Compounds and compositions

Abstract

Disclosed are compounds having the following formula: (I) wherein R is an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: (Ia) wherein Ra and Rb are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3. ##STR00001##

Claims

1. A compound having the following formula: ##STR00037## wherein R is an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: ##STR00038## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3.

2. A compound according to claim 1, wherein R is a saturated linear alkane chain having between 8 and 16 carbon atoms.

3. A compound according to claim 1, wherein R.sub.a and R.sub.b are saturated linear alkane chains having between 8 and 16 carbon atoms.

4. A compound according to claim 1, wherein the one or more anions are selected from chloride anions and bromide anions.

5. A compound according to claim 1, having the following formula: ##STR00039##

6. A compound having the following formula: ##STR00040## wherein R is an alkane chain having between 8 and 20 carbon atoms and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: ##STR00041## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3; and wherein X is 2, 4, 6, 8 or 10.

7. A compound according to claim 6, wherein R is a saturated linear alkane chain having between 8 and 16 carbon atoms.

8. A compound according to claim 6, wherein R.sub.a and R.sub.b are saturated linear alkane chains having between 8 and 16 carbon atoms.

9. A compound according to claim 6, wherein the one or more anions are selected from chloride anions and bromide anions.

10. A compound according to claim 6, having the following formula: ##STR00042## wherein X is 4, 8 or 10.

11. A pharmaceutical composition comprising: the compound of claim 1 or claim 6; and a pharmaceutically acceptable carrier.

12. A method of treatment of Herpes virus, Human Papilloma virus, fungal infections and/or bacterial infections, comprising a step of administering to a subject a compound according to claim 1 or claim 6.

13. A process for producing the compound of claim 1, comprising: i) reacting carvacrol with R.sub.2CH.sub.2COCl to form a compound having the formula: ##STR00043## wherein R.sub.2 is a halogen; ii) reacting a compound having the formula ##STR00044## with the compound having the formula ##STR00045## to form the compound having the formula ##STR00046## wherein R is an alkane chain having between 8 and 20 carbon atoms; or R is a quaternary amine having the following formula: ##STR00047## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms; iii) reacting thymol with R.sub.2CH.sub.2COCl to form a compound having the formula ##STR00048## wherein R.sub.2 is a halogen; iv) reacting the compound having formula ##STR00049## with the compound having the formula ##STR00050## to form the final product having the formula ##STR00051## wherein R is an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: ##STR00052## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3.

14. A process according to claim 13, wherein R is a saturated linear alkane chain having between 8 and 16 carbon atoms.

15. A process according to claim 13, wherein R.sub.a and R.sub.b are each a saturated linear alkane chain having between 8 and 16 carbon atoms.

16. A process according to claim 13, further comprising one or more steps of separation and/or extraction.

17. A process for the production of a compound according to claim 6, comprising reacting the compound having the formula ##STR00053## with bromine to form a compound having the formula ##STR00054## wherein R is an alkane chain having between 8 and 20 carbon atoms and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: ##STR00055## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3; and wherein X is 2, 4, 6, 8 or 10.

18. A compound having the following formula: ##STR00056## wherein R is an alkane chain having between 8 and 20 carbon atoms and A is one or more anions having a total charge of 2; or R is a quaternary amine having the following formula: ##STR00057## wherein R.sub.a and R.sub.b are each an alkane chain having between 8 and 20 carbon atoms, and A is one or more anions having a total charge of 3; and wherein the compound is complexed with bromine.

Description

(1) The present invention will now be described in more detail with reference to the attached drawings, FIGS. 1 to 7, in which

(2) FIG. 1 illustrates an example process for the synthesis of a compound of the invention.

(3) FIG. 2 illustrates an example process for the synthesis of a further compound of the invention.

(4) FIG. 3 illustrates an example process for the synthesis of a further compound of the invention.

(5) FIG. 4 illustrates the results of cytotoxicity tests of a compound of the invention at concentrations between 0 g/mL and 50 g/mL on Vero cells, and the effect of a compound of the invention on Herpes Simplex Virus 1 spread and plaque formation in Vero cells.

