WO1998004584A1 - Decapeptides cycliques utilises comme antibiotiques - Google Patents

Decapeptides cycliques utilises comme antibiotiques Download PDF

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Publication number
WO1998004584A1
WO1998004584A1 PCT/CA1997/000529 CA9700529W WO9804584A1 WO 1998004584 A1 WO1998004584 A1 WO 1998004584A1 CA 9700529 W CA9700529 W CA 9700529W WO 9804584 A1 WO9804584 A1 WO 9804584A1
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WIPO (PCT)
Prior art keywords
phe
tyr
asn
asp
orn
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PCT/CA1997/000529
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English (en)
Inventor
Michael T. Kelly
Raymond J. Andersen
Jeff Gerard
Original Assignee
Seatek Marine Biotechnology, Inc.
University Of British Columbia
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Priority claimed from US08/751,300 external-priority patent/US5962407A/en
Application filed by Seatek Marine Biotechnology, Inc., University Of British Columbia filed Critical Seatek Marine Biotechnology, Inc.
Priority to US09/043,813 priority Critical patent/US6790829B1/en
Priority to AU36164/97A priority patent/AU3616497A/en
Publication of WO1998004584A1 publication Critical patent/WO1998004584A1/fr

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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
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/66—Gramicidins S, C; Tyrocidins A, B, C; Related peptides
    • 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 novel cyclic peptide compounds with potent activity against antibiotic-resistant pathogens.
  • Methicillin-resistant strains of Staphylococcus aureus cause infections that are refractory to standard anti-staphylococci antibiotics, and in many cases vancomycin is the antibiotic of last resort. Consequently, it is of great concern that vancomycin-resistant strains of MRSA may develop.
  • the invention is directed to isolated cyclic decapeptides of the formulas (1), (2) and (3):
  • the decapeptide of Formula (1) is referred to herein as Loloatin A.
  • the decapeptide of Formula (2) is referred to herein as Loloatin B.
  • the decapeptide of Formula (3) is referred to herein as Loloatin C.
  • the invention is directed to the above-identified cyclic decapeptides in an isolated, i.e., substantially purified form, preferably in a quantity of more than about 1 gram, more preferably in a quantity of more than about 10 grams, still more preferably in a quantity of more than about 100 grams, and yet still more preferably in a quantity of more than about 1 kilogram.
  • a substantially purified form is a composition wherein the above-listed cyclic decapeptides constitute at least about 1 weight percent of the composition, preferably at least about 10 weight percent, more preferably at least about 30 weight percent, still more preferably at least about 50 weight percent, yet still more preferably at least about 70 weight percent, and yet still more preferably at least about 95 weight percent, and most preferably at least about 99 weight percent.
  • the invention is also directed to the above-identified cyclic decapeptides in a pharmaceutical composition.
  • a pharmaceutical composition of the invention may not necessarily contain the cyclic decapeptide in a substantially purified form because the composition may contain diluent and/or other materials commonly found in pharmaceutical compositions.
  • the invention is also directed to a method of treating bacterial infection, comprising administering to a patient having a bacterial infection, an amount of the above-identified cyclic decapeptides effective to relieve symptoms associated with or due to the bacterial infection.
  • the invention is directed to various derivatives and analogs of Loloatin A, B and C.
  • Loloatin derivatives of the invention include the solvates, salts (either acid- or base- addition salts, depending on whether the amino acid sidechain being converted to a salt is basic or acidic, respectively), esters (derivatives of amino acid sidechains containing a carboxylic acid group), amines (derivatives of amino acid sidechains containing an amino group), ethers (derivatives of amino acid sidechains containing an hydroxyl group) and amides (derivatives of amino acid sidechains containing either an amine or carboxylic acid group) of Loloatin A, B or C.
  • the invention provides for a salt of a compound selected from the group: cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-
  • the salt may contain at least one negatively charged ion selected from chloride, bromide, sulfate, phosphate, C ⁇ - ⁇ 5 carboxylate, methanesulfonate and p- toluenesulfonate, which are exemplary only.
  • Exemplary Ci-iscarboxylates include acetate, glycolate, lactate, pyruvate, malonate, succinate, glutarate, fumarate, malate, tartarate, citrate, ascorbate, maleate, hydroxymaleate, benzoate, hydroxybenzoate, phenylacetate, cinnamate, salicylate and 2-phenoxybenzoate.
  • the salt may contain at least one positively charged ion selected from lithium, sodium, potassium, beryllium, magnesium, calcium and quaternary ammonium ions, which are exemplary positively charged ions.
  • exemplary quaternary ammonium ions cinlude tetraalkylammonium, and trialkylaralkylammonium ions.
  • the invention also provides for derivatives of a compound selected from cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L- Tyr]; cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L-Asn-L-Asp-L-
  • Trp Trp
  • the derivative may have the amine group of the ornithine sidechain being a secondary, tertiary or quaternary amine group, and other amino acid sidechains are optionally in a derivative form as well.
  • Exemplary ornithine sidechains have the formula -CH 2 -CH 2 -CH 2 -NHR 3 , -CH 2 -CH 2 -CH 2 -N(R 3 ) 2 or -CH 2 -CH 2 -CH 2 - N(R ) 3 wherein R 3 is an alkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities; or an acyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities.
  • the hydroxyl group of one or more tyrosine sidechains has been converted to an ether or ester group, and other amino acid sidechains are optionally in a derivative form as well.
  • at least one tyrosine sidechain may have the formula -Cf-k-C ⁇ Hi-O-R 2 , wherein C ⁇ R- is an aromatic ring and -O-R 2 is in the para position, and R 2 is a C 1 .1 5 alkyl group so as to form an ether, where the alkyl group may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities; or a C ⁇ -15 acyl group so as to form an ester, where the acyl group may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities.
  • the carboxyl group of the aspartic acid sidechain has been converted to an amide or ester group, and other amino acid sidechains are optionally in a derivative form as well.
  • the invention also provides for a pharmaceutical composition comprising a salt, derivative or analog of any of Loloatin A, Loloatin B and Loloatin C, as set forth above, in admixture with a pharmaceutically acceptable carrier.
