阅读说明：本技术 钆和基于pcta的螯合配体的非对映异构体富集的络合物和合成方法 (Diastereomer enriched complexes of gadolinium and PCTA based chelating ligands and methods of synthesis ) 是由 斯瓦兹克·格雷纽 艾伦·切内德 马丁尼·瑟夫 斯特凡·德克龙 布鲁诺·弗朗索瓦 于 2020-01-17 设计创作，主要内容包括：本发明涉及一种由至少80％的非对映异构体过量构成的具有式(II)的络合物,所述非对映异构体过量包含具有下式的异构体II-RRR和II-SSS的混合物：本发明还涉及一种用于制备所述具有式(II)的络合物的方法,并且还涉及两种合成中间体。(The invention relates to a complex having formula (II) consisting of a diastereomeric excess of at least 80%, said diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS having the formula: the invention also relates to a process for preparing said complex having formula (II), and also to two synthetic intermediates.)

1. A gadolinium hexaate complex having the formula (I):

having a diastereomeric excess of at least 95%, including mixtures of isomers I-RRR and I-SSS of the formula:

2. a complex as claimed in claim 1, characterised in that the diastereomeric excess is at least 97%.

3. Complex according to claim 1, characterized in that the isomers I-RRR and I-SSS are present in the mixture in a ratio between 55/45 and 45/55.

4. The complex according to claim 1, characterized in that said diastereomeric excess corresponds to peak 4 of an HPLC profile, characterized by a retention time of about 35.7 minutes, obtained using the following HPLC method:

·WatersRP18-250x4.6mm-5 μm column,

analysis conditions:

-a sample: 10mg/mL of an aqueous solution of a complex having formula (I),

column temperature: at a temperature of 25 c,

-sample temperature: at room temperature (20 ℃ -25 ℃),

-flow rate: 1.0mL/min of the reaction solution,

-sample size: 20 μ L

-UV detection: the particle size of the nano-particles is 200nm,

mobile phase gradient (by volume):

at 0min, 1% acetonitrile and 99% 0.1% v/v H₂SO₄An aqueous solution of a carboxylic acid and a carboxylic acid,

at 10min, 5% acetonitrile and 95% 0.1% v/v H₂SO₄An aqueous solution of a carboxylic acid and a carboxylic acid,

at 40min, 10% acetonitrile and 90% 0.1% v/v H₂SO₄An aqueous solution.

5. A complex having the formula (II):

having a diastereomeric excess of at least 92%, comprising a mixture of isomers II-RRR and II-SSS of the formula:

6. a complex as claimed in claim 5, characterised in that the diastereomeric excess is at least 94%.

7. A complex as claimed in claim 5, characterised in that the diastereomeric excess is at least 97%.

8. The complex according to claim 5, characterized in that isomers II-RRR and II-SSS are present in the mixture in a ratio between 55/45 and 45/55.

9. The complex according to claim 5, characterized in that said diastereomeric excess corresponds to peak 4 of the UHPLC profile, characterized by a retention time of about 6.3 minutes, said profile being obtained using the UHPLC method:

·WatersUPLC T3150x2.1mm-1.6 μm column,

analysis conditions:

-a sample: 2.0mg/mL of an aqueous solution of the complex having formula (II),

column temperature: at a temperature of 40 c,

-sample temperature: at room temperature (20 ℃ -25 ℃),

-flow rate: 0.3mL/min of the water-soluble polymer,

-sample size: 1 μ L

-UV detection: the particle size of the nano-particles is 200nm,

mobile phase gradient (by volume):

at 0min, 1% acetonitrile and 99% 0.0005% v/v H₂SO₄An aqueous solution of a carboxylic acid and a carboxylic acid,

at 3min, 5% acetonitrile and 95% 0.0005% v/v H₂SO₄An aqueous solution of a carboxylic acid and a carboxylic acid,

at 12min, 10% acetonitrile and 90% 0.0005% v/v H₂SO₄An aqueous solution.

10. The complex according to any one of claims 5 to 9, obtained by amidation from the gadolinium hexacarboxylate complex of formula (I) according to any one of claims 1 to 4 with 3-amino-1, 2-propanediol.

11. A complex having the formula (VII):

wherein Y represents a chlorine atom, -OR₁or-O-C (O) -R₂Group, wherein R₁And R₂Independently of each other correspond to (C)₁-C₆) An alkyl group, a carboxyl group,

having a diastereomeric excess of at least 80%, comprising a mixture of isomers VII-RRR and VII-SSS of the formula:

12. a complex as claimed in claim 11, wherein Y represents-OR₁A group, the complex being a triester gadolinium complex having the formula (VIII):

having a diastereomeric excess of at least 80%, comprising a mixture of isomers VIII-RRR and VIII-SSS of the formula:

13. a complex as claimed in claim 12, characterised in that the diastereomeric excess is at least 85%.

14. A complex as claimed in claim 12, characterised in that the diastereomeric excess is at least 90%.

15. A complex as claimed in any one of claims 12 to 14 characterised in that R₁Is a methyl group.

Technical Field

The present invention relates to a novel process for the synthesis of complexes of gadolinium and chelate ligands based on PCTA, which makes it possible to obtain preferentially stereoisomers of said complexes having particularly advantageous physicochemical properties for use as contrast agents in the field of medical imaging, in particular in the field of magnetic resonance imaging. The invention also relates to the diastereoisomerically enriched complex itself and also to two synthetic intermediates optionally containing gadolinium.

Background

Many contrast agents based on chelates of lanthanides (paramagnetic metals), especially gadolinium (Gd), are known, for example as described in US 4647447. These products are generally grouped under the term GBCA (gadolinium based contrast agent). There are several products on the market in which macrocyclic chelates are present, such as meglumine gadolinate based on DOTA (1,4,7, 10-tetraazacyclododecane-N, N ', N ", N'" -tetraacetic acid), gadobutrol based on DO3A-butrol, gadoteridol based on HPDO3A, and also linear chelates, in particular based on DTPA (diethylenetriaminepentaacetic acid) or DTPA-BMA (gadodiamide ligand).

Other products, some of which are under development, represent a new generation of GBCA. They are essentially complexes of macrocyclic chelates, such as the bicyclic polyazamacrocyclic carboxylic acid complex (EP 0438206) or PCTA derivatives (i.e. derivatives comprising at least the chemical structure of 3,6,9, 15-tetraazabicyclo [9,3,1] pentadecane-1 (15),11, 13-triene-3, 6, 9-triacetic acid), as described in EP 1931673.

The complexes of chelating ligands based on PCTA described in EP 1931673 have in particular the following advantages: chemical synthesis is relatively easy and also has a relaxation rate (relaxation rate r) that is superior to the relaxation rates of other GBCAs currently on the market₁Up to 11-12mM in water^-1.s^-1) This relaxation ofThe rates correspond to the efficiency of these products and therefore to their comparative ability.

In organisms, chelates (or complexes) of lanthanides (and in particular gadolinium) are in a state of chemical equilibrium (by virtue of their thermodynamic constant K)_thermCharacterized) which may lead to an undesired release of said lanthanide (see reaction formula 1 below):

the complex chemical equilibrium between the chelate or ligand (Ch) and the lanthanide (Ln) produces the complex Ch-Ln.

