WO2001041534A2 - Promedicaments de phosphate pseudomycine - Google Patents

Promedicaments de phosphate pseudomycine Download PDF

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Publication number
WO2001041534A2
WO2001041534A2 PCT/US2000/030167 US0030167W WO0141534A2 WO 2001041534 A2 WO2001041534 A2 WO 2001041534A2 US 0030167 W US0030167 W US 0030167W WO 0141534 A2 WO0141534 A2 WO 0141534A2
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Prior art keywords
alkyl
hydrogen
prodrug
represented
alkoxy
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PCT/US2000/030167
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WO2001041534A3 (fr
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Shu Hui Chen
Xicheng Sun
Yanzhi Zhang
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Eli Lilly And Company
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Priority to AU30718/01A priority Critical patent/AU3071801A/en
Priority to JP2001542721A priority patent/JP2003515610A/ja
Priority to MXPA02005791A priority patent/MXPA02005791A/es
Priority to BR0016328-7A priority patent/BR0016328A/pt
Priority to CA002396513A priority patent/CA2396513A1/fr
Publication of WO2001041534A2 publication Critical patent/WO2001041534A2/fr
Publication of WO2001041534A3 publication Critical patent/WO2001041534A3/fr
Priority to NO20022798A priority patent/NO20022798L/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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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, phosphate prodrugs of 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-Cl) 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-dihydroxytetradecanoate, pseudomycin A' by 3 , 4-dihydroxypentadecanoate, pseudomycin B by 3-hydroxytetradecanoate, pseudomycin B' by 3-hydroxydodecanoate, pseudomycin C by 3 , 4-dihydroxyhexadecanoate and pseudomycin C ' 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. Therefore, there is a need to identify compounds within this class that are useful for treating fungal infections without the currently observed adverse side effects.
  • the present invention provides a pseudomycin prodrug represented by the following structure which is useful as an antifungal agent.
  • 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 ' are independently hydrogen, halogen, or
  • R b or R b' is amino, alkylamino, - acetoacetate, methoxy, or hydroxy;
  • R c is hydrogen, hydroxy, C 1 -C 4 alkoxy, hydroxy Ci- C 4 alkoxy, 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, C 5 -C 14 alkoxy substituted six-membered aromatic ring, or C 5 -C14 alkyl substituted six-membered aromatic ring, and
  • R f is C 8 -Ci 8 alkyl, or C 5 -C 11 alkoxy; R is
  • R 9 is hydrogen, or C ⁇ -C ⁇ 3 alkyl
  • R 1 is a hydrogen, halogen, or C 5 -C 8 alkoxy, and m is 1, 2 or 3 ; R is
  • R k is C 5 -C 1 alkoxy; or R is - (CH 2 ) -NR- (C ⁇ 3 -Ci 8 alkyl), where R is H, -CH 3 or
  • R 1 is independently hydrogen or a group represented by formula 1(a), 1(b), or 1(c)
  • ,1a is hydrogen, Ci-C ⁇ alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, etc.), benzyl, or -CH 2 CH 2 Si (CH 3 ) 3
  • R lb is hydrogen or C ⁇ -C 6 alkyl, provided that at least one R 1 is a group represented by formula 1(a), 1(b) or 1(c); d R 3 are independently -OR 2a , or -N(R 2b ) (R 2c ) , where
  • R 2 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 (e
  • R 2b is an alkyl carboxylate residue of an aminoacid alkyl ester (e.g., -CH 2 C0 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 ,
  • an aminoacid alkyl ester e.g., -CH 2 C0 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
  • R 2c is hydrogen or Ci-C ⁇ alkyl; and pharmaceutically acceptable salts and solvates thereof.
  • R is represented by the structure
  • R b' is hydroxy
  • R a , R a' , R b , R c , R , and R e are all hydrogen
  • R f is n-octyl
  • a pharmaceutical formulation which includes the pseudomycin prodrug 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 prodrug described above.
  • the use of the pseudomycin prodrug described above in the manufacture of a medicament for use in treating an antifungal infection in an animal is also provided.
  • alkyl refers to a hydrocarbon radical of the general formula C n H 2 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 [2.2.1] heptane, spiro [2.2]pentane, etc.) .
  • the alkane radical may be substituted or unsubstituted.
  • 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 .
  • prodrug refers to a class of drugs which result in pharmacological action due to conversion by metabolic processes within the body (i.e., biotransformation) .
