Complexes of gadolinium and chelating ligands based on diastereomerically enriched pcta and methods of their preparation and purification

Abstract

The present invention relates to a complex of formula (II) consisting of at least 90% diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS of formula: The present invention also relates to a process for the preparation and purification of said complex of formula (II) and also to a composition comprising said complex.

Description

[0001] Related applications

[0002] This invention is a divisional application of patent application No. 202080009625.8, filed on January 17, 2020, entitled “Complex of gadolinium and chelate ligands based on diastereomer-enriched PCTA and its preparation and purification method”. Technical Field

[0003] This invention relates to a novel method for preparing and purifying complexes of gadolinium and diastereomer-enriched PCTA chelate ligands, which preferably yields stereoisomers of the complexes, possessing particularly advantageous physicochemical properties for use as contrast agents in medical imaging, especially in magnetic resonance imaging. The invention also relates to the diastereomer-enriched complexes themselves, compositions comprising the complexes, and a method for preparing the corresponding chelate ligands by decomplexing the complexes, as well as the ligands themselves. Background Technology

[0004] Many contrast agents based on lanthanides (paramagnetic metals), particularly gadolinium (Gd), are known, as described in, for example, US 4647447. These products are generally grouped under the term GBCA (gadolinium-based contrast agents). Several products are marketed, including macrocyclic chelates such as gadolinium gadolinate based on DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid), gadobutrol based on DO3A-butrol, gadoterol based on HPDO3A, and linear chelates, particularly linear chelates 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 polyaza-macrocyclic carboxylic acids (EP 0 438 206) or PCTA derivatives (i.e., derivatives containing at least the chemical structure of 3,6,9,15-tetraazabicyclic[9,3,1]pentadecan-1(15),11,13-triene-3,6,9-triacetic acid), as described in EP 1 931 673.

[0006] The PCTA-based chelate complexes described in EP 1 931 673 have particular advantages: they are relatively easy to synthesize chemically, and they also exhibit a relaxation rate superior to other GBCAs currently on the market (relaxation rate r1 can reach up to 11-12 mM in water). -1 .s -1This relaxation rate corresponds to the efficiency of these products, and therefore to their comparative ability.

[0007] In living organisms, chelates (or complexes) of lanthanides (and especially gadolinium) exist in chemical equilibrium (via their thermodynamic constant K). therm Characterization), which may lead to the undesirable release of the lanthanides (see reaction 1 below):

[0008]

[0009] Chelates or ligands (Ch) with lanthanides (L) n The complexation chemical equilibrium between the two produces the complex Ch-L. n .

[0010] Since 2006, a condition known as NSF (renal systemic fibrosis or fibrotic skin disease) has been linked at least in part to the release of free gadolinium in the body. This condition has led health authorities to issue alerts regarding the sale of gadolinium-based contrast agents to certain patient groups.

[0011] Therefore, several strategies have been developed to address the complexities of patient tolerability in a completely safe manner and to limit or even eliminate the risk of undesirable release of lanthanides after administration. This problem is further complicated by the frequent re-administration of contrast agents, both during diagnostic examinations and in dose adjustments and monitoring of treatment efficacy.

[0012] Furthermore, since 2014, there have been reports of gadolinium deposition in the brain following repeated administration of gadolinium-based products (especially linear gadolinium chelates), particularly gadolinium macrocyclic chelates (e.g. Such deposition has been rarely or never reported. Therefore, given the lack of stability, countries have decided to withdraw most linear chelating agents from the market or significantly restrict their use.

[0013] Therefore, the first strategy to limit the risk of lanthanide release into the body is to select 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 tolerance of lanthanide (especially gadolinium) chelates are described in the prior art. US 5876695, reported more than 30 years ago, describes, for example, formulations containing additional complexing agents in addition to lanthanide chelates, intended to release lanthanides (Gd) through complexation. 3+Additional chelating agents (metal ions) are used to prevent the unwanted release of lanthanides from the body. These additional chelating agents can be introduced into the formulation either in their free form or as weak complexes (typically calcium, sodium, zinc, or magnesium). Although it may differ in composition from the active complex's ligands, it is important that the complex it forms with the released lanthanides is less stable than the active complex, thus preventing transposition reactions between the active complex and the additional chelate. This would particularly have the effect of completely consuming the additional ligand, thus preventing further capture of the released lanthanides. This risk of consuming the additional chelating agent through transposition is more pronounced when it is added in its free form, rather than as, for example, a calcium complex.

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

[0016] However, although PCTA-based chelate ligand complexes containing the pyclene-type structure described in EP 1 931 673 have good kinetic stability, their thermodynamic constants are generally lower than those of other macrocyclic complexes based on cycloalkenes.

[0017] This is especially true for complexes of formula (II) with the following representation:

[0018]

[0019] Specifically, as specifically described in WO 2014 / 174120, the thermodynamic equilibrium constant (also known as the 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 comparative 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 having formula (II) corresponds to several stereoisomers, particularly due to the presence of three asymmetric carbon atoms at the α-position on the side chain of the complex, relative to the nitrogen atom of the macrocycle branched to it. These three asymmetric carbons are marked with an asterisk (*) in formula (II) above.

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

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

[0023] Since each of the three asymmetric carbons can have either an R or S absolute configuration, complexes of formula (II) exist as a family of eight 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, complexes of formula (II) exist as a family of eight diastereomers.

[0024] As mentioned earlier, it is reasonable to use the term "family" because each of these families combines several stereoisomers together, especially due to the presence of asymmetric carbons in the aminopropylene glycol group.

[0025] However, since stereoisomers associated with the asymmetric carbon of the given aminopropylene glycol group will not be considered in the remainder of this 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 without distinction, without specifying that each corresponds to a family of stereoisomers.

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

[0027] They then discovered that these different isomer groups had different physicochemical properties and identified the isomer group, known as iso4, which consisted of a mixture of isomers II-RRR and II-SSS with the following formulas (II-RRR) and (II-SSS), as the most advantageous contrast agent for medical imaging.

[0028]

[0029]

[0030] Therefore, surprisingly, iso4 is characterized by its significantly superior thermodynamic stability compared to the diastereomeric mixtures obtained by the method described in EP 1 931673, which are complexes having formula (II). Specifically, its thermodynamic equilibrium constant K therm iso4 Equal to 10 18.7 (i.e. log(K) therm iso4 ( ) = 18.7), which has been determined by the methods described in Pierrard et al., Contrast Media Mol. Imaging, 2008, 3, 243-252 and Moreau et al., Dalton Trans., 2007, 1611-1620.

