METHOD FOR PRODUCING PURE L-HERCYNINE
The present invention relates to a method for the industrial-scale
production of pure L-hercynine.
This invention relates to a new method for obtaining pure L-hercynine
without any chromatography purification step. More particularly, this invention
relates to a new industrial method for producing pure L-hercynine that includes a
step for electrodialysis purification, as well as a new method for producing Lhercynine
from L-histidine, as well as a new L-hercynine-d9 compound.
Background of the Invention:
Compounds of the betaine (quaternary ammonium) type are among the
most abundant nitrogenous organic molecules in the ground after proteogenic
and non-proteogenic amino acids. Glycine betaine and hercynine are the main
representatives of these betaines.
L-hercynine is the betaine derivative of L-histidine.
L-hercynine is the direct precursor of L-ergothioneine (Y. Ishikawa et al., J.
Biol, Chem.; 1974; 249; 4420-4427) in certain fungi, mycobacteria and
cyanobacteria (Pfeiffer, C. et al.; Food Chemistry; 2011; 129; 4; 1766-1769). It has
therefore naturally been found in these microorganisms (Mahmood, Z.A. et al.;
Pak. J. Pharm. Sci.; 2010; 23; 349-357), but also in some snakes (Ackermann, D. et
3
al.; Z. Physiol. Chem. Hoppe-Seyler’s; 1960; 318; 212-217). Very recently, Lhercynine
was shown for the first time in human red blood cells (Chaleckis, R. et
al.; Mol. Biosystems; 2014; DOI 10.1039/c4mb00 346b).
L-hercynine is a growth regulating substance in fungi such as Agaricus
bisporus (Champavier, Y. et al.; Enz. and Microbial. Technologt; 2000; 26; 2-4;
243-251).
As a betaine, L-hercynine could play an osmolyte role, in particular in plants
(Velisek, J.; Chemistry of Food; J. Wiley & Sons Eds.; 2013). As such, L-hercynine
performs a cyto-protective function in particular by contributing to the correct
folding of proteins in the cells.
Although its biological characteristics are considerable, L-hercynine has
not been studied very much biologically; this is probably due to the fact that it is
not commercially available (Warren, C.R.; New Physiologist; 2013; 198; 476-485).
Regarding its preparation, whether direct (1 step) or indirect (2 steps),
only 4 documents published to date mention it on the laboratory scale.
In 1968, Melville and his team reported the first synthesis of L-hercynine
from L-histidine in 2 steps (Reinhold, V. et al.; J. Med. Chem.; 1968; 11; 258-260).
Diagram 1: Two-step synthesis of L-hercynine according to Melville et al.
During the first step, the L-histidine undergoes methylation under
reducing conditions using formaldehyde in the presence of palladium on carbon
and hydrogen to lead to L-N,N’di-methyl-histidine. After treatment using organic
N,N-dimethylhisitidine
4
solvents, the desired intermediary is obtained by recrystallization with a yield of
82%. The second step consists of methylation using methyl iodide with pH 9. The
L-hercynine is obtained after treatment and purification on ion exchange column,
then recrystallization with a yield of 89%. It should be noted that the conditions
used by Melville are not racemizing. The global synthesis yield of pure Lhercynine
from L-histidine using this procedure is therefore 73%.
Having tested the direct tri-methylation route for L-histidine, Melville mentions
"difficulties in the tests to prepare L-hercynine by direct methylation of L-histidine
in particular due to the concomitant methylation of the imidazole core that
occurs under the conditions used and the corresponding pentamethylated
derivative that forms during the reaction" (Reinhold, V. et al.; J. Med. Chem.;
1968; 11; 260).
More recently, a second document mentions hercynine as anon-isolated
intermediary in the production of urocanic acid, the desired objective for the
authors of this publication (Valeev, F.A. et al., Chemistry of Natural Compounds;
2007; 43(2); 143-148) according to:
Diagram 2: Synthesis of L-hercynine by tri-methylation according to Valeev et al.
The authors mention the "one-step" methylation of L-histidine to obtain, on an
intermediary basis, hercynine, which, in a highly basic medium and hot, leads,
after elimination of the trimethyl amine, to the desired urocanic acid with a
urocanic acid
5
global yield of 66% from the L-histidine. Not having isolated the intermediate
hercynine, or shown the trimethyl amine as a sub-product, the formation of the
latter is only hypothetical. Furthermore, at no time do they mention subproducts
originating from the methylation of the imidazole core, as mentioned
by Melville in his 1968 publication. Lastly, since the chiral center is destroyed
during the reaction sequence leading to urocanic acid, no information is available
in order to determine whether this direct tri-methylation is done with or without
racemization.
In 2012, the preparation (in solution and mixed with the per-methylation
products) of hercynine by direct methylation for analytical studies was done
using a very large excess (16 equivalents) of methylation agent (Chary V. N. et al.,
J. Mass. Spectrom. 2012; 47; 79-88).
Lastly, in a Master's thesis in chemistry, Khonde also mentions the
preparation of L-hercynine by direct methylation of the L-histidine, but without
purifying the desired L-hercynine (Khonde, L.P.; Synthesis of Mycobacterial
ergothioneine biosynthetic pathway metabolites; University of Cape Town, South
Africa; 2013; page 61, diagram 3.19).
