MXPA02000320A - 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

MODIFIED PSEUDOMYCIN COMPOUNDS IN AMINA GROUPS Field of the Invention The present invention relates to pseudomycin compounds, in particular, modified pseudomycin compounds in the amine groups.

BACKGROUND OF THE INVENTION Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae (bacteria associated with plants) and have been shown to have antifungal activities. (see, that is, Harrison, L. and 'collaborators, "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity ", J. Gen. icrobioloqy, 137 (12), 2857-65 (1991) and US patents Nos. 5,576,298 and 5,837,685.) In contrast to the previously described antimycotics of P. syringae ( for example, syringomycins, syringotoxins and syringo-statins), AC pseudomycins contain hydroxyapartic acid, aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid.The peptide portion for pseudomycins A, A ', B, B ', C, C corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp (3-OH) -L-Thr (4-C1) with the terminal carboxyl group that closes a macrocyclic ring in the OH group of REF: 134815 the N-terminal Being. The analogs are distinguished by the N-acyl secondary chain, ie, pseudomycin A is N- 'acylated by 3, -dihydroxytetradecanoyl, pseudomycin A' by 3, -dihydroxypentadecanoyl, pseudomycin B by 3-hydroxytetradecanoyl, pseudomycin B 'by 3-hydroxydecanoyl, pseudomycin C by 3,4-dihydroxyhexadecanoyl and pseudomycin C by 3-hydroxyhexadecanoyl. (see, ie, Ballio, A. et al., "Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins", FEBS Letters, 355 (1), 96-100 (1994) and Coiro, VM 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).) It is known that pseudomycins possess certain adverse biological effects. For example, endothelial destruction of the vein, tissue destruction, inflammation, and local toxicity to host tissues have been observed when pseudomycin is administered intravenously. Since pseudomycins have a 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 that have fewer adverse side effects.

Brief Description of the Invention The present invention provides modified pseudomycin compounds in the amine groups represented by the following structure which are useful as antifungal agents or in the design of antifungal agents. where R is where Ra and Ra 'are independently hydrogen or methyl, or any of Ra or Ra' is alkylamino, taken together with Rb or Rb 'form a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc they form a six-membered aromatic ring; Rb and Rb are independently hydrogen, halogen or methyl or any of Rb or Rb 'is amino, alkylamino, α-acetoacetate, methoxy or hydroxy; Rc is hydrogen, hydroxy, alkoxy of 1 to 4 carbon atoms, hydroxy-alkoxy of 1 to 4 carbon atoms, or taken together with Re forms a 6-membered aromatic ring or a cycloalkyl ring of 5 to 6 carbon atoms; Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, a six-membered aromatic ring substituted with alkoxy of 5 to 14 carbon atoms, or a six-membered aromatic ring substituted with alkyl of 5 to 14 carbon atoms carbon, and Rf is alkyl of 8 to 18 carbon atoms, alkoxy of 5 to 11 carbon atoms or biphenyl; R is where Rg is hydrogen, or alkyl of 1 to 13 carbon atoms, and Rh is alkyl of 1 to 15 carbon atoms, alkoxy of 4 to 15 carbon atoms, (alkyl of 1 to 10 carbon atoms) phenyl, - (CH 2) n-aryl or - (CH 2) n- (cycloalkyl of 5 to 6 carbon atoms), where n = 1 or 2; or R is where R1 is hydrogen, halogen or alkoxy of 5 carbon atoms, and m is 1, 2 or 3; R is where R3 is alkoxy of 5 to 14 carbon atoms or alkyl of 5 to 14 carbon atoms, and p = 0, 1 or 2; R is where Rk is alkoxy of 5 to 14 carbon atoms; or R is - (CH2) -NRm- (alkyl of 13 to 18 carbon atoms), where Rra is H, -CH3 or -C (0) 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 (O) CH (NH2) CH3), an acylazaalkyl (e.g., -C (0) NHCH3 and "-C (0) NHCH (CH3) 2), an acyloxyalkene (e.g. , -C (0) 0CH2CH = CH2), an acyloxyaryl (for example, -C (0) OC6H5), or an acylmethylenecarbamate (for example, the compounds 1 (a) represented below) Ka) wherein Rla is alkyl of 1 to 10 carbon atoms, alkenyl of 1 to 10 carbon atoms, benzyl, or aryl and Rlb is hydrogen or methyl, with the proviso that at least one R1 is not hydrogen; R and RJ are independently -OR 2"a, or (R, 2cc), where R 2a and R 2b are independently hydrogen, alkyl of 1 to 10 carbon atoms (e.g., methyl, ethyl, n-propyl, i- propyl, n-butyl, i-butyl, s-butyl, t-butyl, etc.), cycloalkyl of 3 to 6 carbon atoms (eg, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethylene, methylcyclopentyl, cyclohexyl, etc.) hydroxy-alkyl from 1 to 10 carbon atoms, alkoxy-alkyl of 1 to 10 carbon atoms (for example methoxyethyl), or alkenyl of 2 to 10 carbon atoms, amino-alkyl of 1 to 10 carbon atoms, mono- or di- alkylaminoalkyl of 1 to 10 carbon atoms, aryl-alkyl of 1 to 10 carbon atoms (for example, benzyl), heteroaryl-alkyl of 1 to 10 carbon atoms (for example 3-pyridylmethyl, 4-pyridylmethyl), or cycloheteroalkyl-alkyl of 1 to 10 carbon atoms (for example, N-tetrahydro-1, 4-oxazinylethyl and N-piperazinylethyl), or R2b is an alkyl residue carboxylate of an amino acid alkyl ester (e.g., -CH2C02CH3, -CH (C02CH3) CH (CH3) 2, -CH (C02CH3) CH (phenyl), -CH (C02CH3) CH2OH, -CH (C02CH3) CH2 (p -hydroxyphenyl), -CH (C02CH3) CH2SH, -CH (C02CH3) CH2 (CH2) 3NH2, -CH (C02CH3) CH2 (4- or 5-imidazole), -CH (C02CH3) CHC02CH3, -CH (C02CH3) CH2C02NH2 , and the like), and R is hydrogen or alkyl of 1 to 6 carbon atoms; 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 preferred embodiment of the present invention, there is provided a method of treating a fungal infection in an animal in need thereof, which comprises administering to the animal the pseudomycin compound described above.

