Process for separating stereoisomers of folinic acid

The (6S) stereoisomer of folinic acid is obtained in high optical purity by salification of (R,S) folinic acid with di- or polyamines and subsequent selective crystallization of the desired diasteromeric salt.

The present invention relates to a process for separating stereoisomers of 
folinic acid. 
Folinic acid, i.e. N-(5-formyl-(6R,S)-5,6,7,8-tetrahydropteroyl)-L-glutamic 
acid, when obtained by chemical synthesis, is formed by an equimolar 
mixture of its two (6R) and (6S) diastereomeric forms. 
It is known that only the (6S) isomer, as calcium salt, has the well-known 
pharmacological activity of the product, while the other one is totally 
devoid of it. Therefore, there is a strong need for a process allowing the 
preparation of the optically pure (6S) form. 
Various attempts were made to synthetize (6S) isomer through asymmetrical 
synthesis but they were not fully successful from the industrial point of 
view. 
As far as the separation of both diastereomers from their equimolar mixture 
is concerned, it is worth citing U.S. Pat. No. 2,688,018 and patent 
application WO 88/03844, even if they have not yet provided the ideal 
solution to the problem. 
It has now been found a process allowing the separation and the isolation 
with good yields of the two optically pure (6R) and (6S) forms of folinic 
acid, both free and salified with alkaline-earth metal ions, from the 
equimolar diastereomeric mixture produced by chemical synthesis starting 
from folic acid. 
Another remarkable embodiment of this invention relates to a process for 
the preparation and isolation of salt derivatives of (6R)-folinic acid, 
respectively (6S)-folinic acid, characterised by an excellent optical 
purity (O.P.), with suitable at least dibasic organic amines, which are 
aliphatic, straight, cyclic and/or heterocyclic, from said diastereomeric 
equimolar mixture. 
Another remarkable embodiment of this invention is represented by the new 
salts of (6R)-folinic and/or (6S)-folinic acid with said amines. 
Folinic acid is usually prepared by synthesis starting from folic acid, 
which contains a (S) chiral centre in that part of the molecule 
corresponding to (S)-glutamic acid. By hydrogenating the double bond 
between the 5- and 6- positions of the pterinic residue, a new chiral 
centre in the 6- position is originated with a (6R,S) configuration. The 
subsequent formylation step of nitrogen atom at the 5-position leads to 
the production of (6R,S)-folinic acid. 
It now has been surprisingly found that, by crystallization of 
(6R,S)-folinic acid salts with at least dibasic organic amines from a 
suitable solvent or preferably, from binary or ternary mixtures of 
solvents, mixtures of said salts enriched in their (6R) or (6S) form can 
be obtained, according to the amines used. 
Said optically enriched mixtures can be further enriched in the 
preponderant isomeric form through one or more recrystallizations to 
obtain the corresponding salts with said amines of (6R)-folinic acid, 
respectively (6S)-folinic acid, with an O.P. of at least 99%, usually 
higher than 99%. 
The conversion from these salts to the corresponding salts with 
alkaline-earth metal ions with the same optical purity is very easy. 
By way of example, from the mixture enriched in its (6S)-form, the calcium 
salt (or the corresponding salts with alkaline-earth metal ions) of 
(6S)-folinic acid can be prepared with an O.P. higher than 99%. This 
result can preferably be achieved according to one of the following 
methods: 
a) from the aqueous solution of the (6S) enriched diamino folinate, through 
treatment with an alkaline-earth metal chloride at neutral pH and 
optionally in presence of a precipitating solvent, the alkaline-earth 
metal salt of the (6S) enriched folinic acid is separated. Further 
crystallizations from a suitable solvent or solvent mixtures lead to the 
optically pure (6S) form of the desired alkaline-earth metal folinate, 
b) the (6S) enriched diamino folinate is recrystallized from suitable 
solvent mixtures in order to obtain the optically pure (6S) form. The 
corresponding salt with alkaline-earth metal ions is subsequently obtained 
through exchange, as described in a). 
