WO2001005816A1

WO2001005816A1 - Amine-modified pseudomycin compounds

Info

Publication number: WO2001005816A1
Application number: PCT/US2000/015019
Authority: WO
Inventors: Shu Hui Chen; James Andrew Jamison; Michael John Rodriguez; Xicheng Sun; Venkatraghavan Vasudevan; Mark James Zweifel
Original assignee: Eli Lilly And Company
Priority date: 1999-07-15
Filing date: 2000-06-08
Publication date: 2001-01-25
Also published as: EA200200163A1; CN1361790A; BR0013168A; MXPA02000320A; NO20020194L; HUP0202237A3; NO20020194D0; EP1198472A1; AU5453100A; HUP0202237A2; JP2003505398A; CA2379317A1

Abstract

An amine-modified pseudomycin compound represented by structure (I), where R1 is an acyl linkage is described. The amine-modified pseudomycin derivatives are useful as antifungal agents or in the design of antifungal agents.

Description

AMINE-MODIFIED PSEUDOMYCIN COMPOUNDS

FIELD OF THE INVENTION The present invention relates to pseudomycin compounds, in particular, amine-modified, pseudomycin compounds .

BACKGROUND OF THE INVENTION

Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae (plant-associated bacterium) and have been shown to have antifungal activities. (see i.e., Harrison, L., et al . , "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity, " J. Gen. Microbiology, 137(12), 2857-65 (1991) and US Patent Nos. 5,576,298 and 5,837,685) Unlike the previously described antimycotics from P. syringae (e.g., syringomycins, syringotoxins and syringostatins) , pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid.

The peptide moiety for pseudomycins A, A', B, B' , C, C corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z- Dhb-L-Asp(3-OH) -L-Thr (4-C1) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N- terminal Ser . The analogs are distinguished by the N-acyl side chain, i.e., pseudomycin A is N-acylated by

3 , 4-dihydroxytetradeconoyl, pseudomycin A' by

3 , 4-dihydroxypentadecanoyl, pseudomycin B by

3-hydroxytetradecanoyl, pseudomycin B' by

3-hydroxydodecanoyl, pseudomycin C by

3 , 4-dihydroxyhexadecanoyl and pseudomycin C by

3-hydroxyhexadecanoyl . (see i.e., Ballio, A., et al . ,

"Novel bioactive lipodepsipeptides from Pseudomonas syringae : the pseudomycins," FEBS Letters, 355(1), 96-100,

(1994) and Coiro, V.M. , et al . , "Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from MR data, " Eur. J. Biochem. , 257(2), 449-456 (1998).)

Pseudomycins are known to have certain adverse biological effects. For example, destruction of the endothelium of the vein, destruction of tissue, inflammation, and local toxicity to host tissues have been observed when pseudomycin is administered intraveneously .

Since the pseudomycins have proven antifungal activity and relatively unexplored chemistry, there is a need to explore this class of compounds for other potential compounds that may be useful as antifungal agents having less adverse side affects .

BRIEF SUMMARY OF THE INVENTION The present invention provides amine-modified pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.

wherein R is

where

R^a and R^a are independently hydrogen or methyl, or either R^a or R^a' is alkyl amino, taken together with R^b or R^b' forms a six-membered cycloalkyl ring, a six- membered aromatic ring or a double bond, or taken together with R^c forms a six-membered aromatic ring;

R^b and R^b' are independently hydrogen, halogen, or

methyl, or either R^b or R^b' is amino, alkylamino, α- acetoacetate, methoxy, or hydroxy;

R^c is hydrogen, hydroxy, C₁-C4 alkoxy, hydroxy (Ci- C₄)alkoxy, or taken together with R^e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;

R^e is hydrogen, or taken together with R^f is a six-membered aromatic ring, C₅-C₁₄ alkoxy substituted six-membered aromatic ring, or C₅~Cι alkyl substituted six-membered aromatic ring, and

R^f is Cs-Cis alkyl, C₅-C₁₁ alkoxy, or biphenyl;

where

R^g is hydrogen, or C1-C₁₃ alkyl, and R^h is C₁-C₁₅ alkyl, C₄-Cι₅ alkoxy, (Cι-Cι_C alkyl ) phenyl , - (CH₂)_n-aryl, or - (CH₂)_n- (C₅-C₅ cycloalkyl), where n = 1 or 2 ; or

R is

where

R¹ is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3 ;

R is

where

R³ is C₅-C₁4 alkoxy or C₅-Cι alkyl, and p = 0, 1 or R is

where

R^k is C5-C14 alkoxy; or R is - (CH₂) -NR- (C13-C18 alkyl), where R^m is H, -CH₃ or

