Process for the preparation of pcta-based chelating ligands

Abstract

The invention relates to a process for the preparation of a macrocyclic ligand of formula (L) : involving: i) decomplexation of a complex of formula (II) : which complex consists of at least 80% diastereomeric excess, which diastereomeric excess comprises a mixture of isomers II-RRR and II-SSS of formulae: ii) removal of the gadolinium salt formed in step i) for example by filtration, and iii) recovery of the free macrocyclic ligand of formula (L). The invention also relates to a composition comprising a diastereomeric enriched complex of formula (II) and a macrocyclic ligand of formula (L).

Description

[0001] This invention relates to a novel method for preparing the ligand by discomplexing a gadolinium complex of a PCTA-based chelating ligand, wherein the complex is obtained in advance by a preparation and purification method that allows preferential acquisition of stereoisomers of the complex, which possess particularly advantageous physicochemical properties for use as contrast agents in medical imaging, notably in magnetic resonance imaging. The invention also relates to compositions comprising the diastereomeric enriched complex and its free ligand as chelating excipients.

[0002] Many contrast agents based on lanthanides (paramagnetic metals), particularly gadolinium (Gd), are known, as described in, for example, US 4647447. These products are generally organized under the terms GBCA or GdCA (gadolinium-based contrast agents). Several products are marketed, including macrocyclic chelates such as gadoteric acid meglumine 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, notably linear chelates based on DTPA (diethylenetriaminepentaacetic acid) or DTPA-BMA (gadolinium diamine ligand).

[0003] Other products, some of which are under development, represent a new generation of GdCA. They are essentially complexes of macrocyclic chelates such as bicyclic polyaza-macrocyclic carboxylic acids (EP 0438206) 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 1931673.

[0004] The PCTA-based chelate complexes described in EP 1931673 notably possess the following advantages: relatively easy chemical synthesis, and superior relaxation rates compared to other GdCA currently on the market (relaxation rate r1 can reach up to 11-12 mM in water). -1 .s -1 This relaxation rate corresponds to the efficiency of these products, and therefore to their comparative ability.

[0005] In vivo, chelates (or complexes) of lanthanides (and notably gadolinium) are in chemical equilibrium (via their thermodynamic constant K). therm Characterization), which may lead to the unwanted release of the lanthanides (see Equation 1 below):

[0006]

[0007] Chelates or ligands (Ch) with lanthanides (L) n The complexation chemical equilibrium between the two produces the complex Ch-L. n .

[0008] Since 2006, a pathology known as NSF (renal systemic fibrosis or fibrotic skin disease) has been linked at least in part to the release of free gadolinium into the body. This disease has prompted health authorities to raise concerns about gadolinium-based contrast agents marketed to certain patient groups.

[0009] Therefore, several strategies have been adopted to address the complex issues of patient tolerability in a completely safe manner and to limit or even eliminate the risk of lanthanide release 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.

[0010] Furthermore, since 2014, there have been reports of gadolinium deposition in the brain following repeated administration of gadolinium-based products (especially linear gadolinium chelates), gadolinium macrocyclic chelates, such as... 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.

[0011] 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.

[0012] Other methods for improving the tolerance of lanthanide chelates (notably gadolinium) are described in the prior art. US 5876695, reported over 30 years ago, describes, for example, formulations containing additional complexing agents besides lanthanide chelates, intended to improve the tolerance of leached lanthanides (Gd) by complexation. 3+ (Metal ions) are used to prevent the unwanted release of lanthanides from the body. Additional chelating agents can be introduced into the formulation in their free form or as weak complexes (typically calcium, sodium, zinc, or magnesium).

[0013] Therefore, in both strategies mentioned above, it is important to make the active complex as stable as possible.

[0014] However, although PCTA-based chelate ligand complexes containing the pyclene-type structure described in EP 1931673 have good kinetic stability, their thermodynamic constants are generally lower than those of other macrocyclic complexes based on cycloalkenes.

[0015] For complexes of formula (II) with the following expression, this is noteworthy:

[0016]

[0017] Specifically, as notably 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).

[0018] However, it should be noted that the complex having formula (II) corresponds to several stereoisomers, notably due to the presence of three asymmetric carbon atoms at the α-position on the side chain of this 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.

[0019] Therefore, the synthesis of complexes having formula (II) as described in EP 1931673 results in the production of a mixture of stereoisomers.

[0020] The aminopropylene glycol group in the side chain of the complex having formula (II) also includes 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.

[0021] 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.

[0022] As mentioned earlier, it is reasonable to use the term "family" because each of these families has several stereoisomers, notably due to the presence of asymmetric carbons in the aminopropanediol group.

[0023] 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.

[0024] 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, referred to as iso1, iso2, iso3, and iso4 in the remainder of this specification. By performing the method described in EP 1931673, the respective contents of iso1, iso2, iso3, and iso4 groups in the resulting mixture are as follows: 20%, 20%, 40%, and 20%. They then discovered that these different isomer groups have different physicochemical properties and determined that the isomer group, referred to as iso4, comprising a mixture of isomers II-RRR and II-SSS having the following formulas (II-RRR) and (II-SSS), proved to be the most advantageous medical imaging contrast agent.

[0025]

[0026] Therefore, surprisingly, iso4 is characterized by its significantly superior thermodynamic stability compared to the diastereomeric mixtures obtained by the method described in EP 1 931 673, which are complexes having formula (II). Specifically, its equilibrium thermodynamic constant K therm iso4 Equal to 10 18.7 (i.e. log(K) therm iso4 =18.7), a value determined by the methods described in Pierrard et al., ContrastMedia Mol. Imaging, 2008, 3, 243-252 and Moreau et al., Dalton Trans., 2007, 1611-1620.

[0027] 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 amount of complex to dissociate according to the following decomposition reaction (Equation 2):

[0028]

[0029] Isomer group <![CDATA[T 1 / 2 (pH1.2-37℃)]]> Iso1 18 hours Iso2 6 hours Iso3 8 days Iso4 27 days

[0030] Table 1 Discomplexation kinetics of isomers iso1 to iso4

[0031] For comparative purposes, gadobutrol or gadotate, as macrocyclic gadolinium complexes, exhibited kinetic inertia of 18 hours and 4 days, respectively, under the same conditions, while linear gadolinium complexes (such as gadodiamine or gadopentetate) dissociated instantaneously.

[0032] Furthermore, it is noteworthy that 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 (Equation 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.

[0033]

[0034] The measurements of the relaxation rates of the isomers (i.e. their efficiency as contrast agents) showed that the contrast capabilities of iso1, iso2 and iso4 were relatively comparable, while the efficiency of iso3 was reduced (see Table 2).

