MXPA02000315A - Pseudomycin prodrugs. - Google Patents

Abstract

A pseudomycin prodrug represented by structure (A) where R1 is an acyloxyalkylcarbamate linkage is described. The prodrug demonstrates antifungal activity with less adverse side effects.

Description

PROFARMACOS DE PSEUDO ICINA Field of the Invention The present invention relates to pseudomycin compounds, in particular, prodrugs of pseudomycin compounds.

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, 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 U.S. Patent Nos. 5,576,298 and 5,837,685). Unlike the previously described antimycotics of P. syringae (for example, syringomycins, syringotoxins and syn-estestatins), pseudomycins A-C 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-Cl) with the terminal carboxyl group that closes a macrocyclic ring in the OH group of the N-Being REF: 134820 terminal. The analogs are distinguished by the N-acyl secondary chain, ie, pseudomycin A is N-acylated by 3,4-dihydroxytetradecanoyl, pseudomycin A 'by 3,4-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. Therefore, there is a need to identify compounds within this class that are useful for the treatment of fungal infections without the adverse side effects currently observed.

Brief Description of the Invention The present invention provides a prodrug of pseudomycin represented by the following structure which is useful as an antifungal agent. 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, -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 atoms of carbon, and Rf is alkyl of 8 to 18 carbon atoms or alkoxy of 5 to 11 carbon atoms; 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; where R ^ 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 (0) CH3; R1 is independently hydrogen, an acyloxymethylene-1,3-dioxolen-2-one (for example, the compounds 1 (a) represented below), or an acyloxymethylenecarboxylate (for example the compounds 1 (b) represented below) Ka) Kb) where R is hydrogen, alkyl of 1 to 10 carbon atoms, alkenyl of 2 to 10 carbon atoms, benzyl, or aryl and R 1 is hydrogen or methyl with the proviso that at least one R 1 is an acyloxymethylene-1,3-dioxolen -2-one or an acyloxymethylenecarboxylate; R2 and R3 are independently -0R2a, or -N (R2b) (R2c), wherein R2a and R2b 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-1-alkyl 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-alkylamino- alkyl 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 a residue of alkyl c arboxylate 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) CH2C02CH3, -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 prodrug of pseudomycin 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 prodrug described above. The use of the pseudomycin prodrug described above in the preparation of a medicament for use in the treatment of a fungal infection in an animal is also provided.

Defined ions As used herein, the term "alkyl" refers to a hydrocarbon radical of the general formula CnH2n +? containing from 1 to 30 carbon atoms unless otherwise indicated. 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, methylcyclopentyl, 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 containing 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 group, alkenoyl or alkenoate 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 includes the unsubstituted alkyl moiety . 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 "prodrug" refers to a class of drugs that result in a pharmacological action due to conversion by metabolic processes within the body (ie, biotransformation). In the present invention, the pseudomycin prodrug compounds contain linkers that can be divided by esterases in the plasma to produce the active drug. 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 a prodrug derivative of the natural or semi-synthetic pseudomycin products provides fewer adverse side effects than the corresponding natural products and maintains efficacy in vivo against C. albican, C. neoformans and A. fumiga your The prodrug is produced by the acylation of at least one of the pendant amino groups attached to the lysine units or 2-diaminobutyric acid peptide in the ring system of the pseudomycin cyclopeptide to form a substituent or substituents. The acylating agent (or linker) is generally an acylating compound of acyloxymethylene-1,3-dioxolen-2-one or acyloxymethylenecarboxylate containing a suitable leaving group such that a carbamate bond with the pendant amino group can be formed in the structure of pseudomycin. Suitable leaving groups are well known to those of skill in the art and include groups such as p-nitrophenoxy and N-oxysuccinimide. An acylating compound of acyloxymethylene-1,3-dioxolen-2-one can be synthesized using the synthetic route shown in reaction scheme I below. For illustrative purposes, a specific acylation compound is represented. However, it will be understood by those of experience in the art that one could synthesize a variety of derivatives using the same synthetic, basic method.

Reaction Scheme I For a more detailed description of the synthesis procedures, see below the preparation section of the Examples. The acyloxymethylenecarboxylate acylation compound can be synthesized using the synthetic route shown in the Reaction II scheme below. For illustrative purposes, a specific acylation compound is represented. However, it will be understood by those of experience in the art that one could synthesize a variety of derivatives using the same synthetic, basic method.

