WO1998050022A1

WO1998050022A1 - Diphenylacetic acid derivatives and their use as antiviral agents

Info

Publication number: WO1998050022A1
Application number: PCT/GB1998/001313
Authority: WO
Inventors: David John Garnett
Original assignee: Lovesgrove Research Limited
Priority date: 1997-05-07
Filing date: 1998-05-06
Publication date: 1998-11-12
Also published as: GB2340490A; AU7343398A; GB9926138D0

Abstract

A new broad-spectrum anti-viral compound for the treatment of viral infections, such as human immune deficiency virus. The compound has general formula (I). Ar?1 and Ar2¿ are each either a phenyl group or substituted aromatic group; R1 is a primary, secondary or tertiary alkyl or alkenyl group, a phenyl or hydrogen; R2 is an alkyl group, a primary, secondary or tertiary alkyl amino group or hydrogen; and X is C, O, N or S. Preferred compounds include diethylamino-ethyl-2,2-diphenylvalerate and aminoethyl-2,2-diphenylvalerate.

Description

D IPHENYLACETIC AC ID DER IVATIVES AND THEI R USE AS ANTIVIRAL AGENTS

DESCRIPTION

The present invention relates to a new type of anti-viral compound.

Viruses are small cellular parasites containing an infectious particle, or virion, which replicate by infecting host cells of other organisms such as plants, animals or bacteria. The viruses are only able to infect a limited range of host cells due to the requirement for an appropriate surface receptor to be present in the membrane of the host cell to which the corresponding protein in the outer capsule of the virus can bind. Once the virus binds to the receptor protein the viral DNA or RNA crosses the membrane of the host cell and enters its cytoplasm. The nucleic acid typically ends up in the host cell's nucleus where it is replicated using the host cell's machinery for synthesizing proteins and nucleic acids. The proteins produced by the virus are usually special enzymes required for viral replication, inhibitory factors for preventing cell metabolism or proteins for construction of new virions.

Once a number of virions have been produced inside the host cell, the cell may lyse to release the particles, causing death of the host cell. Alternatively, the viral nucleic acid may become integrated with the host chromosome such that the viral genome is replicated along with the nucleic acid of the host cell thereby passing the viral nucleic acid from generation to generation.

Viral infection of animals, including humans, is one of the most common causes of disease and death in today's society. The vast range of different viruses which exist, such as small pox virus, polio virus, influenza virus, human immune

deficiency virus, coxsackie B virus, T-cell lymphocyte virus I and respiratory syncytial virus, in conjunction with the ability of the virus to mutate at rapid rates has created serious problems in producing drugs to prevent or cure viral infections. Their ability to integrate with the host's own DNA material increases the difficulty of providing a suitable cure since providing drugs to attack the viral genetic material may also result in damage occurring to the host's own DNA, with possible disastrous consequences, such as carcinogenic effects.

An example of a virus which is infecting an increasing proportion of the

human population is the human immune deficiency virus (HIV). This virus enters a human host cell, such as a T4 lymphocyte, by interaction of the glycoprotein gp!20 present in the lipid bilayer membrane of the virus with the plasma membrane receptor CD4 of the host cell. The two membranes are then able to fuse and the viral core is

released, along with its nucleic acid, into the cytosol of the lymphocyte. Eventually, the viral genome reaches the nucleus of the cell where it is able to replicate. Replication enables a large amount of viral glycoprotein to be produced which becomes inserted into the membrane of the host cell, allowing the infected T4 cell to

fuse with other T4 cells having the CD4 receptor thereby resulting in infection of these cells and the prevention of the T-cells carrying out their normal immune function. Hence, HIVJnfected individuals eventually develop acquired immune deficiency syndrome which may lead to the death of the individual.

A large amount of research has been carried out in relation to the prevention of viral infection or for causing selective death of the viral particle once inside the cell. Protease inhibitors have been used to endeavour to block the activity of the proteins produced by the viral particle and DNA analogs have been developed for blocking replication of the viral genome.