[0035]

[0036] Table 2 Relaxation rates of isomer groups iso1 to iso4 at 37°C

[0037] The inventors have successfully developed a novel method for the preparation and purification of complexes having formula (II), which enables the preferential 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.

[0038] When compared to alternatives that involve preparing a mixture of stereoisomers and then attempting to separate the diastereomers using conventional techniques, thereby separating the target isomer using any separation technique known in the art, implementing a method that makes it possible to obtain the target diastereomer in most cases is undoubtedly advantageous. Specifically, in addition to the fact that processes not involving diastereomer separation steps are easier to perform on an industrial scale, not performing separation firstly saves significant time, and secondly allows for increased overall method yield by minimizing the generation of unwanted diastereomers that are ultimately discarded. Furthermore, conventional separation techniques typically involve the use of large amounts of solvents, which exceeds financial costs and is undesirable for environmental reasons. Moreover, given the inherent health risks of professional exposure to silica, silica chromatography, which the International Agency for Research on Cancer classifies as carcinogenic to humans (Group 1), should be particularly avoided.

[0039] 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:

[0040]

[0041] 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 carbons are marked with an asterisk (*) in formula (I) above.

[0042] 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.

[0043] 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 1931673 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 following sections of this specification.

[0044] IsoD crystallizes from 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 with the following formulas (I-RRR) and (I-SSS) representing complexes of formula (I).

[0045]

[0046] It should be noted that the diastereomers I-RRR and I-SSS of the complex having formula (I) are enantiomers of each other.

[0047] The isomer enrichment step of the method of the present invention involves enriching the intermediate gadolinium hexaate complex having formula (I) in isoD.

[0048] The synthesis of the complex having formula (II) notably 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).

[0049] 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.

[0050] Furthermore, when carried out after the method for preparing a complex having the above formula (II), the purification method developed by the inventors enables the acquisition of a complex having the formula (II) with optimized isomer characteristics, and significantly improves the impurity distribution.

[0051] This complex, which has improved stability, can then be formulated using its free macrocyclic ligands for diastereomeric enrichment and purification. WO 2014 / 174120 recommends the formulation of a complex of formula (II), which is obtained as a mixture of isomers of DOTA calcium complex according to the method of EP 1931673.

[0052] Method for preparing macrocyclic ligands of formula (L)

[0053] Therefore, the present invention relates to a method for preparing macrocyclic ligands having the following formula (L):

[0054]

[0055] The method includes:

[0056] i) Discomplexing complexes having formula (II):

[0057]

[0058] The complex consists of at least 80% diastereomer excess, which comprises a mixture of isomers II-RRR and II-SSS having the following formula:

[0059]

[0060]

[0061] ii) For example, by filtering to remove the gadolinium salt formed in step i), and

[0062] iii) Recover free macrocyclic ligands with formula (L).

[0063] The term "decomplexation" is intended to refer to the reverse reaction of a complexation reaction, which in turn corresponds to the formation of a coordination complex between a metal cation and one (or more) ligands. Therefore, a decomplexation reaction corresponds to breaking the coordination bonds between the metal constituting the complex and one or more ligands, resulting in the formation of a free metal cation and ligand material. In the context of this invention, the decomplexation of the complex having formula (II) thus leads to the formation of Gd. 3+ Ions and macrocyclic ligands with formula (L).

[0064] In the context of this invention, the term "diastereomer excess" is intended to represent the fact that, with respect to a complex having formula (II), the complex is primarily present in the form of isomers or groups 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 diastereomer excess is expressed as a percentage and corresponds to the amount expressed 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, by definition, isomers have the same molar mass.

[0065] In one specific embodiment, the complex having formula (II) has an excess of at least 85%, notably 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.

[0066] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers II-RRR and II-SSS.

[0067] Advantageously, the diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0068] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers 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" preferentially represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0069] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture at ratios between 65 / 35 and 35 / 65, notably between 60 / 40 and 40 / 60, and specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture at a ratio of 50 / 50.

[0070] 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.

[0071] For the purposes 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.

[0072] UHPLC method:

[0073] Waters UPLC T3 150x 2.1mm-1.6μm column

[0074] This is a reversed-phase UPLC column with spherical particles consisting of a preferentially very rigid silica core 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%.

[0075] Preferably, the fixed response used is compatible with the aqueous flow phase.

[0076] -Analysis conditions:

[0077]

[0078] -Mobile phase gradient (%v / v):

[0079]

[0080] Preferably, the complex having formula (II) involved in step i) of the method according to the invention is a diastereomeric enriched complex having formula (II), which can be obtained according to the preparation method described in the following part of the specification, and advantageously purified according to the purification method also described in the following part of the specification.

[0081] The decomplexing step i) is typically carried out by contacting the complex having formula (II) with a decomplexing agent such as oxalic acid.

[0082] Step i) typically includes the following successive sub-steps:

[0083] - Dissolve the complex having formula (II) in water, preferably deionized water.

[0084] - Add an anti-complexing agent, such as oxalic acid, to the previously obtained solution, preferably under stirring.

[0085] - The reaction mixture is heated to a temperature advantageously between 60°C and 130°C, notably between 80°C and 110°C, for example at 95°C, typically for a duration between 1 h and 10 h, notably between 3 h and 7 h, preferably with stirring.

[0086] - Cool the mixture to a temperature advantageously between 10°C and 30°C, for example to 20°C.

[0087] Step ii) to remove the gadolinium salts formed during step i) is typically carried out by filtration.

[0088] According to a preferred embodiment, the decomplexing step i) is performed by contacting the complex having formula (II) with oxalic acid, and the salt formed in step i) is typically removed by filtration in step ii) is gadolinium oxalate.

[0089] Then, in step iii), the free macrocyclic ligand having formula (L) is typically recovered in solution form.

[0090] For the purposes of this invention, the term "free ligand" refers to a ligand in its free form, i.e., not complexed with, in particular, metals (including lanthanides and actinides) or alkaline earth metal cations (such as calcium or magnesium).

[0091] The free macrocyclic ligand of formula (L) that is typically recovered in solution form in step iii) can then be purified in step iv).

[0092] The purification step iv) can be performed according to any purification method known to those skilled in the art (e.g., crystallization, preparative chromatography, by ion exchange resin or a combination thereof).

[0093] In a preferred embodiment, step iv) involves passing the free macrocyclic ligand of formula (L) recovered in step iii) through an ion exchange resin.

[0094] For the purposes of this invention, the term "ion exchange resin" refers to a solid material, typically in bead form, consisting 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.

