JPH03123782A - Novel oxazopyrroloquinoline compound - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to novel oxazopyrroquinoline compounds.

[Prior Art 2: Problems to be Solved by the Invention] That is, the present invention is a novel oxazopyrroquinoline compound represented by the formula and a salt thereof.

Therefore, this compound is 5-hydroxymethyl-2,
8,10-) Ricarboxy IH-oxazo[5,4-
h]-pyrrolo[2,3-f]quinoline (hereinafter referred to as hydroxymethyl OPQ).

The hydroxymethyl PQQ salts include alkali metal salts, alkaline earth metal salts, ammonium salts, and substituted ammonium salts (hydroxymethyl OPQ and hydroxymethyl OPQ salts may be collectively referred to as hydroxymethyl OPQs hereinafter). be).

Substituted ammonium salts include, for example, alkyl-substituted ammonium salts and hydroxyalkyl-substituted ammonium salts.

Representative examples of the hydroxymethyl OPQ salts of the present invention include:
These include sodium salts, potassium salts, magnesium salts, calcium salts, ammonium salts, trimethylammonium salts, triethylammonium salts, and triethanolammonium salts.

As a method for obtaining hydroxymethyl OPQ of the present invention,
There are synthetic methods and fermentation methods using microorganisms, but PQ
The method of synthesis from Q and serine is relatively open and practical.

When synthesizing from PQQ and serine, the P used
QQ may be either a fermented product or a synthetic product.

Also, for example, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as magnesium and calcium salts, alkyl-substituted ammonium salts such as ammonium salts, trimethylammonium salts and triethylammonium salts, and triethanolammonium salts. PQQ salts, such as hydroxyalkyl-substituted ammonium salts, can also be used instead of PQQ (PQQ and PQQ salts are collectively called PQ
(Sometimes referred to as Class Q).

Moreover, serine may be any of 0-form, L-form and a mixture thereof.

Commercially available products can be used for both of these PQQs and serine.

The amount of serine to be used may be a stoichiometric amount or more, but in practice, it is usually 1 mole or more, preferably about 1 to 1000 times the mole of PQQIW.

This reaction is carried out in the liquid phase using a solvent.

Typical examples of this solvent include lower aliphatic alcohols such as methanol and water.

The concentration range of PQQton in the reaction solution is not particularly limited, and depending on the type of solvent and reaction conditions, the pH of the reaction solution is usually about 2 to 6, preferably about 2 to 5. If the pH of the reaction solution is outside this range, by-products will significantly increase.

Oxygen is required for the reaction to obtain hydroxymethyl OPQ, and for that purpose, it is necessary to supply oxygen to the reaction solution.8 Supply a sufficient amount of oxygen to the reaction solution to reduce the dissolved oxygen concentration in the reaction solution. Although the reaction can be completed in a relatively short time by maintaining 0 to be greater than 0, it is possible to obtain hydroxymethyl OPQ even under oxygen rate-limiting conditions.

The method for supplying oxygen includes, for example, a method of aerating and mixing oxygen-containing gases such as a mixture of oxygen gas and ozone with other gases such as air, or a method of stirring in the presence of these gases. There are ways to do this.

The reaction temperature is not particularly limited, but in practice it is usually 20
°C or more, preferably in the range of about 20 to too'c,
Particularly preferably, the temperature is about 20 to 80°C.

Hydroxymethyl OPQ salt is generally prepared by adding the desired hydroxymethyl OPQ to a solution containing hydroxymethyl OPQ.
Obtained by adding the corresponding alkaline earth metal to the Q salt.

From the reaction solution thus obtained, hydroxymethyl O
There are various methods for separating and purifying PQs. For example, there are methods using resin carriers that adsorb hydroxymethyl OPQs, organic solvent extraction methods, precipitation methods, washing methods, and ultrafiltration methods.These methods can be used alone or in combination to separate hydroxymethyl OPQs. Can be purified.

