Preparation of dimeric contrast agents

Abstract

The present invention relates to a dimeric contrast agent for use in magnetic resonance imaging (MRI), in particular [μ-[1-[bis[2-(hydroxy-κO)-3-[4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κN 1 ,κN 4 ,κN 7 ,κN 10 The present invention relates to a method for the preparation of the 1-propylamino-1-deoxy-D-glucitrate(6-)-di-gadolinium complex, comprising a preparation step carried out in one pot (without isolation of the resulting intermediate) and precipitation of at least a portion of the excess free gadolinium metal ion.

Description

[Technical field]

[0001] The present invention relates to the manufacture of contrast agents for use in magnetic resonance imaging (MRI). In particular, the present invention relates to dimeric contrast agents, in particular [μ-[1-[bis[2-(hydroxy-κO)-3-[4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κN 1 ,κN 4 ,κN 7 ,κN 10 This invention relates to a novel method for the preparation of the di-gadolinium complex (compound 5). [Background technology]

[0002] Magnetic resonance imaging (MRI) is a well-known imaging technique that is increasingly used in the clinical diagnosis of an ever-growing number of conditions.

[0003] Contrast agents for use in MRI imaging techniques typically contain a paramagnetic metal ion complexed with a cyclic or acyclic chelating ligand, more typically a polyaminopolycarboxylic acid chelating agent. The most important class of MRI contrast agents is represented by Gd(III) chelates, which are currently used in about one-third of clinical examinations. Indeed, Gd(III) is highly paramagnetic with seven unpaired electrons and has a long electronic relaxation time, making it an excellent candidate as a relaxation agent. However, the gadolinium metal ion [Gd(H2O)8] 3+ is toxic to living organisms even at low doses (10-20 micromoles / kg).

[0004] Therefore, to be potentially valuable MRI contrast agents, Gd(III) complexes must exhibit high thermodynamic (and in some cases kinetic) stability to prevent the release of toxic metal ions. Moreover, it would be advantageous for a process for producing Gd(III) complexes to allow for effective and efficient removal of toxic metal ions present in the reaction mixture after the complexation step.

[0005] WO 2017 / 098044 (of the same applicant as the present application) discloses a method for the preparation of a compound of formula [ka] This invention discloses dimeric paramagnetic complexes of the formula:

[0006] Among many specific compounds, this application relates to compounds of the formula [ka] (hereinafter referred to as “dimeric complex Compound 5” or more simply “Compound 5”).

[0007] This complex compound exhibits interesting properties, especially with regard to relaxivity and tolerability, making it suitable for use in in vivo imaging performed with paramagnetic complexes at doses lower than those required by commercially available contrast agents.

[0008] The said international application further includes the following general scheme 1: [ka] A manufacturing method shown in is disclosed.

[0009] The main steps of the disclosed synthetic process are: a) preparation and isolation of DO3A tri-tert-butyl ester (compound 1A), essentially as disclosed in Org. Synth. 2008, 85, 10; b) Preparation of intermediate 2 by alkylation of D-glucamine with epichlorohydrin (molar ratio 1:4.95) in MeOH at 50° C. for 26 hours and isolation of the concentrated product by evaporation of the crude reaction mixture; c) alkylation of DO3A tri-tert-butyl ester with intermediate 2 in DMSO and Et3N, followed by evaporation of the solvent and purification of the crude residue by Amberlite XAD® 1600 to give the protected ligand 3; d) deprotection of ligand 3 using TFA acid and TIPS in dichloromethane, evaporation of the crude reaction mixture and purification of the residue by Amberlite XE 750; e) complexation of ligand 4 by stoichiometric addition of gadolinium chloride hexahydrate in water, filtration of the solution and evaporation of the solvent to obtain a crude product, which is purified on Amberchrome CG161M resin. Includes.

[0010] The process requires synthesis and isolation of each individual intermediate, which is typically performed by evaporating the solvent to the residue. Such isolation steps are not suitable for large-scale production and inevitably reduce the overall yield and efficiency of the process. Furthermore, the prior art processes involve the use of harsh materials such as TFA, TIPS and DCM, which are difficult to handle and may cause corrosion and wear of the synthesis equipment and / or may be unsafe for the health of the workers, making them particularly unsuitable for large-scale implementation (e.g. industrial processes).

[0011] Finally, the complexation steps disclosed in WO2017 / 098044 are not easily reproducible, as they depend on accurate weighing of the reactants of the complexation step and accurate determination of their title. For at least this reason, the robustness of the process disclosed in WO2017 / 098044 could be improved.

[0012] There is therefore a need for a process for the preparation of the Gd(III) complexes disclosed in WO2017 / 098044 that overcomes the above-mentioned problems, in particular a process that is reproducible, robust and advantageous for the large-scale preparation of the dimeric paramagnetic complexes disclosed in WO2017 / 098044. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention generally relates to an optimized process for the preparation of dimeric complex compound 5, which involves a preparation step carried out in one pot, without isolation of the obtained intermediates, allowing both to save time and to improve the overall yield and efficiency, while at the same time obtaining a final product with a low content of toxic free gadolinium metal ions. [Means for solving the problem]

[0014] More specifically, the present invention relates to a dimeric complex compound 5 [ka] A method for producing the above-mentioned compound, comprising the steps of: 1) For example, in an organic solvent, [ka] preparing a solution of DO3A tri-tert-butyl ester represented by the formula: 2) For example, in an organic solvent, [ka] preparing a solution of an intermediate represented by the formula: 3) Mix the solutions prepared in steps 1) and 2) to obtain a solution of formula 3 [ka] obtaining a solution of the protected ligand represented by 4) Removing the tert-butyl protecting group from the protected ligand without isolating the protected ligand from the solution of step 3) to give a compound of formula 4 [ka] obtaining a solution of free ligand represented by 5) adding a molar excess of gadolinium metal ions to the solution of step 4) without isolating the free ligand of formula 4 from the resulting solution to obtain a solution of the dimeric complex of formula 5; 6) adding a precipitating agent to the solution of the dimeric complex of formula 5 to precipitate the free gadolinium metal ions as insoluble gadolinium salts; and 7) Isolating the dimeric complex Includes.

[0015] Interestingly, the process according to claim 1 avoids or greatly reduces the use of hazardous reagents such as trifluoroacetic acid (TFA) and hazardous solvents such as dichloromethane, which are required in prior art processes and are difficult to handle when carried out on a large scale, e.g. in an industrial process.

[0016] Furthermore, the process according to claim 1 includes steps carried out in one pot, making it suitable for implementation on a large scale and not requiring isolation of either the prepared precursor (such as 1A) or intermediates. As a result, in addition to facilitating shorter reaction times and being easily implemented on a large scale as described above, the process advantageously allows for a significant increase in the overall yield of the process, from 10% (obtained in the process disclosed in WO2017098044) to more than 30% (e.g. 33%).

[0017] Moreover, the process of the present invention is robust and therefore particularly suitable for large-scale implementation. Indeed, due to the addition of a molar excess of gadolinium metal ions in step 5), which was not disclosed in the prior art processes, the burden of accurate weighing of reactants and determination of titers of the complexation step is significantly reduced.

[0018] Moreover, step 6) results in the removal of free gadolinium metal ions via precipitation. This purification step is less burdensome than the removal of said ions by purification through a column (performed in the above-mentioned prior art documents). Adding a molar excess of gadolinium metal ions results in a higher amount of free gadolinium metal ions after complexation compared to the addition of stoichiometric or substoichiometric amounts of gadolinium metal ions; however, it has been found that this free gadolinium metal ion can be effectively and efficiently removed by performing the precipitation step 6) of the present invention, i.e., by precipitating the free gadolinium metal ions using a precipitant as disclosed herein. Thus, the combination of steps 5) and 6) results in a particularly effective removal of free gadolinium metal ions from the solution of the dimeric complex of formula 5 via a method that is robust and suitable for large-scale production.

[0019] The preparation method of the present invention is robust, reproducible and efficient (at least in terms of yield) and thus allows for the easy large-scale preparation of dimeric complex 5.

[0020] Step 1) of the present process generally involves the preparation of a solution of DO3A tri-tert-butyl ester 1A. In one embodiment, the solution is prepared in an organic solvent, for example by solubilizing commercially available DO3A tri-tert-butyl ester or DO3A tri-tert-butyl ester prepared by using known synthetic procedures in the solvent.

[0021] In a preferred embodiment, the solution of 1A is prepared by converting a salt of DO3A tri-tert-butyl ester, such as the hydrobromide salt, to the free base 1A immediately before its use. The conversion, which typically involves neutralization of the hydrobromide salt, is preferably carried out in an organic solvent and in the presence of a base or a basic salt (a salt that is the product of neutralization of a strong base with a weak acid, which hydrolyzes by forming a basic solution). Removal of the formed salt (and optionally concentration of the filtrate solution) then gives a solution of DO3A tri-tert-butyl ester 1A in an organic solvent, which is suitable for use directly in the subsequent steps of the preparation process without the need for purification or isolation of the ester.

[0022] Suitable organic solvents preferably include MeCN, propylene carbonate, ethanol, t-butanol, hexane, etc. More preferably, the organic solvent is MeCN.

[0023] For example, suitable bases or basic salts for neutralization of the starting hydrobromide salt include strong bases and anion exchange resins such as Diaion PA308, Amberlite IRA 400, KOH, tBuOK, Na2CO3 and K2CO3, the latter two being preferred. More preferably, neutralization of DO3A tri-tert-butyl ester hydrobromide salt is carried out in the presence of K2CO3.

[0024] Step 2) of the process involves the preparation of a compound of formula 2, which can be obtained by alkylation of D-glucamine with epichlorohydrin. The alkylation is carried out in an organic solvent, such as a dipolar organic solvent, or in an aqueous mixture thereof. Suitable organic solvents include, for example, alcohols, such as DMAC, DMF, MeOH, and mixtures thereof. More preferably, the organic solvent is DMAC. Distillation of the aqueous solvent and / or the excess of epichlorohydrin from the mixture provides a solution of the compound of formula 2 in an organic solvent, which is suitable for use directly in the next step of the process without isolation and / or further purification of the alkylation product.

[0025] Step 3) of the present process essentially involves the condensation (or coupling, as used interchangeably herein) of the intermediate compound of formula 2 with DO3A tri-tert-butyl ester 1A to form the protected ligand of formula 3. The condensation reaction is preferably carried out in the presence of a base, which acts, for example, as an acceptor for the HCl formed. Suitable bases include, for example, anion exchange resins such as Amberlite GC 400, NMM, tBuOK, Et3N, and DIPEA, with Et3N and DIPEA being preferred, and DIPEA being particularly preferred.

