JP5639768B2 - Novel Spoxazomicin A substance and process for producing the same - Google Patents

Description

  The present invention relates to a novel Spoxazomicin A substance effective for animal drugs and pharmaceuticals because it has the growth inhibitory activity of Trypanosoma protozoa and a method for producing the same.

  Trypanosomiasis includes African sleeping sickness prevalent in the African region and Chagas disease prevalent in the South American region. Trypanosomiasis, which is called African sleeping sickness or human African trypanosomiasis, is a reemerging protozoan infection, with an estimated annual infection of more than 18.3 million and approximately 50,000 deaths annually. In particular, Trypanosoma protozoa parasitic on humans are classified into two types: Trypanosoma brucei gambiense and Rhodesia trypanosomes (T. b. Rhodesiens). The former causes chronic sleep disease and the latter causes acute sleep disease. . These infectious diseases are highly infectious diseases in which protozoa are transferred to the central nervous system at the end of infection, and more than 80% of those infected with Trypanosoma protozoa fall into a coma and die. These Trypanosoma protozoa are mediated by tsetse flies that only inhabit Africa.

  Furthermore, the Gambia trypanosomes and Rhodesia trypanosomes are also common animal and livestock parasites, and infest livestock such as cattle, horses and sheep, and wild animals such as gazelle and wildebeest. These animals serve as reservoirs but do not develop. There are also other Trypanosoma protozoa that do not infest humans and infest livestock animals. T. is a protozoan of the subgenus Trypanosoma brucei brucei (Nagana disease), a protozoan of the subgenus Duttonella. There are vivax vivax (Zuma disease) and the like, and when these protozoa are infected, they follow a lethal course of infection depending on the animal species. These are also mediated by tsetse flies.

  The region where tsetse flies inhabit 10 million square kilometers in 36 countries on the west coast from the east coast south of the Sahara Desert on the African continent, and more than 150 million domestic animals have these trypanosomiasis The current situation is exposed to threats. Further, as a tsetse fly non-transmissible trypanosoma protozoan that infects animals by mechanical transmission of blood-sucking insects such as abs and flies, T. is a protozoan of the subgenus Trypanosoma. evansi (Sura disease), T. et al. There is an equiperdum. Surah's disease is particularly prevalent in Africa, Central and South America, Southeast Asia, China, the Middle East and India. Surah's disease is an animal trypanosomiasis that has become more prevalent in recent years and that needs to be most alert to invasion of Japan.

  Existing human antitrypanosomiasis against these trypanosoma protozoa include classical drugs such as suramin (development in 1923), pentamidine (development in 1939), meralsoprole (development in 1953) for the past half century. Chemo-synthetic pharmaceuticals such as Ephronitin (developed in 1978) have long been used.

  Suramin is effective early in the infection of Gambia trypanosomes and Rhodesia trypanosomes but is nephrotoxic.

  Pentamidine is effective in the early stages of Gambia trypanosomiasis infection, but is ineffective against Rhodesia trypanosoma protozoa and has side effects of lowering blood pressure and reducing blood sugar.

  The arsenic agent melalsoprol is effective in the end stage of infection (central neurosis) of Gambia trypanosomes and rhodesia trypanosomes by crossing the blood-brain barrier, but causes encephalopathy due to strong side effects on the central nervous system . In addition, protozoan strains resistant to meralsoprol have also appeared.

  Ephronitin is effective at the end of infection of resistant Gambia trypanosomiasis to which melansoprol does not work by passing through the blood-brain barrier, but is ineffective against Rhodesia trypanosoma protozoa. These drugs are old and their effectiveness is gradually declining. In addition, diminazen, suramin, isometadium, and mutagenic substances such as fomidium have been used for the treatment of trypanosomiasis in animals. In particular, since these drugs have been used in large quantities for a long period of time, drug-resistant protozoa are now appearing in various places, and their usefulness as anti-trypanosoma protozoa has been significantly reduced, which is a big problem.

  Because of the delay in the development of new drugs in African sleeping sickness, existing drugs with strong classical side effects are currently being used for treatment, and there is a need to develop new drugs that are effective globally. ing. Thus, existing human antitrypanosomiasis protozoa have different efficacy depending on the type of protozoa and the stage of infection, and some have emerged drug-resistant protozoan strains. Anti-trypanosoma protozoan, an effective drug regardless of the type of protozoa and the stage of infection, a drug specific to rhodesia trypanosoma protozoa and end stage infection (central neurosis), and has a novel skeleton with few side effects The development of trypanosoma protozoa is desired on a global scale.