(6) FIG. 5 illustrates the results of cytotoxicity tests of a further compound of the invention at concentrations between 0 g/mL and 100 g/mL on Vero cells, and the effect of a further compound of the invention on Herpes Simplex Virus 1 spread and plaque formation in Vero cells.

(7) FIG. 6 illustrates the results of cytotoxicity tests of a further compound of the invention at concentrations between 0 g/mL and 100 g/mL on Vero cells.

(8) FIG. 7 illustrates the results of the effect of compounds of the invention on Vaccinia Virus plaque formation in BSC 40 cells.

PROCESS FOR PRODUCING THE COMPOUNDS OF THE INVENTION

Compound 2

N.SUP.1.-{2-[2-methyl-5-(propan-2-yl)phenoxy]-2-oxoethyl}-N.SUP.1.,N.SUP.1.,N.SUP.10.,N.SUP.10.-tetramethyl-N.SUP.10.-{2-[5-methyl-2-(propan-2-yl)phenoxy]-2oxoethyl}decane-1,10-bis(aminium) dichloride

(9) FIG. 1 shows an example process for synthesising Compound 2, which is a compound of the invention defined by formula II above.

(10) In a first step, 1,10-Dibromodecane (Compound 5) is reacted with 4 molar equivalents of dimethylamine to form 1,10-Bis(dimethylamino)decane (Compound 6). The reaction takes place at 4-5 C. in benzene and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

(11) In a second step, Carvacrol (2-Methyl-5-(1-methylethyl)-phenol) (Compound 7) is reacted with chloroacetyl chloride to form Compound 8. The reaction is carried out at 10 C. for 1 hour and then stirred at room temperature for 5 hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

(12) In a third step, Compound 8 is reacted with 1,10-Bis(dimethylamino)decane (Compound 6) to form Compound 9. The reaction is carried out by boiling Compound 6 and Compound 8 in benzene for 15 minutes and then leaving the reaction mixture at room temperature for 24 hours. Ethyl acetate is then added to the reaction mixture, the upper layer is removed and the lower layer is isolated as a residue. The residue (containing Compound 9) is then used in the fifth step.

(13) In a fourth step, thymol (2-isopropyl-5-methylphenol) (Compound 10) is reacted with chloroacetyl chloride to form Compound 11. The reaction is carried out at 10 C for 1 hour and then stirred at room temperature for 5 hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

(14) In a fifth step, the residue of the third step (containing Compound 9) is reacted with compound 11 to form the final product: Compound 2. The reaction is carried out by boiling Compounds 9 and 11 in benzene for 15 minutes and then leaving the reaction mixture at room temperature for 24 hours. Ethyl acetate is then added to the reaction mixture and the upper layer is removed and the lower layer is isolated as a residue.

(15) The resulting residue is then dissolved in acetone and Compound 2 is precipitated by the addition of diethyl ether.

(16) It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound (Compound 2).

(17) Separation steps may include steps of performing column chromatography, low pressure liquid chromatography, high performance liquid chromatography and the like. Purification steps may include standard purification processes known in the art, for example, filtration, evaporation, liquid-liquid extraction, crystallisation, adsorption, recrystallization, chromatography, distillation and the like.

(18) Compound 2 was isolated as a brown hygroscopic powder, having the following properties.

(19) Appearance: brown hygroscopic powder

(20) Molecular formula: C.sub.38H.sub.62N.sub.2O.sub.4Cl.sub.2

(21) Molecular weight: 681.81 gmol.sup.1

(22) Melting point: 92-96 C

(23) Aqueous solution pH=6

(24) Solubility: soluble in water, acetone, acetonitrile, dimethyl sulfoxide (DMSO); insoluble in diethyl ether, ethyl acetate and hexane

(25) .sup.1H NMR (DMSO/CCl.sub.41/3) 1.18 (d, 6H, CH.sub.3), 1.22 (d, 6H, CH.sub.3), 1.37-1.40 (m, 12H, CH.sub.2), 1.81-1.85 (m, 4H, CH.sub.2), 2.17 (s, 3H, ArCH.sub.3), 2.32 (s, 3H, ArCH.sub.3), 2.87 (q, 1H, CH), 3.04 (q, 1H, CH), 3.47 (s, 12H, +N(CH.sub.3).sub.2), 3.80-3.85 (m, 4H, +NCH.sub.2), 5.33 (s, 4H, (CO)CH.sub.2N+), 6.96-7.02 (m, 4H, ArH), 7.16-7.20 (m, 2H, ArH).

