• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:

    公开号:AU2004248859A1
    申请号:U2004248859
    申请日:2004-06-26
    公开日:2004-12-29
    发明作者:Wayne Best；Gary Dinneen Ewart；Peter William Gage；Anita Premkumar；Lauren Elizabeth Wilson
    申请人:Biotron Ltd；
    IPC主号:A61K31-00
    专利说明:
    WO 2004/112687 PCT/AU2004/000866 -1 ANTIVIRAL COMPOUNDS AND METHODS FIELD OF INVENTION The present invention relates to methods for retarding, reducing or otherwise 5 inhibiting viral growth and/or functional activity. The invention also relates to compounds and compositions suitable for use in the methods. BACKGROUND OF THE INVENTION Currently, there is a great need for the development of new treatments that are effective against viral infections, particularly against viral infections which are 10 associated with high morbidity and mortality, and which impact on sizable populations. Treatments currently available are inadequate or ineffective in large proportions of infected patients. For example, in ameliorating AIDS symptoms and prolonging life expectancy, a measure of success has been achieved with drugs targeting the viral reverse 15 transcriptase and protease enzymes (Miller and Sarver, 1997; Mitsuya, 1992; Moore, 1997; and Thomas and Brady, 1997). However, no single treatment method is completely effective against HIV infection. (Barry et a4, 1998; Deeks, 1998; Miles, 1997; Miles, 1998; Moyle et al, 1998; Rachlis and Zarowny, 1998; Veil et al, 1997; Volberding and Deeks, 1998; and Volberdin, 1998). 20 PCT application PCT/AU99/00872 describes the use of compounds 5-(N,N hexamethylene)-amiloride and 5-(N,N-dimethyl)-amiloride in the treatment of HIV infection. Another virus considered to be a significant human pathogen is the Hepatitis C virus (HCV). This is a significant human pathogen in terms ofboth cost to human 25 health and associated economic costs. HCV causes chronic hepatitis and cirrhosis and is the leading indicator for liver replacement surgery. In 2002 the Centre for Disease Control and Prevention estimated that more- than 4 million people were infected in the USA alone and that approximately 8,000 to 10, 000 die as a result of chronic HCV infection yearly. There is no known cure or vaccine. More effective 30 pharmacological agents arc urgently required.
    WO 2004/112687 PCT/AU2004/000866 -2 A further well-known family of pathogenic viruses are the Coronaviruses. Coronaviruses (Order Nidovirals, family Coronavtridae, Genus Coronavirus) are enveloped positive-stranded RNA viruses that bud from the endoplasmic reticulum Golgi intermediate compartment or the cis-Golgi network (Fischer, Stegen et al. 5 1998; Maeda, Maeda et al. 1999; Corse and Machamer 2000; Maeda, Repass et al. 2001; Kuo and Masters 2003). Coronaviruses infect humans and animals and it is thought that there could be a coronavirns that infects every animal. The two human coronaviruses, 229E and OC43, are known to be the major causes of the common cold and can occasionally 10 cause pneumonia in older adults, neonates, or immunocompromised patients (Peiris, Lai et al. 2003). Animal coronaviruses can cause respiratory, gastrointestinal, neurological, or hepatic diseases in their host (Peiris, Lai et al. 2003). Several animal coronavirus are significant veterinary pathogens (Rota, Oberte et al. 2003). Severe acute respiratory syndrome (SARS) is caused by a newly identified 15 virus. SARS is a respiratory illness that has recently been reported in Asia, North America, and Europe (Peiris, Lai et al. 2003). The causative agent of SARS was identified as a coronavirms. (Drosten, Gunther et al. 2003; Ksiazek, Erdmanu et al. 2003; Peiris, Lai et al. 2003). The World Health Organization reports that the cumulative number of reported probable cases of SARS from 1 November 2002 to 20 the 11 July 2003 is 8,437 with 813 deaths, nearly a 10% death rate. It is believed that SARS will not be eradicated, but will cause seasonal epidemics like the cold or influenza viruses (Vogel 2003). To improve the prospect of treating and preventing viral infections, there is an on-going need to identify molecules capable of inhibiting various aspects of the viral 25 life cycle. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of 30 common general knowledge in the field.
    WO 2004/112687 PCT/AU2004/000866 -3 SUMMARY OF THE INVENTION The inventors have surprisingly found that certain compounds that fall under the classification of substituted acylguanidines have antiviral activity against viruses from a range of different virus families. Without intending to be bond by any $ particular theory or mechanism of action, and despite current dogma, it appears possible that viral replication can be retarded by inhibiting or otherwise down regulating the activity of ion channels expressed in the host cell. Thus, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel 10 relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HICV life cycle. 15 The SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel. As similar E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. 20 The present invention is concerned with novel antiviral compounds that fall under the classification of substituted acylguanidines. It does not include in its scope the use of compounds 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl) amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV. 25 Accordingly, a first aspect of the present invention provides an acylguanidine with antiviral activity. According to a second aspect, the present invention provides an antiviral compound of Formula I 0
    N
    "
    '
    R
    3 R, N N H I 1<4 WO 2004/112687 PCT/AU2004/000866 -4 wherein Ri-R 4 are independently aromatic groups, heteroaromatic groups, alkylaromatic groups, alkylheteroaromatic groups, alkenylaromatic groups, alkenylheteroaromatic groups, cycloalkylaromatic groups, cycloalkylheteroaromatic groups, aryloxyalkyl groups, heteroaryloxyalkyl 5 groups, said groups are mono or polycyclic, and are optionally substituted with one or more substitutonts independently selected from hydrogen, hydroxy, nitro, halo, amino, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, C 1 .alkyl, C 1 . salkyloxy, C3.6cycloalkyl, halo-substituted Ct6salkyl, halo-substituted C 10 6alkyloxy, phenyl, C 1
    .
    6 alkeneyl, C3.
    6 cycloalkeneyl, C 1
    .
    6 alkeneoxy, bezo, H2N N aryl, substituted aryl, PrS, O , or NH2 0 According to a third aspect, the present invention provides an antiviral compound of Formula I 15 0 NN H I or pharmaceutically acceptable salts thereof, wherein, Rb 20 : Nx,; , N ~ C' WO 2004/112687 PCT/AU2004/000866 5, R, , R, and R14 are independently hydrogen, Rn NN ', 0 5 or L RRk 0 0 A I -1> O D'P-F ~Or . , 10 and wherein WO 2004/112687 PCT/AU2004/000866 -6 X = hydrogen, hydroxy, nitro, halo, C6alkyl, C 1
    .
    6 alkyloxy,
    C
    3 c 4 oycloalkyl, halo-substituted C 1
    .
    6 alkyl, halo-substituted C 1 . 6 alkyloxy, phenyl, Ct-6alkeneyl, C 3
    .
    6 cyoloalkeneyl, Cvsalkeneoxy, or benzo; 5s, Rs , , R , 1., Rf, R1, Rk, RL,R, Rn,Ro ,R independently= hydrogen, amino, halo, C 1 salkyl, Ct-salkyloxy, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, H3C %
    H
    2 N N
    NH
    2 0 orPrS; 10g g, Ri independently= hydrogen, hydroxy, halo, or C 1 .s alkyl; R = hydrogen, amino, halo, C..sallkyl, C1-alkyloxy, hydroxy, aryl, substituted aryl, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, PrS, HAN N NY X 0"', or NH 2 0 or 15 Preferably, the compounds of the invention include the following: 5-(N,N-hexamethylene)amiloride comprising the structure 0 NH 2 aN NH2 O NH 20 WO 2004/112687 PCT/AU2004/000866 -7 5-(N,N-Dimethyl)amiloride hydrochloride comprising the structure
    OH
    3 S-(N-methyl-N-isobutyl)smiloride comprising the structure O NH2 Cl N :U NNH 2 11 N N NH 2 5-(N-ethyl-N-isobutopyl)amilorid (hen rfrrd to as EPA), comprising the structure structure 0 NHz Cl N" NI N N
    NH
    2 5-(N-ethyl-N-isopropy1)amiloide (herein referred to as ElA), comprising the structure 0 NB2 Cl: N / N" NU2 1N N NH2 WO 2004/112687 PCT/AU2004/000866 -8 N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N'-phenyl-guanidine, comprising the structure o NH 2 X Cl N NA
    H
    2 N N NH 2 N-Benzyl-N'-(3,5-diamino-6-chloro-pyrzine-2-carbonyl)-guanidine, comprising the 5 structure o NH 2 Cl N N
    H
    2 N N NH 2 3-methoxy.amiloride comprising the structure comprising the structure 0 NH 2 Cl N N. N NH 2
    H
    2 N N OCH3 3-methoxy-5-(N,N-Hexamethylene)-amiloride comprising the structure 0 NH 2 C N N N NH2 G0l 3 10" WO 2004/112687 PCT/AU2004/000866 -9 3-hydroxy-5-hexamnethyleneimino-amiloride comprising the structure O NI 2 CN / OH NH2 Hexamethyleneimino-6-phenyl-2-pyraxinccarboxamide comprising the structure N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide comprising the stmcture S 0 NI 2 NZr S N- N > Nl 2
    H
    2 N N NH2 10 WO 2004/112687 PCT/AU2004/000866 -10 5-(N,N-hexamethylene)amiloride comprising the structure o NE 2 ClN 0 NNH2 N N NH 2 N-amidino-3-amino-5-phnyl-6-chloro-2-pyrazinecarboxamide comprising the 5 structure O NIa Cl N N N NH2 1N NH2 3'4 DichloroBenzamil comprising the structure 0 NH 2 N NI2 10 WO 2004/112687 PCT/AU2004/000866 -11 2'4 DiohloroBenzamil HC comprising the structure 0 N2 Cl1 CN C N NH / NHz N NH 2 Cl 5-(N-methyl-N-guanidinocazbonyl-methyl)amiloride comprising the structure 0 NH 2 C N 'N x N t
    NH
    2 N N Nit 2 N 5 5-(NN-Diethyl)amiloride hydrochloride comprising the structure 0 NH 2 Cl N
    NANH
    2 H3C 3 N NH 2 BHC 5-(N,N-Dimethyl)amiloride hydrochloride comprising the structure N N NH2
    OH
    3 .1 : N N NH 2 CH3 WO 2004/112687 PCT/AU2004/000866 -12 5-tert-butylamino-amilorido comprising the structure O NH 2 Cl N Nl NH2 N NH2 6-Iodoamiloride comprising the structure 0 NH 2 Y N NH 2 5 H 2 N N NH 2 Bodipy-FL Amiloride comprising the structure o NH 2 Cl N 1 N ODUL NH 1 N N NH 2 5-(4-fluorophenyl)amiloride comprising the structure 0 NH 2 N Nit N N NHA 10 WO 2004/112687 PCT/AU2004/000866 -13 1 -napthoylguanidine comprising the structure
    H
    2 N NH 2 O N 2-napthoylguanidine comprising the structure 0 NH 2 . N1 NA NH 2 N 5 N-(2-napthoyl)-N'-phenylguaaidine comprising the structure N N1 0 A N / N : NH2 N,N'-bis(2-napthoyl)gnanidine comprising the structure 0 NH12 0 / NNK H 10 WO 2004/112687 PCT/AU2004/000866 -14 N,N'-bis(1-napthoyl)guanidine comprising the structure / O NB2 O0 NN jiH H N,N'-bis(2-napthoyl)-N"-phenylguanidine comprising the structure 1 0O S N NK NH I H o 5 6-methoxy-2-naphthoylguanidine comprising the structure O NH * N NH 2 IH N-Cinnamoyl-N',N'-dimethylguanidine comprising the structure N CH 3 HI3 WO 2004/112687 PCT/AU2004/000866 -15 3-quinolinoyguanidine comprising the structure O NH 2 A N N N$ 2 . N N cinnamoylguanine comprising the structure O NH 2 NN 5 4-phenylbenzoylguanidine comprising the structure 0 NH
    N
    3
    KH
    2 H N-(cinnamoyl)-N'phenylguanidine comprising the structure o NH N NH 1IH WO 2004/112687 PCT/AU2004/000866 -16 (3-phenylpropanoyl)guanidine comprising the structure 0 NH N " NH 2 NN'-bis-(oinnamoyl)-N"-phenylguanidine camrnpsi g the structure H 5 N '-bis-(cinnamoyl)-N'-ph nylguanidine comprisig the structure ON Ho 4 I 5 N-(bis3phnylpropanoyl)-N-phnylguanidine comprising the structure O NH A NN NN'-bis(3phenylpropanoyl-N"-phec~ylgnanidine comprising the structure O N. 0 NN H H WO 2004/112687 PCT/AU2004/000866 -17 trans-3-fOranacryoylguanidine comprising the structure O NH N, NN 2 0 H N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine comprising the structure O NH N 1 N NH 1OH (4-Phenoxybenzoyl)guanidino comprising the straotre o Nfl 2 O' A' N NH 2 10 N,N-Bis(amidino)napthalene-2,6-dicarboxamide comprising the structure O NH2 / N NB 2 H 2N N N /
    NIH
    2 0 15 WO 2004/112687 PCT/AU2004/000866 -18 6-bromo-2-napthoylguanidine comprising the structure 0 NHI N N NPNH 2 H Br 1-bromo-2-napthoylguanidine comprising the structure Br O NH N NH2 H 2-(2-napthyl)acetoylguanidine comprising the stmucture H NH 10 N"-Cinnamoyl-N,N'-diphenylguanidine comprising the structure A .0 HN O I-IN " (E) N NH N1 (Phenylacetyl)guanidine comprising the structure jI 0 NH N
    NH
    2 H 15 WO 2004/112687 PCT/AU2004/000866 -19 N,N'-Bis(3-phenylpropanoyl)guanidine comprising the structure O NH 0 r NXN it I H Benzoylguanidine comprising the structure 0. Nl N NH2 NR 5 (4-Chlorophenoxy-acetyl]guanidineo comprising the structure O NH C1" 10 N-Bcnzoyl-N'-oinnamoylguanidine comprising the structure o NH 2 0 P N N NN [(E)-3-(4-Dimethylaminophenyl)-2-methylacryloyl]guanidinc comprising the 15 structure .O NH N NH2 H
    N
    WO 2004/112687 PCT/AU2004/000866 -20 (4-Chlorochmamoyl)guanidine comprising the stmoture 0 Nl 2 (E) Cl (4-Bromocinnamoyl)guanidine comprising the structure o Nil 2 N NH 2 5 (4-Methoxycinnamoyl)guanidine comprising the structure 0 NH2 - ( N NH 2 I "O 10 (5-Phcnyl-pcnta-2,4-dicnoy1)guanidine comprising the structure o NH Br 15 (3-Methoxycinnamoyl)guanidine comprising the structure o NH O (E I- N NH2
    H
    WO 2004/112687 PCT/AU2004/000866 -21 (3-Chlorocinnamoyl)guanidine comprising the structure O NH Cl I a (2-Chlorocinnamoy)guanidine comprising the structure O NHl M ( N NH 2 H Cl 5 (2-Bromocinnmmoyl)guanidina comprising the.structure O NH P4 NH 2 Br (2-Methoxycinnamoyl)guanidine comprising the structure 0 NH N0 N NH 2 H 0 10 (trans-2-Phenyloyclopropanecarbonyl)guanidine comprising the structure O NH - N Nh I H [3-(3-Pyridyl)acryloyl]guanidine comprising the structure O NH 5 N NH 2 N H
    N
    WO 2004/112687 PCT/AU2004/000866 -22 (4-Hydroxycinnamoyl)guanidine comprising the structure 0 Nl ( N NH 2 HO (Quinoline-2-carbonyl)guanidine comprising the structure O NIR 2 OA Nil NN / 1N NH2 5 (4-Nitrocinnamoyl)guanidine comprising the structure N N N NH 2 021 H /H 02N 10 (3-Nitrocinnamoyl)guanidine comprising the structure 0 NH N N N N 1 2 H H I . H
    NO
    2 (2-Nitrocinnamoyl)guanidine comprising the structure O NH N N NH 1 H /NH
    NO
    2 15 WO 2004/112687 PCT/AU2004/000866 -23 (a-Methylinnamoyl)guanidine comprising the structure 0 NH N N NA NH 2 H / CH 3 trans-3-(1-napthyl)aoryloylguanidine comprising the structure O NH N N N NEI 2 H x/H 4-phenylcinnamoylguaanidine comprising the structure O NH N "UN NHJ2 / H 3-(trifluoromethyl)cinnamoylguanidine comprising the structure N NH 2 0H H 10
    CF
    3 3-methyloinnamoylguanidine comprising the structure O NH N NH 2 H CII3 WO 2004/112687 PCT/AU2004/000866 -24 4-(trifluoromethyl)cinnamoylguanidine comprising the structure 0 NH N N N2 H /H F3C 2-mothylinnamoylganidine comprising the structure CH 0 NIT N NH 2 /H 5 2-(trifluoronmethyl)innamoylguanidine comprising the structure
    CF
    3 0 NH N N N 1 1 2 H 4-methyloinnamoylguanidine comprising the structure . N N NH 2 H H 10 HzC 4-isopropylcinnamioylguanidine comprising the structure O NH N N N NH 2
    H
    3 C H H
    CI-
    3 15 WO 2004/112687 PCT/AU2004/000866 -25 3-fluorocinnamoylguanidine comprising the structure O NHi N NI-1 I NH H F 2-fluorocinnamoylguanidine comprising the structure o NH H 4-fluorocinnamoylguanidine comprising the structure 0 NH N N NH 2 H F A H 10 3,4-dichlorocinnamoylguanidine comprising the structure O NH N N N NH 2 H cCll Cl 2,4-dichlorocinnamolyguanidine comprising the stmuture o NH N N NA NH 2 III /H 15 WO 2004/112687 PCT/AU2004/000866 -26 2,6-dichlorocinnamoylguanidine comprising the structure Cl 0 NH N N N NH2 H 4-ethoxycinnamoylguanidine comprising the structure O NH N N NH 2 1 H /H
    SH
    3
    CH
    2 CO 3,4-(methylenedioxy)oinamtoylguanidine comprising the structure 0 'NRl Ioca° N N N NH 2 H OH 0 C 10 3-(2-napthyl)acryloylguanidine comprising the sticture O NH N N N NH 2 I H 4-t-butylcinnamoylguanidine comprising the structure 0 NE 1N NHM2
    H
    3 Cr I
    CHU
    3 15 WO 2004/112687 PCT/AU2004/000866 -27 3,4,5-trimethoxycinnamoylguanidine comprising the structure O NH HzCO CO N NH 2 H HsCO )PI OCH3 2-(1-napthyl)acetoylguanidine comprising the structure O NH N NH 2 H NIN 5 2,5-dimethylcinnamoylguanidine comprising the structure O NH 13 N
    NH
    2 H / H
    CH
    3 2,3"difluorocinnamoylguanidine comprising the structure o NH N N N NH 2 HI H /H F F 3-phenyloinmmoylguanidine comprising the structure H 10 WO 2004/112687 PCT/AU2004/000866 -28 3-(trans-hept-1 -en-1-yl)cinnmamoylguanidine comprising the structure O NH N N N NH 2 H 2-ethylcinnamoylguavidine comprising the structure O NH N N NH 2 H 2-chloro-6-fluorocinamoy1guanidine comprising the structure F 0 NH N N
    NH
    2 H /H Cl 10 .3-t-butyleinnmoylguanidine comprising the structure O NIH S NNH 2 I H
    H
    3 C ClH 3
    CH
    3 WO 2004/112687 PCT/AU2004/000866 -29 3,4-difluorocinnamoylguanidine comprising the structure 0 NH N j i
    NH
    2 H F 5-bromno-2-fluorocirmamoylguanidine comprising the structure o NH Br N NH 2 H SF 3-(trifluoromethoxy)cinnamoylguanidine comprising the structure O NH N ) NHz H H
    OCF
    3 2-ethoxyoinnamoylguanidine comprising the structure 0 NH N N N 2 IH / I-I 10 C2 2-t-butyloinnamoylguanidine comprising the structure AJ 0 NH N N N 3
    KNH
    2 CHH S15N %;15
    H
    WO 2004/112687 PCT/AU2004/000866 -30 3-(cyclohex-1-en-1-yl)oinnamoylguanidine comprising the structure O NH N~z N NH H einnamoylguanidine hydrochloride comprising the structure O NH N N 4 H .HCl 2,3,5,6,-tetramethykinnamoylguanidine comprising the structure (Bit 134)
    CH
    3 0 NH HNC
    NH
    2 H H A/H
    CH
    3 CH3 2-eyelohexylcinnamoylguanidine comprising the structure o NH N N~ H H 10 5-bromo-2-methoxycinnmamoylguanidine comprising the structure 0 NH N NN H OCH3 WO 2004/112687 PCT/AU2004/000866 -31 2,3-dimethy1cinnamoy1guanidine comprising the structure O NH N N NANH I H
    CH
    3
    CH
    3 3-ethoxycinnamoylguaidine comprising the structure O NH N NH 2 |H /H
    OCH
    2
    CH
    3 3-isopropylinnamoylguanidine hydrochloride comprising the structure O NH N N N NH H A- H HCI 2-phenylcihnamoyguanidine comprising the structure O NH LN NIHz H |- H 10 2-(cyclohex-1-en-lyl)cinnamoylguanidine comprising the structure o NH
    H
    WO 2004/112687 PCT/AU2004/000866 -32 2,4,6-trimethylcinnamoylguanidine comprising the structure
    CH
    3 0 NH ' N NH2 H 1130 H E3C CH3 (5-Phonyl-penta-2,4-dienoyl)guamidine comprising the structure O NH N
    NH
    2 5H 5-(3'-bromophbenyl)penta-2,4-dienoylguanidine comprising the structure o NH Br 10 5-(2'-bromophenyl)penta-2,4-dienoylguanidine comprising the structure O NH
    NH
    2 ~Br Furmanacryloyl comprising the structure 0 NH 15 WO 2004/112687 PCT/AU2004/000866 -33 Preferably, the compounds of the invention are capable of reducing, retarding or otherwise inhibiting viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family 5 family of viruses. For example, the compounds of the invention exhibit antiviral activity against viruses such as Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Mouse Hepatitis virus (), and Hepatitis C virus (CV). According to a fourth aspect of the present invention, there is provided a 10 pharmaceutical composition comprising an antiviral compound according to any one of the first, second or third aspects, and optionally one or more pharmaceutical acceptable carriers or derivatives, wherein said compound is capable of reducing, retarding or otherwise inhibiting viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against 15 viruses such as those belonging to the Lentivirus family, and the Coronovirus family of viruses. For example, the compounds of the invention exhibit antiviral activity against viruses such as Human Jmmunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus 0043, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine 20 Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. The compositions of the invention may further comprise one or more known 25 antiviral compounds or molecules. According to a fift aspect, there is provided a method for reducing, retarding or otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said virus or exposed to said virus with a compound according to any one of the first, second or third aspects. 30 Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus WO 2004/112687 PCT/AU2004/000866 -34 OC43, Mouse Hepatitis virus (MVHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Mouse Hepatitis virus (MHV), Hepatitis C virus (ICV), or Equine Arteritis Virus (EAV). Mostpreferably, the virus is HV-1, HIV-2, the SARS virus, Coronaviruse 229B, Coronavirus OC43, PRCV, BCV, HCV, or 5 EAV. Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. According to a sixth aspect, there is provided a method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound 10 according to any one of the first, second or third aspects. Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hlepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine 15 Respiratory Coronavirus (PRCV), Mouse Hepatitis virus (MHV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse 229E, Coronavirus OC43, PRCV, BCV, HCV, EAV. Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. 20 According to a seventh aspect of the invention, there is provided a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the administration of a compound according to any one of the first, second or third aspects, to a subject in need of said treatment. Preferably, infection with a virus or exposure to a virus occurs with viruses 25 belonging to:the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229B, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, 30 HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
    WO 2004/112687 PCT/AU2004/000866 -35 Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), 5 companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, dooeer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to a eighth aspect, there is provided a method of down regulating a membrane ion channel functional activity in a cell infected with a virus, comprising 10 contacting said cell with a compound according to any one of the first, second or third aspects, SThe membrane ion channel may be endogenous to the cell or exogenous to the celL Preferably, the membrane ion channel of which functional activity is down 15 regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, the Coronavirus B protein membrane ion channel, and the .$AR$ E protein membrane ion channel. Preferably, infection with a virus or exposure to a virus occurs with viruses 20 belonging to the Leativirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HICV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, 25 HIV-2. SARS, Human Coronavirus 229E, Human Coronavirus 0C43, Hepatitis C virus (HCV), or Equine Arteritis Virus (BAV). According to an ninth aspect of the present invention, there is provided a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a 30 compound according to any one of the first, second or third aspects, wherein said compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell.
    WO 2004/112687 PCT/AU2004/000866 -36 Preferably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), 5 Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2. SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (BAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. 10 Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel. According to an tenth aspect, the present invention provides a method of 15 reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound according to any one of the first, second or third aspects, or a pharmaceutical composition according to thd fourth aspect, wherein said compound or said composition down regulates fumonctional activity of a membrane ion channel 20 expressed in said infected celL Preferably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MEIV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), 25 Hepatitis C virus (-CV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. 30 Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral WO 2004/112687 PCT/AU2004/000866 -37 replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion 5 animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, dooeer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to a eleventh aspect, the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus 10 comprising administering to said subject a compound according to any one of the first, second or third aspects, or a pharmaceutical composition according to the fourth aspect, wherein said compound or said composition down-regulates functional activity of a membrane ion channel derived from said virus. Preferably, infection occurs with a virus belonging to the Lentivirus family, or 15 the Coronovirus family of viruses. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MIV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (BAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SAR, Human Coronavirus 229E, Human 20 Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lntiviruses, and Coronaviruses utilise for mediating viral 25 replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel,. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, oat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, 30 hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human.
    WO 2004/112687 PCT/AU20041000866 -38 According to a twelfth aspect, the invention provides an antiviral compound select ed from the grou consist of: N-35Daia--hooprzn--uoy)N-hnlgaiie N-Benzy1-N'.(3,5-diamin-6-chloro-pyhrzne-2-cbofl)guafldie 5 314 DichloroBenztlil 2'4 DichloroBenzamil, 5-(N-methyl-N-guanidinocarbonyl-methyl)amliloride, 5-4N-MoIthyl-N-isobutyl)fliloride, 5-(N-Egthyl-N-isopropyl)aniiloiide, 10 5.(NN-Dimetliyl)amiloride hydrochlorid;, 5-(NN-hexamethylene)amniloride, 5-(N,N-Diethyl)amidloridc hydrochloride, 6-Iodoamiloride, Bodllpyv-FL amiloride, 15 3-hydrcxy-.5-hexamethiyleneimino-aliloride, 5-(4-fluorapbenyl)aailoride, 5-tert-butylaniino-amiloride, N-mdn--mn- hnt6choo2przncroaie 3-methoxy -5-(NHexamflthyflCn)-amilo~deO, 20 3-nhethoxy-amiloride, hesamethyleneimino-6-phenyl-2-pyizilecarboximide, N-amidino3,5diamio-6-phyl2-pyrSzif8O8XboxBTYid, l-naphthoylguanidine, 2-naphthoylguanidine, 25 N.(2-naphthoyl)-N'.phenylgaidilO 3 N,N-bis(2-naphthoy)guanfidine, N4,N'-bis(l-naphthoyl)guanidiflc, NN'bis(2-naphthoy)-NLphenylguaidilO, 6-methoxy-2-naphthoylguauiddille, 30 3-quinolinoylguanidine, cinnmoylguamdme, 4-phenylbenzoylguanldine, WO 2004/112687 PCT/AU20041000866 N-(cinnanoyl)-N'phenylganidine, (3-phenylpropanoyl)gaanidine, NN-bis-(cinnanioyl)-N"-phenylguanidine, N-(3-plienylpropanoyl)-N'-phenylguanidine, 5 N,-I-bis(3phenylpropMnoy)NM-phenylguanidine, trans-3-ttranaxyoylguariidine, N-(6-Hydroxy-2-naphthoyl)-N'-phenylguanidio, (4-Phenoxybenzoyl)gumnidine, NNf-Bis(anidino)napthalene-2,6-dicarboxamide, 10 N'LCinnamoylNNdiphenylgaanidine, MPheylacety)guane, NN-B3is(3-phenylpropanoyl)guanidine, benzoylgua-didine, (4-Chlorophenoxy-aoetylguanidin;, 15 N-benzoyl-N-innanoylguanidinv, [(PB)-3-(4-Dimethylaminopheny1)-2-methylaaryoy1]guanidine, (4-Chlorocinnamoyl)Suanidino, (4-Bromocinnamoyl)guanidino, (4-Methoxyirmamoyl)guanidine, 20 (5-Phenyl-penta-2A-dienoyl)guanf dine. (3-Bromooimnmoyl)guanidine, (3-Methoxycinnamoy1)Suanidinc, (3-Cbloroeinnaoyl)gumnidino,, (2-Chlarocinnaoyl)guauidine, 25 (2-BRomocinoyl)guanidine. (2-MetoxycmiaoyIguanidine,, (trans-2-Phenylcyclopropanecaxbonyl)guanidifle, [3-(3-Pridyl)acryloyl]guanidine, (4-Hydroxyoirmamuoyl)guai'dine, 30 (Quinoline-2-carbonyl)guaniidine, or pharmaceutically acceptable salts thereof.
    WO 2004/112687 PCT/AU2004/000866 -40 According to a thirteenth aspect, the present invention provides a pharmaceutical composition comprising a compound according to the twelfth aspect, and optionally one or more pharmaceutical acceptable carriers or derivatives. Preferably, the pharmaceutical composition may further comprise one or more 5 known antiviral compounds or molecules. Unless the context clearly requires otherwvise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are tobe construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to", 10 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of plasmids used for expression of Vpu in . colt. A. The amino acid sequence ( < 400 > 1) encoded by the vpu open reading frame (ORF) generated by PCR from an HIV- 1 strain HXB2 eDNA clone. The vpu ORF was cloned in-frame at the 3' end of the GST gene in p2GEX to generate 15 p2GEXVpu (B). It was subsequently cloned into pPAL451 to produce the plasmid pPL + Vpu (C). Figure 2 is a photographic representation of the expression and purification of Vpu in E. colt. A. Western blotting after SDS-PAGE was used to detect expressed Vpu in E, colt extracts. Lanes 1-4 contain samples, at various stages of purity, of Vpu expressed 20 from p2GEXVpu: lane 1, GST-Vpu fusion protein isolated by glutathione-agarose affinity chromatography; lane 2, Vpu liberated from the fusion protein by treatment with thrombin; lane 3, Vpu purified by HPLC anion exchange chromatography; lane 4, Vpu after passage through the immunoaffmnity column. Lanes 5 and 6, membrane vesicles prepared from 42'C induced cells containing pPL+Vpu orpPL451, 25 respectively. B. Silver stained SDS-PAGB gel: lane 1, Vpu purified by HPLC anion exchange chromatography; lane 2, Vpu after passage through the immunoaffinity column. Figure 3 is a graphical representation of ion channel activity observed after exposure of lipid bilayers to aliquots containing purified Vpu. In A and B, the CIS chamber 30 contained 500mM NaCl and the TRANS chamber contained 50mM NaCl; both WO 2004/112687 PCT/AU2004/000866 -41 solutions were buffered at pH 6.0 with 10 mM MES. B shows a current versus voltage curve generated from data similar to that shown in A. Figure 4 is a photographic representation of bacterial cross-feeding assays. For all plates, the Mof, Pro" auxotrophic strain was used to seed a soft agar overlay. Plates A 5 and B contain minimal drop-out medium minus proline; in plate C the medium was minus methionine. To control for viability of the cells in the background lawn, the discs labelled P and M contained added proline or methionine, respectively. The discs labelled C and V were inoculated with Met" , Pro t E, coli cells containing the plasmids pPL451 or pPL+Vpu, respectively. Plates were incubated at 37 0 C (A and C) 10 or 30 0 C (B) for two days and photographed above a black backgroundwith peripheral illumination from a fluorescent light located below the plate. The images were recorded on a Novaline video gel documentation system. Light halos around the discs labelled P or M on all plates and around the disc labelled V on plate A indicate growth of the background lawn strain. 15 Figure 5 is a graphical representation of the screening of drugs for potential Vpu channel blockers. The photograph shows a section of a minimal medium-lacking adenine - agarose plate onto which a lawn of XL-I-blue E. coli cells containing the Vpu expression plasmid pPLVpu has been seeded. Numbers 6-11 are located at the sites of application of various drugs being tested, which were applied in 31 l drops and 20 allowed to soak into the agarose. The plate was then incubated at 37 0 C for 48hr prior to being photographed. The background grey shade corresponds to areas of no bacterial growth. The bright circular area around" 10 "represents bacterial cell growth as a result of application of adenine at that location (positive control). The smaller halo of bacterial growth around "9" is due to the application of 5-(N,N 25 hexamethylene)-amiloride at that location. Figure 6. SARS E protein ion channel activity observed in NaC1 solutions after exposure of lipid bilayer to 3-10g of E protein. A. The closed state is shown as solid line, openings are derivations from the line. Scale bar is 300ms and SpA. The CIS 30 chamber contained 50mM NaCI in 5mM HEPES buffer pI 7.2, the TRANS chamber contained 500mM NaCl in 5mM HIEPES buffer pIH 7.2. The CIS chamber was WO 2004/112687 PCT/AU2004/000866 -42 earthed and the TRANS chamber was held at various potentials between -100 to +100mV. B. Largest single opening events of a single channel. Figure 7. SARS B protein ion channel activity observed in NaCi solutions after 5 exposure of lipid bilayer to 3-10pg of E protein. A. The closed state is shown as solid line, openings are derivations from the line. Scale bar is 300ms and SpA. The CIS chamber contained 50mM NaCI in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in 5mM HIEPES buffer pH 7.2. The CIS chamber was earthed and the TRANS chamber was held at various potentials between -100 to 10 +100mV, B. Largest single opening events of a single channel. Figure 8. Cinnamoylguanidine (Bit036) inhibits SARS E protein ion channel activity in NaC1 solution. A. Representative currents at holding potential of -40mV. Scale bar is 300mS and SpA. E protein ion channel activity and E protein channel activity after the addition of 100gM Bit036. B. All points histogram at holding potential of 15 40inV. B protein ion channel activity before and after the addition of 100pM Bit036. C. Average current (pA), before formation of B protein ion channel, E protein ion channel activity and after addition of 10pM BitO36. Figure 9 229E E protein Ion channel activity in lipid bilayers in KC1 solutions. Figure 10: Part A shows raw currents generated by the 229E-E protein ion channel 20 in a planar lipid bilayer. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100M oinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidino. 25 Figure 11: MHV E protein Ion channel activity in lipid bilayers NaCl solutions. Figure 12: Part A shows raw currents generated by the MHV-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100pM cinnamoylguanidine on 30 channel activity, Part B is a graphical representation of the average current flowing WO 2004/112687 PCT/AU2004/000866 -43 across the bilayer (in arbitrary units), before and after addition of cinnamnoylguanidine. DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the surprising determination that 5 certain compounds that fall under the classification of substituted acylguanidines have antiviral activity against viruses from a range of different virus families. Without intending to be bound by any particular theory or mechanism of action, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel 10 relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HCV life cycle. 15 The SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel. As similar E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. 20 While the present invention is concerned with novel antiviral compounds iffling under the classification of substituted acylguanidines, it does not include in its scope the use of compounds 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl) amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV. 25 It will be understood by those skilled in the art that the compounds of the invention may be administered in the form of a composition or formulation comprising pharmaceutically acceptable carriers and excipients. The pharmaceutical compositions of the invention may further comprise one or more known antiviral compounds or molecules. Preferably, the known antiviral 30 compounds are selected from the group consisting of Vidarabine, Acyclovir, Ganciclovir, Valgancidolovir, Valacyclovir, Cidofovir, Famiolovir, Ribavirin, WO 2004/112687 PCT/AU2004/000866 -44 Amantadine, Rimantadine, Interferon, Oseltamivir, Palivizumab, Rimantadine, Zanamnivir, nucleoside-analog reverse transcriptase inhibitors (NRTI) such as Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine and Abaeavir, non nucleoside reverse transcriptase inhibitors (NNRTI) such as Nevirapine, Delavirdine 5 and Efavirenz, protease inhibitors such as Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, and other known antiviral compounds and preparations. Known antiviral compounds or molecules may in some cases act synergistically with the antiviral compounds of the invention. 10 Table 1 Known coronavlrus isolates Group 1 species Canine coronavirus Canine enteric coronavirus (strain INSAVC-1) Canine enteric coronavirus (strain K378) Feline coronavirus Feline enteric coronavirus (strain 79-1683) Feline infectious peritonitis virus (FLPV) Human coronavirus 229E Porcine epidemic diarrhea virus Porcine epidemic diarrhea virus (strain Brl/87) Porcine epidemic diarrhea virus (strain CV777) Transmissible gastroentex itis virus Porcine respiratory coronavirus Porcine transmissible gastroentermitis coronavirus (STRAIN FS772/70) Porcine transmissible gastroenteritis coronavirus (strain Miller) Porcine transmissible gastroenteritis coronavirus (strain Neb72-RT) Porcine transmissible gastroenteritis coronavirus (STRAIN PURDUE) Group 2 species Bovine coronavirnis Bovine coronavirus (STRAIN F15) Bovine coronavirus (strain G95) Bovine coronavirus (STRAIN L9) Bovine coronavirus (strain LSU-94LSS-051) Bovine coronavirus (STRAIN LY-138) Bovine coronavirus (STRAIN VEBUS) Bovine coronavirus (strain OK-0514-3) Bovine coronavirus (strain Ontario) Bovine coronavirus (STRAIN QUEBEC) Bovine coronavirus (STRAIN VACCINE) Bovine enteric coronavirus (strain 98TXSF-I 10-ENT) Canine respiratory coronavirus WO 2004/112687 PCT/AU2004/000866 -45 Chicklen enteric coronavirus Human coronavirus 0C43 Murine hepatitis virus Murine coronavirus (strain DVIM) Murine hepatitis virus (strain A59) Muine hepatitis virus (strain JHM) Muxino hepatitis virus (strain S) Murine hepatitis virus strain 1 Murine hepatitis virus strain 2 Murine hepatitis virus strain 3 Murine hepatitis virus strain 4 Murine hepatitis virus strain ML-11 Porcine hmagglutinating encephalomyElitis virus Porcine hemagglutinating encephalomyeitis virus (strain 67N) Porcine hemagglutinating encephalomyelitis virus (strain IAF-404) Puffinosis virus Rat coronavirus Rat coronavirus (strain 681) Rat coronavims (strain NJ) Rat sialodacryoadenitis coroavirus Group 3 species Turkey coronaviruas Turkey coronavirus (strain IndiBana) Turkey coronavirus (strain Min Bota) Turkey coronavirus (strain NC95) Avian infectious bronchitis virus Avin infetious bronchitis virus (STRAIN 6/82) Avian infectious bronchitis virus (strain Arkansas 99) Avian infections bronchitis virus (strain Beaudette CK) Avian infectious bronchitis virus (strain BPeaudette M42) Avian infectious bronchitis virus (strain Beaudefte US) Avian infectious bronchitis virus (strain Beaudetto) Avian infectious bronchitis virus (strain D1466) Avian infectious bronchitis virus (strain D274) Avian infectio s bronchitis vims (strain D3896) Avian infectious bronchitis vims (strain D41) Avian infectious bronchitis virus (strain DE072) Avian infectious bronchitis virus (strain GRAY) Avian infectious bronchitis virus (strain H120) Avian infectious bronchitis virus (strain H52) Avian infectious bronchitis virus (strain KB8523) Avian infectious bronchitis virus (strain M41) Avian infectious bronchitis virus strainn PORTUGAL/322/82) Avian infectious bronchitis virus (strain SAIB20) WO 2004/112687 PCT/AU2004/000866 -46 Avian infectious bronchitis virus (strain UK/123/82) Xvian- infectious bronchitis virus (strain UK/ 42Y8 6) Avian infectious bronchitis virus (strain UTK/i 67/84) Avian infetious bronchitis virus (strain Ml /6) Avian infectious bronchitis virus (strain Vi /8/1) Avian infbotious bronchtis virus (sfrai Vic S) Avian infectious IM otxaheitis virus Preliminary Group 4 pees SARS coronaviras SARS coronavirus Boiiu ZY-2003 SARS coronavirus BTIl SARS corounavirus B302 SARS cownavirus BJ03 SARS coronavirus BJ04 SARS coronavirus CURHK-SalO SARS coronavirus CUHX-WI SARS coronavirus Frankfurt 1 SARS coronavirms GZ01 SARS coronavirus HKU-3 9849 SARS coronavirus Hona N2ong ZY-2003 SARS coronavirus Hong Kon/ 0/2003 SAPS coronaviru HISR 1 SARS coronavirn Sin2500 SAPS coronavirus Sin2677 SARS coronavirus Tiwan C SARS coronavirus Tin TC2 SARS coronavirus Tor277 SARS coronavirus Taiwa SARS coronavirus TawCn SARS coronavirus TarbanT SAtRic coronavis T Equin coronaviaTW WO 2004/112687 PCT/AU2004/000866 -47 Equine coronavirus NC99 The present observations and findings now permit the use ofegents such as certain substituted acylguanidines, as anti-viral agents for the therapy and prophylaxis of viral conditions caused by different viruses. The methods and compositions of the 5 present invention may be particularly effective against viruses which rely on ion channel formation fbr their replication, however it will be understood that this is not the only mechanism orelied on by viruses for replication and that the compounds and methods of the present invention are not limited to agents which exert their action by -retarding or inhibiting the function of ion channels. 10 Reference to."membrane ion channel" should be understood as a reference to a structure which transports ions across a membrane. The present invention extends to ion channels which may function by means such as passive, osmotic, active or exchange transport. The ion channel may be formed by intracellular or extracellular means. For example, the ion channel may be an ion channel which is naturally formed 15 by a cell to facilitate its normal functioning. Alternatively, the ion channel maybe formed by extracellular means. Extracellular meas would include, for example, the formation of ion channels due to introduced chemicals, drugs or other agents such as ionophores or due to the fimctional activity of viral proteins encoded by a virus which has entered a cell. 20 The ion channels which are the subject of certain embodiments of the present invention facilitate the transport of ions across membranes. Said membrane may be any membrane and is not limited to the outer cell wall plasma membrane. Accordingly, "membrane" as used herein encompasses the membrane surrounding any cellular organelle, such as the Golgi apparatus and endoplasmic reticulum, the 25 outer cell membrane, the membrane surrounding any foreign antigen which is located within the cell (for example, a viral envelope) or the membrane of a foreign organism which is located extracellularly. The membrane is typically, but not necessarily, composed of a fluid lipid bilayer. The subject ion channel may be of any structure. For example, the Vpu ion channel is formed by Vpu which is an integral membrane 30 protein encoded by HIV-1 which associates with, for example, the Golgi and ndoplasmic reticulum membranes of infected cells, Reference hereinafter to "Vpu WO 2004/112687 PCT/AU2004/000866 -48 ion channels" is a reference to all related ion channels for example P7 HCV and M2 of influenza and the like. Reference to "HIV", "SARS", "Coronavirus" or "HCV" should be understood as a reference to any IRV, SARS, Coronavirus or HCV virus strain and including 5 homologues and mutants, Reference to the "functional activity" of an ion channel should be understood as i a reference to any one or more of the functions which an ion channel performs or is involved in. For example, the Vpu protein encoded ion channel, in addition to facilitating the transportation of Na, K, cr and P0 4 3 ", also plays a role in the 10 degradation of the CD4 molecule in the endoplasmic reticulum. Without wishing to be bound by a particular theory, the Vpu protein encoded ion channel is also thought to play a role in mediating the HIV life cycle. The present invention is iot limited to treating HIV infection via the mechanism of inhibiting the -IV life cycle and, in particular, HIV replication. Rather, the present invention should be understood to 15 encompass any mechanism by which the compounds of the present invention exert their anti-viral activity and may include inhibition of HIV viability or functional activity. This also applies to HCV, Coronaviruses, and to other viruses. Reference to the "functional activity" of a virus should be understood as a reference to any one or more of the functions which a virus performs or is involved 20 in. Reference to the "viral replication" should be understood to include any one or more stages or aspects of the viral life cycle, such as inhibiting the assembly or release of virions. Ion channel mediation of viral replication may be by direct or indirect means. Said ion channel mediation is by direct means if the ion channel 25 interacts directly with the virion at any one or more of its life cycle stages. Said ion channel mediation is indirect if it interacts with a molecule other than those of the virion, which other molecule either directly or indirectly modulates any one or more aspects or stages of the viral life cycle. Accordingly, the method of the present invention encompasses the mediation of viral replication via the induction of a 30 cascade of steps which lead to the mediation of any one or more aspects or stages of the viral life cycle.