  Trypanosomiasis is caused by the growth of protozoan trypanosomes in host cells. Substances that inhibit the growth of Trypanosoma protozoa are expected to be anti-trypanosoma drugs that suppress or reduce the onset of trypanosomiasis.

  Thus, as a result of continuing to search for substances that inhibit the growth of Trypanosoma protozoa from microbial cultures, the present inventors have found that a novel Spoxazomicin A substance produced by actinomycete K07-0460 has its inhibitory activity, The present invention has been completed based on this finding.

The present invention has been completed based on such findings, and includes a novel Spoxazomicin A substance represented by the following formula (I) and Streptosporangium sp. K07- which produces this Spoxazomicin A substance . This is a method for producing Spoxazomicin A substance characterized in that 0460 (NITE P-864) is cultured in a medium, Spoxazomicin A substance is accumulated in the culture, and Spoxazomicin A substance is collected from the culture.

The present invention also relates to a trypanosoma protozoan growth inhibitory active substance comprising the Spoxazomicin A substance according to claim 1 as an active ingredient, and an antitrypanosoma protozoan drug comprising the Spoxazomicin A substance according to claim 1 as an active ingredient.

  As an example of the strain used for producing the novel Spoxazomicin A substance represented by the above-mentioned formula I of the present invention, Streptosporangium sp. Streptosporangium sp.) K07-0460 (NITE P-864).

  Streptosporangium sp. The mycological properties of K07-0460 are as follows.

(I) Morphological properties The strain grew well on oatmeal agar, sucrose / nitrate agar, starch inorganic salt agar, etc., and spore formation was observed on oatmeal agar and sucrose / nitrate agar. A spore sac having a diameter of about 5 μm is observed on the tip of the aerial hyphae or on the lateral spore sac, and its form is spherical.

(II) Properties on various media Methods of EB Shirring and D. Gottlieb (International Journal of Systematic Bacteriology, Vol. 16, page 313) 1966), the culture properties of the production bacteria are shown in Table 1. The color tone was determined using the Color Harmony Manual 4th edition (Container Corporation of America Chicago, 1958) as a standard color, and the code was also written in parentheses along with the color chart name. The following are the results of observation with the naked eye in each medium at 27 ° C. for 2 weeks unless otherwise specified.

(III) Physiological properties (1) Formation of melanin pigment (b) Tyrosine agar negative (b) Peptone yeast / iron agar negative (c) Tryptone yeast liquid negative (d) Glucose / peptone / gelatin medium (21- Negative (2) Reduction of nitrate Negative (3) Liquefaction of gelatin (21-23 ° C) False positive (glucose, peptone, gelatin medium)
(4) Starch hydrolysis Positive (5) Coagulation of skim milk (32 ° C) Negative (6) Peptonization of skim milk (32 ° C) False positive (7) Growth temperature range 13-36 ° C
(8) Utilization of carbon source (Pridham Gotrib Agar)
Utilized: D-glucose, D-xylose, D-mannitol,
D-fructose not used: L-arabinose, raffinose, melibiose,
L-rhamnose, myo-inositol, sucrose (9) Cellulose degradation Negative

(IV) Cell chemical composition The cell wall contains meso-type diaminopimelic acid. Majurose is included as a whole cell sugar. The main menaquinones are MK-9 (H ₂ ) and MK-9 (H ₄ ). Cell wall muramic acid is acetyl type. Mycolic acid is not included.

(V) 16S rRNA gene analysis A sequence of about 1400 bases in the 16S rRNA gene is determined, and a neighbor binding method using data of strains belonging to the genus Streptosporangium and other actinomycetes registered in the DNA database and published As a result of the phylogenetic analysis, it is appropriate to classify this strain into the genus Streptosporangium.

(VI) Conclusion As described above, the bacteriological properties of this bacterium are summarized as follows. Diaminopimelic acid in the cell wall is meso-type, and whole cell sugar contains majurose. Cell wall muramic acid is acetyl type, and main menaquinones are MK-9 (H ₂ ) and MK-9 (H ₄ ). Does not contain mycolic acid. A spherical spore capsule having a diameter of about 5 μm is formed. The colony has a red color tone and does not produce a melanin pigment, but produces a red soluble pigment.

