Synthesis of modified 1,5-imino-d-xylitols as ligands for lysosomal β-glucocerebrosidase

Abstract Modified 1,5-dideoxy-1,5-imino-d-xylitol analogues with different substitution patterns involving position C-1 and/or the ring nitrogen were prepared, which were designed to serve as precursors for the preparation of iminoxylitol-based ligands and tools for the elucidation and modulation of human lysosomal β-glucocerebrosidase. Biological evaluation of the synthesized glycomimetics with a series of glycoside hydrolases revealed that these substitution patterns elicit excellent β-glucosidase selectivities. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00706-019-02427-1) contains supplementary material, which is available to authorized users.


Introduction
Iminoalditols, also termed iminosugars, are natural occurring glycomimetics, in which the ring oxygen of the carbohydrate moiety is replaced by a trivalent basic nitrogen.
Paradigmatic structural scaffolds are polyhydroxylated piperidines 1, pyrrolidines 2, indolizidines 3, and pyrrolizidines 4 ( Fig. 1) [1][2][3][4][5]. The nitrogen in the endocyclic position is responsible for the unique biological behavior of this compound class to interact and modulate active site specifically glycoside-processing enzymes. Since the last decades, such compounds have been of great interest for an interdisciplinary scientific community, including chemists, biochemists, as well as physicians.
Many different naturally occurring structures are known, exceeded by the number of synthetic derivatives, with manifold different modification patterns concerning the carbohydrate scaffold as well as customized derivatisations for different applications. This substance class has been implicated as potential therapeutic agents [6], for example, as immunomodulators [7,8], as antibacterial [9,10], antiviral [11,12], anti-cancer [13], and anti-fungal [14] agents. In addition, iminoalditol-based glycomimetics have been Dedicated to Professor Dr. Heinz Falk on the happy occasion of his 80th birthday anniversary.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0070 6-019-02427 -1) contains supplementary material, which is available to authorized users. identified as plant growth inhibitors [15]. An interesting field of application has emerged when iminoalditols have been applied at sub-inhibitory concentrations to act as proteinfolding templates [16,17] for mutant lysosomal enzymes, thus becoming candidates for the management of lysosomal storage disorders in the pharmacological chaperone therapy [18]. Moreover, this compound class has received great attention as probes for activity-based profiling of glycosideprocessing enzymes [19][20][21].
The d-xylo configuration in the dideoxy iminoalditol scaffold has been shown to have very interesting ligand properties for glycoside-processing enzymes in terms of activity as well as selectivity [22]. Various modifications with respect to substituents as well as positions on the iminoxylitol scaffold have been synthesized and biologically investigated. Basically all of these compounds have been shown to be highly selective ligands for β-glucosidases. For example, fluorinated iminoxylitols carrying an N-alkyl group [23] (Fig. 2), such as compound 5, have been found to exhibit immunosuppressive as well as glycosidase inhibitory activities. Based on Lehmann's early finding [24], iminoxylitols bearing a guanidino or urea function at the ring nitrogen [25,26], for example compound 6, were synthesized and found to be selective inhibitors of human lysosomal β-glucocerebrosidase (GCase) with IC 50 values in the low nm range. A deficiency of this enzyme causes Gaucher disease [27]. We have synthesized iminoxylitols modified at the endocyclic ring nitrogen with functionalized alkyl groups, such as compound structure 7 [28], as well as featuring more sophisticated substituents, including structure 8 [29]. These compounds exhibited inhibitory properties against β-xylosidase from Thermoanaerobacterium saccharolyticum (Xyl Therm. sac.), with K i values in the lower µm range (Table 1).
Martin and co-workers have developed elegant synthetic routes towards 1-C-alkyl imino-d-xylitols 9 (Fig. 3), and showed that the introduction of the substituent at position C-1 improved the ligand properties as well as the  [30][31][32]. In addition, in a structure-activity study, the influence of the position of an alkyl chain has been investigated, showing that a 1-2 shift of the alkyl substituent from C-1 to O-2 (compounds 10a-10b) increased the inhibitory property of the respective compound against GCase by a factor of 2 [33]. The same group has also synthesized 1,5-dideoxy-1,5-iminod-xylitol (DIX) derivatives with alkyl substituents similar to ceramide at position C-1, for example compound 11, and obtained highly potent GCase inhibitors which also showed selective chaperone properties for mutations associated to Types 1 and 2 Gaucher Disease [34]. Compain and co-workers synthesized a library of 1-C-triazolylalkyl side chain-modified DIX analogues (Fig. 4), including compounds 12, by a click chemistry approach, and found that some of these are GCase enhancers for selected Gaucher disease genotype mutants [35,36].
