WO1991008748A1 - Synthesis of rotavirus receptor saccharides - Google Patents

Abstract

Saccharides which are active as rotavirus receptor sites are disclosed. These saccharides are synthetically produced sugar derivatives recognized by the antireceptor of a rotavirus. The molecules are useful in studying the interaction between the rotavirus antireceptor and most receptor sites so as to develop drugs useful in blocking the connection between such viruses and the host cells. Further, the molecules can be modified in order to increase the degree to which they attract the antireceptor sites of the virus and/or modified by binding to them antiviral compounds which are effective in destroying the virus once the receptor molecule binds to the virus. Synthetic receptor molecules recognizable by a rotavirus are obtainable by the synthesis methods disclosed.

Description

SYNTHESIS OF ROTAVIRUS RECEPTOR SACCHARIDES

Cross-Reference

This application is a continuation-in-part of U.S. Patent Application Serial No. 07/450,026 filed 13 December 1989 which application is incorporated herein by reference and to which application is claimed priority under 35 USC Section 120.

Field of the Invention

This invention relates generally to the field of rotavirus receptors. More particularly, this

invention relates to saccharide compounds and their synthesis which compounds are recognized by the

antireceptor of a rotavirus which compounds can be formulated into pharmaceutical compositions to treat and/or prevent rotavirus infection.

Background of the Invention

Rotaviruses are double stranded RNA viruses of the family Reoviridae. These viruses replicate in the intestinal epithelial cells of a wide range of animal species including most mammalian and avian species and are the major etiological agents of several

gastrointestinal disorders in humans and other animals. For example, rotaviruses are responsible for infantile diarrhea and enteritis, causing infant morbidity and mortality. Rotaviruses also cause diarrheal illnesses in calves and piglets, as well as other mammals. These viruses are responsible for debilitating diarrhea in immune-compromised patients such as transplant recipients and AIDS sufferers and have been implicated as a

significant cause of traveler's diarrhea. Currently, there is no effective prophylactic or therapeutic drug available to combat rotaviral disorders and attempts to develop vaccines have been problematic.

In order to infect cells and replicate, viruses bind specific receptors on the target cell surface.

After attachment, the virus fuses with the cell membrane and is internalized where it uses the target cell's own metabolism to replicate. The initial attachment process is therefore essential to successful infection. For several viruses, a glycoprotein on the viral surface interacts with a specific target cell carbohydrate.

Like all viruses, a rotavirus has a virion which attaches to the surface of a host cell. The attachment is brought about by the specific binding of the virion protein (the antireceptor) to a constituent of the cell surface (the receptor). A classic example of an antireceptor is the hemagglutinin of influenza virus (orthomyxovirus). The antireceptors are distributed throughout the surface of viruses which infect human cells. Complex viruses, such as vaccinia (a pox virus) and herpes symplex virus (a herpes virus), may have more than one species of antireceptor molecules. Furthermore, antireceptor molecules may have several domains, each of which may react with a different receptor. Accordingly, complex structures and interactions are clearly involved with respect to receptors and antireceptors.

The details of the initial interaction between rotaviruses and the host cell surface have not been completely elucidated. However, sialic acid appears to be an important component of the rotavirus receptor, Yolken et al., J. Clin. Invest. 79: 148-154 (1987), and asialo GM1 binds rotavirus and inhibits viral replication in plague reduction assays, Willoughby et al., abstract from Proceedings of U.S. -Japan International Rotavirus Meeting, Annapolis, MD, August 1989. Furthermore, bovine submaxillary mucin and chicken ovoinhibitor have been shown to prevent rotavirus gastroenteritis in mice.

Yolken et al., supra. Additionally, it has been shown that rotavirus strains isolated from one species

cross-react with hosts of another species (see e.g. Leece et al., Infect. Immun. 14.: 816-825 (1976); Mebus et al., Infect. Immun. 14: 471-474 (1976); Wyatt et al., Science 207: 189-191 (1980)), suggesting conservation of

rotaviral receptors between species.

Viral binding molecules to which a rotavirus binds could be bound to antiviral drugs and rotaviral binding molecules by themselves could be used to hinder or prevent the subsequent infection of host cells. A distinct advantage of such an approach over traditional methods of preventing viral infections, e.g. vaccines, is that the portion of the viral protein normally binding to the specific cell surface carbohydrate does not mutate. Thus, antiviral agents which act by preventing viral binding to host cells are likely to remain effective in the face of mutations to other parts of the viral genome.

In U.S. patent application Serial No.

07/450,026 filed December 13, 1989, certain glycolipids were extracted from intestinal cells and found to bind to rotavirus. The present invention extends that of the 07/450,026 application by finding saccharides which bind to rotavirus and disclosing such saccharides, their method of synthesis and the formulation of such

saccharides into pharmaceutical compositions useful in treating and/or preventing rotavirus infection. Summary of the Invention

Various saccharide compounds are disclosed which bind rotavirus. The compounds can be used to prevent interaction of the virus with a target cell and thus prevent viral infection. These compounds can be bound to antiviral drugs and advantageously employed to treat rotaviral infections. The discovery and chemical characterization of relatively small sugars able to bind rotavirus allows for the efficient production of

pharmaceutical and/or food compositions useful in the prevention and treatment of rotaviral infections.

A primary object of the present invention is to provide a rotavirus receptor compound.

Another object of the subject invention is directed to a composition for preventing or treating rotaviral infection comprising a therapeutically

effective amount of synthetic rotavirus receptor compound in combination with a pharmaceutically acceptable

carrier.

