Direct synthesis of imino-C-nucleoside analogues and other biologically active iminosugars

Iminosugars have attracted increasing attention as chemical probes, chaperones and leads for drug discovery. Despite several clinical successes, their de novo synthesis remains a significant challenge that also limits their integration with modern high-throughput screening technologies. Herein, we describe a unique synthetic strategy that converts a wide range of acetaldehyde derivatives into iminosugars and imino-C-nucleoside analogues in two or three straightforward transformations. We also show that this strategy can be readily applied to the rapid production of indolizidine and pyrrolizidine iminosugars. The high levels of enantio- and diastereoselectivity, excellent overall yields, convenience and broad substrate scope make this an appealing process for diversity-oriented synthesis, and should enable drug discovery efforts.

I minosugars are naturally occurring carbohydrate mimics that inhibit many enzymes of medicinal interest 1 . Their biological activity is often attributed to a structural resemblance to the oxacarbenium ion-like transition states that occur during the enzymatic hydrolysis of carbohydrates 2 . As such, many iminosugars are potent inhibitors of glycosidases and glycosyltransferases 1 , and have been highlighted as lead candidates for the treatment of a variety of diseases, including cancer, diabetes, viral infections and lysosomal storage disorders (for example, Gaucher and Fabry disease) 1,3 . The most common naturally occurring iminosugars possess a polyhydroxylated pyrrolidine core and may be additionally annulated as in the pyrrolizidines (for example, 2, Fig. 1), indolizidines (for example, 3), or nortropanes 4 . A growing number of unnatural analogues of these compounds have also been reported as leads for drug discovery, including the imino-C-nucleosides developed by Schramm (for example, 1) 5,6 and b-hexosaminidase inhibitors developed by Wong 7,8 . Unfortunately, the incorporation of pyrrolidine iminosugars into chemical screening libraries or diversity-oriented synthesis (DOS) campaigns is problematic, as their syntheses are often lengthy, low-yielding, cost-intensive and limited by reliance on carbohydrate building blocks 9 . Thus, while several such pyrrolidine iminosugars have emerged as clinical candidates or drugs 1 , fundamental tools for their high-throughput synthesis are lacking. In fact, much of the success in imino-Cnucleoside synthesis [10][11][12][13][14] (for example, 1 (ref. 6) and 4 (ref. 5)) has relied on the common building block 5 (refs [14][15][16]. As evidenced by step counts provided in Fig. 1, the synthesis of pyrrolizidineand indolizidine-based iminosugars (for example, 2; (ref. 17) and 3; (ref 18)) also remains a significant synthetic challenge.
We have reported preliminarily that when mixtures of the dioxanone 8, an aliphatic aldehyde 6 and N-chlorosuccinimide (NCS) are treated with (S)-proline, a series of well-orchestrated reactions occur 19 . First, the aldehyde undergoes a-chlorination 20 , producing a racemic mixture of a-chloroaldehydes 7. Second, an enantioselective proline-catalysed aldol reaction occurs between the dioxanone 8 and the a-chloroaldehyde (R)-7. Importantly, proline also catalyses racemization of the a-chloroaldehydes 7 and, consequently, this second step effects a dynamic kinetic resolution (DKR) 19 . Thus, this one-pot reaction transforms commodity chemicals 6 and 8 into carbohydrate building blocks 9 in excellent yield, diastereoselectivity and enanantioselectivity. Considering the spatial relationship between the chloromethine and carbonyl functions in 9, these aldol adducts may also serve as building blocks [21][22][23][24] for the synthesis of polyhydroxypyrrolidines via a reductive amination-annulation sequence (see grey box, Fig. 2). Such a strategy would allow for the conversion of virtually any acetaldehyde derivative 6 into an iminosugar 10 in two straightforward transformations from commodity chemicals, thus enabling their integration with modern high-throughput screening technologies.
Here we demonstrate that the reductive amination of a wide range of ketochlorohydrins 9 provides a rapid route to pyrrolidine iminosugars 8,11,[25][26][27][28][29][30][31][32] , such as those depicted in Fig. 1. Importantly, this unique two-or three-step process requires no cryogenic, anhydrous or otherwise complicated experimental conditions. The demonstration of this strategy in several short syntheses of biologically active imino-C-nucleoside analogues, and indolizidine and pyrrolizidine iminosugars highlights its adaptability for DOS and the rapid preparation of iminosugar-based screening libraries 26 .