    WO 2004/112687 PCT/AU2004/000866 -49 Reference to "down-regulating' ion channel functional activity, should be understood as a reference to the partial or complete inhibition of any one or more aspects of said activity by both direct and indirect mechanisms. For example, a suitable agent may interact directly with an ion channel to prevent replication of a 5 virus or, alternatively, may act indirectly to prevent said replication by, for example, interacting with a molecule other than an ion channel. A further alternative is that said other molecule interacts with and inhibits the activity of the ion channel. Screening for molecules that have antiviral activity can be achieved by the range of methodologies described herein. 10 Reference to a "cell" infected with a virus should be understood as a reference to any cell, prokaryotic or eukaryotic, which has been infected with a virus. This includes, for example, immortal or primary cell lines, bacterial cultures and cells in situ. In a suitable screening system for antiviral compounds, the preferred infected cells would be macrophages/monocytes or hepatocytes/lymphoid cells infected with 15 either HIV or HCV respectively. Without limiting the present invention to any one theory or mode of action, the compounds of the present invention are thought to inhibit viral replication or virion release from cells by causing ion channels, namely VPU of HIV, the E protein of SARS and other Coronaviruses, or P7 of HCV to become blocked. The present 20 invention encompasses antiviral compounds that are substituted acylguanidines. The present invention also includes the use of compounds 5-(N,N hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride in the control of viral replication and/or growth other than HIV. The subject of the viral inhibition is generally a mammal such as but not 25 limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a human or primate. Most preferably, the subject is a human. The method of the present invention is useful in the treatment and prophylaxis 30 of viral infection such as, for example, but not limited to H1V infection, HCV infection and other viral infections. For example, the antiviral activity may be effected in subjects Imknown to be infected with HIV in order to prevent replication of WO 2004/112687 PCT/AU2004/000866 -50 HIV thereby preventing the onset of AIDS. Alternatively, the method of the present invention may be used to reduce serum viral load or to alleviate viral infection symptoms. Similarly, antiviral treatment may be effected in subjects known to be infected with, for example, HCV, in order to prevent replication of HCV, thereby 5 preventing the further hepatocyte involvement and the ultimate degeneration of liver tissue. The method of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as a prophylactic treatment to be applied immediately prior to or for 10 a period after exposure to a possible source of virus. Reference herein to "therapeutic" and "prophylactic" is to be considered in their broadest contexts. The term "therapeutic" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, 15 - therapy and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis"may be considered as reducing the severity of onset of a particular condition. Therapy may also reduce the severity of an existing condition or the frequency of acute attacks. 20 In accordance with the methods of the present invention, more than one compound or composition may be co-administered with one or more other compounds, such as known anti-viral compounds or molecules.. By "co administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by 25 the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two or more separate compounds. The subject antiviral compounds may be administered in any order. Routes of administration include but are not limited to intravenously, 30 intraperitionealy, subcutaneously, intracranialy, intradermally, intramuscularly, intraocularly, intrathecaly, intracerebrally, intranasally, transmucosally, by infusion, WO 2004/112687 PCT/AU2004/000866 -51. orally, rectally, via iv drip, patch and implant. Intravenous routes are particularly preferred. Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of 5 sterile iujectable solutions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, 10 phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the usein the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin, Sterile injectable solutions are prepared by incorporating the active 15 compounds in the required amoumt in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by, for example, filter sterilization or sterilization by other appropriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the 20 required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile Injectable solutions, a preferred method of preparation includes vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution. 25 When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, 30 troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.01 % by weight, more preferably 0.1% by weight, even more preferably 1% by weight of active compound.
    WO 2004/112687 PCT/AU2004/000866 -52 The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 1 to about 99%, more preferably about 2 to about 90 %, even more preferably about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a 5 suitable dosage willbe obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of active compound. The tablets, trochei, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, com starch or gelatin; 10 excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a 15 liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Any material 20 used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be ncorporated into sustained-release preparations and formulations. The present invention also extends to forms suitable for topical application such as creams, lotions and gels. In such forms, the anti-clotting peptides may need to 25 be modified to permit penetration of the surface barrier. Procedures for the preparation of dosage unit forms and topical preparations are readily available to those skilled in the art from texts such as Pharmaceutical Handbook. A Martinddle Companion Volume Ed. Ainley Wade Nineteenth Edition The Pharmaceutleal Press London, 30 CRC Handbook of Chemistry and Physics Ed. Robert C. Weast Ph D. CRC Press Inc.; Goodman and Gilman's; The Pharmacological basis of Therapeutics. Ninth Ed WO 2004/112687 PCT/AU2004/000866 -53 McGraw Hill; Remington; and The Science and Practice ofPharmacy. Nineteenth Ed. Ed. Alfonso R. Gennaro Mack Publishing Co. Easton Pennsylvania. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and 5 absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 10 It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated.to produce the desired therapeutic effect in association with 15 the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding. Effective amounts contemplated by the present invention will vary depending 20 on the severity of the pain and the health and age of the recipient. In general terms, effective amounts may vary from 0.01 ag/kg body weight to about 100 mg/kg body weight. Alternative amounts include for about 0. 1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ng/kg body weight to about 80 mg/kg body weight. 25 The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, eat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a human or primate. Most preferably, the subject is a human. 30 The methods of the present invention is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to IHV infection, HCV infection and other viral infections. For example, the antiviral activity may be WO 2004/112687 PCT/AU2004/000866 -54 effected in subjects known to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS. Alternatively, the methods of the present invention maybe used to reduce serum viral load or to alleviate viral infection symptoms. Similarly, antiviral treatment may be effected in subjects known to be 5 infected with, for example, HCV, in order to prevent replication of HCV, thereby preventing the further hepatocyte involvement and the ultimate degeneration of liver tissue. The methods of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in 10 affected cells or as a prophylactic treatment to be applied immediately prior to or for a period after exposure to a possible source of virus. The present invention will now be described in more detail with reference to specific but non-limiting examples describing studies of viral membrane ion channels and screening for antiviral activity. Some examples involve the use of the SARS 15 virus. It will be clear from the description herein that other lentiviruses, and coronaviruses and other compounds maybe used effectively in the context of the present invention. It is to be understood, however, that the detailed description is included solely for the purpose of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the invention as set 20 out above. Example 1. Synthesis of the Compounds of the Invention. The compounds of the present invention may be made from the corresponding acid chlorides or methyl esters as shown in Scheme 1. Both of these methods are well described in the literature. 0 0 NH 0 R 01 R NH 2 R OMe acid chlorde anylanaidine ester Sdeme 1 25 WO 2004/112687 PCT/AU2004/000866 -55 The following examples show synthetic schemes for some compounds of the invention. Example 2. Synthesis of Cinnamovlignanidine from Cinnamic acid Cinnamovi chloride 5 0 I.(001)blmew tMP N H 2.(H)kQL=anHC( [t NH2 ,r NnqOH I 11,0 t TII To a solution of trans-cinnamic acid (1.50 g, 10.12 mmol) in dry benzene (30mb) containing a drop ofN,N-dimethylformamide was added oxalyl chloride 10 (5.14 g, 40.5 mmol) causing the solution to effervesce. After refluxing for 2 h, the solution was evaporated to dryness under reduced pressure. The resulting solid was dissolved in dry tetrahydrofbran (20mL) and added slowly to a solution of guanidine hydrochloride in 2M aqueous sodium hydroxide (25mL). The reaction was stirred at room temperature for lh then extracted with ethyl acetate (3x50mL). The combined 15 extracts were dried over magnesium sulfate and evaporated to give an orange oil. The crude product was purified by column chromatography. Elution with 10% to 20% methanol in dichloromethano gave Cinnamoylguanidine as a cream solid (0.829 g, 43%). Example 3 20 Synthesis of N-amidino-3-amino-5-phenyvl-6-ehloro-2-pyrazineearboxamide. Part 1 +N 0 , - W f ZO ft l I w To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (0.444 g, 2.0 mmol) in tetrahydrofaran (5 mL) / water (10 mL) / toluene (20 mL) was added 25 phenyl boronic acid (0.536 g, 4
    .
    4 rmmol), sodium carbonate (0.699 g, 6.6 nmol) and tetrakis(triphaylphosphine)- palladium(0) (0.116 g, 0.10 mmol). The reaction was WO 2004/112687 PCT/AU2004/000866 evacuated and purged with nitrogen sevmral times before being refluxed for 6 h. The organic layer was separated and the aqueous layer extracted with toluene (3 x 20 mL). The combined organic extracts were dried over magnesium sulfate, filtered and evaporated under reduced pressure to give methyl 3-amino-6-chloro-5 5 phenyl-2-pyrazinecarboxylate as a yellow solid (0.43 g, 82%). Part 2 To a solution of sodium (0.040 g, 174 mmol) dissolved in methanol (5 mL) 10 was added guanidine hydrochloride (0.258 g, 2.70 mmol) and the mixture refluxed for 30 min after which it was filtered. To the filtrate was added methyl 3-amino-6 ehloro-5-phenyl-2-pyrazinecarboxylate (0.264 g, 1.0 mmol) in NN dimethylfonnramide (5 mL) and the solution heated at 75oC for 12 Ih. The solvent was removed under reduced pressure and the residue chromatographed on silica gel 15 eluting with 1% triethylamnine / 5% methanol I dichloromethane. The resulting solid was suspended in chloroform, filtered and dried under high vacuum to give N Amidino-3-amino-5-pheny--chloro-2-pyrazinecarboxamide as a yellow solid (0.04 g, 14%). Example 4. 20 Synthesis of hexamethyleneimino.6i-Dhenyl-2-.vrazinecarboxamide Part 1 U.H N N NH CIA Nm. ()i N NH 2 To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (1.11 g, 5.0 mmol) in tetrahydrofuran (50 mL) was added hexamethyleneimine (1.49 g, 15.0 25 mmol) and the reaction was refluxed for 1 h. The reaction was allowed to cool and the solid hexamethylencimine hydrochloride removed.by filtration. The filtrate was WO 2004/112687 PCT/AU2004/000866 -57 evaporated and the residue chromatographed over silica gel. Blution with dichloromethane gave methyl 3-amino-6-chloro-5-hexamethyleneimino-2 pyrazineearboxylate as an off-white solid (1.20 g, 85%). 5 Part2 0 MS 0 + 6uso ] To a solution of methyl 3-amino-6-ohloro-5-hexainmethyleneimino-2 pyrazinecarboxylate (0.350g, 1.23 mmol) in dimethylsulfoxide (5 mL) was added phenyl boronic acid (0.166 g, 1.35 mmnol), potassium carbonate (0.511 g, 3.70 mmnol) 10 and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(l)-dichloromethane complex (0.041 g, 0.05 mmol). The reaction was heated at 90oaC for 16 h before being poured into water (50mL) and extracted with ethyl acetate (3 x 50mL). The combined extracts were dried over magnesium sulfate, filtered and evaporated to give a brown oil which was purified by chromatography on silica gel. Elution with 15 diobloromethane followed by 10% ethyl acetate/dichloromethane gave methyl 3 amino-5-hexamethyleneimino-6-phenyl-2-pyrazinecarboxylate as a yellow solid (0.309 g, 77%). Part 3. _ _ n 20 O 0 To a solution of sodium (0.090 g, 6.17 mmol) dissolved in methanol (8 mL) was added guanidine hydrochloride (0.598 g, 6.26 mmol) and the mixture was refluxed for 30 min after which it was filtered. To the filtrate was added methyl 3 amino-5-hexamethyleneimino-6-phonyl-2-pyrazinecarboxylate (0.310 g, 0.95 mmol) 25 in tetrahydrofuran (10 mL) and the solution refluxed for 72 it. The solvent was removed under reduced pressure and the residue chromatographed on silica gel.
    WO 2004/112687 PCT/AU2004/000866 -58 Blution with 5% methanol/dichloromethane gave N-amidino-3-amino-s5 hexamethyleneimino-6-phenyl-2-pyrazinecarboxamide as a yellow solid (0.116 g, 35%). 5 Example 5. Viral Studies Construction of recombinant plasmids containing open reading frames encoding various virus proteins. Complimentary DNA (eDNA) fragments for the various viral proteins listed in Table 2 were obtained either by PCR amplification from a parental virus genome 10 clone, or by direct chemical synthesis ofthe polynucleotide sequence. For example, the open reading frame encoding Vpu (Fig la) was amplified by PCR from a eDNA clone of an Nde I fragment of the HIV-1 genome (isolate HXB2, McFarlane Burnet Centre, Melbourne, Australia) as follows: Native Pfu DNA polymerase (Stratagene; 0.035 U/Ill) was chosen to catalyse the PCR reaction to minimise possible PFCR 15 introduced errors by virtue of the enzyme's proofreading activity. The 5', sense, primer AGTAGGATCCATGCAACCTATACC (<400 >2) introduces aBamH1 site (underlined) for cloning in-frame with the 3' end of the (ST gone in p2GEX (41). This primer also repairs the start codon (bold T replaces a Q of the vpu gone which is 20 a threonine codon in the HXB2 isolate. The 3', antisense, primer TCTGGAATTLTACAGATCAT CAAC (<400 > 3) introduces an EcoR site (underlined) to the other end of the PCR product to facilitate cloning. After 30 cycles of 94CC for 45 sea, 55CC for 1 min and 72C for 1 min in 0.5 ml thin-walled eppendorf tubes in a Parkin-Ehnlmer thermocycler, the 268bp fragment was purified, 25 digested with BamHI and EcoRI and ligated to p2GEX prepared by digestion with the same two enzymes. The resultant recombinant plasmid is illustrated in Fig lb. The entireVpu open reading frame and the BamHill and EcoR ligatiot sites were sequenced by cycle sequencing, using the Applied Biosystems dye-terminator kit, to confirm the DNA sequence. Other oDNAs were synthesised for us using state of the 30 art methods by GenScript Corporation (New Jersey, USA). Codon sequences were optimised for expression in bacterial, insect or mammalian cells, as appropriate. Restriction endonuclease enzyme recognition sites were incorportated at the 5' and 3' WO 2004/112687 PCT/AU2004/000866 -59 ends of the synthetic ODNAs to facilitate cloning into plasmid expression vectors, pcDNA3.1, pFastBac and pPL451 for expression of the encoded virus proteins in mammalian, insect or bacterial cells, respectively. 5 Standard techniques of molecular biology were used in cloning experiments. For example, to prepare the Vpu open reading frame for insertion into the pPL451 expression plasmid, p2GEXVpu was first digested with BamHI and the 5'base overhang was filled in the Klenow DNA polymerase in the presence ofdNTPs. The , Vpu-encoding fragment was then liberated by digestion with EcoR1, purified from an 10 agarose gel and ligated into pPL451 which had been digested with Hpal and EvoRl. Western blots subsequently confirmed that the pPLVpu construct (Fig Ic) expressed Vpu after induction of cultures at 421C to inactivate the cI857 repressor of the PR and PL promoters. Table 2 Source of viral cDNA or peptide sequences. Target Protein Source organism Strain or Sequence Accession number Vpu HIV-1 strain EXB2 SARS-CoV E protein SARS coronavirus P59637 HCVp7 Hepatitus C virus H77 la NP 751922 MUV-E protein Murine hepatitis virus NP 068673 229E E protein Human coronavirus 229E NP 073554 Dengue M protein Dengue virus type 1 Strain Singapore S275/90 Example 6. Purification of Recombinant Vpu from E. Coil Cultures of E. col strain XLI-blue cells containing p2GEXVpu were grown at 301C with vigorous aeration in LB medium supplemented with glucose (6g/L) and 20 ampicillin (50mg/L) to a density of approximately 250 Klett units, at which time IPTG was added to a final concentration of 0.01mM and growth was continued for a further 4hr. The final culture density was approximately 280 Klett units. Since early experiments revealed that the majority of expressed GST-Vpu fusion protein was associated with both the cell debris and 30 membrane fractions, the method of 25 Varadhachary and Maloney (Varadhachary and Maloney, 1990) was adopted to isolate osmotically disrupted cell ghosts (combining both cell debris and membrane fractions) for the initial purification steps. Cells were harvested, washed, weighed and WO 2004/112687 PCT/AU2004/000866 -60 resuspended to O1ml/g wet weight in MTPBS containing DTT (ImM) and MgC12 (10mM). Lysozyme (0.3 mg/ml; chicken egg white; Sigma) was added and incubated on ice for 30 min with gentle agitation followed by 5 min at 37 0 C. The osmotically sensitised cells were pelleted at 12,OOOg and resuspended to the original volume in 5 water to burst the cells. The suspension was then made up to ]xMTPBS/DTT using a 10x buffer stock and the ghosts were isolated by centrifugation and resuspended in MTPBS/DTT to which was then sequentially added glycerol (to 20 % wt/vol) and CHAPS (to 2 % wt/vol) to give a final volume of one quarter the original volume. This mixture was stirred on ice for 1 hr and then centrifuged at 400,000g for lhr to 10 remove insoluble material. The GST-Vpu fusion protein was purified from the detergent extract by affinity chromatography on a glutathione agarose resin (Sigma). The resin was thoroughly washed in 50mM Tris pH 7.5 containing glycerol (5 %), DTT (lmM), and CHAPS (0.5 %) (Buffer A) and then the Vpu portion of the fusion protein was liberated and cluted from the resin-bound GST by treatment of a 50% 15 (v/v) suspension of the beads with human thrombin (100Unlml; 37 0 C for lbr). PMSF (0.5mM) was added to the eluant to eliminate any remaining thrombin activity. This Vpu fraction was further purified on a column of MA7Q anion exchange resin attached to a BioRad HPLC and eluted with a linear NaC1 gradient (0-2M) in buffer A. 20 The Vpu was purified to homogeneity - as determined on silver stained gels - on an immunoaffinity column as follows: HPLC fractions containing Vpu were desalted on a NAP 25 column (Pharmacia) into buffer A and then mixed with the antibody agarose beads for 1hr at room temperature. The beads were washed thoroughlly and Vpu was eluted by increasing the salt concentration to 2M. Protein was quantitated 25 using the BioRad dye binding assay. Example 7. Expression and Pftrification of Vpiu in E.Coli. The plasmid p2GEXVpu (Fig. 1) was constructed to create an in-frame gene fusion between the GST and Vpu open-reading frames. This system enabled IPTG-inducible 30 expression of thc Vpu polypeptide fused to the C-terminus of GST and allowed purification of the fusion protein by affinity chromatography on glutathione agarose.
    WO 2004/112687 PCT/AU2004/000866 -61 Optimal levels of GST-Vpu expression were obtained by growing the cultures at 30 0 C to a cell density of approximately 250-300 Klett units and inducing with low levels of PTG (0.01mM). To purify the GST-Vpu, a combined cellular fraction containing the cell debris and plasma membrane was prepared by lysozyme treatment 5 of the induced cells followed by a low-speed centrifugation. Approximately 50% of the GST-Vpu protein could be solubilised fiom this fraction using the zwitterionic detergent CHAPS. Affinity chromatography using glutathione-agarose beads was used to enrich the fusion protein and thrombin was used to cleave the fusion protein at the high affinity thrombin site between the fusion partners, liberating Vpu (Fig. 10 2A). In fractions elated from the anion exchange column Vpu was the major protein visible on silver stained gels (Fig. 2B1, lane 1). Finally, Vpu was purified to apparent homogeneity on an immunoaffinity column (Fig. 2B, lane 2). The N-terminal amino acidsequence of the protein band (excised from SDS-PAGE gels) corresponding to the immunmodetected protein confirmed its identity as Vpu. 15 Example 8. Reconstitution of Vpu in Phospholipid Vesieles. Proteoliposomnes containing Vpu were prepared by the detergent dilution method (New, 1990). A mixture of lipids (PB:PC:PS; 5:3:2; 1ng total lipid) dissolved in cblorofomnn was dried under a. stream of nitrogen gas and resusp ended in 0.1 ml of 20 potassium phosphate buffer (50mM pH 7,4) containing DTT (ImM). A 25l aliquot containing purified Vpu was added, followed by octylglucoside to a final concentration of 1. 25 % (wt/vol). This mixture was subject to three rounds of freezing in liquid nitrogen, thawing and sonication in a bath type sonieator (20-30 see) and was then rapidly diluted into 200 volumes of the potassium phosphate buffer. 25 Proteoliposomes were collected by centrifugation at 400,000g for lfir and resuspended in approximately 150pl of phosphate buffer. Example 9. Assavinig VaU Ion Channel Activity Purified Vpu was tested for its ability to induce channel activity in planar lipid bilayers using standard techniques as described elsewhere (Miller, 1986; and Piller et 30 al, 1996). The solutions in the CIS and TRANS chambers were separated by a Delhin m plastic wall containing a small circular hole of approximately 100pm WO 2004/112687 PCT/AU2004/000866 -62 diameter across which a lipid bilayer was painted so as to form a high resistance electrical seal. Bilayers were painted from a mixture (8:2) of palmitoyl-oleoly phosphatidyl-ethanolamine and pahnitoyl-oleolyphosphatidyl-choline (Avanti Polar Lipids, Alabaster, Alabama) in n-decane. The solutions in the two chambers 5 contained MES buffer (10mM, pH 6.0) to which various NaCI or KC1 concentrations were added. Currents were recorded with an AxopatchTM 200 amplifier. The electrical potential between the two chambers could be manipulated between +/-200mV (TRANS relative to grounded CIS). Aliquots containing Vpu were added to the CJS chamber either as a detergent solution or after incorporation ofthe protein into 10 phospholipid vesicles. The chamber was stirred until curretnts were observed. Example 10. Vpu Forms Ion Channels in Lipid Bilavers. To assay for ion-channel formation by Vpu, reconstitution into planar lipid bilayers was performed. When samples (containing between 7 and 70ng of protein) of purified recombinant Vpu were added to the Iml of buffer in the CIS chamber of the 15 bilayer apparatus, current fluctuations were detected after periods of stirring that varied from 2 to 30 min (Fig. 3). This time taken to observe channel activity approximately correlated with the amount of protein added to the chamber. No channels were detected when control buffer aliquots or control lipid vesicles were added to the CIS chamber. In those control experiments the chambers could be stirred 20. for more than an hour without appearance of channel activity. Example 11. Properties of The Vu Chanels. Channel activity was observed in over 40 individual experiments with Vpu samples prepared from five independent purifications. In different experiments, the amplitude of the currents varied over a large range and, again, seemed to 25 approximately correlate with the amount of protein added. The smallest and largest channels measured had conductances of 14 pS and 280 pS, respectively. The channels were consistently smaller when lipid vesicles containing Vpu were prepared and fused to the bilayer rather than when purified protein in detergent solution was added. This may be because the former method included treatment with high concentrations 30 of detergent and a dilution step that may have favoured the breakdown of large aggregates into monomers.
    WO 2004/112687 PCT/AU2004/000866 -63 The relationship between current amplitude and voltage was linear and the reversal potential in solutions containing a ten-fold gradient of NaC1 (500mM CIS; 50mM TRANS) was +3OmV (Fig. 3B). A similar reversal potential was obtained when solutions contained KCI instead of NaCL. In 5 experiments with either NaCI or 5 KCI in the solutions on either side of the membrane, the average reversal potential was 31.0 +/-1.2mV (+/-SEM). This is more negative than expected for a channel selectively permeable for the cations alone. Using ion activities in the Goldman Hodgkin-Katz equation gives a PNa/Poj ratio of about 5.5 indicating that the channels are also permeable to chloride ions. An attempt was made to reduce the anion current 10 by substituting phosphate for chloride ions. When a Na-phosphate gradient (150mM Na & 100mM phosphate CIS; 15mM Na & 10mM phosphate TRANS, pH 6.8) was used instead of the NaCl gradient, the reversal potential was 37.1 +/- 0.2 (+/-SEM, n--2) again indicating a cation/anion permeability ratio of about 5. (For calculations involving the phosphate solutions, the summed activities of the mono and bivalent 15 anions were used and it was assumed that the two species were equally permeable). The current-voltage curve now exhibited rectification that was not seen in the NaCl solutions. It can be concluded that the channels formed by Vpu are equally permeably to Na and K+ and are also permeable, though to a lesser extent, to chloride as well as phosphate ions. 20 Example 12. Bacterial Bio-Assay for Screening Potential Ion Channel-Blockinu Drugs This bio-assay is based on the observation that expression of Vpu in E eoli results in an active Vpu channel located in the plasmalemma that dissipates the transmembrane sodium gradient. As a consequence of this Vpu channel activity, 25 metabolites whose accumulation within the cells is mediated by a sodium dependent co-transporter (for example proline or adenine) leak out of the cell faster than they canbe synthesised so that the metabolites' intracellular levels become limiting for growth of the cell. Thereby, an E. coli cell expressing Vpu is unable to grow in minimal drop-out media lacking adenine or proline. However, in the presence of a 30 drug that blocks the Vpu channel, the cell is once again able to re-establish its transmembrane sodium gradient - due to the action of other ion pumps in the membrane - and the leakage of metabolites is prevented enabling growth.
    WO 2004/112687 PCT/AU2004/000866 -64 Experiments to demonstrate that Vpu can form sodium channels in the plasma membrane of E. coil were performed as follows. To express unfused Vpu in E. coli, the vpu open-reading frame was cloned into the plasmid pPL451 to create the recombinant plasmid pPL-Vpu (Fig. Ib). In this 5 vector the strong PL and Pa lambda promoters are used to drive expression of Vpu under control of the temperature sensitive c1 857 represser, such that when grown at 30C expression is tightly repressed and can be induced by raising the temperature to between 370C and 42 0 C. On agar plates, cells containing pP.Vpu grew when incubated at 301C and 37CC but not at 42C, while control strains grew well at 42"C. 10 Liquid cultures of cells containing pPL-Vpu were grown at 300C to OD 6 0 o,=0.84 then moved to grow at 421C for two hours (the final cell density was OD6oo,=0.75). The plasma membrane fraction was prepared and western blotting, using an antibody that specifically binds to the C-terminus of Vpu, detected a single band at approximately 16kDa, indicating that Vpu was expressed and associated with the membranes (Fig. 15 ZA,lane 5). Example 13. Cross-Feeding Experiments Reveal That Proline Leaks Out of Cells Exuressing Vpu, Uptake of proline byE. coli is well characterised and active transport of the amino acid into the cells is known to use the sodium gradient as the energy source 20 (Yamato et al, 1994). To detect whether proline leakage occurs, the following cross feceing assay was used: A lawn of an E. coli strain auxotrophic for proline and methioninte (Mef Pro), was seeded and poured as a soft agar overlay on minimal drop-out media plates lacking proline but containing methionine. Sterile porous filter discs were inoculated with a Met Pro* strain (XL-1 blue) containing either the 25 pPL451 control plasmid or pPL-Vpu and placed onto the soft agar. The plates were then incubated at 37C or 30C for two days. After than time a halo growth of the Met" Pro" strain was clearly visible surrounding the disc inoculated with the cells containing pPL-Vpu incubated at 37 0 C (Fig. 4A). This growth can only be due to the leakage of proline from the Vpu-expressing cells on the disc. No such leakage was 30 apparent from the control strain at 370C nor around either stain on plates grown at 300C (Fig. 4B).
    WO 2004/112687 PCT/AU2004/000866 -65 In contrast to proline transport, the E. coli methionine permease is known to belong to the ABC transporter family (Rosen, 1987) and hence be energised by ATP. Identical erossfeeding experiments to those described above were set us except that the Met Pro strain was spread on minimal drop-out plates lacking methionine but 5 containing proline. N o growth of this strain was evident around any of the discs (Fig. 4C), indicating that methionine was not leaking out of the XL-1 blue cells even when Vpu was being expressed. Example 14. E.Coli Cells Expressing Vpm Require Adenine in the External Medium for Growth. 10 It was observed that, due to an uncharacterised mutation in the adenine synthesis pathway, growth ofE. colt cells of the XLI-blue strain expressing Vpu at 370C was dependant on the presence of admine in the medium. This allowed the development of an even simpler bioassay for Vpu ion-channel activity than the proline cross-feeding assay described above: A lawn of XL1-blue cells containing the 15 pPL-Vpu plasmid is seeded onto an agarose plate lacking adenine in the medium, small aliquots of drugs to be tested for inhibition of the Vpu channel are spotted onto the agarose in discrete locations and the plates are incubated at 37CC for a suitable period of time (12-36 hours). Halos of growth around a particular drug application site indicate that the drug has inhibited expression of the Vpu ion channel activity that 20 prevents growth in the absence of the drug. (Figure 5). Example 15 Assay of Compounds in Planar Lipid Bilavers for Vpa Channel Blockine. Actvity 25 Comrnopunds were characterized for their ability to block Vpu ion channel activity reconstituted into planar lipid bilayers. Vpu N-terminal peptide (residues 1 32) dissolved in trifluoroethanol was added to the CIS chamber of the bilayer apparatus and the solutions was stirred until ion currents were observed, indicating incorporation of one or more Vpu ion channels into the bilayer. After recording the 30 channel activity for a few minutes, drugs were added to the solutions in the CIS and TRANS chambers - with stirring - to a final concentration of 100pM. Channel activity was then recorded for at least a further three minutes and the effect of drug addition on ion current was determined by comparing the channel activity before and WO 2004/112687 PCT/AU2004/000866 -66 after drug addition. For each experiment, drug effect was classified into four categories: "Stong block", if current was inhibited approximately 9 0-100%; "weak block", approx. 50-90% inhibition; "partial block", <50%; and "no effect". Experiments were disregarded if currents larger than :SOpA were generated after 5 addition of Vpu N-peptide because in such cases it is possible that non-native peptide aggregates contribute to bilayer breakdown. Such aggregates, by virtue of their disorganized structure may not be specifically blocked by the drugs at the concentrations tested. 10 Table 3 summarises the results of the bilayer experiments. A novel outcome of these experiments was the strong blocking of Vpu channels observed with Phenamil. Phenamil has a phenyl group derivative at the guanidine group of amiloride. Amiloride itself is not a blocker of Vpu, whereas addition of the hexamethylene group at the 5- position of the pyrazine ring created a structure (HMA) that blocks the 15 channel at concentrations as low as 25gM. These now results with Phenamil, however, now show that a bulky hydrophobic derivative at the opposite end of the molecule can also turn amiloride into an effective Vpu channel blocker. Interestingly, benzamil, with a very similar structure was much less effective at blocking the Vpu channel. 20 Table 3: Summary of Compounds Inhibiting the Vpu Ion Channel in Bilayers Compound No. of Results Expts. Phenamil 3 3x Strong block MIA 2 lx Strong block; lx weak Benzamil 10 3x partial block; 7x no effect EIPA 3 3x weak block; HMA 1 lx Strong block; (5-Phenyl-penta-2,4-dienoyl)guanidine 6 6x strong block 6-methoxy-2-naphthoylguanidine 5 5x strong block (2-Chlorocinnamoyl)guanidine 6 4x strong; 2x partial blocks 3-(trifluoromethyl)cinnamoylguanidine 5 4x strong blocks; lx no effect WO 2004/112687 PCT/AU2004/000866 -67 N-{5-[3-(5-Guanidino-pentyloxymethyl). benzyloxy]-pentyl}-guanidine 4 3x strong block; lx no effect 4-phenylbenzoylguanidine 3 3x strong block 3-methyloinnamoylguanidine 4 2x strong book; 2x partial (3-Chlorooinnamoy)guanidine 4 2x strong block; 2x partial N-(3-phenylpropanoyl)-N' phenylguanidine 1 lx strong blocks (3-Bromocinnamoyl)guanidine 3 3x partial-strong block 5-tert-butylamino-amiloride 3 3x partial block N-amidino-3-anmino-5-phenyl-6-chloro-2 pyrazinccarboxamide 3 3x partial block 3-methoxy -HMA 3 3x partial block 5-(N-Methyl-N-isobutyl)amiloride 1 lx partial block 5-(N-Ethyl-N-isopropyl)amiloride 1 lx partial block 2-napthoylguanidine 7 7x weak block N,N'-bis(3phenylpropanoyl)-N" phenylguanidine 7 7x weak block cinnamoylguanidine 3 3x weak block (5-Phenyl-penta-2,4-dienoyl).uanidine 6 6x strong block Example 16 Compound Screening using the Bacterial Bio-Assay for the Vpu protein. 5 The halos of growth around the site of application of particular drugs- as described in example 14- were given a score between zero and six reflecting the size and density ofthe zone of bacterial cell growth. Scores greater than 3 represent strong inhibition of the Vpu protein; scores between 1.5 and 3 represent moderate inhibition and scores between 0.01 and 1.5 represent fair inhibition. 10 Table 4 lists the scores for inhibition of Vpu protein in the bacterial bio-assay. Table 4 Vpu Inhibition (score / # of times Compound tested) 3-Chlorocinnamoyl)guanidine 4.38/4 3-Bromocinmamoyl)guanidine 4.3/24 2-Chlorocinnamnoyl)guanidine 4.0/4 2-Bromooinnamoyl)guanidine 3.7/2 -(trifluoromethyl)oinnamoylguanidine 3.7/2 WO 2004/112687 PCTAU20011000866 5-bromo-2-fluorocinnanioylguanidine 3.5/2 3-metlylwinemoylgumldine 3.4/2 2-methyloinnamoylguanktine 3.1/2 2,3-dimetlylcinnamoylgauidine 3.1/2 inniamoylguanidine 2. 96/12 6-meffioxy-2-naphflioylguanidine 2.9/4 ta-3-(1-napthyl)acryloylguanidine 2.9/3 A,-dichlorocinnamoylguanidine 2.9/3 2,6-dichlorociinamoylguanidine, 2.88/2 -phenylbenzoylguanidine 2.7S/5 -ethylcinnamoylguanidine 2.73/2 (4-Cblorocinnamoyl)guanidine 2.7/5 -naptoylgwmdine 2.7/111 ,5-dimethylcinnanioylguanidine 2.69/2 1-isopropylcinnamnoylguanidiuc hydrochloride 2.6/2 5-Phenyl-penta-2,4-dienayl)guanidine .2.5 6/2 .- plenylcinoylguani dine 2.54/3 (4-Bromocimnmoyl)gilidine 2.5/4 5-(3 t -bromophenyflpcnta-2,4-dicnoylguanidine 2.5/2 3-(ayclohex-1 -en-1-yl)cinnamoylganidine 2.5/2 3-(Irifluoromethoxy)cinaoylguanidine 2A44/2 2-(tifluoromethyl)oinoanioylguandine - 2.4/2 -ethoxycixmamoylguanidine 2.25/2 -(3-phenylpropanoyl)-N'-phenylguanidine 2.21/3 (trifluoromethyl)cinnamoylguanidine 2.2/2
    -
    Met(ho in aop 1an idi-"-n el a id 2.213 3 -butyleinnatoylguanidine 2.13/2 M etylcnana oylguandine 2.1/2 -fluorocinunoylguanidine 2.1/2 -phenylcinnamoylguanidine 2.1/2 N-(6-Hydroxy-2-napthoyl)-N-phenylguanidin 2.062 3-t-butyleinnamoylgaanidine 2.06/2 3,4-'difluorooinnaxoylguanidine 2.06/2 5-UtN-hexamethylene)amiloride 1.9/31 -fluorooinnainoylguanidine 1.9/2 5-bromo-2-methoxycinnamoylguanidine 1.9/2 3-etboxyeinnmamoyguexdine 1.9/2 A,-(methylenedioxy)cinnamoylguanidine 1.88/2 (2-Me-thoxyoinnamoyl)gumnidine 1.7/4 T4 DichioroBeuzamil ECI 1.7/2 2,3,546-etramethylinamoylguanidine 1.6/2 9-(2-napthyl)acryloylguanidine 1.56/2 2-(1-napthyl)acetoylguanidina 1.5 6/2 ,3-difluorooimiaoylguanidine 1.5 6/2 (3-Methoxycinnamoyl)guanidine 1.52/6 4-isopropylewnnmanoylguenidine 1.4/2 ,4,6-lrimethylcinnarnoylguanidine 14/ WO 2004/112687 PCT/AU2004/000866 -69 N-(cinnamoyl)-Nphenylguidine 1.25/3 2-(cyclohex-1-en-lyl)cinnamoylguanidine 1.2/2 2.(2-napthyl)acetoylguanidine 1.19/2 (4-Hydxroxycinnamoyl)guanidine 1.1/2 4-phenyloinnamoy1guanidine 1.1/2 4-fluorocinnamoylguanidine 1.1/2 ,N'-bis-(cinnamoyl)-N"
    '
    -pheny lgu an idine 0.94/2 (2-Furanacryloyl)gnanidine 0.94/2 PhOnamil mothanesulfonate salt 0.9/5 Benzamil hydrochloride 0.9/3 (3-Nitrocinnamoyl)guanidine 0.9/1 Benzyoylguanidine 0.88/2 (4-Phenoxybenzoyl)guanidinfe 0.81/2 3-(trans-hept-1-en-1-yl)cinnamoylguanidine 0.81/2 5-(N.Mothyl-N-isobutyl)amiloride 0.8/2 2-oyclohexylcinnamoylguanidine 0.8/2 4-ethoxycinnamoylguanidinc 0.69/2 S'4dichlorocinnamolygunidine 0.63/2 5-(N-Ethyl-N-isopropyl)antiloide 0.6/3 N-amidino-3-amino-5-hexamethyleneimino-6-phenyl Z-pyrazinearboxamide 0.6/2 a-Methylcinnamoyl)guanidine - 0.6/2 inmamoylguanidinemo hydrochloride 0.612 [(4-Chlorophenoxy-acotyllguanidine 0.56/2 N-amidino-3-anino-5-phenyl-6-chloro- 2 pyrazinecarboxamide 0.5/11 5-(4-fluorophenyl)amiloride 0.4/6 (trans-2-Phny1cyclopropanecarbony)guanidine 0.4/2 (2-Nitrocinnamoyl)guanidine 0.4/2 trmns-3-Furanacryoylguanidine 0.38/2 1-napthoylguanidine 0.3/2 5-tert-butylamino-amiloride 0.2/7 3-methoxy -HMA 0.2/4 (3-phenylpropanoyl)guanidine 0.2/4 4-t-butyleinnamoylguanidine 0.19/2 5-(N,N-Dimethyl)smiloride hydrochloride 0.1/2 N,N'-Bis(3-phnylpropanoyl)guanidino 01/2 N-Benzoyl-N'-cinnamoylguanidine 0.06/2 1-bromo-2-napthoylguanidine 0.06/2 Examnle 17. Effect of Compounds on HIV Replication in Human Monocvtes and Macrophages. Human monocytes were isolated from peripheral blood and cultured either for 5 24hr (one day old monocytes) or for 7 days to allow differentiation into monocyte derived maophages (1DM). These cells were then exposed to cell-free preparations WO 2004/112687 PCT/AU2004/000866 -70 of HWV isolates and allowed to absorb for 2hr before complete aspiration of the medium, washing once with virs-.free medium and resuspension in fresh medium. The cells were exposed to various concentration of compound either 24 hr prior to infection or after infection. Subsequent HIV replication, at various times after 5 infection, was compared in cells exposed to drugs and in cells not exposed to drugs (controls). The progression and extent of viral replication was assayed using either an HIV DNA PCR method (Fear et al, 1998) or an ELISA method to quantitate p24 in culture supernatants (Kelly et al, 1998). Table 5 provides examples of results obtained using this assay and test 10 antiviral compounds. Table 5 Drug Pertent of Cone. Positive Compound PM Control None - positive control 100% 4-phenylbenzoylguanidine 10 26 5 4. 2.5 9 1. 216 ______cntol0.625 10 None - positive control 100% (3-Bromocinnamoyl)guanidine 10 3 5 1 2.5 9 1.25 59 0.625 116 None - positive control 100% 3-(trifluero- 10 11 methyl)oinnamoylguanidine 5 8 2.5 25 1.25 27 0.625 38 None - positive control 100% 5-(N,N- 10 6 hexamethylene)amiloride 5 21 2.5 50 1.25 19 S 0.625 30 WO 2004/112687 PCT/AU2004/000866 -71 Example 18. SARS Coronavirus. SARS E protein forms an ion channel Peptide Synthesis A peptide corresponding to the full-length SARS-CoV (isolate Tor2 and 5 Urbani) E protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCA YCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV) and a second peptide comprising the first 40 amino acids of the full length E protein which correspond to the transmnembrane domain (MYSFVSEETGTLIVNSVLLFLAFVVF LLVTLAILTALRLC) were synthesized manually using FMOC chemistryand solid 10 phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Cuttin School of Medical Research, ANU, Australia) using a Symphony Peptide Synthesiser from Protein Technologies Inc.(Tucson, AZ, USA) according to the manufacturers instmeructions. Example 19. Peptide purification 15 Mass spectral analysis of the synthetic peptide revealed that the preparation contained significant amounts of material with lower miz ratio than expected for the Sfull-length product. The majority of these are presumably truncmeated peptides generated during the peptide synthesis process. To enrich the full-length E protein, the following procedure was used, which relies on differential solubility of the 20 smaller molecules and full-length peptide. The crude preparation was suspended at 12 mg/ml in 70% CH 3 CN, 0.1%TFA and vortexed for 10 minutes. This suspension was centrifuged at 10,000g for 10 minutes at 2000C. The supernatat was discarded and the insoluble fractions was extracted with 70% CH 3 CN, 0.1% TFA, as above, two more times. The insoluble material containing the E protein was dried using 25 Speedvac an the weight of the final product was used to calculate the yield. The purified peptide was analysed by Bruker Omniflex MALDI-TOF mass spectrometry in HABA matrix at 2.5mg/ml in methanol at a 1:1 ratio and spectra were obtained in the positive linear mode. A clear peat at m/z ratio of 8,360.1 was seen as expected for the calculated molecular weight of full-length B protein and 4422.3 for the N-temnrminal 30 E protein.
