PSEUDOMYCIN AMIDE AND ESTER ANALOGS

Abstract: Acid-modification of the aspartic acid and/or hydroxyaspartic acid units of naturally occurring or semi-synthetic pseudomycin compounds is described as well as methods of treatment against fungal activities.

Description: PSEUDOMYCIN AMIDE & ESTER ANALOGS FIELD OF THE INVENTION The present invention relates to pseudomycin compounds, in particular, acid-modified, semi-synthetic 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, AX 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 NMR 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 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, cc- acetoacetate, methoxy, or hydroxy; R c is hydrogen, hydroxy, C 1 -C4 alkoxy, hydroxy (Ci- C 4 ) 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-C 1 4 alkoxy substituted six-membered aromatic ring, or C5-C 14 alkyl substituted six-membered aromatic ring, and R f is C 6 -Ci8 alkyl, C5-C 11 alkoxy, or biphenyl; R is where R g is hydrogen, or Cι-Cι 3 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl)phenyl , - (CH 2 ) n -aryl, or - (CH 2 ) n - (C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or R is where R 1 is a hydrogen, halogen, or C 5 -C 8 alkoxy, and m is 1, 2 or 3 ; R is where R? is C5-C14 alkoxy or C5-C14 alkyl , and p = 0 , 1 or 2; R is where R k is C 5 -C 14 alkoxy; or R is -(CH 2 )-NR rn -(Cι 3 -Cι 8 alkyl), where R m is H, -CH 3 or -C(0)CH 3 ; R 1 is independently -NH 2 or -NHp-Pg, where p is 0 or 1; R and R J are independently -OR 2a, or -N(R^ D ) (R^ c ) , where R 2a and R 2b are independently hydrogen, C 1 -C10 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, π-butyl, i- butyl, s-butyl, t-butyl, n-amyl, i-amyl, n-hexyl, n- heptyl, n-octyl, n-nonanyl, n-decyl, etc.), C 3 _C 6 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethyl, methylcyclopentyl, cyclohexyl, etc.) haloalkyl (e.g., CF 3 CH 2 -) , hydroxy (C 1 -C 10 ) alkyl, alkoxy (Ci-Cio) alkyl (e.g, methoxyethyl) , allyl, C-Cι 0 alkenyl, amino (Ci-Cio) alkyl, mono- or di-alkylamino (Ci- Cio) alkyl, aryl (Ci-Cio) alkyl (e.g., benzyl), heteroaryl (Ci-Cio) alkyl (e.g., 3-pyridylmethyl, 4- pyridylmethyl) , or cycloheteroalkyl (Ci-Cio) alkyl (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., -CHC0 2 CH 3 , -CH(C0 2 CH 3 )CH(CH 3 ) 2 , -CH (C0 2 CH 3 ) CH (phenyl) , -CH (C0 2 CH 3 ) CH 2 OH, -CH (C0 2 CH 3 ) CH 2 (p-hydroxyphenyl) , -CH (C0 2 CH 3 ) CH 2 SH, -CH (C0 2 CH 3 ) CH 2 (CH 2 ) 3 NH 2 , -CH(C0 2 CH 3 )CH 2 (4- or 5-imidazole) , -CH (C0 2 CH 3 ) CH 2 C0CH 3 , -CH(C0 2 CH 3 )CH 2 C0NH 2 , and the like), and R 2c is hydrogen or Cι-C 6 alkyl, provided that both R 2 and R 3 are not -OH; and pharmaceutically acceptable salts and solvates thereof. In another embodiment of the present invention, a prodrug of a pseudomycin compound is provided having structure I represented above wherein R 2 and R 3 are represented by -OR a , where R a is Cι-C 3 alkyl. In yet another embodiment of the present invention, a 3-amido derivative of a pseudomycin compound is provided where the compound is prepared by the steps of (i) providing a compound having structure I above wherein R 1 is -NH 2 and R 2 and R 3 are both -OH; (ii) protecting the amino groups, R 1 , at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 3 using an o- Benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate as a coupling agent; and (iv) removing the amino-protecting groups. An 8-amido derivative is also provided where the derivative is prepared using the steps described above except using benzotriazol-1-yloxy- tripyrrolidinophosphonium hexafluorophosphate as the coupling agent . In another embodiment of the present invention, a pharmaceutical formulation is provided which includes the pseudomycin compound represented by structure I 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 I described above . Defini tions As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula C n H 2n+ 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 [2.2.1] 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 "alkynyl" refers to an acyclic hydrocarbon containing at least one carbon carbon triple bond. The alkyne radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted. The alkynyl portion of an alkynoxy, alkynoyl or alkynoate 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. The term "heteroaryl" refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, oxazine, pyrimidine, purine, benzimidazole, guinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted. "NHp-Pg" and "amino protecting group" refer to a substituent of the amino group (Pg) commonly employed to block or protect the amino functionality while reacting other functional groups on the compound. When p is 0, the amino protecting group, when taken with the nitrogen to which it is attached, forms a cyclic imide, e.g., phthalimido and tetrachlorophthalimido . When p is 1, the protecting group, when taken with the nitrogen to which it is attached, can form a carbamate, e.g., methyl, ethyl, and 9-fluorenylmethylcarbamate; or an amide, e.g., N-formyl and N-acetylamide . 