DE1112250B - Process for the production of substances with vitamin B activity - Google Patents

Description

Process for the production of substances with vitamin Bl2 effectiveness Benzimidazoles are components of physiologically active substances from the cobalamin series. In nature z. B. the cyanocobalamin (vitamin Bols) and the so-called vitamin Bl2 factor III found.

The first-mentioned substance contains 5,6-dimethylbenzimidazole, the second a 5-oxy-benzimidazole.

These substituted benzimidazoles can be used as chemical precursors or Vitamin formation in the fermentative production of compounds of the Vitamin-Bis series can be added.

It has now been found that substances with vitamin B12 activity can be used can also be obtained by producing fermentations with cobalamin Microorganisms in nutrient solutions carries out the 5,6-methyl-methoxy-benzimidazole contain. The 5,6-methyl-methoxybenzimidazole used for this procedure can obtained by the following process: I. a) Preparation of 2- (4'-nitrobenzolazo) -4-methyl-5-methoxy-aniline 10 g of 3-methoxy-4-methyl-aniline (I) are dissolved in 90 ml of HCl (d = 1.19), 450 ml of water and 180 ml of 90 ° / Oiger formic acid dissolved (solution 1). A p-nitrophenyl diazonium chloride solution is added to this which from 12.6 g of p-nitroaniline, dissolved in 225 ml of 5N HCl, by adding 6.8 g NaNO2 (10% solution). is prepared (solution 2). Shortly after adding the solution 2 to solution 1 begins the excretion of 2- (4'-nitrobenzolazo) -4-methyl-5-methoxy-aniline-chlorohydrate.

After standing for 12 hours at room temperature, the red compound becomes suctioned off and the hydrochloric acid salt in 1200 ml of acetone and 25 ml of conc. Ammonia warm solved. The free solution is precipitated from the alkaline solution by adding 2.5 liters of water Base off, which is collected on a suction filter and washed alkali-free with water will.

The dried base is almost completely pure, but can be obtained from toluene petroleum ether be recrystallized, from which the dye is shiny, greenish-red fluorescent Lamellae crystallized. It melts at 195 ° C (uncorr.) (II). The m-nitrobenzolazo analog melts at 152 ° C, the o-nitrobenzolazo analog at 175 ° C, the p-chlorobenzolazo analog at} 62 ° C. b) Reductive cleavage of 2- (4'-nitrobenzolazo) -4-methyl-5-methoxy-aniline 3 g of the azo base II are dissolved in 260 ml of acetone and added in portions with stirring alternately water (150 ml in total) and also portionwise 50 ml of a 24 ° / Oigen Sodium hydrosulfite solution added.

The originally deep red solution is straw yellow after the reduction. It is acidified with 10 n-H Cl and evaporated in vacuo under nitrogen Volume of about 50 ml, adding amounts of 10 N HCl, so that the solution always remains strong against the Congo. After concentration, the solution is cooled, whereupon p-Nitraniline-HCl separates, which is removed by filtration.

The acidic aqueous solution is then with conc.

Bred NaOH to pH 8.5 to 9.0 and extracted with ether. With K2CO3 dried ethereal solution leaves a brown solid after evaporation Composition representing crude 4-methyl-5-methoxy-1,2-phenylenediamine (III) and without further purification was processed. c) Representation of the 5,6-disubstituted benzimidazole The crystalline base III is dissolved in 5ml 90 ° / Oiger formic acid and boiled Solution for 3 hours under nitrogen in an oil bath (140 ° C). Afterwards the brown one 50 ml of water are added to the solution and the pH of the acidic aqueous solution is adjusted with the aid adjusted from 7N NaOH to pH 7.5 to 8.0. The 5,6-methoxymethyl-benzimidazole then it initially precipitates oily, but solidifies after the mixture has cooled for a short time.

It is taken up in a little chloroform, the chloroform solution with dried with annealed Na2SO4 and evaporated. The remaining 5,6-methoxymethyl -benzimidazole can be purified by distillation, among other things. It distills when it is good Vacuum without decomposition, e.g. B. it can be in the bulb tube at 140 to 150 ° C air temperature and 0.2 mm pressure are obtained as a pale yellowish, rapidly solidifying oil, which after rubbing with ether gives a white powder. Crystallized from ether-petroleum ether the 5,6-methoxy-methyl-benzimidazole (IV) combined in shiny clusters Needles that melt at 118 ° C (uncorrupted, after drying at 80 ° C in the vacuum gun). It is easily soluble in acids and strong alkalis, alcohol, acetone, a little heavier in chloroform and benzene, difficult in ether and insoluble in petroleum ether.

