ANTIVIRAL POLYMERS

Abstract

The present invention relates to polymer compounds that have antiviral activity. The compounds have the structural Formula I defined herein. The present invention also relates to processes for the preparation of these compounds, to compositions comprising them, and to their use in the prevention or treatment of viral infections.

Claims

1. A branched polymer according to Formula I: ##STR00141## wherein ##STR00142## is a polyvalent core structure; X is a monomer residue comprising at least one sulfonate or sulfate substituent; Y is a monomer residue; Z is a capping group; m is greater than or equal to 3; n is 5 to 500; p is 1 to 500; and q is 0 to 200; wherein if q is greater than 1, then at each occurrence, Y may be the same or different residues.

2. A branched polymer according to claim 1, wherein X has a structure according to Formula II: ##STR00143## wherein R.sup.1 is aryl, C.sub.1-20alkyl, C.sub.1-20alkylene-aryl, C(O)OR.sup.4, C(O)NHR.sup.4, OC(O)R.sup.4, NHC(O)R.sup.4, or sulfonate; R.sup.2 and R.sup.3 are independently selected from hydrogen and C.sub.1-4alkyl; and R.sup.4 is C.sub.1-10alkyl or aryl; and wherein each of the aryl, C.sub.1-20alkyl, C.sub.1-20alkylene-aryl, or R.sup.4 groups is substituted with one or more sulfonate or sulfate groups, and is optionally substituted with one or more substituents selected from hydroxy, halo, C.sub.1-4alkyl, C.sub.1-4alkoxy, aryl, and cyano.

3. A branched polymer according to claim 2, wherein R.sup.1 is phenyl, C(O)OR.sup.4, or C(O)NHR.sup.4, wherein R.sup.4 is C.sub.1-10alkyl and each of the phenyl or R.sup.4 groups is substituted with one or more sulfonate groups and is optionally substituted with one or more substituents selected from hydroxy, halo, C.sub.1-4alkyl, C.sub.1-4alkoxy, and cyano.

4. A branched polymer according to claim 2, wherein R.sup.1 is selected from: ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## wherein L .sup.2 is C.sub.1-20alkylene.

5. A branched polymer according to any one of claims 2 to 4, wherein R.sup.2 and R.sup.3 are independently selected from hydrogen and methyl,.

6. A branched polymer according to any one of claims 2 to 4, wherein R.sup.2 is hydrogen or methyl and R.sup.3 is hydrogen.

7. A branched polymer according to any one of claims 1 to 6, wherein n is 5 to 400, such as 5 to 300, 10 to 250, or conveniently 20 to 100.

8. A branched polymer according to any one of claims 1 to 7, wherein Z is selected from hydrogen, hydroxyl, bromo, chloro, or one of the following groups: ##STR00149## ##STR00150## ##STR00151## ##STR00152## wherein: R.sup.5 is hydrogen, halo, cyano, CO.sub.2H, C.sub.1-3haloalkyl, C.sub.1-3alkylene-OH, or C.sub.1-3alkylene-NH.sub.2; R.sup.6 and R.sup.7 are independently chosen from hydrogen and C.sub.1-3alkyl optionally substituted with cyano, halo, or CO.sub.2H; R.sup.3 is S-C.sub.1-15alkyl, S-aryl, NR.sup.9R.sup.10 or aryl, said C.sub.1-15alkyl and aryl groups being optionally substituted with one or more substituents selected from hydroxy, C.sub.1-3alkyl, halo, cyano, CO.sub.2H, and C.sub.1-3haloalkyl; R.sup.9 is hydrogen or C.sub.1-3alkyl; and R.sup.10 is hydrogen, C.sub.1-12alkyl or aryl, said C.sub.1-12alkyl and aryl groups being optionally substituted with one or more substituents selected from hydroxy, C.sub.1-3alkyl, halo, cyano, CO.sub.2H, and C.sub.1-3haloalkyl.

9. A branched polymer according to any one of claims 1 to 7, wherein Z is selected from one of the following groups: ##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159## .

10. A branched polymer according to any one of claims 1 to 9, wherein p is 5 to 400, such as 5 to 300, 10 to 250, or conveniently 20 to 150.

11. A branched polymer according to any one of claims 1 to 10, wherein q is 0, or q is 1.

12. A branched polymer according to any one of claims 1 to 11, wherein m is 3 to 12, such as 4 to 10, or conveniently 4 or 6.

