Piperidine Azasugars Bearing Lipophilic Chains: Stereoselective Synthesis and Biological Activity as Inhibitors of Glucocerebrosidase (GCase)

Piperidine Azasugars Bearing Lipophilic Chains: Stereoselective Synthesis and Biological Activity as Inhibitors of Glucocerebrosidase (GCase) Francesca Clemente, Camilla Matassini, Sara Giachetti, Andrea Goti, Amelia Morrone, Macarena Martínez-Bailén, Sara Orta, Pedro Merino,*and Francesca Cardona* Cite This: J. Org. Chem. 2021, 86, 12745−12761 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: We report a straightforward synthetic strategy for the preparation of trihydroxypiperidine azasugars decorated with lipophilic chains at both the nitrogen and the adjacent carbon as potential inhibitors of the lysosomal enzyme glucocerebrosidase (GCase), which is involved in Gaucher disease. The procedure relies on the preparation of C-erythrosyl N-alkylated nitrones 10 through reaction of aldehyde 8and primary amines 13 followed by oxidation of the imines formed in situ with the methyltrioxorhenium catalyst and urea hydrogen peroxide. The addition of octylMgBr to nitrone 10e provided access to both epimeric hydroxylamines 21 and 22 with opposite configuration at the newly created stereocenter in a stereodivergent and completely stereoselective way, depending on the absence or presence of BF3· Et2O. Final reductive amination and acetonide deprotection provided compounds 14 and 15 from low-cost D-mannose in remarkable 43 and 32% overall yields, respectively, over eight steps. The C-2 R-configured bis-alkylated trihydroxypiperidine 15 was the best ligand for GCase (IC50 =15μM), in agreement with MD simulations that allowed us to identify the chair conformation corresponding to the best binding affinity. 1. INTRODUCTION Iminosugars [e.g., deoxynojirimycin, DNJ (1), Figure 1] are among the most fascinating monosaccharide analogues in which a nitrogen atom replaces the endocyclic oxygen. 1 Together with azasugars, [e.g. isofagomine, IFG (2), or 1,5- dideoxy-1,5-iminoxylitol, DIX (3), Figure 1], which are characterized by a nitrogen atom replacing the anomeric carbon of monosaccharides, iminosugars have been extensively investigated in the last thirty years as glycosidase 2 and glycosyltransferase inhibitors. 3 More recently, imino- and azasugar-based glycomimetics became attractive as potential therapeutic agents toward lysosomal storage disorders (LSDs), following the observation of their counter-intuitive effect in enhancing the enzyme activity, thus acting as chaperones. In the pharmacological chaperone therapy (PTC) of LSDs, these glycomimetics are employed at sub-inhibitory concentration to favor the mutated enzyme correct folding in the endoplasmic reticulum (ER), facilitate its translocation to the lysosomes, and recover some hydrolytic activity, compromised as a consequence of diverse genetic mutations. 4 Gaucher disease (GD), the most common LSD, is determined by mutations in the GBA gene, which encodes for the lysosomal enzyme glucocerebrosidase (GCase). The Received: June 4, 2021 Published: September 1, 2021 Figure 1. Some imino- and azasugars, their N- and C-alkyl derivatives, and glucosylceramide (GlcCer), the natural substrate of GCase. Articlepubs.acs.org/joc © 2021 The Authors. Published by American Chemical Society 12745 https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 Downloaded via UNIV DE SEVILLA on September 21, 2022 at 11:40:11 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. GBA mutations provoke partial deficit of GCase, with consequent loss or reduction of its hydrolytic activity [i.e., hydrolysis of glucosyl ceramide, GlcCer (4), Figure 1,to ceramide and glucose] in the lysosomes. Accumulation of glucosylceramide then leads to organ dysfunctions and severe impairment. Several good candidates behaving as pharmacological chaperones (PC) for GCase have been found, but no drugs are on the market yet. 5 The presence of an alkyl chain either at the nitrogen atom or at the adjacent carbon of imino- and azasugars was found to improve their pharmacokinetic properties, furnishing better PCs for GD. For instance, contrary to DNJ (1), N-nonyl-DNJ (5)(Figure 1) is a potent inhibitor of lysosomal GCase (IC50 = 1μM) which showed a twofold increase in the activity of the N370S mutant enzyme in fibroblasts at 10 μM concentration. However, it did not enhance the intracellular activity of the L444P variant. 