Peptide Antibiotics and Methods For Making Same

Abstract

Novel protected cyclopeptide intermediates are prepared from polymyxin B are used to synthesize new peptide antibiotics. Intermediates are readily derivatized and deprotected to provide new families of antibiotics, which have potent anti-bacterial activity against gram-negative bacteria; but also are useful and potent against gram-positive bacteria.

Description

FIELD OF THE INVENTION

Disclosed herein are novel peptides and novel protected peptides derived from polymyxin and octapeptin peptides, including, e.g., colistin, circulin A, polymyxin A, polymyxin B, polymyxin D, octapeptin B, octapeptin C, and [Ile⁷]polymyxin B₁. The novel peptides and novel protected peptides have antibacterial properties. Also disclosed are pharmaceutical compositions containing the novel peptides and novel protected peptides, as well as methods for preparing the novel peptides and novel protected peptides.

BACKGROUND

Gram-negative bacteria that are resistant to aminoglycoside, β-lactam, and fluoroquinolone antibiotics are increasingly common. These bacteria are often susceptible to the polymyxins and related peptides having antibacterial properties (Refs. 1, 10, 23). As a result, there is interest in the use of polymyxins for multidrug-resistant gram-negative bacterial infections in humans (Ref. 23).

Peptides such as polymyxin B and the related colistin (polymyxin E) have been administered to humans as antibacterial agents. However, their use has been previously restricted because of their toxicity. These peptides comprise a seven amino acid cyclic peptide attached to an exocyclic three amino acid chain, wherein the N-terminal amine of the exocyclic chain is linked to a “side chain” or “tail”. The tail is most commonly an acyl group.

Some renal toxicity has been observed with the recommended dosing of polymyxin B in patients. Neurotoxicity has also been observed in patients with compromised renal functions, with an overall incidence of 7.3% reported in one large study with colistin (Ref. 1). The acyl exocyclic chain and the adjacent N-terminal 2,4-diaminobutanoic acid (Dab) residue can be enzymatically removed from polymyxin, thereby yielding the corresponding nonapeptide. The in vivo toxicity of the nonapeptide of polymyxin B is significantly less than that of polymyxin B itself (Ref. 16). The toxicity of the nonapeptide in cell culture is reduced by about 100-fold relative to polymyxin B; however, the antibacterial activity of the nonapeptide is also reduced by about 2-64 fold relative to polymyxin B (Ref. 11).

Attempts have been made to chemically modify polymyxin and colistin in order to obtain peptides with improved antibacterial properties. For example, the total synthesis of polymyxin B and four analogs was previously accomplished by a combination of solid phase peptide syntheses to obtain linear structures, followed by removal from the resin and condensation in solution at high dilution to obtain the cyclic peptide structure (Ref. 7). The derivatives, however, were less active than polymyxin B. A more recent total synthesis of polymyxin B and a few closely related compounds was accomplished only by solid phase peptide synthesis (Refs. 15, 26). Although both of these solid phase total synthetic approaches can provide new derivatives of polymyxin, these methods appear limited since the quantities of antibiotic produced are small and require large amounts of amino acid precursors. Any scale up of these methods for clinical studies may prove to be difficult and costly.

Accordingly, there remains an ongoing need for new peptide compounds having antibacterial properties, and new methods for preparing such compounds.

SUMMARY

Disclosed herein are novel peptides, such as peptide antibiotics and/or other peptides having antibacterial properties, and methods for preparing the peptides. The compounds disclosed herein can provide structural diversity in the exocyclic region (the exocyclic amino acids and tail) of the cyclic peptide.

DEFINITIONS

“Acyl,” as used herein, refers to a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group. Exemplary acyls include, but are not limited to: (1) “unsubstituted alkanoyl,” which is defined as carbonyl radical attached to an unsubstituted alkyl group; (2) “unsubstituted alkenoyl,” which is defined as carbonyl radical attached to an unsubstituted alkenyl group; (3) “substituted alkanoyl,” which is defined as a carbonyl radical attached to a substituted alkyl group, in which one or more hydrogen atoms is replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido; and (4) “substituted alkenoyl,” which is defined as carbonyl radical attached to an substituted alkenyl group, in which one or more hydrogen atoms is replaced by a substituent group as described above. Nonlimiting examples of acyls include radicals such as acetyl, n-octanoyl, n-nonanoyl, benzoyl, and isonicotinoyl.