  From these results and the analysis result of 16S rRNA gene, this strain was determined to be one strain belonging to the genus Streptosporangium based on the Burseys Manual of Systematic Bacteriology, Vol. 4, 1989. This strain was deposited as Streptosporangium sp. K07-0460 at the Patent Organism Depositary, National Institute of Technology and Evaluation. The deposit date is January 18, 2010, and the deposit number is NITE P-864.

The physicochemical properties of Spoxazomicin A substance are as follows.
(1) Property: white powder (2) Molecular weight: 336
(3) Molecular formula: C ₁₆ H ₂₁ O ₃ N ₃ S
[M + H] ⁺ theoretical value (m / z) 336.1445, measured value (m / z) 336.1375 by fast atom collision ionization
(4) Specific rotation: [α] _D ³¹ = −34.3 ° (c = 0.1, methanol)
(5) Ultraviolet absorption maximum λ _max (in methanol): 304.0 nm (1.16), 260.5 nm (1.14), 251.0 nm (1.36), 243.5 nm (1.43), It has a maximum absorption at 208.0 nm (1.98). In the parentheses, log ε.
(6) Infrared absorption maximum ν _max (KBr tablet): Maximum absorption at 3440, 2921, 1637, 1367, 1076 cm ⁻¹ .
(7) Proton nuclear magnetic resonance spectrum: Table 2 shows the chemical shift (ppm) and spin coupling constant (Hz) in deuterated methanol.
(8) Carbon nuclear magnetic resonance spectrum: Chemical shift (ppm) in deuterated methanol is shown in Table 2.
(9) Solubility in solvents: Easily soluble in chloroform, acetone, methanol and ethyl acetate. Normal hexane, hardly soluble in water.
(10) Color reaction: Positive for Dragendorf reagent.

  In Table 2, s is a single line, d is a double line, m is a multiple line, H is the number of protons, and J is a spin coupling constant (Hz).

  As a result of examining various physicochemical properties and spectral data of Spoxazomicin A substance, it was determined that Spoxazomicin A substance has a structure represented by the aforementioned formula (I).

  As described above, the various physicochemical properties of Spoxazomicin A substance have been described in detail. However, a compound that matches such a property has not been reported so far, and it was determined that Spoxazomicin A substance is a novel substance.

  As described above, the present invention provides a novel Spoxazomicin A substance and a method for producing the substance from Actinomyces Spoxazomicin A strain. This provides a substance that inhibits the growth of Trypanosoma protozoa and a method for producing the same.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

  In producing the Spoxazomicin A substance of the present invention, first, microorganisms having the ability to produce Spoxazomicin A substance belonging to actinomycetes may be cultured, and separated and purified from the culture. As the strains that can be used in the present invention, the above strains, mutants thereof, and all Spoxazomycin A substance-producing bacteria belonging to actinomycetes can be used.

  The nutrient source suitable for the production of Spoxazomicin A substance may be any nutrient source that can be used as a nutrient source for actinomycetes. For example, commercially available peptone, meat extract, corn steep liquor, cotton seed flour, peanut flour, soybean flour, yeast extract, NZ-amine, casein hydrate, nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, glycerin , Carbohydrates such as starch, glucose, galactose, and mannose, or carbon sources such as fat, and inorganic salts such as sodium chloride, phosphate, calcium carbonate, and magnesium sulfate can be used alone or in combination.

  In addition, if necessary, a trace amount of metal salt, vegetation / planting / mineral oil, etc. can be added as an antifoaming agent. Any of these may be used as long as they can play a role in the production of Spoxazomicin A substance using the produced bacteria, and all known actinomycete culture materials can be used. Moreover, the culture temperature of Spoxazomicin A substance can be applied as long as the producing bacteria grow and Spoxazomicin A substance can be produced. Culturing can be carried out by appropriately selecting according to the properties of Spoxazomicin A substance-producing bacterium using the conditions described above.

  Spoxazomicin A substance can be extracted from the culture solution with a water-immiscible organic solvent such as ethyl acetate. In addition to the extraction methods described above, known methods used for collecting fat-soluble substances, such as adsorption chromatography, partition chromatography, gel filtration chromatography, scraping from thin layer chromatography, centrifugal countercurrent distribution chromatography, high speed It can be purely collected by appropriately combining or repeating liquid chromatography.