6 With IFG and DIX derivatives, better results in terms of PC properties were obtained by shifting the alkyl chain from nitrogen to the adjacent carbon. Indeed, 6-nonyl IFG (6) (Figure 1) displayed a remarkable GCase inhibition and PC activity (IC50 = 0.6 nM, 1.5-fold enzyme activity enhancement at 3 nM in N370S GD fibroblasts) 7 and α-1-C-nonyl-DIX (7) showed an IC50 of 6.8 nM toward GCase and a 1.8-fold enzyme activity increase in N370S mutated fibroblasts at 10 nM. 8 Among the most straightforward synthetic strategies to afford polyhydroxylated piperidine imino- and azasugars 9 are the reductive amination (RA) ring-closure starting from nitrogen-containing carbohydrate-based precursors or the double reductive amination (DRA) of dicarbonyl carbohydrate derivatives with an external nitrogen source. 10 By applying the latter strategy on the “masked”dialdehyde 8derived from D- mannose, we have synthesized the N-alkyl trihydroxypiperidine 9(Scheme 1), 11 which showed IC50 =30μM toward GCase and 1.25-fold activity increase in N370S-mutated fibroblasts at 100 μM. 12 The former RA strategy, instead, was applied to the C- erythrosyl nitrone 10a, 13 obtained in turn by condensation of the same “masked”dialdehyde 8with N-benzyl hydroxylamine. 14 Stereoselective Grignard additions to this key nitrone in the presence or absence of a suitable Lewis acid 14,15 afforded, in a stereodivergent manner, both epimers of a series of 2-alkylated trihydroxypiperidines, among which the octyl derivatives 11 and 12 (Scheme 1) showed the most promising biological properties. 16 In particular, the (2R) diastereoisomer 12 showed remarkable PC properties toward fibroblasts bearing the N370S/RecNcil mutation (1.9-fold enzyme recovery at 100 μM) and, more importantly, proved to be responsive with the homozygous L444P mutation (1.80-fold enzyme recovery at 100 μM), which is refractory to most PCs. Remarkably, both compounds 9and 12 performed PC tasks toward wild-type fibroblasts (1.5-fold at 50 μM and 1.4-fold at 100 μM, respectively), which is an important factor for targeting sporadic forms of Parkinson disease. 17 Given the relevance of the presence of a long alkyl chain for inducing good enzyme recognition, and in consideration of the structure of the natural substrate GlcCer (4), we speculated that compounds possessing two alkyl chains might show improved biological properties. We therefore addressed the synthesis of 1,2-dialkyltrihy- droxypiperidines and envisaged that a general synthetic strategy could involve the Grignard addition to C-erythrosyl N-alkyl nitrones 10 followed by RA (Scheme 1). In this work, we report our results on this subject, which include a docking study of the putative 1,2-dialkyltrihydrox- ypiperidines in the GCase catalytic site; a general direct synthesis of nitrones 10 and their conversion to target compounds 14 and 15 and to derivatives containing three alkyl tails; the biological evaluation of the dialkylated trihydroxypiperidines toward commercial glycosidases and human lysosomal enzymes and their in vitro activity on cell lines; a molecular dynamics (MD) simulation of the new compounds within the GCase enzyme active cavity. Scheme 1. Previous rResults and Scope of This Work a a General strategies employed to access C-orN-alkyl trihydroxipiperidines from the “masked dialdehyde”8and nitrone 10a derived thereof. This work:“one-pot”synthesis of C-erythrosyl N-alkyl nitrones 10 and their application to the synthesis of C,N-dialkylated azasugars 14 and 15. The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12746 2. RESULTS AND DISCUSSION 2.1. Preliminary Docking Studies. With the aim of assessing the viability of target compounds 14 and 15 in GCase catalytic site, we carried out preliminary docking studies with acid-beta-glucosidase (PDB ID 2NSX). 18 The protonated forms of compounds 14 and 15 were considered, taking into account that a stereogenic nitrogen atom is formed (for details, see the Supporting Information section). For compound 14, the best pose corresponds to an (R)-configuration at the nitrogen atom adopting a 1C4conformation (score −6.332). The observed orientation for the piperidine ring is different with respect to that observed in a known chaperone [5- hydroxymethyl-3,4-dihydroxypiperidine, IFG (2), Figure 1], thus loosing interactions of hydroxyl groups with Asp127, Trp179, and Asn396 that now form new interactions with Ser237, Asp283, and Gln284. Although the aliphatic chain linked to the nitrogen atom shows hydrophobic interactions with