13. A branched polymer according to any one of claims 1 to 11, wherein ##STR00160## is selected from: ##STR00161## ##STR00162## ##STR00163## wherein T is O, S or NH; R.sup.11 is hydrogen or C.sub.1-4alkyl; and L.sup.1 is selected from one of the following linker groups: ##STR00164## ##STR00165## ##STR00166## ##STR00167## and wherein ##STR00168## is the point of attachment to T and * is the point of attachment to an X residue; R.sup.12 and R.sup.13 are independently chosen from hydrogen, C.sub.1-3alkyl, hydroxy, and halo; r is 1 to 10; and R.sup.14 and R.sup.15 are independently chosen from hydrogen and C.sub.1-3alkyl.

14. A branched polymer according to claim 13, wherein L.sup.1 is selected from: ##STR00169## ##STR00170## and Z is selected from: ##STR00171## ##STR00172## ##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177## .

15. A branched polymer according to claim 13, wherein L.sup.1 is ##STR00178## and Z is: ##STR00179## .

16. A branched polymer according to claim 13, wherein L.sup.1 is ##STR00180## and Z is bromo or chloro.

17. A branched polymer according to any one of claims 13 to 16, wherein T is O.

18. A branched polymer according to claim 1 wherein X is: ##STR00181## n is 10 to 100, q is 0, and m is 4.

19. A composition comprising a branched polymer according to any one of claims 1 to 18, or a salt or salts thereof.

20. A method of sterilisation or viral disinfection, comprising using an effective amount of the composition of claim 19.

21. A device for sterilisation or viral disinfection comprising the composition of claim 19 and means for dispensing the composition.

22. A pharmaceutical composition comprising a branched polymer according to any one of claims 1 to 18, or a pharmaceutically acceptable salt or salts thereof, and one or more pharmaceutically acceptable excipients.

23. A branched polymer according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 22, or a pharmaceutically acceptable salt or salts thereof, for use in the prevention or treatment of viral infections.

24. The branched polymer or pharmaceutical composition for use in the prevention or treatment of viral infections according to claim 23, wherein the viral infection is associated with herpes simplex virus (HSV), adenovirus, adeno-associated virus, human papillomavirus (HPV), respiratory syncytial virus (RSV), dengue virus, norovirus, lentivirus, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), human metapneumovirus (HMPV), human parainfluenza virus type 3 (HPIV-3), coronavirus (such as MERS-CoV, SARS-CoV, or SARS-CoV-2), foot-and-mouth disease virus, hepatitis B virus, hepatitis C virus, Ebola virus, nipah virus, Rift Valley fever virus, West Nile virus, Crimean Congo virus, Toscana virus, ZIKA virus, Chickungunya virus (CHIKV), Akabane virus (AKAV) or Schmallenberg virus (SBV), influenza (such as Influenza A H3N2 or H1N1 virus), adeno-associated virus (AAV), Newcastle disease virus (NDV), or vesicular stomatitis virus (VSV).

25. A material comprising a branched polymer according to any one of claims 1 to 18, or a salt, or salts, thereof.

Description

LIST OF FIGURES

[0245] FIG. 1 shows the .sup.1H-NMR spectrum in D.sub.2O of Example 9.4 (4-arm PSS DP100)

[0246] FIG. 2 shows the HSV-2 plaque forming units/ml (log-scale) following incubation with (A) Example 9.1 (4-arm PSS DP10); (B) Example 9.2 (4-arm PSS DP30); (C) Example 9.3 (4-arm PSS DP50); and (D) Example 9.4 (4-arm PSS DP100) at 5 .Math.g and 15 .Math.g versus the untreated control sample (labelled HSV-2).

[0247] FIG. 3 shows the HSV-2 percentage infection at increasing concentrations of Example 9.1 (4-arm PSS DP10), Example 9.2 (4-arm PSS DP30), Example 9.3 (4-arm PSS DP50) and Example 9.4 (4-arm PSS DP100).

[0248] FIG. 4 shows the SARS-CoV-2 percentage infection at increasing concentrations of Example 9.3 (4-arm PSS DP50).

[0249] FIG. 5 shows the SARS-CoV-2 percentage infection at increasing concentrations of Example 9.3 (4-arm PSS DP50) and Example 9.4 (4-arm PSS DP100) in a separate assay to FIG. 4.

