Complexes of gadolinium and chelating ligands based on diastereoisometrically enriched PCTA and methods of making and purifying same

Abstract

The present invention relates to a complex of formula (II) consisting of a diastereoisomer excess of at least 90%, said diastereoisomer excess comprising a mixture of the isomers II-RRR and II-SSS having the formula: # imgabs0. The present invention also relates to a method for preparing and purifying said complex of formula (II), and to a composition comprising said complex.

Description

[0001] Related Applications

[0002] The present invention is a divisional application of a patent application with an application date of January 17, 2020, an application number of 202080009625.8, and an invention name of "Complexes of gadolinium and chelating ligands based on diastereoisomer-enriched PCTA and methods for their preparation and purification". Technical Field

[0003] The present invention relates to a novel process for the preparation and purification of a complex of gadolinium and a chelating ligand based on diastereomerically enriched PCTA, which makes it possible to preferentially obtain stereoisomers of said complex, which have particularly advantageous physicochemical properties for use as contrast agents in the field of medical imaging, in particular in the field of magnetic resonance imaging. The invention also relates to the diastereomerically enriched complex itself, to a composition containing said complex, and also to a process for the preparation of the corresponding chelating ligand by decomplexing said complex, and to the ligand itself. Background Art

[0004] Many contrast agents based on chelates of lanthanides (paramagnetic metals), in particular gadolinium (Gd), are known, for example as described in US 4647447. These products are usually grouped together under the term GBCA (gadolinium-based contrast agents). Several products are on the market, among which are macrocyclic chelates, such as meglumine gadolinium salt based on DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid), gadobutrol based on DO3A-butrol, gadoteridol based on HPDO3A, and linear chelates, in particular based on DTPA (diethylenetriaminepentaacetic acid) or DTPA-BMA (gadolinium diamide ligand).

[0005] Other products, some of which are under development, represent a new generation of GBCAs. They are essentially complexes of macrocyclic chelates, such as bicyclic polyazamacrocyclic carboxylic acids (EP 0 438 206) or PCTA derivatives (i.e. derivatives containing at least the chemical structure of 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), as described in EP 1 931 673.

[0006] The PCTA-based chelating ligand complexes described in EP 1 931 673 have the advantages, inter alia, of being relatively easy to chemically synthesize and also having a relaxivity superior to that of other GBCAs currently on the market (relaxivity r1 in water up to 11-12 mM -1 .s -1), this relaxation rate corresponds to the efficiency of these products and therefore to their contrast power.

[0007] In living organisms, chelates (or complexes) of lanthanides (and especially gadolinium) are in a state of chemical equilibrium (measured by their thermodynamic constants, K therm Characterization), which may lead to the undesired release of the lanthanide (see reaction 1 below):

[0008]

[0009] Chelate or ligand (Ch) with lanthanide (L n ) produces the complex Ch-L n .

[0010] Since 2006, a disease called NSF (nephrogenic systemic fibrosis or fibrogenic dermopathy), which is at least partly linked to the release of free gadolinium in the body, has led health authorities to issue alerts against the marketing of gadolinium-based contrast agents to certain categories of patients.

[0011] Therefore, several strategies have been adopted to solve the complex problem of patient tolerance in a completely safe manner and to limit or even eliminate the risk of undesired release of lanthanides after administration. This problem is made more difficult by the fact that contrast agents are often repeatedly administered, both during diagnostic examinations and during dose adjustments and monitoring of therapeutic effects.

[0012] In addition, since 2014, it has been mentioned that gadolinium may be deposited in the brain after repeated administration of gadolinium-based products, especially linear gadolinium chelates, especially gadolinium macrocyclic chelates (e.g. ) have been reported little or not at all. Therefore, due to the lack of stability, countries have decided to withdraw most linear chelators from the market or to significantly restrict their indications for use.

[0013] Therefore, a first strategy to limit the risk of lanthanide release into the body is to choose complexes characterized by the highest possible thermodynamic and / or kinetic stability. The reason is that the more stable the complex, the more limited the release of lanthanides will be over time.

[0014] Other methods for improving the tolerability of chelates of lanthanides, in particular gadolinium, are described in the prior art. More than 30 years ago, US Pat. No. 5,876,695 reported, for example, formulations containing, in addition to lanthanide chelates, additional complexing agents, which were intended to release lanthanides (Gd) by complexation. 3+The additional chelator can be introduced into the formulation in its free form or in the form of a weak complex (typically calcium, sodium, zinc or magnesium). Although it may be different from the constituent ligands of the active complex, it is important that the complex it forms with the released lanthanide is less stable than the active complex, thereby preventing a translocation reaction between the active complex and the additional chelator, which will in particular have the effect of completely consuming the additional ligand, which will then no longer capture the released lanthanide. This risk of consuming the additional chelator by translocation is even more pronounced when it is added in free form, rather than in the form of, for example, a calcium complex.

[0015] Therefore, in both strategies described above, it is important to make the active complex as stable as possible.

[0016] However, complexes containing PCTA-based chelating ligands of the pyclene-type structure described in EP 1 931 673, although having good kinetic stability, generally have thermodynamic constants lower than those of other cycloolefin-based macrocycle complexes.

[0017] This is particularly the case for the complexes of formula (II) represented by:

[0018]

[0019] Specifically, as described in particular in WO 2014 / 174120, the thermodynamic equilibrium constant (also called stability constant) corresponding to the reaction for forming the complex having formula (II) is 10 14.9 (i.e. log(K therm )=14.9). For comparison purposes, the stability constant of the gadolinium complex of 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid (DOTA-Gd) is 10 25.6 (i.e. log(K therm )=25.6).

[0020] However, it should be noted that the complex of formula (II) corresponds to several stereoisomers, in particular due to the presence of three asymmetric carbon atoms in the side chain of the complex, located in the alpha position relative to the nitrogen atom of the macrocycle to which the side chain is grafted. These three asymmetric carbons are marked with an asterisk (*) in the formula (II) represented above.

[0021] Thus, the synthesis of the complex of formula (II) as described in EP 1 931 673 results in the production of a mixture of stereoisomers.

[0022] The aminopropanediol group of the side chain of the complex of formula (II) also contains an asymmetric carbon. Therefore, the complex of formula (II) contains six asymmetric carbons in total and therefore exists in the form of 64 configurational stereoisomers. However, in the remainder of this specification, for simplicity, the only source of stereoisomers considered for a given side chain will be the source corresponding to the asymmetric carbon with the carboxylate group, which is marked with an asterisk (*) in the above formula (II).

[0023] Since each of the three asymmetric carbons can have an absolute configuration of R or S, the complexes of formula (II) exist in the form of eight 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 precisely, according to the usual nomenclature in stereochemistry, the complexes of formula (II) exist in the form of eight families of diastereomers.

[0024] As mentioned before, the use of the term "family" is justified since each of these families groups together several stereoisomers, in particular due to the presence of an asymmetric carbon in the aminopropanediol group.

[0025] However, since stereoisomers associated with an asymmetric carbon of a given aminopropanediol group will not be considered in the remainder of the present specification, the terms isomer, stereoisomer or diastereomer II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS will be used indiscriminately without stating that each corresponds to a family of stereoisomers.

[0026] The inventors have succeeded in separating and identifying by high performance liquid chromatography (HPLC) and ultra high performance liquid chromatography (UHPLC) four unresolved peaks or isomeric groups of the complex of formula (II) obtained according to the methods of the prior art, corresponding to four different elution peaks characterized by their retention times on the chromatogram, which will be referred to as iso1, iso2, iso3 and iso4 in the remainder of this specification. By carrying out the method described in EP 1 931 673, the respective contents of the iso1, iso2, iso3 and iso4 groups in the obtained mixture are as follows: 20%, 20%, 40% and 20%.

[0027] They then found that these different isomeric groups had different physicochemical properties and determined that an isomeric group called iso4, comprising a mixture of isomers II-RRR and II-SSS having the formulas (II-RRR) and (II-SSS) represented below, proved to be the most advantageous medical imaging contrast agent.

[0028]

[0029]

[0030] Thus, surprisingly, iso4 is characterized by a thermodynamic stability significantly superior to that of the diastereomeric mixture in the form of the complex of formula (II) obtained by carrying out the process described in EP 1 931673. In particular, its thermodynamic equilibrium constant K therm iso4 Equal to 10 18.7 (i.e. log(K therm iso4 )=18.7), which value has been determined by carrying out the method of Pierrard et al., Contrast Media Mol. Imaging, 2008, 3, 243-252 and Moreau et al., Dalton Trans., 2007, 1611-1620.

[0031] Moreover, iso4 is the isomer group with the best kinetic inertia (also called kinetic stability) among the four groups separated by the inventors. Specifically, the inventors evaluated the kinetic inertia by studying the decomplexation kinetics of the four groups of isomers in an acidic aqueous solution (pH = 1.2) at 37°C. The half-life time values ​​(T 1 / 2 ), which half-life time corresponds to the time when 50% of the amount of complex initially present dissociates according to the following decomplexation reaction (reaction formula 2):

[0032]

[0033]

[0034]

[0035] [Table 1]: Decomplexation kinetics of isomers iso1 to iso4

[0036] For comparison purposes, gadobutrol or gadolinate, which are macrocyclic gadolinium complexes, have kinetic inertias of 18 hours and 4 days, respectively, under the same conditions, whereas linear gadolinium complexes (such as gadodiamide or gadopentetate) dissociate instantaneously.

[0037] In addition, iso4 is chemically more stable than iso3 in particular. The reason for this is that the amide functional group of the complex of formula (II) is susceptible to hydrolysis. The hydrolysis reaction of the amide functional group (reaction formula 3) leads to the formation of a double coupled impurity, accompanied by the release of 3-amino-1,2-propanediol. The inventors studied the hydrolysis reaction kinetics of the complex of formula (II) in an aqueous solution at pH 13 and observed that the amide functional group of iso4 is more stable in terms of hydrolysis than the amide functional group of iso3.

[0038]

[0039] Measurements of the relaxivities of the various groups of isomers (ie, their efficiency as contrast agents) showed that the contrast capabilities of the iso1, iso2, and iso4 groups were relatively comparable, whereas iso3 had reduced efficiency (see Table 2).

