DOI: 10.1002/cbic.201300442

A Systematic Investigation of Iminosugar Click Clusters asPharmacological Chaperones for the Treatment of GaucherDiseaseAntoine Joosten,[a] Camille Decroocq,[a] Julien de Sousa,[a, b] J�r�my P. Schneider,[a]

Emile Etam�,[a] Anne Bodlenner,[a] Terry D. Butters,[b] and Philippe Compain*[a, c]

Introduction

Since its recent discovery, the pharmacological chaperone con-cept has experienced spectacular growth in the field of inherit-ed diseases caused by improperly folded proteins.[1] In particu-lar, promising success has recently been achieved in the treat-ment of glycosphingolipid lysosomal storage disorders(GLSDs),[2, 3] with iminosugar-based chaperones reaching phaseII and phase III clinical trials. GLSDs are a small group of rarediseases characterized by deficiency of glycosidases involved inthe catabolism of glycosphingolipids in the lysosome.[4] Theseinherited enzyme deficiencies result in pathological lysosomalaccumulation of glycolipids, with widespread clinical conse-quences.

Pharmacological chaperone therapy (PCT) is based on theuse of reversible inhibitors of these deficient enzymes capableof enhancing their residual hydrolytic activity at subinhibitoryconcentrations.[2, 3] Since the seminal work of Asano and Fan in1999, which showed that the residual a-galactosidase A activi-ty in lymphoblasts of patients with Fabry disease could be

significantly enhanced by treatment with 1-deoxygalactonojiri-mycin (1 a, Amigal, Scheme 1),[5] hundreds of small molecules,mainly iminosugars, have been evaluated as pharmacologicalchaperones (PCs) for the treatment of GLSDs.[3] The scientificinterest in pharmacological chaperone therapy is also high-lighted by the exponential growth in citations of articles deal-ing with this concept (�200 citations in 2004, more than 2000in 2012). Indeed, use of PCT for the treatment of GLSDs offersmany advantages over the other established therapies,[6] basedeither on substitutive enzymotherapy (enzyme replacementtherapy, ERT)[7] or on inhibitors of glycolipid substrate biosyn-thesis (substrate reduction therapy, SRT).[8, 9] PCT targets thecause of GLSDs by restoring the hydrolytic activity of the mu-tated enzymes, whereas the other two therapies reduce theaccumulation of un-degraded glycosphingolipids either by theadministration of a recombinant protein as an exogenous sub-stitute (ERT) or by the dysregulation of the glycosphingolipidmetabolic pathway (SRT).[10] PCT combines the benefits of

A series of 18 mono- to 14-valent iminosugars with different li-gands, scaffolds, and alkyl spacer lengths have been synthe-sized and evaluated as inhibitors and pharmacological chaper-ones of b-glucocerebrosidase (GCase). Small but significantmultivalent effects in GCase inhibition have been observed fortwo iminosugar clusters. Our study provides strong confirma-tion that compounds that display the best affinity for GCaseare not necessarily the best chaperones. The best chaperoningeffect observed for a deprotected iminosugar cluster has been

obtained with a tetravalent 1-deoxynojirimycin (DNJ) analogue(3.3-fold increase at 10 mm). In addition, our study provides thefirst evidence of the high potential of prodrugs for the devel-opment of potent pharmacological chaperones. Acetylation ofa trivalent DNJ derivative, to give the corresponding acetateprodrug, leads to a pharmacological chaperone that produceshigher enzyme activity increases (3.0-fold instead of 2.4-fold) ata cellular concentration (1 mm) reduced by one order of magni-tude.

Scheme 1. Iminosugar-based pharmacological chaperones.

[a] Dr. A. Joosten, Dr. C. Decroocq, J. de Sousa, J. P. Schneider, E. Etam�,Dr. A. Bodlenner, Prof. P. CompainLaboratoire de Synth�se Organique et Mol�cules Bioactives (SYBIO)Universit� de Strasbourg/CNRS (UMR 7509)Ecole Europ�enne de Chimie, Polym�res et Mat�riaux25 rue Becquerel, 67087 Strasbourg (France)E-mail : philippe.compain@unistra.fr

[b] J. de Sousa, Dr. T. D. ButtersGlycobiology Institute, Oxford UniversitySouth Parks Road, Oxford OX1 3QU (UK)

[c] Prof. P. CompainInstitut Universitaire de France103 Bd Saint-Michel, 75005 Paris (France)

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201300442.

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a small-molecule approach, including oral bioavailability andthe potential to cross the blood–brain barrier, with the specific-ity of an enzyme-directed approach.

Despite all this promise, however, a number of scientificchallenges might impede the development of drugs based onPCT. The first relates to the mechanism by which pharmacolog-ical chaperones increase the residual hydrolytic activity of themutant enzymes involved in GLSDs. There are currently twoopposite views concerning the molecular basis of pharmaco-logical chaperoning activity. According to the more acceptedtheory,[2, 11] the pharmacological chaperone acts at neutral pHin the endoplasmic reticulum (ER) by inducing or stabilizingthe correct conformation of the misfolded but catalyticallyactive enzyme, preventing its degradation by a “quality-con-trol” mechanism (endoplasmic-reticulum-associated degrada-tion, ERAD).[12, 13] In sharp contrast, Wei et al. have very recentlyproposed that the main role of pharmacological chaperonesmight be to increase the resistance of a correctly folded butcatalytically deficient enzyme to proteases in the acidic lyso-somes.[14] Since the report of this theory, which is based in parton crystallographic evidence, few studies have been performedto resolve these conflicting views.[15, 16] However, the debate isstill open, and more research is needed. Beyond its fundamen-tal interest, a clear understanding of the chaperoning effectwould constitute a decisive leap forward towards the rationaldrug design of potent pharmacological chaperones.

A second obstacle to therapeutic application of pharmaco-logical chaperones might be achievement of insufficient levelsof residual cellular activity enhancement. For example, almostall the PCs reported to date for type I Gaucher disease, themost prevalent GLSD, double to triple the residual cellularactivity of N370S b-glucocerebrosidase (GCase) whatever theiraffinity for the mutant enzyme.[2, 3] These enhancement valuesare comparable to that obtained for N-nonyl-1-deoxynojirimy-cin (NN-DNJ, 1 b, Scheme 1), the first example of a PC forGaucher disease, reported in the literature ten years ago.[17]

Treatment with isofagomine (2, Plicera), the leading clinicalcandidate for chaperone therapy for Gaucher disease, led to anincrease in enzymatic activity for all patients enrolled in a pha-se II trial without serious side effects.[18] However, Plicera mightnot move forward to phase III development because significantclinical improvements were observed for only one patient outof eighteen.[18a] This result could indicate that the doubling ofthe residual GCase activity reported in the case of Plicera is notsufficient for a therapeutic application.[19, 20]

In this context, one current question is how to get closer toa second generation of pharmacological chaperones thatwould display improved enhanced activity. Because only veryfew rational criteria for the design of such compounds areavailable, new approaches and concepts have to be explored.

Mechanism-based inactivators[21] or iminosugars bearing car-bonyl groups on their pseudo-anomeric carbon atoms,[22] forexample, have been found to be promising new class of PCs.In connection with our studies on multivalent effect in glycosi-dase inhibition,[23, 24] we have recently explored the potential ofmultivalency for the design of efficient PCs containing fourDNJ–cyclodextrin conjugates.[25] The heptavalent iminosugar

16 (Scheme 2) was found to be a nm inhibitor of recombinantGCase (ceredase), whereas the corresponding monovalent ana-logue displayed inhibition in the mm range. Compound 16increased residual GCase activity in fibroblasts from Gaucherpatients by 53 % at 10 mm, a chaperoning effect comparable tothat observed for the corresponding monovalent iminosugar(60 % increase at 10 mm).[25] The ability of 16 to gain access tothe ER and/or the lysosomes was thus unambiguously demon-strated by the significant enhancement of GCase activity inGaucher fibroblasts. However, in view of the high value of in-hibition in vitro observed for 16, the cellular concentration of10 mm required to achieve a chaperoning effect is relativelyhigh. We hypothesized that this result could be explained by16 having a lower cellular permeability than the correspondingmonovalent analogue.

