Synthesis of tricyclic carbohydrate-benzene hybrids as selective inhibitors of galectin-1 and galectin-8 N-terminal domains

Article abstract of DOI:10.1039/d0ra03144e

As the galactoside binding family of galectin proteins is involved in many physiological and pathological processes, the inhibitors of these proteins are considered to be of significant interest in the treatment of diseases such as cancer and fibrosis. Herein, fused tricyclic carbohydrate-benzene hybrid core structures are reported to be the selective inhibitors of galectin-1 and the N-terminal domain of galectin-8 by a competitive fluorescence polarization assay. The key intermediates mono- or diiodo tricyclic carbohydrate-benzene hybrids were synthesized from protected 2-bromo-3-O-propargyl-d-galactoseviaa domino reaction and subsequently utilized for further derivatization by Stille couplings to achieve derivatives carrying substituents at C10 and/or C11. Several compounds showed affinity for the galectin-1 and galectin-8 N-terminal (8N) domains; however, weak or even no binding was observed for galectin-3. Monosubstituted derivatives at C10 or C11 exhibited better affinity for galectin-8N than di-substituted derivatives at C10 or C11. Especially, a benzyl substituent orp-fluorobenzyl substituent at C11 displayed affinity and selectivity for galectin-1 and galectin-8N over galectin-3. This suggests that tricyclic carbohydrate-benzene hybrids are promising scaffolds for the development of selective galectin-1 and galectin-8N inhibitors.

Full text of DOI:10.1039/d0ra03144e

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Synthesis of tricyclic carbohydrate–benzene
hybrids as selective inhibitors of galectin-1 and
Cite this: RSC Adv., 2020, 10, 19636
galectin-8 N-terminal domains†
a
b
Chunxia Wu,a Can Yong,a Qiuju Zhong,a Zhouyu Wang,
Ulf J. Nilsson
a
*
and Yuanyuan Zhang
As the galactoside binding family of galectin proteins is involved in many physiological and pathological
processes, the inhibitors of these proteins are considered to be of significant interest in the treatment of
diseases such as cancer and fibrosis. Herein, fused tricyclic carbohydrate–benzene hybrid core
structures are reported to be the selective inhibitors of galectin-1 and the N-terminal domain of
galectin-8 by a competitive fluorescence polarization assay. The key intermediates mono- or diiodo
tricyclic carbohydrate–benzene hybrids were synthesized from protected 2-bromo-3-O-propargyl-D-
galactose via a domino reaction and subsequently utilized for further derivatization by Stille couplings to
achieve derivatives carrying substituents at C10 and/or C11. Several compounds showed affinity for the
galectin-1 and galectin-8 N-terminal (8N) domains; however, weak or even no binding was observed for
galectin-3. Monosubstituted derivatives at C10 or C11 exhibited better affinity for galectin-8N than di-
substituted derivatives at C10 or C11. Especially, a benzyl substituent or p-fluorobenzyl substituent at C11
displayed affinity and selectivity for galectin-1 and galectin-8N over galectin-3. This suggests that
tricyclic carbohydrate–benzene hybrids are promising scaffolds for the development of selective
galectin-1 and galectin-8N inhibitors.
Received 7th April 2020
Accepted 4th May 2020
DOI: 10.1039/d0ra03144e
rsc.li/rsc-advances
and cutaneous T cell lymphomas.15–19 As a result, due to their
intimate connection with diseases, galectins have become
1 Introduction
promising drug targets.
Galectins are glycan-binding proteins that selectively bind to
glycoconjugates containing b-D-galactopyranoside residues. To
date, more than 15 members of the galectin family have been
identied, puried, isolated, and characterized, out of which,
only 12 are found in humans.1,2 These galectins are involved in
many biological functions such as cell–cell3 and cell–matrix
interactions,4 immune and inammatory responses,5,6 anti-
apoptosis,7 induction of T cell apoptosis, and regulation of cell
adhesion and migration.8 Among them, galectin-1 is widely
expressed in the human body and is associated with various
neurological diseases,9 HIV-1 viral infectivity,10 and cancer
progression.11 Moreover, galectin-1 has been highlighted as
a diagnostic tumor marker.12 In addition to this, galectin-8 has
attracted attention due to its physiological activity. For example,
it has been evidenced to regulate autophagy13 and lym-
phangiogenesis14 and is highly expressed in many clinical
diseases such as larynx cancer, prostate cancer, breast cancer,
A variety of articial galectin inhibitors have been synthe-
sized in the past two decades. The structure of the inhibitors
were typically based on monosaccharides or oligosaccharides,20
such as D-galactose,21,22 talose,23 lactosamine,24 and thio-
digalactoside,25,26 and with modications mostly at C1 and C3
positions of galactoside. Both 4-OH and 6-OH were always
conserved as they were essential for galectin binding. A majority
of these inhibitors exhibit good affinity for galectin-3,25–28
whereas fewer show potent affinity for other galectins such as 1
shows potent affinity for galectin-1 (ref. 27) and 2 shows potent
affinity for galectin-8N21 (Fig. 1). However, they all show similar
or even more potent affinity for galectin-3. This drawback limits
the application of the abovementioned compounds as galectin-
1 or galectin-8 inhibitors in biological assays or systems.
aSchool of Science, Xihua University, Jinniu District, 610039 Chengdu, China. E-mail:
yuanyuan.zhang@mail.xhu.edu.cn
bCentre for Analysis and Synthesis, Department of Chemistry, Lund University, POB
124, SE-221 00 Lund, Sweden
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03144e
Fig. 1 Structures of the compound 1 and 2.
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Herein, a series of tricyclic carbohydrate–benzene hybrids was
designed and synthesized. The key galectin-binding sites 4-OH
and 6-OH of galactose were preserved, and C1–C3 were con-
strained by two rings to a fused and rigid tricyclic structure as
previously reported by Werz et al.29 This ring structure was
hypothesized to favorably change the hydrophobic/hydrophilic
properties of the inhibitors and enable the incorporation of
substituents into them to optimize their affinity and selectivity
for selected individual galectins among the panel of galectin-
1,3,4 N-terminal (4N), 4 C-terminal (4C), 8N, 9 N-terminal
(9N), and 9 C-terminal (9C) evaluated in the study.