Compound 4

Di-Bromine Complex

N.SUP.1.-{2-[2-methyl-5-(propan-2-yl)phenoxy]-2-oxoethyl}-N.SUP.1.,N.SUP.1.,N.SUP.10.,N.SUP.10.-tetramethyl-N.SUP.10.-{2-[5-methyl-2-(propan-2-yl)phenoxy]-2oxoethyl}decane-1,10-bis(aminium) dichloride

(26) FIG. 2 shows an example process for synthesising Compound 4, which is a further compound of the invention and is defined by formula V above.

(27) Compound 2 was synthesised according to the process set out above for Compound 2.

(28) After formation of Compound 2, Compound 2 was reacted with bromine to form Compound 4.

(29) As set out above for Compound 2, it will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound (Compound 4).

(30) Separation steps may include steps of performing column chromatography, low pressure liquid chromatography, high performance liquid chromatography and the like. Purification steps may include standard purification processes known in the art, for example, filtration, evaporation, liquid-liquid extraction, crystallisation, adsorption, recrystallization, chromatography, distillation and the like.

(31) Compound 4 was isolated as an orange gum, having the following properties.

(32) Appearance: orange gum

(33) Molecular formula: C.sub.38H.sub.62N.sub.2O.sub.4Cl.sub.2Br.sub.4

(34) Molecular weight: 1001.41 gmol.sup.1

(35) Solubility: soluble in dimethyl sulfoxide (DMSO); insoluble in water.

(36) .sup.1H NMR (DMSO/CCl.sub.41/3) 1.18-1.22 (m, 12H, CH.sub.3), 1.37-1.40 (m, 12H, CH.sub.2), 1.81-1.85 (m, 4H, CH.sub.2), 2.17 (s, 3H, ArCH.sub.3), 2.32 (s, 3H, ArCH.sub.3), 2.87 (q, 1H, CH), 3.04 (q, 1H, CH), 3.47 (s, 2H, +N(CH.sub.3).sub.2), 3.80-3.85 (m, 4H, +NCH.sub.2), 5.33 (s, 4H, (CO)CH.sub.2N+), 6.96-7.02 (m, 4H, ArH), 7.16-7.20 (m, 2H, ArH).

Compound 3

Systematic Name: N.SUP.1.-{{2-[2-methyl-5-(propan-2-yl)phenoxy]-(2-oxoethyldimethyl)azaniumyl bromide}decyl}-N.SUP.10.-{2-[5-methyl-2-(propan-2-yl)phenoxy]-2oxoethyl}-N.SUP.1.,N.SUP.1.,N.SUP.1.,N.SUP.10.,N.SUP.10.-tetramethyldecane-1,10-bis(aminium) dichloride

(37) FIG. 3 shows an example process for synthesising Compound 3, which is a compound of the invention defined by formula III above.

(38) In a first step, 1,10-Dibromodecane 5 is reacted with 3 molar equivalents of dimethylamine to form Compound 12. The reaction takes place at 4-5 C. in benzene and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

(39) In a second step, thymol (2-isopropyl-5-methylphenol) (Compound 10) is reacted with chloroacetyl chloride to form Compound 11. The reaction is carried out at 10 C for 1 hour and then stirred at room temperature for 5 hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

(40) In a third step, Compound 11 is reacted with Compound 12 to form Compound 13. The reaction is carried out by boiling Compounds 11 and 12 in benzene for 15 minutes and then leaving the reaction mixture at room temperature for 24 hours. Ethyl acetate is then added to the reaction mixture and the upper layer is removed and the lower layer is isolated as a residue. The residue (containing Compound 13) is then used in the fifth step.

