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Article Synthesis of “All-Cis” Trihydroxypiperidines from a -Derived Ketone: Hints for the Design of New β-Gal and GCase Inhibitors

Maria Giulia Davighi 1, Francesca Clemente 1, Camilla Matassini 1,* , Amelia Morrone 2 , Andrea Goti 1,3, Macarena Martínez-Bailén 4 and Francesca Cardona 1,3,*

1 Department of Chemistry “Ugo Schiff”, University of Firenze, via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy; [email protected]fi.it (M.G.D.); [email protected]fi.it (F.C.); [email protected]fi.it (A.G.) 2 Paediatric Neurology Unit and Laboratories, Neuroscience Department, Meyer Children’s Hospital and Department of Neurosciences, Pharmacology and Child Health, University of Florence, Viale Pieraccini 24, 50139 Firenze, Italy; [email protected]fi.it 3 Associated with Consorzio Interuniversitario Nazionale di Ricerca in Metodologie e Processi Innovativi di Sintesi (CINMPIS), 70100 Bari, Italy 4 Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, García González 1, E-41012 Sevilla, Spain; [email protected] * Correspondence: [email protected]fi.it (C.M.); [email protected]fi.it (F.C.); Tel.: +39-055-457-3536 (C.M.); +39-055-457-3504 (F.C.)  Academic Editors: Ivo Piantanida, René Csuk, Claus Jacob and Adegboyega K. Oyelere  Received: 30 July 2020; Accepted: 30 September 2020; Published: 2 October 2020

Abstract: Pharmacological chaperones (PCs) are small compounds able to rescue the activity of mutated lysosomal enzymes when used at subinhibitory concentrations. Nitrogen-containing glycomimetics such as aza- or iminosugars are known to behave as PCs for lysosomal storage disorders (LSDs). As part of our research into lysosomal sphingolipidoses inhibitors and looking in particular for new β-galactosidase inhibitors, we report the synthesis of a series of alkylated azasugars with a relative “all-cis” configuration at the hydroxy/amine-substituted stereocenters. The novel compounds were synthesized from a common carbohydrate-derived piperidinone intermediate 8, through reductive amination or alkylation of the derived . In addition, the reaction of ketone 8 with several lithium acetylides allowed the stereoselective synthesis of new azasugars alkylated at C-3. The activity of the new compounds towards lysosomal β-galactosidase was negligible, showing that the presence of an alkyl chain in this position is detrimental to inhibitory activity. Interestingly, 9, 10, and 12 behave as good inhibitors of lysosomal β-glucosidase (GCase) (IC50 = 12, 6.4, and 60 µM, respectively). When tested on cell lines bearing the Gaucher mutation, they did not impart any enzyme rescue. However, altogether, the data included in this work give interesting hints for the design of novel inhibitors.

Keywords: synthesis; azasugars; iminosugars; lithium acetylides; lysosomal enzyme inhibitors; lysosomal storage disorders (LSDs); lysosomal sphingolipidoses; Gaucher disease

1. Introduction Lysosomal storage disorders (LSDs) are a group of more than 70 inherited orphan diseases caused by specific mutations in genes encoding lysosomal enzymes and characterized by progressive accumulation of substrates within the lysosomes, leading to organ dysfunctions [1–4]. Defective activity of lysosomal β-galactosidase (β-Gal), which is responsible for the hydrolytic removal of a terminal β-galactose residue from several glycoconjugates, leads to two different types

Molecules 2020, 25, 4526; doi:10.3390/molecules25194526 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 24

Defective activity of lysosomal β-galactosidase (β-Gal), which is responsible for the hydrolytic removal of a terminal β-galactose residue from several glycoconjugates, leads to two different types of rare LSDs, the sphingolipidosis GM1-gangliosidosis and the mucopolysaccharidosis IVB (MPS IVB, also known as Morquio disease type B) [5,6]. Typical substrates of β-Gal are GM1-gangliosides, glycoproteins, oligosaccharides, and glycosaminoglycan keratan sulfate, which accumulate inside the lysosomes due to the malfunctioning enzyme, which usually presents a mutation in its natural amino Moleculesacid sequence.2020, 25, 4526To date, no pharmacological treatment is available for these severe diseases. 2 of 23 Pharmacological chaperone therapy (PCT) is emerging as a promising therapeutic approach for the treatment of LSDs, especially for those that cannot be treated with enzyme replacement therapy of(ERT). rare PCT LSDs, is thebased sphingolipidosis on pharmacological GM1-gangliosidosis chaperones (PCs), and small the mucopolysaccharidosismolecules that can selectively IVB (MPS bind IVB,mutant also enzymes known asin Morquio the endoplasmi diseasec type reticulum B) [5,6]. (ER) Typical and substrates stabilize their of β-Gal correct are GM1-gangliosides,three-dimensional glycoproteins,conformation when oligosaccharides, used at subi andnhibitory glycosaminoglycan concentrations, keratan thus improving sulfate, which lysosomal accumulate trafficking inside and the lysosomesrescuing enzymatic due to the activity. malfunctioning PCs are reversible enzyme, which inhibitors usually and presents can be replaced a mutation by the in itsnatural natural substrate amino acidof the sequence. enzyme Toinside date, nothe pharmacologicallysosomes. Their treatment main pros is available include fororal these admi severenistration, diseases. broad body distributionPharmacological (PCs have chaperone the potential therapy to cross (PCT) the isbl emergingood–brain as barrier), a promising and mino therapeuticr side effects approach [7–11]. for the treatmentNitrogen-containing of LSDs, especially glycomimetics, for those such that cannotas iminosugars be treated with( enzyme replacement analogues therapywith a (ERT).nitrogen PCT atom is based replacing on pharmacological the endocyclic chaperonesoxygen), ar (PCs),e the most small investigated molecules that class can of selectively PCs for LSDs bind mutant[12,13]. enzymes in the endoplasmic reticulum (ER) and stabilize their correct three-dimensional conformationRecently, whenthe first used commercially at subinhibitory available concentrations, PC for treating thus improvinglysosomal Fabry lysosomal disease, traffi Galafoldcking andTM rescuing(1-deoxygalactonojirimycin, enzymatic activity. PCs DGJ, are 1 reversible, Figure 1), inhibitors has been and marketed can be replaced in Europe by the[14]. natural Previous substrate studies of thehave enzyme shown inside that the the lysosomes.N-alkylated Their derivative main pros of DGJ include N-nonyl-DGJ oral administration, (2, Figure broad1) is able body to distribution rescue the (PCsintracellular have the activity potential of mutant to cross β the-Gal blood–brain in GM1-gangliosidosis barrier), and patient minor sidefibroblasts, effects [thus7–11 highlighting]. the potentialNitrogen-containing of PC therapy for glycomimetics, patients with such brain as iminosugars pathologies (carbohydrates [15,16]. In addition, analogues several with azasugars, a nitrogen atomcarbohydrate replacing analogues the endocyclic in which oxygen), nitrogen are the formally most investigated replaces the class anomeric of PCs for carbon, LSDs [12have,13]. shown TM interestingRecently, biological the first activities, commercially as recently available reviewed PC for by treating Simone lysosomal and coworkers Fabry [17]. disease, Galafold (1-deoxygalactonojirimycin,In particular, 4-epi-isofagomine DGJ, 1, Figure (3, Figure1), has 1) been showed marketed moderate in Europe inhibition [ 14].Previous of human studies lysosomal have shownβ-Gal (IC that50 the= 1 NµM)-alkylated [18]. Introduction derivative of of DGJ an alkylN-nonyl-DGJ chain as (in2, compounds Figure1) is able 4 and to rescue 5 (Figure the intracellular1) enhanced activitythe inhibitory of mutant potencyβ-Gal up in to GM1-gangliosidosis IC50 = 10 and IC50 = patient0.4 nM, fibroblasts,respectively. thus These highlighting compounds the were potential able ofto PCrescue therapy mutant for β patients-Gal activity with brainin fibroblasts pathologies from [15 GM1-gang,16]. In addition,liosidosis several patients azasugars, [19,20]. Moreover, carbohydrate the analogues“all-cis” trihydroxypiperidines in which nitrogen formally 6 and replaces 7 (Figure the anomeric1) were carbon,able to haveincrease shown β-Gal interesting activity biological in GM1 activities,gangliosidosis as recently patient reviewed fibroblasts by up Simone to 2–6 and fold coworkers [21]. [17].

FigureFigure 1.1. Structures ofof aa generalgeneral ββ-galactoside andand ofof somesome imino-imino- andand azasugarsazasugars inhibitorsinhibitors ofof ββ-Gal.-Gal.

InA commonly particular, 4-employedepi-isofagomine strategy ( 3for, Figure the design1) showed of a potential moderate PC inhibition is to modify of humana natural lysosomal inhibitor βof-Gal the (ICcompromised50 = 1 µM) [ 18enzyme]. Introduction with the ofaim an of alkyl increasing chain as its in efficacy compounds and 4selectivity.and 5 (Figure However,1) enhanced since thethe inhibitoryinhibitory potencypotency upof a to synthetic IC50 = 10 analogue and IC50 does= 0.4 not nM, always respectively. correlate These with compounds its performance were ableas a toPC, rescue a reliable mutant structure-activityβ-Gal activity relationship in fibroblasts (SARs) from for GM1-gangliosidosis PCs is not easy and patients needs to [19 be,20 demonstrated]. Moreover, theby experimental “all-cis” trihydroxypiperidines evidence [21]. 6 and 7 (Figure1) were able to increase β-Gal activity in GM1 gangliosidosis patient fibroblasts up to 2–6 fold [21]. A commonly employed strategy for the design of a potential PC is to modify a natural inhibitor of the compromised enzyme with the aim of increasing its efficacy and selectivity. However, since the inhibitory potency of a synthetic analogue does not always correlate with its performance as a PC, a reliable structure-activity relationship (SARs) for PCs is not easy and needs to be demonstrated by experimental evidence [21]. Molecules 2020, 25, x FOR PEER REVIEW 3 of 24

Molecules 2020, 25Based, 4526 on our previous experience in the synthesis of imino- and azasugars as potential PCs [22– 3 of 23 24], we envisaged that a new series of “all-cis” trihydroxypiperidines and congeners with potential activity as β-Gal inhibitors could be synthesized starting from the common key ketone intermediate 8 [25] (Scheme 1). We report the synthesis of compounds 9–15 (Scheme 1) and their assessment as Based on our previous experience in the synthesis of imino- and azasugars as potential PCs [22–24], inhibitors of β-Gal and other glycosidases. we envisaged thatKetone a new8 [25] serieswas functionalized of “all-cis” through trihydroxypiperidines three different synthetic and strategies: congeners (i) reduction with potential to the activity as β-Gal inhibitors“all-cis” alcohol could followed be synthesized by Williamson starting reaction fromto afford the the common ether 9; (ii) key reductive ketone amination intermediate of 8 8 [25] (Scheme1).Moleculeswith We dodecyl report 2020, 25 , thexamine FOR synthesis PEER provided REVIEW of the compounds new amino azasugar9–15 (Scheme 10; and1 (iii)) and addition their assessmentof organolithium3 of as 24 inhibitors derivatives followed by proper manipulation allowed access to diversely C-3 functionalized of β-Gal and other glycosidases. compoundsBased on 11 our–15 previous. experience in the synthesis of imino- and azasugars as potential PCs [22– 24], we envisaged that a new series of “all-cis” trihydroxypiperidines and congeners with potential activity as β-Gal inhibitors could be synthesized starting from the common key ketone intermediate 8 [25] (Scheme 1). We report the synthesis of compounds 9–15 (Scheme 1) and their assessment as inhibitors of β-Gal and other glycosidases. Ketone 8 [25] was functionalized through three different synthetic strategies: (i) reduction to the “all-cis” alcohol followed by Williamson reaction to afford the ether 9; (ii) reductive amination of 8 with dodecyl amine provided the new amino azasugar 10; and (iii) addition of organolithium derivatives followed by proper manipulation allowed access to diversely C-3 functionalized compounds 11–15.

Scheme 1.SchemeCompounds 1. Compounds synthesized synthesized in in thisthis work work through through the functionalization the functionalization of ketone 8 of via: ketone (i) 8 via: reduction to alcohol and Williamson reaction to ether 9, (ii) reductive amination with dodecyl amine (i) reduction to alcohol and Williamson reaction to ether 9, (ii) reductive amination with dodecyl amine to access 10, (iii) addition of organolithium derivatives to finally obtain compounds 11–15 . to access 10, (iii) addition of organolithium derivatives to finally obtain compounds 11–15. 2. Results and Discussion Ketone 8 [25] was functionalized through three different synthetic strategies: (i) reduction to the “all-cis” alcohol2.1. Synthesis followed by Williamson reaction to afford the ether 9; (ii) reductive amination of 8 with dodecyl amineKetone provided 8, precursor the new of all amino the new azasugar compounds,10 was; and synthesized (iii) addition as reported of organolithium from 16 derivatives,

followed byderived proper in turn manipulation from inexpensive allowed D-mannose access in to four diversely steps with C-3 a functionalizedhigh overall yield compounds (85%). The 11–15. piperidineScheme skeleton 1. Compounds of 17 was synthesized obtained in through this work a doublethrough reductive the functionalization amination ofprocedure ketone 8 (DRA)via: (i) [25– 27] followedreduction by to protectionalcohol and ofWilliamson the endocyclic reaction nitrogen to ether atom9, (ii) reductivewith a tert amination-butyloxycarbonyl with dodecyl (Boc) amine group. 2. Results and Discussion Oxidationto access of 1017, (iii)with addition Dess Martin of organolithium periodinane derivatives (DMP) togave finally ketone obtain 8, compoundswhich was 11diastereoselectively–15 . reduced to the “all-cis” alcohol 18 with NaBH4 in EtOH (Scheme 2) [25]. 2.1. Synthesis2. Results and Discussion

Ketone2.1.8 Synthesis, precursor of all the new compounds, was synthesized as reported from aldehyde 16, d derived in turnKetone from inexpensive8, precursor of all-mannose the new compounds, in four steps was withsynthesized a high as overall reported yield from (85%). aldehyde The 16,piperidine skeleton ofderived17 was in obtainedturn from throughinexpensive a doubleD-mannose reductive in four steps amination with a high procedure overall yield (DRA) (85%). [25 –The27 ] followed by protectionpiperidine of the skeleton endocyclic of 17 was nitrogen obtained atom through with a double a tert reductive-butyloxycarbonyl amination procedure (Boc) group. (DRA) [25– Oxidation of 27] followed by protection of the endocyclic nitrogen atom with a tert-butyloxycarbonyl (Boc) group. 17 with Dess Martin periodinane (DMP) gave ketone 8, which was diastereoselectively reduced to the Oxidation of 17 with Dess Martin periodinane (DMP) gave ketone 8, which was diastereoselectively all-cis 18 Scheme 2. Previous work to synthesize ketone 8 and the 17 and 18. “ ” alcoholreduced towith the “all-cis NaBH” alcohol4 in EtOH 18 with (Scheme NaBH4 in2 EtOH)[25 ].(Scheme 2) [25]. The “all-cis” ether 19 was obtained by Williamson synthesis following a recently reported procedure on a similar substrate [28]. Treatment of alcohol 18 with sodium hydride in dry DMF, alkylation with 1-nonyl bromide gave ether 19 with a 53% yield. Concomitant deprotection of acetonide and Boc groups under acidic conditions (aqueous HCl in MeOH), followed by treatment with the strongly basic resin Ambersep 900 OH, gave ether 9 with a 70% yield (Scheme 3). In order

SchemeScheme 2. Previous 2. Previous work work to to synthesize synthesize ketone ketone 8 and8 and the alcohols the alcohols 17 and 1718. and 18. The “all-cis” ether 19 was obtained by Williamson synthesis following a recently reported The “procedureall-cis” ether on a19 similarwas obtainedsubstrate [28]. by WilliamsonTreatment of synthesisalcohol 18 with following sodium a hydride recently in reporteddry DMF, procedure on a similaralkylation substrate with [ 281-nonyl]. Treatment bromide ofgave alcohol ether 1918 withwith a sodium53% yield. hydride Concomitant in dry deprotection DMF, alkylation of with 1-nonyl bromideacetonide gaveand Boc ether groups19 underwith acidic a 53% conditions yield. (aqueous Concomitant HCl in MeOH), deprotection followed of by acetonide treatment and Boc groups underwith the acidic strongly conditions basic resin (aqueousAmbersep 900 HCl OH, in gave MeOH), ether 9 followedwith a 70% byyield treatment (Scheme 3). with In order the strongly basic resin Ambersep 900 OH, gave ether 9 with a 70% yield (Scheme3). In order to investigate the role of different configurations at C-3 on biological activity, the diastereomeric alcohol 17 was treated similarly to give protected ether 20 with a 54% yield. Deprotection of 20 as above yielded ether 21 quantitatively (Scheme3). Molecules 2020, 25, x FOR PEER REVIEW 4 of 24

to investigate the role of different configurations at C-3 on biological activity, the diastereomeric Molecules 2020alcohol, 25, 4526 17 was treated similarly to give protected ether 20 with a 54% yield. Deprotection of 20 as 4 of 23 above yielded ether 21 quantitatively (Scheme 3).

SchemeScheme 3. Synthetic 3. Synthetic strategies strategies to to obtainobtain ethers ethers 9 and9 and 21 and21 theand amine the 10 amine. 10.