“Acylamino,” as used herein, refers to an amino group bonded to an acyl group.

“Acyloxy,” as used herein, refers to an oxygen radical substituted with an acyl group. In some embodiments, acyloxy is substituted with an acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, or ureido group.

An “addition reagent,” as used herein, is a compound that can react with an amino group such as the N-terminus of a peptide, thereby chemically modifying the amino group by addition of all, or a component, of the addition reagent to the amino group. For example, an addition reagent may be an acylamino reagent such as R′—(C═O)-LG or a sulfonating reagent such as R′—SO₂-LG, where LG is a leaving group, that can react with an amino group to form an acylamino group or a sulfonamino group, respectively. An addition reagent may also be, for example, an isocyanate, isothiocyanate, activated ester, acid chloride, sulfonyl chloride, activated sulfonamide, activated heterocycle, activated heteroaryl, chloroformate, cyanoformate, thioacylester, phosphoryl chloride, phosphoramidate, imidate, or lactone. An addition reagent may also be an aldehyde or ketone that reacts with an amine under reductive conditions to form an alkylated amine. An addition reagent may also be an activated amino acid, or an amino acid and a peptide coupling reagent, such as, e.g., PyBOP® (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), HBtU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HBtU/HOBt (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/N-hydroxybenzotriazole), or DCC (dicyclohexylcarbodiimide).

“Alkenyl,” as used herein, refers to linear or branched radicals having 2-20 carbon atoms, such as 2-12, 2-10, or 2-6 carbon atoms, and containing at least one carbon-carbon double bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration. Nonlimiting examples of alkenyls include “unsubstituted alkenyl,” which is defined as an alkenyl group that bears no substituent groups. Other nonlimiting examples of alkenyl groups include ethenyl, 2-phenyl-1-ethenyl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-1-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl.

“Alkoxy,” as used herein, refers to an oxygen radical substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl group. Nonlimiting examples include methoxy, tert-butoxy, benzyloxy, and cyclohexyloxy.

“Alkyl,” as used herein, refers to a linear or branched saturated radicals having at least one carbon atom, such as 1-20 carbon atoms, 1-12, 1-10, or 1-6 carbon atoms, or at least 6 carbon atoms, at least 7 carbon atoms, at least 8 carbon atoms, at least 9 carbons atoms, or at least 10 carbon atoms, unless otherwise specified. A “lower alkyl” is defined as an alkyl group containing 1-4 carbon atoms. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting examples of alkyl groups include methyl, butyl, tert-butyl, isopropyl, trifluoromethyl, nonyl, undecyl, octyl, dodecyl, methoxymethyl, 2-(2′-aminophenacyl), 3-indolylmethyl, benzyl, and carboxymethyl. Other exemplary alkyls include, but are not limited to: (1) “unsubstituted alkyl,” which is defined as an alkyl group that bears no substituent groups; and (2) “substituted alkyl,” which denotes an alkyl radical in which one or more hydrogen atoms is replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Exemplary alkyl groups include, but are not limited to, methyl, ethyls such as ethanyl (ethyl), propyls such as propan-1-yl (n-propyl), propan-2-yl (iso-propyl), butyls such as butan-1-yl (n-butyl), butan-2-yl (s-butyl), 2-methyl-propan-1-yl (iso-butyl), 2-methyl-propan-2-yl (tert-butyl), trifluoromethyl, nonyl, undecyl, octyl, dodecyl, methoxymethyl, 2-(2′-aminophenacyl), 3-indolylmethyl, benzyl, and carboxymethyl.

“Alkynyl,” as used herein, refers to linear and branched radicals having from 2-10 carbon atoms, and containing at least one carbon-carbon triple bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Examples of alkynyl groups include, but are not limited to, ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl.