Diagram 3: Synthesis of L-hercynine by tri-methylation according to Khonde et al.
Khonde uses dimethyl sulfate, as methylating agent, in a basic medium
(10% NaOH). Curiously, only 2.6 equivalents of dimethyl sulfate are used,
whereas at least 3 equivalents are theoretically necessary to obtain L-hercynine
1. NaOH/
Me2SO4 (2.6 equivalents)
2. HCl/lyophilization
3. ether trituration
L-hercynine
(subsequently used without purification)
6
from L-histidine. Furthermore, no structural data is provided by the author, who
uses the raw reaction medium for the following step.
Although there are several chemical synthesis methods for L-hercynine in
solution and in mixture with permethylation products, as we saw above, the
obtainment of pure L-hercynine is severely limited by the difficulties
encountered during its purification. Indeed, the most used purification technique
for amino acids, well known by those skilled in the art, i.e., recrystallization, is
not very effective or completely unusable in the presence of large quantities of
salts. Only ion exchange chromatography purification methods make it possible
to obtain L-hercynine with a good purity. In the context of the two-step
preparation described by Melville and already cited in this document (Reinhold,
V. et al.; J. Med. Chem.; 1968; 11; 258-260), to obtain a kilogram of pure Lhercynine,
it would be necessary to use 13.2 kg of ion exchange resins. On an
industrial scale, this represents considerable quantities of resins that must be
eliminated in accordance with environmental regulations. This highlights the
importance of being able to have a purification method that avoids using very
large volumes of resins.
In the Journal of Medicinal Chemistry vol. 11, no. 2, March 1, 1968, pages
258-260, REINHOLD et al. also describe a method for producing Hercynine from
alpha-N,N-dimethyl-L-histidine.Hcl.H2O in the methanol adjusted to 9, through
the addition of NH4OH, and reaction with Mel at room temperature overnight.
The solvent is next evaporated under reduced pressure to yield a solid
white residue that is subsequently treated on an ion exchange column while
being re-dissolved in a minimal amount of water.
Furthermore, in Green Chemistry, 2012, 14, pages 2256-
2265,ERDELMEIER et al. describe a desalination method for the reaction medium
containing the ergothioneine by electrodialysis. ERDELMEIER stresses that
7
electrodialysis has advantages relative to the ion exchange column and leads to a
high purity of the ergothioneine.
However, it was not obvious to replace the ion exchange column
purification method of REINHOLD with the electrodialysis method used by
ERDELMEIER, even though ergothioneine and hercynine have neighboring
structures.
Indeed, first of all, the ERDELMEIER document mentions the use of
electrodialysis, but stresses that this is done after the cysteine precipitation
("cysteine scavenging") step (see page 2259, right column, 2nd§).
Yet it is clearly specified previously on the same page 2259, left column,
that the cysteine precipitation occurs just after adjusting the pH to 4-5.
In the context of the first tests done by the inventors with electrodialysis,
at an initial pH of about 7, losses of 18-25% of the hercynine are observed; see
comparative examples 1 and 2 at the end of this description.
However, surprisingly, it was discovered in the context of the present
invention that if the pH is at least 8, in particular about 9, the hercynine losses
are significantly lower than 5%.
This unexpected drop in hercynine losses was not at all obvious for one
skilled in the art.
It should also be emphasized that in the ERDELMEIER document, nothing
is said about the pH at the beginning of the electrodialysis, and in particular
nothing is said about its relevance to the loss level.
It should also be noted that the ERDELMEIER document mentions a global
yield of 40% in ergothioneine over the two steps (see page 2259, right column
2nd§).
ERDELMEIER thus demonstrates that although the electrodialysisresults
in a very high purity, the product yield, i.e., ergothioneine, is obtained with a
8
global yield of 40%, demonstrating major losses of ergothioneineto one skilled in
the art, which are unacceptable on an industrial scale.
Under these conditions, it was not obvious for one skilled in the art to
find conditions for hercynine with losses < 5%, as claimed in the context of the
present invention.
Although its physiological role is still poorly defined, L-hercynine is a
natural molecule whose significance appears obvious. It is therefore essential to
be able to have a method for effectively producing pure L-hercynine that is easy
to industrialize, economically viable, does not cause racemization, and respects
the environment.
Knowing that recrystallization is not very effective, or iseven completely
unusable, in the presence of a large quantity of salts, several difficulties must be
resolved beyond the problem relative to the stereochemistry of the final
product, namely:
1. the complete separation of the L-hercynine, a very hydrophilic molecule, and
inorganic salts, sub-products of the methylation reaction (direct or indirect)
through an economically viable method that can be industrialized, excluding
ion exchange chromatography;
2. an implementation limiting operational losses of the desired product, i.e., <
5% loss of L-hercynine during the desalination step, and lastly
3. obtaining pure L-hercynine from a mixture containing structurally very close
derivatives such as L-N-mono-methyl-histidine, L-N,N-di-methyl-histidine, or
products from the permethylation as mentioned by Melville.