Ketone 8Ketonewas also 8 was converted also converted to theto the amine amine22 22 throughthrough reductive reductive amination amination employing employing dodecyl dodecyl amine. The reaction was performed in dry MeOH under catalytic on Pd(OH)2/C and amine. Thegave, reaction after purification was performed by flash column in dry ch MeOHromatography under (FCC), catalytic the protected hydrogenation piperidine on 22 Pd(OH)with a 2/C and gave, after55% purification yield. Final bydeprotection flash column under chromatographyacidic conditions followed (FCC), by thetreatment protected with Ambersep piperidine 900 22OHwith a 55% yield. Finalresin deprotection gave the free underamine 10 acidic with an conditions 83% yield (Scheme followed 3). The by treatment“all-cis” configuration with Ambersep in compound 900 OH resin 1 gave the free22 was amine established10 with by ancomparison 83% yield of its (Scheme H-NMR3 spectrum). The “ withall-cis those” configuration of similar compounds in compound with a 22 was different alkyl chain at the exocyclic nitrogen atom [29]. The broad singlet at 4.41 ppm observed for Molecules 2020, 25, x FOR PEER REVIEW1 5 of 24 established4-H by in comparison the 1H-NMR ofspectrum its H-NMR of 22 is consistent spectrum with with eq-ax those rela oftionships similar with compounds both 3-H and with 5-H a different alkyl chainacetylationoccurring at the exocyclicin of its the chair free conformations. nitrogen OH group atom (see [the29 ].Supplem The broadentary singletMaterials) at 4.41(Scheme ppm 4) observedin case of octyl for 4-H in the 1H-NMR spectrummagnesiumThe synthesis ofbromide22 is of consistentaddition.“all-cis” trihydroxypiperidines with eq-ax relationships alkylated at with C-3 by both addition 3-H of and organometallic 5-H occurring in its Given the poor results obtained with alkyl Grignard reagents, we investigated the addition chair conformations.reagents to the of 8 was then addressed. Ketone 8 was first reacted with Grignard reactionreagents with(octylMgBr, less bulky ethylMgBr, sp2 and sp and organometals. methylMgBr, The Scheme reaction 4) underof 8 with conditions vinyl magnesium which had bromide proved The synthesiswassuccessful less sluggish for of Grignard “all-cis and a ” singleadditions trihydroxypiperidines diastereoisomeric to aldehyde 16 adduct and a alkylated nitrone26 (see belowderived at for C-3 thereof structural by addition[23,30]. assignment) of organometallic was reagents toobtained theThe carbonyl additionwith a 51% of group ethyl yield magn of(Scheme8esiumwas 4). thenbromide Hydrogenation addressed. to a simpler of 26 KetoneN in-Boc-protected the presence8 was 4-piperidone firstof Pd/C reacted under has acidic with been Grignard reagents (octylMgBr,conditionspreviously (aqueousreported ethylMgBr, HCl[31]. in However, EtOH) and allowed methylMgBr, Grignard complete additions Schemedeprotection to ketone4) under and 8 proved reduction conditions difficult of the and . which sluggish. Final had proved treatment with Ambersep 900 OH and purification by FCC gave the saturated azasugar 13 with a successfulVariation for Grignard of reaction additions conditions to (temperature aldehyde 16 rangingand a from nitrone −78 °C derived to room thereoftemperature, [23, 30Grignard]. 52%equivalents yield (Scheme ranging 4). from 1 to 1.8, see the Supplementary Materials for further details) did not lead to substantial improvements, and the desired alcohols 23, 24, or 25 were obtained with yields of 12% or less, in complex mixtures difficult to purify. In case of 23 and 24, a single diasteromeric adduct was isolated (as assessed by ESI-MS and 1H-NMR), while with methyl magnesium bromide a mixture of two diastereoisomers (about 12% yield) was observed in the 1H-NMR spectrum. The low yields can be ascribed to formation of the reduction product 18. Reduction of ketones by Grignard reagents through a β-hydride addition is known as an undesired side reaction that can occur with particularly encumbered substrates as a consequence of the reduced rate of nucleophilic addition [32,33]. Formation of 18 was attested by analysis of the 1H-NMR and ESI-MS spectra and confirmed after

3 2 Scheme 4.SchemeAddition 4. Addition reactions reactions of sp of spand3 and sp sp2 Grignard reagents reagents to ketone to ketone 8 and synthesis8 and synthesis of 13. of 13. Encouraged by this result, we turned our attention to the addition of alkynyl organometallic The addition of ethyl magnesium bromide to a simpler N-Boc-protected 4-piperidone has been reagents, namely, lithium acetylides generated in situ by treating terminal alkynes with butyl lithium. previouslyCompared reported to [many31]. C-nucleophiles, However, Grignard acetylides additionsare less basic to and ketone less sensitive8 proved to steric diffi congestioncult and sluggish. Variation of[34]. reaction The results conditions of the addition (temperature of structurally ranging differentiated from lithium78 ◦C toacetylides room temperature,to ketone 8 are Grignard reported in Table 1 −

Table 1. Addition reactions of lithium acetylides to ketone 8.

Entry a Alkyne Time (h) Product Yield (%) 1 Phenylacetylene 2.5 27 77 2 1-octyne 3 28 78 3 3,3-diethoxyprop-1-yne 3 29 65 4 3-ethynylthiophene 4 30 88 5 4-ethynyl-N,N-dimethylaniline 4 31 83 a: All the experiments were carried out in dry THF with temperatures ranging from −78 °C to room temperature, with ketone 8 (1.0 equiv.) and BuLi (1.5 equiv.).

Molecules 2020, 25, x FOR PEER REVIEW 5 of 24 acetylation of the free OH group (see the Supplementary Materials) (Scheme 4) in case of octyl magnesium bromide addition. Given the poor results obtained with alkyl Grignard reagents, we investigated the addition reaction with less bulky sp2 and sp organometals. The reaction of 8 with vinyl magnesium bromide was less sluggish and a single diastereoisomeric adduct 26 (see below for structural assignment) was obtained with a 51% yield (Scheme 4). Hydrogenation of 26 in the presence of Pd/C under acidic conditions (aqueous HCl in EtOH) allowed complete deprotection and reduction of the alkene. Final treatment with Ambersep 900 OH and purification by FCC gave the saturated azasugar 13 with a 52% yield (Scheme 4).

Molecules 2020, 25, 4526 5 of 23 equivalents ranging from 1 to 1.8, see the Supplementary Materials for further details) did not lead to substantial improvements, and the desired alcohols 23, 24, or 25 were obtained with yields of 12% or less, in complex mixtures difficult to purify. In case of 23 and 24, a single diasteromeric adduct was isolated (as assessed by ESI-MS and 1H-NMR), while with methyl magnesium bromide a mixture of two diastereoisomers (about 12% yield) was observed in the 1H-NMR spectrum. The low yields can be ascribed to formation of the reduction product 18. Reduction of ketones by Grignard reagents through a β-hydride addition is known as an undesired side reaction that can occur with particularly encumbered substrates as a consequence of the reduced rate of nucleophilic addition [32,33]. Formation of 18 was attested by analysis of the 1H-NMR and ESI-MS spectra and confirmed after acetylation of the free OH group (see the Supplementary Materials) (Scheme4) in case of octyl magnesium bromide addition. Given the poor results obtained with alkyl Grignard reagents, we investigated the addition reaction with less bulky sp2 and sp organometals. The reaction of 8 with vinyl magnesium bromide was less sluggish and a single diastereoisomeric adduct 26 (see below for structural assignment) was obtained with a 51% yield (Scheme4). Hydrogenation of 26 in the presence of Pd/C under acidic conditionsScheme (aqueous 4. Addition HCl in reactions EtOH) of allowed sp3 andcomplete sp2 Grignard deprotection reagents to andketone reduction 8 and synthesis of the alkene.of 13. Final treatmentEncouraged with Ambersep by this result, 900 OH we and turned purification our attent by FCCion to gave the the addition saturated of alkynyl azasugar organometallic13 with a 52% yieldreagents, (Scheme namely,4). lithium acetylides generated in situ by treating terminal alkynes with butyl lithium. ComparedEncouraged to many by this C-nucleophiles, result, we turned acetylides our attention are less to thebasic addition and less of alkynyl sensitive organometallic to steric congestion reagents, namely,[34]. The lithium results acetylides of the addition generated of in structurally situ by treating differentiated terminal alkynes lithium with acetylides butyl lithium. to ketone Compared 8 are to manyreported C-nucleophiles, in Table 1 acetylides are less basic and less sensitive to steric congestion [34]. The results of the addition of structurally differentiated lithium acetylides to ketone 8 are reported in Table1. Table 1. Addition reactions of lithium acetylides to ketone 8. Table 1. Addition reactions of lithium acetylides to ketone 8.

a EntryEntry a Alkyne Alkyne Time Time (h) (h) Product Product Yield Yield (%) (%) 11 PhenylacetylenePhenylacetylene 2.5 2.5 2727 7777 22 1-octyne1-octyne 3 3 2828 7878 3 3,3-diethoxyprop-1-yne 3 29 65 34 3,3-diethoxyprop-1-yne 3-ethynylthiophene 4 3 3029 6588 45 4-ethynyl-3-ethynylthiopheneN,N-dimethylaniline 4 4 3130 8883 5 4-ethynyl-N,N-dimethylaniline 4 31 83 a: All the experiments were carried out in dry THF with temperatures ranging from 78 C to room temperature, a: All the experiments were carried out in dry THF with temperatures ranging◦ from −78 °C to room with ketone 8 (1.0 equiv.) and BuLi (1.5 equiv.). − temperature, with ketone 8 (1.0 equiv.) and BuLi (1.5 equiv.).

The alkynes were treated with BuLi (1.5 equiv.) at 78 C for 30 min, followed by the addition of − ◦ ketone 8 at 78 C. The reaction mixture was allowed to warm to room temperature and left to react − ◦ for 2.5–3 h, then worked up. In all reported cases, FCC of the crude mixtures afforded good yields (65–88%) of adducts with both simple and functionalized (Table1). A single diastereoisomer was obtained in all cases, which was ascribed the (S) configuration at the newly formed C-3 stereocenter on the basis of the following considerations. Alkynyl lithium derivatives are small nucleophiles, which typically prefer an axial rather than an equatorial attack on cyclohexanones [35–37] in order to avoid torsional strain [38,39]. In Scheme5, the two more stable chair conformations of ketone 8 6 are depicted. Only in the C3 conformation, nucleophilic axial attack at C-3 experiences a stabilizing interaction by the low-lying energy σ* orbital of the antiperiplanar C-O bond at C-4, according to MoleculesMolecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 2424

TheThe alkynesalkynes werewere treatedtreated wiwithth BuLiBuLi (1.5(1.5 equiv.)equiv.) atat −−7878 °C°C forfor 3030 min,min, followedfollowed byby thethe additionaddition ofof ketoneketone 88 atat −−7878 °C.°C. TheThe reactionreaction mixturemixture waswas allowedallowed toto wawarmrm toto roomroom temperaturetemperature andand leftleft toto reactreact forfor 2.5–32.5–3 h,h, thenthen workedworked up.up. InIn allall reportedreported cases,cases, FCCFCC ofof thethe crudecrude mixturesmixtures affordedafforded goodgood yieldsyields (65–88%)(65–88%) ofof adductsadducts withwith bothboth simplesimple andand functionalizedfunctionalized alkenesalkenes (Table(Table 1).1). AA singlesingle diastereoisomerdiastereoisomer waswas obtainedobtained inin allall cases,cases, whichwhich waswas ascribedascribed thethe ((SS)) configurationconfiguration atat thethe newlynewly formedformed C-3C-3 stereocenterstereocenter onon thethe basisbasis ofof thethe followingfollowing consconsiderations.iderations. AlkynylAlkynyl lithiumlithium derivativesderivatives areare smallsmall nucleophiles,nucleophiles, whichwhich typicallytypically preferprefer anan axialaxial ratherrather thanthan anan equatorialequatorial attackattack onon cyclohexanonescyclohexanones [35–[35– 37]37] inin orderorder toto avoidavoid torsionaltorsional strainstrain [38,39].[38,39]. InIn SchemeScheme 5,5, thethe twotwo moremore stablestable chairchair conformationsconformations ofof 6 ketone 8 are depicted. Only in the 6 C33 conformation, nucleophilic axial attack at C-3 experiences a ketoneMolecules 82020 are, 25 depicted., 4526 Only in the C conformation, nucleophilic axial attack at C-3 experiences6 of 23a stabilizingstabilizing interactioninteraction byby thethe low-lyinglow-lying energyenergy σσ** orbitalorbital ofof thethe antiperiplanarantiperiplanar C-OC-O bondbond atat C-4,C-4, accordingaccording toto aa favorablefavorable Felkin–AnhFelkin–Anh modelmodel [40,41[40,41].]. Thus,Thus, basedbased onon stereoelectronicstereoelectronic andand stericsteric considerations,aconsiderations, favorable Felkin–Anh wewe assumedassumed model thatthat [ 40nucleophilicnucleophilic,41]. Thus, attacksattacks based occurred onoccurred stereoelectronic selectivelyselectively andatat thethe steric ReRe faceface considerations, ofof ketoneketone 88,, givingwegiving assumed thethe ““all-cis”all-cis” that nucleophilic 3,3, 4,4, 5-trihydroxypiperidines5-trihydroxypiperidines attacks occurred selectively (Scheme(Scheme 5).5). at A theA carefulcarefulRe face analysisanalysis of ketone ofof the8the, giving 11HH NMRNMR the spectra “spectraall-cis” of3,of 4,derivativesderivatives 5-trihydroxypiperidines 2626––3131 andand thethe productsproducts (Scheme 5 ofof). theirtheir A careful transformationtransformation analysis ofss thesupportssupports1H NMR thisthis structural spectrastructural of assignment derivativesassignment (see26(see–31 below).below).and the products of their transformations supports this structural assignment (see below).

SchemeScheme 5. 5. StereochemicalStereochemical outcome outcome of of the the addition addition of of lithium lithium acetylides acetylides to to ketone ketone 88..

CompoundsCompounds 2727,, 28,28, andand 3030 werewere subjectedsubjected to to acidicacidic treatmenttreatmtreatmentent for for thethe concomitantconcomitant removal removal of of acetonideacetonide andandand Boc-protecting Boc-protectingBoc-protecting groups, groups,groups, leading leadingleading to toto the thethe “all-cis ““all-cisall-cis” trihydroxypiperidines”” trihydroxypiperidinestrihydroxypiperidines32, 33, 3232,,and 33,33, 34andandwith 3434 withgoodwith goodgood yields yieldsyields (51–71%) (51–71%)(51–71%) after treatmentafterafter treatmenttreatment with thewithwith strongly ththee stronglystrongly basic basic resinbasic resin Ambersepresin AmbersepAmbersep 900 OH. 900900 The OH.OH. triple TheThe tripletriple bond bondwasbond subsequently waswas subsequentlysubsequently reduced reducedreduced by catalytic byby catalyticatalyti hydrogenationcc hydrogenationhydrogenation in the inin presence thethe presencepresence of Pd(OH) ofof Pd(OH)Pd(OH)2/C in22/C/C EtOH inin EtOHEtOH to give toto giveazasugarsgive azasugarsazasugars11, 12, 1111and,, 12,12, 14 andand(39–98%) 1414 (39–98%)(39–98%) (Scheme (Scheme(Scheme6). 6).6).

SchemeScheme 6. 6. SynthesisSynthesis ofof trihydroxypiperidines trihydroxypiperidines 1111,,, 1212,,, 14,14, andand 1515...

DueDue to to the the presence presence of of the the additional additional acid-labile acid-labile acetal acetal moiety,moiety, adduct adduct 2929 waswas expected expected to to give give complicationscomplications arising arising fromfrom possible possible interactions interactions ofof thethe free free amine amine with with the the aldehyde aldehyde and and was was not not subjectedsubjected tototo acid-induced acid-inducedacid-induced deprotection. deprotecdeprotection.tion. Unsuccessful UnsuccessfulUnsuccessful deprotection deprotectiondeprotection of 31 with ofof di3131ff erentwithwith acids differentdifferent (CF3COOH, acidsacids CH COOH, and HCl) was ascribed to the higher reactivity of its triple bond. Therefore, hydrogenation 3 of the alkyne under neutral conditions (H2, Pd/C in EtOH) was first carried out to give piperidine 35, which was subsequently deprotected with aqueous HCl in MeOH. Final treatment with the strongly basic resin Ambersep 900 OH gave trihydroxypiperidine 15 with a 56% yield (Scheme6).

2.2. Configuration Assignment Relevant chemical shifts and coupling constants are reported in Tables2 and3 for H-4, H-5, 1 and H-6 in H-NMR spectra in selected compounds of the two series of protected (in CDCl3) and deprotected (in CD3OD) trihydroxypiperidines, respectively. These values show regularities (the same Molecules 2020, 25, x FOR PEER REVIEW 7 of 24

(CF3COOH, CH3COOH, and HCl) was ascribed to the higher reactivity of its triple bond. Therefore, hydrogenation of the alkyne under neutral conditions (H2, Pd/C in EtOH) was first carried out to give piperidine 35, which was subsequently deprotected with aqueous HCl in MeOH. Final treatment with the strongly basic resin Ambersep 900 OH gave trihydroxypiperidine 15 with a 56% yield (Scheme 6).