[0026] In one embodiment, the condensation reaction is carried out by adding a base and an organic solution of DO3A tri-tert-butyl ester 1A recovered directly from step 1) to a solution of compound 2 recovered from step 2) to obtain a crude organic solution comprising the condensation product of formula 3 in an organic solvent mixture. Purification of the crude organic material to obtain a purified product in a water / organic solvent mixture, and optionally a final distillation of the organic solvent, then provides the protected ligand of formula 3 in an aqueous solvent or aqueous solvent mixture, which can be used directly in the next step of the process without isolation and / or further purification of the protected ligand itself.

[0027] Step 4) of the present process essentially comprises the removal of the carboxyl protecting group from the protected ligand of formula 3, resulting in an aqueous solution or mixture of the free ligand of formula 4. Deprotection by hydrolysis of the tert-butyl protecting group can be carried out by using reactants and conditions known to those skilled in the art, both under acidic and basic conditions. In one embodiment, deprotection is carried out by acidification of the aqueous solution or mixture of the protected ligand, directly recovered from step 3) of the present process, resulting in an acidic solution of the free ligand of formula 4. Acidification is preferably carried out by addition of an acid, for example selected from HCl, H2SO4, and H3PO4. Particularly preferred are inorganic acids with counterions having a single negative charge, such as HCl, HBr, etc. In a preferred embodiment, deprotection is carried out by using HCl. In a particularly preferred embodiment, deprotection is carried out by heating and / or maintaining the acidic reaction mixture at a temperature above 40°C, preferably above 40°C to 60°C, more preferably in the range of 45-55°C. Neutralization of the acidic solution, subsequent purification and partial concentration of the resulting mixture then recovers an aqueous solution or mixture of ligand 4, which is used directly in the complexation step without isolation.

[0028] Step 5) involves the complexation of the ligand with a molar excess of gadolinium metal ions to the desired dimeric complex 5. The complexation reaction may be conveniently carried out by adding a suitable Gd(III) derivative, in particular an oxide such as Gd2O3 or a soluble gadolinium salt, to a solution of the ligand. In one embodiment, the complexation reaction is carried out by adding the soluble gadolinium salt GdCl3 to a solution of the ligand directly recovered from step 4) of the present process.

[0029] Step 6) involves precipitating the free gadolinium metal ions present in the solution of the dimeric complex of formula 5. These free ions are present because they were added in molar excess in a previous step, and in some cases because not all of the gadolinium added in the previous step reacted with the ligand 4 to form the desired dimeric complex 5. The precipitation step 6) is carried out by adding a precipitating agent to precipitate the free gadolinium metal ions as an insoluble gadolinium salt. The preferred precipitating agent is phosphate ion (PO4 3- ), monohydrogen phosphate ion (HPO4 2- ), dihydrogen phosphate ion (H2PO4 - ), orthophosphoric acid (H3PO4), oxalate ion (C2O4 2- ), hydrogen oxalate ion (HC2O4 - ), and oxalic acid (H2C2O4). In a preferred embodiment, the precipitating agent is added in an amount of at least 1.1 molar, preferably in an amount of 1.1 to 5 molar, relative to 1 molar amount of dimeric complex 5. In a further preferred embodiment, during and / or after the addition of the precipitating agent according to step 6), the pH is adjusted and / or maintained at a value higher than 4.5, preferably higher than 4.7, and optionally lower than 10.0, preferably lower than 9.0. In a further preferred embodiment, after the precipitation of the insoluble gadolinium salt, a removal step is provided in which the insoluble gadolinium salt is removed from the solution, for example by filtering the resulting solution, thereby isolating the insoluble gadolinium salt from such solution.

[0030] The resulting filtered mixture can then be further purified (through additional purification steps) and concentrated to obtain a solution of the desired dimeric complex 5 having the desired purity.

[0031] Step 7) involves the final isolation of the desired gadolinium complex 5. This step may be conveniently carried out by known procedures. In one embodiment, the solution of purified complex recovered from step 5) is spray dried to obtain the desired product as a white solid meeting the required purity specifications. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] As used herein, unless otherwise specified, the term "intermediate" (as used with respect to, for example, a compound of formula 2 resulting from the alkylation reaction of D-glucamine with epichlorohydrin, or a protected ligand of formula 3) includes within its meaning a molecule produced during the chemical synthesis or preparation steps of the present process that is not itself the end product, but requires one (or more) further reactions, e.g., alkylation / deprotection / complexation reactions, to obtain the end product of the present process, i.e., dimeric complex compound 5.

[0033] Unless otherwise specified, the term "precursor" (e.g., as used with respect to compound 1A) includes within its meaning a molecule that participates in a chemical reaction that facilitates the conversion of said precursor into another molecule that contains or is derived from said precursor.

[0034] As used herein, the term "aqueous solvent" includes water and saline within its meaning, but may contain a small amount of water-miscible organic solvent (e.g., 10% or less, preferably 8% or less, more preferably 5% or less by volume of water-miscible organic solvent, etc.) since the production method of the present invention is carried out without isolating most of the intermediate products, and thus a small amount of organic solvent may remain upstream and throughout the first step of the production method. Preferably, the aqueous solvent is water.

[0035] The expressions "water / organic solvent mixture", or more simply "aqueous solvent mixture", are used interchangeably herein and include within their meaning mixtures of two or more solvents, including aqueous solvents such as mixtures of water and one or more organic solvents, all of which are miscible with each other to form a homogeneous solvent mixture, with the volume percentage of one or more organic solvents being higher than 10%, preferably higher than 15%, more preferably higher than 20%. Suitable examples include mixtures of water and acetonitrile (or water / MeCN), for example used as eluents in the chromatographic purification of the compound of formula 3, or mixtures of water / MeCN / DMAC, for example obtained after dilution with water of the crude mixture resulting from the condensation reaction of step 3). According to a preferred embodiment of the present invention, the one or more organic solvents of the aqueous solvent mixture (as well as in the aqueous solvent, if present) are not highly hazardous solvents or substances; indeed, the aqueous solvent mixture is preferably free of highly hazardous solvents such as TFA, TIPS and / or DCM.

[0036] Similarly, the expressions "aqueous solution" and "aqueous mixture" respectively include within their meaning solutions or mixtures containing water. Suitable examples include solutions of one or more compounds, e.g., reagents, acids, bases or reaction products, respectively, in water (more generally, in an aqueous mixture), or in an aqueous solvent mixture, and mixtures such as aqueous / organic mixtures resulting from the addition of water or an aqueous solution to a reaction mixture in an organic solvent or solvent mixture.

[0037] As used herein, the term "protecting group" (e.g., as used with respect to compounds of formula 3) refers to a protecting group suitable for retaining the function of the group to which it is attached. In particular, the protecting group is used to retain carboxyl functionality. More specifically, the term refers to a tert-butyl group that retains the chelating function of the carboxyl group of a ligand by forming a tert-butyl ester [for a general reference on protecting groups and deprotection conditions, see TW Green and PGM Wuts; Protective Groups in Organic Synthesis, Wiley, NY 1999, third edition].

[0038] As used herein, unless otherwise specified, the term "precipitating agent" refers to an agent that, when added to a solution of dimeric complex 5 according to the manufacturing method of the present invention, at least under the conditions of step 6), is an anion or generates an anion added in step 6. Such an anion can generate an insoluble gadolinium salt as defined herein through ionic bonding with free gadolinium metal ions.

[0039] As used herein, unless otherwise specified, the term "insoluble gadolinium salt" refers to a salt resulting after addition of a precipitating agent as defined herein. An insoluble gadolinium salt is one that contains Gd as the cation. 3+, and an anion as a counterion which is a precipitant or is generated by the precipitant. At least under the conditions of step 6) of the preparation method of the present invention, and preferably also under the conditions of the downstream steps of step 6) up to at least the filtration step, the insoluble gadolinium salt is present in the reaction mixture in a solid and filterable physical form; under these conditions, the parts by weight of the solvent required to dissolve one part by weight of the gadolinium salt is preferably 100 or more, more preferably 1,000 or more, even more preferably 10,000 or more. Examples of insoluble gadolinium salts are gadolinium phosphate and gadolinium oxalate. On the other hand, the term "soluble gadolinium salt" refers to the soluble gadolinium salt (typically GdCl3) added during step 5) to carry out the complexation of the dimeric ligand 4, thereby obtaining the complex 5.

[0040] As used herein, unless otherwise specified, the term "free gadolinium metal ion" refers to the gadolinium metal ion that is present in solution and is not chelated by a dimeric ligand, [Gd(HO)]. 3+ This refers to gadolinium ions such as

[0041] One embodiment of the present invention essentially corresponds to the following general synthetic scheme 2: [ka] The method for producing the dimer compound 5 shown in The method comprises the following main steps: 1) neutralizing the hydrobromide salt of DO3A tri-tert-butyl ester in an organic solvent to obtain a solution of DO3A tri-tert-butyl ester 1A in an organic solvent; 2) reacting D-glucamine with epichlorohydrin to obtain a solution of the compound of formula 2 in an organic solvent such as DMAC and without product isolation; 3) reacting the compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1) in the presence of a base, optionally concentrating the solution to obtain an organic crude, diluting the organic crude with water, a water / organic solvent mixture and / or optionally an aqueous solution to obtain an aqueous / organic crude, purifying the aqueous / organic crude and optionally removing the organic solvent to obtain an aqueous solution or aqueous mixture of the protected ligand of formula 3; and without product isolation; 4) acidifying the solution of the protected ligand of formula 3 from step 3) while heating and / or maintaining the temperature of the reaction mixture at a temperature above 40° C. to obtain an acidic aqueous solution or mixture of the deprotected ligand 4, neutralizing the acidic solution and purifying the resulting neutral solution to obtain an aqueous solution or mixture of the deprotected ligand 4, without isolation of the latter; 5) adding a molar excess of gadolinium metal ions to a solution of ligand 4 to obtain a solution of the corresponding complex compound 5; 6) adding a precipitant to the solution of the corresponding complex compound 5 to precipitate the free gadolinium metal ions as insoluble gadolinium salts, thereby obtaining a solution of the corresponding complex compound 5; and 7) Isolating the complex Includes.

[0042] Process 1 The first step of the process involves preparing a solution of DO3A tri-tert-butyl ester 1A in an organic solvent such as MeCN by directly converting the hydrobromide salt of DO3A tri-tert-butyl ester to the free base in an organic solvent in the presence of a base or a basic salt.

