Purification of Echinocandin cyclopeptide compounds

Details Purification of Echinocandin cyclopeptide compounds Purification of Echinocandin cyclopeptide compounds

2001-12-10

2003-01-14

Bugaisky, Gabrielle (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S011400, C514S009100, C514S002600, C530S317000, C530S344000, C530S345000

Reexamination Certificate

active

06506726

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a process for purification of cyclopeptide compounds containing at least one protonatable amino group, in particular, the process relates to the purification of an Echinocandin-type compound by adsorption onto a hydrophobic, reversed phase chromatographic media and eluting with a continuous nearly linear gradient of increasing acetic acid. A purification process is also provided for selectively removing a tripeptide-aldehyde by-product of the Echinocandin fermentation process to yield a higher purity Echinocandin compound.
BACKGROUND ART
Echinocandin cyclopeptides are natural products that have been shown to have antifungal activities. Included in the Echinocandin cyclopeptide family are natural products such as Echinocandin B (ECB), Echinocandin C, Aculeacin A&ggr;y, Mulundocandin, Sporiofungin A, Pneumocandin A
0
, WF11899A, and Pneumocandin B
0
. The natural products are typically produced by culturing various microorganisms. For example, Echinocandin B is produced from the fermentation of the fungus,
Aspergillus nidulans.
In the search for more active materials, the natural products have been modified in a variety of ways. One of the most common modifications has been the replacement of the N-acyl side chain on the natural product to produce a semi-synthetic derivative. For example, U.S. Pat. Nos. 4,293,489; 4,320,052; 5,166,135; and 5,541,160; and EP 359529; 448353; 447186; 462531; and 561639 describe a variety of N-acyl derivatized Echinocandin compounds that provide varying degrees of antifungal activity.
The N-acyl derivatives are produced by deacylating the natural product followed by reacylation with a different acyl group. The deacylation is typically achieved by means of an enzyme (e.g., deacylase enzyme). The deacylase enzyme may be obtained from the microorganism
Actinoplanes utahensis
or Pseudomonas species. See i.e., U.S. Pat. Nos. 4,293,482; and 4,304,716; and EP 460,882. The deacylated compound is typically referred to as the nucleus of the corresponding natural product (i.e., the deacylated product of Echinocandin B is referred to as the Echinocandin B nucleus (ECBN)). Unfortunately, both the fermentation and deacylation processes produce several by-products that are difficult to remove and decrease the purity of the desired deacylated cyclic peptide nucleus.
U.S. Pat. No. 4,874,843 describes a chromatographic process using non-functional resins in a reversed mode to purify Echinocandin-type products. Even though the process improved the purity of products derived from a fermentation process, further improvements are still needed to remove contaminants that are difficult to separate from both the intermediate deacylated nucleus and the final acylated pharmaceutically active compounds. Since the potency of the final pharmaceutical product is dependent upon the purity of the intermediates used to make the final product, improvements in purity at any stage of the manufacturing process are highly desirable. Ideally, the contaminants are removed at the earliest stage possible in the manufacturing process.
General discussions of non-functional resins and their applications in liquid chromatographic separations may be found in
J. Chromatography,
201, 287-292 (1980) and Grieser, M.D. et al,
Analytical Chemistry,
45, 1348-1353 (1973). The use of either step or continuous gradients are discussed; however, the eluents contain significant amounts of organic solvents. In a manufacturing process, the use of organic solvents raises several concerns such as environmental regulations (e.g., air quality emission standards), special handling requirements (e.g., flammability standards) and disposal limitations (e.g., toxic waste regulations). Therefore, there is a need for an eluent system that minimizes the use of organic solvents yet effectively separates mixtures into their pure components.
DISCLOSURE OF THE INVENTION
The present invention provides a method for separating and purifying a wide variety of fermentation cyclopeptide products containing at least one protonatable amino group (including the deacylated Echinocandin-type compounds) from their fermentation or mixed broths and partially purified process streams by adsorbing the mixture onto a hydrophobic, reversed phase, chromatographic media and eluting with a continuous nearly linear acetic acid gradient ranging from 0.1% acetic acid to 10.0% acetic acid by volume in water, preferably from 0.5% (pH=5.5) to 4.0% (pH=2.5) acetic acid.
In another embodiment of the present invention, a process for purifying Echinocandin-type compounds (including simple derivatives thereof) is provided where an aldehyde by-product (in particular, a tripeptide-aldehyde by-product) in the fermentation mixture or partially purified mixture is reacted with a derivatizing agent. Preferably, the fermentation broth or mixed broth is reacted with the derivatizing agent prior to purification of the corresponding Echinocandin nucleus using the method described above.
As used herein, the term “derivatizing agent” refers to a reagent capable of reacting with the aldehyde functionality of the tripeptide by-product to produce an intermediate that is sufficiently different in hydrophobicity to allow separation of the tripeptide intermediate from the desired Echinocandin-type compound.
The term “protonatable amino group” refers to an amino group that undergoes protonation when subjected to the eluting conditions of the present invention (i.e., 0.1% acetic acid to 10% acetic acid by volume in water).
The term “Echinocandin-type compounds” refers to compounds having the following general structure including any simple derivatives thereof:
wherein R is a hydrogen or —C(O)R′ where R′ is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or heteroaryl group having attached thereon at least one protonatable amino group; R1 is —H or —OH; R2 is —H or —CH
3
; R3 is —H, —CH
3
, —CH
2
CONH
2
or —CH
2
CH
2
NH
2
; R4 is —H or —OH; R5 is —OH, —OPO
3
H
2
, or —OSO
3
H; and R6 is —H or —OSO
3
H. “Echinocandin nucleus” refers to the deacylated Echinocandin compound where R is a hydrogen. “ECBN” refers to the Echinocandin B nucleus where R1, R4 and R5 are hydroxyl groups, R2, R3, and R7 are methyl groups; and R1 and R6 are hydrogens.
The term “alkyl” refers to a hydrocarbon radical of the general formula C
n
H
2n+1
containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e.g. methyl, ethyl, propyl, butyl, etc.), branched (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e.g., bicyclo[2.2.1]heptane, spiro[2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group or alkanoate has the same definition as above.
The term “alkenyl” refers to an acyclic hydrocarbon containing at least one carbon-carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted.
The term “alkynyl” refers to an acyclic hydrocarbon containing at least one carbon-carbon triple bond. The alkene radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted.
The term “aryl” refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted.
The term “heteroaryl” refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that si

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