WO2001005814A1 - Analogues a base de pseudomycine a chaines laterales n-acyle - Google Patents

Analogues a base de pseudomycine a chaines laterales n-acyle Download PDF

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Publication number
WO2001005814A1
WO2001005814A1 PCT/US2000/015017 US0015017W WO0105814A1 WO 2001005814 A1 WO2001005814 A1 WO 2001005814A1 US 0015017 W US0015017 W US 0015017W WO 0105814 A1 WO0105814 A1 WO 0105814A1
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Prior art keywords
pseudomycin
hydrogen
compound
alkyl
mmol
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PCT/US2000/015017
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English (en)
Inventor
Matthew David Belvo
Shu Hui Chen
Christopher William Doecke
Sarah Lynne Hellman
James Andrew Jamison
Lawrence Edward Patterson
Michael John Rodriguez
Xicheng Sun
William Wilson Turner
Venkatraghavan Vasudevan
Mark James Zweifel
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Eli Lilly And Company
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Priority to AU57249/00A priority Critical patent/AU5724900A/en
Priority to JP2001511471A priority patent/JP2003505397A/ja
Priority to MXPA02000321A priority patent/MXPA02000321A/es
Priority to CA002379851A priority patent/CA2379851A1/fr
Priority to EA200200160A priority patent/EA200200160A1/ru
Priority to BR0012447-8A priority patent/BR0012447A/pt
Priority to EP00942655A priority patent/EP1200460A1/fr
Publication of WO2001005814A1 publication Critical patent/WO2001005814A1/fr
Priority to NO20020193A priority patent/NO20020193L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to pseudomycin compounds, in particular, semi-synthetic pseudomycin compounds having novel N-acyl side-chains.
  • Pseudomycins are natural products isolated from liquid cultures of Pseudomonas syringae (plant-associated bacterium) and have been shown to have antifungal activities.
  • Pseudomonas syringae plant-associated bacterium
  • Pseudomycins a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity, " J . Gen . Microbiology, 137(12), 2857-65 (1991) and US Patent Nos . 5,576,298 and 5,837,685
  • syringae e.g., syringomycins, syringotoxins and syringostatins
  • pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, dehydroaminobutyric acid, lysine and diaminobutyric acid.
  • the peptide moiety for pseudomycins A, A', B, B' , C, C corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L- Asp(3-OH) -L-Thr (4-C1) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser.
  • the analogs are distinguished by the N-acyl side chain, i.e., pseudomycin A is N-acylated by 3 , 4-dihydroxytetradeconoyl , pseudomycin A' by 3 , 4-dihydroxypentadecanoyl, pseudomycin B by 3-hydroxytetradecanoyl , pseudomycin B' by 3-hydroxydodecanoyl , pseudomycin C by 3 , 4-dihydroxyhexadecanoyl and pseudomycin C by 3-hydroxyhexadecanoyl . (see i.e., Ballio, A., et al .
  • Pseudomycins are known to have certain adverse biological effects. For example, destruction of the endothelium of the vein, destruction of tissue, inflammation, and local toxicity to host tissues have been observed when pseudomycin is administered intraveneously .
  • the present invention provides pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.
  • R a and R 5a a ' ' are independently hydrogen or methyl, or either R a or R a' is alkyl amino, taken together with R b or R b' forms a six-membered cycloalkyl ring, a six- membered aromatic ring or a double bond, or taken together with R c forms a six-membered aromatic ring;
  • R and R b' are independently hydrogen, halogen, or
  • R b or R b' is amino, alkylamino, ⁇ - acetoacetate, methoxy, or hydroxy provided that R b' is not hydroxy when R a , R b , R d , R e are hydrogen, R c is hydrogen and R f is n-hexyl, n-octyl or n-decyl, or R a , R b , R d , R e are hydrogen, R c is hydroxy and R f is n- octyl, n-nonyl , or ⁇ -decyl; R c is hydrogen, hydroxy, C ⁇ -C alkoxy, hydroxyalkoxy, or taken together with R e forms a 6- membered aromatic ring or C 5 -C 6 cycloalkyl ring;
  • R e is hydrogen, or taken together with R f is a six-membered aromatic ring, Cs-C ⁇ alkoxy substituted six-membered aromatic ring, or C 5 -C ⁇ 4 alkyl substituted six-membered aromatic ring, and
  • R f is Cs-Cia alkyl, C 5 -C 11 alkoxy, or biphenyl ; or R is
  • R g is hydrogen , or C ⁇ -C ⁇ 3 alkyl
  • R 1 is a hydrogen, halogen, or C 5 -Cs alkoxy, and m is 1, 2 or 3 ;
  • R k is C 5 -C ⁇ alkoxy; or R is -(CH 2 )-NR m -(C ⁇ 3 -C ⁇ 8 alkyl), where R m is H, -CH 3 or -C(0)CH 3 ; and pharmaceutically acceptable salts and solvates thereof .