Yet another object of the subject invention is directed to methods of producing molecules able to bind rotavirus.

Still other objects of the subject invention include methods for preventing or treating rotaviral induced disorders by administering to a subject a

therapeutically effective amount of a synthetic rotavirus receptors.

A feature of the present invention is that the molecules are produced in an effective manner by

synthetic means.

An advantage of the present invention is that the receptor molecules can be linked to antiviral drugs to provide effective antiviral compositions. Another advantage of the present invention is that the compounds can be linked to probes such as radioactive probes to form conjugates which can be used to test for the presence of rotavirus and/or the binding effectiveness of compound with respect to a rotavirus.

These and other objects, features and advantages of the present invention will become apparent to those persons skilled in the art upon reading the details of the structure, synthesis and usage as more fully set forth below, reference being made to the accompanying general structural formulae forming a part hereof.

Detailed Description of Preferred Embodiments

Before the present synthetic saccharide

receptor molecules and processes for making and using such are described, it is to be understood that this invention is not limited to the particular sugar residues described. For example, derivatives of such saccharides can be used such as when such saccharides are bound to anti-viral compounds. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

It must be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus for example, reference to "a synthetic receptor molecule" includes mixtures of such molecules, reference to "an

antireceptor" includes mixtures of antireceptors of the type described and reference to "the process for

synthesizing" includes similar processes which will become apparent to those skilled in the art upon reading this disclosure and so forth.

A. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

By "isolated rotavirus receptor glycolipid" is meant any sugar containing lipid that is capable of binding rotavirus. The glycolipid can be isolated from intestinal cells as described in U.S. patent application Serial No. 07/450,026, filed December 13, 1989, which is incorporated herein by reference in its entirety. The term also refers to those glycolipids which bind

rotavirus which glycolipids which can be synthetically produced by applying the present disclosure in

combination with methods well known in the art.

By "rotavirus receptor saccharide" is meant any synthetic saccharide of the invention such as the

saccharide epitopes or analogs therof which are capable of binding rotavirus as determined by standard viral binding assays including but not limited to overlay assays with thin layer chromatography plates and by probing receptor compounds adsorbed to PVC wells with labeled virus. A receptor compound of the invention will bind a rotavirus more avidly than asialo G_M1, as determined by orcinol staining in comparison with known amounts of asialo G_M1.

The glycolipid Ggose Cer (also known as G_A1) will run on thin layer chromatography (TLC) silica plates using chloroform:methanol:water (60:40:10) as solvent to provide a standard. The oligosaccharide moiety of the glycolipid G_A1 is shown below as structural formula III and is Ggose₄ (also known as asialo G_M1). (Note that Ac is COR throughout and R is preferably CH₃ but may be any alkyl containing 1 to 30 carbons - in formulae I, II and III, R is CH₃.

Ill

The oligosaccharide Ggose₃ is the

oligosaccharide moiety of the glycolipid Ggose₃Cer. The compound of structural formula II below is Ggose. (also known as asialo G _M2) .

II

The β- (1-6)-linked isomer of II is shown below as I:

The term "therapeutically effective amount" refers to the amount of a rotavirus receptor saccharide or conjugate of such a compound with an antiviral drug sufficient to prevent, (i.e., treat prophylactically), or treat, (i.e., reduce or eliminate the symptoms of

rotavirus) induced disorders in a recipient subject when administered.

B. Receptor Molecules for Rotavirus

Information regarding receptor sites for the antireceptor of a rotavirus are of particular interest to those involved in the research of the rotavirus and its infectious process. Since a rotavirus will readily attach to a receptor site, the production of receptor sites other than those present on cell surfaces would provide false hosts to the viruses and aid in preventing infection of the host cells to be protected. Further, by providing such receptors, it is possible to link other compounds to the receptors, such as antiviral drugs which can act to destroy the rotavirus after the rotavirus antireceptor has attached. A probe could be attached to or incorporated as part of the receptor so that the

"tagged" receptor could be used to detect the presence of rotavirus. Saccharides presently disclosed can be adapted for all or any of these uses.

This invention relates to: (1) synthetically produced saccharides such as saccharide epitopes or analogs thereof which are recognized by the antireceptor of a rotavirus; (2) pharmaceutical compositions

containing such saccharides in combination with a

pharmaceutical acceptable excipient; and (3) the

saccharides of (1) in combinaton with an antiviral compound or detectable probe. The rotavirus receptor molecules of the invention are structurally small, but functionally similar to the naturally occurring

glycolipid receptor sites which are described further below.

Glycolipids can be isolated from cells receptive to rotavirus using any of several techniques for purifying glycolipids, well known in the art. A particularly useful technique is a modified Folch extraction as developed by Svennerholm and Fredman, Biochim. Biophys. Acta 617: 97-109 (1980), the disclosure of which is incorporated by reference herein in its entirety to disclose such extraction techniques. Isolated

glycolipids known to act as receptors can be used for comparison purposes with the synthetic saccharide receptors of the present invention.

Sugar residues and related compounds can also be synthesized using standard biochemical techniques, well known in the art. For example, the methods

described by Tang et al., Biochem. Biophys. Res. Comm. 132: 474-480 (1985); Schnaar et al., J. Biol. Chem.

253:7940-7941 (1978); and Laine et al., J. Biol. Chem. 249: 4460-4466 (1974), all incorporated herein by

reference in their entirety to disclose techniques which can be used in combination with the present disclosure to synthesize the saccharide compounds of the invention.

