ZA200600650B - Antiviral compounds and methods - Google Patents

Description

-1- Co

ANTIVIRAL COMPOUNDS AND METHODS

FIELD OF INVENTION

The present invention relates to methods for retarding, reducing or otherwise inhibiting viral growth and/or functional activity. The invention also relates to : compounds and compositions suiteble for use in the methoda.

BACKGROUND OF THE INVENTION

Currently, there is groat need for the development of new treatments that are effictive against viral infections, perticularly against viral infections which are © 10. associxted with high mocbidily and mortality, and which ionpact on sizable populations. Treatments currently available ere inadequate or ineéfbotive in large proportions of infected patients. = For example, im ameliarating AIDS symptoans and prolonging life expectency, transcriptase mud protoase eazyoves (Miller and Server, 1997; Mitsuya, 1992; Moors, 1997; and Thomas and Brady, 1997). However, 10 single treatment method is completely effective against HIV infection. (Barry et al, 1998; Decks, 1998; Miles, 1997; Miles, 1998; Moyle ct al, 1998; Rachlis and Zarowny, 1998; Veil etal, 1997;

Volberding and Decks, 1998; and Volbendin, 1998).

PCT application PCT/AU9S/00872 desczibes the use of compounds S-(N.N- hexamethyiene)-amiloride and S-(N;N-dimethyl)-amiloride in the trestrment of HIV

Another viras considered to be a significant human pathogen is the Hepatitis C virus (HCV). This is « significant human pathogen in tarms of both cost to human health and sssocistod econamic costa. HCV cansos chronic hepatitis and cirrhosis and is the leading indicator for liver replacement surgery. Tn 2002 the Centro for

Disease Control and Prevention estimated that mote than 4 million people were infooted in the USA alanc and that spproximately 8,000 to 10, 000 dis as a result of chronic HCV infection yearly. ‘There is no known cure or vaccine. More effective pharmacological agents are urgently required.

oo 2 .

A fiirther well-known family of pathogenic viruses aro the Coronavirsss. :

Co Corooaviruses (Order Nidovirales, family Coronaviridas, Geos Coronavirus) sre soveloped positive-stranded RNA virusca that bud from the endoplasmic retiealurn-

Lo Golgi intermediate compartment or the cts-Golgi network (Fischer, Stegen ct al. 1998; Moda, Maeda et al. 1999; Carse and Machsmer 2000; Masds, Repass of el. o 2001; Kno and Masters 2003)

Coronaviruses infoot bumans and animals and it is thought that there could be a coronavirus that infects every animal. The two human coronaviruses, 229E and : C43, are known to be the major causes of the common cold and can occasionally cause prcumonia in older adults, neonitcs, or immunocompromised patients (Pers,

Lai ct al. 2003). Animal coronaviruses od cause respiratory, gastrointestinal, neurological, or hepatic diseases in their host (Peiris, Lai et al. 2003). Severs] animal _ cononavirns are significant veterinary pathogens (Rota, Obersts ct al. 2003).

Severs acute respiratory syndrome (SARS) is caused by a newly idantified 1S vires. SARS is a respiratory illness that has reocutly been roported in Aste, North

America, and Europe (Peitis, Lai ot al. 2003). The causative agent of SARS was sdentificd as a coronavirus. (Droste, Gunther ot al. 2003; Keiszck, Erdmen ot ol. "2003; Peiris, Lad et al, 2003). The World Health Organization roports that the ounmlative number of reported probable cases of SARS from | November 2002 to the 11% July 2003 is 8,437 with 813 deaths, nearly a 10% dosth rate. Jt is belioved that SARS will not bo eeadicated, but will oanso seasonal epidemics Hike the cold ar infinenza viroses (Vogel 2003).

To improve the prospect of frosting and preventing viral infsctions, thecs is an : on-going need to identify molecules capablo of inhibiting various aspects of the viral life cycle.

Tt is an object of the present invention to overcome or ameliorate st least ano of © the disadvantages of the prior art, ar to provide a usefhl alternative. :

Any discussion of the prior art throughout the specification should in po waybe considered a8 an admission that such prior art is widely known or forms part of common general knowledge in the fiald. :

o

SUMMARY OF THE INVENTION :

Ths inventors have surprisingly found that certain compounds that fall under from a range of different viras Smilies, Without intending to be bound by any puricula theory or mechani of scion, and deep caren dog, appers possible that viral replication oan be retarded by inhibiting ar otherwise down- regulating the scivity of ion cbeonsla expressed in the host oe. Thus, the negative © impact of the compounds of the present invention on viral replication maybe . mediated by the inhibition or otherwise down-regulation of a membrane jon chammel relied upon by the viras for replication. This meenbrans ion chammel may be a viral membrane jon channel (exogenous to the hoat cell) or a host cell ion channel induced aq a result of viral infection (endogenous to the host cell).

As mn 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.

The SARS virus encodes an E protein which is shawn for the first time, by the oe present inventors, to act ss am ion chamel, As similar B peotcing are present in other would have utility in the inhibition and/or treatment of infections by other

Tho prosent tnveation is concerned with novel antiviral compounds that fall under the Classification of subatitited acylgoamidines. It does not include in its soope : amiloride for retarding, reducing or otherwise inhibiting viral growth and/or fonctional activity of HIV. :

Accordingly, a first aspect of the present invention provides an acylguanidine with

Apcording to a second aspect, the pressut invention provides an antiviral compound of Formula I Co . 1 J aaa

Rd 1 wherein R-Ry are independently aromatic groups, heteroaromatic groups, alkenythetoroaromatic groups, cycloalkylaromatic groupe, oycloalkylhsteroaromstic groups, aryloxyalkyl groups, heteroacyloxyalkyl groups, ssid groups are mono or polycyclic, snd ro optionally substituted : with one ar more substitutents independeotly selected from hydrogen, - hydroxy, nitro, halo, amino, substituted amino, alkyk-substituted amino, cycloaTkyl-substituted amino, aryl-subetituted amino, C1.alkyl, Ci. aalkyloxy, Cs.eoycloalkyl, balo-substituted Cy.salkyl, halo-substituted C)- aalkyloxy, phenyl, C1.ealkonoyl, Cs.soycloalkeneyl, Cr.ealkensaxy, beezo, at NE, O aryl, substituted aryl, PrS, » OF

According to a third aspect, the present invention provides an antiviral 3 compound of Formula I :

J SO

R N no

Re I : or pharmaceutically acceptable salts thereof, Co . wherein, )

R= } Ny N ASC \

Bi ) ne ©A :

N Re N E] ’ k Os \ \

COT

Lo oo

IXY : 2 | SE

OY GE

LI "A "a

Re . Ro ? $ [3 n 0

Co ~ Ry R's . } ’ . GY . oe | 2 . ’ [] g » N ’ ’ . N Ye . : } 3 Za \= ; : Ry, Ry and R, are independently hydrogen,

Ry 0

ONO

S. . - 0 0} .

NY 0) ~

Ary a & : X= hydrogen, hydroxy, nitro, halo, Cy.ealkyl, Crealkyloxy,

Cs scyoloalkyl, halo-substitated Ci.salkcyl, halo-sabatitated C,. oo salkyloxy, phenyl, C1 alkeneyl, Cs.goycloalkeneyl, Cs ealleaneoxy, of ’ s Re, Ry, Res Ra, Re, Re. Rs Res Ris Res, Re, Ro, Ry independently = _ hydrogen, amino, halo, Cr.salkyl, Ci salkyloxy, hydroxy, aryl, substitated aryl, substituted amino, mono or dialkyl-substitated

LS 14 ) : | ’

HE

NE: 0 gpm: oo

Rg, Ry independently = hydrogen, hydroxy, halo, or C1 alkyl

Ry = hydrogen, amino, halo, Cy salkyl, Cy salkyloxy, hydroxy, aryl,

RES Wie 4 + oo

Preferably, the compounds of the invention include the following: :

N A | :

SG i: :

. : .7- 3 5.(N,N-DimethyDemilorids hydrochloride comprising the structure .

N A :

OC

: Bon NZ NH; - . 5: (N-methyl: N-isobuityDamiloride comprising the structure Co 0 NB

PSG BE

A

Nw NZ NE, © 5{N-ethyl-N-isopropyDamiloride (hercin referred to a# EIPA), comprising the giructure . ’ :

Coo

A, NZ “NH;

P | oo

-3- J .

N-(3.5-Diamino-6-<horo- pyrazine 2-carbonyl)-N'-pheny'-guamidine, compeising the structure . . le) NH .

DN ~N

HN” "N° "NH; N-Boozyl -N'{3,5-diamino-6-chioro-pyezine-2-carbomyl)-gasxidine, comprising the . 5 structure : 0 Nh ~~ N A a

CCQ -

BENT NT NE oo 0 Nm re

BN” ON" oc, 3-methoxy-5-(N,N-Hexamethylenc)-amiloride comprising the structure 0 NE oo ox : e oO | :

_ = EE 3 hydroxy 5 haxamethylm cimino-amiloride comprising the structure "0 NH oe @ N° on | ; 0 NE

Opa oo ig: @ oo

Co N-ammidino-3,5~Gismino-6- phenyl 2-pyrezinsoarboxamido comprising the structure

Q NH yrhebs

HN ig NH, 1

: 0 NB

ES © GE ao" ’ N iat 3 . 5p 3-6-chl 2 . v . ide » = the lo) NH; . . N .

N A :

Posed

OM

: 3'4 DichloroBenzamil comprising the structure 0 NH - peGRAeE oo

B Cm Nm, cl ]

a. 11 : 2'4 DichloroBenzamil HCl comprising the stmeture 0 NE cl oo POGLAS! - .

Nm” SNP ONE, cl Co $ (% mefiyh N-guanidinocarbonyb methyDemiloride comprising the structure } BCay py NE

NE; O | Co 5.(NN-DiethyDemilocide hydrochloride comprising the structure

RE : Toa SNE : ao 5-ON,N-DimethyDemiloride hydrochloride comprising the structure

Pete

HC. N N? NH, :

5-text butylamino-smiloride comprising the structure 0 NH oo : No A

No GT

N N? NH; Co 6-Iodoamiloride comprising the structure i PO Gls | a s ENTONT ONE oo

Bodipy-FL Amiloride comgrising the structure 0 NE

Ch __N PY _BODFVFL So

Di S N N oo

EN" NT “NE, (4 fhuorophenylaesloride comprising the structure 0 NH

OC oo NON TNR oo Co -13- SE - 1-nepthoylguanidine comprising the structure

A | . : k 2 nspthoyiguenidine comprising the sructare 0 NH 6 oat

N-(2-napthoyl)-N'-phenylgusnidine comprising the stroctare eS og © NN-bis(2-napthoyguanidine comprising the structure eos nee

Sa N B | .

Nin -aaptioyDganidine comping the sts | oo oui

UY ry

N,N-bis(2-papthoyl)-N"-pherylgnenidine comprising the structure

LD jeops¥ 6 methoxy-2-nsphthoyignenidine comprising the structure

Jeon yo

No | : : © N-CtonsmoyhN',N'-dimethylguamidine comprising the structure

IX x Hy Cle oo . Cts ’

-15- oo s-cuinclinoyiguanidt iing the struc 0 NE | » | oo oy p : .

N

0 NE | Co oo YN om s Co 0 NH om .

H oo ' Nc yI)-N'pt 1 idk [3 tho st t & yo

N 16 (3-phenylpropanoyl)guanidine comprising the structure . SE oe

N.N"bis{cinnamoyl)- N'-phenylgusnidins comprising the struoture

SAAS IONE

EY

N-{>phenylwopanoyl) N-pherylgnenidine comprising te structure

PUSS SCN

H H :

N,N'-bis(3phenylpropenoyl)-N"-phenylgnanidine comprising the structure :

H H trans 3-fursnacryoylgnaniding comprising the structure

SE NH; :

N-(6-Hydroxy-2-napthoyl)- N-phenylgosnidine comprising the structure oor ® 0 (4-Phanoxybenzoyl) guanidine comprising tho structuro 0 NH ao :

NN-Bis(amidinojnapthaleno-2,6-dicarboxamide comprising the structure ‘ o NH;

Seon oo

HN. _N : ad

NH; ©

. } : | 18 : ’ ’ 6-1 2 ’ n a idin ining the sl . oo 0 ji :

Cry ha 1-bromo-2-papthoylgnanidine comprising the structure - BR I . 2-(2-napthyl)soetoylgnanidine comprising the stractmre

SONNE

8 - U :

N°Cirmamoy}-N;N-diphenylguanidine comprising the structure

Oo o> ZN (Phenylacetyl)guanidine comprising the structure Qur, oo : Cs oo N,N'-Bis(3-pheylpropanoyl)gnanidine comprising the structure : : Oo NH O .

Benzoylgasnidins comprising the structs © (4-Chlorophenoxy-scctyllgaanidine comprising the structure 0 NH

LT Ay oo 0 NE ©

Or [()3-(4- Dimethlaminophenyl)-2-mefhylaceyloyl)gasnidine comprising the structure

Ps

(4-Chloeocinnamoyguanidine comprising the structure :

QO NB ~

EY N7 NH;

Ci . (4-Bromocimamoyl)guanidine comprising the structure 0 NH SE

Jens a . 5 (4-Methoxyvirnamoyl)guanidine comprising the structure 0 Ni .

Ore oo } H :

No (5-Phenyl-pents-2,4-denoyl)guanidine comprising the stracture 0 NH ~ (3-Bromocinnamoyl)gnanidine comprising the structure 15 .(3-Methoxycinnamoyl)guanidine comprising the structure Co ym

. 21- i . :

SUSE

Cl = . (2-Chlorocinnamoyl) guanidine comprising the structure = : a (oi J

PST

- a (2-Methoxycimamoy!)guanidine comprising the structure

SN

SOR NE

0” : (tvans-2-Pheylcyclopeopancoarbonyl)guanidine comjpuising the structure oo 0 Ji

Neca tl yt NOME

. . | . : : . 22- } | . . oo (4 Hydroxyoimnemoyl)guanidine comprising the stricture : - o Pi ; or yo oo

HO : \ . ( . . 2 idé « the stroctore : 0 NH | :

N PO

> N NH, : . (] ; } : (4-Nitrocimmamoyl)guanidine comprising the strecture oY om . H . ’

ON

(3-Nitrocinnamoyl)guanidine comprising the structure adhd

EN

NO; (2-Nitrocimemoyt)guanidine comprising the structure a o I C

NO; is

: . . (o-Mothyloinnamoyl)granidine comprising the structure : : 0 NH oar oo

H * . ca o xT i

OF

: oo ONY oo $ H ’ . : | 3-(triffuoromethyl)cinnamoylguanidine comprising the structure

H .

4-(triftuoromethyl)immamoylguanidine comprising th structure

SOON TR I

. H : ' kK} 2-metbylcinnanoylguanidine comprising the structure

Ny N NH; . * . u 2triffaoromethylcinnamoylguanidine comprising the structose

SP

N N NH oo . H . : . Co. 4 methylcimamoylgnanidine comprising the structure

O NH

H

. 10 Hy } . ’ . . : NH i Hs : H . | :

oo 25- 3-fncrocinnamoylguanidine comprising the structure :

X

NON

H -

PB ’ . 2 fiuomotonamoylguamidine comprising the structure

LI

Ecos ’ 8 F . : : :

LI

XN oye

H

3,4-dichlorocinnamoylgnanifine comprising the structure oo oo 0 I : }

H Co

Q . . } | . . . y | 2,4-dichlorocinnamolyguanidins comprising the stractars x | oo sete oo

Cl a : .

2,6-dichlorocimamoylgnanidine ocmprising the structure

Co 0 NH cath oo

I a. cl oo 4-cthoxycinnamoylgasnidine comprising tho structure -

Co H s BsCHCO Co 3 A-{mstylencdioxy)cinasmoyl guanidine comprising the structure

H - : oo L.° } : a oo : 3-(2-nepthylacryloylguanidine comprising the stricture x

Econ

NH

SRS SH

HC

3 4,5-trimethoxycimamoylguanidine comprising the structure

H

. H

HyCO

OCH; 2-(1-napthyl)acetoylguanidine comprising the structure

EE : SE . 5 2.5 dimathylcs ° 1 idk PRS the . ,

NH

+ Hy Nv i. Co :

H

H

23 diftn orocin an | idi oe the t = NH, soso lI 3 3-phenylcinmamoylguanidine comprising the structure

H g : .

a. 28 © 3{trane-hept-1-en-1-yl)cinnamwoylguanidine ocmptising the structure rr.

NT ONT ONE

BH . . . 2-cfhylcimumoylguanidine comprising the structure

LAE

S satis

H : s CH, CH : . idi .. the st ture

F 0 NH . coy

H loi} : 10 3.4. butykcinnsmoylgnamidine comprising the structure x i.

H :

BCH

CH,

3 4-diffuorocinpamoylgnanidine comprising the stroture : A y NE, i H oo : 5 bromo-2-fiucrochmamoylguanidine comprising the structure

POSS

Br

COTY NE,

H

5s F 3-(trifinoromethoxy)cirmamoylguanidine comprising the structure

ESN SJ -

H . \ 2-ethoxyimmamoylgnanidine comprising the structure ©

LL

1x

OCH CH, 2-t-butykinnamoylguanidine comprising the structure - me. [bm NH :

AN i — i H :

. ’ -30- : | ’ . 0 NE

Sh I. : H : cirmamoylguanidine hydrochloride comprising the structure - i :

Lr N

SB BI - s oo 2,3,5,6,-tetrsmethylchmamoylgnanidine comprising the structure (Bit 134)

CH 0 XI,

Or

H } id . a the st t . :

O NH

LAS

- H : 5-bromo-2-methoxycinnsmoylgnanidine comprising the structure

Br cy

H

. 2.3 hvicix 1 idi » the t .

Co | NOY Me i}

CEs .

CB

3 efhoxycinamoyl guanidine comprising the structure

Co 0 bid :

H

= om : COL? - : 2-(cyolohex-1-en-1ylcinnamoylguanidine comprising the structure TL. 0 Nd : seth

H

: . Co 32- © 2,4,6-trimethylcinnamoylguainidine comprising the strocture

Joes N° Ng Co (5-Phanyl-pente-2,4-dimoyl) guanidine comprising the structure 0 : shes ol. s .

SAS .

Br | . 5-(2-bromophenyl)penta-2,4-dienoylgnanidine comprising the structure

CO w“ {IC 8) ™N : as oN oo 33- :

Preferably, fhe compounds of the invention are capsble of reducing, retarding or otherwise inhibiting viral growth and/ar replication. _ Prefscably, the antiviral activity of fhe compounds of the invention is against viruses such aa those belonging to the Lentivirus family, and the Coronoviros family

Aunily of virases. For cxsmple, the compounds of the invention exhibit sutiviral NB oo activity againat viruses such as Human Fnmmnodeficiency Virus (HIV), Severe Acute

Respiratory Syndrome virus (SARS), Mouse Hepatitis virus (, and Hepatitis C virus oo HCV). - oo . According to a fisurth aspect of the present invention, there is provideda pharmaceutical composition comprising an antiviral compound according to any ons "of the first, second or third aspects, and optionally one or more pharmecentical acceptable carriers or derivatives, wherein ssid compound is capable of reducing,

Soon retarding or otherwise inhibiting viral growth and/or replication.

Preforably, the antiviral activity of fhe compounds of the invention is against viruses such ss those belonging to the Lentivirus family, and the Coronovirus family - of viruses. For example, the compounds of the invention exhibit antiviral activity ~~ againat viroses such as Human smmmmodeficiency Virus (HIV), Severe Acute

Respiratory Syndrome vires (SARS), Human Coronavirus 2298, Bnman Coronavirus

C43, Mouse Hopatitis virus (MEV), Bovine Coronavirus (BCV), Porno

Respirstory Coronavirus (PRCV), Hepatitis C viros (EICV) and Equine Arteritis

Viras (BAV). :

Other Coronsyiruscs which can be inhibited ar 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 mate known © 25 sntiviral compounds or molecules. "

According to a fifth aspect, there is provided a method for reducing, retarding or otherwise inhibiting growth and/or replication of 8 virus compeiaing contacting cell infected with said virus or exposed to said virus with a compound socording to any one of the first, second or third aspects.

Preferably, tho virus is from the Lentivirus family, of tbe Coronaviras family.

Co Mors preferably, the virus is Fioman Immunodeficiency Virus (HIV), Severe : Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus

34 i. .

Respiratory Coronavirus (PRCV), Mouse Hepatitis virus (MEV), Hepatitis C virus (LCV), ar Equine Asterits Virus (BAV). Most preferably, the virus is HIV-1, HIV 2, the SARS virus, Caropaviruss 2298, Coronaviras OC43, PRCV, BCV, HCV, or . 5 BAV. .

So Other Coroaaviruses which oun be inhibited or thei infections troated by the : compounds of the invention are those listed in Table 1.

According a sixth aspect, tharo is provided a method for preventing the "infection ofa cell exposed to a viras comprising contacting said cell with a compound sccording to any one of the first, second or third aspects.

Preferably, the virus is from the Lentivirus family, or tbe Cosonavirus fkemily.

Mare preforsbiy, the iris is Bumn Inmnodeficiency Vim (IV), Severe

Co Respiratory Syndroens viros (SARS), Human Coroaviras 229%, Homan Coronsviras 18 Respiratory Coronaviras (PRCV), Mouse Hepatitis virus (MEV), Hepatitis C virus - (HCV), or Equine Atteritis Virus (EAV). Most profirebly, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruss 229E, Coronaviras OC43, PRCV, BCV, ECV, BAV.

Other Coronaviruses which can be inhibited or their infections treated by the compounds of tho inveation are those fisted in Table 1.

According to 8 soventh aspect of the inveation, thers is provided a metbod for the therspentic or prophylactic treatment of 8 subject inficted with or exposed to : virus, comprising the adnrinistration of 8 compound according to any ane of the first, second or third aspects, to a subject in need of said treatment. ; Profecably, infoction with & virs or exposure to a virus occurs with viruses = 25 belonging to the Lentivirus emily, or the Coranovirus family. More preferably, infection or axposure cocurs with HIV, SARS, Human Corouavirus 2298, Human Co nL Coronavirus OCA3, Mouse Hepatitis virus (MEV), Bovine Coronavirus (BCV), \ Porcine Respiratory Coronavirus (PRCV), Hepatitis C viras (BCV), or Equine

Arteritis Virus (RAV). Most preferably, infection or exposure occurs with HIV-1,

HIV-2, SARS, Human Caronavirus 2298, Human Coronavirus OCA3, Hepatitis C viros (HCV), ar Equine Arteritis Virus (BAV).

oo 35

Other Coronaviruses which can be inhibited or their infections trestod by the compounds of the invention are those listed in Table 1. . © Tho subject of the virel inhibition is genorally mammal such as bt not imzited to human, primate, livestock animal (e.g. sheep, cow, borss, donkey, pig), y : 5 companion animal (6g. dog, cat), laboratory test unimal (¢.g. mouse, rabbit, re, guirios pig, hamater), captive wild anirial (6.3. fox, door). Preferably, the subject is a "priate, or horse. Most prefirably, the subject is 8 human. oo

According to & eighth aspect, there is provided a method of down regulating 8 . membrane jon chanal fimotiona! sctivity in 8 cell infocted with a virus, compeiking : 10 contacting sxid cell with a compound according to any aoe of the first, second or third . aspects,

The membrane ion channel may be endoganons to the cell or exogenous to the " Proforably, the membeans jon channel of which functional activity is down regulated is that which Lentiviruses, md Corcmaviruses utilise for mediating vinal- replication and incinde, fur exxmpla, the HIV membrane ion channel Vpn, the HCV membescie ion channel P7, the Coronavirus B protein membeanc ion channal, snd the

SARS E protein membrane jon channel.

Preferably, infection with a virus or exposure to 8 vires occurs with viruses belonging to the Lentivirus family, or the Corenovirus family. More preferably, Co

N infection or exposure occurs with HIV, SARS, Haman Coronavirus 2298, Human

Porcine Respiratory Coronavirus (PRCV), Hepatitia C virus (HCV), or Bquine :

Anteritis Virus (RAV). Most preferably, infection or exposure occurs with HIV-1,

HIV-2, SARS, Humen Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Artertis Vira (BAV).

According to an ninth aspect of the present invention, there is provided a method

Co of rédtucing, retarding or otherwise inhibiting growth and/or replication of a viras that " "has infected a cell, said method comprising contacting said infected cell with 8 compound according to amy ane of the first, second or third aspects, wherein said compound down regulates fictional activity of 8 membrane jon channel derived from seid virus and axprossed in said infected cell

=

Preferably, infootion obours with @ virus belonging to the Lentivirus fully, oc the Caromovirus fimily, Mare prefscably, infection or exposure ocours with FIV, © SARS, Human Coronavirus 229E, Human Coramavirus OCA3, Mouse Hepatitia virus (MEV), Bovine Coscnavirus (CV), Borvine Respiratory Corcuavirus (FRCV),

Hepatitis C virus (HCV), of Equine Arteritis Virus (AV). Most prefiably, infoction x axposre occurs with HIV-1, HIV-2, SARS, Human Coronavirus 220E, Hunan

Coronavirus OCA3, Hepatitis C viras (HCV), or Bquine Ateritis Virus (BAV).

Other Coroasvirases which oan be inhibited or their infections treated by the : compounds of the invention sre those listed in Table 1.

Preferably, the membrane ion chamal of which fictional activity is down. nN rogalsted ia that which Leativiruscs, md Coronavirusos utilise for medisting viral replication and include, for example, the HIV membrane ion channel Va , the HCV menmbexne ion channel P7, and te Coronaviras E protein membrans ion chamal

According to an tenth aspect, the present invention provides a method of 1S reducing, retarding or otherwise inhibiting growth and/or repticstion of a virus that has infected 2 ocll in mammal, ssid method comprising administering to said maromel a compoand acconding to any one of the first, second or third spects, or & pharmaceatical composition sooording to Ge fourth speci, wherein sxid compound or ssid composition down regulates functional sotivity of a membrana jon channel expresacd in said infected cell Prefiaably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirns family. Mare preferably, infection or cxposars occurs with HIV,

SARS, Human Coronavirus 2298, Human Coronavirus OCA3, Mouse Hopatitis virus - (MEV), Bovine Coronavirus (BCV), Parcine Respiratory Coronaviros (PRCV),

Hepatitis C virus (HCV), or Equine Arteritis Virus (BAV). Most prafecsbly, infection oc exposure occurs with HIV-1, HIV-2, SARS, Humsn Coronevirus 229K, Human : _ Coronaviras OC43, Hepatitia C virus (HCV), or Bquino Arteritis Virus (BAV).

Other Coronsviruses which cm be inhibited or thelr infections trested by the : compounds of the invention are thoes listed in Table 1. :

Prefecshly, the membrane ian channel of which fictional activity is down regulated is that which Lantiviruses, and Coronaviruses utilise for mediating viral

37- oC replication snd incinds, for example, the FIV meehrane ion channel Vp , the HCV membrane jon channel P7, and the Coronavirus E protein membrane jon channel.

The subject of the viral inhibition is generally 8 mammal such as but not limited to human, primate, livestock anima (e.g. sheep, cow, horse, donkey, pis), companian s animal (e.g. dog, oat), laboratory test anime] (c.g. monse, rabbit, Tat, guinos pig, : haste), captive wild animal (e.g. fox, does), Profecably, the subject is a peizats, or horse. Most preferably, the subject is a human. oo According o a eleventh aspect, the present invention provides method for the therapeatic or prophylactic treatment of a subject infested with or exposed 10.8 virus. _ comprising administering to seid subject a compound according to any cnc of the first, socond or third aspects, or a pharmaceutical composition according to the fourth : spect, wherein exid compound ar sald composition down-regulates fncticnal _ activity of a membrans ion channel derived from ssid virus. : Preforsbly, infection oocurs with a virus belonging to the Lentivirus family, or © 15 the Coronovirus family of viruses. More praficsbly, infootion or exposure oocurs with

HIV, SARS, Human Coronavirus 2298, Human Corenaviras OC43, Mouso Hepetitis

Hepatitis C virus (HCV), or Equine Arteritis Virus (BAV). Most prafucsbly, infection or exposure ocoms with HIV-1, HIV-2, SARS, Human Coronsviras 2298, Human

Coronavirus OC43, Hopatitia C virus (HCV), or Bauine Artes Virus (BAV). : Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. " Prefecsbly, the membrans ion channel of which functional activity is down . regulated is that which Lantivirases, and Coronavirus utiliss for mediating viral replication end inclade, for example, the HIV membrane ion charmel Vpu , the HCV

R membeane jon chamel P7, and the Coronavirus E protein membrane ion channel,

The subject of the viral inhibition is generally a mammal such as but not Hmited ~ to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion anima! (e.g. dog, oat), lsboratory test animal (e.g. mouse, rabbit, rat, guinee pig, "30 hamster), captive wild animal (6.3. fox, deer). Prederably, the subject is a primate, or horse. Most preferably, the subject is a human. Co a8

According bo & twelfth sspect, tho invention provides an sntiviral compotmd selected fram the group consisting of!

N-(3,5-Diamino-6-chloro-pyrazine-2-catbonyl)-N -phenyl- guanidine, © N-Benzyl-N'(3,5-diamino-6-chiloro-pyrazino-2-oarbonyl)-guanidine, 34 DichloroBenzamil, 2'4 DichloroBenzamil, 5.(N-Methy}-N-isobutylyemiloride, 5-(N-Bthy}-N-isopropyl)amiloride, 5 : 10 © 5-(NN-Dimethyf)emiloride hydrochlorid, : : §-(N.N-hexsmethylene)amiloride, 5.(N,N-DiethyDamiloride hydrochloride, 6-Iodoamilorids,

Bodipy-F1. amiloride, : is 3-hydroxy-S-hexametirylencimino-smiloeide, S-{d-fuorophenylsmilozids, : oo | 5-tert-butylamino-amlaride, 3-methoxy-5-(N,N-Hexametirylenc)-smilocde, : = 20 3-methoxy-amiloride, : : N-smidino-3,5-diamino-6-pheayl-2-pyrazinecarboxamide, . 2-naphthoylgnanidine, 3 N-(2-naphtoyl)-N-pheaylguanidine,

NN-bia(2-naphthoyl)guanidins, . . N,N-bis{1-nephthoyl)goanidine, ;

N,N-bis(2-nsphthoyl)-N"-phenylguamidine, ° 3-quinolinoylgnanidine, 4-phenylbenzoylguanidine,

l N-{cimamoyl) Nphenyiguanidine : (3-phenyipropanoyl)gnanidine, © NN-bis-(cinnamoyl)-N"-phanylguatidine,

NG phanyipropamoyi-N'phenylgoanidine,

N;N“bis(3phenyipropanoy!)- N"-pheuyiguanidive, trans-3-furanacryoylgnanidine,

Lo N-(6-Hydroxy-2-naghthoyl)-N-phenyl guanidine, " (-Phenoxybenzoyb)gamnidine, - N,N-Bis(amidino)napthalene-2,6-dicarboxamide,

C10 N"-Cinmamoyl-N,N'<tiphenylgnanidine, :

N-benzoy-N'-ciunamoyl guanidine, Lo oo [(8)-3(4-Dimethrylaminophanyl)-2-methylacryloyi]gnanidine, (4-ChilorocinnamoyDgusnidine, . ( . : (4-Methoxycimamoyl)guatidine, a 2 (5-Phenyk-penta-2,4-dienoyl)guanidine, i ~ (2-Bromocinnamoy!) guanidine, : (2-Methoxycintamoyf)guanidine, © (trans-2-PhenyloyolopropanscasbonyDgumicins, (4-Hydroxyoimamoyl)ganidine, 30 . (Quinoline-2-carbomyl)guanidine,. .- of pharmaceutioally acceptable sats thereof.

According to hirtoenth aspect, the prosent invention provides a pharmaceutical composition comprising a compound according to the twelfth espect, and optionally oo ams or more pharmaoeutios] acceptable carriers or derivatives. ‘Preferably, fhe phamaceutioal composition may further comprise ons Of 1001S mown antiviral compounds or molecules. : loan the contre clearly requires otherwise, throughout tho description and : the claima, the words ‘comprise’, ‘comprising’, and the like are to be construed in an } inclusive sanso 8s opposed 10 an exclusive or exhanstive senses that is to say, in the sense of “including, but not limited to”

BRIEF DESCRIPTION OF THE DRAWINGS

Figare 1 is & schematic representation of plasmids used for expression of Vpu in

E. coli. A. The amino acid sequence < 400 > 1) encoded by the vps open reading * frame (ORF) goncratod by PCR from mn HIV- 1 strain) HXB2 DNA clone. Tho vpw

ORF was cloned in-frams at the 3' and of the GST gene in p2GEX to generate p2GEXVpu(B). Tt was subsequently cloned into pPLAS1 to produce tho plasmid FL . + Vp (CQ). 3

Figure 3 is a photographic represcntxtion of the expreasion and purification of Vu in

E coll. A. Western blotting after SDS-PAGE was used fo dstect expressed Vpu in E. coll axtracts. Lames 1-4 contain samples, st various stages of purity, of Vpu exprosscd from p2GEXVpu: lame 1, GST-Vpu fusion protein isolated by giutathions-agacosc : affinity chromatography: Iane 2, Vir liberated from the fusicn protein by treatment with Srombin; lane 3, Vpu purified by HPLC anion exchange cliromatography; lane 4, Vpu after passage throngh the immunoaffinity colurm. Lanes S and 6, membrane vesiolos prepared from 42'C induced cells containing pPL+Vpu or pPLAS1, respectively. B. Silver stained SDS-PAGE gel: lane 1, Vpu purified by HPLC anion exchange chromatography; lane 2, Vpu after passage through the immumoaffinity :

Figure 3 is a graphical representation of fon channa! activity observed aftar exposure of lipid bilyers to aliquots containing purified Vu. In A and B, the CIS chamber contained S00mM NeCl and the TRANS chamber contained SOmM NaCl; both

Co 4 oo "solutions were buffered at pH 6.0 with 10 mM MES. B shows a current varsus voltage curve generated from dats similar to that shown in A.

Fignre 4 is a photographic representation of bacterial cross-feeding assays. Far all lates, fhe Msf, Pro” sumofropkic srain was neod to sced a soft agar oveciay. Flstes A s snd B contain minimal drop-out medium minus proline; in plate C tho medium wes mine methionine. To control for viability of the calls in the background lawn, the oo isos Iabelisd P and M cantained added profino or methionine, respectively. The discs jabelled C and V were inoculated with Met”, Pro” B. coli cells containing the "plasmids pPLASI or pPL#+Vp, reepectivaly. Plates were incubsted a 37°C (A and C) or 30°C (B) for two days and photographed above s black background with periphersl illumination from a fluoresomt light located below the plate. The images were recorded on a Novaline video gal documentation system. Light halos around the discs

Isbelled P or M an all plates and around the disc labelled V on plate A indicate © gowthofthcbackground lawn strain. oo

Figure § is a graphical representation of the screening of drugs for potential Vpu channe! blocks. The photograph shows a section of a minimal medium-lecking ~~ - adenine - agarose plats onto which a lswn of XL-I-blue E. coll cells containing the

Vpn expression plasmid pPLVpu has been seeded. Numbers 6-11 gre located st tho sites of application of various drugs being toated, which were applied in 31 drops snd allowed to soak into the agarose. The piste was then incubated at 37°C for 48br prior to being photographed. The background grey shade corresponds to areas of zo bacterial growth. The bright circular area around " 10 " represcuts bacterial cell growth as a result of application of adaing at that location (positive control). The smaller halo of bscterial growth around "9" is due to the application of S-(N.N- © 25 hexsmethylens)-amiloride at that location.