    WO 2004/112687 PCT/AU2004/000866 -72 Example 20. Planar Lipid Bflavers The SARS virus B protein was resuspended at 1mg/ml in 2,2,2-trifluoroethanol. The SARS virus E protein's ability to form ion channels was tested on a Warner (Warner instruments, Inc. 1125 Dixwell Avenue, Iamden, CT 06514) bilayer rig as follows; A S lipid mix of 3:1:1, 1-Palmitoyl-2-oleolyl phosphatidyl Ethanolamine: 1-Palmitoyl-2 oleolyl phosphatidyl Serine: 1-Palmitoyl-2-oleolyl phosphatidyl choline in CHC13 was dried under N 2 gas and resuispended to 50mg/ml in n-decane. Bilayers were painted across a circular hole of approximately 100ptmO diameter in a Delin T cup separating aqueous solution in the CIS and TRANS chambers. The CIS chamber 10 contained a solution of 500mM NaC1 or KCI, in a 5mM HEPES buffer pH 7.2, the TRANS chamber contained a solution of 50mM NaCl or KC1, in a 5mM HEPES buffer pH 7.2. Silver electrodes coated in chloride with 2% agarose bridges are placed in the CIS and TRANS chamber solutions. The SARS E protein full-length or N tenninal peptides (3 -10ug) were added to the CIS chamber, which was stirred until 15 channel activity was detected. The CIS chamber was earthed and the TRANS chamber was held at various holding potentials ranging between +100 to -100mV. Currents were recorded using a Warner model BD-525D amplifier, filtered at 1kHz, sampling at 5 kHz and digitally recorded on the hard disk of a PC using software developed in house. 20 Drugs to be tested for their ability to inhibit SARS E protein ion channel activity were made up at 50mM in a solution of 50% DMSO: 50% methanol, For experiments testing the ability of compounds to inhibit E protein ion channel activity, 100 pM to 400 pM of compound was added to the CIS chamber while stirring for 30 seconds. Bilayer currents were recorded before channel activity, during channel 25 activity and after the addition of the drug, Among the compounds tested was oinnamoylguanidine (Bit036), a compound which was shown in earlier experiments to be antiviral and to inhibit ion channel proteins from other viruses. Example 20.1. Polvervlamide gel electrophoresis 30 Purified E protein was dissolved to 1 mg/ml, 5 mg/ml and 10 mg/ml in, 6 M Urea, 10% Glycerol, 5% SDS, 500 mM DTT, 0.002% Bromophenol Blue, 62.5 mM Tris HC1 (pH 8.3). Peptides in solutions were heated at 10000 for 20 minutes before WO 2004/112687 PCT/AU2004/000866 -73 30 pL samples were run on stacking gel 4-20% (Gradipore). SeeBlue- pre-stained standard (Invitrogen) was used for molecular weight markers. Example 20.2 Results To test ifthe SARS E protein forms ion channels the purified synthetic 5 peptide was reconstituted into planar lipid bilayers (21). Typically, 3pg of SARS fi-length B protein was added to the CIS chamber, while stirring. This CIS chamber contained 500 mMNaC1 and the TRANS chamber contained 50 mM NaC1. In 60 experiments, ion currents due to SARS E protein ion channel activity were observed after about 5 -15 minutes of stirring. Activity was detected more rapidly and reliably 10 with a holding potential of approximately-100mV across the bilayer. Currents recorded at -100mV, (A) and at -60mV (B) in one of these experiments are shown in Figure 6. In that experiment the reversal potential was about +48mV and the channel conductances were calculated to be 5 2 pS and 2 6 pS, respectively. This indicates that the current-voltage (IV) relationship is not linear. In ten other experiments, where no 15 protein was added to the CIS chamber, no ion channel activity was detected, even after recording for over 1 hour. Figure 7a shows typical current traces recorded over a range of potentials in NaC1 solutions. In that experiment the direction of current flow reversed at +48mV (Fig 7b). The lV curve shows that at the lower voltages the average current flow across the 20 bilayer is small but at higher potentials there is an increase in average current across the bilayer, resulting in a non-linear IV relationship, In seven independent experiments, the average reversal potential was +48.3 :t 2.3 mV (mean t ISEM), indicating that the channels were about 37 times more permeable to Na+ ion than to CT ions. The reversal potential is close to the Na+ equilibrium potential (+53mV), 25 therefore the channel is selective for Na+ ions. For these 7 experiments the channel conductance varied between 95-164 pS; the average conductance was 130 ± 13 pS. SARS E protein ion channel is slightly less selectivity for K ions than Na t ions. Figure 8b shows recording of currents in KC1 solutions at a range of potentials. In this experiment the currents reversed at +31 mV. In seven similar experiments E 30 protein ion channel average reversal potential was +34.5 ± 2.5 mV. Therefore the SARS E protein ion channel is about 7.2 times more permeable to K ions than CT WO 2004/112687 PCT/AU2004/000866 -74 ions. In seven experiments, the channel conductance varied ranging between 24-166 pS, the average conductance was 83.4 ± 26 pS. Similar results were obtained with a second synthetic peptide, which corresponded to the first forty N-terminal amino acids of the SARS E protein "N-terminal peptide" 5 (21). The average reversal potential in NaCI solution in four experiments was +46.3 S2.5 mV, indicating that the ion channel formed by N-terminal peptide is about 25 times more permeable to Na+ ion than to Cl- ions. The SARS E protein N-terminal peptide was sufficient for the formation of ion channels with properties like those of the fall length SARS E protein. Therefore, the selectivity filter for the SARS E 10 protein is most likely contained within the first forty amino acids of the N-terminal. SARS E protein N-terminal peptide also formed ion channels in KCI solution that were similarly selective for K+- ions compared to the full-length E protein. In five independent experiments the average channel reversal potential was +39.5 ± 3.6 mV, therefore the channel is about 11 times more permeable to K ions than CF ions. 15 SDS-PAGE of the purified fuill-length E protein peptide showed bands corresponding to the full-length E protein (Data not shown). Larger bands of varying size up to about 20 kDa wore detected, suggesting that SARS E protein may form homo oligomers. 20 Example 21. SARS E protein ion channel is blocked by einnamovleuanidine and other compounds E protein ion channel activity in NaC1 solutions was significantly reduced (p 0.01, n=6 experiments) by addition of 100 to 200 pM cinnamoylguanidine to the CIS chamber. The average current across the bilayer was reduced to baseline by 25 100pM cinnanoylguanidine. In experiments when E protein ion channels had higher conductance, 100 to 200 pM cinnamoulguanidine reduced the average current across the bilayer about 4 fold: Similarly, in four other experiments, 100 to 200 pM cinnamoylguanidine blocked channels formed by full-length E protein in KC1 solutions. In two additional experiments, the SARS E protein N-terminal peptide was 30 blocked by 100 to 200 pM cirmnnamoylguanidine, demonstrating that the cinnamoylguanidine drug-binding site is located within the first forty amino acids of WO 2004/112687 PCT/AU2004/000866 -75 the B protein N-terminal domain. Other compounds tested in bilayers for their effect on the SARS E protein are shown it below in Table 6. Table 6 5 % Reducton of avei Compound current by 00uk 5-(N,N-hexamethyloee)amilotide 91 ± 7 6-methoxy-2-naphthoylguanidine 92 ± 16 2'4 DichloroBenzamil HCI 78 & 0 N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine 88 : 6 (3-Bromocinnamoyl)guanidine 87 ± 11 (2-Bromocinmamoyl)guanidine 88 ± 6 trans-3-(1-napthyl)acryloylganidine 66 2 Example 21.1 Results and Discussion. We have shown that SAKS B protein can form ion channels in lipid bilayer 10 membranes. The ion currents reversed at positive potentials, which demonstrates that E protein ion channels are selective for monovalent cations over monovalent anions. E protein Ion channels were about 37 times more selective for Na+ ions over Cl-ions and about 7.2 times more selective for K+ ions over Cl- ions. In over 60 experiments the Na+ conductance of the E protein ion channel varied from as low as 26 pS to as 15 high as 164 pS. SDS-PAGE showed that the E protein forms homo-oligomers, and we surmised that the larger conductances were probably due to aggregation of the E protein peptide leading to larger ion channels or the synchronous opening of many ion channels. Single channel currents were observed in several experiments and from these the channel conductance was calculated to be voltage dependent. 20 The first 40 amino acids of the N-terminal which contains the hydrophobic domain of the SARS virus B protein is sufficient for the formation of ion channels on planar lipid bilayers. The N-terminal E protein ion channel has the same selectivity and conductance as the full-length E protein ion channemol, The SARS virus full length E protein ion channel activity and N-terminal 25 domain B protein ion channel activity on planar lipid bilayers in NaCL and KCl solutions was inhibited by addition of between 100pM to 200pM cirmnnamoylguanidine to the CIS chamber. Inhibition or partialinhibition of the E WO 2004/112687 PCT/AU2004/000866 -76 protein ion channel activity by cinnamoylguanidine has been observed in seven independent experiments in NaCI solution and four independent experiments in KCI solution. All known coronaviruses encode an E protein with a hydrophobic N-terminus 5 transmembrane domain therefore all coronaviruses E proteins could form ion channels on planar lipid bilayers. This indicates that the E protein could be a suitable target for antiviral drugs and potentially stop the spread of coronavirus from infected host cells. Drugs that block the E protein ion channel could be effective antiviral therapy for the treatment of several significant human and veterinary coronavims 10 diseases including SARS and the common cold. Example 22. Bacterial Bi.o-Assay for Screening Potential SARS-CoV E protein Ion Channel-Blocking Drugs. 15 SARS-CoV E protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of SARS-CoV E protein function in bacterial cells was developed. A synthetic eDNA fragment encoding SARS-CoV B protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli 20 cells expressing E protein at 37C was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 23. Compound Sereening using the Bacterial Blo-Assay for SARS coronavirns E protein. 25 The halos of growth around the site of application of particular drugs- as described in example 14 -were scored as decribed in example 15. Table 7 lists the scores for inhibition of SARS-CoV B protein in the bacterial bio 30 assay. Table 7 SARS E protein Inhibition (score / # of times Compound tested) WO 2004/112687 PCT/AU20041000866 -77 ,3-difluorocinaoYlguldine 4,5011 3,44dclilorocinnamoylguanidine 4.15/2 4t-utylcinnaxnoylguanidine 4.00/1 3-(2-napthyl)acryloylguaaidino 3.88/1 '3-Chlorocinnanoyl)guanidie 3.87/3 -(eyclohe'X-1-Qn-lr-yl)cinnMOyguanidine 3.75/1 ~5-dimethy~oim~oylguanidine 3,63/11 ans-3 -(I1-napthyl)acryloylguaidine 3.38 /2 -isopropyleinnamoylguanidine 3.16/2 3-Bromocinnamoyl)gaanf dine 3.15/27 -mcthoxy-2-naphthoyguaidixie 3.13/13 -(N-MethylkN-isohutyl)amiloride 3.13/2 3-phenylminamoylguandine 3.13/1 (2-Chloxocinnamoyl)guanidine 3.1/3 '4 DichloroBenzamil HCl 3.00 /2 4phenylcinnanioylguanidine' 2.75 /2 4(trifluoromeothyl)cinnm~oylguanidine 2.75 /1 3-(frifluoromethoxy)cinnamoylguanidi-ne 2.71/1 3-(triluoromethyl)oinnamoylguanidine 2.67/1 -ethoxycfrmamoylguanidine 2,57/1 innamoylguanidine hydrochloride 2.50/1 *ethoxyeinnamoylguanidine 2A81/2
    (
    2 -Bromocinnainoylguanidine 2.47/3 ,6-dichlorocinnamoylguanidine 22/ ,465-trimellioxytinamoylguanidine 2.25/11 5-text-butyiwnino-amjloride 2. 01 /2 -t-buty1einaioylguaidine 2.00/11 5-brorno-2-fluoroelnaoylgu-nidne 2.00 / 1 (4-Chlorocinnamoyl)gaanidine 1.9412 2-1-butylcinnamoylguenidine 1.86/11 -cyclohoxyloinnmoylguanidine 1.83/1 -(tranm-kept-i-en- 1-yl)cirmiinoylguarjdjne 1.71/ 1 4-B3romocinnmoyl)guanidine 1.6912 (4-HIydroxycfimamnyl)guanidine 1.63/2 -(3-pbienylpropsnoyl)-N'-phenylguanidine 1.57/2 (3-Nitroeinnamoyl)guanidiue 1.51/ 2 3-fluorocinnamoylguanidine 1.50/11 2-C-napthyl~acetaylguanidine 1.50/1 2-ethyleinnamoylguanaidine 1.50/11 '5-(N,N-D~imethyl)amiloride hydrochloride 1.38/2 2-napthoylguanidino 1.38/2 5-(4-fluorphenyl~wniloride 1.38/1 2-(trifluoromethyl)oinnamoylguanidine 1.38/1 N-(6-Hydroxy-2-napthoyl)-N'-phenylgwanidine 1.35/3 (trans-2-Phenyloyclopropanearbonyl)gaanidine 1.34/3 N'-bis(3phenylpropanoyl)-N"-phenylguandine .1.33/3 1-napthoylguanidine 1.32/3 WO 2004/112687 PCT/AU2004/000866 -78 Benzamil hydrochloride 1.32/2 3-methoxy -HIMA 1.25/1 4-methyloinnamoyguanidine 1.25 / 1 4-fluorocinnamoylguanidine 1.25 / 1 3,4-(methylenedioxy)cinnamoylguanidine 1.25 / 1 5-(N,N-hexamethylene)amiloride 1.2 / 3 N-(cinnamoyl)-N'phenylguanidine 1.19/2 5-(N-Ethyl-N-isopropyl)amiloride 1.07/2 3-methylcinnamoylguanidine 1.00 / 1 2-methyleinnamayguanidine 1.00 1 2,3,5,6,-tetramethyleinnamoylguanidine 1.00 / 1 trans-3-Furanaeryoylguanidine 0.88/2 (4-Metboxycinnamoyl)guanidine 0.88 / 2 (2-Furanacryloyl)guanidine 0.8212 (3-phenylpropanoyl)guanidine 0.73/5 2-(2-napthyl)acetoylguanidine 0.71 / 1 iramoylguanidine 0.69/3 (2-Methoxycinnamoyl)guanidine 0.69 / 2 [3-(3-Pyridyl)acryloyl]guanidine 0.67 /3 4-phenylbenzoylguanidine 0.63/2 2,4-dichlorocinnamolyguanidine 0.63 / 2 (3-Methoxycinamoyl)guanidine .0.63 / 2 ,-fluorocinnamoylguanidine 0.63 / 1 (4-Phenoxybenzoyl)guanidine 0.57/2 (a-Mthylcinnamoyl)guanidine 0.50 / 1 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 0.5 / 1 (5-Phenyl-p enta-2,4-dienoyl)guanidine 0.44/2 Quinoline-2-carbonyl)guanidine 0.41/ 1 (enylacetyl)guanidine 0.32 / 3 N,N'-Bis(amidino)napthalene-2,6-dicarboxamide 0.25/2 6-bromo-2-napthoylguanidine 0.25 /1 1t-bromo-2-napthoylguanidine 0.25 / 1 2-ohloro-6-fluorocimnamoylguanidino 0,25 / 1 [(4-Chlorophenoxy-acetyl]guanidine 0.19/2 Phenamilmethanesulfonate salt 0.13 / 2 N-Benzoyl-N'-ci-namaylguanidine 0.13 / 2 N-(2-napthoyl)-N'-phenylguanidipe 0.07/2 Example 24. SARS Antiviral Assay for testing compounds against replication of SARS coronavirus (SARS-CoV). 5 Compounds were tested against SARS-CoV (Hong Kong strain) using virus plaque purified three times in Vero cells. Stock virus was generated by infecting Vero cells at MOI = lx TCIDso 50 per 100 cells. Example 24.1 Screening for anti-viral activity using the virus mierotitre assay WO 2004/112687 PCT/AU2004/000866 -79 Monolayers of Vero cells grown in 25cm 2 flasks were infected at a multiplicity of 1:50 and treated immediately post infection with compounds at two concentrations, 10OuM and 2uM. A control infected monolayer remained untreated. Samples of culture media were taken at 48 hours post infection. Two aliquots from 5 each of the samples (titrations 1 and 2) were serially log diluted and 12 replicates of log dilutions -4 to -7 added to cells in microtitre plates. Four days later, wells in the microtitre plates were scored for cytopathic effect (CPE) and the titration values calculated based on the number of CPE positive wells at the 4 dilutions. Control titres were 4.8 and 5.9 TCIDso 0 x 106 (average 5.35 x 10') 10 Example 25: Effect of compounds in SAIRS CoV antiviral assay: Three selected compounds were tested for activity against SARS-CoV according to the method described in example 21. For trans-3-(1 15 napthyl)acryloylguanidine and cinnamoylguanidine a d crease in virus titre of approximately 80% was observed at a concentration of 10uM and a reduction of approximately 50% was seen to persist at 2pM trans-3-(1-napthyl)acryloylgpanidine. Table 8 provides Virus titration data presented as % of a control (SARS CoV grown 20 for 48 hours in the absence of compounds). Table 8 Compound Average Titre Concentration TCID 5 0 (x (% Name (uM) 101) control) cinnamoylguanidine 10 1.3 24 2 4.4 82 trans-3-(1- 10 1.15 22 napthyl)acryloylguanidine .2 2.45 46 6-methoxy-2-naphthoylguanidine 10 5,95 111 2 635 118 Control 0 5.35 100 25 WO 2004/112687 PCT/AU2004/000866 -80 Example 26. Human 229E Coronavirus SynVthesis and Purification ofa Peptide Corresponding to the 229E-E Protein A peptide corresponding to the full-length 229E-E protein (sequence: 5 MFLKLVDDHALVVNVLLWCVVLIVLLVCITIKLIKLCFTCHMFCNRTVYGPI KNVYT1YQSYMHIDPFPKRVIDF; accession number NP_073554) was synthesized manually using FMOC chemistry and solid phase peptide synthesis. The synthesis was done at the Biomoleoular Resource Facility (John Curtin School of Medical Research, ANU Australia) using a SymphonyR Peptide Synthesiser from Protein 10 Technologies Inuo.(Wobunm, MS, USA) according to the manufacturers instructions to give C-terminal amides, the coupling was done with HBTU and hydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesis cycles used double coupling and a 4-fold excess of the amino acids. Temporary a-N Fmac protecting groups were removed using 20% piperidine in DMF. 15 The crude synthetic peptide was purified using he ProteoPlus m kit (Qbiogene inc. CA), folbilowing manufactures instructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-HC1 pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ± 100 mM 1-mercaptoethanol) and run on 4-20% gradient polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis 20 buffer (25 tmM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the full-longth peptide were excised out of the gel. The gel slice was transferred to the ProteoPLUSTM tube and filled with tris-glycine 25 electrophoresis buffer. The tubes were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for approximately 1 hour. The polarity of the electric current was reversed for 1 minute to increase the amount of protein recovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 minute. The purified peptides were dried in a Speedvao and the weight of the final product was used to 30 calculate the yield.
    WO 2004/112687 PCT/AU2004/000866 -81 Example 27. 229E-E protein forms Ion channels in planar lipid bilavers. Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine 5 (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 gm in the wall of a I ml derin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 10 500 mMNaClor 500mMKCl andthe trans 50 mMNaCl or 5OmMKCl. Thebilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and IS, stored on magnetic disk. The 229E E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TFE) at 0.05mg/ml to 1 mg/ml. 10 gl of this was added to the ois chamber (lml aqueous volume) of the bilTyer apparatus, which was stirred via a magnetic "flea". Ionic currents, indicating channel activity in the bilayer, were typically detected within 15 20 30 min. After channels were detected the holding potential across the bilayer was varied between -100mV and +100mV to characterise the size and polarity of current flow and enable the reversal potential to be determined. In 15 experiments where the cis chamber contained 500mM NaCI solution and the trans chamber contained 50 mM NaCI solution, the average reversal potential 25 of the channel activity was calculated to be 22 ±7 (SEM) mnV. In 13 experiments where the cis chamber contained 500mM KC1 solution and the trans chamber contained 50 mM KCl solution, the average reversal potential of the channel activity was calculated to be 38 ±4 (SEM) mV. These results indicate that the 229B3 protein forms cation selective ion channels that are slightly more selective for K than for 30 Na ions.
    WO 2004/112687 PCT/AU2004/000866 -82 Figure 9 shows examples of raw current data for the 229E E ion channel at various holding potentials (cis relative to trans) in asymmetrical KC1 solutions (500/50 mM). The graph is a representative plot of average bilayer current (pA; y-axis) versus holding potential (mV; x-axis). 5 Example 28. Chemical compounds inhibit the ion channel activity of the 229E E protein synthetic lepeptide. To test compounds for their ability to block or otherwise inhibit the ion channel formed by 229E B protein, small aliquots of solutions containing the 10 compounds were added to the aqueous solutions bathing planar lipids in which the peptide channel activity had been reconstituted and the effect of the compound addition on the ionic currents was recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% 15 methanol and 2 pl of the appropriately diluted compound was added to the cis and/or trans chambers to yield the desired final concentration. In the example shown in Figure 10, addition of 100pM cinnamoylguanidine to the ois chamber greatly reduced current flow through the 229E E ion channel. 20 Example. 29. Bacterial Bio-Assay for Sereening Potential 229E-CoV E protein Ion Channel-Blocking Drugs. 229E-CoV E-protein Ion Channel inhibits Bacterial Cell growth. 25 A bio-assay of 229E-CoV E-protein function in bacterial cells was developed. A synthetic cDNA fragment encoding 229E-CoV E-protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protein at 37 0 C was observed as an indicator of p7 ion channel 30 function dissipating the normal Na+ gradient maintained by the bacterial cells. 35 WO 2004/112687 PCT/AU2004/000866 -83 Example 30 Compound Screenine using the Bacterial Bio-Assay for 229E-CoV E-yrotein. The halos of growth around the site of application of particular drugs - as described in example 14 -were scored as described in example 15. 5 Table 9 list the scores for inhibition of 229E-CoV E-protein in the bacterial bio-assay. Table 9 229E E protein Inhibition Compound (score) 4-isopropylcinnamoylgupanidine 4.9 3,4-dichlorocinnamoylguanidine 4.4 3-(trifluoromethoxy)oinnamoylguanidine 4.1 4-t-butylcimnnamoylguanidine 4.0 3-isopropylcinnamoylguanidine hydrochloride 4.0 3-t-butyloinnamnoylguanidine 3.9 2--butyliinnamoylguanidine 3.9 trans-3-(1-napthyl)acryloylguanidine 3.7 5-bromo-2-methoxycinnamoylguanidine 3.6 2,3-difluorocinnamoylguaoidine 3.3 3-(2-napthyl)acryloylguanidine 3.0 2-phenylcinnamoylguanidine 3.0 3-phenyloinnamoylguanidine 2.9 3-(cyclohex-1-en-l-yl)cinnamoylguanidine 2.4 4-phenylbenzoylguanidine 2.3 3 -(trifluoromethyl)cinnamoylguanidine 2.3 (4-Phenoxybenzoyl)guanidine 2.3 4-(trifluoromethyl)cinnamoylguanidine 2.3 2-(cyelohex-1-en-lyl)cinnamoylguanidine 2.3 (4-Bromocinnamoyl)gnanidine 2.0 5-(N,N-hexamethylene)amiloride 1.9 1-napthoylguanidine 1.9 5-(4-fluorophonyl)amiloridc 1.8 (5-Phcnyl-penta-2,4-dienoyl)guaaidine 1.8 (3-Bromooinnamoyl)guanidine 1.7 2,5-dimethyloinnamoylguanidine 1.6 2-(trifluoromethyl)cinnamoylguanidine 1.5 6-mothoxy-2-naphthoylguanidine 1.4 (4-Chlorocinnamoyl)guanidine 1.4 (3-Methoxycinnamoyl)guanidine 1.4 5-bromo-2-fluorooinnamoylguanidine 1.4 5-(N,N-Dimethyl)amiloride hydrochloride 1.3 WO 2004/112687 PCT/AU2004/000866 -84 cinnamoylguanidine 1.3 (2-Methoxycinnamoyl)guanidine 1.1 (4-Methyleinnamoyl)guanidine 1.0 4-phenylcinnamoylguanidine 1.0 2,6-dichlorocinnamoylguanidine 1.0 (2-Bromocinnamoyl)guatdine 0.9 2,4,6-tdrimethylinnamoylgnanidine 0,9 (trans-2-Phenycyclopropneearbonyl)guanidine 0.8 (3-Chloracinnamoyl)guanidine 0.8 2-(1-napthyl)acetoylguanidine 0.8 2-ethyleinuamoylguanidine 0.8 2<-yclohexylcinnamoylguanidine 0.8 (4-Hydroxyoinnamoy)guanidine 0.6 2-ethoxycminamoylguanidine 0.6 3-methylcinnamoylguanidine 0.5 2-methyloinnamoylguanidinoe - 0.5 3-fluomcinnamoylguanidine 0.5 cinnamoylguanidine hydrochloride 0.5 2,3-dimethyloinnamoylguaWdiae 0.5 2-fluorocinnamoylgoanidino 0.4 4-fluorocinnamoylguauidine 0.4 3,4-difluoroeinnamoylguanidine 0.4 5-tort-butylamino-amiloride 0.3 2-napthoylguanidino 0.3 N,N'-Bis(amidino)napthalene-2,6-dicarboxamniide 0.3 N,N'-Bis(3-phenylpropanoyl)guanidine - 0.3 4-methylcinnamoylguanidine 0.3 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 0.3 2,3,5,6,-totramethycionamoylguanidine 0.3 3-ethoxycinnamoylguanidine 0.3 N,N'-bis(3phenylpropanoyl)-N"-phenylguanidin 0.1 (4-Methoxyoinnamoyl)guanidine .0.1 (2-Chlorocinnamoyl)guanidine 0.1 (3-Nitrocirmamoyl)guanidine 0,1 4-ethoxycinnamoylguanidine 0.1 3,4,5-trimethoxyeinnamoylguanidine 0.1. 2-(2-napthyl)acetoylguanidine 0.1 N-(3-pheny 1propanoyl)-N'-phenylguanidine 0.1 Example 31: Antiviral Assay for testing compounds against replication of human coronavirus 229E (229E). To determine the antiviral activity of compounds against human coronavirus 229E replication (ATCC VR-740), an assay measuring reduction in the number of WO 2004/112687 PCT/AU2004/000866 -85 plaques formed in monolayers of 229E infboted MRC-5 cells (human lung fibroblasts ;ATCC CCL-171) was developed: First, avirus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by exposure to the virus at an MO1 5 of approx. 0.01 pfu/cell for 1 hour at 35 0 C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DIMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35"C (in 5% Co) for 3 -5 days post infection, after which time culture supernatant 10 was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. 15 Table 10 Plaque Reduction (%control / # experiments) Compound 5uM 2.5uM luM 2-t-butyloirnnamoylguanidine 100 / 1 100 / 3 050 / 3 4-isopropyloinamoylguanidine 100 / 1 100 / 2 057 / 2 3,4-dichlorocinnamoylganiadine 100 / 3 099 / 4 086 / 3 3 (trifluoromethoxy)cinnamoylguanidine 100 / 2 098 / 4 077 / 3 2,6-diolorocinnamoylguanidine 100 / 1 097 / 3 066 / 2 2-(cyclohex-l1-en lyl)cinnamoylguanidine 100 / 1 097 / 3 021 /1 2-cylohexyloinnamoylguanidine 070/1 097 / 2 089 / 2 5-bromo-2 methoxycinnamoylguanidine 1001/2 096 / 4 088 / 3 2-phenylcinnamoylguanidine 100 / 1 096 / 3 100 / 1 5-(2'-bromophonyl)penta-2,4 Jienoylgumanidine 100 / 2 095 / 3 079 / 2 4-t-butyloinnamoylguanidine 100 / 1 095 / 3 084 / 3 3-phenyloinnamoylguanidine 094 / 3 077 / 2 3-Bromocinnamoyl)guanidine 100 / 2 093 /3 072 / 2 (4-Bromocinnamoyl)gnanidine 094 / 1 091 /3 073 / 2 5-(N,N-hexamethylene)amiloride 089 / 091 /2 033 / 1 trans-3-(1-napthyl)acryloylguauidine 100 / 1 091 / 2 064 / 2 WO 2004/112687 PCT/AU2004/000866 -86 3-(2-napthyl)aoryloylguanidine 100 / 1 091 /2 062 / 2 2,4-diohlorooinnamolyguanidine 100 / 2 090 / 4 06413 (2-Nitrocinnamoyl)guanidine 085 / 2 090 / 2 046 /2 3 (trifluoromethyl)oinnmoylguanidino 097 / 2 089/4 064 / 3 5-bromo-2-fluorocirnamoylguanidine 100 / 1 088 / 3 063 / 2 4-methylcinnamoylguanidine 091 / 2 087 / 4 063 /2 (3-Chlorocinnamoyl)guanidine 100/ 1 086 / 3 009 / 1 4-Methoxyoinnamoyl)guanidine 100 / 1 085 / 4 057 /3 4-Chlorocinamoyl)guanidine 100 / 2 084 / 2 051 / 2 -fluorooinnamoylguanidine , 095 / 1 083 /3 051 / 2 3-(oyclohex-1-en-1 l)oinnamoylguanidine 100 / 1 082 / 3 063 / 2 a-Methyloinnamoyl)guanidine 023 / 1 082 / 1 036 / 2 23.56.
    etramethylcinnamoylguanidin. 098 / 2 079 / 4 064 / 3 -fluorocinnamoylguanidine 090 / 1 079 / 3 045 /2 trifluoromethyl)cinmamoylguanidine 100 / 1 079 / 1 052 / 1 3-Nitrocinnamoyl)guanidine 100 / 1 079 / 1 045 / 1 ,5-dimethyklinnamoylguanidine 092 / 2 078 / 1 078 /1 -t-butylcinnamoylguanidine 100 / 1 077 / 4 - 030 / 3 (3-Methoxycinnamoyl)guanidine 089 / 1 075 / 2 030 / 1 3-methylcinnamoyljuanidine 095 / 1 074 / 3 044 / 1 3-isopropyloimnamoylguarnidine hydrochloride 089 / 1 074 / 3 014 / 1 (2-Bromocinamoyl)guanidine 095 / 2 072/2 043 / 2 3-ethoxycinnamoylguanidine 100 / 1 072 / 3 057 / 1 (5-Phcnyl-penta-2,4-dienoyl)guanidine 100 / 1 072 / 2 069 /1 (2-Chlorocinnamnoyl)guanidine 095 / 2 072 / 2 040 / 2 4-ethoxycinnamoylguanidine 073 / 1 069 / 2 057 / 1 4-fluorocinnamoylguanidine 100 / 1 067 / 3 034 / 2 3,4-difluorocinnamoylguanidine 085 / 1 065 / 3 042 / 2 N-(3-phenylpropanoyl)-N' phenylguanidine 051 / 1 064/1 000 / 1 2,4,6-trimethylcinnamoylguanidine 075 / 2 063 / 3 062 / 2 2-mothylcinnamoylguanidine 074 / 2 063 / 3 053 / 3 (trans-2 Phenyloyclopropanecarbonyl) guanidine 063 / 2 02211 [(E)-3-(4-Dimethylaminophenyl)-2 methylacryloyl]guanidine 059 / 1 N-Benzoyl-N'-cinnamoylguanidine 056 / 1 4-phenylbenzoylguanidine 076 / 1 055 / 2 071 / 1 trans-3-Furanacryoylguanidine 055 / 2 018 / 1 (4-Phenoxybenzoyl)guanidine 069 / 1 054 / 3 040 /2 2-Methoxyoinnamoyl)guanidine 051 / 1 053 / 2 024/ 1 N-amidino-3-amino-5-pbenyl-6- 074 / 2 052 / 2 038 / 1 WO 2004/112687 PCT/AU2004/000866 -87 chloro-2 pyrazinecarboxamide N-(cinnamoyl)-Nphenylguanidino 084 /1 048 /2 035 / 1 oinnamoylguanidine 095 /2 047 / 2 059 / 1 3,4 (methylenedioxy)cinnamoylguanidine 084 / 1 046/1 019 / 1 N,N'-Bis(amidino)napthalene-2,6 dicarboxamide 045 / 1 2;3-dimethylcinnamoylguanidine 073 / 1 044 / 2 024 / 1 5-(3'-bromophenyl)ponta-2,4 ienoylguanidine 044 / 1 N,N'-Bis(3 phenylpropanoyl)guanidino 041 /1 3-methoxy-amiloride 0291/2 039 / 3 022 /2 2,3-difluorocinnamoylguanidine 036 / 1 1-npthoylguanidine 036 / 1 (3-phenylpropanoyl)guanidine 036 / 1 6-methoxy-2-naphthoylguanidin , 49 / 3 030 / 4 5-(NN-Dimethyl)amiloride hydrochloride 027 / 1 -ethoxycinnamoylguanidine 027/1 -napthoylguanidine 0271/1 3,4,5-trimethoxycinnamoylguanidine 027 1 3-methoxy -HMI-IA 027 / 1 Benzyoylguanidine 026/ 1 2 (trifluoromethyl)cinnamoylguanidine 022 / 1 N-amidino-3,5-diamino-6-phynyl-2 pyrazinecarboxamide 022 / 1 innamoylguanidine hydrochloride ' 02011 Quinoline-2-earbonyl)guanidine 015 / 3 0191 3 006 / 2 (4-Hydroxycianamoyl)guanidine 01911 -(4-fluorophonyl)anmiloride 018 / 1 2-(1-napthyl)acetoylguanidine 018 / 1 (2-Furanacryloyl)guanidine 018/1 3-(3-Pyridyl)acryloyl]gunidine 018 / 1 N-Cinnamoyl-N,N dimethylguanidine 015 / 1 N-(2-napthoyl)-N'-phenylguanidine 011 / 1 2-(2-napthyl)acetoylguanidine 009 / 1 N,N-bis(3phenylpropanoyl)-N" henylguanidine 009 / 1 (Phenylacetyl)guanidine . 009 / 1 WO 2004/112687 PCT/AU2004/000866 -88 Example 32 Human OC43 Coronavirus 0C43 Antiviral Assay for testing compounds against replication of human coronavirus OC43. To determine the antiviral activity of compounds against human coronavirus 5 OC43 replication (ATCC VR-759), an BLISA assay was developed measuring the release of the viral N-protein into culture supernatants from monolayers of OC43 infected MRC-5 cells (human lung fibroblasts;ATCC CCL-171); First a virus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by 10 exposure to the virus at an MOIof approx. 0.01 pful/cell for 1 hour at 35 0 C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35"C (in 5% CO 2 ) for 5 days post infection, 15 after which time culture supernatant was harvested and cellular debris removed by centrifugation at 5000 x g for 10 minutes. For N-antigendetection, 100pl samples of clarified culture supernatant were added to duplicate wells of a 96-well Maxi-Sorb plate; 100pl of RIPA buffer was added per well with mixing and the plate was covered and incubated at 4 0 C overnight to enable protein binding to the plastic wells. 20 The next day, the coating solution was discarded, wells were washed thoroughly with PBST, and blocking of unoccupied protein binding sites was performed by incubation in 1% BSA in PBS for 1.5 hours. The antibody recognising QC43 N-protein was used at 1/800 dilution in PBS (lhr at 37CC) and the secondary antibody (goat-anti mouse alkaline phosphatase) was used for the colour development reaction. Optical 25 density of the wells was read at 405 nm and the effect of compounds determined by comparison of the level of signal in presence of compound to level of signal from the solvent control, Example 33: Effect of compounds in OC43 antiviral assay 30 Compounds were screened for activity against OC43 replication according to the method described in example 22. Results are shown in Table 11.
    WO 2004/112687 PCT/AU2004/000866 -89 Table 11 Virus inhibition at Compound 2.SnM 3-methylcinnamoylguanidine 100 trans-3-(1-napthyl)acryloylguanidine 100 (3-Bromooinnamoyl)guanidine 100 (2-Chlorocinnamoyl)guanidine 96 3,4-dichlorocinnmamoylguanidine 90 3-(trifluoromethyl)oinmamoylguanidine 84 (trans-2-Phonylcyclopropanecarbonyl)guanidino 71 4-isopropylcinnamoylguanidine 68 cinnamoylguanidine 57 6-methoxy-2-naphthoylguanidine 47 2,4-dichlorocinnamolyguanidine 36 (4-Chlorocinnamoyl)guanidine 36 5-(N,N-hexamethylene)amiloride 30 (4-Bromooinnamoyl)guanidine 29 2,6-dichlorocinnamoylguanidine 27 5-bromo-2-methoxycinnamoylguanidine 24 (5-Phenyl-penta-2,4-dienoyl)guanidine - 9 3-(triftioromethoxy)cinnamoylguanidino 4 2-t-butylinnamo ylguaidine 4 5 Example 34. Mouse Hepatitis Virus (MIV). Synthesis and Purification of a Peptide Corresponding to the MIHV-A59 E Protein. A peptide corresponding to the full-length MHV-A59 E protein (sequence: 10 MFNLFLTDTVWYVGQIIFIFAVCLMVTIIVVAFLASIKLCIQLCGLCNTL VLSPSIYLYDRSKQLYKYYNBBMRLPLLEVDDI; accession number NP_068673) was synthesized manually using FMOC chemistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curmtin School of Medical Research, ANU, Australia) using a Symphony Peptide Synthesiser from 15 Protein Technologies Inc.(Wobumrn, MS, USA) according to the manufacturers instructions to give C-terminal amides, the coupling was done with I-IBTU and hydroxybenzotriazolo in N-methylpyrrolidone. Each of the synthesis cycles used double coupling and a 4-fold excess of the amino acids. Temporary a-N Fmoc protecting groups were removed using 20% piperidine in DMF.
    WO 2004/112687 PCT/AU2004/000866 -90 The crude synthetic peptide was purified using the ProteoPlus'TM kit (Qbiogene inc. CA), following manufactures instructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-HCI pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, * 100 mM f-mercaptoethanol) and run on 4-20% gradient 5 polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the full-length peptide were excised out of the gel. The gol slice was transferred to the ProteoPLUST M tube and filled with tris 10 glycine electrophoresis buffer. The tubes were.emerged in tris-glycine electrophoresi buffer and subjected to 100 volts for approximately 1 hour. The polarity of the electric cuttent was reversed for 1 minute to increase the amount of protein recovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 minute. The purified peptides were dried in a Speedvae and the weight of the final product 15 was used to calculate the yield. Example 35: MHV-E protein forms Ion channels in planar lipid bilavers. Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, 20 palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 Upm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to 25 the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 500 mM NaCl or 500mM KC1 and the trans 50 mM NaCl or 50mM KC1. The bilayer formation was monitored electrically by the amplitude of the uemrent pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel 30 activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.