2.2. Configuration Assignment Relevant chemical shifts and coupling constants are reported in Tables 2 and 3 for H-4, H-5, and H-6 in 1H-NMR spectra in selected compounds of the two series of protected (in CDCl3) and deprotected (in CD3OD) trihydroxypiperidines, respectively. These values show regularities (the sameMolecules applies 2020, 25 , xto FOR H-2PEER REVIEW signals), which allowed us to ascribe the same7 of 24 configuration at C-3 for all compounds(CF3COOH, CH(see3COOH, the andSupplementary HCl) was ascribed to Ma the higherterials reactivity for the of its full triple Table).bond. Therefore, More over, the shape of the signals andMolecules hydrogenationtheir2020 coupling, 25 of, the 4526 alkyne constants, under neutral where conditions detectable, (H2, Pd/C in EtOH) are was consistent first carried out with to give the (S) absolute configuration7 of 23 piperidine 35, which was subsequently deprotected with aqueous HCl in MeOH. Final treatment tentativelywith the strongly assigned basic resin(see Ambersep above) 900 on OH the gave basis trihydroxypiperidine of mechanistic 15 with considerations. a 56% yield Indeed, the signal of H-5 appears(Scheme as 6). a broad singlet (or as a narrow multiplet), which is in agreement with its equatorial applies to H-2 signals), which allowed us to ascribe the same configuration at C-3 for all compounds position2.2. Configuration in a preferred Assignment chair conformation, which places the R substituent equatorially, i.e., in the (3S) (see the Supplementary Materials for the full Table). Moreover, the shape of the signals and their configurationRelevant chemical (6C3 alcohol shifts and couplingin Scheme constants 5). are Thereported lack in Tables of large2 and 3 forax-ax H-4, H-5, coupling and constants is confirmed by couplingH-6 in 1H-NMR constants, spectra in where selected detectable,compounds of the are two consistent series of protected with (in the CDCl (S3) and absolute configuration tentatively signals of H-6. For example, in piperidine 13 (R = ethyl), the two hydrogens at C-6 display vicinal assigneddeprotected (see (in CD above)3OD) trihydroxypiperidines, on the basis of respectively. mechanistic These values considerations. show regularities (the Indeed, the signal of H-5 appears couplingsame applies constants to H-2 signals), J = 3.4 which and allowed J = 2.4 us to Hz, ascribe typical the same for configuration ax-eq and at C-3 eq-eq for all relationships, respectively. The as acompounds broad singlet(see the Supplementary (or as a narrow Materials for multiplet), the full Table). whichMoreover, isthe in shape agreement of the signals with its equatorial position in a same applies to the other derivatives when the signals are well resolved, as in compounds 11, 12, 14, preferredand their chaircoupling conformation, constants, where detectable, which are places consistent the with R the substituent (S) absolute configuration equatorially, i.e., in the (3S) configuration tentatively assigned (see above) on the basis of mechanistic considerations. Indeed, the signal of H-5 and 153 . (6Cappearsalcohol as a broad in Scheme singlet (or5 as). a The narrow lack multiplet), of large which ax-axis in agreementcoupling with its constants equatorial is confirmed by signals of H-6. Forposition example, in a preferred in piperidine chair conformation,13 (R which= ethyl), places the the R substituent two hydrogens equatorially, i.e., at in C-6 the (3 displayS) vicinal coupling constants configurationTable 2. Chemical (6C3 alcohol shiftsin Scheme and 5). couplingThe lack of large constants ax-ax coupling of H-4, constants H-5, isand confirmed H-6 of by protected compounds 26 and J = signals3.4 and of H-6. J =For2.4 example, Hz, in typical piperidine for 13 (Rax-eq = ethyl),and the eq-eqtwo hydrogensrelationships, at C-6 display respectively. vicinal The same applies to the coupling35, in CDClconstants3. J = 3.4 and J = 2.4 Hz, typical for ax-eq and eq-eq relationships, respectively. The othersame derivatives applies to the other when derivatives the when signals the signals are wellare well resolved, resolved, as in as compounds in compounds 11, 12, 14, 11, 12, 14, and 15. and 15. H-4 H-5 H-6a H-6b

TableTable 2. 2. ChemicalChemical shifts and shifts coupling and constants coupling of H-4, H-5, constants and H-6 of protected of H-4, compounds H-5, and 26 and H-6 of protected compounds 26 and 3535,, inin CDCl CDCl3. . 3 δ δ δ δ H-4 H-5 H-6a H-6b H-4(ppm) H-5(ppm) H-6a(ppm) (ppm) H-6b Molecules 2020, 25, x FOR PEER REVIEW 8 of 24 δ δ δ δ δ δ δ δ (ppm) (ppm) (ppm) (ppm) Molecules 2020, 25, x FOR PEER REVIEW(ppm) (ppm) (ppm) (ppm)8 of 24 Table 3. Chemical shifts and coupling4.07 constants of H-4,4.33 H-5, and H-6 of protected3.95–3.69 compounds 3.53–3.3232 and R = vinyl, 26 4.07 4.33 3.95–3.69 3.53–3.32 3 4.07 4.33 3.95–3.69 3.53–3.32 Table11R–15R= 3.=,vinyl, vinyl,inChemical CD 2626OD. shifts and (d,coupling J = 6.8 constants Hz) of H-4, (brH-5, s) and H-6 of protected(m) compounds 32 and(m) (d, J = 6.8(d, Hz) J = 6.8 Hz)(br s) (m)(br s) (m) (m) (m) Molecules11– 202015, in, 25 CD, x 3FOROD. PEER REVIEW H-4 H-5 H-6a H-6b 8 of 24 3.98 3.984.30 3.68–3.554.30 3.43–3.10 3.68–3.55 3.43–3.10 (d, J = 6.4 Hz)3.98 (br s) (m)4.30 (m) 3.68–3.55 3.43–3.10 MoleculesR 2020 = , 25, x FOR, 35 PEER REVIEW(d, H-4J = 6.4 Hz) H-5 (br s) H-6a (m) H-6b (m)8 of 24 RTableR == 3. Chemical shifts, 35 and(d, coupling J = 6.4δ Hz)constants of H-4,δ(br H-5, s) and H-6 of protectedδ (m) compoundsδ 32 and(m)

11–15, in 35CD3OD. (ppm)δ (ppm)δ (ppm)δ (ppm)δ MoleculesTable 2020 3. , Chemical25, x FOR PEER shifts REVIEW and coupling constants of H-4, H-5, and H-6 of protected compounds8 of 32 24 and (ppm) (ppm) (ppm) (ppm) 11–15, in CD3OD. H-4 H-5 H-6a H-6b TableTable 3.3. Chemical shifts shifts and andcoupling coupling constants constants of H-4, H-5, of and H-4, H-6 H-5, of protected and H-6 compounds of protected 32 and compounds 32 and H-43.89 3.98–3.91H-5 H-6a 2.86–2.76 H-6b 11–15, in CD3OD. δ δ δ δ 11–15, in CD3OD. (br s) (m) (m) R = , 32 (ppm)3.89 3.98–3.91(ppm) (ppm) 2.86–2.76 (ppm) H-4δ H-5 δ H-6a δ H-6b δ (brH-4 s) (m) H-5 H-6a(m) H-6b R = , 32 (ppm)3.51 (ppm) (ppm)2.96 (ppm) δ δ 3.81 δ δ 2.76–2.70 (br δd, J = 2.2 δ (dd, J = 13.7,δ 4.0 δ R = , 11 (ppm)(ppm)3.893.51 (ppm)3.98–3.91(ppm)(br s) (ppm) 2.96(ppm) 2.86–2.76(ppm) (m) (ppm) Hz) 3.81 Hz) 2.76–2.70 R = , 11 (br (brd, J s) = 2.2 (m) (dd, J = 13.7, 4.0(m) R = , 32 3.893.893.44 3.98–3.913.98–3.91(br3.77 s) 2.92 2.86–2.762.86–2.76 (m)2.68 R = octyl, 12 Hz) Hz) 3.89(br(br s) s) 3.98–3.91(m)(m) 2.86–2.76 (m) (m) RR == , 32 3.513.44(br s) 3.77(br s) (d, J2.962.92 = 12.6 Hz) (d, J2.68 = 12.6 Hz) R = octyl, 12 (br s) (m)3.81 (m) 2.76–2.70 R = , 32 (br d,(br3.51 J s)= 2.2 (br3.81 s) (dd,(d, J J = = 12.6 2.9613.7,2.96 Hz) 4.0 (d, J = 12.62.722.76–2.70 Hz) RR == ,, 1111 (br d, J3.51=3.472.2 Hz) (br(br3.81 s) s) (dd,2.96 J = 13.7, 4.0 Hz) (m) (m) R = ethyl, 13 Hz) 3.81 (dd, Hz)2.96J = 13.6, 3.4 (dd,2.76–2.702.72 J = 13.7, (d,3.51 3.44J = 3.2 Hz) (br3.77 s) 2.96 2.92 2.68 R =R = octyl, 12, 11 (br d,3.47 J = 2.2 3.813.81 (dd, J = Hz)13.7, 4.02.76–2.70 2.4 Hz) R = ethyl, 13 (br d,(br 3.44J = s) 2.2 (br3.77(br s) s) (dd, J(dd, = 13.7, (d,J 2.92= J4.013.6,= 12.6 3.4 Hz) (dd,2.68 (m)J (d,= 13.7, J = 12.6 Hz) RR = = octyl, 12, 11 (d, J =Hz) 3.2 Hz) (br s)(br s) Hz) (m) Hz)(br3.473.53 s) (br3.81 s) (d,Hz) J =Hz) 12.63.022.96 Hz) (d, J2.4 = 12.6 2.79Hz) Hz) 2.72 R = ethyl, 13 3.44 3.773.86 2.92 2.68 R = octyl, 12 (d,(br J3.44=3.53 3.2d, J Hz) = 2.7 3.77 (br s) 2.92(dd,(dd, 2.963.02 J == 13.6, 3.43.8 Hz) 2.68 (br(dd, 2.722.79d, J J == 13.613.7, 2.4 Hz) R R= = octyl, 12 , 14 (br3.47 s) (br3.813.86(br s) s) (d, J = 12.6 Hz) (d, J = 12.6 Hz) R = ethyl, 13 (br(br 3.53d, s)Hz) J = 2.7 (br s) 3.86 (d, J (dd,(dd,= 12.6 JJ Hz)== Hz)13.6,13.6,3.02 3.4(d,3.8 J = 12.6(br(dd, Hz) d, J Hz) J= = 13.7, 13.6 2.79 RR = ,, 1414 (br(d, d, J J== 3.22.7 Hz) Hz) (br(br(br s)s) s) 2.96(dd, 2.96 J = 13.6, 3.8 Hz)2.72 2.72(br d, J = 13.6 Hz) 3.473.47Hz) 3.813.81 Hz) 2.4Hz) Hz) RR = = ethyl,ethyl, 13 3.49 3.80 (dd, J(dd, = 13.6, J = 3.42.9513.6, 3.4(dd, J =(dd, 13.7, J 2.70= 13.7, (d,(d, J =J 3.49=3.23.53 3.2 Hz) Hz) (br (brs) 3.80 s) 3.022.95 2.79 2.70 (br s) 3.86(br s) Hz)(d, JHz) = 13.2 Hz)2.4 Hz)(d, 2.4 J = Hz) 13.8 Hz) RR = , 15 (br(br d,3.49 J s) = 2.7 3.80(br s) (dd, (d,J =2.95 J13.6,= 13.2 3.8 Hz) (br d,2.70 J(d, = 13.6 J = 13.8 Hz) 3.533.53 (br s) 3.02 3.02 2.79 2.79 R = 15 , 14 (brHz) s) 3.86(br3.86 s) (d, J =Hz) 13.2 Hz) (d, J =Hz) 13.8 Hz) R = , 15 (br(br d, d, J =J 2.7= 2.7 (dd, J(dd, = 13.6, J = 3.813.6, 3.8(br d, J (br= 13.6 d, J = 13.6 R = , 14 (br (brs) s) TheR = observed upfield, 14 shiftHz) (0.3–0.5Hz) ppm) of H-4 within theHz) twoHz) series of Hz)compounds Hz) on turning 3.49 3.80 2.95 2.70 fromThe the observed alkynyl to upfield the saturated shift (0.3–0.5substituents ppm) (see of for H-4 instance within 32 the vs.two 11), consistent series ofcompounds with H-4 falling on turning The observed upfield shift (0.3–0.53.49(br s) ppm) of3.80 H-4(br s)within the(d,2.95 twoJ = 13.2 series Hz) of compounds2.70(d, J = 13.8 on Hz) turning fromfromin the thetheR deshielding= alkynylalkynyl to to conethe the, 15 saturated saturatedof the triple 3.49substituents substituents bond in the (see former (see3.80 for forinstance derivatives instance 32 vs.2.9532 when 11 vs.), consistent in11 a), cis consistent relationship, with2.70 H-4 with falling further H-4 falling in R = , 15 (br s) (br s) (d, J = 13.2 Hz) (d, J = 13.8 Hz) theinsupports the deshieldingR deshielding = this assignment. cone cone, 15 ofof the triple triple(br bonds) bond in inthe the former(br former s) derivatives derivatives(d, J = when 13.2 Hz) whenin a cis in(d,relationship, aJ =cis 13.8relationship, Hz) further further supportssupportsThe observedthis assignment. assignment. upfield shift (0.3–0.5 ppm) of H-4 within the two series of compounds on turning The observed upfield shift (0.3–0.5 ppm) of H-4 within the two series of compounds on turning from2.3. theBiological alkynyl Screening to the saturated substituents (see for instance 32 vs. 11), consistent with H-4 falling fromThe the observed alkynyl to upfield the saturated shift (0.3–0.5substituents ppm) (see of for H-4 instance within 32 the vs. two11), consistentseries of compoundswith H-4 falling on turning 2.3.in2.3. the Biological deshieldingCompounds Screening Screening cone 9–15 of, 21 the, 32 triple, and bond 33 were in the first former evaluated derivatives as human when lysosomal in a cis relationship, β-Gal inhibitors further at 1 fromin the the deshielding alkynyl to cone the ofsaturated the triple substituentsbond in the former (see for derivatives instance when 32 vs. in 11a cis), consistentrelationship, with further H-4 falling supportsmM in thishuman assignment. leukocyte homogenates and the results are shown in Table 4 and compared to insupports theCompoundsCompounds deshielding this assignment. 9cone–915–15, of21, ,the21 32, ,triple 32and, and 33bond were33 in werefirstthe former evaluated first derivatives evaluated as human when as lysosomal human in a cis lysosomalrelationship,β-Gal inhibitorsβ further-Gal at 1 inhibitors previously published data. Unfortunately, none of the tested compounds strongly inhibited β-Gal supportsmM in human this assignment. leukocyte homogenates and the results are shown in Table 4 and compared to at2.3. 1 Biological mM in humanScreening leukocyte homogenates and the results are shown in Table4 and compared to previously(only2.3. Biological a moderate published Screening 22% data. inhibition Unfo rtunately,was found none for the of “all-cis”the tested ether compounds 9). These data strongly demonstrate inhibited that β-Gal both previously published data. Unfortunately, none of the tested compounds strongly inhibited β-Gal 2.3.(onlythe Biological Compoundsalkylation aCompounds moderate Screening of 22%9 the–15 ,inhibition, hydroxy 2121, ,32 32, and, and or was 33 the33 were found wereexocyclic first forfirst evaluated the aminevaluated “all-cis”e asgroup human etheras humanand lysosomal9). the These lysosomal introducti dataβ-Gal demonstrate inhibitorsβon-Gal of ainhibitors substituent at 1that both at 1 at (only a moderate 22% inhibition was found for the “all-cis” ether 9). These data demonstrate that both mMtheC-3mM alkylation in ofin thehuman human trihydroxypiperidine of leukocyte the hydroxy homogenateshomogenates or theskeleton exocyclic and and dramatically the theaminresults reesults groupare affect shownare and βshown-Gal thein Tableinhibition. introducti in Table4 and on 4compared andof a substituentcompared to toat Compounds 9–15, 21, 32, and 33 were first evaluated as human lysosomal β-Gal inhibitors at 1 previouslyC-3previously of However,the trihydroxypiperidine publishedpublished the screening data.data. Unfo Unfo rtunately, onskeletonrtunately, a panel nonedramatically noneof of 12 theof commercial thetested affect tested compounds β-Gal compoundsglycosidases inhibition. strongly strongly (see inhibited the inhibited Supplementaryβ-Gal β-Gal mM in human leukocyte homogenates and the results are shown in Table 4 and compared to (onlyMaterials),(onlyHowever, a amoderate moderate showed the 22% screening inhibitionthatinhibition only onwas trihydroxypiperidinewas a found panelfound for offor the 12 the“all-cis” commercial “all-cis” ether10 (bearing ether 9). Theseglycosidases 9). aThese data dodecyl demonstrate data (see chaindemonstrate the thatconnectedSupplementary both that atboth C-3 previouslythe alkylation published of the hydroxy data. Unfo or thertunately, exocyclic noneamine ofgroup the andtested the compounds introduction ofstrongly a substituent inhibited at β-Gal theMaterials),through alkylation a showednitrogen of the thathydroxyatom) only was ortrihydroxypiperidine ablethe exocyclicto inhibit aminβ-glucosidasee 10group (bearing and from thea almonds.dodecyl introducti chainInon particular, of connected a substituent compound at C-3 at (onlyC-3 ofa moderatethe trihydroxypiperidine 22% inhibition skeleton was found dramatically for the “all-cis”affect β-Gal ether inhibition. 9). These data demonstrate that both C-3through10 ofshowed the a trihydroxypiperidinenitrogen an IC50 atom)= 85 µM was towards able skeleton to thisinhibit dramatically enzyme, β-glucosidase which affect prompted fromβ-Gal almonds. inhibition. us to evaluate In particular, compounds compound 9–15 , the21 alkylation, 32,However, and 33 of alsothe the screeningon hydroxy human on or lysosomal athe panel exocyclic of β -glucosidase12 commercialamine group (GCase) glycosidases and (Tablethe introducti (see 4). Pointthe Supplementaryon mutations of a substituent in the gene at 10Materials), showedHowever, anshowed IC the50 = screeningthat 85 µMonly towards trihydroxypiperidine on a panelthis enzyme, of 12 commercial10 which (bearing prompted a glycosidasesdodecyl us chainto evaluate (seeconnected the compounds Supplementary at C-3 9–15, C-3encoding of the trihydroxypiperidine this enzyme cause Gaucher skeleton disease, dramatically the most affect common β-Gal autosomalinhibition. recessive LSD [42,43]. Materials),21through, 32, and a 33nitrogenshowed also on atom)that human onlywas lysosomalable trihydroxypiperidine to inhibit β-glucosidase β-glucosidase 10 (GCase) from(bearing almonds. (Table a dodecyl In4). particular, Point chain mutations compoundconnected in the at gene C-3 However,In keeping the with screening the results on aon panel commercially of 12 commercial available βglycosidases-glucosidase, (see the thebest Supplementary GCase inhibitor throughencoding10 showed a thisnitrogen an enzyme IC50 = atom) 85 causeµM was towards Gaucher able this to disease, inhibitenzyme, β th -glucosidasewhiche most prompted common from us autosomaltoalmonds. evaluate Inrecessivecompounds particular, LSD 9– compound15 [42,43]., Materials), showed that only trihydroxypiperidine 10 (bearing a dodecyl50 chain connected at C-3 10was21 showed, 32,In compound keepingand an33 alsoIC with50 10on= , 85 humanthebearing µM results towardslysosomal a dodecylaminoon commerciallythis β-glucosidase enzyme, substituent which(GCase)available prompted (Table atβ-glucosidase, C-3 4). Point(ICus to mutations =evaluate 6.4the µM, best incompounds TableGCasethe gene 4, inhibitor entry 9–15 2)., through a nitrogen atom) was able to inhibit β-glucosidase from almonds. In particular, compound 21wasRegardingencoding, 32, compound and this33 the also enzyme newly10 on, bearing humancause synthesized Gaucher lysosomala dodecylamino ethers,disease, β-glucosidase the the “all-cis”mostsubstituent common (GCase)9 showed at autosomal C-3 (Table an (IC inhibitory 50 4).recessive = Point 6.4 µM, activitymutationsLSD Table[42,43]. towards 4,in entrythe GCasegene 2). 10 showed an IC50 = 85 µM towards this enzyme, which prompted us to evaluate compounds 9–15, encodingRegardingone orderIn keeping this the of enzyme magnitudenewly with thesynthesized cause results greater Gaucher on than ethers,commercially disease, its theepimer “all-cis” th availablee 21most (IC 9 showedcommon 50β -glucosidase,= 12 µM an autosomal inhibitoryvs. theIC50 best = recessive130 activityGCase µM, inhibitor TabletowardsLSD [42,43]. 4, entries GCase 1 21was, 32, compoundand 33 also 10 on, bearing human a lysosomaldodecylamino β-glucosidase substituent (GCase)at C-3 (IC (Table50 = 6.4 4). µM, Point Table mutations 4, entry in2). the gene onevs. orderIn8). keepingAmong of magnitude withthe compounds the greater results thanonderived commercially its epimer from organome 21 available(IC50 = tal12 βadditionµM-glucosidase, vs. IC reactions,50 = 130 the µM, best the Table GCaselowest 4, inhibitorinhibitoryentries 1 encodingRegarding this the enzyme newly synthesized cause Gaucher ethers, disease, the “all-cis” the 9most showed common an inhibitory autosomal activity recessive towards LSD GCase [42,43]. wasvs.activities 8). compound Among were the 10observed ,compounds bearing for a dodecylaminothienylderived and from dimethylaminophenyl-co organomesubstituenttal at addition C-3 (IC reactions,50ntaining = 6.4 µM, thederivatives Tablelowest 4, inhibitory 14entry and 2). 15 oneIn order keeping of magnitude with the greater results than on its commercially epimer 21 (IC 50available = 12 µM vs.β-glucosidase, IC50 = 130 µM, the Table best 4, GCaseentries 1inhibitor Regardingactivities(Table 4, were entriesthe newly observed 6 and synthesized 7). for Moreover, thienyl ethers, and GCase the dimethylaminophenyl-co “all-cis” was poorly 9 showed inhibited an inhibitory ntainingalso by activitycompoundderivatives towards 13 14, bearing andGCase 15 a wasvs. compound8). Among the 10 compounds, bearing a deriveddodecylamino from organome substituenttal addition at C-3 reactions, (IC50 = the6.4 lowestµM, Tableinhibitory 4, entry 2). one(Table order 4, entries of magnitude 6 and 7). greater Moreover, than its GCase epimer was 21 poorly (IC50 = inhibited12 µM vs. also IC50 by= 130 compound µM, Table 13 4,, bearing entries 1a Regarding activities werethe newly observed synthesized for thienyl ethers, and dimethylaminophenyl-co the “all-cis” 9 showed ntainingan inhibitory derivatives activity 14 towardsand 15 GCase vs.(Table 8). Among 4, entries the 6 andcompounds 7). Moreover, derived GCase from was organome poorly inhibitedtal addition also by reactions, compound the 13 , lowestbearing inhibitory a one order of magnitude greater than its epimer 21 (IC50 = 12 µM vs. IC50 = 130 µM, Table 4, entries 1 activities were observed for thienyl and dimethylaminophenyl-containing derivatives 14 and 15 vs. 8). Among the compounds derived from organometal addition reactions, the lowest inhibitory (Table 4, entries 6 and 7). Moreover, GCase was poorly inhibited also by compound 13, bearing a activities were observed for thienyl and dimethylaminophenyl-containing derivatives 14 and 15 (Table 4, entries 6 and 7). Moreover, GCase was poorly inhibited also by compound 13, bearing a

