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WO1997008334A1 - Cryptophycines obtenues a partir d'une biosynthese aberrante - Google Patents

Cryptophycines obtenues a partir d'une biosynthese aberrante Download PDF

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Publication number
WO1997008334A1
WO1997008334A1 PCT/US1996/014670 US9614670W WO9708334A1 WO 1997008334 A1 WO1997008334 A1 WO 1997008334A1 US 9614670 W US9614670 W US 9614670W WO 9708334 A1 WO9708334 A1 WO 9708334A1
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Prior art keywords
cryptophycin
substimted
amino acid
unit
cryptophycin compound
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Richard E. Moore
Thomas K. Hemscheidt
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University of Hawaii at Manoa
Wayne State University
University of Hawaii at Hilo
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University of Hawaii at Manoa
Wayne State University
University of Hawaii at Hilo
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Priority to JP9510664A priority Critical patent/JP2000507805A/ja
Priority to AU71090/96A priority patent/AU709828B2/en
Priority to EP96932217A priority patent/EP0850316A4/fr
Publication of WO1997008334A1 publication Critical patent/WO1997008334A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates generally to the production of cryptophycin compounds, and, more particularly, to the production of such compounds by the use of bacterial fermentation.
  • the cryptophycins are a group of structurally related compounds originally isolated from cyanobacteria (blue-green algae). This family of compounds has been found to exhibit a broad spectrum of antineoplastic activity similar to presently-used antineoplastic agents, such as vinblastine, taxol, and adriamycin.
  • Cryptophycin 1 (also termed Cryptophycin A) is the major cytotoxin produced by certain Nostoc species of cyanobacteria. It has shown excellent activity against drug-sensitive and drug- resistant solid tumors.
  • This cyclic depsipeptide consists of four units; two hydroxy acid units and two amino acid units. The stoichiometry of the two hydroxy acid units is as follows: (5S, 6S, 7R, SR)-7,8-epoxy-5-hydroxy-6-methyl-8-phenyl- 2-octenoic acid (Unit A) and (25)-2-hydroxy-4-methylvaleric acid (Unit D; leucic acid).
  • the stoichiometry of the two amino acid units is as follows: (2/?)-3-(3-chloro-4-methoxyphenyl)alanine (Unit B) and (2 -3-amino- 2-methylpropionic acid (Unit C).
  • the units are connected in an ABCD sequence.
  • other members of the cryptophycin group have been produced by chemically modifying the isolates.
  • the present invention provides a method for producing cryptophycin compounds as metabolites by the controlled use of metabolic substrates in bacterial fermentation.
  • the present invention comprises culturing bacteria capable of producing cryptophycin compounds in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.
  • novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.
  • such cryptophycin compounds will have a stable macrolide and possess substituent groups which have been shown to provide beneficial activities.
  • Figure 1 provides a general structure of selected cryptophycin compounds of the present invention and the four different acid units, two hydroxy acid groups (A
  • Figure 1 also provides a numbering system for the hydroxy acid units A and D and two amino acid units B and C in selected embodiments; and
  • Figure 2 provides a schematic representation of the fragment ionic species obtained when selected cryptophycin compounds are subjected to electron impact mass spectrometric analysis.
  • the present invention provides a method for producing cryptophycin compounds as metabolites by the controlled use of metabolic substrates in bacterial fermentation.
  • the present invention comprises culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.
  • the present invention thus provides a means for overcoming the problem of producing only a limited number of cryptophycins from native bacterial fermentation.
