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Article Cite This: ACS Omega 2019, 4, 7047−7053
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C1-Galactopyranosyl Heterocycle Structure Guides Selectivity: Triazoles Prefer Galectin‑1 and Oxazoles Prefer Galectin‑3 Alexander Dahlqvist,† Hakon Leffler,‡ and Ulf J. Nilsson*,† †
Centre for Analysis and Synthesis, Department of Chemistry, Lund University, P.O. 124, 221 00 Lund, Sweden Section of Microbiology, Immunology and Glycobiology, Lund University, Sölvegatan 19, 223 62 Lund, Sweden
‡
ACS Omega 2019.4:7047-7053. Downloaded from pubs.acs.org by 91.204.14.164 on 04/18/19. For personal use only.
S Supporting Information *
ABSTRACT: Galectins are carbohydrate-recognizing proteins involved in many different pathological processes, including cancer and immune-related disorders. Inhibitors of galectins have evolved from natural oligosaccharides toward more drug-like truncated galactoside scaffolds that only retain key specific interactions of the galactose scaffolds with the galectin carbohydrate recognition domains. In this context, C1-galactosides are attractive and stable scaffolds, and this work reports that the synthesis of novel C1-galactopyranosyl heteroaryl derivatives as galectin inhibitors, in which galectin selectivity is governed by the composition of the heterocycle and affinity, is driven by the structure of the aryl substituent to give compounds selective for either galectin-1 or galectin-3. The affinities are close to or better than those of lactose and other natural galectin-binding disaccharides, selectivities induced by the C1-heteroaryl groups are superior to lactose, and compound hydrolytic stabilities and drug-like properties are potentially better than those of natural saccharides. Hence, C1galactopyranosyl heteroaryls constitute a class of promising starting scaffolds for galectin inhibition, in which a natural ligand pyranose has been replaced by more than fivefold selectivity-inducing heteroaryl rings leading to affinities of 90 μM toward galectin-3 for a C1-galactopyranosyl naphthyloxazole and 170 μM toward galectin-1 for a C1-galactopyranosyl 2fluorophenyltriazole.
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INTRODUCTION Galectins are a family of carbohydrate binding proteins defined by their specificity for ligands containing β-galactosides, each member having at least one carbohydrate recognition domain (CRD).1 The galectin family is divided into three distinct types: dimeric (or prototypical), tandem, and chimeric. The dimeric galectins include galectins -1, -2, -5, -7, -10, -11, -13, -14, and -15, having two identical CRDs. The tandem group includes galectins -4, -6, -8, -9, and -12, which have two nonidentical CRDs separated by a linker, the N-terminal CRD usually labeled using an N and the C-terminal CRD labeled using a C (galectin-8N and galectin-8C for example). As the single member of the chimeric type, galectin-3 has a single CRD and an extended non-lectin domain.2 While most galectins are only present in a few tissue types, galectin-1 is present in immune, muscle, and neuronal cells and the kidney tissue, and galectin-3 is ubiquitously expressed.3 Intracellular functions of galectins -1 and -3 include the modulation of cell proliferation through independent pathways.4,5 Outside the cell, galectins are involved in crosslinking a variety of cell surface proteins such as VEGFR,6 integrins,7 and EGFR,8 proteins involved in growth signaling and cell adhesion. Their functions involve them in many diseases, among them idiopathic pulmonary fibrosis,7 lymphangiogenesis,9 inflammation,10 and various forms of cancer,11 which makes the inhibition of galectin function via the use of carbohydrate mimetics an attractive route toward novel therapeutics.3,7,9,12,13 © 2019 American Chemical Society
The binding pocket, which is located in a groove formed by curved β sheets on the CRD, can accommodate up to a linear tetrasaccharide.2 Most of the binding pocket is conserved among galectins with a network of amino acids binding to galactose 4 and 6 hydroxyls, as well as the ring oxygen on the galactose, while a tryptophan side chain stacks toward the hydrophobic α-face of the bound galactose (Figure 1). One of the few differences between galectin-1 and galectin-3 is the presence of an extra histidine in galectin-1,13−15 which is situated close to the anomeric position of a saccharide in the galactose binding subpocket of galectin-1, a difference exploitable to discriminate between galectin-1 and other galectins. A variety of galectin inhibitors based on different scaffolds exists, with the trend going from substituted lactoses16 to thiodigalactosides7,17,18 to scaffolds based on monosaccharides.19,20 C1-Galactosides are particularly interesting as galectin-binding compounds due to their comparatively small size and potentially enhanced chemical and enzymatic stability. Giguère and co-workers have developed C1-galactosides, including C1-exomethylene galactosides, with IC50 values down to 313 μM toward galectin-1 in a haemagglutination assay.21,22 While this work included C1thiazole galactosides of lower potency than the C1-methylene Received: February 4, 2019 Accepted: April 3, 2019 Published: April 18, 2019 7047
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Scheme 1. Synthesis of C1-Heteroaryl Galactosides 1−4a
Figure 1. Interactions between the binding pockets of galectin-1 and galectin-3 shown with the natural ligand lacNAc. Differences include Arg-144 in galectin-3 and His-52 in galectin-1.
