CA2379317A1 - Amine-modified pseudomycin compounds - Google Patents

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-Cl) 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 amine-modified pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.
O

O
OH
O~N H
NH H N OH
HO O
NH SCI
O
O O
R'HN O
NH
O O O H R
N NH
O
R2 NHR' R' HN O
wherein R is Rb~ Rd Rf Ra Ra~ ~c ~a R R

where Ra and Ra~ are independently hydrogen or methyl, or either Ra or Ra~ is alkyl amino, taken together with Rb or Rb~ forms a six-membered cycloalkyl'ring, a six-membered aromatic ring or a double bond, or taken together with R~ forms a six-membered aromatic ring;
Rb and Rb~ are independently hydrogen, halogen, or methyl, or either Rb or Rb~ is amino, alkylamino, oc-acetoacetate, methoxy, or hydroxy;
R° is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy (C1-C4)alkoxy, or taken together with Re forms a 6-membered aromatic ring or CS-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, CS-C14 alkoxy substituted six-membered aromatic ring, or C5-C14 alkyl substituted six-membered aromatic ring, and Rf is C$-C18 alkyl , C5-C11 alkoxy, or biphenyl ;
R is Rg Rn O
where Rg is hydrogen, or C1-C13 alkyl, and Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-Clo alkyl ) phenyl , - ( CHZ ) n-aryl , or - ( CH2 ) n- ( CS-Cs cycloalkyl), where n = 1 or 2; or R is R~
m where Ri is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3;
R is OH
a R' where R' is C5-C14 alkoxy or CS-C14 alkyl , and p = 0 , 1 or 2;

R is -N
Rk where Rk is CS-C14 alkoxy; or R is - (CH2) -NRm- (C13-C18 alkyl) , where Rm is H, -CH3 or -C (O) CH3;
R1 is independently hydrogen, formyl, an acylalkyl (e.g., -C ( 0 ) CH3 , -C ( 0 ) CH2CH3 , -C ( O ) CH ( CH3 ) 2 , and -C ( O ) C ( CH3 ) 3 ) , an acylalkylamine (e. g., -C(0)CH(NH2)CH3), an acylazaalkyl (e. g., -C(O)NHCH3 and -C(O)NHCH(CH3)2), an acyloxyalkene ( a . g . , -C ( 0 ) OCH2CH=CH2 ) , an acyloxyaryl ( a . g . , -C ( O ) OC6H5 ) , or an acylmethylenecarbamate (e. g., compounds 1(a) depicted below) O
N O
~R~a R' b O
1 (a) Rla i s C1-Clo alkyl , C1-C1o alkenyl , benzyl , or aryl and R1b is hydrogen or methyl, provided that at least one R1 is not hydrogen;
RZ and R3 are independently -OR2a, or -N(RZb) (R2°) , where RZa and R2b are independently hydrogen, C1-Clo 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 ( C1-Clo ) alkyl , alkoxy ( C1-Clo ) alkyl ( a . g, methoxyethyl ) , or C2-Clo alkenyl , amino ( C1-Clo ) alkyl , mono- or di-alkylamino ( C1-Clo ) alkyl , aryl ( C1-Clo ) alkyl (e.g., benzyl), heteroaryl(C1-Clo)alkyl (e.g., 3-pyridylmethyl, 4-pyridylmethyl), or cycloheteroalkyl(C1-Clo)alkyl (e. g., N-tetrahydro-1,4-oxazinylethyl and N-piperazinylethyl), or RZb is an alkyl carboxylate residue of an aminoacid alkyl ester (e.g. , -CHzCO2CH3, -CH ( COzCH3 ) CH ( CH3 ) 2 , -CH ( COZCH3 ) CH ( phenyl ) , -CH ( COZCH3 ) CH20H, -CH ( C02CH3 ) CH2 (p-hydroxyphenyl ) , -CH ( COZCH3 ) CHzSH, -CH ( C02CH3 ) CHz ( CHZ ) 3NH2 , -CH ( COZCH3 ) CHZ ( 4- or 5-imidazole ) , -CH ( COZCH3 ) CHzCOzCH3, -CH ( COZCH3 ) CHZC02NH2 , and the 1 i ke ) , and R2~ is hydrogen or C1-C6 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 CnH2n+1 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 "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 vitro indications which suggest that the new compounds may be active against C. albican, C, neoformans, and/or A. fumigatus. 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 - acylalkylamine. 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-B1, 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-B1, 7H9-1, and 67 H1 were deposited with the American Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.:
25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67 H1 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 ~g/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 ~,g/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°-C to about 27°-C, 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 50o saturation, more preferably about 30o 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 H1 each produce predominantly pseudomycin A, but also produce pseudomycin B
and C, typically in ratios of 4:2:1. Strain 67 H1 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 H1, strain 25-B1 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 reactions) 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). 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., 10o Pd/C).
Tn~hen 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., HCl, 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 0.1o to 99.90 by weight of active ingredient, more generally from about 100 to about 30o 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 5o 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. lusitaniaw); 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. dermatitidis);
Fusarium spp.; Trichophyton spp., Pseudallescheria boydii, Coccidioides immits, 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-O-C(O)-CBZ = benzyloxycarbonyl , C6HSCH2-O-C ( 0 ) -PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-1-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.
R'.~.HN
N
O O O ,,.N n N N N
O ' Rz NHR' R'~~HN O
II
Detection and Quantification of An ti fungal Activity:
Antifungal activity was determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc-diffusion test. A typical fungus employed in testing antifungal activity is Candida albicans. Antifungal activity is considered significant when the test sample (50 ~,l) causes 10-12 mm diameter zones of inhibition on C.
albicans x657 seeded agar plates.
Tail Vein Toxi ci 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 5.0o 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).
Preparations Compound 1a-2:
O O
/ O N
O-N
O
O
1a-1 Compound 1a-1 is commercially available from Novabiochem (San Diego, CA).
Preparation of Compound 2a-1:
O
N~N~O
N-N
2a-1 Compound 2a-1 is prepared using the procedures described in Admiak, 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, Rz and R3 - -OH).
Synthesis of Compound 1-2:
R1. ~ Ri , , and R1 . , , _ _ C ( O ) CH2NH2 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 10o 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 C57H97C1N15O22 (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.
O