  • a pharmaceutical formulation which includes the pseudomycin compound represented by structure I above and a pharmaceutically acceptable carrier.
  • a method for treating an antifungal infection in an animal in need thereof which comprises administering to the animal the pseudomycin compound I described above.
  • a process for producing the free amine nucleus of a pseudomycin compound which may be acylated to form the compounds represented by structure I above.
  • the process includes the steps of treating a pseudomycin compound which contains an N-acyl alkyl side-chain having at least one gamma or delta hydroxyl group (e.g., pseudomycin A, A' or C) with trifluoroacetic acid or acetic acid.
  • free amine pseudomycin nucleus or “pseudomycin nucleus” refers to the structure I-A below:
  • alkyl refers to a hydrocarbon radical of the general formula C n H2 n + ⁇ containing from 1 to 30 carbon atoms unless otherwise indicated.
  • the alkane radical may be straight (e.g.
  • alkane radical may be substituted or unsubstituted.
  • alkyl portion of an alkoxy group, alkanoyl, or alkanoate have the same definition as above.
  • alkenyl refers to an acyclic hydrocarbon containing at least one carbon carbon double bond.
  • the alkene radical may be straight, branched, cyclic, or multi- cyclic.
  • the alkene radical may be substituted or unsubstituted.
  • the alkenyl portion of an alkenoxy, alkenoyl or alkenoate group has the same definition as above.
  • alkynyl refers to an acyclic hydrocarbon containing at least one carbon carbon triple bond.
  • the alkyne radical may be straight, or branched.
  • the alkyne radical may be substituted or unsubstituted.
  • the alkynyl portion of an alkynoxy, alkynoyl or alkynoate group has the same definition as above.
  • aryl refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., naphthalene, anthracene, phenanthrene , etc.).
  • the aryl groups may be substituted or unsubstituted.
  • heteroaryl refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.) .
  • the aromatic moiety may consist of a single or fused ring system.
  • the heteroaryl groups may be substituted or unsubstituted.
  • NHp-Pg and "a ino protecting group” refer to a substituent of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups on the compound.
  • the amino protecting group when taken with the nitrogen to which it is attached, forms a cyclic imide, e.g., phthalimido and tetrachlorophthalimido .
  • the protecting group when taken with the nitrogen to which it is attached, can form a carbamate, e . g. , methyl, ethyl, and 9- fluorenylmethylcarba ate; or an amide, e . g. , N-formyl and N- acetylamide .
  • alkyl group allows for substitutents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc.
  • group specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety.
  • substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament.
  • Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, a inoalkoxy, hydroxyalkylamino , aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl , and combinations thereof.
  • solvate refers to an aggregate that comprises one or more molecules of the solute, such as a compound of structure I, with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like.
  • pharmaceutically acceptable salt refers to organic or inorganic salts of the compounds represented by structure I that are substantially non-toxic to the recipient at the doses administered.
  • animal refers to humans, companion animals (e.g., dogs, cats and horses), food-source animals (e.g., cows, pigs, sheep and poultry) , zoo animals, marine animals, birds and other similar animal species.
  • companion animals e.g., dogs, cats and horses
  • food-source animals e.g., cows, pigs, sheep and poultry
  • zoo animals e.g., marine animals, birds and other similar animal species.
  • Scheme I illustrates the general procedures for synthesizing Compound I from any one 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 modification of semi-synthetic derivatives of the naturally occurring pseudomycin compounds may be accomplished in a similar manner. In general, four synthetic steps are used to produce Compound I: (1) selective amino protection; (2) chemical or enzymatic deacylation of the N-acyl side-chain; (3) reacylation with a different side-chain; and (4) deprotection of the amino groups.
  • Suitable amino-protecting groups include benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl , p-methoxybenxyloxycarbonyl , p-methoxyphenylazobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl , t-butyloxycarbonyl , cyclopentyloxycarbonyl, and phthalimido.
  • Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl (Alloc) , phthalimido, and benzyloxycarbonyl (CbZ or CBZ) .
  • the deacylation of a N-acyl group having a gamma or delta hydroxylated side chain may be accomplished by treating the amino- protected pseudomycin compound with a 5-20% aqueous acidic solution.
  • Suitable acids include acetic acid and trifluoroacetic acid. A preferred acid is trifluoroacetic acid. If trifluoroacetic acid is used, the reaction may be accomplished at or near room temperature. However, when acetic acid is used the reaction is generally run at about 40°C.