derivatives, no study has explored galactosides carrying different aromatic heterocycles at C1. In this work, we report the synthesis, evaluation of galectin affinities, and computational modeling of galectin inhibitors based on four different C1-heterocycles, triazoles, oxazoles, isoxazoles, and pyrazoles, which led to the discovery that the C1-heterocycle structure and substitutions significantly influence the selectivity and affinity for galectins.
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RESULTS AND DISCUSSION Chemistry. Synthesis of a common key precursor C1alkynyl galactoside (8) began with Dess−Martin periodinane oxidation of the commercially available 2,3,4,6-tetra-O-benzylD-galactopyranoside (5) to the lactone (6). An alkynyl group was added to 6 by nucleophilic addition of an organocerium reagent generated in situ from lithium trimethylsilyl acetylide (Scheme 1). Reduction of the anomeric carbon and basepromoted removal of the trimethylsilyl protecting group yielded the benzyl protected alkyne (7).23 The switching of the benzyl protecting groups to acetyl groups gave the common precursor 8. The synthesis of the triazoles 1a−1j was done in one step using a copper-catalyzed Huisgen cycloaddition, followed by acetate removal with sodium methoxide to give the triazoles (1a−1j) in yields between 16 and 99% over two steps.18,24,25 In general, synthesis of the mand p-substituted aryltriazoles proceeded with acceptable to excellent yields, while o-substituted phenyltriazoles were impossible to obtain with the exception of the 2-fluoro compound (1j). Synthesis of the oxazoles 2a−2i was accomplished using gold-catalyzed [2 + 2 + 1] synthesis with an aryl nitrile as an addition component and 9-methylmorpholine N-oxide as an oxygen source,26 followed by deacetylation to give the oxazoles 2a−2i. The reaction takes place in a neat melt of the reactants, resulting in differing temperature conditions depending on the aryl nitrile melting point, which affects the yield, and thus yields between 4 and 48% were obtained in the oxazole formation step (Scheme 1). Just as the triazoles 1a−1j, the oxazoles 2a−2i were obtained in moderate to good yields except in the case of ortho-substituted phenyloxazoles that did not form. The isoxazoles 3a−3c and pyrazoles 4a−4c all originate from the alkynones 9a−9c, which are obtained via Sonogashira couplings23,27 on alkyne 8 in 64−68% yields. Hydrazine cyclizations of 9a−9c gave the pyrazoles 4a−4c in 40−88%,28
a
Reagents and conditions: (a) Dess−Martin periodinane, DCM, room temperature. (b) CeCl3, THF, −78 °C, lithium trimethylsilylacetylide for 2 h, then 6 h. (c) BF3·Et2O, Et3SiH, ACN/DCM, −10 °C. (d) NaOH, MeOH/DCM for 2 h, then hydrochloric acid (1 M). (e) TMSOTf, Ac2O; (f) Aryl azide, CuI, Et3N, ACN, 50 °C, 1−18 h then NaOMe, MeOH 16−99%. (g) Aryl nitrile, [bis(trifluoromethylsulfone)imidate] triphenylphosphine gold(I), 8-methylmorpholine N-oxide, 4−48%. (h) NaOMe, MeOH, 29−71%. (i) Aroyl acid chloride, triphenylphosphine palladium(II) dichloride, CuI, Et3N, THF, 64−68%. (j) Hydrazine hydrate, Na2CO3, THF, 40− 88%. (k) Hydroxylamine hydrochloride, Na2CO3, THF, 11−40%.