Withers and co-workers developed a thiol-ene reaction sequence for rapid assembly of 1-C-alkyl DIX derivatives containing a sulfur atom between the DIX scaffold and the lipophilic substituent (Fig. 5), such as compounds 13, and also found excellent ligand properties in terms of activity as well as selectivity for GCase furnishing promising potent and selective pharmacological chaperones for GCase mutants [37].
Overkleeft and co-workers included into their structure-activity relationship study of lipophilic glycomimetics various d-xylo configured 1-C-iminosugar glycosides, for example compounds 14 (Fig. 6), and could demonstrate that these glycomimetics significantly exceed in terms of inhibitory activity as well as selectivity for GCase compared to the corresponding of d-gluco as well as l-ido configured analogues [38].
We have developed a convenient synthetic protocol for the modification of the DIX scaffold at position C-1 taking   advantage of the Staudinger/aza-Wittig/nucleophile reaction sequence [39,40]. By this method, we have synthesized a range of simple C-1 alkyl modified DIX analogues 15 ( Fig. 7) and have found the same trend for these compounds, which are highly selective ligands for GCase. All DIX derivatives carrying a substituent at position C-1, 9-15, have been found to be locked in the 1 C 4 conformation when the alkyl substituent is introduced from the β-face at the pseudoanomeric center (B, Fig. 8). The hydroxyl groups at positions O-2, O-3, and O-4 are in an axial orientation and the substituent at position C-1 is equatorial due to a piperidine ring inversion under acidic conditions such as in the lysosomal environment. In contrast, ring nitrogen substituted DIX derivatives, 5-8, are found in the typical 4 C 1 conformation (A, Fig. 8). This might be an explanation for the exceptional ligand properties as well as the selectivity of C-1-substituted DIX derivatives, which has been observed previously by others and us for similar alkyl-iminoxylitols [30,34,[36][37][38][39][40].
We are interested in the synthesis of iminosugar-based glycomimetics as tools for profiling and as ligands for modulating GCase activity. Consequently, we want to develop a simple and convenient approach towards N-modified DIXbased building blocks locked in the 1 C 4 conformation which carry a substituent suitable for further modifications for

Results and discussion
For this study, we had to take two considerations into account. We wanted to investigate which modification pattern is best for ligand properties, modification at position C-1 or at the ring nitrogen. In addition, we were looking for a suitable functional group at the terminus of the handle which would allow further functionalisation for different applications, including introduction of reporter groups such as fluorescent dyes or click chemistry features. We decided to introduce either an ester group or an imidazole residue. Both functional groups have been found to be suitable for ligand properties of GCase [34,36].
For the introduction of the histamine moiety (Scheme 2), imine 18 was protected with a carboxybenzyl group (Cbz) at the ring nitrogen to give compound 20. The terminal ester group was saponified employing NaOH to furnish 1-C-propionic acid derivative 21, which was used without purification for the coupling step employing histamine dihydrochloride, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylaminomorpholinocarbenium hexafluorophosphate (COMU) and N,N-diisopropylethylamine (DIEA) as coupling cocktail to give protected (1R)-1-C-(imidazo-4-yl) ethylaminocarbonylethyliminoxylitol 22 in 75% yield. Final deprotection under hydrogenolytic conditions gave the imidazole-modified iminoxylitol 23 in a yield of 78%. As expected, also this compound features the 1 C 4 conformation according to the NMR analysis, coupling constants of protons along the sugar ring exhibit characteristic values in the range of 3-5 Hz.
To install the same modification patterns, an ester as well as the imidazole group, at the ring nitrogen, the double bond in protected 1-C-ethenyliminoxylitol 16 was reduced employing Pd/BaSO 4 as catalyst under hydrogen atmosphere (Scheme 3). Under the same reaction conditions, the N-Cbz protecting group was cleaved off to give benzyl-protected (1R)-1-C-ethyliminoxylitol 24 in 81% yield. Introduction of the methoxycarbonylpentyl group at the ring nitrogen was achieved by employing methyl 6-iodohexanoate and sodium carbonate as base in DMF to give N-alkylated iminoxylitol 25 in 67% yield. No formation of a quaternary ammonium ion by double alkylation of the ring nitrogen has been observed during this reaction. Final deprotection of the benzyl groups under hydrogenolytical conditions gave (1R)-1-C-ethyl-N-methoxycarbonylpentyliminoxylitol (26) in 88% yield. Also compounds 24-26 were found in the 1 C 4 conformation exclusively, due to NMR analysis. Likely, the ethyl group at the position C-1 is being responsible for this finding.
The introduction of the imidazole moiety was conducted accordingly to the synthesis of compound 23 (Scheme 4). Saponification of the methyl ester of compound 25 followed by coupling of the histamine moiety led to protected imidazole-modified iminosugar derivative 27. Final debenzylation by hydrogenolysis gave (1R)-1-C-ethyl-N-(imidazo-4-yl)ethylethylaminocarbonylpentyliminoxylitol (28) in 86% yield. Accordingly, all compounds in this series were also found to adopt the 1 C 4 conformation by NMR analysis. The coupling constants of protons along the sugar ring exhibit characteristic values in the range of 3-5 Hz as are typical for this conformation.
