Compound, WF002, production thereof and use thereof

Details Compound, WF002, production thereof and use thereof Compound, WF002, production thereof and use thereof

2001-01-18

2003-02-18

Wilson, James O. (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S006100, C435S052000, C435S074000, C435S075000, C435S254100, C435S911000

Reexamination Certificate

active

06521600

ABSTRACT:

TECHNICAL FIELD
This invention relates to a new antimicrobial compound. More particularly, it relates to a new antimicrobial compound that has an antimicrobial activity against pathogenic microorganisms, especially pathogenic fungi, to a process for the preparation thereof and to a pharmaceutical composition comprising the same.
DISCLOSURE OF INVENTION
The new compound, WF002 has the following physicochemical properties:
a) Molecular weight: ESI-MS (negative): m/z 689 (M−H)
−
b) Elemental analysis: C, 61.08; H, 8.83
c) Melting point: 210-211° C.
d) Optical rotation: [&agr;]
D
23
=+58° (c=0.5, methanol)
e) UV absorption spectrum: &lgr; max(&egr;)=260 nm (methanol, 13000)
f) IR absorption spectrum: &ngr; max(KBr)=3430, 2950, 2880, 1710, 1620, 1450, 1370, 1260, 1100, 1080, 1040 cm
−1
g)
1
H-NMR spectrum: (500 MHz, CD
3
OD) &dgr;(ppm): 5.11 (1H, m), 4.98 (1H, s), 4.37 (1H, d, J=8 Hz), 4.34 (1H, m), 4.08 (1H, m), 3.86 (1H, m), 3.67 (1H, m), 3.38 (1H, d, J=10 Hz), 3.32 (1H, m), 3.28-3.20 (3H, m), 3.13 (1H, m), 2.58 (1H, m), 2.28 (1H, m), 2.19 (1H, m), 2.06 (3H, s), 2.06-1.95 (3H, m), 1.81-1.50 (7H, m), 1.49-1.20 (4H, m), 1.22 (3H, d, J=8 Hz), 1.20 (3H, s), 1.13 (3H, s), 1.12 (1H, m), 0.98 (3H, s), 0.97 (1H, m), 0.93 (3H, s), 0.92 (3H, s).
h)
13
C-NMR spectrum: (125 MHz, CD
3
OD) &dgr;(ppm): 211.3 (s), 173.2 (s), 157.3 (s), 136.9 (s), 105.9 (d), 95.2 (d), 89.3 (d), 78.3 (d), 77.8 (d), 75.9 (d), 71.9 (d), 71.7 (d), 63.0 (t), 59.4 (t), 56.4 (d), 52.7 (d), 45.94 (d), 45.90 (t), 45.2 (s), 44.9 (s), 44.4 (t), 44.1 (s), 42.1 (s), 39.5 (s), 35.6 (t), 35.0 (t), 31.3 (d), 30.1 (t), 28.7 (q), 28.1 (q), 24.9 (t), 23.0 (t), 21.7 (q), 21.1 (q), 19.4 (q), 19.2 (t), 18.3 (q), 17.9 (q).
i) Solubility
Soluble: methanol, ethyl acetate, dimethylsulfoxide
Insoluble: water, n-hexane
j) Color reaction
Positive: reactions with iodine vapor and cerium sulfate, and Molisch's test, respectively.
Negative: reactions with ninhydrin, Ehrlich's reagent, Dragendorff reagent and ferric chloride, respectively.
k) Nature of substance: Neutral substance
l) Thin layer chromatography:
Carrier: Silica gel 60 F254 (Merck)
Solvent: dichloromethane:methanol=8:1
Rf=0.21
From the above physicochemical properties and extensive studies, the provisional chemical structure of WF002 was assigned as follows.
According to this invention, the compound, WF002 can be prepared by culturing a WF002-producing strain, especially belonging to the genus Myrothecium in a nutrient medium.
Particulars of microorganisms used for the production of WF002 and production thereof will be explained in the followings.
Microorganism
The microorganism which can be used for the production of WF002 is a WF002-producing strain belonging to the genus Myrothecium, among which
Myrothecium cinctum
No.002 was newly isolated from a material of Japan.
Lyophilized samples of the newly isolated microorganism, the strain No.002 were deposited with an International Depository Authority on the Budapest Treaty, National Institute of Bioscience and Human-Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-0046 Japan under the deposit number FERM BP-6380 on May 26, 1998.
The fungal strain No.002 was originally isolated from a soil sample. This organism grew rather rapidly on various culture media, and formed orange white to yellowish white colonies. The strain produced many conidial structures on the agar media, while it did not formed teleomorph. The conidial structures consisted of hyphal conidiophores and phialidic conidia, and the conidial masses were dark green to dark gray. On Sabouraud dextrose agar, the strain formed sometimes sporodochial conidiomata. Its mycological characteristics were as follows.