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 "solvate" refers to an aggregate that comprises one or more molecules of the solute, such as a compound of structure I, with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like. The term "pharmaceutically acceptable salt" refers to organic or inorganic salts of the compounds represented by structure I that are substantially non-toxic to the recipient at the doses administered. The term "prodrug" refers to a class of drugs which result in pharmacological action due to conversion by metabolic processes within the body (i.e., biotransformation) . In the present invention, the pseudomycin prodrug compounds contain ester functionalities that can be cleaved by esterases in the plasma to produce the active drug. 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 acid functionality attached to the hydroxyaspartic acid and/or aspartic acid units of a 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. fumigatus . Some bis-esters have been shown to act as a prodrug; therefore, these particular compounds have reduced in vi tro activity but show in vivo efficacy. Scheme I below illustrates the general procedures for synthesizing Compound I from any one of the naturally occurring pseudomycins or N-acyl modified derivatives. In general, three synthetic steps are used to produce Compound I: (1) selective amino protection; (2) condensation with the appropriate alcohol or amine to produce the respective ester or amide; and (4) deprotection of the amino groups. Scheme I The pendant amino groups at residues 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 conditions 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 group (s). Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl, phthalimido, and benzyloxycarbonyl (CBZ) . Most preferred is allyloxycarbonyl (Alloc) and benzyloxycarbonyl (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. 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., HCl, TFA, p-toluenesulfonic acid, 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 under anhydrous conditions) . 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, coupling agents such as o-benzotriazol-l-yl-N,N,NXN' -tetramethyluronium tetrafluoroborate (TBTU) and 2 (lH-benzotriazole-1-yl) - 1,1,3,3, -tetramethyluronium hexafluorophosphate (HBTU) favor formation of monoamides at residue 3. Applicants also discovered that the addition of a bulky amine enhances the ratio of monoamides at residue 3. The ratio of amidation at residue 3 vs . residue 8 increased from about 1:1 to about 6:1 and the amount of bis-amides was reduced through the addition of a bulky amine. The term "bulky amine" refers to an amine having multiple and/or large substituents on the nitrogen atom. Any tertiary amine may be used that is compatible with the reaction conditions. Preferred bulky amines include N,N-diisopropylethylamine (DIEA) and N-ethyldicyclohexylamine. The amount of bulky amine added is generally from about 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 5 to 6 equivalents. The reaction is generally ran at temperatures from about room temperature (25°C) to about -20°C. However, Applicants discovered that lower temperatures (from about 0°C to about -20°C) further enhance the formation of monoamides at residue 3. The ratio of amidation at residue 3 vs. residue 8 increased as much as 20:1 by adding a bulky amine and lowering the temperature of the reaction. However, it will be understood by those skilled in the art that the lower temperature limit will depend upon the solubility of the reactive components. Once the acid group (s) are modified, then the amino protecting groups (at positions 2, 4 and 5) may be removed using standard procedures appropriate for the specific protecting group used. For example, CBZ groups are 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 may 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. 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, AX B, B', C, and C) are described below and described in more detail 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/USOO/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 2 C to about 21 -0. , 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 . Each pseudomycin, pseudomycin intermediate 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 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. Alternatively, the amido or ester derivative 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 aspartic acid and/or hydroxyaspartic acid units can be modified prior to deprotecting the amino groups. 