Searching for Glycomimetics That Target Protein Misfolding in Rare Diseases: Successes, Failures, and Unexpected Progress Made in Organic Synthesis

Abstract

This account describes our efforts aimed toward the discovery of original glycomimetics that target protein misfolding to fight rare diseases, with a focus on cystic fibrosis and Gaucher disease. The pursuit of this goal has led to promising leads and strategies, with the first description of a multivalent effect for correcting protein-folding defects in cells, and has also driven unexpected progress in synthetic methodology.

1 Introduction

2 The Therapeutic Targets: Challenges and Stakes

2.1 Glycosphingolipid Lysosomal Storage Disorders

2.2 Cystic Fibrosis

3 Synthetic Targets Met, Potent Leads Gained

3.1 Synthetic Targets Met

3.2 A First Detour via a Substrate Reduction Therapy Approach

3.3 Potent Leads Gained

4 Synthetic Targets Missed, Synthetic Progress Gained

4.1 A New Domino Reaction

4.2 Extending the Scope of C–H Amination

4.3 A New Versatile Amino Protecting Group

5 Multivalency: A New and Promising Approach

5.1 How It Began

5.2 Gaucher Disease

5.3 Cystic Fibrosis

6 Conclusion

Biographical Sketch

Professor Philippe Compain was born in Savigny-sur-Orge, 20 km south of Paris (France). He gained his engineer degree in chemistry at CPE Lyon. In 1998, he was awarded the Dina ­Surdin Prize from the French Chemical Society for his Ph.D. research on the synthesis of spiro alkaloids by way of 1,2-chirality transfer, conducted in the group of Professor J. Goré at the University of Lyon I. After a postdoctoral stay at Montreal (Canada) with Professor S. Hanessian researching on hetero-Diels–Alder reactions, he was appointed Chargé de Recherche at CNRS in the group of Professor O. R. Martin in Orléans. In 2008, he accepted a full professorship at the University of Strasbourg. He is now Professor of Organic Chemistry in that university and at the European Engineering School of Chemistry, Polymers, and Material Science (ECPM), where he heads the Laboratory of Organic Synthesis and Bioactive Compounds (SYBIO). His research interests span from the development of new synthetic methodologies to the synthesis of carbohydrate mimics of therapeutic interest. He is coeditor of the book Iminosugars: From Synthesis to Therapeutic Applications (Wiley-VCH). In 2010, Professor Compain has been made a junior member of the Institut Universitaire de France (IUF).

‘I love fixed ideas, but only if they can be changed.’ ­François Jacob[1]

Introduction

Many genetic diseases are directly caused by defects in trafficking of mutant misfolded proteins, which are recognized in the endoplasmic reticulum (ER) by means of the ER quality-control system (ERQC),[2] and finally degraded by the proteasome. These so-called traffic-related diseases or misfolding diseases are linked to more than 25 disorders,[3] including rare genetic diseases such as cystic fibrosis and glycosphingolipid lysosomal storage disorders (GLSDs). A promising course of action for fighting these disorders is based on the fact that in many cases the mutant protein is functional when it is being trafficked to its correct cellular location. In this context, it has been recently demonstrated that low-molecular-weight compounds, named correctors, can overcome processing defects of mutant proteins by disturbing strategic components of the ERQC machinery.[4] For example, Becq and co-workers reported in 2006 that N-Bu DNJ (1), a potent inhibitor of key trimming ER glucosidases, restores the trafficking and function of misfolded cystic fibrosis transmembrane regulator (CFTR) from the most common mutation that causes cystic fibrosis, F508del-CFTR (Figure [1]).[5]

CFTR is a cyclic adenosine monophosphate (cAMP) dependent and ATP-gated chloride ion channel present at the apical membrane of epithelial cells.[6] The processing defect can also be overcome by the action of small molecules that bind directly to the mutant proteins, as demonstrated brilliantly in the late 1990s by the seminal work of the groups of Fan and Asano in the field of GLSDs.[7] GLSDs are a group of rare diseases characterized by a deficiency of glycosidases involved in the catabolism of glycosphingolipids in the lysosome.[8] The strategy, now referred to as the pharmacological chaperone therapy (PCT), is based on the use of competitive inhibitors of lysosomal glycosidases that are capable of enhancing enzyme residual hydrolytic activity at subinhibitory concentrations.[9] [10] This counterintuitive approach can be rationalized in terms of the fact that although the defective enzymes are predisposed to misfolding and/or instability, they remain catalytically active. Reversible competitive inhibitors alter or stabilize the three-dimensional architecture of the defective proteins, preventing their premature degradation by the ERQC system before trafficking to lysosomes.[9,10] The pharmacological chaperone (PC) concept challenges the imagination of the chemist in that the protein, a massive biomolecule, is ‘rescued’ by its low-molecular-weight ligand, a much smaller molecule.

From a synthetic chemist’s point of view, a common factor between the strategies aimed at fighting cystic fibrosis (CF) and those for fighting GLSDs is the need to design potent and selective glycosidase inhibitors. Depending on the disease being considered, the inhibitors either act directly as template molecules that bind to the misfolded glycosidases (GLSDs), or they act indirectly as inhibitors of trimming glucosidases involved in the ERQC machinery (CF). Glycomimetics are well positioned to provide a rich source of inspiration for the design of potent glycosidase inhibitors.[11] These structurally altered analogues of carbohydrates are designed to simulate the shape and most of the functionalities of the natural substrates in the ground state or the transition state (Figure [1]).[12] Beyond fine-tuning of affinity and selectivity, structural modifications are also performed to improve drug-like properties such as bioavailability or stability towards endogenous degradative enzymes.

In addition to its interest in the context of drug discovery, the field of glycomimetics is a fantastic playground for a synthetic chemist who is interested in biology. First, in terms of design, the glycochemical space is wide open, with imagination being the only limit! Designing new structures that emulate one of the major classes of biomolecules is a strong driving force for synthetic chemists. Art is, indeed, in the DNA of chemistry. As claimed by ­Marcellin Berthelot as early as 1860, ‘Chemistry creates its own object. This creative ability, similar to an art, is the main feature that distinguishes chemistry from natural and humanitarian sciences.’[13] In this way, in addition to rational thinking, the conscious or unconscious quest for molecular beauty[14] constitutes a part of the design process. Beyond consideration of structure–activity relationships, aesthetic criteria,[14] including simplicity[15] and symmetry, are likely to inspire the mind of a chemist building plastic ball-and-stick models in his effort to create new bioactive molecules. The second element that might attract synthetic chemists to the field of glycomimetics is grounded in the intrinsic structural features of carbohydrates. The high density of asymmetric centers and functional groups present in their structures are a fertile ground for accidental discoveries of new synthetic methodologies. These structural features also provide a formidable testing ground for known reactions, the scope of which has usually been defined on simple substrates in the first place. This is even more true in the case of glycomimetics that have additional potentially reactive centers, such as iminosugars in which the endocyclic oxygen atom of the parent glycoside is replaced by a nitrogen atom (Figure [1]).[16] [17] Total synthesis of complex natural products such as vitamin B₁₂ or taxol has always been an exceptional source of innovation in organic chemistry.[18] Unfortunately, in practice such herculean projects involve time-consuming and expensive work that is well beyond the resources of most academic chemistry laboratories. One advantage of glycomimetics as target molecules is that they offer enough structural complexity to permit accidental discoveries in synthesis while being accessible in a reasonable number of synthetic steps.

This account describes our efforts towards the discovery of potent glycomimetics that target protein misfolding to fight rare diseases, with a focus on CF and Gaucher disease, the most prevalent of the GLSDs. Pursuing this worthwhile goal has given a direction to our research during the last twelve years, like a lighthouse in a foggy night. En route to this objective, we have tried to keep our eyes open to unexpected discoveries in synthetic methodology. We have tried as much as possible to maintain a balance between the therapeutic goals of our research and our will to innovate in organic synthesis. Even if a risky target molecule provides fruitful synthetic challenges, if failure occurs no progress will be made in identifying potent lead compounds.

The Therapeutic Targets: Challenges and Stakes

Glycosphingolipid Lysosomal Storage Disorders

As mentioned in the introduction, GLSDs are a group of rare diseases characterized by a deficiency of lysosomal glycosidases. Defects in the catalytic hydrolytic activity of one of the glycosidases lead to the accumulation of undegraded glycosphingolipid substrates in cell lysosomes, causing mild to severe cell and organ dysfunction (Scheme [1]).[8]

For example, Gaucher disease is caused by a deficiency in β-glucocerebrosidase (GCase), which catalyzes cleavage of the β-glycosidic bond of glucosylceramide (GlcCer) to release ceramide and glucose with retention of configuration (Scheme [2]).[19] Gaucher disease has three clinical subtypes that are linked to particular mutations of GCase. In type I Gaucher disease, the most prevalent form, patients suffer from bone pains, skeletal lesions, anemia, and liver or spleen damage.[20] Types II and III are both neuronopathic. In the acute infantile form (type II), most patients die before they are two years old.