[0031] Furthermore, iso4 is the group of isomers with the best kinetic inertia (also known as kinetic stability) among the four groups isolated by the inventors. Specifically, the inventors evaluated its kinetic inertia by studying the decomplexation kinetics of the four isomers in an acidic aqueous solution (pH = 1.2) at 37°C. The half-life time values ​​(T) determined for each group of isomers are listed in Table 1 below. 1 / 2 This half-life corresponds to the time it takes for 50% of the initially present complex to dissociate according to the following decomposition reaction (reaction 2):

[0032]

[0033]

[0034]

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

[0036] For comparative purposes, gadobutrol or gadotate, as macrocyclic gadolinium complexes, exhibit kinetic inertia of 18 hours and 4 days, respectively, under the same conditions, while linear gadolinium complexes (such as gadodiamine or gadopentetate) dissociate instantaneously.

[0037] Furthermore, iso4 is chemically more stable than iso3. This is because the amide functional group of the complex having formula (II) is readily hydrolyzed. The hydrolysis of the amide functional group (reaction formula 3) leads to the formation of a bicoupled impurity, accompanied by the release of 3-amino-1,2-propanediol. The inventors investigated the hydrolysis kinetics of the complex having 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 that of iso3.

[0038]

[0039] Measurements of the relaxation rates (i.e., their efficiency as contrast agents) of the isomers showed that iso1, iso2, and iso4 were relatively comparable in contrast ability, while iso3 was less efficient (see Table 2).

[0040]

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

[0042] The inventors have successfully developed a novel method for the preparation and purification of complexes having formula (II), which enables the preferred acquisition of diastereomers II-RRR and II-SSS of the complexes having particularly advantageous physicochemical properties. The method according to the invention comprises the step of enriching the isomers by converting the least unstable stereoisomer to the most stable stereoisomer. Surprisingly, when carried out on the hexaacid intermediate complex rather than on the final complex, it makes it possible to obtain the most stable isomer of the complex having formula (II) in the vast majority of cases.

[0043] Compared to alternatives to preparing mixtures of stereoisomers, implementing a method that makes it possible to obtain the target diastereomer in most cases, allowing for subsequent attempts to separate the diastereomers using conventional techniques, thereby separating the target isomer using any separation technique known in the art. Specifically, in addition to the ease of implementation on an industrial scale without a diastereomer separation step, the absence of separation saves significant time and allows for increased overall method yield by minimizing the generation of unwanted diastereomers that will ultimately be eliminated. Furthermore, conventional separation techniques typically involve the use of large amounts of solvent, which exceeds financial costs and is undesirable for environmental reasons. Moreover, given the inherent health risks of occupational exposure to silica, which the International Agency for Research on Cancer classifies as carcinogenic to humans (Group 1), silica chromatography should be specifically avoided.

[0044] As previously stated, the method developed by the inventors for preparing complexes having formula (II) is based on the step of enriching isomers of the intermediate gadolinium hexaate complex having the following representation:

[0045]

[0046] The complex having formula (I) corresponds to several stereoisomers due to the presence of three asymmetric carbon atoms at the α-position on the side chain of the complex, relative to the nitrogen atom of the macrocycle branched to it. These three asymmetric carbon atoms are marked with an asterisk (*) in formula (I) above.

[0047] Since each of the three asymmetric carbons containing the carboxylic acid ester functional group can have either an R or S absolute configuration, the complex of formula (I) exists as 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 as four pairs of enantiomers that are diastereomers of each other.

[0048] The inventors have successfully separated and identified four unresolved peaks or isomer groups of complexes of formula (I) obtained according to the method described in EP 1 931 673 by high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), which correspond to four different elution peaks characterized by their retention times on the chromatograms, and will be referred to as isoA, isoB, isoC and isoD in the remainder of this specification.

[0049] IsoD crystallizes in water. X-ray diffraction analysis enabled the inventors to determine the crystal structure of this set of isomers and thus discover that they contain diastereomers I-RRR and I-SSS having the following formulas (I-RRR) and (I-SSS) representing complexes of formula (I).

[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 hexaate complex having formula (I) in isoD.

[0053] The synthesis of the complex having formula (II) specifically involves converting the carboxylic acid functional group of the intermediate hexaacid complex having 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 having formula (I).

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

[0055] Furthermore, the purification method developed by the inventors enables the preparation of complexes having the above formula (II) by means of the preparation method of complexes having the above formula (II), and significantly improves the impurity distribution.

[0056] Therefore, free macrocyclic ligands (e.g., free DOTA) can be used instead of calcium complexes of DOTA to formulate complexes with improved stability for diastereomeric enrichment and purification, as recommended in WO 2014 / 174120. From an industrial perspective, the use of free DOTA is particularly advantageous, in a sense making it possible to eliminate the methodological step for synthetic formulations described in WO 2014 / 174120, namely the addition of CaCl2. Summary of the Invention

[0057] Complexes having formula (II)

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

[0059]

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

[0061]

[0062]

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

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

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

[0066] Advantageously, the 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 encompasses the case where only one isomer is present, whether it is II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

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

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

[0070] Within the meaning of this invention, the term "UHPLC chromatogram" refers to a curve showing the change in concentration over time, as measured by a detector, after a mixture of compounds (in this case, isomers of the compounds) has passed through and been separated on a stationary phase for a given composition and a given eluent flow rate. A UHPLC chromatogram consists of various peaks or unresolved peak characteristics of the analyzed compound or mixture of compounds. Detailed Implementation

[0071] UHPLC method:

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

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

[0074] Preferably, the stationary phase used should be compatible with the aqueous flow 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 having formula (II) comprising at least 80% diastereomer excess, said diastereomer excess comprising a mixture of isomers II-RRR and II-SSS, and

[0082] -Free macrocyclic ligands.

[0083] In this specification, the terms "macrocyclic ligand" or "macrocyclic chelate" may be used indiscriminately.

[0084] In the context of this invention, the term "macrocycle" typically refers to a ring containing at least nine atoms, whether they are carbon atoms or heteroatoms, and the terms "macrocyclic ligand" or "macrocyclic chelate" refer to a polydentate, or at least bidentate, ligand.

[0085] In the context of this invention, the term "free macrocyclic ligand" refers to a macrocyclic ligand in its free form, i.e., one that does not complex with, in particular, metals (including lanthanides and actinides) or alkaline earth metal cations (e.g., calcium or magnesium). Specifically, as described in US 5876695, the free macrocyclic ligand is not in the form of a complex with gadolinium and is not introduced into the composition as a weak complex (typically calcium, sodium, zinc, or magnesium); however, trace amounts of the cation in the composition and thus the corresponding complex are not excluded.

[0086] As previously discussed, formulations of complexes of formula (II) having free macrocyclic ligands, rather than weak complexes of the macrocyclic ligands recommended in EP 1931 673, are made possible by improving the stability of complexes of formula (II) enriched with diastereomers according to the invention.

