under N2 atmosphere. The solvent was
evaporated in vacuo; the crude was co-evaporated with toluene in
vacuo to remove any remaining MeOH. The residue was dried under
a vacuum to give the intermediate as an amorphous white solid.
The dry crude was dissolved in 1,4- dioxane and BuNBr (1.5 equiv.)
and the corresponding halide 14a, 14b, and 14d (1.1e1.2 equiv.) was
added. The reaction mixture was stirred under N2 atmosphere
overnight at 85 ꢁC. The solvent was evaporated in vacuo. The crude
material was purified by flash chromatography. The compounds
were further purified by preparative HPLC prior to galectin binding
assays.
4.4.10. General procedure for compounds 16a-16c
To a suspension of compounds 15a-15c (1 equiv.) in EtOH: H2O
(3:1), KOH was added, and the mixture was stirred at 80 ꢁC for
4e6 h. The volatiles were evaporated in vacuo. The crude material
was purified by preparative HPLC to obtain pure carboxylic acids
16a-16c.
4.4.10.1. Methyl 3-O-((6-carboxy)1-methyl-1H-benzo[d]imidazole-2-
ylmethyl)-methoxy)-b-D-galactopyranoside (16a). Following the
general method, the reaction was performed with 15a (32.5 mg,
0.081 mmol) and KOH (13.2 mg, 0.24 mmol, 2.9 equiv.) in 4 mL
EtOH: H2O (3:1) for 6 h. Compound 16a was obtained as an
amorphous white solid in 52% yield (16.2 mg). 1H NMR (500 MHz,
4.4.9.1. Methyl
imidazole-2-ylmethyl)-methoxy)-
3-O-((6-methoxycarbonyl)1-methyl-1H-benzo[d]
-galactopyranoside (15a).
b-D
Following the general method, the reaction was performed with 7b
(70.2 mg, 0.36 mmol), dry MeOH (3 mL), Bu2SnO (98.8 mg,
0.4 mmol, 1.1 equiv.), anhydrous 1,4-dioxane (3 mL), Bu4NBr
(174.4 mg, 0.54 mmol, 1.5 equiv.), 14a (103.7 mg, 0.43 mmol, 1.2
equiv.). Compound 15a was obtained as a white amorphous solid in
D2O)
d
7.93 (s, 1H, ArH), 7.82 (d, J ¼ 8.5 Hz, 1H, ArH), 7.53 (d,
J ¼ 8.5 Hz, 1H, ArH), 5.13 (d, J ¼ 14.6 Hz, 1H, CH2C9H7N2O2), 5.01 (d,
J ¼ 14.6 Hz, 1H, CH2C9H7N2O2), 4.32e4.26 (m, 1H, H-1), 4.17 (s, 1H,
H-4), 3.79 (s, 3H, NCH3), 3.77e3.67 (m, 2H, H-6a, H-6b), 3.65e3.61
(m, 2H, H-3, H-5), 3.50 (s, 3H, OCH3). 13C NMR (126 MHz, D2O)
77% yield (110.92 mg). 1H NMR (400 MHz, CD3OD)
d 8.23 (s, 1H),
7.97 (d, J ¼ 8.5 Hz, 1H), 7.69 (d, J ¼ 8.5 Hz, 1H), 5.06 (dd, J ¼ 13.1,
6.2 Hz, 2H, CH2C10H10N2O2), 4.17 (d, J ¼ 6.4 Hz, 1H, H-1), 4.14 (d,
J ¼ 3.3 Hz, 1H, H-4), 3.98 (s, 3H, CO2CH3), 3.97 (s, 3H, NCH3),
3.83e3.67 (m, 3H, H-2, H-6a, H-6b), 3.54 (s, 3H, OCH3), 3.53e3.48
(m, 1H, H-5), 3.47 (dd, J ¼ 9.8, 3.2 Hz, 1H, H-3). 13C NMR (101 MHz,
d
171.71, 151.83, 135.47, 133.14, 130.39 (d, J ¼ 8.9 Hz), 125.84, 114.98,
112.80, 103.53, 81.71, 74.78, 69.79, 65.28, 61.92, 60.75, 57.13, 30.56.
HRMS calcd for C17H22N2O8 þ Hþ (M þ H)þ: 383.1454, found:
383.1444.