“Amino,” as used herein, refers to an NR₁R₂ radical, in which R₁ and R₂ may be selected from hydrido, acyl, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, formyl, heteroaryl, heterocyclyl, hydroxy, imino, nitro, oxo, sulfinyl, sulfonyl, and thio. A monosubstituted amino refers to an NR₁R₂ radical wherein R₁ is hydrido and R₂ is selected from acyl, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, formyl, heteroaryl, heterocyclyl, hydroxy, imino, nitro, oxo, sulfinyl, sulfonyl, and thio. A disubstituted amino refers to an NR₁R₂ radical wherein R₁ and R₂ are each independently selected from acyl, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, formyl, heteroaryl, heterocyclyl, hydroxy, imino, nitro, oxo, sulfinyl, sulfonyl, and thio.

An “amino acid,” as used herein, refers to a compound containing a carboxylic acid group and an amino group and having the formula H₂N[C(R)(R′)]_n—C(O)OH, where n is an integer greater than equal to one, and R and R′ are independently selected from hydrogen and amino acid side chains. For example, when n is equal to one, the amino acid of the formula H₂N[C(R)(R′)]C(O)OH is an alpha amino acid, and when n is equal to two, the amino acid of the formula H₂N—C(R₁)(R₁′)—C(R₂)(R₂′)—C(O)OH is a beta amino acid, where R₁, R₁′, R₂, and R₂′ are each independently chosen from amino acid side chains. An “amino acid residue,” as used herein, refers to an amino acid that is part of a peptide or protein, and having the formula —N(H)C(R)(R′)C(O)—. An “amino acid side chain” as used herein, refers to any side chain from a naturally-occurring or synthetic amino acid. For example, methyl may be referred to as an alanine side chain, and 2-amino-1-ethyl may be referred to as the side chain of 2,4-diaminobutanoic acid.

Exemplary amino acids may be chosen from the twenty encoded amino acids and derivatives thereof, as well as from, e.g., other α-amino acids, α-amino acids, γ-amino acids, δ-amino acids, and Ω-amino acids. An amino acid may have R or S chirality at any chiral atom. An amino acid may be chosen from, e.g., alanine, β-alanine, α-aminoadipic acid, 2-aminobutanoic acid, 4-aminobutanoic acid, 1-aminocyclopentanecarboxylic acid, 6-aminohexanoic acid, 2-aminoheptanedioic acid, 7-aminoheptanoic acid, 2-aminoisobutyric acid, aminomethylpyrrole carboxylic acid, 8-amino-3,6-dioxa-octanoic acid, aminopiperidinecarboxylic acid, 3-amino-propionic acid, aminoserine, aminotetrahydropyran-4-carboxylic acid, arginine, asparagine, aspartic acid, azetidine carboxylic acid, benzothiazolylalanine, butylglycine, carnitine, 4-chlorophenylalanine, citrulline, cyclohexylalanine, cyclohexylstatine, cysteine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, dihydroxyphenylalanine, dimethylthiazolidine carboxylic acid, glutamic acid, glutamine, glycine, histidine, homoserine, hydroxyproline, isoleucine, isonipecotic acid, leucine, lysine, methanoproline, methionine, norleucine, norvaline, ornithine, p-aminobenzoic acid, penicillamine, phenylalanine, phenylglycine, piperidinylalanine, piperidinylglycine, proline, pyrrolidinylalanine, sarcosine, selenocysteine, serine, statine, tetrahydropyranglycine, thienylalanine, threonine, tryptophan, tyrosine, valine, allo-isoleucine, allo-threonine, 2,6-diamino-4-hexanoic acid, 2,6-diaminopimelic acid, 2,3-diaminopropionic acid, dicarboxidine, homoarginine, homocitrulline, homocysteine, homocystine, homophenylalanine, homoproline, and 4-hydrazinobenzoic acid.

N-protected α-amino acids for peptide synthesis having L- or D-chirality at Cα are commercially available, e.g., from Novabiochem® (San Diego, Calif.) and Bachem (Bubendorf, Switzerland). The synthesis of chiral α-amino acids and other amino acids is also well known to those of ordinary skill in the art, and is described, e.g., in Arnstein Synthesis of amino acids and proteins, University Park Press, 1975; Enantioselective Synthesis of Beta-Amino Acids, Juaristi et al., Eds., Wiley-VCH: New York, 2005; and Williams Synthesis of optically active α-amino acids, Pergamon Press, 1989.

