Quinoline-galactose hybrids bind selectively with high affinity to a galectin-8 N-terminal domain

Article abstract of DOI:10.1039/c8ob01354c

Quinolines, indolizines, and coumarins are well known structural elements in many biologically active molecules. In this report, we have developed straightforward methods to incorporate quinoline, indolizine, and coumarin structures into galactoside derivatives under robust reaction conditions for the discovery of glycomimetic inhibitors of the galectin family of proteins that are involved in immunological and tumor-promoting biological processes. Evaluation of the quinoline, indolizine and coumarin-derivatised galactosides as inhibitors of the human galectin-1, 2, 3, 4N (N-terminal domain), 4C (C-terminal domain), 7, 8N, 8C, 9N, and 9C revealed quinoline derivatives that selectively bound galectin-8N, a galectin with key roles in lymphangiogenesis, tumor progression, and autophagy, with up to nearly 60-fold affinity improvements relative to methyl β-d-galactopyranoside. Molecular dynamics simulations proposed an interaction mode in which Arg59 had moved 2.5 ? and in which an inhibitor carboxylate and quinoline nitrogen formed structure-stabilizing water-mediated hydrogen bonds. The compounds were demonstrated to be non-toxic in an MTT assay with several breast cancer cell lines and one normal cell line. The improved affinity, selectivity, and low cytotoxicity suggest that the quinoline-galactoside derivatives provide an attractive starting point for the development of galectin-8N inhibitors potentially interfering with pathological lymphangiogenesis, autophagy, and tumor progression.

Full text of DOI:10.1039/c8ob01354c

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ARTICLE
Quinoline-galactose hybrids bind selectively with high affinity to
galectin-8 N-terminal domain
Received 00th January 20xx,
Accepted 00th January 20xx
Kumar Bhaskar Pal,a Mukul Mahanti,a Xiaoli Huang,b Stella Persson,a Anders P. Sundin,a Fredrik
† †
R. Zetterberg,c Stina Oredsson,b Hakon Leffler,d and Ulf J. Nilssona*
DOI: 10.1039/x0xx00000x
Quinolines, indolizines, and coumarins are well known structural elements in many biologically active molecules. In this
report, we have developed straightforward methods to incorporate quinoline, indolizine, and coumarin structures into
galactoside derivatives under robust reaction conditions for the discovery of glycomimetic inhibitors of the galectin family
of proteins that are involved in immunological and tumor-promoting biological processes. Evaluation of the quinoline,
indolizine and coumarin-derivatised galactosides as inhibitors of the human galectin-1, 2, 3, 4N (N-terminal domain), 4C (C-
terminal domain), 7, 8N, 8C, 9N, and 9C revealed quinoline derivatives that selectively bound galectin-8N, a galectin with
key roles in lymphangiogenesis, tumor progression, and autophagy, with up to near 60-fold affinity improvements relative
to methyl β-D-galactopyranoside. Molecular dynamics simulations proposed an interaction mode in which Arg59 had
moved 2.5Å and in which a inhibitor carboxylate and quinoline nitrogen formed structure-stabilizing water-mediated
hydrogen bonds. The compounds were demonstrated to be non-toxic in an MTT assay with several breast cancer cell lines
and one normal cell line. The improved affinity, selectivity, and low cytotoxicity suggest that the quinoline-galactoside
derivatives provide an attractive starting point for development of galectin-8N inhibitors potenitally interfering with
pathological lymphangiogenesis, autophagy, and tumor progression.
www.rsc.org/
leads.6-9
1. Introduction
Quinolines, indolizines, and coumarins and substituted
derivatives thereof are common structures in medicinal and
Galectins are an ancient family of glycan binding proteins
found in most organisms, including 15 mammalian members.1
Galectins are illustrated as a developmental preserved sub-
class of endogenous lectins based on the structures of their
synthetic organic chemistry and frequently display distinct
biological activities.10-13 As galectins specifically bind galactose-
containing glycoconjugates and have been demonstrated to be
inhibited by synthetic galactosides derivatized with aromatic
structures at C-3,14 we hypothesized that combining
carbohydrate recognition domain (CRD) and binding to
β-D-
galactoside-containing ligands.2 Galectins contribute to a large
diversity of biologically important functions including cell–cell
and cell–matrix interactions, immune and inflammatory
responses, induction of apoptosis for T-cells, anti-apoptotic
functions, modulation of cell adhesion and migration.3-5
Dysfunction of such galectin-related biological mechanisms
have been demonstrated to influence cancer biology, such as
tumor cell survival, neoplastic metamorphosis, angiogenesis,
and tumour metastasis. Thus, there is a pressing need for
molecules towards the discovery of galectin-blocking drug
quinolines, indolizines, and coumarins with a β-D-galactoside
core structure could lead to the discovery of new compounds
with enhanced galectin affinities and selectivities. Here we
report the design and synthesis of a series of quinoline-,
indolizine-, and coumarin-carrying galactoside derivatives and
evaluation of them as inhibitors of galectin-1, 2, 3, 4N (N-
terminal domain), 4C (C-terminal domain), 7, 8N, 8C, 9N, and
9C, as well as of their cytotoxic properties.
2. Results and discussion
2.1 Chemistry
The quinoline bromides 7a
while bromides 7d 7i were synthesized (Scheme 1). The 2-
methyl quinolines 6e-f were synthesized from crotonaldehyde
and anilines and . Radical mono-bromination of the 2-
methyl group of quinolines 6d 6i gave quinolinylmethyl
bromides 7d 7i that together with the known bromides 7a 7c
were used in stannylidene-acetal-mediated regioselective
-
7c15-17 are known in the literature,
-
4
5
-
-
-
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Journal Name
R
R
N
Methyl bromoacetate
Dry Acetone
Ultra sound
1. Crotonaldehyde
6N HCl
∆
N
Br
61ꢀ74%
2. Conc. H2SO4
9d R=CO2CH3
9e R=CH3
MeO2C
10a R=H
10b R=COCH3
10c R=CF3
HO2C
NH2
MeO2C
F
N
MeOH, 65 o
C
10d R=CO2CH3
10e R=CH3
4
6e
27%
F
F
BnO
OBn
BnO
Crotonaldehyde, Chloranil
OBn
DIC, DMAP
Propiolic acid
42%
Conc. HCl, 2ꢀButanol
O
O
OMe
OMe
NH2
F
N
∆
64%
HO
O
5
6f
OBn
OBn
11
12
O
R
R2
R1
N
R2
R1
N
R3
R3
1. NBS/AIBN
CCl4, 80 o
C
OH
HO
1. 10aꢀ10e
K2CO3/MeOH, rt
2. Pd(OH)2ꢀC/ H2
25ꢀ34%
Br
R4
41ꢀ49%
R4
N
O
OMe
O
O
R5
R5
MeO2C
OH
6dꢀ6i
7dꢀ7i
= ArCH2Br
2a R=H
2d R=CO2CH3
2b R=COCH3 2e R=CH3
2c R=CF3
7a R1 = R2 = R3 = R4 = R5 = H
7b R1 = R2 = R4 = R5 = H, R3 = F
7c R1 = R2 = R4 = R5 = H, R3 = Cl
Scheme 2 Synthesis of indolizidine-derivatised thiogalactosides 2a-2e.
