ZA200503522B - Novel substance FKI-1033 and process for producing the same - Google Patents
Novel substance FKI-1033 and process for producing the same Download PDFInfo
- Publication number
- ZA200503522B ZA200503522B ZA200503522A ZA200503522A ZA200503522B ZA 200503522 B ZA200503522 B ZA 200503522B ZA 200503522 A ZA200503522 A ZA 200503522A ZA 200503522 A ZA200503522 A ZA 200503522A ZA 200503522 B ZA200503522 B ZA 200503522B
- Authority
- ZA
- South Africa
- Prior art keywords
- fki
- substance
- ryanodine
- microorganism
- activity
- Prior art date
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- JYVHOGDBFNJNMR-UHFFFAOYSA-N hexane;hydrate Chemical compound O.CCCCCC JYVHOGDBFNJNMR-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002000 high resolution fast-atom bombardment mass spectrometry Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960002418 ivermectin Drugs 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229960003439 mebendazole Drugs 0.000 description 1
- BAXLBXFAUKGCDY-UHFFFAOYSA-N mebendazole Chemical compound [CH]1C2=NC(NC(=O)OC)=NC2=CC=C1C(=O)C1=CC=CC=C1 BAXLBXFAUKGCDY-UHFFFAOYSA-N 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 208000014837 parasitic helminthiasis infectious disease Diseases 0.000 description 1
- 238000004810 partition chromatography Methods 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 229960002957 praziquantel Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
®
Novel FKI-1033 substance and production thereof
The present invention relates tonovel FKI-1033 substance, which is effective as agrochemicals, veterinary medicines and pharmaceuticals having an activity of ryanodine binding inhibition, an insecticidal activity and anthelmintic activity and production thereof.
Insecticide has contributed undoubtedly to increase production of food resources and stable supply, however it has brought about large problems such as residual toxicity and destruction of ecological systems.
Parasitosis has been reducing as the results of improvement in environmental hygiene and progress of anthelmintics, but recently afferent parasitosis, zocnotic parasitosis, optimistic parasitosis and parasitosis derived from perishables are increasing, and as a result various parasitoses become an issue. In livestock firming and agriculture, parasitosis causes great economic burden at present. Among parasitoses, with regard to helminth infection, many compounds such as ivermectin, mebendazole, praziquantel, etc. are used.
However, presently used anthelmintic agents are not always satisfactory in view of effectiveness and toxicities, and new agents are still demanded.
®
A ryanodine receptor is an ion channel which induces cat from the intracellular store to the cytoplasm with increased
Ca’" level in the cytoplasm, and was discovered as a receptor of plant alkaloid, ryanodine, that exhibits the insecticidal activity. In mammals, three types of the ryanodine receptor, i.e. type 1 (the skeletal muscular type), type 2 (the myocardial type) and type 3 (the cerebral type), which were coded independently in the gene, are known as a result of gene cloning.
Primary structure of the ryanodine receptor has been elucidated (Takeshima, H. Ann. N.Y. Acad. Sci. 707, 165-177, 1993). It is identified in insects and helminths as the ryanodine receptor which does not belong to the above three types (Takeshima, H.
Proteins, Nucleic Acids and Enzymes, 43: 1603-1609, 1998).
Consequently, it was reported that among substances inhibiting the binding of ryanodine to the ryanodine receptor, those having high selectivity to insects and helminths were expected as insecticides and anthelmintic substances (M.
Schmitt et al. Pesticide Sci. 48: 375-385, 1996).
We have focused to study the ryanodine receptor and continued to explore the ryanodine binding inhibitors from microbial culture products in order to obtain novel drugs, which were satisfied in the effectiveness and toxicity of the drug, and as a result, we have found that a novel compound, FKI-1033 substance, which was produced by the strain of fungus FKI-1033, had an activity of ryanodine binding inhibition as well as having insecticidal activity and anthelmintic activity, and completed the present invention.
The present invention has an aspect for providing novel
FKI-1033 substance, which is effective as agrochemicals,
veterinary medicines and pharmaceuticals having an activity of ryanodine binding inhibition, an insecticidal activity and anthelmintic activity and production thereof.