2.2 Galectin binding
The binding activities of the compounds 8a–e, 9a–d, 5, and 6 for
galectins were tested using a previously described competitive
uorescence polarization assay.30,31 The data of these
compounds and three reference ligands, i.e. methyl b-D-galac-
toside 10, 1,10-sulfanediyl-bis-{{3-deoxy-3-[4-(thiazol-2-yl)-1H-
1,2,3-triazol-1-yl]-b-D-galactopyranoside}} 1, and 3,4-dichlor-
ophenyl
3-O-(7-carboxy-quinolin-2-yl-methyl)-1-thio-a-D-gal-
actopyranoside 2, are provided hereinaer.
A majority of the synthesized compounds exhibited obvious
affinities for galectin-1 and galectin-8N, but no or very weak
affinity for galectin-3. Hence, the selectivity of the tricyclic
carbohydrate–benzene hybrids was signicantly improved for
galectin-1 and galectin-8N over galectin-3, which is rare among
the reported inhibitors. Although the compound 1 is the best
reported synthetic galectin-1 inhibitor to date, its affinity for
galectin-3 was even more potent, which was 10-fold when
compared with that for galectin-1. The compound 2 has been
reported to be the most potent inhibitor for galectin-8N;
however, it has a similar affinity for galectin-3. Compared
with the case of the reference methyl b-galactoside 10, the
affinities of most of the tricyclic compounds were increased by
3–35 folds and more than 3–71 folds for galectin-8N and
galectin-1, respectively, whereas most of the tested compounds
exhibited weak or even no affinity for galectin-4C, 4N, 9C, or 9N
(Table 1).
Structure–affinity analysis revealed that the substituents at
the C10 and C11 positions of the tricyclic carbohydrate–benzene
hybrids play a key role in the affinity of these hybrids for
galectins. In general, the mono-substituted derivatives 6, 8c,
and 9a–9d showed better galectin-8N binding than the di-
substituted compounds 5, 8a, 8b, 8d, and 8e. The affinities of
the disubstituted derivatives carrying aliphatic alkyl groups,
such as ethyl (8a), n-propyl (8b), or propynyl (8d), for galectin-8N
were very weak or could hardly be observed, whereas the affin-
ities of those carrying di-benzyl groups (8e) and di-iodo (5) were
increased to around 1500 and 2100 mM, respectively, which was
more than 3 times that of the reference galactoside 10. The
analysis of the mono-substituted 8c, 9a, 9b, 9c, 9d, and 6
revealed no binding of these derivatives to galectin-3, whereas
their affinity for galectin-8N was changed. Compared to the best
inhibitor 10,11-disubstituted 8e, all the mono-substituted 8c,
9a, 9b, 9c, 9d, and 6 showed improved affinity for galectin-8N.
Similar to di-substituted derivatives, the benzyl derivative 9b
exhibited the best affinity for galectin-8N, along with the mono-
iodo derivative 6. The evaluation of the derivatives with
substituted benzyl groups, i.e. p-methoxybenzyl 9c and p-uo-
robenzyl 9d, revealed that the electron-donating group 4-
methoxybenzyl at C10 (9c) decreased the affinity; thus, the
affinity of 9c was the worst among mono-substituted deriva-
tives; however, the electron-withdrawing group 4-uorobenzyl
at C10 (9d) improved the affinity of the derivative for galectin-8N
down to the dissociation constant of around 180 mM. Mean-
while, regardless of being mono-substituted or di-substituted,
almost all the tricyclic compounds exhibited affinity for
galectin-1. Benzyl group modication exerted positive effects on
2 Results and discussions
2.1 Chemistry
The tricyclic carbohydrate–benzene hybrids were synthesized by
three strategies (Scheme 1).
At rst, the tricyclic carbohydrate–benzene hybrids 8a–c were
acquired by the Pd-catalyzed domino reaction of 3 with different
alkynes and subsequent deprotection using the method re-
ported by Werz.29 Subsequently, the benzylidene-protected di-
trimethylsilyl tricyclic carbohydrate–benzene hybrid 7c was
converted to the diiodo-hybrid 4 by iodine monochloride. The
benzylidene removal of 4 led to 5, which was converted to the
compounds 8d and 8e via palladium-catalyzed Stille couplings
using organotins. Herein, four tricyclic carbohydrate–benzene
hybrids having same substituents at both C10 and C11 and one
compound with the mono-trimethylsilyl group were synthe-
sized. Finally, the deprotected mono-trimethylsilyl-substituted
tricyclic carbohydrate–benzene hybrid 8c was utilized to
prepare the mono-substituted hybrid molecules 9a–9d through
the monoiodo-hybrid intermediate 6 by similar Stille cross-
couplings as the third strategy.
Scheme
1 Synthesis of tricyclic carbohydrate–benzene hybrids.
Reagents and conditions: (a) substituted alkynes, Pd(PPh3)4, diisopro-
pylamine, [(t-Bu)3PH]BF4, DMF/MeCN/NMP, and 100 ꢀC; (b) 0.2 M HCl,
MeOH, and 55 ꢀC; (c) iodine monochloride, DCM, and r.t.; (d) 0.2 M
HCl, MeOH, and 55 ꢀC; (e) substituted tributyl stannane, triethylamine,
CuI, Pd(PPh3)4, DMF, and 75 ꢀC; (f) iodine monochloride, DCM, and r.t.;
and (g) substituted tributyl stannane, triethylamine, CuI, Pd(PPh3)4,
DMF, and 95 ꢀC.