[0250] FIG. 6 shows the effect of Example 9.4 (4-arm PSS DP100) on the viral titre (expressed as log10 TClD.sub.50 titre reduction) for HSV-2 and RSV at different concentrations.

[0251] FIG. 7 shows the HSV-2 plaque forming units/ml (log-scale) following incubation with untreated control sample (NTC); comparative example 11.1 (linear DP100 PSS polymer with CTA), comparative example 11.2 (linear DP100 PSS polymer without CTA); Example 9.4 (4-arm PSS DP100); and comparative example 11.3 (cleaved 4-arm PSS DP100).

[0252] FIG. 8 shows the results of the MTS cell health assay using 50 to 500 .Math.g/mL concentrations of (left to right): cells only; Example 9.1; Example 9.2; Example 9.3; and Example 9.4

EXAMPLES

General Procedures

[0253] Methods for preparing the compounds of this invention are illustrated in the following Examples. Starting materials are made according to procedures known in the art or as illustrated herein, or are available commercially. Commercial reagents were used without further purification. Where no reaction temperature is included, the reaction was performed at ambient temperature which is typically 18-27° C.

[0254] Where compounds described in the invention are characterized by .sup.1H NMR spectroscopy, spectra were recorded on 400 MHz instruments. Where no temperature is included the spectra were recorded at ambient temperature. Chemical shift values are expressed in parts per million (ppm).

TABLE-US-00001 Abbreviations: ACVA 4,4′-azobis(4-cyanovaleric acid) AIBN azobis(isobutyronitrile) ATRP atom transfer radical polymerisation CTA chain transfer agent DCM dichloromethane DP degree of polymerisation EtOH ethanol HSV herpes simplex virus MeOH methanol MWCO molecular weight cut off MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium NMR nuclear magnetic resonance PBS phosphate buffered saline PFU plaque forming units PSS polystyrene-4-sulfonate RAFT reversible addition fragmentation chain transfer polymerisation RO reverse osmosis (purified) RSV respiratory syncytial virus RT room temperature SARS-CoV severe acute respiratory syndrome coronavirus SPMA 3-sulfopropylmethacrylate

Synthesis of Branched Polymers

Example 1: Synthesis of 4-Arm ATRP Initiator

[0255] ##STR00126##

[0256] To pentaerythritol (2.4 g, 1 eq) was added 20 mL pyridine and the mixture was cooled to 0° C. 2-bromoisobutyryl bromide (24.3 g, 6 eq) added drop wise followed by stirring at 0° C. for 30 min and the reaction mixture was allowed to warm to room temperature overnight with stirring. After concentration under reduced pressure DCM (100 mL) was added followed by washing against NaHSO.sub.4 (1 M; 3 × 50 mL), Na.sub.2CO.sub.3 (10%; 3 × 50 mL) and brine (3 × 50 mL). The organic layer was then dried over MgSO.sub.4 before drying under reduced pressure. The resultant solid was recrystallised in MeOH to yield the pure product in quantitative yield.

Example 2: Synthesis of 6-Arm ATRP Initiator

[0257] ##STR00127##

[0258] To di-pentaerythritol (2.54 g, 1 eq) was added 20 mL pyridine and the mixture was cooled to 0° C. 2-bromoisobutyryl bromide (20.7 g, 9 eq) added drop wise followed by stirring at 0° C. for 30 min and the reaction mixture was then allowed to warm to room temperature overnight with stirring. After concentration under reduced pressure DCM (100 mL) was added followed by washing against NaHSO.sub.4 (1 M; 3 × 50 mL), Na.sub.2CO.sub.3 (10%; 3 × 50 mL) and brine (3 × 50 mL). The organic layer was then dried over MgSO.sub.4 before drying under reduced pressure. The resultant solid was recrystallised in MeOH to yield the pure product in quantitative yield.

Example 3: Synthesis of 4-Arm PSS Via ATRP Polymerisation Using 4-Arm ATRP-Initiator

[0259] Sodium 4-vinylbenzenesulfonate (4n mol eq*) was combined with 4-arm ATRP-initiator (Example 1) (44.4 mg, 1 eq)in H.sub.2O:EtOH (3:1) and degassed by N.sub.2 bubbling. Separately CuCl (24 mg) and tris[2-(dimethylamino)ethyl]amine (Me.sub.6TREN) (55.86 mg) were combined in H.sub.2O:EtOH (3:1) and degassed by N.sub.2 bubbling. After approximately 30 min the copper mixture was added to the stirred monomer solution (under N.sub.2). The reaction was left for 24 hrs at room temperature before being precipitated into acetone. The resultant solid was dialysed (1000 MWCO) against a basic ethylenediaminetetraacetic (EDTA) solution followed by dialysis against RO H.sub.2O. *wherein n is the degree of polymerisation or the number of PSS residues. Therefore, when n = 50, 200 mol eqv of sodium 4-vinylbenzenesulfonate are used.