[0095] Step iv) may then involve contacting an aqueous solution of the free macrocyclic ligand having formula (L) with a strong anion exchange resin. The water used may be selected from purified water, distilled water, or deionized water. The water used is preferably purified water or water for injection (WFI). The advantage of using purified water or WFI water is that the free macrocyclic ligand having formula (L) in the aqueous solution can be used without undergoing its solid separation step, thereby, for example, directly preparing the composition according to the invention, as described in the following sections of the specification.

[0096] 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. It is worth mentioning the resins sold by Dow Chemical. FPA900 or IRA458.

[0097] Certain impurities can be removed using a strong anion exchange resin, while free macrocyclic ligands of formula (L) are adsorbed onto the resin. They can then be desorbed using an acetic acid solution.

[0098] Step iv) may also involve contacting an aqueous solution of a free macrocyclic ligand having formula (L) with a weak cation exchange resin. The water used can be selected from purified water, distilled water, or deionized water. The water used is preferably purified water or water for injection (WFI). The advantage of using purified water or WFI is that the free macrocyclic ligand having formula (L) in the aqueous solution can be used without undergoing a solid-state separation step, thereby, for example, directly preparing the composition according to the invention, as described in the subsequent parts of the specification.

[0099] The weakly cationic resin typically includes carboxylic acid ester groups (CO2) as exchange functional groups. - It is worth mentioning the resins sold by Dow Chemical Company. HP336, advantageously protonated (H + )form.

[0100] Removal of any Gd by a weak cationic resin 3+ Residue.

[0101] The free macrocyclic ligand of formula (L), which is typically recovered in solution form in step iii) and optionally purified in step iv), can then be separated in step v).

[0102] The separation step in solid form can be carried out by any method known to those skilled in the art, notably by precipitation, crystallization, lyophilization, atomization from aqueous solution, or centrifugation after precipitation or crystallization of a free macrocyclic ligand having formula (L).

[0103] The present invention also relates to free macrocyclic ligands having formula (L), which can be obtained according to the method of the present invention.

[0104] Compositions comprising a complex having formula (II)

[0105] Secondly, the present invention relates to a composition comprising:

[0106] - 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

[0107] - Macrocyclic ligands with formula (L).

[0108] In the context of this invention, macrocyclic ligands having formula (L) exist in the composition in a free form.

[0109] For the purposes of this invention, the term "free form" refers to a non-complexed macrocyclic ligand, in particular, one that does not complex with the following: metals (including lanthanides and actinides) or alkaline earth metal cations (such as 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.

[0110] In a preferred embodiment, the complex of formula (II) present in the composition of the invention has an excess of at least 85%, notably 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.

[0111] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers II-RRR and II-SSS.

[0112] Advantageously, the diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0113] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers 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" preferentially represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0114] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture at ratios between 65 / 35 and 35 / 65, notably between 60 / 40 and 40 / 60, and specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture at a ratio of 50 / 50.

[0115] 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).

[0116] In this specification, the terms “Gd”, “gadolinium”, and “Gd2” are used interchangeably unless otherwise stated. 3+ "to represent Gd" 3+Ions. By extension, it could also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0117] 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 could also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0118] Free gadolinium is typically measured colorimetrically, usually with xylenol orange or arsene(III). In the absence of metal ions (such as gadolinium), these indicators have specific colors: xylenol orange is yellow at acidic pH, while arsene is pink. In the presence of gadolinium, they turn purple.

[0119] Visually assessing changes in solution color allows for verification of the presence or absence of gadolinium in the solution.

[0120] Furthermore, it is possible to quantitatively measure free gadolinium in solution using back titration, for example, by 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. Because EDTA is a stronger complexing agent than the colored indicator, gadolinium alters the ligand, causing the colored indicator to preferentially complex with EDTA. Therefore, the colored indicator gradually reverts to its non-complexed form.

[0121] 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.

[0122] These methods are well known to those skilled in the art and are noteworthy in Barge et al. (Contrast Media and Molecular Imaging, 1, 2006, 184-188).

[0123] 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.

[0124] Therefore, if necessary, the pH of the sample to be tested should be adjusted to between 4 and 8. It is worth noting that if the pH of the sample is acidic, 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.

[0125] 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, notably with respect to the content of free paramagnetic metal. According to ICH guidelines, observing stability for six months at 40°C is considered a good indicator of stability for 3 years at 25°C.

[0126] In specific embodiments, the composition according to the invention has a concentration in the range of 0.01 to 1.5 mol·L⁻¹. -1 Between, preferably between 0.2 and 0.7 mol·L⁻¹ -1 Between 0.3 and 0.6 mol·L⁻¹, with a higher preference. -1 The concentrations of the complexes with formula (II) between the above.

[0127] The complex having formula (II) is determined by methods known to those skilled in the art. Notably, the determination can be performed after mineralization and determination of the total amount of gadolinium present in the composition by atomic emission spectrometry (also known as ICP-AES or ICP atomic emission spectrometry).

[0128] 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.

[0129] In specific embodiments, the composition according to the invention comprises a macrocyclic ligand of formula (L) in amounts between 0.002 and 0.4 mol / mol%, notably 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).

[0130] The concentration of macrocyclic ligands of formula (L) in a composition is typically measured by back titration with copper, for example using copper sulfate as a copper ion source.

[0131] In this method, well known to those skilled in the art, a solution containing a known initial concentration Q0 of copper sulfate, greater than the amount of free ligand in the solution, is preferentially used. The test solution containing a to-be-determined amount Q1 of a free ligand of formula (L) is added to this copper sulfate solution. The free ligand of formula (L) is a very good copper complexing agent: therefore, the formation of (L)-copper complexes is observed.

[0132] The copper remaining in 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 solution but does not decomplex (L)-copper because the free ligand having formula (L) 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.

[0133] 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 ligands with formula (L) in the test solution Q1.

[0134] Alternatively, HPLC or UHPLC methods with UV or MS detection, well known to those skilled in the art, can be used. In particular, in UHPLC, the non-co-elution of present substances allows for their quantification.

[0135] Preferably, the proportions specified above in this invention are proportions prior to sterilization of the composition.

[0136] Advantageously, the pH of the composition is between 4.5 and 8.5, preferably between 5 and 8, advantageously between 6 and 8, and notably between 6.5 and 8. These pH ranges notably enable the restriction of the presence of certain impurities and promote the complexation of paramagnetic metal ions M.

[0137] 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.

[0138] The compositions used as the subject of this invention are preferably sterile.

[0139] Preferably, the complex having formula (II) included in the previously described composition according to the invention is a diastereomeric enriched complex having formula (II), which can be obtained according to the preparation method described in the following part of the specification, and is advantageously purified according to the purification method also described in the following part of the specification.

[0140] Advantageously, the macrocyclic ligand having formula (L) included in the previously described compositions according to the invention is a free macrocyclic ligand having formula (L), which can be obtained by the purification method according to the invention.