The same process can be applied to the (6R) enriched mixture to obtain the 
corresponding (6R) isomer with an O.P. higher than 99%. 
Preferred metal cations to salify (6R,S)-folinic acid and its optically 
pure diastereomeric forms are those of the alkaline-earth metals, 
especially Ca, Mg, St, Ba. Ca.sup.2+ ion is particularly preferred. 
At least dibasic organic amines suitable for the salification of 
(6R,S)-folinic acid can be selected from straight, cyclic or heterocyclic, 
substituted or unsubstituted, racemic or chiral, aliphatic di- or 
polyamines, which contain at least two amino groups linked to one another 
by at least one substituted or unsubstituted hydrocarbon chain, comprising 
at least 2 or 3 C atoms. 
Particularly preferred amines are diamines of general formulae from I to 
III, 
##STR1## 
wherein: R.sub.1 to R.sub.8 which are the same or different, are H or 
straight or branched C.sub.1-6 alkyl groups, which can be substituted by 
1-4 OH groups and/or by 1-4 alkoxy or hydroxyalkoxy groups, 
R.sub.9 to R.sub.10 which are the same or different, are R.sub.1 or OH, 
n 0-6, 
m 2-8. 
By way of non-limiting example, particularly preferred diamines can be 
selected from: ethylenediamine, 1,2-diamino-propane, 1,3-diamino-propane, 
1,3-diamino-2-hydroxy-propane, (cis)-1,2-diamino-cyclohexane, 
(trans)-1,2-diamino-cyclohexane, piperazine, 1,4-dimethyl-piperazine, 
2-methyl-piperazine, 2,5-dimethyl-piperazine. Equally preferred amines can 
also be polyamino straight derivatives or macrocyclic compounds containing 
from 3 to 6N atoms. 
By way of example, among these last ones, 1,4,7,10-tetraazacyclododecane 
and its derivatives substituted both on ethylene bridges and on nitrogen 
atoms can be cited. 
Suitable solvents for the crystallization of diastereomeric mixtures of 
folinic acid salts with said amines are preferably binary or ternary 
water/dipolar aprotic organic solvent mixtures and/or mixtures of 
water/dipolar aprotic organic solvent/protic organic solvent. 
Preferred protic organic solvents are, i.e., methanol, ethanol, n-propanol, 
isopropanol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 
formamide, N-methyl-formamide. 
Preferred dipolar aprotic organic solvents are, i.e., dimethylformamide 
(DMF), dimethylacetamide (DMAC), dimethylsulphoxide (DMSO), 
N-methyl-pyrrolydone (NMP), hexamethylphosphoramide (HMPT). 
The process of this invention is hereinafter described, employing, by way 
of non-limiting example as starting material the calcium salt of 
(6R,S)-folinic acid and it is performed according to the following steps: 
STEP 1 
Precipitation of (6R,S)-folinic acid from an aqueous solution of its 
calcium salt, by adjusting pH between 1 and 3 with mineral acids at a 
temperature ranging from 0.degree. to 25.degree. C. 
STEP 2 
Formation of (6R,S)-folinic acid salts with diamines/polyamines in a 
dipolar aprotic organic solvent, preferably DMAC. Said salts are obtained 
under a solid form by: 
a) dissolution of (6R,S)-folinic acid in a dipolar aprotic solvent, in a 
weight/volume ratio ranging from 1:5 to 1:20 (w/v); 
b) reaction, at a temperature from 5.degree. to 35.degree. C., with a 
stoichiometric amount, or a molar excess not higher than 20%, of the 
desired amine, dissolved either in the same dipolar aprotic solvent in a 
ratio ranging from 1:5 to 1:50 (w/v) or in a protic organic solvent in a 
ratio ranging from 1:5 (w/v) to 1:15 (w/v); 
c) gradually cooling the reaction medium at a temperature ranging from 
25.degree. C. to 5.degree. C., up to a complete precipitation of the 
desired salt. Precipitation can be facilitated by adding a protic organic 
solvent in a volume ratio ranging from 0.1:1 to 2:1 (v/v) compared with 
the dipolar aprotic solvent. 