Reaction Scheme II For a more detailed description of the synthesis procedures, see below the preparation section of the Examples. As discussed at the beginning, pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinonapeptides that contain a portion of the cyclic peptide closed by a lactone bond and that includes the unusual amino acids 4-chlorotreonine (CIThr), acid 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 below and described in more detail in the PCT Patent Application. PCT / US00 / 08728 Series No. filed by Hilton et al. On April 14, 2000 entitled "Pseudomycin Production by Pseudomonas Syringae", hereby incorporated 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 Pat. Nos. 5,576,298 and 5,837,685, each of which is incorporated herein by reference. Isolated strains of P. syringae that produce one or more pseudomycins are known in the art. The strain of the uncultivated type MSU 174 and a mutant of this strain generated by the 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. Nati 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 and 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. The strain MSU 16H of P. syringae is publicly available from the American Type Culture Collection, Parkla 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 (e.g., ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N '-nitrosoguanin (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 defined minimal 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 methods 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 pseudomycin biosynthesis 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. Bubbling with air, pure oxygen, or mixtures of gases that include oxygen can regulate the concentration of oxygen in the medium. The 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 4. 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 does 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 prodrug can be formed from a semi-synthetic N-acyl compound. 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 Anaiogs" 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 protecting group can be selectively removed at the appropriate point without breaking the remainder of the molecule that includes 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. 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 T.W. Greene, "Protective Groups in Organic Synthesis," John Wiley and Sons, New York, (2nd edition, 1991), in Chapter 7. Deacylation of an N-acyl group having a hydroxylated gamma or delta side chain (e.g. , 3,4-dihydroxytetradeconate) can be made 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 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 Fat Acylase (raw) or 161-16091 Fat Acylase (pure) available from Wako Puré Chemical Industries, Ltd), or ECB Desacilasa. Enzymatic deacylation can be performed using normal deacylation 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. and collaborators Agrie. Biol., Chem., 53, 3245 (1989) and Kimura, Y. et al., Agrie. Biol., Chem., 53, 497 (1989). The deacylated product (also known as the pseudomycin nucleus) is acylated again 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 activating substituents are those that have a net electron removal effect on carbonyl. These groups include, but are not limited to, alkoxy, aryloxy, nitrogen-containing aromatic heterocyclics, or amino groups (eg, 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 that contain one or more pendant groups aryl, alkyl, amino (including primary, secondary and tertiary amines), hydroxy, alkoxy and amido; 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 prodrug is then produced by acylating at least one of the pendant amino groups bound to lysine or the peptide units of 2,4-diaminobutyric acid of the semi-synthetic pseudomycin compound and modified to N-acyl to form the desired carbamate linkage . Other modified prodrug pseudomycin compounds can be synthesized by 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 / 15021, Chen et al., Filed on the same date as the present one, entitled "Pseudomycin Amide &Ester Anaiogs" and incorporated herein by reference. The modified derivatives in the acid groups can be formed by condensation of any of the previously described prodrugs with an appropriate alcohol or amine to produce the respective 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 a protic acid (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 performed using normal amidation procedures 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-benzothirazl-1-yl-N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent favors the formation of monoamides in residue 3. The prodrug of pseudomycin can be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate. The prodrug is prepared by forming at least one acyloxyalkylcarbamate linkage as described more recently. 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 sulphates, phosphonates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkyl alkanoates, arylalkates, adipates, alginates, aspartates, benzoates, fumarates, glycoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates, citrates, , camphorsulfonates, digluconates, trifluoroacetates, and the like. The term "solvate" refers to an aggregate comprising one or more molecules of the solute (ie, compound of pseudomycin prodrug) 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 gener formed by dissolving the prodrug in the appropriate solvent with heat and retarding cooling to generate an amorphous or crystne solvate form. Each pseudomycin, semi-synthetic pseudomycin, pseudomycin prodrug 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 pseudomycin or pseudomycin prodrug 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 high performance liquid chromatography. . The active ingredient (i.e., pseudomycin prodrug) is typic 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 gener from about 10% to about 30% by weight. As used herein, the term "unit dose" or "unit dosage" refers to physic discrete units containing a predetermined amount of the active ingredient calculated to produce a desired therapeutic effect. When a unit dose is administered or or parenter, it is typic 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 animal. The specific dose for a given animal is usu 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, lubricating agents, 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 add ten ml of water for injection (WFI) to 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), prior to 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. glabrata, C. tropicalis, or C. lusitaniaw); Torulopus spp. (ie, T. glabrata); Aspergillus spp. (that is, A. fumiga tus); Histoplasma spp. (ie, H. capsula tum); Cryptococcus spp. (ie, C. neoformans); Blastomyces spp (ie, B. derma ti tidis); Fusarium spp .; Trichophyton spp., Pseudallescheria boydii, Coccidioides immits, 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 pseudomycin prodrug of the present invention with a fungus. A preferred method includes inhibiting the activity of Candida albicans or Aspergillus fumigatus. 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, such as by a mechanism of inhibition. The methods are defined as including the inhibition of parasitic and 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, Cryptococcus neoformans, 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 including 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-OC (0) - CBZ = benzyloxycarbonyl, C6H5CH2-0-C (0) - PyBOP = benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate TBTU = o-Benzotriazol-1-yl- N, N tetrafluoroborate , 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: 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).