  • the invention also provides for a method for the treatment of a patient afflicted with a bacterial infection comprising the administration to said patient of a therapeutically effective amount of a salt, derivative or analog of any of Loloatin A, Loloatin B and Loloatin C, as set forth above
  • the invention also provides for the use of a salt, derivative or analog of any of Loloatin A, Loloatin B and Loloatin C, as set forth above, in the manufacture of a medicament, where the medicament may be used to treat bacterial infection.
  • the Loloatin analogs include cyclic decapeptides having a "non-natural" stereochemistry at one or more of the ⁇ -carbons of the component amino acids, where the "natural" stereochemistry is as indicated by the L- or D- designations preceding the name of each of the amino acids in the formulas for Loloatin A, B and C set forth above.
  • Loloatin analogs are represented by the formulae: cyclo[Val-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr]; cyclo [ Val-Orn-Leu-Tyr-Pro-Phe-Phe- Asn- Asp-Trp] ; and cyclo [Val-Orn-Leu-Tyr-Pro-Trp-Phe- Asn- Asp-Trp] ,
  • the invention is directed to various analogs of the above- identified compounds, where preferred analogs have the formulas listed below.
  • preferred analogs have the formulas listed below.
  • no stereochemistry is designated because the analogs of the invention may have any possible stereochemistry at each atom capable of having more than one stereochemical arrangement of substituents.
  • preferred analogs have the stereochemistry L-, L-, L-, D-, L-, L-, D-, L-, L- and L-.
  • cyclo[Val-Orn-Leu-Tyr-Pro- Trp-Phe-Asn-Asp- ⁇ -Phenylserine] as written below preferably has the stereochemistry cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-T -D-Phe-L-Asn-L-Asp-L- ⁇ -Phenylserine].
  • Preferred analogs have one amino acid residue present in Loloatin A, B or C replaced with a different amino acid residue.
  • Preferred analogs are: cyclo[Butyrine-Orn-Leu-Tyr-Pro-Phe-Phe-Asn-Asp-Tyr] cyclo[Butyrine-Orn-Leu-Tyr-Pro-Phe-Phe-Asn- Asp-Trp]; cyclo[Butyrine-Orn-Leu-Tyr-Pro-Trp-Phe- Asn- Asp-Trp]; cyclo [Val-diaminobutyric aicd-Leu-Tyr-Pro-Phe-Phe- Asn- Asp-Tyr] ; cyclo[ Val-diaminobutyric aicd-Leu-Tyr-Pro-Phe-Phe-Asn- Asp-Trp] ; cyclo[ Val-dia
  • the present invention is also directed to derivatives of the above-listed Loloatin analogs (i.e., analog derivatives), including the solvates, salts (either acid- or base- addition salts, depending on whether the amino acid sidechain being converted to a salt is basic or acidic, respectively), esters (derivatives of amino acid sidechains containing a carboxylic acid group), amines (derivatives of amino acid sidechains containing an amino group), ethers (derivatives of amino acid sidechains containing an hydroxyl group) and amides (derivatives of amino acid sidechains containing either an amine or carboxylic acid group) of the Loloatin A, B and C analogs listed above.
  • analog derivatives including the solvates, salts (either acid- or base- addition salts, depending on whether the amino acid sidechain being converted to a salt is basic or acidic, respectively), esters (derivatives of amino acid sidechains containing a carboxylic acid group),
  • Loloatin A, B and C derivatives, analogs and analog derivatives are referred to herein as compounds of Formula (A), or compounds of the invention.
  • the invention is also directed to the compounds of the invention in an isolated, i.e., substantially purified form, preferably in a quantity of more than about 1 gram, more preferably in a quantity of more than about 10 grams, still more preferably in a quantity of more than about 100 grams, and yet still more preferably in a quantity of more than about 1 kilogram.
  • a substantially purified form is a composition wherein the above-listed compound of the invention constitutes at least about 1 weight percent of the composition, preferably at least about 10 weight percent, more preferably at least about 30 weight percent, still more preferably at least about 50 weight percent, yet still more preferably at least about 70 weight percent, and yet still more preferably at least about 95 weight percent, and most preferably at least about 99 weight percent.
  • the invention is also directed to pharmaceutical compositions comprising compounds of the invention.
  • the invention is also directed to a method of treating bacterial infection, comprising administering to a patient having a bacterial infection, an amount of a compound of the invention effective to relieve symptoms associated with or due to the bacterial infection.
  • the compounds of the present invention contain multiple asymmetric carbon atoms and thus exist as enantiomers and diastereomers. Unless otherwise noted, the present invention includes all enantiomeric and diastereomeric forms of the compounds. Pure stereoisomers, mixtures of enantiomers and/or diastereomers, and mixtures of different compounds of the invention are included within the scope of the present invention.
  • the compounds of the invention may be in the form of a solvate or a pharmaceutically acceptable salt, e.g., an acid- or base- addition salt.
  • Such salts may have at least one negatively charged ion such as chloride, bromide, sulfate, phosphate, C ⁇ -i5carboxylate, methanesulfonate and / oluenesulfonate, where exemplary C ⁇ - ⁇ 5 carboxylate ions are acetate, glycolate, lactate, pyruvate, malonate, succinate, glutarate, fumarate, alate, tartarate, citrate, ascorbate, maleate, hydroxymaleate, benzoate, hydroxybenzoate, phenylacetate, cinnamate, salicylate and 2- phenoxybenzoate
  • the salt may have at least one positively charged ion such as lithium, sodium, potassium, beryllium, magnesium, calcium and quaternary ammonium ions, where exemplary quatern
  • the invention is also directed to the above-identified cyclic decapeptides and derivatives thereof in a pharmaceutical composition.
  • a pharmaceutical composition of the invention may not necessarily contain the cyclic decapeptide or derivative thereof in a substantially purified form because the composition may contain diluent and/or other materials commonly found in pharmaceutical compositions, such that the above- identified cyclic decapeptides and derivatives thereof constitute less that 1 weight percent of the pharmaceutical composition.
