AU2004289375B2

AU2004289375B2 - Bis-cationic compounds and use thereof

Info

Publication number: AU2004289375B2
Application number: AU2004289375A
Authority: AU
Inventors: Katrina Anne Jolliffe; Tania Christine Sorrell; Alfred Werner Widmer; Lesley Catherine Wright
Original assignee: University of Sydney
Current assignee: University of Sydney
Priority date: 2003-11-13
Filing date: 2004-11-12
Publication date: 2010-12-23
Anticipated expiration: 2024-11-12
Also published as: AU2004289375A1

Description

WO 2005/047230 PCT/AU2004/001570 Bis-cationic Compounds and Use Thereof Technical Field The present invention relates to bis-cationic compounds comprising quaternary ammonium groups and/or quaternary phosphonium groups. The invention also relates to 5 the use of bis-cationic compounds as Phospholipase B inhibitors and the use of bis cationic compounds for the treatment or prevention of microbial infection. Background of the Invention There is a need in therapy and industry for improved antimicrobial agents. The phospholipase(s) have attributes of virulence factors and thus, may be a useful target for 10 at least one of treatment, inhibition and prevention of microbial infection, such as fungal, bacterial, viral or parasitic, infection. Hanel et al (Mycosis, 1995, 38:252-264) investigated the role of fungal phospholipases as drug targets in a mouse model of Candida albicans infection. Mice were treated with beta blockers and related compounds which inhibited secretory 15 phospholipase activity on egg yolk plate assays. However, as discussed by Ashraf et al. Microbiology (1997), 143, 331-340, the assays used by Hanel et al are not specific for phospholipases. Some bis-cationic compounds ammonium are known to have anticholinergic and neuromuscular blocking properties (e.g, Merck Index 13 th Edition (2001), MERCK & 20 CO, Inc., Whitehouse Station NJ USA; Entry number 4364, page 772 Gallamine; Entry number 4660, page 828 Hemicholinium; Entry number 9878, page 1746 Tubocurarine). Other bis-cationic ammonium compounds are known to behave as "Gemini" surfactants, as described for example in S. M. Menger and J. S. Keiper, Angew.. Chen. Int. Ed., 2000, 39, 1906-1920. 25 The present invention relates to bis-cationic compounds comprising quaternary ammonium groups and/or quaternary phosphonium groups and their use as antimicrobial agents. Summary of the Invention According to a first aspect of the invention there is provided a compound of 30 Formula (I) R1 R4

R

2

-Y-C(R

7

R

7 .)-(A)-C(R8R 8

)-Y

2

R

5 R3 R6 WO 2005/047230 PCT/AU2004/001570 2 (I) wherein

Y

1 and Y 2 may be the same or different and are independently selected from N and P; 5 R 1 to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 1

.

1 0 alkyl, optionally substituted C 2

-

1 0 alkenyl, optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

-

10 cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl, wherein said substituents are independently 10 selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, hydroxyl, halogen, O(C 1

-

6 alkyl),

C(O)O(C

1

.

6 alkyl), OC(O)(C 1

.

6 alkyl), NO 2 , amino, hydroxyC 1

.

6 alkyl, aryl, OC(O)Ph, and =C(Ph)2; or

R

1 and R 2 together with the Yi group to which they are attached, or R 1 , R 2 and R 3 together with the Yi group to which they are attached may optionally form a 15 heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are attached, or R 4 , R 5 and R 6 together with the Y 2 group to which they are attached may optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, hydroxyl, halogen, O(CI- 6 alkyl), C(O)O(C 1

.

6 alkyl), OC(O)(C 1

.

6 20 alkyl), NO 2 , amino, hydroxy C 1

.

6 alkyl, aryl, OC(O)Ph, and =C(Ph) 2 ;

R

7 , R 7 ,, Rs and R 8 ' may be the same or different and are independently selected from hydrogen, F and Cl; A comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted 25 phenyl, optionally substituted C 5

.

7 cycloalkyl, and -C(O)-, wherein the length of A is from 5 to 18 carbon atoms, and wherein the substituents are independently selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxyl, halogen, NO 2 , C(O)Rio, OR,, CH2OR 1 ,

CH

2

NR

1 2

R

3 , SRI,, NR 1 2

RI

3 , CONR1 2 R1 3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; 30 RIO is selected from OH, OR 1 , C 1

.

6 alkyl;

R

11 is selected from the group consisting of hydrogen, optionally substituted C 1

.

1 0 alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2

.

1 0 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted aryl, and optionally substituted aralkyl, wherein said optional substituents are independently selected from C 1

.

4 alkyl, hydroxyl 35 and halogen; WO 2005/047230 PCT/AU2004/001570 3 R1 2 and R1 3 are independently selected from the group consisting of hydrogen, optionally substituted C 1

.

1 0 alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

-

10 cycloalkyl, optionally substituted aralkyl, optionally substituted alkylheteroaryl, wherein said substituents are 5 independently selected from C14 alkyl, hydroxyl, halogen, amino, and C(O)ORuI; or

R

12 and R 13 , together with the nitrogen atom to which they are attached may form an optionally substituted heterocycloalkyl group, wherein said substituents are independently selected from CI4 alkyl, hydroxyl, halogen, amino, and C(O)ORu; and salts thereof, 10 provided that the compound of formula (I) is not selected from the following: WO 2005/047230 PCT/AU2004/001570 4 3Et f 3iNR RI "NP 2 R I 'Et R E6 25 R R'N'' OH EH OH R1 = R2 =R4 R5 Me, R3 = R6 Pr RI = R2 = R3 = R4 =R5 R6 = Me, Et, Ri = R2 =R4= R5 =Pr, R3 R6 =Me RI = R2 = R4 = R5 = Me, R3 = R6 = Et, Pr M me R1 =R2=R4=R5=Et,R3=R6=Me Me IMe Me M R1 =R2=R4=R5=PrR3=R6=Me OH RI = R2 = R4 = R5 = allyl, R3 = R6 = Me R R1 R R2' I

R

3 R1 = R2 = R3 = R4 = R5 = R6 = Me, Et, Pr, Bu, pentyl, allyl R1 = R2 = R4 = R6 = Me, R3 R6 = Pr, Bu, Decyl R1 = R4 = Me, R2 = R3 =R5 = R6 = Hexyl, ally RI = R4 =Me, R2 R5 = Bu, R3 = R6 = octyl R4

R

1 R R2' R

R

3 Ri = R2 = R3 = R4 = R5 = R6 = n-Bu, t-Bu, octyl PR R 4 R R2- I I1 Rs Mel--N ,Me Me I l'Me Me OH OH Me RI = R2 =R3 = R4 5 = R6 = Me, Et, allyl R1 = R2 = R4 = R5 = Me, R3 =R6 = Pr, pentyl R = Pr, H, pentyl, hexyl, butyl, Me, Et R1 = R2 = R4 = R5 = allyl, R3 = R6 = Et Bu Bu Bu I I Bu Bu Bu R4Me R1 N

P

3 R Me e R1 = R2 = R3 = R4 = R5 = R6 = Me, Pr, penty, butyl, allyl, ethyl, hexyl Et R1 = R2 = R3 = R4 = R5 = R6 = Bu, Et, hexyl, heptyl, pentyl, propyl, decyl, i-Pr, octyl R1 = R4 = Me, R2 = R3 = R5 = R6 = allyl, ethyl Me Et R1 = R2 = R4 = R5= El, R3 = R6 = hexyl El Me OH Me MeQ(p l:Me MeMe Me OH R4 .R2 R1 = R2 = R3 = R4 = R5 = R6 = Me, Et, Bu, octyl MeN@NM R, p 3 Me

R

3 R Me OH Me RI =R2=R3=R4=R5=R6=Me, Et R1=R2=R4=R5=Me, R3=R6=penlyl WO 2005/047230 PCT/AU2004/001570 5 R RI IR R2R Ri =R2 =R3=R4=R5=R6=Bu, Et, Pr Rl = R2 =R4 =R5=Me, R3 =R6 BuEt,heptyl, nony, RI = R2 =R4=R = allyl, R3=R6 =Me, Et RI = R2 =P4 = R5 = hexyl, R3 = R6 = Me

R

4 R ,.R octyl R p +Re Ioctyl

R

3 Bu' I RI =R2 R3 R4 R5 R6octy, butyl B~u Ri® @,.R 4 OH R.N N, Re Me Me 2 1P 6 NV Me' I I Me ti Et RI = R2 = R3 =R4 = R5 = R6 = Me, Et 4 R R2 l Rl =R2 =R3 R4 =R5 =R6 =Me, Et, Pr Ri = R2 = R4 R5 =Me, R3=R6 = -- " Ri = R4 = Me, R2 = R5 = Et, R3 = R6 =Pr

R

4 R Ri =R2=R3=R4=R5=R6=Et NN IR'R RI R2= R3 =R4=R5 =R = Me, Pt, Bu RI R4 =Me, R2 = R5 = Et, R3 =R6 = Pr

R

4 R2R 3 RI = R2 =R3=R4 =R5 = R6=Me, Et, Prallyl RI = R2 =R4 =P5 = Me, R3 = R6 = Et RI = R2 = R4 = R5 = Et, R3 = R6 = Me

P

3 RI=R4R2=R3=P4=PB=PB= Et I I R 5 I% RI =R2=R3=R4=RS=6=eEt RI = R4 = Me, R2 = R5 = Et, R3 = R6 = Pr

R

4 R R% Rl =R2=R3=R4=R5=R=Me,Et Ri = R2 = R4 = P5 = Et, P3 = R6 = Me RI = R4 = Me, R2 = R5 = Et, P3 = R6 = Pr WO 2005/047230 PCT/AU2004/001570 6 Ri 2 I R R3 R 6 Ri R2 =R3 =R4 =R5 =R6 Me, Et

R

4 5 R3 Me Rl =R2 =R3=R4 R5 =R6 =Me. Et, Pr Mel( RI =R4 =Me, R2 R5 Et, R3 =R6 Pr Et N 'E Et

R

3 Me Me +IIIY R3 R6 Me, Bu <C Me Me R., 0R3 = R6 = Me; Ra, Rb =H Rb C Ra R3 =R6 =Me, Ra =Ph, Rb CO 2 Et Rb Me Me 0\--0

R

3 RN R3 R6 Me, Ra H R3 P6B Me, Ra =Me C R6 R3 =R6 =Me, Ra EtB Ra R3=R6 =Me,Ra Ph, Rb C02Et Rb Ph Ra ~ ~ ~ IR3= R6 =Me, Ra, Rb Rb Ph MMe Me Me Me Me "Me me Me Me Ph (DN C /--c rPh Ph

R

3 N R Rc Me Rb NIR.R. Nr rtRa RaeR Me Rd Rc Rb R3=R6 =Me, Ra, Rb Rc,Rd =H R3 = R6 = Me, Ra =Me, Rb, Rc, Rd = H- Rb R3 = R6 = Ra= Rb =Rc =Rd =Me Ra\ Me f oR 3 \N Rc 0-jaMe Rc R Ph PRa Ph Rb R3 =R6 =Me, Ra =Me, Rb =Rc =H Me Ph ~ R3 = R6 = Me, Ra =Et, Rb =~ Rc=H M N R3 =R6 =Et, R = H, Rb =ORRc =H Ph-Me RS=R6=Me,Ra =Rb=Rc=H R3=R6=Me,Ra =Rb=Rc=H PH R3 =R6 =Me, Re = H.Rb =OC(=0)Pr, Rc H Ph R3=R6=Me,Ra =H,Rb=OAc,Rc=H M (D CO 2 Et R3 =R6 =Me, Re=Rb =H,Rc OC(O)Ph Ph- NI Me EtO 2

C

WO 2005/047230 PCT/AU2004/001570 7 Me Ra Me N Ra,Rb = H Ra =Me, Rb= Et Me Ph Me I M, Ph @N @ Me O. Mee O D Me N N Ph S Me M Me E eM Me~ ~ Phr-\ Ph Me (D Me Me o Me MMe + Ph Me en Ph Ph NIn afuthr modmet f h invninY sPadY sP 0D Me Ph Me N SR1 to R6Mmay be the same or different and in one embodiment are independently selected from optionally substituted C2-10 alkyl. In another embodiment, R1 to R6 are independently selected from optionally substituted C2.-8 alkyl. In a further embodiment, R1 to R6 are independently selected from optionally substituted C3-6 alkyl. In one embodiment of the invention, Y1 and Y2 are each N, and R1 to R6 are 10 independently selected fromMC2-1 alkyl. In some embodiments of the invention, the relative lengths of R1, R2 and R3 may vary by 2 to 6 carbons with respect to each other, for example, by 2, 3, 4, 5 or 6 carbons.

WO 2005/047230 PCT/AU2004/001570 8 In other embodiments of the invention, the relative lengths of R 1 , R 2 and R 3 may vary by 3 or 4 carbons. In some embodiments of the invention, the relative lengths of R 4 , R 5 and

R

6 may vary by 2 to 6 carbons with respect to each other, for example, by 2, 3, 4, 5 or 6 carbons. In other embodiments of the invention, the relative lengths of R 4 , R 5 and R 6 may 5 vary by 3 or 4 carbons. In one embodiment of the invention R 1 to R 3 are the same. In another embodiment

R

4 to R 6 are same. In a further embodiment, R 1 to R 6 are the same. In one embodiment of the invention the length of A is from 5 to 9 carbon atoms. In one embodiment, when Y 1 and Y 2 are each N, R 1 to R 6 are each C 1

.

1 o alkyl, R 7 , 10 R 7 ., R 8 , and R 8 , are each hydrogen, then A is not C 10

.

1 8 alkylene substituted with a C1-3 alky1 substituent. In another embodiment of the invention, when Y 1 and Y 2 are each N, R 1 to R 6 are each C 1

-

1 0 alkyl, R 7 , R 7 ,, R 8 , and R8, are each hydrogen, then A is C 1 0 .8 1 alkylene substituted with a C 1

.

3 alkyl substituent and at least one other substituent. In a further embodiment of the invention, when Y 1 and Y 2 are each N; R 3 and R 6 are 15 each C 1

.

1 0 alkyl; R 1 and R 2 together with the N to which they are attached, and R 4 and R 5 together with the N to which they are attached are each a 4- or 5-membered heterocycloalkyl ring comprising one S or 0, substituted with hydroxyC 1

.

3 alkyl or haloC 1

.

3 alkyl; R 7 , R 7 ., R 8 , and R 8 , are each hydrogen, then A is not C 1 0 .8 1 alkylene substituted with a C 1

-

3 alkyl substituent. 20 In one embodiment of the invention, A may comprise one or more alkylene groups "(CR9R9a)n", wherein R 9 and R9a may be the same or different and are independently selected from the group consisting of hydrogen, halogen, C 1

.

6 alkyl, C 2

-

6 alkenyl, C(O)Rio, OR,,, CH 2 ORII, CH 2 OH, SRI,, NR 12

R

13 , CON 12

R

3 , OH, SH, amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl, and wherein n is an integer 25 selected from I to 18. For example, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, provided that the total length of A is from 5 to 18 carbons. In one embodiment, n is an integer from 5 to 18. In one embodiment of the invention, A comprises one or more groups selected from optionally substituted alkylene (including optionally substituted methylene "CR 9

R

9 a"), 30 optionally substituted phenyl, and optionally substituted Cs6 cycloalkyl, wherein said substituents are independently selected from C 1

-

6 alkyl, hydroxyl and halogen. In one embodiment of the invention, A comprises at least one alkylene group "CR 9

R

9 a". In one embodiment of the invention, A comprises at least one phenylene. In another embodiment of the invention, A comprises at least one cyclohexylene.

WO 2005/047230 PCT/AU2004/001570 9 In one embodiment of the invention, R 9 and Ra are both hydrogen. In another embodiment, R 9 is hydrogen and R 9 a is selected from hydrogen, C1.

6 alkyl, C(O)Rio, OR,,, CONRu 1

R

1 2 , NR 1 2

R

13 , OH and SH. For example, in one embodiment of the invention, R 9 is hydrogen and R 9 a is hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, 5 COOH, CO 2 Me, CO 2 Et, CO 2 Bu, CONHMe, CONMe 2 , CONHEt, CONEt 2 ,

CONH(CH

2 Ph), CON(CH 2 Ph) 2 , CO 2 But, OMe, OEt, NMe 2 , or NEt 2 . A may be unsubstituted C 8

-

12 or C 5

.