[0040]

[0041] [Table 2] Relaxation rates of isomeric groups iso1 to iso4 at 37°C

[0042] The inventors have succeeded in developing a novel process for the preparation and purification of a complex of formula (II) which makes it possible to preferably obtain the diastereomers II-RRR and II-SSS of said complex having particularly advantageous physicochemical properties. The process according to the invention comprises a step of enriching the isomers by converting the least stable stereoisomer into the most stable stereoisomer, which surprisingly makes it possible in the vast majority of cases to obtain the most stable isomer of the complex of formula (II) when carried out on a hexaacid intermediate complex rather than on the final complex.

[0043] Compared to the alternative of preparing a mixture of stereoisomers, it is undoubtedly advantageous to implement a process that makes it possible in most cases to obtain the target diastereomer, so as to subsequently attempt to separate the diastereomers according to conventional techniques, thereby isolating the target isomer using any separation technique well known in the art. In particular, in addition to being easier to carry out on an industrial scale a process without a diastereoisomer separation step, not carrying out a separation allows, on the one hand, to save a great deal of time and, on the other hand, to increase the overall yield of the process by limiting as much as possible the production of unwanted diastereomers that will ultimately be eliminated. In addition, conventional separation techniques generally involve the use of large amounts of solvents, which, beyond the financial cost, are undesirable for environmental reasons. In addition, the use of silica chromatography should be particularly avoided, given the health risks inherent in occupational exposure to silica, which the International Agency for Research on Cancer classifies as carcinogenic to humans (group 1).

[0044] As mentioned above, the method developed by the inventors for preparing the complex having formula (II) is based on the step of isomer enrichment of the intermediate gadolinium hexahydrate complex having formula (I) represented below:

[0045]

[0046] The complex of formula (I) corresponds to several stereoisomers due to the presence of three asymmetric carbon atoms in the side chain of the complex, located in the alpha position relative to the nitrogen atom of the macrocycle to which the side chain is grafted. These three asymmetric carbons are marked with asterisks (*) in the formula (I) shown above.

[0047] Since each of the three asymmetric carbons carrying the carboxylate functional group can have an absolute configuration of R or S, the complex of formula (I) exists in the form of eight stereoisomers, hereinafter referred to as I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR and I-SRS. More precisely, according to the usual nomenclature in stereochemistry, the complex of formula (I) exists in the form of four pairs of enantiomers that are diastereomers of each other.

[0048] The inventors have succeeded in separating and identifying by high performance liquid chromatography (HPLC) and ultra high performance liquid chromatography (UHPLC) four unresolved peaks or groups of isomers of the complex of formula (I) obtained according to the method described in EP 1 931 673, corresponding to four distinct elution peaks characterized by their retention times on the chromatogram, which will be referred to in the remainder of this specification as isoA, isoB, isoC and isoD.

[0049] IsoD crystallizes in water. X-ray diffraction analysis allowed the inventors to determine the crystal structure of this group of isomers and thus found that it comprises diastereomers I-RRR and I-SSS of the complex with formula (I-RRR) and (I-SSS) represented below.

[0050]

[0051] It should be noted that the diastereomers I-RRR and I-SSS of the complex having formula (I) are enantiomers of each other.

[0052] The isomer enrichment step of the method of the present invention involves enriching the intermediate gadolinium hexaphosphate complex having formula (I) in isoD.

[0053] The synthesis of the complex of formula (II) specifically involves converting the carboxylic acid functional group of the intermediate hexaacid complex of formula (I) into an amide functional group. This amidation reaction does not change the absolute configuration of the three asymmetric carbon atoms of the complex of formula (I).

[0054] Therefore, when the hexaacid complex having the formula (I) enriched in isoD obtained previously is subjected to an amidation reaction, the complex having the formula (II) enriched in iso4 can be obtained.

[0055] Furthermore, the purification method developed by the inventors enables the complex of formula (II) to be obtained with optimized isomeric characteristics and significantly improved impurity profile when following the preparation method of the complex of formula (II) described above.

[0056] Thus, such diastereoisomerically enriched and purified complexes with improved stability can be formulated with free macrocyclic ligands (e.g. free DOTA) instead of the calcium complex of DOTA, as recommended in WO 2014 / 174120. From an industrial point of view, the use of free DOTA is particularly advantageous in the sense that it makes it possible to eliminate the process step for the synthesis of the preparation as described in WO 2014 / 174120, namely the addition of CaCl2. Summary of the invention

[0057] The complex having formula (II)

[0058] Therefore, the present invention firstly relates to a complex having the formula (II):

[0059]

[0060] Consists of at least 80% diastereomeric excess, comprising a mixture of isomers II-RRR and II-SSS having the following formula:

[0061]

[0062]

[0063] In the context of the present invention, the term "diastereomeric excess" is intended to represent the fact that, with respect to the complex of formula (II), the complex is present predominantly in the form of an isomer or isomer group selected from the following diastereomers: II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. The diastereomeric excess is expressed as a percentage and corresponds to the amount represented by the predominant isomer or isomer group relative to the total amount of the complex of formula (II). It should be understood that the percentage can be based on moles or on mass, since, by definition, isomers have the same molar mass.

[0064] In one embodiment, the complex of formula (II) according to the invention has a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 92%, preferably at least 94%, advantageously at least 97%, more advantageously at least 99% of the mixture comprising isomers II-RRR and II-SSS.

[0065] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of isomers II-RRR and II-SSS.

[0066] Advantageously, said diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0067] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers the situation where only one isomer is present, whether II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all situations where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0068] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture in a ratio of 50 / 50.

[0069] More specifically, the diastereomeric excess as defined previously corresponds to peak 4 (i.e., the fourth unresolved peak of the isomers arranged in order of elution and corresponding to iso4) in the UHPLC profile, which is characterized by a retention time between 6.0 and 6.6 minutes, typically around 6.3 minutes, said profile being obtained using the UHPLC method described below.

[0070] Within the meaning of the present invention, the term "UHPLC profile" refers to the plot of concentration changes over time as measured by the detector after a mixture of compounds (in this case isomers of compounds) has passed and separated on a stationary phase for a given composition and a given eluent flow rate. The UHPLC profile consists of various peaks or unresolved peaks characteristic of the analyzed compound or mixture of compounds. DETAILED DESCRIPTION

[0071] UHPLC Method:

[0072] Waters UPLC T3 150x 2.1mm-1.6μm column.

[0073] This is a reverse phase UPLC column with spherical particles consisting of a preferably very hard core made of silica surrounded by trifunctional C18 (octadecyl) grafted porous silica and silanol treated with a capping agent (end capping). It is also characterized by a length of 150 mm, an inner diameter of 2.1 mm, a particle size of 1.6 μm, porosity and 4.7% carbon content.

[0074] Preferably, the stationary phase used should be compatible with the aqueous mobile phase.

[0075] -Analysis conditions:

[0076]

[0077] -Mobile phase gradient (% v / v):

[0078]

[0079] Compositions comprising a complex having formula (II)

[0080] Secondly, the present invention relates to a composition comprising:

[0081] - a complex of formula (II) consisting of a diastereomeric excess of at least 80%, said diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS, and

[0082] - Free macrocyclic ligand.

[0083] In the present specification, the term "macrocyclic ligand" or "macrocyclic chelate" may be used without distinction.

[0084] In the context of the present invention, the term "macrocycle" denotes a ring typically comprising at least nine atoms, whether they are carbon atoms or heteroatoms, and the terms "macrocyclic ligand" or "macrocyclic chelate" are polydentate, at least bidentate, ligands.

[0085] In the meaning of the present invention, the term "free macrocyclic ligand" refers to the macrocyclic ligand in free form, i.e. not complexed with, in particular, metals (including lanthanides and actinides) or alkaline earth cations (e.g. calcium or magnesium). In particular, as described in US Pat. No. 5,876,695, the free macrocyclic ligand is not in the form of a complex with gadolinium and is not introduced into the composition in the form of a weak complex (typically calcium, sodium, zinc or magnesium), however, the presence of traces of said cations in the composition and thus in the corresponding complex is not excluded.

[0086] As discussed previously, the formulation of the complex of formula (II) with free macrocyclic ligand rather than the weak complex of said macrocyclic ligand recommended in EP 1931 673 is made possible by the improved stability of the diastereomerically enriched complex of formula (II) according to the present invention.

[0087] In a preferred embodiment, the complex of formula (II) present in the composition of the invention has a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 92%, more specifically at least 94%, preferably at least 97%, advantageously at least 99% of the mixture comprising isomers II-RRR and II-SSS.

[0088] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of isomers II-RRR and II-SSS.

[0089] Advantageously, said diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0090] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers the situation where only one isomer is present, whether II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all situations where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0091] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture in a ratio of 50 / 50.

[0092] In an advantageous embodiment, the composition according to the invention has a free gadolinium concentration of less than 1 ppm (m / v), preferably less than 0.5 ppm (m / v).

[0093] In this specification, unless otherwise specified, the terms "Gd", "gadolinium" and "Gd 3+ "To represent Gd 3+ ions. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0094] In the present invention, the term "free Gd" represents the non-complexed form of gadolinium, which is preferably available for complexation. Typically, Gd dissolved in water 3+ ions. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0095] The free form of gadolinium is typically determined by colorimetry, usually xylenol orange or arsenazo (III). In the absence of metal ions (such as gadolinium), these indicators have a specific color: xylenol orange is yellow and arsenazo is pink at acidic pH. In the presence of gadolinium, their color changes to purple.

[0096] Visual inspection of the change in color of the solution allows verification of the presence or absence of gadolinium in the solution.

[0097] In addition, it is possible to quantitatively measure free gadolinium in solution by back titration, for example using EDTA as a "weak" gadolinium chelate. In this assay, a colored indicator is added until a purple color appears. EDTA (gadolinium ligand) is then added dropwise to the mixture. Since EDTA is a stronger complexing agent than the colored indicator, gadolinium changes the ligand and causes the colored indicator to complex preferentially with EDTA. As a result, the colored indicator gradually returns to its non-complexed form.

[0098] When the amount of EDTA added equals the initial amount of free Gd, the colored indicator is completely free and the solution "turns" yellow. Since the amount of EDTA added is known, the initial amount of free Gd in the solution to be tested can be known.

[0099] These methods are well known to the person skilled in the art and are described in particular in Barge et al. (Contrast Media and Molecular Imaging 1, 2006, 184-188).

[0100] Therefore, these colorimetric methods are usually performed in solutions with a pH between 4 and 8. The reason for this is that outside these pH ranges, the accuracy of the measurement may be affected due to an alteration (or even inhibition) of the color change.