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Table 1 Galectin affinities of tricyclic carbohydrate–benzene hybrids (Kd, mM)
Cpd Galectin-1
Galectin-3
Galectin-4C
Galectin-4N
Galectin-8N
Galectin-9C
Galectin-9N
5
6
890 ꢁ 140
1200 ꢁ 120
2900 ꢁ 580
760 ꢁ 35
[6000
1100 ꢁ 320
310 ꢁ 57
840 ꢁ 85
140 ꢁ 14
1500 ꢁ 11
420 ꢁ 61
[2000
[2000
[8000
[2000
840 ꢁ 60
1600 ꢁ 220
3700 ꢁ 650
[3000
[6000
z3000
naa
[1000
900 ꢁ 87
[6000
[3000
z10 000
870 ꢁ 81
na
2100 ꢁ 320
250 ꢁ 22
z10 000
[2000
330 ꢁ 18
[2000
1500 ꢁ 260
440 ꢁ 47
240 ꢁ 49
640 ꢁ 58
180 ꢁ 44
6300
[1000
[2000
z5000
[2000
2800 ꢁ 290
[2000
na
[2000
na
[1000
[1000
8600
[1000
[2000
[6000
[2000
[6000
[2000
na
[2000
na
[1000
[1000
3300
8a
8b
8c
8d
8e
9a
9b
9c
9d
2700 ꢁ 440
[2000
[2000
[2000
[2000
[2000
1100 ꢁ 22
1500 ꢁ 200
na
na
1700 ꢁ 430
z3000
10 000
[1000
[1000
6600
4100 ꢁ 370
10b >10 000
4400
1b
2b
<0.01 (ref. 27)
ꢂ0.0011 (ref. 27)
na
na
na
na
na
48 ꢁ 4.4 (ref. 21) 1.27 ꢁ 0.07 (ref. 21) 43 ꢁ 5.7 (ref. 21) 43 ꢁ 7.1 (ref. 21) 1.5 ꢁ 0.08 (ref. 21) 14 ꢁ 1.3 (ref. 21) 2.06 ꢁ 0.09 (ref. 21)
a
Not available. b Reference compounds.
the binding activities, and 8e and 9b were the best galectin-1
inhibitors. Similar affinity trends were observed for galectin-1;
however, for galectin-8N, p-uorobenzyl 9d was a more potent
inhibitor than p-methoxybenzyl 9c. Moreover, the two
compounds 5 and 6 containing iodine atoms displayed obvious
affinity for galectin-4C and galectin-4N, respectively, with a Kd
value below 1000 mM.
Fig. 3 Comparison between the complexes of 9d with galectin-1 (a),
galectin-3 (b), and galectin-8N (c).
2.3 Molecular docking of 9d with galectin-1 and galectin-8N
In order to analyze the possible binding modes of 9d with galectin-
1, galectin-3, and galectin-8N, molecular docking was performed.
In general, 9d is located in the CRD of galectin-1 and
galectin-8N, and its saccharide structure coincides with the
galactose part of D-lactose (Fig. 2a and c), respectively. Polar
interactions are mainly the hydrogen bonds between the gal-
actopyranose hydroxyl groups at C4 and C6 and the nearby
residues (Fig. 2b and d). The C4 hydroxyl group of 9d forms
hydrogen bonds with the galectin-1 residues Arg-48 and His-44,
whereas the C6 hydroxyl group interacts with the residues Asn-
61 and Glu-71. In the case of galectin-8N, the C4 hydroxyl group
of 9d forms hydrogen bonds with the galectin-8N residues Arg-
45, Arg-69, and His-65, whereas the C6 hydroxyl group interacts
with the residues Asn-79 and Glu-89.
From the docking poses of 9d in its complex with galectin-1, 3,
and 8N (Fig. 3), it can be observed that the benzene parts of the
tricyclic skeleton were close to the electropositive arginine-
containing regions, which could contribute to the cation–p
interactions. The relatively at surface consisted of Glu-71 and
Arg-48, which seemed to favor the stacking of 4-uorobenzyl in
galectin-1 via p–p or cation–p interactions (Fig. 3a). Moreover,
a similar trend could be observed in galectin-8N, and the surface
was composed of Glu-89 and Arg-69 (Fig. 3c). On the contrary,
steric hindrance occurred outside the CRD groove in galectin-3,
which might not be conducive to the p-interactions (Fig. 3b).
Fig. 2 Molecular docking of 9d with galectins. (a) 9d is shown with
green carbons, and lactose is shown with magenta carbons in the
galectin-1 CRD. (b) Interactions between 9d and CRD of galectin-1. (c)
9d is shown with green carbons and lactose is shown with magenta
carbons in the galectin-8N CRD. (d) Interactions between 9d and CRD
of galectin-8N.
3 Experimental
3.1 Synthesis
3.1.1 General methods. All solvents were well-dried before
use according to standard methods, and commercial reagents
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were directly used without further purication. TLC was per- (d, J ¼ 1.4 Hz, 1H, 5-H), 0.46 (s, 9H, CH3), 0.39 (s, 9H, CH3); 13
C
formed on silica-gel 60 F254 aluminum sheets (Merck) with NMR (100 MHz, CDCl3): 157.3, 149.2, 141.0, 137.7, 121.5 (C-2, C-
detection by UV and/or 10% sulfuric acid in ethanol. The puri- 7, C-9, C-11, C-12), 129.2, 128.8, 128.1, 126.9 (C-Ph), 120.7 (C-10),
cation of compounds was carried out by silica-gel column chro- 101.5 (C-1), 77.6 (C-3), 74.5 (C-8), 72.2 (C-4), 69.9 (C-6), 69.2 (C-
matography. A microwave reaction was performed in the Biotage 5), 3.1 (CH3), 3.0 (CH3). HRMS: calculated for C24H32O4Si2 ([M +
Initiator+. 1H NMR, 13C NMR, 2D COSY, HMQC, and HMBC Na]+): 463.1731; found: 463.1740.
spectra were obtained via the Bruker DRX 400 MHz or Bruker DRX
3.1.3 Synthesis of the di-substituted compounds 8a–8c.
500 MHz spectrometer at ambient temperature using CDCl3, The compounds 7a–7c (0.1 mol) were dissolved in methanol (10
DMSO-d6 or CD3OD as a solvent. HRMS was recorded using the mL), and then, 0.2 M HCl was added. The mixture was stirred at
Waters Micromass Q-TOF mass spectrometer.