9 alkylene, or optionally substituted C8- 12 or C5.

9 alkylene. A may be unsubstituted C8- 12 or C 5

.

9 alkenylene or optionally substituted C 8

-

12 or C5.

9 alkenylene. In one embodiment of the invention, A is unsubstituted C 8 -1 2 or C 5 .9 10 alkylene or C 8

-

12 or C 5

-

9 unsubstituted alkenylene. In one embodiment of the invention, n is 8, 9 or 10, each A is CR 9

R

9 a, and R 9 and R9a are each hydrogen. In some embodiments, R 12 and R 13 , together with the nitrogen atom to which they are attached may form an optionally substituted heterocycloalkyl ring. For example, the 15 group NR 1 2

R

13 may optionally be 3-aminopyrrolidinyl, 3-aminoquinuclidinyl, 3 aminopyridinyl, 1-(3-aminopropyl)-2-pipecolinyl groups. In one embodiment of the invention the compound is a compound of Formula (Ia): RI\ R4

R

2

-Y-CH

2 (A) CH2-Y2 Rq

R

3 R6 (Ia) 20 wherein Yi and Y 2 may be the same or different and are independently selected from N and P;

R

1 to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 1

..

10 alkyl, optionally substituted C 2

.

10 alkenyl, 25 optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

..

10 cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl, wherein said substituents are independently selected from C 1

.

6 alkyl,

C

2

.

6 alkenyl, hydroxyl, halogen, O(C 1

.

6 alkyl), C(O)O(C 1

.

6 alkyl), NO 2 , amino, hydroxy

C

1

.

6 alkyl, aryl, and OC(O)Ph; or 30 R 1 and R 2 together with the Yi group to which they are attached may optionally form a heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are attached may optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected WO 2005/047230 PCT/AU2004/001570 10 from C1-6 alkyl, C 2

.

6 alkenyl, hydroxyl, halogen, O(C 1

-

6 alkyl), C(O)O(C 1

.

6 alkyl), amino, hydroxy C 1

.

6 alkyl, and aryl; A comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, and optionally substituted phenyl, wherein the length of 5 A is from 5 to 18 carbon atoms, and wherein the substituents are independently selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

.

6 alkynyl, halogen, C(O)Rio, OR 1 , SR 11 , CH 2 0RII,

CH

2

NR

1 2

RI

3 , NR 12

R

13 , CONR 1 2

R

13 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; Rio is selected from OH, OR,, C 1

-

6 alkyl; 10 Ra is selected from the group consisting of hydrogen, optionally substituted C 1

..

1 0 alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2

..

1 0 alkynyl, and optionally substituted C 3

.

10 cycloalkyl, wherein said optional substituents are independently selected from C 1

.

6 alkyl, C 2

.

6 alkenyl, aryl, and hydroxyl;

R

12 and R 13 are independently selected from the group consisting of hydrogen, 15 optionally substituted C 1

.

1 0 alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted alkylheteroaryl, wherein said substituents are independently selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, aryl, hydroxyl, halogen, amino, and C(O)ORuI; or

R

12 and R 13 , together with the nitrogen atom to which they are attached may form 20 an optionally substituted heterocycloalkyl group or heteroaryl group, wherein said substituents are independently selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, hydroxyl, halogen, amino, and C(O)OR 1 , and salts thereof. Also disclosed herein are compounds of Formula (II): R1\D ( R 4

R

2

-I-C(R

7

R.)-(A)--C(RSRS.)-Y

2 \ R 5 25 R 3 R6 (II) wherein

Y

1 and Y 2 may be the same or different and are independently selected from N and P; 30 R 1 to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 1

.

10 alkyl, optionally substituted C 2

-

1 0 alkenyl, optionally substituted C 2

-

1 0 alkynyl, optionally substituted C 3

..

10 cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and WO 2005/047230 PCT/AU2004/001570 11 optionally substituted heterocycloalkyl, wherein said substituents are independently selected from C 1

.

6 alkyl, C 2

.

6 alkenyl, C 2

.

6 alkynyl, hydroxyl, halogen, O(C 1

.

6 alkyl),

C(O)O(C

1

.

6 alkyl), NO 2 , amino, hydroxy C 1

.

6 alkyl, aryl, OC(O)Ph, and =C(Ph) 2 ; or

R

1 and R 2 together with the Y 1 group to which they are attached, or R 1 , R 2 and R 3 5 together with the Yi group to which they are attached may optionally form an heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are attached, or R 4 , R 5 and R 6 together with the Y 2 group to which they are attached may optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected from C 1

.

6 alkyl, C 2

-

6 10 alkenyl, C 2

-

6 alkynyl, hydroxyl, and halogen, O(C1.

6 alkyl), C(O)O(C1.6 alkyl), NO 2 , amino, hydroxy C 1

.

6 alkyl, aryl, and =C(Ph) 2 ;

R

7 , R 7 ,, R 8 and R 8 , may be the same or different and are independently selected from hydrogen, F and Cl; A comprises one or more groups selected from optionally substituted alkylene, 15 optionally substituted alkenylene, optionally substituted phenyl, optionally substituted

C

5

.

7 cycloalkyl, and -C(O)-, wherein the length of A is from 4 to 18 carbon atoms, wherein the substituents are independently selected from C 1

.

6 alkyl, C 2

-

6 alkenyl, hydroxyl, halogen, nitro, C(O)Rio, OR 11 , CH 2 0R 11 , CH 2 NR1 2 R13, SR 11 , NR 12

R

1 3 ,

CONR

1 2

R

13 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; 20 Rio is selected from OH, OR,,, C 1

-

6 alkyl, optionally substituted amino-C 1

.

6 alkylsulfonate, optionally substituted amino-CI.-alkylphophonate, optionally substituted amino-C 1 .- alkyl-guanidinyl, and optionally substituted amino-C 1

.

6 -alkyl-tri(C- 6 alkyl)ammonium; Ri 1 is selected from the group consisting of hydrogen, optionally substituted CI.o 1 25 alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

-

10 cycloalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted amino-C 1

.

6 -alkylsulfonate, optionally substituted amino-C- 6 alkylphophonate, optionally substituted amino-C 1

.

6 -alkyl-guanidinyl, and optionally substituted amino-C 1 .- alkyl-tri(CI.-alkyl)ammonium, wherein said optional substituents 30 are independently selected from C 1

.

4 alkyl, hydroxyl and halogen

R

12 and R 13 are independently selected from the group consisting of hydrogen, optionally substituted C 1 .. o alkyl, optionally substituted C 2

-

10 alkenyl, optionally substituted C 2 o 10 alkynyl, optionally substituted C 3

-

10 cycloalkyl, optionally substituted arylalkyl, optionally substituted alkylheteroaryl, optionally substituted amino-C 1

.

6 35 alkylsulfonate, optionally substituted amino-Ci--alkylphophonate, optionally substituted WO 2005/047230 PCT/AU2004/001570 12 amino-C1- 6 -alkyl-guanidinyl, and optionally substituted amino-C- 6 -alkyl-tri(C- 6 alkyl)ammonium, wherein said substituents are independently selected from C 1

-

3 alkyl, hydroxyl, halogen, amino, and C(O)ORii; or

R

12 and R 13 , together with the nitrogen atom to which they are attached may form 5 an optionally substituted heterocycloalkyl group, wherein said substituents are independently selected from C 1

.-

3 alkyl, hydroxyl, halogen, amino, and C(O)OR 1 . With reference to Formula (II), the group amino-CI.6-alkylsulfonate includes for example, aminomethyl-sulfonate (i.e a "-NH-CH 2 -SO3' group), aminoethyl-sulfonate and aminopropyl-sulfonate; amino-C 1 .- alkylphosphonate includes for example, 10 aminomethyl-, aminoethyl- and aminopropyl-phosphonate; amino-C 1 -- alkyl-guanidinyl includes for example, 4-aminobutyl-guanidinyl; amino-C 1

-

6 -alkyl-tri(CI.

6 alkyl)ammonium includes for example 1-(3-aminopropyl)trimethylammonium.

R

1 to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 1 .i 0 alkyl, optionally substituted C 2

-

10 alkenyl, 15 optionally substituted C 2

..

1 0 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted aryl and optionally substituted aralkyl, wherein said substituents are independently selected from C 1

.

6 alkyl, hydroxyl and halogen; or

R

1 and R 2 together with the Y 1 group to which they are attached, or R 1 , R 2 and R 3 together with the Y 1 group to which they are attached may optionally form a 20 heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are attached, or R 4 , R 5 and R 6 together with the Y 2 group to which they are attached may optionally form a heterocycloalkyl group; wherein said heterocycloalkyl group may be optionally substituted with one or more groups selected from C 1

.

6 alkyl, hydroxyl and halogen; 25 A may comprise one or more groups selected from optionally substituted methylene groups "CR 9

R

9 a", optionally substituted phenyl, and optionally substituted C 5

.

7 cycloalkyl, wherein the length of A is from 4 to 18 carbon atoms, and wherein said substituents are independently selected from C 1

.

6 alkyl, hydroxyl and halogen;

R

9 and R9a may be the same or different and are independently selected from the 30 group consisting of hydrogen, halogen, C 1

..

6 alkylene, C 1

.

6 alkenylene, C(O)Rio, OR, SRI,, NR 12

R

13 , CON 12

R

3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; RIO is OH or OR,,; Ru, is selected from the group consisting of hydrogen, optionally substituted C 1

.

1 0 35 alkyl, optionally substituted C 2

-

1 0 alkenyl, optionally substituted C 2

..

1 0 alkynyl, optionally WO 2005/047230 PCT/AU2004/001570 13 substituted C 340 cycloalkyl, and optionally substituted aralkyl, wherein said substituents are independently selected from C 1

.

6 alkyl, hydroxyl and halogen;

R

12 and R 13 are independently selected from the group consisting of hydrogen, optionally substituted Ci..o alkyl, optionally substituted C 2 o 10 alkenyl, optionally 5 substituted C 2

-

10 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted heteroaralkyl, and optionally substituted aralkyl, wherein said substituents are independently selected from C 1

.

6 alkyl, hydroxyl, halogen, and C(O)ORII; or R 12 and R 13 together with the N to which they are attached may optionally form a heterocycloalkyl group. 10 In one embodiment of the invention, the compound is a compound of Formula (I) or (II) wherein R 7 , R 7 ,, R 8 , and Rs, are each hydrogen; A is optionally substituted C 8 1 0 alkylene and R 1 to R 3 are each butyl. In another embodiment, the compound is a compound of Formula (I) or (II) wherein R 7 , R 7 ,, R 8 , and R 8 , are each hydrogen, A is unsubstituted C 8

.

1 0 alkylene, and R 1 to R 3 are each butyl. 15 According to a second aspect of the invention there is provided the use of at least one compound of Formula (I) or Formula (II) for the manufacture of a medicament for one or more of treating, inhibiting and preventing a microbial infection. The microbial infection may comprise one or more of bacteria, fungi, viruses, and parasites. 20 According to a third aspect of the invention there is provided a method for one or more of treating, inhibiting, and preventing a microbial infection in a vertebrate, said method comprising administering to said vertebrate an effective amount of at least one compound of Formula (I) or Formula (II). According to a fourth aspect of the invention there is provided a method for one or 25 more of treating, inhibiting, and preventing a microbial infection of a plant, said method comprising contacting said plant with an effective amount of at least one compound of Formula (I) or Formula (II). According to a fifth aspect of the invention there is provided an antimicrobial composition comprising at least one compound of Formula (I) or a compound of Formula 30 (II), together with an industrially, veterinarially, agriculturally, or pharmaceutically suitable carrier, diluent or carrier. With reference to the fifth aspect of the invention, the composition may be one or more of an antifungal, antibacterial, antiviral and antiparasitic composition. In one embodiment the composition is an antifungal composition. In another embodiment the 35 composition is an antibacterial composition.

14 According to a sixth aspect of the invention there is provided a pharmaceutical composition comprising at least one compound of Formula (I) or Formula (1I) together with a pharmaceutically effective carrier, adjuvant or diluent. According to a seventh aspect of the invention there is provided a method of s inhibiting phospholipase in an organism comprising contacting said organism with an effective amount of at least one compound of Formula (I) or at least one compound of Formula (II), or a composition according to the fourth or fifth aspect of the invention. With reference to the seventh aspect of the invention, the organism may be a microbial organism such as bacteria, fungi, virus, or a parasite, including for example 10 protozoa. The phospholipase may be Phospholipase B. According to an eighth aspect of the invention there is provided a method for identifying an antimicrobial agent comprising contacting microbial cells with a compound suspected of having antimicrobial properties, determining whether said compound inhibits a microbial phospholipase enzyme, wherein inhibition of said phospholipase is enzyme indicates antimicrobial activity, and thereby identifying an antimicrobial agent. With reference to the seventh aspect of the invention, the compound may be a compound of Formula (I) or Formula (II) as defined herein. The phospholipase may be Phospholipase B. In accordance with a ninth aspect of the invention there is provided a compound of 20 Formula (1) or a compound of Formula (II) or a composition according to the fifth or sixth aspect of the invention, when used for one or more of treating, inhibiting or preventing a microbial infection or a parasitic infection. In a further aspect there is provided use, in the manufacture of a medicament for one or more of treating, inhibiting, and preventing a bacterial or fungal infection in a 25 vertebrate, of a compound of Formula (I): R1 ( V R4

R

2 - 1-C(R 7

R

7 .)-(A)-C(R8R 8

.)-Y

2

R

5

R

3 R6 (1) wherein Y, and Y 2 may be the same or different and are independently selected from N and P; 30 R, to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 2

-

10 alkyl, optionally substituted C 2

-

1 0 alkenyl, optionally substituted C 2

-

10 alkynyl, optionally substituted C 3

-

10 cycloalkyl, optionally 14a substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl, wherein said optional substituents are independently selected from Ci.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxyl, halogen,

O(CI.

6 alkyl), C(O)O(Ci- 6 alkyl), OC(O)(Ci.

6 alkyl), NO 2 , amino, hydroxy CI- 6 alkyl, s aryl, OC(O)Ph, and =C(Ph) 2 ; or R, and R 2 together with the Y 1 group to which they are attached, or R 1 , R 2 and R 3 together with the Yi group to which they are attached may optionally form an heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are attached, or R4, R 5 and R 6 together with the Y 2 group to which they are attached may 10 optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected from CI- 6 alkyl, C 2 -6 alkenyl, C 2

-

6 alkynyl, hydroxyl, halogen, O(CI.

6 alkyl), C(O)O(CI- 6 alkyl), OC(O)(CI-6 alkyl), NO 2 , amino, hydroxy C 1

-

6 alkyl, aryl, OC(O)Ph, and =C(Ph) 2 ;

R

7 , R., R 8 and R 8 .may be the same or different and are independently selected from is hydrogen, F and Cl; A comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted phenyl, optionally substituted C 5

.

7 cycloalkyl, and -C(O)-, wherein the length of A is from 5 to 18 carbon atoms, wherein the optional substituents are independently selected from 20 C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxyl, halogen, nitro, C(O)R 1 o, OR 1 , CH 2 0R 11 ,

CH

2

NR

1 2 Ri 3 , SRI 1 , NR 12

RI

3 , CONR 2

RI

3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; Rio is selected from OH, OR,,, and CI- 6 alkyl RI, is selected from the group consisting of hydrogen, optionally substituted CI- 1 a 25 alkyl, optionally substituted C 2

-

1 0 alkenyl, optionally substituted C 2

-

1 0 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted aryl, and optionally substituted aralkyl, wherein said optional substituents are independently selected from C 14 alkyl, hydroxyl and halogen;

R

1 2 and R 13 are independently selected from the group consisting of hydrogen, 30 optionally substituted C 1

.

1 0 alkyl, optionally substituted C 2

.

1 0 alkenyl, optionally substituted C 2

-

1 0 alkynyl, optionally substituted C 3

.