Taken as a group, the prevalence of GLSDs is between one in 8000 and one in 16,000 live births.[21] The incidence of type I Gaucher disease is in the range of one in 40,000 to one in 60,000 live births. Notably, the prevalence is particularly high among Ashkenazi Jews (one in 500 to 1000 live births).[22] Treatments are currently available for only very few types of GLSD. Although more studies are required to fully understand the events leading from a glycosidase deficiency to clinical symptoms, the accumulation of undegraded glycosphingolipids is likely to be a key factor in the initiation and progression of GLSDs.[23] The primary focus for therapeutic strategies has therefore been on reducing the cellular concentration of glycosphingolipids within the lysosome.[24] The first option is to replace the deficient glycosidase by administration of a recombinant protein as an exogenous substitute, a technique known as enzyme replacement therapy (ERT).[25] The second option makes use of small molecules to enhance the activity of the residual enzyme (pharmacological chaperone therapy; PCT)[9] [10] or to inhibit glycolipid substrate biosynthesis (substrate reduction therapy; SRT).[23a,26] The first therapeutic breakthrough with GLSDs was achieved thanks to ERT in the early 1990s with the spectacular clinical and financial success of Cerezyme^©, a recombinant form of β-GCase, used to treat type I Gaucher disease.[25] Since then, other enzyme drugs have been developed and approved for the treatment of Fabry disease and other lysosomal storage disorders, such as Pompe disease and mucopolysaccharidoses I, II, and VI.[25] [27] However, because the enzymes supplied do not cross the blood–brain barrier, this type of treatment has no effect on neuronopathic forms of GLSDs, nor can it reach target cells in the central nervous systems in which substrate storage needs to be alleviated in most glycosphingolipidoses. An additional drawback of this form of treatment is its exorbitant cost. Cerezyme^© has been termed the world’s most expensive drug, costing in excess of $150,000 US per year per patient.[28] The SRT approach, which is based on suppression of the GCase substrate by inhibiting GlcCer biosynthesis, has led to the discovery of N-Bu DNJ (miglustat, Zavesca^©; 1), the first orally administered treatment for a GLSD (Gaucher disease). N-Bu DNJ acts as a potent inhibitor of glucosylceramide synthase, the enzyme that catalyzes the transfer of a glucose moiety from UDP-glucose to the primary hydroxy group of ceramide to yield β-GlcCer (Scheme 10).[23a] [26] Despite this indisputable progress, N-Bu DNJ suffers from several drawbacks.[24b,d] Large doses are required (~300 mg daily), and these lead to serious side effects, such as abdominal pains, that arise mainly from the inhibition of digestive glucosidases. In addition, N-Bu DNJ is recommended only for adults with mild-to-moderate type I Gaucher disease, for whom ERT is not an option. As with ERT, SRT does not improve neurological function in patients with type I Gaucher disease who suffer from mild neurological manifestations.[24d] In this context, PCT holds many advantages for the treatment of GLSDs compared with ERT and SRT, the other established therapies.[29] [30] PCT targets the cause of GLSDs by restoring the hydrolytic activity of the mutated enzymes, whereas the other two therapies reduce the accumulation of undegraded glycosphingolipids by dysregulation of the glycophingolipid metabolic pathway (SRT) or by administration of an exogenous GCase substitute (ERT). PCT combines the benefits of a small-molecule approach, including oral bioavailability and the potential to cross the blood–brain barrier, with the specificity of an enzyme-directed approach. However, the rational design of potent PCs is facing a number of scientific challenges. There are currently two opposing views regarding the molecular basis of pharmacological chaperoning activity. According to the most widely accepted theory,[9,10,31] PCs act at neutral pH in the ER by inducing or stabilizing the proper conformation of the misfolded but catalytically active enzyme, preventing its degradation by the ERQC system. In sharp contrast, Wei et al. have proposed in 2011 that the main role of PCs would be to increase the resistance of a correctly folded but catalytically deficient enzyme to the action of proteases in the acidic lysosomes.[32] Since the report of this theory, few studies have been performed to resolve this conflict,[33] and the debate remains open.

A second challenge is related to the level of enhancement of residual cellular activity achieved with PCs. The key questions are, ‘Is the increase in glycosidase activity sufficient to significantly improve the symptoms associated with GLSDs?’[34] and, ‘How should PCs with improved chaperoning activity be designed, as very few rational criteria are available to date?’. For example, almost all the PCs reported for type I Gaucher disease double to triple the residual cellular activity of N370S GCase whatever their affinity for the mutant enzyme.[9] [10] Treatment with isofagomine (Plicera^©; 2), the leading clinical candidate for PCT for Gaucher disease, led to an increase in enzymatic activity for all patients enrolled in a Phase II trial (Figure [2]).[35] However, isofagomine (2) might not be moved forward to Phase III development, as significant clinical improvements were observed in only one patient out of 18.[35a] This result might indicate that the doubling of the residual GCase activity reported for isofagomine is insufficient for therapeutic application.

Our group entered the field of GLSDs research almost accidently at the very beginning of the PCT story. In 1999, Naoki Asano contacted us by email because he was interested in α-homogalactostatin (3), a compound previously synthesized in our group[36a] (Scheme [3]). In collaboration with Jian-Qiang Fan, Naoki Asano had recently published details of the first example of an active site-specific chaperone for the treatment of GLSDs.[7] This pioneering work demonstrated that the activity of residual α-galactosidase A (α-Gal A) in lymphoblasts of patients with Fabry disease could be significantly enhanced by using 1-deoxygalactonojirimycin (DGJ, Amigal^©; 4; Figure [2]). After an impatient search, we were delighted to find in our laboratory freezer a sample of carbamate 3′, a direct precursor of α-homogalactostatin (3). Unfortunately, on weighing the storage vial, we found that only two milligrams of 3′ were available. The reaction was, nevertheless, performed, and the cyclic carbamate was cleaved by using aqueous sodium hydroxide (Scheme [3]). After purification on an anion-exchange resin, one milligram of pure α-homogalactostatin (3) was obtained, and this amount was eventually enough for a biological evaluation.[36b] [c] This was our first chemical reaction in the field of PCT! α-Homogalactostatin (3) produced a 5.2-fold increase in mutant α-Gal A (R301Q) at a cellular concentration of 100 μM.[36b,c] This was not an improvement in comparison with DGJ (14-fold increase at 100 μM), but the seed was planted. Since then, the search for glycomimetics that target protein misfolding in rare diseases has progressively become our main research project. Our first objective was to target Gaucher disease with a simple design approach based on stable GCase substrate analogues.

In view of the result obtained with DGJ in the field of Fabry disease[7] and the well-known affinity of iminosugars for glycosidases,[16] [17] a general route to iminosugar C-glycosides in the d-gluco series was developed. This work and its development, which led to the identification of a potent PC, is described in Section 3. In the course of our study, N-nonyl-1-deoxynojirimycin (NN-DNJ, 5), the first example of a PC for the treatment of Gaucher disease, was reported in the literature by Kelly and co-workers. [37] The PC properties of DGJ and NN-DNJ triggered an enormous amount of interest in imino analogues of carbohydrates. This was the beginning of a second renaissance in the field, more than twenty years after the discovery by Bayer chemists of the biological activity of DNJ as an α-glucosidase inhibitor. Since 1999, hundreds of small molecules (mainly iminosugars) have been evaluated as PCs for the treatment of GLSDs.[10]

Cystic Fibrosis

Cystic fibrosis (or mucoviscidosis) is the most common life-threatening genetic disease among people of European heritage.[38] In North America and European countries, approximately 68,000 individuals have CF.[39] Most patients suffering from CF have the delF508 mutation on at least one CFTR gene allele. In common with some other traffic-related diseases,[3] the mutant protein is functional but is recognized and degraded by the ERQC machinery, leading to a disturbance in the movement of chloride and sodium ions across membranes such as the alveolar epithelia. In the lung, the reduced ion transport causes surface dehydration and abnormal formation of thick mucus resulting in airway obstruction, recurrent bacterial infection, and permanent inflammation.[40] The average life expectancy of a patient with CF, which was around 14 years in the 1970s, has been extended to 40 years.[41] This progress has been achieved mainly by early diagnosis and by improvements in symptomatic therapy.[41] Heart/lung transplantation is currently considered as the only curative treatment option for CF; as a result, there is the strong interest in the disease as a prime target for pharmacological therapy.[38] [42] [43] The most promising clinical candidates are based on small molecules that target the underlying defect in CF by restoring ion-channel density and function at the membrane.[42] Among the candidate drugs, CFTR correctors rescue functional CFTR channels from degradation by the ERQC machinery, thereby improving the protein trafficking and channel density at the cell membrane. In the early 1990s, the discovery that small molecules such as dimethyl sulfoxide or glycerol,[42] as well as incubation at low temperatures,[44] can promote increased CFTR trafficking, has initiated the search for specific correctors for CFTR.[42] The identification of correctors is nevertheless a tough challenge because, despite many efforts, no corrector mechanism of action has yet been entirely resolved.[42b] [c] Consequently, the identification of CFTR correctors has been largely based on high-throughput screening approaches. In 2005, Pedemonte et al. identified the first effective CFTR corrector, Corr-4a (6), by screening 150,000 chemically diverse compounds and more than 1500 analogues of active compounds (Figure [3]).[45]

Shortly after, Becq and co-workers conjectured that the inhibition of trimming ER-glucosidases might rescue misfolded delF508-CFTR protein by preventing its interaction with calnexin.[5] This ER membrane molecular chaperone assists protein folding and quality control, ensuring that only properly folded proteins proceed further along the secretory pathway.[2] [4] The N-glycan parts of newly synthesized glycoproteins are trimmed by ER-glucosidase I and II to form functional appendices that determine the fate of the associated protein. Calnexin indeed binds only monoglucosylated oligosaccharides, i.e. N-linked glycans of the form GlcMan₉GlcNAc₂–, formed by removal of the two outer glucose units from a 14-oligosaccharide core Glc₃Man₉GlcNAc₂– by the sequential action of glucosidases I and II.[46] After a survey of commercially available α-glucosidase inhibitors, N-Bu DNJ (1), was chosen to test the hypothesis proposed by Becq’s group. This compound was already known to act as an orally bioavailable drug (Zavesca^) for the treatment of type I Gaucher disease, which, in the case of success, would be a major advantage in relation to future clinical evaluation. The glucosidase-inhibition hypothesis turned out to be supported by the experimental results. N-Bu DNJ (1) was shown to prevent the interaction of delF508-CFTR with calnexin and to restore the cAMP-activated chloride current in epithelial CF cells with a half-maximal effective concentration (EC₅₀) of 112 μM after two hours of treatment.[5] [47] On the basis of these promising results and its potential for the chemotherapeutic treatment of CF, N-Bu DNJ (1) entered into Phase II clinical trials soon afterwards.

