CN1360593A - Pseudomycin N-acyl side-chain analogs - Google Patents

Abstract

Semi-synthetic pseudomycin compounds having structure (I) are described which may be useful as antifungal agents or intermediates in the design of antifungal agents.

Description

Pseudomycin N-acyl side chain analogs

Field of Invention

The present invention relates to pseudomycin compounds, especially semi-synthetic pseudomycin compounds having novel N-acyl side chains.

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, for example, 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 US5,576,298 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 portion of pseudomycin A, A', B, B', C, C' corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L- with a terminal carboxyl group aThr-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., "Bioactive 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 compound represented by the following structural formula used as an antifungal agent or for antifungal agent design, a pharmaceutically acceptable salt thereof and a solvate thereof,

where R is

in

R ^a and R ^a' are independently hydrogen 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 hydrogen, halogen or methyl, or R ^b or R ^b' is amino, alkylamino, α-acetoacetate, methoxy or hydroxy, with the proviso that when R ^a , R ^b , R ^d , Re are ^hydrogen , R ^c is hydrogen and R ^f is n-hexyl, n-octyl or n-decyl, or R ^a , R ^b , R ^d , ^Re are hydrogen, R ^c is hydroxyl and When R ^f is n-octyl, n-nonyl or n-decyl, R ^b' is not hydroxyl;

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

R ^e is hydrogen, 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; or R is

in

R ^g is hydrogen 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 hydrogen, halogen, or C ₅ -C ₈ alkoxy, and

其中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

R ^k is C ₅ -C ₁₄ alkoxy, or R is -(CH ₂ )-NR ^m -(C ₁₃ -C ₁₈ alkyl), wherein R ^m is hydrogen, -CH ₃ or -C(O)CH ₃ .

In another embodiment of the present invention, a pharmaceutical preparation is provided, which comprises the pseudomycin compound represented by the above structure I 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 the pseudomycin compound I described above.

In another embodiment of the present invention, there is provided a method for preparing the free amine nucleus of a pseudomycin compound, which can be acylated to produce the compound represented by the above structure I. The method comprises the step of treating a pseudomycin compound having at least one N-acylalkyl side chain having at least one gamma or delta hydroxyl group (e.g., pseudomycin A, A' or C) with trifluoroacetic acid or acetic acid .

definition

其中，R’是-NH₂或-NH_p-P_g，其中P_g为氨基保护基及p为0或1。The term "free amine pseudomycin core" or "pseudomycin core" as used herein refers to the structure of the following IA:

Wherein, R' is -NH ₂ or -NH _p -P _g , wherein P _g is an amino protecting group and p is 0 or 1.

The term "alkyl" refers to a hydrocarbon group of the general formula C _n H _2n+1 containing 1 to 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]pentane, 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 may be substituted or unsubstituted. The alkenyl moiety in alkenyloxy, enoyl or enoate has the same definition as above.

The term "alkynyl" refers to an acyclic hydrocarbon containing at least one carbon-carbon triple bond. Alkynyl groups can be straight or branched and substituted or unsubstituted. The alkynyl moiety in the alkynyloxy, alkynoyl or alkynoate group has the same meaning 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.

The term "heteroaryl" refers to an aryl group containing at least one heteroatom in the aromatic ring system (for example, pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc. ). Aryl groups can contain single or fused ring systems. Heteroaryl groups can be substituted or unsubstituted.

"NH _p -P _g " and "amino protecting group" refer to groups commonly used as amino substituents to prevent or protect the amino functionality while other functional groups of the compound are reacting. When p is 0, the amino protecting group together with the nitrogen to which it is attached forms a cyclic imide, such as phthalimide and tetrachlorophthalimide. When p is 1, the amino protecting group can form a carbamate with the nitrogen to which it is attached, such as methyl, ethyl, and 9-fluorenylmethyl carbamate; or amides, such as N-formyl and N- Acetylamide.

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 refers to and allows substitutions on conventional alkyl groups in the art, such as hydroxyl, halogen, alkoxyl, 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 "solvate" refers to an aggregate comprising one or more solute molecules, such as a compound of structure I, and one or more pharmaceutically acceptable solvent molecules, such as water, ethanol, and the like.

The term "pharmaceutically acceptable salt" refers to an organic or inorganic salt of a compound represented by structure I which is substantially nontoxic to recipients at the administered dose.

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

The applicant found that the N-acyl deacylation of the L-serine unit of the pseudomycin compound, followed by re-acylation with a new N-acyl group gave new compounds, and in vitro experiments showed that these new compounds may have anti-Candida albicans ( C. albican), Cryptococcus neoformans (C, neoformans) and/or Aspergillus fumigatus (Aspergillus fumigatus) activity.

Scheme I below shows a general method for the synthesis of compound I from any of the naturally occurring pseudomycins. Although a naturally occurring pseudomycin compound is depicted in Scheme I, those skilled in the art will understand that side chain modifications of semi-synthetic derivatives of naturally occurring pseudomycin compounds can be performed in a similar manner. The method is complete. Generally, the preparation of compound I requires a four-step synthesis: (1) selective amino protection; (2) chemical deacylation or enzymatic deacylation of the N-acyl side chain; (3) re-acylation with a different side chain and (4) deprotection of the amino group. Reaction Scheme I

The side chain amino groups at positions 2, 4 and 5 can be protected by any standard method for protecting amino groups known to those skilled in the art. The particular nature of the amino-protecting group is not critical so long as the derivatized amino group is stable under the conditions of subsequent reactions elsewhere in the intermediate molecule and the protecting group can be removed selectively at an appropriate time without affecting the rest of the molecule, including any other The amino protecting group is enough. Suitable amino protecting groups include benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-methoxyphenylazobenzyloxy ylcarbonyl, p-phenylazobenzyloxycarbonyl, tert-butyloxycarbonyl, cyclopentyloxycarbonyl and phthalimido. Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl (Alloc), phthalimido and benzyloxycarbonyl (CbZ or CBZ). Most preferred are allyloxycarbonyl and benzyloxycarbonyl. Furthermore, 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), Chapter 7.

Deacylation of N-acyl groups with gamma or delta hydroxylated side chains (e.g., 3,4-dihydroxytetradeconoate) can be achieved by treating amino-protected pseudomonads with 5-20% aqueous acid Mycocin compounds to complete. 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 acetic acid is used, the reaction is generally carried out at about 40°C. Solubilization of the pseudomycin compound can be facilitated by a water-soluble organic solvent. Suitable aqueous solvent systems include acetonitrile, water and mixtures thereof. Acetonitrile is particularly useful when deacylating protected pseudomycin compounds. The preferred acid solution for deacylation of the protected pseudomycin compound is 8% trifluoroacetic acid in acetonitrile in water. Organic solvents accelerate the reaction, however, addition of organic solvents can lead to other by-products. Pseudomycin compounds lacking a delta hydroxyl group on the side chain (e.g., pseudomycins B and C') can be enzymatically deacylated. Suitable deacylases include polymyxin acylase (164-16081 fatty acylase (crude product) or 161-16091 fatty acylase (pure product) can be purchased from Wako Pure Chemical Industries, Ltd.), or ECB deacylase (see, e.g., U.S. Patent No. 5,573,936). Enzymatic deacylation can be accomplished using standard deacylation methods well known to those skilled in the art. For example, see Yasuda, N. et al., Agric. Biol. Chem., 53, 3245 (1989) and Kimura, Y. et al., Agric. (1989).

