JPS5838285A - Preparation of 5-methyl-(6rs)-5,6,7,8-tetrahydro-l-folic acid magnesium salt or its free acid - Google Patents

Abstract

PURPOSE:To prepare the titled compound which is a methyl-transfer coenzyme, in high purity, by reacting (6RS)-5,6,7,8-tetrahydro-L-folic acid with formaldehyde, followed by reduction and precipitation. CONSTITUTION:(6RS)-5,6,7,8-Tetrahydro-L-folic acid is made to react with equivalent amount of formaldehyde to obtain 5,10-methylene-(6RS)-5,6,7,8-tetrahydro-L- folic acid, which is reduced with sodium borohydride or potassium borohydride at 0-100 deg.C and 6-8pH. The pH of the resultant reaction liquid is adjusted to 7-9, the precipitated boric acid is removed from the liquid, and a magnesium salt (e.g. MgCl2) is added to the liquid to obtain the objective compound of formula as precipitate.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides 5-methyl-(6R8)-5,6,7°8-
The magnesium salt of tetrahydrofolic acid (hereinafter referred to as 5-methyl-tetrahydrofolic acid 111ml) relates to a novel process for the preparation of the free acid.

5-Methyl-tetrahydrofolic acid is a coenzyme for methyl group transfer represented by the structural formula: It catalyzes important reactions such as the biosynthesis of methionine and thymidine.

4-Amino-4-deoxy-N-methyl-L-folic acid (
(generic name: methotrexate) is widely used as a folic acid 11 antagonist.

These antifolates are 6-formyl-(6R
8) -5,6,7,8-tetrahydro-L-folic acid (generic name, leucovorin) dihydrofolate, an enzyme that catalyzes the biosynthesis of two coenzymes, sP and b-methyl-tetrahydrofolate. Inhibits the synthesis of folate reductase.

In order to minimize the effects of inhibition of the action of dihydrofolate synthetase, readily available leucovorin is administered to patients undergoing treatment with monofolate antagonists.

However, it is known that 5-methyl-tetrahydrofolate has a superior effect on antifolates than leucovorin (T, l, Kalman et al.
and J.C., YalowiCh; R.L.

K15lluk and G, M, Brown's Chemi
stry andBiology%Elservle
r/North-Holland *1nc, 1979
．． 671), it is difficult to produce 5-methyl-tetrahydrofolic acid with high purity, and it has not yet been used clinically.

6-Methyl-tetrahydrofolic acid is generally prepared by preparing 5.10-methylene-tetrahydrofolic acid using a large excess of sodium borohydride)') I7. '(K), but the product seen is of low purity and D
It cannot be used for treatment unless it is purified by time-consuming and costly chromatography on KAE-cellulose. As a result of extensive research, the present inventors found that (6R8)-5,
6,7,8-tetrahydrofolic acid (hereinafter referred to as tetrahydrofolic acid) and formaldehyde are reacted to produce 6,10-methylene-(6R8)-6,6,7,8-tetrahydrofolic acid. Then, this is reduced using lithium borohydride or potassium borohydride, and the resulting reaction solution is adjusted to pH 7 to 9 to precipitate and remove boric acid, and then magnesium salt is added to remove the precipitate. The present invention was completed based on the discovery that highly pure magnesium salt of 5-methyl-tetrahydrofolic acid or its free acid, which does not require purification by chromatography, can be found in the produced kernels.

In the present invention, it is preferable to use the same violet as tetrahydrofolic acid and formaldehyde. It5c tide response is θ~100
The magnesium salt added is preferably magnesium chloride, aWl- Water-soluble ones such as Ygnesium, magnesium nitrate, and magnesium acetate are preferred, and the pH of the solution is preferably about 6 am.

The free acid can be extracted by treating the magnesium salt of 6-methyl-tetrahydroS acid in a conventional manner.

Tetrahydrofolic acid was prepared by folic acid by S, Futterman's method (J, biol, Chemistry 22g, 1
081 (1967)) or the method described in Reference Examples below to obtain 7,8-dihydro a-I,-folic acid, which is further reduced with sodium borohydride or the like.

Next, the manufacturing method of the present invention will be explained with reference to Reference Examples and Examples, but the present invention is not limited to these Examples.

Reference Example 1 (Production of 7,8-dihydro-1N acid) 15g of Scorbine h was dissolved in 800ml of water, and Na was added to the resulting solution.
Add 1510425g and adjust the pH to 4.6 with 2N-NaOH.
Adjusted to. 10g of folic acid to 175mjK of water! ! ! The suspension was made into a solution using 26 ml of 2N NaOH. The two solutions obtained were mixed at room temperature and after 20 minutes ascorbic acid was added for 10 minutes! Slowly add 1g while stirring.9. In that case, other acids may be used instead of ascorbic acid. When the CpH reached 4 to 4.5, the pH was adjusted to 2.8 by further adding 1ON-HCI. The precipitated 7,8-dihydro-L-folic acid was collected and 10
Washed twice with 0 mm water.