Fortuitously, we were informed of the very exciting work of Becq’s group in Poitiers (France) before its publication. The link between our two laboratories was an article published in 2005 in Microscoop, a regional journal of the CNRS devoted to the popularization of science. In this article, our efforts towards iminosugar-based chaperones for GLSDs were presented in a very simple way. While skimming through the journal, Frederic Becq noticed our paper and realized that chemists capable of synthesizing original iminosugars were only two hours by car from his laboratory! After a telephone discussion, we decided to send to Poitiers all the deprotected iminosugars that we had on our shelves. Unfortunately, however, none of the compounds, which were mainly DNJ analogues with one to three alkyl chains, were active. Potent submicromolar CFTR correctors were finally discovered in 2012 after a long and tortuous path with an unexpected detour via the concept of multivalency (see Section 5).

Synthetic Targets Met, Potent Leads Gained

Synthetic Targets Met

As potent glycosidase inhibitors, iminosugars and their analogues appear as privileged structures for the development of correctors and PCs for future oral treatments for CF and GLSDs, respectively. Our first objective in the early 2000s was to develop a general stereodivergent approach to stable analogues of azapyranosides to target ER glucosidases and the various glycosidases involved in the glycosphingolipid degradation pathway (Scheme [1]). We therefore designed a versatile synthetic strategy for synthesizing iminosugar C-glycosides[48] with full stereocontrol (Scheme [4]). Our approach was first tested in the synthesis of α- and β-1-C-substituted 1-deoxynojirimycins.[49] The retrosynthetic analysis took advantage of l-sorbose to provide three stereogenic centers (C-2, C-3, and C-4) of the final targets.

To generate diversity from advanced intermediates, we concentrated on three directions: the aglycon part (R), the control of the R/S configuration at the pseudoanomeric center, and the rapid access to the d-galacto and the d-manno series. Our planned synthetic strategy based on two stereocontrolled steps to generate the C-1 and C-5 asymmetric centers worked nicely.[49] Starting from l-sorbose, the first steps of the synthesis were devoted to the isolation and oxidation of the hydroxy group at C-6 by following a protecting group strategy. The first key step of our strategy involved the highly diastereoselective chain extension of imine 7, which controls the α- versus β-configuration at the pseudoanomeric center in the final product. The stereoselectivity was effectively inverted by adding an external monodentate Lewis acid (si-face addition). At this stage, structural diversity can be introduced at the ‘anomeric’ position by using the wide library of organometallic nucleophiles that are available. Intramolecular reductive amination of the latent keto function of the 6(R)-aminosorbose derivatives 8a proceeded with high diastereoselectivity in favor of the pseudo-α-d-gluco products. The facial selectivity of the reduction step can be rationalized in terms of the attack by the incoming hydride along a preferential axial trajectory on the diastereotopic face of the most favored and/or the most reactive half-chair conformation of the cyclic iminium intermediate, in which all the substituents are in pseudo-equatorial positions except for the R group.[49a] In addition to being sterically unhindered, hydride delivery in the axial direction minimizes torsional strain during the transition to the final chair conformation of the piperidine ring. Access to nojirimycin β-C-glycosides from 6(S)-aminosorbose derivatives 8b by following the same process turned out to be more difficult. The inversion of one asymmetric center in the reductive amination substrate led to a complete loss of diastereoselectivity for R = alkyl, leading to an equimolar mixture of the pseudo-β-d-gluco and pseudo-α-l-ido products. The cyclic iminium ion in the ² H ₃ conformation A is, in this case, partially destabilized because of A^1,2 strain between the C-1 substituent and the N-benzyl group (Scheme [5]).

Hydride addition might therefore also occur in the axial direction on the alternative ³ H ₂ conformation B of the iminium ion, thereby leading to a substantial proportion of the pseudo-α-l-ido product. The decisive influence of A^1,2 strain was nicely demonstrated by the dramatic increase in the diastereomeric excess to 70% in the presence of the slightly less sterically demanding vinyl group (sp² instead of sp³ carbon linked to C-1). On the basis of this model, the solution found to increase the stereoselectivity of the reduction toward the desired d-configuration was to suppress A^1,2 strain effects by removing the N-benzyl group before internal reductive amination. Removal of the benzyl protecting groups of the aminosorbofuranose derivatives 8b by hydrogenolysis, cleavage of the isopropylidene group, and reductive amination under classical conditions provided the expected β-1-C-alkyl-1-deoxynojirimycin 11 in good overall yields and high diastereoselectivity (Scheme [4]). A variation on this strategy using an N-allyl protecting group has been developed to provide access to β-C-glycosides derivatives containing an unsaturated R group, such as 12 (Scheme [6]).[50]

To further increase the structural diversity at the aglycone part, we exploited the metathesis reactivity of α-1-C-allyl DNJ analogues.[49c] [50] The cross-metathesis reaction between 13 and various functionalized alkenes proceeded with excellent E/Z selectivity and gave good to excellent yields (Scheme [7]).

It is noteworthy that the first application of cross-meta­thesis reactions to iminosugar C-glycosides led to the ­development of a new amine protecting group, the 2-naphthylmethyl (NAP) group (see Section 4.3).[51] The general strategy for preparing nojirimycin C-glycosides was then adapted and extended to the synthesis of a diversity of iminosugars. By starting from l-xylose instead of l-sorbose, α-1-C-alkyliminoxylitol derivatives 14 were synthesized (Scheme [8]).[52] [53]

A stereodivergent synthesis of N-alkyl 1-deoxyimino­sugars of various configurations (d-gluco, d-galacto, or d-manno) was also developed (Scheme [9]).[54]

One of the main advantages of our general strategy is that the one-pot sequence of acetal deprotection and reductive amination provides properly protected piperidines with two free hydroxy groups tactically positioned at C-2 and C-4. These two hydroxy groups were efficiently differentiated by using piperidindiol 15. Highly regioselective protection of the less hindered hydroxy group at C-2 was achieved by using an excess of tert-butyl(dimethyl)silyl chloride and imidazole to give piperidinol 16 (Scheme [9]).[54]

From this key intermediate, access to the d-galacto or d-manno series was achieved by way of a two-step sequence involving Swern oxidation followed by diastereo­selective reduction with sodium borohydride or l-selectride. By taking advantage of our general strategy, we synthesized the new iminoglycolipids 17–21 (Figure [4]). A series of iminosugars bearing two or three alkyl groups were designed as ceramide mimics and analogues of N-Bu DNJ (1) by following an SRT approach for the treatment of Gaucher disease (see Section 3.2).[55] We also synthesized iminodisaccharides 21 as substrate mimics of GCase, in the search for efficient PCs (see Sections 3.2 and 3.3).[56]

A First Detour via a Substrate Reduction Therapy Approach

In 2000, we read a very interesting article by Butters et al. in the field of SRT.[57a] They suggested that N-Bu DNJ (1), a potent inhibitor of glucosylceramide synthase, was, somewhat unexpectedly, a mimic of ceramide 22, and not of the glucose moiety (Scheme [10]).[57]

The first evidence supporting this statement was that the inhibition of glucosylceramide synthase by 1 is competitive with respect to ceramide and not UDP-glucose. Molecular modelling had also revealed a strong structural homology between the ceramide structure and 1.[57] The N-alkyl chain and three chiral centers (C-2, C-3, and C-5) of iminosugar 1 show structural similarities with the N-acyl chain and the C-1′–C-3′ backbone, respectively, of ceramide 22. This rational model suggested very clearly that N-Bu DNJ (1) would be a better ceramide mimic if a second alkyl chain were present at O-2 or C-1 to simulate the second hydrophobic chain of the ceramide. By adopting our general synthetic strategy, we prepared a small library of N-alkyl iminoglycolipids 17–20 bearing one or two alkyl chains at C-1, O-2, and/or O-4 (Figure [4]).[55] With this series of compounds, the influence of the position and the length (C₄ or C₈) of the alkyl chains on glucosylceramide synthase inhibition was investigated in the laboratory of Terry Butters at the Oxford Glycobiology Institute. We were eager to see the results of the biological assays because we expected that better ceramide mimics would lead to inhibitors of glucosylceramide synthase that were more potent and more selective than N-Bu DNJ (1). In addition, four years had passed between the idea and the result. It required a long time to obtain funding for the project, hire the Ph.D. student, synthesize the compounds, and obtain the biological results. Disappointingly, biological evaluation of the iminoglycolipids 17–18 clearly indicated that the addition of a second alkyl chain at O-2 or C-1 led to less-potent inhibitors of glucosylceramide synthase (Table [1]).[55]

Table 1 Inhibition of Glucosylceramide Synthase

Imino sugar	n	R1	R2	R3	IC50 (μM)
1 17a 17b 18a 18b 20a 20b 19b	2 2 6 2 6 2 6 6	H H H Bu (CH2)7Me H H H	H Bu (CH2)7Me H H H H (CH2)7Me	H H H H H Bu (CH2)7Me (CH2)7Me	20 NIa 164 609 174 NI 134 NI

^a NI: no inhibition.

