Piperidine homoazasugars: natural occurrence, synthetic aspects and biological activity study

A number of natural and synthetic analogues of homoazasugars, known in the literature, are promising glycosidase inhibitors. The methodologies used for the synthesis of piperidine homoazasugars are: (i) intramolecular reductive amination, (ii) intermolecular double reductive amination, (iii) amino/amido mercuration, (iv) intramolecular nucleophilic substitution, (v) synthesis from non-carbohydrate building block and aza-heterocycles and (vi) enzyme catalyzed intramolecular reductive amination. Homoazasugars showed higher selectivity and potency in the glycosidase inhibitory activity. In this report, natural occurrence, synthetic methodologies and potential application to glycosidase inhibitory activity of homoazasugars will be reviewed.


Introduction
The search for selective and effective inhibitors of oligosaccharide processing enzymes has promoted intense research over the last 20 years in the synthesis of stereochemically welldefined polyhydroxylated piperidines.This class of compounds, commonly called as azasugars or iminosugars, are known to be endowed with a remarkable therapeutic potential in the treatment of diabetes, viral infections (including HIV) and tumor metastasis 1b due to their action as glycosidase inhibitors. 1 The polyhydroxylated piperidine namely nojirimycin (NJ 1, Figure 1) was first isolated from a fermentation broth of Streptomyces roseochromogenes R-468.2a The more stable form of NJ is the 1-deoxynojirimycin (DNJ, 2), also called as moranoline, was first prepared 2b by catalytic hydrogenation of NJ and later on isolated from different species of the plant Morus.Fi 1 The search for promising glycosidase inhibitors led to the discovery of homoazasugars.In general, the homoazasugars are classified in to two categories.In the first type, hydroxymethyl group/polyhydroxylated carbon chain is present at both the carbons (C-1 and C-5) adjacent to the ring nitrogen (type I, Figure 2) and is also known as aza-C-glycoside.While, in other the type carbon homologation is present at C-6 of the piperidine (type II).The homoazasugars are found to be more stable towards chemical and enzymatic degradation than azasugars, while retaining the powerful biological activity of the parent azasugars, the homoazasugars having substituents recognizing the aglycon-binding site of the enzyme are expected to further increase the selectivity.Due to their higher selectivity and potency in the glycosidase inhibitory activity, the homoazasugars are now gaining their own independent identity.A brief review of natural occurrence, synthetic aspects and biological activity of piperidine homoazasugars is described in this article.

Natural occurrence
Homoazasugars are natural alkaloids widely diffused in plants and microorganisms.Interestingly, α-homonojirimycin (α-HNJ 3, Figure 3) was first synthesized in the protected form 4 just before its isolation in 1988. 5The naturally occurring homoazasugars are listed in Figure 3 and their natural sources are summarized in Table 1.

Synthesis of piperidine homoazasugars
4][15][16][17] As the homoazasugars have a close resemblance with five or six carbon sugars, most of the synthetic strategies make use of sugars as the starting materials.The common methodologies used for the synthesis of piperidine homoazasugars are: (i) intramolecular reductive amination, (ii) intermolecular double reductive amination, (iii) amino/amido mercuration, (iv) intramolecular nucleophilic substitution, (v) synthesis from noncarbohydrate building block and aza-heterocycles and (vi) enzyme catalyzed intramolecular reductive amination.

