CN1373770A - Pseudomycin prodrugs - Google Patents

Abstract

This invention describes a pseudomonadin prodrug represented by structure (A), wherein ^R1 is an acyloxyalkyl carbamate bond. The prodrug exhibits antifungal activity and has low side effects.

Description

Pseudomycin prodrugs

Field of Invention

The present invention relates to pseudomycin compounds, especially pseudomycin compound prodrugs.

Background of the Invention

Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium, and have been shown to have antifungal activity. (See, eg, Harrison, L. et al., "Pseudomonas, a family of novel peptides from Pseudomonas syringae with broad-spectrum antifungal activity", J. Gen. Microbiology, 137(12), 2857- 65 (1991) and US Patents US 5,576,298 and 5,837,685). Unlike the antimycotic agents previously described from P. syringae (e.g., syringomycin, P. syringae toxin, and P. syringae inhibin), pseudomycins A-C contain hydroxyaspartic acid , aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid. The peptide part of pseudomycin A, A', B, B', C, C' corresponds to L-Ser-D-DabL-Asp-L-Lys-L-Dab-L-aThr- with terminal carboxyl group Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl), the carboxyl closes the macrocycle at the OH of the N-terminal Ser. These analogs are distinguished by the N-acyl side chain, that is, pseudomycin A is N-acylated with 3,4-dihydroxytetradecanoyl, and pseudomycin A' is 3, 4-dihydroxypentadecanoyl N-acylated, pseudomycin B is N-acylated with 3-hydroxytetradecanoyl, pseudomycin B' is N-acylated with 3-hydroxydodecane Acyl N-acylated, pseudomycin C is 3,4-dihydroxyhexadecanoyl N-acylated, and pseudomycin C' is 3-hydroxyhexadecanoyl N- Acylated. (see for example Ballio, A., et al., "Biologically active lipopeptides from Pseudomonas syringae: pseudomycins," FEBS Letters, 355(1), 96-100, (1994) and Coiro, V.M., et al., "Solution conformation of the phytotoxic lipopeptidepseudomycin A of Pseudomonas syringae MSU 16H determined by computer simulation using geometric distances and molecular dynamics from NMR data," Eur.J.Biochem ., 257(2), 449-456(1998)).

It is known that pseudomycin has certain biological side effects. For example, when pseudomycins are administered intravenously, disruption of the vein endothelium, tissue destruction, inflammation, and local toxicity to host tissues have been observed. Therefore, there is a need to identify compounds from this class that are useful in the treatment of fungal infections without the side effects currently observed.

Brief description of the invention

其中R是其中The present invention provides a pseudomycin prodrug represented by the following structural formula used as an antifungal agent, its pharmaceutically acceptable salt and its solvate,

where R is in

R ^a and R ^a' are independently H or methyl, or R ^a or R ^a' is alkylamino, together with R ^b or R ^b' form a 6-membered cycloalkyl ring, a 6-membered aromatic ring or A double bond, or forms a 6-membered aromatic ring together with ^Rc ;

R ^b and R ^b' are independently H, halogen or methyl, or R ^b or R ^b' is amino, alkylamino, alpha-acetoacetate, methoxy or hydroxy;

R ^c is H, hydroxyl, C ₁ -C ₄ alkoxy, hydroxyl C ₁ -C ₄ alkoxy, or together with R ^e forms a 6-membered aromatic ring or a C ₅ -C ₆ cycloalkyl ring;

R ^e is H, or together with R ^f forms a 6-membered aromatic ring, a C ₅ -C ₁₄ alkoxy substituted 6-membered aromatic ring, or a C ₅ -C ₁₄ alkyl substituted 6-membered aromatic ring ring, and

其中R ^f is C ₈ -C ₁₈ alkyl, or C ₅ -C ₁₁ alkoxy; R is

in

R ^g is H or C ₁ -C ₁₃ alkyl, and

其中R ^h is C ₁ -C ₁₅ alkyl, C ₄ -C ₁₅ alkoxy, (C ₁ -C ₁₀ alkyl) phenyl, -(CH ₂ ) _n -aryl, or -(CH ₂ ) _n - (C ₅ -C ₆ cycloalkyl), wherein n=1 or 2; or R is

in

R ⁱ is H, halogen, or C ₅ -C ₈ alkoxy, and

m is 1, 2 or 3; R is

in

R ^j is C ₅ -C ₁₄ alkoxy or C ₅ -C ₁₄ alkyl and p=0, 1 or 2; R is

in

其中R ^k is C ₅ -C ₁₄ alkoxy, or R is -(CH ₂ )-NR ^m -(C ₁₃ -C ₁₈ alkyl), wherein R ^m is H, -CH ₃ or -C(O)CH ₃ ; ^R is independently H, acyloxymethylene-1,3-dioxol-2-one (such as compound 1 (a) described below), or acyloxymethylene Carboxylate (eg compound 1(b) below)

in

R ^1a is H, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkenyl, benzyl, or aryl, and

R ^1b is H or methyl

with the proviso that at least ^one R is acyloxymethylene-1,3-dioxol-2-one or acyloxymethylene carboxylate; R ^{and R} are independently -OR ^2a , or -N(R ^2b )(R ^2c ),

in

R ^2a and R ^2b are independently H, C ₁ -C ₁₀ alkyl (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc.) , C ₃ -C ₆ cycloalkyl (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethylene, methylcyclopentyl, cyclohexyl, etc.), hydroxy (C ₁ -C ₁₀ )alkane radical, alkoxy(C ₁ -C ₁₀ )alkyl (eg methoxyethyl), or C ₂ -C ₁₀ alkenyl, amino(C ₁ -C ₁₀ )alkyl, mono- or di-alkyl Amino(C ₁ -C ₁₀ )alkyl, aryl(C ₁ -C ₁₀ )alkyl (e.g. benzyl), heteroaryl(C ₁ -C ₁₀ )alkyl (e.g. 3-pyridylmethyl, 4 -pyridylmethyl), or cycloheteroalkyl (C ₁ -C ₁₀ )alkyl (such as N-tetrahydro-1,4-oxazinylethyl and N-piperazinylethyl), or

R ^2b is an alkyl carboxylate residue of an amino acid alkyl ester (eg -CH ₂ CO ₂ CH ₃ , -CH(CO ₂ CH ₃ )CH(CH ₃ ) ₂ , -CH(CO ₂ CH ₃ )CH( phenyl), -CH(CO ₂ CH ₃ )CH ₂ OH, -CH(CO ₂ CH ₃ )CH ₂ (p-hydroxyphenyl), -CH(CO ₂ CH ₃ )CH ₂ SH, -CH(CO ₂ CH ₃ )CH ₂ (CH ₂ ) ₃ NH ₂ , -CH(CO ₂ CH ₃ )CH ₂ (4- or 5-imidazole), -CH(CO ₂ CH ₃ )CH ₂ CO ₂ CH ₃ , -CH( CO ₂ CH ₃ )CH ₂ CO ₂ NH ₂ etc.), and

R ^2c is H or C ₁ -C ₆ alkyl.

In another embodiment of the present invention, a pharmaceutical preparation is provided, which comprises the above-mentioned pseudomycin prodrug and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, there is provided a method of treating an antifungal infection in an animal in need thereof, the method comprising administering to said animal a pseudomycin prodrug as described above. The present invention also provides the use of the above-mentioned pseudomycin prodrug in the production of medicament for treating antifungal infection in animals.