Once synthesized and purified the receptor saccharide compounds can be tested (in their

neoglycolipid form as explained further below) for their ability to bind labeled rotavirus using thin layer chromatography plates, developed in a suitable solvent or by adsorbing the saccharides to PVC wells and

subsequently probing the immobilized receptor compounds with labeled virus. The efficacy of the saccharide binding receptor compounds can be directly tested in vitro in standard plaque reduction assays. Saccharides can also be directly tested for their ability to inhibit a rotavirus from binding to an immobilized glycolipid in PVC wells. These testing procedures are described in detail in the Examples.

Once receptor compounds such as natural

glycolipids with the ability to bind rotavirus have been isolated, the sugar moieties can be cleaved therefrom using enzymes, including but not limited to

neuraminidase, beta-glucuronidase, alpha-galactosidase, beta-galactosidase, endogalactosidase,

beta-hexosaminidase, ceramide glycanase,

alpha-fucosidase, and the coenzymes sulfatase and

phosphatase. The ability of these cleaved carbohydrates to bind to a rotavirus antireceptor can be tested and compared with the binding of synthetic saccharide

compounds of the invention to the same viral

antireceptor.

The cleavage products can be tested for their ability to bind rotavirus as described above and

compounds (such as the various saccharides of the

invention) with binding capability can be further tested in standard rotavirus plaque reduction assays, such as described below. Receptor compounds with inhibitory activity can be used directly, or equivalent receptor compounds can be synthesized by applying methods known in the art in combination with the present disclosure. For example, receptor oligosaccharides can be synthesized by the sequential addition of appropriate single sugar units to a growing chain covalently linked to an insoluble solid support or by "block synthesis" where di- or trisaccharide blocks are synthesized which are then complexed to provide larger size oligosaccharides. The minimum binding and inhibitory carbohydrate epitope can be determined and used. Further, these sugars can be structurally modified to create more avidly binding analogs and/or linked to antiviral drugs or detectable probes.

In order to determine receptor saccharides recognized by a rotavirus certain characteristics of such receptors were taken into consideration. Firstly, it was noted that the conversion of asialo G _M1 into asialo G_M2 did not cause a difference in the ability of a rotavirus to bind to these molecules as measured by the TLC assay. The microtiter well assay showed some decrease in

binding, although G_A2 still bound some virus, in

comparison with other tested glycolipids which did not. G_A2 does support viral binding but greater concentrations are needed when compared with G_A1 in the PVC adsorption assay. This is in contrast to the TLC viral binding experiments in which G_A1 and G_A2 appear to be more equivalent. Further, it was noted that GalNAc by itself is of insufficient size to bind a rotavirus. Lastly, it was noted that the removal of the terminal Gal from

Ggose₄Cer (G_A1 ) generates Ggose₃Cer (G_A2), a glycolipid which retains viral binding activity although at a reduced potency.

Based on the above discoveries, saccharide structures were deduced, synthesized and tested for rotavirus affinity. The following structural synthesis schemes SI, SII and SIII show how oligosaccharides I, II and III respectively are synthesized in accordance with the methods of synthesis of the present invention.

General Structural Formulae

Based on the above Structural Synthesis Schemes SI, SII and SIII, the following general structural formulae for saccharide compounds useful in producing pharmaceutical formulations of the invention can be deduced.

IV wherein each of X¹ to X⁸ is independently H, an acyl, or a benzyl and X¹⁰ is an acylamine or OH wherein any acyl moiety may contain from 2 to 30 carbons. Preferably X¹ to X ⁹ are H and X¹⁰ is acylamine (most preferably -COCH₃) with the proviso that X⁵ may be in the alpha or beta position.

Another general structure is as follows:

wherein each of X¹¹ - X²⁰ is independently H, acyl or benzyl and X ²¹ is -OH or acylamine wherein any acyl can contain from 1 to 30 carbons. Preferably X ¹¹ - X²⁰ are each H and X ²¹ is acylamine (most preferably -COCH₃) with the proviso that X¹⁶ may be in the alpha or beta

position.

Yet another general structure is:

VI wherein each of X²² - X³³ and X³⁵ is independently H, an acyl, or a benzyl and X³⁴ is an acylamine or OH.

Preferably each of X²² - X³³ and X³⁵ is H and X³⁴ is acylamine (most preferably -COCH₃) with the proviso that any acyl moiety may contain from 2 to 30 carbon atoms and

X ²⁹ may be in the alpha or beta position.

General Description of the Synthesis shown in

SI. SII and SIII

Description of Structural Reaction Scheme I - (SI)

Synthesis of β-D-GalNAc-(1-6)-β-D-Gal-(1-4)-D-Glc. I

Condensation of oxazoline 1 [K.L. Matta and O.P. Bahl, Carbohydr. Res., 21 (1972) 460-464] with the lactoside diol 2 [K. Koike et al. Carbohydr. Res., 163 (1987) 189-208] according to established procedures, followed by column chromatographic purification on silica gel gave trisaccharide derivative 3 (72.5%). Compound 3 was deacetylated in methanolic sodium methoxide to give the partially protected trisaccharide 4 (83%).

Hydrogenolysis of the benzyl groups of 4 (H2/Pd-C in glacial acetic acid at room temperature and - 345 kPa) followed by chromatographic purification afforded the title trisaccharide I (72%).