Preparations Preparation of 4-Bromomethyl-5-methyl-1,3-dioxolene-2-one (-1): la-1 A mixture of 0.1 mol of 4,5-dimethyl-1,3-dioxolene-2-one, 0.1 mol of N-bromosuccinimide and 0.1 g of 2,2-azobis (2-methylpropionitrile) in 70 ml of carbontetrachloride (CC14) it was heated to reflux. After six hours, the mixture was cooled with ice and filtered. The filtrate was washed with 2 × 50 ml of water, 2 × 50 ml of a sodium chloride solution and an additional 50 ml of water. The solution was dried over sodium sulfate and evaporated to dryness, and dried under vacuum to yield 16.5 g (85% yield) of an oil having 1H-NMR data consistent with structure la-1.

Preparation of the Compound (-2) la-2 Compound la-2 was synthesized using the procedures described in Synthetic Communication, 22 (9), 1297 (1992) to produce 11.5 g (78% yield) of a crude oil ...

Preparation of Compound la-3: la-3 A mixture of 11.5 g of Compound la-2 (crude oil), 500 ml of 37% HCl and 300 ml of methanol was allowed to stir overnight at 4 ° C. The mixture was then concentrated to form an oil. Purification by column chromatography (ethyl acetate / hexane 1: 1) yielded 5.27 g (33.8%) of the product having the 1H-NMR data consistent with the la-3 structure.

Preparation of Compound -4: la-4 A mixture of 3.0 g of Compound la-3 and 2.02 g of pyridine in 30 ml of chloroform was cooled to 0-4 ° C. A solution of 5.08 g of p-nitrophenyl-chloroformate in 30 ml of chloroform was added to the mixture and allowed to stir for about 4.5 hours. The mixture was washed with cold 1% sodium hydroxide (3x30 ml), IN HCl (2x30 ml), water (2x30 ml) and brine (2x30 ml). The solution was dried over sodium sulfate, filtered and washed with dichloromethane. Removal of the solvent produced an oil which solidified at rest. The solid was taken up in 10 ml of dichloromethane and hexane was added to form a precipitate. The mixture was filtered, washed with hexane and dried under vacuum overnight to yield 6.39 g (94% yield) of the product having the 1H-NMR data consistent with the la-4 structure.

Preparation of Compound lb-1: lb-1 Compound lb-1 can be synthesized using the procedures described in Synthesis, 1159 (1990).

Preparation of Compound lb-2: lb-2 A solution of 4.57 g (30 mmoles) of crude lb-1 in 40 ml of dichloromethane was added at 0 ° C to a solution of 3.91 g (34 mmoles) of N-hydroxy succinimide and 2.7 g (34 mmoles) of pyridine in 100 ml of dichloromethane. After stirring at 0 ° C for 30 minutes, the mixture was allowed to stand at room temperature overnight. The mixture was then washed with water four times and the organic phase was dried over sodium sulfate. In filtration, the solvent was evaporated to give 4.0 g (58% yield) of an oily crude product having the 1H-NMR data consistent with the lb-2 structure.

Preparation of Pseudomycin B Protected with CBZ (2a-l): Dissolve / suspend Pseudomycin B in DMF (20 mg / ml, Alrich Sure Seal). While stirring at room temperature, N- (benzyloxycarbonyloxy) succinimide (6 eq) is added. Allow to stir at room temperature for 32 hours. Monitor the reaction by HPLC (4.6x50 mm, 3.5 μm, 300-SB, C8, Zorbax column). Concentrate the reaction to 10 ml in a high vacuum roto-evaporator at room temperature. Place the material in a freezer until it is ready for preparation by chromatography. Preparative reverse phase HPLC yields a white, amorphous solid (Compound 2a-1) after lyophilization.

Preparation of Compound 2b-1: R R1 and R1 H R = -NH (cyclopropyl) R3 = -OH 2b-l The CBZ-protected pseudomycin B (2a-l) (400 mg, 0.25 mmol) is dissolved in 4 ml of dry DMF. TBTU (79 mg, 0.25 mmol), DIEA (200 μl, 6 equivalents) and cyclopropylamide (14.2 mg, 0.25 mmol) were added sequentially. The reaction was stirred at room temperature under nitrogen while it was monitored by HPLC. Upon completion of the reaction, it was concentrated in vacuo. The crude product was purified by preparative HPLC. Lyophilization produced 209.2 mg (51.1%) of a colorless powder. The 3-amido compound (279.1 mg, 0.169 mmol) was hydrogenified under a 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 taken up in a 1: 1 mixture of water: ACN and then lyophilized to give 208.3 mg (98.6%) of a colorless powder (2b-1). The structure was verified by the H1-NMR.