[0043] In a preferred embodiment, step 1) of the manufacturing method comprises: i) suspending the hydrobromide salt of DO3A tri-tert-butyl ester together with a base or a basic salt such as K2CO3 in an organic solvent such as MeCN to obtain a suspension containing the salt formed; ii) filtering the suspension; and iii) The filtered suspension is collected and optionally concentrated to give a solution of DO3A tri-tert-butyl ester 1A in an organic solvent suitable for direct use in the subsequent condensation reaction, without the need for purification or isolation of the ester. Includes.

[0044] The hydrobromide ester and K2CO3 are preferably suspended in an organic solvent at room temperature to obtain a mixture, which is maintained under stirring at a temperature of 20-30° C., preferably about 25° C., for a period of 16-30 hours, preferably 18-20 hours. The mixture is then preferably treated by filtration to remove the formed salt.

[0045] In a preferred embodiment, the solution obtained by filtration is thermally concentrated, for example by partial distillation of the solvent, to obtain a solution of tri-tert-butyl ester 1A in MeCN having a final concentration of 55-65%, preferably about 60% (w / w), which is suitable for direct use in the subsequent condensation step of the process.

[0046] Process 2 This step involves preparing a solution of the intermediate compound of formula 2 by reacting D-glucamine with epichlorohydrin. In one embodiment, the reaction is carried out in a mixture of solvents, preferably water / DMAC, by using a slight excess of epichlorohydrin over the stoichiometric amount, for example 2-3, more preferably about 2.2 moles of epichlorohydrin per mole of D-glucamine. Preferably, such reaction of step 2) is carried out by using a slight excess of epichlorohydrin over the stoichiometric amount, for example 2-3, more preferably 2.05-2.5, even more preferably about 2.2 moles of epichlorohydrin per mole of D-glucamine.

[0047] In a preferred embodiment, step 2) of the manufacturing method comprises: i) adding an aqueous solution of D-glucamine to a solution of epichlorohydrin in DMAC to obtain an intermediate compound of formula 2 in a water / DMAC solvent mixture; and ii) removing water from the solvent mixture to obtain a solution of the compound of formula 2 in the organic solvent. Includes.

[0048] The addition of D-glucamine to the solution of epichlorohydrin is preferably carried out at room temperature for about 2 hours to obtain a mixture which is kept under stirring at a temperature of 15-30°C, preferably 15-25°C, more preferably 20-25°C, for a period of 16-24 hours, preferably 16-20 hours, more preferably about 17 hours.

[0049] The mixture is then distilled to remove water and possibly epichlorohydrin residues. The distillation is preferably carried out under reduced pressure at a temperature of preferably 40-65° C. to obtain a solution of the desired intermediate compound of formula 2 in DMAC, preferably having a residual water content of <2% w / w. The resulting solution is then used as is in the subsequent condensation reaction without the need for product isolation or purification.

[0050] Process 3 This step essentially involves the condensation of DO3A tri-tert-butyl ester 1A with intermediate 2 in the presence of a base such as Et3N, or more preferably DIPEA. The condensation is preferably carried out by combining a solution of ester 1A in base and MeCN from step 1) with a solution of intermediate 2 in DMAC directly recovered from step 2), to obtain a raw solution (or crude) containing the protected ligand of formula 3 in a mixture of MeCN / DMAC organic solvents, which is then purified.

[0051] In one embodiment, the organic crude solution resulting from the condensation reaction is diluted with water or partially concentrated and then diluted with water or an aqueous solvent mixture, preferably water / MeCN, to obtain an aqueous / organic crude, or aqueous crude, used interchangeably herein.

[0052] In a preferred embodiment, the aqueous / organic crude thus obtained, or preferably the organic crude resulting from the condensation reaction, is added with an aqueous solution which induces the precipitation of reaction salts, including DO3A tri-tert-butyl ester hydrochloride, which is then removed by filtration to obtain an aqueous / organic filtrate solution. The aqueous / organic crude or aqueous / organic filtrate solution is then purified, preferably by chromatography.

[0053] In one embodiment, the aqueous solution used to facilitate precipitation of the hydrochloride salt is aqueous ammonia.

[0054] More specifically, step 3) of the present production method preferably comprises: i) condensing the intermediate compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1) in the presence of a base, preferably DIPEA, to obtain a crude organic solution comprising the condensation product of formula 3 and a reaction salt in an organic solvent mixture, and optionally concentrating the crude organic solution; ii) diluting the organic crude of step i) with water or a water / organic solvent mixture, preferably water / MeCN, to obtain an aqueous / organic crude; iii) optionally adding to the aqueous / organic crude an aqueous solution which induces precipitation of the reaction salt and removing the precipitate by filtration to obtain an aqueous / organic filtrate; or iv) diluting the organic crude of step i) with an aqueous solution which induces precipitation of the reaction salt and removing the precipitate by filtration to obtain an aqueous / organic filtrate solution; v) purifying the aqueous / organic crude product of step ii), or the aqueous / organic filtrate solution of steps iii) or iv), to obtain a solution of the protected ligand of formula 3 in an aqueous / organic solvent mixture, which may be used in the subsequent deprotection step of the process without the need for isolation or further purification of the protected product; and vi) optionally removing the organic solvent from the mixture to give a solution of the protected ligand of formula 3 in water, or more generally in an aqueous solvent, which can be used in the subsequent deprotection step of the process without the need for isolation or further purification of the protected product. Includes.

[0055] The condensation reaction is preferably carried out by adding a solution of ester 1A in MeCN recovered from step 1) of the present process and a base to a solution of the intermediate compound of formula 2 in DMAC recovered from step 2).

[0056] The suitable amounts of base and ester 1A are conveniently determined relative to the amount of D-glucamine reacted. In one embodiment, the condensation reaction is carried out by using 1.6-2.4 moles, preferably about 1.8 moles, of ester 1A and 2-4 moles, preferably about 2.3 moles, of DIPEA per mole of starting D-glucamine reacted.

[0057] The addition is preferably carried out at a temperature of 40-50° C. The condensation reaction is then carried out at a temperature of 50-80° C., preferably 65-75° C., for example for a period of 60-80 hours, preferably 70-75 hours, to obtain a stock solution containing the desired condensation product of formula 3 and the hydrochloride salt in the MeCN / DMAC solvent mixture.

[0058] In one embodiment, the stock solution is then diluted with water, preferably to obtain an aqueous / organic crude having a concentration of about 25-30%, more preferably about 25% (w / w). In a preferred embodiment, the aqueous crude contains an amount of water at least equal, by weight, to the amount of organic solvent in the mixture, particularly MeCN; more preferably, the crude has a water:MeCN ratio of about 60:40.

[0059] The aqueous / organic crude is then preferably purified by chromatography, more preferably on a resin, even more preferably on an adsorptive resin such as Amberlite XAD® 1600. In a preferred embodiment, the aqueous crude is purified on an adsorptive resin such as Amberlite XAD® 1600 by using a water / MeCN mixture as eluent, making it possible to obtain both the unreacted DO3A tri-tert-butyl ester 1A and the pure condensation product as separate fractions in a water / MeCN solvent mixture.

[0060] In another embodiment, the raw solution resulting from the condensation reaction is first concentrated by removing at least MeCN, for example by distillation. The concentrated solution can then be diluted with water or a mixture of water:MeCN to obtain an aqueous / organic crude having the water:MeCN ratio described above, which is then purified by chromatography as described above.

[0061] Optionally, the aqueous / organic crude obtained as described above may be added with an aqueous solution such as aqueous ammonia, which, upon cooling of the mixture, induces the precipitation of unreacted DO3A tri-tert-butyl ester as the hydrochloride salt, which may then be removed by filtration and recycled. The filtrate solution, depleted of most of the chloride salt, is then purified by chromatography on an adsorptive resin such as Amberlite XAD® 1600 resin as described above to obtain the residual DO3A tri-tert-butyl ester 1A and the pure condensation product as separate fractions in an aqueous / organic solvent mixture such as water / MeCN.

[0062] In a preferred embodiment, an aqueous solution, e.g., containing aqueous ammonia, is added directly to the organic stock solution resulting from the condensation reaction, and cooling of the mixture induces precipitation of unreacted DO3A tri-tert-butyl ester as the hydrochloride salt, which may then be removed by filtration and recycled. The filtrate, depleted of most of the chloride salt, is then purified by chromatography on an adsorptive resin, such as Amberlite XAD® 1600 resin, as described above, to obtain the residual DO3A tri-tert-butyl ester 1A and the pure condensation product as separate fractions in an aqueous / organic solvent mixture, such as water / MeCN.

[0063] Optionally, final distillation of organic solvent from the pure fractions, e.g. under reduced pressure, gives the condensation product of formula 3 in aqueous solution with a final concentration of 5-15% (w / w), preferably about 10% (w / w), which is suitable for use as is in the subsequent deprotection step without the need for isolation or further purification of intermediates.

[0064] Interestingly, the steps of the procedure above make it possible to obtain the protected condensation product 3 in an aqueous solvent or aqueous solvent mixture, thus making it possible to carry out its deprotection and complexation to the final complex 5 by using water, and more generally an aqueous solvent or aqueous solvent mixture, as the only or one of the main reaction solvents. The protected condensation product 3 in an aqueous solvent can be obtained by various methods known to those skilled in the art. For example, the organic crude solution of the compound of formula 3, obtained for example by reacting the compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1), as described above, can be diluted with water, an aqueous / organic solvent mixture, or an aqueous solution to obtain the aqueous / organic crude. Before the removal of the organic solvent, the aqueous / organic crude can be purified via chromatography, preferably via a resin, more preferably via an adsorbent resin such as Amberlite XAD® 1600. The organic solvent is then removed to obtain an aqueous solution of the compound of formula 3, for example by distillation, for example under reduced pressure. The protected condensation product 3 in an aqueous solvent mixture can also be obtained by various methods known to those skilled in the art. As an example, the crude organic solution of the compound of formula 3, e.g. obtained by reacting the compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1), can be diluted with water, a water / organic solvent mixture, or an aqueous solution to obtain an aqueous / organic solution, which can be used in a subsequent step without isolation of the product 3.

[0065] Thus, according to a preferred embodiment of the present invention, step 3) may further comprise the further step of converting the solution of the compound of formula 3 into an aqueous solution or into an aqueous mixture of the compound of formula 3. Preferably, this further step is carried out by: (i) diluting the solution of the compound of formula 3 with water, a water / organic solvent mixture, or an aqueous solution, to obtain an aqueous mixture (or a water / organic solution), and (ii) optionally removing the organic solvent, for example by distillation, thus obtaining an aqueous solution.

