AU2013257422A1

AU2013257422A1 - Short fatty acid tail polymyxin derivatives and uses thereof

Info

Publication number: AU2013257422A1
Application number: AU2013257422A
Authority: AU
Inventors: Martti Vaara; Timo Vaara
Original assignee: Northern Antibiotics Oy
Current assignee: Northern Antibiotics Oy
Priority date: 2008-02-08
Filing date: 2013-11-12
Publication date: 2013-11-28
Anticipated expiration: 2029-02-05
Also published as: AU2013257422B2

Abstract

H:\amc\Intenvoven\NRPorbl\DCC\AMC\58O9951_1.DOC-12/11/2013 The present invention relates to a polymyxin derivative wherein the derivative has a total of three positive charges at physiological pH and wherein the terminal moiety (D) of the derivative comprises a total of 1 to 5 carbon atoms; and to a combination product comprising at least two such derivatives. The invention further relates to a method for sensitizing Gram-negative bacteria to an antibacterial agent by administering, simultaneously or sequentially in any order a therapeutically effective amount of said antibacterial agent and a derivative according to the present invention to said subject; to methods for developing novel antibiotics; and for sensitizing clinically important bacteria to a host defense mechanism complement present in serum. The invention also relates to a method of treating a subject for a gram-negative bacterial infection by administering a polymyxin derivative of the invention in combination with a second antibacterial agent. Finally, the invention relates to a process for preparing such polymyxin derivatives.

Description

WO 2009/098357 - 1 - PCT/F12009/050093 SHORT FATTY ACID TAIL POLYMYXIN DERIVATIVES AND USES THEREOF This application is a divisional of Australian Patent Application No. 2009211287, the entire contents of which is incorporated herein by reference. TECHNICAL FIELD The present invention relates to polymyxin derivatives and to uses thereof in the treatment of infections caused by Gram-negative bacteria. The 5 polymyxin derivatives of the present invention are especially useful in sensitiz ing bacteria to enhance the effects of other antibacterial agents. BACKGROUND Sepsis kills more than 215,000 Americans each year. It is estimated that 750,000 Americans are Infected with severe sepsis and 29% of them die 10 from it each year. Sepsis deaths make 9% of all death cases in the U.S. Sep sis kills as many Americans as myocardial infections, even more than traffic accidents. Two to three million Americans acquire a hospital infection each year and 10% of these infections progress to sepsis. More than 90,000 of 15 these patients die from sepsis infected in hospitals. Severe sepsis and septic shock (severe sepsis combined with low blood pressure) took up to 135,000 lives each year in the intensive care units (ICU) in the European Union according to the OECD Health Report of 2000. In Britain, 5,000 out of 100,000 patients who acquired a hospital infection die 20 from sepsis every year in acute care hospitals belonging to the NHS organisa tion. The death toll has increased year after year due to the fact that the number of patients predisposed to sepsis, such as the elderly, premature neo nates, and cancer patients, has increased, not least because many serious 25 Illnesses are more treatable than before. Also the use of invasive medical de vices and aggressive procedures has Increased. Gram-negative bacteria cause more than 40% of all septicemic in fections and many of the Gram-negative bacteria are extremely multiresistant. Gram-negative bacteria provide a harder challenge in therapy than Gram 30 positives, as they possess a unique structure, the outer membrane, as their outermost structure. Lipopolysaccharide molecules located on the outer mem brane inhibit the diffusion of many antibacterial agents deeper into the cell, where their ultimate targets are located. More than 95% of the novel antibacte rial agents isolated from nature or chemically synthesized in 1972-1991 lacked 35 activity against Gram-negatives (Vaara 1993).

WO 2009/098357 PCT/F12009/050093 2 Polymyxins are a group of closely related antibiotic substances pro duced by strains of Paenibacillus polymyxa and related organisms, These cati onic drugs are relatively simple peptides with molecular weights of about 1000. Polymyxins, such as polymyxin B, are decapeptide antibiotics, i.e. they are 5 made of ten (10) aminoacyl residues. They are bactericidal and especially ef fective against Gram-negative bacteria such as Escherichia coli and other spe cies of Enterobacteriaceae, Pseudomonas, Acinetobacter baumanni, and oth ers. However, polymyxins have severe adverse effects, including nephrotoxic ity and neurotoxicity. These drugs thus have limited use as therapeutic agents 10 because of high systemic toxicity. Polymyxins have been used in the therapy of serious infections caused by those bacteria, but because of the toxicity, their use was largely abandoned in the 70's when newer, better tolerated antibiotics were devel oped. The recent emergence of multiresistant strains of Gram-negative bacte 15 ria has necessitated the therapeutic use of polymyxins as the last resort, in spite of their toxicity, and as many of the less toxic antibiotics have already lost their effectiveness against particular strains of the said bacteria, the use of po lymyxins has again increased. Accordingly, polymyxins have now been recalled to the therapeutic 20 arsenal, although, due to their toxicity, on a very limited scale. Their systemic (i.e. non-topical) use is, however, largely restricted to the therapy of life threatening infections caused by multiply resistant strains of Ps. aeruginosa and A. baumannil as well as by carbapenem-resistant enteric bacteria.

WO 2009/098357 PCT/F12009/050093 3 Polymyxins consist of a cyclic heptapeptide part and a linear part consisting of a tripeptide portion and a hydrophobic fatty acid tail linked to the a-amino group of the N-terminal amino acid residue of the tripeptide and may be represented by the general formula: 5 (R7 -- (8>R8 (,)R6 (9)R9 (5)R5 (10)RIO (4)R4 (3)R3 (2)R12 (l)RI R(FA) wherein R1-R3 represent the tripeptide side chain portion; R4-R10 the heptapeptide ring portion -and R(FA) represents the hydrophobic fatty acid 10 tail linked to the a-amino group of the N-terminal amino acid residue of the tripeptide. The polymyxin group includes the following polymyxins: A1, A2, B1 B6, IL-polymyxin B1, C, D1, D2, El, E2, F, K1, K2, M, P1, P2, S, and T (Storm et a. 1977; Srinivasa and Ramachandran 1979). All polymyxins are polyca 15 tionic and possess five (5) positive charges, with the exception of polymyxin D, F, and S which possess four (4) positive charges. It should be noted that modi fied polymyxins that lack the fatty acid part R(FA) but carry R1-R10 have one additional positive charge when compared to the natural polymyxins they de rived from, due to the free a-amino group in the N-terminus of the derivative. 20 Accordingly, for example, such a derivative of polymyxin B or polymyxin E car ries six (6) positive charges in total. The clinically used polymyxin B and polymyxin E differ from each other only in the residue R6, which is D-phenylalanyl residue in polymyxin B and D-leucyl residue in polymyxin E.

WO 2009/098357 PCT/F12009/050093 4 Also circulin A and B are classified as polymyxins (Storm et aL. 1977). They differ from other polymyxins only in carrying isoleucyl residue in the position R7 whereas other polymyxins have either threonyl or leucyl resi due in the said position. For an overview of the structures of some polymyxins, 5 see Table 1. Table 1. The structure of selected polymyxins and octapeptin as well as selected derivatives thereof Compound R(FA) RI R2 R3 R4 R5 RB R7 R8 RI RIO Polymyxin B MO(H)A- Dab. Thy, Dab- "Dab- Dab- 0 Phe. Lou- Dab Dab *Thr Colistin (polymyoin E) MO(H)A. Dab- Thy Dab. *Dab- Dab- 0 Lou- Leu- Dab Dab Thr Colistin sulphomethate MO(H)A- sm-Dab- Thr- sm-Dab- 'Dab. Sm-Dab- 0 Lou- Lou- sm-Dab- sm-Dab- Thr Polymyxin A MO(H)A- Dab- Th. 0 Dab- *Tab- Dab- D Lou- Thy. Dab Dab Irbr Polynyxin M MOA Dab- Thr. Dab- 'Dab- Dab- D Lou- Thr- Dab Dab 'Thr Polymyxn D MO(H)A. Dab- Thr- D-1- *Dab- Dab. D Lou- Thr- Dab Dab Thr Circulln A MOA Dab- Thr- Dab- "Dab. Dab. D Lou- fla. Dab Dab Thr Octapmptln A ODHMDA -- - Dab- *Dab- Dab- D Lou- Lau- Dab Dab Thr D3eacylcollatin (DAC) Dab- Thr- Dab. *Dab- Dab- D Lou- Leu- Dab Dab *Thr Polymyin E nonspptide(PMEN) Thr- Dab- Dab- Dab. D-LeU- Leu- Dab Dab *Thr Docylpoynyxin B (DAPB) Dab- Thr- Dab- *Dab- Dab- D Phe- Lou. Dab Dab *Thr Polymyxin B nonapepUde (PMBtN) Th- Dab- Dab- Dab- D Ph.- Leu- Dab Dab *Thr Polymyxin B octapeptide (PMBO) Dab- *Dab- Dab- D Pha- Lau. Dub Dab *Thr PolymyxIn B huptapeptido (PMHP) 'Dab- Dab- D Pho- Lou- Dab Dab *Thr WO 2009/098357 PCT/F12009/050093 5 Polymyxin B is represented by the following formula: 7)Len -- (s)Dab+

(

6 pPhe (9)Dab+ ,sDab+ (l)Thr

(

4 )Dab ()Dab+ 1 (flDab MHA/MOA Commercially available polymyxin B is a mixture, where R-FA is 5 predominantly 6-methyloctanoyl (6-MOA, in polymyxin B1) but may also be a related fatty acyl such as 6-methylheptanoyl (6-MHA, in polymyxin B2), oc tanoyl (in polymyxin 63), or heptanoyl (polymyxin B4) (Sakura et at 2004). All these variants are equally potent against Gram-negatives such as E. coli (Sa kura et at 2004). Quite analogously, in polymyxin El (colistin A) and in circulin 10 A the R-FA is 6-MOA and in polymyxin E2 (colistin B) and in circulin B the R FA is 6-MHA. Numerous researchers have attached various hydrophobic moie ties including various fatty acyl residues to the N-terminus of polymyxin deriva tives and analogues and have shown that the resulting derivatives have potent antibacterial activity (Chihara et at 1973, Sakura at al. 2004 and in US patent 15 publication 2006004185. Even the derivative that carries the bulky hydrophobic 9-fluorenylmethoxycarbonyl residue as the R-FA is almost as potent as po lymyxin B in inhibiting the growth of E. coli and other Gram-negative bacteria (Tsubery et a. 2001). For biological activity the heptapeptide ring structure is essential 20 (Storm at al 1997). A derivative with an octapeptide ring is significantly less active as an antibiotic.

C:\NRPonbl\DCC\FMf289973.l.DOC-l5/1 1/2019) -6 Multiple modifications of polymyxins and multiple polymyxin-like synthetic molecules have been made, and with certain limits they have preserved their biological activity. The modifications comprise but are not limited to those in the side chain, as well as molecules in which an inherent hydrophobic amino acid residue (such as DPhe or Leu) has been replaced with another hydrophobic amino acid residue or in which the cationic Dab has been replaced with another cationic amino acyl residue, such as Lys, Arg, or ornithine residue (Storm et a/. 1997, Tsubery et al. 2000a, Tsubery et al. 2002, US patent publication 2004082505, Sakura et al. 2004, US patent publication 2006004185). Other modifications that result in microbiologically at least partially active compounds comprise but are not limited to alkanoyl esters where the OH-groups of the threonyl residues form esters with alkanoyls such as propionyl and butyryl (US Patent 3,450,687). Octapeptins are closely related to polymyxins but have a covalent bond instead of the residues R1-R2 (Table 1). In this invention, the R positions are numbered according to those in the natural polymyxins and thus the only amino acyl residue in the side chain of octapeptins is defined as R3. Accordingly, octapeptins are octapeptides whereas all natural polymyxins are decapeptides, and they possess only four (4) positive charges. The R-FA residues among various octapeptins (Al , A2, A3, B1 , B2, B3, Cl ) include the following: 3-OH-8 methyldecanoic acid, 3-OH-8-methylnonanoic acid, and p-OH-6-methyloctanoic acid. Derivatives that possess a fatty acyl residue with 6 to 18 carbon atoms have a potent antibacterial activity against E. coli (Storm et al. 1977). The first target of polymyxins in Gram-negative bacteria is their outer membrane (OM) that is an effective permeability barrier against many noxious agents including large (Mw more than 700 d) antibiotics as well as hydrophobic antibiotics. By binding to the lipopolysaccharide (LPS) molecules exposed on the outer surface of the OM, polymyxins damage the structure and function of the OM and, as a result, permeabilize (i.e. make permeable) the OM to polymyxin itself, as well as to many other noxious agents (Nikaido and Vaara 1985, Vaara 1992, Nikaido 2003). The final and lethal target (the bactericidal target) of polymyxins is believed to be the cytoplasmic membrane (the inner membrane) of bacteria.

WO 2009/098357 PCT/F12009/050093 7 Numerous efforts have been made to reduce the toxicity of polymyx ins. The treatment of polymyxin E (colistin) with formaldehyde and sodium bi sulfite yields collstin sulphomethate, in which the free amino groups of the five diaminobutyric acid residues have partially been substituted by sulphomethyl 5 groups (Table 1). The preparations consist of undefined mixtures of the mono-, di-, tr-, tetra-, and penta-substituted compounds. The sulphomethylated prepa rations, when freshly dissolved in water, initially lack both the antibacterial ac tivity and toxicity of the parent molecule, but when the compounds start de composing in the solution, in the blood or in the tissues to yield less substituted 10 derivatives and free colistin, both the antibacterial activity and the toxicity are partially brought back. Furthermore, the degree of initial sulphomethylation ap parently varies between the commercially available pharmaceutical prepara tions. Many other ways to block all the free amino groups have been pub lished. Examples comprise but are not limited to the formation of unstable 15 Schiff bases with amino acids (Storm et al. 1977). Polymyxin E nonapeptide (PMEN, colistin nonapeptide, Table 1), obtained by treating polymyxn E enzymatically and lacking the R-FA and RI, was shown in 1973 to be less toxic than the parent compound in acute toxicity assay (immediate death presumably due to direct neuromuscular blockade) in 20 mice (Chihara at a. 1973). However, it also lacked the antibacterial activity, as measured as its ability to inhibit bacterial growth (Chirara et at 1973). The role of the linear part may contribute to the antibacterial activity of the polymyxins. Vaara and Vaara, on the other hand, showed, that polymyxin B nonapeptide (PMBN, Table 1) retains the ability to permeabilize the OM of 25 Gram-negative bacteria (Vaara and Vaara 1983a,b,c; US Patent 4,510,132; Vaara 1992). Accordingly, even though it lacks the direct antibacterial activity (i.e. the ability to inhibit bacterial growth), it is able to sensitize (i.e. make sensi tive or, as also termed, make susceptible) the bacteria to many antibacterial agents such as hydrophobic antibiotics as well as large antibiotics and some 30 other noxious agents. PMBN also sensitizes bacteria to the bactericidal activity of the hu man complement system, present in fresh human serum as a first-line defence system against invaders (Vaara and Vaara 1983a, Vaara at aL 1984, Vaara 1992). Furthermore, it sensitizes the bacteria to the joint bactericidal activity of 35 serum complement and human polymorphonuclear white cells (Rose at al. 1999), WO 2009/098357 PCT/F12009/050093 8 PMBN resembles PMEN in being less toxic In the acute toxicity as say in mice than unmodified polymyxins. In further toxicological assays, sev eral criteria proved PBMN to be less toxic than its parent compound, but this polymyxin derivative was still judged to be too nephrotoxic for clinical use 5 (Vaara 1992). PMBN carries five (5) positive charges. Subsequent studies re vealed, quite expectedly, that PMEN, also carrying five (5) positive charges as well as deacylpolymyxin B and deacylpolymyxin E, both carrying six (6) posi tive charges are potent agents to sensitize bacteria to other antibiotics (Vil 10 janen et al. 1991, Vaara 1992). In addition, it has been shown that a structur ally further reduced derivative polymyxin B octapeptide (PMBO) retains a very effective permeabilizing activity while polymyxin B heptapeptide (PMBH) is less active (Kimura at al. 1992). PMBN, PMEN and PMBO have five (5) positive charges while PMBH has only four (4) positive charges. This difference may 15 explain the weaker activity of PMBH. The group of Ofek, Tsubery and Friedkin recently described po lymyxin-like peptides that were linked to chemotactic peptides, such as fMLF, that attract polymorphonuclear leucocytes (US patent publication 2004082505, Tsubery et al. 2005). They described peptides fMLF-PMBN, MLF-PMBN, 20 fMLF-PMEN, fMLF-PMBO and MLF-PMBO, all carrying four (4) positive charges, that sensitize Gram-negative bacteria to antibiotics, even though no comparative studies with increasing concentrations of the compounds were published (Tsubery at al. 2005). In order to study the structures and functional properties of polymyx 25 ins, a few works have disclosed, among other compounds, polymyxin deriva tives having less than four (4) positive charges. Teuber (1970) has described the treatment of polymyxin B with ace tic anhydride that yields a preparation containing polymyxin B as well as its mono-, di-, tri-, tetra-, and penta-N-acetylated forms. Teuber also separated 30 each group and nonquantitatively reported using an agar diffusion assay that penta-acetylated and tetra-acetylated forms lacked the ability to halt the growth of Salmonella typhimurum, whereas di- and monoacetylated forms did have such ability. Triacetylated form had some ability. Srinivasa and Ramachandran (1978) isolated partially formylated 35 polymyxin B derivatives and showed that a diformyl derivative as well as a tri formyl derivative inhibited the growth of Pseudomonas aeruginosa. They did WO 2009/098357 PCT/F12009/050093 9 not disclose the compounds' ability to sensitize bacteria to antibiotics. Fur thermore, in 1980 they showed that the free amino groups of triformyl polymyxin B in residues R1 and R3, as well as the free amino groups of difor mylpolymyxin B in residues R1, R3, and R5 are essential while the free amino 5 groups in R8 and R9 are not essential for the growth inhibition (Srinivasa and Ramachandran, 1980a). A shortened polymyxin B derivative octanoyl polymyxin B heptapep tide has been disclosed by Sakura et al. (2004). The attachment of the oc tanoyl residue to the N-terminus of the residue R4 of the polymyxin B hep 10 tapeptide results in a compound having only three (3) positive charges. Sakura et al. found that octanoyl polymyxin B heptapeptide Inhibits the growth of bac teria only at a very high concentration (128 pg/ml), whereas the other deriva tives such as octanoyl polymyxin B octapeptide and octanoyl polymyxin B nonapeptide, both having four charges (4) were very potent agents to inhibit 15 bacterial growth. US patent publication 2006004185 recently disclosed certain po lymyxin derivatives and intermediates that can be used to synthesize new pep tide antibiotics. The antibacterial compounds described possessed four (4) or five (5) positive charges. 20 Furthermore, closely related polymyxin B and polymyxin B1 com pounds have also been disclosed by Okimura et al. (2007) and de Visser et al. (2003). Okimura et al. have studied the chemical conversion of natural po lymyxin B and colistin to their N-terminal derivatives and de Visser et al. have studied solid-phase synthesis of polymyxin B 1 and analogues via a safety 25 catch approach. The antibacterial compounds described in these works pos sessed four (4) or five (5) positive charges. There is still an urgent need for polymyxin derivatives, which sensi tize bacteria to enhance the effects of other antibacterial agents, for effective treatments for bacterial infections, in particular for the infections caused by 30 multiresistant Gram-negative bacteria. SUMMARY The present invention relates to a polymyxin derivative wherein the total number of positive charges at physiological pH is three and wherein the derivative has a fatty acid tail (i.e., R(FA) or D) comprising I to 5 carbon at 35 oms, It has been found that certain polymyxin derivatives of the invention hav- WO 2009/098357 PCT/FI2009/050093 10 ing fatty acid tails of I to 5 carbon atoms may have improved pharmacokinetic properties as compared to native polymyxins, octapeptins, and polymyxin de rivatives with longer fatty acid tails. Examples of these pharmacokinetic proper ties include, but are not limited to, longer serum half life, increased renal clear 5 ance, and/or increased urinary recovery. The present invention pertains, at least in part, to polymyxin deriva fives of formula (1): RA3

