Method for the production of magnesium pyridoxal-5'-phosphate glutamate and intermediate products obtained thereby

The invention relates to a method for the production of magnesium pyridoxal-5'-phosphate glutamate, in which pyridoxine or an acid addition salt thereof is oxidised with manganese (IV) oxide to pyridoxal; pyridoxal is reacted with p-phenetidine under formation of the Schiff's base p-phenetidyl-pyridoxal; p-phenedityl-pyridoxal is selectively phosphorylated on the 5'-hydroxymethyl group under formation of p-phenetidyl-pyridoxal-5'-phosphate; p-phenetidyl-pyridoxal-5'-phosphate is hydrolysed under formation of an alkali metal salt of pyridoxal-5'-phosphate; the alkali metal ions are removed in order to obtain pyridoxal-5'-phosphate; pyridoxal-5'-phosphate is reacted with a reaction product of a magnesium alcoholate and L-glutarnic acid; and the formed magnesium pyridoxal-5'-phosphate glutamate is isolated. Furthermore, the invention relates to intermediate products obtained with this method.

Magnesium pyridoxal-5'-phosphate glutamate is known from DE 24 61 742. In 
this publication, it is proposed as a medicament for prophylaxis and 
therapy of metabolic disturbances, especially for influencing the lipid 
and cholesterol state. DE 40 16 963 relates to the use of magnesium 
pyridoxal-51'-phosphate glutamate for the reduction of LDL-bound peroxides 
and for prevention of vascular damage. 
Considering the importance of magnesium pyridoxal-5'-phosphate glutamate as 
a medicament, the need exists for a new, simpler and more economic 
synthesis for this substance. Therefore, an object of the invention is to 
provide a synthesis of this type. 
The object is solved according to the invention by providing a synthesis 
for magnesium pyridoxal-5'-phosphate glutamate in which 
pyridoxal-5'-phosphate is obtained as an intermediate product. The 
pyridoxal-5'-phosphate accumulates in the form of an aqueous solution from 
which the pyridoxal-5'-phosphate can be isolated if required. However, the 
obtained pyridoxal-5'-phosphate solution can also be reacted to the end 
product in an advantageous manner without isolation of the 
pyridoxal-5'-phosphate. Therewith, the steps required according to the 
state of the art for purification and drying of the pyridoxal-5'-phosphate 
are omitted. 
A further point of the invention relates to the fact that, starting from 
pyridoxine, and especially an acid addition salt of pyridoxine, such as 
pyridoxine hydrochloride, pyridoxal-5'-phosphate is produced in a high 
yield according to a new method with p-phenetidyl pyridoxal and 
p-phenetidyl-pyridoxal-5'-phosphate as intermediate products. 
Subject matter of the invention is a method for the synthesis of magnesium 
pyridoxal-5'-phosphate glutamate which is characterised in that: 
A) pyridoxine or an acid addition salt thereof is oxidised with 
manganese(IV) oxide to pyridoxal; 
B) pyridoxal is reacted with p-phenetidine under formation of the Schiff's 
base p-phenetidyl-pyridoxal; 
C) p-phenetidyl-pyridoxal is selectively phosphorylated on the 
5'-hydroxymethyl group under formation of 
p-phenetidyl-pyridoxal-5'-phosphate; 
D) p-phenetidyl-pyridoxal-5'-phosphate is hydrolysed under formation of an 
alkali metal salt of pyridoxal-5'-phosphate; 
E) the alkali metal ions are removed in order to obtain 
pyridoxal-5'-phosphate; 
F) pyridoxal-5'-phosphate is reacted with a reaction product of a magnesium 
alcoholate and L-glutamic acid; and 
G) the formed magnesium pyridoxal-5'-phosphate glutamate is isolated. 
A preferred embodiment of the invention is represented in the following 
reaction scheme. 
##STR1## 
Molecular weight of the compounds. 