6d,
7d R1 = R2 = R4 = R5 = H, R3 = CO2CH3
7e R1 = R2 = R3 = R5 = H, R4 = CO2CH3
7f R1 = R2 = R5 = H, R3 = R4 = F
6e,
6f,
alkylation of the corresponding pyridines 9d
-
9e. Treatment of
10d-10e and the known pyridinium salts 10a
-
10c18 in methanol
7g R1 = Cl, R2 = R4 = R5 = H, R3 = CF3
7h R1 = Cl, R2 = R3 = R5 = H, R4 = CF3
7i R1 = Cl, R2 = R3 = R4 = H, R5 = F
6g,
6h,
6i,
and with K2CO3 as a base generated intermediate pyridinium
ylides that were reacted with the propiolate 12 at room
temperature for 12h. The crude products were subjected to
hydrogenolysis to remove benzyl ether protecting groups to
1. Bu2SnO/MeOH
Reflux
R2
R1
HO
OH
O
R3
R4
OH
O
give the indolizines 2a
-
2e in 25-34% yield over two steps.
R
HO
O
OMe
R
HO
OMe
Propargyl bromide
K2CO3
Dry Acetone
OH
N
8
OH
R5
2. ArCH2Br, Bu4NBr
1aꢀ1i
Reflux
65ꢀ74%
O
O
O
HO
O
O
Dry Dioxane, 85 o
71ꢀ81%
C
1a R1 = R2 = R3 = R4 = R5 = H
1b R1 = R2 = R3 = R4 = R5 = H, R3 = F
1c R1 = R2 = R3 = R4 = R5 = H, R3 = Cl
1d R1 = R2 = R4 = R5 = H, R3 = CO2CH3
1e R1 = R2 = R3 = R5 = H, R4 = CO2CH3
1f R1 = R2 = R5 = H, R3 = R4 = F
13a R=H
13c R=CF3
14a R=H
14c R=CF3
13b R=CH3 13d R=Ph
14b R=CH3 14d R=Ph
1g R1 = Cl, R2 = R4 = R5 = H, R3 = CF3
1h R1 = Cl, R2 = R3 = R5 = H, R4 = CF3
1i R1 = Cl, R2 = R3 = R4 = H, R5 = F
1j R1 = R2 = R4 = R5 = H, R3 = CO2H (66%)
1k R1 = R2 = R3 = R5 = H, R4 = CO2H (63%)
Propargyl bromide
K2CO3
Dry Acetone
Reflux
KOH/EtOH/H2O
60 o
HO
O
N
C
O
O
O
O
66%
13e
14e
Scheme 1 Synthesis of quinoline derived methyl β-D-galactosides 1a-1k.
HO
RO
N3
OH
OR
O
O
N
STol
STol
N
alkylation of methyl β-D-galactopyranoside 8 to afford
compound 1a 1i in 71-81% yield (Scheme 1). Compounds 1j
OH
O
OR
15 R=Ac
16 R=H
-
14aꢀ14d
NaOMe
MeOH
95%
CuI, DIPEA
O
O
and 1k were obtained by the hydrolysis of the corresponding
methyl esters 1d and 1e under basic conditions in 63–66%
yield.
Dry DCM, rt
59ꢀ85%
14e
CuI, DIPEA
Dry DCM, rt
61%
R
Recently, Bonte and co-workers reported an efficient way
towards formation of substituted indolizines via cycloaddition
of pyridinium ylides with propiolic esters.18 Inspired by this
3a R=H
3b R=CH3
3c R=CF3
3d R=Ph
HO
OH
report, we treated known methyl 2,4,6-tri-O-benzyl-β-D-
N
N
O
O
galactopyranoside 1119 with propiolic acid under DIC-
promotion to give the 3-O-propiolate 12 as an alkyne source
N
STol
O
O
OH
3e
for the synthesis of indolizine-derivatized galactosides 2a
-2e
Scheme 3 Synthesis of triazole and coumarin derivatised thiogalactosides 3a-3e.
(Scheme 2). Pyridinium salts 10d-10e were prepared by
2 | J. Name., 2012, 00, 1-3
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Coumarins can be easily equipped with an alkyne functionality, particularly efficient inhibition of galectin-8N with down to
which would open up for use in cycloaddition reactions with ≈100 µM affinities reflecting
a
near 60-fold affinity
the 3-azido galactoside 16. The unprotected 3-azido derivative enhancement over the reference
8
. In general, 1a-1k displayed
16 was synthesized via Zemplén de-O-acetylation20 of the somewhat higher affinity for galectin-8N than the reference
8.
known p-tolyl 2,4,6-tri-O-acetyl-3-azido-3-deoxy-1-thio-
galactopyranoside 1521 (Scheme 3). Copper-(I)-catalysed slightly decreased the affinity for galectin-8N when compared
cycloadditions22-24 with 6-alkoxy coumarins 14a
14d and 7- to the unsubstituted 1a. In contrast, the two carboxylic acid
β-D- Substituents on the quinoline (1b-1i) either had no effects or
-
alkoxycoumarin 14e gave the triazoles 3a
-
3e in 59-85% yields.
substituted quinolines 1j and 1k proved to show promising
affinities for galectin-8N with 1j and 1k having dissociation
constants of 250 and 110 µM, respectively. Hence, moving the
2.2 Galectin binding affinities
The quinoline- (1a-1i), indolizine- (2a-2e), and coumarin- (3a-
carboxylate from the quinoline position 6 (1j) to position 7 (1k
)
3e) derived galactosides were evaluated as inhibitors against
the human galectin-1, -2, -3. -4N- and C-terminal domains, -7,
8N- and C- terminal domains, and -9N- and C- terminal
domains in a competitive protein-binding assay based on
doubled the affinity, suggesting the presence of a specific
interaction formed by the 7-carboxylate. Compound 1k is the
best β-D-galactopyranoside-based monosaccharide inhibitor
for human galectin-8N reported hitherto and better than the
fluorescence anisotropy.25-27 Methyl
was included as a reference27-28 (Table 1). Overall, the
indolizines 2a 2e display better inhibition potency for
galectin-3 and two-five-fold selectivity over the other galectins
evaluated. Compounds 2a 2b and 2e showed similar
inhibition with dissociation constants ~300 µM, thus resulting
in 53-fold affinity enhancement over the reference . The
β-D-galactopyranoside 8
methyl glycoside of corresponding natural disaccharide
fragment sialyl-
al.31 have reported coumarin
α
-(2-3)-galactoside 1729-30 (Fig. 1). Rajput et
(
-
)
α
-D-galactopyranoside-based
derivatives with affinities down to ≈200 µM for galectin-8N,
but these compounds differed from 1k in that they were even
better inhibitors of galectin-3 and thus displayed high galectin-
3 selectivity. In order to investigate the role of the quinoline
nitrogen in interactions enhancing the affinities for galectin-
8N, we synthesized the corresponding naphthalene derivative
,
,
8
coumarin-functionalized galactose derivatives 3a-3e bound the
tested galectins with apparently no selectivity and moderate
affinity enhancements.
18 via stannylidene acetal-mediated alkylation of
8 in the same
The quinoline-derivatized methyl β-D-galactosides 1a-1k
revealed a more varying structure-activity relationship and
manner as described for 1a-1i. The naphthalene-derivatized
Table 1 Kd-values (µM)a of binding compounds 1a-1k, 2a-2e, 3a-3e, 8 and 17-18 against human galectin-1, 2, 3, 4N, 4C, 7, 8N, 8C, 9N, and 9C as measured by a fluorescence
polarization assay.