    WO 2004/112687 PCT/AU2004/000866 -91 The MHV E synthetic peptide was dissolved in 2,2,2-trifluorethanol (Tr.PE) at 0.05mg/mi to 1 mg/m1. 10 pl of this was added to the cis chamber (Iml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic "flea", Ionic currents, indicating channel activity in the bilayer, were typically detected within 15 5 30 amin. After channels were detected the holding potential across the bilayer was varied between -100mV and +lOOmV to characterise the size and polarity of current flow and enable the reversal potential to be determined. In 14 experiments where the cis chamber contained 500mM NaC1 solution and the trans chamber contained 50 mM NaC1 solution, the average reversal potential 10 ofthe channel activity was calculated to be 49±1 (SEM) mV. I11 experiments where the cis chamber contained 500mM KCI solution and the trans chamber contained 50 mM K(CI solution, the average reversal potential of the channel activity was calculated to be 13 ±6 (SEM) mV. These results indicate that the MHV E protein forms cation selective ion channels that are more selective for Na than for K ions. 15 Figure 11 shows examples ofraw current data for the MHV E ion channel at various holding potentials (cis relative to trans) in asymmetrical NaCI solutions (500/50 mM), The graph is a representative plot of average bilayer current (pA; y axis) versus holding potential (mV; x-axis). 20 Example 36. Chemical compounds inhibit the ion channel activity of the MIRV E protein synthetie peptide. To test compounds for their ability to block or otherwise inhibit the ion channel formed by MHV E protein, small aliquots of solutions containing the compounds were added to the aqueous solutions bathing planar lipids in which the 25 peptide channel activity had been reconstituted and the effect of the compound addition on the ionic currents was recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% methanol and 2 Il of the appropriately diluted compound was added to the cis and/or 30 trans chambers to yield the desired final concentration.
    WO 2004/112687 PCT/AU2004/000866 -92 In the example shown in Figure 12 below, addition of 100pM cinmamoylguanidine to the cis chamber greatly reduced current flow through the MHV E ion channel. 5 Example 37.Baeterlal Bio-Assay for Screening Potential MHV E-proteln Ion Channel-Blocking Drugs. 10 MIV E-protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of MHV E-protein function in bacterial cells was developed. A synthetic eDNA fragment encoding MV E-protein was cloned into the expression plasmid pPL451, creating a vector in which B protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protein 15 at 37TO was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 38. Compound Screenint usine the Bacterial Bio-Assay for MHV E protein. 20 The halos of growth around the site of application of particular drugs - as described in example 14 -were scored as decribed in example 15. Table 12 lists the scores for inhibition of MIV E protein in the bacterial bio-assay. 25 Table 12 MIHV E protein Inhibition Compound (score) 4-isopropylinnamoylguanidine 4.5 3-isopropylinnamoylguanidine hydrochloride 4.2 4-t-butylcinnamoylguanidine 4.1 3-(trifluoromethoxy)cinnamoylguanidine 4.1 3-t-butyleinnamoylguanidine 4.0 3,4-dichlorocinnamoylguanidine 3.8 2,3-difluorocinnamoylguanidine 3.8 2-t-butylcinnamoylguanidine 3.8 3-phenyleinnamoylguanidine 3.7 2-phenylcinamoylguanidine 3.4 5-bromo-2-methoxyeinnamoylguanidine 3.3 2-(cyclohex-1-en-l1yl)cinnamoylguanidine 3.3 WO 2004/112687 PCT/AU20041000866 -93 3-Qrifhioromethyl)ciamamoylguanidine 2.9 3-(c~yclohex-1-en-1-yl)cinnamoylguanidie 2.9 tans-3-(1-nspthyl)acryloylguanidine 218 4-(frifluoromethyl)cinnamoylguanidine 2.8 -(2-napthyl)aorYlOylguanidine 2.8 2-(trifluoromethyl)cimnnaoylguanicline 2.7 (4-Phenoxybenzoyl)gtwnIdJie 2.4 (3-Bromocinnamoyl)guanidine, 2.4 2,5-dimethylcinnamoylguauidine 2.3 5-bronio-2-fluorocinnamoylguanidinae 2.1 6-methoxy-2-naphthoylguanidbne 1.8 4-phenylhenzoylgnanidine 1.8 (4-Bromocinnamoyl)gnanidine 1.8 1-napthoylgaanidine 1.7 (5-Pheny1-penta-2,4-dienoyl~guanidine 1.4 (2-Bromocimnaoy~guanidine 1.4 (4-Chlorocimnmoyl)guanidlne 1.3 2-methylinnamoyguaidlne 1.2 2,6-diohlorooinnamoylguauidine 1.2 2,4,6-triniethylcinnamoylguanidine 1.2 5-(NN-hoxamethy~enpaiiloodd 1.1 olmnoylguanidine11 cirmamoylguanidine hydrochloride 1.1 (a-Methylcinnanoyl)guanidine 1.0 2,3-dimethylcimnamoylgnanidine 1.0 2--cyclohexylinnamoylguanidine 0.9 -(3-phenylpzopanoyl)-N'-phenylguanidine 0.8 N'-bis(Sphenylpropanoyl)-N"I-phcnylguanidine 0.8 (3-Methoxycinnamoyl)guanidine- 0.8 (2-MetbOxYCinnaioyl)guaid-Lne 0.8 3-fluorocinnamoylguanidino, 0.8 -fluorooixmmoylgnanidine 0.8 2,- didllorocinnamolygnanidine 0.8 2-ethylcinnaioylguanidine 0.8 (2-Chlorocinnamoyl)guanidine 0.7 (4-Hydroxyoinaxnoylguanidine 0.7 2-cthoxycinmoylguanidine 0.7 2-napthoylguandine 0.6 (frans-2-Phenylcyclopropanecarbonyl)guaidine 0.6 5-(NN-Dime-thyamilorido hydrochloride 0.5 5-(4--fluorophenyl)arniloride 0.5 3-methylinnaunoylgaanidine 0.5 (3-Cblorocinnamoyl)guanidine 0.4 4-rnethylchmamoylguanidine 0.4 4-ethoxycinaoylguanidine 0.4 2-(1-napthyl~acetoylguanidine 0.4 WO 2004/112687 PCT/AU2004/000866 -94 3,4-difluorocinnamoylguanidine 0.4 2-(2-napthyl)acetoylgnanidine 0.4 2,3,5,6,-tetramethyleinnamoylguanidine 0.4 (4-Methoxycinnamoyl)guanidine 0.3 3,4-(methylenedioxy)innanmoylguanidine 0.3 3-ethoxycinnamoylguanidine 0.3 4-fluorocinnamoylguanidine 0.2 1-bromo-2-napthoylguanidine 0.2 5-tert-butylamino-amiloride 0.1 (3-Nitrocinnamoyl)guanidine 0.1 3,4,5-trimethoxycinnamoylguanidine 0.1 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 0.1 5 Example 39. MHV Antiviral Assay for testing compounds against replication of mouse hepatitis virus (MRV). To determine the antiviral activity of compounds against MHVIW replication (strain MHV-A59: ATCC VR-764), an assay measuring reduction in the number of plaques formed in monolayers of MHV infected L929 cells (ATCC CCL-a) was 10 developed: First, a virus working stock was prepared by amplification in NCTC clone 1469 cells (ATCC CCL-9.1). This was then used to infect confluent monolayers of L929 cells grown in 6-well tissue culture platesby exposure to the virus at an MOI of 0.01 pfuloell or 1 pfuilell for 30 minutes at 370C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DMEM 15 supplemented with 10% horse serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 37*C (in 5% CO2) for 16 - 24 hours post infection, after which time culture supernematant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques 20 were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. Example 40. Effect of compounds in MHV antiviral assay. Table 13 provides the results obtained from this study.
    WO 2004/112687 PCT/AU2004/000866 -9$ Table 13 Percent reduction in Plaque number / # experiments Compound 20uM IOuM luM 5-(3'tromopheyl)penta-2,4-dionoylguanidine N/D 99 / 2 66 / 1 5-bromo-2-methoxycinnamoylguanidine N/D 100 / 1 66 / 1 3-phonyloinnamoylguanidine Toxic 86 / 2 64 / 3 2,3-difluorocinnamoylguanidine Toxic 92 / 3 64 / 2 3-ethoxycinnamoylguanidine 100 / 1 89 / 2 58 / 1 5-(2'-bromophenyl)penta-2,4-dienoylguanidine Toxic 100 / 1 57/ 1 cinnamoylguanidine hydrochloride 85 / 1 72 / 2 56 / 1 (2-Chlorocinnamoyl)guanidine 95 / 2 88 / 3 53 / 3 cinnamoylguanidine 97 /8 88/ 8 52/7 (4-Bromocinnamoyl)guanidine Toxic /2 98 / 3 52 / 3 (2-Bromocinnamoyl)guanidine 91 / 2 89/3 52 / 3 (4-Methoxycinnamoyl)guanidine 98 / 4 96 / 4 51 / 3 (a-Methylcinnamnoy)guanidine 81 / 2 75 / 3 51 / 2 3,4-dichlorocinnamoylguaoidine 91/2 96 / 1 50 / 2 2-(cy:lobotx-I-en-lyl)cinnamoylguauidine N/D 97 / 1 50 / 1 3,4-difluorocinnamoylguanidine Toxic 91/ 2 50 / 1 3-t-butylcinnamoylguanidine Toxic 94 / 3 49 / 2 2-ethoxycinnamoylguanidine 93 / 2 85 / 3 48/2 WO 2004/112687 PCT/AU2004/000866 -96 trans-3-Furanacryoylguanidine 70 / 1 65 / 1 48 / I N-amidino-3-amino-5-hexamethyleneimino-6- 84/1 52/2 48/ phenyl-2-pyrazinewarboxamide 4 / 52 / 2 8 / 1 (2-Nitrocainnamoyl)guanidine 97 / 1 77 / 2 47 / 1 -(trifluoromethyl)oinnamoylguanidine 97 / 3 95 /3 46 / 3 3,4-(methylenedioxy)cinnamoylguanidiane 93/3 82 /13 45 / 3 5-(N-Methyl-N-isobutyl)amiloride 92 / 1 85 / 1 44 / 2 (4-Chlorocinnamoyl)guanidine . 97/2 88 / 2 43 / 3 2,4-dichlorocinnamolyguanidine .76 / 1 73 / 1 43 / 1 N-(3-phenylpropanoyl)-N'-phenylguanidine 80/1 65 / 1 43 /1 (3-Nitrocinnamoyl)guanidine 95 / 2 77/3 42 / 3 2-phonyloirmnnamoylguanidine N/D 100 / 1 42 / 1 4-isopropylinnamoylg nidine 95 / 3 93 /3 41 / 3 3-(trifluoromethoxy)cinnamoylguanidine 100 / 1 90 / 3 41 / 2 3 -(trifluoromethyl)cinnamoylguanidine 98/1 83 /1 40 / 1 (4-Nitrocinnamoyl)guanidine 97/1 75 / 3 40 / 3 3-(2-napthyl)acryloylguanidine 93 / 1 93 / 1 0 / 1 4-ethoxyoinnamoylguanidine 96 /1 92 / 1 40 / 1 2,6-dichlorocinnamoylguanidine 91 / 1 70 / 1 40/1 2,5-dimethylcinmramoylguanidine 95 /3 91 / 3 39 / 3 (3-Bromocinnamoyl)guanidine 95 / 2 90/3 39 / 3 (3-Chlorocinnamoyl)guanidine 94 / 1 86 / 2 39 / 2 WO 2004/112687 PCT/AU2004/000866 -97 3-mothyloinnamoylguanidino 90 / 1 88 / 1 39/1 (3-Methoxyoinnamoyl)guanidine 92 / 2 87 / 2 373 2-t-butylcinnamoylguanidine N/D 98 / 2 37 / 1 [(E)-3-(4-Dimethylaminophanyl)-2- 56/1 45/ 1 37 / 1 methylacryloyl]guanidine 5/ 4 __7/ N,N'-bis(1-napthoyl)guanidino 58 / 1 52 / 2 35 / 1 3-methoxy -BMA 15 / 1I 31/1 35 / 1 5-tert-butylamino-amiloride 89 / 4 84 / 4 34/ 4 trans-3-(1-napthyl)acryloylguanidine 95 / 2 86 / 3 34 / 3 5-methoxy-2-naphthoylguanidine 88 / 3 56 / 3 3413 2-napthoylguanidine 67/2 36 / 2 34/2 2-ethyloinnamoylguanidino 96/ 1 81 / 2 34/ 1 2,3-dimethyloinnamoylguanidine 95 /1 i 79 / 2 34/1 N"-Cinnamoyl-NN'-diphenylguanidine 97 / 1 72 / 2 34/ 1 3-isopropylinnamoylguanidine hydrochloride N/D 99 / 2 32 / 1 (4-Phenoxybenzoyl)guanidine 73/1 65 / 1 32 / 1 (trans-2-Phenylcyclopropanecarbonyl)guanidino 77 / 2 64 / 2 31/2 3-fluorcinnamoylguanidine 100 / 1 93 / 2 31/ 1 5-bromo-2-fluorocinnamoylguanidine Toxic '81/2 31/ 1 ,N'-bis-(cinnamoyl)-N"-phenylguanidine 16 / 1 38 / 2 31 1 3-quinolinoylguanidine 27 / 1 36 / 2 30 / 1 2,4,6-trimethylcinnamoylguanidine 91/2 61 / 3 27 / 2 WO 2004/112687 PCT/AU2004/000866 -98 1-bromno-2-napthoylguanidine 31 1 27 12 27 / 1 N-amidino-3,5-diamino-6-phynyl-2- 53 / 1 39 / 2 25 / 1 pyrazinecarboxamide N-Cinnamoyl-N',N'-dimethylguanidine 92 / 2 65 / 3 24 / 2 (2-Methoxycinnamoyl)gumanidine 90/2 85 / 2 23 / 2 2-(2-napthyl)aoetoylguanidine 52 / 1 20 / 2 23 / 1 4-phenylcinnamoylguanidine 5311 / 36 / 1 21/3 (3-(3-Pyridyl)acryloyl]guanidine 81 / 2 73 /2 21 / 2 3,4,5-trimethoxycinnamoylguanidine 84 / 1 84 /11 21 / 1 4-methykinnamoylguanidine 93 / 1 89 / 1 20 / 1 4-fluorocilnamoylguanidine 86 / 1 83/1 20 / 1 -m ethylcinnamoylguanidine 1 / 1 82 / 1 20 / 1 6-bromo-2-napthoylganidine 65 / 1 3712 19 / I 5-(N,N-Dimethyl)amiloride hydrochloride 42 4 7/4 17/4 (5-Phenyl-penta-2,4-dienoyl)guanidine 2711 24/1 17 / 1 2-cyclohexylcinnamoylguanidine 100 / 1 74 / 2 16 / 1 5-(4-fluoropheny])amiloride 4 / 1 25 /1 16 / 1 Beonzyoylguanidino 22 / 1 39 / 2 14 1 N-Benzoyl-N'-cinnamoylguanidine 0 / 1 0/ 1 14/ 1 5-(N,N-hexamethylene)amiloride 84 / 2 89 / 1 13/2 N-(cinnamoyl)-N'pherylguanidine 83 1 8 / I 13 / 1 (4-Hydroxycinnamoyl)guanidine 19 / 1 15 / 1 13 / 1 WO 2004/112687 PCTIAU20011000866 -99 2-(trifluoromethyl)cinnamoylguaniidine 19 / 1 1511 131 (Quino~ine-2-carbonygulanidiue 86/1 84 /1 12/11 2-(1-napthy1)acetoy~gtanimdine -19 / 1 02/1 it / 1 2-chloro-&flnorocinn~oylguaflidifle 100 /1 8412 9/1 N-mnidino-3-arnino-5-pheiy1-6-ehloro-2- 20/1 20/2 9 /1 4-phcnylbenzoylguanidine 32/1 24 /1 5/1 N-bis(21apthoylguanidine 5/1 3 /2 3/1 (Phenylaotyl)guanidine 33511 2/1 311 I -napthoylguanidine '71 /3 6213 2/3 N'-bis(Sphenylpropanoyl)-N'-phenylguanidine 67/3 0/4 1/3 3-hydroxy-5-hexarnotliylcnoimino-smiloride 16/1 /212 1/ 1 2'4DicbloroBenzanlilflHCl 12 f2 f13 0/3 2,3,5,6,-tetramcthyloinamoylguanidi-ae NID 68/2 0 /1 Benzamil hydrochlouide 0/1 26/1 0/1 6-fodoamiloride 8/1 21/1 O/l N'-Bis(amidino)napthalene-2,6-dicaxboxamide 19/1 16/.1 0/11 [(4-Chlorophcnoxy-acetyllguanidine 19/1 161/1 0il (3-phenylpropaoyl)guonidine 51 /1 03/1 0/1 2-fluorocinnamoylguanidine 76 /1 73/1 6.bromo-2-uapthoylguanidine 43/1 (2-Furmacyloy1~guaidine 67/2 63 /2 -3 /2 WO 2004/112687 PCT/AU2004/000866 -100 N-(6.Hydroxy-2-napthoyl)-N'-phenylguanidine 43 / 1 39 / 1 -5/ 1 Amiloride.HCI 21 / 1 18 / 1 -5/1 3-(trans-hept-1-en-1-yl)cinnamnoylguanidinfe T / 1 23(T) / 1 -6 / 1 3-methoxy-amiloride 60 / 2 47 /3 -7 / 2 NN'-Bis(3-phenylpropanoyl)guanidine 41/3 301 4 -8 13 3.(cyclohex-1-en-1-yl)cinnamoylguanidine / 1 2 / 2 -191 1 Example 41. Porcine Respiratory Coronavirus (PkCV) Antiviral Assay for testing compounds against replication of porcine respiratory 5 earonavirus (PRCV). To deter mine the antiviral activity of compounds against porcine respiratory coronavirus replication (ATCC VR-2384), an assay measuring reduction in the number of plaques formed in monolayers of PRCV infected ST cells (procine fetal testis cell line, ATCC CRL-1746) was developed: Confluent ST cells in 6 well 10 plates were infected with a quaternary passage of porcine respiratory virus (PRCV) strain AR310 at three dilutions 10, 50 " and 10
    -
    ' in PBSto provide a range of plaques numbers to count. 100ul of diluted virus was added per well in a volume of 1m1 of media. Plates were incubated for one hour on a rocking platform at room temperature to allow virus to adsorb to cells. The viral supernatant was removed and 15 2ml/well of overlay containing 1% Seaplaque agarose in lx MEM, 5% FCS was added to each well. Compounds to be tested were added to the overlay mixture by diluting the compounds from frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound. The volume of compound/solvent never exceeded 0.07% of the volume 20 of the overlay. The solvent used to dissolve compounds was DMSO and methanol mixed in equal proportions. Compounds were tested for anti-plaque forming activity at four concentrations, 0.1uM, luM, 10uM and 20uM. Either duplicates or quadruplicates were performed at each concentration. Controls were performed WO 2004/112687 PCT/AU2004/000866 -101 where the same volume of solvent was added to the overlay. The overlay was allowed to set at room temp for 20 rmins. The plates were then incubated at 37 0 C for 2 days. The monolayers were then fixed and stained overnight at room temperature by adding lml/well of 0.5% methylene blue, 4% formaldehyde. Overlay agarose and 5 stain was theh rinsed off to visualize stained and fixed monolayer Example 42: Effect of compounds in PRCV antiviral assay Compounds were screened for activity against PRCV replication according to 10 the method described in example 29. Table 14 provides EC50 values for some tested compounds. Table 14 Compound ECSO (uM) 5-(N,N-hexamethylene)amiloride 0.06 6-methoxy-2-naphthoylguanidine 0.04 ciramraoylguanidine 0.08 N-(3-phenylpropanoyl)-N'-phenylgUanidine 19 3-methylcinnamoy1guanidine 1.43 (3-Bromocinamoyl)guanidine 112 (trans-2-Phonylcyclopropanecarbonyl)guanidine 17.2 trans-3-(1-napthyl)acryloylguanidine 19.1 2-(2-napthyl)acetoylguanidine 119.6 15 Example 43. Bovine Coronavirus. Antiviral Assay for testing compounds against replication of bovine coronavirus .0L.Zh 20 To detennrmine the antiviral activity of compounds against bovine coronavirus replication (ATCC VR-874), an assay measuring reduction in the number ofplaques fornned in monolayers of BCV infected MDBK cells (bovine kidney cell line ;ATCC CCL-22) was developed: Confluent MDBK cells in 6 well plates were infected with a secondary passage of BCV with serially diluted virus diluted to 10, 5s and 10 in 25 PBS to provide a range of plaques numbers to count. 100ul of diluted virus was WO 2004/112687 PCT/AU2004/000866 -102 added per well in a volume of Imi of media. Plates were incubated for one hr to allow virus to adsorb to cells. The viral supernatant was removed and 2ml/well of overlay containing 1% Seaplaque agaose in lx MEM, 5% FCS was added to each well. Compounds to be tested were added to the overlay mixture by diluting the 5 compounds from a 0.SM frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound. The volume of compounld/solvent never exceeded 0.07% of the volume of the overlay. The solvent used to dissolve compounds was DMSO and methanol mixed in equal proportions. Compounds were tested for anti-plaque forming activity 10 at four concentrations, 0. luM, luM, 10uM and 2OuM.
    -
    Quadruplicates were performed at each concentration. Controls were performed where the same volume of solvent was added to the overlay. The overlay was allowed to set at room temp for 20 mins. The plates were then incubated at 37C for 7 days. The monolayers wore then fixed and stained by adding imlwell of 0.5% methylene blue, 4% 15 formaldehyde.. Example 44: Effect of compounds in BCV antiviral assay Compounds were screened for activity against BCV replication according to the method described in example 31. Table 15 provides EC50 values for some tested 20 compounds. Table 15 Compound EC50 UM (3-Bromocinnamoyl)guaidine 3 3-(trifluoromethyl)cinnmamoylguaniidmin 3 6-methoxy-2-naphthoylguanidine 9 5-(N,N-hexamethylene)=miloride 9 trans-3-(1-napthyl)acryloylguanidine 13 cinnamoylguanidine 42 (5-Phenyl-penta-2,4-dienoyl)guanidine 95 2-(2-napthyl)acetoylguanidine 99 (trang-2-Phenylcyclopropanecarbonyl)guanidine 109 N-(3-phenylpropanoyl)-N'-phenylguanidine 156 4-phenylbanzoylguanidine 190 WO 2004/112687 PCT/AU2004/000866 -103 Example 45 Hepatitis C Virus Ion channel activity of Hepatitis C virus P7 Protein Testing of a Synthetic P7 Peptide for channel activity in artificial linid bilayers A peptide mimicking the protein P7 encoded by the hepatitis C virus (HICV) 5 was synthesised having the following amino acid sequence: ALENLVILNAALAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGMWPLL LLLLALPQRAYA Lipid bilayer studies were performed as described elsewhere (Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl 10 phosphatidylserine and palmitoyl-oleoyl-phosphatidylholine (5:3:2) (Avantl Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 um in the wall ofa 1 ml dehlin cup. The aperture separates two chambers, cis and irans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 15 200 amplifier. Normally the cis chamber contained 500 mM KCI and the trans 50 mM KCL. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz,. digitized at 20 2000 Hz and stored on magnetic disk. The P7 peptide was dissolved in 2,2,2 trifluorethanol (TFE) at 10mg/ml. 10 ul of this was added to the cis chamber of the bilayer which was stirred. Channel activity was seen within 15-20 min. When the P7 peptide was added to the cis chamber and stirred, channel activity was recorded. The potential in the trans chamber was -80 mV and the currents are 25 downwatrds. The currents reversed at +50 mV close to the potassium equilibrium potential in these solutions indicating that the channels were cation-selective. The amplitude of the open-channel peak is 1.7 pA corresponding to a channel conductance of about 14 pS. In most experiments, "single channels" had a much larger size, presumably because of aggregation of the P7 peptide. The currents 30 reversed at about +40 mV in this experiment. In some experiments the solution in the WO 2004/112687 PCT/AU2004/000866 -104 cis chamber was 150 mM KCI and 15 mM KCl in the trans chamber. The P7 peptide again produced currents that reversed. Similar re sults were obtained when both chambers contained Nac. Currents 5 recorded in an experiment when the cis chamber contained 500 mM NaC1 and the trans chamber 50 mM NaCI.. Again the currents reversed between +40 and +60 mV, close to the Na + equilibrium potential indicating that channels were much more permeable to Na than to K. 10 The channels formed by the P7 peptide were blocked by 5-(N,N hexamethylene) amiloride (HMA). Addition of the P7 peptide produced channel activity. Addition of 2 jl of 50) pM HMA to the ois chamber followed by stirring resulted in disappearance of the 15 channel activity. Block of channel activity produced by the P7 peptide with 100 pM HMA was recorded in 4 experiments. In 2 experiments, sodium channels (500/50) were blocked by 500 pM EMA When 10 mM CaCl 2 was added to the cis chamber (K solutions) the reversal 20 potential ofthe currents produced by P7 peptide shifted to more negative potentials indicating a decrease in the PK/PC ratio. When the cis chamber contained 500 mM CaCl 2 and the trans chamber 50 mM CaC12, both positive and negative currents were seen at potentials around +20 mV and 25 it was not possible to determine a reversal potential. Example 46. Recombinant Expression of HCV p7 protein. Two eDNA fragments, each encoding the same polypeptide corresponding to the amino acid sequence of the HCV- a p7 protein, were synthesised commercially 30 by GeneScript. The two oDNAs differed in nucleotide sequence such that in one eDNA ("cDp7.coli") the codons were optimised for expression of the p7 protein in E.coli while in the other eDNA ("cDp7.mam)" codons were biased for expression in WO 2004/112687 PCT/AU2004/000866 -105 mammalian cell lines. cDp7.coli was cloned into the plasmid pPL451 as a BamHI/EcoRI fragment for expression in E.coli. cDp7.mam was cloned into vectors (for example, poDNA3.1 vaccinia virus, pfastBac-1) for expression ofp7 in maminalian cell lines. 5 Example 47. Role of p7 in enhancement of Gag VLP Budding. The budding ofvirus-like particles (VLP) from cultured HeLa cells results from the expression of retroviral Gag proteins in the cells and co-expression of small viral on channels, such as M2, Vpu and 6K, with the Gag protein enhances budding. 10 Interestingly, the viral ion channels can enhance budding of heterologous virus particles. Therefore, to assess budding enhancement by p7 it was co-expressed with the HIV-1 Gag protein in HeLa cells, and VLP release into the culture medium was measured by Gag ELISA. To achieve this, the plasmids pcDNAp7 (pc DNA3.1 = pcDp7.mam as described in Example 20, p 7 expressed under control of the T7 15 promoter) and prDNAGag (HIV-1 Gag protein expressed under control ofthe T7 promoter) were cotransfected into HeLa cells infected with the vaccinia virus strain vTF7.3 (vxpresses T7 RNA polymerase) and culture supernatants were collected for ELISA assay after 16 hours incubation. Example 48, Assay of the ability of compounds to inhibit p7 ion channel 20 functional activity. The two methods of detecting p7 ion channel fUnctional activity, described in Examples 33-35, were employed to assay the ability of compounds to inhibit the p7 channel. In the case of Example 33, compounds were tested for their ability to inhibit p7 channel activity in planar lipid bilayers. In the case of Example 35 compounds 25 were tested for their ability to reduce the number of VJPs released from cells expressing both P7 and HIV-I Gag. 30 WO 2004/112687 PCT/AU2004/000866 -106 Example 49. Bacterial Bio-Assay for Screening Potential HCV V7 protein Ion Channel Blocking Drues. HCV p7 Ion Channel inhibits Bacterial Cell Erowth. 5 A bio-assay of p7 ftmnction in bacterial cells was developed. The p7-encoding synthetic eDNA fragment cDp7.coli was cloned into the expression plasmid pPL451, creating the vector pPLp7, in which p7 expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing p7 at 370C was observed as an indicator of p7 ion channel function dissipating the normal 10 Na+ gradient maintained by the bacterial cells. Example 50 Compound Sereening using the BacterialBio-Assay for HCV p7 protein. The halos of growth around the site of application of particular drags - as described 15 in example 14 -were scored as decribed in example 15, Table 16 lists the scores for inhibition of HCV p7 protein in the bacterial bio-assay. Table 16 HCV p7 protein Ibhibition (score I # of times Compound tested) ,3-dimethyklinnamoylguanidine 3.88/2 2,4,6-trimethylinnamoylguanidine 3.75 / 1 5-bromo-2-fluorooinnamoylguanidine 3.73/6 (4-Bromocinnamoyl)guanidine 3.47/4 ,5-dimethyloinnamoylguanidine 3.43/4 3-(trifluoromethyl)oinnamoylguanidine 3.34/3 4-(trifluoromethyl)cinnamoylguanidine 3,33/5 6-methoxy-2-naphthoylguanidine 3.33 13 (2-Chlorocinnamoyl)guanidine 3.31/6 (4-Chlorocinnamoyl)guaidine 3.16/4 2-Bromocinnamoyl)guanidine 3.00 /3 2,6-dichlorocinnamoylguanidino 3.00 /3 3-Bromocinnamoyl)guanidine 2.92/3 (3-Chlorocinnamoyl)guanidine 2.7513 2-(trifluoromethyl)einnamoylguanidine 2.63/3 (4-Phenoxybenzoyl)guanidine 2.63 11 WO 2004/112687 PCT/AU2004/000866 -107 3,4-dichlorocinnamoylguanidine 2.59/3 4-isopropyloinnamoylguanidine 2.51/2 rans-3-(1-napthyl)acryloylguanidine 2.44/2 4-t-butyloinnamoylguanidine 2.42/2 2-t-butylcinnamoylguanidine 2.36/2 2-othyloinnamoylguanidineo 2.36/2 4-methylcinnamoylguanidine 2.32/2 5-bromo-2-methoxyoinnamoylguanidine 2.32/2 3-(trifluoromethoxy)cinnamoylguanidine 2,26/2 -cyolohexylcinnamoylguanidine 2.26/2 1-napthoylguanidine 2.25/1 3-t-butylcinnamoylguanidine 2.23/2 4-phenylbenzoylguanidine 2.19/2 (5-Phenyl-penta-2,4-dienoyl)guanidine 2.13/1 -(cinnamoyl)-N'phenylguanidine 2.1311 3-isopropylcimamoylguanidine hydrochloride 2.00 / 1 Benzamil hydrochloride 2.0/1 N-(3-phenylpropanoyl)-N'-phenylguanidino 2.0/ 1 N,N'-bis(3phenylpropanoyl)-N"-phenylguanidino 2.0 / 1 3-(2-napthyl)acryloylguanidine 1.93/2 5-(N-Methyl-N-isobutyl)amiloride 1.88/1 2'4 DichloroBenzamil HCI 1.88/1 5-tert-butylamino-amiloride 1.88 11 5-(N-Ethyl-N-isopropyl)amiloride 1.8811 (4-Methoxycinnamoyl)guanidine 1.88/1 -fluorocinnamoylguanidine 1.86/1 3-Nitrocianamoyl)guanidine 1.71/1 4-ethoxyeinnamoylgnanidine 1.63/1 4-Hydroxycinnamoyl)guanidine 1.63/1. trans-2-Phenyleyclopropanecerbonyl)gnanidine 1.63/1 3-ethoxycinnamoylguanidine 1.63 / 1 2,3,5,6,-tetramothylcinnamoylguanidine 1.51/2 4-pheonykinnamoylguanidine 1.511 trans-3-Furanaeryoylguanidine 1.38/1 N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine 1.38/1 (2-Furanacryloyl)guanidine. - 1.38/1 3-(oyolohex-1-en-l-yl)cinnamoylguanidine 1.32/2 ainnamoylguanidine hydrmehloride 1.32/2 5-(N,N-hexamethylene)amiloride 1,28/14 2,3-difluorocinnamoylguanidine 1.24/1 2-(1-napthyl)acetoylguanidine 1.14/1 a-Methylcinnamoyl)guanidine 1.14/1 2-Nitroinnamoyl)guanidine 1.14/1 -lodoamiloride 1.13/1 3,4-(methylenedioxy)cinnamoylguanidine 1.13/1 -ethoxycinnamoylguanidine 1.00/1 innamoylguanidine 1.00/1 -phenyloinnamoylguanidine 1.00 / 1 WO 2004/112687 PCT/AU2004/000866 -108 2-(cyclohex-1-en-lyl)cinnamoylguanidine 1.00 / 1 2-napthoylguanidine 1.0/3 3-phenylcinnamoylguanidine 1.0/1 5-(N,N-Dimethyl)amiloride hydrochloride 1.0/1 5-(4-fluorophenyl)amiloride 1.0/1 3-Methoxyoinnamoyl)guanidine 1.0/1 27:fluorocinnamoylguanidine 1.0/1 5-(3Y-bromophonyl)penta-2,4-dienoylguanidine 1.011 [(4-Chlorophenoxy-acdyl]guanidine 1.0/1 (3-phenylpropanoyl)guanidine 1,0 / 1 2-chloro-6-fluorocinnamoylguanidirie 0.88/1 -fluorocinnamoylguanidine 0.86 11 2-methylcinnamoylguanidine 0.75 11 (2-Methoxycinnamoyl)guanidine .0.75/1 1-bromo-2-napthoylguanidine 0.75/1 3,4,5-trimethoxycinnamoylguaidine 0.71 11 3-mothyloinnanmoylguanidine 0.63/1 3-(trans-hopt-1-en-1-yl)oinnamoylguanidine 0.50/1 Amiloridc.HCl 0.5/2 Phenamnil methanesulfonate salt 0.5 11 2,4-dichlorocinnamolyguanidine 0.3811 (4-Nitrooinnamoyl)guanidine 0.25/1 ,4-difluorocinnamoylguanidine 0.13/1 .(E)-3-(4-Dimethylaminophenyl)-2 ethylacrylyl]guanidine 0.03/4 Example 51: Equine Arteritis Virus (EAV) Antiviral Assay for testing compounds against replication of equine arteritis virus (EAV). 5 1 To determnnine the antiviral activity of compounds against EAV replication (strain Bucyrue; ATCC VR-796), an assay measuring reduction in the number of plaques formed in monolayers of EAV infected BHK-21 cells (ATCC CCL-10) was developed: A virus stock was amplified in RK-13 cells (ATCC CCL-37) and this was then used to infect confluent monolayers of BHKI-21 cells grown in 6-well tissue 10 culture plates by exposure to the virus at an MOI of 5XI0- pfu /c ell for I hour at 37CC 5% CO2. The infective inoculum was removed and nd the cells were overlayed with a 1% sea plaque overlay (Cambrex Bio Science) in MEM containing 10% PCS containing and 10, 5 or 1 pM of compounds to be tested or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 15 37*C (in 5% CO2) for 3 days post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol WO 2004/112687 PCT/AU2004/000866 109 for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. 5 Example 52: Effect of compounds in EAV antiviral assay Compounds were screened for activity against EAV replication according to the method described in example 35. Results expressed as iCSO values are shown in Table 17. 10 Table 17 Compound ICso50 5-(NN-hexamethylen)amiloride 7.5 pM (3-Bromocinnamoyl)guanidine 10 pjM trans-3-(1-napthyl)acrylcylgusnidine 7.5 pM 2-t-butyldinnamoylguanidine 1 pM 2-(cylohea-l -en- yl)oinnamoylguanidine 10 pM Example 53 Dengue Flavivirus Peptide Svathesis of Denque virus M Protein 15 The C- terminal 40 amino acids ofthe M protein of the Dengue virus type 1 strain Singapore S275/90 (Fu et al 1992) (ALRHIIPGFTVIALFLABAIGTSITQKGIIFILLMLVTPSMA) was synthesised using the Fmoo method. The synthesis was done on a Symphony Peptide Synthesiser form Protein Technologies ITne (Tucson, Arizona) as used to give C-terminal amides, the 20 coupling was done with BBTU and hydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesis cycle used double coupling and a 4-fold excess of the amino acids. Temporary e&-N Fmoe-protecting groups were removed using 20% piperidine in DMF. Example 54. Incorporation of Dengue M vlrus protein into lipid bilavers. 25 Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmniitoyl-oleoyl-phosphatidylethanolamine, WO 2004/112687 PCT/AU2004/000866 -110 palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine, (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 pm in the wall of a I ml delrin cup. The aperture separates two chambers, cis and Irans, both containing salt solutions at different Concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KC1 and the trans 50 mM KC1. The bflayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the 10 cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk, The dengue virus M protein C-terminal peptide (DMVC) was dissolved in 2,2,2 trifluorethanol (TFE) at 0.05mgfml to I mg/ml. 10 pl of this was added to the ois 15 chamber of the bilayer which was stirred. Channel activity was seen within 15-30 min, Exmnple 55: Hexamathylene amiloride (MA) to Inhibits ion channel activity of the dengue virus M protein C-terminal peptide. Solutions of 50 mM HMA were prepared by first making a 500 mM solution 20 in DMSO. This solution was further diluted to 50 mM HMA using 0.1 M HCI. 2 itl of the 50 mM HMA was added to the cis chamber after channel activity was seen. The cis chamber contained 1 ml of solution making the final concentration of IMA 100 pIM. 25 Examvle 56. Antiviral Assay for testing compounds against Effects of Dengue flavivirus against cytoproliferation. Compounds were tested at 10, 5, 2.5, 1.25 and 0.625 pM for activity against Dengue 1 strain Hawaii using a cytoproliferation assay which measures the effect of dengue virus infection on proliferation of LLC-MK2, rhesus macaque monkey kidney 30 cells. The LLC-MK2 cells were infected with a predetermined amount of virus, titrated such that cell proliferation in infected cultures would be significantly reduced WO 2004/112687 PCT/AU2004/000866 -Ill compared to uninfected controls. The infected cells were then plated at 1.5x10 3 cells per well in a 96 well plate. Negative controls (no virus, no experimental compound), positive controls (virus, no experimental compound), and cytotoxicity controls (experimental compound, no virus) were run with each drug assay. The cultures were 5 allowed to grow for 7 days and then Alamar Blue, a fluorescent dye that measures the metabolism of the cultures (red/ox), was added to each culture and the fluorescence value for each culture was measured. The negative control without experimental compound or virus was fixed at 100%. The positive controls and the cultures with compound were scored by calculating their average fluoresocance as a percentage of 10 the negative control. At least six replicate wells were measured for each experimental condition. Example 57 Effect of compounds in Dengue antiviral assay: 15 Table 18 Drug AntWiviral Cone. Percent of Drug pM Negative Control cinnoylguanidine Negative control 0 NA Positive control 0 76.5% 10 17.1% 5 38.6% 2.5. 58.3% 125 f72.6% 0.625 76.6% (2-chlorocinnamoyl)ganidine Negative control 0 NA Positive control 0 80.3% 10 8,4% 5 7.7% 2.5. 22.7% 1.25 52.5% 0.625 64.3% Trans-3-(1-naphthyl)acryloylguanidine Negative control 0 NA Positive control 0 80.4% 10 6.8% WO 2004/112687 PCT/AU2004/000866 -112 5 12.4% 2.5. 38.7% 1.25 73.7% 0.625 77.7%/o N.A. - not applicable Example 58: Positive correlation between Bacterial Assay and Anti-viral Assays 5 Example 58.1 Positive correlation between Vpim Bacterial Assay and anti-HIV-1 Data. A correlative study was performed to measure correlation between the activity scores assigned to compounds tested in the Vpu bacterial assay and the ability of these compounds to inhibit HIV-1 in the anti-viral assay. 10 Example 58.2. Methodology The p24-antigen data for twelve compounds representing various substituted acyl guanidines was compared with the activity scores obtained for those compounds in the Vpu bacterial assay. The data from each assay wdas initially rank ordered for effectiveness. The rank order for the Vpu bacterial assay was determined from all i5 activity scores, the highest score indicating the greatest effectiveness. The rank order for the anti-HIV-1 assay was determined based on the overall average value of p24 antigen measured in culture supernatants at all of the drug concentrations tested, with the lowest score indicating the greatest effectiveness. The two rank orders generated were then compared statistically by generating the Spearman's Rank correlation 20 coefficient. Example 58.3. Results and Conclusion The Spearman's correlation coefficient was 0,785 which, by comparison with a statistical table of critical values (for n-1 2), indicates that the two rank orders are 25 significantly positively correlated (P<0.01) (Table 19a). In addition, a different comparison of the Vpu Bacterial assay rank order with a yes/no score for whether the anti-viral data indicated a p24 reduction of at least one order of magnitude, aligned the 'yes' group of compounds with the top 6 compounds by the bacterial assay (Table 19b).