Said salification reaction can also be performed in water, in suspension 
phase, followed by dilution of the resulting aqueous solution with or in a 
water-soluble protic organic solvent, which acts as a precipitating agent. 
STEP 3 
Partial separation of folinic acid diastereomers by crystallization or 
insolubilization of its salts with said amines from a water/dipolar 
aprotic water-soluble solvent, optionally with addition of an organic 
protic solvent. 
The weight/volume (w/v) ratio salt/mixtures of solvents can range from 1:2 
to 1:60, preferably from 1:4 to 1:45. 
The composition of the mixture water/dipolar aprotic solvent (v/v) can 
range from 1:0.5 to 1:20, preferably from 1:1 to 1:15; such mixture can 
also contain a protic organic solvent in amounts up to 70% in volume, 
preferably to 50%, in case said protic organic solvent is needed. 
The salts of (6R,S)-folinic acid with the above mentioned amines can 
directly be dissolved in the desired mixture of solvents at a temperature 
comprised between 25.degree. and 70.degree. C., preferably 30.degree. and 
50.degree. C. 
On the other hand, they can also be dissolved in water. The desired amount 
of dipolar aprotic solvent, optionally together with the required 
additional amount of protic organic solvent, is subsequently added to said 
aqueous solution. 
Then these solutions are gradually cooled to nearly 0.degree. C. while 
crystallization takes place. 
The obtained solid moiety consists of a mixture of (6R) and (6S) isomers of 
amino-folinates, which is enriched in the less soluble form (i.e. R or S, 
depending on the amino compound used as salifying agent). 
For example, crystallization of ethylenediamino (6R,S)-folinate leads to a 
mixture enriched in the (6R) isomer salt. 
On the contrary, from piperazino (6R,S)-folinate, the (6S) form preferably 
crystallizes. 
Usually said enriched solid mixtures show an O.P. ranging from 65 to 98%. 
Crystallization mother liquors are obviously enriched in the other isometic 
form and show an O.P. value usually ranging from 65 to 80%. 
STEP 4 
Further purification of the partially optically purified amino folinates, 
deriving from Step 3, is performed through one or more recrystallizations 
of the same from a mixture water/dipolar aprotic solvent whose composition 
ranges from 1:1 (v/v) to 1:20 (v/v). This crystallizing mixture can 
optionally contain also a protic organic solvent in amounts up to 50% in 
volume. The desired optically pure amino folinate is obtained with an O.P. 
higher than 99%. 
Then said optically pure amino folinate can be transformed into the 
corresponding (6R), respectively (6S), calcium folinate. 
One of the preferred ways consists in crystallizing said amino folinate 
from water (pH=7 and temperature in the range 0.degree.-25.degree.0 C.) in 
the presence of 0.2 to 5, preferably 2 to 4, parts (w/w) of water soluble 
calcium salts, preferably chlorides or nitrates. 
Alternatively the desired optically pure calcium folinate can be 
precipitated from the above mentioned aqueous Ca.sup.2+ containing 
solution by adding a great excess of a protic water-soluble organic 
solvent. 
STEP 5 
The stereoisomers of folinic acid, which are contained in the mother 
liquors resulting from the crystallizations described in Step 3, are also 
recovered from said mother liquors with an O.P. higher than 99%. 