Since 2006, a disease called NSF (nephrogenic systemic fibrosis or fibrogenic skin disease) has been associated at least in part with the release of free gadolinium in vivo. This disease has led health authorities to issue alerts for the sale of gadolinium based contrast agents to certain categories of patients.

Therefore, some strategies have been taken to solve the complex problem of patient tolerance in a completely safe manner and to limit or even eliminate the risk of undesired release of lanthanides after administration. This problem is made more difficult to solve because the administration of contrast agents is often repeated both during the diagnostic examination and during dose adjustment and monitoring of the therapeutic effect.

Furthermore, since 2014, it has been mentioned that gadolinium may be deposited in the brain after repeated administration of products based on gadolinium, in particular linear gadolinium chelates, in particular gadolinium macrocycle chelates (e.g. gadolinium macrocycle chelates)) Little or no such deposition has been reported. Therefore, in view of insufficient stability, countries decide to withdraw most of the linear chelators from the market or greatly limit their instructions for use.

Therefore, a strategy to limit the risk of lanthanide release into the body is to select complexes characterized by as high thermodynamic and/or kinetic stability as possible. The reason for this is that the more stable the complex, the more limited the amount of lanthanide released over time will be.

However, complexes comprising PCTA-based chelating ligands of pylene-type structure as described in EP 1931673, although having good kinetic stability, generally have lower thermodynamic constants than complexes of other cycloalkene-based macrocycles.

This is particularly the case for complexes of formula (II) having the following representation:

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

It should be noted, however, that the complex of formula (II) corresponds to several stereoisomers, in particular due to the presence of three asymmetric carbon atoms in position a on the side chain of the complex with respect to the nitrogen atom of the macrocycle onto which the side chain is grafted. These three asymmetric carbons are marked with an asterisk in formula (II) shown above.

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

The aminopropanediol group having the side chain of the complex of formula (II) also includes an asymmetric carbon. Thus, the complex of formula (II) contains a total of six asymmetric carbons and thus exists as 64 configurational stereoisomers. However, in the remainder of the description, for the sake of simplicity, the only source of stereoisomers considered for a given side chain will be the source corresponding to the asymmetric carbon bearing a carboxylate group, which is marked with an asterisk in formula (II) above.

Since each of these three asymmetric carbons can have the absolute configuration of R or S, the complexes having formula (II) exist in the form of eight stereoisomeric families, hereinafter referred to as II-RRR, II-SSS, II-RRS, II-SSR, II-RSS, II-SRR, II-RSR and II-SRS. More precisely, according to the usual nomenclature in stereochemistry, the complexes having formula (II) exist in the form of eight diastereoisomeric families.

As mentioned before, it is reasonable to use the term "family" because each of these families combines several stereoisomers together, in particular due to the presence of asymmetric carbons in the aminopropanediol group.

However, since the stereoisomers associated with the asymmetric carbons of a given aminopropanediol 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 indiscriminately without necessarily stating that each corresponds to a family of stereoisomers.

The inventors have succeeded in separating and identifying, by High Performance Liquid Chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), the four unresolved peaks or isomeric groups of complexes having formula (II) obtained according to methods of the prior art, corresponding to four different elution peaks characterized by their retention time on the chromatogram, which will be referred to as iso1, iso2, iso3 and iso4 in the remainder of the present description. By carrying out the process described in EP 1931673, the respective contents of groups iso1, iso2, iso3 and iso4 in the resulting mixture are as follows: 20%, 40% and 20%.

Then, they found that these different isomer groups had different physicochemical properties, and determined that the isomer group called iso4, which comprises a mixture of isomers II-RRR and II-SSS having the formulae (II-RRR) and (II-SSS) represented below, proved to be the most advantageous medical imaging contrast agent.

Thus, surprisingly, iso4 is characterized by its thermodynamic stability which is significantly better than that of the mixture of diastereomers in the form of the complex having formula (II) obtained by carrying out the process described in EP 1931673. In particular, its equilibrium thermodynamic constant K_thermiso4 equal to 10^18.7(i.e., log (K)_thermiso4) ═ 18.7), which has been determined by performing the method described in Pierrard et al, Contrast Media mol.imaging,2008,3, 243-.

Furthermore, iso4 is the set of isomers with the best kinetic inertia (also called kinetic stability) among the four groups isolated by the inventors. Specifically, the inventors evaluated the kinetic inertia of four groups of isomers by studying their decomplexation kinetics in acidic aqueous solution (pH ═ 1.2) at 37 ℃. The half-life time values (T) determined for each set of isomers are listed in Table 1 below_1/2) This half-life time corresponds to the time at which 50% of the amount of complex initially present dissociates according to the following decomplexation reaction (equation 2):

(reaction formula 2)

Isomer group	T1/2(pH 1.2-37℃)
		Iso1	18 hours
Iso2	6 hours
		Iso3	8 days
Iso4	27 days

[ Table 1 ]: decomplexation kinetics of isomers iso1 to iso4

For comparison purposes, gadobutrol or gadolinate, which are macrocyclic gadolinium complexes, have kinetic inertias of 18 hours and 4 days, respectively, under the same conditions, whereas linear gadolinium complexes (e.g. gadolinium diamine or gadolinium pentanate) dissociate instantaneously.

Furthermore, iso4 is in particular more chemically stable than iso 3. The reason for this is that the amide function of the complex having formula (II) is easily hydrolyzed. The hydrolysis reaction of the amide function (equation 3) results in the formation of the double-coupled impurity, which is accompanied by the release of 3-amino-1, 2-propanediol. The inventors studied the kinetics of the hydrolysis reaction of the complex of formula (II) in aqueous solution at pH 13 and observed that the amide function of iso4 is more stable in hydrolysis with respect to the amide function of iso 3.

With respect to the relaxivity of the isomers of each group (i.e. their efficiency as contrast agents), measurements were made showing that the contrast capacity of the iso1, iso2 and iso4 groups was relatively comparable, while the efficiency of iso3 was reduced (see table 2).

[ Table 2 ]: relaxation rates at 37 ℃ for the isomeric groups iso1 to iso4

The inventors have successfully developed a new process for the preparation and purification of the complex of formula (II) which makes it possible to obtain preferentially the diastereoisomers II-RRR and II-SSS of said complex with particularly advantageous physicochemical properties. The process according to the invention comprises a step of enriching the isomers by converting the least stable stereoisomer into the most stable stereoisomer, which surprisingly makes it possible to obtain in most cases the most stable isomer of the complex having formula (II) when carried out on the hexa-acid intermediate complex and not on the final complex.

