Stereocontrolled synthesis of new iminosugar lipophilic derivatives and evaluation of biological activities.

Iminosugars' similarity to carbohydrates determines the exceptional potential for this class of polyhydroxylated alkaloids to serve as potential drug candidates for a wide variety of diseases such as diabetes, lysosomal storage diseases, cancer, bacterial and viral infections. The presence of lipophilic substituents has a significant impact on their biological activities. This work reports the synthesis of three new pyrrolidine lipophilic derivatives O-alkylated in C-6 position. The biological activities of our iminosugars' collection were tested in two cancer cell lines and, due to the pharmaceutical potential, in the model yeast system Saccharomyces cerevisiae to assess their toxicity.

An alternative route for tricyclic quinazolinone-iminosugars and their glucosidase inhibitory activities.

A series of novel tricyclic quinazolinone-iminosugars 5 and their derivatives 7 were obtained from the tosylated sugars by three steps. Firstly, the reaction of the isopropylidene protected sugar tosylate 1 and o-aminobenzylamine 2 generated the precursor tricyclic quinazolin-iminosuar 3, which was then oxidized by KMnO4 to produce the corresponding quinazolinone 4. Finally, removal of the isopropylidene group yielded the target tricyclic quinazolinone iminosugars 5. In addition, quinazolinone-iminosugars 4ac, 4bc and 4cc who contain bromine in the aromatic region underwent Suzuki reaction with phenylboronic acid, followed with the removal of the isopropylidene group to afford the derivatives 7. This strategy will help to construct such fused multicyclic quinazolinone-iminosugars efficiently. Some compounds show certain inhibition against α-glucosidase (saccharomyce cerevisiae).

Evaluation of nonnatural L-iminosugar C,C-glycosides, a new class of C-branched iminosugars, as glycosidase inhibitors.

Capitalizing on a previously developed Staudinger/azaWittig/Grignard (SAWG)-ring contraction sequence that furnished protected six-membered L-iminosugar C,C-glycosides bearing an allyl group and various substituents at the pseudoanomeric position, the synthesis and glycosidase inhibition of a small library of six- and seven-membered L-iminosugar C,C-glycosides is reported. Their hydrogenolysis or cyclization by RCM followed by deprotection afforded eleven L-iminosugars including spirocyclic derivatives. All compounds adopt a 1C4 conformation in solution according to NMR data. Compared to previously reported branched L-iminosugars, the L-iminosugar C,C-glycosides reported herein were less potent glycosidase inhibitors. However, some of these compounds showed micromolar inhibition of human lysosome ß-glucocerebrosidase suggesting that such iminosugars could be useful to access potent CGase inhibitors by adjusting the structure/length of the pseudoanomeric substituents.

Synthesis of the Purported Structure of Glyphaeaside C and Proposed Revisions to the Structures of the Glyphaeaside Alkaloids.

The 10 glyphaeaside alkaloids isolated from the roots of Glyphaea brevis were originally purported as piperidine-based 1-C-alkylated iminosugars, with the A-, B-, and C-type glyphaeasides bearing l-DFJ, DGJ, and DNJ ring configurations, respectively. Subsequent investigations have revealed glyphaeaside C as being a pyrrolidine-based iminosugar with a DMDP ring configuration via total synthesis of the revised structure. In this work, side chain diastereomers of the originally purported structure of glyphaeaside C (10) and two related α-1-C-alkylated DNJ derivatives were synthesized from a common precursor, which was prepared in turn via stereoselective Grignard addition to a protected d-glycosylamine, followed by a reductive amination-cyclization sequence. Glycosidase inhibitory activity studies revealed general structure 10 as having potent inhibition against various α-glucosidases and weak inhibition against almond ß-glucosidase in agreement with similar DNJ-based iminosugars and in contrast to natural glyphaeaside C, suggesting that the (1,2-dihydroxy-3-phenyl)propyl moiety does not play a particularly vital role in the inhibitory modes of action of either compound. Furthermore, the absolute configuration of natural glyphaeaside C was proposed as that of d-DMDP, and the structures of the A- and B-type glyphaeasides were revised as 1-deoxy-DALDP and DALDP derivatives, respectively, based on interpretation of their reported NMR spectroscopic data.