Figare 6. SARS B protein jon charmel activity observed in NaCl solutions after : exposure of lipid bilayer to 3-10 of E peotein. A. The closed stato is shown as solid line, openings are derivations from the Ene. Scale ber is 300ms end 5pA. The CIS chamber contained 50mM NeCl in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in SmM HEPES buffer pH 7.2. The CIS chamber was -

: earthed and the TRANS chamber wes held at various potentials between —100 to

Co © 4+100mV. B. Largest single opening cvents of a single chanel. ©

Figure 7. SARS E protein ion chanel activity observed in NaCl solutions after ~~ . 5 exposure of lipid bilayer to 310g of E protsin. A. The closed stato is shown ss solid ) fine, openings are derivations from the line. Soale ber is 300ms and SpA. The CIS chamber containcd S0mM NaCl in SmM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in $M HEPES 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.

Figare 8. Cinnamoylguanidine (Bit036) inhibits SARS B protein ion channel activity : in NaCl solution. A. Representative currants 2t holding pot=utial of 40mV., Scal bar ia 300mS and SpA. B protein ion charmel activity and B protein channel activity after the addition of 1004M Bit036. B. All poins histogram at holding potential of - bo 15 40mV. B protein ion channel activity before and after the addition of 100uM Bit036. 0 C. Average carment (pA), before farmation of E protein jon channel, B protein ion channel activity snd after addition of 100M Bit036. :

Figure 9 2298 E protein Ion channel activity in lipid bilayers in KCI solutions.

Figure 10: Part A shows raw curreats gancrated by the 229E-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drag addition and the lower trace shows the effiect of addition of 1004M cinnemoylgnanidine on : channel activity. Part B is a graphioal representation of the average current flowing oo across the bilayer (in arbitrary units), before end after addition of ‘25 Figure 11: MHV B protein Ion channel activity in lipid bilayers NaCl solutions.

Figure 12; Pact A shows raw currents generated by the MHV-E protein ion charmel 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 1004M cinnamoyl guanidine on . 30 chamnel activity, Part B is a graphical representation of the average corent flowing

: SE = oo © across the bilayer (in arbitrary units), before snd after addition of

DETAILED DESCRIPTION OF THE INVENTION

. The preseot invention is based, in part, on the sarprising determination that certain compounds that fall mder the classification of substituted acylgnanidines have antiviral activity against viruses from a range of different virus families. Without intending to be bound by any particular theory or mechanism of actian, the negative "impact of tho compounds of the present inveation on viral replication may be © medinted by the inhibition or otherwise down-regulation of a membrane ion chamel "10 reliod upon by tbe virus for replication. This membrane ion channsl may be a viral membrane jon channel (exogenous 10 the host cell) or a host coll ion channel induced as result of viral infection (andogenons to the host odll). Co } As an example, the compounds of the present invention may inhibit Vpu or p7 finotion and thereby inhibit the contiirsation of the respective HIV or HCV life cycle.

The SARS virus encodos &n B protein which is shown for the first time, by the prosent inventors, to act as sn don channel. As similar B peoteins are presant in other coronaviruses, the compounds, componitions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other :

While fhe present invention is concerned with novel entiviral compounds falling under the classification of substituted acyigoanidines, it docs not include in its scope the nae of compounds 5-(N,N-hexamethylene)emiiloride and S-(N,N-dimethyl)- amiloide for rotanding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV,

Yt will be undecstood try those skilled in the art that the compounds of the inveattion may be administered in tha form of a composition or formulation

The pharmaceutical compositions of the invention may further comprise ons at : more known antiviral compounds or molecules. Preferably, the known antiviral © 30 compounds are selootnd from the group consisting of Vidarabine, Acyclovir, :

Ganciclovir, Valganciclovir, Valacyvlovir, Cidofovir, Pamciclovir, Ribavirin,

. i } 44-

Zanamivir, mcleoside-amalog reverse transcriptase inhibitors (NRTD) such as : mocleosids reverse transcriptase inhibitors (NNRTT) such 8s Nevirapine, Delavirdine and Efiivirenz, proteass inhibitors such as Saquinavir ir, Ritonavir, Indinavir,

Nelfinavir, Amprensvir, and other known antivirel compounds and preparstions.

Known antiviral compounds or molecules mey in some cases sot synargistioally with "the antiviral compounds of the invention.

Tabiel Known coronavirns isolates

Canine enteric coronavirus (strain INSAVC-1 ’ Porcine ep am diarrhea vine strain er —— [Poros trnamissibie gastroenteritis covonavirns (stain Neb72- KT) & EEL HLL ‘"Boviscoronavirws STRANLS)

Bovine coronavirus (strain LSU-94L8S-051) :

Bovine enteric coronavirus (strain 9§TXSF-110-El :

i mma — a = YY) ER

Cie: I Ae r I oo oo ee coon vis (ers D386)

A etions ronal vine (in E38)

Co [Avian infectious | aryngotrac] Sets vis virus , Pretiminary Groap 4 | DOE] — : .

SARS coronavirus] coronavirus Be Teazvaol | ZY-2003 : : : SARSoomesvimsBmId oo

A7-

The preseat observations and findings now permit the use of agents such 28 © certain gubstituted acylgnanidines, as anti-viral agents for the therapy and prophylaxis of viral conditions caused by different viruses. The methods aud compositions of the pioscat invention may be particularly effective against viruses which rely an ion chanel formation fir their replication, however it will be understood that this is not fhe only mechanism seliad on by virasea for replication nd that the compounds and methods of the present invention are not limited to agents which exert their action by - retarding oc inhibiting the finction of ion channels. I

Refiarence to "membrane jon channel” should be understood a8 a reference to a structure which transports ions across 8 megubrane, The present invention extends to jon channels which may fimction by means such as passive, csmotio, active or exiibangs transport, The ion channol may be farmed by intracellular oc éxtraceliular means. For example, the jon channel may be an ion channel which is naturally formed by a cell to facilitate ita normal fimotioning. Alternatively, the ion channel may be formed by extracellular mesns. Extracellular moans would inchude, fis example, the : formation of ion charmels doe to ttroduced chemicals, drugs or other agants such es jonophores or due to the fimctiona! activity of viral proteins encoded by a vims which "bas entered 2 cell

The ion chamels which are the subject of cetain embodiments of the presont irrventian facilitate tho transport of ions across membranes. Said membrane maybe ary membrane and is not limited to the cuter cell wall plasma membrane.

Accordingly, "membrane” as used herein encompasacs the membrane surrounding : any cellular arganelle, sach as the Golgi apparatus and endoplasmic reticulum, the outer cell membrane, tho membrane surrounding any foreign antigen which is located _ within the cell (foc example, a viral envelope) or the membrane of a foreign organism which is located extracellularly, The membrane is typically, bat not necessarily, a compossd of a fluid lipid bilayer. The subjoot ion channel may be of any structure.

For example, the Vpu ion channel is formed by Vpu which is an integral membrane protein encoded by EIV-1 which associates with, for example, the Golgi and : endoplasmic reticulum membranes of infected cells, Reference hereinafter to “Vpu

- ion chammels™ js 8 reference to all related ion channels for example P7 HCV and M2 of influenza and the like. | oo

Referancs to “HIV”, “SARS”, “Coronavirus” or "HCV" should bo undaestood es a reference to any HIV, SARS, Coronavirus or HCV virus strain and inoinding § homologues and mutants.

Reference to the "functional activity” of an ion channel should be understood 83 i a reforencs o any one oc more of the functions which an jon channel performs or is imvolved in. For example, the Vu protein encoded jon chaonal, in addition to oo Aciliteting the transportation of Na’, K', CI" and PO, also plays arole in the degradation of the CD4 molecule in tho endoplasmic reticulum. Without wishing to : be bound by a particular theory, the Vpu protein encoded ion ohatnsl is also thought : to play a role in mediating the HIV lif cycle, The present invention is not tmited to treating HIV infiction via the mechmism of inhibiting the HIV 1if cycle and, in particular, HIV replication. Rather, the present invention should be understood to oe 15 encompans any mechanism by which the cumpoands of ths present invention excrt tei st-viral tivity sd may icludoibiion f EAV via of fcc activity. This also applies to HCV, Coranaviruses, and to other viruses. :

Refixonce to the “mctional activity” of a viros should be understood as 8 refirancs 0 any one of more of the functions which a virus performs ar is volved

Refbrence to the * viral replication” should be understood to include any ane or more stages or aspocts of the viml life cycle, such as inkibiting the assembly or

Co release of virions. lon chamel mediation of viral replication may be by direct or indirect means. Ssid ion chammel mediation is by direct means if the jon channel : 25 interacts directly with the virion at any anc or more of its lifs cycle stages. Said ion chammel mediation is indirect if it interacts with a molecule other then those of the virion, wisich ofher maloculs either directly ar indirectly modsilates any one or more aspects ar stages of the viral lifo cycle. Accordingly, the method of the prosent invention encompasses the mediation of viral replication via the induction of a : cascade of steps which lead to the mediation of any one or mare aspects or stages of + the viral life cycle.

Refbrence to “down-regnlating' ion channel fimctional activity, should be } mdsamstood as & reference to the partial of complete inhibition of any one or more aspects of said activity by both direct and indirect mechanisms. For example, 8 : suitable agent may interact directly with an jon channel to prevent replication of a Co virus or, aliematively, may act indirectly to prevent said replication by, for czample, interacting with a molecule ofher than an fon charmel. ‘A further attornativo is that

PL gid other molecule interacts with and inhibits tho activity of the jon channel " Soreening for molecules that have antiviral activity oan be achieved by therangs ' ofmolhodologics described herein.

Refizenoe to a “cell” infocted with a virus should be understood as a reference to

A any cell, prokaryotic or eukaryotic, which has been infected with virus. This : incindes, fr example, fmmostsl or primary cell lines, bacterial cultures and cells 5 ) situ. Tn a suitable scroaning syskm for antiviral componnds, the prefered infbcted cella would be macophagewmanocytes of hepatooytewlymaphid cells infhcted with 1S either HIV or HCV respectively. | . oT ‘Without limiting the present invention to any ons theory ar mods of action, fhe compounds of the present invention are thought to inhibit viral replication or virion release from cells by causing ion channels, namsly VPU of HIV, the B protein of SARS and other Coronaviruses, of P7 of HCV to become blocked. The present

The pressat invention also inchudes the use of compounds S-(N,N- oo hexamethylens)arniloride and S-(N;N-dimatiryl) amiloride in the contro] of viral replication and/or growth other than HIV. : : The subject of the viral inhibition is genecally a mammal such as but not imited to human, primate, livestock animal (c.g. sheep, cow, horse, donkey, pig), companion animal (e.g dog, cai), Ibarsory test animal (0.5. moss, Tabbit, Tt, gninea pig, hamster), captive wild animal (6.§. fox, deer). Preferably, the subject is a : : human or primate. Most proforably, the subject is a hnmao. "The method of the present invention is useful in the trestment and propirylaxis 3 of viral infection such as, fbr example, but not limited to HIV infection, ECV infection and other viral infections. For example, the antiviral activity may be : affocted in subjects known to be infected with HIV in order to prevent replication of

-S0-

HIV thercby preventing the cuset of AIDS. Alternatively, the method of the preacnt invention may be nsed to reducs serum vical load or to allevisto viral infoction symptoms. Similarly, antiviral treatoent may be effected in subjects known to be : infocted 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 method of fe present invention may be particularly useful cither in the ‘carly stages of viral infsction to prevent the establishment of a viral reservoir in 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,

Reference herein to "therapeutic® and “prophylactic is to be considered in . their broadest contexts. The term “therapeutic” does not necessarly imply thit & ‘mammal is treated until total recovery. Similarly, "propirylactic™ doos not necossarily meen that the subject will not eventaally contract a disease condition. Accordingly, - therapy snd prophylaxis inclnde 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 8 particular condition. Therapy may also reduce the severity of an exiating condition or the frequency of acute attacks, .20 In accordance with the methods of the present invention, more than one compound ar oumposition may be co-administered with ano or more other compounds, such as known anti-viral compounds or molecules. By "co- administered” is meant sinmltancous administration in the seme formulation or in two different formulations via the same or different routes or soquential administration by the same or different routes. By "saquential® administration is meant a time difference of from seconds, minntes, hours or days between the administration of the two or more separate compounds. The subject antiviral compounds may be administered in

Routes of administration include but are not limited to intravenously, oo 30 intapecitioncaly, subcutaneously, intracranial, intradermally, intrarmascularty, intraocularty, intrathocaly, intracerchrally, intranasally, transmucosally, by infusion,

rt cally, rectally, via tv rip, pate and implant. Intravenous toutes sre particularly

Compositians suitable far injectable use inciuds sterile aquoous solution oo (where water soluble) and sterilo powders for the extemporeneous preparation of 5 . sterile injectable solutions. The carrier can be & solvent or dispersion medium

Co containing, for example, water, ethanol, polyol (for example, gtycerol. propylene - glycol and tiquid polyethylene giyool, and tho likes), suitable mixturos thereof and a vegetable ails, The prevention of the action of microorganisms cin be brought shout by Various stibacterial and antifungal gents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the ike. In many oases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absozption oo of the injectable compositions oan be brought about by the use in the compositions of } agents delaying sbeorption, $o example, akaninam monostcarste and golstin. ; Sterile injectable solutions arc prepared by incorporating the active . o “18 compounds in the required amount in the appropriate solvent with vations of the ofher : ingredients enumerated above, as required, fbllovwed by, for exaropls, filter stocilizstion or sirilization by other sppropriste means. Dispersions aro also contemplated sd these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the fequired other ingredients from those amumerated above. In the case of sterile : powders fox the preparation of sterile injectable solutions, a preferred method of propscation includes vacwan drying and the fieeze-drying technique which yield » oo powder of the active ingredient plus any additional desired ingredient from a proviously steclle-filtered solntion.

When the sctive ingredients are suitably protected, they may be orally : administered, for example, with an inert diluent or with an assimilablo edible carrier, or ft may be enclosed in bard of soft shall gelatin capsule, oc it mR be compressed: oo into tablets. For onl therspeutic administration, the active compound may be © jmsorporated with excipients and used in the frm of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.01 % by weight, mors preferably 0.1% by weight, even mare preferably 1% by weight of active compound.

The percentage of the compositions and preparations may, of course, be varied and say camveniently be between sbaut 1 to sbout 99%, moro prefrably about 2 to shout 90 %, even moro preferably about § to about 80% of the weight of the unit. Tho ammomat of active compound in such therapeutically useful compositions in such that 8 5 suitable dosage will be obtained. Prefrred compositions or preparations according to the present invention are prepared so that an oral dosage unit fim containg between © about 0.1 ng md 2000 mg of active cornpound. : | The tablets, troches, pills, capsules and the like msy also contain the components as listed harcafter: A binder such as gum, acacis, com starch of gelatin; : -10 excipients such as calcium phosphate; a disintegrating agent such as com starch, : potato starch, alginic acid and the Hike; a Inbricant such as magnosium stearate; and a sweetening agent such as sucrose, Iactose ar saccharin may be sdded or & flavouring agent wach ss peppermint, oil of wintergreen, or cheery flavouring. When the dosage unit form is a capsule, it may contain, in addition to matecials of the shove type, & © 15 Tiquid carer. Various other materials may bo present sé boatings or fo otherwise modify the physical form of the dosage unit. Far instance, tablets, pills, of capeules may be odeted with shellac, sugar or both. A syrup or elixir may contain the active . componnd, sacross as sweetening agent, methyl and propyiperabens as : prescrvativos, a dye and flavouring such as cherry or orange flavoor. Ary material used in preparing any dosage unit form should bo pharmaceutically puro and substantially non-toxic in the amounts employed. In addition, the active compoend(s) may be incorporated into sastained-relcase preparations and formulations.

The present invention also extends to forms suitshlo fix topical application such 88 creema, lotions and gals. In such forma, the anti-clotting peptides may need to be modified to permit ponctration of the surface barrier. Procedures for the g proparation of dosage uit fxms and topical preparstions are roadily available to those skilled in the art fram texts such as Pharmaceutical Handbook. A Martindale

Companion Volume Ed. Ainley Wade Nineteenth Edition The Pharmaceutical Press

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

Co $3

McGraw Hill; Remington; and The Science and Practice of Pharmacy. Ninetsenth

Ed. Ed Alfonso R. Gennaro Mack Publishing Co. Easton Pennsylvania. © Phamsceutioally scosptable carriers and/or diluents include suy sad all solveats, dispersion media, coatings, antibacterial and antifimgal agents, isotonic and 5s 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 3 conventional media or agent is inoompatible with the active ingredient, use thereof in the therapeutic compoftions fs contemplated. Supplementary active ingrodiauts on also be incorporated into the compositions.

Jt is capocially advantageous to fornmulate parenteral compositions in dosage anit form for eese of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete inita anited as unitary dosages for the mammalian suhjocts to be tested; each unit containing a predetermined quantity of © active material calculated 10 produce the desired thesapoutio offisct in association with . _ 15 the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dspsndent on (a) the unique’ characteristics of fhe active material anid the particular therapeutic effect $0 bo . achisved and (b) the limitations inherent in the art of compounding.

Effective amounts contemplated by the present invention will vary depending on the severity of the pain and the health and age of the recipient. In general terms, oC effective amounts may vary from 0.01 ng/kg body weight to shout 100 mg/kg body

Ahernative 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. "The subject of the viral inhibition is generally a mammal such as but not timited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), cotnpsanion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamater), captive wild animal (e.g. fox, doer). Profarably, the subjectis a human or primate. Most preferably, the subject is a human. :

The methods of the present invention is usefhl in the treatment und prophylaxis of viral infection such as, fur example, but not limited to HIV infection,

HCV infection and other viral infections. For example, the aotiviral activity maybe.

$4- : effbotad in subjects known to be infected with HIV in order to prevent replication of

HIV thereby proventing the anset of AIDS. Altematively, tho methods of the present invention may be used to reduce serum viral load of to alleviate viral infection symptoms. Similarly, antiviral treatment may be affected in subjects known i be 5 infected with, for example, ICV, in ander to prevent replication of ICV, thersby : preventing the futher hepatocyts involvement and the ultimate degencrstion of iver

The methods of the present invention may be particularly useful either tn the carly stages of viral infection to provent the establishment of a viral reservoirin affbotod cells or 8a a prophylactic trostment to be appliod immediately prior to ar for 2 period after exposure to a possible sours of virus.

I ‘The present invention will now be described in more detail with reference specific but non Hmiting examples describing studies of viral membrane ion channcls

Co and soroening for antiviral activity. Some examples involve the use of the SARS virus. Bt will be clear from the description hecein that other lontiviroses, snd coronaviruses and other compounds may be used cfftctively in the context of th preset invention. J is to be undersioad, however, that the detailed description is included solely for the purpose of cxcmplifying the present invention. It should nat be : understood in ary way ae 8 restriction on thé broad description of the invention as set out above. :

Example 1. Synth mis gl the Compounds of the Jnvsation

The compounds of the present invention may be made from the corresponding acid chlorides ar methyl csters as shown in Schemo 1. Both of these methods are well described in the literature.

Ld l— a

Gi R 7 NH, rR” “OMe acidchlodd acyl gnanidine ester gs To dwell

: 5 Ce

The following examples show synthetic schemes for some compounds of the | a {nvention. :

LC

1. 00CY)y / bowcows / sat. DMF : ———em fi oro 2. (LN), CNBC oe

MeOH /B,0/ THF .

To sbtion of tras-canamic acid (150 102 mmol) in dy benene oo (30mL) containing a drop of N,N-dimethylfirmemide was added oxalyl chloride (5.14 5. 40.5 mmol) causing the solution to effervesco. After refluxing for 21, the solution was evaporated to dryness under reduced pressure. The resulting solid was dissotved in dry tetrahydrofuran (20mL) and added slowly to & solution of guanidine : hydrochloride in 2M aqueons sodizm hydroxide (25mL). The reaction was stirred at : ‘room temperature for 1h then extracted with oy! acetate (3xSeL). The combined extracts were dried over magnesium sulfate ad evaporated to give an ormge oil. The rode prodnct was purified by column chromatography. Elution with 10% to 20% methanol in dichloromethane gave Cixnamoyiguanidine ss a croam solid (0.829 g, ’ 43%). : bo +20 3-aming-5-phexvi-6-chloro-2-pyrazinecarboxamide

Part oo

Nagy NPE), ———————" lie vad ==Fov of

To a sohation of methyl 3-amino-S,6-dichloro-2-pyrainecarboxylato (0444 g, 2.0 mmol) in tetrahydrofuran (5 mL) / water (10 mL) / toluene (20 mL) was added phenyl boronic acid (0.536 g, 4.4 mmol), sodive carbonate 0.699 g, 6.6 mmol) and tetrakis(triphanyiphosphine)- palladium(0) (0.116 g 0.10 mmol). The reaction was

: evacuated and purged with nitrogen soveral times before being refluxed for 61. The oo organic layer was separated and the aqueous layer extracted with tolucne (3 x 20 oil).

Co The combined organic extracts were dried over magnesium sulfite, filtered and evaparstod under reduced prossure to give methyl 3-amino-6-chioro-3- phenyi-2-pyrazinecarbaxylase a8 a yollow solid (0.43 g, 82%).

Part2 : oaderers i

To a sclutian of sodium (0.040 g, 1.74 mumof) dissolved in methanol (5 mL) 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- © chloro-S-phenyl-2-pyrezinecarboxylate (0.264 g, 1.0 mmol) in N.V-

SE dimethyl fhrmammide (5 mL) and tho solution hosted st 75+C fir 12 h. The solvent was removed under reduced prossare and the rosidne chromatographed on sifios gol cluting with 1% triethylamine / 5% methanol / dichloromethane. The resulting solid was suspended in chloroform, filtered and driod under high vacuum to give N-

Amiding-3-amino-S-pharl-6-chloro-2-pyrazinecarboxamide as 8 yellow solid (0.04 £ 14%).

Example 4.

Part 1 “OLY oD) ==) + :

NH, 7 Ni

E : To a solution of methyl 3-amino-5, 6-dichloro-2-pyrazinecarboxylats (1.11 g, : 5.0 mmol) in tetraliyvirofaran ($0 mL) was added hexamethylensimina (1.49 g, 15.0 mmol) and the reaction wes refluxed for 1 h. Tho reaction was allowed to cool and the solid hexamethylensimine hydrochloride removed. by Sltration. The filtrate was

: | | LS a evaporated and the residue chromatographed over silica gel. Elution with dichloromethane gave methyl 3-amino-6-chioro-5-hexamethylensimino-2- ’ a pyrasinecarboxylate ss m off-white solid (1.20 g, 85%).

Part2

HO. OH E00, NOU DCH . & S— : To a solution of methyl 3-amino-6-chloro-5-hexamethyleneintino-2- pyrazincomrboxylsto (0.350g, 1.23 mmol) in dimefhylsulfoxide (5 mL) was added . phenyl boronic acid (0.165 8 1.35 mmol), potsasinm carbonate (0.511 g, 3.70 mmol) and [1,1-bia(diphenyiphosphino)ferrocenc]dichloropelladinm(IT)-dichloromethane : complex (0.041 g, 0.05 mma). The reaction was hoetod st 90:C for 16 h before being poured into water (50mL) and extracted with ethyl acotate (3 x 50L). The combined extracts were dried over magnesium sulfide, filtered and evaporated 10 give a beown oil which was purified by chromatography on silica gel. Elution with ~~ dichloromethane followed by 10% ethyl acetate/dichloromethane gave methyl 3- amino-5-haxamethylaneimino-6-phenyl-2-pyrasinecarbazylate 8s 8 yellow solid oo © (0.309 g, 77%).

Part 3. eta m— : MCs / MON / THF

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 : reflizxed for 30 min after which it was filtered. To the filtrate was aided methyl 3- smino-S-haxsmethylensimino-6-phenyl-2-pyrazincoarboxylate (0.310 g. 0.95 mmol) in tetrahydrofuran (10 mL) and the sohution refhuxed for 72 b. The solvent was removed under reduced pressure and the residne chromatographed on silica gel.

Elution with 5% msthanol/dichloromethane gave N-amidino-3-amino-3- hexamethylenaimino-6-phenyl-2-pyrazinecorboxamids as a yollow solid (0.116 g, 35%). : : 5 Examule$, Viral Studies : Complimentary DNA (cDNA) fragments for the various viral proteina listed © in Table 2 were obtained either by PCR amplification from & parental virus genome clone, or by direct chemical synthesis of the polynucleotide sequenbe. For example, this open reading frame encoding Vim (Fig 1a) was amplified by PCR from a cDNA : clone of an Nde I fragment of the HIV-1 genome (isolate HXB2, McFarlane Bumet

Centre, Molboume, Australis) ss fhllows: Native Pfa DNA polymerase (Siratagens; 0.035 U//II) was chosen to catalyse the PCR reaction to minimise possible PCR introduced ecrors by virtue of the enzyme's proofreading activity. The 5', sense, . AGTAGGATCCATGCAACCTATACC (< 400 > 2) introduoes a BamH] sito (underlined) for cloning in-frame with the 3' end of the GST gene in p2GEX (41).

This primer also repairs the start codon (bold T replaces a Q of the vpu gene which is

Lo 20 a threonine codon in the HXB2 isolate. The 3', sntiscnae, primer

TCTGGAATTLTACAGATCAT CAAC (< 400 > 3) introdooos an BooR1site (nndertined) to the other end of the PCR product to facilitate cloning. After 30 oyoles © of 94°C for 4S sec, 55°C for 1 min and 72°C fir 1 min in 0.5 ml thin-walled eppendorf tubes in a Perkin-Elmer thermocycler, the 268bp fragment was purified, digostod with BamHl and EcoRI and ligated to p2GEX prepared by digestion with the - Co sams two eixzymes. The resultant recombinant plasmid is illustrated in Fig Ib. The entire Vipu open reading frama and the BamH1 and EcoR1 ligation sites were sequenced by cycle sequencing, using the Applied Biosystems dye-terminator kit, to confirm the DNA sequence. Other cDNAs weve synthesised for us using stats of the : 30 art methods by GenScript Corporation (Now Jersey, USA). Codon sequences were © optimised for expression in bacterial, insect or mammalian oells, as sppropriate.

Restriction endomnclense enzyme recognition sites were incorportated et the 5° and 3°

IE | 55. : ends of the synthetic cDNAs to fcilitate clanjog into plasmid expression veotars, pODNA3.1, pFastBac and pPLAS1 for expression of ths encoded viras proteins in :

Standard techniques of molecular biology were used in cloning experiments. For : exemple, to prepare the Vpu open reeding frame for insection into the pPLA51 exproasicn plasmid, p2GEXViu was frat digested with BamHI and the 5' base ovechang was filled in the Klenow DNA potymerass in the presence of dNTPs. The

Vpu-encoding fragment was then libarnted by digestion with BeoRl, purified from an agarose gol and gated into pPLAS1 which had been digested with Hpel and EcoRI.

Westom blots subssquently confirmed that the pPLVpu construct (Fig k) expressed " Vpa after induction of cultures at 42°C to inactivate the cI857 repressor of the FR and : PL promoters.

Table 2 Source of viral cDNA or peptide sequences.

Accession number [SARS-CoV Epwotein | SARScoronavirus _______ |P59637 : | MHV-Eprotin | Mormebepatitisvirs [NP 068673 : Cultures of E. coll strain XLI-bine cells containing p2GEXVpu were grown at 30°C with vigarous aeration in LB medium supplemented with glnoose (6g/L) and ampicillin (S0mg/L) to a density of approximataly 250 Klett units, at which time

IPTG was added to a final concentration of 0.0kmM and growth was continued for a further 4br. The final culture density was spproximately 280 Klett units. Since early experiments revoaled that the majority of expressed GST-Vpu fusion protein was associated with both the cell debris and 30 membrane fractions, the method of

Varadhachary and Maloney (Varsdhachary and Maloney, 1990) was adoptedto isolxte csmotically disrupted cell ghosts (combining both cell debris and membrane fractions) for the initia! purification steps. Cells were harvested, washed, weighed and

© So © romspended to 10ml/g wet weight in MTPBS containing DTT (mM) md MgClz (10mM). Lysozyme (0.3 mg/ml; chicken cgg white: Sigma) was dded and inoubeted on 100 for 30 in with gentle agitation Solow by 5 min st 37°C. The oemsticelty sensitised cells were pelloted at 12,0005 and resuspended to the original volume in § water to burst the cells, The suspension was then made up to BMTPBS/DTT using a © ox buffer stock and the ghosts wars isolated by centrifugation and resuspended in

MTPRS/DTT to which was then sequectially added glycerol (to 20 % witvol) and

CHAPS (to 2 % wtival) to give final volume of one quarter the origina] votama.

This mixture was stirred on ice for 1 br and then oantrifugsd at 400,000 for Ihr to . 10 remove insoluble material. The GST-Vpu fusion protein was purified from the detergent axiract by affinity chromatography on 8 ghitathiooe agarose reein (Sigma).

The rosin was thoroughly washed in S0mM Tris pH 7.5 containing glycerol (5 %),

DTT (mM), sod CHAPS (0.5 %) (Baffhr A) and then the Vp portion of tho fuslon protain was liberated and cittod from the resin-bound GST by treatment of 8 50% (v/v) suapension of the beads with human thrombin (100U/ml; 37°C for the). PMSF © (0.5mM) was added to the eluant to eliminate any remaining fhroebin activity. This

Vp fraction was farther purified on a cotemn of MA7Q anion exchange resin attached to 8 BioRad FPLC and eluted with » linear NaCl gradient (0-2) in buffer

A

The Vu was purified to homogeneity - s determined an silver stained gals - on an immunoaffinity column as follows: HPLC fractions containing Va were desalted on a NAP 25 column (Pharmacia) imo buffer A and thon mixed with the antbody- agarose beads for Thr st room temperature. The beads were washed thoroughly and : Vip was eluted by increasing the ealt concentration to 2M. Protein was quantitated using the BioRad dye binding sassy.

The plasmid p2GEXVpu (Fig. 1) was constructed to crests an in-frame gene fusion between the GST nd Vpu open-roading frames. This system eoshied IPTG-nducible © 30 expression of the Vu polypeptids fused to the C-terminus of GST and allowed © porifioation of the fusion protein by affinity chromatography on ghuathions agarmes..

oo -61- oo Optimal lévels of GST-Vpu expression were obtained by growing the cultures at 30°C to a cell density of spproximataty 250-300 Klett units and induoing with low oo Jovela of IPTG (0.01mM). To purify fhe GST-Vpu, 8 comisined cellular fraction

C- containing the cell dsbeis and plastoa meesbeune was propared by lysozymo trostment "5 ofthe induced cells followed by 2 low-speed centrifigation. Approximately 50% of the GST-Vpu protein coukd be sofubilised fom this fraction using the zwitterionic ased to enrich the fasion protein and thrombin was used to cleave the fisian protein it the high affinity thrombin sito between the fusion partners, tiherating Vpu (Fig. 2A). In fractions eluted fiom the anion exchangs column Via was the major proton visible on silver stained gels (Fig. 2B, lans 1). Finally, Vpu was purified to spperent homogeasity on &n imnmnoaffinity column (Fig. 2B, lane 2). The N-terminal amino acid sequence of the protein band (excised from SDS-PAGR gels) corresponding fo the immumodetected protein confirmed its identity as Vin. :

Protealiposotnas containing Vpo were prepared by the detergent dilution method (New, 1990). A mixture of lipids (PE:PC:PS; 5:3:2; Img total lipid) dissolved in chloroform was dried onder & stream of nitrogen gas end resuspended in 0.1 mi of : 20 potassium phosphate buffer (S0mM pH 7.4) containing DTT (mM). A 25x! aliquot containing purified Vpn wes added, thllowed by octylghcoaide to a final canceirtration of 1. 25 % (wt/vol). This mixture was subject to three rounds of freceing in Hquid nitrogen, thawing ud sorsiostion in beth type sonicator (20-30 sec) amd was then rapidly diluted into 200 volumes of ths potassium phosphate buffer.

Protooliposomes were collected by centrifugation at 400,000g for Lhr end rosuspendod "in approximately 1501 of phosphate buffer. © Purified Vp was tasted forts ability to induce channel activity in planar lipid bilayers using standard techniques as described clscwhere (Miller, 1986; and Piller ot al, 1996). Tho solutions in the CTS and TRANS chambers were separated by a . Delrin™ plastic wall containing a small circular hole of spproximately 100pm -

diametér acroas which a lipid bilayer was painted 80 as to form a high resistance So alectrical seal. Bilayers were painted from a mixture (8:2) of palmitoyi-oleoly- phosphatidyl-ethenolamine end pahnitoyl-oleolyphosphatidyl-choline (Avanti Polar

Lipids, Alsbaster, Alsbams) in n-decane. The solutions in the two chambers contained MES buffir (10mM, pH 6.0) to which vaclous NaCl or KC! concentrations were added. Currents wero recorded with an Axopetch™ 200 amplifier. The elcotrioal potential between the two chambers could be manipulated between +/-200mV (TRANS relative to grounded CIS). Aliquots containing Vipu were added to the CTS chamber cither 8s 8 detergent solution or after incorporation of fhe peotein into phospholipid vesicles: The chamber was stirred until currents weee observed. : To amay fie ion-channel foemation by Vis, reconstitution ioto plankr lipid _ bilayors was performed. When samples (containing between 7 and 70ng of protein) of purified recombinant Vip wece added to the 1m of buffer in the CIS chersber of the bilayer spparstus, current finctustions were detected after periods of stirring that oC varied from 2 to 30 min (Fig. 3). This time taken to observe chammel activity : appeoximately correlated with the amount of protein added to the chamber. No

Co channels were detected when control buffer aliquots or control lipid vesicles ware added 0 the CTS chamber. In those control experiments the chambers could be sired ~~ 20. for more than an hour without appearance of channel activity. Chaxmel 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, socmed to approximately correlate with the smount of protein added. The smallest and largest charmels meesared had conductances of 14 pS and 280 pS, respectively. The chanmels © swere consistently smaller when Hpid vesicles containing Vpu wers prepared and £308 to the bilayer rather than when purified protein in dotergent solution was added.

This may be becanss the former method included treatment with high concentrations of detergent and a dilution step that may have favoured the breakdown of large ~~

- . oo The relationship between current amplitude and voltage was linear and the reversal potential in solutions containing a ten-fold gradient of NaCl (S00mM CIS; $0mM TRANS) was +30mV (Fig. 3B). A similar roversal poteutial yas obtained when solutions comtained KCI instead of NCL In § experiments with either NaCl or

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 pecmeable fir the cations alone, Using ion activities in the Goldman-

Hodgkin-Katz equation gives & P/Py ratio of sbout 5.5 indicating that the channels . gre also permeable to chloride fons. An attempt was made to reduce the anion currect 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, nv) again indicating 8 cation/anion pormesbility ratio of shout S. (For calculations oo involving the phosphate solutions, the summed activities of the mono and bivalent anions were used and it was assumed thet the two spocios were equally permeable). - : The current-voltage curve now exhibited rectification that was not seen in the NaCl solntions. It oan be concluded thet the channels formed by Vpn ars equally parmeshly to Na" and K* and are also pacmesble, thongh 0 & lesser extant, to chiorido ss well as phosphate ions. : oo» "This bio-ssssy is based on the observation that expression of Vpu in E coli : results in an active Vpu channel located in the plasmalemma that dissipetos the transmembranc sodium gradient. As 8 consequence of this Vpu channel activity, metabolites whose accumulation within the oolls is mediated by a sodium dependent co-transposter (for exemple proline or adenine) leak out of the cell faster than they . can'be synthesised 80 that the metabolites’ intracellnlar levels become limiting for growth of the cell. Thereby, an £ cof cell expressing Vpu is unsble to grow in minimal drop-out media lacking adenine or proline. However, in the presence of a dmg that blocks the Vip channel, the call is once again able to ro-cstablish its . transmembrane sodium gradient - due to the action of other jon pumps in the membrane - and the leakage of motabolitos is provented enabling growth.