On the basis of this proof-of-concept preliminary study, wehave performed a systematic investigation of structure–activityrelationships by synthesizing and evaluating a panel of tri- to14-valent systems with different alkyl spacer lengths (C6 or C9).In particular, we have investigated the effects of size, valency,ligand topology, and scaffold structure on GCase binding affini-ty and chaperoning activity (Scheme 2). The two cyclodextrin-based 14-valent systems 19 and 21, for example, were synthe-sized to explore the impact of ligand spatial orientation. Small-er systems of lower valency, as well as acetylated analoguesdesigned as potential prodrugs (10 and 17), were preparedwith the aim of facilitating cellular uptake. Iminosugars 4–7were also synthesized and evaluated as monovalent controlsto assess any possible multivalent effect in GCase inhibitionand/or chaperoning activity. Here we describe the full detailsof our study, from synthesis to cellular assays.

Results and Discussion

Synthesis of multivalent iminosugars

Our synthetic strategy was based on experience gained in ourprevious work on multivalent iminosugars[23, 24] and involvedattachment of the iminosugar moieties onto the scaffolds byCuI-catalyzed azide–alkyne cycloaddition (CuAAC).[26] N-Alkylderivatives of DNJ, the first family of PCs disclosed for Gaucherdisease,[17] were chosen as the peripheral ligands. Multivalentsystems containing analogues of a-1-C-alkyl-iminoxylitols (a-1-C-alkyl-DIXs, 3, Scheme 1), which are highly potent nanomolarinhibitors of human GCase,[27] were also synthesized and evalu-ated.

We started with the multivalent iminosugars in the iminoxy-litol series. The syntheses of tri- and tetravalent iminosugars 11and 14 were performed in five and three steps, respectively,from pentaerythritol (22, Schemes 3 and 4). Trivalent iminoxyli-tol derivative 11 was synthesized by direct microwave CuI-cata-lyzed 1,3-cycloaddition between tripropargyl ether 23, pre-pared in three step from 22,[28] and the corresponding azidoiminosugars precursor 24.[29] Subsequent clean O-deacetyla-tion[24] of adduct 25 by use of Amberlite IRA 400 (OH�) anion-exchange resin afforded the expected trivalent iminosugar ina very good yield (80 % for the two steps).

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Compound 14, the tetravalent analogue of 11, was synthe-sized according to the same convergent strategy (Scheme 4).The tetrapropargyl ether 26,[30] prepared in one step from pen-taerythritol (22), reacted with the azide precursor 24. Subse-quent deprotection provided the desired tetravalent C6 imino-sugar 14.

The same two key reactions—CuAAC reaction followed byO-deacetylation—were again applied for the synthesis of imi-nosugar 20, containing 14 DIX-based ligands (Scheme 5). Thetwo cycloaddition partners were 29, the N-Boc-protected ana-logue of azido iminosugar 24,[29] and per-(2,6-di-O-propargyl)-b-cyclodextrin (28), obtained in only one step from b-cyclodex-trin (b-CD).[31] The protection and deactivation of the endocy-clic nitrogen in 29 was nevertheless found to reduce the yieldof the CuAAC reaction and ultimately the efficiency of thewhole process.

For the synthesis of 18, the heptavalent analogue of imino-sugar 20, the alkyne substrate for the CuAAC was heptakis(2,3-di-O-methyl-6-O-propargyl)cyclomaltoheptaose (31), obtainedin four steps from b-CD (Scheme 5).[24, 30b, c] To complete theseries, hepta- and 14-valent CD-based iminosugar clusters 15–17, 19, and 21 were efficiently synthesized through CuAAc re-actions by our reported strategy,[24] together with tri- and tetra-valent DNJ derivatives 8–10 and 12–13, respectively.[32]

Monovalent controls were also prepared to allow assess-ment of the multivalent effects. Iminosugars 4–6 were synthe-sized as described previously.[24, 32] Iminosugar 33[29] was treatedwith pent-1-yne in the presence of CuSO4·5 H2O and sodium

Scheme 2. Library of multivalent iminosugars and their corresponding monovalent analogues.

Scheme 3. Reagents and conditions: a) CuSO4·5 H2O cat. , sodium ascorbate,DMF/H2O (4:1), MW, 80 8C, to afford 25 (80 %); b) Amberlite IRA 400 (OH�),MeOH/H2O (1:1), to afford 11 (100 %).

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ascorbate to yield the corresponding fully deprotected adduct7 after treatment with TFA (Scheme 6).

Biological results

Having a library of tri- to 14-valent systems and the corre-sponding monovalent controls to hand, we first evaluatedtheir inhibitory activities toward human placental GCase(Table 1). With the exceptions of 14-valent DNJ clusters 15, 19,and 20, which behaved as reversible mixed (competitive/non-competitive) inhibitors, and tetravalent DIX cluster 14, thecomplex inhibition mode of which could not be determined,all the other iminosugars were found to be competitive inhibi-tors of GCase. Less-potent inhibition was observed for multiva-lent systems with the shorter linker (C6) in the DNJ series. Mul-tivalent presentation of the DNJ motif in these compoundswas either detrimental to or irrelevant for inhibition of GCase.

Replacement of the DNJ ligand by the much more potentDIX ligand[27] led to dramatic improvements in the affinities forGCase, as reflected in Ki increases of three orders of magni-tude. In this series (compounds 7, 11, 14, 18, and 20), a smallbut significant[33] multivalent effect was observed for the tetra-valent DIX derivative 14, with inhibition potency increasedapproximately eightfold (�2 on a molar basis) over the corre-sponding monovalent analogue 7.

The influence of linker length on GCase inhibition was evalu-ated by comparison of DNJ click clusters possessing two differ-ent alkyl spacer lengths. Extension of the spacer length bythree carbons (from C6 to C9) in the DNJ series led to improve-ment in inhibition, with Ki decreases of one to three orders ofmagnitude. Again, a small but significant multivalent effectwas observed only for one compound: the heptavalent DNJ

derivative 16, with inhibition potency increased approximately14-fold (�2 on a molar basis) over the corresponding monova-lent analogue 5. It is noteworthy that the multivalent effectobserved for 16 is one order of magnitude lower than that

Scheme 4. Reagents and conditions: a) CuSO4·5 H2O cat. , sodium ascorbate,DMF/H2O (4:1), MW, 80 8C, to afford 27 (83 %); b) Amberlite IRA 400 (OH�),MeOH/H2O (1:1), to afford 14 (100 %).

Scheme 5. Reagents and conditions: a) 29, CuSO4·5 H2O cat. , sodium ascor-bate, DMF/H2O (4:1), MW, 80 8C, to afford 30 (44 %); b) TFA, CH2Cl2, then Am-berlite IRA 400 (OH�), MeOH/H2O (1:1), to afford 20 (66 %); c) 24,CuSO4·5 H2O cat. , sodium ascorbate, DMF/H2O (4:1), MW, 80 8C, to afford 32(94 %); d) Amberlite IRA 400 (OH�), MeOH/H2O (1:1), to afford 18 (92 %).

Scheme 6. Reagents and conditions: a) pent-1-yne, CuSO4·5 H2O cat. , sodiumascorbate, THF/H2O (1:1), to afford 34 (89 %); b) TFA, H2O then IR 120 (H+),to afford 7 (64 %).

