Background of the Invention

This invention relates to a idine, amidrazone and amidox- i e derivatives of monosaccharides which have an unexpectedly broad spectrum glycosidase inhibitory effect. The invention further relates to methods of preparing the derivatives, novel intermediates, methods of preparing the intermediates, and the use of the derivatives to inhibit glycosidases, particularly multiple classes thereof.

Glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds and are essential for the normal growth and development of all organisms. Their vital role is reflected in their wide distribution in nature. They participate in bio¬ logically significant reactions such as the breakdown of car¬ bohydrate foodstuffs, the processing of eucaryotic glycopro- teins, the catabolism of polysaccharides and glycoconjugates, and the like. Certain glycosidase inhibitors have been found to inhibit human immunodeficiency virus (HIV) syncytium forma¬ tion and virus replication, thereby indicating their potential use as antiretroviral agents. And some glycosidases are show¬ ing promise in treating diabetes or as antiviral and antican- cer agents.

Polyhydroxylated piperidines constitute a class of natur¬ ally-occurring glycosidase inhibitors. Examples of such mono¬ saccharide analogs which contain a nitrogen atom (an endocyc- lic nitrogen) in place of the pyranose oxygen include: nojir- imycin (A) and 1-deoxynojiri ycin (B) (Inouye et al., Tetra¬ hedron, 1968, 24, 21252144), 1-deoxymannojirimycin (C) (Fel¬ lows et al, J. Chem. Soc. Chem. Co mun.. 1979, 977-8), and ga- lactostatin (D) (Miyake et al., Agric. Biol. Chem.. 1988, 52, 661-6) . These alkaloids are quite specific inhibitors of their targeted enzymes. The arrangement of the hydroxyl groups apparently determines individual enzyme specificity of these inhibitors. Compounds A and B are potent glucosidase

inhibitors that closely resemble glucose; compound C inhibits mannosidases; and compound D inhibits galactosidases. The inhibitors are believed to function by mimicking the natural substrates. Compound B has been shown to interfere with the infectivity of HIV (Gruters et al., Nature. 1987, 330, 74-7).

Since a number of glycosidases are insensitive to the na¬ turally occurring alkaloids, tremendous effort has been devot¬ ed to the development of synthetic inhibitors which may in¬ hibit those enzymes. Examples include inhibitors of: jack bean α-mannosidase (E) (Eis et al., Tetrahedron Lett. 1985, 26, 5397-8), coffee bean α-galactosidase (F) (Berno- tas et al., Carbohvdr. Res.. 1987, 167, 305-11), 0-N-acetyl- glucoaminidase (G) (Fleet et al., Chem. Lett.. 1986, 1051-4), and 0-hexosaminidase (H) (Bernotas et al., Carbohvdr. Res.. 1987, 167, 312- 6). In these polyhydroxylated piperidines, the ring oxygen in the sugar has been replaced by a nitrogen to form azasugars. Unfortunately, the analogy of using azasugars as glycosidase inhibitors is not always applicable. For example, while polyhydroxylated pyrrolidines are better inhibitors of yeast α-glucosidase, their relative activi¬ ties are reversed for a number of mouse gut disaccharides. Therefore, the actual effectiveness of a given compound as a glycosidase inhibitor of a specific enzyme and the effect on specificity by a given structural change of the compound remain unpredictable.

Other potent glycosidase inhibitors contain at the ano- meric position an exocyclic nitrogen. (Lai et al., Biochem. Biophvs. Res. Commun.. 1973, 54, 463-8) For example, glucosyl- amine inhibits both a- and 3-glucosidases.

The present invention is the result of attempting to com¬ bine into a single compound several specific features which, among many others, have been found to be present in some gly¬ cosidase inhibitors to determine whether the combination com¬ pound would prove beneficial. Thus, the compounds of this in¬ vention contain both endocyclic and exocyclic nitrogens in an

sp -hybridized functional group and also have a flattened, half-chair conformation. While previous glycosidase inhibit¬ ors have generally shown activity only against a single class of glycosidases, i.e. glucosidases, mannosidases, galactosi- dases, etc. , they have not demonstrated broad spectrum activi¬ ty across multiple classes. The a idine, amidrazone, and amid- oxime derivatives of the monosaccharide analogs of the present invention, on the other hand, have been unexpectedly found to exhibit an apparently unique and broad spectrum of glycosidase inhibitory activity which extends across class lines, i.e. in¬ dependent of the sugar analog from which they are formed.

Previous attempts at preparing amidine derivatives of glucose to form possible glycosidase inhibitors were reported by Bird et al., Can. J. Chem.. 1990, 68(2), 317-22. Bird et al. started with glucose and prepared 5-azido-2,3,4,6-tetra- O-benzyl-5-deoxy-D-gluconitrile and 2,3,4,6-tetra-0-benzyl-5- deoxy-5-trifluoroacetamido-D-gluconitrile, but was unable to convert these compounds into a protected 5-amino-5-deoxy-D- gluconitrile and to subsequently cyclize them to an amidine analog of glucose.

Accordingly, it is an object of the present invention to produce glycosidase inhibitors which combine both endocyclic and exocyclic nitrogens into an sp -hybridized functional group and also have a half-chair conformation.

It is a further object to produce novel amidine, amidra¬ zone, and amidoxime derivatives of monosaccharides.

It is a further object to develop a process for produc¬ ing glycosidase inhibiting compounds which process could gener¬ ate amidines, amidrazones or amidoximes of different glycoses by simply changing the configuration of the starting material.

These and still further objects will be apparent from the following detailed description of the invention.

Summary of the Invention

In accordance with the present invention, novel amidine, amidrazone, and amidoxime derivatives of monosaccharides, i.e. hexoses and pentoses, are synthesized from appropriate thiono- lacta precursors. The thionolactam precursors are themselves novel compounds and are prepared by the action of Lawesson's reagent on the appropriate lactams in which the hydroxyl groups have been previously protected/blocked.

Brief Description of the Drawings

Figure 1 shows the synthetic route used to prepare D-glu- coamidine of Example 1.

Figure 2 shows the chemical structures of the amidine compounds of Examples 1-7.

Figure 3 shows the chemical structures of the amidrazone compounds of Examples 8-11.

Figure 4 shows the chemical structures of the amidoxime compounds of Examples 12-15.

Figures 5a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidine of Example 1 against almond 7-glucosidase.

Figures 6a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidine of Example 1 against jackbean alpha-mannosidase.

Figures 7a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucodimethylamidine of Exam¬ ple 2 against almond 0-glucosidase.

Figures 8a and b are the graphs of (a) Hanes-Woolf Plot and (b) Y-intercepts from Hanes-Woolf Treatment plotted vs. [I] for the D-glucodimethylamidine of Example 2 against almond 3-glucosidase.

Figures 9a and b are the graphs of (a) l/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidrazone of Example 8 against almond _/ S-glucosidase.

Figures 10a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidrazone of Example 8 against jackbean α-mannosidase.

Figures 11a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidrazone of Example 8 against bovine 8-galactosidase.

Figures 12a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-mannoamidrazone of Example 9 against almond J-glucosidase.