Molecules 2020, 25, x FOR PEER REVIEW 9 of 24 Molecules 2020, 25, x FOR PEER REVIEW 9 of 24 short ethyl chain (Table 4, entry 5), and by trihydroxypiperidines 11 and 32, bearing the phenylethyl Molecules 2020, 25, x FOR PEER REVIEW 9 of 24 orshort the ethyl phenylethynyl chain (Table substituent, 4, entry 5), resp andectively by trihydroxypiperidines (Table 4, entries 3 and 11 and 9). 32, bearing the phenylethyl Molecules 2020, 25, x FOR PEER REVIEW 9 of 24 orshort the phenylethynyl ethyl chain (Table substituent, 4, entry 5),resp andectively by trihydroxypiperidines (Table 4, entries 3 and 11 9). and 32, bearing the phenylethyl MoleculesTable 2020 4., 25 β,-galactosidase x FOR PEER REVIEW (β-Gal) and β-glucosidase (GCase) inhibition in human leukocytes from9 of 24 shortor the ethyl phenylethynyl chain (Table substituent, 4, entry 5), andresp byectively trihydroxypiperidines (Table 4, entries 3 11 and and 9). 32, bearing the phenylethyl healthyTable 4. donors. β-galactosidase (β-Gal) and β-glucosidase (GCase) inhibition in human leukocytes from orMolecules the phenylethynyl 2020, 25, x FOR PEER substituent, REVIEW respectively (Table 4, entries 3 and 9). 9 of 24 shorthealthy ethyl chain donors. (Table 4, entry 5), and by trihydroxypiperidines 11 and 32, bearing the phenylethyl MoleculesMolecules2020, 25Table 2020, 4526, 254. , βx -galactosidaseFOR PEER REVIEW (β-Gal) and β-glucosidaseβ-Gal (GCase) inhibition inGCase human leukocytes from9 of 24 8 of 23 or the phenylethynylEntry substituent,Compound resp ectively (Table 4, entries 3 and 9). short ethylhealthy chain donors. (Table 4, entry 5), and by trihydroxypiperidinesa 11 and 32, abearing the phenylethylb MoleculesTable 2020 4., 25 β,-galactosidase x FOR PEER REVIEW (β-Gal) and β-glucosidaseInhibitionβ-Gal (GCase) (%) Inhibitioninhibition in (%)GCase human IC leukocytes50 (µM) from9 of 24 or the phenylethynylEntry substituent,Compound resp ectively (Table 4, entries 3 and 9). shorthealthy ethyl chain donors. (Table 4, entry 5), and by trihydroxypiperidinesa 11 and 32, abearing the phenylethylb MoleculesTable 2020 4., 25β,-galactosidase x FOR PEER REVIEW (β-Gal) and β-glucosidaseInhibitionβ-Gal (GCase) (%) Inhibitioninhibition in (%) humanGCase IC leukocytes 50 (µM) from9 of 24 Entry Compound shortor the ethyl phenylethynyl chain (Table substituent, 4, entry 5), resp andectively by trihydroxypiperidines (Table 4, entriesa 3 and 11 and 9). 32, bearinga the phenylethylb the alkylationhealthyTable of 4. thedonors. β-galactosidase hydroxy or (β-Gal) the exocyclicand β-glucosidaseInhibition amineβ-Gal (GCase) group(%) inhibitionInhibition and the inGCase introduction human(%) leukocytesIC50 (µM) of from a substituent at C-3 Entry1 Compound 22 98 12 ± 6 orshort the ethylphenylethynyl chain (Table substituent, 4, entry 5), resp andectively by trihydroxypiperidines (Table 4, entriesa 3 and 11 and9). 32, abearing the phenylethylb of the trihydroxypiperidinehealthyTable 4. donors. β-galactosidase skeleton (β-Gal) and dramatically β-glucosidaseInhibitionβ-Gal a(GCase) (%)ffect β Inhibitioninhibition-Gal inhibition. in (%)GCase human IC leukocytes50 (µM) from or the phenylethynylEntry1 substituent,Compound resp ectively (Table22 4, entries 3 and 98 9). 12 ± 6 healthy donors. Inhibition (%) a Inhibition (%) a IC50 (µM) b Table 4. β1-galactosidase (β-Gal) and β-glucosidaseβ-Gal22 (GCase) inhibition 98 in GCase human leukocytes 12 ± 6 from β Entry Compoundβ β Table 4.healthy-galactosidase donors. ( -Gal) and -glucosidaseInhibition (%) (GCase) a Inhibition inhibition (%) a inIC human50 (µM) b leukocytes from Table 4.1 β-galactosidase (β-Gal) and β-glucosidaseβ-Gal22 (GCase) inhibition 98 inGCase human leukocytes 12 ± 6 from healthy donors.Entry Compound healthy donors. Inhibition (%) a Inhibition (%) a IC50 (µM) b 1 β-Gal22 98 GCase 12 ± 6 Entry2 Compound 0 100 6.4 ± 0.7 Inhibitionβ-Gal (%) a Inhibition (%)GCase a IC 50 (µM) b Entry12 Compound β-Gal220 100 98 GCase 6.4 12 ±± 0.76 Entry Compound Inhibition (%) a Inhibition (%) a IC50 (µM) b 1 2 Inhibition220 (%) a Inhibition 98 100 (%) a 12 6.4 ± ± 6IC 0.7 (µM) b 50 12 220 100 98 6.4 12 ±± 0.76

21 220 100 98 6.4 12 ± ± 0.7 6 1 3 220 36 98n.d. 12 6 ± 23 0 10036 6.4n.d. ± 0.7

2 3 0 0 10036 6.4 ±n.d. 0.7

23 0 10036 6.4n.d. ± 0.7 2 0 100 6.4 0.7 432 0 100 9336 6.4 60n.d. ± 230.7 ±

34 0 36 93 60n.d. ± 23

3 4 0 0 36 93 n.d. 60 ± 23 3 0 36 n.d. 34 0 36 93 60n.d. ± 23

543 30 933036 60n.d. ± 23

45 03 9330 60n.d. ± 23 4 0 93 60 23 ± 4 5 0 3 9330 60 ±n.d. 23

45 03 9330 60n.d. ± 23

654 1630 30 939 60n.d. ± 23 5 3 30 n.d. 56 163 309 n.d.

5 6 316 309 n.d.n.d.

6 56 163 309 9n.d. n.d.

65 163 309 n.d.n.d. 7 6 6 n.d. 6 16 9 n.d. 7 6 6 n.d. 6 16 9 n.d. 7 7 66 6 6n.d. n.d. 6 16 9 n.d. 7 6 6 n.d. 6 16 9 n.d. 7 6 6 n.d. 8 0 100 130 ± 13 78 60 1006 130n.d. ± 13 8 0 100 130 13 Molecules 2020, 725 8, x FOR PEER REVIEW 6 0 6 100 130n.d. ± 13 ±10 of 24

Molecules 2020,78 25 , x FOR PEER REVIEW 60 1006 130n.d. ± 13 10 of 24

Molecules 2020,98 725 , x FOR PEER REVIEW 06 100256 130n.d. ± 13 10 of 24 9 0 25 n.d. 1089 40 1006525 130n.d. ± 13

108 9 04 0 1006525 130n.d. n.d.± 13

1089 04 1002565 130n.d. ± 13 10 4 65 n.d. 98 0 10025 130n.d. ± 13

119 150 9325 c 40n.d. ± 3 c

c c 11119 150 9325 93 c 40n.d. ± 3 40 3 c ± 119 150 9325 c 40n.d. ± 3 c

9 0 25 n.d.

1212 1414 d 100100 d d 29 ± 2 d 29 2 d ± 12 14 d 100 d 29 ± 2 d

12 14 d 100 d 29 ± 2 d

OH HO OH 13 OH 5 d 100 d 1.5 ± 0.1 d HO OH NOH C12H25 13 H 5 d 100 d 1.5 ± 0.1 d HO 38OH N C12H25 13 H 5 d 100 d 1.5 ± 0.1 d 38 N C12H25 H 38 14 31 d 80 d 94 ± 5 d 14 31 d 80 d 94 ± 5 d

14 31 d 80 d 94 ± 5 d a Percentage inhibition of β-galactosidase (β-Gal) and β-glucosidase (GCase) in human leukocytes extractsa Percentage incubated inhibition with of compounds β-galactosidase (1 mM). (β-Gal) b IC 50and values β-glucosidase were determined (GCase) byin humanmeasuring leukocytes GCase activity at different concentrations of each inhibibtor for compounds showing inhibitory activity higher extractsa Percentage incubated inhibition with of compounds β-galactosidase (1 mM). (β-Gal) IC 50and values β-glucosidase were determined (GCase) byin humanmeasuring leukocytes GCase than 70% at 1 mM (n.d. = not determined). c Ref. [22] d Ref. [24]. activityextracts atincubated different concentrationswith compounds of each (1 mM). inhibi btor IC 50for values compounds were determinedshowing inhibitory by measuring activity higherGCase c d thanactivity 70% at atdifferent 1 mM (n.d. concentrations = not determined). of each inhibi Ref.tor [22] for Ref.compounds [24]. showing inhibitory activity higher Conversely compound 33, bearing the triple bond and a longer aliphatic substituent, showed than 70% at 1 mM (n.d. = not determined). c Ref. [22] d Ref. [24]. 65% inhibition,Conversely which compound increased 33, bearingconsiderably the triple after bondhydrogenation and a long toer 12 aliphatic (Table 4, substituent, entry 10 vs. showed4). This latter65% inhibition,Conversely compound which compound showed increased a33 ,moderate bearingconsiderably the IC triple50 after = 60 bondhydrogenation µM, and which a long toeris 12 aliphaticclose (Table to 4, substituent, entrythat observed10 vs. showed4). Thisfor trihydroxypiperidineslatter65% inhibition, compound which showed increasedbearing a anmoderate considerably octyl chain IC 50at after C-2,= 60hydrogenation previously µM, which synthesized tois 12 close (Table into 4,our entrythat group observed10 (vs.37 4).and This 39for, Table 4, entries 12 and 14) [23,24]. trihydroxypiperidineslatter compound showed bearing a anmoderate octyl chain IC 50at C-2,= 60 previously µM, which synthesized is close into our that group observed (37 and for39, TabletrihydroxypiperidinesThese 4, entries data 12overall and bearing14) show [23,24]. that an GCaseoctyl chain inhibition at C-2, high previouslyer than 90%synthesized is guaranteed in our by group the presence (37 and 39 of, aTable longThese 4, alkyl entries data chain 12overall and(8, 9, 14) show or [23,24]. 12 that carbon GCase atoms, inhibition compounds higher 9than, 10 ,90% 12, andis guaranteed 21). This parallelsby the presence previous of studies,a longThese alkyl which data chain showedoverall (8, 9, show orthat 12 thatalkylated carbon GCase atoms, imino- inhibition compounds and high azasugarser 9than, 10 ,90%are 12, strongandis guaranteed 21 ).GCase This parallelsbyinhibitors the presence previous due toof favorablestudies,a long alkyl which interaction chain showed (8, of9, orthatthe 12 alkylalkylated carbon chain atoms, imino- with compounds thaned hydrophobic azasugars 9, 10 ,are 12,domain strongand 21 of ).GCase Thisthe enzyme parallelsinhibitors [24,44,45]. previous due to However,favorablestudies, which interactionin terms showed of ICof 50thatthe values, alkylalkylated a chainremarkable imino- with thdifferenceaned hydrophobic azasugars was foundare domain strong between ofGCase the compounds enzyme inhibitors [24,44,45]. 9 anddue 21to, inHowever,favorable agreement interactionin terms with ofprevious ICof 50the values, alkylobservations a chainremarkable with by Compainthdifferencee hydrophobic and was coworkersfound domain between onof thedifferently compounds enzyme configured [24,44,45]. 9 and 21, ethers derived from 1,5-dideoxy and 1-5-imino-D-xylitol (DIX) [46]. Moreover, good GCase inhibitors inHowever, agreement in terms with ofprevious IC50 values, observations a remarkable by Compaindifference and was coworkersfound between on differently compounds configured 9 and 21, canethersin agreement be derived identified withfrom among 1,5-dideoxyprevious piperidines observations and 1-5-imino- with byonly DCompain -xylitoltwo fr ee(DIX) and hydroxy [46].coworkers Moreov groups oner, (e.g., differentlygood 9 ,GCase 10, andconfigured inhibitors 21), as previously observed with different azasugars [46]. canethers be derived identified from among 1,5-dideoxy piperidines and 1-5-imino- with onlyD-xylitol two fr ee(DIX) hydroxy [46]. Moreov groupser, (e.g., good 9 ,GCase 10, and inhibitors 21), as previouslycan beWe identified then observed assayed among with the strongest pidifferentperidines GCaseazasugars with inhibitors only [46]. two 9 , fr10,ee andhydroxy 12 (IC 50groups lower (e.g.,than 1009, 10, µM) and in human21), as fibroblastspreviouslyWe then derivedobserved assayed from with the Gaucher strongestdifferent patients GCaseazasugars bearinginhibitors [46]. the 9 N370S, 10, and mutation. 12 (IC50 lowerNone thanof the 100 compounds µM) in human gave enzyme rescue when tested at six different concentrations (10, 100, 1, 10, 50, and 100 µM) (see the fibroblastsWe then derived assayed from the Gaucher strongest patients GCase bearinginhibitors the 9 N370S, 10, and mutation. 12 (IC50 lower None thanof the 100 compounds µM) in human gave Supplementaryenzymefibroblasts rescue derived whenMaterials). from tested Gaucher In at particular, six patients different compoundbearing concentr the ations 10N370S showed (10, mutation. 100, remarkable 1, None10, 50, oftoxicity and the compounds100 at µM) the (seehighest gave the concentrationsSupplementaryenzyme rescue (50 whenMaterials). and tested 100 µM). In at particular,six Notably, different thecompound concentr stronger ations 10inhibitory showed (10, 100, activityremarkable 1, 10, of 50,10 toxicitywithand 100respect at µM) the to (seehighest 36 (see the TableconcentrationsSupplementary 4) does not (50 Materials). correspondand 100 µM).In toparticular, Notably, a higher the compoundchaperoning stronger 10 inhibitory activityshowed (indeed, activityremarkable ofcompound 10 toxicitywith respect 36 at wasthe to highest able36 (see to rescueTableconcentrations 4) GCase does activitynot (50 correspondand of 100 1.5-fold µM). to Notably,at a 100higher µM) the chaperoning [22]. stronger inhibitory activity (indeed,activity ofcompound 10 with respect 36 was to able36 (see to rescueTableThe 4) GCase doessame activity notbehavior correspond of is1.5-fold evident to ata by100higher comparing µM) chaperoning [22]. the C-2 activity alkylated (indeed, trihydroxypiperidines compound 36 was 38 andable 37 to: Therescue bestThe GCase sameinhibitor activity behavior was of the is1.5-fold evidentdodecyl at by100 alkylated comparing µM) [22]. (compound the C-2 alkylated 38) but thetrihydroxypiperidines best chaperone was 38 the and octyl 37: The bestThe sameinhibitor behavior was the is evident dodecyl by alkylated comparing (compound the C-2 alkylated 38) but thetrihydroxypiperidines best chaperone was 38 the and octyl 37: The best inhibitor was the dodecyl alkylated (compound 38) but the best chaperone was the octyl

Molecules 2020, 25, x FOR PEER REVIEW 10 of 24

Molecules 2020, 25, x FOR PEER REVIEW 10 of 24

10 4 65 n.d.