R

2 ' I R' m3 M2 Ml 10 (1) wherein: A is a polymyxin ring moiety; D is a terminal moiety comprising 1 to 5 carbon atoms; mi, m 2 , and m 3 are each independently 0 or 1; 15 Q', Q 2 , and Q 3 are each independently CH 2 , C=O, or C=S; WI, W 2 , and W 3 are each independently NR 4 , 0, or S;

R

1 , R 2 -, and R 3 ' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, alkyl, arylalkyl, aryl, alkoxy, alkoxycar bony, aryloxycarbonyl, alkylamino, or alkynyl; and 20 R 4 is hydrogen or alkyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that (1) when A is an octapeptin ring, ml and m 2 are 0, m 3 is 1, W 3 is NH, Q 3 is C=0, and R 3 ' is the side chain of diaminobutyric acid (Dab), then D is not C 2 -C acyl, and (2) when D is acetyl, butanoyl or pentanoyl, then R 3 ' is not the side chain 25 of Dab. The invention is also directed to polymyxin derivatives of formula (11): WO 2009/098357 PCT/F12009/050093 11 0 R3' R 0 R1' H H 11 1 H A CN C N C N D m m2 (Il) wherein: A is a polymyxin ring moiety; 5 D is R 12 -C(=O), R 1 -C(=S), or R"; m mand m 3 are each independently 0 or 1, provided that at least one of mi, M 2 , and m 3 are 1;

R

1 , Rr, and R 3 ' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl, 10 aryloxycarbonyl, alkylamino, or alkynyl; and R1 2 is C 1

-C

4 alkyl, CrC4 alkenyl, or CrC4 alkynyl,

R

12 is C 1

-C

5 alkyl, C 2

-C

8 alkenyl, or C 2

-C

6 alkynyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that (1) when A is an octapeptin ring, ml and m 2 are 0, m 3 is 1, and R 3 ' is the side 15 chain of dlaminobutyric acid (Dab), and D is R' 2 -C=Q, then R1 2 is not C 1 -C alkyl , and (2) when D is acetyl, butanoyl or pentanoyl, then R 3 ' is not the side chain of Dab. In another embodiment, the invention also includes polymyxin de rivatives of formula (111): 20 0 R 3 0 R 2 0 R 1 ' A-i C-I D wherein: A is a polymyxin B or polymyxin E ring moiety; 25 D is R' 2 -C(=Q), R' 2 -C(=S), or R12; ml is 0 or 1;

R

1 , R 2 ', and R 3 ' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl, WO 2009/098357 PCT/F12009/050093 12 aryloxycarbonyl, alkylamino, or alkynyl, wherein at least one of R 2 ' and R comprise a carbamyl, hydroxyl or carboxylate group; and

R

12 s C1-C 4 alkyl,

R

1 ' is C 1

-C

5 alkyl, 5 and pharmaceutically acceptable prodrugs and salts thereof, provided that when D Is acetyl, butanoyl or pentanoyl, then R 3 is not the side chain of Dab. In yet another embodiment, the invention also includes polymyxin derivatives of formula (IV): /M3 M2 M1 H H 1 A- N-C N C i 10 (IV) wherein: A is a polymyxin B or polymyxin E ring moiety; mi is 0 or 1; 15 L 1 , L2 and L 3 are each independently C 1

-C

3 alkyl or a covalent bond;

M

1 , M 2 and M 3 are each independently H, C(=O)NH 2 , C(=O)OH, or -OH;

R

12 is C1-C4 alkyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that 20 when R 2 is methyl, propyl or butyl, then L 3 - M 3 is not the side chain of Dab, and wherein said derivative has three positive charges at physiological pH. In another embodiment, the invention also pertains to polymyxin de rivatives of formula (V): WO 2009/098357 PCT/F12009/050093 13

(

7 )R7 - (8)R8 (6)R6 (9)R19 (s)R5 (l)R10 (4)R4 (3)R3 ()R2 R(FA) (V) wherein R4 is an amino acid residue comprising a functional side chain able to cyclicize the molecule; 5 R6 and R7 are each independently selected optionally substituted hydrophobic amino acid residues; R10 is Leu or any non-hydrophobic amino acid residue; and wherein RI is optional; and wherein R1, R2, R3, R5, R8 and R9 are each independently selected amino acid residues; and wherein R(FA) is an 10 optionally substituted alkanoyl or alkyl residue having a total of I to 5 carbon atoms; or a pharmaceutically acceptable salt or prodrug thereof, provided that (1) when RI and R2 are absent, R3, R4. R5, R8, and R9 are Dab, R6 is D Leu, R7 is L-Leu or L-Phe, and R10 is Thr, or when RI and R2 are absent, R3, R4. R5, R8, and R9 are Dab, R6 Is D-Phe, R7 is L-Leu, and R10 is Thr, then 15 R(FA) is not an unsubstituted alkanoyl residue, and (2) when R(FA) is acetyl, butanoyl or pentanoyl, then R 3 is not Dab. More specifically, the present invention relates to a derivative, wherein R2-R10 is selected from the group consisting of Thr-DSer-cy[Dab Dab-DPhe-Leu-Dab-Dab-Thr-] [= SEQ ID NO: 10] and Thr-DAsn-cy[Dab-Dab 20 DPhe-Leu-Dab-Dab-Thr-] [ = SEQ ID NO: 39]. SEQ ID NO: 10 corresponds to SEQ ID NO:1 and SEQ ID NO: 39 corresponds to SEQ ID NO:2 in the at tached sequence listing.

WO 2009/098357 PCT/F12009/050093 14 The Invention also relates to a combination product comprising two or more of the derivatives according to the present invention, and to a pharma ceutical composition comprising such derivative(s) or a combination thereof and pharmaceutically acceptable carriers and excipients. 5 Furthermore, the present invention relates to a method for sensitiz ing Gram-negative bacteria to an antibacterial agent, comprising administering, simultaneously or sequentially in any order, a therapeutically effective amount of said antibacterial agent and a derivative according to the present invention, wherein said antibacterial agent may be selected from the group consisting of 10 clarithromycin, azithromycin, erythromycin and other macrolides, ketolides, clindamycin and other lincosamines, streptogramins, rifampin, rifabutin, rifa lazile and other rifamycins, fusidic acid, mupirocin, oxazolidinones, vancomy cln, dalbavancin, telavancin, oritavancin and other glycopeptide antibiotics, fluoroquinolones, bacitracin, tetracycline derivatives, betalactam antibiotics, 15 novobiocin, pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, and bacterial efflux pump inhibitors. Also provided are methods for developing novel antibiotics; and for sensitizing clinically important Gram-negative bacteria to a host defense mechanism complement present In the serum. 20 The present Invention also provides uses of a polymyxin derivative according to the present invention in the manufacture of medicament for sensi tizing Gram-negative bacteria, such e.g., Escherichia col, Klebsiella pneumo niae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundi, and Acine tobacter baumannii against antibacterial agents; and for sensitizing Gram 25 negative bacteria to a host defense mechanism complement present in the serum. The present invention also pertains to methods of treating Gram negative infections in a subject comprising administering a derivative of the invention (e.g., a derivative of formulae (I)-(V)) in combination with an antibac 30 terial agent to a subject, such that the subject is treated for the infection. Finally, the present invention relates to a process for preparing a po lymyxin derivative according to the present invention, comprising (A) modify ing a natural or synthetic polymyxin or octapeptin compound or a derivative thereof carrying 4 to 5 positively charged residues and a terminal moiety (D) 35 comprising I to 5 carbon atoms by replacing 1 to 2 of said positively charged residues by neutral residues or a covalent bond, or by converting 1 to 2 of said WO 2009/098357 PCT/F12009/050093 15 positively charged residues into neutral residues in order to obtain a polymyxin derivative of formula (1) carrying 3 positively charged residues and a terminal moiety (D) comprising 1 to 5 carbon atoms, or (B) modifying a natural or syn thetic polymyxin or octapeptin compound or a derivative thereof carrying 4 to 5 5 positively charged residues and a terminal moiety (D) comprising more than 5 carbon atoms by replacing 1 to 2 of said positively charged residues by neutral residues or a covalent bond, or by converting 1 to 2 of said positively charged residues into neutral residues, and by replacing said terminal moiety (D) hav ing more than 5 carbon atoms with a terminal moiety (D) comprising in total I 10 to 5 carbon atoms in order to obtain a polymyxin derivative of formula (1) carry ing 3 positively charged residues and a terminal moiety (D) comprising in total I to 5 carbon atoms, or (C) modifying a natural or synthetic polymyxin or oc tapeptin compound or a derivative thereof carrying 4 to 6 positively charged residues and lacking the terminal moiety (D) by replacing 1 to 3 of said resi 15 dues by neutral residues, or by a covalent bond, or converting I to 3 of said residues into neutral residues, and by introducing a terminal moiety (D) com prising in total 1 to 5 carbon atoms, in order to obtain a polymyxin derivative of formula (I) according to claim 1, carrying 3 positively charged residues and an R(FA) having in total I to 5 carbon atoms. In one embodiment of the invention, 20 the terminal moiety D is R 12 -C(=O), R- 12 C(=S), or R 17 , wherein R1 2 and R 12 ' are defined hereinafter. In another embodiment, the terminal moiety (D) is R(FA), which is an optionally substituted alkanoyl or alkyl residue having a total of 1 to 5 carbon atoms. DEFINITIONS 25 "Physiological pH" as used herein refers to a pH value of more than 7,0 and below 7,6, such as a pH value in the range of from 7.1 to 7.5, for ex ample in the range of from 7.2 to 7.4. "Positive charge" as used herein denote positive charges at the above-defined physiological pH. 30 "Cationic" molecule as used herein refers to a molecule that con tains one or more positive charges. "Amino acid residue" as used herein refers to any natural, non natural or modified amino acid residue, either in L- or D-configuration. "Equivalent residues" as used herein, is intended to include obvious 35 modifications to e.g., amino acids, resulting in non-natural amino acids or de- WO 2009/098357 PCT/F12009/050093 16 rivatives thereof, but retaining the structural and/or functional capacity of the replaced residue. "Natural polymyxin(s)" as used herein, refers to polymyxins and cir culins. 5 "Polymyxin derivative" refers, for the purpose of this invention, to synthetic or semisynthetic derivatives of natural polymyxins or octapeptins, which have a cyclic heptapeptide (or heptapeptide ring) portion R4-R10 and a side chain linked to the N-terminal aminoacyl residue R4. The side chain may consist of an R(FA)-triaminoacyl(R1-R3), an R(FA)-diaminoacyl(R2-R3), an 10 R(FA)-monoamino-acyl(R3), or of R(FA) alone. "R(FA)" or "fatty acid tail" as used herein refers to the fatty acid part, i.e. the alkanoyl part of the polymyxin structure, linked to the N-terminal amino acid residue of the linear peptide part (side chain) of the polymyxin or, in the absence of the linear peptide part, to the amino add residue R4 (the amino 15 acid in 4-position of the cyclic peptide part of the polymyxin). Furthermore, fbr the purpose of the present invention, R(FA) may also be a related hydrophobic group, such as alkyl. In certain embodiments of the invention, the fatty acid tail may, in certain instances, be a terminal moiety selected from the group con sisting of R 12 -(C=O); R 12 -Sor; R- 1 2 (C=NH)-; R' 2 -NH-(C=S)-; R1 2 -NH-(C=O)-; 20 R 12 -NH-(C=NH)-; R -O-(C=S)-; R' 2 -0-(C=O); R 12 -P(O)OH-; R 2 -(C=S); and

R

12 , wherein R 1 and R 1 z are alkyl, alkenyl, alkynyl, aryl, or aryl alkyl. "Compounds" as used herein include all stereochemical isomers of said compound. "Sensitizing activity' or "ability to sensitize" as used herein is in 25 tended to include any ability to increase the sensitivity, make sensitive or make susceptible a bacterium to an antibacterial agent. "Polymyxin ring moiety" or "A" includes the ring portion of polymyxin A, polymyxin B, IL-polymyxin-B 1 , polymyxin D, polymyxin E, polymyxin F, po lymyxin M, polymyxin S, polymyxin T, circulin A, octapeptin A, octapeptin B, 30 octapeptin C, octapeptin D, and derivatives thereof. Examples of derivatives include moieties with modifications which do not substantially effect the ability of the ring moiety to perform its intended function, i.e., as an antibiotic and/or its ability to sensitize bacterium to one or more antibacterial agents. The term "polymyxin B ring moiety" refers to the ring portion of polymyxin B (i.e., cy[Dab 35 Dab-DPhe-Leu-Dab-Dab-Thr-]). Other examples of polymyxin ring moieties include moieties of the formula: WO 2009/098357 PCT/F12009/050093 17 R7-R8 R9 R5. , R10 R4 wherein: 5 R4 is an amino acid residue comprising a functional side chain able to cyclicize the molecule; R5, R8, and R9 are independently selected amino acid residues; R6 and R7 are optionally substituted hydrophobic amino acid resi dues; and 10 RIO is Leu or any non-hydrophobic amino acid residue. Other ex amples of R4-R1 0 are discussed in further detail in Formula (V). The term "octapeptin ring" refers to the ring portion of native oc tapeptin A (i.e., cy[Dab-Dab-DLeu-LLeu-Dab-Dab-Thr-], i.e., compounds wherein R4, R5, R8, and R9 are Dab, R6 is DLeu, R7 Is LLeu, and RI0 is Thr), 15 octapeptin B (i.e., cy[Dab-Dab-DLeu-LPhe-Dab-Dab-Thr-], i.e., compounds wherein R4, R5, R8 and R9 are Dab, R6 is DLeu, R7 is LPhe and R1 0 is Thr), and octapeptin C (i.e., cy[Dab-Dab-DPhe-LLeu-Dab-Dab-Thr-], i.e., com pounds wherein R4, R5, R8 and R9 are Dab, R6 is DPhe, R7 is LLeu, and RIO is Thr). 20 The term "prodrug" includes moieties which are cleaved in vivo to yield an active polymyxin derivative compound of the invention. The prodrugs include moieties which mask or otherwise neutralize the positive charges (e.g., the -NH;* or other protonated species) at physiological pH. Once the prodrug is administered to the subject, the prodrug moieties or charge masking moie 25 ties will be cleaved or other wise removed to yield the active polymyxin deriva tive of the invention, optionally with three positive charges at physiological pH. The term "'charge masking moiety' includes moieties that reversibly neutralize positive charges on the derivatives. Preferably, the moieties are cleaved or otherwise disassociated with the positive charges of the polymyxin 30 compound after being administered to a subject. Examples of charge masking moieties include sulfoalkyl (e.g., sulfomethylated derivatives). Other positive charge masking moieties include, but are not limited to, chloride, bromide, 10- WO 2009/098357 PCT/FI2009/050093 18 dide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, ace tate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, 5 benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'-methylene-bis (2-hydroxy-3-naphthoate)). The term "subject" includes organisms capable of suffering from bacterial infections. Examples of subjects include mammals, e.g., horses, cows, pigs, sheep, goats, cats, dogs, rabbits, ferrets, monkeys, and, prefera 10 bly, humans. ABBREVIATIONS Fatty acids: FA, fatty acyl residue; 6-MOA and MOA, 6-methyloctan oyl residue; 6-MHA and MHA, 6-methylheptanoy residue; MO(H)A, the mixture of 6-methyloctanoyl, 6-methylheptanoyl and related fatty acyl residues occur 15 ring in polymyxin B; OHMDA, 3-OH-8-methyldecanoic acid; OA, octanoyl; DA, decanoyl; Ac, acetyl; Me, methyl. Amino acids: Dab, a,y-diamino-n-butyryl residue; fDab, N-y-formyl diamino-n-butyryl residue; acDab, N-y-acetyldiamino-n-butyryl residue; Abu, a aminobutyryl residue; Asn, aspartyl residue; Thr, threonyl residue; Ser, serinyl 20 residue; Phe, phenylalanyl residue; Leu, leucyl residue; lie, isoleucyl residue; Ala, alanyl residue; sm-Dab, y-sulphomethylated a,y-diamino-n-butyryl resi due. One-letter codes for modified amino acyl residues: X, Dab; Z, Abu; B, N y-fDab; J, N-y-acDab. Peptides: DAPB, deacylpolymyxin B; DAC, deacylcolistin; PMBN, 25 polymyxin B nonapeptide; PMEN, polymyxin E nonapeptide; PMBO, polymyxin B octapeptide; PMHP, polymyxin B heptapeptide. Other: cy, cyclo (to denote the cyclic part of the peptide, enclosed within brackets); f, formyl; ac, acetyl; sm, sulfomethyl; MS, methanesulfonate; LPS, lipopolysaccharide; OM, outer membrane; MIC, minimum inhibitory con 30 centratlon; CFU, colony forming unit. The symbol * is used herein to mark the residues between which the heptapeptide ring portion of the compound is closed leaving the remaining part of the molecule as a side chain. DETAILED DESCRIPTION OF THE INVENTION It has now been found that certain polymyxin-like compounds con 35 taining only three (3) positive charges and having only a short fatty acyl tail WO 2009/098357 PCT/F12009/050093 19 R(FA) or terminal moiety (D) (not more than 5 carbon atoms in total) still pos sess the ability to sensitize Gram-negative bacteria to antibacterial agents such as antibiotics, semisynthetic antibiotics and chemotherapeutic agents as well as to host defence factors such as the complement system of fresh human 5 serum. Because these novel compounds do not have more than three (3) positive charges, they, in analogy with the polymyxin derivatives described in U.S. Patent Application Serial No. 11/891,629, may be less toxic in general and less nephrotoxic In particular than polymyxins and their known derivatives. 10 Similarly, the compounds now Invented may reduce less histamine from the host tissues than and have pharmacokinetic properties advantageous over polymyxin B, colistin, and their previously described derivatives. Furthermore, the short R(FA) or terminal moiety (D) may make the novel compounds less toxic in acute toxicity assays, in analogy with polymyxin B nonapeptide and 15 colistin nonapeptide that lack the entire fatty acyl part. Furthermore, the novel compounds may have pharmacokinetic properties that are advantageous over polymyxin derivatives that have a long R(FA) or a terminal moiety (D) with mo re than five carbon atoms. In one embodiment the invention pertains to polymyxin derivatives 20 of the formula (1): 4 R3' X02R2'W R" A Q3 W\Q1 W1 D -3 M2 mi (1) wherein: A is a polymyxin ring moiety; 25 D Is a terminal moiety comprising 1 to 5 carbon atoms; m, m 2 , and m 3 are each independently 0 or 1; Q1, Q 2 , and Q 3 are each independently CH 2 , C=0, or C=S;