Galectin
Compounds
1
2
3
4Nb
4Cc
7
NBd
8Nb
700±41
700±50
440±46
520±38
630±9
8Cc
2900±38
3100±340
2800±21
4600±760
3100±670
2700±14
3500±440
NBd
9Nb
470±71
510±88
550±110
1300±270
550±53
580±86
1100±10
750±7
390±29
1500±100
300±16
NBd
9Cc
2200±110
NBd
1a
1b
1c
1700±100
1300±130
1900±70
1200±20
1700±20
1200±15
NBd
2600±340
1900±240
2200±100
2000±40
1600±40
1900±600
2000±890
2700±61
1200±19
1700±260
2800±370
1500±140
1200±80
1300±4
620±50
580±22
510±83
710±170
610±2
1000±38
1700±52
1600±280
2300±210
1600±130
1400±50
2100±510
880±160
470±80
NBd
2700±280
2100±520
4000±70
4600±230
NBd
NBd
NBd
2400±770
NBd
1d
1e
1f
NBd
NBd
1900±340
NBd
410±53
820±33
NAd
2800±10
NBd
NBd
1400±60
1000±25
3500±650
640±80
250±10
110±6
1g
1h
1i
NBd
1300±13
NBd
1300±90
1400±130
690±8
2300±280
1700±270
3800±480
2600±270
1700±470
2200±250
3600±1230
870±80
NBd
450±84
250±11
380±11
390±34
360±42
740±29
960±182
300±29
490±67
1370±120
260±41
830±49
480±44
4400
NBd
2400±590
3100±41
3200±1210
NBd
1700±78
930±8
1j
NBd
1k
2a
2b
2c
880±25
NBd
NBd
NBd
3900±150
1800±130
1700±76
2600±54
NBd
1400±370
1200±10
1100±120
830±85
1600±260
650±120
710±150
1300±80
1300±270
840±83
6600
NBd
1000±94
840±22
NBd
1600±220
1100±74
NBd
NBd
3200±165
NBd
NBd
NBd
NBd
2d
2e
3a
3b
3c
500±30
2270±230
>1370
NBd
1300±76
740±39
540±60
480±4
1700±56
1600±76
990±2
NBd
480±34
500±8
750±54
1700±80
NAe
NAe
NBd
1400±47
1100±130
NBd
1600±130
2600±100
NBd
NBd
NBd
640±32
1100±370
NBd
820±100
150±26
500±8
1300±26
890±50
NBd
1000±94
260±84
980±130
950±28
>30000
NAe
2560±560
NBd
NBd
3d
3e
827-28
17
18
620±120
720±76
2000±240
610±110
10000
520±26
360±9
NBd
460±18
>10000
670±125
>4000
NBd
510±13
8600±730
NAe
13000
NAe
4800
NAe
NAe
6300
3300
NAe
NAe
NAe
NAe
150±12
>4000
>6000
730±80
3100±500
2000±40
>4000
1700±110
>3500
c
d
e
a The data are average and standard deviation of 4–8 single-double point measurements. b N-terminal domain. C-terminal domain. Non binding, Not
available.
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respectively). This observation corroborates the recent finding
that -halo-substituted -thiophenyl aglycons greatly
enhances the affinities of galactosides towards galectins.32
However, even larger influence of the -thiophenyl aglycon
m
α
OH
HO
HO2C
O
OH
O
HO
OMe
O
α
HN
OH
HO
was observed for galectin-3 and -9N, as 23a and 23b bound
OH
17
Ac
these two galectins similarly as galectin-8N. Hence, although
the
for galectin-8N, the selectivities observed for 1j and 1k are lost
due to the even larger influence of the -thiophenyl aglycons
α-thiophenyl aglycon as hypothesized increases the affinity
HO
OH
O
OMe
α
O
OH
on galectin-3 and -9N binding. Nevertheless, compound 23b is
clearly the best synthetic monosaccharide-based galetin-8N
inhibitor reported to date.
18
Fig. 1 Reference compound methyl sialyl-
methyl 3-O-naphth-2-yl- -D-metylgalactoside 18.
α
-(2-3)-galactopyranoside 1729-30 and
1. PCl5
BF3.Et2O
β
AcO
AcO
RO
RO
OAc
O
OR
O
2. 3,4ꢀDichlorothiophenol
analog 18 was proved to be more than 36 times worse than 1k
as a galectin-8N inhibitor, which indicates an important role of
the quinoline nitrogen in binding to galectin-8N.
Cl
NaH
AcO
RO
OAc
S
Dry DMF
19
50 o
C
Cl
68%
NaOMe
MeOH
94%
2.3 Affinity Enhancement through combination of 1j and 1k
20 R=COCH3
21 R=H
quinolines with an α-thiophenyl aglycon fragment
R1
R2
HO
In an attempt to further enhance the affinity of the quinoline-
OH
1. Bu2SnO
MeOH
O
derived ligands 1j and 1k, we hypothesised that combining the
O
carboxy-quinolines with α-thiophenyl aglycons, recently
reported to greatly enhance galectin-3 affinities,32 could be a
N
Cl
Reflux
HO
S
2. 5d or 5e
Bu4NBr
viable strategy. Per-acetylated galactose 19 was converted to
Cl
Dry Dioxane
the 3,4-dichlorophenyl
α-thiogalactoside 20, followed by
22a R1=CO2CH3, R2=H
23a R1=CO2H, R2=H
22b R1=H, R2=CO2CH3
23b R1=H, R2=CO2H
KOH
85 o
C
69%
65%
EtOH
H2O
deacetylation and stannylidene-mediated regioselective 3-
O
-
70ꢀ73%
quinolylmethylation to give 22a and 22b. Alkaline hydrolysis of
22a and 22b gave the corresponding carboxy-quinoline
thiogalactosides 23a and 23b (Scheme 4).
60 o
C
Scheme 4 Synthesis of quinoline-derivatized α-thiogalactosides 23a and 23b.
Compounds 23a and 23b showed greatly increased affinity for
galectin-8N as compared to 1j and 1k (128-fold and 73-fold,
Table 2 Kd-values (µM)a of binding compounds 23a and 23b against human galectin-1, 2, 3, 4N, 4C, 7, 8N, 8C, 9N, and 9C as
measured by a fluorescence polarization assay. The high affinities for galectin-3 and 8N are in bold face.
Galectin
4Cc
Compounds
1
2
3
4Nb
7
8Nb
8Cc
9Nb
9Cc
23a
23b
100±5
48±4.4
110±12
59±4
1.2±0.02
1.27±0.07
110±14
43±7.1
43±8
32±5
NAd
1.9±0.1
1.5±0.08
330±48
240±15
8.8±0.5
2.06±0.09
27±7
43±5.7
14±1.3
a The data are average and standard deviation of 5-12 single to double point measurements. b N-terminal domain. c C-terminal domain. d Not available.
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quinoline-, indolizine-, and coumarin-derived galectin
inhibitors 1a-1k 2a-2e 3a-3e 23a, and 23b were investigated
,
,
,
in JIMT-1 and MCF-7 human breast cancer cell lines, as well as
in the human normal-like MCF-10A cell line using an MTT
assay.33-35 The MTT assay is a colorimetric assay that is based
on the reduction of the water-soluble tetrazolium salt MTT to
an insoluble purple formazan in the mitochondria of viable
cells. Thus, the MTT reduction is used to reflect the viable cell
number. None of the inhibitors affect the cell viability at the
concentrations ranging from 0.05 µM to 50 µM in either of the
three cell lines except 3d, which gave the IC50 values 33, 39,
and 27 µM in JIMT-1, MCF-7, and MCF-10A cells, respectively
(Figs S103-108). The results indicate a low cytotoxicity of the
inhibitors.