and hydroxylamine cyclization of 9a−9c gave isoxazoles in 11−35%28,29 over two steps following deacetylations (Scheme 1). Galectin Affinity Evaluation. Initially, affinities of compounds 1a−1c, 2a−2c, 3a−3c, and 4a−4c for galectins -1, -3, -4, -7, -8C, -8N, -9C, and -9N were measured with a fluorescence polarization assay previously published.30 The affinities for galectins -4, -7, -8C, -8N, -9C, and -9N of any of the compounds evaluated were in the millimolar range (data not shown) and similar to the methyl β-D-galactoside for those galectins, typically between 2 and 5 mM. The triazoles 1a−1c turned out to be selective for galectin-1, while the oxazoles 2b and 2c instead displayed selectivity for galectin-3 (Table 1). The isoxazoles 3a−3c had affinities comparable to those of triazoles 1a−1c for galectin-3, but with low to absent selectivity between galectin-1 and galectin-3. The pyrazoles 4a−4c had almost no selectivity between galectin-1 7048
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Table 1. Dissociation Constants (Kd in μM) of 1a−1c, 2a−2c, 3a−3c, and 4a−4c Binding to Galectin-1 and -3 Determined in a Competitive Fluorescense Polarization Assay R
galectin
a
1 3 1 3 1 3
b c
triazole (1) 760 1700 550 1300 290 1600
± ± ± ± ± ±
55 150 50 140 12 110
oxazole (2) 830 1000 490 90 790 230
galectin-1
galectin-3
>10000 190 340 ± 19 400 ± 50 1100 ± 80 680 ± 53 1800 ± 220 540 ± 80 170 ± 28
4400 220 1700 ± 130 1500 ± 300 2300 ± 380 2800 ± 330 1800 ± 170 2000 ± 310 880 ± 140
760 1200 600 1500 430 380
compound 2d 2e 2f 2g 2h 2i
± ± ± ± ± ±
60 100 80 65 200 76
1000 800 1600 1000 1400 1300
± ± ± ± ± ±
140 120 390 140 310 74
galectin-1 870 920 790 890 1900 900
± ± ± ± ± ±
81 160 39 25 120 85
galectin-3 280 670 450 350 600 520
± ± ± ± ± ±
28 3 32 17 130 43
the 1-naphthyloxazole (2b) in terms of affinities or selectivities. Taken together, C1-heteroaryl galactopyranosides are viable monosaccharide scaffolds for galectin inhibitors with improved properties compared to a simple monogalactose ligand and the choice of heterocycle-determining selectivity. As the micromolar affinities of the triazole 1j and 2b compare with those of disaccharide-based galectin ligands, these inhibitors illustrate a strategy for effective replacement of one disaccharide glucose or glcNAc moiety with a heteroaryl ring, which in turn opens up for fine-tuning selectivities with small structural alterations of the heteroaryl ring. Modeling. In an attempt to explain the galectin-1/galectin3 selectivities observed, we performed a conformational analysis of the 2-fluorophenyltriazole (1j) and 1-naphthyloxazole (2b) with the conformational search module, OPLS3 force field, and the mixed torsional/low-mode sampling method implemented in Schrödinger’s MacroModel. The minimum energy conformations of both 1j and 2b adopted a galactose C1-heterocycle bond angle with the galactose H1 and the heterocycle CH in a syn planar arrangement. The structures 1j and 2b were placed in the binding sites of galectin-1 (PDB id 1GWZ) and galectin-3 (PDB id 1KJL), respectively, in a way that the galactose rings of 1j and 2b superposed with and then replaced the lactose and lacNAc ligands of galectin-1 and -3. Molecular dynamic simulations of 200 ns converged toward structures with the triazole of 1j stacking with the His52 side chain of galectin-1 and 2b naphthyl rings stacking onto Arg162 in galectin-3 (Figure 2). The triazole 1j sampled complex structures placed the 2-fluorophenyl moiety in a position with the fluorine atom away from the triazole nitrogen lone pair and close to a His52 NH. The His52 residue is unique to galectin-1 as no other galectins have a corresponding His in the same position, which is why this triazole−His stacking may be inducing galectin-1 selectivity. If the minimum energy conformation of the oxazole 2b with the galactose H1 syn planar to the oxazole CH is placed with its galactose ring superposed with that of a lactose ligand, the naphthyl moiety of 2b will be in a position that would interfere with the galectin-1 His52 side chain of 2b. As mentioned above, galectin-3 lacks a residue corresponding to the galectin-1 His52 and instead
Table 2. Dissociation Constants (Kd in μM) of Triazoles 1d−1j and Control Ligands Methyl β-D-Galactoside and βLactoside Binding to Galectin-1 and -3 Determined in a Competitive Fluorescense Polarization Assay compound
63 300 60 18 54 40
pyrazole (4)
Table 3. Dissociation Constants (Kd in μM) of Oxazoles 2d−2i Binding to Galectin-1 and -3 Determined in a Competitive Fluorescense Polarization Assay
