JPS582680B2 - Hatsukouhou Niyorquinoline -2- Methanol-Oyobiquinaldiyl Acetate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing quinoline-2-methanol and quinaldyl acetate by a fermentation method.

The present inventors are currently researching pharmacologically stable substances produced by actinomycetes, and have discovered a bacterial strain PO-1 belonging to the genus Kitasatoa.
227 strains [Kitasatoa griseophaeus (Kit)
Asatoa griseophaeus)':1 was found to produce quinoline-2-methanol and quinardyl acetate.

These compounds, quinoline-2-methanol and quinardyl acetate, are basic compounds and have the structure represented by the following formula.
8), and quinaldyl acetate is a known substance as reported in Pharmaceutical Journal, Vol. 79, pp. 492-499 (1959).

However, all of the previous reports were obtained by organic synthesis methods, and the present inventors discovered quinoline-2-methanol and quinaldyl acetate for the first time in the metabolites of a new fungal species belonging to the genus Kitasatoa, and cultivated them. These compounds were isolated from

These compounds are basic and positive to Dragendorff's reagent, and fall into the category of so-called alkaloids, have hypoglycemic and analgesic effects, and are expected to be used as pharmaceuticals.

Among the quinoline-2-methanol and quinardyl acetate-producing bacteria used in the present invention, the strain newly isolated by the present inventors from soil (numbered as po-1227 strain) has the following mycological results. have a property.

(1) Morphological characteristics When hyphae grown on a glycerol-asparagine agar medium or an oatmeal agar medium are observed under a microscope, they show the following morphological characteristics.

The hyphae are 0.8 to 1.2 μm in diameter, and their tips extend linearly and branch irregularly.

Spiral branches and whorled branches are not observed, but in addition to many chains of conidia, there are rod-shaped sporangia and motile spores (zoospores).
includes.

The shape of conidia is 1.0 to 1.2 μm. X 0. 6
~0. It has an oval shape of 7 μm, and its surface is smooth.

The shape of zoospores is 2.6-2.7 μm x 0. 7
It is cylindrical with ~0.8ltm.

→((■) Culture properties on various media This strain grows at 15-40℃, but the optimal growth temperature is 2.
The temperature is 5-32°C.

The mycological properties of this bacterial strain PO-1227 are summarized as follows.

The properties of the following various media are as follows:
This is an observation after culturing for ~15 days.

(III) Physiological properties ■ Growth temperature range: 15-40℃ ■ Production of melanin pigment (a) Tryptone/yeast extract solution: negative (mouth) Peptone/yeast/iron agar medium: negative (/chronic Tyrosine agar medium: negative) ■ Starch hydrolysis reaction: Positive ■ Hydrogen sulfide production reaction: Negative ■ Solubility of gelatin: Negative ■ Skim milk conversion: Positive ■ Skim milk coagulation: Negative (IV) Assimilation of various carbon sources (On Pridham-Godlieb agar medium) L-arabinose, D-xylose, D-glucose,
Uses D-fructose and sucrose, but does not use inositol, L-rhamnose, raffinose, or D-mannitol.

(■ Cell wall composition Peiker et al.'s method (Applied Microbiology,
13, pp. 236-243, 1965).

From the above mycological properties, this strain PO-1227 is characterized by containing zoospores in rod-shaped sporangia, in addition to the chain of conidia seen in Streptomyces species.
The genus Kitasatoa established by Matsumae et al.
8th edition, pages 722-723, 1974).

The genus Kitasatoa already includes Kitasatoa purpurea (Kitasatoa).
asatoa purpurea), Kitasatoa diplospo
ra), Kitasatoa kakuienzis (Kitasat
oacaua i ens i s), Kitasatoa nagasak
Four species of B. iensis have been reported.

These standard strains and the present strain PO-1227 were simultaneously cultured and compared, as shown in the following table.

As shown in the table above, strain PO-1227 closely matches the four known species of Kitasatoa in terms of growth on various media, aerial hyphae, soluble pigments, etc., but it is not classified as an actinomycete. Four bacterial species that are known to produce melanin pigment, which is an important indicator, were positive; in other words, they are chromogenic type bacteria, whereas strain PO-1227 does not produce such pigment. , a non-chromogenic type of bacterial species.