-C(0)CH₃; R¹ is independently hydrogen, formyl, an acylalkyl (e.g., - C(0)CH₃, -C(0)CH₂CH₃, -C(0)CH(CH₃)₂, and -C (O) C (CH₃) ₃ ) , an acylalkylamine (e.g., -C (0) CH (NH₂) CH₃) , an acylazaalkyl (e.g., -C(0)NHCH₃ and -C (O)NHCH (CH₃) ₂) , an acyloxyalkene (e.g., -C(0)OCH₂CH=CH₂) , an acyloxyaryl (e.g., -C(0)OC₆H₅), or an acylmethylenecarbamate (e.g., compounds 1 (a) depicted below)

K a) is C1-C10 alkyl , C1-C10 alkenyl , benzyl , or aryl and R il is hydrogen or methyl, provided that at least one R¹ is not hydrogen;

R' and R-³ are independently -OR 2a , or -N (R ,2^b^D v) (R ,2^c^c), , where

R^2a and R^2b are independently hydrogen, Ci-Cio alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i- butyl, s-butyl, t-butyl, etc.), C₃_C₆ cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethylene, methylcyclopentyl , cyclohexyl, etc.) hydroxy (Ci-Cio) alkyl, alkoxy (Ci-Cio) alkyl (e.g, methoxyethyl ) , or C₂-Cιo alkenyl, amino (Ci-Cio) alkyl, mono- or di-alkylamino (Ci-Cio) alkyl, aryl (Ci-Cio) alkyl (e.g., benzyl), heteroaryl (Cι-C₁₀) alkyl (e.g., 3- pyridylmethyl, 4-pyridylmethyl) , or cycloheteroalkyl (Ci-Cio) lkyl (e.g., N-tetrahydro-1, 4- oxazinylethyl and N-piperazinylethyl) , or

R^2b is an alkyl carboxylate residue of an aminoacid alkyl ester (e.g., -CHC0₂CH₃, -CH(C0₂CH₃)CH(CH₃)₂, -CH (C0₂CH₃) CH (phenyl) , -CH (C0₂CH₃ ) CH₂OH, -CH (C0₂CH₃ ) CH₂ (p-hydroxyphenyl ) , -CH(C0₂CH₃)CH₂SH, -CH (C0₂CH₃) CH₂ (CH₂) ₃NH₂, -CH(C0₂CH₃)CH₂(4- or 5-imidazole) , -CH (C0₂CH₃) CH₂C0₂CH₃, -CH(C0₂CH₃)CH₂C0₂NH₂, and the like), and

R^2c is hydrogen or Ci-Cε alkyl; and pharmaceutically acceptable salts and solvates thereof.

In another embodiment of the present invention, a pharmaceutical formulation is provided which includes the pseudomycin compound described above and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, a method is provided for treating an antifungal infection in an animal in need thereof, which comprises administering to the animal the pseudomycin compound described above.

Definitions

As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula C_nH_n+ι containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e.g. methyl, ethyl, propyl, butyl, etc.), branched (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e.g., bicyclo [2.2. l]heptane, spiro [2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group, alkanoyl, or alkanoate have the same definition as above.

The term "alkenyl" refers to an acyclic hydrocarbon containing at least one carbon carbon double bond. The alkene radical may be straight, branched, cyclic, or multi- cyclic. The alkene radical may be substituted or unsubstituted. The alkenyl portion of an alkenoxy, alkenoyl or alkenoate group has the same definition as above .

The term "aryl" refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., naphthalene, anthracene, phenanthrene , etc.). The aryl groups may be substituted or unsubstituted.

Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substitutents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term "group" specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament.

Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.

The term "animal" refers to humans, companion animals (e.g., dogs, cats and horses), food-source animals (e.g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.

