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Title:
COMPOUNDS AND METHODS OF TREATING INFECTIONS
Document Type and Number:
WIPO Patent Application WO/2014/176636
Kind Code:
A9
Abstract:
The invention provides compounds of Formula (I), and methods of treating or preventing a bacterial infection in a subject using a compound of Formula (I). The invention also provides the use of a compound of Formula (I) in the manufacture of a medicament for the treatment of a bacterial infection in a subject. The invention further provides a medical device when used in a method of treating or preventing a bacterial infection in a subject and to a medical device comprising the composition of the invention.

Inventors:
PAGE, Stephen (55 Campbell Street, Newtown, New South Wales 2042, AU)
GARG, Sanjay (University of South Australia, City East CampusFrome Roa, Adelaide South Australia 5001, AU)
KEENAN, Martine (Epichem Pty Ltd, Murdoch University Campus70 South Stree, Murdoch Western Australia 6150, AU)
MCCLUSKEY, Adam (405 Warners Bay Road, Charlestown, New South Wales 2290, AU)
STEVENS, Andrew (1/40 Moira Street, Adamstown, New South Wales 2289, AU)
Application Number:
AU2014/000483
Publication Date:
December 18, 2014
Filing Date:
May 01, 2014
Export Citation:
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Assignee:
NEOCULI PTY LTD (4/25-37 Huntingdale Road, Burwood, Victoria 3125, AU)
PAGE, Stephen (55 Campbell Street, Newtown, New South Wales 2042, AU)
GARG, Sanjay (University of South Australia, City East CampusFrome Roa, Adelaide South Australia 5001, AU)
KEENAN, Martine (Epichem Pty Ltd, Murdoch University Campus70 South Stree, Murdoch Western Australia 6150, AU)
MCCLUSKEY, Adam (405 Warners Bay Road, Charlestown, New South Wales 2290, AU)
STEVENS, Andrew (1/40 Moira Street, Adamstown, New South Wales 2289, AU)
International Classes:
C07C279/02; A61K31/12; A61K31/155; A61K31/341; A61K31/381; A61K31/4045; A61K31/4192; A61K31/44; A61K31/498; A61P31/04; C07C241/04; C07C249/14; C07C275/68; C07C281/08; C07C281/18; C07C337/08; C07D401/12; C07D403/12; C07D407/12; C07D409/12
Attorney, Agent or Firm:
WRAYS (56 Ord Street, West Perth, Western Australia 6005, AU)
Download PDF:
Claims:
CLAIMS

A compound of Formula i, or a stereoisomtsr, iautomer, pharmaceutically acceptable salt, or prodrug thereof:

wherein R, is H. cycioalK i, Formula il, or Formula Hi;

A. >

Formate II F cmuLs lit

wherein 2 is H, NH¾. NHNH2. 0-CHrCHj, H~C(0)-prseny!, NH-chiorophenyl, NH-CH2-c loropheny!f NH-N=CH-cydoalkyJ, Formula IV, Formula V or Formula V'i;

R,5 R.

\

Pocmuia IV Fermu& V FeffiHsSfc Vi wherein Ae is N, C, CH, or A<, is C and Ac is bonded to Rdt wa 2l to form a triazole ring; wherein A, is N, C, NH, =CH-CH= -( (C*H5}C~CH=N-, or Formula VII; R24

R27

Formula VII

A2 is N, C, NH, N-C(0)-phenyl or Formula VII; wherein A3, A4, A5, A8, A7, A8, A , A12, A13, A14, A15, A)8 , A)7, A)8, A19, A20, A21 A23, A24, A25, A26 and A27 are independently C, O, N, NH, S; wherein A9 is C, O, N, NH, N-C(0)-0-CH2-CH3, N-C(0)-0-CH(CH3)2, N-C(0)-NH- CH2-CH3, N-C(0)-NH-CH2-phenyl, N-C(0)-CH2-CH2-CH2-CH2-CH2-CH3, N-C(O)- CH2-furan-2-yl; wherein A10 is C, NH, -N=CH-CH=, -N=CH-C(C6H5)-; wherein A22 is -CH(CH3)-, -N-CH-, -N-C(CH3)-, N-C(CH2OH)-;

R2 is H, COOH, CH2NH2, CH2OH, CH2NHNH2, methyl, ethyl, propyl, butyl, cyclopentyl, or Formula VII and R2 are R4 are bonded together to form a pyrimidine, pyrazine or triazine ring, or R2 and R9 are bonded together to form a pyrrolidinyl oxindole ring; wherein R4 is N, NH, O, S, or R4 and Ao are bonded, via R2, to form a triazole ring, or R4 is N and R4 and R2 are bonded together to form a pyrimidine ring; wherein R7 is H, CI, Br, F, OH, CH3, OCH3, SCH3, CN, CCH, CF3, OCF3, SCF3, N02, butyl, i-butyl, dimethylamino, phenyl, n-propyl, /-propyl, -NH-C(0)-CH3, - CH=CH-COOH, piperazin-1 -yl, or R7 and R8 are bonded together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R6, R8, R14, R16, R25 and R27 are independently H, OH, CI, F, Br, CH3, CN, OCH3, COOH, N02, CF3, R8 and R7 bond together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring, or benzene ring, R14 and R)5 are bonded together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, R8 and R9 are bonded together to form a substituted or unsubstituied, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or R and are bonded together to form a substituted or unsubstituied saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein Rs, f¾: u. R?A and f½ are independently H, O, OH, Ci, F, Br, H2, CHj, CF3, OCHs, CN, NOa, phenyl, -NH-GH(OH)-CH:ii -NH-C{0)-CHs. or 8 and Rs are bonded together to form a substituted or unsubstituied, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or Ri3 and R,A are bonded together to form a substituted or unsubstituied saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R10( R<,, Rfs, R30t £2 and RS5 are independently H, Cf, or Br, or R 0 and R- . are bonded together to form a substituted or unsubstituied , saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or R 3 and RSf, are bonded together to form a substituted or unsubstituied, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or RJJ and R23 are bonded together to form a substituted or unsubstituied, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R«, F½ and R2< are independently H, COON, CH2NH2, CH2OH, methyl ethyl, propyl, butyl, cydopentyl, or Rt z and R< J are bonded together to form a pyfTottdinyi oxindo!e ring; wherein R,£ and R2i; are independently H, CI, Br, F, OH, CH3, OCH3, SCH3l CN, CF;i, OCF?, SCF3, HOz, CCH, n-buryf. r-buiyi, dimethyiamino, phenyl, n-propyi, /- propyl, -NH-C(0)-CH3, -CH=CH-COOH. piperazin-1-yl, or R,% and R„ are bonded together to form a substituted or unsubstituied, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; and wherein "— " is a double bond or a singie bond.

2. A compound of Formula i, or a stereoisomer, tautomer, pharmaceuticaiiy acceptable salt, or prodrug thereof, selected from the compounds presented in Figure 1.

3. A compound of Formula f, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof,

wherein Ao is C; wherein A< is N; or Formula VII; wherein A2 is N; or NH; wherein A3. A4, A6, A7, A,,, A12, A14, A15, are N; or C; wherein A5, A13, A23, A2 , A25, A26 and A27 are C; wherein A8 and A21 are S; wherein A9 is NH; wherein A10 is N; wherein A22 is -N-CH-; -N-C(CH3)-; or -N-C(CH2OH)-; wherein R, is H; Formula II; Formula III; cycloalkyl; wherein Ft2 is H; methyl; ethyl; CH2NHNH2; CH2OH; butyl; cyclopentyl; or Formula VII and R2 is bonded to R4, to form a pyrimidine ring; wherein R3 is NH2; Formula IV; Formula V; Formula VI; NH2, NH-N=CH- cycloalkyl; or 0-CH2-CH3; wherein R4 is NH; O; S; or R4 is N and R and R2 are bonded together to form a pyrimidine ring; wherein R7 is H; F; CI; CF3; methyl; R7 and R8 are bonded together to form an unsubstituted, benzene ring; OH; f-butyl; phenyl; dimethylamino; /-propyl; n- propyl; CN; CCH; n-butyl; SCH3; R7 and R8 are bonded together to form an unsubstituted, unsaturated heterocyclic ring; OCH3; Br; OCF3; piperazin-1-yl; or SCF3; wherein R6, R8, R) , and R)6 are independently H; OH; F; OCH3; CF3; methyl; CI; CN; Br; R8 and R7 are bonded together to form an unsubstituted, benzene ring; R8 and R7 are bonded together to form an unsubstituted, unsaturated heterocyclic ring; R14 and R)5 are bonded together to form an unsubstituted, benzene ring; or R) and R)5 are bonded together to form an unsubstituted, unsaturated heterocyclic ring; wherein R5, R9, R13, and R,7 are independently H; OH; NH2; CI; F; OCH3; OH; - NH-CH(OH)-CH3; wherein R12 is H; methyl; ethyl; CH2OH; or cyclopentyl; wherein R15 is H; ; Ci; CFv. methyl; R15 and P,u are bonded together to form an unsubstituted, benzene ring; OH; f-butyl; phenyl; dimethyfamino; /-propyl; n- propy!; CN; CCH; o-butyi; SCH3; R1S and R1 are bonded together to form an unsubstituted, unsaturated heterocyclic ring; OCH:5; Br; OCF5: piperazin-1-yi; or SCFj; wherein R2< and R2e are independently H; OH; or CI; wherein R2$ and Ri? are independently H; or OH; wherein ¾ is H; Cf+>; Br; Ci; OH; dimethyiamino; -0-P(0){OEt)?.;. CF;5; or F; and wherein *— " is independently a single or a double bond.

A compound according to any one of the preceding claims, wherein the compound is selected from the group comprising: NCLQQ8; NCL009; NCL023; NCL025; NCL026; NCL029; NCL038; NCL037; NCL039; NCL040; NCL050; NCL061 ; NCL064; NCL065; NCL06S; NCL075; NCL076; NCL078; NCL079; NCL0S0: NCL081; NCL08 -, NCL085; NCL086; NCL088; NCL089; NCL090; CL092; NCLG94; NCL09S; NCL097; NCL09S; NCL099; NCL101 ; NCL104; NCL105; NCL106; NCL1G8; NCL111 : CL112: NCL114; NCL115; CL116; NCL118: NCL119: NCL121; NCL122; NCL123; NCL124; NCL125; NCL128; NCL130; NCL131; NCL132; NCL133; NCL135; NCL138; NCL137; NCL138; NCL139; NCL140; NCL141; NCL144; NCL145; NCL146; NCL147; NCL.14S; NCUSO; CL152; NCL153; NCL154; NCL156; NCL157; NCL158; NCL159; CL161 ; CL182; NCL164; NCL165; NCL166; NCL167; NOL168; NCL189; NCL170; NCL171 ; NCL172; NCL173; NCI174, NCL178; NCL177; NCL178; NCL179; NCL180; NCL181 ; NCL183; NCL184; CL185: NCL186; NCL187; NCL188; NCL189; NCL190; NCL1S3; CL194; NCL1 5; NCL196; NCL1S7; NCL198; NCL199; CL200; NCL201 ; NCL202: NCL203; NCL2Q4; NCL205; NCL208; CL207; NCL208; NCL203; NCL210; NCL211 ; NCL212; NCL213; NCL.215; NCL216; NCL217; NCL218; CL219; NCL220; NCL221 ; NCL222; and NCL223.

The compound according to arty one of the preceding claims, wherein the compound is selected from the group comprising; NCL040; NCL078; NCL079; NCL080; NCL081; NCL08 ; NCL.088; NCLQ89; NCL097; NCL099; NCL1 3; NCL148; NCL167; NCL158; NCL177; NCL179; NCL183; NCL193: NCL195; NCL198; NCL197; NCU9S; NCL202; NCL204; NCL205; NCL216; CL216; CL21 ; NCL219, and NCL221.

The compound according to any one of the preceding claims, wherein the compound is selected from the group comprising: CL078; NCL078; NCL080; NCL081; NCL084; NCL089; NCL097; NCL157; NCL158; NCL179; NCL188; NCL193; NCL195; NCL196; NCL199; NCL204; NCL216; NCL217; NCL219; and NCL221.

7. The compound according to any one of the preceding claims, wherein the compound is selected from the group comprising: NCL089; NCL097; NCL157; NCL179; NCL188; NCL193; NCL195; NCL196; NCL216; NCL219; and NCL221.

8. The compound according to any one of the preceding claims, wherein the compound is selected from the group comprising: NCL097; NCL157; NCL179; NCL188; NCL195; and NCL196.

9. The compound according to any one of the preceding claims, wherein the compound is a chloride salt.

10. The compound according to any one of the preceding claims, wherein the compound is not a compound selected from the group comprising: NCL812, NCL001 , NCL002, NCL003, NCL004, NCL005, NCL006, NCL007, NCL010, NCL01 1 , NCL012, NCL013, NCL014, NCL015, NCL016, NCL017, NCL018, NCL019, NCL020, NCL021 , NCL022, NCL024, NCL027, NCL028, NCL030, NCL031 , NCL032, NCL033, NCL034, NCL035, NCL038, NCL041 , NCL042, NCL043, NCL044, NCL045, NCL046, NCL047, NCL048, NCL049, NCL051 , NCL052, NCL053, NCL054, NCL055, NCL056, NCL057, NCL058, NCL059, NCL060, NCL062, NCL063, NCL066, NCL067, NCL069, NCL070, NCL071 , NCL072, NCL073, NCL074, NCL077, NCL082, NCL083, NCL087, NCL091 , NCL093, NCL096, NCL100, NCL102, NCL103, NCL107, NCL109, NCL1 10, NCL1 13, NCL1 17, NCL120, NCL127, NCL128, NCL129, NCL134, NCL142, NCL143, NCL149, NCL151 , NCL155, NCL160, NCL163, NCL175, NCL182, NCL191 , NCL192, and NCL214.

1 1. A method of treating or preventing a bacterial colonisation or infection in a subject, the method including the step of administering a therapeutically effective amount of a compound of any one of claims 1 to 10, or a therapeutically acceptable salt thereof, to the subject, wherein the bacterial infection is caused by a bacterial agent.

12. The method according to claim 1 1 , wherein the subject is an animal most preferably selected from the group comprising: human, canine, feline, bovine, ovine, caprine, porcine, avian, piscine and equine species.

13. The method according to either claim 1 1 or claim 12, wherein the compound is administered to the subject in a dose in the range of 0.1 mg/kg to 250 mg/kg bodyweight.

14. The method according to any one of claims 1 1 to 13, wherein the bacterial agent is gram positive.

15. The method according to claim 14, wherein the bacteria! agent is selected from the group comprising: Abiotrophia detectives, Achol&plas a spp., Actinobaculum suis, Actinomyces bovis, Actinomyces eurapaeus, Actinomyces georgiae, Actinomyces get cseriae, Actinomyces graevenitzii, Actinomyces hordeovuineris, Actinomyces israelii serotype II, Actinomyces israelii, Actinomyces meyeri, A ctino yces naeslundii, Actinomyces neuii, Actinomyces odontoiyticus, Actinomyces radingae, Actinomyces spp, Actinomyces turicensis, Actinomyces viscosus, Al!oscardovia o nicolens, Anaerococcus hydrogenalis. Anaerococcus lactolyticus, Anaerococcus murdoc ii, Anaerococcus octavius, Anaerococcus prevoiii, Anaerococcus tetradius, Anaerococcus vaginalis, Arcanobacterium (Actinomyces) hemardiae, Arcanobacterium (Actinomyces) pyogenes, Arcanobacterium bernatdiae, Arcanobacterium cardiffensis. Arcanobacterium funkei, Arcanobacterium haemolyticum, Arcanobacterium houstonensis, Arcanobacterium iingnae, Arcanobacterium pyogenes (Actinomyces pyogenes), Arihrobacier, Atopobium minutum, Atopobium parvulum, Atopobium rimae, Atopobium spp, Atopobium vaginae. Baciiius anthracis. Bacillus cereus, Bacillus circulans, Bacillus lichenifomvs, Bacillus megaterium, Bacillus melaninogenicus, Bacillus pu ilus, Baciiius sphaericus, Baciiius subiilis, beta aemolytic St&ptococcus spp. Bifidobacteria adoiescentis. Bifidobacteria denfium, Bifidobacteria scardovii, Bifidobacteria, Brevibacillus brevis, Brwibaci!lus laterosporus, Brevibacierium, Bulleidia extructa, Catabacter hongkongensis, CDC corynefonn groups F-1 and G, Clostridium tetani, Clostridium b&ratii, Clostridium bifer entans, Clostridium botulinum (types A, B, C, D, E, F, G), Clostridium botulinum, Clostridium butyricum, Clostridium chauvoei, Clostridium colinum, Clostridium difficile, Clostridium haemolyticum, Clostridium histotyticum, Clostridium novyi type A, Clostridium novyi type B, Clostridium novyi, Clostridium perfringens type A, Clostridium perfringeris types A-E, Clostridium perfringens, Clostridium piliforme, Clostridium ramosum, Clostridium septicum, Clostridium sordeili, Clostridium sphenoides, Clostridium spiroforme, Clostridium spp, Clostridium tertium, Clostridium t&tani, Collinsella aerofaciens, Corynebacterium accolens, Cotynebacterium afermentans afermentans, Corynebacterium afarmentans lipophitum, Cotynebacterium amycolatum, Corynebacterium atyentoratense, Corynebactarium aurimucosum, Corynebacterium auris, Corynebacterium bovis, Corynebacterium confusu , Corynebacterium cystidis, Cotynebacterium diphtheria, Corynebacterium fr&neyi, Corynebacterium glucuronolyticum, Corynebacterium jeikesum.. Corynebacterium kroppenstedtii, Corynebacterium kufscheri, Corynebacterium lipophilofiavum, Cotynebacterium macginleyi, Corynebacterium matruchoiii, orynebacterium minutissimum, Corynebacterium pilosu , Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium riegelii, Corynebacterium simulans, Corynebaciarium striatum, Corynebacterium sundvallense, Corynebacterium thomssensii, Corynebacterium tuberculostearum, Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacterium xerosis, Crossiella equi, Dermabacter, Dermatophilus congolense, Dermatophilus congolensis, Eggerthella brachy, Eggerthella hongkongensis, Eggerthella infirmum, Eggerthella lenta, Eggerthella minutum, Eggerthella nodatum, Eggerthella saphenum, Eggerthella sinensis, Eggerthella sulci, Eggerthella tenue, Eggerthella, Enterococcus avium, Enterococcus bovis, Enterococcus casseliflavus/flavescens, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus hirae, Enterococcus italicus, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pallens, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus sanguinicola, Enterococcus spp, Erysipelothrix rhusiopathiae, Eubacterium, Filifactor alocis, Finegoldia magna, Gallicola barnesae, Gemella asaccharolytica, Gemella bergeri, Gemella cuniculi, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis, Gemella sanguinis, Gordonia spp., Granulicatella adiacens, Granulicatella elegans, Granulicatella para-adiacens, Kytococcus schroeteri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus iners, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus species, Lactobacillus ultunensis, Leifsonia aquatic, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc paramesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes. Listeria seeligeri. Listeria welshimeri, MicrobacteriumM obiluncus curtisii, Mobiluncus mulieris, Mobiluncus spp, Mogibacterium timidum, Mogibacterium vescum, Moryella indoligenes, Mycobacterium senegalense, Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium arupense, Mycobacterium asiaticum, Mycobacterium aubagnense, Mycobacterium avium complex, Mycobacterium avium subsp paratuberculosis, Mycobacterium avium, Mycobacterium bolletii, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacterium conspicuum, Mycobacterium elephantis, Mycobacterium farcinogenes, Mycobacterium florentinum, Mycobacterium fortuitum group, Mycobacterium genavense, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium houstonense, Mycobacterium immunogenum, Mycobacterium interjectum, Mycobacterium intracellular, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium mageritense, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium massiliense. Mycobacterium microti, Mycobacterium montefiorense (eels), Mycobacterium moracertse, Mycobacterium mucogenicu , Mycobacterium nebrasksnse, Mycobacterium neoaumm, Mycobacterium novocastrense, Mycobacterium palustre, Mycobacterium paratubarcuiosis (Johns's Disease), Mycobacterium pamiense, Mycobacterium phiei, Mycobacterium phocaicum, Mycobacterium pinnipedu, Mycobacterium porcinum, Mycobacterium pseudoshotsii (fish), Mycobacterium pseudotuberculosis, Mycobacterium saskstchewanense, Mycobacterium scrofuiaceum, Mycobacterium senue e, Mycobacterium septicum, Mycobacterium si iae, Mycobacterium s egmatis, Mycobacterium spp, Mycobacterium szuigai, Mycobacterium terrae/chromogenicu complex, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium iusciae, Mycobacterium ufcerans, Mycobacterium wo!inskyi, Mycobacterium xenopi, Mycobacterium, Nocardia asteroides, Nocardia brasiliensis, Nocardia farcinica, Nocardia nova, Nocardia otiiidiscaviarun , Nocardia spp, Nocardia transva!ensis, Oerskovia, Olsenelta oral spp, Qiseneila profuse, Olsene!la uli, Oribacterium sinus, Paenibacitlus alvei, Parvimonas micra, Pediococcus, Peptococcus indoiicus, Peptococcus niger, Peptoniphilus asaccharolyticus, Peptoniphilus gorb&c ii, Peptoniphilus harei, Peptoniphilus indoiicus, Peptoniphilus ivorii, Peptoniphilus lacrimalis, Peptoniphilus o/se/w, Peptosireptococcus anaerobius, Peptostreptococcus stomatis, Propionibacierium acnes, Propionibacterium granu!osu , Propionibacterium propionicum, Propionibacterium, Pseudoramibaciar alactolyticus, Rhodococcus equi, Rhodococcus erythwpolis, Rhodococcus fasciens, Rhodococcus rhodochroua, Rothia. Ruminococcus productus, Siackia exigua, Slackia h&liotrinireducens, Solobacierium moorei: Staphylococcus adettae, Staphylococcus aureus subsp. anaerobius, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus capitis, Staphylococcus caprae. Staphylococcus camosus, Staphylococcus caseolyticus, Staphylococcus chromogenes, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus cohnii Staphylococcus condiment!, Staphylococcus deiphini, Staphylococcus epidermidis, Staphylococcus equoru , Staphylococcus felis, Staphylococcus fleur&ttii. Staphylococcus gallinarum, Staphylococcus haemo!yticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus ktoosii. Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus uscae. Staphylococcus nepalensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus piseifermentans, Staphylococcus pseudiniermedius, Staphylococcus pulvereri, Staphylococcus saccharo!yticus, Staphylococcus saprophytics. Staphylococcus schieifari subsp. coaguians, Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus si iae, Staphylococcus simulans, Staphylococcus spp, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri, Staphylococcus xylosus, Staphylococcus vitulinus, Stomatococcus mucilaginosus (reclassified as Rothia mucilaginosa), Streptococcus agalactiae, Streptococcus anginosus species group (Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus), Streptococcus bovis species group (S. gallolyticus subsp. gallolyticus (formerly S. bovis biotype I), Streptococcus bovis, Streptococcus canis, Streptococcus dysgalactiae subsp. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. porcinus, S. canis, S. suis, S. iniae), Streptococcus dysgalactiae subsp. equisimilis, Streptococcus dysgalactiae, Streptococcus equi (Streptococcus equi subsp equi), Streptococcus equi subsp. zooepidemicus, Streptococcus equi, Streptococcus equinus, Streptococcus equisimilis (Streptococcus dysgalactiae subsp equisimilis), Streptococcus gallolyticus subsp. pasteurianus (formerly S. bovis biotype 11/2), Streptococcus infantarius subsp Infantarius, Streptococcus lutetiensis (formerly S. bovis biotype 11/1), Streptococcus mitis species group (S. cristatus , S. infantis, S. mitis, S. oralis, S. peroris, S. orisratti), and Streptococcus mutans species group (S. cricetus, S. downei, S. ferus, S. hyovaginalis, S. macaccae, S. mutans, S. ratti, S. sobrinus, Sanguinis Group, S. gordonii, S. parasanguinis, S. sanguinis), Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pyogenes, Streptococcus salivarius species group (S. alactolyticus, S. hyointestinalis, S. infantarius, S. salivarius, S. thermophilus, S. vestibularis), Streptococcus spp, Streptococcus suis, Streptococcus uberis, Streptococcus zooepidemicus (Streptococcus equi subsp zooepidemicus), Streptococcus zooepidemicus, Trueperella abortisuis, Trueperella bernardiae, Trueperella bialowiezensis, Trueperella bonasi, Trueperella pyogenes (Arcanobacterium pyogenes), Tsukamurella spp., Turicella, and Turicibacter sanguine.

16. The method according to claim 15, wherein the bacterial agent is selected from the group comprising: Staphylococcus aureus, Staphylococcus pseudintermedius, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, and Clostridium difficile.

17. The method according to any one of claims 1 1 to 13, wherein the bacterial agent is gram negative.

18. The method according to claim 17, wherein the bacterial agent is selected from the group comprising: Acetobacteraceae:- Roseomonas cervicalis; Roseomonas fauriae; Roseomonas gilardii. - Aeromonadaceae:- Aeromonas allosaccharophila; Aeromonas aquariorum; Aeromonas caviae; Aeromonas hydrophila (and subspecies); Aeromonas salmonicida; Aeromonas shubertii; Aeromonas veronii biovar sobria (Aeromonas sobria). - Alcaligenaceae:- Achromobacter xylosoxidans; Alcaligenes faecalis; Bordetella ansorpii; Bordetelta avium; Bordeteila bronchiseptica; Bordeteila hinzii; Bordeteila ho! esii; Bordeteila parapertussis; Bordeteila pertussis; Bordeteila petrii; Bordeteila tre atum; Oligella ureolytica; Oligella urethraiis. ~ Anaplasmataceae:- Anaplasma phagoc tophilum; Anaplasma platys; Anaplasma bovis; Anaplasma c&ntrale; Anaplasma marginale; Anaplasma odocoilei; Anaplasrna ovis; Ehrlichia canis; Ehrlichia chaff&en&is; Ehrlichia ewingii; Ehrlichia muris; Ehrlichia ovina; Ehrlichia ruminaniium; Neoehriichia lotoris; Neoehdichia mikurensis; N&orickeiisia helminthoeca; Neorickettsia hsticil; Neorickettsia sennetsu; Wolbachia pipientis. ~ Armatimonadaceae:- Armati onas rosea. - Bacteroidaceae:- Bacieroides forsythus; Bacieroides fragilis: Bacieroides elaninogenicus; Bacieroides ruber; Bacieroides urealtyicus. - Bartonellaceae:- Bartonella a!saitca; Baiioneila australis; Bartonella bacilliformis; Bartonella hiriiesii; Bartonella bovis; Bartonella capreoli; Bartonella chomeiii; Bartonella c!anidgeiae; Bartonella doshiae; Bartonella elizabethae; Bartonella graharnil; Bartonella henselae; Bartonella koehlerae; Bartonella peromysci; Bartonella phoceensls; Bartonella quiniana; Bartonella rattimassiliensis; Bartonella rochaiimae; Bartonella scho buchensis; Bartonella talpae; Bartonella ta iae; Bartonella taylorii; Bartonella tribocorum; Bartonella vinsonii subsp . berkhoffii; Bartonella vinsonii subsp. arupensis; Bartonella vinsonii subsp. vinsonii. -- Bdellovibrionaceae:- Bde!lovsbno spp. - Brachyspiraceae:- Brachyspira spp including Brachyspira ha psonii, Brachyspira hyodyssnteriae, Brachyspira murdochis, Brachyspira piiosicoli. -- Brucellaoeae:- Brucella abortus; Brucella canis; Brucella ceti; Brucella melitensls; Brucella ovis; Brucella pinnipediaiis; Brucella suis; Ochrobacirum anthropi; Ochrobacirum intermedium. ~ Burkholderiaceae:- Burkhoidena at)oris: Burkholderia a bifana (genomovar VII); Burkholderia anihina (genomovar Vili); Burkholderia cenocepacia (genomovar ill}: Burkholderia cepacia (genomovar I); Burkhoidena diffusa; Burkholderia dolosa (genomovar VI); Burkholderia Saians; Burkholderia mallei; Burkholderia metailica; Burkholderia multivorans (genomovar II); Burkholderia psaudomallai; Burkholderia pyrrocinia (genomovar IX); Burkhold&ria seminalis; Burkhoidena stabilis (genomovar IV); Burkholderia ubonensis (genomovar X); Burkholderia vietnamiensis (genomovar V); Cupriavidus paucuiua; Cuph vidus gilardii; Ratstonm pickettii; Ralstonia manniiolilytica; Sphaerotilus hippai; Sphaerotilus ontanus; Sphaerotilus natans. ~ Campylobacterace e:- Arcobacter spp includng Arcobacter skirrowii; Campylobacter coil; Campylobacter concisus; Campylobacter curvus; Campylobacter fetus; Campylobacter gracilis; Campylobacter helveticus; Campylobacter hominis; Campylobacter hyointestinalis; Campylobacter insulaenigrae; Campylobacter jejuni; Campylobacter lanienae: Campylobacter !ari; Campylobacter iahdis; Campylobacter mucosatis; Campylobacter rectus; Campylobacter showae; Campylobacter spulorum; Campylobacter upsaliensis. - Candidates:- Piscichlamydia sal onis. - Cardiobacte aceae:- Cardiohacterium hominis; Cardiobactehum va!varum; Dichelobacter nodosus. - Chlamydiaceae:- Chlamydia spp including Chlamydia avium, Chlamydia gallinacea, Chlamydia muridarum, Chlamydia suis, Chlamydia trachomatis; Chlamydophila spp including Chlamydophila pneumoniae, Chlamydophila pecorum, Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae, and Chlamydophila felis. - Chthonomonadaceae:- Chthonomonas calidirosea. - Comamonadaceae:- Comamonas testosteroni; Verminephrobacter spp. - Coxiellaceae:- Coxiella burnetii. - Cytophagaceae:- Cytophaga columnaris; Cytophaga hutchinsonii; Flexibacter echinicida; Flexibacter elegans; Flexibacter flexilis; Flexibacter litoralis; Flexibacter polymorphus; Flexibacter roseolus; Flexibacter ruber. Desulfovibrionaceae:- Bilophila wadsworthia; Lawsonia intracellularis. Enterobacteriaceae:- Cedecea davisae; Cedecea lapagei; Cedecea neteri; amalonaticus; Citrobacter diversus; Citrobacter freundii; Citrobacter koseri; Cronobacter condimenti; Cronobacter dublinensis; Cronobacter helveticus; Cronobacter malonaticus; Cronobacter muytjensii; Cronobacter pulveris; Cronobacter sakazakii; Cronobacter turicensis; Cronobacter universalis; Cronobacter zurichensis; Edwardsiella ictaluri; Edwardsiella tarda; Enterobacter aerogenes; Enterobacter agglomerans; Enterobacter cloacae; Enterobacter cowanii; Escherichia albertii; Escherichia coli, including AIEC = adherent invasive E. coli, EaggEC = enteroaggregative E. coli; EHEC = enterohemorrhagic E. coli; EIEC = enteroinvasive E. coli; EPEC = enteropathogenic E. coli; ETEC = enterotoxigenic E. coli; ExPEC = extraintestinal pathogenic E. coli, NMEC = neonatal meningitis E. coli, NTEC = necrotoxigenic E. coli, UPEC = uropathogenic E. coli.; Escherichia fergusonii; Ewingella americana; Hafnia alvei; Hafnia paralvei; Klebsiella granulomatis; Klebsiella oxytoca; Klebsiella pneumoniae; Kluyvera ascorbata; Kluyvera cryocrescens; Morganella morganii; Pantoea (formerly Enterobacter) agglomerans; Photorhabdus asymbiotica; Plesiomonas shigelloides; Proteus mirabilis; Proteus penneri; Proteus vulgaris; Providencia alcalifaciens; Providencia rettgeri; Providencia stuartii; Raoultella electrica; Raoultella ornithinolytica; Raoultella planticola; Raoultella terrigena; Salmonella bongori. Salmonella enterica subspecies enterica (many serotypes); Serratia liquifaciens; Serratia marcesans; Shigella boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Yersinia enterocolitica; Yersinia pestis; Yersinia pseudotuberculosis; Yersinia ruckeri. - Fimbriimonadaceae:- Fimbriimonas ginsengisoli. - Flavobacteriaceae:- Bergeyella zoohelcum; Capnocytophaga canimorsus; Capnocytophaga cynodegmi; Capnocytophaga gingivalis; Capnocytophaga granulosa; Capnocytophaga haemolytica; Capnocytophaga leadbetteri; Capnocytophaga ochracea; Capnocytophaga sputigena; Chryseobacterium indologenes; Chryseobacterium piscicola; Elizabethkingia meningoseptica; Flavobacterium branchiophilum; Flavobacterium columnare; Flavobacterium oncorhynchi; Flavobacterium piscicida; Flavobacterium psychrophilum; Myroides odoratus; Myroides odoratimi us; Qrnithobacterium rhinotracheale; Riemerella anatipestifer; Riemerelia columbine; Riernerella columbiphaiyngis; Tenacibacu!um dicentrarchi; Tenacibaculum discolour; Tenacibaculurn gallaicum; Tenacibaculurn maritimum; Tenacibaculurn soieae; Weeksella virosa. -- Fmncisellaceae:- Francisalia tularensis subsp. tularensis; Franciseila tularensis subsp. noiarc!ica; Franciseila tularensis subsp. novicida; Franciseila phiio iragia; Franciseila noatunensis; Franciseila noatunensis subsp, orientalis (also farmed Franciseila asiaiica). ~ Fusobactehaceae:- Fusobacterium spp. including Fusobacterium necrophorum, Fusobacterium nuc!eatum, Fuso-bacierium poiymorphum. Helicobacteraceae:- Helicobacter cinaedi; Helicobacter fennelliaa; Helicobacter pylori. - Legionellaceae;- Legionella pneumophila and other s ecies including; Legionella anisa; Legionella birminghamensis; Legionella boza annii; Legionella cincinnatiensis; Legionella dumoWi; Legionella feeleii; Legionella gor anii; Legionella hackeliae; Legionella jordanis; Legionella iansingensis; Legionella iongbeachae; Legionella maceachemii; Legionella micdadei; Legionella oakridgansis; Legionella parisiensis; Legionella sainthelens; Legionella iusconensis; Legionella wadswotthii; Legionella waiters!!. -- Leptospiraceae:- Leptospira alexanderi (including Leptospira alexanden serovar Hebdo adis, Leptospira alexanderi serovar Manhao 3); Leptospira alstoni (Including Leptospira alstoni serovar Pingchang, Leptospira alstoni serovar Sichuan); Leptospira biflexa (including Leptospira biflexa serovar Ancona, Leptospira biflexa serovar Canela); Leptospira borgp&t&^enii (including Leptospira borgpetersenii serovar Hardjo, Leptospira borgpetersenii serovar Hanip-bovis, Leptospira borgpetersenii serovar Pomona, Leptospira borgpetersenii serovar Tarassovi); Leptospira broo ii (including Leptospira broomi! serovar Hurstbndge); Leptospira fainei (including Leptospira fainei serovar Hurstbndge); Leptospira idonii: Leptospira inadai (including Leptospira inadai serovar Lyme, Leptospira iaadai serovar Malaya); Leptospira interrogans (including Leptospira interrogans serovar Australis, Leptospira inte ogans serovar Autumnaiis, Leptospira interrogans serovar Bratislava, Leptospira interrogans serovar Canicola, Leptospira interrogans san var Grippotyphosa, Leptospira int&nvgans serovar Hardjo, Leptospira interrogans serovar Hardjo-bovis, Leptospira interrogans serovar IcteiOhaemorrhagiae, Leptospira interrogans serovar Pomona, Leptospira interrogans serovar Pyrogenas, Leptospira interrogans serovar Tarassovi); Leptospira kirschneri (including Leptospira kirschneri serovar Buigarica, Leptospira kirschneri serovar Cynopierl, Leptospira kirschneri serovar Grippotyphosa); Leptospira kmetyi; Leptospira licerasi&e; Leptospira meyen (including Leptospira meyeri serovar Sofia); Leptospira noguchii (including Leptospira noguchii serovar Panama, Leptospira noguchii serovar Pomona); Leptospira santarosai; Leptospira terpsirae; Leptospira vanthielii: Leptospira weiiii (including Leptospira weiiii serovar Ce!ledoni, Leptospira weiiii serovar Sarmin); Leptospira wolbachii; Leptospira wotffii; Leptospira S ISA/AU yanagawae. - Leptotrichiaceae:- Leptotrichia buccalis; Streptobacillus moniliformis. - Methylobacteriaceae:- Methylobacterium extorquens group; Methylobacterium fujisawaense; Methylobacterium mesophilicum; Methylobacterium zatmanii. - Moraxellaceae:- Acinetobacter baumannii (genomic species 2); Acinetobacter baylyi; Acinetobacter bouvetii; Acinetobacter calcoaceticus (genomic species 1); Acinetobacter gerneri; Acinetobacter grimontii; Acinetobacter haemolyticus (genomic species 4); Acinetobacter johnsonii (genomic species 7); Acinetobacter junii (genomic species 5); Acinetobacter Iwoffi (genomic species 8/9); Acinetobacter parvus; Acinetobacter radioresistens (genomic species 12); Acinetobacter schindleri; Acinetobacter tandoii; Acinetobacter tjernbergiae; Acinetobacter towneri; Acinetobacter ursingii; Acinetobacter venetianus; Moraxella atlantae; Moraxella boevrei; Moraxella bovis; Moraxella bovoculi; Moraxella canis; Moraxella caprae; Moraxella catarrhalis; Moraxella caviae; Moraxella cuniculi; Moraxella equi; Moraxella lacunata; Moraxella lincolnii; Moraxella macacae; Moraxella nonliquefaciens; Moraxella oblonga; Moraxella osloensis; Moraxella ovis; Moraxella phenylpyruvica; Moraxella pluranimalium; Moraxella porci. - Moritellaceae:- Moritella abyssi; Moritella dasanensis; Moritella japonica; Moritella marina; Moritella profunda; Moritella viscosa; Moritella yayanosii. - Neisseriaceae:- Chromobacterium violaceum; Eikenella corrodens; Kingella denitrificans, Kingella kingae, Kingella oralis, Kingella potus; Neisseria cinerea; Neisseria elongata; Neisseria flavescens; Neisseria gonorrhoeae; Neisseria lactamica; Neisseria meningitidis; Neisseria mucosa; Neisseria polysaccharea; Neisseria sicca; Neisseria subflava; Neisseria weaver; Vitreoscilla spp. - Nitrosomonadaceae:- Nitrosomonas eutropha; Nitrosomonas halophila; Nitrosomonas oligotropha. - Pasteurellaceae;- Actinobacillus actinomycetemcomitans; Actinobacillus equuli; Actinobacillus lignieresii; Actinobacillus pleuropneumoniae; Actinobacillus seminis; Actinobacillus succinogenes; Actinobacillus ureae; Aggregatibacter actinomycetemcomitans, Aggregatibacter segnis, Aggregatibacter aphrophilus; Avibacterium avium; Avibacterium endocarditidis; Avibacterium gallinarum; Avibacterium paragallinarum; Avibacterium volantium; Bibersteinia trehalose; Gallibacterium anatis; Gallibacterium genomospecies 1; Gallibacterium genomospecies 2; Gallibacterium genomospecies 3; Gallibacterium group V; Gallibacterium melopsittaci; Gallibacterium salpingitidis; Gallibacterium trehalosifermentans; Haemophilus aegyptius; Haemophilus avium; Haemophilus ducreyi; Haemophilus haemolyticus; Haemophilus influenzae; Haemophilus parahaemolyticus; Haemophilus parainfluenzae; Haemophilus parasuis; Histophilus somni; Mannheimia caviae; Mannheimia glucosida; Mannheimia granulomatis; Mannheimia haemolytica; Mannheimia ruminalis; Mannheimia varigena; Nicoletella semolina; Pasteurella aerogenes; Pasteurella bettyae; Pasteurella caballi; Pasteurella canis; Pasteurella dagmatis; Pasteurella multocida (subspecies multocida, septicum, gallicida); Pasteurella pneumotropica; Pasteurella stomatis; Pasteurella trehaiosi. ·· Piscirickettsiaceae:- Piscirickettsia salmonis. - Piesiomonadaceae:- Piesiomonas shigeitoides. - Polyangiaceae:- Sorangium cellulosum. Porphyromonadaceae:- Dysgonomonas capnocytophagoides; Dysgonomonas gadei; Dysgonomonas hofstadii: Dysgonomonas ossii; Dysgonomonas oryzarvi; Dysgonomonas wimpennyi; Porphyromonas gingivalis. - Prevoiellaceae - Prevoteiia spp. including Prevoteiia intermedia, Prevoteiia elaninogenica. - Pseudomonadaceae:- Chryseomonas luteola; Pseudomonas aeruginosa; Pseudomonas iuteola; Pseudomonas fluorescens; Pseudomonas putida; Pseudomonas stutzeri; Pseudomonas ory ihabitans.

Rhizobiaceae:- Agrobacterium tumefaciens; Rhizobium radiobacter. - Rickettsiaceae:- Orientia chuto; Orientia tsutsugamushi; Rickettsia aeschlimannii; Rickettsia africae; Rickettsia akari; Rickettsia argasii; Rickettsia asiatica; Rickettsia ausiralis; Rickettsia beiiii; Rickettsia canadensis; Rickettsia conorii; Rickettsia cooieyi; Rickettsia fe!is; Rickettsia heilongjiangensis; Rickettsia helvetica; Rickettsia honei; Rickettsia oogstraaiii; Rickettsia hulinensis; Rickettsia hulinii; Rickettsia japonica: Rickettsia marmionii; Rickettsia martinet; Rickettsia assiiiae; Rickettsia monacensis; Rickettsia ontanensis; Rickettsia monteiroi; Rickettsia moreii; Rick&ttsia parkeri; Rickettsia peacockii; Rickettsia philipii; Rickettsia prowazekii; Rickettsia moultii; Rickettsia rhipicephali; Rickettsia rickettsii; Rickettsia sibirica subgroup; Rickettsia siovaca; Rickettsia iamurae; Rickettsia typhi. - S ewanei!aceae:- S ewanelia putrefaciens. - Sphingo onadaceae:- Sphingobacterium rnulth/onsm; Sphingobacterium spiritivorum; Sphingomonas pauci obilis. - Spirii!aceae:- Spirillum minus: Spirillum volutans; Spirillum winogradskyi. - Spirocbaetaceae:- Borreiia afzeiii; Borreiia anserina; Borreiia bissettii; Borreiia burgdorferi; Borreiia coriaceae; Borreiia duttonii; Borreiia garinii; Borreiia hermsii; Borreiia hispanica; Borreiia japonica; Borreiia lonestari; Borreiia lusitaniae; Borreiia miyamotoi; Bonelia parked; Borreiia persica; Borreiia recurrentis; Borreiia spielmanii; Borreiia iuricatae; Borreiia iuricatae; Borreiia vaiaisiana; Treponema carateu ; Treponema pallidum ssp. endemicum: Treponema patiidum ssp. pallidum; Treponema pallidum ssp. pettenue. -- Succinivibrionaceae:- Anaarobiospirillum spp. - Suttereltaceaa:- Sutterelia spp including Suttere!la wadswortnia. -- Thermaceae:- Meiothermus spp. - Thennotogaceae:- T ermotoga neapoiiiana. ~ Veiiioneliac&ae:- Diaiisier spp; Megamonas spp; Megasphaera spp; Pectinatus spp; Peiosinus spp; Propionispors spp; Sporomusa spp; Veillonelia spp.; Zymophilus spp. ··- Vibrionaceae:- Phoiobacterium damselae; Vibrio adapiatua; Vibrio alginoiyticus; Vibrio ezasii; Vibrio campbeliii; Vibrio cholera; Vibrio damsel: Vibrio fiuvialis; Vibrio furnis ; Vibrio holiisae; Vibrio etchnikovii; Vibrio mi icus; Vibrio parahaemo!yticus; Vibrio vulnificus. - Wolbachiaae:- Woibachia spp. Xanthomonadaceae:- Luteimonas aestuarii: Luteimonas aquatica; Luteimonas composti; Luieimonas htiirnaris; Luteimonas marina; Luieimonas mephitis; Luteimonas vadosa; Pseudoxant omonas bmegbemensis; A/AU Pseudoxanthomonas japonensis; Stenotrophomonas maltophilia; and Stenotrophomonas nitritireducens.

