Manufacture of dimeric contrast agents

Abstract

The present invention relates to a process for the preparation of the dimeric contrast agent [μ-[1-[bis[2-(hydroxy-κO)-3-[4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κN 1 ,κN 4 ,κN 7 ,κN 10 ]propyl]amino]-1-deoxy-D-glucitol(6-)]dodecadodecaacetate for magnetic resonance imaging (MRI), wherein the preparation steps are carried out in one pot without isolation of the resulting intermediates.

Description

Invention Field

[0001] This invention relates to the preparation of contrast agents for magnetic resonance imaging (MRI). In particular, this invention relates to a novel method for preparing dimeric contrast agents, especially [μ-[1-[bis[2-(hydroxy-κO)-3-[4,7,10-tris[(carboxy-κO))methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κN] 1 ,κN 4 ,κN 7 ,κN 10 ]propyl]amino]-1-deoxy-D-glucol(6-)] digadolinium. Background Technology

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

[0003] The powerful expansion of medical MRI has further benefited from the development of a class of compounds, namely MRI contrast agents, which work by causing significant changes in the relaxation rate of nearby water protons in the tissues / organs / fluids where they are distributed, thus adding relevant physiological information to the impressive anatomical resolution typically obtained in uncontrast MRI images.

[0004] Contrast agents used in MRI imaging typically consist of paramagnetic metal ions, which are coupled with cyclic or acyclic chelated ligands, more typically polyamino or polycarboxylic acid chelators. The most important class of MRI contrast agents is represented by Gd(III) chelates, which are currently used in approximately one-third of clinical trials. Indeed, Gd(III) is highly paramagnetic, possesses seven unpaired electrons, and has a long electronic relaxation time, making it an excellent candidate for relaxants.

[0005] WO2017098044 (by the same applicant as this application) discloses a dimer paramagnetic complex of the following formula that can be used as a contrast agent (particularly in magnetic resonance imaging (MRI)).

[0006]

[0007] And the synthetic routes used to prepare them.

[0008] Among many specific compounds, this application discloses a digadolinium complex of the following formula: 1-[bis[2-hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecyl-1-yl]propyl]amino]-1-deoxy-D-glucanol ligand.

[0009]

[0010] The following text is otherwise identified as “dimeric complex compound 5” or more simply as “compound 5”.

[0011] This complex compound exhibits interesting properties, particularly in terms of relaxation and tolerability, making it suitable for in vivo diagnostic imaging with paramagnetic complexes at doses lower than those required by commercial contrast agents.

[0012] The aforementioned international application also discloses a preparation process, the scheme of which is shown in the following general scheme 1:

[0013] Option 1

[0014]

[0015]

[0016] The main steps of the disclosed synthesis method include:

[0017] a) The preparation and isolation of DO3A tritert-butyl ester (compound 1A) were carried out in a manner substantially as disclosed in Org. Synth. 2008, 85, 10;

[0018] b) Intermediate 2 was prepared by alkylating D-glucosamine with epichlorohydrin (molar ratio 1:4.95) in MeOH at 50 °C for 26 hours, and the condensation product was separated by evaporation of the crude reaction.

[0019] c) Alkylation of DO3A tritert-butyl ester with intermediate 2 in DMSO and Et3N, followed by evaporation and reaction in Amberlite. The crude residue was purified at 1600 to obtain protected ligand 3;

[0020] d) Deprotected ligand 3 in dichloromethane with TFA acid and TIPS, evaporated the crude reaction and purified the residue on an Amberlite XE 750;

[0021] e) Ligand 4 was complexed with gadolinium hexahydrate in water and the crude product obtained by filtration and evaporation of the solution was purified on Amberchrome CG161M resin.

[0022] This method requires the synthesis and separation of each individual intermediate, typically by evaporation to the solvent residue. This separation step, besides being unsuitable for large-scale production, inevitably leads to a reduction in the overall yield and efficiency of the method. Furthermore, the existing method is particularly unsuitable for larger-scale production, such as in industrial processes, because it involves the use of harsh and difficult-to-handle materials such as TFA, TIPS, and DCM, which can cause corrosion and thus damage to synthesis equipment and / or may pose health and safety risks to workers. Invention Overview

[0024] This invention generally relates to an optimized method for preparing dimer compound 5, which includes a one-pot preparation step without separating the resulting intermediates, thus saving time and improving overall yield and efficiency.

[0025] More specifically, the present invention relates to a method for preparing a dimer complex compound 5.

[0026]

[0027] As a key step, the method includes:

[0028] 1) Preparation of DO3A tritert-butyl ester of formula 1A, for example, in a solution in an organic solvent;

[0029]

[0030] 2) Preparation of intermediate of formula 2, for example, in a solution in an organic solvent.