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initially measured in our first preliminary study based on IC50

values.[25]

The peracetylated mono- or multivalent DNJ analogues 6,10, and 17, designed as potential prodrugs, were found todisplay no inhibition of GCase. This result might be explainedin part by the fact that these compounds are not deprotectedunder the conditions of the in vitro assays. Cytotoxicity assayswere performed before the investigation of the influence ofthe iminosugar clusters and their monovalent analogues onhuman GCase activity in N370S fibroblasts (Table 1). All thecompounds had CC50 values (the concentration at which thenumber of cells or cell proliferation is reduced by 50 %) greaterthan 20 mm. With the exceptions of compounds 13 and 16, themultivalent deprotected iminosugars are less toxic than orequally toxic as their corresponding monovalent derivatives.These two iminosugar clusters, which displayed higher toxicityvalues, had the longer spacer length.

The assay of N370S Gcase activation was then performed(Table 1). For comparison, a-1-C-nonyl-DIX (3 a) and NN-DNJ(1 b) were also evaluated, and their chaperoning activities werefound to be comparable to those already reported.[17, 27a] Of the14 multivalent iminosugars evaluated, five were found to dis-play weak to no significant chaperoning activity (Table 1). Thebest results were obtained with two DNJ clusters with the C9

spacer, compounds 10 and 13, which demonstrated threefoldand 3.3-fold increases at 1 and 10 mm, respectively, in GCaseactivity.

In the DNJ series with the C6 linker, no improvement in thechaperoning activity was observed, and only compounds 15(heptavalent) and 19 (14-valent) demonstrated effects compa-rable to that of the corresponding monovalent derivative 4 ata cellular concentration of 10 mm (Figure 1).

Interesting results were obtained when the spacer lengthwas increased by three carbon atoms (Figure 2). The presenceof the N-alkyltriazolyl substituent in monovalent control 5

Table 1. Biological evaluation of iminosugars.

Compound Ligand Valency Linker length CC50[a] [mm] Ki

[d] [nm] Fold increase[j]

4 DNJ 1 6 20 1660�210 1.7�0.28 DNJ 3 6 100 23 500�1500 2.5�0.1 at 50 mm

12 DNJ 4 6 100 7630�620 –[l]

15 DNJ 7 6 20 12 000�2000[e] 1.8�0.419 DNJ 14 6 50 14 000�3400[f] 1.7�0.2

7 DIX 1 6 20 35�2 2.4�0.1107�19[g]

11 DIX 3 6 20 29�1 1.7�0.1 at 0.5 mm

14 DIX 4 6 20 14�5[g] 1.2�0.2 at 0.1 mm

18 DIX 7 6 100 50�2 2.1�0.2 at 1 mm

20 DIX 14 6 50 22�11[h] –[l]

3 a DIX 1 9 n.d.[b] 26�3 1.9�0.1 at 10 nm

5 DNJ 1 9 100[32] 758�65 1.2�0.1 at 1 mm

1 b DNJ 1 9 n.d.[b] 300[27a] 3.2�0.26 DNJ (OAc) 1 9 50 n.i.[i] 1.2�0.19 DNJ 3 9 >100[32] 1070�70 2.4�0.1

10 DNJ (OAc) 3 9 >100 n.i.[i] 3.0�0.1 at 1 mm

13 DNJ 4 9 50[32] 284�19 3.3�0.316 DNJ 7 9 50 55�3 2.4�0.117 DNJ (OAc) 7 9 50 n.i.[i] –[l]

21 DNJ 14 9 200 61�6 1.6�0.2 at 0.1 mm

control[k] n.c.[c] 1

[a] HL60 cells were treated with various concentrations (1–500 mm) of compounds for three days, and the compounds’ cytotoxicities were evaluated as de-scribed in the Experimental Section. [b] Not determined. [c] Not cytotoxic. [d] The Ki values were determined for human placental GCase with 4-methylum-belliferyl-b-d-glucoside. Inhibition, when detected, was competitive unless specified otherwise. [e] Mixed inhibition: the Ki value corresponding to the com-petitive term is given, and the noncompetitive contribution had an apparent inhibition constant Ki’= (77 600�40 300) nm. [f] Mixed inhibition: the Ki valuecorresponding to the competitive term is given, and the noncompetitive contribution had an apparent inhibition constant Ki’= (22 900�12 300) nm.[g] IC50 value determined with human placental GCase with 0.5 mm 4-methylumbelliferyl-b-d-glucoside. [h] Mixed inhibition: the Ki value corresponding tothe competitive term is given, and the noncompetitive contribution had an apparent inhibition constant Ki’= (11�10) nm. [i] No inhibition detected at5 mm. [j] Gaucher fibroblasts (N370S) were cultured in the presence of compounds in various concentrations (1 pm–20 mm) for three days, after whichGCase activity was measured. The fold increase in enzyme activity is relative to untreated cells (i.e. , normalized value = 1). The maximum fold increase asshown in the table appeared at 10 mm unless specified otherwise (see the Supporting Information). [k] Control cells (no compound addition) were analyzedafter addition of water or 0.01 % DMSO. [l] No significant chaperoning activity observed.

Figure 1. The influence of iminosugars 4 (^), 15 ( � ), and 19 (&) on b-gluco-cerebrosidase activities in N370S Gaucher fibroblasts. The fold increases inenzyme activity are relative to untreated cells (i.e. , normalized value = 1) andshown as relative enzyme activities. Means�SD obtained from experimentsperformed in triplicate are shown.

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seemed to have a strong negative impact on chaperoning ac-tivity in comparison with N-nonyl-DNJ (1 b). In this series (DNJligand, C9 linker), all of the deprotected multivalent imino-sugars evaluated were found to be better pharmacologicalchaperones than the corresponding monovalent analogue 5,with a maximum 3.3-fold increase in GCase activity at 10 mm

for the tetravalent DNJ derivative 13 (Figure 2). However, thisobserved maximum enzyme activity enhancement was compa-rable to that obtained with 1 b (3.2-fold at 10 mm).

We then evaluated compounds in the DIX series. Tri- andheptavalent iminosugars 11 and 18 with the shorter linker (C6)were found to display lower chaperoning activity (up to 2.1-fold increase) than their corresponding monovalent ana-logue 7, but at lower cellular concentrations (Figure 3). Smallor no significant GCase activity enhancement was observedwith tetra- and 14-valent iminosugars 14 and 20 (Table 1).[34]

The most promising result came from peracetylated trivalentDNJ analogue 10 (Table 1, Figure 4). This molecule, small incomparison with the corresponding heptavalent and 14-valentanalogues,[25] was designed as a prodrug with the aim of facili-tating cellular uptake and permeability. Compound 10 (3.0-foldincrease) displayed enhancement comparable to that seenwith 1 b but at a cellular concentration ten times lower (1 mm).The trivalent cluster 10, which was not an inhibitor of GCase invitro, showed better enhancement than its monovalent ana-logue 6 (1.2-fold increase). The chaperoning effect was lost

with systems of higher valency, as is shown in the case of thecorresponding heptavalent analogue 17.

The positive impact of acylation of the OH group on chaper-oning activity was clearly demonstrated by comparison of theenzyme activity enhancement by 10 (3.0-fold increase at 1 mm)and that by the corresponding deprotected analogue 9 (2.4-fold increase at 10 mm). Taken together, these data support theidea that compound 10 acts as an acetate prodrug of 9 withimproved cellular uptake and permeability. Once inside thecell, endogenous esterases presumably convert 10 into thecorresponding active chaperone 9.[35] However, additional un-known mechanisms of action in addition to this prodrug effectmay not be discounted. To the best of our knowledge, the re-sults obtained with 10 constitute the first demonstration thatthe prodrug concept might be a promising option for thedesign of pharmacological chaperones for GLSDs. It is alsonoteworthy that trivalent cluster 10 is among the less toxiciminosugars evaluated in this study (CC50>100 mm).