10 cycloalkyl, optionally substituted aralkyl, and optionally substituted alkylheteroaryl, wherein said optional substituents are independently selected from C 14 alkyl, hydroxyl, halogen, amino, and C(O)OR 1 ; or 14b

R

1 2 and R 13 , together with the nitrogen atom to which they are attached may form an optionally substituted heterocycloalkyl group, wherein said optional substituents are independently selected from Cia alkyl, hydroxyl, halogen, amino, and C(O)ORii; or a salt thereof. 5 Definitions The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description. 10 Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements. Throughout this specification, unless the context requires otherwise, the word IS "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or WO 2005/047230 PCT/AU2004/001570 15 integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Those skilled in the art will appreciate that the invention described herein is 5 susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. 10 In the context of this specification, the term "amino acid" is defined as having at least one primary, secondary, tertiary or quaternary amino group, and at least one acid group, wherein the acid group may be a carboxylic, sulfonic, or phosphonic acid, or mixtures thereof. The amino groups may be "alpha", "beta", "gamma" ... to "omega" with respect to the acid group(s). The backbone of the "amino acid" may be substituted 15 with one or more groups selected from halogen, hydroxy, guanido, heterocyclic groups. Thus term "amino acids" also includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparte, glutamine, lysine, arginine and histidine, taurine, betaine, N-methylalanine etc. (L) and (D) forms of amino acids are included in the scope 20 of this invention. As used herein, the term "alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 10 carbon atoms, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1-propyl, 25 isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1,2-dimethylpropyl, 1,1 dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3 dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2 30 dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2 trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, and the like. The term "alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon 35 groups having from 2 to 10 carbon atoms, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, WO 2005/047230 PCT/AU2004/001570 16 Examples of alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1 methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-i-propenyl, 2-methyl-1-propenyl, 1 butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, 1-pentenyl, 2-pententyl, 3-pentenyl, 4 pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1 5 hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1 heptenyl, 2-heptentyl, 3-heptenyl, 1-octenyl, 1-nonenyl, 1-decenyl, and the like. The term "alkynyl group" as used herein includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms and having at least one triple bond. 10 Examples of alkynyl groups include but are not limited to ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, 1-methyl-2-butynyl, 3-methyl-1-butynyl, 1-pentynyl, 1-hexynyl, methylpentynyl, 1-heptynyl, 2-heptynyl, 1-octynyl, 2-octynyl, 1-nonyl, 1-decynyl, and the like. The term "cycloalkyl" as used herein refers to cyclic saturated aliphatic groups and 15 includes within its meaning monovalent ("cycloalkyl"), and divalent ("cycloalkylene"), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 carbon atoms, eg, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the 20 like, The term "cycloalkenyl" as used herein, refers to cyclic unsaturated aliphatic groups and includes within its meaning monovalent ("cycloalkenyl") and divalent ("cycloalkenylene"), monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 carbon atoms and having at least one double bond, of either 25 E, Z, cis or trans stereochemistry where applicable, anywhere in the alkyl chain. Examples of cycloalkenyl groups include but are not limited to cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like. The term "heterocycloalkyl" as used herein, includes within its meaning monovalent ("heterocycloalkyl") and divalent ("heterocycloalkylene"), saturated, 30 monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 10 ring atoms wherein 1 to 5 ring atoms are heteroatoms selected from 0, N, NH, or S. Examples include pyrrolidinyl, piperidinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like. The term "heterocycloalkenyl" as used herein, includes within its meaning 35 monovalent ("heterocycloalkenyl") and divalent ("heterocycloalkenylene"), saturated, WO 2005/047230 PCT/AU2004/001570 17 monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 ring atoms and having at least 1 double bond, wherein from 1 to 5 ring atoms are heteroatoms selected from 0, N, NH or S. The term "heteroaromatic group" and variants such as "heteroaryl" or 5 "heteroarylene" as used herein, includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), single, polynuclear, conjugated and fused aromatic radicals having 6 to 20 atoms wherein 1 to 6 atoms are heteroatoms selected from 0, N, NH and S. Examples of such groups include pyridyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, thiophenyl, and the like. 10 The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine. The term "heteroatom" or variants such as "hetero-" as used herein refers to 0, N, NH and S. The term "alkoxy" as used herein refers to straight chain or branched alkyloxy 15 groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like. The term "amino" as used herein refers to groups of the form -NRaRb wherein Ra and Rb are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, 20 and optionally substituted aryl groups. The term "aromatic group", or variants such as "aryl" or "arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like. 25 The term "aralkyl" as used herein, includes within its meaning monovalent ("aryl") and divalent ("arylene"), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight and branched chain alkylene radicals. The term "heteroaralkyl" as used herein, includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), single, polynuclear, conjugated and fused 30 aromatic hydrocarbon radicals attached to divalent saturated, straight and branched chain alkylene radicals. The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl, 35 heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxyl, alkoxy, thioalkoxy, WO 2005/047230 PCT/AU2004/001570 18 alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl, alkylsulfenyl, alkylcarbonyloxy, 5 alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cyano, cyanate, isocyanate, -C(O)NH(alkyl), and -C(O)N(alkyl) 2 . The term "substantially non-polar" as used herein, refers to groups which do not contain more than one polar substituent capable of forming hydrogen bonds and which do 10 not undergo protonation/deprotonation at physiological pH. The term "antimicrobial" includes within its scope antifungal, antibacterial, antiviral and antiparasitic. The term "antifungal" as used herein and unless stated to the contrary, includes within its scope fungistatic activity and fungicidal activity. 15 The term "antibacterial" as used herein and unless stated to the contrary, includes within its scope bacteriostatic activity and bacteriocidal activity. The term "antiviral" as used herein and unless stated to the contrary, includes within its scope virostatic activity and virocidal activity. The term "Phospholipase B" as used herein refers to protein(s) having one or more 20 activities selected from phospholipase B (PLB) activity, lysophospholipase (LPL) activity and lysophospholipase transacylase (LPTA) activity. The protein(s) may or may not have other enzyme activities. This term encompasses cell associated (intracellular and membrane bound) and secretory Phospholipase B enzyme. Thus, in the context of this specification, the tenn "Phospholipase B" refers to a protein which may exhibit one or 25 more of PLB, LPL or LPTA activity. In the context of this invention the tenn "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means. 30 In the context of this specification, the term "vertebrate" includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian.

WO 2005/047230 PCT/AU2004/001570 19 In the context of this specification, the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. 5 In the context of this specification the terms "therapeutically effective amount" and "diagnostically effective amount", include within their meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of 10 the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. 15 Abbreviations "PLB" - Phospholipase B activity "LPL" - lysophospholipase activity "LPTA" - lysophospholipase transacylase activity "MIC" - minimum inhibitory concentration 20 "EDTA" - ethylenediaminetetraacetic acid "EGTA" - ethyleneglycoldiamine tetraacetic acid Brief Description of the Drawings A preferred form of the present invention will now be described by way of example with reference to the accompanying drawings wherein: 25 Figure la. Sites of action of some phospholipases on (di-acyl)phospholipids Figure 1b. The three activities of the cryptococcal phospholipase "PLB". Figure 2. Structures of exemplary bis-cationic compounds in accordance with the present invention. Figure 3. Effects of protein concentration on membrane-associated phospholipase 30 activities. Points shown are the means and SEM of three assays. Figure 4. Effects of substrate concentration on cytosolic phospholipase activities. Points shown are means and SEM of three assays.

WO 2005/047230 PCT/AU2004/001570 20 Figure 5. Effects of pH on membrane-associated phospholipase activities. In A, LPL/LPTA was measured using 30 sec incubation; in C, LPL/LPTA was measured using 10 min incubation. In B, PLB was measured using 10 min incubation. Points shown are means and SEM of three assays. 5 Figure 6. PLA and PLD activity versus pH. Detailed Description of the Invention The present invention relates to bis-cationic compounds, including bis-ammonium compounds, bis-phosphonium compounds and ammonium/phosphonium compounds, and their use as antimicrobial agents, e.g, antibacterial, antifungal, antiviral, antiparasitic 10 and/or antiprotozoal agents. Bis-cationic compounds of Formula () and Formula (II) disclosed herein may display broad spectrum antimicrobial activity. The bis-cationic compounds disclosed herein may be capable of inhibiting microbial phospholipase(s). However the present invention is not limited to bis-cationic compounds which are phospholipase inhibitors. Compounds which are capable of 15 inhibiting intracellular phospholipases, e.g, cytosolic or membrane associated phospholipases may have antimicrobial properties. Inhibition of extracellular phospholipases may have fungistatic properties and/or bacteriostatic properties, and/or virostatic properties. Bis-cationic compounds according to the present invention comprise two cationic 20 head groups substituted with non-polar or substantially non-polar groups. The head groups are linked by a non-polar or substantially non-polar spacer group. Compounds within the scope of the present invention include bis-cationic compounds of Formula (I) and Formula (I) as defined herein. In Formula (I) and Formula (II) the cationic head groups "Y 1

(R

1

R

2

R

3 )" and 25 "Y 2

(R

4

R

5

R

6 )" may be the same or different and are independently selected from a quaternary ammonium group and a quaternary phosphonium group. Thus, the cationic head groups may both be quaternary ammonium groups, one may be a quaternary ammonium group and the other a phosphonium group, or both may be phosphonium groups. 30 The R 1 to R 6 groups attached to the cationic quaternary N or P may be the same or different. In one embodiment of the invention, R 1 to R 3 are the same. In another embodiment, R 4 to R 6 are the same. In a further embodiment, R 1 to R 6 are the same. The R 1 to R 6 groups attached to the cationic quaternary N and P atoms are generally non-polar or substantially non-polar. Thus, the R 1 to R 6 groups may not contain more than WO 2005/047230 PCT/AU2004/001570 21 one polar group which could be involved in hydrogen bonding. Thus, for example, respective R 1 to R 6 groups may each be substituted with one hydroxyl group. In some embodiments, the R 1 to R 6 groups may not contain groups which can become protonated to form positive charges or which can become disassociated to form negative charges in a 5 pH range of about 4 to about 9. R2 to R 6 may be the same or different and in one embodiment are independently selected from optionally substituted C 2

-

10 alkyl. In another embodiment, R 1 to R 6 are independently selected from optionally substituted C 2

-

8 alkyl. In a further embodiment, R1 to R 6 are independently selected from optionally substituted C 3

-

6 alkyl. 10 In one embodiment of the invention, Y 1 and Y 2 are each N, and R 1 to R 6 are independently selected from C 2 -1 0 alkyl. Different groups attached to a respective cationic N or P may vary in length from each other, for example, by 2 to 6 carbons. For example, a cationic head group may comprise: Butyl ~ -N-uty ~ ,Hexl ® ~5Butyl ~ 0 /Ethyl G iHexyl F Propyl /sopentyl N:-Butyl -N;-Butyl P -Propyl FN \isopentyl 15 \Butyl ,Octyl Benzyl isopentyl and the like. The spacer group "C(R 7

R

7 )-A-C(RsRs.)" is a non-polar or substantially non-polar residue and comprises an optionally substituted carbon backbone linking the polar cationic head groups. The spacer portion may be from 7 to 20 carbon atoms in length for 20 Formula (I) compounds, or from 6 to 20 atoms in length for Formula (II) compounds. For example, the spacer may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in length for compounds of Formula (I), or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in length for compounds of Formula (II). In various embodiments of the invention, the spacer may be C 10

.

1 6 , C 1 0.

1 s or C10- 2 0 carbon atoms in 25 length, wherein respective carbon atoms may be optionally substituted. In alternative embodiments, the spacer may be from 6 to 11 or 7 to 11 carbon atoms in length. The spacer group may comprise one or more optionally substituted groups selected from alkylene, alkenylene, alkynylene, cycloalkylene, arylene, and carbonyl groups. Examples of optional substituents include one or more groups selected from halides, 30 alkyl, alkenyl, alkoxy, carboxylic acids, esters, ethers, thioethers, secondary or tertiary amines, secondary or tertiary amides, nitro, hydroxyl, thiol, amino acids, small peptides (eg, 2, 3, 4 or 5 amino acids in length), etc. The groups at respective terminal ends of the WO 2005/047230 PCT/AU2004/001570 22 spacer group which are immediately adjacent the respective cationic groups (sometimes referred to herein as the "cc" and "co" carbons) may be independently selected from -CH 2 -, -CCl 2 -, -CFCl-, and -CF 2 - groups. Compounds of Formula (I) and Formula (II) may comprise one or more amino acids 5 (as defined herein) or oligoamino acids (eg, 2, 3, 4, or 5 amino acids in length), attached to one or more carbons of the spacer group. In the context of this specification, "oligoamino acids" refers to amino acids linked through their respective amino and acid groups to form dipeptidyl, tripeptidyl, tetrapeptidyl, and pentapeptidyl residues. Compounds according to the invention may further optionally include one or more 10 "depsi"(peptides), viz, peptides comprising an ester bond (eg, via a hydroxyl group in an amino acid or hydroxy acid such as glycollic acid, lactic acid, etc). cc, P, or y Amino acids may be used and (L) and (D) isomers may be used. Examples of amino acid substituents include glycinyl, alaninyl, valinyl, leucinyl, isoleucinyl, methioninyl, prolinyl, phenylalaninyl, tryptophanyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, 15 glutaminyl, aspartyl, glutamyl, lysinyl, argininyl and histidinyl. Other amino acids are known to those skilled in the art. The amino acid groups may be N- or C-linked to the spacer portion of the compound. The amino acid group may also be attached via the amino acid side chain, eg, a -COOH substituent of asparatic acid or glutamic acid, a -SH group of methionine or cysteine, etc. 20 In another embodiment of the invention the compound of Formula (I) is: OH 0 NH N In a further embodiment of the invention the compound of Formula (I) is: 0 0 NH N® 25 In another embodiment of the invention the compound of Formula (I) is: WO 2005/047230 PCT/AU2004/001570 23 It In a further embodiment of the invention the compound of Formula (1) is: In another embodiment of the invention the compound of Fonmula (I) is: 01 In a further embodiment of the invention the compound of Formula (I) is: N In another embodiment of the invention the compound of Formula (1) is: In a further embodiment of the invention the compound of Formula (I) is: NN 10N HN

\

WO 2005/047230 PCT/AU2004/001570 24 In other embodiments of the invention, the compound of Formula (I) is selected from: 1,11 -bis-(tributylammonium)undecane, 1,16-bis-(tributylammonium)hexadecane, 5 1,12-bis-(tripentylammonium)dodecane, 1,12-bis-(trihexylammonium)dodecane, 1,12-bis-(trioctylaninonium)dodecane, 1,12-bis-(triisobutylammonium)dodecane, 1,12-bis-(triisopentylammonium)dodecane, and 10 1,12-bis-(1-butylpyrrolidinium)dodecane, and salts thereof. In one embodiment of the invention the compound of Formula (II) is: (N N In another embodiment of the invention the compound of Formula (II) is: 15 In another embodiment of the invention the compound of Formula (II) is: 0 In a further embodiment of the invention the compound of Formula (II) is: (octyl) 3 N () 20 In another embodiment of the invention the compound of Formula (II) is: (hexyl) 3 N ( h In a further embodiment of the invention the compound of Formula (II) is: (isopentyl)3 N(isopentyl) 3 In another embodiment of the invention the compound of Formula (II) is: (pentyl) 3 N ( p 25 N (pentyl) 3 WO 2005/047230 PCT/AU2004/001570 25 In another embodiment of the invention the compound of Formula (II) is: (butyl)3N- - N(butyl)3 In a further embodiment of the invention the compound of Formula (II) is: (butyl)3N b 5 Bis-cationic compounds according to the present invention may inhibit Phospholipase B enzyme(s) in microorganisms, although the present invention is not limited only to compounds having that activity. Phospholipase B enzymes(s) are distinguished by having activity including one or more of phospholipase B (PLB) activity, lysophospholipase (LPL) activity and lysophospholipase transacylase (LPTA) activity. 10 Phospholipase B may be cell associated (i.e., intracellular or membrane bound) enzyme(s), or secretory Phospholipase B enzyme(s). Thus, the class of enzymes is referred to as "Phospholipase B", and Phospholipase B enzymes may exhibit one or more activities, including phospholipase B (PLB) activity. Therefore, in the context of this specification "PLB" refers to the action of the enzyme; "Phospholipase B" refers to the 15 class of enzyme. An overview of the characterisation and activity of Phospholipase B is provided by Wilton et al., in Biochemistry of Lipids, Lipoproteins and Membranes ( 4 th Edition), D.E. Vance and J. E. Vance (Eds.) 2002, pp. 291-314. Synthesis of Compounds Compounds of Formula (I) and Formula (II) may be prepared by methods known to 20 those skilled in the art. Suitable methods are generally described, for example, and intermediates thereof are described, for example, in Houben-Weyl, Methoden der Organischen Chemie; J. March, Advanced Organic Chemistry, 4 h Edition (John Wiley & Sons, New York, 1992); D. C. Liotta and M. Volmer, eds, Organic Syntheses Reaction Guide (John Wiley & Sons, Inc., New York, 1991); R. C. Larock, Comprehensive 25 Organic Transformations (VCH, New York, 1989), H. 0. House, Modern Synthetic Reactions 2 "d Edition (W. A. Benjamin, Inc., Menlo Park, 1972). Those skilled in the art will also appreciate that various protecting groups may be used throughout a synthesis. Examples of protecting groups are known to those skilled in the art and have been described, for example, in Greene et al., Protective Groups in 30 Organic Synthesis; John Wiley & Sons, 2 "d Edition, 1991. Those skilled in the art will also realise that bis-cationic compounds of Formula (I) and Formula (II) may be prepared as salts and may comprise one or more of any suitable counterion. The counterion may be anionic, dianionic or polyanionic. Where more than WO 2005/047230 PCT/AU2004/001570 26 one counterion is present, the counterions may be the same or different. Examples of counterions include, but are not limited to halides (such as Cl~, Br~, I), carboxylates, citrate, acetate, succinate, CF 3 CO2~, tosylate, nitrate, BF 4 -, PF 6 -, and OHf. The counterion(s) may be varied using techniques known to those skilled in the art, for 5 example, ion exchange and crystallisation. The present invention includes within its scope all isomeric forms of the compounds disclosed herein, including all diastereomeric isomers, racemates and enantiomers. Thus, formulae (I) and (II) should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+), and/or (-) forms of the compounds, as appropriate in each case. 10 Compounds of Formula (I) and Formula (II) may be prepared according to the following general Schemes (a) - (f): LG- SPACER LG + 2 YHR 1

