Synthesis of N-Substituted Iminosugar C-Glycosides and Evaluation as Promising α-Glucosidase Inhibitors

A series of N-substituted iminosugar C-glycosides were synthesized and tested for α-glucosidase inhibition. The results suggested that 6e is a promising and potent α-glucosidase inhibitor. Enzymatic kinetic assays indicated that compound 6e may be classified as an uncompetitive inhibitor. The study of structure-activity relationships of those iminosugars provided a starting point for the discovery of new α-glucosidase inhibitors.


Introduction
Diabetes mellitus (DM) is one of the most common chronic diseases with 1.5 million deaths each year [1]. According to the International Diabetes Federation (IDF), the prevalence of DM was estimated to be 783 million by 2045 [2]. Type 2 diabetes mellitus (T2DM) is the most common form of this disease and almost 80-90% of all diabetic patients are T2DM [3]. Generally, diabetic pathogeny mostly focuses on genetic, lifestyle, environmental toxins, and advancing age [4]. Current antidiabetic drugs to treat T2DM include incretin mimetic, biguanides, sulfonylureas, thiazolidinediones, dipeptidyl peptidase 4 inhibitors, sodium-glucose co-transporter 2 inhibitors, glucagon-like peptide 1 agonists, and α-glucosidase inhibitors [5,6]. α-Glucosidase (α-D-glucoside glucohydrolase; EC3.2.1.20) is the glycoside hydrolase specifically hydrolyzing 1,4-α-glucopyranosides bond to produce α-glucose, located in the brush border of the small intestine [7]. α-Glucosidase inhibitors are thought to be the most efficient agents to reduce postprandial hyperglycemia [8]. Thus, it has been considered as one of the most popular targets for the treatment of diabetes [9]. At present, α-glucosidase inhibitors have been marketed as therapeutic drugs for T2DM, including acarbose [10], voglibose [10], and miglitol [11], and have been used in reducing plasma glucose levels and postprandial hyperglycemia ( Figure 1) [12]. However, their widespread application has been limited by the complicated multistep procedures needed for their synthesis and undesirable side effects such as gastrointestinal intolerability, diarrhoea and flatulence [13]. Therefore, the search for new small molecules possessing potent α-glucosidase inhibitory activity and minimal side effects has attracted significant attention for many years.
Iminosugars [14], formed by the replacement of sugar ring oxygen with nitrogen, are well known for their ability to selectively inhibit glycosidases [15]. This kind of scaffold inhibits glycosidases by mimicking the substrate transition states with oxacarbenium ion character during the hydrolysis reaction catalyzed by glycosidases [16]. In the past two decades, more than 100 iminosugars have been isolated from plants and microorganisms [17][18][19]. Besides, hundreds of their analogues and derivatives were synthesized and evaluation of their biological activity was assayed, especially as glucosidase inhibitors [20]. However, various comparative studies on simple glycolipid analogues have demonstrated a marked dependence of the potency of the inhibitors upon the position of the alkyl chain (1-C-or N-alkyl derivatives) [21]. Meanwhile, Butters, T. D. and co-workers also found that the presence of a hydrophobic N-alkyl chain of iminosugars provided an increase in inhibitory potency to glucosidases [22]. As a part of our continuing interest in the synthesis of novel iminosugars and their α-glucosidase inhibition, we report a library of N-substituted iminosugar C-glycosides and their structure-activity relationships against α-glucosidase.
olecules 2022, 27,5517 Figure 1. Important clinically used α-glucosidase inhibitors for the treatment of T2D Iminosugars [14], formed by the replacement of sugar ring oxygen wit well known for their ability to selectively inhibit glycosidases [15]. This ki inhibits glycosidases by mimicking the substrate transition states with oxa character during the hydrolysis reaction catalyzed by glycosidases [16]. In decades, more than 100 iminosugars have been isolated from plants and m [17][18][19]. Besides, hundreds of their analogues and derivatives were synthes uation of their biological activity was assayed, especially as glucosidase i However, various comparative studies on simple glycolipid analogues strated a marked dependence of the potency of the inhibitors upon the p alkyl chain (1-C-or N-alkyl derivatives) [21]. Meanwhile, Butters, T. D. an also found that the presence of a hydrophobic N-alkyl chain of iminosugar increase in inhibitory potency to glucosidases [22]. As a part of our continu the synthesis of novel iminosugars and their α-glucosidase inhibition, we r of N-substituted iminosugar C-glycosides and their structure-activity against α-glucosidase.