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

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

Various acids can be used in the acylation process. Suitable acids include aliphatic acids containing one or more side chain aryl, alkyl, amino (including primary, secondary and tertiary amines), hydroxyl, alkoxy and amino groups; those containing nitrogen or oxygen in the aliphatic chain Aliphatic acids; aromatic acids substituted with alkyl, hydroxy, alkoxy, and/or alkylamino; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy, and/or alkylamino. The acylated products are useful as active antifungal agents or as intermediates for the preparation of active compounds. Even if some compounds are less useful than others, their activity profiles provide valuable information for designing ideas for optimal activity.

Once the amino group is acylated, the amino protecting groups (positions 2, 4 and 5) can be removed by the presence of a hydrogenation catalyst (eg 10% Pd/C). When the amino protecting group is allyloxycarbonyl, the protecting group can be removed with tributyltin hydride and triphenylphosphinepalladium dichloride. The advantage of this particular protection/deprotection route is that it reduces the possibility of hydrogenating the vinyl group of the pseudomycin Z-Dhb unit.

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" filed April 14, serial number PCT/US00/08727, and US 5,576,298 and 5,837,685, which are hereby incorporated by reference Reference.

Isolates of P. syringae that produce one or more pseudomycins are known in the art. The wild-type strain MSU 174 and its mutant MSU 16H (ATCC 67028), produced by transposon mutagenesis, are described in US 5,576,298 and 5,837,685; "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 offfluorescent 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 identity of the culture of the pseudomycin used (Pseudomonas syringae strains such as MSU 174, MSU 16H, MSU 206, 25-B1, 7H9-1) is subject 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

67H1 Accession 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 MSU 16H 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.

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

The pseudomycin compound can be isolated and used as such or in the form of a pharmaceutically acceptable salt or solvate thereof. 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.

The active ingredient (ie, the pseudomycin derivative) 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 formulations may contain from 0.1% to 99.9% wt of 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 administered may vary depending on the physical characteristics of the animal, the severity of the animal's condition, and the mode used for administration. 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 particular method of management used will depend on the type of infection being treated.

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, Pseudallescheria boydii, Coccidioides immature, Sporothrix schenckii, etc.

Therefore, the compounds and formulations of the present invention are useful in the preparation of a medicament useful for combating systemic fungal infections or fungal skin infections. Accordingly, there is provided a method of inhibiting fungal activity comprising contacting a compound I of the invention with a fungus. Preferred methods include inhibiting the activity of Candida albicans, Cryptococcus neoformans 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 comprising inhibiting the activity of fungi 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. The host can be any animal, 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

All chemical reagents were purchased from Aldrich Chemical (Milwaukee, WI) unless otherwise stated.

Biological samples

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 in the American Type Culture Collection on March 23, 2000, and the preservation numbers are as follows:

25-B1 deposit number is PTA-1622

The deposit number of 7H9-1 is PTA-1623

The 67H1 deposit number is the chemical abbreviation of PTA-1621

The following abbreviations are used in the examples used to denote the respectively listed materials:

ACN-acetonitrile

TFA-trifluoroacetic acid

DMF-dimethylformamide

DEAD-Diethyl azodicarboxylate

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)

FMOC = fluorenylmethyloxycarbonyl

                                   

Unless otherwise stated, analytical reversed-phase HPLC operations were performed on a Waters 600E system equipped with a Waters μBondapak (C18, 3.9 x 300mm) column. The eluent used was a 65:35 acetonitrile/0.1% TFA in water solvent system-100% acetonitrile (over 20 minutes) at a flow rate of 1.5 ml/min with UV detection at 230 nm.

Preparative HPLC processing was carried out with a Waters Prep2000 system using a Dynamax 60 Angstrom C18 column, using the same solvent system as the analytical HPLC system, but at a flow rate of 40 ml/min.

Detection and Quantitative Determination of Bioanalytical Antifungal Activity:

Antifungal activity was determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc expansion test. A typical fungus used in the antifungal activity test is Candida albicans. Significant antifungal activity was considered when the inhibition of C. albicans inoculated on agar plates by a test sample (50 μl) resulted in a 10-12 mm diameter area. Tail Vein Toxicity:

Mice were treated with 0.1 ml of test compound (20 mg/kg) administered intravenously (IV) via the lateral tail vein at 0, 24, 48 and 72 hours. Each group included 2 mice. Compounds were formulated in 0.5% dextrose and sterile water for injection. Mice were monitored for 7 days after the first treatment and carefully observed for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss, and other side effects indicative of toxicity.

The mice used in this experiment were outbred, and the average body weight of male ICR mice was 18-20 g (obtained from Harlan Sprangue Dawley, Indianapolis, IN).

           General Procedures for the protection of side chain amino groups at positions 2, 4 and 5 of pseudomycin A, A', B, B', C or C'

The pseudomycin compound (R ¹ =H) was dissolved/suspended in DMF (20 mg/ml, Aldrich Sure Seal). Add N-(benzyloxycarbonyloxy)succinimide (6eq) under stirring at room temperature, stir at room temperature for 32 hours, and pass HPLC (4.6×50mm, 3.5μm, 300-SB, C8, Zorbax column ) to monitor the reaction. Rotary evaporated to 10 ml at room temperature under high vacuum and the material was frozen until ready for chromatographic preparation. An amorphous, white solid was obtained after reverse-phase preparative HPLC and lyophilization. General procedure for chemical deacylation of N-acyl groups of L-serine units.

The protected pseudomycin A was dissolved/suspended in water/acetonitrile (2: ₁ H2O:ACN, about 3.5 mg/ml) and TFA (8% vol) was added slowly at room temperature. The reaction was stirred at room temperature until starting material was consumed. The acetonitrile was removed under vacuum at room temperature and the material was lyophilized. The resulting solid was dissolved in a small amount of DMF (water if necessary, then an equal amount of ACN) and preparative HPLC and lyophilization generally gave a white, amorphous solid (probably TFA salt). Solid-phase acylation of the pseudomycin core with HOBt resin. Myristic acid is used in the following examples; however, the same general procedure can be used for other organic acids.

In a 100 ml reaction tube with glass sintered ends, myristic acid (1.03 g, 3.62 mmol) was dissolved in 50 ml of 1:1 DMF/THF. To this solution was added resin HOBt (1.94 g, 2.9 mmol) and EDCI (0.556 g, 2.9 mmol) and shaken overnight. The solvent was drained off and the resin was washed with 2xDMF, 2xTHF and 2x 1:1 DMF/THF. Dissolve CBZ-protected pseudomycin core (1.0 g, 0.723 mmol) in 50 ml DMF/THF (20 mg/ml) and add to the resin-bound activated ester and place on a rotator or shaker Mix overnight. The product was separated from the resin and the remaining resin was washed with 2x DMF, 2x THF and 2x 1:1 DMF/THF. The combined filtrates were separated by reverse phase HPLC and lyophilized to give (129 mg, 10%) myristanoylated CBZ-protected pseudomycin product. Acylation of pseudomycin nucleus with activated ester (HOBt-mesylate). The following examples use glycine tetradecanoic acid, however this general procedure can be used for other organic acids as well.