DETAILED DESCRIPTION OF THE INVENTION Applicants have discovered that modification of the pendant amino groups attached to the lysine or 2,4- diaminobutyric acid peptide units in the pseudomycin natural product or semi-synthetic derivative provides compounds having in vi tro indications which suggest that the new compounds may be active against C. albican, C, neoformans, and/or A. fu igatus . The amino groups are modified using an acylating agent containing a suitable leaving group such that an amide, carbamate, urea or imide linkage with the pendant amino group on the pseudomycin structure can be formed. Suitable leaving groups are well known to those skilled in the art and include groups such as p-nitrophenoxy and N-oxysuccinimide. Amide linkages are synthesized using conventional chemistry well-known to those skilled in the art. Suitable acylating agents include derivatives of the desired carboxylic acid to produce a pseudomycin compound where R¹ = acylalkyl or amino acid to produce a pseudomycin compound where R¹ - acylalkyla ine . Typically, the acylating agent is formed by replacing the -OH of the carboxylic acid group with a leaving group (e.g., N-oxysuccinimde) . When an amino acid acylating agent is used, the amino group is protected prior to condensation using any conventional amino-protecting group known to those skilled in the art (e.g., benzyloxycarbonyl , p-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl , p-methoxybenxyloxycarbonyl , p- methoxyphenylazobenzyloxycarbonyl , p- phenylazobenzyloxycarbonyl, t-butyloxycarbonyl or cyclopentyloxycarbonyl) . After the amide linkage is formed, then the amino-protecting group is removed using standard hydrogenation chemistry (e.g., Pd/C under a hydrogen atmosphere) . See the Examples below for a more detailed description for forming pseudomycin amide derivatives from amino acids.

As discussed earlier, pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinonapetpides containing a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4- chlorothreonine (ClThr) , 3-hydroxyaspartic acid (HOAsp) ,

2, 3-dehydro-2-aminobutyric acid (Dhb) , and 2,4- diaminobutyric acid (Dab) . Methods for growth of various strains of P. syringae to produce the different pseudomycin analogs (A, A', B, B' , C, and C) are described in PCT

Patent Application Serial No. PCT/US00/08728 filed by

Hilton, et al . on April 14, 2000 entitled "Pseudomycin

Production by Pseudomonas Syringae, " incorporated herein by reference, PCT Patent Application Serial No. PCT/US00/08727 filed by Kulanthaivel, et al . on April 14, 2000 entitled

"Pseudomycin Natural Products, " incorporated herein by reference, and U.S. Patent Nos . 5,576,298 and 5,837,685, each of which are incorporated herein by reference.

Isolated strains of P. syringae that produce one or more pseudomycins are known in the art. Wild type strain

MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H are described in U.S. Patent Nos.

5,576,298 and 5,837,685; Harrison, et al . , "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity, " J . Gen .

Microbiology, 137, 2857-2865 (1991); and Lamb et al . ,

"Transposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease, " Proc . Natl . Acad. Sci . USA, 84, 6447-6451 (1987) .

A strain of P. syringae that is suitable for production of one or more pseudomycins can be isolated from environmental sources including plants (e.g., barley plants, citrus plants, and lilac plants) as well as, sources such as soil, water, air, and dust. A preferred stain is isolated from plants. Strains of P. syringae that are isolated from environmental sources can be referred to as wild type. As used herein, "wild type" refers to a dominant genotype which naturally occurs in the normal population of P. syringae (e.g., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation) . Like most organisms, the characteristics of the pseudomycin- producing cultures employed ( P. syringae strains such as MSU 174, MSU 16H, MSU 206, 25-Bl, 7H9-1) are subject to variation. Hence, progeny of these strains (e.g., recombinants, mutants and variants) may be obtained by methods known in the art.

P. syringae MSU 16H is publicly available from the

American Type Culture Collection, Parklawn Drive,

Rockville, MD, USA as Accession No. ATCC 67028. P. syringae strains 25-Bl, 7H9-1, and 67 Hi were deposited with the American Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.:

25-Bl Accession No. PTA-1622

7H9-1 Accession No. PTA-1623

67 HI Accession No. PTA-1621

Mutant strains of P. syringae are also suitable for production of one or more pseudomycins. As used herein, "mutant" refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, such as radiation (e.g., ultraviolet radiation or x-rays), chemical mutagens (e.g., ethyl methanesulfonate (EMS) , diepoxyoctane, N-methyl-N- nitro-N' -nitrosoguanine (NTG) , and nitrous acid), site- specific mutagenesis, and transposon mediated mutagenesis. Pseudomycin-producing mutants of P. syringae can be produced by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce one or more pseudomycins, that produce one pseudomycin (e.g., pseudomycin B) in excess over other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 μg/ml. Preferred mutants are those that overproduce

pseudomycin B and grow in minimal defined media.

Environmental isolates, mutant strains, and other desirable strains of P. syringae can be subjected to selection for desirable traits of growth habit, growth medium nutrient source, carbon source, growth conditions, amino acid requirements, and the like. Preferably, a pseudomycin producing strain of P. syringae is selected for growth on minimal defined medium such as N21 medium and/or for production of one or more pseudomycins at levels

greater than about 10 μg/ml. Preferred strains exhibit the

characteristic of producing one or more pseudomycins when grown on a medium including three or fewer amino acids and optionally, either a lipid, a potato product or combination thereof .