19. The method of claim 18, wherein the bacterial agent is selected from the group comprising: Acinetobacter species, Aeromonas hydrophila, Citrobacter species, Enterobacter species, Escherichia coli, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

20. The method according to any one of the preceding claims, wherein the compound of Formula I, is administered together with a compound that reduces the integrity of the cell wall of the bacterial agent.

21. The method according to any one of claims 1 1 to 13, wherein the bacterial agent has no cell wall.

22. The method according to claim 21 , wherein the bacterial agent is selected from the group comprising: Mycoplasma spp, Mycoplasma agalactiae, Mycoplasma alkalescens, Mycoplasma amphoriforme, Mycoplasma arginini, Mycoplasma bovigenitalum, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasma buccale. Mycoplasma californicum, Mycoplasma canadense, Mycoplasma capricolum subsp. capricolum. Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma conjunctivae, Mycoplasma cynos, Mycoplasma dispar, Mycoplasma equigenitalium, Mycoplasma faucium, Mycoplasma felis, Mycoplasma fermentans (incognitus str.), Mycoplasma gallisepticum (MG), Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma haemocanis, Mycoplasma haemofelis, Mycoplasma haemosuis (formerly Eperythrozoon suis). Mycoplasma hominis. Mycoplasma hyopneumoniae. Mycoplasma hyorhinis, Mycoplasma hyosynoviae. Mycoplasma iowae meleagridis (MM), Mycoplasma iowae, Mycoplasma leachii, Mycoplasma lipophilum, Mycoplasma meleagridis, Mycoplasma mycoides subsp capri, Mycoplasma mycoides subsp mycoides, Mycoplasma mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP), Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, Mycoplasma penetrans, Mycoplasma pirum. Mycoplasma pneumoniae, Mycoplasma primatum. Mycoplasma putrefaciens, Mycoplasma salivarium, Mycoplasma spermatophilum, Mycoplasma suis, Mycoplasma synoviae (MS), Mycoplasma wenyonii, Mycoplasma, Ureaplasma spp, Ureaplasma parvum, Ureaplasma urealyticum, Ureaplasma, and Ureoplasma diversum.

23. The method according to any one of claims 1 1 to 13, wherein the bacterial agent is gram positive, gram negative or does not have a cell wall and is selected from the group comprising, but not limited to, the following livestock pathogens: Actinchacutuni suis, Actinomyces bovis, Arcanobacteriu pyogenes. Bacillus anthracis, cereus, licheniformis, pumilus, melaninog&nicus, subtllis, Clostridium botufinum, chauvoei ha&molyticu , novyi, perfiingens, septicum, sordeliil, tetani, cctinuni, Coryn&bacterium pseudotuberculosis, renele, Denmtophilus congoiensis, Enterococcus spp (such as E. faecaiis, E. iaecium, E. durans, E. avium, E hirae Etysipefothrix rhusiopathiaa, Listeria ivanovii, grays, innocua, seeligeri, welshimeri, monocytogenes,. Mycobacterium avium, bovis, paratuberculosis (Jo ne's Disease), Mycoplasma (such as capticolum subsp, capripneumoniae, subsp. capricoium, M. mycoides subsp ycoides, M. agaiaciiae, M. ovipn&umoniae, M. conjunctivae, M. arginini, M. tx>vis, and M. puirefaciens) Mycoplasma bovis, dispar, mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP) Mycoplasma gal!isepticum (MG), iowae eleagridis (MM), synoviae (MS) Mycoplasma hae osuis (formerly Eperythrozoon suis), alkatescens, bovigenita!um, bovirhinis, bovoculit caiifomieum, canadense, cynos, equigenitalium, gateae, haemocmis, haemof&lis, hyopneumoniae, hyorhinis, hyosytioviae, iowae, leachii, meleagridis, mycoides subsp capri. wenyonii, suis, Rhodococcus equi, Staphylococcus epidermidis, Staphylococcus $imulans, Staphylococcus felis. Staphylococcus xyiosus, Staphylococcus chromogenes, Staphylococcus wa eri, Staphylococcus haemoiyticus. Staphylococcus sciurs, Staphylococcus S8ptx>phyticuss Staphylococcus homints, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus. Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyiicus, Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus pseudint rmedius, Staphyiococcus delphini. Staphylococcus schleiferi subsp. coaguians, Staphylococcus aureus subsp. anaerobius, Streptococcus ubrnis. Streptococcus can s, Streptococcus agalaciiae, Streptococcus dysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus, Streptococcus equi (Streptococcus equi subsp equi), Streptococcus equisimilis (Streptococcus dysgalactiae subsp equisimilis). porcinus, suis, zooepidemicus, Streptococcus zooepidemicus (Streptococcus equi subsp zooepidemicus). Streptococcus dysgalactiae subsp. equisimilis, Propionibacterium acnes, Propionibactenum granulosu , Eubactenum, Peptococcus indolicus, and Peptostreptococcus anaerobius; Actinobaciilus, Aeromonas, Anaplasma, Arcobacter. Avibacterium, Bacteroides, Bartonella. Botdet&iia, Borre!ia, Brachyspira, Brucella, Campylobacter, Capnocytophaga, Chlamydia, Chlamydophi!a, Chryseobacteriu , Coxselia, Cytophaga, Dicheiobacter, Edwardsie!la, Ehrlichia, Escherichia, F' obactenum, Francisella, Fusobacteriu , Gailibectwium, Haemophilus, Histophi!us, Klebsiella, Lawaonia, Leptospira, Mannheimia, Megasphaera, Moraxella, N&orickettsia, Q iihobacierium, Pasteureila, Photobacterium, Piscichlamydia, Piscirickettsla, SA/AU Porphyromonas, Prevotella, Proteus, Pseudomonas, Rickettsia, Riemerella, Salmonella, Streptobacillus, Tenacibaculum, Vibrio, and Yersinia.

24. The method according to any one of claims 1 1 to 13, wherein the infection or colonisation is caused by a mixture of at least two bacterial agents selected from the group comprising: gram postive bacteria, gram negative bacteria and bacteria with no cell wall.

25. The method according to any one of claims 1 1 to 24, wherein the bacterial agent is resistant to one or more compounds selected from the group comprising: penicillins, cephalosporins, carbapenems, monobactams and other beta lactam antibiotics, fusidanes, aminoglycosides, fluoroquinolones, streptogramins, tetracyclines, glycylcyclines, chloramphenicol and other phenicols, macrolides and ketolides, lincosamides, oxazolidinones, aminocyclitols, polymyxins, glycopeptides, lipopeptides, bacitracin, mupiricin, pleuromutilins, rifamycins, sulphonamides and trimethoprim.

26. The method according to claim 25, wherein the bacterial agent is resistant to one or more compounds selected from the group comprising: beta lactams, glycopeptides, lipopeptides, macrolides, oxazolidinones and tetracyclines.

27. The method according to claims 25 or 26, wherein the bacterial agent is resistant to the compound when the compound is at a concentration range selected from the following: 0.001 pg/mL - 10,000 pg/mL; 0.01 pg/mL - 1000 pg/mL; 0.10 pg/mL - 100 pg/mL; and 1 pg/mL - 50 pg/mL.

28. The method according to any one of claims 1 1 to 27, wherein the bacterial infection or colonisation in the subject substantially causes an indication selected from the group comprising: nosocomial pneumonia caused by Staphylococcus aureus or Streptococcus pneumoniae: complicated skin and skin structure infections caused by Staphylococcus aureus, Streptococcus pyogenes, or Streptococcus agalactiae; invasive pneumococcal diseases including pneumonia, bronchitis, acute sinusitis, otitis media, conjunctivitis, meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess caused by Streptococcus pneumonia; uncomplicated skin and skin structure infections caused by Staphylococcus aureus or Streptococcus pyogenes; community-acquired pneumonia caused by Streptococcus pneumoniae, Staphylococcus aureus or Staphylococcus aureus; bloodstream infections (bacteraemia) caused by methicillin-susceptible and methicillin-resistant isolates; vancomycin-resistant Enterococcus infections; Clostridium difficile associated diarrhea (CDAD); and gram negative, gram positive or mixed bacterial bone and joint infections; central nervous system infections; eye infections; gastrointestinal tract infections; genital infections: intra-abdominal infections; respiratory tract infections; otitis externa; otitis media; sepsis; systemic infections; intra-abdominal infections (lAls); urinary tract infections (UTIs); and bacteremia.

2S. The method according to any one of ciaims 11 to 28, wherein the therapeutically effective amount of compound of Formula i, or a therapeutically acceptable salt thereof, is administered to the subject by oral administration.

30. The method according to any one of claims 11 to 28, wherein the therapeutically effective amount of compound of Formula !, or a therapeutically acceptable sait thereof, is administered to the subject by parenteral administration.

31. The method according to any one of ciaims 11 to 28, wherein the therapeutically effective amount of compound of Formula l, or a therapeutically acceptable salt thereof, is administered to the subject by topical administration.

32. An antibacterial pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, or a therapeutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient or carrier.

33. An antibacterial veterinary composition comprising a therapeutically effective amount of a compound of Formula I. or a therapeutically acceptable salt thereof, and optionally a veterinary acceptable excipient or carrier.

34. The composition according to either claim 32 or claim 33, wherein the composition comprises a further antimicrobial agent selected from the group comprising: antibacterial and antifungal agents.

35. The composition according to either claim 32 or claim 33, wherein the composition is adapted for oral administration.

36. The composition according to either claim 32 or claim 33, wherein the composition is adapted for parenteral administration.

37. The composition according to either claim 32 or claim 33, wherein the composition is adapted for topical administration.

38. Use of a compound of Formula f, or a therapeutically acceptable sait thereof, in the manufacture of a medicament for the treatment of a bacteria! colonisation or infection in a subject.

AU 39. The use according to claim 38, wherein the use comprises administering a therapeutically effective amount of compound of Formula I, or a therapeutically acceptable salt thereof, to the subject.

40. The use according to claim 39, wherein the compound of Formula I is administered to the subject in a dose in the range of 0.1 mg/kg to 250 mg/kg body weight.

41. The use according to any one of claims 38 to 40, wherein the medicament is administered to the subject by oral administration.

42. The use according to any one of claims 38 to 40, wherein the medicament is administered to the subject by parenteral administration.

43. The use according to any one of claims 38 to 40, wherein the medicament is administered to the subject by topical administration.

44. A medical device when used in a method of treating or preventing a bacterial colonisation or infection in the subject, wherein the medical device comprises the composition according to any one of claims 34 to 37.

45. The medical device according to claim 44, wherein the medical device is in a form selected from the group comprising: a plaster, a bandage, a dressing or implant applied to a bacterial or colonisation infection in a subject.

46. A method of killing bacteria, the method including the step of contacting the bacteria with a compound of Formula I, or a therapeutically acceptable salt thereof.

47. Use of a compound of Formula I, or a therapeutically acceptable salt thereof, to kill bacteria, said use comprising the step of contacting the bacteria with a compound of Formula I, or a therapeutically acceptable salt thereof.

A method, a composition, device or a use, substantially as described herein with reference to the accompanying Examples and Figures.

ABSTRACT

The invention provides compounds of Formula I, and methods of treating or preventing a bacterial infection in a subject using a compound of Formula I. The invention also provides the use of a compound of Formula i in the manufacture of a medicament for the treatment of a bacteria! infection in a subject. The invention further provides a medical device w en used in a method of treating or preventing, a bacterial infection in a subject and to a medical device comprising the composition of the invention.

U

Description:
COMPOUNDS AND METHODS OF TREATING INFECTIONS

TECHNICAL FIELD

[OOOt] This invention relates to compounds of Formula ί, methods of treating or preventing a bacterial infection in a subject using 8 compound of Formula t, the use of a compound of Formula i in the manufacture of a medicament for the treatment of a bacteria* infection in a subject, and medical devices when used in a method of treating or preventing s bacterial infection in a subject.

BACKGROUND ART

(0002] A marked increase in prevalence of multi-drug resistance in disease-causing Gram- positive (G-H'S {Staphylococcus aureus, Enierococcus spp. and Streptococcus pneumoniae) ant! Gram negative (G~ve) pathogens (Escherichia coiii, Enterobacter spp., Salmonella spp., Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) has coincided with an unprecedented global decline in investment in new anti-infective drugs. There are few currently registered alternatives for multidrug resistant ( DR) bacterial infections, forcing clinicians to consider older generation drugs such as cofistin with narrow spectrum and considerable potential for toxic side-effects, in addition, there are fewer novel classes of antiinfective therapeutics moving through the drug development pipeline.

(0003] Since the year 2000, a period of almost 15 years, only 5 novel mode of action (MOA) antibacterial agents have been approved by the US FDA - linezo!id (art oxazolidinone) in 2000, daptomycin (a lipopeptide) in 2003, retapamuiin (a pieuromutilin) in 2007, fidaxomicin (a ma olide tiacumicin) in 2011, and bedaquiline (a diarylquinoline) in 2012. Notably, none of these agents has signficiant activity against gram negative bacteria. Ho novel OA antibacterial agents were approved in 2013 and to date in 2014 only tedizoiid and dalbavancin, both analogs of existing classes, have been recommended for approval in the US. While there are more than 300 anti 'infective medicines in various stages of development, the large majority of these medicines are previously approved antibacterial compounds or their derivatives that are undergoing studies for new indications.

|0004] Furthermore, the prevalence of muliidrug-resistance in animal-specific pathogens together with greater regulation of the registration and usage of antimicrobials in animals, has caused veterinarians to become increasingly reliant on the traditional classes of antimicrobial agents. The risk of transfer of MDR zoonotic organisms from animals to humans has also led to calls for further restrictions on the usage of some recently registered antibacterial drugs such as the fluoroquinolones and the third and fourth generation cephalosporins. Epidemiology of antibacterial resistance development in pathogens of humans and animals

[0005] Much of the evolution in resistance development is driven by changes in (he epidemiology of key MDR organisms. Once oniy restricted to human hospitals and aged care facilities, meihieiliin resistant Staphylococcus aureus (MRSA) strains are now being isolated from the community in alarming proportions. Furthermore, communlty-acquirad MRSA strains are more likely to carry the Panton-Vaientine ieukocidin (PVL) toxin, a viruience fac or linked to skin and soft tissue lesions as well as a rapid., fulminating, necrotizing pneumonia with significant associated mortality. Recently MRSA strains have become host-adapted In several key animal species including livestock, horses and companion animals and regular cases of human-to-animal and animai-to-human transfer are being documented. This has important consequences for strain transmission and public health. A recent survey of 751 Australian veterinarians for MRSA nasal carriage found that a remarkable 21.4% of equine veterinarians were MRSA- ositive compared to 4.9% of small animal veterinarians and 0.9% of veterinarians with little animal contact. These ecological shifts of RSA together with the emergence of resistance to new drugs developed specifically for MRSA such as linezolid, confirm that new MRSA anti-infectives are urgentl needed. Furthermore, hospitals that use vancomycin for treating MRSA then have to contend with outbreaks of vancomycin-resistant enterococci (VRE) infections in their patients, once again with limited alternative antimicrobial choices.

[0006] The global emergence and spread within the community of highly virulent MDR Gram- negative (G-ve) bacteria such as £ coli O25b:ST131 confirms that bacterial pathogens can simultaneously evolve both virulence and resistance determinants. Echoing recent MRSA epidemiology, E cols 02Sb:ST131 , a major cause of urinary tract and bloodstream infections in humans, has now been isolated from extraintestinal infections in companion animals, and poultry. The increasing significance of E. coli O25b:ST131 and other MDR Enierobactertaceae with combined resistance to fluoroquinolones and extended spectrum β-lactams and carbapenems is another worrying trend, especially considering there have been few recent breakthroughs in the development of G-ve spectrum anti-infectives apart from Incremental advances in the carbapenem family.

[0007] The World Health Organisation has Identified antibiotic resistance as one of the three major future threats to global health. A recent report from the US Centers for Disease Control and Prevention (CDC) estimated that in the United States, more than two million people are sickened every year with antibiotic -resistant infections, with at least 23,000 dying as a result.' The extra medical costs, in the USA alone, associated with treating and managing a single case of antibiotic-resistant infection are estimated to be between US$16,688 and US$29,089 per year resulting in an overall direct cost to the US health system of over US$20 billion annually, in addition, the cost to US households in terms of lost productivity is estimated at over US$35 billion per annum. Twenty five thousand patients in the European Union (EU) siili die annually from infection with MDR bacteria despite many EU countries having world's best practice hospital surveillance and infection control strategies, The EU costs from health care expenses and lost productivity associated with MDR infections are estimated to be at least€1.5 billion per year.

(0008] There is an unmet clinical need for antibacterial agents with novel mechanisms of action to supplement and replace currently available antibacterial agents, the efficacy of which is increasingly undermined by antibacterial resistance mechanisms. There additionally remains a need for alternative antibacte ais in the treatment of infection by multi-resistant bacteria. However, as reported by the Infectious Diseases Society of America and the European Centre for Disease Control and Prevention, few new drugs are being developed that offer promising results over existing treatments (Infectious Diseases Society of America 2010, Clinical infectious Diseases, 50{8}:1031-1083).

[0009] it is an object of the present invention to overcome at least one of the failings of the prior art.

[0010] The discussion of the background art set out above ss intended to facilitate art understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

SUMMARY OF INVENTION

[0011] According to one aspect of the invention, there is provided a compound of Formula !, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof:

[0012] in one preferred embodiment, R< is H, cydoaikyi, Formula II, or Formula III;

wherein ;; is H, NH 2 , NHNHj , 0-CH CH 3; NH-C{OVphenyi, NH-chiofophenyi, NH-CH 2 -chlorophen t, NH-N-CH-cycioalkyl, Formula IV, Formula V or Formula vi

wherein Ae is , C, CH, or is C and A« is bonded to & Λ , v/a a, to form a iriazoie ring; wherein A, is N, C, NH, *CH « CH»N-, *(C*H S )C-CH=N- , or Formula Vi!;

Aa is N s C, NH, N-C(0)-phertyi, or Formula VII; wherein A3, A«, A¾, Ae, Ar, As. A«, A¾», Α«, A«, Ai$. Ate . Am A«, At«, Α , A 2 t A ¾I , SA, A**, ;¾ Ϊ and s? are independently C, O, N, NH, S; wherein A, is C, O, N, NH, N-C<OK>-CH r CH 3 , N-C(0)-0-CH{CH s }:;, N-CiO)-NH- CH 2 -CH 3 , -C(0)-NH-CH henyl. N-C<0)-CHrCH CH r CHrCH r CH 3 , N-C(Q)~ CH r furan-2-yl; whereto A 10 is C, NH, -N=CH-CH~ -N~CH~C(CsH s }~ wherein A 22 is -CH<CH 3 )-, -N-CH-, -N-C(CH 3 -C{CH 2 OH)

R 2 is H, COOH, CH 2 NH 2 , CH 2 OH. CH 2 NHNH 2 , methyl, ethyl, propyl, butyl, cyc!opentyl, or Formula VII and R 2 are R are bonded together to form a pyrimidine, pynazine or triazine ring, or R 2 and R s are bonded together to form a pyrroiidinyi oxindoie ring; wherein R 4 is N, NH, O, S, or R A and o are bonded, via R 2l to form a t iazole ring, or R is N and * and R 2 are bonded together to form a pyrimidine ring; wherein Rt is H, CI. Br, F. OH, CH¾ OCH¾. SCH 3 , CM, CCH, CF OCF,, SCF 3 , N0 , butyl, f-butyi, dimethylamino, phenyl, n-propy), /-propyl, -NH-C(0}-CH 3 , - CH-CH-GOOH, piperazin-1-yi, or R ? and F¾ are bonded together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R 6 , R«, R , R , R 2S and R 2? are independently H, OH, Ci. F, Br, CH 3 , CM, OCH 3 , COOH, N0 2( CF 3 , R $ and R> bond together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring, or benzene ring, R 14 and R, $ are bonded together to form a substituted or unsubstituted. saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, R a and R $ , are bonded together to form a substituted or unsubstituted, saturated or unsaturated afiphatic ring, heterocyclic ring or benzene ring, or i* and R-.?, are bonded together to form a substituted or unsubstituted saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R s , R s , R, 7 , R 2 and R 2 * are independently H, O. OH, CI, F, Br, H 2 , CH 3 . CFa. OCRs, CN, NO;, phenyl, - H-CH{OH)-CHs, -NH-C(0}-CH 3 , or R 8 and Rs are bonded together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring, or R, a and are bonded together to form a substituted or unsubstituted saturated or unsaturated aliphatic ring, heterocyclic ring or benzene ring; wherein R< 0 , Rn, R<e. Rao, 22 and R 23 are independenUy H, Ci, or Br, or -.<> and R ? are bonded together to form a substituted or unsubstituted, saturated or unsaturated aliphatic ring, heterocyclic ring or bensene ring, or R i5 and RJ are bonded together to form a substituted or unsubstituted, saturated or unsaturated aiiphatic ring, heterocyclic ring or benzene ring, or 2S and R iz are bonded together to form a substituted or unsubstituted, saturated or unsaturated aiiphatic ring, heterocyclic ri g or benzene ring; wherein R«. R« and R ? ,, are independently H, COON, CHjtNH CH E 0H, methyl, ethyf, propyl, butyi, cyctopentyl, or R 2 and R <3 are bonded together to form a pyfToifdinyi oxindo!e ring; wherein R iS and R 26 are independently H, C!, Br. F, OH. CH ? „ OCH 3 , SCH 2 , CN, CF 3( OCFi, SCFs, N0 2 , CCH. n-butyi f-butyi, dimethylamino, phenyl, rt- ropyi, i- propyi, -NH-C(0)-CH 3 , -CH*CH-COOH, piperazin~1-yi, or R iS and R, are bonded together to form a substituted or unsubstituted, saturated or unsaturated aiiphatic ring, heterocyclic ring or benzene ring; and wherein is a double bond or a single bond.

[0013] The compound of Formula I is preferably a chloride salt.

[0014] In another aspect of the invention, there is provided a compound, or a stereoisomer,automer, pharmaceutically acceptable salt, or prodrug thereof, selected from the list of compounds presented in Figure 1. Where a salt is presented in Figure 1, the invention covers both the salt as presented and the freebase of that sait, and stereoisomers, tautomers. other pharmaceutically acceptable salts, and aiso other prodrugs of the freebase.

[0015j Preferably, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof. wfterein Ao is C; wherein Ai is Hi or Formula VII; wherein Aj is H; or NH; wherein A 0 A*, As, A?, A^, A, 2 , A <s . A¾s, are ; or C; wherein As, A, it A23, ¾ <!( A25, A J6 and A*? are C; wherein As and ;i are S; wherein As is NH; wherein A 10 is N; wrtersin k 22 is -N-CH-; -N-C(CH S K or -N-C(CHaOH)-; wherein R, is H; Formula H: Formuia fli; cycfoaikyl; wherein R s is H; methyl; ethyl; CH 2 NHNH ? ; CH 2 OH; butyl; cyclopaniyi; or Formula VII and f¾ is bonded to 4, to form a pyrimidine ring; wherein 3 is H U ; Formula IV; Formula V; Formula VI; NH S , NR-N-CH- cycioaikyi; or 0-CH 2 -CH 3 ; wherein R 4 is NH; O; S; or R ; is H and R 4 and ; are bonded together to form a pyrimidine ring;; wherein R? is H; F; CI; CF a ; methyl: R? and R a are bonded together to fom an unsubstituted, benzene ring; OH; f-fcuiyi; phenyl; dimeihyiamino; / ' -propyl; n- propyl; CN; CCH; n- utyl; SCH 5 ; R? and R s are Ponded together to form an unsubstituted, unsaturated heterocyclic nng; OCH :s ; 8r; OCF ¾ ; piperazin-1-yl; or SCF 3 ; wherein Rg, R s> R M , and i 8 are independently H; OH; F; OCH 3 ; CF 5 ; methyl; Ci; CN; Br; R B and R ? are bonded together to form an unsubstituted, benze e ring; Rs and R? are bonded together to form an unsubstituted, unsaturated heterocyclic ring; R« and 1S are bonded together to form an unsubstituted, benzene hog; or and R<, are bonded together to form an unsubstituted, unsaturated heterocyclic ring; wherein R*, R 3 , «, and R 17 are independently H; OH; NH 2 ; Ci; F, OCH*; OH; - NH-CH<OH)-CH 3 ; wherein R ½ is H; methyl; ethyl; CH 2 OH; or yciopentyl; wherein H is H; F; Cf; CFj; rnethyi; R? and R s are bonded together to form an unsubstituted, benzene ring; OH: f-butyl; phenyl; dimeihyisrnino; /-propyl; ft- propyl; CN; CCH; n-butyl; SCH 3 ; ?s and 4 ate bonded together to form an unsubstituted, unsaturated heterocyclic ring; OCH-<; Br; OCF 3 ; piperazin-1-yl; or SCF¾; wherein R £4 and R JS are independently H; OH; or CI; wherein f½ and R }? are independently H; or OH; wherein R 3S is H; CH ¾ ; Br; C!; OH; dimethyiamino; -0-P{0){OEt) f .; CF ; «; or F; and wherein "— " is independently a single or a double bond.

[0016] More preferably, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, selected from the group comprising: NCL008: NC1009; NCLG23; NCL025; NCL026; NCL029; CL036; NCU 7; NCL039; NCL040; NCL050; NCL061; NCL084; NCL085: r-iCLOSS; NC1075; NCL076; NCL07S; NCL079; NCL080; CL081; CL084; NCL085; NCL.086; NCL088, NCL089; NCL090; NCL092; NCL09 ; NCL095; NCL097; NCL098; NCL099; NCL101; NCL104: NCL105; NCt.106; NCL108; NCL111 ; NCL112; NCL114; NCL115; CL118; NCL1 18; NCL119; NCL121; NCL122; NCL123; NCL124; NCL125; MCL126: NCL130; NCL131; NCL132; CL133; NCL135; CL136; NCL137;

CL138; NCL139; NCL14G; NCL141 ; CL144; NCL145; NCL.146; NCL147; NCL148; NCL150; NCL152; NCL153; CL154; NCL156; NCL157; NCL1S8; NCL159; NCL1S1; NCL162; NCL184; NCL165; NCL166; NCL187; NCL168; CL169; NCL170; NCL171; NCL172; NCL173; NCL174; NCL176; NCL177; NCL178; NCL179; NCL180; NCL181; NCL183; NCL1S4; NCL185; MCL186; NCL187; NCL188; NCL.189; NCL190; NCL193; NCL1S4; NCL195; NCL198; NCL197; NCL198; NCL1 9; NCL200; NCL201 ; NCL202; NCL203; NCL2Q4; NCL205; NCL206; NCL207; NCL208; NCL209; CL210; NCL211; CL212; NCL213: NCL215; NCL216; NCL217; NCL218; NCL219, NCL220; NCL221 ; NCL222; NCL223; NCL224; NCL225; NCL226; NCL227; NCL228; NCL229; and NCL230.

[0017] Even more preferably, the compound is 3 compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, selected from the group comprising: NCL040; NCL078; NCL079; NCL080; CL081; NCL084; CL088; NCL089; NCL097; NCL099; NCL123; NCL146: NCL157; NCL158; CL177; NCL179; NCL188; NCL193; NCI195; NCL196; NCL197; NCL199; NCL202; NCL204; NCL205: MCL215; NCL216; MCL217; NCL219; and NCL221.

[0018] Even more preferably, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, selected from the group comprising: NCL078; NCL079; CL080; CL081; CL084: MCL089; NCL087; NCL157; NCL158; NCL17S; NCL188; NCL193; NCL195; NCL196; NCL199; HCL204; NCL218; NCL217; NCL219; and NCL221.

[0019] Even more preferably, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, selected from the group comprising: NCL089; NCL097; NCL157; NCL179: NCL188; NCL 133; NCL195; NCL196; NCL216; NCL219; and NCL221. 0020] Most preferably, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, selected from the group comprising: CL097; NCL157; NCL179; CL1S8; NCL19S; and NCL196.

[0021] In one preferred embodiment of the invention, the compound is a compound of Formula S, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein the compound is not a compound selected from the group consisting of: NCL812, NCL001 , NCL002, NCL0O3, NCL004, NCL005, NCL006, NCL007. NCL010, NCL01 1 , NCL012, NCL013, NCL014, NCL01S, CL016, NCL017, NCL018, NCL019, NCL020, NCL021 , NCL022, CL024, NCL027, NCL028, MCL030, NCL031 , NCL032. NCL033, NCL034. NCL035, NCL038, NCL041 , NCL042, NCL043, NCL044, NCL045, NCL046, NCL047, NCL048, NCL049, NCL051 , NCL052, NCLD53, CL054. NCLG55, NCL056, NCL057, NCL058, CL059, NCL060. NCL062, NCL063, NCL066, NCL067, NCL069, NCL070. NCL071, NCL072, NCL073, NCL074, NCL077, NCL082, NCL083, NCL087, NCL091 , NCLG93, NCL098, NCL100, NCL102, NCL103. NCL107, NCL109, NCL110, NCL113, NCL117. NCL120, NCL127, NCL128, NCL129, NCL134, NCL142, NCL143, NCL149, NCL151 , NCL155. NCL16G, NCL163, NCL17S, NCL182, NCL191 , NCL192, and NCL214.

(0022] In a preferred aspect of the invention, the compound of Formula I is not robsnidine (also referenced in ihss specification as NCL812 and also known as 1 ,3-bisf{£)-(4- oiiiorophen 4)methyfeneaminojguanidine), which has a structure as follows:

(0023} in one preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein < is Formula IS; R 3 is Formula IV; At and Ato are N; s and A¾ are NH; c, A 3l A4, A S) A¾, A?, n, A«, At* Au and A: S< are C; R 2 , *¾, Re, R 7 , Re, R«, Rrs, R«. Rie, R17 are H; R* is O; Rg and M are CF 3 ; and K — " in Formula I between Ac, and At, all Formula i! and ali Formula iV "— * are double bonds. An example of a compound of this embodiment of the invention includes:

H A t H X *

i j o K [0024] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, iautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R s is Formula II; R :} is NHY, A, is and A 2 is NH; Ao, A ¾ , A l A s , A ¾ , and A? are C; R ? , Rs. R¾. R? > snd f¾, a e H; R, is NH; , is CI; and "— * in Formula I between A © and A, and all Formula il "··— " are double bonds. An example of a compound of this embodiment of the invention includes:

(0025] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, iautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is f rmula H; R 3 is Formula IV; A ¾ and A 5 , are N; j and * are NH; Ao. Α 3 , A Sl s, Ae, AT, A m A n , A r} , A w and A, s , are C; Rj, Rs, R«, *, R*. R 12 , R«, R«, R«, Rr? are H; R* is NH; Rr and are F; and "— * in Formula ! between Ao and Ai, ait Formula II and all Formula I - * are double bonds. An example of a compound of this embodiment of the invention includes-.

{0026] in another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, iautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R ; is Formula II; R 3 is Formula SV; A. and A 0 are N; Aj and A s are NH; As, As, A*, §, As, Ay, A,,, An, A*¾, Ai4 a d A«, are C; R 2 . R s . R«, R?, R S; ¾l R«, R«, R«, Rtr are H; A is NH; R¾ and « are F; and * — " in Formula I between c- and A<, all Formula IS and ail Formula IV - ** are double bonds. An example of a compound of this embodiment of the invention includes;

[0027] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula If; is Formula IV At and A i0 are N; A 2 and * are NH; Ac, A*. A*, As, A*, A?. An, A n , A n . A u and A l8 , are C; R 2 , R ¾ , R 7 , R Sl R«, R«, i 5! i«. Ri? are H; « is NH; R 9 and ;s are OCH ¾ ; and "— * In Formula I between A¾ and A,, all Formula II and ail Formula IV " are double bonds. An example of a compound of this embodiment of the invention includes: 0028] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomar, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R> is Formula IV; At and Aic are N; A : and A ¾ are NH; Ao, A i( A^ A ¾> Ας. A 7 , A„, An, A n , A* arid Α δ , are C; 2( R s , R e , R 7 , R¾, R 12 , R i3 , R< 5 , Rts, iv are H Rs ie NH; Rs and 54 are OCHj; and "— " in Formuia I between A. c and A-, all Formula II and ail Formula IV are double bonds An example of a compound of this embodiment of the invention includes:

|0029] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R> is Formuia 11; R 3 is NH 2 ; A, is ; A 2 is NH; A ¾ . 5 , A>, Α», A*, and A 7 are C; R 2 . R¾, R§, R¾ and R¾ are H; R* is NH; R? is CI, and "— " in Formula I berwaen Ac, and At, and ail Formula U * -·--" are double bonds. An example of a compound of this embodiment of the invention includes: 0030] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formuia If; R 5 is Formuia IV; t and A^ are ; As and A* are NH; g, s, A > , As. As, A ? , A,,, A,2, A ; ): A 14 and A< s , a e C; R 2 , Rs, Re. R*. Rs, ts, Rt», R M , R ie , R< 7 are H; R< is NH; R ? and Rts are CF^; and * * in Formula I between A<> and Ai, aS! Formuia II and Formula I "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0031] in another preferred embodiment of the invention, the compound is a compound oi Formula !, or a stereoisomer, tauiomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula Si; R ¾ is Formula IV; Ai and A t g are N; A 2 arid A , are NH; Α δί A 3 , A», As, As, A?, At,, A,3. At 3 , A« and A 15 . are C; 2 , R 5 , R«, Re, R*. R«, i ¾ R«, R, 8 , Rir are H; ¾ is NH; Rr and R< 5 are methyl; and * — " in Formula I between A^ and A ? , all Formula li and Formula IV "— * are double bonds. An example of a compound of this embodiment of the invention includes:

|Q032] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tauiomer. pharmaceutically acceptable salt, or prodrug hereof, wherein 5 is Formula Ji; R :J is Formula IV; A ¾ and A< a are M; A 2 and * are NH; Ac, A 3l A*, A*, s, A?, A , A«, A» 3 , A M and At$, are C; R¾, R s , R s , R ? . R*, R«, R«, Ris, Rts, R ? are H: R. is NH; Rs and R« are methyl; and "-··-·" in Formula I between Ao and A«„ all Formula li and Formula IV * * are double bonds. An example of a compound of this embodiment of the invention includes:

(00333 In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula !i; ¾ is Formula IV; A< and A 10 are N; A 2 and As are NH; A ?> A 3 , A 4 , As, A*, A ? , An, Ai2. A< 3 , A M and A 15> are C; ¾, s, Rs, R?, Rs, R«, «, is, i 6 and R ? are H; R is NH; R 3 and 4 are methyl; and "— " in Formula ! between A ¾ and A,., all Formula li and Formula IV K -~~ * are double bonds. An example of a compound of this embodiment of the invention includes:

[0034] In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein j is Formula II; R 3 is NH 2 ; A 1 is ; As is NH; A>, A 3> ,;, A Sl $ , and A ? are C; R ¾ , H%. R $ , Rs, and R s are H; R« is NH: R? is CF 3 : and "— * in Formula I between c end A h and ail Formula li * — " are double bonds. An example of a compound of this embodiment of the invention includes:

[0035] in another preferred embodiment of the invention, the compound is a compound of Formula l : or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R ; is Formula II; R-, is NH 2 ; A, is N; A 2 is NH: Ao, As, As, Α», Ae, and A ? are C; R 2> R Sl Rs, Rr, and R§ are H; R_« is NH; R 8 is CF, 5 ; and "—·-" in Formula i between Ao and A i( and all Formula I! K — '· are double bonds An example of a compound of this embodiment of the invention includes:

[0036] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; R 3 is NH 2 ; A, is N; A 2 is NH; A>, A ;i , A«, As, A<., and A r are C; R 2 , Re, e, Rs, and R s are H; R 4 is NH; R ? is methyl; and * * in Formula i between Ao and A,, and all Formula II "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0037] in another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein i is Formula If; is NH 2 ; A, is N; A 2 is NH; Ao. A 3: A*. As, A*, and A 7 are C; R 2 , ¾ R 8 , y, and R 3 are H; R 4 is NH; R s is CI; and "— * in Formula 1 between As and A¾, and alt Formula II "· - *-" are double bonds. An example of a compound of this embodiment of the invention includes: [0038) in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula il; s is Formula !V; A t and A< 0 are N, A z and A s are NH; Α β , A 3 , As, Ag, A, 5 , A?, A,„ A T2 , A,s, AM and A,$, are C; ¾, Rs, R 6) R 7 . R». R«, R u ., R«, R«, i? are H; * is NH; R.¾ and R H are Ci; and i- — " in Formula I between A ¾ and A t , all Formula II and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes.

£0(339] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, iauiamer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R 3 sx; Formula IV; j and A< 0 are N; * and A 9 are NH; A 0 , A¾. A«, A.;, A«. A?, ki A, 2L Air„ A, a d A 1$l are C; R 2 and R n are methyl; R s , R 3 , R 3; R S( R t3 , R«, R 18 , R 1? are H; R4 is NH; R 7 and R are CFj; and " in Formula ί between AQ and At, all Formula I! and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes.

[0040] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ri is Formula II; Rj is Formula IV; A t and A !(l are N; A ? . and As are NH; s, s, A ? , A 5( At„ A?, A<t, A-.j , A 13r AH a d A 1S , are C; R ? and R< ? . are methyl; R 5> R«, R s , *, R i? , «, R½ a d R ; are H: R* is NH; R ? and R-,¾ are CI; and * K in Formula i between Ao and A t , all Formula IS and Formula IV * " are double bonds. An example of a compound of this embodiment of the invention includes:

[0041] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula II; R 3 is NHNH>; A< is ; As is NH; Ao, A 3 , A*, A§, s, and A-,< are C; a ¾s methyl, R s , R«, R 8 and R§ are H, « is NH; R? is CI; and * — " In Formula I between c and A., and ail Formula Si u * are double bands. An example of a compound of this embodiment of the invention includes:

[0042] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R $ is Formula IV; A< and A i0 are N; A 2 and A s are NH; A ¾ A 3 , A*, o, At.. A 7> A !, A,2, A u , A M and A 1S , are C; R 2> R¾, R«, y, R*. i 2 , R«» Rie, «. an ! ? are H; 4 is S; R s and R« are C!; and "— " in Formula ! between Ac, and A¾, aif Formula II and Formula IV --" are double bonds. An example of a compound of this embodiment of the invention includes:

[0043] In another preferred embodiment of the invention, the compound is a compound of Formula S, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R t is Formula II; R 3 is NH 5 ; A, is N;A 2 is NH; Ao, A3, A 4 . As, Α ¾ , and A ? are€; R ¾ , R«., R§, Rs, and R 9 are H; 4 is NH; R 7 is CI; and "— * in Formula I between A 0 and A , and all Formula II "— " are double bonds. An example of a compound of this embodiment of the invention includes:

(0044} in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula if; ¾ is NHNHj; Ai is N; A ? is NH; Ao, A3, A*, As. A«, and A7 are C; 2 is methyl; R¾, R«, R s , and R s are H; FL is NH; R y is CF 3 ; and "— " in Formula I between Q and A,, arid all Formula II "— * are double bonds. An example of a compound of this embodiment of the invention includes:

\QQA5) in another preferred embodiment of the invention, the compound is a compound of Formula i. or a stereoisomer, tautomer, pharmaceutical acceptable salt, or prodrug thereof., wherein i is Formula H, R 3 is Formula IV; A< and A 10 are N; A 2 and A s are NH; A*, A Si A«, A 5 , ., A 7 , ; :, A,2, An, A, 4 a d A 1Bl are C; R 2 , R s , R e , R», R», R {s ., R !3 . R M , and R, e are H; R» is MH; R ? . R«, and R !? are Ci; and in Formula i between A ;! and A,, all Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0046] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R ? is Formula il: R 5 is Formula IV; A, and Α are N; A? and A s are NH; A?., A A.;, Ag, A$, A 7 , A , A,}, An, A„ and A, s , are C; R¾, R t , Re, e, R*. R«, R«, R«, and R« are H; is NH; R? is CI; R,s is CFj; R 17 is F; and "— " in Formula I between A^ and A ¾ , ail Formula If and Formula SV "— * are double bonti&. An example of a compound of this embodiment of the invention includes CNCL078).