Fig 2. Molecular dynamics simulation snapshot of
conformation of 1k in complex with galectin-8N.
a representative low-energy
Conclusions
2.4 Molecular modelling
In conclusion, we have developed simple and efficient,
economically viable methods to synthesize quinoline,
In order to gain understanding of the binding of the quinoline
derivatives for galectin-8N, molecular dynamics simulations
were performed. Low energy conformations of 1k in pbd id
3VKO, with the galactose unit placed in the galectin core
galactose binding site in the same pose as natural galactose-
containing ligands, were generated by rotating the three
bonds between the galactose C-3 atom and the quinoline,
followed by energy minimization with the OPLS3 force
field and the GB/SA solvation method for water. Initial MD
simulations were performed with these low energy structures
(see Supplementary figure S109), which all drifted towards the
same general structure with the quinoline group oriented in
the galactose C3-O3 direction close to Arg45. Subsequently, a
1000 ns molecular dynamics simulation was performed
starting from a ligand pose close to Arg45 and this converged
after 300 ns to a protein-ligand 1k geometry where Gly142
was buried in the protein with hydrogen bonds from Arg59,
indolizine and coumarin derivatised galactosides. 3-O-
(Carboxyquinoline)-derivatized methyl galactosides displayed
particularly good affinity and selectivity against galectin-8N,
while the 3-
moderate affinity enhancements over the reference methyl
D-galactopyranoside. Combining the 3- -carboxyquinoline
moiety with an -3,4-dichlorophenylthio aglycon greatly
O-indolizinyl and coumaryl derivatives showed
β
-
O
α
enhanced affinity for galectin-8N down to 1.5 µM, but also
reduced selectivity over galectin-3 and -9N. Molecular
dynamics simulation of 1k in complex with galectin-8N
suggested that an ideal fit of the quinoline with a subsite near
the galactose site, that allowed Gly142 to be buried in the
protein and directly stabilized through water-mediated ligand
carboxylate-protein hydrogen bonds. Furthermore, a water-
mediated quinoline nitrogen hydrogen bond to galactose HO2
provided optimal steric and electronic complementarity
between 1k and galectin-8N, which may explain the selectivity-
induction and affinity-enhancement of the quinoline moiety.
Finally, the compounds show low cytotoxicity in both tumor
Asp49, His65, Gln47 and
a buried water molecule and
simultaneously Arg59 had both hydrophobic planar sides
removed from solvent (Fig. 2). Furthermore, this interaction
mode led to an altered conformation of the side chain of
Arg59 (the guanidinium group moved about 2.5Å) compared
to the apo structure (2YV8) or corresponding lactose
complexes (2YXS, 3AP4, 3VKL, and 3VKM). The 1k quinoline
moiety was positioned in a subsite adjacent to O3 of the
and normal cell lines, why we argue that the discovery of 3-O-
carboxyquinoline-derivatised galactosides may constitute a
new class of compounds for the discovery of selective galectin-
8 inhibitors. Furthermore, the inhibitory activities of the
quinolines against one of the domains of galectin-8, N-
terminal, is probably enough to have a pharmacological
activity against galectin-8, it has been shown earlier that
inhibition of one of the galectin-8 domains can block the
function of galectin-8.36 This is particularly important in light of
the reported key roles of galectin-8 in autophagy,37
lymphangiogenesis,38 and cancer.39
bound galactopyranose with
a replacement of poorly
coordinated water molecules, while at the same time
positioning the 7-carboxylate for multiple water-mediated
hydrogen bonds with Arg45, Gln47, and Gly142 that would be
less favorable for the corresponding 6-carboxylate 1j
.
Furthermore, in a majority of sampled complex conformations
the quinoline nitrogen formed a water-mediated hydrogen
bond to the galactose HO2, which led to a conformation with
simultaneous ideal steric fit of both the quinoline and
3. Experimental
galactose moieties of 1k
.
3.1 General
2.5 Cytotoxicity evaluation
All reactions were carried out in oven-dried glassware. All
solvents and reagents purchased from commercial sources or
synthesized via literature protocols and used without further
In addition to high galectin-8 affinity and selectivity, a criterion
for compounds to be valuable as inhibitors in biological
experiments is lack of or low toxicity. The cytotoxicity of all the
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purifications. TLC analysis was performed on pre-coated Merck cooled to 0 °C and the solid was collected by filtration and
silica gel 60 F254 plates using UV light and charring solution (10 washed with cold THF (3×15 mL). The solid was stirred in
mL conc. H2SO4/ 90 mL EtOH). Flash column chromatography distilled water (80 mL), the resulting solution made basic with
was done on SiO2 purchased from Aldrich (technical grade, 60 K2CO3 and extracted with EtOAc (3×40 mL). The organic layers
Å pore size, 230-400 mesh, 40-63 μm). Preparative HPLC was were combined, dried over Na2SO4, filtered, and concentrated
performed on an Agilent 1260 infinity system, column to give 2-methyl-6,7-difluoroquinoline 6f as black solid in 64%
SymmetryPrep-C18, and a 17 mL/min H2O-MeCN gradient 10- yield (1.15 g, 6.4 mmol) without any further purification. 1H
100% 15 min with 0.1% formic acid. All NMR spectra were NMR (CDCl3, 400 MHz): 7.96 (d, 1H,
recorded with Bruker DRX 400 MHz spectrometer (400 MHz 1H, 7.6 Hz, 11.6 Hz, Ar ), 7.57 (dd, 1H,
for 1H, 100 MHz for 13C, 376 MHz for 19F) at ambient Ar ), 2.71 (s, 3H, C10H7NF2CH3). 13C
), 7.27 (d, 1H, 8.4 Hz, Ar
temperature using CDCl3, CD3OD or (CD3)2SO as solvents. NMR (CDCl3, 100 MHz): 159.6 (d, 2.8 Hz), 153.6 (d, 15.8 Hz),
Chemical shifts are given in ppm relative to the residual 151.1 (dd, 14.0 Hz, 15.8 Hz), 148.5 (d, 15.7 Hz), 145.0 (d,
solvent peak (1H NMR: CDCl3 δ 7.26; CD3OD δ 3.31; (CD3)2SO δ 9.7 Hz), 135.5 (d,
3.3 Hz), 123.3 (d,
2.50; 13C NMR: CDCl3 δ 77.16; CD3OD δ 49.00; (CD3)2SO δ Hz), 115.1 (d,
16.1 Hz), 112.8 (dd,
J
8.4 Hz, Ar
H), 7.74 (dd,
J
J
H
J
8.8 Hz,
J
10.4 Hz,
H
J
H
J
J
J
J
J
J
J
J
8.4 Hz), 122.3 (d,
J
2.5
J
J
1.4 Hz,
J
17.4 Hz), 25.3 (s,
20.8
20.8 Hz). HRMS calcd. for C10H7F2N+H+ (M+H)+:
multiplet, app = apparent), coupling constants (in Hz) and 180.0625, found: 180.0623.