Example 4: Synthesis of 6-Arm PSS Via ATRP Polymerisation Using 6-Arm ATRP-Initiator

[0260] Sodium 4-vinylbenzenesulfonate (6n mol eq*) was combined with 6-arm ATRP-initiator (Example 2) (46.4 mg, 1 eq) in H.sub.2O—EtOH (3:1) and degassed by N.sub.2 bubbling. Separately CuCl (24 mg) and tris[2-(dimethylamino)ethyl]amine (Me.sub.6TREN) (55.86 mg) were combined in H.sub.2O—EtOH (3:1) and degassed by N.sub.2 bubbling. After approximately 30 min the copper mixture was added to the stirred monomer solution (under N.sub.2). The reaction was left for 24 hrs at room temperature before being precipitated into acetone. The resultant solid was dialysed (1000 MWCO) against a basic ethylenediaminetetraacetic (EDTA) solution followed by dialysis against RO H.sub.2O. *wherein n is the degree of polymerisation or the number of PSS residues. Therefore, when n = 50, 300 mol eqv of sodium 4-vinylbenzenesulfonate are used.

Example 5: Synthesis of 4-Arm CTA-OH

[0261] ##STR00128##

[0262] Pentaerythritol tetra-(3-mercaptopropionate) (0.50 g, 1.02 mmol, 1 eq) was added to a stirred suspension of potassium phosphate (912 mg, 6.70 mmol, 6.6 eq) in 15 ml of acetone and stirred for 45 min at room temperature. Then, carbon disulphide (0.913 g, 11.99 mmol, 11.8 eq) was added to the previous solution and stirred for 45 min at room temperature. 4-chloromethyl(benzoyl) alcohol (689 mg, 4.40 mmol, 4.3 eq) was added and stirred at RT overnight. The reaction mixture was filtered and washed with acetone. Sample was then dried under reduced pressure. The solid was then dissolved in DCM, washed with water and brine several times and then the organic phase dried over magnesium sulphate. Column chromatography in DCM gave the pure product.

Example 6: Synthesis of 6-Arm CTA-OH

[0263] ##STR00129##

[0264] Dipentaerythritol hexakis-(3-mercaptopropionate) (1.00 g, 1.28 mmol, 1 eq) was added to a stirred suspension of potassium phosphate (1.716 g, 12.61 mmol, 9.9 eq) in 20 ml of acetone and stirred for 45 min at room temperature. Then, carbon disulphide (1.748 g, 22.95 mmol, 17.9 eq) was added to the previous solution and stirred for 45 min at room temperature. 4-chloromethyl(benzoyl) alcohol (1.199 g, 7.66 mmol, 6.0 eq) was added and stirred at RT overnight. The reaction mixture was filtered and washed with acetone. Sample was then dried under reduced pressure. The solid was then dissolved in DCM, washed with water and brine several times and then the organic phase dried over magnesium sulphate. Column chromatography in DCM gave the pure product.

Example 7: Synthesis of 4-Arm CTA-COOH

[0265] ##STR00130##

[0266] Potassium hydroxide (505 mg, 4.8 eq) was suspended with vigorous stirring in acetone (30 mL) and water (30 mL), followed by the addition of Pentaerythritol tetra-(3-mercaptopropionate) (1.0 g, 2.04 mmol, 1 eq). This mixture was allowed to stir at room temperature for 1 hour before the addition of carbon disulphide (1.59 g, 4.8 eq). After 1 hr of further stirring at room temperature 2-bromopropionic acid (1.38 g, 4.8 eq) was added, after which the reaction was allowed to stir overnight at room temperature. The reaction was stopped and filtered, the remaining solution was evaporated under reduced pressure, before being re-dissolved in dichloromethane (150 ml) and washed repeatedly against deionised water (3 × 200 ml), the aqueous layers were combined and concentrated under reduced pressure. The concentrated solution was then dialysed against several batches of deionised water, before being evaporated under reduced pressure and finally freeze-dried to recover a solid material. The resultant solid was then acidified with acetic acid before being purified by column chromatography using ethyl acetate:hexane (3:1) with 3% acetic acid as co-eluent.