They can be obtained either as amino or calcium salts, preferably following 
one of the procedures hereinafter described. 
a) From the above mentioned mother liquors the organic protic solvent, if 
present, is distilled off under reduced pressure at temperatures from 
25.degree. to 50.degree. C. and subsequently the water-soluble dipolar 
aprotic solvent is removed by extracting the mixture with a 
water-insoluble organic solvent, preferably methylene chloride (CH.sub.2 
Cl.sub.2). To the resulting aqueous solution CaCl.sub.2 is added in 
amounts corresponding to 0.2-5, preferably to 2-4, parts in weight (w/w) 
in relation with the starting quantity of amino (6R,S)-folinate. Then pH 
is adjusted to a value ranging between 6 and 7 by means of 1N NaOH and, by 
adding ethanol the precipitation of the desired calcium salt is obtained, 
operating at a temperature of 0.degree.-25.degree. C., preferably 
5.degree.-25.degree. C. 
Calcium folinate, enriched in its prevailing diastereomer, with an O.P. 
ranging from 65 to 85%, is recovered through filtration. The optically 
pure calcium folinate (O.P. higher than 99%) is finally obtained through 
one or more crystallizations from 10 to 100 volume of water, with pH 
ranging from 6 to 7 and at temperatures from 0.degree. to 25.degree. C. 
b) To mother liquors, deriving from Step 3, suitable precipitating 
solvents, either dipolar aprotic or protic organic solvents, are added in 
amounts equal to 0.05-5 volumes in relation with the mother liquors 
volume. By cooling to 0.degree.-20.degree. C., the prevailing optical 
isomer of diamino folinate can be preferentially crystallized with an O.P. 
higher than 70%. The desired amine folinate with an O.P. higher than 99% 
is then obtained by one or more crystallizations from water/dipolar 
aprotic solvent mixtures with a ratio ranging from 1:1 to 1:20 (v/v), 
optionally by adding a protic organic solvent, in amounts up to 50% in 
volume. 
Amino (6R,S)-folinate obtained as described in Steps 1 and 2 can 
alternatively be prepared directly from a calcium (6R,S)-folinate aqueous 
solution. To this solution another aqueous solution can be added 
containing, in stoichiometric amounts compared with the starting folinate, 
the desired amine salified with a mineral or organic acid which forms 
insoluble salts with alkaline-earth metal cations, preferably anions of 
sulphuric or oxalic acid. The insoluble salt precipitating, i.e. calcium 
oxalate, is filtered off and to the resulting aqueous solution, containing 
the desired diamino (6R,S)-folinate, the suitable amount of dipolar 
aprotic solvent is subsequently added, optionally added with protic 
organic solvent as previously described. 
Crystallization can be performed at a temperature ranging from 0.degree. to 
25.degree. C. and lasts from 1 h up to five days by operating at a neutral 
or slightly alkaline pH. The procedure described in step 1 can also be 
adopted in order to obtain the optically pure isomers of free folinic acid 
(6R or 6S), with an O.P. higher than 99%, starting from the corresponding 
optically pure salts of the same obtained according to step 4 and 5.

The following examples aim at better disclosing the most remarkable 
features of this invention, and are not a limit to the same for the 
skilled technician. 
EXAMPLE 1 
Preparation of pure (6R,S)-folinic acid. 
100 g of calcium (6R,S)-folinate pentahydrate, dissolved in 750 ml of water 
and 670 ml of 0.5N hydrochloric acid, are added dropwise at the same time 
to 300 ml of water, under stirring and at 5.degree.-20.degree. C. Then the 
obtained suspension, containing (6R,S)-folinic acid, is kept under 
stirring for 1 h at 5.degree.-25.degree. C. After that the precipitate is 
filtered, washed with water and dried. 76 g of (6R,S)-folinic acid are 
obtained. 
HPLC titre: .gtoreq.99% performed under the following chromatographic 
conditions: 
Column: Hibar.RTM. Lichrosphere RP18-5 .mu.m (Merck): 250 mm length; 4 mm 
diameter. 
Mobile phase: A=phosphate buffer pH 7.8; B=25% methanol in phosphate buffer 
pH 7.8 (v/v). 
Flow rate: 1.2 ml/min; column temperature: 35.degree. C.: UV 254 nm 
detector. 