Definitions As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula C n H 2n +? containing from 1 to 30 carbon atoms to less than indicated otherwise. The alkane radical can be straight chain (e.g., methyl, ethyl, propyl, butyl, etc.), branched chain (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, ethylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (for example, bicyclo [2.2.1] heptane, spiro [2.2] heptane, etc.). The alkane radical can be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy, alkanoyl or alkanoate group has the same definition as before. The term "alkenyl" refers to an acyclic hydrocarbon which contains at least one carbon-carbon double bond. The alkene radical can be straight chain, branched chain, cyclic or multi-cyclic. The alkene radical can be substituted or unsubstituted. The alkenyl portion of an alkenoxy, alkenoyl or alkenoate group has the same definition as before. The term "aryl" refers to aromatic portions having individual (e.g., phenyl) ring systems or fused (e.g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups can 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 the compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows substituents that are a classical alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, and the like. The term "group" specifically provides and allows substituents on alkyls that are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamate, etc., as well as also includes the unsubstituted alkyl portion. However, it is generally understood by those of skill in the art that the substituents should be selected so that they do 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-alkylamino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio , carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl and combinations thereof. The term "animal" refers to humans, companion animals (eg, dogs, cats and horses), animals that are food sources (eg, cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.

DETAILED DESCRIPTION OF THE INVENTION Applicants have discovered that the modification of the pendant amino groups attached to the peptide units of 2,4-diaminobutyric acid in the natural product of pseudomycin or semi-synthetic derivative provides compounds having in vi tro indications that suggest that the new compounds may be active against C. albican, C. neoformans and / or A. fumiga tus. The amino groups are modified using an acylating agent containing a suitable leaving group such that an amide, carbamate, urea or imide bond can be formed with the pendant amino group in the pseudomycin structure. Suitable leaving groups are well known to those of skill in the art and include groups such as p-nitrophenoxy and N-oxysuccinimide. Amide bonds are synthesized using conventional chemistry well known to those of skill in the art. Suitable acylating agents include derivatives of the desired carboxylic acid to produce a pseudomycin compound wherein R1 = acylalkyl or amino acid to produce a pseudomycin compound wherein R1 = acylalkylamine. Typically, the acylating agent is formed by replacing the -OH of the carboxylic acid group with a leaving group (e.g., N-oxysuccinimide). 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 of skill in the art (eg, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, -methoxybenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, t-butyloxycarbonyl or cyclopentyloxycarbonyl). After the amide bond is formed, then the amino protecting group is removed using the normal hydrogenation chemistry (eg Pd / C under a hydrogen atmosphere). See the following Examples for a more detailed description to form the pseudomycin amide derivatives from amino acids. As discussed at the outset, pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinone peptides containing a portion of the cyclic peptide closed by a lactone bond and including the unusual amino acids 4-chlorotreonine (CIThr), 3-hydroxyapartic acid (HOAsp), 2, 3-dehydro-2-aminobutyric acid (Dhb), and 2,4-diaminobutyric acid (Dab). Methods for the development 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 US Patent Nos. 5,576,298 and 5,837,685, each of which is incorporated herein by reference. Strains- isolated from P. syringae that produce one or more pseudomycins are known in the art. Strain of the non-cultivated type MSU 174 and a mutant of this strain generated by "transposon mutagenesis, MSU 16H are described in US 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 the 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 Earth, water, air and dust. A preferred strain is isolated from plants. Strains of P. syringae that are isolated from environmental sources can be referred to as non-cultivated type. As used herein, "uncultivated type" refers to a dominant genotype which is of natural origin in the normal population of P. syringae (e.g., strains or isolates of P. syringae found in the nature and are not produced by laboratory manipulation). Like most organisms, the characteristics of the pseudomycin-producing cultures used (strains of P. syringae such as MSU 174, MSU 16H, MSU 