CA2239296A1 - Glycoconjugates as virus cell adhesion inhibitors - Google Patents

Abstract

The host-cell adhesion by human influenza viruses is inhibited by new compounds of the general formula (1), where R1 stands for acyl or a thioacyl group; R2 for hydroxyl, Z-alkyl, substituted Z-alkyl, Z-aryl, substituted Zaryl, and Z corresponds to O, S or NH; R3 stands for an acyl or a thioacyl group; R4 for H or acyl; X for O, S, SH2 or a sugar; and where W is a bifunctional spacer and P a multivalent vector consisting of one of the following substances: polyacrylate, polyacrylamide, N-substituted polyacrylamide, metacrylamide, N-substituted metacrylamide, polyacrylic acid, polycarbonate, polyester, polyamide, polyanhydride, polyiminocarbonate, polyorthoester, polydioxanone, polyphosphazene, polyhydroxy carboxylic acid, polyamino acid, polysaccharide, protein, dextran, chitosan, glucan, liposome, microparticles. The inventive compounds are capable of bonding authentic human viruses from the A (H1 and H3) groups, which have not been adapted through culturing in chicken eggs.

Description

Glycol Coniu~ates as Viral Cell Adhesion Inhibitors Description This invention concerns new multivalent derivatives of Neu5Aca2~Gal~1 4GlcNAc and the production of these compounds. It also concerns the use of these compounds as viral cell adhesion inhibitors of unadapted human influenza viruses.
The first essential step in a viral infection consists of the adhesion of a virus to the surface of the host cell. In the case of type A and B influenza viruses, this occurs through the bonding of viral hemagglutinins (HA) to terminal glycoprotein and glycolipidSialyloligosaccharides on the surface of the host cell. One possible strategy for preventing influenza infections would be to use cell adhesion inhibitors which would bond with viral hemagglutinins and limit their reactions with host cells.
In the literature, there is already a range of known influenza virus adhesion inhibitors. Compounds could be found, especially through the development of multivalent compounds, that can successfully prevent the adhesion of a particular strain of influenza in in vitro tests. (See Table 2).
One disadvantage of these known inhibitors is their restricted effective range, the compounds currently being highly effective only against a single strain and thus not well suited for practical prophylactic or therapeutic use.
A further disadvantage is that these known inhibitors are currently only being shown to be effective against adapted influenza strains that are cultivated from the embryos of hens' eggs. It is known that the cultivation of human influenza viruses from hens' eggs leads to amino acid mutations in the region of the receptor bond area of hemagglutinins (Robertson, 1993). It is true that the inhibitors found in this procedure have a high affinity to the altered hemagglutinins of these mutated viruses but, in many cases, their effectiveness against wild influenza strains is less (see sialoside BGN-PAA
polymers in Table 1).
The purpose of this invention was to find new inhibitors to viral cell adhesion of human influenza viruses. These inhibitors were to have a wide effective range against wild virus strains and thus be as effective as possible.

It has been found that multivalent conjugates of the sialosidligands Neu5Aca2-6Gal~1 4GlcNAc (6'-sialyl-N-acetyllactosamine, 6SLN) are new, highly active viral cell adhesion inhibitors.
The object of the invention are Formula I compounds R4 OyOR~ COOEI

