HK40063676A - Complex of gadolinium and a chelating ligand derived from a diastereoisomerically enriched pcta and preparation and purification process - Google Patents

Description

The present invention relates to a new process for the preparation and purification of a gadolinium complex and a chelating ligand derived from PCTA, which allows the preferential production of stereoisomers of this complex which exhibit physicochemical properties of particular interest for applications as a contrast agent in the field of medical imaging, in particular for magnetic resonance imaging. The process of the invention has also been defined in the claims. Although the following description may also for information purposes include material beyond the scope of the invention, this scope and therefore the scope of protection remain limited to the subject matter of the claims.

Err1:Expecting property name enclosed in double quotes: line 1 column 552 (char 551)

Other products, some of which are under development, represent a new generation of GBCAs. These are mainly macrocyclic chelate complexes, such as bicyclopolyazamacrocyclocarboxylic acid (EP 0 438 206) or PCTA derivatives (i.e. including a minimum chemical structure of 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-115),11,13-triene-3,6,9-triacetic acid), as described in EP 1 931 673.

The chelating ligand complexes derived from PCTA described in EP 1 931 673 have the advantage of being relatively easy to synthesize chemically and, moreover, of having a higher relaxability than other GBCAs (relaxability r1 up to 11-12 mM-1.s-1 in water) currently on the market, which corresponds to the efficacy of these products and therefore to their contrasting power.

In the body, the lanthanide chelates (or complexes) - and in particular gadolinium - are in chemical equilibrium (characterized by its Ktherm thermodynamic constant), which may lead to an unwanted release of the lanthanide (see equation 1 below):

Chemical complexation balance between the chelate or ligand (Ch) and the lanthanide (Ln) to lead to the Ch-Ln complex.

Since 2006, a condition called NSF (Nephrogenic Systemic Fibrosis) has been linked, at least in part, to the release of free gadolinium into the body, leading health authorities to warn against gadolinium contrast agents marketed to certain categories of patients.

Strategies have therefore been developed to completely safely address the complex problem of patient tolerance and to limit or even eliminate the risk of unwanted release of lanthanide after administration, a problem which is particularly difficult to address as contrast agents are often given repeatedly, either during diagnostic examinations or for dose adjustment and monitoring of the effectiveness of a therapeutic treatment.

In addition, since 2014, there have been reports of possible brain deposition of gadolinium following repeated administration of gadolinium products, in particular linear gadolinium chelates, with no or few reports of such deposition with macrocyclic gadolinium chelates, such as Dotarem®. As a result, several countries have decided to either withdraw most linear chelates from the market or to drastically restrict their indications of use, given their estimated inadequate stability.

A first strategy for limiting the risk of lanthanide release into the body is to opt for complexes with the highest possible thermodynamic and/or kinetic stability, since the more stable the complex, the more the quantity of lanthanide released over time will be limited.

Other ways of improving the tolerance of lanthanide chelates (in particular gadolinium) are described in previous art. For example, US 5.876.695, dating back more than thirty years, reports on formulations containing, in addition to lanthanide chelate, an additional complexing agent, intended to prevent an unwanted in vivo release of lanthanide by complexing the re-expanded lanthanide (metal ion Gd3+). The additional chelating agent can be added to the formulation either in free form or as a weak complex, typically in the form of calcium, sodium, zinc or magnesium. This may, however, be a complexing agent when it is not necessary to release the lanthanide in the complex form of a complexing agent.

Thus, in both strategies described above, it is important that the active complex is as stable as possible.

However, PCTA-derived chelating ligand complexes with a pyclen-like structure described in EP 1 931 673 while having good kinetic stability generally have a lower thermodynamic constant than those of other cyclen-derived macrocycle complexes.

This is particularly the case for the formula (II) complex as represented below: - What?

Err1:Expecting property name enclosed in double quotes: line 1 column 445 (char 444)

It should be noted, however, that the complex of formula (II) corresponds to several stereoisomers, in particular because of the presence of the three asymmetric carbon atoms located in the α position on the side chains of the complex, in relation to the nitrogen atoms of the macrocycle on which the said side chains are grafted.

Thus, the synthesis of the formula (II) complex as described in EP 1 931 673 results in a mixture of stereoisomers.

The aminopropanediol groups of the side chains of the formula (II) complex also contain an asymmetrical carbon. Thus, the formula (II) complex comprises a total of 6 asymmetrical carbons, and therefore exists as 64 configuration stereoisomers. However, in the following description, the only source of stereoisomerism considered for a given side chain will, for the sake of simplicity, be the asymmetrical carbon bearing the carboxylate group, marked with an asterisk (*) in the formula (II) shown above.

Since each of these three asymmetric carbons can be of absolute configuration R or S, the formula (II) complex exists as 8 families of stereoisomers, hereinafter referred to as II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. More specifically, according to the usual nomenclature in stereochemistry, the formula (II) complex exists as 8 families of diasteroisomers.

The use of the term family is justified by the fact that each of these families contains several stereoisomers, in particular because of the presence of an asymmetrical carbon in the aminopropanediol group, as mentioned above.

However, since the asymmetrical carbon-linked stereoisomerism of a given aminopropanediol group will not be considered in the following description, we shall speak of isomers, stereoisomers or diastereoisomers II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS, without specifying that each corresponds to a family of stereoisomers.

The inventors succeeded in separating and identifying by high performance liquid phase chromatography (HPLC) and ultra high performance liquid phase chromatography (UHPLC) 4 masses or groups of isomers of formula (II) obtained by the previous method, corresponding to 4 different elution peaks characterized by their retention time on the chromatogram, which will be named iso1, iso2, iso3 and iso4 in the following description.

They then discovered that these different isomer groups had distinct physicochemical properties, and determined that the isomer group called iso4, which includes a mixture of the II-RRR and II-SSS isomers of the formulas (II-RRR) and (II-SSS) shown below, proved to be the most interesting as a contrast agent for medical imaging. - What?

Thus, iso4 is surprisingly distinguished by a thermodynamic stability significantly higher than that of the mixture of diastereoisomers in the form of which the complex of formula (II) is obtained by implementing the process described in EP 1 931 673.

In addition, iso4 is the isomer group that has the best kinetic inertia (also called kinetic stability) among the four groups isolated by the inventors. Indeed, the inventors evaluated the kinetic inertia of the four isomer groups by studying their decomposition kinetics in aqueous acid solution (pH = 1.2), at 37°C. The half-life values (T1/2) that were determined for each of the isomer groups are shown in Table 1 below, the half-life corresponding to the duration after 50% of the amount of the complex initially present has been dissociated, according to the decomposition reaction (equation 2) below: : cinétique de décomplexation des groupes d'isomères iso1 à iso4

Iso1	18 heures
Iso2	6 heures
Iso3	8 jours
Iso4	27 jours

By comparison, gadobutrol or gadoterate, macrocyclic gadolinium complexes, have kinetic inertia of 18 hours and 4 days respectively under the same conditions, whereas linear gadolinium complexes such as gadodiamide or gadopentetate dissociate instantaneously.

In addition, iso4 is more chemically stable than iso3 in particular. The amide functions of the formula (II) complex are indeed susceptible to hydrolysis. The hydrolysis reaction of an amide function (equation 3) results in the formation of a dicoupled impurity, which is accompanied by the release of 3-amino-1,2-propanediol.

As regards the relaxivity of the different isomer groups, i.e. their effectiveness as contrast agents, the measurements made show a relatively equivalent contrasting power for the iso1, iso2 and iso4 groups and a lower efficiency for the iso3 (see Table 2). - What? : relaxivité des groupes d'isomères iso1 à iso4 à 37°C

Iso1	12.6	12.5
Iso2	13.3	12.9
Iso3	8.0	8.1
Iso4	12.9	13.0

The inventors have succeeded in developing a new process for preparing and purifying the formula (II) complex, which allows the diastereisomers II-RRR and II-SSS of the said complex to be obtained preferentially, which have particularly advantageous physicochemical properties.

The implementation of a process which makes it possible to obtain the majority of the diasteroisomers of interest is undoubtedly advantageous over the alternative of preparing the mixture of stereoisomers and then attempting to separate the diasteroisomers according to the usual techniques and thus isolate the isomers of interest using any separation technique well known in the art. In fact, in addition to the fact that it is easier to implement a process without a step of separation of diasteroisomers at industrial scale, the absence of separation on the one hand allows a considerable gain in professional time and, on the other hand, improves the overall health of the process by limiting the inhomogeneity of the diasteroisomers of interest in order to avoid the risks of cancer in the environment.

As indicated above, the method of preparation of the formula (II) complex developed by the inventors is based on an isomeric enrichment step of the gadolinium complex of formula (I) hexacid intermediate as follows: - What?

The complex of formula (I) corresponds to several stereoisomers, due to the presence of the three asymmetric carbon atoms located at the α position on the side chains of the complex, relative to the nitrogen atoms of the macrocycle on which the said side chains are grafted.