  • a water soluble organic solvent may be used to assist in solubilizing the pseudomycin compound.
  • Suitable aqueous solvent systems include acetonitrile, water, and mixtures thereof.
  • Acetonitrile was particularly useful when deacylating a protected pseudomycin compound.
  • a preferred acidic solution for deacylating a protected pseudomycin compound is 8% aqueous trifluoroacetic acid in acetonitrile.
  • Organic solvents accelerate the reaction; however, the addition of an organic solvent may lead to other byproducts.
  • Pseudomycin compounds lacking a delta hydroxy group on the side chain e.g., Pseudomycin B and C
  • Pseudomycin B and C may be deacylated enzymatically .
  • Suitable deacylase enzymes include Polymyxin Acylase (164-16081 Fatty Acylase (crude) or 161-16091 Fatty Acylase (pure) available from Wako Pure Chemical Industries, Ltd.), or ECB deacylase (see, e.g., U.S. Patent No. 5,573,936).
  • the enzymatic deacylation may be accomplished using standard deacylation procedures well known to those skilled in the art. For example, general procedures for using Polymyxin acylase may be found in Yasuda, N., et al, Agric . Biol . Chem . , 53, 3245 (1989) and Kimura, Y., et al . , Agric . Biol . Chem. , 53, 497 (1989).
  • the deacylated product (also known as the pseudomycin nucleus or "PSN") is reacylated using the corresponding acid of the desired acyl group in the presence of a carbonyl activating agent.
  • Carbonyl activating group refers to a substituent of a carbonyl that promotes nucleophilic addition reactions at that carbonyl. Suitable activating substituents are those which have a net electron withdrawing effect on the carbonyl.
  • Such groups include, but are not limited to, alkoxy, aryloxy, nitrogen containing aromatic heterocycles, or amino groups (e.g., oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N- oxysuccinimide, N,N' -dicyclohexylisoure-O-yl , and N-hydroxy- N-methoxyamino) ,- acetates; formates; sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, and p- tolylsulfonate) ; and halides (e.g., chloride, bromide, and iodide) .
  • amino groups e.g., oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N- oxysuccinimide, N,N'
  • a solid phase synthesis may be used where a hydroxybenzotriazole-resin (HOBt-resin) serves as the coupling agent for the acylation reaction.
  • HOBt-resin hydroxybenzotriazole-resin
  • acids may be used in the acylation process .
  • Suitable acids include aliphatic acids containing one or more pendant aryl, alkyl, amino (including primary, secondary and tertiary amines) , hydroxy, alkoxy, and amido groups; aliphatic acids containing nitrogen or oxygen within the aliphatic chain; aromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups; and heteroaromatic acids substituted with alkyl, hydroxy, alkoxy and/or alkyl amino groups.
  • the acylated product may be useful as an active antifungal agent or as an intermediate for the production of an active compound. Even though some compounds were not as useful as others, the activity profiles provide valuable insight into the design trends needed to achieve optimum activity.
  • the amino protecting groups may be removed by hydrogenation in the presence of a hydrogenation catalyst (e.g., 10% Pd/C) .
  • a hydrogenation catalyst e.g. 10% Pd/C
  • the amino protecting group is allyloxycarbonyl
  • the protecting group may be removed using tributyltinhydride and triphenylphosphine palladium dichloride.
  • pseudomycins are natural products isolated from the bacterium Pseudomonas syringae that have been characterized as lipodepsinonapetpides containing a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4-chlorothreonine (ClThr) , 3-hydroxyaspartic acid (HOAsp) , 2 , 3-dehydro-2-aminobutyric acid (Dhb) , and 2 , 4-diaminobutyric acid (Dab).
  • MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H are described in U.S. Patent Nos. 5,576,298 and 5,837,685; Harrison, et al . , "Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity, " J. Gen. Microbiology, 137, 2857-2865 (1991); and Lamb et al . , "Transposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease," Proc . Natl . Acad. Sci . USA, 84, 6447- 6451 (1987) .
  • a strain of P. syringae that is suitable for production of one or more pseudomycins can be isolated from environmental sources including plants (e.g., barley plants, citrus plants, and lilac plants) as well as, sources such as soil, water, air, and dust.
  • a preferred stain is isolated from plants.
  • Strains of P. syringae that are isolated from environmental sources can be referred to as wild type.
  • wild type refers to a dominant genotype which naturally occurs in the normal population of P. syringae (e.g., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation) .
  • wild type refers to a dominant genotype which naturally occurs in the normal population of P. syringae (e.g., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation) .