    WO 2004/112687 PCT/AU2004/000866 These results are indicative that a positive correlation exists between bacterial assays and the antiviral assays as performned-according to the present invention. The bacterial assay may therefore be a useful tool in screening for compounds that exhibit anti-viral activity. 5 Table 19a. Comparison of Rank order of efficacy of 12 substituted acyl guanidines in the Vpu bacterial assay a nd antl-HIIV assay. Compound Bacterial assay p24 djA2 ___________________ rank order rank order (3-bromocinnarnoyDguanidinq 3-(trifluoromethyl)einnamoyLguanjidine 2 2 0 3 -meth ylnn no y]gu anidin e 3 30 -cimmanoylguandin 4 4 0 Uras -3-(I-r@PlIhYi)acryloYlguaniaiie 5.5 7 2.25 6-rnotloxy-2-naphthoylguanidine 5.5 -5 0.25 4-phenylenzoylguanidine 7 1116 (5-phenyl-penta-2,4-dienoy1guanidi 8 9 1 N-(3-phenylprqpanoyl)-N'- 9 .12 9 pheaylguanidine Hexamethylene amiloride1066 Sum diA2 61.5 Spearman correlation coefficient 0.785 Table 19b. Compounds Vpu Bacterial- 'I t ltxlog Ordered by p24 raik order Bacterial assay rank Ireduction seen in score order Jp 2 4 assay (3brmoinamylguaniin 4.3 1 yes 3-(tIM22uorocth ylrofinmpylguanidine 3.7 2 1 yes 37itylcmol anidine 3.4 3 yes oinnmoylguanidine 3.0 4 yes trns--(naty)acxyoyguwiidine 2.9 5.5 yes 6-metboxy-2-naphtho lguat-ddin6 2.9 5.5 Y_____S _ 4-phenylbenpoylguandine 2.8 7 no (5p jy-peqnta-2,4-dienoy1)gg2Ridine 2.6 8no N-(3-phenylpropanoyl)-N'- 2.2 9 no phenylguanidine ____ _____ Hexamethylene amilorde 1.9 I 10 n -2-napthylacetoy 1 anidine 1.2.I 11 _____ WO 2004/112687 PCT/AU2004/000866 -114 (trans-2- 0.4 12 no phenyloyclopropanecarbonyl)guanidine 5 Example 58.4. Correlation Between Percent inhibition of MHV plaque formation and MHV-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to compounds when tested in the Mouse Hepatitis Virus E-protein bacterial bie-assay 10 and the percent inhibition exhibited by those compounds in the Mouse Hepatitis Virus plaque assay. Example 58.5. Method: MHV plaque reduction activity data for 96 compounds screened were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of 15 compounds. This was performed for the data generated by exposure to both 10pM and IpM concentrations of the compounds, giving rise to two rank order lists. Similarly, a rank order list was generated for the MRVE bacterial bioassay scores for the same 96 compounds. Where one or more compounds had the same score, the rank values for that group were averaged. 20 Spearman's statistical test for [as described in "Mathematical Statistics with Applications" ( 2 M edn): Mendenhall, W., Scheaffer, RL.,& Wackerly, DD. Duxbury Press, Boston Massachusetts - 1981] was used to compare rank orders. Briefly, this involved calculating the Sum of squares (SS) of the differences between rank values for each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) 25 according to the formula: Rs - 1-(6.SS/n(n -1)), where n is the number of compounds ranked (96 in this case). Rs is then compared to a Table of critical values to determine statistical significance (P values). Example 58.6. Summary of Results: 30 This table summarises the Rs and P values generated as a result of the indicated pairhwise comparisons between rank orders.
    WO 2004/112687 PCT/AU2004/000866 -115 Table 20 RS +ive correlatf'or Comparison PIve correlate Plaque at 10pM Plaque at 1pM 0.689 >99.5 Yes Bacterial Plaque at 10M 0,444 >99 Yes Plaque at I M 0.406 >98.5 Yes Randomised order -0.382 n/s No Example 58.7. Conclusions: The rank order comparison of 96 compounds assayed in the bacterial bio 5 assay and the antiviral assay show that MHVE bacterial assay rank order for the compounds tested is significantly positively correlated with the rank orders generated by the MHV plaque reduction assay. The significant correlation between the assays is highly indicative that either assay may be utilised to identify compounds that may be useful. The bacterial assay may thereby be a useful tool in screening for 10 compounds that exhibit anti-viral activity. Example 58.8. Correlation Between Percent inhibition of 229E plaque formation and 229E-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to 15 compounds when tested in the Human Coronavirus 229E E-protein bacterial bio assay and the percent inhibition exhibited by these compounds in the Human Coronavirus 229E plaque assay. Example 58.9. Method: 20 229E plaque reduction activity data for 97 compounds screened against 2.5 pM compound concentration were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of compounds. Similarly, a rank order list was generated for the 229E E bacterial bioassay scores for the same 97 compounds. Where one or more compounds had the same score, the rank values for that group were averaged. 25 Spearman's statistical test for [as described in "Mathematical Statistics with Applications"
    (
    2 n a edn): Mendenhall, W., Scheaffer, RL.,& Wackerly, DD. Duxbury Press, Boston WO 2004/112687 PCT/AU2004/000866 -116 Massachusetts - 1981] was used to compare rank orders. Briefly, this involves calculating the Sum of squares (SS) of the differences between rank values for each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) according to the formula: Rs = 1-(6.S/u( 2 -1)), where n is the number of compounds ranked (97 in this 5 case). Rs is then compared to a Table of critical values to determine statistical significance (P values). Example 58.9.1. Summary of Results and Conclusions 10 This table summarises the Rs and P values generated as a result of the indicated pairwise comparisons between rank orders. Table 21 Rs P +ive correlatio Comparison Plaque at 2.5p1 Bacterial 0.584 >99.5 Yes randomized -0.382 s No The results above indicate that the 229E bacterial assay rank order for the 15 compounds tested is significantly positively correlated with the rank orders generated by the 229E plaque reduction assay. This result combined with that shown in Examples 49.1 and 49.4, provides strong evidence that either assay may be utilised to identify compounds that may be useful. The bacterial assay may thereby be a useful tool in screening for compounds that exhibit anti-viral activity. 20 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications The invention also includes all of the steps, features, compositions and compounds 25 referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
    WO 2004/112687 PCT/AU2004/000866 -117 BILIOGRAPHY Barry, M., Mulcahy, F. and Back, D - J., Br J Clin Pharmacol, 45: 221 (1998) Decks, S.G., West I Mod, 168:133 (1998) Miles, S.A., J Acquir Immune Deft Syndr Hum Rotrovirol, 16 SuppI 1: S36 (1997) 5 Miles, S.A., J. Acquir Immune Defic Syndr Hum Ratrovirol, 16 Suppl 1: S I(1998) Moyle, G.J., Gazzard, B.G., Cooper, D.A. and Gatell, J., Drugs, 55:383 (1998) Rachlis, A.R. and Zarowny, D.P., Cmaj, 158:496 (1998) Vella, S., Fragola, V. and Palmisano, L., J Biol Regul Homeost Agents, 11:60 (1997) Volberding, P.A. and Deeks, S.G., Jama, 279:1343 (1998) 10 Volberding, P.A., Hosp Pract (Off Ed), 33:814, 87-90, 95-6 passim. (1998) Miller, R.H. and Sarver, N., Nat Med, 3:389 (1997) Mitsuanya, H., Enzyme Inhibition, 6:1 (1992) Moore, J.P., Science, 276:51 (1997) Thomas, M. and Brady, L., Trends Biotechnol., 15:167 (1997) 15 Balliet, JW., Kolson, D.L., Eiger, G., Kim, F.M., McGann, K.A., Srinivasan, A. and Collman, R., Virology, 200:623 (1994) Wcstervclt, P., Henkel, T., Trowbridge, D.B., Orenstein, J., Heuser, J., Gendelman, H.E.and Ratner, L, J Virol, 66:3925 (1992) Ewart, G.D., Sutherland, T., Gage, P.W. and Cox, G.B., 3 Virol, 70:7108 (1996) '20 Schubert, U., Henkledin, P., Boldyreff, B., Wingender, B., Strebel, K. and Porstmann, T. I Mol Biol, 236;16 (1994) Friborg, J., Ladha, A., Gottlinger, H., Heseltine, W.A. and Cohen, E.A., Journal of Acquired Immune Deficiency Syndromes & Human Retrovirology, 8:10 (1995) FuJ., TanB.H., Yap,E.H., Chan,Y.C. and Tan,Y.H. (1992) Full-length eDNA 25 sequence of dengae type vi is (Singapore strain 5275/90). Virology 188 (2), 953 958 Love, C.A., Lilley, P.E. and Dixon, N.E., Escherichia coli. Gene, in press. (1996) Yamato, I., Kotani, M., Oka, Y. and Anraku, Y., Escherichia col. Journal of Biological Chemistry, 269:5729 (1994) 30 Rosen, B.R., ATP-coupled solute transport systems. Escherichia coli and Salmonella typhimurium: Cellular and molecular biology 1: (1987) Editor: Neidhardt, P.C.
    WO 2004/112687 PCT/AU2004/000866 -118 American Society for Microbiology. Piller, S.C., Ewart, G.D., Premkumar, A., Cox, G3. and Gage, P,W., Proceedings of the National Academy of Sciences of the United States of America, 93:111 (1996) Lu, Y.A., Clavijo, P., Galantino, M., Shbon, Z.Y., Liu, W. and Tam, J.P., Molecular 5 Immunology, 28:623 (1991) Harlow, E. and Lane, D., (1988) Antibodies: A laboratory manual. (ed). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 'Vol:'. Varadhachary, A. and Maloney. P -C., Molecular Microbiology, 4:1407 (1990) - 44 New, R.C.C., (1990). Liposomes: A practical approach. The Practical Approach 10 Series. Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH. (2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Coll. 2002 Mar 8;108(5):717-25. 15 Rickwood, D., Harries, B. D. (eds). IRL Press, Oxford. Miller, C., (1986) Ion channel reconstitution. (ed). Plenum Press, New York and London. Fear, W., Kesson, A.M., Naifl, H., Lynch, G.W. and Cunningham, A.L., J. Virol, 72:1334 (1998) 20 Kelly, MD., Naif, H., Adams, S.L., Cunningham, A.L. and Lloyd, A.R., J. Immunol, 160:3091 (1998) New, RC.C. (ed.), Liposomes: apractical approach. IRL Press, Oxford (1990) rice, A.L., Kerr, I.D. and Sansom, M.S., FEBSLett, 405(3):299-304 (1997) Moore, P.B., Zhong, Q., Husslein, T. and Klein, M.L., FEBS Left, 431(2):143-148 (1998) 25 Schubert, U., Bour, S., Ferrermonflel, A.V., Montal, M., Maldarelli, F. and Strebel, K..Joumal of Virology, 70(2):809-819 (1996a) Suastrom NA, Prenmkumar LS, Premkumar A, Ewart 0, Cox GB, Gage PW (1996), Ion channels formed by NB, an influenza B virus protein. J Membr Biol. 1996 Mar150(2):127-32 WO 2004/112687 PCT/AU2004/000866 -119 Willbold, D., Hofftnann, S. and Rosch, P., Eur J Biochem, 245(3):581-8 (1997) Wray, V., Kinder, R., Federau, T., Henklein, P.. Bechinger, B. and Schubert, U., Biochemistry, 38(16):5272-82 (1999) 5
    权利要求:
    Claims (168)
    [1] 1. An acylguanidine with antiviral activity.
    [2] 2. An antiviral compound of Fornmula I O N ,R2 R N N H I 5R4I. wherein RI-R 4 are independently aromatic groups, heteroaromatic groups, alkylaromatic groups, alky1heteroaromatic groups, alkenylaromatic groups, alkenylh teroaromatic groups, cycloalkylaromatic groups, cycloalkylheteroaromatic groups, aryloxyalkyl groups, heteroaryloxyalkyl 10 groups, said groups are mono or polycyclic, and are optionally substituted with one or more substitutents independently selected from hydrogen, hydroxy, nitro, halo, amino, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, Cs.alkyl, Cj. S 6 alkyloxy, Cs .cycloalkyl, halo-substituted C m . 6 alkyl, halo-substituted C. 15 0 alkyloxy, phonyl, C],alkeneyl, C.cyeloalkeneyl, C 1 . 6 alkeneoxy, benzo, H2N N aryl, substituted aryi, PrS, O, or NH2 0
    [3] 3. An antiviral compound of Formula I R3 O N Ri N N H I 20 I or pharmaceutically acceptable salts thereof, wherein, R, WO 2004/112687 PCT/AU20011000866 R, NN: (E)) ,N NN or00I. R27 R3ad7 r neednl yrgn WO 2004/112687 PCT/AU2004/000866 -122 ~0 0 ' N, " O O A A AN, O A I II or BODWYFL 5 and wherein X -- hydrogen, hydroxy, nitro, halo, C1. 6 alkyl, C 1 .6alkyloxy, C,.6cycloalkyl, halo-substituted C. 6 alkyl, halo-substituted Ct 6alkyloxy, phonyl, C 1 . 6 alkeneyl, C_ 6 cycloalkeneyl, Ct_alkeneoxy, or benzo; 10 Ra, Rb, Ro, Rd, R., RT, Rh, Rk, RL, Rtm, R, Ro, R1p indepeodently-= hydrogen, amino, halo, C t.saIkyl, C1.salkyloxy, hydroxy, aryl, substituted ary!, substituted amino, mono or dialkyl-substituted amino, oycloalkyl-substituted amino, aryl-substituted amino, 11 3 C%. S 1 2 NN n2 0 or PrS; 15 R s , R independently = hydrogen, hydroxy, halo, or C 1 , 4 alkyl; Rj = hydrogen, amino, halo, CI.salkyl, C1.salkyloxy, hydroxy, aryl, substituted aryl, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, PrS, HzN YN ,or NH 2 0 20 WO 2004/112687 PCT/AU2004/000866 -123
    [4] 4. A pharmaceutical composition comprising an antiviral compound according to any one of claims 1 to 3, and optionally one or more pharmaceutical acceptable carriers or derivatives, 5
    [5] 5. The pharmaceutical composition according to claim 4, further comprising one or more known antiviral compounds or molecules.
    [6] 6. A method for reducing, retarding or otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said virus 10 or exposed to said virus with a compound according to any one of claims 1 to 3.
    [7] 7. The method according to claim 6, wherein said virus is a Lentivirus. 15
    [8] 8. The method according to claim 7, wherein said Lentivirus is Human Immunodeficiency Virus (HV).
    [9] 9. The method according to claim 8, wherein said compound is selected from the group consisting of: (3-Chlorocinnamoyl)guanidine, (3-Bromocinnamoyl)guanidine, (2-Chlorocinamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)oinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methylcinnamoylguanidine, 2-methylcinnamoylguanidine, 2,3-dimethylcinnamoylguanidine, Cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, trans-3-(1 -napthyl)aoryloylguanidine, 3,4-dichlorocinnamoylguanidine, 2,6-dichlorocinnamoylguanidine, 4-phenylbenzoy]guanidine, 2-ethyloinnamoylguanicdine, (4-Chlorooimnnamoyl)guanidine, 2-napthoylguanidine, 2,5-dimethylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, WO 2004/112687 PCT/AU20041000866 -12 4 ( 5 -PhenYl-Penta-2,4-dienoyl)guanidine, 3 -phenylc-innaxnaylguanidin;, ( 4 -Bromooinnamoyl)guanidine, $5($'bromopbenyl)penta2,4.dinoylgunidine, 3-(cydloheX-I-en-1 -yl)oinnamoylguanidine, 3 -(ffluoromethioxy)oizmamoylguanidin;, Z-(trifluoromcthyl)cinnaxnoylguanidine, N,'N-~bis(3phonylpropanoyl)-Nh1-phenyganidine, 2-ethoxycinm~oylguanidjne, N-(3-phenylpropanoyl)-N-phenylguaxiidine, 4 -(friflnoronethyl)cinnaraoylguanidin;, ( 4 -Methoxycinnamoyl)guanidin; 2-t-bntyleinnamoylgaanidine, 4-methylCi=naOylguanIdine, 2-:fluorocinnamoylguanjdine, 2-phenylcimamoylgumidine, N-(6-H4ydroxy-.2-napthoyl)-N'-phenylguanidin;, 3-t-butyleinmnoylguanidine, 3,-difluorooinnamoylguanidine,' 5-(N,N-hexaniethyonq)axniloride, 3-fluorovinnamoylguanidino, S-bromo-2-xnethoxycinnamoylguanidine, 3-etboxycirmmoylguanildine, 3,4-(methaylenedioxy)cinnamoylgu~nidine, (2-Methoxycinm~oyl)guanidine, 24 Dicloro~enzanil RdI, 2 ,3,5,%6,-tetramctylcinnamoylguanidine, 3-(2-naptbyl)acryloylguauidine,' 2-(1-napthyl)acetoylguanidin;, 2,3-diluorocizmamoylguanidino, (3-Moffioxyciimamoyl)guanidino, 4-isopropyloinnamoylganidine, 2,4,6-trimethylc-innsmoylguanidn; N-(ainnaaOy1)-N~phenyIguanidine, 2-(cyolohex-l-en-lyl)cinnamoylguanjdjne, 2-(2-napthyl)aoetoylguanidine, (A-Hydroxycinnamoyl)guani din;, 4-phenylaimnamoylguanidine, 4-fluorocinnamoylgumando, NN-bis-(rinnamoyl)-W-phenylguanidinc, (2J-uraacryloyl)guanidine, Phenandil methanesulonate salt, Benzamil hydrochloride, (3-Nitrocinamoyl)guanidin; Bonzyaylguanidinc, (4-Phenoxybenzoyl)guanidine, 3-(trans-hept-1"eta-4-yl)cinnainoylguanidine, 5-(N-Methy1-N-isobutyI)=ioride, WO 2004/112687 PCT/AU2004/000866 -125 2-cyclohexyloinnamoylguanidine, 4-ethoxycinnamoylguanidine, 2,4-dichlorocinnamolyguanidine, 5-(N-Ethyl-N-isopropyl)amiloride, N-amidino-3-amino-5-hoxamethyloneimino-6-phenyl 2-pyrazinecarboxamido, (a-Methylcinnamoyl)guanidino, cinnamoylguanidine hydrochloride, [(4-Chlorophenoxy-acetyl]gnanidine, N-amidino-3-amino-5-phenyl-6-chloro-2 pyrazinecarboxamide, 5-(4-fluorophenyl)amiloride, (trans-2-Phonylcyelopropanecarbonyl)guanidine, (2-Nitrocinnamoyl)guanidine, trans-3-Furanacryoylguanidine, 1-napthoylguanidine, 5-tert-butylamino-amiloride, 3-methoxy- HlA, (3-phonylpropanoyl)gnanidine, 4-t-butyloinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, N,N'-Bis(3-phenylpropanoyl)guanidine, N-Benzoyl-N'-cinnamoylguanidine and 1-bromo-2-napthoylguanidine.
    [10] 10. The method according to claim 8, wherein said compound is selected from the group consisting of 4-phenylbenzoylguanidine, (3 bromocinmamoyl)guanidine, 3-(trifluoromethyl)cinnamoylguanidine, 5 5 (N,N-hexamethylenc)amiloride, and (5-Phcnyl-penta-2,4 dienoyl)guanidine.
    [11] 11. The method according to any one of claims 8 to 10, wherein said I-IV is HIV-1. 10
    [12] 12. The method according to claim 6 wherein said virus is a Coronavirus.
    [13] 13. The method according to claim 12, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS). 15 WO 2004/112687 PCT/AU2004/000866 -126
    [14] 14. The method according to claim 13, wherein said compound is selected from the group consisting of 2,3-difluorociamnoylguanidine, 3,4-dichlorocinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, (3-Chlorocinnamoyl)guanidine, 3-(oyclohex-1-en-1-yl)cinnamoylguanidine, 2,5-dimetby1einnamoy1guanidine, trans-3-(1-napthyl)acryloylguanidine, 4-isopropyloinnamoylgnanidine, (3-Bromocinnamoyl)guanidine, 6-methoxy-2-naphthoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenylcinnamoylguanidine, (2-Chlorocinnamoyl)guanidine, 2'4 DichloroBenzamil C1, 4-ph'enyloinnamoylguanidine, 4-(trifluoromethyl)cinnamoylguanidine, 3-(trifluoromethoxy)oinnamoylguanidine, 3-(frifluoromethyl)cinnamoylguanidine, 2-ethoxycianamoylgnanidine, cinnamoylguanidine hydrochloride, 4-ethoxyoinnamoylguanidine, (2-Bromocinuamoyl)guanidine, 2,6-dichlorocinnamoylguanidine, 3,4,5-trimethoxycinnamoylguanidine, 5-tert-butylanino-amiloride, 3-t-butyloinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Chlorocinnamoyl)guanidino, 2-t-butylcinnamaylguanidine, 2-cyclohexylcinnamoylguanidine, 6-Iodoamiloride, 3-(trans-hept-l-on-1-yl)oinuamoylgaanidine, (4-Bronocinnamoyl)guanidine, (4-Hydroxycinnamoyl)guanidino, N-(3-phenylpropanoyl)-N'-phenylguanidine, (3-Nitrocinnamoyl)guanidine, 3-fluorocinnamoylguanidine, 2-(1 -napthyl)acctoylguanidine, 2-ethylcinnamoy1guanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, 2-napthoylguanidine, 5-(4-fluorophenyl)amiloride, 2-(trifluoromethyl)cinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 -127- N-(6-flydroxy-.2-napthoyl)-N-phenylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, N2W-bis(3phenylpropanoyl)-'l-pbenyguwnidin;,, I -napthoylgunnidine, Bcnzamil hydrochfloride, 3-naethoxy -HM4A, 4-methylcinnamoylguanidinc, .4-fluorocinnamoylgundine, 3,4-(nefthylenedioxy)einnamoylguanidino, 5-(NN-hexamethylene)amilorid, N-(cinnamoyl)-N'phcnylguanidin;, 5-(N-Ethy1-N-isopropyl)amiloride, 3-methyloinnamoylguanidine, 2-mnethyoimxamoylgaanicliue, 2,3,5.,6,-.tcfamethylcinnamoylguanidine, trans-3-Faranacryoylguamidine, * (4-Methoxycinnamoyl)guanidine, (2-Furanacryloyl)guanidine, (3-phenylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidine, Cinnaroylguanidine, (2-Methoxyeinaoyl)guanidine, [3-(3-Pydyl)aryloyl]gunidiic, 4-phenylbenzoylguanidine, 2,4-dichlorocinnamolyguanidine, (3-Methoxycinnarmoyl)guanidine, 2-fluorocinnamoylguanidine, (4-Phenoxybenzoyl)guanldine, (a-Methylcinnamoyl)guanidinc, 5-(3'-bromophenyl)penla-2,4-dienoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, (Quinolie-2-carbonyt)gnandine, (Phenylaeetyl)guoAndine, N,N'-Bi 's(arnidino)nthalene-2,6-dioarboxamide, 6-bromo-2-napthoylguanidine, 1-brorno-2-napthoylguanidin;, 2-ehloro-6-fluorocinnainoylguanidinc, [(4-Chlorophenoxy-acetyflguanidinie, Phenandi methanesulfonate salt, N'-Ilenzoy1-N'-cinnamoylguanidine and N-(2-napthoyl)-N-phienylguanidine.
    [15] 15. The method according to claim 13, wherein said compound is selected from the group consist Of CinnaMOYlguanidine, trans-3-(1 napthyl)acryloylguanidine, and 6-methoxy-2-naphthoylguanidine. WO 2004/112687 PCT/AU2004/000866 -128
    [16] 16. The method according to claim 12, wherein said Coronavirus is human Coronavirus 229E.
    [17] 17. The method according to claim 16, wherein said compound is selected 5 from the group consisting of 4 -isopropylkinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)oinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-isopropylciunamoylguanidine hydrochloride, 3-t-butyinnamoylguanidine, 2-t-butylcinnamoylguanidine, trans-3-(-napthyl)aeryloylguanidine, 5-bromo- 2-m 4tho xyoin n amoylguanidine, 2,3-difluorocinnamoylguanidine, 3-(2-napthyl)aciyloylguanidine, 2-phenylcinnamoylguanidine, 3-phonyloinnamoylguanidine, 3-(cyclohex--en-1-yl)cinnamoylguanidine, 4-phonylbenzoylguanidine, 3-(trifluoromethyl)oinnamoylguanidine, (4-Phonoxybenzoyl)ganidine, 4-(trifluoromethyl)oinnamoylguanidine, 2 -(cyclohex-1-en-lyl)cinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 5-(N,N-hexaethylene)amiloride, I-napthoylguanidine, 5-(4-fluorophenyl)amiloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bromooinnanoyl)guanidine, 2,5-dimethylchmamoylguanidine, 2 -(trifluoromeffiyl)cinnmnoylguanidine, 6-mothoxy-2-naphthoylguanidine, (4-Chlorocinnamoyl)guanidine, (3-Methoxycinnamoyl)guanidine, 5-bromo-2-fluorocinnamoylguanidine, 5-(NN-Dimethyl)amiloride hydrochloride, Cinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, (a-Methylcinnamoyl)guanidine, 4-phenylcinnamoylguanidine, 2,6-dichlorocismamoylguatidine, (2-Bromooinnamoyl)guanidine, 2,4,6-trimethylcinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 -129. (frans- 2 -Phe-nylcyclopropanecarbonyl)guanjj 11 , ( 3 -Cblorocinimoyl)gunnidine, 2 -(l-napthyI)acetoYiguanpiin;, 2 -0411aylimoy1guanidine, 2-oyclohexykcimamoyiganidinj, ( 4 -Hydroxycinnamoy)gunidinre, 2 -ethoxycnniamoylguanjiin;, 3-medlylcinnamoylguanidine, 2 -meffiyicbnamaylgaanjcjjne, 3 -fluorocinnamoylguidine, cinnamOYlgusnidine hydrochloride, 2 ,3-dimethiykinnamoyjguanjijin;, 2 -fluorocinm~oylguaniaine, 4 -fluorocinnamoylguamidin;, 3 , 4 -difuorocinnamoyguanidine; S-teft-butylamino-ailoride, 2-napthoylguanidin;, NN'-Bis43-phenylpropanoy)gpji; 4 -methylcbmiamoylguanidjne 5- 34 -romophoniy)penta-2,4dienoygiidin~, 2 , 3 5 56 ,-etefmethycinnamoylgaadjdjne J-e-thoxycinnmoylguanidin;, N,N-big(3PhnYlPropanoy)'phnyg lu i c; ( 4 -Mothoxycinnamoyl)guanidjne, ( 2 -Chlomcchmnoy)guncdjne ( 3 -Nitrocinnmoyl)guaiine, 4 -ethoxcnnmoylguaidin, 3 , 4 ,-hnetaoxyinnamoylguanidine, 2-(2-nphylpaoyl)gNanidji luaniin S-( 2 '-broinopheuy)penta.2,4. dienoylualndme, ( 4 -Bronlocirmanoyl)guantidine, ( 2 -Nitrocinnamoy1)gua~njai, ( 3 -Chlorocinnamoyoguajcijne 5 ( 4 -Metlioxyeirmamoy1)garsjn* f(B)- 3 -(4-Dilnethylaminaphenyl)y2 methylacryloyl]guwiddn;, N-Benzoy14'frcinnamoy~guanidine, 4 -pbenylbenoylguanidine, frans-3-4 uanacryoyIguandine, N-amidino-3-aminfo--pheny1-6..csoro-2 Pyrazinecarboxamide, WO 2004/112687 PCTAU20011000866 -i30 N-(cinnamoyl)-N'phcnylguanidine, Cim~oylguanidine, 3-methoxy-amiloride, ( 3 -phenylpropanoyl)guanicline, 3-methoxy-HMA, Benzyoylguanidine, N-amnidino-3,5-dianmino-6-phyny-2 Pyrainecartoxamide, (QUinoline-2-carbonyl)guandin, I 3-(3-Pyiidyl)acryloyl]guanidine, N-Cinnamnoy-N',N -dinefiylguafldine, N-(2-napthoyl)-N'-phenylguanidinc and (Phenylacetyl)gua~nide.
    [18] M8 The method according to claim 16, wherein'said compound is selected froMl the group consisting of 2-t-butylcinmamoylguaniclino, 4-isopropyleinnamoyguariidine, 3A4-dachlorocinnamoylgaanidine, 3-(trifluomomethoxy)cinnamaylguanidine, 2,6-dichlorocinnamoylgaanidine, 2 -(cyclohex-1-en-IyI)chmwnmoylguanidiue, 2-o-yclohexylcinnamoylguanidine, S-brcnno-2-methoxycinnamoylguanidine, 2-phenylcinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-phenylcinnamoylguanidine, (3-Bromocfinnmoyl)guanidine, 5-Q'tN-bexametbylone)amiloride, frans-3-(1-napthy)aoryloylguanldine, 3-(2-napthyl)=oyloylguanidine, 2,4-dichlorocinnamlgandn, 3-(trifluoromethyl)cinnamoylguanidin; 5-bromo-2-fluorocinnamoylguanidin;, 4-inethylcinnamoylguanidine, (4-Chlorocinnamoyl)guaaidiae, 3-fluorocinnamoylgaanidine, 3-(cyolohex-1-cn-1 -yl)chmnamoylguanidine, (a-Methylcinnatoyl)guanidine, 2,3,5,6,-tetraxethyloinmoylguanidine, 2-f luorocinnamoylguanidine, (3-Nitrocinnamoyl)guanldlne, 2,5-dimethylcinmamoylguanldline, 3--utyloinnamoylguanidino, (3-Methoxycinnarnoyl)guanidine, 3-methylcinnamoylguamdmc, 3-isopropylcinnaoylguanidine hydrochloride, WO 2004/112687 PCTAU20011000866 (2-Bromocnnaoyl)uatidine, 3-ethoxycinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidiine,, (2-Cfidoroohnn~oy1)guanidine, 4-ethoxycinnamoylguanidine, 4-fluorocinzmxoylguanidin;, 3,4-difluorocinnamnylguanidin;, N-(3-phcnylpropanoyl)-N Phenylguidine, 2A4,6-trimcthylcinnatnoylguanidinae, 2-mothylcimtamoyguanicline, (trans-2-Phenylcyclopropanecarbonyl) guanidine, (4-Phenoxybenzoyl)guanidine, (2-Methoxycha:=noy])guanidIne, Cinmamoylguanidine, 3,4-(methylenedioxy)cinnanoylguanidine, NW,13-is(anmino)napthalene-2,6 Dicarboxamide, 2,3-dimethylinnamoyguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, NN'-Bis(3-phenylpropanoyl)gaanidinc, 2,3-difuoroinmamoylguanidinp, 1-napthoylguanidine, 6-rnethoxy-2-uaphthoylguanidine, 5-(N,N-flimethyl)andloxide hydrochloride, 2-ettioxycinnamoylguanidine, 2-naptboylguanicline, 3,45-trimethoxycinoemoylguaidie, 2-(frifluoromethyl)cinnamoylguanidine, cinnmaylguanidine hydrochloride, (4-Hydroxycinnamoyl)ganidIne 5-(4-fluorophenyl)sniiloride, 2-(1-napthyl~aoetoylguanidine, (2-Furanaoryloyl)guanidine, N-Cinnamoyl-N',N'-dimnethylgmnidine, 2-(2-niptlayl)acctoylguanidine and' NN-bis(3pbenypropanoyl)-N"-. Phenylguanidine.
    [19] 19. The method according to claim 12, wherein said Coronavirus is human Coronavirus 0C43. WO 2004/112687 PCT/AU2004/000866 -132
    [20] 20. The method according to claim 19, wherein said compound is selected from the group consisting of 3-methylinnamoylguanidine, trans-3-(1-napthyl)acryloylguanrdine, (3-Bromocinnamoyl)guanidino, (2-Chlorocinnamoyl)guanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethyl)oinnamoylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, 4-isopropylcinnamoylguanidine, Cinnamoylgaanidine, 6-methoxy-2-naphthoylguanidine, 2,4-dichlorocinnamolyguanidine, (4-Chlorocinnamoyl)guanidine, 5-(N,N-hexamethylene)amiloride, (4-Bromocinnamoyl)guanidinc, 2,6-dichloraocinnamoylguaaidine, 5-bromo-2-methoxycimanamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-(trifluoromethoxy)cinnamoylguanidine and 2-t-butylcinnamoylguanidine.
    [21] 21. The method according to claim 12, wherein said Coronavirus is porcine 5 respiratory Coronavirus (PRCV).
    [22] 22. The method according to claim 21, wherein said compound is selected from the group consisting of 5-(N,N-hexamethylene)amiloride, 6-methoxy-2-naphthoylguanidine, Cinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phonylguanidine, 3-methylcinnamoylgaanidino, (3-Bromocinnamoyl)guanidine, (trans-2-Phenyloyolopropaneearbonyl)guanidine, trans-3-(1-napthyl)aoryloylguanidine and 2-(2-napthyl)acetoylguanidino. 10
    [23] 23. The method according to claim 12, wherein said Coronavirus is bovine Coronvirns (BCV). WO 2004/112687 PCT/AU2004/000866 -133
    [24] 24. The method according to claim 23, wherein said compound is selected from the group consisting of (3-Bromooinnamoyl)guanidine, 3-(trifluoromethyl)cinnamoylgoanidiue, 6-methoxy-2-naphthoylguauidiue, 5-(N,N-hexamethycne)amiloride, trans-3-(l1-napthyl)acryloylguanidine, Cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2-(2-napthyl)acetoylgoanidine, (trahs-2-Phenyleylopropanecarbonyl)guanidine, N-(3-pheny1propanoyl)-N'-phnylguanidino and 4-phenylbenzoylganidine.
    [25] 25. The method according to claim 12, wherein said Coronavirus is any one of 5 the known coronavirus isolates listed in Table 1.
    [26] 26. The method according to claim 25, wherein said compound is selected from the group consisting of 4-isopropylchinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)ohamnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-isopropyleinnamoylguanidine hydrochloride, 0
    [27] 27. The method according to claim 6, wherein said virus is the Hepatitis C viras.
    [28] 28. The method according to claim 27, wherein said compound is selected from the group consisting of 2,3-dimethylkinnmamoylguanidine, 2,4,6-trimethyloinnamoygpanidine, 51omo.2-fluoroeinnamoylganidine, (4-Bromocinnamoyl)guanidine, 2,5-dimethylinnamoylguanidine, 3-(trifluoromethy)oianamoylguanidine, 4-(trifluoromethyl)oinnamoylguanidine, 6-methoxy-2-naphthoylguallidine, (2-Chlorocinnamoyl)guanidine, (4-Chlorocinnamoyl)guanidine, (2-Bromocinmnamoyl)guanidine, WO 2004/112687 PCT/AU20011000866 2,6-diehlorocinuamoylguauiidine, (3-Bromocimnamoyl)guanidin; (3-Chloroci-nnamnoyl)guianidine, 2-(trifluoromethyl)einnamoylguianidine, (4-Phenoxyberizoyl)guanidino, 3,4-dichlorooinnnoylgaanidine, 4-isopropykinnamnylguanidine, trans-3-(l-napthyl)acryloylguanidine, 4-t-butylinnamoyguanidine., 2-t-butykinnamoylguanidine 2-cthykin-namoylguanidinc, 4-methylcinnamoylguanidine, 5-bromo-2-methoxycinnaioylguanidine, 3-Qrifluorometboxy)chwamoylguanidine, 2-oyclohoxykinnamoylgluandi 1-napfthylguanidin;, 3-t-biztylcinnamoylgmnidine, 4-phenylbenzoylguidine, (5-Phanyl-penta-2,4-dienoyl)guanidine, N-(ciimanoyl)-N'phenylguanidino, 3-isopropyloinxzamoylguanidinc hydrochloride 3 Benzamil hydrochloride, N-(3-phc-nylpropanoyl)-N'-phcnylguanidino, NN-bis(3pbenylpropanoyl)-N"41-phenylguanidine, 3-(2-nlapthyl)aoryloylguanidine, 5-(N-Methyl-N-isobutyl)arnilorido, 2'4 DichioroBenzani EdC, S-tert-butylw-aniloride, 5-(N-Bthyl-N-isopropyl)aniiloride, (4-Methoxyclnnamoyl)guanldie, 4-Ifluorooinnamoylguanidine, (3-Nitrocinnmoylguanidine, 4-ethoxyeinnamoylguanidin;, (4-Hydroxyoinnamoyl)gusndine, (frans-2-Phenyloyolopropanecarbonyl)guanidine, 3-cthoxycinnamoylguanidine, 2,3,5,6,-tctramcthykoinnaoylguanidine, 4-phenylcinnamoylguanidine. trans-3-Pmianacryoylguanidin;. N-(6-Hydroxy-.2-napthoyl)-N-phenylguaiiidine, (2-Fiwanacryloyl)auanidine, 3-(cyclohex-l -en-l-yl)cinnanaoylguanidin; cinnamoylguandine hydrochloride, 5-(NN-hexuauethylene)andlodde, 2,3-ifhilorochnmnmoylguanidine, 2-(1-napthyl)acetoylguanaidin;, (a-Methy'rcinnamoylguanidine, (2-.Nitrocinnanoyl)guanidine, WO 2004/112687 PCT/AU2004/000866 -135 6-lodoamiloride, 3,4-(methylnedioxy)cinnamoylguanidine, 2-ethoxycinnamoylguanidine, CinnamoylguanMidino, 2-phenyloinnamoylguanidine, 2-(cyolohex-1-en-l1yl)ohinnamoylguanidine, 2-napthoylguanidine, 3-phonyloinnamoylguanidine, 5-(N,N-Dimcthyl)amiloride hydrochloride, 5-(4-fluorophenyl)anamiloride, (3-Methoxycinnamoyl)guanidine, 2-fluorocinnamoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, (3-phenylpropanoyl)guanidine, 2-chloro-6-fluorocinnamoylganidine, 3-fluorocinnamoylguanidine, 2-methyleinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, 1-bromo-2-napthoylguanidine, 3,4,5-trimethoxycinnamoylguanidine, 3-inethylcinnamoylguanidine, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, Phenamil methanesulfonate salt, 2,4-dichlorosinmamolyguanidine, (4-Nitrocinnamoyl)guandine, 3,4-difluorocinnamoylguanidine and [(E)-3-(4-Dimethylaminophenyl)-2 methylacryloyl]guanidine.
    [29] 29. The method according to claim 6, wherein said virus is Equine Arteritis virus. 5
    [30] 30. The method according to claim 29, wherein said compound is selected from the group consisting of 5-(N,N-hexamethylone)amiloride, (3-Bromocinnamoyl)guanidine, trans-3-(1-napthyl)aoryloylguaniditeo, 2-t-butyIcinnamoylguanidine and 2-(oyclohox-1-en-lyl)cinnamoylguanidine.
    [31] 31, A method according to any one of claims 6 to 30, wherein said compound is provided as a pharmaceutical composition according to claim 4 or claim 10 5. WO 2004/112687 PCT/AU2004/000866 -136
    [32] 32. A method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to any one of claims 1 to 3. 5
    [33] 33. The method according to claim 32, wherein said virus is a Lentiviras.
    [34] 34. The method according to claim 33, wherein said Lentivirus is Human Immunodeficiency Virus (HIV). 10
    [35] 35. The method according to claim 34, wherein said compound is selected from the group consisting of (3-Chlorocinnamyl)guanidine. (3-Bromocinnamoyl)guanidinc, (2-Chlorocimnnamoyl)guanidine, (2-Bromooinnamoyl)guanidine, 3-(trifluoromethyl)oinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methyloimnuamoylguanidine, 2-methyloinnamoylguanidine, 2,3-dimethyloinnamoylguanidine, Cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 3,4-dichlorocimnnamoylguanidine, 2,6-dichlorocinnamoylguanidine, 4-phenylbenzoylguanidine, 2-ethyleinnamoylguanidine, (4-Chlorocinnamoyl)guanidine, 2-napthoylguanidine, 2,5-dimethyloinnamoylguanidine, 3-isopropylkinnamoylguanidine hydrochloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-phenylcinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dionoylguanidine, 3-(oyolohox-1-en-l-yl)cinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 2-(trifluoromethyl)oinnamoylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, 2-ethoxycinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, WO 2004/112687 PCT/AU20041000866 4-Qtrifluoromethiyl)ofnnamoylguanidine, (4-Methoxycinnamoyl)guanidin;, 2-tbutycinnamnoylguanidine, 4-methylcinnamoylguanidine, 2-fluorocinnamoylguanidine, 2-phenylcinnamoylgaawidine, N-(6-Hydroxy-62-napthoyl)-N 1 -phouylguanidiie, 3-t-bulyloinnamoylguanidine, 3,4-difluorochmnmoyguaidi-ne, 5-(NN-hexamethyene)amiorile. 3-fluorocinnamoylguanidinc, S-bromo-2-methoxychmaanoylguanidin;, 3-othoxycinnamoylguanidin;, 3,4-(methylonedioxy)cinnamoylguanidin;, (2-Mcthoxycinnanoy])guanidn, 2-4 DichloroBenzamil HO, 2,3,5,6,-teframethylcirmamoylguanidinc. 3-(2-napthid)aeryloylguanidine, 2-(1-napthyl~acotoylguanidine, 2,3-difluorocinmoylguanidine, (3-Methoxychmnamoyl)guanidine, 4-isopropylcinnamoylguanidine, ZA4,6-trmethylcinnamoylguanidine, N-(dnnamoyl)-N~phenylguanidine, 2-(oyclohex-l-an-lyl)oinnxnoylguanidine, 2-(2-napthy1)acetoyLguanidino, (4-I-ydroxycinamoyl)guanidine, 4-phenyleinnamoylgunidine, 4-fluorocinamoylguaaidine, NNbis(cinnamoy).N"-phenylganidine, (2-Fnranacryloyl)guanidine, Phenainil methanesulfoinate salt , Benzarnil hydrochloride, (3-Nltrocinnamoyl)guanidine, Benzyoy1juanidine, (4-Phcnoxybenzoyl)guanidie, 3-(trans-hept-1-en-1-yl)cinnamoylguunidino, 5-(N-Metbyl-N-isobutyl)nilaride, 2-cyclohexylclnnamoylguanidine, 4-.ethoxyoinnanioylguanldine, 2,4-dichlorocinnamolyguauidine, 5-{N-Ethyl-N-isopropyl)aniiloride, N-amidino-3-amino-5-hexamethyleineo-6-pheny 2-pymainecarboxaide, (a-Methylcinnwmoyl)guanidine, cinnamoylguanidinAe hydrochloride, [(4-Cblorophenoxy-acetyllguanidine, N-aznidino-3-ainino-5-phonyl-6-hloro-2- WO 2004/112687 PCT/AU2004/000866 -138 pyrazinecarboxamide, 5-(4-fluorophonyl)amiloride, (trans- 2 -Phenylcyclopropanecarbonyl)guanidine, (2-Nitrocinnamoyl)guanidine, trans-3-Furanacryoylguanidine, 1-napthoylguanidine, 5-tert-butylamino-amiloride, 3-methoxy-HMA, (3-phenylpropanoyl)gnanidine, 4-t-butylcinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, N,N'-Bis(3-phenylpropanoyl)guanidine, N-Benzoyl-N'-cinnamoylguanidine and I -bromo-2-napthoylguanidine.