Example 8: Synthesis of 6-Arm CTA-COOH

[0267] ##STR00131##

[0268] Potassium hydroxide (487 mg, 7.2 eq) was suspended with vigorous stirring in acetone (30 mL) and water (30 mL), followed by the addition of dipentaerythritol hexakis-(3-mercaptopropionate) (1.0 g, 1.28 mmol, 1 eq). This mixture was allowed to stir at room temperature for 1 hour before the addition of carbon disulphide (1.53 g, 7.2 eq). After 1 hr of further stirring at room temperature 2-bromopropionic acid (1.33 g, 7.2 eq) was added, after which the reaction was allowed to stir overnight at room temperature. The reaction was stopped and filtered, the remaining solution was evaporated under reduced pressure, before being re-dissolved in dichloromethane (150 ml) and washed repeatedly against deionised water (3 × 200 ml), the aqueous layers were combined and concentrated under reduced pressure. The concentrated solution was then dialysed against several batches of deionised water, before being evaporated under reduced pressure and finally freeze-dried to recover a solid material. The resultant solid was then acidified with acetic acid before being purified by column chromatography using ethyl acetate:hexane (3:1) with 3% acetic acid as co-eluent.

Example 9: Synthesis of 4-Arm PSS via RAFT Polymerisation Using 4-Arm CTA-COOH

[0269] ##STR00132##

[0270] 4-arm CTA-COOH (43.7 mg, 1 mol eq), 0.5 M sodium carbonate solution, sodium 4-vinylbenzenesulfonate (4n mol eq*) and 4,4′-azobis(4-cyanopentanoic acid) (ACVA) (1.13 mg) were combined in H.sub.2O (4 ml) and sealed and degassed. The reaction mixture was then stirred at 70° C. for 4 hr. After complete polymerisation the reaction mixture was rapidly cooled and the polymer precipitated into acetone, before being dialysed (1000 MWCO) against H.sub.2O and dried to yield pure polymer. *For example, for the 4-arm PSS DP10, 10 mol eqv of sodium 4-vinylbenzenesulfonate were used for each arm, therefore 40 mol eqv were used relative to the number of moles of CTA.

[0271] The branched polymers prepared according to Example 9 are summarised in Table A below. FIG. 1 shows a .sup.1H-NMR spectrum of Example 9.4.

Example 10: Synthesis of 4-Arm SPMA via RAFT Polymerisation Using 4-Arm CTA-COOH

[0272] ##STR00133##

[0273] 4-arm CTA-COOH (36.6 mg, 1 mol eq), 3-sulfopropyl methacrylate (4n mol eq*) and 4,4′-azobis(4-cyanopentanoic acid) (ACVA) (0.95 mg) were combined in H.sub.2O (4 ml) and sealed and degassed. The reaction mixture was then stirred at 70° C. for 4 hr. After complete polymerisation the reaction mixture was rapidly cooled and the polymer precipitated into acetone, before being dialysed (1000 MWCO) against H.sub.2O and dried to yield pure polymer. *For example, for the 4-arm SPMA DP30, 30 mol eqv of 3-sulfopropyl methacrylate were used for each arm, therefore 120 mol eqv were used relative to the number of moles of CTA.

[0274] The branched polymers prepared according to Example 10 are summarised in Table A below.

TABLE-US-00002 Ex. No. Description [00134] X n q Z.sup.d m 9.1 4-arm PSS DP10 A PSS.sup.b 10 0 Z1 4 9.2 4-arm PSS DP30 A PSS.sup.b 30 0 Z1 4 9.3 4-arm PSS DP50 A PSS.sup.b 50 0 Z1 4 9.4 4-arm PSS DP100 A PSS.sup.b 100 0 Z1 4 10.1 4-arm SPMA DP10 A SPMA.sup.c 10 0 Z1 4 10.2 4-arm SPMA DP30 A SPMA.sup.c 30 0 Z1 4 10.3 4-arm SPMA DP50 A SPMA.sup.c 50 0 Z1 4 10.4 4-arm SPMA DP100 A SPMA.sup.c 100 0 Z1 4 .sup.a Cores:[00135] .sup.b PSS = polystyrene-4-sulfonate,[00136] .sup.c SPMA = 3-sulfopropylmethacrylate,[00137] .sup.d Capping Groups:[00138]