Synthesis of Iminosugar C-Glycosides
The synthesis of the compound 1-C-Acetylmethyl-5-deoxy-5-amino-α-D-ribopyranoside was based on our PREVIOUS synthetic strategy (Scheme 1) [23]. Nucleophilic substitution of known mesylate 1 with NaN 3 generated 5-azido-C-riboside 2. Reduction of the azido group to amine after terminal olefin oxidation, which was immediately treated with the saturated sodium methoxide-methanol solution at room temperature overnight. Purified the crude with silica gel flash column chromatography (ethyl acetate/methanol, 2:1, R f = 0.2) to afford the compound 4.
Based on previous work, the inhibitory potency of C-glycosides 4 has been demonstrated for the α-glucosidase [23]. To obtain higher activity compounds, the compound 4 was structurally modified by introducing alkyl side chains on its nitrogen atom. The reaction conditions were screened (Table 1). Under the conditions of −20 • C-r. t., NaBH(OAc) 3 and MeOH, good yield was obtained by reductive amination. Finally, a variety of aldehydes could be attached to the structure 4 through reductive amination and obtained N-substituted iminosugar C-glycosides 5 (Scheme 2, Figure 2). Based on previous work, the inhibitory potency of C-glycosides 4 h strated for the α-glucosidase [23]. To obtain higher activity compounds, was structurally modified by introducing alkyl side chains on its nitrog action conditions were screened (Table 1). Under the conditions NaBH(OAc)3 and MeOH, good yield was obtained by reductive aminati riety of aldehydes could be attached to the structure 4 through reductiv obtained N-substituted iminosugar C-glycosides 5 (Scheme 2, Figure 2).  Based on previous work, the inhibitory potency of C-glycosides 4 has been demonstrated for the α-glucosidase [23]. To obtain higher activity compounds, the compound 4 was structurally modified by introducing alkyl side chains on its nitrogen atom. The reaction conditions were screened (Table 1). Under the conditions of −20 °C-r. t., NaBH(OAc)3 and MeOH, good yield was obtained by reductive amination. Finally, a variety of aldehydes could be attached to the structure 4 through reductive amination and obtained N-substituted iminosugar C-glycosides 5 (Scheme 2, Figure 2). Based on previous work, the inhibitory potency of C-glycosid strated for the α-glucosidase [23]. To obtain higher activity compou was structurally modified by introducing alkyl side chains on its n action conditions were screened (Table 1). Under the condit NaBH(OAc)3 and MeOH, good yield was obtained by reductive am riety of aldehydes could be attached to the structure 4 through red obtained N-substituted iminosugar C-glycosides 5 (Scheme 2, Figur  Figure 2. Synthesized N-substituted iminosugar C-glycosides (5a-5π).