  • the invention is also directed to a method of employing the compounds of the invention as an antibiotic.
  • the invention is directed to isolated cyclic decapeptides having one of formulas (1), (2) or (3) shown below, and are named Loloatin A, Loloatin B and Loloatin C, respectively.
  • Loloatin C Written in different terms, Loloatin A, B and C have the amino acid sequences set forth in Table A below, where the first listed amino acid is joined through a peptide bond to the last listed amino acid so as to form a cyclic structure. TABLE A
  • the invention is directed to cyclic compounds represented by the shorthand structure shown below, denoted Formula (A).
  • AA' through AA 10 are generic symbols, each representing an amino acid residue as defined herein, or a salt or derivative thereof.
  • Each line between neighboring (attached) AA'-AA 10 residues represents an amide (also known as a peptide) bond formed between neighboring AA'-AA 10 residues, as well as the isosteres thereof.
  • "Isostere” means a modified form of the normal peptide bond ( — C(O)NH — ) between attached amino acid residues, such as — CH 2 NH — (reduced), C(O)N(CH 3 ) (N-methylamide), — COCH 2 — (keto), — CH(OH)CH 2 — (hydroxy), — CH(NH 2 )CH 2 — (amino), — CH 2 CH 2 — (hydrocarbon), or — NHC(O)— (inverted normal peptide bond).
  • the compounds of the present invention are not in isosteric forms.
  • AA'-AA 10 represent residues from the following specific amino acids or other listed compounds, where stereochemical designations are preferred only, and the specifically named amino acid or other listed compound may be in either the L or D form AA 1 L-valine, butyrine;
  • AA 10 L-tryptophan, L-tyrosine, />-fluorophenylalanine, phenylalanine, thienylalanine, ⁇ -phenylserine.
  • the compounds of Formula (A) specifically include salts and other derivatives of the amino acids listed above.
  • amino acid derivative is intended to include the solvates, salts (either acid- or base- addition salts, depending on whether the amino acid sidechain is basic or acidic, respectively), esters (derivatives of amino acid sidechains containing a carboxylic acid group), amines (derivatives of amino acid sidechains containing an amino group), ethers (derivatives of amino acid sidechains containing an hydroxyl group) and amides (derivatives of amino acid sidechains containing either an amine or carboxylic acid group) of the unmodified cyclic compound
  • the compounds of the invention may be referred to as compounds of Formula (A)
  • Preferred compounds of the invention have one of formulas (4), (5) or (6) shown below
  • R represents a hydrogen atom; or an alkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities, or an aryl group
  • R 2 represents a hydrogen atom; or an alkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities, or an acyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities
  • R * represents a hydrogen atom; or an alkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities, or an acyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities
  • R is C ⁇ - 5 alkyl, which may be branched or linear, or C 3 -5cycloalkyl, R 5 is -(CH 2 ) n -N(R 15 ) 2 wherein n is 1, 2, 3 or 4 and R ]5 is independently hydrogen or Ci.isalkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities; or a Ci-isacyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities;
  • R 6 is C ⁇ - 7 alkyl, which may be branched or linear, or C 3 . 7 cycloalkyl;
  • R 7 is -(CH 2 ) m -C 6 H 4 -OR]6 wherein m is 1, 2 or 3 and Rie is hydrogen atom; or a C ⁇ - ⁇ 5 alkyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities; or a Ci- ⁇ acyl group that may be of straight chain, or where possible, of cyclic or branched structure and may contain one or more alkene, alkyne, or aromatic functionalities, and CeFL- is an aromatic ring, and -ORie is in a para relationship to the (CH 2 ) m group; R 8 and R 9 in combination form a 5, 6 or 7 membered ring, which may contain 1 or 2 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur, but otherwise contains
  • R 10 is -(CH 2 ) m -C 6 - ⁇ oaryl, or wherein the aryl and heteroaryl may be monocyclic or bicyclic, a heteroaryl contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur in the ring system, where R 10 specifically includes the sidechain from phenylalanine and tryptophan;
  • R" is -(CH 2 ) n -C6- ⁇ oaryl and -(CH 2 ) n -C 5 .oheteroaryl where a heteroaryl contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur in the ring system, and n is 1, 2, 3 or 4;
  • the compounds of the present invention may be prepared in vitro, using solid phase or solution peptide synthesis techniques, or may be prepared in vivo, from microorganism ATCC 55797 Solution phase techniques as set forth in K Okamato, K et al Bull. Chem. Soc. Jpn 50 231-236 (1977), Ohno, M et al. J. Am. Chem. Soc 88(2).376-377 and Kosui, N et al. Int. J. Peptide Protein Res. 18: 127-134 (1981) may be modified to prepare the cyclic decapeptides of the present invention, merely by appropriate substitution of the suitably protected amino acids.
  • Ion exchange techniques can be used to prepare the various salts of the invention, where such techniques are well known in the art
  • hydrochloric acid may be added to a neutral compounds of the invention to prepare the hydrochloride salt thereof
  • Dialysis techniques may also be employed to effect ion exchange and so obtain a desired salt of the invention from another salt of the invention
  • Loloatin A, B and C may be prepared simply by using the corresponding derivatized amino acid in the synthesis of the cyclic decapeptide
  • a Loloatin A derivative having tyrosine methyl ether at the AA 10 position is desired, such a cyclic decapeptide may be prepared by the techniques described below, by substituting tyrosine methyl ether for tyrosine
  • Other derivatives may be made analogously by techniques known in the art
  • Appropriate derivatived amino acid may be prepared by techniques known in the art, or they may be purchased from any of several chemical supply houses, for example, Sigma Chemical and Bachem as identified elsewhere herein
  • Loloatins and compounds of the invention have utility as antibiotics, and may be used and administered in a manner analogous to antibiotics known in the art, to provide the beneficial effects desired of antibiotics
  • the use is in the veterinary or, more preferably, the pharmaceutical field
  • the invention extends to the use of any compound of Formula (A or B) or Loloatin A, B or C for the manufacture of a medicament for use in therapy.
