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Synthesis of Galectin Inhibitors by Regioselective 3′-O-Sulfation of Vanillin Lactosides Obtained under Phase Transfer Catalysis

Vanillin-based lactoside derivatives were synthetized using phase-transfer catalyzed reactions from per-O-acetylated lactosyl bromide. The aldehyde group of the vanillin moiety was then modified to generate a series of related analogs having variable functionalities in the para- position of the aromatic residue. The corresponding unprotected lactosides, obtained by Zemplén transesterification, were regioselectively 3′-O-sulfated using tin chemistry activation followed by treatment with sulfur trioxide-trimethylamine complex (Men3N-SO3). Additional derivatives were also prepared from the vanillin’s aldehyde using a Knoevenagel reaction to provide extended α, β-unsaturated carboxylic acid which was next reduced to the saturated counterpart.

In this paper, we aimed to combine both modifications to a lactopyranoside scaffold by incorporating aromatic aglycons simultaneously to an O-3′ sulfate group (Figure 1). This combined choice was dictated by the fact that a few galectin members have been shown to bind preferentially to sulfated glycans. This was particularly through for Gal-1 [23,24], -3 [15], -4 [25,26], and -8 [27]. In the latter case, recent modeling and X-ray experiments demonstrated that the beneficial interactions were due to favorable electrostatic interactions with arginine residues (Arg-45 and Arg-59) [4,27]. Furthermore, even though several hydrophobic aglycons have been advantageous in the binding events using affinity measurements by ITC, we choose the natural vanillin as aglycon because of its wellestablished lack of toxicity, the presence of a large amount of vanillin glucoside (glucovanillin) in foodstuffs [42], and the well-known antioxidant properties of phenolic glycosides.

Results and Discussion
Over the years, Phase Transfer Catalysis (PTC) has emerged as an efficient and practical methodology for stereoselective glycosidation [43,44] and related anomeric substitutions. Under our previously optimized conditions for phenolic glycosides (TBAHS, EtOAc, 1M Na 2 CO 3 ), we treated peracetylated lactosyl bromide 1 [43] with vanillin derivatives 2, 3, and 4 to afford lactosides 5, 7, and 8 in 80, 66, and 58% yields, respectively (Scheme 1). The major by-product of the reactions was the usual 2-acetoxy-lactal ( 1 H-NMR of H1 at δ 6.61 ppm) resulting from the HBr S N 2 elimination process [43,44]. The disappearance of the anomeric doublet of the bromide 1 at δ 6.52 ppm with a J 1,2 coupling constant of 4.0 Hz was replaced by a new doublet at δ 5.10 ppm with a distinctive trans coupling constant of J 1,2 of 7.4 Hz in compound 5. The same held for methyl ester 7 (H1: δ 5.05, J 1,2 = 7.4 Hz) and tert-butyl ester 8 (H1: δ 5.03, J 1,2 = 7.5 Hz). As previously demonstrated [42][43][44], these PTC conditions afforded complete stereoselectivity in favor of anomeric inversion from the α-bromide 1 to β-glycosides 5, 7, and 8 exclusively. Since we also wanted to explore the role played by the carboxylic function in the para-position of the vanillin residue, coupled to the fact that we could not hydrolyze the acetate protecting groups in derivative 7 and 8 without losing the esters (vide infra), we opted for the oxidation of the native vanillin's aldehyde in 5 using permanganate treatment (KMnO 4 , H 2 O, 80 • C, 20 min) which afforded acid 6 in 52% yield, showing disappearance of the aldehyde proton at δ 9.91 ppm. All compounds were fully characterized by NMR spectroscopy ( 1 H, 13 C), mass spectrometry, and the datasets agreed with literature data when known (see experimental section).