“Amino protecting group,” as used herein, refers to any substituent that may be used to prevent an amino group on a molecule from undergoing a chemical reaction while chemical change occurs elsewhere in the molecule. An amino protecting group can be removed under the appropriate chemical conditions. Numerous amino protecting groups are known to those skilled in the art, and examples of amino protecting groups, methods for their addition, and methods for their removal can be found in “Protective Groups in Organic Synthesis” by Theodora W. Greene,

John Wiley and Sons, New York, 1991, the disclosure of which is incorporated herein by reference. Nonlimiting examples of amino protecting groups include phthalimido, trichloroacetyl, STA-base, benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl, chlorobenzyloxycarbonyl, and nitrobenzyloxycarbonyl. Other exemplary amino protecting groups include “carbamate amino protecting groups,” which are defined as a carbonyl containing protecting group that when bound to an amino group forms a carbamate. Nonlimiting exemplary amino carbamate protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), carbobenzyloxy (CBZ), and t-butoxycarbonyl (Boc) protecting groups. Other exemplary protecting groups include 9-fluorenylmethoxycarbonyl (Fmoc) substituted with acidic substituents, such as 2-sulfo-9-fluorenylmethoxycarbonylcarbonyl, 2-carboxymethyl-9-fluorenylmethoxycarbonyl, and 4-carboxy-9-fluorenylmethbxycarbonyl.

“Amino protecting group reagents,” as used herein, refer to addition reagents that can react with an amino group such as the N-terminus of a peptide, thereby chemically modifying said amino group by addition of an amino protecting group.

“Aryl,” as used herein, refers to a mono-, bi-, or other multi-carbocyclic, aromatic ring system. The aryl group can optionally be fused to one or more rings selected from aryls, cycloalkyls, and heterocyclyls. Aryls can have from 5-14 ring members, such as from six to ten ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, azido, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting examples of aryl groups include phenyl, naphthyl, biphenyl, and anthracenyl.

“Aryloxy,” as used herein, refers to an oxygen radical substituted with an aryl or heteroaryl group. An exemplary aryloxy includes, but is not limited to, phenoxy.

“Carbamoyl,” as used herein, refers to a nitrogen radical of the formula

—N(R^x2)—C(O)—OR^x3; wherein R^x2 is selected from hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and R^x3 is selected from alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

“Carboalkoxy,” as used herein, refers to a carbonyl radical bonded to an alkoxy or aryloxy group.

“Carboxy,” as used herein, refers to a COOH radical.

“Carboxyamino,” as used herein, refers to a CONH₂ radical.

“Carboxyamido,” as used herein, refers to a carbonyl radical bonded to a monosubstituted amino or disubstituted amino group.

“Cycloalkyl,” as used herein, refers to a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members, such as a ring system having from three to seven ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

“Fmoc” is a 9-fluorenylmethoxycarbonyl group.

“Halo,” as used herein, refers to a bromo, chloro, fluoro or iodo radical.

“Heteroaryl,” as used herein, refers to an aromatic radical having from one to four hetero atoms or hetero groups selected from O, N, NH, S, or SO in a single or fused heterocyclic ring system, having from five to fifteen ring members, such as a heteroaryl ring system having from six to ten ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting examples of heteroaryl groups include indolyl, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazolyl, triazolyl, and pyrrolyl groups.

“Heterocyclyl” or “heterocyclic,” as used herein, refers to a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH, N(alkyl, such as lower alkyl), S, SO or SO₂, in a single or fused heterocyclic ring system having from three to twelve ring members, such as a heterocyclyl ring system having from three to seven ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, cycloalkyl, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido. Nonlimiting examples of heterocyclyl groups include morpholinyl, piperidinyl, pyrrolidinyl and succinimidyl.

“Hydroxy,” as used herein, refers to —OH.

“Iminoamino,” as used herein, refers to —N(H)C(═NR^x26)R^x27, wherein R^x26 and R^x27 are selected from hydrido, alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

“Phosphonamino,” as used herein, refers to

wherein R^x13 and R^x14 are independently selected from alkoxy, alkyl, amino, aryl, aryloxy, cycloalkyl, disubstituted amino, halo, heteroaryl, heterocyclyl, hydroxy, monosubstituted amino, and thio.

“Sulfinyl,” as used herein, refers to —S(═O)OH.

“Sulfo,” as used herein, refers to —SO₃H.

