AU709828B2

AU709828B2 - Cryptophycins from aberrant biosynthesis

Info

Publication number: AU709828B2
Application number: AU71090/96A
Authority: AU
Inventors: Thomas K. Hemscheidt; Richard E Moore
Original assignee: University of Hawaii
Current assignee: University of Hawaii
Priority date: 1995-08-30
Filing date: 1996-08-30
Publication date: 1999-09-09
Anticipated expiration: 2016-08-30
Also published as: EP0850057A1; JP2000501067A; BR9610214A; WO1997007798A1; AU7109096A; EP0850316A4; CA2246117A1; WO1997008334A1; CA2230540A1; AU6904896A; EP0850316A1; JP2000507805A; EP0850057A4

Description

Cryptophycins from Aberrant Biosynthesis Technical Field 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.

Background of the Invention 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.

Of the previously known cryptophycins, 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 stereochemistry of the two hydroxy acid units is as follows: (5S, 6S, 7R, 8R)-7,8-epoxy-5-hydroxy-6-methyl-8-phenyl-2-octenoic acid (Unit A) and (2S)-2-hydroxy-4-methylvaleric acid (Unit D; leucic acid). The stereochemistry of the two amino acid units is as follows: (2R)-3-(3-chloro-4-methoxyphenyl)alanine (Unit B) and (2R)-3-amino-2-methylpropionic acid (Unit C).

The units are connected in an ABCD sequence.

In addition to the cryptophycin isolates of Nostoc spp., other members of the cryptophycin group have been produced by chemically modifying the isolates. Moreover, additional cryptophycins have been produced by total synthesis. Many of these non-bacterial cryptophycins demonstrate activities and in vivo stability which could prove beneficial in therapeutic applications. However, fermentation is far t eeo [n:\Iibc]04092:MEF more cost-effective for producing a compound in a commercial setting than the use of partial or total chemical synthesis. Unfortunately, the variety of cryptophycins obtained via native bacterial fermentation is limited.

Thus, it is considered desirable to provide a means for obtaining both known and Snovel cryptophycin compounds by bacterial fermentation.

It is also considered desriable to produce novel cryptophycin compounds via fermenation which demonstrate improved proeprties of stability and activity when compared to compounds obtained via native bacterial fermenation.

Disclosure of the Invention Io The present invention provides a method for producing cryptophycin compounds as metabolitcs by the controlled use of metabolic substrates in bacterial fermentation. In one aspect, the present invention comprises culturing bacteria capable of producing cryptophycin compounds in the presence of a substituted amino acid under conditions sufficient to produce the desired cryptophycin compound.

I Also provided in accordance with an aspect of the present invention are novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substituted amino acid under conditions sufficient to produce the desired cryptophycin compound.

*Typically, such cryptophycin compounds will have a stable macrolide and possess 20 substituent groups which have been shown to provide beneficial activities.

According to a first embodiment of the invention, there is provided a method for producing a cryptophycin compound comprising culturing at least one strain of bacteria capable of producing cryptophycins in the presence of a substituted amino acid under conditions sufficient to produce the desired cryptophycin compound.

.2 According to a second embodiment of the invention, there is provided a cryptophycin compound obtained by culturing at least one strain of bacteria capable of producing a native cryptophycin compound in the presence of a substituted amino acid under conditions sufficient to produce at least one cryptophycin compound which is distinct from any such native cryptophycin compound produced by the bacteria under standard culture conditions.

Brief Description of the Drawings 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 D) and jd two amino acid groups (B of which cryptophycins are characteristically comprised.

0 I \DAYLIB\Iibfl\22882 docsak 0 0 0 0 0 0 00 00 0 0 0 0 2a 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.

oo ee oo g oo oo oo oe oo oooo a. a. I \DAYLIB\libff\22882.docsak WO 97/08334 PCT/US96/14670 -3- Detailed Description of the Invention The present invention provides a method for producing cryptophycin compounds as metabolites by the controlled use of metabolic substrates in bacterial fermentation. In one aspect, the present invention comprises culturing bacteria capable of producing cryptophycins in the presence of a substituted 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. By the use of the present invention one can obtain pre-determined cryptophycins produced by fermentation of a bacteria capable of producing a cryptophycin compound. Typically, such pre-determined cryptophycin compounds will have a stable macrolide and possess substituent groups which have been shown to provide beneficial activities.

Also provided in accordance with the present invention are novel cryptophycin compounds produced by culturing bacteria capable of producing cryptophycins in the presence of a substituted amino acid under conditions sufficient to produce the desired cryptophycin compound.

Selection of Desirable Crvptophvcin Compounds It has been found that, in addition to Cryptophycin 1, over 20 different cryptophycin compounds can be isolated from one strain of cyanobacteria (Nostoc spp.). Moreover, numerous additional cryptophycin compounds have been produced by total chemical synthesis or semi-synthesis. Many of these compounds have been subjected to structure-activity relationship (SAR) studies in order to determine the structural features of the class of compounds which are most important in providing the beneficial therapeutic properties.

These SAR studies have indicated that in vivo activity is largely premised upon an intact macrolide. Also significant are the epoxide group, the chloro group in Unit B and the methyl group in Unit C. The SAR studies further showed that the lack of the methyl group in Unit C, as in Cryptophycin 21, enhances the lability of the ester bond between Units C and D (thereby decreasing the probability that the macrolide will remain intact in vivo prior to reaching the tumor site). This ester SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTfUS96/14670 -4bond was in excess of 100 times more labile in Cryptophycin 21 compared to Cryptophycin 1.

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.

Thus, based upon the results of such studies, desirable features of cryptophycin compounds can be engineered into the metabolites produced in bacterial fermentation by controlled selection of metabolic substrates.