10 4 65 n.d.

11 15 93 c 40 ± 3 c

11 15 93 c 40 ± 3 c Molecules 2020, 25, 4526 9 of 23

12 14 d 100 d 29 ± 2 d Table 4. Cont. 12 14 d 100 d 29 ± 2 d

β-Gal GCase

Entry CompoundOH a a b Inhibition (%) Inhibition (%) IC50 (µM) HO OH 13 OH 5 d 100 d 1.5 ± 0.1 d HO OH N C12H25 13 H 5 dd 100 d d 1.5 ± 0.1 d d 13 38 5 100 1.5 0.1 N C12H25 ± H 38

1414 3131 dd 80 80d d 94 ± 5 d 94 5 d ± 14 31 d 80 d 94 ± 5 d

a Percentagea Percentage inhibition inhibition of β-galactosidase of β-galactosidase (β-Gal) (β and-Gal)β -glucosidaseand β-glucosidase (GCase) (GCase) in human in human leukocytes leukocytes extracts incubated with compounds (1 mM). b IC values were determined by measuring GCase activity at different concentrations extracts incubated with 50compounds (1 mM). b IC50 values were determined by measuring GCase of eacha inhibitor Percentage for inhibition compounds of β-galactosidase showing inhibitory (β-Gal) activityand β-glucosidase higher than (GCase) 70% atin 1human mM (n.d. leukocytes= not determined). c activityd at different concentrations of each inhibibtor for compounds showing inhibitory activity higher Ref. [22extracts] Ref. incubated [24]. with compounds (1 mM). IC50 values were determined by measuring GCase than 70% at 1 mM (n.d. = not determined). c Ref. [22] d Ref. [24]. activity at different concentrations of each inhibitor for compounds showing inhibitory activity higher than 70% at 1 mM (n.d. = not determined). c Ref. [22] d Ref. [24]. However,Conversely the screening compound 33 on, bearing a panel the oftriple 12 bond commercial and a long glycosidaseser aliphatic substituent, (see the showed Supplementary Materials),65% inhibition,Conversely showed which thatcompound increased only 33 trihydroxypiperidine, bearingconsiderably the triple after bondhydrogenation and10 (bearing a long toer 12 aliphatic (Table a dodecyl 4, substituent, entry chain 10 vs. showed 4). connected This at C-3 latter65% inhibition, compound which showed increased a moderate considerably IC50 after = 60hydrogenation µM, which tois 12 close (Table to 4, entrythat observed10 vs. 4). Thisfor throughtrihydroxypiperidines a nitrogen atom) bearing was able an octyl to inhibit chain atβ C-2,-glucosidase previously synthesized from almonds. in our group In particular, (37 and 39, compound latter compound showed a moderate IC50 = 60 µM, which is close to that observed for µ 10 showedtrihydroxypiperidinesTable an 4, entries IC50 = 1285 and Mbearing14) towards[23,24]. an octyl this chain enzyme, at C-2, previously which prompted synthesized us in our to evaluate group (37 and compounds 39, 9–15, 21, 32, andTableThese33 4, alsoentries data on 12overall humanand 14) show [23,24]. lysosomal that GCase β inhibition-glucosidase higher (GCase)than 90% is (Table guaranteed4). Point by the mutations presence of in the gene encodinga long thisThese alkyl enzyme data chain overall cause(8, 9, show or Gaucher 12 that carbon GCase disease,atoms, inhibition compounds the high moster 9than common, 10 ,90% 12, andis guaranteed autosomal 21). This parallelsby recessive the presence previous LSD of [42,43]. studies, which showed that alkylated imino- and azasugars are strong GCase inhibitors due to Ina keeping long alkyl with chain the (8, results 9, or 12 oncarbon commercially atoms, compounds available 9, 10β, -glucosidase,12, and 21). This the parallels best GCase previous inhibitor was studies,favorable which interaction showed of thatthe alkylalkylated chain imino- with thaned hydrophobic azasugars are domain strong ofGCase the enzyme inhibitors [24,44,45]. due to compound 10, bearing a dodecylamino50 substituent at C-3 (IC = 6.4 µM, Table4, entry 2). Regarding favorableHowever, interactionin terms of ICof the values, alkyl a chainremarkable with thdifferencee hydrophobic was found domain50 between of the compounds enzyme [24,44,45]. 9 and 21, in agreement with previous observations by Compain and coworkers on differently configured the newlyHowever, synthesized in terms of ethers, IC50 values, the “all-cis” a remarkable9 showed difference an was inhibitory found between activity compounds towards 9 and GCase 21, one order ethers derived from 1,5-dideoxy and 1-5-imino-D-xylitol (DIX) [46]. Moreover, good GCase inhibitors of magnitudein agreement greater with than previous its epimerobservations21 (IC by50 Compain= 12 µ Mand vs. coworkers IC50 = on130 differentlyµM, Table configured4, entries 1 vs. 8). can be identified among piperidines with only two free hydroxy groups (e.g., 9, 10, and 21), as ethers derived from 1,5-dideoxy and 1-5-imino-D-xylitol (DIX) [46]. Moreover, good GCase inhibitors Amongpreviously the compounds observed derivedwith different from azasugars organometal [46]. addition reactions, the lowest inhibitory activities were observedcan be identified for thienyl among andpiperidines dimethylaminophenyl-containing with only two free hydroxy groups derivatives (e.g., 9, 10, and14 21and), as 15 (Table4, previouslyWe then observed assayed with the strongestdifferent GCaseazasugars inhibitors [46]. 9, 10, and 12 (IC50 lower than 100 µM) in human fibroblasts derived from Gaucher patients bearing the N370S mutation. None of the compounds gave entries 6 andWe 7 then). Moreover, assayed the GCasestrongest was GCase poorly inhibitors inhibited 9, 10, and also 12 (IC by50 compoundlower than 10013 µM), bearing in human a short ethyl chain (Tablefibroblastsenzyme4 ,rescue entry derived when 5), from andtested Gaucher by at trihydroxypiperidinessix patients different bearing concentr theations N370S (10, mutation.11 100,and 1, 32,None10, 50,bearing of and the compounds100 the µM) phenylethyl (see gave the or the Supplementary Materials). In particular, compound 10 showed remarkable at the highest phenylethynylenzyme rescue substituent, when tested respectively at six different (Table concentr4, entriesations 3(10, and 100, 9). 1, 10, 50, and 100 µM) (see the concentrations (50 and 100 µM). Notably, the stronger inhibitory activity of 10 with respect to 36 (see ConverselySupplementary compound Materials).33 In, particular, bearing thecompound triple bond10 showed and remarkable a longer aliphatictoxicity at the substituent, highest showed concentrationsTable 4) does not (50 correspondand 100 µM). to Notably, a higher the chaperoning stronger inhibitory activity (indeed,activity ofcompound 10 with respect 36 was to able36 (see to 65% inhibition,Tablerescue 4) GCase does which activitynot correspond increased of 1.5-fold to at considerablya 100higher µM) chaperoning [22]. after activity hydrogenation (indeed, compound to 12 (Table36 was 4able, entry to 10 vs. 4). The same behavior is evident by comparing the C-2 alkylated trihydroxypiperidines 38 and 37: This latterrescue compound GCase activity showed of 1.5-fold aat moderate100 µM) [22]. IC50 = 60 µM, which is close to that observed for The best inhibitor was the dodecyl alkylated (compound 38) but the best chaperone was the octyl trihydroxypiperidinesThe same behavior bearing is evident an octylby comparing chain atthe C-2, C-2 alkylated previously trihydroxypiperidines synthesized in 38 our and group37: (37 and

39, TableThe4, best entries inhibitor 12 andwas the 14) dodecyl [23,24 ].alkylated (compound 38) but the best chaperone was the octyl

These data overall show that GCase inhibition higher than 90% is guaranteed by the presence of a long alkyl chain (8, 9, or 12 carbon atoms, compounds 9, 10, 12, and 21). This parallels previous studies, which showed that alkylated imino- and azasugars are strong GCase inhibitors due to favorable interaction of the alkyl chain with the hydrophobic domain of the enzyme [24,44,45]. However, in terms of IC50 values, a remarkable difference was found between compounds 9 and 21, in agreement with previous observations by Compain and coworkers on differently configured ethers derived from 1,5-dideoxy and 1-5-imino-d-xylitol (DIX) [46]. Moreover, good GCase inhibitors can be identified among piperidines with only two free hydroxy groups (e.g., 9, 10, and 21), as previously observed with different azasugars [46]. We then assayed the strongest GCase inhibitors 9, 10, and 12 (IC50 lower than 100 µM) in human fibroblasts derived from Gaucher patients bearing the N370S mutation. None of the compounds gave enzyme rescue when tested at six different concentrations (10, 100, 1, 10, 50, and 100 µM) (see the Supplementary Materials). In particular, compound 10 showed remarkable toxicity at the highest concentrations (50 and 100 µM). Notably, the stronger inhibitory activity of 10 with respect to 36 (see Table4) does not correspond to a higher chaperoning activity (indeed, compound 36 was able to rescue GCase activity of 1.5-fold at 100 µM) [22]. Molecules 2020, 25, 4526 10 of 23

The same behavior is evident by comparing the C-2 alkylated trihydroxypiperidines 38 and 37: The best inhibitor was the dodecyl alkylated (compound 38) but the best chaperone was the octyl derivative 37 [24]. The inefficacy of dodecyl alkylated compounds 10 and 38 as PCs can be ascribed to the cytotoxicity imparted by the 12-carbon atom alkyl chains. These data are also consistent with other reports on amphiphilic N-alkylated iminosugars, which suggest that cytotoxicity is strongly chain-length dependent and that potent inhibitors with chains longer than C8 can be toxic when assayed in cell lines [47,48].

3. Materials and Methods

3.1. General Experimental Procedures for the Syntheses Commercial reagents were used as received. All reactions were carried out under magnetic stirring and monitored by TLC on 0.25 mm silica gel plates (Merck F254). Column chromatographies were carried out on silica gel 60 (32–63 µm) or on silica gel (230–400 mesh, Merck, Kenilworth, NJ, USA). Yields refer to spectroscopically and analytically pure compounds unless otherwise stated. 1H-NMR spectra were recorded on a Varian Gemini 200 MHz, a Varian Mercury 400 MHz, or on a Varian 13 INOVA 400 MHz instrument at 25 ◦C (Agilent Technologies, Santa Clara, CA, USA). C-NMR spectra were recorded on a Varian Gemini 200 MHz or on a Varian Mercury 400 MHz instrument (Agilent 13 Technologies, Santa Clara, CA, USA). Chemical shifts are reported relative to CDCl3 ( C: δ = 77.0 ppm) 13 or to CD3OD ( C: δ = 49.0 ppm). Integrals are in accordance with assignments, coupling constants are given in Hz. For detailed peak assignments 2D spectra were measured (COSY, HSQC, NOESY, and NOE as necessary). IR spectra were recorded with a IRAffinity-1S SHIMADZU system spectrophotometer (Shimadzu Italia S.r.l., Milan, Italy) ESI-MS spectra were recorded with a Thermo Scientific™ LCQ fleet ion trap mass spectrometer (Thermo Fisher Scientific,Waltham, MA, USA). Elemental analyses were performed with a Thermo Finnigan FLASH EA 1112 CHN/S analyzer (Perkin-Elmer, Waltham, MA, USA). Optical rotation measurements were performed on a JASCO DIP-370 polarimeter (JASCO, Easton, MD, USA).

3.1.1. Synthesis of (3R, 4S, 5S)-3, 4-O-(1-Methylethylidene)-5-Nonyloxy-N-Boc-Piperidine (19) NaH (7 mg, 0.3 mmol, 60% on mineral oil) was added to a solution of 18 [25] (22 mg, 0.08 mmol) in dry DMF (1.2 mL) at 0 ◦C. The mixture was stirred at room temperature for 30 min, then 1-bromononane (54 µL, 0.28 mmol) was added, and the reaction mixture was stirred at room temperature for 72 h, until the disappearance of the starting material was observed via TLC (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1). Then, water was slowly added, and the reaction mixture was extracted with AcOEt (3 3 mL). × The combined organic layer was washed with saturated NaHCO3 and brine and concentrated after drying with Na2SO4. The crude residue was purified by flash column chromatography on silica gel (hexane/AcOEt 8:1) to give 17 mg of 19 (Rf = 0.3, hexane/AcOEt 8:1, 0.04 mmol, 53%) as a colorless oil. [α ]21 1 19: D = +17.7 (c = 0.75, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm = 4.51–4.44 (m, 1H, H-3), 4.28 (br s, 1H, H-4), 3.85–3.07 (m, 7H, H-2, H-6, H-5, H-10), 1.66–1.56 (m, 2H, H-20), 1.49 (s, 3H, Me), 1.45 (s, 9H, t-Bu), 1.36 (s, 3H, Me), 1.34–1.20 (m, 12H, H-30, H’40, H-50, H-60, H-70, H-80), 0.90–0.85 13 (m, 3H, H-90). C-NMR (50 MHz, CDCl3) δ ppm = 155.3 (s, 1C, NCOO), 109.8 (s, 1C, OC(CH3)2), 80.0 (s, 1C, OC(CH3)3), 73.4 (d, 1C, C-5), 72.7 (d, 2C, C-3, C-4), 70.4 (t, 1C, C-10), 44.0, 42.8, 41.8, 41.5 (t, 2C, C-2, C-6), 32.0, 29.9, 29.7, 29.6, 29.4, 27.2, 26.1, 25.3, 22.8 (t, 7C, C-20, C-30, C-40, C-50, C-60, C-70, C-80 and q, 2C, OC(CH3)2), 28.6 (q, 3C, OC(CH3)3), 14.2 (q, 1C, C-90). IR (CDCl3): ν = 2959, 2928, 2857, 2249, 1 1686, 1375, 1261, 1165, 1101, 1172, 1008 cm− .C22H41NO5 (399.56): calcd. C, 66.13; H, 10.34; N, 3.51; found C, 66.23; H, 10.32; N, 3.45. MS-ESI (m/z, %) = 422.16 (100) [M + Na]+, 820.89 (17) [2M + Na]+. Molecules 2020, 25, 4526 11 of 23

3.1.2. Synthesis of (3S, 4R, 5R)-4, 5-Dihydroxy-3-(Nonyloxy) Piperidine (9) A solution of 19 (15 mg, 0.04 mmol) in MeOH (3 mL) was left stirring with 12 M HCl (60 µL) at room temperature for 18 h. The crude mixture was concentrated to yield 9 as the hydrochloride salt. The corresponding free amine was obtained by dissolving the residue in MeOH (4 mL), then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration and the crude product was purified on silica gel by flash column chromatography (DCM/MeOH/NH4OH (6%) 10:1:0.1) to afford 7 mg of 9 (Rf = 0.2, DCM/MeOH/NH4OH (6%) 10:1:0.1, 0.03 mmol, 70%) as a pale-yellow oil. 9: [α ]22 = 4.6 (c = 0.54, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 4.01 (br s, 1H, H-4), D − 3 3.60–3.45 (m, 3H, H-5, H-10), 3.38–3.32 (m, 1H, H-3), 2.84–2.66 (m, 4H, H-2, H-6), 1.63–1.54 (m, 2H, 13 H-20), 1.40–1.25 (m, 12H, H-30, H’40, H-50, H-60, H-70, H-80), 0.90 (t, J = 6.9 Hz, 3H, H-90). C-NMR (100 MHz, CD3OD) δ ppm: 78.5 (d, 1C, C-3), 70.5 (d, 1C, C-5), 70.4 (t, 1C, C-10), 70.3 (d, 1C, C-4), 47.5, 44.7 (t, 2C, C-2, C-6), 33.1, 31.0, 30.8, 30.7, 30.6, 30.4, 27.2, 23.7 (t, 7C, C-20, C-30, C-40, C-50, C-60, C-70, C-80), 14.4 (q, 1C, C-90). C14H29NO3 (259.38): calcd. C, 64.83; H, 11.27; N, 5.40; found C, 64.85; H, 11.13; N, 5.60. MS-ESI (m/z, %) = 260.12 (100) [M + H]+, 282.29 (58) [M + Na]+, 540.91 (25) [2M + Na]+.