W

1 , W 2 , and W 3 are each independently NR 4 , 0, or S;

R

1 , R 2 , and R 3 ' are each independently side chains of natural or 30 unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R

4 is hydrogen or alkyl, WO 2009/098357 PCT/FI2009/050093 20 and pharmaceutically acceptable prodrugs and salts thereof, pro vided that (1) when A is an octapeptin ring, m 1 and m 2 are 0, m 3 is 1, W 3 Is NH,

Q

3 is C=O, and R 3 ' is the side chain of diaminobutyric acid (Dab), then D is not

C

2 -C, acyl, and (2) when D is acetyl, butanoyl or pentanoyl, then R3 is not the 5 side chain of Dab. In certain embodiments, the compounds of the invention (e.g., de rivatives of any one of formulae (l)-(V)) may have at least two but no more than three positive charges at physiological pH. In another embodiment, the com pounds have three positive charges at physiological pH. 10 Examples of prodrugs of these derivatives include those with charge masking moieties which neutralize the three positive charges when adminis tered to the subject which are removed in vivo to yield the compound with three positive charges. Examples of charge masking moieties include sulfoal kyl moieties such as sulfomethyl. 15 Preferably, the derivatives have three positive charges at physio logical pH, as defined above. In certain embodiments of the invention, R", R2', and R 3 do not comprise positively charged functional groups at physiological pH. R 1 , R 2 , and R" may comprise, for example, one or two or more hydroxyl, carboxylate, carbamyl, thiol, sulfate, sulfonyl, or phosphate groups. 20 In one embodiment, ml is 0 and m 2 and m 3 are each 1. In another,

Q

2 and Q 3 are each C=O and W 2 and W 3 are each NH. In certain embodiments, R 2 is substituted with one or more groups selected from hydroxyl, carbamyl, carboxylate, thiol, sulfate, sulfonyl, or phos phate groups. Preferably, R 2 is substituted with a carbamyl, hydroxyl or car 25 boxylate group. Examples of R 2 include substituted alkyl and the side chains of alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either the D- or L- configura tion. Preferably, R 2 ' is D-alanine, L-serine, or L-threonine. In certain embodiments, R 3 ' is substituted with one or more groups 30 selected from carbamyl, hydroxyl, carboxylate, thiol, sulfate, sulfonyl, or phos phate. Preferably, R 3 ' is substituted alkyl and maybe substituted with a carba myl, hydroxyl or carboxylate group. R'may be the side chain of alanine, ami nobutyric add, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either the D- or L- configuration. Preferably, 35 R 3 is D-asparagine, L- or D-serine.

WO 2009/098357 PCT/F12009/050093 21 Examples of A include the ring moiety of polymyxin B (i.e., cy[Dab Dab-DPhe-Leu-Dab-Dab-Thr-] and polymyxin E (i.e., cy[Dab-Dab-DLeu-Leu Dab-Dab-Thr-]). In a further embodiment, the terminal moiety is selected from the 5 group consisting of R 12 -(C=0); R 12 -S0 2 -; R 12 -(C=NH)-; R1 2 -NH-(C=S)-;

R'

2 .- NH-(C=0)-; R- 12 NH-(C=NH)-;R1 2 -0-(C=S)-; R- 12 -0(C=Q); R' 2 -P(O)OH-; R-_(C=S); or R 12 ', wherein R 12 and R 1 2 ' are each alkyl, cycloalkyl, alkenyl, al kynyl, aryl, or aryl alkyl. In certain embodiments, D is R 12 -(C=Q) or R 12 -(C=S) and R 12 is methyl, ethyl, propyl, or butyl. Specific examples of D include acetyl, 10 propionyl, butanoyl, and pentanoyl. In another embodiment, the invention also pertains to polymyxin de rivatives of formula (I1): 0 Ra' R' O Rl' H H H A C N C N C N D 15 (II) wherein: A is a polymyxin ring moiety; D is R 2 C(=0), R'-C(=S) or R m , m 2 , and ma are each independently 0 or 1, provided that at least 20 one of mi, m 2 , and m 3 are 1;

R

1 , R 2 , and R' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R

1 2 is C1-C4 alkyl, C2C4 alkenyl, or C2-C4 alkynyl, 25 R 12 is C1-C0 alkyl, C2-C alkenyl, or 02-C alkynyl, and pharmaceutically acceptable prodrugs and salts thereof, pro vided that (1) when A is an octapeptin ring, ml and m 2 are 0, m 3 is 1, and R 3 ' is the side chain of diaminobutyric acid (Dab), and D Is R- 12 C=0, then R 12 is not C1-Ce alkyl, and (2) when D is acetyl, butanoyl or pentanoyl, then R 3 ' is not the 30 side chain of Dab. Preferably, the derivative of formula (II) has three positive charges at physiological pH. In a further embodiment, ml may be 0 and/or m 2 and m 3 WO 2009/098357 PCT/F12009/050093 22 may each be 1. In a further embodiment, R 2 ' and/or R' may each independ ently be substituted alkyl (e.g., substituted with a carbamyl, hydroxyl or car boxylate group). Furthermore, R 2 ' and/or R 3 ' may each be the side chain of serine or threonine (including both D and L configurations). Examples of R 2 5 include the side chains of D-alanine, L-serine and L-threonine. Examples of R 8 include the side chains of D-asparagine, L- and D- serine. In a further embodiment, R 12 is alkyl and D may be acetyl, propionyl, butanoyl, or pentanoyl. In another further embodiment, the invention also pertains to po 10 lymyxin derivatives of formula (Ill): O R 0 R 2 ' 0 RV A--C_ N-C N C I D wherein: 15 A is a polymyxin B or polymyxin E ring moiety; D is R- 12 C(=0), R 1 2 -C(=S) or R1; ml is 0 or 1;

R

1 , R 2 ', and R are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl, 20 aryloxycarbonyl, alkylamino, or alkynyl, wherein at least one of R 2 and R 3 comprise a carbamyl, hydroxyl or carboxylate group; and

R

12 is C 1

-C

4 alkyl,

R

12 ' is C1-Ce alkyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that 25 when D is acetyl, butanoyl or pentanoyl, then R 3 ' is not the side chain of Dab. Preferably, the compounds of the invention have three positive charges at physiological pH, ml is 0, R 2 and R 3 ' are both substituted alkyl, and/or D is acetyl, propionyl, butanoyl, or pentanoyl. In yet another embodiment, the invention also features a polymyxin 30 derivative of formula (IV): WO 2009/098357 PCT/F12009/050093 23 0 L 3 / L2 O L' O A-C _--_ N (IV) wherein: A is a polymyxin B or polymyxin E ring moiety; 5 ml is 0 or 1; L', L 2 and L2 are each independently CrC3 alkyl or a covalent bond; M', M 2 and M 3 are each independently H, C(=O)NH 2 , C(=O)OH, or -OH;

R

12 is CrC4 alkyl, 10 and pharmaceutically acceptable prodrugs and salts thereof, pro vided that when R 12 is methyl, propyl or butyl, then L 3 - M 3 is not the side chain of Dab. Preferably, ml is 0. Examples of L 2 include branched alkyl (e.g.,

-CH(CH

3 )-). Examples of M 2 include OH. Other examples of L 2 include -CH 2 15 and other examples of M 2 include OH and H. In another embodiment, L 3 is

-CH

2 - and M 3 is OH. In yet another embodiment, L 3 is -CHrCHr- and M 3 is

C(=O)NH

2 . Preferably, the compounds of formula (IV) have three positive charges at physiological pH.

WO 2009/098357 PCT/F12009/050093 24 The present invention thus relates to a polymyxin derivative which may be represented by the general formula (V): R7)R7 - (8)R8 (6)R16 (9R9 (s>R5 ajo)R10 (4)R4 (3A13 ()R2 (I)R1 R(FA) ) 5 wherein R4 is an amino acid residue comprising a functional side chain able to cyclicize the molecule; R6 and R7 are an optionally substituted hydrophobic amino acid 10 residues; RIO is Leu or any non-hydrophobic amino acid residue; and wherein RI may be absent; and wherein R1, R2, R3, R5, R8 and R9 are each independently se lected amino acids; and 15 wherein R(FA) is an optionally substituted alkanoyl or alkyl residue, having in total 1 to 5 carbon atoms; or a pharmaceutically acceptable prodrug or salt thereof provided that (1) when R1 and R2 are absent, R3, R4. R5, R8, and R9 are Dab, R6 is D-Leu, R7 is L-Leu or L-Phe, and R10 is Thr or when R1 and R2 are absent, 20 R3, R4. R5, R8, and R9 are Dab, R6 is D-Phe, R7 is L-Leu, and R10 is Thr, then R(FA) is not an unsubstituted alkanoyl residue and (2) when R(FA) is ace tyl, butanoyl or pentanoyl, then R 3 is not Dab.

WO 2009/098357 PCT/FI2009/050093 25 In a derivative according to the present invention, R(FA) may be any residue that has small molecular weight and 1 to 5 carbon atoms. The major role of a short R(FA) is to block the free N-terminal amino group of the peptide and thus eliminate one positive charge of the peptide. 5 Preferably, the compounds of formula (V) may have three positive charges at physiological pH. Furthermore, R1, R2, R3, R5, R8 and R9 may be specifically selected such that the compounds have three positive charges at physiological pH. The R(FA) is preferably selected from the group consisting of car 10 boxylic acid residues, i.e. alkanoyl groups, or alkyl groups, having in total 1 to 5 carbon atoms. R(FA) is preferably selected from the group consisting of methyl, formy and acetyl residues. Other useful residues may be selected from propanoyl, butanoyl, isobutanoyl, valeroyl, and isovaleroyl residues. The residues may be branched, straight-chained or cyclic. 15 R(FA) may also be an unsaturated residue, containing one or more double or triple bonds. R(FA) may be substituted with substituents readily recognizable by one skilled in the art, provided that R(FA) has no more than 1 to 5 carbon at oms. The substituents may include alkyl, hydroxy and alkoxy. Alkyl is prefera 20 bly methyl, ethyl, or propyl. Alkoxy is preferably methoxy, ethoxy, or propoxy. A person skilled In the art may readily recognize equivalents of these preferred R(FA) residues and substituents thereof. In natural polymyxins and octapeptins, R1 is Dab or absent (i.e. re placed by a covalent bond). Examples of known derivatives that have antibac 25 terial activity include those wherein R1 is Ala or a covalent bond. In a derivative according to the present invention R1, if present, may be any amino acid residue, provided that the total number of positive charges in said derivative does not exceed three and that the total number of positive charges in the side chain portion does not exceed one, and is preferably ab 30 sent. In natural polymyxins and octapeptins, R2 is Thr or absent (i.e. re placed by a covalent bond). Examples of known derivatives that have antibac terial activity Include those wherein R2 is O-acetyl-Thr, O-propionyl-Thr, 0 butyryl-Thr or a covalent bond. 35 In a derivative according to the present invention, R2 may be any amino acid residue, preferably hydrophilic or relative hydrophilic, provided that WO 2009/098357 PCT/F12009/050093 26 the total number of positive charges in said derivative does not exceed three and that the total number of positive charges in the side chain portion does not exceed one. Examples of R2 include alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threo 5 nine in either D- or L- configuration, A person skilled in the art may also recog nize an equivalent residue of Thr to be Ser. In natural polymyxins and octapeptins, R3 is Dab, DDab or DSer. In a derivative according to the present invention, R3 may be any amino acid residue, preferably hydrophilic or relatively hydrophilic, provided 10 that the total number of positive charges in said derivative does not exceed three and that the total number of positive charges in the chain portion does not exceed one, and is selected from the group consisting of alanine, amino butyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either D- or L- configuration. 15 A person skilled in the art may readily recognize other hydrophilic or relatively hydrophilic residues than these preferred residues R1, R2 and R3, and may select such from a group consisting of e.g. arginine, N,-methyl argin ine, a-methylaspartate, cysteine, histidine, hydroxylysine, lysine, methionine, ornithine, penicilamine, proline, phosphoserine, phosphothreonine, and tyro 20 sine. A person skilled in the art may readily note that one of the residues R1, R2 and R3 may not be hydrophilic or relatively hydrophilic, provided that the other two residues are so. Accordingly, R1, R2 and R3 may be selected from a group consisting of e.g. a covalent bond, alanine, 2-aminoadipic acid, 25 a-n-butyric acid, N-(4-aminobutyl)glycine, a-aminobutyric acid, y-aminobutyric acid, a-amino-caproic acid, aminocyclopropanecarboxylate, aminoisobutyric acid, aminonorbornylcarboxylate, c-amino-n-valeric acid, arginine, N.-methyl arginine, asparagine, a-methylaspartate, aspartic acid, N-benzylglycine, N-(2 carbamylethyl)glydne, N-(carbamylethyl)glycine, 1-carboxy-1(2,2-diphenyl ethyl 30 amino)cyclopropane, cysteine, Ne-methyldiamino-n-butyric acid, Ny-acetyldiamino n-butyric acid, Ny-formyldiamino-n-butyric acid, Ny-methyldiamino-n-butyric acid, N-(N-2,2-diphenylethyl)carbamylmethyl-glycine, N4N-3,3-diphenyl prop yl) carbamyimethyl(1)glycine, N-(3,3-diphenylpropyl) glycine, glutamic acid, glutamine, glycine, t-butylglycine, 2-amino-4-guanidinobutyric acid, N-(3-guani 35 dinopropyl)glycine, histidine, homophenylalanine, isodesmosine, isoleucine, leucine, norleucine, hydroxylysine, Nemethyllysine, lysine, Ne-methylhydr- WO 2009/098357 PCT/F12009/050093 27 oxylysine, Nemethyllysine, Nsacetylhydroxylysine, Neacetyl lysine, N 8 formyl hydoxylysine, Ne 0 formyllysine, Nemethylhydroxylysine, Nrmethyllysine, me thionine, a-methyl-y-aminobutyrate, a-methyl-amin oisobutyrate, a-methylcyclo hexylalanine, a-napthylaianine, norleucine, norvaline, a-methylornithine, N' 5 methylornithine, Na-acetylornithine, NS-formyl-ornithine, N 8 -methylomithine, ornithine, penicilamine, phenylalanine, hydroxyproline, proline, Nm-methyldi amino-n-propionic acid, Np-acetyldiamino-n-propionic acid, Np-formyldiamino n-propionic acid, N-methyldiamino-n-propionic acid, phosphoserine, serine, phosphothreonine, threonine, tryptophan, tyrosine, norvaline, and valine. 10 In natural polymyxins and octapeptins, R4 is Dab. Examples of syn thetic derivatives that have antibacterial activity include those wherein R4 is Lys. In a derivative according to the present invention R4 is an amino acid residue comprising a functional side chain able to cyclicize the molecule, 15 and may be selected from the group of equivalent residues consisting of Lys, hydroxylysine, ornithine, Glu, Asp, Dab, diaminopropionic acid, Thr, Ser and Cys, preferably Dab. In natural polymyxins and octapeptins, R5, R8 and R9 are Dab. Ex amples of synthetic derivatives that have antibacterial activity include those 20 wherein R5, R8, and R9 may be Lys or 2-amino-4-guanidino butyric acid. In a derivative according to the present invention R5, R8 and R9 may be a positively charged or a neutral amino acid residue, preferably Dab, provided that the total number of positive charges in said derivative does not exceed three. 25 A person skilled in the art, may readily recognize equivalent resi dues of these preferred residues, and may select such from a group consisting of e.g. diaminobutyric acid, diaminopropionic acid, lysine, hydroxylysine, or nithine, 2-amino-4-guanidinobutyric acid, glycine, alanine, valine, leucine, iso leucine, phenylalanine, D-phenylalanine, methionine, threonine, serine, a 30 amino-n-butyric acid, a-amino-n-valeric acid, a-amino-caproic acid, Neformyl lysine, Ne-acetyllysine, Nermethyllysine, Nrfbrmylhydroxylysine, Ne-acetyl hy droxylysine, Nrmethyhydroxylysine, L-Nemethylhydroxylysine, Nrformyl dia mino-n-butyric acid, Nracetyldiamino-n-butyric acid, N-methyldiamino-n butyric acid, Np-formyldiamino-n-proplonic acid, D-Np-formyldiamino-n-prop 35 ionic acid, Np-acetyldiamino-n-propionic acid, Np-methyldiamino-n-propionic acid, Na-formylornithine, Na-acetylornithine and N-methylornithine.