206, 25-B1, 7H9-1) are subject to variation. Therefore, the progeny of these strains (e.g., recombinants, mutants and variants) can be obtained by methods known in the art. MSU strain 16H of P. syringae is publicly available from the American Type Culture Collection, Par la n Drive, Rockville, MD, USA as Accession No. ATCC 67028. Strains of P. syringae 25-B1, 7H9-1 and 67 Hl were deposited with the American Type Culture Collection on March 23, 2000 and assigned the following Access. Nos: 25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67 Hl Accession No. PTA-1621 Mutant strains of P. syringae are also suitable for the production of one or more pseudomycins. As used herein, "mutant" refers to an unexpected hereditary change in the phenotype of a strain, which may be spontaneous or induced by known mutagenic agents, such as radiation (e.g., ultraviolet radiation or x-rays). ), chemical mutagens (eg, ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N '-nitrosoguanine (NTG), and nitrous acid), site-specific mutagenesis, and transposon-mediated mutagenesis . Mutants that produce the pseudomycin of P. syringae can be produced by treating the bacteria with an amount of an effective mutagenic agent to produce mutants that overproduce one or more pseudomycins, which produce a pseudomycin (e.g., pseudomycin B) in excess of other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of the mutagenic agent that is used can vary, a preferred method is to serially dilute the 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 the selection of desirable characteristics of the growth habit, source of growth medium nutrients, carbon source, growth conditions, amino acid requirements and the like. . Preferably, a strain that produces the pseudomycin of P. syringae is selected for growth in a defined, minimal medium such as the N21 medium and / or for the production of one or more pseudomycins at levels greater than about 10 μg / ml. Preferred strains exhibit the characteristic of the production of one or more pseudomycins when grown in a medium that includes three or fewer amino acids and optionally, either a lipid, a potato product or a combination thereof. Recombinant strains can be developed by transforming strains of P. syringae, using procedures known in the art. Through the use of recombinant DNA technology, strains of P. syringae can be transformed to express a variety of genetic products in addition to the antibiotics that these strains produce. For example, one can modify the strains to introduce multiple, copies of the endogenous genes for the biosynthesis of pseudomycin to achieve a higher yield of pseudomycin. To produce one or more pseudomycins of a non-cultivated or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium that includes an effective amount of three or less amino acids, preferably glutamic acid, glycine, histidine, or combinations thereof. Alternatively, glycine is combined with one or more of a potato product and a lipid. The culture is conducted under conditions effective for the development of P. syringae and the production of the desired pseudomycin or pseudomycins. Effective conditions include temperatures from about 22 ° C to about 270 ° C, and a duration from about 36 hours to about 96 hours. The control of the concentration of oxygen in the medium during the cultivation of P. syringae is advantageous for the production of a pseudomycin. Preferably, the oxygen levels are maintained at about 5 to 50% saturation, more preferably about 30% saturation. The bubbling with air, pure oxygen, or mixtures of gases that include oxygen, can regulate the concentration of oxygen in the medium. Control of the pH of the medium during the cultivation of P. syringae is also advantageous. Pseudomycins are labile at the basic pH, and significant degradation can occur if the pH of the culture medium is above about 6 by more than about 12 hours. Preferably, the pH of the culture medium is maintained between 6 and. The strain of P. syringae can produce one or more pseudomycins when grown in batch culture. However, semi-continuous feeding or by a feeding bath of glucose and optionally, an acid or a base (eg, ammonium hydroxide) to control the pH increases production. The production of pseudomycin can be further improved by using continuous culture methods in which glucose and ammonium hydroxide are fed automatically. The selection of the P. syringae strain may affect the amount and distribution of the pseudomycin or pseudomycins produced. For example, strains MUS 16H and 67 Hl each predominantly produce pseudomycin A, but also produce pseudomycin B and C, typically at 4: 2: 1 ratios. Strain 67 Hl typically produces levels of pseudomycins about three to five times larger than those produced by strain MSU 16H. Compared to the MSU 16H and 67 Hl strains, strain 25-B1 produces more pseudomycin B and less pseudomycin C. The characteristic of strain 7H9-1 is the predominant production of pseudomycin B and a larger amount of pseudomycin B than the other strains For example, this strain can produce pseudomycin B in at least a tenfold excess over any