  • pre-determined cryptophycins produced by fermentation of a bacteria capable of producing a cryptophycin compound.
  • pre-determined cryptophycin compounds will have a stable macrolide and possess substituent groups which have been shown to provide beneficial activities.
  • novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substimted amino acid under conditions sufficient to produce the desired cryptophycin compound.
  • Cryptophycin 21 differs from Cryptophycin 1 only in that it does not contain the methyl group in Unit C. Although Cryptophycin 1 and Cryptophycin 21 show essentially the same cytotoxicity in vitro, Cryptophycin 21 was found to be inactive in vivo. This suggested that the C-D ester bond of the macrolide was being broken prior to the compound reaching the tumor site.
  • desirable feamres of cryptophycin compounds can be engineered into the metabolites produced in bacterial fermentation by controlled selection of metabolic substrates.
  • Cryptophycins are a group of structurally related compounds originally isolated from cyanobacteria (blue-green algae).
  • Cryptophycin 1 also termed Cryptophycin A
  • Cryptophycin A is the major cytotoxin produced by certain Nostoc species of cyanobacteria.
  • a limited number of other cryptophycin compounds can be obtained by native bacterial fermentation.
  • the present invention provides that a bacteria capable of producing a cryptophycin compound, for example a Nostoc sp., may be cultured under appropriate conditions and that novel cryptophycin metabolites, as well as previously disclosed cryptophycin metabolites, may be isolated from this culture.
  • a bacteria capable of producing a cryptophycin compound for example a Nostoc sp.
  • novel cryptophycin metabolites as well as previously disclosed cryptophycin metabolites, may be isolated from this culture.
  • GSV 224 is the strain which is cultivated and from which are isolated both previously known and novel cryptophycins.
  • the method of the present invention is directed to any strain of the Nostoc sp. and preferably to the Nostoc sp. GSV 224 strain to produce non- naturally occurring cryptophycin compounds.
  • the GSV 224 strain of Nostoc sp. was deposited on October 7, 1993 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. under ATCC Accession No. 55483.
  • Other strains of Nostoc sp. in particular strain MB 5357 previously deposited by Merck and Co. under ATCC Accession No. 53789, are strains contemplated to be utilized to practice the present invention.
  • Nostoc sp. the characteristics of Nostoc sp. are subject to variation.
  • recombinants, variants, or mutants of the specified strains may be obtained by treatment with various known physical and chemical mutagens, such as ultraviolet ray, X-rays, gamma rays, and N-memyl- N'-nitro-N-nitrosoguanidine. All natural and induced variants, mutants, and recombinants of the specified strains which retain the characteristic of producing a cryptophycin compound are intended to be within the scope of the claimed invention.
  • the cryptophycin compounds of the present invention can be prepared by culturing a strain of Nostoc sp. under submerged aerobic conditions in a suitable culture medium until substantial antibiotic activity is produced. Other culture techniques, such as surface growth on solidified media, can also be used to produce these compounds.
  • the culture medium used to grow the specified strains can include any of one of many nitrogen and carbon sources and inorganic salts that are known to those of ordinary skill in the art. Economy in production, optimal yields, and ease of product isolation are factors to consider when choosing the carbon and nitrogen sources to be used.
  • the customary soluble salts capable of yielding iron, potassium, sodium, magnesium, calcium, ammonium, chloride, carbonate, phosphate, sulfate, nitrate, and like ions.
  • Essential trace elements which are necessary for the growth and development of the organisms should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organisms. It may be desirable to add small amounts (i.e. 0.2mL/L) of an antifoam agent such as polypropylene glycol (M.W. about 2000) to large scale cultivation media if foaming becomes a problem.