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. The acid-modification of the protected N-acyl semi- synthetic compound is then accomplished by reacting at least one of the pendant carboxyl groups attached to the aspartic or hydroxyaspartic peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide or ester linkage (s). The protecting groups are then removed as described earlier. The pseudomycin compound 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., 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 compound in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form. The active ingredient (i.e., pseudomycin compound) 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 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 an animal 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 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 may be any 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 Unless indicated otherwise, all chemicals can be acquired from Aldrich Chemical (Milwaukee, WI) . 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, (CH 3 ) 3 C-0-C (0) - CBZ = benzyloxycarbonyl , C 6 H 5 CH 2 -0-C (0) - PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate DIEA = N,N-diisopropylethylamine HPLC Conditions Unless indicated otherwise, analytical reverse-phase HPLC work was done using the Waters 600E systems equipped with Waters μBondapak (C18, 3.9 X 300 mm) column. The eluent used was 65:35 acetonitrile/0.1% aqueous TFA solvent system to 100% acetonitrile over 20 minutes with a flow rate of 1.5 ml/minute and using UV detection at 230 run. Preparative HPLC work was performed with a Waters Prep 2000 system using Dynamax 60 angstrom C18 column and identical solvent systems as used in the analytical HPLC system but with a flow rate of 40 ml/min. Biological Analysis Detection and Quantification of Antifungal Activi ty: 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 Toxic ity: 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) . General Procedures CBZ-Protected Pseudomycin : General procedures used to protect the pendant amino groups at posi tions 2, 4 and 5 of Pseudomycin A, A ' , B, B ' , C or C wi th CBZ. Dissolve/suspend pseudomycin compound (R 1= H) in DMF (20 mg/ml, Aldrich Sure Seal) . While stirring at room temperature add N- (Benzyloxycarbonyloxy) succinimide (6 eq) . Allow to stir at room temperature for 32 hours. Monitor reaction by HPLC (4.6x50 mm, 3.5 μm, 300-SB, C8, Zorbax column) . Concentrate reaction to 10 ml on high vacuum rotovap at room temperature. Put material in freezer until ready to prep by chromatography. Reverse phase preparative HPLC yields an amorphous, white solid after lyophilization (R 1 = CBZ in structure II below) . Alloc-Protected Pseudomycin : General procedures used to protect the pendant amino groups at posi tions 2, 4 and 5 of Pseudomycin A, A ' , B, B ' , C or C wi th Alloc . Diallyl pyrocarbonate (558 mg, 3.0 mmol) was added to a solution of Pseudomycin A (1.22 g, 1.0 mmol) in 600 ml DMF . The reaction was stirred at room temperature overnight. The solvent was removed in vacuo to afford an oily residue which was washed with ether three times. The oily residue was redissolved in a mixture of water and ACN (-1:1) and lyophilized to provide an alloc-protected psuedomycin A compound in 90% yield. The alloc-protected pseudomycin B compound was prepared using the same procedures in 90% yield (R 1 = alloc in structure II below) . General procedures used to remove CBZ protecting groups at posi tion 2, 4 and 5 by hydrogenation . Dissolve CBZ-protected acylated-derivative in a cold 1% to 10% acetic/methanol solution (5 mg/ml) and add an equivalent amount of 10% Pd/C. Charge the reaction with hydrogen by degassing reaction and replacing volume with H 2 ,4-7 times. Allow reaction to proceed at room temperature. Monitor the reaction by HPLC every hour until starting material is consumed. When the reaction is complete, remove balloon and filter reaction with 0.45 μm filter disk (Acrodisk GHP, GF by Gelman) . Concentrate to about l/10 th volume and prep by HPLC. Lyophilize fractions containing product . General procedures used to remove Alloc protecting groups at posi tion 2, 4 and 5 wi th tr ibutyl tinhydride and triphenylphosphine palladium dichloride. Acetic acid (1 ml) was added to a suspension of alloc- protected pseudomycin B (0.05 mmol) in 5 ml methylene chloride. After degassing under vacuum, the solution was treated with 6.0 mg PdCl 2 (PPH 3 ) 2 (0.008 mmol) and 0.40 ml tri-n-butyltin hydride (1.5 mmol) at room temperature for 2 hours . The solvent was evaporated in vacuo and the residue dissolved in water/ACN (-1:1) and filtered. The resulting solution was purified by preparative HPLC to afford the desired pseudomycin B compound in 93% yield. Alternatively, 5 ml tetrahydrofuran and 0.1 ml acetic acid may be used as the solvent instead of 5 ml methylene chloride and 1.0 ml acetic acid. The following structure II will be used to describe the products observed in Examples 1 through 27. Although a specific pseudomycin natural product (pseudomycin B) was used in the Examples below, those skilled in the art will appreciate that other pseudomycin natural products or semi- synthetic derivatives may be used as starting materials. II Examples 1-3 illustrate the formation of bis-esters at residues 