Aims of the invention:
One of the aims of the present invention is to propose a simple and
reproducible method for producing pure L-hercynine, or its enantiomer, Dhercynine,
or its racemic mixture, or one of its isotopically marked derivatives, in
9
particular including a purification step allowing easy separation of the Lhercynine
from the formed inorganic salts, before the elimination of the organic
sub-products.
These aims are achieved owing to the present invention, which is based
on a method for separating L-hercynine from the inorganic salts present in the
reaction medium, by electrodialysis, irrespective of the mode used for
methylation of the L-histidine or one of its methylated derivatives, followed by
purification by recrystallization. This is exemplified in the present invention.
Brief description of the invention:
The present invention therefore aims to resolve the new technical
problem consisting of providing pure L-hercynine, or its enantiomer,D-hercynine,
or its racemic mixture, or one of its isotopically marked derivatives, according to
a solution that encompasses a step for purification of the L-hercynine other than
ion exchange chromatography.
The Applicant has developed a new method for producing pure Lhercynine
that meets all of these criteria, for the first time simultaneously
resolving all of the stated technical problems, very easily and having many
advantages over the existing methods. In particular, the invention makes it
possible to manufacture large quantities allowing use of the method on an
industrial scale. An industrial scale refers to a scale ≥ 1 kg, in particular greater
than 5 kg, still better greater than 10 kg.
Thus, in its first aspect, the present invention relates to a method for
purifying L-hercynine, or its enantiomer,D-hercynine, or its racemic mixture, or
one of its isotopically marked derivatives from a reaction mixture resulting from
the reaction of L-histidine, or its enantiomer D-histidine or its racemic mixture, or
one of its isotopically marked derivatives or one of its α-N-methylated
derivatives, under controlled pH conditions, with a methylation agent MeX in a
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polar solvent or a mixture of polar solvents, at room temperature, characterized
in that it comprises at least one step for separating the organic products from
the inorganic salts formed during the reaction by electrodialysis; and in that the
electrodialysisis conducted on a reaction medium adjusted to a pH comprised
between 8.5 and 9.5, at the beginning of the electrodialysis.
According to one particular embodiment, the invention relates to a
purification method characterized in that it comprises an additional step for
recrystallization of the L-hercynine, or its enantiomer,D-hercynine, or its racemic
mixture, or one of its isotopically marked derivatives or one of its methylated
derivatives, to eliminate the organic impurities.
According to a second aspect, the invention relates to a method for the
industrial-scale production of L-hercynine, or its enantiomer,D-hercynine, or its
racemic mixture, or one of its isotopically marked derivatives, or one of its α-Nmethylated
derivatives, characterized in that it comprises the following steps:
 reacting the L-histidine, or its enantiomer D-histidine or its racemic
mixture, or one of its isotopically marked derivatives or one of its α-Nmethylated
derivatives, under controlled pH conditions, with a
methylation agent MeX in a polar solvent or a mixture of polar solvents,
at room temperature; followed by
 separating the organic products from the inorganic salts formed during
the reaction by electrodialysis and in that the electrodialysis is done on a
reaction medium adjusted to a pH comprised between 8.5 and 9.5, upon
starting the electrodialysis; followed by
 re-crystallizing the L-hercynine to eliminate the organic impurities.
The α-N-methylated derivatives of L-histidine refer to L-N-mono-methylhistidine
or L-N,N’dimethylhistidine or one of their salts.
Room temperature refers to a temperature comprised between 20°C and
35°C.
11
For each aspect of the invention, it is possible to carry out the following
particular embodiments:
According to one particular embodiment, the method according to the
invention is characterized in that the electrodialysisis done on a reaction medium
adjusted to about 9, at the beginning of the electrodialysis. The pH can be
adjusted traditionally by adding an inorganic base such as sodium hydroxide,
potassium or lithium hydroxide.
It has in fact surprisingly been discovered that hercynine losses during
electrodialysismay be reduced to < 5% if the pH of the aqueous medium is
adjusted to 8.5 to 9.5 at the beginning of the electrodialysis, whereas outside
this range, for example at about 7, hercynine losses of 18-25% are observed.
The electrodialysisaccording to the invention also makes it possible to
achieve complete desalination. Complete desalination refers to the separation of
the salts from the organic product in aqueous solution until a salt proportion is
obtained of less than 1% by weight, which generally corresponds to a final
conductivity of < 200 μS/cm.
It is equally surprising for one skilled in the art that the
electrodialysismethod is applicable to the complete separation of the
inorganic/mineral salts obtained during the purification or preparation step of
the L-hercynine, as betaine, or its enantiomer D-hercynine or its racemic mixture,
or of one of its isotopically marked derivatives, and without significant product
loss (< 5%). Although electrodialysisis known by those skilled in the art as a highperforming
and very cost-effective desalination method, significant losses by
transmembrane migration have nevertheless been observed for certain organic
compounds, such as amino acids (Eliseeva T.V. et al., Desalination; 2002; 149;
405-409, Sato et al., J. Membrane Sci.; 1995; 100; 209-216), and in particular for
betaines (EP 2,216,088; WO 9808803).