  • the characteristics of the pseudomycin- producing cultures employed employed (P.
  • syringae strains such as MSU 174, MSU 16H, MSU 206, 25-Bl, 7H9-1) are subject to variation. Hence, progeny of these strains (e.g., recombinants, mutants and variants) may be obtained by methods known in the art .
  • P. syringae MSU 16H is publicly available from the
  • Mutant strains of P. syringae are also suitable for production of one or more pseudomycins.
  • mutant refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, such as radiation (e.g., ultraviolet radiation or x-rays), chemical mutagens (e.g., ethyl methanesulfonate (EMS) , diepoxyoctane, N-methyl-N- nitro-N' -nitrosoguanine (NTG) , and nitrous acid), site- specific mutagenesis, and transposon mediated mutagenesis.
  • EMS ethyl methanesulfonate
  • NTG N-methyl-N- nitro-N' -nitrosoguanine
  • nitrous acid nitrous acid
  • syringae can be produced by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce one or more pseudomycins, that produce one pseudomycin (e.g., pseudomycin B) in excess over other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 ⁇ g/ml . Preferred mutants are those that overproduce pseudomycin B and grow in minimal defined media.
  • a mutagenic agent effective to produce mutants that overproduce one or more pseudomycins, that produce one pseudomycin (e.g., pseudomycin B) in excess over other pseudomycins, or that produce one or more pseudomycins under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to serially dilute NTG to levels ranging from 1 to 100 ⁇ g/ml . Preferred mutants are
  • Environmental isolates, mutant strains, and other desirable strains of P. syringae can be subjected to selection for desirable traits of growth habit, growth medium nutrient source, carbon source, growth conditions, amino acid requirements, and the like.
  • a pseudomycin producing strain of P. syringae is selected for growth on minimal defined medium such as N21 medium and/or for production of one or more pseudomycins at levels greater than about 10 ⁇ g/ml .
  • Preferred strains exhibit the characteristic of producing one or more pseudomycins when grown on a medium including three or fewer amino acids and optionally, either a lipid, a potato product or combination thereof .
  • Recombinant strains can be developed by transforming the P. syringae strains, using procedures known in the art. Through the use of recombinant DNA technology, the P. syringae strains can be transformed to express a variety of gene products in addition to the antibiotics these strains produce. For example, one can modify the strains to introduce multiple copies of the endogenous pseudomycin- biosynthesis genes to achieve greater pseudomycin yield. To produce one or more pseudomycins from a wild type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or a combination thereof.
  • an aqueous nutrient medium including an effective amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or a combination thereof.
  • glycine is combined with one or more of a potato product and a lipid. Culturing is conducted under conditions effective for growth of P. syringae and production of the desired pseudomycin or pseudomycins. Effective conditions include temperatures from about 22 9 C to about 27 2 C, and a duration of about 36 hours to about 96 hours. Controlling the concentration of oxygen in the medium during culturing of P. syringae is advantageous for production of a pseudomycin. Preferably, oxygen levels are maintained at about 5 to 50% saturation, more preferably about 30% saturation. Sparging with air, pure oxygen, or gas mixtures including oxygen can regulate the concentration of oxygen in the medium.
  • Controlling the pH of the medium during culturing of P. syringae is also advantageous.
  • Pseudomycins are labile at basic pH, and significant degradation can occur if the pH of the culture medium is above about 6 for more than about 12 hours.
  • the pH of the culture medium is maintained between 6 and 4.
  • P. syringae can produce one or more pseudomycins when grown in batch culture.
  • fed-bath or semi-continuous feed of glucose and optionally, an acid or base (e.g., ammonium hydroxide) to control pH enhances production.
  • Pseudomycin production can be further enhanced by using continuous culture methods in which glucose and ammonium hydroxide are fed automatically. Choice of P.
  • strains MSU 16H and 67 HI each produce predominantly pseudomycin A, but also produce pseudomycin B and C, typically in ratios of 4:2:1.
  • Strain 67 HI typically produces levels of pseudomycins about three to five fold larger than are produced by strain MSU 16H.
  • strain 25-Bl produces more pseudomycin B and less pseudomycin C.
  • Strain 7H9-1 are distinctive in producing predominantly pseudomycin B and larger amount of pseudomycin B than other strains . For example, this strain can produce pseudomycin B in at least a ten fold excess over either pseudomycin A or C .
  • Each pseudomycin, pseudomycin intermediate and mixtures can be detected, determined, isolated, and/or purified by any variety of methods known to those skilled in the art.
  • the level of pseudomycin activity in a broth or in an isolate or purified composition can be determined by antifungal action against a fungus such as Candida and can be isolated and purified by high performance liquid chromatography.