[0101] Therefore, if necessary, the pH of the sample to be measured is adjusted to between 4 and 8. In particular, if the pH of the sample is acidic, and in particular less than 4, the pH is advantageously adjusted by adding a base and the free Gd measurement is then carried out on the sample at the adjusted pH.

[0102] Thus, the composition according to the invention is stable over time, i.e. its composition remains compliant with the regulations regarding the concentration of free gadolinium (in particular its free Gd concentration remains less than 1 ppm (m / v)), in particular with respect to the content of free paramagnetic metal, for a period of at least 3 years, preferably at least 4 years or more preferably at least 5 years. According to the ICH guidelines, observing a stability of 6 months at 40°C is considered to be a good indication of a stability of 3 years at 25°C.

[0103] In one embodiment, the composition according to the present invention has 0.01 and 1.5 mol.L -1 Between, preferably between 0.2 and 0.7 mol.L -1 Between, more preferably between 0.3 and 0.6 mol.L -1 The concentration of the complex having formula (II) is between .

[0104] The complex of formula (II) is determined by methods known to those skilled in the art. In particular, it can be determined by atomic emission spectroscopy (also known as ICP-AES or ICP atomic emission spectroscopy) after mineralization and determination of the total amount of gadolinium present in the composition.

[0105] The content of the complex having formula (II) enables the composition to have an optimal contrast capability while having a satisfactory viscosity. Specifically, less than 0.01 mol.L -1 The above-mentioned complex having formula (II) has unsatisfactory performance quality as a comparative product, and when the concentration is higher than 1.5 mol.L -1 When the viscosity of the composition becomes too great, it becomes difficult to handle.

[0106] In a particular embodiment, the composition according to the invention 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 % and more preferably between 0.05 and 0.15 mol / mol % of free macrocyclic ligand relative to the complex of formula (II).

[0107] Advantageously, the macrocyclic ligand is selected from the group consisting of DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA and derivatives thereof.

[0108] Preferably, it is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

[0109] The concentration of free DOTA in the composition is typically measured by back titration with copper, for example using copper sulfate as the copper ion source.

[0110] In this method, which is well known to the person skilled in the art, preferably a solution is used which contains a known initial concentration Q0 of copper sulfate, which is greater than the amount of free ligand in the solution. A test solution containing the amount Q1 of free DOTA to be determined is added to this copper sulfate solution. DOTA is a very good copper complexing agent: therefore the formation of DOTA-copper complexes is observed.

[0111] The free copper remaining in the solution is then advantageously back-titrated by a potentiometer. For this purpose, EDTA is added dropwise to the mixture, for example. EDTA complexes the free copper in the solution, but does not decomplex the DOTA-copper, since DOTA is a stronger complexing agent than EDTA. When the amount of EDTA added, Q2, equals the amount of free copper in the solution, a sudden drop in the solution potential is observed.

[0112] Given the initial amount of copper Q0 and the amount of EDTA added Q2, the two values ​​are subtracted Q0-Q2 to obtain the amount of free DOTA Q1 in the test solution.

[0113] Alternatively, HPLC methods may be used, in particular HILIC LC-UV methods.

[0114] These measurement methods, in particular potentiometric measurement methods, are carried out in solutions having a pH advantageously between 4 and 8. Therefore, if necessary, the pH of the sample to be measured is adjusted to between 4 and 8. In particular, if the pH of the sample is acidic, and in particular less than 4, the pH is advantageously adjusted by adding a base such as meglumine, and the free DOTA measurement is then carried out on the sample at the adjusted pH.

[0115] Preferably, the ratios in the present invention, in particular as specified above, are ratios before the composition is sterilized.

[0116] Advantageously, the pH of the composition is between 4.5 and 8.5, preferably between 5 and 8, advantageously between 6 and 8, in particular between 6.5 and 8. These pH ranges make it possible in particular to limit the presence of certain impurities and to promote the complexation of the paramagnetic metal ion M.

[0117] In particular, the composition according to the invention may be buffered, i.e. it may also comprise a buffer selected from the common buffers established for a pH range of 5 to 8, preferably lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, Tris (tris (hydroxymethyl) aminomethane), HEPES (2-[4-(2-hydroxyethyl) -1-piperazine] ethanesulfonic acid) and MES (2-morpholineethanesulfonic acid) buffers and mixtures thereof, and preferably a buffer selected from Tris, lactate, tartrate, carbonate and MES buffers and mixtures thereof. Advantageously, the composition according to the invention comprises a Tris buffer.

[0118] The compositions which are the subject of the present invention are preferably sterile.

[0119] Process for preparing a complex having formula (II)

[0120] The present invention also relates to a method for preparing a complex having formula (II), comprising the following consecutive steps:

[0121] a) a hexacarboxylic acid having the following formula (III):

[0122]

[0123] complexed with gadolinium to obtain a gadolinium hexacarboxylate complex having formula (I) as defined above,

[0124] b) isomerizing the hexaoxato gadolinium complex of formula (I) by heating it in aqueous solution at a pH between 2 and 4 to obtain a diastereoisomerically enriched complex consisting of at least 80% of diastereoisomer excess, the diastereoisomer excess comprising a mixture of isomers I-RRR and I-SSS of the hexaoxato gadolinium complex of formula (I), and

[0125] c) Starting from the diastereomerically enriched complex obtained in step b), forming the complex of formula (II) by reaction with 3-amino-1,2-propanediol.

[0126] In this specification, unless otherwise specified, the terms "Gd", "gadolinium" and "Gd 3+ "To represent Gd3+ ions. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0127] In the present invention, the term "free Gd" represents the non-complexed form of gadolinium, which is preferably available for complexation. Typically, Gd dissolved in water 3+ ions. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide.

[0128] ■Step a)

[0129] During this step, a complexation reaction occurs between the hexaacid of formula (III) and gadolinium, which makes it possible to obtain the hexaacid gadolinium complex of formula (I) as defined previously.

[0130] According to one specific embodiment, step a) comprises the reaction between the hexaacid of formula (III) and a source of free Gd in water.

[0131] In a preferred embodiment, the source of free Gd is GdCl3 or Gd2O3, preferably Gd2O3.

[0132] Preferably, the reagents used in step a), ie the source of gadolinium (typically gadolinium oxide), the hexaacid of formula (III) and water, are as pure as possible, in particular with respect to metallic impurities.

[0133] Thus, the source of gadolinium will advantageously be gadolinium oxide, preferably with a purity greater than 99.99%, even more preferably greater than 99.999%.

[0134] The water used in the process preferably contains less than 50 ppm calcium, more preferably less than 20 ppm and most preferably less than 15 ppm calcium. Typically, the water used in the process is deionized water, water for injection (injection grade water) or purified water.

[0135] Advantageously, the amounts of reagents (hexacid of formula (III) and gadolinium) used in this step a) correspond to or are close to the stoichiometric proportions, as determined by the equilibrium equation of the complexation reaction occurring in this step.

[0136] The term "close to the stoichiometric ratio" means that the difference between the molar ratio of the introduced reagents and the stoichiometric ratio is less than 15%, in particular less than 10%, preferably less than 8%.

[0137] In particular, the introduction of gadolinium may be slightly excessive relative to the stoichiometric ratio. Then, the ratio of the amount of material introduced as gadolinium to the amount of material introduced as hexaacid having formula (III) is greater than 1, but typically less than 1.15, particularly less than 1.10, and advantageously less than 1.08. In other words, relative to the amount of hexaacid having formula (III) introduced (which itself corresponds to 1 equivalent), the amount of gadolinium introduced is greater than 1 equivalent (eq.), but typically less than 1.15 eq., particularly less than 1.10 eq., and advantageously less than 1.08 eq. In a preferred embodiment in which the source of free gadolinium is Gd2O3, relative to the amount of hexaacid having formula (III) introduced (1 eq.), the amount of Gd2O3 introduced is typically greater than 0.5 eq., but less than 0.575 eq., particularly less than 0.55 eq., and advantageously less than 0.54 eq.

[0138] According to a specific embodiment, step a) comprises the following consecutive steps:

[0139] a1) preparing an aqueous solution of a hexaacid of formula (III), and

[0140] a2) adding a source of free gadolinium to the aqueous solution obtained in step a1).

[0141] In this embodiment, the content of hexaacid of formula (III) in the aqueous solution prepared in step a1) is typically between 10 and 60% by weight, in particular between 15 and 45%, preferably between 20 and 35%, advantageously between 25 and 35% and even more advantageously between 25 and 30% by weight relative to the total weight of the aqueous solution.

[0142] Preferably, steps a) and b) are carried out according to a one-pot embodiment, ie in the same reactor without intermediate steps of separation or purification.

[0143] Therefore, in this preferred embodiment, the hexacarboxylic gadolinium complex of formula (I) formed in step a) is directly subjected to the isomerization step b) without isolation or purification and is carried out in the same reactor as used in step a).

[0144] ■Step b)

[0145] The hexaacid gadolinium complex of formula (I) formed by the complexation reaction between the hexaacid of formula (III) and gadolinium in step a) is first obtained as a mixture of diastereomers.

[0146] Step b) involves enriching the mixture of diastereomers in the isomers I-RRR and I-SSS to obtain a diastereomerically enriched gadolinium hexahydrate complex having formula (I), which consists of a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 95%, preferably at least 97%, advantageously at least 98%, more advantageously at least 99% of the mixture comprising the isomers I-RRR and I-SSS.

[0147] In the context of the present invention, the term "diastereomeric excess" is intended to represent the fact that the hexa-acid gadolinium complex having formula (I) is present mainly in the form of an isomer or isomer group selected from the following diastereoisomers: I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR and I-SRS. The diastereomeric excess is expressed as a percentage and corresponds to the amount represented by the main isomer or isomer group relative to the total amount of the hexa-acid gadolinium complex having formula (I). It should be understood that the percentage can be based on moles or on mass, since, by definition, isomers have the same molar mass.

[0148] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of the isomers I-RRR and I-SSS.

[0149] Advantageously, said diastereomeric excess consists of a mixture of the isomers I-RRR and I-SSS.

[0150] In fact, the inventors have found that factors such as pH and temperature of the solution of the gadolinium hexahydrate complex of formula (I) obtained at the end of step a) have an influence on the ratio of the various isomers of the complex of formula (I) present in the mixture of diastereomers. Over time, the mixture tends to be enriched in a group of isomers that surprisingly contains the most thermodynamically stable but also the most chemically stable isomers, in this case the isomers I-RRR and I-SSS.