Comparative Example 11: Linear Polymers and Branched Cleaved Polymer

[0275] To evaluate the antiviral activity of comparative non-branched polymers, the following materials were prepared:

Comparative Example 11.1 - Linear DP100 (n = 100) PSS Polymer Incorporating Chain Transfer Agent

[0276] ##STR00139##

Comparative Example 11.2 - Linear DP100 (n = 100) PSS Polymer Without Chain Transfer Agent

[0277] ##STR00140##

Comparative Example 11.3 - Branched Cleaved DP100 (n = 100) PSS Polymer

[0278] Example 9.4 branched polymer (4-arm PSS DP100), prepared as described hereinabove, was subjected to chemical cleavage of the trithiocarbonate moieties to yield a derivative wherein the sulfonated residues have been cleaved from the 4-arm core structure. This was achieved by heating Example 9.4 for 3 hours at 75° C. in the presence of N-ethylpiperidine hypophosphite and AIBN in water. After cooling, the cleaved material was dialysed for 3 days with twice daily changes of water.

Biological Assays

Inhibition Assay With HSV-2

[0279] The effect of antiviral branched polymers on HSV-2 infection was evaluated by a plaque reduction assay. Vero cells were seeded 24 hours in advance in 24-well plates at a density of 10.sup.5 cells per well. Increasing concentrations of antivirals were incubated with HSV-2 [multiplicity of infection (MOI), 0.0003 plaque-forming units (PFU)/cell] at 37° C. for 1 hour, and then the mixtures were added to the cells. Following virus adsorption (2 hours at 37° C.), the virus inoculum was removed and the cells were washed with medium and then overlaid with a medium containing 1.2% methylcellulose. After incubation with HSV-2 for 24 hours, respectively, at 37° C., cells were fixed and stained with 0.1% of crystal violet in 20% ethanol and viral plaques were counted. The concentration of compound producing 50% reduction in plaque formation (EC.sub.50) was determined using Prism software by comparing drug-treated and untreated wells.

Evaluation of Virucidal Activity Against HSV-2 and RSV-A

[0280] Viruses (10.sup.5 PFU for HSV-2) and antivirals (15 .Math.g/ml) were incubated for 1 hour at 37° C., and then the virucidal effect were investigated with serial dilutions of the mixtures over a 96 well plate seeded with Vero cells (95% confluent). Following incubation, virus/antiviral mixtures were diluted 1:10, 1:100 and 1:1000 and then 50 .Math.L from each (in duplicate) was added per well (already containing 100 .Math.L of 2% FBS media), before being serially diluted down the plate. Viral titers were calculated at dilutions at which the antiviral was not effective.

Inhibition Assay With SARS-CoV-2 (A)

[0281] The effect of the branched polymers Example 9.3 and Example 9.4 on SARS-CoV-2 infection was evaluated by a plaque reduction neutralisation assay.

[0282] Test compound (Example 9.3 or Example 9.4) was serially diluted, in duplicate and each dilution was incubated with approximately 40 PFU of wild type SARS-CoV-2 (2019-nCoV/Victoria/1/2020) virus, for 1 h at 37° C. The samples were then allowed to absorb for 1 hour at 37° C. on Vero E6 [Vero 76, clone E6 (ECACC 85020206), European Collection of Authenticated Cell Cultures, UK] monolayers in 24-well plates. Afterwards plaque assay overlay media was added and the samples were incubated for at 37° C. for 5 days. Then plates were fixed overnight with 20% (w/v) formalin/PBS, washed with tap water and stained with methyl crystal violet solution (0.2% v/v) and the viral plaques were counted.

[0283] An internal negative control using serial dilutions of test compound with Vero E6 cells only, was run in parallel for 5 days to monitor cell monolayer integrity/toxicity caused by test compound. An internal positive control, was run in parallel, using a sample of human MERS convalescent serum known to neutralise SARS-CoV-2 (National Institute for Biological Standards and Control, UK).

Inhibition Assay With SARS-CoV-2 (B)

[0284] The effect of the branched polymers Example 9.3 and Example 9.4 on SARS-CoV-2 infection was evaluated by an alternative plaque reduction assay.