[0031]

[0032] 3) Mix the solutions prepared according to steps 1) and 2) to obtain a solution of the protected ligand of formula 3.

[0033]

[0034] 4) Without separating the protected ligand from the solution of step 3), remove the tert-butyl protecting group from the protected ligand to obtain a solution of the corresponding free ligand of formula 4.

[0035]

[0036] 5) Without separating the free ligands of Formula 4 from the resulting solution, gadolinium metal ions are added to the solution of step 4) to obtain a solution of the corresponding dimer complex of Formula 5; and

[0037] 6) Separate the dimer complex.

[0038] In a preferred aspect of the invention, the reaction solvent in all steps following the preparation of compound 3 is an aqueous solvent or a mixture of aqueous solvents. Advantageously, the aqueous solvent or mixture of aqueous solvents does not contain harsh materials such as TFA, TIPS, and / or DCM.

[0039] Step 1) of this method typically involves preparing a solution of DO3A tri-tert-butyl ester 1A. In one embodiment, the solution is prepared in an organic solvent, for example by dissolving commercial DO3A tri-tert-butyl ester in a solvent or by using DO3A tri-tert-butyl ester prepared by a known synthetic method.

[0040] In a preferred embodiment, the solution of 1A is prepared prior to its use by converting a salt of DO3A tritert-butyl ester, such as hydrobromide, to the corresponding free base 1A. The conversion, typically involving hydrobromide neutralization, is preferably carried out in an organic solvent and in the presence of a base or basic salt (as a salt of a product of the neutralization of a strong base and a weak acid, which hydrolyzes to form an alkaline solution). Removal of the formed salt and optional concentration and filtration of the solution allow for the acquisition of a solution of DO3A tritert-butyl ester 1A in an organic solvent, suitable for direct use in subsequent steps of the method without requiring any purification or separation of the ester.

[0041] Suitable organic solvents preferably include MeCN, propylene carbonate, ethanol, tert-butanol, hexane, etc. More preferably, the organic solvent is MeCN.

[0042] Suitable bases or basic salts for neutralizing the initial hydrobromide include, for example, strong bases and anion exchange resins such as Diaion PA308, Amberlite IRA 400, KOH, tBuOK, Na2CO3, and K2CO3, with the latter two being preferred. More preferably, the neutralization of DO3A tri-tert-butyl hydrobromide is carried out in the presence of K2CO3.

[0043] Step 2) of the method comprises preparing compound 2, which can be obtained by alkylation of D-glucosamine with epichlorohydrin. Alkylation is carried out in an organic solvent such as a dipolar organic solvent or an aqueous mixture thereof. Suitable organic solvents include, for example, DMAC, DMF, alcohols such as MeOH, and mixtures thereof. More preferably, the organic solvent is DMAC. Any aqueous solvent and / or excess epichlorohydrin are distilled off from the mixture, yielding a solution of compound 2 in an organic solvent, which is suitable for direct use in the next step of the method without separation and / or further purification of the alkylation product.

[0044] Step 3) of this method essentially involves the condensation (or coupling, as used interchangeably herein, of the intermediate compound of Formula 2 with DO3A tritert-butyl ester 1A to form the protected ligand of Formula 3. The condensation reaction is preferably carried out in the presence of a base, for example, acting as an acceptor for the formed HCl. 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.

[0045] In one embodiment, a condensation reaction is carried out by adding an organic solution of DO3A tritert-butyl ester 1A collected directly from step 1 to a solution of compound 2 collected from step 2, to obtain a crude organic solution containing the condensation product of formula 3 in an organic solvent mixture. The crude organic product is then purified, resulting in an aqueous / organic solvent mixture of the purified product, and optionally, any organic solvent is finally distilled, such that the protected ligand of formula 3 in an aqueous solvent or an aqueous solvent mixture can be obtained, which can be used directly in a next step of the method without separation and / or further purification of the protected ligand itself.

[0046] Step 4) of the method essentially involves removing the carboxyl protecting group from the protected ligand of Formula 3 to obtain an aqueous solution or aqueous mixture of the corresponding free ligand of Formula 4. Deprotection by hydrolysis of the tert-butyl protecting group can be carried out under acidic and alkaline conditions using reaction reagents and conditions known to those skilled in the art. In one embodiment, deprotection is carried out directly from the aqueous solution or aqueous mixture of the protected ligand collected in step 3) of the method by acidification to obtain an acidic solution of the free ligand of Formula 4. Acidification is preferably carried out by adding an acid, such as selected from HCl, H2SO4, and H3PO4. In a preferred embodiment, deprotection is carried out by using HCl. The acidic solution is then neutralized, followed by purification and partial concentration of the resulting mixture, resulting in the collection of an aqueous solution or aqueous mixture of ligand 4, which can be used directly in the coordination step without separation.