(41) In a fourth step, Carvacrol (2-Methyl-5-(1-methylethyl)-phenol) (Compound 7) is reacted with chloroacetyl chloride to form Compound 8. The reaction is carried out at 10 C. for 1 hour and then stirred at room temperature for 5 hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

(42) In a fifth step, the residue of the third step (containing Compound 13) is reacted with Compound 8 to form the final product: Compound 3. The reaction is carried out by boiling Compounds 8 and 13 in benzene for 15 minutes and then leaving the reaction mixture at room temperature for 24 hours. Ethyl acetate is then added to the reaction mixture and the upper layer is removed and the lower layer is isolated as a residue.

(43) The residue is then dissolved in acetone and Compound 3 is precipitated by the addition of diethyl ether.

(44) It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound (Compound 3).

(45) Separation steps may include steps of performing column chromatography, low pressure liquid chromatography, high performance liquid chromatography and the like. Purification steps may include standard purification processes known in the art, for example, filtration, evaporation, liquid-liquid extraction, crystallisation, adsorption, recrystallization, chromatography, distillation and the like.

(46) Compound 3 was isolated as a brown hygroscopic powder, having the following properties.

(47) Appearance: brown hygroscopic powder

(48) Molecular formula: C.sub.50H.sub.88N.sub.3O.sub.42ClBr

(49) Molecular weight: 946.06 gmol.sup.1

(50) Melting point: 75-78 C.

(51) Aqueous solution pH=7.2

(52) Solubility: soluble in water, acetone, dimethyl sulfoxide (DMSO); insoluble in diethyl ether and ethyl acetate.

(53) Displays Surfactant Activity

(54) .sup.1H NMR (DMSO/CCl.sub.41/3) 1.18-1.23 (m, 12H, CH.sub.3), 1.38-1.42 (m, 24H, CH.sub.2), 1.80-1.84 (m, 8H, CH.sub.2), 2.18 (s, 3H, ArCH.sub.3), 2.34 (s, 3H, ArCH.sub.3), 2.90-3.10 (m, 2H, CH), 3.45-3.50 (m, 18H, +N(CH.sub.3).sub.2), 3.75-3.80 (m, 8H, +NCH.sub.2), 5.22 (s, 4H, (CO)CH.sub.2N+), 6.96-7.02 (m, 4H, ArH), 7.16-7.20 (m, 2H, ArH),

Example 1

(55) Compounds 2, 3 and 4 were tested for anti bacterial and anti fungal activity.

(56) Minimal inhibitory concentration (MIC) of each compound was tested by broth dilution assay.

(57) Equipment

(58) McFarland standard 0.5 Falcon round-bottom 5 ml tubes Disposable loops (1 l and 10 l) Graduated pipettes (20 l-1000 l) Disposable Petri dishes
Media Sterile normal saline TSB (Tryptic Soy Broth) TSA (Tryptic Soy Agar)
Bacterial and Fungal strains Salmonella enterica serovar Typhimurium ATCC 14028 Staphylococcus aureus ATCC 6538 Candida albicans ATCC 10231

(59) Compounds 2, 3 and 4 were diluted in dimethyl sulfoxide (DMSO) at 10 mg/ml and were future 2-fold diluted for testing in TSB.

(60) Method

(61) Day 1

(62) Standardisation of Inoculum

(63) From a pure o/n culture, material from at least 3-4 colonies was chosen and suspended totally in 4 ml saline in tubes. The suspension was mixed.

(64) The turbidity of inoculum was adjusted to match that of standard by comparing visually with the McFarland 0.5 standard using white paper with black lines as background.

(65) The McFarland 0.5 suspension were diluted as follows for the species tested at this course:

(66) Gr-neg.: 10 l McFarl. 0.5 into 10 ml broth

(67) Gr-pos.: 50 l McFarl. 0.5 into 10 ml broth

(68) The suspensions were used for inoculation within 15 minutes.

(69) Inoculation and Incubation

(70) The Falcon round-bottom 5 ml tubes were inoculated with 500 l of the inoculum suspension with 500 l two-fold dilutions of antimicrobial agent using a graduated pipette

(71) Tubes were sealed and incubated at 37 C. for 18-22 hours. This was done to avoid losing growth media and to avoid cross contamination.