12
In general, these losses, caused by diffusion, facilitated electromigration
or osmotic pressure, are even greater if complete desalination (< 1% salts) is
sought, characterized by low final conductivity (< 200 μS/cm) and limit current
(Shaposhnik V.A. et al., J. Membrane Sci. 1999; 161; 223-228)values. For the
industrial desalination of betaines such as 4-butyobetaine by electrodialysis, it
has even been proposed to combine the electrodialysiswith pretreatment of the
effluents in order to recover the lost betaine (EP 2,216,088).
According to another specific embodiment, the method according to the
invention is characterized in that the methylating agent MeX is chosen from
among methyl halogenide, methyl sulfate, methyl carbonate or methyl or
trifluoromethyl or tosyl sulfonate.
According to still another specific embodiment, the method according to
the invention is characterized in that the polar solvent or the mixture of polar
solvents is chosen from among water; a C1-C6 alcohol, for example methanol,
ethanol, propanol or propanol-2, butanol; ethyl acetate. One particular polar
mixture is a water/alcohol, water/propanol or propanol-2, methanol/ethyl
acetate mixture.
According to another specific embodiment of the method according to
the invention, the controlled pH conditions are chosen to result directly in a trimethylation
of the L-histidine or its D-histidine enantiomer or its racemic
mixture, or one of its isotopically marked derivatives or one of its α-Nmethylated
derivatives, or indirectly from the di-methylated derivative.
According to another specific embodiment of the method according to
the invention, the controlled pH conditions are chosen in the interval pH = 9-13
by adding an inorganic base such as sodium hydroxide, potassium or lithium
hydroxide. It is important to note that a lower pH, for example pH = 8.5, must be
avoided subject to significantly lower selectivity of the reaction and a high yield
of per-methylated hercynine ≥ 20%.
13
According to another specific embodiment of the method according to
the invention, the mixture of polar solvents could be a methanol/water mixture.
According to another advantageous embodiment of the method
according to the invention, the electrodialysisis done by using different
membranes such as anion-selective membranes (for example, PC SA) and cationselective
membranes (for example, PC MV) in particular available from PC Cell
(Germany).
The subsequent reactions making it possible to purify and recrystallize
the L-hercynine to separate it from the organic impurities, from the
electrodialysisfiltrate, are done under traditional conditions, known by those
skilled in the art.
According to another alternative embodiment, the method according to
the invention is characterized in that it is possible to produce the L-hercynine-d9
from a reaction mixture resulting from the reaction of the L-histidine with a trideuterated
(d3) methylation agent, such as iodomethane-d3.
According to a third aspect, the invention provides a new pure Lhercynine-
d9 compound, with formula
, in particular as obtained by the method defined in the preceding description or
the following description including the examples.
It is known from the Foster document in "Advances in Drug Research,
Academic Press, London, GB, Vol. 14, pp. 1-40, listed under no. XP009086953,
ISSN 0065-2490" to deuterate pharmaceutical compounds. However, Foster only
14
shows that deuterated medicaments make it possible to identify the metabolites
during biodegradation of the modules.
In the context of the present invention, hercynine-D9 is specifically
prepared, and for the first time, in which the D atoms are only on the part
bonded to the nitrogen atom in the alpha position. The selective preparation in a
tri-methylation step with iodomethane d3 followed by electrodialysismakes it
possible to obtain the product easily, cost-effectively and with a better
performance, compared to the alternative two-step preparation. This two-step
preparation would be obvious for one skilled in the art by analogy with the
preparation of non-deuterated hercynine according to the REINHOLD et al.
protocol, in Journal of Medicinal Chemistry vol. 11, no. 2, March 1, 1968, pages
258-260. However, this two-step preparation is not economically viable because
it requires the use of three reagents containing deuterium: 1. reducing amination
with formaldehyde-D2 is necessary, using gaseous deuterium D2 followed by
quaternization with iodomethane-D3.Hercynine-D9 also has its own intrinsic
properties.
The method according to the present invention, according to each of its
aspects, has the advantage of not using dangerous reagents such as hydrogen, or
using large quantities of ion exchange resins, as in the method of the prior art
(Reinhold, V. et al.; J. Med. Chem.; 1968; 11; 258-260). It can also be done on a
large scale, in non-toxic solvents such as water or a water/alcohol mixture. Aside
from the feasibility aspect, these advantages considerably reduce not only the
implementation costs, but also the negative impact on the environment.
The technical problems set out above are resolved for the first time all at
once by the present invention, very easily and cost-effectively, the method for
producing said new compounds being very easy to implement while providing
good yields.
Description of the figures:
15
Figure 1: Chromatogram (HPLC) of the L-hercynine obtained in example 2 on an
industrial scale after desalination and recrystallization (ELSD Universal detection)
Figure 2: Chromatogram (HPLC) of the L-hercynine-d9 obtained in example 3
after desalination and recrystallization (ELSD Universal detection)
Examples
The examples below, as well as the figures, are provided simply as an
illustration and cannot in any way limit the scope of the invention, but are
instead an integral part of the invention, in their general means.