Since each of the 3 asymmetric carbons carrying a carboxylate function can be of absolute configuration R or S, the formula (I) complex exists as 8 stereoisomers, hereinafter referred to as I-RRR, I-SSS, I-RRS, I-SSR, I-SRR, I-RSR and I-SRS. More specifically, according to the usual nomenclature in stereochemistry, the formula (I) complex exists as 4 pairs of enomers, diastantioisomers between them.

The inventors succeeded in separating and identifying by high performance liquid phase chromatography (HPLC) and ultra high performance liquid phase chromatography (UHPLC) 4 masses or groups of isomers of the formula (I) complex obtained by the process described in EP 1 931 673, corresponding to 4 different elution peaks characterized by their retention time on the chromatogram, which will be named isoA, isoB, isoC and isoD in the following description.

X-ray diffraction analyses have allowed the inventors to determine the crystal structure of this group of isomers, and thus to discover that it includes the diastereoisomers I-RRR and I-SSS of the complex of formula (I), of the formulas (I-RRR) and (I-SSS) represented below. - What?

It should be noted that the I-RRR and I-SSS diastroeosiomers of the formula (I) complex are enantiomeric to each other.

The isomeric enrichment step of the invention process is intended to enrich the gadolinium complex of hexacid intermediate formula (I) into isoD.

The synthesis of the formula (II) complex involves, inter alia, the conversion of the carboxylic acid functions of the intermediate hexacid complex of formula (I) into an amide function.

Thus, when the amidification reaction is carried out on the previously obtained isoD enriched formula (I) hexacid complex, the iso4 enriched formula (II) complex is obtained.

Furthermore, the purification process developed by the inventors, when implemented as a result of the preparation process of the above-mentioned formula (II) complex, allows the formula (II) complex to be obtained with an optimized isomer profile, but also a significantly improved impurity profile.

This diasteroisomerically enriched and purified complex with improved stability can therefore be formulated with a free macrocyclic ligand, such as free DOTA, instead of a calcium DOTA complex, the use of which was recommended in WO 2014/174120.

The following is the list of the products covered by the scheme:

The purification process according to the invention is therefore applied to the formula (II) complex: - What? consisting of at least 80% of a diastero-isomeric excess comprising a mixture of II-RRR and II-SSS isomers of the formulae:

Diastereoisomeric excess means, for the purposes of this invention, and with respect to the complex of formula (II), that the complex is predominantly present as an isomer or group of isomers selected from the diastereoisomers II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. This excess is expressed as a percentage and is the percentage of the isomer or group of isomers that is the majority in relation to the total amount of the complex of formula (II).

In a particular embodiment of the purification process of the invention, the complex of formula (II) has at least 85%, including at least 90%, in particular at least 92%, preferably at least 94%, preferably at least 97%, more preferably at least 99% of the diastroeisomeric excess comprising the mixture of II-RRR and II-SSS isomers.

Preferably, such excess diasteroisomeric content shall be at least 70%, including at least 80%, preferably at least 90%, preferably at least 95% of the mixture of II-RRR and II-SSS isomers.

The advantage of this is that the diastereoisomeric excess consists of the mixture of II-RRR and II-SSS isomers.

The term mixture of II-RRR and II-SSS isomers also covers, by extension, the case where only one of the isomers, whether II-RRR or II-SSS, is present.

In a preferred embodiment of the purification process of the invention, the II-RRR and II-SSS isomers are present in the mixture in a ratio of 65/35 to 35/65, in particular 60/40 to 40/60, in particular 55/45 to 45/55.

In particular, the diastroeisomeric excess as defined above corresponds to peak 4 of the UHPLC trace (i.e. the fourth mass of isomers in the order of elution and corresponding to the iso4), characterised by a retention time of between 6,0 and 6,6 minutes, typically about 6,3 minutes, which is obtained by implementing the UHPLC method described below.

UHPLC trace means, for the purposes of this invention, the concentration profile measured by the detector after passing and separating a mixture of compounds (in the case of isomers of a compound) over a steady phase as a function of time for a given composition and eluent flow rate.

The following is the UHPLC method:

The test chemical is used to determine the concentration of the test chemical in the test medium.

It is a spherical UPLC column with a reverse phase particle composed of a preferably very hard silica core surrounded by a porous silica with a three-functional C18 (octadecyl) graft, and whose silanols have been treated with end-capped agents.

Preferably, the stationary phase used shall be compatible with the moving aqueous phases. - What? - conditions of analysis: - What?

Echantillon	Solution aqueuse du complexe de formule (II) à 2,0 mg/mL
Température colonne	40°C
Température échantillon	Température ambiante (20-25°C)
Débit	0,3 mL/min
Volume d'injection	1 µL
Détection UV	200 nm

- What? - What? - phase gradient of the moving phase (% v /v): - What?

Temps (min)	acétonitrile (100 %)
0	1	99
3	5	95
12	10	90

Composition including formula (II) complex

The diasteroisomerically enriched and purified complex of formula (II) according to the present invention can be formulated in a composition including: The compound formula (II) consists of at least 80% diasteroisomeric excess comprising a mixture of II-RRR and II-SSS isomers and a free macrocyclic ligand.

In this description, the terms macrocyclic ligand or macrocyclic chelate may be used interchangeably.

For the purposes of this invention, the term macrocycle means a cycle typically comprising at least 9 atoms, whether carbon atoms or heteroatoms, and the macrocyclic ligand or macrocyclic chelate is a polydentate ligand, at least bidentate.

free macrocyclic ligand means, for the purposes of this invention, the macrocyclic ligand in free, i.e. uncomplexed form, particularly to metals - including lanthanides and actinides - or alkaline earth cations such as calcium or magnesium. In particular, the free macrocyclic ligand is not in complex form with gadolinium, and is not introduced into the composition as a weak complex, typically calcium, sodium, zinc or magnesium, as described in US 5.876.695, the presence of these cations in the composition at trace, and therefore not excluding non-corresponding complexes.

As discussed above, the formulation of the formula (II) complex with a free macrocyclic ligand, and not a weak complex of said macrocyclic ligand as recommended in EP 1931 673, and made possible by the improved stability of the diasteroisomerically enriched formula (II) complex of the invention.

Such a composition typically has a free gadolinium concentration of less than 1 ppm (m/v), preferably less than 0,5 ppm (m/v).

In this description, unless otherwise stated, the terms Gd , gadolinium and Gd3+ are used interchangeably to refer to the Gd3+ ion. By extension, it can also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

In the present invention, free Gd refers to uncomplexed forms of gadolinium, preferably available for complexation. This is typically the water-soluble ion Gd3+. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

Free-form gadolinium is typically measured by colorimetric dosing, usually xylenol orange or Arsenazo (III). In the absence of a metal ion (such as gadolinium), these indicators have a specific color: at acid pH, xylenol orange has a yellow color, while Arsenazo has a pink color.

The visual determination of the colour change of the solution allows the presence or absence of gadolinium to be verified.

In addition, it is possible to quantitatively measure the free gadolinium in the solution via a feedback assay, for example using EDTA as chelate weak of Gadolinium. In such a assay, the colored indicator is added until a purple color is obtained. Then from EDTA, a ligand of gadolinium is added to the mixture by drip. Since EDTA is a stronger complexant than the colored indicator, the gadolinium will change from ligand and will leave the colored indicator to complex in preference to EDTA. The colored indicator will therefore gradually return to its uncomplex form.

Err1:Expecting ',' delimiter: line 1 column 195 (char 194)

These methods are well known to the professional, and are described in particular in Barge et al. (Contrast Media and Molecular Imaging 1, 2006, 184-188).

These colorimetric methods are thus usually applied to a solution with a pH between 4 and 8. Outside these pH ranges, the accuracy of the measurement may be affected by a change (or even a suppression) of the color curve.

Thus, if necessary, the pH of the sample to be dosed is adjusted to be between 4 and 8. In particular, if the pH of the sample is acidic, and in particular below 4, the pH is favourably adjusted by adding a base, and then the free Gd is measured on the sample at the adjusted pH.

The composition comprising the diastereoisomerically enriched and purified formula (II) complex according to the present invention thus has a long-term stability, i.e. its composition remains in accordance with the specifications in terms of free gadolinium concentration (in particular its concentration in free Gd remains below 1 ppm (m/v)) over a period of at least 3 years, preferably at least 4 years or more preferably at least 5 years, including in terms of free paramagnetic metal content.

Such a composition typically has a concentration between 0.01 and 1.5 mol.L-1, preferably between 0.2 and 0.7 mol.L-1, and preferably between 0.3 and 0.6 mol.L-1 in the formula (II) complex described above.

The formula (II) complex is dosed by methods known to the professional, in particular after mineralization and a dosing of the total gadolinium present in the composition, by optical emission spectrometry (also called ICP-AES or ICP Atomic Emission Spectrometry).

The formula (II) complex content allows this composition to have optimal contrasting power while still having a satisfactory viscosity. Indeed, below 0.01 mol.L-1 of formula (II) complex described above, the performance as a contrast product is less satisfactory, and at a concentration above 1.5 mol.L-1, the viscosity of this composition becomes too high for easy manipulation.