R

2 : R 1

R

2

Y

1 -- SPACER YR 1

R

2 12R 3 -LG 0 @

R

3

R

2

R

1 Y SPACER YR 1

R

2

R

3 Scheme (a) 6R 1 -LG (

HY

2 SPACER HY 2 A R 1

R

1

R

1 Y- SPACER -YR 1

R

1

R

1 15 Scheme (b) 2YR 1

R

2

R

3 LG- SPACER LG - R 3

R

2

R

1 Y SPACER YR 1

R

2

R

3 Scheme (c) 20 WO 2005/047230 PCT/AU2004/001570 27 LG-- SPACER LG HYR 1

R

2 LG- SPACER -YR 1

R

2

HYR

3

R

3 R4R3R3 Y- SCYR1R2R4 2 R 4 -LG R 3

R

3 Y SPACER YR 1

R

2 Scheme (d)

YR

1

R

2

R

3 0 LG SPACER LG LG- SPACER -YR 1

R

2

R

3

YR

4

R

5

R

6

R

6

R

5

R

4 Y SPACER YR 1

R

2

R

3 Scheme (e) 5

HNR

1

R

2

HO

2 C-- SPACER LG

R

2

R

1 NH-C SPACER LG

HYR

3

R

4 0 Reduction I I

R

2

R

1

NH-CH

2 SPACER YR 3

R

4

R

2

R

1 NH-C- SPACER -YR 3

R

4 2R 5 -LG

R

2

R

1

R

5

NH-CH

2 SPACER

YR

3

R

4

R

5 Scheme (f) With reference to the above Schemes (a)-(f), "Y" represents N or P. "LG" 10 represents a leaving group which can be displaced by a nucleophilic amine or phosphine. Examples of suitable leaving groups are known those skilled in the art and include, for WO 2005/047230 PCT/AU2004/001570 28 example, chloro, bromo, iodo, mesylate, tosylate and triflate groups. The various leaving groups in the above Schemes (a)-(f) may be the same or different and may be varied as appropriate to modify the reactivity. Similarly, different leaving groups and nucleophiles (e.g, HYNRR) may be employed at different stages of the synthesis. In the above 5 Schemes, the groups R 1 , R 2 , R 3 , R 4 , R 5 and R 6 represent non-polar or substantially non polar groups as defined above for Formula (I) and Formula (II), for example, alkyl groups, cycloalkyl groups, alkenyl groups, aralkyl groups such as benzyl, heteroaryl groups, heteroalkyl groups, etc. Respective R 1 , R 2 , R 3 , R 4 , R 5 and R 6 groups may be the same or different and each may be optionally substituted, e.g, with one or more groups 10 selected from C 1

-

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, hydroxyl, halides, O(C 1

.

6 alkyl),

C(O)O(C

1

.

6 alkyl), NO 2 , amino, hydroxyC1.

6 alkyl, aryl, OC(O)Ph, or =C(Ph) 2 . Scheme (a) illustrates a synthetic route for a symmetrical bis-cationic compound. However, those skilled in the art will realise that by varying the stoichiometric ratios and reaction conditions, or by varying the R 1 , R 2 , and R 3 , groups, asymmetric compounds is may also be prepared according to the above route. With reference to Scheme (f), suitable reducing agents are known to those skilled in the art and include, for example, H2, Pd/C, LiALH 4 , NaBH 4 , Zn/HC1, Sn/HCL, etc. With reference to Schemes (a) - (f) above, suitable reagents, reaction conditions etc, will be known to those skilled. By way of example, suitable solvents may include 20 relatively polar solvents, such as acetonitrile, ethanol, ethers, methylisobutyl alcohol, methylisobutyl ketone, and the like. The reaction temperature(s) may be adjusted as appropriate to control the rate of reaction. By way of example, suitable reaction temperatures may be above room temperature, eg, greater than 50'C. The bis-cationic compounds of the invention may be prepared as salts. The 25 counterions may be varied using methods known to those skilled in the art, including for example, ion exchange columns and crystallization techniques. Compounds according to the present invention may be useful for the treatment, inhibition or prevention of microbial infection, such as fungal, bacterial, viral and protozoal infection. Thus, compounds according to the present invention may have 30 therapeutic applications, including pharmaceutical and veterinary applications. The compounds according to the present invention may also have industrial applications and for example, may be useful as disinfectants. Compounds according to the present invention may also have agricultural applications and for example, may be used to treat, inhibit or prevent microbial infection of plants and crops. The compounds may be 35 formulated as composition in an appropriate manner for the intended use.

WO 2005/047230 PCT/AU2004/001570 29 Thus, the present invention also relates to the use of at least one compound of Formula (I) or Formula (II) defined herein for the manufacture of a medicament for one or more of treating, inhibiting and preventing a microbial infection. The microbial infection may be on a surface, such as the surface of an article, e.g, a 5 surgical article or device, a floor, benchtop, or industrial work surface. The microbial infection may be an infection of a vertebrate, e.g, a systemic infection, or an infection on the surface of the skin of a vertebrate. The microbial infection may comprise one or more of bacteria, fungi, viruses, and parasites. 10 The present invention also relates to a method for one or more of treating, inhibiting, and preventing a microbial infection in a vertebrate, said method comprising administering to said vertebrate an effective amount of at least one compound of Formula (I) or Formula (II) defined herein. The present invention also relates to a method of treating, inhibiting, and preventing is a microbial infection of a plant, said method comprising contacting said plant with an effective amount of at least one compound of Formula (I) or Formula (II) defined herein. A compound of Formula (1) or Formula (II) may be contacted with any part of a plant, including leaves, fruit, flowers, stem, tuber, or root. The plant may be an agriculturally or horticulturally important plant. In one embodiment, the infection is a 20 fungal infection. The present invention also relates to an antimicrobial composition comprising at least one compound of Formula (I) or a compound of Formula (II) defined herein, together with an industrially, veterinarially, agriculturally, or phannaceutically suitable carrier, diluent or carrier. 25 The composition may be one or more of an antifungal, antibacterial, antiviral and antiparasitic composition. In one embodiment the composition is an antifungal composition. In another embodiment the composition is an antibacterial composition. The present invention also relates to a pharmaceutical composition comprising at least one compound of Formula (I) or Formula (II) together with a pharmaceutically 30 effective carrier, adjuvant or diluent. The present invention also relates to a method of inhibiting phospholipase in an organism comprising contacting said organism with an effective amount of at least one compound of Formula (I) or at least one compound of Formula (II), or a composition thereof.

WO 2005/047230 PCT/AU2004/001570 30 The organism may be a microbial organism such as bacteria, fungi, virus, or a parasite, including for example protozoa. The phospholipase may be Phospholipase B. The present invention also relates to a method for identifying an antimicrobial agent comprising contacting microbial cells with a compound suspected of having antimicrobial 5 properties, determining whether the compound inhibits a microbial phospholipase enzyme, wherein inhibition of said phospholipase enzyme indicates antimicrobial activity, and thereby identifying an antimicrobial agent. The compound may be a compound of Formula (I) or Formula (II) as defined herein. The phospholipase may be Phospholipase B. 10 In one embodiment of the invention, when used for the treatment, prevention or inhibition of a fungal infection, the compound is not a compound of Formula (1) or Formula (II) wherein Y 1 and Y 2 are each N; R 7 , R 7 2, R 8 , R 8 , are each hydrogen; A is unsubstituted C8.

10 alkylene; and R 1 to R 6 are each methyl or R 1 to R 6 are each isobutyl. In another embodiment of the invention, when used for the treatment, prevention or 15 inhibition of a parasitic infection selected from malaria and babesiosis, the compound is not a compound of Formula (I) or Formula (II) wherein Y 1 and Y 2 are each N; R 7 , R 7 ,, R8, Rs, are each hydrogen; A is C 10 .18 alkylene substituted with a C 1

-

3 alkyl group; and R 1 to R6 are independently C 1

.

10 alkyl. In a further embodiment of the invention, when used for the treatment, prevention 20 or inhibition of a parasitic infection selected from malaria and babesiosis the compound is not a compound of Formula (I) or Formula (II) wherein Y 1 and Y 2 are each N; R 3 and R 6 are each C 1

.

1 0 alkyl; R 1 and R 2 together with the N to which they are attached, and R4 and

R

5 together with the N to which they are attached are each a 5-membered heterocycloalkyl ring comprising one S or 0, substituted with hydroxyC 1

.

3 alkyl or 25 haloC 1

.

3 alkyl; R 7 , R 7 ,, Rg, and Rs. are each hydrogen, and A is C 10

.

1 8 alkylene substituted with a C 1

..

3 alkyl substituent. In another embodiment of the invention, when used for the treatment, prevention or inhibition of a fungal infection, the compound is not a compound of Formula (I) or Fonnula (II) wherein Y 1 and R 1 to R 3 together are quinuclidine; Y 2 and R 4 to R 6 together 30 are quinuclidine; R 7 , R 7 ,, R 8 , R 8 , are each hydrogen; and A is unsubstituted Cs.

10 alkylene. Types and classes of microbes are known in the art. Classes of microbes are listed, for example, in Manual of Clinical Microbiology, 7 th Edition, 1999, American Society of Microbiology, the entire contents of which are incorporated herein by reference. Some general examples of microorganisms include the following, however it is to be understood 35 that the scope of the invention is by known means limited to these microorganisms: WO 2005/047230 PCT/AU2004/001570 31 Bacteria including: Gram positive cocci, such as Staphylococcus spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp.; gram positive rods, such as Coryneform,Listeria spp., Erysipelothrix spp.,and Kurthia spp., Bacillus spp., Mycobacterium spp.; Gram negative bacteria, such as Enterobacteriaceae sp., 5 Escherichia spp., Shigella spp., Salmonella spp., Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Aeromonas spp. and Plesiomonas spp., Pseudomonas spp.; Acinetobacter spp., Alcaligenes spp., Moraxella spp. and Methylobacterium spp; Actinobacillus spp., Capnocytophaga spp., Eikenella spp., Kingella spp., such as Legionella spp., Neisseria spp., Branhamella spp.; anaerobic bacteria, including 10 Clostridium spp., Peptostreptococcus spp., Propionibacterium spp., Lactobacillus spp., Actinomyces spp.; Bacteroides spp.,, Porphyromonas spp., Prevotella spp., Fusobacterium spp., and other anaerobic gram negative cocci; curved and spiral-shaped gram negative rods, including Helicobacter spp., Borrelia spp.; Mycoplasmas and obligate intracellular bacteria, such as Mycoplasma spp., Ureaplasma spp., Chlamydia 15 spp., Coxiella spp.; Viruses including Human Immunodeficiency viruses (HIV), Human T-Cell Lymphotropic Virus Types I and II, Herpes Simplex Viruses, Human cytomegalovirus, Varicella-Zoster Virus, Human Herpesviruses 6, 7 and 8 and Herpes B virus, Measles Virus, Mumps virus, Adenoviruses, Rhinoviruses, Rotaviruses, Hepatitis B and D viruses, 20 Hepatitius C and G viruses, Human papillomavirus; Fungi include Candida spp., Cryptococcus spp. and medically important yeasts, Pneumocystis carini spp., Aspergillus spp., Fusarium spp. and other Moniliaceous fungi, Rhizopus spp., Rhizomucor spp., Absidia spp. and other agents of systemic and subcutaneous Zygomycoses, Trichophyton spp., Microsporum spp., Epidermophyton spp., 25 Bipolaris spp., Exophiala spp., Scedosporium spp., Sporothrix spp. and other dematiaceous fungi, fungi causing Eumycotic Mycetoma; Collectitrichum coccodes, fungi causing powdery mildew, downy mildew, botrytis, black dot and black scurf (Rhizoctonia) in plants; Parasites including Plasmodium spp., Babesia spp., Leishmania spp., Trypanosoma 30 spp., Toxoplasma giardia spp., pathogenic and opportunistic free-living Amebae, intestinal and urogenital Amebae, Flagellates and Ciliates; Cryptosporidium, Cyclospora, Isospora, Microsporidia, and intestinal Helminths.

WO 2005/047230 PCT/AU2004/001570 32 Formulations In accordance with the present invention, when used for the treatment or prevention of microbial infection, compound(s) of the invention may be administered alone. Alternatively, the compounds may be administered as a pharmaceutical, veterinarial, 5 agricultural, or industrial formulation which comprises at least one compound according to the invention. The compound(s) may also be present as suitable salts, including pharmaceutically acceptable salts. In accordance with the present invention, the compounds of the invention may be used in combination with other known treatments or antimicrobial agents, including 10 antifungal treatments, antibiotics, disinfectants, etc. Suitable agents are listed, for example, in the Merck Index, An Encyclopoedia of Chemicals, Drugs and Biologicals, 12 th Ed.,1996, the entire contents of which are incorporated herein by reference. Combinations of active agents, including compounds of the invention, may be synergistic. 15 By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. 20 For instance, suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically 25 acceptable salts of the compounds of the present invention therefore include acid addition salts. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the 30 free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, WO 2005/047230 PCT/AU2004/001570 33 hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naplithalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, 5 toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine 10 and the like. Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. Depending on the route of administration, the formulation and/or compound may be coated with a material to protect the compound is from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The compound may also be administered parenterally or intraperitoneally. Dispersions of the compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary 20 conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable 25 under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi. In one embodiment of the invention, the compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. 30 The compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Suitably, such compositions and preparations 35 may contain at least 1% by weight of active compound. The percentage of the WO 2005/047230 PCT/AU2004/001570 34 compound(s) of formula (I) and/or (II) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 5 35% to about 45%, of the weight of the dosage unit. The amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained. The language "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically 10 active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease 15 of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound(s) may be formulated for convenient and effective administration in effective 20 amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are detennined by reference to the usual dose and manner of administration of the said ingredients. In one embodiment, the carrier may be an orally administrable carrier. 25 Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration. Also included in the scope of this invention are delayed release formulations. Compounds of the invention may also be administered in the form of a "prodrug". A prodrug is an inactive form of a compound which is transformed in vivo to the active 30 form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound. In one embodiment, the compound may be administered by injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid 35 polyetheylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The WO 2005/047230 PCT/AU2004/001570 35 proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those 5 skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, 10 for example, aluminium monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic 15 dispersion medium and the required other ingredients from those enumerated above. Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, 20 lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain 25 the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations. 30 Preferably, the phannaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof. Single or multiple administrations of the pharmaceutical compositions according to 35 the invention may be carried out. One skilled in the art would be able, by routine WO 2005/047230 PCT/AU2004/001570 36 experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable. 5 Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests. Generally, an effective dosage per 24 hours may be in the range of about 0.0001 mg 10 to about 1000 mg per kg body weight; suitably, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 15 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight. Alternatively, an effective dosage may be up to about 500mg/m 2 . For example, 20 generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , about 25 to about 350mg/m 2 , about 25 to about 300mg/m 2 , about 25 to about 250mg/m 2 , about 50 to about 250mg/m 2 , and about 75 to about 150mg/m 2 . Examples The invention will now be described in more detail, by way of illustration only, 25 with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification. Example 1 30 Synthesis and Characterisation of Bis-cationic Compounds 1,12-Bis-(quinuclidinium)dodecane dibromide To 1,12-dibromododecane (0.50 g, 1.52 mmol) in methyl isobutyl ketone (1 ml) was added quinuclidine (0.34 g, 3.0 mmol). The mixture was deoxygenated by WO 2005/047230 PCT/AU2004/001570 37 freeze/thaw and stirred at reflux for 24 h, at which time precipitate was formed. The crude mixture was cooled, the precipitate filtered off to yield the desired compound as a white solid (0.45 g, 54 %). 1 H NMR (200 MHz, CDCl 3 ): 5 3.77 - 3.73 (12H, in,), 3.57 - 3.48 (4H, in), 2.31 - 2.28 (2H, in), 2.22 - 2.17 (12H, in), 1.78 - 1.74 (4H, in), 1.37 - 1.15 5 (18H, in). 1 3 C NMR (300 MHz, CDCl 3 ): 5 55.2, 28.7, 28.6, 28.4, 24.4, 24.3, 22.7, 2 signals obscured or overlapping; m/z 195 [M-2Bf]2+ (100%), 470 [M-Br]+ (75). Found [M-2Bf]2 195.1979, [C 26