(i) Intramolecular reductive amination strategy
Cipolla and co-workers have synthesized the protected α-allyl-C-glycoside of nojirimycin 13 by sequential reductive amination (Scheme 1). 18Thus, reaction of perbenzylated We have recently reported two different strategies for the synthesis of 6-homoazasugars 17 and 20.The first method 19a relies on the reductive amination followed by diastereoselective intramolecular conjugate addition of the in situ generated benzylamine in the formation of desired piperidine ring (Scheme 2).Thus, D-glucose was converted to α,β-unsaturated ester that on 1,2-acetonide cleavage afforded hemiacetal 18.The reaction of 18 with benzylamine forms imine that was reduced to sugar benzylamine.The concomitant in situ addition of amine to α,βunsaturated ester followed by domino lactonization gave lactone 19a with the required homoazasugars ring skeleton.Reduction of the lactone functionality and removal of the protecting group by hydrogenation afforded 1-deoxy-L-ido-homonojirimycin 17.In the next report, we have described an efficient and practical strategy for the synthesis of N-hydroxyethyl-1-deoxy-homonojirimycins 27 and 28 with full stereocontrol (Scheme 4). 23The key step involved is the intermolecular conjugate addition of benzylamine to D-glucose derived α,β-unsaturated ester 29 to afford D-gluco-and L-ido-configured β-amino esters 30a and 30b.The sequential N-alkylation of 30a and 30b with ethyl bromoacetate, reduction with LAH, acetylation, hydrogenation and selective protection with -Cbz group afforded 31a and 31b, respectively.Separate removal of 1,2-acetonide functionality, hydrogenation and deacetylation of 30a and 30b afforded N-hydroxyethyl-D-gluco-1-deoxyhomonojirimycin (27) and Nhydroxyethyl-L-ido-1-deoxyhomo-nojirimycin (28), respectively.The glycosidase inhibition activity of compounds 27 and 28 was evaluated using sweet almond seed as a rich source of different glycosidases.
Transmetalation of the hydroxy protected stannylmethanol derivative 32 with butyllithium is an effective source of the hydroxymethyl carbanion, which undergoes efficient nucleophilic addition to carbonyl compounds and has been utilized for one carbon chain extension of carbohydrate lactones. 23Shilvock and co-workers have exploited this strategy for the synthesis of β-homogalactonojirimycin (β-HGJ) 33 (Scheme 5). 24,25Thus, hydroxy-methylation of protected 5-azido-L-manno-1,4-lactone 34 gave 35, which on hydrogenation afforded the piperidine derivative 36 as a single diastereomer.De-protection of the acetonide and MOM groups yielded 33 in good yield.The same strategy was extended for the synthesis of a variety of homoazasugars by changing the sugar lactones.(ii) Intermolecular double reductive amination methodology Saavedra and Martin 26 used two different approaches for the synthesis of β-homonojirimycin (4).In the first approach (Scheme 6), tetra-O-benzyl-D-glucono-1,5-lactoe 43 was treated with (methoxymethoxy)methyllithium and the resulting heptulopyranose derivative 44 was reduced to alcohols 45a and 45b (~1:1).The oxidation of the mixture of 45a and 45b using DMSO-TFAA gave heptodiulose 46, which was immediately subjected to double reductive amination using HCOONH 4 in the presence of NaBH 3 CN to give 47 as a single stereoisomer.The high degree of stereoselectivity observed in this double reductive amination reaction, probably involves cyclic intermediate and the stereoselective hydride addition (axial attack) appeared due to torsional effects. 27,28In the subsequent step, removal of the benzyl and MOM protecting groups provided In the second approach (Scheme 7), 26 one carbon extension by Wittig reaction of perbenzylated α-D-glucopyranose 14 followed by dihydroxylation afforded 49a and 49b (10:1, the major isomer was predicted from Kishi's empirical rule 29 ) in which the primary hydroxyl group was protected as silyl ether.Oxidation of secondary alcohol functionality in 50a/50b afforded 1,5-diketone.Subsequently, double reductive amination using ammonium formate and sodium cyanoborohydride gave β-homonojirimycin derivative 57 that on removal of the protecting groups yielded 4. β-Homogalactonojirimycin 33 was also synthesized in six steps from heptenitol 52 by Martin and co-workers. 30The synthetic route consisted in forming the piperidine ring by way of double reductive amination process of diketone 53 using ammonium formate and sodium cyanoborohydride (Scheme 8).The intramolecular amido mercuration strategy was also exploited by P.S. Liu in the total synthesis of 7-O-β-D-glucopyranosyl-α-homonojirimycin 10 (MDL 25637). 4The 2,3,4,6-tetra-Obenzyl-D-glucopyranose 14 was converted to the oxime 56 (via Wittig olefination and oxidation of secondary hydroxyl group to ketone), which on reduction with LAH and Cbz protection gave D and L amino sugars in the ratio 6:1, respectively.The major D-gluco-isomer 57 on treatment with mercuric acetate in THF underwent stereospecific cyclization to give exclusively α-mercuriomethyl 48 that on demercuration oxygenation 31 gave 58.The high stereoselectivity of the cyclization can be accounted for by the chelation effect of mercury with the vicinal αbenzyloxy group, resulting in the preferential addition of the carbamate (nucleophile) from the opposite side of the olefin.The coupling with 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl bromide furnished 59a (19%) and 59b (24%).Separation and de-protection in 59b afforded βglucoside 10 (Scheme 10).