    [36] 36. The method according to claim 34, wherein said compound is selected from the group consisting of 4-phenylbenzoylguanidine, (3 bromocinnamoyl)guanidine, 3-(trifluoromethyl)oimnnamoylguanidine, 5 5 (N,N-hexamethylene)amiloride, and (5-Phenyl-penta-2,4 dienoyl)guanidine.
    [37] 37. The method according to any one of claims 34 to 36, wherein said HIV is HIV-1. 10
    [38] 38. The method according to claim 32 wherein said virus is a Coronavirus.
    [39] 39. The method according to claim 38, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS). 15
    [40] 40. The method according to claim 39, wherein said compound is selected from the group consisting of 2,3-difluorocinnamoylguanidine, 3,4-dichlorocinnamoylguanidinc, 4-t-butyloinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, (3-Chlorocitmamoyl)guanidine, 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, 2,5-dimethylcinnamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 4-isopropylcinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 (3-Bromooimuamoyl)gnnldine, 6-mothoxy-2-naphthoylgugmjdjne, 5-fN-Methyl-N-isobutyl)arniloridc, 3-phonylcinananoylguaidin;, (2-Chloroclimamoyl)giiandine, 2'4 DiobloroBeuzamil HCi, 4-phenylcinnamoylguanidine, 4-(Iriflueromethyl)cinuaxnoylguanidine, S-(trifluoromethoxy)cinnamoylgnanidine, 3-(tziflnoromethyl)cinnatnoylguanidine, 2-ethoxycinnmoylgiuiine, oinnanioylguanidine hydrochloride, 4-effioxyomnnanoyguanidinb (2-Bromooinnamoyl)guanidine, 2.6-dichlorocinnainoylguanidine, 3,,-trimethoxyoinnmoYlguanidine, 5-tert-butylamino-ainiloride, 3-tbutylcinnmaoylguanidine, 5-bromo-2-fluorocinm~oylguanidine, (4-Chlorocbwm~oyl)guanidine, 2-t-butylcinnamoylguanidine, 2-oyclohexykoinnmoylguanidine, 6-Iodoamiloride, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine (4-Bromocinnamoyl)guanidine, (4-Hydroxycinnamoyl)guanidiue, N-(3-phcnylpropauoyl)-N'-phenylgua-nidine, (3-Nitrocinnamoyl)guanidine, 3-fluorocinnamoylguanidine, 2-(1-napthyl)acetoylguanidine, 2--etbyloinnmoylguanidine, 5-(NN-Dimethyl)amiloride hydrochloride, 2-napthoylguanidine, 5-(4-fluorophenyl)ainiloride, 2-(trifluoromothyl)oizmamoylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidiue, (trans-2-Phenylcyclopropancoaronyl)guanidinc, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine,, l-napthoylgaanidinc, Eenzainil. hydrochloride, 3-inetboxy -4HMAk, 4-inethylcinamaoylguanidin;, 4-fluorocirm~oylguanidine, 3,4-(methylenedioxy)cinnamoylguanidine, 5-(NN-hexamethylene)amniodide, N-(cinnamoyl)-N'phenylguanidine. 5-(N-Ethyl-N-isopropyl)amilordo, 3-methylcinnaxnoylguanidine, WO 2004/112687 PCT/AU2004/000866 -140 2-methylcinnamoylguaidine, 2,3,5,6,-tetramethyklcinnamoytguanidine, trans-3-Furanacryoylguanidine, (4-Methoxycinnamoyl)guanidine, (2-Furanaoryloyl)guanidine, (3-phenylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidine, Cinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, [3-(3-Pyridyl)acryloyl]guanidine, 4-phenylbenzoylguanidine, 2,4-dichlorocinnamolyguanidine, (3-Mothoxycinnamoyl)guanidinc, 2-fluorocimamoylguanidine, (4-Phonoxybenzoyl)guanidine, (a-Methylcinnamoyl)guanidine, 5-(3'-bromophenyl)ponta-2,4-dienoylguanidine, (5-Phenyl-penta-2,4-dienoy1)guanidine, (Quinoline-2-carbonyl)guanidine, (Phenylacetyl)guanidine, N,N'-Bis(amidino)napthalone-2,6-dicarboxanide, 6-bromo-2-napthoylguanidine, 1-bromo-2-napthoylguanidine, 2-chloro-6-fluorocinnamoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, Phenamil methanesulfonate salt, N-Benzoyl-N'-cinnamoylguanidite and N-(2-napthoyl)-N'-phenylguanidine.
    [41] 41. The method according to claim 39, wherein said compound is selected from the group consisting of oinnamoylguanidine, trans-3- (1 5 napthyl)acryloylguanidine, and 6-methoxy-2-naphthoylguanidinc.
    [42] 42. The method according to claim 38, wherein said Coronavirus is human Coronavirus 229E 10
    [43] 43. The method according to claim 42, wherein said compound is selected from the group consisting of 4-isopropylcinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 -241 3-(tilluo-romethoxy)cinnanaoylguanidine, 4t-butycinnaMoDYlguanidinq, 3-isapropylcinnamoylguanidine hydrochloride, 3-t-butyldimamoylguanidine,, 2-t-butylchmnmnoyguanidinw, traus-3-QI-nathyl)acryloylginidine, 5-bromo-2-methoxyoinnatoylguanidine, 2,3-difluorocinnamoylguanidi-ne, 3-(2-napthyl)acryloyguanidie, 2-phenylcinnamoylguanidine, 3-phenylcinnamoylguanidin;, 3-(ayclohex-l-en-1-yl)cinoaxnoylguanidine, 4-phenylbenzoygrnnidine, 3-(trifluoromethyl)cinnamoylguanidine, (4-Pheuoxybenzoyl)guanidine 4-(trifiuorornethyl)cinnamoylguandine, 2-(cyclohex-1-en-lyl)cinnemoylguanidine, (4-Bromocinnamoyl)gnanidine, 5-(NN-hexamethylene)aniioride, 1-napthoylguanidine, S-(4-fluoroplienyl)anailorido, (5-Pbhnyl-penta-2,4-dienoyl)guanidine, (3-B3romocinnamoyl)guandine, 2,S-dimeffiylcinnamoylguanidine, 2-(trifluoromethyl)oinnaznoylguanidine, 6-metxy-2-naphthoylguanidin;, (4-Chkxrocinnamoyl)guanidine, (3-Methoxycinnamoyl)guendine, 5-bromo-2-fluoroeinnamoylguandine, 5-(NN-Dimethyl~arilorido hydrochloride, Cinnaxoylgpanidine, (2-M~etlioxycinnaoyl)guanidine, (a-Methylcinnarnoyl)gaanidine, 4-phenylcinnamoylguanidinc, 2,6-dilorocinnamoylguanidine, (2-Bromocinvanoyl)guanidine, 2,4,6-tdmethyliimamoylgtianidine, (trans-2-Phenyleyelopropanecarbonyl)guaidine, (3-Chlorocinnamoyiguanidine, 2-(1-napthyl~acetaylguanidine, 2-ethylcinnanioylguanidine, 2-oyolohoxylkinnaxnoylgu.-nidine, (4-Hydroxyoinnamoy])guanidine, 2-ethoxycinnwnoylguanidine, 3-naethylcipnmoylguanidin;, 2-naethylcinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 -142 3-fluarocinnamoylguanidine, ainnamoylguanidino hydroohloiidc,, 2,3-diniethylcinnarnoylguaidine, 2-fluorocinnamoylguanidine, 4-fluoroeinnaoylguanidine, 3A-difluorocinnamoylauniddine, 5-tert-butylamino-aunilorde, 2-naptoylguanidine, NN-Bis(amidino)naptbalene-2,6-dicarboxamide, NN-Bis(3-phenylpropanoyl)guanidine, 4-methyleinanaoylguanidin;, 5-(3'-bronaophanyl)pent a-2,4-dienoylguanidine, 3-etoxycimamaylguanidine, N,N'-bis(3phenylpropanoyl)-N t -phenylguauidine, (4-Methoxycinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (3-Nitrocinnamoyl)gaanidine, 4-ethoxyoinnanoylguanidine, 3,4,5-trimeboxychmiamylguanidine, 2-(2-napty)acetoylgua~idin, N-(3-phenylpropanoyl)-N-phenylgaanidine, 5-(2'-bromophenyl)penta-2,4 dionoylguanidin; (4-1Bromocinnamoy~guanidic, (2-Nitrooinnamoyl)guanidine, (3-Chloroeinnmoyl)guianidine, (4-Mcthioxycinnainoyl)guauidine. 4-(trilluoromethyl)cinnanoylganidine, [(E-)-3-(4-Dinacthylaminophcnyl)-2 naethylacryloyljguanidin;, N-Be oyl-N'-oin~omoylguanidine, 4-pbenylbenoylguanidle, trans-3-Fanryoylguanidine, N-amidino-3-amino-5-phenyl-6-chloro-2 Pyrainecaboxaniide, N-(ci=maoyl)-N'phenylguauidine, Cinnainoylguanidine, 3-mnetloxy-anaikoride, (3-phonylpropanoyl)guaidne, 3-methoxy-JIMA, Bcuzyoylguwnidine, N-aniidino-3,5-diamino-6-phynyl-2 Pyrazinecarboxamide, (Quinoline.-2-carbonyl)guanidine, [3-(3-Pyridyl)acryloyl]guaidine, WO 2004/112687 PCT/AU2004/000866 -143-' N-Cinnamoyl-N',N'-dimethylguanidine, N-(2-napthoyl)-N-phenylguanidine and (Phenylacetyl)guanidine.
    [44] 44. The method according to claim 42, wherein said compound is selected from the group consisting of 2-t-butylcinnamoylguanidine, 4-isopropy1cinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(tdfluoromethoxy)cinnamoylguanidine, 2,6-dichlorocinnamoylguwaidine, 2-(eyclohex-1-en-lyl)cinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, 5-bromo-2-methoxyeinnamoylguanidine, 2-phenyleinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-phenylcinanoylguanidine, (3-Bromacinnamoyl)ganidine, 5-(N,N-hexamethylene)amiloride, trans-3-(1-napthyl)acryloylguanidine, 3-(2-napthyl)acryloylguanidine, 2,4-dichlorocinnamolyguanidine, 3-(trifluoromethyl)einnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 4-methylcinmamoylguanidine, (4-Cblorocinnamoyl)guanidine, 3-f luorooinnamoylguanidine, 3-(cyclohex-1-en-1-yl)oinnamoylguanidine, (a-Mothyloinnamoyl)guanidine, 2.3,5,6,-tetramethylcinnamoylguanidine, 2-fluorocinnamoylguanidine, (3-Nitrocinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-t-butylcinnamoylguanidine, (3-Methoxycinnamoyl)guanidine, 3-methylkinnamoylguanidine, 3-isopropylcinnamoylguauidine hydrochloride, (2-Bromocinnamoyl)guaaidine, 3-ethoxycinnamoylguanidine, (5-Phonyl-penta-2,4-dienoyl)guanidino, (2-Chlorocinnamoyl)guanidine, 4-ethoxycinnamoylguanidine, 4-fluorocinnamoylguanidine, 3,4-difluorocinnamoylguanidine, N-(3-phenylpropanoyl)-N' Phenylguanidine, 2,4,6-trimethyleinuamoylguanidine, WO 2004/112687 PCT/AU2004/000866 -144 2-methylkinnamoylguanidine, (trans-2-Phonyloyclopropanecarbonyl) guanidine, (4-Phenoxybenzoyl)guanidine, (2-Methoxycinnamoyl)guanidine, Cinnamoylguanidine, 3,4-(methylenedioxy)cionamoylguanidine, N,N'-Bis(mnidino)napthalene-2,6 Dicarboxamide, 2,3-dimethylcinnamoylguanidine, 5-(3'-bromophcnyl)penta-2,4-dienoylguanidine, N,N-Bis(3-phenylpropanoyl)guanidine, Z,3-difluorojninamoylguanidine, lnapthoylguanidine, 6-methoxy-2-naphthoylguanidine, 5-(NN-Dimethyl)amiloride hydrochloride, 2-ethoxycinnamoylgpanidine, 2-napthoylguanidine, 3,4,5-trimothoxycimnnamoylguanidine, 2-(trifluoromethyl)oinnamoylguanidine, cinnamoylguanidine hydrochloride, (4-Hydroxycinnamoyl)gnanidine, 5-(4-fluorophonyl)amilorido, 2-(l1-napthyl)acetoylguanidine, (2-Furanacryloyl)guanidine, N-Cinmamoyl-N',N'-dimethylguanidine, 2-(2-napthyl)acetoylguanidine and NN'.-bis(3phenylpropanoyl)-N" Phenylguanidine.
    [45] 45. The method according to claim 38, wherein said Coronavirus is human Coronavirus 0C43. 5
    [46] 46. The method according to claim 45, wherein said compound is selected from the group consisting of 3-methylcinamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, (3-Bromooinnamoyl)guanidine, (2-Chlorooinnamoyl)guanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, (trans-2-Phenylcyclopropanecarbonyl)guanidine, 4-isopropyloinnamoylguanidine, Cinnamoylguanidino, 6-methoxy-2-naphthoylguanidine, WO 2004/112687 PCT/AU2004/000866 -145 2,4-dichlorocinnamolyguanidine, (4-Chlilorocinnamoyl)guanidine, 5-(NN-hexamethylene)amiloride, (4-Bromocinnamoyl)guanidine, 2,6-dichlorooinnamoylgnanidine, 5-bromo-2-methoxycinnamoylguanidine, (5-Phenyl-penta-2,4-dimenoyl)guanidine, 3-(trifluoromethoxy)cinnaamnoylguanidine and 2-t-butylcinnamoyguanidine.'
    [47] 47. The method according to claim 38, wherein said Coronavirus is porcine respiratory Coronavirus (PRCV). 5
    [48] 48. The method according to claim 47, wherein said compound is selected from the group consisting of 5-(NN-hexamcthylcn)amiloridq, 6-methoxy-2-naphthoylguanidine, Cinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 3-methykcinnamoylguanidine, (3-Bromocinnamoyl)guanidine, (trans-2-Phenylyclopropanecarbonyl)guanidine, trans-3-(1-napthy ) acry l oy l guanidine and 2-(2-napthyl)acetoylguanidino.
    [49] 49. The method according to claim 38, wherein said Coronavirus is bovine o10 Convirus (BCV).
    [50] 50, The method according to claim 49, wherein said compound is selected from the group consisting of (3-Bromoeinnamoyl)guanidine, 3-(trifluoromethyl)oinnamoylguanidine, 6-mothoxy-2-naphthoylguanidino, 5-(NN-hexamethylene)amiloride, trans-3-(l-napthyl)acryloylguanidine, Cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2-(2-napthyl)acetoylguanidino, (trans-2-Phenylcyclopropanecarbonyl)guanidine, N-(3-phcnylpropanoyl)-N'-phenylguanidine and WO 2004/112687 PCT/AU2004/000866 -146 4-phenylbenzoylguanidine,.
    [51] 51. The method according to claim 38, wherein said Coronavirus is any one of the known Coronavirus isolates listed in Table 1, 5
    [52] 52. The method according to claim 51, wherein said compound is selected from the group consisting of 4-isopropylkinnamoylguanidine, 3,4-dichlorooinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylganidine, 4-t-butylcinnamoylgaanidine, 3-isopropyleinnamoylguanidine hydrochloride,
    [53] 53. The method according to claim 32, wherein said virus is the Hepatitis C virus. 10
    [54] 54. The method according to claim 53, wherein said compound is selected from the group consisting of 2,3-dimethylcinnamoylguanidiae, 2,4,6-trimethylcinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-(trifluoromthyl)cinnamoylguanidine, 4-(trifluoromethyl)oinnamoylguanidine, 6-methoxy-2-naphthoylguanidine,, (2-Chlorocinnamoyl)guanidine, (4-Chlorocimnnamoyl)guanidine, (2-Bromoinnamnoyl)guanidine, 2,6-dichlorocinnamoylguanidine, (3-Bromocinnamoyl)guaaidine, (3-Chlorocinnamoyl)guanidine, 2-(trifluoromethyl)cinnamoylgoanidine, (4-Phenoxybenzoyl)guanidine, 3,4-dichlorocinnamoylguanidine, 4-isopropylcimnamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 4-t-butyloinnamoylguanidine, 2-t-butylcinnamoylguanidine, 2-ethylinnamoylguanidine, 4-methylcinnamoylguanidine, 5-bromo-2-niethoxycinhmamoylguanidinoe, WO 2004/112687 PCT/AU20041000866 -147 3-(trifluowomethoxy)cinnamoylguanidine, 2-oyclohexylcinnaniylganidine, 1-napthoylguanidine, 3-t-butylvinnamoylganidine. 4-phenylbenzoylguanidinp, (5-Pbonyl-penta-2,4-dienoyl)guanidine, N-(cinnamoyl)-N'p-henylguanidina; 3-isopropylcinnamoylguaniine hydrorhloride, Benzaxnil hydrochloride, N-(3-phenylpropanoyl)-N'-phenylguonidine, bN'-bis(3phenylpropanoyl)-N"-phenylguanidine, 3-(2-napthyl~aoyloylgnanidine, 5-(N-Mthyl-N-isobutyl)tiiloride, 2'4 DiohloroBonzamil HOI, 5-tert-butylamnino-ainilorid, 5-(N-Ethyl-N-isopropyl)axniloride, (4-Methoxyoinnazoyl)guanidine, 4-ffuorochmaanoylguanidine, (3-Nitrocuraioyl)guknidine, 4-ethoxycinnamoylguanidine, (4-Hydroxycinnamoyl)guanidinc, (urnc-2-Phenylcyolopopancaronyl)guanidine, 3-ethoxycinm~oylguanicline,; 2,3,5,6,-tetramethykinm~oylguandine, 4-phenylinnamoyguanidino. trana-3-Furnacryoylguandin, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, (2-Furanacryloyl)guanidine, i-(cyclohex-l-en--y)cinnamoylguanidine, cinnanoylguanidine hydrochloride, 5-<NN-hexamethylene)amiloride,' 2,3-difluorocivnaioyguanidine, 2-(l -napthyflacetoylgmidin., (a-Methykinnanoyl)guanidine, (2-Nitrocinnamyl)guanidino, 6-IodoamilorTide, 3,4-(mothylcnedoxy)climanioylguanidin;, 2-ethoxycinm~oylgauidine, Cinnanicylguanidine, 2-phenyleinnamoylguanidine., 2-(eyclohex-1-en-1y~cinnaxnoylguanidine, 2-napthoylguanidfin; 3-phenyleinnamoylganfidine, 5-(NN-DMetyl)aniiloride hydrochloride, 5-(4-fluorophenyl)amilorid, (3-Methoxyuhmamoyl)guanidino, 2-f luorocinnamoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, WO 2004/112687 PCT/AU2004/000866 -148 [(4-Chlorophenoxy-acetyl]jguanidine, (3-phenylpropanoyl)guanidine, 2-chloro-6-fluorocinnamoylguanidine, 3-fluorocinnamoylguanidine, 2-methyloinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, 1-bromo-2-napthoylguanidine, 3,4,5-trimethoxycinnamoylguanidine, 3-methylcinnamoyguanidino, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, Phenamil methanesulfonate salt, 2,4-dichlorocinnamolyguanidine, (4-Nitrooimnnamoyl)guanidine, 3,4-difluorocinnamoylguanidine and [(E)-3-(4-Dimethylaminophenyl)-2 methylacryloyl]guanidine.
    [55] 55, The method according to claim 32, wherein said virus is the Equine Arteritis virus. 5
    [56] 56. The method according to claim 55, wherein said compound is selected from the group consisting of 5-(N,N-hexamethylene)amiloride, (3-Bromocinnamoyl)guanidine, trans-3-(1-napthyl)aoryloylguanidine, 2-t-butyloinnamoylguanidine and 2-(cyclohex-1-en-lyl)cinnamoylguanidine.
    [57] 57. The method according to any one of claims 32 to 56, wherein said compound is provided as a pharmaceutical composition according to claim 10 4 or claim 5.
    [58] 58. A method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the administration of a compound according to any one of claims 1 to 3 to a subject in need of 15 said treatment.
    [59] 59. The method according to claim 58, wherein said virus is a LeAtivirus. WO 2004/112687 PCT/AU2004/000866 -149
    [60] 60. The method according to claim 59, wherein said Lentivirus is Human Immunodeficiency Virus (HIV).
    [61] 61. The method according to claim 60, wherein said compound is selected from the group consisting of (3-Chlorocinnamoyl)guanidine, (3-Bromocinnamoyl)guanidine, ( 2 -Chlorocinnamoyl)guanidine, ( 2 -Bromooinnamoyl)guanidine, 3-(trif luoromethyl)cinnamoyguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methylcinnamoylguanidine 2 -methylcinnamoylguanidine, 2,3-dimethyloinnamoylguanidise, Cinnamoylguanidine, 6-methoxy-2-naphthoy1guanidine, tras-3-(1-napthy)acryloylguanidine, 3,4-dichlorocinnamoylguanidine, 2,6-dichlorocinnamoylguanidine, 4 -phenylbenzoylguanidine, 2-ethylcinnmoylguanidine, ( 4 -Chlorocinnamoyl)guanidine, 2-napthoylguanidine, 2 ,5-dinethylkinnamoylguanidine, 3-isopropykinnamoylganiine hydrochloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-phonyloionamoylguanidine, (4-Bromooinnamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 3-(cyclohex-l-en-1-yl)ennamoylganidine, 3-(trif luoromethoxy)cinagmoylguatidine, 2 -(trifluoromethyl)cinnamoylguanidine, NN'-bis(3phenylpropanoyl)-N"-phenylguanidine, 2-ethoxycinnamoylguanidine, N-(3-phenylpropenoyl-N-phenylguanidine, 4 -(trifluoromethyl)ohamoylguanidine, (4-Methoxycinsnmoyl)guanidine, 2-t-butycinamoyguanidine, 4 -methyloinnamoylguanidine. 2-fluorocinnamoylguanidine, 2-phenyloinnamoy1guanidine, N-( 6 -Hydroxy-2-napthoyf-N'-phenylguanidine, 3-t-butylkinnarnoylguanidine, 3,4-difluorocinnamoylguanidine, 5-(NN-hexamethylene)amiloride, 3-ftluorocinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 -150 5-bromo-2-methoxycbmanoylguanidine, 3-ethOxycinm~oylguqAndjne, 3,4-(mcthylenedioxy)oinnarnoylguanidinc, (2-MethoxycinnamOyl)guanidine, 2'4 Dichlorcflenil UCI,, 2,3,5,6,-tettamethylcinnamoylguanidine, 3-(2-napthyl)acryloylg~uanidine, 2-(h-napthyl)acetoylgusidine, 2.3-difluorooinnanoylguanidine, (3-Mehoxycinnamoyl~guaiddine, 4-isoprOpylcirma~oylguanidine, 2,4,&tdnaethylcinnamoylguan~dinie, N-(v-innamoyl)-N'phenylguanidin, 2-(oyclohex-l-en-lyl)innmciylguanidine, 2-(2-napthyl)acctoylguanidiuc, (4-Hydroxydinamoyl)guanidine, 4-phenykinim~oy1guanidin; 4-fluoroclnnatoylguanidine, NJW-bis-(dinamoyl)-N"-phenylguanidine, .(2-Fnraacyloyt)guanidine, Phenamil methanculfonate salt, Benzaxnil hydrochloride, (3-Nitrooinnamoyl)gmaidine, Benzyoylgumidine, (4-Plienoxybmwzyl)guanIdine, 3-(frs-het--en--yl)cimamoylganidine,. 5-(N-Mothyl-N-isobutyl)ainiloride, 2-cyclohexylcinnamoylgua-nidine, 4-ethoxycinnamoylguanidine, 2,4-dicblorocinnamolyguanidine, S-(N-Ethyl-N-isoprapyl)amilorid;, N-amidino-3-amino-5-hexamethyleneiinino-6-phenyl 2-pymainacarboxamide, (a-Methylcimamoyl)guanidine, cinnamoylgumAdIne hydrochloride, [(4-Chlorophenoxy-aoetyllguanidine, pyrazinecarboxainide, 5-(4-fluorophenyl)amiloride, (trans-2-PhenylcyvloProPaneoarbonyl)guanidine, (2-Nitrocinnamoyl)guanl dine, trans-3-Furanacryoylgnanidinc, l-napthoylguanidine, 5-tert-butylaminp-amiloride, 3-methoxy.-HMA, (3-phenylpropanoyl)guanidine, 4-1-butylcinaoylguanidine, 5-(N,N-Dimethyl)andiloride hydrochloride, WO 2004/112687 PCT/AU2004/000866 -151 NN'-Bis(3-phenylpropanoyl)guanidine, N-Benzoyl-N'-oinnamoylguanidine and 1-bromo-2-napthoylguanidine.
    [62] 62. The methods according to claim 60, wherein said compound is selected from the group consisting of 4-phenylbenzoylguanidine, (3 bromocinnamoyl)guanidine, 3-(trifluoromethyl)oimnnamoylguanidine, 5 5 (NN-hexamethylene)amiloride, and (5-Phonyl-penta-2,4 dienoyl)guanidine.
    [63] 63, The method according to any one of claims 60 to 62, wherein said HIV is HIV-1. 10
    [64] 64. The method according to claim 58 wherein said virus is a Coronavirus.
    [65] 65. The method according to claim 64, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS). 15
    [66] 66. The method according to claim 65, wherein said compound is selected from the group consisting of 2,3-difluorocinnamoylguanidine, 3,4-dichlomeinnamoylguanidine, 4-t-butyloinnamoylguanidine, 3-(2-napthyl)acryloylguanidine (3-Chlorocinnamoyl)guanidine, 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, 2,5-dimethycinnamnoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 4-isopropylcinnamoylguanidive, (3-Bromocinnamoyl)guanidine, 6-mcthoxy-2-naphthoylguanidino, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenylinnamoylguanidine, (2-Chlorocinnamoyl)guanidine, 2'4 DichloroBenza il HCI1, 4-phenyloinnamoylguanidine, 4-(trifluoromethyl)cinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 3-(trifluoromethyl)oinnamoylguanidine, 2-ethoxycinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 cinamylguanidine hydrochloride, 4-atboxyejinn~oyjpmandi:Oe, C2-Bromocinnaznoyl)guanidine, 2,6-dichlorocinnamoy1guanidine, 3 , 4 ,S-trixne-thoxycinnaanoylgaaudino,, 5-tert-butylamino-amiloride, 3-t-butylcinnamoylguanidinc, 5-bromo-2-fluorocinnamoylguanidine, (4-Cblorocinnamoyl)guanidlue, 2-t-bulylcinnamoylguanidine, 2 -cyclohexylcinnamoylguanidiue, 6-lodoamiloride, 3-(trans-hept-l'-en--ylcinnainoylguanjdjne, (4-&tcaooinoyl)guanidine, (4-Hydroxycinnamoyl)guandine, N-(S-phenylpropaioyl)-N'-pheniylguatiidin;, (3-NitrocinmoyI)guanidino, 3-fluoroocinnmoylguanidine, 2-(l-napthy)acetoyguanidinc, 2-ethylinnamoylguanidine, :5-(NN-Dimethy)amilordea hydrochloride, 2-napthoylguanidine, 5-(4-fluorophenyl~amiloride, 2-(triflnoromethyl)cinnamoylguanidine,, N-(6-Hydroxy-2 napthoy)-N'-phenylgganidin, (trans-2-Phenylcyelopropanecarbonyl)guanidin;, N,-~s3hypoaol)N-hnlundn, I -napthoylguanidine, Beuzamifl hydrochilide, 3-methoxy -HMA, 4-methyleinnanoylguanidine, 4-fluorocinnanoylguanidine, 3,4-(methyleoedioxy)rinoamoylgnanidine, 5-{NN-hexamethiy~ee)amijoride, N-(cinnamoyl)-Nphenylgnanidine, 5-(N-Ethyl-N-isoprcpy)arrdoride, 3-metyloinnaoylguanidine, 2-methylcinnamoylgnanidine, 2,3,5,6,-tetramethylcinnanoylguanjdjno, trms-3-Furanaeryoylganidine, (4-MethoxyinnamoyIlgnanidino, (2-Furanaerylayl)guanidine, (3-phenylpropanoyl)guanidine, 2-(2-napthyl)aeetoylguanidinc,, Ciom~oylguanidine, (2-Methoxycimnnoyl)guandine, [3-(3-Pyridyl)acryloyl]guanidine, 4-phenylbeazylguanidine, WO 2004/112687 PCT/AU2004/000866 -153 2,4-dichlorocinnamolyguanidine, (3-Methoxyeinnamoyl)guanidine, 2-fluoroinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, (a-MethyloinMamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, (Quinoline-2-carbonyl)guanidine, (Phenylacetyl)guanidine, NN-Bis(amidino)napthalene-2,6-dicarboxamide, 6-bromo-2-napthoylguanidine, 1-bromo-2-napthoylguanidine, 2-chloro-6-fluorocinnamoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, Phenamil methanesulfonate salt, N-Benzoyl-N'-cinnamoylguanidine and N-(2-napthoyl)-N'-phenylguanidine.
    [67] 67. The method according to claim 65, wherein said compound is selected from the group consisting of cinnamoylguanidino, trans-3-(1 napthyl)aeryloylguanidine, and 6-methoxy-2-naphthoylguanidine, S
    [68] 68. The method according to claim 64, wherein said Coronavirus is human Coronavirmus 229E.
    [69] 69. The method according to claim 68, wherein said compound is selected 10 from the group consisting of 4-isopropycinnamoylguanidino, 3,4-dichlorocinnamoylguanidine, 3 -(trifluoromethoxy)cinnamoylguanidine, 4-t-butyloinnamoylguanidine, 3-isopropyleinnamoylguanidine hydrochloride, 3-t-butyloinnamoylguanidine, 2-t-butyloinnamoylguanidine, trans-3-(1-napthyl)aeryloylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 2,3-difluorocinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, 2-phenylcinnamoylguanidine, 3-phenylcinnamoylguanidine, 3-(cyclohex-1-en-1-yl)einnamoylguanidine, 4-phenylbenzoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, WO 2004/112687 PCT/AU20011000866 (4-Phenoxybenzoyl)guani'dine, 4<tffiuoromothy1)cinnamoylganidine, 2 -(ayolohex-1-en-lyl)oinnamoylgualaidin;, (4-B3romocinnaxnoyl)guanidln;, 5IIN-Nexamethylene)aniIoride, 1-napthoylgaanidine, 5-(4-fluorophenyl~amiloride, (5-Pheuay1-penta-2,4-dienoy~guanidine, (3-Bromocinnainoyl)guanidine, 2,5-dinethylcinnamoylguanidine, 2-(frifiuoromethyl)cinnmxoylguanidin;, 6-mcthoxy-62-naphthoylguanjidine, (4-ChowcinnsmOyl)guaidine, (3-MethOxYcinmOyl)guanidine, 5-broino-2-fluorocinnmoylguanidinc, 5-(NN-Dhnethylamilofide hydrochloride, Ciimamoy1lanidine, (2-MetbioxYcinnamoyl)guanidin;, (a-Metlcinnamoyl~gnanidine, 4-phenylcinnamoylguanidie, 2,6-dicblorcinnanoylguanidine, (2-Bromocinnamoyl)guandicne 2 ,4,6-trimethylcinnaznoylaniadine, (trens-2-Thenylcyclopropanccarbonyl)guanidine, (3-Chlorooinnaoyl)Suanidine, 2-(1-napthyl~acetoylguanidin;, 2-ethiylcinnamoylguanidind, 2-cyclohexylcinamoylgumnidin;, (4-H-ydroxycinnamoyl)guanidinc, 2-ethoxyirmamoylguanidine, 3-mcthylcinmamoylguawidine, 2-methylc-innmnpoylguanidine, 3-fluorcinnamoylguanldine, Cinnanaoylguanidine hydrochloride, 2,3-dimuthylczmamoylguaidine, 2-fluorocinnanicylgaenidine, 4-fluorocinnainoyguanidin, 3,4-difluorcinnamaylpuanidine,, 5-tert-butylaniino-ainiloride, 2-napthoylguenidine, NJP-Bis(amidino)napthalene-2,,6-dicarboxamide, N,N'-]Bis(3-phenylprapanoy)guanidine, 4-methylcinnainaylguanidine, 5-(3'-bro -mopheny1)p~nta-2,4-dienoylguanidinc, 2,3,5.6,-tctamethyloinnamoylguanidine, 3-ethoxyoinnamoylguanidine, WO 2004/112687 PCT/AU20041000866 INN-bis(3phenylpropanoyl).N"-phenytguanidine, ( 4 -MethoxYOinneoy)guanicinc, (2-ChlorOOinnanoyl)gaanidine, (3-Nifrocinnamoyl)guanidinc, 4-ethoxycinnaxnoylguanidin;, 3 ,4,5-trimehxycinnamoylgusnidine, 2-(2-napthyl)acctoylguanidine, N-( 3 -phenylpropsnoyl)-N'-phenylgumljdine, S-(2Lbromopheny1)penta-2,4 dienoylguanidine, (4-Bromocinnanaoyl)guanidine; (2-Nitrocinnamnoyl)guanidine, (3-Chlorocinnamoylguanidine, (4-Methoxycinusmoyl)guanidie, 4 -(frifluoromethyl)cinnamoylgulanidine, [(F)-3-(4-Dimet'yaminopheny)-2 methylacryloyljjguanidine, N-Benzoyl-N-cixmamoylguanidine, 4-phanylbenzoylguanidine, trans-3-Furanacryoylguanidine, N-rAmidino-3-ami-no-5-phenyl-6-chloro-2 Pyrazinecarboxamide, N-(chnaMoy)-N'henyguanidinae, Cinunoylguanidine, 3-methoxy-ainiloride, (3-phenylpropanoyl)guaidine, 3-metbioxy -JMA, Benzyoylguanidine, N-axnidino-3,5-diainino'-6-phynyl-2 Pyrazinecarboxamide, (Quinoline-2-carbotnyl)guanidine, (3-(3-Pyridyl)aaryloyl]guanidine, N-Cinnamoyl-N',N'-dimethylguaniline, N-(2-napthoyl)-N'-phwiylguanridine, and (Phenylacctyl)guanidine.
    [70] 70. The method according to claim 68, wherein said compound is selected from the group consisting of 2-t-butyleinnanioylguanidine, 4-isopropylcinnainoylguanidine, JA4-dichlorocinnmoylguanidine, 3-(trdfluoromethoxy)cinnamoylguanidine, 2,6-dircblorocinnamoylguanine, '2-(cyclohex-l-en-lyl)chmamoylguanidine, 2-cyclohcxylcinnamaoylguaridiue, WO 2004/112687 PCT/AU20041000866 5 -bromo-2-methoxycirmaxnaylguanidIe, 2-phenylciunamoylguanidine, 4-t-butylcinnamoylguanidine 3-phenylclimsmoyguanidno, (3-Bromocinnamoyl)guanidine, S-(N 1 N-hexamethylene)amiloride, U3-(-napthyl)acroylgaidine, 2,4-dicblorocinuamolyguarnidbe, 3-(trifluoromthyl)cinnamoylgunine, 5-bromo-2-fluorocinnamoylgnanidinc, 4-wethykinnroylguanidine (4-Chlorocinnaolgaiie 3-fluorocinnamoylgaanidin;, 3-(cyclohex-1-mn-1-yl)cinnarnoylguanidinv,, (a-MethyICirnamoy)guanidin; 2 1 3,5,6.-tetramhethylcinnamoylguandine, 2-fluorooinnamoyguanicline, (3-Nitroofizmmoyl)guanidino, 2,5-dimcthylcimiarnoylguianidin;, 3-t-butylcinnamoyguinidinc, (3-MethoxycinnamoyI)guandine, 3-methyloinnemoylguatidne, 3 -isopropylcixmanoylguanidine hydrochloride, (2-Bromocinnamoyl)guianidine, 3-etboxy naoy~gundne, (5-PhenYl-penta-2,4-dienoyl)guanidine, (2-Chlorocinnamoyl)guanidine, 4-othoxycinuwnoylguanidine, 4-fluorocinnamoylguanidine, 3,4-difkuorocinnamoyguanidin, N-(3-phenylpropanoyl)-N' Phenylguanidine, 2,46-trimeth-ylcinuamoylguanidine, 2-mothyloinnainoylguanlidine, (trans-2-Thenylrcyopropanecarbony) guaidine, (4-Phenoxybenzoyl)gumnidie, (2-Methoxycinnamoyl)guanidine, Cinnamoylguanidine, 3,4-(methylenediosy)oinnamoylguianidino, N,N'-Bis(amidino)napthalenc-2,6-' Dicarboxamide 3 2,,3-cineftlinnamoylgnanidine, 5-(3'-bromophanyl)penta-2,4-dienoylguanidin;, N,N'-Bis(3-phenylpropanoyl)guanidine, 2,3-difluorocinnamoylguanjdine, 1 -napdioylguanidine, WO 2004/112687 PCT/AU2004/000866 -157 6-methoxy-2-naphthoylguaniidine, 5-(N,N-Dimethyl)amiloride hydrochloride, 2 -ethoxycinnamoylguanidine, 2-napthoylguanidine, 3 , 4,5-trimethoxycinnamoylguanidine, 2 -(trifluoromethyl)oinnamoylguanidine, cinnamoylguanidine hydrochloride, ( 4 -Hydroxycinnamoyl)guanidin, 5-(4-fluorophenyl)amiloride, 2-(1-napthyl)acetoylguanidine, (2-Furanacryloyl)guanidine, N-Cinnamoyl-N',N'-dimethylguanidine, 2 -(2-napthyl)acetoylguanidino and N,N'-bis(3phenylpropanoyl)-N" Phenylguanidine.
    [71] 71. The method according to claim 64, wherein said Coronavirus is human Coronavirus OC43, 5
    [72] 72. The method according to claim 71, wherein said compound is selected from the group consisting of 3-methylcismamoylguanidine, trans-3-(1-napthyl)acryloylguarn dine, (3-Bromocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, 3,4-dichlorocinnamoylguanidine, 3 -(trifluoromethyl)cinnamoylguanidine, (trans- 2 -Phenyloyolopropaneooarbonyl)guanidine, 4 -isopropylcinnamoylguanidine, Cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, 2,4-dichlorocinnamolyguanidine, (4-Chlorocinnamoyl)guanidine, 5-(N,N-hexamethylene)amiloride, (4-Bromocinnamoyl)guanidine, 2,6-dichlorooinnamoylguanidine, 5-bromo-2-methoxycinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-(trifluoromethoxy)cinnamoylguanidine and 2-t-butylcinnamoyguanidine.
    [73] 73. The method according to claim 64, wherein said Coronavirus is porcine respiratory Coronavirus (PRCV). WO 2004/112687 PCT/AU2004/000866 -158
    [74] 74. The method according to claim 73, wherein said compound is selected from the group consisting of 5-(N,N-hexamethylene)amiloride, 6-methoxy-2-naphthoylgaanidine, Cinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 3-methyleinnamoylguanidine, (3-Bromocinnamoyl)guanidine, (trans-2-Pheiylcyclopiopanecarbonyl)gumidine, trans-3-(1-napthyl)acryloylguanidine and 2-(2-napthyl)acetoylguanidine. 5
    [75] 75. The method according to claim 64, wherein said Coronavirus is bovine Coronvirus (BCV).
    [76] 76. The method according to claim 75, wherein said compound is selected from the group consisting of (3-Bromocinnamoyl)guanidine, 3-(ttifluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, 5-(NN-hexamethylene)amiloride, trans-3-(1-napthyl)acryloylguanidine, Cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2-(2-napthyl)acetoylguanidine, (trans-2-Phonyloyolopropanecarbonyl)guanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine and 4-pheaylbenzoylguanidine. 10
    [77] 77. The method according to claim 64, wherein said Coronavirus is any one of the known Coronavirus isolates listed in Table 1.