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

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

[0089] Advantageously, the 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 encompasses the case where only one isomer is present, whether it is II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0091] In a preferred embodiment, isomers II-RRR and II-SSS are present in the mixture at a ratio between 65 / 35 and 35 / 65, particularly between 60 / 40 and 40 / 60, and specifically between 55 / 45 and 45 / 55. Advantageously, isomers II-RRR and II-SSS are present in the mixture at 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, the terms “Gd”, “gadolinium”, and “Gd2” are used interchangeably unless otherwise stated. 3+ "to represent Gd" 3+ Ions. By extension, it can also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

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

[0095] Free gadolinium is typically determined by colorimetry, usually using xylenol orange or azoarsine(III). In the absence of metal ions (such as gadolinium), these indicators have specific colors: xylenol orange is yellow at acidic pH, while azoarsine is pink. In the presence of gadolinium, they turn purple.

[0096] Visually assessing changes in solution color allows for verification of the presence or absence of gadolinium in the solution.

[0097] Furthermore, it is possible to quantitatively measure free gadolinium in solution by back titration, for example, using EDTA as a “weak” gadolinium chelate. In this determination, a colored indicator is added until a purple color appears. Then, EDTA (the gadolinium ligand) is added dropwise to the mixture. Since EDTA is a stronger complexing agent than the colored indicator, gadolinium alters the ligand, causing the colored indicator to preferably complex with EDTA. Thus, the colored indicator gradually reverts to its non-complexed form.

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

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

[0100] Therefore, these colorimetric methods are typically performed in solutions with a pH between 4 and 8. This is because outside these pH ranges, the accuracy of the measurement may be affected by changes (or even suppression) in color change.

[0101] Therefore, if necessary, the pH of the sample to be tested should be adjusted to between 4 and 8. In particular, if the pH of the sample is acidic, and specifically less than 4, the pH can be advantageously adjusted by adding alkali, and then the free Gd in the sample can be measured at the adjusted pH.

[0102] Therefore, the compositions according to the invention exhibit stability over time, i.e., their composition remains consistent with the requirements regarding the concentration of free gadolinium (specifically, the concentration of free Gd remains less than 1 ppm (m / v)) for at least 3 years, preferably at least 4 years, or more preferably at least 5 years, particularly with respect to the content of free paramagnetic metal. According to ICH guidelines, observing stability at 40°C for 6 months is considered a good indicator of stability at 25°C for 3 years.

[0103] In one specific embodiment, the composition according to the invention has 0.01 and 1.5 mol·L⁻¹ -1 Between, preferably, 0.2 and 0.7 mol·L⁻¹ -1 Between, more preferably, 0.3 and 0.6 mol·L⁻¹ -1 The concentrations of the complexes with formula (II) between the above.

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

[0105] The content of the complex of formula (II) gives the composition optimal contrast while maintaining a satisfactory viscosity. Specifically, below 0.01 mol·L⁻¹. -1 The aforementioned complex of formula (II) is unsatisfactory in performance and quality as a comparative product, especially when the concentration is above 1.5 mol·L⁻¹. -1 At this point, the viscosity of the composition becomes too high and it becomes difficult to handle.

[0106] In one specific embodiment, the composition according to the invention comprises a free macrocyclic ligand in amounts between 0.002 and 0.4 mol / mol%, particularly 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%, relative to the complex having formula (II).

[0107] Advantageously, the macrocyclic ligands are selected from the group consisting of: DOTA, NOTA, DO3A, BT-DO3A, HP-DO3A, PCTA, DOTA-GA and their derivatives.

[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, well known to those skilled in the art, a solution containing a known initial concentration of copper sulfate, Q0, is preferably used, the concentration being greater than the amount of free ligand in the solution. A test solution containing a to-be-determined amount of free DOTA, Q1, is added to this copper sulfate solution. DOTA is an excellent copper complexing agent: therefore, the formation of a DOTA-copper complex is observed.

[0111] The free copper in the solution is then advantageously titrated using 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 mixture, as 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 solution potential is observed.

[0112] Given the initial amount of copper Q0 and the amount of added EDTA Q2, subtracting these two values ​​Q0-Q2 gives the amount of free DOTA in the test solution Q1.

[0113] Alternatively, HPLC methods, particularly HILIC LC-UV methods, can be used.

[0114] These measurements (specifically, potentiometric measurements) are performed in solutions with a pH advantageously between 4 and 8. Therefore, if necessary, the pH of the sample to be tested is adjusted to between 4 and 8. In particular, if the sample's pH is acidic, and specifically less than 4, the pH is advantageously adjusted by adding a base (e.g., meglumine), and then the free DOTA in the sample is measured at the adjusted pH.

[0115] Preferably, the proportions specified above in this invention are proportions prior to sterilization of the composition.

[0116] Advantageously, the pH of the composition is between 4.5 and 8.5, preferably between 5 and 8, advantageously between 6 and 8, and particularly between 6.5 and 8. These pH ranges particularly enable the restriction of the presence of certain impurities and promote the complexation of paramagnetic metal ions M.

[0117] Specifically, the compositions according to the invention can be buffered, i.e., they can also contain buffers selected from 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-morpholinoethanesulfonic acid) buffers and mixtures thereof, and preferably buffers selected from Tris, lactate, tartrate, carbonate and MES buffers and mixtures thereof. Advantageously, the compositions according to the invention contain a Tris buffer.

[0118] The composition used as the subject of this invention is preferably sterile.

[0119] Method for preparing complexes having formula (II)

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

[0121] a) A hexaacid having the following formula (III):

[0122]

[0123] Complexing with gadolinium yields a gadolinium hexaate complex having the previously defined formula (I).

[0124] b) Isomerization of the gadolinium hexaate complex of formula (I) in an aqueous solution with a pH between 2 and 4 to obtain a diastereomeric complex consisting of at least 80% diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS of the gadolinium hexaate complex of formula (I), and

[0125] c) Starting with the diastereomeric enriched complex obtained in step b), a complex having formula (II) is formed by reacting it with 3-amino-1,2-propanediol.

[0126] In this specification, the terms “Gd”, “gadolinium”, and “Gd2” are used interchangeably unless otherwise stated. 3+ "to represent Gd"3+ Ions. By extension, it can also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0127] In this invention, the term "free Gd" refers to the non-complexed form of gadolinium, which is preferably used for complexation. Typically, this refers to Gd dissolved in water. 3+ Ions. By extension, it can 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 hexacid having formula (III) and gadolinium, which makes it possible to obtain a hexacid-gadolinium complex having formula (I) as defined above.

[0130] According to one specific embodiment, step a) includes a reaction between a hexaic acid having 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), namely the source of gadolinium (typically gadolinium oxide), the hexa-acid having formula (III), and water, are as pure as possible, especially involving metallic impurities.

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

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

[0135] Advantageously, the amount of reagents (hexaic acid and gadolinium having formula (III)) used in step a) corresponds to or is close to the stoichiometric ratio, as determined by the equilibrium equation of the complexation reaction that occurs in this step.