Such a composition typically comprises between 0,002 and 0,4% mol/mol, in particular between 0,01 and 0,3% mol/mol, preferably between 0,02 and 0,2% mol/mol, more preferably between 0,05 and 0,15% mol/mol of free macrocyclic ligand compared to the complex of formula (II).

The macrocyclic ligand is preferably selected from the group consisting of DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA and their derivatives.

The preferred method is DOTA (tetra-acetic acid 1,4,7,10-tetraazacyclododecane-1,4,7,10).

The concentration of free DOTA in the composition is typically measured by a copper back-dose, using for example copper sulphate as the source of the copper ion.

In this method, which is well known to the professional, a solution containing a known initial concentration of copper sulphate Q0 is preferably used, this concentration being higher than the amount of free ligand in the solution.

The solution is then potentiometrically re-doseed with EDTA, which will complex the free copper into solution without uncomplexing the DOTA-copper, as DOTA is a stronger complexant than EDTA. When the amount of EDTA added Q2 is equal to the amount of free copper in solution, the potential of the solution drops sharply.

Knowing the initial amount of copper Q0 and the amount of EDTA added Q2, subtracting these two values Q0 - Q2 gives the amount of free DOTA in the dosing solution Q1.

Alternatively, HPLC methods may be used, including the HILIC LC-UV method.

These measurement methods (in particular potentiometric methods) are used on solutions with a pH advantageously between 4 and 8. Thus, if necessary, the pH of the sample to be measured is adjusted to be between 4 and 8. In particular, if the pH of the sample is acidic, and in particular below 4, the pH is advantageously adjusted by adding a base such as meglumine, and then the free DOTA is measured on the sample at the adjusted pH.

Preferably, the proportions specified in this description and in particular above are proportions before sterilisation of the composition.

The pH of the composition is advantageously between 4.5 and 8.5, preferably between 5 and 8, and preferably between 6 and 8, especially between 6.5 and 8.

In particular, the composition comprising the diasteroisomerically enriched and purified formula (II) complex according to the present invention can be buffered, i.e. it can further include a buffer selected from the buffers established for use in the pH range 5 to 8 and preferably from the buffers lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, Tris (Tris (Tris)) hydroxymethyl) aminomethane), HEPES (2-[4-(Hydroxyethyl)-1-piperazine] sulfonic acid), MES (2-morpholine ethanol sulfonic acid) and their mixtures, and preferably a buffer selected from the buffers Tris, Tris, Tris and Tris, carbonate and the mixture of MES, depending on the composition.

Such a composition is preferably sterile.

Method of preparation of the formula (II) complex

The purification process according to the present invention is typically implemented on the formula (II) complex prepared by a process comprising the following successive steps: (a) Complexity of hexacid of formula (III) as follows: - What? with gadolinium to obtain the formula (I) hexacid gadolinium complex as defined above, (b) Heating isomerization of the formula (I) hexacid gadolinium complex in aqueous solution at pH between 2 and 4 to obtain a diasteroisomeric enriched complex consisting of at least 80% of a diasteroisomeric excess comprising a mixture of the isomers I-RRR and I-SSS of the said formula (I) hexacid gadolinium complex, etc.

In this description, unless otherwise stated, the terms Gd , gadolinium and Gd3+ are used interchangeably to refer to the Gd3+ ion. By extension, it can also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

In the present invention, free Gd refers to uncomplexed forms of gadolinium, preferably available for complexation. This is typically the water-soluble ion Gd3+. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide.

▪ Step a)

During this step a complexation reaction between the hexacid of formula (III) and gadolinium takes place, which results in the gadolinium complex of hexacid of formula (I) as defined above.

Depending on the particular embodiment, step (a) involves the reaction between the hexacid of formula (III) and a source of free Gd in water.

In a preferred embodiment, the source of free Gd is GdCl3 or Gd2O3, preferably Gd2O3.

Preferably, the reagents used in step (a), i.e. the source of gadolinium (typically gadolinium oxide), formula (III) hexa acid and water, are as pure as possible, particularly with respect to metallic impurities.

Thus, the source of gadolinium will preferably be gadolinium oxide, preferably with a purity of more than 99,99%, and even more preferably more than 99,999%.

The water used in the process preferably contains less than 50 ppm of calcium, preferably less than 20 ppm, and preferably less than 15 ppm of calcium.

It is advantageous that the quantities of the reactants (hexa (III) formula acid and gadolinium) used in this step (a) correspond to, or are close to, the stoichiometric ratios as dictated by the balance equation of the complexation reaction taking place in this step.

close to the stoichiometric ratios means that the difference between the molar ratios into which the reagents are introduced and the stoichiometric ratios is less than 15%, in particular less than 10%, preferably less than 8%.

The ratio of the amount of material introduced into gadolinium to the amount of material introduced into hexacid of formula (III) is then greater than 1, but typically less than 1.15, and less than 1.10 and advantageously less than 1.08. That is, the amount of gadolinium introduced is greater than 1 equivalent (equivalent), but typically less than 1.15 equivalent, and less than 1.10 equivalent, and advantageously less than 1.08 equivalent, compared to the amount of hexacid of formula (III) introduced, which corresponds to 1 equivalent. In the free production mode according to which the amount of gadolinium GdO2 is preferred to GdO3 of the equivalent, the amount introduced is therefore more than 0.55, but advantageously less than 0.55, in relation to the amount of hexacid of formula (III) equivalent.

Depending on the particular mode of implementation, step (a) comprises the following successive steps: a1) Preparation of an aqueous solution of hexacid of formula (III), eta2) Addition of a free source of gadolinium to the aqueous solution obtained in step a1).

In this embodiment, the hexacid content of formula (III) in the aqueous solution prepared in step a1) is typically between 10% and 60%, in particular between 15% and 45%, preferably between 20% and 35%, preferably between 25% and 35%, and even more preferably between 25% and 30% by weight in relation to the total weight of the aqueous solution.

Preferably, steps (a) and (b) are performed in a monotope (or one-pot) embodiment, i.e. in the same reactor and without an intermediate isolation or purification step.

Thus, in this preferred embodiment, the gadolinium complex of formula (I) hexaacid formed in step (a) is directly subjected to step (b) isomerization, without being isolated or purified, and in the same reactor as used for step (a).

▪ Stage b)

The formula (I) hexacid gadolinium complex formed by the complexation reaction between formula (III) hexacid and gadolinium in step (a) is initially obtained as a mixture of diastereoisomers.

Step (b) shall be to enrich the diasteroisomer mixture into the I-RRR and I-SSS isomers to obtain the diasteroisomer-enriched formula (I) hexacid gadolinium complex consisting of at least 85%, including at least 90%, including at least 95%, preferably at least 97%, preferably at least 98%, more preferably at least 99% of a diasteroisomer excess comprising the mixture of the I-RRR and I-SSS isomers.

Diasteroisomeric excess means, for the purposes of this invention, and with respect to the formula (I) hexacid gadolinium complex, that the said complex is predominantly present as an isomer or group of isomers selected from the diasteroisomers I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR and I-SRS. This excess diasteroisomeric is expressed as a percentage, and corresponds to the amount of the isomer or group of isomers representing the majority in relation to the total amount of the isomer of formula (I) hexacid gadolinium complex. This percentage may, of course, be the same as the molar mass of the isomers, whereas, by definition, it may be the molar mass of the isomers.

Preferably, such excess diasteroisomeric content shall be at least 70%, including at least 80%, preferably at least 90%, preferably at least 95% of the mixture of I-RRR and I-SSS isomers.

The advantage of this is that the diastereoisomeric excess consists of the mixture of the I-RRR and I-SSS isomers.

The inventors have found that factors such as the pH and temperature of the formula (I) hexaacid gadolinium complex solution obtained at the end of step (a) have an influence on the ratio in which the various isomers of the formula (I) complex are present in the diastereoisomer mixture.

The term mixture of I-RRR and I-SSS isomers also covers, by extension, the case where only one of the isomers, either I-RRR or I-SSS, is present.

However, in a preferred embodiment, the I-RRR and I-SSS isomers are present in the mixture in a ratio of 65/35 to 35/65, in particular 60/40 to 40/60, in particular 55/45 to 45/55.

The step (b) isomerization of the gadolinium complex of formula (I) hexaacid in an aqueous solution is typically carried out at a pH between 2 and 4, particularly between 2 and 3, preferably between 2.2 and 2.8.

The pH is preferably adjusted with an acid, preferably an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid or phosphoric acid, e.g. with hydrochloric acid.

It is quite surprising that under such pH conditions, enrichment of the mixture into particular isomers, in this case the isomers I-RRR and I-SSS, occurs, since it is known in the art that gadolinium chelates are characterized by low kinetic inertia in acidic medium. Indeed, the higher the concentration of H+ ions in the medium, the greater the probability that a proton will be transferred to one of the donor atoms of the sand, thus leading to the dissociation of the complex.