  • the pseudomycin prodrug compounds contain linkers that can be cleaved by esterases in the plasma to produce the active drug .
  • 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.
  • a prodrug derivative of the pseudomycin natural or semi-synthetic products provide less adverse side effects than the corresponding natural products and maintains in vivo efficacy against C. albican, C. neoformans , and A . fumigatus .
  • the prodrug is produced by acylating at least one of the pendant amino groups attached to the lysine or 2 , 4-diaminobutyric acid peptide units in the pseudomycin cyclopeptide ring system to form an acyl phosphate substituent (s) .
  • the phosphate acylating agent is generally a compound having one of following formulae la-L, lb-L or lc-L
  • L is a suitable leaving group such that a carbamate linkage with the pendant amino group on the pseudomycin structure can be formed.
  • Suitable leaving groups are well known to those skilled in the art and include groups such as p-nitrophenoxy and N-oxysuccinimide .
  • R la and R lb are as defined above.
  • the benzyl phosphate linkers (la-L) may be synthesized using the synthetic route shown in scheme I below.
  • scheme I For illustrative purposes, a specific para-phenylmethylene substituted acylating compound having a p-nitrophenoxy leaving group is depicted.
  • a variety of derivatives including the ortho substituted derivative (lb-L)) using the same basic synthetic method.
  • the phosphate linker (la-L or lb-L) where R la is -CH 2 CH 2 Si (CH 3 ) 3 may be prepared by oxidative coupling of jis- teocphosphite with 4-hydroxybenzyl alcohol in carbon tetrachloride as described in Li , J., et al . , Bioorg. Med.
  • Bis-teocphosphite may be prepared from 2-2-trimethylsilylethanol and PC1 3 as described in McCombie, et al . , J. Chem. Soc . , 381 (1945).
  • the methylene phosphate linker (lc-L) may be synthesized using the synthetic route shown in scheme II below. For illustrative purposes, a specific acylating compound with an N-succinimide leaving group is depicted. However, it will be understood by those skilled in the art that one could synthesize a variety of derivatives using the same basic synthetic method.
  • phosphate triesters (methyleneoxy and benzyloxy derivatives) may be converted to their phosphate monoester derivatives via hydrogenation (e.g., hydrogen over Pd/C in methanol) .
  • 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) .
  • Methods for growth of various strains of P 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
  • Isolated strains of P. syringae that produce one or more pseudomycins are known in the art. Wild type strain MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H (ATCC 67028) 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 .
  • 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) .
  • P . syringae strains such as MSU 174, MSU 16H, MSU 206, 25-Bl, 7H9-1
  • progeny of these strains may be obtained by methods known in the art .
  • 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.:
  • 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.
  • 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.
  • 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.
  • 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 greater than about 10 ⁇ g/ l .
  • 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.
  • 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.
  • 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 2 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.
  • 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 .
  • the prodrug 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 Application No. PCT/US00/15017 filed by Chen, et al . on June 8, 2000 entitled “Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference.
  • 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 group (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 a 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 40°C.
  • Suitable aqueous solvent systems include acetonitrile, water, and mixtures thereof. Organic solvents accelerate the reaction; however, the addition of an organic solvent may lead to other by-products .
  • 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 prodrug is then produced 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 carbamate linkage.
  • modified prodrug pseudomycin compounds may be synthesized 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 Application No. PCT/US00/15021 filed by Chen, et al . on June 8, 2000 entitled “Pseudomycin Amide & Ester Analogs” and incorporated herein by reference.