FKI-1033 substance can be obtained by culturing a microorganism belonging to fungi having ability to produce
FKI-1033 substance in a medium, accumulating FKI-1033 substance in the cultured mass and isolating FKI-1033 substance from the cultured mass.
An object of the present invention is to provide novel
FKI-1033 substance represented by the following chemical structure:
CH,
CH
N_ CHa 0
A 0 pe CH, 0] 0) 0]
H;C—N 0] 0
N-CHa, o. 0 NA 0)
HiC < CHa
N 0) : HC
CHs
CH,
Another object of the present invention is to provide a process for production of novel FKI-1033 substance comprising culturing a microorganism having ability to produce FKI-1033 substance in a medium, accumulating FKI-1033 substance in the cultured medium and isolating FKI-1033 substance from said
® cultured mass.
Further object of the present invention is to provide a microorganism Verticillium sp. FKI-1033 FERM BP-8291 belonging to genus fungi.
Further object of the present invention is to provide the microorganism Verticillium sp. FKI-1033 having ability to produce FKI-1033 substance and belonging to genus fungi.
Further object of the present invention is to provide the microorganism Verticillium sp. FKI-1033 FERM BP-8291 having ability to produce FKI-1033 substance and belonging to genus fungi.
Further object of the present invention is to provide the process for production of novel FKI-1033 substance wherein the microorganism having ability to produce FKI-1033 substance is
Verticillium sp. FKI-1033 FERM BP-8291 or its mutant strain having ability to produce FKI-1033 substance.
Further object of the present invention is to provide
FKI-1033 substance having an activity of ryanodine binding inhibition and FKI-1033 substance having insecticidal activity and anthelmintic activity.
Further object of the present invention is to provide use of FKI-1033 substance for production of agrochemicals, veterinary medicines and pharmaceuticals having insecticidal activity and anthelmintic activity among substances inhibiting ryanodine binding to the ryanodine receptor.
Further object of the present invention 1s to provide
FKI-1033 substance for production of agrochemicals, veterinary medicines and pharmaceuticals having insecticidal activity and anthelmintic activity among substances inhibiting ryanodine
® binding to the ryanodine receptor.
The microorganism having ability to produce novel FKI-1033 substance represented by the formula hereinabove (hereinafter designates as “FKI-1033 substance producing microorganism”) is fungus, and can be the microorganism having ability to produce
FKI-1033 substance of the present invention without specific limitation. Preferable example of the strain used for production of FKI-1033 substance of the present invention is the strain Verticillium sp. FKI-1033, which was newly isolated froma soil sample collected at Kagoshima Pref. by the inventors of the present invention. Taxonomical properties of the strain are as follows. (1) Morphological characteristics
The strain showed relatively good growth on potato glucose agar medium, cornmeal agar medium, Miura's agar medium, Malt extract agar medium and catmeal agar medium. Conidiogenesis on each medium was moderate.
Microscopic observation of colonies grown on the malt extract agar medium was shown hyphae with septa, and conidiophores were born from the aerial mycelia with branch in some times. The phialide was observed directly from aerial hyphae or alone or two to four verticillations in the middle part or the top of conidophores. Length of the phialide was 25 - 80 pm with slightly swelling in the base (2.0 - 2.8 um) and narrowing with the cuneate shape of the top. Conidia were suglobose to oval (2.5 ~- 4.0 X 2.0 - 3.0 um) and formed viscous conidial mass from the top of phialide.
(2) Culture properties on various agar media
Macroscopic observations of the strain culturedon various agar media at 25°C for 7 days are shown in Table 1.
