JPH0797357A - Malonic (p-nitrobenzyl alcohol) ester-amide and its production - Google Patents

Abstract

PURPOSE:To obtain the subject novel compound which is useful as an intermediate for medicines or the like. CONSTITUTION:Malonic (p-nitrobenzyl alcohol) ester-amide of the formula. This compound is obtained by allowing a bacterium of Pseudomonas such as NKN 003 strain or of Rhodococcus such as NKN 113 strain to act on p- nitrobenzyl cyanoacetate to effect hydrolysis of the cyano group. The compound of the formula is obtained also by converting mono-p-nitrobenzyl malonate into its acid chloride with a chlorinating agent such as thionyl chloride, phosphorus trichloride or phosphorus oxychloride and by allowing the obtained product to react with gaseous ammonia or aqueous ammonia.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is a novel compound,
Malonic acid (P-nitrobenzyl alcohol) ester-
The present invention relates to an amide and a method for producing the same.

[0002]

2. Description of the Related Art This compound is a compound not described in the literature and its production method is not known.

[0003]

PROBLEM TO BE SOLVED BY THE INVENTION The present invention provides a novel compound useful as an intermediate for pharmaceuticals and the like, and an efficient method for producing the same.

[0004]

[Means for Solving the Problems] Malonic acid (P-nitrobenzyl alcohol) ester-amide and malonic acid (P-nitrobenzyl alcohol cyanoacetate) are prepared by hydrolyzing a cyano group by allowing a microorganism to act on the ester. −
A method for producing a nitrobenzyl alcohol) ester-amide is provided.

To obtain this compound, P-nitrobenzyl alcohol-malonic acid half-ester was added to thionyl chloride, 3
There is a method of reacting with a chlorinating agent such as phosphorus chloride or phosphorus oxychloride to form an acid chloride, and reacting with ammonia gas or ammonia water. It can also be obtained by subjecting P-nitrobenzyl alcohol cyanoacetic acid ester to an enzymatic hydrolysis reaction.

The chemical reaction method is as follows. First, P-nitrobenzyl alcohol-malonic acid half ester is dissolved or dispersed in a solvent which is stable against chlorinating agents such as benzene, toluene, xylene, dichloromethane and dichloroethane.
If necessary, a catalyst such as dimethylformamide is added, and the above chlorinating agent is dropped to react. The reaction temperature is preferably room temperature to 150 ° C. A reaction time of 1 to 5 hours is sufficient. It can be synthesized by blowing ammonia gas at 0 ° C to 80 ° C or adding an excess of aqueous ammonia to the solution containing the acid chloride of P-nitrobenzylalcohol malonic acid half ester thus obtained. I can.

The target product thus produced becomes a precipitate, which can be filtered off and washed with methanol, ethanol or the like to obtain a high-purity product.

Malonic acid (P-nitrobenzyl alcohol-
The ru) ester-amide can be converted to a P-nitrobenzyl alcohol-malonic acid half ester by further enzymatic hydrolysis.

The production method using the microorganism of the present invention will be described in detail. The reaction is carried out by culturing cells of the microorganisms or treated cells (crushed cells, crude / purified enzyme, immobilized cells / enzyme, etc.) in a solvent as the raw material P-nitrobenzyl alcohol cyano. It is carried out by contacting with acetic acid ester. The amounts used and the reaction conditions will be described in detail below.