Conclusions

In summary, we report a systematic structure–activity relation-ship study in which we have synthesized 14 iminosugar clus-ters and evaluated their inhibition and chaperoning activitiestoward GCase. Small but significant multivalent effects inGCase inhibition were observed for two iminosugar clusters.Our study provides strong confirmation that compounds thatdisplay the best affinities for GCase, as judged by their in vitroinhibition potencies, are not necessarily the best chaperonesof the mutant enzyme. This observation, recently also madefor a series of amphiphilic DNJ derivatives,[36] is strongly accen-tuated in the case of multivalent iminosugars such as 20 and13. The lack of correlation between inhibition and chaperoningactivity is an additional issue for the rational design of pharma-cological chaperones because no prediction based on struc-ture–inhibition relationship studies can be made. The bestchaperoning effect for a deprotected iminosugar cluster (3.3-fold increase at 10 mm) was observed in the DNJ series withtetravalent compound 13, a sub-micromolar GCase inhibitor.

Our study also provides the first evidence of the great po-tential of prodrugs for the development of potent pharmaco-logical chaperones. Acetylation of the trivalent iminosugar 9,

Figure 2. The influence of iminosugars 5 (^), 9 ( � ), 13 (&), and 16 (*) on b-glucocerebrosidase activities in N370S Gaucher fibroblasts. The fold increas-es in enzyme activity are relative to untreated cells (i.e. , normalized value =

1) and shown as relative enzyme activities. Means�SD obtained from ex-periments performed in triplicate are shown.

Figure 3. The influence of iminosugars 7 (^), 11 ( � ), and 18 (&) on b-gluco-cerebrosidase activities in N370S Gaucher fibroblasts. The fold increases inenzyme activity are relative to untreated cells (i.e. , normalized value = 1) andshown as relative enzyme activities. Means�SD obtained from experimentsperformed in triplicate are shown.

Figure 4. The influence of iminosugars 6 (^), 10 ( � ), and 17 (&) on b-gluco-cerebrosidase activities in N370S Gaucher fibroblasts. The fold increases inenzyme activity are relative to untreated cells (i.e. , normalized value = 1) andshown as relative enzyme activities. Means�SD obtained from experimentsperformed in triplicate are shown.

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giving the corresponding peracetate prodrug 10, led to a phar-macological chaperone that displayed higher enzyme activityincreases (3.0-fold instead of 2.4-fold) at cellular concentrationsreduced by one order of magnitude. Future studies combiningprodrug and multivalent strategies might provide a new gen-eration of potent pharmacological chaperones for the treat-ment of lysosomal diseases.

Experimental Section

General methods : CH2Cl2 was distilled over CaH2 under Ar. All re-actions were performed in standard glassware or microwave reac-tor vials purchased from Biotage. Microwave reactions were carriedout with a Biotage Initiator microwave synthesizer. Column chro-matography: silica gel 60 (230–400 mesh, 0.040–0.063 mm) waspurchased from E. Merck. Flash chromatography was carried outwith a Grace Reveleris flash system equipped with UV/Vis andELSD detectors. TLC was performed on aluminum sheets coatedwith silica gel 60 F254 (purchased from Merck). IR spectra (cm�1)were recorded on a PerkinElmer Spectrum One Spectrophotome-ter. NMR spectra were recorded on a Bruker AC 300 or AC 400 withsolvent peaks as references. Carbon multiplicities were assigned bydistortionless enhancement by polarization transfer (DEPT) experi-ments. The 1H signals were assigned by COSY and HSQC experi-ments. ESI-HRMS spectra were obtained on a Bruker MicroTOFspectrometer. Specific rotations were determined at room temper-ature (20 8C) with a PerkinElmer 241 polarimeter and the use ofsodium light (l= 589 nm).

General procedure A—CuAAC reaction : Alkyne, azide,CuSO4·5 H2O (0.1 equiv per alkyne), and sodium ascorbate(0.2 equiv per alkyne) in DMF/H2O (4:1, 2.5 to 5 mL) were placedsuccessively in a 5 mL microwave reactor vial. The resulting sus-pension was heated under microwave irradiation at 80 8C for30 min, water was added (10 mL), and the aqueous phase was ex-tracted with AcOEt (3 � 12 mL). The organic phases were combined,dried over Na2SO4, and concentrated in vacuo. Traces of coppersalts were removed by filtration over a short pad of silica gel withelution with CH3CN/H2O/NH4OH (15:0.5:0.5), and the residue wasthen purified by flash chromatography (SiO2, CH2Cl2/MeOH 100:0to 98:2) to give the desired iminosugar.

General procedure B—deacetylation reaction : Amberlite resinIRA 400 (OH�) (6 g per mmol Ac) was added to a solution of acety-lated iminosugar in a mixture of MeOH and H2O (1:1), and the solu-tion was stirred for 4 h with the aid of a rotary evaporator (at at-mospheric pressure). The resin was then filtered off and washedwith methanol and water. The solvents were then removed underreduced pressure to give the desired deprotected iminosugar.

a-1-C-(6-Azidohexyl)-2,3,4-tri-O-acetyl-1,5-dideoxy-1,5-tert-but-oxycarboxylimino-d-xylitol (29): Acetic anhydride (1.7 mL) and di-methylaminopyridine (3.3 mg, 0.027 mmol, 0.15 equiv) were addedto a solution of a-1-C-(6-azidohexyl)-1,5-dideoxy-1,5-tert-butoxy-carboxylimino-d-xylitol (33,[29] 64 mg, 0.178 mmol) in pyridine(1.7 mL). The solution was stirred overnight. Water (2 mL) wasadded at 0 8C. Saturated NaHCO3 solution (a few mL) was added,and the phases were separated. The aqueous phase was extractedwith AcOEt (3 � 5 mL). The combined organic layers were washedwith saturated NaHCO3 solution, dried over Na2SO4, filtered, andconcentrated. The residue was purified by automatic flash columnchromatography (AcOEt/petroleum ether 1:5 to 1:3) to afforda pale yellow oil (67 mg, 77 %) as a mixture of rotamers accordingto NMR data. [a]20

D =++10.5 (c = 1, CHCl3) ; 1H NMR (300 MHz, CDCl3):

d= 1.11–1.39 (m, 6 H; H-7, H-8, H-9), 1.39–1.48 (m, 9 H; H-3’), 1.48–1.66 (m, 4 H; H-6, H-10), 1.94–2.06 (m, 9 H; C(O)CH3), 2.69 (br q, J =11.3 Hz, 1 H; H-5b), 3.23 (t, J = 6.6 Hz, 2 H; H-11), 4.09–4.65 (m, 2 H;H-5a, H-1), 4.68–5.01 (m, 2 H; H-4, H-2), 5.25 ppm (t, J = 9.8 Hz, 1 H;H-3); 13C NMR (75 MHz, CDCl3): d= 20.8, 20.9 (C(O)CH3), 24.7, 25.2,25.4, 26.5, 26.7, 28.3 (C-3’), 28.9, 39.0, and 40.1 (C-5), 51.4 (C-11),52.5 and 53.5 (C-1), 69.3, 69.8, 70.4, 71.0, 71.06 (C-2, C-3, C-4), 81.1(C-2’), 153.3 and 153.4 (C-1’), 169.4, 169.6, 169.76, 169.83, 169.9,170.4 ppm (C(O)CH3); IR (neat): n= 2098 (N3), 1753, 1699 cm�1 (C=O); HRMS (ESI): m/z calcd for C22H36N4NaO8 : 507.243 [M+Na]+ ;found: 507.239.

a-1-C-(6-Azidohexyl)-2,3,4-tri-O-acetyl-1,5-dideoxy-1,5-imino-d-xylitol (24): Trifluoroacetic acid (1.2 mL) was added to a solution of29 (136 mg, 0.280 mmol) in CH2Cl2 (4.5 mL), and the mixture wasstirred for 2 h at RT. It was then concentrated to dryness, the resi-due was dissolved in CH2Cl2 (10 mL), and the organic phase waswashed with saturated NaHCO3 solution (2 � 10 mL), dried overNa2SO4, filtered, and concentrated. The residue was purified byflash column chromatography (CH2Cl2/MeOH 100:0 to 95:5) toafford 24 (83 mg, 77 %) as a pale yellow oil. [a]20