R4 Oll~' / ~~J~ X

/2 R4 (~ . . o R4 R4 o~R4~ YW
N ~3 where R' means acyl or thioacyl groups, primarily acetyl, thioacetyl, propionyl or thiopropionyl group; R2H means hydroxyl, Z-alkyl, substituted Z-alkyl, Z-aryl or substituted Z-aryl, and Z O co~ I esponds to S or NH; R3 means an acyl or thioacyl group; R4represents H or acyl; X O means S or a linear C,-C4 alkyl; Y NH is O, S, CH2 or a sugar;
W is a bifunctional spacer;
P a multivalent carrier consisting of one of the following substances: polyacrylate, polyacrylamide, N-substituted polyacrylamide, Metacrylamide, N-substituted metacrylamide, polyacrylic acid, polycarbonate, polyester, polyamide, polyanhydride, polyaminocarbonate, polyorthoester, polydioxanon, polyphosphazene, polyhydroxycarbic acid, polyamino acid, polysaccharide, protein, dextran, chitosan, glucan, liposome, microparticles.
P can also be a submolecular multivalent carrier such as C(CH2NH-An-)4, where A
is a neutral or negatively charged amino acid.
2s Surprisingly, it also happens that Formula I compounds very efficiently inhibit cell adhesion in all known type A and B human influenza strains. These inhibitors areparticularly effective against authentic influenza viruses in which the structure of the receptor bond area of the hemagglutins of influenza viruses is unaltered. In order to ensure an unaltered hemagglutinin receptor bond area structure, influenza viruses from clinical isolates were used, cultivated solely from MDCK (Madin-Derby canine kidney) cells. As a PCR comparison of viral RNA sequences from MDCK cultivated cells with RNA sequences from viruses from clinical isolates has indicated, the MDCK cultivated viruses show no mutation in the region of hemagglutinin receptor bonds (Robertson, 1 993).
Table 1 shows a comparison of the inhibition of viral cell adhesion through the new 6SLN-PM compound and through the glycol conjugate BGN-PM, which were developed earlier against H3N2 A/Sichuan/1/87 type hen egg cultivated strains. While this 6SLN-PM compound is an outstanding inhibitor of all authentic influenza strains, BGN-PM has a narrower effective range and has very little eMect on many strains.
The advantages of the invention-related Formula I compounds lie particularly in their effective range as inhibitors of viral cell adhesion in all known Type A and B human influenza viruses and in the possibilities these compounds open up for prophylactic and therapeutic purposes for counteracting influenza infections.
A further advantage lies in the easy ~ccess of Formula I compound derivatives, which carry an additional marker, examples of suitable markers being biotin, dyes, fluorescent dyes or radioactive markers. These derivatives can be used as components of new,hitherto inaccessible screening systems for the discovery of new viral cell adhesion inhibitors.

Table 1 Inhibition of influenza viral cell adhesion through the polymers sialoside BGN-PM
and 6SLN-PM, measured through the inhibition of viral bonding to fetuin, as in Example 8 as described by Gambaryan and Matrosovich in 1992.
BGN is Neu5Aca 2-OCH2C5H4NHCOCH2NH-, 6SLN is Neu5Aca2-6Gal,B1 4GlcNAc,B1 -NHCOCH2NH-1. Clinical Human Isolates, Reproduced in MDCK Cells Virus Affinity of the bonding on Virus Kaff ~M Neu5Ac AINIBI47/89M 0.03 0.01 A/NIB/3/90 M 0.01 0.01 A/NIB/44/9OM 0.01 0.01 AlEngland/157/83 M >100 0.03 A/NIB/12/89 M >6 0.1 AINIB/23/89 M >6 0.02 A/N IB/50189 M >5 0.02 B
B/England/222/82 M 0.05 0.04 B/NIB/48/90 M 0.05 0.04 B/NIBI15189 M 0.03 0.02 Il. By Cultivation in Hen Egg Adapted Strains X31 (AlAichil2168 100 3 A/Englandl321/77 0.03 0.01 AIUSSRJ3185 0.06 0.1 AlSichuan/1/87 0.02 0.1 A/England/12/64 10 0.1 AIUSSR/90177 6 0.3 A/Chile/1/83 30 6 B

B/Ann Arbor/1/86 >100 2 BlYamagata/11/88 >100 100 The following is a more detailed explanation of the invention using examples.