and galectin-3, with noteworthy affinities toward any galectin. Taken together, the composition of the heterocycle in the C1heteroaryl galactopyranosides significantly influences galectin selectivity, as well as the affinity for galectins -1 and -3. Furthermore, galectin-1 preferred 2-naphthyl as the aryl substituent, while galectin-3 preferred 1-naphthyl. The aryl substituents of the C1-heteroaryl galactopyranosides 1a−1c, 2a−2c, 3a−3c, and 4a−4c had influence on affinity, which was why further evaluations of aryl substituent effects were done with triazoles 1d−1j and oxazoles 2d−2i. Among the triazoles 1a−1j, both 3-methyl (1f) and 4methyl (1g) substituents resulted in lower affinity toward galectin-3 and for 1f, a lower affinity toward galectin-1, as compared to the unsubstituted 1a, while not significantly affecting selectivity. The 3-chloro 1e and 4-chloro 1d enhanced both affinity and selectivity when compared to 1a. The fluoro substituents follow a peculiar pattern: the 4-fluoro 1h had lower affinity for galectin-1 than the unsubstituted phenyltriazole 1a and no selectivity over galectin-3, while the 3-fluoro 1i regained affinity and good selectivity for galectin-1, and 2fluoro 1j has the highest affinity for galectin-1 of all the phenyltriazoles together, with a good selectivity of over fivefold preference for galectin-1. Importantly, the 2-fluoro derivative 1j shows an at least 50-fold improved affinity as compared to the reference methyl β-D-galactopyranoside and is about as potent as methyl β-lactoside (Table 2).
Me β-D-galactoside31 Me β-lactoside31 1d 1e 1f 1g 1h 1i 1j
± ± ± ± ± ±
isoxazole (3)
The substituted oxazoles 2c−2i displayed less spectacular affinity and selectivity patterns than the triazoles 1c−1j (Table 3). The 4-chloro- (2d) and 4-methylphenyloxazoles (2f) are similar with the threefold galectin-3 selectivity of 2d. The 3methyl-substituted 2g has a twofold higher affinity for galectin3 over galectin-1, which is better than the isosteric 3-chloro 2e. The fluoro oxazoles 2h and 2i are both lackluster inhibitors when it comes to affinity and selectivity. None of the substituted phenyloxazoles 2d−2i managed to outperform 7049
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Figure 3. Summary of the structure−activity relationships of triazoles (1) and oxazoles (2).
to 170 μM and a sixfold selectivity for a 2-fluorophenyltriazole galactopyranoside (1j), while the oxazoles were selective for galectin-3 over galectin-1 with 1-naphthyloxazole galactopyranoside (2b) having an affinity of 90 μM and a sixfold galectin3 selectivity. The isoxazoles have good affinities but poor selectivity, while the pyrazoles have poor affinity and poor selectivity. Apart from guiding selectivities, these compounds also possess higher affinities than other known C1-glycosides in the literature.21,22 Hence, the C1-heteroaryls are inhibitors of galectin-1 and galectin-3 built on a scaffold of only one galactopyranoside, preserving only the parts of the molecule necessary for galectin recognition of, for example, lactose. Although none of the compounds display exceptionally high affinities, as seen for doubly derivatized galactosides,20 and thus not as such represent promising drug leads; they demonstrate that the strategy of replacing a galectin-interacting monosaccharide unit of di- or oligosaccharide-based ligands by heteroaryl structural motifs is viable. Furthermore, this strategy offers the additional and important advantage that galectin selectivity can be fine-tuned with the choice of heteroaryl structure.
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EXPERIMENTAL SECTION General. Chemicals were used without further purification, unless stated in the procedure. Chemicals were obtained from Sigma-Aldrich except 1-naphthyl azide and 2-naphthyl azide, which were obtained from Enamine and 2,3,4,6-tetrabenzyl-Dgalactopyranoside, which was obtained from Carbosynth. All chemicals were used without further purification. NMR spectra were collected with a Bruker Ultrashield Plus/Avance II 400 MHz spectrometer. 1H spectra were recorded at 400 MHz and 13 C spectra at 100 MHz with a residual solvent signal as reference. All final compounds were purified using preparative HPLC on an Agilent 1260 Infinity system with a Symmetry Prep C18, 5 μM, 19 mm × 100 mm column using a gradient (water with 0.1% formic acid and acetonitrile), 0−20 min with 10−100% acetonitrile and 20−23 min with 100% acetonitrile. Monitoring and collection were based on UV−vis absorbance at 210 and 254 nm. Purity analysis was performed using UPLC/MS with UV−vis detection on a Waters Acquity UPLC and Waters XEVO-G2 system using a Waters Acquity CSH C18, 1.7 μm, 2.1 mm × 100 mm column. Samples were run using a gradient with water (0.1% formic acid) and acetonitrile using a flow rate of 0.50 mL/min and a column temperature 60 °C. Gradient parameters: 0−0.7 min, 40% acetonitrile; 0.7− 10.0 min, 40−99% acetonitrile; 10.0−11.0 min, 99% acetonitrile; 11.0−11.1 min, 99−40% acetonitrile; 11.1−13 min, 40% acetonitrile; 3 or 6 μL injection, and detection at 190−300 nm. MS parameters: cap voltage of 3.0 kV, cone
Figure 2. (A) MD simulation snapshot of the triazole 1j in complex with galectin-1. The triazole-stacked His52 is shown with a transparent surface. (B) MD simulation snapshot of the oxazole 2b in complex with galectin-3. The naphthyl-stacked Arg162 is shown with a transparent surface.