In the examples described below, all percentages are given by weight, the
temperature is room temperature or is given in degrees Celsius, and the pressure
is the atmospheric pressure, unless otherwise stated. Room temperature refers
to a temperature comprised between 20°C and 35°C.
The reagents used are commercially available from international
suppliers such as Sigma-Aldrich France (France), Alfa Aesar, Fisher Scientific, TCI
Europe, Bachem (Switzerland), except the following compounds, which have
been produced according to the cited protocol: N,N-Dimethyl-L-histidine
hydrochloride hydrate (Reinhold, V. et al.; J. Med. Chem.; 1968; 11; 258-260).
The desalination was done by electrodialysisusing cells made up of
alternating anion-selective (for example, PC SA) and cation-selective (for
example, PC MV) membranes in particular available from PC Cell (Germany),
using desalination units like the ED 8002-001 unit (micro-unit for scale up to 10
g), the B-ED 1-3/ED 200 unit (up to 200 g) and the P20 unit (industrial scale), also
available from PC Cell (Germany).
The NMR-1H analyses were recorded at 400 MHz in D2O or a D2O/DCI
mixture, using the HOD signal (4.79 ppm) as internal reference. The chemical
displacements are noted in ppm, and the multiplicity of the signals indicated by
the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), and m
16
(multiplet). The coupling constraints are noted in Hertz (Hz). The NMR-13C
analyses are recorded at 75 MHz in the D2O.The mass analyses are obtained by
atmospheric pressure chemical ionization (APCI-MS). The melting points were
measured with a device by the company Stuart Scientific. The HPLC analyses
were done on an Acquity (Waters) device, using a C8 250x4.6 (5 μm) column. The
mobile phase used is a water/acetonitrile mixture (98:2) in 12 min. and a flow
rate of 0.6 mL/min. The detection is done with an ELSD (Sedere) universal
detector.
I. Examples according to the invention of the method for producing
pure compounds
The following examples illustrate a new, simple and reproducible method
for producing pure L-hercynine, or its enantiomer, D-hercynine, or its racemic
mixture, or one of its isotopically marked derivatives that is based on a method
for separating inorganic salts, present in the reaction medium, by
electrodialysisof an aqueous solution adjusted to pH = 9, irrespective of the
methylation mode used for the L-histidine or one of its methylated derivatives,
followed by purification via recrystallization.
Example 1 of the invention: Isolation of the pure L-hercynine from a reaction
mixture obtained by tri-methylation of the L-histidine
275 g (1.3 mol) of the hydrochloride hydrate of the L-histidine is
solubilized in 2.7 L of a water/methanol mixture (2:1). The pH is adjusted to 9-13
with a 3M potassium hydroxide solution. Next, 645 g (4.55 mol) of iodomethane
2. ED and recrystallization
17
is added under agitation, and the mixture thus obtained is agitated for 18 hours
at 25-35°C while keeping the pH between 9-13. Next, the mixture is neutralized
by adding 37% aqueous HCl.
After evaporation of the methanol, the pH of the aqueous solution,
containing the raw product in mixture with the potassium chloride and iodide
salts, is adjusted to 9 with a solution of NaOH (30%), then the solution is
desalinated by electrodialysisuntil a conductivity of 50-100 μS is obtained. A test
for the presence of halogen with silver nitrate, done on a withdrawn sample,
confirms the absence of chloride and iodide ions.
The aqueous solution, obtained after electrodialysis, is dry evaporated.
The raw product is purified by recrystallization in aqueous isopropanol, and the
L-hercynine is obtained after filtration and drying (192 g; 73%) in the form of a
white powder.
*α+D = +44.5° (c = 1.0; 5N HCl)
pF: 238°C (dec.)
NMR-1H (D2O, 400 MHz): δ (ppm) = 3.24 (m+s, 11H), 3.90 (dd, J = 10 Hz, J = 5 Hz,
1H), 6.98 (s, 1H), 7.69 (s, 1H).
NMR-1H(D2O/DCI, 400 MHz): δ (ppm) = 3.31 (s, 9H), 3.44 (dd, J = 14 Hz, J = 12 Hz,
1H), 3.55 (dd, J = 14 Hz, J = 4 Hz, 1H), 4.13 (dd, J = 12 Hz, J = 4 Hz, 1H), 7.37 (s,
1H), 8.66 (s, 1H).
NMR-13C (D2O, 75 MHz): δ (ppm) = 25.7; 52.4; 78.9; 116.7; 132.3; 136.5; 171.2.
HPLC-MS (AP+): m/z = 198 (MH+)
Purity by HPLC (ELSD detection): 100%
Example 2 of the invention: Isolation of the pure L-hercynine from a reaction
medium obtained by tri-methylation of the L-histidine on the industrial scale
18
48.0 kg (229 mol) of the hydrochloride hydrate of the L-histidine is
solubilized in 475 liters of a water/methanol mixture (2:1). The pH is adjusted to
9-13 with a 10% sodium hydroxide solution. Next, 113.8 kg (801 mol) of
iodimethane is added under agitation, and the mixture thus obtained is agitated
for 18 hours at 25-35°C while keeping the pH between 9-13. Next, the mixture is
neutralized by adding 37% aqueous HCl.