(v) Synthesis from non-carbohydrate building block and aza-heterocycles
The first synthesis of (+)-α-homonojirimycin (4) was reported by S. Aoyagi et al. 33 via a noncarbohydrate based approach utilizing suitably protected allylic alcohol 62 (Scheme 12).The allylic alcohol 62 was converted to syn epoxide 63 by the Sharpless asymmetric epoxidation.Regio-and diastereoselective ring opening of the epoxide was effected by using dialkylaluminium amide followed by selective amino group protection to give amino alcohol 64 as a single diastereomer, which on MOM protection and desilylation furnished 65.Swern oxidation of 65 gave the aldehyde 66 that on Wittig reaction followed by hydroxylation provided 2.5:1 diastereoselectivity in favor of the desired anti-diol 67.Compound 67 was then converted to homonojirimycin 4 (Scheme 12) by conversion of secondary alcohol to mesylate and hydrogenation wherein de-protection of N-Cbz lead to an amine that subsequently undergoes intramolecular nucleophilic substitution to give piperidine ring skeleton.Johnson and Johns 34 synthesized a number of β-1-C-aryl-mannonojirimycin analogues of type 68 using asymmetric strategy.The polyhydroxylated piperidine ring was constructed using vinyl bromide 70, which was synthesized in six steps from bromobenzene by microbial oxidation to get bromo diol 69. 35A palladium-catalyzed Suzuki cross-coupling of vinyl bromide 70 and the corresponding arylboronic acid reaction served as the key pseudoanomeric C-C bond forming step.Ozonolysis and selective reduction of the resultant carbonyl functions followed by reductive amination produced the azasugar ring (Scheme 13).The stereochemical outcome of the reductive amination resulted into β-oriented aryl group as the hydrogen delivery resulted from the α-face opposite to that of the adjacent β-oriented acetonide group.The homoaminoazasugars 71 and 72 were synthesized by C.-H. Wong and co-workers 36 from commercially available nojirimycin bisulfite and mannonojirimycin bisulfite, respectively.The reaction of bisulfite adducts 73 and 74 with potassium cyanide in the presence of Ba(OH) 2 and ethanolic HCl afforded the corresponding α-nitriles 75 and 76, which were converted to the homoaminoazasugars 71 and 72 by a palladium-catalyzed reduction under acidic conditions (Scheme 14).Tri-O-acetyl imino glucal 78, prepared from D-glucal 77 was utilized for the synthesis of iminosugar C-glycosides. 37

(vi) Enzyme catalyzed intramolecular reductive amination strategy
Enzymes are increasingly recognized as useful catalyst for the organic syntheses.The synthesis of monosaccharides and related compounds via enzymatic aldol addition reaction, catalyzed by aldolases, has been proven to be very useful and several successful examples have been described in recent years. 38In addition, the use of enzyme aldolases with azido substrate followed by hydrogenation of the azido-sugar produces various five-, six-and seven-membered azasugars 27,39,40 (Figure 4).The same strategy is also extended for homoazasugars.For example, Wong and co-workers prepared β-L-homofuconojirimycin (β-HFJ) 12 by an aldolase-based strategy. 41The acceptor substrate (±)-threo-azidoaldehyde 82 was synthesized from commercially available 2-butyn-1-al diethyl acetal 81 (Scheme 16).Aldolase catalyzed aldol condensation of 82 with dihydroxyacetone phosphate (DHAP) followed by dephosphorylation with acid phosphatase afforded the desired enantiomerically pure azidoketose 83.Hydrogenation of 83 in the presence of 10% Pd/C produced β-HFJ 12 as the only product.The complete diastereoselectivity is due to the delivery of hydrogen from the less hindered side of the possible cyclic imine intermediate during the reductive amination process.The same group also utilized the azidoketose 83 for the synthesis of 2-aminomethyl -HFJ 84. 42Amino-homoazasugar was prepared by a novel chemoenzymatic strategy in which azido sugar 83 was constructed by enzymatic aldol reaction under acidic hydrogenation condition (Scheme 17).K. E. Holt et al. 44 and C.-H. Wong et al. 45 used RAMA for the synthesis of a number of naturally occurring homoazasugars.The four stereoisomers of the four-carbon azido sugar 89 have been stereoselectively synthesized by a route involving Sharpless epoxidation and these compounds were considered as substrates for rabbit muscle fructose 1,6-bisphosphate aldolase, giving (after treatment with phosphatase) 6-azido-6-deoxyheptuloses 90a-d, respectively.Further hydrogenation gave corresponding homoazasugars 6, 4, 3 and 5 respectively (Scheme 19).