  From Streptosporangium sp. K07-0460 (NITE P-864) cultured in a liquid medium, starch 2.4%, glucose 0.1%, peptone [manufactured by Kyokuto Pharmaceutical Co., Ltd.] 0 Liquid medium (pH 7) consisting of 0.3%, skipjack extract [manufactured by Kyokuto Pharmaceutical Co., Ltd.] 0.3%, yeast extract [manufactured by Oriental Yeast Co., Ltd.] 0.5%, calcium bicarbonate 0.4% 1.0) was inoculated into a 500 ml Erlenmeyer flask containing 100 ml, and cultured with shaking at 27 ° C. for 5 days. The obtained seed culture solution was starch 2.4%, glucose 0.1%, peptone [manufactured by Kyokuto Pharmaceutical Co., Ltd.] 0.3%, skipjack extract [manufactured by Kyokuto Pharmaceutical Co., Ltd.] 0.3%, Yeast extract [Oriental Yeast Industry Co., Ltd.] Inoculate 1 ml each into 60 500 ml Erlenmeyer flasks containing 100 ml of liquid medium (pH 7.0) consisting of 0.5% and calcium bicarbonate 0.4%. And cultured with shaking at 27 ° C. for 5 days.

  100 ml of ethanol was added to each of 60 500 ml Erlenmeyer flasks that had been cultured, and stirred vigorously for 1 hour. Next, ethanol in the extract was distilled off under reduced pressure, and the pH of the aqueous solution obtained was adjusted to 10 with aqueous ammonia, and then dissolved in the order of 3000 ml of butyl acetate and 15000 ml of 0.1 M hydrochloric acid. After adjusting the pH of the insoluble matter to 10 with aqueous ammonia, extraction with an equal amount of ethyl acetate and concentration to dryness gave 116.9 mg of crude substance 1. The crude substance 1 was placed on a silica gel column (φ17 × 50 mm) packed with chloroform, eluted with chloroform-methanol (100: 2), and concentrated under reduced pressure to obtain 16.6 mg of crude substance 2. Crude substance 2 was dissolved in methanol, injected into an octadecylsilyl column (φ20 × 250 mm) by high performance liquid chromatography, and eluted with acetonitrile-water (45:55). A peak with a retention time of 25.1 minutes was collected, and concentrated under reduced pressure to obtain 3.9 mg of Spoxazomicin A substance represented by the following formula I as a white powder.

  The activity of the Spoxazomicin A substance of the present invention for inhibiting the growth of Trypanosoma protozoa in vitro is described below.

As the test protozoa, Trypanosoma brucei brucei GUTat 3.1 strain (provided by a lecturer from Yoshitoshi Tsuji, Nagoya City University School of Medicine) was used. The maintenance passaging of the protozoa is performed according to the method of Yabu Y, Koide T, Ohota N, Nose M, Ogihara Y. et al. Continuous growth of bloodstream forms of Trypanosoma brucei brucei in axious culture system concentrating of the consortium. Southeast Asian J. et al. Top. Med. Public Health, 29: 591-595 (1998)] was slightly modified. That is, in each well of 24 well plate, using 10% non-immobilized fetal bovine serum (FBS), antibiotics and various supplemented IMDM medium, at 37 ° C. under 5% CO ₂ -95% air. Culture was performed, and continuous culture was performed by exchanging the medium every 1 to 3 days.

In vitro measurement of the anti-trypanosoma protozoan activity of this compound was carried out by the method of Otoguro et al. Selective and Potent in vitro antipanosomal activities of ten micrometabolites. J. et al. Antibiotics, 61: 372-378 (2008)]. That is, 95 μl of a protozoan suspension (adjusted to a number of protozoa of 2.0 × 10 ^{4 to} 2.5 × 10 ⁴ / ml) pre-cultured in each well of 96 well plate and 5 μl of a compound solution (50% ethanol aqueous solution) After adding and mixing, the cells were cultured at 37 ° C. under 5% CO ₂ -95% air for 72 hours.