3.1.3. Synthesis of (3R, 4S, 5R)-3, 4-O-(1-Methylethylidene)-5-Nonyloxy-N-Boc-Piperidine (20) NaH (11 mg, 0.46 mmol, 60% on mineral oil) was added to a solution of 17 [25] (57 mg, 0.21 mmol) in dry DMF (3 mL) at 0 ◦C. The mixture was stirred at room temperature for 30 min, then 1-bromononane (140 µL, 0.73 mmol) was added, and the reaction mixture was stirred at room temperature for 40 h, until the disappearance of the starting material was observed via TLC (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1). Then, water was slowly added and the reaction mixture was extracted with AcOEt (3 5 mL). × The combined organic layer was washed with saturated NaHCO3 and brine and concentrated after drying with Na2SO4. The crude residue was purified by flash column chromatography on silica gel (hexane/AcOEt 10:1) to give 45 mg of 20 (Rf = 0.4, hexane/AcOEt 7:1, 0.11 mmol, 54%) as a colorless oil. [α ]27 1 20: D = +1.8 (c = 0.96, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 4.31–4.26 (m, 1H, H-3), 4.13 (br s, 1H, H-4), 3.92–3.82 (m, 1H, Ha-2), 3.58–3.28 (m, 6H, Hb-2, H-5, H-6, H-10), 1.59–1.48 (m, 2H, H-20), 1.45 (s, 12H, Me, t-Bu), 1.33 (s, 3H, Me), 1.32–1.17 (m, 12H, H-30, H’40, H-50, H-60, H-70, H-80), 13 0.87 (t, J = 6.2 Hz, 3H, H-90). C-NMR (50 MHz, CDCl3) δ ppm: 155.8 (s, 1C, NCOO), 109.1 (s, 1C, OC(CH3)2), 79.8 (s, 1C, OC(CH3)3), 74.8 (d, 1C, C-5), 74.5 (d, 1C, C-4), 72.6 (d, 1C, C-3), 69.6 (t, 1C, C-10), 43.0, 42.0 (t, 1C, C-2), 42.0–40.8 (t, 1C, C-6), 32.0, 30.0, 29.7, 29.6, 29.4, 27.3, 22.8 (t, 7C, C-20, C-30, C-40, C-50, C-60, C-70, C-80), 28.6 (q, 3C, OC(CH3)3), 26.3, 25.0 (q, 2C, OC(CH3)2), 14.2 (q, 1C, C-90). 1 IR (CDCl3): ν = 2930, 2859, 1686, 1416, 1375, 1260, 1165, 1101, 1063, 1016 cm− .C22H41NO5 (399.56): calcd. C, 66.13; H, 10.34; N, 3.51; found C, 66.15; H, 10.40; N, 3.60. MS-ESI (m/z, %) = 422.17 (100) [M + Na]+, 821.05 (96) [2M + Na]+.

3.1.4. Synthesis of (3R, 4R, 5R)-4, 5-Dihydroxy-3-(Nonyloxy) Piperidine (21) A solution of 20 (54 mg, 0.14 mmol) in MeOH (6 mL) was left stirring with 12 M HCl (150 µL) at room temperature for 18 h. The crude mixture was concentrated to yield 21 as hydrochloride salt. The corresponding free amine was obtained by dissolving the residue in MeOH (5 mL), then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration to give 36 mg of 21 (0.14 mmol, 100% yield) as a pale-yellow oil. 21: [α ]25 = 39.6 (c = 0.48, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 3.84–3.77 (m, 1H, H-5), D − 3 3.67–3.60 (m, 1H, H-4), 3.60–3.52 (m, 2H, H-10) 3.46–3.39 (m, 1H, H-3), 3.02 (d, J = 13.4 Hz, 1H, Ha-2), 2.82 (dd, J = 6.1, 13.2 Hz, 1H, Ha-6), 2.65 (d, J = 13.3 Hz, 1H, Hb-6), 2.48–2.38 (m, 1H, Hb-2), 1.62–1.51 (m, 2H, H-20), 1.41–1.20 (m, 12H, H-30, H’40, H-50, H-60, H-70, H-80), 0.90 (t, J = 5.9 Hz, 3H, H-90). 13 C-NMR (50 MHz, CD3OD) δ ppm: 78.6 (d, 1C, C-3), 73.2 (d, 1C, C-4), 71.2 (t, 1C, C-10), 69.5 (d, 1C, C-5), 49.3 (t, 1C, C-6), 47.1 (t, 1C, C-2), 33.0, 31.2, 30.7, 30.6, 30.4, 27.2, 23.7 (t, 7C, C-20, C-30, C-40, C-50, Molecules 2020, 25, 4526 12 of 23

C-60, C-70, C-80), 14.4 (q, 1C, C-90). C14H29NO3 (259.38): calcd. C, 64.83; H, 11.27; N, 5.40; found C, 64.50; H, 11.38; N, 5.30. MS-ESI (m/z, %) = 260.18 (100) [M + H]+.

3.1.5. Synthesis of (3R, 4S, 5S)-5-Dodecylamino-3, 4-O-(1-Methylethylidene)-N-Boc-Piperidine (22) Ketone 8 [25] (63 mg, 0.23 mmol) and dodecylamine (65 mg, 0.35 mmol) were dissolved in MeOH (3 mL), and molecular sieves (3 Å pellets; 25 mg) were added. The reaction mixture was stirred at room temperature for 1 h and then Pd(OH)2/C (30 mg) was added. The mixture was further stirred at room temperature under hydrogen atmosphere for 51 h. The catalyst and the molecular sieves were removed by filtration, the obtained compound was washed several times with MeOH, and the solvent was evaporated under vacuum. The crude residue was purified by flash column chromatography on silica gel (gradient eluent from hexane/AcOEt 5:1 to 2:1) to afford 56 mg of 22 (Rf = 0.4, hexane/AcOEt 2:1, 0.13 mmol, 55%) as a colorless oil. [α ]27 1 22: D = +1.9 (c = 0.90, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 4.41 (br s, 1H, H-4), 4.29 (br s, 1H, H-3), 3.85–3.40 (m, 2H), 3.40–3.22 (m, 1H), 2.99–2.84 (m, 1H), 2.83–2.75 (m, 1H, H-5), 2.74–2.54 (m, 2H, H-10), 1.44 (s, 12H, Me, t-Bu), 1.33 (s, 3H, Me), 1.31–1.17 (m, 20H, H-20, H-30, H-50, H-60, H-70, 13 H-80, H-90, H-100, H-110), 0.86 (t, J = 6.6 Hz, 3H, H-120). C-NMR (100 MHz, CDCl3) δ ppm: 155.4 (s, 1C, NCOO), 108.9 (s, 1C, OC(CH3)2), 79.8 (s, 1C, OC(CH3)3), 72.5 (d, 2C, C-3, C-4), 53.7 (d, 1C, C-5), 47.2 (t, 1C, C-10), 43.6, 43.3, 42.4, 42.3 (t, 2C, C-2, C-6), 32.0, 30.6, 29.8, 29.7, 29.6, 29.5, 27.4, 27.1, 25.1, 22.6 (t, 10C, C-20, C-30, C-40, C-50, C-60, C-70, C-80, C-90, C-100, C-110 and q, 2C, OC(CH3)2, 28.6 (q, 3C, OC(CH3)3), 14.2 (q, 1C, C-120). IR (CDCl3): ν = 3300, 2958, 2927, 2854, 1686, 1414, 1373, 1260, 1165, 1 1098, 1007 cm− .C25H48N2O4 (440.66): calcd. C, 68.14; H, 10.98; N, 6.36; found C, 68.30; H, 10.78; N, 6.33. MS-ESI (m/z, %) = 441.28 (100) [M + H]+, 463.23 (91) [M + Na]+, 903.11 (51) [2M + Na]+.

3.1.6. Synthesis of (3R, 4S, 5S)-3, 4-Dihydroxy-5-(Dodecylamino) Piperidine (10) A solution of 22 (45 mg, 0.10 mmol) in MeOH (6 mL) was left stirring with 12 M HCl (150 µL) at room temperature for 18 h. The crude mixture was concentrated to yield 10 as hydrochloride salt. The corresponding free amine was obtained by dissolving the residue in MeOH (5 mL), then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration to give 25 mg of 10 (0.08 mmol, 83%) as a white solid. 10: M.p. = 92–94 C. [α ]24 = 5.2 (c = 0.85, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 4.00 ◦ D − 3 (br s, 1H, H-4), 3.58–3.51 (m, 1H, H-3), 2.80–2.51 (m, 7H, H-2, H-6, H-5, H-10), 1.55–1.47 (m, 2H, H-20), 1.37–1.26 (m, 18H, H-30, H-40, H-50, H-60, H-70, H-80, H-90, H-100, H-110), 0.90 (t, J = 6.0 Hz, 3H, H-120). 13 C-NMR (50 MHz, CD3OD) δ ppm: 70.9 (d, 1C, C-3), 69.8 (d, 1C, C-4), 58.7 (d, 1C, C-5), 47.4, 45.4 (t, 3C, C-10, C-2, C-6), 33.1, 30.9, 30.7, 30.5, 28.5, 23.7 (t, 10C, C-20, C-30, C-40, C-50, C-60, C-70, C-80, C-90, C-100, C-110) 14.4 (q, 1C, C-120). C17H36N2O2 (300.48): calcd. C, 67.95; H, 12.08; N, 9.32; found C, 67.96; H, 12.03; N, 9.53. MS-ESI (m/z, %) = 301.28 (100) [M + H]+.

3.1.7. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-Vinyl-N-Boc-Piperidine (26) Vinyl magnesium bromide (288 µL, 0.29 mmol) was added to a dry THF solution (1 mL) of ketone 8 (52 mg, 0.19 mmol), dropwise at 0 ◦C under nitrogen atmosphere. The solution was stirred at 0 ◦C for 5 h when the disappearance of 8 was attested by a TLC control (hexane/AcOEt 2:1). A saturated aqueous NH4Cl solution was added at 0 ◦C, and the mixture was stirred for 10 min. The reaction mixture was extracted with AcOEt (3 3 mL). The combined organic layer was washed with water, × saturated NaHCO3, and brine and concentrated after drying with Na2SO4. The crude residue was purified by flash column chromatography on silica gel (gradient eluent from hexane/AcOEt 5:1 to 2:1) to give 29 mg of 26 (Rf = 0.2, hexane/AcOEt 5:1, 0.10 mmol, 51%) as a pale-yellow oil. 26: [α ]25 = 22.9 (c = 1.00, CHCl ). 1H-NMR (400 MHz, CDCl ) δ ppm: 5.85 (dd, J = 10.8, 17.2 D − 3 3 Hz, 1H, H-10), 5.49 (dd, J = 0.8, 17.2 Hz, 1H, H-20trans), 5.28 (d, J = 10.8 Hz, 1H, H-20cis), 4.33 (br s, 1H, H-5), 4.07 (d, J = 6.8 Hz, 1H, H-4), 3.95–3.69 (m, 1H, Ha-6), 3.53–3.32 (m, 1H, Hb-6), 3.46 (d, J = 13.0 Hz, 1H, Ha-2), 3.25 (d, J = 13.2 Hz, 1H, Hb-2), 2.67 (br s, 1H, OH), 1.52 (s, 3H, Me), 1.44 (s, 9H, t-Bu), 1.37 Molecules 2020, 25, 4526 13 of 23

13 (s, 3H, Me). C-NMR (100 MHz, CDCl3) δ ppm: 155.1 (s, 1C, NCOO), 139.3 (d, 1C, C-10), 116.8 (t, 1C, C-20), 109.5 (s, 1C, OC(CH3)2), 80.2 (s, 1C, OC(CH3)3), 77.2 (d, 1C, C-4), 71.9 (d, 1C, C-5), 71.3 (s, 1C, C-3), 48.4, 47.5 (t, 1C, C-2), 43.4, 42.5 (t, 1C, C-6), 28.5 (q, 3C, OC(CH3)3), 27.0 (q, 1C, OC(CH3)2), 25.0 (q, 1C, OC(CH3)2). IR (CDCl3) ν = 3555, 2984, 2932, 2251, 1688, 1410, 1385, 1373, 1252, 1213, 1163, 1070 1 cm− .C15H25NO5 (299.36): calcd. C, 60.18; H, 8.42; N, 4.68; found C, 60.37; H, 8.60; N, 4.67. MS-ESI (m/z, %) = 322.03 (100) [M + Na]+, 620.85 (40) [2M + Na]+, 338.03 (12) [M + K]+.

3.1.8. Synthesis of (3S, 4R, 5R)-3-Ethyl-3, 4, 5-Trihydroxypiperidine (13) Compound 26 (26 mg, 0.09 mmol) was dissolved in EtOH (5 mL) and HCl 12 M (150 µL) and Pd/C (13 mg) were added. The reaction mixture was stirred at room temperature under hydrogen atmosphere for 24 h. The catalyst was removed by filtration through Celite, and the filtrate was concentrated under vacuum to give the hydrochloride salt of 13. The corresponding free amine was obtained by dissolving the residue in MeOH, then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration and the crude product was purified on silica gel by flash column chromatography (gradient eluent from DCM/MeOH/NH4OH (6%) 10:1:0.1 to 1:1:0.1) to give 8 mg of 13 (Rf = 0.2, DCM/MeOH/NH4OH (6%) 10:1:0.1, 0.05 mmol, 52%) as a colorless oil. 13: [α ]24 = 5.7 (c = 0.75, CD OD). 1H-NMR (400 MHz, CD OD) δ ppm: 3.81 (br s, 1H, H-5), 3.47 D − 3 3 (d, J = 3.2 Hz, 1H, H-4), 2.96 (dd, J = 3.4, 13.6 Hz, 1H, Ha-6), 2.79 (br d, J = 13.6 Hz, 1H, Ha-2), 2.72 (dd, J = 2.4, 13.7 Hz, 1H, Hb-6), 2.55 (d, J = 13.6 Hz, 1H, Hb-2), 1.68–1.55 (m, 2H, H-10), 0.90 (t, J = 7.6 Hz, 13 3H, H-20). C-NMR (50 MHz, CD3OD) δ ppm: 75.1 (s, 1C, C-3), 72.1 (d, 1C, C-4), 70.7 (d, 1C, C-5), 53.0 (t, 1C, C-2), 50.3 (t, 1C, C-6), 29.6 (t, 1C, C-10), 7.6 (q, 1C, C-20). C7H15NO3 (161.20): calcd. C, 52.16; H, 9.38; N, 8.69; found C, 52.06; H, 9.46; N, 8.71. MS-ESI (m/z, %) = 184.15 (100) [M + Na]+.

3.1.9. General Procedure for the Addition of Lithium Acetylides to Ketone 8 To a dry THF solution (0.24 M) of alkyne (2 eq.), n-BuLi (1.5 eq.) was added dropwise over 5 min at –78 ◦C under nitrogen atmosphere. The solution was allowed to warm to 0 ◦C over 1 h and held at 0 ◦C for an additional 30 min. The solution was then recooled to –78 ◦C and ketone 8 (1 eq.) was added in one portion. The solution was allowed to warm to room temperature and stirred at room temperature until the disappearance of ketone 8 was attested by a TLC control (EtP/AcOEt 2:1). A saturated aqueous solution of NH4Cl was added and the reaction mixture was extracted with AcOEt. The combined organic layer was washed with water, saturated NaHCO3, and brine and concentrated after drying with Na2SO4. The crude compound was purified by flash column chromatography.