pseudomycin A or C. Alternatively, the pseudomycin compounds modified in the amine groups of the present invention can be formed from a semi-synthetic compound of N-acyl. Semi-synthetic pseudomycin compounds can be synthesized by changing the N-acyl group in the L-serine unit. Examples of various N-acyl derivatives are disclosed in PCT Patent Application Serial No., Belvo et al., Filed therein, hereby entitled "Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference. reference. In general, as many steps of synthesis are used to produce the semi-synthetic compounds from pseudomycin compounds of natural origin: (1) selective protection of amino; (2) chemical or enzymatic deacylation of the N-acyl side chain; (3) reagelation with a different side chain; and (4) deprotection of the amino groups. The pendant amino groups at positions 2, 4 and 5 can be protected using any normal means known to those skilled in the art for amino protection. The exact genus and species of the amino protecting group employed is not critical since the derivatized amino group is stable in the condition of the subsequent reaction (s) in other positions of the intermediate product molecule and the The protecting group can be selectively removed at the appropriate point without breaking "the rest of the molecule" including any other amino protecting group (s) Suitable amino protecting groups include benzyloxycarbonyl, P-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl , p-methoxybenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, t-butyloxycarbonyl, cyclopentyloxycarbonyl and phthalimido The preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl (Alloc), phthalimido and benzyloxycarbonyl (CbZ or CBZ). Additional examples of suitable protecting groups are described in TW Greene, "Protective Groups i Organic Synthesis," John Wiley and Sons, New Yo. rk, N.Y. (2nd edition, 1991), in chapter 7. Deacylation of an N-acyl group having a hydroxylated gamma or delta side chain (eg, 3,4-dihydroxytetradeconolate) can be performed by treating the protected pseudomycin compound with amino 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 can be carried out at or near room temperature. However, when the acetic acid is used, the reaction is generally carried out 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 can lead to other by-products. Pseudomycin compounds lacking a delta or gamma hydroxy group in the side chain (e.g., Pseudomycin B and C) can be enzymatically deacylated. Suitable desacylase enzymes include Polimixin Acylase (164-16081 Fatty Acylase (raw) or 161-16091 Fatty Acylase (pure) available from Wako Pure Chemical Industries, Ltd), or ECB deacylase. Enzymatic deacylation can be performed using normal deacetylation procedures well known to those of skill in the art For example, general procedures for using polymyxin acylase can also be found in Yasuda, N. et al. ., Chem., 53, 3245 (1989) and Kimura, Y. et al., Agrie. Biol., Chem., 53, 497 (1989). The deacylated product (also known as the pseudomycin nucleus) turns to acylating using the corresponding acid of the desired acyl group in the presence of a carbonyl activating agent. "Carbonyl activation group" refers to a carbonyl substituent that promotes nucleophilic addition reactions in that carbonyl. suitable are those that have a net electron removal effect on carbonyl.These groups include, but are not limited to, alkoxy, "aryloxy, aromatic heterocyclics that contain nitrogen, or amino groups (for example, oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N, N '-dicyclohexyloureou-O-yl, and N-hydroxy-N-methoxyamino); acetates, formates, sulfonates (for example, methanesulfonate, ethanesulfonate, benzenesulfonate and p-tolylsulfonate); and halides (for example, chloride, bromide and iodide). A variety of acids can 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 alkylamino groups; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy and / or alkylamino groups. Alternatively, a solid phase synthesis can be used where a hydroxybenzotriazole-resin (HOBt-resin) serves as the coupling agent for the acylation reaction. Once the amino group is deacylated and re-acylated (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. Pd 10% / C). When the amino protecting group is allyloxycarbonyl, then the protecting group can be removed using tributyltin hydride and triphenylphosphine palladium dichloride. This particular reaction scheme for protection / deprotection has the advantage of reducing the potential for hydrogenation of the vinyl group of the Z-Dhb unit of the pseudomycin structure. The modification in the amine groups of the semi-synthetic N-acyl compound is then carried out by the acylation of at least one of the pendant amino groups bound to the lysine or the 2,4-diaminobutyric acid peptide units of the pseudomycin compound semi -synthetic and modified in N-acyl to form the desired amide, urea, carbamate or imide bond. The modified pseudomycin compounds in the amine groups can be further modified by the amidation or esterification of the pending carboxylic acid group of the aspartic acid and / or