  • a vegetative inoculum For production of substantial quantities of the cryptophycin compounds, submerged aerobic cultivation in tanks can be used. Small quantities may be obtained by shake-flask culmre. Because of the time lag in metabolite production commonly associated with inoculation of large tanks with the organisms, it is preferable to use a vegetative inoculum.
  • the vegetative inoculum is prepared by inoculating a small volume of culture medium with fragments of the vegetative trichome or heterocyst-containing form of the organism to obtain a fresh, actively growing culmre of the organism. The vegetative inoculum is then transferred to a larger tank.
  • the medium used for the vegetative inoculum can be the same as that used for larger cultivations or fermentation, but other media can also be used.
  • the bacterial organisms may be grown at temperatures between about 20 °C and 30 °C and at an incident illumination intensity of from about 100 to about 200 ⁇ mol photons m 2 Sec "* (photosynthetically active radiation).
  • carbon dioxide gas is introduced into the culmre by addition to the sterile air stream bubbled through the culmre medium.
  • the proportion of carbon dioxide should be about 1% (at 24°C and one atmosphere of pressure).
  • Cryptophycin compound production can be followed during the cultivation by testing samples of the broth against organisms known to be sensitive to these antibiotics.
  • One useful assay organism is Candida albicans.
  • cryptophycin compounds of the invention can be recovered from the culmre and from the culmre media by methods known to those of ordinary skill in this art. Recovery is generally accomplished by initially filtering the culmre medium to separate the algal cells and then freeze-drying the separated cells. The freeze-dried alga can be extracted with a suitable solvent such as a mixmre of acetonitrile and dichloromethane. The cryptophycins can be separated by subjecting this extract, as well as the culmre media, to rapid chromatography on reversed-phase column. The cryptophycins can be purified by reversed-phase high-performance liquid chromatography (HPLC).
  • HPLC reversed-phase high-performance liquid chromatography
  • novel cryptophycin compounds of the present invention and the previously disclosed cryptophycin compounds can be therapeutically employed as anti-neoplastic agents and thereby used in methods to treat neoplastic diseases.
  • Five cryptophycin compounds, designated Cryptophycins 1, 3, 5, 13 and 15, were disclosed in U.S. Patent Nos. 4,946,835, 4,845,085, 4,845,086, and 4,868,208, such compounds either having been isolated from a strain of Nostoc sp. designated MB 5357 or having been synthesized from such an isolated compound.
  • the present invention provides methods of producing these compounds via aberrant biosynthesis. Additional Cryptophycins, not to mention the disclosure of their use as anti ⁇ neoplastic agents, are disclosed in U.S. Application Serial No.
  • L-phenylalanine is the precursor of the phenyl and epoxide carbons of Unit A, a polyketide assembled from phenylacetate and three equivalents of acetate.
  • L-Tyrosine is the precursor of Unit B with the O-methyl group arising from the methyl group of S-adenosyl-L-methionine.
  • L-Tyrosine incorporated well into Unit B
  • L-O-methyltyrosine is also incorporated well into Unit B, indicating that O-methylation of tyrosine occurs first and this is followed by assimilation of O-methyltyrosine or the chlorinated O-methyltyrosine into the depsipeptide.
  • Cryptophycin 1 (25 * , 5R)-3-methylaspartic acid is the precursor of Unit C.
  • (2R)-2-Methyl- ⁇ -alanine is also incorporated well into Unit C, suggesting that (2S, 5R)-3-methylaspartic acid is decarboxylated into (2R)-2-methyl- ⁇ -alanine first and this is followed by assimilation of the (2i?)-2-methyl- ⁇ -alanine into the depsipeptide.
  • L-leucine is the precursor of Unit D and is inco ⁇ orated well into this unit.