It should be noted that the pH range recommended by EP 1 931 673 for the complexation of formula (III) hexaacid, i.e. 5.0 - 6.5, does not allow the formula (I) complex enriched with its isomers I-RRR and I-SSS to be obtained.

Step (b) is typically carried out at a temperature between 80°C and 130°C, in particular between 90°C and 125°C, preferably between 98°C and 122°C, preferably between 100°C and 120°C, typically for a duration of between 10 and 72 hours, in particular between 10 and 60 hours, preferably between 12 and 48 hours.

Contrary to expectations, such temperature conditions, which, when combined with the above pH conditions, should promote the instability of gadolinium chelate, do not result in its decomposition or formation of any other impurity, but in its isomerization into I-RRR and I-SSS.

In a particular embodiment, the aqueous solution of step (b) contains acetic acid, and step (b) is then advantageously carried out at a temperature of 100 to 120 °C, in particular between 110 and 118 °C, typically for a period of 12 to 48 hours, in particular between 8 and 30 hours, in particular between 24 and 26 hours.

Acetic acid is preferably added before heating the formula (I) gadolinium complex solution of hexacid obtained in step (a) in such quantity that the acetic acid content is between 25% and 75%, including between 40% and 50% by mass of the formula (III) hexacid used in step (a).

When the aqueous solution is heated to a temperature advantageously between 100°C and 120°C, typically between 110°C and 118°C, acetic acid is added as the water evaporates to maintain a constant volume of solution.

In a preferred embodiment, at the end of step (b), the diasteroisomerically enriched complex is isolated by crystallization, preferably by crystallization by seeding.

In this mode of implementation, step (b) comprises the following successive steps: (b1) Isomerization by heating of the hexacid formula (I) gadolinium complex in aqueous solution at pH between 2 and 4 to obtain a diasteroisomerically enriched complex consisting of at least 80% of the excess diasteroisomeric including the mixture of the isomers I-RRR and I-SSS of the hexacid formula (I) gadolinium complex, and (b2) Isolation by crystallization of the diasteroisomerically enriched complex, preferably by crystallization by seeding.

The purpose of step (b2) of crystallization is, first, to remove impurities which may be present in the aqueous solution, which may result from previous steps, in order to obtain a more pure, discolored product in the form of crystals, and, secondly, to continue the diasteroisomeric enrichment of the formula (I) hexacid gadolinium complex in order to obtain an excess diasteroisomeric composed of the mixture of the isomers I-RRR and I-SSS of this complex in excess of that obtained at the end of step (b1).

The fact that the I-RRR and I-SSS isomers, in which the complex tends to enrich during step (b) (and this is quite unexpected given the conditions in which it is carried out), are the only isomers of the complex to crystallize in water is a completely unexpected result.

It should also be noted that the water crystallization of the isomers of interest of the gadolinium complex of hexacid of formula (I) avoids the addition of solvent as described in Example 7 of EP 1931 673, which involves a precipitation step in the ethanol of the trisodium salt of the complex.

Step (b2) is best performed at a temperature between 10°C and 70°C, in particular between 30°C and 65°C, in particular between 35°C and 60°C.

Crystallisation by seeding , also called crystallisation by initiation , involves the introduction into the reactor in which crystallisation is carried out (also called crystallisoire) of a known quantity of crystals, called seed or amorce . This allows to reduce the crystallization time.

The diastereoisomerically enriched hexa acid (I) formula gadolinium complex crystals are then typically isolated by filtration and drying, using any technique well known to the art.

The purity of the diasteroisomerically enriched formula (I) hexaacid gadolinium complex isolated at step (b2) is more than 95%, including more than 98%, and more than 99% as expressed as a percentage by mass of the formula (I) complex by the total mass obtained at step (b2).

In a particular embodiment, the diasteroisomerically enriched complex of step (b) isolated by crystallization is again purified by recrystallization, to obtain a diasteroisomerically enriched and purified complex.

In this embodiment, step (b) includes, in addition to the successive steps (b1) and (b2) described above, a step (b3) of purification by recrystallization of the isolated diasteroisomerically enriched formula (I) hexaacid gadolinium complex.

The purpose of step b3) of recrystallization, as with step b2) of crystallization, is, on the one hand, to obtain a higher purity product and, on the other hand, to continue the diasteroisomeric enrichment of the gadolinium complex of formula (I) hexaacid in order to obtain a diasteroisomeric excess comprising the mixture of the isomers I-RRR and I-SSS of this complex greater than that obtained at the end of step b2).

Step b3) typically comprises the following successive sub-steps: in suspension of the diasteroisomerically enriched hexacid formula (I) gadolinium complex isolated in step (b2) in aqueous solution, preferably in water,solubilization of this complex by heating to a temperature preferably between 80°C and 120°C, for example at 100°C,recrystallization, preferably by seeding, at a temperature preferably between 10°C and 90°C, in particular between 20°C and 87°C, in particular between 55°C and 85°C, typically for a period of 2 to 20 hours, in particular between 6 and 18 hours, and isolation of diasteroisomerically purified crystals of diasteroisomeric formula (I) gadolinium complex between 2 and 20 hours, for example by filtration and sealing.

The degree of purity of the diasteroisomerically purified formula (I) hexa-enriched gadolinium complex isolated at step (b3) is typically greater than 98%, including greater than 99%, and preferably greater than 99,5%, expressed as a percentage by mass of the formula (I) complex by the total mass obtained at step (b2).

In another embodiment, the diasteroisomerically enriched complex of step (b) is further enriched by selective decomposition of the diasteroisomers of the formula (I) complex other than the diasteroisomers I-RRR and I-SSS, i.e. by selective decomposition of the diasteroisomers I-RSS, I-SRR, I-RSR, I-SRS, I-RRS and I-SSR.

In this embodiment, step (b) includes, in addition to the successive steps (b1) and (b2) described above, a step (b4) of selective decomposition of diastereoisomers of the formula (I) complex other than diastereoisomers I-RRR and I-SSS. In this variant, step (b) may also include step (b3) described above, with said step (b3) being implemented between steps (b2) and (b4), or after step (b4).

The purpose of step b4) of selective decomposition is to continue the diasteroisomeric enrichment of the gadolinium complex of formula (I) hexaacid to obtain a diasteroisomeric excess comprising the mixture of the isomers I-RRR and I-SSS of that complex greater than that obtained at the end of step b2) or at the end of step b3), when this is implemented prior to step b4).

Step (b4) typically comprises the following successive sub-steps: in suspension of the diasteroidal hexa-enriched formula (I) gadolinium complex isolated at step (b2) or step (b3) in water,addition of a base, e.g. soda,heating to a temperature of preferably 30°C to 60°C, in particular 35°C to 55°C, e.g. 40°C, typically for a period of 2 to 20 hours, in particular 10 to 18 hours,cooling to a temperature of preferably 10 to 30°C, e.g. 30°C, and isolation of the diasteroidal formula (I) gadolinium complex, e.g. filtration and purification.

The step b4) is made possible by the fact that the I-RRR and I-SSS isomers are most stable in the base medium. Such base conditions favor the formation of gadolinium hydroxide, and consequently the decomposition of the less stable isomers. Thus, it should be noted that, surprisingly, the I-RRR and I-SSS isomers are more stable both in the acid medium, allowing the b1) isomerization step, and in the base medium, allowing the b4) selective decomposition step.

In a preferred embodiment, the diasteroisomerically enriched complex obtained at the end of step (b) in any of the variants described above shall be at least 85%, including at least 90%, including at least 95%, preferably at least 97%, preferably at least 98%, preferably at least 99% of the diasteroisomeric excess comprising the mixture of the I-RRR and I-SSS isomers.

Preferably, such excess diasteroisomeric content shall be at least 70%, including at least 80%, preferably at least 90%, preferably at least 95% of the mixture of I-RRR and I-SSS isomers.

The advantage of this is that the diastereoisomeric excess consists of the mixture of the I-RRR and I-SSS isomers.

The term mixture of I-RRR and I-SSS isomers also covers, by extension, the case where only one of the isomers, either I-RRR or I-SSS, is present.

In a preferred embodiment, the I-RRR and I-SSS isomers are present in the mixture in a ratio of 65/35 to 35/65, in particular 60/40 to 40/60, in particular 55/45 to 45/55.

▪ Stage c)

Step (c) is to form the formula (II) complex from its precursor, the diastereoisomerically enriched formula (I) hexacid gadolinium complex obtained in step (b).

During this step, the three carboxylic acid functions of the hexacid complex of formula (I) carried by the carbon atoms at the γ position on the side chains of the complex, relative to the nitrogen atoms of the macrocycle on which the said side chains are grafted, are converted into amide functions by amidification reaction with 3-amino-1,2-prediopanol, in racemic or enantiomerically pure form, preferably in racemic form.

This amidification reaction does not alter the absolute configuration of the three asymmetric carbon atoms in the α position on the side chains, relative to the nitrogen atoms of the macrocycle on which the said side chains are grafted. Therefore, step (c) gives the formula (II) complex with a diasteroisomeric excess comprising a mixture of II-RRR and II-SSS isomers identical to the diasteroisomeric excess comprising a mixture of I-RRR and I-SSS isomers with which the diasteroisomeric gadolinium complex of formula (I) diasteroisomers is obtained at the step (b) which is obtained at the rate of less than 80%.