H

5 oN 2 ]2+ requires 195.1982. 1,12-Bis-(1-methylmorpholinium)dodecane dibromide 1,12-Dibromododecane (0.50 g, 1.52 mmol) was dissolved in N-methylmorpholine 10 (0.61 g, 6.08 mmol). The mixture was deoxygenated by freeze/thaw and stirred at 80'C for 24 h to yield the desired compound as a white hygroscopic solid which was collected by filtration and washed with dry diethyl ether (0.52 g, 65 %) . 1H NMR (200 MHz, MeOD): 5 4.10 - 4.09 (8H, in), 3.68 - 3.46 (12H, in), 3.36 (6H, s, 2 x CH 3 ), 2.08 - 2.05 (4H, br.s), 1.44 - 1.35 (18H, in). 13C NMR (300 MHz, MeOD): 5 70.1, 65.0, 64.2, 51.9, 15 33.6, 33.3, 30.1, 25.9, 1 signal obscured or overlapping; n/z 185 [M-2Bri]2 (100%). 1,11-Bis-(tributylammonium)undecane dichloride 1,1 1-Dibromoundecane (0.10 g, 0.32 mmol) was dissolved in tributylamine (0.24 g, 1.27 mmol). The resulting mixture was deoxygenated by freeze/thaw and was stirred at 130*C for 20 h. The solvent was removed under reduced pressure and the crude mixture 20 was purified by flash chromatography (90:10:1 CH 2 Cl 2 / MeOH/ NH 3 (aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (CI-). The resulting fractions were combined and the solvent removed under reduced pressure to give the above compound as a light brown oil (0.13 g, 69 %). 1H NMR (200 MHz, CDCl 3 ): ( 3.52 - 3.02 (12H, in), 2.92 - 2.89 (4H, in), 1.68 - 1.58 (16H, in), 1.40 - 1.26 25 (26H, in), 0.95 - 0.86 (18H, in, 6 x CH 3 ). 1 3 C (300 MHz, CDCl 3 ): 60.0, 54.6, 30.7, 27.8, 27.3, 21.3, 14.4, 3 signals obscured or overlapping. 1,12-Bis-(tributylammonium)dodecane dichloride 1,12-Dibromododecane (1.0 g, 3.05 mmol) was dissolved in methyl isobutyl ketone (5 ml). Tributylamine (1.13 g, 6.09 mmol) was added and the resulting mixture was 30 deoxygenated by freeze/thaw. The mixture was stirred at reflux for 24 h and the solvent was removed under reduced pressure. The crude mixture was purified by flash chromatography (90:10:1 CH 2 C1 2 / MeOH/ NH 3 (aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (CI~). The resulting fractions were WO 2005/047230 PCT/AU2004/001570 38 combined and the solvent removed under reduced pressure to give the above compound as a yellow oil (0.5 g, 27 %). 'H NMR (200 MHz, CDCl 3 ): 8 3.37 - 3.29 (12H, in), 3.02 2.94 (4H, in), 1.78 - 1.65 (16H, in), 1.32 - 1.23 (28H, in), 0.89 - 0.82 (18H, m, 6 x CH 3 ). 13 C (300 MHz, CDCl 3 ): 59.6, 31.6, 26.5, 22.8, 22.7, 14.3, 4 signals obscured or 5 overlapping; m/z ESI (positive ion) Found [M-Cl-] 573.5865, [C 36

H

78

N

2 Cl]* requires 573.5852. 1,12-Bis-(tributylammonium)dodecane dibromide To 1,12-diaminododecane (0.25 g, 1.25 mmol) in absolute ethanol (5 ml) was added butyl bromide (3.08 g, 22.5 mmol) and K 2 C0 3 (1.04 g, 7.49 mmol). The mixture was 10 stirred at reflux for 3 days to yield the desired compound as a yellow oil (0.76 g, 87 %) which had identical 1 H and 13 C spectroscopical data to those described above; n/z ESI (positive ion) 270 [M-2B]2+ (100 %), 619 [M-Br~]+ (70). 1,16-Bis-(tributylammonium)dodecane dichloride 1,16-Dibromohexadecane (0.15 g, 0.39 mmol) was dissolved in tributylamine (0.14 15 g, 0.78 mmol). The resulting mixture was deoxygenated by freeze/thaw and was stirred at 130*C for 3 days. The crude mixture was purified by flash chromatography (80:18:2

CH

2 Cl 2 / MeOH/ NH 3 (aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (CI~). The resulting fractions were combined and the solvent removed under reduced pressure to give the above compound as a brown oil (0.13 g, 48 20 %). 1 H NMR (200 MHz, CDC1 3 ): 5 3.37 - 3.29 (4H, in), 3.02 - 2.94 (12H, in), 1.78 1.65 (16H, in), 1.32 - 1.23 (26H, in), 0.89 - 0.82 (18H, m, 6 x CH 3 ). 1 3 C (300 MHz, CDCl 3 ): 59.6, 31.6, 26.5, 22.8, 22.7, 14.3, 6 signals obscured or overlapping; m/z ESI (positive ion) 297 [M-2Cl~] 2 + (27 %). Found [M-2Cl~] 2 + 297.3397, [C 4 oH 86

N

2

]

2 + requires 297.3396. 25 1,12-Bis-(tripentylammonium)dodecane dibromide To 1,12-dibromododecane (0.50 g, 1.52 mmol) was added triisobutylamine (1.38 g, 6.08 mmol) was added and the resulting mixture was deoxygenated by freeze/thaw. The mixture was stirred at reflux for 3 days. The crude product was washed with ether to yield the above compound as a brown oil (0.95 g, 80 %). 1 H NMR (200 MHz, CDCl 3 ): 6 3.29 30 3.27 (12H, in), 2.96 - 2.83 (4H, in), 1.67 - 1.61 (16H, in), 1.30 - 1.19 (40H, in), 0.84 0.79 (18H, m, 6 x CH 3 ). 13 C (300 MHz, CDCl 3 ): 6 59.5, 52.9, 29.3, 29.2, 26.7, 26.1, 22.5, 14.1, 3 signals obscured or overlapping; m/z ESI (positive ion) 311 [M-2Bf] 2 + (100 %), 703 [M-Br~] (76). Found [M-2Br~] 2 + 311.3541, [C 42

H

9 oN 2

]

2 + requires 311.3547.

WO 2005/047230 PCT/AU2004/001570 39 1,12-Bis-(trihexylammonium)dodecane dichloride 1,12-Dibromododecane (0.50 g, 1.52 mmol) was dissolved in methyl isobutyl ketone (5 ml). Trihexylamine (0.82 g, 3.05 mmol) was added and the resulting mixture was deoxygenated by freeze/thaw. The mixture was stirred at reflux for 4 days and the 5 solvent was removed under reduced pressure. The crude mixture was purified by flash chromatography (90:10:1 CH 2 C1 2 / MeOH/ NH 3 (aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (CI-). The resulting fractions were combined and the solvent removed under reduced pressure to give the above compound as a yellow oil (0.14 g, 12 %). 'H NMR (200 MHz, CDCl 3 ): 8 3.37 - 3.29 (12H, m), 3.02 10 - 2.94 (4H, m), 1.78 - 1.65 (16H, m), 1.32 - 1.23 (52H, m,), 0.89 - 0.82 (18H, m, 6 x

CH

3 ). 13 C (300 MHz, CDCl 3 ): 8 59.6, 31.6, 26.5, 22.8, 22.7, 14.3, 6 signals obscured or overlapping; m/z ESI (positive ion) 354 [M-2Cl-]2+ (100 %). 1,12-Bis-(trioctylammonium)dodecane dichloride 1,12 Dibromododecane (0.52 g, 1.52 mmol) was dissolved in methyl isobutyl 15 ketone (5 ml). Trioctylamine (1.12 g, 3.17 mmol) was added and the resulting mixture was deoxygenated by freeze/thaw. The mixture was stirred at reflux for 7 days and the solvent was removed under reduced pressure. The crude mixture was purified by flash chromatography (90:30:1 CH 2 Cl 2 / MeOH/ NH3(aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (Cl-). The resulting fractions were 20 combined and the solvent removed under reduced pressure to give the above compound as a yellow oil (0.11 g, 7 %). 1H NMR (200 MHz, CDCl 3 ): 8 3.37 - 3.29 (12H, m), 3.02 2.94 (4H, m), 1.78 - 1.65 (16H, m), 1.32 - 1.23 (76H, m), 0.89 - 0.82 (18H, m, 6 x CH 3 ). 1 3 C (300 MHz, CDCl 3 ): 59.6, 31.6, 26.5, 22.8, 22.7, 14.3, 8 signals obscured or overlapping; m/z ESI (positive ion) 909 [M-Cl~]+ (100 %). 25 1,12-Bis-(triisobutylammonium)dodecane dibromide To 1,12-Dibromododecane (0.50 g, 1.52 mmol) was added triisobutylamine (1.2 g, 6.10 mmol) and the resulting mixture was deoxygenated by freeze/thaw. The mixture was stirred at reflux for 48 h. The mixture was cooled and the above compound was obtained as a white precipitate (0.014 g, 2 %). 'H NMR (200 MHz, CDCl 3 ): 5 3.40 (4H, dd), 1.89 30 1.78 (12H, m), 1.74 - 1.63 (6H, m), 1.45 - 1.28 (20H, m), 0.95 - 0.85 (36H, m, 12 x

CH

3 ); n/z ESI (positive ion) 618 [M-Bf]* (27).

WO 2005/047230 PCT/AU2004/001570 40 1,12-Bis-(triisopentylammonium)dodecane dichloride 1,12-Dibromododecane (0.56 g, 1.71 mmol) was dissolved in methyl isobutyl ketone (5 ml). Triisopentylamine (0.78 g, 3.42 mmol) was added and the resulting mixture was deoxygenated by freeze/thaw. The mixture was stirred at reflux for 48 h and 5 the solvent was removed under reduced pressure. The crude mixture was purified by flash chromatography (90:10:1 CH 2 Cl 2 / MeOH/ NH 3 (aq)). The combined fractions were then passed a down a column of Lewatit MP-64 anion resin (CI~). The resulting fractions were combined and the solvent removed under reduced pressure to give the above compound as a pale yellow oil (0.37 g, 31 %). 1 H NMR (200 MHz, CDCl 3 ): 8 3.20 - 3.12 (12H, in), 10 2.87 - 2.82 (4H, in), 1.58 - 1.35 (10H, in), 1.26 - 1.04 (28H, in), 0.79 - 0.71 (36H, m, 12 x CH 3 ). 1 3 C (300 MHz, CDCl 3 ): 59.0, 57.7, 52.8, 51.1, 31.7, 30.6, 29.1, 26.6, 22.5, 1 signal obscured or overlapping; n/z ESI (positive ion, before ion exchange) 703 [M-Bf]* (45). 1,12-Bis-(tripropylammonium)dodecane dibromide 15 1,12-Dibromododecane (0.50 g, 1.52 mmol) was dissolved in tripropylamine (1.72 g, 12.1 mmol). The resulting mixture was deoxygenated by freeze/thaw and stirred at 80'C for 48 h. The solvent was removed under reduced pressure and the resulting oil was triturated with ether to give the above compound as dark brown oil (0.70 g, 75 %) 'H NMR (200 MHz, CDC1 3 ): 8 3.55 - 3.29 (12H, in), 3.00 - 2.96 (4H, in), 1.41 (16H, br. s.), 20 1.45 - 1.12 (16H, in), 0.99 - 0.90 (18H, in). 13 C NMR (200 MHz, CDCl 3 ): 8 61.1, 54.6, 29.4, 23.7, 16.3, 11.3; n/z ESI (positive ion) 227 [M-2Bf] 2 + (100). 1,12-Bis-(triethylammonium)dodecane dibromide 1,12-Dibromododecane (0.50 g, 1.52 mmol) was dissolved in triethylamine (0.92 g, 9.12 mmol). The resulting mixture was deoxygenated by freeze/thaw and stirred at 80'C 25 for 24 h. The solvent was removed under reduced pressure and the resulting oil was triturated with ether to give the above compound as a hygroscopic white solid (0.65 g, 81 %). 1,12-Bis-(trimethylammonium)dodecane dibromide Sodium hydroxide (0.25 g, 6.1 mmol) was added to a solution of trimethylamine 30 hydrochloride in dry methanol (10 ml). The mixture was filtered and 1,12 dibromododecane (0.50 g, 1.52 mmol) was added to the filtrate. The mixture was refluxed for 12 h and the solvent removed under reduced pressure. The crude product was WO 2005/047230 PCT/AU2004/001570 41 recrystallised from methanol/ ether to yield the above compound as a white solid (0.64 g, 94 %). Data same as J Med. Chem., 1997, 40, 3557-3566 1,12-Bis-(1-methylpyrrolidinium)dodecane dibromide 1,12-Dibromododecane (0.50 g, 1.52 mmol) was dissolved in 1-methylpyrrolidine 5 (0.52 g, 6.08 mmol). The resulting mixture was deoxygenated by freeze/thaw and was stirred at 80'C for 20 h. The solvent was removed under reduced pressure to yield the above compound as a white hygroscopic solid (0.49 g, 64 %). 1H NMR (200 MHz, CDCl 3 ): E 3.83 - 3.63 (12H, in), 3.28 (6H, s, 2 x CH 3 ), 2.32 - 2.21 (8H, in), 1.85 - 1.82 (4H, in), 1.27 - 1.26 (16H, in). 10 1,12-Bis-(pyrrolidinium)dodecane 1,12 Dibromododecane (0.5 g, 1.52 mmol) was dissolved in pyrrolidine (0.44 g, 6.08 mmol). The mixture was stirred at 90'C for 20 h. The crude mixture was purified by flash chromatography, eluting with 80:18:2 CH 2 C1 2 / MeOH/ NH3(aq) to yield the above compound as a yellow solid (0.33 g, 71 %). 'H NMR (200 MHz, CDCl 3 ): 5 2.51 - 2.36 15 (12H, in), 1.80 - 1.74 (8H, in), 1.53 - 1.47 (4H, in), 1.27 - 1.26 (16H, in); m/z ESI (positive ion) 310 [M+H]* (100). 1,12-Bis-(N-butylpyrrolidinium)dodecane dibromide To 1,12-bis-(pyrrolidinium)dodecane (0.33 g, 1.08 mmol) in methyl isobutyl ketone (2 ml) was added butyl bromide (0.88 g, 6.46 mmol). The resulting mixture was stirred at 20 reflux for 20 h. The crude mixture was purified by flash chromatography, eluting with 80:18:2 CH 2 Cl 2 / MeOH/ NH3(aq) to yield the above compound as a pale yellow oil (0.45 g, 71 %). 1H NMR (200 MHz, CDCl 3 ): 6 3.42 - 3.31 (16H, in), 1.80 - 1.74 (4H, in), 1.45 - 1.20 (32H, in,), 0.86 (6H, t, 2 x CH 3 ); in/z ESI (positive ion) 211 [M-2Br-] 2 + (100). Example 2 - Characterisation of Enzyme Activity 25 Cryptococcus neofornians is the most common cause of fungal meningitis, which is fatal if untreated. Pathogenic strains of cryptococci produce a number of so-called "virulence factors", one of which is a secreted phospholipase, termed phospholipase B (EC 3.1.1.5). This phospholipase is a single protein containing three separate activities, including (i) phospholipase B activity (PLB), which removes both acyl chains 30 simultaneously from phospholipids, (ii) lysophopholipase (LPL), which removes the single acyl chain from lysophospholipids, and (iii) lysophospholipase transacylase (LPTA), which adds an acyl chain to lysophospholipids to form phospholipids (see Figs. la, 1b).