α-Glucosidase Inhibitory Assays
The inhibitory potency of the synthesized N-substituted iminosugar C-glycoside was assessed by testing the compounds in assays for the α-glucosidase from yeast [2 Compared to the positive control Acarbose (Glucobay ® ), the lead compound (4) sugge weak inhibitory activity against α-glucosidase ( Figure 3). To improve the inhibitory p tency against α-glucosidase of the lead compound, construction of the N-functionaliz iminosugars was carried out. Installation of the N atom with aliphatic chain (5a, 5b, showed no improvement of α-glucosidase inhibition, where shortened or lengthened alkylated chain was invalid. Alternatively, N atom was modified with various benzald hydes, giving a library of iminosugar C-glycosides containing phenyl. It was fascinati to discover that the introduction of a hydroxyl group at C4-position (5λ) of the aroma ring improved the enzyme inhibition, whereas introducing a hydroxyl at C2 or C3 (5 5π) reduced the inhibitory potency significantly. This indicates that a hydroxy in C4-p sition of the phenyl contributed to improving the enzyme inhibitory activity. These fin ings demonstrate that subtle changes in the iminosugar N-functionalized region may

α-Glucosidase Inhibitory Assays
The inhibitory potency of the synthesized N-substituted iminosugar C-glycosides 5 was assessed by testing the compounds in assays for the α-glucosidase from yeast [24]. Compared to the positive control Acarbose (Glucobay ® ), the lead compound (4) suggests weak inhibitory activity against α-glucosidase ( Figure 3). To improve the inhibitory potency against α-glucosidase of the lead compound, construction of the N-functionalized iminosugars was carried out. Installation of the N atom with aliphatic chain (5a, 5b, 5c) showed no improvement of α-glucosidase inhibition, where shortened or lengthened N-alkylated chain was invalid. Alternatively, N atom was modified with various benzaldehydes, giving a library of iminosugar C-glycosides containing phenyl. It was fascinating to discover that the introduction of a hydroxyl group at C4-position (5λ) of the aromatic ring improved the enzyme inhibition, whereas introducing a hydroxyl at C2 or C3 (5θ, 5π) reduced the inhibitory potency significantly. This indicates that a hydroxy in C4-position of the phenyl contributed to improving the enzyme inhibitory activity. These findings demonstrate that subtle changes in the iminosugar N-functionalized region may result in remarkable enzyme specificity. To identify more potent inhibitors, we also introduced halogen atoms onto the aromatic ring, and discovered that positioning Cl or Br at C2 (5r, 5v) enhanced the enzyme inhibition. However, the location of the halogen atom at C3 or C4-position (5q, 5t, 5u, 5w, 5x, 5y, 5z) of the phenyl doesn't show any significant improvement for the inhibitory activity on the enzyme. The same result was obtained when introducing electron-donating groups as CH 3 (5j, 5k), OCH 3 (5o, 5p) or electron-withdrawing groups such as CF 3 (5l), NO 2 (5n), CN(5m).
les 2022, 27, 5517 5 when introducing electron-donating groups as CH3(5j, 5k), OCH3(5o, 5p) or elec withdrawing groups such as CF3(5l), NO2(5n), CN(5m). Given the above results, and in order to ascertain whether hydroxyl and halog oms would have a synergistic effect on the enhancement of the enzyme inhibitio synthesized a series of iminosugar C-glycosides 6 containing hydroxyl at C4-positi phenyl and introducing halogen atoms at different positions (Scheme 3, Figure 4) assay of their inhibition against α-glucosidase was listed in Table 2. Compared with pound 5λ, the installation of halogen atoms at C3-position (6a, 6b, 6c and 6i) can sli improve the inhibition potency on the basis of the presence of hydroxyl at C4 pos However, introducing an alkoxyl at C3-position (6f, 6g) severely diminishes the in tion, which may be due to the fact that the substituents are too large to allow the gu fit into the enzyme pocket. Clearly, iminosugars substituted with chlorine at the C position (or at C2) provide greater enhancement of inhibition than others, such which has a 2-fold stronger inhibitory potency than the positive control Acarbose reason for this improvement in inhibition provided by additional chlorine atoms at position (or C2-position) and hydroxyl at C4-position on phenyl is not known but m due to structural and spatial features of the enzyme, which allow the hydroxyl to with active site and chlorine atoms matching the pocket well. Given the above results, and in order to ascertain whether hydroxyl and halogen atoms would have a synergistic effect on the enhancement of the enzyme inhibition, we synthesized a series of iminosugar C-glycosides 6 containing hydroxyl at C4-position on phenyl and introducing halogen atoms at different positions (Scheme 3, Figure 4). The assay of their inhibition against α-glucosidase was listed in Table 2. Compared with compound 5λ, the installation of halogen atoms at C3-position (6a, 6b, 6c and 6i) can slightly improve the inhibition potency on the basis of the presence of hydroxyl at C4 position. However, introducing an alkoxyl at C3-position (6f, 6g) severely diminishes the inhibition, which may be due to the fact that the substituents are too large to allow the guest to fit into the enzyme pocket. Clearly, iminosugars substituted with chlorine at the C2, 6-position (or at C2) provide greater enhancement of inhibition than others, such as 6e, which has a 2-fold stronger inhibitory potency than the positive control Acarbose. The reason for this improvement in inhibition provided by additional chlorine atoms at C2, 6-position (or C2-position) and hydroxyl at C4-position on phenyl is not known but may be due to structural and spatial features of the enzyme, which allow the hydroxyl to bind with active site and chlorine atoms matching the pocket well.