In a preferred embodiment, the formula (II) complex obtained at the end of step (c) shall be at least 85%, including at least 90%, including at least 92%, preferably at least 94%, preferably at least 97%, more preferably at least 99% of the diastroeisomeric excess comprising the mixture of II-RRR and II-SSS isomers.

Preferably, such excess diasteroisomeric content shall be at least 70%, including at least 80%, preferably at least 90%, preferably at least 95% of the mixture of II-RRR and II-SSS isomers.

The advantage of this is that the diastereoisomeric excess consists of the mixture of II-RRR and II-SSS isomers.

The term mixture of II-RRR and II-SSS isomers also covers, by extension, the case where only one of the isomers, whether II-RRR or II-SSS, is present.

In a preferred embodiment, the II-RRR and II-SSS isomers are present in the mixture in a ratio of 65/35 to 35/65, in particular 60/40 to 40/60, in particular 55/45 to 45/55.

The amidification reaction may be carried out by all methods well known to the professional, in particular in the presence of an agent activating carboxylic acid functions and/or in acid catalysis.

It may be carried out in particular by the methods described in patent EP 1 931 673, in particular in paragraph [0027] of that patent.

In a particular embodiment, step (c) involves the activation of the carboxylic acid (-COOH) functions of the hexacid complex of formula (I) carried by the carbon atoms at the γ position on the side chains of the complex, relative to the nitrogen atoms of the macrocycle on which the said side chains are grafted, in the form of derived functions having a carbonyl group (C=O), such that the carbon atom of the carbonyl group is more electrophilic than the carbon atom of the carbonyl group of the carboxylic acid functions.The active forms that can lead to an amide bond are well known to the professional and can be obtained, for example, by all the methods known in peptide chemistry to create a peptide bond. Examples of such methods are given in the publication Synthesis of peptides and peptidomimetics vol.E22a, p425-588, Thiben-Weyl et al., Goodman Editor, Hou-Stuttgart-New York (2004), and, among these examples, the methods of activation of carboxylic acids via a nitrogenous (acetic acid) reaction, for example, a deactivating agent such as diphenylephtylphosphate (DPPA), which is commonly referred to as the acetylated reaction.the use of carbodiimides alone or in the presence of catalysts (e.g. N-hydroxysuccinimide and its derivatives), the use of a carbonyldiimidazole (1,1'-carbonyldiimidazole, CDI), the use of phosphonium salts such as benzotriazol-1-yloxy-trisodioxide (commonly referred to as BOP) or uroniums such as 2-(1H-benzotriazolyl)-1--1--1,1,3,3-tetramethylthuronium hexafluorophosphate (commonly referred to as HBTU).

Preferably step (c) includes activation of the above mentioned carboxylic acid (-COOH) functions as ester, acyl chloride or acid anhydride functions.

This method of achievement is preferred to peptide coupling by activation of the carboxylic acid function using a coupling agent such as EDCI/HOBT as described in EP 1931 673. Indeed, such coupling results in the formation of an equivalent of 1-ethyl-3-[3-dimethylamino) propyl]urea, which must be removed, in particular by silica chromatography or liquid/liquid extraction by adding a solvent.

ester function is used to mean a grouping -C (O) O. This may include a grouping -C (O) O-R1 in which R1 is an (C1-C6) alkyl group.

For the purposes of this invention, group (C1-C6) alkyl means a saturated, linear or branched hydrocarbon chain containing 1 to 6 carbon atoms, preferably 1 to 4. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, dry-butyl, tert-butyl, pentyl or hexyl groups.

acyl chloride function , also called acid chloride function , means, for the purposes of this invention, a -CO-Cl group.

acid anhydride function means, for the purposes of this invention, a -CO-O-CO group. This may include a -CO-O-CO-R2 group, in which R2 corresponds to a (C1-C6) alkyl group.

The reactions for conversion of a carboxylic acid function into an ester, acyl chloride or acid anhydride function are well known to the professional, who can carry them out by any usual method with which he is familiar.

The complex of formula (Il) is then obtained by aminolysis of activated carboxylic acid functions as esters, acyl chlorides or acid anhydrides, including acid esters or anhydrides, preferably esters, by reaction with 3-amino-1,2-propanediol, in racemic or enantiomerically pure form, preferably in racemic form.

Preferably, the activation steps of carboxylic acid functions and aminolysis are performed in a monotope mode, i.e. in the same reactor and without an intermediate step of isolation or purification of the intermediate containing the activated carboxylic acid functions as ester functions, acyl chlorides or acid anhydrides, especially acid esters or anhydrides, preferably esters.

Depending on the particular mode of implementation, step (c) comprises the following successive steps: (c1) formation of an activated formula complex (VII), - What? where Y is a chlorine atom, a -OR1 or -O-C(O) -R2 group, preferably Y is an -OR1 or -O-C(O) -R2 group, with R1 and R2 corresponding independently to a group (C1-C6) alkyl, etc2) aminolysis of the activated complex of formula (VII) with 3-amino-1,2-propanediol.

As will be obvious to the professional, the reaction to form the activated formula complex (VII) does not alter the absolute configuration of the three asymmetrical carbon atoms in the α position on the side chains, in relation to the nitrogen atoms of the macrocycle on which the said side chains are grafted.

In the case where Y is a chlorine atom, step c1) is typically achieved by reaction between the diasteroisomerically enriched hexacid (I) gadolinium complex obtained in step b) and thionyl chloride (SOCl2).

In the case where Y represents an -O-C ((O) -CH3 group, step c1) is typically achieved by reaction between the diasteroisomerically enriched hexacid (I) formula gadolinium complex obtained in step b) and acetyl chloride.

In an advantageous embodiment, step (c) includes activation of the above mentioned carboxylic acid (-COOH) functions in the form of ester functions.

Depending on the method of implementation, step (c) may include in particular the following successive steps: (c1) formation of a triester of formula (VIII), where R1 is a (C1-C6) alkyl group, etc2) aminolysis of the triester of formula (VIII) with 3-amino-1,2-propanediol.

Step c1) is typically performed in the alcohol of formula R1OH, which acts as both a solvent and a reagent in the presence of an acid such as hydrochloric acid.

The step c2) is also typically performed in the alcohol of formula R1OH, in the presence of an acid such as hydrochloric acid.

The reaction medium is then cooled to a temperature below 10°C, preferably between 10 and 20 hours. The reaction is cooled to a temperature below 10°C, preferably between 0 and 5 degrees C. The reaction is cooled to a temperature below 10°C, preferably between 0 and 5 degrees C. The reaction is cooled to a temperature below 10°C, preferably between 0 and 5 degrees C.

Thus, steps c1) and c2) can be easily implemented in a monotope embodiment mode (or one-pot in English).

However, in order to promote the aminolysis reaction, during step c2), the alcohol of formula R1OH is preferably removed by vacuum distillation.

vacuum distillation means, for the purposes of this invention, the distillation of a mixture at a pressure between 10 and 500 mbar, particularly between 10 and 350 mbar, preferably between 10 and 150 mbar, particularly between 50 and 100 mbar.

Similarly, in order to promote the aminolysis reaction, in step c2), 3-amino-1,2-propanediol is introduced in large excess.Typically, the amount of 3-amino-1,2-propanediol introduced is more than 4 equals, including more than 7 equals, advantageously more than 10 equals, compared to the amount of diasteroisomerically enriched formula (I) hexaacid gadolinium complex material initially introduced in step c), which is 1 equivalent.

Surprisingly, despite the acid conditions typically employed during the c1) and c2) stages, which should increase the kinetic instability of the gadolinium complexes, no decomposition or isomerisation of the triester of formula (VIII) is observed.

It should also be noted that, in general, direct ester-amine amidification reactions are very poorly described in the literature (see K. C. Nadimpally et al., Tetrahedron Letters, 2011, 52, 2579-2582).

In a preferred mode of implementation, step (c) comprises the following successive steps: (c1) formation of a methyl triester of formula (IV), - What? In particular, by reaction in methanol in the presence of an acid such as hydrochloric acid, etc. (2) aminolysis of methyl triester of formula (IV) with 3-amino-1,2-propanediol, in particular in methanol in the presence of an acid such as hydrochloric acid.

Advantageously, the methyl triester of formula (IV) is not isolated between the c1 and c2 steps.

In a preferred embodiment, during step c2), methanol is removed by vacuum distillation to a temperature typically above 55°C, in particular between 60°C and 65°C, and the reaction medium is maintained at this vacuum temperature for a period typically above 5 hours, in particular between 10 and 20 hours, before being cooled to room temperature and diluted with water.

The purification process according to the present invention may be implemented on the formula (II) complex prepared by any combination of the particular, advantageous or preferred embodiments described above in connection with each step of the preparation process.