Calculated ... C 66.610 ", H 6.23 ° / o, N 17.29 ° / o; found ... C 66.5%, H 6.34 / 0, N 17.32 / 0.

2. a) Preparation of 2-methoxy-4-acetamino-5-nitro-toluene (I) In a four-necked flask with a stirrer, which from the outside with an acetone-carbonic acid mixture is cooled, the cooled mixture of 290 ml of nitric acid (HNO3 content 64%) and 105 ml conc.

Sulfuric acid and then uses 48 g finely powdered 2-methoxy-4-acetaminotoluene in portions to. It is best to work at a temperature of a little below -5 ° C, by no means but above 0 ° C. After stirring for 2 hours, the mixture is poured onto 1 kg of crushed meat Ice cream. This gives about 40 to 50 g of a yellow to reddish yellow product which consists for the most part of (I) and through crystallization from dilute alcohol, but advantageous by filtering the benzene solution through an activated Tube filled with aluminum oxide, which can be cleaned after washing with benzene. The main product (I) of the nitration runs through the column in a pale yellow color Solution that is evaporated to dryness. The product is pure enough for further use Processing (mp. I 51 ° C). Melts after recrystallization from dilute alcohol it at 152.5 ° C. Yield 30 to 40 g.

If the column is washed further with methanol-containing benzene, so is obtained 2 to 5 g of a red substance which can be readily crystallized from acetone, melting point 147 bis 148,5'C, which was identified as 2-oxy-4-acetamino-5-nitro-toluene.

Calculated ... N 12.59%; found ... N 12.5 ° / 0. b) Manufacturing 22 g of the 2-methoxy-4-amino-5-nitro-toluene (II) are dissolved in 260 ml of alcohol and add 8 g of Na OH in 130 ml of water to the solution. The mixture will last for I hour boiled in a water bath, then 130 ml of distilled water are added. When cooling down II excretes in large, orange-colored needles that are collected on a suction filter and washed out with diluted alcohol and water.

Compound II melts at 156.5 to 157.5 ° C.

C8HloO3N2 Calculated: C 52.7%, H 5.94%; found ... C 52, 7 ° / a, H 5, 74 ° / o.

The constitution of the nitroamine of m.p. 156.5 to 157.5 ° C was through Elimination of the amino group with Cu2O according to Hodgson and co-workers (J. Chem.

Soc., London, 1942, p. 748). This produced 2-methoxy-5-nitro-toluene, M.p. 64 ° C. c) Preparation of the 3,4-methoxymethyl-1,2-phenylenediamine (III) I g des Nitroamine (II) is dissolved in 100 ml of 50% alcohol and, after addition of 5 g Sodium hydrosulfite (70%) boiled under reflux on a water bath. After about 20 The reduction is complete in minutes and the solution is completely decolorized.

6 ml of conc. Hydrochloric acid, evaporates to dryness in vacuo one, dissolves the residue in 15 ml of water and treated the aqueous solution with 10% Sodium carbonate solution to a pH of 8.0. The phenylenediamine (III) separates turns into white crystals and is shaken out with ethyl acetate. To Evaporation of the ethyl acetate in vacuo gives 0.8 g of the base, which is obtained from an ether-petroleum ether mixture crystalline in fine needles. and melts at 110 to 112 ° C with decomposition.

CHi, ON, (152.1) Calculated ... C 64.0%, H 8.07 ° / o, N 18.68 ° / o; Found ... C 63.6 ° / o, H 8.01 ° / o, N 18.80 ° / o.

The dichlorohydrate of the compound decomposes between 125 and 130 ° C.

C8H14ON2CI2 (225) Calculated ... HCl 35.51%; found ... HCI 31, 8 ° / 0.

The phenylenediamine thus obtained can then, as described in 1. c), be converted into the benzimidazole. But this can also be obtained as follows are: d) Preparation of 5,6-methyl-methoxybenzimidazole (IV) 4 g of 2-methoxy-4-amino-5-nitro-toluene are mechanically mixed with 10 g of zinc powder and 30 ml of water in a four-necked flask suspended and treated dropwise with 40 ml of 90% formic acid. The contents of the flask is then heated for 4 hours in an oil bath at an external temperature of 110 ° C. Afterward 160 ml of water are poured in and the solution is filtered through a thin layer of Hyflow Supercell on a porcelain suction cup. To remove the zinc, this is about 100 ml 20% sodium sulfide solution precipitated. The filtered solution is with conc.

NH3 solution added up to pH 7.5. The benzimidazole (IV) falls as a colorless oil that soon solidifies. This is extracted with chloroform and evaporated the chloroform and the crystalline residue crystallized from an ether-petroleum ether mixture around.