Preparation of Compound 3a-1: R1 R1 and R1 = H Rz = -OCH3 3a-l A 50 ml round bottom flask was charged with 10 ml of pure ethanol and 251.7 mg of CBZ protected pseudomycin B (2a-l) (0.156 mmole). To this mixture was added ~ 1 ml of acidified ethanol (previously acidified using HCl gas) and the reaction was allowed to stir at room temperature overnight. The solvent was then removed in vacuo and the residue was taken to the next step without further purification by dissolving it in a solution of 10 ml of MeOH / 1.5 ml of glacial AcOH. Normal hydrogenolysis using 249.7 mg of Pd 10% / C for 30 minutes, removal of the catalyst by means of filtration and purification by means of preparative HPLC yielded 120.9 mg of Compound 3a-l after lyophilization. MS (Ionic Spray) calculated for C55H96CIN12O1C) (M + H) + 1264.89, found 1264.3.

Preparation of the Teral C-18 chain (4a-l) To a dichloromethane solution (190 mL) of chiral acetal 4a-l (6.22 g, 19.1 mmol) was added at -78 ° C trimethylallylsilane (10.9 mL, 68.69 mmol), followed by pure TiCl 4 (2.94 mL, 26.71 mmol). The reaction was stirred at -78 ° C for 1 hour and then at -40 ° C for 2 hours. At this point, the reaction was quenched with methanol (15 ml) and diluted with dichloromethane (200 ml). The resulting reaction mixture was washed with IN HCl (2 x 50 mL), water and brine. The organic layer was dried and concentrated in vacuo to give a residue, which was purified by silica gel chromatography (10% EtOAc / Hexanes) to give 5.51 g (78%) of the desired product 4b-1. To a dichloromethane solution (155 mL) of 4b-l (8.56 g, 23.3 mmol) was added PCC (10.0 g, 46.5 mmol).

The reaction was stirred at room temperature for 18 hours, and then filtered through a pad of Celite. The filtered products were concentrated in vacuo to give a reddish residue, which was purified by silica gel chromatography (10% EtOAc / Hexanes) to give 8.36 g (80%) of the methyl ketone intermediate (structure not shown) . The intermediate product obtained in the present (8.36 g, 22.8 mmol) was dissolved in THF (60 mL) and MeOH (30 mL). To this solution was added 7.5 M KOH (15 mL). After stirring for 3 hours at room temperature, the solvent was partially removed. The remaining reaction mixtures were diluted with EtOAc / Et20 (ratio 3: 1, 350 mL). The organic layer was washed with water (3 x 50 mL) and brine. The resulting organic layer was dried and concentrated in vacuo to give a residue, which was purified by silica gel chromatography (10% EtOAc / Hexanes) to give 6.22 g (96 amp.;) of the desired product 4c-l as white solids. Carbinol 4c-l (6.22 g, 22.0 mmol) was dissolved in a solution of aqueous THF (5.5 mL of water and 55 mL of THF). To this solution was added NMO (4.42 g, 33.0 mmol), followed by Os04 (280 mg dissolved in THF, 1.10 mmol). The reaction was stirred at room temperature overnight. At this time, sodium bisulfide (4 g) was added. The reaction was stirred for 2 hours and then diluted with EtOAc (300 mL). The whole mixture was washed with water (2 x 40 mL) and brine. The resulting organic layer was dried and concentrated in vacuo to give the corresponding triol intermediate. This material was dissolved in MeOH (200 mL) and water (40 mL). To this solution was added NaI04 (10.6 g, 49.5 mmol). After stirring at room temperature for 1 hour, the reaction was filtered through Celite and purified by silica gel chromatography on a short column (30% EtOAc / Hexanes) to give ~ 10 g (> 100% ) of crude beta-hydroxyl aldehyde. The impure aldehyde obtained in this manner was dissolved in t-BuOH (100 mL) and cyclohexene (14 mL). To this solution at room temperature was added an aqueous solution (50 L) of NaC102 (15.97 g, 176 mmol) and KH2P04 (17.8 g, 132 mmol). The reaction was stirred at room temperature for 6 hours and then rapidly cooled to 0 ° C with 5N HCl to pH = 4. The reaction was extracted with a mixture of EtOAc / Et20 3: 1 solvents (3 x 250 mL). The organic layer was washed with brine and dried and concentrated to provide 7.3 g (> 100%) of crude acid 4d-1, which was used directly for the coupling reaction.