▪ Preparation of hexacid of formula (III)

Formula (III) hexacid, which is involved in step (a) of the preparation process of the formula (II) complex, can be prepared by all methods already known and in particular by the methods described in patent EP 1 931 673.

However, in a preferred method of manufacture, hexa acid of formula (III) is obtained by alkylation of pyclen of formula (V): - What? with a compound formula R3OOC-CHGp-(CH2)2-COOR4 (IX), in which: R3 and R4 independently represent a (C3-C6) alkyl group, in particular a (C4-C6) alkyl group such as a butyl, isobutyl, dry-butyl, tert-butyl, pentyl or hexyl group, andGp represents a leaving group such as a tosylate, triphlate group, or a halogen atom, preferably a bromine atom, to obtain the hexaester of formula (X) - What? followed by a hydrolysis step, leading to hexacid of formula (III).

In a preferred embodiment, R3 and R4 are identical.

In an advantageous manufacturing process, hexa acid of formula (III) is obtained by alkylation of pyclen of formula (V): - What? with dibutyl 2-bromoglutarate to obtain the butyl hexaester of formula (VI): - What? followed by a hydrolysis step, leading to hexacid of formula (III).

The dibutyl 2-bromoglutarate used is in racemic or enantiomerically pure form, preferably in racemic form.

The use of dibutyl 2-bromoglutarate is particularly advantageous, compared with that of ethyl 2-bromoglutarate described in EP 1931 673. Commercial diethyl 2-bromoglutarate is a relatively unstable compound, which degrades over time and under the influence of temperature. More specifically, this ester tends to hydrolyze or cyclically lose its bromine atom. Attempts to purify commercial diethyl 2-bromoglutarate or to develop new synthesis pathways to obtain it with improved purity, and thus prevent its degradation, have not been successful.

The alkylation reaction is typically carried out in a polar solvent, preferably in water, especially deionized water, preferably in the presence of a base such as potassium or sodium carbonate.

The use of water is preferred in particular to that of acetonitrile, described in EP 1 931 673, for obvious reasons.

The reaction is best conducted at a temperature of 40 to 80 °C, typically 50 to 70 °C, particularly 55 to 60 °C, for a duration of 5 to 20 hours, especially 8 to 15 hours.

The hydrolysis step is carried out advantageously in the presence of an acid or a base, advantageously of a base such as soda. The hydrolysis solvent can be water, an alcohol such as ethanol, or a water/alcohol mixture. This step is conducted advantageously at a temperature between 40°C and 80°C, typically between 40°C and 70°C, especially between 50°C and 60°C, typically for a duration between 10h and 30h, especially between 3h and 25h.

Process of purification of the formula (II) complex

The present invention therefore concerns a process for purification of the following formula (II): with a diastroeisomeric excess of at least 80% and containing a mixture of II-RRR and II-SSS isomers of formula: - What? including: 1) the combination of the following two steps: (b) ion exchange resin transfer, which involves the contact of an aqueous solution of the formula (II) complex with a strong anionic resin, and (c) ultrafiltration of the complex, and (2) isolation of the purified complex thus obtained in solid form.

It is preferable that the complex of formula (II) containing at least 80%, preferably at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 97%, preferably at least 98%, preferably at least 99%, of a diasteroisomeric excess comprising a mixture of II-RRR and II-SSS isomers has been previously obtained by the preparation process described above.

In a preferred embodiment, the diasteroisomeric enriched complex on which the purification process is implemented shall contain at least 85%, including at least 90%, including at least 92%, preferably at least 94%, preferably at least 97%, more preferably at least 99% of the diasteroisomeric excess comprising the mixture of II-RRR and II-SSS isomers.

Preferably, such excess diasteroisomeric content shall be at least 70%, including at least 80%, preferably at least 90%, preferably at least 95% of the mixture of II-RRR and II-SSS isomers.

The advantage of this is that the diastereoisomeric excess consists of the mixture of II-RRR and II-SSS isomers.

The term mixture of II-RRR and II-SSS isomers also covers, by extension, the case where only one of the isomers, whether II-RRR or II-SSS, is present.

In a preferred embodiment, the II-RRR and II-SSS isomers are present in the mixture in a ratio of 65/35 to 35/65, in particular 60/40 to 40/60, in particular 55/45 to 45/55.

▪ Combination of steps 1 (b) and 1 (c)

Steps 1 (b) and 1 (c) are intended to purify the formula (II) complex by removing any impurities that may be present from the production process.

Such impurities may include, in particular, 3-amino-1,2-propanediol and/or a dicoupled impurity.

As described above, the amidification reaction may involve the activation of the three carboxylic acid functions carried by the carbon atoms in the right position on the side chains of the formula (I) complex, compared to the macrocycle nitrogen atoms on which the amino acids are converted, followed by a 3-amino-acetyl-3-amino-1,2-amino-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-acetyl-ac

Di-coupled impurity means a complex of formulae (II-dc-a), (II-dc-b), (II-dc-c) as shown below or a mixture thereof: - What?

The dicoupled impurity may result from the hydrolysis reaction of an amide function of the formula (II) complex, or from incomplete activation of the carboxylic acid functions of the formula (I) complex (activation of two of the three functions) or incomplete aminolysis of the activated carboxylic acid functions (aminolysis of two of the three functions), when the process of preparation of the formula (II) complex involves such steps, as in the process of preparation of the formula (II) complex described above.

▪ Step 1 (b) is the transfer of ion complexes of formula (II) to diasteroisomerically enriched resin exchange resins as described above.

ion exchange resin means, for the purposes of the present invention, a solid material generally in the form of balls composed of a polymer matrix on which positively charged functional groups (anionic resin) or negatively charged functional groups (cationic resin) are grafted, which will enable anions or cations to be trapped by adsorption respectively.

Step 1 (b) involves the contacting of an aqueous solution of the formula (II) complex diastereoisomerically enriched with a strong anionic resin.

Err1:Expecting ',' delimiter: line 1 column 171 (char 170)

The passage over a strong anionic resin allows the decoupled impurities to be removed at least in part.

Step 1 (b) may also involve the contacting of an aqueous solution of the diasteroisomerically enriched formula (II) complex with a weak cationic resin.

This low cationic resin typically has as its functional groups the carboxylate (CO2-) groups, including the IMAC® HP336 resin marketed by Dow Chemical, which is preferred as H+.

The passing over of the weak cationic resin allows at least partial removal of the 3-amino-1,2-propanediol and any residues of Gd3+.

It should be noted that step 1b) of ion exchange is made possible by the improved stability of the diasteroisomerically enriched formula (II) complex, the integrity of which is therefore preserved during this step.

▪ Step 1 (c) is the ultrafiltration of the diasteroisomerically enriched formula (II) complex as described above.

Ultrafiltration means, for the purposes of this invention, a filtration method through a semi-permeable, mesoporous membrane, the pores of which are generally between 1 and 100 nm in diameter, particularly between 2 and 50 nm, particularly between 10 and 50 nm (mesopores), under the influence of forces such as pressure gradients, typically between 1 and 10 bar, and possibly concentration.

In the purification process of the invention, ultrafiltration is particularly advantageous for the removal of endotoxins.

The ultrafiltration membrane used in step 1 (c) has an advantageous cut-off threshold of less than 100 kD, in particular less than 50 kD, in particular less than 25 kD, typically a cut-off threshold of 10 kD.

Preferably, in step 1 (c), the transmembrane pressure is between 1 and 5 bars, especially between 2.25 and 3.25 bars.

▪ In a particular embodiment, steps 1 (b) and 1 (c) are further combined with a step 1 (a) nanofiltration.

Nanofiltration means, for the purposes of this invention, a filtration method through a semi-permeable, porous membrane, the pores of which are generally between 0.1 and 100 nm in diameter, in particular between 0.1 and 20 nm, in particular between 1 and 10 nm, under the influence of forces such as pressure gradients, typically between 1 and 50 bar, and possibly concentration.

Step 1a of nanofiltration allows the removal of most of the excess 3-amino-1,2-propanediol (possibly in the form of salt, particularly hydrochloride, or derivatives, particularly acetamide derivative) and mineral salts.

In this particular embodiment, the nanofiltration step can be performed directly on the raw diasteroisomerically enriched formula (II) complex as obtained by the previously described preparation process, without the need to precipitate the diasteroisomerically enriched formula (II) complex previously prepared by solvent addition.

The advantage is that the nanofiltration membrane used in step 1a) has a cut-off threshold below 1kD, notably below 500 Daltons, notably below 300 Daltons, typically a cut-off threshold of 200 Daltons.

Preferably, in step 1a), the transmembrane pressure is between 10 and 40 bar, especially between 2 and 30 bar.

In particular, the temperature of the solution of the formula (II) complex undergoing ultrafiltration in step 1a) is between 20 and 40 °C, including 25 and 35 °C.

In an alternative to this particular embodiment, step 1 (b) does not involve contacting an aqueous solution of the diastereoisomerically enriched formula (II) complex with a weak cationic resin.

In a particular manufacturing method, steps 1a) where one is present, 1b and 1c are performed in this order.