The aforementioned treatments do have their drawbacks. The compounds tend to be highly specific to one particular virus and may also produce adverse side effects. The ability of viruses to mutate as such a rapid rate also means that the virus may quickly become immune to such compositions.

It is an object of the present invention to provide a new type of anti-viral agent for preventing the infection of host cells.

It is a further object of the present invention to provide an improved anti-viral agent which may act against a wider range of viruses.

Another object of the present invention is to provide an anti-viral agent which may be used in conjunction with other treatment to provide a combined effect.

Yet another object of the present invention is to provide an anti-viral agent which is less toxic and has fewer in vivo side effects.

Accordingly, to a first aspect of the present invention provides an anti-viral compound of a given general formula for use in the treatment of viral infections, the general formula being :-

wherein

Ar¹ and Ar² are each either a phenyl group or substituted aromatic group;

R¹ is a primary, secondary or tertiary alkyl or alkenyl group, a phenyl or hydrogen;

R² is an alkyl group, a primary, secondary or tertiary alkyl amino group or hydrogen; and

X is C, O, N or S.

Preferably, Ar¹ and Ar² are both phenyl groups. Preferred substituents for Ar¹ and Ar² when Ar is a substituted aromatic group selected from halide and nitrate groups.

Preferably, R¹ is a primary alkyl group or hydrogen. Preferably, R¹ is an alkyl group having 1 to 4 carbon atoms, such as a methyl group or a propyl group.

Preferably, R² is a primary, secondary or tertiary amino ethyl group, especially being a monoethyl amino ethyl group, a diethylamino ethyl group or an amino ethyl group. Alternatively R² may be hydrogen.

Preferred anti-viral compounds of the present invention have the general formula :-

wherein

R¹ is a primary, secondary or tertiary alkyl group or hydrogen; and

R² is an alkyl group, a primary, secondary or tertiary amino alkyl group or hydrogen. A more preferred antiviral compound of the present invention has the formula:-

The compounds of the present invention may be used as an anti-viral agent against, for example, the human immune deficiency virus, the respiratory syncytial virus, paramyxoviruses, slow viruses and arenaviruses, such as pichinide and lymphocytic choriomeningitis virus.

Preferably, the compound is administered in doses ranging from l/ g/ml to 100_/zg/ml, preferably 2/-ιg/ml to 50/ig/ml. The compound may be administered orally or by other suitable means, such as injection. The compound may be administered in conjunction with a carrier material.

The anti-viral compounds of the present invention may be used in combination with other anti-viral agents, such as ribavirin which act at a different stage of the viral infection cycle. The compound may also be administered in conjunction with other substances, such as those acting to reduce inhibition of glucoronyl transferases, monoamine oxidase or cholesterol biosynthesis. According to a second aspect of the present invention there are provided novel diphenylvalerate compounds of the general formula :-

wherein

R¹ is a primary, secondary or tertiary alkyl group or hydrogen; and

R² is an alkyl group, a primary, secondary or tertiary amino alkyl group or hydrogen.

In particular, the present invention provides a novel compound of the formula:-

^ NH

CH ₃ A large number of compounds of the general formula given below have surprisingly been found to inhibit the infectivity of viruses :-

R'

wherein Ar¹ and Ar² are each either a phenyl group or a substituted aromatic group; R¹ is a primary, secondary or tertiary alkyl or alkenyl group, a phenyl or hydrogen;

R² is an alkyl group, a primary, secondary or tertiary amino alkyl group or hydrogen; and X is C, O, N or S.