Compared to the alternative of preparing mixtures of stereoisomers, it is clearly advantageous to carry out a process which makes it possible in most cases to obtain the desired diastereoisomers, in order subsequently to attempt to separate the diastereoisomers according to conventional techniques, in order to isolate the desired isomer. In particular, the absence of separation allows, on the one hand, to save considerable time and, on the other hand, to increase the overall yield of the process by limiting as much as possible the production of undesired diastereoisomers which will eventually be eliminated, in addition to making it easier to carry out the process on an industrial scale, which does not involve a step of separation of the diastereoisomers. Furthermore, conventional separation techniques typically involve the use of large amounts of solvent, which exceeds financial costs, which is undesirable for environmental reasons. Furthermore, silica chromatography should be avoided in particular in view of the health risks inherent in occupational exposure to silica, which international cancer research institutes classify as carcinogenic for humans (group 1).

As previously mentioned, the method developed by the inventors for the preparation of the complex having formula (II) is based on the step of isomer enrichment of the intermediate gadolinium hexaate complex having formula (I) represented below:

the complexes of formula (I) correspond to several stereoisomers, since there are three asymmetric carbon atoms in the alpha position on the side chain of the complex with respect to the nitrogen atom of the macrocycle onto which the side chain is grafted. The three asymmetric carbons are marked with an asterisk in formula (I) shown above.

Since each of the three asymmetric carbons bearing a carboxylate functionality may have the absolute configuration R or S, the complexes having formula (I) exist in the form of eight stereoisomers, hereinafter referred to as I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR and I-SRS. More precisely, according to the usual nomenclature in stereochemistry, the complex having formula (I) exists in the form of four pairs of enantiomers (enantiomers) as diastereoisomers of each other.

The inventors have succeeded in isolating and identifying, by High Performance Liquid Chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), the four unresolved peaks or isomeric groups of complexes having formula (I) obtained according to the method described in EP 1931673, corresponding to four different elution peaks characterized by their retention time on the chromatogram, which will be referred to as isoA, isoB, isoC and isoD in the remainder of the present description.

IsoD was crystallized in water. X-ray diffraction analysis enabled the inventors to determine the crystal structure of this set of isomers and thus found that it comprises the diastereoisomers I-RRR and I-SSS of formulae (I-RRR) and (I-SSS) represented below of the complex having formula (I).

It should be noted that the diastereoisomers of the complex having formula (I), I-RRR and I-SSS, are enantiomers of each other.

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

The synthesis of the complex having formula (II) in particular involves the conversion of the carboxylic acid function of the intermediate hexa-acid complex having formula (I) into an amide function. This amidation reaction does not change the absolute configuration of the three asymmetric carbon atoms of the complex having formula (I).

Thus, when amidation reaction of the hexaacid complex of formula (I) enriched in isoD previously obtained, the complex of formula (II) enriched in iso4 can be obtained.

Disclosure of Invention

Gadolinium hexamate complexes having formula (I)

Accordingly, the present invention relates firstly to a gadolinium hexaate complex having formula (I):

it consists of a diastereomeric excess of at least 80%, comprising a mixture of isomers I-RRR and I-SSS having the formula:

in the context of the present invention, the term "diastereomeric excess" is intended to represent the fact that with respect to a gadolinium hexacarboxylate complex having formula (I), said complex is predominantly present in the form of an isomer or group of isomers of a diastereoisomer selected from: I-RRR, I-SSS, I-RRS, I-SSR, I-RSS, I-SRR, I-RSR, and I-SRS. The diastereomeric excess is expressed as a percentage and corresponds to the amount represented by the major isomer or group of isomers relative to the total amount of gadolinium hexacarboxylate complex having formula (I). It is to be understood that the percentage can be on a molar basis or on a mass basis, since, by definition, the isomers have the same molar mass.

In a particular embodiment, the complex of formula (I) according to the invention has a diastereomeric excess of at least 85%, in particular at least 90%, in particular at least 95%, preferably at least 97%, advantageously at least 98%, more advantageously at least 99% of the mixture comprising isomers I-RRR and I-SSS.

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

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

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

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

More specifically, the diastereomeric excess as defined before corresponds to peak 4 in the HPLC profile (i.e. the fourth peak in elution order and corresponding to isoD), which is characterized by a retention time of between 33.9 and 37.5 minutes, typically about 35.7 minutes, obtained using the HPLC method described below.

Within the meaning of the present invention, the term "HPLC profile" refers to the profile of the concentration measured by the detector as a function of time after the passage and separation of a mixture of compounds (in this case isomers of compounds) on the stationary phase, for a given composition and a given eluent flow rate. The HPLC profile consists of various peaks or unresolved peak features of the compound or mixture of compounds analyzed.

HPLC method:

Watersc18-250x4.6mm-5 μm column.

This is a reversed phase UPLC column containing spherical silica particles having C18 (octadecyl) graft and silanol that has been treated with an end-capping agent (end-capping). It is also characterized by a length of 250mm, an inner diameter of 4.6mm, a particle diameter of 5 μm,Porosity and a carbon content of 19%.

Preferably, the fixation used is compatible with aqueous mobile phase.

Analysis conditions were as follows:

mobile phase gradient (by volume):

complexes of formula (II)

Secondly, the present invention relates to a complex having the formula (II):

it consists of a diastereomeric excess of at least 80%, comprising a mixture of isomers II-RRR and II-SSS having the formula:

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

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

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

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

By extension, the term "mixture of isomers II-RRR and II-SSS" also covers the case where only one isomer is present, whether II-RRR or II-SSS is present. However, the term "mixture of isomers II-RRR and II-SSS" preferably represents all cases in which each of the isomers II-RRR and II-SSS is present in variable but non-zero amounts.

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

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

Within the meaning of the present invention, the term "UHPLC profile" refers to the profile of the concentration measured by the detector as a function of time after the passage and separation of a mixture of compounds (in this case isomers of compounds) on the stationary phase, for a given composition and a given eluent flow rate. The UHPLC plots consist of various peaks or unresolved peak features of the compound or mixture of compounds analyzed.

UHPLC method:

WatersUPLC T3150x2.1mm-1.6 μm column.

This is a reversed phase UPLC column containing spherical particles consisting of a preferably very hard core made of silica surrounded by a trifunctional C18 (octadecyl) grafted porous silica, and the silanols have been treated with a blocking agent (end-blocking). It is also characterized by a length of 150mm, an inner diameter of 2.1mm, a particle diameter of 1.6 μm,Porosity and carbon content of 4.7%.

Preferably, the fixation used is compatible with aqueous mobile phase.

Analysis conditions were as follows:

mobile phase gradient (% v/v):

in a preferred embodiment, the complex according to the invention having formula (II) is obtained by: starting from the complex according to the invention of formula (I) as defined above, amidation is carried out with 3-amino-1, 2-propanediol in racemic or enantiomerically pure form, preferably in racemic form.

Within the meaning of the present invention, the term "amidation" refers to a reaction for converting a carboxylic acid function into an amide function by reaction with an amine function.

This reaction can be carried out in particular after activation of the carboxylic acid function, as detailed hereinafter in the description.

Process for preparing a complex having formula (II)

The invention also relates to a process for preparing a complex having formula (II), comprising the following successive steps:

a) reacting a hexa acid having the following formula (III):

complexing with gadolinium to obtain a gadolinium hexaate complex of formula (I) as defined above,

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

c) starting from this diastereoisomerically enriched complex obtained in step b), a complex of the formula (II) is formed by reaction with 3-amino-1, 2-propanediol.