[0136] The term "close to stoichiometry" means that the difference between the molar ratio of the introduced reagent and the stoichiometry is less than 15%, particularly less than 10%, and 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 a hexacarboxylic acid 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, the amount of gadolinium introduced is greater than 1 equivalent (eq.) relative to the amount of hexacarboxylic acid having formula (III) introduced (which itself corresponds to 1 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 where the source of free gadolinium is Gd₂O₃, 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., relative to the amount of hexacarboxylic acid having formula (III) introduced (1 eq.).

[0138] According to one specific implementation, step a) includes the following sequential steps:

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

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

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

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

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

[0144] ■Step b)

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

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

[0147] In the context of this invention, the term "diasteremeric excess" is intended to represent the fact that, in relation to gadolinium hexacarboxylate complexes having formula (I), the complexes are primarily present in the form of isomers or groups of isomers selected from the following diastereomers: I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR, and I-SRS. The diasteremeric excess is expressed as a percentage and corresponds to the amount expressed by the predominant isomer or group of isomers relative to the total amount of gadolinium hexacarboxylate complexes having formula (I). It should be understood that this percentage can be based on moles or mass, since, by definition, isomers have the same molar mass.

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

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

[0150] In fact, the inventors discovered that factors such as pH and temperature of the solution of the gadolinium hexaate complex of formula (I) obtained at the end of step a) influence the ratio of various isomers of the complex of formula (I) present in the mixture of diastereomers. Over time, the mixture tends to enrich a set of isomers, which contain the most thermodynamically stable but also chemically stable isomers, in this case isomers I-RRR and I-SSS.

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

[0152] However, in a preferred embodiment, the isomers I-RRR and I-SSS are present in the mixture in ratios between 65 / 35 and 35 / 65, particularly between 60 / 40 and 40 / 60, and 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 hexa-acid gadolinium complex of formula (I) in aqueous solution is typically carried out at a pH between 2 and 4, particularly between 2 and 3, and advantageously between 2.2 and 2.8.

[0154] Preferably, an acid is used, preferably an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid, for example, using hydrochloric acid to adjust the pH.

[0155] Completely surprising, under these pH conditions, a mixture occurred, specifically an enrichment of isomers, in this case I-RRR and I-SSS, since gadolinium chelates are known in the art to be characterized by low kinetic inertia in acidic media. Specifically, the medium contains H... + The higher the concentration of the ions, the greater the likelihood of a proton transferring to one of the donor atoms of the ligand, leading to the dissociation of the complex. Therefore, those skilled in the art would expect that placing the gadolinium hexaate complex having formula (I) in an aqueous solution with a pH between 2 and 4 would result in the dissociation of the complex, rather than isomerization to I-RRR and I-SSS.

[0156] It should be noted that the pH range recommended in EP 1 931 673 for the complexation of hexa acids having formula (III), namely 5.0-6.5, cannot yield complexes having formula (I) enriched with their isomers I-RRR and I-SSS.

[0157] Step b) is typically carried out at a temperature between 80°C and 130°C, particularly between 90°C and 125°C, preferably between 98°C and 122°C, advantageously between 100°C and 120°C, for a time typically between 10 hours and 72 hours, particularly between 10 hours and 60 hours, advantageously between 12 hours and 48 hours.

[0158] Contrary to all expectations, such temperature conditions used in conjunction with the pH conditions described above should favor the instability of gadolinium chelates, preventing them from decomplexing or forming any other impurities, and instead isomerizing them into I-RRR and I-SSS.

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

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

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

[0162] According to a preferred embodiment, at the end of step b), the diastereomer-enriched complex is separated by crystallization, preferably by seed crystallization.

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

[0164] b1) Isomerization of the gadolinium hexaate complex of formula (I) in an aqueous solution with a pH between 2 and 4 is performed to obtain a diastereomeric complex consisting of at least 80% diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS of the gadolinium hexaate complex of formula (I), and

[0165] b2) The diastereomeric complexes enriched by crystallization are separated, preferably by seed crystallization.

[0166] Crystallization step b2) first involves removing any impurities present in the aqueous solution, which may be due to previous steps, thereby obtaining a decolorized product of higher purity in crystalline form. Secondly, it involves continuing the diastereomeric enrichment of the hexacarboxylic acid gadolinium complex of formula (I) to obtain an excess of diastereomeric isomers comprising a mixture of the isomers I-RRR and I-SSS of the complex, higher than that obtained at the end of step b1). Specifically, the isomers I-RRR and I-SSS of the hexacarboxylic acid complex of formula (I) crystallize from water. On the other hand, the hexacarboxylic acid gadolinium complex of formula (I) without enrichment of the isomers does not crystallize.

[0167] The fact that the isomers I-RRR and I-SSS, which tend to be enriched in step b) (and contrary to all expectations, depending on the conditions under which they are carried out), are the only isomers of the complex that crystallize from water is a completely unexpected result. Therefore, the synergistic effect of isomerization and crystallization contributes to the enrichment of isomers I-RRR and I-SSS, and thus to the overall efficiency of the method according to the invention.

[0168] Furthermore, it should be noted that the crystallization of the target isomer of the gadolinium hexaate complex having formula (I) from water makes it possible to avoid the addition of solvent as described in Embodiment 7 of EP 1 931 673, which involves the step of precipitating the trisodium salt of the complex from ethanol.

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

[0170] According to one variation, after lowering the temperature of the aqueous solution to within the aforementioned range, a crystallization process is induced by seed crystals. "Crystallization by seed crystals," also known as "crystallization by seed crystals," involves introducing a known amount of crystals (referred to as "seeds" or "primers") into a reactor (also called a crystallization vessel) in which crystallization takes place. This allows for a reduction in crystallization time. Crystallization by seed crystals is well known to those skilled in the art. In the method according to the invention, using primers, in the present case crystals of a diastereomer-enriched gadolinium hexaate complex of formula (I) added to an aqueous solution of a diastereomer-enriched complex that has been pre-cooled, crystallization is carried out to enable nucleation, thereby initiating crystallization. The duration of crystallization by seed crystals is advantageously between 2 hours and 20 hours, and preferably between 6 hours and 18 hours; typically 16 hours.

[0171] Crystals of the hexa-acid gadolinium complex enriched with the diastereomer of formula (I) are then separated by filtration and drying, typically using any technique known to those skilled in the art.

[0172] Advantageously, at the end of step b2), the purity of the diastereomeric gadolinium hexaate complex of formula (I) enriched with a purity greater than 95%, particularly greater than 98%, and advantageously greater than 99%, is expressed as a mass percentage of the complex of formula (I) relative to the total mass obtained at the end of step b2).

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

[0174] In this embodiment, in addition to the previously described consecutive steps b1) and b2), step b) further includes step b3, which involves purification of the diastereomeric gadolinium hexaate complex enriched by recrystallization of formula (I).