After evaporation of the methanol, the pH of the aqueous solution,
containing the raw product in mixture with the sodium chloride and iodide salts,
is adjusted to 9 with a solution of NaOH (30%), then this solution is desalinated
by electrodialysisuntil a conductivity of 50-100 μS is obtained. A test for the
presence of halogen with silver nitrate, done on a withdrawn sample, confirms
the absence of chloride and iodide ions.
The desalinated aqueous solution, obtained after electrodialysis, is
concentrated. After recrystallization in aqueous isopropyl, the L-hercynine is
obtained after filtration and drying (32.2 kg; 72%) in the form of a white powder.
*α+D: + 44° (c = 1.0; 5N HCl)
pF: 242°C (dec.)
The NMR-1H and NMR-13C and mass spectrums are identical to those of
the L-hercynine obtained in example 1.
Purity by HPLC (ELSD detection): 99.2% (see Figure 1).
Example 3 of the invention: Isolation of the pure L-hercynine-d9 from the
reaction mixture obtained by tri-methylation of the L-histidine
2. ED and recrystallization
19
31.4 g (150 mmol) of the hydrochloride hydrate of the L-histidine is
solubilized in 300 mL of a water/methanol mixture (2:1). The pH is adjusted to 9-
13 with a 3M sodium hydroxide solution. Next, 86.9 g (600 mmol) of
iodomethane-d3 is gradually added under agitation, while keeping the pH
between 9-13. After 18 hours of agitation at 30°C, the mixture thus obtained is
neutralized by adding 37% aqueous hydrochloric acid.
After evaporation of the methanol, the pH of the aqueous solution,
containing the raw product in mixture with the sodium chloride and iodide salts,
is adjusted to 9 with a NaOH solution (30%), then the solution is desalinated by
electrodialysisuntil obtaining a conductivity of 50-100 μS. A test for the presence
of halogen with silver nitrate, done on a withdrawn sample, confirms the
absence of chloride and iodide ions.
The aqueous solution, obtained after electrodialysis, is dry evaporated.
The obtained raw product is purified by recrystallization in the aqueous
isopropanol, and the L-Hercynine-d9 is obtained after filtration and drying (23.8
g; 77%) in the form of a white powder.
NMR-1H (D2O/DCI, 400 MHz): δ (ppm) = 3.14 (m, 2H); 3.83 (dd, J = 10 Hz, J = 5 Hz,
1H);6.92 (s, 1H), 7.62 (s, 1H).
HPLC-MS (AP+): m/z = 207 (MH+).
Purity by HPLC (ELSD detection): 100% (see Figure 2).
2. ED and recrystallization
20
Example 4 of the invention: Isolation of the pure L-hercynine from a reaction
mixture obtained by quaternization of the N,N-dimethyl-histidine with
iodomethane
The N,N-dimethyl-histidine is quaternized as described by Reinhold, V. et
al.; J. Med. Chem.; 1968; 11; 258-260. In short, 48.5 g (200 mmol) of the
hydrochloride hydrate of the N,N-dimethyl-histidine is solubilized in 1 liter of
methanol. The pH is adjusted to 9 with a 20% ammonia aqueous solution. Next,
37.3 g (16 mL, 1.3 equivalents) of iodomethane is added under agitation. The
solution thus obtained is agitated for 18 hours at 30°C (room temperature). After
evaporation of the methanol, the raw product, in the reaction mixture containing
the ammonium chloride and iodide salts (88 g), is solubilized in 500 mL of water,
the pH is adjusted to 9 with a NaOH (30%) solution, then the solution is
desalinated by electrodialysisuntil a conductivity of 43 microsiemens (μS) is
obtained. A test with silver nitrate, making it possible to estimate the presence
of halogenide ions, done on a sample taken from the aqueous solution, confirms
the absence of chloride and iodide ions.
The aqueous solution, obtained after electrodialysis, is dry evaporated.
The raw product obtained is purified by recrystallization, and the L-hercynine is
obtained after filtration and drying (36.6 g; 93%) in the form of a white powder.
*α+D = + 44° (c = 1.0; 5N HCl)
pF: 241°C (dec.)
3. ED and recrystallization
21
NMR-1H (D2O, 400 MHz): δ (ppm) = 3.16 (m+s, 11H), 3.85 (dd, J = 10 Hz, J = 5 Hz,
1H), 6.93 (s, 1H), 7.64 (s, 1H).
NMR-13C (D2O, 75 MHz): δ (ppm) = 25.7; 52.4; 78.9; 116.7; 132.3; 136.5; 171.2.