Bacteria Capable of Producing Crvptophvcin Compounds The cryptophycins are a group of structurally related compounds originally isolated from cyanobacteria (blue-green algae). Cryptophycin 1 (also termed Cryptophycin A) is the major cytotoxin produced by certain Nostoc species of cyanobacteria. As disclosed above, a limited number of other cryptophycin compounds can be obtained by native bacterial fermentation.

The morphological characteristics of the Nostoc sp. of cyanobacteria, as described in U.S. Patent No. 4,946,835, are well known, and the basis for the identification of a Nostoc sp. is described in detail in J. Gen. Micro., 111:1-61 (1979).

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. In an embodiment of the present invention, the Nostoc sp. strain designated GSV 224 is the strain which is cultivated and from which are isolated both previously known and novel cryptophycins.

In one aspect, 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 nonnaturally occurring cryptophycin compounds. To that end, 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 SUBSTITUTE SHEET (RULE 26) PCTUS96/14670 WO 97/08334 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.

As is the case with other organisms, the characteristics of Nostoc sp. are subject to variation. For example, 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-methyl- 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. Among the nutrient inorganic salts which can be incorporated in the culture media are 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 0.2mL/L) of an antifoam agent such as polypropylene glycol about 2000) to large scale cultivation media if foaming becomes a problem.

For production of substantial quantities of the cryptophycin compounds, submerged aerobic cultivation in tanks can be used. Small quantities may be SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 WO 97/08334 -6obtained by shake-flask culture. 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 culture 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 and 30 0 C and at an incident illumination intensity of from about 100 to about 200miol photons m 2 Sec' (photosynthetically active radiation).

As is customary in aerobic submerged culture processes of this type, carbon dioxide gas is introduced into the culture by addition to the sterile air stream bubbled through the culture medium. For efficient production of the cryptophycin compounds, the proportion of carbon dioxide should be about 1% (at 24 0 C and one atmosphere of pressure).

The prior art, specifically U.S. Patent No. 4,946,835, the contents of which are hereby incorporated by reference, provides methods of cultivating Nostoc sp.

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.

Following their production under fermentation culture conditions, cryptophycin compounds of the invention can be recovered from the culture and from the culture media by methods known to those of ordinary skill in this art.

Recovery is generally accomplished by initially filtering the culture 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 mixture of acetonitrile and dichloromethane. The cryptophycins can be separated by subjecting this extract, as well as the culture media, to rapid chromatography on reversed-phase column. The cryptophycins can be purified by reversed-phase high-performance liquid chromatography

(HPLC).

The novel cryptophycin compounds of the present invention and the previously disclosed cryptophycin compounds can be therapeutically employed as SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -7anti-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 antineoplastic agents, are disclosed in U.S. Application Serial No. 08/400,057 filed March 7, 1995, International Application Serial No. PCT\US94\14740 filed December 21, 1994, U.S. Application Serial No. 08/249,955 filed May 27, 1994 and U.S. Application Serial No. 08/172,632, filed December 21, 1993. These patent applications are incorporated herein by reference.

Selection of Metabolic Substrates In the biosynthesis of Cryptophycin 1, 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. Not only is 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. In the biosynthesis of Cryptophycin 1, (2S, 3R)-3-methylaspartic acid is the precursor of Unit C. (2R)-2-Methyl-p-alanine, however, is also incorporated well into Unit C, suggesting that (2S, 3R)-3-methylaspartic acid is decarboxylated into (2R)-2-methyl-p-alanine first and this is followed by assimilation of the (2R)-2-methyl-p-alanine into the depsipeptide.

In the biosynthesis of Cryptophycin 1, L-leucine is the precursor of Unit D and is incorporated 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.

SUBSTITUTE SHEET (RULE 26)

I

In order to produce desirable cryptophycin compounds which have the properties noted previously, an intact macrolide, an epoxide group, a chloro group in Unit B and/or a methyl group in Unit C, selected substituted amino acids can be employed as metabolic substrates for fermentation cultures of bacteria capable of producing cryptophycin compounds. In the broadest sense, a substituted 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 substituted amino acids will include amino acids which provide the substituent groups in the appropriate positions to form a desirable cryptophycin compound as a metabolite of a bacterial fermentation culture. Such amino acids include, for example, substituted a-amino acids, substituted phenylalanines, substituted tyrosines, substituted O-methyltyrosines and substituted p-alanines.

Typically substituent groups will be chosen so as to provide the desired structural features in the cryptophycin metabolites. For example, as halogens attached to aryl groups have been shown to be desirable features in Unit B of the cryptophycin compounds, amino acids substituted with at least one halogen moiety will prove to be of use. Commonly, alanine will be the amino acid of choice in this regard, typically substituted with a halogen and/or alkoxy-substituted aryl group.

Similarly, a methyl group in Unit C can be included as a feature of an appropriately substituted p-alanine.

Thus in many cases, the amino acid of choice is substituted with at least one substituent selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halosubstituted derivatives thereof.

Thus, 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 culture one or more of the following pre-selected compounds: a substituted phenylalanine, a substituted p-alanine, substituted tyrosine or O-methyltyrosine and a substituted a-amino acid.