presents a small pocket in the same region above the β-face of a bound galactopyranose ligand. This small pocket above is unique to galectin-3, and the naphthyl rings in 2b fit into it in the converging MD structures of 2b in complex with galectin3. Thus, the 1j triazole stacking with the unique His52 side chain in galectin-1 and the 2b naphthyl occupation of the unique galectin-3 subsite above the β-face of the bound galactose ring may, at least partly, explain the galectin-1/ galectin-3 selectivity profiles of 1b and 2b.
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CONCLUSIONS In conclusion, a panel of ten aryltriazole galactopyranosides, nine aryloxazole galactopyranosides, three arylisoxazole galactopyranosides, and three arylpyrazole galactopyranosides were synthesized and evaluated for their galectin affinities (Figure 3). The triazoles were found to be generally selective for galectin-1 over galectin-3 with dissociation constants down 7050
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voltage of 40 kV, Ext 4, source temp of 120 °C, desolvation temp of 500 °C, cone gas of 50 L/h, desolvation gas of 800 L/ h, centroid resolution mode, m/z interval of 50−1200, LockSpray. Calibration: Leu-enkephalin (m/z 556.2771), 0.25 s every 30 s, three reference scans are averaged for lock mass correction. Samples were dissolved in an appropriate solvent to a concentration of 2−10 mg/mL, and polarimetry was performed on a PerkinElmer Model 341 Polarimeter using a sodium lamp at 589 nM with a 90 mm-long 1 mL cell at 20 °C. Synthetic Procedures. Synthesis procedures for the phenyl variants (a) of each reported compound family are presented as typical procedures; complete synthesis procedures are given in the Supporting Information. 1-Phenyl-4-(1-deoxy-β-D-galactopyranosyl)-1H-1,2,3-triazole 1a. (2,3,4,6-Tetra-O-acetyl-1-deoxy-β-galactopyranosyl)ethylene (8) (25 mg, 0.070 mmol) was dissolved in dry acetonitrile (2 mL) with copper(I) iodide (3 mg, 0.014 mmol). Phenyl azide (0.15 mL, 0.074 mmol, 0.5 M in tert-butyl methyl ether) and triethylamine (10 μL, 0.140 mmol) were added. The reaction was left overnight at room temperature, then poured into ethyl acetate (20 mL) and washed with brine (20 mL). The brine was extracted twice with ethyl acetate (20 mL); the organic phases were pooled, dried with anhydrous sodium sulfate, and evaporated. The crude was dissolved in dry methanol (2 mL) containing sodium methoxide (23 mg, 0.420 mmol) and left for 3 h. The reaction was quenched with Amberlite IR-120 (H+) until pH 7, filtered, and evaporated. The crude was purified with preparative HPLC (20 min gradient from 10% acetonitrile/90% water with 0.1% formic acid to 100% acetonitrile) to give 1a (12 mg, 58%). 1H NMR (400 MHz, CD3OD): δ 8.57 (s, 1H, triazole CH), 7.87−7.82 (m, 2H), 7.63−7.55 (m, 2H), 7.53−7.41 (tt, J = 7.5 Hz, 1.2 Hz, 1H), 4.48 (d, J = 9.4 Hz, H1), 4.05−3.97 (m, 2H), 3.82 (dd, J = 11.7 Hz, 8.2, 1H, H6), 3.78−3.71 (m, 2H), 3.67 (dd, J = 9.5 Hz, 3.4 Hz, 1H, H3). 13C NMR: 147.19, 137.03, 129.57, 128.69, 121.58, 120.17, 79.63, 74.93, 74.83, 70.85, 69.58, 61.52. HRMS: M + H; 308.1249 found, 308.1246 calculated. [α]D20 = 26° (c = 0.21101 in methanol.). Purity by HPLC (UV−vis detector 254 nm): 99.9%. 2-Phenyl-5-(1-deoxy-β-D-galactopyranosyl)oxazole 2a. Compound 10a (25 mg, 0.052 mmol) and sodium methoxide (17 mg, 0.314 mmol) were dissolved in dry methanol (4 mL) under nitrogen. After 3 h, the reaction was quenched with Amberlite IR-120 (H+) to pH 7, filtered, and evaporated. Preparative HPLC (20 min gradient from 10% acetonitrile/ 90% water with 0.1% formic acid to 100% acetonitrile) gave 2a (11 mg, 71%). 