3.1.10. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-(Phenylethynyl)-N-Boc-Piperidine (27) Application of the general procedure to ketone 8 (60 mg, 0.24 mmol) with phenylacetylene (54 µL, 0.49 mmol) and n-BuLi (150 µL, 0.37 mmol, 2.5 M) gave, after purification by flash column chromatography on silica gel (hexane/AcOEt 4:1), 69 mg of 27 (Rf = 0.2, 0.18 mmol, 77%) as a pale-yellow oil. [α ]27 1 27: D = +9.4 (c = 1.05, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 7.40–7.34 (m, 2H, Ar), 7.30–7.21 (m, 3H, Ar), 4.52–4.40 (m, 1H, H-5), 4.36 (d, J = 6.8 Hz, 1H, H-4), 3.92–3.70 (m, 2H, Ha-2, Ha-6), 3.67–3.55 (m, 1H, Hb-6), 3.37–3.28 (m, 1H, Hb-2), 3.09–2.97 (m, 1H, OH), 1.50 (s, 3H, Me), 1.40 13 (s, 9H, t-Bu), 1.36 (s, 3H, Me). C-NMR (50 MHz, CDCl3) δ ppm: 155.2 (s, 1C, NCOO), 132.0, 129.0, 128.4 (d, 5C, Ar), 121.9 (s, 1C, Ar), 109.9 (s, 1C, OC(CH3)2), 88.1, 86.4 (s, 2C, C-10, C-20), 80.2 (s, 1C, OC(CH3)3), 77.1 (d, 1C, C-4), 72.5 (d, 1C, C-5), 67.4 (s, 1C, C-3), 48.3, 47.5 (t, 1C, C-6), 42.7, 41.9 (t, 1C, C-2), 28.5 (q, 3C, OC(CH3)3), 27.0 (q, 1C, OC(CH3)2), 25.0 (q, 1C, OC(CH3)2). IR (CDCl3) ν = 3541, 2982, 1 2934, 2251, 1688, 1600, 1260, 1215, 1163 cm− .C21H27NO5 (373.44): calcd. C, 67.54; H, 7.29; N, 3.75; found C, 67.40; H, 7.33; N, 3.76. MS-ESI (m/z, %) = 769.01 (100) [2M + Na]+, 396.11 (21) [M + Na]+. Molecules 2020, 25, 4526 14 of 23

3.1.11. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-(oct-1-yn-1-yl)-N-Boc-Piperidine (28) Application of the general procedure to ketone 8 (120 mg, 0.45 mmol) with 1-octyne (131 µL, 0.89 mmol) and n-BuLi (267 µL, 0.67 mmol, 2.5 M) gave, after purification by flash column chromatography on silica gel (hexane/AcOEt 4:1), 134 mg of 28 (Rf = 0.2, 0.35 mmol, 78%) as a colorless oil. [α ]27 1 28: D = +5.5 (c = 1.03, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 4.47–4.34 (m, 1H, H-5), 4.24 (d, J = 6.9 Hz, 1H, H-4), 3.74–3.54 (m, 3H, Ha-2, H-6), 3.22 (d, J = 12.3 Hz, 1H, Hb-2), 2.87 (br s, 1H, OH), 2.19–2.12 (m, 2H, H-30), 1.48 (s, 3H, Me), 1.43 (s, 9H, t-Bu), 1.35 (s, 3H, Me), 1.34–1.15 (m, 8H, H-40, 13 H-50, H-60, H-70), 0.91–0.79 (m, 3H, H-80). C-NMR (100 MHz, CDCl3) δ ppm: 155.2 (s, 1C, NCOO), 109.6 (s, 1C, OC(CH3)2), 87.5, 79.4 (s, 2C, C-10, C-20), 80.0 (s, 1C, OC(CH3)3), 77.2 (d, 1C, C-4), 72.4 (d, 1C, C-5), 66.8 (s, 1C, C-3), 48.3, 47.5 (t, 1C, C-2), 42.5, 41.6 (t, 1C, C-6), 31.3, 28.6, 28.4, 22.6 (t, 4C, C-40, C-50, C-60-C-70) e 26.9 (q, 1C, OC(CH3)2), 24.9 (q, 1C, OC(CH3)2), 28.5 (q, 3C, OC(CH3)3), 18.8 (t, 1C, C-30), 14.1 (q, 1C, C-80). IR (CDCl3) ν = 3543, 2961, 2932, 2862, 2249, 1730, 1458, 1412, 1375, 1215, 1167, 1 1142, 1045 cm− .C21H35NO5 (381.51): calcd. C, 66.11; H, 9.25; N, 3.67; found C, 66.34; H, 9.38; N, 3.73. MS-ESI (m/z, %) = 785.02 (100) [2M + Na]+, 404.12 (32) [M + Na]+.

3.1.12. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-(3, 3-Diethoxyprop-1-yn-1-yl)-N-Boc-Piperidine (29) Application of the general procedure to ketone 8 (60 mg, 0.22 mmol) with 3, 3-diethoxyprop-1-yne (64 µL, 0.44 mmol) and n-BuLi (208 µL, 0.33 mmol, 1.6 M) gave, after purification by flash column chromatography on silica gel (EtP/AcOEt 2:1), 57 mg of 29 (Rf = 0.3, 0.14 mmol, 65%) as a pale-yellow oil. [α ]25 1 29: D = +19.8 (c = 1.00, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 5.25 (s, 1H, H-30), 4.47–4.36 (m, 1H, H-5), 4.30 (d, J = 6.8 Hz, 1H, H-4), 3.78–3.49 (m, 7H, Ha-2, H-6, H-40, H-60), 3.32 (d, J = 12.6 Hz, 1H, Hb-2), 3.05–2.96 (m, 1H, OH), 1.50 (s, 3H, Me), 1.44 (s, 9H, t-Bu), 1.36 (s, 3H, Me), 13 1.19 (t, J = 7.1 Hz, 6H, H-50, H-70). C-NMR (50 MHz, CDCl3) δ ppm: 155.4 (s, 1C, NCOO), 110.2 (s, 1C, OC(CH3)2, 91.6 (d, 1C, C-30), 84.9, 82.1 (s, 2C, C-10, C-20), 80.6 (s, 1C, OC(CH3)3), 78.1 (d, 1C, C-4), 72.7 (d, 1C, C-5), 67.1 (s, 1C, C-3), 61.5, 61.4 (t, 2C, C-40, C-60) 48.4, 47.8 (t, 1C, C-2), 42.9, 42.2 (t, 1C, C-6), 28.8 (q, 1C, OC(CH3)2), 27.2 (q, 1C, OC(CH3)2), 25.3 (q, 3C, OC(CH3)3), 15.5 (q, 2C, C-50, C-70). 1 IR (CDCl3) ν = 3750, 3649, 3545, 2982, 2933, 2252, 1689, 1456, 1371, 1215, 1163, 1118, 1080, 1051 cm− . C20H33NO7 (399.48): calcd. C, 60.13; H, 8.33; N, 3.51; found C, 60.35; H, 8.20; N, 3.61. MS-ESI (m/z, %) = 422.14 (100) [M + Na]+, 821.08 (68) [2M + Na]+.

3.1.13. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-(3-Thienylethynyl)-N-Boc-Piperidine (30) Application of the general procedure to ketone 8 (30 mg, 0.11 mmol) with 3-ethynylthiophene (22 µL, 0.22 mmol) and n-BuLi (67 µL, 0.17 mmol, 2.5 M) gave, after purification by flash column chromatography on silica gel (hexane/AcOEt 4:1), 37 mg of 30 (Rf = 0.2, 0.10 mmol, 88%) as a pale-yellow oil. [α ]22 1 30: D = +44.3 (c = 0.60, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 7.48–7.41 (m, 1H, Ar), 7.26–7.22 (m, 1H, Ar), 7.10–7.04 (m, 1H, Ar), 4.55–4.42 (m, 1H, H-5), 4.39 (d, J = 6.8 Hz, 1H, H-4), 3.90–3.60 (m, 3H, Ha-2, H-6), 3.38 (d, J = 12.6 Hz, 1H, Hb-2), 2.96 (s, 1H, OH), 1.54 (s, 3H, Me), 1.45 13 (s, 9H, t-Bu), 1.40 (s, 3H, Me). C-NMR (100 MHz, CDCl3) δ ppm: 155.2 (s, 1C, NCOO), 130.0, 129.9, 125.6 (d, 3C, Ar), 120.9 (s, 1C, Ar), 109.9 (s, 1C, OC(CH3)2), 87.7, 81.6 (s, 2C, C-10, C-20), 80.3 (s, 1C, OC(CH3)3), 77.0 (d, 1C, C-4), 72.3 (d, 1C, C-5), 67.4 (s, 1C, C-3), 48.3, 47.5 (t, 1C, C-2), 42.7, 41.8 (t, 1C, C-6), 28.5 (q, 3C, OC(CH3)3), 27.0 (q, 1C, OC(CH3)2), 25.0 (q, 1C, OC(CH3)2). IR (CDCl3) ν = 2960, 3702, 1 3660, 2980, 2252, 1687, 1460, 1406, 1381, 1215, 1161, 1091, 941 cm− .C19H25NO5S (379.47): calcd. C, 60.14; H, 6.64; N, 3.69; S, 8.45; found C, 60.40; H, 6.58; N, 3.81; S, 8.56. MS-ESI (m/z, %) = 780.96 (100) [2M + Na]+, 402.13 (65) [M + Na]+. Molecules 2020, 25, 4526 15 of 23

3.1.14. Synthesis of (3S, 4R, 5R)-3-Hydroxy-4, 5-O-(1-Methylethylidene)-3-((4-(Dimethylamino) Phenyl) Ethynyl)-N-Boc-Piperidine (31) Application of the general procedure to ketone 8 (50 mg, 0.18 mmol) with 4-ethynyl-N, N-dimethylaniline (53 mg, 0.37 mmol) and n-BuLi (110 µL, 0.28 mmol, 2.5 M) gave, after purification by flash column chromatography on silica gel (hexane/AcOEt 5:1), 62 mg of 31 (Rf = 0.4, hexane/AcOEt 2:1, 0.15 mmol, 83%) as orange oil. [α ]22 1 31: D = +50.4 (c = 1.00, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 7.25 (d, J = 8.2 Hz, 2H, Ar), 6.55 (d, J = 8.8 Hz, 2H, Ar), 4.49–4.42 (m, 1H, H-5), 4.35 (d, J = 6.8 Hz, 1H, H-4), 3.90–3.57 (m, 3H, Ha-2, H-6), 3.33 (d, J = 12.3 Hz, 1H, Hb-2), 3.08 (br s, 1H, OH), 2.93 (s, 6H, N(CH3)2), 1.51 13 (s, 3H, Me), 1.43 (s, 9H, t-Bu), 1.38 (s, 3H, Me). C-NMR (50 MHz, CDCl3) δ ppm: 155.3 (s, 1C, NCOO), 150.6 (s, 1C, Ar), 133.1, 111.8 (d, 4C, Ar), 109.7 (s, 1C, OC(CH3)2), 108.7 (s, 1C, Ar), 87.7, 85.9 (s, 2C, C-10, C-20), 80.0 (s, 1C, OC(CH3)3), 77.3 (d, 1C, C-4), 72.6 (d, 1C, C-5), 67.4 (s, 1C, C-3), 48.5, 47.6 (t, 1C, C-2), 42.7, 41.9 (t, 1C, C-6), 40.2 (q, 2C, N(CH3)2), 28.6 (q, 3C, OC(CH3)3), 27.0, 25.0 (q, 2C, OC(CH3)2). IR (CDCl3) ν = 3541, 2984, 2934, 2249, 2222, 1687, 1607, 1522, 1476, 1452, 1412, 1368, 1215, 1165, 1134, 1 1072, 943 cm− .C23H32N2O5 (416.51): calcd. C, 66.32; H, 7.74; N, 6.73; found C, 66.18; H, 7.68; N, 6.81. MS-ESI (m/z, %) = 855.20 (100) [2M + Na]+, 439.17 (20) [M + Na]+.

3.1.15. General Procedure for the Synthesis of Trihydroxypiperidines 32, 33, and 34 A solution of protected compound in MeOH was left stirring with 12 M HCl at room temperature for 18 h. The crude mixture was concentrated to yield the deprotected compound as hydrochloride salt. The corresponding free amine was obtained by dissolving the residue in MeOH, then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration and the crude product, if necessary, was purified on silica gel by flash column chromatography to afford the 3-substituted trihydroxypiperidine as free base.

3.1.16. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-(Phenylethynyl)-Piperidine (32) Application of the general procedure to 27 (48 mg, 0.13 mmol) with HCl (150 µL) in MeOH (7 mL) furnished, after treatment with Ambersep 900 OH and purification by column chromatography on silica gel (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1), 16 mg of 32 (Rf = 0.2, 0.07 mmol, 53%) as waxy white solid. 32: [α ]24 = 59.1 (c = 0.65, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 7.51–7.43 (m, 2H, Ar), D − 3 7.38–7.27 (m, 3H, Ar), 3.98–3.91 (m, 1H, H-5), 3.89 (br s, 1H, H-4), 3.01 (d, J = 13.1 Hz, 1H, Ha-2), 13 2.86–2.76 (m, 3H, Hb-2, H-6). C-NMR (50 MHz, CD3OD) δ ppm: 132.7, 129.7, 129.4 (d, 5C, Ar), 123.8 (s, 1C, Ar), 90.7, 87.0 (s, 2C, C-10, C-20), 74.8 (d, 1C, C-4), 71.1 (s, 1C, C-3), 69.9 (d, 1C, C-5), 52.7 (t, 1C, C-2), 48.0 (t, 1C, C-6). C13H15NO3 (233.26): calcd. C, 66.94; H, 6.48; N, 6.00; found C, 66.80; H, 6.33; N, 6.07. MS-ESI (m/z, %) = 234.00 (100) [M + H]+.

3.1.17. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-(oct-1-yn-1-yl)-Piperidine (33) Application of the general procedure to 28 (120 mg, 0.30 mmol) with HCl (150 µL) in MeOH (7 mL) furnished, after treatment with Ambersep 900 OH and purification by column chromatography on silica gel (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1), 51 mg of 33 (Rf = 0.2, 0.21 mmol, 71%) as a colorless oil. 33: [α ]25 = 23.8 (c = 0.42, CHCl ). 1H-NMR (400 MHz, CD OD) δ ppm: 3.90–3.84 (m, 1H, H-5), D − 3 3 3.75 (br s, 1H, H-4), 2.87 (d, J = 13.0 Hz, 1H, Ha-2), 2.78–2.70 (m, 2H, H-6), 2.62 (d, J = 13.0 Hz, 1H, Hb-2), 2.25 (t, J = 6.8 Hz, 2H, H-30), 1.57–1.25 (m, 8H, H-40, H-50, H-60, H-70), 0.92 (t, J = 6.8 Hz, 3H, 13 H-80). C-NMR (50 MHz, CD3OD) δ ppm: 88.0, 81.9 (s, 2C, C-10, C-20), 75.1 (d, 1C, C-4), 70.7 (s, 1C, C-3), 69.8 (d, 1C, C-5), 52.7 (t, 1C, C-2), 47.7 (t, 1C, C-6), 32.4, 29.6, 23.6 (t, 4C, C-40, C-50, C-60-C-70), 19.4 (t, 1C, C-30), 14.4 (q, 1C, C-80). C13H23NO3 (241.33): calcd. C, 64.70; H, 9.61; N, 5.80; found C, 64.94; H, 9.49; N, 5.50. MS-ESI (m/z, %) = 504.92 (100) [2M + Na]+, 242.02 (94) [M + H]+, 264.14 (47) [M + Na]+. Molecules 2020, 25, 4526 16 of 23

3.1.18. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-(3-Thienylethynyl))-Piperidine (34) Application of the general procedure to 30 (85 mg, 0.22 mmol) with HCl (120 µL) in MeOH (6 mL) furnished, after treatment with Ambersep 900 OH and purification by column chromatography on silica gel (gradient eluent from DCM/MeOH/NH4OH (6%) 20:1:0.1 to 10:1:0.1), 27 mg of 34 (Rf = 0.2, DCM/MeOH/NH4OH (6%) 10:1:0.1, 0.11 mmol, 51%) as white waxy solid. 34: [α ]22 = 69.2 (c = 1.00, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 7.63–7.57 (m, 1H, Ar), D − 3 7.43–7.38 (m, 1H, Ar), 7.16–7.12 (m, 1H, Ar), 3.95–3.89 (m, 1H, H-5), 3.87 (br s, 1H, H-4), 2.99 (d, J = 13.1 13 Hz, 1H, Ha-2), 2.85–2.74 (m, 3H, Hb-2, H-6). C-NMR (50 MHz, CD3OD) δ ppm: 130.8, 130.4, 126.7 (d, 3C, Ar), 122.7 (s, 1C, Ar), 90.2, 82.2 (s, 2C, C-10, C-20), 74.8 (d, 1C, C-4), 71.2 (s, 1C, C-3), 69.9 (d, 1C, C-5), 52.8 (t, 1C, C-2), 48.1 (t, 1C, C-6). C11H13NO3S (239.29): calcd. C, 55.21; H, 5.48; N, 5.85; S, 13.40; found C, 55.23; H, 5.61; N, 5.93; S, 13.34. MS-ESI (m/z, %) = 261.99 (100) [M + Na]+.

3.1.19. General Procedure for the Reduction of Triple Bond to Piperidines 11, 12, 14

To a solution of the deprotected compound in EtOH, Pd(OH)2/C was added under nitrogen atmosphere. The mixture was stirred at room temperature under hydrogen atmosphere until a 1H-NMR analysis attested the presence of aliphatic hydrogens at C-10 and C-20 (6–23 h). The catalyst was removed by filtration, the obtained compound was washed several times with EtOH, and the solvent was evaporated under vacuum. The crude product, if necessary, was purified on silica gel by flash column chromatography to afford the corresponding completely reduced 3-substituted trihydroxypiperidine.

3.1.20. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-(2-Phenylethyl)-Piperidine (11)

Application of the general procedure to 32 (13 mg, 0.05 mmol) with Pd(OH)2/C (7 mg) in EtOH (1 mL) furnished, after purification by column chromatography on silica gel (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1), 5 mg of the corresponding reduced trihydroxypiperidine 11 (Rf = 0.2, 0.02 mmol, 39%) as orange oil. 11: [α ]24 = 13.1 (c = 0.36, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 7.27–7.19 (m, 4H, Ar), D − 3 7.18–7.10 (m, 1H, Ar), 3.81 (br s, 1H, H-5), 3.51 (br d, J = 2.2 Hz, 1H, H-4), 2.96 (dd, J = 4.0, 13.7 Hz, 1H, Ha-6), 2.88 (d, J = 13.1 Hz, 1H, Ha-2), 2.76–2.70 (m, 1H, Hb-6), 2.70–2.62 (m, 2H, H-20), 2.59 (d, J =13.1 13 Hz, 1H, Hb-2), 1.97–1.88 (m, 1H, Ha-10), 1.86–1.76 (m, 1H, Hb-10). C-NMR (100 MHz, CD3OD) δ ppm: 143.9 (s, 1C, Ar), 129.4, 129.3, 126.7 (d, 5C, Ar), 74.8 (s, 1C, C-3), 72.7 (d, 1C, C-4), 70.8 (d, 1C, C-5), 54.8 (t, 1C, C-2), 53.5 (t, 1C, C-6), 39.6 (t, 1C, C-10), 30.7 (t, 1C, C-20). C13H19NO3 (237.29): calcd. C, 65.80; H, 8.07; N, 5.90; found C, 65.53; H, 8.25; N, 5.81. MS-ESI (m/z, %) = 238.09 (100) [M + H]+, 260.14 (65) [M + Na]+, 497.20 (16) [2M + Na]+.

