Data from both our own and literature studies of the biochemistry and inhibition of influenza virus endonuclease was combined with data on the mechanism of action and the likely active site mechanism to propose a pharmacophore. The pharmacophore was used to design a novel structural class of inhibitors, some of which were found to have activities similar to that of known influenza endonuclease inhibitors and were also antiviral in cell culture.

Catalysis by glycogen phosphorylase involves a mechanism in which binding of one substrate tightens the binding of the other substrate to produce a productive ternary enzyme-substrate complex. In this work the molecular basis for this synergism is probed in crystallographic studies on ternary complexes in which the glucosyl component is substituted by the putative transition state analogue nojirimycin tetrazole, a compound which has been established previously as a transition state analogue inhibitor for a number of glycosidases. Kinetic studies with glycogen phosphorylase showed that nojirimycin tetrazole is a competitive inhibitor with respect to glucose 1-phosphate and uncompetitive with respect to phosphate. Ki values for the phosphorylase-AMP-glycogen complex and the phosphorylase-AMP-glycogen-phosphate complexes are 700 microM and 53 microM, respectively, indicating that by itself norjirimycin tetrazole has poor affinity for glycogen phosphorylase but that phosphate substantially improves the binding of norjirimycin tetrazole. X-ray crystallographic binding studies to 2.4 A resolution with T state phosphorylase b crystals showed that nojirimycin tetrazole binds at the catalytic site and promotes the binding of phosphate through direct interactions. Phosphate binding is accompanied by conformational changes that bring a crucial arginine (Arg569) into the catalytic site. The positions of the phosphate oxygens were definitively established in X-ray crystallographic binding experiments at 100 K to 1.7 A resolution using synchrotron radiation. X-ray crystallographic binding studies at 2.5 A resolution with R state glycogen phosphorylase crystals showed that the protein atoms and water molecules in contact with the nojirimycin tetrazole and the phosphate are similar to those in the T state. In both T and R states the phosphate ion is within hydrogen-bonding distance of the cofactor pyridoxal 5'-phosphate group and in ionic contact with the N-1 atom of the tetrazole ring. The results are consistent with previous time-resolved structural studies on complexes with heptenitol and phosphate. The structural and kinetic results suggest that nojirimycin tetrazole in combination with phosphate exhibits properties consistent with a transition state analogue and demonstrate how one promotes the binding of the other.

The tetrazole 1 is a new 8-glucosidase inhibitor (IC50 = 8 . lo-' M, Emulsin), obtained (92%) by deprotection of 22, the product of an intramolecular cycloaddition of the azidonitrile 20. This azidonitrile was formed as an intermediate by treating the L-idu-bromide 14 or the L-idu-tosylate 19 with NaN, at 1 1 0 -1 2 5 O . It was isolated in a separate experiment. The yield of 22 from 19 reached 70%; 21 was formed as by-product (10 %). The bromide 14 (42%) and the iodide 15 (30-35%) were obtained from the nitrile 13, together with the 2,5-anhydro-~-idononitrile 16, which was formed in cu. 35-45 %. The tosylate 19 was obtained from 18 (97%). To obtain 18, the nitrile 13 was oxidized according to Swern (+ 17, 92%) and then reduced (NaBH4, CeCI,), leading to 18 and 13 (92%, 18/13 93 :7). Reduction of the tetrahydropyridotetrazole 22 with LiAIH, afforded 83% of the piperidine 23, which was deprotected to (+)-1-deoxynojirimycin hydroacetate (2. AcOH, 86 %) and further converted into the corresponding hydrochloride and into the free base 2.Introduction. -A number of glycosidase inhibitors, some of them with promising biological properties, have been isolated or designed. Among them are not only polyhydroxylated piperidines (e.g. 1-deoxynojirimycin (2) [I], nojirimycin (3) [2], and related compounds), polyhydroxylated pyrrolidines (e.g. DAB 1 [3]) and indolizidines (e.g. castanospermine (4) [4] and swainsonine [5]), but also analogues of glyconolactones (glucono-1,5-lactone [6], nojirilactame 5 [7], the amidine 6 [8], acylated hydroximolactones 7 [9], and lactone hydrazones 8 [lo]). 2 R = H 3 R = O H HO OH 7 R=OCONHPh

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