Definition

Unless otherwise specified, the term "alkyl" as used herein refers to a hydrocarbon group of the general formula _{CnH2n +1} containing 1-30 carbon atoms. Alkyl groups can be straight chain (such as methyl, ethyl, propyl, butyl, etc.), branched (such as isopropyl, isobutyl, tert-butyl, neopentyl, etc.), cyclic (such as cyclopropyl group, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or polycyclic (such as bicyclo[2.2.1]heptane, spiro[2.2]heptane, etc.). The alkyl groups may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy, alkanoyl or alkanoate has the same definition as above.

The term "alkenyl" refers to an acyclic hydrocarbon containing at least one carbon-carbon double bond. The alkenyl group may be linear, branched, cyclic or polycyclic. The alkenyl group can be substituted or unsubstituted. The alkenyl moiety in alkenyloxy, alkenoyl or alkenoate has the same definition as above.

The term "aryl" refers to an aromatic moiety having a single ring system (eg, phenyl) or a condensed ring system (eg, naphthalene, anthracene, phenanthrene, etc.). The aromatics can be substituted or unsubstituted.

In the field of organic chemistry, and in particular organic biochemistry, it is broadly understood that effective substitution of compounds is permissible, or even useful. For example, in the present invention the term alkyl is permissible to include typical alkyl substituents such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl and the like. The term "group" specifically assumes and allows substitutions on alkyl groups conventional in the art, such as hydroxyl, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., and includes no Substituted alkyl moieties. However, it is generally understood by those skilled in the art that substituents should be chosen so as not to adversely affect the pharmacological properties of the compound, or to interfere negatively with the use of the drug. Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halogen, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di- Alkylamino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamoyl, carbonyl, carboxyl, glycolyl, glycyl, hydrazino, amidino, and combinations thereof.

The term "prodrug" refers to a class of drugs that are transformed in vivo by metabolic processes (ie, biotransformation) to produce their pharmacological effects. In the present invention, the pseudomycin prodrug compound contains a linker that can be cleaved by esterase in plasma to produce the active drug.

The term "animal" refers to humans, pets (such as dogs, cats, and horses), food-source animals (such as cattle, pigs, sheep, and poultry), zoo animals, marine animals, birds, and other similar animal species.

Detailed description of the invention

Applicants have found that prodrug derivatives of natural or semi-synthetic products of pseudomycin have lower side effects than the corresponding natural products and retain anti-Candida albicans (C.albican), Cryptococcus neoformans (C.neoformas) in vivo and Aspergillus fumigatus (A.fumigatus). The prodrug is produced by acylation of at least one of the pendant amino groups attached to a lysine or 2,4-diaminobutyric acid peptide unit in the pseudomycin cyclic peptide ring system to form an acyl substituent. The acylating agent (or linker) is usually an acyloxymethylene-1,3-dioxol-2-one or an acyloxymethylene carboxylate acyl compound so that a carbamate linkage to a pendant amino group on the pseudomycin structure can be formed. Suitable leaving groups are well known to those skilled in the art and include groups such as p-nitrophenoxy and N-oxysuccinimide.

Acyloxymethylene-1,3-dioxol-2-one acylated compounds can be synthesized using the synthetic route shown in Scheme I below. For purposes of illustration, a specific acylated compound is described. However, it will be understood by those skilled in the art that one can synthesize various derivatives using the same basic synthetic method.

For a more detailed description of these synthetic steps, see the Preparations section of the Examples below.

Acyloxymethylene carboxylate acylated compounds can be synthesized using the synthetic route shown in Scheme II below. For purposes of illustration, a specific acylated compound is described. However, it will be understood by those skilled in the art that one can synthesize various derivatives using the same basic synthetic method.

For a more detailed description of these synthetic steps, see the Preparations of the Examples below.

As discussed earlier, pseudomycins are natural products isolated from Pseudomonas syringae characterized as lipodepsinonapetpides, which contain cyclic peptide moieties closed by lactone bonds and include the unusual Amino acids 4-chlorothreonine (ClThr), 3-hydroxyaspartic acid (HOAsp), 2,3-dehydro-2-aminobutyric acid (Dhb) and 2,4-diaminobutyric acid (Dab). Methods for growing different strains of Pseudomonas syringae to produce different pseudomycin analogs (A, A', B, B', C, and C') are described below and described in more detail in Hilton et al., 2000 PCT patent application entitled "Pseudomycin Production by Pseudomonas Syringae" filed April 14, 2000, with serial number PCT/US00/08728, Kulanthaivel et al. PCT patent application entitled "Pseudomycin Natural Products", serial number PCT/US00/08727, and US 5,576,298 and 5,837,685, filed April 14, 2009, all of which are hereby incorporated Incorporated by reference.

Isolates of P. syringae that produce one or more pseudomycins are known in the art. The wild-type strain MSU 174 and the mutant MSU16H of this strain produced by transposon mutagenesis are described in US 5,576,298 and 5,837,685; "Pseudomycins, a family of novel peptides from Pseudomonassyringae possessing broad-spectrum antifungal activity," by Harrison et al. .Gen.Microbiology, 137, 2857-2865 (1991); and "Transposonmutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease," Proc.Natl.Acad.Sci.USA, 84, 6447-6451 (1987).

Strains of Pseudomonas syringae suitable for the production of one or more pseudomycins can be isolated from environmental sources including plants such as barley plants, citrus plants and clove plants, as well as sources such as soil, water, air and dust . Preferred strains are isolated from plants. A strain of P. syringae isolated from an environmental source may be referred to as wild type. As used herein, "wild-type" refers to a dominant genotype naturally occurring in the normal flora of P. syringae (e.g., a P. syringae strain or isolate found in nature and not things). As with most organisms, the characteristics of the culture of the pseudomycin used (Pseudomonas syringae strains such as MSU174, MSU 16H, MSU 206, 25-B1, 7H9-1) are prone to variability. Accordingly, progeny (eg recombinants, mutants and variants) of these strains can be obtained by methods known in the art.

Pseudomonas syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA, under accession number ATCC 67028. Pseudomonas syringae strains 25-B1, 7H9-1 and 67 H1 were deposited with the American Type Culture Collection on March 23, 2000 and have the following accession numbers respectively:

25-B1 Deposit No. PTA-1622

7H9-1 Accession No. PTA-1623

67 H1 Deposit No. PTA-1621

Mutant strains of Pseudomonas syringae are also suitable for the production of one or more pseudomycins. As used herein, "mutant" refers to a sudden heritable change in the phenotype of a strain, which may be spontaneous or induced by a known mutagen, such as radiation (e.g. ultraviolet radiation or x-rays), chemical Mutagens (such as ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N'-nitroguanine (NTG) and nitrous acid), position-specific mutagens mutagenesis and transposon-mediated mutagenesis. A pseudomycin-producing Pseudomonas syringae mutant can be produced by treating the bacterium with an amount of a mutagen effective to produce a mutant that produces one or more pseudomycins in excess , production of one pseudomycin (eg, pseudomycin B) over other pseudomycins, or production of one or more pseudomycins under favorable growth conditions. Although the type and amount of mutagen used may vary, the preferred method is to serially dilute NTG to levels of 1-100 [mu]g/ml. Preferred mutants are those that overproduce pseudomycin B and grow in minimally defined media.

Environmental isolates of Pseudomonas syringae, mutant strains and other desired Pseudomonas syringae Strains are selected. Preferably, a strain of Pseudomonas syringae is selected that grows on a minimally defined medium, such as N21 medium, and/or produces one or more pseudomycins in an amount greater than about 10 μg/ml of pseudomycin. Preferred strains exhibit the property of producing one or more pseudomycins when grown on media containing three or fewer amino acids, optionally containing lipids, potato products, or combinations thereof.