3.1.21. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-Octyl-Piperidine (12)

Application of the general procedure to 33 (40 mg, 0.17 mmol) with Pd(OH)2/C (20 mg) in EtOH (2 mL) furnished 41 mg of the corresponding reduced trihydroxypiperidine 12 (0.17 mmol, 98%) as orange oil. 12: [α ]23 = 9.0 (c = 1.00, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 3.77 (br s, 1H, H-5), 3.44 D − 3 (br s, 1H, H-4), 2.92 (d, J = 12.6 Hz, 1H, Ha-6), 2.77 (d, J = 13.6, 1H, Ha-2), 2.68 (d, J = 12.6 Hz, 1H, Hb-6), 2.50 (d, J = 13.6 Hz, 1H, Hb-2), 1.67–1.51 (m, 2H, H-10), 1.36–1.25 (m, 12H, H-20, H-30, H-40, H-50, H-60, 13 H-70), 0.90 (m, 3H, H-80). C-NMR (50 MHz, CD3OD) δ ppm: 75.1 (s, 1C, C-3), 72.5 (d, 1C, C-4), 70.9 (d, 1C, C-5), 53.6 (t, 1C, C-2), 50.7 (t, 1C, C-6), 37.3 (t, 1C, C-10), 33.1, 31.5, 30.7, 30.4, 23.9, 23.7 (t, 6C, C-20, C-30, C-40, C-50, C-60-C-70), 14.4 (q, 1C, C-80). C13H27NO3 (245.36): calcd. C, 63.64; H, 11.09; N, 5.71; found C, 63.78; H, 11.01; N, 5.92. MS-ESI (m/z, %) = 246.15 (100) [M + H]+, 268.26 (22) [M + Na]+, 513.00 (10) [2M + Na]+. Molecules 2020, 25, 4526 17 of 23

3.1.22. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-((2-(3-Thienyl) Ethyl))-Piperidine (14)

Application of the general procedure to 34 (22 mg, 0.09 mmol) with Pd(OH)2/C (11 mg) in EtOH (1 mL) furnished, after purification by column chromatography on silica gel (CH2Cl2/MeOH/NH4OH (6%) 5:1:0.1), 16 mg of the corresponding reduced trihydroxypiperidine 14 (Rf = 0.2, 0.07 mmol, 73%) as a white solid. 14: M.p. = 73–75 C. [α ]20 = 19.9 (c = 0.75, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: ◦ D − 3 7.32–7.24 (m, 1H, Ar), 7.06–7.00 (m, 1H, Ar), 7.00–6.94 (m, 1H, Ar), 3.86 (br s, 1H, H-5), 3.53 (br d, J = 2.7 Hz, 1H, H-4), 3.02 (dd, J = 3.8, 13.6 Hz, 1H, Ha-6), 2.92 (d, J = 13.5 Hz, 1H, Ha-2), 2.79 (br d, J = 13.6 Hz, 1H, Hb-6), 2.75–2.61 (m, 3H, Hb-2, H-20), 2.04–1.92 (m, 1H, Ha-10), 1.92–1.80 (m, 1H, Hb-10). 13 C-NMR (100 MHz, CD3OD) δ ppm: 143.9 (s, 1C, Ar), 129.1, 126.3, 120.8 (d, 3C, Ar), 74.6 (s, 1C, C-3), 72.3 (d, 1C, C-4), 70.4 (d, 1C, C-5), 53.2 (t, 1C, C-2) 50.4 (t, 1C, C-6) 38.3 (t, 1C, C-10), 24.6 (t, 1C, C-20). C11H17NO3S (243.32): calcd. C, 54.30; H 7.04; N, 5.76; S, 13.18; found C, 54.11; H, 7.15; N, 5.88; S, 13.33. MS-ESI (m/z, %) = 243.99 (100) [M + H]+, 266.02 (31) [M + Na]+, 508.78 (30) [2M + Na]+.

3.1.23. Synthesis of (3S, 4R, 5R)-3-hydroxy-4, 5-O-(1-methylethylidene)-3-(2-((4-(dimethylamino) phenyl) ethyl)-N-Boc-piperidine (35) To a solution of 31 (75 mg, 0.18 mmol) in EtOH (2 mL), Pd/C (37 mg) was added under nitrogen atmosphere. The mixture was stirred at room temperature under hydrogen atmosphere until a 1 H-NMR analysis attested the presence of aliphatic hydrogens at C-10 and C-20 (2 h). The catalyst was removed by filtration, the obtained compound was washed several times with EtOH, and the solvent was evaporated under vacuum. The crude product was purified on silica gel by flash column chromatography (hexane:AcOEt 2:1) to afford 54 mg of the corresponding reduced piperidine 35 (Rf = 0.3, 0.13 mmol, 71%) as a colorless oil. [α ]22 1 35: D = +6.3 (c = 0.60, CHCl3). H-NMR (400 MHz, CDCl3) δ ppm: 7.07 (d, J = 8.3 Hz, 2H, Ar), 6.69 (d, J = 8.3 Hz, 2H, Ar), 4.30 (br s, 1H, H-5), 3.98 (d, J = 6.4 Hz, 1H, H-4), 3.92–3.70 (m, 1H, Ha-2), 3.68–3.55 (m, 1H, Ha-6), 3.43–3.10 (m, 2H, Hb-2, Hb-6), 2.90 (s, 6H, N(CH3)2), 2.78–2.64 (m, 2H, 13 H-20), 1.81–1.66 (m, 2H, H-10), 1.54 (s, 3H, Me), 1.47 (s, 9H, t-Bu), 1.38 (s, 3H, Me). C-NMR (100 MHz, CDCl3) δ ppm: 155.0 (s, 1C, NCOO), 149.2 (s, 2C, Ar), 129.0, 113.3 (d, 4C, Ar), 109.4 (s, 1C, OC(CH3)2), 80.3 (s, 1C, OC(CH3)3), 77.9 (d, 1C, C-4), 72.0 (d, 1C, C-5), 70.7 (s, 1C, C-3), 47.2, 46.4 (t, 1C, C-6), 43.7, 42.9 (t, 1C, C-2), 41.0 (q, 2C, N(CH3)2), 40.5, 39.6 (t, 1C, C-10), 28.6 (q, 3C, OC(CH3)3), 28.0 (t, 1C, C-20) 27.4 (q, 1C, OC(CH3)2), 25.3 (q, 1C, OC(CH3)2). IR (CDCl3) ν = 3561, 2984, 2934, 2803, 2247, 1684, 1614, 1 1520, 1456, 1416, 1375, 1346, 1246, 1215, 1165, 1136 1059 cm− .C23H36N2O5 (420.54): calcd. C, 65.69; H 8.63; N, 6.66; found C, 65.58; H, 8.78; N, 6.50. MS-ESI (m/z, %) = 443.10 (100) [M + Na]+.

3.1.24. Synthesis of (3S, 4R, 5R)-3, 4, 5-Trihydroxy-3-(2-((4-(Dimethylamino) Phenyl) Ethyl)-Piperidine (15) A solution of 35 (53 mg, 0.13 mmol) in MeOH (6 mL) was stirred with 12 M HCl (120 µL) at room temperature for 18 h. The crude mixture was concentrated to yield 15 as hydrochloride salt. The corresponding free amine was obtained by dissolving the residue in MeOH, then the strongly basic resin Ambersep 900 OH was added, and the mixture was stirred for 45 min. The resin was removed by filtration and the crude product was purified on silica gel by flash column chromatography (CH2Cl2/MeOH/NH4OH (6%) 10:1:0.1) to afford 36 mg of the 3-substituted trihydroxypiperidine 15 (Rf = 0.1, 0.13 mmol, 100% yield) as a waxy colorless solid. 15: [α ]20 = 21.8 (c = 1.00, MeOH). 1H-NMR (400 MHz, CD OD) δ ppm: 7.06 (d, J = 8.2 Hz, 2H, D − 3 Ar), 6.72 (d, J = 8.2 Hz, 2H, Ar), 3.80 (br s, 1H, H-5), 3.49 (br s, 1H, H-4), 2.95 (d, J = 13.2 Hz, 1H, Hb-6), 2.91–2.75 (m, 7H, Ha-2, N(CH3)2), 2.70 (d, J = 13.8 Hz, 1H, Hb-6), 2.63–2.44 (m, 3H, Hb-2, H-20), 13 1.94–1.71 (m, 2H, H-10). C-NMR (100 MHz, CD3OD) δ ppm: 150.6, 132.7 (s, 2C, Ar), 129.8, 114.9 (d, 4C, Ar), 74.9 (s, 1C, C-3), 72.6 (d, 1C, C-4), 70.8 (d, 1C, C-5), 53.6 (t, 1C, C-2), 50.6 (t, 1C, C-6), 41.6 (q, 2C, N(CH3)2), 39.7 (t, 1C, C-10), 29.2 (t, 1C, C-20). C15H24N2O3 (280.36): calcd. C, 64.26; H, 8.63; N, 9.99; found C, 64.10; H, 8.33; N, 9.63. MS-ESI (m/z, %) = 582.84 (100) [2M + Na]+, 281.04 (59) [M + H]+. Molecules 2020, 25, 4526 18 of 23

3.2. Preliminary Biological Screening towards Commercial Glycosidases 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 and rice, amyloglucosidase EC 3.2.1.3 from Aspergillus niger, β-glucosidase 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, β-N-acetylglucosaminidases EC 3.2.1.52 from Jack beans and bovine kidney) purchased from Sigma-Aldrich (St. Louis, MO, USA) or Megazyme Ltd. (Bray, Co. Wicklow, Ireland) was screened. The percentage (%) of inhibition towards the corresponding glycosidase was determined in quadruplicate in the presence of 100 µM of the inhibitor on the well. Each enzymatic assay (final volume 0.12 mL) contains 0.01–0.5 units/mL of the enzyme (with previous calibration) and 4.2 mM aqueous solution of the appropriate p-nitrophenyl glycopyranoside (substrate) buffered to the optimal pH of the enzyme. Enzyme and inhibitor were preincubated for 5 min at room temperature, and the reaction started by addition of the substrate. After 20 min of incubation at 37 ◦C, the reaction was stopped by the addition of 0.1 mL of sodium borate solution (pH 9.8). The p-nitrophenolate formed was measured by visible absorption spectroscopy at 405 nm (Asys Expert 96 spectrophotometer, Biochrom Ltd., Cambridge, England). Under these conditions, the p-nitrophenolate released led to optical densities linear with both reaction time and concentration of the enzyme (for more details, see the Supplementary Materials). The IC50 value (concentration of inhibitor required for 50% inhibition of enzyme activity) was determined from plots of % inhibition vs. different inhibitor concentrations. Each point of the graph is the average of four measures. The standard deviation for each point has been also represented in the graph.

3.3. Biological Screening towards Human Lysosomal β-Galactosidase (β-Gal) and β-Glucosidase (GCase) All experiments on biological materials were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments. In keeping with ethical guidelines, all blood and cell samples were obtained for storage and analyzed only after written informed consent of the patients (and/or their family members) was obtained, using a form approved by the local Ethics Committee (Project ID code: 16774_bio, 5 May 2020, Comitato Etico Regionale per la Sperimentazione Clinica della Regione Toscana, Area Vasta Centro, Florence, Italy). Controls and patients’ samples were anonymized and used only for research purposes. The new compounds were screened at 1 mM concentration towards β-galactosidase (β-Gal) and β-glucosidase (GCase) in leukocytes isolated from healthy donors (controls). Isolated leukocytes were disrupted by sonication, and a Micro BCA Protein Assay Kit (Sigma–Aldrich, St. Louis, MO, USA) was used to determine the total protein amount for the enzymatic assay, according to the manufacturer’s instructions.

3.3.1. Human Lysosomal β-Galactosidase (β-Gal) Activity β-Gal activity was measured in a flat-bottomed 96-well plate. Azasugar solution (3 µL), 4.29 µg/µL leukocytes homogenate 1:10 (7 µL), and substrate 4-methylumbelliferyl β-d-galactopyranoside (1.47 mM, 20 µL, Sigma–Aldrich) in acetate buffer (0.1 M, pH 4.3) containing NaCl (0.1 M) and sodium azide (0.02%) were incubated at 37 ◦C for 1 h. The reaction was stopped by addition of sodium carbonate (200 µL; 0.5 M, pH 10.7) containing Triton X-100 (0.0025%), and the fluorescence 4-methylumbelliferone released by β-galactosidase activity was measured in SpectraMax M2 Microplate Reader (λex = 365 nm and λem = 435 nm; Molecular Devices LLC, San Jose, CA, US). Inhibition is given with respect to the control (without azasugar). Data are mean SD (n = 3). Molecules 2020, 25, 4526 19 of 23

3.3.2. Human Lysosomal β-Glucosidase (GCase) Activity GCase activity was measured in a flat-bottomed 96-well plate. Compound solution (3 µL), 4.29 µg/µL leukocytes homogenate (7 µL), and substrate 4-methylumbelliferyl-β-d-glucoside (3.33 mM, 20 µL, Sigma–Aldrich Chemie Gmbh, Munich, Germany)) in citrate/phosphate buffer (0.1:0.2, M/M, pH 5.8) containing sodium taurocholate (0.3%) and Triton X-100 (0.15%) at 37 ◦C were incubated for 1 h. The reaction was stopped by addition of sodium carbonate (200 µL; 0.5 M, pH 10.7) containing Triton X-100 (0.0025%), and the fluorescence of 4-methylumbelliferone released by β-glucosidase activity was measured in SpectraMax M2 Microplate Reader (λex = 365 nm and λem = 435 nm; Molecular Devices LLC, San Jose, CA, US). Percentage GCase inhibition is given with respect to the control (without compound). Data are mean SD (n = 3). For compounds showing GCase inhibitory activity higher than 80% at 1 mM concentration, the IC50 values were determined by measuring the initial rate with 4-methylumbelliferyl-β-d-glucoside (3.33 mM). Data obtained were fitted by using the appropriate equation (for more details, see the Supplementary Materials).

3.4. Pharmacological Chaperoning Activity of Compound 10 Anonymized patient fibroblasts cell lines for research purposes were provided by “Cell Line and DNA Biobank from Patients Affected by Genetic Diseases” (G. Gaslini Institute)-Telethon Genetic Biobank Network (Project No. GTB07001A). Fibroblasts with the N370S/RecNcil mutation from patients with Gaucher disease were obtained from the “Cell line and DNA Biobank from patients affected by Genetic Diseases” (Gaslini Hospital, Genova, Italy). Fibroblasts cells (15.0 104) were seeded in T25 flasks with DMEM supplemented with × fetal bovine serum (10%), penicillin/streptomycin (1%), and glutamine (1%) and incubated at 37 ◦C with 5% CO2 for 24 h. The medium was removed, and fresh medium containing compound 10 was added to the cells and left for 4 days. The medium was removed, and the cells were washed with PBS and detached with trypsin to obtain cell pellets, which were washed four times with PBS, frozen, and lysed by sonication in water. Enzyme activity was measured as reported above. Reported data are mean S.D. (n = 2).

4. Conclusions In conclusion, with the aim of searching for new human lysosomal β-Gal inhibitors, we synthesized a series of trihydroxypiperidines and congeners with the “all-cis” configuration at the carbons bearing the hydroxy/amino groups. The reported syntheses exploited the reaction of ketone intermediate 8, which was functionalized at C=O through ketone reduction followed by alcohol alkylation (9, 21), reductive amination (10), or via the stereoselective addition of various lithium acetylides (11–15, 32, and 33). Unfortunately, none of the reported compounds showed β-Gal inhibition. However, these data give useful pointers, suggesting that the presence of three “all-cis” hydroxy groups at C-3, C-4, and C-5 of the piperidine ring is essential for β-Gal inhibitory activity, but that the introduction of an alkyl chain at C-3, either through oxygen (or nitrogen) or directly to carbon (as in compound 12), hampers interaction with the enzyme active site. Interestingly, newly synthesized compounds 9, 10, and 12 showed moderate to good inhibition against human lysosomal β-glucosidase (GCase). For this enzyme, our data confirmed that the presence of an alkyl chain of at least eight carbon atoms is essential to impart GCase inhibitory activity. However, we show that the alkyl chain can be placed at the C-3 position either maintaining the three hydroxyl groups (as in compound 12) or via alkylation of the hydroxy group at C-3 (as in ether 9). Compound 10, the best GCase inhibitor synthesized in this work, failed to rescue the enzyme activity on cell lines likely due to its cytotoxicity. Nevertheless, this study provided useful hints for the design of novel inhibitors for both GCase and β-Gal. Molecules 2020, 25, 4526 20 of 23

Supplementary Materials: Figures S1–S44 containing 1H and 13C-NMR of new compounds, Table S1: Addition of sp3 Grignard reagents to ketone 8. Table S2: Configuration assignment. Table S3: Biological screening towards commercial glycosidases. Figure S45: IC50 for compound 10 towards β-glucosidase from almonds. Figure S46 and Figure S47: Biological screening towards human lysosomal β-Gal and GCase. Figures S48–S51: IC50 for compounds 9, 10, 12, and 21 towards GCase. Figure S52 to Figure S54: Chaperoning activity assay of compounds 9, 10, and 12. Author Contributions: F.C. (Francesca Cardona), C.M., and A.G. planned the synthetic strategy and wrote the manuscript; M.G.D. and F.C. (Francesca Clemente) made the syntheses; F.C. (Francesca Clemente) contributed to the N.M.R. characterization of the new compounds; A.M., F.C. (Francesca Clemente), and M.M.-B. performed the biological evaluation. All authors have read and agreed to the published version of the manuscript. Funding: The research was funded by Cassa di Risparmio di Pistoia e Pescia (Bando [email protected] scientifica 2017) through a fellowship to C.M. and financial support by Fondazione CR Firenze (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), and by Fondazione CR Firenze (grant N◦2018.0942). We thank MIUR-Italy (“Progetto Dipartimenti di Eccellenza 2018–2022”) allocated to the Department of Chemistry “Ugo Schiff”, Università di Firenze. Fondazione CR Firenze is also acknowledged for a fellowship to M.G.D. (grant number 2017.0734). M.M.-B. thanks the Ministerio de Economía y Competitividad of Spain (CTQ2016-77270-R) for financial support. F.C. (Francesca Cardona) and A.M. also thank 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. A.M. also thanks AMMeC (Associazione Malattie Metaboliche Congenite, Italy) for financial support. Acknowledgments: We thank Paolo Paoli (Università di Firenze) for helpful discussions. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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