Multivalent Pyrrolidine Iminosugars: Synthesis and Biological Relevance.

Recently, the strategy of multivalency has been widely employed to design glycosidase inhibitors, as glycomimetic clusters often induce marked enzyme inhibition relative to monovalent analogs. Polyhydroxylated pyrrolidines, one of the most studied classes of iminosugars, are an attractive moiety due to their potent and specific inhibition of glycosidases and glycosyltransferases, which are associated with many crucial biological processes. The development of multivalent pyrrolidine derivatives as glycosidase inhibitors has resulted in several promising compounds that stand out. Herein, we comprehensively summarized the different synthetic approaches to the preparation of multivalent pyrrolidine clusters, from total synthesis of divalent iminosugars to complex architectures bearing twelve pyrrolidine motifs. Enzyme inhibitory properties and multivalent effects of these synthesized iminosugars were further discussed, especially for some less studied therapeutically relevant enzymes. We envision that this comprehensive review will help extend the applications of multivalent pyrrolidine iminosugars in future studies.

Bambus[4,6]urils as Dual Scaffolds for Multivalent Iminosugar Presentation and Ion Transport: Access to Unprecedented Glycosidase-Directed Anion Caging Agents.

Bambusurils, BU[4] and BU[6], were used for the first time as multivalent scaffolds to link glycosidases inhibitors derived from 1-deoxynojirimycin (DNJ). Two linear DNJ ligands having six or nine carbon alkyl azido linkers or a trivalent DNJ dendron were grafted onto octapropargylated BU[4] and dodecapropargylated BU[6] using copper-catalyzed cycloaddition (CuAAC) to yield corresponding neoglycobambus[4] and neoglycobambus[6]urils bearing 8 to 24 iminosugars. The inhibition potencies of neoglycoBU[4], neoglycoBU[6] and neoglycoBU[6] caging anions were evaluated against Jack Bean α-mannosidase and compared to monovalent DNJ derivatives. Strong affinity enhancements per inhibitory head were obtained for the clusters holding trivalent dendrons with inhibitory constants in the nanomolar range (Ki = 24 nM for BU[4] with 24 DNJ units). Interestingly, the anion (bromide or iodide) encapsulated inside the cavity of BU[6] does not modify the inhibition potency of neoglycoBU[6], opening the way to water-soluble glycosidase-directed anion caging agents that may find applications in important fields such as bio(in)organic chemistry or oncology.

Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Recent results from non-basic glycosidase inhibitors: How structural diversity can inform general strategies for improving inhibition potency.

This review covers the literature in the past 15 years on glycosidase inhibitors lacking a basic nitrogen (for example iminosugars/azasugars) with a focus on natural terpenoids, and mono- and polycyclic aromatic hydrocarbons. From quite diverse structures, insight into inhibitor structural features that may be applicable to optimisation of all glycosidase inhibitors including iminosugars are identified.

Discovery of human hexosaminidase inhibitors by in situ screening of a library of mono- and divalent pyrrolidine iminosugars.

Two libraries of mono- and dimeric pyrrolidine iminosugars were synthesized by CuAAC and (thio)urea-bond-forming reactions from the respective azido/aminohexylpyrrolidine iminosugar precursors. The resulting monomeric and dimeric compounds were screened for inhibition of ß-N-acetylglucosaminidase from Jack beans, the plant ortholog of human lysosomal hexosaminidases. A selection of the best inhibitors of these libraries was then evaluated against human lysosomal ß-N-acetylhexosaminidase B (hHexB) and human nucleocytoplasmic ß-N-acetylglucosaminidase (hOGA). This evaluation identified a potent (nM) and selective monomeric inhibitor of hOGA (compound 7A) that showed a 6770-fold higher affinity for this enzyme than for hHexB. The corresponding dimeric derivative (compound 9D) further remarkably improved the selectivity in the inhibition of hOGA (2.7 × 104 times more selective for hOGA over hHexB) and the inhibition potency (by one order of magnitude). Docking studies were performed to explain the selectivity of inhibition observed in compound 7A.