After completion of the culture, protozoan growth was measured by adding 10 μl of Alamar Blue reagent (Sigma-Aldorich, USA) to each well of 96 well plate, mixing, and mixing at 37 ° C. under 5% CO ₂ -95% air. After culturing for ˜6 hours, the redox potential of the protozoa is measured by measuring the fluorescence intensity at an excitation wavelength of 528/20 nm and a fluorescence wavelength of 590/35 nm with a fluorescence microplate reader (Bio-Tek, USA). The presence or absence of proliferation was colorimetrically determined. The 50% protozoa growth inhibitory concentration (IC ₅₀ value) of this compound was determined from the compound concentration action curve of KC-4 (manufactured by Bio-Tek, USA) of software attached to a fluorescent microplate reader. The antitrypanosome protozoan activity of this compound and known antitrypanosome protozoan agents against cultured trypanosoma protozoa was as shown in Table 3 below.

  As known anti-trypanosoma protozoan agents for cultured Trypanosoma protozoa, suramin and eflonitin (provided by (Prof. R. Brun, Swiss Tropical Institute, Basel, Switzerland)) were used, respectively.

Spoxazomicin A material of the present invention is T.P. b. b. Antitrypanosoma protozoa activity (IC ₅₀ value) against GUTat 3.1 strain is 0.11 μg / ml, and its activity is 14-21 times superior to that of existing antitrypanosoma protozoan agents compared with suramin and efronitin. It showed protozoan activity.

  The cytotoxicity test of the Spoxazomicin A substance of the present invention was carried out by the method of Otoguro et al. [Otoguro K, Kohana A, Manabe C, Shiyama A, Ui H, Shiomi K, Yamada H, Omura S. et al. Potential axial activities of polymer antibiotic, X-206. J. et al. Antibiotics, 54: 658-663, (2001)]. That is, human fetal lung-derived normal fibroblast MRC-5 cells [Dr. L. Maes (available from Tibotec NV, Mechelen, Belgium)] was maintained in 10% fetal calf serum (FCS) and MEM medium supplemented with antibiotics, and subcultured.

The human fetal lung-derived normal fibroblast MRC-5 cells were adjusted to 1 × 10 ³ cells / ml with 10% FCS-MEM, and 100 μl was added to 96 well plate and mixed. After incubating under 5% CO ₂ -95% air for 24 hours, add 90 μl of 10% FCS-MEM and 10 μl of this compound solution (50% ethanol aqueous solution) to each well, mix, Cultivation was carried out for 7 days. The presence or absence of proliferation of MRC-5 cells was colorimetrically determined by the MTT method. The 50% cell growth inhibitory concentration (IC ₅₀ value) of this compound was determined from the compound concentration action curve. The results are shown in Table 4 below.

The cytotoxicity (IC ₅₀ value) of Spoxazomicin A substance of the present invention to human fetal lung-derived normal fibroblast MRC-5 cells is 27.84 μg / ml, and the selective toxicity ratio (cytotoxic IC of cytotoxicity) with antitrypanosome protozoan activity. ₅₀ value / IC ₅₀ value of antitrypanosome protozoan activity) was 253, indicating high selective toxicity.

  The antibacterial activity of Spoxazomicin A substance of the present invention is as follows. 10 μl each of 1 mg / ml methanol solution of Spoxazomicin A substance was immersed in a filter paper disc (Advantech, diameter 6 mm), air-dried for a certain period of time to remove the solvent, and affixed to the test bacteria-containing agar plate of Table 5. Table 5 shows the diameters of the growth inhibition circles formed around the filter paper disc after culturing at 35 ° C. for 24 hours.

  The Spoxazomicin A substance of the present invention showed little antibacterial activity against the microorganisms in Table 5. Therefore, the Spoxazomicin A substance of the present invention can be used as a drug that inhibits the growth of Trypanosoma protozoa or an anti-trypanosoma protozoan agent.

Claims (5)

Spoxazomicin A substance, which is a compound represented by the following formula I:

Streptosporangium sp. K07-0460 (NITE P-864) , which produces Spoxazomicin A substance according to claim 1, is cultured in a medium, Spoxazomicin A substance is accumulated in the culture, A method for producing Spoxazomicin A substance, which comprises collecting Spoxazomicin A substance from a culture. Streptosporangium sp.
) K07-0460 (NITE P-864) strain. A Trypanosoma protozoan growth inhibitory active substance comprising the Spoxazomicin A substance according to claim 1 as an active ingredient. An antitrypanosoma protozoan drug comprising the Spoxazomicin A substance according to claim 1 as an active ingredient.