CD3OD)
d
167.37, 154.55, 144.34, 135.59, 124.83, 123.43, 118.06,
4.4.10.2. Methyl 3-O-((5-carboxy)1-methyl-1H-benzo[d]imidazole-
112.00, 104.55, 82.09, 75.04, 70.18, 65.52, 63.25, 61.03, 55.88, 48.24,
48.03, 47.81, 47.60, 47.39, 47.18, 46.96, 29.49. HRMS calcd for
C
2-ylmethyl)-methoxy)-b-D-galactopyranoside (16b). Following the
general method, the reaction was performed with 15b (41.3 mg,
0.1 mmol) and KOH (15.4 mg, 0.27 mmol, 2.7 equiv.) in 4 mL EtOH:
H2O (3:1) for 4 h. Compound 16b was obtained as an amorphous
18H24N2O8 þ Hþ (M þ H)þ: 397.1611, found: 397.1608.
4.4.9.2. Methyl 3-O-((5-ethoxycarbonyl)1-methyl-1H-benzo[d]imid-
azole-2-ylmethyl)-methoxy)- -galactopyranoside (15b).
white solid in 56% yield (17 mg). 1H NMR (400 MHz, CD3OD)
d 8.18
b
-D
(s, 1H, AreH), 7.94 (dd, J ¼ 8.5, 1.6 Hz, 1H, Ar-sH), 7.67 (d, J ¼ 8.5 Hz,
1H, AreH), 5.05 (q, 2H, J ¼ 13.2, 6.14 Hz, 2H, CH2C9H7N2O2), 4.17 (d,
J ¼ 7.7 Hz, 1H, H-1), 4.14 (d, J ¼ 2.4 Hz, 1H, H-4), 3.83e3.67 (m, 3H,
H2, H-6a, H-6b), 3.54 (s, 3H, OCH3), 3.51 (m, 1H, H-5), 3.46 (dd,
Following the general method, the reaction was performed with 7b
(100.5 mg, 0.51 mmol), dry MeOH (3 mL), Bu2SnO (143 mg,
0.57 mmol, 1.1 equiv.), anhydrous 1,4-dioxane (3 mL), Bu4NBr
(250.1 mg, 0.77 mmol, 1.5 equiv.), 14b (156 mg, 0.62 mmol, 1.2
equiv.). Compound 15b was obtained as a white amorphous solid in
J ¼ 9.6, 3.2 Hz, 1H, H-3). 13C NMR (126 MHz, D2O)
d 172.41, 151.74,
136.01, 132.99, 130.87, 126.13, 116.88, 111.39, 103.52, 81.65, 74.77,
69.76, 65.31, 62.01, 60.74, 57.13, 30.71. HRMS calcd for
65% yield (137.3 mg). 1H NMR (400 MHz, CD3OD)
d 8.27 (d,
J ¼ 1.5 Hz, 1H, ArH), 7.97 (dd, J ¼ 8.6, 1.6 Hz, 1H, ArH), 7.53 (d,
J ¼ 8.6 Hz, 1H, ArH), 5.04 (q, J ¼ 13.2, 6.3 Hz, 2H, CH2C8H7N2), 4.39
(q, J ¼ 7.1 Hz, 2H, CO2CH2CH3), 4.18 (d, J ¼ 7.7 Hz, 1H, H-1), 4.14 (d,
J ¼ 2.4 Hz, 1H, H-4), 3.93 (s, 3H, NCH3), 3.84e3.68 (m, 3H, H2, H-6a,
H-6b), 3.54 (s, 3H, OCH3), 3.53e3.50 (m, 1H, H-5), 3.46 (dd, J ¼ 9.7,
3.2 Hz, 1H, H-3), 1.43 (t, J ¼ 7.1 Hz, 3H, CO2CH2CH3). 13C NMR
C
17H22N2O8 þ Hþ (M þ H)þ: 383.1454, found: 383.1448.