Recombinant strains can be grown by transforming Pseudomonas syringae strains using methods known in the art. In addition to the antibiotics produced by these strains, Pseudomonas syringae strains can be transformed to express different gene products through the use of recombinant DNA techniques. For example, one can modify these strains to introduce multiple copies of endogenous pseudomycin biosynthesis genes to obtain greater pseudomycin production.

For the production of one or more pseudomycins from Pseudomonas syringae wild-type or mutant strains, the organism contains effective amounts of three or fewer amino acids, preferably glutamic acid, glycine, histidine Stirring culture in the aqueous nutrient medium of its combination. Alternatively, glycine is combined with one or more potato products and lipids. The culture is carried out under conditions under which Pseudomonas syringae can grow efficiently and produce the desired pseudomycin. Effective conditions include a temperature of about 22°C to about 27°C for a time of about 36 hours to about 96 hours. Controlling the oxygen concentration in the medium during the cultivation of Pseudomonas syringae is beneficial for the production of pseudomycin. Preferably the oxygen level is maintained at about 5-50% saturation, more preferably about 30% saturation. The oxygen concentration in the medium can be adjusted by sparging with air, pure oxygen, or a gas mixture containing oxygen.

It is also beneficial to control the pH of the medium during the cultivation of Pseudomonas syringae. Pseudomycin is unstable at alkaline pH and can undergo significant degradation if the pH of the culture medium is above about 6 for a period of more than about 12 hours. Preferably the pH of the medium is maintained between 6-4. Pseudomonas syringae can produce one or more pseudomycins when grown in batch culture. However, fed-bath or semi-continuous addition of glucose and, optionally, acid or base (eg, ammonium hydroxide) to control pH can improve yield. Pseudomycin production can also be increased using continuous culture with automated addition of glucose and ammonium hydroxide.

The choice of P. syringae strain can affect the amount and distribution of pseudomycins produced. For example, strains MSU 16H and 67 H1 each primarily produce pseudomycin A, but also produce pseudomycins B and C in a ratio typically 4:2:1. The amount of pseudomycin produced by strain 67 H1 is typically about 3-5 times higher than that produced by strain MSU 16H. Strain 25-B1 produced more pseudomycin B and less pseudomycin C than strains MSU16H and 67 H1. Strain 7H9-1 is characterized by mainly producing pseudomycin B, and the production of pseudomycin B is higher than that of other strains. For example, the strain can produce pseudomycin B that is at least 10 times less pseudomycin A or C.

Alternatively, the prodrugs may be formed from N-acyl semi-synthetic compounds. Semisynthetic pseudomycin compounds can be synthesized by exchanging the N-acyl group on the L-serine unit. Examples of various N-acyl derivatives are described in PCT patent application serial number entitled "Pseudomycin N-Acyl Side-Chain Analogs" filed concurrently by Belvo et al. _____, which is incorporated herein by reference. In general, naturally occurring pseudomycin compounds are produced as their semi-synthetic compounds using the following four synthetic steps: (1) selective amino protection; (2) chemical or enzymatic modification of the N-acyl side chain Facilitated deacylation; (3) reacylation with a different side chain; and (4) deprotection of the amino group.

Pendant amino groups at positions 2, 4 and 5 can be protected using any standard method known to those skilled in the art of amino protection. The precise nature of the amino-protecting group employed is not critical, provided that the derivatized amino group is stable at other positions in the intermediate molecule relative to the subsequent reaction conditions and that the protecting group can be selectively removed at an appropriate time, for any group including It is sufficient that the remaining part of the molecule of the other amino protecting group is not destroyed. Suitable amino protecting groups include benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, p- Phenylazobenzyloxycarbonyl, tert-butoxycarbonyl, cyclopentyloxycarbonyl and phthalimido. Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl (Alloc), phthalimido and benzyloxycarbonyl (CbZ or CBZ). Further examples of suitable protecting groups are described in T.W. Greene, "Protective Groups in Organic Synthesis," John Wiley and Sons, New York, N.Y., (2nd ed., 1991), at chapter 7.

Deacylation of N-acyl groups with gamma or delta hydroxylated side chains (eg, 3,4-dihydroxytetradecanoate) can be achieved by acid treatment of amino-protected pseudomycin compounds in aqueous solvents. Suitable acids include acetic acid and trifluoroacetic acid. A preferred acid is trifluoroacetic acid. The reaction can be carried out at or near room temperature if trifluoroacetic acid is used. However, when acetic acid is used, the reaction is usually carried out at about 40°C. Suitable aqueous solvent systems include acetonitrile, water, and mixtures thereof. Organic solvents increase the rate of this reaction; however, addition of organic solvents may result in other by-products. Pseudomycin compounds lacking a delta or gamma hydroxyl group on the side chain (such as pseudomycins B and C') can be deacylated enzymatically. Suitable deacylases include polymyxin acylase (164-16081 fatty acyltransferase (crude) or 161-16091 fatty acyltransferase (pure), available from Wako Pure Chemical Industries, Ltd.), or ECB deacylase. This enzymatic deacylation can be performed using standard deacylation procedures well known to those skilled in the art. For example, general procedures for using polymyxin acylase can be found in the following documents: Yasuda, N. et al., Agric. Biol. Chem., 53, 3245 (1989) and Kimura, Y. et al., Agric. Biol. Chem., 53, 497 (1989).

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

Various acids can be used in this acylation process. Suitable acids include fatty acids containing one or more pendant aryl, alkyl, amino (including primary, secondary and tertiary amines), hydroxyl, alkoxy and amido groups; fatty acids containing nitrogen or oxygen within the fatty chain; Alkyl, hydroxy, alkoxy and/or alkylamino substituted aromatic acids; and alkyl, hydroxy, alkoxy and/or alkylamino substituted heteroaromatic acids.

Alternatively, solid-phase synthesis can be used in which hydroxybenzotriazole resin (HOBt-resin) is used as a coupling agent for the acylation reaction.

Once the amino group has been deacylated and reacylated (as described above), the amino protecting groups (of positions 2, 4 and 5) can be removed by hydrogenation in the presence of a hydrogenation catalyst (eg 10% Pd/C). When the amino protecting group is allyloxycarbonyl, the protecting group can be removed using tributyltin hydride and triphenylphosphine palladium dichloride. The advantage of this particular protection/deprotection scheme is that the vinyl hydrogenation potential of the Z-Dhb unit in the pseudomycin structure is reduced.

The prodrug is then produced by acylation of at least one of the lysine or the pendant amino group attached to the 2,4-diaminobutyric acid peptide unit in the N-acyl-modified semi-synthetic pseudomycin compound to form Urethane linkage required.

Other modified prodrug pseudomycin compounds can be synthesized by amidating or esterifying the pendant carboxylic acid groups of the aspartate and/or hydroxyaspartate units in the pseudomycin ring. Examples of various acid-modified derivatives are described in PCT Patent Application Serial No. _____, entitled "Pseudomycin Amide & Ester Analogs," filed contemporaneously by Chen et al., which is incorporated herein by reference. These acid-modified derivatives can be formed by condensing any of the aforementioned prodrugs with a suitable alcohol or amine to produce the corresponding ester or amide.

The formation of these ester groups can be accomplished using standard esterification procedures well known to those skilled in the art. Esterification under acidic conditions typically involves dissolving or suspending the pseudomycin compound in a suitable alcohol in the presence of a protic acid (eg, HCl, TFA, etc.). Under basic conditions, the pseudomycin compounds are usually reacted with a suitable alkyl halide in the presence of a mild base such as sodium bicarbonate and potassium carbonate.

Formation of amide groups can be achieved using standard amidation procedures well known to those skilled in the art. However, the choice of coupling agent provides selective modification of the acid groups. For example, the use of benzotriazol-1-yloxy-tripyrrolidinylphosphonium hexafluorophosphate (PyBOP) as a coupling agent allows one to simultaneously isolate pure monoamides at the 8-position of residues and (certain case) pure diamide. However, the use of o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) as a coupling agent favors the formation of a monoamide at the 3-position of the residue.