Description of Structural Reaction Scheme II - (SII)

Synthesis of β-D-GalNAc-(1-4)-β-D-Gal-(1-4)-D-Glc. II

Imidate 5 was prepared and reacted according to Schmidt's procedure [R.R. Schmidt and G. Grundler, Angew. Chem. Int. Ed. Engl., 22. (1983) 776-777] with compound 6 [K. Koike et al. Carbohydr. Res., 163 (1987) 189-208] to give trisaccharide 7 which was subjected to

hydrazinolysis (NH₂.NH₂.H₂O in ethanol) followed by acetylation (2:1 Pyridine-acetic anhydride) to give the fully protected trisaccharide 8 (65%, based on lactoside acceptor 6). Zemplen transesterification of 8 gave the partially protected derivative 9 (84%). Compound 9 was hydrogenolyzed in the usual manner to provide the title compound II (51.3%).

Description of Structural Reaction Scheme III - (SIII)

Synthesis of β-D-Gal-(1-3)-β-D-GalNAC-(1-4)-β-D-Gal- (1-4)-D-Glc. Ill

The benzylated trisaccharide 9 (scheme II) was converted into its 4",6"-O-benzylidene acetal 10 (84%) by reaction with benzaldehyde-zinc chloride complex.

Compound 10 was condensed with acctobromogalactose 111 under Helferich conditions to give a tetrasaccharide derivative which was O-deacetylated (NaOMe-MeOH) and then deacetalated (80% agueous acetic acid, 90) to afford 12 (50%, based on trisaccharide 10). Hydrogenolysis of the benzyl groups of 12 (50%, based on trisaccharide 10). Hydrogenolysis of the benzyl groups of 12 then afforded the desired tetrasaccharide III (44.5%).

DETAILED PROTOCOLS

Detailed Description of Reaction Scheme I-III

SI - SIII

The following detailed protocols are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to carry out Structural Reaction Schemes I, II and III and are not intended to limit the scope of what the inventors regard as their invention or as being the only methods of synthesizing such molecules. Efforts have been made to insure accuracy with respect to numbers used (e.g., amounts, temperature), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, temperate is in degrees centigrade, and pressure is at or near

atmospheric. In these detailed synthesis, reference can be made to SI-SIII where Ac is acetate, i.e., Ac is -COCH₃. Detailed Description of Structural Reaction Scheme I Synthesis of β-GalNAc-(1-6)-β-Gal-(1-4)-D-Glc

(SI)

Benzyl 0-(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D- galactopyranosyl)-(1-6)-0-(2 ,6-di-0-benzyl-β-D

galactopyranosyl)-(1-4)-2,3,6-tri-0-benzyl-β-D- glucopyr anos ide

A mixture of benzyl 0-(2,6-di-0-benzyl-β-D- glucopyranoside 2. (0.88g, 1 mmol), 2-methyl (3,4,6-tri- 0-acetyl-1,2-dideoxy-α-D-galactopyrano)-[2,1-d]-2- oxazoline 1 (0.66g, 2 mmol) and 4-toluenesulfonic acid (25 mg) in 1,2-dichloroethane (15 mL), protected from moisture, was heated (~72°) with stirring for 16h.

T.L.C. (3:2 ethyl acetate-hexane) revealed the presence of a major product faster-migrating than 2.; some

unchanged 2., as well as some slower-migrating

contaminants (presumably due to the decomposition of oxazoline 1) were also revealed by T.L.C. After cooling, a few drops of pyridine were added, the mixture

evaporated to dryness, and the residue dissolved in a small volume of dichloromethane and applied to a column of silica gel (~100g). The column was eluted first with1:2 and then 1:1 ethyl acetate-hexane. On evaporation, the first fractions gave unchanged 2 (0.1g). Fractions corresponding to the product gave a residue (0.9 which was dissolved in a small volume of ethyl acetate).

Addition of ether-hexane caused the precipitation of 3. 0.87g (72.5%), [α]_D ²⁰-3° (c 0.43, chloroformm.

Benzyl 0-(2-acetamido-2-deoxy-β-D-qalactopyranosyl (1- 6)-0-(2,6-di-0-benzyl-β-D-galactopyranosyl)-(1-4)-2,3,6- tri-0-benzyl-β-D-glucopyranoside

Trisaccharide derivative 3 (0.8g) was suspended in methanol (30mL) containing a catalytic anion of sodium methoxide and stirred at room temperature. The suspended 3 quickly dissolved, and, in a few minutes,

crystallization ensued. The stirring was continued overnight at room temperature, the base neutralized with a few drops of glacial acetic acid and the precipitation was filtered and thoroughly washed with cold methanol to give 4 ( 0. 6g, 83%) , [α]_D ²⁰ + 2 .7 ° (c 4. 6 , chloroform) .

0-(2-Acetamido-2-deoxy-β-D-galactopyranosyl) (1-6)-0-β- D-galactopyranosyl-(1-4)-D-glucopyranose

Compound 4 (0.42g) in glacial acetic (25 mL) was shaken under H₂ at ~345 kPa for 3 days at room temperature in the presence of 10% palladium-on-carbon (0.4 g). The suspension was filtered (a bed of Celite) and thoroughly washed with glacial acetic acid. The acetic acid was evaporated under diminished pressure and the residue was applied to a column of silica gel.