In a 500 ml round bottom flask, glycine tetradecanoic acid (0.309 g, 1.1 mmol) was dissolved in 100 ml DMF. To this solution was added HOBt-mesylate (0.229 g, 1.1 mmol) and triethylamine (0.081 g, 0.8 mmol). The solution was stirred rapidly under 1 atm _N2 overnight and dried under high vacuum to remove DMF and TEA. The residual oil was azeotroped three times with toluene until a white solid formed. To this solid was added 100 ml DMF and 1 g CBZ-protected pseudomycin core. The solution was stirred overnight and dried under high vacuum. The product was purified by reverse phase HPLC and lyophilized to give (233 mg, 20%) myristyl acylated CBZ-protected pseudomycin product. General procedure for deprotection of side chain amino groups at positions 2, 4 and 5 via hydrogenation

The CBZ-protected activated derivative was dissolved in cold 13% acetic acid/methanol solution (5 mg/ml) and an equivalent of 10% Pd/C was added. Charge the reaction with hydrogen by degassing and displacing the volume with _H 4-7 times. The reaction was performed at room temperature and monitored every 15 minutes by HPLC and mass spectrometry until the starting material was consumed. When the reaction was complete, the balloon was removed and the reaction was filtered through a 0.45 μm filter disc (Acrodisk GHP, GF by Gelman), concentrated to about 1/10 volume and purified by preparative HPLC, and product-containing fractions were lyophilized. Preparation of side chain precursor (1c)

To a 250 ml round bottom flask containing 100 ml of degassed ACN was added m-bromobenzaldehyde (5.000 g, 27.02 mmol), triethylamine (5.490 g, 54.25 mmol) and 1-dodedecyn (5.000 g, 30.06 mmol). To this mixture was added _PdCl2 (243.1 mg, 1.370 mmol), triphenylphosphine (718.8 mg, 2.740 mmol) and CuI (173.8 mg, 0.9120 mmol). It was then heated to reflux and reacted overnight. The reaction was then cooled to room temperature and the solvent was removed in vacuo, the resulting residue was treated with dichloromethane and washed with 2 x 1N HCl and 1 x brine. The organic layer was dried with _MgSO4 . The drying agent was filtered off, the solvent was removed in vacuo and purification by column chromatography on silica gel eluting with 3% EtOAc/hexanes afforded 3.73 g of the title compound as a brown oil. Spectral data are consistent with the structure of m-(l-dodeynyl)benzaldehyde (la).

To 100 mL of EtOAc was added the above compound (1.00 g, 3.70 mmol) and 0.1 g of 5% Pd/Al ₂ O ₃ . The reaction mixture was treated under 50 psi of _H2 at room temperature for 1 h. The reaction mixture was filtered through celite to remove the catalyst and the celite was rinsed with copious amounts of EtOAc. EtOAc was removed by rotary evaporator to give 882.1 mg of product. This product was used in the next step without further purification. Spectral data are consistent with m-dodecylbenzaldehyde (lb).

Into an oven-dried 50ml round-bottomed flask under a nitrogen atmosphere at -78°C, 6.0ml of anhydrous THF was added followed by lithium diisopropylamide (1.2mL of a 2M heptane/THF/ethylbenzene solution, 2.41 mmol). To this was added tert-butyl acetate (0.331 ml, 2.45 mmol) and the resulting solution was warmed to about -40°C and maintained at this temperature for 1 hour. Then a solution of the above compound (501.9 mg, 1.83 mmol) dissolved in 4 mL of anhydrous THF and precooled to -40°C was added dropwise to the anion. The reaction mixture was stirred for 1 hour, then quenched with 2 mL of saturated aqueous ammonium chloride and 10 mL of water. The reaction mixture was partitioned between diethyl ether and water, the organic layer was washed once with brine and dried over sodium sulfate. The drying agent was filtered off, the solvent was removed in vacuo, and purification by column chromatography on silica gel with 5% EtOAc/hexanes gave 238.1 mg of a yellow oil. Spectral data were consistent with tert-butyl 3-hydroxy-3-(m-dodecylbenzyl)propionate.

Precooled (0° C.) 4 ml TFA solution was added to a 50 ml round bottom flask containing crude tert-butyl 3-hydroxy-3-(m-dodecylbenzyl)propionate. The reaction was stirred at this temperature for 25 min, TLC (10% EtOAC/hexanes) showed complete consumption of the starting ester. TFA was removed under vacuum to give oil (1c). Preparation of side chain precursor (2c):

Compound 2a was synthesized in the same way as compound 1a above, substituting 1-octyne for 1-dodedecyne. Compounds 2b and 2c were synthesized in the same way as the synthesis of 1b and 1c above. Preparation of side chain precursor (3b):

To a 500 mL round bottom flask containing 100 mL of acetone was added m-hydroxybenzaldehyde (5.00 g, 40.94 mmol), 1-bromoundecane (9.65 g, 41.02 mmol) and K ₂ CO ₃ (8.48 g, 61.36 mmol). The reaction mixture was heated to reflux and reacted for 10 hours. The reaction was cooled and the acetone was removed under vacuum. The resulting residue was partitioned between ether/water and the organic layer was washed twice with saturated aqueous NaHCO ₃ and once with brine. The organic layer was dried over _MgSO4 , then the desiccant was filtered off and the solvent was removed in vacuo, purified on a silica gel column eluting with 3% EtOAc/hexanes to give 1.6876 as a yellow oil. Spectral data were consistent with compound 3a, and the aldehyde was then prepared into compound 3b by the same method as described for the preparation of 1c. Preparation of side chain precursor (4c):

Compound 4a was synthesized in the same manner as Compound 1c above, except that Compound 1a was not hydrogenated prior to condensation with tert-butyl acetate.

In a 50ml round bottom flask was added tert-butyl 3-hydroxy-3-(m-dodeynylbenzyl)propionate 4a (346.8 mg, 0.897) and dissolved in 10 ml MeOH/1 mL glacial AcOH. Standard hydrogenation with 10% Pd/C (304.5 mg) for 24 h, the catalyst was removed by filtration, the solvent was removed under vacuum to give 302.4 mg tert-butyl 3-(m-dodecylbenzyl) propionate, which was then treated with TFA to remove tert-butyl Butyl esters afford compound 4b. Preparation of side chain precursor (5a):

To a 50 ml round bottom flask containing 5 mL THF was added sec-butyllithium (1.56 mmol, 1.2 mL of a 1.3 M solution in cyclohexane) at -78 °C. To this mixture was added dropwise tert-butyl bromoisobutyrate (in 2ml THF at -78°C), the reaction was stirred for 30min, and compound 1b (318.9mg, 1.16mmol in 2ml THF, - 78°C). The resulting reaction mixture was stirred at -78 °C for 30 min, then warmed to 0 °C over 30 min and stirred at this temperature for 1.75 h, then quenched with 2 ml of saturated aqueous ammonium chloride and allowed to warm to room temperature. The reaction mixture was partitioned between ether/water and the organic layer was washed once with brine and dried over _MgSO4 . The drying agent was then filtered off and the solvent was removed in vacuo to give 370.5 mg of crude product as a racemic mixture. The tert-butyl ester was then removed by TFA to afford compound 5a. Preparation of side chain precursor (6a):