Preparation of Pseudomycin C-18 (Compound 4b-2): 4a-2 R = cbz 4b-2 R = H The crude acid 4d-1 (2.1 g, 6.99 mmol) was dissolved in dry THF (20 mL) and DMF (20 mL). To this solution was added HOBt (1.23 g, 9.08 mmol) and EDCI (1.74 g, 9.08 mmol). After stirring at room temperature for 8 hours, the core of the CBZ-protected pseudomycin (3.87 g, 2.80 mmol) was added. The reaction was stirred at room temperature for 2 days. At this point, the solvent was partially removed. The reaction mixture was loaded in a preparative reverse phase HPLC system for purification (4 injections). With lyophilization, 550 mg (12%) of the C18 acyl derivative protected with CBZ 4a-2 was isolated together with the side chain ester activated with HOBt (1 g). Then the recovered side-chain ester (1.0 g, 2.40 mmol) was reacted with the core of the CBZ-protected pseudomycin (1.33 g, 0.96 mmol) in dry THF and DMF (10 mL each). After the same purification procedure just mentioned, additional amounts of the C18 derivative protected with CBZ 4a-2 (606 mg, 38%) were obtained. To a solution of HOAc 10% / MeOH (55 mL) of the C18 derivative protected with CBZ 4a-2 (550 mg, 0.33 mmol) was added at -78 ° C Pd / C (550 mg, palladium content 10%). The reaction was subjected to hydrogenation under 1.5 atmospheric pressure for 40 minutes. The progress of the reaction was monitored by analytical HPLC. Upon completion, the catalyst was filtered and the filtrates were concentrated in vacuo at 30 ° C. The resulting residue was redissolved in aqueous acetonitrile 1: 1 and lyophilized to give 250 mg (60%) of the desired product 4b-2. 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 The following example demonstrates the formation of prodrugs with a mono-, di- and tri-substituted acyloxyalkylcarbamate bond of pseudomycin C (n = 12, R2 and R3 = -OH). To a solution of DMF (1 liter) containing Pseudomycin C (1.5 g, 1 eq.) Is added 1.5 eq. of Compound la-4 and the mixture was stirred at room temperature for about 3 days. The solvent was partially removed and the residue was purified by reverse phase HPLC (Waters * 111, Delta Pak C18 column) to produce the following products and the product mixture: 86 mg of mono-substituted pseudomycin C, pure (1- 1); 87 mg of a mono-substituted pseudomycin C mixture (1-2); 177 mg of a di-substituted pseudomycin C mixture (1-3); 132 mg of di-substituted pseudomycin C, pure (1-4); and 248 mg of tri-substituted pseudomycin C (1-5). No irritation of the tail vein was observed by the tri-substituted prodrug or the mixture of di-substituted prodrugs. Some irritation was observed with the mono-substituted, pure mixture of mono-substituted and di-substituted, pure prodrug samples. In comparison, unsubstituted pseudomycin C and unsubstituted pseudomycin B clearly showed tail vein irritation. All samples indicated a significant efficacy in vivo except the pure, disubstituted prodrug sample (ED50> 20 mg / kg × 4). Samples of mono-, di- and tri-substituted prodrugs of structure II above, where R1 ', R1"and / or R1 are and n is equal to 10, 12 and 14, they have also been made using the same procedure described above. Compounds 1-1 and 1-5 exhibited similar efficacy in vivo as pseudomycin C of the parent compound. No evidence of tail vein irritation was observed for Compound 1-5 and an improved profile of tail vein toxicity was observed for Compound 1-1. Surprisingly, in vivo efficacy for Compound 1-4 was not observed.

Ejexaplo 2 The following examples illustrate the formation of prodrugs with a mono-, di- and tri-substituted acyloxyalkylcarbamate linkage of pseudomycin B (n = 10, R2 and R3 = -OH). To a solution of Pseudomycin B (2.0 g, 1.65 mmol) dissolved in 500 ml of dimethylformamide was added 574 mg (2.47 mmoles) Compound lb-2. The mixture was stirred at room temperature overnight. The solution was then concentrated to approximately 50 ml and the products were purified by HPLC using a gradient elution scheme: 0-30% TFA / ACN in 5 minutes and 30-70% TFA / ACN in 40 minutes. A combined yield of 59% was observed. Three of the isolated products (mono-substituted prodrug (Compound 2-1) where R1 'and R1"= H and R1'" = -C (O) OCH2OAc, di-substituted prodrug (Compound 2-2) where R1 and R1 -C (0) OCH2OAc and R1"= H and tri-substituted prodrug (Compound 2-3) where R and R and R 1 -C (O) OCHOAc) were all tested and demonstrated efficacy in vivo against systemic murine candidiasis. However, the toxicity of the tail vein was positive.