Elution first with 13:6:1 and then 4:5:1 chloroform- methanol-water, and evaporation of the fractions corresponds to the product gave a residue (0.18g) which was dissolved in methanol, filtered, and concentrated. It was redissolved in water and lyophilized to give the desired trisaccharide (I) (0.152g, 72.4%) [α]_D ²⁰ + 23.4 (c 0.32, methanol). Detailed Description of Structural Reaction Scheme II Synthesis of β-GalNAc-(1-4)-β-Gal-(1-4)-D-Glc

(SII)

Benzyl 0-(2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D- galactopyranosyl)-(1-4)-0-(2,3,6-tri-0-benzyl-β-D- galactopyranosyl)-(1-4)-2,3,6-tri-0-benzyl-β-D- grlucopyranoside

A mixture of benzyl 0-(2,3,6-tri-0-benzyl-β-D- galactopyranosyl) - (1-4) 2,3, 6-tri-0-benzyl-β-D- glucopyranoside 7 (0.64g, 0.66 mmol), ionidate 5 (0.77g, 1.3 mmol) and powdered molecular sieves (1-5g) in dry dichloromethane (15 mL), protected from moisture, was stirred for ~0.5h. at room temperature. It was then cooled (-20°, bath) and treated with boron trifluoride etherate (0.7 mL) and stirred for 0.5h. at -20°. It was then allowed to warm to room temperature and the stirring was continued for an additional lh. T.L.C. (4:1 toluene- acetone) revealed the presence of a major product, slower-migrating than acceptor 6; a trace of 6. and some slower-migrating contaminants (presumably due to the decomposition of 5) were also revealed by T.L.C. The mixture was diluted with dichloromethane (100 mL) and filtered (Celite) and the solution washed with water, dried, and concentrated. The residue was applied to a column of silica gel (~100g) and eluted with 10:1

toluene-acetone. On evaporation, fractions corresponding to the product gave 7 (0.75), 82%) which was slightly contaminated with a faster-migrating compound. This material was heated for 15 min. at ~70° in a mixture of ethanol (15 mL) and 85% hydrazine hydrate (7.5 mL). The mixture was evaporated, and several portions of ethanol were added to, and evaporated from the residue. The residue was thoroughly dried in vacuo and mixed with acetic anhydride (8 mL) and pyridine (16 mL) and heated for 20 min. at ~90°. The acetic anhydride and pyridine were removed under reduced pressure and the crude product was taken in chloroform, filtered and concentrated to a small volume. The concentrate was applied to a column of silica gel (~150g) and eluted with (10:) toluene-acetone (~1L). On evaporation, the fractions corresponding to the product gave 8 0.56g; 65% yield based on lactoside acceptor.

Benzyl 0- (2-acetamido-2-deoxy-β-D-galactopyranosyl (1- 4) -0- (2,3,6-tri-0-benzyl-β-D-galactopyranosyl)-(1-4)- 2,3,6-tri-0-benzyl-β-D-glucopyranoside

Compound 8 (0.55g) was stirred overnight at room temperature in methanol (30 mL), containing a catalytic amount of sodium methoxide. The base was neutralized with a few drops of glacial acetic acid, the mixture concentrated, and taken into chloroform. The chloroform solution was washed twice with water, dried and concentrated to give a residue which was dissolved in a small volume of dichloromethane. Addition of ether- hexane caused the precipitation of 9 (0.42g, 84%).

0-(2-Acetamido-2-deoxy-β-D-galactopyranosyl) (1-4)-0-β- D-galactopyranosyl-(1-4)-D-glucopyranose

The benzylated trisaccharide 9 (0.38g) in glacial acetic acid (30 mL) was shaken under H₂ at ~345 kPa for 3 days at room temperature in the presence of 10% palladium-on-carbon (0.38g). T.L.C. (4:5:1 chloroform- methanol-water) indicated the presence of a major slower- migrating product as well as some faster-migrating contaminants which were faintly detectable in u.v. light. The latter contaminants are presumably due to incomplete debenzylation. The solids were removed by filtration and thoroughly washed with glacial acetic acid and then with 1:2 water-methanol. After concentration, the residue was applied to a column of silica gel and eluted successfully with 13:6:1, 5:4:1 and 4:5:1 chloroform-methanol-water. On evaporation, the earlier fractions gave 100 mg. of the faintly u.v. visible contaminants. The fractions

corresponding to the product yielded 95mg of material which was dissolved in methanol and filtered (Celite). It was concentrated, dissolved in water and lyophilized to give the desired trisaccharide (II) (87.9 mg, 51.3%), [α]_D ²⁰ + 21.5° (c 0.26, methanol). Detailed Description of Structural Reaction Scheme III Synthesis of β-Gal-(1-3)-β-GalNac-(1-4)-β-Gal-(1-4)- D-Glc

(SIII)

Benzyl 0- (2-acetamido-2-deoxy-β-D-galactopyranosyl 4,6- 0-benzylidene-β-D-galactopyranoside-(1-4)-0-(2,3,6-tri- 0-benzyl-β-D-galactopyranoside)-(1-4)-2,3,6-tri-0-benzyl- β-D-glucopyranoside

Zinc chloride (1g) was added quickly with stirring, to benzaldehyde (5 mL) and the mixture was stirred for ~20 min. Trisaccharide 9 (1g) was then added and the stirring continued for 4h. at room temperature. The mixture was taken in chloroform (150 mL) and washed with water, dried and concentrated to a small volume. The concentrate was applied to a column of silica gel and eluted first with 1:1 chloroform-hexane (150 mL) to remove excess benzaldehyde, and then with 4:1 chloroform- acetone to give 10 (0.9g, 84%), [α]_D ²⁰ + 20.8° (c 0.53, chloroform). Condensation of Acetobrogalactose with the Trisaccharide Acceptor 10