__________________________________________________________________________ 
manganese(IV) oxide 
MnO 86,9 g/mol 
para-phenetidine (7) 
C.sub.8 H.sub.11 NO 
137,0 g/mol 
para-phenetidyl-pyridoxal 
(4) 
C.sub.16 H.sub.18 N.sub.2 O.sub.3 
286,0 g/mol 
para-phenetidyl-pyridoxal-5'-phosphate 
(3) 
C.sub.16 H.sub.19 N.sub.2 O.sub.6 P 
366,0 g/mol 
pyridoxal (5) 
C.sub.8 H.sub.9 NO.sub.3 
167,0 g/mol 
pyridoxal-5'-phosphate 
(2) 
C.sub.8 H.sub.10 NO.sub.6 P 
247,0 g/mol 
pyridoxal-5'-phosphate monohydrate 
(2') 
C.sub.8 H.sub.12 NO.sub.7 P 
265,0 g/mol 
pyridoxine hydrochloride 
(6) 
C.sub.8 H.sub.12 ClNO.sub.3 
205,5 g/mol 
magnesium pyridoxal-5'-phosphate 
(1) 
C.sub.3 H.sub.11 Mg.sub.2..sub.5 N.sub.2 O.sub.9 
432,0 g/mol 
glutamate 
magnesium pyridoxal-5'-phosphate glutamate 
(1') 
C.sub.13 H.sub.15 Mg.sub.2..sub.5 N.sub.2 O.sub.11 
P 464,0 g/mol 
dihydrate 
__________________________________________________________________________ 
It has been shown to be advantageous to carry out the individual synthesis 
steps discontinuously in suitable temperature and pH controlled units 
under light and/or air exclusion. 
In order to be able to monitor the chemical reaction at any time, it is 
possible to carry out in-process controls for example. In-process controls 
of this type can comprise analyses, such as HPLC analyses, which can be 
reliably carried out in a short amount of time (15 min). 
Furthermore, it has been shown to be suitable to release the product of 
each step for further processing first after carrying out an analysis and 
a purity test. 
The individual steps of the synthesis are illustrated in detail in the 
following. 
According to the invention, pyridoxine, preferably in form of an acid 
addition salt such as pyridoxine hydrochloride (6), is first oxidised to 
pyridoxal (5). A suitable oxidation agent is manganese(IV) oxide which is 
suitably used in activated form. The oxidation is then preferably carried 
out in sulfuric acid solution under controlled pH and temperature 
conditions as well as under light exclusion. The degree of reaction can be 
continuously examined by means of a suitable analytical system (for 
example, HPLC). It is particularly preferred to stop the reaction as soon 
as the entire amount of pyridoxine was oxidised. As opposed to known 
methods of oxidation of pyridoxine hydrochloride (6), the amount of 
oxidation side products formed can be diminished and the yield of 
pyridoxal (5) can be increased thereby. 
The described oxidation of a pyridoxine acid addition salt to pyridoxal (5) 
with manganese(IV) oxide is typically carried out in the acidic range 
without adherence to particular pH or temperature conditions. However, 
oxidation with manganese(IV) oxide is carried out in a particularly 
advantageous manner at a constant pH value of 5,1 and a constant 
temperature of 14.degree. C. for further increasing the yield and 
minimising the amount of side products formed. 
The reaction mixture can subsequently be subjected to processing for 
separation, for example by filtration, of precipitated material such as 
reacted or non-reacted oxidation agent, for example manganese salts. 
Thereby, it is also possible to recover non-reacted oxidation agent, such 
as manganese(IV) oxide and newly add this later to the process. 
Isolation of pyridoxal (5) can be carried out by the person skilled in the 
art according to new methods without difficulty. 
However, according to the invention, isolation is not required and the 
obtained pyridoxal solution can be directly used as an aqueous pyridoxal 
solution in the next reaction step. 
An aqueous solution of pyridoxal (5), preferably relating to the reaction 
solution obtained in the oxidation, is brought to a weakly acidic pH value 
and preferably a pH value of 4.5. After that, p-phenetidine (7) is added 
preferably in slight excess. After conclusion of the reaction, the formed 
precipitate is separated (preferably filtered), for example by washing 
with water and/or an organic solvent, purified, and preferably dried, 
whereby p-phenetidyl-pyridoxal (4) is obtained. 
Excess p-phenetidine (7) can be recovered. If ions originating from the 
oxidation agent, such as manganese(II) ions, are contained in the solution 
obtained after the separation of the precipitate, these can also be 
recovered by precipitation with the aid of lye for example. 
The Schiff's base p-phenetidyl-pyridoxal (4) is subjected to a treatment 
for selective phosphorylation of the 5'-hydroxymethyl group under 
formation of p-phenetidyl-pyridoxal 5'-phosphate (3). The phosphorylation 
occurs through treating p-phenetidyl-pyridoxal (4) with a suitable 
phosphorylation agent. For example, reaction with polyphosphoric acid is 
suitable. 
In this case, phosphorylation preferably occurs under controlled 
temperature conditions (4 to 25.degree. C.) as well as light exclusion. 
