11-hydroxysordarin and a process for producing it using...

Details 11-hydroxysordarin and a process for producing it using... 11-hydroxysordarin and a process for producing it using...

1999-10-26

2001-05-08

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C514S025000, C549S417000, C435S252100

Reexamination Certificate

active

06228622

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is directed toward the synthesis of a novel antifungal agent prepared by biotransformation of known compound, sordarin.
Sordarin is an antifungal antibiotic isolated from the ascomycete
Sordaria araneosa
(see GB 1,162,027 and
Helvetica Chimica Acta,
1971, 51:119-20). Other compounds having the sordarin skeleton have also been reported as antifungal agents. Japanese Kokai J62040292 discloses the compound zofimarin isolated from
Zopfiela marina
sp.; Japanese Kokai J06157582 discloses the compound BE-31405 isolated from Penicillium sp.; and SCH57404 is reported in
J. Antibiotics,
1995, 48:1171-1172. Semi-synthetic sordarin derivatives are reported in PCT Applications WO96/14326 and WO96/14327. The compounds exhibit antifungal activity against fungi including
Saccharomyces cerevisiae, Candida albicans, C. glabrata
and
C. tropicalis.
SUMMARY OF THE INVENTION
The present invention is directed toward the synthesis of the novel antifungal compound of the formula
This compound is prepared by biotransformation of sordarin. The compound has antifungal activity against a number of pathogenic fungi including Candida spp., but is significant because it allows access to a series of sordarin derivatives that are chemically inaccessible.
This invention also relates to a process for the preparation of the compound by fermentation of Actinomyces spp. MA7235, ATCC No. 202103 in the presence of the substrate compound sordarin.
The invention also relates to pharmaceutical compositions containing a therapeutically effective amount of compound I in combination with a pharmaceutically acceptable carrier or excipient.
Additionally, the invention relates to a method of treatment of diseases caused by certain fungal pathogens.
DETAILED DESCRIPTION OF THE INVENTION
There is disclosed compound I of the formula
or a pharmaceutically acceptable salt or hydrate thereof which can be produced by a biotransformation process. The compound of the present invention is prepared by fermentation of the microorganism Actinomyces spp. MA7235, ATCC No. 202103 in the presence of the substrate compound, sordarin, of the formula:
under appropriate conditions.
A sample of the microorganism Actinomyces sp. has been deposited under the Budapest Treaty in the culture collection of the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, on Apr. 1, 1998 and assigned accession number ATCC 202103.
MA7235 can be generally described as follows. Observations of growth, and general cultural characteristics were made in accordance with the methods of Shirling and Gottleib (International J. System. Bacteriol. 16:313-340). Chemical composition of the cells was determined using the methods of Lechevalier and Lechevalier (in Actinomycete Taxonomy, A. Dietz and D. W. Thayer, Ed. Society for Industrial Microbiology, 1980). Whole Cell Fatty Acids were derivatrized and analyzed as methyl esthers (Fames) by Gas Chromatography by the procedure of Miller and Berger using as MIDI Microbial Identification system (Microbial Identification Systems, Newark, Del.). Coloration of the culture was determined by comparison with color standards in the Munsell color charts (Macbeth Division of Kollmorgen Instruments Corp. P.O. Box 230 Newburgh, N.Y. 12551-0230).
Analysis of Whole Cell Extracts
Peptidoglycan contains Meso-diaminopimelic acid and whole cell extracts contain arabinose, galactose, and rhamnose.
General Growth Characteristics
Good growth on yeast malt extract agar (YME) and Czapeks agar (CZ). Moderate growth on Oatmeal Agar and Glycerol Asparagine agar (Gas). Poor growth on Inorganic Salt Starch agar (ISS) and water agar supplemented with NZ-Amine A (table 1).
Colony Morphology (Oatmeal Agar at 21 Days)
No aerial mycelium. The substrate mycelium is orange in color (7.5YR/7/8) and has a leathery texture.