Phe128, Trp179, Tyr313, Leu314, and Ala238, the other aliphatic chain is clearly exposed to the solvent (see the Supporting Information section). The (S)-isomer shows a close value in the docking score (−6.071) for the same conformation and, also in this case, the piperidine ring is oriented in the wrong way with respect to IFG. A similar situation was found for protonated 15. In this case, the (S)- isomer in a 1C4conformation showed the best docking value (−6.614) due to hydrophobic interactions of the aliphatic chains in the binding site, the (R)-isomer in the same conformation being very close (docking score = −6.354). Again, in both cases, the piperidine ring is oriented in the wrong way when compared with the original structure bound to IFG. For the (R)-isomer, the chain bonded to the nitrogen atom gives interactions with Phe128, Trp179, Phe246, and Trp381. The similar orientation of the piperidine ring to that observed for 14 gives rise to the same interactions of the hydroxyl groups with Ser237, Asp283, and Gln284 (Figure 2). These preliminary results should be considered with caution due to the predicted orientations of the piperidine ring in a rigid protein. Even though the calculations predict a good binding to the enzyme, the particular interactions and orientations of the ligands will be confirmed (or rejected) through further molecular dynamics studies that will allow conformational changes in the protein (vide infra). 2.2. Synthesis and Structural Assignment. The aldehyde 8was synthesized in four steps from D-mannose on gram scale as reported. 11,19 The synthesis of nitrones 10 from 8by condensation in analogy to the reported 10a would require the corresponding N-monosubstituted hydroxylamines, compounds not readily available in general. However, nitrones can also be obtained in several alternative ways, 20 among which the oxidation of imines developed by some of us 21 was selected as the most appropriate for our purposes. This procedure was established first on preformed imines employing methyltrioxorhenium (MTO) as the catalyst and urea hydrogen peroxide complex (UHP) as a mild stoichiometric oxidant. The methodology has the great advantage over other oxidation methods (on secondary amines 22 or N,N-disubstituted hydroxylamines) 20a of forming a single nitrone, since the double bond is previously installed during the imine formation. Moreover, a convenient one-pot version has been subsequently implemented, where imines are formed in situ from inexpensive or readily available primary amines and aldehydes. 23 In this work, we employ the one-pot condensation/oxidation strategy to our carbohydrate-derived “masked”dialdehyde 8 with primary amines 13a−hfor the synthesis of several new C- carbohydrate N-alkyl nitrones 10 (Scheme 1). We also demonstrate that such nitrones undergo stereodivergent and highly stereoselective Grignard reagent additions in the presence or absence of a suitable Lewis acid. The following reductive amination (RA) of the formed adducts directly gives access to new C,N-dialkyl trihydroxypiperidines, as demonstrated with the synthesis of compounds 14 and 15 (Scheme Figure 2. Molecular docking for protonated 15 with an (S)-configuration at the nitrogen atom. Left: 2D schematic view of interaction in the binding site. Right: Visualization of the ligand, represented as cyan CPK at the binding site. The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12747 1). The target compounds were designed as to better mimic the two chains of the GlcCer (4) natural substrate. The one-pot condensation/oxidation was investigated starting from aldehyde 8with structurally diversified primary Table 1. Condensation/Oxidation Strategy for the Synthesis of Nitrones 10a−h from Aldehyde 8 and Primary Amines 13a−h a a Concerning the synthesis of nitrone 10a (Table 1, entry 1), this method compares very well with the previously reported condensation procedure of aldehyde 8with N-benzyl hydroxylamine (85%), 14 especially considering the difference of costs with benzylamine (13a). The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12748 amines 13a−hfollowed by addition of UHP and catalytic MTO, and the results are shown in Table 1. The reaction afforded the corresponding N-alkyl nitrones 10a−hwith good yields and in an operationally very simple manner. The procedure was optimized using benzylamine (13a) for the preparation of the known nitrone 10a. The best results were obtained by stirring a solution of the aldehyde 8with 1.2 equiv of the appropriate amine 13 in MeOH at room temperature in the presence of anhydrous Na2SO4, until disappearance of the starting aldehyde 8at an 1H NMR control (3−5 h, depending on the amine), which attested the formation of the corresponding imine. After cooling of the reaction mixture at 0°C, addition of UHP (3 equiv) and MTO (4 mol %) caused the solution to turn yellow, indicating the formation of the catalytically active peroxorhenium species. 