In the present specification, the terms "Gd", "gadolinium" and "Gd" are used indiscriminately, unless otherwise stated³⁺"to represent Gd³⁺Ions. By extension, it may also be a source of free gadolinium, such as gadolinium chloride (GdCl)₃) Or gadolinium oxide (Gd)₂O₃)。

In the present invention, the term "free Gd" represents the uncomplexed form of gadolinium, which is preferably available for complexation. Typically Gd dissolved in water³⁺Ions. By extension, it may also be a source of free gadolinium, for exampleGadolinium chloride (GdCl)₃) Or gadolinium oxide.

■ step a)

During this step, a complexation reaction occurs between the hexa-acid of formula (III) and gadolinium, which enables to obtain a gadolinium hexa-acid complex of formula (I) as defined before.

According to a specific embodiment, step a) comprises a reaction between the hexa-acid having formula (III) and a source of free Gd in water.

In a preferred embodiment, the source of free Gd is GdCl₃Or Gd₂O₃Preferably Gd₂O₃。

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

Thus, the source of gadolinium will advantageously be gadolinium oxide, the purity of which is preferably greater than 99.99%, even more preferably greater than 99.999%.

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

Advantageously, the amount of reagent (hexa-and gadolinium with formula (III) used in this step a) corresponds to or is close to the stoichiometric ratio, as determined by the equilibrium equation of the complexation reaction that takes place in this step.

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

It is noted that the gadolinium may be introduced in a slight excess with respect to the stoichiometric ratio. The ratio of the amount of material introduced as gadolinium to the amount of material introduced as hexa-acid having formula (III) is then greater than 1, but typically less than 1.15, in particular less than 1.10, advantageously less than 1.08. In other words, the gadolinium introduced is such that it is relative to the amount of hexa-acid of formula (III) introduced (which itself corresponds to 1 equivalent)The amount is greater than 1 equivalent (eq.), but is typically less than 1.15eq, in particular less than 1.10eq, advantageously less than 1.08eq. Wherein the source of free gadolinium is Gd₂O₃In a preferred embodiment, the Gd (lll) is introduced relative to the amount (1eq.) of the hexa-acid of formula (III) introduced₂O₃The amount of (d) is typically greater than 0.5eq, but less than 0.575eq, in particular less than 0.55eq, advantageously less than 0.54eq.

According to a particular embodiment, step a) comprises the following successive steps:

a1) preparing an aqueous solution of a hexa-acid having formula (III), and

a2) adding a source of free gadolinium to the aqueous solution obtained in step a 1).

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

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

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

■ step b)

The gadolinium hexaate complex having the formula (I) formed by the complexation reaction between the hexa acid having the formula (III) and gadolinium in step a) is first obtained as a mixture of diastereomers.

Step b) involves enriching the mixture of diastereomers in the isomers I-RRR and I-SSS to obtain a diastereoisomerically enriched gadolinium hexaacid complex of formula (I) consisting of a diastereoisomeric excess of the mixture comprising the isomers I-RRR and I-SSS of at least 85%, in particular at least 90%, in particular at least 95%, preferably at least 97%, advantageously at least 98%, more advantageously at least 99%.

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

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

In fact, the inventors have found that factors such as pH and temperature of the solution of gadolinium hexacarboxylate complex of formula (I) obtained at the end of step a) have an effect on the ratio of the various isomers of the complex of formula (I) present in the mixture of diastereoisomers. Over time, the mixture tends to enrich a set of isomers, which comprises the isomers that are surprisingly most thermodynamically stable but also chemically stable, in this case the isomers I-RRR and I-SSS.

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

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

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

The pH is preferably adjusted with an acid, preferably a mineral acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, for example with hydrochloric acid.

It is entirely surprising that under such pH conditions an enrichment of the mixture, in particular the isomers, in this case the isomers I-RRR and I-SSS, takes place, since this is the state of the artGadolinium chelates are known to be characterized by low kinetic inertia in acidic media. In particular, H in the medium⁺The higher the concentration of the ion, the greater the probability of proton transfer to one of the donor atoms of the ligand, resulting in complex dissociation. Thus, one skilled in the art would expect that placing the gadolinium hexaate complex of formula (I) in an aqueous solution at a pH between 2 and 4 results in dissociation of the complex, rather than isomerization to I-RRR and I-SSS.

It should be noted that EP 1931673 does not make it possible to obtain complexes of formula (I) enriched in its isomers I-RRR and I-SSS for the pH range recommended for the complexation of the hexaacids of formula (III), i.e. 5.0-6.5.

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

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

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

Preferably, acetic acid is added in an amount such that the content of acetic acid is between 25% and 75% by mass, in particular between 40% and 50% by mass, with respect to the mass of the hexa-acid of formula (III) used in step a), before heating the solution of gadolinium hexa-acid complex of formula (I) obtained in step a).

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

According to a preferred embodiment, at the end of step b), the diastereoisomerically enriched complex is isolated by crystallization, preferably by crystallization from seed crystals.

In this embodiment, step b) comprises the following successive steps:

b1) isomerisation is carried out by heating a gadolinium hexaacid complex having formula (I) in an aqueous solution having a pH between 2 and 4 to obtain a diastereoisomerically enriched complex constituted by a diastereoisomeric excess comprising a mixture of isomers I-RRR and I-SSS of said gadolinium hexaacid complex having formula (I) of at least 80%, and

b2) the diastereoisomerically enriched complex is isolated by crystallization, preferably by crystallization from seed crystals.

Crystallization step b2) firstly involves removing any impurities present in the aqueous solution, which may have resulted from the previous step, thus obtaining a decolorized product of higher purity in the form of crystals, and secondly involves continuing the diastereoisomeric enrichment of the gadolinium hexacarboxylate complex having formula (I) to obtain a diastereoisomeric excess of the mixture comprising the isomers I-RRR and I-SSS of said complex, which is higher than that obtained at the end of step b 1).

In particular, the isomers I-RRR and I-SSS of the hexaacid complex having formula (I) are crystallized from water. On the other hand, gadolinium hexaate complexes of formula (I) not enriched in the isomers do not crystallize.

The fact that the isomers I-RRR and I-SSS in which the complex tends to be enriched during step b) (and contrary to all expectations, depending on the conditions under which it is carried out) are the only isomers of the complex crystallized from water is a totally unexpected result. Thus, the isomerization and crystallization synergy contributes to the enrichment of the isomers I-RRR and I-SSS and thus to the overall efficiency of the process according to the invention.

Furthermore, it should be noted that the crystallization in water of the target isomer of gadolinium hexamate complex of formula (I) makes it possible to avoid the addition of solvents as described in example 7 of EP 1931673, which involves the step of precipitating the trisodium salt of the complex from ethanol.

Step b2) is advantageously carried out at a temperature between 10 ℃ and 70 ℃, in particular between 30 ℃ and 65 ℃, in particular between 35 ℃ and 60 ℃.