Recombinant strains can be developed by transforming the P. syringae strains, using procedures known in the art. Through the use of recombinant DNA technology, the P. syringae strains can be transformed to express a variety of gene products in addition to the antibiotics these strains produce. For example, one can modify the strains to introduce multiple copies of the endogenous pseudomycin- biosynthesis genes to achieve greater pseudomycin yield. To produce one or more pseudomycins from a wild type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or a combination thereof. Alternatively, glycine is combined with one or more of a potato product and a lipid. Culturing is conducted under conditions effective for growth of P. syringae and production of the desired pseudomycin or pseudomycins. Effective conditions include temperatures from about 22 ^aC to about 27 ^SC, and a duration of about 36 hours to about 96 hours. Controlling the concentration of oxygen in the medium during culturing of P. syringae is advantageous for production of a pseudomycin. Preferably, oxygen levels are maintained at about 5 to 50% saturation, more preferably about 30% saturation. Sparging with air, pure oxygen, or gas mixtures including oxygen can regulate the concentration of oxygen in the medium.

Controlling the pH of the medium during culturing of

P. syringae is also advantageous. Pseudomycins are labile at basic pH, and significant degradation can occur if the pH of the culture medium is above about 6 for more than about 12 hours. Preferably, the pH of the culture medium is maintained between 6 and 4. P. syringae can produce one or more pseudomycins when grown in batch culture. However, fed-bath or semi-continuous feed of glucose and optionally, an acid or base (e.g., ammonium hydroxide) to control pH, enhances production. Pseudomycin production can be further enhanced by using continuous culture methods in which glucose and ammonium hydroxide are fed automatically.

Choice of P. syringae strain can affect the amount and distribution of pseudomycin or pseudomycins produced. For example, strains MSU 16H and 67 Hi each produce predominantly pseudomycin A, but also produce pseudomycin B and C, typically in ratios of 4:2:1. Strain 67 HI typically produces levels of pseudomycins about three to five fold larger than are produced by strain MSU 16H. Compared to strains MSU 16H and 67 Hi, strain 25-Bl produces more pseudomycin B and less pseudomycin C. Strain 7H9-1 are distinctive in producing predominantly pseudomycin B and larger amount of pseudomycin B than other strains. For example, this strain can produce pseudomycin B in at least a ten fold excess over either pseudomycin A or C.

Alternatively, the amine-modified pseudomycin compounds of the present invention can be formed from an N- acyl semi-synthetic compound. Semi-synthetic pseudomycin compounds may be synthesized by exchanging the N-acyl group on the L-serine unit. Examples of various N-acyl derivatives are described in PCT Patent Application Serial

No. , Belvo, et al . , filed evendate herewith entitled "Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference. In general, four synthetic steps are used to produce the semi-synthetic compounds from naturally occurring pseudomycin compounds: (1) selective amino protection; (2) chemical or enzymatic deacylation of the N-acyl side-chain; (3) reacylation with a different side-chain; and (4) deprotection of the amino groups .

The pendant amino groups at positions 2, 4 and 5 may be protected using any standard means known to those skilled in the art for amino protection. The exact genus and species of amino protecting group employed is not critical so long as the derivatized amino group is stable to the condition of subsequent reaction (s) on other positions of the intermediate molecule and the protecting group can be selectively removed at the appropriate point without disrupting the remainder of the molecule including any other amino protecting grou (s) . Suitable amino- protecting groups include benzyloxycarbonyl, p- nitrobenzyloxycarbonyl , p-bromobenzyloxycarbonyl , p-methoxybenxyloxycarbonyl , p-methoxyphenylazobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl , t-butyloxycarbonyl , cyclopentyloxycarbonyl, and phthalimido. Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl (Alloc) , phthalimido, and benzyloxycarbonyl (CbZ or CBZ) . Further examples of suitable protecting groups are described in T.W. Greene, "Protective Groups in Organic Synthesis," John Wiley and Sons, New York, N.Y., (2nd ed. , 1991), at chapter 7.

The deacylation of an N-acyl group having a gamma or delta hydroxylated side chain (e.g., 3 , 4-dihydroxytetra- deconoate) may be accomplished by treating the amino- protected pseudomycin compound with acid in an aqueous solvent. Suitable acids include acetic acid and trifluoroacetic acid. A preferred acid is trifluoroacetic acid. If trifluoroacetic acid is used, the reaction may be accomplished at or near room temperature. However, when acetic acid is used the reaction is generally ran at about

40°C. Suitable aqueous solvent systems include

acetonitrile, water, and mixtures thereof. Organic solvents accelerate the reaction; however, the addition of an organic solvent may lead to other by-products.