The pseudomycin prodrug can be isolated and used as such or in the form of a pharmaceutically acceptable salt or solvate thereof. As noted above, the prodrug is prepared by forming at least one acyloxyalkylcarbamate linkage. The term "pharmaceutically acceptable salts" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, hydrogensulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphates, monohydrogenphosphates, phosphoric acid Dihydrogen salt, metaphosphate, pyrophosphate, alkanoate, naphthenic alkanoate, aryl alkanoate, adipate, alginate, aspartate, benzoic acid Salt, fumarate, glucoheptanate, glycerophosphate, lactate, maleate, nicotinate, oxalate, palmitate, pectinate, picrate, neopentyl salt, succinate, tartrate, citrate, camphorate, camphorsulfonate, digluconate, trifluoroacetate, etc.

The term "solvate" refers to an aggregate containing one or more molecules of a solute (ie, a pseudomycin prodrug compound) and one or more molecules of a pharmaceutically acceptable solvent such as water, ethanol, or the like. When the solvent is water, the aggregate is called a hydrate. Solvates are generally formed by dissolving the prodrug in a suitable solvent with heating and cooling slowly to produce an amorphous or crystalline solvate form.

Each pseudomycin, semi-synthetic pseudomycin, pseudomycin prodrug, and mixtures thereof can be detected, identified, isolated and/or purified by any of the various methods known to those skilled in the art. For example, the activity level of a pseudomycin or a pseudomycin prodrug in a broth or isolate or purified composition can be determined by antifungal activity against fungi such as Candida and can be Separation and purification by high performance liquid chromatography.

The active ingredient (ie, the pseudomycin prodrug) is typically formulated into a pharmaceutical dosage form that provides an easily controlled dosage of the drug and presents the patient, physician or veterinarian with an elegant and easy-to-handle product. The preparation may contain from 0.1% to 99.9% wt active ingredient, more typically from about 10% to about 30% wt.

As used herein, the term "unit dose" or "dosage unit" refers to a physically discrete unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect. When the unit dosage is administered orally or parenterally, it is typically presented in the form of tablets, capsules, pills, powder packets, topical compositions, suppositories, wafers, ampoules, or assay units in multidose containers, etc. . Alternatively, the unit dose may be administered in the form of a dry or liquid aerosol that can be inhaled or sprayed.

The dosage may vary depending on the physical characteristics of the animal, the severity of the animal's disease, the method for administration and the animal species. The specific dosage for a given animal will often be determined by the judgment of the physician or veterinarian.

Suitable carriers, diluents and excipients are well known to those skilled in the art and include the following materials: carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water wait. The particular carrier, diluent or excipient employed will depend upon the manner and purpose for which the active ingredient is to be used. The preparation may also include wetting agents, emollients, surfactants, buffers, enhancers, fillers, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, fragrances, flavoring agents, and combinations thereof.

Pharmaceutical compositions can be administered using a variety of methods. Suitable methods include topical (eg ointment or spray), oral, injection and inhalation. The specific treatment used will depend on the type of injection being performed.

When administered parenterally intravenously, these preparations are typically diluted or reconstituted (if lyophilized), and further diluted, if necessary, prior to administration. An example of reconstitution instructions for a lyophilized product is to add 10 ml of water for injection (WFI) to a vial and stir gently to dissolve. Typical reconstitution times are less than 1 minute. The resulting solution is then further diluted in an infusion fluid such as 5% dextrose in water (D5W) prior to administration.

Pseudomycin compounds have been shown to have antifungal activity, including, for example, inhibition of the growth of infectious fungi of: Candida species (i.e., C. albicans, C. parapsilosis ), C. krusei, C. glabrata, C. tropicalis, or C. lusitania); Various (i.e. T. glabrata); Aspergillus species (i.e. A. fumigatus); Histoplasma species (i.e. H. capsulatum) various species of Cryptococcus (i.e., C. neoformans); various species of Blastomyces (i.e., B. dermatitidis); various species of Fusarium; various species of Trichophyton, Pseudallescheriaboydii, Coccidioides immosa, Sporothrix schenckii, etc.

Therefore, the compounds and formulations of the present invention are useful in the preparation of a medicament for combating systemic fungal infections or fungal skin infections. Accordingly, there is provided a method of inhibiting fungal activity comprising contacting a pseudomycin prodrug of the invention with a fungus. Preferred methods include inhibiting the activity of Candida albicans or Aspergillus fumigatus. The term "contacting" includes association or conjugation of the compound of the invention with the fungus, or surface contact or mutual contact. The term does not confer any other limitation on the method, for example by mechanism of inhibition. These methods are defined as encompassing the inhibition of parasitic and fungal activity through the action of these compounds and their intrinsic antifungal properties.

Also provided is a method of treating a fungal infection comprising administering to a host animal in need of such treatment an effective amount of a pharmaceutical formulation of the invention. Preferred methods include treating Candida albicans, Cryptococcus neoformans or Aspergillus fumigatus infections. The term "effective amount" refers to the amount of active compound capable of inhibiting fungal activity. The dosage administered will vary with factors such as the nature and severity of the infection, the age and general health of the host, the tolerance of the host to antifungal agents and the species of the host. The specific dosage regimen will also vary depending on these factors. The drug can be administered in a single daily dose or in multiple doses during the day. The regimen can extend from about 2-3 days to about 2-3 weeks or longer. A typical daily dosage (administered in single or divided doses) will contain dosage levels of the active compound from about 0.01 mg/kg to 100 mg/kg body weight. Preferred daily dosages are generally about 0.1 mg/kg-60 mg/kg, more preferably about 2.5 mg/kg-40 mg/kg. Hosts are generally animals including humans, pets (such as dogs, cats, and horses), food source animals (such as cattle, pigs, sheep, and poultry), zoo animals, marine animals, birds, and other similar animal species.

Example

The following abbreviations are used throughout the examples to refer to the respective listed materials:

ACN-acetonitrile

TFA-trifluoroacetic acid

DMF-dimethylformamide

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

BOC = tert-butoxycarbonyl, (CH ₃ ) ₃ COC(O)-

CBZ = benzyloxycarbonyl, C ₆ H ₅ CH ₂ -OC(O)-

PyBOP = benzotriazol-1-yloxy-tripyrrolidinylphosphonium hexafluorophosphate

TBTU = o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate

DIEA=N,N-Diisopropylethylamine

抗真菌活性的检测和定量：The products obtained in Examples 1-7 are described using Structure II below.

Detection and quantification of antifungal activity:

Antifungal activity is determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc diffusion test. A typical fungus used in the determination of antifungal activity is Candida albicans. Antifungal activity was considered significant when a sample (50 [mu]l) was assayed to produce inhibition in a 10-12 mm diameter area on C. albicans x657 inoculated agar plates. Tail Vein Toxicity:

Mice were treated intravenously (IV) with 0.1 ml of assay compound (20 mg/kg) via the lateral tail vein at 0, 24, 48 and 72 hours. There were 2 mice in each group. Compounds were formulated in 5.0% dextrose and sterile water for injection. Mice were monitored for 7 days after the first treatment and closely observed for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss and any other signs of toxic side effects.