Gradient: from 0 to 2 min B=12%, from 2 to 20 min straight gradient up to 
B=80% then maintenance for 3 min, then inverse gradient from 23 to 35 min 
up to B=12%. 
Following the same procedure the following free acid compounds have been 
obtained: 
(6E)-folinic acid with an O.P. higher than 99%, starting from calcium 
(6S)-folinate of Example 6. 
(6R)-folinic acid with an O.P. higher than 99%, starting from 
ethylenediamino (6R)-folinate of Example 6. 
EXAMPLE 2 
Preparation of ethylenediamino (6R,S)-folinate. 
75 g of (6R,S)-folinic acid are dissolved in 750 ml of dimethylacetamide 
(DMAC). 10.5 g of ethylenediamine, dissolved in 400 ml of the same 
solvent, are added at a temperature of 5.degree.-35.degree. C. The 
precipitated salt suspension is kept under stirring for abut 1 h, at 
5.degree.-25.degree. C., then is filtered. After drying, 82 g of 
ethylenediamino (6R,S)-folinate are obtained. 
HPLC titre: .gtoreq.98.5% according to the chromatographic method of 
Example 1. 
EXAMPLE 3 
Diastereomer separation of ethylenediamino (6R,S)-folinate. 
80 g of ethylenediamino (6R,S)-folinate are dissolved in 740 ml of water. 
920 ml of DMAC are added under stirring at a temperature of 
25.degree.-40.degree. C. Then temperature is lowered to 5.degree. C. and 
the product crystallizes, under stirring, for 24 h. After that the 
precipitate is filtered off and washed with ethanol. After drying, 40.2 g 
of ethylenediamino folinate are obtained, containing the 80% of (6R) 
diasteroisomer. The (6R) form content is determined by performing a chiral 
HPLC under the following conditions: 
Column: length 250 mm; diameter 4 mm; manually fed with Chiral Si 300 BSA 
(SERVA), 5 .mu.m stationary phase. 
Mobile phase: 0.25M solution of NaH.sub.2 PO.sub.4 in water. 
Flow rate: 1.0 ml/min; column temperature 40.degree. C.; detector: UV 280 
nm (310 nm). 
Mother liquors, after filtration, contain ethylenediamino (6S)-folinate 
with a 60% diastereomeric excess (determined with chiral HPLC under the 
above mentioned conditions). 
Chromatographic conditions disclosed in Examples 1 and 3 are also used for 
controls and determinations in the remaining examples. 
EXAMPLE 4 
Ethylenediamino (6S)-folinate isolation and purification. 
To the mother liquors derived from the crystallization described in Example 
3 and mostly containing ethylenediamino (6S)-folinate, with a 60% 
diastereomeric excess, 170 ml of DMAC are added. The solution is kept 
under stirring for 48 h at 10.degree. C. The resulting precipitate is 
filtered, washed with ethanol and dried. 15 g of ethylenediamino 
(6S)-folinate are obtained with an O.P. higher than 99% (chiral HPLC 
control was performed according to Example 3). 
HPLC titre: .gtoreq.58.5; [.alpha.].sup.20.sub.D =-14.8.degree.(c=2.1% 
H.sub.2 O). 
EXAMPLE 5 
Preparation of calcium (6S)-folinate from ethylenediamino (6S)-folinate. 
To 10 g of ethylenediamino (6S)-folinate (obtained as disclosed in Example 
4 ), dissolved into 100 ml of water, 10 g of calcium chloride and 400 ml 
of ethanol are added under stirring at 5.degree.-25.degree. C. The 
separated precipitate is filtered, washed with aqueous ethanol (95%) and 
dried. Then, the product is dissolved again in water at pH 6-6.5, at 
50.degree.-60.degree. C. One part in weight of calcium chloride is added 
and pH is adjusted to 7 with NaOH 1N. The desired compound crystallizes at 
0.degree.-25.degree. C. for 16 h. 