[0175] The recrystallization step b3) is similar to the crystallization step b2), firstly involving obtaining a product of higher purity, and secondly involving continuing the diastereomeric enrichment of the gadolinium hexaate complex having formula (I) to obtain an excess of diastereomeric isomers comprising a mixture of 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 includes the following consecutive sub-steps:

[0177] ● The diastereomeric gadolinium hexaate complex enriched with formula (I) separated in step b2) is suspended in an aqueous solution, preferably in water.

[0178] ● The complex is dissolved by heating to a temperature advantageously between 80°C and 120°C, for example, up to 100°C.

[0179] ● Recrystallization, preferably by seed crystal, is advantageously carried out at a temperature between 10°C and 90°C, particularly between 20°C and 87°C, specifically between 55°C and 85°C, typically lasting between 2 hours and 20 hours, particularly between 6 hours and 18 hours.

[0180] ●Isolation of diastereomers enriched and purified crystals of hexa-acid gadolinium complex of formula (I), for example by filtration and drying.

[0181] At the end of step b3), the purity of the purified diastereomeric gadolinium hexaate complex of formula (I) isolated is typically greater than 98%, particularly greater than 99%, and advantageously greater than 99.5%, said purity being expressed as a mass percentage of the complex of formula (I) relative to the total mass obtained at the end of step b2).

[0182] In another embodiment, the complex enriched from the diastereomers in step b) is further enriched by selective decomplexation of the diastereomers of the complex having 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.

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

[0184] The selective decomplexing step b4) involves continuing the diastereomeric enrichment of the gadolinium hexaate complex having formula (I) to obtain a diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS of the complex, which, when performed prior to step b4), is higher than the diastereomeric excess obtained at the end of step b2) or at the end of step b3).

[0185] Step b4) typically includes the following consecutive sub-steps:

[0186] ● The diastereomeric gadolinium hexaate complexes enriched with formula (I) isolated in step b2) or step b3) are suspended in water.

[0187] ● Add an alkali, such as sodium hydroxide.

[0188] ● Heat to a temperature advantageously between 30°C and 60°C, particularly between 35°C and 55°C, such as 40°C, typically for a duration between 2 hours and 20 hours, particularly between 10 hours and 18 hours.

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

[0190] ● Separation of diastereomers, enrichment and purification of gadolinium hexaate complexes of formula (I), for example by filtration and drying.

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

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

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

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

[0195] By extension, the term "mixture of isomers I-RRR and I-SSS" also encompasses the case where only one isomer is present, whether it is I-RRR or I-SSS. However, the term "mixture of isomers I-RRR and I-SSS" preferably represents all cases where each of the isomers I-RRR and I-SSS is present in a variable but non-zero amount.

[0196] In a preferred embodiment, the isomers I-RRR and I-SSS are present in the mixture in ratios between 65 / 35 and 35 / 65, particularly between 60 / 40 and 40 / 60, and 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 a complex of formula (II) from a gadolinium hexaate complex enriched from its precursor, i.e., the diastereomer obtained in step b).

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

[0200] The amidation reaction does not change the absolute configuration of the three asymmetric carbon atoms at the α-position of the nitrogen atom on the grafted macrocycle. Therefore, step c) enables the acquisition of a complex of formula (II) with a diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS, which is identical to a diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS. The diastereomeric gadolinium hexaate complex of formula (I) obtained at the end of step b) is acquired with at least 80% of this diastereomeric excess comprising the mixture of isomers I-RRR and I-SSS.

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

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

[0203] Advantageously, the 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 encompasses the case where only one isomer is present, whether it is II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

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

[0206] Amide reactions can be carried out by any method known to those skilled in the art, particularly in the presence of a reagent for activating carboxylic acid functional groups and / or by acid catalysis.

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

[0027] of the patent.

[0208] In one specific embodiment, step c) comprises activating a carboxylic acid (-COOH) functional group carried by a carbon atom at the γ position on the side chain of the hexacarboxylic acid complex of formula (I), relative to the nitrogen atom of the macrocycle to which a side chain is grafted, in the form of a derived functional group comprising a carbonyl (C=O) group, such 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. Thus, according to this specific embodiment, the carboxylic acid functional group may in particular be activated in the form of an ester, acyl chloride, or anhydride functional group, or in any activated form capable of generating an amide bond. Activated forms capable of generating amide bonds are well known to those skilled in the art and can be obtained, for example, by a set of methods known in peptide chemistry for forming peptide bonds. Examples of such methods are found in the publication *Synthesis of peptides*. In andpeptidomimetics, Vol. E22a, pp. 425-588, Houben-Weyl et al., edited by Goodman, Thieme-Stuttgart-New York (2004), and in these examples, the method of activating carboxylic acids by azides (acyl azides) can be particularly mentioned, for example by the action of a reagent (e.g., diphenylphosphohydrazide (usually abbreviated as DPPA)), alone or in the presence of a catalyst (e.g., N-hydroxysuccinimide and its derivatives), using carbonyl diimazole (1,1'-carbonyl diimazole, CDI), using phosphonium salts (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (usually abbreviated as BOP)), or other ureon, such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate (usually abbreviated as HBTU).

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

[0210] This embodiment is more preferred than that described in EP 1 931 673, which uses a coupling agent (e.g., EDCI / HOBT) to activate the carboxylic acid functional group for 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 liquid / liquid extraction with the addition of a solvent. As previously discussed, such purification methods are not desirable, independent of the increased complexity of the method resulting from such additional steps. Furthermore, the use of HOBT is problematic in itself because it is an explosive product.

[0211] Within the meaning of this invention, the term "ester functional group" is intended to represent a -C(O)O- group. Specifically, it can be the group -C(O)O-R1, where R1 corresponds to a (C1-C6) alkyl group.

[0212] Within the meaning of this invention, the term "(C1-C6)alkyl group" refers to a straight-chain 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 this invention, the term "acyl chloride function," also known as "acid chloride function," is intended to represent the -CO-Cl group.

[0214] Within the meaning of this invention, the term "anhydride functional group" is intended to represent a -CO-O-CO- group. Specifically, it can be the group -CO-O-CO-R2, where R2 corresponds to a (C1-C6) alkyl group.

[0215] Reactions for converting carboxylic acid functional groups into ester, acyl chloride, or anhydride functional groups are well known to those skilled in the art, who will be able to carry out the reactions according to any conventional methods they are familiar with.

[0216] Then, by aminolysis with racemic or enantiomerically pure form, preferably racemic form, of a carboxylic acid functional group, specifically an ester or anhydride functional group, preferably an ester form, the complex having formula (II) is obtained.

[0217] Preferably, the activation of carboxylic acid functional groups and the aminolysis steps are carried out according to a one-pot method, which is an intermediate step in the same reactor without the separation or purification of intermediates, the intermediates including carboxylic acid functional groups activated in the form of ester, acyl chloride or anhydride functional groups, particularly esters or anhydrides, preferably esters.