3 and 8. Example 1 Synthesis of Bis-Ethyl ester 1 -1 : R = H R^ = -OCH 2 CH 3 R 3 = -OCH 2 CH 3 1-1 A 50 ml round bottom flask was charged with 10 ml of absolute ethanol and CBZ-protected pseudomycin B(251.7 mg, 0.156 mmol) . To this mixture was added - 1 ml of acidified ethanol (previously acidified using HC1 gas) and the reaction was allowed to stir at room temperature overnight. The solvent was then removed in vacuo and the residue was carried on to the next step without further purification by dissolving it in a solution of 10 ml MeOH/1.5 ml glacial AcOH. Standard hydrogenolysis using 249.7 mg of 10% Pd/C for 30 minutes, removal of the catalyst via filtration and purification via preparatory HPLC led to Compound 1-1 (120.9 mg) after lyophilization. MS (Ionspray) calcd for C 55 H 96 ClNι 2 Oi 9 (M+H) + 1264.89, found 1264.3. The mono-esters may be isolated by following the reaction carefully by HPLC. The reaction is stopped at the appropriate time when the ratio of starting material: mono ester (s): bis ester is greatest. The methodology remains the same. The resulting mixture of mono esters is isolated where some ester is formed on the aspartic acid residue and some on the hydroxy aspartic acid residue. This mixture of CBZ-protected, mono esters is hydrogenated using standard methodology to yield a mixture of mono ethyl esters of Pseudomycin B. Compounds 1-2 and 1-3 were synthesized using the same procedures described above. R = -H R = -H R2 = -0CH 3 R 2 = -OCH(CH 3 ) 2 R3 = -0CH 3 R 3 = -OCH(CH 3 ) 2 1-2 1-3 Example 2 illustrates the synthesize of bis-esters using basic conditions. Example 2 Synthesis of Bis-propyl ester 2-1 : R = -H R 2 = -OCH 2 CH 2 CH 3 R 3 = -OCH 2 CH 2 CH 3 2-1 CBZ-protected pseudomycin B (247.3 mg, 0.154 mmol) was dissolved in 5 ml DMF . A large excess of propyl iodide and an excess of NaHC0 3 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 147.6 mg of the protected bis ester. Hydrogenolyis of this compound under standard condition using 149.3 mg of 10% Pd/C yielded 78.9 mg of Compound 2-1 after HPLC purification and lyophilization. Example 3 R = -H R = -H R 2 = -0(CH 2 ) 4 CH 3 R 2 = -OH R 3 = -OH R 3 = -0(CH 2 ) 4 CH 3 3-1 3-2 CBZ-protected pseudomycin B (282.3 mg, 0.175 mmol) was dissolved in 5 ml DMF. A large excess of n-pentyl iodide and an excess of NaHC0 3 were then added. The reaction was allowed to stir for 10 h at room temperature. Purification via preparatory HPLC followed by lyophilization provided 49.1 mg of the mixture of protected mono pentyl esters. Hydrogenolyis of this mixture under standard condition using 47.3 mg of 10% Pd/C yielded 30.6 mg of Compounds 3-1 and 3-2 after HPLC purification and lyophilization. R = -H R = -H R = -H R 2 = -0(CH 2 ) 3 CH 3 R 2 = -0(CH 2 ) 3 CH 3 R 2 = -OH R 3 = -0(CH 2 ) 3 CH 3 R 3 = -OH R 3 = -0(CH 2 ) 3 CH 3 3-3 3-4 3-5 Substitution of the propyl iodide with n-butyl iodide afforded the bis-butyl ester (3-3) , a mixture of mono esters (3-4 + 3-5) and a mixture of mono ester + the following cyclic imide compound 3-6 : 3-6 Example 4 Synthesis of cyclopentylmethyl ester 4-1 : R = -H R 2 = -0CH 2 (cyclopentyl] R 3 = -OH 4-1 CBZ-protected pseudomycin B, a large excess of p- toluenesulfonic acid and cyclopentanemethanol are mixed and allowed to stir overnight. An additional 10 equivalents of alcohol was added the next day. The CBZ-protected ester was isolated via preparatory HPLC and then hydrogenated using standard methodology to produce Compound 4-1. Each of the compounds synthesized in Examples 1-4 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized. Simple esters (bis-methyl, bis- ethyl and mono-ethyl) were active and efficacious; however, the larger esters exhibited less efficacy (e.g., propyl esters and larger) . ADME has shown that Compounds 1-1 and 2-1 quickly cleave to the parent pseudomycin B compound. Examples 5-11 illustrate the synthesis of amide derivatives at residue 3. Example 5 Synthesis of Compound 5-1 : R = -H R 2 = -NH 2 R 3 = -OH 5-1 CBZ-protected pseudomycin B (1.12 g) and 224 mg TBTU, 0.56 ml DIEA and 1.0 g deprotected rink amide resin (4- (2 ' , 4 ' -dimethoxyphenyl-aminomethyl ) -phenoxy resin, available from Advance ChemTech, Inc., Louisville, KY) were mixed for 3 days. The mixture was filtered and the resin washed 3x with DMF and 3x with dichloromethane. The resin was treated with 5% water in 1:1 TFA/CH 2 C1 2 for 3 hours. The mixture was filtered and the resin washed 3x with TFA. The filtrate was collected and concentrated in vacuo. Upon purification by HPLC, 60 mg (5.3%) of the CBZ-protected amido product was isolated. The protected amido compound (60 mg) was dissolved in 6 ml of 1% AcOH in methanol and 60 mg of 10% Pd/C was added. The mixture was stirred for 30 minutes under hydrogen at room temperature. After filtering, the solution was concentrated in vacuo. The residue was dissolved in 50% ACN/water and lyophilized to yield 45 mg (90%) yield of Compound 5-1. Example 6 Synthesis of Compound 6-1 : R = -H R 2 = -NH ( cyclo