  • the acid-modified derivatives may be formed by condensing any of the previously described prodrugs with the appropriate alcohol or amine to produce the respective ester or amide. Formation of the ester groups may be accomplished using standard esterification procedures well-known to those skilled in the art.
  • Esterification under acidic conditions typically includes dissolving or suspending the pseudomycin compound in the appropriate alcohol in the presence of a protic acid (e.g., HC1, TFA, etc.).
  • a protic acid e.g., HC1, 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) .
  • 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 jis amides simultaneously.
  • the use of o- benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent favors formation of monoamides at residue 3.
  • TBTU o- benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate
  • the pseudomycin prodrug may be isolated and used per se or in the form of its pharmaceutically acceptable salt or solvate.
  • the prodrug is prepared by forming at least one phosphate carbamate linkage as described earlier.
  • 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, citrates, camphorates, camphorsulfonates, digluconates, trifluoroacetates, and the like.
  • 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 prodrug in the appropriate solvent with heat and slowing cooling to generate an amorphous or crystalline solvate form.
  • pseudomycin, semi-synthetic pseudomycin, pseudomycin prodrug 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 pseudomycin prodrug 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 derivative
  • 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 taniaw) ; Torulopus spp. (i.e., T. Glabrata) ;
  • Aspergillus spp. i.e., A . Fumigatus
  • Histoplasma spp. i.e., H. Capsulatu
  • Cryptococcus spp. i.e., C. Neoformans
  • B . Dermati tidis a group consisting of Coccidioides
  • Fusarium spp. a group consisting of Trichophyton spp., Pseudallescheria boydii , Coccidioides immi ts , Sporothrix schenckii , etc.
  • a method for inhibiting fungal activity comprising contacting the pseudomycin prodrug of the present invention with a fungus.
  • a preferred method includes inhibiting Candida albicans 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 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 parasitic and 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,
  • 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 and the tolerance of the host to the antifungal agent.
  • 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 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.
  • CBZ benzyloxycarbonyl
  • C 6 H 5 CH 2 -0-C (0) - PyBOP benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate
  • TBTU o-Benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate
  • DIEA N,N-diisopropylethylamine
  • Antifungal activity in the following examples were determined in vi tro by obtaining the minimum inhibitory concentration (MIC) of the compound using a standard agar dilution test or a disc-diffusion 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.
  • 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. The 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) .
  • mice were x-irradiated with 400 r 24 hr prior to infection with a Gamacell 40 (Atomatic Energy of Canada Limited Commercial Products, Ottawa, Canada) .
  • Mice were infected by an intravenous (IV) injection of 0.1 mL (containing 2 x 10 6 blastoconidia per mouse) in the lateral tail vein. Untreated controls were moribund within 3-4 days post-infection. Mice were dosed four times at 0, 4, 24 and 48 hr post-infection with 0.2 mL of testing compounds which were given at 20, 10 and 5 mg/kg.
  • Compounds were formulated in 4.0% hydroxypropyl cyclodextrin and sodium acetate, pH 7.0 buffer and 1.75% dextrose.
  • Infected sham-treated mice (10 animals) were dosed with vehicle alone. Morbidity and mortality were recorded for 7 days. The 50% effective doses (ED 5 o) were determined using the method of Reed and Muench. Statistical differences in treated groups compared to untreated infection controls were determined using the Student's t test.
  • the phosphate triester prodrug 1A-1 may be converted to the phosphoric acid monoester prodrug IB via hydrogenation.
  • the phosphate triester prodrug 2A-1 may be converted to the phosphoric acid monoester prodrug 2B via hydrogenation.
  • the purified product 2A-1 was dissolved 5 ml methanol . 50 mg of 10% Pd/C was added under nitrogen. Hydrogen was applied and the reaction mixture was stirred for one hour at room temperature. The mixture was filtered and the solvent was removed. The product was dissolved in water/acetonitrile (1/1) and lyophilized to give 32 mg (90% yield) of the desired product 2B.