Reference Example 2 (Production of Tetrahydrofolic Acid) The wet precipitate of 7,8-dihydro-L-folic acid obtained in Reference Example 1 was added directly to the solution obtained by dissolving 45 g of NIBH in 150 m# of water, and the mixture was stirred at room temperature. Stirred for 45 minutes. 0
After refrigerating at 8C, 10N- was added until the pH reached 7.5.
HCI was slowly added and the mixture was washed twice with Tetotsu.

Example: The wet precipitate of tetrahydrofolic acid obtained in Reference Example 2 was diluted with 2N
-NaOH18mj K @ m1 seen with turbidity
The Il solution was brought into solution very slowly by stirring at 22 cC. This solution was dissolved in P using hydrochloric acid.
6.10-methylene-(6R3)-5,6,7 by adding 37% formaldehyde at 22°C.
, 8-tetrahydro-L-folic acid. After 10 minutes, 1.5 g of NmBH was added.

After leaving this at 22 cCf for 12 to 15 hours,
Carefully adjust the pH of the solution to ~4% to 8. The mixture was adjusted to OK and a portion of the precipitated borate was collected. See the nine-liquid lN-11c
After adjusting the pH to 6.0 Kli and decolorizing with activated carbon, the activated carbon was removed. Seen 9-liquid 2~
When a solution of 8 g of magnesium chloride dissolved in 8 mA water was added and left at θ to 4°C for 16 hours, a precipitate formed.9.

This was collected, washed with a small amount of cold water, and then recrystallized from 80 B of water to obtain 8.8 g of magnesium salt (tetrahydrate) of 6-methyl-tetrahydrofolic acid (yield 26.8%).
.

The results of analysis of this substance were as follows.

Elemental analysis value (0-Mu-fuki) 06・4H2O) Calculated value (4
): 04! , 6 BS, 6 Seal 17.7 Actual value (%
): 04B, 1 ink 115.65 117-89TJ
v Spectral analysis value = (Using this sphate buffer, p
H7) Jmax (290mm): 28-11X10
”8 towers 11m (245 Yuho) Knee 7.78X103
1H→jMl spectrum analysis value: (100MHss
0ff3000D) 8.0 and 7.5 (Amu-J
Part A and Part 1 of the IB-System, J W 91i
,,4 H), 4.9-5.2 (ha-a(g))
, 4-6 to 4-2 (1kH-c(6)k4.2 to 3.7
(xH2-CX7) and II, 1! -0(9)), L
A! (s, Hg0-summer (5)), 2.8-2.5 (xH
χ-〇(β) and Hg-Oke)) 13o-n is spectrum analysis: Fig. 1 shows a spectrum chart. In addition, J, Mu, Ly
on, 11. Dstmlop and P,D,1lli
s, J, Magnetia Rvs, j@, 29B (1
97! Please refer to S).

The resulting magnesium salt was treated in a conventional manner to obtain 5-methyl-tetrahydrofolic acid.The reactions in Reference Examples and Examples were carried out under an atmosphere of nitrogen or argon gas.

[Brief explanation of the drawing]

FIG. 1 is an O-IMII spectrum chart of the magnesium salt (tetrahydrate) of 5-methyluptrahydrofolic acid.

Claims (1)

[Claims] 1 (6R8)-5,6,7,8-tetrahydro, reacted with monofolic acid and formaldehyde to form 5°10-
Methylene-(6R5)-5,6,7,8-nato2hydro-L-folic acid is obtained, which is reduced using aqueous sodium boron or potassium borohydride, and the resulting reaction solution is adjusted to pH 7-9. 5-Methyl-(6R5)-5,6゜7.8-, which is characterized in that the borate is precipitated and removed, and then a magnesium salt is added to cause precipitation.
A method for producing a magnesium salt of tetrahydrofolic acid or its free acid. 2. The method according to claim 1, wherein (6R8)-5,6,7,8-tetrahydro-folic acid and formaldehyde are reacted in equivalent amounts. 8. The production method according to claim 1, wherein the reduction reaction is carried out at θ-100OC and pH 6-8. 4. The production method according to claim 1, wherein the reduction reaction is carried out at room temperature and pH 7. 6. The method according to claim 1, wherein the precipitation of boric acid is carried out at pH 8 and room temperature. 6 Add magnesium salt and precipitate at pH 6.
The manufacturing method according to claim 1, which is carried out in 7. The manufacturing method according to claim g61 or vj6, wherein the magnesium salt is water-soluble magnesium chloride, magnesium sulfate, magnesium nitrate, or magnesium acetate.