Table 1
Medium Growth Color of | Color of | Soluble condition on | surface of | reverse pigment the medium | colony side of (diameter of colony colony)
Potato - | Moderate White Pale None glucose (42-44 mm), yellowish agar Floccose - brown velvety,
Entire smooth penumbra
Cornmeal Moderate White Pale None agar (39-42 mm), yellowish
Powdery - brown velvety,
Entire smooth penumbra
Miura's Moderate White White None agar (44-45 mm),
Powdery - velvety,
Entire smooth penumbra
Malt Moderate White White - | None extract (42-46 mm), Pale agar Floccose - yellowish velvety, brown
Entire smooth penumbra
Oatmeal Moderate White White None agar (43-46 mm),
Floccose - velvety,
Entire smooth penumbra (3) Physiological properties (1) Optimum growth condition
Optimum growth conditions of the strain were pH 5 - 7 and 18.5 =- 29.0°C. (2) Growth range
The pH range for growth of the strain was 4 - 10, and the temperature range was 6.5 - 31°C. (3) Nature: aerobic
As a result of comparison with the morphological properties, culture properties and physiological properties of the strain FKI-1033 and known strains, the present strain was identified as a strain belonging to genus Verticillium, and was referredtoVerticillium sp. FKI-1033. The strain was deposited as Verticillium sp. FKI-1033 in the International Patent
Organism Depositary, National Institute of Advanced Industrial
®
Science and Technology of AIST Tsukuba Central 6, 1-1, Higashi l-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan. Date of depository was October 21, 2002 as deposition No. FERM BP-8219.
Production of FKI-1033 substance of the present invention can be performed by culturing FKI-1033 substance producing microorganism belonging to fungi in a medium, isolating from the cultured mass thereof and purifying the same. Since general taxonomical properties of microorganisms are very easily mutated and are not stable, not only the natural mutants but also the artificial mutants obtained by conventional ultraviolet irradiation or treatment with mutant inducer such as N-methyl-N’-nitro-N-nitrosoguanidine, including all
FKI-1033 substance producing strains belonging to fungi can be used in the present invention.
Nutrient sources suitable for production of FKI-1033 substance can be nutrient sources of fungi. For example, the nutrient sources can be used are commercially available nitrogen sources such as peptone, meat extract, corn steep liquor, cotton seed powder, peanuts powder, soybean meal, yeast extract,
NZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate and ammonium sulfate, carbohydrates such as glycerin, starch, . glucose, galactose and mannose, or carbon sources such as fatty acids, and inorganic salts such as sodium chloride, phosphate, calcium carbonate and magnesium sulfate, alone or in combination thereof.
If necessary, trace metal salts, animal, vegetable and mineral cil as an antifoaming agent can be added. These are preferable substances which can be used by the producing
® microorganism and useful for production of FKI-1033 substance, and all known materials for culturing fungi can be used. In a mass production of FKI-1033 substance, liquid culture is preferably applied. Culturing temperature can be applied within a range of the growth of the producing strain and the production of FKI-1033 substance. Culturing condition hereinabove described can be optionally selected depending on properties of FKI-1033 substance producing microorganism.
FKI-1033 substance can be extracted by using water immiscible organic solvent such as chloroform and ethyl acetate from the cultured liquid. In addition to the above extraction, known isolation methods used for collection of fat-soluble substance such as adsorption chromatography, partition chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal <counter-current chromatography and high performance liquid chromatography can be applied in combination or repeatedly to obtain purified substance.
Physicochemical properties of FKI-1033 substance of the present invention are as follows. (1) Nature: colorless oil (2) Molecular weight: 875.5397 (M+Na, high resolution fast atom bombardment mass spectrometry) (3) Molecular formula: C44H76N4015 (4) Specific rotation: [a]p?®> = -53.0° (c = 0.2, methanol) (5) Ultraviolet absorption spectrum (in methanol): maximum absorption at 202 nm (eg = 19700) (6) Infrared abscrption spectrum (KBr): maximum absorption at 3467, 2956, 2933, 2862, 1743, 1662, 1464, 1416, 1205, 1084 cm
® (7) 'H proton NMR: Chemical shift in deuterated chloroform and spin coupling constant were measured by using Unity Inova 600
NMR spectrometer, Varian Inc., the U.S. Chemical shift (ppm) and spin coupling constant (Hz) are shown in Table 2. (8) 13C NMR: Chemical shift in deuterated chloroformwas measured by using Unity Inova 600 NMR spectrometer, Varian Inc., the U.S.
Chemical shift (ppm) is shown in Table 2. (9) Solubility in solvent: Soluble in methanol, ethanol, ethyl acetate and chloroform. Hardly soluble in water and n-hexane. (10) Color reaction: Positive for sulfuric acid.