The strongest inhibition was found for N-octyl DNJ derivatives bearing a second octyl chain at C-1, O-2, or O-4, which showed IC₅₀ values in the hundreds of micromolar range (seven- to ninefold less potent than N-butyl DNJ!).[58] Quite surprisingly, comparison of the IC₅₀ values of the dialkylated iminosugars 17b, 18b, and 20b showed that the glucosylceramide synthase active site does not discriminate among inhibitors differing in the position of the second octyl chain (α-C-1, O-2, or O-4). Two years later, by following a PCT approach, we further challenged the hypothesis of N-Bu DNJ (1) being a ceramide mimic by designing the iminodisaccharides 21a and 21b as substrate-like inhibitors of GCase (Figure [5]).[56] Iminoglycolipid 17a, considered in this study as a mimetic of the ceramide moiety of the GCase substrate, displayed no inhibitory activity. Addition of a glucosyl moiety at C-4 to give 21a (designed as an analogue of glucosylceramide) resulted in a marked improvement in affinity for GCase (IC₅₀ = 56 μM). In contrast, although N-butyl DNJ (1) displayed better inhibition than 17a towards GCase,[59] the pseudodisaccharide 21b showed no inhibitory activity at 100 μM against GCase. The biological results obtained with compounds 17a and 21a partly validated our initial design hypothesis and demonstrated that the conjugation of a d-glucose moiety with an iminosugar-based mimetic of ceramide should enhance binding toward GCase. However, as discussed in Section 3.3, our attention then became focused on nanomolar GCase inhibitors that were identified by means of studies on structure–activity relationships.

Potent Leads Gained

Having developed a stereocontrolled strategy for the synthesis of imino-C-glycosides (see Section 3.1), we began our search for potent inhibitors of GCase on the basis of structure–activity relationship studies, taking N-Bu DNJ (1) as a starting point for optimization (Table [2]).

Table 2 Evaluation of DNJ and Analogues as Glycosidase Inhibitors

			IC50 (μM)
Enzyme	DNJ (23)	N-Bu-DNJ (1)	R = Bu (11a)	R = Bu (10a)	R = (CH2)8Me (10b)
α-glucosidase
rice	0.05	0.42	61	11	1.5
yeast	190	NIa	NI	465	56
maltaseb	0.36	2.1	210	12	4.0
sucraseb	0.21	58	155	3.6	2.5
isomaltaseb	0.3	2.7	115	0.15	0.0035
β-glucosidase
GCase	240	270	NI	100	0.27
sweet almond	81	NI	NI	NI	150
Caldocellum saccharolyticum	55	NI	NI	NI	80

^a NI = less than 50% inhibition at 1 mM.

^b From rat intestine

The results clearly showed that a simple 1,2-shift of the alkyl chain from the endocyclic nitrogen to the ‘anomeric’ carbon having a β-configuration was detrimental to the inhibition towards glycosidases. In contrast, α-1-C-butyl DNJ was found to be a slightly more selective glycosidase inhibitor than N-Bu DNJ and to display better inhibition towards GCase and rat intestinal isomaltase.[49c] [52] We were then pleased to observe that the potency of α-1-C-alkyl 1-deoxynojirimycins as GCase inhibitors increased regularly with the length of the alkyl chain, reaching IC₅₀ values of the order of hundreds of nanomoles for α-1-C-nonyl DNJ (10b; Table [2]).[49c] [52] To summarize these first results in a simple way, the addition of a nonyl chain having an α-configuration to the ‘anomeric’ carbon of DNJ led to a 890-fold more potent GCase inhibitor, with increased selectivity towards all glycosidases other than rat intestinal isomaltase. On the basis of this observation, we evaluated simple analogues of DNJ 23 and we found that 1,5-dideoxy-1,5-imino-d-xylitol (24, DIX) is about 100-fold more potent than DNJ, with an IC₅₀ of 2.3 μM (Scheme [11]).[52]

In addition, preliminary biological studies indicated that DIX (24) was a very specific inhibitor of GCase and lacked inhibitory activity against α-glucosidases. This latter point is very important as the main side-effects associated with iminosugar-based drugs arise from the inhibition of digestive α-glucosidases. On the basis of these findings, we were eager to synthesize α-1-C-nonyl-iminoxylitol (14a). If biology is viewed through the lens of simple mathematics, combining the two effects (DIX scaffold plus nonyl chain at C-1) should have led to an inhibitor 89,000-fold more potent than DNJ, displaying IC₅₀ values in the low nanomolar range. α-1-C-Nonyl-iminoxylitol (14a) was prepared quite quickly by taking advantage of a strategy for the preparation of 1,6-dideoxy-l-nojirimycin, developed a short time previously by our group (Scheme [8]).[60] Compound 14a and its analogues, including N-nonyl DIX (25) and α-1-C-dodecyl DIX (14b), were sent to the laboratory of Naoki Asano in Japan. A few weeks later we received the biological results, and these turned out exactly as we had expected (or, rather, had hoped). Iminosugar 14a was found to be a potent nanomolar inhibitor of GCase (K _i = 2.2 nM), displaying no significant inhibitory activity towards all the α-glucosidases evaluated (Scheme [12]).[52] This compound is still one of the strongest known inhibitors of GCase.[61] Extension of the alkyl chain by three additional methylene groups (from nonyl to dodecyl) to give 14b slightly weakened the inhibition of GCase (K _i = 14 nM). We hypothesized that the qualitative leap in inhibitory potency and selectivity between 10b and 14a might be due to a piperidine ring inversion from a classical ⁴ C ₁(D) conformation to a ¹ C ₄(D) conformation. NMR spectroscopy showed that compound 14a exists predominantly in an inverted ¹ C ₄(D) conformation in which all the hydroxy groups are axial and the alkyl chain is equatorial, whereas the conformation of its N-nonyl analogue 25 is ⁴ C ₁(D). This observation might explain the large difference in inhibitory activity between the two regioisomers 14a and 25. This difference is indeed absent in the case of N-nonyl DNJ (5) and α-1-C-nonyl-1-DNJ (10b), both of which have the same ⁴ C ₁(D) conformation (Scheme [12]).

With the hope of further improving the inhibitory activity of our lead compounds, we prepared analogues of 14a having an l-ido configuration, as the inverted ¹ C ₄(D) conformer is even more strongly favored in this series.[62] Biological evaluation showed clearly that the iminoiditols 26 were much weaker inhibitors than their analogues 14 in the d-xylo series. For example, β-1-C-hexyl-1-deoxyimino-l-iditol (26b) is about 2300-fold less potent on GCase than is its analogue 14d in the d-xylo series (Table [3]).[62]

Table 3 Evaluation as GCase Inhibitors of 1-C-Alkyl Iminosugars in the l-ido, d-gluco, and d-xylo Series

	IC50 (μM)
	n = 2 26a	n = 5 26b	n = 2 10c	n = 5 10d	n = 2 14c	n = 5 14d
GCase	487	43	400	4.2	0.56	0.019

These results might be explained by a detrimental effect of the hydroxymethyl chain on the affinity for GCase, as β-1-C-alkyl iminoiditols 26 and α-1-C-alkyl DNJs 10 are both significantly less active than the corresponding analogues 14 in the d-xylo series. The chaperoning activity of our lead compound, α-1-C-nonyl-DIX (14a), was then evaluated. This compound does indeed act as a pharmacological chaperone and is able to double the residual cellular activity of GCase in N370S fibroblasts from Gaucher patients at an extremely low concentration (10 nM).[52] To get closer to therapeutic application without loss of affinity for human GCase, we decided to simplify our lead structure by shifting the alkyl chain from C-1 to the O-2, O-3, or O-4 hydroxy group of 1,5-dideoxy-1,5-imino-d-xylitol (24). Removal of one stereogenic center (C-1) did indeed result in shorter synthetic sequences and avoided the need for problematic C–C bond-forming steps (Scheme [8]).

In addition, our aim was also to evaluate derivatives with shorter alkyl chains, as cytotoxicity associated with long chains (arising mainly from membrane insertion and pore formation) has been observed with N-alkylated iminosugars.[63] A series of O-alkyl iminoxylitol derivatives were first synthesized from diacetone d-glucose.[64] A structure–activity study showed a dramatic influence of the position of the alkyl chain (α-C-1, O-2, O-3, or O-4) on inhibition of human GCase (Table [4]).

Table 4 Influence of the Position of the Alkyl Chain on the Inhibition of GCase[64]

	R1	R2	R3	R4	R5	IC50 (nM)
	(CH2)5Me H H H H	H (CH2)5Me H H H	H H (CH2)5Me H H	H H H (CH2)5Me H	H H H H (CH2)5Me	19[ 62 ] 9 404,000 1600 >150065

Remarkably, a 1,2-shift of the alkyl chain from C-1 to O-2 was found to maintain both a high inhibitory potency towards GCase and a chaperone activity at subinhibitory concentrations (1.6-fold increase at 10 nM).

In conclusion to this section of the account, our first efforts towards the discovery of potent drugs for treating Gaucher disease led to a new class of promising pharmacological chaperones (Table [5]).

By using a structure–activity relationship approach and taking N-butyl DNJ as a starting point, progress has been made little by little from nonselective micromolar chaperones to iminoxylitol-based compounds that are active at a cellular concentration reduced by three orders of magnitude.

Synthetic Targets Missed, Synthetic Progress Gained

A New Domino Reaction

In connection with the results obtained with iminoxylitol-based pharmacological chaperones (Part 3.3), one of my first objectives when I started a new research group in Strasbourg was to synthesize bicyclic spiranic iminosugars 27 based on 2-azaspiro[3.3]heptane skeleton (Figure [6]). The aim was to further challenge the ‘inverted chair hypothesis’ with constrained analogues of our leads in the d-xylo series. For example, compounds 28 can be viewed as constrained analogues of 14 having a blocked ¹ C ₄ conformation.