  • L-Leucic acid is not incorporated into Unit D, suggesting that L-leucine is taken up into the depsipeptide synthase (the multifunctional enzyme that carries out the assembly of the depsipeptide) and the nitrogen in L-leucine is lost and replaced by an oxygen during the assembly of the Unit A, B, C, and D precursors into the depsipeptide.
  • desirable cryptophycin compounds which have the properties noted previously, e.g., an intact macrolide, an epoxide group, a chloro group in Unit B and/or a methyl group in Unit C, selected substimted amino acids can be employed as metabolic substrates for fermentation cultures of bacteria capable of producing cryptophycin compounds.
  • a substimted amino acid is considered to be any amino acid other than the protein amino acids which ordinarily form the basis for bacterial fermentation cultures. More usually, such substimted amino acids will include amino acids which provide the substiment groups in the appropriate positions to form a desirable cryptophycin compound as a metabolite of a bacterial fermentation culmre.
  • Such amino acids include, for example, substimted ⁇ -amino acids, substituted phenylalanines, substimted tyrosines, substimted O-methyltyrosines and substimted ⁇ -alanines.
  • substiment groups will be chosen so as to provide the desired strucmral feamres in the cryptophycin metabolites.
  • halogens attached to aryl groups have been shown to be desirable feamres in Unit B of the cryptophycin compounds
  • amino acids substimted with at least one halogen moiety will prove to be of use.
  • alanine or phenylalanine will be the amino acids of choice in this regard, typically substimted with a halo-substituted aryl compound.
  • a methyl group in Unit C can be included as a feamre of an appropriately substimted ⁇ -alanine.
  • the amino acid of choice is substimted with at least one substiment selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo-substituted derivatives thereof.
  • the present invention provides methods of producing previously known cryptophycins and new cryptophycins through the culturing of a strain of the Nostoc sp. and introducing into the culmre one or more of the following pre-selected compounds: a substimted phenylalanine, a substimted ⁇ -alanine, substimted tyrosine or O-methyltyrosine and a substimted ⁇ -amino acid.
  • the present invention provides a method for producing previously disclosed Cryptophycin-52, Cryptophycin- 110 and Cryptophycin-115 by aberrant biosynthesis, as well as the novel cryptophycin compounds described hereafter.
  • Cryptophycin-52 is produced, along with other cryptophycins, when the cyanobacterium is grown in the presence of 2 , 2-dimethyl- ⁇ -alanine :
  • Cryptophycin-110 and Cryptophycin-115 are produced when the cyanobacterium is grown in the presence of DL-p-fluorophenylalanine:
  • the present method can be used to produce novel cryptophycins that differ in the aryl group of Unit A of Figure 1 by growing the cyanobacterium in the presence of the appropriately substimted phenylalanine.
  • the present method can be used to produce novel cryptophycins that differ (1) in Unit B by growing the cyanobacterium in the presence of the appropriately substimted tyrosine or
  • O-methyltyrosine (2) in Unit C by growing the cyanobacterium in the presence of the appropriate substimted ⁇ -alanine, and (3) in Unit D by growing the cyanobacterium in the presence of the appropriately substimted ⁇ -amino acid.
  • Cryptophycin- 189 also called Cryptophycin B-8 which is produced by growing the cyanobacterium in the presence of DL-3-(3-methyl-4-methoxyphenyl)alanine:
  • Cryptophycin- 190 also called Cryptophycin B-l
  • Cryptophycin-210 which are produced by growing the cyanobacterium in the presence of DL-3-(3-fluoro-4-methoxyphenyl)alanine:
  • Cryptophycin B-2 Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-2 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(3-bromo-4-methoxyphenyl) alanine or (2R)-3-(3-bromo- 4-hydroxyphenyl)alanine :
