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EP1198473A1 - Analogues amides et esters de pseudomycine - Google Patents

Analogues amides et esters de pseudomycine

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Publication number
EP1198473A1
EP1198473A1 EP00942656A EP00942656A EP1198473A1 EP 1198473 A1 EP1198473 A1 EP 1198473A1 EP 00942656 A EP00942656 A EP 00942656A EP 00942656 A EP00942656 A EP 00942656A EP 1198473 A1 EP1198473 A1 EP 1198473A1
Authority
EP
European Patent Office
Prior art keywords
alkyl
alkoxy
aromatic ring
membered aromatic
hydrogen
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Withdrawn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
EP00942656A
Other languages
German (de)
English (en)
Inventor
Shu Hui Chen
Christopher Stanley Galka
Sarah Lynne Hellman
John Leonard Krstenansky
Michael John Rodriguez
Xicheng David Sun
Alexander Ya. Usyatinsky
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
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Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1198473A1 publication Critical patent/EP1198473A1/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Withdrawn legal-status Critical Current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
    • 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, acid-modified, semi-synthetic 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, AX 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 . Since the pseudomycins have proven antifungal activity and relatively unexplored chemistry, there is a need to explore this class of compounds for other potential compounds that may be useful as antifungal agents having less adverse side affects. BRIEF SUMMARY OF THE INVENTION 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 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 methyl, or either R b or R b' is amino, alkylamino, cc- 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, C5-C 1 4 alkoxy substituted six-membered aromatic ring, or C5-C 14 alkyl substituted six-membered aromatic ring, and
  • R f is C 6 -Ci8 alkyl, C5-C 11 alkoxy, or biphenyl; R is
  • R g 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 14 alkoxy; or R is -(CH 2 )-NR rn -(C ⁇ 3 -C ⁇ 8 alkyl), where R m is H, -CH 3 or
  • R 1 is independently -NH 2 or -NHp-Pg, where p is 0 or 1;
  • R and R J are independently -OR 2a, or -N(R ⁇ D ) (R ⁇ c ) , where R 2a and R 2b are independently hydrogen, C 1 -C10 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, ⁇ -butyl, i- butyl, s-butyl, t-butyl, n-amyl, i-amyl, n-hexyl, n- heptyl, n-octyl, n-nonanyl, n-decyl, etc.), C 3 _C 6 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethyl, methylcyclopentyl, cyclohexyl, etc.) haloalkyl (e.g., CF 3 CH 2 -)
  • a prodrug of a pseudomycin compound having structure I represented above wherein R 2 and R 3 are represented by -OR a , where R a is C ⁇ -C 3 alkyl.
  • a 3-amido derivative of a pseudomycin compound is provided where the compound is prepared by the steps of (i) providing a compound having structure I above wherein R 1 is -NH 2 and R 2 and R 3 are both -OH; (ii) protecting the amino groups, R 1 , at positions 2, 4 and 5 with an amino-protecting group; (iii) forming an amide linkage at position 3 using an o- Benzotriazol-l-yl-N,N,N' ,N' -tetramethyluronium tetrafluoroborate as a coupling agent; and (iv) removing the amino-protecting groups.
  • An 8-amido derivative is also provided where the derivative is prepared using the steps described above except using benzotria
  • 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 .
  • alkyl refers to a hydrocarbon radical of the general formula C n H 2n+ ⁇ 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, oxazine, pyrimidine, purine, benzimidazole, guinoline, etc.).
  • the aromatic moiety may consist of a single or fused ring system.
  • the heteroaryl groups may be substituted or unsubstituted.
  • amino protecting group refers to a substituent of the amino group (Pg) 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-fluorenylmethylcarbamate; 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, aminoalkoxy, 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.
  • 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 ester functionalities 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.
  • 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 or N-acyl modified derivatives.
  • three synthetic steps are used to produce Compound I: (1) selective amino protection; (2) condensation with the appropriate alcohol or amine to produce the respective ester or amide; and (4) deprotection of the amino groups.