Our hope was that these strained derivatives, with the hydroxy groups in a pseudo axial orientation, might display highly specific inhibition in the picomolar range towards GCase, even with shorter alkyl chains. In addition, because the spiranic compounds that were designed constitute an unprecedented class of iminosugars, we wanted to evaluate the impact of constrained conformations on the inhibition of various carbohydrate-processing enzymes. In terms of chemistry, we were also excited by the number of synthetic challenges present in these small molecules, with their four contiguous asymmetric centers, two small cycles, an azaspiranic skeleton, and a high density of functional groups. We were therefore convinced that these structures would drive innovation in synthetic methodology. More-subjective motivations were also at play, because we were attracted by the beauty of such small molecules, which can be described as an attractive mix of symmetry, apparent structural simplicity, and synthetic complexity. We first envisioned a rapid but risky synthetic strategy to access 2-azaspiro[3.3]heptane derivatives by way of a Dieckmann condensation from diester 29.[66] The formation of four-membered cycles by such a process is disfavored by ring strain and has almost no precedent.[67] Our first attempts, performed under various conditions (sodium hydride in tetrahydrofuran or sodium in benzene[67]) failed to give the desired 2-azaspiro[3.3]heptane derivative. Finally, we took advantage of a cationic variant of the Dieckmann reaction involving a silyl ketene acetal generated by treatment of enolizable ester 29 with trimethylsilyl triflate and triethylamine.[68] In our first attempt, the reaction of 29 following Hoye’s protocol[68a] did not lead to the formation of the Dieckmann product but instead gave the functionalized 5-azaspiro[2.4]heptane derivative 30 in good yield and as a single diastereoisomer (Scheme [13]). The structure of 30 and the relative configuration of the two asymmetric centers were unambiguously assigned by X-ray crystallographic analysis of the primary alcohol 31 obtained by selective reduction of the ester group (Scheme [13]).[66] In this efficient one-step process, two rings (a cyclopropane and a γ-butyrolactam) and two asymmetric centers are created. It is noteworthy that the 5-azaspiro[2.4]heptane skeleton thus generated is a motif present in various biologically active molecules including antibacterial and anti-autoimmune agents.[69]

Because of the synthetic interest of this novel domino process, we performed a mechanistic study to rationalize the rather unexpected behavior of 29 under Hoye’s conditions. We believe that the key step of the process is an S_N2-type ring opening[70] of the trimethylsilyl triflate-activated azetidine ring by the silyl ketene acetal generated by treatment with trimethylsilyl triflate and triethylamine (Scheme [14]). The amino ester 32 thus obtained finally undergoes an intramolecular cyclization to afford the 5-membered lactam 30 by reaction of the amine function with the ester group in the γ-position. This reaction proceeds with high regioselectivity, as no formation of a six-membered lactam is detected. The proposed mechanism was supported by experimental evidence.[66] For example, to confirm the S_N2 ring-opening of the azetidine, we have shown that the spirocyclization reaction performed from the enantioenriched azetidine (–)-29 (88% ee) proceeded without loss of the enantiomeric excess.

The spirocyclization process was found to be particularly sensitive to structural variations in the substrate because of the high density of potential reactive centers present in its structure. Interestingly, the addition of a carbonyl group to the azetidine ring to give a β-lactam led to a complete change in reactivity. Under typical spirocyclization conditions, 2-azetidinone 33 provided the 6-azabicyclo-[3.2.0]heptane derivatives 34 and 35 (Scheme [15]). The rather complex structures of 34 and 35 were confirmed at the stage of ketone 36, obtained as a single diastereomer after a two-step sequence. The structure of 36 and the relative configuration of the two asymmetric centers were determined unambiguously by X-ray crystallographic analysis.[66b] The fused bicyclic system might reasonably be formed by a reaction sequence involving the formation of an O-silyl imidate in situ, followed by intramolecular addition to the sterically less hindered ester group (Scheme [15]).

This Mukaiyama aldol-like reaction pathway becomes predominant with β-lactam 33, because the deactivation of the endocyclic nitrogen atom blocks the key azetidine trimethylsilyl triflate-activated ring-opening step. N-tert-Butoxycarbonyl or N-tosyl analogues of azetidines 29 are, indeed, not substrates of the spirocyclization reaction.[66] Remarkably, to our knowledge, the synthesis of 34 and 35 represents the first examples of Mukaiyama aldol-like reactions of N,O-ketene acetals in which the carbonyl reactant is an ester. The limited substrate scope of the spirocyclization reaction was overcome by exploiting the reactivity of the tandem reaction product 30. A range of 5-azaspiro[2.4]heptane derivatives were prepared in one step by way of chemoselective reduction of 30. Reduction of the ester group by treatment with Superhydride^® afforded primary alcohol 31 in high yield, whereas the fully reduced pyrrolidine analogue 37 was easily obtained by using lithium aluminum hydride (Scheme [16]).[66] Primary alcohol 31 was efficiently converted into the methylenecyclopropane derivative 39 via the corresponding bromide 38 in 71% yield (two steps).[66b] [71] The search for a synthetic route to original constrained iminosugars eventually led to spiranic compounds, but not the ones initially foreseen. We had therefore missed our synthetic target,[72] but had discovered a novel, highly stereoselective, tandem intramolecular azetidine ring-opening/closing cascade reaction. In addition, this reaction represents a rare example[73] of a nucleophilic ring-opening of an azetidine without formation of a quaternary azetidinium salt by N-alkylation or the use of N-electron-withdrawing groups.[70]

Extending the Scope of C–H Amination

In 2001, two fascinating research papers in the field of C–H functionalization attracted our attention.[74] [75] On the basis of the pioneering studies of Breslow[76] and the discovery of efficient in situ generation of iminoiodinanes,[74,77] Du Bois and co-workers developed a powerful process for the intramolecular amination of nonactivated C–H bonds by using carbamate or sulfamic ester substrates (Scheme [17]).[74] [75] This was a boom time for the field, with significant mechanistic and synthetic studies independently performed by the groups of Che, Dauban and Dodd, Lebel, He, Müller, and others.[78]

The main advantage of intramolecular C–H amination using carbamate or sulfamic ester substrates resides in the high level of regio- and stereoselectivity that is usually observed for this methodology. The reaction process is stereospecific; intramolecular aminations of enantiopure carbamate or sulfamate substrates proceed with complete retention of configuration at the insertion site.[78] When we began our study in 2002, only five-membered ring product had been obtained from carbamates (Scheme [17]). Amination reactions performed with sulfamate esters generally led to the formation of the corresponding six-membered ring insertion products. The highly favored formation of the oxathiazinane ring can be rationalized in terms of the elongated S–N and S–O bond lengths and the N–S–O angle of the sulfamate (~103°), which match the metrical parameters of the heterocycle.[78] [75] The formation of the oxathiazolidine ring would require an unfavorable compression of this angle to 95°. In the beginning of the 2000s, various studies had shown that electronic factors also play a decisive role in the regioselectivity of the reaction and might influence the size of the ring formed. Benzylic, allylic, and tertiary C–H bonds, as well as sites adjacent to electron-donating groups, were found to be favored. The order of reactivity for C–H bond insertion was roughly formulated as follow: allylic > α-ethereal ≈ 3° > benzylic > 2° >> 1°.[78] Our interest in sulfamic esters as substrates for C–H amination reactions was further increased by the formation of the cyclic oxathiazinane products. These electrophilic azetidine equivalents offer many opportunities for the diversity-oriented synthesis of 1,3-amino-functionalized products.[79] With the aim of extending the scope of PCs to other lysosomal diseases, such as Tay–Sachs or Sandhoff diseases (Scheme [1]), we wanted to develop a general access to mimetics of N-acetylhexosaminidase substrates I based on iminosugar C-glycosides (Scheme [18]). On the basis of the first paper published by Du Bois et al. and the guidelines known at that time for predicting the regioselectivity of the reaction,[74] [75] the intramolecular amination of a sulfamic ester appeared to us to be promising as a double-shot weapon to achieve our synthetic goal.

First, this methodology should theoretically have allowed the regio- and stereoselective formation of the key C–N bond of targets I by amination of the C-2 position of the 2-deoxyiminosugar advanced intermediates II (Scheme [18]). Secondly, the oxathiazinane generated might be subsequently opened by various nucleophiles after activation of the NH moiety,[79] permitting the introduction of structural diversity at the aglycone part. Another advantage of this approach is the late introduction of the amino group at C-2, because it is well known that the intrinsic reactivity of this functional group can be a decisive issue in the preparation of 2-amino-C-glycosides.[80] It is noteworthy that the synthesis of 2-amino imino-C-glycosides is a difficult synthetic challenge. Despite the biological relevance of GlcNAc- and GalNAc-containing glycans and the accumulated expertise in the synthesis of iminosugar C-glycosides,[48] there is still no general synthesis for this class of compounds.[81] To achieve a rapid evaluation of the potential of our synthetic strategy (Scheme [18]), we first performed a model study from piperidine 40 (Figure [7]). The only compound isolated after treatment with (diacet­oxyiodo)benzene, magnesium oxide, and 5 mol% dirhodium tetraacetate was confidently identified to be the oxathiazinane derivative 41 (Figure [7]).

Our interpretation of the NMR spectra was detrimentally influenced by the fact that, at that time (2002), we were convinced that the insertion could occur only at the C-3 position (with formation of a favored six-membered ring) or the C-2 position (with formation of a five-membered ring by insertion into a favored tertiary α-amino C–H bond). This misinterpretation was corrected when we returned to the piperidine series after a model study performed with C-glycosides (Scheme [19]). This mistake served as a good lesson: beware of dogma and wishful thinking; be open to the unexpected! On the basis of the erroneously determined regioselectivity for the C-H amination of the sulfamic ester 40, we decided to evaluate our strategy in more detail with more-complex test substrates, the C-glycosides III (Scheme [19]).