hydroxyapartic acid units of the pseudomycin ring. Examples of various derivatives modified in the acid groups are described in PCT Patent Application Serial No. PCT / US00 / 15019, Chen et al., Filed on the same date as the present one, entitled "Pseudomycin Amide & amp;; Ester Analogs "and incorporated herein by reference Derivatives modified in the acid groups can be formed by the condensation of any of the pseudomycin compounds modified in the amine groups previously described with the appropriate alcohol or amine to produce the ester or amide The formation of the ester groups can be carried out using normal esterification procedures well known to those of skill in the art, Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of an acid. protic (eg, HCl, TFA, etc.) Under basic conditions, the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (eg, sodium bicarbonate and potassium carbonate). The formation of the amide groups can be done using pr Normal amidation processes well known to those of skill in the art. However, the selection of the coupling agents provides selective modification of the acid groups. For example, the use of benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as the coupling agent allows the pure monoamides to be isolated at residue 8 and (in some cases) the pure bis amides simultaneously. Whereas, the use of o-benzothirazol-1-yl-N, N, ', N' -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent favors the formation of mono-amides in residue 3. Amide derivatives of pseudomycin can 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, sulphites, bisulfites, arylsulfonates, alkyl sulfates, phosphonates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanadates, arylalkates, adipates, alginates, aspartates, benzoates, fumarates, glycoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, camhorates, camphorsulfonates, digluconates, trifluoroacetates, and the like. The term "solvate" refers to an aggregate comprising one or more molecules of the solute (ie, pseudomycin compound modified in the amine groups) 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 retarding cooling to generate an amorphous or crystalline solvate form. Each compound of pseudomycin, semi-synthetic pseudomycin derivatives, and mixtures can be detected, determined, isolated and / or purified by any variety of methods known to those of skill in the art. For example, the level of activity of the modified pseudomycin or pseudomycin in the amine groups in a broth or in an isolated or purified composition can be determined by the antifungal action against a fungus such as Candida and can be isolated and purified by liquid chromatography. high resolution The active ingredient (ie, pseudomycin compound of the present invention) is typically formulated into pharmaceutical dosage forms that provide an easily controllable dosage of the drug and that give the patient, physician or veterinarian an elegant and easy to handle product. The formulations may comprise from 0.1% to 99.9% by weight of the 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 containing a predetermined amount of the 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 packets, topical composition, suppository, wafer, units measured in ampules or in containers for multiple doses, and so on. Alternatively, a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed. The dosage that is administered may vary depending on the physical characteristics of the animal, the severity of the animal's symptoms, the means used to administer the drug, and the species of the animal. The specific dose for a given animal is usually established by the judgment of the doctor or veterinarian who is carrying out the treatment. Suitable carriers, diluents and excipients are well known to those of skill in the art and include materials such as carbohydrates, waxes, soluble and / or expandable polymers in water, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent or excipient used will depend on the medium and purpose for which the active ingredient is being applied. The formulations may also include wetting agents, lubricants, surfactants, buffers, tonicity agents, bulking agents, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, perfume agents, flavoring agents and combinations thereof. A pharmaceutical composition can be administered using a variety of methods. Suitable methods include topical administration (e.g., ointments or sprays), oral, injection and inhalation. The particular treatment method used will depend on the type of infection to which it is directed. In parenteral iv applications, the formulations are typically diluted or reconstituted (if they are dehydrated by "freezing) and further diluted if necessary, prior to administration." An example of reconstitution instructions for the freeze-dried product is to adhere to ten ml of water for injection (WFI) into the vial and shake gently to dissolve Typical reconstitution times are less than one minute The resulting solution is then further diluted in an infusion solution such as 5% dextrose in water (D5W), before the administration.