Figures 13a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs fl] for the D-mannoamidrazone of Example 9 against jackbean α-mannosidase.

Figures 14a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-glucoamidoxime of Example 12 against almond 3-glucosidase.

Figures 15a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs [I] for the D-mannoamidoxime of Example 14 against jackbean α-mannosidase.

Description of the Preferred Embodiments

The compounds of the present invention combine a half- chair, i.e. flattened anomeric, conformation with both exocyc¬ lic and endocyclic nitrogens to form an sp ² -hybridized func¬ tional group in a monosaccharide which conformation and group produce broad spectrum glycosidase inhibitory activity.

Specific compounds of this invention are derivatives of monosaccharides and have the general formula:

wherein R is selected from hydrogen, alkyl, substituted alkyl, alkaryl, aryl, substituted aryl, aralkyl, and Q is selected from R, -NR ₂ , and -OH; wherein when Q is R the two R groups may be joined together to form a ring containing at least 2 carbon atoms; and wherein the -N=C- is part of a monosaccha¬ ride azaanalog ring.

Preferably, -NRQ together with the carbon atom to which it is attached and said nitrogen atom form a functional group selected from amidines, amidrazones, and amidoximes.

The amidines have the general formula:

wherein each R is hydrogen or a hydrocarbon group. Suitable hydrocarbon groups are generally selected from alkyl, substi¬ tuted alkyl, alkaryl, aryl, substituted aryl, aralkyl, or the two R groups are joined together to form a ring containing at least 2 carbon atoms. Preferably each R is hydrogen or alkyl

'1-6'

The amidrazones have the general formula:

wherein R ¹ and R ² are each hydrogen or a hydrocarbon group. Suitable hydrocarbon groups are generally selected from alkyl, substituted alkyl, alkaryl, aryl, substituted aryl, aralkyl, or the two R ² groups are joined together to form a ring containing at least 2 carbon atoms.

The amidoximes have the general formula:

wherein R ³ is hydrogen or a hydrocarbon group. Suitable hy¬ drocarbon groups are generally selected from alkyl, substitut¬ ed alkyl, alkaryl, aryl, substituted aryl, and aralkyl.

Preferred numbers of carbon atoms and substituents for the various hydrocarbon groups in each of the above formulae include: alkyl from 1 to about 18 carbon atoms, substituted alkyl from about 1 to 18 carbon atoms and wherein the substitu¬ ents do not substantially interfere with the glycosidase inhib¬ itory action of the compounds, alkaryl from 7 to about 18 car¬ bon atoms and which may also be substituted, aryl from 6 to 18 carbon atoms, substituted aryl from 6 to about 18 carbon atoms and wherein the substituents do not substantially interfere with the glycosidase inhibitory action of the compounds, aral¬ kyl from 7 to about 18 carbon atoms. Specific examples of use¬ ful substituents include: hydroxypropyl, hydroxybutyl, naph- thyl, 2-furyl, imidazolyl, p-methoxyphenyl, p-fluorophenyl, shingosinyl, phenacylmethyl, and the like.

The terminal N of the amidrazones is reactive and thus, either before or after the formation of the amidrazone, it may be reacted with acid halides, anhydrides, esters, isocyanates, isothiocyanates, and the like in a conventional manner. Simi¬ larly, the -OH group which is part of the amidoxime functional-

ity of the amidoximes (as opposed to the monosaccharide hydrox- yls) is reactive and may be etherified or esterified either be¬ fore or after formation of the amidoxime.

In the azaanalog derivatives of monosaccharides of this invention, the —N=C— portion of the hexose or pentose ring (in which the usual oxygen atom of a monosaccharide has been replaced by a nitrogen atom) also forms part of the amidine, amidrazone, or amidoxime functional group.

Monosaccharides on which the present analog derivatives are based are hexoses or pentoses which form cyclic struc¬ tures. Suitable monosaccharides include the glucose and ri- bose families as well as the stereoisomers, deoxy and substi¬ tuted derivatives thereof. Specific monosaccharides upon which the azaanalog derivatives of this invention may be based include, for example, (i) aldohexoses: altrose, allose, man- nose, glucose, galactose, talose, gulose, idose, fucose, 2-de- oxy-2-aminoglucose, 2-deoxy-2-aminogalactose, and the like; (ii) aldopentoses: ribose, arabinose, 2-deoxyribose, 2-deoxya- rabinose, and the like. Preferred such monosaccharides are mannose, glucose, galactose, fucose, 2-deoxy- 2-aminoglucose, 2-deoxy-2-aminogalactose, ribose, arabinose, 2-deoxyribose, and 2-deoxyarabinose. The currently most preferred monosaccha¬ rides are glucose, mannose, and galactose due to the ready availability of lactams thereof which are starting materials for the preparative part of this invention.

The preparation of the compounds of the invention will be described herein commencing from the lactams of a monosac¬ charide. Lactams are organic compounds which contain an -NH-CO- group within a ring. They are generally formed by the elimination of water from neighboring carboxyl and amino groups. The monosaccharide lactams of the present invention are therefore cyclic amides having the general structure:

The reactions used herein to convert a lactam to the corre¬ sponding amidines, aβidraxonββ, and anidoxiaea of the inven¬ tion are independent of both the -stereochemistry and the sice of the balance of the monoβaccharida ring. Thus the present invention is applicable to any nonosaccharide. Hunerous refer¬ ences to the preparation of lactams of nonosaccharides have been reported in the recen chemical literature. The refer¬ ences include: Inoύye et al.. Tetrahedron. 1968, 24(5), 2125- 44; Hane ^β sian, J. Ore. Cham.. 1969, 34(3), 675τ81; Ito et al. _f JP 46-024,382. 19 ij Tsuruoka et al. _f Meiii Seika Kenkyu Hssα≥a, 1973, Mo. 13, 80-4; Hiva et al., U.S. Pat. 3,956,337; T ^β uruoka et al-, Jt> 50-129,572; Miwa et al. , J. Antibiot.. 1984, 37(12), 1579+86; Ehata et ali, JP 61-280,472? Miyaike et al., fterric. Biol. fehein. _r 1988, 52(3), 661-6; Shing, J. Chem. Soc. Chem. GoBθtinn _fl . 1988, 18, 1221; Fleet et al., Tetrahedron. 1989, 45(1) 319-26J Tβ ruoka et al., JP 63-258,421 and JP 63- 216,867; Fleet et al., Tetrahedron Lett.. 1990, 31(3), 409~12.

λ particularly suitable procedure for preparing D-gluco- nolactam, i.e. the lactam of D-glucpse, by the oxidation of no- jirimycin is described in Example 1 below. This procedure re¬ presents an improved oxidation procedure over previous proced¬ ures in that the yield obtained ie si «gnificantly higher than the published yield, i.e. about 50% vs. about 30*.

Other monosaccharide lactams may be prepared in accord¬ ance vith one or more preparative procedure cited above.