These compounds were structurally close to the key intermediates II, but were more readily accessible. In addition, this model study was interesting in itself. Amination of the C-2 position might open a general route from IV to hexosamine C-glycosides,[80] whereas insertion of a nitrogen atom into the pseudo C–H anomeric bond might yield glycomimetics V containing N,O-acetal structures that might serve as surrogate iminium ions.[82] The study was first performed with sulfamic esters 42 obtained in six steps from tri-O-benzyl-d-glucal (Scheme [20]).[83]

No product corresponding to insertion of a nitrogen atom at C-2 to give a six-membered ring was observed. In addition, C–H amination reactions of sulfamic esters 42 were found to be strongly dependent on the ‘anomeric’ configuration. Under typical C–H amination conditions, oxathiazolidine 43 was obtained in 63% yield from 42β after treatment with di-tert-butyl dicarbonate in pyridine.[84] In sharp contrast, exposure of the α-epimer to (diacetoxyiodo)benzene, magnesium oxide, and dirhodium tetraacetate catalyst gave a mixture of unidentifiable products. The starting material 42α was the only compound that could be isolated by chromatography on silica gel (~12% yield). The results observed for the sulfamate esters 42 prompted us to explore the reactivity of the corresponding carbamates. In sharp contrast to the findings for sulfamates 42, both epimers of carbamate 44 provided the expected insertion products in similar yields (60–63%) (Scheme [21]).[83] Conversion of 44β afforded the spirooxazolidine 45α with an anomeric α-configuration, whereas the corresponding epimer 44α gave a mixture of the two diastereoisomers 45α and 45β. In this latter reaction, because of the small anomeric effect expected for nitrogen substituents and the favorable equatorial position of the carbamoyloxymethyl group, the carbamate 45α was probably generated through equilibration of the initially formed epimer 45β.[83]

The model study performed on C-glycosides 42 and 44 led finally to two puzzling results. Why did the two ‘anomers’ of sulfamic esters 42 react so differently under typical C–H amination conditions (Scheme [20])? Our first thought was to explain this result in terms of an increase in the reactivity of axial anomeric C–H bonds in comparison to equatorial C–H bonds,[85] as this had been shown to occur in a number of reactions, such as radical hydrogen abstraction. However, this rationale did not stand up in the light of the results obtained for the conversion of carbamates 44 (Scheme [21]). The second puzzling point was the distinct regioselectivity obtained in the pyran series and in the piperidine series (Figure [7] and Schemes 20 and 21). In addition, this result called into question our initial strategy for accessing 2-amino imino-C-glycosides I (Scheme [18]). We carefully reexamined our NMR data and the structure of the C–H amination product of sulfamic ester 40, originally proposed as oxathiazinane 41 (Figure [7]), was revised to that of the oxathiazepane 41′ (Scheme [22]). Evidence for structure 41′, which corresponds to the insertion product at C-6, was obtained unambiguously by X-ray crystallographic analysis.[86] Compound 41′ was the first seven-membered ring ever obtained by way of the ­Du Bois reaction. Remarkably, the amination process could be extended to the synthesis of eight-membered rings, as demonstrated by the reaction of sulfamate ester 46 (Scheme [22]).

To rationalize this unexpected result, we decided to study the influence of various structural parameters (Table [6]). Ring size was found to play a key role, because no trace amount of an oxathiazepane product could be detected from pyrrolidine 48 (Table [6], entry 1). Instead, compound 49 corresponding to the open-chain imine form of the C-2 insertion product was isolated in 29% yield from 48. The nature of the endocyclic heteroatom was also found to be critical. In confirmation of the regioselectivity observed with C-glycosides 42 and 44, the C–H amination reaction of the simple pyran and furan derivatives 50 and 52 led exclusively to products corresponding to insertion into the C–H bond at C-2 (entries 2 and 3).

Table 6 Study of the Influence of Various Structural Parameters in Rh-Catalyzed C–H Insertion Reactions of Sulfamate Esters

Entrya	Substrate	Product	Yield (%)
1	48 X = NTs; n = 0	49 X = NTs; n = 0	29
2	50 X = O; n = 0	51 X = O; n = 0	65
3	52 X = O; n = 1	53 X = O; n = 1	76b
4	54		45

^a Reaction conditions: CH₂Cl₂, 40 °C, PhI(OAc)₂:MgO:Rh₂(OAc)₄ = 1:1:2.3:0.05.

^b Compound 53 was obtained along with 22% of the corresponding cyclized spiran product.

The amination reaction was then performed with compound 54, which was designed to strongly favor the formation of the six-membered ring (Table [6], entry 4). On the basis of the then-current guidelines for prediction of the regioselectivity of the C–H amination reaction, we expected that insertion into the ethereal C^α–H bonds would be favored. Even in this case, the oxathiazinane derivative 55 was not obtained as the major product, as judged by NMR analysis of the crude reaction mixture (ratio 55/56 ≈ 1:1). In view of the results obtained, it became clear that the regioselectivity of the C–H amination reaction is not controlled solely by electronic factors. This is nicely highlighted by the distinct regioselectivities obtained in the pyran series and the piperidine series. Effectively, according to electronic effect, the most activated C–H bond is the tertiary C–H bond at C-2 in the position α to the endocyclic heteroatom. Regardless of whether we have a nitrogen or an oxygen atom, the observed regioselectivity for the C–H amination product should be the same: insertion at C-2. To explain the marked difference in the observed regioselectivity, we hypothesized that it might be controlled by conformational effects. As predicted and later demonstrated by selective-irradiation NMR experiments, pyran 52 adopts a chair conformation with the sulfamoyl­oxymethyl substituent occupying an equatorial position (Figure [8]).[86]

This conformation makes the addition into the reactive axial C–H bond at C-2 highly favorable and it prevents amination at the C-6 position. In contrast, the conformation of the piperidine ring of 40 is likely to be close to a chair in which the sulfamoyloxymethyl substituent occupies a quasiaxial position to minimize the pseudo A[1] [3] strain caused by the partial double-bond character of the S–N sulfonamide bond.[86,87] This conformation favors insertion into the reactive axial C–H bond at C-6 to form a seven-membered ring in which the observed values for the S–N and S–O bond lengths and the N–S–O angle (~106°) are very close to those obtained for oxathiazinane derivatives.[75] Amination at C-2 is penalized by the formation of a less favored five-membered ring and by the fact that the insertion would occur into a much less reactive equatorial C–H bond.[85] By analogy with the study performed on cyclic ethers by Malatesta and Ingold,[85b] we assume that stereoelectronic weakening of the C–H bond adjacent to the amide nitrogen is maximal when the dihedral angle Φ between the C–H bond and the p-type lone-pair orbital on the nitrogen is 0° and minimal when this angle is 90° (Figure [8]). To confirm our model, we performed the C–H amination reaction with acyclic substrates 57 and 58 to reduce the conformational control of the reaction selectivity (Figure [9]). As expected, compounds corresponding to the formation of a five-membered ring and, more importantly, a seven-membered ring were obtained from the amination reaction of ether 57. Similar results were obtained with tosyl amide 58.[86]

The decisive influence of conformational effects was further highlighted with piperidines and pyrrolidines in which the sulfamoyloxymethyl group was attached to the endocyclic nitrogen with an appropriate linker (Table [7]).[88]

Table 7 Study of the Influence of Various Structural Parameters in C–H Insertion Reactions of Sulfamic Esters 59

Entry	R1	R2	n	Substrate	Product	Yield (%)
1	H	H	1	59a	60a	20
2	–(CH2)2–		1	59b	60b	24
3	H	H	0	59c	60c	47
4	–(CH2)2–		0	59d	60d	86

The better yields obtained in the pyrrolidine systems compared with the piperidine systems was explained by the fact that the pyrrolidine five-membered ring is more conformationally flexible and therefore better able to accommodate the transition state for C–H insertion.[88] As supported by X-ray crystallographic analysis and ¹H NMR analysis, the more flexible pyrrolidine ring can adopt an averaged planar conformation in which the two geminal α-amino C–H bonds are both activated, with an average Φ value of ~30° (Figure [10a]).

The piperidine ring of compounds 59a and 59b is expected to adopt a well-defined chair conformation. Additional conformational constraints are introduced by the amide conjugation, which induces a planar arrangement of the NC=O group. This stabilized conformation is likely to place the nitrene center in an unfavorable position with respect to the more reactive axial C–H bond at C-2 (Figure [10b]). The yield of C–H amination increased to 86% in the pyrrolidine series on introducing a cyclopropyl group in the spacer arm between the endocyclic nitrogen and the sulfamoyloxy group. This improvement was rationalized in terms of the reactive rotamer concept.[88] Results presented in Table [7] and obtained with tetrahydropyridine 59e, a close analogue of 59a, remarkably highlight the decisive influence of conformational effects, which can dominate electronic effects. The introduction of an electronically favored allylic site was indeed less effective in improving the insertion process (Scheme [23]).

In the light of our studies performed with azacycloalkanes, the puzzling results obtained from the C-glycoside 42β and its α-epimer (Scheme [20]) make more sense. In 42β, the sulfamoyloxymethyl substituent occupies an equatorial position and therefore the oxathiazolidine is readily obtained by C–H insertion into the reactive axial C–H bond at C-1. In 42α, this reacting group is shifted to an axial position and the insertion can occur into two activated α-oxygenated axial C–H bonds at C-3 and C-5, leading to unstable hemiaminal derivatives and, subsequently, degradation. Such regioselectivity could not have been foreseen when we began our study because there was no precedent for the formation of a seven-membered ring by the Du Bois reaction.