4.4.10.3. Methyl 3-O-((5-carboxy)1H-benzo[d]imidazole-2-
ylmethyl)-methoxy)- -galactopyranoside (16c). Following the
b-D
general method, the reaction was performed with 15c (21.9 mg,
0.056 mmol) and KOH (10.1 mg, 0.18 mmol, 3 equiv.) in 4 mL EtOH:
H2O (3:1) for 6 h. Compound 16c was obtained as an amorphous
(101 MHz, CD3OD)
d 167.03, 153.65, 140.57, 139.11, 124.52, 124.08,
11

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
white solid in 37% yield (7.6 mg). 1H NMR (500 MHz, D2O)
d
7.82 (s,
2), 4.29 (t, J ¼ 6.1 Hz, 2H, H-4), 4.26 (d, J ¼ 3.0 Hz, 1H, H-5), 4.00 (s,
3H, NCH3), 3.79e3.64 (m, 3H, H-3, H-6a, H-6b). 13C NMR (126 MHz,
1H, ArH), 7.70 (dd, J ¼ 8.6, 1.5 Hz, 1H, ArH), 7.38 (d, J ¼ 8.6 Hz, 1H,
ArH), 5.03 (d, J ¼ 14.8 Hz,1H, CH2C8H5N2O2), 4.91 (d, J ¼ 14.8 Hz,1H,
CH2C8H5N2O2), 4.29 (d, J ¼ 7.4 Hz,1H, H-1), 4.15 (d, J ¼ 3.1 Hz,1H. H-
4), 3.78e3.56 (m, 5H, H-2, H-3, H-5, H-6a, H-6b), 3.50 (s, 3H, OCH3).
CD3OD)
d
168.92, 154.21, 144.29, 135.43 (d, J ¼ 18.1 Hz), 132.91,
131.98, 131.20, 130.62, 130.15, 123.63, 117.87, 111.99, 89.66, 79.54,
72.06, 67.50, 65.94, 63.10, 60.87, 29.31. HRMS calcd for
13C NMR (126 MHz, D2O)
d
168.74, 152.37, 135.15, 132.50, 129.26,
C
22H22Cl2N2O7S þ Hþ (M þ H)þ: 529.0603, found: 529.0602.
125.63, 115.56, 113.54, 103.50, 81.80, 74.74, 69.85, 65.24, 62.83,
60.76, 57.14. HRMS calcd for C16H20N2O8 þ Hþ (M þ H)þ: 369.1298,
found: 369.1289.
4.4.14. 3,4-Dichlorophenyl 3-O-((5-carboxy)1H-benzo[d]imidazole-
2-ylmethyl)-1-thio- -galactopyranoside (19b)
a-D
Compound 18b (106.8 mg, 0.20 mmol) and LiOH (30 mg,
1.25 mmol, 6 equiv.) were dissolved in 4 mL EtOH: H2O (3:1), and the
reaction was run at 80 ꢁC overnight. The solvent was then evapo-
rated in vacuo, and compound 19b was purified by preparative HPLC.
White amorphous solid (43.1 mg, 41% yield). 1H NMR (400 MHz,
4.4.11. 3,4-Dichlorophenyl 3-O-((5-methoxycarbonyl)1-methyl-1H-
benzo[d]imidazole-2-ylmethyl)-1-thio-a-D-galactopyranoside (18a)
Compound 17 [17] (100 mg, 0.29 mmol), Compound 14a
(83.4 mg, 0.35 mmol, 1.2 equiv.), Bu2SnO (80.3 mg, 0.32 mmol, 1.1
equiv.) and n-Bu4NI (270.7 mg, 0.73 mmol, 1.2 equiv.) were dis-
solved in 4.8 mL toluene: MeCN (5:1) in a 2e5 mL biotage micro-
wave vial. The reaction was run at 120 ꢁC for 1 h under normal
absorption of the microwave. The volatiles were evaporated in
vacuo. The mixture was purified with flash column chromatog-
raphy (DCM: MeOH: 97: 3% to 95: 5%) followed by preparative HPLC
to provide 18a as a white amorphous solid (50.6 mg, 31% yield). 1H
CD3OD)
d
8.18 (s, 1H, ArH), 7.87 (dd, J ¼ 8.5, 1.6 Hz, 1H, ArH), 7.64 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.50 (d, J ¼ 8.4 Hz, 1H, ArH), 7.42e7.31 (m, 2H,
ArH), 5.61 (d, J ¼ 5.6 Hz, 1H, H-1), 4.94 (q, J ¼ 14.0 Hz, 2H,
CH2C8H5N2O2), 4.34 (dd, J ¼ 10.1, 5.6 Hz, 1H, H-2), 4.24e4.17 (m, 2H,
H-4, H-5), 3.71e3.57 (m, 3H, H-3, H-6a, H-6b). 13C NMR (101 MHz,
CD3OD) d 135.36, 133.04, 132.10, 131.32, 130.77, 130.26, 123.97, 89.57,
80.08, 72.08, 67.73, 66.14, 64.63, 60.95. HRMS calcd for
21H20Cl2N2O7S þ Hþ (M þ H)þ: 515.0447, found: 515.0451.
