WF14573 or its salt, production thereof and use thereof

Details WF14573 or its salt, production thereof and use thereof WF14573 or its salt, production thereof and use thereof

2000-09-18

2004-05-04

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Cyclic peptides

C514S011400, C514S016700, C530S329000

Reexamination Certificate

active

06730776

ABSTRACT:

TECHNICAL FIELD
This invention relates to a new antimicrobial compound, WF14573 or its salt. More particularly, it relates to a new antimicrobial compound, WF14573 or its salt which has an antimicrobial activity against pathogenic microorganisms, especially pathogenic fungi, a process for the preparation thereof and a pharmaceutical composition comprising the same.
DISCLOSURE OF INVENTION
The new compound, WF14573 is represented by the following formula:
wherein R
1
is hydrogen or methyl and R
2
is hydrogen or palmitoyl.
In this specification, the following designations of the specific compound are conveniently used.
Compound name
R
1
R
2
WF14573 A
—H
—CO(CH
2
)
14
CH
3
WF14573 B
—CH
3
—CO(CH
2
)
14
CH
3
Deacyl WF14573 A
—H
—H
Deacyl WF14573 B
—CH
3
—H
The new compound, WF14573A has the following physico-chemical properties:
a) Molecular weight: ESI-MS(negative)m/z 1143 (M−H); b) Elemental analysis: C 48.39; H 7.15; N 8.95; c) Melting point: 230-240° C. (dec.); d) Optical rotation: [&agr;]
D
23
=−12 (c 0.5, methanol); e) UV spectrum: &lgr; max(&egr;)=276 nm (methanol); f) IR spectrum: &ngr; max(KBr)=3360, 2920, 2830, 1670, 1630, 1540, 1440, 1270, 1240, 1050 cm
−1
; g)
1
H-NMR spectrum: (500 MHz, CD
3
OD) &dgr; (ppm): 7.18 (1H, d, J=8 Hz), 6.80 (1H, d, J=2 Hz), 6.67 (1H, dd, J=8, 2 Hz), 5.30 (1H, d, J=3 Hz), 5.10 (1H, d, J=4 Hz), 4.98 (1H, d, J=3 Hz), 4.64 (1H, m), 4.58~4.52 (3H, m), 4.47~4.43 (2H, m), 4.40~4.33(2H, m), 4.27 (1H, d, J=3 Hz), 4.02~3.95 (3H, m), 3.82~3.76 (2H, m), 2.70 (1H, m), 2.63~2.55 (2H, m), 2.48~2.40 (2H, m), 2.30~2.17 (3H, m), 2.05~1.93(4H, 1.57 (2H, m), 1.35~1.23 (24H, m), 1.15 (3H, d, J=6 Hz), 0.89 (3H, t, J=7 Hz). h)
13
C-NMR spectrum: (125 MHz, CD
3
OD) &dgr; (ppm): 176.8 (s), 175.9 (s), 174.4 (s), 174.1 (s), 172.9 (s), 172.7 (s), 172.5 (s), 169.3 (s), 150.3 (s), 140.1 (s), 137.6 (s), 124.0 (d), 121.9 (d), 119.4 (d), 74.2 (d), 74.0 (d), 73.9 (d), 71.3 (d), 70.7 (d), 70.5 (d), 69.8 (d), 68.3(d) 62.4 (d), 58.5 (d), 58.0 (d), 57.2 (t), 55.4 (d), 51.2 (d), 47.0 (t), 41.1 (t), 39.7 (t), 38.9 (t), 36.7 (t), 35.0 (t), 34.6 (t), 33.1(t), 30.80 (t×5), 30.76 (t), 30.7 (t), 30.6 (t), 30.5 (t), 30.3 (t), 27.0 (t). 23.7 (t), 19.5 (q), 14.4 (q). i) Solubility; Soluble: water, methanol. dimethylsulfoxide; Insoluble: n-hexane, chloroform; j) Thin layer chromatography: Carrier: Silica gel 60 F254 (Merck); Solvent: 1-butanol: acetic acid: water=4:1:2; Rf=0.39.
The new compound, WF14573B (as its sodium salt), has the following physico-chemical properties:
a) Molecular weight: ESI-MS(negative)m/z 1157 (M−H); b)Elemental analysis: C 48.79; H 7.34; N 8.96; S 2.80; Na 1.69; c) Melting point: 220-225° C. (dec.); d)Optical rotation: [&agr;]
D
23
=15° (c 0.9, methanol); e) UV spectrum: &lgr; max(&egr;)=276 nm (methanol); f) IR spectrum: &ngr; max(KBr)=3360, 2940, 2830, 1670, 1630, 1530, 1440, 1270, 1240, 1050 cm
−1
; g)
1
H-NMR spectrum: (500 MHz, CD
3
OD) &dgr; (ppm): 7.18 (1H, d, J=8 Hz), 6.80 (1H, d, J=2 Hz), 6.67 (1H, dd, J=8, 2 Hz), 5.29 (1H, d, J=3 Hz), 5.07 (1H, d, J=4 Hz), 4.99 (1H, d, J=4 Hz), 4.64 (1H, m), 4.58~4.51 (3H, m), 4.48~4.36 (4H, m), 4.16 (1H, m), 4.07 (1H, m), 4.02~3.97 (2H, m), 3.79 (1H, br. d, J=11 Hz), 3.37 (1H, m), 2.69 (1H, m), 2.60~2.40 (5H, m), 2.22 (2H, m), 2.05~1.98 (3H, m), 1.57 (2H, m), 1.35~1.24 (24H, m), 1.15 (3H, d, J=6 Hz),1.06 (3H, d, J=7 Hz), 0.89 (3H, t, J=7 Hz). h)
13
C-NMR spectrum: (125 MHz, CD
3