[0218] According to one specific implementation, step c) includes the following sequential steps:

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

[0220]

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

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

[0223] It will be readily apparent to those skilled in the art that the reaction forming the activated complex of formula (VII) does not alter the absolute configuration of the three asymmetric carbon atoms at the α-position of the nitrogen atom on the side chain relative to the grafted side chain. Therefore, step c1) enables the obtaining of an activated complex of formula (VII) with a diastereomeric excess comprising a mixture of isomers VII-RRR and VII-SSS having the following representations (VII-RRR) and (VII-SSS), which is identical to a diastereomeric excess comprising a mixture of isomers I-RRR and I-SSS, and the diastereomeric gadolinium hexaate complex of formula (I) obtained at the end of step b) has at least 80% of this diastereomeric excess comprising the mixture of isomers I-RRR and I-SSS.

[0224]

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

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

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

[0228] According to this implementation, step c) may more specifically include the following sequential steps:

[0229] c1) forms a trimer having formula (VIII),

[0230]

[0231]

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

[0233] c2) Amine hydrolysis 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 (e.g., hydrochloric acid) in an alcohol having the formula R1OH, which acts as both a solvent and a reagent.

[0235] Step c2) is also typically carried out in the presence of an acid (e.g., hydrochloric acid) in an alcohol having the formula R1OH.

[0236] In the first stage, the gadolinium hexaate complex having formula (I) and the 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 an acidic solution of the 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), typically for more than 5 hours, preferably 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] Therefore, according to the one-pot method, steps c1) and c2) can be easily performed. Advantageously, the triester having formula (VII) is not separated between steps c1) and c2).

[0238] However, in order to promote the aminolysis reaction, in step c2), alcohols having the formula R1OH are preferably removed by vacuum distillation.

[0239] Within the meaning of this invention, the term "vacuum distillation" refers to the distillation of a mixture at a pressure between 10 and 500 mbar, particularly between 10 and 350 mbar, preferably between 10 and 150 mbar, and especially between 50 and 100 mbar.

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

[0241] Surprisingly, despite the acidic conditions typically employed in steps c1) and c2) increasing the kinetic instability of the gadolinium complex, no decomplexing or isomerization of the triester having formula (VIII) was observed. The desired triamide was obtained with very good conversion, and the absolute configuration of the three asymmetric carbon atoms at the α-position on the side chain relative to the nitrogen atom of the macrocycle was preserved.

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

[0243] In a preferred embodiment, step c) includes the following sequential steps:

[0244] c1) Forms a methyltriester having formula (IV),

[0245]

[0246] Especially in the presence of acid (such as hydrochloric acid), the reaction takes place in methanol, and

[0247] c2) Amine hydrolysis of methyltriesters of formula (IV) with 3-amino-1,2-propanediol, especially in the presence of an acid (e.g., hydrochloric acid), in methanol.

[0248] Advantageously, the methyltriester having formula (IV) is not separated between steps c1) and c2).

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

[0250] The present invention includes all combinations of the specific, advantageous or preferred embodiments described above associated with each step of the method.

[0251] ■Preparation of hexaacids with formula (III)

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

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

[0254]

[0255] With formula R3OOC-CHG p Alkylation of compounds with -(CH2)2-COOR4(IX)

[0256] in:

[0257] -R3 and R4 independently represent (C3-C6) alkyl groups, especially (C4-C6) alkyl groups, such as butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl groups, and

[0258] -Gp represents a detaching group, such as a toluenesulfonate or trifluoromethanesulfonate group, or a halogen atom, preferably a bromine atom.

[0259] To obtain a hexaester having formula (X)

[0260]

[0261] Then a hydrolysis step is performed to obtain the hexaacid having formula (III).

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

[0263] According to an advantageous embodiment, a hexaic acid having formula (III) is obtained by: a pyclene having formula (V):

[0264]

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

[0266]

[0267] Then a hydrolysis step is performed to obtain the hexaacid having formula (III).

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

[0269] The use of dibutyl 2-bromoglutarate is particularly advantageous compared to the use of ethyl 2-bromoglutarate as described in EP 1 931 673. Specifically, commercially available diethyl 2-bromoglutarate is a relatively unstable compound that degrades over time and with the influence of temperature. More precisely, the ester has a tendency to be hydrolyzed or cyclized, thus losing its bromine atom. Attempts to purify commercially available diethyl 2-bromoglutarate, or to develop new synthetic routes to obtain the ester in higher purity and thus prevent its degradation, have been unsuccessful.

[0270] Alkylation reactions are typically carried out in polar solvents, preferably in water, specifically in deionized water, and advantageously in the presence of a base such as potassium carbonate or sodium carbonate.

[0271] For obvious reasons, water is preferred, and acetonitrile is particularly preferred, as described in EP 1 931 673.

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

[0273] The hydrolysis step is advantageously carried out in the presence of an acid or a base, particularly a base (e.g., sodium hydroxide). The hydrolysis solvent can be water, an alcohol (e.g., ethanol), or a water / alcohol mixture. The step is advantageously carried out at temperatures between 40°C and 80°C, typically between 40°C and 70°C, and especially between 50°C and 60°C, typically lasting between 10 hours and 30 hours, and specifically between 15 hours and 25 hours.

[0274] Method for purifying complexes 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 an excess of at least 80% diastereomers, comprising 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) Separate the purified complex thus obtained in solid form.

[0284] Advantageously, the complex having formula (II) is obtained prior to the preparation method described above, having an excess of at least 80%, preferably at least 85%, particularly at least 90%, specifically at least 95%, more specifically at least 97%, preferably at least 98%, and advantageously at least 99% of the diastereomeric excess of the mixture of isomers II-RRR and II-SSS.

[0285] In a preferred embodiment, the complex enriched by the diastereoisomers thereon, having undergone the purification process, has a diastereoisomer excess of at least 85%, particularly at least 90%, specifically at least 92%, preferably at least 94%, advantageously at least 97%, and more advantageously at least 99% of the mixture of isomers II-RRR and II-SSS.

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

[0287] Advantageously, the 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 encompasses the case where only one isomer is present, whether it is II-RRR or II-SSS. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

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

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

[0291] Steps 1b) and 1c) involve purifying the complex having formula (II) by removing impurities that may be present due to its production process.

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

[0293] Specifically, 3-amino-1,2-propanediol may be present in the final product obtained during the process of preparing the complex having formula (II), typically when the complex having formula (I) and 3-amino-1,2-propanediol are used to obtain the complex having formula (II) by amidation. This is particularly true for the method of preparing the complex having formula (II) according to the invention. As detailed above, the amidation reaction may comprise the activation of three carboxylic acid functional groups carried by the carbon atom at the γ position on the side chain of the complex having formula (I) relative to the nitrogen atom of the macrocycle to which the side chain is grafted, followed by aminolysis of the activated carboxylic acid functional groups by reaction with 3-amino-1,2-propanediol. An excess of 3-amino-1,2-propanediol is then advantageously used to ensure good conversion to the amide functional groups of the three activated carboxylic acid functional groups.