For an optimal reaction course, it has been shown to be favourable to 
bring the dried p-phenetidyl-pyridoxal (4) to a particle size of 
.ltoreq.500 .mu.m before the reaction. Furthermore, it is advantageous to 
mix p-phenetidyl-pyridoxal (4) and polyphosphoric acid as intimately as 
possible for reaction, for example in a kneading unit. The degree of 
reaction can be constantly examined by means of a suitable analytical 
system (for example, HPLC). It is particularly preferred to stop the 
reaction as soon as the entire amount of p-phenetidyl-pyridoxal (4) is 
phosphorated. 
According to a particularly preferred embodiment, polyphosphoric acid and 
p-phenetidyl-pyridoxal are used in the ratio of 4-6 parts to 1 part. 
In the above described reaction, p-phenetidyl-pyridoxal-5'-polyphosphates 
form. Then, through the subsequent addition of water and acid, a partial 
hydrolysis occurs under formation and precipitation of 
p-phenetidyl-pyridoxal-5'-phosphate (3). The precipitation can be promoted 
by addition of lye. The compound obtained is purified in the customary 
manner, for example by washing with water and/or an organic solvent. It 
can be dried and isolated, but is suitably processed as a moist 
precipitate for the purpose of further synthesis. 
The phosphate accumulating due to the excess of polyphosphate can be 
reacted with calcium hydroxide to calcium phosphate and can be optionally 
easily stored with low risk and/or further processed or disposed. 
p-phenetidyl-pyridoxal-5'-phosphate (3) is then hydrolysed to pyridoxal 
5'-phosphate (2) and p-phenetidine (7) by treatment with an aqueous alkali 
solution, preferably at a pH value of more than 12.5. The formed 
p-phenetidine and the alkali metal salt of pyridoxal 5'-phosphate can be 
separated according to any suitable method. p-phenetidine (7) can be 
separated from the aqueous solution by extraction with a suitable organic 
solvent, for example an aliphatic or aromatic hydrocarbon such as toluol. 
Alternatively, the p-phenetidine (7) can be separated according to a 
preferable embodiment by means of a liquid-liquid separator unit. A 
separation of this type is more time-saving, more ecological, more 
efficient, easier and therewith more economic than the extraction. 
The p-phenetidine (7) recovered in this reaction step can be purified by 
means of distillation for example and then newly used in the first 
reaction step. 
The alkali metal salt of pyridoxal-5'-phosphate (2) can be precipitated 
from the obtained aqueous solution by concentration. The precipitate can 
be further purified by washing or recrystallization for example. 
In order to produce magnesium pyridoxal-5'-phosphate glutamate (1) from the 
alkali metal salt of pyridoxal-5'-phosphate (2), the alkali metal ions 
must first be removed. For this purpose, any suitable method can be 
employed. For example, the obtained aqueous alkali metal salt solution of 
pyridoxal-5'-phosphate can be subjected to an ion exchange treatment. The 
solution obtained thereby can --after optional concentration--be directly 
used for synthesis of magnesium pyridoxal-5'-phosphate glutamate (1). It 
is also possible to isolate free pyridoxal-5'-phosphate (2) from the 
aqueous solution and to purify the compound according to customary methods 
known to the person skilled in the art. For the next synthesis step, an 
aqueous solution of previously isolated and purified pyridoxal 
-5'-phosphate (2) can be used in this case. 
The aqueous solution of pyridoxal-5'-phosphate (2) is added to a solution 
which was obtained by reaction of a magnesium alkolholate with water and 
addition of glutamic acid (8). magnesium ethylate (9) is particularly 
preferred as a magnesium alcoholate because ethanol is then released by 
the reaction which does not encumber the pharmaceutical quality of the 
produced active ingredient with any undesired residues. The mixture is 
then added to a suitable solvent, preferably ethanol. The reaction is 
preferably carried out in the cold under air and light exclusion in as 
much as this is conducive to no side products forming. Magnesium 
pyridoxal-5'-phosphate glutamate (1) can be separated from the suspension 
by filtration for example. The product obtained in this manner can be 
purified by washing and/or recrystallization and subsequently dried. 
By using magnesium ethylate and ethanol, a solution is obtained after 
separation of magnesium pyridoxal-5'-phosphate glutamate (1) which 
consists of water and ethanol. The solution can be simply disposed of or 
can be separated by distillation into its components. 