  • the pseudomycin compound may be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate.
  • pharmaceutically acceptable salt refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulfates, sulfites, bisulfites, arylsulfonates, alkylsulfates, phosphonates, monohydrogen- phosphates, dihydrogenphosphates , metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanoates, arylalkonates, adipates, alginates, aspartates, benzoates, fumarates, glucoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartarates,
  • solvate refers to an aggregate that comprises one or more molecules of the solute (i.e., pseudomycin prodrug compound) with one or more molecules of a pharmaceutical solvent, such as water, ethanol, and the like.
  • a pharmaceutical solvent such as water, ethanol, and the like.
  • the solvent is water, then the aggregate is referred to as a hydrate.
  • Solvates are generally formed by dissolving the compound in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.
  • the active ingredient i.e., pseudomycin derivative
  • the active ingredient is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the physician, patient, or veterinarian an elegant and easily handleable product.
  • Formulations may comprise from 0.1% to 99.9% by weight of active ingredient, more generally from about 10% to about 30% by weight.
  • unit dose refers to physically discrete units that contain a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect.
  • a unit dose is typically provided in the form of a tablet, capsule, pill, powder packet, topical composition, suppository, wafer, measured units in ampoules or in multidose containers, etc.
  • a unit dose may be administered in the form of a dry or liquid aerosol which may be inhaled or sprayed.
  • the dosage to be administered may vary depending upon the physical characteristics of the animal, the severity of the animal's symptoms, and the means used to administer the drug. The specific dose for a given animal is usually set by the judgment of the attending physician or veterinarian.
  • Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.
  • the particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied.
  • the formulations may also include wetting agents, lubricating agents, surfactants, buffers, tonicity agents, bulking agents, stabilizers, emulsifiers, suspending agents, preservatives, sweeteners, perfuming agents, flavoring agents and combinations thereof.
  • a pharmaceutical composition may be administered using a variety of methods.
  • Suitable methods include topical (e.g., ointments or sprays), oral, injection and inhalation.
  • topical e.g., ointments or sprays
  • oral injection and inhalation.
  • the particular treatment method used will depend upon the type of infection being addressed.
  • the formulations are typically diluted or reconstituted (if freeze-dried) and further diluted if necessary, prior to administration.
  • An example of reconstitution instructions for the freeze-dried product are to add ten ml of water for injection (WFI) to the vial and gently agitate to dissolve. Typical reconstitution times are less than one minute.
  • the resulting solution is then further diluted in an infusion solution such as dextrose 5% in water (D5W) , prior to administration .
  • infusion solution such as dextrose 5% in water (D5W)
  • Pseudomycin compounds have been shown to exhibit antifungal activity such as growth inhibition of various infectious fungi including Candida spp. (i.e., C. albicans, C. parapsilosis, C. krusei , C. glabrata, C. tropicalis, or C. lusi tania) ; Torulopus spp. (i.e., T. glabrata) ; Aspergillus spp. (i.e., A . fumigatus) ; Histoplas a spp. (i.e., H. capsulatum) ; Cryptococcus spp. (i.e., C.
  • Candida spp. i.e., C. albicans, C. parapsilosis, C. krusei , C. glabrata, C. tropicalis, or C. lusi tania
  • Torulopus spp. i.e., T.
  • the compounds and formulations of the present invention may be useful in the preparation of medicaments for use in combating either systemic fungal infections or fungal skin infections. Accordingly, a method is provided for inhibiting fungal activity comprising contacting Compound I of the present invention with a fungus .
  • a preferred method includes inhibiting Candida albicans, Cryptococcus neoformans, or Aspergillus fumigatus activity.
  • the term "contacting" includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a f ngus. The term does not imply any further limitations to the process, such as by mechanism of inhibition.
  • the methods are defined to encompass the inhibition of fungal activity by the action of the compounds and their inherent antifungal properties.
  • a method for treating a fungal infection which comprises administering an effective amount of a pharmaceutical formulation of the present invention to a host in need of such treatment is also provided.
  • a preferred method includes treating a Candida albicans, Cryptococcus neoformans, or Aspergillus fumigatus infection.
  • the term "effective amount” refers to an amount of active compound which is capable of inhibiting fungal activity.
  • the dose administered will vary depending on such factors as the nature and severity of the infection, the age and general health of the host, the tolerance of the host to the antifungal agent and the species of the host.
  • the particular dose regimen likewise may vary according to these factors.
  • the medicament may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer.
  • a typical daily dose (administered in single or divided doses) contains a dosage level between about 0.01 g/kg to 100 mg/kg of body weight of an active compound.
  • Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg.
  • the host may be any animal including humans, companion animals (e.g., dogs, cats and horses), food-source animals (e.g., cows, pigs, sheep and poultry), zoo animals, marine animals, birds and the like .
  • P. syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA as Accession No. ATCC 67028.
  • P. syringae strains 25-Bl, 7H9-1, and 67 Hi were deposited with the American Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.: 25-Bl Accession No. PTA-1622
  • Preparative HPLC work was performed with a Waters Prep 2000 system using Dynamax 60 angstrom C18 column and identical solvent systems as used in the analytical HPLC system but with a flow rate of 40 ml/min.
  • Antifungal activity was determined in vi tro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a discdiffusion test.
  • a typical fungus employed in testing antifungal activity is Candida albicans .
  • Antifungal activity is considered significant when the test sample (50 ⁇ l) causes 10-12 mm diameter zones of inhibition on C. albicans x657 seeded agar plates.
  • Tail Vein Toxici ty
  • mice were treated intravenously (IV) through the lateral tail vein with 0.1 ml of testing compound (20 mg/kg) at 0, 24, 48 and 72 hours. Two mice were included in each group. Compounds were formulated in 5.0% dextrose and sterile water for injection. The mice were monitored for 7 days following the first treatment and observed closely for signs of irritation including erythema, swelling, discoloration, necrosis, tail loss and any other signs of adverse effects indicating toxicity.
  • mice used in the study were outbred, male ICR mice having an average weight between 18-20 g (available from Harlan Sprangue Dawley, Indianapolis, IN).
  • Solid phase acylation of the Pseudomycin nucleus using HOBt- resin uses myristoyl acid; however, the same general procedure may be used with other organic acids .
  • glycine myristoyl acid (0.309 g, 1.1 mmol) was dissolved in 100 ml of DMF.
  • HOBt-mesylate (0.229 g, 1.1 mmol )
  • triethylamine (0.081g, 0.8mmol )
  • the solution was stirred rapidly overnight under 1 atm N .
  • DMF and TEA were dried off using the high vacuum.
  • the residual oil was azeotroped 3x with toluene till a white solid formed.
  • To the solid was added 100 ml of DMF and lg of CBZ-protected Pseudomycin nucleus.
  • the solution was stirred overnight, and dried on the high vacuum.
  • the product was isolated by reverse-phase HPLC and lyophilized to yield (233 mg, 20%) Myristoyl acylated CBZ-protected Pseudomycin product.
  • reaction mixture was allowed to stir for 1 hour before quenching with 2 ml of saturated aqueous ammonium chloride and 10 ml water.
  • the reaction mixture was partitioned between ether and water.
  • the organic layer was washed 1 X brine and dried over sodium sulfate.
  • the drying agent was then filtered off and the solvent was removed in vacuo .
  • Purification on a silica gel column eluting with 5% EtOAc/hexanes yielded 238.1 mg of a yellow oil.
  • the spectral data was consistent with t-Butyl 3-hydroxy-3- (m- dodecylbenzyl) propionoate.
  • Compound 2a is synthesized using the same procedures as described above for Compound la using 1-octyne instead of 1- dodecyne .
  • Compounds 2b and 2c are synthesized using the same procedures described above for lb and lc, respectively.
  • Compound la is not hydrogenated prior to condensation with t-butyl acetate.
  • reaction was then quenched with 2 ml of saturated aqueous ammonium chloride and allowed to warm to room temperature.
  • the reaction mixture was the partitioned between ether/water and the organics were washed lx brine and dried over MgS0 4 .
  • the drying agent was then filtered off and the solvent was removed in vacuo to yield 370.5 mg of crude material as a racemic mixture.
  • the t-butyl ester was then removed by treatment with TFA to produce Compound 5a.
  • R is n-CgH ⁇ 3 , n-C 7 H ⁇ 5 , n-CsH ⁇ 7 , n-CgHig, n- C ⁇ oH ⁇ , and n-C ⁇ H 2 g were also made using the same procedures as described above.
  • acetal 11a (6.22 g, 19.1 mmol) was added at -78°C tri ethylallylsilane (10.9 mL, 68.69 mmol), followed by neat TiCl (2.94 mL, 26.71 mmol). The reaction was stirred at - 78°C for 1 hr and then at -40°C for 2 hr . At this point, the reaction was quenched with methanol (15 mL) and diluted with dichloromethane (200 mL) . The resulting reaction mixture was washed with IN HCl (2 x 50 mL) , water and brine. The organic layer was dried and cone, in vacuo to give a residue, which was purified by silica gel chromatography (10%
  • Carbinol lie (6.22 g, 22.0 mmol) was dissolved in an aqueous THF solution (5.5 mL water and 55 mL THF) .