WO 2009/098357 PCT/F12009/050093 28 In natural polymyxins and octapeptins, R6 is DPhe or DLeu and R7 is Leu, Ile, Phe or Thr. Synthetic derivatives that have antibacterial activity in clude those wherein R6 is DTrp and wherein R7 is Ala. In a derivative according to the present invention, R6 is an optionally 5 substituted hydrophobic amino acid residue, preferably DPhe or DLeu, and R7 is an optionally substituted hydrophobic residue, preferably Leu, Thr or lie. A person skilled in the art may readily recognize equivalent residues of these preferred hydrophobic residues, and may select such from a group consisting of e.g. phenylalanine, a-amino-n-butyric acid, tryptophane, leucine, 10 methionine, valine, norvaline, norleucine, isoleucine and tyrosine. A person skilled in the art may also recognize the equivalent residue of threonine to be serine. In natural polymyxins and octapeptins, R10 is Thr and Leu. Exam ples of known derivatives that have antibacterial activity include those wherein 15 R10 is O-acetyl-Thr, O-propionyl-Thr or O-butyryl-Thr. In a derivative according to the present invention, R10 is Leu or any non-hydrophobic amino acid residue, provided that that the total number of positive charges in said derivative does not exceed three. Preferably R10 is Thr or Leu. 20 A person skilled in the art may also recognize the equivalent residue of threonine to be serine. The three (3) positive charges present in the derivatives according to the invention can be located in the heptapeptide ring portion; or two (2) posi tive charges can be located in heptapeptide ring portion while the remaining 25 one positive charge is located in the side chain. In one embodiment, derivatives according to the present invention can be selected from the group of derivatives wherein R2-R1 0 is selected from the group consisting of Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. SEQ ID NO: 10; and Thr-DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. 30 SEQ ID NO: 39. In other embodiments, derivatives according to the present inven tion can be selected from the group consisting of: acetyl-Thr-DSer-cy[Dab Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. Ac-SEQ ID NO: 10; and acetyl-Thr-DAsn cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. Ac-SEQ ID NO: 39.

WO 2009/098357 PCT/F12009/050093 29 As shown in the example section herein, the compounds according to the present invention carrying only three (3) positive charges and having an R(FA) containing 1 to 5 carbon atoms only can be very potent agents to sensi tize Gram-negative bacteria to antibacterial agents. 5 For sensitizing activity at least two (2) and more preferably three (3) positive charges are located in the heptapeptide ring part. The works of Teuber (1970), Srinivasa and Ramachandran (1980a), and Sakura et al. (2004) disclose, among other polymyxin derivatives, deriva tives having only two (2) or three (3) positive charges. However, the com 10 pounds carry a fatty acid part R(FA) longer than 5 carbon atoms. On the other hand, polymyxin B nonapeptide and colistin nonapeptide, both previously known effective agents to sensitize Gram-negative bacteria to antibiotics, lack the entire R(FA) part but carry five (5) positive charges. In certain embodiments of the invention, the polymyxin derivatives 15 of formulae I-V may be administered to a subject in prodrug form. The prodrug may comprise one or more charge masking moieties which mask the positive charges of the compound until after it is administered to the subject. The present invention in one aspect provides new polymyxin deriva tives carrying three (3) positive charges only and an R(FA) containing 1 to 5 20 carbon atoms only and being capable of sensitizing one or more Gram negative bacterial species to an antibiotic or antibacterial agent. The susceptibility of bacteria to an antibacterial agent may be de termined by two microbiological methods. A rapid but crude procedure uses commercially available filter paper disks that have been impregnated with a 25 specific quantity of the antibacterial agent. These disks are placed on the sur face of agar plates that have been inoculated with a suspension of the organ ism being tested, and the plates are observed for zones of growth inhibition. A more accurate technique, the broth dilution susceptibility test, involves prepar ing test tubes containing serial dilutions of the drug in liquid culture media, then 30 inoculating the organism being tested into the tubes. The lowest concentration of drug that inhibits growth of the bacteria after a suitable period of incubation is reported as the minimum inhibitory concentration (MIC).

WO 2009/098357 PCT/FI2009/050093 30 Derivatives according to the present invention may sensitize clini cally important Gram-negative bacteria to antibacterial agents, where said Gram-negative bacteria may be those belonging to the genus of Acinetobacter, Aeromonas, Alcaligenes, Bordetella, Branhamella, Campylobacter, Citrobac 5 ter, Enterobacter, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Kiebsiella, Legionella, Moraxella, Pasteurella, Plesiomonas, Pseudomonas, Salmonella, Serratia, Shigella, and Yersinia species. The bac teria may be, for example, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, other species of En 10 terobacter, Citrobacter freundi, Acinetobacter baumannl, Pseudomonas aeruginosa and other Pseudomonas species as well as many other species of non-fermentative Gram-negative bacteria. The bacteria also include Helico bacterpylori, as well as other clinically important Gram-negative bacteria. The bacterial infections to be treated include, for example, bactere 15 mia, septicemia, skin and soft tissue infection, pneumonia, meningitis, infec tions in the pelveoperitoneal region, foreign body infection, fever in hemato logical patient, infection associated with an intravenous line or other catheter, canyl and/or device, infection in gastrointestinal tract in the eye, or in the ear, superficial skin infection, and colonization of gastrointestinal tract, mucous 20 membranes and/or skin by potentially noxious bacteria. The bacterial infectious diseases include (but are not limited to) se vere hospital-acquired infections, infections of the immunocompromised pa tients, infections of the organ transplant patients, infections at the intensive care units (ICU), severe infections of burn wounds, severe community 25 acquired infections, infections of cystic fibrosis patients, as well as infections caused by multi-resistant Gram-negative bacteria. The present invention is also directed to combinations of two or more derivatives according to the present invention for combination treatment. The combinations may include derivatives having a capability to sensitize dif 30 ferent species or strains of Gram-negative bacteria to antibacterial agents. Another aspect of the present invention is directed to pharmaceuti cal compositions comprising polymyxin derivatives according to the present invention, their prodrug and salt forms, selected combinations thereof, and op tionally an antibacterial agent formulated together with one or more pharma 35 ceutically acceptable carriers and excipients. They facilitate processing of the active compounds into preparations which can be used pharmaceutically and WO 2009/098357 PCT/FI2009/050093 31 include e.g. diluting, filling, buffering, thickening, wetting, dispersing, solubiliz ing, suspending, emulsifying, binding, stabilizing, disintegrating, encapsulating, coating, embedding, lubricating, colouring, and flavouring agents as well as absorbents, absorption enhancers, humefactants, preservatives and the like, 5 well-known to a person skilled in the art. Pharmaceutical compositions include compositions wherein the ac tlive ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount in connection with the present invention means an amount of compound effective to sensitize 10 Gram-negative bacteria to antibacterial agents. Determination of a therapeuti cally effective amount is well within the capability of those skilled in the art of medicine. Compositions may be produced by processes well known in the art, e.g. by means of conventional mixing, dissolving, encapsulating, entrapping, 15 lyophilizing, emulsifying and granulating processes. The proper formulation is dependent upon the route of administration chosen, and the pharmaceutical composition can be formulated for Immediate release or slow release (e.g. in order to prolong the therapeutic effect and/or improve tolerability). Further more, the formulations may conveniently be presented in unit dosage form by 20 methods known in the art of pharmacy. Pharmaceutical compositions according to the present invention in dude (but are not limited to) those intended for intravenous, intramuscular, oral, or topical administration as well as those being administered as a sup positorum or as an inhalable aerosol. The compositions include intravenous, 25 intramuscular, intraperitoneal, subcutaneous, intramedullary, intrathecal, intra ventricular, intranasal, or intraocular injections, inhalable aerosols as well as those intended for rectal, oral, intravaginal, transmucosal or transdermal deliv ery. For parenteral administration (e.g. by bolus injection, fast running in 30 fusions, or slow infusions), the compounds according to this invention as well as the combinations described above may be formulated as their suitable salt or ester forms in sterile aqueous solutions, preferably physiologically compati ble fluids such as saline, 5% dextrose, Ringer's solution, and Hank's solution. The fomiulation may also include organic solvents such as propylene glycol, 35 polyethylene glycol, propylene glycol or related compounds as well as pre servatives and surfactants.

WO 2009/098357 PCT/F12009/050093 32 Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic 5 acid, glycolic acid, pyruvic acid, oxalic acid, malic add, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene sulfonic acid, salicylic acid, and the like, In addition, the pharmaceutical compositions for parental admini 10 stration may be suspensions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispers ing agents. Suitable lipophilic vehicles and solvents include fatty oils such as natural and/or synthetic fatty acids enters, such as ethyl oleate and triglyc erides, or liposomes. The suspensions may contain substances, which in 15 crease the viscosity of the suspension, such as sodium carboxymethyl cellu lose, sorbitol or dextran. The parenteral compositions can be presented in unit-dose or multi dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liq 20 uid excipient, for example, water, for injections, immediately prior to use. For oral administration, solid form preparations include e.g. pow ders, tablets, pills, dragees, lozenges, capsules, cachets, and microgranular preparations. Pharmaceutical preparations can be made using a solid excipi ent, optionally grinding the resulting mixture, and processing the mixture of 25 granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. A solid carrier/excipient can be one or more substances which may also act as diluents, solubilizers, lubricants, suspending agents, binders, preserva tives, flavouring agents, wetting agents, tablet disintegrating agents, or an en capsulating material. Suitable carriers Include, but are not limited to, magne 30 sium carbonate, magnesium stearate, talc, dextrose, lactose, pectin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Liquid preparations suitable for oral administration include e.g. aqueous solutions, syrups, elixirs, aqueous suspensions, emulsions and gels. 35 Aqueous solutions can be prepared by dissolving the active component in wa ter and adding suitable stabilizing and thickening agents as well as colorants WO 2009/098357 PCT/FI2009/050093 33 and flavours. Aqueous suspensions can be prepared by dispersing the finely divided active component In water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Emulsions may be prepared in solutions 5 in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate or acacia. The compounds according to the invention or combinations de scribed above may also be formulated for topical administration. The active compounds are admixed under sterile conditions with pharmaceutically ac 10 ceptable carriers/excipients, including any needed buffering agents and pre servatives. Ointments, creams and lotions may, for example, be formulated with an aqueous or oily base with the addition of suitable emulsifying, dispers ing, suspending, thickening, stabilizing, or coloring agents. Commonly used excipients include animal and vegetable fats and oils, waxes, paraffins, starch, 15 cellulose derivatives, tragacanth, and polyethylene glycol. Other topical formulations include, but are not limited to, ear-drops, eye-drops and transdermal patches. For transdermal as well as transmucosal administration, penetrants generally known in the art may be used in the formulation. 20 For administration by inhalation, the compounds according to this invention and the combinations described above are delivered in the form of an aerosol spray presentation from a ventilator, pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichloro fluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a 25 pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds according to this invention and the combinations 30 described above may also be formulated in rectal compositions such as reten tion enemas or suppositories, using conventional suppository bases such as cocoa butter, other glycerides, polyethylene glycol, or a suppository wax. The present invention also relates to a method for using the present polymyxin derivatives or a combination of such derivatives as a part of the 35 clinical treatment of (or a preventive prophylactic regimen for) human or animal subjects suffering of an infectious disease (i.e., a Gram-negative bacterial in- WO 2009/098357 PCT/FI2009/050093 34 fiction), and comprises administering to said subject an therapeutically effec tive dose of at least one derivative according to the present invention, in com bination with an antibacterial agent. The present invention also relates to a method of sensitizing Gram 5 negative bacteria to an antibacterial agent, wherein the derivative according to the present invention is administered simultaneously, or sequentially in any order, with a therapeutically effective amount of said antibacterial agent, The derivative of the present invention and the antibacterial agent may be administered together as one formulation or by different routes. For 10 example, the polymyxin derivative may be administered intravenously while the antibacterial agent Is administered intramuscularly, intravenously, subcutane ously, orally or intraperitoneally. Alternatively, the derivative may be adminis tered Intramuscularly or intraperitoneally while the antibacterial agent is admin istered intravenously, intramuscularly or intraperitoneally, or the derivative may 15 be administered in an aerosolized or nebulized form while the antibacterial agent is administered, e.g., intravenously. The derivative and the antibacterial agents may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both to achieve effective concentra tions at the site of infection. 20 "Therapeutic effectiveness" is based on a successful clinical out come, and does not require that a derivative according to the present inven tion, in combination with an antibacterial agent, kills 100% of the bacteria in volved in an infection. Successful treatment depends on achieving a level of antibacterial activity at the site of infection, sufficient to inhibit the bacteria in a 25 manner that tips the balance in favor of the host. When host defenses are maximally effective, the antibacterial effect required may be modest. Reducing organism load by even one log (a factor of 10) may permit the host's own de fenses to control the infection. In addition, augmenting an early bacteri cidal/bacteriostatic effect can be more important than long-term bacteri 30 cidal/bacteriostatic effect. These early events are a significant and critical part of therapeutic success, because they allow time for host defense mechanisms to activate. Increasing the bactericidal rate may be particularly Important for infections such as meningitis, bone or joint infections. The therapeutic effectiveness of an antibacterial agent depends on 35 the susceptibility of the bacterial species to said antibacterial agent at the clini cally relevant concentration of the derivative according to this invention. The WO 2009/098357 PCT/FI2009/050093 35 effect of compounds according to the present Invention to improve the thera peutic effectiveness of antibacterial agents in vivo may be demonstrated in vivo animal models, such as mouse peritonitis or rabbit bacteremia assays, and may be predicted on the basis of a variety of in vitro tests, including (1) 5 determinations of the minimum inhibitory concentration (MIC) of an antibacte rial agent required to inhibit growth of a Gram-negative bacterium for 24 hours, (2) determinations of the effect of an antibacterial agent on the kinetic growth curve of a Gram-negative bacterium, and (3) checkerboard assays of the MIC of serial dilutions of antibacterial agent alone or in combination with serial dilu 10 tions of compound(s). Exemplary models or tests are well known in the art. Using in vitro determinations of MIC at 24 hours, a derivative ac cording to the present invention may be shown to reduce the MIC of the anti bacterial agent. With this result, It is expected that concurrent administration of the compound in vivo will increase susceptibility of a Gram-negative bacterium 15 to the antibacterial agent. A compound according to the present invention may also be shown to reduce the MIC of an antibacterial agent from the range in which the organism is considered clinically resistant to a range in which the organism is considered clinically susceptible. With this result, it is expected that concurrent administration in vivo of the one or more compound(s) accord 20 ing to the present invention with the antibacterial agent will reverse resistance and effectively convert the antibiotic-resistant organism into an antibiotic susceptible organism. By measuring the effect of antibacterial agents on the in vitro growth curves of Gram-negative bacteria, in the presence or absence of a compound 25 according to the present invention, the compound may be shown to enhance the early antibacterial effect of antibacterial agents within a period of preferably less than 24 hours. Enhancement of early bactericidal/growth inhibitory effects is important in determining therapeutic outcome. In a checkerboard assay, the combination of a compound according 30 to the present invention with antibacterial agents may result in a "synergistic" fractional inhibitory concentration index (FICI). The checkerboard method is based on additivity, which assumes that the result observed with multiple drugs is the sum of the separate effects of the drugs being tested; according to this system a FICI of less than 0.5 is scored as synergy, 1 is scored as additive, 35 and greater than 1 but less than 2 Is scored as indifferent.