(72) McFarland 0.5 is approximately 10.sup.8 CFU/ml. Standardization of inoculum is essential because the interpretation of the results is based on a certain inoculum.

(73) Each tube contained approximately 510.sup.5-110.sup.6 CFU/ml after inoculation of bacteria and 55 510.sup.3-110.sup.4 CFU/ml of yeast.

(74) Day 2

(75) Purity of the inoculum suspension was checked.

(76) Growth in the 3 positive control tubes was checked.

(77) Minimal inhibitory concentration (MIC) was recorded as the lowest concentration of antimicrobial agent with no visible growth.

(78) Results are set out in the table 1 below.

(79) In summary, all compounds tested demonstrated potent anti fungal activity against Candida albicans, as well as anti bacterial activity against Staphylococcus aureus and Salmonella enterica serovar Typhimurium.

(80) TABLE-US-00001 TABLE 1 Antimicrobial activity of different samples tested in indented experiments (n = 4) by broth dilution method Bacterial/ yeast cells count Sample's MIC (g/ml).sup.1 Cultures (cfu/ml)# Compound 2 Compound 3 Compound 4 Candida albicans 6 10.sup.3 62.5-125* 31.25-62.5 31.25-62.5 ATCC 10231 3 10.sup.3 62.5-125 31.25-62.5 31.25-62.5 6 10.sup.3 62.5-125 31.25-62.5 31.25-62.5 6.5 10.sup.3 62.5-125 31.25-62.5 31.25-62.5 Staphylococcus 2.1 10.sup.6 31.25-62.5 15.6-31.25 15.6-31.25 aureus 1.2 10.sup.6 31.25-62.5 15.6-31.25 31.25-62.5 ATCC 6538 2 10.sup.6 31.25-62.5 15.6-31.25 15.6-31.25 1.4 10.sup.6 31.25-62.5 15.6-31.25 15.6-31.25 Salmonella enterica 5 10.sup.3 62.5-125 31.25-62.5 31.25-62.5 serovar 62.5-125 31.25-62.5 31.25-62.5 Typhimurium 62.5-125 31.25-62.5 31.25-62.5 ATCC 14028 62.5-125 31.25-62.5 31.25-62.5 #Represents bacterial/yeast cells count (CFU/ml) tested in 2 repeats in 4 indented experiments. *numbers in bold were from seeding for detection of final titer of bacterial/yeast cells. This means that numbers in bold represent the bactericide concentration, whereas non bold numbers are the bacteristatic concentration of compounds in the range of the MIC. .sup.1MIC determination by broth dilution using Sensititre. 4th Ed. April 2003 Edited by: Rene S. Hendriksen (DFVF)

Example 2

(81) The antiviral effect and cytotoxicity of the compounds of the invention were investigated by testing compounds 2, 3 and 4 in different mammalian cell lines.

(82) Material and Methods

(83) Cells and Viruses

(84) Herpes Simplex virus-1 (HSV 1) and vaccinia virus (W) were both tested, and cell lines used included HeLa, BSC 40 and Vero cells.

(85) HeLa, Vero and BSC 40 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) in a 5% CO.sub.2 incubator at 37 C.

(86) The Western Reserve strain of Vaccinia virus (WR-VV) was amplified in BSC40 cells, titrated and stored at 80 C.

(87) Herpes Simplex virus 1 (HSV-1) was amplified in Vero cells, titrated and stored at 80 C.

(88) Compounds and Reagents

(89) Compounds 2, 3 and 4 (in powder form) were dissolved in absolute ethanol to obtain stock solutions at a concentration of 0.2 g in 1 ml. Aliquots were stored at 20 C. Prior to the experiments, fresh serial dilution was carried out in serum free growth media (DMEM).