Therefore, the PO-1227 strain was distinguished from any of the four known bacterial species, and was determined to be a new bacterial species belonging to the genus Kitasatoa.
Kitasatoa griseophaeus (Ki) based on the color of its growth.
tasatoa griseophaeus).

This Kitasatoa griseophaeus has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the microorganism accession number 3077. Ru.

The present invention was completed based on the above findings, and involves culturing quinoline-2-methanol and quinaldyl acetate-producing bacteria belonging to the genus Kitasatoa in a medium, and producing quinoline-2-methanol and quinaldyl acetate from the culture. Quinoline-2 characterized by being collected
- A method for producing methanol and quinardyl acetate.

Kitasatoa griseophaeus is one specific example of the bacterium used in the present invention, and a mutant strain obtained by mutating this bacterium by artificial mutagenesis means using, for example, ultraviolet rays, X-rays, radiation, chemical agents, etc. Of course, all bacteria belonging to the genus Kitasatoa and having the ability to produce quinoline-2-methanol and quinardyl acetate can be used in the present invention.

As the culture medium used in the present invention, a culture medium commonly used for culturing microorganisms containing a carbon source, a nitrogen source, inorganic substances, etc. is widely used.

The carbon source may be any assimilable carbon compound, such as glucose, maltose, lactose, sucrose, starch, dextrin, glycerin, and molasses.

The nitrogen source can be any available nitrogen compound, such as soybean flour, corn steep liquor, or cottonseed? , peptone, meat extract, yeast extract, yeast, casein hydrolyzate, ammonium salts, nitrates, etc. are used.

Other salts such as phosphate, magnesium, potassium, calcium, sodium, iron, and manganese are used as necessary.

Cultivation is usually carried out aerobically, and aerated agitation culture is preferred.

The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce quinoline-2-methanol and quinaldyl acetate, but a particularly preferred temperature is 25 to 30°C.

Cultivation time varies depending on various conditions, but is usually 90 to 2
The culture is terminated at an appropriate time, which is about 00 hours, and the time required for quinoline-2-methanol and quinardyl acetate to reach the maximum titer is determined.

After completion of the culture, quinoline-2-methanol and quinardyl acetate are collected from the culture.

For example, the culture is separated into bacterial cells and P solution, and the pH of the P solution is made alkaline with aqueous ammonia, etc., and the bacterial cells are eluted with aqueous ammonia, which is usually used in the extraction and purification of alkaloids. By a known method,
Recover quinoline-2-methanol and quinardyl acetate.

The physicochemical properties of quinoline-2-methanol and quinaldylacetate obtained according to the present invention, which will be described below, were consistent with quinoline-2-methanol and quinaldylacetate-1. obtained by organic synthesis.

(1) Quinoline-2-methanol ■ Elemental analysis (as picrate) Measured values: C: 49.47%H: 3.41%, N: 1
3.70% Theoretical value: (C1oH, NO-C6H3N30, C
: 49.52%, H: 3.43%, N: 13.84%) ■ Molecular weight The rV/e of free quinoline-2-methanol measured by mass spectrometry is 159.0690.

Theoretical value is 159.0696 as molecular formula C1oH9NO
It is.

■ Melting point = 67~68℃? Specific optical rotation [α) -0 (C = 1% MeOH) ■ Ultraviolet absorption spectrum λMeOH nm (a:): 230 (47700), 23
3 (44500), 277 (4000) 284 (380
0), 290 (3700), 296 (3300), 30
2 (4000), 308 (2500), 317 (500
0), (Figure 3) ■ Infrared absorption spectrum measured by KBr method: 3200, 1600, 1500, 1420, 13
It shows relatively strong absorption at 10, 1080, 820, 780, and 750.

(Fig. 4) ■ Solubility It is easily soluble in methanol, ethanol, chloroform, and benzene, and slightly soluble in water.

■ Color reaction: Positive for Dragendorff, Mayer, and Wagner reagents, and negative for Ehrrich and ninhydrin reagents.