25 Specifically, 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. For example, Cryptophycin-52 is produced, along with other o °o

I

PCT[US96/14670 WO 97/08334 -9cryptophycins, when the cyanobacterium is grown in the presence of 2 ,2-dimnethyl- I0 -alanine: CRYPTOPHYCIN-52 SUBSTITUTE SHEET (RULE 26) WO 97/08334 Cryptophycin-] 110 and Cryptophycin- 115 are produced when the cyanobacterium is grown in the presence of DL-p-fluorophenylalanine: 0 HN 0 N) 0 OCH 3 CRYPTOPHYCIN-1 150 0 HN c F 0 N1 0 CH

H

CRYPTOPHYCIN-1 PCTIUS96/14670 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -11- 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 substituted phenylalanine. Likewise, the present method can be used to produce novel cryptophycins that differ in Unit B by growing the cyanobacterium in the presence of the appropriately substituted tyrosine or O-methyltyrosine, in Unit C by growing the cyanobacterium in the presence of the appropriate substituted p-alanine, and in Unit D by growing the cyanobacterium in the presence of the appropriately substituted a-amino acid.

An example of a novel cryptophycin compound of the present invention is 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: k O HN. 0 H

OCHS

CRYPTOPHYCIN-189 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTJUS96/14670 -12- Two further examples of novel cryptophycin compounds of the present invention are Cryptophycin-190 (also called Cryptophycin B-i) and Cryptophycin-210 which are produced by growing the cyanobacterium in the presence of DL-3-(3-fluoro-4-methoxyphenyl)alanfle: 0 0 0 H N F 0 0N 0 0CH 3

"CH

CRYPTOPHYCIN-1 0 0 H N F 100 N 0O OCH 3 kCH CRYPTOPHYCIN-21 0 SUBSTITUTE SHEET (RULE 26) 13 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 (DL)-3-(3-bromo-4methoxyphenyl) alanine or (DL)-3-(3-bromo-4-hYdroxyphenyl)alanine: 0 0 HNBr 0 N 0 OCH 3

H

B-2 [N:\LIBC]04092:MEF 14 Another example of a novel cryptophycin compound of the present invention is Cryptophycin 13- 3 which would be produced by growing the cyanobacterium in the presence of dimethoxyphenyl)alanine or (DL)-3-(3-hydroxy-4-methoxyphenyl)alanine, or dihydroxyphenyl)alanine: 00H 0 N 0 OCH 3

H

B -3 [N :\LIBC]04092:MEF WO 97/08334 PCT/US96!14670 Two further examples of novel cryptophycin compounds of the present invention are 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: 0 0, 00 HN 10 )CN 0>

H

CRYPTOPHYCI N-208 lo00H 0 N 0~

"ICH

CRYPTOPHYCIN-209 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT[US96/1 4670 -16- Another example of a novel cryptophycin compound of the present invention is Cryptophycin-21 1 which is produced by growing the cyanobacteriumn in the presence of DL-3-(3-fluoro-4-hydroxyphenyl)alanine: 00 0 N 0 O0H 3 H0 F CRYPTOPHYCIN-21 1 SUBSTITUTE SHEET (RULE 26) 17 Another example of a novel cryptophycin compound of the present invention is Cryptophycin Bwhich would be produced by growing the cyanobacterium in the presence of (DL)-3-(4-methoxy-2pyridyl) alanine or (DL)-3-(4-hydroxy-2-pyridyl)alanine: Q 0 0 HIN N 0 IN 0 0CH 3

H

B [N:\LIBC]04092:MEF

I

18 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 (DL)-3-(4-methoxy-3pyridyl)alanine: 0D 00 HN 0QN 0 N 0 r OCH 3

H

B-6 [N:\LIBC]04092:MEF 19 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 ethoxyphenyl)alanine: 0 0 HNCl 0 IN 0 OCH 2

CH

3

H

B- 7 [N:\LIBC]04092:MEF WO 97/08334 PCTIUS96/14670 Another example of a novel cryptophycin compound of the present invention is Cryptophycin-213 which is produced by growing the cyanobacterium in the presence of (3S)-3-aminobutanoic acid: 0 o0,0 HN, ci 0 0 N 0 OCH 3

H

CRYPTOPHYCIN-21 3 SUBSTITUTE SHEET (RULE 26)

I

WO 97/08334 PCT/US96/14670 -21- 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- P-alanine: 0- HN

CI

0 H OCH 3 C-1 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -22- 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- p-alanine: 0 N O H OCH 3 C-2 SUBSTITUTE SHEET (RULE 26) WO 97/08334 -2-PCTIUS96/14670 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- p-alanine: 0 0 0H N 0

I~

H 0CH C-3 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -24- 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- p-alanine: o /o ,O SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 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-dimnethylaminomethyl- 1-alanine: 00 0 N 0 H OCH 3 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -26- 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- 1-alanine: HN CI N 0 OCHs C-6 SUBSTITUTE SHEET (RULE 26) WO 9708334PCTIUS96/14670 -27- 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(methoxylmethyl)- p-alanine: 0 0- 0 0

HN

0O N)0 1 H 0CH 3

OCH

3

OCH

3 C-7 SUBSTITUTE SHEET (RULE 26) S97/08334 PCT/US96/14670 WO 97/08334 -28- 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: 0 0 HN O H

OCH

3 C-8 SUBSTITUTE SHEET (RULE 26) PCTIUS96/14670 WO 97/08334 -29- Two further examples of novel cryptophycin compounds of the present invention are Cryptophycin-214 and Cryptophycin-215 which are produced by growing the cyanobacterium in the presence of (2S)-2-aminobutyric acid: 0 0 0 n HN, Ci 0 0 N 0 OCH 3 ,rH CRYPTOPHYCI N-21 4 CRYPTOPHYCIN-21 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 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-a-alanine: 0 OHN O N 0CI H OCH 3 D-1 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 -31- 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-ct-alanine: 00 0 0 H N C I 0O N 0 H OCH 3 D-2 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 -32- 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-a-alanine: 0 uk0 HN cN 0 Il 0N 0 H OCH 3 D-3 SUBSTITUTE SHEET (RULE 26) Experimental In the experimental disclosure which follows, all weights are given in grams milligrams micrograms nanograms picograms (pg) or moles (mol), all concentrations are given as percent by volume molar millimolar micromolar nanomolar or picomolar normal and all volumes are given in litres millilitres (mL) or microlitres (lpL), and measures in millimetres unless otherwise indicated.