Example 1 Production of Neu5Aca2-6Gal,~1-4GlcNAc~1-NHCOCH2NH2 (modified method based on Likhosherstov et al.,1986; and Manger et al., 1992) At room temperature, 13.8 mg of 6-Sialyllactosamine NH4-Selz (20 ,uM, isolated from human urine) was dissolved in a 1 ml saturated ammonium bicarbonate solution and stirred for 170 hours during the incubation, solid ammonium bicarbonate was added 10 several times to the solution until it was saturated. The reaction solution was diluted with 2 ml water, frozen and Iyophilized. 13.7 mg of residue was obtained (the weight of the obtained product was greater than the saccharide used, so the residue was again absorbed in 2 ml of water and Iyophilized), this was absorbed in 0.6 ml 1 M NaHCO3. After stirring at 0~C, 34 mg of chloracetic acid anhydride (200 I~M) in 0.34 ml ethylacetate were 15 added. After 1 hour, the solution was neutralized with acetic acid and concentrated in a vacuum. The residue thus obtained was dissolved in a small quantity of water andchromotographed using a Sephadex G-25 column (1x50 cm). The sugarbearing part was concentrated and the residue was absorbed in 1 ml of saturated ammonium carbonate.
After 48 hours, the solution was diluted with 2 ml of water, frozen and Iyophilized. The 20 residue was dissolved in 2 ml 1 % acetic acid and, after 15 hours, processed using a column with 3 ml Dowex AG 50W-X4 (H~ form). The column was washed with 30 ml water and then eluted with a 1 M ammonium hydroxide solution. After concentration of the ammonium hydroxide eluate, 9.8 mg (67%) of the 6-Sialyllactose N-glycyl derivate was obtained.
'H-NMR-spectrum (D2O, o, ppm): 5.16 (d, 1H, J2 9 HZ, H-1 Gal), 4.47 (d, 1H, J2 8 HZ, H-1 GlcNAc), 4.00-3.55 (Gal, GlcNAc, Neu5Ac), 3.46 (s, 2H, COCH2NH2), 2 69 (dd, 1 H, J3ax1 12 HZ, J4 4.5 HZ, H-3eq Neu5Ac), 2.04 (s, 6H, 2CH3CO), 1.72 (dd, 1 H, J4 12.5 Hz, H-3ax Neu5Ac).

The reaction process was monitored by thin-layer chromotography (silica gel 60, Merck):
Eluant 1: 2-Propanol/AcetoneNVater 4:3:2:
6-SLN R1, 0.61 6-SLN-NH2 0.49 ninhydrin positive 6-SLN-NHCOCH2CI 0.66 6-SLN-NHCOCH2NH2 0.09 ninhydrin positive Eluant 2: Methanol/AcetoneNVater 1:1:1;
6-SLN-NHCOCH2NH2 0.73 ninhydrin positive Example 2 Production of Neu5Aca2~Gal~14GlcNAc,~1-NHCOCH2NHCO(CH2~4COO-p-C6H,,NO2 At room temperature, 39 mg Bis(4-nitrophenyl)-adipat (100 ~M) in 0.6 ml DMF was added to a 7.3 mg solution of Neu5Aca2~Gal~14GlcNAc~1-NHCOCH2NH2 (10 ,uM) from Example 1 in 0.2 ml DMSO. After 3 hours, the reaction mixture was chromatographed using a Sephadex LH-20 column (1.8x30 cm, eluant - acetonitrile/water 1:1). The sugar bearing part was concentrated, absorbed in water, frozen and Iyophilized. 7.4 mg of the glycoside (78%) was obtained.

Example 3 Production of Tetrakis (N-tert-butyloxycarbonyl-~lycyl-amidomethyl) methane A mixture of 1 g tetrakis (aminomethyl) methane (Fleischer et al., 1971 ) tetrahydrochloride (3.6 mmol), 8.529 BocGlyONph (28.8 mmol) and NEt3 (2.5 ml, 18'mmol) in 2 ml DMF was stirred for 120 hours at room temperature. The reactionmixture was concentrated, suspended in 100 ml ethylacetate, and washed in a 2% H2SO4, water, saturated NaHCO3 solution and water and dried using Na2SO4. The solution was concentrated, the residue was dissolved in trifluorethanol, a mixture of CHCI3/EtOAc/MeOH (9:3:1 ) and Et2O was added. 2.1 g of a crystalline product was obtained (78%).
DC: R,=0.5 in CHCI3/EtOAc/MeOH/AcOH 9:3:3:0.2 (ninhydrin positive, after 5 minutes processing of the plate with HCI-Gas).

'H-NMR-spectrum in D8-DMSO (o, ppm): 7.97 (br t, 1 H, CCH2NH), 7.37 (t, 1 H, NHGIY), 3.49 (d, 2H, JNH 6 Hz, CH2G'7, 2.76 (br d, 2H, CCH2),1.37 (s, 9H, OCMe3). Mass spectrum: 783 (M+Na).