[0285] The media was aspirated from the cells (max 6 wells per time, to avoid drying) and 200 .Math.l of a mixture of SARS-CoV-2 virus and test compound (Example 9.3 or Example 9.4) was added per well (each dilution in duplicate). The 24-well plate was incubated for 1 h at 37° C.

[0286] The solution was aspirated (max 6 wells per time, to avoid drying) and 500 .Math.l of Avicel-rich DMEM solution (containing 0.33% of Avicel3515 and 5%FBS) was added. The plates were covered with a film and incubated at 37° C. for 48 h.

[0287] The solution was aspirated (max 6 wells per time, to avoid drying) and 500 .Math.l of formalin (4% PFA) was added. After 15-30 minutes the solution was aspirated (max 6 wells per time, to avoid drying) and 500 .Math.l of Crystal Violet solution was added. After 15-30 minutes the solution was aspirated and 500 .Math.l of water was added to wash.

[0288] The solution was aspirated again and then incubated at 70° C. for 30-60 min. The viral plaques were then counted.

TCID.SUB.50 Viral (HSV-2/RSV) Titre Reduction Assay Using Example 9.4

[0289] A flask of Vero cells was split with trypsin for seeding 96 well plates. Plates were seeded with Dulbecco’s modified eagle’s media (DMEM) and cells at a density of 5×10.sup.5/ml. All wells of the plate contained 100 .Math.l of cell and media mixture, before an additional 80 .Math.l of cell and media mixture was added to the first four wells of the left-most column.

[0290] Once seeded, plates were left for ninety minutes for cells to adhere. One plate in each experimental batch was utilised as a positive control (virus + sterile deionised water). Subsequently, using sterile Eppendorf tubes, 55 .Math.l of virus stock solution was mixed together with 55 .Math.l of Example 9.4 solution (or water) for five minutes contact time at room temperature. Mixtures were pipetted up and down to ensure virus stock and test solution were thoroughly mixed. 20 .Math.l of the mixture was then added to the first four wells of the first column on the appropriate plate (bringing the total volume within the well to 200 .Math.l).

[0291] These four wells were then titrated across the plates using a 1-in-2 serial dilution (100 .Math.l from the first set of four wells into the second set, 100 .Math.l from the second set into the third set, continuing across the plate but leaving the last column untouched as a negative control). Dilutions from the top four wells of the penultimate column were transferred to the bottom four wells of the first column and serial dilution continued.

[0292] Finally, cytopathic effects were scored after an appropriate length of time for the relevant virus (7 days for RSV, 2 days for HSV-2) and titres calculated from these observations using the Spearman-Karber method for determining endpoint dilution.

MTS Assay for Cell Health Using Examples 9.1-9.4

[0293] HeLa cells were cultured in a 96-well microtiter plate until confluent (16,000 cells per well). Media was discarded and 50.Math.l of fresh minimum essential medium Eagle (MEME) was added. 50 .Math.l of test material (Example 9.1, 9,2, 9.3 or 9.4) was added to give 100 .Math.l final volume in each well with a concentration range from 50 .Math.g/mL to 500 .Math.g/mL.

[0294] The plate was incubated for 24 h and then 20 .Math.l of CellTiter 96® AQueous One Solution Reagent was pipetted into each well. The plate was incubated at 37° C. for 4 hours in a humidified, 5% CO2 atmosphere and then the absorbance at 490 nm was recorded using a 96-well plate reader.

Biological Assay Results

HSV-2 Assay Results

[0295] The antiviral activity of the branched polymers tested against HSV-2 can be seen in Table B and FIGS. 2 and 3.

TABLE-US-00003 Example Number Description HSV-2 IC.sub.50 (ng/ml) HSV-2 IC.sub.90 (ng/ml) 9.1 4-arm PSS DP10 138.1 4899 9.2 4-arm PSS DP30 113.6 690.9 9.3 4-arm PSS DP50 137.4 281.2 9.4 4-arm PSS DP100 138.1 288.0

[0296] It can be seen that all the 4-arm PSS polymers tested demonstrated antiviral activity against HSV-2. FIG. 2 shows that Examples 9.2 (4-arm PSS DP30), 9.3 (4-arm PSS DP50) and 9.4 (4-arm PSS DP100) gave greater than 2 log units decrease in HSV-2 PFU/mL at both 5 .Math.g and 15 .Math.g incubation. A greater than 2 log units decrease in viral PFU/mL compared to control is typically seen as being indicative that an agent has virucidal activity. Therefore, the DP30-100 PSS branched polymers exhibit virucidal activity against HSV-2.