Example 1 General Procedure for Feeding Substituted Amino Acids For the production of cryptophycin compounds by bacterial fermentation utilising substituted amino acids, the selected amino acid is dissolved in 0.5N HCI to a concentration in the range of 25mg/mL. Typically, a 0.5mL portion of the solution is added to each of 2-4 carbouys of the bacterial culture in two-day intervals beginning on day 7-10 after bacterial innoculation. After 7-9 additions of the amino acid solution, the cultures are allowed to grow for an additional 3-5 days, and then harvested.

Example 2 Identification of Cryptophycins by El Mass Spectrometry Mass spectra, including high resolution mass measurements are determined in the electronimpact mode on a VG-70SE Instrument. Cryptophycin-1, 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.

a a a a a.

[N:\LIBC]04092:MEF WO 97/08334 PCT/US96/14670 -34- Example 3 Aberrant Biosynthesis of Cryptophycin 52 To each of nine carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added 2,2-dimethyl-P-alanine (400mg) in four equal portions of 100mg each on days 6, 8, 14 and 22 after inoculation. The cultures were harvested 30 days after inoculation and lyophilized to yield 103.6g of dried alga.

Extraction was carried out in 52g batches with 2L of 4:1 acetonitrile/dichloromethane. The extract was evaporated in vacuo and the residue fractionated by C-18 flash chromatography. The crude cryptophycin fraction (300mg) eluted with 65:35 acetonitrile/water was subjected to HPLC on C-18 (Econosil, 101, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction eluting after 66.5 min was purified by normal phase HPLC on silica (Econosil, 5 p, 250 x 4.6mm column, 3:2 EtOAc/hexanes, 2.5mL/min). The fraction eluting after 21.4 min, which was a mixture of Cryptophycin-52 and Cryptophycin-28, was purified further by HPLC on C-18 (Econosil, 101p, 250 x 10mm, 85:15 methano/water, 2mL/min). Cryptophycin-52 (2mg), identical with authentic material by spectroscopic comparison, eluted after 18.4 min.

The spectral data for Cryptophycin 52 is as follows: +19.90 (c 0.5, CHC1,); EIMS m/z 668/670 445 244 227 (22, ion 195/197 (66/27, ion 184 155/157 (38/10), 91 (100, benzyl ion tropylium ion); HREIMS m/z 668.2873 (C 3

,H,

4

N

2 0 8 35 C1, A -0.9 mmu), 445.2497 (C2H, 3 NO,, A -3.3 mmu); UV (MeOH) X 204 (35100), 218 (20900)nm; IR (NaC1) v. 3415, 3270, 2960, 1748, 1721, 1650, 1536, 1504, 1260, 1192, 1150, 1066, 1013, 800, 698 'H NMR (CDC1 3 8 Unit A 7.33-7.38 (11-H/12-H/13-H; bm, W,,-25 Hz), 7.24 (10-H/14-H; m, W,,=15 Hz), 6.76 ddd, 15.1/10.8/4.3), 5.71 dd, 15.1/1.7), 5.20 ddd, 11.0/5.0/1.8), 3.68 (8-H; d, 2.92 dd, 2.57 (4-Hb; ddd, 2.45 ddd, -14.6/11.0/10.8), 1.78 bm, W,,=15 Hz), 1.14 (6-Me; d, Unit B 7.18 d, 7.04 dd, 6.83 d, 5.56 (NH; d, 4.73 ddd, 3.87 (OMe; 3.09 dd, 3.05 dd, Unit C 7.20 (NH; dd, 3.41 (3-Hb; dd, 3.10 dd, 1.22 1.15 Unit D 4.82 dd, 10.2/3.5), 1.73 (3-Hb; bm, W,,=20 Hz), 1.66 bm, W,,=20 Hz), 1.31 ddd, 0.84' (4-Me; d, 0.82' d, "C NMR (CDC13) 8 Unit SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 A 164.9 141.8 136.7 128.7 (11/13), 128.3 125.6 (10/14), 124.7 75.9 63.0 59.0 40.7 36.9 13.5 Unit B 170.3 154.1 130.9 129.5 128.5 122.6 112.4 56.1 (7-OMe), 54.3 35.3 Unit C 178.0 46.5 42.8 22.8 22.8 Unit D 170.5 71.2 39.3 24.6 22.7t 21.2' SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/1 4670 -36- Example 4 Aberrant Biosynthesis of Crvptoohvcin-110 and Crvptophvcin-115 To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(4-fluorophenyl)alanine (150mg) as described in the general procedure. After harvesting, the freeze-dried alga (26g) was extracted with 1L of 5:1 CH 3 CN/CHCL, for 24 hours, and the extract (Ig) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75

CH

3

CN/H

2 0 (250mL), 50:50 CH 3 CN/HO (250mL), 65:35 CH 3 CN/HO (250mL), 80:20 CH 3 CN/H,O (500mL), CH 3 OH (600mL), and CH,Cl 2 (500mL). The fractions that were eluted with 65:35 CH,CN/H 2 0 (98mg) and 80:20 CH 3

CN/H

2 0 were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction containing Cryptophycin-115 eluted at 45min t, <59min and the fraction containing Cryptophycin-110 eluted at 75min <90min. Purification was achieved by normal-phase HPLC (Econosil silica, 5 250 x 4.6mm column, 50:50 EtOAc/hexanes, 3mL/min) to give Cryptophycin-110 (tR 14 min, 0.1mg) and Cryptophycin-115 (18min<tR<23min, 0.7mg) The 'H-NMR spectra of the isolated cryptophycins were identical to those of synthetic samples.