[0066] Process 4 This step involves the deprotection of the protected ligand of formula 3, by removing the carboxyl protecting group, to obtain an aqueous solution or mixture of the free ligand 4. This reaction is preferably carried out by acidification of the aqueous solution or mixture of the protected ligand of formula 3, directly recovered from step 3) of the present process, and even more preferably by heating and / or maintaining the temperature of the reaction mixture at a temperature above 40° C. during and / or after acidification of the solution.

[0067] In one embodiment, step 4) of the manufacturing method comprises: i) adding an acid to the aqueous solution or mixture of compound of formula 3 recovered from step 3) and heating and / or maintaining the temperature of the solution at a temperature above 40°C, preferably above 40°C to 60°C, more preferably within the range of 45-55°C, following and / or during such addition, to obtain an acidic solution of free ligand 4; ii) adding a base to the acidic solution to obtain a substantially neutralized solution of Ligand 4; and iii) Optionally, the neutralized solution is purified (e.g., via distillation) and then optionally concentrated to obtain an aqueous solution or mixture of free ligand 4, which is suitable for direct use in a subsequent complexation reaction without the need for isolation of the ligand. Includes.

[0068] In one embodiment, a solution of the protected compound of formula 3 is acidified by the addition of an acid, preferably an inorganic acid, more preferably an inorganic acid containing a mono-negative counterion, such as 34% aqueous HCl. Inorganic acids containing a mono-negative counterion are particularly preferred, as these acids tend not to interact with the free gadolinium metal ion and can be more easily removed during downstream purification processes, such as nanofiltration, which may be performed optionally.

[0069] This deprotection step 4) is highly advantageous, particularly when carried out within the above temperature range, since it makes it possible to obtain very short deprotection times, specifically reaction times, of less than 24 hours, for example 8 to 20 hours, preferably 12 to 18 hours, more preferably 16 hours.

[0070] Moreover, this deprotection step 4) provides for deprotection of the protected dimeric ligand by using low amounts of acid, especially when carried out within the temperature ranges mentioned above. Indeed, the acidification is carried out by using an amount of acid, such as those mentioned above, preferably HCl, for example 10-45 moles, preferably 10-35 moles, more preferably 15-25 moles, per mole of protected compound 3. This brings the benefit of saving reagents as well as reducing the formation of salts during the deprotection step. For example, when HCl is used to acidify the solution, using a low amount of HCl reduces the amount of NaCl salt formed when NaOH is subsequently used to neutralize the deprotected dimeric ligand; a similar example can also be given when other acids and bases are used in the step.

[0071] As mentioned above, during and / or preferably after the addition of the acid, the temperature of the solution is preferably heated and / or maintained at a temperature above 40° C., more preferably above 40° C. to 60° C., even more preferably within the range of 45-55° C. The resulting solution is then maintained under stirring within the above mentioned temperature range for less than 24 hours, for example within the range of 8-20 hours, preferably within the range of 12-18 hours, more preferably for 16 hours, following deprotection of the ligand, for example by chromatography.

[0072] The acidic solution is then cooled (e.g. to 25° C.) and then neutralized by the addition of a base, preferably NaOH, to obtain a raw solution having a final pH of 4-7, preferably 5-6, more preferably 5.3-5.7, even more preferably up to 5.5, which may then be purified.

[0073] By carrying out the deprotection step 4) as disclosed herein, a solution containing the corresponding deprotected dimeric ligand 4 and alcohol (t-butanol) is obtained. Thus, according to a further preferred embodiment, preferably before the complexation step 5), tBuOH is removed from the solution containing the deprotected dimeric ligand 4, preferably by distillation of the solution. According to a preferred embodiment, the solution containing the dimeric ligand 4 is distilled until it contains a final concentration of dimeric ligand 4 in the range of 8% to 12% (w / w), more preferably 9% to 11% (w / w), even more preferably 10% (w / w).

[0074] Advantageously, the above procedure involves the use of water alone or as one of the main reaction solvents, and avoids or reduces the use of organic solvents, particularly the highly hazardous solvents such as DCM, which are required in the above-mentioned prior art processes, and the highly hazardous reactants such as TFA and TIPS. These highly hazardous materials are difficult to handle and therefore not suitable for use in large-scale production. Moreover, the process provides the desired ligand in an aqueous solution or aqueous mixture, which can be used directly in the complexation reaction without the need for isolation.

[0075] Process 5 The process involves complexing the dimeric ligand of formula 4 with gadolinium ions to obtain an aqueous solution or mixture of the desired chelating complex 5.

[0076] More specifically, this step preferably involves adding a molar excess of a soluble gadolinium salt, such as GdCl3, to a solution of the ligand recovered from step 4) to obtain a mixture containing the dimeric chelated complex 5.

[0077] Since the deprotected dimeric ligand of formula 4 has two chelating moieties and one dimeric ligand can chelate two gadolinium metal ions, the term "molar excess" refers to a molar amount of gadolinium metal ions that is more than twice the molar amount of the dimeric ligand of formula 4 when referring to step 5) of the preparation method of the present invention. Thus, the term "molar excess" refers to more than 2 moles of gadolinium metal ions per mole of dimeric ligand when referring to step 5) of the preparation method of the present invention. For example, 2.05 moles or more, preferably 2.05 to 2.50 moles, more preferably up to 2.20 moles, even more preferably up to 2.12 moles of gadolinium metal ions per mole of the deprotected dimeric ligand of formula 4 are added to the solution of step 4).

[0078] This reaction is preferably carried out by adding GdCl3 directly to a solution of the ligand recovered from the previous step of the process. The addition is preferably carried out at a temperature in the range of 20-50°C, more preferably 30-45°C, even more preferably 37-43°C. After adding the gadolinium ions according to step 5), the reaction mixture is preferably maintained for a period of 1-5 hours, more preferably 2-4 hours, for example in the temperature ranges described above, before carrying out the subsequent steps.

[0079] After the addition, the pH of the resulting mixture is adjusted, for example by addition of a base, preferably NaOH, to a value in the range of 5.0 to 7.0, more preferably 5.0 to 6.0, for example for a time and / or temperature as described above.

[0080] As mentioned above, step 5) is particularly advantageous because the addition of a molar excess of gadolinium metal ions significantly reduces the burden of accurate weighing and titration of the reactants of the complexation step, thereby improving the robustness of the entire process. The present method of manufacture is particularly robust, reproducible and efficient, making it easier to implement in large-scale production.

[0081] According to a preferred embodiment, after step 5) and optionally before step 6), the preparation method of the invention comprises a further step of desalting the solution of the dimeric complex of formula 5, preferably via nanofiltration. This desalting (for example nanofiltration) step makes it possible to remove salts generated in the complexation step, such as those generated after the addition of soluble gadolinium salts, as well as those generated in the deprotection step 4). The desalting step does not remove either the free gadolinium metal ions or the mono-gadolinized complexes, but is useful for removing salts in order to improve the subsequent step for removing the precipitating agent.

[0082] The desalting step may be carried out until the conductivity value of the solution is below 5.0 mS / cm, preferably below 1 mS / cm, even more preferably below 0.8 mS / cm.

[0083] Process 6 Step 6) of the method of the invention involves the addition of a precipitant to the solution of the previous step to precipitate the free gadolinium metal ions present in the solution. In fact, this step makes it possible to precipitate the free gadolinium metal ions, in particular the majority of them: as shown in the experimental section below, the precipitation step 6) makes it possible to precipitate the majority of the free gadolinium metal ions present after the complexation step 5), thereby reducing the content of free gadolinium metal ions from approximately tens of thousands of ppm (relative to the amount of dimeric complex 5) to only 100 ppm or even tens of ppm. In particular, this step provides for the production of a solution containing dimeric complex 5, in which the amount of free gadolinium metal ions relative to the amount of gadolinium complex 5 is less than 350 ppm, preferably less than 150 ppm, more preferably less than 100 ppm. According to the present invention, high ppm values ​​of free gadolinium metal ion, such as the ppm values ​​before the precipitation step or after the addition of potassium hydrogen tartrate in Example 8, or generally ppm values ​​above 4000 ppm, are determined by conventional complexometric titration with EDTA in the presence of xylenol orange, while low ppm values ​​of free gadolinium metal ion, such as the ppm values ​​after the precipitation step, or generally ppm values ​​below 4000 ppm, are determined by carrying out procedure 5 described in the experimental section below.

[0084] To precipitate the free gadolinium metal ions while avoiding the formation of mono-gadolinium complexes, the precipitating agent is preferably phosphate ions (PO4 3- ), monohydrogen phosphate ion (HPO4 2- ), dihydrogen phosphate ion (H2PO4 - ), orthophosphoric acid (H3PO4), oxalate ion (C2O4 2- ), hydrogen oxalate ion (HC2O4 - ), and oxalic acid (H2C2O4). Preferably, the precipitant is at least one selected from the group consisting of phosphate ions (PO4 3- ), oxalate ion (C2O4 2- ), and monohydrogen phosphate ion (HPO4 2-), more preferably monohydrogen phosphate ion (HPO4 2- ).

[0085] Mono-gadolinized complexes are Gd(III) complexes in which the dimeric ligand 5 chelates only one gadolinium ion instead of two. These mono-gadolinized complexes do not show the favorable relaxometric properties of the di-gadolinized Gd(III) complexes 5. As the mono-gadolinized complexes have very similar physical characteristics to the di-gadolinized complexes, it is troublesome and difficult to efficiently remove these mono-gadolinized complexes from the solution obtained from the complexation step. Therefore, the preferred embodiment of step 6) avoids as much as possible the formation of mono-gadolinized complexes during or after the complexation step; this is especially true in comparison with the processes disclosed in the prior art mentioned above, which result in a solution of dimeric gadolinium complexes containing more mono-gadolinized complexes.