WO 2009/098357 PCT/F12009/050093 36 Antibacterial agents suitable for use in combination with derivatives according to the present invention, include e.g. macrolides, such as clarithro mycin, azithromycin, and erythromycin, ketolides, lincosamines, such as clin damycin, streptogramins, rifamycins, such as rifampin, rifabutin an-d rifalazlle, 5 fusidic acid, mupirocin, oxazolidinones, glycopeptide antibiotics, such as van comycin, dalbavancin, telavancin and oritavancin, fluoroquinolones, tetracy cline derivatives, hydrophobic derivatives of penicillins, cephalosporins, mono bactams, carbapenems, penems and other betalactam antibiotics, novobiocin, pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, and bacterial 10 efflux pump inhibitors. A person skilled in the art of treating Gram-negative in fections may easily recognize additional, clinically relevant antibacterial agents that may be useful. Preferably said antibacterial agents are selected from a group of hydrophobic or moderately hydrophobic antibacterial agents against which the outer membrane of Gram-negative bacteria acts as an effective 15 permeability barrier. The invention also includes the use of the present compounds or combinations thereof to sensitize clinically important bacteria listed herein to the host defence mechanism complement (present in the fresh human and animal serum) by subjecting said bacteria to the action of such compounds 20 during a clinical infection or a suspected infection. The host defence can be exerted, e.g., by the combined action of complement and polymorphonuclear leucocytes. Those skilled in the art of medicine can readily optimize effective dosages and administration regimens for the compounds according to the pre 25 sent invention as well as for the antibiotics in concurrent administration, taking into account factors well known in the art including type of subject being dosed, age, weight, sex and medical conditIon of the subject, the route of administra tion, the renal and hepatic function of the subject, the desired effect the par ticular compound according to the present invention employed and the toler 30 ance of the subject to it. Dosages of all antimicrobial agents should be ad justed in patients with renal impairment or hepatic insufficiency, due to the re duced metabolism and/or excretion of the drugs in patients with these condi tions. Doses in children should also be reduced, generally according to body weight. 35 The total daily dose of a derivative according to the present inven tion administered to a human or an animal can vary, for example, in amounts WO 2009/098357 PCT/F12009/050093 37 from 0.1 to 100 mg per kg body weight, preferably from 0.25 to 25 mg per kg body weight. It will also be recognised by one skilled in the art that the optimal course of treatment, i.e., the number of doses given per day for a defined 5 number of days, will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular pa tient being treated, and that such optimums can be determined by conventional techniques. There is also provided a method for assaying a compound accord 10 ing to the present invention, said compound being a derivative of a natural po lymyxin or octapeptin, wherein said derivative has a only 3 positive charges and a terminal moiety (D) comprising I to 5 carbon atoms, in contrast to the naturally occurring compound from which it is derived, for the ability to sensi tize a harmful Gram-negative to antibacterial agents and/or the complement 15 present in the serum, said method comprising the step of contacting the bacte rium with said derivative of a natural polymyxin or octapeptin, and identifying derivatives possessing sensitizing activity towards said bacterium. In a further aspect there Is provided a method for developing novel antibiotics comprising the steps of 20 (a) providing a natural polymyxin or octapeptin compound, or a de rivative thereof, carrying a total of 4 to 6 positive charges and a terminal moiety (D) comprising 1 to 5 carbon atoms, (b) replacing from 1 to 3 residues carrying one or more positive charges with a residue not having a positive charge, or with a covalent bond, 25 thereby generating a polymyxin derivative carrying 3 positive charges and a terminal moiety (D) comprising 1 to 5 carbon atoms, (c) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to antibacterial agent; and (d) selecting compounds having the ability to sensitize Gram 30 negative bacteria to an antibacterial agent. In one embodiment of the method of the invention, the terminal moi ety (D) is R'-C(=Q), R' 2 -(C=S), or R', wherein R 1 and R 12 are as defined above. In another embodiment of the invention, the terminal moiety (D) is R(FA), which is an optionally substituted aikanoyl or alkyl residue having a total 35 of I to 5 carbon atoms.

WO 2009/098357 PCT/F12009/050093 38 In a still further aspect of the invention there is provided a method for developing novel antibiotics comprising the steps of (a) providing a natural polymyxin or octapeptin compound, or a de rivative thereof, carrying a total of 4 or 5 positive charges, or a total of 6 posi 5 tive charges, as In deacylpolymyxins, and a terminal moiety (D) comprising more than 5 carbon atoms, (b) replacing from I to 3 residues carrying one or more positive charges with a residue not having a positive charge, or with a covalent bond, thereby generating a derivative of a polymyxin compound having 3 positive 10 charges, (c) replacing a terminal moiety (D) comprising more than 5 carbon atoms with a terminal moiety (D) comprising I to 5 carbon atoms, thereby gen erating a derivative of a polymyxin compound carrying 3 positive charges and an a terminal moiety (D) comprising 1 to 5 carbon atoms, 15 (d) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to antibacterial agent; and (e) selecting compounds having the ability to sensitize Gram negative bacteria to an antibacterial agent. In one embodiment of the method of the invention, the terminal moi 20 ety (D) is R 12 -C(=O), R 12 -(C=S), or R, 12 wherein R 12 and R17 are as defined above. In another embodiment of the invention, the terminal moiety (D) is R(FA), which is an optionally substituted alkanoyl or alkyl residue having a total of 1 to 5 carbon atoms. In a still further aspect of the invention there is provided a method 25 for developing novel antibiotics comprising the steps of a) providing a polymyxin or octapeptin compound, or a derivative thereof, having a total of 4 to 6 positive charges and lacking the terminal moi ety (D), b) replacing from I to 3 residues carrying one or more positive 30 charges with a residue not having a positive charge, or with a covalent bond, thereby generating a derivative of a polymyxin compound carrying 3 positive charges; c) introducing a terminal moiety (D) comprising I to 5 carbon atoms, thereby generating a polymyxin compound carrying 3 positive charges and a 35 terminal moiety (D) comprising of 1 to 5 carbon atoms; WO 2009/098357 PCT/F12009/050093 39 e) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to antibacterial agent; and f) selecting compounds having the ability to sensitize Gram-negative bacteria to an antibacterial agent. 5 In one embodiment of the method of the invention, the terminal mol ety (D) is R" 2 -C(=O), R' 2 -(C=S), or R, 12 wherein R 1 2 and R" are as defined above. In another embodiment of the invention, the terminal moiety (D) is R(FA), which Is an optionally substituted alkanoy or alkyl residue having a total of 1 to 5 carbon atoms. 10 There is also provided in accordance with the present invention a semisynthetic polymyxin derivative obtainable by treating chemically or enzy matically naturally-occurring polymyxins or octapeptins, respectively, or those variants thereof which are manufactured by genetically modified organisms. Chemical treatments include, but are not limited to, those with acetanhydride, 15 formic acid, hydrazine, and oxalic acid. Enzymatic treatments Include, but are not limited to, with enzymes such as polymyxin deacylase, ficin, papain, bro melain, subtilopeptidases, subtilisin, colistin hydrolase, and Nagarse. Preferred compounds according to one embodiment are less cati onic than natural polymyxins or octapeptins, carry three (3) positive charges 20 only and an R(FA) having 1 to 5 carbon atoms, and are: (a) able to sensitize Gram-negative bacteria such as Escherichia coll, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citro bacter freundii, and Acinetobacter baumannil to antibiotics, and/or (b) less toxic than clinically used polymyxins, as evidenced in in vivo 25 animal model, and/or (c) less nephrotoxic than clinically used polymyxins, as evidenced in an animal model and/or in an in vitro test that measures affinity of the com pounds to kidney structures, and/or (d) able to cause less histamine liberation from the tissues than 30 clinically used polymyxins when administered topically or when inhaled as an aerosol, and/or (e) pharmacokinetically more favorable, such as having a longer se rum half life, increased renal clearance, increased urinary recovery and/or by being less inactivated by polyanionic tissue and pus constituents than clinically 35 used polymyxins.

WO 2009/098357 PCT/F12009/050093 40 In a further embodiment, the compounds of the invention have one or more more pharmacokinetically favorable properties as compared to native polymyxins or octapeptins (e.g., polymyxin A, polymyxin B, IL-polymyxin-Bi, polymyxin D, polymyxin E, polymyxin F, polymyxin M, polymyxin S, polymyxin 5 T, circulin A, octapeptin A, octapeptin B, octapeptin C, or octapeptin D). Exam ples of such pharmacokinetically favorable properties include a longer serum half life, increased renal clearance, or increased urinary recovery as compared to native polymyxins or octapeptins (such as polymyxin E). In a further embodiment, the compounds of the invention may have 10 a greater percent urinary recovery of an administered dose over 24 hours than polymyxin E (colistin). In another further embodiment, the urinary recovery, based on experiments with rats is about 1% or greater, about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 35% or greater, about 40% or greater, 15 about 45% or greater, or about 50% or greater. In contrast, the urinary recov ery of polymyxin E (colistin) was determined to be about 0.18 ± 0.14 % of dose in 24 hours (Li et al., 2003), using the same dose and procedure. In another further embodiment, the compounds of the invention may have a greater renal clearance than polymyxin E (colistin) when administered 20 using the same route and dosing. In a further embodiment, the compounds of the invention have a renal clearance, based on experiments with rats greater than about 0.1 mI/min/kg, greater than about 0.5 mi/min/kg, greater than about 1.0 mI/min/kg, greater than about 2.0 mI/min/kg, greater than about 2.5 mI/min/kg, greater than about 3.0 m/min/kg, or greater than about 25 3.5 ml/min/kg. In another further embodiment, the renal clearance of the com pounds of the invention may be at least 10 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least 300 times that of polymyxin E, when administered at the same dose and ad ministration route. 30 In another further embodiment, the compounds of the invention may also have one or more pharmacokinetically favorable properties as compared to similar compounds with longer fatty acid tails (i.e., a terminal moiety or R(FA) having more than five carbon atoms). As shown in Example 8, NAB741 has increased renal clearance and increased urinary recovery as compared to 35 NAB739. The compounds are chemically identical except that NAB741 has an acetyl terminal moiety and NAB739 has an octanoyl terminal moiety.

WO 2009/098357 PCT/F12009/050093 41 Methods for synthesising compounds according to the present in vention include but are not limited to the following described below. For a spe cific compound to be synthetised, an expert in the art Is able to choose the ap propriate method. 5 1. Semisynthetic derivatives of polymyxins and octapeptins that carry an unchanged heptapeptide part and a modified acyl-aminoacyl side chain can be made by the procedures described as follows: Protection of the free amino groups in the starting material (po lymyxin or octapeptin, or modifications thereof) by methods known to those 10 skilled in the art. The protection can be achieved by the use of residues such as t-butoxycarbonyl (tBoc), fluorenylmethoxycarbonyl (Fmoc), benzyloxycar bony[ (CBZ, Z), allyloxycarbonyl (ALOC), 3-pyridyl-N-oxide-methoxycarbonyl (as described in patent publication GB 1323962), by using Schiff bases such as benzaldehyde by the method described in Japanese Patent publication 15 7115630/1971 or the like which can be removed by conventional conditions compatible with the nature of the product. In conditions where the poor water solubility occasionally poses a problem in the sub-sequent steps, the protection can be made by using nega tively-charged blocking groups such as a sulfonic acid derivative of Fmoc or a 20 carboxylic acid derivative of Fmoc, the method being described in US patent publication 2006004185. The water solubility can also be enhanced by linking a suitable, removable, negatively charged, very hydrophilic blocking group to the OH-group of threonine. Thereafter, the compound is subjected to an enzymatic treatment 25 with enzymes such as polymyxin deacylase, polymyxin hydrolase, papain, fi cin, bromelain, subtilopeptidase, Nagarse or other enzymes that remove a terminal part of the side chain or even the entire side chain of polymyxin or octapeptin compounds. This treatment can optionally be followed by the Ed man degradation procedure. The resultant compound lacks the entire side 30 chain and consists of the cyclic heptapeptide part only, but has a free N terminal alpha amino group. Alternatively, polymyxins and octapeptins that have amino groups protected by acid-stable groups such as benzyloxycarbonyl can be treated by oxalic acid or formic acid to yield protected deacylderivatives, the method be 35 ing described by Kurihara et aL (1974). The procedure is followed by further WO 2009/098357 PCT/F12009/050093 42 enzyme treatment as above and/or by Edman degradation to yield a heptapep tide. Thereafter, a suitable residue is linked to the free alpha-amino posi tion of the heptapeptide ring portion. The residue might contain an. acyl or re 5 lated residue (R(FA) having in total 1 to 5 carbon atoms), such as methyl, ace tyl, proplonyl, butanoyl, isobutanoyl, valeroyl, or isovaleroyl residue) as well as amino acid residues, up to three and preferably two residues. For instance, one semisynthetic compound with an acyl group and two amino acid residues can be prepared by adding to the above-described heptapeptide a synthetic N 10 (acyl)-threonyl-Dthreonyl residue. This can be achieved by conventional gen eral techniques known to those familiar with the art of organic chemistry, these techniques including the use of N-hydroxysuccinimide-linked residues as de scribed in US 2006004185. In this particular synthesis the procedure may in volve the use of N-acetylthreonyi-Dserinyl-N-hydroxysuccinimide. 15 2. Acylated polymyxin nonapeptides carrying three (3) free amino groups. Polymyxin D possesses only four (4) positive charges and has DSer In the position R3. The free amino groups of polymyxin D can be protected by the means described above. This is followed by an enzymatic treatment and an optional Edman degradation step, to yield a nonapeptide, which can then be 20 acylated by acylisotiocyanate (by the method well-known to a person skilled In the art and described in US 2006004185, by acyl chloride (by the method well known to a person skilled in the art and described in Chihara et aL. 1974), or by using residues linked to N-hydroxysuccinimide (by the method well-known to a person skilled in the art and described in US 2006004185). Finally, the protec 25 tive groups are removed. The acylated polymyxin D nonapeptide carries only three (3) free amino groups, all in the heptapeptide ring portion. In an analogous manner, acylated polymyxin S nonapeptide can be made. It carries only three (3) free amino groups. 3. Totally synthetic polymyxin and octapeptin derivatives can be 30 made by the very conventional methods known for those skilled in the art. Such methods include the liquid-phase synthesis procedures as well as the solid-phase synthesis procedures described for instance by Sakura et al. (2004), Tsubery et al. (2000a, 2000b, 2002, 2005), and Ofek et aL (2004). The methods include e.g. the use of protecting agents such as Fmoc, tBoc, and 35 CBZ at strategic positions, as well as the cyclisation step where DPPA (di phenyl phosphorazidate) or a mixture of benzotrizole-1-yl-oxy-tris-pyrrolidino- WO 2009/098357 PCT/F12009/050093 43 phophonium hexafluorophosphate (PyBop), N-hydroxybenzotriazole (HoBt), and N-methylmorpholine (NMM) is used. Fmoc derivatives of many non-trivial as well as D-amino acids are commercially available. The amino terminus of the last amino acid residue is left unprotected to enable direct reaction in the 5 acylation procedure with acids such as propionic acid, butyric acid, Isobutyric acid, valeric acid, and isovaleric acid. 4. Acylation of the free N-terminal alpha-amino group of the inter mediate compounds described above (paragraphs 1-3) can also be performed by using anhydrides such as acetic anhydride (see Example 1), propionic an 10 hydride, butyric anhydride, and valeric anhydride by using conditions well known to a person skilled in the art. N-formylation can be performed by using p-nitrophenyl formate in N-methyl pyrrolidine and conditions well-known to a person skilled in the art. N-methylation can be performed by using a mixture of formic acid and acetic anhydride in dimethylformamide and conditions well 15 known to a person skilled in the art. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the 20 scope of the present invention and are covered by the following claims. The contents of all references, patents, and patent applications cited throughout this application are hereby incorporated by reference. The appropriate compo nents, processes, and methods of those patents, applications and other docu ments may be selected for the present invention and embodiments thereof.

WO 2009/098357 PCT/F12009/050093 44 LIST OF REFERENCES All references cited in the present application are hereby incorpo rated by reference in their entirety. 5 Chihara S, Tobita T, Yahata M, Ito A, Koyama Y. 1973. Enzymatic degradation of colistin. Isolation and identification of a-N-Acyl a,y-diamlno butyric acid and colistin nonapeptide. Agr Biol Chem 37:2455-2463. Chihara S, Ito A, Yahata M, Tobita T, Koyama Y. 1974. Chemical synthesis, isolation and characterization of a-N-fattyacyl colistin nonapeptide 10 with special reference to the correlation between antimicrobial activity and car bon number of fattyacyl moiety. Agric Biol Chem 38:521-529. de Visser PC, Kriek NMAJ, van Hooft PAV, Van Schepdael A, Filip pov DV, van der Marel GA, Overkleeft HS, van Boom JH, Noort D. 2003. Solid phase synthesis of polymyxin B 1 and analogues via a safety-catch approach. 15 J. Peptide Res. 61:298-306. Kimura Y, Matsunaga H, Vaara M. 1992. Polymyxin B octapeptide and polymyxin B heptapeptide are potent outer membrane permeability increasing agents. J Antibiot 45:742-749. Kurihara T, Takeda H, Ito H, Sato H, Shimizu M, Kurosawa A. 1974. 20 Studies on the compounds related to colistin. IX. On the chemical deacylation of colistin and colistin derivatives. Yakugaku Zasshi 94:1491-1494. Li J, Milne RW, Nation RL, Turnidge JB, Smeaton TC, and Coult hard K. 2003. Use of high-performance liquid chromatography to study the pharmacokinetics of colistin sulfate in rats following intravenous administration. 25 Antimicrob Agents Chemother 47:1766-1770. Li J, Milne RW, Nation RL, Turnidge JB, Smeaton TC, and Coult hard K. 2004. Pharmacokinetics of colistin methanesulphonate and colistin in rats following an intravenous dose of colistin methanesulphonate. J Antimicrob Chemother 53:837-840. 30 Nagai J, Saito M, Adachi Y, Yumoto R, Takano M. 2006. Inhibition of gentamicin binding to rat renal brush-border membrane by megalin ligands and basic peptides. J Control Release 112:43-50. Nikaido H. 2003. Molecular basis of bacterial outer membrane per meability revisited. Microbiol Molec Biol Rev 67:593-656. 35 Nikaido H, Vaara M. 1985. Molecular basis of bacterial outer mem brane permeability. Microbiol Rev 49:1-32.