According to a variant, after lowering the temperature of the aqueous solution so that it is within the above-mentioned range, the crystallization process is induced by seeding. "crystallization by seeding", also known as "crystallization by seeding", comprises introducing a known amount of crystals (referred to as "seeds" or "primers") into a reactor (also referred to as a crystallization vessel) in which crystallization is carried out. This enables 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, the crystallization, in the present case crystals of the diastereoisomerically enriched gadolinium hexacarboxylate complex of formula (I) added to an aqueous solution of the diastereoisomerically enriched complex previously reduced in temperature, using a primer, enables nucleation to be obtained, thus initiating crystallization. The duration of the crystallization by seeding is advantageously between 2 and 20 hours, and preferably between 6 and 18 hours; typically 16 hours.

The crystals of the diastereoisomerically enriched gadolinium hexacarboxylate complex having formula (I) are then typically isolated by filtration and drying by any technique well known to those skilled in the art.

Advantageously, the purity of the diastereoisomerically enriched gadolinium hexacarboxylate complex having formula (I) isolated at the end of step b2) is greater than 95%, in particular greater than 98%, advantageously greater than 99%, expressed as a percentage by mass of the complex having formula (I) with respect to the total mass obtained at the end of step b 2).

In one embodiment, the diastereoisomerically enriched complex from step b) isolated by crystallization is purified again by recrystallization to obtain a diastereoisomerically enriched and purified complex.

In this embodiment, step b) comprises, in addition to the successive steps b1) and b2) previously described, a step b3) of purifying the diastereoisomerically enriched gadolinium hexacarboxylate complex of formula (I) isolated by recrystallization.

The recrystallization step b3) is, like the crystallization step b2), firstly directed to obtaining a product of higher purity and secondly directed to continuing the diastereoisomeric enrichment of the gadolinium hexaacid complex having formula (I) in order to obtain a diastereoisomeric excess of the mixture comprising the isomers I-RRR and I-SSS of said complex, which is higher than that obtained at the end of step b 2).

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

● the diastereoisomerically enriched gadolinium hexacarboxylate complex of the formula (I) isolated in step b2) is suspended in an aqueous solution, preferably in water,

● the complex is dissolved by heating to a temperature advantageously between 80 ℃ and 120 ℃, for example to a temperature of 100 ℃,

● recrystallization, preferably by seeding, advantageously at a temperature between 10 ℃ and 90 ℃, in particular between 20 ℃ and 87 ℃, in particular between 55 ℃ and 85 ℃, typically for a time between 2 hours and 20 hours, in particular between 6 hours and 18 hours, and

● isolating the diastereoisomerically enriched and purified crystals of gadolinium hexacarboxylate complex of formula (I), for example by filtration and drying.

The purity of the purified diastereoisomerically enriched gadolinium hexacarboxylate complex of formula (I) isolated at the end of step b3), expressed as the percentage by mass of the complex of formula (I) with respect to the total mass obtained at the end of step b2), is typically greater than 98%, in particular greater than 99%, advantageously greater than 99.5%.

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

In this embodiment, step b) comprises, in addition to the successive steps b1) and b2) previously described, a step b4) of selective decomplexing the diastereomer of the complex having formula (I) except for the diastereoisomers I-RRR and I-SSS). In this variant, step b) may also comprise the previously described step b3), said step b3) being performed between steps b2) and b4) or after b 4).

The selective decomplexation step b4) involves continuing the diastereoisomeric enrichment of the gadolinium hexacarboxylate complex having formula (I) so as to obtain a diastereoisomeric excess comprising a mixture of the isomers I-RRR and I-SSS of said complex, which is higher than the diastereoisomeric excess obtained at the end of step b2) or at the end of step b3) when said step is carried out before step b 4).

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

● the diastereoisomerically enriched gadolinium hexacarboxylate complex of the formula (I) isolated in step b2) or step b3) is suspended in water,

● adding a base, such as sodium hydroxide,

● to a temperature advantageously between 30 ℃ and 60 ℃, in particular between 35 ℃ and 55 ℃, for example 40 ℃, typically for a time between 2 hours and 20 hours, in particular between 10 hours and 18 hours,

● to a temperature advantageously between 10 ℃ and 30 ℃, for example to 30 ℃, and

● isolating the diastereoisomerically enriched and purified gadolinium hexacarboxylate complex of formula (I), for example by filtration and drying.

The fact that the isomers I-RRR and I-SSS are the most stable in alkaline medium makes step b4) possible. Such basic conditions promote the formation of gadolinium hydroxide, thus promoting the decomplexation of the least stable isomer.

It should therefore be noted that, surprisingly, the isomers I-RRR and I-SSS are both more stable in an acidic medium allowing the isomerization step b1) to be carried out and in a basic medium allowing the selective decomplexation step b4) to be carried out.

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

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

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

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

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

■ step c)

Step c) involves forming a complex having formula (II) from its precursor, i.e. the diastereoisomerically enriched gadolinium hexacarboxylate complex having formula (I) obtained in step b).

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

This amidation reaction does not change the absolute configuration of the three asymmetric carbon atoms alpha to the side chain relative to the nitrogen atom of the macrocycle onto which the side chain is grafted. Thus, step c) makes it possible to obtain a complex of formula (II) with a diastereomeric excess comprising a mixture of isomers II-RRR and II-SSS, which is the same as the diastereomeric excess of a mixture comprising isomers I-RRR and I-SSS, with at least 80% of the diastereomeric excess of this mixture comprising isomers I-RRR and I-SSS obtaining the diastereomerically enriched gadolinium hexaacid complex of formula (I) obtained at the end of step b).

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

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

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

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

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

The amidation reaction may be carried out according to any method known to the person skilled in the art, in particular in the presence of a reagent for activating the carboxylic acid function and/or by acid catalysis.

It can be carried out in particular according to the method described in EP 1931673, in particular in paragraph [0027] of said patent.

In a particular embodiment, step C) comprises activating a carboxylic acid (-COOH) functional group carried by the carbon atom located in position γ on the side chain of the hexa-acid complex having formula (I), relative to the nitrogen atom of the macrocycle on which the side chain is grafted, in the form of a derivatised 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 embodiment, the carboxylic acid function can be activated in particular in the form of an ester, an acid chloride or an anhydride function, or in any activated form that can give rise to an amide bond. Activated forms that can give rise to 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 the formation of peptide bonds. Examples of such processes are given in the publications Synthesis of peptides and peptides, volume E22a, page 425-588, Houben-Weyl et al, Goodman's editor, Thieme-Stuttgart-New York (2004), and mention may in these examples be made in particular of processes for activating carboxylic acids by means of an azide (acylazide), for example by the action of an agent, such as diphenylphosphoryl azide (commonly abbreviated to DPPA), by means of a carbodiimide, alone or in the presence of a catalyst, such as N-hydroxysuccinimide and derivatives thereof, by means of carbonyldiimidazole (1, 1' -carbonyldiimidazole, CDI), by means of a phosphonium salt, such as benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (commonly abbreviated to BOP), or other uronium salts, such as 2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate (commonly abbreviated as HBTU).