1H NMR (400 MHz, CD3OD): δ 8.09−8.03 (m, 2H, phenyl H), 7.55−7.50 (m, 3H, phenyl H), 7.31 (s, 1H, oxazole CH), 4.39 (d, J = 10.3 Hz, 1H, H1), 4.09 (t, J = 9.6 Hz, 1H, H2), 4.00 (d J = 3.2 Hz, 1H, H6), 3.83−3.69 (m, 3H), 3.62 (dd, J = 9.3 Hz, 3.3 Hz, 1H, H3). 13C NMR: 161.88, 150.07, 130.52, 128.65, 128.13, 126.92, 126.60, 126.02, 79.64, 74.84, 73.93, 69.39, 69.00, 61.40. HRMS: M + H; 308.1135 found, 308.1134 calculated. [α]D20 = 16° (c = 0.43650 in methanol.). Purity by HPLC (UV−vis detector 254 nm): 99.8%. 3-Phenyl-5-(1-deoxy-β-D-galactopyranosyl)isoxazole 3a. Compound 9a (43 mg, 0.093 mmol) was dissolved in dry THF (3 mL)with hydroxylamine hydrochloride (16 mg, 0.230 mmol) and sodium carbonate (30 mg, 0.188 mmol) followed by refluxing overnight under nitrogen. The reaction was poured into ethyl acetate (30 mL) and washed with brine. The brine was extracted two times with ethyl acetate (30 mL); the
organic phases were pooled, dried with anhydrous sodium sulfate, and evaporated. The crude was dissolved in dry methanol (2 mL) containing sodium methoxide (16 mg) under nitrogen. After 3 h, the reaction was quenched by addition of Amberlite IR-120 (H+) until pH 7, filtered, and evaporated. The crude was purified first with column chromatography (5:1 dichloromethane/methanol) and then with preparative HPLC (20 min gradient from 10% acetonitrile/90% water with 0.1% formic acid to 100% acetonitrile) to give 3a (10 mg, 35%). 1H NMR: 7.87−7.83 (m, 2H), 7.55−7.48 (m, 3H), 6.98 (s, 1H, isoxazole CH), 4.37 (d, J = 10.0 Hz, 1H, H1), 4.01 (d, J = 3.1 Hz, 1H, H4), 3.89 (t, J = 9.4 Hz 1H, H2), 3.81 (dd, J = 10.0 Hz, 4.3 Hz, 1H, H5), 3.77−3.68 (m, 2H), 3.64 (dd, J = 9.4 Hz, 3.3 Hz, 1H, H3). 13C NMR: 169.80, 163.69, 130.00, 128.82, 127.37, 125.32, 98.31, 79.72, 75.13, 74.65, 70.11, 69.42, 61.45. HRMS: M + Na; 330.0962 found, 330.0954 calculated. [α]D20 = 29° (c = 0.29721 in methanol.). Purity by HPLC (UV−vis detector 254 nm): 99.7%. 3-Phenyl-5-(1-deoxy-β-D-galactopyranosyl)-1H-pyrazole 4a. Compound 9a (130 mg, 0.282 mmol) was dissolved in THF (6 mL) followed by addition of sodium carbonate (120 mg, 1.128 mmol) and 64% hydrazine hydrate (130 μL, 1.694 mmol). The reaction mixture was refluxed overnight, cooled to room temperature, filtrated, and evaporated. Column chromatography (5:1 dichloromethane/methanol) followed by preparative HPLC (20 min gradient from 10% acetonitrile/ 90% water with 0.1% formic acid to 100% acetonitrile) gave 4a (34 mg, 40%). 1H NMR: 7.78−7.72 (m, 2H), 7.45−7.38 (m, 2H), 7.33 (tt, J = 7.3 Hz, 1.2 Hz, 1H), 6.77 (s, 1H, pyrazole CH), 4.33 (d, J = 9.9 Hz, 1H, H1), 3.98 (d, J = 3.0 Hz, 1H, H4), 3.90 (t, J = 9.6 Hz 1H, H2), 3.83 (dd, J = 11.2 Hz, 7.1 Hz, 1H, H5), 3.77−3.68 (m, 2H), 3.61 (dd, J = 9.0 Hz, 3.3 Hz, 1H, H3). 13C NMR: 148.24, 146.97, 131.81, 128.44, 127.65, 125.23, 101.12, 79.45, 75.70, 74.89, 70.95, 69.56, 61.57. HRMS: M + H; 307.1298 found, 307.1294 calculated. [α]D20 = 46° (c = 0.3363 in methanol). Purity by HPLC (UV−vis detector at 254 nm): 99.0%. 1-Phenyl-3-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)prop-2-yn-1-one 9a. (2,3,4,6-Tetra-O-acetyl-1-deoxy-βgalactopyranosyl)ethylene (8) (150 mg ,0.421 mmol), bis(triphenylphosphine) palladium(II) dichloride (15 mg, 0.021 mmol) and copper(I) iodide (12 mg, 0.063 mmol) were dissolved in dry THF (5 mL) at room temperature. Benzoyl chloride (54 μL, 0.463 mmol) and triethylamine (0.120 mL, 0.842 mmol) were added. The reaction mixture goes from pale yellow to a deep orange upon triethylamine addition. After 1 h, the reaction mixture was poured into ethyl acetate (40 mL), washed with brine (40 mL); the brine was extracted twice with ethyl acetate (40 mL); the organic phases were pooled, dried with sodium sulfate, and evaporated. Column chromatography (1.5:1 heptane/ethyl acetate) gave 9a (130 mg, 67%). 