NMR (500 MHz, CD3OD)
d
8.23 (s, 1H, ArH), 7.97 (dd, J ¼ 8.5, 1.6 Hz,
C
1H, ArH), 7.73 (d, J ¼ 2.0 Hz, 1H, ArH), 7.71 (d, J ¼ 8.5 Hz, 1H, ArH),
7.49e7.42 (m, 2H, ArH), 5.70 (d, J ¼ 5.6 Hz,1H, H-1), 5.08 (q, J ¼ 13.6,
2.2 Hz, 2H, CH2C10H10N2O2), 4.41 (q, J ¼ 10.1, 5.6 Hz, 1H, H-2), 4.29
(t, J ¼ 6.2 Hz, 1H, H-4), 4.26 (d, J ¼ 2.2 Hz, 2H, H-5), 3.98 (s, 3H,
4.5. Molecular dynamics simulations
Molecular dynamics simulations were performed using the
CO2CH3), 3.97 (s, 3H, NCH3), 3.79e3.62 (m, 3H, H-3, H-6a, H-6b). 13
C
€
OPLS3 force field in Desmond implemented in Schrodinger Release
NMR (126 MHz, CD3OD)
d 167.29, 144.52, 135.34, 132.90, 131.98,
2020-1, using default settings except for the length of the simula-
tion, and the use of light harmonic constraints (1 kcal molꢀ1 Åꢀ2) on
all stranded backbone atoms and the galactose O4 atom. To keep
galectin-8N simulations stable, the coulombic cut-off radius had to
be increased from the default 9 Å to 10 Å. Galectin-3 complexes
were constructed based on the structure with the PDB code 5GZD,
while galectin-8N complexes on the structure with the PDB code
7AEN, and the ligands were placed by the superposition of the
corresponding galactose parts.
131.19, 130.62, 130.15, 124.71, 123.30, 118.07, 111.89, 89.66, 79.57,
72.05, 67.49, 65.93, 63.10, 60.86, 51.19, 29.36. HRMS calcd for
C
23H24Cl2N2O7S þ Hþ (M þ H)þ: 543.0760, found: 543.0753.
4.4.12. 3,4-Dichlorophenyl 3-O-((5-methoxycarbonyl)1H-benzo[d]
imidazole-2-ylmethyl)-1-thio- -galactopyranoside (18b)
a-D
Compound 17 (305.5 mg, 0.90 mmol), Compound 14d (350 mg,
1.08 mmol, 1.2 equiv.), Bu2SnO (245.1 mg, 0.98 mmol, 1.1 equiv.) and
n-Bu4NI (496 mg, 1.34 mmol, 1.5 equiv.) were dissolved in 12 mL
toluene: MeCN (5:1) in a 10e20 mL biotage microwave vial. The
reaction was run at 120 ꢁC for 90 min under normal absorption of
the microwave. The volatiles were evaporated in vacuo. The mixture
was purified with flash column chromatography (DCM: MeOH: 95:
5% to 90: 10%) followed by preparative HPLC to provide 18b as a
white amorphous solid (166 mg, 34% yield). 1H NMR (400 MHz,
Accession codes
Protein Data Bank: 7AEN, 7ALS.
Declaration of competing interest
CD3OD)
d
8.28 (s, 1H, ArH), 7.97 (dd, J ¼ 8.5, 1.2 Hz, 1H, ArH), 7.75 (d,
F.R.Z. is an employee of, and H.L. and U.J.N. are shareholders in
Galecto Biotech AB, a company developing galectin inhibitors. The
other authors have no conflicts to declare.
J ¼ 2.0 Hz, 1H, ArH), 7.63 (d, J ¼ 8.5 Hz, 1H, ArH), 7.54e7.40 (m, 2H,
ArH), 5.73 (d, J ¼ 5.5 Hz, 1H, H-1), 5.06 (q, J ¼ 14.1 Hz, 2H,
CH2C9H7N2O2), 4.47 (dt, J ¼ 9.9, 5.5 Hz, 1H, H-2), 4.36e4.27 (m, 2H,
H-4, H-5), 3.95 (s, 3H, CO2CH3), 3.87e3.66 (m, 3H, H-3, H-6a, H-6b).