55 ꢀC for 6 h. Aer being cooled to room temperature, the
All the nal compounds were of >95% purity according to the reaction mixture was neutralized by the addition of a saturated
HPLC analysis (Agilent Series 1100 system, Zorbax Eclipse XDB- NaHCO3 solution. The resulting mixture was extracted with
C18 column, and H2O–MeCN gradient 5–95% with 0.1% TFA). ethyl acetate (3 ꢃ 15 mL), and then, the combined organic
3.1.2 Synthesis of the protected di-substituted tricyclic layers were washed with water and brine. Aer being dried over
compounds 7a–7c. The compound 3 (ref. 29) (115 mg, 0.33 Na2SO4, the solvent was removed by rotary evaporation. The
mmol) and substituted alkyne (3.3 mmol) were dissolved in residue was puried by column chromatography (hepta-
a mixture of DMF/MeCN/NMP (10 mL/10 mL/1.4 mL) solution, ne : ethyl acetate ¼ 3 : 5) to afford 8a–8c.
1
and then, the catalytic reagent Pd(PPh3)4 (41 mg, 35 mmol), [(t-
8a. White solid; yield: 32%; H NMR (400 MHz, DMSO-d6):
Bu)3PH]BF4 (10 mg, 35 mmol), and diisopropylamine (184 mL, d ¼ 6.63 (s, 1H, 9-H), 5.01–4.96 (m, 2H, 7-Ha, 3-H), 4.87 (t, J ¼
1.32 mmol) were added. The reaction mixture was stirred in 5.6 Hz, 1H, OH), 4.79–4.76 (m, 2H, 7-Hb, OH), 4.19 (t, J ¼ 1.4 Hz,
a microwave-reactor (absorption level was set as very high) for 1H, 4-H), 4.05 (t, J ¼ 6.4 Hz, 1H, 5-H), 3.76–3.70 (m, 2H, 6-Ha, 6-
3 h at 120 ꢀC. The reaction was stopped by the addition of brine, Hb), 2.69–2.67 (m, 2H, CH2), 2.34–2.33 (m, 2H, CH2), 1.14 (t, J ¼
and the aqueous layer was extracted with EtOAc (3 ꢃ 30 mL). 7.5 Hz, 3H, CH3), 1.04 (t, J ¼ 7.5 Hz, 3H, CH3); 13C NMR (100
The combined organic layer was washed with water and brine MHz, CDCl3): d ¼ 149.2, 143.4, 138.7, 124.9, 118.8 (C-1, C-2, C-8,
and then dried over anhydrous Na2SO4. Aer the solvent was C-10, C-11), 114.3 (C-9), 77.8, 76.9 (C-3, C-5), 74.2 (C-7), 65.2 (C-
removed by rotary evaporation, the residue was puried by 4), 63.5 (C-6), 24.9, 18.1 (CH2), 16.8, 14.5 (CH3); HRMS: calcu-
column chromatography (heptane : ethyl acetate ¼ 3 : 1) to lated for C15H20O4 ([M + Na]+): 287.1254; found: 287.1254.
afford the compounds 7a–7c.
8b. White solid; yield: 40%; 1H NMR (400 MHz, CD3OD): d ¼
1
7a. White solid; yield: 44%; H NMR (400 MHz, CDCl3): d ¼ 6.65 (s, 1H, 9-H), 5.10–5.07 (m, 2H, 3-H, 7-Ha), 4.87–4.84 (m, 1H,
7.25–7.16 (m, 5H, Ph), 6.68 (s, 1H, 10-H), 5.62 (s, 1H, 7-H), 5.22– 7-Hb), 4.33 (dd, J ¼ 3.4 Hz, 0.8 Hz, 1H, 4-H), 4.11 (t, J ¼ 6.4 Hz,
5.16 (m, 2H, 8-Ha, 3-H), 4.94 (dd, J ¼ 11.6 Hz, 1.6 Hz, 1H, 8-Hb), 1H, 5-H), 3.92 (d, J ¼ 6.3 Hz, 2H, 6-H), 2.63–2.54 (m, 4H, CH2),
4.60–4.56 (m, 2H, 6-Ha, 4-H), 4.20 (dd, J ¼ 12.0 Hz, 1.2 Hz, 1H, 6- 1.59–1.50 (m, 4H, CH2), 0.97 (t, J ¼ 7.4 Hz, 3H, CH3), 0.96 (t, J ¼
Hb), 4.12 (d, J ¼ 1.4 Hz, 1H, 5-H), 2.76–2.61 (m, 4H, CH2), 1.23 (t, 7.4 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): d ¼ 150.7, 143.8,
J ¼ 7.6 Hz, 3H, CH3), 1.18 (t, J ¼ 7.6 Hz, 3H, CH3); 13C NMR (100 140.0, 125.5, 121.2 (C-1, C-2, C-8, C-10, C-11), 115.0 (C-9), 79.8,
MHz, CDCl3): 149.3, 144.2, 137.7, 137.6, 119.2, 113.4, 101.2 (C- 79.4 (C-3, C-5), 74.8 (C-7), 64.8 (C-4), 62.6 (C-6), 36.4, 28.2, 26.2,
Ph, C-1, C-2, C-7, C-9, C-10, C-11, C-12), 129.0, 128.0, 126.6 (C- 24.5 (CH2), 14.6, 14.5 (CH3); HRMS: calculated for C17H24O4 ([M
Ph), 77.3 (C-3), 74.4 (C-8), 72.4 (C-4), 70.2 (C-6), 69.0 (C-5), + Na]+): 315.1567; found: 315.1579.
1
26.2 (CH2), 18.5 (CH2), 16.2 (CH3), 14.6 (CH3). HRMS: calcu-
8c. White solid; yield: 43%; H NMR (400 MHz, CDCl3): d ¼
lated for C22H24O4 ([M + Na]+): 375.1567; found 375.1570.