The hydrσxyl groups of the monosaccharide lactams are then protected froή reaction vith ^" Lawesson's reagent which is subsequently used to form thionolactaπs which are then reacted

with NH-containing compounds to yield the desired functional compounds. Suitable blocking/protecting groups are those that, for a particular lactam, do not participate in neighbor¬ ing group interactions which result in the formation of unde- sired compounds. Preferably, the groups will also be remova¬ ble under relatively mild conditions. Examples of such suit¬ able groups include trimethylsilyl, acetyl, t-butyldimethyl- silyl, trityl, methoxymethyl , benzyl, and benzoyl.

The protecting groups may be incorporated onto the lac¬ tams by any conventional suitable manner. For example, tri¬ methylsilyl groups may be produced by suspending the lactam in a non-solvent, such as dry pyridine; adding a mixture of chlor- otrimethylsilane and hexamethyldisilazane; stirring at room temperature; and then recovering the resulting persilylated lactam. Acetyl groups may be provided by peracetylation with Ac ₂ 0, NaOAc, at room temperature for extended periods.

The protected hydroxyl group-containing lactam is then converted to the corresponding thionolactam by reaction with Lawesson's reagent (p-methoxyphenylthionophosphine sulfide dimer) in a conventional manner, i.e. by forming a solution of the protected lactam in a solvent such as benzene under an in¬ ert gas such as argon at room temperature and then heating to reflux. Lawesson's reagent and its use are described in Schei- bye et al., Bull. Soc. Chem. Belq. , 1978, 87, 229-38. The re¬ action with Lawesson's reagent converts the C=0 of the lactam to a C=S of the thionolactam without disturbing the protecting groups.

The protecting groups are then removed in a conventional manner such as by acidic hydrolysis. Any sufficiently strong acid which does not effect the balance of the compounds may be used. Suitable such acids include hydrochloric acid, sulfuric acid, perchloric acid, fluoroboric acid, p-toluenesulfonic acid, and the like. Other methods of removing protecting groups include hydrogenolysis, alcoholysis, and fluoride treat¬ ment. Alternatively, when the final products are sufficiently

stable the protecting groups may be allowed to remain on the thionolactam during reaction with the NH-compound and then re¬ moved as discussed.

The thionolacta s of the monosaccharides produced are be¬ lieved novel compounds themselves. They are useful intermedi¬ ates in the preparation of the glycosidase inhibitors. Specif¬ ically, depending upon the particular NH-containing compound with which they are reacted, they may yield amidine, amidra¬ zone, and amidoxime derivatives.

Thus, the glycosidase inhibitor compounds of the present invention are ultimately synthesized by reaction of monosaccha¬ ride thionolactam precursors of the general formula:

with an NH-containing compound, preferably in solution. Suit¬ able solvents include water, alcohols, acetonitrile, and the like. Preferably the solvent is methanol, ethanol, isopropyl alcohol or acetonitrile. Suitable temperatures will range from about O'C. to reflux.

The amidine derivatives are readily prepared by reacting the thionolactam with ammonia or the appropriate mono- or di- substituted amine. Suitable such amines or ammonia are those of the formula NHR ₂ wherein each R is independently selected from hydrogen, alkyl, substituted alkyl, alkaryl, aryl, substi¬ tuted aryl, aralkyl, or the two R groups are joined together to form a ring contain at least 2 and generally less than about 8 carbon atoms.

The amidrazone derivatives are prepared in like manner to the amidines but with the thionolactam being reacted with

hydrazine or a hydrazine derivative, generally of the formula NHR ¹ -NR ² ₂ wherein R ¹ and each R ² are individually se¬ lected from hydrogen, alkyl, substituted alkyl, alkaryl, aryl, substituted aryl, aralkyl, or the two R ² groups are joined together to form a ring contain at least 2 carbon atoms and generally less than about 8 carbon atoms. Either before or af¬ ter formation of the amidrazone, the terminal N of the hydra¬ zine compound may be reacted with an acid halide, anhydride, ester, isocyanate, isothiocyanate or the like.

The amidoxime derivatives are prepared in like manner to the amidines and amidrazones but with the thionolactam being reacted with a hydroxyamine of the general formula NHR OH wherein R ³ is selected from hydrogen, alkyl, substituted alkyl, alkaryl, aryl, substituted aryl, and aralkyl. Either before or after formation of the amidoxime, the OH-group may be esterified or etherified by reaction with an acid halide, anhydride, ester, isocyanate, isothiocyanate or the like.

While the degree of glycosidase inhibitory activity on a particular glycosidase enzyme or series of glycosidase enzymes is likely to at least partially depend upon the specific R groups which are provided on specific compounds, the princi¬ ples of this invention have not been found dependent thereon. Preferred numbers of carbon atoms and substituents for the var¬ ious groups in each of the above formulae include: alkyl from 1 to about 10 carbon atoms, substituted alkyl from about 1 to 10 carbon atoms and wherein the substituents do not substan¬ tially interfere with the glycosidase inhibitory action of the compounds, alkaryl from 7 to about 18 carbon atoms and which may also be substituted, aryl from 6 to 18 carbon atoms, sub¬ stituted aryl from 6 to about 18 carbon atoms and wherein the substituents do not substantially interfere with the glycosi¬ dase inhibitory action of the compounds, aralkyl from 7 to about 18 carbon atoms. Examples of specific substituents in¬ clude: halogens, alkyl, aryl, alkoxy, fluoro, cyano, carboal- koxy, reto, thio, nitro, and the like.

The glycosidase inhibitors of this invention are general¬ ly useful with a broad spectrum of glycosidase enzyme classes as opposed to a single class. While the general inhibitory strength of a particular compound will vary depending upon its particular structure and the enzyme, the compounds of this invention have exhibited potent inhibitory effects against glu- cosidases, mannosidases, and galactosidases. Other classes of glycosidase enzymes for which the inhibitors are likely to be useful include one or more of: L-fucosidases, sialidases, glu- cosaminidases, chitinases, lysozyme, cellulases, and the like. Specific glycosidase enzymes which have been inhibited by one or more of the claimed compounds include yeast α-glucosi- dase, asper illus niqer amyloglucosidase, almond 3-glucosi- dase, jackbean α-mannosidase, green coffee bean α-ga- lactosidase, and bovine liver 3-galactosidase.

While the D-glucoamidines have been found to be potent inhibitors against gluco, manno and galactosidases, they have also proved to be unexpectedly labile, undergoing rapid hydrol¬ ysis above pH 7. The amidrazone and amidoxime derivatives are much more stable and they were unexpectedly found to be gener¬ ally even more potent inhibitors, further emphasizing the im¬ portance of binding interactions with the anomeric region of the glycosyl cation. The amidoximes represent the first near- neutral monosaccharide analogs possessing the half-chair con¬ formation of the glycosyl intermediates.