[004?] In another preferred embodiment of the invention, the compound is a compound of Formula l s or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein ¾ is Formula II; R ;s is Formula IV; A, and A !Cl are ; A 2 and A» are NH; Ac, ?, A«, A», A$, A?, A , Ai2, A«, Au and Α«, are C; Rj, R$, R§, R«, R*. R«, R , Ru, is a d R 17 are H; R« is NH; R/ is Ci; Ri S is F; and *— " in Formula ! between Α« and Ai, all Formula I! and Formula IV are double bonds. An example of a compound of this embodiment of the invention includes (NCL079):

{0048] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof. wherein Ri is Formula M; R; is Formula IV; A¾ and Aw are N; A 2 and A¾ am NH; A¾, A¾. A*, As, A< it A ? « A„. A.;;, A !3> Aw and A«. are C; R 2 , R 5 , R«, R«, R», R sa , R ?4 , R-e and R 17 are H; R., is NH; Rr is Ci; 1? is methyl; R< ¾ is CF S ; and * — " in Formula i between A and A|, ali Formula II and Formula IV "-- ·" are double bonds. An example of a compound of this embodiment of the invention includes (NCL080):

[0049] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein i is Formula H; R¾ is Formula IV; At and A« e are N; A 2 and ? are NH; As. A 4 , As, As, A 7 , An, A,*. A, 3 , A M and A«, are C; R 2 , R s , e, R«. Re, R«, «, R<e a d R<? are H; * is NH; R 7 and R it are CI; R« is methyl; and "— " in Formula I A¾ and A t> all Formula il and Formula IV are double bonds. An example of a compound of this embodiment of the invention includes:

{0050} in anothe preferred embodiment of the invention, the compound is a compound of Formula i : or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein i is Formula II; R-, is Formula IV; A, and Aio a e N; A 2 and A¾ are NH; Ac, As, A 4 , A¾, A*, A?, A«, Aw, n, A« and A* 8 , are C; R 2 , R s , e, Re, R*. R«, i¾ Ru, is. a d R 18 are H; R* is NH; R 7 and R« y are Ci; and in Formula I between Ac and A,, ali Formula II and Formula IV —" are double bonds. An example of a compound of this embodiment of the invention includes:

[0051] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula II; s is Formula IV; A¾ and Aio are N; A ? and Α» are NH; A->, As, A*, A 5 , A s , A r , An, A S , At 3 , A M and Ai S , are C; R 2> Rs, Re, s, Rs, i?, Ri*. Ru, and R are H: R 4 is NH; Ryand R <5 are Ci; R« ? is F; and "··-" in Formula I between Ac and A !s all Formula II and Formula IV are double bonds. An example of a compound of this embodiment of the invention includes (NCL08 ):

[0052] in another preferred embodiment of the invention, the compound is a compound of " Formula l : or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ri is Formula Ii; R s is Formula IV; A« and A = a e N; A 3 and A? are NH; A*. Α 5; A 4 , A 5 . A< ; , A 7t A 1 ( Ai2, A,,, AM and A, SL are C; R 2 , R s , R ¾ , R ¾ , R* R R, 3 , R, s , R S , and R t7 are H; is NH; R? is Ci; R., s is CM; and "— " in Formula I between Ao and A<, ail Formula If and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0053] In another preferred embodiment of the invention, the compound is a compound of Formula !, or a stereoisomer, tautomer, phamiaeeutica!ly acceptable salt, or prodrug thereof, wherein R< is Formula ii; R 3 is Formula IV; A, and A<o are N, A 2 and A 9 are NH; A 3 , A,;, *., Ae. A?, An, A-J2, A T3 , A?.: and A«, are C; R 2 , R $ , ¾, R«, R¾, i 2 , R, 3; R w , R-,¾, and R« are H; R» is NH; R 7 is CI; R r ? is F; and * * in Formula I between Ao and A 1 t ali Formula II and Formula IV are double bonds. An example of a compound of this embodiment of the invention includes;

(00541 In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodnjg thereof, wherein R ; is Fomiuia II; R 3 is Formula IV; ^ and 0 are N; A-. and A 8 are NH; A¾, A 3 , A*, s, As, Ar, Att, Au, A 53 , A w end Ate, are C; R≥ and R are methyl; R¾, R e , R g , ^, R W , M, R ¾, and R¾v are H; R.< is NH; R ? ls ; R«5 is CF 3 ; and "— " in Formula I between Ag and A lt ali Formula S! and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes ( CL089):

[0055] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; R ;5 is Formula IV; A, and A I& are N; 2 and A 3 are NH; A A ¾ . A«, A s , Ae, Ay, A (1 l A 12 , A, a , A 14 and A«, are C; R 2 , R 5 , Re, R S: Rs ¾ , i ¾< R«, and R 1? are H; R* is NH; R? and H are bonded together to form an unsu instituted, benzene ring; R« and R 1S a e bonded together to form an unsubstituted, benzene ring: and " in Formula ί between Ao and A<, all Formula II and Formula IV "— * ' are doubles bonds. An example of a compound of this embodiment of the invention includes:

[0056] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein A* is C; A< is N; A 2 is NH; Ri is cyciohexyi; R :} is NH-N=CH-cyeionexyl; is NH; R 2 is H; and a — " in Formula I between Ac. and A*, is a double bond. An example of a compound of this embodiment of the invention includes:

[00573 In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrxsg thereof, wherein R< is Formula II; R 3 is Formula IV; A< and A, 0 are N; A 2 and A B are NH; A<5, A 3 , A*, A 5 , As, A r , , A 2, A , A« and A 1S , are C; R 2 , R 5 , R«, R«, R« and R 1? are H; R* is NH; R 6 , R ?s R¾, R«, Rst. and « are OH; and "— " in Formula I between A and A<, all Formula I! and Formula IV --" are double bonds. An example of a compound of this embodiment of the invention includes <NCL097):

[0058] lit another preferred embodiment of the invention, the compound is a compou d of Formula i, or a stereoisomer, tautonter, pharmaceutically acceptable salt, or prodrug thereof, wherein ; is Formula ίϊ; 5 is Formula IV; Ai and io are N; Aj and Aa are NH; A¾, 3, A*, s- As, A 7 . A^, Ai2, A,3, A 14 and A, s , are C; it Ρ½. R s , R«, R 9( R 2 . Rr > . Rt«. « a d R 17 are H; R 4 is NH; R ? and R< s are f-buty!; and in Formula I between A<; and A,, all Formula il and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

(0069} In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer; tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein R-; is Formula II; R 3 is Formula IV; A, and A< {? are N; ; and A ¾ are NH: A 0? A 3 , As, As, Ae, AT. An, A ¾L A13, A,* and A,¾, are C; Ra, R ? , Re, R», R¾ R«, R«, and R t8 are H; F is NH; Re. Re, Rt*. and R : ? are OH; and "— " in Formula ! between A 0 and A,, all Formula If and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

(0060] In another preferred embodiment of the invention, the compound is a compound of Formula !, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R 3 is Formula IV; A< and Α !0 are N; A s and A 3 are NH; Ao, A ? „ A*, A Si A*, A?, An. A t2 . A,3, A„ and A 1S . are C; Rs. R«, R* R t2> Ru, and R 1? are H; is NH; R*. R ? , R s , Ri3, R15, a d ig are OH; and in Formula I betwee Ao and A<, all Formula ii and Formula IV *— " are double bonds. An example of a compound of this embodiment of the invention includes:

J0061J In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula It; ; is Formula tV; A* and A-,a are N; A 2 and A s are NH, A», As, A*. A 5l A*. Ay. A,-,, A ;2 . A,3, A-,* and are C; R 2 , R s , R s , «, R, 6 , and R« are H; Β Λ is NH; R , R 8 , R e , R 1S> R 14 , and R, 5 are OH; and "-····" in Formula I between A 0 and A 1( all Formula II and Formula

IV " w are double bonds. An example of a compound of this embodiment of the invention includes (NCL097):

[0062] In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, iautomer, pharmaceutically acceptable salt, or prodrug thereof, svherein R< is Formula H; R ; < is Formula IV; A, and Α ; are N; A-. and A s are NH; As, A}, A^ A 5> Ag ; A 7 , it, ?< A< 3 , A and A* s , are C; Rj, R«, R?, s, s, <2, R ( ,¾.- ^, is. and t S are H; R,, is NH; R 5 and R r? are OH; and * — " in Formula t between A $ and A,, ail Formula if and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

fOQ63] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R^ is Formula II; 3 is Formula IV; At and A¾ are N; A 2 and 9 are NH; Ac, Aj, A.., A§, s, A 7 l An, A- ;i! Ais, Au and A«, are C; R 2 , R 5 , R?, &, Rs. R«, 13, R«, R<s. and R i7 are H; * is NH; F¾ and R !t: are OH; and "-··-··" in Formula I between Ao and A 1 t all Formula II and Formula IV *— * are double bonds. An example of a compound of this embodiment of the invention includes:

{0064] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula ti; R 3 is Formula IV; A t and A Ui are N; A 2 and s are NH; A^, A i( A$, A«„ Ag, A , A«, A«, Ai¾ A,,, and A, $ . are C; R z , R 8 , R*. R ¾! R 1∑ , R 3 , Rn, ami R, y are H; 4 Is NH; R ¾ . R 7l Rt« : and R-si are OH; and "— K in Formula i between t and Ai, aii Formula i! and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0065] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R¾ is Formula II; R 3 is Formula IV; A, and A, e are N; A 2 and A ¾ are NH; Ay, A 3 , A*, A s , At, A?, A, ,, A 2 , Ai 3 . A 1 and A«, are C; R 2 . Rs. R*., R ¾ , R>. R«, R«, R«, R s , an R 7 are H; R 4 is NH; R? and R 1S are phenyl; and "— * in Formula i between Ao and A<. ail Formula It and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes:

{0066] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein < is Formula !i; R 3 is Formula IV; A- and <Q are N; A 2 and A s are NH; Ao, A J( A 4 , A*. *, A?, At-, A 1Sl 3 , AM and A<¾. are C; R 2) R s , R 5 , s. s. R«. R«. is, and i 7 are H; * is NH; R r and R, s are dimethylamino; and "— " in Formula i between A ¾ and A<, all Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0067} In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R t is Formula !l; R ¾ is Formula IV; Ai and A 1 are N; A 2 and A 3 are NH; Ac, Aj, *. A s , As, A 7 , An, A,2, A t3 , A 1 and Ai§, are C; R ¾ , R 8 , R g , R», R 12 , R S , R 14 , and R ? are H; R 4 is NH; R e and R 16 are OCH ;i ; R ? and R !& .are OH; and * * in Formula I between Α> and A-„ all Formula II and Formula IV are double bonds. An example of a compound of this embodiment of the invention includes:

[0068] in another preferred embodiment of the inventson. the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula H; R¾ is Formula IV; Ai and A<Q are N; A? and A 3 are NH; % A$. *, A 5> As, Ay, A^, Ai2, A , AW and A, Ci are C; R 2 , Rs, Re, Re, R». «. «, R M , i«, and R 17 are H; R. ; is NH; R 7 and i 5 are /-propyl; and "— " in Fon-rsula I between and A<, all Formula H and Formula IV !< K are double bonds. An example of a compound of this embodiment of the invention includes;

[0069] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R ? is Formula !V; A : and A< 0 are N: A* and A S are NH; o, A 3 , A*, As, A¾ A», A«, Α«, A«, A M and A 14 , are C; R 2 , s, g, R 8> R.j. Rn. R«, RIB, and are H; R« is NH; R ? and R 15 are n-propyi; and "— " in Formula I between A¾ and A ( ail Formula \i and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0070] In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula II; R :i is Formula IV; A< and A 5{1 are N; A 2 and A 3 are NH; g, A;,, A<s, As, Ae, A?, An, A«, A«, A t4 and A S , are C; R 2 , R 5 . R s , R R«, R 13 , R M , and R 17 are H; R* is NH; R«, Rr. Ri5. and R<* are F; and "— " in Formula I between A 0 and A ! t ail Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes: 0071] in another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, iautomer, pharmaceutically acceptable sait, of prodrug thereof, wherein R, is Formula Si; R 3 Is Formuia IV; A ( and A 10 are A ; .. and A 3 are NH; Ao, A 3 . A*, As, A« } A?, AH, An, A«, A M end A 15< are C; f¾, », R$, a, R¾, R , R«, Ru, i«. and ¾ 7 are H; R, is NH; R? and R¾ s are CCH; and "--·-" in Formuia I between ho and i, all Formula li and Formula IV "-— * ' are double bonds. An example of a compound of this embodiment of the invention includes:

[0072] in another preferred embodiment of the invention., the compound is a compound of Formula L or a stereoisomer, iautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; R 3 is Formula IV; A, and A !0 are ; A 2 end A S are NH; Ay, A 5 , A»„ As, A¾, A?, AH, A^, A 1i( H and Ai 8 , are C; j, R$, R?, s, Rs, Rn, i3 ; R«, R¾s, and R 17 are H; 4 is NH; R s and !& are Br. and * — " in Formula i between s and A,, all Formula M and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0073] In another preferred embodiment of the invention, the compound is a compound of Formuia I, or a stereoisomer, tauiomer, pharmaceutically acceptable sait. or prodrug thereof, wherein R< is Formuia ii; R 3 is Formula IV; A. and A v3 are N; Aj and A§ are NH; A-,, A S> A 4) A S , Ac, A?, A*,, A 2 , A t3 , A 14 and A iS( are C; R 2 , R s , R 6) Rg, R>, R !2 , R t3< R > , R ie . and R, r are M; R, is NH; R 7 and R S are butyl; and * — " in Formuia i between Ag and At, all Formula li and Formuia iV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0074] in another preferred embodiment of the invention, the co pound is a compound of Formuia 1, or a stereoisomer, tautomer, pharmaceutically acceptable saH, or prodrug thereof, wherein R< is Formula H; R s is Formula IV, Ai is -C(Ct.H 5 )-CH~N- and A-. a is -N^CH-Ct'CeiHs)-; Ai and A s are NH; Ao, A¾, A 4 , As, Ag, A ?t A*,, A i3> A«, ,.< and A«, are C; 2i ¾, R¾ R?, R$, R 9 , Ri2, ;¾, -,*. «.s ; Ris, a -,7 ere H; R,, is NH; and "— " in Formula I between end A¾, ail Formula ii and Formuia IV "— " are double bonds. An example of a compound of this embodiment of the invention irrciudes:

(0075] in another preferred embodiment of the invention, the compound is a compound of Formuia I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein i is Formuia ii: s is Formula IV; A¾ and A« are ; A 5 and A§ are NH; Ac, A3.. A*.. As, Ai., AT, An, A n , A<3, « and A 1;<( are C; R Sl Rs, e, R¾. R$. R«, R1.1, R M , Ris, and R, ? are H; 4 is NH; R ? and R s are CH ? S; and "— " in Formuia i between A¾ and Ai, aff Formuia Ii and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes:

[0075] in another preferred embodiment of the invention, the compound is a compound of Formuia !, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula Hi; R s is Formula VI; A <5 is C: R ? and R 2i are H; A^ and A 20 are ; A z and 1 S are NH; ^ and A 21 are S; R« is NH; R !( i and are bonded together to form a substituted benzene ring; f3 and R ?3 are bonded together to form a substituted benzene ring; and "— " in Formuia ! between Ao and A-,, and ail Formuia HI and Formula VI * * are double bonds. An example of a compound of this embodiment of the invention includes:

[0077j In another preferred embodiment of the invention, the compound is a compound of Formula \, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R s is Formula if; R> is Formula IV; A, and Ai Q are ; A 3 and A s are NH; Ao, A¾, A*, A s , As, A?, A.-„ A ; , A< 3 , A,* and A,*, are C; R ? and R< « - are methyl; ¾ R s , R?, R s , R>, R«, Rt , i*« ! S) and R ? are H; R 4 is NH; and "— " in Formula I between Ao and Ai, all Formula II and Formula iV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0078) In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula il; R 3 is Formula IV; < and A t e are ; * and A* are NH; o, A s , A . As. s, Ar, An. Ai2, A Wl A t and A< 5 , are C; R 2 . R 5 , R«, Rs, R«, Ris, R i8 . and R 1T are H; is NH; R 7 and Rs are bonded together to form an unsubstttuted, heterocyclic ring; R ;4 and R 1S are bonded together to form an unsubstituted, unsaturated heterocyclic ring; and * — " in Formula I between Ao and A<, ail Formula II and Formula I K — " are double bonds. An example of a compound of this embodiment of the invention includes:

{0079} In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ri is Formula il; ¾ is Formula IV; ^ is -CH-CH=N- and A, s is-N~(CH} ; * and A¾ are NH; Ac, Aa, A*, A s> As, A?, A v „ , A w , A w and A, s> are C; R 2< Rs, s, s, R», R<2, «, RM > Rie, and , Y are H; ¾ is NH; R ? and R, 0 are OCH 3 ; and K — " in Formula I between o and A<, all Formula il and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes:

{0080} In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Rj i≤ Formula ίί; R 5 is Formula IV; A« and A ie s»"<s ; j and A s are NH; Ao, A 3l Α·,, A ¾l fc, A7.An, A ?, Ai), Aw and A 4 , are C; R 2 , R s< R«, R«, R», R«, R 13 , R«. R^. and R 17 are H; R, is NH; R T and Ri 5 are OH: and — " in Formula I between Ac and A ail Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0081] In another preferred embodiment of the invention, the compound is a compound of Formula I. or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R ; is Formula !i; R 3 is Formula IV; A, and A ?0 are N; A 2 and A 3 are NH: A A 3 , A*, Ag, As, A 7 , An, A f2 , A j3 , Α and A« s , are C; R 2 and R, y . are ethyl; R s , R 5 , R s< R s , «, R t4 , R«, and ,7 are H; R< is NH; R 7 and R 5 are CI; and 4 — " in Formula i between A* and A t> all Formula II and Formula IV * — " are double bonds. An example of a compound of this embodiment of the invention includes:

[0082] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ri is Formula Si; R 3 is Formula IV; A 1 and A K> are N; A 2 and A s are NH; Ac, A¾, *, A 5> Ae, A ? , A-t : Ai 2 , A«, Aw and A, Sl are C; Rj and R ½ are methyl; R«, R§, e, R», R !5 , R M , R ? «, and R,r af« H; R« is NH; R 7 and R< are Br; and "— " in Formula I between o and Aj, all Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes:

[0083} in another preferred embodiment of the invention, t e compound is a compound o? Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein < is Formula SI; R 3 is Formula IV; A< and An are ; A 2 and A ¾ are NH; Ay, A3, A<, As, A*, A;, A<<, Ats, A , A (4 and A, s , are C; R 2 , R s . R«, R«. R«, R„, , e , and R ; r are H; R. is NH; R 7 and R, s are Ci; R 3 and R 3 are NH ? .; and :i — " in Formula ! between A 0 and A,, all Formula II and Formula IV ,: — " are double bonds. An example of a compound of this embodiment of the invention includes (NCL167):

[QQ84] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R is Formula i!; R ;i is Formula IV; A< and A 50 are N; A s and A 3 are NH; A>, A ¾ , A^, As, As, A?, An, A, 2 , A . A« and A !S , are C; R 2 and R« are eihyi; R 5 and R.. r are OH; g, e, R s , n, R H , and Rn are K, R 4 is NH; R? and R< 5 are Ci; and * — " in Formula i between A3 and A : , ail Formula II and Formula IV "— " are double bonds. An example of a compound of this embodiment of the invention includes (NCL158):

10085] In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; is Formula IV; A ; and Α* δ are N; A 2 and A ¾ are NH; Ao, A ;; , A*, A s , g, A r , An, tS; n, 4 and A 15 . are C; R 2 and are cyciopentyl; R 5 and R iy are OH; R*, R 3l Rs, 13. Ri . and R, $ are H; * is NH; R 7 and R 1S are Ci; and "— " in Formula I between A e and A<, all Formula IE and Formula IV "— " are double bonds An example of a compound of this embodiment of the invention includes-.

[0086] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein , is Formula if; R.« is Formula IV; A; and A< 0 are ; A ? and A¾ are NH; A ¾ « A>, 4, A$, A s , A 7 , A„, A i3> A, a , A,^ and A«, are C; R ¾ , R $ , R 6 , R*. R¾. R 12 , R, 3 , R», R and R 17 are H; R» is NH; R 7 and R 1S are OCF 3 ; and * — " in Formula I between Ac and A ¾ , ati Formula II and Formula IV '— are double bonds. Ars example of a compound of ibis embodiment of the invention includes:

[00S7] in another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable sail, or prodrug thereof, wherein R, is Formula II; R 3 is Formula IV; A< and 10 are N; A } and A 9 are NH; Ae, As, A4, A$, As, A ? . AH, A , A i . A« and A< s , are C; R :: and < 2 are methyl; R s> R>, R«, R St «, R^, Ri¾ and R,r are H: « is NH; R and R,¾ are piperazin-1-yl; and "· -·-" ' in Formula i between Α δ and A<, all Formula H and Formula IV K are double bonds. An example of a compound of this embodiment of the invention includes:

[0088] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; > is 0-CH 2 -CH 3 ; A, is N: A 2 is NH; Ac, A 3 , As As, A* and A ? ars C; R 2 is methyl; R... R 5> R s , and R a are H; R« is NH; R 7 is C!; and !! — " in Formula I between A ¾ and Α·.. and all Formula II "-··-- * are double bonds. An example of a compound of this embodiment of the invention includes;

[0089] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R ; is Formula H; R 3 is Formula IV; Ai and <s are N; A 2 and A s are NH; £<>.. As.. 4, A5, As, Ar, A rtl An, A«, A H and A«, are C; R?, R 5 , R e> R s , R». R< 2 , Ri¾ Rtx, R?e a d R, 7 ars H; ¾ is NH; R 7 and R iS are SCF 3 ; and "— * in Formula I between Ac. and A< ; all Formula II and Formula IV "·— " are double bonds. An example of a compound of this embodiment of the invention includes:

{0090] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable sait, or prodrug thereof, wherein , is Formula if; R$ is Formula IV; A, and A s;i are N; A; and A$ are NH; Ao, A-¾, A*, A S( As, A 7 , A„, A , A, 3f A w and A 1S , are C; R 2< R«, R s , R 8l R, a , R«, R«, and R,« are H; R, is NH; R r and R-s are CI; ¾ and are -NH-CH(OH -CH s ; arid · ' '-—" in Formula I between Ao and A,, all Formula It and Formula IV ' — " are double bonds. An example of a compound of this embodiment of the invention includes:

(0091) In another preferred embodiment of the invention., the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodaig thereof, wherein A 3 is C. Ri is H; A 2 and R« are N; R s is MHj; Ai is Formula VII; R 2 is Formula VII and R 2 is bonded to R, (l forming a pyrimidine ring; "— " in Formula ί between s and Ao, and between A-, and A 2 are double bonds; A is -N-CH-; R 2 4, Ras, Rzr and ^ are H; A ¾ , A 2 r ϊδ! Ass and A ? .? are C; and ? e is Ci. An example of a compound of this embodiment of the invention includes {NCL179);

[0092] in another preferred embodiment of the invention, the compound is a compound of Formula f, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R, is Formula II; ¾ is NH 2 ; A; is N; A ? and F are NH; Ac, A3, A*, A 5 , At, and Ay are C; Rs, Re. Rs. and 3 are H; Rj is butyl; R? is C!; and "— * in Formula ί between * and A,, and ail Formula !i "— " are double bonds. An example of a compound of t is embodiment of the invention includes {NCL188}-.

[0093] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein A ¾ is C; R< is H; As and R$ are N; R ;i is NH 2 ; A ; is Formula VII; R ? is Formula VII and 2 is bonded to forming a pyrimidine ring; "— * in Formula I between R 2 and A 0l and between As and A 2 are double bonds; A 22 is -N-CH-; R 2 *, R 2 - and K; are H; A 23t 2 .*, A 2 « and A;.'? are C; and F½ is CH 3 . An example of a compound of this embodiment of the invention includes {NCL1S5}:

|0094] in another preferred embodiment of the invention, the compound is a compound of Formula ! ; or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ao is C; R< is H; A 2 and R* are N; R ;5 is H 2 ; , is Formula VII; R 2 is Formula VII and R 2 is bonded to R*, forming a pyrimidine ring; * — " in Formula I between R 2 and Ag, and between i and A? are double bonds; r is -N-CH-; R J(il Rj¾, R 2 ? and R¾ are H; A23, As*, A 2S , A¾ and A 2 ; are C; and ^ is OH. An example of a compound of this embodiment of the Invention includes (NCL186): 0095] In another preferred embodiment of the invention, the compound is a compound of Formula i, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ao is C; R< is H; A 2 and R4 are N; R 2 is NH 2 ; A, is Formula VU; R 2 is Formula VII and £ is bonded to R ii; forming a pyrimidine ring; "— " in Formula between R 2 and A ¾ and between A t and A 2 are double bonds; A 22 is -N-CH-; R 24 , R 2St R 2 ? and R il5 are H; K , A 34 , A i5l A 33 and 2 ? are C; arid R is Br. An example of a compound of this embodiment of the invention includes {NCL193):

£0096} In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein Ao is C; i is H, A 2 and R« are H; R 3 is NH 2 ; A, is Formula Vii; R 2 is Formula VI! and R : is bonded to ,, forming a pyrimidine ring; : '— " in Formula I between R 2 and Ao, and between A< and A 2 are double bonds; A^ is -N-CH-; R 24 , R 5 «., R 2Sl R 2? and R 23 are H; and A 23 . A 24> A 2til A sg and A ? ? are C. An example of a compound of this embodiment of the Invention includes (NCL199):

(0097} In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer. pharmaceutically acceptable salt, or prodrug thereof, wherein Ao is C; i is H; A 2 and R< are N; Rj is NH ? ; A is Formula VII; j is Formula VI! and R? Is bonded to .i, forming a pyrimidine ring; "— :< in Formula I between R 2 and A 0 , and between At and A 2 are double bonds; A 22 is N-C(CH$K Rw. Rzr and R 2a are H; A 23 , A i;i , A : .* ( A i5 and Aj7 are C; and R 26 is CI. An example of a compound of this embodiment of the invention includes (NCL204):

(0 98| In another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein R< is Formula li; R ¾ is Formula IV; A, and A 1C , are ; A 2 arid A* are NH; Ay. A;, A 4 , A s , A¾, A 7 , A,,, At2, A«, Ai4 and A ?S , are C; 2l R s , e, R», R«. R , R M , and R,<. are H; R« is NH; R r and R, s are Ci; R«. and R< ? are F; and '— " in Formula I between A c . and A¾, ail Formula II and Formula IV "— 11 are double bonds. An example of a compound of mis embodiment of the invention includes (NCL216).

[0099] in another preferred embodiment of the invention, the compound is a compound of Formula !, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein < is Formula II; R 3 is Formula IV; A< and A- l0 are N; A 2 and A ¾ are NH; AQ, AJ, A*, As, A<., AT, A«, A 12 , Ai3, A s< and A»s, are- C; R* and R 2 are methyl; R s , R s , R 8 , R s , R 13 , R 14 , t e and R 17 are H; R is NH; R? and R iS are CH ; and in Formula I between o and A !t ail Formula II and Formula IV * — " are double bonds. An example of a compound of this embodiment of the Invention includes (NCL217):

[00 00] in another preferred embodiment of the invention, the compound is a compound of Fon-nula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein is Formula II; 3 is Formula IV; A< and m are N; 2 and A¾ are NH; A>. A ¾ , A*. A$, As, A 7> H, A , Α ϊ5ί A u and A , are C; R¾ and ¾ are methyl; R St s, ¾ R< ; „ « t ¾ and R» are H; R is NH; R y and R it . are f-buty!; and "— " in Formula I between Ao and A«, all Formula It and Formula IV "— * are double bonds. An example of a compound of this embodiment of the invention includes (NCL219):

[00101] in another preferred embodiment of the invention, the compound is a compound of Formula I, or a stereoisomer, tautomer, pharmaceutically acceptable salt, or prodrug thereof, wherein As is C: Rt is H; A 2 and * are N; s is H ; A< is Formula V'H; R E is Formula VII and R £ is bonded to R*, forming a pyrimidine ring; · " in Formula I etween R ; , ; and Ao, and between

A, and A 2 are double bonds; A 22 is -N-CH-; R 2 <, R&, 2y and R are H; Α ≥δ , A ¾ , A 2 =, A ¾ ? and A 2 r are C; and f½ is CF 3 . An example of a compound of this embodiment of the invention includes {NCL221):

(00102} According to another aspect of the invention, there is provided a method of treating or preventing a bacterial colonisation or infection in a subject, the method comprising the step of administering a therapeutically effective amount of a compound of Formula S, or a therapeutically acceptable salt thereof, to the subject. In this aspect, the bacterial infection is caused by a bacteria! agent. The method of treating or preventing a bacteria! infection or colonisation in a subject, may also comprise the administration of the pharmaceutical or veterinary compositions of the invention.

[00103] According to a further aspect of the invention, there is provided the use of a compound of Formula I, or a therapeutically acceptable salt thereof, in the manufacture of medicament the treatment of a bacterial colonisation or infection in a subject. In this aspect, the bacterial infection is caused by a bacterial agent.

{00104} The subject may be any subject capable of colonisation and infection by bacteria. The subject may be mammalian, or may be piscine or avian. Preferably, the subject is selected from the group comprising, but not limited to, human, canine, feline, bovine, ovine, caprine, other ruminant species, porcine, equine, avian, or piscine.

(00105) The compound of Formula i may be administered to the subject in a dose selected from the group comprising 0.1 mg/kg to 250 mg/kg body weight, preferably 1 mg kg to 100 mg/kg body weight, and more preferably 5 mg/kg to 50 mg/kg body weight. The compound of Formula I may be administered to the subject using a dosing schedule selected from the group consisting of: hourly, 3 times daily; twice daily; daily; every second day; twice weekly; once weekly; once fortnightly; once monthly; once ever two months or by constant rate or variable rate infusion. Preferably, the compound of Formula I is administered until colonisation or the signs and symptoms of infection or colonisation have at least been partially treated or alleviated. [00106] In one embodiment., the concentration of compound of Formula \ (or a metabolite) in the subject's blood after treatment is within a range selected from the group comprising, but not limited to: between 0.1 and 10 ug/mL at 2 hours, 1 and 200 ug/mL. after 12 hours; between 0.1 and 5 ug/mL after 24 h: between 0.01 and 2 ug/mL after 48 hours; between 0.0001 and 1 ug/mL after 72 h. Preferably, the concentration is selected from the group comprising, but not limited to: less than 200 ug/mL after 12 hours; less than 5 ug/mL after 24 hours; less than 1 ug/L after 48 hours and less than 0.5 ug/mL after 72 hours.

[00107} The agent causing the bacterial infection is a bacterial agent, in one preferred embodiment, the agent is not a protozoan species. In one preferred embodiment, the agent is not a coccidsan protozoan. More preferably, the agent is not Clostridium perfring s nor a heterotrophic bacterial species present in soil samples collected by Hansen et al from Jyndevad Denmark as discussed in the following papers: Hansen ef a/. 2012, Chamosphere. 86:212-215; and Hansen et al. 2009, Environmental Pollution 57:474-480.

[00108] In another embodiment, the bacterial agent is gram negative, in another embodiment, the bacterial agent is gram positive, in another embodiment, the bacterial agent has no ceil wall, in another embodiment, the bacterial infection is caused by a mixture of at least two agents selected from the group consisting of; gram negative, gram positive and bacterial agents with no ceil wall.

[00109] The bacterial agent causing the bacterial infection may be a gram positive bacterial agent selected from the group comprising, but not limited to, Staphylococcus spp, Streptococci, nterococcus spp, Leuconostoc spp. Coryn0t>soterium spp, Arcanobacteria spp, Trueperelia spp, Rhodococcus spp, Bacillus spp, Anaerobic Cocci, Anaerobic Gram-Positive Nonsporulaiing Bacilli, Actinomyces spp, Clostridium spp, Nocardia spp, rysipelothnx spp. Listeria spp, Kytococcus spp, Mycoplasma spp, Ur&apfasma spp, and Mycobacterium spp.

[00110] In one embodiment, the bacterial agent is gram positive and is selected from the group comprising, but not limited to, Staphylococcus spp. Examples of Staphylococcus spp include Staphylococcus epider idis, Staphylococcus haemolyticus, Staphylococcus lugdunansis, Staphylococcus saprophytics, Staphylococcus auricu!arls, Staphylococcus capitis, Staphylococcus caprae. Staphylococcus camosus, Staphylococcus coh ii, Staphylococcus hominis. Staphylococcus pesteuri, Staphylococcus pe enkof&ri, Staphylococcus pulvemri, Staphylococcus sacchatoiyticus, Staphylococcus simufans. Staphylococcus schleifeti Staphylococcus wameri. Staphylococcus xylosus, Staphylococcus ariaitae, Staphylococcus casQo!yticus, Staphylococcus chro og n&s, Staphylococcus condiments, Staphylococcus de!phini, Staphylococcus ^quorum, Staphylococcus feiis, Staphylococcus flaurettii, Staphylococcus gallinarum, Staphylococcus hyicm. Staphylococcus intermedius, Staphylococcus kioosii, Staphylococcus ientus, Staphylococcus iu ae, Staphylococcus uscae, Staphylococcus n&palensis, Staphylococcus ptscifermentans, Staphylococcus pseudinterrnedius, Staphylococcus sciuh, Staphylococcus simiae, Staphylococcus succinus, and Staphylococcus vitulinus.

[00111} in another embodiment, the bacteria! agent is gram positive and is selects*! from the group comprising, but not limited to.. Streptococcus spp. Examples of Streptococcus spp include Streptococcus agalactiae, Streptococcus alactoiyticus, Streptococcus anginosos, Streptococcus canis, Streptococcus constellates,. Streptococcus chcetus, Streptococcus chstaius , Streptococcus downei Streptococcus dysgatactiae subsp. dysgalactiae, Streptococcus dysgalactiae subsp. equisi ilis.. Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepide icus, Streptococcus ferus. Streptococcus gallolyticus subsp. gallolyticus (formerly Streptococcus bovis biotype i), Streptococcus gallolyticus subsp. pasteurianus (formerly Streptococcus bovis biotype ii/2), Streptococcus gordonii, Streptococcus hyointesfinaiis. Streptococcus hyovaginalis, Streptococcus infantarius. Streptococcus infantarius subsp infantarius, Straptowccus infantis, Streptococcus iniae. Streptococcus intermedius, Str&ptococcus luietiensis (formedy Streptococcus bovis biotype Streptococcus macaccae. Streptococcus mstis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis. Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus. Streptococcus pseudintermedius, Streptococcus pyogenes, Streptococcus rath, Streptococcus saiivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus ihermophilus, Streptococcus vestibularis, and Nutritionally Variant (Deficient) Streptococci (Abiairophia defective, Granulicateila adiacens, Granulicateila elegans, and Granulicateila para-adiacens) and related species such as Rothia muci!aginosa (formerly Sto atococcus mucilaginosus ) and Pediococcus.

[00112] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Enterococcus sp Examples of Enterococcus spp include Enterococcus faecalis, Enterococcus faeciu , Enterococcus gallinanim, Enterococcus durans, Enterococcus avium, Enterococcus raffinosus, Enterococcus pallens, Enterococcus gilvus, Enterococcus cecorurn, Enterococcus malodoraius, EntetOcocc italicus, Enterococcus sanguinico!a, Enterococcus mundtii, Enterococcus casse!ifiavus/ffavescens, Entefococcus dispar, Enterococcus hiraa, Enterococcus pseudoavium, and Enterococcus bovis.

[00 13] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Leuconostoc spp. Examples of Leuconostoc spp include Leuconostoc esenteroides, Leuconostoc pseudomesenteroides, Leuconostoc paramesenieroides, Leuconostoc citreum, and Leuconostoc lactis. [00114] in another embodiment, the bacteria! agent is gram positive and selected from the group comprising, but not limited to, Corynebacterium spp. Examples of Corynebacterium spp include noniipophilic, fermentative Corynebacterium spp such as Corynebacterium ulc&rans. Coiynebacierium pseudotuberculosis. Corynebacterium xerosis, Corynebacierium striatum, Corynebactenum minutissimum, Corynebacterium amycolatum, Corynebact&rium glucurono!yticu , Corynebacterium argentoraiense, Corynebacterium matrvchotii, Corynebacterium riegeiii, Corynebactenum confusum, Corynebacterium cystkHs, Corynabacterium diphtheria Corynebacterium simu!ans, Corynebacterium sundvaliansa, Corynebacierium ihomssensii, Corynebacterium freneyi, and Corynebacierium aurimucosum, noniipophilic, nonfef entative Corynebacterium spp such as Corynebacterium afennentans afermentans, Corynebaciatium auris, Corynebacterium pseudodiphtheriticu x and Corynebacterium propinquum end lipophilic Corynebacterium spp such as Corynebacterium jeikeium, Corynebacterium u ealyticum, Corynebactenum afennentans Rpophilum, Corynebacienum accolens, Corynebacterium macginiayi, Corynebacterium tubercutosiearum, Corynebact&rium kroppenstadtsi, Corynebacterium kutscheri, Corynebacterium pi!osu , Corynebacterium bovis, CDC coryneform groups F-1 and G, and Corynebacterium fipophilofiavum, and other Corynebacterium spp such as Turicalla, Arthrobaeter, Brevibacterium, Dermabacter, Rothia, Oerskovia, Microbact&rium, and Leifsonia aquatica.

[00115] In another embodiment, the bacteria! agent is gram positive and selected from the group composing, but not limited to, Arcanobacteria spp. Exam les of Arcanobacteria spp include A haemolyticu , A. pyogenes (now known as Trueperelia pyogenes, originally known as Actinomyces pyogenes), and A, bemardiae. 00116] in another embodiment, the bacteria? agent is gram positive and selected from the group comprising, but not limited to, Rhodococcus spp. Examples of Rhodococcus spp include Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus fasciens, and Rhodococcus rhodochrous. 00117} in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Gordonia spp.

[00118] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Tsukamureiia spp.

[00119] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Achofaplasma spp.

[00120] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Actinobacteria such as Crossiei!a equi. [00121] In another embodiment, the bacteria! agent is gram positive ¾nd selected from the group comprising, but not limited to, Bacillus spp. Examples of 8ae///u$ spp inciude Bacillus anthracis, Bacillus cereus. Bacillus circulans, Bacillus licheniforrnis, Bacillus egai&rium, Bacillus pumiius, Bacillus sphaaricus, Bacillus subtilis, Br&vibacil!us brevis, Brevibaci!lus laterosporus, and Paenibacil!us alvei.

[00122] In another embodiment, the bacteria! agent is gram positive and selected from the group comprising, but not iimited to, Anaerobic Cocci. Examples of Anaerobic Cocci inciude Anaatococcus murdochii, Anaetococcus prevail!, Anaerococcus tetradius, Anaerococcus octavtus, Anaerococcus hydrOgenalis, Anaerococcus lactolyticus, Anaerococcus vaginalis, Atopobium parvulu , Finagoidia magna, Galiicoia bamesae, Ge ella asaccharolytica, Gemalla hergari, Gemalla cuniculi, Gemalla haamolysans, Gemalla orbi!lorum, Gambia paiaticanis, Ga ella sanguinis, Parvimonas icra, Peptococcus niger, Peptoniphilus asacc arolyticus, Peptoniphilus gorbachii, Peptoniphilus indolicus, Peptoniphilus arai, Peptoniphilus t ' vorii, Paptoniphilus lacrimalis, Peptoniphilus olsenii, Peptostreptococcus sfomatis. Peptostrepiococcus anaerobius, Ruminococcus producius. Slackia heliotrinireducens, and Staphylococcus saccharotyiious. 00123] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not !imited to, Anaerobic Gram-Positive Nonspoat!ating Bacilli. Examples of Anaerobic Gram-Positive Nonsporulating Baciiii inciude AlSoscardovia omnicolens, Atopobium species (such as Atopobium minutum, Atopobium hmae, Atopobium parvuiu , and Atopobium vaginae), Bifidobacteria (such as Bifidobacteria adolescantis, Bifidobacteria d&ntium, Bifidobacteria scardovii), Catabacter hongkongensis, CollinseHa aarcfaciens, Eggerthei!a (such as Eggert afia lenta, Eggarthella hongkongensis and Eggerthella sinensis), Eubacierium and related species (such as Eubacterium nodatum, Eubacterium fanue. Eubacterium brac y, Eubacterium infirmu , Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium sulci, Fiiifactor alocis, Mogibacterium iimidum, Mogibacterium vescum, Pseudoramibacter alactolyticus, Buileidia extmcta, and Solobacterium oorai), Lactobacillus species (such as Lactobacillus rha nosus, Lactobacillus case/ ' , i ctobacillus far entum, Lactobacillus gasseri, Lactobacillus planiarum, Lactobacillus acidophilus, Lactobacillus inars and Lactobacillus ultunensis), Mobiiuncus species (such as Mobiiuncus curtisii, Mobiiuncus mulieris}, Moryella indoligenas, Olsaneila oral species (such as Qisenella uli and Qisene!ia profuse), Oribaciariu sinus, Propionibacterium (such as Propionibacterium acn&s and Propionibacterium pfopionicum), Slackia axigua, and Turicibactar sanguine.

[00124] in another embodiment, the bacteiai agent is gram positive and selected from the group comprising, but not limited to, Actinomyces spp. Examples of Actinomyces spp inciude Actinomyces israelii, Actinomyces naaslundii, Actinomyces vtecosus, Actinomyces odontoiyticus, Actinomyces rneyeri, and Actinomyces gerencseriae (formerly Actinomyces israelii serotype II), Actinomyces europaeus, Actinomyces neuii, Actinomyces radingae, Actinomyces gmevenitzii, Actinomyces hordeovulneris, Actinomyces turice sis, Actinomyces georgiaa, Arcanobacterium (Actinomyces) pyogenes, Arcanobacterium {Actinomyces} bemardiae, Actinomyces funkei, Actinomyces iingnae, Actinomyces oustonensis, and Actinomyces cardiffensis.

100125} in another embodiment, t ' ns bacterial agent is gram positive and selected from the group comprising, but not limited to, Clostridium spp. Examples of Clostridium spp include Clostridium baraiii, Clostridium bifermentans, Clostridium boiuimum, Clostridium botulinu (types A, 8, C, D, E, F, G), Clostridium butyiicum, Clostridium difficile, Clostridium histolyticum, Clostridium novyi (type A), Clostridium novyi (type 8), Closthdium perfringens, Clostridium perfringens (types A-E}, Clostridium ramosum, Clostridium septicum, Clostridium sordeiti, Clostridium sph noides, Clostridium tedium, and Clostiidium tetani.

[00126} in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Nocardia spp. Examples of Nocardia spp include Nocardia asteroides. Nocardia brasiliensis, Nocardia farcinica, Nocardia nova, Nocardia otHidiscaviaru , and Nocardia transva!ensis. 00127] in another embodiment, the bacteria! agent is gram positive and selected from the group comprising, but not limited to, Etysipelot rix spp, such as Erysipelothrix riiusiopat iae

(00 28] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Listeria spp, such as Listeria monocytogenes.

(00128] in another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Kytococcus spp. such as Kytococcus schroetari.

[00130] In another embodiment, the bacteria! agent is gram positive and selected from the group comprising, but not limited to, Mycooacfenum spp. Examples of Mycobacterium spp include Mycobacterium abscessus, Mycobacterium arupanse, Mycobacterium asiaticum, Mycobacterium subag ense, Mycobacterium avium complex, Mycobacterium boiietii, Mycobacterium boiietii, Mycobacterium branderi, Mycobacterium caneUH, Mycobacterium caprae, Mycobacterium ceSatum, Mycobacterium c eionae, Mycobacterium chimaera, Mycobacterium colo biense, Mycobacterium concepiion&nse, Mycobacterium conspicuum, Mycobacterium ataphantis, Mycobacterium farcinoganes, Mycobacterium fioreniinum, Mycobacterium fotiuitum group, Mycobacterium ganavense, Mycobacterium goodii, Mycobacterium h&emophi!um, Mycobacterium heckeshomense, Mycobacterium heidelbergensa, Mycobacterium houstonense, Mycobacterium immumgenum, Mycobacterium inten ' ectum, Mycobacterium intracellular®.. Mycobacterium senegalens , Mycobacterium africanu , Mycobacterium avium subsp paratubercuiosis, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium ieniiflavum, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium mageritense, Mycobacterium maimoense, Mycobacterium marinum, Mycobacterium t ssiliense, Mycobacterium microti, Mycobacterium montefiorense (eels), Mycobacterium moracense, Mycobacterium mucogenicum, Mycobact rium nebraskense, Mycobacterium neoauru , Mycobacterium novocastrense, Mycobacterium paiustre, Mycobacterium pannense, Mycobacterium ph!ei, Mycobacterium phocaicu , Mycobacterium pinnipedii, Mycobacterium p rcinum, Mycobacterium pseudoshottsii (fish), Mycobacterium pseudotuberculosis, Mycobacterium saskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senuense, Mycobacterium septicu , Mycobacterium simiae, Mycobacterium smegmatis, Mycobacterium szulgai, Mycobacterium tarrae/ohromogenicum complex, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium tusciae, Mycobacterium u!ce ns, Mycobacterium woiinskyi, and Mycobacterium xenopi.

[00131] in another embodiment, the bacteria! agent is gram positive and selected from the group comprising, but not limited to, Trueperatia spp. Examples of Truepereiia spp include Truepereiia abortisuis, Truepereiia be nardiae. Truepereiia tiaiowiezensis, Truepereiia bonasi, Tivepereila pyogenes (Arcanobacteriu pyogenes).