Cryptophycin-110: 'H NMR (CDCI 3 6 (carbon position, multiplicities, J in Hz) Unit A 7.29 (10-H/14-H; dd, 8.6, 6.99 (11-H/13-H, dt, 8.6, 6.68 ddd, 15.3, 9.7, 6.38 d, 15.8), 5.83 dd, 15.8, 5.78 d, 15.3), 5.00 ddd, 10.8, 7.3, 2.53 2.63 m), 1.13 d, Unit B 7.21 d, 7.07 dd, 8.4, 6.84 d, 5.68 (NH, d, 4.82 3.87 (OMe, 3.14 dd, 5.6, 3.04 dd, 7.2, Unit C 6.95 (NH, bdd, 6.8, 3.50 td, 4.4, 3.28 ddd, 6.8, 6.7, 2.72 1.23 (2-Me, d, Unit D 4.82 1.65 1.35 ddd, 4.5, 3.8, 0.78 d, 0.74 d, 6.4).

Cryptophycin-115: EIMS m/z (relative intensity 672 412 (5.8, ion 280 (10, ion 245 (17, ion 195 (52, ion 155 141 135 109 (100, p-fluorobenzyl ion fluorotropylium ion); high-resolution

EIMS

668.2853 (calcd for C 3 5

H

4 2 C1FN 2 Os, A +3.4mmu, SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 -37- 'H NMR (CDC1 3 6 (carbon position, multiplicities, J in Hz) Unit A 7.22 (10-H/14-H; ddt, 8.7, 5.2, 7.01 (11-H/13-H, ddt, 8.7, 8.5, 6.68 (3-H, ddd, 15.2, 9.7, 5.74 dd, 15.2, 5.15 ddd, 11.2, 5.0, 1.8), 3.67 d, 2.88 dd, 7.4, 2.54 dtd, 5.2, 1.8, 2.44 ddd, 11.2, 9.7, 1.79 1.13 (6-CH 3 d, Unit B 7.21 d, 7.06 dd, 8.3, 6.83 d, 5.63 (NH, d, 4.80 ddd, 8.4, 7.2, 3.87 (OMe, 3.14 dd, 5.4, 3.03 dd, 7.2, Unit C 6.94 (NH, bdd, 6.7, 3.48 ddd, 5.0, 3.7, -13.4), 3.30 ddd, 6.8, 6.7, 2.72 1.22 (2-Me, d, Unit D 4.83 dd, 9.9, 1.70 1.35 0.87 d, 0.85 d, SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 -38- Example 5 Aberrant Biosynthesis of Cryptophvcin-189 To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3-methyl-4-methoxyphenyl)alanine (120mg) as described in the general procedure. After harvesting, the freeze-dried alga (20g) was extracted with 1L of 5:1 CH 3

CN/CH

2 CL for 24 hours, and the extract g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75

CH

3

CN/H

2 0 (250mL), 50:50 CH 3 CN/H,O (250mL), 65:35 CH 3 CN/HO (250mL), 80:20 CH 3

CN/H

2 0 (250mL), CH 3 OH (500mL), and CHC 2 (500mL). The fractions that were eluted with 65:35 CH 2 CN/HO (178mg) and 80:20 CH 3

CN/H

2 0 were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction containing Cryptophycin-189 eluted at 50min t, <64min. Normal phase HPLC (Econosil silica, 10p, 250 x 10mm column, 60:40 EtOAc/hexane, 3mL/min) of this fraction (150mg) afforded 2.8mg of the pure compound (tR 28 min).

To facilitate the isolation and identification of Cryptophycin-189, [O-methyl- 2

H

3 ]-DL-3-(3-methyl-4-methoxyphenyl)alanine was fed to the alga. The 2 H,-labeled Cryptophycin-189 displayed the following spectral properties: 'H NMR (500MHz, CDC1 3 6 (carbon position, multiplicity) for Unit B 3.74 (7-OC0H 3 +27.80 (CHCl 3 c IR (neat) AL 3412, 3272, 2959, 1749, 1725, 1667, 1503, 1263, 1176 EIMS m/z (relative intensity 637 (13, 395 ion 178 (43, ion 91 (100); high resolution EIMS m/z (rel.

intensity) 637.3477 (calcd for CHN 3

DN

2 Os, A-3.5mmu); 'H NMR (CDCI,) 6 (carbon position, multiplicities, J in Hz) Unit A 5.70 dd, 15.1, 6.71 (3, ddd, 16.2, 8.4, 2.45 ddd, 14.5, 12.6, 2.55 br. dd, 12.1, 2.1), 5.20 ddd, 11.2, 4.9, 1.78 1.14 (6-Me, d, 2.92 dd, 3.68 d, 7.24 (10/14, 7.33-7.29 (11/12/13, Unit B 4.75 (2, dd, 13.1, 3.09 dd, 14.5, 3.03 dd, 14.4, 7.07 2.16 (6-Me, 6.71 d, 6.95 dd, 10.5, 5.57 (NH, d, Unit C 2.68 1.23 (2-Me, d, 3.34 dt, 13.7, 3.45 6.71 (NH, br. m); Unit D 4.82 dd, 10.0, 1.70 1.32 1.70 0.85 d, 0.83 d, SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 -39- 3 C NMR (CDCI1) 6 (carbon position) Unit A 165.1 125.1 141.2 36.7 76.0 40.6 13.5 63.0 59.0 136.8 125.6 (10/14), 128.7 (11/13), 128.5 Unit B 171.2 54.0 35.4 127.8 131.5 126.9 16.1 (6Me), 156.8 110.1 127.3 Unit C 176.1 38.1 14.3 40.7 Unit D 170.6 71.3 39.4 24.5 22.9 21.3 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTIUS96/14670 Example 6 Aberrant Biosynthesis of CrvDtophycin-190 and Cryptophycin-210 To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL- 3 3 -fluoro-4-methoxyphenyl)alanine (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (32g) was extracted with 1.5L of 5:1 CHCN/CHCL, for 24 hours, and the extract (1.2g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75