Glyphaeaside C- enriched extract of Glyphaea brevis restored the antioxidant and reproductive integrity of 1,4-Dinitrobenzene-intoxicated rats.

This study assessed the fertility potential of methanol leaf extract of Glyphaea brevis (MGB) in rats exposed to 1,4-Dinitrobenzene (DNB), an environmental reprotoxicant. Male Wistar rats were orally exposed to 50 mg/kg DNB and administered 750 mg/kg MGB, 1500 mg/kg MGB or 300 mg/kg vitamin E for 21 days after 48 h of DNB exposure. Determination of serum reproductive hormone levels by enzyme-linked immunosorbent assays, evaluation of hematologic profile, computer-assisted sperm analyses (CASA) of sperm kinematics and morphology, assessment of testicular and spermatozoan antioxidant systems, and histopathological evaluation of reproductive tissues were performed. HPLC-DAD analysis identify Glyphaeaside C as the major component of the extract. In rats toxified with 50 mg/kg DNB, testicular and epididymal weights, serum levels of luteinizing hormone, testosterone and follicle-stimulating hormone, and packed cell volume, haemoglobin concentration, and white blood cell counts were decreased. There was altered sperm kinematics which reflected in increased sperm abnormalities. Treatment with the Glyphaeaside C -enriched MGB counteracted all DNB-induced changes and corrected DNB-induced aberrations in kinematic endpoints. Also, testicular and epididymal antioxidant systems were disrupted and there was damage to tissue histoarchitecture. Furthermore, our molecular docking study revealed that Glyphaeaside-C exhibited high binding affinities to the binding pocket of some free radical generating enzymes. Conclusively, the results indicated that Glyphaeaside C-enriched extract of Glyphaea brevis leaf enhanced the quality of semen and improved the functional capabilities of spermatozoa following exposure of rats to DNB which could translate to enhanced fertility.

Structural variation of the 3-acetamido-4,5,6-trihydroxyazepane iminosugar through epimerization and C-alkylation leads to low micromolar HexAB and NagZ inhibitors.

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.

Synthesis, conformational analysis and glycosidase inhibition of bicyclic nojirimycin C-glycosides based on an octahydrofuro[3,2-b]pyridine motif.

A set of bicyclic iminosugar C-glycosides, based on an octahydrofuro[3,2-b]pyridine motif, has been synthesized from a C-allyl iminosugar exploiting a debenzylative iodocycloetherification and an iodine nucleophilic displacement as the key steps. The halogen allowed the introduction of a range of aglycon moieties of different sizes bearing several functionalities such as alcohol, amine, amide and triazole. In these carbohydrate mimics the fused THF ring forces the piperidine to adopt a flattened 4C1 conformation according to NMR and DFT calculations studies. In their deprotected form, these bicycles were assayed on a panel of 23 glycosidases. The iminosugars displaying hydrophobic aglycon moieties proved to be superior glycosidase inhibitors, leading to a low micromolar inhibition of human lysosome ß-glucosidase (compound 11; IC50 = 2.7 µM) and rice α-glucosidase (compound 10; IC50 = 7.7 µM). Finally, the loose structural analogy of these derivatives with Thiamet G, a potent OGA bicyclic inhibitor, was illustrated by the weak OGA inhibitory activity (Ki = 140 µM) of iminosugar 5.

N-Substituted Valiolamine Derivatives as Potent Inhibitors of Endoplasmic Reticulum α-Glucosidases I and II with Antiviral Activity.

Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.

Synthesis and Evaluation of Novel Iminosugars Prepared from Natural Amino Acids.

Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,ß-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.

Toward the Identification of Novel Antimicrobial Agents: One-Pot Synthesis of Lipophilic Conjugates of N-Alkyl d- and l-Iminosugars.