Example 4 Production of Tetrakis (N-tert-butyloxycarbonyl-tri~lycl-amidomethyl) methane A solution of 0.76 9 tetrakis (N-tert-butyloxycarbonyl-glycyl-amidomethyl)-methane (1 mmol) from Example 3 was stirred into 8 ml of CF3COOH for 2 hours at room temperature. The reaction mixture was mixed with 16 ml toluol and concentrated. The residue was absorbed in 3 ml water, 0.35 ml of concentrated HCI (12M) was added, the solution was concentrated and the residue was vacuum dried. The tetraamine thus formed was suspended in 18 ml DMF and 1.45 9 BocGlyGlyONsu (4.4 mmol) and 0.6 El3N
was added. The reaction solution was stirred for 24 hours at room temperature, concentrated in a vacuum (0.5-1 Torr) and the residue thus obtained was chromatographed using a silica gel column (silica gel 60, Merck). Elution with Me2CO/MeOH/H2O between 30:1:1 and 15:1:1 gave 0.94 9 (77%) of the [illegible] product.
DC: R,=0.79 in Me2CO/MeOH/H2O 15: 1 :1 (ninhydrin positive, after 5 minutes treatment of the plate with HCI gas).

'H NMR spectrum in D5-DMSO (o, ppm): 8.53 (t, 1 H, NHGIY3), 7.98 (t, 1 H, CCH2NH), 7.81 (t,1 H, NHG'Y2), 6.98 (t,1 H, NHG'Y'), 3.85 (d, 2H, JNH 5.5 HZ, CH2GIY2), 3.73 (d, 2H, JNH 5 5 Hz, CH2GIY3), 3.59 (d, 2H, JNH 6 Hz, CH2GIY'), 2.69 (br. d, 2H, JNH 6.5 Hz, CCH2), 1.38 (s, 9H, OCMe3). Mass spectrum: 1239 (M + Na).

Example 5 Poly-N-(2-Hydroxethyl) - acrlyamide with 20% mol, 6SLN (6SLN-PM) 1.32 mg of poly(4-nitrophenylacrylate) (6.84 I~M) and 7 ml Et3N in 0.132 ml DMF
were added to a 1 mg solution of 6SLN-Gly (1.37 ~M) from Example 1 in 0.1 ml DMSO).
The reaction mixture was incubated at 40~C (The conjugation process was monitered by DC: the disappearance of sugar spots of the educt), after 24 hours 23 ~l of ethanolamine 5 were added. After a further 15 hours at room temperature, the reaction solution was chromatographed using a Sephadex LH-20 column (1.5x25, eluent + acetonitril/water 1: 1).
The sugar bearing portion was concentrated, absorbed in water, frozen and Iyophilized.
1.6 mg of conjugate were obtained (90%).

10 Example 6 Polyacryl acid (Ne+ - salt) With 20 % mol 6SLN

1.32 mg of poly(4-nitrophenylacrylate) (6.84 ,uN) in 0.132 ml DMF and Et3N
(7 ,ul) were added to a solution of 1 mg 6SLN-Gly (1.37 ,uM) in 0.1 ml of DMSO. The 15 reaction mixture was incubated at 40~C (The conjugation process was monitored by DC:
the disappearance of sugar spots of the educt), after 24 hours, 230 ,ul of 0.1 M NaOH
were added. After 15 hours at room temperature, 15 ~l of 1 M HCI were added and the reaction solution was chromatographed using a Sephadex LH-20 column (1.5x25 cm, eluent - acetonitril/water 1: 1). The sugar bearing portion was concentrated, absorbed in 20 water, frozen and Iyophilized. 1.5 mg of conjugate were obtained (93%).

Example 7 (Neu5Aca2~Gal~1 4GlcNAc~1 -NHCOCH2NHCO(CH2)~CO(NHCH2CO)3NHCH

A 1.2 mg solution of tetrakis (N-tert-butyloxycarbonyl-triglycyl-amidomethyl) methane (1 IJM) from Example 4 in 0.1 ml CF3COOH was stirred at room temperature.
After 15 hours, 1 ml of toluol was added and the solution was concentrated. The residue was dissolved in 0.5 ml of water, 5 ,ul of concentrated HCI was added, the solution was concentrated and the residue was vacuum dried. The tetraamine thus obtained was suspended in 0.2 ml DMF and a 4.9 mg solution of Neu5Aca2~Gal,B1 4GlcNAc,~1 -NHCOCH2NHCO(CH2)4COO-p-C6H4NO2(5 ~M) in 0.3 ml DMF and 5 ,ul of NEt3 was added.
The reaction mixture was stirred for 15 hours at 20~C. 10 ,ul of concentrated ammoniac solution was added and after 1 hour the reaction solution was processed using a Sephadex G-50 (1.2x50 cm) (eluant 0.05 M ammoniac solution).
The sugar bearing portion was concentrated, absorbed in water, frozen and Iyophilized.
2.9 mg of conjugate were obtained (68%).