  • the invention further provides the use of any compound of Formula (A or B) or Loloatin A, B or C for the manufacture of a medicament for use in the treatment of methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and Streptococcus pneumomae infections in a mammal
  • the cyclic decapeptides of the invention may be used against gram negative and gram positive bacteria.
  • compositions may be in any form used for oral, topical, vaginal, parenteral, rectal and inhalatory application
  • a pharmaceutical or veterinary composition comprising also a pharmaceutically or veterinarily acceptable carrier, and optionally, one or more other biologically active ingredients
  • Such compositions may be in any form used for oral, topical, vaginal, parenteral, rectal and inhalatory application
  • the compositions may be provided in discrete dose units
  • the carriers may be particulate, with compositions being, for example, tablets or powders, or liquid, with the compositions being, for example, oral syrups or injectable liquids, or gaseous, for inhalatory application
  • an excipient and/or binder may be present.
  • examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose Coloring and/or flavoring agents may be present
  • a coating shell may be employed.
  • oleaginous bases may be employed, for example, lanolin or cocoa butter.
  • buffers, stabilizers and isotonic agents may be included
  • the optimal dosage of the compounds of Formula (A or B), or Loloatin A, B or C may depend on the weight and physical condition of the patient; on the severity and longevity of the illness; and on the particular form of the active ingredient, the manner of administration and the composition employed
  • a compound of Formula (A or B), or Loloatin A, B or C in chemotherapy can involve such a compound being bound to an agent, for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound to the site of infection.
  • an agent for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound to the site of infection.
  • the invention relates further to a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising an effective amount of compound of Formula (A or B), or Loloatin A, B or C in association with a carrier.
  • the present invention provides a method for the treatment of a patient afflicted with a bacterial infection comprising the administration thereto of a therapeutically effective amount of a compound of Formula (A or B), or Loloatin A, B or C.
  • a therapeutically effective amount refers to an amount which is effective, upon single or multiple dose administration to the patient, in providing relief of symptoms associated with bacterial infections.
  • "relief of symptoms" of a bacterial infection refers to a decrease in severity over that expected in the absence of treatment and does not necessarily indicate a total elimination or cure of the infection or condition caused thereby.
  • a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific infection involved; the degree of or involvement or the severity of the infection or condition arising therefrom; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
  • a therapeutically effective amount of a compound of Formula (A or B), or Loloatin A, B or C is expected to vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts are expected to vary from about 0.5 to about 10 mg/kg/day.
  • a compound of Formula (A or B), or Loloatin A, B or C can be administered in any form or mode which makes the compound bioavailable in effective amounts, including oral, aerosol, and parenteral routes.
  • compounds of Formula (A or B), or Loloatin A, B or C can be administered orally, by aerosolization, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, topically, and the like.
  • Oral or aerosol administration is generally preferred
  • One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected, the condition to be treated, the stage of the condition, and other relevant circumstances. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990).
  • the compounds can be administered alone or in the form of a pharmaceutical composition in combination with pharmaceutically acceptable carriers or excipients, the proportion and nature of which are determined by the solubility and chemical properties of the compound selected, the chosen route of administration, and standard pharmaceutical practice.
  • the present invention provides compositions comprising a compound of Formula (A or B), or Loloatin A, B or C, in admixture or otherwise in association with one or more inert carriers.
  • compositions are useful, for example, as assay standards, as convenient means of making bulk shipments, or as pharmaceutical compositions.
  • An assayable amount of a compound Formula (A or B), or Loloatin A, B or C is an amount which is readily measurable by standard assay procedures and techniques as are well known and appreciated by those skilled in the art.
  • Assayable amounts of a compound Formula (A or B), or Loloatin A, B or C will generally vary from about 0.001% to about 75% of the composition by weight.
  • Inert carriers can be any material which does not degrade or otherwise covalently react with a compound of Formula (A or B), or Loloatin A, B or C,.
  • suitable inert carriers are water; aqueous buffers, such as those which are generally useful in High Performance Liquid Chromatography (HPLC) analysis; organic solvents, such as acetonitrile, ethyl acetate, hexane and the like; and pharmaceutically acceptable carriers or excipients.
  • the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula (A or B), or Loloatin A, B or C, in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art.
  • the carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art.
  • the pharmaceutical composition may be adapted for oral, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like.
  • the compounds of the present invention may be administered orally, for example, with an inert diluent or with an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
  • These preparations should contain at least 4% of the compound of the invention, the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit.
  • the amount of the compound present in compositions is such that a suitable dosage will be obtained.
  • compositions and preparations according to the present invention are prepared so that an oral dosage unit form contains between 5.0-300 milligrams of a compound of the invention.
  • the tablets, pills, capsules, troches and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
  • binders such as microcrystalline cellulose, gum tragacanth or gelatin
  • excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like
  • lubricants such as magnesium
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
  • a liquid carrier such as polyethylene glycol or a fatty oil.
  • Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
  • tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
  • a syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • the compounds of the present invention may be incorporated into a solution or suspension These preparations should contain at least 0 1% of a compound of the invention, but may be varied to be between 0.1 and about 50% of the weight thereof.
  • the amount of the inventive compound present in such compositions is such that a suitable dosage will be obtained
  • Preferred compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 5.0 to 100 milligrams of the compound of the invention.
  • the compounds of Formula (A or B), or Loloatin A, B or C, of the present invention may also be administered by aerosol
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients. Aerosols of compounds of Formula (A or B), or Loloatin A, B or C, may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like Preferred aerosols are able to be determined by one skilled in the art
  • the compounds of Formula (A or B), or Loloatin A, B or C, of this invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers
  • Topical formulations may contain a concentration of the Formula (A or B), or Loloatin A, B or C, compound of from about 0.1 to about 10% w/v (weight per unit volume).
  • the solutions or suspensions may also include one or more of the following adjuvants sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, buffers such
  • the cyclic decapeptides of the invention may be combined with one or more known antibiotics to provide a synergistic composition.