In the following, the invention is illustrated further by an example. In 
this example, the synthesis of magnesium pyridoxal-5'-phosphate glutamate 
(1) occurs with the aid of the following equipment: 
a) temperature controlled mixing vessel (0 to 100.degree. C.) with pH 
monitoring and metering unit; 
b) temperature controlled kneading unit (0 to 100.degree. C.) with strong 
gearing in the low rotational speed range; 
c) extraction unit (aqueous/organic) and/or liquid-liquid separator; 
d) cation exchanger unit; 
e) filtration unit and/or solid-liquid separator; and 
f) drying unit. 
HPLC analyses were conducted under the following conditions: 
HPLC Conditions 
1. System 
a) chromatographic system: 
Shimadzu-unit comprising: LC-10AS liquid chromatograph; SIL-10A Auto 
Injector; SPD-10AV (UV-VIS; spectrophotometric detector); CBM-10A 
Communications Bus Module;

PRODUCTION EXAMPLE 
1st Step 
Production of p-phenetidyl-pyridoxal (4) from pyridoxine hydrochloride (6) 
100 g pyridoxine hydrochloride (6) are dissolved in 1500 g water and 
adjusted to a pH of 5.10 with cold 4N sodium hydroxide solution. The 
obtained solution is brought to a temperature of 14.degree. C. and added 
to 75 g activated manganese(IV) oxide under strong stirring. 
The reaction starts immediately after manganese dioxide addition and the pH 
value increases if it is not permanently adjusted. Through addition of 
100% sulfuric acid in portions, the pH value of the reaction suspension is 
held constant at 5.10.+-.0.10 by means of a constant pH constant holder 
under intensive stirring and temperature control (14.+-.2.degree. C.). The 
course of the reaction is followed by means of HPLC (system 1) in the form 
of in-process controls. After approximately ten hours, the entire amount 
of pyridoxine hydrochloride was converted into pyridoxal. After that, the 
excess manganese dioxide--non-reacted and contaminated with pyridoxal--is 
filtered, washed three times with approximately 300 g deionized water and 
dried at 120.degree. C. 25 g of manganese dioxide is recovered which can 
be used again. The combined aqueous solutions are first subjected to a 
membrane filtration (0.8 .mu.m) and then adjusted to pH 4.5 with 10% 
sulfuric acid. Then, 80 g freshly distilled para-phenetidine is added in a 
portion to the pyridoxal solution under very intensive stirring. Thereby, 
the pH value of the reaction mixture increases to about 4.7 and the 
reaction product abruptly precipitates. This is further stirred for half 
an hour and subsequently filtered. The para-phenetidine containing therein 
is recovered by means of a liquid-liquid separator from the filtrate which 
is to be disposed. 
The precipitate is suspended three times each in 700 g deionized water and 
filtered. Subsequently, it is suspended and filtered by means of an 
Ultra-Turax in 700 g n-heptane. The n-heptane filtrate is redistilled and 
the collected para-phenetidine residues are stored until conclusion of the 
third step. 
The residue obtained in this manner is dried at 120.degree. C. and sifted 
to a particle size of .ltoreq.500 .mu.m. 124.8 g (=89.7% theoretical 
yield) of a fine, very light orange-yellow powder of 
para-phenetidyl-pyridoxal (4) is obtained. 
2nd Step 
Production of para-phenetidyl-pyridoxal-5'-phosphate (3) from 
para-phenetidyl-pyridoxal (4). 
1000 g of Polyphosphoric acid are placed into a temperature controlled 
kneading unit and cooled to 10.degree. C. 250 g para-phenetidyl-pyridoxal 
(4) are slowly applied in small portions to the Polyphosphoric acid under 
intensive cooling. The Schiff's base of the pyridoxal slowly dissolves in 
the phosphorylation reagent under red coloration. The reaction heat formed 
thereby must be intensively carried off in such a manner that the 
temperature of the reaction mixture does not exceed 25.degree. C. A 
temperature increase to about the double of this leads to approximately 
20% additional loss of yield. After the entire para-phenetidyl-pyridoxal 
mass was applied, the reaction mixture is further stirred under intensive 
cooling (.ltoreq.25.degree. C.) overnight. On the following day, no more 
para-phenetidyl-pyridoxal is to be identified by means of an in-process 
control with the aid of HPLC (system 1). The phosphorylation step is 
concluded therewith. Subsequently, approximately 2125 g ice are added into 
the mixing chamber and the reaction suspension is stirred further under 
intensive cooling for three hours. A homogeneous mustard-coloured mass 
forms while doing so. It is slowly added to 75 g 95-97% cold sulfuric 
acid, homogenised and heated for approximately 30 min at 80.degree. C., 
wherein the polyphosphates are hydrolysed. The hydrolysis is followed by 
means HPLC (system 2), whereby the exact moment of the stop of hydrolysis 
is recognised (based on the complete disappearance of the Polyphosphate 
peak). The end of hydrolysis is brought about by rapid cooling of the 
reaction solution to 10.degree. C. Thereby, the phosphorylated product 
begins to precipitate. Then, approximately 7500 g of 2N sodium hydroxide 
solution pre-cooled to approximately 10.degree. C. are slowly added under 
intense stirring and cooling until the pH value of the reaction suspension 
is 2.10. The orange-brown reaction product precipitated thereby is 
filtered well and washed three times each with approximately 500 g 
deionized water. The well filtered precipitate is directly used for the 
third step of the synthesis. 