Acknowledgements
13C NMR (101 MHz, CD3OD)
d 167.59, 135.34, 133.04, 132.10, 131.32,
130.78, 130.26, 124.31, 123.69, 89.57, 80.08, 72.08, 67.73, 66.14,
64.62, 60.94, 51.21. C22H22Cl2N2O7S þ Hþ (M þ H)þ: 529.0603,
found: 529.0610.
This project has received funding from the European Union's
Horizon 2020 research and innovation program under the Marie
Skłodowska-Curie grant agreement No 765581, Galecto Biotech
Inc., and the Royal Physiographic Society in Lund. The authors
kindly thank Barbro Kahl Knutson for assistance with the fluores-
cence polarisation measurements.
4.4.13. 3,4-Dichlorophenyl 3-O-((5-carboxy)1-methyl-1H-benzo[d]
imidazole-2-ylmethyl)-1-thio-a-D-galactopyranoside (19a)
Compound 18a (23.3 mg, 0.042 mmol) and KOH (9.8 mg,
0.174 mmol, 4 equiv.) were dissolved in 4 mL EtOH: H2O (3:1), and
the reaction was run at 80 ꢁC for 6 h until the TLC indicated the
complete consumption of the starting material. The solvent was
evaporated in vacuo, and compound 19a was purified by prepara-
tive HPLC. White amorphous solid (7 mg, 31% yield). 1H NMR
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113664.
(500 MHz, CD3OD)
d
8.26 (d, J ¼ 1.5 Hz, 1H, ArH), 8.00 (dd, J ¼ 8.5,
References
1.6 Hz, 1H, ArH), 7.74 (d, J ¼ 2.0 Hz, 1H, ArH), 7.71 (d, J ¼ 8.5 Hz, 1H,
ArH), 7.50e7.41 (m, 2H, ArH), 5.70 (d, J ¼ 5.6 Hz, 1H, H-1), 5.09 (q,
J ¼ 13.3, 2.0 Hz, 2H, CH2C9H7N2O2), 4.41 (dd, J ¼ 10.1, 5.6 Hz, 1H, H-
[1] H. Leffler, S. Carlsson, M. Hedlund, Y. Qian, F. Poirier, Introduction to galectins,
Glycoconj.
J.
19
(2004)
433e440,
https://doi.org/10.1023/B:
12

M. Hassan, S. van Klaveren, M. Håkansson et al.
European Journal of Medicinal Chemistry 223 (2021) 113664
[24] I. Cumpstey, S. Carlsson, H. Leffler, U.J. Nilsson, Synthesis of a phenyl thio- -d-
GLYC.0000014072.34840.04.
b
[2] L. Johannes, R. Jacob, H. Leffler, Galectins at a glance, J. Cell Sci. 131 (2018),
https://doi.org/10.1242/jcs.208884.
[3] C. Boscher, J.W. Dennis, I.R. Nabi, Glycosylation, galectins and cellular
signaling, Curr. Opin. Cell Biol. 23 (2011) 383e392, https://doi.org/10.1016/
J.CEB.2011.05.001.
galactopyranoside library from 1,5-difluoro-2,4-dinitrobenzene: discovery of
efficient and selective monosaccharide inhibitors of galectin-7, Org. Biomol.
Chem. 3 (2005) 1922, https://doi.org/10.1039/b502354h.
€
[25] C. Diehl, O. Engstrom, T. Delaine, M. Håkansson, S. Genheden, K. Modig,
H. Leffler, U. Ryde, U.J. Nilsson, M. Akke, Protein flexibility and conformational
entropy in ligand design targeting the carbohydrate recognition domain of
galectin-3, J. Am. Chem. Soc. 132 (2010) 14577e14589, https://doi.org/
10.1021/ja105852y.
[4] Y. Levy, S. Auslender, M. Eisenstein, R.R. Vidavski, D. Ronen, A.D. Bershadsky,
Y. Zick, It depends on the hinge: a structure-functional analysis of galectin-8, a
tandem-repeat type lectin, Glycobiology 16 (2006) 463e476, https://doi.org/
10.1093/glycob/cwj097.