[0086] According to a preferred embodiment of step 6), the precipitating agent is preferably added in at least a stoichiometric amount relative to the free gadolinium metal ions in the solution of the dimeric complex of formula 5 obtained from step 5). Advantageously, the precipitating agent is added in an amount of at least 1.1 moles, preferably between 1.1 and 5 moles, more preferably between 1.2 and 3 moles, even more preferably between 1.4 and 2.5 moles, most preferably between 1.4 and 1.6 moles relative to 1 mole of gadolinium metal ions in the solution of the dimeric complex of formula 5 obtained from step 5). As also demonstrated by the comparative examples in the experimental section, the addition of these preferred amounts of precipitants results in a solution containing both low amounts of free gadolinium metal ions (i.e. lower amounts of free gadolinium metal ions than those specified above) and low amounts of mono-gadolinized complexes (i.e. lower amounts of complexes than 550 ppm, preferably lower than the LoQ of the method used to determine the amount of mono-gadolinized complexes (<400 ppm relative to the amount of gadolinium complexes)) after the optional filtration step and before the optional further purification step. Conversely, if the precipitants are added in step 6) in amounts higher than the above preferred amounts, the resulting solution may contain high amounts of mono-gadolinized complexes (i.e. higher than 600 ppm). These ppm values ​​of mono-gadolinized complexes, as well as all ppm values ​​of mono-gadolinized complexes in the present invention, are determined by carrying out procedure 6 presented in the experimental section below.

[0087] When step 6) of the process of the present invention is carried out by adding the preferred amount of precipitating agent as defined above, the process of the present invention preferably comprises the further step of determining the amount of free gadolinium metal ions in the solution of dimeric complex of formula 5 obtained from step 5) before adding the precipitating agent, whereby the precipitating agent can be added in the preferred amount as defined above. This determination step can be carried out by known methods for determining the amount of free gadolinium metal ions, for example by the methods disclosed herein.

[0088] Step 6) is preferably carried out by maintaining the temperature of the solution in the range of 15-40° C., more preferably 20-30° C. After adding the precipitating agent, the reaction mixture is preferably maintained, for example, in the above temperature range for 1-4 hours, preferably 1.5-3 hours, more preferably 2 hours, before carrying out any subsequent steps.

[0089] In a preferred embodiment, during and / or after the addition of the precipitating agent according to step 6), the pH is adjusted and / or maintained at a value of 4.5 or more, preferably 4.7 or more, more preferably 4.9 or more, even more preferably 5.5 or more, for example for the times and / or temperatures described above. Preferably, this pH is maintained at least until the precipitated insoluble gadolinium salts are filtered off from the solution of gadolinium complexes. As also demonstrated in the experimental section below by comparative examples, the applicant has surprisingly found that by precipitating free gadolinium metal ions whilst adjusting and / or maintaining the pH at these values, after the optional filtration step and before the optional further purification step, a solution containing a particularly low amount of mono-gadolinized complex (e.g. a solution containing less than 550 ppm, preferably less than 400 ppm, relative to the gadolinium complex) is obtained.

[0090] According to a further preferred embodiment, during and / or after the addition of the precipitating agent according to step 6), the pH can be adjusted and / or maintained higher than the values ​​indicated above and at or below 10.0, preferably at or below 9.0, more preferably at or below 8.5, even more preferably at or below 7.5, most preferably at or below 6.5, for example for the times and / or temperatures described above. Preferably, this pH is maintained at least until the precipitated insoluble gadolinium salts are filtered off from the solution of gadolinium complexes. The applicant has surprisingly found that by operating below these pH values, the amount of free gadolinium metal ions in the solution after the precipitation step and before any further purification steps is particularly reduced.

[0091] According to a more preferred embodiment, during and / or after the addition of the precipitating agent according to step 6), the pH may be adjusted and / or maintained in the range of 4.5-9.0, more preferably 4.7-8.5, even more preferably 4.9-7.3, most preferably 6-6.5 or 5.5-6.5, for example for the times and / or temperatures described above. Preferably, this pH is maintained at least until the precipitated insoluble gadolinium salts are filtered off from the solution of gadolinium complexes. The Applicant has surprisingly found that adjusting and / or maintaining the pH within the ranges indicated above makes it possible to obtain a solution with a particularly low content of free gadolinium metal ions and mono-gadolinized complexes (e.g. a solution with a lower content compared to the same process in which the pH is not adjusted and / or maintained at such a pH) after the optional precipitation step and before the optional further purification step.

[0092] The pH adjustment can be performed by adding a suitable acid, such as, for example, HCl, or a suitable base, such as, for example, NaOH, to the solution. This adjustment is particularly useful to offset the pH change that may occur due to the addition of a precipitating agent. It is clear that no pH adjustment is required if the addition of the precipitating agent does not cause a change in pH that would cause the pH of the resulting solution to fall outside the preferred ranges disclosed above (e.g., because the pKa of the precipitating agent is within the preferred values ​​above and / or because the precipitating agent is added in a low amount such that the pH of the resulting solution does not fall outside the preferred ranges disclosed above).

[0093] Preferably, the pH according to the preferred values ​​disclosed above is maintained at least until the precipitated insoluble gadolinium salts are filtered out from the solution of gadolinium complexes.

[0094] In a further preferred embodiment, after step 6), preferably before step 7), the preparation method of the present invention further comprises a step of filtering the solution of gadolinium complex 5 obtained after the addition of the precipitating agent and removing the precipitated insoluble gadolinium salt from the solution, whereby the precipitated insoluble gadolinium salt is separated from such solution. This filtering step can be carried out by filtering methods known in the art, for example by using a pharmaceutical membrane filter.

[0095] In a further preferred embodiment, the process of the invention comprises a further step of treating the solution of gadolinium complexes obtained after step 6) to remove any precipitant (if any) that has not reacted with the free gadolinium metal ions to form insoluble gadolinium salts, which treatment step does not remove either the free gadolinium metal ions or the mono-gadolinized complexes.

[0096] This treatment step can be carried out, for example, by loading the solution of gadolinium complex onto at least one ion exchange resin, preferably at a flow rate of 1-3 BV / h. Alternatively, or together with loading the complex onto at least one ion exchange resin, the treatment step can be carried out by (A) adding to the solution of dimeric complex 5 a cation that is a precipitant or capable of precipitating an anion generated by the precipitant, thereby precipitating at least such anion as a salt together with such cation, and (B) removing the salt so formed by a filtration step such as that disclosed above. Advantageously, only one filtration step can be carried out to remove both the precipitated insoluble gadolinium salt and the salt formed by the precipitant and the precipitating cation.

[0097] In one embodiment, the present invention comprises at least one further purification step after step 6), preferably after the treatment step for removing the precipitating agent (if performed). This further purification step is useful for further reducing the residual amount of free gadolinium metal ions present after the precipitation of step 6), in order to obtain a solution of gadolinium complexes with the lowest possible content of free gadolinium metal ions. Specifically, according to this embodiment of the production method of the present invention, the majority of free gadolinium metal ions are removed by the precipitation step 6) (wherein the amount of free gadolinium metal ions is reduced from several thousand ppm to several hundred or even tens of ppm relative to the gadolinium complex 5), and a smaller portion is removed by at least one further purification step.

[0098] Further purification steps are preferably carried out by chromatography, preferably on a resin such as an adsorptive resin.

[0099] In one embodiment, a further purification step involves elution of the mixture resulting from the complexation reaction on a polymeric resin, preferably Amberlite XAD® 1600 resin.

[0100] In another embodiment, purification comprises a first elution of the mixture resulting from the complexation reaction with a chelating resin, for example selected from Hi Trap IMAC FF, Lewatit MonoPlus TP 260, Lewatit TP 208, IRC748I, DIAION CR11, SiliaMets AMPA and SiliaMets DOTA, preferably Diaion CR11 and Amberlite IRC748, to minimize the free gadolinium content and to allow further purification of the collected eluate with a polymeric resin, such as Amberlite XAD® 1600 resin.

[0101] In practical practice, the mixture, adjusted to an approximately neutral pH value, is suitably purified by elution on Amberlite XAD® 1600 resin.

[0102] Alternatively, the mixture resulting from the adjustment of the solution pH to a lower value (e.g. 5-5.6) is preferably first eluted with a chelating resin such as Amberlite IRC748 or Diaion CR11 resin. The collected eluate is then preferably readjusted to a pH value of about 5.5-6 and concentrated, preferably under vacuum at 50° C., to obtain an aqueous solution or mixture of dimeric complexes, preferably with a concentration of about 25% (w / w), which is then purified with Amberlite XAD® 1600 resin.

[0103] The collected fractions may then be treated with activated charcoal and filtered. The resulting filtered solution is then preferably concentrated, for example by distillation under vacuum at 45-55° C., to obtain a solution of dimeric complex 5 having a final concentration of about 25% (w / w).

[0104] Process 7 The dimeric complex of formula 5 is then isolated. The complex may be isolated from the aqueous solution or mixture from step 6) by, for example, lyophilization or by spray drying. In a preferred embodiment, the desired dimeric complex is obtained as a white solid by spray drying the solution recovered directly from step 6 of the present process.

[0105] The overall yield of this preparation, as free base DO3A tri-tert-butyl ester 1A, determined from limited reactants, is greater than 30%, such as 33%.

[0106] Interestingly, the method includes steps carried out in one pot, which is suitable for large-scale implementation and does not require isolation of either the prepared precursor (such as the compound of formula 1A) or the reaction intermediates. As a result, the objective of the synthetic approach of the present invention makes it possible to obtain a final product with a >3-fold increase in overall yield compared to the process disclosed in WO2017098044.

[0107] Additionally, the lack of isolation of intermediates allows for a reduction in overall process time.

[0108] Furthermore, the proposed process includes the use of water, or more generally an aqueous solvent or mixture of aqueous solvents, as the reaction solvent in all steps following the preparation of the coupling product 3. In particular, if the compound of formula 3 is prepared in an organic solvent in step 3), for example by reacting the compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1), the organic solvent is replaced by an aqueous solvent or mixture of aqueous solvents by methods known to those skilled in the art, for example by first diluting the organic solution of the compound of formula 3 with water, a water / organic solvent mixture, or an aqueous solution, and then optionally removing the organic solvent to obtain an aqueous solution of the compound of formula 3. The use of an aqueous solvent or mixture of aqueous solvents as the reaction solvent in all steps following the preparation of the coupling is highly advantageous, especially in terms of cost, environmental impact, and ease of implementation on an industrial scale. Indeed, the process disclosed in WO2017098044 uses solvents such as DCM and substances such as TFA and TIPS, which are expensive and may be difficult and unsafe to handle, especially when the process is on an industrial scale. The problems of the prior art processes are solved by the present invention since the process of the present invention avoids or greatly reduces the use of organic solvents by using aqueous solvents or aqueous solvent mixtures in all steps subsequent to the preparation of the compound of formula 3, the latter being suitable and easy to implement for working on a large scale, e.g. for implementation in an industrial process. Moreover, the process of the present invention surprisingly leads to very high yields of isolated dimeric complex, in particular higher than the yields of the prior art processes, even when it involves the use of only aqueous solvents or aqueous solvent mixtures subsequent to the preparation of the compound of formula 3.