D =�3.0 (c = 1,CHCl3) ; 1H NMR (300 MHz, CHCl3): d= 1.17–1.47 (m, 8 H; H-6, H-7,H-8, H-9), 1.47–1.58 (m, 2 H; H-10), 1.58–1.68 (m, 1 H; NH), 1.94–2.08 (m, 9 H; C(O)CH3), 2.88 (dd, J = 14.3, 5.1 Hz, 1 H; H-5a), 2.93–2.98 (m, 1 H; H-1), 3.03 (dd, J = 14.3, 3.7 Hz, 1 H; H-5b), 3.20 (t, J =6.8 Hz, 2 H; H-11), 4.64 (br q, J = 4.7 Hz, 1 H; H-4), 4.72 (dd, J = 5.6,3.5 Hz, 1 H; H-2), 5.05 ppm (t, J = 5.5 Hz, 1 H; H-3); 13C NMR(75 MHz, CHCl3): d= 20.8, 21.0 (3 C; C(O)CH3), 25.9, 26.6, 28.8 (2 C),29.1 (C-6, C-7, C-8, C-9, C-10), 44.1 (C-5), 51.4 (C-11), 53.7 (C-1), 68.3(C-3), 68.7 (C-4), 70.2 (C-2), 169.3, 169.8, 169.9 ppm (C(O)CH3); IR(neat): n= 3342 (N�H), 2096 (N3), 1744 cm�1 (C=O); HRMS (ESI): m/zcalcd for C17H29N4O6 : 385.208 [M+H]+ ; found: 385.209.

a-1-C-[6-(4-Propyl-1 H-1,2,3-triazol-1-yl)hexyl]-1,5-dideoxy-1,5-tert-butoxycarboxylimino-d-xylitol (34): Pent-1-yne (48 mL,0.488 mmol, 5 equiv) was added to a solution of a-1-C-(6-azidohex-yl)-1,5-dideoxy-1,5-tert-butoxycarboxylimino-d-xylitol (33,[29] 35 mg,0.098 mmol) in THF (1 mL), followed by copper sulfate pentahy-drate (3.3 mg, 0.010 mmol, 0.1 equiv) and sodium ascorbate(5.8 mg, 0.03 mmol, 0.2 equiv) dissolved in water (1 mL). Fourhours later, the reaction mixture was filtered over celite and thesolvents were evaporated. The residue was purified by flashcolumn chromatography (CH2Cl2/MeOH 90:10 and 1 % NH4OH) toafford 34 as a pale yellow oil (37 mg, 89 %) and as a mixture of ro-tamers according to NMR data. [a]20

D =++2.5 (c = 1, MeOH); 1H NMR(300 MHz, MeOD): d= 0.96 (t, J = 7.3 Hz, 3 H; H-16), 1.17–1.39 (m,6 H; H-7, H-8, H-9), 1.38–1.50 (m, 10 H; H-6a, H-3’), 1.61–1.81 (m,3 H; H-15, H-6b), 1.88 (br quintuplet, J = 6.7 Hz, 2 H; H-10), 2.49–2.70(m, 1 H; H-5a), 2.66 (t, J = 7.6 Hz, 2 H; H-14), 3.22–3.32 (m, 1 H; H-4),3.35–3.44 (m, 2 H; H-3, H-2), 3.91–4.15 (m, 1 H; H-5b), 4.17–4.30 (m,1 H; H-1), 4.35 (t, J = 7.1 Hz, 2 H; H-11), 7.72 ppm (s, 1 H; H-12);13C NMR (75 MHz, MeOD): d= 14.0 (C-16), 23.8 (C-15), 24.7 and24.8, 26.6 and 26.9, 27.5, 28.3 (C-14), 28.6 (C-3’), 29.6 and 29.7, 31.3(C-6), 43.0 and 44.2 (C-5), 51.2 (C-11), 55.8 and 57.1 (C-1), 71.6 and71.9, 72.8 and 73.0, 75.9 (C-2, C-3, C-4), 81.5 (C-2’), 123.1 (C-12),149.0 (C-13), 156.7 ppm (C-1’) ; IR (neat): n= 3380 (O�H), 1686 cm�1

(C=O); HRMS (ESI): m/z calcd for C21H38N4NaO5 : 449.273 [M+Na]+ ;found: 449.274.

a-1-C-[6-(4-Propyl-1 H-1,2,3-triazol-1-yl)hexyl]-1,5-dideoxy-imino-d-xylitol (7): Trifluoroacetic acid (0.3 mL) was added dropwise at0 8C to a suspension of 34 (37 mg, 0.087 mmol) in water (0.6 mL).The mixture was stirred for 5 h at RT and then concentrated to dry-ness. The product was dissolved in water, and IR 120 (H+) resinwas added. After 15 min of stirring, the resin was filtered and

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washed with water and methanol. The resin was then eluted withNH4OH solution (6 %) and concentrated, and 7 was obtained asa white solid (18 mg, 64 %). [a]20

D =�8.8 (c = 1, MeOH); 1H NMR(400 MHz, MeOD): d= 0.96 (t, J = 7.4 Hz, 3 H; H-16), 1.27–1.52 (m,7 H; H-7, H-8, H-9, H-6a), 1.52–1.62 (m, 1 H; H-6b), 1.68 (sextuplet,J = 7.4 Hz, 2 H; H-15), 1.90 (br quintuplet, J = 7 Hz, 2 H; H-10), 2.66(t, J = 7.5 Hz, 2 H; H-14), 2.76–2.87 (m, 1 H; H-5a), 2.92 (br t, J =6.8 Hz, 1 H; H-1), 2.99–3.12 (m, 1 H; H-5b), 3.52–3.61 (m, 2 H; H-2, H-4), 3.74–3.82 (m, 1 H; H-3), 4.36 (t, J = 7.1 Hz, 2 H; H-11), 7.72 ppm(s, 1 H; H-12); 13C NMR (100 MHz, MeOD): d= 14.0 (C-16), 23.8 (C-14), 26.7, 27.3, 28.3 (C-15), 30.1, 31.2, 47.5 (C-5), 51.2 (C-11), 55.6 (C-1), 70.3, 70.9, and 71.5 (C-2, C-3, C-4), 123.1 (C-12), 149.0 ppm (C-13); IR (neat): n= 3321 cm�1 (O�H); HRMS (ESI): m/z calcd forC16H31N4O3 : 327.240 [M+H]+ ; found: 327.238.

Preparation of 25 : Compound 25 (32.5 mg, 80 %) was preparedaccording to General procedure A, starting from 2,2-bis(prop-2ynyloxymethyl)-3-(prop-2-ynyloxy)propan-1-ol (23,[28] 7.1 mg,0.029 mmol) and iminosugar 24 (36.1 mg, 0.094 mmol, 3.3 equiv).[a]20

D =�2.0 (c = 1, CHCl3) ; 1H NMR (300 MHz, CHCl3): d= 1.19–1.51(m, 24 H; H-6, H-7, H-8, H-9), 1.82–1.94 (m, 6 H; H-10), 2.05 (s, 9 H;C(O)CH3), 2.06 (s, 9 H; C(O)CH3), 2.07 (s, 9 H; C(O)CH3), 2.84–3.01 (m,6 H; H-1, H-5a), 3.05 (dd, J = 14.4, 3.6 Hz, 3 H; H-5b), 3.50 (s, 6 H; H-15), 3.61 (s, 2 H; H-17), 4.31 (t, J = 7.2 Hz, 6 H; H-11), 4.57 (s, 6 H; H-14), 4.67 (br q, J = 4.7 Hz, 3 H; H-4), 4.74 (dd, J = 5.5, 3.4 Hz, 3 H; H-2), 5.07 (t, J = 5.4 Hz, 3 H; H-3), 7.51 ppm (s, 3 H; H-12); 13C NMR(75 MHz, CHCl3): d= 21.0, 21.1 (3 C; C(O)CH3), 26.0, 26.5, 28.9, 29.0,30.3 (C-6, C-7, C-8, C-9, C-10), 44.2 (C-5), 45.3 (C-16), 50.4 (C-11),53.7 (C-1), 64.6 (C-17), 65.1 (C-15), 68.3 (C-3), 68.7 (C-4), 70.2 (C-2),70.4 (C-14), 122.3 (C-12), 145.2 (C-13), 169.3, 169.9, 170.0 ppm(C(O)CH3); IR (neat): n= 1742 cm�1 (C=O); HRMS (ESI): m/z calcd forC65H103N12O22 : 1403.730 [M+H]+ ; found: 1403.751.