Cultural characteristics on various agar media are summarized in Table 1. Culture on potato dextrose agar grew fairly rapidly, attaining 3.5-4.5 cm in diameter two weeks later at 25° C. This colony surface was plane to raised, felty to cottony, sulcate or wrinkly, exudate, sometimes sectoring, orange white to pale orange at the center and the margin, and gray at the middle. Many conidial structures were formed on the media. The reverse color was pale orange to light orange. Colonies on corn meal agar grew restrictedly, attaining 1.5-2.5 cm in diameter under the same conditions. The surface was plane, thin, sometimes sectoring, white to orange white at the center and the margin, and brownish gray to dark gray at the middle. The reverse was white to orange white. Many conidial structures were formed.
The morphological characteristics were mainly determined from the cultures on a Miura's LCA plate (Mura, K. and M. Kudo: Trans. Mycol. Soc. Japan, 11:116-118, 1970). The conidiophores were erect from vegetative or aerial hyphae. They were semi-macronematous, hyaline, smooth to roughened, repeatedly branched, and formed a whorl of 2-4 phialides at the tips. The phialides were discrete, acrogenous, hyaline, roughened to granulate, cylindrical, with differentiated collarettes, (9-)16-28(-34)×(1.5-)2-3 &mgr;m in size, and producing conidia in slimy drops. Conidia were enteroblastic, phialidic, subhyaline to dark green, oblique or longitudinal striate, one-celled, fusiform to lentiform, and 8.5-12×2.5-3.5(-4.5) &mgr;m. Vegetative hyphae were smooth, septate, hyaline and branched. The hyphal cells were cylindrical and 1.5-5 &mgr;m in width. Chlamydospores were not observed. Sporodochia on Sabouraud dextrose agar were composed of loosely aggregations of hyphae and conidiophores, and 100-300 &mgr;m in diameter.
Strain No.002 was able to grow at the temperature range from 6 to 33° C., with the growth optimum at 19 to 22° C. These temperature data were determined on potato dextrose agar (made by NISSUI).
On the basis of comparing the morphological characteristics with fungal taxonomic criteria by von Arx (J. A. von Arx: The Genera of Fungi—Sporulating in Pure Culture. 3rd ed., pp.315, J. Cramer, Vaduz. 1974) and Barron (G. L. Barron: The Genera of Hyphomycetes from Soil. pp.364, Williams & Wilkins, Baltimore, 1968), strain No.002 was considered to belong to the hyphomycete genus Myrothecium Tode (1790). Moreover, above characteristics were corresponded the species description of
Myrothecium cinctum
(Code) Sacc. (1886) by Domsch et al. (K. H. Domsch, W. Gams and T.-H. Anderson: Compendium of Soil Fungi. vol. 1, p.482, Academic Press, London, 1980), with few exceptions. Thus, we identified this isolate as one strain of
Myrothecium cinctum,
and named it
Myrothecium cinctum
No.002.
TABLE 1
Cultural characteristics of strain No.002.
Media
Cultural characteristics
Malt extract agar*
G: Rather restrictedly, 2.5-3.5 cm
S: Irregular, plane, felty, formed some conidial
structures, yellowish white (2A2) at the center,
white to orange white (5A2) at the margin,
and olive gray (1F2) at the middle
R: Pale yellow (4A3) to grayish yellow (4B3),
and olive brown (4E4) at the middle
Potato dextrose
G: Fairly rapidly, 3.5-4.5 cm
agar (Difco 0013)
S: Circular, plane to raised, felty to cottony,
sulcate or wrinkly, exudate, sometimes sectoring,
formed many conidial structures, orange white
(6A2) to pale orange (6A3) at the center and
the margin, and gray (1E1) at the middle
R: Pale orange (5A3-6A3) to light orange
(5A4-6A4)
Czapek's solution
G: Rather restrictedly, 2.5-3.5 cm
agar*
S: Circular, centrally raised to umbonate, zonate,
felty, radiately sulcate, exudate, formed few
conidial structures, and yellowish white (3A2)
to orange white (6A2)
R: Pale yellow (4A3) to pale orange (6A3)
Sabouraud dextrose
G: Rather rapidly, 3.0-4.0 cm
agar (Difco 0190)
S: Circular to irregular, centrally raised to
umbonate, felty, radiately sulcate or wrinkly,
exudate, grayish yellow (4B3) to orange
white (6A2), and formed some grayish dots of
sporodochia
R: Grayish orange (5B5) to brownish orange
(5C4)
Emerson Yp Ss agar
G: Rather rapidly, 3.0-4.0 cm
(Difco 0739)
S: Circular, plane, felty, hygroscopic, formed
many conidial structures, dark green
(27F4-27F5), and orange wh

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