  • the pendant amino groups at residues 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 conditions 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).
  • Preferred amino protecting groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl, phthalimido, and benzyloxycarbonyl (CBZ) . Most preferred is allyloxycarbonyl (Alloc) and benzyloxycarbonyl (CBZ) .
  • 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., HCl, TFA, p-toluenesulfonic acid, etc.).
  • a protic acid e.g., HCl, TFA, p-toluenesulfonic acid, etc.
  • the pseudomycin compound is generally reacted with the appropriate alkyl halide in the presence of a weak base (e.g., sodium bicarbonate under anhydrous conditions) .
  • 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.
  • coupling agents such as o-benzotriazol-l-yl-N,N,NXN' -tetramethyluronium tetrafluoroborate (TBTU) and 2 (lH-benzotriazole-1-yl) - 1,1,3,3, -tetramethyluronium hexafluorophosphate (HBTU) favor formation of monoamides at residue 3.
  • TBTU o-benzotriazol-l-yl-N,N,NXN' -tetramethyluronium tetrafluoroborate
  • HBTU 1,1,3,3, -tetramethyluronium hexafluorophosphate
  • a bulky amine enhances the ratio of monoamides at residue 3.
  • the ratio of amidation at residue 3 vs . residue 8 increased from about 1:1 to about 6:1 and the amount of bis-amides was reduced through the addition of a bulky amine.
  • the term "bulky amine” refers to an amine having multiple and/or large substituents on the nitrogen atom. Any tertiary amine may be used that is compatible with the reaction conditions.
  • Preferred bulky amines include N,N-diisopropylethylamine (DIEA) and N-ethyldicyclohexylamine.
  • the amount of bulky amine added is generally from about 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 5 to 6 equivalents.
  • the reaction is generally ran at temperatures from about room temperature (25°C) to about -20°C. However, Applicants discovered that lower temperatures (from about
  • the amino protecting groups may be removed using standard procedures appropriate for the specific protecting group used. For example, CBZ groups are 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. This particular protection/deprotection scheme has the advantage of reducing the potential for hydrogenating the vinyl group of the Z-Dhb unit of the pseudomycin structure.
  • 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 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/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.
  • 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 21 -0. , 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. 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 .
  • 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 amido or ester derivative 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 No. , Belvo, et al . , filed evendate herewith entitled "Pseudomycin N-Acyl Side-Chain Analogs" and incorporated herein by reference.
  • 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 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 acid-modification of the protected N-acyl semi- synthetic compound is then accomplished by reacting at least one of the pendant carboxyl groups attached to the aspartic or hydroxyaspartic peptide units of the N-acyl modified semi-synthetic pseudomycin compound to form the desired amide or ester linkage (s).
  • the protecting groups are then removed as described earlier.
  • 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 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 compound
  • the active ingredient is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient, physician or veterinarian an elegant and easy to handle 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, 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,
  • Torulopus spp. i.e., T. glabrata
  • Aspergillus spp. i.e., A . fumigatus
  • H. capsulatum i.e., H. capsulatum
  • Cryptococcus spp. i.e., C. neoformans
  • Blastomyces spp. i.e., B . dermati tidis
  • a method for inhibiting fungal activity comprising contacting the pseudomycin compound 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 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 an animal host in need of such treatment is also provided.
  • a preferred method includes treating a Candida albicans 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 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 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 other similar animal species.
  • 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. albicans x657 seeded agar plates.
  • Tail Vein Toxic ity 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.
  • 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) .
  • Alloc-Protected Pseudomycin General procedures used to protect the pendant amino groups at posi tions 2, 4 and 5 of
  • Diallyl pyrocarbonate (558 mg, 3.0 mmol) was added to a solution of Pseudomycin A (1.22 g, 1.0 mmol) in 600 ml DMF .
  • the reaction was stirred at room temperature overnight. The solvent was removed in vacuo to afford an oily residue which was washed with ether three times. The oily residue was redissolved in a mixture of water and ACN (-1:1) and lyophilized to provide an alloc-protected psuedomycin A compound in 90% yield.