[00132] in another embodiment, the bacteria! age t is gram positive, gram negative or does not have a eel! wail and selected from the group comprising, but not limited to, livestock pathogens. Examples of livestock pathogens include Actinobacuium suis, Actinomyces bovis, Arcmcbacteriu pyogenes, Bacillus anthracis, c&reus, iicheniformis, pumtlus, elariinogenicus, subtilis, Clostridium botuiinum, chauvoei, haemolyticum, novyi, p&rfringens, septicu , sordellii, tetani, olinum, Corynebacteriu pseudotuberculosis, renaie, Dermstophilus congolensis, Enierococcus spp (such as E. faeca!is, E. faeciu n, E. durans, E. avium, E. hirae), Erysipelothrix rhusiopathiae, Listeria iva vii, grayi, innocua, seeftgen, welshimeri, monocytogenes, Mycobacterium avium, bovis, paratubercuiosis (Johne's Disease), Mycoplasma (such as capricolum subsp. capripneumoniae, subsp. capricolum, M. mycoides subsp mycoides, M. agalactiae, M. ovipmu oniae, M. conjunctivae, M. arginini, M. bovis, and M. putrefaciens) Mycoplasma bovis, dispar, mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia C8PP) Mycoplasma gaiiisepticum (MG), iowae m&teagridis (MM), synoviae (MS) Mycoplasma haemosuis (formerly Eperythrozoon suis),. alkalescens, bovlgen alum, bovirhinis, bovoculi, catifornicum, canadense, cynos, equigenitatium, gateae, haernocanis, ha&mofeiis, hyopneumoniae, hyorhinis, hyosynoviae, iowae, ieachii, meieagridis, mycoides subsp capri, w&nyonii, suis, Rhodococcus equi, Staphylococcus epideimidis, Staphylococcus simulans, Staphylococcus fells. Staphylococcus xy!osus, Staphylococcus chromogenes, Staphylococcus wameri, Staphylococcus haemoiyticus, Staphylococcus sciuri, Staphylococcus saprophyticus. Staphylococcus hominis, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hyicus, Staphylococcus a re s, Staphylococcus pseudintermedius, Staphylococcus delphini, Staphylococcus sch!eiferi subsp, coagulans. Staphylococcus aureus subsp, ar erobius. Streptococcus uberis, Streptococcus canis, Streptococcus agalactiae, Streptococcus dysgalactsae, Streptococcus pyogenes. Streptococcus bovis, Streptococcus equi subsp. Zooapidemicus, Streptococcus equinus, Streptococcus equi (Streptococcus equi subsp equi}, Streptococcus equisimi ' lis (Streptococcus dysgalactiae subsp equisimilis), porcinus, suis, zooepidemicus, Streptococcus zooepidemicus (Streptococcus equi subsp zooepidernicus), Streptococcus dysgalactiae subsp, equisimilis. Propionibactehum acnes, Prophnibacteriu granulosus. Eubacterium, Peptococcus indolic s, and Peptostrepiococcus anaercbius; and various species of the following Gram negative genera: Aciinobacillus, Aeromonas, Anaplasma, Arcobacter, Avibacterium, Bacter ides. Bartonella, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Capnocytophaga, Chlamydia, Chlamydophila, Chryseobacterium, Coxiella, Cyiophaga. Dichslobacter, Edwardsiel!a, Ehrlichia, Escherichia, Flavobactehum, Francise!la, Fusobacteiium, Gailibacterium, Haemophilus, Histophilus, Klebsiella. Lawsonia, Leptospira, Marmheimia, M&gasphaera, Moraxelle, Neotickettsia, Nico!etelia, O iihobacterium, Pasteurella, Photobacteriu , Piscichlamydia, Ptscihckettsia, Porphyromonas, Prevotella, Proteus, Pseudomonas, Rickettsia, Riemeretla, Salmonella, Streptobacitlus, Tenacibaculu , Vibrio, and Yersinia.

[00133] in another embodiment, the bacteria! agent is selected from the group comprising, but not limited to, pathogens of companion animal species such as cats, dogs and horses. Examples of such pathogens include equine pathogens such as Sfreptococcus equi. Streptococcus zooepidernicus, Rhodococcus equi, Clostridium difficile, Clostridium perfringens, Corynebactehum pseudotuberculosis, Clostridium psliforme, Actinomyces bovis, Staphylococcus aureus, fi-ha&molytic Stepiococcus spp, Demiatophil congolense, Clostridium letani, and Clostridium botu!inum. Further examples include pathogens of clogs and cats such as Staphylococcus spp, Streptococcus spp, Clostridium spp, Actinomyces spp, Enterococcus spp, Nocardia spp, Mycoplasma spp, and Mycobacterium spp.

[00134] In another embodiment, the bacterial agent is gram negative and selected from the group consisting of the following representee families and species: Acetobacteraceae:- Roseomonas cervicalis; Roseomonas faunae; Roseo onas gilardii. - Aeromonad&ceae:- Aeromonas allosaccharophila; Aeromonas aquahorum; Aeromonas caviae: Aeromonas hydrophila (and subspecies): Aerotnonas salmonicida; Aeromonas shubertii; Aeromonas veronii biovar sobria A&romonas sohria). - Alca!igenaceae:- Achromobacter xylosoxidans; Alcalig&nes faecalis; Bordetelta ansorpii; Bordeteila avium; Bordetelta bronchiseptica; Bordetelia hinzii; Botdeieiia holmesii; Bordefelie parapertussis; Bordeteiia pertussis; Bordeteiia petrii; Bordeteila trematum; Glige!la ureoiytica; Oiigella urethrals. - Anaplasmataceae:- Anapiasma phagocytophilum; Anapiasma platys; Anapiasma bovis; Anapiasma centrals, Anapiasma marginale; Anapiasma odocoilei; Anapiasma ovis; Ehrlichia canis; Ehriichia chaffeensis; Ehttichia ewingii; Ehriichia maris; Ehrlichia ovina; Ehriichia rumlt niium; Neoehriichia fotoris; Neoehriichia mikurensis; Neorickettsia heiminthoeca; Neorickettsia risticii: Neorickettsia senneisu; Wolbachia pipientis. Armatimonadaceae:- Armatirrionas rosea. ~- Bactemidaceae:- Bacleroides forsythus; Bacteroides fragilis; Bacteroides melaninogenicus; Bacteroides ruber; Bacteroides urealtyicus. - Bartonellaceae:- Bartonella a!satica; Bartonella australis; Bartonella baciliifomiis; Bartonella birttesii; Bartonella bovis; Bartonella capceoti; Bartonella chom&Hi; Bartonella clamdgeiae; Bartonella doshiae; Bartonella e!izabethae; Bartonella graha ii; Bartonella henselae; Bartonella koehleraa; Bartonella peromysci; Bartonella phoceansis; Bartonella quintana; Bartonella tattimassiliensis; Bartonella rochalimae; Bartonella schoenbuchensis; Bartonella talpae, Bartonella ta iae; Bartonella taylorii; Bartonella tribocoru ; Bartonella vinsonii subsp berkhoffii; Bartonella vinsonii subs ar pensis; Bartonella vinsonii subsp. vinsonii. ~ Bdellovibrionaceae:- Bdetfovihrio spp. - Brachyspi aceae:- Brachyspira spp including Brachyspira hampsoniL Brachyspira hyodysenteriae, Brachyspira murdochii, Brachyspira pilosicoli. - Brucellaceae:- Brucella abortus; Brucella canis; Brucella ceti; Brucella tnetiiensis; Brucella ovis; Brucella pinnipedialis; Brucella suis; Qchrobacttvm anthropi; Ochrobactrum intermedium. -- Burkhoideriaceae: Burkholderia aboris: Burkholderia ambifaha (genomovar VII); Burkholderia anthina (genomovar Vll!); Burkholderia cenocepacia (geno ovar ill): Burkholderia cepacia (genomovar I); Burkholderia diffusa; Burkholderia dolosa (genomovar VI); Burkhoid&ria tatens; Burkholderia mallei; Burkholderia metallica; Burkholderia ultivo ns (genomovar II); Burkholderia pseudamait&i; Burkholderia pyrrocinia (genomovar IX); Burkholderia se inalis; Burkholderia stabiiis (g&nomovar IV); Burkholderia ubonensis (genornovar X); Burkholderia vieinamiensis (genomovar V); Cupriavidus paucuim; Cupriavldus gilardii; Ralstonia pickettii; Ratstonia mannitolilytica; Sphaeroti!us hippei; Sphaerotilus montanus; Sphaerotilus nat s. ~ Campylobacteraceae:- Arcobacter spp includng Arcobacter ski vwii; Campylobacter coil; Campylobacter concisus; Campylobacter cutvus, Campylobacter fetus: Campylobacter gracilis; Campylobacter heiveiicus; Campylobacter hominis; Campylobacter hyointestinalis: Campylobacter insulaenigrae; Campylobacter jejuni; Campylobacter lanienae; Campylobacter lari; Campylobacter !aridis; Campylobacter mucosal! ; Campylobacter rectus; Campylobacter showae; Campylobacter sputorum; Campylobacter upsaliensi - Candidatus:- Piscichlamydia saimoni - Cardiobacteriaceae:- Cardiobacterium hominis: Cardiobacterium valvaru ; Dichelobacter nodosus. - Chlamydiaceae:- Chlamydia spp including Chlamydia avium, Chlamydia gaiiinacea, Chlamydia uridarum, Chlamydia suis f Chlamydia trachomatis; Chlamydophiia spp including Chlamydophiia pneumoniae, Chlamydophiia pecorum, Chlamydophiia psitiaci, Chlamydophiia abortus, Chlamydophiia caviae, and Chlamydophila felis. ~ Chthonomonadaceae:- Chihonomonas calidirosea. ···· Comamonadaceae:- Comamonas iestosteroni; Verminephrobacter spp. Coxiellaceae:- Cexiella burnetii. Cyiophagaceae:* Cytophaga colu mris; Cy ophaga hutchinsonii; Fiexibaci&r echinictda; Ftexihacier e!egans; Flexibactet flexilis; Fiaxihacter iitoiaiis; Flexibacter polymorphic; Flexibacter roseolus; Flexibacter ruber. --- Desulfovibrionaceae;- Bi!ophila wadsworthia; Lawsonia intraceiluiaris. - Enterobacteriaceae:- Cedecea davisae, Cedecea lapagei; Cedecea neteri; amaionaticus; Citmbact&r diversus; Citrobacter freundii; itiohacter koseri; Cronobacter condiments; Cronobacter dublinensis; Cronobacter helveticus; Cronobacter ma nattcus; Cronobacter niuyijensii; Cronobacter puiveris; Cronobacter sakazakii; Cronobacter turicensis; Cronobacter universalis; Cronobacter zu ichensis; Edwardsielta ictalurt; Edwardsiella tarda; Enterobacter aerogenes; Enterobacter aggiomerans; Enterobacter cloacae; Enterobacter cowanii; Escherichia albetlii; Escherichia coli, including AIEC ~ adherent invasive E. coli, EaggEC - enteroaggregative E. coli; EHEC = enter hemorrtiagic E. coli; EIEC - enter invasive E. co//; EPEC ~ enteropat ogenic E, coli; ETEC - enterotoxigenic E. coli: ExPEC - extraintestinal pathogenic E. coli, NMEC - neonatal meningitis E. co!i, NTEC - necrotoxigenic E. coli, UPEC ~ uropathogenic E. coli.; Escherichia fergusonii; Ewingella americana; Hafnia a!vei; Hafnia paralvei; Klebsiella granulomatis; Klebsiella oxytoca, Klebsiella pneumoniae; Kluyvera ascorbata; Kluyvera ctyocrescens; Morganeila morganii; Pantoea (formally Enterobacter) aggiomerans; Photorhahdus asymbiotica: Plesiomonas s igeiioides; Proteus mirabilis; Proteus penneri; Proteus vulgaris; Piwidencia alcalifaciens; Ptovidencia rettgeri; Providencia stuartii; Raou!te!ia electrica; Raoutieila ornithsnolytica; Raouttella pianticola; Raoultelia temg&na; Salmonella bongori. Salmonella enterica subspecies enterica (many serotypes); Serratia liquifaciens; Serratia marcessns; Shigella boydii; Shigella dysenteriae; Shigella f!exneri; Shigella sonn&i: Yersinia enterocolitis; Yersinia pesiis; Yersinia pseudotuberculosis: Yersinia ruckeri. Fimbrii nonadaceae:- Fimbriimonas ginsengisoii. - Flavobacteriaceae:- Bergeyei!a zoohelcum; Capnocytophaga canimorsus: Capnocyiophaga cynodegmi; Capnocytophaga glngivalis; Capnocytophaga granulosa; Capnocytophaga haemolytica; Capnocytophaga leadbetteri; Capnocytophaga ochraces; Capnocytophaga sputigena; Chryseobacterium indologenes; Chryseebact&rium piscicola; Elizabethkingia eningoseptica; Flavobacterium branchiophilum; Flavobacterium co!umnare; Flavobacterium oncorhynchi; Flavobacterium piscicida; Flawbacterium psychrophilum; Myroides odoratus; Mymides odonstimimus; O ithobacterium rhinot cheale; Ri&m&rella anatipestifer; Riemerelia columbina; Riemerefia columbipharyngis; Tenacibaculum dicentrarchi; Tenacibaculum discolour; Tenacibaculum galialcum; Tenacibaculum maritimum; Tenacibaculum soleae; Weekselia vimsa. - Francisellacaae:- Franciselia tulai-ensis subsp. tuiarensis; Franciselia tuiar sis subsp. holamtica; Franciselia tuiarensis subsp. novicida; Franciselia phi!otrmagia; Francisella noatunensis; Francisella noatunensis subsp. orieniatis (also termed Fraricisei!a asiatica). Fusobacteriac&ae:- Fusobacterium spp. including Fusobacterium necrophorum, Fusobacterium nucleatum, Fuso-bacteriu poly orphu . Helicobacteraceae:- Helicobacter cinaedi; Helicobacter fennelliae; Helicobacter pylori. - Legione!laceae:- Legionella pneumophila and other species including; Legionella anisa; Legionella birminghamensis: Legionella bozemannii; Legionella cincinnatiensis; Legionella dumoffii; Legionella feeieii; Legionella gormanii; Legionella hackeliae; Legionella jordanis; Legionella iansingensis; Legionella longbeachae; Legionella maceachernii; Legionella micdadei; Legionella oakridgensis; Legionella paiisiensis; Legionella sainthelens; Legionella tusconenm; Legionella wadsworthii; Legionella waitersii. - Leptospir&ceae:- Leptospira alexanderl (including Leptospira a!exanderi serovar Hebdomadis, Leptospira al&xanderi serovar Manhao 3); Leptospira alstoni (including Leptospira alstoni serovar Pingcbang, Leptospira alstoni serovar Sichuan); Leptospira biflexa (including Leptospira biflexa serovar Ancona. Leptospira hifiexa sesvvar Canela); Leptospira borgpetersenii (including Leptospira borgpetersenii serovar Hardjo, Leptospira borgpetersenii serovar Hardjo-bovis, Leptospira borgpetersenii serovar Pomona, Leptospira borgpetersenii serovar Tarassovi); Leptospira broomii (including Leptospira broomii s&rovar Hurstbndge): Leptospira fainei (including Leptospira fainei serovar Hurstbridge); Leptospira idonii Leptospira inadai (including Leptospira inadai serovar Lyme, Leptospira inadai serovar Maiaya); Leptospira interrogans (including Leptospira interrogans serovar Ausiralis, Leptospira interrogans serovar Autumna!is, Leptospira interrogans setovar Bratislava, Leptospira interrogans serovar Canico!a, Leptospira interrogans serovar Grippoiyphosa, Leptospira interrogans serovar Hardjo, Leptospira interrogans serovar Hardjo-bovis, Leptospira interrogans serovar icierohaemorrhagiae. Leptospira interrogans serovar Pomona, Leptospira interrogans serovar Pyrogenes, Leptospira Interrogans serovar Tarassovi): Leptospira kirschneri (including Leptospira kirschneri serovar Bulgarica, Leptospira kirschneri serovar Cynopteri, Leptospira kirschneh serovar Grippotyphosa); Leptospira kmetyi; Leptospira licerasiae; Leptospira rneyeri (including Leptospira rneyeri serovar Sofia); Leptospira noguchii (including Leptospira noguchii serovar Panama, Leptospira noguchii serovar Pomona); Leptospira santarosai; Leptospira terpstrae; Leptospira vanthieiii; Leptospira weilii (including Leptospira weilii serovar Cetledoni, Leptospira weilii serovar Sarmin): Leptospira wolhachii; Leptospira weiffii; Leptospira yanagawae. - Leptotnchiaceae;- Lepiotrichia buccalis; Streptobacilius moniliformis. - Methylobacterlaceae:- Methylobacterium extorquens group; Methylobacterium fujisawaense; Methylobacten ' um mesophilicum; Methylobacterium zatmanii. - Moraxellaceae:- Acinetobacter baumannii (genomic species 2); Acinetobacter bayiyi: Acinetobacter bouvetii; Acinetobacter calcoaceticus (genomic species 1): Acinetobacter gemeri; Acinetobacter grimontii; Acinetobacter haemolyticus (genomic species 4); Acinetobacter johnsomi (genomic species 7); Acinetobacter junii (genomic species 5): Acinetobacter Iwoffi (genomic species 89}; Acinetobacter parvus; Acinetobacter radioresistens (genomic species 12); Acinetobacter schindleh; Acinetobacter tandoii; Acin&tobacier ijernbergiae; Aeinetobacier towneri; Aeinetobacier ursingii; Aeinetobacier veneiianus; Moraxeila atiantae; Moraxeila boevrei; Moraxeila bovis; Moraxeila bovoculi: Moraxeila cants; Moraxeila caprae, Moraxeila catarr alis; Morsxella caviae; Moraxeila cunicuii; Moraxeila equi; Moraxeila !acunata; Moraxeila lincolnii; Moraxeila acacae; Moraxeila nonliquefaciens; Moraxeila oblonga; Moraxeila osloensis; Moraxeila avis; Moraxeila phenylpyruvica; Moraxeila p!uranimaiium; Moraxeila porci. -- Moriteliaceae:- Moritelia abyssi; Moritelia dasanensis; Mohiella japonica; Moriielia marina; Moritelia pro-funda; Moritelia viscosa; Moritelia yayanosii. ~ Neisseriaceae:- Chrome-bacterium violaceum; Eikenel!a cotrodens; KingelSa denitrif l ans, Kingeila kingae. Kirigelia oralis. Kingeila potus; Neisseria cinerea; Neisseria elongate; Neisseria fia%'escens; Neisseria gonorrhoeae; Neisseria lactamica; Neisseria meningitidis; Neisseria mucosa; Neisseria polysaccharea; Neisseria sicca; Neisseria subflava; Neisseria weaver; Vitreoscilla spp. - Nitrosomonadace&e:- Niimsormnas eutropha; Nitroso onas halophiia; Nitrosomonas oligotropha. Pasteurellaceae:- Actinobacillus actinomycetemcomitans; Actinobacillus equuli; Actinobacillus tignieresii; Actinobacillus pleuropneumonias: Actinobacillus seminis; Actinobacillus succinogenes; Actinobacillus ureae; Aggregatibacter actinomycetemcomitans, Aggregatibacter segnis, Aggregatibacter aphrophilus; Avibacterium avium; Avibacterium endocarditidis; Avibactehu gallinarum; Avibacterium paragallinaru ; Avibacterium volantium; Bibersteinia trehalose; Gallibscteriu anatis: Gallibacterium genornospecies 1; Gallibacterium genomospecies 2; Gallibacterium genornospecies 3; Gallibacterium group V; Gallibacterium melopsittaci; Gallibacterium saipingitidis; Gaiiibacterium treha!osifermentans; Haemophilus aegyptius; Hae,mophi!u$ avium; Haemophilus ducreyi; Haemophilus haemoiyticus; Haemophilus influenzae; Haemophilus parahae o!yticus; Haemophilus parainiluenzae; Haemophilus parasuis; Hisiophiius somni; Mannhei ia caviae; Mannheimla giucosida; Mannheimia granulomatis: Mannheimia haemoiytica; Mannheimia ruminalis; Mannheimia vahgena; Nicotet&lla semolina; Pasteurella aerogenes; Pasteurella bettyae; Pastewella cabaili; Pasteurella canis; Pasteure!la dagmaiis; Pasteurella multocida (subspecies muitooida, septicum, galticida); Pasteurella pneumotropica; Pasteurella sto atis; Pasteurella trehalosi - Piscirickettsiaceae:- Piscisickettsia sal onis. - Plesiomonadaceae: · Ples monas shige!ioides. - - Poiyangiaceae:- Sorangium cellulosum. - Porphyromonadaceae:- Dysgonomonas capnocytophagoides; Dysgonomonas gadei; Dysgono onas hofstadii; Dysgonomonas mossis; Dysgonomonas oryzarvi; Dysgonomonas wimpennyi; Porphyromonas gingiva!is. Prevotel!aceae:- Prevote!la spp.including Prevotel!a intermedia, Prevotel!a melaninogenica. - Pseudo onadaceae:- Chryseomonas lut&ola; Pseudomonas aeruginosa; Pseudomonas iuteola; Pseudomonas fluorescens; Pseudomonas putida; Pseudomonas stuizeri; Pseudomonas oryzihabitans. - Rhizobiaceae:- Agrobacterium tumefaciens; Rhizobium radiobacier. -- Rickeitsiaceae:- Qrientia chuto; Ghentia isutsugamushi; Rickettsia aeschli annii; Rickettsia africae; Rickettsia akan: Rickettsia argasii; Rickettsia asiatica; Rickettsia australis; Rickettsia bellii; Rickettsia canadensis; Rickettsia conorii; Rickettsia cooleyi; Rickettsia felis; Rickettsia heilongjiangensis: Rickettsia helvetica; Rickettsia honei; Rickettsia hoogstraalii; Rickettsia hulinensis; Rickettsia hulinii; Rickettsia iaponica; Rickettsia amiionii; Rickettsia martinet: Rickettsia massiiiae; Rickettsia mortacensis; Rickettsia ontanensis; Rickettsia monteiroi; Rickettsia moreli; Rickettsia patke i; Rickettsia peacockn; Rickettsia philipii; Rickettsia piQwazekii; Rickettsia ra uliii; Rickettsia rhipicephali; Rickettsia rickettsii; Rickettsia sibirica subgroup; Rickettsia slovaca; Rickettsia ia urae; Rickettsia typhi. ·- Shewanellaceae:- Shewaneila putrefaciens. - Sphmgomonadaceae:- Sphingobacteiium muttivorum; Sphingobacierium spiritivorum; Sphingomonas pauci obilis. ~~ Spiriiiaceae:~ Spirillum minus; Spirillum volutins; Spirillum winogradskyi. ~ Spirochaeiaceae:- Borrelia afzeiii; Borrelia anserine; Borrelia bissettii; Borrelia burgdorferi; Borrelia co aceae; Ekirrelia duttonii; Borre!ia garinii; Borrelia hermsii; Boir&lia hispsnica; Borrelia japonic®; Borrelia lonestari; Borrelia lusiianiae; Bofrelia iyamotoi; Borrelia parkeri; Borrelia persica; Soirelia recurrentis; Borrelia spielmanii; Bonelia turicaise; Borrelia turicatae; Borrelia va!aisiana; Treponema oarateum; Treponema pallidum ssp. endemicu ; Treponema pallidum ssp. pallidum; Treponema pallidum ssp. pertenue. -- Succinivibrionaceae:- Anaerobiospirillum $pp. - Sutterel!aceae:- Suttereifa spp including Sutterella wadsworthia. ~~ Thermaceae:- Meiothermus spp. - Themiotogaceae:- Ther otoga neapolitana. ~ Veillonellaceae:- Dialister spp; Megamonas spp; Megasphae spp; Pectinatus spp; Pelosinus spp; Propionispora spp; Spotomusa spp; Veil nelia spp.: Zymophilus spp. ~ Vihrionaceae:- Photobacteriu n da selae; Vibno adaptatus; Vibrio alginolyticus; Vibrio azasii; Vibrio campbeltii; Vibrio cholera; Vibrio damsel; Vibrio tluvialis; Vibrio fu isii; Vibrio hoSiisae; Vibho metchnikovii; Vibno mimicus; Vibrio parahae olyticus; Vibrio vulnificus. ~ Wolbachieae:- Wolbachia spp. - Xantho onadaceae:- Lutei onas a&stuarii; Luteimonas aquatics; Luteimonas composti; Luteimonas iutlmans; Luteimonas marina; Luteimonas mephitis; Luteimonas vadosa; Pseudoxanthomonas broegbemensis; Pseudoxanthomonas japonensis; Stenotrophomonas aitophilia; Stenotrophomonas nitritireducens.

[00135] Most preferably, the bacterial agent causing the bacteria! infection ts gram negative and is selected from the group comprising-. Acinetobacter species, Aeromonas hydrop iia, Ciirobacter species, Ente becter species, Escherichia coli, Klebsiella pneumoniae, Morganetla morganii, Pseudomonas aeruginosa, and Stenotrophomonas maitophilia.

{00136} in another preferred embodiment, the bacteria agent causing the bacterial colonisation or infection is resistant to a conventional antibiotic used to treat the colonisation or infection. In one preferred embodiment, the bacteria! agent is resistant to a compound selected from the group comprising: one or more of aminoglycosides (for example geniamicin, tobramycin, amikacin, or netilmicin); anti-MRSA cephalosporins (for example ceftaroiine); amtpseudoroonal penicillins *· β-lactamase inhibitors (for example ticarci!iin-clavu!anic acid or piperacillin-tazobactam); carbapenems (for example ertapenem, imipenero. meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for e ample oefazolin or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (for example cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefoietan); fluoroquinolones (for example ciprofloxacin); folate pathway inhibitors (for example trimethopri -sulpbamethoxazole); giycyieyciines (for example tigecyciine); monobacta s {for example aztreonam); penicillins (for example ampicillin); penicillins + β- lactamase inhibitors (for example amoxiestiin-cSavulanic acid or ampiei!tin-sulpactam}; phenico!s (for example chloramphenicol); phosphonic acids (for example fosfomycin); polymyxins (for example colistin); and tetracyclines (for example tetracycline, doxycycline or minocycline. Preferably, the bacterial agent resistant to these compounds is gram negative.

(00137] Preferably, the bacterial agent is resistant to a compound selected from the group comprising: penicillins, cephalosporins, carbapenems, monobactams and other (5-lactam antibiotics, fusidanes, aminoglycosides, fluoroquinolones, streptogramins, tetracyclines, giycyieyciines, chloramphenicol and other phenicols, m3crolides and ketolidss, fincosamides, oxazolidinones, aminocyciitois, polymyxins, g!ycopeptides, lipopeptides, bacitracin, m piricin, pleuromutilins, rifamycins, sulphonamides and trimethoprim. Preferably, the compound is selected from the group comprising: beta lactams, glycopepiides, !ipopeptides, mactolides, oxazo!idinones and tetracyclines. Preferably, the bacterial agent is resistant to the compound when the compound is at a concentration range selected from the following: 0.001 pg mL - 10,000 pg/rnL; 0.01 pg/mL - 1000 pg.'mL; 0.10 pg/ml - 100 pg mL; and 1 pg/mL - 50 pg/ml.

[00133] in another preferred embodiment, the bacterial agent causing the bacterial infection is selected from the group comprising, but not. limited to, gram positive bacteria. The microbe is preferably a gram positive bacterial agent selected from the group comprising Staphylococcus aureus, Staphylococcus pseudinter edius, Streptococcus pneumoniae. Streptococcus pyogenes, Streptococcus agalactia®, Streptococcus uteris, Enterococcus faeck , £nfer©eoc «s faecalis, and Clostridium difficile.

[00 39] In one preferred embodiment, the bacteria! agent has no cell wail. Preferably, the bacteria! agent is selected from the group comprising: Mycoplasma spp, My o las a agalactia®, Mycoplasma alka!escens, Mycoplasma amp oriforme, Mycoplasma arginini, Mycoplasma bovig&nitsium, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovocull, Mycoplasma buccals. Mycoplasma californicum. Mycoplasma canadanse, Mycoplasma capricolum subsp. capricolum, Mycoplasma capricolu subsp, capripneumoivas, Mycoplasma conjunctivae, Mycoplasma cynos, Mycoplasma dispar. Mycoplasma aquigmitaiium, Mycoplasma faucium, Mycoplasma felis. Mycoplasma fermantans (incognitas str.}. Mycoplasma gallisepticum (MG), Mycoplasma gaiese, Mycoplasma genitalium, Mycoplasma haemocanis. Mycoplasma haemofe!is, Mycoplasma haemosuis (formerly Eperythrozoor) suis), Mycoplasma hominis, Mycoplasma hyopneumoniae. Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma iowae meleagridis (MM), Mycoplasma iowae, Mycoplasma ieachii, Mycoplasma lipophilutn, Mycoplasma meleagridis, Mycoplasma mycoides subsp caph, Mycoplasma ycQides subsp mycoides, Mycoplasma mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP), Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, Mycoplasma penetrans, Mycoplasma piru , Mycoplasma pneumoniae, Mycoplasma primatum, Mycoplasma putrefaciens, Mycoplasma salivariuni Mycoplasma spe atophllum. Mycoplasma &ui$, Mycoplasma synoviae (MS), Mycoplasma wenyonil, Mycoplasma, Unsaplasma spp, Ureaplasma parvum, Ureaplasma umaiytlcum, Ureaplasma, and Ureopla$ma diversurn.

(00140] in another most preferred embodiment, the bacteria! agent is Staphylococcus aureus.

(00141] In another preferred embodiment, the bacteria! agent is resistant to a compound selected from the group comprising: one or more of aminoglycosides (for example gentamicm); ansamycins (for example rifampicin); anti- RSA cephalosporins (for example ceftaroiine); anti- staphyiococcai ^-lactams (or cephamycins) (for example oxacillin or cefoxitin); carbapenems (for example ertapenern, imipenem, meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for example cefazo!in or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (t r examp!e cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefotetan); fluoroquinolones (for examp!e ciprofloxacin or moxifloxacin); folate pathway inhibitors (for example trimethoprim - sulphamethoxa2oie); fucidanes (tor example fusidic acid); g!ycopepttdes (for example vancomycin, iescopianin or tefavancin); gfycylcyciines {for example tigecyciine); Hncosamides (for example clindamycin); tipopeptides (for example daptomycin); macro!ides (for example erythromycin); oxazoltdtnones (for example iinezoiid or tedixolid); pbenicols (for example chloramphenicol; phosphonic acids (for example fosfomycin); streptogramins (for example quinupristin-da fopristin); and tetracyclines (for example tetracycline, doxycydine or minocycline). Preferably, the bactena! agent resistant to these compounds is gram positive.

[00142] In another most preferred embodiment, the bacterial agent is Streptococcus pneumoniae. The Streptococcus pneumoniae may be a strain that is resistant to one or more of β-iactams and macrolides.

[00143] in another most preferred embodiment, the bacteria! agent is Streptococcus pyogenes. (00144} In another most preferred embodiment; the bacteria! agent is Streptococcus agalactia*}.

[00145] In another most preferred embodiment, the bacterial agent is either Enterococcus faecium or Enterococcus faecaiis. The Enterococcus faecium or Enterococcus faecaiis may be a strain that is resistant to aminoglycosides (for example gentamicin (high level) or streptomycin (for example streptomycin (high level}); carbapenems (for example imipenem, meropenem or doripenem); fluoroquinolones (for example ciprofloxacin, levofioxacin or moxifloxacin); giycopeptides (for example vancomycin or teicopianin); giycyicyclinss (for example tigecycSine); lipopeptides {tor example daptomycin); oxazolidinones (for example linezolid); penicillins (for example ampiciiitn); streptogramins (for example quinupristin-dalfopristin); tetracycline (for example doxycycline or minocyciine).

[00146] in another most preferred embodiment, the bacterial agent is Clostridium difficile.

[00147] The bacteria! infection in the subject may cause a disease selected from the group comprising, but not iimited to, nosocomial pneumonia caused by Staphylococcus a reus (MDR, XOR, PDR or methiciilin-susceptibie or -resistant strains), or invasive pneumococcal diseases such as pneumonia, bronchitis, acute ssnusitis, otitis media, conjunctivitis, meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess caused by Streptococcus pneumoniae (including multi-drug resistant strains [ DRSP] such as those resistant to β-iactams and macroiides), complicated skin end skin structure infections, including diabetic foot infections, with or without concomitant osteomyelitis, caused by Staphylococcus aureus (methiciilin-susceptibie and -resistant strains), Streptococcus pyogenes, or S aptococcus agalactiae, uncomplicated skin and skin structure infections caused by Staphylococcus aureus (methiciilin-susceptibie and -resistant strains) or Streptococcus pyogenes, community-acquired pneumonia caused by Streptococcus pneumoniae (including multi-drug resistant strains [MDRSF], including cases with concurrent bacteraemia, or Staphylococcus aureus (methiciilin-susceptibie and -resistant strains) and Staphylococcus aureus bloodstream infections (bacteraemia), including those with right-sided infective endocarditis, caused by methiciilin-susceptibie and methiciliin-resistant isolates, vancomycin- resistant enterococcus infections, including cases with concurrent bacteraemia, and treatment of Clostridium <f V/c//e- associated diarrhea (CDAD).

[00148] Gram negative organisms are important causes of many infectious diseases in humans and other animal species. Bone and joint infections (Gram-negative organisms or mixed bacteria, are an important cause of vertebra! osteomyelitis and septic arthritis), cardiovascular system infections (including endocarditis caused by the HACEK group - He o ilus parainfluenzas, Haemophilus aphrophilus, Aggregstihacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingeiia kingae), central nervous system infections (the commonest causes of bacterial meningitis are Neisseria meningitidis. Streptococcus pneumoniae and, in nonvaccinated young children, Haemophilus influenzae type b (Hib), in neonates and infants less than 3 months of age, Streptococcus agaiacliae (group B streptococcus), Escherichia co!i and other aerobic Gram-negative rods are important pathogens, brain abscess or subdural empyema, the infecting organism(s) vary with the underlying predisposing cause but where the likely site of origin is the ear, enteric Gram- negative bacilli are commonly involved), eye infections (common pathogens include Haemophilus influenza. Neisseria gonorrhoeae or Chlamydia trachomatis), gastrointestinal tract infections (a wide range of pathogens are implicated including enterotoxigenic Escherichia coli (ETEC), Salmonella. Campylobacter, Shigella, Vibrio cholera and Yersinia anterocolitica), genital infections (bacterial vaginosis is a polymicrobial clinical syndrome with high concentrations of anaerobic (eg Mobiiuncus species) and other fastidious bacteria (including Gardnerella vaginalis and Atopobiu vaginae), and Mycoplasma hominis; non-sexuaily acquired pelvic inflammatory disease (P!D) is usually caused by mixed vaginal flora, including anaerobes, facultative Gram-negative bacteria and Mycoplasma horninis, while sexually acquired PID is usually initiated by C. trachomatis or N. gonorrhoeae with growing evidence that M. gentta!ium infection is involved in a significant minority of cases), intra-abdominal infections (peritonitis due to perforated viscus is usually a polymicrobial infection wit aerobic and anaerobic bowsi flora while spontaneous bacterial peritonitis (SBP) is usually caused by enteric Gram-negative bacjlli, such as Escherichia coli and Klebsiella species, Klebsiella pneumoniae is an increasingly identified cause of liver abscess), community-acquired pneumonia (Mycoplasma pneumoniae, Chiamydophi!a (Chlamydia) pneumoniae, Chiamydophila (Chlamydia) psittaci, Haemophilus influenza, aerobic Gram-negative bacilli including Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkhoideria pseudomallei), otitis xXe a (including acute diffuse) (bacterial cultures commonly yield Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus and Klebsiella species), otitis media (including acute) (common bacteria! pathogens include Streptococcus pneumoniae, Haemophilus influenzae and Morax&lla catan-haiis), sepsis (including severe) (including Acinetobacter baumannii, disseminated gonococcal sepsis, Gram-negative enteric bacteria, Neisseria meningitidis (meningococcal sepsis) and Pseudomonas aeruginosa), Systemic infections (Spotted fevers (Rickettsia) and scrub typhus (Orientia), Brucellosis, Cat-scratch disease and other Bartonella infections, Leptospirosis, Lyme disease, Melioidosis, Q fever, Typhoid and paratyphoid fevers (enteric fevers), urinary tract infections (acute cystitis, acute pyelonephritis, recurrent urinary tract infections and atheter-associated bacteritiria and urinary tract infections).

{00149] In humans gram negative bacteria are common causes of intra-abdominal infections (lAls), urinary tract infections (UTIs), hospital acquired pneumonia, and bacter mia. Escherichia coii (£. coii). Klebsiella pneumoniae ( . pneumoniae), and Pseudomon& aeruginosa (P. aeruginosa) are important pathogens in the hospital setting, accounting for 27% of aii pathogens and 70% of ail Gram-negative pathogens causing healthcare-associated infections iSieveri DM, Ricks P, Edwards JR, et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention. 2009-2010. Infect Control Hosp Epidemiol 2013;34:1-14.].

(00150} Grafts negative bacteria are showing rising rates of resistance to current therapies. The production of extended-spectrum β-lactamase (ESBL) enzymes is a common mechanism of resistance. Rates of ESBL-producing £. coii and K. pneumoniae have risen substantially, with the result that these bacteria are increasingly resistant to widely used antimicrobials.

[00151] P. aeruginosa is the most common Gram-negative cause of nosocomial pneumonia and the second most common cause of catheter-related UT!s in the U.S.

[00152] £ co/ is the most common cause of UTIs. Cases of UT! caused by ES8L- producing £. coii and K. pneumoniae as well as P. aeruginosa., including MDR strains, are increasing. ESBL-producing £. coii and K. pneumoniae are also frequently isolated in patients with complicated !Ai (ciAi).

(00153] P. aeruginosa is a clinicaliy challenging and virulent pathogen that can be a cause of common infections in humans such as nosocomial pneumonia, UTI, lAi, and bloodstream infections. P. aeruginosa is the mast common Gram-negative organism causing ventilator associated pneumonia and the second most common cause of catheter-associated UTIs.

[00154} The increase in the number of infections caused by Gram-negative bacteria is being accompanied by rising rates of resistance. Treatment options to meet this challenge are increasingly limited. There is a critical need for new antibiotics to meet the needs of patients now and in the future.

[00155] in one preferred aspect, more than one compound of the invention is administered to the subject.

[00156] in another preferred embodiment, a compound of the invention, or a therapeutically acceptable salt thereof, is administered together wi h a compound or agent that removes or substantially removes or reduces the integrity of the cell wail of the bacteria! agent. As an example, the compound is selected from the group consisting of: β lactams, fosfomycin. l sozyme. polymyxins and chelating agents such as ethyienediaminetetraacetic acid (EOT A). As an example, the agent is an immunological agent (such as an antibody or vaccine) thai reduces the integrity oi the eel! wall. In one preferred embodiment, the compound, or a therapeutically acceptable sail thereof, is administered together with a compound that removes or substantially removes or weakens the integrity of t e outer eel! wall of a gram negative bacteria! agent.

{00157} According to another aspect of the invention, there is provided an antibacterial pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I, or a therapeutically acceptable salt thereof. Preferably, the composition is an antibacterial pharmaceutical composition.

(00158) According to another aspect of the invention, there is provided an antibacterial veterinary composition comprising a therapeutically effective amount of a compound of Formula I. or a therapeutically acceptable salt thereof. Preferably, the composition is an anti-bacterial veterinary composition.

[00159] The pharmaceutical composition may optionally include a pharmaceutically acceptable excipient or carrier. The veterinary composition may optionally include a veterinary acceptable excipient or carrier.

[00160] The pharmaceutical or veterinary composition of the invention preferably contains a compound of Formula or a pharmaceutically acceptable salt, at a concentration of selected from the groupd consisting of: 1 mg/g to 500 mg/g; 5 mg to 400 mg/g; 10 mg g to 200 mg/g; 20 mg/g to 100 mg/g; 30 mg/g to 70 mg/g; and 40 mg/g to 60 mg/g.

(00161} In another embodiment, the pharmaceutical or veterinary composition comprises impurities, wherein the quantity of impurities as a percentage of the total weight of the composition is selected from the group consisting of: less than 20% impurities (by total weight of the composition); less than 15% impurities; less than 10% impurities; less than 8% impurities; less than 5% impurities; less than 4% impurities; less than 3% impurities; less than 2% impurities; less than 1% impurities; less than 0.δ% impurities; less than 0.1% Impurities, in one embodiment, the pharmaceutical or veterinary composition comprises microbial impurities or secondary metabolites, wherein the quantity of microbial impurities as a percentage of the total weight of the composition is selected from the group consisting of; less than 5%: less than 4%; less than 3%; iess than 2%; less than 1%; less then 0.5%; less than 0.1%; less than 0.01%; less than 0.001%. In one embodiment, the pharmaceutical or veterinary composition is sterile and stored in a sealed and sterile container. In one embodiment, the pharmaceutical or veterinary composition contains no detectable level of microbial contamination. [00182] The pharmaceutical or veterinary composition of the invention ma comprise a further antimicrobial agent. The further antimicrobial agent may be an antifungal agent or antibacterial agent The method of treating or preventing a bacteria! infection or colonisation in a subject, may also comprise the administration of a compound of the invention with a further antimicrobial agent

[00163] The pharmaceutical or veterinary composition of the invention may comprise more than one compound of the invention. For example, a combination of compounds. The method of treating or preventing a bacterial infection or colonisation in a subject, may also comprise the administration of more than one compound of the invention.

[00164] In one embodiment, the antifungal agent is selectees from the group comprising, but not limited to naturally occurring agents including Eehinocandins (Ans ulafungin, Caspofungin, Micafungin), Polyenes {Amphotericin B, Candicidin, Fi!ipin, fungichromin (Pentamycin), Machimycin, Hamycin, Lucensomycin. Mepartricin, Natamycin, Nystatin, Pecilocin, Panmycin), and other naturally occurring antifungal agents including Grsseofu!vin, OSigomycins. Pyn-o!nitrin, Siccanin, and Viridtn. The antifungal agent may be a synthetic compound selected from the group comprising, but not limited to Aily!amines (Butenafine, Naftifine, Terbinafine) Imidazoles (Bif nazole, Butoconazoie, Chiormidazo!e, Ciimbazoie, Croconazoie (Cioconazole), Clotrimazole, Eberconazoie, Econazole, Enilconazole, Fenticonszole.. Flutrimazote, Fosftuconazole, !soconazoie, etoconazole, Lanoconazole, Luliconazote, Miconazole, Neticonazoie, Qmoconazo!e, Oxlconazo!e Nitrate, Parconazole, Sertaconazole, Su!conazoie, Tioeonazole), Thiocarfcamates (Liransftate, Tofciclate, Toiindate, Toinaftate), Triazotes (Fluconazole, isavueonazoie, itraconazole, Posaconazote, Ravuconazoie, Saperconazoie, Terconazoie, Voriconazole),and other synthetic agents such as Acnsorcin, Amorolffne, Biomosalicyichloranilide {Bromochlorosalicyianilide}. Buciosaroide, Calcium Propionate, Chlorphenesin, CicSopirox, Cloxyquin (Cloxiquine}, Coparaffinate, Exalamide, Flucytosine, Hatoprogin, Hexetidsne, Lof!ucarban, Nifuratel, Nifuroxiroe, Piroctone, Potassium Iodide, Propionic Acid, Pynthione, Saiicyianitide, Sodium Paraclilorobenzoate, Sodium Propionate, SulbenUne. Tenonitrozole, Triacetin, Thmetrexate, Undecyfenic Acid {Undecenoic Acid), and Zinc Propionate.