Molecules 2022, 27, 5517 5 when introducing electron-donating groups as CH3(5j, 5k), OCH3(5o, 5p) or elect withdrawing groups such as CF3(5l), NO2(5n), CN(5m). Given the above results, and in order to ascertain whether hydroxyl and haloge oms would have a synergistic effect on the enhancement of the enzyme inhibition synthesized a series of iminosugar C-glycosides 6 containing hydroxyl at C4-positio phenyl and introducing halogen atoms at different positions (Scheme 3, Figure 4). assay of their inhibition against α-glucosidase was listed in Table 2. Compared with c pound 5λ, the installation of halogen atoms at C3-position (6a, 6b, 6c and 6i) can slig improve the inhibition potency on the basis of the presence of hydroxyl at C4 posi However, introducing an alkoxyl at C3-position (6f, 6g) severely diminishes the in tion, which may be due to the fact that the substituents are too large to allow the gue fit into the enzyme pocket. Clearly, iminosugars substituted with chlorine at the C position (or at C2) provide greater enhancement of inhibition than others, such a which has a 2-fold stronger inhibitory potency than the positive control Acarbose. reason for this improvement in inhibition provided by additional chlorine atoms at C position (or C2-position) and hydroxyl at C4-position on phenyl is not known but ma due to structural and spatial features of the enzyme, which allow the hydroxyl to with active site and chlorine atoms matching the pocket well.   The inhibitory mechanisms of glucosidases were divided into two distinct mech nisms: reversible inhibitors that have a high affinity for the enzyme, and irreversible hibitors that react with carboxylic acid of the active site of the enzyme [25]. To determ which mechanism 6e belonged to, we removed the unreacted inhibitor from the enzym solution by ultrafiltration [26], and examined whether the activity would be recovered not by the method described in the literatures. When an enzyme solution containing was subjected to ultrafiltration and then redissolved with the same concentration, the glucosidase activity was substantially recovered ( Figure 5A). From this result, we can co clude that 6e is a reversible inhibitor against α-glucosidase.
Further analysis of the compound 6e, in order to reveal the type of enzyme inhibiti on α-glucosidase, was done with different substrate concentration [7]. Based on L eweaver-Burk plots ( Figure 5B), compound 6e may be classified as an uncompetitive hibitor with Ki = 8.6 μM. These results indicate that 6e may reversibly combine with on the E-S (enzyme-substrate) complex and inhibit the activity of this enzyme. It is spec cally mentioned that uncompetitive inhibitors have a benefit over competitive inhibito such as therapeutic drugs, since the inhibition is not overcome even when the substr concentration reaches saturation.  The inhibitory mechanisms of glucosidases were divided into two distinct mechanisms: reversible inhibitors that have a high affinity for the enzyme, and irreversible inhibitors that react with carboxylic acid of the active site of the enzyme [25]. To determine which mechanism 6e belonged to, we removed the unreacted inhibitor from the enzyme solution by ultrafiltration [26], and examined whether the activity would be recovered or not by the method described in the literatures. When an enzyme solution containing 6e was subjected to ultrafiltration and then redissolved with the same concentration, the α-glucosidase activity was substantially recovered ( Figure 5A). From this result, we can conclude that 6e is a reversible inhibitor against α-glucosidase.