  • Acetic acid (1 ml) was added to a suspension of alloc- protected pseudomycin B (0.05 mmol) in 5 ml methylene chloride. After degassing under vacuum, the solution was treated with 6.0 mg PdCl 2 (PPH 3 ) 2 (0.008 mmol) and 0.40 ml tri-n-butyltin hydride (1.5 mmol) at room temperature for 2 hours . The solvent was evaporated in vacuo and the residue dissolved in water/ACN (-1:1) and filtered. The resulting solution was purified by preparative HPLC to afford the desired pseudomycin B compound in 93% yield. Alternatively, 5 ml tetrahydrofuran and 0.1 ml acetic acid may be used as the solvent instead of 5 ml methylene chloride and 1.0 ml acetic acid.
  • Examples 1-3 illustrate the formation of bis-esters at residues 3 and 8.
  • the mono-esters may be isolated by following the reaction carefully by HPLC. The reaction is stopped at the appropriate time when the ratio of starting material: mono ester (s): bis ester is greatest. The methodology remains the same. The resulting mixture of mono esters is isolated where some ester is formed on the aspartic acid residue and some on the hydroxy aspartic acid residue. This mixture of CBZ-protected, mono esters is hydrogenated using standard methodology to yield a mixture of mono ethyl esters of Pseudomycin B.
  • R2 -0CH 3
  • R 2 -OCH(CH 3 ) 2
  • R3 -0CH 3
  • R 3 -OCH(CH 3 ) 2
  • Example 2 illustrates the synthesize of bis-esters using basic conditions.
  • R 2 -OCH 2 CH 2 CH 3
  • R 3 -OCH 2 CH 2 CH 3
  • R -H
  • R -H
  • R 2 -0(CH 2 ) 4 CH 3
  • R 2 -OH
  • R 3 -OH
  • R 3 -0(CH 2 ) 4 CH 3
  • R 2 -0(CH 2 ) 3 CH 3
  • R 2 -0(CH 2 ) 3 CH 3
  • R 2 -OH
  • R 3 -0(CH 2 ) 3 CH 3
  • R 3 -OH
  • R 3 -0(CH 2 ) 3 CH 3
  • R 2 -0CH 2 (cyclopentyl]
  • R 3 -OH 4-1
  • CBZ-protected pseudomycin B a large excess of p- toluenesulfonic acid and cyclopentanemethanol are mixed and allowed to stir overnight. An additional 10 equivalents of alcohol was added the next day.
  • the CBZ-protected ester was isolated via preparatory HPLC and then hydrogenated using standard methodology to produce Compound 4-1.
  • the protected amido compound (60 mg) was dissolved in 6 ml of 1% AcOH in methanol and 60 mg of 10% Pd/C was added. The mixture was stirred for 30 minutes under hydrogen at room temperature. After filtering, the solution was concentrated in vacuo. The residue was dissolved in 50% ACN/water and lyophilized to yield 45 mg (90%) yield of Compound 5-1.
  • R 2 -NH ( cyclo ropyl )
  • Compound 6-1 can also be made from the Alloc-protected pseudomycin B using the following procedures.
  • the alloc-protected intermediate (117 mg, 0.078 mmol) was dissolved in 15 ml of methylene chloride and 1 ml of acetic acid. After degassing the reaction mixture with dry nitrogen, 30 mg of (PPh 3 ) PdCl 2 and 1 ml of tributyltinhydride was added to the mixture. The progress of the reaction was monitored by HPLC. Upon completion, the reaction mixture was purified by reverse phase preparative
  • CBZ- protected Pseudomycin B (0.5 g, 0.311mmol) was dissolved in 25 mL of DMF. To this solution was added TBTU(0.2 g, 0.622 mmol), 3- (aminomethyl)pyridine (0.067 g, 0.622mmol), and N- ethyldicyclohexylamine (0.391 g, 1.87 mmol). The solution was stirred for three hours and then concentrated down. The product was isolated by reverse-phase preparatory HPLC, and lyophilized to yield, (96 mg, 18% yield) CBZ-protected amide.