ropyl ) R 3 - -OH 6-1 CBZ-protected pseudomycin B (400 mg, 0.25 mmol) is dissolved in 4 ml dry DMF. TBTU (79 mg, 0.25 mmol), DIEA (200 μl, 6 equivalents) and cyclopropylamine (14.2 mg, 0.25 mmol) were added sequentially. The reaction was stirred at room temperature under nitrogen while being monitored by HPLC . Upon completion the reaction was concentrated in vacuo . The crude product purified by preparative HPLC. Lyophilization yielded 209.2 mg (51.1%) of a colorless powder . The 3-amido compound (279.1 mg, 0.169 mmol) was hydrogenated under hydrogen balloon catalyzed by 10% Pd/C in 1% HOAc/MeOH for 45 minutes. The reaction was filtered and concentrated in vacuo . The residue was picked up in a 1:1 mixture of water :ACN and then lyophilized to give 208.3 mg (98.6%) of a colorless powder. The structure was verified by H^NMR. Compound 6-1 can also be made from the Alloc-protected pseudomycin B using the following procedures. 1-Hydroxybenzotriazole hydrate (136 mg, 1.0 mmol) and EDCI (211 mg, 1.1 mmol) was added to a solution of alloc- protected pseudomycin B (730 mg, 0.50 mmol) in 7 ml of DMF. After stirring overnight, cyclopropylamine (85.6 mg, 1.5 mmol) was added. The progress of the reaction was monitored by HPLC. Upon completion, the alloc-protected pseudomycin derivative (334 mg, 50% yield) was isolated via preparative HPLC and lyophilization. The alloc-protected intermediate (117 mg, 0.078 mmol) was dissolved in 15 ml of methylene chloride and 1 ml of acetic acid. After degassing the reaction mixture with dry nitrogen, 30 mg of (PPh 3 ) PdCl 2 and 1 ml of tributyltinhydride was added to the mixture. The progress of the reaction was monitored by HPLC. Upon completion, the reaction mixture was purified by reverse phase preparative HPLC to provide 88 mg (91% yield) of Compound 6-1. Table I below lists other 3-amido derivatives that were synthesized using the same general procedures described above using the appropriate corresponding amine starting material . Table I Example 7 Synthesis of 3 -amido compound 7-1 : R = -H R J = -OH 7-1 In a 500 mL oven dried round bottom flask, CBZ- protected Pseudomycin B(0.5 g, 0.311mmol) was dissolved in 25 mL of DMF. To this solution was added TBTU(0.2 g, 0.622 mmol), 3- (aminomethyl)pyridine (0.067 g, 0.622mmol), and N- ethyldicyclohexylamine (0.391 g, 1.87 mmol). The solution was stirred for three hours and then concentrated down. The product was isolated by reverse-phase preparatory HPLC, and lyophilized to yield, (96 mg, 18% yield) CBZ-protected amide. The deprotection of the CBZ groups was performed by adding slowly an equivalent mass of 10%Pd/C to a cold 1% acetic/methanol solution of CBZ-protected amide. The solution was allowed to warm to rt and stirred rapidly for 3.5 hours under 1 atm H 2 . After removal of the catalyst via filtration, purification on reverse phase HPLC and lyophilization yielded 40 mg , 55% yield of Compound 7-1. MS data Calculated for C57 H93 Cl N14 018 Mol . Wt . = 1296.6 Found ES+ 1297.15, ES- 1294.95 Example 8 Synthesis of 3 -amido compound 8-1 : R = -H R 3 -OH 8- -1 The same general procedures as described in Example 7 may be used. When no base is added, a mixture of 8 and 3 amido substituted compounds are observed. Example 9 Synthesis of 3-amido compound 9-1 : R = -H R = -H R 2 = -NH (benzyl ) R 2 = -NH (benzyl ) R 3 = -OH R 3 = -NH (benzyl ) 9-1 9-2 The same general procedures as described in Example 7 may be used. When no base is added, a mixture of Compounds 9-1 and 9-2 are observed. Example 10 Synthesis of 3 -amido compound 10-1 : R = -H 10-1 The same general procedures as described in Example 7 are used to synthesize Compound 10-1 using the appropriate corresponding amine starting material . Example 11 Synthesis of 3 -amido compound 11 -1 : R -H R 3 = -OH 11-1 The same general procedures as described in Example 7 are used to synthesize Compound 11-1 using 4- (aminomethyl) pyridine as the amine starting material. Example 12 Synthesis of 3 -amido Compound 12-1 : R = -H R 2 = -N (CH 3 ) 2 R 3 = -OH 12-1 CBZ-protected pseudomycin B (260 mg, 0.16 mmol), 51.8 mg TBTU and 152 μl DIEA were dissolved in 3 ml DMF and 320 ml dimethylamine (0.16 mmol) in THF (2 molar solution). The reaction was stirred at room temperature for 20 minutes and the then purified via HPLC. The product was lyophilized to give 172 mg (66% yield) of the desired CBZ-protected amide. The CBZ-protected amide was hydrogenated using the general procedure described above to provide Compound 12-1. Example 13 illustrates the synthesis of pseudomycin compounds where the carboxylic acid group is reacted with a variety of amino acid alkyl esters. Example 13 Synthesis of 3 -amido Compound 13 -1 : R = -H R 2 = -NHCH (C0 2 CH 3 ) CH 2 CH 2 CH 2 CH 2 NH 2 R 3 = -OH 13-1 CBZ-protected Lysine methyl ester (164 mg, 0.49 mmol) was added to a solution of CBZ-protected pseudomycin B (800 mg, 0.49 mmol), TBTU (158 mg, 0.49 mmol) and 400 ml DIEA (2.51 mmol) in 8 ml DMF. The reaction was allowed to stir at room temperature