Example 3 Using the same general procedures described above in Example 2, the mono-, di- and tri-substituted prodrugs of Pseudomycin B (n 10, R2 and R3 -OH) were prepared where R1 ', R1"and / or R1 '"-C (O) OCH2OC (O) C (CH3) 3. The following five samples were isolated: 3-1 R1'" mono-substituted 3-2 R1 'and R1"mono-substituted, mixed 3- 3 R1"'+ R1' and R1 '" + R1"di-substituted, mixed 3-4 R1' + R1" di-substituted 3-5 R1 '+ R1"+ R1'" tri-substituted Samples 3-1 , 3-3 and 3-5 each showed negative toxicity in the tail vein. All five samples demonstrated efficacy in vivo against systemic murine candidiasis.

Example 4 Using the same general procedures described above in Example 2, the mono-, di- and tri-substituted prodrugs of Pseudomycin C were prepared (n = 12, R2 and R3 = -OH) where R1 ', R1"and / or R1'" = -C (O) OCHOC (0) C (CH3) 3. The following five samples were isolated: 4-1 R1 '"mono-substituted 4-2 R1' and R1" mono-substituted, mixed 4-3 R1 '" + R1 'and R1' "+ R1" di-substituted, mixed 4-4 R1 '+ R1"di-substituted 4-5 R1' + R1 '+ tri-substituted Rx Sample 4-1 was not tested. 4-3, 4-4 and 4-5 all showed negative toxicity in the tail vein. Samples 4-2, 4-3, 4-4 and 4-5 all demonstrated efficacy in vivo against systemic murine candidiasis.

Example 5 Using the same general procedures described above in Example 2, mono-, di- and tri-substituted prodrugs of Pseudomycin B were prepared (n- = 10) where R1 ', R1"and / or R1'" = -C (O) OCH (CH3) OC (O) CH3. Only the tri-substituted derivative (Compound 5-1) was tested. The "tri-substituted compound showed negative toxicity in the tail vein.

EXAMPLE 6 Using the same general procedures described above in Example 2, mono-, di- and tri-substituted prodrugs of Pseudomycin B (n- = 10, R2 and R3 = -OH) where R1 ', R1"and / or R1 '"= -C (O) OCH2OC (O) CH2CH3. Only the tri-substituted derivative (Compound 6-1) was tested. The trisubstituted compound demonstrated good efficacy in vivo against systemic murine candidiasis without irritation of the tail vein.

Example 7 Using the same general procedures described above in Example 2, mono-, di- and tri-substituted prodrugs of Pseudomycin B were prepared (n = 10, R2 and Rd = -OH) where R1, R1 and / or R1 -C (O) OCH2OC (O) CH (CH3) CH3. Only the tri-substituted derivative (Compound 7-1) was tested. The trisubstituted compound demonstrated good efficacy in vivo against systemic murine candidiasis without irritation of the tail vein.

Example 8 Examples 8 and 9 illustrate the synthesis of prodrugs prepared from the semi-synthetic pseudomycin compounds where the N-acyl group pending the L-serine unit of the pseudomycin structure has been modified. Using the same general procedures described above in Example 2, mono-, di- and trisubstituted prodrugs of Pseudomycin C-18 (n = 14, R2 and R3 - -OH) (4b-2) were prepared where R1 ', R1"and / or R1 '" = -C (O) OCH2OC (O) C (CH3) 3. Only the tri-substituted derivative (Compound 8-1) was tested. The trisubstituted compound showed negative toxicity in the tail vein.

Example 9 Using the same general procedures described above in Example 2, mono-, di- and tri-substituted prodrugs of Pseudomycin C-18 (n = 14, R2 and R3 = -OH) (4b-2) were prepared where R1 ', R1"and / or R1'" = -C (0) OCH2OC (0) CH (CH3) CH3. Only the tri-substituted derivative (Compound 9-1) was tested. The trisubstituted compound showed negative toxicity in the tail vein.

Example 10 Example 10 illustrates the further modification of the prodrugs described above, wherein the carboxylic acid group of the aspartic acid unit of the pseudomycin ring is modified to form a 3-monoamido derivative.

Synthesis of Compounds 10-1, 10-2, 10-3, 10-4 and 10-5: Rx R? R1 -C (0) OCH2OC (0) C (CH3) 3 R "= -NHCH2CH2N (CH3) 2 RJ = -OH 10-1 To a solution of DMF (9 ml) of the prodrug 3-5 (864 mg, 0.52 mmol) was added l-dimethylamino-2-aminoethane (57.9 μl, 0.52 mmole) and TBTU (168.6 mg, 0.52 mmole), followed by diisopropylethylamine (423 μl). After stirring at room temperature for 20 minutes, the reaction mixture was purified by reverse phase HPLC (ACN: 0.1% TFA / Water). Lyophilization produced 295 mg (34%) of Compound 10-1.