[0109] Also, the overall process of the present invention is more robust, faster, safer, has higher yields and gives a purer product compared to the prior art process of WO 2017 / 098044.

[0110] Considering all the above mentioned advantages, it can be seen that the preparation method of the present invention can be easily implemented for large scale synthesis of dimeric complex 5.

[0111] All solvents and starting materials, including reactants such as epichlorohydrin, D-glucamine, and DO3A tri-tert-butyl ester hydrobromide salt, are either commercially available or can be obtained by known procedures.

[0112] In a preferred embodiment, the hydrobromide salt of DO3A tri-tert-butyl ester used as starting material for the preparation of the solution of ester 1A is prepared by using the preparation method described in co-pending patent application EP19215900.2 (of the same applicant as the present application) and exemplified below in the experimental section of this specification, and stored until use.

[0113] For the purpose of illustrating the invention in more detail, without limiting its scope, non-limiting examples of preferred embodiments of the manufacturing method of the invention are described in the following paragraphs.

[0114] Experimental Part Definitions of Abbreviations and Terms DO3A tri-tert-butyl ester: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester DO3A tri-tert-butyl ester-HBr: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate tri-tert-butyl hydrobromide TAZA: 1,4,7,10-tetraazacyclododecane tBuOK Potassium tert-butoxide DMAC N,N-Dimethylacetamide DMC Dichloromethane DIPEA N,N-Diisopropylethylamine HCl Hydrochloric acid MeCN Acetonitrile NaOH Sodium hydroxide NH3 Ammonia MRI Magnetic Resonance Imaging MeCN Acetonitrile NMM N-Methylmorpholine K2CO3 Potassium Carbonate TFA Trifluoroacetic acid TIPS Triisopropylsilane FLD Fluorescence Detector UV / Vis Ultraviolet / Visible

[0115] HPLC characterization of the obtained compounds and determination of the free gadolinium metal ion (free-Gd) or mono-gadolinium complexes (mono-Gd) Step 1: HPLC characterization of DO3A-tri-tert-butyl ester and determination of analytical results Chromatography conditions HPLC System A liquid chromatograph (eg Agilent 1100) equipped with a solvent delivery system, autosampler, column thermostat, degasser and a diode array or variable wavelength detector (or equivalent). Stationary phase: Zorbax Eclipse XDB-C8, 5μm, 150×4.6mm Column temperature: 45℃ Mobile phase: A: 0.01M K2HPO4, 0.017M H3PO4 B: Acetonitrile Elution: Gradient Time (min) %B 0 5 30 80 35 80 38 5 45 5 Flow rate 1mL / min Temperature 45℃ Detection UV, 210nm, Bw=8nm; Reference 360nm, Bw=100nm Injection volume 10μL Downtime 35 minutes Reference peak DO3A 3tBu Retention time DO3A 3tBu approximately 14~15 minutes

[0116] Procedure 2: HPLC method to monitor the formation of intermediate 2 This method is utilized to monitor the mixture upon completion of alkylation of D-glucamine and after distillation of water. Chromatography conditions HPLC system: a liquid chromatograph (e.g., Agilent 1100) equipped with a solvent delivery system, autosampler, column thermostat, degasser, and a diode array detector or variable wavelength detector (or equivalent). Stationary phase: SeQuant ZIC-cHilic 3μm, 150×2.1mm (Merck PN1.50658.0001) Column temperature: 40℃ Mobile phase: Gradient elution Eluent A = 5 mM ammonium acetate Eluent B=ACN / MeOH, 75 / 25 Elution: Gradient Time (min) %B 0 97 5 97 30 20 40 20 45 97 60 97 Flow rate: 0.25mL / min Detection: UV, 210~240nm Injection volume: 10μL Duration: 60 minutes Dilute solution ACN / MeOH, 75 / 25 Sample preparation: Add 200 μL of 5 mM ammonium acetate solution to 75 μL of the mixture and dilute to 5 mL with diluent solution.

[0117] Procedure 3: HPLC method to monitor the formation and purification of intermediate 3 General Procedure This method is used to monitor the process of formation and purification of intermediate 3. Analysis conditions HPLC system Liquid chromatograph Agilent 1100 Stationary phase: Gemini, 5 μm, 250 × 4.6 mm (Phenomenex, item 00G-4435-EO) Column temperature: 40℃ Mobile phase: A: Mobile phase A B:MeCN Elution: Gradient Time (min) %B 0 40 5 40 30 90 35 90 36 40 45 40 Flow rate 0.7mL / min Detection UV / 210nm Injection volume 10μL Downtime 45 minutes INT 2 R t 21 minutes

[0118] Mobile phase A Preparation of solutions Accurately weigh 2.0 g of ammonium acetate into a 1000 mL volumetric flask, then dilute to volume with water. Transfer 600 mL of ammonium acetate solution and 300 mL of methanol to a 1000 mL volumetric flask. Sonicate for 30 minutes.

[0119] Procedure 4: HPLC method to monitor the formation and purification of chelating ligand 4 General Procedure The formation and purification of dimeric ligand 4 was monitored by reverse-phase HPLC with UV detection at 210 nm. Analysis conditions HPLC system Liquid chromatograph Agilent 1260 Infinity Stationary phase: Synergi Polar-RP, 4 μm, 150 x 4.6 mm (Phenomenex, item 00F-4336-EO) Column temperature: 40℃ Mobile phase: A: 10 mM KH2PO4 B: Methanol Elution: Gradient Time (min) %B 0 0 5 0 35 60 40 60 41 80 46 80 47 0 60 0 Flow rate 0.8mL / min Detection UV / 210nm Injection volume 10μL Downtime 60 minutes Compound 4 R t 2.4 minutes

[0120] Step 5: Determination of the amount of free gadolinium metal ion (free-Gd) The amount of free gadolinium metal ion relative to the amount of compound 5 is determined by reversed-phase HPLC (high performance liquid chromatography) with FLD (fluorescence detector) detection. Free Gd(III) present in the sample is formed into a Gd(EDTA) complex using EDTA (ethylenediaminetetraacetic acid) in the mobile phase. Chromatography conditions Instrumentation: Agilent 1100 liquid chromatograph equipped with solvent delivery system, cooled autosampler at 5°C, column thermostat, degasser and fluorescence detector or equivalent. Column: YMC-PACK ODS-AQ, 250×4.6 mm, 5 μm particle size (YMC, cod.AQ12S05-2546WT) Temperature: 40℃ Mobile phase: A: CH3COONH4 (1.5g / L), EDTA (0.55g / L) B: Methanol Flow rate: 1mL / min Detection (FLD): Wavelength excitation = 275 nm Wavelength emission = 314 nm Duration: 25 minutes Acquisition time: 6 minutes Injection volume: 20μL Reference peak: Gd(EDTA) Elution: Gradient Time (min) %B 0 0 5 0 10 50 15 50 16 0 25 0

[0121] Solution preparation mobile phase Accurately weigh 1.5 g of ammonium acetate into a 1000 mL measuring flask, dissolve in purified water, add 0.70 g of ethylenediaminetetraacetic acid disodium salt anhydrous, and then dilute to volume with purified water. Dilute Solution Accurately weigh 3 g of ammonium acetate into a 1000 mL measuring flask, dissolve in purified water, add 1.4 g of ethylenediaminetetraacetic acid disodium salt anhydrous, and then dilute to volume with purified water. Blank solution Transfer 0.5 mL of purified water to the vial and add 0.5 mL of the dilution solution. Mix well and inject directly into the chromatography system. Reference Solution Weigh 0.32 g of gadolinium acetate hydrate (expressed on an anhydrous basis; determine water content prior to use) into a 50 mL volumetric flask and dilute to volume with mobile phase. The concentration of gadolinium is 3 mg / mL. Transfer 0.1 mL of this solution to a 100 mL volumetric flask and dilute to volume with mobile phase. The concentration of gadolinium is 0.003 mg / mL. LOQ solution Transfer 1 mL of the reference solution to a 5 mL volumetric flask and dilute to volume with mobile phase. The concentration of gadolinium is 0.0006 mg / mL. Test Solution Accurately weigh 600 mg of the test sample (expressed on an anhydrous basis) into a 10 mL volumetric flask and dilute to volume with purified water. The concentration of dimeric gadolinium complex 5 is approximately 60 mg / mL. Transfer 0.5 mL of this solution to a vial and add 0.5 mL of the diluted solution. Mix the sample well. Once diluted, immediately place the sample in a cooled autosampler (5-8 °C) and inject the sample within 5 min of dilution. The final concentration of compound 5 is approximately 30 mg / mL.

[0122] Analysis sequence Blank n=1 LOQ solution n=1 Reference solution n=6 Test solution n=6 Reference solution n=1

[0123] System Suitability Testing A system suitability test (SST) is performed each time this manufacturing method is applied. - After equilibrating the chromatographic system, inject one blank solution to verify the absence of interfering peaks. - Inject LOQ solution once If the Gd(EDTA) peak has a S / N≧10, the results of the analytical sequence are informative. - Inject six times the first reference solution and verify that the following requirements for the Gd(EDTA) peak are met: - Area repeatability of Gd(EDTA) expressed as a percentage Relative standard deviation (RSD%, n=6) ≦10% - Retention time reproducibility of the Gd(EDTA) peak expressed as a percentage Relative standard deviation (RSD%, n=6) ≦2% - Symmetry factor for the Gd(EDTA) peak, T, Calculated using formula 1 0.7~2.0 T=w 0.05 / 2f expression 1 During the ceremony: w 0.05 = width at 1 / 20th of peak height (min) f = the distance (in minutes) between the perpendicular line drawn from the peak maximum and the rising edge of the peak at 1 / 20th of the peak height

[0124] calculation Calculate the percentage content, free Gd%, by Equation 2: Free Gd% = [(A t ×W RS ×(100-K)×20) / (A std ×w×(100-K1)×50×1000×2.126)]×100 Formula 2 A t = Peak area of free Gd in the test solution A std = Average area of Gd(EDTA) in the SST reference solution injection (n = 6) W RS = mg of Gd in the reference solution w = Weight (mg) of the compound 5 sample for preparing the test solution K = % of H2O content in gadolinium acetate hydrate K1 = % of H2O content in the sample 2.126 = Correction factor between gadolinium acetate and gadolinium The limit of quantification for free Gd is 0.002% (w / w). Values lower than the LOQ limit must be indicated as <LOQ or n.q. (not quantifiable). The percentages calculated as described above can be converted to ppm of free gadolinium with respect to compound 5 by multiplying such percentages by 10,000.