  • NMO 4.42 g, 33.0 mmol
  • Os0 280 mg dissolved in THF, 1.10 mmol
  • the reaction stirred at rt overnight.
  • sodium bisulfide (4 g) was added.
  • the reaction was stirred for 2 hr, and then diluted with EtOAc (300 mL) . The whole mixture was washed with water (2 x 40 mL) and brine. The resulting organic layer was dried and cone . in vacuo to give the corresponding triol intermediate.
  • the methyl ester 12b from above (499.7 mg/1.84 mmol) was dissolved in a 1:1 mixture of acetic anhydride/pyridine in a 50 mL round bottom flask. The reaction was allowed to stir overnight at which time the solvent was removed in vacuo . The resulting oil was taken up in CH 2 C1 2 and washed 2x IN HCl, lx water, lx brine and dried over MgS0 4 . The drying agent was then filtered off and the solvent was removed in vacuo to yield 319.9 mg of a colorless oil (13a) which was used without purificaton.
  • Glycine side-chain precursors (14-a) For example the following procedure describes the synthesis of tridecanoyl-glycine-acid from Fmoc-glycine-wang resin and tridecanoic acid.
  • Fmoc-glycine-wang resin (lOg, 4.4 mmol) was added to 50 mL of 30% Piperdine/DMF. The reaction was shaken for 20 minutes, and was washed 3x with DMF, 3x with isopropanol, and 3x with DMF. To the resin was added a solution of tridecanoic acid (4.708g, 22 mmol) in 50 mL DMF. To this mixture was added HOBt (2.97 g, 22 mmol) and DIC (2.77 g,
  • Diastereomer 1-2 (152.8 mg, 0.089mmol) was subjected to hydrogenolyis as described for diasteromer 1-1 using 152.8 mg of 10% Pd/C for 30 min.
  • HPLC indicated consumption of starting material and formation of two product peaks which, after preparatory HPLC and lyophilization, were found to be Compound 1-4 (18.0 mg ) and Compound 1-5 (11.3 mg) .
  • Compound 1-4 MS (Ionspray) calcd for C 58 H 94 ClN ⁇ 2 Oi 9 (M+H) + 1297.89, found 1297.8.
  • Compound 1-5 MS (Ionspray) calcd for C 58 H 92 ClN ⁇ 2 Oi8 (M+H) + 1279.63, found 1281.7.
  • R 1 is a hydrogen .
  • alkyl chain is increased progressively from Cll to C15 show significant increased activity.
  • Side chains of this class may be prepared using the preparation described in preparation 14-a.
  • Example 16 illustrates the attachment of a Beta-amino substituted side-chain.
  • Example 17 illustrates the attachment of a chiral side- chain.
  • binaphthol-Ti catalyst 17b (0.42 mmol) was added at -78°C a THF solution containing trimethylsilyldimethylketene acetal (0.43 mL, 2.1 mmol) and the unsaturated aldehyde 17a (500
  • the reaction was then extracted with EtOAc (75 mL) .
  • the organic layer was washed with water and brine.
  • the organic layer thus obtained was dried and cone, in vacuo to afford 222 mg (90%) of the crude acid 17d, which was used directly for side chain coupling reaction.
  • Example 18 illustrates the attachment of a chiral alkenyl side-chain.
  • each of the compounds listed in the Examples showed measurable activity against Candida Albicans, Cryptococcus neoformans, Aspergillus Fumigatus, Candida Parapsilosis , or Histoplasma capsulatum .
  • the stereochemistry of the ⁇ -hydroxy group is preferably R. Longer alkyl chain lengths (i.e., C ⁇ -C 0 ) tend to have higher activities than shorter alkyl chains (e.g., ⁇ Cu) regardless of stereochemistry or unsaturation levels.
  • alkyl side-chains represented by the following structure are preferred for antifungal treatment:
  • R a and R a' are independently hydrogen or methyl, or either R a or R a' is alkyl amino, taken together with R b or R b' forms a six-membered cycloalkyl ring, a six- membered aromatic ring or a double bond, or taken together with R c forms a six-membered aromatic ring;
  • R b and R b' are independently hydrogen or methyl, and either R b or R b' is hydroxy provided that R b' is not hydroxy when R a , R b , R d , R e are hydrogen, R c is hydrogen and R f is n-hexyl, n-octyl or n-decyl, or R a , R b , R d , R e are hydrogen, R c is hydroxy and R f is n-octyl, n-nonyl, or n-decyl; R c is
  • R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C ⁇ 4 alkoxy substituted six-membered aromatic ring, or C 5 -C ⁇ alkyl substituted six-membered aromatic ring, and
  • R f is C 8 -C ⁇ alkyl, or C 5 -Cn alkoxy.