Nevertheless, should the product of this step be dried, this can occur at 
105.degree. C. 263.4 g (=82.3% theoretical yield) of an orange-brown fine 
powder of para-phenetidyl-pyridoxal-5'-phosphate (3) would be obtained. 
3rd Step 
Production of pyridoxal-5'-phosphate (2) from 
para-phenetidyl-pyridoxal-5'-phosphate (3). 
100 g and/or the corresponding amount of the moist precipitate of 
para-phenetidyl-pyridoxal-5'-phosphate (3) obtained in the second step are 
added to 700 g 2N sodium hydroxide solution. The Schiff's base is 
hydrolysed to pyridoxal-5'-phosphate and para-phenetidyl at pH 
.gtoreq.12.5. In the case that the above given amount of lye is not 
sufficient in order to obtain a pH .gtoreq.12.5--on account of the moist 
precipitate --the necessary amount of additional 2N sodium hydroxide 
solution is used. By extracting three times each with 300 g toluol, the 
para-phenetidine is removed from the solution. The toluol solution is 
redistilled. As an alternative to toluol extraction, an 
organic-aqueous-separator for continuous operation can be used for the 
separation of para-phenetidine. The collected para-phenetidine residues 
from the first and third steps can be purified by means of distillation 
and newly used. 
The pyridoxal-5'-phosphate is present at the end of this separation 
operation as a sodium salt in the aqueous solution. By means of 800 ml 
cation exchanger, for example Amperlite Type IR-120 (1.9 mVal/ml), the 
sodium ion is removed from pyridoxal-5'-phosphate. A pure aqueous solution 
of pyridoxal-5'-phosphate is obtained. 2500 ml eluate contain 57-63 g 
(78.6-86.9% theoretical yield) pyridoxal-5.sup.1 -phosphate (2). This is 
concentrated under vacuum at max. 40.degree. C. to approximately 500 ml 
and used for MPPG production. The Pyridoxal-5'-phosphate synthesised in 
this manner can also be isolated at this point (new synthesis of 
pyridoxal-5'-phosphate). 
4th Step 
Production of magnesium pyridoxal-5'-phosphate glutamate (1) from 
pyridoxal-5'-phosphate (2). 
540 g deionized water are placed into a reaction vessel and cooled to 
0-3.degree. C. Under nitrogen atmosphere and light exclusion, 62.58 g 
magnesium ethylate (9) are added and brought into solution by stirring. 
Thereby, the temperature increases to approximately 10.degree. C. This is 
cooled to 3.degree. C., 31.85 g glutamic acid (8) are added, and this is 
stirred for a further 10 min. 
Then, 500 ml pyridoxal-5'-phosphate solution of the third step 
(.ident.11.6% solution) is added in small portions within 60-90 min. to 
the magnesium glutamate solution just produced. Thereby, attention is paid 
that the pH value of the solution remains within the range 8-9. 
Subsequently, this is further stirred for 3-5 hours and the resulting 
solution is filtered. 
2000 ml ethanol is placed into a mixing vessel and cooled to 0.degree. C. 
under nitrogen atmosphere and light exclusion. Subsequently, the solution 
produced above is added drop-wise under stirring. The suspension attained 
in this manner is further stirred for approximately 15 hours, then 
filtered, and washed with a total of 200 ml ethanol. After that, the 
entire product is suspended in approximately 100 ml ethanol and newly 
filtered. The ethanol-moist product is dried in a drying cabinet at max. 
60.degree. C. 94.5-99.5 g (94-99% theoretical yield) of magnesium 
pyridoxal-5'-phosphate glutamate (1) is obtained.