A mixture of trisaccharide 10. (0.6g, 0.5 mmol) and mercuric cyanide (0.18g, 0.75 mmol) in 1:1 benzene- nitro methane (50 mL) was boiled until -25 mL of the solvent had distilled off. The temperature was then adjusted to 45° and treated with 2 , 3 , 4 , 6-tetra-0-acetyl α-D-galactopyranosyl bromide 11 (0.4g, 1 mmol) and the stirring was continued at 45-50° overnight. T.L.C. (20:1 chloroform-methanol) revealed the formation of a product, marginally faster-migrating than acceptor 10; some 10 and some faster-migrating contaminants (presumably due to decomposition of bromide 11) were also revealed by T.L.C. The mixture was concentrated and the residue taken in chloroform (~100 mL). The suspended material was

filtered off and washed with chloroform. The chloroform solution was washed with 10% potassium iodide solution and water, dried and evaporated to dryness. The residue was taken in methanol (50 mL) containing a catalytic amount of sodium methoxide and stirred for 5h at room temperature. After deionization with Amberlite 1R-120 (H⁺) the mixture was filtered and concentrated. T.L.C. 6:1 chloroform-methanol indicated the presence of a slower-migrating product; some unchanged trisaccharide as well as slower-migrating contaminants were also revealed by T.L.C. The mixture was taken in chloroform (~150 mL) and washed with water. T.L.C. then showed the

disappearance of the slower-migrating contaminants.

After concentration, the crude product mixture was applied to a column of silica gel (~130g) and eluted first with 20:1 and 6:1 chloroform-methanol. Evaporation of the earlier fraction gave unchanged trisaccharide 10 (0.2g). On evaporation, the fractions corresponding to the product gave 0.37g of an amorphous white solid. Yield 54.4% (86% based on reacted trisaccharide).

Benzyl 0-ß-D-galactopyranosyl-(1-3)-0-(2-acetamido-2- deoxy-β-D-galactopyranosyl-(1-4)-0-(2,3,6-tri-0-benzyl- β-D-galactopyranosyl)-(1-4)-2,3,6-tri-0-benzyl-β-D- glucopyranoside

The benzylidenated tetrasaccharide derivative 0.32g, in 80% aqueous acetic acid (25 mL) was stirred for lh. at ~90°. The acetic acid was evaporated and several portions of toluene were added to, and evaporated from the residue to give the compound 12 0.27g (90%), [α]_D20 + 18.8° (c 0.43, CHCl₃).

0-β-D-Galactopyranosyl-(1-3)-0-(2-acetamido-2-deoxy-β-D- galactopyranosyl-(1-4)-0-β-D-galactopyranosyl-(1-4)-D- glucopyranose

(III)

Compound 12 (0.21g) was hydrogenolyzed as described for 9 and then purified by column

chromatography to give the desired tetrasaccharide in (44.5%), [α]_D ²⁰ + 16.7° (c 0.24, methanol).

USE and ADMINISTRATION

The above disclosed saccharide epitopes and analogs thereof can be administered to a subject either prophylactically or after rotaviral infection. The saccharide receptor molecules are administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a solid carrier and I.V. administration a liquid salt solution carrier. The method of choice, can be accomplished using a variety of excipients including, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose,

magnesium carbonate, and the like. These oral compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. Particularly useful is the administration of the subject carbohydrate receptor molecules directly in infant nutrient formulae or

rehydration formulae, given to replenish electrolytes lost in severe bouts of diarrhea.

Alternatively, the receptor molecules of the invention can be administered orally, linked to supports such as beads, resins, or natural or synthetic polymers. Methods for binding substances to such supports are well known in the art. The receptor saccharides may also be administered in small lipid particles comprising these receptor saccharides, such as in vesicles, micelles, or liposomes.

A sufficient amount of receptor saccharide molecules should be administered to bind to a substantial portion of the rotavirus expected to cause or actually causing infection so that infection can either be

prevented or ameliorated. Typically, the oral composi tions of the instant invention will contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%. Preferably, between about 12 μg and 1.2 mg will be administered to a child and between about 200 μg and 10 mg will be administered to an adult. The frequency of administration will be determined by the care giver based on patient responsiveness. Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials

establishing dose response curves.

Other modes of administration will also find use with the subject invention. For instance, the rotavirus receptor saccharides can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.

Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and

benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

The receptor saccharides formulations of the instant invention may also be administered as

injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active

ingredient encapsulated in liposome vehicles. The receptors saccharides can be mixed with compatible, pharmaceutically acceptable excipients.

Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the receptor saccharides adequate to achieve the desired state in the subject being treated.

The various receptor saccharides of the present invention can be used by themselves or in combination with pharmaceutically exceptable excipient materials as described above. However, it is more preferable to use the receptor compounds of the invention as conjugates wherein the compounds of the invention are linked in some manner to an antiviral drug. By forming such conjugates, the receptor saccharides of the invention act as

biochemical delivery systems for the antiviral drugs. It is well known that many antiviral drugs are extremely toxic. Accordingly, it is desirable to provide such drugs to a patient in extremely small amounts. The smaller the amount of the drug delivered, the less toxic affects. However, the decrease in toxic affects must be balanced against the need to provide sufficient amounts of the drug in order to sufficiently destroy the virus. The receptor saccharides of the invention aid in solving the problem by providing the biochemical delivery system. By forming conjugates of the receptor saccharides of the invention with antiviral drugs, the conjugates readily attach to the viruses and destroy them. Thereby, smaller amounts of the antiviral drug need be delivered to a patient to obtain efficatious results .