To a 250 mL round bottom flask containing 100 mL of MeOH and 1 mL of concentrated _H2SO4 was added m-bromo-p-methoxybenzoic acid (5.00 g, 21.6 _mmol ). The resulting mixture was heated to reflux for 24 hours. The reaction was cooled and the solvent was removed under vacuum, the resulting solid was treated with diethyl ether, the organic layer was washed twice with water, once each with saturated aqueous NaHCO ₃ and brine and dried over MgSO ₄ . The desiccant was then filtered off and the solvent was removed under vacuum to give 4.72 g of crude product which was used without further purification. This product was then condensed with 1-tetradecyne and hydrogenated to give olefins similar to the previous examples. The methyl ester was converted to formic acid by the following steps: the methyl ester (538.4mg, 1.48mmol) was suspended in 20ml of 30% HBr in AcOH solution and heated to reflux. After reflux for 24h, the solution was poured into 150ml of water and washed with 2×200ml CH _{2 Cl2} extraction. The organic phase was washed with copious amounts of water and dried over _MgSO4 , then the desiccant was filtered off and the solvent was removed in vacuo to yield 398.7 g of crude 6a, which was used without further purification. Preparation of side chain precursor (7a):

Compound 6a (385.2 mg) was added to the pyridinium hydrochloride solid. The two solids were heated to ~220°C to melt and the mixture was maintained for 3 hours, then the reaction was cooled and partitioned between _CH2Cl2 /1N HCl _. The organic phase was then washed 5 times with 1 N HCl and dried over MgSO ₄ , then the desiccant was filtered off and the solvent was removed in vacuo to give 158.8 mg of crude (7a), which was used without further purification. Preparation of side chain precursor (8b):

A 250 ml round bottom flask was charged with biphenylboronic acid (2.00 g, 10.1 mmol) and Pd(PPh) ₄ (980.0 mg, 0.848 mmol) in 60 mL toluene/30 mL 2 M Na ₂ CO ₃ aqueous solution. To this slurry was added m-bromobenzaldehyde (in 10 mL MeOH). The reaction mixture was heated to reflux and maintained for 20 h. The reaction was cooled, the organic layer was washed twice with water, once with brine and dried over _MgSO4 , then the desiccant was filtered off and the solvent was removed in vacuo. The resulting solid was rinsed with cold hexane to remove any residual m-bromobenzaldehyde, then the solid was slurried in hot hexane and filtered hot to remove any solid. The filtrate was freed of solvent under vacuum to afford the desired aldehyde 8a. Conversion of compound 8a to 8b using the same procedure as described for the preparation of 1c afforded the inseparable diastereomers. Preparation of side chain precursor (9a):

A solution of tert-butyl acetate (2.02ml, 0.015mol) in anhydrous THF (25ml) was cooled to -78°C and n-butyllithium (1.6M in hexane) (9.35ml, 0.015mol) was added dropwise. After 45 min, a THF solution of 2-tridecanone (2.0 g, 0.01 mol) was added dropwise, stirring was continued at low temperature for 1 h, and then the reaction was allowed to warm to room temperature over 15 min. Excess 1 N HCl was added to quench the reaction, the aqueous solution was extracted with ether (2x), the ether layer was dried over _MgSO4 and concentrated in vacuo to give a crude oil. Purification by silica gel column chromatography (5% ethyl acetate/hexane) afforded 1.01 g (32% yield) of tert-butyl ester. NMR was consistent with the desired product. Then the tert-butyl ester was treated with trifluoroacetic acid to decompose the tert-butyl ester quantitatively to obtain compound 9a. Preparation of side chain precursor (10e): R _{＝ nC5H11}

To a solution of m-hydroxybenzaldehyde (1.00 g, 8.20 mmol) in THF (16 mL) was added DEAD (1.29 mL, 8.20 mmol), PPh ( _2.15 g, 8.20 mmol) and n-pentanol (723 mg, 8.20 mmol) at room temperature ). The mixture was stirred overnight at room temperature and purified on silica gel to afford 1.02 g (65%) of the desired product 10a.

To a solution of 10a (6.70 g, 34.90 mmol) in THF was added _Ph3P =CHO (10.6 g, 34.90 mmol) at 0 °C. After stirring overnight at room temperature, the reaction mixture was filtered and concentrated in vacuo to give a residue which was purified by silica gel chromatography to afford 3.57 g (47%) of the desired unsaturated aldehyde 10b.

A solution of 10b (3.37 g, 15.5 mmol) in EtOAc (30 mL) was hydrogenated (1.5 atm) with 10% Pd/C (1.64 g, 1.55 mmol). The reaction was stirred overnight and the reaction mixture was filtered through a pad of Celite. The filtrate and rinses were concentrated in vacuo, and the residue was purified by column chromatography on silica gel (10% EtOAc/hexanes) to afford 2.37 g (70%) of 10c.

To a solution of aldehyde 10c (1.26 g, 5.71 mmol) in dichloromethane (23 mL) was added allyltrimethylsilane (0.91 mL, 5.71 mmol) followed by TiCl ₄ (0.63 mL, 5.71 mmol). After stirring at 0°C for 1 hour, the reaction was quenched with saturated NaHCO ₃ solution, the mixture was diluted with dichloromethane (75 mL), and the organic layer was washed successively with saturated NaHCO ₃ , water and brine. The organic layer was dried, concentrated and purified by silica gel chromatography (10% EtOAc/hexanes) to afford 0.91 g (61%) of the desired allyl alcohol 1Od.

Allyl alcohol 10d (0.91 g, 3.47 mmol) was dissolved in aqueous acetone (7 mL each), and to this solution was added NMO (704 mg, 5.21 mmol) and OsO ₄ (44 mg, 0.17 mmol) in THF. After stirring at room temperature for 2 hours, the reaction was quenched with _NaHSO3 (750 mg) to kill excess oxidant. The reaction mixture was diluted with brine (10 mL) and extracted with EtOAc (3 x 50 mL), the combined organic layers were dried and concentrated in vacuo to afford 850 mg (82%) of the desired triol intermediate. The triol thus obtained (850 mg, 2.87 mmol) was dissolved in MeOH (30 mL) and water (6 mL), the solution was treated with NaIO ₄ (1.38 g, 6.46 mmol) at room temperature and after 1 hour the reaction mixture was washed over Celite Plate filter and carefully concentrate the filtrate under vacuum to give the corresponding β-hydroxyaldehyde. The aldehyde was dissolved in t-BuOH (14 mL) and cyclohexane (2 mL), and to the solution was added KH ₂ PO ₄ (2.33 g, 17.7 mmol) and NaClO ₂ (2.08 g, 23.0 mmol) in water ( 15 mL H ₂ O) solution, the reaction was stirred at room temperature for 5 hours, then acidified to pH=3 with 1N HCl, the reaction mixture was extracted with EtOAc (3×50 mL), and the combined organic phases were washed with water and brine. The organic layer was dried and concentrated in vacuo to give crude acid 10e (1.5 g, ~3.4 mmol).