The precipitated crystalline product is filtered, washed with cold water 
and aqueous ethanol and dried. 11 g of calcium (6S)-folinate containing 
about 20% of crystallization water are obtained with an O.P. higher than 
99% (through chiral HPLC). 
MPLC titre: &gt;99%; [.alpha.].sup.20.sub.D =-15.degree.(c=1% H.sub.2 O). 
EXAMPLE 6 
Purification of ethylenediamino (6R)-folinate. 
35 g of ethylenediamino (6R)-folinate (obtained as described in Example 3), 
with 80% O.P., are recrystallized from 500 ml of water/DMAC with a 
volumetric ratio of 1/1.25. Temperature and crystallization time are the 
following: 0.degree.-25.degree. C. and 48 h. The crystallized product is 
filtered, washed with ethanol and dried. 25 g of ethylenediamino 
(6R)-folinate with an O.P. higher than 99% (chiral HPLC) are obtained. 
[.alpha.].sup.20.sub.D =+44.4.degree.(c=2.1% H.sub.2 O). 
EXAMPLE 7 
Calcium (6S)-folinate isolation and purification from an ethylenediamino 
folinate mixture enriched in the (6S) isomer. 
The mother liquors deriving from the crystallization described in Example 3 
and mostly containing ethylenediamino (6S)-folinate with a 60% 
diasteroisometic excess are extracted with 5.times.400 ml methylene 
chloride. To the resulting aqueous solution, 50 g of calcium chloride are 
added under stirring at 0.degree.-25.degree. C., then pH is adjusted to 7 
with NaOH 1N and 250 ml of ethanol are added. The solid separated product 
is filtered, washed with aqueous ethanol and dried. The resulting product 
is recrystallized twice from water, at pH 7, at 0.degree.-25.degree. C. 
and in the presence of 3 parts in weight of calcium chloride and washing 
the filtered product every time, the first time with cold water and then 
with ethanol. 
17 g of calcium (6S)-folinate are obtained with an O.P. higher than 99% 
(through chiral HPLC) and with about 20% crystallization water content. 
HPLC titre: &gt;99%; [.alpha.].sup.20.sub.D =-15.degree. (c=1% H.sub.2 O). 
EXAMPLE 8 
Piperazino (6R,S)-folinate preparation. 
80 g of (6R,S)-folinic acid, prepared as described in Example 1, are 
dissolved in 950 ml of DMAC. 17.5 g of piperazine dissolved into 2000 ml 
of ethanol are added thereto under stirring at a temperature of 
5.degree.-30.degree. C. The resulting suspension is kept under stirring 
for 3 h at room temperature and then filtered. The wet product is washed 
with ethanol, and then dried. 90.5 g of piperazino (6R,S)-folinate are 
obtained. 
HPLC titre: &gt;99%. 
EXAMPLE 9 
Diastereomer separation of piperazino (6R,S)-folinate. 
85 g of piperazino (6R,S)-folinate are suspended in 3600 ml of a mixture 
water/DMAC (1:10.5 v/v). Temperature is raised to 35.degree. C., until the 
solid compound is completely dissolved. Crystallization takes 3 days at 
5.degree. C., under stirring. The obtained precipitate is filtered, washed 
with ethanol and dried. 35 g of piperazino (6S)-folinate with an O.P. of 
.gtoreq.97% are recovered. 
EXAMPLE 10 
Piperazino (6S)-folinate purification. 
10 g of piperazino (6S)-folinate, obtained as described in Example 9, are 
recrystallized from a mixture water/DMAC (1:10.5 v/v) following the 
procedure of Example 6. After filtering, washing and drying the 
crystalline product, 8 g of piperazino (6S)-folinate with an O.P. higher 
than 99% (chiral HPCL control) are obtained. 
EXAMPLE 11 
Piperazino (6R)-folinate isolation and purification. 