Results and Discussion
Over the years, Phase Transfer Catalysis (PTC) has emerged as an efficient and practical methodology for stereoselective glycosidation [43,44] and related anomeric substitutions. Under our previously optimized conditions for phenolic glycosides (TBAHS, EtOAc, 1M Na2CO3), we treated peracetylated lactosyl bromide 1 [43] with vanillin derivatives 2, 3, and 4 to afford lactosides 5, 7, and 8 in 80, 66, and 58% yields, respectively (Scheme 1). The major by-product of the reactions was the usual 2-acetoxy-lactal ( 1 H-NMR of H1 at δ 6.61 ppm) resulting from the HBr SN2 elimination process [43,44]. The disappearance of the anomeric doublet of the bromide 1 at δ 6.52 ppm with a J1,2 coupling constant of 4.0 Hz was replaced by a new doublet at δ 5.10 ppm with a distinctive trans coupling constant of J1,2 of 7.4 Hz in compound 5. The same held for methyl ester 7 (H1: δ 5.05, J1,2 = 7.4 Hz) and tert-butyl ester 8 (H1: δ 5.03, J1,2 = 7.5 Hz). As previously demonstrated [42][43][44], these PTC conditions afforded complete stereoselectivity in favor of anomeric inversion from the α-bromide 1 to β-glycosides 5, 7, and 8 exclusively. Since we also wanted to explore the role played by the carboxylic function in the para-position of the vanillin residue, coupled to the fact that we could not hydrolyze the acetate protecting groups in derivative 7 and 8 without losing the esters (vide infra), we opted for the oxidation of the native vanillin's aldehyde in 5 using permanganate treatment (KMnO4, H2O, 80 °C, 20 min) which afforded acid 6 in 52% yield, showing disappearance of the aldehyde proton at δ 9.91 ppm. All compounds were fully characterized by NMR spectroscopy ( 1 H, 13 C), mass spectrometry, and the datasets agreed with literature data when known (see experimental section). The peracetylated intermediates 5, 7, 8 were next de-O-acetylated under the classical Zemplén conditions (NaOMe, MeOH) in essentially quantitative yields in all cases to give free lactosides 9, 13, and 15 (Scheme 2). Following sequential treatment of the unprotected lactosides with dibutyltin oxide (Bu2SnO, DMF, PhMe, 90° C, 6h) and regioselective sulfation [45] using sulfur trioxide-trimethylamine complex (Me3N . SO3), the 3′-O-sulfated lactosides 10, 14, 16 (Scheme 2) were obtained in good to excellent yields (81%-quantitative). The regioselectivity of this transformation is well-known and has been explained through the formation of a cyclic stannylene complex at the unique cis-3′,4′-dihydroxyl groups of the galactoside moiety [46,47]. The aldehyde functions of vanillin lactoside 9 and 3′-O-sulfated lactoside 10 were reduced using NaBH4 in MeOH (rt, 3h) to give vanillin lactoside analogs 11 and 12 in excellent 95% and 88% yields, respectively. The position of the sulfate groups were readily confirmed on the basis of the H-3′ downfield shift from ~3.66 ppm to ~4.28 ppm (SI) together with the characteristic 13 C-NMR chemical shift displacement of the 3′-carbon, which usually appears ~7 ppm downfield (~ δ 80 ppm) from The peracetylated intermediates 5, 7, 8 were next de-O-acetylated under the classical Zemplén conditions (NaOMe, MeOH) in essentially quantitative yields in all cases to give free lactosides 9, 13, and 15 (Scheme 2). Following sequential treatment of the unprotected lactosides with dibutyltin oxide (Bu 2 SnO, DMF, PhMe, 90 • C, 6h) and regioselective sulfation [45] using sulfur trioxide-trimethylamine complex (Me 3 N . SO 3 ), the 3'-O-sulfated lactosides 10, 14, 16 (Scheme 2) were obtained in good to excellent yields (81%-quantitative). The regioselectivity of this transformation is well-known and has been explained through the formation of a cyclic stannylene complex at the unique cis-3',4'-dihydroxyl groups of the galactoside moiety [46,47]. The aldehyde functions of vanillin lactoside 9 and 3'-Osulfated lactoside 10 were reduced using NaBH 4 in MeOH (rt, 3h) to give vanillin lactoside analogs 11 and 12 in excellent 95% and 88% yields, respectively. The position of the sulfate groups were readily confirmed on the basis of the H-3' downfield shift from~3.66 ppm tõ 4.28 ppm (SI) together with the characteristic 13 C-NMR chemical shift displacement of the 3'-carbon, which usually appears~7 ppm downfield (~δ 80 ppm) from the unsubstituted precursors at~δ 73 ppm [47]. Dept 135 13 C-NMR analysis was required to ensure that the regioselective sulfation was unequivocally performed at the C-3' position in order to show the absence of signals attributed to C-6 and C-6's modifications (usually at δ 59-62 ppm [47]. NMR COSY and HSQC experiments were also used to unambiguously correlate the sulfation process and regioselectivity.
the unsubstituted precursors at ~δ 73 ppm [47]. Dept 135 13 C-NMR analysis was required to ensure that the regioselective sulfation was unequivocally performed at the C-3' position in order to show the absence of signals attributed to C-6 and C-6's modifications (usually at δ 59-62 ppm [47]. NMR COSY and HSQC experiments were also used to unambiguously correlate the sulfation process and regioselectivity.