Example 8 Cultivation of influenza viruses and determination of the inhibition of viral bondin~ to fetuin The unadapted human influenza viruses AlEngland/157/83 M, AINIB/12189 M, AINIB/23/89 M, AINIB/50/89 M (H1N1), AJNIB/47/89 M, AINIB/3/90/M, A/NIB/44/90 M
(H3N2), B/England/222/82 M, B/NIB/48/90 M and B/NIB/15/89 M were obtained from the National Institute for Biological Standardisation and Control (NIBSC, Potters Bar/UK).
They were isolated from clinical samples and cultivated exclusively in MDCK cells.
The influenza viruses that were cultivated in hen eggs were obtained from the virus collection of the D.l. Ivanovsky Institute for Virology in Moscow. These viruses were incubated in 9-10 day old embrionic hens' eggs.
For the study of viral bonding, the virus bearing culture stocks and the allentoissh fluids were separated from any cell fragments by centrifugation. These solutions were, without further cleaning, either immediately used or stored for up to 4 weeks at 4~C.
The coating of the microlilralion plates with cow fetuin (Fluka, CH) was carried out as follows: the cavities of a 95er EIA polystyrol micro titration plates (Flow, USA) were incubated for 2 hours at 37~C with 0.1 ml of a fetuin solution in PBS (10 mg/ml). The plate was washed with a 0.01 % Tween 20 (Serva, D) solution in PBS and then with distilled water, and either used directly or air-dried and stored at -20~C until used. In order for the virus to be adsorbed specifically to the feutin coated plates, the viral cultures were diluted with PBS to a hemagglutinin titration of 1 :50-1 :200. The plates were then incubated with 0.1 ml of this solution per cavity for 2 hours at 4~C.
The inhibition of the bonding of the horseradish-peroxidase-fetuin conjugate (produced according to the standard Perjodat activation method) to the adsorbed viruses was determined as follows:
The plates were washed with a 0.01% Tween 20 solution in PBS, then 0.1 ml of a 0.02 ~M solution of the peroxidase-fetuin conjugate along with various concentrations of the inhibitors to be tested were added. After a 1-hour incubation period at 4~C, the plates were washed with a cold PBStTween solution.
The peroxidase activity was determined by the addition of 0.1 ml of a substrate solution (0.4 mg/ml o-phenylenediamine plus 0.02 % H2O2 in a 50 mM Natriumacetalbuffer, pH 5.5). After the addition of the substrate solution, the plates were incubated for 30 minutes in the dark at room temperature, then 0.05 ml of 5% H2SO4 was added and the absorption was determined at 492 nm with a Titertek Multiscan Reader (Flow, Finland).
For the determination of unspecified bonding, several cavities without the addition of viruses were incubated on each plate. Only very low levels of unspecified bonding were found (A492 values of 0.05-0.2).
For the determination of the maximum bonding Ama,~, the bonding of the peroxidase-fetuin conjugate without inhibitor was measured for each plate. As a further control, each separate virus strain was replaced with a peroxidase-fetuin conjugate that had previously been replaced with neurominidase from vibrio choleras: in all cases the conjugate lost its affinity to viruses, thus proving that the conjugate-virus interaction is determined by sialyloligosaccharide.
This bonding affinity was calculated according to the following formula:

Cl x Al (Ama~(-Ao) ~ff Ama,~ (A1-Ao) where KaffiS the dissociation constant of the virus-inhibitor complexes; AmaX is the meaured absorption in the absence of inhibitors; C, and A1 are the inhibitor concentrations used and the corresponding measured absorption; Ao is the absorption 30 measured at inhibitor saturation.

The results of the inhibition of the cell adhesion of the new conjugate 6SLN-PM
from Example 5 are summarized in Tables 1 and 2.