[0125] Procedure 6: Determination of the amount of mono-gadolinium complex (mono-Gd) The content of mono-Gd impurities in the dimer complex of compound 5 is quantified by the reverse-phase HPLC method using any FLD detector in the same chromatographic run. The quantification of the specified impurity mono-Gd (specifically, the mono-Gd complex of ligand 4 having only one gadolinium metal ion) is performed by FLD detection using a reference sample mono-Gd as the sodium salt. The mono-Gd sodium salt (reference sample) can be obtained by complexing the dimer ligand 4 with less than stoichiometric amounts of gadolinium ions to obtain mono-Gd, adjusting to neutral pH with NaOH, and then concentrating to the residue.

[0126] Chromatography conditions Instrumentation: HPLC Agilent 1100 equipped with solvent delivery system, autosampler, column thermostat, degasser, UV diode array detector and fluorescence detector 2475 Waters or equivalent. Column: Xselect® HSS T3, 3.5 μm, 150×3.0 mm (Waters, Part No. 186004781) Temperature: 40℃ Mobile phase: Solvent A: Mobile phase A (40 mM potassium phosphate-0.02 mM EDTA in water, pH 6.2) Solvent B: Mobile phase B (solvent A / acetonitrile, 60 / 40 v / v) Flow rate: 0.35mL / min Detection (FLD): Wavelength excitation (λex) = 275 nm Wavelength emission (λem) = 314nm Detection (UV): Wavelength = 210nm / Bw: 8nm; Ref. Wavelength = 480nm / Bw: 80nm Analysis time: 50 minutes Acquisition time: 32 minutes Injection volume: 10μL Eluition: Gradient Time (min) %B 0 0 4 0 20 15 25 15 30 100 40 100 43 0 50 0

[0127] Solution preparation Mobile phase A Into a 2000 mL volumetric flask: - 8.56 g of potassium dihydrogen phosphate - 3.97 g dipotassium hydrogen phosphate trihydrate - 0.015 g Titriplex® III (EDTA disodium salt) Accurately weigh out and then dilute to volume with purified water. Filter through a 0.22 μm membrane filter. Mobile phase B Transfer 600 mL of Mobile Phase A to a 1000 mL volumetric flask and dilute to volume with acetonitrile. Mix well. CaCl 2 Solution of Accurately weigh 165 mg of CaCl2 (expressed on an anhydrous basis) into a 50 mL volumetric flask and dilute to volume with purified water. The concentration is approximately 3.3 mg / mL. Stock solution of mono-Gd Accurately weigh 25 mg of mono-Gd sodium salt (denoted on anhydrous basis and purity) into a 50 mL volumetric flask and dilute to volume with purified water. The concentration of mono-Gd is approximately 0.5 mg / mL. Weight of mono-Gd = weight of mono-Gd sodium salt × 1140.31 / 1162.29 Mono-Gd reference solution Accurately transfer 0.45 mL of the mono-Gd stock solution into a 5 mL volumetric flask. Add 1 mL of CaCl2 solution and dilute to volume with purified water. The standard concentration is 0.045 mg / mL. LoQ solution of mono-Gd Accurately transfer 0.1 mL of the stock solution of mono-Gd into a 5 mL volumetric flask. Add 1 mL of CaCl2 solution and dilute to volume with purified water. The concentration of mono-Gd is approximately 0.01 mg / mL. Blank solution Transfer 0.8 mL of the aqueous solution to a vial and add 0.2 mL of the CaCl2 solution. Mix well. Test Solution Accurately weigh 125 mg of test sample (expressed on an anhydrous basis) into a 5 mL volumetric flask. Add 1 mL of CaCl2 solution and dilute to volume with purified water. The concentration of compound 5 is approximately 25 mg / mL.

[0128] Analysis sequence Blank n=1 LoQ solution n=1 Reference solution n=6 Test solution n=6 Reference solution n=1

[0129] Calculation The percentage content of mono-Gd (w / w) is calculated by the following formula (3) through FLD acquisition. C モノGd % = [(A T × V S × W S × (100 - KF) × a × f) / (A R × w T × 50 × 100 × (100 - KF 化合物5 ))] × 100 Formula 3 In the formula: A T : Peak area in the test solution (if present, the total area of the mono-Gd-1 / 2 / 3 / 4 peaks) A R : Peak area in the injection of the reference solution (average value n = 6) (total area of the mono-Gd-1 / 2 / 3 / 4 peaks) w T : Weight of the sample in the test solution (mg) V S : Volume of the mono-Gd stock solution taken out to prepare the reference solution (mL) W S : Weight of the mono-Gd sodium salt used to prepare the stock solution (mg) KF: % of H2O content in the mono-Gd sodium salt KF 化合物5 : % of H2O content in Compound 5 a: % analysis result of the mono-Gd sodium salt f: Correction factor for molecular weight: 0.98 The limit of quantification for mono-Gd (total of four peaks) is 0.04% (w / w). Values lower than the LOQ limit must be indicated as <LOQ or n.q. (not quantifiable). The percentage calculated as described above can be converted to ppm of the mono-gadolinium complex with respect to the complex of Compound 5 by multiplying such percentage by 10,000.

Example

[0130] Example 1: Synthesis of DO3A-tri-tert-butyl ester hydrobromide The synthesis of the starting material DO3A tri-tert-butyl ester hydrobromide was carried out by using the procedure of co-pending patent application EP19215900.2 (of the same applicant as the present application). Specifically: To a suspension of commercial TAZA (14.39 kg; 83.53 mol) and sodium acetate (21.58 kg; 263.12 mol) in DMAC (98.07 kg; 104.33 L) was added a solution of tert-butyl bromoacetate (51.32 kg; 263.12 mol) in DMAC (50.72 kg; 53.96 L) over 2.5 hours at 10° C. The temperature was then raised to 25° C. and the mixture was stirred at this temperature for 24 hours. Water (57.56 kg) was then added over 0.5 hours and after 2 hours the mixture was centrifuged and washed with water (2×57 kg). The wet solid was dried under vacuum to give 36.62 kg; 61.48 mol of DO3A tri-tert-butyl ester hydrobromide (73.6% yield). The assay of the product, determined by HPLC (against a standard), is 100% w / w; the assay, determined by NMR (against a standard), is 99.86% w / w.

[0131] Example 2: Preparation of dimeric compound 5 The dimeric complex compound 5 is obtained by using the synthetic procedure shown below. [ka] Includes: a) Neutralization of the Hydrobromide to Give DO3A Tri-tert-Butyl Ester 1A DO3A tri-tert-butyl ester-HBr (36.60 kg; 61.4 mol) obtained as described in Example 1 and potassium carbonate (16.99 kg; 122.9 mol) are suspended in MeCN (72.50 kg; 91.77 L) at room temperature (i.e. 25° C.) and the mixture is kept under stirring at 24° C. for 19 hours. The resulting salts are then filtered off and the filtrate is partially distilled under vacuum at 50° C. to obtain a solution of DO3A tri-tert-butyl ester 1A (31.07 kg; 60.4 mol) in MeCN with a final concentration of about 56% w / w, which is used in the next step without further isolation. The analysis of 1A in the solution is determined by HPLC-UV method. b) Synthesis of intermediate 2 D-glucamine (6 kg; 33.1 mol) in water (15.1 kg) is added dropwise to a solution of epichlorohydrin (6.75 kg; 73.0 mol) in DMAC (6.2 kg; 6.60 L) at room temperature for 2 h. The mixture is kept under stirring for 16 h. The mixture is then diluted with DMAC (12.2 kg; 12.98 L) and water is distilled under vacuum at 55-60° C. to a solution of intermediate 2 in DMAC with a residual water content of <2.0%, which is used in the next step without purification. c) Alkylation of intermediate 2 with DO3A tri-tert-butyl ester 1A to give the protected ligand 3 A solution of DIPEA (9.80 kg; 75.8 mol) and substrate 1A recovered from step a) is added to the solution of intermediate 2 heated to 50° C., and the resulting mixture is then stirred at 70° C. for 80 h while monitoring the conversion by HPLC-UV method. The mixture is then partially concentrated under vacuum at 60° C. The residue is cooled to 25° C. and diluted with a previously prepared mixture of water (12.2 kg) and MeCN (19.0 kg; 24.05 L), then with 25% aqueous ammonia solution (21.7 kg; 24.03 L) to obtain a mixture that is kept under stirring for 15 h to obtain the precipitation of the hydrochloride salt, which is filtered. The filtrate is then collected and purified by chromatography on Amberlite XAD® 1600 (450 L; eluent: water / MeCN gradient). Pure fractions (HPLC area %≧90) are then collected, the organic solvent is distilled and the aqueous residue is concentrated under vacuum at about 50° C. to give a solution of the protected ligand 3 in water (at a concentration of about 10% w / w) suitable for direct use in the next step without further isolation. d) Deprotection of protected ligand 3 to obtain deprotected ligand 4 To a mixture (219 kg) of protected dimeric ligand 3 at a concentration of 20% (w / w), the theoretically calculated product in water, 34% w / w aqueous hydrochloric acid (53.3 kg, 50.0 mol, 15 mol / mol with respect to protected ligand 3) is added while maintaining the temperature at 30° C. At the end of the addition, the mixture is heated to 50° C. and kept under stirring for 16 hours. After complete deprotection, 30% w / w aqueous sodium hydroxide is added to pH 5.6 to obtain the deprotected ligand 4. The t-butanol formed as a by-product is removed by distillation. The solution containing ligand 4 is concentrated by distillation under vacuum at 50° C. to a final concentration of about 10% (w / w). e) Complexation of ligand 4 to give dimeric complex 5 The solution of dimeric ligand 4 obtained by the previous step d) is placed in a first reactor and heated to 40°C. A gadolinium chloride solution (2.05 mol for 1 mol of ligand 4) is added while maintaining the temperature in the range of 37-43°C. At the end of the addition, the pH is adjusted to 5.5 by addition of 10% w / w aqueous sodium hydroxide solution. The mixture is maintained at 40°C for 3 hours. A solution containing gadolinium complex 5 is thus obtained and the amount of mono-gadolinized complex (mono-Gd) and free gadolinium metal ion (free Gd) is measured. The salts formed in example 1 and in the complexation step are then removed by nanofiltration; diafiltration is carried out until the conductivity value is lower than 1.0 mS / cm. At the end of the nanofiltration, the mixture is concentrated to 10÷12% w / w and 1.5 mol / mol of Na2HPO4 for free gadolinium metal ion (free Gd) is added to the solution. After addition of Na2HPO4, the pH of the solution is measured for each test and reported in Table I below (column "pH Start"); the formation of a white precipitate is observed. The pH is then adjusted to the value reported in column "pH End" of Table I. The mixture is kept under stirring for 2 hours. Finally, the suspension is filtered and the amount of mono-gadolinized complex (mono-Gd) and free gadolinium metal ion (free Gd) is measured. These amounts, as well as the amounts obtained after complexation, are shown in Table I below. [Table 1] Based on the results shown in Table I, a significant reduction in the amount of free Gd can be observed for all of the tests 1-9 shown above. All solutions of tests 1-5 and 7-11 contained non-quantifiable amounts of mono-Gd, i.e., less than 400 ppm of mono-Gd; test 6 shows that at pH 4.54, there is an amount of mono-Gd higher than the LoQ, i.e., 538 ppm for compound 5. Table I also shows that while maintaining the pH within the range of 4.9-8.3, less than the LoQ amount of mono-Gd and low amounts of free Gd can be obtained. f) Removal of residual phosphate The solution of test 1 obtained in step e) above is loaded onto an ion exchange resin (Diaion PA 308, pre-activated) at a flow rate of 1-3 BV / h. Most of the residual phosphate is thus removed from the solution. g) Further purification and isolation of the dimeric gadolinium complex The solution from step f) is placed in a second reactor, the pH of the solution is adjusted to 5.7-6.3 by addition of dilute HCl, and water is distilled under vacuum at 45-55°C until the gadolinium complex assays about 20-25% w / w. The concentrated solution is loaded onto pre-activated Amberlite XAD® 1600 (amount of resin: 30 mL / g of product) at a flow rate of 0.5 BV / h. Purification is carried out using water and a mixture of isopropanol and water. The fraction with high purity (assessed by HPLC-FLD / UV) is placed in a separate reactor. After preconcentration, it is treated with activated charcoal. The suspension is filtered to remove the activated charcoal, the solution is concentrated under vacuum at 45-55 °C to a concentration of 25% w / w, and the gadolinium complex is isolated by spray drying as a white powdery solid with a final titration analysis of 99% (w / w%; anhydrous basis) (14.1 kg, 12.7 kg on anhydrous basis, 9.83 mol). Overall yield determined from DO3A tri-tert-butyl ester 1A: 33%.