Preferably, step c) comprises the activation of the above carboxylic acid (-COOH) functional groups in the form of ester, acid chloride or anhydride functional groups.

This embodiment is more preferred than the peptide coupling described in EP 1931673 using a coupling agent (e.g. EDCI/HOBT) to effect peptide coupling by activating the carboxylic acid function. In particular, this coupling results in the formation of one equivalent of 1-ethyl-3- [3- (dimethylamino) propyl ] urea, which must be removed, in particular by silica gel chromatography or by liquid/liquid extraction with the addition of a solvent. As previously discussed, it is not desirable to use such purification methods independent of the increased complexity of the method resulting from such additional steps. Furthermore, the use of HOBT is inherently problematic because it is an explosive product.

Within the meaning of the present invention, the term "ester function" is intended to represent a-C (O) O-group. It may in particular be a group-C (O) O-R₁Wherein R is₁Corresponds to (C)₁-C₆) An alkyl group.

Within the meaning of the present invention, the term "(C)₁-C₆) Alkyl group "means a straight or branched saturated hydrocarbon chain containing from 1 to 6, preferably from 1 to 4, carbon atoms. Examples which may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl groups.

Within the meaning of the present invention, the term "acid chloride function", also referred to as "acid chloride function", is intended to represent a-CO-Cl group.

Within the meaning of the present invention, the term "anhydride function" is intended to represent a-CO-O-CO-group. It may in particular be a group-CO-O-CO-R₂Wherein R is₂Corresponds to (C)₁-C₆) An alkyl group.

The reactions for converting carboxylic acid functions into ester, acid chloride or anhydride functions are well known to the person skilled in the art and they will be able to carry out the reaction according to any of the conventional methods with which he is familiar.

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

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

According to a particular embodiment, step c) comprises the following successive steps:

c1) forming an activated complex having the formula (VII),

wherein Y represents a chlorine atom, -OR₁or-O-C (O) -R₂A group; preferably, Y represents-OR₁or-O-C (O) -R₂Group, wherein R₁And R₂Independently of each other correspond to (C)₁-C₆) An alkyl group, and

c2) aminolysis of an activated complex having formula (VII) with 3-amino-1, 2-propanediol.

It is very obvious to the person skilled in the art that the reaction to form the activated complex of formula (VII) does not change the absolute configuration of the three asymmetric carbon atoms located alpha to the side chain with respect to the nitrogen atom of the macrocycle onto which said side chain is grafted. Thus, step c1) enables obtaining an activated complex of formula (VII) with a diastereomeric excess comprising a mixture of isomers VII-RRR and VII-SSS with the formulae (VII-RRR) and (VII-SSS) represented below, which is the same as the diastereomeric excess of a mixture comprising isomers I-RRR and I-SSS, the diastereomeric excess of this mixture comprising isomers I-RRR and I-SSS being at least 80% of the diastereomeric excess of the diastereomeric enriched gadolinium hexaacid complex of formula (I) obtained at the end of step b).

In the case where Y represents a chlorine atom, step c1) is typically carried out by obtaining the diastereoisomer having formula (I) in step b)Body-enriched gadolinium hexa-carboxylate complex with thionyl chloride (SOCl)₂) To proceed with the reaction between them.

In which Y represents-O-C (O) -CH₃In the case of radicals, step c1) is typically carried out by reaction between the diastereoisomerically enriched gadolinium hexacarboxylate complex of formula (I) obtained in step b) and acetyl chloride.

In an advantageous embodiment, step c) comprises the activation of the above carboxylic acid (-COOH) functional group in the form of an ester functional group.

According to this embodiment, step c) may more particularly comprise the following successive steps:

c1) to form a triester having the formula (VIII),

wherein R is₁Is represented by (C)₁-C₆) An alkyl group, and

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

Step c1) is typically carried out in the presence of an acid (e.g. hydrochloric acid) in the presence of a solvent and a reagent both of formula R₁In OH alcohol.

In a first stage, a gadolinium hexaate complex having the formula (I) and an alcohol R₁OH is put into the reactor. The reaction medium is then cooled to a temperature of less than 10 ℃, in particular less than 5 ℃, typically to 0 ℃, and the alcohol R is then added gradually₁Acidic solution of OH, typically hydrochloric acid, in R₁Solution in OH. The reaction medium is kept stirred at room temperature (i.e. at a temperature between 20 ℃ and 25 ℃) for a period typically greater than 5 hours, preferably between 10 hours and 20 hours. Prior to step c2), the reaction medium is cooled to a temperature below 10 ℃, in particular between 0 ℃ and 5 ℃.

Step c2) is also typically carried out in the presence of an acid (e.g. hydrochloric acid) in a solvent of formula R₁In OH alcohol.

Thus, according to a one-pot embodiment, steps c1) and c2) can be easily performed. Advantageously, the triester of formula (VII) is not isolated between steps c1) and c 2).

However, in order to facilitate the aminolysis reaction, in step c2), the removal of the compound of formula R is preferably carried out by vacuum distillation₁Alcohol of OH.

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

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

Surprisingly, no decomplexing or isomerization of the triester of formula (VIII) was observed, although the acidic conditions typically employed in steps c1) and c2) increased the kinetic instability of the gadolinium complex. The desired triamide is obtained with a very good degree of conversion and the absolute configuration of the three asymmetric carbon atoms located in the alpha position on the side chain is preserved with respect to the nitrogen atom of the macrocycle.

Furthermore, it should be noted that amidation reactions by direct reaction between esters and amines are often described only rarely in the literature (see, for this subject, k.c. nadimpaly et al, Tetrahedron Letters,2011,52, 2579-.

In a preferred embodiment, step c) comprises the following successive steps:

c1) to form a methyl triester having the formula (IV),

especially in the presence of an acid, such as hydrochloric acid, in methanol, and

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

Advantageously, the methyl triester of formula (IV) is not isolated between steps c1) and c 2).

In a preferred embodiment, in step c2), methanol is removed by vacuum distillation until a temperature typically above 55 ℃, in particular between 60 ℃ and 65 ℃, is reached and the reaction medium is kept at this temperature under vacuum for a time typically greater than 5 hours, in particular between 10 and 20 hours, before cooling to room temperature and dilution with water.

The invention includes all combinations of the specific, advantageous or preferred embodiments described above in connection with each step of the method.

The invention also relates to a triester gadolinium complex having formula (VIII):

it consists of a diastereomeric excess of at least 80%, comprising a mixture of isomers VIII-RRR and VIII-SSS having the formula:

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

In a particular embodiment, the triester gadolinium complex having formula (VIII) according to the invention has a diastereomeric excess of the mixture comprising isomers VIII-RRR and VIII-SSS of at least 85%, in particular at least 90%, in particular at least 95%, preferably at least 97%, advantageously at least 98%, more advantageously at least 99%.

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

Advantageously, the diastereomeric excess consists of a mixture of the isomers VIII-RRR and VIII-SSS.