  • a composition comprising a cyclic decapeptide of the invention and a known antibiotic may have greater efficacy against bacteria than would be expected based on the individual efficacies of the cyclic decapeptide and the known antibiotic.
  • the term "patient” refers to a warm-blooded animal such as a mammal which is afflicted with a particular inflammatory disease state. It is understood that guinea pigs, dogs, cats, rats, mice, horses, cattle, sheep, and humans are examples of animals within the scope of the meaning of the term.
  • Trp refers to tryptophan
  • Tyr refers to tyrosine
  • Val refers to valine
  • Z benzyloxycarbonyl
  • ⁇ g refers to micrograms
  • ⁇ L refers to microliters
  • ⁇ M' refers to micromolar.
  • the cyclic decapeptides of the present invention may be prepared in vitro, using solid phase or solution peptide synthesis techniques, or may be prepared in vivo, from microorganism ATCC 55797. Solution phase techniques as set forth in K
  • Chem. Soc 88(2):376-377 and Kosui, N. et al. Int. J. Peptide Protein Res 18.127-134 (1981) may be modified to prepare the cyclic decapeptides of the present invention, merely by appropriate substitution of the suitably protected amino acids.
  • the / nitrobenzophenone oxime polymer described by DeGrado and Kaiser may be used as a solid support in preparing cyclic decapeptides of the present invention. See DeGrado, W.F.; Kaiser, E.T., J. Org. Chem. 45: 1295-1300 (1980).
  • the peptide chain may be assembled by consecutive addition of the following N ⁇ -Boc-am ⁇ no acids, which have the L-configuration unless otherwise noted BocOrn(Z)OH, BocValOH, BocTyr(2,6-Cl 2 -Bzl)OH, BocAsp( ⁇ - Bzl)OH, BocAsnOH, Boc-D-PheOH, BocPheOH, BocProOH and BocTyr(2,6-Cl 2 - Bzl)OH, all according to the BOP peptide coupling procedure of Fournier, A , Wang, C T., Felix, A M., Int. J. Pept., Prot. Res., 31 86-97 (1988)
  • Boc protecting groups may be removed by treatment with 25% TFA/DCM solution for 30 minutes After the appropriate washing steps, Boc-amino acids and BOP reagent may be added in 5-fold excess in DMF solution followed by the same excess of DIEA After a 2-hour reaction time, the completeness of each coupling may be monitored by the Kaiser test See Kaiser, E , Colescott, R L , Cook, P I , Anal Biochem , 34 595-598 (1970) Coupling of BocAsnOH may be repeated with 2 5 molar equivalent reagent to insure a high yield of the product
  • the amino group may be liberated from its TFA salt by addition of DIEA (1 5 equivalents)
  • DIEA 1 5 equivalents
  • the free amino group may cleave the peptide from the polymer support by intrachain aminolysis in DCM at room temperature
  • the product may be obtained from the solution phase by filtration
  • Protecting groups of the peptide may be removed with TMSOTf in TFA in the presence of thioanisole, according to the procedure of Fujii, N et al , J Chem Soc, Chem.
  • Hydrolysis of the partly silylated product by NRtOH may be followed by gel permeation chromatography, for example, using Sephadex G-10 column (eluent, e.g.,. 2 M acetic acid in H 2 O/MeOH, 4/1 [v/v])
  • Final purification may be carried out by RP-HPLC on, for example, a Vydac Cig Proteins semi-preparative column eluted at, e.g , 4 mL/min with a linear gradient of 25%-80% acetonitrile in 0.1% (v/v) TFA over 45 minutes
  • N ⁇ Boc-amino acid starting materials may be purchased from chemical supply houses, for example, Sigma Chemical Company, PO Box 14508, St Louis, Missouri 63178 (Sigma's "Peptides and Amino Acids” catalog provides a convenient listing) and Bachem, 6868 Nancy Ridge Dr., San Diego, C A 92121.
  • Loloatin B follows the synthesis of Loloatin A as described in Example Al, with the exception that BocTyr(2,6-Cl 2 -Bzl)OH is replaced with BocTrp(Z)OH or other sidechain amine-protected L-tryptophan.
  • Loloatin C follows the synthesis of Loloatin B as described in Example A2, with the exception that BocPheOH is replaced with BocTrp(Z)OH or other sidechain amine-protected L-tryptophan.
  • EXAMPLE A4 Solid phase peptide synthesis according to the method originally described by Merrifield, J. Am Chem. Soc. 55:2149-2154, 1963, the disclosure of which is hereby incorporated by reference, may be used to prepare the linear analogs of the cyclic decapeptides of the invention. Alternatively, solution synthesis may be used to prepare these linear peptide analogs. Generally, peptides may be elongated by deprotecting the ⁇ -amine of the C-terminal residue and coupling the next suitably protected amino acid through a peptide linkage using the methods described.
  • This deprotection and coupling procedure is repeated until the desired sequence is obtained.
  • This coupling can be performed with the constituent amino acids in stepwise fashion, or by condensation of fragments (two to several amino acids), or combination of both processes, or by solid phase peptide synthesis as stated above. Thereafter, the linear peptides may be cyclized by well known peptide cyclization techniques, to prepare the cyclic decapeptides of the invention.
  • the C-terminal carboxylic acid is attached to an insoluble carrier (usually polystyrene).
  • insoluble carriers contain a group which will react with the ⁇ -carboxyl group to form a bond which is stable to the elongation conditions but readily cleaved later. Examples of which include, chloro- or bromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many of these resins are commercially available with the desired C-terminal amino acid already incorporated. Many of the suitably protected amino acids used in the present invention are also available commercially from Sigma Chemical Company and Bachem. Alternatively, compounds of the invention can be synthesized using automated peptide synthesizing equipment.
  • Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester ( -nitrophenyl ester, N-hydroxy-succinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-Cl, or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1 -hydroxybenzotriazole. These coupling reactions can be performed in either solution (liquid phase) or solid phase.
  • the functional groups of the constituent amino acids must be protected during the coupling reactions to avoid formation of undesired bonds.