WO 2005/047230 PCT/AU2004/001570 42 Secreted phospholipase B is involved in the survival of cryptococci in macrophages, destruction of lung tissue and production of eicosanoids, which modulate phagocytic activity. This Phospholipase B is also related to virulence in other medically important fungi such as Candida albicans and Aspergillus funigatus. Consequently, secreted 5 Phospholipase B enzyme may be a potentially useful target for treatment of microbial infection, including, for example, antibacterial, antifungal, antiviral and antiparasitic treatments. The cell-associated (membrane and cytosolic) phospholipase B activities in C. neoformans, were characterised. 10 Materials and Methods Fungal isolates and media. A virulent clinical isolate of C. neoformans var. grubii (serotype A), H99, which produces high levels of secreted phospholipase B activity was used for cell-associated phospholipase characterisation and inhibition of phospholipase activities. Isolate H99 was kindly supplied by Dr. Gary Cox (Duke University Medical 15 Center, Durham, NC, USA), and subcultured onto Sabouraud dextrose agar (SDA) at 30 0 C. Preparation of supernatants containing secreted phospholipase activities. Isolate H99 was grown to confluence on SDA in 16 cm diameter Petri dishes for 72 h at 30*C in air. Cells scraped from 10-20 dishes were washed sequentially with isotonic 20 saline and imidazole buffer (10 mM imidazole, 2 mM CaCl 2 , 2 mM Mg C1 2 , 56 mM D Glucose, made up in isotonic saline, pH 5.5), resuspended in a volume of this buffer of about 10% of the cell volume, and incubated for 24 h at 37*C. The cell-free supernatant was separated by centrifugation as previously described and stored at -70'C. Cellular disruption to prepare membrane and cytosolic fractions. The cell 25 pellet from the preparation of the supernatant, above, was also frozen at -70'C. After washing twice with imidazole buffer, it was disrupted in the presence of a protease inhibitor cocktail (P 8215 for fungal and yeast cells; 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 100mM, 1,10-phenanthroline, 500mM, pepstatin A, 2.2mM, E-64, 1.4 mM; Sigma) in a MiniBeadbeater-8 Cell Disrupter (MBB-8; Daintree Scientific, 30 Tasmania, Australia) for three cycles of 1 min, alternating with a 1 min cooling period on ice. The homogenate was centrifuged at 14,000g for 15 min to obtain the membrane (pellet) and the cytosolic (supernatant) fractions. The cytosolic enzyme activities were stable during storage at -70*C for up to 3 months, whereas the membrane associated activities were less stable (maximum 5 weeks).

WO 2005/047230 PCT/AU2004/001570 43 Radiometric assay method for phospholipases. Enzyme activities were measured as described previously in a final volume of 125 pL at 37*C. For the determination of secreted PLB activity, carrier dipalmitoyl phosphatidylcholine (DPPC, final concentration 800 ptM) and 1,2-di[l- 1 4 C] palmitoyl phosphatidylcholine (20,000 dpm) were dried under 5 nitrogen and suspended in 125 mM imidazole acetate buffer (assay buffer, pH 4.0) by sonication using a Branson 450 sonifier. The reaction time was 22 min, using 1 pg total protein and PLB activity was determined by the rate of decrease of the radiolabelled PC substrate, with appearance of the label in free fatty acid. Variations to these conditions for the cytosolic and membrane fraction assays are shown in Table 1. This assay also 10 simultaneously allows for the determination of phospholipase A, C and D activities. These activities were measured by the appearance of radiolabel from PC in Lyso PC, diacylglycerol and phosphatidic acid, respectively. Secreted LPL and LPTA activities were measured simultaneously in a reaction mixture containing l-[ 1 4 C]palmitoyl lyso-PC (25,000 dpm) and carrier lyso-PC (final 15 concentration 200 ptM) in assay buffer. The reaction time was 15 s with 1 tg of total protein and LPL activity was measured by the rate of loss of l-[14C]palmitoyl lyso-PC with release of radiolabelled fatty acids. LPTA activity was estimated from the rate of formation of radiolabelled PC. Variations to these conditions for membrane and cytosolic fractions are presented in Table 1. 20 All reactions were terminated by adding 0.5 mL of chloroform: methanol (2:1 v/v). The reaction products were extracted by the method as disclosed in Bligh, E.C. and W.J. Dyer. 1959 'A rapid method of total lipid extraction and purification.' Can. J. Biochem. Physiol. 37:911-917. , separated by TLC and quantified as previously described. In the case of PLC activity, the TLC plates were developed in petroleum ether (BP 60-80'C): 25 diethyl ether: acetic acid (90:15:1, v/v/v) instead of chloroform: methanol: water (65:25:4, v/v/v). Table 1: Optimal conditions for H99 cell-associated and secreted phospholipases Cytosolic Membrane Secreted Activity LPL/LPTA PLB LPL/LPTA PLB LPL/LPTA PLB Protein (pg) 1 4 80 120 1 1 Time 20s 18 min 30s 18 min 15s 22 min Substrate pm) 200 1000 600 800 200 400 PH 4.0 4.0 4.0 4.0 4.0 4.0 WO 2005/047230 PCT/AU2004/001570 44 Characterisation of enzyme activities All experiments were carried out in duplicate or triplicate. The effects of pH on the various phospholipase activities were measured using 50 mM final concentration of imidazole-acetate buffer (pH range 3-5), MES buffer (pH range 6-8) and glycine buffer 5 (pH 9-10). Controls for non-enzymic breakdown of substrates were included at all pH values. Cations, Triton X-100 and metal chelators were made up as stock solutions in water and diluted to the final concentration in the appropriate assay buffer. Protein assays. Total protein estimations were performed using a Coomassie Blue binding assay 10 (for supernatant containing secreted enzymes) or the bicinchoninic acid (BCA kit) for cell-associated fractions, with BSA as standard (Pierce Chemical Co., IL, USA). Identification of PLB, LPL and LPTA in cell-associated fractions. Assays of cell-associated phospholipase activities were performed at pH 4.0, using the substrates palmitoyl lyso-phosphatidylcholine (Lyso-PC) and DPPC, since these 15 compounds were preferred substrates of the secreted enzyme in both its natural and purified states and enzyme activity was maximal at pH 4.0. Hydrolysis of the substrate lyso-PC by the cryptococcal membrane and cytosolic fractions, resulted in the formation of free fatty acids and PC only, indicating the presence of both LPL and LPTA, as found for the secreted enzyme. Similarly, DPPC, radiolabelled in both acyl chains, was 20 degraded to produce free fatty acids, only, indicating that the activity was due to PLB at pH 4.0. Effect of protein concentration and time on phospholipase activity. Activity of the cytosolic fraction with increasing protein concentration was linear only to 1 tg for LPL/LPTA and 4 pg for PLB, similar to values for the secreted enzyme 25 (not shown). In contrast, membrane-associated LPL and LPTA activities were linear with increasing protein concentration to 80 ptg, after which no further increase occurred (Fig. 3A). The PLB activity was linear to 160 jig protein (Fig. 3B). The time course of both the cytosolic and membrane activities resembled that of the secreted enzyme, with linearity of LPL/LPTA only to 20-30 sec, beyond which no further 30 increase occurred. Membrane-associated PLB activity was linear to 30 min, whereas cytosolic activity was linear to 22 min (not shown). Effect of substrate concentration on enzyme activity. Cytosolic LPL/LPTA activities reached a maximum between 50-200 pM Lyso-PC, after which there was a rapid decline in activity with increasing substrate concentration 35 (Fig. 4A). Cytosolic PLB activity reached saturation at 400 pM DPPC, and declined after WO 2005/047230 PCT/AU2004/001570 45 1000 tM (Fig. 4B). Membrane-associated LPL/LPTA reached a maximum around 50 pM Lyso-PC, but maintained the same level of activity until 600 tM, after which it decreased (not shown). The membrane-associated PLB activity reached a maximum at 200 piM DPPC, and declined after 800 pM (not shown). 5 Effects of pH on enzyme activity. Optimal conditions for further studies were selected from Table 1. Variations in cell-associated phospholipase activity were observed over the pH range 3-10. Cytosolic LPL/LPTA activities exhibited two pH optima, at 4.0 and 6.0, with activity decreasing to zero at pH 9.0 (not shown). The cytosolic phospholipase B pH profile (not shown) was 10 similar to that of membrane-associated PLB (Fig. 5B), though less sharp. The pH optimum was 3-4 for both membrane-associated LPL and LPTA, and decreased gradually to zero at pH 9.0 (Fig 5A). The optimum pH for membrane PLB (pH 4.0) was very narrow (Fig. 5B), with virtually no activity at pH 7.0. The most obvious difference between the cell associated (cytosolic and membrane 15 associated) activities and those of both the crude and purified secreted enzymes was the greater activity of the cell-associated enzymes at pH 6.0 (PLB) and 7-8 (LPL/LPTA). Interestingly, when the incubation time for the LPL/LPTA assay was extended from 20 30 sec to 10 min, the activity was increased over the range pH 7-9, but not at lower pH values, in both membrane and cytosolic enzyme preparations (Fig. 5 C, membrane 20 associated enzyme). Cellular distribution of phospholipase B activities. Taking 4.0 as the optimal pH for all three activities (Table 1), it is clear that the distribution of PLB differs from that of LPL and LPTA, in that the greatest percentage of the total activity is secreted (Table 2). With LPL and LPTA most of the activity is 25 cytosolic. The specific activities and percentages of all three activities were lowest in the membrane fraction (Table 2).

WO 2005/047230 PCT/AU2004/001570 46 ~1~00 Sc* N ~ ., 0 *Z ; trrn

-P

00 ~ 4i. *1Et WO 2005/047230 PCT/AU2004/001570 47 Modifying agents and cell-associated activities. There was no stimulation of any of the cytosolic or membrane-associated activities by 10 mM calcium or magnesium at pH 4.0 (Table 3A). Triton X-100 was the only notable inhibitor at pH 4.0, with cytosolic and membrane-associated LPTA and PLB most 5 affected (Table 3A). Table 3. Effects of modifying agents on cytosolic and membrane-associated activities. (A) Assayed at pH 4.0" Modifier Cytosolic Membrane-associated 10 LPL LPTA"PLB LPLb LPTAb PLBb Calcium 100 100 100 100 96 83 Magnesium 73 70 100 97 91 79 EDTA 100 100 100 98 94 87 15 EGTA 86 88 100 100 90 77 FeC1 3 81 78 79 97 75 92 Triton X-100 62 39 54* 71 52 19 (B) Assayed atpH 7. 0' 20 LPLb LPTbPLB LPL LPTAPLB Calcium 236' 206 591* 166* 140* 533* Magnesium 96 85 477* 145* 123* 467* EDTA 30 32 100 96 100 67 25 EGTA 26 29 163 100 100 200 FeC 3 22 21 61 61* 45* 2* Triton X-00 0.9 0 59 4* 2* 33* "Activities are expressed as percentages of the control, taken as 100%. Values are the means of triplicate 30 or duplicate assays. Final concentrations of modifying agents were 10mM, except for Triton X-100, which was 0.1%(w/v). *Significantly different from the controls, P <0.01, by the Dunnett Multiple Comparisons Test. When assayed at pH 7.0, all three of both cytosolic and membrane-associated 35 activities were stimulated by calcium (Table 3B). Cytosolic PLB, and all three of the membrane-associated activities were also stimulated by magnesium (Table 3B). This was reflected in lower activity in the presence of EDTA and EGTA for cytosolic LPL and WO 2005/047230 PCT/AU2004/001570 48 LPTA. Both FeCl 3 and Triton X-100 were inhibitors of all three activities, from both cytosolic and membrane-bound fractions. Triton X-100 was less effective in inhibiting the PLB, than the LPL and LPTA activities (Table 3B). 5 Phospholipases A, C, and D. Secreted forms of Phospholipases A, C, and D have not been identified in cryptococcal supernatants. High levels of phospholipase A (PLA) were detected by the formation of radiolabelled Lyso-PC from DPPC at pH 7-8 in membrane preparations (Fig. 6A), but only trace amounts of PLA were found in the cytosolic fraction (not shown). 10 Small amounts of phospholipase D (PLD) activity were detected by the formation of radiolabelled phosphatidic acid from both cytosolic and membrane fractions at pH 7-8 (Fig. 6B, membrane PLD). No evidence of PLC was found at any pH value using DPPC as substrate. Under the assay conditions tested, phospholipase B was the major phospholipase is present in the cytosol and secreted from C. neoformans. In contrast, phospholipases A and D were membrane-associated, with some PLD activity in the cytosol. The pH optimum for PLB activity, whether secreted, membrane-bound or cytosolic, was always acidic (pH 4.0), whereas the cytosolic LPL/LPTA was bimodal (pH 4.0 and 6.0). PLA and PLD activities were detected only at pH 7-8. These observations are consistent with the role of 20 secreted PLB activities in cryptococcal virulence since the putative sites of action of Phospholipase B are the acidic vacuoles of macrophage-like cell lines, and mouse macrophages in vivo. The membrane-associated LPL/LPTA activities were stimulated by both calcium and magnesium (Table 3). The unexpected stimulation of cell-associated PLB activity by 25 calcium and magnesium at pH 7.0 might be due in part to PLA activity. Example 3 Selection and Testing of Potential Phospholipase Inhibitors MATERIALS AND METHODS 30 Selection of potential phospholipase inhibitors. Selection of potential inhibitors was based on the traditional approach of testing compounds that are structurally related to the substrate, i.e. phospholipids. Commercially available compounds were initially used containing the two dominant features in phospholipids (one or two hydrophobic alkyl WO 2005/047230 PCT/AU2004/001570 49 chains and a tetra-alkylated strongly positively charged nitrogen atom), which would be metabolically stable and sufficiently water-soluble to avoid use of solvents in the assays. Preparation of inhibitors and use in assays. The following compounds were tested (for structures, see Fig. 2): compound 1, 1,12-bis(tributylphosphonium)dodecane 5 dibromide (Fluka AG, Buchs, Switzerland); compound 2, 1,10-bis(tributylammonium) decane dibromide; compound 3, 1,12-bis(tributylammonium)do-decane dibromide (both synthesised in-house); compound 4, 1,10-bis(trimethylammonium)decane dibromide ["decamethonium"] ( Sigma, St.Louis MO, USA). All compounds were prepared as stock solutions of 700 yM in assay buffer containing 5 mM EDTA, which was then diluted 10 serially with buffer to obtain solutions of 70, 7, 0.7 and 0.07 yM. In each assay, 45 tL of these solutions was used, and the final volume of 125 yL was made up of substrate, enzyme and buffer. The radiometric assay was carried out as above. Inhibition was calculated as the percent of substrates (DPPC or Lyso-PC) remaining in the case of PLB and LPL activities, or of DPPC produced, in the case of the LPTA activity. The amounts 15 converted, or produced, in the inhibitor-free control were normalised to 100%, and the inhibition calculated against it. All assays were done in triplicate. Pancreatic phospholipase assay. Porcine pancreatic phospholipase A 2 suspension in 3.2M ammonium sulfate (2.9 mg protein/mL, Sigma St.Louis MO, USA) was used. One part of well mixed enzyme suspension was added to 4 parts of buffer (10 mM 20 Tris/HCl, pH 8.2; 10 mM CaCl 2 . Activity and inhibition by test compounds was then measured by the radiometric method described in Example 2 for fungal PLB activity. However, 25 AL enzyme solution was used, and the reaction time was 1 hour. These conditions result in ~ 60% substrate conversion in the inhibitor-free control. Antifungal susceptibility testing. The antifungal activity of the compounds was 25 measured by a standard microdilution method. The minimal inhibitory concentration of each compound (MIC) was defined as that which produced no visible growth after 48 h of culture (Candida) and 72 h (Cryptococcus) at 35'C. The fungal strains tested included Cryptococcus neoformans H99, Cryptococcus neoformans ATCC 90112 and Candida albicans ATCC 10231. All tests were performed in duplicate. 30 Ammonium- and phosphonium compounds were examined. The structures and common names of four compounds studied are shown in Figure 2. These compounds each have a strong positive charge and fatty acid-like hydrophobicity. Assays were performed at pH 4 in the absence of added cations, using the optimised conditions summarised in Table 1. Under these conditions only PLB, LPL and LPTA 35 activities (both secreted and cell-associated) were measured. Initially, compounds deemed WO 2005/047230 PCT/AU2004/001570 50 to be potential inhibitors were assayed at 25 and 250 AM. Those showing some inhibition were then also assayed at 2.5 and 0.25 tM. This bis-tributylphosphonium compound (1) inhibits PLB activity more so than LPL or LPTA. Importantly it inhibits the cytosolic as well as the secretory enzyme, while 5 it has no effect on the membrane-bound enzyme (Table 4). Interestingly, it inhibits the porcine pancreatic PLA2 more strongly than the fungal secretory Phospholipase B (Table 5). This is in contrast to the two bis-tributylammonium compounds (2 and 3) which inhibit the fungal enzyme more strongly, and thus form a platform to achieve even higher selectivity. The fungicidal activity of these three tributyl bis-cationic compounds is quite 10 strong, the best having an MIC of 2.5 ptMolar (Table 6). There is a sharp drop in MIC as the chain length is increased by two CH 2 groups, and, importantly this increase in chain length also increases the inhibition potency (Table 5). Compound (4), which has only methyl-alkylation at the quaternary nitrogen, shows neither enzyme inhibition nor antifungal activity (Tables 4 and 5).