GENERAL EXPERIMENTAL INFORMATION Proton NMR spectra were taken on a Bruker WM-300 spec¬ trometer. All chemical shifts were reported on the S scale in parts per million downfield from Me ₄ Si. Spectra taken in CDC1 ₃ were referenced to either Me ₄ Si (0.00 for compounds with aromatic protons) or residual CHC1 ₃ (7.24, for compounds without aromatic protons) . Spectra taken in D ₂ 0 were referenced to HOD (4.67), and those in CD ₃ OD were referenced to CHD ₂ 0D (3.30). Carbon-13 NMR spectra were taken on a Varian XL-400 (100 MHz) , Bruker WM-300 (75 MHz)

spectrometer and referenced to p-dioxane (66.5 ppm) or CH ₃ OH (49.0 ppm). Infrared spectra were taken on a Mattson Galaxy Model infrared specto eter. Ultraviolet absorption spectra were measured on a Hewlett-Packard HP 8451 A Diode Array Spec¬ trometer. Mass spectra were obtained from a Finnigan 3300 mass spectrometer. Chemical ionization spectra were obtained using isobutane as reagent gas; electron impact spectra were run at 70 e V ionizing voltage. Fast atom bombardment spectra were obtained in glycerol matrix on a Kratos MS-890 Spectrom¬ eter. Optical rotations were measured on a Perkin Elmer 241 polarimeter. Sample concentrations were expressed in grams of sample per 100 cc of solvent. High performance liquid chroma¬ tography was performed with an Eldex 9600 system using a μ- Porasil column (internal diameter 7.8 mm).

Anhydrous methanol was prepared by distillation from Mg(OCH ₃ ) ₂ . Benzene, pyridine, chlorotrimethylsilane and hexamethyldisilane were dried and distilled from CaH ₂ . All enzymes were obtained from Sigma and used as it.

In the following non-limiting examples, all parts and percents are by weight unless otherwise specified. Also, the compounds produced were generally isolated as their quaternary acetate salts, though other salts may be substituted in a con¬ ventional manner.

EXAMPLE 1 PREPARATION OF GLUCOAMIDINE

Oxidation of (+)-Noiirimycin to (3R,4S,5R,6R'ι-6-Hydroxγ- methyl-3,4.5-trihydroxy-2-piperidone TD- (+)-Gluconolactam

To a stirred suspension of nojirimycin bisulfite addi¬ tion product (1.02 g, 4.22 mmol; Baeyer AG) in distilled water (25 mL) was added activated Dowex 1 x 2-200 resin (HO-form, 10 g, Aldrich) to make the pH 8-10. After stirring 30 min at room temperature, the resin was filtered, rinsed with distill¬ ed water (160 mL) and the combined filtrates lyophilized to afford a crude sample of (+)-nojirimycin (0.89 g, R _f 0.58 in

4:1 ethanol:H ₂ 0) which was dissolved in distilled Water (15 mL) and used immediately in the next step.

The magnetically stirred aqueous nojirimycin solution was treated with alternating portions of 0.1 M I ₂ -0.5 M KI solution (83 mL; 2 mL aliquots) and 0.1 M NaOH (100 mL, 2.5 mL aliquots) at room temperature slowly over a period of 90 min. After 24 hours, the brown solution was decolorized by addition of aqueous NaHS0 ₃ (1 M, 4 mL) , then Amberlite IR-120 (H+) resin was added (0.5 g, Aldrich) to bring the pH to 1. After stirring 4 hr at room temperature, the resin was filtered and rinsed with water (50 mL) . The combined filtrates were then neutralized with Dowex MWA-1 (Serva Corp.), the resin filtered and washed, and the combined filtrates (ca. 400 mL) concentrat¬ ed at the rotary evaporator to afford the crude lactam as a white solid. A small portion was chromatographed and recrys- tallized from ethanol:water to afford pure lactam whose mp (203 ^β C) and chiroptical properties were identical with publish¬ ed values.

Silylation of Lactam

Crude (5-6 g) was suspended in dry pyridine (20 mL) , then (trimethylsilyl) ₂ NH(5 mL) and trimethylsilylchloride (3 L) was added and the dark brown reaction mixture stirred at room temperature for 90 minutes. Concentration in vacuo af¬ forded a brownish foam (1.6 g) which was flash chromatographed on Si0 ₂ (40 mm x 4.5 inch column; 7:1 hexanes:ethyl acetate; 8 mL fractions collected) to furnish the persilylated glucono- lactam (1.14 g, 2.46 mmol, 50%): R _f 0.27 (1:7 hexanes:ethyl acetate); [α] _D + 71° (c=0.54, CHC1 ₃ ) ; ^■ '"H-NMR δ

(CDC1 ₃ ) 5.88 (br. s, 1H) , 3.87 (d, 1H, J=8.6 Hz) , 3.78 dd, 1 H, J=8.3, 1.8 Hz), 3.69 (dd, 1 H, J=8.7, 8.6 Hz) , 3.43 (dd, 1 H, J=8.4, 8.4 HZ), 3.35-3,25 (m, 2H) , 0.18 (s, 9 H) , 0.14 (s, 18 H) , 0.09 (s, 9 H) ; ¹³ C-NMR (CDCI ₃ ) 170.9, 76.2, 74.0, 71.4, 63.7, 57.0, 0.91, 0.73, -0.70; IR (film) 3210, 3110, 2980, 2905, 1690, 1320, 1250, 1130, 950, 840 cm ^"1 ; CIMS (methane) m/e 478 (M+2, 65%) 466 (M+l, 50%) 450 (M ⁺ -CH ₃ , 100%) .

Formation of D-Glucothionolactam

(3R,4S,5R,6R)-6-Hydroxymethyl-3,4,5-trihvdroxy-2-thiono- pjperidone

To a solution of the persilylated lactam of above (0.284 g, 0.61 mmol) in benzene (15 mL) under argon at room tempera¬ ture was added Lawesson's reagent (p-methoxyphenylthionophos- phine sulfide dimer, 0.148 g, 0.6 equiv) and the suspension was warmed. At 65°C. the reaction became homogeneous and was brought to reflux for 30 in. After concentrating in vacuo, the residue was dissolved in CH3OH (16 mL) , acidified (1:9 cone. HC1:CH ₃ 0H, 10 drops) and stirred for 35 minutes at room temperature. Concentration afforded a white solid (0.25 g) which was flash chromatographed over Si0 ₂ (20 mm x 6 inch column; 7:3:1 CH ₂ C1 ₂ : CH ₃ OH:NH ₄ OH, 3 mL fractions) to afford slightly impure glucothionolactam (0.105 g) which was further purified as follows.

The thionolactam (0.105 g) was dissolved in distilled water (10 mL) and stirred with Norit A (1.0 g) for 30 minutes. The Norit was filtered, rinsed with water (10 mL, discarded) , then eluted with 1:1 ethanol:H ₂ 0 and the eluant concentrated in vacuo to give analytically pure thionolactam (74 mg, 0.38 mmol, 63%): R _f 0.33 (7:3:1 CH ₂ Cl ₂ :CH ₃ OH:NH ₄ OH) mp 126-128°C; [α] _D + 31°(c=0.72, CH3OH) -"Η-NMR S (D ₂ 0) 3.90 (d, 1 H, J=9.5 Hz) 3.78 (dd, 1 H, J=12.3, 2.4 Hz), 3.74 (dd, 1 H, J=9.8, 8.7 Hz), 3.67 (dd, 1 H, J=12.4, 4.1 HZ), 3.60 (dd, 1 H, J= 9.7 Hz) , 3.33 (m, 1 H) ; ¹³ C-NMR (D ₂ 0, acetone ref.) 214.8, 74.2, 72.6, 67.4, 61.6, 59.7; IR ( Br) 3360, 2940, 2900, 1660, 1560, 1450, 1292, 1075, 1030 cm ^""1 .