for 20 minutes and then purified via HPLC to yield 260 mg (32% yield) of the CBZ-protected amide. The CBZ-protected amide was hydrogenated using the general procedures described above to produce Compound 13-1. The compounds 13-2 through 13-4 listed in Table II may be synthesized using the same general procedures as described above using the appropriate corresponding aminoacid ester. Table II Examples 14-16 illustrate the synthesis of amide derivatives at residue 8. Example 14 Synthesis of 8-amido Compound 14 -1 : R = -H R 2 = -OH R 3 = -NH 2 14-1 Compound 14-1 is synthesized using the same procedures as described for compound 6-1 using a rink amide resin with the exception that PyBOP is used as the coupling agent instead of TBTU. Example 15 Synthesis of 8-amido Compound 15-1 : R = -H R 2 = -OH R 3 = -NH ( CH 2 ) 3 CH 3 15-1 n-Butyl amine (45.4 mg, 0.62 mmol) was added to a solution of CBZ-protected pseudomycin B (1000 mg, 0.62 mmol) and PyBop (323 mg, 0.62 mmol) dissolved in 10 ml of DMF. The reaction was stirred at room temperature for 1 hour and then purified via HPLC. The product was lyophilized to give 280 mg (27% yield) of the CBZ-protected amide. The CBZ-protected amide (280 mg, 0.17 mmol) was hydrogenated under hydrogen catalyzed by 10% Pd/C in 1% acetic acid/methanol for 45 minutes. The reaction mixture was filtered and the solvent removed in vacuo . The residue was dissolved in 50% ACN in water and lyophilized to give 189 mg (89% yield) of Compound 15-1. The 8-amido compounds listed in Table III may be synthesized using the same general procedures described above using the appropriate corresponding amine starting material . Table III Example 16 Synthesis of 8-amido Compound 16-1 : R = -H -OH 16-1 In a 100 ml round bottom flask, alloc-protected Pseudomycin B(0.25 g, 0.171 mmol) was dissolved in 25 ml of DMF. To this solution was added Pybop (0.089g, 0.171 mmol)and 4- (2-Aminoethyl)morpholine (0.022 g, 0.171 mmol). The solution was stirred rapidly overnight under 1 atm N 2 . The solution was concentrated down, and the product was isolated by reverse-phase HPLC, and lyophilized to yield (140 mg, 0.089 mmol, 52%) alloc-protected Psuedomycin B Morpholine derivative. The deprotection of the alloc groups was performed by adding Bu 3 SnH( 0.648 g, 2.23 mmol), and (Ph 3 P) 2 PdCl 2 (0.009g, 0.013 mmol) to a 1% acetic/ dichloromethane solution of alloc-protected Psuedomycin B Morpholine derivative (10 mg/mL) . Reaction time was 30 minutes. Reaction was monitored by HPLC. The solution was concentrated down, and the product was isolated by reverse phase HPLC prep, and lyophilized to yield 38 mg, 32% of Compound 16-1. MS data: Calculated for C57 H99 CI N14 019 Mol. Wt. 1318.7 Found ES+ 1320.0, ES- 1318.0 The 8-amido compounds listed in Table IV were synthesized using the same general procedures described above using the appropriate corresponding amine starting material . Table IV Each of the compounds synthesized in Examples 5-16 showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum. However, the following basic trends in activity were observed based on the compounds synthesized. When the 8-amido derivatives were assayed against C. albicans, several trends were apparent from the data. The in vi tro potency decreases in the following order of R 3 substitution: -NH 2 > -NHCH 3 > -NHCH 2 CH 3 > -NH(CH 2 ) 2 CH 3 > -NH(CH 2 ) 3 CH 3 ; -NHCH 2 CH 2 N (CH 3 ) 2 > -NH (CH 2 ) 3 N (CH 3 ) 2 ; and -NH(GlyOMe) > -NH(PheOMe) . In general, better activities were realized with amido groups having smaller alkyl groups. The free amide group was found to be the most active of the series. In addition, the cycloalkyl amides demonstrated better activity than the corresponding straight chain alkyl groups. Alkyl groups having a polar substitution on the end of the alkyl chain showed less activity than the corresponding natural product. Unlike the parent natural product, none of the 8-amido derivatives showed tail vein irritation. The 3-amido derivatives demonstrated a similar trend as observed with the 8-amido derivatives in comparison with the parent natural product (e.g., amide substituents at R 2 having shorter alkyl chains were more active than longer alkyl chains) . Unlike the 8-amido derivatives, the 3-amido derivatives did not show a significant decrease in in vi tro activity against C. albicans until the alkyl chain reached 7-carbons or longer (3-amido PSB compound where R 2 = -NH(CH 2 ) 6 CH 3 had a MIC = 20 μg/ml) versus 4-carbons or longer for the 8-amido derivatives (8-amido PSB compound where R 3 = -NH(CH 2 ) 3 CH 3 had a MIC = 20 μg/ml) . Most of the 3-amido derivatives tested showed an improvement in tail vein irritation. The exceptions being R 2 = -NH (iso-amyl) , -NH(n- hexyl), -NH(CH 2 ) 2 N(CH 2 CH 3 ) 2 , and -NH(CH 2 ) 3 N(CH 3 ) 2 . Although formation of an amide bond at residues 3 and 8 demonstrated an improved toxicity profile in comparison with the corresponding natural product (Pseudomycin B) , in vivo efficacy generally decreased.