Mice used in this study were outbred male ICR mice (available from Harlan Sprangue Dawley, Indianapolis, IN) weighing an average of 18-20 g. Preparation Preparation of 4-bromomethyl-5-methyl-1,3-dioxol-2-one (1a-1):

Reflux heating 0.1mol 4,5-dimethyl-1,3-dioxol-2-one, 0.1mol N-bromosuccinimide and 0.1g 2,2-azobis(2-methyl propionitrile) in 70 ml of carbon tetrachloride (CCl ₄ ) mixture. After 6 hours, the mixture was cooled with ice and filtered. The filtrate was washed with 2 x 50 ml of water, 2 x 50 ml of sodium chloride solution and another 50 ml of water. The solution was dried over sodium sulfate and evaporated to dryness, and dried under vacuum to afford 16.5 g (85% yield) of an oil with 1H-NMR data consistent with structure 1a-1.

Preparation of compound (1a-2):

Compound 1a-2 was synthesized using the method described in Synthetic Communication, 22(9), 1297 (1992) to obtain 11.5 g (78% yield) of crude oil.

Preparation of Compound 1a-3:

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

Preparation of Compound 1a-4:

A mixture of 3.0 g of compound 1a-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 stirred for about 4.5 hours. The mixture was washed with cold 1% NaOH (3x30ml), 1N HCl (2x30ml), water (2x30ml) and brine (2x30ml). The solution was dried over sodium sulfate, filtered and washed with dichloromethane. Removal of the solvent yielded an oil which solidified on standing. 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 afford 6.39 g (94% yield) of product whose 1H-NMR data was consistent with the structure of 1a-4.

Preparation of compound 1b-1: Compound 1b-1 can be synthesized using the method described in Synthesis, 1159 (1990). Preparation of compound b-2:

A solution of 4.57 g (30 mmol) of crude product 1b-1 in 40 ml of dichloromethane was added to a solution of 3.91 g (34 mmol) of N-hydroxysuccinimide and 2.7 g (34 mmol) of pyridine in 100 ml of dichloromethane at 0°C. After stirring at 0°C for 30 minutes, the mixture was allowed to stand overnight at room temperature. The mixture was then washed four times with water and the organic phase was dried over sodium sulfate. After filtration, the solvent was evaporated to obtain 4.0 g (58% yield) of crude oil, whose 1H-NMR data was consistent with the structure of 1b-2. Preparation of CBZ-protected pseudomycin B (2a-1):

Pseudomycin B was dissolved/suspended in DMF (20 mg/ml, Aldrich Sure Seal). N-(benzyloxycarbonyloxy)succinimide (6 eq) was added while stirring at room temperature. Stir at room temperature for 32 hours. The reaction was monitored by HPLC (4.6 x 50 mm, 3.5 μm, 300-SB, C8, Zorbax column). The reaction was concentrated to 10 ml on a high vacuum rotary evaporator at room temperature. Place the material in the freezer until ready for preparative chromatography. An amorphous white solid (compound 2a-1) was obtained after reverse phase preparative HPLC purification and lyophilization. Preparation of compound 2b-1:

R ^1' , R ^1" and R ^1'' = H

R ² =-NH(cyclopropyl)

R ³ =-OH

2b-1

CBZ-protected pseudomycin B (2a-1) (400 mg, 0.25 mmol) was dissolved in 4 ml dry DMF. TBTU (79 mg, 0.25 mmol), DIEA (200 μl, 6 equiv) and cyclopropylamine (14.2 mg, 0.25 mmol) were added sequentially. The reaction was stirred at room temperature under nitrogen while monitoring by HPLC. After completion the reaction was concentrated under vacuum. The crude product was purified by preparative HPLC. Freeze drying afforded 209.2 mg (51.1%) of a colorless powder.

The 3-acylamino compound (279.1 mg, 0.169 mmol) was catalytically hydrogenated with 10% Pd/C in 1% HOAc/MeOH under a hydrogen balloon for 45 min. The reaction was filtered and concentrated under vacuum. The residue was treated with a 1:1 water:ACN mixture, then lyophilized to afford 208.3 mg (98.6%) of a colorless powder (2b-1). The structure was confirmed by H ¹ -NMR. Preparation of compound 3a-1:

R ^1' , R ^1" and R ^1'' = H

R ² =-OCH ₃

R ³ =-OCH ₃

3a-1

To a 50 ml round bottom flask was added 10 ml absolute ethanol and 251.7 mg CBZ-protected pseudomycin B (2a-1 ) (0.156 mmol). To this mixture was added approximately 1 ml of acidified ethanol (pre-acidified with HCl gas) and the reaction was stirred overnight at room temperature. The solvent was then removed in vacuo and the residue was carried on to the next step without further purification by dissolving it in a 10 ml MeOH/1.5 ml glacial AcOH solution. Using 249.7 mg of 10% Pd/C standard hydrogenolysis for 30 minutes, the catalyst was removed by filtration, purified by preparative HPLC, and 120.9 mg of compound 3a-1 was obtained after lyophilization. MS ( _Ionspray ) calcd for _{C55H96ClN12O19} (M ₊ H) ⁺ 1264.89, found _1264.3 . Preparation of C-18 side chain (4a-1):

To a solution of chiral acetal 4a-1 (6.22 g, 19.1 mmol) in dichloromethane (190 mL) was added trimethylallylsilane (10.9 mL, 68.69 mmol) at -78 °C, followed by neat _TiCl4 (2.94 mL, 26.71 mmol). The reaction was stirred at -78°C for 1 hour, 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 1N 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 solution of 4b-1 (8.56 g, 23.3 mmol) in dichloromethane (155 mL) was added PCC (10.0 g, 46.5 mmol). The reaction was stirred at room temperature for 18 hours, then filtered through a pad of Celite. The filtrate was concentrated in vacuo to give a reddish residue which was purified by silica gel chromatography (10% EtOAc/hexanes) to yield 8.36 g (80%) of the methyl ketone intermediate (structure not shown). The intermediate obtained here (8.36 g, 22.8 mmol) was dissolved in THF (60 mL) and MeOH (30 mL). To this solution was added 7.5M KOH (15 mL). After stirring at room temperature for 3 hours, part of the solvent was removed. The remaining reaction mixture was diluted with EtOAc/ _Et2O (3:1 ratio, 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 afford 6.22 g (96%) of the desired product 4c-1 as a white solid.

Methanol 4c-1 (6.22 g, 22.0 mmol) was dissolved in aqueous THF (5.5 mL water and 55 mL THF). To this solution was added NMO (4.42 g, 33.0 mmol) followed by _OsO4 (280 mg in THF, 1.10 mmol). The reaction was stirred overnight at room temperature. At this point sodium disulfide (4 g) was added. The reaction was stirred for 2 hours, 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 under vacuum to give the corresponding triol intermediate. This material was dissolved in MeOH (200 mL) and water (40 mL). To this solution was added _NaIO4 (10.6 g, 49.5 mmol). After stirring at room temperature for 1 hour, the reaction was filtered through Celite and purified by short column silica gel chromatography (30% EtOAc/Hexanes) to yield approximately 10 g (>100%) of crude β-hydroxyaldehyde. The impure aldehyde thus obtained was dissolved in tert-butanol (100 mL) and cyclohexene (14 mL). To this solution _was added an aqueous solution (50 mL) of _NaClO2 (15.97 g, 176 mmol) and _KH2PO4 (17.8 g, 132 mmol) at room temperature. The reaction was stirred at room temperature for 6 hours, then quenched with 5N HCl at 0°C to pH=4. The reactant was extracted with a mixed solvent of 3:1 EtOAc/Et ₂ O (3×250 mL). The organic layer was washed with brine, dried and concentrated to afford 7.3 g (>100%) of crude acid 4d-1, which was used directly in the coupling reaction. Preparation of pseudomycin C-18 (compound 4b-2):

                                                           

                                                                        

Crude acid 4d-1 (2.1 g, 6.99 mmol) was dissolved in anhydrous 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, CBZ-protected pseudomycin core (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 onto a preparative reverse phase HPLC system for purification (4 injections). After lyophilization, 550 mg (12%) of the CBZ-protected C18 acyl derivative 4a-2 with HOBt activated side chain ester (1 g) were isolated. The recovered side chain ester (1.0 g, 2.40 mmol) was next reacted with the CBZ-protected pseudomycin core (1.33 g, 0.96 mmol) in anhydrous THF and DMF (10 mL each). Following the same purification steps as above, an additional amount of CBZ-protected C18 derivative 4a-2 (606 mg, 38%) was obtained.