HPLC-MS (AP+): m/z = 198 (MH+)
Example 5 of the invention: Isolation of the pure D-hercynine from a reaction
mixture obtained by quaternization of the D-N,N-dimethyl-histidine with
iodomethane
The D-N,N-dimethyl-histidine is quaternized by analogy with example 4
describing the synthesis and purification of the L-hercynine. In short, 21.3 g (90
mmol) of the hydrochloric hydrate of the D-N,N-dimethyl-histidine is solubilized
in 0.45 liters of methanol. The pH is adjusted to 9 with a 20% ammonia aqueous
solution. Next, 15.3 g (6.7 mL, 1.2 equivalents) ofiodomethane is added under
agitation. The solution thus obtained is agitated for 18 hours at 25-30°C. After
evaporation of the methanol, the raw product, in the reaction mixture containing
the ammonium chloride and iodide salts, is solubilized in 300 mL of water. After
adjustment of the pH of the solution to 9 with a NaOH (30%) solution, the
mixture is desalinated by electrodialysisuntil a conductivity of 70 μS is obtained.
A silver nitrate test, making it possible to estimate the presence of halogenide
ions, done on a sample taken from the aqueous solution, confirms the absence
of chloride and iodide ions.
3. ED and recrystallization
22
The aqueous solution, obtained after electrodialysis, is dry evaporated.
The raw product obtained is purified by recrystallization with a methanol/ethyl
acetate mixture, and the L-hercynine is obtained after filtration and drying (16.7
g; 93%) in the form of a white powder.
*α+D: -44° (c = 1.0; 5N HCl)
The NMR-1H and NMR-13C and mass spectrums are identical to those of the Lhercynine
obtained in example 1.
Example 6 of the invention: Isolation of the pure L-hercynine from a reaction
mixture obtained by quaternization of the N,N-dimethyl-histidine with the
methyl sulfate
The N,N-dimethyl-histidine is quaternized by analogy with the procedure
described by Reinhold, V. et al.; J. Med. Chem.; 1968; 11; 258-260, replacing the
iodomethane with methyl sulfate. In short, 4.85 g (20 mmol) of hydrochloride
hydrate of the N,N-dimethyl-histidine is solubilized in 100 mL of methanol. The
pH is adjusted to 9 with a 20% ammonia aqueous solution. Next, 3.03 g (2.28 mL,
24 mmol, 1.2 equivalents) of methyl sulfate is added under agitation. The
solution thus obtained is agitated for 18 hours at 30°C. The NMR-1H analysis of a
sample indicates that the conversion is not complete. 1.0 g (0.75 mL, 8 mmol, 0.4
equivalents) of methyl sulfate is added to the reaction mixture. After agitation
for 3 hours, the solvent is evaporated, and the raw product, in the reaction
3. ED and recrystallization
23
mixture containing the ammonium chloride and iodide salts, is solubilized in 50
mL of water. After adjusting the pH to 9, desalination by electrodialysisis done
until a conductivity of 50 μS is obtained, a silver nitrate test making it possible to
estimate the presence of halogenide ions, done on a sample taken from the
aqueous solution, and an analysis by HPLC-ELSD confirms the absence of salts.
The aqueous solution, obtained after electrodialysis, is dry evaporated.
After recrystallization of the raw product with a methanol/ethyl acetate mixture,
the L-hercynine is obtained after filtration and drying (3.0 g; 76%) in the form of
a white powder.
The NMR-1H spectrum and the rotatory power are identical to those
obtained for the isolated product in example 1.
II. Comparative examples of the electrodialysismethod
Comparative example 1: Evaluation of the separation of the inorganic salts
from the L-hercynine at pH 7 and pH 8
A) 2000 mL of an aqueous solution containing 113 g (0.57 mol) Lhercynine,
300 g (3.5 equiv.) of sodium iodide and 33 g (1 equiv.) of sodium
chloride (1.3 mol) at pH 7 and a conductivity of 70 mS/cm are placed in the
"diluent" compartment of an electrodialysisdevice, for example the B-ED 1-3/ED
200 unit, equipped with an ED 200 cell. 2000 mL of demineralized water is placed
in the "concentrate" compartment. 8000 mL of an aqueous solution of sodium
sulfate (0.5-1%) is used as electrolyte. The circulation flow rate of all of the
solutions is 100/120 L/h, by applying a voltage of 10-15 V, until the conductivity
of the "diluent" is < 0.1 mS/cm. A test for the presence of halogen with the silver
nitrate, done on a withdrawn sample, confirms the absence of chloride and
iodide ions. The "diluent" contains 86 g of L-hercynine, corresponding to a loss of
24%.
B) 2000 mL of an aqueous solution containing 113 g (0.57 mol) of Lhercynine,
300 g (3.5 equiv.) of sodium iodide and 33 g (1 equiv.) of sodium
24
chloride (1.3 mol) at pH 8 and a conductivity of 75 mS/cm is electrodialyzed (as
described in A) up to a conductivity of the "diluent" of 0.02 mS/cm. A test for the
presence of halogen with silver nitrate, done on a withdrawn sample, confirms
the absence of chloride and iodide ions. The diluent contains 98.3 g of Lhercynine,
corresponding to a loss of 13%.