High Resolution FAB-MS:

Calculated for C ₆ H _1;L N0 ₄ S: 193.0409

Found: 193.0403.

Analysis for C ₆ H ₁₁ N0 ₄ S:

Calculated: C, 37.29; H, 5.74; N, 7.25; S, 16.56

Found C, 36.17; H, 5.87; N, 7.22; S, 13.94

Preparation of D-Glucoamidine

Saturated anhydrous NH ₃ in methanol (1.5 mL) was added dropwise to a stirred solution of the thionolactam (14 mg, 0.072 mmol) in anhydrous CH ₃ OH (1 mL) at room temperature under argon. Thin layer chromatographic monitoring indicated that the thionolactam disappeared in 11 hours and two new spots were visible (baseline and R _f 0.08 in 7:3:1 CH ₂ Cl ₂ :CH ₃ OH:NH OH) . The solution was concentrated in vacuo. redissolved in fresh CH ₃ OH (2.5 mL) and acidified to pH 3.5 with anhydrous HC1- CH ₃ OH (0.5 mL, prepared from 0.3 mL AcCl in 8 mL CH3OH) . Concentration in vacuo afforded a brown oil which was purified by Sio ₂ flash chromatography (8 mm x 2.5 inch column; 20:4:1 CH ₃ CN:H 0:HOAc, 2 mL frac¬ tions) to afford the amidine HOAc (11.6 mg, 68%): R _f 0.14 (20:4:1 CH ₃ CN:H ₂ 0:H0AC) ; [α] _D +27° (c=0.17, CH3OH) ; ¹ H-NMR δ (D ₂ 0) 4.24 (d, 1 H, J=9.6 Hz), 3.77-3.56 (m, 4 H) , 3.34 (m, 1 H) 1.94-1.76 (S, e H) , ¹³ C-NMR (d ₂ 0, diox- ane ref), 179.0, 167.5, 72.0, 68.2, 67.3, 60.1, 59.3, 21.9; IR (KBr) 3390, 3240, 2930, 1685, 1575, 1555, 1415, 1075 cm ^"1 .

High Resolution FAB-MS:

Calc. for C ₆ H ₁₃ N ₂ 0 ₄ : 177.0875;

Found: 177.0875.

EXAMPLE 2 PREPARATION OF D-GLUCO-N.N-DIMETHYLAMIDINE Anhydrous dimethylamine was bubbled into dry CH3OH (5 mL) under argon at 0°C. until the volume of the solution doub¬ led. Four milliliters of this solution was transferred by Teflon cannula to the thionolactam of Example 1 (11 mg, 0.057 mmol) and the resulting solution was stirred 10 minutes at 0°C before warming to room temperature. Thin layer chromatograph¬ ic monitoring indicated that the thionolactam disappeared in 8 hours and one new spot was visible (R _f 0.17 in 20:4:1 CH ₃ CN:H ₂ 0:HOAc) . The solution was concentrated in vacuo. redissolved in fresh CH ₃ 0H (3 L) and acidified under argon to pH 3.0 with anhydrous HCI-CH ₃ OH (0.4 mL, prepared from 0.3 mL AcCl in 8 mL CH3OH) . After concentrating in vacuo

the residue was purified by Si0 ₂ flash chromatography (8 mm x 2.5 inch column; 20:4:1 CH ₃ CN: H ₂ 0:HOAc, 2 mL fractions) to afford the amidine-HOAc (10.6 mg, 71%): R _f 0.21 (20:4:1

CH ₃ CN:H ₂ 0:H0AC) ; [α] _D + 15° (c=0.57, CH ₃ OH) ;

^ ^• H-NMR δ (D ₂ 0) 4.46 (d, 1 H, J=7.0 Hz), 3.86 (dd, 1 H,

J=11.6, 9.0 HZ), 3.77 (dd, 1 H, J=9.0, 7.0 Hz) , 3.71 (dd, 1 H,

J=12,2, 3.8 Hz) 3.60 (dd, 1 H, J=11.6, 9.0 Hz), 3.45 (m, 1 H) ,

3.21 (s, 3H) , 3.04 (s, 3H) ; ¹³ C-NMR (D ₂ 0, dioxane ref)

179.7, 163.2, 74.5, 68.9, 65.9, 58.9, 58.6, 41.7, 39.4, 22.2;

IR (KBr) 3320, 2925, 1660, 1565, 1420, 1060 cm ^"1 .

High Resolution FAB-MS:

Calculated for C ₈ H ₁₆ N ₂ 0 ₄ : 204.1111; Found: 204.1110.

EXAMPLE 3 PREPARATION OF D-MANNOAMIDINES L-gluconolactone was converted to D-mannolactam by the procedure of Fleet (1989) . As described in detail in Example 9 below, hydroxyl groups of the lactam were blocked with tri- ethylsilyl groups, and the lactam converted to the thionolac¬ tam. Then the amidine was formed by reaction of the thionolac¬ tam with ammonia in methanol at room temperature and the block¬ ing groups removed, all as in Example 1 for D-gluconolactam.

A portion of the D-mannothionolactam is also reacted with diethylamine to produce the corresponding D-manno-N,N-di- ethylamidine.

EXAMPLE 4 PREPARATION OF D-GALACTOAMIDINES The procedure of Example 1 is repeated to produce two D-galactoamidines by reacting D-galactothionolactam (produced from galactostatin) in one case with ammonia and in the other with benzylamine. The resulting compounds are D-galactoamid- ine and D-galacto-N-benzylamidine.

EXAMPLE 5 PREPARATION OF L-IDOAMIDINE The procedure of Example 1 is repeated to produce two L-idoamidines by reacting L-idothionolactam (produced by the procedure of Ito et al., JP 46-024382) in one case with ammonia and in the other with diphenylamine. The resulting compounds are L-idoamidine and L-ido-N,N-diphenylamidine.

EXAMPLE 6 PREPARATION OF D-GLUCOEPIMINOAMIDINE The procedure of Example 1 is repeated to produce D-glu- coepiminoamidine by reacting D-glucothionolactam with ethylene- imine in ethanol. The resulting compound is D-glucoepiminoami- dine which has the exocyclic nitrogen atom in a three membered ring with two carbon atoms.

EXAMPLE 7 PREPARATION OF RIBOAMIDINES The basic procedure of Example 1 is repeated to produce two D-riboamidines by reacting D-ribothionolactam (produced by the procedure of Hanessian, J. Orq. Chem. , 1969, 34 (3), 675- 81) in one case with ammonia and in the other with dimethyl- amine. The resulting compounds are D-riboamidine and D-ribo- N,N-dimethylamidine.