: | “ . Experiments to demonstrate that Vip oun form sodium chanmels in the plasma membrane of B, coli were performed as follows.

Co : To express unfised Vpu in E. coli, the vpu open-rosding frame wes cloned into the plasmid PLAS] to creatn the recombinent plasenid pPL-Vpn (Fig. Th). In this © 5 vector th strong Py, and Py lamba promotars are used to drive expression of Vpn under control of the temperature sensitive c1857 reprosser, such that when grownst 30°C exprossion ls tightly repressed and can be indnood by rising the tempersture to between 37°C and 42°C. On agar plates, cells containing pPL-Vpu grew when incubated at 30°C snd 37°C but not at 42°C, while control strains grew well at 42°C.

Liquid cultures of cells contsiting pPL-Vpu were grown at 30°C to ODax=0.84 then moved to grow at 42°C fur two hoara (the final cell density was ODexn=0.75). The plasma membre fraction was prepared and western hiotting, using an antibody that specifically binds to the C-terminms of Vpn, detectad 8 single band at spproximately : 16kDa, indicating that Vpu was expressed and associated with the membrancs (Fig. 2A,lm08).

Exacuminz You. : : Uptake of proline by B. coll is well characterised nd active transport of the amine acid imo the cells is known to use the sodium gradient as the energy source ©. 20 (Yamato tal, 1994). To detoot whther proline leakage occurs, the fllowing croms- " focing assay was sad: A lswn of an E. coli strain suxotropbic for proling snd meiionine (Met Pro’), was seeded and poured as a soft agar overlay on minimal drop-out media plates lacking proline but containing methionine. Steril porous filter discs were inoculated with a Met* Pro* strain (XL-1 blue) containing either the - pPLAS] control plasmid or pPL-Vpu and placed nto the soft agar. The plates ware "then incubated at 37°C ar 30°C for two days. After than time a halo growth of the © Met Pro’ strain was clearly visible surrounding the disc inoculsted with the cells. containing pPL-Vpn incubated st 37°C (Fig. 4A). This growth can ouly be due to the ‘Ioaknge of proline from the Vpu-axpressing cells an the disc. No such leakage was apparent from the control strain at 37°C nor around cither strain on plates grown at 30°C (Fig. 4B). oo

Tn contrast to proline transport, the B. coli methionine permease is known to CC belong to fhe ARC transporter fumily (Rosen, 1987) and hance be energised by ATE.

Identical crossfoeding expériments 10 those described above were get us except that © the Met Pro’ strain wes spread on minimal drop-out pistes lacking methionine but "5 containing proline. No growth of this strain was cvident sround any of the discs (Fig

Co 4C), indicating that methicnine was not leaking out of the XL-1 blus cells oven when

Vpu was being expressed.

It was observed that, due to an uncharecterised mutstion in the adenine ‘syntheéls pattrway, growth of E. colt cells of the XLi-blue strain expressing Vpu at 37°C was dependant on tho prosence of adanine in the medium. This allowed the " dowelopment of an even simpler bioassay Sor Vpn ioo-channel activity than the proline croas fooding sissy described sbove: A lw of XL1-bine oells containing the ©. 15 pPL-Vpa plamid is seeded onto un agares plats lacking sdmine in the médium, © small aliquots of drags to be tested for inhibitian of the Vip channel are spotted onto the agarose in discrete locations snd the plates are incubated at 37°C for a suitable peciod of time (12-36 hours). Halos of growth around a particular drug application gitn indicate that the drag has inhibited axpreasion of the Vpt ion channel activity that - © 20 provents growth in the seas of the drug. (Figur 5).

Assay of Compounds in Plaga Lipid Bflavers for Vou Channc] Blocking.

Comopunds were characterized foe their ability to block Vu jon channel activity roocmatituted info planar lipid bilayers. Vpu N-terminal peptide (residuse 1- 32) dissolved in trifinorethanol was added to the CIS chamber of the bilxyer © apparatus and the solutions was stirred until ion currents were observed, indicating . incorporation of one or mors Vpu jon channels into the bilayer. After recording the : 30 chennel activity for a fow minutes, drugs were added to the solutions in the CIS and

SE TRANS chambers — with stirring - to a final concentration of 100uM. Charmel . activity was then recorded for at losst & further three minutes and the effect of dmg addition on jon current was determined by comparing the chmnnel activity before and after drug addition. For each experiment, drug effbct was classified into four : categories: “Stang block”, if current was inhibited approximately 50-100%; “week block”, appeox. 50-90% inhibition; “partial block”, <50%; and “no effect”. te Bxpeximents were disrogarded if currents larger than +50pA were genaraicd after addition of Vipu N-peptide beoanse in such cases it ia possible that non-native peptide © aggregates contribute to bilsyer breakdown. Such aggregates, by virtue of their disorganized structure may not be specifically blosked by the drugs at the

Table 3 summarises {ho results of the bilayer experiments. A novel cutoome of thess experiments was the strorig blocking of Vpu channels observed with Phenamil.

Phensail has a pheayl group derivative at the gnamidine group of anlotide. : © Amiloride itself is not a blocker of Vpu, whereas addition of the hexamethylane : _ goup at the 5- position of the pyrazine ring reatod 8 structure (HMA) that blocks the channel at concentrations as low as 2SuM. Thesé now results with Phenamil, - howevec, now show that 8 bulky hydrophobic derivative at the opposite and of the . molecule can also torn smilorids into an effective Vip channel blocker. Toterestingly, benzamil, with a very similar strocture was much loss effective at blocking the Vpn

Tabi: Summary of Compounds Inhibiting the Va Ion Channel ia Bilyers N a : Compound No.of Resits

Pm 3 3xSuomgblock

MIA : 2 1x Strong block; 1x weak ’ Benzamil 10 3x partial block; 7x no effect :

EIPA | 3 3xweakblck

HMA | 1 1x Strong blook; ~ (S-Phenyl-pents-2,4-dienoyl)guamidine 6 6xstrong block 6-methoxy-2-naphthoylgusnidine § Sxstrong block (2-Chlorocinnamoyl) guanidine 6 4x. strong; 2x partial blocks 3-{trifluoromethyl)cinnamoylguanidine ~~ 5 4x strong blocks; 1x no effect

CL N-{5-[3~(5-Gumidino-pentyloxymethryi)- Co benzyloxy]-pentyl}-gnsnidine 4 3x strong block; 1x no effect 4-phenylbenzoylguanidine 3 3x strong block ‘ 3-methyleinmamoylguanidine 4 2x strong block; 2x partial

N-(3-phenylpropanoyl)}-N- phenylgusnidive 1 1x strong blocks (3-Bromocinnamoy{)gumidine 3 3x partial-strong block © S-tert-butylamino-amiloride 3 - 3x partial block

N-umidino-3-amino-5-pheny}-6-chloro-2- = pynzinccarboxamide 3 3x pertial block 3-methoxy -HMA 3 3x partial block 5 (N-Methyl-N-isobutyDamiloride 1 1x partial block 5-(N-Ethyl-N-isopropyDemiloride 1 1x partial block 2-napthoygnanidine Co 7 = Txweakhlock

N,N"-bis(Ipheryipropsmoyl)-N"- phenyignanidine 7 Tx weskblock cirmemoylguaidine 3 3xweskblock (S-Fhenyl-penta-24-disncy guanidine 6 6x strong blook oo : s The halos of growth around the site of application of particular drugs — es descaibed in example 14— were given a scare between zaro and six reflocting the size : and density of the zone of bacterial ocll growth. Scores greator than 3 represent strong inhibition of the Vpu protein; scores betwoon 1.5 and 3 represent moderate : inhibition and scores between 0.01 and 1.5 represent fiir inhibition, : 10 . .

Table 4 lists the scores for inhibition of Vpu protein in the bacterial bio-assay.

Table 4

Vpa Inkibition (score / # of times } ’ Chlorocinnamovi)onanidin 4738/4

Bromocimamoyl)guanidine 4.3/24

Chlorocinnsmoyl)guanidine 4.0/4

Bromocinnamoyl)guanidine 3.72 3-(triffucromethyl)cinnamoylguanidine F372

: 5-hromo-2-finorocinnamoyignanidine 3.512 .methylcipnsmoylguanidine 3.4/2 2-methylcinnamoylguanidine Hr ’ di sthylcimmamoyl guanidine T . - hd . Sin mo ‘tl anidin - ’ 2.96/12 6-mothoxy-2-naphthoylguanidine 2.9/4 rans 3-(1-napthylactyloyl guanidine 2983 . 3.4-dichl s novi idi 2.93 ' }

R,6-dichlorocimnamoylguanidine 2.8822" : 4-phenyibenzoylgnsnidine 2.75/5 : has Srnanidine S21 4-Chlorocinnamoyl) guanidine : 2.7/5

R-napthoylguanidine 2,711

R,5-dimethylcimamoylguanidine 2.69/2 : 3-isopropylcinamoyligusnidine hydrochloride 2.62 : S-Phenyl-pents-2 4-dienoyl)gnanidine 2.5612

B-phenylcinnamoyignanidine 2.5473 4-Bromocinnamoyl)guanidine 2.54 . . "-bramophenyl)pents 2.4 den oyl guanidine > 252 3-(cyclohex-1-en-1-yl)cinnamoylgnanidine 2.52

B-{irifinoromethoxvicinnamovisnanidme . ’ 2442

R-(triftluoromethylcinnamoylguanidine 24/2

WN'-bis(3phenylpropanoyl)-N"-phenylgumidine 22513 - 4 OL OX YCIIINEINO VAR MI OINO 2252 (3-phenyipropanoy!)-N'-phenylgeanidine : 22173 4-(txiflnoromethyl)cinnamoyignanidine 22/2 4-Methoxycinnamoyl)guanidine ; 2.133 2-t-butyloinnamoyiguanidine 2.132 : ¢-mothylcinnamoylgoanidine = 2.12 } 2-flnorocimnamoyigoanidine 212 . R-ohenvicinnamovignsmidin . ' 212 6-Hydroxy-2-napthoyl)-N'-phenyignanidine 2.0672 4 » ’ Henmnamoy guanine 2.06/2 : 3,4-diffuorocinnamoylignanidine 2.0672 5-(N,N-hoxamethyleno)amiloride 1931 3-flnorocinnamoylguanidine : 1.972 5-hramo-2-methoxycinnamoylgnanidine 1.972 : } Y [3-SOXVCIINaemo RUALOINES 1.922 .

B,4-(mothylenedioxy)cinnamoylguanidine 1.882

Methoxycimamoyl) guanidine 1.7/4 2'4 DichloroBenzamnil HC] : 1.72 : »3,6,-tetramethylchnnamoyignanidine 1.62 : 3-(2-napthyDacryloylguanidine 1.5672 2-(1-napthyDectoyiguanitine 15672 [4 1 INOroctnniy oylgnanidine : 1.5612 )

Methoxycinnamoyl)guanidine oo 1.52/6 4-isopropylcinnemoyl guanidine 1.422 2,4,6-trimethylkinnamoyigumidine 1472

’ . £9- : . .

N-(cizmamoyl)- Npbanylgaanidins IR bh (cyolobex-1-en-1yl)cimmamoylguanidine 122 2-(2-naptiylacetoylgusnidine + L192

Wirndn “rmamovigusnidin . 1.122 honamil metbenceolfousto 68 1 oss

Benzamil hydrochloride 0.9/3

Nitrocinnamoy]guanidine - 0.9/1

Benzyoylgusnidine | 0.8372 4.Phenoxybenzoyl)guanidine : 0.812 : OB trans-hept-1-en-1-yl)cimamoyiguanidine 0.8172 (N-Motlyk-N-iscobutylpemiloride | 082 ine on - B 4-dichiomcinnamaolygnanidine 0.632 [7 0 rinecarboramide ’ 0.672 . » Methylcionsmoyf)gnaniding 0.6/2 finnamoylguanidine hydrochloride 0.62 (4-Charophenoxy-acetyl] goanidine 0.5672 5-(4-flnora phenyl) ms Onoe 0.4/6 ’ trans-2-Phenyicyclopropanecatbanyl)gasaidine 0472

Nitrocinnamoyf)gnanidine 042 rans-3-Fusnacryoylguanidine 0.382 1-napthoylguanidine 0.372 5-00 soy TENO-ED1 IOC 02/7 : 3.methaxy -HMA 0.2/4 3 (N,;N-Dimethylamiloride hydrochloride “012

N,N'-Bis(3-pheaylpeopanoyl) guanidine 0.12

N-Benzoyl-N'-cinnamoylgumidine 0.0672 1-bromo-2-ns pthoylguaniame 0.0672 Exxmula 17, Eftect of Compounds on FIV Replication iu Hazan Mogocvies and

Human monocytes were isolated from peripheral blood and cultured either for 5 24k (one dary old monocytes) or for 7 days to allow differentiation into monocyte dexived macrophages (MDM), Thess celle wers then exposed to cell-free pecparations :

=70- © of HIV isolates and allowed 1 sbearb for Zhr before ocmplote aspiration of the medium, washing onoo with virus-free medium and resuspension in freeh 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 infection, was compared in cells exposed fo drags and in cells not exposed to drugs (controls). The progression snd extent of viral replication was assayed using either an

HIV DNA PCR method (Fear ot 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 teat antiviral compounds, oo

Table 3

Dreg Peréoat of

ORIDONAL Control 5 [4 9 1. [216 oo Nooe—positivecontol | [100% 5 11 25 [9 : 125 [8% 0625 [116 | } (Nome —positivecontrol |. 100% 3-{uifivere [10 fn] methylcimamoylgosnidine | 5 [8 0625 {38

Nons—positivecontrol | [100%

S(NN- | 10 |6 |] hexmmethylene)amiloride 5 Jar 125 19 [ 0625 [30

Co n-

SARS E qproteie forme sx joa chaxnel

A peptide corresponding to the full-lagth SARS-CoV (isolate Tos2 and . 5 Utbani) B protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLATLTALRLCA

YCCNIVNVSLVEPTVYVYSRVEKNLNSSBGVPDLLV) and a second peptide "comprising the first 40 amino acids of the fill langth RB protein witich correspond to the transmembrane domain (MYSFVSEETGTLIVNSVLLFLAFVVF

LLVTLAILTALRLC) were synthesized manually using FMOC ohernistry sod solid phase peptido synthesis The synthesis was done at the Biomolecular Resource : Facility (John Curtin School of Madical Research, ANU, Australis) using 2

Symphony® Peptide Synthesiser from Protein Technologies Inc.(Tucson, AZ, USA) according to the manmfacturers instroctiona,

Example. Fepfidopurification

Maas spectral analysis of the synthetic poptide revealed that the preperstion contained significant mounts of material with Jowe m/z ratio than expected for the " full-length prodnct. The majority of these are presumably truncated peptides generated during the peptide synthesis process. To enrich the fall-length B protein, the following prooeduro wes waed, which relies on differential solubility of the smaller molecules and full-length peptide. The crude preparation was suspended at 12 mg/ml in 70% CEyCN, 0.19% TFA and vortexed for 10 minutes. This suspension ws centrifuged at 10,000g for 10 minutes at 20°C. The supernatant was discarded and the insolnble fractions was extracted with 70% CB;CN, 0.1% TFA, as above, two more times. Tha insoluble material containing the B protein was dried using

Speedvac en the weight of the final product was used to caloulate the yield. The purified peptide was analysed by Bruker Omniflex MALDI-TOF mass spectrometry in HABA matrix at 2.5mg/m1 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 tha calculated molocular weight of full-length B protein and 4422.3 for the N-terminal

B protein. So

The SARS virus B protein was resuspended at 1mg/ml in 2,2,2-triftuoroethanol. The

SARS virns E protein's shility to form jon channels wes tested on a Warner (Wamer instruments, Enc. 1125 Dixwell Avene, Hamden, CT 06514) bilayer rig as follows; A lipid mix of 3:1:1, 1-Palmitoyl-2-olealyl phosphatidyl Ethenolamine: 1-Palmitoyl-2- oleolyl phosphatidyl Serine: 1-Palmitoyl-2-oleolyl phosphatidyl choline in CHCl : was dried under Nz gas and resuspended to 50mg/ml in n-decane. Bilxyery wees painted across a circular hole of approximately 100m dismeter in a Delrin™ cup separating aqueous solution in the C18 and TRANS chambers. The CIS chamber © 10 contsined a solution of 500mM NaCl or KCl, in a SmM HEPES buffer pH 72, the "TRANS chamber contained a solution of S0mM NaCl or KCI, in a SmM HEPES buffer pH 7.2. Silver eleotrodas coated in chloride with 2% agarose bridges sreplaced in the CIS and TRANS chamber solutions. The SARS B protein full-langth or N- terminal peptides (3-10ug) were added to the CIS chamber, which wes stirred until channel activity was detected. The CIS chamber was carthed and the TRANS chamber was held at various holding potentials ranging between +100 to ~100mV. " Cumaarts were recorded using a Wamer modal BD-525D amplifier, filtered at 1kfiz, sampling at 5 kfiz and digitally recorded on the hard disk of a PC using software developed in house. So "20 Drugs to be tested for their shility to inhibit SARS E protein jon chammel activity were mado up at S0mM in a solution of 50% DMSO: 50% methanol, For oo experiments testing the shillity of compounds to inhibit B protein jon channel activity, 100 1M 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 activity and after the addition of the drug.

Among the compounds tested was cinnamoylguanidine (Bit036), a compound which was shown in esctier expeciments 10 be antiviral and to inhibition channel ~~ 2 Purified E protein was dissotved to 1 tng/eal, § 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 HCI (pH 8.3). Peptides in solutions were heated at 100°C for 20 minutes before

. "30 iL samples were run on stacking gel 4-20% (Gradipare). SeaBine® pro-stained tender (Tvitrogen) was used for molecular weight markers.

Example 20.2 Results

B To teat ifthe SARS B protein forms ion chamels the purified synthetic peptide was reconatituted into planar lipid bilayers (21). Typically, 3pg of SARS full-length B protein was added to the CIS chamber, while stirring. This CIS chamber contained 500 mM NaCl anid fhe TRANS chamber contained 50 mM NaCl. In 60

SE expeciments, ion currents due to SARS E protein ion channel activity ware observed after about 5 15 mites of stirring. Activity was detected moro rapidly and reliably with a holding potential of approximately —100mYV across the bilayer. Currents recarded at —100mV, (A) and at -60mV (B) in one of these experiments are shown in . Figure 6, In that experiment the reversal potantial was about +48mV and the channel : conductances wees calculated to be 52pS and 26pS, respectively. This idicates that the current-voltage (TV) relationship is not near. In ten other experiments, whereno protein was sdded fo tho CIS chamber, no jon channel activity was deteated, sven after recording for over 1 hour.

Pigire 7a shows typical current traces recorded over a range of potantials in NaCl solutions. Fn that experiment the direction of current flow reversed at +48mV (Fig 7h). The IV curve shows that at the lower voltages the sverage current flow across the bilayer is small but at higher potentials there is mm increase in average current actoss the bilayer, resulting in a non-linear IV relationship. In seven independent experiments, the average reversal potential was +483 + 2.3 mV (mean + 1SEM), indicating that the channels were about 37 times more permesblo to Na+ jon than to

CT ions. The reversal potential is close to the Ne+ oquilibrium potential (+53mV), . | 25 thorefore the channel ia selective for Na+ ions. For thee 7 experiments the channel oonductence varied between 95-164 pS; the average conductance was 130 + 13 pS.

SARS E protein lon channel is slighty cas selectivity for K* ions han Na fone. : Figure 8b shows recording of currents in KCl solutions at a range of potatisls. Fa this experiment the currents reversed at +31 mV. In sven similar experiments B protein ion channel average reversal potential was +34.5 & 2.5 mV. Therefore the : SARS E protein jon chammel is about 7.2 times more parmeshle to K* ions than CT

C4 jons. In seven experiments, the channel conductance varied ranging betwoen 24-166 : pS, the averags conductance was 83.4 + 26 pS. "Similar results were obtained with a sooond synthetic peptide, which corresponded to the first forty N-teeminal amino acids of the SARS E protein “N-terminal peptide”

Co S (21). The average reversal potential in NaCl solution in four experiments was +46.3 © $2.5mV, indicating that tha ion channel formed by N-terminal peptids is about 25 ; . times more permeable to Na+ ion than to Cl- ions. The SARS E protein N-terminal poptide was sufficient for the formation of ion channels with properties like those of the full length SARS B fxotein, Therefore, the selectivity filter for the SARS B protein is most likely containad within the first forty amino acids of the N-terminal.

SARS E protein N-terminal peptide also formed jon chermels in KCI solution that wero similarly selective for K+ ions compared to the full-length E protain. In five : independant experiments the average channel reversal potential was +39.5 + 3.6 mV, . therefore the channal is about 11 times more permeable to KX" ions tian CI ions. :

SDS-PAGE ofthe purified full-length E protein peptide showed bands corresponding to the full-length E protein (Data not shown). Larger bands of varying sive up to about 20 kDa were detected, suggesting thet SARS E protein may frm homo- oligomers. gthex sommonnds : : E protein ion channel activity in NaCl sobntions was significantly reduced ~~ . (= 0.01, n=6 experiments) by addition of 100 to 200 pM cinnamoyignanidine to the :

CIS chamber. The average cuirent acroes the bileyer was reduced to baseline by 100uM cinnamoylgnanidine. In experiments when B protein ion channels had higher ‘conductance, 100 to 200 uM cimmamoulgnanidine reduced the average currant across the bilayer sbout 4 fold: Similarly, in four other experiments, 100 to 200 WM cinnamoylgusnidine blocked channels fixrmed by full-length E protein in KCl : solutions. In two additional experiments, the SARS E protein N-terminal peptide was blocked by 100 to 200 pM cimamoylgnanidine, demonstrating that the otmamoyi guanidine drog-binding sits is located within the first forty amino acids of :

: tho B protein N-terminal domain. Other compounds tested in bilayers for their effect on the SARS E protein are shown iri below in Table 6.

Table 6 :

EP

"N,N-bis(3phanyipropanoyl)-N"phenylgnanidine ~~ [8826 FI

Examole 21.1 Boralts snd Discuss y

We have shown that SARS B protein can fem jon charmels in lipid bilayer ) 10 membrana. The ion currents reversed at positive potentials, which demonstrates that E protein jon channels ave selactive for monovalent cations over monovalent anions. . * B protein Ion charmels were aboat 37 times more selective for Na+ ions over Cl-ions and about 7.2 times mare selective for K+ ions over Cl- ions. In over 60 experiments the Na+ condnctance of the E protein ion channel varied from as low as 26 pS to as high as 164 pS. SDS-PAGE showed that the E protein forms homo-cligomers, and wo surmised that the larger conductances were probably due to aggregation of the E protein peptide leading to larger ion charmels or the synchronous opening of many jon channels, Singio channel currects were cbsarved in several experiments and from . these the channel conductance was calculated to be voltago dependent.

The first 40 amino acids of the N-terminal which contain the hydrophobic domain of the SARS virus B protein is sufficient for the formation of fon channels on planar lipid bilayers, Tho N-teeminal B protein ion channel has the seme solootivity snd conductance as the full-length E protein ion channel,

The SARS virus hil length E protein jon channel activity and N-terminal domain B protein ion channel activity on planar Hpid bilayers inNaCl and KCI solutions was inhibited by addition of between 1004 to 200uM cimamoylguanidine to the CIS chamber. Inhibition or partial inhibition of the B protein jon channel activity by cinmamoylgusnidine has been observed in seven independent experiments in NaCl solution and four independent experiments in KCl gohion. _ All known coronaviruses encode an E protsin with a Irydrophobic N-terminus : 5 transmembrane domain therefore all coronaviruses B proteins could form jon chamels an planar lipid bilayers. This indicates that the E protein could be 8 suitable ‘target for antivirel drugs end poientially stop the spread of coronavirus from infected host cells. Drugs that block the B protein jon channel could be effective antiviral therapy for the treatment of several significant human and veterinary coronavirus diseases including SARS and the common cold. .

A bioassay of SARS-CoV E protein fimotion in bacterial cells was developed. A "synthetic cDNA fragment encoding SARS-CoV B protein was cloned into the " expression plasmid pPLAS1, creating a vector in which E protein expression is temperature indncible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protain at 37°C was observed as an indicator of p7 ion channel function dissipating the normal Net+ gradient maintained by the bacterial oclls. . sorepavirss ¥ protein.

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-

Co 0 assay.

Table 7 | oo : SARS E protein

Inhibition

: : : -T7- Co : a di filnorocinnAmMOyiguamain 450/1

EE 3,4~dichlorocimamoylgnanidine 415/2 4-t-butylcinnamoylguanidine 4001 (2 napthyl)acryloylguanidine . 388/1 + f3-Chiorocinnamoyl)guimidine 3.8713 , 3-(oyolohax-1-en-1-yl)cinnemoyignanidine 375/71 i’. dineth Tens tee oylguanidine 363/1 rems-3-(1 Jactyloylguanidine 338/2 timovopylosmamoy guanine 3182 : Bromocimsmoy))guanidine C3157 6-methoxy-2-nsphthoylguanidine 3.135 5-(N-MethykN-isobutyl)amiloride 3.132 : B-phenylcinmamoylguanidine 3.13/1 : / Chlorocinmam oyl) guanidine ’ 3.1/3

R'4 DichloroBenzamil HCl ~~ 3.002

Lo . : -pRenvicinn moylguanidine . . : 2757/2 4-(trifluoromothylcimmamoylguanidine 2751/1 3-(triflucromethoxy)cirmamoylguanidine 2771 3-(trifinoromethycinnamoylgoamidine } 267/71

R-ethoxycinnamoyl guanidin 257171 . ~ hinnamoylgnanidine hydmchlaride ~250/1 © R,6-dichlorocimamoylgumnidine 225/1 3,4, 5-trimathoxycirmamoylgnanidine 225/1 5-tert-butylamino-amiloride 20172 4 DA CINNRAEMNO RT nidin - 2007/1 bromo-2-flucrocinnamoyliguanidine : 1200/1 4-Chlorocinnemoyl)goanidine 1.9472 [2 bntvicionasnov Eman . ' | 1.86/1 ’

D-cyclohexylcinnamoyignanidic . 1.83/1 ) §-Jodosmiloride a 1.7572 :

B-(trans-hopt-1-an- 1 -yl)cinnemoyiguanidine | 1.7171 4-Bromocinnamoyl)gnanidine 1.692 ‘4-Hydroxycinnamoyl)guanidine | 163/2

N-(3-phenylpropanoyl)-N'-phenylgnanidine 15772 :

Nitrociunsmoyl)guanidine 1.5172

B-fluorocinnamoyigoanidine 1.50/1 : 2~(1-napthyl)acctoylguanidine : 1.50/1

R-ethyicimamovi gusnidin '1.50/1 s{N,N-DimethyDamiloride hydrochloride ~~ - 1382 . R-napthoylguanidine 13872 : - p-(4-fluorophenyl)amilotide 1.381

R-(triflnoromsthyf)cinmamoylgnanidine . 1387/1 . N<(6-Hydroxy-2-napthoyl)-N"-phenylgnanidine 135/3 trans-2-Phenyloyclopropsnecarbonyl)gnanidine 134/3 ,N'-bis(3phenyipropanoyl)-N"-phecylgnanidine 13373 1-napthoyignanidine 13273

SE 78-

Benzamil hydrochloride . 1.3272 : 3 methoxy -HMA. ’ 1251 : 4-methyicimnamoylgusnidine 125/1 : 4 fiuorocinnamoylguanidine 125/1 3,4-(methylenedioxy)cinnamoylgumidine 12571 : : 5-(N,N-haxamethylane)amiloride Co 12/3 a

N-(cinnamoyl)-Npheaylgoanidine Le (N-Bthyl-N-isopropyDamiloride 1.0712 ’ IS=rnetm riMEmOVIET midine . 1.00/1 2-methylcinnsmoylgnanidine 1.0041 2.3,5,6,-tetramathylcinnamoyl guanidine 1.00/1 © jrans-3-Furanacryoylgnaniding 0.882 4-Methoxycinnamoyl)goanidine 0.88/2

Furanacryloyl) guanidine 0.822 phenylpropencyl)geanidine Co 0.73/75 . -(2-nspthyl)acetoyiguamidine 0717/1 rinman 0 onanidine = ' ) 0.69/13 ol (2-Methoxycinnamoyl)guanidine : 069/2 (3-PyridyDecryloyl Jgnanidine 0.67/3 h A-dichlorocinnamolyguanidine : 0.63/2

MethoX cionsmoyl)guanidine .063/2 : 2-flucrocinmamoylguanidine 0.63/1 . {4-Fhenoxybenzoyf)gnanidine a. 0.5722 : a-Methylcinnsmoyl)granidine : 0.50/1 5-(3-bromophenryf)pents-2. 4-dienoylgnanidine 05/1

S Phenyl-ponta-2,4-dienoyl)guanidine 0.44 2 : ‘Quinoline-2-carbouyl)gusnidine 041/1 : . {Phenylacctyl)goanidine 032/3 -Bis(amidino)napthalene-2 6-dicarboxamide 0.25 2 5-hromo-2-napthoylgnanidine 0.25/1 1-bromo-2-napthoyignanidine 025/1

D-ohkiro-6-finorocinsemoylgmenidine 0258/1 (4-Chlorophenoxy-acetyl]gnanidine 01972 : Phenxmil methsnosolfonate salt ~~ 0.13/2 : : Banzoyl-N'-cinnamoyligusnidine : 0.13/2

N-(2-napthoyl)- N*-phenylgnanidine 0.0772

SARS coronavirus (SARS-CoV).

S Compounds ware 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 = 1x TCIDso per 100 cells.

oo | 7

Monolayers of Vero calls grown in 25cm? flesks were infested at 2 multiplicity of 1:50 and treated immediately post infection with compounds a two conpentrations, 10uM and 2uM, A control infected monolayer remained untreated.

Samples of culture media were taken at 48 hours post infection. Two aliquots from each of the samples (titrations 1 and 2) were serially log diluted snd 12 replicates of

Jog dilutions -4 to -7 added to cells in microtitre plates. Four days later, wells in the ". microtitre plates were scored for cytopathic effet (CPB) and the titration valuce - calculated based on the number of CPE positive wells at the 4 dilutions. Control : titros were 4.8 and 5.9 TCIDsy x 10° (average 5.35 x 10°) . 10 ’ )

Three selscted cotnpounds were fosted for activity against SARS-CoV sooording to the method described in example 21, For trans-3-(1- napthyDacryloylgnanmidine and cinnamoylgoanidin a decrease in virus titre of . approximately 80% was observed at a conoentration of 10uM and a reduction of approximately 50% was seen to persist at 2M trans-3-(1-napthyDacryloyignanidine.

Table 8 provides Virus titration dats presented as % of 2 control (SARS CoV grown for 48 hours in the absence of compounds).

Table 8 oo oo : Concentration x (%

EE el Mv A 2

EE en me au 2 1 44 | 8

Fer I ee a.” ar :

GuetioyJamttoyigunidine | 10 398 J : | 2 |] 635 | 118 (Control 1 0 [| 535 | 100 oo

A peptide corresponding to the full-length 229E-E protein (sequence: s MFLRKLVDDHALVVNVLLWCVVLIVILLVCITIKLIKLCFTCHMFCNRTVYGPT -

KNVYHIYQSYMHIDPFPKRVIDF; accession mimber NP_073554) was synthesized oo menmally using FMOC chemistry and solid phase peptide synthesis. The synthesis was done at the Biomolecular Resource Facility (Jobn Curtin School of Medical

Research, ANU, Anstralia) zing a Symphony Peptide Synthesiser from Protein

Tecimologios Ins.(Wobum, MS, USA) according to the mamufscturers instractions to give C-terminal amides, the coupling was done with HBTU and hydroxybensotriasole in N-methylpyrrolidone. Each of the synthesis cycles nsed double coupling and a 4-fold excess of the amino acids. Temporary o-N Fmoc-

The cade synthetic peptide was purified using the ProseoPles™ kit (Qbiogene inc.

CA), following mammfhoturos instructions. Briefly, the peptides wero diluted in loading buff (600M Tris-HCl pH 8.3, GM ures, 5% SDS, 10% glycerol, 02%

Bromophenol bius, + 100 mM P-mercaptosthanol) and ran cn 4-20% gradient © polyacrytsmide gels (Gradipors, NSW, Australis) in tris-glycine electrophoresis 20 . buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel cods blue (Promega, NSW) and the bands corresponding to the full-length peptide : were excised ont of the gel. © The gel slice was transferred to the ProtocPLUS™ taibe and filled with tris-gtysine ; . 25 electrophoresis buffer. The tubes were emerged in tris-glycine electrophoresis buffer and mabjected to 100 volts far approximately 1 hour. The polarity of the electric current was reversed for 1 minnte to increase the amount of protein recovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 mime. The purified peptides were dried in a Speedvac and the weight of the final product was used to calculate the yield

So | 81- - = oo Lipid bilayer stadies were performed as doscribed elsewhere (Sunstrom, 1996; "Miller, 1986). A lipid mixture of paimitoyl-oleoyl-phosphatidylethanolsmine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:32) (Avanti Polar Lipids, Alebastcr, Alsbams) was uscd. The lipid mixture was painted onto an aperture of 150-200 pm in the wall of a 1 ml delrin cup. The aperture © separstos two chambers, cis and trans, both containing salt sofutions at different . concentrations. The cis chamber wes connected to ground and the trans chamber to the tput of an Axopatuh 200 amplifier. Normally the cis chamber contained either © 10 500 mM NaCl or 500mM KC! an tho trans 50 mM NaCl or 50mM KCL Tho bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chember with respect to 'tho ois. The synthetic paptidc was added to the cis chamber and stirred until channel . activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Ez and 15. sored on magnetic disk. oo

The 229R B synthetic peptide was dissolved in 2,2,2-trifluorethano] (TFE) at 0.05mg/ml to 1 mg/ml. 10 ul of fis was added to the cis chamber (1m! 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- 30min After channels were detected the holding potential across the bilayer was varied between ~100mV end +100mYV to characterise the size md polarity of current "Sow nd ausble the reversal potential to be determined. i 15 experiments where the cis chamber contained S00mM NaCl solution and the trans chamber coutainsd 50 mM NaCl solution, the average reversal potential of the channel activity was calculated to be 22 +7 (SEM) mV. In 13 experiments

B whero the cis chamber contained S00mM KC 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. Theso results indicate that the 229E E protein forms cation selective ion channels that are slightly mare sclective for X* than for ~~~ 3% Ne‘ioms - } '

. ’ : . i -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 KCl solutions (500/50 mM).

The graph is a roproeantative plot of average bilayer current (pA; y-axis) versus : holding potential (mV; x-axis).