[26] S. Tahlan, S. Kumar, B. Narasimhan, Pharmacological significance of hetero-
cyclic 1H-benzimidazole scaffolds: a review, BMC Chem. 13 (2019) 1e21,
https://doi.org/10.1186/s13065-019-0625-4.
[27] R.S. Keri, M.R. Patil, S.A. Patil, S. Budagupi, A comprehensive review in current
developments of benzothiazole-based molecules in medicinal chemistry, Eur.
€
[5] S. Carlsson, C.T. Oberg, M.C. Carlsson, A. Sundin, U.J. Nilsson, D. Smith,
R.D. Cummings, J. Almkvist, A. Karlsson, H. Leffler, Affinity of galectin-8 and its
carbohydrate recognition domains for ligands in solution and at the cell
surface, Glycobiology 17 (2007) 663e676, https://doi.org/10.1093/glycob/
cwm026.
J.
Med.
Chem.
89
(2015)
207e251,
https://doi.org/10.1016/
~
[6] A.J. Cagnoni, M.F. Troncoso, G.A. Rabinovich, K.V. Marino, M.T. Elola, Full-
j.ejmech.2014.10.059.
length galectin-8 and separate carbohydrate recognition domains: the whole
is greater than the sum of its parts? Biochem. Soc. Trans. 48 (2020)
1255e1268, https://doi.org/10.1042/BST20200311.
[28] S. Kakkar, S. Tahlan, S.M. Lim, K. Ramasamy, V. Mani, S.A.A. Shah,
B. Narasimhan, Benzoxazole derivatives: design, synthesis and biological
evaluation, Chem. Cent. J. 12 (2018) 1e16, https://doi.org/10.1186/s13065-
018-0459-5.
ꢂ
ꢂ
[7] S. Bertuzzi, J.I. Quintana, A. Arda, A. Gimeno, J. Jimenez-Barbero, Targeting
galectins with glycomimetics, Front. Chem. 8 (2020) 593, https://doi.org/
10.3389/fchem.2020.00593.
[8] W.S. Chen, Z. Cao, S. Sugaya, M.J. Lopez, V.G. Sendra, N. Laver, H. Leffler,
U.J. Nilsson, J. Fu, J. Song, L. Xia, P. Hamrah, N. Panjwani, Pathological lym-
phangiogenesis is modulated by galectin-8-dependent crosstalk between
podoplanin and integrin-associated VEGFR-3, Nat. Commun. 7 (2016) 1e17,
https://doi.org/10.1038/ncomms11302.
ꢂ
[29] G. Bastug, C. Eviolitte, I.E. Marko, Functionalized orthoesters as powerful
building blocks for the efficient preparation of heteroaromatic bicycles, Org.
Lett. 14 (2012) 3502e3505, https://doi.org/10.1021/ol301472a.
[30] L. Ballell, J.A.F. Joosten, F.A. el Maate, R.M.J. Liskamp, R.J. Pieters, Microwave-
assisted, tin-mediated, regioselective 3-O-alkylation of galactosides, Tetra-
hedron
Lett.
45
(2004)
6685e6687,
https://doi.org/10.1016/
J.TETLET.2004.06.113.
[9] Y. Vinik, H. Shatz-Azoulay, Y. Zick, Molecular mechanisms underlying the role
of galectin-8 as a regulator of cancer growth and metastasis, Trends Glycosci.
Glycotechnol. 30 (2018) SE119eSE128, https://doi.org/10.4052/tigg.1742.1SE.
[10] J.F. Sampson, A. Suryawanshi, W.S. Chen, G.A. Rabinovich, N. Panjwani,
Galectin-8 promotes regulatory T-cell differentiation by modulating IL-2 and
[31] T. Delaine, P. Collins, A. MacKinnon, G. Sharma, J. Stegmayr, V.K. Rajput,
S. Mandal, I. Cumpstey, A. Larumbe, B.A. Salameh, B. Kahl-Knutsson, H. van
Hattum, M. van Scherpenzeel, R.J. Pieters, T. Sethi, H. Schambye, S. Oredsson,
H. Leffler, H. Blanchard, U.J. Nilsson, Galectin-3-Binding glycomimetics that
strongly reduce bleomycin-induced lung fibrosis and modulate intracellular
glycan recognition, Chembiochem 17 (2016) 1759e1770, https://doi.org/
10.1002/cbic.201600285.