It has been shown that pseudomycin compounds exhibit antifungal activity such as growth inhibition of various infectious fungi including Candida spp. (ie C. albicans, C. parapsilosis, C. krusei, C. glabra ta, C. tropicalis, or C. lusi taniaw); Torulopus spp. (ie, T. glabrata); Aspergillus spp. (ie, A. fumigatus); Histoplasma spp. (ie, H. capsula tum); Cryptococcus spp. (ie, C. neoformans); Blastomyces spp (ie, B. derma titidis); Fusarium spp.; Trichophyton spp., Pseudallescheria boydii, Coccidioides iüimi ts, Sporothrix schenckii, etcetera. Accordingly, the compounds and formulations of the present invention are useful in the preparation of medicaments for use in the combat of either systemic fungal infections or fungal skin infections. Accordingly, there is provided a method for inhibiting fungal activity comprising contacting the modified pseudomycin compound in the amine groups of the present invention with a fungus. A preferred method includes inhibiting the activity of Candida albicans or Aspergillus fumiga tus. The term "contacting" includes a junction, or an apparent touch or mutual tangency of a compound of the invention with a fungus. The term does not imply any additional limitation to the process, as by a mechanism of inhibition. The methods are defined as including the inhibition of fungal activity by the action of the compounds and their inherent antifungal properties. A method for treating a fungal infection comprising 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 an infection of Candida albicans or Aspergillus fumiga tus. The term "effective amount" refers to an amount of the active compound that is capable of inhibiting fungal activity. The dose administered will vary depending on factors such 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 may vary in the same way according to these factors. The medication can be given in a single daily dose or in multiple doses during the day. The regimen can last from approximately 2-3 days to approximately 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 that includes humans, companion animals (eg dogs, cats and horses), animals useful as a food source (eg cows, pigs, sheep and poultry), zoo animals, marine animals, birds and other similar animal species.