  • Cryptophycin B-3 Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-3 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(3,4-dimethoxyphenyl)alanine or (2R)-3-(3-hydroxy- 4-methoxyphenyl)alanine, or (2R)-3-(3,4-dihydroxyphenyl)alanine:
  • Cryptophycin-208 and Cryptophycin-209 also called Cryptophycin B- 4 which are produced by growing the cyanobacterium in the presence of DL-3-(3,4-methylenedioxyphenyl)alanine:
  • Cryptophycin-211 which is produced by growing the cyanobacterium in the presence of DL-3-(3-fluoro-4-hydroxyphenyl)alanine:
  • Cryptophycin B-5 Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-5 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-methoxy-2-pyridyl)alanine or (2R)-3-(4-hydroxy- 2-pyridyl)alanine:
  • Cryptophycin B-6 Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-6 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-methoxy-3-pyridyl)alanine:
  • Cryptophycin B-7 Another example of a novel cryptophycin compound of the present invention is Cryptophycin B-7 which would be produced by growing the cyanobacterium in the presence of (2R)-3-(4-ethoxyphenyl)alanine:
  • Cryptophycin-213 which is produced by growing the cyanobacterium in the presence of (35)-3-aminobutanoic acid:
  • Cryptophycin C-1 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-1 which would be produced by growing the cyanobacterium in the presence of (2R)-2-ethyl- ⁇ -alanine:
  • Cryptophycin C-2 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-2 which would be produced by growing the cyanobacterium in the presence of (2R)-2-isopropyl- ⁇ -alanine:
  • Cryptophycin C-3 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-3 which would be produced by growing the cyanobacterium in the presence of (2R)-2-t-butyl- ⁇ -alanine:
  • Cryptophycin C-4 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-4 which would be produced by growing the cyanobacterium in the presence of (2R)-2-dimethylamino- ⁇ -alanine:
  • Cryptophycin C-5 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-5 which would be produced by growing the cyanobacterium in the presence of (2R)-2-dimethylaminomethyl- ⁇ -alanine:
  • Cryptophycin C-6 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-6 which would be produced by growing the cyanobacterium in the presence of 2, 2-diethyl- ⁇ -alanine:
  • Cryptophycin C-7 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-7 which would be produced by growing the cyanobacterium in the presence of 2, 2-di(methoxy Imethy 1)- ⁇ -alanine :
  • Cryptophycin C-8 Another example of a novel cryptophycin compound of the present invention is Cryptophycin C-8 which would be produced by growing the cyanobacterium in the presence of 1-aminomethylcyclopropane-l -carboxylic acid:
  • Cryptophycin-214 and Cryptophycin-215 which are produced by growing the cyanobacterium in the presence of (2 ⁇ S)-2-aminobutyric acid:
  • Cryptophycin D-1 Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-1 which would be produced by growing the cyanobacterium in the presence of 3-cyclopropyl- ⁇ -alanine:
  • Cryptophycin D-2 Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-2 which would be produced by growing the cyanobacterium in the presence of 3-t-butyl- ⁇ -alanine:
  • Cryptophycin D-3 Another example of a novel cryptophycin compound of the present invention is Cryptophycin D-3 which would be produced by growing the cyanobacterium in the presence of 3-vinyl- ⁇ -alanine:
  • Example 1 General Procedure for Feeding Substimted Amino Acids
  • the selected amino acid is dissolved in 0.5N HCl to a concentration in the range of 15-25mg/mL.
  • a 0.5mL portion of the solution is added to each of 2-4 carbouys of the bacterial culmre in two-day intervals beginning on day 7-10 after bacterial innoculation.
  • the culmres are allowed to grow for an additional 3-5 days, and then harvested.
  • Mass spectra, including high resolution mass measurements are determined in the electron-impact mode on a VG-70SE Instrument (source ??).
  • Cryptophycin-1, -2, -3, and -4 were found to give characteristic fragmentation patterns as shown in Figure 2.
  • Ion c is useful for identifying analogs that differ in the aryl group found in Unit A.