Pseudomycin compounds lacking a delta or gamma hydroxy group on the side chain (e.g., Pseudomycin B and C) may be deacylated enzymatically . Suitable deacylase enzymes include Polymyxin Acylase (164-16081 Fatty Acylase (crude) or 161-16091 Fatty Acylase (pure) available from Wako Pure Chemical Industries, Ltd.), or ECB deacylase. The enzymatic deacylation may be accomplished using standard deacylation procedures well known to those skilled in the art. For example, general procedures for using polymyxin acylase may be found in Yasuda, N. , et al, Agric Biol . Chem. , 53, 3245 (1989) and Kimura, Y. , et al . , Agric . Biol . Chem. , 53, 497 (1989) .

The deacylated product (also known as the pseudomycin nucleus) is reacylated using the corresponding acid of the desired acyl group in the presence of a carbonyl activating agent. "Carbonyl activating group" refers to a substituent of a carbonyl that promotes nucleophilic addition reactions at that carbonyl. Suitable activating substituents are those which have a net electron withdrawing effect on the carbonyl. Such groups include, but are not limited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles, or amino groups (e.g., oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N,N'- dicyclohexylisoure-O-yl, and N-hydroxy-N-methoxyamino) ; acetates; formates; sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, and p-tolylsulfonate) ; and halides (e.g., chloride, bromide, and iodide).

A variety of acids may be used in the acylation process. Suitable acids include aliphatic acids containing one or more pendant aryl, alkyl, amino (including primary, secondary and tertiary amines) , hydroxy, alkoxy, and amido groups; aliphatic acids containing nitrogen or oxygen within the aliphatic chain; aromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups.

Alternatively, a solid phase synthesis may be used where a hydroxybenzotriazole-resin (HOBt-resin) serves as the coupling agent for the acylation reaction.

Once the amino group is deacylated and reacylated (described above) , then the amino protecting groups (at positions 2, 4 and 5) can be removed by hydrogenation in the presence of a hydrogenation catalyst (e.g., 10% Pd/C) . When the amino protecting group is allyloxycarbonyl, then the protecting group can be removed using tributyltinhydride and triphenylphosphine palladium dichloride. This particular protection/deprotection scheme has the advantage of reducing the potential for hydrogenating the vinyl group of the Z-Dhb unit of the pseudomycin structure.

The amine-modification of the N-acyl semi-synthetic compound is then accomplished by acylating at least one of the pendant amino groups attached to the lysine or 2,4- diaminobutyric acid peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide, urea, carbamate or imide linkage.

The amine-modified pseudomycin compounds may be further modified by amidation or esterification of the pendant carboxylic acid group of the aspartic acid and/or hydroxyaspartic acid units of the pseudomycin ring. Examples of various acid-modified derivatives are described in PCT Patent Application Serial No. , Chen, et al . , filed evendate herewith entitled "Pseudomycin Amide & Ester Analogs" and incorporated herein by reference. The acid- modified derivatives may be formed by condensing any of the previously described amine-modified pseudomycin compounds with the appropriate alcohol or amine to produce the respective ester or amide.

Formation of the ester groups may be accomplished using standard esterification procedures well-known to those skilled in the art. Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of a protic acid (e.g., HCI, TFA, etc.) . Under basic conditions, the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (e.g., sodium bicarbonate and potassium carbonate) .

Formation of the amide groups may be accomplished using standard amidation procedures well-known to those skilled in the art. However, the choice of coupling agents provides selective modification of the acid groups. For example, the use of benzotriazol-1-yloxy- tripyrrolidinophosphonium hexafluorophosphate (PyBOP) as the coupling agent allows one to isolate pure mono-amides at residue 8 and (in some cases) pure bis amides simultaneously. Whereas, the use of o-benzotriazol-1-yl- N,N,N' ,N' -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent favors formation of monoamides at residue 3.

The pseudomycin amide derivatives may be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate. The term "pharmaceutically acceptable salt" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphonates, monohydrogen-phosphates, dihydrogenphosphates , metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanoates, arylalkonates, adipates, alginates, aspartates, benzoates, fumarates, glucoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, camphorates, camphorsulfonates, digluconates, trifluoroacetates, and the like.

The term "solvate" refers to an aggregate that comprises one or more molecules of the solute (i.e., amine- modified pseudomycin compound) with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like. When the solvent is water, then the aggregate is referred to as a hydrate. Solvates are generally formed by dissolving the pseudomycin derivative in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.