The term "mixture of isomers VIII-RRR and VIII-SSS" also covers the case where only one isomer is present, whether VIII-RRR or VIII-SSS is present. However, the term "mixture of isomers VIII-RRR and VIII-SSS" preferably represents all cases in which each of the isomers VIII-RRR and VIII-SSS is present in variable but non-zero amounts.

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

In a preferred embodiment, the triester gadolinium complex having formula (VIII) according to the invention is a trimethyl gadolinium complex, i.e. wherein R is₁Is a methyl group (CH)₃) The triester gadolinium complex having formula (VIII).

■ preparation of hexaacids having formula (III)

The hexa-acid of formula (III) can be prepared according to any known method, in particular according to the method described in EP 1931673, which participates in step a) of the process for preparing the complex of formula (II) according to the invention.

However, according to a preferred embodiment, the hexa-acid having formula (III) is obtained by: pylene having formula (V):

and has the formula R₃OOC-CHG_p-(CH₂)₂-COOR₄(IX) alkylating the compound of (IX),

wherein:

-R₃and R₄Independently of one another represent (C)₃-C₆) Alkyl radicals, especially (C)₄-C₆) An alkyl group, for example a butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl group, and

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

to obtain the hexaesters of formula (X)

A hydrolysis step is then carried out to obtain the hexa-acid of formula (III).

In a preferred embodiment, R₃And R₄Are the same.

According to one advantageous embodiment, the hexa-acid having formula (III) is obtained by: pylene having formula (V):

alkylation with dibutyl 2-bromoglutarate to give butyl hexaester having the formula (VI):

a hydrolysis step is then carried out to obtain the hexa-acid of formula (III).

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

The use of dibutyl 2-bromoglutarate is particularly advantageous compared with the use of ethyl 2-bromoglutarate as described in EP 1931673. In particular, the commercially available diethyl 2-bromoglutarate is a relatively unstable compound which degrades over time and under the effect of temperature. More precisely, the ester has a tendency to be hydrolyzed or cyclized and thus to lose its bromine atom. Attempts to purify the commercially available diethyl 2-bromoglutarate, or to develop new synthetic routes to obtain this ester in higher purity and thus to prevent its degradation, have not been successful.

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

For obvious reasons, water is preferably used, particularly acetonitrile is preferably used, as described in EP 1931673.

The reaction is advantageously carried out at a temperature of between 40 ℃ and 80 ℃, typically between 50 ℃ and 70 ℃ and in particular between 55 ℃ and 60 ℃, for a time of between 5 hours and 20 hours, in particular between 8 hours and 15 hours.

The hydrolysis step is advantageously carried out in the presence of an acid or a base, advantageously a base (for example sodium hydroxide). The hydrolysis solvent may be water, an alcohol (e.g. ethanol) or a water/alcohol mixture. This step is advantageously carried out at a temperature of between 40 ℃ and 80 ℃, typically between 40 ℃ and 70 ℃ and in particular between 50 ℃ and 60 ℃, typically for a time of between 10 hours and 30 hours, in particular between 15 hours and 25 hours.

Furthermore, the present invention relates to butyl hexaesters having formula (VI):

in particular, the hexaesters are distinguished by a significantly improved stability relative to esters with shorter alkyl chains, in particular relative to ethyl hexaester as described in EP 1931673.

Drawings

FIG. 1: degradation of the isomeric groups iso1 to iso4 of the complexes having formula (II) under basic conditions, expressed as area percentage of a given isomeric group over time.

Detailed Description

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

Separation by HPLC of the isomeric groups isoA, isoB, isoC and isoD of the gadolinium hexacarboxylate complex of formula (I)

An HPLC machine consisting of a pump system, an injector, a chromatography column, a UV spectral brightness detector and a data processing and control station was used. The chromatography column used was a C18-250X4.6mm-5 μm column (from Waters)Range).

-a mobile phase:

route a: 100% acetonitrile and route B: 0.1% v/v H₂SO₄(96%) aqueous solution

-preparation of test solutions:

10mg/mL of a gadolinium hexaacid complex of formula (I) in purified water

-analysis conditions:

-a gradient:

Time	％Acn	％H2SO4 0.1％
			0	1	99
10	5	95
			40	10	90
50	25	75
			55	1	99
60	1	99

% Acn: % v/v of acetonitrile in the mobile phase

％H₂SO₄0.1%: 0.1% v/v H in the mobile phase₂SO₄% v/v of solution

Four main peaks were obtained. Peak 4 (i.e., isoD) of the HPLC plot corresponds to a retention time of 35.7 minutes. Isolation of the isomeric groups isoA, isoB, isoC and isoD of the gadolinium hexacarboxylate complex of formula (I) by UHPLC

Using a UHPLC machine consisting of a pump system, a sample injector, a chromatography column, a UV detector and a data stationA device. The chromatography column used was a UHPLC 150X2.1 mm-1.8 μm column (Waters Acquity UPLC HSS T3 column). This is a reversed phase UPLC column containing spherical particles composed of silica grafted with trifunctional C18 (octadecyl) and silanol that has been treated with an end-capping agent (end-capping). It is also characterized by a length of 150mm, an inner diameter of 2.1mm, a particle diameter of 1.8 μm,Porosity and a carbon content of 11%.

Preferably, the fixation used is compatible with aqueous mobile phase.

-a mobile phase:

route a: 100% acetonitrile and route B: 0.1% v/v H₂SO₄(96%) aqueous solution

-preparation of test solutions:

a solution of 0.8mg/mL gadolinium hexacarboxylate complex of formula (I) in purified water

-analysis conditions:

-a gradient:

Time	％Acn	％H2SO4 0.1％
			0	1	99
14	8	92
			20	11	89
25	25	75
			27	1	99
32	1	99

four main peaks were obtained. Peak 4 (i.e., isoD) of the UHPLC plot corresponds to a retention time of 17.4 minutes. Isolation of the isomeric groups iso1, iso2, iso3 and iso4 of the complex having formula (II) by UHPLC

A UHPLC machine consisting of a pump system, sample injector, chromatography column, UV detector and data station was used. The chromatography column used was a UHPLC 150X2.1mm-1.6 μm column (Waters)UPLC T3 column).

-a mobile phase:

route a: 100% acetonitrile and route B: 0.0005% v/v H₂SO₄(96%) aqueous solution

-preparation of test solutions:

2mg/mL of a solution of the complex of formula (II) in purified water

-analysis conditions:

-a gradient:

four main peaks were obtained. Peak 4 of the UHPLC plot (i.e., iso4) corresponds to a retention time of 6.3 minutes.

Relaxation rate measurement

By heating at 20MHz (0.47T), 60MHz (1.41T) and 37 deg.CRelaxation times T were determined by standard procedures on a mq20 machine (Bru ker)₁And T₂. Measuring longitudinal relaxation time T using recovery sequence₁And measuring the transverse relaxation time T by CPMG (Carr-Purcell-Meiboom-Gill) technique₂。

At 37 ℃ for different concentrations of total metals in aqueous solution (range from 0.5x 10)^-3To 5x10^-3mol/L) calculating the relaxation rate R₁(＝1/T₁) And R₂(＝1/T₂). R as a function of concentration₁Or R₂The correlation between them is linear and the slope represents the relaxation rate r₁(R₁C) or r₂(R₂/C) in (1/sec) x (1/mMol/L), i.e. (mM)^-1.s^-1) And (4) showing.