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Abstract

L'invention concerne des composés semi-synthétiques à base de pseudomycine représentés par la structure (I), qui peuvent être utiles comme agents antifongiques ou comme intermédiaires dans l'élaboration d'agents antifongiques.
PCT/US2000/015017 1999-07-15 2000-06-08 Analogues a base de pseudomycine a chaines laterales n-acyle WO2001005814A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU57249/00A AU5724900A (en) 1999-07-15 2000-06-08 Pseudomycin n-acyl side-chain analogs
JP2001511471A JP2003505397A (ja) 1999-07-15 2000-06-08 プソイドマイシンのｎ−アシル側鎖アナログ
MXPA02000321A MXPA02000321A (es) 1999-07-15 2000-06-08 Analogos de cadena lateral de n-acilo de pseudomicina.
CA002379851A CA2379851A1 (fr) 1999-07-15 2000-06-08 Analogues a base de pseudomycine a chaines laterales n-acyle
EA200200160A EA200200160A1 (ru) 1999-07-15 2000-06-08 Аналоги псевдомицина с n-ацильной боковой цепью
BR0012447-8A BR0012447A (pt) 1999-07-15 2000-06-08 Análogos de pseudomicina de cadeia lateral de n-acila
EP00942655A EP1200460A1 (fr) 1999-07-15 2000-06-08 Analogues a base de pseudomycine a chaines laterales n-acyle
NO20020193A NO20020193L (no) 1999-07-15 2002-01-14 Pseudomycin N-acyl side-kjede analoger

Applications Claiming Priority (2)

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US14398999P 1999-07-15 1999-07-15
US60/143,989 1999-07-15

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006042655A1 (fr) * 2004-10-20 2006-04-27 Aicuris Gmbh & Co. Kg Desoxo-nonadepsipeptides
US7718611B2 (en) 2004-11-05 2010-05-18 Aicuris Gmbh & Co. Kg Cyclic nonapeptide amides
US7786079B2 (en) 2004-10-20 2010-08-31 Aicuris Gmbh & Co. Kg. Substituted nonadepsipeptides
US8076285B2 (en) 2006-04-13 2011-12-13 Aicuris Gmbh & Co. Kg Lysobactin amides
US20120088784A1 (en) * 2009-05-04 2012-04-12 Boulos Zacharie Substituted Aromatic Compounds and Pharmaceutical Uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5837685A (en) * 1992-11-30 1998-11-17 Research And Development Institute Inc. At Montana State University Peptides from Pseudomonas syringae possessing broad-spectrum antibiotic activity

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5837685A (en) * 1992-11-30 1998-11-17 Research And Development Institute Inc. At Montana State University Peptides from Pseudomonas syringae possessing broad-spectrum antibiotic activity

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006042655A1 (fr) * 2004-10-20 2006-04-27 Aicuris Gmbh & Co. Kg Desoxo-nonadepsipeptides
US7727956B2 (en) 2004-10-20 2010-06-01 Aicuris Gmbh & Co. Kg Deoxonadepsipeptides
US7786079B2 (en) 2004-10-20 2010-08-31 Aicuris Gmbh & Co. Kg. Substituted nonadepsipeptides
US7718611B2 (en) 2004-11-05 2010-05-18 Aicuris Gmbh & Co. Kg Cyclic nonapeptide amides
US8076285B2 (en) 2006-04-13 2011-12-13 Aicuris Gmbh & Co. Kg Lysobactin amides
US20120088784A1 (en) * 2009-05-04 2012-04-12 Boulos Zacharie Substituted Aromatic Compounds and Pharmaceutical Uses thereof
CN102438975A (zh) * 2009-05-04 2012-05-02 普罗米蒂克生物科学公司 取代的芳香化合物及其药物用途
US8927765B2 (en) * 2009-05-04 2015-01-06 Prometic Biosciences Inc. Substituted aromatic compounds and pharmaceutical uses thereof
US9938221B2 (en) 2009-05-04 2018-04-10 Prometic Pharma Smt Limited Substituted aromatic compounds and pharmaceutical uses thereof
US10815183B2 (en) 2009-05-04 2020-10-27 Prometic Pharma Smt Limited Substituted aromatic compounds and pharmaceutical uses thereof

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CN1360593A (zh) 2002-07-24
NO20020193L (no) 2002-03-14
BR0012447A (pt) 2002-04-02
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AU5724900A (en) 2001-02-05

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