39.52) with multiplicity (b = broad, s = singlet, d = doublet, t = 3H, C10H7NF2CH3). 19F NMR (CDCl3, 376 MHz): -131.9 (d,
J
triplet, q = quartet, quin = quintet, hept = heptet, m = Hz), -137.0 (d,
J
integration. High-resolution mass analyses were obtained 3.4. General Method for the Preparation of Substituted-2-
using Micromass Q-TOF mass spectrometer (ESI). Purities of (Bromomethyl)benzoquinolines 7d-7i
final compounds were determined by UPLC (Waters Acquity
A mixture of substituted 2-methylquinolines 7d-7i (1.0 eq),
UPLC system, column Waters Acquity CSH C18, 0.5 mL/min
H2O-MeCN gradient 5-95% 10 min with 0.1% formic acid).
Analytical data is given if the compound is novel or not fully
characterized in the literature.
NBS (1 eq) and AIBN (0.2 eq) in CCl4 (5 mL for 1 mmol of 7d
was stirred at 80 °C for 6 h. H2O (20 mL for 1 mmol of 7d
-
-
7i
)
)
7i
and DCM (20 mL for 1 mmol of 7d-7i) were added to the
mixture and the layers were separated. The aqueous layer was
extracted with DCM (20 mL×2). The combined organic layers
3.2. Methyl 2-methylquinoline-7-carboxylate 6e
To 3-aminobenzoic acid
was added and the mixture was refluxed for
Crotonaldehyde (1.8 mL, 21.9 mmol) was added dropwise over chromatography
4
(2.5 g, 18.24 mmol), 6N HCl (37 mL) were washed with brine and dried over anhydrous Na2SO4. The
h. organic layers were concentrated and purified by column
to afford substituted 2-
See Supplementary
2
45 min. After 4 h, the reaction mixture was cooled to 0°C and (bromomethyl)benzoquinolines 7d-7i.
the pH adjusted to 3-5 with aqueous ammonia solution. The information for physical data.
solid suspended in the aqueous layer, was dissolved by adding 3.5. General Method for the Preparation of Quinoline Derived
DCM. The organic layer was collected, dried over Na2SO4, Galactosides 1a-1i and 23a-23b
filtered and dried under vacuum. Recrystallization from EtOH
Methyl
β-D-galactopyranoside, 8 (1eq) was dissolved in dry
and washing (EtOH, 7×50 mL) afforded 2-methylquinoline-7-
carboxylic acid in 46% yield (1.56 g, 8.34 mmol) as white solid,
which was dissolved in methanol (15 mL). Sulfuric acid (1.5 mL)
was added dropwise at 0°C and then stirred at 65°C for 12 h.
The reaction mixture was concentrated and to the residue
dichloromethane and aqueous sodium carbonate solutions
were added. The organic layer was collected, dried with
Na2SO4 and concentrated to afford 6e (1.0 g, 4.97 mmol,
59.6%) as a white solid. 1H NMR (CDCl3, 400 MHz): 9.09 (d, 1H,
MeOH (1 mL per 15 mg) and Bu2SnO (1.1 eq) was added to the
solution. The mixture was stirred under reflux condition for 3
hours under N2 atmosphere. The reaction mixture became
transparent after 1 h. After 3 h, the solvent was evaporated
under reduced pressure and also the crude was co-evaporated
with toluene to remove any remaining MeOH. The residue was
dried under vacuum to give the intermediate as an amorphous
white solid. The dry crude was dissolved in 1,4-dioxane and
Bu4NBr and corresponding bromide 7a-7i (1.5 eq.) were
J
J
8.8 Hz, Ar
H
), 8.09 (dd, 2H,
), 7.26 (d, 1H,
J
2.8 Hz,
J
6.8 Hz, Ar
H), 7.57 (t, 1H,
added. The reaction was stirred under N2 atmosphere for
overnight at 85 °C. TLC showed no starting material remained.
The solvent was removed in vacuo and the crude material was
purified by flash chromatography to give quinolyl galactosides
as a white amorphous solid. Compounds were further purified
with preparative HPLC prior to galectin binding and cell assays.
All tested compounds were >95% pure according to analytical
HPLC analysis. See Supplementary information for physical
8.0 Hz, Ar
H
J
8.8 Hz, ArH
), 3.98 (s, 3H, CO2CH3),
2.64 (s, 3H, C11H8NO2CH3). 13C NMR (CDCl3, 100 MHz): 166.9
(CO2CH3), 159.2, 147.9, 134.2, 134.1, 129.8, 127.8, 126.4,
125.2, 123.4, (CH2Ar), 52.1 (CO2CH3), 34.0 (C11H8NO2CH3).
HRMS calcd. for C12H11NO2+H+ (M+H)+: 202.0868, found:
202.0867.
3.3. 2-Methyl-6,7-difluoroquinoline 6f
To a refluxing solution of 3,4-difluoroaniline 5 (1 mL, 10 mmol), data.
tetrachloro-1,4-benzoquinone (2.5 g, 10 mmol), and 3.6. General Method for the hydrolysis of methyl
concentrated hydrochloric acid (2.6 mL) in 2-butanol (20 mL) carboxylates 1d, 1e, 22a, and 22b
was added crotonaldehyde (1 mL, 12 mmol). After 2.5 h, the
To a suspension of methyl carboxylates (1 eq.) in EtOH-H2O
reaction mixture was concentrated and the resulting residue
(3:1, 1 mL per 10 mg of sugar derivative), KOH (2 eq.) was
was stirred in warm (50 °C) THF (15 mL). The mixture was
added and it was stirred at 60 °C for 6 hours. Volatiles were
6 | J. Name., 2012, 00, 1-3
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evaporated under reduced pressure and the crude material crude), without any further purification. The crude was
was purified by preparative HPLC to obtain pure carboxylic dissolved in EtOAc (3 mL for 0.1 mmol of crude) and
acids 1j, 1k, 23a, and 23b as sole isolated product at a yield isopropanol (9 mL per 0.1 mmol. of crude) and the solution
66%, 63%, 69% and 65% respectively. All tested compounds was stirred under hydrogen atmosphere at room temperature
were >95% pure according to analytical HPLC analysis. See for 12 h. After the completion of reaction (as indicated by TLC),
Supplementary information for physical data.
3.7. General Method for the Synthesis of Pyridinium Salts
10d-10e
the reaction mixture was filtered through a Celite bed and
washed with methanol. The filtrate was concentrated under
reduced pressure and purified through flash column to get the
desired compound 2a-2e at a yield 25-34% over two steps.
The pyridine derivatives 9d-9e (1 eq.) and the alkylating
Finally, the compounds were purified by preparative HPLC
before galectin binding and cell assays. All tested compounds
were >95% pure according to analytical HPLC analysis. See
Supplementary information for physical data.
reagent (1.5 eq.) were dissolved in dry acetone (2 mL for 1
mmol of pyridine derivative). The reaction mixture was stirred
in an ultra-sound bath for 12 h. The temperature of the bath
was kept under 50 °C by adding ice occasionally. Et2O (5 mL for
1 mmol of pyridine derivative) was added and the quaternary
3.10. General Method for the Preparation of Substituted
Propargylated Coumarins 14a-14e
salt 10d-10e precipitated, was filtered off, and washed with
Et2O. See Supplementary information for physical data.
To a solution of hydroxyl coumarin, 13a-13e (1 eq) in dry
acetone (20 mL per 1 mmol of hydroxyl coumarin) were added
K2CO3 (2 eq) and propargyl bromide (80 wt% in toluene) (1.5
eq.). The resulting mixture was stirred at 60 °C for overnight.