6.99 (s, 1H, 11-H), 6.90 (s, 1H, 9-H), 5.22–5.18 (m, 1H, 7-Ha),
1
7b. White solid; yield: 38%; H NMR (400 MHz, CDCl3): d ¼ 5.12–5.11 (m, 1H, 3-H), 4.96 (dd, J ¼ 11.9 Hz, 1.8 Hz, 1H, 7-Hb),
7.25–7.15 (m, 5H, Ph), 6.66 (s, 1H, 10-H), 5.61 (s, 1H, 7-H), 5.22– 4.41 (dd, J ¼ 3.6 Hz, 0.8 Hz, 1H, 4-H), 4.19–4.12 (m, 2H, 5-H, 6-
5.15 (m, 2H, 8-Ha, 3-H), 4.94 (dd, J ¼ 11.6 Hz, 1.8 Hz, 1H, 8-Hb), Ha), 4.06 (dd, J ¼ 11.2 Hz, 4.2 Hz, 1H, 6-Hb), 0.25 (s, 9H, CH3);
4.59 (dd, J ¼ 3.6 Hz, 1.4 Hz, 1H, 4-H), 4.57 (dd, J ¼ 12.3 Hz, 13C NMR (100 MHz, CDCl3): d ¼ 151.1, 144.4, 141.8, 122.7 (C-1,
1.8 Hz, 1H, 6-Ha), 4.20 (dd, J ¼ 12.3 Hz, 1.3 Hz, 1H, 6-Hb), 4.10 C-2, C-8, C-10), 118.2, 117.0 (C-9, C-11), 77.8, 77.2, 65.3 (C-3, C-4,
(d, J ¼ 1.3 Hz, 1H, 5-H), 2.72–2.52 (m, 4H, CH2), 1.66–1.58 (m, C-5), 74.4 (C-7), 63.5 (C-6), ꢄ0.9 (CH3); HRMS: calculated for
4H, CH2), 0.99 (t, J ¼ 7.4 Hz, 3H, CH3), 0.97 (t, J ¼ 7.4 Hz, 3H,
C
14H20O4Si ([M + Na]+): 303.1023; found: 303.1024.
3.1.4 Synthesis of the benzylidene-protected di-iodo tricy-
CH3); 13C NMR (100 MHz, CDCl3): 149.6, 143.2, 137.8, 137.5,
125.0, 119.2, 114.2, 101.3 (C-Ph, C-1, C-2, C-7, C-9, C-10, C-11, C- clic compound 4. A solution of 7c (123 mg, 0.28 mmol) in DCM
12), 129.2, 128.2, 126.8 (C-Ph), 77.5 (C-3), 74.5 (C-8), 72.4 (C-4), (5 mL) was stirred in an ice bath, and iodine monochloride (29
70.3 (C-6), 69.1 (C-5), 35.6 (CH2), 27.5 (CH2), 25.1 (CH2), 23.4 mL, 0.56 mmol) in 2 mL DCM was added to the abovementioned
(CH2), 14.6 (CH3), 14.3 (CH3). HRMS: calculated for C24H28O4 solution over 10 min. The mixture was allowed to stir at room
([M + Na]+): 403.1880; found: 403.1890.
temperature (r.t.) for 1–2 h and then quenched by a saturated
7c. Colorless oil; yield: 43%; 1H NMR (400 MHz, CDCl3): d ¼ KHSO4 solution. The organic layer was separated and then dried
7.28–7.22 (m, 5H, Ph), 7.17 (s, 1H, 10-H), 5.64 (s, 1H, 7-H), 5.23– over anhydrous Na2SO4. Aer the solvent was removed by rotary
5.18 (m, 2H, 8-Ha, 3-H), 4.94 (dd, J ¼ 13.0 Hz, 3.0 Hz, 1H, 8-Hb), evaporation, the crude compound 4 was afforded by column
4.62 (dd, 1H, J ¼ 3.6 Hz, 1.3 Hz, 4-H), 4.58 (dd, J ¼ 12.4 Hz, chromatography (heptane : ethyl acetate ¼ 3 : 1) as a white solid
1.8 Hz, 1H, 6-Hb), 4.20 (dd, J ¼ 12.4 Hz, 1.4 Hz, 1H, 6-Hb), 4.13 (72 mg, 39%). 1H NMR (400 MHz, CDCl3): d ¼ 7.44 (s, 1H, 10-H),
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7.28–7.27 (m, 3H, Ph-H), 7.23–7.21 (m, 2H, Ph-H), 5.63 (s, 1H, 7- poured into saturated KHSO4. The resulting mixture was
H), 5.16–5.14 (m, 2H, 3-H, 8-Ha), 4.94 (dd, J ¼ 12.2 Hz, 2.2 Hz, extracted with DCM (20 mL ꢃ 3) and dried over anhydrous
1H, 8-Hb), 4.66 (dd, J ¼ 12.4 Hz, 1.6 Hz, 1H, 6-Ha), 4.62 (dd, 1H, J Na2SO4. Aer the solvent was removed by rotary evaporation,
¼ 3.6 Hz, 1.2 Hz, 4-H), 4.26–4.22 (m, 2H, 5-H, 6-Hb); 13C NMR the residue was puried by column chromatography (hepta-
(100 MHz, CDCl3): d ¼ 151.9, 142.7, 137.2, 129.2, 128.1, 126.5, ne : ethyl acetate ¼ 3 : 1) to obtain the white solid 6 (33 mg,
124.8, 121.7, 108.8 (C-2, C-7, C-9, C-11, C-12, C-10, C-Ph), 101.2 66%). 1H NMR (400 MHz, MeOD): d ¼ 7.19 (s, 1H, 11-H), 7.02 (s,
(C-1), 77.0 (C-3), 73.6 (C-8), 71.9 (C-4), 71.4 (C-5), 69.8 (C-6); 1H, 9-H), 5.12–5.05 (m, 2H, 3-H, 7-Ha), 4.90 (m, 1H, 7-Hb), 4.33
HRMS: calculated for C C18H14I2O4 ([M + Na]+): 570.8874; (dd, J ¼ 3.6, 0.8 Hz, 1H, 4-H), 4.16 (t, J ¼ 6 Hz, 1H, 5-H), 3.95–
found: 570.8871.