It has been found that these membrane active compounds can inhibit the infectivity of viruses respirator}' syncytial A2 and N2 (RS-A2 and RS-N2

respectively), human immune deficiency virus 1 (HIV-1), paramyxoviruses, slowviruses and arenaviruses. For example, the compounds (i) diethylamino-ethyl-

2,2-diphenylvalerate (SKF-525-A) (ii) 2-(ethylamino)-ethyl-2,2-diphenylvalerate

(LR#3) and the double N-dealkylated derivative (iii) aminoethyl-2,2-diphenylvalerate

(SKF-26754A) have been found to have anti-viral properties, as has the product (iv) of the acid hydrolysis of the latter compound (iii). The structures of the compounds are as follows :-

(iϋ) (iv) SKF-525A (DEDV) and SKF-26754-A (AEPV) have both shown profound inhibition of cytopathic effects following chronic exposure of live cells to low and high viral loads

Additionally, the related compounds (v) 2-dιethylamιnoethyl 2,2-

diphenylethanoate (LR#4) and (vi) 2-dιethylamιnoethyl 2,2-dιphenylpropanoate (LR#5) have been found to have anti-viral activity with reduced toxic effects, the structures of which are given below -

(v ) (vi)

The following are examples ot additional compounds which have been shown to exhibit anti-viral activity -

(ix) (x)

(XV) (xvi)

(xvii)

The present invention will now be further illustrated by means of the following Examples in which Examples 1 to 4 relate to the preparation of preferred anti-viral compounds of the present invention and Examples 5 to 8 illustrate the anti-viral activity of preferred compounds of the present invention with reference to the accompanying drawings in which :-

Figure 1 is a graph illustrating the results of an antiviral assay carried out on lymphoblastoid cells infected with HIV IIIB treated with DEDV; and

Figure 2 is a graph illustrating the cytoxicity levels of lymphoblastoid cells infected with HIV IIIB treated.with DEDV.

Example 1 - Preparation of 2Jethylamino ethyl 2.2-diphenylvalerate and 2- fdiethylamino ethyl 2_r2-diρhenylvalerate.

The synthesis of 2-(ethylamino)ethyl 2,2-diphenylvalerate and 2- (diethylamino)ethyl 2,2-diphenylvalerate were readily achieved from the common starting material diphenylacetic acid 1. Treatment of 1 with two equivalents of butyl

lithium leads to the formation of the dianionic species 2 which readily reacts with allyl bromide to afford the alkylated product 3 after completion (Scheme 1 below).

Scheme 1

Esterification was accomplished by the reaction of 3 with thionyl chloride to give the acid chloride 4, followed by stirring the crude acid chloride with the appropriate aminoalcohol overnight (Scheme 2 below). The final procedure requires hydrogenation over a palladium catalyst to reduce the double bond and in the case of the mono-ethyl derivative, remove the benzyl protecting group.

H2 P /C Me OH

Step 1. Preparation of 2,2-diphenylpent-4-enoic acid.

A solution of n-butyl lithium in hexanes (2.5M, 83ml) was added dropwise to a solution of diphenylacetic acid (20g, 94mmol) in tetrahydrofuran and cooled to - 78°C by a dry ice-acetone bath. The resultant solution was allowed to stir for 1 hour before 3-bromopropene (16.3ml, 188 mmol) was added dropwise. The solution was stirred at -78°C for 1 hour and was then warmed to room temperature and stirred overnight.

Water (100ml) was then added to the mixture, followed by dilute hydrochloric acid (2M, 100ml) and the aqueous phase was extracted with diethyl ether (3 x 100ml). The combined ethereal extracts were dried with magnesium sulphate and the solvent removed in vacuo to give an off-white solid. The solid was recrystallised using ethanol to give 2,2-diphenylpent-4-enoic acid as colourless crystals (19. Og, 80%).

Step 2. Preparation of N-benzyl-N-ethylethanolamine.