Measurement of the kinetic inertia of the isomeric group of complexes having formula (II) in an acidic medium

At 37 deg.C, pH 1.2, in hydrochloric acid solution under pseudo-first order kinetic conditionsThe dissociation of gadolinium complexes present in the four unresolved peaks of isomers iso1 to iso4 (C ═ 8 × 10) was investigated by monitoring the release of gadolinium into solution without controlling the ionic strength (C ═ 8 × 10)^-6M). After addition of the solution of azoarsine III, the amount of free gadolinium was determined by spectroscopy at 654nm (C ═ 5.3 × 10^-4M)。

Half-life (T) determined for each of the isomeric groups_1/2) Collated in the following table:

isomer group	T1/2(pH 1.2-37℃)
		Iso1	18 hours
Iso2	6 hours
		Iso3	8 days
Iso4	27 days

Investigating the degradation of the isomeric group of complexes of the formula (II) under basic conditions

The complex having formula (II) is referred to as AP in the remainder of this example.

The degradation kinetics of the unresolved peaks of isomers iso1 to iso4, referred to as the generic term isoX, were assessed by measuring HPLC purity and by monitoring the area of each unresolved peak of an isomer over time. Thus, the magnitude measured is:

-P_HPLC(time), and

- [ mathematical formula 1]

The degradation conditions chosen were the following: [ AP ] ═ 1mM in 0.1N sodium hydroxide. At these dilution conditions, the effect of AP degradation on the experimental media was lower. The degradation products do not change the pH of the medium, a parameter which is critical in the study of degradation kinetics. This was confirmed experimentally by measuring the initial pH and the pH at the end of degradation (72 hours, 37 ℃):

the method for preparing the solution is described below:

-weighing about 0.05g of each product, an appropriate amount of 10mL of mQ water, to obtain a solution a, such that [ AP]_A＝5mM，

-dilution: 2mL of solution A, an appropriate amount of 10mL of NaOH (0.1N) to obtain solution B, [ AP ] was allowed to stand]_B1mM and [ NaOH]＝0.08M，

-aliquoting the solution in an HPLC flask, and

-incubation of HPLC flasks containing AP solution in NaOH at the study temperature (37 ℃).

For each spot, an aliquot was taken and analyzed by HPLC without diluting the sample (ammonium acetate method).

The results obtained are given in figure 1.

Preparation of butyl hexaester having formula (VI)

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

Preparation of the Hexaoic acid having formula (III)

113kg (121mol) of butyl hexaester were placed in a reactor together with 8kg of ethanol. The medium is brought to 55. + -. 5 ℃ and then 161kg (1207.5mol) of 30% sodium hydroxide (m/m) are added over 3 hours. The reaction mixture was held at this temperature for about 20 hours. The butanol is then removed by decanting the reaction medium. The hexa-acid obtained as sodium salt having formula (III) was diluted with water to obtain an aqueous solution of about 10% (m/m). The solution is treated on an acidic cationic resin. The hexa acid having formula (III) in aqueous solution was obtained in about 90% yield and 95% purity.

Preparation of gadolinium hexamate complexes having formula (I)

■ protocol

● complexation and isomerization

-is free of acetic acid

418kg (117kg of pure hexa-acid of the formula (III)/196 mol) of a 28% by weight aqueous solution of hexa-acid of the formula (III) were placed in a reactor. The pH of the solution was adjusted to 2.7 by addition of hydrochloric acid, followed by addition of 37kg (103.2mol) of gadolinium oxide. The reaction medium is heated at 100 ℃ and 102 ℃ for 48 hours to achieve the desired isomer distribution of the hexaacid of formula (III).

-containing acetic acid

Gadolinium oxide (0.525 molar equivalents) was suspended in a solution of the hexa-acid of formula (III) at 28.1% by mass.

99-100% acetic acid (50% by mass/pure hexa-acid of formula (III)) was poured into the medium at room temperature.

The medium is heated to reflux and subsequently distilled up to 113 ℃ by gradually refilling the medium with acetic acid by mass while removing water. Once a temperature of 113 ℃ was reached, a sufficient amount of acetic acid was added to reach the starting volume.

The medium was kept at 113 ℃ overnight.

● crystallizing and recrystallizing

-crystallization

The gadolinium hexaate complex of formula (I) in solution is cooled to 40 ℃, the primers are added, and the reagents are allowed to contact for at least 2 hours. The product was then isolated by filtration at 40 ℃ and washed with permeate water.

-recrystallization

180kg of the previously obtained gadolinium hexacarboxylate complex of formula (I) (solids content about 72%) were suspended in 390kg of water. The medium was heated to 100 ℃ to dissolve the product and then cooled to 80 ℃ for pretreatment by addition of a small amount of primer. After cooling to room temperature, the gadolinium hexaate complex having the formula (I) was isolated by filtration and drying.

● Selective decomplexation

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

■ content of diastereoisomeric excess of mixture comprising diastereoisomers I-RRR and I-SSS

The ratio of the various isomers of the complex having formula (I) present in the mixture of diastereomers depends on the conditions under which the complexation and isomerization steps are carried out, as seen in table 3 below.

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

The additional steps of recrystallization and selective decomplexing enable an increase in the diastereomeric excess of the I-RRR and I-SSS mixture (see Table 4).

[ Table 4 ]: content of diastereoisomeric excess of mixture comprising I-RRR and I-SSS after crystallization/recrystallization/Selective decomplexing

Preparation of a Complex having the formula (II)

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

Testing of the isomer conversion starting from a complex having the formula (II)

The isomers of the complex having formula (II) were synthesized from the isomeric groups isoA, isoB, isoC and isoD of the hexaacid complex having formula (I) separated by preparative HPLC. The four isomeric groups were separated and then amidated with R and S3-amino-1, 2-propanediol (APD). Eight isomers were thus obtained:

isoA + APD (R) and isoA + APD (S),

isoB + APD (R) and isoB + APD (S),

isoC + APD (R) and isoC + APD (S), and

isoD + APD (R) and isoD + APD (S).

Each of these isomers is subjected to conditions which allow isomerization of the gadolinium hexaate complex having formula (I).

Thus, it is possible to provideA solution of HCl pH 3 was prepared by diluting 1mL of 1N HCl in 1 liter of water. The isomer was added at a concentration of 1mM to a HCl solution at pH 3. 10mg of the powder was dissolved in 10mL of the solution. The eight solutions obtained were heated to 100 ℃ and then at T₀And T₀At +23 hours by HPLC analysis.

The purity percentages measured by HPLC are given in the table below.

The reason for the loss of purity is the chemical degradation of the product (hydrolysis of the amide function) due to the conditions resulting from the isomerization reaction.

The complex of formula (II) obtained according to the process described in EP 1931673 cannot be directly isomerized in a clean and selective manner, since the conditions which allow the isomerization of the various compounds lead to a significant chemical degradation of the product by hydrolysis of the amide function.