(00165] The composition of the invention may comprise an antibiotic adjunct selected from the group comprising, but not limited to, β-Lactamase Inhibitors (Avibactam, Ciavuianic Acid, Sulbactam, Sultamidilin, Tazobactam), Renal Dspeptidase Inhibitors (Ciiastatin). and Renal Protectant (Betamipron).

(00166] In one embodiment, the composition of the invention comprises a further antibiotic selected from the group comprising, but not limited to, 2,4-DIA!vtiNOPYRi iDSNES, including Baqui!oprim, Brodimoprim, fciaprim. Ormetoprim, Pyrimethamine, Tetroxoprim, Trimethoprim; AMINOCOU A iNS, including Novobiocin; A INOCYCLITOLS, including Spectinomycin,; AMINOGLYCOSIDES, including Amikacin, Apramycin, Arbskacin, Bekanamycin. Sutirosin, Dibekacin, Dihydrostreptomycin, Etimicin, Fortimicins (Astromicin), Framycetin, Geniamicin, Hygromycin B, isepamicin, anamycin, Micranomicin, Neomycin, Netilmicin, Paromomycin, P!azomicin, " Rifoostamycin, Sisomicin, Streptomycin, Tobramycin. Verdamicin; AMSNOMETHYLCYCLINES, including Ornadacyciine; AMPHENiCOLS. including Azidamfenicol, Chloramphenicol, Florfenicol, Thiamphsnicoi; A SA YC!NS, including Rifabutin, Rif amide, Rifampin (Rifampicin), Rifamycin, Rifapentine, Rifaximin; ANTISEPTIC AGENTS, including Acridine derivatives (including acriflavine, aminoacridina, ethaeridine. proflavine}, Sispyridines (including oetenidine dihvdrochionde), Brominatsd salicyianilides (including bromsalans), Cniorhexidsne, Phenol derivatives (including thymol and tridosan), Quaternary ammonium compounds (including A!kyldi ethylethy!benzyi Ammonium Chloride, benzalkoniu chloride, cetyipyridinium chloride, benzeihonium chloride, cetrimonium); ANTITUBERCULAR AGENTS, including Cycloserine, Delamanid, hambutol, Ethionamide, Isoniazid (Ftivazide), Morinamide, p- minosalicylic Acid (PAS), ProUonamide, Pyrazinamida, Terizidone, Thtoacetazone, Tiocarlide; ARSENiCALS, including Arsaniiic Acid, Roxarsone; BACTERIOCINS, including Nisin. Srilacidin (P X-30063); β- LACTAM CARBACEPHE S, including Loracarbef; β- LACTAM CARBAPENEMS, including Biapenem, Doripenem, Ertapenem. Faropenem. imipenem, Meropenam, Panipenem, Razupenem, Ritipenem, Suiopenem, Tebipenem, Tomopenem; (J-LACTAM CEPHALOSPORINS, including Ceiacetriie, Cefaclor. Cefadroxii. Cefatexin, Cefaiogiycin. Cefaionium, Cefaioridine, Cefaiotbin, Cefamandcie, Cefapirin, Cefatrizine. Cefazaffur, Cefazedone, Cefazoltn, Cefcapene,. Cefdinir, Ceftfitoren, Cefepime, Cefetamet, Cefixima, Cefmenoxime, Cefodizime, Cefonieid, Cefoperazone, Ceforanide, Cefosaiis, Cefotaxime, Qsfotiam, Cefovecin, Csfozopran, Cefpimizote, Cefpiramide, Cefpirome, Cefpodoxime, Cafprozil, Cefquinome, Cefradine, Cefroxadine, Cefsulodin, Cefieroline, Ceftazidime, Ceftersm, Caftezofe, Ceftibuten, Ceitiofur, Ceffeoxime, Ceftobiprole, Geftolozane, Ceftradine, Ceftrezo!e, Ceftriaxone. Ceftroxadsne, Cefuroxime, Cefuzonam, Pivcefa!exin; β-LACTAM CEPHA YCiNS, including Cefb perazone, Cefmetazole, Cefminox, Cefotetan, Cefoxitin; β-LACTAM MONOBACTAMS, including Aztreonam, Carumonam, Tigernonam; β- LACTAM OXACEPHEMS, including Flomoxef, Latamoxef, Moxalactam; β-LACTAM PENICILLINS, including Amdinocillin (Meciilinam), Amoxicillin, Ampicilfin, Apaici iin, Amoxicillin, Azidoci!lin, Azlociffin, Bacampiciflin, Carbenieiiiin, Carindaciltin, Cidaciiiin. C!emizoie Penicillin, C!ome!oci!iin, CioxacilUn, Cyclacillin, DidoxacsfSin, Epicitlin, Fenheniciliin, Fioxaei iin (FludoxaclHin), Hetaci!lin, Lenampiciilin, Meciilinam, MeiampiciSlin, Methiciilin Sodium, Mezlocillin, Nafci!lin, Oxacillin, Penameciliin, Penethamate Hydriodide, Penicillin G, Penicillin G Benzathine, Penicillin G Procaine, Penicillin , Penicillin O, Penicillin V, Phenethiciilin Potassium, Piperacillin.. PivampiciSiin, Pivmeciliinam. Propicillin, Guinaciiiin, Suf niciitin. Sultarniciilin, Taiampici!iin, Tsmociliin, Ticarciilin; BiCYCLOMVCiNS, including Bicozamycin; BORON CONTAINING ANTIBACTERIAL AGENTS, including AN3365 {a inomethyjben QxabofOies}, GSK2251052 (teucy!-tRNA synthetase inhibitors); CYCLIC ESTERS, including Fosfomycin; FATTY ACID SYNTHESIS INHIBITORS (Fabl), AFN-1252, ΜυΤ05 β 399, FA8-001;FLUOROQUINOLO ES, including Avaroftoxacin, BaSof!oxactn, Sesifloxacin, Chinfloxadn, Cinoxacin, Ciprofloxacin, Qtnafloxacin, Oanof!oxacin, Delafloxacin, Difloxacin. Enoxacin, ErsrofSoxacin, Finatioxadn, Fleroxaein. Fiumeguine, Garenoxacin, Gattftoxacin, Gemifioxacin, Grepafioxacin, ibafloxacm, Levofloxadn, Lomefloxacin, fvlarbafioxacin, Miioxacin, Moxifioxacin, Nadifioxacin, Norfloxacin, Ofloxacin, Orbifioxacin, Pazufioxacin, Pefioxacin, Pradofioxacin, Pruiifloxacin, Rosoxacin, Rtsfioxactn, Safafioxacin, Sitafloxacin, Sparfloxacin. Temaf!oxacin, Tosufioxacin, Trovafloxadn, abofioxacin; FUSIDANES. including Fusidic Acid; GLYCOLIPODEPS!PEPTIDE, including Ramoplanin; GLYCOPEPTIDES, including Avoparcsn, Dalbavancin, Norvancomycin, Oritavancin, Taicoplanin, TeSavancin, Vancomycin,; GLYCOPHOSPHOLIP!DS, including Barnbarmycins (bambermy in, moenomycins. fiavophospholipoJ); GLYCYLCYCL!NES , including Tigacyctine; HYBRIDS, Cadazoitd (Oxazoiidinone-quinoione), TD- 7S2 (giycopapisde-cepha!osporin); LINCOSAMiDES, including Clindamycin, Uncomycin, Piriirnycin; LIPOPEPTIDES, including Daptomycin, Surotomycin; MACROLIDES, including Azithromydn, Carbomycin, Cethromycin, Clarithromycin, Dirithromycin, Erythromycin, Fldaxoroicm, Fiunthromycin, Gamithromycin, Josamycin, Ksiasarnycin, Leucomycin. eleumycin, Midecamycins, iokamycin, Mirosamycin, Oleandomycin, Primycirt, Rokitamycin, Rosaramictn, Roxithromycin, Sedecarnycin. Soiithromycin, Spiramycin, Teiithromycin, Terdecamycin, Tiidipirosin, Tilmicosin, Troteandomycin, Tulathrornycin, Tylosin, Tyivaiosin; NITROFURANS, including Furaltadone, Furazidin, Furazolidone. Furazoiium Chloride, Nifuratei, Nffurfoitne, Nifuroxazide, Nifurpirinoi, Nifustotnoi, Nifurzide. Nitrofural, Nitrofurantoin, Nitrofurazone; NITROIMIOAZOLES, including Dimetfidazole, Metronidazole. Ornidazole, Ronidazole, Secnsdazole, Tinidazole; OLIGOSACCHARIDES, including Avilamycin, Eveminomicin; OTHER ANTIBACTERIAL AGENTS, including Auriciossne, Chioroxine, Chlorquinaidol, Clioquinol, Ctofoctoi, Halquinol, Lotilibcin, andelic Acid, Methenamine (hexamine). Nitazole, itroxoline, Perchlozone. Taurotidine, Thenoic Acid, Xibornoi; OXAZOL!DINONES, including Eperezolid, Linezolid, Posizolid, Radezolid, Sutezolid, Tedizolid (Torezolid); PEPTIDE DEFORMYLASE INHIBITORS, including GSK1322322; PEPTIDES, including Omiganan, Pexiganan; PLEURO UTILINS, including Reiaparnulin, Tiamulin, Vainemulin; POLYETHER IO OPHORES, including laidiomycin. Lasalocid, Maduramicin, onensin. Narasin, Salinomycin, Semduramicin; POLYMYXINS, including Coii? in, Polymyxin B; POLYPEPTIDES, including Amphomycin, Bacitracin, Capreomydn, Enduracidin, Enramycin, Enviomycin, Fusaftmgins, Gramicidin(s), Iseganan, Magainins, Nosiheptide, Ristocetin, Thiostrapton, Tuberactinomycin, Tyrocidine, Tyfothricin, Vkxnydn; PSEUDOMONIC ACIDS, including Mupirocin; QUINOLONES, including Nalidixic Acid. Nemonoxacin, Oxolinic Acid, Ozenoxacin, Plpemidic Acid, Piiosnidic Acid; GUINOXALINES, including Carbadox, Otequindox; RIMINOFENAZINES, including Clofazimine, STATINS, including At fvastatin, Fluvastatin, Lovastatm, Mevastatin, Pravastatin, Pravastatin, Rosuvastatirf, Simvastatin; STREPTOGRAMi S, including OaJfopristin, opristin, Linopristin.. Pristinamycin, Quinuphstin, Virginiamycin; STREPTOTHRICINS, including Mcurseot ricin; SULFONAMIDES, including Acetyl Sulfaroeihoxypyrazine, Chloramine-B, Chloramine-T, Dichioramine T, Formosuifsthsazo!e, Mafenide. N4-Sulfanjly!su!fanitamide, Nopryisu!famide, N- Suifanilyl-3,4-xylamide, Ormaosulfaihiazole. Phthaf !suifaceiamide, PhthalyfsuffaihiaEOie.. Salazosuifadimidine, Succinyisulfatniaxoie, SuSfabenzamide, Suifacarbamide, Sulfacetamide, Sutfachlorpyridazine, Sulfachrysoidine, Sulfacloztne, Sutfacytine, Sulfadiazine, Sulfadicramide, Sulfadimathoxine, Sulfadimidine, Suifadoxine, Suifaethidole, Sulfaguanidine. Sujfaguanoie, Sulfaiene, Suifaioxic Acid, Suifaniera2ine, Suifameter. Sulfamethazine. Suifamelhizoje, Suitamethomidine, Sulfamethoxazole, Sulfamethoxypyridazine.. Sulfamethyjthtazole, Suifametopyrazine, Sulfametrole, Suifamidochrysoidine, Su!famonomsihoxine, Suifamoxole, Sulfanilamide, SuSfani!ylurea, Sulfaperine, Suifaphenazote, Sulfaproxyline, Sulfapyrazine, Suifapyndine, Suifaquinoxaiine, Sulfathiazo!e, Suifathiourea, Sutfatroxaz le, Sulfisomidine, Su!tisoxazoie (Suifafurazole); SULFO ES, including Acediasuffone. Dapsone, G!ucosuifone Sodium, p-Suifanilylbenzyiamine, Succisuifona, Suifaniltc Acid. Su!foxone Sodium, Thiazotsulfone; TETRACYCLINES, including Chiorieiracyc!ine, Clomocyciine, Deroeclocyciine, Doxycyeiine, Eravacycline, Guamecycline, Lymec dine, Mectoeyciine, Methac ciine, Minocycline, Oxytetracycline, Penimepicydine, Pipacycline, Rolitetraeycline. Sarecyeline. and Tetracycline.

[0018?] The composition of the invention may further comprise an excipient selected from the group comprising, but not limited to, binders and compression aids, coatings and films, colouring agents diluents and vehicles dhsintegrants. emulsifying and so!ubitising agents, flavours and sweeteners, repellents, giidanis and lubricants, plasticisers. preservatives, propei!ants, solvents, stabilisers, suspending agents and viscosity enhancers.

[001681 According to a further aspect of the invention, there is provided a medical device when used in a method of treating or preventing a bacteria! infection in the subject.

[00169] According to further aspect of the invention, there is provided a medical device comprising the composition of the invention. The composition of the invention may be any slow release form, dlor in the form of a coating of the medical device.

[00170] The medical device may be in a form selected from the group comprising: an implant, a plaster, a bandage, and other dressing applied to a bacteria! infection in a subject. JOG 171] According to further aspect of the invention, there is provided a method of killing bacteria, the method including the step of contacting the bacteria with a compound of the invention, or a therapeutically acceptable salt thereof.

(001 2] According to further aspect of the invention, there is provided the use of a compound of the invention, or a therapeutically acceptable salt thereof, to kill bacteria, said use comprising the step of contacting the bacteria with a compound of the invention, or a therapeutically acceptable salt thereof.

[00173] Terms used herein will have their customary meanings in the art unless specified. As used herein, the term robenidine, NCL812 (also known as 1 ,3-bis[{£>{4- chiorophenyi)me^ ieneamino{guanidine) refers to a compound having the following chemical structure:

BRIEF DESCRIPTION OF THE DRAWINGS

(00174] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

Figure 1 presents the chemical name and chemical structure of the compounds CL001 to NCL230;

Figure 2 shows a graph of the effect of NCL812 on DNA macromoiecular synthesis in

Staphylococcus aureus according to example 2;

Figure 3 shows a graph of the effect of NCL81 on RMA macromoJecu!ar synthesis in

Staphylococcus aureus according to example 2;

Figure 4 shows a graph of the effect of NCL.812 on protein macromolecuSar synthesis in

Staphylococcus aureus (ATCC29218) according to example 2;

Figure 5 shows a graph of the effect of NCL812 on cell wall macrornolecular synthesis in

Staphylococcus aureus (ATCC29213) according to example 2; Figure 8 shows a graph of the effect of NCL512 on lipid macromoiecular synthesis in Staphylococcus aureus (ATCC23213} according to example 2;

Figure 7 shows a graph summarising the effect of NCL812 or) macromoiecular synthesis in Staphylococcus aureus (ATCC29213) according to example 2;

Figure 8 shows a graph of the effect of NCL812 on ATP release from Staphylococcus aureus (ATCC2S213) according to example 3;

Figure 9 shows a graph showing the average melting point peaks for the negative derivative dF/dT after real-time polymerase chain reaction of the mecA gene in methiciiiin-fssistant S. aureus isolates grouped by msc gene complexes, A <n=4), B (rv~10). C2 (n-4) and unclassified (n=2). Groups indicated with different superscripts are significantly different (P«0.O5), according to example 4;

Figure 10 shows a graph of the optical densities of the unsupplemented growth control, ampidliin and different concentrations of antibacterial agent CL812 against methictllin-suscepUbie S. aureus ATCC 49775 using broth microdilution methodology according to example 4. The concentrations of NCL812 tested were at the MIC and four times the MIC determined under test conditions, up to 24 h incubation. Ampidliin was tested at the MIC- Bactericidal activity was tested at 0, 1. 2. 4, 8, 12, and 24 h for antibacterials;

Figure 1 shows a graph of kill kinetic curves for methiciliin-susceptible S. aureus ATCC

49775 demonstrating bactericidal activity of MCL8 2 using the Clinical and Laboratory Standards Institute macrodilution methodology in a 10 ml viai according to example 4. The concentrations of antibacterials tested were at 1* and 4x the MiC determined under test conditions. Bactericidal activity was determined at 0, i, 2, 4, 8, 2 and 24 h after antibacterial addition. Bactericidal activity was defined as a 3log ½ (99.9%) decrease in the number viable bacteria from the initial inoculum size;

Figure 12 shows a graph indicating the change of pH during macro-broth dilution assay for S. pneumoniae strain D39 exposed to 4 g/mL in NCL812 and 0.0023 ijg/mL ampiciilin according to example 5;

Figure 3 shows a graph illustrating the 48-hour time-kill of S. ptmumoniae strain D39 treated with NCL812 according to example 5; Figure 1 shows a graph illustrating the 48-hour time-Kill of S. pneumoniae strain D39 treated with NCL.082 according to example 5:

Figure 15 shows graph illustrating in the 14-hour time-kill of S. pneumoniae strain 039 treated with NCL812 according to example 5;

Figure 16 shows a graph illustrating in the 1 -hour time-kiit of S. pneumonia strain D39 treated with NCL062 according to example 5;

Figure 17 shows a graph illustrating the 14-hour time -kill of S. pneumoniae strain D39 treated with ampiciflin according to example 5;

Figure 18 shows a graph illustrating the 12-hour time-kill of S. pneumoniae strain D39 treated with NCL812, adopted from the Figure 43. according to example 5;

Figure 19 shows a graph illustrating the 12-hour time-ki!E of S. pneumoniae strain D39 treated with NCL062, adopted from the Figure 44, according to example 5;

Figure 20 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with ampic iltn according to example 5;

Figure 21 shows a raph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with erythromycin according to example 5;

Figure 22 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with NCL812 and 5% choline chloride;

Figure 23 shows a graph illustrating the 12-hour time-kill of S. pneumoniae strain D39 treated with NCL812 and 5% choline chloride according to example 5;

Figure 24 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with NCLQ62 and 5% choline chloride according to example 8;

Figure 25 shows a graph illustrating the 12-hour time-kill of S. pneumoniae strain D39 treated with NCL062 and 5% choline chloride according to example 5;

Figure 26 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with ampiciilin and choline chloride according to example 5;

Figure 27 shows a graph of the relative BC of D39 treated with MCL812 or NCL082 for

48 hours according to example 5: Figure 28 shows a graph illustrating the relative minima! bactericidal concentration ( 8C) of S. pneumoniae strain D39 treated with ampicifiin over a 48 h time period according io example 5;

Figure 29 shows a graph illustrating the relative MBC for S. pneumoniae strain D39 treated with erythromycin over a 48 h time period according to example S;

Figure 30 shows a graph illustrating the viable count (!og 10 CFU ml} of S. pneumoniae strain P39 treated with NCL812 from a macro-broth dilution of time-kill over 24 hours according to example 5;

Figure 31 shows a graph illustrating the viable count Ciog^CFU/ml) of S. pneumoniae strain DSD treated with ampictllin from a macro-broth dilution of time-kill over 24 hours according to example 6;

Figure 32 is a bar graph illustrating the mean cell membrane thickness of treated and untreated D39 according to example 5;

Figure 33 is a bar graph illustrating the mean width of periplasms space of treated (16 pg/mL NCL812) and untreated 039 samples according to example 5;

Figure 34 shows the kill kinetics of MRSA 580 isolate obtained at different concentrations of NCL81 over a period of 8 hours according to example 7;

Figure 35 shows the kill kinetics of IvIRSA 580 in different concentrations of NCL812 over a period of 24 h according to example 7;

Figure 38 shows the kill kinetics of MRSA 698 in different concentrations of NCL812 over a period of 24 h according to example 7;

Figure 37 shows the kill kinetics of VRE 26c(dc) at different concentrations of NCL812 over a period of 24 h according to example 7;

Figure 3$ shows the kill kinetics of VRE iSc(dc) at different concentrations of NCLS12 over a period of 24 h according to example 8;

Figure 39 shows the kill kinetics assay of Staphylococcus aureus KC01 at different concentrations of NCL8i , up to 24 h incubation according to example 8;

Figure 40 shows the kill kinetics assay of Enterococcus feeca!is USA01 at different concentrations of CL812, up to 24 h incubation according to example 8; and Figure 41 is a graph illustrating the cumulative release of NCL812 and NCLOSS from Formulation B according to example 10.

DESCRIPTION OF EMBODIMENTS

General

100175] Before describing the present invention in detail, it is to be understood that the invention is not limited to particular exemplified methods or compositions disclosed herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[00176] All publications referred to herein, including patents or patent applications, are incorporated by reference in their entirety. However, applications thai are mentioned herein are referred to simply for the purpose of describing and disclosing the procedures, protocols, and reagents refen-ed to in the publication which may have been used in connection with t e; invention. The citation of any publications referred to herein is not to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[00177] In addition, the carrying out of the present invention makes use of, unless otherwise indicated, conventional microbiological techniques within the skill of the art. Such conventional techniques are known to the skilled worker.

100178] As used herein, and in the appended claims, the singular forms "a\ 'an * , and "the" include the plural unless the context clearly indicates otherwise.

[00179] Unless otherwise indicated * ail technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any matehais and methods similar to, or equivalent to, those described herein may be used to carry out the present invention, the preferred materials and methods are herein described.

[00180] The invention described herein may include one or more ranges of values {e.g. size, concentration, dose etc). A range of values will be understood to include all values within the range, including the values defining the range, and vaiues adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which define the boundary of the range.

(00181] The pharmaceutical or veterinary compositions of the invention may be administered in a variety of unit dosages depending on the method of administration, target site, physiological state of the patient, and other medicaments administered. For example, unit dosage form suitable for oral administration include solid dosage forms such as powder, tablets, pills, and capsules, and liquid dosage forms, such as elixirs, syrups, solutions and suspensions. The active ingredients may also be administered parenterally in sterile liquid dosage forms. Gelatin capsules may contain the active ingredient and inactive ingredients such as powder carriers, glucose, lactose, sucrose, mannitol. starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate, and the like.

[00182] The phrase "therapeutically effective amount" as used herein refers to an amount sufficient to inhibit bacterial growth associated with a bacterial infection or colonisation. That is, reference to the administration of the therapeutically effective amount of a compound of Formula f according to the methods or compositions of the invention refers to a therapeutic effect in which substantial bacteriocidal or bacteriostatic activity causes a substantia! inhibition of bacterial infection The term "therapeutically effective amount" as used herein, refers to a sufficient amount of the composition to provide the desired biological, therapeutic, and/or prophylactic result. The desired results include elimination of bacterial infection or colonisation or reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system An effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In relation to a pharmaceutical or veterinary composition, effective amounts can be dosages that are recommended in the modulation of a diseased state or signs or symptoms thereof. Effective amounts differ depending on the composition used and the route of administration employed. Effective amounts are routinely optimized taking into consideration pharmacokinetic and pharmacodynamic characteristics as well as various factors of a particular patient, such as age. weight, gender, etc and the area affected by disease or disease causing microbes.

100183) As referred to herein, the terms "treatment" or "treating" refers to the full or partial removal of the symptoms and signs of the condition. For example, in the treatment of a bacterial infection or colonisation, the treatment completely or partially removes the signs of the infection. Preferably in the treatment of infection, the treatment reduces or eliminates the infecting bacterial pathogen leading to microbial cure. 00184} As referred to herein, the term "bacteria" refers to members of a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals and can be present as individual cells or present in linear chains or clusters of variable numbers and shape. Preferably the terms "bacteria" and its adjective "bacterial" refer to bacteria such as the Gram positive Staphylococcus spp, Streptocccus spp, Bacillus spp, &iferococc s spp, Listeria spp, and WO 2014/176636 PCT/AU2O14/O0O48J

63

anaerobic bacteria; Gram negative Escherichia coli. Enterohacter spp. Klebsiella spp and Pseudomonas spp; and the cell wall free bacteria such as Mycoplasma spp and Ureaplasma spp. The terms may refer to an antibiotic-sensitive strain or an antibiotic-resistant strain. In a preferred embodiment, the terms refer to MRSA or MRSP. In another preferred embodiment, the terms refer to MDR Staphylococcus spp, Streptococcus spp, Enterococcus spp, Clostridium difficile, Escherichia coii, Enreraoacferspp, Klebsiella spp and Pseudomonas spp.

(00185] Referred to herein, the term "meihicillln-resistant bacteria" (such as methiciflin- resistant Staphylococcus) refers a bacteria isolate that demonstrates resistance at any dose to all β-lactams including penicillins, carbapenems and first to fourth generation cephalosporins, but not to the fifth generation antt-MRSA cephalosporins {for example ceftaroline). Multidrug* resistant (MDR) is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, extensively drug-resistant (XDR) is defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and pandrug-resistant (PDR) is defined as non- susceptibiliiy to all agents in ail antimicrobial categories currently available.

[00186] An example of susceptible, MDR, XDR and PDR bacteria includes the following. Wild type, antibacterial unexposed isolates of Staphylococcus aureus that are likely to be susceptible to all of the following antibacterial categores (and agents): aminoglycosides (for example gentamicin); ansamycins (for example rtfampicin); anti-MRSA cephalosporins (for example ceftaroline); anti-staphylococcal β-lactams {for example oxacillin or cefoxitin): carbapenems {for example ertapenem, imipenem, meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for example cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (for example cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefotetan); fluoroquinolones (for example ciprofloxacin or moxifloxacin); folate pathway inhibitors (for example thmethoprim-sulphamethoxazole); fucidanes (for example fusidic acid); g!yco eptides (for example vancomycin, teicoplanin or telavancin); glycylcyclines (for example tigecycline); lincosamides (for example clindamycin); lipopeptides (for example daptomycin); macrolides (for example erythromycin); oxazolidtnones (for example linezoHd or tedizolid): phenicols (for example chloramphenicol); phosphonic acids (for example fosfomycin); streptogramins (for example quinupristin-dalfopristin); and tetracyclines (for example tetracycline, doxycyc!ine or minocycline), isolates that are non-susceptible to more than one agent in more than three antimicrobial categories are classified as MDR (afi MRSA, for example, meet the definition of MDR). Isolates that are non-susceptible to more than one agent in ail but one or two antimicrobial categories are classified as XDR. Isolates that are non-susceptible to all listed antibacterial agents are PDR.

RECTIFIED SHEET

(Rule 91) ISA/AU (00187) Pharmaceutically and veterinary acceptable salts include salts which ret in the biological effectiveness and properties of the compounds of the present disclosure and which are not biologically or otherwise undesirable. In many sses, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carbexyi groups or groups similar thereto. Acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as by way of example only, aikyi amines, dialkyl amines, In ' aiky! amines, substituted alkyl amines, di(subsrituted aikyi) amines, iri{substituted alky!) amines, alkenyi amines, dialkeny! amines, trialkenyj amines, substituted alkenyi amines. di{ substituted alkeny!) amines, ^{substituted alkenyi) amines, cycioalkyl amines, di(cycloalky!) amines, tri(cycloaikyi) amines, substituted cycioalkyl amines, disubstituted cycioalkyl amines, tnsubstituted cycfoalkyf amines, cycloalkenyi amines, di(cycioaikenyi) amines, tri(cycloaikenyi) amines, substituted cycloalkenyi amines, disubstituted cycloalkenyi amines, trisubstituted cycloalkenyi amines, ary! amines, diaryJ amines, triaryt amines, heteroaryi amines, diheteroaryl amines, triheteroaryS amines, heterocyclic amines, diheterocyclic. amines, triheterocyciie amines, mixed dt- and tri-amirses where at least two of the substituents on the amine are different and are selected from the group consisting of alkyf, substituted aikyi, alkenyi, substituted alkenyi, cycioalkyl, substituted cycioalkyl, cycloalkenyi. substituted cycloalkenyi, ary!, heteroaryi, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the ami o nitrogen, form a heterocyclic or heteroaryi group. 00188] Pharmaceutically and veterinary acceptable acid addition salts may be prepared from inorganic and organic acids. The inorganic acids that can be used include, by way of example only, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. The organic acids that can be used include, by way of example only, acetic acid, propionic acid, glycoiic acid, pyruvic acid, oxalic acid, malic acid, maionic acid, succinic acid, maieic acid, fumaric acid, tartaric add, crtric acid, benzoic acid, cinnamic acid, mandelic acid, methanesuifonic acid, ethanesuifonic a d, p-toluenesulfonic acid, salicylic acid, and the like.

[00189] The pharmaceutically or veterinary acceptable salts of the compounds useful in the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanoi, isopropanol, or acetonitriie are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences. 17th ed.. Mack Publishing Company, Easton, Pa. (1985), p. 1418, the disclosure of which is hereby incorporated by reference. Examples of such acceptable sails are the iodide, acetate, phenyl acetate, trif!uoroacetate, aery! ate, ascorbate, benzoate, chSorobenzoate, dinitrobenzoata, hydroxybenzoate. methoxybxnzoate, methyibenzoate, o-acetoxybenzoate, naphthaiene-2- benzoate, bromide, isobutyrate, p senyibuiyrate, y-rtydroxybutyrate, (S-hydroxybutyrate, utyne- 1,4-dioate, hexyne-i,4-dsoate. hexyne- 1,6-dioate, caproate, caprylate, chloride, cinnamate, citrate, decanoaie, formate, fumarate, glycoliate, heptanoate, hippurate, lactate, malate, maieate, hydroxyma!&ate, roalonate, mandelate, mesylate, nicotinate, isonscotinate, nitrate, oxalate, phtha!ate, terephtha!ate. phosphate, monohydrogenphosphate, dihydrogenphosphate, netaphosphate, pyrophosphate, propioiate, propionate, phenylpropionate, salicylate, sehaeaie, succinate, suberate, sulfate, bisuifate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesuifonate. p-bromopheny!sulfonate, ch!orobenzenesulfonate, prcpanesulfonate, ethanesu!fonste, 2- hydroxyethanesuifonate, merhanesultonate, naphthalene -l-su!fonate, naphthalene -2-sulfonate, p-toluenesuifonate, xy!enesuifonate, tariarate. and the like.

[00i§0] The pharmaceutical or veterinary compositions of the invention may be formulated in conventional manner, together with other pharmaceutically acceptable excipients if desired, into forms suitable for oral, parenteral, or topical administration. The modes of administration may include parenteral, for example, intramuscular, subcutaneous and intravenous administration, oral administration, topical administration and direct administration to sites of infection such as intraocular, intraaural, intrauterine, intranasal, intramam nary, intraperitoneal, intralesiona!, etc.

[00191] The pharmaceutical or veterinary compositions of the invention may be formulated for oral administration. Traditional inactive ingredients may be added to provide desirable colour, taste, stability, buffering capacity, dispersion, or other known desirable features. Examples include red iron oxide, silica gel, sodium laurel sulphate, titanium dioxidss, edible white ink, and the like. Conventional diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as sustained-retease compositions for the continual release of medication over a period of time. Compressed tablets may be in the form of sugar coated or film coated tablets, or enteric-coated tablets for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colouring and/or flavouring to increase patient compliance. As an example, the oral formulation comprising compounds of the invention may be a tablet comprising any one, or a combination of, the following excipients: calcium hydrogen phosphate dehydrate, mi ocrystalfine cellulose, lactose, hydroxypropy! methyl cellulose, and talc.

}0C192] The compositions described herein may be in the form of a liquid formulation. Examples of preferred liquid compositions include solutions, emulsions, injection solutions, solutions contained in capsules. The liquid formulation may comprise a solution that includes a therapeutic agent dissolved in a solvent. Generally, any solvent that has the desired effect may be used in which the therapeutic agent dissolves; and which can be administered to a subject. Generally, any concentration of therapeutic agent that has the desired effect can be used. The formulation in some variations is a solution which is unsaturated, a saturated or a supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components, in some variations the solution formed is a in situ gelling formulation. Solvents and types of solutions that may be used are well known to those versed in such drug delivery technologies.

(00193) The composition described herein may be in the form of a liquid suspension. The liquid suspensions may be prepared according to standard procedures known in trie art. Examples of liquid suspensions include micro-emulsions, the formation of completing compounds, and stabilising suspensions. The liquid suspension may be in undiluted or concentrated form. Liquid suspensions for oral use may contain suitable preservatives, antioxidants, and other excipients known in the art functioning as one or more of dispersion agents, suspending agents, thickening agents, emulsifying agents, wetting agents, solubilising agents, stabilising agents, flavouring and sweetening agents, colouring agents, and the like. The liquid suspension may contain glycerol and water.

(00194) The composition described herein may be in the form of an oral paste. The oral paste may be prepared according to standard procedures known in the art.

(00195) The composition is described herein may be in the form of a liquid formulation for injection, such as intra-muscuiar injection, and prepared using methods known in the art. For example, the liquid formulation may contain polyvinylpyrrolidone K30 and water.

(00196) The composition is described herein may be in the form of topical preparations. The topical preparation may be in the form of a lotion or a cream, prepared using methods known in the ah. For example, a lotion may be formulated with an aqueous or oily base and may include one or more excipients known in the art, functioning as viscosity enhancers, emulsifying agents, fragrances or perfumes, preservative agents, chelating agents, pH modifiers, antioxidants, and the like. For example, the topical formulation comprising one or more compounds of the invention may be a gel comprising anyone, or a combination of, the following excipients: PEG 8000, PEG 4000, PEG 200, glycerol, propylene glycol. The NCL812 compound may further be fonnuiated into a solid dispersion using SoluPSus (BASF, www.soluplus.c m) and formulated with anyone, or a combination of, the following excipients: PEG 8000, PEG 4000, PEG 200, glycerol, and propylene glycol. |00197] For aerosol administration, the composition of the invention Is provided in a finely divided form together with a non-toxic surfactant and a propeiiant. The surfactant is preferably soluble in the propeiiant. Such surfactants may include esters or partial esters of fatty acids.

[00198} The compositions of the invention may alternatively be formulated for delivery by injection. As an example, the compound is delivered by injection by any one of the following routes: intravenous, intram scuiar, intradermal, intraperitoneal, and subcutaneous.

100199] The compositions of the invention may alternatively be formulated using nanotechnoiogy drug delivery techniques such as those known in the art. Nanotechno!ogy- based drug delivery systems have the advantage of improving bioavailability, patient compliance and reducing side effects.

[00200] The formulation of the composition of the invention includes the preparation of nanoparticles in the form of nanosuspensions or nanoe ulsions, based on compound solubility. Nanosuspensions are dispersions of nanosized drug particles prepared by bottom-up or top- down technology and stabilised with suitable excipients. This approach may be applied to the compounds of the invention which cars have poor aqueous and lipid solubility, in order to enhance saturation solubility and improve dissolution characteristics. An example of this technique »s set out in Sharma arid Garg (2010) {Pure drug and polymer-based nanotecbnoiogies for the improved solubility, stability, bioavailability, and targeting of anti-HIV drugs. Advanced Drug Delivery R&vi&ws, 62. 491-502). Saturation solubility will be understood to be a compound-specific constant that depends on temperature, properties of the dissolution medium, and particle size (<1~2 pm).

[00201] The composition of the invention may be provided in the form of a nansuspension. For nanosuspensions, the increase in the surface area may lead to an increase in saturation solubility. Nanosuspensions are colloidal drug delivery systems, consisting of particles belo 1 pm. Compositions of the invention may be in the form of nanosuspensions including nanocrystaliine suspensions, solid lipid nanoparticles (SLNs), polymeric nanoparticles, nanocapsu!es, polymeric micelles and dendrimers. Nanosuspensions may be prepared using a top-down approach where larger particles may be reduced to nanometre dimensions by a variety of techniques known in the art including wet-milling and high-pressure homogenisation. Alternatively, nanosuspensions may be prepared using a bottom -up technique where controlled precipitation of particles may be carried out from solution.

{0020 ] The composition of the invention may be provided in the form of a nanoemulsion. Nanoemulsions are typically clear oiMn-water or water-in-oif biphasic systems, with a droplet size in the range of 100-500 nm, and with compounds of interest present in the hydrophobic phase. The preparation of nanoemulsions may improve the solubility of the compounds of the invention described herein, leading to better bioavailability. Nanosized suspensions may include agents for electrostatic or steric stabilisation such as polymers and surfactants. Compositions in the form of SLNs may comprise biodegradable lipids such as triglycerides, steroids, waxes and emuisifiers such as soybean lecithin, egg lecithin ; and poioxamers. The preparation of a SLN preparation may involve dissolving/dispersing drug in melted lipid followed by hot or cold homogenisation. If hoi homogenisation is used, the melted fipidic phase may be dispersed in an aqueous phase and an emulsion prepared. This may be solidified by cooling to achieve SLNs. !f co!d homogenisation is used, the lipidic phase may be solidified in liquid nitrogen and ground to micron size. The resulting powder may be subjected to high-pressure homogenisation in an aqueous surfactant solution. 00203] The Compounds of Formula ! as described herein may be dissolved in oils/liquid lipids and stabilised into an emulsion formulation. Nanoemu!sions may be prepared using high- and low-energy droplet reduction techniques. High-energy methods may include high-pressure homogenisation, ulirasonication and nicrofiuidisation. if the low-energy method is used, solvent diffusion and phase inversion will generate a spontaneous nanoemu!sion. Lipids used in nanoemuisions may be selected from the group comprising triglycerides, soybean oil, safflower oil, and sesame oil. Other components such as emuisifiers, antioxidants, pH modifiers and preservatives may aiso be added.

|00204] The composition may be in the form of a controlled -release formulation and may include a degradabSe or non-degradabie polymer, hydrogei, organogel, or other physical construct that modifies the release of the compound, it is understood that such formulations may include additional inactive ingredients that are added to provide desirable colour, stability, buffering capacity, dispersion, or other known desirable features. Such formulations may further include liposomes, such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use in the invention ma be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and a sterol, such as cholesterol.

[00205] The formulations of the invention may have the advantage of increased solubility and/or stability of the compounds, particularly for those formulations prepared using nanotechnology techniques. Such increased stability and/or stability of the compounds of Formula I may improve bioavailability and enhance drug exposure for oral and/or parenteral dosage forms.

[00208] Throughout this specification, unless the context requires otherwise, the word "comprise'' or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

EXAMPLES

EX PLE 1 he minimum inhibitory concentrations (MiC) for NCL812 in methicillin- resistant Staphylococcus aureus (MRSA), vancomycin-resisiant Enterococcus spp. (VRE) and Streptococcus pneumoniae.

Specific

{00207] As is apparent from the preceding summary of the invention, the invention relates to compounds of Formula I, methods of treatment of a bacterial infection, uses and medical devices.

[00208] This study was undertaken to determine minimum inhibitory concentrations {MIC } for antibacterial agent NCL812. The antibacterial agent represents a potentially new class of drug with a perceived narrow spectrum of activity against bacteria and a novel mechanism of action. This study focused on recent isolates of three major opportunistic pathogens of humans where the development of antibacterial resistance to existing antibacterial classes is problematic; methicHlin-resistant Staphylococcus aureus (MRSA), vanco ycin-resistant Enterococcus spp. (VRE) and Streptococcus pneumoniae.

[00209] in this example, NCL812 minimum inhibitory concentrations (MICs) were determined for 01 Australian clinical isolates (comprised of 21 MRSA, 20 putative YRE and 20 S. pneumoniae isolates). The MiC profiles for CL312 were found to be remarkably consistent, with !G JS and tCgn values of A pg/mL recorded for each of the species tested.

Materials and Methods

Bacierisi Isolate Collection and Identification

[00210] Sixty one test isolates were sourced from clinical diagnostic microbiology laboratories. The MRSA isolates were originally cultured on selective Brilliance MRSA Chromogenic Agar (Oxoid). Suspect colonies were selected on the basis of their colony appearance on this agar and identification as Staphylococcus aureus was determined using colony characteristics on non-selective Sheep Blood Agar (SBA) and phenotypic characteristics such as Gram stain, positive catalase test, positive coaguiase test (tube coagulase test using rabbit plasma) and dumping factor (agglutination with the Oxoid Stapbytect latex test), positive Voges Proskauer test, and the ability to produce acid from trehalose. A positive cefoxitin resistance screen confirmed the isolates as MRSA. All Enterococcus isolates underwent a standard biochemical identification. Biochemical profiling provisionally identified fou of the VRE isolates as Enteroc ccus faecaiis and the remainder as Enteroooccus faecium. All S. pneumonia® isolates were identified on the basis of standard biochemical profiling.

Preparation of AntibacteriaSs

[00211] Analytical grade NCL812 (batch 20081214} with a defined potency of 1000 mg/g {is 100%) was obtained. The powder was stored at a temperature of -20 *C. Aiiquots (1 ml) of stock solution {25.0 mg/mL) were prepared in DMSO and stored at -SO * C and defrosted immediately before use.

Preparation of 96 we// microtitre plates for broth microdiiution MIC test using NCL812

[00212] Cation adjusted Mueller Hinton Broth (CAMHB) was prepared using 100 ml sterile Mueller Hinton broth (pH adjusted). To each 100 mL volume, 125 μΐ of calcium stock solution (10 mg Ca 2* per ml) and 43 μΐ magnesium stock solution {10 rng g a* per ml) was aseptically added. Sufficient broth is made up for daily use, with unused portions being stored at 4 overnight.

[00213] Microdiiution trays with 4% lysed horse blood in CAMHB was prepared by lysing horse blood (Oxokt) by repeated freezing and thawing {3-4 times) and asepticaity mixing the lysed horse b!ood (LH8) 50:50 with sterile distilled water. A ceil free suspension was obtained by centrifuging 50% LHB at 16.000*g (7000 rpm) for 20 min. The supernatant was decanted, recentrtfuged and stored frozen. 50% LHB was diluted with CAMHB to obtain a final concentration of 4% (7 mL LHB into S3 mL CAMHB). 4% LHB-CA HB was used instead of CAMHB in all steps in the preparation of the microdiiution trays and preparation of antimicrobial solutions for Streptococcus species. 00214] A stock antibiotic working solution of NCL8 2 was prepared to a concentration of 25 60 mg/mL. Potency was described as 1000 mg/g or 100%. The powder was dissolved in 10 rnL DMSO and 1 mL volumes were aiiquoted into eppendorf tubes and stored at -80 X. When added to CAMHB, a fine cloudy precipitate formed., and was shaken well before and during aliquoting.

[00215] A stock solution of ampictllin was prepared to the concentration of 25.60 mg/mL. Aropiciliin was used for internal quality control. The powdered ampicii!in was dissolved in 4 mL of phosphate buffer pH 8.0, 0.1 mo!/L. then diluted in 6 mL phosphate buffer pH 8 0, 0.1 mo!/L. 1 mL volumes were aiiquoted into eppendorf tubes and stored at -SO "C.

[00216] For Staphylococcus aureus, a working solution of 256 pg/mL was prepared by diluting stock solutions as described above 1 :100 in CAMHB (100 uL into 9.8 mL). When 90 pL was added to each well 12, there was a 1 :2 dilution so thai well 2 had 128 pg/mL of antibiotic. The range of antimicrobial was calculated as 0.25 pg/mt. (well 3} to 128 pg/mL {well 12).

(00217] For Enterococcus species, a working solution of 64 pg mL was re ared by diluting stock solutions as described above 1 :400 in CAIVSHB (100 pL into 9.9 rnl, then further dilute this 1 :4}. When 90 pL was added to well 12 there was a 1 :2 dilution so that well 12 had 32 pg/mL of antibiotic.