A mixture of 2-(ethylamino)ethanol (20ml, 0.21mol), benzylbromide (24.4ml, 0.21 mol), potassium carbonate (29. Og, 0.21 mol) and dichloromethane were heated under reflux for 15 hours. Water (100ml) was added to the cooled reaction mixture, followed by extraction with ether (3 x 100ml). The organic extracts were dried with magnesium sulphate and the solvent removed under reduced pressure to give the amine as a light brown oil. The amine was purified by distillation to give N-benzyl- N-ethylethanolamine as a colourless liquid. (28.6g, 76%)

Step 3. Synthesis of 2-ethylamino-ethyl 2,2-diphenylvalerate.

2,2-Diphenylpent-4-enoic acid (9.50g, 37.7 mmol) was refluxed with thionyl chloride (20ml) for 8 hours after which the excess thionyl chloride was distilled off to give the crude acid chloride. N-benzyl-N-ethylethanolamine (6.75g, 37.7 mmol) dissolved in pyridine (10ml) and DMF (10ml) was added to the acid chloride and the mixture stirred at room temperature for 24 hours. Water (100ml) was added and the

crude product was extracted with ether (3 x 50ml). The organic extracts were dried with magnesium sulphate and the solvent removed under vacuum.

The resultant product was dissolved in methanol (100ml) and 10% palladium on carbon was added. The mixture was hydrogenated at 1 atmosphere of hydrogen

for 15 hours and the solution filtered. The solvent was removed under reduced pressure to give 2-ethylamino 2,2-diphenylvalerate (7.92g, 65%).

Step 4. Preperation of 2-diethylamino-ethyl 2,2-diphenylvalerate.

2,2-Diphenylpent-4-enoic acid (9.50g, 37.7 mmol) was refluxed with thionyl chloride (20ml) for 8 hours and any excess thionyl chloride was distilled off to give the crude acid chloride. 2-(diethylamino)ethanol (4.42g, 37.7 mmol) dissolved in

pyridine (10ml) and DMF (10ml) was added to the chloride and the mixture was stirred at room temperature for 24 hours. Water (100ml) was added and the crude product was extracted with ether (3 x 50ml). The organic extracts were washed with water (3 x 50ml), dried with magnesium sulphate and the solvent removed under vacuum.

The resultant product was dissolved in methanol (100ml) and 10% palladium on carbon was added. The mixture was then hydrogenated at 1 atmosphere of hydrogen for 15 hours and the solution filtered. The solvent was removed under reduced pressure to give the product, 2-diethylamino-ethyl 2,2-diphenylvalerate (9J0g, 68%).

Example 2 - Synthesis of Aminoethyl 2.2-diphenylvalerate.

The synthesis of aminoethyl 2,2-diphenylvalerate was carried as described by Anders, M. W. et al in Mol. Pharmacol. 2 [1966] 328-334. 6Jg of ethanolamine was added to 21.0g of sodium bicarbonate in 125ml of water. This was left at room temperature for 1 hour. 17.7g of carbobenzoxychloride was then added to the mixture and the reaction vessel was stirred for a further hour. Acidified 5M hydrochloric acid was then added dropwise until no further gas was evolved. The resultant mixture was then cooled to -5°C for 24 hours.

Crystals formed and were collected from the bottom of the flask and the solution was concentrated to approximately half its volume and allowed to cool. Chlorobenzoxy ethanolamine was produced and, when re-crystallised from benzene, had a melting point of 64°C. An approximately 90% yield was obtained.

6.35g of 2,2-diphenylvaleric acid was refluxed for 8 hours with lOcc of thionyl chloπde using a reflux condenser incorporating a calcium chloride drying

tube Afterwards, excess thionyl chloride was vacuumed off to give a yield of 90% of the predicted amine

3 7g of chlorobenzoxyethanolamine was then dissolved in 2ml ot pyridine and 6 lg of acid chloπde, pre-dissolved in 2ml of pyridine, was added The mixture was stirred thoroughly and allowed to stand at room temperature for 24 hours It was then refluxed for a further 24 hours and then cooled The mixture was then added to 10ml of cold 1M hydrochloπc acid The resultant product was then extracted with two portions of ether (2 \ 50ml extractions) and washed twice with hydrochloric acid (2 x 10ml) The acid was then neutralised with 5 % sodium bicarbonate solution (2 x 10ml)