In the effort to improve the antimicrobial activity of iminosugars, we report the synthesis of lipophilic iminosugars 10a-b and 11a-b based on the one-pot conjugation of both enantiomeric forms of N-butyldeoxynojirimycin (NBDNJ) and N-nonyloxypentyldeoxynojirimycin (NPDNJ) with cholesterol and a succinic acid model linker. The conjugation reaction was tuned using the established PS-TPP/I2/ImH activating system, which provided the desired compounds in high yields (94-96%) by a one-pot procedure. The substantial increase in the lipophilicity of 10a-b and 11a-b is supposed to improve internalization within the bacterial cell, thereby potentially leading to enhanced antimicrobial properties. However, assays are currently hampered by solubility problems; therefore, alternative administration strategies will need to be devised.

Iminosugars: A host-targeted approach to combat Flaviviridae infections.

N-linked glycosylation is the most common form of protein glycosylation and is required for the proper folding, trafficking, and/or receptor binding of some host and viral proteins. As viruses lack their own glycosylation machinery, they are dependent on the host's machinery for these processes. Certain iminosugars are known to interfere with the N-linked glycosylation pathway by targeting and inhibiting α-glucosidases I and II in the endoplasmic reticulum (ER). Perturbing ER α-glucosidase function can prevent these enzymes from removing terminal glucose residues on N-linked glycans, interrupting the interaction between viral glycoproteins and host chaperone proteins that is necessary for proper folding of the viral protein. Iminosugars have demonstrated broad-spectrum antiviral activity in vitro and in vivo against multiple viruses. This review discusses the broad activity of iminosugars against Flaviviridae. Iminosugars have shown favorable activity against multiple members of the Flaviviridae family in vitro and in murine models of disease, although the activity and mechanism of inhibition can be virus-specfic. While iminosugars are not currently approved for the treatment of viral infections, their potential use as future host-targeted antiviral (HTAV) therapies continues to be investigated.

Kinetic analysis of inhibition of α-glucosidase by leaf powder from Morus australis and its component iminosugars.

Mulberry leaves contain iminosugars, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 2-O-α-D-galactopyranosyl deoxynojirimycin (GAL-DNJ) that inhibit α-glucosidase. In this study, we quantified iminosugars in Morus australis leaves and made the kinetic analysis in the hydrolysis of maltose by α-glucosidase. By LC-MS/MS, the concentrations of 1-DNJ, fagomine, and GAL-DNJ in the powdered leaves were 4.0, 0.46, and 2.5 mg/g, respectively, and those in the roasted ones were 1.0, 0.24, and 0.73 mg/g, respectively, suggesting that the roasting process degraded iminosugars. Steady-state kinetic analysis revealed that the powdered and roasted leaves exhibited competitive inhibition. At pH 6.0 at 37ºC, the IC50 values of the extracts from the boiled powdered or roasted leaves were 0.36 and 1.1 mg/mL, respectively. At the same condition, the IC50 values of 1-DNJ, fagomine, and GAL-DNJ were 0.70 µg/mL, 0.18 mg/mL, and 2.9 mg/mL, respectively. These results suggested that in M. australis, 1-DNJ is a major inhibitor of α-glucosidase. ABBREVIATIONS: 1-DNJ: 1-deoxynojirimycin; GAL-DNJ: 2-O-α-D-galactopyranosyl-DNJ.

Synthesis of multimeric pyrrolidine iminosugar inhibitors of human ß-glucocerebrosidase and α-galactosidase A: First example of a multivalent enzyme activity enhancer for Fabry disease.