In the method using a resin carrier that adsorbs hydroxymethyl OPQs, any resin carrier that can adsorb and desorb hydroxymethyl OPQs can be used without particular restrictions. As a representative example of this resin carrier, an anion exchange resin carrier is a polysaccharide-based carrier D[! AE-Sephadex A-2
5 (manufactured by Pharmacia), hydrophilic polymer resin Df
! Examples include AE-Toyo Pearl 650 (manufactured by Tosoh) and Amberlyst^-21 (manufactured by Rohm and Haas). In adsorption/separation type resin carriers, the hydrophobic polymer resin Diaion HP series (manufactured by Mitsubishi Kasei) and Amberlite XAD series (manufactured by Rohm & Co., Ltd.)
(manufactured by Haas Corporation), as well as silica, octadecyl silica, and alumina. Hydrophobic chromatographic resin carriers include butyl-Toyopearl 650 and phenyl-Toyopearl 650 (manufactured by Tosoh), which are wood-philic polymer resins.
and so on.

The organic solvent extraction method can be carried out either under acidic or alkaline conditions; however, as the extractant, a low polar solvent is used under acidic conditions, and a polar solvent is used under alkaline conditions.

Water and organic solvents and mixtures of water and organic solvents are respectively used as extractants. As the organic solvent, organic solvents having low compatibility with water are preferred, such as n-
Aliphatic alcohols having 4 or more carbon atoms such as butanol are preferably used.

For example, when a mixture of water and n-butanol is used as an extractant, if left to stand after extraction, the extract will separate into two layers, an aqueous layer and an n-butanol layer, and the reaction product will be acidic. Depending on the alkalinity, the target hydroxymethyl O
PQ[ is dissolved in either the n-butanol layer or the aqueous layer.

As a precipitation method, for example, the reaction product solution is acidified with inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acids such as trichloroacetic acid, trifluoroacetic acid, and trifluoromethanesulfonic acid, and hydroxymethyl OP
Acidic precipitation method to precipitate Qs, salts to precipitate hydroxymethyl OP Qs by adding alkali metal salts such as sodium chloride and potassium chloride, alkaline earth metal salts such as calcium chloride and magnesium chloride to the reaction product solution There is a solvent precipitation method in which a reaction product solution is mixed with a solvent in which hydroxymethyl OPQ has low solubility, such as acetone, and precipitated.

In these precipitation methods, the lower the liquid temperature is, the higher the recovery rate of hydroxymethyl OPQ is.

A powder of hydroxymethyl OPQs obtained by a separation and purification method such as the precipitation method described above or a precipitate containing hydroxymethyl OPQs is mixed with hydroxymethyl OPQiQ such as acetone, ether, and acidic water.
The purity of hydroxymethyl OPQs can be further improved by applying a cleaning method in which hydroxymethyl OPQs are washed with a solvent that is difficult to dissolve.

In the production of hydroxymethyl 0PQi by fermentation, ultrafiltration can be used to remove polymeric impurities from the culture solution.

Ultrafiltration methods include methods using gel filtration compatible resin carriers such as Sephadex G-10 (manufactured by Pharmacia) and Toyo Pearl IIW series (manufactured by Tosoh), methods using various ultrafiltration membranes and ultrafiltration hollow fibers, etc. can be applied.

〔Example〕

The present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example I L-serine 30. og was dissolved in 600 rrdl of distilled water and adjusted to pH 3.0 with 6N hydrochloric acid to obtain an L-serine solution. P Q Q 604m in the serine solution
g, and with strong mechanical stirring while aerating air,
This reaction product solution was diluted with 6N hydrochloric acid at 30°C for 20 hours.
Adjust to 12.0 and add CaC1z・11□012g to this.
was added, the solution was cooled with ice water, and hydroxymethyl O
PQ was precipitated.

This precipitate was collected by centrifugation, and 60% of water was added to the precipitate.
(Add lnf and pl+ this solution with 5N Na011
7.8 to dissolve the precipitate.

To the obtained solution, 25100 mQ of DEAE-Sephadex, which is an anion exchange gas, was added and thoroughly stirred. After adsorbing hydroxymethyl OPQ to this carrier, it was allowed to stand, and the carrier was separated by removing the upper layer.・Recovered.