Biological activity
Glycosidases are involved in several important biological processes such as digestion, biosynthesis of glycoproteins and the lysosomal catabolism of glycoconjugates.Therefore, glycosidase inhibitors have many potential medical applications, for example, diabetes type 2, 46 cancers, 47 viral infection, 48 and heredity lysosomal storage diseases. 49Homoazasugars are selective and in some cases better glycosidase inhibitors 50 and thus have been used for the treatment of a number of carbohydrate mediated diseases.

Glycosidase inhibition
The IC 50 values of a number of piperidine homoazasugars, shown in Table 2, was evaluated by Fleet and co-workers. 9α-HNJ 3 inhibited α-glucosidases and trehalase to a similar extent as DNJ 2, failing to have any activity toward other glycosidases tested.Thus, the hydroxymethyl group at the anomeric position of 2 contributed to a greater selectivity.β-HNJ 4 was very specific inhibitor of α-glucosidase (IC 50 = ~10 µM).Similar selectivity and potency was found in case of α-HMJ (5) against human liver α-mannosidases. 51β-HMJ 6 is a potent inhibitor of rice and rat α-glucosidases and human α-L-fucosidase (K i = 4.5 mM). 51β-4,5-Di-epi-HNJ (7) showed potent inhibitory activity toward all α-glucosidases tested and was found much better α-galactosidase inhibitor than that of β-galactosidase.Thus, the epimerization at C-4 of 6 definitely enhances its inhibition toward α-glucosidases and α-galactosidase.N-alkylation of 3 enhances the potency and selectivity against the glycosidases tested.It has been proposed that the C-6 OH axial conformation of the N-alkyl derivatives of DNJ best fit the active site of ER α-glucosidase I 52 or glucoamylase from Aspergillus awamori 53 and is responsible for strong inhibitory activity.In fact Me-HNJ 91 (Figure 5) with this preferred conformation is more potent inhibitor of αglucosidase I than 3.  Homoazasugars, due to the remarkable glycosidase inhibitory activity, were studied against diabetes.For example, compound 10 (MDL 25637) was reported to effectively reduce ISSN 1424-6376 Page 129 © ARKAT USA, Inc postprandial elevations of blood glucose and plasma insulin in animals when administered 30-60 min before a sucrose load. 54

Antiviral activity
Glycoproteins are often essential proteins in that they are required in the viral life cycle, either in viron assembly and secretion and/or infectivity.As processing of these glycoprotein occurs through the cellular machinery, processing glycosidase inhibitors have been used to study the role of N-linked oligosaccharides in several viral systems including human immunodeficiency virus (HIV), 55 human hepatitis B virus (HBV), 56 human cytomegalovirus (HCMV), 57 influenza virus, 58 Sinvis virus 59 and VSV. 60α-Glucosidase inhibitors are potent inhibitors of HIV replication and HIV-mediated syncytium formation in vitro. 55,61,62Whereas, N-linked oligisaccharide processing inhibitors have no effect on the secretion of infectious virus. 62,63α-HNJ 3 and its N-methyl derivative 91 are more potent against α-glucosidase I both in vitro and in cell culture. 64So it is expected that these homonojirimycins would show excellent anti-HIV activity.Surprisingly, both HNJ 3 and Me-HNJ 91 showed no significant anti-HIV activity even at concentrations of 500 µg/mL. 9

Conclusions
In conclusion, we have described the natural occurrence of piperidine homoazasugars.The synthetic aspects of homoazasugars using chiron, asymmetric approaches including chemoenzymatic methods have been discussed.The structural basis for the specificity of glycosidase inhibition and biological applications are also discussed.