Table 2 13¢ 4 171.2s 171.0s 170.8s x 4 170.0s 169.9s x 2 169.8s 169.3s x 3 169.2s 71.6d 5.11dd (1H, J=2.5, 10.6) 71.3d x 3 5.42m (3H) 71.0d 5.45dd (1H, J=5.3, 8.4) 70.8d 5.36dd (1H, J=5.3, 9.0) 70.7d 5.32dd (1H, J=3.3, 10.1) 54.4d 4.56g (1H, J=7.3) 51.94d x 3 5.40qg (3H, J=7.2) 51.85d 5.54q (1H, J=7.4)
( 51.76d 5.30g (1H, J=7.4) 51.5d 5.53q (1H, J=7.3) 31.5q 3.18s (3H) 31.40t x 5 1.31m (10H)
31.36t x 2 1.30m (4H) 31.2¢t 1.78m (2H) 31.1t 1.78m (2H) 31.0t x 3 1.78m (6H) 30.98t 1.78m (2H)
30.97q 2.96s (3H) 30.92t 1.78m (2H) 30.73q x 3 2.91s (9H) 30. 66g 3.01s (3H) 29.4q 2.89s (3H) 25.1t 1.30m (2H) 24.83t 1.30m (2H) 24.82t 1.30m (2H) 24.76t x 4 1.30m (8H) 22.42t x 4 1.30m (8H) 22.39t x 3 1.30m (6H) 15.99 1.59d (3H, J=7.3) 15.0q 1.41d (3H, J=7.4) 14.8q 1.454 (3H, J=7.3) 14.2g x 3 1.38d (9H, J=7.2) 14.0q 1.39d (3H, J=7.4) 13.93q x 3 0.88m (9H) 13.90q 0.88m (3H) 13.88g x 3 0.88m (9H)
In the table, each symbol shows: s: singlet, d: doublet, t: triplet, g: quartet, m: multiplet, H: number of proton, J: coupling constant (Hz).
As a result of detailed discussion and examination of various physicochemical properties and spectral data of
FKI-1033 substance, chemical structure of FKI-1033 substance was determined to be represented by the following formula. In the proton and °C NMR spectra of FKI-1033 substance, 1.75 times signal strength above the molecular formula were shown. This may be due to that FKI-1033 substance may exist in the form of two types of the conformational isomer with a ratio of 3 : 4.
In one conformational isomer, the signal number can only be observed in 1/4 thereof. This suggests that the conformation is four-fold rotation symmetry.
CH,
CH o N. CHa
NA 0 3 CHa, 0) 9) 0]
HsC—N 0] 0
N—CHj; oO 0 NA oO : H,C < CHs
N 0) : HC |}
CH,
CHj
As described hereinabove, various physicochemical
_ properties of FKI-1033 substance are described in detail, and no compound identical with these properties has been reported, consequently FKI-1033 substance was determined as a novel substance.
The ryanodine binding inhibition activity of FKI-1033 substance of the present invention will be explained in detail as follows. (1) Preparation of the crude ryanodine receptor fraction of the insect was performed as follows.
The thorax and legs of ten adult insects of Periplaneta americana were frozen with liquid nitrogen and finely cut, and homogenized three times in 15 ml of 50 mM Tris HCl buffer (pH 7.4) containing 4 pg/ml leupeptin and 3 ug/ml pepstatin by using a knife homogenizer at 0°C, 24, 000 rpm for 30 sec. The homogenate was filtered using double layered cotton cloth and the filtrate was centrifuged at 4°C, 3,000 rpm for 20 minutes. The obtained supernatant was centrifuged at 4°C, 30,000 rpm for 30 minutes and the precipitate suspended in 1 ml of 20 mM Tris HCl buffer (pH 8.0) containing 0.3 M sucrose, 500 uM calcium chloride and 1.5 M potassium chloride and homogenized with glass potter to obtain crude ryanodine receptor fraction. The crude ryanodine . receptor fraction was used by diluting about 7 times with the buffer A (20 mM Tris-HCl buffer (pH 8.0) containing 4 ug/ml aprotinin, 4 pg/ml leupeptin, 0.3 M sucrose and 500 uM calcium chloride) immediately before use. (2) Preparation of mammalian crude ryanodine receptor fraction was performed as follows.