Water is preferably used as the reaction solvent, but physiological saline or a buffer solution can be used to keep the pH of the solvent stable. Type of buffer is phosphate, trisaminomethane-HC
1, citrate, glycine soda, etc. can be used, but are not particularly limited. Buffer concentration is 1M-0.001
Although M can be used, it may be within the range where the effect as a buffer is exhibited. When water is used, the reaction is accelerated by adding a metal chelating agent such as EDTA. Further, when using the treated product of the above bacterial cells, the addition of an antioxidant such as dithiothreitol or mercaptoethanol enhances the reaction efficiency. In addition, a mixed solvent of water and methanol, ethanol, acetone, acetonitrile or the like can be used. The pH should be kept at pH 5-8. The reaction temperature is
It is good to carry out in the range of 0 ° C to 80 ° C, preferably 5 ° C to 40 ° C. The reaction time requires about 30 minutes to 2 days depending on the type of enzyme and the concentration of the raw material. Since the starting material, p-nitrobenzyl alcohol cyanoacetic acid ester, is unstable in neutral to alkaline water, it is desirable that the reaction be completed in a short time. Raw material P-nitrobenzyl alcohol
The concentration of lucyanoacetic acid ester is 0.1% to 3 with respect to the solvent.
Good results can be obtained with 0% (% by weight), preferably 0.3% to 10%. After the reaction is completed, when the isolated enzyme is used, it can be taken out by filtration. When the microbial cells are used as they are, it is preferable to add an organic solvent to dissolve the target substance, remove the microbial cells by filtration or centrifugation, and then distill off the solvent to separate the deposited precipitate by filtration.
The microorganism used in the present invention can be cultured according to a known culture method. The medium is glucose that can be assimilated,
A normal medium containing a carbon source such as glycerin, a nitrogen source such as ammonium sulfate, inorganic phosphorus, and inorganic nutrients essential for growth can be used. Further, it is also possible to use a medium to which a natural medium such as yeast extract or meat extract is added. Isovaleronitrile, benzonitrile, p at a concentration that does not significantly inhibit growth during the early to mid-stage of culture
-By adding nitriles such as nitrobenzonitrile and cyanoacetic acid, amides such as ε-caprolactam, dimethylacetamide and nicotinamide and metals such as iron, cobalt, manganese and zinc as enzyme inducers, high enzyme activity can be obtained. You can get it. The medium to be used may have a pH of 5 to 9 and a culture temperature of 5 to 40 ° C, preferably 25 to 35 ° C. Culture is 1-7
It should be performed aerobically for about a day and continued until the desired enzyme activity is maximized.

The microorganism used in the present invention is, for example, a novel Pseudomonas bacterium NKN.
Bacterial 003 (Deposit No. FERM P-13850
No.) and a new genus Rhodococcus
s) Bacteria NKN113 bacteria (Ministry of Engineering Research Deposit No. FERM
P-13865). All of these have been deposited at the Institute of Microbial Technology, Institute of Industrial Technology. The characteristics of these two bacteria are shown in Table 1 below.

[0012]

[Table 1]

Based on the above-mentioned mycological properties, BERGEY'S M
ANUAL of Systematic Bacte
As a result of examining the known strains and their differences according to riology Volume 1 and 2, the above 2 strains were as follows.

NKN003 bacterium (Ministry of Engineering Research Deposit No. FERM
P-13850) is an aerobic, non-auxotrophic bacillus of catalase plus having Gram-staining negativeness, motility, and polar hairs as flagella, and produces a fluorescent dye. The characteristics of this bacterium are Pseudomonas (Ps
eudomonas), which is almost the same as the P. putida species in the above-mentioned document except that the somatic cell size of this strain is a little small and acid is generated from D-galactose. From the above points, this bacterium was estimated to be a species closely related to P. putida species. The malonic acid (P
-Nitrobenzyl alcohol) ester-amide is useful as a protective group in the production of carbapenem antibacterial agents and the like.

NKN113 bacteria (Ministry of Industrial Science deposit number FERM
P-13865) is positive for Gram stain, has no motility,
It belongs to the genus Rhodococcus because it has polymorphism, is aerobic, has a catalase plus bacillus, has a GC content of 69.1% in DNA, and has diaminopimelic acid and mycolic acid as cell wall components. Also, the nitrate reduction plus, no acid is generated from sugar, and the test results described above are shown in Rhodococcus equi (R. e.
qui) Matches the description of the species. From the above points, this bacterium was presumed to be related to Rhodococcus equi (R. equi).

Next, a detailed description will be given with reference to examples.

Example 1 A 100 ml flask was charged with 10 g of P-nitrobenzyl alcohol-malonic acid half ester and 60 ml of toluene, and 0.5 g of dimethylformamide was added. The temperature was raised to 75 ° C., and a product prepared by dissolving 6 g of thionyl chloride in 5 ml of toluene was added dropwise over 0.5 hour. After reacting at the same temperature for 1 hour, the reaction mixture was gradually added dropwise to 25 ml of 28% ammonia water stirred at room temperature. A precipitate formed and was filtered. After drying, the yield of malonic acid (P-nitrobenzyl alcohol) ester-amide was 3.9 g. The purity measured by liquid chromatography is
It was 90%. Disperse this in methanol and mix at room temperature for 1
After stirring for a time, the mixture was filtered and dried. The purity by liquid chromatography was improved to 97,2%. Melting point is 127
Was ~ 129 ° C. The IR spectrum shows that the absorption based on the NH of the primary amide is at wave numbers 3400 and 3190, and the absorption based on the carbonyl group of the primary amide is
In addition, the carbonyl-based absorption of the ester bond has a wavenumber of 1
Observed at 730 as a characteristic absorption. In the 1H-NMR spectrum, the absorption based on methylene hydrogen of malonic acid at 3.45 ppm, the absorption based on hydrogen of benzylmethylene at 5.30 ppm, and the ortho position aroma of the nitro group at 7.5 to 7.6. Group hydrogen doublets, 8.3-8.4
A doublet absorption of aromatic hydrogen at the meta position of the nitro group was observed. Further, in the CI-mass spectrum using isobutane, a mass number of 239 peak with a molecular weight of +1 was observed.