Compounds wherein R is nC ₆ H ₁₃ , nC ₇ H ₁₅ , nC ₈ H ₁₇ , nC ₉ H ₁₉ , nC ₁₀ H ₂₁ and nC ₁₄ H ₂₉ can also be prepared in the same manner as above. Preparation of side chain precursor (11d):

To a solution of chiral acetal 11a (6.22 g, 19.1 mmol) in dichloromethane (190 mL) was added trimethylallylsilane (10.9 mL, 68.69 mmol) followed by neat _TiCl4 at -78 °C (2.94 mL, 26.71 mmol). The reaction mixture was stirred at -78 °C for 1 hour, then at -40 °C for 2 hours, at which time the reaction was quenched with methanol (15 mL) and diluted with dichloromethane (200 mL), and the resulting reaction mixture was washed with 1 N HCl (2 x 50 mL), Washed with 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 11b. ¹ H NMR (CDCl ₃ ) of 11a: δ4.73 (m, 1H), 4.21 (m, 1H), 3.86 (m, 1H), 1.75 (m, 1H), 1.60-1.10 (m, 35H), 0.80 (m, 3H). ¹ H NMR (CDCl ₃ ) of 11b: δ5.81(m, 1H), 5.05(m, 2H), 4.12(m, 1H), 3.86(m, 1H), 3.41(m, 1H), 2.22(m , 2H), 1.67-1.18 (m, 36H), 0.88 (m, 3H).

To a solution of 11b (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 (8.36g, 22.8mmol) was dissolved in THF (60mL) and MeOH (30mL), 7.5M KOH (15mL) was added to the solution, and after stirring at room temperature for 3 hours, part of the solvent was removed, The remaining reaction mixture was diluted with EtOAc/ _Et2O (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 afford 6.22 g (96%) of the desired product 11c , as a white solid. ¹ H NMR (CDCl ₃ ) of 11c: δ5.75 (m, 1H), 5.06 (m, 2H), 3.56 (m, 1H), 2.23 (m, 1H), 2.07 (m, 1H), 1.75-1.17 (m, 28H), 0.80 (m, 3H).

Methanol 11c (6.22 g, 22.0 mmol) was dissolved in aqueous THF (5.5 mL of water and 55 mL of THF), and to this solution was added NMO (4.42 g, 33.0 mmol) followed by OsO ₄ (280 mg in THF, 1.10 mmol ). The reaction was stirred overnight at room temperature, at which 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, and the resulting organic layer was dried and washed in Concentration in vacuo gave the corresponding triol intermediate. It was dissolved in MeOH (200 mL) and water (40 mL), NaIO ₄ (10.6 g, 49.5 mmol) was added to the solution, and after stirring at room temperature for 1 hour, the reaction mixture was filtered through Celite and purified by short silica gel column chromatography ( 30% EtOAc/Hexanes) afforded -10 g (>100%) of crude β-hydroxyaldehyde. This obtained impure aldehyde was dissolved in t-BuOH (100 mL) and cyclohexane (14 mL), and to this solution were added NaClO ₂ (15.97 g, 176 mmol) and KH ₂ PO ₄ (17.8 g, 132mmol) (50mL) in water. The reaction was stirred at room temperature for 6 hours, then quenched with 5N HCl at 0°C to pH 4. Extracted with a 3:1 mixture of EtOAc/ _Et2O (3 x 250ml), the organic layer was washed with brine, dried and concentrated to afford 7.3g (>100%) of crude acid 11d, which was directly used in the coupling reaction. ¹ H NMR (CDCl ₃ ) of 11d: δ 3.95 (m, 1H), 2.60-2.35 (m, 2H), 1.40-1.10 (m, 28H), 0.82 (m, 3H). Preparation of side chain precursor (12c):

In a 1 liter round bottom flask were added tridecanal (5.00 g, 25.2 mmol) and Gly-Ome hydrochloride (12.66 g, 100.8 mmol) in 600 ml anhydrous MeOH. To the reaction mixture was added _NaCNBH3 (1.787 g, 28.44 mmol) and the reaction was stirred overnight at room temperature. The solid was filtered off and the solvent was removed in vacuo _, the resulting residue was partitioned between _CH2Cl2 /sat aq. _NaHCO3 . The organic layer was washed twice with NaHCO ₃ solution and dried over MgSO ₄ , then the desiccant was filtered off and the solvent was removed in vacuo, purified on a silica gel column eluting with 50% EtOAc/hexanes to give 2.94 g of white solid (12a).

In a 50 ml round bottom flask was added glycine derivative 12a (504.6 mg, 1.86 mmol), triethylamine (224.6 mg, 2.22 mmol) in 10 ml dry THF. To this was added (BOC) _2O (494.3mg, 2.26mmol) in one portion, the reaction was stirred for 18 hours, then the solvent was removed under vacuum, the resulting oil was treated with EtOAc and washed twice with 1N HCl, once each with water and brine and washed with MgSO ₄ was dried, then the desiccant was filtered off and the solvent was removed in vacuo to give 463.3 mg of a colorless oil (12b), which was used without purification.

To a 50 ml round bottom flask containing 5 ml THF was added methyl ester 12b (463.3 mg, 1.25 mmol), to which was added 1.8 ml 1N LiOH solution and the resulting reaction mixture was stirred overnight. The reaction was quenched by adding 1.8 ml of 1N HCl solution, then THF was removed under vacuum and _the resulting aqueous layer was extracted twice with _CH2Cl2 . The organic phase was dried over _MgSO4 , then the desiccant was filtered off and the solvent was removed in vacuo to give 246.2 mg of a colorless oil (12c), which was used without purification. Preparation of side chain precursor (13a):

In a 50 mL round bottom flask, the methyl ester 12b obtained above (499.7 mg/1.84 mmol) was dissolved in a 1:1 acetic anhydride/pyridine mixture, and the reaction was stirred overnight. The solvent was then removed under vacuum and the resulting oil was treated with _CH2Cl2 and washed twice with 1N HCl, once each with water and brine and dried over _MgSO4 , then the desiccant was filtered off and the solvent removed under vacuum to yield 319.9 _mg Colorless oil (13a), which was used without purification. General preparation of the glycine side chain precursor (14-a):

For example the following procedure describes the synthesis of tridecanoyl-glycine using Fmoc-glycine-wang resin and tridecanoic acid.

In a 100mL reaction tube capped with glass at both ends, Fmoc-glycine-wang resin (10g, 4.4mmol) was added to 50mL 30% piperidine/DMF, shaken for 20 minutes and washed 3 times with DMF, washed with isopropyl Washed 3 times with alcohol and 3 times with DMF. A solution of tridecanoic acid (4.708 g, 22 mmol) in 50 mL of DMF was added to the resin and to this mixture were added HOBt (2.97 g, 22 mmol) and DIC (2.77 g, 22 mmol). The reactor was placed on a shaker overnight. The resin was then washed with the following solvents: DMF 3 times, dichloromethane 3 times, MeOH 3 times, THF 3 times and dichloromethane 3 times. The resin beads were dried in a vacuum oven for 1 hour, 100 mL of 95% TFA/H ₂ O was added to the resin, the reaction was shaken for 1.5 hours and the non-resin products were washed with TFA and collected. Drying in a vacuum oven gave a white residue, which was azeotropically distilled with toluene to give tridecanoyl-glycine (1.14 g, 95%) ¹ H NMR (THF) 0.82-0.92 (t, J=7.2, 3H), 1.2- 1.4(s, 18H), 1.52-1.62(m, 2H), 2.10-2.17(t, J=7.2, 2H), 3.83-3.89(d, J=7.1, 2H), 7.04-7.17(s, 1H) , 10.8-10.9 (s, 1H).

