JPS6345274A - Production of amide derivative of ai-77 compound and salt thereof - Google Patents

Abstract

PURPOSE:To obtain the titled compound having excellent antiulcer activity in high purity in a mass on an industrial scale with simple chemical synthesis, by using AI-77-B as a raw material, dehydrating the material under acidic condition and reacting the resultant 5-membered lactone with ammonia. CONSTITUTION:AI-77 strain (Agency of Industrial Science & Technology Stock No.4066) separated from soil and identified as a strain belonging to Bacillus pumilus is cultured in a medium under aerobic condition. The AI-77-B of formula accumulated in the cultured liquid is dissolved in a dried organic solvent such as ethanol, added with an acid such as hydrochloric acid and subjected to dehydrative cyclization under acidic condition to obtain a 5-membered lactone of formula II. The lactone is dissolved in a dried organic solvent such as anhydrous methanol, dried ammonia gas is introduced into the solution and the lactone is cleaved by ammonolysis reaction to obtain the objective compound of formula II having antibacterial activity in addition to antiulcer activity. The compound is used as a medicine preferably in the form of stable amino salt.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing an amide derivative of the AI-77 type represented by the following formula (1) and a salt thereof having anti-T:R wounding activity.

(Prior Art) As a technique for producing this substance, the Hatsuno method is known (Japanese Patent Publication No. 57-6913). That is, it is produced by culturing a bacterial strain belonging to the genus Bacillus under aerobic conditions, and then going through a complicated purification process that combines various glue atomography and solvent extraction methods from the culture solution.

(Problem to be solved by the invention) In the above conventional method, the production amount of r 1 fermentation is 200 to 25
It is extremely low at 0 mcg/-, and the full production process is complicated. Therefore, there is a need for a production method that can industrially supply a single substance in large quantities with high purity.

(Means for solving the problem yk) Compared to the above-mentioned piggyback method, the present inventors have conducted an in-depth study on a manufacturing method that can industrially supply the compound W with high purity as a pharmaceutical in large quantities. , compared to the compound, K in the culture medium
AI-77-B is produced in much larger quantities during fermentation, and due to its crystallinity, it can be easily isolated from the culture solution. Complete the method to get the following.

That is, in the present invention, a 6A JJk :) chthonic body represented by the following formula (II) is produced by dehydration under AI-77-Bifi conditions, and ammonia is added to the 6A JJk :) chthonic body to form the following formula ( 1) This is a method for producing A (-77 type amide visiting conductor and its salt) as shown in the following.

AI-77-B was isolated from soil by the inventors and was identified as a strain belonging to Bacillus pumilus. By culturing, it accumulates in the positive culture solution and can be collected from the culture solution.

Next, the above-described characteristics of AI-77-B will be described.

Mass spectrometry value, ultraviolet absorption value, infrared absorption 1 of AI-77-H
Direct, NMR@ is shown in Table 1, and the solubility is in water.

Easily soluble in methanol, dimethylformamide, dimethyl sulfoxide, other lower alcohols, dioxane,
It is sparingly soluble in tetrahydrofuran, and almost insoluble in ethyl acetate, chloroform, ether, hexane, and benzene. Regarding stability, the water bath solution is unstable to alkalis, and the lactone de-ice gradually progresses even at room temperature.

For culturing, the method described by Ichifune Tsurichi, which concerns the cultivation of a strain of the genus Bacillus, can be applied. i.e. nitrogen source, carbon source.

Inorganic salts, vitamins, amino acids, nucleic acid-related substances, etc.

25 to 40% in a medium containing substances necessary for the growth of the microorganism.
It is carried out aerobically at C. To carry out cultivation on a large scale,
It is advantageous to carry out an aerated agitation culture.

The bacterial character of strain AI-77 (Agency of Industrial Science and Technology No. 4066) is as follows.

Mycological properties a) Growth status on each culture medium ■ Juice: The medium is almost uniformly turbid and the film does not peel.

■Meat juice agar medium: The area around the colony is mostly smooth.
It exhibits h-type, but at 1 o'clock it becomes rough-type.

Spreads flat. The color is opaque or semi-transparent, with an eye-poly
The color changes from white to cream color.

No dyes are produced.

■Glucose juice agar medium: Growth is better than meat juice agar medium, and the appearance is the same as meat juice agar medium.

■(Latin medium; liquefies slowly.

■Peptone water: Growth is slightly suppressed and the whole becomes slightly cloudy.

■ Lidmus milk; milk does not harden. I don't even use peptone. No discoloration of lid mass was observed.

■Potato medium: Grows in a flame yellowish brown color.

Causes fine wrinkles.