3.87 (m, 2H, 6-Ha, 6-Hb); 13C NMR (100 MHz, MeOD): d ¼ 153.6,
3.1.5 Synthesis of the di-iodo tricyclic compound 5. The 145.5, 124.6 (C-1, C-2, C-8), 123.7, 122.0 (C-9, C-11), 94.2 (C-10),
compound 4 (72 mg, 0.13 mmol) was dissolved in 4 mL meth- 80.3 (C-5), 79.6 (C-3), 74.3 (C-7), 64.7 (C-4), 62.6 (C-6); HRMS:
anol, and 0.2 M HCl solution was added to it to adjust the pH < calculated for C11H11IO4 ([M + Na]+): 356.9594; found: 356.9606.
3. The reaction was maintained at 55 ꢀC for 10 h. Aer cooling to
3.1.8 Synthesis of the mono-substituted compounds 9a–
r.t., the saturated NaHCO3 solution was added. The mixture was 9d. The compound 6 (33 mg, 0.10 mmol), substituted benzyl(-
extracted by EtOAc (15 mL ꢃ 3). Then, the combined organic tributyl)stannane (0.20 mmol), and triethylamine (35 mL) were
phase was washed with brine (25 mL ꢃ 2) followed by drying dissolved in 10 mL DMF, and then, catalytic amount of CuI and
over anhydrous Na2SO4. Aer the solvent was removed by rotary Pd(PPh3)4 were added. Aer being stirred under a nitrogen
evaporation, the residue was puried by column chromatog- atmosphere at 95 ꢀC for 14 h, the mixture was cooled and
raphy (heptane : ethyl acetate ¼ 1 : 2) to obtain the white solid 5 concentrated to dryness. The crude product was puried by
(34 mg, 57%). 1H NMR (400 MHz, DMSO-d6): d ¼ 7.46 (s, 1H, 9- column chromatography (heptane : ethyl acetate ¼ 3 : 1) to
H), 5.11 (br, 1H, OH), 5.04 (d, J ¼ 2.7 Hz, 1H, 3-H), 4.99–4.95 (m, afford the compounds 9a–9d.
1
2H, OH, 7-Ha), 4.80 (dd, J ¼ 12.7 Hz, 1.8 Hz, 1H, 7-Hb), 4.22–4.19
9a. White solid; yield: 32%; H NMR (400 MHz, CDCl3): d ¼
(m, 2H, 6-Ha, 4-H), 3.77–3.68 (m, 2H, 5-H, 6-Hb); 13C NMR (100 6.87 (s, 1H, 11-H), 6.77 (s, 1H, 9-H), 5.22–5.05 (m, 2H, 3-H, 7-Ha),
MHz, DMSO-d6): d ¼ 152.0, 144.1, 124.0, 123.9, 108.7 (C-1, C-2, 4.92 (d, J ¼ 11.9 Hz, 1H, 7-Hb), 4.40 (d, J ¼ 3.2 Hz, 1H, 4-H), 4.24–
C-8, C-10, C-11), 92.8, 80.6, 78.1 (C-9, C-3, C-5), 72.4 (C-7), 61.8 4.13 (m, 3H, 5-H, 6-Ha, 6-Hb), 2.04 (d, J ¼ 5.6 Hz, 3H, CH3); 13
C
(C-4), 59.7 (C-6); HRMS: calculated for C11H10I2O4 ([M + Na]+): NMR (100 MHz, CDCl3): d ¼ 151.1, 141.9, 126.4, 121.7 (C-1, C-2,
482.8561; found: 482.8562.
C-8, C-10), 117.3, 115.8 (C-9, C-11), 85.7, 79.8 (C^C), 77.8 (C-3),
3.1.6 Synthesis of the di-substituted compounds 8d–8e. To 77.5 (C-5), 74.2 (C-7), 65.3 (C-4), 63.5 (C-6), 4.5 (CH3); HRMS:
a mixture of compound 5 (46 mg, 0.10 mmol), substituted ben- calculated for C14H14O4 ([M + Na]+): 269.0784; found: 269.0789.
1
zyl(tributyl)stannane (0.35 mmol), and triethylamine (50 mL) in
9b. White solid; yield: 29%; H NMR (400 MHz, CDCl3): d ¼
10 mL DMF, catalytic amount of CuI and Pd(PPh3)4 were added. 7.31–7.27 (m, 2H, Ph), 7.22–7.18 (m, 3H, Ph), 6.68 (s, 1H, 11-H),
Aer being stirred under a nitrogen atmosphere at 75 ꢀC for 20 h, 6.59 (s, 1H, 9-H), 5.17–5.10 (m, 2H, 3-H, 7-Ha), 4.85 (dd, J ¼
the mixture was cooled to r.t. and concentrated to dryness. The 11.6 Hz, 1.6 Hz, 1H, 7-Hb), 4.40–4.39 (m, 1H, 4-H), 4.17–4.11 (m,
crude product was puried by column chromatography (hepta- 2H, 5-H, 6-Ha), 4.08–4.04 (m, 1H, 6-Hb), 3.96–3.85 (s, 2H, CH2);
ne : ethyl acetate ¼ 3 : 1) to afford the compounds 8d–8e.
13C NMR (101 MHz, MeOD): d ¼ 151.4, 144.8, 142.3, 129.1,
8d. White solid; yield: 43%; 1H NMR (400 MHz, CD3OD): d ¼ 126.4, 120.5 (C-1, C-2, C-8, C-10, C-Ph), 114.5 (C-11), 113.0 (C-9),
6.84 (s, 1H, 9-H), 5.09–5.05 (m, 2H, 7-Ha, 3-H), 4.86–4.83 (m, 1H, 77.7, 77.4 (C-3, C-5), 74.4 (C-7), 65.4 (C-4), 63.6 (C-6), 42.4 (CH2);
7-Hb), 4.35 (dd, J ¼ 3.3 Hz, 0.8 Hz, 1H, 4-H), 4.18 (t, J ¼ 6.4 Hz, HRMS: calculated for C18H18O4 ([M + Na]+): 321.1097; found:
1H, 5-H), 3.99–3.91 (m, 2H, 6-Ha, 6-Hb), 2.09 (s, 3H, CH3), 2.05 321.1101.