S :

To test compounds for their ability to block or otherwise inhibit the jon * chiel formed by 2298 B protein, small aliquots of solutions containing the compounds wero 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 wars typically propered at 500 mM in DMSO. This oo Bh solution was farther diluted to 50 mM, or lower concentration in 50% DMSO/50% - 15 methanol and 2 pl of the approguiately dilated compound was added to the cis and/or - n the example shown in Figare 10, addition af 100M cinnemoylgnanidine to the cis chember gresily reduced current flow through the 2298 E jon chammel.

Ion Chamncl-Blocking Drugs. ) * A bioassay of 229E-CoV B-protein function in bacterial cells was developed. A synthetic cDNA fragment encoding 229B-CoV E-protein was cloned into the expression plasmid pPLAS], cresting a vector in which B protein expression is temperature inducible, as doscribed in Exaraple 4. Inhibition of the growth of E.coli : cells expressing E protein at 37°C was observed as an indicator of p7 jon channel function dissipating the normal Na+ gradient maintained by the bacterial cells,

. : * -£3- . . . 7 The halos of growth around the site of application of particular drugs — as described in example 14 — were scored as decribed in example 15. . S ) :

Table 9 list the scores for iithibition of 229B-CoV E-protein in the bacterial bio-assay.

Table 9 : 229E E protein

Tnhibition : : : I— (score) -B,4-dichlorocinnamoylguanidine : 4.4 - p~(trifinoromethoxy)cinnamoylgoanidine : 4.1 . -butylcinnamoylguanidine 40

B-isopropylcinnamoylgnanidine hydrochloride 4.0 3=t-DlY Gin MOVIST nidine 3.9 4 » ul CIAO BoaaIns 39 - rans-3-(1-nepthyDacryloylgasnidine 3.7 : .[P=-troma methoxveimmamovigeanidine 3.6 ra . (NINO cinnam OYRSALA dine . 3.3 3 2-naptiyDacryloyl gnanidine 3.0 [2-0 CIN no Tad midin B . 3.0 " . B{cyclohex-1-en-1-yl)cinnamoylgusnidine 24

PCN YIDENZDYIRK amido . 23 . . 3=-Omifinommeiyl )emnsmg guanidine 23 . 4-Phenoxybenzoyl) guanidine 23 4-(trifluoromethyl)cinnamoyignanidine 23 2-(cyclohex-1-en-1yl)cinnamoylguanidine 23 4-Bromocinnamoyl)gusnidine : 20

N-hexamethyleno)amiloride 19 : -napthoylguanidine 19 5-(4-flunorophenyl)amiloride 18

S-Phenyl-penta-2,4-dianoyl) guanidine 1.8

BromocInnamoy puaniamse ) 1.7 2,5-dimethylcirmamoylgusnidine 1.6 2-(trifluoromethyl)cinmamoyignanidine 1.5 6 mothoxy-2-naphthoylgusnidine 14 4-Chlarocinnemoyl)gnanidine 14 , -Methoxycinnamoy{) guanidine 1.4 :

S-hromo-2-flnorocinnamoylgnanidine ’ 14 ‘ 5-(N,N-Dimethyfjamilodids hydrochloride 13 cinnamoyiguanidin 13 -Meathoxycinnemoyl)gnanidine 11 »-Methylcinnamoyl)gnanidine 1.0 : 4-phenylcinnamoylguanidine oo 1.0

Bromocinnamoyl)guanidins 0.9 B.4,6-trimethykinnamoylguanidine 09 ) trans-2-Phenyicyalopropanecarbonyl) guanidine 0.8 3.Chlorocinnamoyf)guanidine 0.8 2-(1-napthyDacetoylguanidine 08 2-ethylcinnamoylguanidine 0.8 : 2-cyclohexylcinmamoylguanidine 0.8 4-Hydroxyoinnermoyl)gusnidine 06 : h-ethoxycininamoylgnanidine 0.6 : . [4 methylcinnamoylgnanidine 0.35 2-methylcinnamoylgusnidine * - 0.5 3-flaorocirmsmoyignsnidine 0.5 2,3-dimethykinnamoylgusvidine 0.5 2-finorocinnemoyigoanidine 04 : finorocimmamoylgusnidine 04 : 3,4-difinorocinnamoylignenidine 04 .|5-tect-butylamino-amiloride : 03 ’ 2-napthoylgnanidine 0.3 'N'-Bis(smidinonepthalane-2,6-dlcarboxamide 0.3

IN,N'-Bis(3-phenylpropanoyl)guanidine - 03 4-methyloinnamoyigusnidine 03 : "bromophenyl)penta-2,4-dienoyl guanidine 03 23,5,6,-tetramethylcinnamoylgnanidine 0.3 3. sthoxyveinnamoyl goemidine : 0.3

IN. N'-bis(3phexryipropanoyl)-N"-phonylgaanidine 0.1 :

A-Methoxycinnamoyl)guanidine 01 + (2-Chlorocinnamoyf)guanidine . ol : Nitrocimmamoy) guanidine . 01 4-ethoxycimmamoyiguanidine 0.1 3,4, 5-trimethoxycirmsmoylgnanidine 0.1. :

Co 2-(2-napthyl)acetoylguanidine : 0.1

N-(3-phenylpropanoyl)-N'-phenyiguamidne 01

Lo 8 :

To determine the antiviral activity of compounds against human coronavirus 229 replication (ATCC VR-740), an assay measuring reduction in tho member of

Co 85- : plaques firmed in monolayers of 229E infected MRC-S cells (lumen lung fibroblasts oo

Co JATCC CCL-171) was developed: 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 exposure to the virus at an MOI : 5 of approx. 0.01 pfi/cell for 1 hour at 35°C in SHCOs. The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf scrum) containing various test concentrations of contpounds or the appropriate level of solvent used for the corapounds (control). Plates were subsequently ircubated at . 35°C (in 5% CO) for 3 —5 days post infection, after which time culture supernatant was rémoved and he calls were stained with 0.1% crystal violet solution in 20% : a "7 ethanol for 10 minutos. Plaques wero counted in all wells and the percentage reduction in plaque number compared to solvent control was calculsted. : Measurements were pecformad in duplicate to quadraplicate wells.

Table10 a oo

Plague Reduction (Y%control / # 2-t-butylcinnamoyiguanidine 100/1 100/3 050/3 4-isopropylcinnemoyignenidine 100/1 100/2 057/2 . 3 4-dichlorocinnemoyigaanidine 100/3 099/4 086/3 trifluoromethoxy)cimnamoylgnaniding 100 /2 098/4 077/3 : h 6-dichlorocinnemoylgnanidine 100/1. | 097/3 066/2

R2-{cyclohex-1-an- 1 yDeinnamoylguanidine 100/1 097/3 021/1 2 -cvolohexyicimmamo guanidine 070/1 - 09712 089/2" . ETN cing amoylguanidine 100/2 09/4 088/3 .

Co 2-phenylcimmamoylguanidine 1600/1 096/3 100/1 5-(2'-bromophenyl)penin-2,4- . |ienoylguenidine Co 100/2 | 1095/3 079/2 4-t-butyloinnamoylgnanidine 100/1 | 095/3 084/3 } } 3-phex cinnamoylgusnidine } 094/3 0777/2 . i BrOInNOCINMAMO }gu mame 100/2 093/3 072/12 4-Bromocinnamoyl)guanidine 0954/1 091/3 0737/2 : 5-(N,N-hexamethylene)amilorids 089/ 091/2 033/1

Tan -napthylacryloylgusnidine | 100/1 .091/2 064/2

3-(2-napthyl)acryloylguanidine 100/1 {| 091/2 062/2

D 4-dichlorocinnamolygusnidine 100/2 090/4 064/3

Nitrocinnamoyl)guanidine 085/2 090/2 046/72 [3 : triflucromethyl)cimnamoylguanidine | 097/2 089/4 06473 hromo-2-fhiorocinnemoylgnanidine { 100/1 088/3 063/2

Lrmethvicimmamoylguanidine 091/2 087/4 0683/2 :

Olly: nCInEY 9) guanidine 100/1 086/3 009/1 4-Mathoxycinnamoyl)gnanidine 100/1 085/4 057/3 4-Chloroctonamoyl)gusnidive 100/2 084/2 051/2 : 3-fluorocinnamoyl guanidine .095/1 083/3 051/2

B{oyclohex-1-en-1- a vI)cinnamoylgnanidine 100/1 082/3 063 12 © (a-Methylcinnamoy{)guanidine 023/1 082/1 036/2 4 3:0 . : etramethyicinnamoyignanidine 098 /2 079/4 064/3

D-finorocinnamoylgnanidine 090/1 079/73 0457/2 triftuoromethylcinnamoylgusnidine | 100/1 79/1 | 0582/1

NitrocinmamoyT)goanidine 100/1 79/1 045/1

DR 5-dimethyloimnemoylgusnidine 1 092/72 078/1. 078/1 . 2 IX CINNENOYISATLIOING 100/1 077/74 = 030/3 -Mathoxycinnsmoyl)gnanidine 089/1 | 075/2 30/1 3.methyicinnamoyignanidine 095/1 074/3 044/1

Promremmepeei®on | goiy | oss | own . 12-Rromocint MOV EREMQINS 095/2 072/2 043/2 . [5-GLIOXY CII tee mamidine 100/1 0727/3 0571/1 5.Phenyl-ponta-2 A-dicnoyl)guanidinel 100/1 {| 072/2 | 069/1

Chlorocinnamoyi)gusnidine 095/2 072/2 040/2 sthoxycinnemoyionanidine 0731/1 0689/2 05771 finorocinnamoyiguanidine 100/1 067/3 034/2 3, 4-difluorocirmemoyigonanidine 085/1 0565/3 | O042/2 : : shenvinropanayi}-N'- shenvignaniding 051/1 064/1 000/1 : 2 A,6-trimethyleinnamoylguanidine 075/2 063/3 062/2

R-methyloinmamoylgnanidine 074/2 063/3 053/3 . trans-2-

Phenyloyolopropanecarbonyl)- : manidine : 063/2 022/1 (B)-3-(4-Dimethylaminophenyl)-2- : methytacryloyl]gnanidine : 0389/1

Benzoyl-N'-cinnamoyigusnidine 056/1 -phens benzoylgnanidine } . 076/1 055/2 0711/1 rans-3-Furtnacryoylguanidine 055/2 018/1 4-Phenoxybenzoyl)gnanidine 069/1 054/3 040/2 :

Methoxycimamoyl) guanidine 051/1 053/2 024/1

N-axridino-3-emino-S-phenyl-6- 074/2 052/2 038/1 vallege 2 . cinnsmoyf)-N'phenyignanidine 084 /1 048/2 035/1 sinnamoylgnenidine 095/2 047/2 059/1 3,4- , motirylenedioxy)cinnamoylgranidine 084/1 046/1 019/1

N.N'-Bis(amidino)napthalece-2,6- : : dicarboxamide : 045/1 . [4 dimethy sInamoyigy midin 073/1 044/2 024/1 : 5:(3-bromophenyl)perta-2,4-

Hen “a mid [10 - 044/1 © N,N-Bis(3-- I ’ nhenvipropanovi)gu midine 041/1 : 3-methoxv-amiloride 029/2 039/3 022/2 ’. diflnorocinnameo gusnidine 036/1 . 1-napthoylgnanidine 036/1 :

PRCIYIDTOPRIN goanidine 036/1 : } _ [p-methoxy-2-naphthoylgnenidine 49/3 030/4 wdrochloride : 0227/1

D-cthoxycinnamoylguanidine 027/1 - D-napthoviguenidine : 027/1- ’ 3.4 rmnsSoxvemaamo FORCING 027/71 : - B-methoxy -HMA 1 027/71

Deozyoyignarisine 026/1 trifluoromethyl )cinnamoylguanidine 2/1 iding.3 S-diamino-6.phynyl-2-

Quinoline-2-carhonyl)guanidine 015/3 0195/3 | 006/2 ne 4-Hydroxycinnamoyl)gnanidine 019/1 a : 5-(4-fluorophenylamiloride 018/1

D-(1-napthyDacetoylguanidine 018/1. -Fursnacryloyl)gnamidine 018/1 3-(3-Pyridyfacryloyf]gnanidine 018/1

IN-Cinnamoyl-N',N- : pimethylgaanidine 015/1 -(2-napthoy!)-N'-phenylguanidine 011/1 2-2-capthylaoctoyignamicine, : Co 009/1 : phenyignanidine : 009/1 (Phentylacetyguanidine . _009/1 s : :

Example 32 Human OC43 Coronavires ’ : soronsvirus C43. :

To determine the antiviral activity of ocempounds against rnin coronavirus

C43 roplisstion (ATCC VR-759), an ELISA assay was doveloped measuring the roloase of the viral N-protein into culture supernatants from monolayers of OC43- © infacted MRC-S cells (human hg fibroblasts ;ATCC CCL-171); First, a virus working stock wes prepared by amplification in MRC-S cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by "10 expose to the virus at an MOI of approx. 0.01 pfu/oell for 1 hour at 35°C in S%CO;. © The infective inoculum was recoved and replaced with fresh madium (DMEM supplemented with 10% fetal calf serum) containing various test comoentrations of "compounds or the spproprixte lovel of solvent used for tho compounds (control). " Plaics were subscquontly incubated at 35°C (in 5% CO) for 5 days post infection, after which time culture supernatant was harvested and cellular debris removed by : oentrifogation st 5000 x g for 10 mimutes. For N-antigen detection, 100i] samples of clarified culture supernatent were added to dupliets wells of & 96-well Maxi-Sorb plats; 100p1 Gf RIPA buffer was added par well with mixing and the plate was covered and incubated at 4°C overnight to enshle protein binding to the plastic wells.

The next day, the costing solution was discarded, weils were washed thoroughly with . PBST, snd blocking of unoccupicd protein binding sites was performed by incubation - in 1% BSA in PBS fir 1.5 hours. Tho antibody recognising OC43 N-protein was : used at 1/800 dilution in PBS (1kr at 37°C) and the secondary antibody (goat-anti- mone alkaline phosphatase) was used for the colour development reaction. Optical density of the wells was read af 405 mm 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. : 30 .

Cotupounds were screened thr activity against OC43 replication according to the method described in cxsmple 22. Results aro shown in Table 1.

oo Table 11 : -

EN ER hall

A DHIDOY 2.5sM ’ * ’ 3.methylcinnamoyignanidine 1 100 trans-3-(1-napthyDacryloylguanidine 100 -Bromocimamoyl)guanidine 100 ’ : Chiorocimmamo T)gnanidine 96 ! 3,4 dichlorocint yig i . 20 3-(trifluoremetirylcinnamoylgoanidine ‘84 ; trans-2-Pheinyloyolopropanocarbonyl) guanidine nn : 4-isopropyleimmamoyignanidine 68 cinmamo marnidine ’ 57 oo . Co - 5-methoxy-2-naphthoylguanidine 47 _. 1 4-dichicrooirman plyguanidine . 36 - 4-Chlorocinnamoyl)guanidine : 36 5-(N,N-haxamethylens)amilocide 30 (4-Bromocinnamoyi)guanidine 29 : .2,6-dichlorocimamoylgnanidine 1 27 tromo-2-methoxycirnemoylgammidine ‘ 24

S-Phenyl-pents-2 4-dienoyl)gnanidine - 9 [4 wi fing DIRDOXY Namo NANIIng 4 . 2-t-butyicinnanoyigu radia 4 . 5 - :

A poptide ocxresponding to the full-length MHV-AS9 E protein (sequence: : 10 MPNLFLTDTVWYVGQIFIFAVCLMVTIIVVAFLASIKLCIOLCGLCNTL

VLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDL 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 Curtin School of

Modioal Resoarch, ANU, Australis) uaing 8 Symphony Peptide Synthesiser Som

Protein Technologies Inc.(Woburn, MS, USA) according to the manufacturers ~ - _ instructions to give C-terminal amides, the coupling was done with HBTU and hydroxybenzotriszolo in N-methyipyrrolidono. Bach of the synthesis cycles used double coupling and a 4-fold excess of the amino acids. Temporary a-N Fmoo- protecting groups were removed using 20% piperidine in DMF. :

The orads synthe peptide was purified using the ProteaPIus™ kit (Qbiogene inc.

CA), following memmfactaros instructions. Briefly, the peptides were diluted in loading buffer (60cM Tris-HCI pH 8.3, GM ures, 5% SDS, 10% glycerol, 0.2%

Bromophanol bins, 100 aM B-mercaptosthancl) end run on 4-20% gradient polyacrylamide gels (Gradipore, NSW, Australis) in tris-giycine electrophioresls : © puffer (25 mM Tes, 250 mM glycine, 0.1% SDS). The peptides were stained with gel ood luo (Prommoga, NSW) a the bands correspanding 0 the full length peptide were excised out of the gel. Co : : The gol slice was transferred to. the ProtecPLUS™ tube snd filled with tris-

Jiyvine eleciropharesis buffer. The tubes waro emarged in tris-glyeine electrophoresis oo buffer and subjected to 100 volts for approximately 1 hour. The polarity of tho electric cutrent was reversed for 1 minuto to increase the amount of protein zecovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 mimte.

The purified peptides were dried in a Speedvac and tho weight of the final product was used to calculate the yield : : © Lipid bilayer stucfies were perfhrmed as described elsowhere (Sunstrom, 1996;

Miller, 1986). A lipid mixturo of palmitoy-oleoyl-phosphatidylethanolamine, - palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine

Co (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) wes used. The pid mixture was painted cuto an aperture of 150-200 jum n the wall of a | m! delrin cup. The aperture : separates two chambers, cis and trans, both containing selt solutions at different Co concentrations. The cis chamber was conneoted to ground and the trans chamber to © 25 fhe input of an Axopatoh 200 suplifier. Normally the cis chamber contained cither 500 mM NaCl or 500mM KC] and the trans 50 mM NaCl or 50mM KCL The bilayer formation was monitored electrically by the amplitnde of the current pulse goncratod by a current ramp. The potentials were measured in the trans chexnber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents ware filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk. i o :

The MEV E sythefio poptide was dissolved in 2,2,2-triffuorethsnol (TFE) at © 0.05mg/ml to 1 mg/mL. 10 ul of this was added to the cis chamber (Lm! 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- 30min Afer channels wees detocted the holiing potential across the bilayer was : - varied between ~100mYV and +100mV to charscteriso the size sud polarity of current © flow and enable the reversal potential to be determined.

I 14 experiments where the cis chamber contained 500mM NaCl solution and fhe trans chamber conteined 50 mM NaCl solution, the average reversal potential of the channel activity was calculated to bo 49 11 (SEM) mV. In‘11 experiments where tho cis chamber contained 500mM KCl solution and the trans chamber . contained 50 mM KCl solution, the average reversal potential of the channel activity was calculsted to be 13 +6 (SEM) mV. These results indicate that the MEV Eprotein forms cation selective ion channels that are mare selective for Na than for K* ions. : iS Figure 11 shows examples of raw curreat data for the MEV B ion chammel at various holding potentials (cis relative to trans) in asymmetzical NaCl solutions (500/50 mM). The graph ia a representative plot of average bilayer current (PA; y- axis) versus holding potential (mV; x-axis). protein svathetic pentide.

To test compounds for their ability to block or otherwise inhibit the jon * channal formed by MHV E protein, small aliquots of solutions containing the compounds were added to the aqueous solutions bathing plenar lipids in which the poptide chanel 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 sotuticn was further diluted 10 50M, or lower concentration fn $0% DMSO/50% methanol and 2 pl of the appropriately diluted compound wes added to the cis and/or trans chambers to yield the desired final conoentration.

In the example shown in Figure 12 below, addition of 100uM cirmamoylgnanidine to the cis chamber greatly reduced current flow through the . MHVEionchmnel : og | E

A bio-assay of MEV E-protain function in bacterial cells was developed. A synthetic cDNA fragment encoding MEV E-protein was cloned into the expreasion plasmid : _ pPLAS), creating a vector in which B protein expression is temperature inducible, 43 described in Example 4. Inhibition of the growth of E.coli cells expressing Eprotein ~~ at 37°C waa observed 2s an indicator of 7 om channel fimetion dissipating the _ normal Na+ gradient maintained by the bacterial cells. oo

Rrwisin 2

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 MHV E protein in the bacterial bio-assay.

Table 12

MHYV E protein

Tohibiti

Om oRnda (score) . isopropylcimamoylguanidine 45 3-isopropylcimnamoylguanidine hydrochloride 42 4-t-butylcinnamoyl guanidine 4.1

B-(trifluoromethoxy)cinnamoyl guanidine 41 3-t-butylcinnamoyiguanidine 4.0 3,4-dichlorocinnamoyignenidine 38 2,3-diffluorochmamoylguanidine 38

R-t-butylcimamoylguanidine 38 3-500 CINNRAMO ROSIN 3.7 2-phenylcinnamoyiguanidine 34 $-bromo-2-methoxycinmsmoylgnanidine 3.3 2-{cyclohex-1-en-1yl)cinnamoyignanidine 33

"© B(trifinoromethyl)cinnamoyiguanidine 29 -(cyclohex-1-e-1-yl)oinmamoylguanidine 2.9 rane-3-(1-neptiyscryloylguanidine 28 : d-(trifinoromethyl)cinmamoylguanidine 2.8 3 nanthvilacrvioylgnanidine } 2.8 . : ’ trifluoromethyl )cimmamoylgeanidine 2.7 a 4-Phenoxybenzoyl) guanidine _ 24 3-Bromocinnamoyl)gusnidine : 2A ’. gimeth cinmamoylguanidine - i ’ 23 : . ’ 5-bromo-2-flnorocinnamoylguanidine 2.1 . |6-methoxy-2-naphthoylgnanidine 1.8 4-phenylbenzoylguanidine : 1.8 : 4-Bromocinnamoyl)guanidine 1.8 -nspthoylgeanidine =. 1.7

So S-Phenyl-penta-2,4-dicnoyl)guanidine i. : 14

Bromocinnsmoyf)guanidine 14° : 4-Chlorocinnemoyl)gnanidine 1.3 2-tethyicmnamovigoataing . 1.2 2 6-dichlc OCIINAINOY RU mndine 1.2 : 2,4,6-trimethylcimmamoyignanidine 1.2 5- hexamethylene)amiloride 11 cinnamoylgnanidine hydrochloride 11 a-Mathylcinnamoyl)gnanidine 1.0 [4 meth VICIInNamon PRaniaIne 1.0 : ’ phexyicinn moylguanidine 09

N-(3-phexryipropanoyl)- N-phenylguanidine 0.8

IN,N'™-bis(3phenyipropanoy])-N"-phenylguanidine 0.8

Med . ) idine: 0.8 ’

Methoxycinnsmoyl)gnanidine 0.8 p-floorocinnemoyigoanidine 0.8 : 2-finorocinnamoylgnanidine 08 } 2,A-dichlorocinnamatygnani fine 0.8 2-ethylcinnamoylignanidine 0.8 : + {Z2-Chlorocimamo guanidine 07 To. 4-Hydroxycimmamoyl)guanidine 0.7 : 2-ethoxycinnamoyiguanidine 0.7

R-napthoylgnanidine 0.6 : trans-2-Phenyloyclopropenecarbonyl)guanidine : 0.6 5-(N,N-Dimethyl)amiloride hydrochloride 05 4-fluorophenyfamiloride 0.5 3-mothylcirnamoyiguanidine 0.5 :

Lalo DCI moy{)gnanidine 04 4 mothylcinnamoylgnanidin 04 sthoxycinnamoyignanidine “04 2-(1-napthyl)acetoyigusnidine 04

© B,A-difluorocinnsmoylgusmidine 04 R2-napthyfacetoylguanidine 0.4

SE 2,3,5,6,tetrmmethyloinnamoylguanidine 04

Co 4-Msthoxycinnamoyl)guanidine 0.3. : 4 4 ‘meth anadio nn moylguanidine i . 0.3 3-ethoxyeinnemovige niding . 03 ’ 4-fluorocinnamoyl guanidine 02 . B-tert-butviamino-anmiionas . : . : 3-Nitrocinnamoyl) guanidine 0.1 3,4,5-trimethoxycinnamoylgnanidine 0.1 ’ 5-(3'-brompophen yl)penta-2.4 dienc ylgns nidm . 0.1

To determine the sntiviral activity of compounds against MHV replication : (strain MHV-A59: ATCC VR-764), an assay mossuring rednction in the number of plaques formed tn monolayers of MEV infected L929 cells (ATCC CCL) was developed: First, a virus working stock was prepared by smplification in NCTC clone 1469 calls (ATCC CCL-5.1). This was then used to infect confluent monolayers of L929 oclls grown in 6-well tissus culture plates by exposure to the "vias st an MOI of 0.01 pf/ool] or 1 pfiv/oell for 30 minutes at 37°C in SHCO;. The supplemented with 10% horse scrum) 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% CO) for 16 — 24 hours post stained with 0.1% crystal violet solution in 20% ethanol for 10 mites. Plaques were counted in all wells and the percentage reduction in plaque number compared to . solvent control was calculated. Measarements were performed in duplicato to quadruplicate wells. :

Table 13 provides the results obtained from this stody. p

. Table1d : : . | Percent reduction in Plaque

Compemd 200M | 10uM | Tum © lsbromo-2-methoxycinnamoylguenidine ~~ [ND 00/1 l66/1 © boheaylcinmsmoylguanidine oxic [86/2 64/3 : 2 3-diffluorocinnamoylgnanidine oxic 92/3 [64/2 3 . B-cthoxychmamoylguanidir 100/1 [89/2 58/1

Ck tromopheaylpents 2 A-dienoytgmnidine mic [100/71 $7/1 ne gusnidine hydrochloride CBs 2/2 "p6/1 hlccocinnamoy)goaidin bs/2 lgss3 Is3/3 cinmamovyignanidine 07/8 88/8 52/7 4-Bromocinnamoyl) guanidine | oxic/2 [p8/3 3.

Bromocinnamoyf)gnanidine 1/2 [89/3 [52/3 4-Methoxycimamoyl)granidine 98/4 P6/4 [51/3 - » MethylinnamoyDguanidioe B12 psr3 p12 3 4-dicklorocionamoylgaanidios bua ben bor2 hb (oyolobin-l-eo-lyl)cimmemoylgomidine ~~ ND B7/1 sors B-difluorocinnamoylgusnidine [Toxic § 2 0/1 : 3.t-butylinnamoylgusnidine oxic [94/3 ®I12 2-ethoxyinnamoygnanidine 93/2 85/3 8/2 rans 3-Pursnacryoylguanidine 0/1 [65/1 /1

Syontitioois donb fstuplgimaigioes. 84/1 /2 Nn

Nitrocimmamoyl)guanidine 07/1 12 7/1 " -(finorometyl)oimamoylguanidine ~~ 97/3 PS /3 3 ba stytonetionyirmemoy lgousidine 93/3 82/3 WS/3

Ts Methyl N-isobutyDermilaride Co I /1 /2 '(4-Chlorocimamoyl)gusnidine 5 2 2 3/3 b 4 dichlorosinnamolyguanidine nen /n /1 ©. N«3-phenytpropanoyf)-N'-phenylgnanidine 80/1 /1 /1 trocitmemoyl)guanidine ) 05 / 2 13 13 . h-chenvicimamoylgnanidine N/D 00/1 /1 + sopropylcimamoylgnaniding CTE YER VER -(trifimoromethoxy)cinnamoylgnanidine 00/1 90/3 1/2 oo 3-((rifinoromethyl)cimemoylguanidin 8/1 3/1 /1 4-Nitrocinnamoyl)guanidine oo O11 [15/3 [40/3 ¢ 2 negthyDaceyloylganidine oo 03 /1 Nn /1 {totmonyeimmaoylgnanidine be nolan lon h 6-dichlorooinnamoyignanidine O1/1 10/1 11 oo ’ fimathylion moylgamidine 95/3 1/3 9/3

Bromocimamoyi)guanidine 05/2 90/3 9/3

Chiorocinnamoyf)gnanidine bas 2 PB9/2 on 3 mothylcimamoylguanidine | 00/1 88/1 PpB9/1

Methoxycinnamoyl)guanidine 02/2 g7/2 [1/3 bit cinpamoylguunidine ND 98/2 BT/1 2) (+ Dinetslemogheny)2- 611 sin pit

N,N'-bis(1-napthoyl)gusmidine I" 3 methoxy EMA 15/1 P1/1 Psi 5. tert-butylamino-amilaride lose lara fare oo rang-3-(1-nepthylacryloylgoenidine 5/2 [86/3 34/3 ) mathvscy-J-aaphtboylgaanidine 83/3 [56/3 [34/3 ’ uapthoylguanidine 57/2 © B6/2 | 4/2 Potiyloinmamoylguanidine 06/1 BL/2 [4/1 2,3-dimethylcinnamo smaniding 9s / | 5/2 [Berl -Clrmamoy}-NN"diphenylgaanidine 57/1 riz Best 7 isopropykcinnamoylgumnidine hydrochloride | [ND 99/2 B2/1 (+-PhencxybenzoyDguanidine. nls pan trans-2-PhenyloyolopropmecarbonyDguanidine [77/2 [64/2 B1/2 :

B p-loorocimenoyignanidine w/1 siz prin 5 hromeo-2- fluorocinnamoylgnanidine ode 81/2 P1717

N,N"-bis-(cinmamoyf)- N"-pherrylgnamidine 16/1 [8/2 B1/1 ' 'p-quinolinoylguanidine | 27/1 [36/2 Bo/1 2.48 wimothylsinnamoylguanidine 51/2 61/3 p12 oo -98- oo 1-bromo-2-aagthoylgasniding IVI 2 bin

Pamition 3,5 damino-6 hyn 2- | 11 per2 psn

N-Ginnamoyl-N'N'-dimethylgnanidine sa sia paz

Methrycinosmay godine 90/2 B5/2 2 12 b(2-napthylecetoylgnanidine | ls271 hora 3/1 t phenylcimamoylgnanidine 53/1 6/1 P1/3 (3-(3-Pyridylacryloyl]guanidine | g1/2 [13/2 p12 3,4, 5-trimethoxycinnamoyl guanidine barn Barn pn mefhykcimamoyl guanidine 03/1 [9/1 Ro/1 foorocimamoylgomidine 86/1 [83/1 p0/1 6-bromo-2-napthoylgnanidine 55/1 pr/2 19/1

Dimethylamikrido hydrochloride ~~ 42/4 [1/4 [11/4

Phenyl-penta-2, A-diahoyl)ghanidine 7/1 PAIL 17/1 bcyclohexylcinnamoylgnsnidine ~ hoost harz fern 5 (4-foorophenyl)amilorids /1 Pps/1 hen

Benzyoylguanidine 2/1 Po/2 [14/1 © N-Bacizoyl-N'cimamoyiguanidine 0/1 rr j4r1 (NN-hexamethylene)amiloride 84/2 89/1 3/2 © IN<(cirmamoy)}N'phoaylguanidine Cn ls [i 4-Hydroxycinnamoylguanidine 191 hs fn bo ctiftuorometiycimamoylgnamidine Wo /1 srt past oD

Quiooline2-catvanyDpusaiding gs/1 pal 12/1 1 napthylecetoylguanidine 19/1 2/1 jus : -chloro-6-finorocinnamoylguanidine 100/1 [4/2 P/1 [elo me Sha oiood: yt poz BIL ¢phenylbenzoylguamidine ~~ - 2/1 bar [S11

NN-bis(2-nspthoyDguenidine bin plz pi ecylootyDguanidine hs/1 mann Bit © honaphoylgnmidine Co 13 2/3 pI3 oo

Co bisC3phenyipropanoyl)-N'-phenylgnanidine [67/3 0/4 [1/3 hs todo S haxmmotyleimino- amiloride [16/1 3/2 [1/1 2% DichloroBenzamil HCI 2/2 pr p13 oo © hase-ttametykimanoyiguanidine [ND 8/2" pi cosa hydrochloride 0/1 pI pi 7 llodoumilorias hs/1 pin pri

NN" Bis(amidino)nspthalens 2, 6-dcarbexamide 19/1 [16/1 0/1 4-Chlorophencay-soetyllgoanidine 19/1 jie/1 pn (3-phenytpropsnoytygnanidine s1/1 [03/1 U1 h- fiacrocionamoylguanidine | 6/1 [B/1 bromo mipboylgomidine. | 3/1 -Furanacryloyl)guanidine 57/2 [63/2 3/2 oo N(6-Hydroxy-2-napthoyl)-N'-phemylgumnidine. 43/1 B9/1 5/1 ; amiloride HCI | p1/1 [18/1 ksi g . b-(trans-hept-1-en-1-yl)cinnamoyiguanidine /1 Ram/1 6/1 :

S-=1TReEN00 amiloride 60/2 47/3 1712 . © INN-Bis(3-phenylpropencyD guanidine s1/3 P0/4 |873 } 3 cyclohex-1-en-1-yl)cinmamoyiguanidine AN 2/2 ‘ 19/1

SER To determine the antiviral activity of compounds against porcine respiratory : oaronsvirus rephication (ATCC VR-2384), mn assty measuring reduction in the rmmiber of plaques formed in monolayers of PRCV infected ST cells (procine fetal teatis ocll line, ATCC CRL-1746) was developed: Confluent ST cells in 6 well plates were infected with a quaternary passage of porcine respiratory virus (PRCV) : strain AR310 at throe dilutions 10”, 50" and 10 in PBS to provide s range of plaques nombers to count. 100ul of diluted virus was added per-well in a volume of {mi of media Plates were incubated for one hour on a rocking platform at room ©. temperature to allow viras to adsorb to cells. The viral supernatant was removed and 2mlfwell of ovedsy ocntaining 1% Scaplaque agarose in 1x 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/scivent would be added to the overlay for each concentration of } compound. The volums of compotnd/sclvent never exceeded 0.07% of the volume ofthe 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, 0.1aM, 1uM, 10uM and 20uM. Rither duplicates ot

Co quadraplicates wate pecformed at each concentration. Controls were performed :

: where the same volume of solvent wes added to the overlay. The overlsy was : allowed to set st room temp for 20 mins. The plates were then incubated at 37°C for 2days. The monolayers were then fixed and stained overnight at 100m tentpecsiure . by aiding Lmiwell of 0.5% methylens blue, 4% formaldetryde. Overlay sgerose and "5 stgin was then rinsed off to visualize stained and fixed monolayer

Compounds were screcncd for activity against PRCV replication according to tho method described in example 29, Table 14 provides BC50 values for somo tested

Table 14 :

Er RR I

Rope) phage

Err RE [CPomammobpedme | uz

Es Poeeepopesontonyhgids | 112

Ct I NR

Tot | 16 : ,

To determine the sutiviral activity of compounds against bovine coronsvirus replication (ATCC VR-874), xn asesy measuring reduction in the umber of plaques formed in monolayers of BCV infected MDEK oells (bovine kidney cell line ;ATCC

CCL-22) was developed: Confluent MDEK cells in 6 well plates wers infectod with a secondary passage of BCV with serially diluted virus diluted to 10°, 5° and 10 in

PBS to provide 2 range of plaques mumbers to cout. 100ul of diluted virus was added per well in a volume of 1m of media. Plates were incubated for one hr to allow viros bo adaceb fo cells. The viral supemstant was removed arid Zmliwell of : overlay containing 1% Seeplique agarose in 1x MEM, 5% FCS was added to each well. Compounds to be tested wero added to the overlay mixture by dituting the compounds frm a 0.5M frazen stock to a concentration so that the sams volume of oo compound/solvent would be added to the oveclay for’ each concentration of compound. The volume of compound/solvent never exceeded 0.07% of the volume of the overlay. The solvent used to dissolve compaunds was DMSO smd miothanol mixed in equal proportions. Compounds were tested for antl-plaque forming activity mt four concentrations, 0.1uM, 1uM, 10uM and 20uM. - Quadruplicates were © pecfiormed at vach concentration, Controls were performed where the same volume : of solvent was added to the overiny. The overlay was allowed to set at room temp for mins. The plates were then incubated at 37°C for 7 days. The monolayers were oo thea fixed snd stained by adding 1mlwell of 0.5% methylene blue, 4% : 15 female. | E

Exammle 44: Effcet of compounds in BCV gativirs] apsay . Compounds were screened thr activity against BCV replication acoording to the method described in example 31. Table 15 provides BCS0 values for some tested . . 20 compounds ‘ ce . [Cro ———

Hifuommclylcimanoylgaaniine | 3

Cr rN a

Eo I I ana prog | ©

Co [Comoyigeifine | 42 (Er A

ME ER WL

SC

[Fphmybomoylguamidine | 190

SE | 10% | oo

Co is Hepatita C V1 : A peptide mimicking the protein B7 encoded by fhe hepatitis C virus (HCV) was synthesised having the following amino acid soquence: .