24 Upon completion of the oxidation at room temperature, a simple work-up consisting of solvent removal under reduced pressure followed by CH2Cl2addition allowed filtering offthe undissolved urea and Na2SO4to give, after evaporation of the solvent, the crude nitrones which were purified by flash column chromatography on silica gel. Alternative addition of UHP and MTO since the beginning of the reaction (prior to imine formation) was found to be unpractical and afforded complex mixtures of products deriving from competitive oxidation of the primary amines. The scope listed in Table 1 demonstrates the versatility of this method, which gives comparable results with complete conversions and good yields (70−80%) for benzylamines 13a−c(entries 1−3), homobenzylamine 13d (entry 4), and linear aliphatic amines 13e−f(entries 5−6). Decrease in reaction yields with the α-branched (methyl)benzyl 13c (entry 3) and aliphatic amines 13g and 13h (entries 7−8) suggests that the oxidation reaction is somewhat sensitive to steric effects. However, the corresponding nitrones 10c and 10g−h were still obtained in overall satisfactory yields (60−75%) through the two-step process. All the nitrones 10 were obtained exclusively as the more stable Z-diastereoisomer, in agreement with previous results (see also the Supporting Information section for 1D NOESY experiments on nitrone 10f). 21 Nitrones 10 turned out to be excellent starting materials for the straightforward synthesis of N-substituted trihydroxy piperidines. Indeed, treatment of the nitrones 10e,10f, and 10h in MeOH under H2atmosphere (balloon) over Pd/C or Pd(OH)2/C as the catalyst afforded the N-substituted piperidines 16, 11 17, and 18, respectively, in excellent yields (Scheme 2). The overall efficiency of this one-pot process is remarkable, considering that it results from a cascade of several reactions, consisting of nitrone reduction, debenzylation, condensation with sugar aldehyde and iminium or enamine reduction (RA). Application of the same procedure to nitrone 10a afforded the N-unsubstituted piperidine 19 11 in quantitative yield. It is worth to note that the yields obtained using this strategy were by far superior to those previously reported for the synthesis of piperidines 16 and 19 employing condensation of aldehyde 8with primary amines and subsequent reduction to amine and RA-cyclization step, or, in alternative, employing a DRA strategy with NaBH3CN as the reducing agent on aldehyde 20, in turn derived from 8. 11 (Scheme 3). For comparison, N-dodecyl trihydroxypiperidine 17, obtained in 90% yield from nitrone 10f (Scheme 2) was obtained also via DRA of 20 with dodecylamine in a modest 38% yield (Scheme 3). The synthetic strategy based on the addition of Grignard reagents to nitrone 10e followed by intramolecular RA provided access to the desired 1,2-octyl trihydroxypiperidines 14 and 15 (Scheme 1). The length of the chains (C8) was chosen on the basis of the data previously obtained from biological assays. We had previously observed that the addition of Grignard reagents to the carbohydrate-derived nitrone 10a proceeded with opposite diastereofacial preference in the presence or absence of a suitable Lewis acid. The addition reaction, followed by RA, allowed the introduction of different alkyl groups with opposite configurations at the piperidine C-2, Scheme 2. Reduction/Ring-Closure/RA Sequence to Trihydroxy Piperidines 16−19 from Nitrones 10 Scheme 3. Previously Reported Strategies for the Synthesis of 16 and 19 Starting from the Aldehyde 8 and Alternative Synthesis of the Novel N-Dodecyl Trihydroxypiperidine 17 The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12749 affording two diastereomeric series of 2-alkyl trihydroxy piperidines. 