(2) Quinardyl acetate ■ Elemental analysis (as picrate) Measured values: C: 52.74%, H: 3.60%, N: 12
．． 51% Theoretical value = (C1H1, NO-06H307N3:
C: 50.76%, H: 3.50%, N: 13.90% ■ Molecular weight The rrVe of free quinardyl acetate measured by mass spectrometry is 201.078 (theoretical value is based on the molecular formula C, 2H, , NO2 is 201.078).

■ Melting point: 145-148°C ■ Specific optical rotation [α] = 0 (C = 1% MeOH) ■ Ultraviolet absorption spectrum λMeOH nm (ε) = 230 (47700), 233
(44500), 277 (4000), 284 (380
0), 290 (3700), 296 (3300), 30
2 (4000), 308 (250), 317 (5000
) (Figure 1) ■ Infrared absorption spectrum When measured with CHC13 2960, 1740, 1240, 1050, 8
20 shows relatively strong absorption.

(Fig. 2) ■ Solubility Easily soluble in methanol, ethanol, chloroform, and benzene, slightly soluble in water.

■ Color reaction: Positive for Dragendorff, Mayer, and Wagner reagents, and negative for Ehrrich and ninhydrin reagents.

Next, various pharmacological effects were investigated using quinoline-2-methanol hydrochloride, and remarkable effects were observed in hypoglycemic and analgesic effects.

The results regarding blood sugar lowering effect are shown below.

Wista male rat under constant breeding conditions, weight 110
The effect of quinoline-2-methanol hydrochloride on fasting normoglycemic levels was investigated using samples of around 9.

That is, the animals were fasted 18 hours before the start of the experiment, quinoline-2-methanol was orally administered, and blood sugar levels were measured before drug administration and 2 hours after administration.

Quantification of blood sugar is carried out using Hoffman's method (Journal of Biological Chemistry, Vol. 120, p. 51, 193).
7 years) and was quantified as reducing sugar using an autoanalyzer.

Next, examples of the present invention will be shown, but these are merely examples and do not limit the present invention.

Example (1) Curcose 2%, peptone 0.5%, meat extract 0
．． Prepare a liquid medium containing 5% dry yeast, 0.3% dry yeast, 0.5% salt, and 0.3% calcium carbonate, and adjust the pH to 7.5% by adding concentrated caustic soda solution. After correcting to 0, dispense 100 ml into 500-capacity Sakaguchi flasks and sterilize them.
Mycelia and spores of Kitasatoa griseophaeus are inoculated as a seed fungus, and cultured at 27°C for 2 days to prepare a seed fungus solution.

Next, glucose 1%, starch 2%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.
Correct the pH of quinoline-2-methanol and quinardyl acetate production medium 20.a containing 4% to 7.0, place it in a 30 t jar fermenter and sterilize it, and then add the above seed culture solution at a ratio of 1% to the production medium. Inoculated with 27
Culture with aeration and agitation at ℃ for 3 to 4 days.

15 t of the culture solution containing quinoline-2-methanol and quinardyl acetate thus obtained was corrected to pH 10 with concentrated ammonia water, and 4 t of butyl acetate was added to
Extraction was performed twice.

Subsequently, quinoline-2-methanol and quinardyl acetate from the butyl acetate layer were added to 500,172 liters of 0. IN
Extract with hydrochloric acid solution three times, then extract the hydrochloric acid solution with concentrated ammonia water.
H10 and extracted twice with 500 ml of chloroform.

This chloroform layer was added to 300 ml of 0. 2 with IN hydrochloric acid solution
Extract twice, adjust to pl-110 with concentrated ammonia water,
Extraction was carried out three times with 0ml of ethyl ether.

This ether layer was washed with water, and 51 anhydrous sodium sulfate l-IJ
After drying over 100 ml, it was concentrated and passed through a 15 g silica gel column, using 170 ml of a benzene-acetone solvent system to first elute the quinaldyl acetate, followed by 300 ml of a penzene-acetone mixed solvent to elute the quinoline-acetate.
Elute 2-methanol and collect each 30I71
50 rnl of a saturated picric acid solution in ether were gently added dropwise to obtain a picrate of quinardyl acetate of ~240 and a picrate of quinoline-2-methanol of ~220.