To a solution of CBZ-protected C18 derivative 4a-2 (550 mg, 0.33 mmol) in 10% HOAc/MeOH (55 mL) was added Pd/C (550 mg, 10% palladium content) at -78 °C. The reaction was hydrogenated at 1.5 atmospheres for 40 minutes. The progress of the reaction was monitored by analytical HPLC. After completion, the catalyst was filtered off and the filtrate was concentrated under vacuum at 30°C. The resulting residue was redissolved in 1:1 aqueous acetonitrile 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 a starting material; however, one skilled in the art will recognize that, in addition to starting with a pseudomycin compound with a different N-acyl group, using the same Steps can synthesize other N-acyl derivatives.

Example 1

The following examples illustrate the preparation of mono-, di- or tri-substituted acyloxyalkylcarbamate prodrugs of pseudomycin C' (n = 12, ^R2 and ^R3 = -OH).

To a DMF solution (1 L) containing pseudomycin C' (1.5 g, 1 eq) was added 1.5 eq of compound 1a-4 and the mixture was stirred at room temperature for about 3 days. Partial removal of the solvent and purification of the residue by reverse phase HPLC (Waters ^™ , Delta Pak C18 column) afforded the following products and mixtures of products:

86 mg pure monosubstituted pseudomycin C' (1-1);

87 mg of a mixture of monosubstituted pseudomycin C' (1-2);

177 mg mixture of disubstituted pseudomycin C' (1-3);

132 mg of pure disubstituted pseudomycin C'(1-4); and

248 mg trisubstituted pseudomycin C'(1-5). No irritation of the tail vein was observed for either the trisubstituted prodrug or the disubstituted prodrug mixture. Some stimulation was observed with pure mono-substituted, mixture of mono-substituted and pure di-substituted prodrug samples. In comparison, unsubstituted pseudomycin C' and unsubstituted pseudomycin B clearly showed stimulation of the tail vein. All samples showed significant in vivo efficacy except the pure disubstituted prodrug samples ( _ED50 > 20 mg/kgx4).

Monosubstituted, disubstituted or trisubstituted prodrug samples of structure II above were also prepared using the same method as above, wherein R ^1′ , R ^1″ and/or R ^1′′ are

And n=10, 12 and 14.

Compounds 1-1 and 1-5 exhibited similar in vivo efficacy to the parent compound pseudomycin C'. No evidence of tail vein irritation was observed for Compound 1-5, and increased tail vein toxicity was observed for Compound 1-1. Surprisingly, no in vivo efficacy of compounds 1-4 was observed.

Example 2

The following examples illustrate the preparation of mono-, di- or tri-substituted acyloxyalkylcarbamate prodrugs 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 mmol) of compound 1b-2. The mixture was stirred overnight at room temperature. The solution was then concentrated to about 50 ml and the product was purified by HPLC using the following gradient elution scheme: 0-30% 0.1% TFA/ACN in 5 minutes and 30-70% TFA/ACN in 40 minutes. Overall yield 59%.

For the three isolated products (monosubstituted prodrug (compound 2-1), where R ^1' and R ^1" = H and R ¹ '' = -C(O)OCH ₂ OAc, disubstituted prodrug (compound 2-2 ), wherein R ^1′ and R ^1″ =-C(O)OCH ₂ OAc and R ^1 = H and a trisubstituted prodrug (compound 2-3), wherein R ^1′ , R ^1″ and R ^1 = -C(O) _OCH2OAc ) were both assayed and demonstrated in vivo efficacy against murine systemic Candida. However, tail vein toxicity was positive.

Example 3

Monosubstituted, disubstituted and trisubstituted prodrugs of pseudomycin B (n = 10, R ² and R ³ = -OH) were prepared using the same general method as in Example 2 above, wherein R ^1′ , R ^1″ and/or R ^1'' = -C(O)OCH ₂ OC(O)C(CH ₃ ) ₃ . The following 5 samples were isolated:

3-1- Substitution of R ^1

3-2 mixed-substituted R ^1' and R ^1″

3-3 Mixed disubstituted R ^1 + R ^1′ and R ^1 + R ^1″

3-4 Disubstituted R ^1′ + R ^1″

3-5 Trisubstituted R ^1′ + R ^1″ + R ^1

Samples 3-1, 3-3, and 3-5 each showed negative tail vein toxicity. All 5 samples showed in vivo efficacy against murine systemic Candida.

Example 4

Monosubstituted, disubstituted and trisubstituted prodrugs of pseudomycin C' (n = 12, R ² and R ³ = -OH) were prepared using the same general method as in Example 2 above, where R ^1' , R ^1″ and/or R ^1’’ =-C(O)OCH ₂ OC(O)C(CH ₃ ) ₃ . The following 5 samples were isolated:

4-1-substituted R ^1

4-2 Mixed-substituted R ^1' and R ^1″

4-3 Mixed disubstituted R ^1 +R ^1′ and R ^1 +R ^1″

4-4 Disubstituted R ^1′ + R ^1″

4-5 Trisubstituted R ^1′ + R ^1″ + R ^1

Sample 4-1 was not assayed. Samples 4-3, 4-4 and 4-5 were all negative for tail vein toxicity. Samples 4-2, 4-3, 4-4 and 4-5 all showed in vivo efficacy against murine systemic Candida.

Example 5

Monosubstituted, disubstituted and trisubstituted prodrugs of pseudomycin B (n=10) were prepared using the same general method as in Example 2 above, wherein R ^1′ , R ^1″ and/or R ^1′′ =-C(O)OCH( _CH3 )OC(O) _CH3 . Only the trisubstituted derivative (compound 5-1) was assayed. The trisubstituted compound showed negative tail vein toxicity.

Example 6

Monosubstituted, disubstituted and trisubstituted prodrugs of pseudomycin B (n = 10, R ² and R ³ = -OH) were prepared using the same general method as in Example 2 above, wherein R ^1′ , R ^1" and/or R 1 ^'' = -C(O)OCH ₂ OC(O)CH ₂ CH ₃ . Only the trisubstituted derivative (compound 6-1) was determined. The trisubstituted compound showed good In vivo efficacy of anti-murine systemic Candida without tail vein irritation.

Example 7

Monosubstituted, disubstituted and trisubstituted prodrugs of pseudomycin B (n = 10, R ² and R ³ = -OH) were prepared using the same general method as in Example 2 above, wherein R ^1′ , R ^1" and/or R ^1'' = -C(O)OCH ₂ OC(O)CH(CH ₃ )CH ₃ . Only the trisubstituted derivative (compound 7-1) was determined. The trisubstituted compound Good in vivo efficacy against murine systemic Candida was shown without tail vein irritation.

Example 8

Examples 8 and 9 describe the synthesis of prodrugs from semisynthetic pseudomycin compounds in which the N-acyl group of the L-serine unit in the pseudomycin structure is altered.