[0297] From FIG. 3 and Table B it can be seen that all of the 4-arm PSS polymers exhibited similar IC.sub.50 values against HSV-2 in the range 114-138 ng/ml. However, 90% inhibition (IC.sub.90) correlated with the number of PSS residues, whereby the polymers having longer blocks of sulfonated residues (Example 9.3 - DP50 and Example 9.4 -DP100) gave more effective inhibition (IC.sub.90 280-290 ng/ml) than the polymers having shorter blocks of sulfonated residues, namely Example 9.1 - DP10 (IC.sub.90 4900 ng/ml) and Example 9.2 - DP30 (IC.sub.90 691 ng/ml).

[0298] The 4-arm SPMA polymers (Examples 10.1 to 10.4) were tested in the same HSV-2 assay and were all found to have at least virustatic activity.

SARS-CoV-2 Assay Results

[0299] The antiviral acivity of the 4-arm PSS DP50 (Example 9.3) branched polymer was tested against SARS-CoV-2 using the inhibition assay (A) protocol, as can be seen in Table C.

TABLE-US-00004 Viral plaque count Sample 3 .Math.g/ml 6 .Math.g/ml 12.5 .Math.g/ml 25 .Math.g/ml 50 .Math.g/ml 100 .Math.g/ml Ex. 9.3 0 0 0 0 0 0 Ex. 9.3 0 0 0 0 0 0 Ex 9.3 + Virus 13 9 13 14 13 4 Ex 9.3 + Virus 10 9 8 10 8 2 Virus - 34 35 37 31 36

[0300] The vero cell monolayers in the test compound-only negative control samples were healthy and no viral plaques were observed as expected. The number of plaques in the samples containing both test compound and virus were lower than the virus-only control wells, indicating that the test compound (Example 9.3) has antiviral activity against SARS-CoV-2.

[0301] From the data in Table C it can be seen that compared to an average viral plaque count from the virus-only control samples of 34.6, for the samples containing both test compound and virus, the average % inhibition from duplicate runs was 33% at 3 .Math.g/ml test compound, 26% at 6 .Math.g/ml, 30% at 12.5 .Math.g/ml, 35% at 25 .Math.g/ml, 30% at 50 .Math.g/ml and 9% at 100 .Math.g/ml. This % inhibition data against SARS-CoV-2 is shown in FIG. 4.

[0302] The antiviral acivity of the 4-arm PSS DP50 (Example 9.3) and 4-arm PSS DP100 (Example 9.4) branched polymers was also tested against SARS-CoV-2 using the inhibition assay (B) protocol; the results can be seen in FIG. 5. Both branched polymers showed good inhibition of the SARS-CoV-2 virus with the DP100 polymer giving the greatest inhibition (IC.sub.50 = 0.21 .Math.g/mL).

HSV-2/RSV Titre Reduction Assay Results

[0303] The antiviral activity of the 4-arm PSS DP100 (Example 9.4) branched polymer was tested against HSV-2 and RSV and the results can be seen in FIG. 6. The polymer exhibited virucidal activity against both viruses, with a 2 log unit reduction in viral titre being observed for HSV-2 at less than 100 .Math.g/mL, and for RSV at approximately 500 .Math.g/mL.

HSV-2 PFU Assay - Effect of Branched Versus Non-branched PSS Polymers

[0304] FIG. 7 shows that only the branched sulfonated polymer according to the present invention (Example 9.4 - DP 100) possessed virucidal activity (at least 2 log unit reduction in viral PFU). When linear sulfonated polymers having DP 100 were tested in the same assay they showed only virustatic activity. The presence (comparative example 11.1) or absence (comparative example 11.2) of the chain transfer agent in the structure of the linear polymer was shown to have no significant effect on the antiviral activity.

[0305] To further corroborate this finding, after Example 9.4 had been subjected to cleavage of the branches (comparative example 11.3), the virucidal activity was lost.

[0306] This data indicates that in order to provide sulfonated polymers having virucidal activity, a branched polymer having a core surrounded by a high density of anionic moieties (sulfonate or sulfate) is needed.

Cell Health Assay Results

[0307] FIG. 8 demonstrates that compared to the control (cells only) none of the branched polymers tested (Examples 9.1 to 9.4) showed any cytotoxic activity, indicating their potential usefulness for clinical applications.