Each picrate was suspended in 100 rrLl of chloroform, and 10-20 rnL of 20% potassium hydroxide aqueous solution was added.
．． After adding and stirring, the chloroform layer was washed with water, and 15 g
Each was dehydrated and dried over anhydrous sodium sulfate, concentrated to dryness under vacuum, and crystallized with 10 mN-hexane to give 120 Tny of quinal diacetate and 140 Tny of quinoline-2-methanol, respectively. Needle crystals were obtained.

(2) Prepare a liquid medium containing 2.0% glucose, 0.5% peptone, 0.5% meat extract, 0.3% dried yeast, 0.5% salt, and 0.3% calcium carbonate, and add caustic soda solution. to pH 7. After correcting to 0, 500rn
One platinum loopful of Kitasatoa griseophaeus was inoculated into each 100,171 liter volume sterilized Sakaguchi flask and cultured at 27° C. for 2 days to prepare a seed culture.

Next, 20 tons of medium containing 2% Chryserol, 2% soya bean meal, and 0.3% salt was adjusted to pH 7.0, placed in a 30-ton jar fermenter, sterilized, and the above seed culture was added to 1%
The cells were inoculated at a ratio of 1, and cultured with aeration at 27°C for 20 to 24 hours.

This was further used as a seed culture, and 200 t of the same medium as the jar fermenter was put into a 400 t tank, and after sterilization, 20 t of the jar fermenter culture solution was inoculated, and the culture was incubated at 27°C for 2 hours.
Perform aerated agitation culture for days.

200 t of the culture solution of Quixatoa griseophaeus containing quinoline-2-methanol and quinaldyl acetate obtained in this way was brought to pH 10 with concentrated ammonia water, and 4 (l) of butyl acetate was added to the culture solution containing quinoline-2-methanol and quinaldyl acetate. Extract the acetate, extract the butyl acetate layer once with 10 t of 0.IN hydrochloric acid solution, once with 5 t of 0.IN hydrochloric acid solution, adjust the 15 t of hydrochloric acid solution to pH 10 with concentrated aqueous ammonia, and extract with 5 t of chloroform. Two extractions were performed.

Add this 10t chloroform layer to 3t 0. Extract twice with IN hydrochloric acid solution, adjust to pH 10 with concentrated aqueous ammonia, and reduce to 70
Extraction was performed three times with 0rd ethyl ether.

This 2.1 ton layer was washed twice with 200 ml of distilled water, dried over anhydrous sodium sulfate, concentrated, and
Through a 50 g silica gel ram, 1.6 t of benzene-acetone solvent system was used to first elute quinardyl acetate, followed by 3 t of benzene-acetone mixed solvent to elute quinoline-2-methanol.

Each was collected, concentrated to 100 rnl, and diluted with picric acid in saturated ether! Gently drop 200 wLl of 1.7 g of quinaldiyl acetate picrate and 1.

4 g of quinoline-2-methanol picrate was obtained.

Each picrate was suspended in 250 rttl of chloroform, and 50-70 WLl of 20% potassium hydroxide aqueous solution was added.
After adding and stirring, the chloroform layer was washed with water, and 150g
Each was dehydrated and dried over anhydrous sodium sulfate, concentrated to dryness under vacuum, and crystallized with 100 ml of n-hexane to give white needle-shaped crystals of sooy quinardyl acetate and 650 ml of quinoline-2-methanol, respectively. Obtained.

[Brief explanation of the drawings] Figure 1 is the ultraviolet absorption spectrum of quinaldiyl acetate, Figure 2 is the infrared absorption spectrum of quinaldiyl acetate 1, and Figure 3 is the ultraviolet absorption spectrum of quinoline-2-methanol. , FIG. 4 shows the infrared absorption spectrum of quinoline-2-methanol.

Claims (1)

[Claims] 1. A quinoline-2-methanol and quinaldyl acetate-producing bacterium belonging to the genus Quixatoa is aerobically cultured in a medium, and quinoline-2-methanol and quinaldyl acetate are collected from the culture. Method for producing methanol and quinardyl acetate.