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Abstract

La présente invention concerne un promédicament de pseudomycine, représenté par la structure (A), dans laquelle R1 est une liaison benzyloxycarbamate de phosphate ou méthylèneoxycarbamate de phosphate. Ces promédicaments de phosphate présentent une activité antifongique, avec moins d'effets indésirables que le composé de pseudomycine parent.
PCT/US2000/030167 1999-12-13 2000-11-29 Promedicaments de phosphate pseudomycine WO2001041534A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU30718/01A AU3071801A (en) 1999-12-13 2000-11-29 Pseudomycin phosphate prodrugs
JP2001542721A JP2003515610A (ja) 1999-12-13 2000-11-29 シュードマイシンホスフェートプロドラッグ
MXPA02005791A MXPA02005791A (es) 1999-12-13 2000-11-29 Profarmacos de fosfato de pseudomicina.
BR0016328-7A BR0016328A (pt) 1999-12-13 2000-11-29 Pró-drogas de fosfato de pseudomicina
CA002396513A CA2396513A1 (fr) 1999-12-13 2000-11-29 Promedicaments de phosphate pseudomycine
NO20022798A NO20022798L (no) 1999-12-13 2002-06-12 Pseudomycinfosfatprodrug

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17046699P 1999-12-13 1999-12-13
US60/170,466 1999-12-13

Publications (2)

Publication Number Publication Date
WO2001041534A2 true WO2001041534A2 (fr) 2001-06-14
WO2001041534A3 WO2001041534A3 (fr) 2001-11-22

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PCT/US2000/030167 WO2001041534A2 (fr) 1999-12-13 2000-11-29 Promedicaments de phosphate pseudomycine

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JP (1) JP2003515610A (fr)
AU (1) AU3071801A (fr)
BR (1) BR0016328A (fr)
CA (1) CA2396513A1 (fr)
MX (1) MXPA02005791A (fr)
NO (1) NO20022798L (fr)
WO (1) WO2001041534A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10933069B2 (en) 2018-01-05 2021-03-02 Cybrexa 1, Inc. Compounds, compositions, and methods for treatment of diseases involving acidic or hypoxic diseased tissues
US11634508B2 (en) 2019-07-10 2023-04-25 Cybrexa 2, Inc. Peptide conjugates of cytotoxins as therapeutics

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002542257A (ja) * 1999-04-15 2002-12-10 イーライ・リリー・アンド・カンパニー シュードマイシン天然産物
AU2020311925A1 (en) 2019-07-10 2022-02-03 Cybrexa 3, Inc. Peptide conjugates of microtubule-targeting agents as therapeutics

Citations (2)

* 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
WO2001005817A1 (fr) * 1999-07-15 2001-01-25 Eli Lilly And Company Analogues amides et esters de pseudomycine

Patent Citations (2)

* 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
WO2001005817A1 (fr) * 1999-07-15 2001-01-25 Eli Lilly And Company Analogues amides et esters de pseudomycine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10933069B2 (en) 2018-01-05 2021-03-02 Cybrexa 1, Inc. Compounds, compositions, and methods for treatment of diseases involving acidic or hypoxic diseased tissues
US11634508B2 (en) 2019-07-10 2023-04-25 Cybrexa 2, Inc. Peptide conjugates of cytotoxins as therapeutics

Also Published As

Publication number Publication date
BR0016328A (pt) 2002-08-27
AU3071801A (en) 2001-06-18
JP2003515610A (ja) 2003-05-07
NO20022798D0 (no) 2002-06-12
CA2396513A1 (fr) 2001-06-14
NO20022798L (no) 2002-06-12
WO2001041534A3 (fr) 2001-11-22
MXPA02005791A (es) 2002-09-30

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