1H NMR: 8.14−8.09 (m, 2H), 7.69−7.64 (m, 1H), 7.55−7.50 (m, 2H), 5.56 (t, J = 10.2 Hz, 1H, H2), 5.49 (dd, J = 3.4 Hz, 1.0 Hz, 1H, H4), 5.10 (dd, J = 10.2 Hz, 3.3 Hz, 1H, H3), 4.49 (d, J = 9.8 Hz, 1H, H1), 4.21−4.16 (m, 2H), 4.03−3.98 (m, 1H, H5), 2.22 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H). 13C NMR: 176.87, 170.46, 170.20, 170.05, 169.35, 136.09, 134.62, 129.72, 128.71, 86.42, 83.58, 74.99, 71.39, 69.07, 67.71, 67.22, 61.55, 20.74, 20.70, 20.61. [α]D20 = −6° (c = 0.84958 in chloroform). HRMS: M + H; 461.1455 found, 461.1448 calculated. 7051
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2-Phenyl-5-(2,3,4,6-tetra-O-acetyl-1-deoxy-β- D galactopyranosyl)oxazole 10a. (2,3,4,6-Tetra-O-acetyl-1deoxy-β-galactopyranosyl)ethylene (8) (50 mg, 0.140 mmol), 8-methylquinoline N-oxide (29 mg, 0.182 mmol) and [bis(trifluoromethanesulfonyl)imidate](triphenylphosphine)gold(I) 2:1 toluene adduct (11 mg, 0.007 mmol) were dissolved in benzonitrile (1.4 mL), heated to 75 °C under nitrogen, and left for 2 days. Additional 8-methylquinoline Noxide (22 mg) and [bis(trifluoromethanesulfonyl)imidate](triphenylphosphine)gold(I) 2:1 toluene adduct (11 mg) were added after 2 days, and the mixture was left for another 5 days. The reaction mixture was poured into a solution of brine (30 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were pooled, dried with anhydrous sodium sulfate, and evaporated. Column chromatography (2:1 then 1:1 heptane/ethyl acetate) resulted in 28 mg of yellow powdered 10a for a yield of 42%. 1H NMR: 8.10−8.05 (m, 2H), 7.51− 7.47 (m, 3H), 7.28 (s, 1H, oxazole CH), 5.69 (t, J = 9.8 Hz, 1H, H2), 5.55 (dd, J1 = 3.5 Hz, J2 = 1.0 Hz, 1H, H4), 5.20 (dd, J1 = 9.8 Hz, J2 = 3.4 Hz, 1H, H3), 4.66 (d, J = 10.3 Hz, 1H, H1), 4.20−4.09 (m, 3H), 2.25 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 1.94 (s, 3H). 13C NMR: 170.47, 170.35, 170.16, 169.11, 130.77, 128.82, 128.02, 127.06, 126.62, 74.70, 72.70, 72.17, 67.42, 66.82, 61.59, 20.79, 20.74, 20.63, 20.59. HRMS: M + H; 476.1554 found, 476.1557 calculated. [α]D20 = −10° (c = 0.31549 in chloroform). Competitive Fluorescence Polarization Assay. Human galectin-132 and galectin-333 were expressed and purified as earlier described. Competitive fluorescence polarization experiments were performed on a PheraStar FS plate reader with software PHERAstar Mars version 2.10 R3 (BMG, Offenburg, Germany) with the fluorescent probe (3,3′-dideoxy-3-[4(fluorescein-5-yl-carbonylaminomethyl)-1H-1,2,3-triazol-1-yl]3′-(3,5-di-methoxybenzamido)-1,1′-sulfanediyl-di-β-D-galactopyranoside.32 Specific experimental conditions were a galectin1 concentration of 0.5 μM together with the fluorescent probe at a concentration of 20 nM for the galectin-1 assay and a galectin-3 concentration of 0.2 μM together with the fluorescent probe at a concentration of 20 nM for the galectin-3 assay. Inhibitors were dissolved in dimethylsulfoxide (analytical grade) to a concentration of 20 mM, diluted with PBS for three to six different concentrations, and tested in duplicates, twice for galectin affinity using a competitive fluorescence polarization assay as earlier described.30 The highest inhibitor concentrations tested were 1 mM due to solubility limitations at a higher concentration. Dissociation constant averages and SEM were calculated from two to eight