EXAMPLE 8 PREPARATION OF D-GLUCOAMIDRAZONE Anhydrous NH ₂ NH ₂ (70 μL, 2.208 mmol, distilled from NaOH) was added dropwise to a stirred solution of D-gluco- nothionolactam (20.5 mg, 0.106 mmol) in anhydrous CH ₃ OH (3 mL) in an ice-H 0 bath under Ar. TLC monitoring (CH ₃ CN:- OAc:H ₂ 0 20:1:4) indicated that the thionolactam disappeared after 90 min. The solution was concentrated in vacuo and the residue (24 mg) was purified by Si0 ₂ chromatography (3.5 in x 12 mm column; 25:3:1 CH ₃ CN:H ₂ 0:HOAc, 2 L fractions) to afford D-glucoamidrazone HOAc (19.3 mg, 73%): Rf=0.33 (CH ₃ CN:AcOH:H ₂ 0 10:1:4) j ] _D + 15.6" (c=0.45, MeOH) ; ¹ H-NMR δ (D ₂ 0) 4.27 (m, 1 H, J=2.3, 6.9 Hz) , 3.81 (dd.

1 H, J=2.8, 12.2 Hz), 3.72-3.67 (m, 3 H) , 3.41 (m, 1 H) , 1.78 (s,3 H) ; ¹³ C-NMR (D ₂ I, dioxane ref.), 179.1, 164.4, 72.4, 67.7, 60.2, 59.1, 22.2, IR (KBr) , 3310, 2920, 1700, 1665, 1560, 1410, 1110, 1070, 1020 cm ^"1 .

High Resolution FAB-MS:

Calc. for C ₆ H ₁₄ 0 ₄ N ₃ : 192.0984

Found: 192.0989

EXAMPLE 9 PREPARATION OF D-MANNOAMIDRAZONE Preparation of D-Mannonothionolactam

D-Mannolactam (0.203 c, 1.14 mmol) was dissolved in dry pyridine (15 mL) , then (TMS) ₂ NH (5 mL) and TMSC1 (2.5 mL) were added dropwise. The resulting suspension was stirred at room temperature under Ar for 90 min. Concentration in vacuo afforded a white residue (613 mg) which was triturated with hexanes (60 mL in 3 mL fractions) . The combined triturants were concentrated using a rotary evaporator to yield the cor¬ responding persilylated lactam (0.533 g, 93%): Rf=0.20 (hex- ane:EtOAc 7:1); ¹ H-NMR δ (CDC1 ₃ ) 6.11 (s, 1 H, broad) 4.3 (d, 1 H, J=2.7 Hz), 3.76 (dd, 1 H, J=4.5, 2.7 Hz), 3.7 (m, 1 H) , 3.56-3.51 (m, 2 H) , 3.26 (m, 1 H) , 0.12-0.0 (m, 36 H) ; IR (CHC1 ₃ ), 3390, 2970, 1675, 1460, 1310, 1250, 1100 cm ^"1 ; ¹³ C-NMR δ (CDCI ₃ ) 170.5, 74.9, 69.3, 68.8, 64.1, 60.6, 0.10, 0.05, -0.21, -0.87. To a solution of the persilylated mannolacta (176 mg, 0.378 mmol) in benzene (12 mL) under Ar was added Lawesson's reagent (136 mg, 0.337 mmol) and the sus¬ pension was heated to reflux for 1 h. After concentrating the homogeneous reaction mixture in vacuo, the residue was suspend¬ ed in CH3OH (10 mL) , acidified [8 drops from a solution of CH ₃ OH (10 L) and AcCl (0.3 mL) ] whereupon it became homogen¬ eous after 90 minutes at room temperature. Concentration af¬ forded a white solid (230 mg) which was triturated with CHCI ₃ (30 L in 2 mL portions). The residue (81.2 mg) was purified by Si0 ₂ chromatography on a 5 inch x 15 mm column eluting with CH ₃ CN:H ₂ 0:AcOH 200:4:1 (3 mL fractions) to afford D-mannothionolactam (38 mg, 53%): Rf=0.30 (CH ₂ C1 ₂ :

CH ₃ OH:NH ₄ OH 7:3:1); [α] _D + 53.3° (c=0.69, MeOH) ;

^" " ^• "H-NMR δ (D ₂ 0) 4.29 (d, 1 H, J=3.7 Hz) , 3.94 (dd, 1

H, J=3.8, 5.4 Hz), 3.8 (dd, 1 H, J=12.0, 3.6 Hz), 3.77 (dd, 1

H, J=5.3, 7.1 HZ), 3.66 (dd, 1 H, J=5.6, 12.0 Hz) , 3.32 (m, 1

H) ; ¹³ C-NMR δ (D ₂ 0, dioxane ref.) 202.8, 72.7, 72.0,

67.7, 60.9, 60.5; IR (KBr) 3390, 2910, 1620, 1540, 1390, 1120,

1060; CIMS 194 (M+l, 100%); EI-MS 193 (M ⁺ , 100%), 157 (96%),

140 (29%), 139 (26%), 111 (54%), 102 (32%).

Preparation of D-Mannoamidrazone Anhydrous NH ₂ NH ₂ (60μL, 1.89 mmol, distilled from NaOH) was added dropwise to a stirred solution of the D-manno-thionolactam above (14 mg, 0.072 mmol) in anhydrous CH3OH (3 mL) in an ice-H 0 bath under Ar. TLC monitoring (CH ₃ CN:AcOH:H 0 20:1:4) indicated that the starting materi¬ al disappeared in 90 minutes. The solution was concentrated in vacuo and the residue (16 mg) was purified by Si0 ₂ chroma¬ tography (17 mm x 6 cm column; 25:3:1 CH ₃ CN:H ₂ 0:AcOH 2 mL fraction) to afford D-mannoamidrazone HOAc (14 mg, 75%) : Rf= 0.35 (CH ₃ CN:H ₂ 0:AcOH 10:4:1); [α] _D + 10.9° (c=0.46, CH3OH) ; ¹ H-NMR δ (D ₂ 0) 4.64 (d, 1 H, J=3.4 Hz) , 3.97 (dd, 1 H, J=3.6, 4.8 HZ), 3.89 (dd, 1 H, J=4.9 Hz) , 3.78 (dd, 1 H, J=4.5, 11.8 HZ) 3.67 (dd, 1 H, J=5.9, 11.8 Hz) , 3.37 (m, 1 H) , 1.84 (s, 3 H) ; ¹³ C-NMR δ (D ₂ 0, dioxane ref.) 179.1, 164.1, 70.8, 67.5, 64.7, 61.1, 58.2, 22.1; IR (KBr) 3290, 2930, 1705, 1575, 1410, 1350, 1120, 1065, 101 cm ^"1 .

High Resolution FAB-MS:

Calc. for C ₆ H ₁₄ 0 ₄ N ₃ : 192.0984

Found: 192.0980

EXAMPLE 10 PREPARATION OF D-GALACTO-N.N-DIMETHYLAMIDRAZONE The procedure of Example 4 is repeated to produce D-ga¬ lactothionolactam which is reacted with 1, 1-dimethylhydrazine as in Example 8 to produce D-galacto-N,N-dimethylamidrazone.