[0132] Example 3: Further testing of precipitation with precipitants Test 12 The content of mono-gadolinium complex (mono-Gd) and free gadolinium metal ion (free Gd) is determined for a 10% w / w solution of complex 5 containing free gadolinium obtained by the procedure of steps a) to d) of Example 2. K3PO4 is then added to the solution (1.5 mol per mol of free Gd). After the addition of phosphate, the pH rises from 5.42 to 9.00 and the formation of a white solid is observed immediately. The mixture is maintained at pH 9.00 under stirring at room temperature for 2 hours, and then the solid is filtered to obtain a solution. The contents of free gadolinium metal ion (free Gd) and mono-gadolinized complex (mono Gd) are measured and are shown in Table II below. [Table 2] Based on the results shown in Table II, a significant reduction in the amount of free Gd can be observed in the precipitation using phosphate as the precipitating agent. Furthermore, the solution contains an unquantifiable amount of mono-Gd, i.e., less than 400 ppm of mono-Gd.

[0133] Test 13 The content of mono-gadolinium complex (mono-Gd) and free gadolinium metal ion (free Gd) is measured for a 10% w / w solution of complex 5 containing free gadolinium obtained by the procedure of steps a) to d) of Example 2. Disodium oxalate is then added to the solution (2.25 mol / mol with respect to free Gd). After the addition of oxalate, the pH rises from 5.56 to 7.78 and the formation of a white solid is observed immediately. The pH is then lowered to 6.43 using 1N HCl. At the end of mixing, the mixture is maintained at room temperature for 2 hours. After filtering the suspension, the content of mono-gadolinium complexes and free gadolinium metal ions is measured. The content of mono-gadolinium complexes (mono-Gd) and free gadolinium metal ions (free Gd) is shown in Table III below. [Table 3] Table III clearly shows that tests carried out as described above, especially those using the preferred amount of oxalate as the precipitant, result in solutions containing very low amounts of free Gd and mono-Gd below the LoQ.

Claims

1. formula 【Chemistry 1】 A method for producing a dimeric complex compound 5 represented by the following formula: 1) A compound of formula 1A in an organic solvent 【Chemistry 2】 Preparing a solution of DO3A tri-tert-butyl ester represented by the formula: 2) in an organic solvent, 【Chemistry 3】 preparing a solution of a compound represented by 3) Mix the solutions prepared in steps 1) and 2) to obtain a solution of formula 3 【Chemistry 4】 obtaining a solution of a compound represented by 4) Removing the tert-butyl protecting group from the compound of formula 3 without isolating the compound from the solution of step 3) to give a compound of formula 4 【Chemistry 5】 obtaining a solution of free ligand represented by 5) adding a molar excess of gadolinium metal ions to the solution of step 4) without isolating the free ligand of formula 4 to obtain a solution of the dimeric complex of formula 5; 6) adding a precipitating agent to the solution of the dimeric complex of formula 5 to precipitate the free gadolinium metal ions; and 7) Isolating the dimeric complex wherein the reaction solvent in all steps subsequent to the preparation of the compound of formula 3 is an aqueous solvent.

2. 2. The process according to claim 1, wherein the solution of DO3A tri-tert-butyl ester 1A in step 1) is prepared from DO3A tri-tert-butyl ester hydrobromide.

3. Step 1) is: i) suspending the hydrobromide salt of DO3A tri-tert-butyl ester together with a base or a basic salt in an organic solvent to obtain a suspension; ii) filtering the suspension; and iii) recovering the filtered suspension and optionally concentrating it to obtain a solution of DO3A tri-tert-butyl ester 1A in an organic solvent; The method of claim 1 or 2, comprising:

4. Step 2) comprises reacting D-glucamine with epichlorohydrin to obtain a solution of the compound of formula 2 in an organic solvent, preferably comprising the following steps: i) adding an aqueous solution of D-glucamine to a solution of epichlorohydrin in an organic solvent to obtain a compound of formula 2 in a water / organic solvent mixture; preferably the amount of epichlorohydrin is in slight excess over the stoichiometric amount; ii) removing water from the mixture to obtain a solution of the compound of formula 2 in the organic solvent. The method of claim 1 or 2, comprising:

5. The method according to claim 4, wherein the organic solvent is dimethylacetamide (DMAC).

6. Step 3) is a. reacting the compound of formula 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1) in the presence of a base to obtain an organic crude solution; b. diluting the resulting organic crude solution with water, a water / organic solvent mixture, or an aqueous solution to obtain an aqueous / organic crude product; c. purifying the aqueous / organic crude to obtain the compound of formula 3 in an aqueous / organic solvent mixture; and d. Removing the organic solvent from the mixture to obtain an aqueous solution of the compound of formula 3. The method of claim 1 or 2, comprising:

7. 7. The process of claim 6, wherein the purification of the aqueous / organic crude in step c. is carried out by chromatography.

8. Step 4) is: i) adding an acid, preferably an inorganic acid comprising a counterion having a single negative charge, to the aqueous solution of the compound of formula 3 from step 3) and heating and / or maintaining the temperature of the solution at a temperature above 40° C., preferably from above 40° C. to 60° C., more preferably within the range of 45-55° C., following and / or during such addition, to remove the tert-butyl protecting group to obtain an acidic aqueous solution of the free ligand of formula 4; ii) adding a base to the acidic solution to obtain a substantially neutral aqueous solution of the free ligand; iii) purifying the resulting neutral solution and then optionally concentrating it to obtain an aqueous solution of the free ligand of formula 4. The method of claim 1 or 2, comprising:

9. Step iii) is distilling the neutral solution to remove the formed t-butanol; and Optionally concentrating the aqueous solution The method of claim 8, comprising:

10. 3. The method according to claim 1 or 2, wherein in step 5), gadolinium metal ions are added to the solution of step 4) in an amount of 2.05 moles or more per mole of the deprotected dimeric ligand of formula 4.

11. 3. The process according to claim 1 or 2, wherein during and / or after the addition of the precipitating agent according to step 6) the pH is adjusted and / or maintained at a value of 4.5 or higher.

12. 12. The process according to claim 11, wherein during and / or after the addition of the precipitating agent according to step 6) the pH is adjusted and / or maintained at a value of 4.7 or higher.

13. 13. The process according to claim 12, wherein during and / or after the addition of the precipitating agent according to step 6) the pH is adjusted and / or maintained at a value of 4.9 or higher.

14. 14. The process according to claim 13, wherein during and / or after the addition of the precipitating agent according to step 6) the pH is adjusted and / or maintained at a value of 5.5 or higher.

15. The process according to claim 1 or 2, wherein the precipitating agent is added in an amount of 1.1-5 moles per mole of gadolinium metal ion in the solution of the dimeric complex of formula 5 obtained from step 5).

16. The process according to claim 15, wherein the precipitating agent is added in an amount of 1.2-3 moles per mole of gadolinium metal ion in the solution of the dimeric complex of formula 5 obtained from step 5).

17. The process according to claim 16, wherein the precipitating agent is added in an amount of 1.4-2.5 moles per mole of gadolinium metal ion in the solution of the dimeric complex of formula 5 obtained from step 5).

18. The process according to claim 17, wherein the precipitating agent is added in an amount of 1.4-1.6 moles per mole of gadolinium metal ion in the solution of the dimeric complex of formula 5 obtained from step 5).

19. The precipitant is phosphate ion (PO ４ ３－ ), monohydrogen phosphate ion (HPO ４ ２－ ), dihydrogen phosphate ion (H ２ P.O. ４ － ), orthophosphoric acid (H ３ P.O. ４ ), oxalate ion (C ２ O ４ ２－ ), hydrogen oxalate ion (HC ２ O ４ － ), and oxalic acid (H ２ C ２ O ４ 3. The method according to claim 1, wherein the at least one selected from the group consisting of:

20. 3. The method according to claim 1 or 2, further comprising the step of filtering the solution of gadolinium complex 5 obtained after the addition of the precipitating agent and removing the precipitated insoluble gadolinium salt from said solution after step 6) and before step 7).

21. 3. The process of claim 1 or 2, wherein step 7) comprises isolating the dimeric complex of formula 5 as a white solid by spray drying the solution directly recovered from step 6).

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