  • the protecting groups that can be used are listed in Greene, "Protective Groups in Organic Chemistry,” John Wiley & Sons, New York (1981) and “The Peptides: Analysis, Synthesis, Biology,” Vol. 3, Academic Press, New York (1981), the disclosures of which are hereby incorporated by reference.
  • the ⁇ -carboxyl group of the C-terminal residue is usually protected by an ester that can be cleaved to give the carboxylic acid.
  • Protecting groups which can be used include (1) alkyl esters such as methyl and t-butyl, (2) aryl esters such as benzyl and substituted benzyl, or (3) esters which can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters
  • acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl
  • aromatic carbamate types such as benzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyls, l-(p-biphenyl)-l, -methylethoxy-carbonyl, and 9- fluorenylmethyloxycarbonyl (Fmoc)
  • aliphatic carbamate types such as tert- butyloxycarbonyl (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl
  • cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl
  • alkyl types such as tripheny
  • the ⁇ -amino protecting group is cleaved prior to the coupling of the next amino acid
  • the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HC1 in dioxane
  • the resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or dimethylformamide
  • the reagents of choice are piperidine or substituted piperidine in dimethylformamide, but any secondary amine or aqueous basic solutions can be used
  • the deprotection is carried out at a temperature between 0°C and room temperature Any of the amino acid bearing side chain functionalities must be protected during the preparation of the peptide using any of the above-described groups Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities depends upon the amino acid and presence of other protecting groups in the peptide.
  • protecting groups are important in that it must not be removed during the deprotection and coupling of the ⁇ -amino group.
  • Boc is used as the ⁇ -amino protecting group
  • p-toluenesulfonyl (tosyl) moieties can be used to protect the amino side chain of Orn.
  • Fmoc is chosen for the ⁇ -amine protection usually /er/-butyl based protecting groups are acceptable.
  • Boc can be used for ornithine.
  • the peptide is cleaved from the resin usually simultaneously with the protecting group removal.
  • Boc protection scheme is used in the synthesis, treatment with anhydrous HF containing additives such as dimethyl sulfide, anisole, thioanisole, or p-cresol at 0°C is the preferred method for cleaving the peptide from the resin.
  • the cleavage of the peptide can also be accomplished by other acidic reagents such as trifluoromethanesulfonic acid/trifluoroacetic acid mixtures.
  • the Fmoc protection scheme is used, the N-terminal Fmoc group is cleaved with reagents described earlier.
  • the other protecting groups and the peptide are cleaved from the resin using a solution of trifluoroacetic acid and various additives such as anisole, etc.
  • the linear peptide is cyclized using conventional procedures such as by treatment with triethylamine and diphenylphosphorylazide in dimethylformamide.
  • any remaining protecting and functional group precursors are removed or transformed into the desired group.
  • Example A4a Synthesis of cyclo[L-Val-L-Orn-L-Leu-D-Tyr-L-Pro-L-Phe-D-Phe-L- Asn-L-Asp-L-Tyr] (Loloatin A)
  • Loloatin A The linear analog of Loloatin A may be synthesized by standard solid phase methods using an Applied Biosystems (850 Lincoln Centre Dr., Foster City,
  • Boc-amino acids may be used with the following side chain protection Orn(Tos), Tyr(BrZ), Asp(Bzl)
  • Boc-Val- PAM resin (0 5 mmol, Applied Biosystems) may be deprotected with trifluoroacetic acid (2% anisole) and coupled in NMP with the HOBt esters of Boc-amino acids (4 equivalents).
  • the HOBt esters of Boc-amino acids may be formed by the reaction of the Boc-amino acid with DCC and HOBt Couplings may be carried out for 30 minutes and the resin subsequently washed with NMP and DCM Any unreacted amine may be acylated with acetic anhydride The deprotection and coupling may be repeated until complete assembly of the protected peptide resin is achieved
  • the linear peptide may be simultaneously deprotected and removed from the resin with anhydrous hydrogen fluoride (10 mL) at 0°C for 30 minutes in the presence of anisole (5%) The peptide may then be extracted with 50% acetic acid, water and aqueous acetonitrile, and lyophilized
  • the crude linear peptide prepared above may be dissolved in DMF ( ⁇ 5 ⁇ mol ml) and cyclized with DPPA (5 equivalents) TEA to adjust the pH to 9 After completion of the reaction (4-48 hours), the solvent may be removed and the crude cyclic peptide lyophilized from acetonitrile/water The cyclic peptide may then be desalted by gel filtration in, e.g , 70% acetic acid over a Spectragel GF05 column (2 5 x 55 cm), and purified by reversed phase preparative HPLC (Dynamax Cig, 21 4 x 250 mm, Rainin) using various gradients of 0 1% aqueous TFA and acetonitrile
  • the purified peptide may then be characterized by Analytical HPLC (Vydac 218TP54, 4 6 x 250 mm), FAB-MS and amino acid analysis, and these characterizations compared to theoretical values when available
  • Example A5 The procedure of Example A4a may be modified to prepare Loloatin B Thus, when Tyr(BrZ) is replaced with Trp(Z) or other amine-protected L-tryptophan, Loloatin B may be prepared.
  • Example A6 The procedure of Example A5 may be modified to prepare Loloatin C Thus, when Phe is replaced with Trp(Z) or other amine-protected L-tryptophan, Loloatin C may be prepared.
  • Loloatin A, B and C may be isolated after fermentation of ATCC 55797 as described below.
  • the marine bacterial isolate MK-PNG-276A tentatively identified as a Bacillus laterosporus by MIDI analysis of cellular fatty acids, was obtained from the tissues of an unidentified tube worm collected at -15 m off of Loloata Island, Papua, New Guinea. MK-PNG-276A has been deposited with the American Type Culture Collection as ATCC 55797.
  • MK-PNG-276A was cultured on trays of solid tryptic soy agar supplemental with NaCl to a concentration of 1%. Twenty-six 400 mL trays (9" x 15" x 1/4" deep agar) were cultured for five days after which the combined cells and agar were lyophilized. The lyophilization product, (61.5 g dry weight) was extracted with three 600 mL portions of methanol that were combined, filtered, and reduced in vacuo to give a brown/gray tar. The tar was dissolved in 750 mL of MeOH/H 2 O (1/4) and sequentially extracted with hexanes (3 x 250 mL) and EtOAc (3 x 250 mL).