Morphological properties on glucose broth agar medium are 0.5
~Q,9 It exhibits a short culm shape with a size of 2.0 to 5.0μ, and produces elliptical to cylindrical spores that are gram-like. The spores are generally located in the center, and the bulge is not distinct.

b) Physiological properties ■Optimal growth conditions: pH 5 to pH 8, 27 to 35'C aerobic.

(!) Conditions that allow raw hydrogen; pH 5.0 to 10.0.1
0-5C 1, ]) Gram staining; df staining.

(Anti-acidity; no λ) Methyl red test: Positive ■Voges-Puchskauer reaction; Positive■No formation of indole was observed.

■Hardly any generation of arsenic hydrogen is observed.

■No ammonia production was observed.

◎Reduction of nitric salt is not allowed.

■ Catalase production is vigorous.

■ Gently mix the gelaten with the liquid.

[Phase] Does not hydrolyze starch.

[Co.] Use citric acid.

■Grows in a 7-width salt culture solution.

) Do not reduce methylene blue.

■Usability of urea is weak.

C) Utilization of carbon sources At is produced using fructose, saccharose, glucose, and mannitol. Mannose.

A small amount of trehalose is also used to generate acid.

Arabinose, xylose, galactose, lactose, maltose, raffinose, sorbitol.

Inosit, glycerol, α-methylglucosinod,
Does not use inulin, texturin, starch, or cellulin. No gas generation was observed from either carbon source.

The above properties 1kBergey's manual o
f Determinative Bacteriolo
When comparing the 7th and 8th editions of Gy, there are some differences, such as the single box does not produce mt from arabinose and xylose, and does not cause peptone reaction or placement in milk. It corresponds to the characteristics of Pumilus.

Therefore, we determined that the single circle was a strain belonging to Bacillus pumilus, but since there was no known identical strain, we named it Bacillus pumilus AI-77. This strain has been deposited with the Agency of Industrial Science and Technology VtI Research Institute, No. 4066.
It has been deposited as an issue. In the present invention, of all the natural and artificial mutant strains of Bacillus pumilus AI-77, all strains capable of producing AI-77FA can be used.

In the method of the present invention, the steps of producing AI-77-B by culturing and collecting it from a field culture are carried out using a 1% constant method. That is, AI-77-Bij,
The physicochemical properties of AI-77-B [k utilization Then, it is collected from the culture solution using a combination of purification methods.

Nutrient sources used in fermentation include carbon sources, nitrogen sources, inorganic salts, and, if necessary, vitamins, amino acids, and various nutritional factors necessary for the growth of the bacteria used. Examples of carbon sources include glucose, sucrose,
Fructose, mannitol, organic acids, molasses, fi powder, glycerin, etc.

Examples of nitrogen sources include corn steep liquor,
Soybean flour, Famamedia, yeast extract, meat extract, protein ice melt such as peptone and casamino acid.

Inorganic salts such as amino acids and ammonium salts.

For example, metal ions such as sodium, potassium, calcium, magnesium, iron, and ammonium.

Salts containing ions such as phosphoric acid, sulfuric acid, chlorine, carboxylic acid, etc. are used. The culture medium must also contain essential nutrients and vitamins necessary for the growth of the producing bacteria.

Amino acids, nucleic acids related! : Elements are added as necessary.

The AI-77-B production garden is grown under woodcutter conditions.

For example, the microorganism can grow on a variety of media having a wide range of initial pH't-. However, the pH of the medium to start fermentation is 6-8. Preferably at pH 6-7.

Generally, the pH of a culture medium changes somewhat depending on the growth of microorganisms. Culture temperature is 25-40C aerobically for 60 hours.
It will be held for 96 hours.

The AI-77-producing bacteria of the present invention can be cultured on an agar slant, in a shaking flask, in a medium pot or in a large fermentation tank. For mass-produced ICs, fermentation conditions of aeration and stirring are preferred. Under aerobic culture conditions with aeration and stirring, adding an antifoaming agent such as silicone or polypropylene glycol derivatives to the medium may be effective in increasing the accumulation 1k of the AI-77-B component. Regarding the progress of fermentation AI-77-
The production amount of H is culture i! It can be tracked by the increase in ultraviolet absorption at 314 mμ of the tear fluid.

7tAI-77-B produced under aerobic fermentation conditions.