WO 2009/098357 PCT/F12009/050093 45 Okimura K, Ohki K, Sato Y, Ohnishi K, Uchida Y, Sakura N. 2007. Chemical conversion of natural polymyxin B and colistin to their N-terminal de rivatives. Bull. Chem. Soc. Jpn. 80 (No. 3):543-552. Rose F, Heuer KU, Sibelius U, Hombach-Klonisch S, Ladislau K, 5 Seeger W, Grimminger F. 1999. Targeting lipopolysaccharides by the non toxic polymyxin B nonapeptide sensitizes resistant E. coli to the bactericidal effect of human neutrophils. J Infect Dis 182:191-199. Sakura N, Itoh T, Uchida Y, Ohki K, Okimura K, Chiba K, Sato Y, Sawanishi H. 2004. The contribution of the N-terminal structure of polymyxin B 10 peptides to antimicrobial and lipopolysaccharide binding activity. Bull Chem Soc Jpn 77:1915-1924. Srinivasa BD, Ramachandran LK. 1978. Chemical modification of peptide antibiotics: Part VI - Biological activity of derivatives of polymyxin B. Ind J Biochem Blophys 14:54-58. 15 Srinivasa BD, Ramachandran LK. 1979, The polymyxins. J Scient Industry Res 38:695-709. Srinivasa BD, Ramachandran LK. 1980. Essential amino groups of polymyxin B. Ind J Biochem Biophys 17:112-118. Storm DR, Rosenthal KS, Swanson PE. 1977. Polymyxin and re 20 lated peptide antibiotics. Annu Rev Biochem 46:723-63. Teuber M. 1970. Preparation of biologically active mono-N acetyl(14C)-derivatives of the membrane-specific polypeptide antibiotic po lymyxin B. Z Naturforsch 25b: 117. Tsubery H, Ofek I, Cohen S, Fridlkin M. 2000a. Structure-function 25 studies of polymyxin B nonapeptide: Implications to sensitization of Gram negative bacteria. J. Med Chem 43:3085-3092. Tsubery H, Ofek I, Cohen S, Fridkin M. 2000b. The functional asso ciation of polymyxin B with bacterial lipopolysaccharide is stereospecific: Stud ies on polymyxin B nonapeptide. Biochemistry 39:11837-11844. 30 Tsubery H, Ofek I, Cohen S, Frldkin M. 2001. N-terminal modifica tions of polymyxin B nonapeptide and their effect on antibacterial activity. Pep tides 22:1675-1681. Tsubery H, Ofek I, Cohen S, Elsenstein M, Fridkin M. 2002. Modula tion of the hydro-phobic domain of polymyxin B nonapeptide: effect on outer 35 membrane permeabilization and lipopolysaccharide neutralization. Molecular Pharmacology 62:1036-42.

WO 2009/098357 PCT/F12009/050093 46 Tsubery H, Yaakov H, Cohen S, Giterman T, Matityahou A, Fridkin M, Ofek 1. 2005. Neopeptide antibiotics that function as opsonins and mem brane-permeabilizing agents for gram-negative bacteria. Antimicrob Agents Chemother 49:3122-3128. 5 Vaara M. 1992. Agents that increase the permeability of the outer membrane. Microbiol Rev 56:395-411. Vaara M. 1993. Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium. Antimicrob Agents Chemother 37:2255 2260. 10 Vaara M, Vaara T. 1983a. Sensitization of Gram-negative bacteria to antibiotics and complement by a nontoxic oligopeptide. Nature (London) 303:526-528. Vaara M, Vaara T. 1983b. Polycations sensitize enteric bacteria to antibiotics. Antimicrob Agents Chemother 24:107-113. 15 Vaara M, Vaara T. 1983c. Polycations as outer membrane disorganizing agents. Antimicrob Agents Chemother 24:114-122. Vaara M, Vijanen P, Vaara T, Makeli P. 1984. An outer membrane disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action. J Immunol 132:2582-2589. 20 Viljanen P, Matsunaga H, Kimura Y, Vaara M. 1991. The outer membrane permeability-increasing action of deacylpolymyxins. J Antibiotics 44:517-523.

WO 2009/098357 PCT/F12009/050093 47 EXAMPLES The following examples illustrate certain embodiments of the pre sent invention and should not be construed as limiting the scope of the inven tion. 5 Example I Peptide synthesis Polymyxin derivatives ("NAB peptides" or "NAB compounds") were synthesized by conventional solid phase chemistry, using the standard Fmoc protection strategy. The amino acid at the C-terminus is commercially available 10 as pre-attached to the solid phase and when cleaved off the resin with acid, yields a C-terminal carboxylic acid. The strategy in the protection was to use three levels of orthogonal protection, temporary Fmoc protection for the alpha amino functions, groups which are removed during the acid cleavage stage, and semi-permanent pro 15 tection to cover reactive side chain functions while the cyclisation reaction takes place. After cleavage of the peptide from the resin, the C-terminal car boxylic acid is reacted with an amino function on the side chain of one of the amino acids to form a cyclic peptide. After the cyclisation step, the semi permanent protection groups are removed to yield NAB peptide. 20 Accordingly, the alpha amino function of the amino acid was pro tected by fluorenyl-methoxycarbonyl (Fmoc) and Fmoc was removed by 20% piperidine in dimethylformamide (DMF) at every cycle. The amino acid that is involved with cyclisation, i.e. diaminobutyric acid, was protected by t butoxycarbonyl (tBoc), an acid labile group which was removed at the cleav 25 age step. The functional group of asparagine was protected by tritylation. All the other amino acids which have functional side chain groups were protected by a group that is stable to the acid cleavage stage, i.e. benzyloxycarbonyl (Z). Amino acids phenylalanine and leucine naturally needed no side chain protec tion. The amino terminus was not protected; this enabled direct reaction in the 30 acylation procedure. The synthesis steps were performed in a commercial automatized synthesizer that employed O-(6-Chlorobenzotriazol-1 -yl)-N, N, N', Ntetram ethyl uronium hexafluorophosphate (HCTU) as activator.

WO 2009/098357 PCT/F12009/050093 48 The acylation was performed by using a four-fold molar excess of each amino acid or the fatty acid, four-fold molar excess of the activator HCTU (see above), and an eight-fold molar excess of N-methyl morpholine. The reac tion time was 30 min. 5 The amino acids were purchased already protected from standard suppliers. The peptide was removed from the resin by reaction with a solution of 95% trifluoroacetic acid and 5% water for 2 hours at room temperature, to yield the partially protected product. The resulting peptide was precipitated with 10 diethyl ether. The cyclisation mixture used was benzotriazole-1-yl-oxy-tris-pyrroll dino-phosphonium hexafluorophosphate (PyBop), N-hydroxybenzotri-azole (HoBt), and N-methyl morpholine (NMM) at the molar excess of 2, 2, and 4, respectively. The peptide was dissolved in dimethylformamide, the cyclisation 15 mix was added and allowed to react for 2 hours. The cyclised, protected pep tide was precipitated by the addition of cold diethyl ether. Any residual PyBop was removed by washing the peptide with water. Acetylation for performed by using acetic anhydride - diisopropyl ethylamine - DMF (1:1:18 by vol.). 20 The remaining side chain protection groups (Z) were removed by catalytic dehydrogenation. The peptide was dissolved in acetic acid-methanol water (5:4:1), under an atmosphere of hydrogen and in the presence of a pal ladium charcoal catalyst. The peptide was purified by reverse phase chromatography using 25 conventional gradients of acetonitrile:water:trifluoroacetic acid. The product was dried by lyophilisation. The yield was 10-20 mg representing approx. 10%-20% of the theo retical, calculated from the molar amount (approx. 100 micromoles) of the first amino acyl residue bound to the resin. 30 The purity, as estimated by reversed phase HPLC was more than 90%. Within experimental error, the masses obtained were those expected from the theoretical values.

WO 2009/098357 PCT/F12009/050093 49 Example 2 Activity of the compounds against Escherichia coli and Pseudomonas aeruginosa Peptides synthesized in Example 1, both carrying only three (3) 5 positive charges, were studied for their ability to sensitize E. coli to the model antibiotic rifampin. This was tested employing LB agar (LB Agar Lennox, Difco, BD, Sparks, MD, U.S.A) plates that contain increasing concentrations (0.1 pg/ml, 0.3 pg/ml, 1 pg/ml) of rifampin (Sigma-Aldrich, St. Louis, MO, U.S.A) as well as by using LB agar control plates that did not contain rifampin. 10 The indicator organism E. coli IH3080 (K1:018) was an encapsu lated strain originally isolated from a neonate suffering from meningitis (Vaara et al. 1984) and obtained from National Public Health Institute, Helsinki, Finland. From an ovemight-grown culture of IH3080 on LB agar, a suspen 15 sion of approx. 108 cells/mI was prepared in 0.9% NaCl. Aliquots of this sus pension were then pipetted on the agar plates and the plates were gently shaken to spread the suspension evenly on the entire surface of the plate. Thereafter, the unabsorbed part of the suspension was removed by using a Pasteur pipette. After the surface had drIed, small wells (diameter, 2 mm) were 20 drilled on the plates (five wells per plate) by using a sterile sharp-edged narrow metal tube, single-use pipette tip, and vacuum suction. Alternatively, a swab was used to spread the inoculum. Samples (4 pl and 10 pl) of the peptide solu tion in 0.9% NaCI (at concentrations of 1 pg/ml and 0.1 pg/ml) were then pipet ted to the wells and the sample fluids were allowed to absorb. Controls in 25 eluded 0.9% NaCI solution without the compound to be tested. The plates were then incubated for 18 h at 37*C whereafter the diameters of growth inhibition zones around each well were measured; the diameter of the well itself was not reduced. Finally, the diameters were converted to surface areas of growth in hibition (in square mm). 30 Table 2 shows the activity of the novel compounds against E. col IH3080 as compared with that of control compounds. Even though both lacked the direct antibacterial activity of NAB739, they sensitized at a concentration of 4 pg/mI the target to a concentration of rifampln as low as 0.1 pg/mI. Interes tingly, NAB747 was directly antibacterial against P. aeruginosa ATCC 27853. 35 In a well containing 10 lag of the peptide, it caused a zone of inhibition with the surface area of 50 sq mm. At 4 pg, the corresponding value was 20 sq mm.

WO 2009/098357 PCT/F12009/050093 50 Table 2. Structure of the novel compounds and their activity against E .cpli IH3080 Compound Compound SEQ Structure* Positive Direct Activity w. actv group name ID Peptide sequence charges Ity" rifampin*" side NO. FA-part chain cyclic part total (cyclic) Control cy [XXfLXX compounds Polymyxin B MO(f)A XTX TI 5 (3) 79 95 Deacyl- cy [XXfLXX polymyxin B - +XTX TI 6 (3) 57 79 cy [XX1LXX Deacylcolistin - +xTX T3 6(3) 79 87 Polymyxin B cy [xXfLXX nonapeptide - +TX TI 5(3) 0 20 Polymyxin B cy EXX!LXX heptapaptide - + TI 4 (4) 0 0 cy[XXfLXX NAB704 - +T1 T] 4(3) 0 0 cy[fxfLXX NAB705 - +ZTZ T] 4(3) 0 0 cy[XXfLZZ NAB701 - +TX TI 3(1) 0 0 cy[XXELBB NAB 702 - +TX Ti 3(1) 0 0 cy XXfLar NAB703 - +TX TI 3(1) 0 0 Octanoyl cy (XXfLXX PMBH oA - TI 3(3) 0 0 NAB730 DA - [XXfEXXT] 3(3) 0 113 cy[XXfLXX NAB739 OA To TI 3(3) 133 177 cy[XXfLXX NAB 740 DA Ta TI 3(3) 95 95 cy[XxfLXX NAB 7061 OA TZ TI 3(3) 0 113 Novel cy [EXfLXx compounds NAB 741 10 Ac To T] 3(3) 0 95 cy [XXfLXX NAB745 39 Ac Tn TI 3(3) 0 50 cy[XXfLXX NAB747 10 Me To TJ 3(3) 0 28 * One-letter codes for amino acyl residues: F, Phe; L,Leu; N, Asn; S, Ser; T, Thr; 5 X Dab; Z, Abu; B, N-gammaformyl-Dab; J, N-gamma-acetyl-Dab. Small letters indicate resi dues that are in D-configuration. + indicates the positive charge of the alpha-amino group in the free N-terminus of the peptide. Abbreviations: MO(H)A, the mixture of 6-methyloctanoyl, 6-methylheptanoyl and related fatty acid residues occurring In polymyxin B; OA, octanoyl; DA, decanoyl; Ac, acetyl; 10 Me, methyl. " Antibacterial activity measured as the growth Inhibition (In square millimeters) around a well containing 4 micrograms of the compound on LB plates. ** Antibacterial activity measured as the growth inhibition (In square millimeters) around a well containing 4 micrograms of the compound on a LB plate containing rifampin 15 (0.1 micrograms/mi).

WO 2009/098357 PCT/F12009/050093 51 Example 3 NAB741 sensitizes E. coli, Klebsiella pneumoniae, and Enterobacter clo acae to a broad range of antibacterial agents The minimum inhibitory concentrations (MIC) of a representative set 5 of clinically used antimicrobial agents were determined for two strains of E coll (ATCC25922 and 1H3080), K. pneumoniae ATCC13883, and E. cloacae ATCC23355 by using Mueller-Hinton agar medium (product no LabO39; LabM Ltd., Bury, Lancs, U.K.) in the presence of NAB741 (4 pg/ml) as well as in its absence. MICs were determined by using E-strips (Biodisk Ltd., Solna, Swe 10 den) according to the manufacturer's instructions. The NAB741 concentration used did not itself inhibit the growth of the target bacteria. The MIC of NAB741 for all these strains was >16 pg/ml. The results are shown in Table 3. NAB741 at a concentration of 4 pg/mi was able to sensitize the tested strains to rifampin by a factor ranging 15 from >64 to >2000. Sensitization factor is defined as the ratio of the MIC of an antibiotic in the absence of NAB741 to that in the presence of 4 pg/ml of NAB741. Extremely high sensitization factors were observed also to clarithro mycin (24-340), mupirocin (8-192), azithromycin (16-32), for some of the strains to fusidic acid (128-170), and for E. cloacae to vancomycin (170). All 20 these antibacterial agents are notably hydrophobic or large (vancomycin) and are known to be excluded by the intact OM of Gram-negative bacteria but penetrate the damaged OM.

WO 2009/098357 PCT/F12009/050093 52 Table 3. Sensitization factors* to selected antibacterial agents at NAB 741 concentration of 4 pg/mi E. coli E. coli K. pneum. E. cloacae ATCC IH 3080 ATCC ATCC 25922 13883 23355 Rifampin 750 250 >64 >2000 Clarithromycin 340 96 24 96 Muplrocin 128 64 8 190 Azithromycin 24 32 32 16 Fusidic acid 170 130 >5 >130 Vancomycin >16 16 >2 170 *Sensitization factor is the ratio of the MIC of the antibiotic in the absence of NAB 741 5 to that in the presence of 4 pg/mI of NAB 741 Example 4 Susceptibility of seven different strains of Gram-negative bacteria to ri fampin and clarithromycin in the presence of NAB741 (4 pg/mi) The minimum inhibitory concentrations (MIC) of rifampin and 10 clarithromycin for a representative set of different strains of clinically relevant Gram-negative bacteria were determined by the E-test method as in Example 3 and by using Mueller-Hinton agar with or without NAB741 (4 pg/mI). This concentration of NAB741 did not itself inhibit the growth of the target bacteria. Five of the strains originated from ATCC. Acinetobacter baumannil F264 was 15 purchased from Mobidiag Ltd., Helsinki, Finland. The source of E. coil H3080 has been given in Example 2. The results are shown in Table 4. It shows that NAB 741 is re markably active even against Acinetobacter baumannii.

WO 2009/098357 PCT/F12009/050093 53 Table 4. The ability of NAB 741 to sensitize Gram-negative bacteria to model antibiotics (rifampin and clarithromycin) MIC (pg/ml) of Sensltlzation MIC (pg/mi) of SenstIzation Bacterial strain rifampin in the factor* clarlthromycin in factor* presence of the presence of . 4 pg/ml to rifampin 4 pg/mI to clarithromycin of NAB 741* of NAB 741 E. coil ATCC25922 0,016 750 0,125 340 E. coil IH3080 0,047 250 0,125 96 K. pneumoniae ATCC13883 0,5 >64 1 24 E. cloacae ATCC23355 0,016 2000 0,5 96 Ac. baumannil ATCC19606 0,19 16 0,5 32 Ac. baumannll F264 0,125 64 0,5 32 P. aeruginosa ATCC27853 16 2 64 2 *The ratio of iifampin MIC In the absence of NAB 741 to that in the presence of NAB 741 (4 pg/ml). ** The ratio of clarithromycin MIC In the absence of NAB 741 to that In the presence of NAB 741 (4 pg/ml). 5 Example 5 NAB741 sensitizes a meropenem-resistant strain of Acinetobacter baumannil to meropenem The minimum inhibitory concentrations (MIC) of meropenem for two strains of A. baumannil were determined by the E-test method as in Example 4 10 and by using Mueller-Hinton agar with or without NAB741 (4 pg/mI). This con centration of NAB741 did not itself inhibit the growth of the target bacteria. Sensitization factor was defined as in Example 4. The results are shown In Ta ble 5. NAB7061 sensitized the meropenem-resistant strain F264 to merope men by a factor >4.