General Synthetic Methods
All reactions in organic medium were performed in standard oven dried glassware under an inert atmosphere of nitrogen using freshly distilled solvents. Solvents and reagents were deoxygenated, when necessary by purging with nitrogen. All reagents were used as supplied without prior purification unless otherwise stated, and obtained from Sigma-Aldrich Chemical Co. Ltd.(St. Louis, MO, USA) Reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel 60 F254 precoated plates (E. Merck (Darmstadt, Germany)) and compounds were visualized with a 254 nm UV lamp, a mixture of iodine/silica gel and/or mixture of ceric ammonium molybdate solution (100 mL H 2 SO 4 , 900 mL H 2 O, 25 g (NH 4 ) 6 Mo 7 O 24 H 2 O, 10 g Ce(SO 4 ) 2 ), and subsequent spots development by gentle warming with a heat-gun. Purifications were performed by silica gel flash column chromatography using Silica (60 Å, 40-63 µm) with the indicated eluent. NMR spectroscopy was used to record 1 H-NMR and 13

General Synthetic Procedure B: Zemplén Transesterification Reaction
To a solution of lactosides 5, 6, 7, 8, 17 and 19 in dry methanol was added a solution of sodium methoxide (1 M in MeOH, 0.1 equiv.). After stirring at room temperature for 1-2 h, the reaction was completed and then neutralized by addition of ion-exchange resin (Amberlite IR 120 H + ). The solution was filtered and evaporated in vacuo to afford the de-O-acetylated lactosides as white powders (yield 95%-quant.)

General Synthetic Procedure C: Preparation of 3'-O-sulfated Lactosides
A mixture of deacetylated lactosides (1 equiv.) and dibutyltin oxide (Bu 2 SnO, 1.15 equiv.) in DMF/toluene (6 mL/3 mL) was stirred at 90 • C for 6 h. The solution was then concentrated and sulfur trioxide-trimethylamine complex (Me 3 N . SO 3 ) (1.3 equiv.) and dry DMF (6 mL) were added. After stirring at room temperature for 17 h, the reaction was quenched with water and evaporated under vacuum. The residue was purified through a column of DOWEX Marathon C (Na + ) and eluted with H 2 O to obtain the pure 3'-O-sulfated lactosides as white powder after lyophilization (yields 82%-quantitative). To a solution of per-O-acetylated lactose [49] (14.2 g, 21 mmoL) in anhydrous CH 2 Cl 2 (63 mL) was added hydrobromic acid (33% in AcOH, 47.9 mL). The reaction mixture was stirred at room temperature for 1 h, then neutralized with saturated aqueous NaHCO 3 and washed with brine. The organic layer was dried over Na 2 SO 4 and concentrated under reduced pressure to give lactosyl bromide 1 (13.6 g, 93%) as a white solid. Its spectroscopic data agreed well with those of the literature [49].

Methyl-3-methoxy-4-(3'-O-sulfo-β-D-lactopyranosyloxy)benzylic Alcohol, Sodium Salt (12)
The reduction of compound 10 by NaBH 4 (1.2 equiv.) in methanol gave the compound 12 after 3 h of agitation at room temperature; compound 12 was obtained as a white powder  Figure 1 DS Biovia Discovery Studio 2020 (https://www.3ds.com) was used for the docking experiments. Galectin-8 N-terminal crystallographic data were obtained from the Protein Data Bank as PDB 3AP6 in which lactose 3'-O-sulfate had been co-crystallized. The crystallographic data of p-nitrophenyl lactoside were next obtained from https://pubchem.ncbi. nlm.nih.gov/ as accession no CID 11812612. The two sugar derivatives were superimposed using the tool of the DS software by the tether method and the aglycon (PNP) was next modified into vanillin.

Conclusions
A series of extended aromatic lactosides harboring vanillin pharmacophores at the anomeric position together with their corresponding O-3'-sulfated analogs were efficiently prepared using PTC and tin acetal-catalyzed stereo-(β-anomer) and regioselective (O-3'-position) transformations, respectively. The para-position of the aromatic moiety was further adjusted to afford additional modifications, which would allow evaluating the detailed role of this area upon binding to various galectin family members, especially those (Gal-1, -3, and -8) shown to be particularly sensitive to the presence of sulfation. As previously seen in the case of E. coli FimH inhibitors, the design of glycomimetics with optimized aglycons permitted to generate drug-like mannosides of real therapeutic interests [50]. In the present case, the numerous challenges raised for the identification of potent and selective galectin ligands of therapeutic interest are even more exacerbated by the 15 family members having their own physiological function and subtle structural differences. Moreover, the fact that galectins possess extended and shallow binding sites capable of accommodating longer oligosaccharides offers several opportunities in the design of improved ligands. When the actual double modification strategy will be coupled to the third one involving multivalent presentation, further improvement in affinity and selectivity would be achievable, as recently seen when the TD139 clinical phase 2 candidate [10][11][12][13] was coupled to a multivalent protein scaffold [41]. Preliminary data with Gal-3 have been obtained for compounds 13 and 14, which clearly indicated their potential [15]. Analogous modifications using the N-acetyllactosamine scaffold are in preparation and await comparative studies.