The mono-, di- and tri-substituted pros of pseudomycin C-18 (n=14, R ² and R ³ =-OH) (4b-2) were prepared using the same general procedure as in Example 2 above. where R ^1' , R ^1" and/or R ^1'' = -C(O)OCH ₂ OC(O)C(CH ₃ ) ₃ . Only the trisubstituted derivative (Compound 8-1) was assayed. The trisubstituted compounds showed negative tail vein toxicity.

Example 9

The mono-, di- and tri-substituted pros of pseudomycin C-18 (n=14, R ² and R ³ =-OH) (4b-2) were prepared using the same general procedure as in Example 2 above. where R ^1' , R ^1" and/or R ^1'' = -C(O)OCH ₂ OC(O)CH(CH ₃ )CH ₃ . Only the trisubstituted derivative (compound 9-1) was determined The trisubstituted compounds showed negative tail vein toxicity.

Example 10

Example 10 describes a further modification of the above prodrug 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:

R ^1′ , R ^1″ , R ^1 ＝-C(O)OCH ₂ OC(O)C(CH ₃ ) ₃

R ² =-NHCH ₂ CH ₂ N(CH ₃ ) ₂

R ³ =-OH

10-1

Add 1-dimethylamino-2-aminoethane (57.9 μl, 0.52mmol) and TBTU (168.6mg, 0.52mmol) in the DMF solution (9ml) of prodrug 3-5 (864mg, 0.52mmol), then Diisopropylethylamine (423 μl) was added. After stirring at room temperature for 20 minutes, the reaction mixture was purified by reverse phase HPLC (ACN: 0.1% TFA/water). Freeze drying afforded 295 mg (34%) of compound 10-1.

R ^1′ , R ^1″ , R ^1 ＝-C(O)OCH ₂ OC(O)C(CH ₃ ) ₃

R ² =-NH(cyclopropyl)

R ³ =-OH

10-2

Compound 10-2 was synthesized using the same method as above, but using 0.052 mmol of cyclopropylamine instead of 1-dimethylamino-2-aminoethane.

R ^1′ , R ^1″ , R ^1 ＝-C(O)OCH ₂ OC(O)C(CH ₃ ) ₃

R ² =-NHCH ₂ (CO ₂ CH ₃ )

R ³ =OH

10-3

Compound 10-3 was synthesized using the same method as above, but using glycine methyl ester instead of 1-dimethylamino-2-aminoethane.

R ^1′ , R ^1″ , R ^1 ＝-C(O)OCH ₂ OC(O)CH(CH ₃ ) ₂

R ² =-NHCH ₂ CH ₂ N(CH ₃ ) ₂

R ³ =-OH

10-4

Compound 10-4 was synthesized using the same method as above, but using 0.052 mmol of prodrug 7-1 instead of prodrug 3-5.

R ^1′ , R ^1″ , R ^1 ＝-C(O)OCH ₂ OC(O)CH(CH ₃ ) ₂

R ² =-NH(cyclopropyl)

R ³ =-OH

                             

Compound 10-5 was synthesized using the same method as above, but using 0.052 mmol of prodrug 7-1 instead of prodrug 3-5 and 0.052 mmol of cyclopropylamine instead of 1-dimethylamino-2-aminoethane .

Example 11

Example 11 describes the synthesis of prodrugs of pseudomycin compounds in which the carboxylic acid group of the aspartic acid unit of the pseudomycin ring is modified to generate a 3-amido derivative. Synthesis of 3-monoamido derivatives 11-1:

R ^1′ , R ^1″ and

R ² =-NH(cyclopropyl)

R ³ =-OH

                                                                                         

To a DMF solution (1 L) containing compound 2b-1 (1 eq) was added 1.5 eq of compound 1a-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 ^™ , Delta Pak C18 column) to obtain compound 11-1 and other mono- and di-substituted products.

Example 12

Example 12 describes the synthesis of prodrugs in which the carboxylic acid groups on aspartate and hydroxyaspartate are modified to generate diester derivatives. Synthesis of diester 12-1:

R ^1′ , R ^1″ and

R ² =-OCH ₃

R ³ =-OCH ₃

                                                                                                           

To a DMF solution (1 L) containing compound 3a-1 (1 equivalent) was added 1.5 equivalents of compound 1a-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 ^™ , Delta Pak C18 column) to obtain compound 12-1 and other mono- and di-substituted products.

Claims (16)

1. Pseudomycin prodrugs, its pharmacologically acceptable salt and solvate thereof with following structure,

Wherein R is

Wherein

R ^aAnd R ^{A '}Be H or methyl, perhaps R independently ^aOr R ^{A '}Be alkylamino, with R ^bOr R ^{B '}Form 6-unit cycloalkyl ring, 6-unit's aromatic ring or two key together, perhaps with R ^cForm 6-unit aromatic ring together;

R ^bAnd R ^{B '}Be H, halogen or methyl, perhaps R independently ^bOr R ^{B '}Be amino, alkylamino, α-acetylacetic ester, methoxyl group or hydroxyl;

R ^cBe H, hydroxyl, C ₁-C ₄Alkoxyl group, hydroxy alkoxy base or and R ^eForm 6-unit's aromatic ring or C together ₅-C ₆Cycloalkyl ring;

R ^eBe H, perhaps with R ^fBe 6-unit aromatic ring, C together ₅-C ₁₄6-unit's aromatic ring or C that alkoxyl group replaces ₅-C ₁₄6-unit's aromatic ring that alkyl replaces and

R ^fBe C ₈-C ₁₈Alkyl, C ₅-C ₁₁Alkoxyl group or xenyl; R is

Wherein

R ^gBe H or C ₁-C ₁₃Alkyl, and

R ^hBe C ₁-C ₁₅Alkyl, C ₄-C ₁₅Alkoxyl group, (C ₁-C ₁₀Alkyl) phenyl ,-(CH ₂) _n-aryl or-(CH ₂) _n-(C ₅-C ₆Cycloalkyl), n=1 or 2 wherein; Perhaps R is

Wherein

R ⁱBe H, halogen or C ₅-C ₈Alkoxyl group,

And m is 1,2 or 3; R is

Wherein

R ^jBe C ₅-C ₁₄Alkoxyl group or C ₅-C ₁₄Alkyl, and

P=0,1 or 2; R is

Wherein

R ^kBe C ₅-C ₁₄Alkoxyl group, perhaps R is-(CH ₂)-NR ^m-(C ₁₃-C ₁₈Alkyl), R wherein ^mBe H ,-CH ₃Or-C (O) CH ₃

R ¹Be H, acyloxy methylene radical-1 independently, 3-dioxole-2-ketone or acyloxy methylene radical carboxylicesters, condition is at least one R ¹Be acyloxy methylene radical-1,3-dioxole-2-ketone or acyloxy methylene radical carboxylicesters;

R ²And R ³Be independently-OR ^2a, or-N (R ^2b) (R ^2c),

Wherein

R ^2aAnd R ^2bBe H, C independently ₁-C ₁₀Alkyl, C ₃-C ₆Cycloalkyl, hydroxyl (C ₁-C ₁₀) alkyl, alkoxyl group (C ₁-C ₁₀) alkyl, C ₂-C ₁₀Alkenyl, amino (C ₁-C ₁₀) alkyl ,-or two-alkylamino (C ₁-C ₁₀) alkyl, aryl (C ₁-C ₁₀) alkyl, heteroaryl (C ₁-C ₁₀) alkyl, the assorted alkyl (C of ring ₁-C ₁₀) alkyl, perhaps

R ^2bBe the alkyl carboxylates residue and the R of aminoacid alkyl ester ^2cBe H or C ₁-C ₆Alkyl.

2. the prodrug of claim 1, wherein said acyloxy methylene radical-1,3-dioxole-2-ketone is represented by following formula 1 (a):

R wherein ^1aBe C ₁-C ₁₀Alkyl, C ₁-C ₁₀Thiazolinyl, benzyl or aryl, and R ^1bBe H or methyl.