Structure II below is used to describe the products obtained in Examples 1-17.

The synthesis of embodiment 1 diastereomer 1-1 and 1-2:

Hydroxybenzotriazole (55.8 mg, 0.413 mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (82.4 mg, 0.430 mmol) were added to compound 1c in 4ml dry DMF. The reaction mixture was stirred at room temperature for 10 hours, after which Z-PSN (375.2 mg, 0.271 mmol) was added. After 5 hours HPLC showed that the CBZ-protected pseudomycin nucleus (Z-PSN) was consumed. The solvent was removed in vacuo and the resulting residue was treated with 1:1 ACN/ _H2O and purified by preparative HPLC, which gave 2 larger peaks whose mass spectrum indicated that these peaks corresponded to the 2 diastereoisomers 1 -1 (88.3 mg) and 1-2 (166.3 mg). Deprotection of Diastereomer 1-1:

Add 10ml MeOH/1ml glacial AcOH and diastereomer 1-1 (82.0mg, 0.048mmol) in a 50ml round bottom flask, add 89.1mg 10% Pd/C to the reaction mixture after degassing ₂ Hydrogenation for 30 minutes. Purification by preparative HPLC and lyophilization afforded 21.7 mg of compound 1-3 (R ¹ =H) after removal of the catalyst by filtration. MS ₍ Ionspray) calcd for _{C58H94ClN12O19} (M+H) ⁺ _1297.64 , found _1297.8 . Deprotection of Diastereomer 1-2:

Diastereomer 1-2 (152.8 mg, 0.089 mmol) was hydrogenated with 152.8 mg of 10% Pd/C for 30 min as described for Diastereomer 1-1, HPLC showed complete consumption of starting material and formation of two The product peaks were purified by preparative HPLC and freeze-dried to obtain compound 1-4 (18.0 mg) and compound 1-5 (11.3 mg). Compound 1-4: MS (Ionspray) calculated for C ₅₈ H ₉₄ ClN ₁₂ O ₁₉ (M+H) ⁺ 1297.89, found 1297.8. Compound 1-5: MS (Ionspray) calcd. for C ₅₈ H ₉₂ ClN ₁₂ O ₁₈ (M+H) ⁺ 1279.63, found 1281.7.

The compounds listed in Table 1 below were synthesized using the same acylation and deprotection procedures as above, using the indicated side chain precursors. For each compound listed in Table 1, ^R1 is hydrogen.

*虽然该化合物和无N-甲基的化合物表现出很低的活性，但其中烷基链连续从C11增加到C15的化合物，其活性明显升高。这一类侧链可通过在14-a的制备中描述的制备方法制备。Table 1

*Although this compound and the compound without N-methyl group showed very low activity, the compound in which the alkyl chain was continuously increased from C11 to C15 showed a marked increase in activity. Such side chains can be prepared by the preparation described in the preparation of 14-a.

The synthesis of embodiment 15 compound 15-1:

Add 31.3 mg (0.0237 mmol) of compound 12-1 and 5 ml of TFA (pre-cooled to 0° C.) into a 50 ml round bottom flask. The reaction mixture was stirred at this temperature for 15 min, TFA was removed under vacuum, then the residue was treated with water and lyophilized to give 24.3 mg of compound 15-1. No further purification was required.

Example 16 Example 16 illustrates the preparation of linker compounds 16a, 16b, 16c and 16d with β-amino substituted side chains:

A solution of tert-butoxycarbonylmethylenetriphenylphosphorane (5.0 g, 13.3 mmol) and dodecanal (2.2 mL, 10 mmol) in toluene (50 mL) was heated at reflux for 1 hour and 20 minutes. The solution was filtered through a plug of silica gel to remove the phosphorus reagent, and then concentrated under vacuum to give a crude oil, which was purified on a silica gel column and eluted with 2% ethyl acetate in hexane to give 2.36 g (84% yield ) compound 16a. MS-283.4 (M+1) NMR was consistent with the structure.

Butyllithium (1.6M in hexane) (1.19ml, 1.9mmol) was added slowly to (R)-benzylmethylbenzylamine (0.42ml, 2.0mmol) in THF ( 5ml) solution, then a THF solution of 16a (500mg, 1.77mmol) was added dropwise. The mixture was stirred at -78°C for 1 hour, then the reaction mixture was poured into saturated _NH4Cl solution and extracted with diethyl ether (2x). The ether solution was dried over _MgSO4 and concentrated in vacuo to afford 0.95 g of 16b as a crude oil which was used in the next step without purification.

A solution of 16b (0.95 g) in ethanol (25 ml) and Pd(OH) ₂ /C (0.47 g) was placed under 60 psi H ₂ at 55° C. for 18 h. The suspension was filtered and then concentrated under vacuum to afford 280 mg (53% yield) of crude 16c, which was used directly in the next step.

Compound 16c (280 mg, 0.93 mmol) and N-benzyloxycarbonyloxysuccinimide (274 mg, 1.1 mmol) were mixed in THF (10 ml) and stirred overnight. The solvent was removed in vacuo and the oily product was purified by column chromatography on silica gel using 5% ethyl acetate in hexanes as eluent to afford 268 mg (66% yield) of tert-butyl ester 16d. NMR was consistent with the target structure. The ester (97mg, 0.224mmol) was dissolved in trifluoroacetic acid (2ml) at 0°C for 0.5h to remove the tert-butyl ester (16d) in quantitative yield. Synthesis of compound 16-1:

Compound 16d was dissolved in DMF (2ml), and hydroxybenzotriazole (36.4mg, 0.269mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride were added salt (47.1 mg, 0.246 mmol) and the solution was stirred for 18 hours. CBZ protected pseudomycin nucleus (Z-PSN) (277 mg, 0.185 mmol) was added and the reaction was stirred for an additional 18 hours. The reaction product was purified by HPLC and lyophilized to give 136.3 mg (yield 42%) of acylated CBZ-protected pseudomycin derivative as a white solid. MS-1743 (M) and NMR were consistent with the expected structure.

A solution of the acylated compound (130 mg, 0.0746 mmol) in methanol (10 ml) and acetic acid (1.5 ml) with 10% Pd/C (120 mg) was placed under a balloon of hydrogen for 20 min. The solution was filtered and purified by preparative HPLC and lyophilized to afford 23 mg (18% yield) of the trifluoroacetate salt of 16-1 as a white solid. MS-1206.8 (M) and NMR was consistent with the expected structure. Compound 16-1 had little or no activity against Candida albicans and Cryptococcus neoformans, and its activity was significantly reduced compared with the hydroxyl analogs.

Example 17 Example 17 illustrates the preparation of the attached side chain precursor 17d of the chiral side chain:

To a solution of (S)-binaphthol (240 mg, 0.84 mmol) in THF was added 4A molecular sieves (4 g), followed by neat Ti(OPr-I) ₄ (0.25 mL, 0.84 mmol). The reaction mixture turned red quickly and remained red, and the chiral catalyst (17b) thus prepared was used in the following reaction.