Preparation of 11: Compound 11 (23.3 mg, 100 %) was preparedaccording to General procedure B, starting from 25 (32 mg,0.023 mmol). [a]20

D =�6.2 (c = 1, MeOH); 1H NMR (300 MHz, MeOD):d= 1.21–1.63 (m, 24 H; H-6, H-7, H-8, H-9), 1.84–1.96 (m, 6 H; H-10),2.74 (dd, J = 13.5, 3.9 Hz, 3 H; H-5b), 2.80–2.87 (m, 3 H; H-1), 3.00(dd, J = 13.5, 2.4 Hz, 3 H; H-5b), 3.44 (s, 6 H; H-15), 3.47–3.56 (m,8 H; H-2, H-4, H-17), 3.71–3.77 (m, 3 H; H-3), 4.39 (t, J = 7.1 Hz, 6 H;H-11), 4.53 (s, 6 H; H-14), 7.93 ppm (s, 3 H; H-12); 13C NMR (75 MHz,MeOD): d= 26.9, 27.4, 30.1, 30.7, 31.2 (C-6, C-7, C-8, C-9, C-10), 46.7(C-16), 47.5 (C-5), 51.3 (C-11), 55.5 (C-1), 62.4 (C-17), 65.4 (C-15),70.2 (C-14), 70.7, 71.4, 71.8 (C-2, C-3, C-4), 123.1 (C-12), 149.0 ppm(C-13); IR (neat): n= 3292 cm�1 (O�H); HRMS (ESI): m/z calcd forC47H85N12O13: 1025.635 [M+H]+ ; found: 1025.6441.

Preparation of 27: Compound 27 (33.3 mg, 83 %) was prepared ac-cording to General procedure A, starting from tetrakis(prop-2ynyloxymethyl)methane (26,[30] 6.3 mg, 0.022 mmol) and iminosu-gar 24 (37.2 mg, 0.097 mmol, 4.4 equiv). [a]20

D =�7.0 (c = 1, CHCl3);1H NMR (300 MHz, CHCl3): d= 1.21–1.58 (m, 32 H; H-6, H-7, H-8, H-9), 1.80–1.95 (m, 8 H; H-10), 2.04–2.10 (m, 36 H; C(O)CH3), 2.92–3.14(m, 8 H; H-5, H-1), 3.45 (s, 8 H; H-15), 4.30 (t, J = 7.2 Hz, 8 H; H-11),4.54 (s, 8 H; H-14), 4.66–4.74 (m, 4 H; H-4), 4.75–4.81 (m, 4 H; H-2),5.09 (t, J = 5.1 Hz, 4 H; H-3), 7.55 ppm (s, 4 H; H-12); 13C NMR(75 MHz, CHCl3): d= 20.9, 21.0, 21.1 (3 C; C(O)CH3), 25.9, 26.5, 28.8,29.0, 30.3 (C-6, C-7, C-8, C-9, C-10), 44.1 (C-5), 45.4 (C-16), 50.3 (C-11), 53.8 (C-1), 65.2 (C-15), 67.9 (C-3), 68.3 (C-4), 69.3 (C-14), 69.7 (C-2), 122.5 (C-12), 145.4 (C-13), 169.2, 169.89, 169.93 ppm (C(O)CH3);IR (neat): n= 1742 cm�1 (C=O); HRMS (ESI): m/z calcd forC85H133N16O28 : 1825.947 [M+H]+ ; found: 1825.940.

Preparation of 14: Compound 14 (13.4 mg, 100 %) was preparedaccording to General procedure B, starting from 27 (18 mg,

0.01 mmol). [a]20D =�6.2 (c = 1, MeOH); 1H NMR (300 MHz, MeOD):

d= 1.28–1.64 (m, 32 H; H-6, H-7, H-8, H-9), 1.86–1.98 (m, 8 H; H-10),2.76 (dd, J = 13.5, 3.7 Hz, 4 H; H-5b), 2.82–2.89 (m, 4 H; H-1), 3.02(dd, J = 13.5, 2.5 Hz, 4 H; H-5b), 3.45 (s, 8 H; H-15), 3.50–3.57 (m,8 H; H-2, H-4), 3.73–3.80 (m, 4 H; H-3), 4.40 (t, J = 7.0 Hz, 8 H; H-11),4.51 (s, 8 H; H-14), 7.93 ppm (s, 4 H; H-12); 13C NMR (75 MHz,MeOD): d= 26.9, 27.4, 30.2, 30.8, 31.3 (C-6, C-7, C-8, C-9, C-10),46.4 (C-16), 47.5 (C-5), 51.3 (C-11), 55.5 (C-1), 65.4 (C-15), 69.9 (C-14), 70.7, 71.4, 71.8 (C-2, C-3, C-4), 124.9 (C-12), 146.1 ppm (C-13);IR (neat): n= 328 cm�1 (O�H); HRMS (ESI): m/z calcd forC61H108N16NaO16: 1343.802 [M+H]+ ; found: 1343.801.

Preparation of 30 : Heptakis(2,6-di-O-propargyl)cyclomaltohep-taose (28,[30b, c] 19.9 mg, 0.012 mmol), 29 (89 mg, 0.184 mmol, 1.1 �14 equiv), CuSO4·5 H2O (4.2 mg, 0.017 mmol, 0.1 � 14 equiv), andsodium ascorbate (6.6 mg, 0.033 mmol, 0.2 � 14 equiv) in DMF/H2O(2.2:0.5 mL) were placed in a 5 mL microwave reactor vial. The re-sulting suspension was heated under microwave irradiation condi-tions at 80 8C for 25 min. Water (a few mL) was added, and theaqueous phase was extracted with AcOEt (3 � 10 mL). The com-bined organic layers were washed with water (10 mL), dried(Na2SO4), and concentrated. Because the crude mixture showeda mixture of partially clicked product, further iminosugar (2 equiv)was added in the presence of copper sulfate and sodium ascor-bate, as described above. The mixture was again heated under mi-crowave irradiation conditions at 70 8C for 20 min. Water (a fewmL) was added, and the aqueous phase was extracted with AcOEt(3 � 10 mL). The combined organic layers were washed with water(10 mL) and were then dried (Na2SO4) and concentrated. The resi-due was purified by flash chromatography (CH2Cl2/MeOH 98:2 to90:10). In order to remove copper from the product, the residuewas dissolved in CH2Cl2 (1 mL). NH4OH (10 % solution, 3 mL) wasadded, and the mixture was stirred for 15 min. The phases wereseparated, and the aqueous phase was extracted with CH2Cl2 (2 �1 mL). The combined organic layers were dried over Na2SO4 andconcentrated. The residue was again purified by flash chromatog-raphy (CH2Cl2/MeOH 98:2 to 90:10) to give 30 (44.2 mg, 44 %) asa yellow solid and as a mixture of rotamers according to NMRdata. [a]20