[002 8] For Sfrepiococcus pneumoniae, a working solution of 64 pg/mt was prepared by diluting stock solutions as described above 1 :400 in 4% LHB-CAMH8 ( 100 pL into 9.9 mL, then further diluting this 1 :4}. When 00 pL Mars added to well 12 there was a 1 :2 dilution so that well 12 had 32 pg/rol of antibiotic.

[00219] Serial dilutions were prepared in 96 well plates were set up in a safety cabinet according to methods standard in the art. Bhfe!y: 90 pL of the working antibiotic solution was added to each well in Column 12 of the plate, and mixed well, before SO pt was transferred to column 1 1. The solutions were mixed again, and then transferred to the next column as before, continuing the dilutions through to column 3. Mixing the well requires the pick up and expulsion of 90 pL in each well 3-4 times before picking up and transferring the 90 pL to the next welt. Column 2 (bacterial positive control) and column 1 (negative control) did not form part of the serial dilution. The trays were set up as follows: 2 strains were tested in duplicate in one tray, such that strain 1 was located in rows A to D, strain 2 was located in rows E to H, etc. The MSC (pg/mL) interpretive Standard for Ampicillin using Control strains is shown in Tabie 1 below. Staphylococcus aureus ATCC 29213 Acceptable MIC range for Ampicillin - 0.5 to 2 pg mL Efi erococci/s faecalis ATCC 29212 Acceptable MIC range for Ampicillin - 0.5 to 2pg/mL Streptococcus pneumoniae ATCC 49619 Acceptable MIC range for Ampicillin - 0.06 to 0.25 pg/ nL.

Table 1 : MIC (pg/mL) Interpretive Standard lor Ampicillin using Control strains according to Example .

Preparation of bacteria! suspension for broth icrodiiution MIC method

[00220] Fresh cultures of bacteria were prepared for testing on sheep blood agar (SBA), and overnight incubation at 37 "C as follows; -3 colonies of each strain in 7 ml sterile saline, and the ODsoe measured as an indication of the density (approx. 0.5x 108 CFU ' mL or 0.5 cFartand Standard). The bacterial suspension was adjusted to a final absorbance of 0.08 to 0.100, using saline to achieve correct density, and as the blank. Within 15 minutes of preparation., the adjusted bacterial suspension with sterile saline 1 :20 (1 roL into 19 ml sterile saline) to achieve a final bacterial concentration of 4 to 5* 10 s CFU/mL The bacterial solution was placed into a sterile trough and 10 μΐ of bacterial solution added into wells 2 through to 12 on each required row {dilution of 1 :10, with final concentration of bacteria in weiis ~ 5*105 CFU/mL}. The tray was sealed and incubated at 37 U C for 18-24 h. The purity of bacterial suspension was confirmed by streaking out 50 μΐ of the 1.20 dilution onto a S8A plats, which was incubated for 37 *C for 8 h and examined. Viable counts were carried out to ensure that the correct concentration of bacteria and been added to the wells. The diluted bacteria! solution {4 to 5 * 10 e CFU/mL) was diluted 1 :10 downwards by adding 100 pL to 900 it of steriie saline in sterile tubes, and the serial dilutions continued 1 : 10 for S tubes. 100 pL (4-5 drops) of the 4* and 5 ih dilutions {tube 4 = 105 and tube 5 = 106 CFU/ml) was plated around in duplicate on pre- dried PCA agar plates and incubated at 37"C overnight. The following day the number of colonies on the plates was counted and the average count in 100 μΐ obtained. The study was multiplied by 0 to obtain a viable bacterial count per mL.

Description and identification of isolates

[0022 J The sVIRSA isolates were originally cultured on selective Brilliance MRSA Chfomogenic Agar (Oxoid). Suspect colonies were selected on the basis of their colony appearance on this agar and identification as Staphylococcus aureus was determined using colony characteristics on non-selective S8A and phenoiypic characteristics such as Grafts stain, positive cata!ase test, positive coagu!ase test (tube coagulase test using rabbit plasma) and dumping factor {agglutination with the Oxoid Staphytect latex test), positive Voges Proskauer test, and the ability to produce add from trehalose. A positive cefoxitin resistance screen confirmed the isolates as MRSA.

[00222] MRSA clonal complexes were determined by rapid molecular typing. Two of the strains could not be typed using the rapid method, as shown in Table 2 below.

Table 2: a table showing the MRSA clonal complexes according to Example .

Organism / Sample no. j Clonal complex

Staph, aureus ATCC 29213 j HA

MRSA 71 δ COB

MRSA 741 j CC8

MRSA 580 ! CC8

MRSA 622 j CC22

MRSA 815 j CC88

NA: No Applicab e; TBD: Isolates could not be ΐ method and are currently being identified using traditional methodology.

[00223] Alt Enterococcus isolates underwent a simplified biochemical identification based on Quinn et. ai. (1994, Clinical Veterinary Microbiology, osby Ltd, New York) Biochemical profiling provisionally identified four of the VRE isolates as Enterococcus fa&ca!is and the remainder as possibly Enterococcus faecium. All S. pneumonia® isolates were identified on the basis of standard biochemical profiling.

Test ptvduci and storage

[00224] Analytical grade NCL812 (batch 20081214) with a defined potency of 1000 rng/g (ie 100%) was obtained and the powder svas stored at a temperature of -20 C C. Aliquots (1 rnt) of stock solution (25.6 mg/mL) were prepared in DMSQ and stored at -80 °C and defrosted immediately before use.

Minimum inhibitory conc&niration determination

[00225] Minimum inhibitory concentrations {Mg/mL} were determined using the broth microdiiution method recommended by the Clinical and Laboratory Standards institute (CLSI) {Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Seventh Edition. CLSi 7-A7, 2006; Performance Standards lor Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria isolated from Animals; Approved Standard - Second Edition. CLSI 31--A2, 2002; Performance Standards for Antimicrobial Susceptibility Testing; CLS! M2-A9, 2006). 100228] The MIC was regarded as the the lowest concentration o» an antimicrobial agent that completely inhibited growth of the organism in the microdilution wells as detected by the unaided eye. MIC breakpoints were determined by visual assessment and then confirmed using an ELISA plate reader, measuring absorbance levels at 450 nrn. Bacterial growth (turbidity) in the wells with antimicrobial was compared with the amount of growth (turbidity) in the growth- control well (containing no antimicrobial). All isolates were tested in duplicate, if there was a difference of greater than one two-loW dilution in the results, the test was repeated a third time. The purity of the isolates was closely monitored during testing by subcuituring the prepared bacterial inoculum onto SSA. Control organisms {Enterococcus faeca!is strain ATCC 29212, S. aureus strain ATCC 29213 and S. pneumoniae strain ATCC 49619) were used throughout the testing to monitor qualit control The MICs of the control strains for the antimicrobial ampiciifin (range 1.0, 2.0 and 0.06 μρ,/mL, respectively) were determined for each testing run as an interna! quality control. The U\C , MIC & c and MIC range (minimum and maximum) were calculated for each of the bacterial groups.

Results

(00227) Ampicillin MIC values obtained for the ATCC control strains were within the normal range expected on the basis of CLSf recommendations. The NCL812 and ampicillin MiC values for each isolate are indicated in Table 3 (MRSA isolates), Table 4 (VRE isolates) and Table S (S. pneumoniae isolates) below. ICso, MIC¾„ MIC mode and MIC range for each of the species of bactena tested are shown in Table 6 below. The MiC«, is considered to be the lowest concentration which inhibits visible growth for 50% of the isolates. The MI so is considered to be the lowest concentration which inhibits visible growth for 90% of the isolates. The MIC mode is the most commonly occurring MIC value and MIC range the minimum and maximum MiC values obtained.

Table 3: The Minimum Inhibitory Concentrations for the individual Staphylococcus aureus isolates according to Example 1.

¾*iC Testing - A**P,'NC 12 T*st 2 19 t 1 Test 2

IC«0 aitrsS

Table 4: The Minimum inhibitory Concentrations for the in tividua! Entsrococcus isolates according to Example 1 ,

Table 5: The Minimum inhibitory Concentrations for the individual Streptococcus pneumoniae isolates according to Example 1.

Table 6: The NCL812 M!Cse, MICso, MIC mode and MIC range for Australian isolates of MRSA, VRE and S. pneumoniae.

[00228} NCL812 MIC values were consistent within and between each of the three species. MIC*; and MICso values were both equal (4 Mg/m!) for MRSA, VRE and S. pneumoniae isolates, with less than 10% of isolates showing MIC values either 1-2 dilutions below or only one dilution above this figure.

[00229] On the baste of these results, NCL812 represents a new antibacterial. EXAMPLE 2: Effect of NCL812 on Staphylococcus aureus Macromolecular Synthesis Materials and Methods

Te$t compounds

[00230] Test compound NCL812 was transported to the experimental facility under conditions of ambient temperature and then stored at 2-8 * C until assayed. Stock solutions were made by dissolving CL812 dry powder in 100% DMSO to a concentration of 6,400 pg mL Vancomycin (Cat. # 1134335), Rifampin (Cat. # R-7382) and Cerutenin {Cat. # C-2389) were all obtained from Sigma, Ciprofloxacin was obtained from USP (Cat. # 1134335} and Linezolid was obtained from ChemPacific {Cat. # 35710).

Minimal inhibitory Concentration Testing

[00231] The MIC assay method followed the procedure described by the Clinical and Laboratory Standards Institute, or CLSi (Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bact&ria That Grow Aerobicaiiy; Approved Standard— Eighth Edition. CLSi document M07-A8 [ISBN 1-56238-689-1}. Clinical and Laboratory Standards Institute, 040 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-13898 USA, 200S), and employed automated liquid handlers to conduct serial dilutions and liquid transfers. The medium employed for the MIC assay was Mueller Hinton II Broth {MHB II- Becton Dickinson, Sparks, D; Cat No 212322; Lot 9044411). S aureus ATCC 29213 served as the quality control strain, and linezolid was utilized as the quality control antibiotic to validate the assay. NCL812 and iinezoiid were both dissolved in 100% DMSO before addition to the growth medium.

Macromol&cutar Synthesis Assays

Bacteria and growth conditions

[00232] The effect of NCL812 on whole ceii DHA, RNA, cell wall, protein and lipid synthesis was investigated using S. aureus ATCC 29213. Ceils were grown at 35 °C overnight on TrypBcase Soy agar. A colony from the plate was used to inoculate 10 mi of Mueller Hinton broth il {MHB!I}, and the culture was grown to early exponential growth phase {OD«oo = 0 2 to 0.3) while incubating in a shaker at 35 * C and 200 rpm.

DMA, RNA, and protein synthesis

[00233] When ceils reached early exponential phase, 100 uL of culture was added to triplicate welis containing various concentrations of test compound or control antibiotics (5 pL) at 20* the final concentration in 100% DMSO. A 5% DMSO treated culture served as the "no drug" control for ail experiments. Ceils were added in HBH at 105% to account for the volume of drug added to each reaction or in M9 minima! medium for protein synthesis reactions. Following 15 min incubation at room temperature, either H] thymidine (DNA synthesis}.. H] uridine (RNA synthesis) or fHj leucine (protein synthesis) was added at 0.5-1.0 jjCi per reaction., depending on the experiment. Reactions were allowed to proceed at room temperature for 15-30 min and then stopped by adding 12 pL of cold 5% trichloroacetic acid (TCA) or 5% TCA 2% easamino acids (protein synthesis only). Reactions were incubated on ice for 30 min and the TCA precipitated material was collected on a 25 mm GF/A filter. After washing three times with 5 mi of cold 5% TCA, the filters were rinsed two times with 5 mL 100% ethanoi. allowed to dry, and then counted using a Beckman LS3801 liquid scintillation counter.

Ce/7 wall synthesis

[QG234J Bacterial cells in early exponential growth phase were transferred to M9 minimal medium and added to 1.5 mL eppendorf tubes (100 pt tube) containing various concentrations of test compound or control antibiotics (5 μί_) at 20* the final concentration in 100% DMSG as described above. Following a 5 min incubation at 37 t: C, [^CjN-aceiy!giucosarnine (0.4 pCi/reaction) was added to each tube and incubated for 45 min in a 37 "C heating block. Reactions were stopped through the addition of 100 uL of 8% SDS to each tube. Reactions were then heated at 95 for 30 min in a heating block, cooled, briefly centrifuged, and spotted onto pre-wet HA filters (0.45 μ ). After washing three times with 5 mL of 0.1% SDS, the filters were rinsed two times with 5 m! of leionized water, allowed to dry, and then counted using a Beckman LS3801 liquid scintillation counter.

Lipid synthesis

[00235] Bacterial cells were grown to early exponential growth phase in MHBil broth and added to 1.5 mL eppendorf tubes (in triplicate) containing various concentrations of test compound or control antibiotics as described above. Following a 5 min incubation at room temperature, ( 5 H) glycerol was added at 0.5 pCi per reaction.

[00238] Reactions were allowed to proceed at room temperature for 15 min and then stopped through the addition of 375 ΐ chSorofonrs methanol {1 :2} followed by vortexing for 20 seconds after each addition. Chloroform (125 pL) was then added to each reaction, vortexed, followed by the addition of 125 uL dHjO and vortexing. Reactions were centrifuged at 13,000 rprn for 10 min, and then 150 of the organic phase was transferred to a scintillation vial and allowed to dry in a fume hood for at least 1 hr. Samples were then counted via li uid scintillation counting. Results

(00237} Susceptibility testing was conducted with NCL812 and S. aureus ATCC 29213 to determine the concentrations of drug needed in the rnacromoiecular synthesis assays.

[00238] Table 7 shows that the MiC for NCL812 was 4 pg/mL while the quality control agent iinezoiid was within the CLSI-estabiished quality control range (Clinical and Laboratory Standards institute. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth informational Supplement. CLSI document 100-320 [ISBN 1-56238-716-21- Clinical and Laboratory Standards institute, 940 West Vaiiey Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2010). Precipitation of NCL8 2 was observed at≥B pg/mL in plates that were prepared in an identical fashion, but did not receive an inoculum of S. aureus. Macromoiecular synthesis inhibition studies were performed using concentrations of NCL8 2 that were equivalent to 0, 0.25. 0.5, 1, 2, 4 or 8-foid the MIC value {4 Mg/mi) for S. aureus ATCC 29213 (Figures 11-16).

Table 7: Minimum inhibitory Concentrations values foi NCL812 (robenidine) and Iinezoiid against Staphylococcus aureus ATCC29213 according to Example 2.

[00239] Figure 2 shows the effect of NCL812 on DNA synthesis. NCL812 demonstrated no inhibition at 0.25 fold the MIC, 40% inhibition at 0.5 fold, and approximately 95% inhibition at the MiC. This is compared to the control ciprofloxacin which showed approximately 51% at 8 fold the MIC (0.5 μα, roL). The results for NCL812 inhibition of RNA synthesis were very similar to the DNA synthesis study, with rifampicin serving as the positive control (Figure 3. It should be noted that precipitation was observed at 4 to 8 fold the MiC in the Mueller Hinton broth II utilized in the DNA and RNA synthesis assays.

(00240] Protein synthesis was inhibited in a dose dependent manner at 0.25, 0.5, and 1 fold the MIC value of NCL812 showing up to 97% inhibition at the MIC (Figure 4. Unezo!id demonstrated approximately 61% inhibition of protein synthesis at 8 fold the MIC (2 pg ml). Precipitation of NCL812 occurred at 4 and 8 fold the MiC in the protein synthesis assay.

[00241] In Figure 5 NCL812 also showed s somewhat dose-dependent inhibition of ce!S wall synthesis, though there was a large increase in inhibition from 1 to 2 fold the MIC. However, inhibition dropped to approximately 68% and 52% at 4 fold and 8 fold the MIC, respectively. Precipitation of NCL812 occurred at 2, 4. and 8 foid the MIC in the M9 minimal medium used for the cell wail synthesis assay, a d that is the likely cause of the decline in inhibition, in comparison, the positive control vancomycin showed 96% inhibition at 8 fold the MIC (2 g/ml.}. NCL812 demonstrated a similar inhibition profile against iipid synthesis as that shown for DMA and RNA synthesis, reaching approximately 90% inhibition at the MIC {Figure 6). The positive control inhibitor cerulenin demonstrated 72% inhibition at S fold the MIC (32 ug/rnL).

[00242] Figure 7 represents a composite of all five macromoiecular synthesis reactions. It can be observed thai the inhibition curves were similar fo each pathway, suggesting a global inhibition of several pathways simultaneously by CL81 . St is possible thai NCL812 targets the cell membrane, causing leakage of essential ions and/or metabolites, thereby leading to a global shutdown of the cell synthesis pathways.

[00243] In summary, CL812 inhibited D A, RNA, protein, cell wa!i, and Iipid pathways in a growing culture of S. aureus. Though some instances of dose-dependent inhibition of pathways was observed, all five macromoiecular synthesis reactions were similarly sensitive to CL812.

EXAMPLE 3: Effect of NCL812 on ATP Release from Staphylococcus aureus

Materials and Methods

Test compounds

(00244] The test compound NCL812 was shipped under conditions of ambient temperature and then stored at 2-8 "C until assayed. Stock solutions were made by dissolving NCL812 dry powder in 100% DMSO to a concentration of 1,600 μδ/mL. Polymyxin 8 was obtained from Sigma (Cat. # P-4932).

Test Organism

[00245] S. aureus ATCC 29213 was originally acquired from the American Type Culture Collection (Manassas, VA).

ATP Release Assay

(00248) The CellTiter-Gio Luminescent Ceil Viability Assay (Promega) was utilized to measure the leakage of ATP from bacteria. Cultures were grown to early exponential phase (0.2 - 0.3 QF½c) in ueller-Hinton Broth il and then treated with seven different concentrations of either NCL812 or polymyxin B (positive control) utilizing the MIC for each compound as a guide {0, 0.25, 0.5, 1 , 2, 3, 4, or 8 fofd the MIC). The negative control received 2% DM80, which represented the final DMSO concentration in each assay. After a 30 min exposure to drug, ceils were segmented by centrifugation a id the supernatant as analyzed for the presence of ATP. Results; were expressed as ATP concentration released to the medium (pM).

Results

[00247) The MIC for NCL812 has been previously determined to be 4 ug'mL. The ATP release assay is conducted by growing S. aureus to exponential phase and then adding drug at multiples of the MIC in an effort to detect a dose-dependent response.

(00248} As shown in Figure 8 the positive control polymyxin 6 released ATP from S. aureus ceils in a dose-dependent fashion with maximal release of approximately 0.34 Μ ATP at 8 fold the MIC (256 M /rnL). ATP release in the presence of NCL8 2 was dose-dependent at 0.5-1 fold the MiC. resulting in maximal release {0.33 uM) observed at the MIC {4 pg/ml). ATP release actually decreased thereafter at 2 to 8 fofd the MIC. It should be noted that in previous studies precipitation of NCL812 was observed at 4 to 8 fold the MiC in Mueller Hinton broth SI.

[00249] In summary, NCL812 demonstrated dose-dependent release of ATP from actively growing S. aureus cells. ATP release from the cells into the growth medium reached maximum levels at the MiC value, and this was followed by a decrease in ATP release at higher doses. The data indicated that NCL812 may interact with the ceil membrane of S. aureus, causing leakage of vita! metabolites such as ATP.

EXAMPLE 4: in vitro antibacterial activity of CL812 against methicillin-resistanl and methicil!in-susceptible Staphylococcus aureus

Materials and Methods

Antimicrobial agents

[00250] AKquots of stock solution of CL812 (25.6 mg/rnl) was prepared in DMSO, stored at -80 °C and defrosted immediately before use. Ampicsitin stock was obtained from Sigma- Aidrich (Australia). Antimicrobial discs were obtained from Thermo Fisher Scientific (Australia).

Microorganisms

[00251] Clinical isolates of MRSA that represented the most common sequence types of both hospital-acquired (HA) and community-associated (CA) MRSA in Australia were obtained and are described in Table 8 below. The S. aureus control organism ATCC 49775 was used, isolate identification was confirmed by conventional phenotypic methodologies, including the stide coagu!ase test, Vogues-Proskauer test, polymyxin B sensitivity {300 units), and Siaphytect Plus Protein A iatex slide agglutination (Thermo Fisher Scientific Australia). Bacteria were stored at -80 !: C in 40% glycerol broth and routinely grown from stock on sheep blood agar (SBA) incubated at 37 C 'C in subsequent experiments, only fresh cultures «24 h were used.

Tabte 8; Staphylococcus au eus cione/isoiate name, type, source, sni i gram, clindamycin resistance status, multMocus sequence type (MLST) staphylococcal cassette chromosome (SCCmec) type, clonal complex, Panton-Valentine leukocidtn status (PVL), and spa type for isolates used according to example 4

o

0\

00 fv4SSA; methsdllin-suscepisble S. aumus. HA-MRSA. hospital-acquired rnei icf!!in-restetent S. aureus. CA-MRSA: community-associated metnidiiin- resisiani S. aumus, Ern; Er/i roroycin. Ci; Ciprofloxacin. Gn; Genlamicin. Tm; Trimethoprim. Te; Tetracycline. FA; Fusidlc Acid. f; Riiampldn. Up. Mupirocin

H

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© © © 4-. oe

Isolate resistotyping

{00252] Antibiotic-susceptibility profiling of the isolate collection was undertaken using Kirby-Bauer disc diffusion, as recommended by the Clinical and Laboratory Standards institute (CLSI) on f lueiier-Hinton agar, isolates were grown overnight on S8A at 37 * C. Colonies were suspended in physiological saline. Turbidity was adjusted to a 0.5 McFariand standard and suspensions were spread over the medium. Antibiotic discs according to Table 9 below were transferred onto the inoculated medium and analysed after 2.4 h incubation at 37 "C. Isolates labelled as RSA that were not β-lactam-resistant on the basis of the Kirby-Bauer lest were grown from stock on plate count agar supplemented with 5 pg/ml arnpiciilin and subject to repeat testing, as P8P2a expression can be induced by exposure to β-!actarn antimicrobials.

Table 9: Antibacterial agent zone diameter interpretive sizes for Kirby-Bauer disc diffusion, as used in Example 4.

Molecular detection of the protein A end mecA enes to confirm MRSA status

[00253] Isolate identities were confirmed genotypical!y using a duplex polymerase chain reaction (PGR) test targeting the s a (protein A) and mscA {methiciin resistance) genes, in addition, the isolates were tested in a mecA and spa Sybr green real-time PGR. Approximately ten colonies of each overnight bacterial subculture was suspended in 1 * phosphate buffered saline (pH 7.4} and vortexed. Isolates were subject to D A extraction using the Q!Aamp® DNA Mini Kit (Qiagen, Australia) following the manufacturers protocols. Template DNA was e!uted in 50 ΐ of eltrtion buffer and either used directly in PCR, or stored at -20 °C prior to DNA amplification using the spa forward {5 -TGATAGAGTAAATGACATTG-3'} and reverse (5 - TTCTTATCAACAACAAGTTC-3') primers and mecA forward (5'- TTCGTGTCTTTTAATAAGTGAGG-3 ! ) and revere (6'-ATGAAGTGGTAAATGGTAATATCG-3') primers (invitrogen, Australia). Conventionai PCR amplification was performed in a 20 pL volume containing 0 pL HotStarTaq Plus Master i (Qiagen, Australia), 0.5 μΜ of each spa primer, 0.2 μ of each mecA primer, and 3 ul. of extracted DNA. An automated thermal cycler (T100 Thermal Cycler, Bio-Rad) was used for PCR amplification of the spa and mecA genes according to the following conditions: PCR Stage (Enzyme activation at 95 e C for 300 s s followed by 38 amplification rounds of 94 °C for 30 s (denaturation), 50 °C for 30 s (annealing) and 72 C C for 38 s (extension) and then a cooling stage of 20 °C until required): Real-time PCR Stage (Enzyme activation at 95 *C for 300 s, followed by 40 amplification rounds of 95 ¾ C for 15 s (denaturation), 50 °C for 20 s (annealing) and 70 °C for 40 s (extension), a single round at 95 °C for S s, a sinige round at 55 °C for 20 s, continuous melting curve from SS C C to 0 *C and a cooling period of 40 °C for 30 s. The mecA and spe amplified products of 325 and 120 bp, respectively, were detected by GeiRed staining foliowed by electrophoresis in 2% agarose gels.

Minimum inhibitory concentration testing

[00254] The in vitro activities of NCL812 and ampici!lin as a positive control were determined by broth microdiSution as recommended by the CLSi in cation- adjusted Mueller- Hinton H broth. Micrati!er piates containing two-fold dilutions of each antimicrobial agent were inoculated with - 10* CFU/ml of each isolate in a 100 uL final volume. Plates were incubated for 24 h at 37 . Turbidity (absorbance at OD ia ») was measured using a Bio-Rad Benchmark Pius microplate spectrophotometer in Microplate Managed version 5.2.1 (Bio-Rad). Minimum inhibitory concentration (MIC) endpotnts were defined as the lowest antimicrobial concentration assessed by the spectrophotometer that inhibited bacterial growth. ATCC 4977S was included in the isolate collection as a control organism using breakpoints defined by the CLSf. The ΜΙΰεο, MICso (concentrations that inhibited growth of the lower 50% and 90% of tola! organisms, respectively), and MIC range (minimum and maximum) were calculated to profile the antimicrobial susceptibility of the isolate collection.

Bactericidal activity

[00255] The bactericidal activity of CL812 was established by determination of the minimum bactericidal concentration (MBC) and time-kill analyses using CLSi guidelines. The MBC was defined as the lowest drug concentration at which 99.95% of the original inoculum was eliminated.

[00256] Tirne-kill assays for ATCC 49775 were performed in cation-adjusted Mueller- Hinton If broth in Microtiter plates and again in 10 mi volumes for macrodilution assays at antimicrobial concentrations equivalent to 1 * and 4* the !C. Bactericidal activity in macrodilution assays was identified as a 3iog<o, decrease from the initial inoculum si2e. Bacteria were cultured overnight at 37 on S8A. Colonies were suspended in broth and the turbidity was adjusted to a 0.5 McFartand standard to obtain a bacterial suspension of - 0 s CFU/ml. Bacterial suspensions were incubated at 37 "C with shaking. Aliquots were removed at 0, , 2, 4, 8, 2, and 24 h after antimicrobial addition, diluted, plated onto SBA and incubated for 48 h at 37 for viable count determination. Turbidimetric growth curves for S. aureus were obtained for Microtiter plate assays by monitoring optical density changes using a Bio-Rad Benchmark Plus microp!ate spectrophotometer at 600 nm. Optical densities were measured at 0, 1, 2, 4, 8, 12, and 24 h after antimicrobial addition.

Statistical methodology

{00257} Microbiological data was interpreted using CLSI guidelines. Data was examined using the student's West, Fisher's exact test, analysis of variance, and a generalized linear model for tests of between- subjects effects where appropriate. Differences were considered significant at the 0.05 level in IBM SPSS® version 19.0.

Results

Confirmation of Staphylococcus aureus identity and r cA status [00258] Latex agglutination tests confirmed that all 30 isolates were protein A positive. The isolates tested positive for coagu!ase activity using slide agglutination. Voges-Pfoskauer and polymyxin B resistance tests confimie that ail isolates were S, aureus except for a single methicillin-susceptible isolate; MSSA DE-25, as shown in Table 10 below. Based on spa gene PGR amplification, this isolate was not identified as a S. aureus isolate despite testing positive in the protein A latex agglutination and slide coagutase tests. This canine-origin Staphylococcus spp. was identified as Staphylococcus pssudintermedius based on biochemical characteristics. mecA conventional and real-time PGR results confirmed that 86.66% of the isolates were classified as methiciSlin-resistant on the basis of possession of the mecA gene. There were no significant differences between the ability of conventional and real-time PCR to detect the mecA gene (P>0.05).

Tabte 10: Percentage of presumptively identified S. aureus isolates reporting positive to selected phenotypic and genotypic tests according to Example 4.

HA-M SA; hospital-acquired S. aureus . CA-MRSA; community-associated S. aureus . S. aureus isolates svere identified as testing positive to protein A latex agglutination ( rol& A), slide coagulase, Voges-Proskawer and polymyxin B resistance tests, as wefi as testing positive for polymerase chain reaction {PCR} and real-time PCR amplification of the spa gene. Met icil!in-resistarit S. aureus isolates we e identified as isolates testing positive to the criteria described above, as weii as positive for PCR and reel-time PCR of the mec gens.

Tabie 11: Resistance of S. aureus isolates to antibacterial agents using the Kirby-Bauer disc diffusion method according to example 4

HA-MRSA; hospital-acquired rrtethicillin-resislani S. aureus. CA-MRSA; community-associated meinieiiiin-resisiant S. aureus

Staphylococcus aureus antimicrobial susceptibility profiles

[00269] Antimicrobial susceptibility assays revealed that HA- RSA isolates had the highest mean prevalence of resistance to multiple antimicrobial classes (P<0.000). CA-MRSA isolates were ne t most resistant {Ρ 0.007), followed by methici!!in-susceptibie staphylococci (P<0.O37), as shown in Table i 1 above. Oxaciiiin resistance was expressed in only 80.00% and 10.00% of HA-MRSA and CA-MRSA isolates, respectively. Cefotetan resistance was expressed in 80.00% and 20.00% of HA-MRSA and CA-MRSA isolates, respectively. Although oxacillin and cefotetan did not significantly differ in their ability to detect RSA (P>0.05). detection was significantly improved when using the mecA PGR when compared to disc diffusion (P<0.013). The majority of HA-MRSA isolates expressed resistance to amoxiciflin-ciavuianic acid, cefotetan, cephalexin, clindamycin, erythromycin, oxacillin, and peniciliin-G, whereas the majority of CA-MRSA isolates were resistant to only clindamycin, erythromycin, and peniciiiin-G. None of the isolates tested were vancomycin resistant. Overall, the most prevalent resistance pheriotypes were penicillin-G (83.33%), erythromycin (73.33%), and clindamycin (43.33%), whilst only single isolates (3.33%) were resistant to trimethoprim-sulfamethoxazole and rifampicin. &c gene complex interactions

[00260] All MRSA isolates belonging to mec gene complex A expressed resistance to both oxacillin and cefotetan, as shown in Table 1 below. However, oniy 20% of &c gene complex B MRSA Isolates were phenotypica!iy resistant to these antimicrobials. Of the MRSA isolates belonging to mec gene complex C2, only a single isolate expressed methicillin resistance to oxacillin and only two isolates expressed resistance to cefotetan. Unclassified MRSA isolates expressed full resistance to oxacillin and cefotetan.

Table 12: Number and percentage of identified mec gene complexes in 20 S. aureus strains classified as methiciliin-resistant according to example 4

Respective staphylococcal cassette chromosome (SCCmec) complexes and types expressing phenotypic resistance to oxacillin and cefotetan are indicated as well as real-time eoA status, and the average negative dF/dT peak obtained from melting point analysis from real-time PCR of the m&cA gene

(00261] Melting point peaks for the rnecA real-time PCR negative derivative plot -dF/dT differed between rnec gene complex (P<0.003) (Figure 8. On average, mec gene complex B and unclassified isolates demonstrated higher melting point peaks than other SCCmec types (P<0.012).

Physical properties of test antimicrobials end comparison of minimum inhibitory conc&ntmiion results from initial analogue testing

[00262} Test antimicrobials were selected on the basis of solubility and antimicrobial activity from preliminary studies. Cloudy precipitates were observed when both NCL812 were dissolved in cation-adjusted ueller-Hinton !f broth, as shown in Table 13 be!ow. Following initial structure-activity testing on each synthesized analogue, NCL812 was found to have consistent MIC values in this present study.

Table 13: Characteristics of antibacterial NCL812 and the β-!actam antibacterial ampicilfin according to Example 4.

e a l ng acte i l solubility in dimethyl sulfoxide (D SO), solubility in cation-adjusted Mueller-Hinton U broth (CAMHB), and average minimum inhibitory concentrations (MIC) (pg mi at 24 h) against metrnciili -resistant S. aureus (MRSA) determined from preliminary studies and those determined during this present study. ATCC 49775; methici!!in-susceptible S. aur us isolate and ATCC controi strain. MRSA580; methiciiltn-resistant S. aureus isolate #580. RSA698; methicillin- resistant S. aureus isolate #698 in vitro antibscteria! activities: minimum inhibitory concentrations

(00263] MICsc and MfC¾o values for lead compound CL812 (4- and 4-8 pg mL) are shown in Table 14 below. MIC values differed by S. aureus classification (susceptible, HA- or CA-MRSA) (P<0.00S). In many cases, NCL812 had significantly increased activity against CA- MRSA and methi ilin-susceptib!e staphylococci by one dilution when compared to HA-MRSA (P 0.002 and P<0.020 ( respectively), however there were no significant differences between MIC values for mefhicillin-suseeptifele staphylococci and CA-MRSA (P>0.05). Ampicilisrs MIC values obtained for the ATCC control strain were within the normal range expected on th© basis of CLS! guidelines

Table 14: In vitro activities of the novel antibacterial NCL812 and the β-lactarn antibacterial ampiciilin against S. aureus clinical isolates according to Example 4.

HA-MRSA; hospital-acquired methiciiiin-resistani S. aureus. CA-MRSA; community-associated methiciilin-resistant S. aureus. MIC; minimum inhibitory concentration (pg/mi). MBC; minimum bactericidal concentration (pg/rnl). MIC MSCrange; minimum and maximum MIC/MBC for all isolates. MlC/MBC«>; MIC/MBC at which 50% of isolates are inhibited. MJC/ BCan; MIC/MBC at which 90% of isolates are inhibited In vitro antibacterial activities: minimum bactericidal concentrations

100264] The MBCs determined from NCL812 were equivalent to the MIC for 93.33% and 83.33% of S. aureus isolates, respectively (Table 14). in aif remaining cases, MBCs were one dilution higher. For NCL812, MBCs ranged from 2-8 pg/ml and 4-16 pg/mL, respectively.

Time-kill studies

JG0265] in comparison to the turbidirnetrtc growth curve of ATCC 4977S, no visible bacterial growth was observed when ATCC 49775 was inoculated into cation-adjusted Mueller Hinton if broth supplemented with NCL812 at 1* and 4x the K IC in microdilution assays (P<0.033 and P 0.038, respectively) (Figure 10).

(00266} When analysed in 10 ml., rnacrodilution assays, broth supplemented with antimicrobials at ί χ and 4* the MIC and inoculated with ATCC 49775 displayed significantly reduced viable counts for both NCL812 concentrations when compared to the growth control (0.000<P<0.008) (Figure 11). Additionally, the time-kill profiles of each concentration of NCL812 did not significantly differ (P>0.05). Both concentrations remained bactericidal until approximately 8-12 h after antimicrobial addition, where bacterial regrowth was observed. Considerable variation in the killing activity of NCL812 was observed from 8-24 h. Although NCL812 was no longer bactericidal by 24 h, viable counts observed at 1 * the MIC remained significantly lower than those obtained from unsupp!emented broth <P<0 046).

(00267] In summary, the example set out above demonstrates bactericidal activity against both methici!lin-susceptibie staphylococci and MRSA. MIC and M8C values were consistently lo across the selection of isolates (MIC, S; 5f; 2-8 ug/mL). NCL8 2 retained good in vitro antimicrobial activity against common, mu!iidrug-resistant MRSA isolates, including the epidemic UK E RSA-15. EM SA-16, and E RSA-17, Irish EMRSA-1, AUS EMRSA-3. NY/JAPAN HA-MRSA, and predominant CA-MRSA clones. ?^iCL812 was also active against one S. pseudintermedius isolate that was originally identified as a S. aureus strain.

[00268] Preliminary testing suggests that CL812 targets the S. aureus cell membrane, causing dose-dependent release of vital metabolites such as AT Disruption of the bacterial membrane bilayer or proteins that are integral to membrane function in bacteria is a target for numerous large antimicrobials which are ubiquitous in nature; including g!ycoiipids, lipopeptides, lipoproteins, fatty acids, neutral lipids, phospholipids, and biosurfactants. Although NCL812 is a low molecular mass (≤500 Da) synthetic compound, it does appear to exert bactericidal activity in a similar manner to other antimicrobials which target the Gram-positive ceil membrane, including the high molecular weight cyclic lipodepsipeptide antimicrobial agent daptomycin, or the low molecular mass quino!one-derived HT61 , whose chemical structure is not currently available. Many of these lipophilic antibacterial agents are also not effective against Gram- negative microorganisms due to the presence of the outer lipid bi!ayer membrane, which contains narrow porin channels reducing the net penetration of some compounds into the ceil.

(002693 The insolubility of NCL812 at even iow concentrations in microbiological media may reflect the amphipathic and oiigomerie nature of this antimicrobial and suggests that the real MIC may be much lower than observed, as it is likely that it is only NCL812 in solution that is biologically active. In time-kiii studies, CL812 exerted rapid in vitro bactericidal activity against ATCC 49776 Again, these findings are consistent with a time-kill profile of cell membrane function inhibitors such as daptomycin and HT81.

[00270] Importantly, the apparent short in vitro half-life of this antimicrobial resulted in bacterial regrowth observed at 12 h after antimicrobial addition. This suggests that if a viable bacterial population survives the initial exposure to NCL812 prior to antimicrobial inactivation, bacteria! regrowth will occur. The development of resistance to F-4CL812 in these studies was ruled out as test bacteria remained susceptible to NCL812 following harvesting, washing and fvISC testing. Whilst the apparent short in vitro half-life of NCL812 may be a desirable characteristic for future in vivo application, it does suggest that MCL812 should be administered every 8 h in future in vivo safety and efficacy experiments to maintain adequate systemic concentrations, though it would appear from the time-kill profile that the NCL compound series are concentration-dependent rather than time-dependent antimicrobials.

[00271] To overcome the methicillin-susceptibie phenotype, extending disc diffusion incubation time from 24 to 48 h compensates for the slow derepression of the m&cR gene. Although the effects of longer incubation were not examined, and the small sample size of RSA isolates prevented further investigation into mec complex interactions; genetic techniques were of significantly improved sensitivity when compared to phervotypic methods for confirmation of the mecA status of the isolates in this study. Although genetic techniques are not always employed as a routine method for detecting MRSA, real-time PCR identification of the presence of the mecA gene in a Staphylococcus spp. isolate remains the diagnostic gold standard. EXAMPLE 5: in vitro pharmacodynamics of a new antimicrobial agent for Streptococcus pneumoniae.

Materials and Methods

Pneumococcal antimicrobial susceptibility Pneumococcal strains and growth conditions

(00272] Twenty pneumococcal isolates that comprised six characterised laboratory strains and 14 clinical isolates were the subject of this study (P9/6A, P21 3, WCH16/8A, WCH43/4, WCH48/4, WCH57/8, WCH77/5, WCH86/4, WCHB9 7, VVCH92/4, WCH137/8A, WCH158/18F, WCH184 19F and WCH211/11 ; strain/serotype, respectively). Other isolates used in this example were: A68.1/3 (Francis ef a!., 2001. infect immun. 69: 3350-2358); EF3030/1 F (Briies et ai. r 2003 J. Infec. Diseases. 188:339-348); L82016/68 (Briies ei a/., 2000 infect Immun. 68:796-800); TIG 4/4 (Tetfelin ef a!., 2001 Science 293:498-506); and WU2/3 {Briies e a/., 1981 J. Exp Med. 153:694-705). See Table 15 below for the phenoiypic characteristics of the isolates used in this study. The National Collection of Type Cultures (NCTC) control strain 039 (Avery et a/., 2010 Nature Reviews Microbiology 8:280-271 ) was used as a growth control for a!! MIC and MBC assays. D39 was later designated for kill kinetics, point of resistance assays and transmission electron microscopy (TEM) studies as ii is a well documented laboratory strain with a defined in vivo pathogenesis (Table 18) that displayed consistent NCL812 ICs and MBCs.

Table 15: Pneumococcal isolates and their phenotypic description according to Example 5.

1002.73] For ail /n vr/o assays, fresh pneumococcal isolates were grown overnight (O/N) on horse biood agar (NBA) plates {39 g/L Columbia blood agar base JOxoidj 5% [v/vj defribinated horse biood (Oxoid} at 37 X with 5% supplemented CO?). ueller-Hinton blood agar with 5% defib inated sheep blood (MHSBA Roseworthy Media and Blood Service) was used for disk diffusion analysis as directed by Clinical Laboratory Standards Institute {CLSI? standards. Pneumococci ware routinely g wth in broth consisting of 4% lysed horse blood (LHB) with Cation Adjusted Muei!er Hinton Broth (CAHMB. |Difco]) at 37 i: C with 5% supplemented C<¾. Horse serum broth (HSB, 10% (v/v) donor horse serum in nutrient broth [10 g/L peptone, 10 g/L Lab Lemco (Oxiori) and 5 g/L NaCI}} was also used i some MIC assays. Isolates were stored in HSB at -80 'C.

Antibiotic stocks and reagents

(00274} NCL812 was provided in dry powder form. A total of 258 mg was dispensed into 10 ml of 100% DMSO to make a stock of 25.6 mg/mL which was then diluted 1:100 in CAHMB to make a final working stock of 258 pg mL. Ampidilin dry powder was from Sigma A0168. The original 25.6 mg/mL stock was diluted in saline 1.100, 1:4, 1:20 and finally 1:16 in CAMH8 to make a final working stock of 0. 8 pg/mL. Erythromycin was purchased from Sigma Aldrich and choline chloride was from Roche Diagnostics. Twenty micro litres of 0.05 pg/mL erythromycin was diluted 1:25 in 4.980 mL of CAMHB to give a final working stock of 0.2 pg/mL. Choline chloride (0.5%) was added to 4% LH8:CAMH8 for specific kill kinetic assays.

Defining antimicrobial susceptibility of pneumococcal isolates

(00275] Isolate susceptibility to 12 different antimicrobials ( able 16} was determined by CLS! and European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Antimicrobials were selected based upon the CLSI and EUCAST guidelines. Standardised bacteria! suspensions were spread onto HSSA using a sterile cotton swab. Bacterial suspensions from of Streptococcus pneumoniae were standardised to an OD¾;¾ between 0.08 and 0.1 using a spectrophotometer and then diluted 1:20. Bacterial colonies were taken from an O/' horse blood agar plate. To ensure the purity of the 1:20 bacterial suspension, 50 pL was spread piated onto horse biood agar and incubated O/N at 37 X with 5% COj. The CPU was calculated and compared to the initial plate counts. Antibiotic disks (Purchased from Sigma Aldrich) were placed using a disk dispenser (Purchased from Oxoid) according to CLSI standards. MHS8A plates were incubated for 16 h - 24 at 37 *C in 5% C(¾. Zones of complete inhibition were measured in triplicate to the nearest millimetre using a ruler on natural light-reflected growth, and the mode was represented as the diameter for each isolate. Pneumococcal isolates were categorised as sensitive, intermediate (!) or resistant (R) by CLSI standards and quality control (QC) ranges (Table 16).

Table 16: Antibacterials used for disc diffusion analysis with interpretive standards of zone diameters (mm) according to Example 5.

(0 one ame ers or an m cro a s o er an pro oxac n or . pneumon ae were determined by CLSI standards .

* Zone diameters for Ciprofloxacin antimicrobial susceptibility to S. pneumoniae were determined bv EUCAST .