“Sulfonamino,” as used herein, refers to an amino radical of the formula

wherein each of R^x24 is selected from hydrido, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, and R^x25 is selected from alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

“Sulfonyl,” as used herein, refers to a hexavalent sulfur radical substituted with two oxo substituents and a third substituent selected from alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

“Thio,” as used herein, refers to a radical containing a substituent group independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to a divalent sulfur atom, such as methylthio and phenylthio.

“Thioacyl,” as used herein, refers to a thiocarbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl or heteroaryl group.

“Thioacylamino,” as used herein, refers to an amino radical bonded to a thioacyl group.

“Thioacylester,” as used herein, refers to a thiocarbonyl radical attached to an alkoxy group.

“Thioureido,” as used herein, refers to a nitrogen radical of the formula

—N(R^x5)—C(S)—N(R^x6)(R^x7), wherein each of R^x5 and R^x6 is independently selected from hydrido alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and R^x7 is selected from an alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

“Ureido,” as used herein, refers to a nitrogen radical of the formula —N(R^x21)—C(O)—NR^x22R^x23, wherein each of R^x21 and R^x22 is independently selected from hydrido, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; and R^x23 is selected from an alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl.

The compounds of the present invention may be used in the form of salts or pharmaceutically-acceptable salts derived from inorganic or organic acids. The present invention includes all such salts and all crystalline forms of such salts. By “pharmaceutically-acceptable salt” is meant those salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, and allergic response, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically-acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically-acceptable salts in J. Pharm. Sci., 1977, 66:1-19. All of these salts may be prepared by conventional means from the corresponding compound of the invention by treating, for example, the compound with the appropriate acid or base.

The salts or pharmaceutically-acceptable salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reaction of a free base function with a suitable acid. For example, basic addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by combining a carboxylic acid-containing group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically-acceptable metal cation, or with ammonia or an organic primary, secondary, or tertiary amine.

Nonlimiting examples of organic acids may be selected from aliphatic, cycloaliphatic, aromatic, aryl, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid. Representative organic acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate addition salts. Examples of acids that may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid, and citric acid.

Claims

1. A method for preparing a compound, comprising: (a) treating a peptide having at least one sidechain comprising an amino group with an amino protecting group reagent to form a protected peptide,wherein the peptide comprises a cyclic heptapeptide attached to an exocyclic peptide chain comprising an acyl group, and the protecting group comprises at least one acidic substituent; and(b) treating the protected peptide with a deacylating agent to form a protected, deacylated peptide.

2. The method according to claim 1, wherein the peptide in (a) has a structure of formula (A):

3. The method according to claim 2, wherein R6 and R7 are each independently chosen from isopropyl, benzyl, isobutyl, sec-butyl, 1-hydroxy-1-ethyl, and hydroxymethyl and R10 is chosen from isopropyl, isobutyl, sec-butyl, 1-hydroxy-1-ethyl, and hydroxymethyl.

4. The method according to claim 2, wherein Y1, Y2, and Y3 are each independently selected from 2,4-diaminobutanoic acid residue, threonine residue, and serine residue.

5. The method according to claim 2, wherein T is chosen from 6-methyloctanoyl, 6-methylheptanoyl, octanoyl, heptanoyl, nonanoyl, and 3-hydroxy-6-methyloctanoyl.

6. The method according to claim 1, wherein the peptide in (a) is selected from polymyxin A, polymyxin B, [Ile7]-polymyxin B, polymyxin C, polymyxin D, colistin, polymyxin F, polymyxin M, polymyxin P, polymyxin S, polymyxin T, circulin A, octapeptin A, octapeptin B, octapeptin C, and octapeptin D.

7. The method according to claim 1, wherein the peptide in (a) is selected from polymyxin B, polymyxin A, polymyxin D, [Ile7]-polymyxin B, colistin, circulin A, octapeptin B, and octapeptin C.

8. The method according to claim 1, wherein the peptide in (a) is polymyxin B.

9. The method according to claim 1, wherein the peptide in (a) is selected from polymyxin A, polymyxin D, [Ile7]-polymyxin B, colistin, circulin A, octapeptin B, and octapeptin C.

10. The method according to claim 1, wherein the at least one acidic substituent is selected from carboxy, carboxylate, sulfo, sulfate, phosphate, phosphonate, and salts thereof.