CH

3

CN/H

2 0 (250mL), 50:50 CH 3

CN/H

2 0 (250mL), 65:35 CH 3

CN/H

2 0 (250mL), 80:20 CH,CN/H 2 0 (500mL), CH 3 OH (500mL), and CH 2 C1 (500mL). The fractions that were eluted with 65:35 CHCN/H 2 0 and 80:20 CH 3 CN/HO were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10P, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction containing Cryptophycin-190 eluted at 44min t, 50min and the fraction containing Cryptophycin-210 eluted at t, 96min. Further purification of these two fractions by normal-phase HPLC (Econosil silica, 51, 250 x 4.6mm column, 2.5mL/min) afforded 4.3mg of Cryptophycin-190 (45:55 EtOAc/hexane, t, 22 min) and 1.5 mg of Cryptophycin-210 (50:50 EtOAc/hexane, t, 14.4min).

To facilitate the isolation and identification of Cryptophycin-190 and Cryptophycin-210, [O-methyl-'H,]-DL-3-(3-fluoro-4-methoxyphenyl)alanine was fed to the alga. The 2

H

3 -labeled Cryptophycin-190 displayed the following spectral properties: 2 H NMR (500MHz, CDC1,) amino or hydroxy acid 6 (carbon position, multiplicity) Unit B 3.74 (7-OC 2 'F-NMR (CDC1 3 6 -135.6 [a]D +16.8* (CHC,, c 0.64); IR (neat) A. 3416, 2959, 1748, 1651, 1517, 1276, 1244, 1177, 1126 EIMS m/z (relative intensity 641 399 (17, ion 267 (13, ion 227 (39, ion 182 (72, ion 91 (100); high resolution EIMS m/z 641.3198 (calcd for C 3 5

HD

3 FNO, A-0.6mmu); 'H NMR (CDCI 3 6 (carbon position, multiplicities, J in Hz) Unit A 5.73 d, 15.8), 6.68 ddd, 15.2, 9.8, 2.45 ddd, 14.0, 11.1, 2.55 dd, 14.1, 5.16 ddd, 11.2, 4.6, 1.80 1.14 (6-Me, d, 2.92 (7, dd, 7.5, 3.69 d, 7.25 (10/14, 7.32-7.39 (11/12/13, Unit B 4.80 3.03 dd, 14.4, 6.84-6.97 3.85 (7-OMe, 7.32- 7.39 5.67 (NH, d, Unit C 2.70 1.22 (2-Me, d, 3.30 (3, SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -41dt, 13.6, 3.46 dt, 13.6, 6.83-6.97 (NH, Unit D 4.82 m), 1.66-1.74 1.66-1.74 0.86 d, 0.84 d, 6.6).

3 C NMR (125MHz, CDC13) 8 (carbon position) Unit A 165.3 125.3 141.0 36.7 76.2 40.7 13.5 63.0 59.0 136.8 125.6 (10/14), 128.7 (11/13), 128.5 Unit B 170.9 53.6 35.2 not determined 117.5 not determined 141.1 59.0 113.6 124.9 Unit C 175.6 38.3 14.1 41.1 Unit D 170.7 71.3 39.4 24.5 22.9 21.3 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -42- Example 7 Aberrant Biosynthesis of CrvptoDhvcin-208 and Crvptophycin-209 To each of two carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL- 3 3 ,4-methylenedioxyphenyl)alanine (1 2 0mg) as described in the general procedure. After harvesting, the freeze-dried alga (25g) was extracted with 1L of 5:1 CH,CN/CH 2 CL, for 24 hours, and the extract 5 10mg) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CHCN/HO (250mL), 50:50 CH 3

CN/H

2 0 (250mL), 65:35 CH,CN/HO 2 (250mL), 80:20 CHCN/HO (250mL),

CH

3 OH (500mL), and CH 2

C

2 (500mL). The fractions that were eluted with 65:35 CH,CN/H 2 0 (204mg) and 80:20 CH 3 CN/HO were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 101, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction containing Cryptophycin-209 eluted at 42min 48.5min and the fraction containing Cryptophycin-208 eluted at 69min<t,< 88.5min. Normal phase HPLC (Econosil silica, 5p, 250 x 4.6mm column, 45:55 EtOAc/hexane, 3mL/min) of the fraction containing Cryptophycin-209 afforded <0.1mg of the pure compound (tR 16.5 min). HPLC (Econosil silica, 5p, 250 x 4.6mm column, 50:50 EtOAc/hexane, of the fraction containing Cryptophycin-208 afforded <0.1mg of the pure compound (tR 12 min).