WO 2005/047230 PCT/AU2004/001570 51 * * C1 -k .' *I '1 't 0 kn CAI 00 00 0 0 In C14 CD 0 0 - WZ cq0 2 0i ti 0 0 NZ! 2c u tzl ?E N ;tz)! 0 0 WO 2005/047230 PCT/AU2004/001570 52 0 -p-d oc C4 00 m Cu~ ot oh Cn 00 cq u WO 2005/047230 PCT/AU2004/001570 53 Example 4 - Antifungal Activity Compounds 1-4 referred to in Example 3 were assayed for antifungal activity in a standardised serial dilution sensitivity test against two strains of C. neoformans and one strain of Candida albicans (Table 6). The two stronger phospholipase inhibitors (1 and 3) 5 were quite potent, with MIC in the 2.5 to 10 AM range, whereas the non-inhibitory decamethonium compound (4) had a much higher MIC (88pM - 350 yM) (Table 6). Example 5 - Antifungal Activity A range of cationic compounds were assayed for antifungal activity in a 10 standardised serial dilution sensitivity test against C. neoformans (ATCC 90122) and C. Albicans (ATCC 10231). Results are presented in Table 7. A good correlation was observed between inhibition and antifungal activity.

WO 2005/047230 PCT/AU2004/001570 54 C-C) 00 00 000 C-n 4, 6% C) N 1 00 WO 2005/047230 PCT/AU2004/001570 55 C)0 0 6 0 nC 11~l l 111 1 1 1 r.00 cli U,.- I II I II I if if =L =L =L =L in Al- '0 M c'n -' U Cl C CMin C14 Al~ CD C C 0)4 -nk W 0z :2 (D &1 ci) 4 ~ ~* - WO 2005/047230 PCT/AU2004/001570 56 =L =L L = = 'n kn Ci il 1111 *~C -qnll ,111 111111~ In i~I~I =L kn tn1 nI I inii Ii kn 11 kn 00n0 tnq kn 0 k i C: 0 00 W) In :: - 00 kn 00 00M 00 w Al Al CO L cl L 0 co m L ®ZCN 00 00 00 C) kn 00 G Q U No (@ N ( :3m m o 0 00 00 0j3 o ~0 C0o -~~o0 ,v0~ Fl s =~00 CO d WO 2005/047230 PCT/AU2004/001570 57 C) lii o 1,kn 0 f C C d ' kn Cli q ", Cl 01 1111 11i If kn ~ l C li V C11 Nl Cl 0.1 Cl 00o ]I 0 Cl =L =fl CD kn X c )V)k 1 C-4 c, cq DCC1 80C 00 ' oC N> Nf ] D i I I knClC kn 00)r 004 Cli o.,r<i 000 l WO 2005/047230 PCT/AU2004/001570 58 ~C II= IIi I C) tn tn= Ln- kn cq 0 kn in i -I Cl 0 Cl, 41 CA m~ - n , V) ~ - 0 kn 00 W -lm f; r 0 f ,n , m 0~00 ~ S~CoU00 WO 2005/047230 PCT/AU2004/001570 59 kn cl Cl C CDC CD CDlC in l c on 00 m 0 0 0 mm00 r Al Al Al ZO~-Z M C- C,~E~ 0 0 0 / z -0 F Gz 00 0~ 0~ ~ u Cd WO 2005/047230 PCT/AU2004/001570 60 Example 6 Haemolytic activity assay Human blood was collected in 10 mL Vacutainer tubes containing potassium EDTA as anticoagulant. The blood from each vacutainer was transferred to a 50 mL 5 centrifuge tube and the cells washed three times with calcium- and magnesium-free phosphate buffered saline (PBS) (Gibco) by centrifugation at 2000 x g for 15 min. The third supernatant was clear and colourless. Cells were stored in PBS for up to two weeks. The cell suspension in PBS (0.5 mL) was mixed with a stock solution of test substance (0.5 mL) at the appropriate concentration (final erythrocyte concentration of approx. 0.5 x 10 10 9 per mL). The mixtures were incubated at 37 "C for 1 h with gentle shaking, then centrifuged at 2000 x g for 10 min. The supernatant was diluted 10-fold with PBS and the optical density measured at 540 nm. The values for 0% and 100% lysis were determined by incubating cells with PBS or 0.1% Triton X-100 (in water), respectively. Assays were carried out in triplicate. Results are shown in Table 7. Haemolytic activity of potent 15 antifungal compounds was found to be negligible at concentrations fifty or more times above MIC. Example 7 - In vitro Antibacterial Activity Two bis-cationic compounds were tested for antibacterial activity. The assay used was that published by the National Committee for Clinical Laboratory Standards. 2003. 20 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically: Approved Standard - Sixth Edition. NCCLS document M7-A6.Villanova, Pa., USA. The results are shown in Table 8. Example 8 - In vitro Antifungal Activity Two bis-cationic compounds were tested for antifungal activity. 25 The assay used for filamentous fungi (Aspergillus; Scedosporium; Fusarium) was that published by the National Committee for Clinical Laboratory Standards. 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard. NCCLS document M38-A.Villanova, Pa., USA. The assay used for yeasts (Candida; Cryptococcus) is that of the National 30 Committee for Clinical Laboratory Standards. 1997. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. NCCLS document M27 A.Villanova, Pa., USA as published in Ghannoum, M. A., A. S. Ibrahim, Y. Fu, M. C. Shafiq, J. E Edwards, Jr. and R. S. Criddle. 1992. Susceptibility testing of Cryptococcus neoformans: A microdilution technique. J. Clin. Microbiol. 30:2881-2886. The results are 35 shown in Table 9.

WO 2005/047230 PCT/AU2004/001570 6 1 CIS ~~00

I,

I~ 00 Cl) 0 o co W~ W Q) - ) r Ii 2 .0 0 0 0 42.). co - WO 2005/047230 PCT/AU2004/001570 62 pqq ~~Cl C) Cl 0 w)c~ CCinlq 0 Cdl o q c?. 00 C? o4 12 Cl 03 U I Cl z = L~~'- ij rQ 9I, -,o bo C 12 "Iz C H WO 2005/047230 PCT/AU2004/001570 63 40 "d -6b 'C 40 -d d ~0

OC

0 04', 0 0 0, c3 110 WO 2005/047230 PCT/AU2004/001570 64 Example 9 Susceptibility of Dermatophytes to Phospholipase Inhibitors Isolates were tested using the method described in the National Committee for Clinical Laboratory Standards (NCCLS) document M38-P for filamentous fungi, with 5 dermatophyte specific modifications described by Fernandez -Torres et al, Current topics in Medical Mycology, 14-19 Feb 1999. MATERIALS and METHODS Test organisms Five dermatophyte isolates were, namely; T. rubrun, T. mentagrophytes, T 10 tonsurans, M canis and M. gypseum, all of which produce lipases and some, phospholipase. These isolates were tested by the broth microdilution method according to NCCLS document M38-P, for activity against selected antifungal agents. In addition, ten randomly selected isolates of T. tonsurans were also tested. All isolates were maintained on SDA at 28 *C. Candida parapsilosis (ATCC 20019) was used as the reference control 15 organism. Antifungal compounds 1,12bis(Tributylammonium)dodecane,dibromide (VS 1-32) - below: Bu NBus

BU

3 N -. 2Br Itraconazole (ITRA) (Janssen ,Cilag, Beerse, Belgium) and Terbinafine (TRB) 20 (Novartis, Basel, Switzerland) were supplied by manufacturers as standard powders, and used as reference agents. Broth medium The microdilution plates used were a single lot of RPMI 1640 basal medium (JRH bioScience Inc. Lenexa, Kansas, USA) with L- glutamine and without sodium 25 bicarbonate, buffered to pH 7.0, at 25 *C, with 0.165 M (MOPS) morpholinepropanesulfonic acid (Sigma). Broth microdilution procedure Preparation of stock solution Preparation of stock solution was performed according to the guidelines set out in 30 NCCLS M38-P, with some modifications, or according to manufacturer's directions. Powdered drugs were dissolved in 100% dimethyl sulfoxide (DMSO), at concentrations 100 times higher than the final test concentration. All compounds were prepared in 100% (DMSO). For ITRA and TRB, two-fold drug dilutions were prepared WO 2005/047230 PCT/AU2004/001570 65 in 100% DMSO at 100 times the final concentration, followed by further dilution 1:50 in RPMI medium, to yield 2 x the final strength required for the test. Itraconazole 10,000 tg/ml solution was further diluted 1:10 to give 1:1000 concentration. Small aliquots of the drug were frozen at -70 *C. .On the day of testing, an 5 aliquot was thawed and further diluted to make a 2 x drug concentration of 32 ptg/ml (e.g. 0.25 mg of 1:1000 drug solution in 7.56 ml of RPMI). Test range of concentrations for ITRA was 0.03 tol6 pg/ml. Terbinafine, 1000 ptg/ml solution was also frozen in aliquots at -70 "C. On the day of testing, it was further diluted 1:50 to give 20 pg/ml concentration (e.g. 0.5 ml in 24.5 ml of RPMI). Finally, it was again diluted 1:10 to give 2 ptg/ml 10 concentration (e.g. 1 ml in 9 ml of RPMI). The range of concentrations tested for TRB was 0.0019 to 1 pg/ml. The range of concentration for 1,12bis(Tributylammonium)dodecane,dibromide was 0.45 to 244 pg/ml Preparation offungal inocula 15 Fungal cultures were subcultured onto Potato Dextrose Agar and grown at 28 'C for 7-10 days. Fungal suspensions were covered with 5 ml of distilled water, and then colonies were scraped off with cotton tipped swabs. The resulting mixture of conidia and hyphae elements was withdrawn, transferred to a sterile tube and then vortexed with glass beads. After the particles settled, the upper homogeneous suspension was collected and its 20 density adjusted by a densitometer to a 0.5 McFarland standard at the wavelength of 530 nm (80-85% transmittance). These stock suspensions were then diluted 1:50 with RPMI medium to obtain final working inoculum concentration ranging from 0.3 x 104 to 9 x 104 CFU/ml. Inoculum quantification was performed by plating 100 gl of the 1:1000 dilution of the adjusted inoculum on SDA plates, incubated at 28 "C, and after 7 days of 25 incubation, the colonies were counted. Test procedure Testing was based on NCCLS M38- P document for testing filamentous moulds. Testing was carried out in sterile 96-well flat bottomed microtitre plate. Columns 1 and 3 to 12 were filled with 100 pl of RPMI medium, leaving column 2 unfilled. Columns 1, 2 30 and 3 were filled with 100 pl of 2 x drug concentration. Serial dilutions were then made by transferring 100 gl from column 3 to each column up to column 11, and 100 pl from column 11 was discarded. Column 12 served as drug free growth control. Finally, 100 pl of inoculum suspension was added to all wells except column 1, which acted as sterility control (Columns 1 and 12 served as sterility and growth control respectively). Plates WO 2005/047230 PCT/AU2004/001570 66 were incubated at 28 "C for 7 days, and read at 3 days and 7 days of incubation. All testing was performed in duplicate, on three separate occasions. Determination of MIC end points Growth in microtitre trays were read by visual inspection by using an illuminated 5 reversed mirror. Growth in each well was compared with the "growth control "drug free well. The MIC end point for all the drugs was chosen to be the first well with 100 % growth inhibition (i.e. the lowest drug concentration inhibiting growth). RESULTS The MICs ranges for ITRA were from 0.03 to 2.0 ig/ml, and from 0.007 to 10 0.25p.ig/ml for TRB (Tables 10 and 11). 1,12-Bis(tributylammonium)dodecane,dibromide, VS1-32, had an MIC range of 1.9-7.6 ptg/ml to T.tonsurans and Mgypseuin. The lowest GM was against T.tonsurans. The highest GM had M.canis (Tables 10 and 11). 15 Table 10. MIC values for selected antifungal compounds against dennatophyte isolates. Antifungal MIC T.rubrum T.mentagrophyte T.tonsurans M.gypseum M.canis agent (ig/ml) ITRA Range 0.03 - 1.0 0.06 - 1.0 0.25 -2.0 0.25 -2.0 0.125 - 2.0 Mean 0.276 0.355 1.06 0.93 0.63 GM 0.171 0.260 0.84 0.74 0.42 TRB Range 0.007 - 0.06 0.03 - 0.06 0.015 - 0.06 0.015 - 0.25 0.03 Mean 0.032 0.035 0.03 0.122 0.03 GM 0.026 0.034 0.024 0.077 0.03 VS1-32 Range 3.8 - 7.6 3.8 1.9 -7.6 1.9 - 7.6 3.8 -7.6 Mean 5.7 3.8 3.8 4.75 6.33 GM 5.37 3.8 3.38 3.8 6.032.