EXAMPLE 11 PREPARATION OF RIBOAMIDRAZONE

The procedure of Example 8 is repeated to produce D-ri¬ bothionolactam which is reacted with hydrazine as in Example 8 to produce D-riboamidrazone.

EXAMPLE 12 PREPARATION OF D-GLUCOAMIDRAZONE-N-PHENYLUREA The procedure of Example 8 is repeated except that the hydrazine is replaced by an equivalent amount of 4-phenylsemi- carbazide (NH ₂ -NH-CONHPh and the solvent is acetonitrile. Reaction readily occurs to produce the D-glucoamidrazoneN-phen- ylurea.

EXAMPLE 13 PREPARATION OF D-GLUCOAMIDOXIME Anhydrous hydroxylamine (200 μL of a 1.25 M CH ₃ OH solution) was added under Ar to a stirred solution of D-gluco- nothiolactam (10 mg, 0.052 mmol) in CH3OH (2.5 mL) . After 14 hours at room temperature, the lactam was completely consumed and a new spot had appeared at Rf 0.14 (CH ₂ C1 ₂ : CH ₃ OH:NH ₂ OH 7:3:1). The solution was concentrated in vacuo and the residue (17 mg) was purified by Si0 ₂ chromatography (3" x 16 mm column; CH ₃ CN:H ₂ 0:HOAc 30:3:1, 2 mL fractions) to afford D-glucoamidoxime HOAc (10 mg, 75%) : Rf 0.41 (CH ₃ CN:H ₂ OHOAc 10:4:1); [α] _D + 62°(c=0.39, CH3OH) ; ---H-NMR δ (D ₂ 0) 4.49 (d, 1 H, J=10.1 Hz), 4.18 (dd, 1 H, J=2.2 Hz) , 3.86 (dd, 1 H, J=2.3, 10.1 Hz), 3.69 (m, 3 H) , 1.83 (s, 3 H) ; ¹³ C-NMR δ (D ₂ 0, CH3OH ref.) 179.1, 156.4, 74.3, 68.4, 60.9, 57.8, 22.0,; IR (KBr) 3400, 2920, 1660, 1590, 1570, 1420, 1335, 1110, 1020 cm ^"1 .

High Resolution FAB-MS C Caallcc.. ffoorr ^c C6 ₆ ^H Hi ₁ 3 ₃ °05 ₅ N ₂ : 193.0824 Found: 193.0821

EXAMPLE 14 PREPARATION OF D-GLUCO-N-METHYLAMIDOXIME The procedure of Example 13 is repeated except that the hydroxylamine is replaced by an equivalent amount of N-methyl- hydroxylamine to produce the D-gluco-N-methylamidoxime.

EXAMPLE 15 PREPARATION OF D-MANNOAMIDOXIME Anhydrous hydroxylamine (prepared as above, 280 μL) was added under Ar to a stirred solution of mannonothiolactam IX (12.2 mg, 0.063 mmol) in CH3OH (2.5 mL) . After 24 hours at room temperature, the D-mannolactam was completely consumed and a new spot had appeared at Rf 0.10 (CH ₂ C1 ₂ : CH3OH:- NH ₂ OH 7:3:1). The solution was concentrated in vacuo and the residue (16 mg) was purified by Si0 ₂ chromatography (2" x 16 mm column: CH ₃ CN:H ₂ 0:HOAc 30:3:1, 1.5 mL fractions) to afford D-mannoamidoxime HOAc (11.6 mg, 73%): Rf 0.60 (CH ₃ CN:H ₂ 0:HOAc 10:4:1); [α] _D -1.0° (c=0.4, CH ₃ OH) ; ^■ ' ^■ H-NMR δ (D ₂ 0) 4.64 (d, 1 H, J=2.2 Hz) , 3.38 (m, 1 H) , 1.95 (s, 3 H) ; ¹³ C-NMR δ (D ₂ 0, CH ₃ OH ref.) 178.8, 156.0, 71.6, 66.5, 66.4, 61.7, 58.1, 21.8,; IR (KBr) 3390, 2930, 1660, 1560, 1545, 1420, 1340, 1100, 1055, 1010 cm ^"1 .

High Resolution FAB-MS:

Calc. for C ₆ H ₁₃ 0 ₅ N ₂ : 193.0824

Found: 193.0825

EXAMPLE 16 PREPARATION OF L-IDOAMIDOXIME The procedure of Example 5 is repeated to produce L-ido- thionolactam which is reacted with hydroxylamine as in Example 12 to produce L-idoamidoxime.

EXAMPLE 17 PREPARATION OF D-GLUCOAMIDOXIME-N-PHENYLURETHANE The D-glucoamidoxime of Claim 13 is reacted with phenyl isocyanate in acetonitrile at a temperature ranging from 0°C. to room temperature to substantially quantitatively produce

the D-glucoamidoxime-N-phenylurethane.

GENERAL BIOLOGICAL PROCEDURES

A. Preparation of Solutions for Enzyme Assays Phosphate Citrate Buffers — Phosphate-citrate buffers at constant ionic strength (0.5 M) were prepared according to a published procedure. (2)

HOAc/NaOAc Buffer — To an aqueous solution of acetic acid (0.4 M, 100 mL) was added aqueous sodium acetate (0.4 M, 100 mL) . The resulting solution was adjusted to pH 5.0 by addition of aqueous sodium hydroxide (6.0 M) .

NaCl/NaOAc Buffer — A mixture of aqueous NaCl solution (0.1 M, 2.0 mL) and HOAc/NaOAc buffer (0.75 mL) was diluted with deionized water (17.25 mL) to yield NaCl/NaOAc buffer (pH 5.0) .

Glvcine Buffer — Glycine (22.47 g, 0.299 mol) was dis¬ solved in deionized water (705 L, final concentration: 0.42 M) . The resulting solution was adjusted to pH 10.4 by aqueous NaOH (6.0 M) .

pH for Enzyme Assays — Yeast α-glucosidase and green coffee bean α-galactosidase were assayed at pH 6.6. Almond ,9-glucosidase and jackbean α-mannosidase were as¬ sayed at pH 5.0. Bovine liver j8-galactosidase was assayed at pH 7.0.

Working Enzyme Solutions — Typical enzyme concentra¬ tions in the assay buffer for inhibitor screening were as fol¬ lows: yeast α-glucosidase, 10 μL of enzyme suspension in 2.00 L pH 6.6 phosphate-citrate buffer; almond /3-glucosi- dase, 25 μL of enzyme solution (0.4 mg solid enzyme in 70 μL pH 5.0 NaCl/NaOAc buffer) in 5.00 mL pH 5.0 NaCl/NaOAc buffer; jackbean α-mannosidase, 5-8 μL of enzyme sus-

pension in 2.00 mL NaCl/NaOAc buffer; green coffee bean α galactosidase, 15 μL of enzyme suspension in 1.2 mL NaCl/- NaOAc buffer; bovine liver /3-galactosidase, 1.7 mg solid en¬ zyme in 850 μL NaCl/NaOAc buffer.