[0151] By extension, the term "mixture of the isomers I-RRR and I-SSS" also covers the situation where only one isomer is present, whether I-RRR or I-SSS.

[0152] However, in a preferred embodiment, the isomers I-RRR and I-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the mixture of isomers I-RRR / I-SSS is a racemic (50 / 50) mixture.

[0153] Step b) of isomerization of the hexaacid gadolinium complex of formula (I) in aqueous solution is typically carried out at a pH between 2 and 4, in particular between 2 and 3, advantageously between 2.2 and 2.8.

[0154] The pH is preferably adjusted with an acid, preferably an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, for example with hydrochloric acid.

[0155] It is completely surprising that under such pH conditions, an enrichment of the mixture, in particular of the isomers, occurs, in this case the isomers I-RRR and I-SSS, since it is known in the art that gadolinium chelates are characterized by low kinetic inertia in acidic media. + The higher the concentration of ions, the greater the probability that a proton will be transferred to one of the donor atoms of the ligand, thereby causing the complex to dissociate. Therefore, one skilled in the art would expect that placing the hexacarboxylic acid gadolinium complex of formula (I) in an aqueous solution at a pH between 2 and 4 would cause the complex to dissociate, rather than isomerize to I-RRR and I-SSS.

[0156] It should be noted that the pH range recommended by EP 1 931 673 for the complexation of the hexaacid of formula (III), ie 5.0-6.5, does not allow the complex of formula (I) to be obtained enriched in its isomers I-RRR and I-SSS.

[0157] 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, advantageously between 100°C and 120°C, typically for a time between 10 hours and 72 hours, in particular between 10 hours and 60 hours, advantageously between 12 hours and 48 hours.

[0158] Contrary to all expectations, such temperature conditions used in combination with the above mentioned pH conditions should favour the instability of the gadolinium chelate, not leading to its decomplexation or formation of any other impurities, but rather to its isomerization into I-RRR and I-SSS.

[0159] In a particular embodiment, the aqueous solution of step b) comprises acetic acid.Step b) is then advantageously carried out at a temperature between 100°C and 120°C, in particular between 110°C and 118°C, typically for a time between 12 hours and 48 hours, in particular between 20 hours and 30 hours, in particular between 24 hours and 26 hours.

[0160] Preferably, an amount of acetic acid is added before heating the solution of the hexaacid gadolinium complex of formula (I) obtained in step a) so that the content of acetic acid is between 25% and 75% by mass, in particular between 40% and 50% by mass, relative to the mass of the hexaacid of formula (III) used in step a).

[0161] When the aqueous solution is heated to a temperature advantageously between 100 and 120° C., typically between 110 and 118° C., acetic acid is gradually added as the water evaporates so as to maintain a constant volume of solution.

[0162] According to a preferred embodiment, at the end of step b), the diastereomerically enriched complex is isolated by crystallization, preferably by seeded crystallization.

[0163] In this embodiment, step b) comprises the following consecutive steps:

[0164] b1) isomerizing the hexaoxato gadolinium complex of formula (I) by heating it in aqueous solution at a pH between 2 and 4 to obtain a diastereoisomerically enriched complex consisting of at least 80% of diastereoisomer excess, the diastereoisomer excess comprising a mixture of isomers I-RRR and I-SSS of the hexaoxato gadolinium complex of formula (I), and

[0165] b2) isolating the diastereomerically enriched complex by crystallization, preferably by seeded crystallization.

[0166] The crystallization step b2) involves firstly removing any impurities present in the aqueous solution, which may be caused by the previous step, so as to obtain a decolorized product of higher purity in the form of crystals, and secondly, continuing the diastereoisomer enrichment of the hexaacid gadolinium complex of formula (I) to obtain a diastereoisomer excess of a mixture containing the isomers I-RRR and I-SSS of the complex, which is higher than that obtained at the end of step b1). Specifically, the isomers I-RRR and I-SSS of the hexaacid complex of formula (I) are crystallized from water. On the other hand, the hexaacid gadolinium complex of formula (I) not enriched in said isomers does not crystallize.

[0167] The fact that the isomers I-RRR and I-SSS in which the complex tends to be enriched during step b) (and contrary to all expectations, according to the conditions under which it is carried out) are the only isomers of the complex that crystallize from water is a completely unexpected result. Isomerization and crystallization therefore act synergistically to contribute to the enrichment of the isomers I-RRR and I-SSS and therefore to the overall efficiency of the process according to the invention.

[0168] Furthermore, it should be noted that the crystallization of the target isomer of the gadolinium hexaoxide complex of formula (I) from water allows avoiding the addition of solvent as described in embodiment 7 of EP 1 931 673, which involves a step of precipitating the trisodium salt of the complex from ethanol.

[0169] Step b2) is advantageously carried out at a temperature between 10 and 70°C, in particular between 30 and 65°C, specifically between 35 and 60°C.

[0170] According to a variation, after reducing the temperature of the aqueous solution so that it is within the above range, the crystallization process is induced by seeding. "Crystallization by seeding", also known as "crystallization by seeding", includes introducing a known amount of crystals (referred to as "seeds" or "primers") into a reactor (also referred to as a crystallization vessel) in which crystallization is performed. This makes it possible to reduce the crystallization time. Crystallization by seeding is well known to those skilled in the art. In the method according to the present invention, a primer, in the present case a crystal of a six acid gadolinium complex with formula (I) enriched in diastereomers added to an aqueous solution of a diastereomer-enriched complex whose temperature has been previously reduced, is used to crystallize so that nucleation can be obtained, thereby initiating crystallization. The duration of crystallization by seeding is advantageously between 2 hours and 20 hours, and preferably between 6 hours and 18 hours; typically 16 hours.

[0171] The diastereomerically enriched crystals of the gadolinium hexahydrate complex having formula (I) are then isolated by filtration and drying, typically by any technique known to those skilled in the art.

[0172] Advantageously, the diastereomerically enriched gadolinium hexahydrate complex of formula (I) isolated at the end of step b2) has a purity greater than 95%, in particular greater than 98%, advantageously greater than 99%, expressed as a percentage by mass of the complex of formula (I) relative to the total mass obtained at the end of step b2).

[0173] In one embodiment, the diastereomerically enriched complex from step b) separated by crystallization is purified again by recrystallization to obtain a diastereomerically enriched and purified complex.

[0174] In this embodiment, in addition to the previously described consecutive steps b1) and b2), step b) further comprises a step b3) of purifying the diastereomerically enriched gadolinium hexahydrate complex having formula (I) separated by recrystallization.

[0175] The recrystallization step b3) is, like the crystallization step b2), firstly concerned with obtaining a product of higher purity and secondly with continuing the diastereomeric enrichment of the gadolinium hexaoxide complex of formula (I) in order to obtain a diastereomeric excess of a mixture comprising the isomers I-RRR and I-SSS of the complex which is higher than that obtained at the end of step b2).

[0176] Step b3) typically comprises the following successive sub-steps:

[0177] suspending the diastereomerically enriched gadolinium hexahydrate complex of formula (I) separated in step b2) in an aqueous solution, preferably in water,

[0178] • dissolving the complex by heating to a temperature advantageously between 80° C. and 120° C., for example to a temperature of 100° C.,

[0179] recrystallization, preferably by seeding, advantageously at a temperature 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 time between 2 hours and 20 hours, in particular between 6 hours and 18 hours, and

[0180] - Isolating the diastereomerically enriched and purified crystals of the gadolinium hexaoxide complex of formula (I), for example by filtration and drying.

[0181] The purity of the purified diastereomerically enriched gadolinium hexahydrate complex of formula (I) isolated at the end of step b3) is typically greater than 98%, in particular greater than 99%, advantageously greater than 99.5%, expressed as a percentage by mass of the complex of formula (I) relative to the total mass obtained at the end of step b2).

[0182] In another embodiment, the diastereoisomerically enriched complex from step b) is further enriched by selective decomplexation of diastereoisomers of the complex having formula (I) other than the diastereoisomers I-RRR and I-SSS, i.e., by selective decomplexation of the diastereoisomers I-RSS, I-SRR, I-RSR, I-SRS, I-RRS and I-SSR.

[0183] In this embodiment, in addition to the previously described consecutive steps b1) and b2), step b) also comprises a step b4) of selective decomplexation of diastereomers of the complex of formula (I) other than the diastereomers I-RRR and I-SSS). In this variant, step b) may also comprise the previously described step b3), which is carried out between steps b2) and b4) or after b4).

[0184] The selective decomplexation step b4) involves continuing the diastereomeric enrichment of the gadolinium hexaoxide complex of formula (I) so as to obtain a diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS of the complex, which is higher than the diastereomeric excess obtained at the end of step b2) or at the end of step b3) when said step is carried out before step b4).

[0185] Step b4) typically comprises the following successive sub-steps:

[0186] ● suspending the diastereomerically enriched gadolinium hexahydrate complex of formula (I) separated in step b2) or step b3) in water,

[0187] ● Adding a base, such as sodium hydroxide,

[0188] heating to a temperature advantageously between 30°C and 60°C, in particular between 35°C and 55°C, for example 40°C, typically for a time between 2 hours and 20 hours, in particular between 10 hours and 18 hours,

[0189] - cooled to a temperature advantageously between 10°C and 30°C, for example to 30°C, and

[0190] - Separation of the diastereomerically enriched and purified gadolinium hexahydrate complex of formula (I), for example by filtration and drying.

[0191] The fact that the isomers I-RRR and I-SSS are the most stable in alkaline medium makes step b4) possible. Such alkaline conditions promote the formation of gadolinium hydroxide and therefore the decomplexation of the least stable isomer. It should therefore be noted that, surprisingly, the isomers I-RRR and I-SSS are more stable both in an acidic medium that allows the isomerization step b1) and in an alkaline medium that allows the selective decomplexation step b4).

[0192] In a preferred embodiment, the diastereomerically enriched complex obtained at the end of step b) according to any of the above-described variants has a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 95%, preferably at least 97%, advantageously at least 98%, more advantageously at least 99% over the mixture comprising the isomers I-RRR and I-SSS.

[0193] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of the isomers I-RRR and I-SSS.