[0017]

Example 2 Glycerin 1%, Polypeptone 0.5%,
Well water containing 0.3% malt extract and 0.3% yeast extract was sterilized with an autoclave, and 0.1% isovaleronitrile was added to the medium.
The NKN003 strain was inoculated and the culture was started at 30 ° C. On the first day of culture, 0.1% isovaleronitrile was added, and on the second day of culture, 4 ml of a culture solution was obtained. Bacteria were separated by centrifugation and dispersed in 20 ml of physiological saline containing 10 mM EDTA. To this, 1257 mg of P-nitrobenzyl alcohol cyanoacetic acid ester was added to carry out cyano group hydrolysis. Adjust the pH to 7 with 10% sodium hydroxide.
The mixture was stirred at room temperature for 4 hours while maintaining the temperature at 5 to 8.0. After completion of the reaction, analysis by liquid chromatography revealed that the formed malonic acid (P-nitrobenzyl alcohol) ester-amide was 83.3%, the P-nitrobenzyl alcohol-malonic acid half ester was 2.1%, and P -Nitrobenzyl alcohol was 2.7% and unreacted P-nitrobenzyl alcohol cyanoacetic acid ester was 12%. After the reaction was completed, 20 ml of acetonitrile was added to dissolve the target substance, and then the bacteria were centrifuged, and a part of acetonitrile was distilled off from the filtrate. The deposited precipitate was separated by filtration and dried to obtain 800 mg of malonic acid (P-nitrobenzyl alcohol) ester-amide. The purity by liquid chromatography is 9
It was 7.0%. The melting point is 127 ° C to 129 ° C,
The 1H-NMR spectrum and IR spectrum were the same as those obtained in Example 1.

[0018]

[Reference Example] In Example 2, when the reaction time was extended to 15 hours, it was confirmed that P-nitrobenzyl alcohol-malonic acid half ester, which was further enzymatically hydrolyzed, was produced as the main product. Confirmed in.

[0019]

Example 4 The NKN113 strain was cultured in the same medium as in Example 2, and the same culture was started, and 0.1% isovaleronitrile was added on the second day of the culture. On the 3rd day of culture, 5 ml of culture solution was obtained. Bacteria were separated by centrifugation and 5 ml of 0.
It was dispersed in 1M phosphate buffer (pH = 7.0). 25 mg of P-nitrobenzyl alcohol cyanoacetic acid ester was added thereto and stirred for 4 hours. After completion of the reaction, analysis by liquid chromatography revealed that the produced malonic acid (P-nitrobenzyl alcohol) ester-amide was 17.1 mg. P-nitrobenzyl alcohol malonic acid half ester was produced in an amount of 5.3 mg, and P-nitrobenzyl alcohol was produced in an amount of 3.9 mg.

[0020]

The novel compound, malonic acid (P-nitrobenzyl alcohol) ester-amide, is a microorganism
It can be efficiently obtained by the hydrolysis reaction of -nitrobenzyl alcohol cyanoacetic acid ester.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:01)

Claims (3)

[Claims] 1. Malonic acid (P-nitrobenzyl alcohol) ester-amide represented by the following chemical formula 1 2. P-nitrobenzyl alcohol cyanoacetate is added to Pseudomonas.
s) or malonic acid (P-nitrobenzyl alcohol) characterized by being hydrolyzed by the action of a bacterium belonging to the genus Rhodococcus (Phodococcus)
Method for producing esteramide 3. The genus Pseudomonas
The bacterium belonging to as) is NKN003, and the bacterium belonging to Rhodococcus is NK.
The method according to claim 2, which is N113 bacteria.