This was then added to 20g ot glacial acetic acid and the resultant solution was saturated with hydrogen bromide under nitrogen and left for 3 hours until carbon

dioxide ceased to e\ol\e 100ml of ether was then added to precipitate the hydrogen bromide salt The salt product was then re crystallised from hot toluene/chloroform (9 1) giving a 41 % vield ot the h\drogen bromide salt having a melting point ot 146°C

Example 3 - Preparation of 2-(N N-dιethylamιno)ethyl 2.2 -diphenyiethanoate

A mixture of 2,2-dιphenylacetιc acid (5.00g, 23 6 mmol) and thionyl chloride

(10ml) was heated to reflux for 1 hour before the excess thionyl chloride was removed by distillation A solution of 2-dιethylamιnoethanol (5 53g, 47 2 mmol) in dichloromethane (10ml) with triethylamine (2ml) was added cautiously and the resultant mixture was stirred at room temperature tor 2 hours The reaction mixture was diluted with dichloromethane (50ml) and washed with saturated bicarbonate solution (3 x 20ml) with water ( 1 \ 20ml) The organic layer was dπed (Na₂C0₃) and the solvent removed under reduced pressure to give the amine as a light brown liquid (6 30g, 86%)

The hydrochloride salt ol the above amine was ieadily prepared by passing dry

HC1 gas through an ethereal solution of the compound The resultant solid was collected by filtration and washed well with ether to giv e an oft white solid

Example 4 - Preparation of 2-(N N dιethylamιno)ethyl 2.2-dιphenylpropanoate

2-(N,N-dιethylamuιo)ethyl 2 2-dιphenylproponoate was prepared by the same method as 2-(N,N-dιethylamιno)ethyl 2,2-dιphenylethanoate only using 2,2 diphenylpropiomc acid (5 OOg, 22 1 mmol) as the starting material to give the amine as a light brown liquid (5 85g, 86%) Examples 5 and 6.

Source of Materials:

Two respiratory syncytial virus isolates from the University Hospital at Aberdeen were used to determine anti-viral activity and are designated RS-A2 and RS-N2. The cell lines used were all sourced from the P.H.L.S, Porton Down, U.K. Media (Minimal Eagles Medium) containing 2% fetal calf serum, and all other media and reagents were, unless otherwise specified, from Sigma Aldrich Ltd, Dorset, U.K. The HIV-IIIb isolate was supplied by St. Bartholomews Hospital, London.

Example 5 - RSV

The compound DEDV synthesised according to the method described above was tested against each RSV viral isolate at the final concentrations 18.8 μg/ml and 50 μg/ml. Sub-confluent HEp-2 cell monolayers were infected with virus and each virus was allowed to absorb for 30 minutes at room temperature before adding fresh media containing either no drug or the drug at the above concentrations. The infected cell monolayers were not washed to remove inoculum (harmless) virus.

Plates were then incubated at 37°C and the infected cell cultures were monitored for the appearance of cytopathic effects at 24 hours and 42.5 hours. The results of which are shown in Table 1 below. TABLE 1

¹ Virus cpe was determined by visual inspection of the monolayers and scored on a scale of -(no cpe) to 4+ (all cells showing cpe).

² The incubation was terminated at 42.5 hrs since the uninfected HEp-2 cells in the absence of the DEDV were beginning to show signs of non-specific deterioration.

Infectious viral litres were removed at 42.5 hours for analysis in further plaque assay . Uninfected HEp-2 cells were incubated in parallel in the presence of the agent with no detectable effects on the appearance of HEp-2 cells at the concentrations assayed.