[0194] Advantageously, said diastereomeric excess consists of a mixture of the isomers I-RRR and I-SSS.

[0195] By extension, the term "mixture of isomers I-RRR and I-SSS" also covers the situation where only one isomer is present, whether I-RRR or I-SSS. However, the term "mixture of isomers I-RRR and I-SSS" preferably represents all situations where each of the isomers I-RRR and I-SSS is present in variable but non-zero amounts.

[0196] In a preferred embodiment, the isomers I-RRR and I-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the mixture of isomers I-RRR / I-SSS is a racemic (50 / 50) mixture.

[0197] ■Step c)

[0198] Step c) involves the formation of the complex of formula (II) from its precursor, ie the diastereomerically enriched gadolinium hexacarboxylate complex of formula (I) obtained in step b).

[0199] In this step, the three carboxylic acid functional groups of the hexaacid complex of formula (I) carried by the carbon atom located in the γ position on the side chain of the complex relative to the nitrogen atom of the macrocycle to which the side chain is grafted are converted into amide functional groups by an amidation reaction with 3-amino-1,2-propanediol in racemic or enantiomerically pure form, preferably in racemic form.

[0200] This amidation reaction does not change the absolute configuration of the three asymmetric carbon atoms located in the alpha position on the side chain relative to the nitrogen atom of the macrocycle grafted with the side chain. Thus, step c) makes it possible to obtain a complex of formula (II) with a diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS, which is identical to the diastereomeric excess of a mixture comprising isomers I-RRR and I-SSS, the diastereomeric enriched gadolinium hexahydrate complex of formula (I) obtained at the end of step b) being obtained with a diastereomeric excess of at least 80% of the mixture comprising isomers I-RRR and I-SSS.

[0201] In a preferred embodiment, the complex of formula (II) obtained at the end of step c) has a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 92%, preferably at least 94%, advantageously at least 97%, more advantageously at least 99% of the mixture comprising isomers II-RRR and II-SSS.

[0202] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of isomers II-RRR and II-SSS.

[0203] Advantageously, said diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0204] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers the situation where only one isomer is present, whether II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all situations where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0205] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture in a ratio of 50 / 50.

[0206] The amidation reaction can be carried out according to any method known to those skilled in the art, in particular in the presence of reagents for activating the carboxylic acid function and / or by acid catalysis.

[0207] It can be carried out in particular according to the method described in EP 1 931 673, in particular in paragraph

[0027] of said patent.

[0208] In one embodiment, step c) comprises activating the carboxylic acid (-COOH) functional group carried by the carbon atom located in the γ position on the side chain of the hexaacid complex of formula (I) relative to the nitrogen atom of the macrocycle on which the side chain is grafted, in the form of a derivative functional group, including a carbonyl (C=O) group, so that the carbon atom of the carbonyl group is more electrophilic than the carbon atom of the carbonyl group of the carboxylic acid functional group. Therefore, according to this embodiment, the carboxylic acid functional group can be activated in particular in the form of an ester, acyl chloride or anhydride functional group, or in any activated form that can produce an amide bond. Activated forms that can produce amide bonds are well known to those skilled in the art and can be obtained, for example, by a group of methods known in peptide chemistry for forming peptide bonds. Examples of such methods are given in the publication Synthesis of peptides and Peptidomimetics, Vol. E22a, pp. 425-588, Houben-Weyl et al., ed. Goodman, Thieme-Stuttgart-New York (2004), and among these examples mention may be made in particular of methods of activating carboxylic acids by azides (acyl azides), for example by the action of reagents such as diphenylphosphoryl azide (commonly abbreviated to DPPA), the use of carbodiimides, alone or in the presence of catalysts such as N-hydroxysuccinimide and its derivatives, the use of carbonyldiimidazoles (1,1′-carbonyldiimidazole, CDI), the use of phosphonium salts such as benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (commonly abbreviated to BOP), or other uroniums, for example 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (commonly abbreviated to HBTU).

[0209] Preferably, step c) comprises activating the above-mentioned carboxylic acid (—COOH) functional group in the form of an ester, acid chloride or anhydride functional group.

[0210] This embodiment is more preferred than the use coupling agent (e.g. EDCI / HOBT) described in EP 1 931 673 by activating the carboxylic acid functional group to carry out peptide coupling. Specifically, this coupling results in the formation of an equivalent of 1-ethyl-3-[3-(dimethylamino)propyl]urea, which must be removed, particularly by silica gel chromatography or by adding a solvent to carry out liquid / liquid extraction to remove. As previously discussed, independent of the increased complexity of the method caused by such additional steps, it is not desirable to use such purification methods. In addition, the use of HOBT itself is problematic because it is an explosive product.

[0211] Within the meaning of the present invention, the term "ester function" is intended to represent a -C(O)O- group. It may in particular be a group -C(O)O-R1, wherein R1 corresponds to a (C1-C6)alkyl group.

[0212] Within the meaning of the present invention, the term "(C1-C6)alkyl group" refers to a linear or branched saturated hydrocarbon chain containing 1 to 6, preferably 1 to 4, carbon atoms. Examples that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl groups.

[0213] Within the meaning of the present invention, the term "acyl chloride function", also called "acid chloride function", is intended to represent a -CO-Cl group.

[0214] Within the meaning of the present invention, the term "anhydride function" is intended to represent a -CO-O-CO- group. It may in particular be a group -CO-O-CO-R2, wherein R2 corresponds to a (C1-C6)alkyl group.

[0215] Reactions for converting a carboxylic acid function into an ester, acid chloride or anhydride function are well known to the person skilled in the art who will be able to carry out the reaction according to any conventional method with which he is familiar.

[0216] The complex of formula (II) is then obtained by aminolysis of the activated carboxylic acid function in the form of an ester, chloride or anhydride function, in particular an ester or anhydride, preferably an ester, by reaction with 3-amino-1,2-propanediol in racemic or enantiomerically pure form, preferably in racemic form.

[0217] Preferably, the steps of activating the carboxylic acid function and the aminolysis are carried out according to a one-pot embodiment, i.e. in the same reactor, without intermediate steps of isolating or purifying intermediates comprising the carboxylic acid function activated in the form of an ester, chloride or anhydride function, in particular an ester or anhydride, preferably an ester.

[0218] According to a specific embodiment, step c) comprises the following consecutive steps:

[0219] c1) forming an activated complex having formula (VII),

[0220]

[0221] wherein Y represents a chlorine atom, an -OR1 or an -OC(O)-R2 group; preferably, Y represents an -OR1 or -OC(O)-R2 group, wherein R1 and R2 independently of one another correspond to a (C1-C6)alkyl group, and

[0222] c2) Aminelysis of the activated complex of formula (VII) with 3-amino-1,2-propanediol.

[0223] It is obvious to a person skilled in the art that the reaction to form the activated complex of formula (VII) does not change the absolute configuration of the three asymmetric carbon atoms located in the alpha position on the side chain relative to the nitrogen atom of the macrocycle grafted with the side chain. Thus, step c1) makes it possible to obtain an activated complex of formula (VII) with a diastereomeric excess comprising a mixture of isomers VII-RRR and VII-SSS having the formula (VII-RRR) and (VII-SSS) represented below, which is the same as the diastereomeric excess of a mixture comprising isomers I-RRR and I-SSS, and the diastereomeric enriched gadolinium hexahydrate complex of formula (I) obtained at the end of step b) has a diastereomeric excess of at least 80% of the mixture comprising isomers I-RRR and I-SSS.

[0224]

[0225] In the case where Y represents a chlorine atom, step c1) is typically carried out by reaction between the diastereomerically enriched gadolinium hexahydrate complex of formula (I) obtained in step b) and thionyl chloride (SOCl2).

[0226] In the case where Y represents a -OC(O)-CH3 group, step c1) is typically carried out by reaction of the diastereomerically enriched gadolinium hexahydrate complex of formula (I) obtained in step b) with acetyl chloride.

[0227] In an advantageous embodiment, step c) comprises activating the above-mentioned carboxylic acid (—COOH) functions in the form of ester functions.

[0228] According to this embodiment, step c) may more specifically comprise the following consecutive steps:

[0229] c1) forming a triester having the formula (VIII),

[0230]

[0231]

[0232] wherein R1 represents a (C1-C6)alkyl group, and

[0233] c2) Aminelysis of the triester of formula (VIII) with 3-amino-1,2-propanediol.

[0234] Step c1) is typically carried out in the presence of an acid, such as hydrochloric acid, in an alcohol of formula R1OH acting both as solvent and reagent.

[0235] Step c2) is also typically carried out in the presence of an acid, such as hydrochloric acid, in an alcohol of formula R1OH.

[0236] In the first stage, a hexa-acid gadolinium complex of formula (I) and an alcohol R1OH are placed in a reactor. The reaction medium is then cooled to a temperature below 10°C, particularly below 5°C, typically to 0°C, and then an acidic solution of alcohol R1OH, typically a solution of hydrochloric acid in R1OH, is gradually added. The reaction medium is kept stirred at room temperature (i.e., at a temperature between 20°C and 25°C) for typically more than 5 hours, preferably for a time between 10 hours and 20 hours. Prior to step c2), the reaction medium is cooled to a temperature below 10°C, particularly between 0°C and 5°C.

[0237] Thus, according to the one-pot embodiment, steps c1) and c2) can be easily performed. Advantageously, the triester of formula (VII) is not isolated between steps c1) and c2).

[0238] However, in order to promote the aminolysis reaction, in step c2), the alcohol of formula R1OH is preferably removed by vacuum distillation.

[0239] Within the meaning of the present invention, the term "vacuum distillation" refers to the distillation of the mixture carried out at a pressure between 10 and 500 mbar, in particular between 10 and 350 mbar, preferably between 10 and 150 mbar, in particular between 50 and 100 mbar.

[0240] Similarly, in order to promote the aminolysis reaction, in step c2), a large amount of 3-amino-1,2-propanediol is introduced. Typically, relative to the amount of material of the diastereoisomer-enriched gadolinium hexahydrate complex of formula (I) initially introduced in step c) (which itself corresponds to 1 equivalent), the amount of material of the introduced 3-amino-1,2-propanediol is greater than 4 eq., particularly greater than 7 eq., advantageously greater than 10 eq.