WO 2009/098357 PCT/F12009/050093 54 Table 5. Sensitization of the meropenem-resistant strain of Acinetobacter baumannll to meropenem in the presence of NAB 741 (4 pg/ml) Strain MIC (pg/ml) of meropenem at the indicated conan (pg/mi) of NAB 741 0 4 A. baumannlATCC19606 0,75 0,5 A. baumannl F264 >32 8 Example 6 NAB741 sensitizes E. coi to the complement In fresh normal serum 5 The ability of NAB741 to sensitize encapsulated, smooth strain of E. col to the bactericidal action of normal guinea pig serum (GPS) was studied by the method described by Vaara et at (1984). E. co/i IH3080 (018,K1) was grown in LB broth (LB broth Lennox, Difco, BD, Sparks, MD, U.S.A) at 37 0 C in a rotary shaker into early logarithmic growth phase, washed with PBS (phos 10 phate-buffered saline, 8.0 g of NaC, 0.2 g of KCI, 1.44 g of Na 2

HPO

4 x2H 2 0 and 0.2 g of KH 2

PO

4 per liter) and resuspended in PBS, to approx. 100 cells/mI). GPS was used as complement source. It was stored at -70"C before use. To inactive the complement, serum was incubated at 56"C for 30 min. The experimental procedure was as follows. 10% GPS in PBS was 15 inoculated with approx. 500 CFU (colony forming units) of bacteria per ml and pipetted in 0.2 ml aliquots into wells of microtiter plates. The wells already con tained increasing amounts of NAB7061 in 0.020 ml of 0.9% NaCl. The plate was incubated at 370C for 2 h whereafter each well was emptied onto LB plates. The plates were incubated overnight at 37*C and the developed colo 20 nies were counted. The results are shown in Table 6. NAB741 itself did not significantly reduce CFU count in the absence of GPS or in the presence of heat-inactivated 10% GPS. However, as low a concentration of NAB741 as 2 pg/ml was sufficient to reduce CFU count by a factor of approx. 100 in the presence 10% fresh 25 GPS. Accordingly, NAB741 acts synergistically with the bactericidal comple ment machinery present in fresh serum, as does PMBN, the agent well known to have this property.

WO 2009/098357 PCT/FI2009/050093 55 Table 6. The synergistic bactericidal activity of NAB741 and 10% guinea pig serum (OPS) against E. coil IH3080 (018:Kl)* Concentration of NAB741 (pg/ml) 0 1 2 4 none (PBS) 100 71 86 58 10% GPS >200 5 7 0 10% GPS, heat inactivated >200 >200 >200 125 * measured as % survival after 2-hour treatment at 37 0 C 5 Example 7 Preparation and biological activity of NAB 739 methanesulfonate sodium salt NAB 739 acetate (100 mg) was dissolved in water (2 ml) and neutral formaldehyde solution (400 microliters of 30% aqueous formaldehyde [brought 10 to pH 7.2 with I N Na-HCQ3]) was added. Then, I N NaHCO3 solution (2 ml) was added, and the precipitated NAB 739 formaldehyde derivative was filtered and washed with water. The moist solid was suspended in water (5 ml), and sodium metabisulphite (100 mg) was added. A clear solution was obtained af ter a few minutes and was freeze-dried. The flocculent white solid was ex 15 tracted with warm acetone (7.5 ml) and dried in vacuo. The yield was 56 mg. Analysis of the product by ESI mass spectrometry revealed a predominant peak with the molecular mass of 1075.3 indicating that most of the derivative was sulfomethylated at each of the three Dab residues of the NAB 739 com pound. A minor peak representing the NAB 739 blocked randomly at two of the 20 three Dab residues was also visible. For the measurement of the antibacterial activity of aqueous solu tions of NAB 739 methanesulfonate sodium, three different solutions were made: 1) A solution (1mg/ml) made in 0.9% NaCl immediately prior to the ex periment, 2) a solution (1mg/ml) made in 0.9% NaCI 24 h prior to the experi 25 ment and kept at 37"C, 3) a solution (1mg/ml) made in 0.9% NaCl 48 h prior to the experiment and kept at 37 0 C. A freshly made solution of NAB 739 acetate served as the control compound.

WO 2009/098357 PCT/FI12009/050093 56 Table 7. The bactericidal activity of NAB 739 methanesulfonate as com pared with that of NAB 739 against E. coli IH3080* Compound Solution age 0 1 2 4 NAB739 MS** fresh 100 70 62 16 NAB 739 MS** 24 h 55 34 6 NAB 739 MS** 48 h 35 11 0 NAB 739 fresh 21 2 0 * measured as % survival after 2-hour treatment at 37 degrees centigrade. **MS, methanesulfonate 5 The test bacterium was E. coli IH3080. It was grown in LB broth (LB broth Lennox, Difco, BD, Sparks, MD, U.S.A.) at 37 0 C in a rotary shaker into early logarithmic growth phase, washed with PBS, and resuspended in PBS to approx. 10e9 cells/mi. PBS was inoculated with approx. 500 CFU (colony form ing units) of bacteria per ml and pipetted in 0.2 ml aliquots into wells of a mi 10 crotiter plate. The plates already contained increasing concentrations of NAB 739 methanesulfonate or the control compound in 0.020 ml of 0.9% NaCl. The plate was incubated at 370C for 1 h whereafter each well was emptied onto LB plates. The plate was incubated overnight at 37*C and the developed colonies were counted. 15 The results are shown in Table 7. The fresh solution of NAB 739 methanesulfonate was much less antibacterial than the control compound NAB 739. Keeping the NAB 739 methanesulfonate solution at 37 0 C for 24 h prior to use slightly increased the activity whereas keeping for 48 h resulted in activity almost equal to that observed with the control compound. These results indi 20 cate that NAB 739 methanesulfonate, in analogy with colistin methanesul fonate, slowly decomposes in aqueous solutions to yield antibacterially more active substances, i.e. less sulfomethylated substances and eventually free NAB 739. Similarly, methanesulfonate derivatives of NAB 741, NAB 745, NAB 25 747 and other compounds described as herein are prepared. These prodrugs decompose in vivo to yield compounds which possess the ability to sensitize target bacteria to other antibacterial agents and serum complements.

WO 2009/098357 PCT/F12009/050093 57 Example 8 Comparison of basic pharmakokinetic properties of NAB 741 and NAB 739 The studies were principally performed by using the methods de 5 scribed by Li et al. (2003, 2004). Each rat (n=4 for both compounds, Sprague Dawley, male) was anaesthetized using isoflurane, and a polyethylene cannula was inserted Into the jugular vein. Each rat was placed into a metabolic cage and allowed to recover from the procedure overnight. The test compound (ace tate, 1 mg/kg) was administered as a bolus (in 200 pl sterile 0.9% saline) 10 through the cannula, followed by washing with 0.8 ml of saline. Nine blood samples (0, 10, 20, 30, 60, 90, 120, 180, and 240 min), each 200 pi, were manually collected through the cannula. When collecting samples, the first 100 pi of blood was withdrawn and kept in the syringe. After collecting the actual sample with another syringe, the content of the first syringe was returned to the 15 rat together with 400 pi of heparinized saline. Blood samples were centrifuged to obtain plasma. Urine samples were collected in 0-4 h, 4-6 h, and 6-24 h in tervals. Plasma and urine samples were stored at -80'C. The samples were analyzed using liquid chromatography and mass spectrometry with electrospray ionization Interface (LC/electrospray ionization 20 MS). To a 100-pl sample, 10 pl of internal standard (NAB 739, 80 pg/mI) and 200 pl (plasma samples) or 100 pi (urine samples) of acetonitrile was added, the mixture was vortex-mixed for 1 min, and centrifuged at 10.000 g for 10 min. The chromatography employed the HPLC C18 column (50 x 2 mm), 0.1% for mic acid as the solvent A, 0.1% acetonitrile as the solvent B, flow rate of 0.2 25 mI/min, and the following gradient: 5%-30% B in 6 min, 30%-90% B in 0.5 min, 90% B held for 2.5 min, 90%-5% B in 1 min. The eluent between 5.90-7.00 min and 9.00-10.1 min was directed to the MS system using a switching valve. The positive protonated molecular ions of NAB 741 at m/z 496.7 and 331.4 and of NAB 739 at m/z = 538.8 and 359.6 and were monitored. NAB 741 was 30 eluted at 6.65 ± 0.05 min and NAB739 was eluted at 9.45 ± 0.05 min. Non compartmental analysis of the compounds in plasma was performed using WinNonlin software (version 4.0, Mountain View, CA, USA), with the model of NA201 (i.v.'bolus input for plasma data). The basic pharmacokinetic parameters determined for NAB 741, 35 were as follows: half-life (min), 32.7 ± 2.41; volume of distribution (ml/kg), 243 58 ± 24.0; clearance (mlmin/kg), 7.39 ± 0.85; urinary recovery (% of dose in 24 h), 50.9 ± 13.6; and renal clearance (ml/min/kg), 3.78 ± 1.11. The basic pharmacokinetic parameters determined for NAB 739, a compound otherwise identical to NAB 741 but having octanoyl residue instead 5 of acetyl residue as its terminal inoiety, were as follows: half-life (min), 69;0 ± 21.9; volume of distribution (ml/kg), 222 ± 20.5; clearance (milmin/kg), 2.63 ± 0.54; urinary recovery (% of dose in 24 h), 19.4 ± 7.38; and renal clearance (ml/min/kg), 0.53 ± 0.30. The corresponding parameters for colistin, as determined by Li et al. 10 (2003) by using an identical dosing and administration procedure, are the fol lowing: half-fife (min), 74.6 ± 13.2; volume of distribution (ml/kg), 496 t 60; clearance (ml/minlkg), 5.2 ± 0.4; urinary recovery (% of dose in 24 h), 0.18 i 0.14; and renal clearance (ml/minkg). 0.010 ± 0.008. The reference in- this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

1. A polymyxin derivative of formula (1): R' R1 A Q 3 W3 ) 2 W2 ai W1 D 5 m (I) wherein: A Is a polymyxin ring moiety; D is a terminal moiety comprising I to 5 carbon atoms; 10 m , m 2 , and m 3 are each independently 0 or 1; Q1, Q 2 , and Q 3 are each independently CH 2 , C=O, or C=S; W 1 , W 2 , and W 3 are each independently NR 4 , 0, or S; R 1 , R 2 , and R 3 ' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, alkyl, arylalkyl, aryl, alkoxy, alkoxycar 15 bonyl, aryloxycarbonyl, alkylamino, or alkynyl; and R 4 is hydrogen or alkyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that (1) when A is an octapeptin ring, ml and m 2 are 0, m' is 1, W 3 is NH, Q 3 is C=0, and R 3 ' is the side chain of diaminobutyric acid (Dab), then D Is not CrC 5 acyl, 20 and (2) when D is acetyl, butanoyl or pentanoyl, then R 3 ' is not the side chain of Dab.

2. The polymyxin derivative of claim 1, wherein ml is 0.

3. The polymyxin derivative of claim 1 or 2, wherein m 2 and m 3 are each 1. 25

4. The polymyxin derivative of any one of claims 1-3, wherein Q2 and Q 3 are each C=0.

5. The polymyxin derivative of any one of claims 1-4, wherein W 2 and W3 are each NH.

6. The polymyxin derivative of any one of claims 1-5, wherein R 1 , 30 R T , and R 3 'do not comprise positively charged functional groups at physiologi cal pH. WO 2009/098357 PCT/F12009/050093 60

7. The polymyxin derivative of any one of claims 1-6, wherein R", R T , and R 3 ' comprises one or more hydroxyl, carbamy, amidyl, carboxylate, thiol, sulfate, sulfonyl, or phosphate groups.

8. The polymyxin derivative of claim 7, wherein R', R-, and R 3 ' 5 comprise two or more hydroxyl, carboxylate, thiol, amidyl, carbamyl, sulfate, sulfonyl, or phosphate groups.

9. The polymyxin derivative of any one of claims 1-8, wherein R is substituted with one or more groups selected from hydroxyl, carbamyl, amidyl, carboxylate, thiol, sulfate, sulfonyl, or phosphate groups.

10 10. The polymyxin derivative of claim 9, wherein R' is substituted with a carbamyl, hydroxyl or carboxylate group.

11. The polymyxin derivative of claim 9 or 10, wherein R' is substi tuted alkyl.

12. The polymyxin derivative of claim 9, wherein R' is the side 15 chain of alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either D- or L- configura tion.

13. The polymyxin derivative of claim 12, wherein R 2 ' is the side chain of D-alanine, L-serine, or L-threonine. 20

14. The polymyxin derivative of any one of claims 1-13, wherein R 3 ' is substituted with one or more groups selected from hydroxyl, amidyl, carba myl, carboxylate, thiol, sulfate, sulfonyl, or phosphate groups.

15. The polymyxin derivative of claim 12, wherein R 3 ' is substituted with a carbamyl, hydroxyl or carboxylate group. 25

16. The polymyxin derivative of claim 14 or 15, wherein R- is substi tuted alkyl.

17. The polymyxin derivative of claim 1, wherein R' is the side chain of alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either D- or L- configura 30 tion.

18. The polymyxin derivative of claim 17, wherein Ra is the side chain of D-asparagine, L-serine, or D-serine,

19. The polymyxin derivative of any one of claims 1-18, wherein A is a polymyxin ring moiety selected from that of polymyxin A, polymyxin B, IL 35 polymyxin-B1, polymyxin D, polymyxin E, polymyxin F, polymyxin M, polymyxin WO 2009/098357 PCT/F12009/050093 61 S, polymyxin T, circulin A, octapeptin A, octapeptin B, octapeptin C, octapeptin D, or derivatives thereof.

20. The polymyxin derivative of claim 19, wherein A is a polymyxin ring moiety of polymyxin B or polymyxin E. 5

21. The polymyxin derivative of any one of claims 1-20, wherein D is R 12 -(C=O); R 12 -S0 2 -; R 12 (C=NH)-; R 12 -NH-(C=S)-; R" 2 -NH-(C=O)-; R 12 -NH (C=NH)-;R 2 -O-(C=S)-; R 12 -O-(C=0); R" 2 -P(O)OH-; R 12 -(C=S); or R 12 ', wherein R 12 and R 17 are alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or aryl alkyl.

22. The polymyxin derivative of claim 21, wherein D is R 2 -(C=O) or 10 R- 12 (C=S).

23. The polymyxin derivative of claim 21 or 22, wherein R 12 is methyl, ethyl, propyl, isopropyl, cyclopropyl, or butyl.

24. The polymyxin derivative of claim 22, wherein D is acetyl, propionyl, butanoyl, or pentanoyl. 15

25. A polymyxin derivative of formula (II): 0 R& R2 O R' A NC D wherein: 20 A is a polymyxin ring moiety; D is R- 12 C(=O), R 2 -C(=S), or R m , m 2 , and m 3 are each independently 0 or 1, provided that at least one of mi, M 2 , and m 3 are 1; R', R 2 ', and RT are each independently side chains of natural or 25 unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbony, aryloxycarbonyl, alkylamino, or alkynyl; and R 2 Is C 1 -C 4 alkyl, C 2 -C 4 alkenyl, or C 2 -C 4 alkynyl, R 12 ' is CrC alkyl, C 2 -C 5 alkenyl, or C 2 -C 5 alkynyl, and pharmaceutically acceptable prodrugs and salts thereof, provided that (1) 30 when A is an octapeptin ring, ml and m 2 are 0, m 3 is 1, and R 3 ' is the side chain of diaminobutyric acid (Dab), and D is R' 2 -C=O, then R 12 is not C-Cs WO 2009/098357 PCT/F12009/050093 62 alkyl, and (2) when D is acety, butanoyl or pentanoyl, then R' is not the side chain of Dab.

26. The polymyxin derivative of claim 25, wherein ml is 0.

27. The polymyxin derivative of claim 25 or 26, wherein.m 2 and m 3 5 are each 1.

28. The polymyxin derivative of any one of claims 25-27, wherein R 7 is substituted alkyl.

29. The polymyxin derivative of claim 28, wherein R 2 ' is substituted with a carbamyl, hydroxyl or carboxylate group. 10

30. The polymyxin derivative of claim 28, wherein R 2 is the side chain of alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, or threonine in either D- or L- configura tion.

31. The polymyxin derivative of claim 30, wherein R 2 ' is the side 15 chain of D-alanine, L-serine, or L-threonine.

32. The polymyxin derivative of any one of claims 25-31, wherein R 3 ' is substituted alkyl.

33. The polymyxin derivative of claim 32, wherein R 3 ' is substituted with a carbamyl, hydroxyl or carboxylate group. 20

34. The polymyxin derivative of claim 33, wherein R 3 ' is the side chain of alanine, aminobutyric acid, asparagine, aspartic acid, diaminobutyric add, glutamic acid, glutamine, serine, or threonine in either D- or L- configura tion.

35. The polymyxin derivative of claim 34, wherein R 3 - is the side 25 chain of D-asparagine, L-serine, or D-serine.

36. The polymyxin derivative of any one of claims 25-35, wherein A is a polymyxin ring moiety of polymyxin B or polymyxin E.