EXAMPLES The following abbreviations are used in all examples to represent the respective materials listed: ACN - acetonitrile TFA - trifluoroacetic acid DMF - dimethylformamide EDCI - 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride BOC = t-butoxycarbonyl , (CH3) 3C-0-C (O) - CBZ = benzyloxycarbonyl, C6H5CH2-0-C (O) - PyBOP = benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-1-yl- N tetrafluoroborate , N, N ', N' -tetramethyluronium DIEA = N, N-diisopropylethylamine The following structure II will be used to describe the products observed in Examples 1 through 7.

II Detection and Quantification of Antifungal Activity: The 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 disk diffusion test. A typical fungus used in a test of the antifungal activity is Candida albicans. The antifungal activity is considered significant when the test sample (50 μl) causes zones of inhibition of 10-12 mm in diameter on agar plates seeded with C. albicans x657.

Tail Vein Toxicity: -The mice were treated intravenously (IV) through the lateral tail vein with 0.1 ml of the test compound (20 mg / kg) at 0, 24, 48 and 72 hours. Two mice were included in each group. The compounds were formulated in 5.0% dextrose and sterile water for injection. The mice were monitored for 7 days after the first treatment and signs of irritation including erythema, swelling, discoloration, necrosis, loss of tail and any other signs of adverse effects indicating toxicity were observed closely. The mice used in the study were male ICR mice, multiplied by exogamy having an average weight between 18-20 g (available from Harlan Sprangue Dawley, Indianapolis, IN).

Prepare the Composite -1 la-1 Compound la-1 is commercially available from Novabiochem (San Diego, CA).

Preparation of Compound 2a-l: 2a-1 Compound 2a-l was prepared using the procedures described in Admiak, R.W. and collaborators, Tetrahedron Lett. , No. 22, 1935-1936 (1997). In each of the following Examples, a specific pseudomycin compound was used as the starting material; however, those of skill in the art will recognize that other N-acyl derivatives can be synthesized using the same procedures, except by starting with a pseudomycin compound having a different N-acyl group.

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

Synthesis of Compound 1 -1.

R R1 and R1 = -C (0) CH2NH2 1-1 A 50 ml round bottom flask was charged with 10 ml of anhydrous DMF, pseudomycin B (250.6 mg, 0.181 mmol) and the acylating agent la-1 (343.0 mg, 1.12 mmol). The reaction was allowed to stir at room temperature for 24 hours. The solvent was then removed in vacuo and the residue was taken up in ACN and purified by preparative 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 10 ml MeOH / 1.5 ml glacial AcOH solution. Normal hydrogenolysis using 129.6 mg of 10% Pd / C for 20 minutes, removal of the catalyst by filtration and purification by preparative HPLC yielded 74.8 mg of Compound 1-1 after lyophilization. MS (Ionspray) calculated for C57H97ClN? 5022 (M + H) + 1378.65, found 1378.9, The following pseudomycin amide derivatives (1-2 and 1-3) can be synthesized using the same procedures described above and the appropriate amino acid for form the acylating agent. 1-2 1-3 Example 2 Example 2 illustrates the synthesis of the acyloxyaryl derivatives of pseudomycin B (n = 10, R2 and R3 = -OH).

Synthesis of Compound 2-1: 2-1H) Compound 2-1 was synthesized using the same procedures described in Example 1, except that Compound 2a-l is used as the acylating agent. Compound 2-1 can 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 was diluted with 100 ml of DMF and pseudomycin B (1.0 g, 0.83 mmol) was added. The mixture was allowed to stir overnight. The solvent was then removed in vacuo and the residue was purified by HPLC to yield 430 mg (33% yield) of Compound 2-1.

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

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

Synthesis of Compound 3-2: R1 ', Ri ", and R * - - (CH3) 3 3-2 Compound 3-2 is synthesized using the same procedures described in Example 1, except that trimethylacetic anhydride is used as the acylating agent.

Example 4 Example 4 illustrates the synthesis of acylazaalkyl derivatives (ie, urea linkage) of pseudomycin B (n = 10, R2 and R3 = -OH).

Synthesis of Compound 4-1: R1 ', R1 * and Ra * T? HCH, 4-1 - Compound 4-1 is synthesized using the same procedures described in Example 1, except that methyl isocyanate is used as the acylating agent.

Example 5 Example 5 illustrates the synthesis of a formyl derivative of pseudomycin B (n = 10, R and R 3 _ = -OH) 5-1 Compound 5-1 is synthesized using the same procedures described in Example 1, except that 4-nitrophenylformate is used as the acylating agent.