14,16,17 The addition of octylMgBr to nitrone 10e was first carried out in THF at −78 °C for 3 h without any Lewis acid. The reaction gave smoothly the hydroxylamine 21, with the Sabsolute configuration at the newly formed stereocenter accordingly to our prior work in good yield (75%) and excellent stereoselectivity (>98%) (Scheme 4). The configuration at the newly created stereocenter was unambiguously confirmed by 1H NMR and 1D NOESY spectra at a later stage of the synthesis (vide infra). The addition of octylMgBr to nitrone 10e in the presence of BF3·Et2Oat−30 °C for 2 h resulted in a complete reversal of the diastereoselectivity in favor of the epimeric hydroxylamine 22, which was obtained with excellent stereoselectivity (>98%) and good yield (70%) with an Rconfiguration at the newly formed stereocenter (Scheme 4). Albeit the observed stereochemical outcome of the additions of octylMgBr to the nitrone 10e in the absence or presence of BF3·Et2O is in agreement with the results obtained previously in the additions of several Grignard reagents to nitrone 10a,we were delighted to see that in this case a complete diastereoselectivity was reached. Instead, in the Grignard additions to 10a, we had observed diastereomeric ratios in the range 1.4−5.6:1 without Lewis acids and 3−9:1 with BF3·Et2O in favor of the (S) and (R) configured adducts, respectively. 16,17 A magnesium Cram-chelate transition state (TS) may account for the preferred nucleophilic attack to the Si diastereoface of nitrone 10e in the absence of Lewis acid, resulting in the exclusive formation of the hydroxylamine 21 with the Sconfiguration at the newly formed stereocenter (Figure 3A). Conversely, when an equimolar amount of BF3· Et2O is added, chelation is prevented, thus favoring the nucleophilic attack to the more accessible Re face of nitrone 10e, which leads to the hydroxylamine 22 with the R configuration at the newly formed stereocenter (Figure 3B). 14,17 The hydroxylamines 21 and 22 showed high tendency to oxidize spontaneously in air to the corresponding aldonitrones 23 and 24 (Scheme 5), according to the behavior observed for other hydroxylamines obtained by addition of Grignard reagents to the N-benzyl nitrone 10a; notwithstanding the CN bond does not benefit of conjugation in the present case. 16,17 In particular, spontaneous partial oxidation of 21 and 22 occurred with 50% conversion, as evaluated by 1H NMR spectroscopy. The exclusive formation of aldonitrones instead of ketonitrones was providential, since it did not trigger any loss of configurational integrity at the newly formed stereocenter and was not detrimental for our synthetic purposes (see below). However, a certain regioselectivity in favor of the aldonitrones was expected based on our previous studies. Due to the low stability of hydroxylamines 21 and 22, they were characterized only by 1H NMR and MS analyses immediately after purification by column chromatography. Characterization of pure nitrones 23 and 24 was carried out after complete oxidation of the hydroxylamines 21 and 22 with the hypervalent iodine reagent 2-iodoxy benzoic acid (IBX), which is the reagent of choice to promote regioselective oxidation of N,N-disubstituted hydroxylamines in favor of the corresponding aldonitrones. 13,25 In this case, it provided the corresponding nitrones 23 and 24 in excellent yields and with complete regioselectivity (Scheme 5). The final reductive amination step was performed on the hydroxylamine/nitrone mixtures by employing 2 equiv of acetic acid under an H2atmosphere (balloon) and Pd/C as the catalyst in MeOH (0.015 M solution), followed by treatment with a strongly basic resin to give the free amines 25 and 26 in excellent 90 and 80% yield, respectively (Scheme 6). Careful analysis of the 1H NMR, 2D NMR (COSY, HSQC), and 1D NOESY spectra of piperidines 25 and 26 allowed us to establish unambiguously their configuration, thus validating the stereochemical outcome of the additions of octylMgBr reported in Scheme 4. The two chair conformations of each compound are reported in Figure 4. The 1H NMR spectrum of (2S) piperidine 25 showed a small coupling constant between 3-H and 4-H (3J3−4= 4.0 Hz), suggesting a high preference for its 1C4conformation where both 3-H and 4-H are equatorial. The 1C4is expected to be the absolute minimum energy conformation for the S-configured piperidine at C-2, with the octyl chain lying in equatorial position (Figure 4). Accordingly, the 1D NOESY spectrum of 25 did not show the nOe correlation peak between 2-H and 4-H. For the diastereomeric piperidine 26, the opposite Rconfiguration at C-2 shifted the equilibrium to a preferred 4C1conformation in order to accommodate again the octyl chain in an equatorial position, as Scheme 