ALENLVI-NAASLAGTHGLVSFLVFFCPAWYLEGRWVPGAVYAFYGMWPLL _ LLLLALPQRAYA -

Lipid bilayer studios wee peciad sa desaribed elsswhors (Mill, 1986). & tipid mixture of palmitoyl-olsoyl phophatidylethanolaming, palmitoyl-oleoyl- phosphatidylserine and palmitoyl-oleoyl-phosphatidyloholine (5:3:2) (Avautl Polar _ Lipids, Alabaster, Alabama) was used. The lipid mixture was painted oto an aperiure of 150-200 wn in the wall of a 1 m1 delrin cup. The aperture separates two chambers, ’ cis and trans, both containing salt solutions st diffrent concentrations. The cis chambéc was cornsoind to ground and the trans chamber to the input of sn Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KC1 and the trans 50 : mM ECL. The bilayer formation was monitored electrically by the amplitude of the cutrect pulse genseated by 8 current ramp. The potentials were measured in the rans chamber with respect $0 tho cis. The protein was added to the cis chamber and stirod : until channel activity was seen. The currents were filtered at 1000 Hz, digitized st 2000 Hz and stored on magnetic disk. The P7 peptide was disscived in 2.2.2 © trifiuorsfhanoi (TFE) at 10mg/nl. 10 ul of fils wes added to tho cis chamber of the bilayer which was stirred. Channel activity was seen within 15-20 nin.

When the P7 peptide wes added to tho cis chamber and stirred, channel activity was recorded. The potential in the trans chamber was -80 tV and the currents are downwards, The currents reversed at +50 mV close to the potassium equilibcum potential in these sohtions indicating that the channels were cation-sclective. The amgplituds of the open-channel peak is 1.7 pA corresponding to & chanel conductance of about 14 pS. In most expeciments, “single channels” had a much . jargec size, presumably because of aggregation of the P7 peptide. The currents : 30 reversed at sbout +40 mV in this experiment. In some experiments the solution in the cia chamber was 150 mM KCI and 15 mM KCl in the trans chamber. The P7peptide

Similar rosalts were obtained when both chambers contained NaCl. Currents a 5 rocarded in an experiment when the cis chamber contained 500 mM NaCl and the trans chamber 50 mM NaCL. Again the currents reversed between +40 and +60mV, close to the Na* equilibrium potential indicating that charmels were much mars permeable to Na* than to K*.

The channels formed by the P7 peptide were blocked by 5-(N,N- bexamethylens) amiloride (HMA). J Co

Addition of the P7 peptide produced chamel activity: Addition of 2 pl of 50 uM HMA to the cis chamber followed by stirring resulted in disappearances of the - chennel activity. Block of channel activity produced by the P7 peptide with 100 uM " HMA was recorded in 4 experiments. In 2 experiments, sodium channels (S00/50) : were blocked by 500 uM BMA : When 10 mM CaCl; was added to the cis chamber (K solutions) the reversal potential of the currents produced by P7 peptide shifted to more negative potentials indicating a decrosse in the Px/Pc; ratio. ’ When the cis chamber contained 500 mM CaCl; and the trans chamber 50 mM "CaCl, both positive and negative currents were soen at potentials around +20 mV and

N 35 it was not possiblo to determine a roversal potential,

Two cDNA fragments, cach encoding the same polypeptide cocrespanding to the amino acid sequence of the HCV-1a p7 protein, were synthesised commercially by GeneScript. The two cDNAs differed in nuclestide sequence such thet in ono cDNA (“cDp7.coli”) the codons were optimised for exprossion of the p7 protein in.

E.ooli while in the other cDNA (“cDp7.mmam)” codons were biased for expréssion in

Co aes Co "mammalian cell lines. aDp7.coli was clooed into the plasmid pPLAS1 as a * BamBI/BooRI fragment for expression in B.coll. cDp7.mam was cloned into vectors (for example, poDNA3.1 veocinia virus, pfastBac-1) for expression of p7 in

Example 47. Role of p7 in enhancement of Gag VL.P Bu pdding, .

The budding of virus-like particlés (VLP) from cultured HeLa cella results © from the expression of retroviral Gag proteins in the calls and co-expression of small

Tnsoeoatingly, tho viral ion channels can eabance budding of heterologous virus particles. Therefore, to assees budding enhanosment by p7 it was co-expressed with the HIV-1 Gag protein in Hela cells, snd VLP raleass into the culture medium was : moesured by Gag ELISA. To achicve this, the plasmids pcDINAp7 (pe DNA3.1 = -

Co pcDp7.mam 8s described in Example 20, p7 expressed under control of the T7 Co 1S promoter) and pcDNAGsg (HIV-1 Gag protein expressed under control of the T7 promoter) were cotranafactad into HeLa cells infocted with the vaccinia virus strain =~

VIF7.3 (cxprosscs T7 RNA polymersac) and culture supernatants were collected for ’

ELISA asszy after 16 hours incobetion. : fumetionsl activity,

The two methods of detecting p7 ion channel functional activity, described in

Examples 33-35, were employed to assxy the shility of compounds to inhibit thep? : 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 2s were tested for their ability to reduce the umber of VLPs released from oolls expressing both p7 and HIV-1 Gag. :

C30

: | 06 oo “ S Abio-assay of p7 function in bacterial cells was developed. The p7-encoding - synthetic cDNA fragment cDp7.coli was cloned into the exprossion plasmid pP1451, creating the vector pPLp7, in which p7 exproasion is temperature inducible, as described in Example 4. Inhibition of the growth of B.coli cells expressing p7 at "37°C was observed as m indicator of p7 ion channel function dissipating the normal oo 10 Na+ gradient maintained by the bacterial cells. -

The halos of growth sround the site of application of particular drugs — as described in example 14 — were scored ss decribed in example 15. :

Tablo 16 iets tho scores for inhibition of HCV p7 protain in the bacterial hio-sssay.

Table 16

HCV p7 protein ‘Bokbition : (score / # of thmes & DOWN heated] : ’ ~dimethylcimamoylgnamidine 388/72

D 4,6-trimethylcinnamoyignanidine 3.7571 5-bromo-2-flucrocinnamoylgusnidine 3.3/6 ‘4-Bromocinnamoyl) guanidine : 3.47/4 . ] ’, dimethy kinnamoylguanidine . 3.43 /4 R

B-trifluorometyDeinmemoylgaanidine C3348

ST 4-(triflucromethyl)cirmemoyl guanidine 333/5 :

G-mathoxy-2-naphthoylgnanidine : 333/73

Lalorocinnamo 1 nidine 331/66 .

Bromocimamoyl)guanidine 3.003 1 6-dichlorocinnamoylguanidine 3.00/3

Bromocinnamoyl)gnanidine 20273

Uh OCINNAMO Jed nidir : } 27513 : R-(trifinoromethylcinnamoyl guanidine 26313

BE 4-Phenoxybensoyl) guanidine 26311 . * - " .

B,4-dichlorocinnamoylgnanidine 2593 ¢-isopropylcimemoylguamidine 2.5172 : _jrans-3-()-napthyDecryloylguanidine 24412 4-t-butyloinmamoylgnanidine 2422

D-t-butylcinnsmoylgumnidine 23612 :

R-othylcimamoylgoanidine 23612

A methylcimmamoylgnanidine 23212 5-hromo-2-methoxyoinnamoylguanidine 2322 3-(triftnoromsthoxy)cimamoylgnanidine : 22612 = 2-cyolohexyloinnamoylguanidine 2261/2 1 -nspthoylguanidine oo 22511. 3-t-butylcimamoyignanidine 2232 4-phenylbenzoylgusnidine 219722

S-Phenyl-penta-2 4-dienoyl)guanidine 213/1

N~(cinnamoyi)-N'phenyl guanidine | x 2.131

B-isopropylcinnamoyignanidine hydrochloride 200/1 . Banzamil hydrochloride . ’ . 201 ) phenylpropanoy!)-N'-phenyignanidine : 20/1 -bis(3pbenyipropanoy)-N"-pharyl guanidine 20/1

B3-(2-napthyDecryloylgnanidine 1.932

Co 5-(N-Methyl-N-isobutyl)amiloride 1.881 ) 2'4 DichloroBenzamnil HC! 1.88/71 [5-tert-butylamino-smiloride . Co 1.881 ‘ . ny V-isopropyDemiloride 1.881 4-Methoxycmnamoyf)gnanidine : : 1.88/1 © #-fluorocinnamoylignanidine 1.86/1 :

Nitrocinnemoyl)guenidine - ) I) V) BE 'Y-ethoxveinnamo vigoenidin } . 1.631 . : 4-Hydroxycinnamoyl)gnanidine 1.63/11. rans-2-Phenyicyclopropenecarbonyl)gnanidine 1.6311 3-ethoxycinnamoyliguanidine 1.63/1 2.3,5.6,-totramethyicinnamoylguanidine 1512

BOR YIIINAO YIZaname ’ 1.5/1 rans-3-Furanscryoylgnanidine : 1.381 . IN{(6-Hydroxy-2-nepthoyl)-N'-phenylgusmidine 1381

FursoscryloyD guanidine : 1.3871 3-(cyclohex-1-en-1-yl)cinnamoylguanidine 13272 dmemoylgnanidine hydrochloride 13222 “ hao methylene)amiloride . 128/74 } ’ gifluorooinnamo guanidine 1.241 ’ 2-(1-napthyDaoetoylgnanidine | L141 a-Methrylcirmamoyl) guanidine 1.141

Nitrocinuamoyl) guanidine 1.1411 §-Iodosmiloride 1.131 3,4-(methylenodioxy)cinmamoyl guanidine 1.1371 ’. NL CEERI IN) LAMY] . 1.001 ’ cinnamoylguanidine 1.0071 :

Z-phRen yiciam moylgusnidine . 1.00/1 :

h<(cyclohex-1-en-1ylcinnamoylgnanidine 1.00/71 [$-pherTVICINNAINU guanidino ’ - 1.041 (N,N-Dimethyamiloride hydrochloride : 1 ten 8 (4-finorophenyl)amiloride : 1.01 : sthoxyoimmamoyl)guanidine : 1.01 . h. fuorocinnamoylguanidine 1.04 : : 5_(3'-bromophenyl)penta-2,4-dienoylgoanidine 1.01 (4-Chlorophenoxy-scctyl] guanidine 1.01 phenyipropanoyl) guanidine 10/1 . ~ p-chloro-6-flucrocinnamoylgnanidirie 0.88/1 : B-fluorocinnamoylguanidine 0.86 /1 ) 2-Te) cinnamoylguanidine Co 0.75/1 : -Methoxycinnamoyl)guanidine oC 0.751 ~bromo-2-napthoylgnanidine 0.751 © BAS-trimefroxycinnamoylguanidine 0.7111 3-methyvicinnamoyigoanidin 0.6311 . 3 (trans-hepi-1-00-1-ylcimamoylgnanidine . 0501

MOC HC1 : . : 0522

Phenamil methanesulfonate salt 0s : 4-Nitrocinmamoyl)guanidine 02511 : 3 4-diffoorocimamoyl guanidine 0.1311 :

J (B)-3-(A-Dimetrylaminophenyf)-2- methylacryloyl]goanidir 0.03/4 ma (RAV) 5s To determine the antiviral activity of compounds against EAV replication (strain Buoys; 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 smplified in RK-13 cells (ATCC CCL-37) and this was

So then used to infect confluent monolayers of BEK-21 cells grown in 6-well tissuc . culture plates by exposare to the virus at an MO! of SX10° pftvoell for 1 hour at : a 37°C 5% CO;. The infactive inoculum was removed snd nd the cells wore overlayed “with a 1% sou plaque overlay (Cambrex Bio Science) in MEM containing 10% FCS : containing and 10, § or 1 WM of compounds to be tasted of the appropriate level of

BE solvent used for the compounds (control). Plates were subsequently incubated at 37°C (in 5% CO) for 3 days post infection, after which time culture supernatant was : removed and the cells were stained with 0.1% arystal violet sofution in 20% ethanol

Co y 109 for 10 minutes. Plaques were counied in ll wells and the percentage reduction in plaque smber compered to solvent control was calculated. Measurements were ' performed in duplicate to quadraplicate wells. oo 5

Compounds were screened for activity against BAV replication accanding to the method described in example 35. Results expressed as ICS0 valuos are shown in iE Table 17. } : : 10° Tablel17 Lo mem | mm

C [FoNmambycentonide | 7s 0

Sobuiyidmencylgomadine | 1M [Coroeieiiemcygeite | 10g "1s The C- terminal 40 amino acids of the M protein of the Dengus virus type 1 struin Singapore 5275/50 (Fu ct al 1952) oo : (ALRHPGFTVIALFLAHAIGTSITQRGIFILLMLVTPSMA) wes synthesised using : the Fmoc method, The synthesis was done on a Symphony Peptido Synthesiser form

Protein Technologies nc (Tucson, Arizona) as used to give C-terminal amides, the coupling was dons with HBTU and hydroxybenzotriazole in N-methylpyrrolidone.

Rach of the synthesis cycle used doublo coupling and a 4-fold excess of the amino acids. Temporary o-N Fimoo-protecting groupe ware removed using 20% piperidine in

Exammla $4, Jucarveration of Dengue M virus Droteln into Hpi bilsver, : 25 Lipid bilayer stodies were performad as described elsewhere (Sunstrom, 1996; : Miller, 1986). A lipid mixture of palmitoyl-olcoyl-phosphatidylethanolamine,

: oo -110- palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylchotine . (5:3:2) (Avanti Polar Lipids, Alsbester, Alabama) was used. The lipid mixture was painted onto an sperture of 150-200 pm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing sait 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 S00 mM © KCland the trans 50 mM KCL The bilayer formation was monitored electrically by the amplitnde of the current pulse generated by a cutrent ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the - 10 cis chaniber and stirred until channal activity was seen. Tho currents wero filtered at "1000 He, digitized at 5000 Hz and stored on magnetic disk.

The dengue virus M protein C-teriinal peptide (DMVC) was dissolved in2.22- .triffuorethanol (TFE) at 0.05mg/ml fo 1 mg/ml. 10 al of this was added 0 the cis . 15 chamber of the bilayer which was stirred. Channel activity was seen within 15-30

Solutions of 50 mM HMA were prepared by firit making 2 500 inM solution in DMSO. This sohution was further diluted to 50 mM HMA using 0.1 M HCL 2 ul of the 50 mM HMA was added to the cis chamber after charmed activity was seen. The cis chamber contained 1 ml of solution making the final concentration of HMA 100 mM ‘Compounds were testod ot 10, 5, 2.5, 1.25 and 0.625 uM for activity against ~~

Dengue 1 strain Hawaii using a oytoproliferstion assy which measures the effect of dengne virus infection on proliftration of LLC-MK2, thes macaque monkey kidney cells. Tho LLC-MK2 cells were infacted with a predetermined amoant of vie, titrated such that cell proliferation in infected cultures would be significantly reduced a1 "compared to uninfected controls. The infected cells were then plated at 1.5x10° cells per-well in a 96 well plate. Negative controls (no virus, no experimental compound), positive controls (vires, no experimental compound), and cytotoxicity controls : - ©. (experimental compound, no virus) were run with each drug assay. The cultures were allowed to grow for 7 days and then Alxmar Blve, a fluorescent dye that measures the oo metabolism of the cultures (red/ox), was added to cach culture and the fluorescence vatue for cach 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 calenlating their average fluorescence as a percentage of the nogstive control. At least six replicate wells were measured for each experimental

Teble1s : - Drug Antiviral .

Convo. | Percent of

Drug pM | Negative

Cantrol [Negativecontrol | 0 | NA

Positivecontrol | 0 | 76.5% lio] 171% |] 1s | 386% : L125 | 583% loss] 76.6% - | ___(2-chlorocimamoyl)gnanidine [Negativooontrol | 0 | NA | - : | Positivecontrol | 0 | 803%

I

125s | 2% 1125] 525%

Joes] 643% |.

Nogetivecontrol | 0 | NA : 1 10] 68% | :

25 | 387% | CL

N.A. — not applicable =. s

A correlative study was performed to messure correlation between the activity scores assigned to compounds tested in the Vpu bacterial assay and the shility of * hess compounds to inkibit HIV-1 in the anti-viral assay.

Example S82. Methodology

The p24-stigen data fox twelve compounds representing various substituted scyl- gosnidines was compared with the activity scores obtained for those compounds in the Vpu bacterial assay. The dats from cach asesy wis initially rank ordered for : effectiveness. The rank order for the Vpu bacterial assxy was determined from all

I 15 activity scores, the highest score indicating the grestest effectivencss. The rank order . for the anti-HIV-1 assay was determined based on the overall average value of p24 : antigen mossared in culture supemstants at all of the drug concentrations tested, with were then compared statistically by generating the Spearman’s Rank correlation . ~~ cocfficient. oo ~The Spearman’s correlation coefficient was 0.785 which, by comparison with a statistical tals of critical valuos (for n=12), indicates that the two rank ordécs are .. 25 significantly positively cotrelated (P<0.01) (Table 19%). : Tn addition, a different comparison of the Vpu Bacterial assxy rank order with a yes/no scare for whether the anti-viral data indicated a p24 reduction of st least one onder of magnitude, aligned the ‘yes’ group of compounds with the top 6 compounds by the bacterial assay (Table 19b). :

Cam ‘Thess rosults are indicative that a positive correlation exists betwoen bacterial assays and the antiviral aseays as performed according to tho present invention. The - bacterial assay may therefore be & useful tool in screening for compounds that exhibit "Table 19a. Comparison of Rank order of efficacy of 12 substituted acyl guanidines in the Vpu bacterial assay and anti-HIV assy. nn J 9 kl rank order rank order [Emery 2oaphhoyignenidine | 55 | 5 | 025 _ piu EA EE [A VA PIR IRIE [Oosmethyimesmilosde [10 | 6 | 16 [=r phenylcyclopropanccarbanyl) guanidine oe sumdi2]| 615 ee Spearmancomelstioncoefficient| 0.785 a Pvalme]| >001

Tables 19h. ‘ : Compounds Vpu Bacterial | Atlenst 1xleg . Ordered by pa4 rank order Bacterial assay rank redaction seen In [4phenybenmoylgnaniding | 28 | 7 | mo

PR Ca ell EE

IGUL vy ASC SLIM

[meta

Co. nhenyieyclopropanecarbor yuan '-

A positive correlation was seen between the activity scores assigned to + compounds when testad in the Mouse Hepatitis Virus E-protein bacterial bio-assay _ 10 and the percent inhibition exhibited by these compounds in the Mouse Hepatitis Virus plaque assay.

MHV plaque reduction activity data for 96 compounds screened were sorted from ‘greatest to lonst percent plaqus reduction and rank orders were assigned to the list of compounds. This was perfrmed for the data generated by exposure to both 104M and 12M concentrations of the compounds, giving rise to two rank order lists.

Similarly, a rank order list was generated for the MEVE bacterial bicassxy soores for the _ same 96 compounds. Where one or more compounds had the same socre, the rank values . fbr that greup wers averaged.

Spearman's statistical test for [as described in “Mathematical Statistios with

Applications” (2™ edn): Mendsnhall, W., Schaffer, RL.,& Waokerly, DD. Duxbury

Press, Boston Massachusetts — 1981] was used to compere 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) according to the formula: Rs «= 1-(6.88/n(n?-1)), where n is the number of compounds to ranked (96 in this cass). Rs is then compared to a Table of critical values to determine statistical significance (P values). ©

This table summarises the Rs snd P values geneated as a reqult of the indicated oo | Table 20

I EE EE RE RE oo "The rank order comparison of 96 compounds assayed in the bacterial bio- assay and the antiviral assay show that MHVE bacterial assay rank order for tho compounds tested is significantly positively correlated with the rank orders generated by the MEV plaque reduction assay. The significant correlation between the sseays : is highty indicative that either assay may be utilised to identify compounds that may : be useful. The bacterial assay may tharcby be a useful tool in screaming for compounds that exhibit anti-viral activity. . :

A positive correlation was seen between the activity scores assigned to - 13 compounds when tested in the Human Coronavirus 229E B-protein bacterial bio- : assay and the percent inhibition exhibited by these compounds in the Human : : 20 2298 plaque reduction activity data for 97 compounds screened against 2.5 uM ) compound concentration wero sortad from greateat to least percent plaque reduction and rank orders were assigned to the list of compounds. Similarly, a rank order list was generated for the 220 E bacterial bioassay soares Sr the sams 97 compounds. ‘Where one or more compounds had the same score, the rank values for that group were averaged.

SE :

Spearman’s statistical test for [as described in “Mathematical Statistics with Applications” (2* edn): Mendenhall, W., Scheaffer, RL.,& Wackerty, DD. Duxbury Press, Boston

"Massachusetts — 1981] was used to compare rank orders. Briefly, this involves calculating the Sum of squares (SS) of the differences between rank values for cach compound, snd : then generating the Spearman's Rank Cocrelation coefficient (Rs) according to the formula: Re = 1-(6.88/a(n’-1)), where n is the number of compounds ranked (97 in this : 'S case). Rsis then campared to a Tshls of critical vahics to determine statistical significance (P values)

This table summarises the Rs and P valnes genarsted as a result of the indicated pairwise Table21 fom [OT © |Paem2S0fBacial | 04] 5 | 2 Ye

HE

I Ce J I

The results above indicate that the 229F bacterial assay rank order for the : 15 compounds tostod is significantly positively correlated with the rank orders generated by the 229 plaque reduction assay. This result combined with that shown in

Examples 4.1 and 49.4, provides strong evidence that either assay may be utilised to identify compounds that may bo usefl. The bacterial assay may thereby bea useful ~~ - tool in screening for compounds that exhibit anti-viral ectivity.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. © is tobe understood thet the invention includes all such variations and modifications : The invention also includes all of the steps, features, compositions and compounds 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. .

BE a7-

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Co

Claims (1)

1. . : -120- | ’ : © THE CLAMS DEFINING THE INVENTION ARE AS POLLOWS:-

   1. Anscylgnmidine with antiviral activity. © © 2. "An antiviral compound of Forumula I

   : . : JR Co :

   p . . Ry | I wherdin R;-R, are independsntly aromatic groups, heteroaromatic groups, : alkylaromatic groups, alkylhoteroaromatic groups, alkenylaromatio groups, g alkenylheterocromatic groups, cycloalkylaromatic groups, cycloalkylheteroaromatic groups, aryloxyalkyl groupe, heteroaryloxyalkyl groups, said groups are mono or polycyclic, and sre optionally substitirtad fxydiraxy, nitro, halo, emino, mibstitated amino, alkyl-substituted amino, cycloalkyl-sbstituted imino, aryl-substitated amino, Cretlkyl, C- : : oC aalkyloxy, Cs soycloalkyl, halo-substituted C).ealkyl, halo-substituted C.- alkyloxy, phenyl, C,.calkaneyl, Cy gcycloalkeneyl, Ci ¢alkeneoxy, benzo, : : Ol “IY ary, substituted ar, PS, 0X NRO I * An antiviral compound of Formula I :

   Re . . NZ °N . . he HH 'Y PY I or pharmaceutically soceptablo salts thereof, R=

   EEE > CR . Ny nN xX SY : | JE ) Cr Ug SC BE ale icad on () | 3 ko Ry Rg ) RNR, : . (0) <5 ECR alers

   Cs. , Be Re ~~ Sn CO : ’ Ny ‘ ’ N° ’ : CNUs Oo Re, Rg and Ry are independently hydrogen,

   : | 122 Co oo

   } R. (0 J a 0 to ’ . ’ rt \ iN } ) J OJ FS I can lau cH

   , . " ’ } or 5BOD oo So , i» oo a. © X= hydrogen, hydroxy, nitro, halo, C.ealkyl, Crealkyloxy, ; Csscycloalkyl, halo-substituted C.salkyl, halo-substitated Cy. alkyloxy, phenyl, C,.alkeneyl, Cy scycloatkeneyl, Cy ealkeneoxy, or benzo; Ra,By, Ro, Ry, Ra, Re, Bas Ra, Re, Rin, Ra, Ro, Ry independently = hydrogen, amino, halo, C1.salkyl, C1salkyloxy, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl-substituted a iy NE; 0 or P1S; oo Ry, Ri independently = hydrogen, hydroxy, halo, or Cys alkyl; Co Ry = hydrogen, amino, halo, C.salkyl, C) salkyloxy, hydroxy, aryl, ENN} Oe LT pe

   C = 4, A pharmaceutical composition comprising an antiviral compound : according to arty one of claims 1 to 3, and optionally ons or more : pharmaceutical acceptable carriers or detivatives, : _

   5. The pharmaceutical composition acooeding to claim 4, finther comprising : one or more known antiviral compounds or molecules. 6 Amethod for reducing, retarding ot otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with seid virus or exposed to said viros with a componnd scoording to any ane of claims 1 ‘ to 3.

   © 7. The mood siconding to claim 6, wherein said vims isa Lentivirus. . 18 - 8. . Themethod according to claim 7, wherein said Lentivirus is Human

   9. The method according to claim 8, wherein said compound is selected from the group consisting of : (2-Chlorocinnamoyf)gnanidine, : (2-Bromocinnemoylguanidine, 3-(triflooromethyl)cinnamoyiguanidine, So 5-bromo-2-floorocinnamoylgoanidine, : : 3-methyicinnamoyignanidine, 2-methylcinnamoylgnanidine, i

   2 3. dimethyl Py——y Cinnamoylguanidine, 6-methoxy-2-naphthoylguanidins, : trane-3-(1-napthylacryloylgnanidine, 3,4-dichlorocinnamoyignanidine, 2,6-dichlorosics tomamidi (4-Chlorocinnamoyl) guanidine, oo 2-napthoyliguanidine, Co 3-isopropylcinnamoylguanidine hydrochlorida, Co

   Co au (5-Phenyl-penta-2,4-dienoyl)guanidine, : : 3-phenyicinngmoylguanidine, - : (4-Bromocinnamoy)guanidine, LL 5-(3'-bromophenyl)penta-2,4-dienoyiguanidine, oC 3-{cyclohex-1-en-1-yl)cinnamoylguanidine, ’ : N,N"-bia(3phenytpropanoyl)-N"-phenyignanidine, Co 2-ethoxycinnamoylguanidine, +G-phomy] 0yT)-N-pheerylguanidi '(4-Methoxycinramoyl) guanidine, 4-methyléinnamoylgnanidine, 2- ; i : : 2 fox Wn Viguanidine, yr

   ~. N{6-Hydroxy-2-napthoyl)-N"-phenyiguanidine, : 3--butylcinnamoyignanidine, S-(IN,2N-hexamethylene)amiloride, ‘ 3-floorocinuamoylguanidine, : S-beomo-2-methoxycinmamoylguanidine, 3 & § A [3 ii N 3,4-(methylenedioxy)cinnamoylgnanidine, Co (2-Methoxycinnamoyl)gnanidine, : oo 2'4 DichloroBenzemil HCl, 2,3,5,6,-tetramathyicinnamoyignanidine, " 23 diftnorociniemoy iguanidine, : ¢ Mothuxy a guaridine, i 24,6-trimethylcinnamoyignanidine, 2-{cyclohex-1-en-1yl)cinmamoyignanidine, (4-Hydroxycimnsmoyf)guanidine, oo 4-fuorocinnamoylgnanidine, iy any paitim, : . Phenamil methanesulfonate salt, (3-Nitrooinnsmoy1) guanidine, (4-Phenoxybanzoyl)guanidine, CL 3-(trave-hept-1-en-1-yl)cinnamoyiguanidine, : oo 5-(N-Mothyl-N-isobutyl)amiloride,

   - as : 2,4-dichlorocirmamolyguanidine, (N-Ethy}-N-isopropyl)amiloride, : oC N-amidine-3-amino-5-hexansthyleneimino-6-phenyl- : 2-pyrezinecarboxamide, (e-Mothyloinnemoyl)guanidine,

   i. cimamoylgnanidine irydrochloride, - [(4-Chlorophencxy-acetyl] guanidine, oo : N-amidino-3-amino-5-phenyl-6-chloro-2- . 5-(4-finorophenyfamiloride, : 1-napthoyignanidine, . S-tert-butylamino-amiloride, : oo 3-methoxy -HMA, ~ N;N-Bis(3-phenylpropanoyl)gnanidine, N-Benzoyl-N'-cinnamoylgnsnidine snd oo . 1-bromo-2-napthoyignanidine. © 10. The method according to claim 8, wherein said compound is selected from the group consisting of 4-phenyfbenzoylguanidine, (3- bromocinnamoyl)guanidine, 3-(triflucromethyf)cinnamoylguanidine, 5- ES) (NN-hexamethrylene)arniloride, and (5-Phanyl-pents-2.4-

   11. The method soconding to any one of claims 8 to 10, wherein seid HIV is : HIV-1. ) oo :

   12. The method sccording to claim 6 wherein said virus is a Coronavims. : | 13. The method scoarding to claim 12, wherein said Caronaviras is the Severs Acute Respiratory Syndrome virus (SARS). :

   14. The method according to claim 13, wherein said compound is selected oo from the group consisting of oo . 3 4-dichlorocimmamoyiguanidine, . 4tbotykimamoylgoanidine, 3-(2-napthyl)acryloylgnamidi (3-Chlorocinnamoyl)guanidine, 3-(oyclobex-1-en-1-yDcinnamoylguamidine, 2,5-dimethrylcinnamoyignanidine, trans-3-(1-napthyl)acryloylguanidina, } it enanidt : (3-Bromocinnamoyl)gnanidine, oo : 6-methoxy-2-naphthoylguanidine, : 5-(N-Methyl-N-isobutylamiloride, 3nhenyled \snamidi 3 2'4 DichloroBenzamil HCl, {oheylci PY 3-(hrif thoxy)ed \gnanidi 3-(triflnoromethyl)cinmamoyignanidine, 2 oft » p——p : (2-Bromocirmamoy])guanidine, 2,6-dichlorocimamoyiguanidine, 34, 5-trimethoxycinnemoylgnanidine, S-tert-butylamino-amiloride, * 3 t-but } 3 | idk mnie . 2-cyclohexylcinnamoylguanidine, . 6-1 3 il id * C in Sone 4-Bromocinnamoyl)guanidine, (4-Hydroxycinnamoyl) guanidine @ Nitrocinnamoyf) guatidine, flucrocinnamoyigoanidine, 2-(1-napthyljacctoylguanidine, 2-athyloinmamoylgumnidine, CL 5-(N,N-Dimethyl)amiloride rydrochloride, : p beayl)amilorid 2~(trifiucromethyl)cimamoylguanidine,

   NA(6 Hydroxy-2-napthoyl)-N-phenylgumidine, oo (trans-2-Pheayicyclopropanscarbonyl)gnamidine, - os N,Nbis(3phenyipropanoyl)-N"-phenylgnanidine,, 1-napthoylgoaniding, : oo CL . 3-methoxy -HMA, j 4-methykinnam oylgnanidine, : 4-fluotocinnamoylgusnidine, : 3 A-(methylenedioxy)cinnamoylguanidine, . °° Ne{cimamoyl)-Nphenylguanidine, 2-methylcimamoyiguanidine, 2,3,5,6,-totramoftrylcinnamoylguanidine, Co revere Ach. ur : (3-phonylpropenoyl)guanidine, : Ci fdmiey - (2-Methoxycinnamoylguanidine, 4-phenylbenzoyl guanidine, 2 A-dichloroct : di (3-Methoxycinnamoyf)guanidine, :

   2.8 ; longmidi (4-Phenoxybenzoyl)gumnidine, ruin mi i =bromophenyljpante-2.4-dienoylgusnidine, (5-Phenyl-panta-2 4-dienoyl)gnenidine, (Quinaline-2-cachonyl)gnanidine, (Phenylacetyl)gnanidine, N N-Bis(amidino)napthalens-2,6-dicarbox amide, 6-bromo-2-nepthoylgnenidine, 1-bromo-2-napthoylgnanidine, [(4-Chlorophenoxy-acetyllguanidine, : Phenemil methanosnlfonate salt, N-Benzoy-N'-cimamoylgnanidine and N-(2-napthoy])-N'-phenyignanidine.

   15. The mothod according to claim 13, wherein said compound is selected from the group consisting of cinnamoylgnanidine, trans-3-(1-

   : .

   a | -128- : © + 16. Themathod according to claim 12, wherein said Coronavirus is luman

   17. * The method according to claim 16, wherein seid compo is selected RB from the group consisting of . . 4 ino on ;! : 1 - H * : 3,4-dichlorocinnamoylguanidine, 3(triflnoromethoxy)cirmamoylgnanidine, } + 3.4 | tyl Ll | o ki trans-3-(1-aapthyDacryloylgnanidine, + 3<2-naptirylecryloylgusmidine, : 2mhenvici anemic 3-phenylcinnamoylguenidine, ) 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, (4-Phenoxybenzoyl)gnanidine, I 4-(triftooromethyl)cimamoylguanidine, . : : 2-(cyclohex-1-en-1ycinmamoylguanidine, (4-Bromocinnamoyl)guanidine, : S-(N;N-haxamethylens)emiloride, 1-napthoyiguanidine, + 5-(4-fiuorophenyl)emiloride, B A Deuanidi | oo : 2-(triftooromethyDcirmamoylguanidine, 6~-meothoxy-2-naphthoylgnanidine, : . (4-Chlorocinnsmoyf)gnanidine, : : (3 Meth 0 : 1) . $ . : S-bromo-2-fluorocinnamoylguanidine, . _ 5-(N,N-Dimethyl)arilaride hydrochloride, : Cimamoyiguanidine, (s-Methykinnamoyl)gnanidine, 4-phenylcinnamoylgnanidine, (2-Bromocimamoyl)guanidine, :

   _ CL - oo =]28- | : oo (trs-2-Phenyleyolopropanecarbony]) guanidine, (3-Chlorocinnamoyl)guanidine, : 2-(1-napthyl)acetoyiguanidine, + 2~gthylcinnamoyignanidine, Co 2-cyclohexylcinnamoylguanidine, (4-BydroxycimamoyT)guanidine, 2-ethoxycinnamoylguanidine, . : Co 3-methylcinnamoylguanidine, 3-finorocinnamoyl guanidine, - oo ~ 2-ilnorocinnamoyliguanidine, 4-fluorockmamoyignanidine, 3,4-diflnorocinnamoyignanidine, ‘ N,N'-Bis(smidino)napthalene-2,6-dicarboxamide, N,N'-Bis(3-phenylpropanoyl)gnanidine, oC 4-methyicinnamoylgnanidins,

   2.3,5,6,-totramothylcinnamoyiguanidine, siphon fpopeny)N- ghar grain, - (2-Chlorocimmamoyl)gusuidine, 34,5-trimethoryes LL I N-(3-phenylpropancy])-N'-phanyl guanidine, * 54(2-bromophenyDpents-2,4- fienovlanarridi: (4-Bromocinnamoyf)gnanidine, (3-ChlorocinnamoyT)guanidine, : (4-Mothoxycimamoyl)gomidine, ! - Htiflocrometyl)oimamoyl guanidine, [(B)-3-(4-Dimethylaminopherryl)-2- . :

   _ . mothylacryloyl]guanidine, N-emidino-3-amino-5-phenyi-6-chloro-2- Cd : : !