R1 ', R1", R1'" = -C (0) OCH2OC (0) C (CH3) 3 R2 = -NH (cyclopropyl) RJ = -OH 10-2"Compound 10-2 was synthesized using the same procedures described above, except that 0.052 mmol of cyclopropylamine was used in place of 1-dimethylamino-2-aminoethane.

R? R1 = -C (0) OCH2OC (0) C (CH3) 3 R "" = -NHCH2 (C02CH3) RJ -OH 10-3 Compound 10-3 was synthesized using the same procedures described above, except that glycine methyl ester was used instead of 1-dimethylamino-2-aminoethane.

R R1 R1 = -C (0) OCH2OC (0) C (CH3) 2 R¿ = -NHCH2CH2N (CH3) 2 RJ = -OH 10-4 Compound 10-4 is synthesized using the same procedures described above, except that 0.052 mmoles of prodrug 7-1 was used in place of prodrug 3-5.

Ra Rx R1 = -C (0) OCH2OC (0) CH (CH3) 2 R = -NH (cyclopropyl; RJ = -OH 10-5 Compound 10-5 is synthesized using the same procedures described above, except that 0.052 mmol of prodrug 7-1 was used in place of prodrug 3-5 and 0.052 mmol of cyclopropylamine was used in place of l-dimethylamino-2-aminoethane.

EXAMPLE 11 Example 11 illustrates the formation of the prodrug of the pseudomycin compounds where the carboxylic acid group of the aspartic acid unit of the pseudomycin ring has been modified to form a 3-amido derivative.

Synthesis of the 3-monoamido derivative 11 -1: R1, R1 and R1 R2 = -NH (cyclopropyl) RJ -OH 11-1 To a solution of DMF (1 liter) containing Compound 2b-1 (1 eq.) Was added 1.5 eq. of Compound la-4 and the mixture was stirred at room temperature for about 3 days. The solvent was partially removed and the residue was purified by reverse phase HPLC (WaterMR, Delta Pak C18 column) to yield Compound 11-1 as well as the other mono- or disubstituted products.

Example 12 Example 12 illustrates the synthesis of a prodrug where the carboxylic acid group of both the aspartic acid and hydroxyapartic acid units have been modified to form a bis-ester derivative.

Synthesis of Bis-ester 12-1: R2 = -0CH3 RJ = -OCH3 12-1 To a solution of DMF (1 liter) containing Compound 3a-1 (1 eq.) Was added 1.5 eq. of Compound la-4 and the mixture was stirred at room temperature for about 3 days. The solvent was partially removed and the residue was purified by reverse phase HPLC (Waters1, Delta pak C18 column) to yield Compound 12-1 as well as the other mono- and disubstituted products.