[0141] It should be noted that in this advantageous embodiment, when the free macrocyclic ligand having formula (L) acts as a chelating excipient in the formulation, i.e., when it captures gadolinium released from the complex in the formulation, a complex is formed that corresponds to the complex having formula (II), typically having RRR and / or SSS configurations. Therefore, the diastereoisomeric excesses of the complex having formula (II) II-RRR and II-SSS are stable over time in the formulation.

[0142] Method for preparing complexes having formula (II)

[0143] Complexes having formula (II) can be prepared by a method comprising the following sequential steps:

[0144] a) A hexaacid having the following formula (III):

[0145]

[0146] Complexing with gadolinium yields a gadolinium hexaate complex having the previously defined formula (I).

[0147] b) Isomerization of the gadolinium hexacarboxylate 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 hexacarboxylate complex of formula (I), and

[0148] 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.

[0149] In this specification, the terms “Gd”, “gadolinium”, and “Gd2” are used interchangeably unless otherwise stated. 3+ "to represent Gd" 3+ Ions. By extension, it could also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide (Gd2O3).

[0150] 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 could also be a source of free gadolinium, such as gadolinium chloride (GdCl3) or gadolinium oxide.

[0151] ■ Step a)

[0152] 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.

[0153] 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.

[0154] In a preferred embodiment, the source of free Gd is GdCl3 or Gd2O3, preferably Gd2O3.

[0155] 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, with particular attention to metal impurities.

[0156] Therefore, the source of gadolinium will advantageously be gadolinium oxide, with a purity preferably greater than 99.99%, and even more preferably greater than 99.999%.

[0157] The water used in this method preferably contains less than 50 ppm of calcium, more preferably less than 20 ppm, and even more 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.

[0158] Advantageously, the amount of reagents used in step a) (hexaic acid and gadolinium having formula (III)) corresponds to or is close to the stoichiometric ratio, as determined by the equilibrium equation of the complexation reaction that occurs in this step.

[0159] The term "close to stoichiometry" means that the difference between the molar ratio of the introduced reagent and the stoichiometry is less than 15%, notably less than 10%, and preferably less than 8%.

[0160] It is worth noting that the introduction of gadolinium may be slightly excessive relative to the stoichiometric ratio. The ratio of the amount of material introduced as gadolinium to the amount introduced as a hexacarboxylic acid having formula (III) is then greater than 1, but typically less than 1.15, notably 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 equivalent), but typically less than 1.15 equivalents, notably less than 1.10 equivalents, and advantageously less than 1.08 equivalents. In a preferred embodiment where the source of free gadolinium is Gd₂O₃, the amount of Gd₂O₃ introduced is typically greater than 0.5 equivalents, but less than 0.575 equivalents, notably less than 0.55 equivalents, and advantageously less than 0.54 equivalents, relative to the amount of hexacarboxylic acid having formula (III) introduced (1 equivalent).

[0161] According to one specific embodiment, step a) includes the following sequential steps:

[0162] a1) Prepare an aqueous solution of a hexaacid having formula (III), and

[0163] a2) Add a source of free gadolinium to the aqueous solution obtained in step a1).

[0164] 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, notably between 15% and 45%, preferably between 20% and 35%, advantageously between 25% and 35%, and even more advantageously between 25% and 30%.

[0165] Preferably, steps a) and b) are carried out according to the one-pot method embodiment, i.e., in the same reactor without intermediate steps of separation or purification.

[0166] 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).

[0167] ■ Step b)

[0168] 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.

[0169] Step b) involves enriching a mixture of diastereomers of the I-RRR and I-SSS isomers to obtain a diastereomer-enriched gadolinium hexaate complex of formula (I), which consists of at least 85%, noteworthyly 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.

[0170] In the context of this invention, the term "diasteremeric excess" is intended to represent the fact that, with respect 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.

[0171] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers I-RRR and I-SSS.

[0172] Advantageously, the diastereomer excess consists of a mixture of isomers I-RRR and I-SSS.

[0173] 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) affect 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.

[0174] By extension, the term "mixture of isomers I-RRR and I-SSS" also covers the case where only one isomer is present, whether it is I-RRR or I-SSS.

[0175] 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, notably 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.

[0176] Step b) of the isomerization of the hexa-acid gadolinium complex of formula (I) in aqueous solution is typically carried out at pH between 2 and 4, notably between 2 and 3, and advantageously between 2.2 and 2.8.

[0177] Acids are preferred, especially inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid, for example, hydrochloric acid is used to adjust the pH.

[0178] 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 a 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.

[0179] It should be noted that the pH range recommended in EP 1931673 for the complexation of hexa acids of formula (III), namely 5.0-6.5, cannot yield complexes of formula (I) enriched with their isomers I-RRR and I-SSS.

[0180] Step b) is typically carried out at a temperature between 80°C and 130°C, notably 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, notably between 10 hours and 60 hours, advantageously between 12 hours and 48 hours.

[0181] 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 discomplexing or forming any other impurities, and instead isomerizing them into I-RRR and I-SSS.

[0182] In a 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, notably between 110°C and 118°C, typically for a time between 12 hours and 48 hours, notably between 20 hours and 30 hours, and specifically between 24 hours and 26 hours.

[0183] 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), and noteworthyly between 40% and 50%.

[0184] 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.

[0185] According to a preferred embodiment, at the end of step b), the diastereomer-enriched complex is separated by crystallization, preferably by crystallization via seed crystals.

[0186] In this embodiment, step b) includes the following consecutive steps:

[0187] 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

[0188] b2) The diastereomeric complexes enriched by crystallization are separated, preferably by seed crystallization.

[0189] 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 having 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 having formula (I) crystallize from water. On the other hand, the hexacarboxylic acid gadolinium complex having formula (I) without enrichment of the isomers does not crystallize.

[0190] The fact that the isomers I-RRR and I-SSS, which tend to be enriched during 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.

[0191] Furthermore, it should be noted that the crystallization of the target isomer of the gadolinium hexaate complex having formula (I) in water makes it possible to avoid the addition of solvent as described in Example 7 of EP 1931673, which involves the step of precipitating the trisodium salt of the complex from ethanol.

[0192] Step b2) is advantageously carried out at a temperature between 10°C and 70°C, notably between 30°C and 65°C, and specifically between 35°C and 60°C.

[0193] 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.

[0194] 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.

[0195] 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%, notably 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).

[0196] In one specific embodiment, the complex enriched from the diastereomers of step b) separated by crystallization is further purified by recrystallization to obtain a complex enriched and purified from the diastereomers.

[0197] 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).

[0198] 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 isomers I-RRR and I-SSS of the complex, which is higher than that obtained at the end of step b2).