  • Comparison of ions a, b and d is useful for identifying analogs that differ in Unit B, Unit C and/or Unit D.
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (500mL), CH 3 OH (600mL), and CH 2 C1 2 (500mL).
  • Cryptophycin-110 'H NMR (CDC1 3 ) ⁇ (carbon position, multiplicities, J in Hz) Unit A 7.29 (10-H/14-H; dd, 8.6, 5.6), 6.99 (11-H/13-H, dt, 8.6, 8.5), 6.68 (3-H; ddd, 15.3, 9.7, 5.6), 6.38 (8-H; d, 15.8), 5.83 (7-H; dd, 15.8, 8.8), 5.78 (2-H, d, 15.3), 5.00 (5-H, ddd, 10.8, 7.3, 1.3), 2.53 (4-H/6-H, m), 2.63 (4-H ⁇ m), 1.13 (6-CH 3 , d, 6.8); Unit B 7.21 (5-H; d, 1.8), 7.07 (9-H, dd, 8.4, 1.8), 6.84 (8-H; d, 8.4), 5.68 (NH, d, 8.5), 4.82 (2-H, m), 3.87 (OMe, s
  • EIMS m/z (relative intensity %) 672 (1.9, M + ), 412 (5.8, ion a), 280 (10, ion b), 245 (17, ion c), 195 (52, ion d), 155 (31), 141 (23), 135 (15), 109 (100, -fluorobenzyl ion fluorotropylium ion); high-resolution EIMS 668.2853 (calcd for C 35 H 42 ClFN 2 O 8 , ⁇ +3.4mmu, M + ).
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (250mL), CH 3 OH (500mL), and CH 2 C1 2 (500mL).
  • EIMS m/z (relative intensity %) 637 (13, M + ), 395 (3, ion a), 178 (43, ion d), 91 (100); high resolution EIMS m/z (rel.
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (500mL), CH 3 OH (500mL), and CH 2 C1 2 (500mL).
  • the fractions that were eluted with 65:35 CH 2 CN/H 2 O and 80:20 CH 3 CN/H 2 O were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10 ⁇ , 250 X 22mm column, 65:35 MeCN/H 2 O, 6mL/min).
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (250mL), CH 3 OH (500mL), and CH 2 C1 2 (500mL).
  • Cryptophycin-209 [methy lenedioxy- 2 HJ-DL-3-(3 ,4-methylenedioxyphenyl) alanine was fed to the alga.
  • the 2 H 2 -labeled Cryptophycin-208 displayed the following spectral properties: ⁇ NMR (500MHz, CHC1 3 ) ⁇ (carbon position, multiplicity) Unit B 5.88 (6,7-OC ⁇ 2 O, s); EIMS m/z (relative intensity, assignment) 620 (5, M + ), 394 (68, ion a), 393 (49), 262 (12, ion b), 227 (45, ion c), 177 (100, ion d), 91 (79); high resolution EIMS m/z 620.3090 (calcd for s H ⁇ NA, ⁇ -2.3mmu error).
  • the 2 H 2 -labeled Cryptophycin-209 displayed the following spectral properties: ⁇ NMR (500MHz, CHC1 3 ) ⁇ (carbon position, multiplicity) Unit B 5.85 (6,7-OC ⁇ 2 O, S); EIMS m/z (relative intensity, assignment) 636 (M + , 17), 394 (6, ion a), 262 (5, ion b), 193 (35), 177 (50, ion d), 91 (79); high resolution EIMS m/z 636.3031 (calcd for H ⁇ NA, ⁇ -1.5mmu).
  • Example 8 Aberrant Biosynthesis of Crvptophvcin-211
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (600mL), CH 3 OH (600mL), and CH 2 C1 2 (500mL).
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (250mL), CH 3 OH (500mL), and CH 2 C1 2 (500mL).
  • the crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH 3 CN/H 2 O (250mL), 50:50 CH 3 CN/H 2 O (250mL), 65:35 CH 3 CN/H 2 O (250mL), 80:20 CH 3 CN/H 2 O (250mL), CH 3 OH (600mL), and CH 2 C1 2 (500mL).
  • Cryptophycin-52 (IC 50 43pM) and Cryptophycin- 115 (IC 50 39pM) are slightly less cytotoxic than Cryptophycin- 1 (IC 50 9-29pM) against the human tumor cell line KB.
  • Cryptophycin-110 (IC 50 4.6nM) has a cytotoxicity against KB comparable to that of Cry ⁇ tophycin-3 (IC 50 3.1-4.6nM).
  • the cytotoxicities (MIC's) of the novel sytrene-type Cryptophycins 208, 210 and 214 against KB are in the range of 10-lOOnM.
  • the cytotoxicities of Cryptophycins 189, 190, 209, 211, 213 and 215 against KB are ⁇ lnM.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédés de production de composés à base de cryptophycine par fermentation bactérienne et nouvelles compositions obtenues à partir desdits composés.
PCT/US1996/014670 1995-08-30 1996-08-30 Cryptophycines obtenues a partir d'une biosynthese aberrante Ceased WO1997008334A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP9510664A JP2000507805A (ja) 1995-08-30 1996-08-30 変形生合成からのクリプトフィシン
AU71090/96A AU709828B2 (en) 1995-08-30 1996-08-30 Cryptophycins from aberrant biosynthesis
EP96932217A EP0850316A4 (fr) 1995-08-30 1996-08-30 Cryptophycines obtenues a partir d'une biosynthese aberrante