▪ Stage 2

Step 2 is to isolate the purified formula (II) complex obtained by combining steps 1 (b) and 1 (c) and optionally combined further in step 1 (a) into solid form.

This solid form isolation step can be carried out by any method well known to the professional, in particular by atomisation, precipitation, freeze-drying or centrifugation, preferably atomisation.

In a preferred embodiment, step (2) includes atomization.

The solid form of the formula (II) complex purified by atomization is thus possible without the use of precipitation solvents.

The temperature of the air entering the atomizer is then typically between 150°C and 180°C, in particular between 160°C and 175°C, preferably between 165°C and 170°C. The temperature of the air leaving the atomizer is typically between 90°C and 120°C, preferably between 105°C and 110°C.

The degree of purity of the formula (II) complex diastereoisomerically enriched in the purified and isolated mixture of II-RRR and II-SSS isomers at step 2 is preferably greater than 95%, including more than 97%, preferably greater than 97,5%, more preferably greater than 98%, preferably more than 99%, expressed as a percentage by mass of the formula (II) complex in relation to the total mass obtained at step 2.

Examples

The following examples are provided for illustrative purposes and not as a limitation of the invention.

Separation of iso1, iso2, iso3 and iso4 isomer groups from the formula (II) complex by UHPLC

A UHPLC apparatus consisting of a pumping system, an injector, a chromatographic column, a UV detector and a data station is used.

- The mobile phase:

Pathway A: 100% acetonitrile and Pathway B: aqueous solution of H2SO4 (96%) at 0,0005% v/v

- Preparation of test solutions:

Solution of the formula (II) complex at 2 mg/mL in purified water

- Conditions of analysis:

Température colonne	40°C
Température échantillon	Température ambiante (20-25°C)
Débit	0,3 ml/min
Volume d'injection	1 µl
Détection UV	200 nm
Durée d'analyse	20 min

- Gradient is:

Temps	% Acn
0	1	99
3	5	95
12	10	90
15	25	75
16	1	99
20	1	99

- What? The peak 4 of the UHPLC trace, i.e. iso4, corresponds to a retention time of 6.3 minutes.

Preparation of butyl hexaester of formula (VI)

In a reactor, 184 kg (570 moles) of dibutyl 2-bromoglutarate and 89 kg (644 moles) of potassium carbonate are mixed and heated to 55-60°C. An aqueous solution of 29.4 kg (143 moles) of pyclen in 24 kg of water is added to the previous preparation. The reaction mixture is maintained at 55-60°C and then reflux heated for about ten hours. After reaction, the medium is cooled, diluted with 155 kg of toluene and then washed with 300 litres of water. The butyl hexaester is extracted in the aqueous phase with 175 kg (1340 moles) of phosphoric acid (75%). It is then washed with approximately 150 times toluene. The hexaester is diluted in the aqueous phase with a pH of 165 kg (55%), followed by a dilution of toluene to a concentration of 85% (or less) of toluene.

Preparation of hexacid of formula (III)

In a reactor, 113 kg (121 moles) of butyl hexaester is loaded and 8 kg of ethanol is added. The medium is brought to 55+/-5°C and then 161 kg (1207.5 moles) of 30% (m/m) sodium is poured in 3 hours. The reaction mixture is maintained at this temperature for about 20 hours. The butanol is then removed by settling from the reaction medium.

Preparation of the gadolinium complex of hexacid of formula (I) ▪ The experimental protocol • Complexation and isomerization - No acetic acid

In a reactor 418 kg (117 kg of pure formula (III) hexacid / 196 moles) of an aqueous solution of formula (III) hexacid at 28% by weight are loaded. The pH of the solution is adjusted to 2.7 by addition of hydrochloric acid, then 37 kg (103.2 moles) of gadolinium oxide are added. The reaction medium is heated to 100-102 °C for 48H to achieve the expected isomeric distribution of formula (III) hexacid.

- With acetic acid

Gadolinium oxide (0.525 molar units) is suspended in a hexacid solution of formula (III) at 28.1% by weight.

The acetic acid 99-100% (50% by weight/pure formula (III) hexa acid) is poured into the medium at room temperature.

The medium is heated at reflux and then distilled to 113°C by mass by recharging the medium with acetic acid as the water is removed.

The medium is kept at 113°C for one night.

• Crystallisation and recrystallisation - Crystallisation

The gadolinium complex of formula (I) hexaacid in solution is cooled to 40°C, the primer is added, left in contact for at least 2 hours, then isolated by filtration at 40°C and washed with osmosis water.

- Recrystallization

180 kg of the previously obtained formula (I) hexacid gadolinium complex (dry extract at approximately 72%) is suspended in 390 kg of water. The medium is heated to 100°C to solubilize the product, then cooled to 80°C to be initiated by adding some amortizer. After cooling to room temperature the formula (I) hexacid gadolinium complex is isolated by filtration and drying.

• Selective decomplexing

The dry product is loaded into the reactor with osmosis water/ at 20°C. The added water mass is twice the theoretical formula (I) gadolinium hexacid complex mass. 30.5% (m/m) (6.5 eq) of soda is poured over the medium at 20°C. The medium is left in contact at 50°C at the end of the addition of NaOH for 16h. The medium is cooled to 25°C and the product filtered on a Clarcel bed.

▪ Content of diasteroisomers I-RRR and I-SSS in the mixture

The ratio of the various isomers of the formula (I) complex to the diastereoisomer mixture depends on the conditions under which the complexation and isomerisation steps are performed, as shown in Table 3 below. - What? 3 : teneur en le mélange I-RRR et I-SSS en fonction des confitions de complexation / isomérisation

5,7	80°C	40 %	3h	19 %
3,5	90°C	50 %	10h	49 %
3,0	101°C	40 %	10h	68 %
2,7	101°C	28 %	48h	98,04 %

The additional steps of recrystallization and selective decomplexation allow the diastroeisomeric excess in the I-RRR and I-SSS mixture to be increased (see Table 4). - What? : teneur en le mélange I-RRR et I-SSS après cristallisation / recristallisation / décomplexation sélective

98,04 %

99,12 %

99,75 %

Preparation of the formula (II) complex

In a reactor, 90 kg (119 moles) of formula (I) hexacid complex and 650 kg of methanol are loaded. The mixture is cooled to about 0 °C and then 111 kg (252 moles) of a solution of methanol hydrochloric acid (8.25% HCI in methanol) is poured by maintaining the temperature at 0 °C. The reaction medium is brought to room temperature and then kept agitated for 16 hours. After cooling to 0-5 °C, 120 kg (1319 moles) of 3-amino-1,2-propanediol are added. The reaction is then heated by dilution of the methanol formula in a vacuum to a temperature of 60-65 °C. The complex is cooled for 16 hours.

Purification of the formula (II) complex • Nanofiltration

The nanofiltration membrane used has a cutting threshold of 200 Daltons (Koch Membran System SR3D). - What? The nanofilter is then filled with the solution. The pump is first started at low flow to purge the system, then the flow rate of the nanofilter pump is gradually increased to the desired recirculation rate (1.0 m3/h for a 2.5 X 40 inch membrane). The system is then fully recirculated at 30 °C for at least 2 hours to establish a polarization layer. The medium is then diafiltrated at 30 °C at 25 bar by maintaining the constant volume by adding pure water until a conductivity of the diaphragm less than 1000 μS is achieved. At the end of retrotraction, the medium is concentrated until a concentration of about 40 % (m/m) is obtained.

• Resin treatment

The solution of formula (II) complex from nanofiltration is diluted with purified water by stirring to obtain a 15% solution (m/m). This solution is elevated in series on 50 litres of strong anionic resins (FPA900) as OH- and then on 50 litres of weak cationic resins (HP336) as H+ at an average elevation rate of 2 V/V/H (2 volumes of solution per volume of resin per hour). The resins are then rinsed with approximately 450 litres of purified water until a refractive index of less than 1.3335 is reached.

The solution of the formula (II) complex is then concentrated by heating to 50-60°C under a vacuum of 20 mbar to a concentration of 35% (m/m).

• Ultrafiltration

The ultrafiltration membrane is a UF 10KD Koch Spiral membrane.

The ultrafilter is fed by the previous 35 per cent solution of formula (II) complex heated to 40 °C. Ultrafiltration is applied at a flow rate of 3 m3/H at a transmembrane pressure of 2.5-3 bar. Several rinse-offs of the system with 13 litres of purified apyrogenic water are carried out until a final dilution of formula (II) complex of 25 per cent (m/m) is reached.

• Atomisation

The formula (II) complex is obtained as a powder by atomisation of the previous solution of formula (II) complex concentrated at 25%.

The atomization shall be carried out as follows: - What? The pure apyrogenic water atomizer is balanced by setting the inlet temperature to 165°C - 170°C and adjusting the feed rate so that the outlet temperature is between 105°C and 110°C.

Then the solution of the formula (II) complex is added and the flow rate is adjusted to maintain the above parameters.