Table 2:
Inhibition of viral cell adhesion in influenza viruses using sialoside inhibitors. The literature data has been su"lnlari~ed and the highest inhibiting activity described above has been explained. The activity is expressed in ,uM concentrations on Neu5Ac inhibitor groups that cause 50% inhibition.
The test system used: Hemagglutination inhibition (HAI), inhibition of virus adsorption to erythrocyte (Al), decrease in viral reproduction in cell cultures (Ml), decrease in viral bonding to fetuin (FBI).
For comparison purposes, the 6SLN-PM bonding data from Example 5 are presented at the bottom of the table.

Bonding type Test ~uM Neu5Ac groups Reference at 50% inhibition BivalentSialoside HAI30(X31) Glick& Knowles, 1991 HAI180(X31) Sabesanetal., 1992 Cluster Sialoside HAI20 (X31 ) Roy et al., 1993 Liposome sialoside HAI 0.02 (X31 ) Kingery-Wood et al., 1992 HAI 0.33(unknown) Spevaketal., 1993 ~ 11 Sialylpolymer FBI 0.3 (A/TX177, AIBK/79) Matrosovich etal., 1990 HAI 0.2 (X31 ) Spaltstein & Whitesides, HAI 0.1 (X31 ) Sparks et al., 1993 HAI 0.2 (X31) Lees et al., 1994 Ml 0.2(X31, H3N2) Itoh etal., 1995 5 (WSN/33, H1 N1 ) Itoh et al., 1995 ~400 (SG/57, H2N2) Itoh et al., 1995 FBI >50 (X31, H3N2) Mochalova et al., 1994 0.03(USSR185, H3N2) Mochalovaetal., 1994 10 (Chile/83, H1 N1 ) Mochalova et al. , 1994 10 (B/USSR~83) Mochalova et al., 1994 Ml ~8 (X31, H3N2) Mochalovaetal., 1994 0.2 (USSR/85, H3N2) Mochalova et al., 1994 4 (Chile/83, H1N1) Mochalova et al., 1994 HAI 0.001 (X31, H3N2) Maman etal., 1995 6SLN-PM as FBI 0.02 (A/NIBI23189M, H1 N1 ) described in Example 0.01 (A/NIBI44/9OM, H3N2) 5 of this invention 0.02 (BINIBI15/89M) .
- L~-terature - Bovln, N.V., Byramova, N.E., ~uzikov, A.B., Mstrosovich, M.N., Mioch~lova, i .V., and Gambaryan, A.S. (1992) Viral attachrnent inhlbi~ors~ Patent PCT No.
5 PCT/US92109746.
Byramova, N.E., Mochalovsl L.Y., B~lyanchikov, I.M., ~atrosovich~ t~i.N., and ~ovln, N.V. ~1991 ) Synthecls of slalic acid pseudopolysaccharides by coupling of cpacer-connected NsuSAc with actlvat~d polymer. J.Carbohydr.C~em. 10, 691-700.
- Fl~ischer, E.i3., Gebala, A.E.~ LOYOY~ A., Tasker, P.~. (1971). Conversion of alipi~atic and ~licyclic polyalcohols to ths corr~sponding primary polyamlnes. J.Organic Chem., Vol. 36, No. 20, 3042-3044.
Gambaryan~ A.S. and Matrosovlch~ M.N. (1992) A solid-phase enzyrne-llnked assay for influenza vlrus receptor-bindlng actlvily. J.Virol,Methods, 39, 111-123, Gllck, G.D., 2nd Knowlss, J.R. (199~). Molecular recognition of blval~nt sialosides by infiu~nza ~Irus. J.Amer.Chem.Soc. 113, 47014103.
Gottsche!k, A., Belyavln, G., i3iddl~, F. (1972). Glycoprot~ins as influenz~ virus haemagglutlnln inhibitors and as cellular virus recep~ors. In "Glycoproteins. Thelr composition, structure and function", (A.Gottschalk, Ed.), 2-d edition, part B, pp.1082-1096. ~1tevier Publishing Company, Amsterdam-~ondon-Ne~4-York.
Itoh, M., i~etterlchj P., Isecke, R., Btossmer, R., and Kl~nk, ti~-D. (1995).
- Suprcssion of influen~a virus infection by an N-thioacetylneuraminic aoid acrylamlde copolymer~reslstant ~o neurzminidase. ~./lrology 212, 340-347 (1995).
Kln~ery-Wood, J.E., \Nilllams, K.W" Sl~al~ G.B., and White~ldes, G.M~, ~1992).
The sgglytlnatlon of erythrocytes ~y influ~nza virus is strongly inhibited by llposomcs - incorpora~in~ an analog of siàlyl ~arigliosides. J.Amer.Chem.Soc. 114, 7303-7305.
Krizanova, O., and RatnoYa, V. t1969). Serum inhibitors of myxoviruses.
Curr.~op.Microblol.lmmunot. 47,'125-151.
Lees, W.J., Spaltenstein, A., Kln~ery-Wood, J.E., and \~Yhi~eslde9, G.M. (1994).Polyacrylamldes b~arin3 pendant a-si~loside gro~lps strongly Inhlbl~ agglu~ination of erythrocytes by Influenza A virus: m~ ltlvalency ~nd steric st~bili a~lon of partlculate blolo~ical syst~ms. J.Med.Chem. 3~, 3419~3433.