Each pseudomycin compound, semi-synthetic pseudomycin derivatives, and mixtures can be detected, determined, isolated, and/or purified by any variety of methods known to those skilled in the art. For example, the level of pseudomycin or amine-modified pseudomycin activity in a broth or in an isolate or purified composition can be determined by antifungal action against a fungus such as Candida and can be isolated and purified by high performance liquid chromatography .

The active ingredient (i.e., pseudomycin compound of the present invention) is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient, physician, or veterinarian an elegant and easy to handle product. Formulations may comprise from 0.1% to 99.9% by weight of active ingredient, more generally from about 10% to about 30% by weight.

As used herein, the term "unit dose" or "unit dosage" refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. When a unit dose is administered orally or parenterally, it is typically provided in the form of a tablet, capsule, pill, powder packet, topical composition, suppository, wafer, measured units in ampoules or in multidose containers, etc. Alternatively, a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed.

The dosage to be administered may vary depending upon the physical characteristics of the animal, the severity of the animal's symptoms, the means used to administer the drug and the animal species. The specific dose for a given animal is usually set by the judgment of the attending physician or veterinarian.

Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied. The formulations may also include wetting agents, lubricating agents, surfactants, buffers, tonicity agents, bulking agents, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, perfuming agents, flavoring agents and combinations thereof.

A pharmaceutical composition may be administered using a variety of methods. Suitable methods include topical (e.g., ointments or sprays), oral, injection and inhalation. The particular treatment method used will depend upon the type of infection being addressed.

In parenteral iv applications, the formulations are typically diluted or reconstituted (if freeze-dried) and further diluted if necessary, prior to administration. An example of reconstitution instructions for the freeze-dried product are to add ten ml of water for injection (WFI) to the vial and gently agitate to dissolve. Typical reconstitution times are less than one minute. The resulting solution is then further diluted in an infusion solution such as dextrose 5% in water (D5W) , prior to administration .

Pseudomycin compounds have been shown to exhibit antifungal activity such as growth inhibition of various infectious fungi including Candida spp. (i.e., C. albicans, C. parapsilosis, C. krusei , C. glabrata, C. tropicalis, or C. lusi taniaw) ; Torulopus spp. (i.e., T. glabrata) ; Aspergillus spp. (i.e., A . fumigatus) ; Histoplasma spp. (i.e., H. capsulatum) ; Cryptococcus spp. (i.e., C. neoformans) ; Blastomyces spp. (i.e., B. dermati tidis) ; Fusarium spp.; Trichophyton spp., Pseudallescheria boydii , Coccidioides immi ts, Sporothrix schenckii , etc.

Consequently, the compounds and formulations of the present invention are useful in the preparation of medicaments for use in combating either systemic fungal infections or fungal skin infections. Accordingly, a method is provided for inhibiting fungal activity comprising contacting the amine-modified pseudomycin compound of the present invention with a fungus. A preferred method includes inhibiting Candida albicans or

Aspergillus fumigatus activity. The term "contacting" includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a fungus. The term does not imply any further limitations to the process, such as by mechanism of inhibition. The methods are defined to encompass the inhibition of fungal activity by the action of the compounds and their inherent antifungal properties.

A method for treating a fungal infection which comprises administering an effective amount of a pharmaceutical formulation of the present invention to a host in need of such treatment is also provided. A preferred method includes treating a Candida albicans or

Aspergillus fumigatus infection. The term "effective amount" refers to an amount of active compound which is capable of inhibiting fungal activity. The dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host, the tolerance of the host to the antifungal agent and the species of the host. The particular dose regimen likewise may vary according to these factors. The medicament may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer. A typical daily dose (administered in single or divided doses) contains a dosage level between about 0.01 mg/kg to 100 mg/kg of body weight of an active compound. Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg. The host is generally an animal including humans, companion animals (e.g., dogs, cats and horses), food-source animals (e.g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species .

EXAMPLES

The following abbreviations are used through out the examples to represent the respective listed materials:

ACN - acetonitrile

TFA - trifluoroacetic acid

DMF - dimethylformamide

EDCI - 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride

BOC = t-butoxycarbonyl, (CH₃) ₃C-0-C (0) -

CBZ = benzyloxycarbonyl , C₆H₅CH₂-0-C (0) -

PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate

TBTU = o-Benzotriazol-l-yl-N,N,N' ,N'- tetramethyluronium tetrafluoroborate DIEA - N,N-diisopropylethylamine

The following structure II will be used to describe the products observed in Examples 1 through 7.