These operating conditions shall be maintained throughout the atomization process, while ensuring that the powder behaves properly in the atomization chamber and out of the atomizer, in particular ensuring that the product does not stick.

At the end of the solution supply to the atomizer, rinse the container of this formula (II) complex and the atomizer with pure apyrogenic water until the maximum recovery of the powder.

This results in a formula (II) complex that is 99.6% pure.

This degree of purity was determined by reverse phase liquid chromatography.

Composition according to the invention and results of studies on it • Example of manufacturing process according to the invention

The manufacturing process of a composition according to the invention is carried out by following the following steps: (a) 485.1 g (or 0.5 M) of formula (II) complex is dissolved in water (qs 1 litre) by heating the tank to a temperature of 39 to 48°C and by making a strong agitation of the solution until the solution is completely dissolved in water. The solution is then cooled to about 30°C. (b) 0.404 g (or 0.2 % mole/mol relative to the proportion of complex added in step (a)) of DOTA (Simafex, France) is added by stirring to the solution obtained in step (a) via a DOTA solution at 10 m/v. (c) Dulamol (Tris) is added to the solution obtained in step (b) by stirring. The solution is then adjusted between pH 7.19 and 7.7 g/d and a target concentration of 1.19 g/dl (0.7 g/dl) is added by stirring to a concentration of 1.19 g/dl.

The liquid composition is then filtered on a polyethersulfone membrane and placed in its final container, which is finally sterilised at 121°C for 15 minutes.

• Example of composition according to the invention

The process described above gives the following formula:

Complexe de formule (II)	485,1 g (0,5 M)
DOTA**	0,404 g (1 mM soit 0,2% mol/mol vs complexe)
NaOH ou HCI	Qs pH 7,2 à 7,7
Trométamol	1,211 g
Gadolinium libre*	< 1 ppm m/v
Eau ppi (prête pour injection)	Qs 1 L

* Mesure effectuée par méthode colorimétrique au xylénol orange **exprimé sur une base anhydre et pure

• Tests of formulation carried out

Different concentrations of tromethamol from 0 to 100 mM were tested and the results of these tests showed that a content of 10 mM (0.12% w/v) was sufficient to ensure the pH stability of the formulation while limiting the formation of degradation impurities.

Different concentrations of DOTA from 0 to 2.5 mM were tested and the results of these tests showed that a concentration of 1 mM, which corresponds to 0.04% m/v or 0.2% mol/mol, ensures that no free Gd is released during the process and during the product life.

• Accelerated stability studies of a composition of the invention

The formula in the previous example is analysed immediately after manufacture (To) and after storage at 40°C for 6 months after manufacture (T+6 months).

A T0: Purity as assessed by chromatography*: 99.6% Gd-DOTA concentration: 0.007% (m/V) Gd concentration: below 0.0001% (m/V) pH: 7.5%

A T+6 months: Purity as assessed by chromatography*: 97.2% Concentration in Gd-DOTA: 0.014% (m/V) - 0.25 mMConcentration in Gd: below 0.0001% (m/V) pH: 7.5 * reverse phase liquid chromatography

These results demonstrate that this formulation has good stability over time.

• Comparative stability studies

The stability of the following compositions has been evaluated over time. Unoptimized PA refers to the active substance, namely the formula (II) complex, obtained by the process described in EP 1931673 Optimized PA refers to the diasteroisomerically enriched and purified formula (II) complex obtained by the process of the invention. - What?

PA (0,5 M)	[DOTA] % mol/mol	Trométamol mM
C1	Non optimisé	0,3	-	5,0
C2	Optimisé	0,2	-	7,5
C3	Optimisé	0,1	-	7,5
C4	Optimisé	0,2	10	7,5
C5	Optimisé	0,1	10	7,5
C6	Optimisé	0,2	-	5,0
C7	Optimisé	0,1	-	5,0

	Gd libre en ppm m/v (xylénol)		DOTA-Gd en % mol/mol (LC formiate*)
	T 0	T 6 mois 40°C	T 0	T 6 mois 40°C
C1	< LOD	0,18	0,27	0,3
C2	< LOD	< LOD	0,02	0,05
C3	< LOD	< LOD	0,02	0,05
C4	< LOD	< LOD	0,02	0,05
C5	< LOD	< LOD	0,02	0,08
C6	< LOD	< LOD	0,03	0,03
C7	< LOD	< LOD	0,02	0,07

* LC formiate : méthode chromatographique mettant en jeu une détection fluorimétrique. La séparation s'effectue sur une colonne chromatographique greffée C18 en phase inverse avec une élution en mode gradient.

The results reported above indicate that the formulation of unoptimized PA with free DOTA is not possible, as the chelation excipient is completely consumed by the trans-binding reaction between the formula (II) complex and DOTA, and can therefore no longer fulfill its role as a trapping agent for the released Gd3+.

In contrast, the diasteroisomerically enriched and purified formula (II) complex obtained by the process of the invention can be formulated with free DOTA. The absence of free Gd in the composition is observed at 6 months, 40°C, regardless of the pH of the formulation and whether buffering species are present or not.

Claims (13)

1. Process for purifying the complex of formula (II) below: constituted of at least 80% of a diastereoisomeric excess comprising a mixture of isomers II-RRR and II-SSS of formulae: comprising:

   1) the combination of the following two steps:

   1b) passage through ion-exchange resin(s), this step involving placing an aqueous solution of the complex of formula (II) in contact with a strong anionic resin, and

   1c) ultrafiltration of said complex, and

   2) the isolation of the purified complex thus obtained in solid form.
2. Process according to Claim 1, characterized in that the complex of formula (II) on which the purification process is performed has at least 90% of the diastereoisomeric excess comprising the mixture of isomers II-RRR and II-SSS.
3. Process according to Claim 1 or 2, characterized in that the complex of formula (II) on which the purification process is performed has at least 94% of the diastereoisomeric excess comprising the mixture of isomers II-RRR and II-SSS.
4. Process according to Claim 1, characterized in that step 1b) also involves placing an aqueous solution of the complex of formula (II) in contact with a weak cationic resin.
5. Process according to any one of Claims 1 to 4, characterized in that steps 1b) and 1c) are also combined with a nanofiltration step 1a).
6. Process according to any one of Claims 1 to 5, characterized in that the steps 1a), when it is present, 1b) and 1c) are performed in this order.
7. Process according to any one of Claims 1 to 6, characterized in that step 2) comprises atomization.
8. Process according to any one of Claims 1 to 7, characterized in that the complex of formula (II) constituted of at least 80% of a diastereoisomeric excess comprising a mixture of isomers II-RRR and II-SSS on which the purification process is performed was prepared previously via the following successive steps:

   a) complexation of the hexaacid of formula (III) below: with gadolinium to obtain the hexaacid gadolinium complex of formula (I) below:

   b) isomerization by heating the hexaacid gadolinium complex of formula (I) in an aqueous solution at a pH of between 2 and 4, to obtain a diastereoisomerically enriched complex constituted of at least 80% of a diastereoisomeric excess comprising a mixture of theisomers I-RRR and I-SSS of said hexaacid gadolinium complex of formula (I), of formulae: and

   c) formation, starting with the diastereoisomerically enriched complex obtained in step b), of the complex of formula (II), by reaction with 3-amino-1,2-propanediol.
9. Process according to Claim 8, characterized in that, on conclusion of step b), the diastereoisomerically enriched complex is isolated by crystallization and purified by recrystallization.
10. Process according to Claim 8 or 9, characterized in that step c) comprises the following successive steps:

    c1) formation of a triester of formula (VIII),in which R₁ represents a (C₁-C₆) alkyl group,

    notably by reaction in the alcohol of formula R₁OH in the presence of an acid such as hydrochloric acid, and

    c2) aminolysis of the triester of formula (VIII) with 3-amino-1,2-propanediol,

    notably in the alcohol of formula R₁OH in the presence of an acid such as hydrochloric acid,

    the triester of formula (VIII) not being isolated between steps c1) and c2).
11. Process according to any one of Claims 8 to 10, characterized in that step c) comprises the following successive steps:

    c1) formation of a methyl triester of formula (IV) notably by reaction in methanol in the presence of an acid such as hydrochloric acid, and

    c2) aminolysis of the methyl triester of formula (IV) with 3-amino-1,2-propanediol, in methanol in the presence of an acid such as hydrochloric acid, during which the methanol is removed by vacuum distillation, until a temperature greater than 55°C is reached, the reaction medium being maintained at this temperature under vacuumfor a time typically greater than 5 hours, before being cooled to room temperature and diluted with water.
12. Process according to any one of Claims 8 to 11, characterized in that the hexaacid of formula (III) as defined in Claim 8 is obtained by alkylation of the pyclene of formula (V): with dibutyl 2-bromoglutarate, to obtain the butyl hexaester of formula (VI) followed by a step of hydrolysis, leading to said hexaacid of formula (III).
13. Process according to any one of Claims 8 to 12, characterized in that it comprises a step 1a) of nanofiltration, and in that it is performed directly on the complex of formula (II) as obtained on conclusion of step c).