single-point measurements showing between 20 and 80% inhibition. Molecular Modeling. Conformational searches for compounds 1b−4b were performed with the OPLS3 force field in MacroModel (Schrödinger Release 2017−3: MacroModel, Schrödinger, LLC, New York, NY, 2017) using default settings and with the mixed torsional/low-mode sampling method. Molecular dynamics simulations were performed with the OPLS3 force field in Desmond (Schrödinger Release 2017−3: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2017; Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2017) using default settings except for the length of the simulation and with the use of light harmonic constraints (1 kcal/mol/Å) on all stranded backbone atoms and the galactose O4 atom. Nonminimized conformations of 1j were positioned to replace
lactose in the binding site of galectin-1 (PDB id 1GWZ) with the galactose ring in an orientation identical to that in lactose and subjected to short 10 ns molecular dynamics simulations, which drifted toward a complex geometry where the 1j triazole CH was parallel to the galactose H1. Subsequently, a starting conformation with the 1j triazole CH parallel to the galactose H1 was subjected to a 200 ns molecular dynamics simulation. Nonminimized conformations of 2b were positioned to replace lacNAc in the binding site of galectin-3 (PDB id IKJL) with the galactose ring in an orientation identical to that in lacNAc and subjected to short 10 ns molecular dynamics simulations, which also drifted toward a complex geometry where the 2b oxazole CH was parallel to the galactose H1. Subsequently, a starting conformation with the 2b oxazole CH parallel to the galactose H1 was subjected to a 200 ns molecular dynamics simulation.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00320. Synthetic procedures and physical data for compounds 1b−1j, 2b−2i, 3b, 3c, 4b, 4c, 9b, 9c, and 10b−10i; 1H and 13C NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hakon Leffler: 0000-0003-4482-8945 Ulf J. Nilsson: 0000-0001-5815-9522 Author Contributions
A.D. performed the experiments. U.J.N. and H.L. conceptualized the work and designed the project. A.D. wrote the manuscript with contributions from U.J.N. and H.L. All authors have given approval to the final version of the manuscript. Funding
The Swedish Research Council (Grant No. 621−2012-2978 and 621−2016-03667), Royal Physiographic Society, Lund, Sweden, a project grant awarded by the Knut and Alice Wallenberg Foundation (KAW 2013.0022), and Galecto Biotech AB, Sweden. Notes
The authors declare the following competing financial interest(s): H.L. and U.N. are shareholders in Galecto Biotech AB.
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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council (grant no. 621-2012-2978 and 621-2016-03667), Royal Physiographic Society, Lund, Sweden, Knut and Alice Wallenberg Foundation (KAW 2013.0022), and Galecto Biotech AB, Sweden. The authors kindly thank Barbro Kahl Knutson for the fluorescence polarization measurements and Sofia Essén for high-resolution mass spectrometry and purity determinations. 7052
DOI: 10.1021/acsomega.9b00320 ACS Omega 2019, 4, 7047−7053
ACS Omega
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Article
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ABBREVIATIONS CDR, Carbohydrate recognition domain; lac, lactose; lacNAc, N-Acetyllactosamine; VEGFR, vascular endothelial growth factor receptor; EGFR, epidermal growth factor receptor
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DOI: 10.1021/acsomega.9b00320 ACS Omega 2019, 4, 7047−7053