Example 6 Example 6 illustrates the synthesis of an acyloxyalkenyl derivative of pseudomycin B (n = 10, R2 and R3 = -OH). 6-1 • Compound 6-1 is synthesized using the same procedures described in Example 1, except that diallyl pyrocarbonate is used as the acylating agent. 77% yield.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (6)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A modified pseudomycin compound in the amine groups having the following structure: characterized because R is. where Ra and Ra 'are independently hydrogen or methyl, or any of Ra or Ra' is alkylamino, taken together with Rb or Rb 'form a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc they form a six-membered aromatic ring; Rb and Rb 'are independently hydrogen, halogen or methyl or any of Rb or Rb' is amino, alkylamino, α-acetoacetate, methoxy or hydroxy; Rc is hydrogen, hydroxy, alkoxy of 1 to 4 carbon atoms, hydroxy-alkoxy of 1 to 4 carbon atoms, or taken together with Re forms a 6-membered aromatic ring or a cycloalkyl ring of 5 to 6 carbon atoms; Rd is hydrogen; Re is hydrogen, or taken together with Rf is a six-membered aromatic ring, a six-membered aromatic ring substituted with alkoxy of 5 to 14 carbon atoms, or a six-membered aromatic ring substituted with alkyl of 5 to 14 carbon atoms. carbon, and Rf is alkyl of 8 to 18 carbon atoms or alkoxy of 5 to 11 carbon atoms; R is where Rg is hydrogen, or alkyl of 1 to 13 carbon atoms, and Rh is alkyl of 1 to 15 carbon atoms, alkoxy of 4 to 15 carbon atoms, (alkyl of 1 to 10 carbon atoms) phenyl, - (CH2) n-aryl or - (CH2) n- (cycloalkyl of 5 to 6 carbon atoms), where n = 1 or 2; or 5 R is wherein 0 R1 is hydrogen, halogen or alkoxy of 5 carbon atoms, and m is 1, 2 or 3; R is Where Rj is alkoxy of 5 to 14 carbon atoms or alkyl of 5 to. 14 carbon atoms, and p = 0, 1 or 2; R is where R is alkoxy of 5 to 14 carbon atoms; or R is - (CH2) -NRm- (alkyl of 13 to 18 carbon atoms), where Rm is H, -CH3 or -C (O) CH3; R1 is independently hydrogen, formyl, an acylalkyl, an acylalkyl ina, an acylazaalkyl, an acyloxyalkene, an acyloxyaryl, or an acylmethylenecarbamate, with the proviso that at least one R1 is not hydrogen; R2 and R3 are independently -OR2a or -N (R2) (R2c), wherein R2a and R2b are independently hydrogen, alkyl of 1 to 10 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, hydroxy-alkyl of 1 to 10 carbon, alkoxyalkyl or alkenyl atoms of 2 to 10 carbon atoms, amino-alkyl of 1 to 10 carbon atoms, mono- or di-alkylamino-alkyl of 1 to 10 carbon atoms, aryl-alkyl of 1 to 10 atoms of carbon, heteroaryl-alkyl of 1 to 10 carbon atoms, cycloheteroalkyl-alkyl of 1 to 10 carbon atoms, or R2b is an alkyl carboxylate residue of an alkyl amino acid ester and R2c is hydrogen or alkyl of 1 to 6 carbon atoms; and pharmaceutically acceptable salts and solvates thereof.

2. The modified pseudomycin compound in the amine groups according to claim 1, characterized in that the acylmethylenecarbate is represented by structure 1 (a): Ka) where Rla is alkyl of 1 to 10 carbon atoms, alkenyl of 1 to 10 carbon atoms, benzyl, or aryl and Rlb is hydrogen or methyl.

3. The modified pseudomycin compound in the amine groups according to claim 1, characterized in that the alkyl carboxylate residue of an alkyl amino acid ester is represented by -CHC02CH3, -CH (C02CH3) CH (CH3) 2, -CH (C02CH3) CH (phenyl), -CH (C02CH3) CH2OH, -CH (C02CH3) CH2 (p-hydroxyfenyl), -CH (C02CH3) CH2SH, -CH (C02CH3) CH2 (CH2) 3NH2, -CH (C02CH3) CH2 (4-imidazole), -CH (C02CH3) CH2 (5-imidazole), -CH (C02CH3) CH2C02CH3, or -CH (CO2CH3) CH2C02NH2.

4. The use of a compound according to any of the preceding claims in the preparation of a medicament for use in the combat of either systemic fungal infections or fungal skin infections.

5. A pharmaceutical formulation, characterized in that it comprises the modified pseudomycin compound in the amine groups according to claim 1 or the pharmaceutically acceptable salt or solvate thereof, in combination with a pharmaceutically acceptable carrier, buffer, diluent or excipient.

6. The use of a compound according to claim 1 for the manufacture of a medicament for treating an infection in an animal.