The receptor saccharides of the invention could also be used as laboratory probes to test for the

presence of a rotavirus in a sample. Such probes are preferably labeled such as with a radioactive or

fluorescent labels. C. Experimental

The following examples are provided so as to provide those of ordinary skill in the art with a

complete disclosure and description of how to test the receptor saccharides of the invention and are not

intended to limit the scope of what the inventors regard as their invention or as being the only methods of testing such molecules. Efforts have been made to insure accuracy with respect to numbers used (e.g., amounts, temperature), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, parts or parts by weight, temperate is in degrees

centigrade, and pressure is at or near atmospheric.

Example 1

Rotavirus Thin Layer Overlay Binding Assay

The ability of any of the compounds of the invention to bind rotavirus can be tested in the

following manner. Rotavirus SA11 for use in the following

experiment can be grown in and isolated from MA104 cells, available from Whittaker Bioproducts, Walkersville MD., using previously described methods. See, e.g. Yolken et al., J. Clin. Invest. 79: 148-154 (1987) and Kabcenell et al., J. Virol. 62: 2929 (1988), the disclosures of which are incorporated herein by reference in their entirety. The virus can be iodinated using solid state iodobeads as reagent as described by Markwell, M.A., Analyt. Biochem. 125: 427-432 (1982), incorporated herein by reference.

Approximately 25 ug of gradient purified virus (by protein assay) can be incubated with 2 mCi of Na ¹²⁵-I and the virus subsequently purified by gel exclusion on a

PD-10 column.

Silica 60 high performance thin layer chromatography plates (Merck) can be spotted with the isolated glycolipids and from 50 to 500 pmoles of asialo

GM1 as standard. The plates can be run in

chloroform:methanol:water 60:40:10 (v:v:v). The plates can be probed with 10 ⁷ dpm of ¹²⁵I-labeled rotavirus per

100 sq.cm. using a modification of the technique of

Magnani et al., Anal. Biochem. 109: 399-402 (1980).

After probing, the plates can be treated with iodine and sprayed with orcinol (Alltech). Following autoradiography of the plates , it can then be determined if the molecules bind rotavirus, and whether they bind to a higher degree than asialo GM1 which normally binds virus if present in 50 pmoles in a 2 mm streak.

The above binding assay can be used for testing cleaved or synthesized sugar moieties and derivatives thereof after their attachment to lipids by means well known in the art of neoglycolipid synthesis. For example, the Ten Feizi method can be used whereby oligosaccharides are attached to phosphatidylethanolamine by reductive animation to make neoglycolipids.

Example 2

Rotavirus Binding to Adsorbed Glycolipids

The ability of synthetic saccharides which are believed to be receptors to bind rotavirus can be tested by synthesizing the neoglycolipid form of the saccharide and using the following technique. Saccharides (in their neoglycolipid form) to be tested are first adsorbed to

PVC wells by evaporating the saccharide be tested out of chloroform:methanol:water (4:8:3, v:v:v) and resuspending by sonication in an appropriate volume of 2.6 uM

phosphatidyl choline/2.6 uM cholesterol in absolute ethanol. The resuspended compounds being tested should then be diluted with an equal volume of water to give stock solutions between 0 and 20 uM of test lipid. Prior to use, the PVC 96 well microtiter plates should be washed by immersion and agitation in n-butanol for 30 minutes, rinsed in absolute ethanol and air dried. 50 ul of test solution should be added to the microtiter wells to yield between 0 and 1 nmole of test lipid with 130 pmoles each of phosphatidyl choline and cholesterol. The PVC plates should be allowed to stand at room temperature for 80 minutes or just before the meniscus of the

solution contacts the well bottom. Microtiter plates should then be washed three times with water and stored with water in each well until probed with labeled virus.

The putative receptor saccharides (in their neoglycolipid form) can be probed as follows:

¹²⁵ -Rotavirus is to be diluted in complete PBS supplemented with 10 mg/ml BSA (Fraction V), pH 7.4, to give approximately 20,000 cpm/100ul. This volume should be added to each microtiter well and the plates incubated at ⁴⁰C. for 3 hours with gentle agitation. Following incubation, plates are to be washed three times with ice cold PBS to remove unbound virus. The bottom half of each well should then be clipped off and placed in vials for measurement of bound radioactivity. This procedure will indicate if the tested compound demonstrates the ability to bind rotavirus.

Soluble rotavirus binding substances can also be tested (for their ability to inhibit binding to an immobilized glycolipid) with this technique by first adding the putative rotavirus binders to microtiter wells in 50 ul volumes of PBS-BSA at twice the desired final concentration. Labeled rotavirus should then be added to the wells at approximately 20,000 cpm/50 ul and the radioactivity in the wells determined as above.

Example 3

Rotavirus Plague Reduction Assay

The ability of the above-described synthesized sugar moieties to inhibit rotavirus infection can be tested in vitro using the following plaque reduction assay. SA11 rotavirus (at 100 pfu) is to be added to 6 well plates containing confluent MA104 cells. The cells are to be incubated for one hour at 37°C., the inoculum removed and the cells washed once with EBSS medium. The cells are then to be overlaid with 3 mis of medium containing 0.7% agarose and the compounds to be tested. Known inhibitors, such as ovalbumin and bovine

submaxillary mucin and substances known not to inhibit rotavirus, such as globoside and trihexosyl ceramide, can be added as positive and negative controls, respectively. After the overlay gels, the plates are to be incubated for 48 hours and the number of plaques counted. The instant invention is shown and described herein in what is considered to be the most practical, and preferred embodiments. It is recognized, however, that departures may be made therefrom which are within the scope of the invention, and that obvious

modifications will occur to one skilled in the art upon reading this disclosure.