. CA 02239296 1998-06-02 .

Likhoshorsto~, L.M., ~Jovikova. O.S., I:)ereYitskaJa, V.A, l~ochetkov, N K (1986).
A n~w si~pl~ synthesis of amino s-l~ar b-D-~lycosylarnines. Carbohydralo ~search, 146, C1 -C5.
Mammen, M., Dahmann, G.~ Whltesldes, G.M. (1995) Effective inhlbltors of 5 hemagglutln~tlon by tnfluenza virus syn~hesized from polymers havin~ active ester ~ groups; Insl~hl Into mechanism of Inhibitlon. J.Mod.Chem, 38, 4179~190.
Manger, I.~)., Rademacher, T.W., Dwek, R.A. (1992). 1-N-Glycyl b-~- oligosaecharide deriYa~lves as stabl~ intermediates for the formatlon of glycoconjugate probes Eiochemistry, 31, 10724-10732. .
Matrosovlch, M.N., l~iochalova, L.V., Marinina, V.P., Byramo~a~ N.E., and Bovin,N.~/. (1993). Syn~hetlc polymerlc sialosid~ inhlbitors of the Influenza Yirus recepior-bindin~ activity FEBS Letters 272, 209-212.
Matrosovlch M.N., Gambaryan A S, Tuzikov A.B., Byramova N E, Mochalova L.V., Golbralkh A.A., Shenderovlch l\l.D., Finne J., and BoYin, N.V. (1993) Probing of ~ 15 ~hc reccpIor-binding si~es of the 1 11 end H3 influenza A ~nd.influcnza B virus hemag~lutinin~ by synthetlc and.naturei sialosldes. Virolo~y 196, 111~121.
Mochalo~a, L.V., TuzikoY, A.B., Marinina, V.P., Gambaryen, A.S., Byramo~.~a, N E., Bo-~ln, ~I V., 2nd Matroso~.~lc~l, M.N, (1994) Synthctic potymeric Inhibi'~ors of influenza vlrus receptor-binding activity suppress virus replication. Antiviral Research 20 23, 1 79-1 90 Robertson, J.S. ~9g3). Clinlcal Influenza virus and the embryonated hen's eg~
~ ReY~Med;Virol. 3, 97-106.- .
Roy, R.! and Lafcrrlere, C.A. (1988). Synlh~sis of antl~enic copolymers of t~i-acetyl~n~uremlnic acid and their bindlng to wheat çerm a~lutinin and antlbodies.25 Carbohydr.Res. 177, c1 -c4.
Roy, R., 2~nlnl, D., M~unier, S.J., and Romanowska, A. (1993). Solld-phase synthesis of dendrl~lc slaloside tnhibi~ors of Influ~nza A virut~ herna~glutinin.
J.Chem.Soc., Ch~m. Comrnun. 1993,1869-1872.
Sabesan, S., DUUS, J.O., ~elra, S., Domaille, P., Kelm, S., Palllson, J.C, and 30 ~ock, K. (1992). Clu3ter aialoside inhibitors for Influenza virus: synth~sls, NMR, and blolo~ical studles. J.Am~r.Chem Soc 114, 8363-8375.