   -130- a Cinnamoylguanidine, : 3-methoxy-amilorids, (3-phenylpropanoyl)guanidine, : 3-methoxy -HMA, oo . Benzyoylguanidine, N-amidino-3,5-diamino-6-phynyi-2- (Quinoline-2-carbony!) guanidine, [3-(3-PyridyDacryloyl]guanidine, _ N-Cinnamoyl-N',N'-dimethyiguanidine, So N-z-nspthoy!)-N-pheaylguanidine md

   18. “The method according to olsim 16, wherein said compound is selected from the group consisting of 4-isopeopylcinnamoyignenidine, 34-dichlorocinnamoylguaniding, 3-(triftnoromethoxy)cinnamoylguanidine, 2<(cyclohex-1-en-1yl)cinnamoylignanidine, 2 cvelohexvic \euanidi ' §-bromo-2-msthoxycinnamoylignanidine, 2-ubenvici tansnidi :

   2 . 3 +butyloimamo vigneidine, ii S-(N,N-hexametbylene)amiloride, truns-3-(1-nepthyDacryloyiguanidine, h 2 ¢ py 4 dew Yioygranidi, # . 5-bramo-2-fluorocinnamoyiguanidine, : 4-methylcinnamoyliguanidine, © 3{cyclohex-1-cn-1-yl)cinnamoyl ganidine, (a-Methylcinnamoy!)guanidine, 2,3,5.6,-tetramothylctonamoylgnanidine, 2-flucrocimamoylguanidine, : + (3-NitrocimnamoyDguanidine, 3-t-butylcimnamoylguanidine, (3-Methoxycinnamoyl)guanidine, _ 3methylcinnamoylguanidi Ce 3-isopropylcinnamoyigumnidine hydrochloride,

   Co -131- | : -BromociunammoyD guanidine . 3-cthoxysimamnoylguamidi —- (5-Phenyl-penta-2,4-dienoyl) guanidine, - dif . iganidine, 2,4,6-trimethylcinnamoylgnanidine, 2-mothylkcinmamoylgoanidine, (trams-2-Phenyloyelopropsuooarbomyl)- oo Meothoxycinnamoy)guanidine, Gi 3,46 oy pum dine, fioxy)es lanamiding N,N'-Bis(smidino)napthalene-2.6- Dicarboxamide, 2,3-dimethylcinnamoyiguanidine, - 543“-txomopbmyl)pents-2.4-dienoylgnanidine, 1-napthoylguanidine, oo S-(N,N-DimethyDamiloride hydrochlorids, 2-napthoylgnanidine, 3,4,5-trimefhoxycinnamoylgnanidine, : 2 temeidine kydrochlorid 5-(4-finorophenyl)amiloride, AC a ce : Appi bears. Ar N,N'bia(3pbenylpropmoyl}N*- Phenyignanidins. :

   19. “The method socaxding to olsim 12, wherein ssid Coronavirus is bumen s

   : : ~132- TL } 20. ‘The method according to chim 19, wherein said compound is selected from the group consisting of - 3-methylcinnamoyiguanidine, trans-3-(1-napthyDacryloylguanidine, O- fd ¢ pier - Ay 4-dichlorocinnamoyiguanidine, if chyToi eunnidi (trans-2-Fhenylcyclopropanecarbonyl)gusnidine, P ci anid Co 6-methoxy-2-naphthoylgnanidine, : 2, 4-dichloroci i pe : _ (4-Chlorocinnamoyl) guanidine, rn Co -Bromocinnamoy]) guanidine, pet ne Yiguunidne, din (5-Phenyl-penta-2 4-dienoyl) guanidine, 3(triflnoromethoxy)cinnamoyignenidine end

   21. “The mathod according to claim 12, wherein said Coronavirus is porcine

   22." The method according to claim 21, wherein said compound is selected : from the group consisting of ' 54(N,N-hexamethylencemiloride, oo 6-methoxy-2-naphthoy]ganidine, Cinnamoylguanidine,

   _. N{3-phemyipropanoyl)-N-pheaylguamidine, 3methid teuanidi : (3-Bromocinnamoyl)guanidine, Co } . (trane-2-Phenyloyolopropanecarbonyf)guanidine, trans-3-(1-napthyDecryloylguanidine and

   23. Themed sccording to claim 12, wherein said Coronavirus is bovine

   24. The method according to claim 23, wherein aid compound is se.ccted from the group consisting of

   : . Denacic trificoromety)cinnamoylgoanidine, 6-methoxy-2-naphthoylgoanidine, 5~(N,N-hexametirylenc)amilonide, trans-3-(1-nspthylacryloylgoanidine, (5-Phenyl-pents-2,4-dienoyl)gusnidine, 2-(2-nspthylacetoyiguanidine, (trana-2-Phenyicyclopropanecarbosyl) gustidive, N-(3-phenyipropanoy])-N'-phenyl guanidine and . 4-phenylbenzoyl guanidine. oo

   25. Tho method according to claim 12, wherein said Coronavirus is amy ans of 8 the known coronavirus isolates fisted in Table 1. Co 26. The method according to claim 25, wherein sxid compound is selacted : from the group consisting of : i 3 A-dichlorocinnemoylignanidine, 3 (aif shory)cinnamoylgoanidi 4-t-butylcinnamoylgnanidine, ai ici tonanidine Iydrochlorid 0 27 The method according to claim 6, wherein ssid vir is the Hepatitis C virus, :

   2. "The method according to olsim 27, wherein said covxpoand is selected from the group consisting of . : ’ . ‘ «as . : . — hus guanidine, mi tans rid dic enanidi triflnoromethyl)cirmamoyiguanidine,

   a. 5 methoxy-2-naphthoylgoaridi (2-Chlorocinnemoyl)guanidine, . (2-Bromocintamoyl)guanidine,

   oo ae . 2,6-dichlorocinnamoylgusnidine, : (3-BromocinnamoyDgaanidine, : 2{trifluoromethyl)citnamoylgnanidine, : (+ Prenaxsbonoylgmaridine : 4-dichloroci PE EK tras 3(1-vapthyaceyloyiguanidine batvic enanidi 5-bromo-2-methoxycinnamoyignanidine, : © 3{triftuoromethoxy)cinnamoyiguanidine, 1-napthoyignanidine, ’ : 3-t-butylcinnamoylgnanidine, 4-pbenyfbeozoylgnanidine, (5-Phonyl-pents-2,4-dieaoy!)guanidine, N-(cimemoyl)-N'pbenyl guanidine, : N-(3-phanylpropanoyl)-N'-phenylgnanidine, - NN-bis(3pbenylpropanoyl)-N"-phenyignanidine, 3-(2-naptivyDecryloyignanidi © S-(N-Methyl-N-iscbutyl)amiloride, 2'4 DichloroBenzamil HCI, : - 5-tert-butylamino-amiloride, Aru te ering 4-Methoxycinnamoyl)gnanidine, 4-finorocinnamoylgnanidine, : (3-Nitrocinnemoyf)gnanidine, (4-Hydroxycinnsmoyl)gusnidine, (trans-2-Phenryloyclopropanecarbonyl) guanidine, 3-ethoxycinnamoyignanidine, ol 2,3,5,6,-totramethylcinmamoyl guanidine, trans-3-Furanacryoylgaanidine, N+(6-Hydroxy-2-napthoyl)-N'-phenylgaanidine, © (2-Furanacryloylgusnidina, 3-(cyclohex-1-en-1-yl)cinnamoylgnanidine, + 5<(N.N-bexamethylene)amiloride, 2,3-difluorocinnamoylgnanidine, : 2-(1-napthyl)acetoylguanidine, @ Nirotmumey pits, Nitrocinnamoyl) guanidine,

   6-Iodoamiloxide, Aananidi Co 2-(cyclobex-1-en-1ylcinnamoylgaanidine, 2-napthoylguanidine, Co havc lquamidine, 5-(N,N-Dimethyljamiloride hydrochloride, ethoxychmamoyi)guanidine, . . 2-fi . | - gk : [(4-Chlotophenoxy-acetyl]guanidine, (3-phenylpropanoyl) guanidine, : Co 2-chloro-6-fincrocinnamoyiguanidine,

   34 . Ap—y 2-methylcinnamoyiguanidine, n . | 2-Met . )guanidi . : 1-bromo-2-napthoyignanidine,

   3.4, 5-trimethoxycinnamoylguanidine, i. 3-methyicimamoyignanidine, 3-(trans-hept-1-en-1-yl)cinnamoylguanidine, _ Phenamil methanesulfonats salt , 2 4-dichlorocs 1 dine, (4-Nitrocinnamoyl)guanidine, . 3 4-difinorocinnamoyiguanidine and [(@)-3-(4-Dimethylaninophenyl)-2- 29, The method acocrding 0 claim 6, wherein end viru i Bquine Atait

   S30. Themsthod according fo claim 29, wherein said compound is selected from the group consisting of : 5-(N,N-hexamethylene)emiloride,

   : . (3-Bromocinnamoyl)guanidine, 2-t-butyicinnamoyliguanmdine and : : 2{oyclohex-1-en-1yl)cimmamoyl guanidine.

   31." A method according to sary one of claims 6 to 30, wherein said compound : : is provided as a pharmaceutical composition acoording to claim 4 ar claim . 10 5.

   : I 136- | oo

   32. A method for preventing the infection of a cell exposed to virus - i comprising contacting said cell with & compound according to amy ons of claims 1103, Co

   ’ 3. The method according to claim 32, wherein seid virus is a Lentivirus.

   34. Thomethod according to claim 33, wherein ssid Lentiviras is Hizmen Immunodeficiancy Vires (HIV). : 10 © 35, Themethod according to olsim 34, wherein ssid compound is selested - from the group conaisting of (3-Bromocinnemoyl)guamidine, . }

   QB . Dauanidi . 3<(triffucromethyleinnamoyigasnidine, : 5-bromo-2-finorocinnamoyiguanidine, Smethviimsmovignanidi : © 23-dimethyle {guanidine : 6-methoxy-2-naphthoylguanidine, trane-3-(1-napthyl)acryloylguanidine, : : -3,4-dichlorocinnemnoyiguanidine, 2,6-dichlorocinnemoyignanidine, 4-phenylbenzoyiguanidine, 2-gthyicinnamoylgnanidine, (4-Chlorocinnamoyl)gnanidine, : 2-napthoyigusnidine, 2 5_dimetiici lgnanidi . (5-Phenyl-penta-2 A-diencyl)guanidine, oo 3-phenyicinnamoyignanidine,

   4B . fguinidi 5-(3"-bromophenyi)pents-2,4-diencylguanidine, 3-(oyclohex-1-en-1-yl)cinnamoylgusnidine, 3<(triflnoromethoxy)cimnamoyl guanidine, » 2(triflnoromethryl)cirmamoylgnanidine, N,N'-bis(3phenyipropanoyl)-N"-pbetyl guanidine, 2-ofhaxycinnamoylguanidine, nL

   ~ | BS k 8 (4-Msthoxycinnamoyl)gnanidine, . : : 2-+-butyicinnamoylgnanidine, \ 0 | N{cHykory2nephoy)N phenyiguaidine, © . 3 4} t 1 . i id . . . i 3, 4-difluorocinnamoylguaniding, CL 3 ethene oamiding, - 3,4<(methylenedioxy)cinnamoylgnanidine, (2-Methoxrycinnamoyl)guanidine,

   : . 2'4 DichloroBenzamil HCl, : : 2,3,5,6,-tetramethylcinnamoyl guanidine,

   : ©. 2{1-naptryDacetoylgusnidine, (3-Mothoxycimainoylguamid © Ne(cinnamoyl)-N'phenyignanidine, : 2-(cyclohex-1-en-1ylcinnamoylgnanidine, (4-Bydroxycinnamoy/)guanidine, : N,N'-bis-(cimnamoy])-N"-phenyl guanidine, Phenamil methanesnifonste salt , (4-Phenoxybenzoyl)guanidine, 3-(trans-hept-1-en-1-yl)cimmamoylgunidine, 5-(N-Ethyl-N-isopropyl)amiloride, CC Namidino.3emino-S.3 2-pyrszinacarboxamide, Co (a-Methylcinnamoyl)gnanidine, [(4-Chlorophencxy-acetyl]gnanidine, oo oo N-amidino-~3-amino-5-phenyl-6-chloro-2-

   5-(4-flnorophenylamiloride, : - (trans-2-Phenyleyclopropanecarbonyl)gnanidine, (2-Nitrocinnamoyt)guanidine, ~ trans-3-Furansacryoylguanidine, 1 navthoviznmid : 5-tert-butylamino-amiloride, © 3-mothoxy-HMA, : (3-phenyipropanoyl)guanidine, 4-t-butylcinnamoylgnanidine, So 5<(N,N-DimethyDamiloride hydrochloride, oo N,N'-Bis(3-phenyipropanoyl)guanidine, : Co N-Benzoyl-N'-cirmamoylgusnidine and " 1-bromo-2-napthoylguanidine, : © 36. Thomethod according to claim 34, wherein ssid compound is selected from the group consisting of 4-phenylbenzoyigamnidine, (3- CT : hromocinnamoyl)guanidine, 3-(triftuoromethyfoimamoyiguanidine, 5-

   (N.N-boxamethyiene)emfloride, and (S-Phenyl-penta-24-

   37. The method according to any ane of claims 34 to 36, wherein seid HIV is HIV-1.

   38. The method according to claim 32 wherein said virus is a Coronavirus.

   39. The method according to claim 38, wherein said Coronavirus ia the Severe : - Acute Respiratory Syndrome virus (SARS). g Cs

   40. The method according to claim 39, wherein ssid compound is selected from the group consisting of . oo . . 23-Sifluoreimamoylguniiie, andi 4-t-butyleinnamoylgnanidina, 3-(2-upthylacryloylguanidine, : (3-Chlorocinnamoyf) guanidine, : 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, } 2,5-dimethyicirnamoyiguanidine, * trans-3-(1-napthyDacryloylgusnidine,

   (3-Bromocimmamoyl)guanidine, 6-methoxy-2-nsphthoylguanidine, : 5-(N-Metiry}-N-isobulylemiloride, L . } (2 Chi [3 1) - die 2'4 DichloroBenzamil HCl, {tif dryci leusmidine, : 3-(triflnoromethylcinnamoyiguanidine, 2-ethoxycinnamoylguanidine, rcth . Ixaaridi 2,6-dichlorocinnamoylignanidine, : 3 A4,5-trimethoxycinnamoylguanidine, 3-¢-butyicinnamoyignanidine, (4-Chlorocimnamoyl) guanidine, 2-t-butylcinnamoyiguanidine, oe 2-cyckoheny i rd yiguanidine, 3-(trans-hapt-1-en-1-yl)cinnamoylgnanidine, (4-Bromocinnamoy!) guanidine, : (4-Hydroxycimamoyl)gusnidine, N-(3-phonyipropencyl)-N'-phentylgusnidine, : : ~ (3-Nitrocinnamoy{)gnanidine, 3-finorocinnamoyignanidine, 2-(1-napthryf)acetoylgnmidine, S44 tuoroghenyamikride Co N-(6-Hydroxy-2-napthoyl)-N'-phenylgnanidine, (trans-2-Phenyloyclopropanecarbonyi)guanidine, N,N™-bis(3phenylpropanoy])- N"-phenylguanidine,, Lo 1-napthoyiguanidine, Benzamil hydrochloride, 3-methoxy -HMA, + mefhvici 48 ylcinnamoylgnanidine, g 3,4-(methylenedioxy)cionamoylgnanidine, 5-(NN-hexamethylene)amilorids, 5-(N-Ethyl-N-isopropyl)emiloride,

   : | | © 60 oo 2,3,5,6,-tetramethylcinnamoyignanidine, : trans-3-Furanacryoylgnanidine, ) of unsaid SR Furanacryloyl)guanidine, G iguanidine, [3-(3-Pyridyl)acryloyl]guanidine, : . 4-phenylbenzoylguenidine, SE (4-Phenoxybenzoyl)guanidine, ; © (a-Methylcinmamoyl)gnanidine, : : : 5-(3'-tromophenylpenta-2,4-dienoyiguanidine, : (5-Phenyl-penta-2,4-dienoyl guanidine, (Quinoline-2-carbonyl)gusnidine, (Phextylacotyl)guanidine, : N,N-Bis(smidino)napthalenc-2,6-dicarboxamide, . ¢-bromo-2-napthoyignanidine, . 1-bromo-2-napthoylgnanidine, 2-chioro-6-flucrocinnamoyignanidina, : : [(4-Chlorophenaxy-acetyljgnanidine, Phensmnil methanesalfonate salt , N-Benzoyl-N'-cinnamoyligoanidine and N~(2-napthoyl)-N'-phenylguamidine.

   41. The method scconding ® claim 39, wherein seid oormpound is slecied s . ~~ mapthyDecryloylguanidine, and 6-methoxy-2-naphthoyignanidine.

   42. The method acconting fo clsim 38, wherein seid Coronavirus is human Coronavirus 2298

   43. The method according to claim 42, wherein said compound is sslected from the group consisting of ’ 3 ,4-dichlorocinnamoyignanidine,

   : Co . 41 . 3(triftncromethoxy)cimamoylguanidine, 3-isopropyloimnamoyiguanidine hydrochloride, : 3-t-butykinnamoylguanidine, SE 2-+batylcinnamoylguanidine, SL trans-3-(1-napthyDacryloylguanidine, 5-bromo-2-methoxycinnamoylguanidins, } co rir ri : 2-napthyf)acryloyl guanidine, : 2-phenylcinnsmoylguanidine, Co - 3-phenylcinnamoylgnanidine, 3-(6yclohex-1-a0-1-yl)cimnamoyiguanidine, : . 4 pl 0 il 1 id : : 3-{triffuorometiryT)cinnamoyignanidine, (4-Phenoxybenzoyl)guanidine, naan, : 5-(N,N-hexamethylane)amiloride, - l-nepthoylguanidine, - 5-(4-flocrophenyDariloride, : - : : (5-Phenyl-pents-2 4-dienoylguanidine, : trifinoroenethyoirmemoyigiaanidine, Methoxycinnamoy)gnanidine, (2-Mothoxycinnamoyl)gusnidine, (a-Methylcinnamoyi)goanidine, | . 4-phenylcirmamoyiguanidine, 2,6-dichloroc P— . (2-Bro mo cl ) 0) . 4 . 2,4,6-trimethyloinnemoylguanidine, : (trens-2-FPhenyloyclopropanecarbonyl)gnanidine, 2-ethylcinnemoylguanidine, 2-sthoxycinnamoylgusmidine, | : 3-mathylcipmamoylgnsnidino, : 2-methyleinnemoyiguanidine,

   . ‘3 fluarocinnamoyignanidine, : pain 3d Vigumnidius . ig dime, . 2. . . dine, . : . pier Vigan ip 3,4-difluorocinnamoyiguanidine, © 2-pspthoylgusnidine, : I NN-Bis(amidino)napthalene-2,6-dicarboxamids, NN'-Bia(3-phexylpropanoyl)guanidine, Lo a 5-(3"bromophenyl)penta-2,4-dienoylguanidine, Co + 2,3,5,6,:tetramethylcinnamoylgnanidins, ) prea s Methoxycinnamoyf)guanidine, / , di ot . \opanidi } 3,4,5-trimethoxycinnemoylguanidine, rol iguanidi oo. N-3-phomyipropanoy)-N-phenylguanidie, : 5-(2"-bromophenyl)penta-2,4- | : . } Bromocirmamoyf)guanidine, (2 Nitrocd oy adi 4-Mothoxycinnmmoyl)goamidine, - A-(triftunoromet yl)cimamoy 1goemidine, i . [(E)-3-(4-Dimcthyiaminopherryf)-2- : } methylacryloyl)gnanidine, N-Benzoyl-N'-cinnamoyl guanidine, N-amidino-3-emino-S-phenyi-6-chioro-2- Pasi : id N-{cinnamoyl)}- N'phenylgnanidine, : Cimamoylguanidine,

   3.methoxy-amiloride, 3-methoxy -FIMA, : N-amidino-3,5-dlemino-S-phynyl-2- Pyrazinecart ia (Quinoline-2-carbonyl) guanidine, [3-(3-PyridyDacryloyl]guanidine,

   aw : N-(2-napthoyl)-N'-phenrylgaanidine and oo (Phenylacetyl)gnanidine. : N 44, The method according to claim 42, wherein ssid compound 1s sslected from the group consisting of 3,4-dichlorocinmamoylignanidine, oo 3-(trifiuoromethoxy)cinmamoyignanidine,

   2.6-dtivhloroct FJ— : 2-(cyclobex-1-an-1yIcimmamoylguanidine, 5-bromo-2-methoxycinnamoylgnanidine, 2-phenyici anand 4t-dutylcinnamoylguanidine, :

   3.phenyicimnamoylguanidine, 3-(2-napthyDecryloylguanidine, ) . . Fr mir dry) Bosnidine, adi © 5bromo-2-finorocinnamoyliguamidine, ¢ methyl p— : (4-Chlorocirmamoyl)gnanidine, Co 3-finorocinnamoyliguenidine, 3-(cyclohex-1-en-1-yl)cinnamoyiguanidine, (a-Methylcinnemoyl)gaanidine, : Co 2,3,5,6,-setramethyicinnamoyignanidine,

   2 . <q Lo Aen — ri ia : 2,5-dimethylcinnemoylgnenidine . 3¢-butyici emai + 3 Joely wr gnanidiue, {dine hydrochlorid © (5-Pheayl-pents-2,4-dicnoyDgnumidine, toll . \enanidi 4-fluorocinmamoylguanidine, : ] 3 A-dif} . Vignanids N-(3-phenylpropencyl)-N'- Phenylguanidi : 2,4,6-trimethrylcinnamoyiguanidine,

   : Methoxyei f)ganidi CC Cinnamoylguanidine, 3 A-(methylenedioxy)cinnamoylguanidine, : - ~ N.N'-Bis(amidino)nspthalens-2,6- : ] 2,3-dimethylcirmamoylgaanidine, N,N-Bia(3-phenylpropanoyl)guanidine,

   23. difluoroc lemaridt 1-napthoyiguanidine, . : 6-methoxy-2-naphthoyiguanidine, - 3 4 5. trimofoxyc amid 4-Hydroxycinnamoyf)gnanidine, : 5-(4-fiuorophenyl)amilotide, N 2-(1-naptirylacesoyignanidine, | : : N-Cinnamoyl-N',N'-dimethyignanidine, 2-(2-napthyecetoyignaniding and SE N,N'-bis(3phenyipropanoyl)-N"- : 45. Thomethod according to claim 38, wherein ssid Coronaviros is humm ~~~ s 46. Themathod according to claim 45, wherein said compound is selected from the group consisting of 3-methylcinnamoylguanidine, : trans-3-(1-napthyDacryloylguanidine, : (3-Bromocimmemoyl)gusnidine, . (2-Chloroct T)guanidi . 3,4-dichlwocimmamoyigusnidine, 3-(triflnoromathyl)cinnamoyignanidine, (trans-2-Phenylcyclopropanscarbonyi)guanidine, 4-isopropykirmamoylguanidine, no pe \epaidins,

   (4-Chilorocs Demand | : NE S-(N.N-hexamethylenelamiloride, : chloro iomiding, (5-Phemyl-penta-2,d-tisnoyl) guanidine, . SE 7} The method according to clsim 38, wherein said Coronavirus is porcine oo - 4% The method scconding to claim 47, wherein sald compound is selected ~~ from the group consisting of 5-(N.N-hexamethylenc)amilaride, : oo 3-mathylcinnamoyignanidine, . trans-2-Phenryloyolopropanecarbonyl)guanidine, " trans-3(1-napthyDecryloylguanidine and

   4. The method according to claim 38, wherein seid Coranavirus i bovine Caronvires (BCV). | : EE © 50." Themethod scoording fo claim 49, wherein siid compound is selected } from tho group consisting of . . trifinoromethyl)cinnamoyl guamdine : 6-methoxy-2-naphthoylguanidine, . trana-3-(1-napthylacryloylgusnidine, : Cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)gnanidine, 2-Phentylcyclopropanecarbonyl)guanidine, N-(3-pheayipropanoyl)-N'-phenrylgnanidine and

   : | : as 4phoybesoylgomidine. : st. The method socording to claim 38, wherein said Coronaviras is sny one of : the known Coronavirus isolates listed in Table 1. : .

   5. The method according to claim 51, wherein said oompound is sslectsd : 3 4-diohl (] A adi L . 3-(triffnoromethoxy)cinnamoylguanidine, . 3 1c Jauanidine hydrochlorid

   53. Themefhod soconding to claim 32, wherein seid virus is the Hopatits C oS

   54. The method according to claim 53, wherein suid compound is selocted from ths group consisting of * 5-bromo-2-fluorocinnamoylguanidine, . (4-Bromocirnamoyl)guanidine, 3(tiftuorométhyDcimnamoyi guanidine, - (2-Chloroci T)guamidi (4-Chlorocinnamoy!)gnanidine,

   2B . f)gaanids © 2,6-dichlorocinnamoyignanidine, GB ; Deuanidine, (4-Fhenoxybenzoyl) guanidine, : 3 4-dichloroei enanidi 4-isopropylcinnamoyl guanidine, trans-3-(1-napthyNacryloyiguanidine, : 4-t-butylcirmamoylguanidine, 2-t-butylcimnamoylgnanidine, ’ 4-methylcinmamoyiguanidine,

   N -147- ’ ) . 3 (t ifl & ) 1] 1 i ‘i 1-napthoylguanidine, 3-t-butylcinnsmoyiguanidine, (5-Phenyl-penta-2,4-dicnoyl)gumnidine, N-(cinnamoyl)-N'phenyiguanidine, : 3-isopropylcinnamoyignanidine hydrochloride, : Benzamil hydrochloride, N-(3-phenyipropanoyl)-N'-phenrylgnanidine, 5-{N-Methyl-N-isobutyl)amiloride, - 2'4 DichloroBemzamil HC), . flucrocinmamoylgnanidine, . @ o idt - qe : (irate 2 Phen eyed neces gua dine, . -cthoxychmamoylguanidine, 2,3,5,6,-tetramethyicinnamoylgnanidine, 4-phenryicinnamoyl guanidine, trang-3-Fursnacryoylgnanidine, N-(6-Hydroxy-2-napthoy{)-N'-phenylguanidine, 3-(cyclohex-1-en-1-ylimmamoyignanidine, 2,3-diftucrocinnemoyignanidine, 2-(1-napthyDecetoylgnanidine, (a-Methylsinnamoyl)guanidine, SE 6-Indosmiloride, 3,4-(methyleoodioxy)cinnamoyl guanidine, . 2 } 1 i 1 » 3 ' . ) 2-(cyclohex-1-en-1yloinnamoylgusnidine, : 2-napthoylgusnidine, : 3-phenyicinnamoyiguanidine, 5-(N.N-Dimethyl)amiloride hydrochloride, 5-(4-fluorophenyl)amilorids, (3-Methoxycinnamoyl)guanidine, 2-fluorocimnarnoylgnamidine,

   - | 148 N oo | | [(4-Chlorophenoxy-cetyljguanidine, (3-phenylpropanoyl)guanidine, : 2-chloro-6-fluorocinnamoylgusnidine,

   20 . Pp—y 2-Med N Dgusnidine, . 1-bromo-2-napthoyiguanidine, 3,4,5-triméthoxycinmamoylguenidine, 3-(trans-hept- 1-en-1-yl)cinmamoyiguanidine, Phenamil methanesulfonste salt , 2,4-dichlorocinnamolyguanidine, (4-Nitrocirmemoyl)guanidine, 3 4-diffuorocirmeamoylgnanidine and : : [(8)-3~(4-Dimcthylaminophenyl)-2- -

   Se . mefhylacryloyl]gnanidine. © 55 Themethod according to claim 32, wherein ssid virus is fhe Equine

   SE. 56. The method acooeding to claim 55, wherein said compound is sclested from the group consisting of Co -Bromocinnamoyl)guanidine, . : trans-3-(1-napthylaceyloylguanidine, ‘ } 2-t-butylcirnamoylgoanidine and 2-(cyclohex-1-en-1ylcinnamoylgusnidine. © 57. Tho method according fo say one of claims 32 10 56, wherein ssid compound is provided es a pharmaceutical compoaition sccording to claim : 4 ar claim 5. } 58, A method for the therapentic or prophylactic treatment of a subject oo "infected with or exposed to a virus, comprising the administration of a Bn compound according to any ene of clxims 1 to 3 to a subject in noed of said treatment,

   59. The method according to olsim 58, wherein said virus is a Lentivirus ae

   60. Tho method according to claim 59, wherein said Lentivirns is Human oo " 61. The method according to claim 60, wherein said compound is selected from the group consisting of © (3-Bromocimamoyl)guanidine, oo (2-Chlorocinnamoyl)guanidine, (2-Bromocinnamoyl) guanidine, i : S-bromo-2-fiucrocinnamoyiguanidine, ~ 2,3-dimathylcinnamoylgusnidine, : 6-methoxy-2-naphthoylgnanidine, : trans-3-(1-napthylucryloyigosnidine, : 3,4-dichlorocirnamoyignanidine, ( ’ 2-napthoylgnanidine, - (5-Phenyk-penta-2,4-dienoylgnanidine, ae: : _ 3-(cyclohex-1-en-1-yl)einnamoylgnanidine, : N,N'-bis(3phenylpropanoy!)-N"-phenyl guanidine, N-(3-phenyipropanoyl)-N'-phenylguanidine, 4-(tifuoromethyl)oinnamoylgnaniding, : -butylcinnamoylguanidine, : 4-mettiylcinnamoylguanidine, 2 fooroct guanidine Hydtury. 2 napthoyl)-N' phenyiguenidine, : 34-diflnorocinnamoyiguanidine, $-QUN-hexamethylenejamiloride, oo eth » dine. . 2'4 DichloroBenzamil HCL, . Lo © 2,3,5,6,-tettamethylcimamoylgusnidine, 3-2-neptiryNacryloylgnanidine, + 2-(1-napthyDacetoylgnauidine, 23 Ef p Jguanidine, oo . 2,4,6-trhmethylcinnamoylguanidine, Co mr Li wt fry) Adomrys } (4-Bydroxycimmamoyl)gnanidine, 4-finorocinnemoyiguanidine, } NN N-bis~(cirmamnoy])-N"-phenylguanidine, : (2-Puranacryloyl)gnamidine, = - Phenemil methanosuifonate salt , ' Benzamil hydrochloride, . (3-Nitrocinnamoyl)goanidine, 4-Phenoxybenzoyl)guanidine, 3-(trane-heps-1-en-1-y)cimmamoyignanidine, 5-(N-Mothyl-N-isobutyl)amiloride, eq : = yooh icimmao yignanidine, ” N-saidino-3-amino-5.4 cirvieneiinino-6-phenyl 2-pyrezinecarboxantide, N-amidino-3-arhino-S-phenyl-6-chloro-2- : © pyrazinecarboxamide, 5-(4-finorophenyDemiloride, (trana-2-Phenyicyclopropanecarbonyl) guanidine, (2-Nitrocinmsmoy{)gnanidine, trans-3-Furanacryoylgnanidine, ~ 3-methoxy—HMA, (3-phenylpropencyl)gnanidine, 3 | 5-(N,N-Dimethyamiloride hydrochloride,

   oo =13a- NN-Bis(3-phenylpropanoyDguanidine, N-Benzoyl-N'-cinnamoylguanidine and : 1-bromo-2-napthoyigusnidine. Co

   62. The methods according to claim 60, wherein ssid compound is selected from the group consisting of 4-phenyibenzoylguanidine, (3- . bromocinnamoyf)gusnidine, 3-(trifluoromethyl)oinnamoylguanidine, S- : 5 (NN-hexamethylene)amiloride, and (S-Phenyl-penta-2,4-

   63. Thomefhod according to any one of elaima 60 to 62, wherein seid HIV is HIV-1. - :

   64. The method according to claim 58 wherein said virus is a Coronavirus.

   65. The method according to claim 64, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS). Co 15

   66. The method sccording to claim 65, wherein said compound is selected from the group consisting of

   2.3 difh 20roci 1 1 id i 3 2 2 - ge 3-(2-napthyDecryloyiguanidine, 3-(cyclohex-1-en-1-ylcinnamoylgnmidine, trans-3(1-napthylacryloylgnanidine, 4-isopropylcinnamoylgnanidine, © (3-Bromocimnamoyl)guanidine, 6-methoxy-2-nasphthoylguanidine, 5-(N-Methyl-N-fsobutylamilaride, 3-phenyloi nid (2-Chlorocinnamoyl)guanidine, : 2'4 DichloroBenzamil HCL, 4-pheuyloinmamoyiguanidine, . 4-{rifiuoramothyl)oinaamoyiguanidine, 2-ethoxycinnamoylgumidine,

   ~i52- 4-othoxyoinnamoylguanidine, (2-Bramocinnamoyl)gnanidine, 3,4,5-trimethoxycinnamoylguanidine, ; yl ylamino {gum rg 5-bromo-2-flucrocinnamoylguanidine, : (4-Chlorochmamoyl) guanidine, CL So 2-cyclohexylcinnamoylguanidine, _ - 6-Todoamiloride, .