To facilitate the isolation and identification of Cryptophycin-208 and Cryptophycin-209, [methylenedioxy- HJ-DL-3-(3,4-methylenedioxyphenyl) alanine was fed to the alga. The 2 H,-labeled Cryptophycin-208 displayed the following spectral properties: 2 H NMR (500MHz, CHCI,) 6 (carbon position, multiplicity) Unit B 5.88 (6,7-OC 2

H

2 0, EIMS m/z (relative intensity, assignment) 620 394 (68, ion 393 262 (12, ion 227 (45, ion 177 (100, ion 91 high resolution EIMS m/z 620.3090 (calcd for C 3

,HD

2

,N

2 A-2.3mmu error).

The 2 H-labeled Cryptophycin-209 displayed the following spectral properties: 2 H NMR (500MHz, CHC1 3 8 (carbon position, multiplicity) Unit B 5.85 (6,7-OC'H20, EIMS m/z (relative intensity, assignment) 636 17), 394 (6, ion 262 ion 193 177 (50, ion 91 high resolution EIMS m/z 636.3031 (calcd for C 3 H D 2

N

2 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT[US96/1 4670 -43- Example 8 Aberrant Biosynthesis of Crvptophvcin-211 To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added DL-3-(3-fluoro-4-hydroxyphenyl)alanine (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (34g) was extracted with 1.5L of 5:1 CH 3

CN/CH

2 CL, for 24 hours, and the extract (1.1g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75

CH

3 CN/H,0 (250mL), 50:50 CH 3 CN/HO (250mL), 65:35 CH 3 CN/HO (250mL), 80:20 CH3CN/HO (600mL), CH 3 OH (600mL), and CH,C1 2 (500mL). The fractions that were eluted with 65:35 CH,CN/H 2 0 (302mg) and 80:20 CH 3 CN/HO (49mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction containing Cryptophycin-211 eluted at tR 66 min. Further purification by normal-phase HPLC (Econosil silica, 5p, 250 x 4.6mm, 50:50 EtOAc/hexane, 3mL/min) afforded 3.5mg of the pure compound (tR 5.5 min).

Cryptophycin-211: 9 F-NMR (CDC13) 6 -128.1 11); EIMS m/z (relative intensity) 674/672 3/6), 432/430 (5/12, ion 300/298 (8/19, ion 227 (40, ion 215/213 (15/39, ion 91 (100); high resolution EIMS m/z 672.2580 (calcd for C 3

H

4 2 C1FN 2

O,,

A +3.4mmu error); 'H NMR (CDC13) 6 (carbon position, multiplicities, J in Hz) Unit A 5.75 dd, 16.4, 6.66 ddd, 15.3, 9.4, 2.46 dd, 10.3, 2.56 dd, 14.2, 5.14 ddd, 6.5, 4.8, 1.81 1.14 (6-Me, d, 2.93 dd, 8.4, 3.69 d, 7.25 (10/14, 7.30-7.40 (11/12/13, Unit B 4.81 3.13 dd, 14.4, 3.00 dd, 14.5, 7.01 3.92 (7-OMe, 6.90 dd, 11.4, Unit C 2.72 1.22 (2-Me, d, 3.22 m), 3.54 Unit D 4.83 1.71 1.37 1.71 0.87 d, 0.85 d, 7.2).

3 C NMR (125MHz, CDC1,) 6 (carbon position) Unit A 165.5 125.4 141.0 36.7 76.3 40.6 13.5 63.0 58.9 136.7 125.6 (10/14), 128.7 (11/13), 128.5 Unit B 170.8 53.3 35.3 133.4 126.2 127.4 141.4 61.4 (7-OMe), 155.8 d 248Hz), 116.4 d, SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCTfUS96/1 4670 -44- 28Hz); Unit C 175.3 38.3 14.0 41.4 Unit D 170.7 71.3 39.4 24.6 22.9 21.3 SUBSTITUTE SHEET (RULE 26) Example 9 Aberrant Biosynthesis of Cryptophycin-213 To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added S-3-aminobutanoic acid (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (31g) was extracted with 1.5L of 5:1 CH 3

CN/CH

2

CL

2 for 24 hours, and the extract (1g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODScoated silica column and subjected to flash chromatography with 25:75 CH 3

CN/H

2 0 (250mL), 50:50

CH

3 CN/H20 (250mL), 65:35 CH 3

CN/H

2 0 (250mL), 80:20 CH 3

CN/H

2 0 (250mL), CH 3 0H (500mL), and CH 2 C1 2 (500mL). The fractions that were eluted with 65:35 CH 2

CN/H

2 0 (205mg) and 80:20

CH

3

CN/H

2 0 (108mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10p, 250 X 22mm column, 65:35 MeCN/H 2 0, 6mL/min). The fraction (192mg) containing Cryptophycin-213 eluted at 52min tR 69min. Further purification by normal phase HPLC (Econosil silica, 5j., 250 x 4.6mm column, 50:50 EtOAc/hexane, 2.5mL/min) afforded 0.1mg of the pure compound.

To facilitate the isolation and identification of Cryptophycin-213, [methyl- 2

H

3 aminobutanonic acid was fed to the alga. The 2

H

3 -labeled Cryptophycin-213 displayed the following spectral properties: 2 H NMR (500MHz, CHCl 3 8 (carbon position, multiplicity) Unit C 1.11 (C 2

H

3 on C-2).

o** o •9 9 [N:\LIBC]04092:MEF WO 97/08334 PCTIUS96/1 4670 -46- Example 10 Aberrant Biosynthesis of Crvntophvcin-214 and Crvtophycin-215 To each of three carboys (20L) of Nostoc sp. GSV 224 growing under standard conditions was added S-2-aminobutanoic acid (180mg) as described in the general procedure. After harvesting, the freeze-dried alga (41g) was extracted with 1.7L of 5:1 CHCN/CH,CL, for 24 hours, and the extract (1g) was concentrated in vacuo to give a dark green solid. The crude extract was applied to an ODS-coated silica column and subjected to flash chromatography with 25:75 CH,CN/H,O (250mL), 50:50 CH 3