II

Detection and Quantification of Antifungal Activity: Antifungal activity was determined in vi tro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a discdiffusion test. A typical fungus employed in testing antifungal activity is Candida albicans . Antifungal activity is considered significant when the test sample (50 μl) causes 10-12 mm diameter zones of inhibition on C.

albicans x657 seeded agar plates. Tail Vein Toxici ty:

Mice were treated intravenously (IV) through the lateral tail vein with 0.1 ml of testing compound (20 mg/kg) at 0, 24, 48 and 72 hours. Two mice were included in each group. Compounds were formulated in 5.0% dextrose and sterile water for injection. The mice were monitored for 7 days following the first treatment and observed closely for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss and any other signs of adverse effects indicating toxicity.

The mice used in the study were outbred, male ICR mice having an average weight between 18-20 g (available from Harlan Sprangue Dawley, Indianapolis, IN).

Prepare, t i ons

Compound la-1 :

la-1 Compound la-1 is commercially available from Novabiochem

(San Diego, CA) .

Preparation of Compound 2a-l :

2a-l

Compound 2a-l is prepared using the procedures described in Ad iak, R.W., et al . , Tetrahedron Lett . , No. 22, 1935-1936 (1997) .

In each of the following Examples a specific pseudomycin compound is used as the starting material; however, those skilled in art in the art will recognize that other N-acyl derivatives may be synthesized using the same procedures except starting with a pseudomycin compound having a different N-acyl group.

Example 1

Example 1 illustrates the formation of acylalkylamine derivatives of pseudomycin B (n = 10, R² and R³ = -OH) . Synthesis of Compound 1 -1 :

R¹', R¹" and R¹''' = -C(0)CH₂NH₂ 1-1 A 50 ml round bottom flask was charged with 10 ml of anhydrous DMF, pseudomycin B (250.6 mg, 0.181 mmol) and the acylating agent la-1 (343.0 mg, 1.12 mmol) . The reaction was allowed to stir at room temperature for 24 hours. The solvent was then removed in vacuo and the residue was taken up in ACN and purified via preparatory HPLC to yield 172.5 mg of the tri-substituted protected amine after lyophilization. 134.4 mg of tri-substituted protected amine was dissolved in a solution of 10 ml MeOH/1.5 ml glacial AcOH. Standard hydrogenolysis using 129.6 mg of 10% Pd/C for 20 minutes, removal of the catalyst via filtration and purification via preparatory HPLC yielded 74.8 mg of Compound 1-1 after lyophilization. MS (Ionspray) calcd for C₅₇H₉₇ClNι₅0₂₂ (M+H)⁺ 1378.65, found 1378.9.

The following pseudomycin amide derivatives (1-2 and 1-3 ) may be synthesized using the same procedures as described above and the appropriate amino acid to form the acylating agent .

1-2

1-3 Example 2

Example 2 illustrates the synthesis of acyloxyaryl derivatives of pseudomycin B(n = 10, R² and R³ = -OH) . Synthesis of Compound 2-1 :

2-1

Compound 2-1 is synthesized using the same procedures as described in Example 1 except Compound 2a-l is used as the acylating agent .

Compound 2-1 may be alternatively synthesized by adding phenyl chloroformate (389 mg, 2.48 mmol) to a solution of HOBT (37.5 mg, 2.48 mmol) and DIEA (322.8 mg,

399 ml, 2.48 mmol) at 0-4°C. The mixture is diluted with 100 ml DMF and pseudomycin B (1.0 g, 0.83 mmol) is added. The mixture is allowed to stir overnight. The solvent is then removed in vacuo and the residue purified by HPLC to yield 430 mg (33% yield) of Compound 2-1. Example 3

Example 3 illustrates the synthesis of acylalkyl derivatives of pseudomycin B(n = 10, R² and R³ = -OH) . Synthesis of Compound 3-1 :

3-1

Compound 3-1 is synthesized using the same procedures as described in Example 1 except acetic anhydride is used as the acylating agent . Synthesis of Compound 3-2 :

O

R¹', R¹", and R¹' ^■ C(CH₃)₃

3-2

Compound 3-2 is synthesized using the same procedures as described in Example 1 except trimethylacetic anydride is used as the acylating agent.

Example 4

Example 4 illustrates the synthesis of acylazaalkyl (i.e., urea linkage) derivatives of pseudomycin B(n = 10, R² and R³ = -OH) . Synthesis of Compound 4-1 :

4-1

Compound 4-1 is synthesized using the same procedures as described in Example 1 except methyl isocyanate is used as the acylating agent .

Example 5

Example 5 illustrates the synthesis of a formyl derivative of pseudomycin B(n = 10, R² and R³ = -OH) .