The mixture was cooled and the solvent was removed under
reduced pressure. The residue was treated with of water (10
mL per 1 mmol. of hydroxyl coumarin) and extracted with
ethyl acetate. The combined organic phase was washed with
water, dried over Na2SO4 and evaporated in vacuum, and the
residue was purified through flash column chromatography to
3.8. Methyl 2,4,6-tri-O-benzyl-3-O-propynoyl)-
β-D-
galactopyranoside 12
Compound 11 (4.0 g, 8.62 mmol) was dissolved in 20 mL of dry
THF. Propiolic acid (0.8 mL, 12.9 mmol), and DMAP (5 mg,
0.043 mmol) were added and the temperature lowered to 0
°C. DIC (2 mL, 12.925 mmol) was slowly added to the cold
reaction mixture. The mixture was slowly allowed to warm to
room temperature and stirred overnight. The reaction mixture
was filtered through a fritted funnel to remove the urea
precipitate. The THF mixture was exposed to reduced pressure
to remove the solvent and excess propiolic acid and DIC.
Compound 12 was purified by flash chromatography
(heptane/EtOAc, 8:1 to 4:1) at a yield of 67% (2.98 g, 5.77
afford 14a-14e. See Supplementary information for physical
data.
3.11. p-Tolyl 3-azido-3-deoxy-1-thio-
β
-D-galactopyranoside
16
p
-Tolyl
2,4,6-tri-O-acetyl-3-azido-3-deoxy-1-thio-β-D-
mmol) containing small amounts of DIC and DIU impurities. galactopyranoside, 15 (700 mg, 1.6 mmol) was dissolved in
25 +12.4 ( 1.4, CHCl3). 1H NMR (CDCl3, 400 MHz): 7.36-7.26 MeOH (7.5 mL). NaOMe (2 mL, 0.5 M in MeOH) was added and
), 5.02 (dd, 1H, J2,3 10.0 Hz, J3,4 2.8 Hz, H-3), 4.85 the solution was stirred at room temperature for overnight.
11.2 Hz, CH2Ph), 4.71 (d, 1H,
11.2 Hz, CH2Ph), 4.66 (d, The solution was neutralized with DOWEX 50 W H+ resin,
[α
]
D
c
(m, 15H, Ar
(d, 1H,
H
J
J
1H,
1H,
J
J
11.2 Hz, CH2Ph), 4.50 (d, 1H,
11.6 Hz, CH2Ph), 4.43 (d, 1H,
J
J
11.2 Hz, CH2Ph), 4.49 (d, filtered and the solvents were evaporated under reduced
11.6 Hz, CH2Ph), 4.33 (d, pressure and the crude, 16 was used without any further
1H, J1,2 7.6 Hz, H-1), 3.98 (d, 1H, J3,4 2.8 Hz, H-4), 3.79 (dd, 1H, purification. The crude product was obtained as a white
25
1
J1,2 7.6 Hz, J2,3 10.0 Hz, H-2), 3.67 (m, 1H, H-5), 3.64-3.60 (m, amorphous solid (458 mg, 92%). [
2H, H-6a, H-6b), 3.55 (s, 3H, OCH3), 2.99 (s, 1H, COCC
). 13C NMR (CD3OD, 400 MHz): 7.44 (dd, 2H,
NMR (CD3OD, 100 MHz): 152.2 ( OCCH), 138.3, 137.81, 7.10 (d, 1H, 7.6 Hz, Ar ), 4.58 (d, 1H, J1,2 9.6 Hz, H-1), 3.95 (d,
137.79, 128.5, 128.4, 128.0, 127.9, 127.8, 127.7 (Ar ), 104.8 1H, J3,4 2.8 Hz, H-4), 3.80 (t, 1H, J1,2 J2,3 9.6 Hz, H-2), 3.74 (dd,
(C-1), 76.9, 76.7, 76.0, 75.0, 74.8, 73.7, 73.5, 73.0, 57.1 (O H3). 1H, J5,6a 6.8 Hz, J6a,6b 11.2 Hz, H-6a), 3.68 (dd, 1H, J5,6b 5.2 Hz,
HRMS calcd. for C31H32O7+NH4 (M+NH4)+: 534.2492, found: J6a,6b 11.6 Hz, H-6b), 3.55 (m, 1H, H-5), 3.36 (dd, 1H, J2,3 9.6 Hz,
534.2496
J3,4 2.8 Hz, H-3), 2.28 (s, 3H, SC6H4CH3). 13C NMR (CD3OD, 100
3.9. General Method for the Preparation of Indolizine Derived MHz): 138.4, 132.8, 131.5, 130.5 (Ar ), 90.9 (C-1), 80.6, 69.3,
α
]
D
+8.9 (
c
1.6, CH3OH). H
H
J
1.6 Hz,
J
6.4 Hz, Ar ),
H
C
J
H
C
,
C
+
C
Galactosides 2a-2e
69.2, 68.3, 62.3, 21.1(SC6H4CH3). HRMS calcd. for C13H17N3O4S-
H+ (M-H)+: 310.0862, found: 310.0858.
The reaction was performed with 1 eq. of the quaternary salt
3.12. General Method for the Preparation of Coumarin
Derived Thiogalactosides 3a-3e
(
10a
-10e), 1 eq. of the propiolate galactoside derivative, 12
and 1 eq. of K2CO3 in methanol (4 mL for 0.1 m.mol. of 12). The
reaction was stirred for 4 h at room temperature under air A solution of the azide 16 (1 eq.) in dichloromethane (1 mL for
atmosphere. After that reaction mixture was quenched with 10 mg of sugar azide), propargylated coumarin 14a 14e (1.5
-
water and collected in EtOAc. The EtOAc layer was washed eq.), CuI (10 mol% with respect to sugar azide) and DIPEA (1.5
successively with brine solution and water. Organic layer was eq) were added and the mixture was stirred at room
collected, dried over Na2SO4, and evaporated under reduced temperature for 24 h, until TLC showed no trace of the starting
pressure. The crude material was obtained, subjected for sugar azide. The solvent was removed under reduced pressure,
hydrogenolysis with Pd(OH)2-C (10% wt., 1 mg for 4 mg of and the residue was dissolved in EtOAc and the solution was
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washed with brine, dried over Na2SO4 and concentrated in 1H, J2,3 10.8 Hz, J3,4 3.2 Hz, H-3), 4.58 (m, 1H, H-5), 4.02 (dd, 1H,
vacuo The product was purified by flash column J5,6a 5.6 Hz, J6a,6b 11.6 Hz, H-6a), 3.98 (dd, 1H, J5,6b 7.2 Hz, J6a,6b
chromatography give 11.6 Hz, H-6b), 2.06 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 1.92 (s,
.
(DCM/MeOH,
25:1-10:1)
to
corresponding triazoles as white amorphous solid. The 3H, COCH3), 1.89 (s, 3H, COCH3). 13C NMR (CDCl3, 100 MHz):
compounds were further purified with preprarative HPLC prior 170.1, 169.84, 169.79, 169.6, 133.1, 132.7, 131.9, 131.0, 130.5
to galectin binding and cell assays. All tested compounds were (Ar
C
), 85.2 (C-1), 67.8, 67.7, 67.4, 61.8, 20.6 (CO
H3), 20.39 (CO H3). HRMS calcd. for C20H22Cl2O9S+NH4
(M+NH4)+: 526.0705, found: 526.0705.
3.15. 3,4-Dichlorophenyl 1-thio- -D-galactopyranoside 21
CH3), 20.40 (2×
+
>95% pure according to analytical HPLC analysis. See
Supplementary information for physical data.