TGF
b signaling, Immunol. Cell Biol. 94 (2016) 213e219, https://doi.org/
10.1038/icb.2015.72.
[11] M.V. Tribulatti, J. Carabelli, C.A. Prato, O. Campetella, Galectin-8 in the onset of
the immune response and inflammation, Glycobiology 30 (2020) 134e142,
https://doi.org/10.1093/glycob/cwz077.
[32] K.B. Pal, M. Mahanti, H. Leffler, U.J. Nilsson, A galactoside-binding protein
tricked into binding unnatural pyranose derivatives: 3-deoxy-3-methyl-
gulosides selectively inhibit galectin-1, Int. J. Mol. Sci. 20 (2019), https://
doi.org/10.3390/ijms20153786.
[33] G. Winter, D.G. Waterman, J.M. Parkhurst, A.S. Brewster, R.J. Gildea, M. Gerstel,
L. Fuentes-Montero, M. Vollmar, T. Michels-Clark, I.D. Young, N.K. Sauter,
G. Evans, DIALS: implementation and evaluation of a new integration package,
Acta Crystallogr. Sect. D Struct. Biol. 74 (2018) 85e97, https://doi.org/10.1107/
S2059798317017235.
[34] W. Kabsch, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 125e132,
https://doi.org/10.1107/S0907444909047337.
[35] P. Evans, G. Marshudov, How good are my data and what is the resolution?
Acta Crystallogr. Sect. D Biol. Crystallogr. 69 (2013) 1204e1214, https://
doi.org/10.1107/S0907444913000061.
[12] S. Carlsson, M.C. Carlsson, H. Leffler, Intracellular sorting of galectin-8 based
on carbohydrate fine specificity, Glycobiology 17 (2007) 906e912, https://
doi.org/10.1093/glycob/cwm059.
ꢂ
[13] T.L.M. Thurston, M.P. Wandel, N. Von Muhlinen, A. Foeglein, F. Randow,
Galectin 8 targets damaged vesicles for autophagy to defend cells against
bacterial invasion, Nature 482 (2012) 414e418, https://doi.org/10.1038/
nature10744.
[14] M.H. Bohari, X. Yu, C. Kishor, B. Patel, R.M. Go, H.A. Eslampanah Seyedi,
Y. Vinik, I.D. Grice, Y. Zick, H. Blanchard, Structure-based design of a mono-
saccharide ligand targeting galectin-8, ChemMedChem 13 (2018) 1664e1672,
https://doi.org/10.1002/cmdc.201800224.
[15] B. Patel, C. Kishor, T.A. Houston, H. Shatz-Azoulay, Y. Zick, Y. Vinik,
[36] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni,
R.J. Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007)
658e674, https://doi.org/10.1107/S0021889807021206.
H. Blanchard, Rational design and synthesis of methyl-b- d -galactomalonyl
phenyl esters as potent galectin-8 N antagonists, J. Med. Chem. 63 (2020)
11573e11584, https://doi.org/10.1021/acs.jmedchem.0c00602.
ꢂ
[37] G.N. Murshudov, P. Skubak, A.A. Lebedev, N.S. Pannu, R.A. Steiner,
[16] C. Wu, C. Yong, Q. Zhong, Z. Wang, U.J. Nilsson, Y. Zhang, Synthesis of tricyclic
carbohydrate-benzene hybrids as selective inhibitors of galectin-1 and
galectin-8 N-terminal domains, RSC Adv. 10 (2020) 19636e19642, https://
doi.org/10.1039/d0ra03144e.
[17] K.B. Pal, M. Mahanti, X. Huang, S. Persson, A.P. Sundin, F.R. Zetterberg,
S. Oredsson, H. Leffler, U.J. Nilsson, Quinolineegalactose hybrids bind selec-
tively with high affinity to a galectin-8 N-terminal domain, Org. Biomol.
Chem. 16 (2018) 6295e6305, https://doi.org/10.1039/C8OB01354C.
[18] F.R. Zetterberg, K. Peterson, R.E. Johnsson, T. Brimert, M. Håkansson,
D.T. Logan, H. Leffler, U.J. Nilsson, Monosaccharide derivatives with low-
nanomolar lectin affinity and high selectivity based on combined
R.A. Nicholls, M.D. Winn, F. Long, A.A. Vagin, REFMAC5 for the refinement of
macromolecular crystal structures, Acta Crystallogr. Sect. D Biol. Crystallogr.