Further analysis of the compound 6e, in order to reveal the type of enzyme inhibition on α-glucosidase, was done with different substrate concentration [7]. Based on Lineweaver-Burk plots ( Figure 5B), compound 6e may be classified as an uncompetitive inhibitor with Ki = 8.6 µM. These results indicate that 6e may reversibly combine with only the E-S (enzyme-substrate) complex and inhibit the activity of this enzyme. It is specifically mentioned that uncompetitive inhibitors have a benefit over competitive inhibitors such as therapeutic drugs, since the inhibition is not overcome even when the substrate concentration reaches saturation.
As shown in Figure 6, compound 6e forms hydrogen bonds with amino acid residues Lys155, Phe157, Asn241 and Arg312, as well as hydrophobic interactions with amino acid residues Leu176, Phe177, Leu218, His239, Pro240 and Pro309, which were close to the catalytic active site (Asp214) [38] of α-glucosidase, supporting the experimental conclusion that it is a noncompetitive inhibitor.
As shown in Figure 6, compound 6e forms hydrogen bonds with amino acid residues Lys155, Phe157, Asn241 and Arg312, as well as hydrophobic interactions with amino acid residues Leu176, Phe177, Leu218, His239, Pro240 and Pro309, which were close to the catalytic active site (Asp214) [38] of α-glucosidase, supporting the experimental conclusion that it is a noncompetitive inhibitor.

In Silico Analysis
Because some compounds showed good inhibitory activities, we chose to con silico researches to evaluate their drug-likeness and pharmacokinetic properties tha carried out using the SwissADME [39] and the admetSAR [40] platforms. Satisfyin compounds were found to have excellent obedience (75-100%) with different dru ness filters (Lipinski [41], Ghose [42], Veber [43], Egan [44], and Muegge [45]) (Tab   All reactions sensitive to air or moisture were carried out under nitrogen or argon with anhydrous solvents. All reagents were purchased from commercial suppliers and used without further purification unless otherwise noted. TLC was performed by using silica gel GF254 precoated plates (0.20-0.25 mm thickness) with a fluorescent indicator. Visualization of TLC plates was achieved by UV light (254 nm) and a typical TLC indicator solution (10 % sulfuric acid/ethanol solution). Column chromatography was performed on silica gel 90, 200-300 mesh. Optical rotations were measured with a Perkin-Elmer M341 digital polarimeter. 1 H and 13 C NMR spectra (600 and 150 MHz, respectively) were recorded with a Bruker Avance 600 spectrometer. 1 H NMR chemical shifts are reported in ppm (δ) relative to tetramethylsilane (TMS) with the solvent resonance employed as the internal standard (DMSO-d 6 or CD 3 OD). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constants [Hz]. 13 C NMR chemical shifts are reported in ppm from TMS with the solvent resonance as the internal standard (DMSO-d 6 or CD 3 OD). ESI-HRMS data were recorded with a BioTOF Q instrument.

Synthetic Procedures
A suspension of compound 4 (40 mg, 0.2 mmol) containing activated 4Å molecular sieves in anhydrous methanol (3 mL) was stirred at room temperature for 10 min. After cooling to −20 • C, the corresponding aldehydes (0.6 mmol, 3eq) and NaBH(OAc) 3 (0.6 mmol, 3eq) were added, and the solution was stirred for 3 h under an argon atmosphere. The reaction solution was concentrated in vacuo and purified by silica gel flash column chromatography (dichloromethane/methanol, 25:1→5:1) to afford colorless syrupy products.