_
Muscles of the hind legs of ICR mouse were finely cut, and 3.0 g thereof was homogenized at 0°C, 1,000 rpm for 3 x 30 sec using glass homogenizer in 15 ml of buffer B (20 mM Tris-HCl buffer (pH 8.0) containing 4 pg/ml aprotinin and 4 ug/ml leupeptin). The homogenate was transferred into the micro test tube, and the glass homogenizer was also washed with the 7.5 ml of buffer B, and the washed solution was also added to the micro test tube, which was stirred, then centrifuged by using ultracentrifuge cooled at 4°C, 25,000 x g for 40 min. 2.4 ml of the buffer B was added to the obtained precipitate and suspended to obtain the crude ryanodine receptor fraction of mouse origin. (3) Binding activity of crude ryanodine receptor of Periplaneta americana was assayed as follows. 70 pl of the buffer A, 10 pl of ethanol solution of 0.1 nM ryanodine and 10 pl of 20 nM (9,21-2H] (N) ryanodine were added into the 96 well microplate (Corning Inc. the U.S.). 10 nl of crude ryanodine receptor fraction of Periplaneta americana was added to initiate the reaction, and shaken at room temperature for 90 min. by using microplate mixer. The reaction mixture was filtered with glass fiber filter (Wallac Inc, Printed Filtermat
B) using harvester 96°Mach III M (Tomtech Inc.), and washed three times with 300 pl of 20 mM Tris HCl buffer (pH 8.0) at low temperature. The filter was driedwith using the microwave oven.
A scintillator sheet (Wallac Inc. the U.S., MeltiLex™ B/HS) was overlaid thereon and the sheet was fused on the hot plate.
Radioactivity was measured by measuring fluorescence intensity of the filter with using scintillation counter (Wallac Inc. the
®
U.S., MicroBeta™ TriLux)
The ryanodine binding inhibition activity of FKI-1033 substance was assayed by adding FKI-1033 substance initially in the assay of ryanodine binding activity and calculated by the following equation as compared with the control ryanodine binding activity.
Activity of ryanodine binding inhibition = (1 - (Radioactivity at addition of FKI-1033 substance/
Radioactivity of control) )x100
According to the calculated result of the above equation,
FKI-1033 substance showed the ryanodine binding inhibition activity, ICsp = 4.2 uM, against the ryanodine receptor of
Periplaneta americana origin. (4) Binding activity of crude mammalian ryanodine receptor was assayed as follows. 70 pl of the buffer A, 10 pl of ethanol solution of 0.1 nM ryanodine and 10 pl of 20 nM [9,21-3H] (N) ryanodine were added into the 96 well microplate (Corning Inc. the U.S.). 10 ul of crude ryanodine receptor fraction of mouse origin was added to initiate the reaction, and shaken at room temperature for 90 ) min. by using microplate mixer. The reaction mixture was filtered with glass fiber filter (Wallac Inc, Printed Filtermat
B) using Harvester 96°Mach ITI M (Tomtech Inc.), and washed three times with 300 pl of 20 mM Tris HCl buffer (pH 8.0) at low temperature. The filter was dried with using the microwave oven.
BA scintillator sheet (Wallac Inc. the U.S., Meltilex® B/HS) was overlaid thereon and the sheet was fused on the hot plate.
Radioactivity was measured by measuring fluorescence intensity of the filter with using scintillation counter (Wallac Inc. the
U.S., MicroBeta™ TriLux).
The ryanodine binding inhibition activity of FKI-1033 substance was assayed by adding FKI-1033 substance initially in the assay of ryanodine binding activity and calculated by the following equation as compared with the control ryanodine binding activity.
Activity of ryanodine binding inhibition = (1 - (Radioactivity at addition of FKI-1033 substance/
Radioactivity of control))x100
According to the calculated result of the above equation,
FKI-1033 substance showed the ryanodine binding inhibition activity, ICsp = 53.9 uM, against the ryanodine receptor of mouse origin.
Consequently, according to the above assay results,
FKI-1033 substance was confirmed to exhibit more than ten times potent ryanodine binding inhibition activity against the insect ryanodine receptor than the mammalian ryanodine receptor. (5) Antimicrobial activity of FKI-1033 is shown as follows.