Claims

1. A composition for preventing or treating rotaviral infection, comprising:

a pharmaceutically acceptable carrier; and a therapeutically effective amount of a

rotavirus receptor having the following structural formula III:

wherein each of X¹ to X⁸ is independently H, an acyl, or a benzyl and X¹⁰ is acylamine or OH.

2. A composition for preventing or treating rotaviral infection as claimed in claim 1, wherein X¹, X² and X³ are acyl, X⁴, X⁵, X⁶, X⁷, X⁸ and X⁹ are benzyl and

X ¹⁰ is acylamine.

3. A composition for preventing or treating rotaviral infection as claimed in claim 1, wherein X¹ - X³ are each H, X⁴ - X⁹ are each benzyl and X¹⁰ is -NHCOR and R is an alkyl containing 1 to 30 carbon atoms.

4. A composition for preventing or treating rotaviral infection as claimed in claim 1, wherein each of X¹ - X⁹ is H, X¹⁰ is NHCOCH₃ with the proviso that X⁵ may be in the alpha or beta positions.

5. A composition for preventing or treating rotaviral infection, comprising:

a pharmaceutically acceptable carrier; and a therapeutically effective amount of a

rotavirus receptor having the following structural formula IV:

wherein each of X¹¹ - X ²⁰ are independently H, acyl or benzyl and X²¹ is -OH or acylamine.

6. A composition for preventing or treating rotaviral infection as claimed in claim 5, wherein each of X¹¹ - X¹³ is acyl, each of X¹⁴ - X²⁰ is benzyl and X²¹ is NHCOR and R is an akyl containing 1 to 30 carbon atoms.

7. A composition for preventing or treating rotaviral infection as claimed in claim 5, wherein each of X¹¹ - X¹³ is OH, each of X¹⁴ - X²⁰ is benzyl and X²¹ is NHCOR and R is an alkyl containing 1 to 30 carbon atoms.

8. A composition for preventing or treating rotaviral infection as claimed in claim 5, wherein each of X ¹¹ - X ²⁰ is H and X²¹ is NHCOCH₃ with the proviso that X¹⁶ may be in the alpha or beta position.

9. A composition for preventing or treating rotaviral infection, comprising:

a pharmaceutically acceptable carrier; and a therapeutically effective amount of a

rotavirus receptor having the follwoing general

structural formula V:

wherein each of X²² - X³³ and X³⁵ is independently H, an acyl, or a benzyl and X³⁴ is OH or an acylamine.

10. The composition for preventing or treating rotaviral infections as claimed in claim 9, wherein each of X²² - X³³ and X³⁵ is H and X³⁴ is -NHCOCH₃ with the proviso that X²⁹ may be in the alpha or beta position.

11. A composition for preventing or treating rotaviral infection as claimed in claim 1 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

12. A composition for preventing or treating rotaviral infection as claimed in claim 11, wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

13. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 11.

14. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 12.

15. A composition for preventing or treating rotaviral infection as claimed in claim 5 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

16. A composition for preventing or treating rotaviral infection as claimed in claim 15 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

17. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a

composition according to claim 15.

18. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a

composition according to claim 16.

19. A composition for preventing or treating rotaviral infection as claimed in claim 9 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

20. A composition for preventing or treating rotaviral infection as claimed in claim 19 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

21. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 19.

22. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 20.

23. A composition for preventing or treating rotaviral infection as claimed in claim 10 wherein the pharmaceutically acceptable carrier is a carrier for oral delivery.

24. A composition for preventing or treating rotaviral infection as claimed in claim 21 wherein the pharmaceutically acceptable carrier is an infant nutrient formula.

25. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 21.

26. A method for preventing or treating rotavirus induced disorders comprising administering to a subject a therapeutically effective amount of a composition according to claim 22.

27. A method of synthesizing β-D-GalNAc-(1- 6)-β-D-Gal-(1-4)-D-Glc comprising the steps of:

reacting the compound of formula 1 with the compound of formula 2

n

to obtain the condensation product of formula 3.

deacetylating the compound of formula 3 and carrying out hydrogenolysis of the benzyl groups to obtain β-D-GalNAc-(1-6)-β-D-Gal-(1-4)-D-Glc.

28. A method of synthesizing β-D-GalNAc-(1- 4)-β-D-Gal-(1-4)-D-Glc, comprising the steps of:

reacting the compound of formula 5 with the compound of formula 6

to obtain the compound of formula 7

carrying out hydrazinolysis and acetylation of the compound of formula 7 to obtain the compound of formula 8

carrying out transesterifaction of the compound 8 followed by hydrogenolysis to obtain β-D-GalNAc-(1-4)- β-D-Gal-(1-4)-D-Glc.

29. A method of synthesizing β-Gal-(1-3)-β- GalNAc-(1-4)-β-Gal-(1-4)-D-Glc, comprising the steps of:

reacting the compound of formula 9 with a benzaldehyde-zinc chloride complex to obtain the compound of formula 10

condensing the compound of formula 10 with acctobromogalactose followed by deacetylation,

debenzylidenation and hydrogenation to obtain β-Gal-(1- 3)-β-GalNAc-(1-4)-β-Gal-(1-4)-D-Glc.