Example 6 - HIV

HIV IIIB (lOTCID's) was used to infect T-lymphoblastoid cells (2 x 10⁵

C8166 cells) for 90 minutes. Unabsorbed virus was removed by washing and the compounds were then added to the culture. After 72 hours, supernatant was removed and assayed for HIV p24 antigen, following standard ELISA method (described in Patience et al 1991). Figures 1 and 2 of the accompanying drawings illustrate respectively the results of the antiviral assay and the cytoxicity levels of the lymphoblastoid cells.

The target cells C8166 were incubated in the presence of the agent DEDV for 72 hours. The compound was removed and the labelled protein hydrolysate (¹⁴C) was added to the culture for 8 hours. Cells were then harvested and the labelled protein uptake was measured on a scintillation counter. Cut-off for the assay is 50% uptake (CC50) when compared to the positive control, nonoxynol-9. The results are shown below in Table 2 and in Figures 1 and 2 of the accompanying drawings.

TABLE 2

Examples 5 and 6 indicate that the compounds of the present invention can inhibit the infectivity of the viruses RS-A2, RS-N2 and HIV-1. The compound DEDV and its double N-dealkylated derivative (AEPV) show profound inhibition of cytopathic effects following chronic exposure to live cells to both low and high viral loads. It is likely that the DEDV is metabolically de-alkylated to the -NH₃ moiety in the liver.

The toxicity results indicate that the compounds may be provided in non-toxic doses from 2μg/ml to 50μg/ml dependent upon the cell line used. In vivo acute toxicity of the compounds has not been experienced by experimenters using rats and mice given oral doses of between 50 and 500 mg/kg [Fouts and Brodie 1956]. The compounds also remain active in vivo [Axelrod et al 1954] and in studies with male rats dosed i.p. for three days at 50mg/kg the animals did not display signs of acute toxicity [Fernandez et al 1978], although certain in vivo effects have been noted such as the inhibition of glucoronyl transferases, monoamine oxidase and cholesterol biosynthesis. However, these effects may be counteracted by means of a suitable treatment regime. Indeed, DEDV has been used as an anti-cholesterol drug prior hereto but its use as an anti -viral agent has never before been realised.

Transient fatty acid filtration of the liver has also been noticed post-mortem although this effect was rapidly reversible on withdrawal of the treatment [Anders

1971] . It is clear that at the suggested dose for HIV treatment (10 to 50 mg orally) no significant toxicity should be apparent.

Example 7 - Simian Virus 5 (Paramyxovirus).

The anti-viral compounds SKF-525-A (DEDV), LR#3, LR#4 and LR#5 were tested to establish their anti viral activity in relation to the Simian Virus 5 acquired from St. Andrews University stock. All the analogues were tested with and without Ribavirin in combination therewith.

A six well Linbro plate was seeded with 500,000 Vero cells/well in 10% NBCS medium. After 24 hours the anti-viral compounds were added and left for two hours, prior to the inoculation. A dilution series of the stock viral culture was prepared containing 2% NBCS, medium was removed from Linbro plate and inoculated with 1 ml per well of each dilution onto separate wells.

The virus was allowed to absorb for 2-3 hours at 37°C in a CO₂-gassed box set on a rocking platform. Inoculum was then removed and 10ml of medium containing 2% NBCS 0.5% Methocel and anti-viral agent was added to each well. The residual inoculum was also washed to remove any interferon which the Vero cells may be sensitive to. The plates were then incubated for 10 to 12 days at 37°C in a CO₂ incubator. The medium was then removed and the cells fixed and incubated with 5% formaldehyde, 2% sucrose in PBS for 10 minutes and incubated with 0.5% NP40, 10% sucrose in PBS for 5 minutes. The cells were washed 3 times with PBS containing 1 % calf serum.

It was then possible to visualise plaques simply by staining with Crystal violet or Coomassie Blue. Plaque levels were quantified using a scanner directly viewing the plates with quantification of plaque density read using "iphotoplus" program.