[0294] The term "double-coupled impurity" is intended to represent complexes having the following formulas (II-dc-a), (II-dc-b), (II-dc-c), or mixtures thereof:

[0295]

[0296] The dual-coupling impurities are particularly likely to be caused by the hydrolysis of the amide functional group of the complex having formula (II). When the preparation of the complex having formula (II) involves such a step, this may also be due to incomplete activation of the carboxylic acid functional group of the complex having formula (I) (two of the three functional groups are activated) or incomplete ammonolysis of the activated carboxylic acid functional group (two of the three functional groups are ammonolyzed). This is particularly true for the method of preparing the complex having formula (II) according to the present invention.

[0297] ■ Step 1b) corresponds to passing a complex of formula (II) enriched with diastereomers as previously described through one or more ion exchange resins.

[0298] Within the meaning of this invention, the term "ion exchange resin" refers to a solid material, typically in bead form, composed of a polymer matrix, grafted with positively charged functional groups (anion exchange resin) or negatively charged functional groups (cation exchange resin), which makes it possible to capture anions or cations, respectively, through adsorption. The adsorption of anions or cations on the resin occurs through ion exchange between counterions of the initially present functional groups, ensuring the electroneutrality of the resin and the anions or cations to be captured.

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

[0300] The strong anion exchange resin typically includes ammonium groups (N(RR'R") as exchange functional groups. + (where R, R', and R" are the same or different (C1-C6) alkyl groups). Of particular note are the resins sold by Dow Chemical. FPA900, preferably HO - form.

[0301] The use of strong anion exchange resins enables at least partial removal of double-coupled impurities.

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

[0303] The weakly cationic resin typically includes carboxylic acid ester groups (CO2) as exchange functional groups. - One product that deserves special mention is the resin sold by Dow Chemical. HP336, advantageously H + form.

[0304] The use of a weak cationic resin enables at least partial removal of 3-amino-1,2-propanediol and possible Gd. 3+ Residues.

[0305] It should be noted that step 1b) via one or more ion exchange resins is made possible by improving the stability of the complex of formula (II) enriched with diastereomers according to the invention, thus maintaining the integrity of the complex in this step.

[0306] ■ Step 1c) corresponds to ultrafiltration for enriching diastereomers of complexes having formula (II) as previously described.

[0307] In this invention, the term "ultrafiltration" refers to a method of filtration through a mesoporous semipermeable membrane, under the influence of a force (e.g., a pressure gradient typically between 1 and 10 bar) and, as desired, a concentration gradient, wherein the pores of the mesoporous semipermeable membrane typically have a diameter (mesopore) between 1 and 100 nm, specifically between 2 and 50 nm, particularly between 10 and 50 nm. Thus, this is a membrane separation process by which particles larger than the pore size in a solution or suspension are retained by the membrane and separated from the liquid mixture containing them.

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

[0309] Advantageously, the ultrafiltration membrane used in step 1c) has a cutoff threshold of less than 100 kD, particularly less than 50 kD, specifically less than 25 kD, and typically 10 kD.

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

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

[0312] In this invention, the term "nanofiltration" refers to a method of filtration using a porous, semi-permeable membrane, where the pores of the membrane typically have a diameter between 0.1 and 100 nm, specifically between 0.1 and 20 nm, and particularly between 1 and 10 nm, under the influence of force (e.g., a pressure gradient typically between 1 and 50 bar) and, if desired, a concentration gradient. Thus, this is a membrane separation process by which particles larger than the pore size in a solution or suspension are retained by the membrane and separated from the liquid mixture containing them.

[0313] The nanofiltration step 1a) enables the removal of most excess 3-amino-1,2-propanediol (as needed, in the form of a salt (specifically hydrochloride) or a derivative, particularly an acetamide derivative) and mineral salts.

[0314] In this specific embodiment, the crude diastereomer-enriched complex of formula (II) obtained according to the previously described preparation method can be directly subjected to a nanofiltration step. In particular, it is not necessary to precipitate the previously prepared diastereomer-enriched complex of formula (II) by adding a solvent.

[0315] Advantageously, the nanofiltration membrane used in step 1a) has a cutoff threshold of less than 1 kD, particularly less than 500 audendons, specifically less than 300 audendons, and typically 200 audendons.

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

[0317] Specifically, the temperature of the solution containing the complex of formula (II) that is ultrafiltered in step 1a) is between 20°C and 40°C, particularly between 25°C and 35°C.

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

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

[0320] ■Step 2)

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

[0322] The separation step of the solid form can be carried out by any method known to those skilled in the art, particularly by atomization, by precipitation, by freeze-drying or by centrifugation, with atomization being advantageous.

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

[0324] Specifically, the separation and purification of the complex of formula (II) in solid form by atomization makes it possible to eliminate the need for precipitation solvents.

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

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

[0327] The present invention also relates to the enrichment and purification of diastereomers of complexes having formula (II), which can be obtained by the purification method according to the present invention.

[0328] Preferably, the complex of formula (II) included in the previously described compositions according to the invention is a diastereomer-enriched and purified complex of formula (II) that can be obtained by the purification method according to the invention.

[0329] Example

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

[0331] isomers iso1, iso2, iso3 and iso4 of the complex of formula (II) were separated by UHPLC.

[0332] A UHPLC system consisting of a pump system, injector, chromatography column, UV detector, and 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 of 96% aqueous solution of H2SO4

[0335] -Preparation of test solution:

[0336] A solution of a complex of formula (II) at a concentration of 2 mg / mL in pure water

[0337] -Analysis conditions:

[0338]

[0339]

[0340] -gradient:

[0341] 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 (iso4) in the UHPLC chromatogram corresponds to a retention time of 6.3 minutes. Butyl hexaester with formula (VI) was prepared.

[0343] In a reactor, 184 kg (570 mol) of dibutyl 2-bromoglutarate and 89 kg (644 mol) of potassium carbonate were mixed 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 formulation. The reaction mixture was maintained at 55-60 °C and then refluxed for approximately 10 hours. After the reaction, the medium was cooled, diluted with 155 kg of toluene, and then washed with 300 L of water. Butyl hexaester was extracted into the aqueous phase with 175 kg (1340 mol) of phosphoric acid (75%). This was then washed three times with 150 kg of toluene. Butyl hexaester was then extracted into the toluene phase again by dilution with 145 kg of toluene and 165 kg of water, and then alkalized with 30% sodium hydroxide (m / m) to achieve 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 approximately 85%.