The synthesis of a chemical library of multimeric pyrrolidine-based iminosugars by incorporation of three pairs of epimeric pyrrolidine-azides into different alkyne scaffolds via CuAAC is presented. The new multimers were evaluated as inhibitors of two important therapeutic enzymes, human α-galactosidase A (α-Gal A) and lysosomal ß-glucocerebrosidase (GCase). Structure-activity relationships were established focusing on the iminosugar inhitope, the valency of the dendron and the linker between the inhitope and the central scaffold. Remarkable is the result obtained in the inhibition of α-Gal A, where one of the nonavalent compounds showed potent inhibition (0.20 µM, competitive inhibition), being a 375-fold more potent inhibitor than the monovalent reference. The potential of the best α-Gal A inhibitors to act as pharmacological chaperones was analyzed by evaluating their ability to increase the activity of this enzyme in R301G fibroblasts from patients with Fabry disease, a genetic disorder related with a reduced activity of α-Gal A. The best enzyme activity enhancement was obtained for the same nonavalent compound, which increased 5.2-fold the activity of the misfolded enzyme at 2.5 µM, what constitutes the first example of a multivalent α-Gal A activity enhancer of potential interest in the treatment of Fabry disease.

Synthesis of tricyclic benzimidazole-iminosugars as potential glycosidase inhibitors via a Mitsunobu reaction.

A series of tricyclic benzimidazole-iminosugars 1(a-f) and 2(a-f) were synthesized and evaluated for their their inhibitory activities against five glycosidases. The synthesis initiated from the benzyl protected sugar (aldehyde) 5 that reacted with 1,2-diaminobenzene to afford aldo-benzimidazole 6 by the iodine-induced oxidative condensation. Then, tricyclic compound 7 was obtained in high yields of 73%-87% by the key Mitsunobu reaction through intramolecular cyclization of the unprotected OH and the NH in 6. After removal of the benzyl group in CF3SO3H, the target tricyclic benzimidazole-iminosugars 1 and 2 were achieved. The protocol was effective for the preparation of the tricyclic iminosugar in satisfactory yield. The results of the glycosidase inhibitory activities of 1 and 2 showed that three compounds derived from d-ribose exhibited specific and good inhibitory effects on ß-glucosidase. Among them, 1e-1 was the best one with IC50 value of 5.37 µM. All hydroxyl groups on ß-position would be favourable to the inhibitory activity of such tricyclic benzimidazole-iminosugars against ß-glucosidase.

Synthesis of polyfluoroalkyl sp2-iminosugar glycolipids and evaluation of their immunomodulatory properties towards anti-tumor, anti-leishmanial and anti-inflammatory therapies.

Immunomodulatory glycolipids, among which α-galactosylceramide (KRN7000) is an iconic example, have shown strong therapeutic potential in a variety of conditions ranging from cancer and infection to autoimmune or neurodegenerative diseases. A main difficulty for those channels is that they often provoke a cytokine storm comprising both pro- and anti-inflammatory mediators that antagonize each other and negatively affect the immune response. The synthesis of analogues with narrower cytokine secretion-inducing capabilities is hampered by the intrinsic difficulty at controlling the stereochemical outcome in glycosidation reactions, particularly if targeting the α-anomer, which seriously hampers drug optimization strategies. Here we show that replacing the monosaccharide glycone by a sp2-iminosugar glycomimetic moiety allows accessing N-linked sp2-iminosugar glycolipids (sp2-IGLs) with total α-stereocontrol in a single step with no need of protecting groups or glycosidation promotors. The lipid tail has been then readily tailored by incorporating polyfluoroalkyl segments of varied lengths in view of favouring binding to the lipid binding site of the master p38 mitogen activated protein kinase (p38 MAPK), thereby polarizing the immune response in a cell-context dependent manner. The compounds have been evaluated for their antiproliferative, anti-leishmanial and anti-inflammatory activities in different cell assays. The size of the fluorous segment was found to be critical for the biological activity, probably by regulating the aggregation and membrane-crossing properties, whereas the hydroxylation profile (gluco or galacto-like) was less relevant. Biochemical and computational data further support a mechanism of action implying binding to the allosteric lipid binding site of p38 MAPK and subsequent activation of the noncanonical autophosphorylation route. The ensemble of results provide a proof of concept of the potential of sp2-IGLs as immunoregulators.