    [78] 78. The method according to claim 77, wherein said compound is selected 15 from the group consisting of 4-isopropylcinnamoylguanidine, 3,4-dichlorocinnamnoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 4-t-butylinnamoylguanidine, 3-isopropylcimnamoylguanidine hydrochloride, WO 2004/112687 PCT/AU2004/000866 -159
    [79] 79. The method according to claim 58, wherein said virus is the Hepatitis C
    [80] 80. The method according to claim 79, wherein said compound is selected 5 from the group consisting of 2,3-dinethylcinamoylguanidie, 2,4,6-trimethylcinnamoylguanidine, 5-bromo-2-fluorooinnamoylguaidine, ( 4 -Bromocinnamoyl)guanidine, 2, 5 -dimethylinnamoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, 4 -(trifluoromethy)citmamoylguanidine, 6-methoxy-2-naphthoylguanidjine, ( 2 -Chlorocinnamoyl)gnanidine, (4-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 2,6-dichlorocinnamoylguantdine, (3-Bromocinnamoyl)guanidine, (3-Chlorocinnamoyl)guanidine, 2 -(trifluoromethyl)cinnamoylguanidine, (4-Phenoxyhenzoyl)guanidine, 3 ,4-dichlorocinnamoylguanidine, 4 -isopropykiInnamoylguanidine, trans- 3 -(1-napthyl)aryloylguanidine, 4-t-butylkinnamoylguanidine, 2-t-butykcinnamoyguanidine, Z-ethylciuzamoylguanidine, 4-methylinnamoylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 3 -(trifiuoromethoxy)cinnamoylguanidine,, 2 -cyclohexylcinnamoylguanidine, 1-napthoylguanidine, 3-t-butylinnamoylguanidine, 4-phenybenzoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guaidine, N-(cinnamoyl)-N'phenylguanidine, 3-isopropylcinnamoylgnanidine hydrochloride, Benzamil hydrochloride, N-(3-phonylpropanoyl)-N-phenylguanidine, NN-bis(3phenylpropanoyl)-N"-phenylguanidine, 3-(2-napthyl)aoryloylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 2'4 DichloroBcnzamil HCI, 5-tert-butylamino-amiloride, 5-(N-Ethyl-N-isopropyl)amiloride, (4-Methoxycinnamoyl)guanidine, WO 2004/112687 PCTAU20011000866 4-fluorocinnamoylguanidfiae, (3-'Nitrocixinamoyl)guanidine, 4-athoxycinnamoylguanaidine, (4-Hydroxycinnaioy1)gua~dine, (trans"-2-Phenylroyclopropanecarbonyt)guanidine, 3-ethoxycinnamoylgtianidine, 2,3,5,6,-teframethylkinnamaylguanidine, 4-phenyle-iimamoylguanidine, tras-3-Furanacryoylgumidine, N-(6-R-ydroxy-2-napfioyl}.N-phenylguoidine, (2-Pumanacryloylgumnidine, 3-(cyclohex-1-en-17yl)cinnamoylguanidine, cinnamoylguanidine hydrochloride, 5 2 {NN-hexamethylene)aniiloride, 2.3-difluorocinnaioylguaaidine, 2-(1-napthyl)aoetoylguanidine, (a-Mcthylcinnamoyl)guanidin:e, (2-Nitrocbmamoyl)guaxidine, 6-Iodoaxnilorlde, 3,4-(methylenedioxy)oinnamoylguanidine, 2-ethoxycinnamoylguaniclinc, Cimianoylguanidine, 2-phenylcinnamoylguanidine, 24coyclohex-1-en-1 yl)oinmoylguanidine, 2-napthoylguaanidine, 3-phenylcinnanioylguanidine, 5-(NN-Dimethyl)amiloride hydrochloride, 5-(4-fiuorophenyl)aniiloride, (3-Mcthoxycinnainoyl)guanidi-ne, 2-fluorocinnanaoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanjdine, ((4-Chlorphenoxyi-acetyl]guanidine, (3-p-beylpropanoyl)guanidine, 2-chloro-6-fluorocinnamoylguanidine, 3-fluoracinnamoylguaoidie, 2-inethylcinnamoylguamidine, (2-Methoxycinnamoyl)guanidine, I -brorno-2-napthoylguanidne, 3,4,5-trimethoxyoinnaaoylguanidine, 3-methyldimamoylguanidine, 3-(trans-hept-l-cn-1-y)cimnamoylguanlidin,, Phenamil nietbanesullbnata salt, 2,4-dicblorcinnaiolyguanidine, (4-Nitocinnaioyl)guanidine, 3,4-difluorocinnamcylguanidine and [(B)-3-(4-Dimothylamnoheny1)-2 methylacryloyl]gnanidine. WO 2004/112687 PCT/AU2004/000866 -161
    [81] 81. The method according to claim 58, wherein said virus is the Equine Arteritis virus.
    [82] 82, The method according to claim 81, wherein said compound is selected S from the group consisting of 5-(NN-hexa nothylene)amiloride, (3-Bromocinnamoyl)guanidine, trans-3-(l-napthyl)acryloylguanidine, 2-t-butyleinnamoylguanidine and 2-(oyclohex-l-en-l yl)cirmnamoylguaidine.
    [83] 83. The method according to any one of claims 58 to 82, wherein said compound is provided as a pharmaceutical composition according to claim 4 or claim 5. 0to
    [84] 84. " A method of down regulating a membrane ion channel funotional activity in a cell infected with a virus, comprising contacting said cell with a compound according to any one of claims 1 to 3. 15
    [85] 85. The method according to claim 84, wherein said virus is a Lentivirs.
    [86] 86. The method according to claim 85, wherein said Lentivirus is Human Immunodeficiency Virus (IV). 20
    [87] 87. The method according to claim 86, wherein said membrane ion channel is the IV Vpu membrane ion channeL
    [88] 88. The method according to claim 87, wherein said compound is selected from the group consisting of (3-Chlorocinnamoyl)gusnidine, (3-Bromocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)cinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methyleinnamoylguanidine, WO 2004/112687 PCT/AU20011000866 -162 2-meffiylcinnmoylguaniue, 2,3-dimeffiylinru oylgundinle, Cinnoylgmnidine, 6-mothoxy-2-naphthoylguanidine, trans-3-(I -napthyl)aryloylguanidin;, 3 9 4-dicfflorocinnaioyguanidine, 2,6-diohloroointimoylguanidine, 4-phenylbenzoylguaifidine 2-etycinnamoyguanidine> (4-Chlorooiimanoyl)guaxidinc,, 2-napthoylgvanidine, 2,5-dimethylcinamoylguanidizwc, 3-isopropylcinnamoylgumAndife hydroobloride, (5-Phenyi-penta-2,4-dienoylgumnidine, 3-phenyiineoylguanidin;, (4-Bromocinnanioy1)guandine, 5.%3L-bromophenylpenta-2A.-diefloylguanidine, 3-(cyclohex-1 -en- 1-y1)oinnamoylgunnidine, 3-(trifluorometoxy)cinnamoylguanidine, 2-4trifiuoroaehyl)eihnamoylguanidin;, N,N'-bis(Jphenylpropaioyl)-N"-phenylguanicline, 2-ethoxyoinnamoylguanin N-(3-phenylpropanoyl)-N'-phenylguaaidi-ne, 4-(trtfuoromethyl)cinnamoylgnanidinae, (4-Mefhoxyo;inamvyl)guaniqin 2-t-butylinnamoy1guanidine, 4-mothyloiimoylguanidine, 2-fluorocinamoylgaanidine, 2-phenyloinnamoylguanidine, N-(r-Hkydroxy-2-napthoyl)-N'-phe-nylguanidifl 3-t-butyloinnwnoylIguarnidin, 3A4-difluorocinnamoylguanidin;, 5-(NN-hexamcthyleno)amiloride, 3-fluorocinnamoylguanidine, 5-bromo-2-inothoxycinnamoylgumnidin; 3-ethoxyrinnamoylguanidine, 3,4-(methylenedioxy)einnaroylua-nidine, (2-Mcthoxycinnamoyl)guanidiine. 2'4 DicblordBenzanail HCI, 2,3,5,6,-tewaanethylinnamoylguanidin;e 3-(2-napthylacryloyguanidin;, 2-(1-naptylacetoylguanidin, 2,3-diilluorocinnamoylguanidine,. (3-Motlioxyclimamoylguanidine, 4-isopropyloizmamoylgflaridine, 2A46-trixncthylcinnanoylguanidine, N-(ci==aoy1)-NphenylgumAndin;, 2-(clolaex--en-lyl)dinnamoylguanidinc. WO 2004/112687 PCT/AU2004/000866 -163 2-(2-napthyl)acvetoylguanidine, (4-Hydroxycinnamoyl)guanidine, 4-phonylcinnamoylguanidine, 4-fluorocinnamoylguanidine, NN'-bis-(oinnamoyl)-N"-phnylguanidine, (2-Furanacryloyl)guanidine, Phenamil methanesulfonate salt, Benzamil hydrochloride, (3-Nitrocinnamoyl)guanidine, Benzyoylguanidine, (4-Phcnoxybonzoyl)guanidine, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 2-cyclohexylcinnamoylguanidie, 4-ethoxyciunamoylguanidine, 2,4-dichlorocinnamolygufnidine, 5-(N-Ethyl-N-isopropyl)amiloride, N-amidino-3-amino-5-hexamethyleneiminao-6-phenyl 2-pyrazinecarboxamide, (a-Methyloinnamoyl)guanidine, cinnamoylguanidine hydrochloride, [(4-Chlorophenoxy-acetyliguanidine, N-amidino-3-amino-5-phenyl-6-ohloro-2 pyrazinecatboxamide, 5-(4-fluorophenyl)amiloride, (trans-2-Phenyloyclopropanecarbonyl)guanidine, (2-Nitrocinnambyl)guanidine, trans-3-Furanacryoyguanidine, 1-napthoylguanidine, 5-tert-butylamnino-amiloridt, 3-methoxy-HMA, (3-phenylpropmnoyl)guanidine, 4-t-butylcinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, N,N'-Bis(3-phenylpropanoyl)guanidine, N-Benzoyl-N'-cinnamoylgnanidine and 1-bromo-2-napthoylguanidine.
    [89] 89. The method according to any one of claims 86 to 88, wherein said HIV is HIV-1. 5
    [90] 90. The method according to claim 84, wherein said virus is a Coronavirus.
    [91] 91. The method according to claim 90, wherein said membrane ion channel is the Coronavirus E protein. WO 2004/112687 PCT/AU2004/000866 -164
    [92] 92. The method according to claim 91, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virs (SARS). 5
    [93] 93. The method according to claim 92, wherein said compound is selected from the group consisting of 2,3-difluorocinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, (3-Chlorocinnamoyl)guanidine, 3-(oyclohex-1-n-1-yl)oilnnamoylguanidine 2,5-dimethyleinnamoy1guanidine, trans-3-(1-napthyl)acryloylganidine, 4-isopropycimamoylguanidine, (3-Bromoinnamoyl)guanidine, 6-mothoxy-2-naphthoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenylkinnamnoyguanidine, (2-Chlorocinnamoyl)gnanidine, 2'4 DichloroBenzamil HC0, 4-phonylcinnamoylguanidine, 4-(trif lnoromethyl)einnamoylguanidine, 3-(Irifloromethoxy)innamoylguanidine, 3-(trifluoromethyl)cinoamoylgs1idine, 2-ethoxycinnamoylgUalidine, cinnamoylguanidine hydrochloride, 4-ethoxycinnamoylguanidine, (2-Bromocinnamoyl)guanidine, 2,6-dicblorocinnamoylguanidine, 3,4,5-trimethoxycinamWoylgWidinfe, 5-tert-butylamino-amiloride, 3-t-butylcinnamoy1gnanidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Chlorocinnamoyl)ganidine,. 2-t-butyleinnamoylgaanidine, 2-cyclobexykoinnamoylguanidine, 6-lodoamiloride, 3-(trans-hept--en-1-yl)cinamoy1ganidine, (4-Bromooimnamoyl)guanidine, (4-Hydroxycinnamoyl)guanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, (3-Nitrooionamoyl)guanidine, 3-f luoreeinnamylgusidine, 2-(1-napthyl)acetoylguanidine, WO 2004/112687 PCTAU20011000866 2-ethylefmaylgaunidine, S-(N,N-Dimethyl)aqmiloride hydrochloride, 2-napthoylguanidie, .5-(4-fluorophenyl)amiloride, 2-(trifluoromethyl)o~mawoylguidinO, N-(6-Hiycroxy-2-nhoyl)-N'-penylguanidine, (tran-2-Phenylcyclopropanecarbony)guanidine, N,N-bis(3phonylpropanoyl)-N"-phenylguanidin;, 1l-napthoylgnanidine, Benramil hydrochloride, 3-mcthoxy -JIMA, 4-methylcinnamoylguanidine, 4-fluorociunmoylguanidine, 3,4-(methylenadioxy)einnamoylguanidine,, :5-(Nb,N-hexanethylenefturilodrde 3 N-(cinnamoyl)-N'pheny~guanidine, 5-Q4-Ety1-N-isopwopyamiloride 3-methylcimmamoylguanidine, 2-meffiylcinnamoylguabe, 2,3,5,6,tWranietyli2floflyguafidifl6, trans-3-F uranauoyl~uanidine, -(4-Mothoxycbmuamoyl)guanidine, (2-FurnaCryloDYl)guanidin;. (3-phenylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidina, Cinnamoylgnaidiue, (2-Methoxycinaoyflganidie, f3.(3-Pyiddyl)aciyloyllguanidine, 4-phenylbenzoylguanidine, 2,4-dichblorocinnamolyguanidine, (3-MethoxychmarnoylgumnidIne, 2-f luorocinnamoylgamidine, (4-Phenoxyhenzoyl)guunidie (a-Methylcinmnoyl)ganidina, 5-(3'-bromophenyl~enta-2,4-dienoylgusnidine, (Quinoline-2-~a-rbonyl)gugnidino, (Phenylacetyl)guanidine, N,N'-Ths(amidino)napthalene -2,6-dicarboxamide, 6-bromo-2-napthoylguanidine, l-bronao-2-napthoylguanidine, 2-chloro-&-fluorocinnamoylgaauidine, [(4-Chlorophenoxy-acetyl]guanidine, Phenamil methanesulfonate sal, N-Benzciyl-N'-cinnaxoylguanidine and N-(2-napthoyl)-N'-phenylguanidine. WO 2004/112687 PCT/AU2004/000866 -166
    [94] 94. The method according to claim 91, wherein said Coronavirus is human Coronavirus 229E.
    [95] 95. The method according to claim 94, wherein said compound is selected 5 from the group consisting of 4-isopropyeiamoylgualidifne, 3,4-diehlorocinnamoylguanidine, 3-(trifluoromethoxy)innamoylguanidine, 4-t-butylcinnamoylguanidine, 3-isopropycinnamoy1guaidine hydrochloride, 3-t-butylkinnarnoylguanidine, 2-t-butycinnamoylgumiIdine, trans-3-(1-napthyl)aeryoy1ganidine, 5-bromo-2-methoxycinnamoylgaandine, 2,3-difluorocinnamoylguanidine, 3-(2-napthyl)aoryloy1guanidine, 2-phenylcinnamoylguanidine, 3-phenycinnamoylganidine, 3-(cyclohex--en-1-yl)cinnamoylguanidine, 4-phenylbenzoylguanidine, 3-(trifluoromethyl)eiam~oyguanidine, (4-Phenoxybenzoy)guanidine, 4-(trifluoromethyl)cinmoy1ganidine, 2-(cyclohex-1-en-1yl)cinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 5-(NN-hexamethylene)amiloride, 1-napthoylguanidine, 5-(441uorophenyl)amiloride, (5-Phenyl-penta-2,4-dienoyl)gaidine, (3-Bromocinnamoyl)guanidine, 2,5-dimethylcimamoylguanidine, 2-(trifluoromethyl)cinnanoylguanidine, 6-nethoxy-2-naphthoylguanidine, (4-Chlorocinnhmoy1)guanidinie, (3-Methoxycinnamoyl)uanidinz, 5-bromo-2-fluoroonafmoylgnanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, Cinnanoylguanidine, (2-Methoxycitnamoyl)ganidine, (a-Methylkinnamoyl)guanidine, 4-phenykinnamoyguanidine, 2,6-dichlorocinamoylpanidine, (2-Bromocimamoyl)guanidine, 2,4,6-trhmethykinnamoylguanidie, WO 2004/112687 PCT/AU2004/000866 -167 (trans-2-Phenylcyclopropancarbonyl)guanidine, (3-Chlorocinnamoyl)guanidine, 2-(1-napthyl)acetoylguanidine, 2-ethyloinnamoylguanidinfe, 2-eyclohexycinnamoylguanidine, (4-Hydroxycimamoyl)guanidine, 2-ethoxycinnamoylguanidine, 3-methyleinnamoylguanidine, 2-methyleinnamoyguanidine, 3-fluorocinnamoylguanidine, cinnamoylguanidine hydrochloride, 2,3-.dimoehyloinnamoylguanidine, 2-fluorocinnamoylguanidine, 4-fluorocionamoylgnanidine, 3,4-difluorocinnamoylguanidine 5-tert-butylamnino-amiloride, 2-napthoylguanidine, NN'Bis(amidino)napthalzn-2,6-dicarboxamide, N,N'-Bis(3-phenylpropanoyl)ganidine, 4-methylcinnamoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 2,3,5,6,-teramethylinnamoylguanidine, 3-ethoxycinnmamoylguanidine, N,N'-bis(3phnylpropanoyl)-N"-phonylguanidine, (4-Methoxycinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (3-Nitrooinnamoyl)guanidine, 4-etboxycinnamoylguanidine, 3,4,5-trimethoxyinnamoylguanidine, 2-(2-napthyl)acetoylguanidine and N-(3-phenylpropanol)-N-phenylgaanidine.
    [96] 96. The method according to claim 91, wherein said Coronavirus is any one of the known Coronavinrs isolates listed in Table 1. S
    [97] 97. The method according to claim 96, wherein said compound is selected from the group consisting of 4-isopropylcinnamoylguanidine, 3,4-dichlorocinnamoylguanidine. 3-(trifluoromethoxy)rinnamoylguanidine, 4-t-butyleinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, WO 2004/112687 PCT/AU2004/000866 -168
    [98] 98. The method according to claim 84, wherein said virus is the Hepatitis C virs. 5
    [99] 99. The method according to claim 98, wherein said membrane ion channel is the Hepatitis C virus p7 membrane ion channel.
    [100] 100. The method according to claim99, wherein said compound is selected from the group consisting of 2,3-dimethyloinnamoylguanidine, 2,4,6-trimethylcinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-(trifluoromethyl)cimamoylguanidine,. 4-(trifluoromethyl)cinnamoylguanidino, 6-methoxy-2-naphthoylguaidine, (2-Chlorocinnamoyl)guanidine, (4-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 2,6-dichlorochmaimoylguanidine,. (3-Bromooinnamoyl)guanidine, (3-Chlorocinnamoyl)guanidine, 2-(trifluoromethyl)cinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, 3,4-dichlorocirmnnamoylguanidine, 4-isopropylcinnamoylguanidine, trans-3-(1-napthyl)acryloylguanidino, 4-t-butyloinnamoylguanidine, 2-t-butyloinnamoylguanidine, 2-ethylcinnamoylguanidine, 4-methylcinnamoylguanidine, 5-bromo-2-methoxycinnammoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, 1-napthoylguanidine,. 3-t-butylcinnamoylguanidine, 4-phenylbenzoylguanidine. (5-Phenyl-penta-2,4-dienoyl)guanidine, N-(cinnamoyl)-N'phenylguanidine, 3-isopropyloinnmamoylguanidine hydrochloride, Benzamil hydrochloride, N-(3-phanylpropanoyl)-N'-phenylguanidine, N,N'-bis(3pbenylpropanoyl)-N"-phenylguanidine, WO 2004/112687 PCT/AU20041000866 -169 3-(2-napthyl)acryloylguanlldjne, 5-tiN-Mthy-N-isobutypanii'lorid;, 2'4 DichloroBcnzamil 'HCl, 5-tert-butylamino-annloide, 5-(N-Ethyl-I4-isopropyl)amiloride, (4-Methoxycinnaanoyl)guanidin;, 4-fluorocinnamoylgmaidine, (3-Nitrocinnarnoyl)guanidfine, 4 -ethoxycinnamoylguanidmao, (4-Hydroxycinnamy)guanicune, (trans-2-PhenYlcyclopopamcabony1)guancdne, 3-ethoxyeinnmnoylguanidine, 2,3,5,6,-tetmthylocbnamoylpuani dine, 4-phanylcinnamoy1guanidine, trans-3-Furanaoryygumuidine, N-(6-Hydroxy-2-napthoyl)-N-pheuylguanidine, (2-Furanacryloyl)guanicline, 3 -(eyclchex-l-en-1-y1)cinnamoylgpanidine, cinnamoylguanidire hydrochloride, 5-(N;N-hexamethyleue)amiloride, 2 ,3-difluorocinnamoylguanidinc,, 2-(I'-napthyl)acetoylguanidine, (a-Methylcinanoyl)guanidine, (2-Nitroc-innamoyl)guanldinc, 6-fodomAanloride, 3 5 4-(methylenedioxy)cinnamoylguanidine, 2-othoxyoinnanoylgvanidine, 'Cinamoylguanidine, 2-phenylcinnamoyguanicline, 2 -(cyvclohex-1-en-lyl)oinnamoylguanidie, 2-napthoylguaaiidine, 3-'phenykinnmoylguanf dine, 5-(N,N-Dimethyl)unioride hydrochloride, 5-(4-fluorophenyiamilvride, (3-MethoxycinnaoyIlgugnidlno, 2-fuorocinnamoylguanidine, 5-(3Y-bromopbenyl)penta-2,4-dinoylguanidine, [(4-Clorophenoxy-acetyljguanidine, (3-phcnylpropanoyl)guaidine, 2 -cbloro-6-fluorocinnamoylguanidine, 3-finorocinnamoylguanidine, 2-methylclimiaoyguanidiine, (2-Methoxycinnamoy)guaudne, 1-bromo-2-napthoylguanidiue, 3A45-trimethoxycinnainoylguanidine, 3-methylciumamoylguazidine, 3 -(trans-hept-l-en-1-yl)cinnamoylguanidine, Phenamil methanesulfonato salt, WO 2004/112687 PCT/AU2004/000866 -170 2,4-dichlorocinnamolyguanidine, (4-Nitrocinnamoyl)guanidine, 3,4-difluorocinnamoylguanidine and ((E)-3-(4-Dimethylaminophonyl)-2 methylacryloyl]guanidine.
    [101] 101. The method according to any one of claims 84 to 100, wherein said compound is provided as a pharmaceutical composition according to claim 5 4 or claim 5.
    [102] 102. A method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to any one of 10 claims 1 to 3, wherein said compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell,
    [103] 103. The method according to claim 102, wherein said virus is a Lentivirus. 15
    [104] 104. The method according to claim 103, wherein said Lentivirus is Human Immunodeficiency Virus (HIV).
    [105] 105. The method according to claim 104, wherein said membrane ion channel 20 is the HIV Vpu membrane ion channel.
    [106] 106. The method according to claim 105, wherein said compound is selected from the group consisting of (3-Chlorocinnamoyl)guanidine, (3-Bromocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine., 3-(trifluoromethyl)cinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methylinnamoylguanidine, 2-methylcinnamoylguanidineo, 2,3-dimnethyleinnrmamoylguanidine, WO 2004/112687 PCT/AU20041000866 4171 Cinm~oylguanidine, 6-niothoxy-2-naphthoylguanidinae, trans-3-(1 -napthyl)aaryloyguandine,, 3,4-diolilorocirmamnoylguanidine, 2 3 6-dicllorocinnanioylguanidine, 4-phenylbenzoylguanidine, 2-ethylcinuamoylguanldine, (4-Chlorooinnamoyl)guanidine,, 2-napthoylgaanidine, 2,S-dimothylci=naoylguanidine, 3-isopropylcinnanoylguanidine hydrochloride, (5-IPhenyl-pcnta-2,4-dinoyl)guanidine, 3-pheriylcinnamoylguanidine; (4-Bromocinm~oyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 3-(cyclohex-l-en-l-yl)cinnmoylgaanidine, 3-(trif luoiomethoxy)cinnamoylguanidine, 2-(frifiuoromethyl)cinnsmoylgumnidine, NN-bis(Sphenylpropanoyl)-N'-phenylguanidin;, 2-ethoxycinnamoylguauidine, N-(3-phenylpropanoyl)-N-pbenylguianidie, 4-(trif luormethyl)cinamoylguanidine, (4-Methoxycinnamoyl)guanidie, 2-t-butylei=naoylguanidine, 4-xnetykinm~oylguanidine, 2-fluorocinnainoylguanidine, 2-phenylcinnamoylguanidine, N-(6-Hydroxy-62-napthoyl)-N-phcnylguariidine, 34-butylolimamoylguanidine, 3,4-diflnorocinnanoylguanidine, 5-(N,N-hexaiethylene)amilotile, 3-luorocinnamoylgauidine, 5-broina-2-methoxycinnamoylguanidinao, 3-otoxycinnioygumnidine, 3,4-(mcth-ylcncdioxy)cinnanoylguanidine, (2-Methoxyo-innamoyl)guanidine, 2'4 Dicliloroflcnzainil EdI, 2,3,5,6,-tetramethylinunamoylguanidine, 3-(2-napthyl)acryloylguanidine, 2-(l -naptbyl)avetoylguaidine, 2,3-difluoroeinniamoylguanidine, (3-Methoxycinnm~oylguazdina, 4-isopropylaimnnaoy1guanidine, 2,4,6-tdimethyleinnamoylguanidine_, N-(oinnamoyl)-N'phenylguaniidinc, 2-(cyrlohex-l-en-lyl)cinnamoylguanidine, 2-(2-napthyl)aoetoylguanidine, (4-Hydroxyoinnamoyl)guanidie. WO 2004/112687 PCT/AU2004/000866 -172 4-phonylcienamoylguanidine, 4-fluorocinnamoylguanidine, NN'-bis-(cinnamoyl)-N"-phenylguanidine, (2-Furmanacryloyl)guanidine, Phenamil methanesulfonate salt, Beuzamil hydrochloride, (3-Nitrocinnamoyl)guanidine, Benzyoylguanidine, (4-Phenoxybenzoyl)guanijijne, 3-(trans-hept-1-en-1-yl)innamoylganidine, 5-(N-Methyl-N-isobutyl)amiloride, 2-cyclobexyleinnamoylguanidine, 4-ethoxycinnamoylguanidine, 2,4-dichlorocinnamolyguanidine, 5-(N-Ethyl-N-isopropyl)amiloride, N-amidino-3-amino-5-hexamethyleneimino-6-phenyl 2-pyrazinecarboxamide, (a-Methylcinnamoyl)guanidine, cinnamoylguanidine hydrochloride, [(4-ChtoWrophenoxy-acetyl]guanidine, N-amidino-3-amino-5-phenyl-6-chloro-2 pyrazinearboxamide, 5-(4-fluorophonyl)amiloride, (trans-2-Phenylcyclopropanecarbonyl)guanidine, (2-Nitrocinnamoyl)guanidino,. trams-3-Furanacryoylguanidine, 1-napthoylguanidine, 5-tert-butylamino-amiloride, 3-m ethoxy -HMA, (3-phenylpropanoyl)guanidine, 4-t-butylcirmamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, N,N'-Bis(3-phenylpropanoyl)guanidine, N-Benzoyl-N'-cinnmamoylguanidine and 1-bromo-2-napthoylguanidine.
    [107] 107. The method according to any one of claims 104 to 106, wherein said NIV is HIV-1.
    [108] 108. The method according to claim 102, wherein said virus is a Coronavirus.
    [109] 109. The method according to claim 108, wherein said membrane ion channel is the Coronavirus E protein. WO 2004/112687 PCT/AU2004/000866 -173
    [110] 110. The method according to claim 109, wherein said Coronavims is the Severe Acute Respiratory Syndrome virus (SARS). 5
    [111] 111. The method according to claim 110, wherein said compound is selected from the group consisting of 2,3-difluorocinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 4-t-butyloinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, (3-Chlorooinnamoyl)guanidine, 3-(oyclohex-1-on-1-y)cinmamoylguanidine, 2,5-dimethyloinnamoylguanidin, trans-3-(l-napthyl)aoryloylguanidine, 4-isopropylcinnamoylguanidine (3-Bromocinnamoyl)guanidine, 6-methoxy-2-naphthoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenylcinnamoylguanidine, (2-Chlorocinnamoyl)guanidino, 2'4 DichloroBenzamil HICI, 4-phenyloinnamoylguauidine, 4 -(trifluoromethyl)oinnammoylguanidine, 3 -(trifluoromethoxy)cinnamoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, 2-othoxycinnamoylguanidine, cinnamoylguanidine hydrochloride, 4-ethoxycinnamoylguanidine, (2-Bromoeinnamoyl)guanidine, 2,6-dichlorocinnamoy1guanidine, 3,4,5-trimethoxycinnamoylguanidine, 5-tort-butylamino-amiloride, 3-t-butylcinnamoylguanidine, 5-bromo-2-fluoroeinnamoylguanidine, (4-Chlorocinnamoyl)guanidine, 2-t-butyloinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, 6-lodoamiloride, 3-(wtrans-hept-1-en-1-yl)cinnamoylguanidine, (4-Bromocinnamoyl)guanidine, (4-Hydroxycinnamoyl)gaanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, (3-Nitrocinnamoyl)guanidine, 3-fluorocinnamoylguanidine, 2-(1-napthyl)acetoylguanidine, WO 2004/112687 PCT/AU20041000866 -174 2-pthykminamoyguanidiuc. S-(N,N-Dixnethyl)amiloyjde hydrochloride, 2-napthoylguanidine, 5-(4-fluorophenY1)aniIoiide, 2 -(frfuoromthy)limasnoylgua~dine, N-( 6 -Hydroxy-.2-napthoylj-N'-phenylguanidine, (trans- 2 -Thenylcyclopropanecarbonyl)guanidin, NNbs3hnlrpny)*'-hnlu-die 1-naapthoylguanidne Beuzaroil hydrochloride, 3 -methoxy -INA, 4-methylainnamoylgaanidine, 4 -fluorooinnaaoylguanidine, 3 ' 4 -(meth 'y1encdioxy)cinmoylguaicflne, S-(N,N-hexaxnethylene)ainilorido, N-(cim~oy)-Vphenylguanidine, 5-(N-Ethyl-N-isopropyl)amnilorTide, 3-methycnaxoyguanjne, 2 -methyohmaxnoylguanjdine, 2 , 3 ,S, 6 ,$.etramethylinnamoylguancune, trans-3-Furaacryoylguanidin; .(4-Methoxycinnamoyl)guaiidine,, (2Tvuranaeryloyl)guanidine, (3-pb aylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidine, Cinn amoylguanidine, (2-Methoxycinnanioyl)guanidine, 13-(3-Pyridyl)aoiyloyl]guanld mc, 4-phenylbenzoylguanidin;, 2 , 4 -dichlorccinnamolyguanidine, (S-Methoxycinnamoyl)guanidine, 2-fluorocinnmtoylgusnidine, (4-Phenoxybenoy)guaxddino, (a-Methylcinnanoyl)guanclhne. S-(Y -bromophenyl)penta-2,4-dienoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidlne, (Quinolino-2--carbonyl)guanidine, (Phenylacetyl)guanidine, NN-Bis(amidino)napthaene-2,&-dioarboxade, 6-bromo-2-napthoylgaanidine, 1-brorno-2-napthoylguanidine, 2 -chloro- 6 -fluorooinnamoylguanjdi-ne, E( 4 -Chlorophenoxy-acetyl]guanid in;, Phmnil methaesulfonate salt, N.]Benzoyk-Mteinamoylganidmno and N-(2-napthoyl)-N-phenylgancjnc. WO 2004/112687 PCT/AU2004/000866 -175
    [112] 112. The method according to claim 109, wherein said Coronavirms is human Coronavirus 229E.
    [113] 113. The method according to claim 112, wherein said compound is selected 5 from the group consisting of 4 -isopropylcinnamoylguanidine, 3,4-dichlorocinnamoylguaidine, 3 -(trifluoromethoxy)oinnamoylguanidine, 4+butyloinnamoylguanidine, 3-isopropylomnnamoylguanidine hydrochloride, 3-t-butylkinnamoylguanidine, 2 -t-butyloinnamoylguanidine, trans- 3 -(l1-napthyl)acryloylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 2,3-difluorocinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, 2-phenylcinnamoylguanidine, 3-phonylcinnamoylguanidine, 3-(cyclohex-1-on-1-yl)clhmamoylguanidine, 4-phenylbenzoylguanidine, 3-(trifluomromethyl)cinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, 4-(trifluoromethyl)cintamoylguanidine, 2 -(cyclohex-l-en-lyl)cinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 5-(N,N-hexamethylene)amiloride, 1-napthoylguanidine , 5-(4-fluorophenyl)amiloride, (5-Phenyl-penta-2,4-dienoyl)guanidinc, (3-Bromooinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 2 -(trifluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, (4-Chlorocinmamoyl)guanidine, (3-Methoxycinnamoyl)guanidine, S-bromo-2-fluorocinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, Cinnamoylguanidine, (2-Methoxycimnnamoyl)guanidine, (a-Methylcinnamoyl)guanidine, 4-phenyloinnamoylguanidine, 2,6-dichlorocinnamoylguanidine, (2-Bromocinnamoyl)guanidine, 2,4,6-trimethylcinnamoylguanidine, WO 2004/112687 PCT/AU2004/000866 -176 (trans-2-Phenylcyclopropanecarbonyl)guanidine 3 , (3-Chlorocinnamoyl)gnanidine, 2-(1-napthyl)acetoylgnaidine, 2-ethylcinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, (4-Hydroxycinamoyl)guanidino, 2-ethoxycinnamoylguanidine, 3-methylcinnamoylguanidine, 2-methyloinnamoylguanidine, 3-fluorocinnamoylguanidine, cinnamoylguanidine hydrochloride, 2,3-dimethylcinnamoylguanidine, 2-fluorocinnamoylguanidine, 4-fluorocinnamoylgunidine, 3,4-difluorocinnamoylguanidine, 5-tert-butylamino-amiloride, 2-napthoylguanidine, N,N-Bis(amidino)napthalone-2,6-dicarboxamide, NN-Bis(3-phnylpropanoyl)guanidine, 4-methylinuamoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 2,3,5,6,-tetramethyloinnamoylguanidine, 3-ethoxycinnamoylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, (4-Methoxycinnamoyl)gumanidine, (2-Chlorocinamoyl)guanidino, (3-Nitrocinnamoyl)guanidine, 4-ethoxycinnamoylguanidine, 3,4,5-trimethoxycimnMoylguanidine, 2-(2-napthyl)acetoygunidine and N-(3-phenylpropanoyl)-N'-phenylguanidineo.,
    [114] 114. The method according to claim 109, wherein said Coronavirus is any one of the known Coronavirus isolates listed in Table 1. 5
    [115] 115. The method according to claim 114, wherein said compound is selected from the group consisting of 4-isopropyloinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)cinanamoylguanidine, 4-t-butylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, WO 2004/112687 PCT/AU2004/000866 -177
    [116] 116. The method according to claim 102, wherein said virus is the lRepatitis C vin's.
    [117] 117. The method according to claim 116, wherein said membrane ion channel 5 is the Hepatitis C virus p7 membrane ion channel.
    [118] 118. The method according to claim 117, wherein said compound is selected from the group consisting of 2,3-dimethyleinnamoylguanidine, 2,4,6-trimethyloinnamoylgnanidine, 5-bromo-2-flumorocinnamoylguanidine, (4-Bromocinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-(trifluoromethyl)cinnamoylgnuanidine, 4-(trifluoromethyl)oinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, (2-Chlorocinnamoyl)guanidine, (4-Chlorocinnrmamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 2,6-dichlorocirnamoylguanidine, (3-Bromocinnamoyl)guanidine, (3-Chlorocinnamoyl)guanidine, 2-(trifluoromethyl)cimnamoylguanidine, (4-Phenoxybtenzoyl)guanidine, 3,4-dichlorocinnamoylguanidine, 4-isopropylcinnamoylguanidine, trans-3-(1-napthyl)acryloylgnanidine, 44butylcinnamoylgnanidine, 2-t-butylcinnamoylguanidine, 2-ethyloinnamoylguanidine, 4-methylcinnamoylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 3-(tifluoromethoxy)cimamoylguanidine, 2-eyclohexyloinnamoylguanidine, 1-napthoylguanidine, 3-t-butylcinnamoylguanidine, 4-phenylbenzoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, N-(oinnamoyl)-Nphenylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, Benzamil hydrochloride, N-(3-phenylpropanoyl).N'-phenylguanidine, N,1-bis(3phenylpropanoyl)-N"-phnylguanidine, 3-(2-napthyl)acryloylguanidine, WO 2004/112687 PCT/AU2004/000866 5-(N-Methyl-N-isobntyl)amlloride, 2'4 DiohloroBenzamil 1101, 5-tart-butylamino-amiloride, 5-(N-Ethyl-N-isopropyl)amiiloide, (4-Methoxycinnamoyl)guanidine, 4-fluorocinnamoylgaanidine, (3-Nitrocinnamoyl)guauidine, 4-ethOxYCimoylguandie, (4-Hydroxyczinnamoyl)guanidine, (trans-2-Phenylcyoclopropanecarbonyl)guanidine, 3-ethoxycinnamoylgaanidin;, 2,3,5,6,-tetramethylcinnamoylguanidine, 4-phenyloinnamoylguanidine, tmis-3-Furnacryoylguamdim, N-(6-Hydroxy-2-napthoyfl4PM-phenylguanidine, (2-Furmnaoryloyl)guanidine, 3-(cyclohex-1-en-1-yl)cinanaznoylgnanidine, clnnamoylgtwnidino hydrochloride, 5-(NN-h==mehyene)amilozide, 2,3-difluorocinnamoylgaidin;, 2-(1-napthyl)acetoyiguanidin;, (a-Methyloinnamoy])guanidine, (2-Nifrooinrmoyl)guauidine. 6-Iciloamiloride, 3,4-(methylenedioxy)cinnaroylguanidin; 2-ethoxyeinnamoylguanidine, Ciinamoylguanidine, 2-phenylcinnamoylguanldine, 2-(oyclohex- 1-en-I yl)oinmoylgaidine, 2-napthoylgumnidine, 3-phonylcinuamoylguanidine, 5-KNN-Dimetbyl~amilodde hydrochloride, 5-(4-fluorophenyl)arniloride, (3-Methoxyoinaoyl)guanidiae, 2-flnorainnnoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, ((4-ChtoroPhcnOXY-aeetyl~guanidine, (3-phenylpropanoyl)guanidine, 2-ohloro-6-fluowinmnoylguanidine, 3-fluorocfrnamoylgnaniin; 2-nethy~cim~oylguanidineI (2-Metoxyinnamoyl)gusnidine, 1 -bromo-2-napthoylguaidine, 3,4,5-tiimethoxyainnamoylguanidine, 3-mothylcirmainoylguanidine, 3-(frans-hpt-I -en-I-yI)dnnaaoylguidie, Phenamhl aetbanesufonate salt, 2,4-dicbloroeinnamolyguanidine WO 2004/112687 PCT/AU2004/000866 -179 (4-Nitrocinnamoyl)guanidine, 3,4-difluorooinuamoylguanidine and [(E)-3-(4-Dimethylaminophonyl)-2 methylacryloyl]guanidine.
    [119] 119. The method according to any one of claims 102 to 118, wherein said compound is provided as a pharmaceutical composition according to claim 4 or claim 5. 5
    [120] 120. A method of reducing, retWarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound according to any one of claims 1 to 3, wherein said compound down regulates functional 10 activity of a membrane ion channel expressed in said infected cell.
    [121] 121. The method according to claim 120, wherein said virus is a Lentivirus.
    [122] 122. The method according to claim 121, wherein said Lentivirus is Human 15 Immunodeficiency Virus (HIV).
    [123] 123. The method according to claim 122, wherein said compound is selected from the group consisting of (3-Chlorocirnnamoyl)guanidine, (3-Bromooinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)cinnamoylguanidine, 5-bromo-2-fluorocinnamoylguanidine, 3-methylcinnamoylguanidine, 2-mcthylinnamoylguanidine. 2,3-dimethylcinnamoylguanidinie, Cinnamoylguanidino, 6-methoxy-2-naphthoylgnanidine, trans-3-(l-napthyl)acryloylguanidine, 3,4-dichlorocinnamoylguanidine, 2,6-dichlorodinnamoylguanidine, 4-phenylbenzoylguanidine, 2-ethylinnamoylguanidine, (4-Chlorocinnamoyl)guanidine,, WO 2004/112687 PCTIAU20011000866 2-napthoylguaW41nn; 2,5-dimethylcinnamnoylgaanidine, 3-isopropykoinnamoylguanidine hydrochloride, (5-Pheny1-penta-2A-dienoy)gpaidhin0 3-phenylinnamoygumaidine. (4-Bromacimnoyl)guanidin;, 5-(3'-broxnophcnyl)penta-2,4-dienoylgaanidine, 3-(cyelohex-1-en-1-yl)cinnamoylguanidine, 3-(trifluoromethoxy)innamoylguanidfle, 2-(triflnromethyl)cinm~oylguanidine NN'bis(3phenylpropanaylD-N"-phenylguanidinc. 2-ethoxycinnamoylguaiiidifle, N-(3-pheniylpxopnoyl)44'-phenylguanidine, 4-(trifluoroniethyl)cinnamoylguanidine, (4-MeffioXyoinnamoylguainidine, 2-t-butylcinnamoygumnidine, 4-mcthylc-innaxnoylguanidin, 2-fiuoraoinnamoylgmiiidifle, 2-phenylcinnmtoylguanidine. N-(6-Hydroxy-2-napthoyl)-N-pbcnylguanidinc, 3-t-butylcinnamoylguanidine, 3,4-difluorocimimoylguanildine, 5-(NL-hexmethyleDne)ami~oride, 3-fluorooimamoylguaniidifle, S-bromo-2-metkoxyvhmaoylguanidine. 3-ethoxycinnmncylguanidine, 3,4-(methylenedioxy)cinnaioylguankline, 2'4 DichloroBenzamil HCI, 2,3,5,6,-tetrainethyloinnhznoylguanidine, 3-(2-naptbyl)acryloylgaanidine, 2.{1-napthyl)acetoylguandin, 2,3-difluorooinnamoylguanidino, (3-Methoxycinnmoyl)guanidine, 4-isopropyIrnnamOygu~ndife, 2,4,6-trimethyloinnamoylguanidine,. N-(cinamoyl)-N'phenylguanidinc, 2-(oyolohex--en-lyl)cinnamoylguanidin;, 2-42-napthyl)acetoylguanidime (4-Hydroxycinnaoyl)guauidineu, 4-phenylcinnamoylguanidn; 4-fluorocimnamoylgnanidine, N,-i-cnawl-NIpeygaiie (2-Furanacryloyl)guanidine, ,Phnmjil motbhasulfonate salt, BcnzajI hydrochloride, (3-Nitroinamoyl)guanidilo, Beoylguaidine, WO 2004/112687 PCT/AU2004/000866 -181 (4-Phonoxybenzoyl)guanidine, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 2-cyclohbexyloinnamoylguanidine, 4-ethoxycinnamoylguanidine, 2,4-dichlorocinnamolyguanidine, 5-(N-Ethyl-N-isopropyl)amiloride, N-amidiso-3Lamino-5-hexamethylnaimino-6-phenyl 2-pyrazinocarboxamide. (a-Methyloinnamoyl)guanmidine, cinnamoylguanidine hydrochloride, [(4-Chlomrophenoxy-acetyl]guanidine, N-amidino-3-amino-5-phenyl-6-chloro-2 pyrazinecarboxamide, 5-(4-fluorophenyl)amiloride, (trans-2-Phbenylcylopropanecarbonyl)guanidine, (2-Nitrodcinnamoyl)guanidine, trans-3-Furanacryoylguanidine, 1-napthoylguanidine, 5-tert-butylamino-amiloride, 3-methoxy -HMA, (3-phenylpropanoyl)gnanidine, 4-t-butyloinnamoylguanidine, 5-(N,N-Dimcthyl)aniloride hydrochloride, NN'-Bis(3-phnylpropanoyl)guanidine, N-Benzoyl-N'-cinnamoylguanidine and 1-bromo-2-napthoylguanidine.
