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EP1198472A1 - Compose de pseudomycine modifie par amine - Google Patents

Compose de pseudomycine modifie par amine

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Publication number
EP1198472A1
EP1198472A1 EP00939447A EP00939447A EP1198472A1 EP 1198472 A1 EP1198472 A1 EP 1198472A1 EP 00939447 A EP00939447 A EP 00939447A EP 00939447 A EP00939447 A EP 00939447A EP 1198472 A1 EP1198472 A1 EP 1198472A1
Authority
EP
European Patent Office
Prior art keywords
alkyl
pseudomycin
alkoxy
hydrogen
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00939447A
Other languages
German (de)
English (en)
Inventor
Shu Hui Chen
James Andrew Jamison
Michael John Rodriguez
Xicheng David Sun
Venkatraghavan Vasudevan
Mark James Zweifel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1198472A1 publication Critical patent/EP1198472A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61P17/00—Drugs for dermatological disorders
    • 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, amine-modified, pseudomycin compounds .
  • 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
  • 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 amine-modified pseudomycin compounds represented by the following structure which are useful as antifungal agents or in the design of antifungal agents.
  • 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, halogen, or
  • R b or R b' is amino, alkylamino, ⁇ - acetoacetate, methoxy, or hydroxy;
  • R c is hydrogen, hydroxy, C 1 -C4 alkoxy, hydroxy (Ci- C 4 )alkoxy, or taken together with R e forms a 6-membered aromatic ring or C5-C6 cycloalkyl ring;
  • R e is hydrogen, or taken together with R f is a six-membered aromatic ring, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 ⁇ C ⁇ alkyl substituted six-membered aromatic ring, and
  • R f is Cs-Cis alkyl, C 5 -C 11 alkoxy, or biphenyl
  • R g is hydrogen, or C1-C 13 alkyl
  • R 1 is a hydrogen, halogen, or C5-C8 alkoxy, and m is 1, 2 or 3 ;
  • R k is C5-C14 alkoxy; or R is - (CH 2 ) -NR- (C13-C18 alkyl), where R m is H, -CH 3 or
  • an acylalkyl e.g., - C(0)CH 3 , -C(0)CH 2 CH 3 ,
  • K a) is C1-C10 alkyl , C1-C10 alkenyl , benzyl , or aryl and R il is hydrogen or methyl, provided that at least one R 1 is not hydrogen;
  • R' and R- 3 are independently -OR 2a , or -N (R ,2 ⁇ b D v) (R ,2 ⁇ c c ), , where
  • R 2a and R 2b are independently hydrogen, Ci-Cio alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i- butyl, s-butyl, t-butyl, etc.), C 3 _C 6 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethylene, methylcyclopentyl , cyclohexyl, etc.) hydroxy (Ci-Cio) alkyl, alkoxy (Ci-Cio) alkyl (e.g, methoxyethyl ) , or C 2 -C ⁇ o alkenyl, amino (Ci-Cio) alkyl, mono- or di-alkylamino (Ci-Cio) alkyl, aryl (Ci-Cio) alkyl (
  • R 2b is an alkyl carboxylate residue of an aminoacid alkyl ester (e.g., -CHC0 2 CH 3 , -CH(C0 2 CH 3 )CH(CH 3 ) 2 , -CH (C0 2 CH 3 ) CH (phenyl) , -CH (C0 2 CH 3 ) CH 2 OH, -CH (C0 2 CH 3 ) CH 2 (p-hydroxyphenyl ) , -CH(C0 2 CH 3 )CH 2 SH, -CH (C0 2 CH 3 ) CH 2 (CH 2 ) 3 NH 2 , -CH(C0 2 CH 3 )CH 2 (4- or 5-imidazole) , -CH (C0 2 CH 3 ) CH 2 C0 2 CH 3 , -CH(C0 2 CH 3 )CH 2 C0 2 NH 2 , and the like), and
  • an aminoacid alkyl ester e.g., -CHC0 2 CH 3 ,
  • R 2c is hydrogen or Ci-C ⁇ alkyl; and pharmaceutically acceptable salts and solvates thereof.
  • a pharmaceutical formulation which includes the pseudomycin compound described 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 described above.
  • alkyl refers to a hydrocarbon radical of the general formula C n H n+ ⁇ containing from 1 to 30 carbon atoms unless otherwise indicated.
  • the alkane radical may be straight (e.g. methyl, ethyl, propyl, butyl, etc.), branched (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e.g., bicyclo [2.2.
  • 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 .
  • 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.
  • 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, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
  • 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.
  • the acylating agent is formed by replacing the -OH of the carboxylic acid group with a leaving group (e.g., N-oxysuccinimde) .
  • amino acid acylating agent When an amino acid acylating agent is used, the amino group is protected prior to condensation using any conventional amino-protecting group known to those skilled in the art (e.g., benzyloxycarbonyl , p-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl , p-methoxybenxyloxycarbonyl , p- methoxyphenylazobenzyloxycarbonyl , p- phenylazobenzyloxycarbonyl, t-butyloxycarbonyl or cyclopentyloxycarbonyl) .
  • any conventional amino-protecting group e.g., benzyloxycarbonyl , p-nitrobenzyloxycarbonyl, p- bromobenzyloxycarbonyl , p-methoxybenxyloxycarbonyl , p- methoxyphenylazobenzyloxycarbonyl , p- phen
  • the amino-protecting group is removed using standard hydrogenation chemistry (e.g., Pd/C under a hydrogen atmosphere) . See the Examples below for a more detailed description for forming pseudomycin amide derivatives from amino acids.
  • 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) ,
  • MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H are described in U.S. Patent Nos.
  • 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) . Like most organisms, the characteristics of the pseudomycin- producing cultures employed ( P.
  • syringae strains such as MSU 174, MSU 16H, MSU 206, 25-Bl, 7H9-1) are subject to variation.
  • progeny of these strains e.g., recombinants, mutants and variants
  • 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.
  • 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
  • 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 a C to about 27 S 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.
  • 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. Preferably, 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. However, 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.
  • an acid or base e.g., ammonium hydroxide
  • Choice of P. syringae strain can affect the amount and distribution of pseudomycin or pseudomycins produced.
  • 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.
  • the amine-modified pseudomycin compounds of the present invention can be formed from an N- acyl semi-synthetic compound.
  • Semi-synthetic pseudomycin compounds may be synthesized by exchanging the N-acyl group on the L-serine unit. Examples of various N-acyl derivatives are described in PCT Patent Application Serial
  • the pendant amino groups at positions 2, 4 and 5 may be protected using any standard means known to those skilled in the art for amino protection.
  • the exact genus and species of amino protecting group employed is not critical so long as the derivatized amino group is stable to the condition of subsequent reaction (s) on other positions of the intermediate molecule and the protecting group can be selectively removed at the appropriate point without disrupting the remainder of the molecule including any other amino protecting grou (s) .