Beyond its mechanistic interest, the C–H amination reaction provides access to bicyclic aminals such as 41′ or 60. These compounds, as potential precursors of N-tosyliminium ions, can react with various nucleophiles. In addition to the stereocontrolled formation of a new bond at C-6, the major advantage of this process is the regeneration of the sulfamoyloxy group that can be reused for a further intramolecular C–H amination. On the basis of this starting point, we have devised unique bond-construction strategies in nitrogen-containing heterocycles involving iterative multifunctionalization of non-activated C–H bonds.[89] In this process, the sulfamoyloxymethyl group is used several times as a molecular activating arm, permitting the formation of a C–C or C–N bond or a C=C double bond. We have, for example, designed a strategy in which the sulfamoyloxy activating arm was directly or indirectly involved in the functionalization of every saturated methylene group of a monosubstituted piperidine (Scheme [24]).[89] This approach exploited the reactivity of cyclic enamine 61, which was readily obtained from 41′ by reaction in acetic acid. Iodomethoxylation of the α,β-unsaturated N-tosylpiperidine 61 with molecular iodine and sodium methoxide in methanol, followed by 1,8-diazabicyclo[5.4.0]undec-7-ene-mediated dehydroiodination provided the desired 6-methoxy-tetrahydropyridine 62 with a double bond tactically positioned at C(4)–C(5).

A second rhodium-catalyzed amination to functionalize the allylic C–H bond at C-3 was then performed and gave an 87% yield of oxathiazinane 63 as an advanced intermediate for the general synthesis of polysubstituted piperidines. Nucleophilic additions to the N,O-acetal 63 were found to proceed with good to high diastereoselectivity and with complete regioselectivity. The versatility of bicyclic intermediate 64 was further probed by stepwise functionalization into more-complex structures such as 65 (in racemic form).[89] This amino iminosugar, obtained after a journey in the fascinating world of C–H functionalization, was not the product we originally desired. The synthetic target I was missed this time, but relevant progress was made in the field of metal-catalyzed nitrene insertion reactions. It was shown, for the first time, that seven- or eight-membered rings could be obtained by this methodology, and that a combination of stereoelectronic and conformational effects could be used to control the regioselectivity of the reaction. In 2010, Davis and co-workers provided further examples that highlighted the influence of such effects on the regioselectivity of nitrene insertion reactions of 1,6-anhydropyranose substrates.[90] The first examples of Du Bois reactions leading to the formation of nine- and ten-membered rings have recently been reported.[91] In 2009, despite the well-established preference of carbamates to afford oxazolidinones, the formation of six-membered ring-insertion products was described for the first time by Ranatunga and Del Valle.[92] The unexpected regioselectivity observed with piperidine 40 led eventually to a new original bond-construction strategy based on iterative multifunctionalization of nonactivated C–H bonds in nitrogen-containing hetero­cycles.[89] [93]

A New Versatile Amino Protecting Group

Our general strategy for preparing iminosugar C-glycosides by way of a cross-metathesis reaction from the advanced intermediate 9a (R = allyl) required replacement of the endocyclic amine by a less coordinating group to avoid chelation with the catalyst metal center and the consequent formation of unproductive complexes (Schemes 4 and 7).[50] [94] After various attempts, we found that the N-benzyl group in intermediate 9a could be removed by using four equivalents of cerium(IV) ammonium nitrate in a tetrahydrofuran/water two-phase system[95] to give a 35–50% yield of the iminosugar 66 (Scheme [25]).[50] [94] This was a disappointing result, especially considering that the advanced intermediate 9a was synthesized in a similar yield (~40%), but over 11 steps!

We therefore needed to find a new protecting group for the endocyclic amine that would fulfill the following requirements according to our general synthetic strategy (Scheme [4]): introduction through an imine function, stability under strongly acidic conditions, and chemoselective removal in the presence of alkene and of benzyloxy groups. To achieve this aim, we successfully applied our initial strategy to the 2-naphthylmethyl (NAP)-protected imine obtained from 67, instead of the corresponding N-benzyl derivative 7 (Schemes 4 and 25). Investigation of the chemoselective deprotection of the endocyclic amine in 68 showed that the N-NAP protecting group could be efficiently removed in 89% yield by treatment with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in dichloromethane–methanol (4:1) at room temperature.[51] Further studies on the scope of the deprotection process indicated that the NAP group could be cleaved in the presence of various functionalities such as hydroxy, acetal, alkene, ester, benzyloxy, or N-benzyl groups (Scheme [26]).[51]

In connection with our studies on lysosomal diseases, we were interested in the synthesis of azide-armed α-1-C-alkyl-imino-d-xylitol derivatives to generate multivalent pharmacological chaperones (See Section 5.2). Again, our synthetic strategy was based on a cross-metathesis reaction, and an efficient deprotection of the N-NAP-protected intermediate 69 was required. To our surprise, the removal of the N-NAP protecting group proved difficult.[96] Under typical deprotection conditions (3 equivalents of DDQ in dichloromethane–methanol), the expected amine 70 was obtained in low yield (28%) along with compound 71, formed by a side reaction that had never been observed in our original investigations of the scope of the oxidative cleavage of the N-NAP group (Scheme [27]).[51]

After various attempts, we optimized the cleavage conditions, and the best results were obtained by using a smaller quantity of DDQ (2 equiv) in the presence of water instead of methanol as the nucleophilic cosolvent. Under these conditions, the secondary amine 70 was obtained in 86% yield and could be protected with a tert-butoxycarbonyl group to provide the cross-metathesis substrate and complete the synthesis of 72 (Scheme [27]).[96]

Multivalency: A New and Promising Approach

How It Began

When I arrived in Strasbourg at the end of 2008, my new office was located next door to the laboratory of Jean-François Nierengarten. At that time, his research group was developing an efficient approach for the preparation of fullerene hexakis adducts bearing twelve trimethylsilyl-protected alkyne groups.[97] [98] Multivalency has always attracted the interest of chemists, especially those working in the field of glycoscience.[99] In glycobiology, many key recognition events are indeed mediated by interactions between multimeric carbohydrate-binding proteins, called lectins, and their matching carbohydrate ligands. On the basis of numerous studies over the last decades,[99] chemists have obtained spectacular results with synthetic glycoclusters that exhibit enhancements in affinity of up to six orders of magnitude over those of the corresponding monovalent ligands,[100] an increase known as the cluster or multivalent effect. It was highly tempting to take advantage of C₆₀ fullerene scaffolds to prepare, by means of click chemistry, unique globular platforms presenting twelve iminosugar motifs. In addition, for subjective reasons, we were strongly attracted by the beautiful structure of fullerene iminosugar balls. These symmetrical molecules are like the collision of two different worlds: the third form of carbon and highly polar glycomimetics! Despite the considerable interest in iminosugars as potent inhibitors of glycosidases and the publication of a number of studies in this field,[11] [16] [17] there were very few examples of multivalent analogues of this class of compounds in the literature before 2009.[101] In contrast to lectins, glycosidases can appear to be unpromising targets for multivalent binding, as they usually possess a single substrate-binding site. This causes the major mechanism underlying the multivalent effect, the so-called chelate effect, to become inoperative, as this effect involves the binding of multivalent ligands to oligomeric receptors.[102] However, in 1999, in a paper mainly devoted to the synthesis of a combinatorial library of iminosugars, Bols and co-workers reported the first attempt to investigate the effect of multivalency on glycosidase inhibition.[101c] A promising affinity enhancement was obtained with tetravalent aza­fagomine 74 for the inhibition of sweet almond β-glucosidase (Figure [11]). Unfortunately, this effect could not be quantified because 73, the corresponding monovalent analogue of 74, was an extremely weak inhibitor of the enzyme (K _i > 1000 μM).[101c]

At the beginning of 2009, the first systematic evaluation of multivalent iminosugars as glycosidase inhibitors was reported by Gouin, Kovensky, and co-workers.[103] This work led to a demonstration of a small but quantifiable inhibitory multivalent effect. Among the panel of di- to trivalent analogues of DNJ screened, compound 77 (Figure [12]) was found to display the best enhancement, reaching a relative inhibition potency toward jack bean α-mannosidase ­(JBα-Man) of six compared with the corresponding monovalent analogue (two on a molar basis).[103]

On the basis of the simplistic idea that the multivalent effect should increase with increasing valency and because of the literature precedents, we were even more eager to evaluate the inhibition of C₆₀-based dodecavalent iminosugars. An efficient synthesis of the DNJ cluster 78 [104] by way of copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) was finally completed, and biological evaluation with a panel of commercial glycosidases began in the laboratory of Carmen Ortiz Mellet at the University of Sevilla. A first exciting result was obtained with baker’s yeast isomaltase (BKisomal) for which an increase in inhibitory potency of almost two orders of magnitude (90-fold) compared with the monovalent model 75 was observed (Figure [12]). We hoped that the best was yet to come, as inhibition studies on jack bean α-mannosidase remained to be done. Shortly after this preliminary evaluation, we received very good news from the University of Sevilla: the 12-valent DNJ cluster 78 was indeed found to display a very strong binding enhancement to α-mannosidase (2150-fold) relative to the corresponding monomer 75.[104] To improve the multivalent effect further and to probe the influence of architectural parameters, we synthesized seven- to fourteen-valent iminosugar derivatives based on a cyclodextrin (β-CD) core.[105] The best compound of the series, the 14-valent DNJ-cyclodextrin conjugate 79 with a C₉ linker, was found to be a nanomolar inhibitor of JBα-Man (Figure [12]). Interestingly, it has been reported very recently that tetravalent DNJ clusters based on a rigid porphyrin scaffold also show a strong multivalent effect (over 200 on a molar basis).[106c] So far, the strongest affinity enhancement observed with binding enhancement close to four orders of magnitude (over 600 on a molar basis) has been obtained with the 14-valent DNJ-cyclodextrin conjugate 79 (Table [8]).