[0241] Surprisingly, despite the acidic conditions typically employed in steps c1) and c2) which increase the kinetic instability of the gadolinium complex, no decomplexation or isomerization of the triester of formula (VIII) was observed. The desired triamide was obtained with a very good degree of conversion, and the absolute configuration of the three asymmetric carbon atoms located in the alpha position of the side chain relative to the nitrogen atom of the macrocycle was preserved.

[0242] Furthermore, it should be noted that amidation reactions via direct reaction between esters and amines are generally rarely described in the literature (on this subject see KC Nadimpally et al., Tetrahedron Letters, 2011, 52, 2579-2582).

[0243] In a preferred embodiment, step c) comprises the following consecutive steps:

[0244] c1) forming a methyl triester of formula (IV),

[0245]

[0246] In particular, the reaction is carried out in methanol in the presence of an acid such as hydrochloric acid, and

[0247] c2) Aminelysis of the methyl triester of the formula (IV) with 3-amino-1,2-propanediol, in particular in the presence of an acid such as hydrochloric acid in methanol.

[0248] Advantageously, the methyl triester of formula (IV) is not isolated between steps c1) and c2).

[0249] In a preferred embodiment, in step c2), the methanol is removed by vacuum distillation until a temperature of typically above 55°C, in particular between 60°C and 65°C is reached, and the reaction medium is kept at this temperature under vacuum for a period of typically greater than 5 hours, in particular between 10 and 20 hours, then cooled to room temperature and diluted with water.

[0250] The present invention includes all combinations of the above-mentioned particular, advantageous or preferred embodiments in connection with each step of the method.

[0251] ■ Preparation of hexacarboxylic acid having formula (III)

[0252] The hexaacid of formula (III) which participates in step a) of the process for preparing the complex of formula (II) according to the invention can be prepared according to any known method, in particular according to the method described in EP 1 931 673.

[0253] However, according to a preferred embodiment, the hexaacid of formula (III) is obtained by: a pyclene of formula (V):

[0254]

[0255] With the formula R3OOC-CHG p -(CH2)2-COOR4(IX) compounds are alkylated,

[0256] in:

[0257] - R3 and R4 independently of one another represent a (C3-C6)alkyl group, in particular a (C4-C6)alkyl group, for example a butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl group, and

[0258] -Gp represents a leaving group, such as a tosylate or triflate group, or a halogen atom, preferably a bromine atom,

[0259] To obtain hexaester having formula (X)

[0260]

[0261] A hydrolysis step is then carried out to obtain the hexaacid of formula (III).

[0262] In a preferred embodiment, R3 and R4 are the same.

[0263] According to an advantageous embodiment, the hexaacid of formula (III) is obtained by: a pyclene of formula (V):

[0264]

[0265] Alkylation with dibutyl 2-bromoglutarate to give the butyl hexaester having the formula (VI):

[0266]

[0267] A hydrolysis step is then carried out to obtain the hexaacid of formula (III).

[0268] The dibutyl 2-bromoglutarate is used in racemic or enantiomerically pure form, preferably in racemic form.

[0269] Compared with using the 2-bromoglutarate ethyl ester described in EP 1 931 673, using 2-bromoglutarate dibutyl ester is particularly advantageous. Specifically, the commercially available 2-bromoglutarate diethyl ester is a relatively unstable compound, which degrades over time and under the effect of temperature. More precisely, this ester has a tendency to be hydrolyzed or cyclized and therefore lose its bromine atom. Purification of the commercially available 2-bromoglutarate diethyl ester or developing new synthetic routes to obtain this ester with higher purity and therefore prevent the attempt of its degradation are all unsuccessful.

[0270] The alkylation reaction is typically carried out in a polar solvent, preferably in water, in particular in deionized water, advantageously in the presence of a base such as potassium carbonate or sodium carbonate.

[0271] For obvious reasons, preference is given to using water and particular preference to using acetonitrile, as described in EP 1 931 673.

[0272] The reaction is advantageously carried out at a temperature between 40 and 80°C, typically between 50 and 70°C and in particular between 55 and 60°C, for a time between 5 and 20 hours, specifically between 8 and 15 hours.

[0273] The hydrolysis step is advantageously carried out in the presence of an acid or a base, advantageously in the presence of a base such as sodium hydroxide. The hydrolysis solvent may be water, an alcohol such as ethanol or a water / alcohol mixture. The step is advantageously carried out at a temperature between 40° C. and 80° C., typically between 40° C. and 70° C. and in particular between 50° C. and 60° C., typically for a time between 10 hours and 30 hours, in particular between 15 hours and 25 hours.

[0274] Method for purifying a complex having formula (II)

[0275] The present invention also relates to a method for purifying the following complex having formula (II):

[0276]

[0277] The complex has a diastereomeric excess of at least 80% and comprises a mixture of isomers II-RRR and II-SSS having the following formula:

[0278]

[0279] The method includes:

[0280] 1) A combination of the following two steps:

[0281] 1b) through one or more ion exchange resins, and

[0282] 1c) ultrafiltration of the complex, and

[0283] 2) Isolating the purified complex thus obtained in the form of a solid.

[0284] Advantageously, the complex of formula (II) is previously obtained according to the preparation process described previously, having a diastereomeric excess of at least 80%, preferably at least 85%, in particular at least 90%, specifically at least 95%, more specifically at least 97%, preferably at least 98% and advantageously at least 99% of a mixture comprising isomers II-RRR and II-SSS.

[0285] In a preferred embodiment, the diastereoisomerically enriched complex on which the purification process is carried out has a diastereomeric excess of at least 85%, in particular at least 90%, specifically at least 92%, preferably at least 94%, advantageously at least 97%, more advantageously at least 99% of the mixture comprising isomers II-RRR and II-SSS.

[0286] Preferably, the diastereomeric excess consists of at least 70%, in particular at least 80%, advantageously at least 90%, preferably at least 95% of the mixture of isomers II-RRR and II-SSS.

[0287] Advantageously, said diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0288] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers the situation where only one isomer is present, whether II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all situations where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0289] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture in a ratio between 65 / 35 and 35 / 65, in particular between 60 / 40 and 40 / 60, specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture in a ratio of 50 / 50.

[0290] ■ Combination of steps 1b) and 1c)

[0291] Steps 1b) and 1c) involve purifying the complex of formula (II) by removing impurities that may be present as a result of its production process.

[0292] The impurities may include in particular 3-amino-1,2-propanediol and / or double coupling impurities.

[0293] In particular, 3-amino-1,2-propanediol may be present in the final product obtained during the implementation of the method for preparing the complex of formula (II), typically when the complex of formula (II) is obtained by amidation starting with the complex of formula (I) and 3-amino-1,2-propanediol. This is particularly true for the method for preparing the complex of formula (II) according to the invention. As previously detailed, the amidation reaction may comprise activating three carboxylic acid functions carried by carbon atoms located in the γ position on the side chain of the complex of formula (I) relative to the nitrogen atom of the macrocycle on which the side chain is grafted, and then amination of the activated carboxylic acid functions by reaction with 3-amino-1,2-propanediol. 3-amino-1,2-propanediol is then advantageously used in excess to ensure good conversion into amide functions of the three activated carboxylic acid functions.

[0294] The term "doubly coupled impurity" is intended to represent a complex having the formula (II-dc-a), (II-dc-b), (II-dc-c), or a mixture thereof as shown below:

[0295]

[0296] The double coupling impurities may be caused in particular by the hydrolysis reaction of the amide functional groups of the complex of formula (II). When the preparation of the complex of formula (II) involves such a step, this may also be caused by incomplete activation of the carboxylic acid functional groups of the complex of formula (I) (two of the three functional groups are activated) or incomplete aminolysis of the activated carboxylic acid functional groups (two of the three functional groups are aminolyzed). This is particularly true for the process according to the invention for the preparation of the complex of formula (II).

[0297] ■ Step 1b) corresponds to passing the diastereomerically enriched complex of formula (II) as previously described over one or more ion exchange resins.

[0298] Within the meaning of the present invention, the term "ion exchange resin" refers to a solid material, generally in the form of beads, consisting of a polymer matrix, onto which are grafted positively charged functional groups (anion resins) or negatively charged functional groups (cation resins), which make it possible to capture anions or cations, respectively, by adsorption. The adsorption of anions or cations on the resin takes place by ion exchange between the counterions of the functional groups initially present, so as to ensure the electrical neutrality of the resin and the anions or cations to be captured.

[0299] Step 1b) involves contacting the diastereomerically enriched aqueous solution of the complex of formula (II) with a strong anionic resin. The water used is preferably purified water.

[0300] The strong anion resin typically includes ammonium groups (N(RR'R") as exchange functional groups. + , in which R, R' and R" are identical or different (C1-C6)alkyl groups). Mention may be made in particular of the resins sold by Dow Chemical FPA900, preferably HO - form.

[0301] Passage through a strong anionic resin enables at least partial removal of the doubly coupled impurities.

[0302] Step 1b) may also involve contacting the diastereomerically enriched aqueous solution of the complex of formula (II) with a weak cationic resin. The water used is preferably purified water.

[0303] The weak cationic resin typically includes carboxylate groups (CO2 - Mention may be made in particular of the resins sold by Dow Chemical. HP336, advantageously H + form.

[0304] Passage through a weak cationic resin enables at least partial removal of 3-amino-1,2-propanediol and possibly Gd 3+ Residue.

[0305] It should be noted that step 1b) is made possible by increasing the stability of the diastereomerically enriched complex of formula (II) according to the invention by passing it through one or more ion exchange resins, thus maintaining the integrity of the complex during this step.

[0306] ■ Step 1c) corresponds to an ultrafiltration of the diastereomerically enriched complex of formula (II) as previously described.

[0307] In the present invention, the term "ultrafiltration" refers to a method of filtering through a mesoporous semipermeable membrane, the pores of which typically have a diameter (mesopores) of between 1 and 100 nm, in particular between 2 and 50 nm, especially between 10 and 50 nm, under the action of a force (e.g. a pressure gradient typically between 1 and 10 bar) and, if necessary, a concentration gradient. Thus, this is a membrane separation process by which particles in a solution or suspension whose size is greater than the pore size are retained by the membrane and separated from the liquid mixture containing them.

[0308] In the context of the purification method according to the invention, ultrafiltration is particularly advantageous for removing endotoxins.

[0309] Advantageously, the ultrafiltration membrane used in step 1c) has a 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.

[0310] Preferably, in step 1c), the transmembrane pressure is between 1 and 5 bar, in particular between 2.25 and 3.25 bar.