Mainly by utilizing the physical and chemical properties of each fermentation medium,
Isolation can be achieved by a combination of chromatography using ion exchange resins, macroreticular resins, gel filtration agents, adsorbents, solvent extraction, and the like. That is, after culturing aerobically for 48 to 96 hours, culture P liquid fz black reticular resin 1. For example, Amberlite XAD-2 manufactured by Rohm and Haas Co., Ltd.
-4 or Mitsubishi Kasei's Diaion HP- system, and bathed using water and an organic solvent or a mixed solvent of both. As an organic solvent, C, which has a particular affinity with water, is used.
1 to C4 alcohols, acetone, dioxane, tetrahydrofuran, etc. can be used. For example, AI-77-
Since 33 is desorbed from the macroreticular resin XAD-2,
This eluate is concentrated under reduced pressure to obtain a crude sample of AI-77-H. Highly pure crystals of AI-77-H can be obtained by repeated purification using macroreticular resin VC or by recrystallization from hot water or methanol.
Obtainable.

Next, from AI-77-B, the following formula side The production of AI-77-Br-lactone is as follows.

When the organic solvent 1 obtained by thoroughly drying AI-77-B is dissolved in alcohols, tetrahydrofuran, etc. and made acidic by adding an acid, it easily undergoes dehydration and ring closure to form an r-lactone form. When the solvent is distilled off.

AI-77-Br-lactone is obtained as a salt of a primary amine. Free amine is this salt? It is obtained by dissolving in an organic solvent, washing with diluted sodium bicarbonate solution, and drying the organic solvent.

The compound of formula (1) is produced from the compound of formula (1) above.

2. AI-77-B-γ-lactone was dried, then dissolved in an organic solvent, and dried ammonia gas was blown into it.

The r-lactone is ring-opened by the ammonolysis reaction to obtain compound 2 (1). The compound of formula (I) is.

When this is dissolved in an organic solvent, 6 amounts of acid are added and the solvent is distilled off to form an amine salt, a more stable compound is obtained. For use as a medicine, it is preferably used as a stable amine salt.

(Effects of the Invention) The compound of formula CI) obtained according to the present invention has excellent anti-ulcer activity, as is clear from the results of the anti-corrosion test shown in Table 2, and also has antibacterial activity. have.

Anti-ulcer test M was carried out as follows.

Rats (Wistar, 9 males, approximately 1502 mm) were fasted for 24 hours before the start of the test. Using physiological saline as a solvent, a solution or suspension of the test compound at a predetermined concentration is prepared and administered orally to animals. Rats in a wire mesh cage 1 hour after drug administration? The specimen was fixed and immersed in 21C water up to the xiphoid bone for 6 hours. Animals are analogous and dislocated after layer killing nine, blue? It was removed, an incision was made along the large bay, and the inner wall of the stomach was dissected.

0 indicates that almost no ulcer is observed.

Depending on the degree of ulceration, the score is 0.5.1.2.3. Calculate the threat suppression rate using the formula below.

Table 2 shows the formula (dosage of 11 compounds and their ulcer inhibition rate?
show.

Table 2 (Example) Examples of the present invention are shown below.

T to the light minute! i, 7tA dried! -77-810 mmol was dissolved in 10011 I/INHC6-ethanol on a water bath. After 7'5'y, remove the solvent under reduced pressure (pass @2
0-50C) and then dry the residue using a vacuum pump. Next, 50-0.5 mol of 1-sodium carbonate solution was added to this residue, and extraction was performed using 600-mol of ethyl acetate.9. After washing the ethyl acetate layer with saturated saline, add Glauber's salt and leave it overnight for 7 liters.

When ethyl is removed from vinegar under reduced pressure, 3,9
89 (yield 98%) of AI-77-Br-lactone was obtained.

100 mmol of the above AI-77-Br-lactone
Dissolve the fg in anhydrous methanol and boil on a water bath (10-15
C) Blowing dry ammonia gas up to 50 mmol,
After sealing and stirring for 2 hours.

The solvent was removed under reduced pressure, and the solution was dissolved by adding 50 methanol. Supercell 5? After stirring, the solvent was removed under reduced pressure, and the residue was subjected to a titanium ray.

This residue was pulverized using a mortar and then filled with chloroform and placed on a silica gel column (200j'). First, the column was washed with 500-ml chloroform.
Separate the raw Fy, w' with 500 ml of chloroform:methanol; 3:1. Fractions 51 to 150 were solidified to 4 concretions, and when a small amount of methanol was added to the residue and allowed to stand, a single crystal precipitate was formed. The precipitate was washed and collected with ethyl acetate, and the solvent was removed under reduced pressure to obtain 0.519 of the desired compound 18.

Claims (1)

[Claims] The following formula (I) is characterized in that AI-77-B is dehydrated under acidic conditions to form a 5-membered ring lactone, and this is then reacted with ammonia. ▲ Numerical formulas, chemical formulas, tables, etc. ▼(I) A method for producing AI-77 amide derivatives and their salts.