Besides optimization and the rationalization[106] of the inhibitory multivalent effect, our next logical step was to target glycosidases of therapeutic interest with a focus on GCase and ER-glucosidases in connection with our therapeutic goals (see Section 3.3).

Gaucher Disease

We first evaluated the GCase inhibitory potency of seven- to fourteen-valent DNJ click clusters based on a β-CD core with two different alkyl spacer lengths (C₆ or C₉) (Figure [13]).[107] The best results were obtained with the seven-valent iminosugar 81 with the longer spacer. This compound was found to be 150-fold more potent as GCase inhibitor than the corresponding monomer 83, as judged from the IC₅₀ values (IC₅₀ = 8 nM versus IC₅₀ = 1200 nM). In addition, DNJ cluster 81 was found to act as a pharmacological chaperone, increasing residual GCase activity by 53% in N370S fibroblasts from Gaucher patients at 10 μM. Although the chaperoning activity of the corresponding monovalent ligand 83 is slightly higher (60% increase at 10 μM), the ability of seven-valent DNJ cluster 81 to access the ER and/or to the lysosomes was unambiguously demonstrated by the significant enhancement of GCase activity in Gaucher fibroblasts. This was the first example of a multivalent pharmacological chaperone.

Based on this promising preliminary study, a library of three- to fourteen-valent systems with various ligands (DNJ or DIX), alkyl spacer lengths (C₆ or C₉), and scaffolds (β-CD or pentaerythritol) were synthesized by way of CuAAc.[108] Systems of lower valency (three- to four-valent), as well as acetylated analogues designed as potential prodrugs, were prepared with the aim of facilitating cellular uptake. Small but significant multivalent effects in GCase inhibition (~2 on a molar basis) were observed for only two of the iminosugar clusters: the four-valent DIX cluster 84 and the DNJ–cyclodextrin conjugate 81.[109] However, in this study, the best chaperoning effect observed for a deprotected iminosugar cluster (a 3.3-fold increase at 10 μM) was obtained in the DNJ series with the tetravalent compound 87, a submicromolar GCase inhibitor (Figure [14]). This systematic structure–activity relationship study also provided the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of the trivalent iminosugar 85 to give the corresponding peracetylated prodrug 86 gave a pharmacological chaperon with higher enzyme activity increases (3.0-fold instead of 2.4-fold) at cellular concentrations reduced by one order of magnitude.[108] On the basis of these preliminary results, we hope that future studies combining prodrug and multivalent strategies will provide a new generation of potent pharmacological chaperones for the treatment of lysosomal diseases.

Cystic Fibrosis

The impact of multivalency on F508del-CFTR defective trafficking was evaluated with a library of three- to fourteen-valent DNJ clusters with various alkyl spacer lengths (C₆ or C₉) and scaffolds (C₆₀, β-CD, or pentaerythritol). In the design part of this project, we liked to consider these multivalent iminosugars as ‘super miglustat’ molecules. These compounds indeed gave ‘super’ results, far beyond our initial expectations. The best correcting effects were obtained for tri- to tetravalent DNJ clusters.[110] For the trivalent iminosugar 85, the multivalent potency enhancement was found to be 75- and 330-fold per DNJ unit, as compared with N-Bu DNJ (1) and the corresponding monovalent model 76, respectively (Scheme [28]).

By virtue of multivalency, a clinical candidate displaying an EC₅₀ value in the high micromolar range, had therefore been transformed into a submicromolar CFTR corrector. Western blot analysis showed unambiguously that trivalent iminosugar 85 and its monovalent analogue 76 restored the mature fully glycosylated CFTR to the plasma membrane via the Golgi apparatus. Studies were performed to get a first picture of the mechanisms underlying the impressive multivalent effect observed in the CFTR-correction activity.[110] On the basis of our initial design hypothesis, we logically first evaluated whether the strong efficiency enhancement observed with the trivalent iminosugars 85 was due to a multivalent effect in inhibition of ER-glucosidases I and II. Quite surprisingly, the trivalent iminosugars 85 was found to be significantly less ­potent than the corresponding monovalent inhibitor 76 or N-Bu DNJ (1) in cellular assays.[110] Competition experiments were then performed to assess a possible different mechanism of action for the trivalent iminosugar 85 and the corresponding monovalent analogue 76. The absence of potentiation observed when cells were co-treated with 85 and 76 indicated that the two iminosugars share a similar mode of action in correction. Further competition experiments with several known inhibitors of the biosynthetic pathway and of the ER quality machinery demonstrated that the mono- and trivalent iminosugar-based correctors 85 and 76 both show a calnexin-dependent mechanism of action.[110]

The evaluation of a library of DNJ clusters has led to the first description of a multivalent effect for correcting protein-folding defects in cells. We hope that these results will provide the basis for a new paradigm in the field, as they demonstrate that multivalency can be a simple and powerful option for producing potent misfolded protein correctors.

Conclusion

The search for small molecules that target protein misfolding in lysosomal diseases and cystic fibrosis has led to promising leads and new strategies. In the field of Gaucher disease, nanomolar pharmacological chaperones have been identified by adopting a classical structure–activity relationship approach.[111] Removal of the 5-CH₂OH group of N-alkyl-DNJ and a shift of the alkyl chain from the endocyclic nitrogen atom to C-1 or O-2 have led to powerful and selective inhibitors of N370S GCase. Extension of the scope of this structural modification to develop PCs of other lysosomal glycosidases is a promising avenue for future research. Interest in α-1-C-substituted iminoxylitol derivatives has been heightened by the recent disclosure by Martin and co-workers of the best PC identified to date for L444P GCase in Gaucher patients, a mutation associated with severe neurological forms.[112]

Multivalency surprisingly turned out to be a fruitful option for the design of pharmacological chaperones and CFTR correctors, a result that was unexpected at the start of our studies. In the field of Gaucher disease, small multivalent effects have been observed in the inhibition of GCase, and the first examples of multivalent chaperones have been described. This study has also demonstrated the potential of the prodrug strategy in PCT.[108] To date, the best results by far have been obtained with multivalent CFTR correctors that were found to be up to three orders of magnitude more efficient than the corresponding monovalent analogues, including the clinical candidate N-Bu DNJ (1).[110] This study provides a new potential answer to cystic fibrosis, but also raises many fundamental questions, especially concerning the molecular basis of the strong multivalent effect that is observed. This is just a beginning. Much exciting work remains to be done in optimizing, understanding, and extending the scope of the inhibitory multivalent effect.[113]

In terms of chemistry, our pursuit of potent original glycomimetics targeting protein misfolding has inevitably led to synthetic failures. The preparation of some challenging targets has not yet been completed, which is a rather unpleasant feeling for a synthetic chemist. Nevertheless, unexpected reactivities and troubles met on the way have led to progress in organic synthesis. A tandem intramolecular azetidine ring-opening/closing cascade reaction has been disclosed, and a new amino-protecting group has been developed. The synthetic scope of intramolecular C–H amination has been extended through conformational control of the regioselectivity of the reaction. This study has given rise to innovative strategies for the general synthesis of piperidines by means of iterative multifunctionalization of nonactivated C–H bonds. In this novel concept, a sulfamoyloxy group is used several times as a molecular-activating arm.

In my introduction to this account, I mentioned that the search for small molecule rescuers of misfolded proteins has guided our research efforts during the past twelve years like a lighthouse in a foggy night. Because of the exploratory nature typical of academic research projects, this metaphor does not actually stand up to scrutiny. The lighthouse can move erratically in unpredictable directions according to new results published in the literature, and the fog generated by puzzling experimental outcomes can suddenly become very thick. As a consequence, the journey reported in this account is a further example of how synthetic chemistry projects move forward through paradoxes, fruitful failures, and fortunate accidents. Failure by an organic chemist to synthesize the desired molecular targets can eventually lead to progress in synthetic methodology. Sometimes, a molecule designed on the basis of a wrong working hypothesis displays outstanding biological activity; very often, however, the opposite is likely to be true. Most unexpectedly though, some molecules designed to be nanomolar inhibitors on the basis of structure–activity relationships study do indeed display nanomolar inhibitory activity! For all these reasons, we have really enjoyed the hectic journey from subtle conformational effects in nitrene insertions to the cell’s quality-control machinery. One of the most rewarding lessons learned along the way is that the research objective is more a bridge to the new than an end in itself.

Acknowledgment

The chemical story presented here began when I was a young CNRS researcher in Orléans (France) and has been continued after my move to the University of Strasbourg (Unistra) as a full professor. My first thanks go to Professor Olivier R. Martin for introducing me to the fascinating world of glycomimetics and for our exciting collaboration. The work described in this account was made possible because of the dedication of enthusiastic students and co-workers; my thanks go to all of them. I am also grateful to all the co-authors from various fields with whom I had the privilege off collaborating during the past years. Financial support from the Institut Universitaire de France (IUF), the CNRS, the Universities of Strasbourg and Orléans, the Agence Nationale de la Recherche (ANR, grant numbers 08JC-0094-01 and 11-BS07-003-02), the Centre International de Recherche aux Frontières de la Chimie (FRC), the associations Vaincre les Maladies Lysosomales and AFM (Association Française contre les Myopathies) is gratefully acknowledged.

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