[0311] ■ In one embodiment, steps 1b) and 1c) are also combined with the nanofiltration step 1a).

[0312] In the present invention, the term "nanofiltration" refers to a method of filtering through a porous semipermeable membrane, the pores of which typically have a diameter of between 0.1 and 100 nm, in particular between 0.1 and 20 nm, especially between 1 and 10 nm, under the action of a force (e.g. a pressure gradient typically between 1 and 50 bar) and, if necessary, a concentration gradient. Thus, this is a membrane separation process by which particles in a solution or suspension whose size is greater than the pore size are retained by the membrane and separated from the liquid mixture containing them.

[0313] The nanofiltration step 1a) makes it possible to remove most of the excess 3-amino-1,2-propanediol, if desired in the form of a salt, in particular the hydrochloride, or in the form of a derivative, in particular the acetamide derivative, and mineral salts.

[0314] In this embodiment, the crude diastereomerically enriched complex of formula (II) obtained according to the previously described preparation method can be directly subjected to the nanofiltration step. In particular, there is no need to precipitate the previously prepared diastereomerically enriched complex of formula (II) by adding a solvent.

[0315] Advantageously, the nanofiltration membrane used in step 1a) has a cut-off threshold of less than 1 kD, in particular less than 500 Daltons, specifically less than 300 Daltons, typically a cut-off threshold of 200 Daltons.

[0316] Preferably, in step 1a), the transmembrane pressure is between 10 and 40 bar, in particular between 2 and 30 bar.

[0317] In particular, the temperature of the solution of the complex of formula (II) subjected to ultrafiltration in step 1a) is between 20 and 40°C, in particular between 25 and 35°C.

[0318] In an alternative to this embodiment, step 1b) does not involve contacting the diastereomerically enriched aqueous solution of the complex of formula (II) with a weak cationic resin.

[0319] In one particular embodiment, steps 1a) (when present), 1b) and 1c) are carried out in this order. This advantageous embodiment makes it possible in particular to minimize the amount of resin used and therefore the industrial manufacturing costs.

[0320] ■Step 2)

[0321] Step 2) involves isolating in solid form the purified complex of formula (II) obtained at the end of the combination of steps 1b) and 1c) (and optionally also with step 1a).

[0322] This solid form isolation step can be carried out according to any method well known to a person skilled in the art, in particular by nebulization, by precipitation, by lyophilization or by centrifugation, advantageously by nebulization.

[0323] In a preferred embodiment, step 2) comprises atomization.

[0324] In particular, the isolation of the purified complex of the formula (II) in solid form by atomization makes it possible in particular to dispense with the use of precipitation solvents.

[0325] The air inlet temperature in the atomizer is then typically between 150 and 180° C., in particular between 160 and 175° C., advantageously between 165 and 170° C. The outlet temperature itself is typically between 90 and 120° C., preferably between 105 and 110° C.

[0326] Advantageously, the purity of the purified complex of formula (II) of the mixture of diastereomerically enriched isomers II-RRR and II-SSS separated at the end of step 2) is greater than 95%, in particular greater than 97%, preferably greater than 97.5%, more preferably greater than 98%, advantageously greater than 99%, expressed as a percentage by mass of the complex of formula (II) relative to the total mass obtained at the end of step 2).

[0327] The present invention also relates to a diastereomerically enriched and purified complex of formula (II), which is obtainable according to the purification process of the present invention.

[0328] Preferably, the complex of formula (II) comprised in the composition according to the invention described previously is a diastereomerically enriched and purified complex of formula (II), obtainable according to the purification method of the invention.

[0329] Examples

[0330] The examples given below are presented as non-limiting illustrations of the invention.

[0331] The isomeric groups iso1, iso2, iso3 and iso4 of the complex having formula (II) are separated by UHPLC

[0332] A UHPLC machine consisting of a pump system, an injector, a chromatography column, a UV detector, and a data station was used. The chromatography column used was a UHPLC 150×2.1 mm-1.6 μm column (Waters UPLC T3 column).

[0333] -Mobile phase:

[0334] Route A: 100% acetonitrile and Route B: 0.0005% v / v H2SO4 (96%) in water

[0335] -Preparation of test solution:

[0336] 2 mg / mL solution of the complex of formula (II) in purified water

[0337] -Analysis conditions:

[0338]

[0339]

[0340] -gradient:

[0341] time %Acn <![CDATA[%H2SO4 0.0005%]]> 0 1 99 3 5 95 12 10 90 15 25 75 16 1 99 20 1 99

[0342] Four main peaks were obtained. Peak 4 (i.e., iso4) of the UHPLC graph corresponds to a retention time of 6.3 minutes. Preparation of butyl hexaester having formula (VI)

[0343] 184 kg (570 mol) of dibutyl 2-bromoglutarate and 89 kg (644 mol) of potassium carbonate were mixed in a reactor and heated to 55-60° C. An aqueous solution of 29.4 kg (143 mol) of pyclene in 24 kg of water was added to the aforementioned preparation. The reaction mixture was maintained at 55-60° C. and then refluxed for about 10 hours. After the reaction, the medium was cooled, diluted with 155 kg of toluene, and then washed with 300 liters of water. Butyl hexaester was extracted into the aqueous phase with 175 kg (1340 mol) of phosphoric acid (75%). It was then washed 3 times with 150 kg of toluene. The butyl hexaester was extracted into the toluene phase by diluting with 145 kg of toluene and 165 kg of water, and then alkalized with 30% sodium hydroxide (m / m) to reach a pH of 5-5.5. The lower aqueous phase was removed. Butyl hexaester was obtained by vacuum concentration to dryness at 60° C. with a yield of about 85%.

[0344] Preparation of hexacarboxylic acid having formula (III)

[0345] 113 kg (121 mol) of butyl hexaester was placed in a reactor together with 8 kg of ethanol. The medium was raised to 55 ± 5 ° C, and then 161 kg (1207.5 mol) of 30% sodium hydroxide (m / m) was added within 3 hours. The reaction mixture was kept at this temperature for about 20 hours. Butanol was then removed by decanting the reaction medium. The hexaacid of formula (III) obtained in the form of a sodium salt was diluted with water to obtain an aqueous solution of about 10% (m / m). The solution was treated on an acidic cationic resin. The hexaacid of formula (III) in an aqueous solution was obtained with a yield of about 90% and a purity of 95%.

[0346] Preparation of gadolinium hexahydrate complex having formula (I)

[0347] Experimental plan

[0348] ● Complexation and isomerization

[0349] - No acetic acid

[0350] 418 kg (117 kg of pure hexaacid of formula (III) / 196 mol) of a 28% by weight aqueous solution of hexaacid of formula (III) are placed in a reactor. The pH of the solution is adjusted to 2.7 by adding hydrochloric acid, and then 37 kg (103.2 mol) of gadolinium oxide are added. The reaction medium is heated at 100-102° C. for 48 hours to achieve the desired isomer distribution of hexaacid of formula (III).

[0351] -Contains acetic acid

[0352] Gadolinium oxide (0.525 molar equivalents) was suspended at 28.1% by mass in a solution of the hexaacid of formula (III).

[0353] 99-100% acetic acid (50% by mass / pure hexaacid of formula (III)) is poured into the medium at room temperature.

[0354] The medium is heated to reflux and then distilled up to 113° C. by gradually refilling the medium by mass with acetic acid while removing the water. Once a temperature of 113° C. has been reached, sufficient acetic acid is added to reach the starting volume.

[0355] The medium is kept at 113° C. overnight.

[0356] ●Crystallization, recrystallization

[0357] -crystallization

[0358] The hexa-acid gadolinium complex of formula (I) in solution is cooled to 40°C, the primer is added, and the reagents are allowed to contact for at least 2 hours. The product is then isolated by filtration at 40°C and washed with permeate water.

[0359] - Recrystallization

[0360] 180 kg of the previously obtained hexacarboxylic acid gadolinium complex of formula (I) (solid content of about 72%) are suspended in 390 kg of water. The medium is heated to 100° C. to dissolve the product and then cooled to 80° C. to pre-treat by adding a small amount of primer. After cooling to room temperature, the hexacarboxylic acid gadolinium complex of formula (I) is isolated by filtration and drying.

[0361] ●Selective decomplexation

[0362] The dried product is placed in a reactor together with 20° C. permeate water. The mass of water added is equal to twice the theoretical mass of the hexaacid gadolinium complex of formula (I). 30.5% sodium hydroxide (m / m) (6.5 equivalents) is poured into the medium at 20° C. At the end of the addition of NaOH, the medium is kept in contact at 50° C. for 16 hours. The medium is cooled to 25° C. and the product is filtered on a Clarcel pad.

[0363] ■Content of the mixture of diastereoisomers I-RRR and I-SSS

[0364] The ratios in which the various isomers of the complex having formula (I) are present in the mixture of diastereoisomers depend on the conditions under which the complexation and isomerization steps are carried out, as can be seen in Table 3 below.

[0365]

[0366] [Table 3]: Content of I-RRR and I-SSS mixtures as a function of complexation / isomerization conditions

[0367] Additional steps of recrystallization and selective decomplexation made it possible to increase the diastereomeric excess of the mixture of I-RRR and I-SSS (see Table 4).

[0368]

[0369] [Table 4]: Content of I-RRR and I-SSS mixture after crystallization / recrystallization / selective decomplexation Preparation of complex having formula (II)

[0370] 90 kg (119 mol) of the hexaacid complex of formula (I) and 650 kg of methanol are placed in a reactor. The mixture is cooled to about 0°C and then 111 kg (252 mol) of a methanol solution of hydrochloric acid (8.25% HCl in methanol) are poured, while maintaining the temperature at 0°C. The reaction medium is allowed to reach room temperature and stirring is then continued for 16 hours. After cooling to 0-5°C, 120 kg (1319 mol) of 3-amino-1,2-propanediol are added. The reaction medium is then heated while methanol is distilled off under vacuum until a temperature of 60-65°C is reached. The concentrate is kept under vacuum at this temperature for 16 hours. At the end of the contact, the medium is diluted with 607 kg of water while cooling to room temperature. The solution of the crude complex of formula (II) is neutralized with 20% hydrochloric acid (m / m). 978.6 kg of a solution having a concentration of 10.3% is thus obtained, representing 101 kg of material. The yield obtained was 86.5% and the purity of the complex of formula (II) was 92.3% (HPLC s / s). The amount of double coupled impurity was 6.4% (HPLC s / s).