For the biological evaluation of the synthesized DIX derivatives 19, 23, 26, and 28, we have probed a series of standard glycoside hydrolases, including β-glucosidase from Agrobacterium sp. (ABG), β-galactosidase from E. coli, Fabrazyme (commercial recombinant human lysosomal α-galactosidase), α-glucosidase S. cerevisiae, and human β-glucocerebrosidase GCase, to investigate ligand activity as well as selectivity. All compounds were found highly selective inhibitors of β-glucosidases and showed practically no detectable interaction with α-glucosidase (S. cer.), β-Gal (E. coli), as well as human α-Gal (Fabrazyme), respectively, confirming the findings of other groups mentioned above. Both imidazole-modified compounds, 23 (K i value 1.1 µM) as well as 28 ( GCase compared to the ester-modified iminoxylitols 19 (K i value 5.1 µM) and 26 (57 µM). Concerning our question regarding the modification pattern, we have obtained a very clear picture: 1-C-modified iminoxylitols 19 and 23 did not distinguish in their ligand properties between β-Glu from ABG and GCase with K i values in the same low µM range. In contrast, the ring nitrogen-modified compounds 26 and 28 showed excellent selectivity, with K i values of 57 and 4.1 µM, respectively, for GCase. No detectable inhibition of 26 as well as 28 was found with the other enzymes investigated, including β-Glu ABG. This increase in selectivity might be explained by the fact that compounds 26 and 28 combine the advantages of both features, the ethyl group at position C-1 locking the structure in the 1 C 4 conformation as well as the lipophilic substituents at the ring nitrogen. The former has been implied for favorable ligand properties and the fitting into the active site of GCase. The latter interacts with the lipophilic entrance to the active site of GCase mimicking the ceramide residue of the natural substrate glucosyl ceramide. Compounds 26 and 28 will serve as building blocks for further functionalisation as proposed.

Conclusion
We have investigated which position of substitution at the iminoxylitol scaffold for the introduction of further modifications is favorable, the ring nitrogen or position C-1. Therefore, we have synthesized two compounds in both patterns, one carrying a terminal ester group, compounds 19 and 26, and the other presenting an imidazole motif, compounds 23 and 28, for further modification. All four compounds were biologically evaluated with a series of standard glycosidases including human lysosomal β-glucocerebrosidase (GCase). Compounds 19 and 23, with the modification at position C-1 of the DIX scaffold, showed excellent selectivity towards β-glucosidases; however, both did not discriminate β-Glu from ABG and human lysosomal GCase, with K i values found in the low µM range. Compounds 26 and 28, carrying the modifications at the ring nitrogen and additionally an ethyl group at position C-1, turned out to interact exclusively with human lysosomal GCase with K i values of 57 and 4.1 µM, respectively. No detectable inhibition for any other enzyme included in this study has been observed. Thus, DIX-based scaffolds 26 and 28 are excellent building Kinetic studies were performed at 37 °C in an appropriate buffer using a known concentration of enzyme (specific conditions depicted below). K i determinations were performed using the corresponding 4-nitrophenyl α-or β-Dglycopyranoside as substrate. In a typical assay, the enzyme was incubated with different inhibitor concentrations for up to 5 min before initiating the reaction by the addition of substrate. The initial reaction rate was measured by monitoring the increase in absorbance at 400 nm for up to 10 min. K i determinations were performed using at least two different substrate concentrations. For each inhibitor, a range of four-to-six inhibitor concentrations bracketing the K i value ultimately determined was used for each substrate concentration. Dixon plots (1/v vs. [I]) were constructed to validate the use of the competitive inhibition model. The data were then fitted using non-linear regression analysis with Grafit 7.0. Specific assay conditions for each enzyme: Agrobacterium sp. β-glucosidase was expressed and purified recombinantly in E. coli as previously described [43]: 50 mM sodium phosphate buffer (pH 7) using 1.85 × 10 −4 mg/cm 3 of enzyme (K m = 4.1 mM) [41,42]; E.coli lac z β-galactosidase (Sigma-Aldrich): 50 mM sodium phosphate, 1.0 mM MgCl 2 (pH 7) using 6.4 × 10 −4 mg/cm 3 of enzyme (K m = 60 μM); Fabrazyme (acid α-galactosidase, generously gifted by Dr Lorne Clarke, Department of Medical Genetics, University of British Columbia): 20 mM sodium citrate, 50 mM sodium phosphate, 1.0 mM tetrasodium EDTA, 0.25% v/v Triton X-100 ® , and 0.25% w/v taurocholic acid buffer (pH 5.5) using 5 × 10 −5 mg/cm 3