[0199] Step b3) typically includes the following consecutive sub-steps:

[0200] ● The diastereomeric gadolinium hexaate complex enriched with formula (I) separated in step b2) is suspended in an aqueous solution, preferably in water.

[0201] ● The complex is dissolved by heating to a temperature advantageously between 80°C and 120°C, for example, up to 100°C.

[0202] ● Recrystallization, preferably by seed crystal, is advantageously carried out at temperatures between 10°C and 90°C, notably between 20°C and 87°C, specifically between 55°C and 85°C, typically for a duration between 2 hours and 20 hours, notably between 6 hours and 18 hours, and

[0203] ●Isolation of diastereomers enriched and purified crystals of hexa-acid gadolinium complex of formula (I), for example by filtration and drying.

[0204] At the end of step b3), the purified diastereomeric gadolinium hexaate complex of formula (I) isolated typically has a purity greater than 98%, notably greater than 99%, and advantageously greater than 99.5%, which is expressed as a mass percentage of the complex of formula (I) relative to the total mass obtained at the end of step b2).

[0205] 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.

[0206] In this embodiment, in addition to the previously described consecutive steps b1) and b2), step b) further includes step b4) 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).

[0207] The selective decomplexing step b4) involves continuing the diastereomeric enrichment of the gadolinium hexacarboxylate 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).

[0208] Step b4) typically includes the following consecutive sub-steps:

[0209] ● The diastereomeric gadolinium hexaate complexes enriched with formula (I) isolated in step b2) or step b3) are suspended in water.

[0210] ● Add an alkali, such as sodium hydroxide.

[0211] ● Heat to a temperature advantageously between 30°C and 60°C, notably between 35°C and 55°C, such as 40°C, typically for a duration between 2 hours and 20 hours, notably between 10 hours and 18 hours.

[0212] ● Cool to a temperature advantageously between 10°C and 30°C, for example to 30°C, and

[0213] ● Separation of diastereomers, enrichment and purification of gadolinium hexaate complexes of formula (I), for example by filtration and drying.

[0214] 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).

[0215] 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%, notably 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.

[0216] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers I-RRR and I-SSS.

[0217] Advantageously, the diastereomer excess consists of a mixture of isomers I-RRR and I-SSS.

[0218] By extension, the term "mixture of isomers I-RRR and I-SSS" also covers 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" preferentially represents all cases where each of the isomers I-RRR and I-SSS is present in a variable but non-zero amount.

[0219] In a preferred embodiment, the isomers I-RRR and I-SSS are present in the mixture in ratios between 65 / 35 and 35 / 65, notably 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.

[0220] ■ Step c)

[0221] 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).

[0222] 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.

[0223] 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.

[0224] In a preferred embodiment, the complex of formula (II) obtained at the end of step c) has an excess of at least 85%, notably 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.

[0225] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers II-RRR and II-SSS.

[0226] Advantageously, the diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0227] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers 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" preferentially represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0228] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture at ratios between 65 / 35 and 35 / 65, notably between 60 / 40 and 40 / 60, and specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture at a ratio of 50 / 50.

[0229] The amidation reaction can be carried out by any method known to those skilled in the art, notably in the presence of a reagent for activating the carboxylic acid functional group and / or by acid catalysis.

[0230] It is noteworthy that this can be carried out according to the method described in EP 1931673, and noteworthy in paragraph

[0027] of the patent.

[0231] In a 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 can noteably 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, it is noteworthy to mention the method of activating carboxylic acids by azides (acyl azides), for example by the action of reagents such as diphenylphosphohydrazides (commonly abbreviated as DPPA) or by the use of carbodiimides alone or in the presence of catalysts such as N-hydroxysuccinimides and their derivatives, the use of carbonyl diimazoles (1,1'-carbonyl diimazole, CDI), the use of phosphonium salts such as benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (commonly abbreviated as BOP) or other ureonium, such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate (commonly abbreviated as HBTU).

[0232] 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.

[0233] This embodiment is more preferable than the use of coupling agents (such as EDCI / HOBT) to activate carboxylic acid functional groups for peptide coupling as described in EP 1931673. Specifically, this coupling results in the formation of an equivalent of 1-ethyl-3-[3-(dimethylamino)propyl]urea, which must be removed, notably by silica gel chromatography or by liquid / liquid extraction with the addition of a solvent. As previously discussed, the increased complexity of the method, independent of such additional steps, is not desirable. Furthermore, the use of HOBT is problematic in itself, as it is an explosive product.

[0234] For the purposes 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.

[0235] For the purposes of this invention, the term "(C1-C6)alkyl group" refers to a straight-chain or branched saturated hydrocarbon group 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.

[0236] For the purposes of this invention, the term "acyl chloride function," also known as "acid chloride function," is intended to represent the -CO-Cl group.

[0237] For the purposes 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.

[0238] 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.

[0239] Then, by ammonolysis with racemic or enantiomerically pure form, preferably racemic form, of ester, acyl chloride or anhydride functional group, notably ester or anhydride, or preferably ester form of activated carboxylic acid functional group, a complex having formula (II) is obtained.

[0240] Preferably, the steps of activating the carboxylic acid functional group and ammonolysis are carried out according to the one-pot method embodiment, which is an intermediate step in the same reactor without separation or purification of intermediates, including carboxylic acid functional groups activated in the form of esters, acyl chlorides or anhydrides, noteworthyly esters or anhydrides, preferably esters.

[0241] According to a specific embodiment, step c) includes the following sequential steps:

[0242] c1) Formation of an activated complex having formula (VII),

[0243]

[0244] 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

[0245] c2) Ammonolysis of the activated complex of formula (VII) with 3-amino-1,2-propanediol.

[0246] 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 of the macrocycle with 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.

[0247]

[0248] 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).

[0249] 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.

[0250] In an advantageous embodiment, step c) comprises activating the above-mentioned carboxylic acid (-COOH) functional group in the form of an ester functional group.

[0251] According to this embodiment, step c) may more specifically include the following successive steps:

[0252] c1) forms a trimer having formula (VIII),

[0253]

[0254] Where R1 represents a (C1-C6) alkyl group, and

[0255] c2) The triester having formula (VIII) is subjected to ammonolysis with 3-amino-1,2-propanediol.

[0256] Step c1) is typically carried out in the presence of an acid (such as hydrochloric acid) in an alcohol having the formula R1OH, which acts as both a solvent and a reagent.

[0257] Step c2) is also typically carried out in the presence of an acid (such as hydrochloric acid) in an alcohol having the formula R1OH.

[0258] 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, notably below 5°C, typically to 0°C, and then 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, notably between 0°C and 5°C.