  • 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) . Further examples of suitable protecting groups are described in T.W. Greene, "Protective Groups in Organic Synthesis," John Wiley and Sons, New York, N.Y., (2nd ed. , 1991), at chapter 7.
  • the deacylation of an N-acyl group having a gamma or delta hydroxylated side chain may be accomplished by treating the amino- protected pseudomycin compound with acid in an aqueous solvent.
  • Suitable acids include acetic acid and trifluoroacetic acid.
  • a preferred acid is trifluoroacetic acid. If trifluoroacetic acid is used, the reaction may be accomplished at or near room temperature. However, when acetic acid is used the reaction is generally ran at about
  • Suitable aqueous solvent systems include
  • Pseudomycin compounds lacking a delta or gamma hydroxy group on the side chain 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.
  • 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) 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'- dicyclohexy
  • 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.
  • 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
  • the amino protecting groups (at positions 2, 4 and 5) can 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 can be removed using tributyltinhydride and triphenylphosphine palladium dichloride.
  • the amine-modification of the N-acyl semi-synthetic compound is then accomplished by acylating at least one of the pendant amino groups attached to the lysine or 2,4- diaminobutyric acid peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide, urea, carbamate or imide linkage.
  • the amine-modified pseudomycin compounds may be further modified by amidation or esterification of the pendant carboxylic acid group of the aspartic acid and/or hydroxyaspartic acid units of the pseudomycin ring.
  • Examples of various acid-modified derivatives are described in PCT Patent Application Serial No. , Chen, et al . , filed evendate herewith entitled "Pseudomycin Amide & Ester Analogs" and incorporated herein by reference.
  • the acid- modified derivatives may be formed by condensing any of the previously described amine-modified pseudomycin compounds with the appropriate alcohol or amine to produce the respective ester or amide.
  • Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of a protic acid (e.g., HCI, TFA, etc.) .
  • a protic acid e.g., HCI, TFA, etc.
  • the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (e.g., sodium bicarbonate and potassium carbonate) .
  • amide groups may be accomplished using standard amidation procedures well-known to those skilled in the art.
  • the choice of coupling agents provides selective modification of the acid groups.
  • the use of benzotriazol-1-yloxy- tripyrrolidinophosphonium hexafluorophosphate (PyBOP) as the coupling agent allows one to isolate pure mono-amides at residue 8 and (in some cases) pure bis amides simultaneously.
  • the use of o-benzotriazol-1-yl- N,N,N' ,N' -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent favors formation of monoamides at residue 3.
  • the pseudomycin amide derivatives 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, tartarate
  • solvate refers to an aggregate that comprises one or more molecules of the solute (i.e., amine- modified pseudomycin 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 pseudomycin derivative in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.
  • Each pseudomycin compound, semi-synthetic pseudomycin derivatives, 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 or amine-modified 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 active ingredient i.e., pseudomycin compound of the present invention
  • 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, the means used to administer the drug and the animal species.
  • 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. 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.
  • WFI water for injection
  • the resulting solution is then further diluted in an infusion solution such as dextrose 5% in water (D5W) , prior to administration .
  • 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 taniaw) ; Torulopus spp. (i.e., T. glabrata) ; Aspergillus spp. (i.e., A . fumigatus) ; Histoplasma 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 taniaw
  • Torulopus spp. i.e., T.
  • Blastomyces spp. i.e., B. dermati tidis
  • Fusarium spp. Fusarium spp.
  • Trichophyton spp. Pseudallescheria boydii , Coccidioides immi ts, Sporothrix schenckii , etc.
  • the compounds and formulations of the present invention are 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 the amine-modified pseudomycin compound of the present invention with a fungus. A preferred method includes inhibiting Candida albicans or
  • Aspergillus fumigatus activity includes a union or junction, or apparent touching or mutual tangency of a compound of the invention with a fungus.
  • 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 or
  • 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 mg/kg to 100 mg/kg of body weight of an active compound.
  • Preferred daily doses are generally between about 0.1 mg/kg to 60 mg/kg and more preferably between about 2.5 mg/kg to 40 mg/kg.
  • the host is generally an animal including humans, companion animals (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 .
  • CBZ benzyloxycarbonyl , C 6 H 5 CH 2 -0-C (0) -
  • TBTU o-Benzotriazol-l-yl-N,N,N' ,N'- tetramethyluronium tetrafluoroborate DIEA - N,N-diisopropylethylamine
  • 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.
  • 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).
  • Compound 2a-l is prepared using the procedures described in Ad iak, R.W., et al . , Tetrahedron Lett . , No. 22, 1935-1936 (1997) .
  • R 1 ', R 1 " and R 1 ''' -C(0)CH 2 NH 2 1-1
  • a 50 ml round bottom flask was charged with 10 ml of anhydrous DMF, pseudomycin B (250.6 mg, 0.181 mmol) and the acylating agent la-1 (343.0 mg, 1.12 mmol) .
  • the reaction was allowed to stir at room temperature for 24 hours.
  • the solvent was then removed in vacuo and the residue was taken up in ACN and purified via preparatory HPLC to yield 172.5 mg of the tri-substituted protected amine after lyophilization. 134.4 mg of tri-substituted protected amine was dissolved in a solution of 10 ml MeOH/1.5 ml glacial AcOH.
  • pseudomycin amide derivatives (1-2 and 1-3 ) may be synthesized using the same procedures as described above and the appropriate amino acid to form the acylating agent .
  • Compound 2-1 is synthesized using the same procedures as described in Example 1 except Compound 2a-l is used as the acylating agent .
  • Compound 2-1 may be alternatively synthesized by adding phenyl chloroformate (389 mg, 2.48 mmol) to a solution of HOBT (37.5 mg, 2.48 mmol) and DIEA (322.8 mg,
  • Compound 3-2 is synthesized using the same procedures as described in Example 1 except trimethylacetic anydride is used as the acylating agent.
  • Compound 4-1 is synthesized using the same procedures as described in Example 1 except methyl isocyanate is used as the acylating agent .
  • Compound 5-1 is synthesized using the same procedures as described in Example 1 except 4-nitrophenylformate is used as the acylating agent.