A 50 mmφ column was filled with new DEA [i-Cefazo Nx^-25100-], and the recovered carrier was packed thereon.

After washing this column with 300 mf of distilled water,
．． 5M saline solution 3400- was poured, followed by further 0
．． 2,500 ml of 7H saline solution was poured. At this time, the trace amount of unreacted PQQ accompanying hydroxymethyl OPQ was 0.
Hydroxymethyl OP was also added to the elution fraction of 5M saline.
Q was present in the 0.7M saline elution fraction.

The eluted fraction of 0.7M saline was adjusted to pH 2.0 with 6N hydrochloric acid, and then cooled to 5°C to precipitate hydroxymethyl OPQ.

The precipitate thus obtained was collected by centrifugation, washed with dilute hydrochloric acid, then washed with dimethyl ether, dried in a vacuum oven at 60°C, and dried at 330°C.
+ng of hydroxymethyl OPQ was obtained.

The color of the hydroxymethyl OPQ thus obtained was orange, and it gradually decomposed at 210-220°C and did not exhibit a clear melting point. Also, this hydroxymethyl OPQ is easily soluble in water, extremely soluble in neutral and alkaline conditions, and also soluble in lower alcohols, but what about acetone and dimethyl ether? I couldn't take it.

The color of this aqueous solution of hydroxymethyl OPQ varies depending on the concentration of hydroxymethyl OPQ and the pi of the aqueous solution, but the color of the aqueous solution of hydroxymethyl OPQ varies depending on the concentration of hydroxymethyl OPQ and the pi of the aqueous solution.
The PQ aqueous solution was pale yellow when the aqueous solution was neutral to alkaline, and reddish and pale yellow when the aqueous solution was acidic.

Next, the elemental analysis value of this hydroxymethyl OPQ, Il
'l spectrum,') I-N? 1R spectrum and visible/ultraviolet spectrum are shown.

■) Elemental analysis value: C+bl-1qN30g (Msu37
1.25) Theoretical value (χ): C51,76N2.44 N
11.32 Actual value (χ): C50,5882,63N1
1.082) IR spectrum (C□9 value + Cm-'):
(KBr) 2500b'''', 1680s"・", 1
575"'-s, 1510', 150"1095s
'-'', 700'', 650°3) 'II-NMR spectrum (δ value, ppm): (DMSO-db, internal standard: TMS) 4.98 (s, 2H, CHz-011), 7.27 ( d
, ltl, pyrrole ring C-11, J = 1.98Hz)
, 8.07 (s, LH, pyridine ring C-1+).

14.60 (d, 11!, pyrrole ring N-H, J=0
．． 44Hz) 4-) Visible/ultraviolet spectrum (λmaX
value, nm): (10mM potassium phosphate buffer p
if 7.0) 254.269'', 416 Example 2 40d of Log/l L-serine aqueous solution adjusted to p)I 1.5-9 with hydrochloric acid or sodium hydroxide, 1
40 mg of PQQ was added to each Erlenmeyer flask.

An Erlenmeyer flask with a vented stopper was stirred using a rotary bowl and reacted at 30°C for 1 day.

The pl+ of the reaction product solution was adjusted to 8.0, and the amount of hydroxymethyl OPQ produced was examined using liquid high performance chromatography.

The conditions for liquid high performance chromatography are shown.

・Measuring equipment Pump LC-64 manufactured by Shimadzu Corporation Detector 5PD-6AV Column constant temperature bath CTO-6AS (40°C)・Column ODS A-3024, manufactured by YMC Corporation, 6n+mφX150mm・Measurement wavelength 259nn+・Flow rate 1. 5mf/a+in ・Eluent composition 0. IM KHiPO4,0,1M H
Cl0410χCH, CN aqueous solution pH 2,5 (NaOI
l) The results are shown in Table 1.

Table 1

Claims (1)

[Scope of Claims] A novel oxazopyrroquinoline compound represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼ and a salt thereof.