  • Loloatin B (2) gave a (M+H) ion in the HRFABMS at m/z 1296.64232 appropriate for a molecular formula of C6 7 H 8 5 ⁇ 3 Oi 4 ( ⁇ M+0.46 ppm).
  • Detailed analysis of the 'H, 13 C, COSY, HOHAHA, HMQC, HMBC and ROESY data for Loloatin B (2) identified the ten amino acids residues indicated in Table 1.
  • the amino acid sequence in (2) was determined by analysis of HMBC and ROESY data HRFABMS and MS/MS studies supported the amino acid sequence derived from the NMR data.
  • the MS/MS data was consistent with initial cleavage of the ring at the TYR-CO/PRO-N bond to give a linear decapeptide that sequentially loses LEU-TYR (m/z 1019), ORN-LEU-TYR (m/z 905) and TRP-VAL-ORN-LEU-TYR (m/z 621).
  • FABMS peaks at m/z 245 and 377 could be assigned to the protonated fragments PRO-PHE1 and PHE2-ASN-ASP, respectively.
  • Fermentation techniques with the microoganism ATCC 55797 may be used to obtain the cyclic decapeptides of Formulas 1, 2 and 3 as described in Example
  • the cyclic decapeptides of Formulas 1, 2 and 3 may be derivatized to form salts (either acid- or base- addition salts, depending on whether the amino acid sidechain is basic or acidic, respectively), esters (from amino acid sidechains containing a carboxylic acid group), amines (from amino acid sidechains containing an amino group), ethers (from amino acid sidechains containing an hydroxyl group) and amides (from amino acid sidechains containing either an amine or carboxylic acid group) of the invention.
  • salts either acid- or base- addition salts, depending on whether the amino acid sidechain is basic or acidic, respectively
  • esters from amino acid sidechains containing a carboxylic acid group
  • amines from amino acid sidechains containing an amino group
  • ethers from amino acid sidechains containing an hydroxyl group
  • amides from amino acid sidechains containing either an amine or carboxylic acid group
  • N-acetyl loloatin B methyl ester (276bs2) Loloatin B (276bsl) (100 mg) was acetylated under argon, at 23 °C for 16 hours, using 2 mL of acetic anhydride and 1 mL anhydrous pyridine (freshly distilled).
  • Loloatin B (2) gave a (M+H) ion in the HRFABMS at m/z 1296 64232 appropriate for a molecular formula of C 67 H8 5 N ⁇ 3 Oi 4 ( ⁇ M+0 46 ppm)
  • L-ornithine L-leucine, D-tyrosine, L-proline, L-phenylalanine, D-phenylalanine,
  • ROESY correlations observed in (7) between amino acid ⁇ -methine protons and adjacent residue NH protons unambiguously identified the following five amino bonds: ORN-CO/LEU-NH ( ⁇ 5.36/8.22), ASP-CO/TRP-NH ( ⁇ 4.40/8.72), ASN-CO/ASP-NH ( ⁇ 4.55/8.36), PHE2-CO/ASN-NH ( ⁇ 5.73/9.13) and PRO-CO/PHE1-NH ( ⁇ 4.18/7.39).
  • the latter ROESY correlation suggests the possibility of a ⁇ -bulge in the TRP/VAL region of the cyclic peptide as shown in Figure 1. See, e.g., Kuo, M. et al., J. Am. Chem. Soc. 102:520-24, 1980; Eggleston, D.S.
  • HMBC correlations from both the VAL ⁇ -methine proton resonance at ⁇ 2.12 and the ORN-NH resonance at ⁇ 8.81 to the well resolved carbonyl resonance at ⁇ 169.6 in derivative 7 identified the VAL-CO/ORN-NH amide bond
  • HMBC correlations from both the LEU ⁇ methylene proton resonance at ⁇ 1.25 and the TYR-NH resonance at ⁇ 9.21 to the carbonyl resonance at ⁇ 171.8 in 2 identified the LEU-CO/TYR-NH amide bond.
  • the final PHE 1-CO/PHE2-NH amide bond was required to complete the macrocyclic ring.
  • EXAMPLE A9 Precursor directed biosynthesis, wherein a culture media containing the microorganism isolated in Example A7 is supplemented with a replacement amino acid at fairly high concentrations, may be used to prepare cyclic decapeptides of the invention See, e.g., Katz, E. and Demain, A L , "The Peptide Antibiotics of Bacillus Chemistry Biogenesis, and Possible Functions," Bacteriological Reviews, June 1977, pp 449-474
  • Example A7 The culture media described in Example A7 can be employed in precursor directed biosynthesis to prepare the analogs of Loloatin A, B and C by providing to the microorganism ATCC 55797 fairly high concentrations of the following amino acids
  • Butyrine may replace valine
  • L-diaminobutyric acid may replace ornithine
  • Any of L-isoleucine, L-alloisoleucine, L-norvaline, L-cyclopropylalanine and norleucine may replace leucine
  • Any of / fluorophenyalanine, tryptophan and thienylalanine may replace tyrosine
  • Any of azetidine-2-carboxylic acid, pipecolic acid, trans-3- methylproline and tr ⁇ «5-4-fluoroproline may replace proline.
  • Any of tyrosine, p- fluorophenyalanine, tryptophan, thienylalanine and ⁇ -phenylse ⁇ ne may replace phenylalanine. Any of tyrosine, / fluorophenyalanine, phenylalanine, thienylalanine and ⁇ -phenylserine may replace tryptophan
  • Linear precursors were prepared, and then those precursors were cyclized to provide Loloatin B.
  • the structure of the linear precursor is selected in order that the geometry of the linear sequence will bring together the N- and C- termini, thus favoring the cyclization reaction
  • the linear precursor was prepared by standard solid phase peptide synthesis (i.e., typical coupling and cleavage conditions), using a standard peptide acid resin (Wang), to provide
  • the complex reaction mixture from Method C was chromatographed on reverse phase HPLC, and a microtiter plate assay was run throughout the entire chromatogram to identify active materials. Only one fraction, fr 73, (1 mg yield of starting 100 mg) exhibited the desired biological activity.