To a THF solution (4 mL) of freshly prepared (S)-binaphthol-Ti catalyst 17b (0.42 mmol) was added a trimethylsilyldimethylketene acetal at -78°C for more than 15 minutes. (trimethylsilyldimethylketene acetal) (0.43 mL, 2.1 mmol) and unsaturated aldehyde 17a (500 mg, 2.1 mmol) in THF. The reaction was stirred at -78°C for 1 hour, then at room temperature overnight. The reaction was quenched with saturated NaHCO ₃ solution and extracted with EtOAc (100 mL). The organic layer was washed with NaHCO ₃ , brine and dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo, the residue was purified by silica gel column chromatography (10% EtOAc/Hexane) to give 257 mg (36%) of the desired product 17c. ¹ H NMR (CDCl ₃ ) of 17c: δ5.29(m, 2H), 3.65(s, 3H), 3.53(t, J=7.3Hz, 1H), 2.33(d, J=6.3Hz, 1H, 3 '-OH), 1.97 (m, 4H), 1.65-1.22 (m, 20H), 1.13 (s, 3H), 1.12 (s, 3H), 0.84 (m, 3H).

A THF solution (5 mL) of 17c (259 mg, 0.76 mmol) was treated with aqueous NaOH (0.30 mL, 5N, 1.52 mmol) and the reaction was heated at 50 °C overnight. The reaction mixture was cooled to 0 °C and acidified to pH = 3 with 5N HCl, then extracted with EtOAc (75 mL), the organic layer was washed with water and brine, dried and concentrated in vacuo to give 222 mg (90%) of crude acid 17d, which was used directly in the side chain coupling reaction. ¹ H NMR (CDCl ₃ ) of 17d: δ 5.28 (m, 2H), 3.56 (m, 1H), 1.95 (m, 4H), 1.60-1.10 (m, 26H), 0.83 (m, 3H). Preparation of 17-1 and 17-2:

A THF solution (7 mL) of crude acid 17d (240 mg, 0.74 mmol) was treated with HOBt (90.5 mg, 0.67 mmol) and EDCI (128 mg, 0.67 mmol) at room temperature. After stirring for 1.5 hours DMAP (41 mg, 0.33 mmol) was added. After stirring for another 2 hours, CBZ-protected pseudomycin core (614 mg, 0.44 mmol) in DMF (4 mL) was added and the reaction was stirred overnight at room temperature. The reaction mixture was purified by preparative HPLC and freeze-dried to obtain (160 mg, 21%) the target product.

A solution of the CBZ-protected compound (53.4 mg, 0.032 mmol) in acetonitrile (2 mL) was treated with TMSI (77 mg, 0.38 mmol) at 0°C. After 100 minutes, the reaction was terminated with 1:1 CH ₃ CN/H ₂ O, and the resulting reaction mixture was purified by reverse-phase preparative HPLC to obtain 25.8 mg (65%) of the desired final product 17-1.

Compound 17-2 was prepared in the same manner as above using an appropriate starting material in which R ^* is hydrogen.

Example 18 Example 18 illustrates the preparation of the attached side chain precursor 18d of the chiral alkenyl side chain:

To a solution of 18a (993 mg, 3.73 mmol) in dichloromethane (25 mL) was added methyltrimethylsilyl dimethylketene acetal (2.27 mL, 11.2 mmol) at -78 °C and pure _TiCl4 (0.49 mL, 4.48 mmol). After 2 hours, the reaction was quenched with MeOH (5 mL) at -78 °C. The reaction mixture was extracted with dichloromethane (3×40 mL), and the combined organic layers were washed with NaHCO ₃ and brine. The organic layer was dried and concentrated in vacuo to give a residue which was chromatographed (15-20% EtOAc/hexanes) to afford 837 mg (61%) of the desired product 18b. ¹ H NMR (CDCl ₃ ) of 18a: δ6.28-5.87(m, 2H), 5.62-5.43(m, 2H), 4.75(m, 1H), 4.22(m, 1H), 3.87(m, 1H) , ¹ H NMR (CDCl ₃ ) of 18b: 5.98-5.90 (m, 2H), 5.55-5.47(m, 2H), 4.08(m, 1H), 3.86(m, 1H), 3.61(s, 3H), 3.50(m, 1H), 2.00-1.96(m, 2H ), 1.74-1.63 (m, 3H), 1.50-1.00 (m, 24H).

To a solution of 18b (837 mg, 2.27 mmol) in dichloromethane (22 mL) was added PCC (0.98 g, 4.54 mmol). The reaction was stirred at room temperature for 18 hours, then filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give a pink residue, which was purified by silica gel chromatography (20% EtOAc/hexanes) to afford 441 mg (53%) of the desired methyl ketone 18c. ¹ H NMR (CDCl ₃ ) of 18c: δ5.92-5.87(m, 2H), 5.48-5.43(m, 2H), 3.88(m, 1H), 3.56(s, 3H), 3.49(m, 1H) , 2.59(dd, J=6.4, 14.7Hz, 1H), 2.29(dd, J=5.9, 15.2Hz, 1H), 2.06(s, 3H), 1.96(m, 2H), 1.63(m, 2H), 1.40-0.90 (m, 20H).

To a solution of 18c (440 mg, 1.20 mmol) in THF (4 mL) and methane (2 mL) was added 7.5M KOH (1 mL) at room temperature. After stirring at room temperature for 4 hours, it was acidified to pH=3 with 1N HCl, the reaction mixture was extracted with EtOAc (3×30 mL), the combined extracts were washed with water (2×10 mL) and brine, the organic layer was dried and dried under vacuum Concentration afforded 388 mg (>100%) of crude acid 18d, which was used directly in the coupling reaction of the side chain. Preparation of compound 18-1:

To a THF solution (10 mL) of crude acid 18d (-1.66 mmol) was added HOBt (244 mg, 1.66 mmol) and EDCI (318 mg, 1.66 mmol). After stirring at room temperature for 5 hours, a DMF solution (5 mL) of Alloc-protected pseudomycin nucleus (614 mg, 0.50 mmol) was added, and after stirring at room temperature for several days, DMAP (61 mg, 0.50 mmol) was added to the reaction mixture ). After stirring for an additional 12 hours, the reaction mixture was purified by reverse phase HPLC and lyophilized to yield 225 mg (30%) of the alloc-protected acylated derivative.

To a solution of degassed alloc-protected acylated derivative (240 mg, 0.16 mmol) in THF (20 mL) and HOAc (1 mL) at room temperature was added PdCl ₂ (PPh ₃ ) ₂ (23 mg, 0.032 mmol) and Bu ₃ SnH (0.87 mL, 3.23 mmol). After 1.5 hours, the reaction mixture was purified by preparative HPLC to afford 33 mg (17%) of compound 18-1.

In addition to the compounds listed in the above examples, the following N-acyl derivatives were prepared which showed limited or insignificant activity. Even if these compounds cannot be used as fungicides, they can provide valuable guidance for designing compounds with optimal activity.