Determination of NCL8i2 C», MICss, MIC range and MBCs , MBC^ MBC range

100277] !Cs for NCL812 for a!l isolates listed in Table 15 were determined by measuring OD^o {Spectramax spectrophotometer, Molecular Devices Corporation) as an indicator of bacterial growth using 96 well microtitre trays after incubation for 24 h at 37 in 5% CO 2 . IMicro-broth dilutions and 96 welt trays are prepared by the following method: 90 pL of 4% LH8:CA H8 is aiiquotted into all wells using a multichannel pipette. 90 \il of working antimicrobial stocks were no serial diluted down the tray by a 1:2 dilution. Negative broth controls and dilution control were taken into account when planning the set up of a 98 well tray.] 10 μΐ of bacterial suspension was then added to the appropriate wells in the 96 well tray. Appropriate positive (no antimicrobial}, negative (no antimicrobial or bacteria) and negative dilution (a serial dilution controi of antimicrobial and broth) controls were included in each assay. MBC and plate counts for Kill kinetic assays were determined by aliquotting 20 μΐ_ from each well of the 96 well microtitre tray onto NBA, and incubating at 37 "C with 5% CO z . The MBC was determined by a 99.95% inhibition of S. pneumoniae, taking into account the dilution factor. MICs and MBCs were determined in quadruplicate and the mode was taken as the representative value. The iC 5 . :5> lCso and MIC range and 8C ¾ . MSCso and MBC range were determined according to CLSI standards. The MIC¾ snd MfC¾o, or M8C r ,o and MBCw. are defined by the lowest concentrations which, when a!i the MICs and MBCs of the isolates are arranged from lowest to highest, inhibited the 50th and 90th percentile of the total amount of isolates, respectively.

Micro-broth dilution time kill studies with NCL812 using strain D39

(002783 Bacteria! suspensions were added in triplicate to a 98 well microtitre tray containing NCL812 With a starting concentration of 128 g ml. and serially diluted 1:2 sequentially to a concentration of 0.25 pg mL. Negative dilution controls were subtracted from the median growth value to obtain a suitable indicator of overall bacterial production. The 96 well tray was incubated at 37 "C in 5% C0 2 and OD f; o read every 2 h for the first 12 h followed by final reads at 24 and 48 h. To further supplement this data, a separate experiment in which a 96 well tray was read automatically at half hourly intervals using a spectrophotometer (Spectramax spectrophotometer. Molecular Devices Corporation) for 14 h was performed to confirm the trends in growth curves observed from original micro-broth dilution studies.

MBC time kill studies with NCL812 using strain P39

[00279] MBC kill kinetics assays Involved the preparation of three 96 well microtitre trays. At specific time points, ajiquots obtained from these trays provided viable counts following incubation at 37 "C in 5% CO ¾ on HBA, and the MBC was determined after 24 h of growth.

MmiD-hfoth dilution time kill studies of D39 with NCL812

[00280] Bacterial suspensions and working antibiotic stocks were prepared as described above. {For preparing macro-broth dilutions, 20 ml tubes were filled each with 9 ml of 4% LBB.CAMMB. 9 ml of a working antimicrobial stock was diluted 1 :2 when added to to one of the tubes, and then serial diluted down from a high to low concentration of antimicrobial. 1 ml of S. pneumoniae bacteria) suspension was added to the appropriate tubes, including the positive control. Tubes were incubated at 37 with 5% CO ; ; with gentle manual tilting of the tubes treated with NCI.812 every 10 min for the first 12 . At every 2-3 h during the first 12 h of growth and then at 24 h and 48 h, 50 μΐ of each bacterial suspension was spread plated onto H8A and incubated at 37 * C with 5% CO ? . for 16-24 h.j

[00281] Table 17 below indicates the concentrations used for each antimicrobial. Cultures were* incubated at 3? in 5% CC with gentle manual tilting every 10 min for the first 12 h Viable counts from 50 pL aiiquots of each concentration were read following incubation af 37 *C in 5% CO* for 24 h. The pH of each sample was measured at specific time points using pH indicator strips. Confluent growth was defined when more than 1000 colonies were counted per piate. A bactericidal effect was defined as a f 000 fold reduction (99.9%) of the original cell suspension determined at 24 h for each concentration.

Table 17: Antibacterial agent concentrations used in macro-broth dilution assays according to Example 5.

Point of resistance assay for NCL81

[00282} Macro-broth dilutions were prepared as above. Broth cultures of strain 039 (10 mL) were incubated in the presence of 2 pg/mL and 4 pg/mL of NCL812, and 0.022 pg/mL of Ampicillin for S h at 37 C C in 5% CO*. Samples were centrifuged ai a relative centrifugal force (RCF) of 101.45xg for 10 min and washed in 60 ml of phosphate buffered saline (PBS) twice to remove any residual antimicrobial, and/or bacterial end products and media. Washed bacteria were resuspended and M!Cs were performed.

Effect of NCL812 on D39 cell membrane ultra-structure

Transmission Electron Microscopy

{00283] Morphological appearance and morphomei ic analysis of the cell membrane was determined using transmission electron microscopy (TEM). Bacterial suspensions and 10 mL cuitures of D39 were prepared as before. Samples were incubated ai 37 *C in 5% C0 2 with gentle manual tilting of the cultures every 10 rntn. Cultures were exposed to either 1 pg roL, 4 pg/mL or 16 ug/mL of NCL812 and harvested at 6 or 12 h by centnfugation at RCF of 10 45*g for 20 rnin and washed twice in 50 nr-L of PBS. Critical time points for Ύ Μ work were determined by analysing trends in the growth curves produced from the kilt kinetics studies. Samples were resuspended in PBS containing 20% glycerol and stored at -80 °C until requited. Before fixation 20% glycerol was removed by centrifugation and washing on ice three times in SO rnL of PBS.

[00284j Samples were fixed using modified protocols defined by a previous study examining cell wall uttrastructure of S. pneumoniae (Hammerschmidt, S. er si. 2005. Infect Immun 73:4653-4667). A lysine acetate-based formaldehyde-glutaraldehyde ruthenium red- osmium fixation procedure involved fixing the bacterial pellets with a cacodyiate buffer solution containing 2% formaldehyde, 2.5% giutarakfehycte, 0.075% ruthenium red and 0.075 of lysine acetate for 1 h. After washing with cacodyiate buffer containing 0.075% ruthenium red three times, a second fixation in cacodyla e buffer solution containing 2% formaldehyde, 2.5% glutaraldehyde and 0.075% ruthenium red was undertaken for 1.5 . Ceils were subsequently washed three times with cacodyiate buffer containing 0.075% ruthenium red and underwent a final fixation in 1% osmium tetroxide in cacodyiate containing 0.075% ruthenium red for 1 h. The samples were then washed three times in cacodyiate buffer containing 0.075% ruthenium red only.

(00285) Samples were washed and dehydrated using a graded series of ethanol (70, 90, 05 and 100%) for 10-20 min, two times for each step. Samples were infiltrated using 50:50 LR White resin in 100% ethanol for 1 h. and subsequently washed with 100% LR White resin for 1 h and left O N in a third change of 100% LR -white to ensure adequate infiltration of resin. The samples were then embedded in fresh LR White resin and incubated at 50 for 48 h. Sections were cut to 1 um using a glass knife, stained with Tofuidene Blue and viewed under a light microcrope 400* to identify the presence of stained pneumoeocci. At least four ultra-thin sections were then cut to 90 nm using a diamond knife and placed on matrix grids, one section per grid. Ultra-thin sections were then stained with uranyl acetate and lead citrate alternatively at 5 min intervals, followed by three washes with distilled water in-between each exposure. Stained sections were then placed on grids and viewed between 25000* and 130000* on a Philips C 00 Transmission Electron Microscope, images - e e obtained at 130000* magnification and analysed using anaiySIS {Olympus Soft Imaging Systems}.

Statistical analysis

[00286] Statistical analyses were conducted using statistics program GraphPad Prism (5th ed, GnaphPad Software Inc.) for Windows. For growth curves, data presented were the mean and standard error of mean (SEM) (represented as error bars) for each data point except for macro-broth dilution studies where multiple replicates could not be obtained due to the high costs involved in this assay. Two tailed, unpaired Mests were performed.

Results

Pharmacodynamics of NCL812 in S. pneumoniae

Quality control disk diffusion analysis for 20 S. pneumoniae isolst&s

(00287] Although nine out of the twelve antimicrobials used for disk diffusion analysis had established QC ranges by EUCAST, QC ranges were not defined for amoxtcil!in-ciavutanate, clarithromycin and clindamycin (Table 18 and Table 1S>. WCH16 and WCH184 were both resistant to at least two antimicrobials whereas EF3030 and WCH137 were intermediate and resistant to trimethoprim-sulphamethoxazoie respectively (Table 19). The other remaining sixteen isolates were sensitive to all twelve antimicrobials. Sensitivity to ampiciiisn was confirmed for each isolate, enabling the use of ampicillin as a positive control in later micro- broth dilution assays (Table 18)

Table 18: Antibacterial susceptibility of 20 S. pneumoniae isolates for six different aniibacteria!s according to Example 5.

Blue eet!s represent sensitive isolates: range ceils re resent intermediate l) isolates; grsen ceils represent resistant ( ) isolates. Tabie 18: Antibacterial susceptibility of 20 S. pneumoniae isolates for six. different antibacterials according to example 5 j Arst nicrobial Erythromycin 0¾a !«in Rifampin Tetracaine j ί rimethopnm- j Vancomycin

j suipharnethoxaz ,

Solubility and activity f NCL812 and NCL062 in different media

[00288] CL812 visually appeared to have higher solubility in 100% DMSO compared to NCI..062 and only developed turbidity when it was further diluted into CAMHB or PBS (Table 20). Although a CAMHB diluent for NCL002 appeared to be transparent by visual inspection (Table 20), further studies on CL062 with a CAMHB diluent resulted in complete confluence in microbroth dilution assays for six S. pn0umoniae isolates in comparison to growth with the DMSO diluents (Table 21 and Table 22).

Table 20: Visual analysis of NCt.812 and NCL062 and ampiciilin solubility according to Example

5.

Table 21: Individual MICs of NCL062 for each pneumococcal isolate according to Example 5

Table 22: Difference in activity of NCL812 and NCL062 In different media using micro-broth dilution to obtain an MIC as a predictor according to Example 5. CL662 j AmpTciiiiri j

(00289] Growth of S. pneumoniae strain 039 in an MIC assay for MCL812 and NCL062 using 10% HSB (220 mL of horse serum is filtered to 0% in 80 rot of Lemco nutrient broth) resulted in a threefold increase in the MIC for D39 treated with NCLD12 and NCL062 (Table 23) with a twofold increase for the positive ampiciiiin control. There was no notable change in MIC for 030 with differing storage conditions of pre-prepared 96 well microtitre trays (Table 24). During macro-broth dilutions, the pH of the media did not change compared to appropriate controls (Figure 12.

Table 23: Growth of S. pneumoniae strain 039 in an MIC assay for CL812 and NCL062 using horse serum supplemented broth

100290] Tabie 24: Storage of prepared microliter trays for nicro-broth dilution doss not change MIC of D39 according to Example S.

Determination of S. pneumoniae in vitro susceptibility to NCL812 and NCL0$2 D&i&mvnaiion of NCL812 and NCL062 MiCsc, M C a MIC range

1002913 NCL812 exhibited a M1C« and MiC¾o IC» of 8 μ ΙπιΙ and iC range of 4-8 pg/mL whereas for NCL062 these values were higher and more variable (Table 25 and Table 26). The MIC for ampicillin was comparable to recent published findings using micro-broth dilution as an endpoint for antimicrobial resistance sn pneumococcal isolates, thus confirming the accuracy of SCs obtained for NCL812 and NCL062 {Tables 25 to 26 and Figure 13).

Table 25: MiCsa. MIC*,. BC» t BC» and MIC ange for elf isolates treated with NCL812, NCL0S2, and ampicilltn according to Example 5.

Table 26: ICs of NCL812 of each pneumococcal isolate according to Example 5.

Determination o†NCL$12 and NCL0$2 MBC$c, MBC$& MBC range

[00292] Minimum bactericidal concentrations { BC»> r BCso a d MBC rang© respectively) were determined for NCL812 and a npiciiiin for all twenty isolates (Tables 25 to Table 26). The MBC 5a! BC¾ 0 . and MBC range was io ver and more consistent for NCl.812 compared with NCL.062 (Table 2S). Micro-broih dilution time kiii studies of D39 treated with NCL81 and NCL062

[00293] D39 exposed to sub-inhibitory concentrations (& 2 pg/ini) of NCL812 or NCL062 grew similarly to unexposed controls over a 48 h period ( igure 13 and 14. Higher concentrations of NCLS12 and NCL062 (≥ 16 pg/mL) resulted in no bacterial growth for 48 h (Figure 14 and 15. Those growth characteristics were validated by a micro-broth kill kinetic study using a Spectrarnax spectrophotometer, which measured growth (represented as OD¾¾ 5 ) at haif-hourty intervals for 14 h for NCL8 2, NCL062 and ampiciiiin (Figures 15 to 17. There was an approximate six hour difference between the commencement of exponential growth for D39 treated with NCL.812 and D33 treated with NCL062 (Figures 13, 4, 18 and 13}.

[002S4] The growth of D39 treated with NCL812 or NCL062 was compared to D33 treated with ampiciiiin or erythromycin over 48 h (Figures 20 and 21). D33 treated with ampiciiiin exhibited similar growth to D39 exposed to NCL812 or NCL062 over 4$ h (Figure 20). Er thromycin-treeted D38 produced very different growth curves from NCL812 and NCL062 where a larger difference in growth between concentrations was observed (Figure 21). The addition of 5% choline chloride to the media over a 48 h period resulted in no significant difference in growth for NCL812 and CL062 compared to positive and growth controls (Figures 22 to 26).

Point of resistance testing

[00295] D39 treated with≤4 pg/mL NCL812 entered a log phase of growth at 8 h (Figure 13 and 18), as shown in four independent experiments. The possibility of antimicrobial resistance to CL812 between 5 and 6 h was investigated by determining further MSCs on 03S exposed to 2 pg/mL NCL812, 4 ug/mL NCL8 2 and 0.0225 pg/rnL ampiciiiin for 6 h. Results showed no significant increase in MIC for alt samples of D39 exposed to NCL81 compared to growth controls, and ampiciiiin (Table 27).

Table 27: WiCs of D39 exposed to 2 pg/mL or 4 igiml of NCL8 2 for 6 h according to Example

!

"D39 growth control: S. pneumoniae strain D39 grown for β hrs in %LHB:CAMH8.

* * D39 growth2 control: S. pneumoni e strain Q39 on HBA O N, resuspended in saiine (0.1 OD«o) and diluted 1/20 in sterile saiine.

Micro-broth dilutions by measuring relative MBC at specific time points

[00296] Relative MBCs were determined at specific time intervals from using broth dilution assays incubated for 48 h for CL812 and NCL062 (Figure 27) and control antimicrobials amplciiiin and erythromycin (Figures 20 and 21). MlCs of ampicillin and erythromycin for D39 were determined (Tables 26 and 28). The comparative features of the growth of ampicillin, and erythromycin are described {Figures 28 and 29). Ampicillin and erythromycin demonstrated a time-dependent reduction in bacteria. NCL062 exhibited rapid bactericidal action, with an immediate (within the first 10 min of administration) MBC of 8 pg mL (F gure 2?}. Although there were inconsistencies in the MBCs for NCL062 between 5 and 12 h, MCL062 maintained a constant bactericidal concentration (4 ug/mL) between 24 and 48 h. NCL812 exhibited fast bactericidal action, evidenced by an approximate 3 fold decrease in MBC within 5 h (Figure 27). A consistent bactericidal concentration (8 pg/mL) was maintained for the full 48 h for CL812.

[002973 Table 28: MfC and MBC for erythromycin with D39 according to Example 5.

?dacro~btx>ih dilution time kill studie of D39 with NCL812 and NCLQS2

[00298} Viable counts for each time point were represented as a !og c . CFU/mL reduction for NCL812 (Figure 30) and ampicillin (Figure 31). Consistent confluent growth (determined by a limit of 2*104 CFU) was observed for unexposed controls and 2 pg/mL NCL812. Complete bactericidal activity (defined by a 3Sog,<, reduction in CFU) for 128 pg/rnL of NCL812 was observed by a 4iogi 0 reduction of CFU in 3 h and concentrations between 16 pg/mL and 64 pg/mL NCL812 were effective at eliminating bacterial growth within 8 h (Figure 30). NCL812 at 4 pg/mL and 8 pg/mL appeared to be inactivated at 11 h post-exposure, as increased growth of strain D39 after this time point was observed (Figure 20 and 31). The viable counts of strain D39 treated with ampicillin demonstrated consistency for this particular assay by showing a constant diminished time-dependant killing over 48 h (Figure 31),

Transmission electron microscopy [00299] Morphometry analysts revealed significant changes to the cei! membrane in strain D39 exposed to 16 pg mL NCL812 for 6 h compared to growth controis. Samples treated with 4 pg mL as well as 12 h cultures we e not considered for morphometry analysis due to the lack of bacteria! cells available in each section. Treated samples possessed significantly thicker cell membranes (6.43 ± 0.29 nm) compared to untreated samples (4.35 i 0.24 nm) (p«0.G0Q1) (Figures 32 and 2S). The peripiasmic space (intracellular space between the cell membrane and the cell wall) of 039 treated with 16 pg/mL NCL812 was significantly wider (4.54 ±0.096 nm) compared to untreated samples (3.91 ± 0.14 nm) (p<0.001 ) (Figures 29 and 33).

[00300] Table 29: Morphometry studies o the ultra structures of D39 treated with NCL812 for 8 hours according to Example 5.

(00301] In summary, NCL812 produced highly consistent MlCs and equivalent BCs for the S. pneumoniae strain collection, confirming that it is bactericidal against this organism. In kill kinetics experiments, which measured the relative MBC over a 48 h period, a consistent bactericidal effect was elicited in D3S after 6 h from initial exposure to NCL812.

[00302} This demonstration of bactericidal activity is the first to be observed in S. pneumoniae. This demonstrates that r-4CL812 -is effective against pneumococcal in vitro.

[00303] Competitive binding between components in blood, serum or broth decreased the antimicrobial activity of NCL This was reflected in the increase of MIC observed between different broth types and diluents. Following the completion of these studies, recent independent research confirmed precipitation of CL8 2 in PBS and reported complete solubilit in water containing 4% DMSO. following initial dilution in 100% DMSO. A water-soluble MCL812 will greatly improve in vivo bioavailability and negative interaction between blood or serum proteins.

[00304} Based on the findings of this study, CL.812 exhibits a mechanism of action against S. pneumoniae that is different from β -lactam or macroiide classes, as it appears to exhibit concentration-dependent bactericidal activity as opposed to time-dependant qualities. Identifying the maximum pharmacokinetic serum concentration of NCL812 in vivo will assist confirmation of its concentration-dependant pharmacodynamic activity. Furthermore, the addition of choline chloride to the media confirmed that the mechanism of action for CL is not associated with the affinity to cell wall choline binding proteins, and therefore may not be cell wall associated.

[00305] Morphometry analysis of the cell membrane and periplasmic space of D39 treated with 16 ug/mL NCL812 for 6 h showed that the eel! membrane and periplasms space was larger in treated samples, compared to control samples. The apparent increase in membrane size could be due to an accumulation of electron dense intracellular material beneath the cell membrane. The increase in the size of the periplasms space may be have been due to disruption of the cell membrane, potentially by depoiarisation or ATP inhibition. The mechanism of action of NCL812 may not be calcium-dependant as i appears that no competitive binding between NCL812 and ruthenium red, a calcium channel inhibitor of lipid biSayers, was observed in electron micrographs.

[00306] in conclusion, this in vitro study has demonstrated that NCl.812 has many desirable characteristics as a fast-acting concentration-dependent bactericidal antimicrobial that appears to target the cell membrane of S. pneumoniae. These characteristics are desirable to treat acute pneumococcal infections. As NCL812 may possess a mechanism of action that targets the cell membrane, it will act much more quickly than time-dependent antimicrobials such as β lactams and macro!ides and potentially more effective than other bactericidal concentration-dependent antimicrobials such as fluoroquinolones which have intracellular targets.

EXAMPLE 6: Characterization of ethiciilin-susceptible and methicillin-resistant isolates of

Staphylococcus pseudintermedius from Australia and preliminary in vitro efficacy of a new anti-staphylococcal compound

Materials nd Methods

Sample coilect&n and identification of methici!iin susceptible Staphylococcus pseudintetm dius (MSSP) and methicillin resistant Staphylococcus pseudkitetm&dius (MRSP)

[00307] A total of 23 Staphylococcus pssudintemmdius isolates were obtained from dogs (Table 30). [00308} Ten metnicillin susceptible and 13 methlciflin resistant Staphylococcus pseudintermedius were collected for the study, Isolates were phenotypica!iy classified as metnicillin ressstani on the basis of in vitro resistance to oxacillin and genetically for the presence of &cA gene according to standard procedures.

[00309] Oxacillin and cefoxitin susceptibility testing using disk diffusion technique and Epsiiometer testing were performed, identification of mecA gene was performed using polymerase chain reaction (PGR)

(003103 CLSI disk diffusion susceptibility testing was performed on the 23 Sp. isolates for the following antimicrobials: penicillin, amoxicillin, erythromycin, gentamictn, clindamycin, ciprofloxacin, cephalexin, chloramphenicol, tetracycline, oxytetracycline, vancomycin, cefotetan, moxiftoxacin and rifampin.

[00311} Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) testing was undertaken using CLSI methodology for NCL812 and included ampictilin as a control. Anti -staphylococcal compounds were than tested against all 23 isolates and minimum inhibitory concentrations (MIC) were determined according to standard protocols. After tire MICs were determined, the minimum bactericidal concentrations were performed to determine if these compounds are bacteriostatic or bacteriocidal. esults

[00312] The ecA gene was present in 13 isolates of MRSP and negative in 10 MSSP (Tables 30 and 31). All M SP isolates were resistant to oxacillin based on disc diffusion { 7 mm) and E-test MIC (≥0.S mg L).

Table 31: Staphylococcus pseodinterm&dius isolates tested according to Example 8.

(00313] When cefoxitin resistance breakpoint was set at≤24 mm, 3/13 (23%) and 5/13 (38%) of MRSP tested respectively were susceptible to cefoxitin. When cefoxitin resistance break point was set at≤30 mm, only 1/13 (7.7%) of MRSP tested was susceptible (Tables 30 and 31).

[00314] The MRSP isolates were resistant to multiple antibiotic classes. Of the 13 MRSP isolates, all 13 were susceptible to rifampin. 3/13 (23%) were susceptible to chloramphenicol; 10/13 (77%) were susceptible to vancomycin (Tables 30 and 31).

(003 5) Interestingly, 3/13 {23%} of the MRSP isolates were susceptible to amoxicillin. 8/13 {62%) were susceptible to cephalexin; 12/13 (92%) susceptible to cefotetan and 12/13 (82%) susceptibe to moxifloxacin (Tables 30 and 31).

[00316] All 23 isolates were susceptible to NCL812 based on MICs. In addition, NCL8 2 has been shown to be bactericidal based on minimal bactericidal concentrations (MBC).

(0031 ] The MIC range of NCL812 against the Staphylococcus pseudirttennsdius isolates was found to be between 1 pg/mL and 4 pg/mL {Table 32). The ICso and MiC¾j of NCL.8 2 against the Staphylococcus pseudintetmedius isolates was found to be 2 pg/rol and 4 pg/mL respectively (Table 33). The MIC mode and MIC range of NCL812 against the Staphylococcus pseudintermedius Isolates was found to be 2 pg/mL and 1-4 pg/mL respectively (Table 33).

Table 32: MICs of CL81 and ampicil!in against Staphylococcus p$$udinterm 1iu$ isolates according to Example 6.

Table 33: MIC50, MIC90, the MIC mode, an MIC range of NCL812 against Staphylococcus pseudintermedius isolates according to Example 6.

(00318] Methiciiiin resistant Staphylococcus pssudinierm&dius ( SP) is an emerging problem in dogs, cats and horses. Two major clonal MRSP lineages have been reported from dogs in Europe (ST 71 } and North America (ST 68). There were also reports of MRSP affecting dogs in Japan and a single case o MRSP in veterinary personnel in Hong Kong.

[00319] In this study, MRSP isolates were determined using a combination of presence of ecA gene and in vitro resistance to oxacillin. Cefoxitin susceptibility has been used as a substitute for oxacillin for methiciiiin resistant Sfap y/ococcus aureus. However, cefoxitin disk diffusion tests using interpretive guidelines recommended for human isolates of methiciiiin resistant Staphylococcus aureus and coagulase negative staphylococci are unreliable in identifying MRSP. A cefoxitin breakpoint resistance of ≤30 mm~resistant and≥3 Insusceptible has been proposed by Semis et ai, 2012[8emis, D. A., R. D. Jones, ef el. (2012). "Evaluation of cefoxitin disk diffusion breakpoint for detection of methiciiiin resistance in Staphylococcus pseudintermedius isolates from dogs." Journal of Veterinary Diagnostic Investigation 24(5); 964- 867], This study is in agreement that this breakpoint may be more reliable in predicting methiciiiin resistant Staphylococcus pseudintermedius.

[00320} MRSP isolates are generally resistant to multiple antibiotic classes. Bacterial culture end antibiotic susceptibilities are therefore recommended for ail suspect MRSP infections to a!fovv appropriate selection of antibiotics. A limitation noted in this study is the apparent in vitro susceptibility of MRSP isolates to amoxicillin and cephalosporins (csphalothtn and cefotetan).

[00321] NCL812 was effective against ail 23 isolates of both MSSP and MRSP. A larger scale study is warranted to confirm the effectiveness of NCL812 against Staphylococcus pseudintermetjius as it may provide a safe alternative antibiotic option for emerging MRSP infections in domestic animals.

EXAMPLE 7: Preparation and testing of NCL812 analogues (also known as compounds of the invention).

Materials and Methods

NCL812

Analytical grade NCL8 2 with a defined potency of S8G mg/g (i.e. 96%) was obtained. The powder was stored in a sealed sample container out of direct sunlight and at room temperature at the study site. Aiiquots (1 ml) of stock solution (containing 25.6 mg mL of NCL812 in DMSO) were prepared and stored at -80 * C and defrosted immediately before use.

$ynth&$i$ing and Testing of NCL812 Analogues

(00322} Analogues CLQ01 to NCL230, as identified in Figure 1, were synthesised using standard methods in the art. As an example, the methods used to manufacture compounds NCLG97; NCL157; NCL179; NCL188; CL195; and NCL19S are as follows:

NCL 0S7 ^^.. -bis iS t.S-trihydroxyphenylJmethylenelcarbonimidtc dihydrazide hydrochloride)

(00323) A suspension of 3,4,S-trihydroxyben2aldehyde {412.0 mg, 2.673 mmoi, 2,21 eq.) and N.rV ' -diaminoguanidine hydrochloride (152.0 mg, 1.211 mmol) in EtOH (5 mL) was subjected to microwave irradiation ( 50 W) at 100 °C for 10 min. The reaction was then allowed to cool to ambient temperature. The resulting precipitate was collected and washed with chilled EtOH (5 mL) and Et 5 0 (5 mL) to afford the carbonimidic ihydrazide (369.0 mg, 77%) as a pale brown solid.M.P. 292°C (Oecomp.^H NMR (300 MHz, D SO-d6) δ 9.08 < r s, 6H), 8.25 - 8.01 (m. 4H). 6.83 (s, 4H). 13 C NMR (75 MHz, D SO-d6) δ 152.2, 49.7. 46.2, 136.5, 23.7, 107.4. LR S(ES ): 361.95 [ + 1f.

NCL157 2,2'-bis[(2-amino-4-chlorophenyl)methylene]carbc»nimidic dihydrazide hydrochloride) [00324] Synthesis of 2-aminO"4 hlora-N~methoxy~N~m&thyibenzamid$. To a solution of 2-amino-4-c orobenzoic acid (5.6891 Q, 33.041 mrnoi), A .O dimethythydroxylamine hydrochloride (5.7504 g, 58.954 mmol, 1.78 eq.), /V-{3-dimetriylaminopropyl)-<V- ethyicarbodiimide hydrochloride (7.7925 g, 40.649 mrnoi, 1.23 eq.) and W-hydroxybenzotriazo!e hydrate (5.2371 g, 38,793 mrnoi (anhydrous basis), 1.17 eq.) in D F (100 ml) was added diisopropytetbySa nine (18.0 mL, 13.4 g, 104 mrnoi, 3.15 eq.) and the brown solution stirred at ambient temperature for 7 h. The reaction was then concentrated in vacuo before dilution with 1M NaOH (100 mL) and extracting with CH s Ci a (3 * 100 mL) The combined organic extracts were washed with 1 HO (100 mL) before drying over MgSO* and concentrating in vacuo to afford a brown syrup. This oil was then further dried at 60 "C Kinder high vacuum to afford the crude Weinreb amide (7.021 g, 99%) as a brown syrup that crystallised on standing. The crude material was used without further purification. 1 H NMR (400 M z, CDC ) o 7.24 (ct, J = 8.4 Hz, 1H), 6.62 (d, J « 18 Hz, 1H), 6.54 (dd, J ~ 8.4, 1.9 Hz, 1H), 4.75 Cs, 2H), 3.48 (s, 3H). 3.24 (s, 3H). ? C NMR (101 MHz, CDCi 5 ) δ 163.2, 148.4, 137.1 , 130.6, 116.6, 16.1 , 115.0, 81.1 , 34.0.

(00325] Synthesis Qf 2-amirio-4-ch!orob&nz8idehyde. Crude 2-amino-4- « chloro-W- methoxy-W- ethyibenzamide (751.1 mg, 3.532 mrnoi) was broken up into ca. 120 mg batches and each dissolved in THF (10 ml) and cooled to 0 <: C before LiAfH* (2M in THF, 0.5 mL) was added to each and the solutions stirred for 16 h, allowing the reactions to achieve room temperature. The reactions were quenched with saturated NH 4 Ci (1 mL) before being combined, diluted with saturated NaHC0 3 (160 mL) and extracted with CHCI> (2 * 150 mL, 1 * 75 mL). The combined organics were dried over MgSO* and concentrated in vacuo to afford the crude benzakiehyde (463.3 mg, 85%) as yellow/orange crystals. The material was used without further purification. -H (400 MHz, CD 3 OD) 9.77 (d, J - 0.7 Hz, 1H), 7.46 (d, J ~ 8.3 Hz, 1H), 8.83 - 6.71 (m, 1H), 8.63 (dd, J ~ 8.4, 1.9 Hz. 1H). U C NMR (101 MHz, CDjOD) δ 194.6, 153.0, 142.5, 138.4, 118.3, 116.8, 16.1.

100326} Synthesis of 2,2'*bisi(2~mvno-4-ch!orophenys}methyte^^

dihydrazide hydrochloride. A suspension of 2- amino- 4- ehiorobenzaSdebyde (128.0 mg, 0.823 mrnoi, 1.78 eq.) and Λ ,ΛΓ-diaminoguanidine hydrochloride (58.0 mg, 0.462 mrnoi) in EtOH (2 ml) was subjected to microwave irradiation (100 W) at 60 n C for 5 minutes. Most solvent was then removed in vacuo, EtOH (1 mL) was added and the flask was transferred to the freezer to effect crystallisation. The resulting precipitate was collected and washed with EtOH (1 ml.) to afford the carboni nidicdihydrazide (21.0 mg, 13%) as a pa!e yellow solid. * H NMR (400 MHz, DMSO-d*) δ 11.71 (br s, 2H), 8.40 (s, 2H), 8.37 (s, 2H}, 7.29 (d, J * 8.4 Hz, 2H), 6.87 (d. J * 2.0 Hz. 2H), 6.73 (br s, 4H), 6.59 (dd, J = 8.3, 2.0 Hz, 2H). Vi C NMR (101 MHz, DMSO-d*) δ 152.1, 151.5, 148.9. 136.0, 1:34.7, 115.1 , 114.5, 112.8.

NCL179 (4 < 6-hls(2 (£)-4-chlorobenzyiidene)hydrazinyS)py imidirv2-amins) {00327] A suspension of 2-amino-4,6-dihydi -52!nylpyrimidfne (67.3 mg, 0,434 mmoi) and 4-chtorobsnzaicla yde (198.8 mo,, 1.414 mmol, 3 26 eq.) in EtOH (25 rnL) was heated ai reflux for 16 h. After this time, the condenser was removed and the solution concentrated to approx. 1 ml and the resulting precipitate filtered hot and washed with Et 2 0 (10 ml) to afford the aminopyrimidine (42.8 mg, 25%) as an off-white amorphous powder. MP. 275 X (Decomp.). H HUR (400 MHz, D SO) 6 10.70 (5, 2H>. 8.02 (s. 2H), 7.67 (d, J - 8.4 Hz, 4H), 7.52 (d, J ~ 8.4 Hz, 4H), 6.28 is, 1H), 5.85 (s. 2H). :i C N R (101 MHz, DMSO) δ 162.6, 162.6, 138.8, 134.1 , 133.1. 128.S, 127.6, 73.5.

NCL188 ((£)~2-(l H;4-chioroph€nyf)pentylio:ene hydraane-1-carfooxirr«idamtde hydrochloride)

{00328} A suspension of 1-(4-chlorophenyl)penianone (1.831S g, 9.3146 mmol, 1.95 eq.) and aminoguanidine hydrochloride (527.6 mg, 4.773 mmol) in EtOH (15 rnL) was heated at 65 * C for 6 h. The crude was cooled to ambient temperature before being diluted with Et s O (60 ml) and cooled to 0 * C to precipitate unreached aminoguanidine hydrochloride (174.5 mg). The mother liquors were then concentrated in vacuo and the residue dissolved in Ht s O (20 mL). The solution was then boiled and hexanes (10 mL) added to afford the caboximidamide as a cream solid. H NMR (400 MHz, DMSO} δ 11.54 (s, 1H). 7.99 (d, J * 8.7 Hz, 2H), 7.90 (s, 3H), 7.47 (d, J » 8.6 Hz, 2H), 2.91 - 2.82 (m. 2H), 1.48 - 1.32 (m, AH), 0.89 - 0.84 (m, 3H). 13 C NMR (101 MHz, DMSO) δ 156.2, 153.8, 134.8, 134.4, 128.7, 128.4. 28.1 , 26.8, 22.0, 13.8

NCL195 (4,6-bis(2-((£)^-methy!ben2yltdene) ydJ¾zin> )pyrirnidin-2-amine5

A suspension of 2-amino-4,6-dihydrazinapyrimidine (68.9 mg, 0.380 mmol) and 4- methy!benzatdehyde (0.10 mL, 100 mg, 0.832 mmol, 2.19 eq.) in EtOH (4 ml) was heated at reflux for 16 h. The reaction mixture was cooled to ambient temperature before collecting the pellet-like precipitate, washing with Ei 2 0 (20 mL). The 'pellets' were then crushed and the solid further washed with Et 2 0 (10 ml.) to afford the pyrimidine (85.8 mg, 63%) as a white 'fluffy' powder. MP. 274-276 *C. 'H NMR (400 MHz, DMSO) δ 10.51 (s, 2H), 8.00 (s, 2H). 7.54 (d, J * 8.0 Hz, 4H), 7.26 (d, J = 7.9 Hz, AH). 6.26 (s, 1H), 6.77 (s, 2H), 2.34 (s. 6H). 3 C NMR (101 MHz, DMSO) δ 162.8, 162.6, 140.1, 138.4, 132.5, 129.4, 126.0, 73.3, 21.0.

NCL196 (4.4 * -((f£,r£M(2^minopyrimW

y'idene))bis(methariyfylider!e))dipS eno!)

[00329J A suspension of 2-amino-4,6-dihydrazinopyrimtdine (70.4 mg. 0.454 mmol) and 4-hydroxybenzaidehyde (140 3 mg, 1.149 mmol, 2.53 eq.) in EiOH (3 mL) was heated at reflux for 16 h. The reaction mixture was cooled to ambient temperature before collecting the precipitate, washing with Et 2 0 (25 mL), to afford the pyrimidine (91.4 mg, 55%) as an off-white powder. M.P. 298 *C (Decomp.). Ή NMR (400 MHz. DMSO) δ 10.31 (s, 2H), 9.74 (s, 2H), 7.94 (s, 2H). 7.48 (d, J - 8.6 Hz, 4H), 6.S3 id, J * 8.6 Hz, 4H), 6.20 is, 1H), S.?0 (s, 2H>. 13 C (101 MHz. D SO) δ 162.7, 162.5, 158.3, 40.5, 127.7, 128-3, 115.7. 73.0.

MIC tests

{00330} Minimum inhibitory concentrations (MQ/ml) were determined using ihe broth microdiiution method recommended by the Clinical and Laboratory Standards institute (CLSI). MIC breakpoints were determined by visuai assessment and then confirmed using an EL!SA plate reader, measuring absorbance levels at 600 nm. Bacterial growth (turbidity) in the wells with antimicrobial was compared with the amount of growth (turbidity) in the growth-control well (containing no antimicrobial). Ail isolates were tested in duplicate, if there was a difference of greater than one two-fold dilution in the results, the test was repeated a third time. The purity of the isolates was closely monitored during testing by subculturing the prepared bacterial inoculum onto S8A (sheep blood agar). The fVtlCs of the control strains for the antimicrobial ampictllin were determined for each testing run as an internal quality control, The M\C $ o, A C^, and iC range (minimum and maximum) were calculated for each of the bacterial groups.

Activity of NCL812 and MiC against Gram-negative bacteria

[00331] The activity of NCL812 against Gram-negative bacteria was assessed using the broth microdiiution method recommended by the Clinical and Laboratory Standards institute (CLSI), and the MICs (pg/ml) for CL812 and ampici!Htt were detemnined.

Determination of Minimum Bactericidal Concentration (MBC)

CLSI methodology

[00332] Briefly, 10 pi. of the contents of each well starting at the MIC was inoculated on to a Columbia SBA plate and incubated at 37 °C for 48 h. Plates were examined at 24 and 48 h and the MBC was recorded as the lowest concentration of CL812 at which no colonies of bacteria wera observed on the plate (or significant inhibition of growth was observed compared to the control) (CLSI 2005).

Kill kinetics assays (MRSA & VRE)

(00333] MRSA/VRE were grown overnight on Columbia SBA at 3? * C. A few colonies of bacteria were then suspended in CAMHB and adjusted to an optical density of 0.08 to 0.10. The bacterial suspension was diluted 1:10. One rnillilitre of the bacteria was added to 9 mL of CAMHB containing various concentrations (up to 4 x MIC) of NCL812, to achieve a final bacterial concentration of 1 to 3x 0 s CFU mi. The tubes were incubated at 37 °C. In order to determine the number of viable bacteria present at various time points, a 100 μΐ aliquot was removed from each tube and diluted in normal saline. Then, 100 μΐ of each dilution was spread onto colony count agar, in duplicate, and incubated for 48 h at 37 * C. After 24 h the numbers of colonies present on each plate were counted and therefore the number of viable bacteria present in the original suspension enumerated. Plates were re-checked after 48 hours.

Synergy studies with other classes of antimicrobial agent.

[00334] The checkerboard method (Gunics et at., 2000 Int. J. Antimicrob. Agents. 14:23S- 42) was used to find interactions {synergy, antagonism, no effect) of NCL812 in combination with tetracycline, chloramphenicol, erythromycin (macroiide), ampscillin (β-!actam broad- spectrum), geniarnicin (aminoglycoside), ciprofloxacin {fluoroquinolone), sulfamethoxazole (sulphonamide), or penicillin G φ-iactarn narrow-spectrum), fo initial experiments, a laboratory strain of Staphylococcus aureus T3-129 was used, however this strain gave inconsistent results for some of the antimicrobials and a new strain of Staphylococcus spp. designated MK1 (definitive species identification currently in progress) that was sensitive to alt tested antimicrobials was used in subsequent tests.

[00335] Firstly, the M!C of each antibiotic atone was determined in accordance to CIS! standard guidelines. Secondly., the combination of NCL812 with each of above antibiotics was tested in duplicate. To evaluate the effect ot the combination the fractional inhibitory concentration (FIG) was calculated for each antibiotic as follows:

FSC of tested antibiotic = M\C of tested antibiotic in combination / MIC of antibiotic alone.

FIC of NCL812 = MIC of NCL612 in combination / MIC of NCL812 alone.

FIC, - FIC index = FIC of NCL812 + RC of each tested antibiotic.

(00336] According to the checkerboard guidelines. Synergy (S) was defined as an FICi<0.5. No effect (NE) was defined as 0.5<FICi«4. Antagonism (A) was defined as a 4<FiC t .

Testing of NCL&12 Analogues

(00337] NCL812 analogues were stored ai 4 *C until assayed. MiCs were determined against two RSA strains, two VRE strains and one strain each of E. ©off and Pseudomonas aensginosa.

Results

Determination of Minimum inhibitory Concentration (MIC) [00338] The comparative NCL812 and ampiciiisn MiC values (gg/rnL) for 21 SA isolates were obtained. The results for the original experiments (phase 5)· an< * repeat testing (phase !!) are s ove in Tabie 34. Each MiC test was performed in duplicate.

Table 34: NCL812 and ampici!!in MIC values (pg/mi) for 2t RSA isolates obtained according to Example 7.

Phase ! j Phase !f

f " 0t¾¾nism j CLS zlsTT wCLSl nd sTTAm ictHin 2nd j ^CL812 1st | NCL8122nd T ~ Ampiciilm 1st j AropiciWn 2nd j

MRSA S16 j 44 p pgg//mmii [ i 44 p pgg//mmii ! 32 μ¾ι#ηί j 32j g/m? | j S ^ _J 4 pg/mi j 32 pg/mi i 18 pg/mi

M SA 570 4 pg/mi j 4 pg/ml .8 pg/mi Ί28 pg/ i 4 pg/m< 4 pg/mi >128 |jg/rrji M28 pg/ml MRSA 580 4 pg/mi j >?28 pg rni_ j_ j 128_pg m? j 4 pg ntf 4 ug/mt >128 pg/mi j 128 pg/mi

4 pg/mi 4 pg/mi j__ 32 pg mi j 32 pg/tnt I 4 pg/mi 4 pg/mi 32 pg/mi 32 pg/mi

— f

MRSA 610 j 4 pg/.mi 4 pg/mi 128 pg/ml I28 pg/ms | ug/mi ■ P3'ft»' 32 pg/mi j IWRSA S1S ! 3 u≠n\ 4 pg/mi I 64 pg/mi 64 pg/mi j 4 pg/mi j 4 pg/mi j 84 pg/mi 64 pg/mi

MRSA 622 j 4 pg/mi 4 pg/ml j 64 pg/mi 84 ug/roi j 4 pg/mi j 4 pg/mi j 32 pg/mi ! 32 P9/^

MRSA 688 j 4 pg/rni 4 pg/mi ! 64 pg/m! 84 ug/irsl i 4 pg/mi 4 pg/mi j 8 pg/mi e pg/mi

MRSA 713 ί 4 pg/mi 4 pg/mi >128 pg/mi >128 pg/mi I 4 pg ml 4 pg/mi j 128 pg/mi '«28 pg/mi

MRSA 718 f 4 pg/mi 8 pg/ml j >128 pg/mi >128 pg/mi j 4 pg/mi 4 pg/mi i 64 pg/mi 64 pg/mi

MRSA 728 j 4 pg/mi 4 pg/mi I 84 pg/mi 64 pg/mi j 4 pg/mi 4 pg/mi 32 pg/mi 32 pg/mi ί t MRSA 734 j■ r 4 pg/mi 4 pg/mi j >128 pg/mi j 128 pg/mi j 4 pg/mi 4 pg/mi J 84 pg/mi 84 pg/mi

MRSA 741 4 pg/mi 4 pg/mi j 8 pg/mi 8 Mg/mi I 4 pg/mi I 4 pg/mi j 4 pg/mi 4 pg/mi

" MRSA 747 j 8 pg mJ 4 pg/mi I > i28 pg/mi j 128 pg/mi I 4 pg/mi ί 4 pg/mi j S4 pg/mi 64 pg/mi

MRSA 773 4 pg/mi 4 pg/ml i 64 pg/mi i 64 pg/mi j 4 pg/mi 4 pg/mi j 64 pg/mi 84 pg/mi

MRSA 78 j ~ 4 pg/mi 4 pg/mi ; >128 pg/mi >126 pg/mi [ 4 pg/mi 4 pg/mi j 1 8 pg/mi 128 pg/mi

SSRSA 786 I j 4 ug/mi 4 pg/mi j 32 pg mi 32 pg/mi j 4 pg/mi 4 pg/mi ί 32 pg/mi 32 pg/mi

MRSA 787 j 4 pg/mi 4 pg/mi j >12S pg/mi >128 pg/mi j 4 pg/mi 4 pg/mi ! 64 pg/mi 64 pg/ml

MRSA 815 t 4 pg/mi j 4 pg/mi 1 84 pg/mi 64 pg/mi I 4 pg/mi 4 pg/mi j 32 pg/mi 32 pg/mi

MRSA 823 j 4 pg/mi j 4 pg/mi >128 pg/mi j >128 pg mi j 4 pg/mi 4 pg/mi T~ 12S pg/mi 128 pg/mi t MRS .A 344 ί 4

: pg/mi j 4 pg/mi j 128 pg/m: 128 pg/ml ] pg/mi j 4 pg/mi J 64 ug/mi 32 pg/mi

c

.εί 8 ' t So

5

«5

Ω.