5-1

Compound 5-1 is synthesized using the same procedures as described in Example 1 except 4-nitrophenylformate is used as the acylating agent.

Example 6

Example 6 illustrates the synthesis of an acyloxyalkenyl derivative of pseudomycin B(n = 10, R² and R³ = -OH) .

6-1

Compound 6-1 is synthesized using the same procedures as described in Example 1 except diallylpyrocarbonate is used as the acylating agent. Yield 77%

Claims

WE CLAIM :

1. An amine-modified pseudomycin compound having the following structure:

wherein

R is

where

R^a and R^a' are independently hydrogen or methyl, or either R^a or R^a' is alkyl amino, taken together with R^b or R^b' forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with R^c forms a six-membered aromatic ring;

R^b and R^b' are independently hydrogen, halogen, or methyl, or either R^b or R^b' is amino,

alkylamino, α-acetoacetate, methoxy, or hydroxy;

R^c is hydrogen, hydroxy, Cχ-C alkoxy, hydroxy(Cχ-C ) alkoxy, or taken together with R^e forms a 6-membered aromatic ring or C₅-C₆ cycloalkyl ring;

R^e is hydrogen, or taken together with R^f is a six-membered aromatic ring, C5-C₁₄ alkoxy substituted six-membered aromatic ring, or C5-C₁₄ alkyl substituted six-membered aromatic ring, and

R^f is C₈-Ci8 alkyl, or C5-C11 alkoxy;

R is

where

R⁹ is hydrogen, or C₁-C₁₃ alkyl , and R^h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl ) phenyl , - (CH₂)_n-aryl, or - (CH₂)_n- (C₅-C₆ cycloalkyl), where n = 1 or 2 ; or

R is

where

R¹ is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3 ;

where

R^D is C₅-C14 alkoxy or C₅-C₁₄ alkyl, and p = 0, 1 or 2;

where

R^k is C₅-C14 alkoxy; or

R is -(CH₂)-NR^m-(Ci3-Ci8 alkyl), where R is H, -CH₃ or -C(0)CH₃; R¹ is independently hydrogen, formyl, an acylalkyl, an acylalkylamine, an acylazaalkyl, an acyloxyalkene, an acyloxyaryl, or an acylmethylenecarbamate, provided that at least one R¹ is not hydrogen;

R² and R³ are independently -0R^2a or -N(R^2b) (R^2c) , where

R^2a and R^2b are independently hydrogen, Ci-Cio alkyl, C₃-C6 cycloalkyl, hydroxy (C1-C1₀) alkyl, alkoxyalkyl, or C₂-Cι₀ alkenyl, amino (C1-C1₀) alkyl, mono- or di-alkylamino (C1-C10) alkyl, aryl(Cι- C10) alkyl , heteroaryl (C1-C10) alkyl , cycloheteroalkyl (C1-C10) alkyl, or

R^2b is an alkyl carboxylate residue of an aminoacid alkyl ester and R^2c is hydrogen or Ci-Cε alkyl ; and pharmaceutically acceptable salts and solvates thereof.

2. The amine-modified pseudomycin compound of Claim 1 wherein said acylmethylenecarbamate is represented by the structure 1(a)

Ka) where R is Ci-Cio alkyl, Ci-Cio alkenyl, benzyl, or aryl and

R ,1b is hydrogen or methyl,

3. The amine-modified pseudomycin compound of Claim 1 wherein said alkyl carboxylate residue of an aminoacid alkyl ester is represented by -CH₂C0₂CH₃, -CH (C0₂CH₃) CH (CH₃) ₂, -CH (C0₂CH₃ ) CH (phenyl ) , -CH (C0₂CH₃ ) CH₂OH, -CH (C0₂CH₃ ) CH₂ (p- hydroxyphenyl ) , -CH (C0₂CH₃) CH₂SH, -CH (C0₂CH₃) CH₂ (CH₂) ₃NH₂, -CH(C0₂CH₃)CH₂(4-imidazole) , -CH (C0₂CH₃) CH₂ (5-imidazole) , -CH(C0₂CH₃)CH₂C0₂CH₃, or -CH (C0₂CH₃) CH₂C0₂NH₂.

4. The use of a compound as claimed in any one of the preceding claims in the preparation of a medicament for use in combating either systemic fungal infections or fungal skin infections.

5. A pharmaceutical formulation comprising an amine- modified pseudomycin compound of Claim 1 and a pharmaceutically acceptable carrier.

6. A method for treating an antifungal infection in an animal in need thereof, comprising administering to said animal an amine-modified pseudomycin compound of Claim 1.