C
OC
C
3.13. Methyl 3-O-(napth-2-ylmethyl)-
β
-D-galactopyranoside
β
17
Compound 20 (1.2 g, 2.36 mmol) thus obtained was dissolved
in MeOH (15 mL). NaOMe (5 mL, 0.5 M in MeOH) was added
and the solution was stirred at room temperature overnight.
The solution was neutralized with DOWEX 50 W H+ resin,
filtered and the solvents were evaporated under reduced
pressure and the crude was used without any further
purification. Compound 21 was obtained as a colourless oil
The reaction was performed with
8 (103 mg, 0.53 mmol) and
2-bromomethylnapthalene (176 mg, 0.795 mmol), and 2-
bromomethyl naphthalene (176 mg, 0.795 mmol) following
general method 4.5. Methyl 3-O-(napthalen-2-ylmethylene)-β-
D-galactopyranoside, 17 was obtained in 88% yield (166 mg,
25
1
0.467 mmol) as a colourless oil. [
NMR (CD3OD, 400 MHz): 8.33 (s, 1H,
(m, 3H, 8.4 Hz, Ar ), 7.58 (dd, 1H,
7.48-7.42 (m, 2H, Ar ), 4.91 (d, 1H,
(d, 1H, 12.0 Hz, CH2C10H7), 4.15 (d, 1H, J1,2 7.6 Hz, H-1), 4.07
α
]
+42.7 (
8.4 Hz, Ar
1.6 Hz, 8.4 Hz, Ar
J 12.0 Hz, CH2C10H7), 4.81
c
1.2, CH3OH). H
), 7.85-7.80
),
D
(755 mg, 94%). [
MHz): 7.70 (d, 1H,
Hz, Ar ), 7.40 (d, 1H,
α] c
25 +3.4 (
1.3, CH3OH). 1H NMR (CD3OD, 400
2.0 Hz, Ar ), 7.45 (dd, 1H, 2.0 Hz, 8.4
8.4 Hz, Ar ), 5.66 (d, 1H, J1,2 5.6 Hz, H-
J
H
D
J
H
J
J
H
J
H
J
J
H
H
J
H
J
1), 4.27 (m, 1H, H-5), 4.19 (dd, 1H, J1,2 5.6 Hz, J2,3 10.4 Hz, H-2),
3.80 (dd, 1H, J3,4 3.2 Hz, J4,5 1.2 Hz, H-4), 3.74 (dd, 1H, J5,6a 5.2
Hz, J6a,6b 11.2 Hz, H-6a), 3.69 (dd, 1H, J5,6b 6.8 Hz, J6a,6b 11.2 Hz,
H-6b), 3.65 (dd, 1H, J2,3 10.4 Hz, J3,4 3.2 Hz, H-3). 13C NMR
(CD3OD, 100 MHz): 137.0, 134.2, 133.3, 132.5, 131.9, 131.5
(dd, 1H, J3,4 3.2 Hz, J4,5 0.4 Hz, H-4), 3.80-3.68 (m, 3H, H-2, H-
6a, H-6b), 3.53 (s, 3H, OCH3), 3.44 (m, 1H, H-5), 3.41 (dd, 1H,
J2,3 9.6 Hz, J3,4 3.2 Hz, H-3). 13C NMR (CD3OD, 100 MHz): 137.3,
134.7, 134.5, 129.0, 128.9, 128.6, 127.6, 127.1, 127.0, 126.9
(Ar
(O
C
), 105.9 (C-1), 82.4, 76.5, 72.6, 71.8, 67.1, 62.4, 57.2
(ArC), 91.1 (C-1), 73.5, 72.1, 70.6, 69.7, 62.3. HRMS calcd. for
C12H14Cl2O5S+Na+ (M+Na)+: 362.9837, found: 362.9835.
C
H3). HRMS calcd. for C18H22O6+H+ (M+H)+: 335.1495, found:
335.1495.
3.16. 3,4-Dichlorophenyl 3-O-(6-methoxycarbonyl-quinolin-2-
3.14. 3,4-Dichlorophenyl 2,3,4,6-tetra-O-acetyl-1-thio-
galactopyranoside 20
α-D-
yl-methyl)-1-thio-
The reaction was performed with 21 (64 mg, 0.19 mmol)
following the general method 4.5 Compound 22a was
obtained in 73% yield (74 mg, 0.14 mmol) as a white
α-D-galactopyranoside 22a
To a stirred suspension of 19 (2.0 g, 5.13 mmol) and PCl5 (1.17
g, 5.64 mmol) in dry DCM (20 mL), BF3.Et2O (32 µL, 0.26 mmol)
was added. After stirring for 30 min TLC analysis
(heptane:EtOAc, 1:1) showed complete consumption of the
starting material. The reaction mixture was diluted with DCM
(100 mL) and then washed with ice-cold water (50 mL),
saturated ice-cold NaHCO3 solution (2×50 mL), and again ice-
cold water (2x30 mL). The organic layer was dried over
anhydrous Na2SO4 and evaporated under reduced pressure.
The residue was co-evaporated with toluene colorless oil,
which solidified slowly and was used without further
purification. NaH (221 mg, 9.23 mmol) was added in dry DMF
(15 mL) in a separate flask. 3,4-Dichlorobenzenethiol (1.83 g,
10.25 mmol) was added in the reaction mixture and the
mixture was stirred at room temperature for 30 min. Then the
.
25
amorphous solid. [
((CD3)2SO, 400 MHz): 8.71 (d, 1H,
8.8 Hz, Ar ), 8.22 (dd, 1H, 2.0 Hz,
8.8 Hz, Ar ), 7.92 (d, 1H, 8.4 Hz, Ar
), 7.53 (d, 1H, 8.4 Hz, Ar ), 7.44 (dd, 1H,
), 5.91 (d, 1H,
α
]
+58.8 (c
0.7, CH3OH). 1H NMR
D
J
2.0 Hz, Ar
8.8 Hz, Ar
), 7.74 (d, 1H,
2.0 Hz,
H
), 8.61 (d, 1H,
J
J
H
J
J
H), 8.05 (d, 1H,
H
J
H
J
2.0 Hz,
8.4 Hz,
Ar
H
H
J
H
J
J
Ar
J
4.4 Hz, OH), 5.76 (d, 1H, J1,2 5.2 Hz, H-1),
6.0 Hz, OH), 5.01 (d, 1H, 14.8 Hz, CH2C11H8NO2),
14.8 Hz, CH2C11H8NO2), 4.64 (t, 1H, 5.6 Hz, OH),
5.06 (d, 1H,
4.92 (d, 1H,
J
J
J
J
4.30 (m, 1H, H-2), 4.15 (t, 1H, J4,OH 4.4 Hz, H-4), 4.00 (t, 1H,
J5,6a J5,6b 6.4 Hz, H-5), 3.93 (s, 3H, CO2CH3), 3.59 (m, 1H, H-6a) ,
,
3.52 (dd, 1H, J2,3 10. Hz, J3,OH 6.0 Hz, H-3), 3.41 (dd, 1H, J5,6a 6.0
Hz, J6a,6b 10.8 Hz, H-6b). 13C NMR ((CD3)2SO, 100 MHz): 166.5
(
C
O2CH3), 160.3, 146.7, 136.3, 134.1, 133.7, 131.9, 131.2,
crude 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl chloride was
130.8, 130.5, 130.1, 129.0, 128.7, 126.8, 125.2, 121.3 (ArC),
added dissolved in 15 mL DMF and added to the reaction
mixture. The mixture was heated to 50 °C for 12 h. When TLC
showed that all starting material was consumed, the mixture
was diluted with DCM (100 mL) and water (50 mL). The organic
phase was washed with water (30 mL×3) and concentrated.