Comparative example 2: Evaluation of the separation of the inorganic salts
from the L-hercynine at pH 7 (comparative) and pH 9 (according to the
invention)
A) The reaction mixture obtained by tri-methylation of the L-histidine, as
described in example 2 of the invention, after neutralization with the
hydrochloric acid HCl, containing 5% (w/w) of the hercynine mixed with reaction
sub-products, as well as the sodium chloride and iodide salts, is desalinated in
portion in 2000 mL by electrodialysisas described in comparative example 1A,
until a conductivity of 50-100 μS is obtained. The L-hercynine content is assayed
by HPLC. The procedure has been applied on 5 lots numbered 1 to 5, and the Lhercynine
losses (from 22-25%) are listed in table 1.
B) Under the same conditions, with the exception that the pH is adjusted
to 9, according to the invention, with 30% sodium hydroxide in the "diluent"
compartment upon starting the electrodialysis, 5 lots numbered 6 to 10 are
desalinated until obtaining a conductivity of 50-100 μS. The L-hercynine content
in the "diluent" is assayed by HPLC. The procedure was applied on 5 lots, and the
L-hercynine losses, going from only 1.5-2.5%, are listed in Table 1.
Table 1: L-hercynine losses
Example
2A: pH = 7
loss
Lot 1 Lot 2 Lot 3 Lot 4 Lot 5
24% 22% 25% 22% 23%
25
Example
2B: pH = 9
loss
Lot 6 Lot 7 Lot 8 Lot 9 Lot 10
2.5% 2.3% 1.9% 2% 1.5%
Comparative examples 1 and 2 therefore indeed show that the losses
during electrodialysiscan be limited very significantly by adjusting the pH of the
aqueous solution to 8.5-9.5, ideally to around 9, at the beginning of the
electrodialysis, according to the invention.

WE CLAIM:
1. A method for purifying L-hercynine, or its enantiomer, D-hercynine, or its
racemic mixture, or one of its isotopically marked derivatives from a reaction
mixture resulting from the reaction of L-histidine, or its enantiomer D-histidine
or its racemic mixture, or one of its isotopically marked derivatives or one of its
α-N-methylated derivatives, under controlled pH conditions, with a methylation
agent MeX in a polar solvent or a mixture of polar solvents, at room
temperature, characterized in that it comprises at least one step for separating
the organic products from the inorganic salts formed during the reaction by
electrodialysis; and in that the electrodialysis is conducted on a reaction medium
adjusted to a pH comprised between 8.5 and 9.5, at the beginning of the
electrodialysis.
2. The purification method according to claim 1, characterized in that it
comprises an additional step for recrystallization of the L-hercynine, or its
enantiomer, D-hercynine, or its racemic mixture, or one of its isotopically marked
derivatives or one of its methylated derivatives, to eliminate the organic
impurities.
3. A method for the industrial-scale production of L-hercynine, or its enantiomer,
D-hercynine, or its racemic mixture, or one of its isotopically marked derivatives,
or one of its α-N-methylated derivatives, characterized in that it comprises the
following steps:
 reacting the L-histidine, or its enantiomer D-histidine or its racemic
mixture, or one of its isotopically marked derivatives or one of its α-Nmethylated
derivatives, under controlled pH conditions, with a
27
methylation agent MeX in a polar solvent or a mixture of polar solvents,
at room temperature; followed by
 separating the organic products from the inorganic salts formed during
the reaction by electrodialysis; and in that the electrodialysis is done on a
reaction medium adjusted to a pH comprised between 8.5 and 9.5, upon
starting the electrodialysis; followed by
 re-crystallizing the L-hercynine to eliminate the organic impurities.
4. The method according to one of claims 1 to 3, characterized in that the
electrodialysis is done on a reaction medium adjusted to about 9, at the
beginning of the electrodialysis.
5. The method according to one of claims 1 to 4, characterized in that the
methylating agent MeX is chosen from among methyl halogenide, methyl sulfate,
methyl carbonate or methyl or trifluoromethyl or tosyl sulfonate.
6. The method according to one of claims 1 to 5, characterized in that the polar
solvent or the mixture of polar solvents is chosen from among water; a C1-C6
alcohol, for example methanol, ethanol, propanol or propanol-2, butanol; ethyl
acetate. One particular polar mixture is a water/alcohol, water/propanol or
propanol-2, methanol/ethyl acetate mixture.
7. The method according to one of claims 1 to 6, characterized in that the
controlled pH conditions are chosen to result directly in a tri-methylation of the
L-histidine or its D-histidine enantiomer or its racemic mixture, or one of its
isotopically marked derivatives or one of its α-N-methylated derivatives, or
indirectly from the di-methylated derivative.
28
8. The method according to one of claims 1 to 7, characterized in that the
controlled pH conditions are chosen in the interval pH = 9-13 by adding an
inorganic base such as sodium hydroxide, potassium or lithium hydroxide.
9. The method according to one of claims 1 to 8, characterized in that the LHercynine-
d9 is prepared from a reaction mixture resulting from the reaction of
the L-Histidine with a methylation-d3 agent, in particular iodomethane-d3.
10. L-Hercynine-d9, with formula
in particular as obtained using the method according to one of claims 1 to 9.