Micromorphology
Highly branched vegetative hyphae. No aerial hyphae was observed and no spore structures were observed.
Whole Cell Fatty Acid Analysis
FAME analysis revealed that whole cell extracts contained major amounts of 16:0 ISO, 16:0 ISO 2 OH, and 17:1 Cis 9 fatty acids (table 2).
Conclusions
Whole cell analysis reveals that MA7235 has a type IV cell wall. Microscopic observations reveal that MA7235 is filamentous but produces no distinguishing characteristics. Those strains that have type IV cell wall are reported to belong to the Nocaridaceae and Nocardioform bacteria (7). MA7235 does not contain Tuberculasteric acid and thus can not be considered a member of the Nocardiaceae. MA7235 does not share any phenotypic properties with the Nocardioforms and does not group with them by fatty fatty acid analysis. Therefore, an identification of Actinomycetes sp. will be assigned to MA7235.
TABLE 1
Cultural Characteristics of MA7235 at 21 days
Aerial mycelium
Reverse
Medium
Growth
Spore structure
color
Color
Yeast Malt
good
no spore structure
no aerial
Orange
Extract agar
observed
mycellium
(7.5YR/6/8)
Glycerol
moderate
no spore structure
no aerial
Orange
Asparagine
observed
mycellium
(7.5YR/6/8)
agar
Inorganic
poor
no spore structure
no aerial
Orange
Salt Starch
observed
mycellium
(10YR/7/8)
agar
Oatmeal
moderate
no spore structure
no aerial
Orange
agar
observed
mycellium
(10YR/7/8)
Czapeks
good
no spore structure
no aerial
Orange
agar
observed
mycellium
(7.5YR/6/8)
Water agar
poor
no spore structure
no aerial
Orange
observed
mycellium
ND- No colors to report
TABLE 2
Percent of fatty acids in whole cell extracts
Percent in
Fatty Acid
whole cell extracts
14:0 ISO
 0.88%
15: ISO
 4.73%
15:0 Anteiso
 0.68%
15:1 B
 1.99%
15:0
 2.56%
16:1 ISO H
 2.85%
16:0 ISO
33.13%
16:1 CIS 9
 1.54%
16:0
 1.31%
16:0 10 methyl
 1.40%
17:0 ISO
 1.75%
17:0 Anteiso
 3.12%
17:1 CIS 9
12.54%
16:0 ISO 2OH
14.74%
17:0
 5.79%
17:0 ISO 10 Methyl
 1.62%
17:0 10 Methyl
 7.86%
17:0 Anteiso 2 OH
 1.31%
The present invention can be practiced with any strain of Actinomycetes sp. capable of producing compound I and particularly preferred is the ATCC No. 202103 strain.
In general, compound I may be produced by culturing the above described microorganism in the presence of an appropriate concentration of substrate compound sordarin in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen.
Substrate compound sordarin can be obtained as previously described or by synthetic organic procedures.
The compound has antimicrobial properties and may be useful for controlling systemic and superficial fungal infections in humans. Additionally, the compound exhibits activity against certain plant fungal pathogens and may be useful as a broad spectrum crop antifungal agent.
The compound of this invention has antimicrobial properties and is especially useful as a new platform for antifungal agents and organisms causing systemic human pathogenic mycotic infections such as Candida sp. and
Cryptococcus neoformans.
These properties may be effectively utilized by administering compositions containing an antifungal amount of the compound to an area, object or subject, on or in which fungi are to be controlled. Thus, compositions containing an antifungally effective amount of the compound and their use for the control of fungi are aspects of the present invention. Especially preferred aspects of the present invention are compositions in a pharmaceutically acceptable carrier and their use for the control of mycotic infections by administering a therapeutically effective amount of the compound
Compound I may be useful as an antifungal agent, especially as an antimycotic agent, which may be demonstrated with the compound in a broth microdilution assay for the determination of minimum inhibitory concentration (MIC). The compound is found to be effective in the assay against fungi selected for their resistance/susceptibility to known compounds, animal virulence, source and clinical importance, at concentrations comparable to an established antifungal agent, amphotericin B.
In the microbroth dilution assay, mic

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