[0259] Therefore, according to the one-pot method embodiment, steps c1) and c2) can be easily performed. Advantageously, the triester having formula (VII) is not separated between steps c1) and c2).

[0260] However, in order to promote the ammonolysis reaction, in step c2), it is preferable to remove the alcohol having the formula R1OH by vacuum distillation.

[0261] For the purposes of this invention, the term "vacuum distillation" refers to the distillation of a mixture at a pressure between 10 and 500 mbar, notably between 10 and 350 mbar, preferably between 10 and 150 mbar, and particularly between 50 and 100 mbar.

[0262] Similarly, to facilitate the ammonolysis 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 equivalents, notably greater than 7 equivalents, and advantageously greater than 10 equivalents, 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).

[0263] 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.

[0264] 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).

[0265] In a preferred embodiment, step c) includes the following sequential steps:

[0266] c1) Forms a methyltriester having formula (IV),

[0267]

[0268] It is worth noting that the reaction takes place in methanol in the presence of an acid (such as hydrochloric acid), and...

[0269] c2) Ammonolysis of methyltriesters of formula (IV) with 3-amino-1,2-propanediol, notably in the presence of an acid (such as hydrochloric acid), in methanol.

[0270] Advantageously, the methyltriester having formula (IV) is not separated between steps c1) and c2).

[0271] In a preferred embodiment, in step c2), methanol is removed by vacuum distillation until a temperature typically above 55°C, notably 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, notably between 10 and 20 hours, after which it is cooled to room temperature and diluted with water.

[0272] This invention includes all combinations of the specific, advantageous, or preferred embodiments described above associated with each step of the method.

[0273] ■ Preparation of hexaacids having formula (III)

[0274] The hexaacid having formula (III) can be prepared according to any known method, notably according to the method described in EP 1931673, which participates in step a of the method for preparing the complex having formula (II) according to the present invention.

[0275] However, according to a preferred embodiment, a hexaacid having formula (III) is obtained by: a pyclene having formula (V):

[0276]

[0277] With formula R3OOC-CHG p Alkylation of compounds with -(CH2)2-COOR4(IX)

[0278] in:

[0279] -R3 and R4 independently represent (C3-C6) alkyl groups. It is worth noting (C4-C6) alkyl groups, such as butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl groups.

[0280] -G pThis indicates a leaving group, such as a toluenesulfonate or trifluoromethanesulfonate group, or a halogen atom, preferably a bromine atom.

[0281] To obtain a hexaester having formula (X)

[0282]

[0283] Then a hydrolysis step is performed to obtain the hexaacid having formula (III).

[0284] In a preferred embodiment, R3 and R4 are the same.

[0285] According to an advantageous embodiment, a hexaic acid having formula (III) is obtained by: a pyclene having formula (V):

[0286]

[0287] Alkylation with dibutyl 2-bromoglutarate yields butyl hexaester having formula (VI):

[0288]

[0289] Then a hydrolysis step is performed to obtain the hexaacid having formula (III).

[0290] The 2-bromoglutarate dibutyl ester used is in racemic or enantiomeric pure form, preferably in racemic form.

[0291] The use of dibutyl 2-bromoglutarate is particularly advantageous compared to the use of ethyl 2-bromoglutarate as described in EP 1931673. 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.

[0292] Alkylation reactions are typically carried out in polar solvents, preferably in water, specifically in deionized water, and advantageously in the presence of bases such as potassium carbonate or sodium carbonate.

[0293] For obvious reasons, water is preferred, and acetonitrile is particularly preferred, as described in EP 1931673.

[0294] The reaction is advantageously carried out at temperatures between 40°C and 80°C, typically between 50°C and 70°C, and notably between 55°C and 60°C, for a duration between 5 hours and 20 hours, specifically between 8 hours and 15 hours.

[0295] The hydrolysis step is advantageously carried out in the presence of an acid or a base, particularly a base (such as sodium hydroxide). The hydrolysis solvent can be water, an alcohol (such as 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 notably between 50°C and 60°C, typically lasting between 3 hours and 30 hours, more preferably between 3 hours and 15 hours, and even more preferably between 6 hours and 10 hours.

[0296] Method for purifying complexes having formula (II)

[0297] A complex of formula (II) having a diastereomer excess of at least 80% (a mixture of isomers II-RRR and II-SSS) can be purified according to a method comprising the following sequential steps:

[0298] 1) A combination of the following two steps:

[0299] 1b) Through one or more ion exchange resins, and

[0300] 1c) Ultrafiltration of the complex, and

[0301] 2) Separate the purified complex thus obtained in solid form.

[0302] In a preferred embodiment, the complex enriched by the diastereoisomers thereon, having undergone the purification process, has a diastereoisomer excess of at least 85%, notably 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.

[0303] 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%, noteworthyly 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.

[0304] Preferably, the diastereomeric excess consists of a mixture of at least 70%, notably at least 80%, advantageously at least 90%, and preferably at least 95% of the isomers II-RRR and II-SSS.

[0305] Advantageously, the diastereomeric excess consists of a mixture of isomers II-RRR and II-SSS.

[0306] By extension, the term "mixture of isomers II-RRR and II-SSS" also covers 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" preferentially represents all cases where each of the isomers II-RRR and II-SSS is present in a variable but non-zero amount.

[0307] In a preferred embodiment, the isomers II-RRR and II-SSS are present in the mixture at ratios between 65 / 35 and 35 / 65, notably between 60 / 40 and 40 / 60, and specifically between 55 / 45 and 45 / 55. Advantageously, the isomers II-RRR and II-SSS are present in the mixture at a ratio of 50 / 50.

[0308] ■ A combination of steps 1b) and 1c)

[0309] Steps 1b) and 1c) involve purifying the complex having formula (II) by removing impurities that may be present due to its production process.

[0310] Notably, the impurities may include 3-amino-1,2-propanediol and / or bicoupled impurities.

[0311] 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 as a starting point to obtain the complex having formula (II) by amidation. This is noteworthy 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 ammonolysis 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.

[0312] 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:

[0313]

[0314] Notably, bis-coupling impurities may arise from the hydrolysis of the amide functional groups 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 groups of the complex having formula (I) (two of the three functional groups are activated) or incomplete ammonolysis of the activated carboxylic acid functional groups (two of the three functional groups are ammonolyzed). This is noteworthy for the method of preparing the complex having formula (II) according to the present invention.

[0315] ■ Step 1b) corresponds to passing a complex of formula (II) enriched with diastereomers as previously described through one or more ion exchange resins.

[0316] For the purposes of this invention, the term "ion exchange resin" refers to a solid material, typically in bead form, consisting 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.

[0317] 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.

[0318] 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.