4. Addition of Octyl Magnesium Bromide (1.8 equiv) to Nitrone 10e in the Presence or Absence of BF3· Et2O (1 equiv) Figure 3. Transition state models proposed for the nucleophilic attack to nitrone 10e: (A) cram-chelate TS in the absence of Lewis acid; (B) TS in the presence of BF3·Et2O. The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12750 attested by the increase of the coupling constant between 3-H and 4-H (3J3−4= 8.0 Hz) in agreement with their ax−ax relationship (Figure 4). The set of strong nOe correlation peaks observed in the 1D-NOESY spectrum of 26 among all axial protons (2-H with 4-H and 6-Hband 4-H with 6-Hb2-H, see the Supporting Information section) substantiated this assignment. Final deprotection of the acetonide protecting groups under acidic conditions (aqueous HCl in MeOH) followed by treatment with the strongly basic resin Ambersep 900-OH gave the target 1,2-dioctyl trihydroxypiperidines 14 and 15 as free amines in excellent yields (Scheme 6). We envisaged that the intriguing formation of the aldonitrones 23 and 24 by easy and regioselective oxidation of hydroxylamines 21 and 22 would furnish the chance for further extending our study. Indeed, another alkyl chain might be introduced via iteration of the Grignard addition to these nitrones to provide final piperidines possessing three lipophilic tails. This hypothesis was proven with nitrone 24. In order to avoid formation of a further stereogenic center resulting in a mixture of diastereoisomers, heptyl magnesium bromide was chosen as the appropriate Grignard reagent, but the addition turned out to be sluggish. In the absence of the promoter at different temperatures (−30 °C, 0 °C, rt), in THF, it failed to give any product. Addition of BF3·Et2O (1.0 equiv) resulted in the formation of the desired hydroxylamine 27 after 16 h (Scheme 7), as attested by the peak at m/z: 618.31 ([M + H]+) at an ESI-MS analysis. The hydroxylamine 27 was Scheme 5. Oxidation of Hydroxylamines 21 and 22 with IBX: Synthesis of Nitrones 23 and 24 Scheme 6. Ring-Closure/Reductive Amination to 25 and 26 and Their Final Deprotection to 1,2-Dioctyl Trihydroxypiperidines 14 and 15 Figure 4. Chair conformations of piperidines 25 and 26 (R = nC8H17). Double-ended arrows show the observed diagnostic nOe correlation peaks. The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12751 unstable and presumably underwent mainly rapid oxidation to the corresponding ketonitrones 28 and 29, as suggested by the presence of two TLC spots with very similar Rfand a peak at 638.55, corresponding to [M + Na]+, in the ESI-MS spectrum (Scheme 7). The lack of regioselectivity in the oxidation of 27 is a major drawback in view of our synthetic target, since the ketonitrone 29 has lost the stereochemical information to be installed at C- 2 in the final piperidine. In order to prevent the rapid oxidation of the hydroxylamine 27, the adduct 30 was reduced in situ to the corresponding amine 31 by direct addition to the crude reaction mixture of indium powder in slightly acidic aqueous ethanol. 26 This one-pot Grignard addition/reduction afforded the amine 31 in 50% yield without any loss of stereochemical integrity (Scheme 7). The RA of amine 31 with H2as the reducing agent in the presence of catalytic Pd/C and acetic acid (2 equiv) in MeOH provided the partially protected trihydroxy piperidine 32 in 55% yield (Scheme 7). Final deprotection of the acetonide performed under acidic conditions (aqueous HCl in MeOH) led to the desired trihydroxy piperidine 33 as the hydrochloride salt (Scheme 7). 27 2.3. Biology and Molecular Dynamics. 2.3.1. Preliminary Biological Evaluation toward Commercial Glycosidases. Preliminary biological evaluation of compounds 14 and 15 (at 100 μM inhibitor concentration) was performed toward a panel of 12 commercial glycosidases (α-L-fucosidase EC 3.2.1.51 from Homo sapiens,α-galactosidase EC 3.2.1.22 from coffee beans, β-galactosidases EC 3.2.1.23 from Escherichia coli and Aspergillus oryzae,α-glucosidases EC 3.2.1.20 from yeast and rice, amyloglucosidase EC 3.2.1.3 from Aspergillus niger,βglucosidase EC 3.2.1.21 from almonds, α-mannosidase EC 3.2.1.24 from Jack beans, β-mannosidase EC 3.2.1.25 from snail, and β-N-acetylglucosaminidases EC 3.2.1.52 from Jack beans and bovine kidney). No remarkable inhibitory activity was found at this concentration for compounds 14 and 15 toward any of these enzymes apart from a 30% inhibition of β-glucosidase from almonds by compound 14 (see the Supporting Information section). Even if moderate, this value was encouraging, since we have previously noticed compounds with a low inhibitory activity toward this commercial enzyme that turned out to be stronger inhibitors of human lysosomal GCase. 