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 (16)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A prodrug of pseudomycin 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, -acetoacetate, methoxy or hydroxy; Rc is hydrogen, hydroxy, alkoxy of 1 to 4 carbon atoms, hydroxyalkoxy, or taken together with Re forms a 6-membered aromatic ring or a cycloalkyl ring of 5 to 6 carbon atoms; Re is hydrogen, Rd 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 5 to 14 carbon atoms, and Rf is alkyl of 8 to 18 carbon atoms, alkoxy of 5 to 11 carbon atoms or bi-enyl; 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 R is where 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 Rk 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 (0) CH3; R1 is independently hydrogen, an acyloxymethylene-1,3-dioxolen-2-one or an acyloxymethylenecarboxylate with the proviso that at least one R1 is an acyloxymethylene-1,3-dioxolen-2-one or an acyloxymethylenecarboxylate; R2 and R3 are independently -OR2a or -N (R2b) (R2c), wherein R2a and R are independently hydrogen, alkyl of 1 to 10 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, hydroxy-alkyl of 1 to 10 carbon atoms, alkoxy-alkyl of 1 to 10 carbon atoms, alkenyl of 2 to 1 carbon atoms, amino-alkyl of 1 to 10 carbon atoms, mono- or di-alkylamino-alkyl of 1 to 10 carbon atoms carbon, aryl-alkyl of 1 to 10 carbon atoms, 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 ester of amino acid and R 2c is hydrogen or alkyl of 1 to 6 carbon atoms; and pharmaceutically acceptable salts and solvates thereof. 2. The prodrug according to claim 1, characterized in that acyloxymethylene-1,3-dioxolen-2-one is represented by structure 1 (a): l. { to) where Rla is alkyl of 1 to 10 carbon atoms, alkenyl of 1 to 10 carbon atoms, benzyl, or aryl and R, 1b is hydrogen or methyl. 3. The prodrug according to claim 1, characterized in that the acyloxymethylenecarboxylate is represented by the structure Kb): Kb) where Rla is alkyl of 1 to 10 carbon atoms, alkenyl of 2 to 10 carbon atoms, benzyl or aryl and Rlb is hydrogen or methyl. 4. The prodrug according to claim 2, characterized in that R is represented by the structure where Rb 'is hydroxy, Ra, Ra', Rb, Rc, Rd and Re are all hydrogen and Rf is n-octyl. 5. The prodrug according to claim 3, characterized in that R is represented by the structure where Rb 'is hydroxy, Ra, Ra', Rb, Rc, Rd and Re are all hydrogen, and Rf is n-octyl. 6. The prodrug according to claim 1, characterized in that the alkyl carboxylate residue of an alkyl amino acid ester is represented by -CH2C02CH3 / -CH (C02CH3) CH (CH3) 2, -CH (C02CH3) CH (phenyl) , -CH (C02CH3) CH20H, -CH (C02CH3) CH2 (p-hydroxyphenyl), -CH (C02CH3) CH2SH, -CH (C02CH3) CH2 (CH2) 3NH2, -CH (C02CH3) CH2 (4-imidazole), -CH (C02CH3) CH2 (5-imidazole), -CH (C02CH3) CH2C02CH3, or -CH (C02CH3) CH2C02NH2. 7. A prodrug of pseudomycin 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 'forms a six-membered cycloalkyl ring, a six-membered aromatic ring or a double bond, or taken together with Rc it forms 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, hydroxyalkoxy, or taken together with Re forms a 6-membered aromatic ring or a cycloalkyl ring of 5 to 6 carbon atoms; Re is hydrogen, Rd 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 5 to 14 carbon atoms, 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, - ( CH2) n-aryl or - (CH2) n- (cycloalkyl of 5 to 6 carbon atoms), where n = 1 or 2; or R is where R is hydrogen, halogen or alkoxy of 5 to 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 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 (0) CH3; R1 is independently hydrogen, an acyloxymethylene-1,3-dioxolen-2-one or an acyloxymethylenecarboxylate, with the proviso that at least one R1 is an acyloxymethylene-1,3-dioxolen-2-one or an acyloxymethylenecarboxylate; R2 and R3 are independently -OR2a or -N (R2b) (R2c), where 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 atoms, alkoxy-alkyl of 1 to 10 carbon atoms, alkenyl 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 carbon atoms, heteroaryl-alkyl of 1 to 10 carbon atoms, cycloheteroalkyl-alkyl of 1 to 10 carbon atoms, or R2 is an alkyl carboxylate residue of an alkyl ester of amino acid and R2c is hydrogen or alkyl of 1 to 6 carbon atoms; and pharmaceutically acceptable salts and solvates thereof. 8. The prodrug according to claim 7, characterized in that acyloxymethylene-1,3-dioxolen-2-one is represented by structure 1 (a): 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. 9. The prodrug according to claim 7, characterized in that the acyloxymethylenecarboxylate is represented by structure 1 (b): Kb) 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. The prodrug according to claim 8, characterized in that R is represented by the structure where Rb 'is hydroxy, Ra, Ra', Rb, Rc, Rd and Re are all hydrogen and Rf is n-octyl. 11. The prodrug according to claim 9, characterized in that R is represented by the structure where Rb 'is hydroxy, Ra, Ra', Rb, Rc, Rd and Re are all hydrogen, and Rf is n-octyl. 12. The prodrug according to claim 7, characterized in that the alkyl carboxylate residue of an alkyl amino acid ester is represented by -CH2C02CH3, -CH (C02CH3) CH (CH3) 2, -CH (C02CH3) CH (phenyl) , -CH (C02CH3) CH2OH, -CH (C02CH3) CH2 (p-hydroxyphenyl), -CH (C02CH3) CH2SH, -CH (C02CH3) CH2 (CH2) 3NH2, -CH (C02CH3) CH2 (4-imidazole), -CH (C02CH3) CH2 (5-imidazole), -CH (C02CH3) CH2C02CH3, or -CH (C02CH3) CH2C02NH2. 13. The use of a compound according to any of the preceding claims in the preparation of medicaments for use in combating either systematic fungal infections or fungal infections of the skin. 14. A pharmaceutical formulation, characterized in that it comprises the prodrug of pseudomycin or the pharmaceutically acceptable salt or solvate thereof according to claim 1 and a pharmaceutically acceptable carrier, buffer, diluent or excipient. 15. A medicament for the treatment of a fungal infection in an animal, characterized in that the medicament comprises the prodrug of pseudomycin or the pharmaceutically acceptable salt or solvate thereof according to claim 1. 16. The use of the prodrug of pseudomycin or the A pharmaceutically acceptable salt or solvate thereof according to claim 7 for the manufacture of a medicament for treating a fungal infection in an animal.