[0319] The use of strong anion exchange resins enables at least partial removal of double coupling impurities.

[0320] 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.

[0321] 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.

[0322] The use of a weak cationic resin enables at least partial removal of 3-amino-1,2-propanediol and any Gd. 3+ Residue.

[0323] 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.

[0324] ■ Step 1c) corresponds to ultrafiltration for enriching diastereomers of complexes having formula (II) as previously described.

[0325] In this invention, the term "ultrafiltration" is intended to refer to a method of filtration through a mesoporous semipermeable membrane, under the influence of a force (such as a pressure gradient typically between 1 and 10 bar) and an optional 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, and notably 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.

[0326] In the context of the purification method according to the present invention, ultrafiltration is particularly advantageous for removing endotoxins.

[0327] Advantageously, the ultrafiltration membrane used in step 1c) has a cutoff threshold of less than 100 kD, notably less than 50 kD, specifically less than 25 kD, and typically 10 kD.

[0328] Preferably, in step 1c), the transmembrane pressure is between 1 and 5 bar, specifically between 2.25 and 3.25 bar.

[0329] ■ In a specific embodiment, steps 1b) and 1c) are also combined with nanofiltration step 1a).

[0330] In this invention, the term "nanofiltration" refers to a method of filtration through a porous, semi-permeable membrane, under the influence of a force (such as a pressure gradient typically between 1 and 50 bar) and an optional concentration gradient, wherein the pores of the porous, semi-permeable membrane typically have a diameter between 0.1 and 100 nm, specifically between 0.1 and 20 nm, and notably between 1 and 10 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.

[0331] The nanofiltration step 1a) enables the removal of most excess 3-amino-1,2-propanediol (optionally in the form of a salt, specifically a hydrochloride, or in the form of a derivative, notably an acetamide derivative) and mineral salts.

[0332] 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. Notably, it is not necessary to precipitate the previously prepared diastereomer-enriched complex of formula (II) by adding a solvent.

[0333] Advantageously, the nanofiltration membrane used in step 1a) has a cutoff threshold of less than 1 kD, notably less than 500 Daltons, specifically less than 300 Daltons, and typically 200 Daltons.

[0334] Preferably, in step 1a), the transmembrane pressure is between 10 and 40 bar, specifically between 2 and 30 bar.

[0335] 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, and noteably between 25°C and 35°C.

[0336] In an alternative to this specific embodiment, step 1b) does not involve contacting an aqueous solution of a complex of formula (II) enriched with diastereomers with a weak cationic resin.

[0337] In a specific embodiment, steps 1a (when present), 1b, and 1c are performed in this order. This advantageous embodiment particularly enables the minimization of resin usage and thus minimizes industrial manufacturing costs.

[0338] ■ Step 2)

[0339] Step 2) involves separating the purified complex of formula (II) obtained at the end of the combination of steps 1b) and 1c) (and optionally also in combination with step 1a) in solid form.

[0340] The separation step of the solid form can be carried out by any method known to those skilled in the art, notably by atomization, by precipitation, by freeze-drying or by centrifugation, with atomization being advantageous.

[0341] In a preferred embodiment, step 2) includes atomization.

[0342] 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.

[0343] The air inlet temperature in the atomizer is typically between 150°C and 180°C, notably 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, preferably between 105°C and 110°C.

[0344] Advantageously, at the end of step 2), the purified complex of the mixture of diastereomer-enriched isomers II-RRR and II-SSS separated by the complex of formula (II) has a purity greater than 95%, notably greater than 97%, preferably greater than 97.5%, more preferably greater than 98%, and advantageously greater than 99%, said purity being expressed as a mass percentage of the complex of formula (II) relative to the total mass obtained at the end of step 2).

[0345] Example

[0346] The examples given below are presented as a non-limiting illustration of the present invention.

[0347] isomers iso1, iso2, iso3 and iso4 of the complex of formula (II) were separated by UHPLC.

[0348] A UHPLC system consisting of a pump system, injector, column, UV detector, and data station was used. The column used was a 150x2.1mm-1.6μm UHPLC column (Waters). UPLC T3 column).

[0349] - Mobile phase:

[0350] Pathway A: 100% acetonitrile and Pathway B: 0.0005% v / v of 96% aqueous H2SO4 solution

[0351] - Preparation of test solution :

[0352] A solution of a complex of formula (II) at a concentration of 2 mg / mL in pure water

[0353] - Analysis conditions:

[0354]

[0355]

[0356] - gradient:

[0357] time %Acn <![CDATA[%H2SO40.0005%]]> 0 1 99 3 5 95 12 10 90 15 25 75 16 1 99 20 1 99

[0358] Four main peaks were obtained. Peak 4 (iso4) in the UHPLC chromatogram corresponds to a retention time of 6.3 minutes.

[0359] Preparation of butyl hexaester having formula (VI)

[0360] 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%.

[0361] Preparation of hexaacids having formula (III)

[0362] 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.

[0363] Preparation of gadolinium hexaate complex with formula (I)

[0364] ■Experimental Plan

[0365] ● Complexation and isomerization

[0366] - Contains no acetic acid

[0367] 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).

[0368] - Contains acetic acid

[0369] Gadolinium oxide (0.525 molar equivalents) was suspended at 28.1% by mass in a solution of a hexaacid having formula (III).

[0370] Pour 99-100% acetic acid (50% by mass of pure hexa-acid of formula (III)) into the medium at room temperature.

[0371] 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.

[0372] The medium was kept at 113°C overnight.

[0373] ● Crystallization, recrystallization

[0374] -crystallization

[0375] 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.

[0376] -Recrystallization

[0377] 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.

[0378] ● Selective decomplexation

[0379] 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.

[0380] ■ Content of the mixture of diastereomers I-RRR and I-SSS

[0381] 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.

[0382]

[0383] Table 3 Content of I-RRR and I-SSS mixture as a function of complexation / isomerization conditions

[0384] The additional steps of recrystallization and selective decomplexation enable the addition of diastereomeric excesses in I-RRR and I-SSS mixtures (see Table 4).

[0385]

[0386] Table 4 Content of I-RRR and I-SSS mixture after crystallization / recrystallization / selective decomplexation

[0387] Preparation of complexes having formula (II)

[0388] 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).

[0389] Purification of complexes having formula (II)

[0390] ● Nanofiltration

[0391] The nanofiltration membrane used has a retention threshold of 200 Daltons (Koch Membrane System SR3D). The treatment was performed as follows:

[0392] 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 the polarization layer. The medium was then percolated at 30°C and 25 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).

[0393] ● Treatment on resin

[0394] 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), and then 50 liters of H + 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.

[0395] 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.