67 (2011) 355e367, https://doi.org/10.1107/S0907444911001314.
[38] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of
Coot, Acta Crystallogr. Sect. D Biol. Crystallogr. 66 (2010) 486e501, https://
doi.org/10.1107/S0907444910007493.
[39] V.B. Chen, W.B. Arendall, J.J. Headd, D.A. Keedy, R.M. Immormino, G.J. Kapral,
L.W. Murray, J.S. Richardson, D.C. Richardson, MolProbity: all-atom structure
validation for macromolecular crystallography, Acta Crystallogr. Sect. D Biol.
Crystallogr. 66 (2010) 12e21, https://doi.org/10.1107/S0907444909042073.
[40] K.V. Kutonova, M.E. Trusova, P. Postnikov, V.D. Filimonov, J. Parello, A simple
and effective synthesis of aryl azides via arenediazonium tosylates, Synthesis
45 (2013) 2706e2710, https://doi.org/10.1055/s-0033-1339648.
fluorineeamide, phenylearginine, sulfurep, and halogen bond interactions,
ChemMedChem
13
(2018)
133e137,
https://doi.org/10.1002/
cmdc.201700744.
[41] P.S. Gribanov, M.A. Topchiy, Y.D. Golenko, Y.I. Lichtenstein, A.V. Eshtukov,
V.E. Terekhov, A.F. Asachenko, M.S. Nechaev, An unprecedentedly simple
method of synthesis of aryl azides and 3-hydroxytriazenes, Green Chem. 18
(2016) 5984e5988, https://doi.org/10.1039/c6gc02379g.
ꢂ
ꢁ
[19] V. Denavit, D. Laine, T. Tremblay, J. St-Gelais, D. Giguere, Synthetic inhibitors
of galectins: structures and syntheses, Trends glycosci. Glycotechnol. 30
(2018) SE21eSE40, https://doi.org/10.4052/tigg.1729.1SE.
[20] K. Barral, A.D. Moorhouse, J.E. Moses, Efficient conversion of aromatic amines
into azides: a one-pot synthesis of triazole linkages, Org. Lett. 9 (2007)
1809e1811, https://doi.org/10.1021/ol070527h.
[42] N.P. Pera, U.J. Nilsson, N. Kann, Intermolecular PausoneKhand reactions on a
galactose scaffold, Tetrahedron Lett. 49 (2008) 2820e2823, https://doi.org/
10.1016/j.tetlet.2008.02.114.
[21] H. Xu, B. Ren, W. Zhao, X. Xin, Y. Lu, Y. Pei, H. Dong, Z. Pei, Regioselective mono
and multiple alkylation of diols and polyols catalyzed by organotin and its
applications on the synthesis of value-added carbohydrate intermediates,
Tetrahedron 72 (2016) 3490e3499, https://doi.org/10.1016/j.tet.2016.04.076.
[43] H. An, T. Wang, V. Mohan, R.H. Griffey, P. Dan Cook, Solution phase combi-
natorial chemistry. Discovery of 13- and 15-membered poly-
azapyridinocyclophane libraries with antibacterial activity, Tetrahedron 54
(1998) 3999e4012, https://doi.org/10.1016/S0040-4020(98)00131-8.
[44] N. Teno, K. Gohda, K. Wanaka, Y. Tsuda, M. Akagawa, E. Akiduki, M. Araki,
A. Masuda, T. Otsubo, Y. Yamashita, Novel type of plasmin inhibitors:
providing insight into P4 moiety and alternative scaffold to pyrrolopyr-
imidine, Bioorg. Med. Chem. 23 (2015) 3696e3704, https://doi.org/10.1016/
j.bmc.2015.04.013.
€
[22] P. Sorme, B. Kahl-Knutson, U. Wellmar, U.J. Nilsson, H. Leffler, Fluorescence
polarization to study galectineligand interactions, in: Methods Enzymol.,
2003, pp. 504e512, https://doi.org/10.1016/S0076-6879(03)01033-4.
€
[23] P. Sorme, B. Kahl-Knutsson, M. Huflejt, U.J. Nilsson, H. Leffler, Fluorescence
polarization as an analytical tool to evaluate galectineligand interactions,
Anal. Biochem. 334 (2004) 36e47, https://doi.org/10.1016/j.ab.2004.06.042.
13