(s, 3H, CH3); 13C NMR (100 MHz, CD3OD): d ¼ 153.2, 142.1,
9c. White solid; yield: 33%; 1H NMR (400 MHz, CD3OD): d ¼
129.8, 123.8, 112.2 (C-1, C-2, C-8, C-10, C-11), 118.0 (C-9), 94.1, 7.11–7.08 (m, 2H, Ph), 6.83–6.79 (m, 2H, Ph), 6.66 (s, 1H, 11-H),
89.4, 80.5, 74.6 (C^C), 79.9, 79.7 (C-3, C-5), 74.8 (C-7), 64.4 (C- 6.47 (s, 1H, 9-H), 5.10–5.07 (m, 2H, 3-H, 7-Ha), 4.85 (m, 1H, 7-
4), 62.2 (C-6), 4.4, 4.0 (CH3); HRMS: calculated for C17H16O4 ([M Hb), 4.30 (dd, J ¼ 3.3 Hz, 0.8 Hz, 1H, 4-H), 4.12 (t, J ¼ 6.2 Hz, 1H,
+ Na]+): 307.0941; found: 307.0948.
5-H), 3.94–3.85 (m, 4H, CH2, 6-Ha, 6-Hb), 3.75 (s, 1H, OCH3); 13
C
1
8e. White solid; yield: 42%; H NMR (400 MHz, CDCl3): d ¼ NMR (101 MHz, MeOD): d ¼ 152.8, 146.1, 143.5, 134.8 (C-1, C-2,
7.31–7.05 (m, 10H, Ph), 6.63 (s, 1H, 9-H), 5.19–5.16 (m, 2H, 7-Ha, C-8, C-Ph), 130.8 (C-Ph), 122.0 (C-10), 114.8 (C-Ph, C-11), 113.4
3-H), 4.94–4.92 (m, 1H, 7-Hb), 4.43–4.42 (m, 1H, 4-H), 4.18 (t, J ¼ (C-9), 79.8, 79.6 (C-3, C-5), 74.9 (C-7), 65.0 (C-4), 62.7 (C-6), 55.7
4.8 Hz, 1H, 5-H), 4.06–3.88 (m, 6H, CH2, 6-Ha, 6-Hb); 13C NMR (OCH3), 42.2 (CH2); HRMS: calculated for C19H20O5 ([M + Na]+):
(100 MHz, CDCl3): d ¼ 149.9, 142.2, 140.8, 140.6, 140.0, 128.9, 351.1202; found: 351.1202.
1
128.6, 128.5, 128.3, 126.2, 126.0, 123.4, 120.3 (C-1, C-2, C-8, C-
9d. White solid; yield: 40%; H NMR (500 MHz, MeOD): d ¼
10, C-11, C-Ph), 115.8 (C-9), 78.2 (C-3), 77.5 (C-5), 74.4 (C-7), 7.21–7.18 (m, 2H, Ph), 6.97 (t, J ¼ 8.5 Hz, 2H, Ph), 6.67 (s, 1H, 11-
65.6 (C-4), 63.7 (C-6), 39.4, 31.1 (CH2); HRMS: calculated for H), 6.48 (s, 1H, 9-H), 5.10–5.08 (m, 2H, 3-H, 7-Ha), 4.31 (d, J ¼
C
25H24O4 ([M + Na]+): 411.1567; found: 411.1566.
3.3 Hz, 1H, 4-H), 4.13 (t, J ¼ 6.2 Hz, 1H, 5-H), 3.93–3.86 (m, 4H,
3.1.7 Synthesis of the mono-iodo tricyclic compounds 6. To CH2, 6-Ha, 6-Hb); 13C NMR (126 MHz, MeOD): d ¼ 162.8 (C-Ph, J
a stirred solution of 8c (42 mg, 0.15 mmol) in 5 mL DCM in an ¼ 244 Hz), 152.9, 145.3, 143.7, 138.8 (C-1, C-2, C-8, C-Ph), 131.5
ice bath, ICl (49 mg, 0.30 mmol) in 6 mL DCM was added over (C-Ph, J ¼ 7.7 Hz), 122.2 (C-10), 115.9 (C-Ph, J ¼ 21 Hz), 114.9 (C-
10 min. The mixture was allowed to stir at r.t. for 1 h and then 11), 113.4 (C-9), 79.8, 79.6 (C-3, C-5), 74.9 (C-7), 65.0 (C-4), 62.7
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(C-6), 42.1 (CH2); HRMS: calculated for C18H17FO4 ([M + Na]+): 4)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1–3)-b-D-galactopyr-
339.1003; found: 339.1009.
anosyl-(1–4)-b-D-glucopyranoside at 0.1 mM.
3.3 Molecular docking
3.2 Galectin binding evaluations
The X-ray crystal structures of human galectin-1, galectin-3, and
N-terminal domain of galectin-8 with D-lactose (PDB ID 1gzw,32
3aye33 and 2yxs) were used to investigate the binding modes of
these galectin proteins with the compound 9d. In brief, the
proteins were prepared using Autodock Tools 1.5.6 by deleting
water molecules, adding polar hydrogens, and assigning Gas-
teiger charges. The original ligand D-lactose was used to dene
the binding site, and the box was set using D-lactose as a grid
center (galectin-1: center_x ¼ 55.497, center_y ¼ 26.987, cen-
ter_z ¼ 23.331; size_x ¼ 15, size_y ¼ 15, size_z ¼ 15; galectin-3:
center_x ¼ ꢄ13.334, center_y ¼ 7.803, center_z ¼ ꢄ26.075;
size_x ¼ 15, size_y ¼ 15, size_z ¼ 15; galectin-8N: center_x ¼
14.183, center_y ¼ 24.913, center_z ¼ ꢄ7.492; size_x ¼ 15,
size_y ¼ 15, size_z ¼ 15). Autodock vina 1.1.2 (ref. 34) was used
to perform the docking, and the exhaustiveness value was set
to 20.