12,17 2.3.2. Inhibitory Activity of GCase. Compounds 14,15, and 33·HCl were then tested at 1 mM for GCase inhibition in human leukocyte homogenates. The percentages of inhibition, together with the corresponding IC50 values, are shown in Table 2. The results obtained were compared with data of previously reported compounds 9,11, and 12. 12,16,17 Our results show that both the newly synthesized trihydroxypiperidines 14 and 15 were able to strongly inhibit GCase, imparting 100% inhibition of the enzyme at 1 mM concentration (Table 2, entries 4−5). However, a remarkable difference emerged between the two compounds from measurement of their IC50, which followed the same trend observed for the C-2 mono-alkylated azasugars 11 and 12. Indeed, the 1,2-dioctyl azasugar 15 with the Rconfiguration at C-2 was a stronger inhibitor than its S-configured diaster- Scheme 7. Addition of heptylMgBr to Nitrone 24, One-Pot Addition/Reduction to Amine 31, and Synthesis of the Three- Tailed Trihydroxypiperidine 33·HCl The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12752 eoisomer 14 (IC50 = 15.0 μMvs IC50 = 100 μM, Table 2 entry 5vs 4). In this latter case, the difference was even more pronounced than for the corresponding monoalkylated congeners 12 and 11. It is also worth to note that the dialkylated piperidine 15 is twofold more active than its monoalkylated counterpart 12. Apparently, the presence of a second octyl chain at the nitrogen atom imparted beneficial interactions within the enzyme active site only in the case of the 2R-configured pair. Conversely, the 1,2-dialkylated azasugar 33·HCl, although maintaining the octyl chain at C- 2 with Rconfiguration is a much less potent GCase inhibitor than 15 (IC50 = 130.0 μMvs IC50 = 15.0 μM, Table 2 entry 6 vs 5), suggesting that the presence of a further alkyl chain is detrimental. 2.3.3. Molecular Dynamic Studies. We carried out MD simulations for (R) and (S) protonated forms of compounds 14 and 15, considering both conformations 4C1and 1C4as starting points and taken into consideration the best poses obtained from the docking studies and also the best ones in which the piperidine ring is correctly oriented as IFG. In all cases, MD converged to stable complexes which were reached after 10 ns of simulation and continued to be stable for 250 ns (all MD simulations were replicated four times). The complexes showed the ligand in the binding site establishing different interactions with key residues Asp128, Trp180, Asn235, Glu236, Tyr314, and Glu341 and with the correct orientation of the piperidine ring (see the Supporting Information section). 28 The overall RMSD for the protein system appeared to have reached equilibrium in the first ns, and the stabilization of the protein−ligand complex after 20 ns keeping the interactions of the complexes constant during the rest of the simulation. The ligands showed a complete stability of chair conformations and tend to arrange the aliphatic chains toward the external part, exposed to the solvent, in order to keep interactions of the hydroxyl groups of the piperidine ring. The presence of the alkyl chains induces some visible conformational change of the protein in the area close to the binding site. Interestingly, similar protein conformations were observed for (R)-14H+4C1and (S)−14H+1C4, whereas a loop formed by residues 345−349 is partially opened with (R)- 14H+1C4and completely opened with (S)-14H+4C1(see the Supporting Information section). In the case of compound 15H+, the protein shows a higher flexibility with the loop formed by residues 345−349 adopting different orientations. The mobility of the loop, rich in hydrophobic residues (Met347, Phe348, and Trp349) can be attributable to hydrophobic interactions with alkyl chains that stabilizes the complex as expected. The sole inspection of the interactions in the binding site shows that (R)-15H+4C1is the complex having the highest stability (Figure 5). With the aim of obtaining more accurate information on the stability of the complexes (rather than the qualitative observation of the interactions), we carried out additional calculations to evaluate the binding energy. For the purpose of comparison both MM/GBSA and MM/PSBA calculations were performed. 