TABLE 4

Results :-

TABLE 5

TABLE 6

ANTI-VIRAL TESTED CYTOPATHIC EFFECT (CPE)

CPE scored visually : - + + + = Total CPE, -/+ = No detectable CPE

Example 8 - Arenaviruses: Pichinde and Lymphocytic choriomeningitis virus (LCMV).

The growth of the arenaviruses Pichinde and LCMV in the presence of the anti -viral compound ethylaminoethyl-2,2-diphenylvalerate was investigated by the Department of Biochemistry, Microbiology and Immunology at the University of Ottawa, Canada.

Five x 104 Vero cells/well were seeded in 48 well tissue culture plates. After twenty four hours, the cells were counted in one extra well which had 9.7 x 10⁴ cells. The cells were infected at a MOI of 0J by adding 50 μl of virus at 2 x 105 /ml to each well and 50 μl of the viral compound to each well at various concentrations to give final concentrations of 1.25 - 10 μg/ml.

Triplicate wells of each virus/drug combination were set up, together with three wells which received virus and medium only as a control. The wells were incubated for 1 hour at 37°C, the inoculum was removed and media added back having the appropriate concentration of the anti viral compound. The wells were incubated for a further 48 hours at 37°C and harvested by collecting supernatants. The supernatants were assayed for infectious virus by plaque assay on Vero cell monolayers. Dilutions of the virus were added to confluent cells in 12-well plates, incubated for 1 hour and then removed. The monolayers were overlaid with 199 medium containing 0.6% Oxoid agar and incubated for 3 days. The plaques were counted on day 4 after overlay of plates with 0.3 % neutral red in PBS.

The results for the growth of Pichinde virus and Lymphocytic choriomenigitis virus (Armstrong strain) are summarised in Tables 7 and 8 respectively.

TABLE 7

TABLE 8

The inventor proposes that the anti-viral activity of the compounds of the present invention is due to the reduced availability of the cell surface receptor sites which are normally present on the surface of a host cell to enable the virus to bind thereto and penetrate the cell. It is believed that the anti-viral compounds perturb the lipid bilayer of the cell membrane increasing the fluidity thereof to allow conformational changes in the receptor sites thereby preventing recognition and attachment of the viral envelope. DEDV is known to be surface active in artificial membranes [Florence 1970] and has been studied in the context of therapeutic enhancement. Hence, this supports the contention that the compounds act upon the membrane of the host cell.

The production of an anti-viral agent which acts on the cell membrane enables blockage of viral infection at the early stages of viral attachment. This is advantageous since the agent is a useful and complimentary mechanism for controlling viral infection. Blockage at this stage during viral infection has been largely unexploited in the clinical environment with the possible exception of Amantidine™ and its interference with the uncoating processes of Influenza A virus [Lamb 1989].

Additional plaque assay and viral titre experiments have been performed using the compounds of the present invention in relation to Coxsackie B virus (CBV) which showed no activity in either viral plaque assay or infected viral titre experiments. The ability of the compounds to act against certain viral agents and not others is explained by the difference in the cell membrane receptor utilised by the virus for attachment to the host cell. As mentioned above, the HIV virus binds to the large CD4 protein which is part of the immunoglobin superfamily [Weiss 1994] . It is proposed that this protein, and the alternative cellular attachment site galactoside ceramide [Bhat et al 1991 ; Fantini et al 1993] both require stable membrane conditions to be present before interaction with a viral glycoprotein, such as that of the HIV envelope, is possible. In contrast, Coxsackie B virus uses a smaller integrin as its viral receptor [Roivainen et al 1994] which may be unaffected by change to its conformation or orientation at the membrane surface.