Rl . ~ Rl , . ~ and Ri , . , O

Rl , ~ Rl , . ~ and Rl , , .

Exempla 2 Example 2 illustrates the synthesis of acyloxyaryl derivatives of pseudomycin B(n = 10, RZ and R3 - -OH).
Synthesis of Compound 2-1:
O
R1, ~ R1. . ~ and Ri . , . - ~O

Compound 2-1 is synthesized using the same procedures as described in Example 1 except Compound 2a-1 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, Rz and R3 - -OH).
Synthesis of Compound 3-2:
O
R1, ~ R1, , ~ and Ri , . . -_CH

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. ~ Rl . . ~ and Rl . , , - ~
_C ( CH ) 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, RZ
and R3 - -OH ) .

Synthesis of Compound 4-2:
O
R1, ~ R1, . ~ and R1, , . - ~
" _NHCH

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, Rz and R3 - -OH).
O
R1, ~ R1, . ~ and R1, , . -H

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, Rz and R3 - -OH ) .

O
Rl ' , Rl ' ' , and R1, .
O

Compound 6-1 is synthesized using the same procedures as described in Example 1 except diallylpyrocarbonate is used as the acylating agent. Yield 770

Claims (6)

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, .alpha.-acetoacetate, methoxy, or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy(C1-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-C14 alkyl substituted six-membered aromatic ring, and R f is C8-C18 alkyl, or C5-C11 alkoxy;
R is where R g 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;
R is where R j is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or 2;
R is where R k is C5-C14 alkoxy; or R is - (CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3 or -C(O)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 -OR2a or -N(R2b)(R2c), where R2a and R2b are independently hydrogen, C1-C10 alkyl, C3-C6 cycloalkyl, hydroxy(C1-C10)alkyl, alkoxyalkyl, or C2-C10 alkenyl, amino(C1-C10)alkyl, mono- or di-alkylamino(C1-C10)alkyl, aryl(C1-C10)alkyl, heteroaryl(C1-C10)alkyl, cycloheteroalkyl(C1-C10)alkyl, or R2b is an alkyl carboxylate residue of an aminoacid alkyl ester and R2c is hydrogen or C1-C6 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) where R1a is C1-C10 alkyl, C1-C10 alkenyl, benzyl, or aryl and R1b 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 -CH2CO2CH3, -CH(CO2CH3)CH(CH3)2, -CH(CO2CH3)CH(phenyl), -CH(CO2CH3)CH2OH, -CH(CO2CH3)CH2(p-hydroxyphenyl), -CH(CO2CH3)CH2SH, -CH(CO2CH3)CH2(CH2)3NH2, -CH(CO2CH3)CH2(4-imidazole), -CH(CO2CH3)CH2(5-imidazole), -CH(CO2CH3)CH2CO2CH3, or -CH(CO2CH3)CH2CO2NH2.

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.