    [124] 124. The method according to any one of claims 120 to 123, wherein said membrane ion channel is the HIV Vpu membrane ion channel. S
    [125] 125. The method according to any one of claims 122 to 124, wherein said HIV is HIV-1.,
    [126] 126. The method according to claim 120, wherein said virus is a Coronavirus. 10
    [127] 127. The method according to claim 126, wherein said Coronavirus is the Severe Acute Respiratory Syndromc virus (SARS). WO 2004/112687 PCTIAU20011000866
    [128] 128. The method according to claim 121, wherein said compoud is selected flom the group consisting of 2,3-difluorooinnsmoylguanidine, 3,4-dicblorocinnamoyguaflidie, 4-t-butyloinnamoylguanidine, 3-42-napthyl)aoryloylguanidine, (3-Chloroinnamoylguanidine, 3-(cyclohex-1-en-l-yl)cinnamoylguanicline, 2,5-diiethyloinmoylguanidine, trans-3-(1-napthyl)acryloylgaanidin;. 4-isopropylcinnanoylgnanidine, 6-methoxy-62-naphthoylguanidine, S-{N-Methyl-N-isobutyl)amiloride, 3-phenylClnnaloylgFandie, (2-Chloroc~innamoyl)guanidine, 2'4 DichioroBerizmil HCJ, 4-phenyloinnainoylguanidine, 4-(trifluoromethyl)cinnamoylguanidine, 3-(frifluoromcthoxy)oinxiamoylglianidine, 3.(trifluoromnethy1)cinnatuoylguanidine, 2-ethoxycairmnoylguanidine, cinnaoylganidine hydrochloride, 4-etboxycoinnamoylgaanidine, (2-Bromocinnanioy1)gvanidine, 2,6-dichloroolimamoylguanidine, 3,4,5-trimethoxycinnoylguanldile, 5-tefl-butylamino-amiloride, 3-t-butylcinamoyguanidIne, 5.bromo-2-fluorociimdlmoylgnanidine, (4-Cblorovinnamoyl)gn~Anide, 2-t-hutylcinnamoylguanidine, 2-o~yrlohexyleinnamoylguaridifle, 6-Iodoamiloride, 3-(tram-hept-l -en-I -yI)cinnamoylguanidine, (4-Bromocinnnoyl)guanidilie, (4-Hydroxycinnamoyl)guanidine, N-(3-phenylpropanoyl)-N'-phonylguaflidhnl, (3-Nitkoeinn~unoy1)guanidine, 3-flnorocinnaniylguanidine, 2-(l-napthy])aceoylguanidin;, 2-ethyleixmaoylguanidine, 5-(NN-DimethyIamiloride hydrochloride, 2-napthoylguanidine, S-(4-tluorophenyi)awiloride, 24tdfluoromehiy)oinnamoylguanidfle, N-(6-Hydroxy-2-napthoy1)-N'-phenylguanidile, WO 2004/112687 PCT/AU2004/000866 -183 (trans-2-Phenylyclopropanecarbonyl)guanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, 1-napthoylguanidine, Benzamil hydrochloride, 3-methoxy -JHMA, 4-methyloinnamoylguanidine, 4-fluorochmanoylguanidine, 3 , 4 -(methylenedioxy)einnamoylguanidine, 5-(NN-hexamethylene)amiloride, N-(cianamoyl)-N'phenylguanidine, 5-(N-Ethyl-N-isopropyl)amiloride, 3-methyloinnamoylguanidine, 2-methylcimamoylguanidine, 2 ,3,5,6,-tetrwmethylcinnamoylguanidine, trans-3-Furanacryoylguanidine, (4-Methoxycinnamoyl)guanidine (2-Furanaoryloyl)guanidine, (3-phenylpropanoyl)guanidine, 2-(2-napthyl)acetoylguanidine, Cinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, [ 3 -(3-Pyridyl)acryloyl~guanidine, 4-pheny1benzoylguanidine, 2,4-dichlorocinnamolyguanidine, (3-Methoxycinnamoyl)guanidine, 2-fluorocinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, (a-Methyleinnamoyl)guanidine, 5-(3'-bromophony)pnta-2,4odienoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, (Quinoline-2-carbonyl)guanidine, (Phenylacetyl)guanidine, NN-Bis(mdino)oaptaen 26-dcarboamide, 6-bromo-2-napthoylguanidine, 1 -bromo-2-napthoylguanidinle, 2-chloro-6-fluorocinnamoylguanidine, [(4-Chlorophenoxy-acetyl]ganidine, Phenamil metbanesulfonate salt, N-Benzoyl-N'-cinnamoylguanidine and N-(2-napthoyl)-N'-phenylguanidine.
    [129] 129. The method according to any one of claim 126 or 128, wherein said membrane ion channel is the Coronavirus E protein. 5 WO 2004/112687 PCT/AU2004/000866 -184
    [130] 130. The method according to claim 126, wherein said Coronavirus is human Coronavirus 229E.
    [131] 131. The method according to claim 130, wherein said compound is selected 5 from the group consisting of
    [132] 132. The method according to claim 131, wherein said compound is selected from the group consisting of 4-isopropyloinamoylgusanidine, 3,4-dichlorocinnamoylguanidine, 3-(tiif uoromethoxy)cinm oylguamidin e, 4-t-butylcinnamoylguanidine, 3-isopropyleinnamoylguanidine hydrochloride., 3-t-butyleibnamoylgnanidine, 2-t-butylcinnamtoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 5-bromo-2-methoxycinnamoylguanidine, 2,3-difluorocinnamoylguanidine, 3-(2-napthyl)aeryloylguanidine, 2-phonylinnamoylguanidine, 3-phenylcinnamoylguanidine, 3-(cyclohox-1-en-1-yl)cinnamoylgnanidine, 4-phenylbenzoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, 4-(trifluoromethy)oinnamoylgnanidine~, 2-(cyclohex-1-en-lyl)cinnamoylguanidine, (4-Bromooinnamoyl)guanidine, 5-(N,N-hexamethylene)amiloride, 1-napthoylguanidine, 5-(4-fluorophonyl)amiloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bromocinnamoyl)guanidine, 2,5-dimethyloinnamoylguanidine, 2-(trlfluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylgpanidine, (4-Chlorocinnamoyl)guanidine, (3-Methoxycinnmamoyl)guanidin, 5-bromo-2-fluorocinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, Cinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, (a-Methylcinnamoyl)guanidine, WO 2004/112687 PCT/AU2004/000866 -185 4-phenylcinnamoylguanidine, 2,6-dichlorocinnatuoylguanidine, (2-Bromocinnamoyl)guanidine, 2,4,6-trimethylcinnamoylguanidine, (trans-2-Phenylcyolopropanecarbonyl)guanidineo, (3-Chlorocirmnnamoyl)guanidine, 2-(1-uapthyl)acetoylguanidine, 2-othylinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, (4-Hydroxycinnamoyl)guanidine, 2-ethoxycinnamoylguanidine, 3-methylcinnamoylguanidine, 2-methylcinnamoylguanidine, 3-fluorocinnamoylguanidine, cinnamoylguanidine hydrochloride, 2,3-dimethylcinnamoylguanidine, 2-fluorocinnammoylguanidine, 4-fluorocinnamnoylguanidine, 3,4-difluorocinnamoylguanidine, 5-tert-butylamino-amiloride, 2-napthoylguanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, NN-Bis(3-phonylpropanoyl)guanidine, 4-methylcinnamoylguanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 2,3,5,6,-tetramethylcinnamoylguanidin, 3-ethoxycinnamoylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, (4-Methoxycinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (3-Nitrooinnamoyl)guanidine, 4-ethoxycinnmamoylguanidine, 3,4,5-trimethoxyoihmamoylguanidine, 2-(2-napthy)acetoylguanidine and N-(3-phonylpropanoyl)-N'-phenylguanidine.
    [133] 133. The method according to anyone of claims 130 or 132, wherein said membrane ion channel is the Coronavirus E protein. 5
    [134] 134. The method according to claim 126, wherein said Coronavirus is any one of the known Coronavirus isolates listed in Table 1. WO 2004/112687 PCT/AU2004/000866 -186
    [135] 135. The method according to claim 134, wherein said compound is selected fom the.group consisting of 4-isopropylinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)oinnamoylguanidine, 4-t-butyloinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride,
    [136] 136. The method according to claim 134 or claim 135, wherein said membrane S5 ion channel is the Coronavirus E protein.
    [137] 137. The method according to claim 120, wherdin said virus is the Hepatitis C virus. 10
    [138] 138. The method according to claim 137, wherein said compound is selected from the group consisting of 2,3-dimethylcinnamoylguanidine, 2,4,6-trimethylcinnamoyguanidine, 5-bromo-2-fluorooinnamoylguanidine, (4-Bromocinnamoyl)guanidine. 2,5-dimethylcinnamoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine. 4-(trifluoromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, (2-Chlorocinnamoyl)guanidine, (4-Chlorocinnamoyl)guanidine, (2-Bromocinnanoyl)guanidine, 2,6-dichlorocinnamoylguanidine, (3-BromccinnamoyI)guanidine, (3-Chlorociunamoyl)guanidine, 2-(trifluoromethyl)oinnamoylguanidine, (4-Phenoxybenzoyl)guanidinc. 3,4-dichlorocinmamoylguanidine, 4-isopropyloinnamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 4-t-butylcinnamoylguanidine, 2-t-butylcinnhmamoyguanidine, 2-ethylcinnamoylguanidine, 4-methyleinnamoylguanidine, 5-bromo-2-methoxyeinnamoylguanidine, 3-(trfluoromiethxy)cinnamoylgualidine, 2-oyclohexyloimnamoylguanidine, 1-napthoylguanidine, WO 2004/112687 PCTAU20011000866 3-1-butyleimiamoylguanidin;, 4-phenylbenzoylgwonidine, (5-Phenyl-penta-2,4-dienoyl)guaniddtne, N-(cinnamoyl)-N'phenylguonidine, 3 -isopropylnnmoyguandjne hydrooblarido, Benzamil hydrochloride, N-(3-phenylpropanoyl)-N-phenylguanidinc, N,N-bis(3phenylpropanoy1)-N"l-phenylguanidine, 3-(2-napthyl)aoryloylguanoidine. 5-(N-Methyl-N-isobutyl)ainiilaride, 274 DichlonoBenzamil HOI, 5-tert-butylamino-aniilorkle, 5-(N-Bthyl-N-isopropyl)amnioride, (4-Methoxyoinnamoyl)guanidlne, 4-fluorocinnamoylguanidine, (3-Nitrochmainoyl)guanidin;, 4-ethoxycinmoylguaidine, (4-HydrosycinnamOy)9unndine (tras- 2 -Phenylc-yclopropanearbonyl)guanidine, 3-ethoxycirmnamoylgnanidine, 2,3,546-tlramthyliamoylguanidine, 4-phenylcinnamoy~guanidime, trans-3-Furanacryoylguanidine, N-(6-Hydroxy-2-napthoy)-N'-phenylgunidin, (2-Furanacryloyl)guanidine, 3-(oyclohex-l-en-1 -yl)cinnamoylguanidine, cinnamoylguanidine hydrochloride, 5-(NN-hexamethylene~axnloride, 2,3-difluoroeinnamoylguanidine, 2-(I-naptby)acetoylguanidine, (A-Methylcimamoy)gaIwftie, (2-Nltrocinnamoyl)guanidine, 6-Iodoamiloride, 3,4-(methylenedioxy)cinnamoylguarnidine, 2-ethoxycinmnoylguanidin;, Cinnamoylguandine, 2-phcaylcinnamoylguanidine, 2-(cyc~lohex-1-en-lyl)cinnamoylguanidine, 2-napthoylguanidin;, 3-phenylcinnatoylguanidin;, 5-(N,N-Dhnethyl)amiloride, hydrochloride, 5-(4-fluoropheny)amiloride, (3-MethoxyOi==~oy1)guan~idine, 2-fluorocimiamoylguanidine, 5-(3'-bromophenyl)ponta-2,4-dienoylguanidine, [(4-Cilorophenoxy-acewfllguanidine, (3-phenylpropanoyl)guanidine, 2-chloro-6&fluorooinnamoylguanidine, WO 2004/112687 PCT/AU2004/000866 -188 3-fluorocinnamoylguanidine, 2-methylinnamoylguanidine, (2-Methoxycinmamoyl)guanidine, 1-bromo-2-napthoylguanidine, 3,4,5-trimethoxycinnamoylguanidine, 3-methylcinnamoylguanidine, 3-(trans-hopt-1-en-1-yl)oimamoylguanidine, Phenamil methanesulfonate salt, 2,4-dichlorocinnamolyguanidine, (4-Nitrocinnamoyl)guanidine. 3,4-difluorocinnamoylguanidine and [(E)-3-(4-Dimethylaminophenyl)-2 methylacryloylJguanidine.
    [139] 139. The method according to claim 138, wherein said membrane ion channel is the Hepatitis C virus p7 membrane ion channeL 5
    [140] 140. The method according to any one of claims 120 to 133, wherein said mammal is a primate.
    [141] 141. The method according to any one of claims 137 to 139, wherein said mammal is a primate. 10
    [142] 142. The method according to claim 140 or claim 141, wherein said primate is human.
    [143] 143. The method according to any one of claims 120 to 142, wherein said 15 compound is provided as a pharmaceutical composition according to claim 4 or claim 5.
    [144] 144. A method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising administering to said 20 subject a compound according to any one of claims 1 to 3, wherein said compound down-regulates functional activity of a membrane ion channel derived from said virus.
    [145] 145. The method according to claim 144, wherein said virus is a Lentivirus. WO 2004/112687 PCT/AU2004/000866 -189
    [146] 146. The method according to claim 145, wherein said Lentivirus is Human Immunodeficiency Virus (HIV). 5
    [147] 147. The method according to claim 146, wherein said membrane ion channel is the HIV Vpu membrane ion channel.
    [148] 148. The method according to claim 147, wherein said compound is selected from the group consisting of (3-Chloracinnamoyl)guoanidine, (3-Bromocinnamoyl)guanidine, S(2-Chlorocitmamoyl)guanidine, (2-Bromocinnamoyl)guanidine, 3-(trifluoromethyl)cinamoylguanidine, 5-bromo-2-fluorocinnamoylgualidine, 3-methyloinnamoylguanidine, 2-methylcinnamoylguaidine, 2,3-dimcthylcinnamoylguanidine, Ciniamoylguanidine, 6-methoxy-2-naphthoylguanidine, trans!-3-(1-napthyl)acryloylguanidine, 3,4-dicbhlorocinnamoylguanidine, 2,6-dichlorocinnamoylgumnidine,. 4-phenylbenzoylguanidine, 2-ethyleinnamoyguanidine, (4-Chlorocinnamoyl)guanidine,, 2-napthoylguanidine, 2,5-dimethylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, (5-Phenyl-ponta-2,4-dienoyl)guanidine, 3-phenylcinnamoylguanidino, (4-Bromocinnrmamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, 3-(tdrlfluoromethoxy)cinnamoylguanidine, 2-(trifluoromethyl)oinnamoylguanidine, N,N'-bis(3phonylpropanoyl)-N"-phenylguanidinc, 2-ethoxycinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 4-(trifluoromethy])cinnamoylguanidine, (4-Methoxyciznamoyl)guanidine, 2-t-butylinnamoylguanidine, 4-methykinnamoylguaniiidine, 2-fluorociamoylguanidine, WO 2004/112687 PCTIAU20011000866 2-phenylchmamoylgaanidine, N-(6-HydrToxy-2-napthoyl)-N-phenylguanidn% 3-t-bulyleinnamoylgu&andine, 3,4-difluorocinrnoylguanidine; 5-4N,'N-hpxamethylono)ainiloride, 3-fluorochmamoylguWidiao, 5-bromo-2-methoxycinnamoylguanidne, 3-ethoxyoinmnmoylgumnidiue,. 3,4-(metliylenedioxy)cinnamoylgaanid ine, (2-Methoxycinnainoyl)guanidin; 214 DichloroBenzamil ECI, 2,3,5,6,-tetnimethyloinuaxnoylguanildine, 3-(2-napthyl)acryloylguanidine,, 2-(I-ziapthyl)acetoylguanidine, 2,3-difluorocinnamoylgusnidine, (3-Metoxycinnamoyl)guanidine; 4-isopropylcinnmoylguanidine, 2,4,6-trimethylcinnamoylguanidine, N-(cinnamoyl)-N~pheylguaidin; 2-(eYCloliosl'-en-l yl)cinnamoylguiaidine, 2-42-napthyl)acotoylguanidine, (4-Hydroxycinnmnoyl)guanidine. 4-phenylcirmmnoylguanidine, 4-fluoroeinnamoylgaanidinc, N,N'-bis-(oinnanoyl)-W-phenylguanidine, (2-Furanaoryloylguankinile, Phenewnil methanesulfonate salt, Benzmil hydrochloride, (3-Nitrooinamoyl)guanidin; Benzyoylguanidine, (4-Phenoxybenzoyl)guanidine, 3-(trans-hept- 1-en-1-yl)oinnaioylguanf dine, 5-(N-Methyl-N-isobutyl)ainiloride, 2-cyctohexylcirmamoylgaanidine, 4-othoxycinnamoylganidine, 2,4-dichlorocinnamolyguanidine, 5-(N-Ethy1-N-isepropy1)amiloride-, N-arnidino-3-amino-5-hexamethyleneimino-6-phenyl 2-pyrazineoarhoxanid;, (a-Methylreiuinoyl)gua-nidine, cimamoylguanidine hytochoride, [(4-Chlorphenoxy-acetylguanidine, N-amidino-3-aniino-5-phenyl-6-ch~oro-2 pyrazineoaboxamnide,. 5-(4-:fluorophenyl)amiloride, (tras-2-Thenylclopropanecazbonyl)guanidino, (2-Nitrooinnmoyl)guanidin; trans-3-Fu~aaoryoylguamidfl, WO 2004/112687 PCT/AU2004/000866 -191 1-napthoylguanidine, 5-tert-butylamino-amilorido, 3-methoxy-HMA, (3-phenylpropanoyl)guanidine, 4-t-butyloinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, N,N'-Bis(3-phonylpropanoyl)guanidine, N-Benzoyl-N'-cinnamoylguanidine and 1-bromo-2-napthoylguanidine.
    [149] 149. The method according to any one of claims 146 to 148. wherein said HIV is HIV-1, S
    [150] 150. The method according to claim 144, wherein said virus is a Coronaviras.
    [151] 151. The method according to claim 150, wherein said membrane ion channel is the Coronavirus E protein. 10
    [152] 152. The method according to claim 151, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS).
    [153] 153. The method according to claim 152, wherein said compound is selected 15 from the group consisting of 2,3-difluorocinnamoylguanidine, 3, 4 -dichlorocinnamoylguanidine, 4-t-butyloinnamoylgumnidine, 3-(2-napthyl)acryloylganmidine, (3-Chlorocinnamoyl)gpanidine, 3-(cyclohOXl-on-1-yl)cinnamoylguanidine, 2,5-dimethylcinnamoylguanidine, trans-3-(1-napthyl)aryloylguanidine,. 4-isopropylcinnamoylguanidine, (3-Bromocionamoyl)guanidine, 6-methoxy-2-naphthoylguanidine, 5-(N-Methyl-N-isobutyl)amiloride, 3-phenyleirmamoylguanidin%, (2-Chloroinnamoyl)guanidine, 24 DichloroBenzamil HCI, 4-phenyleinnamoylguanidine, 4-(trifluoromethyl)cinnamoylguanidine, WO 2004/112687 PCT/AU20011000866 -192 3-(trifluoromethoxy)cinnamoylguandine, 3-(trifiuoromethyloinnamoylnaidin0, 2-etlioxycinnainoylguanidine,' cinnamoylguanidine hydrochloride, 4-ethoxycinna-moylguanicline, (2-Rromocinnamoyl)guanidine, 2,6-dichoracihmanoylguaidxae, 3,4,5-trimethoxycinnamoylguanidine, 5-tert-butylamino-amiloride,' 3-t-butylcinnaioylguandine, 5-bromo-2-fluorocinnamnoylguanidine, (4-Choroeinnamnoyl)guaidine, 2-t-butyleinamoylgiianidie, 2-cyolohexylcinm~oylguanidine, 6-Iodoaznilojride, 3-(t-ns-hcpt-1-en-l-yI)cinnamoylguaaidine, (4-Bromocinamoyl)guanidin, (4-Ilydroxycinnainoyl)guanidne, N-(3-phanylpropanoy])-N'-phenylguanidinc, (3-Nifrocinnmoyl)guanidine, 3-fluorccinnanoylgvauidine, 2-(l -napthyl)acetoylguanIdiu:e. 5-(NN-Dimethyl)amiloride hydrochloride, 2-napthoylguanidine, $-(4-fluorophenyl)amiloride, 2-Ctt fluetshyflel namoylguanidinle, N-(6-Hydroxy-2-napthoy)-Nh-phenylguanidine, (frans-2-Phenyloyoloproptmccarbonyl)guanidinc,' N,N'-bis(3phenylpropanoyl)-'N"-phenylguanidine, 1-napfthylguanidiic, Benzatnil hydrochloride, 3-methoxy -HM6A, 4-methylinnamoylguanidine, 4-f luorocinnamoylguanidine, 3,4-(methylenedioxy)cinnamoylguanidine, 5-(N,N-bexamethylene)amilorid;, 'N-(lnnamoyl)-Nphenylguanidine, 5-(N-Ethyl-N-isopropyl)ainiloride, 3-methykinnmoylguanidine, 2-zncthylcinnoylguaive, 2,3,S,6,-tetrarnethylcinnamoylguandine, trans-3-Furanacryoylguanidine, (4-Mothexyeinnmoyl)guanidine, (2-Furanacryloyl)guanidine, (3-phenylpropanoyl)guanidin;, 2-(2-napthyI)acetoy~guanidine, Cimnmoylguanidlne, WO 2004/112687 PCT/AU2004/000866 -193 (2-Methoxycinnamoyl)guanidine, [3-(3-Pyridyl)acryloyljguanidine, 4-phenybenzoylguanidine, 2,4-dichlorocinnamolyguanidine, (3-Methoxyoinnamoyl)guanidine, 2-fluorocinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, (a-Methylcinnamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, (Quinoline-2-carbonyl)guanidine, (Phonylacotyl)guanidine, NN'-Bi(amidino)napthalone-2.6-dicarboxamide, 6-bromo-2-napthoylguanidine, 1-bromo-2-napthoyguanidine, 2-chloro-6-fluorocinnamoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, Phenamil methanesulfonate salt, N-Beuzoyl-N'-oinnamoylguanidine and N-(2-napthoyl)-N-phenylguanidine.
    [154] 154. The method according to claim 151, wherein said Coronavirus is human Coronavirus 229E. 5
    [155] 155. The method according to claim 154, wherein said compound is selected from the group consisting of 4-isopropyleinnamoylguanidine, 3,4-dichlorocinnamoylguanidine, 3-(trifluoromethoxy)cinnamoylguanidiine, 4-t-butyleinamoylguanidine, 3-isopropyloinnamoylguanidine hydrocbloride, 3-t-butycinnamoyiguanidine, 2-t-butycinnamoyguanidine, trans-3-(1-napthyl)acryloylguanidine, 5-bromo-2-methoxychmamoylguanidine, 2,3-difhnorocinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, 2-phenylkinnamoylguanidine, 3-phonyloinnamoy]guanidine, 3-(cyclohex-1-enl-yl)cimamoylguanidine, 4-phenylbenzoylguanidine, 3-(trifluoromethyl)cinnamoylguanidine, (4-Phenoxybenzoyl)guanidine, WO 2004/112687 PCTIAU20011000866 -194 4-(trifluorome-thyl)cinmnmoylguanidine, 2-QPylohex-J -en-I yI)roimwnoylgnanidine, (4-Bromoehamoy1)guanidinO, 5-(NN-hexarnethylenD)axilorid;, l-napthoylguanidine, S-(4-fluorophenyl)amiloride, (5-Phenyl-penta-2,4-dienoyl)giaidin;, (3-Bromooinm~oyl)guandine,. 2,5-dimcthylcimiamdoylganidine, 2-(Uiiftuoromethy)rinnamoylpuanidin, 6-mietloxy-2-naphthoylguanidine, (4-Cblorociniamoyl)gaanidine, (3-Mefhoxycinnamoy)guaidie,. 5-bwmio-2-fluoroo-innamoylganidine, S-(N,N-DImethyl)arniloride hydrochloride, Ginnamoylguanidine, (2-Methoxycinnamoyl)guanidine, (a-Mothyc.innmoy1)guanidine. 4-phenylcinnmoylguanidinae, 2,6-diohlorocinmamoylguanidin;, (2-B3romocizmamoyIguanidine, 2,4,&timethykinnamoylguamidine, (trans-2-Phenylcyclopropauecarbonyl)guankline, (3-Mhorooinnmnoyl)guanidine, 2-(l-.napthy)actoyguaidine, 2-ethykinnamoy~guanidine.. 2-cyclohexyInnanoylguanidine, (4-Hydroxyoinmnoylguanidiue, 2-elhoxycinnamoylgaanidine, 3-naethyleinnmoylgnanidine, 2-methylcinm~oylguanIdinv. 3-fluorocbmmoylguanidine, cinmnoylguanidine hydrochoride, 2,3-dimethylehnaoylguanidino, 2-f luorochnnmoylguanidine, 4-fluorocinnamoylguaidine, 3,4-difluorooinnamoylguanidine, 5-tert-butylaniino-amiloride, 2-napffhoylguaniine, NN-Bis(amicdno)napthalene-2,6-dicarboxamide% N,'-]Bis(3-phenylpropanoyl)guanidine, 4-methylcininaroylguanidine, 5-(3-bromophenyl)penita-2,4-dienoylguanidiiie, 2,3,5,6,-itetxanaethylcimnoylguanidine, 3-ethoxycinnamoylguanidine, N,N-bis(3phenylpropanioyl)-N"-phenylguaflidinI; WO 2004/112687 PCT/AU2004/000866 -195 (4-Methoxycinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, (3-Nitroiniamoyl)guanidine, 4-ethoxycinnamoylguanidine, 3,4,5-trimethoxycinnamoylgumlidin, 2-(2-napthyl)acetoyguanidine and N-(3-phenylpropanoyl)-N'-phenylguanidine.
    [156] 156. The method according to claim 151, wherein said Coronavirs is any one of the known Coronavirus isolates listed in Table 1. 5
    [157] 157. The method according to claim 156, wherein said compound is selected from the group consisting of 4-isopropyloinanamoy1guanidine, 3,4.dichlorocinnamoylguanidine, 3 - (ifuo o methoxy)ei nmoylguanidine, 4-t-butyloinnamoylguanidine, 3-isopropycinnamoyguanidine hydrochloride,
    [158] 158. The method according to claim 144, wherein said virus is the Hepatitis C virus. 10
    [159] 159. The method according to claim 158, wherein said membrane ion channel is the Hepatitis C vius p7 membrane ion channeL
    [160] 160. The method according to claim 159, wherein said compound is selected 15 from the group consisting of 2,3-dimethylcinnmamoy1guanidinc, 2,4,6.trimethylcinnamoy1guanidine, 5-bromo-2-fluorocinuamoylgnanidine, (4-BJromoihamoyl)guanidine 2,5-dimthylnnamnoylguanidine, 3-(3ifluoromethyl)innamoylguanidine,. 4-(trifluoromethyl)innamoylguanidino 6.methoxy-2-naphthoylguaniditie, (2-Chlorocinnamoyl)guanidine, (4.Cghlorocinnamoyl)guanidine, (2-Bromooinnamoyl)guanidine, 2,6-dichloroinnamoylguanidin, (3-Bromooinnamoyl)guanidine, WO 2004/112687 PCT/AU20041000866 -196 (3-Chlrocinnamoyl)guanidine, 2-(tif iuaromethyl)oixnmoylguanidine, (4-Pbenoxybenoyl)guaidine, 3,44dihlorocinnanxoylguanidine, 4-isopropylcinnamoylguanidine, trans-3-(1-napthyl)aciyloylguanidino, 4-t-butylcinaxnoylguanidie, 2-t-butyloinnamoylguanidine, 2-ethylcinnamoylguanidine, 4-niethyloinnamoylguanidine, 5-bwomo-2-inethoxycinnamoylgna~dine, 3-(trifluoromethoxy)einnamoylguaidine, 2-cyclohexylcinnamoylguanidine, 1-naptboylgtiandine, 3-t-but34cinnamoylguanidine, 4-phenylbenzoylguanidine, (5-Phenyl-penta-2.4-dienoyl)guanicline. N-(cimiamoy1)-NXphenylguanidine, 3-iaopropylcnm~oylguanidine hydrochioid;, Benzwnfl hydrochloride, N-(3-phenylpropanoyl)-N-phertylguanidine, N,N-bis(3phenylpropanoyl)-N"-phenyguanidiue,, 3-(2-napthYl)acryloylguanidine, 5-(N-Methyl-N-isobutyl)aauloride, 2'4 DicbloroBenzaxnil 1101, 5-tert-butylainino-atniloride, 5-(N-]Ethyl-N-isopropyl)amiloride, (4-Metboxyc-innamoyt)gusnidine, 4-f luorocinm~oylguanidine. (3-Nitrooinnmoyl)guanidine, 4-ethoxycinnamoylgunidine, (4-Hydroxyrinnamnoyl)guanidiu;, (trans-2-Phenylcyelopxoplnecarbanyl)guanidine, 3-othoxycinnamoylguandine, 2,3,5.6,-tetramiethylcinnamoylguandine, 4-phcnylcinazoylguanidine, twas-3-Furanacryoylguanidine, N.-(6-Hydroxy-2-napthoyl)-N-phenylguanidi-ne, (2-Furanacryloyl)guanidine, 3-(cyclohex-1 -en- 1-yl)cinnamoylguanidine, cinnamoylguanidine hydrochloride, 5-(N,1'-hexamethylene)amiloride, 2,3-difluoroinnamoylguanidina, 2-(1-napthyl)acetoylgnanidinc, (a-Methylcinamoyl~guanidine, (2-Nitrocinnanoyl)g uanidine, 6-Jodoaniloride, 3A4-(methylenedioxy)cinnamoylguanidin;, WO 2004/112687 PCT/AU2004/000866 -197 2-cthoxycinnamoylguanidlne, Cinnamaylguanidine, 2-phenylcinnamoylguanidine, 2-(cyolohex-1-en-l1yl)cinnamoylguanidine, 2-napthoylguanidine, 3-phenylcinnamoylguanidine, 5-(N,N-Dimethyl)amiloride hydrochloride, 5-(4-fluorophenyl)amiloride, (3-Methoxycinnamoyl)guanidine, 2-fluorocinnamoylguanimdine, 5-(3'-bromophnyl)penta-2.4-dienoylguanidine, [(4-Chlorophenoxy-acetyl]guanidine, (3-phenylprppanoyl)guanidine, 2-chloro-6-fluorocinnamnoylguanidine, 3-fluorocinnamoylguanidine, 2-methylcinnamoylguanidine, (2-Methoxycinnamoyl)gnanidine, 1-bromo-2-naptboylguanidine, 3,4,5-trimethoxycinnamoylguanidine, 3-methyloinnamoylguanidine, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, Phenamil methanesulfonate salt, 2,4-dichlorocimnamolyguanidine, (4-Nitroominamnoyl)guanidine, 3,4-difluorocinnamcylguanidine and [(E)-3-(4-Dimethylaminophenyl)-2 methylacryloyl]guanidine.
    [161] 161. The method according to any one of claims 144 to 155, wherein said mammal is a primate. 5
    [162] 162. The method according to any one of claims 158 to 160, wherein said mammal is a primate.
    [163] 163. The method according to claim 161 or claim 162, wherein said primate is human. 10
    [164] 164. An antiviral compound selected from the group consisting of N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N'-phenyl-guanidine, N-Benzyl-N'-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)-guanidine, 3'4 DichloroBenzamil, WO 2004/112687 PCT/AU20011000866 2'4 DioblarolRmizaif, 5-(N-methyl-N-guanidinooaxbonyl-methyl)arniloride. 5-(N-Mothy1-N-isobutyil)amiloride, 5-(N-Ethyl-N-isopropyl)aniiloride, 5 5-(,N-hehy)amiloridc hydrochloride, 5-(N{N-hexamethyken)amiloide 4 5-(N,N-Diethyl)anff loride hydrooode, 6-lodoaxiloride, Bodipy-EL amWioride, 10 3-hydroxy-.5-hexamethyleneimino-aniiloride, 5-(4-fluorophenylazilorida, 5-tert-butyano-mmiloride, N-amidino-3-amnino-5-phenyl-6-cloro-2-pyrazinecarboxamide, 3-mcthioxy-5-(NN-Hexamethylene)-aniiloride, is 3-methoxy-=moide, hexanethyleneimino-6-phenyl-2-pyrazinecarboximide, N-amidino-3 ,5-diamino-6-phenyl-2-pyrazinecarboxamide, 1-napthoylguanidine, 2-napthoylguanidine, 20 N-2nphy)N-peygaiie NN-bis(2--napthoy1)Suanidin;, N,N-bie(1-napthoyl)guanidine, N,N'-bis(2-napthoyl)-N"-phienylguanidin;, 6-methoxy-2-naphtboylguanidin;, 25 3-quinolinoylguanidine, cinnamoylguanidine, 4-phenylbenzoylguanidine, N-cfmamoy)-Nhenyguanike, (3-phenylpropanoyl)gnanidino, 30 NW-bis-(cinnamaoyl)-N"-phenylguanidine, N-(3-phertylpropanoyl)-N-phenylguanidine. N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, WO 2004/112687 PCT/AU2004/000866 -199 trans-3-furanacryoylguanidine, N-(6-Hydroxy-2-uapthoyl)-N'-phenylguanidino, (4-Phnoxybenzoyl)guanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, 5 N"-Cinnamoyl-NN'-diphenylguanidine, (Phenylacetyl)guanidine, N,N'-Bis(3-phenylpropanoyl)guanidine, benzyoylguanidine, (4-Chlorophbenoxy-acetyl]guanidine, 10 N-benzoyl-N'-oinnamoylguanidine, [(E)- 3 -(4-Dimethylaminophenyl)-2-methylacryloyl]guanidine, (4-Chlorocitnnamoyl)guanidine, (4-Bromooinnamoyl)guanidine, (4-Methoxycinnamoyl)guanidine, 15 (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bmromocinnamoyl)guanidine, (3-Methoxycinnamoyl)guanidine, (3-Chlorocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, 20 (2-Bromocinnamoyl)guanidine, (2-Methoxycinnmamoyl)guanidine, (trans-2-Phenyloyolopropanecarbonyl)guanidine, [3-(3-Pyridyl)acryloyl]guanidine, (4-Hydroxycinnamoyl)guanidine, 25 (Quinolinc-2-carbonyl)guanidine, or pharmaceuticoally acceptable salts themof.
    [165] 165. A pharmaceutical composition comprising a compound according to claim 164, and optionally one or more pharmaceutical acceptable carriers or 30 derivatives. WO 2004/112687 PCT/AU2004/000866 -200- '
    [166] 166. The pharmaceutical compositionaccording to claim 165, further comprising one or more known antiviral compounds.
    [167] 167. The method according to any one of claims 6 - 8,12,13, 16, 19,21, 23, 25, 5 27, 29, 31, 32 to 34, 37 to 39, 42, 45, 47,49, $1, 53, 55, 57, 58 to 60, 63 to .65, 68, 71, 73, 75, 77, 79, 81, 83 to 87, 89 to 92, 94 to 96, 98, 99, 101, 102, 103 to 105, 107 to 110, 112 to 114, 116, 117, 119, 120-122, 124-127, 129-131, 133-134, 136, 137, 139-147, 149-152, 154, 156, 158, or 159, wherein said compound is selected from the antiviral compounds 10 according to claim 164.
    [168] 168. The method according to anyone of claims 6, 31,58,84,102 or 144, wherein said virus is Dengue virus and said compound is selected from the group consisting of cinnamoylguanidine, ( 2 -chlorocinnamoyl)guanidine or 15 trans -3-(l1-napthyl)acryloylguanidine.
    类似技术:
    公开号 | 公开日 | 专利标题
    US11192863B2|2021-12-07|Antiviral compounds and methods
    CA2222972A1|1996-12-19|Reversible cysteine protease inhibitors
    Ngo et al.2015|Identification and mechanism of action of a novel small-molecule inhibitor of arenavirus multiplication
    JP2008543836A|2008-12-04|Antiviral compositions comprising heterocycle-substituted phenylfurans and related compounds
    Park et al.2008|High throughput screening of a small molecule one-bead-one-compound combinatorial library to identify attenuators of p21 as chemotherapy sensitizers
    Weng et al.2017|Identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors
    Shankar et al.2016|Small-molecule fusion inhibitors bind the pH-sensing stable signal peptide-GP2 subunit interface of the Lassa virus envelope glycoprotein
    Du Pont et al.2019|Status of antiviral therapeutics against rabies virus and related emerging lyssaviruses
    Singethan et al.2010|N-|-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity
    US20070099968A1|2007-05-03|Antiviral compounds and methods
    AU2004248859B2|2011-08-04|Antiviral acylguanidine compounds and methods
    JP2009242247A|2009-10-22|Human immunodeficiency virus infection inhibitor and aids-treating or preventing drug
    Kühl et al.2021|Beyond basicity: Discovery of nonbasic DENV-2 protease inhibitors with potent activity in cell culture
    CN101111475B|2012-09-05|Antiviral compounds and methods
    WO2005066205A2|2005-07-21|Antiviral compositions which inhibit paramyxovirus infection
    KR20200012894A|2020-02-05|Peptides and Uses thereof as Antiviral Agents
    US20120122896A1|2012-05-17|2,1,3-benzoxadiazol derivatives for the inhibition of influenza a and b virus and respiratory syncytial virus replication
    US20180086749A1|2018-03-29|Inhibitors of viruses
    EP3377519B1|2021-06-16|Labyrinthopeptins as anti-viral agents
    US10173968B2|2019-01-08|Bishydrazone-based antifungal agents
    Liu2016|Dissecting the Novel Roles of TRIM56 in Antiviral Innate Immunity
    Hoffmann2020|Discovery and Design of Broad-spectrum Antiviral Peptides
    Vasou2016|Development of a novel cell-based screening platform to identify inhibitors of viral interferon antagonists from clinically important viruses
    Pattabhi2014|Exploring RIG-I like receptor signaling: Regulation by DHX15 and RIGing the pathway for broad-spectrum antiviral immunity
    Forafonov et al.2010|Targeting Disease with Small Molecule Inhibitors of Protein–Protein Interactions
    同族专利:
    公开号 | 公开日
    AU2004248859C1|2011-11-24|
    AU2004248859B2|2011-08-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2017118582A1|2016-01-06|2017-07-13|Atotech Deutschland Gmbh|1-acylguanidine compounds and the use of said compounds in electroless deposition of nickel and nickel alloy coatings|NL299931A|1962-10-30||||
    IN177137B|1992-12-11|1996-11-16|Hoechst India||
    US6011059A|1997-12-24|2000-01-04|Bristol-Myers Squibb Company|Acyl guanidine sodium/proton exchange inhibitors and method|
    法律状态:
    2011-08-04| DA2| Applications for amendment section 104|Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT( S) FILED 20 JUL 2011. |
    2011-11-24| DA3| Amendments made section 104|Free format text: THE NATURE OF THE AMENDMENT IS AS SHOWN IN THE STATEMENT(S) FILED 21 JUL 2011 |
    2011-12-01| FGA| Letters patent sealed or granted (standard patent)|
    优先权:
    申请号 | 申请日 | 专利标题
    AU2003903251||2003-06-26||
    AU2003903251A|AU2003903251A0|2003-06-26|2003-06-26|Antiviral compounds and methods|
    AU2003903850A|AU2003903850A0|2003-07-25|2003-07-25|Anti-coronavirus compounds and methods|
    AU2003903850||2003-07-25||
    AU2003904692A|AU2003904692A0||2003-08-29|Anti-flavivirus compounds and methods|
    AU2003904692||2003-08-29||
    AU2004902902||2004-05-31||
    AU2004902902A|AU2004902902A0||2004-05-31|Anti-flavivirus compounds and methods|
    PCT/AU2004/000866|WO2004112687A2|2003-06-26|2004-06-26|Antiviral acylguanidine compounds and methods|
    AU2004248859A|AU2004248859C1|2003-06-26|2004-06-26|Antiviral acylguanidine compounds and methods|AU2004248859A| AU2004248859C1|2003-06-26|2004-06-26|Antiviral acylguanidine compounds and methods|
    [返回顶部]