   : 3-(trans-hept-1-en-1-ylcinnamoylguanidine, (4-BramocinnamoyDguanidine, : Lo (4-Hydroxycimnemoyl)guanidine, ~~ en paki, Nitrocinnamoylgusnidine, . 3-flucrocinmamoylguanidine, a. : LW wicowi wen trifluoromethyl)cinnamoylguanidine, : N-(6-Hydroxy-2-napthoyl)-N'-phenyignanidine, (trans-2-Phenyicyclopropanccarbonyl)goanidine, } N;N'-bis(3pheayipropanoyl)-N"-phanyignanidine,, 1-napthoylguanidine, finorocinnamoylgnanidine, - 5-(N,N-hexamethyleme)amiloride, : N-(cinnamoy])-N'phenylgnanidine, S-(N-Rehryl-N-isopropyl)amiloride, 3-mechylcinnamoyignanidine, 2,3,5,6,-tetramethylcimamoylguanidine, trams-3-Fursnsoryoyi guanidine, (4-Methoxycirmamoyi)gusnidine, . (2-FurenacryloyD guanidine, 2-(2-napthylacetoylguanidine, ) - (2-Methoxycinnamoyl)guanidine, 4-phenylbanzoylguanidine,

   2, i i y i ir . -fiuorocimnamoylguanidine, Mi al ao o Methylo: Dguanidine, '5-(3'-bromophenyl)pents-2,4-dionoylguanidine, . (5-Phenyl-penta-2,4-dicnoyDguastidine, (Phenylacetyl)guanidine,

   N.N'-Bis(amidino)napthalene-2,6-dicarboxamide, 1-hromo-2-napthoylgusnidine, . [(4-Chlorophenoxy-acetyl]guanidine, } Phenamil methanesulfonato salt , : N-Benzoyh-N'~cimamoylguanidine and :

   67. The method according to claim 65, wherein said compound is selected © from the group consisting of cinnamoylguanidine, trans-3-(1- napthyl)acryloyiguanidine, and 6-methoxy-2-niphthoylguanidine, : BE

   68. The method according to claim 64, wherein said Coronavirus is inmman

   69. The mettiod according alsin 68, wherein sad compormd is selected fron the group consisting of 4 isopropylci 1 . $ 3,4-dichlorocinnamoylgnanidine, : trans-3-(1-napthyfecryloylgnanidine, S-bromo-2-methoxycinnamoyl guanidin e, 2,3-diflmoroc fomanid 2-phenylcinnamoylgusoidine, : 3-phentylcinmamoylguaniding, Co 3-(oyclohex-1-en-1-yl)cinnamoylguanidine, oo - 3-(trifluoromethyl)cinnemoylgusnidine, oC

   . : ’ | . -154- Co 4-(trifinoromethyDcinnamoylguanidine, 2-(cyclohex-1-en-1yl)cirmeamoylguanidine, (4-Bromocinnamoyl)gnanidine, * l-napthoylgnanidine, : 5{4-fluorophenyleimiloride, ~~ - : © (5-Phenyl-penta-2,4-diencyl guanidine, (3-Bromocinnamoyl)guanidine, \ 2-(triflnoromethyl) (4 | idi . 6-methoxy-2-naphthoylguanidine, _ (4-Chlorocinnsmoyl)gnanidine, Lo (3-Mothoxycimnamoyl)guanidine, ! 5-bromo-2-finorocirmamoyi guanidine, (»-Methylcimamoyl)guanidine, : = Perris: . ,6 trim efhryloi A id } . © (trne-2 Phenyloyclopropmocarbomy)gumidine, 2-(1-naptiryl)acotoylguanidine, - 2-othrvici fenamidi (4-Hydroxycinnemoyl) guanidine, B . . 2-ethoxycinnamoyignanidine, ! 3-methylcionamoyiguanidine, : 2-methvick Jenamidi cinnamoylgusnidine hydrochloride,

   : . ok as ome " panic , 4-finorocimnamoylguamiding CL 3,4-difluorocinnamoyignanidine, 5-tart-butylami tlocid 2-oapthoylgustidine,

   N.N-Bis(amidino)napthaleno-2,6-dicerboxamide,

   N.N-Bis(3-phenylpropanoyl) guanidine, 4-methylcinnamoyiguanidine, 5-(3'-bromophenyf)penta-2,4-dienoylgnanidine, 2,3,5,6,-tetramethyicinnamoyignanidine,

   : + N,N"-bis(3phenylpropanoyl)-N"-phenylgnanidine, (4-Mechoxycinnamoyl)guanidine, : (2-Chlorocimmamoyl)guanidine, (3-Nitrocinnamoyl) guanidine, ,S-trimathoxycinnamoylgnanidine, 2-(2-napthyDacetoylgoanidine, N-(3-phenyipropanoyl)-N'-phearylguanidine,

   5.@-bromophenyDpentad,d- : prides Bromocinnamoyl)guanidine, (2-Nitrochmamoyl)guanidine, (3-Chloroci — : (4-Methoxycimemoyf)guanidine, [(B)-3-(4-Dimethylaminophenyl)-2- N-Benzoy}-N'-cinnamoyignanidine, trans-3-Furansoryoylgnanidine, . : Nesniding-3-amitio-5.pheay! .. Pyrazinecarboxamide, N-(cinnamoyl)-N'phenylguanidine, Cirnamoyl gnanidine, N-amidino-3,5-dismino-6-phymyi-2- Pyrazinecarhaxamide, [3-(3-Pyridyljacryloyl] guanidine, N-Cinnamoyl-N'N'-dimethylguanidine, CL * N«{(2-napthoyl)-N'-phenyliguanidine and : (Phenrylacetyl) guanidine.

   70. The method according to claim 68, wherein said compound is aclected from the group consisting of: : 2-t-butylcinmamoylignanidine . » E : i : 3 4-dichlorocinnamoylgnanidins, : 3-(trifiu d - SO oeemoxy) guanidine v ’ : 2+(cyclobhex-1-en-1yl)cinnsmoyiguanidine, : 2-oyolohexylci tami pa

   4-t-butylcimamoylgnanidine, (3-Bromocinnamoyl)gnanidine, 5-(N.N-hexamethylens)amiloride, trans-3-(1-napthylacryloyignanidine, 3-(2-napthyDaceyloylguanidine, 2,4-dichlorocirmamolyguanidine, S5-bromo-2-finorocinmamoyiguanidine, : : 4-methylcinnamoyignanidine, : 3-fluorocinmemoylgnanidine, I 3-(cyclohex-1-en-1-yl)cinnamoylgnanidine, (a-Methylkinnemoyl) guanidine, : : 23:5, tdtraethylccomatioy guanidine, floorocinnemoyignanidine, . 3 Nitro , 3-t-butylcinr A [ idi 3 (3-Methoxycinnamoyl)guanidine, . : 3-mothylcimamoyigusnidine, : (2-Bromocinnamoyl)gnanidine, 3-sthoxycinnamoylgnanidine, Co (5-Phenyl-penta-2,4-dienoyl)gusnidine, (2-Chlorocirnamoyl)guanidine, ) 4-othoxycinnemoylgusnidine, 4-fluorocimnamoyignanidine, : 3,4~diflnorocinnamoyignanidine, . SE N-(3-phenryipropanoyl)-N'- guanidt 4, 6-trimethyicimamoyiguanidine, " (trans-2-Phenylcyclopropanecarbonyl)- Phenoxybenzoylguanidine, Co 3,4-{(methylenedioxy)cinnamoylgusmidine, N,N'-Bis(smidino)napthalenc-2,6- Co Dioatboxamidse, - NN DC Ras. it : ~ 1-napthoylguanidine,

   . a : . . 157- 2 (tit thyi)ci fgmanidi . Fursnacryloyl)guanidine, } N-Cirnamoy}-N'N'-dimethylgusnidine, NN-bla(iphenyipropanoy)}-N"

   ©. 71. The method according to claim 64, whersin said Coronavirus is barman . S72 Themethod according claim 71, wherein seid compound is selected © fromthe group oonsistingof : 3 methvici tenanid: : : trane-3-(1-nspthylacryloyignanidine, . (3-Bromocimamoyf)gnamidine, @-Chlcwociungmoy)guanidite, : : JA-dichlorocimamoylguanidine, : : : 2,4-dichlorocinnamolyguanidine, + (4-Chlorocinnamoyl)guanidine, : : 5-(NN-bexamethrylene)emiloride, oo (4-Bromocinnamoyf)guanidine, : : " 2,6-dichlorocinnamoyiguanidine, : 5-bromo-2-methoxycimmemoyigoanidine, : (5-Phenyl-pents-2,4-dienoyl) guanidine, 2-t-butylcinnamoylgnanidine.

   73. The method socording to claim 64, wherein said Coronavirus is porcine §

   Co -158-

   74. The method according to claim 73, wherein said compound is selected : 5-(N)N-hexamethylene)amiloride, 6-methoxy-2-naphthoyl guanidine, N-(3-phenyipropanoyl)-N'-phenyl guanidine, 3-methyicinnamoylguanidine, (trems-2-PhenyloyolopropanccarbonyDguanidine, © trane-3-(1-napthyDecryloylguanidine and oo 2-(2-napthyl)acetoylguanidine. 75 Themathod sccoeding1o clsim 64, wherein ssid Coronavirus is bovine

   76. The method sccceding fo clan 75, wherein sid compound is selected h from the group consisting of - 3(triftnoromefhryi)cinnamoyignanidine, 6-methoxy-2-naphthoylgnanidine, (trans-2-Phentyloyclopropanecarbonyf) guanidine, : 0 Co

   77. The method according to claim 64, wherein sxid Coronavirus is sary ons of - the known Coronavirus isolates listed in Table 1. oo 78. Tho method according to claim 77, Wherein said compound is selected from the group consisting of 4-isopropylcinnamoylguanidine, : 3,4-dichlorocirmamoyiguatidine, 3-(triflnoromethoxy)chmamoyiguanidine, 3-jsopropylcinnamoyiguanidine hydrochloride,

   79. The method according to claim 58, wherein ssid virus is the Hepatitis C virus, :

   80. The method according to claim 79, wherein said compound is selected

   5 . from the group consisting of : 2 3 dimethyics — 5-bromo-2-flucrocinnamoyiguanidine, -dimethylcinnamoylguamidine, : if hyf)ct lgnanidi 6-methoxy-2-nsphthoylguanidine, 2-Chloroc Dguanidi (4-Chilorocinnamoyl)gnanidine, oo (2-Bromocinnamoy{)gnanidine, 2,6-dichlorocinnamoyiguanidine, (3-Chlorocimamoyl)gusmidine, } A N me irituorome un poVlgaamidine, : isopropylcirmamoylgaanidine, trane-3-(1-nepthylecryloyiguanidine, 24¢-butylcinnamoylgnanidine, 2-etdylcinnsmoylgnanidine, 4-methylolonamoyignanidine, 2-cyclohexykinn ax yignani SYiganidine, ® : t-butylcimamoylguanidine, 4-phenylbenzoylguanidine, : rh po mn aii, ‘N+(cinnamoyl)-N'phenylgnenidine, Benzamil hydrochloride, © N-(3-phenylpropanoyl)-N'-phenyl guanidine, N,N-bis(3phenyipropanoyl)-N"-phenylguanidine, ce 3~(2-napthyl)acryloylguanidine, ; 5-(N-Methyl-N-isobutylemilorids, 2'4 DichloroBenzamil HC], 5-tert-butylamino-amiloride, 3-(N-Bthyi-N-isopropyl)amiloride, (4-Methoxycinnamoyl)gusnidine,

   (3-Nitrociznamoyl)guanidine, (+ Hydroxycimamoyguenidine, (trans-2-Phenyloyclopropanscarbonyl)guenidine, : 2,3,5,6,-tetramethyloinnamoylguanidine, . trams 3 o : Furanacryoylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenyiguanidine,. : 3-(cyclohex-1-en-1-yl)cinnamoylgusnidine, : : | . lgnanidine hydrochlosid a RE

   2 3.diff . 1 " : .° 2-(1-napthyDacetoylguanidine, : : (a-Methylcimnamoyl)gnanidine, (2-Nitocinnamoyl) guanidine, 344 a (metyienediny aiid lpanidioe, 2-phenylcinnamoylgusnidine, Co 2-(cyclohex-1-en-1yeinnamoylgnanidine, 2-napthoylgnanidine, finorocinnamoyiguanidine, 4-Chlorophenaxy-acetyl]guanidine, oo 3-finorocimemoylgnanidine, 2-methylcirmemoylguanidine, Ceo 3 map tine, 34,5 trimethoxyoinnemoylgnucidios, methylcinnamoylguanidine, . 3(trans-hept-1-en-1-yl)cimamoyiguaniding, Phanamil methanesulfonato salt, : ’ (4-Nitrocinnamoyf)guanidins, 3,4-difluorocinnamoylgnanidine and [(E)-3-(4-Dimethylaminopheryl)-2-

   CL 161- Co

   81. The method according to claim 58, wherein said virus is the Equine oo 22. The method scoording to claim 81, wherein said compound is selected Cs from fhe group consisting of : 5-(N,N-hexemethrylono)emiloride, : (3-Bromootnnamoyl)guenidine, . : : trans-3-(1-napthyDacryloylguanidine, 2-t-butyicinnamoylguanidine and 2-(cyclohex-1-ca-1yl)cinnemoyl guanidine. : 8 "The method according fo say one of claims 58 to 82, wharein ssid : coutpound is provided 8s 8 pharmaceutioal composition scoording to claim . #4 orclaim §. : _ 84. * A method of down regulating a membrane jon channel finctional activity : in a oell infected with a virus, comprising contacting said cell with a compound according to ary one of claims 1 to 3. 45 8S. The method according tn claim 84, wherein sxid virus is a Lentiviras. © 8 Themethod according to cli 85, wherein said Lentivirae is Homan Immunodeficiency Viros (HIV). Co :

   87. Themethod according to claim 86, wherein ssid membrane ion chanel i

   88. The method socording to claim 87, wherein said compound is selected . oo fon dear of * _ (3-Bromocimamoyl)guanidins, (2-Chlorocinnamoyf)guanidine, trifinoromethyl)cirmamoyiguanidine, ~ : 5-bromo-2-flnorocinnamoyigasnidine, :

   . . : nN 162

   Ll . . 4-dichlorocinnamoylguanidine, . Co 2-ethylcinnamoyignanidine, : (4-Chlorocinnamoyf)guanidine,, 2-napthoylguanidine, 2,5-dimethylchmamoyiguanidins, as © 3-isopropylcimnamoylgnanidine hydrochloride, - (5-Phenyi-penta-2,4-dienoyl)guanidine, . (4-Bromocinmamoyl)guanidine, Co : 5-(3-bromophayl)penta-2A-diepoyl guanidine, 3-{cyclobex-1-en-1-yl)cinnamoylgnanidine, 3: ia ehoxyet \gnmidine, trifinoromathyl)cinnamoyignanidine,

   N.N-bis(phenyipropanoyl)-N"-pheayl guanidino, : 2-sthoxyvinnamoylguanidine, N-(3-phenyipropanoyl)-N-phenylgoanidine, : 4 Methoxyoinnamoyf)gnanidine, : 2-4-butyicimamoylguanidine, _ 4-meothylcinnamoylguanidine, . - | ! 2 prop ei Vago id 3-t-butyloinnamoyiguanidine, - : finorocinmamoylignanidine, bromo-2-methoxycinnamoylgusnidine, 3,A-(mothylenedioxy)cimamoyl guanidine, Co (2-Methoxycinnamoyl)gnanidine, : 2'4 DichloraBenzamil HCI, 2,3,5,6,-tetcamethylcirmamoylgnanidine, uorocinnamoylguanidine, (3-Mothoxychnamoy]) guanidine, 4-isopropylcimamoyiguanidive, 2-(cyclohex-1-en-1yl)cinnemoylignanidine,

   Sana PE it ou fluorocinmnamoylguanidine, (2-Fursnacryloyf)gnanidine, : Phenamil methancsulfonato salt, prem hydrochloride, ‘Benyoylgiamidtoe Phenoxybonzoyl)gusmnidine, tans hopt-1-ct I-yDcimumoylguamidine, 5-(N-Mathyl-N-isobutylamiloride, 2-cyolohexyloimamoyiguanidine, . 4-cthoxycirnamoylgusnidine, : (a-Methylcinnamoyl)guanidine, ‘4-Chiorophenoxy-acetyl]gnanidine, N-emidino-3-emino-5-phenyl-6-chlaro-2- pyrazinecarboxamide, : . S<{4-floorophenyDemiloride, (trans-2-Phenyloyclopropanccarbonyl)guanidine, (2-Nitrocimamoyl)guanidine, trans-3-Furmacryoylguenidine, 1-napthoylgnanidine, S-tert-butylamino-amiloride, 3-methoxy -HMA, (3-phenyipropanoyl)guanidine, 4-t-butylcinnsmoylgnanidine, : 5-(N;N-DimethyDamiloride hydrochlaride, : N,N'-Bis(3-pheaylpropanoyl)gnanidine, N-Benzoyl-N*-cinnamoyignanidine and . 1-bromo-2-napthoylgusnidine. : 80. The method according to any one of claims 86 to 88, wherein sald HIV is HIV-1.

   90. The method according to claim 84, wherein seid virus is 4 Coronavirus, . 91. The method according to claim 90, wherein said membrane jon channel is the Coronavirus E protein. Co

   92. The method according to claim 91, whortin said Coronavirus is the Severo © Acute Respiratory Syndrome virus (SARS).

   93. Tho method according to claim 52, whaesin said compound is selected "from the group consisting of : 2 3-diffucrocinnamoylguanidine, 3,4 dichloroci tener 3«(2-napthyDacryloylguanidine, . (3-Chlorocinnamoyl)guanidine, : Hoaber trang-3-(1-napthyDecryloylguanidine, : : 4-isopropylcinnamoyiguanidine, (3-Bromocinnamoyl)guanidine,

   ©. S{N-Motiyl-N-isobutyDamileride,

   3.phenylcinnamoyiguanidine, : 3-{t iff gd ) . 1 idi . 3-(triftnoromethyf)cinnsmoyiguanidine, 4-cthoxycinnamoylgnanidine, (2-Bromocinnamoyf)gnanidine, . . rye — guanidine, oh S-gert-butylamino-amiloride, : 3-t-butylcinnamoylgnanidine, eat +-Chioroci Deguanidine, 2-t-butyleinnamoylgnanidine, 6-lodoamilaride, Cuma Aor 4-Bromocimamoyl)guanidine, (4-Hydroxycinnsmoyl)guanidine, ~ No(3-phenylpropanoyl)-N'-phenyiguanidine, - 3-fluorocinnamoylguanidine, 2-(1-apthyl)acetoylguanidine,

   2-napthoyiguanidine, : 5-(4-finoropheny)amiloride, . 2-(triffluoromethyDcinnamoylgnanidine, N+(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, (trans-2-Phenylcyclopropanecarbonyl) guanidine, N,N-bis(3pbenylpropanoyl)-N"-phenylgoanidine, Co 1-nspthoylgnanidine, Beozamil hydrochloride,

   3.mothoxy -HMA, 4-methyicinnamoylguanidine, : ) 4-fluorocinnamoylguanidine, 3,4-(methylenadioxy)cinnamoylgnanidine, . 5-(N,N-baxamethylene)amilorids, N-{cinnamoyl)-N'phenyiguanidine, © 5-(N-Bthyl-N-isopropyDamiloride, 3-methylcinnamoyignsnidine, : 2,3.5,6,-totramethylcinnamoyignanidine, Cinnamoyignsnidine, (2-Mothoxycinmamoyl)guaidine, [3-(3-PyridyDucryloyl]gusnidine, 2,4-dichlorocinnamolygoanidine, (3- Mott : nani 2-finorociznemoylgnenidine, (4-Phenoxybensoyf)gusnidine, : (»-Meshylcinnamoyf)gnanidine, 5-(3"-bromophenylpenta-2,4-dienoylgnanidina, (5-Pheny}-penta-2,4-diencyl)guanidine, (Quinoline-2-oarbonyl) guanidine, : (Phenylacctyl)guanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamida, } _ 6-bromo-2-napthoyignanidine, 1-bromo-2-napthoyignanidine, 2-chloro-6-fluarocinnamoylguanidine, [(4-Chlorophenoxy-acetyl]goanidine, Pheoami] methanesulfonats salt , N-Benzoyl-N'-cinnamoylguanidine and : N-(2-napthoyl)-N'-phenylgoanidine.

   aes

   %4. ho method scoording to claim 91, wherein ssid Corsnavirns is Bs. Coronavirus 2298. ~

   95. TR "from the group consisting of 3,4-dichlarocinnamoylguanidine, 3-{nifhormethony mam Lari, 4--butylcinnemoyl gum ari rar hydrochloride, 2-tbutyloinnamoylgnanidivs, trane-3-(1-napthyDecryloylgnanidins, 5 -bromo-2-Methoxycinsmoylgnanidins, 2-phenylinnsmoyigeanidine,

   3.ghenyloinnamoyigeanidios, 3 {cyclobex-1-60-1-yi)cinnamoylguanidins, 4 chenyl} apanidi 3{uiffuoromethyf)cinnamoyigaanidine, a (4-Phencxybenzoyguanidios, 2{cyclobex-1-em-1yDcimmumoylguamidine, (4 BromocinnemoyDgnanidine, 5-(NN-hexamethylencjamlocide, 1-napthoylgnmidine, : 5-{4-fivorophenyfamiloride, (S-Phonyl-pats-2 A-dienoylgoamidine, 2-{trifivoromethylcinmamoylgnanidins, Ct pita, Mott . 1) guaid CO eens 2 forocineamoylgnanidine,

   5.(N,N-Dimethylamiloride hydrochloride, Cinpamoylguanidine, (s-MethykcinnamoyT)guanidine, 4-phenyloinnamoylguanidine, . 2,6-diohlorocipnamoylgnanidine, (2-Bromocinnamoylguanidine, 2,4,6-trimethylcinnamoylguaniding,

   (trate-2-Phenylcyclopropanecmbanyl) guanidine, (3-Chlorocinnsmoyl)guanidine, reymcheryich meygratiion di (4-BydroxycipnamoyDgnanidins, 3-methylcimamoylguanidine, . . 2-msthylcirnsmoylgnanidine, 3-finorocinnamoyiguanidine, cinmamoyiguanidine hydrochloride, Ar fn . A euanidi A nas : if ; enanidi } NB _Bis(3-pheryipropanoyl)guanidine, : methyl \enanidi . 5-(3"bromopheryNpents-2,4-dienoylgamidine, 3 sthoxycinnamoylgnanidine . Pre Mp a . (3-Nitrocimamoyl)guanidine, y ppv — Yigaaridine, i. 2-(2-napthyDacctoyignanidine and NC3pheay? 2037) N-phenylgnanidin

   96. The method according to claim 91, wherein said Coronaviras ia any onc of fhe known Coronavirus isolates listed in Table 1. : :

   97. The method sccarding to claim 96, wharein said compound ia selected from the group consisting of 4-isopropyloinnamoylguanidine, 3,4-dichlorocinnamoylgusnidine, 3-(trifluoromethoxy)cinnamoylguanidine, - _ 3-isopropylcimamoylguanidine hydrochlorids,

   98. - The matiod according t claim 84, wherein sid virus i the Hepatita C s 99. Themefhod accanding to claim 98, wherein ssid membrane ion chamel is "the Hepatitis C virus p7 merabrano jon chenmel. : "100. Tho method socording to claim 99, wherein ssid compound is selected 5-bromo-2-flucrocinnamoyiguanidine, : (4-Bromocinnamoyl)gnanidine, . > 5 dimetiryici tomanidi 3(trifinoromethyf)cinnamoylguanidine, A(triftooromethyl)cinmamoylgneniding, - : 6-methoxy-2-naphthoyl guanidine, . 2,6-dichiorocinamoylguanidine, triffuoromethyl)cinnamoylignanidine, : (4-FhenoxybenzoyDguanidine, 3,4-dichlorocimnamoylgusnidine, _ : teonconvict AI 2-tbutylcinnamoyiguanidine, 2-ethylcinnamoylguanidine,

   _ 5.bromo-2-methoxycimamoylguanidine, : 3 if} i ) ¢ 1 nidi . 2-cyolobaxyloinnamoyignanidine, : 1-napthoyiguanidine, 3:4 utylaimamny guste, (5-Phenyl-pents-2,4-dienoyl)guanidine, N-(cinnamoy)-N'phanylgnanidine, : 34 rd teaanidine hydrochlorid

   : | -169-

   _. " 2'4 DichloroBenzamil ry 5~(N-BEthyl-N-isopropylJamiloride, (3-Nitrocinnamoyl) guanidine, :

   ) . i . . ethos —— ys di (trans-2-Phenylcyclopropanccarbonyl) guanidine, 2,3,5,6,-tetramethylcinnamoylgusnidine, . 4-phenylcimamoylgnanidine, : 3-7 vl . ii . - oo © N+{(6-Hydroxy-2-napthoy!)-N'-phenryl guanidine, (2-FurenacryloyDguanidine, : oy 3-(cyclohex-1-m-1-yl)cinnamoylgnanidine, . {smanidine hvdrochlorid Mottvict Dgnanidin oo - (2-NitrocinnemoyT) guanidine, } S Todoamilorid * 3 A-{(methylensdioxy)cinnamoylgnsnidine, aed tenenidi Cinnamoylguanidine, : 2-phenylcinmmmoyignanidine, 2-(cyclobex-1-en-1ylcinnamoyiguanidine, : 2-napthoyigasnidine, . 3-phenylcinnsmoyignanidine, : 2-flucrocinnamoylguanidine, : Se A ey aciiion 4 Chlorord gnanid (3-phenylpropencyl)guanidine, aE 2 hloro-6-2 : ieuanidi . finorocinnemo yigusnidine, 0 2-methyicinnamoyigusnidine, - (2-Methoxycinnamoy)guznidine, ,5-trimethoxycinnemoyigusnidine, . 3-(trans-hept-1-en-1-yl)cinnamoyignanidine, : Phenamil molhencsalfonate salt,

   -170- oo (4-Nitrociomamoyl)guanidine, p 3 4-diffiuorocinmamoyignanidine and : [(E)-3(4-Dimethylmminophenyl)}-2-

   101. The method scoording 10 any one of claims 84 to 100, wherain said : : ‘compound is provided as a pharmaceutical composition recording to claim - 4 orolaim S.

   102. A method of reducing, rotarding or otherwise inkibiting growth and/or oo replication of 8 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 ssid compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in suid infected oell

   103. The mathod according to claim 102, whevein said virus is a Lentivirua. : Co

   104. The method according to claim 103, wherein said Lentivirus is Human Imnmnodeficiency Viras (HIV).

   105. The method according to claim 104, wherein ssid membrane ion channel is the HIV Vpu membrane ion charmel. ~ © 106. The method according to claim 105, wherein said compound is sclested from the group consisting of Chlorock guanidi (2-Bromocinnamoyl)guanidine, {rif yc tenamidt : ‘S-bramo-2-fluorocinnamoyl guanidine, 3-methyloimmamoylgnanidine, 2 methict enanidi k | AT - - : 6-mothoxy-2-naphthoylgvamidine, - trans-3-(1-napthylacryloylgnanidine, 3,4-dishlorocinnamoylgusnidine, } 26 y hic [3 1 ids i (4-Chlorocinnamoyl)gusmnidine,, : 3-isopropylcinnamoylgnanidine hydrochloride, (5-Phenyl-pents-2, 4-dienoyl) guanidine, (4-Bromocinnamoyl)gusnidine,

   o .. 3(cyolohex-1-en-1-yl)cinnamoyignanidine, g 2-{trifluoromethyl)cinnamoyignanidine, 2-othoxycinnamoylgnanidine, N-(3-phenylpropanoyl)-N'-phenyignanidine, . _ 2-+butylcinnamoylgnanidi on bg pu thylcimnamo Vigeuidicn, " N+(6-Hydroxy-2-napthoyl)-N'-phenyignanidine, - . 3-tdutykinnemoylgnanidine, : 3 A-diflnorocinnamoyignanidine, SVN -hoxamothyienelerilocide,

   a . Ars S-bromo-2-methoxycinnamoyignanidino, . 3 oth OXY in i . dt : Methoxycinnemoyl)guatidine, 2'4 DichloroBanzamil HC), > 2<(1-napthryDacetoylgumnidine, : 2,3-difluorocinnamoylgnanidine, . (3-Methoxycinnamoyl) guanidine, : 4-isopropylcirmamoylguanidine, : 2,4,6-trimethylcimamoylguanidine, Neo 1)-N'phenylguamidi _° 24{cyclohex-1-en-1yl)cinnamoylguanidine, 2-2-napthyl)acetoylgnanidine, . (4-Bydroxycinnamoyl)guanidine,

   AT 4 he Act 1 idi 5 4-flucrocinnamoylgnanidine, . :

   : . N,N'-bis-(cinnamoyl)-N"-phenylgnanidine, - (2-Fursnacryloyl) guanidine, (4-Phenoxybenzoyl) guanidine, : 3-(trans-hopt-1-en-1-yl)cirmamoyigaanidine, - oo SHO Math isobar, -cyclobexylcinnamoyiguanidine, : tol ; teuanidi 2 4-dichlorocinmamolygranidine, : SRR 5-(N-Ethryl-N-isopropyDamilorids, 2-pyrazinocartboxamids, : : (s-Methylcnnemoyl) guanidine, 4-Chlarophenoxy-acetyl]gaanidine, . pyrszinecarboxamide, _ 5~(4-finorophenrylamilaride, (tras-2-Phanyloyclopropanecarbonyl)gnanidine, : } (2-Nitr 4 ) id . trans-3-Porsnacryoyignanidine, -napfhoylguanidi

   : . S-taxt-butylamino-amiloride, . (3-phenylpeopancyl)gramidine -Bis(3-phenyipropenoyf)gnanidine, N-Benzoyl-N'-cimnamoyignanidine and : : 1-bromo-2-napthoylgnsmidine. © 107. ‘Themethod according to say cue of claims 104 to 106, wherein said HIV : is HIV-1. SE : | :

   108. The method according to claim 102, wherein said virus is a Coronavirus.

   109. The method according to claim 108, wherein sed menbeums fon chara oo is the Coronsvirus B protein.

   110. The method according to claim 109, wherein said Coronavirus is the Severo Acute Respiratory Syndrome virus (SARS).

   nt. The method 4 according ing to claim 110, wherein ssid compound is selected from the group consisting of utvict Inamidine. 3~2-napthyDacryloylgnanidine, (3-Chlorocinnamoyl)gnanidine, : 3-(cyclohex-1-en-1-yDcinamoyiguanidine,

   2 5. dimetiyict Ay trans-3-(1-napthyl)acryloylgusnidine, GB " Dgnanidi ’ 6-methoxy-2-naphthoyiguanidine, 5-(N-Mothry}-N-isobuitylemfloride, i : ” 2' DichloroBenzamil HCI, ’ . . : per mare ovimim nami 3-(triflnoromethyl)cinnamoyigusnidine, 2-othoxycinnamoylgnanidine, . \emanidine kvdrochiorid . ~ . } -cthoxycinamoyl guanidine, @ rar . me i - 34,5-trimethoxycinnamoylguanidine, S-tert-butylamino-emileride, Co . 3-tbutylcinnamoyignanidine, dine 4-Chlorocinnamoyl)guanidine, . 2tbutylcinnamoylguanidine, 2-cyclohexykinmamoylgusnidine, 6-Jodosmiloride, : : 3-(trane-hept-1-en-1-yl)cimamoylgnanidine, (4-Bromocinnamoyl)guanidine, (4-Hydroxycinnamoyf)guanidine, Co N-(3-phenyipropanoyl)-N'-phenyignanidine, " (3-Nitrocinmemoyl)gnanidine, 3-finorocinnamoyiguanidine, 2-(1-napthyl)acetoylguanidins, :

   oo | AT (4 foorophenylemilocide, oo (tane-2.Phecylcyclopropanscarbos A. > Dgaanidin ine, N)N“-bis(3phenyipropanoyl)}-N"-phenyignanidine, 1-napthoylguanidine, 3-methoxy ~HMA, : 4-fluorocinnamoylguanidine, 3,4-(methylenedioxy)cinnamoylguanidine, - : 5-(NN-hexamothylano)amiloride, ~ 5-(N-Bthyi-N-isopropyl)amiloside, 2-methylcimamoyigoanidine, - trens-3-Fursnacryoylgeanidine, : (4-Methoxycinnamoyf)gnanidine, - (2-Furanacryloyl)guanidine, 2-2-nsptirylymcetoyignaridine, (2-Methoxycinnamoyl)gnanidine, + [3-(3-PyridyDacyloyl]guanidine, : 2 A-dichlorochmamolygnanidine, 2-finorocinnamoylgnanidine (4-Pt ” I iF (e-Methylcimmamoyl)guanidine, 5-(3'-bromophenyl)penta-2,4-dienoylguanidine, - (5-Phanyl-pents-2,4-dienoyl)guanidine, + (Quinoline-2-carbonyl)gnanidine, (PhenylacetyDgnanidine, N,N'-Bis(amidino)napthalens-2,6-dicarboxanside, 6-bromo-2-nepthoylguanidine, : : 1-bromo-2-napthoylguanidine, 2-chloro-6-flucrocirmamoylgomnidine, 4-Chloropbenoxy-scetyl]gnanidine, fc py N-Benzoyl-N'-cinnamoyiguanidine and :

   ans a

   12. The mefhod according to clio 109, wherein ssid Coronavirus 12 suman

   113. The method acoording to claim 112, wherein ssid compound is selected 8 * from the group consisting of - : 4-1 i . | idi : 3,4-dichlorocinnamoylgnanidins, + butvici temanidi isopropytclnuuiyigusnidine bydrochlocid,

   a. atv auanidi oo . . 2 tt 4 1 [] 1 idk 8 . . trans-3-{1-napthyl)acryloylgmenidine,

   _ 5.bromo-2-methoxycinnamoylguanidine, 23-dift ; Isuacidi 3-phenylcinnamoylgganidine, : 3-{cyolohex-1-en-1-yl)cinnamoylguanidine, : 3{(txiflnoromethyl)cinnamoyigmenidine, (4-Pt : — . 4{triflucromethyl)cinnamoyignanidine, : 2-(cyclohex-1-an-1yl)cimmamoylguanidine, } (4-Bromocinnamoy{)gnanidine, 5-(N,N-hexamethylene)smiloride, : 1-papthoyignanidine, 5-{4-finorophenyfamiloride, (5-Phanyl-pents-2 4-diencyl)gnanidine, (3-Bromocinnamoyf)guanidine, : frm dry) iamidi 6-methoxy-2-naphthoylguamidine,

   } . (4-Chlorocinnamoyi)gnanidina, . S-bromo-2-fincrocinmamoyiguanidine, : 5-(N,N-DixnethyDamiloride hydrochloride, Gi lenamidi oo -* (2-Methoxycinnamoyl)guanidine, (a-Methylcinnamoyl)guanidins, 4-phenyicinnamoylgnanidine, 2,6-dichlorocinnamoyignanidine,

   Co | 176- (3-Chlorocimnamoyl)gnamidine, 2-(1-napthylacetoylgninidine, Le neylguai ] -methylcinnamoylguanidine, : Zstyldmamog ie cinnamoylgnsnidine hydrochlarida,

   a . Tami 4-finorocinnsmoyignanidine, 3 4-diffoorocimmamoylgnanidine, s tert botylsmino-amilorids, © “Bia(3-pheayipropanoylguanidine, tmnofiryici taamidfine, . © §{3bromophenyl)pente-2,4-dienoylguanidine, 2,3,5,6,-tetramethykinnsmoylguanidine, Pg X Jumanidt Co tn, (2-Chlorocinnemoyl)guanidine, . (3-Nitrocinnamoyf)goanidine, hott . nari 3,4,5-tri # [ : 1 id : 2{2-naptrylaccioyignemidine and

   114. The method accarding to claim 109, wherein said Coronaviras is any one of the known Coronavirus isolates listed in Table 1.