[0371] Purification of the complex having formula (II)

[0372] Nanofiltration

[0373] The nanofiltration membrane used had a cut-off threshold of 200 Daltons (Koch Membran System SR3D). The treatment was carried out in the following manner:

[0374] The solution of the crude complex of formula (II) was heated to 30°C. The nanofilter was filled with the solution. The pump was first turned on at a low rate to purge the system, and then the nanofilter pump rate was gradually increased to the desired recirculation rate (1.0 m / s for a 2.5×40 inch membrane). 3 / h). The system is then placed in full circulation at 30°C for at least 2 hours to establish the polarization layer. The medium is then diafiltered at 30°C and 2.5 bar, while keeping the volume constant by adding pure water, until the conductivity of the retentate is less than 1000 μS. At the end of the diafiltration, the medium is concentrated to obtain a concentration of about 40% (m / m).

[0375] ●Resin treatment

[0376] The solution of the complex of formula (II) obtained from the nanofiltration was diluted with purified water under stirring to obtain a 15% solution (m / m). - form of strong anion resin (FPA900), then 50 liters of H + The series of elutions were carried out on a weak cation resin (HP336) in the form of 2V / V / H (2 volumes of solution / resin volume / hour). The resin was then washed with about 450 liters of pure water until a refractive index of less than 1.3335 was obtained.

[0377] The solution of the complex of formula (II) was then concentrated by heating to 50-60° C. under a vacuum of 20 mbar to reach a concentration of 35% (m / m).

[0378] Ultrafiltration

[0379] The ultrafiltration membrane was UF 10KD Koch spiral membrane.

[0380] The ultrafilter was fed with a 35% solution of the complex of formula (II) previously heated to 40°C. 3 The ultrafiltration was carried out at a flow rate of 1 / h and a transmembrane pressure of 2.5-3 bar. The system was flushed several times with 13 liters of pyrogen-free pure water until a final dilution of 25% (m / m) of the complex of formula (II) was reached.

[0381] Atomization

[0382] The complex of formula (II) is obtained in powder form by atomizing a solution of the complex of formula (II) previously concentrated to 25%.

[0383] Nebulization is performed in the following manner:

[0384] The nebulizer was equilibrated with pyrogen-free purified water by setting the inlet temperature to 165-170°C and adjusting the feed rate to bring the outlet temperature between 105 and 110°C.

[0385] The concentrated solution of the complex having formula (II) is then added and the flow rate is adjusted to maintain the above parameters.

[0386] These operating conditions are maintained throughout the atomization process, while ensuring good behavior of the powder in the atomization chamber and at the atomizer outlet. In particular, it should be ensured that there is no product adhesion.

[0387] At the end of the delivery of the solution to the nebulizer, the container of the complex of formula (II) and the nebulizer are rinsed with pyrogen-free purified water until maximum powder recovery is achieved.

[0388] The complex of formula (II) was obtained in a purity of 99.6%.

[0389] The purity was determined by reverse phase liquid chromatography.

[0390] Composition according to the present invention and study results thereof

[0391] ●Example of the manufacturing method according to the present invention

[0392] The method for producing the composition according to the invention is carried out according to the following steps:

[0393] a) Dissolve 485.1 g (i.e. 0.5 M) of the complex of formula (II) in water (q.s., 1 liter), heat the pot to a temperature between 39° C. and 48° C., and stir the solution vigorously until the complex is completely dissolved in the water. The solution is then cooled to about 30° C.

[0394] b) 0.404 g (ie 0.2 mol / mol % relative to the complex added in step a)) of DOTA (Simafex, France) were added to the solution obtained in step a) via a 10% m / v DOTA solution under stirring.

[0395] c) Adding tromethamine (Tris) to the solution obtained in step b) under stirring. The pH is then adjusted to a value between 7.2 and 7.7 by adding hydrochloric acid solution under stirring.

[0396] d) The target concentration (0.5 mol / L) was obtained by adding water for injection in two steps until a density value between 1.198 and 1.219 g / mL was reached.

[0397] The liquid composition was then filtered through a polyethersulfone membrane and placed in its final container, which was terminally sterilized at 121°C for 15 minutes.

[0398] • Examples of compositions according to the invention

[0399] The following preparations were obtained by the above method:

[0400]

[0401]

[0402] *Measured by colorimetry with xylenol orange

[0403] **Expressed in anhydrous pure form

[0404] ●Testing of formulations performed

[0405] Various concentrations of tromethamine were tested, ranging from 0 to 100 mM. The results of these tests indicated that a level of 10 mM (0.12% w / v) was sufficient to ensure pH stability of the formulation while limiting the formation of degradation impurities.

[0406] Various concentrations of DOTA from 0 to 2.5 mM were tested. The results of these tests show that a content of 1 mM (corresponding to 0.04% m / v or 0.2 mol / mol%) makes it possible to ensure that no free Gd is released during the process and during the service life of the product.

[0407] ● Stability studies of the compositions according to the invention under accelerated conditions

[0408] The formulations of the foregoing examples were analyzed after manufacture (T0) and after storage at 40°C for 6 months after manufacture (T+6 months).

[0409] At T0:

[0410] -Purity assessed by chromatography*: 99.6%

[0411] -Gd-DOTA concentration: 0.007% (m / V)

[0412] -Gd concentration: less than 0.0001% (m / V)

[0413] -pH: 7.5

[0414] At T+6 months:

[0415] -Purity assessed by chromatography*: 97.2%

[0416] -Gd-DOTA concentration: 0.014% (m / V) - 0.25 mM

[0417] -Gd concentration: less than 0.0001% (m / V)

[0418] -pH: 7.5

[0419] * Reversed phase liquid chromatography

[0420] These results demonstrate that the formulation has good stability over time.

[0421] ●Comparative stability study

[0422] The stability of the following compositions was evaluated over time. The term "non-optimized AP" stands for the active ingredient, i.e. the complex of formula (II) obtained according to the method described in EP 1 931 673. The term "optimized AP" stands for the diastereoisomerically enriched and purified complex of formula (II) obtained by the method according to the invention.

[0423]

[0424]

[0425] *LC Formate: Chromatographic method involving fluorescence detection. Separation was performed on a reverse phase C18 grafted column eluted in gradient mode.

[0426] The results reported above indicate that it is not possible to formulate unoptimized APs with free DOTA. The reason is that the chelating excipient is completely consumed by the trans-ligation reaction between the complex of formula (II) and DOTA and is therefore no longer available to capture the released Gd 3+ role.

[0427] On the other hand, the diastereomerically enriched and purified complex of formula (II) obtained according to the process of the invention can be formulated with free DOTA. In particular, it was observed that no free Gd was present in the composition at 40°C for 6 months, regardless of the pH of the formulation and the presence or absence of buffer substances. In addition, the consumption of chelating excipients is very low, since it does not exceed 0.08 mol / mol%.

Claims

1. A complex having the following formula (II): Consists of at least 90% diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS having the formula:

2. A composition comprising the complex according to claim 1, and free macrocyclic ligand, and advantageously having a free gadolinium concentration of less than 1 ppm (m / v).

3. The composition according to claim 2, characterized in that The composition comprises between 0.002 and 0.4 mol / mol % of free macrocyclic ligand relative to the complex having formula (II).

4. The composition according to claim 2 or 3, characterized in that The free macrocyclic ligand is selected from the group consisting of DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA and derivatives thereof.

5. The composition according to claim 4, characterized in that The free macrocyclic ligand is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

6. A method for purifying a complex having the following formula (II): The complex is constituted by a diastereomeric excess of at least 80%, said diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS having the following formula: The method comprises: 1) A combination of the following two steps: 1b) through one or more ion exchange resins, and 1c) ultrafiltration of the complex, and 2) Isolating the purified complex thus obtained in the form of a solid.

7. The method according to claim 6, characterized in that Steps 1b) and 1c) are also combined with the nanofiltration step 1a).

8. The method according to claim 6 or 7, characterized in that: When step 1a) is present, steps 1a), 1b) and 1c) are performed in this order.

9. The method according to any one of claims 6 to 8, characterized in that Step 2) includes atomization.

10. The method according to any one of claims 6 to 9, characterized in that The complex of formula (II) consisting of at least 80% diastereomeric excess, comprising a mixture of isomers II-RRR and II-SSS, on which the purification process is carried out is previously obtained by the following successive steps: a) a hexacarboxylic acid having the following formula (III): Complexed with gadolinium to obtain a gadolinium hexahydrate complex having the following formula (I): b) isomerizing the hexacarboxylic acid gadolinium complex of formula (I) by heating in aqueous solution at a pH between 2 and 4 to obtain a diastereomerically enriched complex consisting of at least 80% of a diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS of the hexacarboxylic acid gadolinium complex of formula (I), and c) starting from the diastereomerically enriched complex obtained in step b), forming the complex of formula (II) by reaction with 3-amino-1,2-propanediol.

11. The method according to claim 10, characterized in that: - at the end of step b), the diastereomerically enriched complex is separated by crystallization, purified by recrystallization and further enriched by selective decomplexation of diastereomers of the complex of formula (I) other than the diastereomers I-RRR and I-SSS, i.e. by selective decomplexation of the diastereomers I-RSS, I-SRR, I-RSR, I-SRS, I-RRS and I-SSR, and - Step c) comprises the following consecutive steps: c1) forming a triester having the formula (VIII), wherein R1 represents a (C1-C6) alkyl group, In particular by reacting in the presence of an acid such as hydrochloric acid in an alcohol of formula R1OH, and c2) aminolysis of a triester of formula (VIII) with 3-amino-1,2-propanediol, Especially in the presence of an acid such as hydrochloric acid in an alcohol of formula R1OH, The triester of formula (VIII) is not isolated between steps c1) and c2).

12. A complex having formula (II): It consists of a diastereomeric excess of at least 80% comprising a mixture of isomers II-RRR and II-SSS having the following formula: It can be obtained by the method according to any one of claims 6 to 11.

13. The complex according to claim 12, characterized in that Its purity is greater than 95%.

14. A composition comprising the complex according to claim 12 or 13, and free DOTA, and advantageously having a free gadolinium concentration of less than 1 ppm (m / v).

15. The composition according to claim 14, characterized in that The composition comprises between 0.002 and 0.4 mol / mol % DOTA relative to the complex having formula (II).

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