11. The method according to claim 1, wherein the protecting group comprises an aryl or heteroaryl substituted with the at least one acidic substituent.

12. The method according to claim 11, wherein the at least one acidic substituent is selected from carboxy, carboxylate, sulfo, sulfate, phosphate, phosphonate and salts thereof.

13. The method according to claim 1, wherein the protecting group is a sulfonic acid of 9-fluorenylmethoxycarbonyl.

14. The method according to claim 13, wherein the protecting group is 2-sulfo-9-fluorenylmethoxycarbonyl.

15. The method according to claim 1, wherein each amino group of the peptide in (a) is protected with the protecting group.

16. The method according to claim 1, wherein the deacylating agent is an enzyme.

17. The method according to claim 16, wherein a source of the enzyme is Actinoplanes utahensis.

18. The method according to claim 1, further comprising: (c) forming from the protected deacylated peptide compounds having the following formulae:

19. The method according to claim 18, wherein the forming comprises treating the protected peptide with a reagent having the formula A-LG, wherein LG is a leaving group.

20. The method according to claim 18, wherein the forming comprises treating the protected peptide with a reagent selected from isocyanate, thioisocyanate, lactone, an activated heterocycle, an activated heteroaryl, imidate, ketenamine, an aldehyde and a reducing agent, and a ketone and a reducing agent.

21. The method according to claim 18, wherein the forming comprises treating the protected peptide with an acylating reagent selected from acyl halides, acyl cyanides, esters, lactones, and anhydrides.

22. The method according to claim 18, wherein the forming comprises treating the protected peptide with a sulfonating reagent selected from sulfonyl chloride and activated sulfonamides.

23. The method according to claim 18, wherein the forming comprises subjecting the protected deacylated peptide to an N-terminal amino acid hydrolysis reaction such that x is 0 and each of y and z is independently 1.

24. The method according to claim 23, wherein the forming comprises subjecting the protected deacylated peptide to a second N-terminal amino acid hydrolysis reaction such that each of x and y is independently 0 and z is 1.

25. The method according to claim 23, wherein the forming comprises subjecting the protected deacylated peptide to a third N-terminal amino acid hydrolysis reaction such that each of x, y and z is 0.

26. A method for preparing a compound, comprising: (a) treating a peptide having at least one sidechain comprising an amino group with an amino protecting group reagent to form a protected peptide,wherein the peptide comprises a cyclic heptapeptide attached to an exocyclic peptide chain comprising an acyl group, and the protected peptide is water soluble; and(b) treating the protected peptide with a deacylating agent to form a protected, deacylated peptide.

27. A compound having a structure chosen from formulae (I)-(VII):

28. The compound according to claim 27, wherein P is a 9-fluorenylmethoxycarbonyl group substituted with at least one acidic substituent.

29. The compound according to claim 28, wherein P is a 2-sulfo-9-fluorenylmethoxycarbonyl group.

30. The compound according to claim 27, wherein x and y are each independently 0, z is 1, and X3 is:

31. The compound according to claim 27, wherein x is 0, each of y and z is independently 1, and X2-X3 is:

32. The compound according to claim 27, wherein x, y, and z are each independently 1, and X1-X2-X3 is:

33. The compound according to claim 27, wherein each of x, y, and z is independently 0 and A is hydrogen.

34. The compound according to claim 27, wherein the compound is a prodrug.

35. A compound having a structure chosen from formulae (I)-(VII):

36. A compound of formula (1):

37. The compound of claim 36, wherein if X3 is 2,4-diaminobutanoic acid, x and y are independently selected from 0 and 1, and A is R′—(C═O)—, then R′ is selected from C9-20 unsubstituted alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heterocyclyl and substituted alkyl, wherein at least one hydrogen is replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkynyl, amino, substituted aryl, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, and ureido.

38. A compound of formula (2):

39. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—O—(C═O)— and R′ is phenyl.

40. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is n-C9-alkyl.

41. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is an alkyl substituted with a group selected from N—(C1-10-alkyl)-4-aminophenyl and a benzyloxy group.

42. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═O)— and R′ is selected from n-C8-alkyl, C2-alkyl substituted with a 3-indolyl group, cyclohexyl, unsubstituted phenyl, benzyl, 4′-biphenyl, and phenyl substituted with a group selected from 4-C10-alkyl, 4′-phenyloxy, and 4-chloro.

43. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═S)— and R′ is phenyl.

44. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is selected from phenyl, 4-pyridinyl, and alkyl substituted with a 2-naphthoxy group.

45. The compound according to any one of claims 30, 36 or 38, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—SO2— and R′ is 4-methylphenyl.

46. The compound according to any one of claims 30, 36 or 38, wherein x is 0, y and z are each 1, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═O)— and R′ is n-C8-alkyl.

47. The compound according to any one of claims 30, 36 or 38, wherein x is 0, each of y and z is independently 1, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═S)— and R′ is phenyl.

48. The compound according to any one of claims 30, 36 or 38, wherein each of x and y is independently 0, z is 1, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═O)— and R′ is n-C9-alkyl.

49. The compound according to any one of claims 30, 36 or 38, wherein X1 is glycine, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—SO2— and R′ is n-C10-alkyl.

50. The compound according to any one of claims 30, 36 or 38, wherein X1 is lysine, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is n-C9-alkyl.

51. The compound according to any one of claims 30, 36 or 38, wherein X1 is phenylalanine, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is n-C9-alkyl.

52. A compound of formula (3):

53. The compound according to claim 52, wherein if x is selected from 0 or 1, each of y and z are independently 1, X1 is an amino acid residue, X2 is threonine, X3 is 2,4-diaminobutanoic acid and A is R′—(C═O)—, then R′ is selected from unbranched unsubstituted C1-4 alkyl, cycloalkyl, and heterocyclyl.

54. The compound according to claim 52, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—NH—(C═O)— and R′ is n-C8-alkyl.

55. The compound according to claim 52, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—SO2— and R′ is 4-methylphenyl.

56. A compound of formula (4):

57. The compound according to claim 56, wherein A does not comprise at least one amino acid residue.

58. A compound of formula (5):

59. The compound according to claim 58, wherein A does not comprise at least one amino acid residue.

60. A compound of formula (6):

61. The compound according to claim 60, wherein A does not comprise at least one amino acid residue.

62. The compound according to claim 60, wherein if each of x and y is independently 0, z is 1, X3 is 2,4-diaminobutanoic acid, and A is R′—(C═O)—, then R′ is selected from substituted C1-7-alkyl, substituted C12-20-alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, and heterocyclyl.

63. A compound of formula (7):

64. The compound according to claim 63, wherein A does not comprise at least one amino acid residue.

65. A compound of formula (1):

66. The compound according to any one of claims 30 or 65, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is an Nα-(n-C9-alkanoyl)lysine.

67. A compound of formula (2): wherein: A is selected from R′—(C═O)—, R′—SO2—, R′—(C═NH)—, R′—NH—(C═S)—, R′—NH—(C═O)—, R′—NH—(C═NH)—, R′—O—(C═O)—, R′—O—(C═S)—, R′—P(O)OH—, R′—(C═S)—, R′-alkyl-, R′-, and hydrogen;R′ is selected from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, and heterocyclyl;X1, X2, and X3 are each independently selected from amino acid residues; andx, y, and z are integers independently selected from 0 and 1,with the proviso that if x, y and z each equals 1, X1 is 2,4-diaminobutanoic acid, X2 is threonine, X3 is 2,4-diaminobutanoic acid, and A is R′—(C═O)—, then R′ is not branched C8-alkyl.

68. A compound of formula (3):

69. The compound according to any one of claims 30, 65, 67 or 68, wherein X1 is 2,4-diaminobutanoic acid, X2 is threonine, and X3 is 2,4-diaminobutanoic acid, and wherein A is R′—(C═O)— and R′ is an Nα-(n-C9-alkanoyl)phenylalanine.

70. A method for treating an infection in a subject by administering a therapeutically-effective amount of a pharmaceutical composition comprising the compound of any one of claims 27 to 38, 52 to 65, 67 and 68, and a pharmaceutically acceptable carrier.

71. A method for preparing an intermediate for use in the synthesis of a new peptide antibiotic, including the steps of: (a) Protecting the amino groups of the polymyxin B, colistin, [Ile7]-polymyxin B1, circulin, and an octapeptin with (2-sulfo)-9-fluorenylmethoxy-carbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl;(b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate; and(c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate by reducing the number of amino acids in the side chain of the protected peptide intermediate; and(d) Purifying the protected peptide intermediate by chromatography.