Results
Reductive amination of a-chlorination-DKR aldol products. The utility of the synthetic strategy outlined in Fig. 2 relies intimately on a diastereoselective reductive amination of aldol adducts 9. Enders has reported 27 that the reductive amination of related aldol adducts that lack a chloromethine function were non selective (dro2:1) using NaB(OAc) 3 H. Likewise, Madsen found similar selectivities in the reductive amination of the corresponding syn-aldol adduct 33 . Bearing this in mind, we began by screening solvents and reducing agents, as well as the addition of acetic acid to the reductive amination of ketochlorohydrin 11 (Table 1) (ref. 19). In all cases, an excess of amine was required for complete imine formation and avoidance of competing ketone reduction (entry 1). As indicated in entry 2, the conditions reported by Enders 27 delivered the amino alcohols 12a and 12b in good yield (82%), albeit low diastereoselectivity. The relative stereochemistry of 12a was assigned based on analysis of 3 J H,H coupling constants and NOESY spectra recorded on the cyclic carbamate derived from the reaction of 12a with carbonyldiimidazole. Use of NaB(CN)H 3 resulted in an improved diastereomeric ratio of these products (dr B6:1) in both CH 2 Cl 2 and MeCN (entries 3 and 4) and in tetrahydrofuran (THF) the 1,3-syn amino alcohol 12a was produced as the only detectable diastereomer in near quantitative yield (entry 5). As summarized in entries 6 and 7, this optimized protocol proved general and also provided access to the corresponding N-allyl and N-propargyl amines 13a and 14a, in excellent yield and diastereoselectivity.
Synthesis of pyrrolidine iminosugars. While the aminochlorohydrin 12a did not cyclize directly, its high-yielding conversion into the pyrrolidine iminosugar 15 simply required heating in methanol, which also promoted acetonide removal (Fig. 3). Alternatively, this cyclization could be effected by heating 12a in toluene with excess NaHCO 3 , which provided the orthogonally protected iminosugar 16. Anticipating that the increased reactivity of a benzylchlorohydrin would favour a one-pot reductive amination-annulation process, the readily available aldol adduct 17 (ref. 19) was also treated with NaB(CN)H 3 in a mixture of THF/HOAc. Following this optimized procedure, the orthogonally protected iminosugar 18 was produced directly and in excellent yield. Removal of both the acetonide and benzylprotecting groups by hydrogenolysis in acidic methanol gave the imino-C-nucleoside analogue 4. Considering the aldol adduct 17 is available in one step from phenyl acetaldehyde 19 , this threestep synthesis of 4, a potent (K i ¼ 170 nM) transition-state analogue inhibitor of nucleoside hydrolase 5 , represents a significant advance.
Short syntheses of polyhydroxy pyrrolizidines and indolizidines. Figure 5 highlights the further application of this convenient strategy to the rapid preparation of several structurally complex polyhydroxy indolizidine and pyrrolizidine alkaloids, including analogues of the glycosidase inhibitors hyacinthacine and steviamine. While several strategies could be exploited for  Figure 3 | Synthesis of pyrrolidine iminosugars and imino-C-nucleoside analogues. A highly diastereoselective reductive amination of chlorohydrin aldol adducts followed by brief heating in methanol or toluene with NaHCO 3 provides rapid access to native or differentially protected iminosugars. Reductive amination of the benzyl chloride-containing aldol adduct 17 leads directly to the protected iminosugar 18, a precursor to the potent nucleoside hydrolase inhibitor 4.
The reductive amination strategy was explored in short syntheses of the hyacinthacine and steviamine analogues 2 (ref. 17) and ent-3 (ref. 18). In both cases, the ketone function in the readily available pyrrolidines 34 and 35 was unveiled in concert with hydrogenolytic cleavage of the N-benzyl group, and the resulting iminium species (not shown) was reduced in situ to afford the products depicted as single diastereomers. Importantly, each of the total syntheses depicted in Fig. 5 requires 5 steps or less, originates with inexpensive and readily available chemicals, and is completed in a matter of days, which compares well with the reported syntheses of these and related compounds (see for example, Fig. 1).

Discussion
In summary, a highly convergent synthesis of iminosugars has been developed that converts a wide range of acetaldehyde derivatives into polyhydroxypyrrolidines in two or three straightforward reactions and does not rely on carbohydrate building blocks. The application of this cost-effective process to the rapid synthesis of indolizidine and pyrrolizidine iminosugars also highlights its utility for the preparation of more structurally complex natural products and their analogues. Importantly, the excellent overall yields, diastereoselectivity and enantioselectivity, coupled with tunability of pharmacophoric features make this process well suited for chemical screening library and DOS campaigns.

Methods
Representative example of reductive amination/annulation sequence. Preparation of the imino-C-nucleoside analogue 4. A solution of 18 (20 mg, 0.059 mmol) and pyridinium p-toluenesulfonate (15 mg, 0.059 mmol) in 1:1 H 2 O/ MeOH (4.0 ml) was added to a microwave vial. The vial was sealed in a CEM Discover LabMate microwave reactor and the resulting mixture was heated at 100°C (as monitored by a vertically focused infrared temperature sensor) for 30 min. The resulting solution was concentrated under reduced pressure and the crude product was used in the next reaction without further purification. A solution of the crude iminocyclitol p-toluenesulfonate salt in MeOH (20 ml) was passed twice through an H-Cube continuous-flow reactor using a 30 mm 10% Pd/C cartridge. Conditions: temperature ¼ 35°C; flow rate ¼ 0.8 ml min À 1 ; H 2 pressure ¼ 40 bar. The resulting mixture was stirred with DOWEX 1X8-100 (HO À form) for a further 30 min and the resin was removed by filtration. Concentration and purification of the crude product by flash chromatography on C 18 silica gel (H 2 O) afforded iminoribitol 4 (10 mg, 83% yield over 2 steps) as a colourless oil.