OD) &dgr; (ppm): 176.7 (s), 176.0 (s), 174.3 (s), 174.1 (s), 172.8 (s), 172.5 (s), 172.5 (s), 169.3 (s), 150.3 (s), 140.1 (s), 137.6 (s), 124.0 (d), 122.0 (d), 119.4 (d), 75.9 (d), 74.0 (d), 73.9 (d), 71.3 (d), 70.7 (d), 70.5 (d), 70.2 (d), 68.2 (d), 62.4 (d), 58.6 (d), 58.0 (d), 57.2 (t), 55.5 (d), 52.9 (t), 51.3 (d), 41.1 (t), 39.8 (t), 39.1 (d), 38.9 (t), 36.7 (t), 34.9 (t), 33.0 (t), 30.8 (t×5), 30.7 (t), 30.7 (t), 30.5 (t), 30.4 (t), 30.3 (t), 27.0 (t), 23.7 (t), 19.5 (q), 14.4 (q), 11.1 (q). i) Solubility; Soluble: water, methanol, dimethylsulfoxide; Insoluble: n-hexane, chloroform; j) Thin layer chromatography: Carrier: Silica gel 60 F254 (Merck); Solvent: 1-butanol: acetic acid: water=4:1:2; Rf=0.45.
In the result of the above physicochemical properties and extensive investigations, the chemical structures of WF14573A and B are identified as mentioned above, respectively.
BEST MODE FOR CARRYING OUT OF THE INVENTION
According to this invention, the compound, WF14573A and B can be prepared by culturing a WF14573A and/or B-producing strain belonging to the genus Coleophoma in a nutrient medium.
Particulars of microorganisms used for the production of WF14573A and B and production thereof will be explained in the followings.
Microorganism
The microorganism which can be used for the production of WF14573A and B is a WF14573A and/or B-producing strain belonging to the genus Coleophoma, among which
Coleophoma empetri
No.14573 was newly isolated from a decayed leaf sample, collected at Mitsushima-cho, Kamiagata-gun, Nagasaki-ken, Japan.
Lyophilized samples of the newly isolated microorganism,
Coleophoma empetri
No. 14573 was 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, Japan under the deposit number FERM BP-6252 on Feb. 12, 1998.
It is to be understood that the production of the new compound, WF14573A and/or B is not limited to the use of the particular organism described herein, which is given for illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF14573A and/or B including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means, such as genetic engineering, X-ray, ultraviolet radiation, treatment with N-methyl-N′-nitro-N-nitrosoguanidine and the like.
The strain No.14573 has the following morphological, cultural and physiological characteristics.
The strain grew rather restrictedly on various culture media, and formed grayish colonies. The strain produced pycnidial to stromatic conidiomata on the autoclaved leaf segments affixed on agar media, while it formed neither teleomorph nor anamorph on or in the media. The conidiomata were convex to discoid, dark brown to black, and formed ampulliform to lageniform conidiogenous cells on the lower cells of their inner walls. Conidia were hyaline, one-celled and cylindrical. 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 by Sutton (B. C. Sutton: The Coelomycetes—Fungi Imperfecti with Pycnidia, Acervuli and Stroma., pp.696, Commonwealth Mycological Institute, Kew, 1980), strain No.14573 was considered to belong to the coelomycete genus Coleophoma Höhn. 1907 (Sphaeropsidales). Its mycological characteristics were as follows.
Cultural characteristics on various agar media are summarized in Table 1. Culture on potato dextrose agar grew restrictedly, attaining 1.5-2.5 cm in diameter two weeks later at 25° C. This colony surface was plane to raised, felty to cottony, light gray to dark gray, and yellowish white at the margin. The reverse color was olive. Conidial structures were not observed. Colonies on corn meal agar grew rather restrictedly, attaining 2.5-3.5 cm in diameter under the same conditions. The surface was plane to centrally raised, dark gray and lustrous. At the colony center was felty to cottony and purplish gray to dark purple. Mycelium near the margin was submerged and white. The reverse was dark gray to dark green, and vellowish white at the margin. Conidial structures were not produced.
The morphological characteristics were determined from the cultures on the sterile leaf segments affixed

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