3. the prodrug of claim 1, wherein said acyloxy methylene radical carboxylicesters is represented by following formula 1 (b):

R wherein ^1aBe C ₁-C ₁₀Alkyl, C ₁-C ₁₀Thiazolinyl, benzyl or aryl, and R ^1bBe H or methyl.

4. the prodrug of claim 2, wherein R is represented by following structure:

R wherein ^{B '}Be hydroxyl, R ^a, R ^{A '}, R ^b, R ^c, R ^dAnd R ^eAll be H, and R ^fIt is n-octyl.

5. the prodrug of claim 3, wherein R is represented by following structure: R wherein ^{B '}Be hydroxyl, R ^a, R ^{A '}, R ^b, R ^c, R ^dAnd R ^eAll be H, and R ^fIt is n-octyl.

6. the prodrug of claim 1, the alkyl carboxylates residue of wherein said aminoacid alkyl ester is by following expression :-CH ₂CO ₂CH ₃,-CH (CO ₂CH ₃) CH (CH ₃) ₂,-CH (CO ₂CH ₃) CH (phenyl) ,-CH (CO ₂CH ₃) CH ₂OH ,-CH (CO ₂CH ₃) CH ₂(p-hydroxybenzene) ,-CH (CO ₂CH ₃) CH ₂SH ,-CH (CO ₂CH ₃) CH ₂(CH ₂) ₃NH ₂,-CH (CO ₂CH ₃) CH ₂(4-imidazoles) ,-CH (CO ₂CH ₃) CH ₂(5-imidazoles) ,-CH (CO ₂CH ₃) CH ₂CO ₂CH ₃, or-CH (CO ₂CH ₃) CH ₂CO ₂NH ₂

7. Pseudomycin prodrugs, its pharmacologically acceptable salt and solvate thereof with following structure, Wherein R is Wherein

R ^aAnd R ^{A '}Be H or methyl, perhaps R independently ^aOr R ^{A '}Be alkylamino, with R ^bOr R ^{B '}Form 6-unit cycloalkyl ring, 6-unit's aromatic ring or two key together, perhaps with R ^cForm 6-unit aromatic ring together;

R ^bAnd R ^{B '}Be H, halogen or methyl, perhaps R independently ^bOr R ^{B '}Be amino, alkylamino, α-acetylacetic ester, methoxyl group or hydroxyl;

R ^cBe H, hydroxyl, C ₁-C ₄Alkoxyl group, hydroxy alkoxy base or and R ^eForm 6-unit's aromatic ring or C together ₅-C ₆Cycloalkyl ring;

R ^eBe H, perhaps with R ^fBe 6-unit aromatic ring, C together ₅-C ₁₄6-unit's aromatic ring or C that alkoxyl group replaces ₅-C ₁₄6-unit's aromatic ring that alkyl replaces and

R ^fBe C ₈-C ₁₈Alkyl, C ₅-C ₁₁Alkoxyl group or xenyl; R is Wherein

R ^gBe H or C ₁-C ₁₃Alkyl, and

R ^hBe C ₁-C ₁₅Alkyl, C ₄-C ₁₅Alkoxyl group, (C ₁-C ₁₀Alkyl) phenyl ,-(CH ₂) _n-aryl or-(CH ₂) _n-(C ₅-C ₆Cycloalkyl), n=1 or 2 wherein; Perhaps R is

Wherein

R ⁱBe H, halogen or C ₅-C ₈Alkoxyl group,

And m is 1,2 or 3; R is

Wherein

R ^jBe C ₅-C ₁₄Alkoxyl group or C ₅-C ₁₄Alkyl, and

P=0,1 or 2; R is

Wherein

R ^kBe C ₅-C ₁₄Alkoxyl group, perhaps R is-(CH ₂)-NR ^m-(C ₁₃-C ₁₈Alkyl), R wherein ^mBe H ,-CH ₃Or-C (O) CH ₃

R ¹Be H, acyloxy methylene radical-1 independently, 3-dioxole-2-ketone or acyloxy methylene radical carboxylicesters, condition is at least one R ¹Be acyloxy methylene radical-1,3-dioxole-2-ketone or acyloxy methylene radical carboxylicesters;

R ²And R ³Be independently-OR ^2a, or-N (R ^2b) (R ^2c),

Wherein

R ^2aAnd R ^2bBe H, C independently ₁-C ₁₀Alkyl, C ₃-C ₆Cycloalkyl, hydroxyl (C ₁-C ₁₀) alkyl, alkoxyl group (C ₁-C ₁₀) alkyl, C ₂-C ₁₀Thiazolinyl, amino (C ₁-C ₁₀) alkyl ,-or two-alkylamino (C ₁-C ₁₀) alkyl, aryl (C ₁-C ₁₀) alkyl, heteroaryl (C ₁-C ₁₀) alkyl, the assorted alkyl (C of ring ₁-C ₁₀) alkyl, perhaps

R ^2bBe the alkyl carboxylates residue and the R of aminoacid alkyl ester ^2cBe H or C ₁-C ₆Alkyl.

8. the prodrug of claim 7, wherein said acyloxy methylene radical-1,3-dioxole-2-ketone is represented by following formula 1 (a):

R wherein ^1aBe C ₁-C ₁₀Alkyl, C ₁-C ₁₀Thiazolinyl, benzyl or aryl, and R ^1bBe H or methyl.

9. the prodrug of claim 7, wherein said acyloxy methylene radical carboxylicesters is represented by following formula 1 (b):

R wherein ^1aBe C ₁-C ₁₀Alkyl, C ₁-C ₁₀Thiazolinyl, benzyl or aryl, and R ^1bBe H or methyl.

10. the prodrug of claim 8, wherein R is represented by following structure: R wherein ^{B '}Be hydroxyl, R ^a, R ^{A '}, R ^b, R ^c, R ^dAnd R ^eAll be H, and R ^fIt is n-octyl.

11. the prodrug of claim 9, wherein R is represented by following structure: R wherein ^{B '}Be hydroxyl, R ^a, R ^{A '}, R ^b, R ^c, R ^dAnd R ^eAll be H, and R ^fIt is n-octyl.

12. the prodrug of claim 7, the alkyl carboxylates residue of wherein said aminoacid alkyl ester is by following expression :-CH ₂CO ₂CH ₃,-CH (CO ₂CH ₃) CH (CH ₃) ₂,-CH (CO ₂CH ₃) CH (phenyl) ,-CH (CO ₂CH ₃) CH ₂OH ,-CH (CO ₂CH ₃) CH ₂(p-hydroxybenzene) ,-CH (CO ₂CH ₃) CH ₂SH ,-CH (CO ₂CH ₃) CH ₂(CH ₂) ₃NH ₂,-CH (CO ₂CH ₃) CH ₂(4-imidazoles) ,-CH (CO ₂CH ₃) CH ₂(5-imidazoles) ,-CH (CO ₂CH ₃) CH ₂CO ₂CH ₃, or-CH (CO ₂CH ₃) CH ₂CO ₂NH ₂

13. each compound of aforementioned claim is used for resisting the purposes of the medicine of systemic fungal infection or fungal skin infections in preparation.

14. a medicinal preparations contains Pseudomycin prodrugs and pharmaceutically acceptable carrier of claim 1 or 7.

15. a medicine for the treatment of the animal anti-fungal infection, wherein said medicine contains the compound of claim 1 or 7.

16. a method for the treatment of the animal anti-fungal infection that needs treatment comprises to described animals administer claim 7,8,9,10 or 11 Pseudomycin prodrugs.