Claims: WE CLAIM : 1. A pseudomycin compound having the following structure I 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, C 1 -C 4 alkoxy, hydroxy (C 1 -C 4 ) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C 14 alkyl substituted six-membered aromatic ring, and R f is Cs-Cis alkyl, or C 5 -Cn alkoxy; R is where R g is hydrogen, or Cι-Cι 3 alkyl , and R h is C1-C15 alkyl , C4-C 15 alkoxy, (C 1 -C10 alkyl ) phenyl , - (CH 2 ) n -aryl , or - (CH 2 ) n - (C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or R is where R 1 is a hydrogen, halogen, or C 5 -C 8 alkoxy, and m is 1, 2 or 3 ; R is where ? is C 5 -C14 alkoxy or C 5 -C 14 alkyl, and p = 0, 1 or 2; R is where R k is C 5 -C 14 alkoxy; or R is - (CH 2 )-NR m -(Cι 3 -Ci8 alkyl) , where R m is H, -CH 3 or -C(0)CH 3 ; R 1 is independently -NH 2 or -NHp-Pg, where p is 0 or 1; R z and R J are independently -OR 2 a, or -N(R^ D ) (R where R 2a and R 2b are independently hydrogen, Ci-Cio alkyl, C 3 .C 6 cycloalkyl, hydroxy (Ci-Cio) alkyl, alkoxy (Ci-Cio) alkyl, C 2 -Cι 0 alkenyl, amino (Cι~ Cio) alkyl, mono- or di-alkylamino (Ci-Cio) alkyl, aryl (Ci-Cio) alkyl, heteroaryl (Ci-Cio) alkyl, or cycloheteroalky1 (Ci-Cio) alkyl , or R 2b is an alkyl carboxylate residue of an aminoacid alkyl ester, and R 2c is hydrogen or Cι-C 6 alkyl, provided that both R 2 and R 3 are not -OH; and pharmaceutically acceptable salts and solvates thereof. 2. A pseudomycin prodrug having the following structure wherein R is where R 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, C 1 -C 4 alkoxy, hydroxy (C1-C 4 ) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C 14 alkyl substituted six-membered aromatic ring, and R f is C 8 -Ci8 alkyl, C 5 -C 11 alkoxy or biphenyl; R is where R 9 is hydrogen, or Cι-Cι 3 alkyl, and R h is C1-C15 alkyl, C4-C1 5 alkoxy, (C1-C 10 alkyl ) phenyl , - ( CH 2 ) n -aryl , or - ( CH 2 ) n - ( C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or where R 1 is a hydrogen, halogen, or C 5 -C 8 alkoxy, and m is 1, 2 or 3 ; where R 3 is C 5 -C 14 alkoxy or C 5 -C 14 alkyl, and p = 0, 1 or 2; R is where R k is C 5 -C 14 alkoxy; or R is - (CH 2 )-NR m -(Cι 3 -C 18 alkyl), where R m is H, -CH 3 or -C(0)CH 3 ; R 1 is independently -NH 2 or -NH p -Pg, where p is 0 or 1; R 2 and R 3 are -OR 2a , where R 2a is Cι-C 3 alkyl; and pharmaceutically acceptable salts and solvates thereof. 3. A 3-amido derivative of a pseudomycin compound prepared by the steps of (i) providing a pseudomycin compound having the following structure I 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 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, C 1 -C4 alkoxy, hydroxy (C 1 -C 4 ) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C 1 4 alkyl substituted six-membered aromatic ring, and R f is C 6 -Ci8 alkyl, C 5 -C 11 alkoxy or biphenyl ; where R g is hydrogen, or Cι-Cι 3 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C 10 alkyl ) phenyl , - (CH 2 ) n -aryl, or - (CH 2 ) n - (C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or R is where R 1 is a hydrogen, halogen, or Cs-Cj alkoxy, and m is 1, 2 or 3 ; R is where R j is C5-C14 alkoxy or C 5 -C 14 alkyl , and p = 0 , 1 or 2 ; R is where R k is C5-C14 alkoxy; or R is - (CH 2 )-NR-(Cι 3 -Cι 8 alkyl), where R m is H, -CH 3 or -C(C)CH 3 ; R 1 is -NH 2 ; R 2 and R 3 are -OH; and pharmaceutically acceptable salts and solvates thereof; (ii) protecting the amino groups, R 1 , at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 3 using o- benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate or 2 (lH-benzotriazole-1-yl) - 1,1,3,3, -tetramethyluronium hexafluorophosphate as a coupling agent; (iv) removing said amino-protecting groups. 4. The 3-amido derivative of Claim 3 wherein step (iii) forming an amide linkage is accomplished in the presence of a bulky amine. 5. The 3-amido derivative of Claim 3 wherein step (iii) forming an amide linkage is accomplished in the presence of a bulky amine and at a temperature between about 0°C and -20°C. 6. An 8-amido derivative of a pseudomycin compound prepared by the steps of (i) providing a pseudomycin compound having the following structure wherein R is where R a and R ' are independently hydrogen or methyl, or either R 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, C 1 -C 4 alkoxy, hydroxy (C 1 -C4) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C14 alkyl substituted six-membered aromatic ring, and R f is C 6 -C18 alkyl, C5-C 11 alkoxy or biphenyl ; where R 9 is hydrogen, or Cι-Cι 3 alkyl , and R h is C1-C15 alkyl , C4-C1 5 alkoxy, (C1-C1 0 alkyl ) phenyl , - ( CH 2 ) n -aryl , or - ( CH 2 ) n - ( C 5 -C 6 cycloalkyl ) , where n = 1 or 2 ; or R is where R 1 is a hydrogen, halogen, or C 5 -C 5 alkoxy, and m is 1 , 2 or 3 ; R is where R D is C5-C14 alkoxy or C 5 -C14 alkyl , and p = 0 , 1 or 2 ; where R k is C 5 -C 14 alkoxy; or R is - (CH 2 )-NR m -(Cι 3 -Ci8 alkyl), where R m is H, -CH 3 or -C(C)CH 3 ; R 1 is -NH 2 ; R 2 and R 3 are -OH; and pharmaceutically acceptable salts and solvates thereof; (ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent; (iv) removing said amino-protecting groups. 