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US293595P 1995-08-30 1995-08-30
US60/002,935 1995-08-30

Publications (1)

Publication Number Publication Date
WO1997008334A1 true WO1997008334A1 (fr) 1997-03-06

Family

ID=21703268

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US1996/014670 Ceased WO1997008334A1 (fr) 1995-08-30 1996-08-30 Cryptophycines obtenues a partir d'une biosynthese aberrante
PCT/US1996/013855 Ceased WO1997007798A1 (fr) 1995-08-30 1996-08-30 Composes pharmaceutiques

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US1996/013855 Ceased WO1997007798A1 (fr) 1995-08-30 1996-08-30 Composes pharmaceutiques

Country Status (6)

Country Link
EP (2) EP0850057A4 (fr)
JP (2) JP2000507805A (fr)
AU (2) AU709828B2 (fr)
BR (1) BR9610214A (fr)
CA (2) CA2246117A1 (fr)
WO (2) WO1997008334A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046581A1 (fr) * 1997-04-11 1998-10-22 Eli Lilly And Company Methode synergique de traitement du cancer
WO1998046221A1 (fr) * 1997-04-11 1998-10-22 Eli Lilly And Company Methode de traitement de mycoses
EP0869786A4 (fr) * 1995-12-22 1999-04-14 Lilly Co Eli Composes pharmaceutiques
EP0957912A4 (fr) * 1996-08-30 1999-12-22
US6103913A (en) * 1998-10-16 2000-08-15 Eli Lilly And Company Process for preparing enollactone derivatives
EP0934065A4 (fr) * 1996-08-30 2000-10-11 Lilly Co Eli Composes pharmaceutiques
US6143909A (en) * 1997-02-26 2000-11-07 Eli Lilly And Company Selective epoxidation process for preparing pharmaceutical compounds
US6180679B1 (en) * 1998-04-07 2001-01-30 Eli Lilly And Company Method for treating fungal infections
EP0850057A4 (fr) * 1995-08-30 2001-04-11 Lilly Co Eli Composes pharmaceutiques
US6376230B1 (en) 1998-10-16 2002-04-23 Eli Lilly And Company Stereoselective process for producing intermediates of cryptophycins
US6680311B1 (en) 1996-08-30 2004-01-20 Eli Lilly And Company Cryptophycin compounds
EP2266607A2 (fr) 1999-10-01 2010-12-29 Immunogen, Inc. Des immunoconjugués pour le traitement des cancers.
CN103724290A (zh) * 2013-11-15 2014-04-16 浙江大学 一种环肽化合物clavatustide A及其产生菌、制备方法和应用

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JP2001500484A (ja) * 1996-08-30 2001-01-16 イーライ・リリー・アンド・カンパニー 医薬化合物の製造法
WO1998009974A1 (fr) * 1996-09-06 1998-03-12 Eli Lilly And Company Procede et nouveaux intermediaires
AU6338698A (en) * 1997-02-26 1998-09-18 Eli Lilly And Company Tripeptide and tetrapeptide pharmaceutical compounds
AU2002238939A1 (en) * 2002-03-18 2003-09-29 Nihon University Cyclic etheramine derivatives as medicaments for malignant tumors
US7229814B2 (en) 2004-05-05 2007-06-12 Regents Of The University Of Minnesota Nucleic acids and polypeptides involved in the production of cryptophycin
US20110098477A1 (en) * 2005-06-28 2011-04-28 Chugai Seiyaku Kabushiki Kaisha Method Of Producing Compound Having Anti-Hcv Activity

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US4946835A (en) * 1988-07-15 1990-08-07 Merck & Co., Inc. Antifungal fermentation product and method