B. General Assay Procedure

An acceptable concentration of enzyme, upon hydrolysis of the corresponding glycoside, should give an absorbance read¬ ing between 0.4 and 2.0 at 400 nm. A control assay was first performed to determine whether the enzyme concentration was ac¬ ceptable. The assays involved addition of working enzyme solu¬ tion (0.09 mL) and deionized water (0.18 mL) to buffer at the appropriate pH (0.09 mL, phosphate-citrate). The resulting so¬ lution was incubated at 37°C for 5 minutes. Aliquots (0.1 mL) of this preincubation mixture was then added to three separate test tubes. Substrate (the corresponding p-nitrophenyl-D-gly- coside, 0.1 mL, 10 mM in appropriate buffer), which had also been incubated at 37 ^β C, was then added to each tube at thirty- second intervals. The hydrolyses were allowed to continue for 0.25 hours and then quenched by addition of glycine buffer (2.5 mL) . The absorbance of the resulting solution was meas¬ ured at 400 nm. Once this control run of the system gave an absorbance within 0.4-2.0, the potential inhibitors were assay¬ ed against this working enzyme solution at final inhibitor and substrate concentrations of 1 and 5 mM, respectively. In the inhibitor assays, deionized water was replaced by the inhibi¬ tor (4 mM solution in deionized water) . All inhibitors were tested in triplicate, with a control (by substituting the in¬ hibitors with deionized water) and a standard.

C. General Procedure for K _j Determination

Five substrate solutions (usually 2-20 M) and five in¬ hibitor solutions (usually 0-200 μM) were prepared. To a 3-mL test tube were added buffer at the appropriate pH (0.28 L, phosphate-citrate), working enzyme solution (0.28 mL) and inhibitor solution (0.56 mL) . The solution was incubated at 37°C for 5 minutes. Aliquots (0.1 mL) of this solution were then pipetted into ten separate test tubes. Substrate solu-

tion (0.1 mL) was then added to each tube at 30 second inter¬ vals. Duplicate reactions were terminated by addition of gly- cine buffer (2.5 mL) at the end of 3, 6, 8, 10, and 12 min¬ utes. The amound of released p-nitrophenolate was measured spectrophotometrically at 400 nm. The process was repeated for all combinations of inhibitor and substrate concentra¬ tions.

The velocities (V) of substrate hydrolysis at every combination of inhibitor [I] and substrate [S] concentrations were determined by plotting the absorbance values with respect to time and then calculating the slopes of the lines. Double reciprocal plots of 1/V versus 1/[S] at different [I] were then generated. The slopes of each of these lines were then plotted against [I] and the data were fitted to a straight line. The [I]-intercept gave the enzyme-inhibitor dissocia¬ tion constant. K _j values were also calculated from Hanes- Woolf plots of [S]/V vs. [S] by replotting the [S]/V inter¬ cepts versus [I] and ascertaining the [I] intercept. K val¬ ues in the Table represent an average of the two calculations.

EXAMPLE 18 EVALUATION OF INHIBITORY EFFECTS In accordance with the above General Biological Proced¬ ures various of the above prepared amidines, amidrazones, and amidoximes were evaluated to determine their glycosidic enzyme inhibitory capacity.

A summary of the results of the assays are provided in Table I below in which the compounds which were tested are identified by the example above by which they were prepared and, in some cases, in graphical form in Figures 5-15. In the Table, "potent" means a K _χ of less than about 100 μM, "mod" is moderate which means a K of from about 100 to 1,000 μM, and "weak" means a K _j of greater than about 1,000 μM.

TABLE I Results of Bioassavs in oM

Com- 9-Glu α-Glu amylo-Glu α-man 0-gal α-gal pound pH 5.0 pH 6.6 pH 5.0 pH 5.0 pH 7.0 pH 6.6

Nojir¬ 380 13 imycin

1 8 none

2 83 none

8 8.4±0.9

9 205±25 13 13.8±3 15

/9-glu is almond ,9-glucosidase α-glu is yeast α-glucosidase amylo-glu is A. niger amyloglucosidase α-man is jackbean α-mannosidase /9-gal is bovine /3-galactosidase α-gal is coffee bean α-galactosidase

COMPARATIVE EXAMPLE A An attempt to prepare D-glucoamidine (Example 1) was made by aminolysis of the corresponding D-glucoiminoether, an alternative synthetic route starting from D-glucolactam as in Example 1. The hydroxyl groups of the lactam were protected by being peracetylated (NaOAc, Ac ₂ 0, room temperature, 44 hr) to produce the tetra-O-acetyl lactam which was then treat¬ ed with Meerwein's salt (Borch, Tetrahedron Lett. _f 1968, 61- 65) (triethyloxonium tetrafluoroborate, CH ₂ C1 ₂ , room tem¬ perature, 36 hr) to produce the D-glucoiminoether. Exposure of the iminoether to excess ammonia in methanol regenerated the starting D-glucolactam in 72-84% yield. Attempted aminol¬ ysis by treatment with concentrated ammonium hydroxide, anhy¬ drous liquid ammonia, or ammonium chloride also produced the starting lactam as the sole reaction product.

The re-formation of the lactam could be rationalized by postulating initial attack of ammonia on the C-3 acetate to

generate an intermediate which could cyclize with loss of etha- nol to form an acetamide ketal structure which, upon prolonged exposure to methanolic ammonia would produce the starting lac¬ tam. Thus an alternative hydroxyl protecting group, i.e. tri- methylsilyl, which would not enter into such neighboring-group participation was tried as described in Example 1. Attempts at preparing the D-glucoiminoether by O-alkylation were unsuc¬ cessful. Instantaneous desilylation occurred when the blocked lactam was treated with triethyloxonium tetrafluoroborate, even when buffered with Na ₂ HP0 ₄ .

Other literature procedures for O-alkylation of lactams to produce an iminoether (ethyl chloroformate (Suydam et al., J. Org. Chem.. 1969, 34, 292-6), dimethylsulfate (Benson, et al., In Organic Syntheses Collective Volume IV. Rabjohn, Ed., John Wiley: New York, 1963, pp 588-590), diazomethane (Nishi- yama et al., Tetrahedron Lett.. 1979, 48, 4671-4)) also failed to produce the corresponding iminoether.

EXAMPLE 19 ALTERNATIVE PREPARATION OF D-MANNOAMIDINE The procedure of the first paragraph of Comparative Exam¬ ple A was repeated but starting with the iminoether of D-man- nose rather than of D-glucose. Specifically, to a solution of D-mannoiminoether (27 mg, 0.072 mmol, 1.0 equiv) in methanol at -25°C was added NH ₃ -MeOH solution (4.6 M, 3.5 mL) . The resulting solution was stirred at -25 ^β C for 24 hr, at -5"C for 24 hr, and finally at room temperature for 12 hr. The solu¬ tion was concentrated in vacuo and chromatographed (7:3:1 CH ₂ Cl ₂ :CH ₃ OH:NH OH) to afford D-mannoamidine.

Various other examples will be apparent to the person skilled in the art after reading the present disclosure with¬ out departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.