[0396] ● Ultrafiltration

[0397] The ultrafiltration membrane is a UF 10KD Koch spiral membrane.

[0398] 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.

[0399] ● atomization

[0400] The complex of formula (II) in powder form is obtained by atomizing a previously concentrated solution of the complex of formula (II) to 25%.

[0401] Atomization is performed in the following manner:

[0402] 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.

[0403] Then add a concentrated solution of the complex having formula (II) and adjust the flow rate to retain the above parameters.

[0404] Maintain these operating conditions throughout the atomization process, while ensuring good powder performance in the atomization chamber and at the atomizer outlet. It is important to ensure that no product adheres.

[0405] At the end of the solution delivery to the atomizer, the container and atomizer of the complex of formula (II) are rinsed with pyrogen-free pure water until maximum powder recovery is achieved. A complex of formula (II) with a purity of 99.6% is obtained.

[0406] The purity was determined by reversed-phase liquid chromatography.

[0407] Preparation of macrocyclic ligands with formula (L) :

[0408] 20 g (0.02 mol) of the complex of formula (II) was dissolved in 64 mL of deionized water. 4.88 g (0.039 mol) of oxalate dihydrate was added to the solution with stirring. The medium was heated to 95 °C and stirred for 5 hours, then cooled to 20 °C and filtered to remove gadolinium oxalate. The resulting mixture was cooled to room temperature and then filtered and washed with 10 mL of deionized water. This yielded an aqueous solution of the free ligand of formula (L).

[0409] The composition according to the invention

[0410] Examples of the preparation of compositions according to the present invention

[0411] A method for manufacturing the composition according to the present invention is performed according to the following steps:

[0412] 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.

[0413] b) Add 0.816 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 macrocyclic ligand solution of formula (L).

[0414] c) Add tromethamine (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.

[0415] 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.

[0416] 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.

[0417] ● Examples of compositions according to the present invention.

[0418] The following preparations were obtained using the above method:

[0419] Element Proportion in the composition Complexes having formula (II) 485.1g (0.5M) Macrocyclic ligands with formula (L) 0.816 g (1 mM, i.e., 0.2 mol / mol% relative to the complex) NaOH or HCl Appropriate amount, pH 7.2 to 7.7 Tromethamine 1.211g Free gadolinium* <1ppmm / v Water for injection (injection grade) Appropriate amount, 1L

[0420] *Measured using xylenol orange by colorimetric method.

[0421] **Expressed in anhydrous pure form

[0422] ● Preparation testing

[0423] Various concentrations of tromethamine 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.

[0424] Macrocyclic ligands of formula (L) at various concentrations from 0 to 2.5 mM were tested. The results of these tests indicate that a concentration of 1 mM (corresponding to 0.08% m / v or 0.2 mol / mol%) ensures that no free Gd is released during the process and throughout the product's lifespan.

Claims

1. A method for preparing macrocyclic ligands having the following formula (L): The method includes: a) A hexaacid having the following formula (III): Gadolinium complexation yields a gadolinium hexaate complex having the following formula (I): b) Isomerization of the gadolinium hexacarboxylate complex of formula (I) in an aqueous solution with a pH between 2 and 4 is carried out to obtain a diastereomeric complex consisting of at least 80% diastereomeric excess, which is a mixture of diastereomeric excesses I-RRR and I-SSS of the gadolinium hexacarboxylate complex of formula (I), step b) being carried out at a temperature between 90°C and 125°C. c) Starting with the diastereomeric enriched complex obtained in step b), the complex having formula (II) is formed by reacting it with 3-amino-1,2-propanediol: The complex consists of at least 80% diastereomer excess, which is a mixture of diastereomers II-RRR and II-SSS having the following formula: i) The complex having formula (II) is decomplexed by contacting the complex having formula (II) with a decomplexing agent. ii) Remove the gadolinium salt formed in step i), and iii) Recover the free macrocyclic ligand with formula (L).

2. The method as described in claim 1, wherein, The mixture of isomers II-RRR and II-SSS obtained in step c) has a molar ratio between 65 / 35 and 35 / 65.

3. The method as described in claim 2, characterized in that, - At the end of step b), the complex enriched by the diastereomer is separated by crystallization and purified by recrystallization, and Step c) includes the following sequential steps: c1) forms a trimer having formula (VIII), Where R1 represents a C1-C6 alkyl group, and c2) The triester having formula (VIII) is subjected to ammonolysis with 3-amino-1,2-propanediol. The triester of formula (VIII) was not separated between steps c1) and c2).

4. The method as described in claim 3, characterized in that, The triester of formula (VIII) is formed in step c1) by reacting it in an alcohol having formula R1OH in the presence of an acid.

5. The method as described in claim 3 or 4, characterized in that, The ammonolysis in step c2) is carried out in an alcohol having the formula R1OH in the presence of an acid.

6. The method as described in claim 1 or 2, characterized in that, The complex having formula (II) thereof, consisting of at least 80% diastereomer excess, is purified prior to step i) by the following steps: 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.

7. The method as described in claim 6, characterized in that, Steps 1b) and 1c) are also combined with step 1a) of nanofiltration.

8. The method as described in claim 6, characterized in that, Steps 1b) and 1c) are performed in this order.

9. The method as described in claim 7, characterized in that, Steps 1a), 1b), and 1c) are performed in this order.

10. The method as described in claim 6, characterized in that, Step 2) includes atomization.

11. The method as described in claim 1 or 2, characterized in that, The anti-complexing agent is oxalic acid.

12. The method as described in claim 11, characterized in that, Step i) includes the following consecutive sub-steps: - Dissolve the complex having formula (II) in water, - Add the anti-complexing agent to the previously obtained solution. -Heat the reaction mixture to a temperature advantageously between 60°C and 130°C, and - Cool the mixture to a temperature advantageously between 10°C and 30°C.

13. The method as described in claim 12, characterized in that, Step i) includes heating to a temperature between 80°C and 110°C.

14. The method as described in claim 12, characterized in that, Step i) includes heating for a duration between 1 hour and 10 hours.

15. The method as described in claim 13, characterized in that, Step i) includes heating for a duration between 1 hour and 10 hours.

16. A composition comprising: - A complex having formula (II) as defined in claim 1, comprising at least 80% diastereomer excess, wherein the diastereomer excess is a mixture of diastereomers II-RRR and II-SSS, and - Macrocyclic ligands with the following formula (L): in, Relative to the complex having formula (II), the composition contains between 0.01 and 0.3 mol / mol% of free macrocyclic ligands. The free gadolinium concentration in this composition is less than 1 ppm m / v.

17. The composition of claim 16, characterized in that, The composition contains, relative to the complex having formula (II), 0.02 to 0.2 mol / mol% of free macrocyclic ligands.

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