The paper discs (Advantech Inc., Japan, diameter 6 mm) were soaked with 10 pl of methanol solution (1 mg/ml) of FKI-1033 substance, and the solvent was removed with drying for constant time. Then the discs were put on the agar plate containing each test microorganism. Agar plates were incubated at 35°C for time. 24 hours, and the diameter of growth inhibition zone surrounding
_ the paper disc was measured. Results are shown in Table 3.
Table 3
Test microorganism Inhibition zone diameter (mm)
Staphylococcus aureus ATCC 6538p -
Bacillus subtilis ATCC 6633 -
Micrococcus luteus ATCC 9341 -
Mycobacterium smegmatis ATCC 607 -
Escherichia coli NIHJ -
Escherichia coli NIHJ JC-2 (IFO 12734) -
Pseudomonas aeruginosa IFO 3080 -
Xanthomonas campestris pv. oryzae KB 88 -
Bacteroides fragilis ATCC 23745 -
Acholeplasma laidrawii KB 174 -
Candida albicans KF1 -
Saccharomyces cerevisiae KF 26 -
Pyricularia oryzae KF 180 -
Aspergillus niger ATCC 6275 -
Mucor racemosus IFO 4581 -
As shown in Table 3 hereinabove, FKI-1033 substance exhibited no growth inhibitory activities against various microorganisms. (6) Anti-nematode and anti-arthropod activities of FKI-1033 substance will be explained in detail as follows.
Methanol solution of FKI-1033 substance was added into 96 well plate (Corning Inc., the U.S.), removed off methancl in vacuo, added 250 pul of test medium (lecithin 0.01%, sodium
® hydrogen carbonate 7.5 mM, potassium chloride 7.5 mM, calcium chloride dihydrate 7.5 mM and magnesium sulfate heptahydrate 7.5 mM) and shaken for 15 min. 50 pl of buffer containing several nauplii of Artemia salina, which were hatched off in the buffer, was added thereto, and about ten nematodes, Caenorhabditis elegans, which were bred on agar medium for growth of nematode, were added thereto.
Appearance of these organisms were observed after 2 days under microscope, and FKI-1033 substance inhibited growth of the arthropods and nematodes at 20 pg/ml.
Consequently, FKI-1033 substance «can be used as pharmaceuticals such as ryanodine binding inhibitor, insecticide or anthelmintic agent.
Best mode for carrying out the invention
The present invention is explained with an example but is not construed as limiting within the example.
Each loopful strain of Verticillium sp. FKI-1033 FERM
BP-8219 cultured on the agar slant medium was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liguid medium (pH 6.0) consisting of glucose 2.0%, Polypepton (Nihon Seiyaku
Co., Japan) 0.5%, yeast extract (Oriental Yeast Co., Japan) 0.2%, agar 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05% and water, and shake cultured at 27°C for 3 days. Each 20 ml of this seed culture liguid was added into each one of 4 flasks of 500 ml Erlenmeyer flask containing each 80 ml of sterilized and deionized water and diluted. Each 10 ml of the diluted seed culture was inoculated into 24 Roux flasks (capacity 1 lit.) containing 240 g of production medium
® (Italian rice 150 g, tap water 90 ml, iron (II) sulfate heptahydrate 0.9 mg, copper (II) sulfate pentahydrate 0.9 mg and cobalt chloride hexahydrate 0.9mg), and cultured statically at 27°C for 13 days.
A mixed solution of methanol : water = 2 : 1 (200 ml) was added to each of the 24 Roux flasks (1 1it.), vigorously agitated and allowed to stand for 3 hours. Methanol was removed in vacuo, and the obtained aqueous solution was extracted twice with equal amount of ethyl acetate. Using anhydrous sodium sulfate, the ethyl acetate solution was dehydrated and dried in vacuo to obtain 2.53 g of dark brown oily substance. This was charged on a silica gel column (p 2.8 x 14.0 cm) packed with hexane, washed with a mixture of hexane - ethyl acetate (100: 75), eluted with a mixture of hexane ~ ethyl acetate (100 : 150) and concentrated in vacuo to obtain 183 mg of dark brown oily substance. The substance was charged on Sephadex LH-20 column: (p 2.7 x 92 cm) and eluted with methanol to obtain 86.5 mg of
FKI-1033 substance as colorless oily substance.