Unless otherwise stated, each compound in the examples exhibited appropriate activity against C. albicans, C. neoformans, A. fumigatus, C. parapsilosis or Histoplasma capsulatus. However, the following basic trends in activity were observed from these compounds synthesized. The stereochemistry of the β-hydroxyl group is preferably R; irrespective of the stereochemistry and degree of unsaturation, longer alkyl chain lengths (i.e., C ₁₂ -C ₂₀ ) tend to favor shorter alkyl chain lengths (e.g., <C ₁₁ ) have higher activity; removal of β-hydroxyl, α, α-disubstitution, reduction of alkyl chain length in alkyl and alkoxy substituents, special rigidity and increased branching in side chains all tend to have Less active than long flexible chains.

其中Therefore, alkyl side chains represented by the following structures are preferred for fungicidal treatment:

in

R ^a and R ^a' are independently hydrogen or methyl, or one of R ^a or R ^a' is alkylamino, and together with R ^b or R ^b' form a six-membered cycloalkyl ring, a six-membered aromatic ring or Double bond, or form a six-membered aromatic ring together with R ^c ;

R ^b or R ^b' is independently hydrogen or methyl, and R ^b or R ^b' is hydroxyl, provided that when R ^a , R ^b , R ^d , ^Re are hydrogen, R ^c is hydrogen and R ^f is n-hexyl , n-octyl or n-decyl, or when R ^a , R ^b , R ^d , ^Re are hydrogen, R ^c is hydroxyl and R ^f is n-octyl, n-nonyl or n-decyl, R ^b' is not Hydroxy;

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

R ^e is hydrogen, or together with R ^f is a six-membered aromatic ring, a C ₅ -C ₁₄ alkoxy substituted six-membered aromatic ring or a C ₅ -C ₁₄ alkyl substituted six-membered aromatic ring;

R ^f is C ₈ -C ₁₄ alkyl, or C ₅ -C ₁₁ alkoxy.

Claims (15)

1. compound and its pharmacologically acceptable salt and its solvate of representing of a structure I

Wherein R is

Wherein

R ^aAnd R ^{A '}Independent is hydrogen or methyl or R ^aOr R ^{A '}Be alkylamino and R ^bOr R ^{B '}Form hexa-atomic cycloalkyl ring, hexa-atomic aromatic nucleus or two key or and R together ^cForm hexa-atomic aromatic nucleus together;

R ^bAnd R ^{B '}Independent is hydrogen, halogen or methyl, perhaps R ^bOr R ^{B '}Be amino, alkylamino, α-acetylacetic ester, methoxyl group or hydroxyl, condition is to work as R ^a, R ^b, R ^d, R ^eBe hydrogen, R ^cBe hydrogen and R ^fBe n-hexyl, n-octyl or positive decyl, or R ^a, R ^b, R ^d, R ^eBe hydrogen, R ^cBe hydroxyl and R ^fDuring for n-octyl, n-nonyl or positive decyl, R ^bIt is not hydroxyl;

R ^cBe hydrogen, hydroxyl, C ₁-C ₄Alkoxyl group, hydroxy alkoxy base, or and R ^eForm hexa-atomic aromatic nucleus or C together ₅-C ₆Cycloalkyl ring;

R ^eBe hydrogen or and R ^fForm hexa-atomic aromatic nucleus, C together ₅-C ₁₄Hexa-atomic aromatic nucleus or C that alkoxyl group replaces ₅-C ₁₄The hexa-atomic aromatic nucleus that alkyl replaces and

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

Wherein

R ^gBe hydrogen or C ₁-C ₁₃Alkyl and

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

R ⁱBe hydrogen, 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; Or R is-(CH ₂)-NR ^m-(C ₁₃-C ₁₈Alkyl), R wherein ^mFor H ,-CH ₃Or-C (O) CH ₃

2. the compound of claim 1, wherein structure I has following stereochemistry:

3. the compound of claim 1, wherein R is

Wherein

R ^aAnd R ^{A '}Independent is hydrogen or methyl, perhaps R ^aOr R ^{A '}Be alkylamino, with R ^bOr R ^{B '}Form hexa-atomic cycloalkyl ring, hexa-atomic aromatic nucleus or two key together, or and R ^cForm hexa-atomic aromatic nucleus together;

R ^bAnd R ^{B '}Independent is hydrogen, halogen or methyl or R ^bOr R ^{B '}Be amino, alkylamino, α-acetylacetic ester, methoxyl group or hydroxyl, condition is: work as R ^a, R ^b, R ^d, R ^eBe hydrogen, R ^cBe hydrogen and R ^fBe n-hexyl, n-octyl or positive decyl, or R ^a, R ^b, R ^d, R ^eBe hydrogen, R ^cBe hydroxyl and R ^fDuring for n-octyl, n-nonyl or positive decyl, R ^{B '}It is not hydroxyl;

R ^cBe hydrogen, hydroxyl, C ₁-C ₄Alkoxyl group, hydroxy alkoxy base or and R ^eForm hexa-atomic aromatic nucleus or C together ₅-C ₆Cycloalkyl ring;

R ^eBe hydrogen, or and R ^fBe hexa-atomic aromatic nucleus, C together ₅-C ₁₄Hexa-atomic aromatic nucleus or C that alkoxyl group replaces ₅-C ₁₄The hexa-atomic aromatic nucleus that alkyl replaces; With

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

4. the compound of claim 3, wherein R ^{B '}Be hydroxyl, condition is: work as R ^a, R ^b, R ^d, R ^eBe hydrogen and R ^fDuring for n-hexyl, n-octyl or positive decyl, R ^cBe not hydrogen or work as R ^fR during for n-octyl, n-nonyl or positive decyl ^cIt is not hydroxyl.

5. aforementioned claim in each claimed compound be used for the treatment of purposes in the medicine of whole body fungi infestation or fungal skin infections in preparation.

6. pharmaceutical composition, it comprises pseudobactin compound and pharmaceutically acceptable carrier of claim 2.

7. the method for a treatment anti-fungal infection in the animal of needs treatments, it comprises the step to the pseudobactin compound in the described animals administer claim 2.

8. the method for preparing pseudobactin nuclear, it comprises provides pseudobactin compound and described pseudobactin compound with the N-acyl group alkyl group side chain that comprises at least one γ or δ hydroxyl to prepare the step that described pseudobactin is examined with a kind of acid-respons.

9. the method for claim 8, wherein said pseudobactin nuclear is represented by structure I-A.

Wherein R ' is-NH ₂Or-NH _p-P _g, P wherein _gFor amino protecting group and p are 0 or 1.

10. the method for claim 8, wherein said pseudobactin compound with the N-acyl group alkyl group side chain that comprises at least one γ or δ hydroxyl is selected from pseudobactin A, pseudobactin A ' and pseudobactin C.

11. the method for claim 8, wherein said acid are trifluoroacetic acid and acetate.

12. the method for claim 11, wherein said acid are trifluoroacetic acid.

13. pseudobactin nuclear, it is by the method preparation of claim 8.

14. the pseudobactin of claim 13 nuclear, wherein said nuclear is represented by structure I-A. Wherein R ' is-NH ₂Or-NH _p-P _g, P wherein _gFor amino protecting group and p are 0 or 1.

15. examine by the pseudobactin that structure I-A represents.

Wherein R ' is-NH ₂Or-NH _p-P _g, P wherein _gFor amino protecting group and p are 0 or 1.