72. A method for producing an antibiotic active against gram-negative and gram-positive bacteria, including strains resistant to clinically used antibiotics, comprising the steps of: (a) Protecting the amino groups of the polymyxins or other related antibiotics chosen from the group consisting of colistin, [Ile7]-polymyxin B1, circulin, and an octapeptin, with (2-sulfo)-9-fluorenylmethoxycarbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl;(b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate;(c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate peptide by reducing in size one to three amino acids in the exocyclic peptide side chain of the protected peptide.(d) Chemically modifying the intermediate to produce a protected antibacterial derivative; and(e) Removing the acidic protecting groups to produce the antibiotic.

73. An intermediate, which is a chemically protected form of a peptide derived from the polymyxins, [Ile7)]-polymyxin B1, octapeptins, colistin, circulins, or related antibiotics and selected from a group consisting of the following, or their corresponding salts: 1) H— (X1) (X2) (X3)-peptide-[(2-sulfo)-9-Fmoc]n 2) H— (X2) (X3)-peptide-[(2-sulfo)-9-Fmoc]n 3) H— (X3)-peptide-[(2-sulfo) 9-Fmoc]n 4) H-peptide-[(2-sulfo)-9-Fmoc]3 wherein for Case 1) H—(X1) (X2) (X3)-peptide-[(2-sulfo) Fmoc]n: H is hydrogen, X1 is L-Dab or another amino acid, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid and n=3-6;for Case 2) H— (X2) (X3)-peptide-[(sulfo)-9-Fmoc]n: H is hydrogen, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, an n=3-5;for Case 3) H— (X3)-[peptide-[(2-sulfo)-9-Fmoc]n: H is hydrogen, X3 is L-Dab or D-Dab or another amino acid and n=3-4; andfor Case 4) H-peptide-[(sulfo)-9-Fmoc]3: H is hydrogen.

74. Acidic protected peptide intermediates, derived from the corresponding protected polymyxin B, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably (2-sulfo)-9-Fmoc and the protected peptide intermediates have the structure:

75. Acidic protected peptide intermediates, derived from the corresponding protected [Ile7]-polymyxin B1, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably (2-sulfo)-9-Fmoc and the protected peptide intermediates have the structure:

76. Acidic protected peptide intermediates, derived from the corresponding protected colistin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably (2-sulfo)-9-Fmoc and the protected peptide intermediates have the structure:

77. Acidic protected peptide intermediates, derived from the corresponding protected circulin A, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably (2-sulfo)-9-Fmoc and the protected intermediates have the structure:

78. Acidic protected peptide intermediates, derived from the corresponding protected octapeptin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably (2-sulfo)-9-Fmoc and the protected peptide intermediates have the structure:

79. The protected peptide intermediate of claim 8, wherein another component of the octapeptin antibiotic includes L-phenylalanine instead of L-leucine at the 5-position and wherein the component forms a similar, but alternative, protected peptide intermediate.

80. An antibacterial compound or protected compound prepared from a chemically protected form of PBpeptides, Cpeptides or ILpeptides having the following structure where P equals the protective group (2-sulfo)-9-Fmoc or hydrogen:

81. An antibiotic prepared from an intermediate, which is a chemically protected form of a peptide derived from the polymyxins, octapeptins, colistin, circulin, [Ile7]polymyxin B1, said antibiotic selected from a group consisting of the following, or their corresponding salts:

82. Peptide antibiotics having the following structure and minimum inhibitory concentrations for use against gram-negative and gram-positive bacteria:

83. A process for preparing a water-soluble, stable, solid form of a deacylase enzyme including the steps of; (a) Fermenting a strain of Actinoplanes utahensis to obtain cells of the organism.(b) Washing the cells with water to remove impurities.(c) Extracting the washed cells with an aqueous base, pH 8-11.(d) Adjusting the extract to pH 7-8 and freeze drying the solution to obtain the solid form of said enzyme.

84. A process according to claim 83 wherein step (e) is: Thereafter further purifying said enzyme by use of chromatography.