The provision of an anti-viral agent which alters the fluidity of the host cell membrane rather than by binding and altering a specific protein results in the agent being able to prevent penetration of the cell membrane by a wider range of viruses, thus providing a broad spectrum anti-viral agent. Furthermore, due to the fact that the other treatments, such as protease inhibitors and DNA analogs, act on a different stage of viral infection, the agent of the present invention may be used to compliment other treatments. The agent has also been shown to produce low levels of toxicity with the recommended dosages and shows few in vivo effects. In particular, the agent

LR#4 is as effective anti-viral agent and demonstrates a low level of toxicity and therefore would appear to be a very useful therapeutic agent.

Claims

1. An anti-viral compound of the general formula :-

R'

wherein

Ar¹ and Ar² are each either a phenyl group or substituted aromatic group;

X is C, O, N or S.

2. An anti-viral compound as claimed in claim 1, wherein Ar¹ and Ar² are both phenyl groups.

3. An anti-viral compound as claimed in claim 1 wherein, when Ar is a substituted aromatic group, the substituent is selected from a halide or nitrate group.

4. An anti- viral compound as claimed in claim 1,2 or 3, wherein R¹ is a primary alkyl group having 1 to 4 carbon atoms.

5. An anti-viral compound as claimed in claim 4, wherein R¹ is a methyl group, ethyl or propyl group.

6. An anti- viral compound as claimed in any one of 1 to 5, wherein R² is a monoethyl amino ethyl group, a diethylamino ethyl group or an amino ethyl group.

7. An anti-viral compound as claimed in claim 1 having the general formula:-

wherein

R¹ is a primary, secondary or tertiary alkyl group or hydrogen; and

8. An anti-viral compound as claimed in claim 7 of the formula :-

1

CH - CH .

An anti-viral compound as claimed in claim 7 of the formula :-

NK

CH - 0 An anti-viral compound as claimed in claim 7 of the formula

WH

1 An anti-viral compound as claimed in claim 7 ot the formula

2 An anti-viral compound as claimed m claim 7 ot the formula

O

13 An anti-viral compound as claimed in claim 7 ot the formula -

14 An anti-viral compound as claimed in any one of the piecedmg claims, wherein the compound is administered in a dose of 1 μg/ml to lOOμg/ml

15. An anti-viral compound as claimed in claim 14, wherein the compound is administered in a dose of 2μg/ml to 50μg/ml.

16. An anti-viral compound as claimed in any one of the preceding claims, wherein the compound is used in the treatment of human immune deficiency virus.

17. An anti-viral compound as claimed in any one of claims 1 to 15, wherein the compound is used in the treatment of respiratory syncytial virus.

18. An anti-viral compound as claimed in any one of claims 1 to 15, wherein the compound is used in the treatment of paramyxovirus.

19. An anti-viral compound as claimed in any one of claims 1 to 15, wherein the compound is used in the treatment of slow viruses.

20. An anti-viral compound as claimed in any one of claims 1 to 15, wherein the compound is used in the treatment of arenaviruses.

21. An anti-viral compound as claimed in any one of the preceding claims, wherein the compound is provided in tablet form for oral administration.

22. An anti-viral compound as claimed in claim 21 , wherein the compound is mixed with a carrier material.

23. An anti-viral compound as claimed in any one of claims 1 to 20, wherein the compound is provided in liquid form for administration by infusion.

24. An anti-viral compound as claimed in any one of the preceding claims, wherein the compound is administered in conjunction with an alternative anti-viral agent.

25. An anti-viral compound as claimed in any one of the preceding claims, wherein the compound is administered in conjunction with other substances for reduction of any possible side effects.

26. An anti-viral compound as claimed in claim 25, wherein the substance reduces the inhibition of glucoronyl transferases, monoamine oxidase or cholesterol biosynthesis.

27. A novel diphenylvalerate compound of the general formula:-

R¹

0 . O wherein R^'

R¹ is a primary, secondary or tertiary alkyl group or hydrogen; and

28. A compound as claimed in claim 27 of the formula :-

29. A compound substantially as hereinbefore described with reference to the accompanying Examples.