7. 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. 8. A process for making a 3-amido derivative of a pseudomycin compound comprising the steps of (i) providing a pseudomycin compound having the following structure wherein R is where R 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, C 1 -C 4 alkoxy, hydroxy (C 1 -C 4 ) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C 14 alkyl substituted six-membered aromatic ring, and R f is C 6 -Ci 8 alkyl, C 5 -Cn alkoxy or biphenyl ; R is where R g is hydrogen, or Cι-Cι 3 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C 10 alkyl ) phenyl , - (CH 2 ) n -aryl, or - (CH 2 ) n - (C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or R is where R 1 is a hydrogen, halogen, or Cs-Cg alkoxy, and m is 1, 2 or 3 ; R is where R D is C 5 -C 14 alkoxy or C 5 -C 14 alkyl , and p = 0 , 1 or 2 ; R is where R k is C5-C 14 alkoxy; or R is -(CH 2 )-NR m -(C 13 -Ci8 alkyl), where R m is H, -CH 3 or -C(C)CH 3 ; R 1 is -NH 2 ; R 2 and R 3 are -OH; and pharmaceutically acceptable salts and solvates thereof; (ii) protecting the amino groups, R 1 , at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 3 using o- benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate or 2 (lH-benzotriazole-1-yl) - 1,1,3,3, -tetramethyluronium hexafluorophosphate as a coupling agent in the presence of a bulky amine and at a temperature between about 0°C and -20°C; (iv) removing said amino-protecting groups. 9. A process for making an 8-amido derivative of a pseudomycin compound comprising the steps of (ii) providing a 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 ' 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 ' 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, C 1 -C 4 alkoxy, hydroxy (C 1 -C 4 ) alkoxy, or taken together with R e forms a 6-membered aromatic ring or C 5 -C 6 cycloalkyl ring; R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C 14 alkyl substituted six-membered aromatic ring, and R f is C 6 -Ci8 alkyl, C 5 -C 11 alkoxy or biphenyl ; R is where R g is hydrogen, or Cι-Cι 3 alkyl, and R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10 alkyl ) phenyl , - (CH 2 ) n -aryl, or - (CH 2 ) n - (C 5 -C 6 cycloalkyl), where n = 1 or 2 ; or where R 1 is a hydrogen, halogen, or C 5 -C ε alkoxy, and m is 1, 2 or 3 ; R is where R D is C5-C14 alkoxy or C 5 -C 14 alkyl, and p = 0, 1 or 2; R is where R is C 5 -C 14 alkoxy; or R is -(CH 2 )-NR π, -(Cι 3 -Ci8 alkyl), where R m is H, -CH 3 or -C(C)CH 3 ; R 1 is -NH 2 ; R 2 and R 3 are -OH; and pharmaceutically acceptable salts and solvates thereof; (ii) protecting the amino groups at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 8 using benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate as a coupling agent; (iv) removing said amino-protecting groups. 10. A pharmaceutical formulation comprising a compound of Claim 1 and a pharmaceutically acceptable carrier. 11. A pharmaceutical formulation comprising a prodrug of Claim 2 and a pharmaceutically acceptable carrier. 12. A method for treating an antifungal infection in an aminal in need thereof, which comprises administering to said animal a pseudomycin compound of Claim 1. 13. A method for treating an antifungal infection in an animal in need thereof, which comprises administering to said animal a prodrug of Claim 2.

Inventors: CHEN SHU HUI, GALKA CHRISTOPHER STANLEY, HELLMAN SARAH LYNNE, KRSTENANSKY JOHN L, RODRIGUEZ MICHAEL JOHN, SUN XICHENG DAVID, USYATINSKY ALEXANDER YA, VASUDEVAN VENKATRAGHAVAN, ZWEIFEL MARK JAMES, CHEN, SHU, HUI, GALKA, CHRISTOPHER, STANLEY, HELLMAN, SARAH, LYNNE, KRSTENANSKY, JOHN, L., RODRIGUEZ, MICHAEL, JOHN, SUN, XICHENG, DAVID, USYATINSKY, ALEXANDER, YA., VASUDEVAN, VENKATRAGHAVAN, ZWEIFEL, MARK, JAMES
Applicants: CHEN SHU HUI, GALKA CHRISTOPHER STANLEY, HELLMAN SARAH LYNNE, KRSTENANSKY JOHN L, LILLY CO ELI, RODRIGUEZ MICHAEL JOHN, SUN XICHENG DAVID, USYATINSKY ALEXANDER YA, VASUDEVAN VENKATRAGHAVAN, ZWEIFEL MARK JAMES, CHEN, SHU, HUI, ELI LILLY AND COMPANY, GALKA, CHRISTOPHER, STANLEY, HELLMAN, SARAH, LYNNE, KRSTENANSKY, JOHN, L., RODRIGUEZ, MICHAEL, JOHN, SUN, XICHENG, DAVID, USYATINSKY, ALEXANDER, YA., VASUDEVAN, VENKATRAGHAVAN, ZWEIFEL, MARK, JAMES
Application year: 2000
Main classification: C07K7
IPC codes: A61K38, A61P31, C07K7