I

o O

Φ

[00339] The comparative NCL81 and ampiciiiin MIC values (ug mt) for 13 V E isolates were obtained. The results for the original experiments (phase I), and repeat testing (phase II) are shown in Table 35. Each MIC test was performed in duplicate.

100340] NCL812 MfCg , MIC 9 0, MIC mode and MIC range were obtained for Australian isolates of MRSA and VRE, as shown in Table 6 Comparative MIC values for ampiciiiin are shown in parentheses.

Activity of NCLB12 and MIC against Gram-negative bacteria

[00341] Comparative NCL812 and ampiciiiin MIC values (pg/mL) for Escherichia co!i, Pseudo onas aeruginosa and Salmonella arizonae were obtained, as shown in Table 36. Each MIC test was performed in duplicate.

Table 36: Comparative NCL81 and ampiciiiin MIC values {pQ/m!} for Escherichia co/ , Pseudomonas aeruginosa and Salmonella arizonae obtained according to Example 7.

[00342] The antimicrobial ' activity of NCL812 against the selected Gram-negative bacteria was 128 pg ml.

Determination of Minimum Bactericidal Concentration (MBC)

[00343] MBC results for MRSA isolates are shown in Table 3? which shows NCL812 MBC values (pg mL) for 20 MRSA isolates. Each MBC test was performed in duplicate starting from the MCL812 MIC concentration to 16 times the M!C. For all isolates, the MBC was equal to the MIC. However, inconsistent growth on agar plates was recorded for some concentrations.

Table 37: NCL812 MBC values (pg/ml) for 20 MRSA isolates according to Example 7.

NCL812 BC

Organism / Sample No.

I pg mlft p¾/mli

0 0

MRSA 1

0 ! 0 0

0 0 0 _ ΤΓ.

MRSA 2

" 0 0 ' N

0 ' GB*

MRSA 3 ϋ

-V "" 0 0 H

0

MRSA 4 .9.

- 2 na ί 0 0 _L ! _£ 0 11 • 1 S ' j

MRSAS ?_ i o I 0 i 0 i N

o ! 0 ! " o Γ .... o.... ' " N

} ·· i

0 I

MRS A 6 o ! o Γ " N j

-2 NRT ! 0 0 ! 0 I 0 I""" N

0 o ;

MRSA? <·>«! Γ o

- Jfc i 0 GB I 0 i N

! 0 ! N .

GB

MRSAS o ! 0 }

I , Ϊ N i

-2 M I 0 ' 0 -I ~ Q \ 0

5 N

0 o N

MRSAS S 0 i

-2' i o ' !

" o -8 b ! o " r 0 Ϊ N

0 s 0 : N !

MRSA 1Q

0 0 0 i 0 I N

t 0 0

MRSA 11 "" :? sT o |_ c N

- : 2_ Β s Β r T Γ~ GB o 0 i

0 o 1 0 ■

MRSA 12 N !

-2 M ! 0 GB „.... L 0 N

MRSA 0

13 "' : ' ΐ κ ΐ " 0 0 i 1

N ;

-2 Η * ! 0 0 o F " 0 N

} Ί

MRSA 14 - i 0 6

a r ~ 0 ! 0 .....i.... N

•2 n ....o...... . . " . " .. " .( 0 } 0 I 0 N

I 0 c

MRS 516 J 0 i

0 d ! 0 ' 0

0 0 0 1

I o

MRSA 570 0 —† 1

-2"" I "' 0 0 0 1 0

I 0 0

MRSA 580 " c { 0 0

-2 fte I 0 0 " 0

- FT A. o

I MRSA 606 0 0 i

-2 Λ * Γ ~0 I 0 0 0 t I 0 GB

0 0

~ - 1* ¾' T 0 0 0 1

MRSA 0

610 "}"""

-2"" i 0 GB c i 0 c

GB * Bacterial Growth on Sheep Blood Agar

N" - Not cultured on Sheep Blood Agar

100344] The results for 10 VRE isolates are shown in Table 38. Each MBC test was performed in duplicate starting from NCL82 M!C concentration to 32 times the MIC. As with the MRSA isolates tested, the MBC appears to be equal to the MIC. However, with the VRE isolates, an anomaly was observed at higher NCL812 concentrations. There is significant inhibition of growth at concentrations near to the MIC, but as the NCL812 concentration increases, bacteria appear to be less subject to inhibition. High numbers of bacteria were observed on the plates at NCL812 concentrations≥16 g/mL.

Table 38: NCL812 MBC values {ug/ml) for 10 VRE isolates according to Example 7

I NCL812 MBC

Organism / Sample No.

2 yrj/mf i 4 &'ml 1 8j*gf»l 16|*g/ml $2 Mg/mi 64 ug/mS j 12S j - *' 00 *

VRE 26c(dc) j 2 4000 M U 1 " M

-2 n " b " "' j 70 3500 [ M j M _ 1 j

-1 ft * 500 20 250

VRE 3?c j 100 M LT M 1 M

-2 na M j 50 100 i ' ioo 1400 " Γ M 1 M

-1" 0

VRE 35* 1 o 0 720 0 I o j 0

-2 ηΰ 0 i o 0 0 10 Ζ 20 1 i ' o j VRE 16c(dc.J_ .1* 80 j 330 0 M M I M ί M VRE 12c

-Number of ; acteria growing after 24 ours per ml of sample (CFU/mi); = many cactena growing on the psate (too many to count)

[00345] NCL812 was found to be bactericidal against Gram-positive bacteria at concentrations equivalent to the iC.

Kill kinetics assays (MR$A & VRE)

[00346] In preliminary experiments, colony counts were performed at t ~ 15, 3D, 45 and 80 win. No significant changes in the bacterial concentration were observed at these time points, suggesting that NCL812 is not rapidly bactericidal (by comparison, the lipoglycopeptide oritavancin (McKay ei al. (2009) J. AniJmicrob. Chemother, 83 (6): 1181-1199) caused a 3log,o reduction in viable count within an hour of exposure to a concentration equivalent to the C ms:< ). Therefore, for future experiments, sampling time points were extended out to one arid then two hour intervals,

|00347| In the initial experiments, for MRSA, at 4 h a reduction of at least 2.5log, c CFU/mL was observed in comparison to the growth control. At. 8 h there was at least a 3.5log 1s CFU/mL difference between the control and the bacteria exposed to NCL812. After 24 h the numbers of bacteria present in all NCL8 concentrations was not significantly different to the control. There was a consistent reduction in bacterial numbers at NCLS12 concentrations from 4-16 pg/ml, up to 8 h, but the same was not observed for concentrations greater than 18 ug/mL. By comparison, most bactericidal antimicrobial agents, used or being developed for the treatment of MRSA end VRE (oritavancin, daptomycin, vancomycin) are rapidly bactericidal achieving similar log reductions within 1 h of exposure in a concentration dependent manner (McKay ei a/., 2009) in kii! kinetics experiments bacteriostatic antimicrobials recommended for the treatment of MRSA and VRE infections (teicoplanin and linezoltd only marginally decrease the viable count and growth).

[00348} For VRE the observed decrease in the CFU/mL of bacteria exposed to CL812 was less than for MRSA. At 4 h there was approximately a 2iog 10 reduction in the viable count compared to the control, and at 8 h there was approximately a 2.5!og 10 reduction. However, at 24 h the growth of bacteria was no longer as significantly reduced in comparison to the control. Bacterial numbers increased after 8 h incubation and this affect appeared to be more pronounced with increasing concentrations of NCL812.

[00349] The kill kinetics of MRSA 580 were obtained at different concentrations of CLS12 over a period of 8 h, as shown in Figure 34 The kill kinetics of RSA 580 in different concentrations of NCL812 over a period of 24 h are shown in Figure 35 After 4 h of incubation the media was changed to fresh media containing the same concentration of NCLS12.

[00350] The kill kinetics of MRSA 698 in different concentrations of NCL812 over a period of 24 h is shown in Figure 36 After 4 h of incubation the media was changed to fresh media containing the same concentration of CL812.

{00361} The kill kinetics of VRE 28c{dc) at different concentrations of NCL812 over a period of 24 h are shown in Figure 37.

[00352] The kill kinetics of VRE 16c(dc) at diffejent concentrations of CL81 over a period of " 24 h are shown in Figure 38.

Test for bacterial resistance to NCL812

{00353] Preliminary tests were carried out to determine whether bacterial resistance may account for t e observations of bacterial growth at higher concentrations of NCI..312 and the increase in bacterial numbers in Rill kinetics experiments at 24 h incubation. Bacteria (MRSA) growing at high concentrations of NCLB12, in the S6 welt micro titre tray, were subcultured on to SBA and incubated for 24 h, then MIC testing was performed. There was no change in the MIC of these bacteria. Bacteria growing in broth used for kill kinetics experiments was also tested for any change in the MIC. No change was observed.

(00354] Additionally, bacteria exposed to NCL812 at high concentrations were then subcultured on to plate count agar containing CLS12 {64 pg/ml and 128 pg/ml.) and incubated for 24 h at 37 : 'C. Bacteria growing on the piste were then used for running an MIC test. There was still no change in the MIC of the bacteria

[00355] In summary, NCLS12 has bactericidal activity against MRSA which is less pronounced against VRE strains. The bactericidal effect is not rapid in comparison to bactericidal antimicrobials developed for MRSA and VRE infections (daptomycin, oratovancin, vancomycin}. Aberrant bactericidal results at higher concentrations of NCLS12 are not indicative of resistance development, but may be suggestive of loss of activity. Stability testing of the compound in broth media should therefore be undertaken before exploring these interesting but currently unexplained Jesuits. This will include a detailed examination oi the literature to determine if this henomenon is observed in other classes of antimicrobial agent. In this case, closer examination of the kill kinetics between 8 and 24 h will be required. NCL812 kill curves for RSA and V E suggest more bactericidal activity in comparison to bacteriostatic antimicrobials {linesoli l. tescoplanin). Kill curves should now be generated for Streptococcus pneumoniae once stability issues of NCL812 are investigated, as for example with linezo!id, as some antibacterial agents can be bacteriostatic against some bacteria a rut bactericidal against others.

Synergy studies with other da$s&$ of tsmicrohlai egent.

{00366] !Cs, FICs, FiCs and the interaction between NCL812 and eight antibiotics is shown in Table 39. None of the eight tested compounds, representing distinct classes of antimicrobial agent showed either positive (synergism) or negative (antagonism) interaction with NCL812 consistent with an additive effect when antibacterial agents are added to NCL812.

Table 39: MICs, FICs, FSC- and the interaction between NCL812 and eight antibiotics according to Example 7.

Ciprofloxacin 2 ^ulfame lm oie 7 Penicillin G'

S. aureus strain 13-23

1 Staphylococcus spp. Strain MK1

FiCi = MIC of anitbi tio in combination with NCL812/MSC of antibiotic alone

FH¾ - MIC of NCL812 in combination with aotibiotte/MtC of NCL812 alone

FIG, * FiC i dex

Testing ofNCL812 Analogues

[00357] The chemical structures of analogues NCL001 to NCL230 are shown in Figure

1.

100358} !Cs for NCL8 2 and analogues NCL001-070 are shown in Table 40.

(003593 iCs for analogues CL071 to 171 are shown in Table 41

[00360] M!Cs for analogues NCL1 1 to 230 are shown in Table 42.

131

.c

8

CM

*■

«B

t

u

i

CM

a.

5

c

3

O a

E o

u

00

—I

O Λ

O

©

RECTIFIED SHEET 132

RECTIFIED SHEET 133

RECTIFIED SHEET 134

RECTIFIED SHEET Table 41 : MICs for analogues NCL071 -170 according to Example 7.

Table 42: M!Cs for analogues NCL 171 -230 according to Example 7.

M\ ί& \) at 24 hours

NCL ,0>i*tp¾u?xS

VREi VRS2 6. ca

32 >128 128 >12

NCL 71

28 32 128 >128 128 128 64 >128 i 12S >128 >128

NCL 172 — ..

128 64 128 >128 128 28

...... « |"

16 1« 4 • » 128 >128

NCL 173 "Y"

16 18 ■>128 12.8

32 128 :>12S

NCL 174

32 32 32 128 > 28 84 128 > 28 >128

NCL 175

84 32 123

>128 >128 >128

NCL 176

28 28 >1 8 t23 128 8 >128 »128

NCL 77

~~ 8 128 128

E ess$ ' 64 >64 >64

NCL 78

> 4 >6·4

4 >64 64

NCt. 7S

>&4 >64 >64 64 >64 >64

NCL 180

>64 64 >64 >64

>64 >64 >64 >64

NCL 181

>64 >64 >64 >64

>64 >64 >64 >64

NCL 182

>64 >64 >64 >64

>64 >64 >64 >64

NCL 183

>64 >64 >64 >64

>64 64 >64 >64

NCL 184

>64 64 >64 >64

>64 >64 >64 >64

NCL 185

>64 >64 >64 >64

>64 >64 >64 >64

NCL 186

>64 >64 >64 >64

>64 >64 >64 >64

NCL 187

>64 >64 >64 >64

32 32 64 >64

NCL 188

32 32 64 >64

>64 >64 >64 >64

NCL 189

>64 >64 >64 >64

32 64 >64 >64

NCL 190

64 64 >64 >64

>64 >64 >64 >64

NCL 191

>64 >64 >64 >64

>64 >64 >64 >64

NCL 192

>64 >64 >64 >64

MIC (pg/ml) at 24 hours

NCL Compound

Code

MRSA 1 MRSA 2 VRE 1 VRE 2 E.∞li P. aeruginosa

2 2 4 4 >128 >128

NCL193

2 4 4 8 >128 >128

>128 >128 >128 >128 >128 >128

NCL194

>128 >128 >128 >128 >128 >128

2 2 4 2 >128 >128

NCL195

2 2 4 4 >128 >128

32 64 32 64 64 64

NCL196

32 32 64 64 64 128

8 8 8 8 >128 >128

NCL197

8 8 4 8 >128 >128

>128 >128 >128 >128 >128 >128

NCL198

>128 >128 >128 >128 >128 >128

2 4 >128 >128 >128 >128

NCL199

4 4

>128 >128 >128 >128 >128 >1 8 128 r 12 * 123 > 28

..j

128 >128 >128 • •• 128 v1 8 >128

NCL201 32 16 > 28 >128 >12S > 28

64 82 >V2B >128 128 M28

NCL2G2 8 S δ 8 12a >128

8 8 8 >128 >128

NCL203 32 64 32 32 >128 M28

32 64 64 32 >128 >128

NCL204 4 4 64 >128 128 >m

8 8 64 >128 >128 >

NCL203 4 [ 4 64 >12B >128 128

8 > 8 64 1 8 >12S >128

NCL206 >V28 -•128 > 23 M28 M28 128

>12δ • 128 > 28 >128 >128 128

NCL207 16 32 >126 >128 >128 >128

32 32 128 >128 >128 >128

NCL208 > 28 128 v128 >128 > 28 28

>128 >123 >12S >128 8

NCL209 M26 >1 6 123 >128 > 28 28

128 128 >128 > 8 > 28 ¾28

NCl.210 >128 128 28 32 > 8 >128

>128 V128 28 54 28 >128

NCL211 > ?.S >128 > >128 >128 28

>128 >128 >V2 123 8 --128

„„,

NCL212 >126 >123 32 16 >128 >128

128 128 32 18 >V 128

NCL2 3 > 28 123 >123 128 >128 >1 8

12S 28 128 >1 8 M28 > 28

NCL214 >128 >128 28 >128 >128 >12S

128 >128 >128 >128 > 28 >128

NCL215 8 3 2. 4 > 8 12S

16 3 4 8 :>12S »128

NCL216 2 2 2 4 >128 128

4 2 4 12S •> 28

NCL21? 4 2. 4 >128 >128

4 4 2 4 28 >128

NCL218 >128 > 28 16 ■»128 > 28 128

>128 >128 16 128 > 28 28

NCL219 2 2 16 16 >128 >128

2 2 16 16 > 28 •"128

NCl.2.20 16 16 32 32 >128 28

16 16 32 32 128 128

NCI221 4 64 54 >128 > 28

4 2 64 1 *L_ >128 > 8 NCL222 >128 >128 >128 >128 >128 >128

>128 >128 >128 >128 >128 >128

NCL223 >128 >128 >128 >128 >128 >128

>128 >128 >128 >128 >128 >128

NCL224 >128 >128 >128 >128 >128 >128

NCL225 >128 >128 >128 >128 >128 >128

NCL226 >128 >128 >128 >128 >128 >128

NCL227 >128 >128 >128 >128 >128 >128

NCL228 >128 >128 >128 >128 >128 >128

NCL229 >128 >128 >128 >128 >128 >128

NCL230 >128 >128 >128 >128 >128 >128

(00361] The NCL analogues showing the highest level of Gram-negative antibacterial activity included CL030 {especially Ps&ud monas), NCLG41, CL043, NCLQ44 {especially PsBudo or)8$}, NCL0S2 (especially Ps&udomonas), and NCL053 {especially Pseudomo s), NCL097 {especially Pseudomonas), NCL196 and CL188 (especially £ ooff).

(00362} The NCL analogues showing greatest activity against MRSA included: NCL 021; MCl.023; NCL029; NCL030; NCL03S; NCL038; NCL039; NCL040: NCL041 ; NCLG43; CL044; NCL052; NCL054; NCl.062; NCL069; NCL072; NCL073; NCL074; NCL078; CL07Q; NCL080; NCL081 ; NCL082; NCL084; NCL088; NCL089; CL093; NCL094; NCL097; NCL099; NCL101; NCL104; NCL107; CL108: NCL111 ; NCL113; NCL117; NCL120: NCL121; NCL123; NCL136: NCL138; CL140; NCL143; NCL148; NCL150; NCL151: NCL153: NCL155; NCL157; NCL1S8; NCL159; NCL160; NCL161; NCL166; NCL168; NCL169; NCL171; NCL172; NCL173; NCL1 4;

CL177; NCL178; NCL179; NCL180; NCL181 ; CL182; NCL183; NCL184; NCL185; NCL186; NCL187; NCL 188; NCL 189; NCL 90; NCL191; NCL192; NCL193; NCL195; NCL 196; CL197; NCL1 9; NCL201; NCL202; NCL203 NCL204; NCL205; NCL207; NCL215; NCL21d; NCL217; NCL21 ; NCL220; and NCL221.

100383} The NCL analogues showing greatest activity against VRE included: NCL011; NCL021 ; CL023; NCL029; NCL030; NCL035; NCL038: NCL039; NCL040; NCL041 ; CL043: NCL044; NCL052; NCL054; CL061 ; NCL062; NCL089; NCL070; NCL072; NCL073; NCL074; NCL078; NCL079; NCL080; NCL081 ; NCL082; NCL084; NCL088; NCL093; NCL094; NCL097; CL099; NCL101; CL105; NCL107; NCL108; NCL111 ; NCL112; NCL113; NCL117; MCL120; CL121 ; NCL123; NCL128; NCL131 ; NCL138; NCL1 0; NCL141; NCL143; NCL146; NCL1S1; NCL153; NCL155; HCL.157; NCL158; NCL159; NCL160; NCL161; NCL166: NCL188; NCL 189; NCL171 ; NCL173; NCL174; NCL175; NCL177: NCL178; NCL179; NCL180; NCL181; NCL182; NCL183; NCL 184; NCL185; NCL188; NCL187; NCL188; NCL189; NCL190; NCL191; NCL192; NCL193; NCL195; NCL 198; NCL1S7; NCL202; NCL203: NCL204; NCL205; NCL2 0: NCL212; NCL215; NCL216; NCL217; NCL218; NCL219; NCL220; and NCL221.

[00364J The bioassay ranking of the analogues tested is shown in Table 43.

Table 43: The bioassay ranking of the analogues tested according to Example 7. r Γ !

' I G- M+R M+R MRSA MRSA VRE

Rsnk_ NCL ID ! m¾g rr * t___ rank fflcg n? rank NCL ED mcg/nsi k ..renK_

J^CL¾?1 1 MCL062 \ 2.2 * 1 NCL157 "J 1 NCL0S9 0.75

MCLl ' Sel 1 f CL157 j 2 NCL1 S 2 2 1.5 2

^CL03Q ! 1 NCL812 1 2.375 ' 3 NCL179 1 2 3 NCL812 1.75 3

NCL041 i. NCL195 I 2.75 __4 NCL219 * «>. 4 NCL084 4 _

NCL043 1 1 NCL216 I 2.75 NCL21€< 2.5 ! 5 NCL157 2.5

1 Ι...ΪΓ.

NCL044 [ 2.875 " β NCL 93 2.5 ! β I NCL081 2.5 " ~ δ J1CL052 :j 1 " NCL0S4 T _NCL062 _3 JNCL216. i _1 7 NCL053

EXAMPLE 8: The effects of NCL812 on antimicrobial sensitive isolates of Staphylococcus aureus and Enterococcus faecalis

Materials and Methods

Strain information {00366] Two Staphylococcus aureus isolates were used in the following experiments; S. aureus MK01 a human skin strain, and S. aureus KCOl an equine skin strain. These isolates were identified by Gram stain and biochemical methods, including the RemsS Staphs urex commercial kit. One Enterococcus faecaiis isolate (USA01 ), was not identified as a V E strain. As this isolate has previously bean speciated, it was not subjected to further testing, except for observation of pure, characteristic growth on blood agar.

Investigation of Minimum Bactericidal Concentration (MBC)

CLSI methodology

{00366] As in previous experiments, 10 μΐ. of the contents of each well starting at the MlC was inoculated on to a Columbia SBA plate and incubated at 37 °C for 48 h. Plates were examined at 24 an i 48 h and the MBC was recorded as the lowest concentration of NCL812 at which no colonies of bacteria were observed on the plate {or significant inhibition of growth was observed compared to the control) (CLSI 2005).

Kill kinetics assays for S. aureus KC01 & E. faecaiis USA01 Method

[00367] S. aureus KC01 and E. fa&calis USAG1 , not determined to be SA or VRE, respectively, were grown overnight on Columbia SBA at 37 "C. A few colonies of bacteria were then suspended in CAMHB (cation-adjusted Mueller Hinton broth) and adjusted to ODm of 0.08 to 0.10. The bacterial suspension was diluted 1 :10. One miil litre of the bacteria were added to 9 mL of CAWHB containing various concentrations (up to 4xMIC) of HCL, to achieve a ftnai bacterial concentration of 1 to 3*106 CFU/mL The tubes were incubated at 37 *C. with constant shaking. In order to determine the numbe of viable bacteria present at various time points, a 100 pL aliquot was removed from each tube and diluted. Then, 100 pi of each dilution were spread onto colony count agar, in duplicate, and incubated for 48 h at 37 °C. After 24 h the numbers of colonies present on each plate were counted and therefore the number of viable bacteria present in the original suspension enumerated. Plates were re-checked after 43 hours.

Results

Minimum Inhibitory Concentration (MIC)

[00368] The NCL812 MIC for isolates S. aureus MK01 and C01 , and E. faecalis USA01 was investigated. The results were: S. aureus K01 = 4-8 ug/mL, S. aureus C01 - 2 pg mL, £ faecaiis USA 01 - 4 pg/mL

[00369] S. aureus isolates MK01 and KC01 were investigated and no growth, or growth only at low concentrations of NCL812 (2 pg ml). was observed, indicating that CL812 is bactericidal against S. aureus. For the E. faecalis isolate tested (USA01) however, growth of bacteria was observed at all concentrations of NCL812 tested. There was an obvious reduction in the number of bacteria with increasing concentration, but growth was present compared with no growth for S. aureus. A summary of these results can be seen in Table 45. Table 45 shows the results for NCL812 MBC tests on two non-MRSA S. aureus isolates and one non-VRE E. faecalis isolate. Each MBC test was performed in duplicate. No change in the results was observed at 48 h. Table 37 shows NCL812 MBC values (pg/mL) for 20 MRSA isolates. Each MBC test was performed in duplicate starting from NCL812 MIC concentration to 16 times of MIC. Table 38 shows NCL812 MBC values (pg/ml) for 10 VRE isolates. Each MBC test was performed in duplicate starting from NCL812 MIC concentration to 32 times the MIC.

Table 45: NCL812 MBC tests on two non-MRSA Staphylococcus aureus isolates and one non- VRE Enterococcus faecalis isolate according to Example 8.

+ = Growth on Sheep Blood Agar; 0 = No Growth on Sheep Blood Agar; N = Not Cultured; Numbers in Parenthesis are the Number of Bacteria Growing after 24 hours per ml of sample (CFU/ml)

Kill kinetics assays for S. aureus KC01 & E. faecalis USA01 Method

[00370] Colony counts were performed at t = 0, 120, 240, and 360 min, then again at 24 h. At the 2 h time point S. aureus KC01 showed a minimum of a 2.5logi 0 reduction in bacterial numbers from initial numbers, and greater than a 3logi 0 reduction in comparison to the control at the same time point. A minimum of a 2log 10 reduction was still evident at 6 h incubation, however after 24 h the numbers of bacteria present had increased and this was not significantly different to the control.

[00371] Similar results were obtained with E. faecalis USA01 , however the reduction in bacterial numbers observed was less than for S. aureus KC01. A 2log 10 reduction in CFU/mL was observed at 2 h, compared to the growth control. However, the reduction in CFU/mL compared to the original bacterial numbers was only just greater than 1logi 0 . At concentrations of 4-16 pg/mL of NCL812 this reduction in bacterial numbers remained consistent until the 6 h time point. At concentrations of 32 and 64 pg/mL however, there was approximately a 1logi 0 rise in bacterial numbers over the same time period. At 24 h bacterial numbers at all concentrations had increased to almost the same level as the growth control

(00372] The results observed with these strains of S. aureus and £ faecalis are consistent with the results observed for the kill kinetics assay for ali SA and VRE isolates tested. The kiil kinetics assay of Staphylococcus aureus KC01 at different concentrations of CL812, up to 24 h incubation are shown in Figure 39. The kill kinetics assay of Entemcoccus faecalis USA01 at different concentrations of CL812, up to 24 h incubation are shown in Figure 40.

EXAMPLE 9: Formulations of Compounds

{00373} The following formulations were prepared using standard methods in the art.

Formulation A · - Topical Formulation - PEG-based Gel with compounds of the invention

4.0g PEG 4000:

S.Sg PEG 200;

0.8g propylene glycol;

1 9g water; and

0.204ο, of Compound (fo example, CL099)

100374] PEG 4000, PEG 200 and propylene glycol were mixed and heated to 150 °C and until ail solid crystals were dissolved. Compound was added to water and sonicated for 30 minutes until fully suspended. The Compound solution and gel solutions were mixed and allowed to cool and solidify. Formulation A will likely demonstrate acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.

Formulation B - Topical Formulation - PEG-based Gel with compounds of the invention

3.0g PEG 4000;

1.0s PEG 8000;

3.0g PEG 200;

1 -0g propylene glycol;

1 ,Sg water; and

0.202g of Compound (for example. NCL099)

(00375) PEG 4000 ; PEG 8000, PEG 200 and propylene glycol were mixed and heated to 150 *C and until all solid crystals were dissolved. Compound {for example, NCL099) as added to water and sonicated for 30 minutes until fully suspended. The Compound solution and gel solutions were mixed and allowed to cool and solidify. Formulation B demonstrated acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.

Formulation C - Topical Formulation - PEG-hased G&t with Comoound-Soluplus 2.5g PEG 4000;

4.0g PEG 200;

2.5g propylene glycol;

1.0g water; and

1.8g solid dispersion of Compound-SoluPlus.

[00376] Soluplus was purchased from BASF (www.soluplus.com). Compound-SoluPlus was prepared using standard methods in the art. PEG 4000, PEG 200, Compound-SoluPlus and propylene glycol were mixed and heated to 150 °C and until all solid crystals were dissolve. Water was added and then the solution was sonicated. The solution was allowed to cool and solidify. Formulation C demonstrated acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.

Formulation D - Tablet Formulation

30mg Calcium hydrogen phosphate dehydrate;

80mg Microcrystalline cellulose;

50mg Lactose;

8mg Hydroxypropyl methyl cellulose

1.5mg Talc

10mg of compound (for example NCL099)

[00377] The excipients were weighed and mixed for 5 minutes. The mixture was fed into a feed hopper of a tablet press machine and the machine was operated according to standard procedures in the art. Formulation D demonstrated acceptable tablet hardness, disintegration and frability.

Formulation E - Oral Suspension

2.0 ml Glycerol;

1.5ml Absolute ethanol;

600mg NCL812; and

To 60ml Vehicle (Ora Sweet and Ora Plus, 1:1).

[00378] NCL 812 powder was sieved through a 75 pm sieve. 600 mg of sieved NCL 812 was mixed with 2.0 ml glycerol and 1.5 ml absolute ethanol. The mixture was placed in a mortar and manually milled until all NCL 812 was suspended uniformly. The suspension was sonicated for 30 minutes. Vehicle (55 ml of Ora Sweet and Ora Plus mixture) was then added to the suspension and milled for another 10 minutes. Volume was made up with the Ora plus and Ora sweet mixture to 60 ml by transferring to a measuring cylinder

[00379] Formulation E demonstrated acceptable suspension and demonstrated acceptable short term stability. Formulation F -· Intramuscular Injection

2Gmg/mf Polyvinylpyrrolidone 30 (PVPK3G);

0.09mg mi NCL812; and

50ml water.

{00380} Two percent of w/v PVP K30 solution was prepared by the addition of 1.0 g of PVP K30 to 50 mi of iltiQ water. The solution was then placed in a sonicator for 30 minutes to equilibrate and 4.5 mg of NCL 812 was added to the PVP soiution nd placed on an incubator shaker at a maximum speed of 10 rpm over a period of 24 hours, with controlled temperature of 25±1 °C. Solution was transferred to 5 ml vsa!s and checked for clarity, appearance. pH and short-term stability. The pH of solution was 7.25.

[00381] Formulation F demonstrated acceptable transparency and short term stability.

EXAMPLE 10: Release of NCL812 and NCL0S9 f om Formulation 8.

[00382] The objective of this study was to measure the release of NCL812 and NCL099 from Formulation B prepared in Example 9.

[00383] Franz diffusion cells were utilized to quantify the release rate of NCL 812 and NCL099 from its topical formulations. Five milii!itres of absolute ethanol, which was chosen as the desired release medium, was loaded into the receptor chamber. Temperature of the receptor fluid was kept constant, at 32±1 * C using a water jacket. Acetyl cellulose membranes, with pore size of 0.45 um (Pall Corporation) was selected and placed between donor and receptor chamber. Followed by that, a number of test samples (Formulation 8} was loaded into the donor chamber. One mtlls!itre of receptor fluid was collected at regular time intervals of 0.25. 0.50, 0.75, 1, 2, 3 t 4, 5, 6, 7, 8 and 24 hours through the sampling port. One niltiiitre of fresh absolute ethanol was immediately returned to the receptor chamber. UV-RPLC was utilized to analyse the content of the receptor fluids attained.

[00384] Figure 41 presents the cumulative release of NCL812 and NCL099 over lime. This study demonstrates that Formulation B provides an acceptable release profile for NCL812 and NCL099.

EXAMPLE 11: R Specroscopy Lists of Compounds NCL812, NCL00 -NCL230 [00385] NMR Spectroscopy was performed on compounds NCL812, NCL001-NCL230 using standard methods in the art. The lists of the NMR spectroscopy are presented in Table 46.

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O O 0 0 O O O O O 0 0 0 0 O 0 z Z Z Z Z Z Z Z Z z z z Z Z Z EXAMPLE 12. Treatment of a bacterial infection in vivo by the administration of NCI812 or CL099.

[00386] The objective of fhis study was to determine t e efficacy of an investigational Veterinary Product containing NCL812 or NCL099 in the treatment of a skin infection in mice

[00387] Summary of the Mode!: A useful animal mode! system should be clinically relevant, experimentally robust, ethically acceptable, convenient to perform and should provide reliable and reproducible results. There are many animal models of topical skin infection that have been described including the croion oil-inflamed skin model (Akiyama, H„ H. anzaki, Y. Abe, J. Tada and J. Arata (1994). "Staphylococcus aureus infection on experimental croton oil- inflamed skin in mice." Journal of Dermatologicai Science 8(1 ): 1-10), the burnt skin model (Stierite, 0. O., A. Bondi, 0. McDermott and E. B. Michaels (1982). "A burned mouse mode! to evaluate anti pseudomonas activity of topical agents." Journal of Antimicrobial Chemotherapy 9(2): 133-140), the skin suture-wound model ( cRipley, . J. and R. R. Whitney (1976). "Characterization and Quantitation of Experimental Surgical-Wound infections Used to Evaluate Topical Antibacterial Agents." Antimicrobial Agents and Chemotherapy 10(1): 38-44), the skin tape-stripping model (Kuge!berg, £., T. Norstrdm, T. K. Petersen, T. Duvoid, O. I. Andersson and D. Hughes (2005). "Establishment of a Superficial Skin Infection Model in Mice by Using Staphylococcus aureus and Streptococcus pyogenes " Antimicrobial Agents and Chemotherapy 48(8): 3435-3441) and the linear full thickness scalpel cut method (Guo, Y., R. I. Ramos, J. 8. Cho, N. P. Donegan, A. L. Cheung and L. S. Miller (2013). "in Vivo Biotuminescence imaging To Evaluate Systemic and Topical Antibiotics against Community-Acquired Metftsciliin-Ressstani Staphylococcus aureus-Jnfecied Skin Wounds in Mice." Antimicrobial Agents and Chemotherapy 57(2): 855-863)

[00388] Preliminar studies prior to the conduct of the current study established a new method of skin infection arising from a detailed study of the models mentioned above. Briefly, study mice are anaesthetised, a patch of dorsal skin is clipped to reveal the skin and a circular area of skin is removed with a hand held punch, leaving a wound on the dorsum with a central cavity. The wound is infected with a known number of the challenge organism. Approximately four to six hours after infection, the wound is either treated topically w th a vehicle formulation or an active formulation. The infected skin wound is retreated every 12 hours for a total of 14 treatments. Mice are humanely euthanased, the area of the original infected wound is dissected and removed and its bacterial content quantified by standard microbiologic tests. In this way, the change in bacterial concentration due to treatment with the active formulation can be readily determined by examining the reduction in bacterial burden compared with the vehicle control. Materials and Methods

Preparation of Infection Inoculum

[00389] Fresh cultures of bacteria (Staphylococcus aureus) were grown on Sheep Blood Agar at 37 °C for 16 - 18 hours. A few typical colonies were selected and suspended in 10 ml of Tryptic Soy Broth and incubated overnight in a shaking incubator (240 rpm) at 37 °C. The overnight suspension was vortexed and diluted (1 :100) in fresh Tryptic soy broth (100 μΙ [0.1 ml] in 9.9 ml broth). The fresh suspension was incubated for 3 hours in a shaking incubator (as above) in order to obtain mid-logarithmic phase bacteria. Bacteria were pelleted through centrifugation at 7,500 rpm for 10 mins. Broth supernatant was removed and bacteria suspended in 10 ml Phosphate Buffered Saline (PBS). These steps were repeated a further two times. The density of the suspension was checked by measuring absorbance at 600 nm, using a spectrophotometer with saline as a blank, to confirm the target density at a reading of approximately 0.100, consistent with a bacterial density of 2.5 x 10 7 CFU/ml. The suspension was placed into a rack placed into a lockable transport box with an ice brick to maintain refrigeration during transport, followed by storage in cool room upon arrival at the mouse skin infection laboratory. Final suspension was mixed thoroughly before inoculating the skin wounds created in mice.

[00390] In order to ensure the purity and accuracy of the suspension, the following steps were performed prior to placement into lock box.

[00391] Purity of bacterial suspension ensured by spreading 100 μΙ of the final suspension onto a SBA (sheep blood agar) plate which was incubated at 37 °C for 18 hours and examined to confirm uniform growth of one colony type. Viable counts were performed on final suspension by prepping saline in Eppendorf tubes (approximately 900 μΙ per tube), removing 100 μΙ sample and adding to first Eppendorf tube, vortexing the mixture and repeating using 2 nd Eppendorf tube containing saline. This process was continued for 5 - 6 tubes. Finally, 100 μΙ of 5 ,h and 6 ,h dilutions were plated out on plate count agar, incubated at 37 °C for 18 hours and colony counts performed to confirm that the CFU/ml was approximately 2.5 x 10 7 . Following inoculation of the wounds, this process was repeated to ensure that no contamination or decrease in viable counts had occurred during the time of the surgery.

Skin Wound Surgical Procedure

[00392] Each mouse was placed into induction chamber and anaesthesia induced using 2% isoflurane. Eyes of each anaesthetised mouse were covered with veterinary eye lubricant in order to prevent corneal dehydration. Each mouse removed from induction chamber and placed onto surgical area, in front of individual aesthetic nose cone. While under anaesthesia each mouse was monitored for assessment of depth of anaesthesia (response to pain, blink reflex, skeletal muscle tone) and respiratory and cardiac function. Back skin hair was shaved from surgical area with mechanics! clippers. Shaved area was cleaned using 70% ethanol applied to paper towel followed by 10% w v povidone -iodine solution. Once the iodine solution svas dry, s subcutaneous injection of the nonsteroidal anti-inflammatroy agent meioxicarn was administered. Dorsal skin was pinched gently to allow creation of a circular full-thickness wound using ear punch/biopsy punch. Vehicle control and CL812 and NCL099 mice had wounds inoculated with 10 pi of bacterial suspension using a micropipette (2.5 x 10 s CFU/10 μΙ). Once the bacterial suspension was dry, mice were placed into individual recover boxes labelled with the mouse number. The time of inoculation was recorded. Initial body weights of each mouse were recorded on the appropriate score sheet. Mice recovered to full consciousness within 5 minutes. Recovered mice were returned to individual housing and monitored hourly for postsurgical or anaesthetic complications.

Post-Surgicei Care (4 hours post-surgery) 00393} Mice were assessed for post-surgica! complications and observations were recorded on clinic record sheet. Each mouse was carefully removed from IVC and placed into an assessment container, avoiding excessive handling or touching of the surgical site. Once the mouse was inside assessment container, it was assessed and the observations recorded on the post-surgical clinical record sheet. Whenever the suggested wellness breakpoints were reached, post-operative analgesia was administered and recorded on the clinical record sheet.

Animal Monitoring and Daily Care

(00394] Antibiotic Administration (7am and 8pm). The first administration of vehicle or NCL812 or NCL099 ointment occurred 4 hours post-surgically. Each ointment container was weighted prior to administration and the weight recorded. Each mouse was carefully restrained. Ointment (vehicle or NCL812 or NCL099) was applied to the lesion area and the treated mouse was returned to !VC where each mouse was observed to ensure ointment was not immediately removed by grooming. The weight of the ointment container post-administration was recorded. The vehicle and active NCL products were applied to the skin wound each 12 hours following the first administration for a total of 14 consecutive treatments. Both the NCL812 and NCL099 products (Formulation 8, as presented in Example 9) contained their respective active ingredients at a concentration of 20 mg/g. Approximately 0.1-0.2 g of ointment was applied on each occasion, delivering a total topical dose of NCL812 or NCL099 between 28 and 56 mg to mice weighing between 18 g and 25 g. [00395] Daily Monitoring. Monitoring of each mouse took place once daily at around 12pm. Each mouse carefully removed from IVC and placed into observation container, avoiding excessive handling or touching surgical site. The coat, posture, eyes, behaviour, vocalisation and activity whilst in the container were carefully assessed and observations recorded on assessment sheet. Mouse faeces (either on floor of cage or in container) were checked for consistency and observations recorded. The weight of each mouse was determined whilst it was in the container and change in body weight calculated and recorded. The observation container was disinfected with ethanol and set aside to dry while a fresh container was used for the next mouse. For every second day, mice were again anaesthetised using 2% isoflurane and photographed using a ruler for size referencing. These photos were used to assess lesion size and infection progression during the trial period.

Tissue analysis and assessment of antibacterial efficacy

[00396] At the end of the 7 day skin wound assessment period, all test mice were euthanased prior to wound collection for post mortem examination. The skin wound was dissected from the dorsum of each mouse. The sample was placed in a sample tube and weighed before 1 ml PBS and sterile tissue homogenisation beads were added. Tissue samples were homogenised for 10 mins using a tissue homogeniser (Next Advance Bullet Blender) and then vortexed for approximately 30 seconds. 100 μΙ of supernatant was removed and placed into an Eppendorf tube containing 900 μΙ of PBS. This procedure was repeated using serial dilutions for a total of 8 dilutions. Finally, 100 μΙ of each dilution was pipetted onto a plate count agar in duplicate and incubated overnight at 37 °C. Ten microlitres of original suspension was placed onto sheep blood agar to assess culture purity and incubated overnight at 37 °C. The following day, viable counts were performed using incubated plate count agar plates and the identity of Staphylococcus aureus (the challenge organisms) as the harvested strain was confirmed.

Results

[00397] The mean colony count per g of tissue observed in vehicle treated group was 5,888,436 (6.77 logi 0 ). The mean colony count per g of tissue observed in NCL812 group was 141 ,254 (5.15 logi 0 ). The mean colony count per g of tissue observed in NCL099 treated mice was 1 ,318 (3.12logio). The logi 0 colony forming units per gram of tissue and % reduction are summarised in the following table. Table 47: Log< g colony forming units per gram of tissue and percentage reduction following topics! administration of vehicle and treatment.

Treatment j LoQ 1f) {CFU/g) %reducison

Vehicle

NCL312 5.15 97.6 CL099 3.12 99.98

£00398] it is clear from this table that treatment with either NCL812 or NCL099 leads to high level reductions in the number of infecting Staphylococcus aureus. These results demonstrate effective treatment of a bacteria! colonisation or infection in vivo by administration of compounds of the invention.