  • Fraction 73 was identified as Loloatin B by mass spectroscopy and NMR spectroscopy. Fraction 73 did not have a free N-terminal amino group.
  • Loloatin B was also prepared from the following precursor, which was prepared by standard solid-phase synthesis techniques: dF NDWVOL dY PF-COOH
  • Loloatin B was found to be selectively antimicrobial, with the minimum inhibitory concentrations listed in Table 2 below Antimicrobial activity was determined by macrobroth dilution antimicrobial susceptibility testing A solution of Loloatin B was prepared in tryptic soy broth Initially, a 100 ⁇ g/ml solution of the peptide was tested If inhibition of a target microorganism was detected, serial two-fold dilutions (in broth) of the Loloatin B solutions were tested to determine the minimal inhibitory concentration (MIC) of Loloatin B for each target organism Target organisms tested are identified in Table 2 Turbidity standardized suspensions of each organism were prepared according to accepted protocols using a 0 5 McFarland turbidity standard, and these standardized suspensions were used to inoculate a tube containing Loloatin B Activity of Loloatin B was indicated by lack of
  • Pseudomonas aeruginosa 100 ⁇ g/mL
  • Enterobacter cloacae > 100 ⁇ g/mL
  • Xanthomonas maltophilia > 100 ⁇ g/mL
  • EXAMPLE B2 Antimicrobial activity was determined by macrobroth dilution antimicrobial susceptibility testing Solutions of the cyclic decapeptides (Loloatin A, B and C) and a control (vancomycin) were prepared in tryptic soy broth. Initially, a 100 ⁇ g/ml solution of each compound was tested.
  • MIC minimal inhibitory concentration
  • Target organisms tested are listed below in Table 3, and included methicillin resistant Staphylococcus aureus, vancomycin resistant Enterococcus sp., Escherichia coli, multiply drug resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Candida albicans.
  • Turbidity standardized suspensions of each organism were prepared according to accepted protocols using a 0.5 McFarland turbidity standard, and these standardized suspensions were used to inoculate the tubes containing the compounds Activity of a compound was indicated by lack of growth (turbidity) of one or more of the target organisms. See Woods, G.L. and Washington, J.A , "Antibacterial susceptibility tests: dilution and disk diffusion methods, Manual of Clinical Microbiology (6 th Ed.), Murray, Baron, Pfaller, Tenover and Yolken (Eds.), ASM Press, Washington DC, 1 95, pp. 1327-1341. Other compounds of the invention may be screened and evaluated for antibiotic activity in the same manner.
  • Stenotrophomonas maltophilia >100 >100 >100

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Abstract

La présente invention porte sur diverses décapeptides cycliques ayant une activité antibiotique. Les décapeptides sont efficaces aussi bien contre les bactéries gram-positives que contre les bactéries gram-négatives.
PCT/CA1997/000529 1996-07-26 1997-07-25 Decapeptides cycliques utilises comme antibiotiques WO1998004584A1 (fr)

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WO2001068675A2 (fr) * 2000-03-17 2001-09-20 Seatek Marine Biotechnology Inc. Traitement de l'acne rosacee
US6784283B2 (en) * 2000-02-25 2004-08-31 The University Of British Columbia Peptide antibiotics
DE102005049537A1 (de) * 2005-10-17 2007-04-26 Forschungszentrum Karlsruhe Gmbh Zyklische Peptide und Verfahren zu deren Herstellung
EP2067867A1 (fr) 2007-12-03 2009-06-10 Siemens Aktiengesellschaft Procédé de concentration de molécules d'acide nucléique
CN101906149A (zh) * 2010-07-13 2010-12-08 暨南大学 一种线性多肽及其合成方法和应用
CN109354604A (zh) * 2018-09-04 2019-02-19 广州药本君安医药科技股份有限公司 一种线性假多肽及其制备方法与用途

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US4874850A (en) * 1986-08-07 1989-10-17 Medice Chem.-Pharm. Fabrik Putter Gmbh & Co. Pharmaceutical preparations

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6784283B2 (en) * 2000-02-25 2004-08-31 The University Of British Columbia Peptide antibiotics
WO2001068675A2 (fr) * 2000-03-17 2001-09-20 Seatek Marine Biotechnology Inc. Traitement de l'acne rosacee
WO2001068675A3 (fr) * 2000-03-17 2001-12-13 Seatek Marine Biotechnology In Traitement de l'acne rosacee
US7153826B2 (en) 2000-03-17 2006-12-26 Seatek Marine Biotechnology, Inc. Treatment of rosacea
DE102005049537A1 (de) * 2005-10-17 2007-04-26 Forschungszentrum Karlsruhe Gmbh Zyklische Peptide und Verfahren zu deren Herstellung
DE102005049537B4 (de) * 2005-10-17 2010-04-22 Forschungszentrum Karlsruhe Gmbh Verfahren zur Herstellung zyklischer Peptide und Verwendung der derart hergestellten zyklischen Peptide
EP2067867A1 (fr) 2007-12-03 2009-06-10 Siemens Aktiengesellschaft Procédé de concentration de molécules d'acide nucléique
WO2009071404A1 (fr) 2007-12-03 2009-06-11 Siemens Aktiengesellschaft Procédé de concentration de molécules d'acide nucléique
US8975017B2 (en) 2007-12-03 2015-03-10 Boehringer Ingelheim Vetmedica Gmbh Process for concentrating nucleic acid molecules
CN101906149A (zh) * 2010-07-13 2010-12-08 暨南大学 一种线性多肽及其合成方法和应用
CN101906149B (zh) * 2010-07-13 2012-10-17 暨南大学 一种线性多肽及其合成方法和应用
CN109354604A (zh) * 2018-09-04 2019-02-19 广州药本君安医药科技股份有限公司 一种线性假多肽及其制备方法与用途

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