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US5952298A (en) * 1993-12-21 1999-09-14 The University Of Hawaii Cryptophycins
DE69633667T2 (de) * 1995-03-07 2006-03-02 University Of Hawaii, Honolulu Neue synthetische cryptophycine
JP2000507805A (ja) * 1995-08-30 2000-06-27 ユニバーシティ オブ ハワイ 変形生合成からのクリプトフィシン

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US4868208A (en) * 1988-07-15 1989-09-19 Merck & Co., Inc. Antifungal agent and method
US4946835A (en) * 1988-07-15 1990-08-07 Merck & Co., Inc. Antifungal fermentation product and method

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Title
J. AM. CHEM. SOC., 01 June 1994, Vol. 116, No. 11, TRIMURTULU et al., "Total Structures of Cryptophycins, Potent Antitumor Depsipeptides from the Blue-Green Alga Nostoc sp. Strain GSV 224", pages 4729-4737. *
J. AM. CHEM. SOC., 08 March 1995, Vol. 117, No. 9, BARROW et al., "Total Synthesis of Cryptophycins. Revision of the Structures of Cryptophycins A and C", pages 2479-2490. *
J. AM. CHEM. SOC., 13 December 1995, Vol. 117, No. 49, TRIMURTULU et al., "Structure Determination, Conformational Analysis, Chemical Stability Studies and Antitumor Evaluation of the Cryptophycins. Isolation of 18 New Analogs from Nostoc sp. Strain GSV 224", pages 12030-12049. *
See also references of EP0850316A4 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0850057A4 (fr) * 1995-08-30 2001-04-11 Lilly Co Eli Composes pharmaceutiques
EP0869786A4 (fr) * 1995-12-22 1999-04-14 Lilly Co Eli Composes pharmaceutiques
EP0934065A4 (fr) * 1996-08-30 2000-10-11 Lilly Co Eli Composes pharmaceutiques
US6680311B1 (en) 1996-08-30 2004-01-20 Eli Lilly And Company Cryptophycin compounds
EP0957912A4 (fr) * 1996-08-30 1999-12-22
US6143909A (en) * 1997-02-26 2000-11-07 Eli Lilly And Company Selective epoxidation process for preparing pharmaceutical compounds
WO1998046581A1 (fr) * 1997-04-11 1998-10-22 Eli Lilly And Company Methode synergique de traitement du cancer
WO1998046221A1 (fr) * 1997-04-11 1998-10-22 Eli Lilly And Company Methode de traitement de mycoses
US6180679B1 (en) * 1998-04-07 2001-01-30 Eli Lilly And Company Method for treating fungal infections
US6103913A (en) * 1998-10-16 2000-08-15 Eli Lilly And Company Process for preparing enollactone derivatives
US6376230B1 (en) 1998-10-16 2002-04-23 Eli Lilly And Company Stereoselective process for producing intermediates of cryptophycins
EP2266607A2 (fr) 1999-10-01 2010-12-29 Immunogen, Inc. Des immunoconjugués pour le traitement des cancers.
EP2289549A2 (fr) 1999-10-01 2011-03-02 Immunogen, Inc. Des immunoconjugués pour le traitement des cancers.
CN103724290A (zh) * 2013-11-15 2014-04-16 浙江大学 一种环肽化合物clavatustide A及其产生菌、制备方法和应用
CN103724290B (zh) * 2013-11-15 2015-04-29 浙江大学 一种环肽化合物clavatustide A及其产生菌、制备方法和应用

Also Published As

Publication number Publication date
AU709828B2 (en) 1999-09-09
AU6904896A (en) 1997-03-19
WO1997007798A1 (fr) 1997-03-06
EP0850316A4 (fr) 2000-08-23
EP0850057A4 (fr) 2001-04-11
BR9610214A (pt) 1999-06-15
JP2000501067A (ja) 2000-02-02
CA2246117A1 (fr) 1997-03-06
AU7109096A (en) 1997-03-19
CA2230540A1 (fr) 1997-03-06
EP0850316A1 (fr) 1998-07-01
JP2000507805A (ja) 2000-06-27
EP0850057A1 (fr) 1998-07-01

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