WO 2005/047230 PCT/AU2004/001570 67 Table 11. MIC values for ten T tonsurans isolates Organism MIC ITRA TRB VSI-32 pig/ml T. tonsurans 1 Range 0.03- 0.125 0.015 - 0.06 1.9-3.8 Mean 0.21 0.037 3.17 GM 0.12 0.034 3.02 T. tonsurans 2 Range 0.125 - 2.0 0.015 - 0.06 0.45 - 7.6 Mean 0.604 0.041 2.83 GM 0.397 0.036 1.15 T. tonsurans 3 Range 0.06 - 0.5 0.015 - 0.125 0.45 - 3.8 Mean 0.27 0.051 2.68 GM 0.196 0.038 1.866 T tonsurans 4 Range 0.5 0.015 - 0.06 0.45 - 3.8 Mean 0.5 0.0275 2.68 GM 0.5 0.024 1.866 Ttonsurans 5 Range 0.25 - 0.5 0.03 - 0.06 0.45 - 7.6 Mean 0.46 0.05 5.216 GM 0.44 0.048 2.96 T.tonsurans 6 Range 0.25 - 2.0 0.03 - 0.125 0.45 -15.25 Mean 0.96 0.071 6.94 GM 0.71 0.061 4.32 T.tonsurans 7 Range 0.5 - 1.0 0.015 - 0.06 0.45 - 3.8 Mean 0.58 0.045 2.683 GM 0.56 0.038 1.866 T.tonsurans 8 Range 0.25 - 2.0 0.06 - 0.125 0.45 - 3.8 WO 2005/047230 PCT/AU2004/001570 68 Mean 1.08 0.07 3.13 GM 0.79 0.068 2.48 T. tonsurans 9 Range 0.125 - 1.0 0.06 - 0.125 3.8- 15.25 Mean 0.52 0.081 7.61 GM 0.445 0.077 6.039 T.tonsurans 10 Range 0.25 - 1.0 0.015 -0.06 3.8-7.6 Mean 0.5 0.035 4.75 GM 0.445 0.03 4.52 VS1-32 inhibits Cryptococcus neoformans H99 and C. albicans (ATCC10231) phospholipases, and has also fungicidal activity against those yeasts. In the present study, the mean MICs for VS1-32 were relatively high across all the isolates (2.625-7.61gg/ml). 5 Example 10 - Pharmaceutical Formulations Antimicrobial compounds of the present invention may be administered alone, although they may also be administered as a pharmaceutical fonnulation. For instance, the active ingredient may comprise, for topical administration, from 0.001% to 10% by 10 weight, and more typically from 1% to 5% by weight of the formulation, although it may comprise as much as 10% by weight. By way of illustration, specific examples of pharmaceutical compositions in accordance with the present invention are outlined below. The following are to be construed as merely illustrative examples of formulations and not as a limitation of the 15 scope of the present invention in any way. Example 10(a) - Topical Cream Composition A typical composition for delivery as a topical cream is outlined below: Compound of Formula (I) 1.0 g Polawax GP 200 25.0 g 20 Lanolin Anhydrous 3.0 g White Beeswax 4.5 g Methyl hydroxybenzoate 0.1 g Deionised & sterilised Water to 100.0 g WO 2005/047230 PCT/AU2004/001570 69 The polawax, beeswax and lanolin are heated together at 60 0 C, a solution of methyl hydroxybenzoate is added and homogenisation achieved using high speed stirring. The temperature is then allowed to fall to 50'C. The compound of Formula (I) is then added and dispersed throughout, and the composition is allowed to cool with slow speed stirring. 5 Example 10(b) - Topical Lotion Composition A typical composition for delivery as a topical lotion is outlined below: Compound of Formula (I) 1.2 g Sorbitan Monolaurate 0.8 g Polysorbate 20 0.7 g 10 Cetostearyl Alcohol 1.5 g Glycerin 7.0 g Methyl Hydroxybenzoate 0.4 g Sterilised Water about to 100.00 ml The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of the water at 15 750C. The sorbitan monolaurate, polysorbate 20 and cetostearyl alcohol are melted together at 75 0 C and added to the aqueous solution. The resulting emulsion is homogenised, allowed to cool with continuous stirring and the compound of Formula (I) is added as a suspension in the remaining water. The whole suspension is stirred until homogenised. 20 Example 10(c) - Eye Drop Composition A typical composition for delivery as an eye drop is outlined below: Compound of Formula (I) 0.3 g Methyl Hydroxybenzoate 0.005 g Propyl Hydroxybenzoate 0.06 g 25 Purified Water about to 100.00 ml. The methyl and propyl hydroxybenzoates are dissolved in 70 ml purified water at 75'C, and the resulting solution is allowed to cool. The compound of Fonnula (I) is then added, and the solution sterilised by filtration through a membrane filter (0.22 tm pore size), and aseptically packed into sterile containers. 30 Example 10(d) - Aerosol Composition For an aerosol container with a capacity of 20-30 ml: a mixture of 10 ng of a compound of Formula (I) with 0.5-0.8% by weight of a lubricating agent, such as polysorbate 85 or oleic acid, is dispersed in a propellant, such as freon, and put into an WO 2005/047230 PCT/AU2004/001570 70 appropriate aerosol container for either intranasal or oral inhalation administration or topical application. Example 10(e) - Composition for Parenteral Administration A pharmaceutical composition of the present invention for intramuscular injection 5 could be prepared to contain 1 mL sterile buffered water, and 1 mg of a compound of Fonrula (I). Similarly, a pharmaceutical composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mg of a compound of Formula (I). Example 10(f) - Capsule Composition A phannaceutical composition of a compound of Formula (I) in the form of a 10 capsule may be prepared by filling a standard two-piece hard gelatin capsule with 50 mg of a compound of Formula (I), in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate. Example 10(g) - Injectable Parenteral Composition A pharmaceutical composition of this invention in a form suitable for 15 administration by injection may be prepared by mixing 1% by weight of a compound of Formula (I) in 10% by volume propylene glycol and water. The solution is sterilised by filtration. Example 10(h) - Ointment Composition A typical composition for delivery as an ointment includes 1.0g of a compound of 20 Formula (I), together with white soft paraffin to 100.0 g, dispersed to produce a smooth, homogeneous product. Example 10(i) - Gel Composition A percutaneous gel can be prepared as outlined below: Compound of Formula (I) 1.0 g 25 Propylene glycol 5.0 g Isopropyl alcohol 20.0 g Carboxyvinyl polymer 2.0 g Aqueous ammonia q.s. Purified water 72.0 g 30 The compound of Formula (I) is dissolved into a mixed solvent of propylene glycol and isopropyl alcohol. The Carboxyvinyl polymer is added to the solution after swelling in purified water. After stirring, the mixture is adjusted to pH 7 with the aqueous ammonia.

WO 2005/047230 PCT/AU2004/001570 71 Example 10(j) - Powder Composition A powder composition can be prepared by mixing 6.0 g of sodium caseinate, 3.0 g of xanthan gum and 60.0 g of water for 30 minutes at room temperature. To this aqueous phase 2.0 g of a compound of Fonnula (I) is added, while agitating the solution to form an 5 emulsion, having a pH around 7. The emulsion is homogenized and dehydrated by heating the emulsion to form dry powder particles. The dried powder is then sieved to obtain fine powder particles of mesh size 120-180pm.

Claims

1. Use, in the manufacture of a medicament for one or more of treating, inhibiting, and preventing a bacterial or fungal infection in a vertebrate, of a compound of Formula (I): R1 T R4 R 2 -YG C(R 7 RT)-(A)-C(R8R8.)-Y 2 -R 5 R 3 R6 5(I wherein Yj and Y 2 may be the same or different and are independently selected from N and P; R, to R 6 may be the same or different and are independently selected from the io group consisting of optionally substituted C 2 - 10 alkyl, optionally substituted C 2 - 1 0 alkenyl, optionally substituted C 2 - 10 alkynyl, optionally substituted C 3 - 10 cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heterocycloalkyl, wherein said optional substituents are independently selected from C 1 - 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxyl, halogen, is O(C 1 - 6 alkyl), C(O)O(CI- 6 alkyl), OC(O)(CI. 6 alkyl), NO 2 , amino, hydroxy C 1 - 6 alkyl, aryl, OC(O)Ph, and =C(Ph) 2 ; or R, and R 2 together with the Yi group to which they are attached, or RI, R 2 and R 3 together with the Yi group to which they are attached may optionally form an heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they are 20 attached, or R 4 , R 5 and R 6 together with the Y 2 group to which they are attached may optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected from CI- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxyl, halogen, O(CI- 6 alkyl), C(O)O(C 1 - 6 alkyl), OC(O)(Ci. 6 alkyl), NO 2 , amino, hydroxy Cl- 6 alkyl, aryl, OC(O)Ph, and =C(Ph) 2 ; 25 R 7 , Rr, R 8 and R 8 .may be the same or different and are independently selected from hydrogen, F and Cl; A comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted phenyl, optionally substituted C 5 . 7 cycloalkyl, and -C(O)-, wherein the length of A is from 30 5 to 18 carbon atoms, wherein the optional substituents are independently selected from Ci- 6 alkyl, C2-6 alkenyl, C2-6 alkynyl, hydroxyl, halogen, nitro, C(O)Rio, ORI, CH 2 0R 1 , 73 CH 2 NR 1 2 RI 3 , SRI,, NR 12 RI 3 , CONR 2 RI 3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; RIO is selected from OH, OR,,, and CI- 6 alkyl R 11 is selected from the group consisting of hydrogen, optionally substituted CI.io s alkyl, optionally substituted C 2 1 0 alkenyl, optionally substituted C 2 - 10 alkynyl, optionally substituted C 3 - 10 cycloalkyl, optionally substituted aryl, and optionally substituted aralkyl, wherein said optional substituents are independently selected from C 1 . 4 alkyl, hydroxyl and halogen; R 12 and R 13 are independently selected from the group consisting of hydrogen, io optionally substituted Ci-io alkyl, optionally substituted C 2 10 alkenyl, optionally substituted C 2 1 0 alkynyl, optionally substituted C 310 cycloalkyl, optionally substituted aralkyl, and optionally substituted alkylheteroaryl, wherein said optional substituents are independently selected from CI4 alkyl, hydroxyl, halogen, amino, and C(O)ORu 1 ; or R 1 2 and R 13 , together with the nitrogen atom to which they are attached may form is an optionally substituted heterocycloalkyl group, wherein said optional substituents are independently selected from C14 alkyl, hydroxyl, halogen, amino, and C(O)OR 1 ; or a salt thereof.

2. Use according to claim 1, wherein Yi and Y 2 are each N.

3. Use according to claim 1, wherein Yi and Y 2 are different. 20

4. Use according to claim 1, wherein Yi and Y 2 are each P.

5. Use according to claim 1, wherein R, to R 6 are independently selected from the group consisting of optionally substituted C 2 . 1 0 alkyl, optionally substituted Ci- 10 alkylene, optionally substituted aryl, and optionally substituted heterocycloalkyl, or R, and R 2 together with the Y, group to which they are attached, or RI, R 2 and R 3 25 together with the Yi group to which they are attached form a heterocycloalkyl group; and R4 and R 5 together with the Y 2 group to which they are attached, or R 4 , R 5 and R 6 together with the Y 2 group to which they are attached form a heterocycloalkyl group; wherein said optional substituents are independently selected from CI- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, hydroxyl, halogen, O(CI. 6 alkyl), C(O)O(CI- 6 alkyl), OC(O)(Ci-6 30 alkyl), NO 2 , amino, hydroxy CI. 6 alkyl, and aryl.

6. Use according to claim 1, wherein A comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted phenyl, and -C(O)-, wherein the optional substituents are independently 35 selected from CI- 6 alkyl, C 2 - 6 alkenyl, hydroxyl, halogen, NO 2 , C(O)Rio, OR, 1 , CH 2 0RI 1 , 74 CH 2 NR 12 Ri 3 , SRII, NR 12 RI 3 , CONR 2 Ri 3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl.

7. Use according to claim 1, wherein the length of A is from 5 to 9 carbon atoms. 5

8. Use according to claim I wherein the compound is of Formula (la): R1\v D R4 R 2 -Y 1 CH 2 -- (A)-CH 2 2-R 5 R 3 R 6 (1a) wherein Yj and Y 2 may be the same or different and are independently selected from N and 10 P; R, to R 6 may be the same or different and are independently selected from the group consisting of optionally substituted C 2 - 10 alkyl, optionally substituted C 2 - 1 0 alkenyl, optionally substituted C 2 - 1 0 alkynyl, optionally substituted C 3 -10 cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted 15 heterocycloalkyl, wherein said optional substituents are independently selected from CI-6 alkyl, C 2 - 6 alkenyl, hydroxyl, halogen, O(Ci- 6 alkyl), C(O)O(CI. 6 alkyl), NO 2 , amino, hydroxy Ci- 6 alkyl, aryl, and OC(O)Ph; or R, and R 2 together with the Yi group to which they are attached may optionally form a heterocycloalkyl group; and R 4 and R 5 together with the Y 2 group to which they 20 are attached may optionally form a heterocycloalkyl group; wherein each of said heterocycloalkyl groups may be optionally substituted with one or more groups selected from Ci- 6 alkyl, C 2 - 6 alkenyl, hydroxyl, halogen, O(CI- 6 alkyl), C(O)O(CI- 6 alkyl), amino, hydroxy CI- 6 alkyl, and aryl; A comprises one or more groups selected from optionally substituted alkylene, 25 optionally substituted alkenylene, and optionally substituted phenyl, wherein the length of A is from 5 to 18 carbon atoms, and wherein the optional substituents are independently selected from CI- 6 alkyl, C 2 .6 alkenyl, C 2 - 6 alkynyl, halogen, C(O)RIO, OR,,, SRI, CH 2 0RII, CH 2 NR 12 R 13 , NR 1 2 R 3 , CONR 2 RI 3 , amino acids, dipeptidyl, tripeptidyl, tetrapeptidyl and pentapeptidyl; 30 RIO is selected from OH, OR, 1 and C 1 - 6 alkyl; R I is selected from the group consisting of hydrogen, optionally substituted Ci.-o alkyl, optionally substituted C 2 - 10 alkenyl, optionally substituted C 2 . 1 0 alkynyl, and 75 optionally substituted C 3 . 10 cycloalkyl, wherein said optional substituents are independently selected from C 1 . 6 alkyl, C 2 - 6 alkenyl, aryl and hydroxyl; R 1 2 and R 13 are independently selected from the group consisting of hydrogen, optionally substituted CI-io alkyl, optionally substituted C 2 - 1 0 alkenyl, optionally 5 substituted C 2 - 10 alkynyl, optionally substituted C 3 . 1 0 cycloalkyl, optionally substituted alkylheteroaryl, wherein said optional substituents are independently selected from Ci-6 alkyl, C 2 - 6 alkenyl, aryl, hydroxyl, halogen, amino, and C(O)ORI 1 ; or R 12 and R 1 3 together with the nitrogen atom to which they are attached may form an optionally substituted heterocycloalkyl group, wherein said optional substituents are io independently selected from C 1 . 6 alkyl, C 2 - 6 alkenyl, hydroxyl, halogen, amino, and C(O)OR 11 , or a salt thereof.

9. Use according to any one of the preceding claims wherein R, to R 6 are the same or different and are independently selected from the group consisting of optionally is substituted C 2 - 8 alkyl, wherein the optional substituents are as defined in claim 1.

10. Use according to any one of the preceding claims wherein R, to R 3 are the same and/or wherein R4 to R 6 are the same.

11. Use according to claim 10 wherein R, to R 6 are all the same.

12. Use according to claim 1, selected from 1,11-bis 20 (tributylammonium)undecane, 1,1 6-bis-(tributylammonium)hexadecane, 1,1 2-bis (tripentylammonium)dodecane, 1,1 2-bis-(trihexylammonium)dodecane, 1,1 2-bis (trioctylammonium)dodecane, 1,1 2-bis-(triisobutylammonium)dodecane, 1,12-bis (triisopentylammonium)dodecane and 1,12-bis-(I-butylpyrrolidinium)dodecane, or a salt thereof. 25

13. Use according to any one of the preceding claims wherein the infection is a fungal infection.

14. Use according to any one of claims 1 to 12 wherein the infection is a bacterial infection.

15. A compound of Formula (I) as defined in claim I when used in a method of 30 treatment, inhibition or prevention of a bacterial or fungal infection in a vertebrate.

16. A compound according to claim -1-3-15_wherein Formula (I) is as further defined in any one of claims 2 to 12.

17. A method of inhibiting phospholipase in an organism, comprising contacting said organism with an effective amount of at least one compound of Formula (I) as 35 defined in claim 1. 76

18. A method according to claim 17 wherein Formula (I) is as further defined in any one of claims 2 to 12.

19. The method according to claim 17 or 18 wherein the organism is selected from bacteria, fungi, virus, and parasite. 5

20. The method according to any one of claims 17 to 19 wherein the phospholipase is Phospholipase B.

21. A method for identifying an antimicrobial agent comprising contacting microbial cells with a compound of Formula (I) as defined in claim 1, the compound being suspected of having antimicrobial properties; determining whether said compound io inhibits a microbial phospholipase enzyme, wherein inhibition of said phospholipase enzyme indicates antimicrobial activity; and thereby identifying an antimicrobial agent, with the proviso that the method is not carried out in vivo.

22. A method for one or more of treating, inhibiting, and preventing a microbial infection in a vertebrate or in a plant, said method comprising administering to said is vertebrate, or contacting said plant with, an effective amount of a compound of Formula (I) as defined in claim 1. Dated 29 November, 2010 The University of Sydney Patent Attorneys for the Applicant/Nominated Person 20 SPRUSON & FERGUSON