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Abstract

L'invention concerne un composé de pseudomycine modifié par amine, qui est représenté par la structure (I), dans laquelle R1 est une liaison d'acyle. Les dérivés de pseudomycine modifiés par amine sont utiles en tant qu'agents antifongiques ou pour la préparation d'agents antifongiques.
EP00939447A 1999-07-15 2000-06-08 Compose de pseudomycine modifie par amine Withdrawn EP1198472A1 (fr)

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US14383999P 1999-07-15 1999-07-15
US143839P 1999-07-15
PCT/US2000/015019 WO2001005816A1 (fr) 1999-07-15 2000-06-08 Compose de pseudomycine modifie par amine

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EP1198472A1 true EP1198472A1 (fr) 2002-04-24

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EP (1) EP1198472A1 (fr)
JP (1) JP2003505398A (fr)
CN (1) CN1361790A (fr)
AU (1) AU5453100A (fr)
BR (1) BR0013168A (fr)
CA (1) CA2379317A1 (fr)
EA (1) EA200200163A1 (fr)
HU (1) HUP0202237A3 (fr)
MX (1) MXPA02000320A (fr)
NO (1) NO20020194L (fr)
WO (1) WO2001005816A1 (fr)

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US5576298A (en) * 1992-11-30 1996-11-19 Research And Development Institute, Inc. At Montana State University Peptides from pseudomonas syringae possessing broad-spectrum antibiotic activity

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See references of WO0105816A1 *

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JP2003505398A (ja) 2003-02-12
MXPA02000320A (es) 2002-06-21
AU5453100A (en) 2001-02-05
NO20020194L (no) 2002-03-14
BR0013168A (pt) 2002-04-02
CA2379317A1 (fr) 2001-01-25
NO20020194D0 (no) 2002-01-14
HUP0202237A3 (en) 2002-12-28
EA200200163A1 (ru) 2002-06-27
HUP0202237A2 (hu) 2002-11-28
CN1361790A (zh) 2002-07-31

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