D =++20.0 (c = 1.0, CHCl3) ; 1H NMR (400 MHz, CDCl3) d=1.11–1.38 (m, 84 H; H-7’, H-8’, H-9’), 1.44 (s, 126 H; C(CH3)3), 1.50–1.71 (m, 28 H; H-6’), 1.77–1.94 (m, 28 H; H-10’), 1.99 (s, 42 H;C(O)CH3), 2.01 (s, 42 H; C(O)CH3), 2.03 (s, 42 H; C(O)CH3), 2.70 (br q,J = 12.0 Hz, 14 H; H-5’a), 3.40 (t, J = 8.6 Hz, 7 H; H-4), 3.46 (br d, J =9.6 Hz, 7 H; H-2), 3.54–3.84 (m, 21 H; H-5, H-6), 3.91 (t, J = 8.9 Hz,7 H; H-3), 4.10–4.35 (m, 42 H; H-5’b, H-11’), 4.40–4.66 (m, 28 H; H-1’,H-7 or H-10), 4.69–5.07 (m, 49 H; H-1, H-2’, H-4’, H-7 or H-10), 5.25(t, J = 9.8 Hz, 14 H; H-3’), 7.48–7.87 ppm (m, 14 H; H-9, H-12);13C NMR (100 MHz, CDCl3) d= 20.8, 20.87, 20.94 (C(O)CH3), 24.7 (C-6’), 25.4 and 26.7 (C-7’, C-8’), 28.3 (C(CH3)3), 28.9 (C-9’), 30.5 (C-10’),39.0 and 40.2 (C-5), 50.4 (C-11), 51.4 and 52.6 (C-1), 64.9 and 65.0(C-7, C-10), 68.7 (C-6), 69.4 (C-2’ or C-4’), 69.8 (C-5), 70.4 (C-2’ or C-4’), 71.0 (C-3’), 73.4 (C-3), 79.0 (C-2), 81.1 (�C(CH3)3), 83.0 (C-4),101.7 (C-1), 122.9 and 123.7 (C-9, C-12), 144.0 and 144.7 (C-8, C-11),154.3 and 154.5 (N�C=O), 169.5, 169.6, 169.8, and 169.9 ppm(C(O)CH3); IR (neat): n= 3431 (OH), 1750 and 1696 cm�1 (C=O); MS(MALDI-TOF): m/z calcd for C392H603N56O147: 8447.15 [M+H]+ ; found:8447.6.

Preparation of 20 : Compound 30 (44.2 mg, 5.23 mmol) was dis-solved in CH2Cl2 (1.3 mL), and TFA (0.3 mL) was added. The solutionwas stirred for 5 h and concentrated to dryness. The residue wasfiltered through a short silica gel column with CH2Cl2/MeOH (9:1 +1 % Et3N), and the solvent was evaporated. This residue was thendissolved in MeOH/H2O (1:1, 3 mL), and Amberlite resin IRA 400

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(OH�), (1.4 meq mL�1, 0.81 g) was added. The mixture was stirredfor 4.5 h at 27 8C with the aid of a rotary evaporator (at atmospher-ic pressure). After concentration, the product was dissolved againin water and washed with ether to remove minor impurities. Thephases were separated, and the solvent was evaporated to give 20(18 mg, 66 % over two steps) as a white solid. [a]20

D =++16.5 (c = 1,MeOH); 1H NMR (400 MHz, MeOD): d= 1.30–1.48 (m, 98 H; H-7’ toH-9’, H-6’a), 1.48–1.64 (m, 14 H; H-6’b), 1.81–1.99 (m, 28 H; H-10’),2.76 (br d, J = 13.0 Hz, 14 H; H-5’a), 2.81–2.90 (m, 14 H; H-1’), 3.01 (d,J = 13.4 Hz, 14 H; H-5’b), 3.40 (t, J = 8.9 Hz, 7 H; H-4), 3.47–3.56 (m,35 H; H-2’, H-4’, H-2), 3.69–3.98 (m, 42 H; H-6, H-5, H-3’, H-3), 4.33(t, J = 6.2 Hz, 14 H; H-11’), 4.42 (t, J = 6.9 Hz, 14 H; H-11’), 4.48–4.64(m, 14 H; H-7 or H-10), 4.80–4.91 (m, 14 H; H-7a or H-10a, H-1), 5.00(d, J = 12.3 Hz, 7 H; H-7b or H-10b), 7.91 (s, 7 H; H-9 or H-12),8.07 ppm (s, 7 H; H-9 or H-12); 13C NMR (100 MHz, MeOD): d= 26.9,27.4, 30.2, 30.7 (C-6’ to C-9’), 31.3 (C-10’), 47.5 (C-5’), 51.3 (C-11’),55.6 (C-1’), 64.4 (C-11’), 65.2 and 66.1 (C-7, C-10), 70.5 (C-6), 70.7 (C-2’ or C-4’), 71.4 (C-5), 71.9 (C-2’ or C-4’), 73.9 (C-3’), 74.6 (C-3), 81.2(C-2), 84.2 (C-4), 102.5 (C-1), 125.1 and 125.7 (C-9, C-12), 145.3 and145.9 ppm (C-8, C-11); IR (neat): n= 3352 cm�1 (OH); MS (MALDI-TOF): m/z calcd for C238H407N56O77: 5285.11 [M+H]+ ; average found:5286.20.

Preparation of 32 : Compound 32 (108.5 mg, 94 %) was preparedaccording to General procedure A, starting from heptakis(2,3-di-methyl-6-propargyl)cyclomaltoheptaose (31, 43 mg, 0.027 mmol)and iminosugar 24 (80 mg, 0.208 mmol, 1.1 � 7 equiv). [a]20

D =++42.0(c = 1.0, CHCl3) ; 1H NMR (300 MHz, CDCl3): d= 1.12–1.52 (m, 56 H;H-6’, H-7’, H-8’, H-9’), 1.72–1.89 (m, 14 H; H-10’), 2.03 (s, 21 H;C(O)CH3), 2.05 (s, 42 H; C(O)CH3), 2.82–2.99 (m, 14 H; H-5’a, H-1),3.04 (dd, J = 14.2, 3.7 Hz, 7 H; H-5’b), 3.11 (dd, J = 9.8, 3.2 Hz, 7 H; H-2’), 3.35–3.42 (m, 28 H; H-3, OMe), 3.52–3.72 (m, 35 H; H-4, H-6a,OMe), 3.78 (d, J = 8.3 Hz, 7 H; H-5), 3.92 (d, J = 7.6 Hz, 7 H; H-6b),4.26 (t, J = 7.3 Hz, 14 H; H-11’), 4.52 (d, J = 12.2 Hz, 7 H; H-7a), 4.57–4.70 (m, 14 H; H-4’, H-7b), 4.73 (dd, J = 5.6, 3.5 Hz, 7 H; H-2’), 4.97–5.11 (m, 14 H; H-3’, H-1), 7.62 ppm (s, 7 H; H-9); 13C NMR (75.5 MHz,CDCl3): d= 20.9, 21.0 (C(O)CH3), 25.9, 26.5, 28.7, 29.0 (C-6’ to C-9’),30.3 (C-10’), 44.0 (C-5’), 50.2 (C-11’), 53.6 (C-1’), 58.6 (OMe), 61.5(OMe), 64.9 (C-7), 68.2 (C-3’), 68.7 (C-4’), 69.2 (C-6), 70.2 (C-2’), 71.1(C-5), 80.2 (C-4), 81.8 (C-3), 82.1 (C-2), 99.0 (C-1), 122.7 (C-9), 144.9(C-8), 169.2, 169.8 and 169.9 ppm (C(O)CH3); IR (neat): n= 3336(NH), 1746 cm�1 (C=O); MS (MALDI-TOF): m/z calcd forC196H309N28O77: 4287.11 [M+H]+ ; found: 4287.50.