The residue was purified by column chromatography
(Heptane:EtOAc, 6:1 to 5:2) to give 20 (1.77 g, 68%) as a white
88.7 (C-1), 79.7, 72.7, 71.0, 66.9, 64.8, 60.1, 52.5 (CO2CH3).
HRMS calcd. for C24H23Cl2NO7S+H+ (M+H)+: 540.0651, found:
540.0648.
3.17. 3,4-Dichlorophenyl 3-O-(7-methoxycarbonyl-quinolin-2-
yl-methyl)-1-thio-
The reaction was performed with 21 (55 mg, 0.162 mmol)
following the general method 4.5 Compound 22a was
obtained in 67% yield (58.mg, 0.108 mmol) as a white
α-D-galactopyranoside 22b
.
solid over two steps. [
400 MHz): 7.48 (d, 1H,
Ar ), 7.20 (dd, 1H,
α
]
25 +28.6 (
c
1.2, CHCl3). 1H NMR (CDCl3,
2.0 Hz, Ar ), 7.27 (dd, 1H, 8.4 Hz,
8.4 Hz, Ar ), 5.90 (d, 1H, J1,2 5.6
D
J
H
J
25
amorphous solid. [
((CD3)2SO, 400 MHz): 9.18 (d, 1H,
2H, 8.4 Hz, Ar ), 7.95 (d, 1H, 9.2 Hz, Ar
α
]
+59.1
(
c
0.6, CH3OH). 1H NMR
9.2 Hz, Ar ), 8.24-8.21 (m,
), 7.87 (dd, 1H, 7.2
D
H
J
2.0 Hz,
J
H
J
H
Hz, H-1), 5.25 (dd, 1H, J1,2 5.6 Hz, J2,3 10.8 Hz, H-2), 5.15 (dd,
J
H
J
H
J
8 | J. Name., 2012, 00, 1-3
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Hz,
Hz, Ar
OH), 5.76 (d, 1H, J1,2 5.6 Hz, H-1), 5.05 (bs, 1H, OH), 5.00 (d, 1H, MCF-7 cell lines and 3000 cells for MCF-10A cell line per well in
14.4 Hz, CH2C11H8NO2), 4.91 (d, 1H, 14.4 Hz, CH2C11H8NO2), 180 μl medium) and the plates were incubated for 24 h before
J
8.4 Hz, Ar
H
), 7.74 (d, 1H,
J
2.0 Hz, Ar
H), 7.54 (d, 1H,
J 8.4 used. Accordingly, control was treated with 0.1% DMSO in PBS.
H), 7.44 (dd, 1H,
J
2.0 Hz,
J
8.4 Hz, Ar
H), 5.88 (bs, 1H, Cells were seeded in 96-well plates (5000 cells for JIMT-1 and
J
J
4.62 (bs, 1H, OH), 4.30 (dd, 1H, J1,2 5.6 Hz, J2,3 10.0 Hz, H-2), addition of compound. After 72 h of treatment, MTT solution
4.14 (bs, 1H, H-4), 4.00-3.95 (m, 4H, H-5, CO2CH3), 4.58 (dd, (20 μl of 5 mg/mL in PBS) was added to each well and the plate
1H, J5,6a 6.0 Hz, J6a,6b 10.8 Hz, H-6a) , 3.51 (dd, 1H, J2,3 10.0 Hz, was incubated for 1 h. Thereafter, the medium was removed
J3,4 3.2 Hz, H-3), 3.41 (m, 1H, H-6b). 13C NMR ((CD3)2SO, 100 and the purple formazan product was dissolved by the
MHz): 165.9 (
130.8, 130.5, 129.1, 128.8, 128.7, 127.1, 126.4, 120.7 (Ar
88.8 (C-1), 79.8, 72.7, 71.2, 66.9, 64.8, 60.2, 52.4 (CO2CH3). cells. Absorbance was monitored at 540 nm using
HRMS calcd. for C24H23Cl2NO7S+H+ (M+H)+: 540.0651, found: Labsystems iEMS Reader MF (Labsystems Oy, Helsinki, Finland)
C
O2CH3), 162.7, 148.5, 138.0, 136.3, 132.0, 131.3, addition of 100 μl of 100% DMSO per well. The plates were
C
), swirled gently for 10 min to dissolve the precipitate and the
a
540.0653.
and the software DeltaSoft II v.4.14 (Biometallics Inc.,
Princeton, NJ, USA). The software program GraphPad Prism
was used to analyze the data and plot dose response curves.
3.20. Molecular dynamic simulations
Molecular dynamic simulations were performed with the
OPLS3 force field in Desmond implemented in Schrödinger
Release 2017-3 using default settings except for length of the
simulation and the use of light harmonic constraints (1 kcal
mol-1 Å-2) on all strand backbone atoms and on the galactose
O4 atom. A series of energy-minimized starting conformations
of 1k with different dihedral angles of the three rotable bonds
linking galactose C3 to the quinoline ring system of 1k was
placed in the published structure of galectin-8N in complex
Conflicts of interest
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.
Acknowledgements
with sialyl-α-(2-3)-lacNAc (pdb id 3VKO) and subjected to
molecular dynamics simulation. All simulations drifted towards
a complex geometry where the quinoline of 1k was close to
Arg45, why a starting conformation with the 1k quinoline close
to Arg45 was subjected to a 1000ns molecular dynamics
simulation.
This work was supported by the Swedish Research Council
(Grants No. 621-2012-2978), the Royal Physiographic Society,
Lund,
a project grant awarded by the Knut and Alice
Wallenberg Foundation (KAW 2013.0022), and by Galecto
Biotech AB, Sweden.
3.21. Cell lines and cell culture
The human breast cancer cell line JIMT-1 (ACC589) was
purchased from the German Collection of Microorganisms and
Cell Cultures (DSMZ) and was routinely maintained in
Dulbecco’s modified Eagle’s medium/nutrient mixture Ham’s
F12 medium (VWR, Lund, Sweden). The human breast cancer
cell line MCF-7 (HTB-22) and human normal-like breast
epithelial cell line MCF-10A (CRL-10317) were obtained from
American Type Culture Collection (Manassas, VA, USA) and
were cultured in RPMI1640 medium (VWR). The JIMT-1 and
MCF-7 cell lines were cultured with the addition of 10% fetal
calf serum (FCS) (VWR), nonessential amino acids (1 mM)
(VWR), insulin (10 µg/mL) (Sigma-Aldrich), penicillin (100
U/mL) (VWR), and streptomycin (100 g/mL) (VWR). The MCF-
10A cells were cultured with the addition of 10% heat-
inactivated FCS, nonessential amino acids (1 mM), insulin (10
µg/mL), penicillin (100 U/mL), streptomycin (100 g/mL),
epithermal growth factor (20 ng/mL) (Sigma-Aldrich), cholera
toxin (50 ng/mL) (Sigma-Aldrich), and hydrocortisol (250
ng/mL) (Sigma-Aldrich). All cell lines were maintained at 37°C
in a humidified incubator with 5% CO2.
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10 | J. Name., 2012, 00, 1-3
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