To the mother liquors derived from the crystallization described in Example 
9, mostly containing piperazino (6R)-folinate, 1800 ml of ethanol are 
added. Crystallization takes place at 5.degree. C., for 64 h. The 
precipitate is then filtered, washed with ethanol and dried. 30 g of 
piperazino (6R)-folinate, with an O.P. of .gtoreq.94% (chiral HPCL 
control) are recovered. 
Said salt is then recrystallized from a mixture water/DMAC/ethanol (1:10:5 
v/v/v), following the procedure of Example 6, to give the desired product 
with an O.P. higher then 99%. 
EXAMPLE 12 
Preparation of calcium (6S)-folinate from piperazino (6S)-folinate. 
Calcium (6S)-folinate with an O.P. higher than 99% is obtained in yield of 
80% from the piperazino (6S)-folinate deriving from Example 10 following 
the procedure described in Example 5. 
EXAMPLE 13 
Preparation of 1,4-dimethylpiperazino (6R,S)-folinate 
80 g of (6R,S)-folinic acid, prepared as described in Example 1, are 
dissolved into 800 ml of DMAC. 21 g of 1,4-dimethyl-piperazine, dissolved 
in 460 of DMAC, are added under stirring at a temperature of 
5.degree.-25.degree. C., then 700 ml of ethanol are added. The solution is 
kept under stirring for 5 days at 5.degree. C. to obtain a precipitate 
which is filtered, washed with ethanol and dried. 
90 g of 1,4-dimethylpiperazine (6R,S)-folinate are obtained. 
(HPLC titre: &gt;99%). 
EXAMPLE 14 
Preparation of 1,3-diamino-2-hydroxy-propane (6R,S)-folinate. 
80 g of (6R,S)-folinic acid, prepared as described in Example 1, are 
dissolved into 800 ml of DMAC. 16.8 g of 1,3-diamino-2-hydroxy-propane 
dissolved into 420 ml of DMAC are added under stirring at 
5.degree.-25.degree. C. The solution is kept under stirring for 1.5 h at 
the same temperature to obtain a precipitate which is filtered, washed 
first with DMAC and then with ethanol, and then dried. 89 g of 
1,3-diamino-2-hydroxy-propane (6R,S)-folinate are obtained (HPLC titre 
&gt;99%). 
EXAMPLE 15 
Partial separation of 1,3-diamino-2-hydroxy-propane (6R,S)-folinate 
diastereomers, 
87 g of 1,3-diamino-2-hydroxy-propane (6R,S)-folinate are dissolved into 
600 ml of water. 780 ml of DMAC are added under stirring and at a 
temperature of 25.degree.-40.degree. C. The product crystallizes at 
5.degree. C. from the solution kept under stirring for 2 days, then is 
filtered. The wet product is washed with ethanol. After drying, 50 g of 
1,3-diamino-2-hydroxy-propane folinate containing about 70% of 
diastereomer (6R) (chiral HPLC), are obtained. In the residual 
crystallization mother liquors, the (6S) diastereomer is present in about 
55% diastereomeric excess. 
EXAMPLE 16 
1,3-diamino-2-hydroxy-propane (6S)-folinate isolation and purification. 
160 ml of DMAC are added to the mother liquors deriving from the fractional 
crystallization described in Example 15, and containing 
1,3-diamino-2-hydroxypropane (6S)-folinate, with a 55% diastereomeric 
excess. The solution is kept under stirring for 30 h at 5.degree. C. to 
obtain a precipitate which is filtered and washed with ethanol. After 
drying, 20 g of 1,3-diamino-2-hydroxy-propane (6S)-folinate are obtained 
with 95% O.P. This product is recrystallized from 300 ml of water/DMAC 
mixture in a volumetric ratio of 1/1.5 at a 0.degree.-25.degree. C. 
temperature. The resulting precipitate is filtered and washed with 
ethanol. After drying, 16 g of 1,3-diamino-2-hydroxy-propane (6S)-folinate 
are obtained with an O.P. higher than 99% (determined through chiral 
HPLC), titre .gtoreq.98.5%.