(90) Cytotoxicity

(91) The cytotoxic effect of the chemicals was tested on HeLa, BSC40 and Vero cells. Cells were seeded in 12 well plates, such that on the following day the plates had 80% confluence. The compounds 2, 3 and 4 were applied to the cells at varying concentrations. After 30 minutes of pre-treatment, growth media was added to the cells in the presence of the same concentration of compounds 2, 3 and 4. Cells were monitored every day, fixed and stained at 48 hours or 72 hours after treatment, and then photographed. All samples were fixed with 4% formaldehyde in H.sub.2O for 20 minutes at room temperature and subsequently stained with Crystal Violet for 30 minutes at room temperature.

(92) Virus Infections and Plaque Assay

(93) Freshly confluent cells were infected with WR-VV and HSV-1 (approximately 200 to 300 PFU per well in a 6-well plate or 12 well plate) at 37 C. for 1 hour. The cells were washed and cultured in growth medium containing 1% agarose, and fixed at 2 days post infection.

(94) Alternatively, cells were pre-treated with serum free Dulbecco's Modified Eagle Media (DMEM) or DMEM containing a different concentration of the compounds prior to infection, and compounds 2 and 3 remained in cultures throughout the experiment until cells were 50 fixed as described above. In some experiments, a liquid plaque assay was performed without agar.

(95) Results

(96) Cytotoxicity of Compounds in Different Mammalian Cell Lines

(97) In order to test the antiviral effect of the compounds 2, 3 and 4, an appropriate dilution of the compounds was chosen, such that the compounds were not toxic to the cells and such that cell viability was not affected. A variety of compound concentrations were tested in different mammalian cell lines such as HeLa, BSC40 and Vero cells.

(98) Overnight cultured cells were pre-treated with high to low doses of compounds 2, 3 and 4 in serum free media for 30 minutes. The medium was then replaced with normal growth media containing the compounds 2, 3 and 4 throughout the experiment. Cells were monitored daily for up to 72 hours then fixed, stained and photographed. As shown in FIGS. 4, 5 and 6, this experiment revealed that for all 3 compounds, the cells tolerate the compounds 2, 3 and 4 at concentrations from 25 to 100 g/ml. Rows marked C in each of FIGS. 4 and 5, and FIG. 6 illustrate the results of treatment of Vero cells, which were not infected with HSV 1, and treated with compounds 2, 3 and 4.

(99) Spread of HSV 1 and Virus Plaque Formation.

(100) The inhibitory effect of compounds 2 and 3 was monitored on Vero cells. Cells were pre-treated with different concentrations of compounds 2 and 3, subsequently treated with buffer or buffer containing HSV 1. After two days of infection, cells were fixed stained and photographed. To determine whether the compounds can inhibit HSV 1 viral spreading or plaque formation, a plaque assay was performed in liquid to determine spreading (Row A in FIGS. 4 and 5) as well as in agar to determine plaque number (Row B in FIGS. 4 and 5). A strong inhibitory effect was observed by compound 2 and 3, as shown in FIGS. 4 and 5 respectively. Both compounds effectively reduce HSV 1 spreading as well as plaque number in Vero cells in a dose dependent manner.

(101) Compounds 2 and 3 had No Inhibitory Effect on Vaccinia Virus Plaque Formation.

(102) To test the specificity of compounds 2 and 3 on the HSV 1 virus, the compounds were also tested on the Vaccinia virus, which is a further DNA virus. BSC 40 cells were kept untreated or were pre-treated with the compounds at a concentration of 50 mg/ml and cells were then infected with Vaccinia virus and monitored side by side throughout the experiment. As shown in FIG. 7, none of compounds 2 or 3 inhibited Vaccinia virus (VV) plaque formation which indicates that the inhibitory effect of compounds 2 and 3 on HSV 1 infection is highly specific.

(103) From the experiments mentioned above, it can be concluded that all cells tested can tolerate the compounds 2, 3 and 4 in concentrations from 0 to 100 g/ml. Compound 2 and 3 both demonstrate a strong antiviral effect against HSV-1 infected Vero cells, both in terms of virus spreading and plaque formation. In the presence of 501 g/ml of the compounds, infectivity was reduced by 50%. In the presence of 100 g/ml of the compounds, infectivity was reduced by 80%. None of the tested compounds are active towards the Vaccinia virus.