   115. The method according to claim 114, wherein said compound ia selected from the group consisting of 3(triflnoromethoxy)cinnamoylguanidine, 4 butvici Hy an

   116. The method socording to claim 102, wherein said virus is the Hepatitis C © 417. The method according to clsim 116, Wherein said membrane jon chanel : is tho Hepatitis C virus p7 membrane jon channel

   118. The method according to claim 117, wherein said compound is selected 2,4,6-trimethylcinnamoylgnanidine, : ilo is

   B . Dua 2,5-dimsthylcinnamoylgnanidine, : 3-(triffuoromethyl)oinnamoyignanidins, id 4-(triftoorometirylJcimamoylgenidine, 2 : or ) aye eure wo . -Bromocinnamoy)gnanidine, ° ; . 2 6 of } } i i id : [3 [yO } Co i N——— i cm 4 (4 Phaxsbenoyl)goenicon . adi . ae pane r— Mgaanicine. trans-3-(1-napthyl)acryloylgnanidinc, : 4-t-butylcinnamoylgnanidine, 2-t-butyicinmamoylgusnidine, - . 2-cthylcinnamoylguanidine, : 5-bromo-2-methoxycinmamoyiguanidine, TY 1 X 3-t-butylcinnsmoylgnanidine, (5-Phenyl-pents-2,4-diencyl)gnsnidine, N-(cinuamoyl)-N'phenylguanidine, oo 3-isopropylcinnamoylgnanidine hydrochloride, Benzami] hydrochloride, - N~(3-phenylpropanoy!)-N'-phenylguanidine, : N,N-bis(3phenylipropanoyl)-N"-phenylguanidine,

   24 DighloroBenzamil HCI, : Co 5-tert-butylamino-amilarids, : - 5-(N-Bthryl-N-isopropyDamiloride, : finorocinnamoylgnanidine, (-NiosimemoyDgunidie (4-H i ydroxycinnamoylguanidine, (trans-2-Phenyloyolopropanscurbonyl)guanidine, 23,5 tetramettycionamol guanidine, trans-3-Furanacryoylguanidine, : N-(6-Hydroxy-2-nspthoyl)-N'phenylguanidine, 3-(cyclohex-1-en-1-yl)cipnamoylgnanidine, cinnamoylgusnidine hydrochloride, : > 3 dif . namic ion, } Nitrocinnamoyl)gusnidine, : 6-Indoamiloride, ‘ E 3,4-(methylenedioxy)cinnamoyl guanidine, 2-ethoxycimamoyignanidine, Cinnsmoyignsnidine, 2-phenylcinnamoyiguanidine, : " 2+{oyclohex-1-cn-1yloinnamoylgnanidine, 3-phenylcinoamoyiguanidine, 5-(N.N-DimethyDamiloride hydrochloride, ’ 5-{4-fluorophenyl)amiloride, (3-Met . Dguanidi : 2-ft ° 4 id 5-(3'-bromophenyl)penta-2,4-disnoylgnanidine, : [(4-Chiocophenoxy-acetyljguanidine, (3-phenylpropanoyl)guanidine, Co 3-finorocirmsmoyiguanidine, . : (2-Mott C 2 0) . ‘ 1-bromo-2-napthoyiguanidine, 3,4,5-trhnethoxycirmamoylguanidine, 3 etic Jananidt a 3-{trans-hept-1-en-1-yDcimamoylguanidins, Phonamil methanesulfonsjo salt , 2,4-dichlorocinnamolygeanidine,

   } Co . -179- . (A-Nitrocimamoyl)goatidine, : 3 4-difilucrocinnamoyl guanidine and : oo [(E)-3-{4-Dimetirylaminophenyi)-2- a methylacryloyl]gaanidine. oo : 119, Tho method sccending 0 my coe af clas 1020 118, wherein sid hE compound ia peovidsd as a pharmaoeutical composition according to claii dorclaim §. }

   5 .

   120. A method of reducing, retarding or otherwise inhibiting growth and/or "7 replication of a virus that has infected 8 cell in a mammal, said method compeising administering to s2id mamma! a compound according to any one of claims 1 to 3, wherein said compound down regalates functional activity of a membrane jon channel expressed in said infocted oll C121 The method soocding clam 120, wherein sid vir a Lestivirun . © 122. The method acconting to claim 121, wherein ssid Lentivirus is Human Trmumodeficiency Virus (HIV).

   123. The method according to claim 122, wherein said compound is selected : : (3-Chlorocinanemoyl)guanidine, eB cyDgaanidi 3-(triftuoromethyf)cinnamoylgusnidine, 5-bromo-2-finorocinnamoylguanidine, 2 methvicimmarnoylgoamids 6-methoxy-2-naphthoylgnanidine, trans-3~(1-napthyl)scryloylguanidine, : : 3 A-dichlorocinnamoylgnanidine, 2 &dichlaroc euanidine, -athylcinnamoylgnanidins, (-Chlorocinnamoyl)guenidine

   2 thovl id ’ . ’ (5-Phanyl-penta-2,4-dienoyD) guanidine, . 3-phenyloinnamoylguanidine, .. (4-Bromocinnamoyl) guanidine, Co 5-(3-bromophenyl)pentn-2,4-dienoyl guanidine, 3-(cyclohex-1-en-1-y])cinnamoyl guanidine, : NN -inpbenypropenoyl IV" pheigrmidie. 2a ; {omni N-(3-pheaylpropanoyl)-N'-pheoyiguanidine, 4-(trifinoromethy)cinnamoyl guanidine, (4-MothoxysimamoyT)gnanidine, z ns primi . Ngaio, di 2-finorocinnamoyignanidine, : 2-pheayicineamoyiguanidine, (6-day. tpt T- phe arin butylcinnsmoylgusnidine, 3,4-48\ fhaorocinnemoyiguanidine, SOON hesmnetiolemajetiiionids fraorocinnamoyignanidine, : §-beamo-2-methoxycinnemoyigusnidine, 3-cthoxycimamoylgnmnidine, 3,4-(methylenedioxy)cinnamoyignanidinc, (2-Mathoxycinnamoyl)gusnidine, 2'4 DichloroBenzamil HCl, 3-(2-napthyl)acryloyignanidine, 2,3-difluorocinnemoyiguanidine, (3-MethoxycinnamoyDgusnidine, . re oi \enarridine, 2,4,6-trimethylcinmsmoylguanidine, N-(cinnamoy!)-N'phanylgnanidine, Pa Sm er -napthylecetoylguanidine, (4-Bydroxycinnsmoyl)gusnidine, 4-phenyichmamoyignanidine,

   of . Atsmery : Nbr (cian) phenyl gues - Furnacryloyl)gnanidins, Phonami) methanesulfonats salt , Bry got: ] Natroci Dguanid

   : .

   (4-Phenoxybenzoyl)guanidin, 3-(grans-hept-1-en-1-yDeimnamoylgnanidine,

   5.(N-Mothyl- N-issbutyDsmilorids, 2-oyclohexyloinnemoylguanidine, 4-cthoxycinnamoylguanidine, * 2 4-dichlaroc 1 id oo 5-(N-Bthyt-N-isopropyamilocide, N-smidino-3-amino-S-hexamethylansimino-6-phenyl- 2-pyrazinecarboxamide, ’ (a-MethylcinnamoyI)gnanidine, : [(4-Chlorophenoxy-acetyl]guanidine, - N-smidino-3-amino-5-phenyl-6-chloro-2- : imooart id (trans-2-Phenylcyclopropanecarbonyl) guanidine, @-Nikocimmamoy hunt 3-Furanacryoylguaniding, : 1-napthoyiguanidine, Stet butyiam} florid 3-methoxy -HMA, fie mica Sarr t-butylcimamoylgnanidine, 5-(NN-Dimethyl)amilaride hydrochloride, N,N'-Bis(3-phenylpropsncyl) guanidine, N-Benzoyl-N'-cimnamoyliguanidine and 1-bromo-2-nepthoylgnanidine.

   124. The method according to any one of claims 120 to 123, wherein said membrana ion channel is the HIV Vpu membrana jon chanel. . 5

   125. The method scconding to any ons of claims 122 to 124, wherein said HIV is HIV-1. :

   126. The method according to claim 120, wherein said virus is a Coronavirus.

   127. ‘The method according to claim 126, wherein said Coronavirus is the Severs Acute Respiratory Syndrome virus (SARS).

   128. The method according to claim 127, wherein said compound is selected from the group consisting of » 3 ft . —— 3 4dichlorocinnamoyigumidine, 3-(2-napthylacryloylgnanidine, (3-ChlorocimamoyT)guanidine, 3-(cyclohex-1-cn-1-y)oitmamoylguanidine, 2,5-dimethyloinnamoylguenidine, : 4-isopropylcinnamoylgnanidine, (3-Bromocinnamoyl)guanidine, Co 6-methoxy-2-naphthoylguanidine, oo 5(N-Methyl-N-isobutylamilorids, 3-phenyicinnamoylgnanidine, (3-Ch . oul ” : } . 2'4 DichloroBenzamil HCl, _ } 4 i 3 ° 1 idk . . . . 3<riftuoromethoxy)cinnamoyl guanidine, oC 3-(triftuorcmethyl)cinnamoylgnanidine, 4-ethoxycinnamoylgnanidine, oa goanidin 6 dichloro ananidi 3 A, S-trimethoxyciimemoylguanidine, sect buctylami Horid S-bromo-2-flucrocinnamoylgnanidine, +-Chloroc] — 2-cyclohexylcinnamoylgoanidine, 6-lodoamiloride, 3-(truns-hept-1-en-1-yi)cinmamoylguanidine, (4-Bromocinnamoyl)guanidine, (4-Hydroxycinnamoyl)goanidins, N-(3-phanyipropanoyl)-N'-phonylgnaridine, - (3-Nitrociomamoyf)gnanidine, ra thyT)acetoylguamids 2-cthyleinnamoyliguanidine, 2-napthoylgnanidine, 5-(4-fluorophenyl)amiloride, 2-(trifluoromethyf)cirmamoylguanidine, N+(6-Hydroxy-2-napthoyl)-N'-phenylgnanidine,

   . aE BE <2 - (trans-2-Phenylcyclopropanscarbonyl)guanidine, © NN'-bis(3phenyipropanoyl)-N*-phenylguanidins, 1-napthoylguanidine, © © Benzamil hydrochloride, 3-methoxy ~HMA, 4-methylcirnamoylguanidine, : 4-fiuorocinnamoylguanidine, : : N-(cinnamoyl)-N'pheayignanidine, : 5-(N-Behyl-N-isopropylamiloride, : ,6y-tetramethyicinnamoylguanidine, trans-3-Furanacryoylgwanidine, : : (4 Mlheaycimamoy)guacidioe, P ioyDgoanidi : (2-Mothoxycimamoyl)gnanidine, - 24 dick 2 focrod guanidine, (4-Phenoxybenzoyl)gnanidine, _ 5<(3“bromophenyl)penta-2,4-dienoylguanidine, (5-Phemyl-pants-24-dicnoyl)guanidine, - (Quinoline-2-carbomryl)gnmnidine, " 1-bromo-2-papthoylguanidine, 4-Chlorophenoxy-acctyl]gnanidine, Phenzmil methanasulfonate salt, : .. N-2-napthoyl)-N'-phenyignanidine.

   129. Tha method according to any ons of claim 126 or 128, wherein said 5 membrane fon channel is the Coronavirus E protein. s

   -184- co Co

   130. The method according to claim 126, wherein ssid Coronavirus is human Co Coronavirns 229E.

   131. The method sccording to claim 130, wherein ssid compound is selected from the group consisting of © 132. The method scoording to claim 131, wherein ssid compound is seloctod from the group consisting of oo | : Co ¢-dichloroci ‘mandi oo 3(triflooromefhoxy)cinnamoyigusnidine, : Cag feimamoyl dios hydrochloride, 2 uty Janenidi tran 3-(L sup Decryioyiguenicms,

   2.3 diflmoroci {snamids 3-(2-oapthyDecryloylgoanidine, 2-phenylcimamayl guanidine, : 3 3 oi 1 id R 3{(cyclobex-1-en-1-yl)cinnamoylgnanidine, 4-phenylbenzoyiguanidine, : 3-(irif ylicE anid a (4-Phedioxybanzoyl)gesnidine, So 2-(cyclohex-1-an-1 yl)cinnamoylgnanidine, : (4-Bromocinnamoyl)guanidine, 1-napthoyignsnidine, ~ 5-(4-finorophenylamiloride, (5-Phenyl-peats-2,4-dienoy])goacidine, Co 2-(trifinoromethyl)cinmamoylguanidine, (4-ChlorocinnamoyT) guanidine, 5-bromo-2-fluorocimamoylgnanidine, 5-(N,N-Dimethylyamiloride hydrochloride, ch amidine Cima ee icine (s-MothylcimamoyT)gnanidine,

   4-phenyicinnemoylguanidine, 2,6-dichlorocinnamoylgnanidine, - (2-Bromocinnarnoyf)guaidine,

   2.4,6-irimethylciimamoylguanidine, (trans-2-Phenyloyclopropanecerbonyl) guanidine, Oe oon, g a ich enenidi 2-cyclohexylcirnamoylgoanidine, (4-Hydroxycinnamoyl)guanidine, 2-ethoxychmamoylgnanidine,

   3 . : sdine, Smal — Yigeanid di 3-finorocinnamoylignanidine, Sine i Chute . dine iydrochi o de, 2-finorochmemoyigumnidine, 4-finorocipnamoylgnanidine, 3 A-difincrocinnamoylguanidine, S-texrt-butylmmino-emiloride, © 2-mpthoylguanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, . 4-methrylocinnamoyignanidine,

   $.(3-bromophenyl)pents-2,4-dienoylgusnidine, © 2,3,5,6,-tetramethylcinnamoylguanidine, Sot . — NN-bis(3phenylpropanoyl)-N"-pheaylgnanidine, mn mn foi -Chlorocinnamoyl)guanidine, 4-othoxycinnamoylguanidine, ] 3,4, S-trimethoxycinnamoylgnanidine, N-(3-phenyipropenoyl)-N-phenylgnanidine.

   133. The method according to any one of olaima 130 or 132, wherein said membranes ion chanel is the Coronavirus E protein. 134, The method according to claim 126, wherein ssid Coronavirus is ary amo. : of the known Coronavirus isolates listed in Table 1.

   oo 135. The method according to claim 134, wherein suid compound is selected from the group consisting of : . 4isopropylcinnamoylgnanidine, Tu 34 dicht a im ids : . . : 3-(trifimoromathoxy)cinnamoylgnanidine, * 4-t-butyloimamoylguanidine, 3-isopropylcimamoylgnanidine hydrochloride, : © 136. The method according bo claim 134 or claim 135, Whetein ssid membrane ‘8 jon channel is the Coronavirus B protein.

   137. The method according to claim 120, whetsin said virus is the Hepatitis C 138 The method according to claim 137, wheeein ssid compound is selected

   23. thylod | id 5-bromo-2-finarocionamoyignanidine, (4-Bromocinnamoy{gusnidine, 2 5-dimetiryici amid Auf yl) namic 6-methoxy-2-naphthoylgnanidine, (2-Chiorocinnamoyl)guanidine, } Bromocinnamoy)guanidine, iin, . ) Dgusnidi (3-Chlorocimamoyf)gnanidine, (4-Phanoxybensoyl)guanidine, : oo 3 4-dichlorocimnamoyignanidine, Co trans-3-(1-neptiylJecryloyl guanidine, 2-ethylcinnsgmoylgnanidine, 4-methylcinnamoylignanidine, : 51 py . — : 3-(triflnoromethaxy)cinnamoyiguanidine, 1-napthoylgnanidine,

   oo 87 . . . . ao . : (5-Phenyl-pente-2,4-dienoyl)guanidine, - N-(cirmamoyl)-N'phenylguanidine, .- 3-isopropylkinnamoylguanidine hydrochloride, " Benzamil hydrochloride,

   : N-(3-phenyipropanoyl)-N'-phenylguanidine, N,N-bis(3phenyipropanoyi)-N"-pheaylguanidine, 3-(2-nspthyDacryloylguanidine,

   2'4 DichloroBanzamil HCI, : 5-(N-Bthyl-N-isopropyl)amiloride, (3-Nitrocinnamoyl)gnanidine, ( 4-Hydroxycinnamoyl)gusnidine, (trans-2-Phenyicyclopropanecarbony)guanidine, 3-ethoxycinnamoylgnanidine, . 2,3,5,6,-tetramethyicinnamoyl guanidine, 4-phenylcinnamoylguanidine, . trans-3-Fursnacryoylguanidine, So N-(6-Hydroxy-2-napthoyl)-N'-phanylguanidine, (2-Fursnacryloyl)gusidine, 3-(cyclohex-1-en-1-yl)cinnamoylgnanidine, cimamoylguanidine hydrochloride, 5-(N,N-hexsmethylene)amilaride, 2(1-oaptiylacetoyiguanidine, (a-Mathylcinoamoyl)gnanidine, (2-Nitrocinnamoyl)guanidive, 6-lodosniloride, 3,4-(methylenedioxy)oimamoyignanidine, 2-athoxycinnamoylguanidine,

   : 2-phenylcinnamoylgnanidine, 2<(cyolohex-1-en-1yl)cinnamoylguanidine, 2-napthoyi guanidine, 3-phenylcimsmoyignanidine, 5-NA-Dimethyemloride ydroclocds finorophenyl)amilorides, . 2-flucrocinnamoyigusnidine, 1 i A 4-Chlorophenoxy-acetyl]guanidine, (3-phenylpropanoyl)guanidine, =~ 2-chioro-6-fiuorocinnamoyignanidine,

   ppv ylcinnamoylgusnidine, (2-Methoxycinnamoyl)guanidine, 1-bromo-2-napthoylguanidine, 3 4 S-trimethoxycinnamoylgeenidine, 3-(trans-hopt-1-en-1-y)cinnemoylguanidine, : 2,4-dichiorocinnamolygusnidine, (4-Nitrocinnamoyl)guanidine, 3 A-diffaorocimsmoylguanidine and : [(8)-3-4-Dimethylaminophenyl)-2- methylacryloyliguanidine. :

   139. . The method sccording to claim 138, wherein said mernbrane ion channel is the Hepatitis C virus p7 mambrans ion channel. 5s 140. The method according to any one of claims 120 to 133, wherein ssid - . mammalisaprimste. i

   141. The method acoording 10 any ane of claims 137 to 139, wharain esid mammal is a primate. : } : ) -. "142. Tho method scoarding to claim 140 or clsim 141, wherein seid primato ia

   143. . The method according to any coe of claims 120 t 142, wherein ssid compound is provided as a pharmaceutical composition according to claim 4 or claim 5. Eg

   144. A method for the fhecapeutic or prophylactic treatment of a subject infected with or exposed to 8 virus comprising administering to said subject a compound according to any one of claims 1 to 3, wherein said : compound down-regulates functional activity of 8 membrane jon channel © 145. The method acoading to claim 144, wherein ssid virus is a Lentivirus.

   146. Tho method according to claim 145, wherein said Lentivirus is Hue Immunodeficiency Virus (HIV). oo 5 147. The method according to claim 146, wherein said membrane ion channel is the HIV Vpu membrane ion chamel.

   Co 148. The method according to claim 147, wherein said compound is selected B from the group consisting of mole (riflacractotts foie Yignanidine, % 2,3-dimethyloinnamoylgnanidine, Ci ixoanidi A-dichlorocinnamoylignamidine, 2,6 Sichlorocinsamoylgumidine, 4-phenylbenzoylguani 4Chioroci f)goanidi . ’ 2 2 py 5 Agssmidion . — (5-Phenyl-pents-2 A-dienoyDgnanidine, - 3-phenyicinnamoylgnanidine, : : (4B - 1) ids 3-(cyclohex-1-en-1-yl)cinnamoyigaanidine, rif yi eranidi ~ethoxycirmamoyl guanidine, : N+(3-phenyipropencyl)-N'-pheatyl guanidine, (4 Mathorysiomamoy hgeniting, -butylcinnamoylgnanidine, : A mothylolmemoy gui fiucrocinnamoylgnanidine,

   2-phenrylcimamoyigneanidine, CL N-{6-Hydroxy-2-napthoyl)-I'-phenylguamdme, 4-difluorocinnamoyignanidine, 3-fluorocinnamoyiguanidine, 5-bromo-2- metboxyclunamoylguaaidios, 3 el . \easmiding, 3 4-(methylenediaxy)cinnamoylguanidine, (2-Methoxycinnamoyl)guanidine, : 24 DichloroBenzamil HCl,

   2,3.5,6.-tetramothyloimamoylguanidine, : es nyDacryloylguanidi -napthylacetoylguanidine, : 23d \ jemanidi isopropylcimamoyl guanidine, } ey Nbr ylgoanitive. 2-(cyclohex-1-en-1yNcinnamoylguanidine, 2-)-naptirylecctoylguanidi (4-Hydroxycinnsmoyl)gnanidine, 4-phenrylcinnamoyignsnidine, } 4-fiuorocinnamoylgnanidine, Ney ne guaridine Fursnacryloyl)guanidine, Phanxmil mothanesulfonate salt , Nitroci 1 ” p | id . 4-Phenoxybenzoyl) guanidine, 3-(trsns-hept-1-on-1-yl)cimnamoylgnanidine, 5-(N-Mathyl-N-iaobuty]) florid 2-cyclohexylcimamoyliguanidine, 2,A-dichlorocinnamolyguanidine, 5-(N-Ethryl-N-isopropyDemilotide, -pyrzinscathoxamids, (a-Msthylcinnemoyl)guanidine, [(4~Chlorophenoxy-acetyljguanidine, : N-amidino-3-amino-5-phenyi-6-chloro-2- 5<(4-finorophanyDemilaride, : (trxoe-2-Phenyicyclopropancoarbomyl)guanidine, " (2-Nitrocinmamoy))guanidine, trens-3-Furanacryoyl guanidine,

   1-uapthoylguanidine, oo ~ (3-phanylpropanoyl)gusnidine, © 4-t-butylchnmamoylguanidine, : ’ 5-(N.N-Dimothyl) amiloride hydroobloride, N,N-Bis(3-phenylpropenoyl)guanidine, : : N-Banzoyl-N'-cinnamoylgpenidine and | : 1-bromo-2-napthoyl guanidine. : 149. ‘The method according to arty ons of claims 146 to 148, wherein sxid HIV : SHIV-1,

   150. The method socording to claim 144, wherein said virus is a Corongvirus. © 151. Tho method scoonding to clsim 150, wherein said mermrane on chennel oo is the Coronavirus E prosein.

   10. oo . 152. The method according to claim 151, wherein said Coronavirus is the : Severe Acute Respiratory Syndrome virns (SARS).

   153. ‘The method acconting to claim 152, wherein said compound is selected from the group consisting of : | ri . Viguanidine, ii AL Cl aman ry " - 3<cyclohex-1-cn-1-yDeinnamoyl guanidine, . ” i byl i EL yigo id J trans-3-(1-napthyDacryloyiguanidine, ~ (3-Bromocionamoyl)gnanidina : G-methoxy-2-naphthoylgusnidine, : 3 Q¥Methyl-N-lsobutyDemiloride, : : -phenylcimamoylgnanidine, _ 2'4 DichloroBenzamil HCI,

   : ~192-. 3(uift thoxy)cim sgnanidin 3(trifluoromethycinnamoyl, goanidine, 2-ethoxycinnamoylguanidine, 4-ethoxycinhamoylguanidine, : (2-Bromocinnamoyl) guanidine, : 2,6-dichlorocinnamoylguanidine, 34,5-trimet . - uemidine 5-tert-dutylamino-amiloride, 3-t-butylcionamoylgnanidine, 5-bromo-2-flucrocinnamoylguanidine, . ( [3 & GO amor ici gudin % 2 Y! \ i . 3! ich 6-Iodosmiloride, : 3-(trane-bept-1-cn-1-yl)cinnamoylguanidins, CC N-(3-phenyipropanoy])-N'-phenylgnanidine, finorocinnemoylgomniding, 2-(1-napthyDecctoylguamidine, _ 2-ethylcinnamoylignanidine, : 2-napthoyignanidine, $-(4-fluoropherrylamiloride, 2-(trifimoromathyf)cimnsmoyigranidine, N-(6-Hydroxy-2-napthoyl)-N'phenylgoanidine, (trene-2-Phenylcyclopropanecarbonyl)guenidine, NN"-bis(3phenyipropanoy)-N"-phenylgnanidine, 1-napthoyigaanidine, Benzamil hydrochloride, . 3-mothoxy -FIMA, fluorocinnamoylguanidine, 5-(N,N-hexamethylenc)emiloride, 0% Bayh isopeopyemalocide methylcirmamoylguanidins, : 2-methykcimamoylguanidive, 2,3,5,6,-tetramethylcinnamoylguarnidine, (4 Mothoxycinnamoyl)gnanidine, SE (3-phenylpropanoyl)guanidire,

   (2-Methoxycinnamoyl)gnanidine, [3-(3-PyridyDacryloyl]gnanidine, tyme. i A-dichiorocimamolyguanidine, (3-Msthoxytinnamoylgnanidine, (a-MethylcinramoyT)guanidine, ]

   5.(3“beomophenyl)pents-2,4-dienoylguanidine, (5-Phemyl-penta-2,4-dienoyf)guanidine, (Quinoline-2-carbanyl)gnanidine, Co N,N'-Bis(amidino)napthalone-2,6-dicatboxantide,

   : . G-bromo-2-napthoylguenidine, 1-bromo-2-napthoylgusnidine, 2-chloro-§-finorocimamoylgnanidine, {(4-Chilorophenoxy-acetyl]gusnidine, Phenamil methanesulfonate salt, . N-Banzoyl-N'-cimnemoylgnanidine and

   154. The method according to claim 151, wherein said Coronavirus is tuman Coronavirus 229E. oo } :

   155. The method according to claim 154, wherein said compound is selected from the group consisting of 3,4-dichlorocinnamoyiguanidine, 3{trifincromethoxy)cinnamoyignanidine, : t Tr . cYipmoidie sine hydrochlorid -+-butyicinnamoyignanidine, 2-t-butylcinnamoylguavidine, - trans-3-(1 -napthyDecryloylgusnidine, oo 5-bromo-2-methoxycinnamoyignanidine, 3~2-napthyljacryloyignanidine, -phenyicimamoylguanidine, © 3-(cyclohex-1-en-1-yl)cinnamoylguamidine, 4-phenylbenzoylguasidine,

   2-(cyclohex-1-en-1yl)cinnamoylgnaniding, PA Dani: " 5(N,N-hexamethrylene)amiloride, ~ 1-papthoylgnamidine, 5-(4-flucrophenyl)amilorids, (5-Phonyl-pents-2,4-dienoyl)guamidine, N (3-Bromocinnemoy()guanidine, 2<(triftucromethyl)cinnamoylguanidine, 6-methoxy-2-naphthoylguanidine, (4-Chlorocinnamoyl)gpanidine, (3-Msthoxycinnamoyl)guaniding, 5-(N.N-Dimethyl)amilaride hydrochloride, (2-Methoxycinnamoyf)guanidine, (»-Methykcitnamoyl)goanidine, 4-phenylcimamoylgoanidine, 2,6-dichloroolnnamoylgusnidine, © (2-Bromocinnamoyf)gnenidine,

   2.4,6-trimethylcinnamoylguanidine, : (tran 2 id (G-Chl Tha loyslopopeebory Dey 2-(1-napthyDacetoyignanidine, 2-ethylcinnamoylgoanidine, 2-cyclohexylcinmamoylguanidine, (4-Hydroxycinnamoyl)gusnidine, 3-methylcinnemoyigasnidine, 3-fluorocimemoyigoaidine, 2,3 , ddtin -dimethylcinnamoylguanidine, 2-flnorocimamoylguanidine, 4-flucrocinmamocyignanidine, 3,4-diflnorocinnamoylguanidine, S-tert-butylamino-amiloride, 2-napthoylguanidine,

   N.N-Bis(amidino)napthalene-2,6-dicarboxamide,

   N.N'-Bis(3-phenylpropanoyf)guanidine, 5-(3"-bromophenyl)pants-2,4-diencylgnanidine, 2,3,5,6,Aetramathylcinnemoyl guanidine, 3 oft . Aguanidi N,N'-bis(3phenytpeopanoyl)-N"-phenyignanidine,

   . To -195- ) : (4-Methoxycinnamoyl)guanidine, : : (3-NitrocintismoyDgnacidine, 3 4 5-rimethaxycimmsmoyl guanidine, 2-(2-aspthylacetoylgnanidine sud

   N.(3-phenyipropanoyl)-N'-pheaylguanidine.

   156. The method according 10 claim 151, wherein said Coranavirus 8 &1y 000 } of {ho known Coronavirus isolates listod in Teblo 1. s 157. os ato csoting ole 155 where ei compound i ected from the group consisting of Co phi pigunidin, idine ld Hlonid

   158. Tho macthiod scccrdisg 1 claim 144, wherein seid virus is the Hepaiitis C 10 : : ’ 159, The method according to clin 158, wherein seid maabrans ice chanel ia the Hepatitis C virus p7 membrane ion channel.

   160. The method according to claim 159, wherein ssid compound is sclectad 15 from the group consisting of 2,4,6-trimethylcinnamoylgnanidine, - bromo-2-finorocinnsmoylignanidine, (4-Bromocinnamoyl)guanidine, 3(trifinoromethylcimamoylgnanidine, {if cyl) fgoanid (2-Chlorocinnamoyl)guanidine, oo {4-Chiorocinnamoyf)guanidine, (2-Bromocipnamoyl)guanidine,

   oo 196 (3-Chlarocinnamoyl)guanidine, 2(triftluoromethylcinnamoylguanidine, - (4-Phenoxybenzoyl)guanidine, isopropyicimamoylgnanidine, : trans-3-(1-napthyl)scryloylgnanidine, - : 4-t-butylcinnamoylguanidine, 2-sfiryicinnemoylguanidine, : 4-methyloinnamoylguanidine, : 3-(trifmoromethoxy)cinnemoylgnanidine, ' : 1-napthoylgnanidine, : 3-t-butykimamoylguanidine, : (5-Phenyk-pante-2,4-dienoyD guanidine, Benzamil hydrochloride, : N-(3-phenylpropanoyl)-N'phenylgnanidine, Co 2'4 DichlaroBenzamil HCI, oo : S-ert-butylamino-emiloride, finorocinnsmoylgnanidine, (4-Hydroxycinnamoyf) guanidine, : 3-ethoxycinnamoylgnanidine, 2,3,5,6,~totramothylcinnamoylgusnidine, Co 4-phanylcimamoylguanidine, oo trane-3-Furanacryoylgnanidine, : i eyipomidic, Fursnacryloyl)guanidine, 3-(cyclohex-1-an-1-yDcinnamoyigusnidine, 23 5a . PT -pspthyl)acctoylgnanidine, (+-Mothylcipnamoyl)gnanidine, 6-lodoamiloride, : 3 A-(methylenadioxy)cinnamoyiguanidine, :

   - Cinnsmoylgnanidine, 2-phenyl cinnamoylguanidine, 2-{cyclohes-1-en-1ylcinnamoylgnanidine, 5-(N N-Dimethyl)amiloride hydrochloride, 5-(4-fluorophenyl)amiloride, S-(3"bromophenyl)pents-2,4-dicooylguanidine, [(4-Chlorophenoxy-acetyl)guanidine, 3 finorocimamoylgusanidine, . 2-methvici ry . * 1-beomo-2-napthoylgnsnidine, 3,4,5-trimethoxycinnamoylgosnidine, 3-(trme-hept-1-co-1-yhoimamoyiguanidine, Phensmil methancsulfonste salt , : * 2 A-dichlorocinnemolyguanidine,

   @* . vo 3 a Tguaniding dine and [(E)-3-(4 Dimethylaminopheny1)-2- methylacryloyl]gasnidine.

   161. The method according to any one of claims 144 to 155, wherein ssid mammal is a primate. 5s 162. The method according to sty ane of claims 158 to 160, wherein suid ‘mammal is a primate. :

   16s. The method according to claim 161 or claim 162, wherein said primate is Imman,

   164. An mtiviral compound selected from the group consisting of © NY(3,5-Diamino-6~chloro-pyrazine-2-cxrbonyl)-N’-phenyl-guanidine, N-Benzyl-N’~(3,5~diamino-6-chioro-pyrazine-2-carbonyl)-gusnidine, : - 34 DichloraBenzamil,

   24 DichloroBenzamil, 5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride, 5-(N-Miothy}-N-isobutylamilorids, oo [ . 5.(N,N-Dimethylamiloride hydrochloride, 5-(N,N-hexamethylene)emiloride, 5-QN,N-Dicthyl)amiloride hydrochloride, 6-lodoarniloride, 3-hydroxy-5-hexametirylencimino-amiloride, 54 finorophenylemilorids, . 5-tert-butylamino-amiloride, : 3-methoxy-5-(N,N-Hexamethylene)-amiloride, -

   15 . 3-methoxy-amiloride, N-amidino-3, 5-dixmine-6-pheayl-2-pyrasinecarboxamide, . 1-nepthoyignanidine, 2-napthoylgnanidine, N-(2-nupthoy])-N'-pheaylgnanidine, : N;N-bis(1-nepthoyl)gumnidine, EU N,N'-bis(2-nepthoyl)-N"-phenylgnanidine, 6-methaxy-2-naphfhoylgnanidine, 23 3-quinolinoyignanidine, 4-phanyihenzoyignanidine, N~(cimmamoyf)-N'phenylgnanidine, (3-phenyipropanoy) guanidine, N;N'-bis-(cirmamoyl)-N"-pheay|gnsnidine, N,N-bis(3phenyipropancyl)-N" phenyiguanidine,

   : | . -199- . NA{(6-Hydroxy-2-napthoy) N'phenylgnmnidine, ~~ (4-Phenoxybenzoyl)guanidine, I N,N'-Bis(amidino)napthalens-2,6-dicarboxamide, N"-Cinnamoyi-N,N'-diphenrylgnanidine, taootyDgueniding oo N,N-Bis(3-phenylpropanoyl)gnanidine, . benzy oylguanidin i 2 » : (4-Chiorophenoxy-scctyl guanidine, N-benzoyl-N'-cinnamoylgusnidine, oo [(B)-3-(4-Dimethrylaminophenyl)-2-metylacryloyi]gnanidine, {(4-Chlorocinnamoyl) guanidine, SE (4-Bromocinnamoyf) guanidine, : (4 Métbaxycinoamoyf)gusnidine,

   a. 15 (S-Phanyl-penta-2,4-dienoyf) guanidine, (3-Bromocinnamoyf)goanidins, (3-Methoxycimnamoy])guanidive, (3-Chlorocimmamoyl)guanidine, (2-Chlococinnamoyl) guanidine, » 2 Bromo guanid; oo oo (trne-2-Phemyloyclopropanecarbanyl)gnanidine, - | 7 EH 3 oC ) idi i us (Quinoline-2-carbonyl)guemids or pharmaooutically acceptable salts thereof. }

   165. . A pharmaceutical composition comprising a compound according to claim 164, and optionally one cr mare pharmaoeuticsl acceptable carriers or ‘derivatives. :

   166. ‘The phamacentical composition according to claim 165, further

   167. The method according to any one of claims 6 - 8,12,13, 16, 19, 21, 23, 25, © 27,29,31,3210 34,37 1039, 42, 45, 47, 49, 51, 53, 55, 57, 58 10 60, 63 to 65, 68,71, 73,75, 77,79, 81, 8 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, } 120-131, 133-134, 136, 137, 139-147, 149-152, 154, 156, 158, or 159, wherein said compound is selected fram the antiviral compounds C10 according to claim 164, : :

   168. The method according to any ons of claims 6, 31,584,102 or 144, } wherein said virus is Dengne virus and said compound is selected from the group consisting of cinnamoylgoanidine, (2-chlotocinnamoyl)guanidine or trans —3-(1-napthyljacryloylgusnidine. i