The product is based on the 5,6-methyl-methoxy-benzimidazole produced according to the main patent identical. Melting point and mixed melting point 118 ° C.

Yield 3g.

For the processes specified here for the preparation of the benzimidazole derivatives no protection is claimed.

The new 5,6-methyl-methoxy-benzimidazole-cobalamin, which is called vitamin B12 factor S is to be designated, arises z. B. If you start fermentation with Bacillus propionicus Freudenreichii in the presence of 5,6-methyl-methoxy-benzimidazole. The yield is the same as in parallel experiments with 5,6-dimethylbenzimidazole. Vitamin B12 factor S can be found in its pure form in beautifully developed ruby red Needles isolate. Its solubility in dilute acetone is slightly greater than that of vitamin B12.

He is one of those cobalamines that are used in paper chromatography with butanol as the eluent, migrate as slowly as the natural vitamin B12. A mixture of vitamin B12 and vitamin B12 factor S can be obtained by means of a Separate filter paper soaked with 9% aqueous KH2PO4 solution; the speed of migration the S factor is 1.3 times greater than that of vitamin B12.

The absorption spectrum of the new factor is different from that one of vitamin B12 in that the inflection characteristic of vitamin B12 the wavelengths 305 to 308 mp are absent.

In the vitamin Bl2 test with E. Coli 133-3 and Ochromonas Malhamensis A high growth substance effect of the new vitamin B12 factor S can be determined. In tests with the latter microorganisms, it is about two thirds of that of the natural vitamin B12, in the Coli test there is no difference between the natural and artificial vitamin B12.

Example The biosynthesis of factor S takes place z. B. with Bac. propionicus Freudenreichii in the fermentation approach according to the method of Leviton and Hargrove (Ind. Eng. Chem., 44.2651-5.1952). Mix to prepare the appropriate nutrient solution one 81 torula yeast cooking juice with a solution of cysteine hydrochloride .......... 4.0 g peptone, solub .................... 200, 0 g lactic acid, 75% .............. 1200, 0 g sodium hydroxide ................ 200.0 g glycose ....................... 200.0 g Calcium carbonate ................ 200.0 g CoCl2 6 H2O 160.0 g in 101 tap water and adds 1 1 mineral salt solution, which is crystalline. MgSO4 40g crystall. (NH4) 2SO4 40 g crist. CaCl2 ...................... 4 g crystall. Zn S 04 0.08 g crystall. FeSO-7 HO .............. contains 0.4 g. The mixture is filled after adding 120 mg of 5,6-methyl-methoxy-benzimidazole, dissolved in 200 ml of 0.1 n hydrochloric acid, with tap water to 401 and sets the pH of the mixture to 7.6.

Sterilize in a 50 l round bottom flask and cool to 27 ° C Flask contents under sterile conditions with a B. propionicus Freudenreichii inoculum inoculated and for about 7 days with intermittent stirring of the contents of the flask Subjected to fermentation. Once the vitamin B12 activity of the fermenting liquid no longer increases significantly (which is the case with boiled samples of the well-stirred Bacterial suspension is detectable), the fermented liquid is replaced by a high-speed clarifying centrifuge driven (10,000 to 15,000 revolutions / min.). The centrifuged Bacterial mass is suspended in water containing 0.01% sodium cyanide and heated to 100 to 110 ° C in an autoclave. After cooling to 20 ° C, the engine revs up centrifuged and the slightly cloudy solution with lead acetic acid, then with aluminum hydroxide (Addition of aluminum sulfate and ammonia) clarified. After discontinuing the resulting Precipitation creates a clear solution colored pink from the dissolved cobalamin, from which this can be extracted with a phenol-chloroform solution (1: 4).

The cobalamin is driven out of the phenol-containing solution by adding it of ether and water into the aqueous phase. The aqueous solution thus obtained contains 30 to 50y vitamin Bl2 factor S per milliliter, which is produced in various ways can be further enriched or brought to crystallization. For example If one adsorbs on active charcoal, the adsorbate is washed with phenol-containing water and pure water, the factor S is eluted from the diluted alcohol containing benzene Coal off, the solvent evaporates in a vacuum and crystallizes in the dry residue remaining vitamin B12 factor S from aqueous acetone (1: 8). The yield is 20 to 40 mg vitamin B12 factor S from 401 fermentation fluid.

Claims (1)

PATENT CLAIM. Process for the production of substances with vitamin Bi2 effectiveness, characterized in that fermentations with cobalamin-producing microorganisms in nutrient solutions that contain 5,6-methyl-methoxy-benzimidazole. Documents considered: German Auslegeschrift No. 1 004 343; Zentralblatt für Bakteriologie, Section II. 109.325 to 329.1956.