  • Example 7 The same general procedures as described in Example 7 may be used. When no base is added, a mixture of 8 and 3 amido substituted compounds are observed.
  • Example 10 The same general procedures as described in Example 7 may be used. When no base is added, a mixture of Compounds 9-1 and 9-2 are observed.
  • Example 10
  • Example 11 The same general procedures as described in Example 7 are used to synthesize Compound 10-1 using the appropriate corresponding amine starting material .
  • Example 11 The same general procedures as described in Example 7 are used to synthesize Compound 10-1 using the appropriate corresponding amine starting material .
  • Example 7 The same general procedures as described in Example 7 are used to synthesize Compound 11-1 using 4- (aminomethyl) pyridine as the amine starting material.
  • R 2 -N (CH 3 ) 2
  • R 3 -OH
  • Example 13 illustrates the synthesis of pseudomycin compounds where the carboxylic acid group is reacted with a variety of amino acid alkyl esters.
  • R 2 -NHCH (C0 2 CH 3 ) CH 2 CH 2 CH 2 CH 2 NH 2
  • the CBZ-protected amide was hydrogenated using the general procedures described above to produce Compound 13-1.
  • the compounds 13-2 through 13-4 listed in Table II may be synthesized using the same general procedures as described above using the appropriate corresponding aminoacid ester.
  • Examples 14-16 illustrate the synthesis of amide derivatives at residue 8.
  • R -H
  • R 2 -OH
  • R 3 -NH ( CH 2 ) 3 CH 3
  • Pseudomycin B (0.25 g, 0.171 mmol) was dissolved in 25 ml of DMF. To this solution was added Pybop (0.089g, 0.171 mmol)and 4- (2-Aminoethyl)morpholine (0.022 g, 0.171 mmol). The solution was stirred rapidly overnight under 1 atm N 2 . The solution was concentrated down, and the product was isolated by reverse-phase HPLC, and lyophilized to yield (140 mg, 0.089 mmol, 52%) alloc-protected Psuedomycin B Morpholine derivative.
  • the deprotection of the alloc groups was performed by adding Bu 3 SnH( 0.648 g, 2.23 mmol), and (Ph 3 P) 2 PdCl 2 (0.009g, 0.013 mmol) to a 1% acetic/ dichloromethane solution of alloc-protected Psuedomycin B Morpholine derivative (10 mg/mL) . Reaction time was 30 minutes. Reaction was monitored by HPLC. The solution was concentrated down, and the product was isolated by reverse phase HPLC prep, and lyophilized to yield 38 mg, 32% of Compound 16-1. MS data: Calculated for C57 H99 CI N14 019 Mol. Wt. 1318.7 Found ES+ 1320.0, ES- 1318.0
  • the in vi tro potency decreases in the following order of R 3 substitution: -NH 2 > -NHCH 3 > -NHCH 2 CH 3 > -NH(CH 2 ) 2 CH 3 > -NH(CH 2 ) 3 CH 3 ; -NHCH 2 CH 2 N (CH 3 ) 2 > -NH (CH 2 ) 3 N (CH 3 ) 2 ; and
  • the 3-amido derivatives demonstrated a similar trend as observed with the 8-amido derivatives in comparison with the parent natural product (e.g., amide substituents at R 2 having shorter alkyl chains were more active than longer alkyl chains) .

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Abstract

Cette invention concerne la modification acide d'unités d'acide aspartique et/ou d'acide hydro-aspartique d'un composé de pseudomycine naturel ou semi-synthétique ainsi que des méthodes de traitement antifongiques.
EP00942656A 1999-07-15 2000-06-08 Analogues amides et esters de pseudomycine Withdrawn EP1198473A1 (fr)

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HUP0202267A2 (en) 2002-10-28
JP2003505399A (ja) 2003-02-12
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