CN/HO

2 0 (250mL), 65:35 CH 3 CN/H,O (250mL), 80:20 CHCN/HO (250mL), CH30H (600mL), and CH,Cl, (500mL). The fractions that were eluted with 65:35 CH 2 CN/H,O (529mg) and 80:20 CHCN/H,O (59mg) were evaporated and further separated by reversed-phase HPLC (Econosil C-18, 10i, 250 X 22mm column, 65:35 MeCN/HO, 6mL/min). The fraction containing Cryptophycin-214 eluted at 87min<t, and the fraction containing Cryptophycin-215 eluted at 53min t, <68min. Further separation was achieved by normal phase HPLC to afford <1mg of impure Cryptophycin-214 (Econosil silica, 5P, 250 x 4.6mm column, 45:55 EtOAc/hexane, 2.5mL/min) (17min< 19.5min) and <1mg of impure Cryptophycin-215 (Econosil silica, 10p, 250 x 10mm, 60:40 EtOAc/hexane, 3mL/min) (10min<t,< 12min).

To facilitate the isolation and identification of Cryptophycin-214 and Cryptophycin-215, [methyl- 2

H,

3 1-S-2-aminobutanoic acid was fed to the alga. The 2 'H,-labeled Cryptophycin-214 displayed the following spectral properties: 2 H NMR (500MHz, CHCl) Unit D 6 (assignment) 0.76 (4- 2 The 2 H,-labeled Cryptophycin-215 displayed the following spectral properties: 'H NMR (500MHz, CHCl 3 Unit D 8 (assignment) 0.76 SUBSTITUTE SHEET (RULE 26) WO 97/08334 PCT/US96/14670 -47- Example 11 Cytotoxicity of Cryptophycins Produced by Aberrant Biosynthesis Cryptophycin-52 (ICo 43pM) and Cryptophycin-115 (ICo 39pM) are slightly less cytotoxic than Cryptophycin-1 (ICs 9-29pM) against the human tumor cell line KB. Cryptophycin-110 (ICso 4.6nM) has a cytotoxicity against KB comparable to that of Cryptophycin-3 (ICo 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 10-100nM. The cytotoxicities of Cryptophycins 189, 190, 209, 211, 213 and 215 against KB are lnM.

All publications and patent applications cited in this specification are hereby incorporated by reference as if they had been specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

SUBSTITUTE SHEET (RULE 26)

Claims

1. A method for producing a cryptophycin compound comprising culturing at least one strain of bacteria capable of producing cryptophycins in the presence of a substituted amino acid under conditions sufficient to produce the desired cryptophycin compound.

2. A method according to claim 1 wherein said bacteria is a cyanobacteria.

3. A method according to claim 2 wherein said bacteria is at least one species from the genus Nostoc.

4. A method according to claim 3 wherein said bacteria is at least one strain selected from the group consisting of Nostoc sp. strain GSV 224 (ATCC Accession No. 55483) and strain MB 5357 (ATCC Accession No. 53789). A method according to claim 1 wherein said substituted amino acid is an a-amino acid.

6. A method according to claim 1 wherein said substituted amino acid is at least one amino acid selected from the group consisting of substituted phenylalanine, substituted tyrosine, substituted O-methyltyrosine and substituted p-alanine.

7. A method according to claim 1 wherein said substituted amino acid is substituted with at least one halogen moiety.

8. A method according to claim 1 wherein said substituted amino acid is substituted with at least one substituent selected from the group consisting of halo- substituted aryl compounds. SUBSTITUTE SHEET (RULE 26)

9. A method according to claim 1 wherein said substituted amino acid is substituted with at least one substituent selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo-substituted derivatives thereof. A method for producing a cryptophycin compound, substantially as hereinbefore described with reference to any one of the Examples.

11. A cryptophycin compound produced by the method of any one of claims 1 to

12. A cryptophycin compound obtained by culturing at least one strain of bacteria capable of producing a native cryptophycin compound in the presence of a substituted amino acid under conditions sufficient to produce at least one cryptophycin compound which is distinct from any such native cryptophycin compound produced by the bacteria under standard culture conditions.

13. A cryptophycin compound according to claim 12 wherein said bacteria is a cyanobacteria.

14. A cryptophycin compound according to claim 12 wherein said bacteria is at least one species from the genus Nostoc.

15. A cryptophycin compound according to claim 12 wherein said bacteria is at least one strain selected from the group consisting of Nostoc sp. strain GSV 224 (ATCC Accession No. 55483) and strain MB 5357 (ATCC Accession No. 53789).

16. A cryptophycin compound according to claim 12 wherein said substituted amino acid is an a-amino acid.

17. A cryptophycin compound according to claim 12 wherein said substituted amino acid is at least one amino acid selected from the group consisting of substituted phenylalanine, substituted tyrosine, substituted O-methyltyrosine and substituted p-alanine.

18. A cryptophycin compound according to claim 12 wherein said substituted amino acid is substituted with at least one halogen moiety. 25 19. A cryptophycin compound according to claim 12 wherein said substituted amino acid is substituted with at least one substituent selected from the group consisting of halo-substituted aryl compounds.

20. A cryptophycin compound according to claim 12 wherein said substituted amino acid is substituted with at least one substituent selected from the group consisting of alkyl, alkoxy, alkaryl and alkoxyaryl compounds and halo-substituted derivatives thereof.

21. A cryptophycin compound, substantially as hereinbefore described with reference to any one of the Examples. Dated 9 September, 1998 University of Hawaii a 35 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:\LIBC]04092:MEF