3.2.1 Competitive uorescence polarization experiments.
Human galectin-1, galectin-3, galectin-4N, galectin-4C, galectin-
8N, galectin-9N, and galectin-9C were expressed and puried as
previously described.30 Fluorescence polarization experiments
were performed using the PHERAstar FS plate reader (soware
version 2.10 R3), and the uorescence anisotropy of uorescein-
tagged probes was measured by excitation at 485 nm and
emission at 520 nm. The Kd values were determined using
GraphPad Prism under specic conditions for each galectin as
described below. The synthesized compounds were dissolved in
neat DMSO at 100 mM and diluted in PBS to 3–6 different
concentrations, and each concentration was tested in duplicate.
Methyl b-D-galactoside was used as a reference. Experiments
were performed 3–10 times, and the average values of Kd and
SEM were calculated from 10 to 30 single-point measurements,
showing 10–90% inhibition.
Galectin-1 affinities. Experiments were conducted at 20 ꢀC
using galectin-1 at 0.50 mM and the uorescent probe 3,30-
dideoxy-3-[4-(uorescein-5-yl-carbonylaminomethyl)-1H-1,2,3-
triazol-1-yl]-30-(3,5-dimethoxy-benzamido)-1,10-sulfanediyl-di-b-
D-galactopyranoside at 0.10 mM.
Galectin-3 affinities. Experiments were performed at 20 ꢀC
using galectin-3 at 0.20 mM and the uorescent probe 3,30-
dideoxy-3-[4-(uorescein-5-yl-carbonylaminomethyl)-1H-1,2,3-
triazol-1-yl]-30-(3,5-dimethoxybenzamido)-1,10-sulfanediyl-di-b-
D-galactopyranoside at 0.02 mM or with galectin-3 at 1.0 mM and
2-(uorescein-5/6-yl-carbonyl)-aminoethyl 2-acetamido-2-deoxy-
a-D-galactopyranosyl-(1–3)-[a-L-fucopyranosyl-(1–2)]-b-D-gal-
actopyranosyl-(1–4)-b-D-glucopyranoside at 0.10 mM.
4 Conclusions
Herein, a series of tricyclic carbohydrate–benzene hybrids were
synthesized from D-galactose, and derivatizations at C10 and
C11 were implemented via iodo-intermediates. Furthermore,
their galectin binding activities were evaluated by the compet-
itive uorescence polarization assay, and the results indicated
that most of the hybrid derivatives exhibited selective affinity for
galectin-1 and galectin-8N over galectin-3, 4C, 4N, 9C, and 9N.
Structure–activity analysis revealed that the C10-mono-
substituted compounds displayed better affinity for galectin-
8N when compared with the C10, C1-di-substituted, and
compounds with (substituted) benzyl groups were good for
activity enhancement when compared with other aliphatic alkyl
groups. The relatively at surface near the groove of galectin-1
or galectin-8N CRD was benecial to the binding since the
residues might interact with the benzyl group via cation–p or p–
p stackings. Hence, the C10-benzyl tricyclic carbohydrate–
benzene hybrids are promising scaffolds for the discovery of
selective galectin-1 and galectin-8N inhibitors.
Galectin-4C affinities. Experiments were conducted at 20 ꢀC
using galectin-4C at 0.50 mM and the uorescent probe 2-
(uorescein-5/6-yl-carbonyl)-aminoethyl 2-acetamido-2-deoxy-a-
D-galactopyranosyl-(1–3)-[a-L-fucopyranosyl-(1–2)]-b-D-gal-
actopyranosyl-(1–4)-b-D-glucopyranoside at 0.1 mM.
ꢀ
Galectin-4N affinities. Experiments were carried out at 20 C
using galectin-4N at 3.0 mM and the uorescent probe 3,30-
dideoxy-3-[4-(uorescein-5-yl-carbonylaminomethyl)-1H-1,2,3-
triazol-1-yl]-30-(3,5-dimethoxy-benzamido)-1,10-sulfanediyl-di-b-
D-galactopyranoside at 0.10 mM.
Conflicts of interest
ꢀ
Galectin-8N affinities. Experiments were performed at 20 C
UJN is a shareholder in Galecto Biotech AB, Sweden.
using galectin-8N at 0.40 mM and the uorescent probe 2-
(uorescein-5-yl-carbonylamino) ethyl b-D-galactopyranosyl-(1–
4)-2-acetamido-2-deoxy-b-D-glucopyranosyl-(1–3)-b-D-galactopyr-
anosyl-(1–4)-b-D-glucopyranoside at 0.1 mM.
Acknowledgements
Galectin-9C affinities. Experiments were conducted at 20 ꢀC This work was supported by International Cooperation Project
using galectin-9C at 2.0 mM and the uorescent probe 3,30- of Science and Technology Department of Sichuan Province
dideoxy-3-[4-(uorescein-5-yl-carbonylaminomethyl)-1H-1,2,3-
triazol-1-yl]-30-(3,5-dimethoxy-benzamido)-1,10-sulfanediyl-di-b-
(2016HH0075), the European Community's Seventh Framework
Program (FP7-2007-2013) under the grant agreement no.
HEALTH-F2-2011-256986 – project acronym PANACREAS and
D-galactopyranoside at 0.10 mM.
ꢀ
Galectin-9N affinities. Experiments were performed at 20 C Ministry of Education Chunhui Project of China (Z2016164). We
using galectin-9N at 1.0 mM and the uorescent probe 2- thank Mrs Barbro Kahl-Knutsson for providing assistance with
(uorescein-5-yl-carbonylamino)-ethyl b-D-galactopyranosyl-(1– the uorescence polarization experiments.
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