As explained hereinabove, FKI-1083 substance is produced by culturing the microorganism having ability to produce
FKI-1033 substance in the medium, accumulating FKI-1033 substance in the medium, and isolating FKI-1033 substance from the cultured mass. FKI-1033 substance has ryanodine binding inhibition activity, insecticidal activity and anthelmintic activity, and 1s expected as useful novel FKI-1033 substance for agricultural chemicals, veterinary drugs and medicaments which can be satisfactorily on the point of effectiveness,
toxicity, etc.
Claims (22)
1. A FKI-1033 substance represented by the formula: CH CH o N. CHa oo {8 3 CHy o) d Q HC—N 0} 0) N-CH, oi® 0 9A 0) HC < CH, N 0) : HC CH, CHa
2. A process for production of FKI-1033 substance comprising culturing a microorganism belonging to fungi and having ability to produce FKI-1033 substance in a medium, accumulating FKI-1033 substance in the cultured medium and isclating FKI-1033 substance from the cultured mass.
3. A microorganism which is Verticillium sp. FKI-1033 FERM . BP-821% belonging to fungi.
4. Amicroorganismof Verticillium sp FKI-1033 having ability to produce FKI-1033 substance of claim 1 and belonging to fungi.
5. The microorganism according to claim 4 wherein the microorganism has ability to produce FKI-1033 substance and is
Verticillium sp. FKI-1033 FERM BP-8219.
6. The process for production of FKI-1033 substance wherein the microorganism having ability to produce FKI-1033 substance is Verticillium sp. FKI-1033 FERM BP-8219 belonging to fungi or mutant thereof having ability to produce FKI-1033 substance.
7. FKI-1033 substance according to claim 1 which has ryanodine binding inhibition activity.
8. FKI-1033 substance according to claim 1 which has insecticidal activity and anthelmintic activity.
9. A ryanodine binding inhibitor comprising FKI-1033 S15 substance as an active ingredient.
10. An insecticide and anthelmintic agent comprising FKI-1033 substance as an active ingredient.
11. The ryanodine binding inhibitor, insecticide and anthelmintic agent comprising FKI-1033 substance as the active ingredient.
12. Use of FKI-1033 substance for production of agrochemicals, veterinary drugs and medicaments having insecticidal activity and anthelmintic activity in substances inhibit ryanodine binding to the ryanodine receptor.
13. FKI-1033 substance for production of agrochemicals,
PCT/JP02/11777 S$ veterinary drugs and medicaments having insecticidal activity and anthelmintic activity in substances inhibit ryanodine binding to the ryanodine receptor.
14. A substance or composition for use in a method for inhibiting ryanodine binding to the ryanodine receptor, said substance or composition comprising FKI-1033, and said method comprising administering said substance or composition.
15. A compound according to claim 1, substantially as herein described and illustrated.
16. A process according to claim 2, or claim 6, substantially as herein described and illustrated.
17. A microorganism according to claim 3, or claim 4, substantially as herein described and illustrated.
18. An inhibitor according to claim 9, or claim 11, substantially as herein described and illustrated.
19. An insecticide and anthelmintic agent according to claim 10, or claim 11, substantially as herein described and illustrated.
20. Use according to claim 12, substantially as herein described and illustrated.
21. A substance or composition for use in a method of treatment or prophylaxis according to claim 13, or claim 14, substantially as herein described and illustrated. - 23 - AMENDED SHEET
PCT/JP02/11777 @
22. A new compound, a new process for producing a compound, a new microorganism, a new inhibitor, a new insecticide and anthelmintic agent, a new use of FKI- 1033, or a substance or composition for a new use in a : method of treatment or prophylaxis, substantially as herein described. - 24 - AMENDED SHEET
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ZA200503522A ZA200503522B (en) | 2005-05-03 | 2005-05-03 | Novel substance FKI-1033 and process for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ZA200503522A ZA200503522B (en) | 2005-05-03 | 2005-05-03 | Novel substance FKI-1033 and process for producing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200503522B true ZA200503522B (en) | 2006-07-26 |
Family
ID=38181274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200503522A ZA200503522B (en) | 2005-05-03 | 2005-05-03 | Novel substance FKI-1033 and process for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| ZA (1) | ZA200503522B (en) |
-
2005
- 2005-05-03 ZA ZA200503522A patent/ZA200503522B/en unknown
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