~A 02239296 1998-06-02 ' , Sauter, N.K., Hanson, J.E., Gilck, G.D., Brown, J.H., Crowther, R.L., Par~ S.-J., . Skehel, J.J., and Wiley~ ~.C. (1992) Bindin~ of influenza vlrus h~mag~lutlnln to - analogs of Its cell-surfece recoptor, slalic 2cid; analysis by proton nuclear ma~netlc resonancs spectloscopy and X-ray crystallo~r2phy. Biochemistry 31, 9609-962l.
Spaltenstein, A r ~nd Whltesides, G ~l. (1931). Polyecryamides b~aring pendanl a-sialosld~ ~1fOUpS stron~ly inhii~lt a~giutination of erythrocytes by Influenza vlrus.
J.Amer.Ch~m.Soc; 113, 686~87.
Spsvak, W., Na~y, J.O., Charychl D.i~., Schaefer, ~.E ~ Gilbort, J.~., end 8ednarskl, M D. (~993). Polymeri2ed llposome containin~ C-~lycosides of si~lic acld:
potent inhlbltors of influen~a YlrUS in vitro infectlvl~y. .I.Am~r.Chem.Soc. 115, 1146-1147.
Too~ood, P.L, Gallikerl P.K., Glick, G.D. and Knowles, J.R. (19g1) Monovalen~
slalosides ~hat bind tightly to influen~a A vlrus. J.Med.Chem. 34, 3138-3140.
Watowloh, S.J., Skehel, J.J., and Wiiey, D.C. (1994) Crystal s~ruclures of influenza Yirus hemagslLJtinln In complex wlth hl~h-affinlty receptor analo~s. Structure 2, 719-~31.
W~lnhold, E.G. and Knowl2s, J.R. (1992) Design and evzluation of a tlgh~ly bindin~ fluorescent li~and for In~luenza A h~ma~glutlnin. J.Am.Chern Soc. 114, 9270-~27S.
Wlley, D.C. and Skehel, J.J. (1987) The structure and functlon of the hemagglu~lnin ~ ,niirar,e Qlycopro~sin of influsnza virus. Ann.Rev.eiochem. 56, 365-394.

Claims (12)

1. Compounds of the general formula wherein R1 means an acyl group or a thioacyl group;
R2 -means H, Hydroxyl, Z-alkyl, substituted Z-alkyl, Z-aryl, substituted Z-aryl, and Z
corresponds to O, S or NH;

R3 -means an acyl group or a thioacyl group;

R4 -represents H or acyl;

X -means O, S or a linear C1-C4 alkyl;
Y -is NH, O, S, CH2 or a sugar;
W -is a bifunctional spacer;
P -is a multivalent carrier consisting of one of the following substances:
polyacrylate, polyacrylamide, N-substituted polyacrylamide, metacrylamide, N-substituted metacrylamide, polyacryl acid, polycarbonate, polyester, polyamide, polyanhydride, polyiminocarbonate, polyorthoester, polydioxanon, polyphosphazen, polyhydroxycarbon acid, polyamino acid, polysaccharide, protein, dextran, chitosan, glucan, liposome, microparticles.

2. Formula I compounds according to claim 1, characterized by the fact that the multivalent carriers represent C(CH2NH-An-)4; where A is a neutral or negatively charged amino acid.

3. Formula I compounds according to claim 1 or claim 2 where P is marked with biotin, a dye, a flourescent dye or a radioactive marker.

4. Use of Formula I compounds in accordance with claim 1 or claim 2 for virus bonding.

5. Use of Formula I compounds in accordance with claim 1 or claim 2 for influenza virus bonding.

6. Use of Formula I compounds in accordance with claim 1 or claim 2 for human influenza virus bonding.

7. Use of Formula I compounds in accordance with claim 3 as components of test systems for screening viral cell adhesion inhibitors.

8. Use of Formula I compounds in accordance with claim 1 or claim 2 for bonding to influenza virus hemagglutinin.

9. Use of Formula I compounds in accordance with claim 1 or claim 2 as inhibitors of viral cell adhesion to natural receptors.

10. Use of Formula I compounds in accordance with any one of claims 4 to 8 in combination with neuraminidase inhibitors.

11. Use of Formula I compounds in accordance with claim 1 or claim 2 as for prophylactic or therapeutic treatment of infections.

12. Use of Formula I compounds in accordance with claim 1 or claim 2 for prophylactic or therapeutic treatment of viral infections in combination with neuraminidase inhibitors.