Preparation of 18 : Compound 18 (75 mg, 92 %) was prepared ac-cording to General procedure B, starting from 32 (102.5 mg,0.024 mmol). [a]20

D =++54.0 (c = 1, MeOH); 1H NMR (300 MHz,MeOD): d= 1.07–1.68 (m, 56 H; H-6’ to H-9’), 1.77–2.02 (m, 14 H; H-10’), 2.75 (dd, J = 13.4, 3.5 Hz, 7 H; H-5’a), 2.81–2.90 (m, 7 H; H-1’),3.01 (d, J = 12.6 Hz, 7 H; H-5’b), 3.11 (br dd, J = 9.0, 2.8 Hz, 7 H; H-2),3.42–3.56 (m, 42 H; OMe, H-2’, H-4’, H-3), 3.57–3.70 (m, 35 H; OMe,H-6a, H-4), 3.71–3.86 (m, 14 H; H-3’, H-5), 3.86–4.04 (m, 7 H; H-6b),4.37 (t, J = 6.9 Hz, 14 H; H-11’), 4.54 (d, J = 11.6 Hz, 7 H; H-7a), 4.61(d, J = 12.2 Hz, 7 H; H-7b), 5.05 (br s, 7 H; H-1), 8.01 ppm (s, 7 H; H-9); 13C NMR (75.5 MHz, MeOD): d= 27.0, 27.6, 30.3, 30.7, 31.4 (C-6’to C-10’), 47.5 (C-5’), 51.4 (C-11’), 55.5 (C-1’), 59.0, (OMe), 65.2 (C-7),70.5 (C-6), 70.7 (C-2’ or C-4’), 71.5 (C-3’ or C-5), 71.8 (C-2’ or C-4’),72.3 (C-3’ or C-5), 80.8 (C-4), 83.0 (C-2), 83.4 (C-3), 99.6 (C-1), 125.2(C-9), 145.8 ppm (C-8); IR (neat): n= 3378 cm�1 (OH and NH); MS(MALDI-TOF): m/z calcd for C154H266N28NaO56: 3426.87; averagefound: 3426.51.

Fibroblast culture : Type I GD fibroblasts (GM00372) were estab-lished from a patient with GD homozygous for N370S GCase (1226A>G) mutation and obtained from Coriell Cell Repositories,

Camden, NJ. Fibroblasts were cultured in minimum essentialmedium (MEM) with Earle’s salts and nonessential amino acids(GIBCO) supplemented with fetal bovine serum (FBS, 15 %, IrvineScientific) and l-glutamine (2 mm), penicillin (100 U mL�1), andstreptomycin (100 mg mL�1, Irvine Scientific) at 37 8C under CO2

(5 %) in air. Monolayers were passaged on reaching confluencywith trypsin–EDTA (Irvine Scientific). The culture medium was re-placed every two to three days, and all cells used in this studywere between the fifth and the 15th passages.

HL60 culture : HL60 cells were cultured in RPMI1640 mediumsupplemented with FBS (10 %), l-glutamine (2 mm), penicillin(100 U mL�1) and streptomycin (100 mg mL�1) at 37 8C under CO2

(5 %). The culture medium was replaced every two to three days,and all cells used in this study were between the fourth and the16th passages.

b-Glucocerebrosidase inhibition assay : Human placental b-gluco-cerebrosidase was purified from tissue, and activity against gluco-sylceramide was determined as described previously.[22a] Enzymeactivity was measured in the well of a flat-bottomed 96-well(300 mL) plate: inhibitor solution (1 mL), enzyme solution (2 mL) and47 mL substrate solution of 4-methylumbelliferyl-b-d-glucoside incitrate/phosphate buffer (pH 5.2, 0.1 m) containing 0.25 % sodiumtaurocholate, 0.1 % TX100) at 37 8C for 30 min of incubation. Thereaction was stopped by addition of a solution of sodium carbon-ate (0.5 m, 200 mL), and fluorescence was measured by use of anexcitation wavelength of 355 nm and an emission wavelength of460 nm. Inhibition constants were generated for placental b-gluco-cerebrosidase [Km for 4-MU-b-d-glucoside (1.9�0.3) mm] with useof 0.25 to 2 mm substrate concentrations for Ki determinations and0.5 mm substrate concentration for IC50 determinations. All experi-ments were performed in triplicate, and nonlinear regression wasperformed with Prism software. Standard deviations were obtaineddirectly from Prism software for Ki values.

Determination of IC50 values : Percentage inhibition was plottedagainst the log of the inhibitor concentration, and a nonlinear re-gression was fitted with the aid of Prism software. The IC50 valuefor each compound was calculated from the value of the log ofthe inhibitor concentration at 50 % inhibition of enzyme activity foreach three series of data, and the mean value of IC50�SD wasdetermined with Microsoft Excel.

Determination of Ki values : Ki values were determined by use ofa suitable range of substrate and inhibitor concentrations. Velocityvalues were plotted against [S] and subjected to nonlinear regres-sion by use of Prism software. The different inhibition type modelswere tested on the substrate/velocity curves, and the inhibitionmode fitting best with experimental data was selected, providingKi�SD. Lineweaver–Burk plots (1/v against 1/[S]) were also drawnwith the aid of Prism software reflecting Km and Vm values foundfrom the nonlinear regression to show the inhibition mode visually.

Cytotoxicity assays : A CellTiter 96 aqueous non-radioactive cellproliferation assay kit (Promega) was used to measure the effect ofeach compound on cell proliferation by the manufacturer’s proto-col. HL60 cells were seeded in 96-well plates at 5 � 104 cells perwell and were incubated for three days in the presence of variousconcentrations of inhibitor. An aliquot (40 mL) of MTS-phenazinemethosulfate mixture (20:1) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-oxymethoxyphenyl)-2-(4-sulfophenyl-2H tetrazolium) was added toeach well, and the samples were incubated for 3–4 h. The absorb-ance was read at 490 nm. Experiments were performed in tripli-cate, and the cytotoxic concentrations (CC50s), at which absorbance

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at 490 nm was 50 % of that of an untreated control, were deter-mined.

b-Glucocerebrosidase activation assays : Having been distributedover 96-well plates, the cells were incubated for three days withvarious inhibitor concentrations up to 50 mm. Cells were washedtwice in phosphate-buffered saline and incubated for three extrahours with fresh medium. All enzyme activation measurementswere performed by adding homogenate substrate solution [50 mLper well, 4-methylumbelliferyl-b-d-glucoside (5 mm) in citrate phos-phate buffer (pH 5.2, 0.1 m) containing 0.25 % sodium taurocholate,0.1 % TX100)] at 37 8C for 3 h incubation. The reaction was stoppedby addition of glycine (1 m, 50 mL) in sodium hydroxide solution(1 m) before measurement of the b-glucocerebrosidase activity. Thefluorescence was measured with use of an excitation wavelengthof 355 nm and an emission wavelength of 460 nm. Enzyme activa-tion is defined as the fold increase in enzyme activity in treatedcells relative to untreated cells. Results were plotted with Prismsoftware. All experiments were performed in triplicate, and themean values�SDs were determined with the aid of Prism soft-ware.

Acknowledgements

This work was supported by the Institut Universitaire de France(IUF), the CNRS (UMR 7509), the University of Strasbourg, theAgence Nationale de la Recherche (ANR, grant numbers 08JC-0094-01 and 11-BS07-003-02), and doctoral fellowships from theFrench Department of Research to C.D. The authors express theirgratitude to Michel Schmitt for NMR measurements.

Keywords: Gaucher disease · iminosugars · multivalency ·pharmacological chaperones · prodrugs · structure–activityrelationships

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Received: July 5, 2013

Published online on && &&, 0000

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FULL PAPERS

A. Joosten, C. Decroocq, J. de Sousa,J. P. Schneider, E. Etam�, A. Bodlenner,T. D. Butters, P. Compain*

&& –&&

A Systematic Investigation ofIminosugar Click Clusters asPharmacological Chaperones for theTreatment of Gaucher Disease

The right combination : Combining pro-drug and multivalent strategies has ledto an unprecedented pharmacologicalchaperone. A trivalent acetylated imino-sugar is able to increase mutant N370Sb-glucocerebrosidase (GCase) activitylevels by therapeutically relevantamounts, by as much as threefold incells, and at lower concentrations thanrequired with the corresponding depro-tected analogue.

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