37. The polymyxin derivative of any one of claims 25-36, wherein R 12 is alkyl. 30

38. The polymyxin derivative of claim 37, wherein D is acetyl, propionyl, butanoyl, or pentanoyl.

39. A polymyxin derivative of formula (Ill): WO 2009/098357 PCT/F2009/050093 63 0 R 3 ' 0 R 2 ' 0 R t A 1_1_H 1H 1 H A-- N--C N C - -N D III wherein: A is a polymyxin B or polymyxin E ring moiety; 5 D is R"-C(=O), R' 2 -C(=S), or R m 1 is 0 or 1; R 1 , R2', and R 3 ' are each independently side chains of natural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryi, alkoxy, alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl, wherein at least one of R 2 and R 3 10 comprise a carbamyl, hydroxyl or carboxylate group; R 12 is C-C 4 alkyl; R2' is C 1 -Cs alkyl; and pharmaceutically acceptable prodrugs and salts thereof, provided that when D is acetyl, butanoyl or pentanoyl, then RW is not the side chain of Dab. 15

40. The polymyxin derivative of claim 39, wherein m is 0.

41. The polymyxin derivative of claim 39 or 40, wherein R 2 and R 3 are each substituted alkyl.

42. The polymyxin derivative of any one of claims 39-41, wherein D is acetyl, propionyl, butanoyl, or pentanoyl. 20

43. The polymyxin derivative of any one of claims 39-42, wherein R 1 , R and R' comprise at least two carbamyl, hydroxyl and carboxylate groups.

44. A polymyxin derivative of formula (IV): /M3 /M2 Mi / / / O La a L2 O Li 0 H H H A-C N-C N C N 25 m (IV) wherein: A is a polymyxin B or polymyxin E ring moiety; WO 2009/098357 PCT/F12009/050093 64 ml Is 0 or 1; L 1 , L2 and L' are each independently C-C3 alkyl or a covalent bond; M 1 , M 2 and M 3 are each independently H, C(=0)NH 2 , C(=O)OH, or OH; 5 R 12 is Cr-C4 alky, and pharmaceutically acceptable prodrugs and salts thereof, provided that when R 2 is methyl, propyl or butyl, then 12- M 3 is not the side chain of Dab.

45. The polymyxin derivative of claim 44, wherein ml is 0.

46. The polymyxin derivative of claim 44 or 45, wherein L 2 is 10 -CH(CH3)- and M 2 is OH; L 2 is -CH 2 - and M 2 is H; or L 2 is -CH 2 - and M 2 is OH.

47. The polymyxin derivative of any one of claims 44-46, wherein L 3 is -CH 2 - and M 3 is OH or; L 3 is -CH 2 -CH 2 - and M 3 is C(=0)NH 2 .

48. A polymyxin derivative of the general formula (V), (7)R17 -(8)R8 (6)R6 ( 9 )R9 (s)R5 (l)RIO (4)R4 (3)R13 15 R(FA) (V) wherein R4 is an amino acid residue comprising a functional side chain able to cyclicize the molecule; R6 and R7 are each independently selected optionally substi 20 tuted hydrophobic amino acid residues; RI0 is Leu or any non-hydrophobic amino acid residue; and wherein R1 is optional; and wherein R1, R2, R3, R5, R8 and R9 are each independently selected amino acid residues; and WO 2009/098357 PCT/F12009/050093 65 wherein R(FA) is an optionally substituted alkanoyl or alkyl resi due having a total of 1 to 5 carbon atoms; or pharmaceutically acceptable prodrugs and salts thereof, pro vided that (1) when R1 and R2 are absent, R3, R4. R5, R8, and R9 are Dab, 5 R6 is D-Leu, R7 is L-Leu or L-Phe, and RIO is Thr or when R1 and R2 are ab sent, R3, R4. R5, R8, and R9 are Dab, R6 is D-Phe, R7 is L-Leu, and RIO is Thr, then R(FA) is not an unsubstituted alkanoyl residue and (2) when R(FA) is acetyl, butanoyl or pentanoyl, then R 3 is not Dab.

49. The derivative according to claim 48, wherein R(FA) is a residue 10 having 1 to 3 carbon atoms.

50. The derivative according to any one of claims 48 or 49, wherein R(FA) is acetyl.

51. The derivative according to any one of claims 48-50, wherein RI-R1O is selected from the group consisting of Thr-DSer-cy[Dab-Dab-DPhe 15 Leu-Dab-Dab-Thr-] and Thr-DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-].

52. The derivative according to claim 51, selected from the group consisting of Ac- Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], Ac- Thr DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-, and Me- Thr-DSer-cy[Dab-Dab DPhe-Leu-Dab-Dab-Thr-. 20

53. The polymyxin derivative of any one of claims 1-52, wherein said derivative has at least two positive charges at physiological pH.

54. The polymyxin derivative of claim 53, wherein said derivative has three positive charges at physiological pH.

55. The polymyxin derivative according to claim 53 or 54, wherein 25 said positive charges are selected from the group consisting of free, unsubsti tuted amino groups and other cationic groups.

56. The derivative according to any one of claims 1-54, wherein said derivative is a prodrug comprising one or more positive charge masking moie ties. 30

57. The derivative according to claim 56, wherein said positive charge masking moieties are cleaved in vivo.

58. The derivative according to claim 56, wherein said positive charge masking moieties reduce toxicity.

59. The derivative according to claim 56, wherein said positive 35 charge masking moieties are sulfoalkyl. WO 2009/098357 PCT/F12009/050093 66

60. The derivative of claim 59, wherein said sulfoalkyl moieties are sulfomethyl.

61. The derivative of any one of claims 1-60, wherein said derivative has lower toxicity than polymyxin B. 5

62. The derivative of any one of claims 1-61, wherein said derivative increases bacterial sensitivity to a second antibacterial agent or serum com plement.

63. The derivative of any one of claims 1-62, wherein said derivative increases bacterial sensitivity to a second antibacterial agent by a factor of five 10 or greater.

64. The derivative of claim 63, wherein said derivative increase bac terial sensitivity to a second antibacterial agent by a factor of ten or greater.

65. The derivative of any one of claims 62-64, wherein said second antibacterial agent is rifampin, clarithromycin, mupirocin, azithromycin, fusidic 15 acid, or vancomycin.

66. The derivative of any one of claims 1-65, wherein said derivative has one or more more pharmacokinetically favorable properties as compared to native polymyxins, octapeptins, or polymyxin derivatives with terminal moie ties with more than five carbon atoms. 20

67. The derivative of claim 66, wherein said pharmacokinetically fa vorable property is a longer serum half life, increased renal clearance, or in creased urinary recovery as compared to native polymyxins, octapeptins, or polymyxin derivatives with terminal moieties with more than five carbon atoms.

68, The derivative of claim 67, wherein said urinary recovery is 25 greater than about 10% of an administered dose of said derivative over 24 hours.

69. The derivative of claim 68, wherein said urinary recovery is greater than about 30% of an administered dose of said derivative over 24 hours. 30

70. The derivative of claim 67, wherein said renal clearance of said derivative is greater than about 0.5 ml/min/kg.

71. The derivative of claim 70, wherein said renal clearance of said derivative is greater than about 2 m/min/kg.

72. The derivative of any one of claims 66-71, wherein said native 35 polymyxin or octapeptin is polymyxin A, polymyxin B, IL-polymyxin-B 1 , po- WO 2009/098357 PCT/F12009/050093 67 lymyxin D, polymyxin E, polymyxin F, polymyxin M, polymyxin S, polymyxin T, circulin A, octapeptin A, octapeptin B, octapeptin C, or octapeptin D.

73. The derivative of claim 72, wherein said native polymyxin or oc tapeptin is polymyxin E. 5

74. A combination product comprising two or more of the derivatives according to any of claims 1 to 73.

75. A pharmaceutical composition comprising at least one derivative according to any one of claims 1 to 73, and at least one pharmaceutically ac ceptable carrier and/or exciplent. 10

76. The pharmaceutical composition according to claim 75, further comprising a second antibacterial agent.

77. A method for sensitizing Gram-negative bacteria to an anti bacterial agent, comprising administering, simultaneously or sequentially in any order, a therapeutically effective amount of said antibacterial agent and a 15 derivative according to any one of claims 1 to 73 or a combination according to claim 74.

78. The method according to claim 77, wherein said anti-bacterial agent is selected from the group consisting of clarithromycin, azithromycin, erythromycin and other macrolides, ketolides, clindamycin and other lincosa 20 mines, streptogramins, rifampin, rifabutin, rifalazile and other rifamycins, fusidic acid, mupirocin, oxazolidinones, vancomycin, dalbavancin, telavancin, orita vancin and other glycopeptide antibiotics, fluoroquinolones, bacitracin, tetracy cline derivatives, betalactam antibiotics, novobiocin, pleuromutilins, folate syn thesis inhibitors, deformylase inhibitors, and bacterial efflux pump inhibitors. 25

79. The method according to claim 78, wherein said anti-bacterial agent is selected from the group consisting of: clarithromycin, azithromycin, erythromycin, clindamycin, the streptogramin combination quinupristin dalfopristin, rifampin, fusidic acid, mupirocin, the oxazolidinone linezolid, van comycin, the fluoroquinolone moxifloxacin, and the folate synthesis inhibitor 30 trimetoprim.

80. The method according to claim 77, wherein said bacterium is se lected from the group consisting of: Escherichia coi, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundi, and Acinetobac ter baumanni. 35

81. A method for developing novel antibiotics comprising the steps of WO 2009/098357 PCT/F12009/050093 68 a) providing a natural polymyxin or octapeptin compound, or a de rivative thereof, having a total of 4 to 6 positive charges and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms; b) replacing from 1 to 3 residues carrying one or more positive 5 charges with a residue not having a positive charge, or with a covalent bond, thereby generating a derivative of a polymyxin compound having 3 positive charges and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms; c) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to an antibacterial agent; and 10 d) selecting compounds having the ability to sensitize Gram negative bacteria to an anti-bacterial agent.

82. A method for developing novel antibiotics comprising the steps of a) providing a natural polymyxin or octapeptin compound, or a de 15 rivative thereof, having a total of 4 to 6 positive charges and a terminal moiety (D) having more than 5 carbon atoms; b) replacing from I to 3 residues carrying one or more positive charges with a residue not having a positive charge, or with a covalent bond, thereby generating a derivative of a polymyxin compound having 3 positive 20 charges; c) replacing said terminal moiety (D) having more than 5 carbon at oms with a terminal moiety (D) comprising a total of 1 to 5 carbon atoms, thereby generating a derivative of a polymyxin compound having 3 positive charges and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms; 25 d) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to an antibacterial agent; and e) selecting compounds having the ability to sensitize Gram negative bacteria to an anitbacterial agent.

83. A method for developing novel antibiotics comprising the steps 30 of: a) providing a polymyxin or octapeptin compound, or a derivative thereof, having a total of 4 to 6 positive charges and lacking the terminal moi ety (D), b) replacing from I to 3 residues carrying one or more positive 35 charges with a residue not having a positive charge, or with a covalent bond, WO 2009/098357 PCT/F12009/050093 69 thereby generating a derivative of a polymyxin compound having 3 positive charges; c) introducing a tenninal moiety (D) comprising a total of 1 to 5 car bon atoms, thereby generating a polymyxin compound having 3 positive 5 charges and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms; e) assaying said polymyxin derivative for the ability to sensitize Gram-negative bacteria to an antibacterial agent; and f) selecting compounds having the ability to sensitize Gram-negative bacteria to an anitbacterial agent. 10

84. A method for sensitizing clinically important Gram-negative bac teria to a host defense mechanism complement present in the serum, wherein said bacteria are subjected to the action of a derivative according to any one of claims I to 73 during a clinical infection.

85. The method according to claim 84, wherein said bacteria are se 15 lected from the group consisting of; Escherichia coi, Klebsiella pneumoniae, Kiebsiella oxytoca, Enterobacter cloacae, Citrobacter freundi, and Acinetobac ter baumannl.

86. Use of a derivative according to any one of claims 1 to 73 for the manufacture of a medicament for sensitizing Gram-negative bacteria against 20 antibacterial agents.

87. The use according to claim 86, wherein said bacteria are se lected from the group consisting of: Escherichia coli, Kiebsiella pneumoniae, Kiebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, and Acinetobac ter baumanni. 25

88. The use according to claim 87, wherein said antibacterial agent is selected from the group consisting of clarithromycin, azithromycin, erythro mycin and other macrolides, ketolides, clindamycin and other lincosamines, streptogramins, rifampin, rifabutin, rifalazile and other rifamycins, fusidic acid, mupirocin, oxazolidinones, vancomycin, dalbavancin, telavancin, oritavancin 30 and other glycopeptide antibiotics, fluoroquinolones, bacitracin, tetracycline derivatives, betalactam antibiotics, novobiocin, pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, and bacterial efflux pump inhibitors.

89. The use according to claim 88, wherein said antibacterial agent is selected from the group consisting of: clarithromycin, azithromycin, erythro 35 mycin, clindamycin, the streptogramin combination quinupristin-dalfopristin, WO 2009/098357 PCT/F12009/050093 70 rifampin, fusidic acid, mupirocin, the oxazolidinone linezolid, vancomycin, the fluoroquinolone moxifloxacin, and the folate synthesis inhibitor trimetoprim.

90. Use of a derivative according to any one of claims I to 73 for the manufacture of a medicament for sensitizing Gram-negative bacteria to a host 5 defense mechanism complement present in the serum.

91. The use according to claim 90, wherein said bacteria are se lected from the group consisting of: Escher/chia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundli, and Acinetobac ter baumannl. 10

92. A process for preparing a polymyxin derivative of formula (I) as defined in claim 1 comprising (A) modifying a natural or synthetic polymyxin or octapeptin com pound or a derivative thereof having 4 to 5 positively charged residues and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms by replacing 1 to 15 2 of said residues by neutral residues, or by a covalent bond, or converting I to 2 of said residues into neutral residues in order to obtain a polymyxin deriva tive of formula (1) according to claim 1, having 3 positively charged residues and a terminal moiety (D) comprising a total of I to 5 carbon atoms; or (B) modifying a natural or synthetic polymyxin or octapeptin com 20 pound or a derivative thereof having 4 to 5 positively charged residues and a terminal moiety (D) having more than 5 carbon atoms by replacing 1 to 2 of said residues by neutral residues, or by a covalent bond, or converting I to 3 of said residues into neutral residues, and by replacing said terminal moiety (D) comprising more than 5 carbon atoms with a terminal moiety (D) comprising a 25 total of 1 to 5 carbon atoms, in order to obtain a polymyxin derivative of for mula (1) according to claim 1, having a total of 3 positively charged residues and a terminal moiety (D) comprising a total of 1 to 5 carbon atoms, or (C) modifying a natural or synthetic polymyxin or octapeptin com pound or a derivative thereof having 4 to 6 positively charged residues and 30 lacking the terminal moiety (D) by replacing 1 to 3 of said residues by neutral residues, or by a covalent bond, or converting 1 to 3 of said residues into neu tral residues, and by introducing a terminal moiety (D) comprising a total of 1 to 5 carbon atoms, in order to obtain a polymyxin derivative of formula (1) accord ing to claim 1, having 3 positively charged residues and a terminal moiety (D) 35 comprising a total of I to 5 carbon atoms. WO 2009/098357 PCT/F12009/050093 71

93. The process according to claim 92 comprising carrying out the process as a total synthetic process.

94. The process according to claim 92 comprising carrying out the process as a semisynthetic process. 5

95. The process according to claim 94 comprising the steps of: a) subjecting a natural or synthetic polymyxin or octapeptin com pound or a derivative thereof to cleavage in order to remove the side chain of said polymyxin compound, and recovering the cyclic part of said compound, and 10 b) coupling to the cyclic part obtained in step a) a synthetically pre pared side chain in order to obtain a polymyxin derivative of formula (1) accord ing to claim 1.

96. The process according to claim 94 comprising carrying out the cleavage In step a) enzymatically. 15

97. The process according to claim 94 comprising carrying out the cleavage in step a) chemically.

98, The process according to claim 94 comprising carrying out the cleavage in step a) using a combination of both chemical and enzymatic treat ments. 20

99. A method for treating a Gram-negative bacterial infection in a subject, comprising administering an effective amount of a derivative of any one of claims 1-73 in combination with a second antibacterial agent, such that the bacterial infection is treated.

100. The method of claim 99, wherein said second antibacterial 25 agent is clarithromycin, azithromycin, erythromycin, a macrolide, a ketolide, clindamycin, a lincosamine, streptogramin, rifampin, rlfabutin, rifalazile, a rifa mycin, fusidic acid, mupirocin, oxazolidinone, vancomycin, dalbavancin, tela vancin, oritavancin, glycopeptide antibiotics, fluoroquinolones, bacitracin, a tetracycline derivative, a betalactam antibiotic, novobiocin, a pleuromutilin, a 30 folate synthesis inhibitor, a deformylase inhibitor, or a bacterial efflux pump inhibitor.

101. The method of claim 99 or 100, wherein said subject is a mammal.

102. The method of claim 101, wherein said subject is a human. 35

103. The method of any one of claims 99-102, wherein said bacte rial infection Is an infection of Escherichia coli, Kiebsiella pneumoniae, Kleb- WO 2009/098357 PCT/F12009/050093 72 siella oxytoca, Enterobacter cloacae, Citrobacter freundiY, or Acinetobacter baumannil.

104. The method of any one of claims 99-103, wherein said deriva tive and said second antibacterial agent are administered in combination with a 5 pharmaceutically acceptable carrier. 2013257422 12 Nov 2013 2013257422 12 Nov 2013