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Seven pyranoses and three furanoses with a nitrogen in the ring were prepared by chemical synthesis, microbial conversion, and isolation from plants to investigate the contribution of epimerization, deoxygenation, and conformation to the potency of inhibition and specificity of mammalian glycosidases. The seven pyranoses are 1-deoxynojirimycin (1), the D-manno (2), D-allo (3), and D-galacto (4) isomers of 1, fagomine (1,2-dideoxynojirimycin, 5), and the D-allo (6) and D-galacto (7) isomers of 5, while the three furanoses are 2,5-dideoxy-2,5-imino-D-mannitol (8), 1,4-dideoxy-1,4-imino-D-arabinitol (9), and 1,4-dideoxy-1,4-imino-D-ribitol (10). The 2-deoxygenation and/or 3-epimerization of 1 enhanced the potency for rat intestinal lactase and bovine liver cytosolic beta-galactosidase. Especially compound 6 showed a potent inhibitory activity against both enzymes, and compound 8, a mimic of beta-D-fructofuranose, was a potent inhibitor of both beta-galactosidases as well. Compound 4, which has been known as a powerful alpha-galactosidase inhibitor, exhibited no significant inhibitory activity for most of mammalian beta-galactosidases. In addition, compound 6 fairly retained a potency of 1 toward rat intestinal isomaltase. In this study, compound 8, known as a processing alpha-glucosidase I inhibitor in cell culture, has been found to have no effect on processing alpha-glucosidase II, whereas 9 has been shown to be a good nonspecific inhibitor of intestinal isomaltase, processing alpha-glucosidase II, Golgi alpha-mannosidases I and II, and porcine kidney trehalase. It has been speculated that glycosidase inhibitors have structures which resemble those of the respective glycosyl cations. This Broad inhibitory activity of 9 toward various glycosidases suggest that it superimposes well on the various glycosyl cations.

The conformations of nitrogen-in-the-ring sugars and their N-alkyl derivatives were studied from 1H NMR analyses, mainly using 3J(H,H) coupling constants and quantitative NOE experiments. No significant difference was seen in the ring conformation of 1-deoxynojirimycin (1), N-methyl-1-deoxynojirimycin (2), and N-butyl-1-deoxynojirimycin (3). However, it was shown that the C6 OH group in 1 is predominantly equatorial to the piperidine ring, while that in 2 or 3 is predominantly axial, and its N-alkyl group is oriented equatorially. In the furanose analogues 1,4-dideoxy-1,4-imino-D-arabinitol (4) and its N-methyl (5) and N-butyl (6) derivatives, the five-membered ring conformation differed significantly by the presence or absence of the N-substituted group and the length of the N-alkyl chain. Compound 3 reduced its inhibitory effect on almost all glycosidases, resulting in an extremely specific inhibitor for processing alpha-glucosidase I since N-alkylation of 1 is known to enhance both the potency and specificity of this enzyme in vitro and in vivo. This preferred (C6 OH axial) conformation in 2 and 3 appears to be responsible for their strong alpha-glucosidase I activity. Compound 4 is a good inhibitor of intestinal alpha-glucohydrolases, alpha-glucosidase II, and Golgi alpha-mannosidases I and II, but its N-alkyl derivatives 5 and 6 markedly decreased inhibitory potential for all enzymes tested. In the case of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP, 7), which is a potent beta-galactosidase inhibitor, its N-methyl (8) and N-butyl (9) derivatives completely lost potency toward beta-galactosidase as well. N-Alkylation of compounds 4 and 7, known well as potent yeast alpha-glucosidase inhibitors, resulted in a serious loss of inhibitory activity toward yeast alpha-glucohydrolases. Activity of these nine analogues against HIV-1 replication was determined, based on the inhibition of virus-induced cytopathogenicity in MT-4 and MOLT-4 cells. Compounds 2 and 3, which are better inhibitors of alpha-glucosidase I than 1, proved active with EC50 values of 69 and 49 micrograms/mL in MT-4 cells and 100 and 37 micrograms/mL in MOLT-4 cells, respectively, while none of the furanose analogues exhibited any inhibitory effects on HIV-1. The change in potency and specificity of bioactivity by N-alkylation of nitrogen-in-the-ring sugars appears to be correlated with their conformational change.

Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion-exchange column chromatographies. Their structures have been determined from the 'H-and I3C-NMR spectral data, and two of the compounds were identified as calystegins A, and B2, which have been isolated from the roots of Calystegia sepium (Convolvulaceae). Two of the remaining three were found to be la,3a,4P-trihydroxy-nortropane and la,2a,3a,4P-tetrahydroxy-nor-tropane and given the trivial name calystegins A, and B,, respectively. The last calystegin was assigned as la,2P,3a,6a-tetrahydroxy-nor-tropane, which was the same as the relative configuration proposed in the literature for calystegin B, isolated from C. sepium. However, the I3C-NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the I3C-NMR chemical shifts of calystegin B, in the original paper had been erroneous. Since their corrected 13C-NMR data of calystegin B, and its 'H-NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and I? alkekengi are identical.Calystegin B, has been known to be a potent competitive inhibitor of almond P-glucosidase (K, = 1.2 pM) and coffee bean a-galactosidase (K, = 0.86 pM). In this study calystegin B, (la,2/3,3a,6a-tetrahydroxynor-tropane) proved to be a potent competitive inhibitor of almond P-glucosidase (Kl = 1.9 pM) and bovine liver P-galactosidase (K, = 1.6 pM), but not an inhibitor of a-galactosidases. Calystegin A, was found to be a weaker inhibitor compared to calystegin B, but with the same inhibitory spectrum. Calystegin A,, a 2-deoxy derivative of calystegin B,, showed no activity against any glycosidases tested. Since calystegin B,, a 2-epimer of calystegin B2, also exhibited only a weak inhibitory activity, it was concluded that the equatorially oriented OH group at C2 is the essential feature for recognition and strong binding by the active site of glycosidases. Based on the structure/activity relationships for the five calystegins isolated from P: alkekengi var. francheti and calystegin C, from Morus alba,we propose that the OH group at C6 of calystegin B, or C,, in place of the p-glycoside oxygen, is protonated by an acidic group in the active site of the P-glycosidase.Keywords. Calystegins ; polyhydroxy-nor-tropanes ; glycosidase inhibitors ; structure/activity relationships ; mechanism.Recently, a new structural type of polyhydroxylated alkaloids has been added to the known five structural types of naturally occurring glycosidase inhibitors : polyhydroxylated piperidines, pyrrolidines, pyrrolines, indolizidines, and pyrrolizidines. These new compounds are polyhydroxy-nor-tropanes and have been given the trivial names calystegins. Calystegins were first discovered as secondary metabolites of plants and were imp...

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