29 In both cases, higher values were obtained for compound 15, the best values being found for (R)-15H+ 4C1(Ebinding =−57.9 kcal/mol with MM/GBSA and −49.4 kcal/mol with MM/PBSA) corroborating the observed interactions and in good agreement with experimental results (see the Supporting Information section). 2.3.4. Preliminary Biological Screening toward Human Lysosomal Glycosidases. In order to avoid undesired inhibitory side effects of a potential new drug, it has to be selective for a given target. The selectivity of the newly synthesized compounds 14,15, and 33·HCl toward GCase was then investigated, evaluating their inhibition at 1 mM concentration toward six other lysosomal glycosidases (namely, α-and β-mannosidases, α- and β-galactosidases, α-fucosidase, and α-glucosidase) in cell homogenates (leucocytes or lymphocytes) isolated from healthy donors. The results, Table 2. GCase Inhibition and IC50 Values in Human Leukocytes from Healthy Donors entry compound GCase inhibition [%] a IC50 (μM) b 1998 30.0 ±1.0 c 211 80 93.5 ±5.3 d 312 100 29.3 ±1.8 d 414 100 100.0 ±9.0 515 100 15.0 ±4.0 633·HCl 75 130 ±15 a Percentage inhibition of GCase in human leukocytes extracts incubated with azasugars (1 mM). b IC50 values were determined by measuring GCase activity at different concentrations of each inhibitor. c Ref 12 (IC50 value obtained using the same experimental protocol and substrate concentration of this manuscript). d Refs 16 and 17 (IC50 value obtained using the same experimental protocol and substrate concentration of this manuscript). Figure 5. Close-up view of GCase in complex with compounds (R)- 15H+4C1(cyan) with (R) configuration at C-2 and (R)-14H+4C1 (green) with (S) configuration at C-2. Dashed yellow and magenta lines indicate H-bond interactions of (R)-15H+4C1and (R)-14H+ 4C1, respectively. It can be appreciated that the former has more polar contacts with the residues of the binding site than the latter. The Journal of Organic Chemistry pubs.acs.org/joc Article https://doi.org/10.1021/acs.joc.1c01308 J. Org. Chem. 2021, 86, 12745−12761 12753 Facultad de Química, Universidad de Sevilla, E-41012 Sevilla, Spain Sara Orta −Unidad de Glicobiología, Instituto de BiocomputaciónyFísica de Sistemas Complejos (BIFI), Universidad de Zaragoza, 50009 Zaragoza, Spain Complete contact information is available at: https://pubs.acs.org/10.1021/acs.joc.1c01308 Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS We thank Fondazione Cassa di Risparmio di Pistoia e Pescia (Bando Giovani@Ricerca scientifica 2017) for a fellowship to C.M. and financial support. We thank MIUR-Italy (“Progetto Dipartimenti di Eccellenza 2018-2022”allocated to the Department of Chemistry “Ugo Schiff”), Universitadi Firenze and Fondazione CR Firenze for financial support (“Bando congiunto per il finanziamento di progetti competitivi sulle malattie neurodegenerative”. Project: A multidisciplinary approach to target Parkinson’s disease in Gaucher related population, Acronym: MuTaParGa). Fondazione CR Firenze (grant no 2018.0942) is also acknowledged for financial support. F.Ca also thanks Regione Toscana (Bando Salute 2018) for the project: Late onset Lysosomal Storage Disorders (LSDs) in the differential diagnosis of neurodegenerative diseases: development of new diagnostic procedures and focus on potential pharmacological chaperones (PCs), Acronym: LysoLate. M.M.-B. thanks The Spanish MINECO (CTQ2016- 77270-R) for financial support. P.M. and S.O. thank the Spanish MINECO (PID2019-104090RB-100) and Government of Aragón (Grupos Consolidados, E34_20R) for financial support. S.O. thanks also Government of Aragón for a pre-doctoral contract. The authors thankfully acknowledge the resources from the supercomputers “Memento”and “Cierzo”, technical expertise and assistance provided by BIFIZCAM (Universidad de Zaragoza, Spain), and the Biological and Computational Research Group (BIFI, Universidad de Zaragoza) for GPU facilities. We also thank Prof. Paolo Paoli (Dipartimento di Scienze Biochimiche, Sperimentali e Cliniche Mario Serio, Universitadi Firenze) for the help in the analysis of biological data. ■DEDICATION Dedicated to Prof. Alberto Brandi on the occasion of his 70th birthday. ■REFERENCES (1) Compain, P.; Martin, O. R. Iminosugars: From Synthesis to Therapeutic Applications; Wiley VCH: New York, 2007. (2) (a) Brás, N. F.; Cerqueira, N. M.; Ramos, M. J.; Fernandes, P. A. 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