[0344] Preparation of hexaacids having formula (III)

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

[0346] Preparation of gadolinium hexaate complex with formula (I)

[0347] Experimental protocol

[0348] ●Complexation and isomerization

[0349] - Contains no acetic acid

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

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

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

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

[0356] ● Crystallization, recrystallization

[0357] -crystallization

[0358] The gadolinium hexaate complex of formula (I) in solution was cooled to 40°C, primers were added, and the reagents were contacted for at least 2 hours. The product was then separated by filtration at 40°C and washed with permeate water.

[0359] -Recrystallization

[0360] 180 kg of the previously obtained gadolinium hexaate complex of formula (I) (solid content approximately 72%) was suspended in 390 kg of water. The medium was heated to 100 °C to dissolve the product, and then cooled to 80 °C for pretreatment by adding a small amount of primer. After cooling to room temperature, the gadolinium hexaate complex of formula (I) was separated by filtration and drying.

[0361] ●Selective decomplexation

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

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

[0364] The ratio of various isomers having a complex of formula (I) in a mixture of diastereomers depends on the conditions under which the complexation and isomerization steps are performed, as shown in Table 3 below.

[0365]

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

[0367] The additional steps of recrystallization and selective decomplexation enable the addition of diastereomeric excesses in I-RRR and I-SSS mixtures (see Table 4).

[0368]

[0369] [Table 4]: Preparation of complexes with formula (II) by the content of I-RRR and I-SSS mixture after crystallization / recrystallization / selective decomplexation

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

[0371] Purification of complexes having formula (II)

[0372] ●Nanofiltration

[0373] The nanofiltration membrane used has a retention threshold of 200 Daltons (Koch Membran System SR3D). The treatment was performed as follows:

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

[0375] ●Resin treatment

[0376] The solution of the complex of formula (II) obtained from nanofiltration was diluted with purified water under stirring to obtain a 15% solution (m / m). This solution was then subjected to 50 L of OH... - The form of strong anion exchange resin (FPA900), then 50 liters of H2O. + A series of elutions were performed on a weakly cationic resin (HP336) at an average elution flow rate of 2V / V / H (2 volumes of solution / resin volume / hour). The resin was then rinsed with approximately 450 liters of purified water until a refractive index less than 1.3335 was obtained.

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

[0378] ●Ulfiltration

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

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

[0381] ●Atomization

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

[0383] Atomization is performed in the following manner:

[0384] By setting the inlet temperature to 165℃-170℃ and adjusting the feed rate to keep the outlet temperature between 105℃ and 110℃, the atomizer is balanced with pyrogen-free pure water.

[0385] Then add a concentrated solution of the complex having formula (II) and adjust the flow rate to retain the above parameters.

[0386] Maintain these operating conditions throughout the atomization process, while ensuring good powder performance in the atomization chamber and at the atomizer outlet. In particular, ensure no product adheres.

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

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

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

[0390] The composition according to the invention and the results of the study

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

[0392] A method for manufacturing the composition according to the present invention is performed according to the following steps:

[0393] a) Dissolve 485.1 g (i.e. 0.5 M) of the complex of formula (II) in water (appropriate amount, 1 liter), heat the container to a temperature between 39°C and 48°C, and stir the solution vigorously until the complex is completely dissolved in the water. Then cool the solution to about 30°C.

[0394] b) Add 0.404 g (i.e., 0.2 mol / mol% relative to the complex added in step a) to the solution obtained in step a) with stirring via a 10% m / v DOTA solution.

[0395] c) Add glycerol (Tris) to the solution obtained in step b) with stirring. Then adjust the pH to a value between 7.2 and 7.7 by adding hydrochloric acid solution with 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 achieved.

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

[0398] ● Examples of compositions according to the present invention

[0399] The following formulations were obtained using the methods described above:

[0400]

[0401]

[0402] *Measured using xylenol orange by colorimetric method.

[0403] **Expressed in anhydrous pure form

[0404] ● Formulation testing conducted

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

[0406] Various concentrations of DOTA, ranging from 0 to 2.5 mM, were tested. The results of these tests indicate that a concentration of 1 mM (corresponding to 0.04% m / v or 0.2 mol / mol%) ensures that no free Gd is released during the process and throughout the product's lifespan.

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

[0408] The formulations of the aforementioned examples were analyzed after manufacturing (T0) and after being stored at 40°C for 6 months after manufacturing (T+6 months).

[0409] At T0:

[0410] Purity as 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] In T+6 months:

[0415] Purity as 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 exhibits good stability over time.

[0421] ●Comparative stability study

[0422] The stability of the following compositions was evaluated over time. The term "unoptimized AP" refers to 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" refers to the complex of formula (II) obtained by enrichment and purification of diastereomers obtained according to the method of the present invention.

[0423]

[0424]

[0425] *LC formate: A chromatographic method involving fluorescence detection. Separation is achieved by gradient elution on a reversed-phase C18 grafted chromatography column.

[0426] The results reported above indicate that it is impossible to formulate unoptimized AP using free DOTA. This is because the chelating excipient is completely consumed by the trans-linking reaction between the complex of formula (II) and DOTA, and therefore cannot capture the released Gd. 3+ Its function.

[0427] On the other hand, the complex with diastereomer enrichment and purification of formula (II) obtained according to the method of the present invention can be formulated together with free DOTA. Specifically, it was observed that no free Gd was present in the composition after 6 months at 40°C, regardless of the pH of the formulation and the presence or absence of buffer substances. Furthermore, the consumption of chelating excipients is very low, as it does not exceed 0.08 mol / mol%.

Claims

1. A method for purifying a complex having the following formula (II): (II) The complex has at least 80% diastereomer excess of a mixture of isomers II-RRR and II-SSS, wherein the isomers II-RRR and II-SSS have the following formula: (II-SSS), (II-RRR), The method includes: 1) A combination of the following two steps: 1b) Through one or more ion exchange resins, and 1c) Ultrafiltration of the complex, and 2) Separate the purified complex thus obtained in solid form.

2. The method according to claim 1, wherein, The purification process involves a complex of formula (II) having a diastereomeric excess of at least 90% of a mixture of isomers II-RRR and II-SSS.

3. The method according to claim 1, wherein, The complex of formula (II) undergoing the purification process has a diastereomeric excess of at least 94% of the mixture of isomers II-RRR and II-SSS.

4. The method according to claim 1, wherein, Step 1b) involves contacting an aqueous solution of a complex having formula (II) with a strong anion exchange resin.

5. The method according to claim 4, wherein, Step 1b) further includes contacting an aqueous solution of the complex of formula (II) with a weak cationic resin.

6. The method according to claim 1, wherein, Steps 1b) and 1c) are also combined with step 1a) of nanofiltration.

7. The method according to claim 1, wherein, If step 1a) exists, proceed with steps 1a), 1b), and 1c in this order.

8. The method according to claim 1, wherein, Step 2) includes atomization.

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