[0047] Step 5) involves the ligand coordinating with gadolinium metal ions to obtain the desired dimer complex 5. The coordinating reaction can be conveniently carried out according to known methods, for example by stoichiometric addition of a suitable Gd(III) derivative, particularly an oxide such as Gd₂O₃ or a gadolinium salt, to the ligand solution. In one embodiment, the coordinating reaction is carried out by adding GdCl₃ directly to the ligand solution collected from step 4) of the method. The resulting mixture is adjusted to a pH of approximately 5 to approximately 7 and maintained under stirring to obtain an aqueous solution or aqueous mixture of gadolinium complex 5, which is then purified and concentrated to obtain a solution of the desired dimer complex 5 with the desired purity.

[0048] Step 6) involves the final separation of the desired gadolinium complex 5. This step can be conveniently performed according to known methods. In one embodiment, the solution of the purified complex collected in step 5) is spray-dried to obtain the desired product as a white solid meeting the required purity specifications.

[0049] Interestingly, the methods described above avoid or greatly reduce the use of harsh reagents, such as trifluoroacetic acid (TFA), and unpleasant solvents, such as dichloromethane, which are required in existing methods and difficult to handle when produced on a larger scale, such as in industrial processes.

[0050] Furthermore, it includes a one-pot process step, which is suitable for large-scale implementation and does not require separation of any of the precursors (e.g., 1A) or intermediates used in the preparation. Therefore, in addition to facilitating a reduction in process time and ease of implementation on a larger scale as described above, this process advantageously allows the overall process yield to increase significantly from 10% (obtained using the process disclosed in WO2017098044) to at least 20%, preferably 25%, typically about 28%, advantageously exceeding 30% overall yield. Invention Details

[0052] In this specification, unless otherwise stated, the term "intermediate" (e.g., used to refer to a compound of formula 2 derived from the alkylation reaction of D-glucosamine with epichlorohydrin, or a protected ligand of formula 3) includes, within its meaning, a molecule produced during the chemical synthesis or preparation steps of this method that is not itself a final product but requires one (or more) further reactions, such as alkylation / deprotection / coordination reactions, to obtain the final product of this method, namely the dimer complex compound 5.

[0053] Unless otherwise stated, the term "precursor" (e.g., used to refer to compound 1A) includes, within its meaning, a molecule that participates in a chemical reaction that promotes its conversion into another molecule, which includes or is derived from the precursor.

[0054] In this description, the term "aqueous solvent" includes, within its meaning, water and aqueous solutions of brine, and may include small amounts of water-miscible organic solvents, such as 10% or less by volume, preferably 8% or less, more preferably 5% or less, for example, because the method of the present invention is carried out without separating most of the intermediate products, so small amounts of organic solvents can be introduced through upstream and preceding steps of the method. Preferably, the aqueous solvent is water.

[0055] The expression "water / organic solvent mixture," or more simply, "aqueous solvent mixture," includes, within its meaning, a mixture of two or more solvents comprising an aqueous solvent, such as water, and a mixture of one or more organic solvents miscible with each other, to obtain a homogeneous solvent mixture, wherein the volume percentage of said one or more organic solvents is greater than 10%, preferably greater than 15%, more preferably greater than 20%. Suitable examples include, for example, a mixture of water and acetonitrile (or water / MeCN) used as an eluent in the chromatographic purification of compounds of Formula 3, or a mixture of water / MeCN / DMAC, for example, obtained by diluting the crude mixture obtained from the condensation reaction of step 3 with water. According to a preferred aspect of the invention, the one or more organic solvents in the aqueous solvent mixture (and in the aqueous solvent, if present) are not harsh solvents or materials; in fact, said aqueous solvent mixture preferably does not contain harsh solvents or materials such as TFA, TIPS, and / or DCM.

[0056] Similarly, the expressions “aqueous solution” and “aqueous mixture” respectively include, in their meaning, solutions or mixtures containing water. Suitable embodiments respectively include solutions of one or more compounds, such as reagents, acids, bases, or reaction products, in water (more generally, in aqueous mixtures) or in aqueous solvent mixtures and mixtures, such as water / organic mixtures obtained by adding water or an aqueous solution to a reaction mixture in an organic solvent or solvent mixture.

[0057] In this description, the term "protecting group" (e.g., used for compounds of Formula 3) refers to a protecting group suitable for retaining the function of the group bound to it. Specifically, a protecting group is used to retain the carboxyl functional group. More specifically, the term refers to tert-butyl, which retains the chelating function of the ligand carboxyl group by forming tert-butyl esters [see General Reference on Protecting Groups and Deprotection Conditions, TW Green and PGM Uts; Protective Groups in Organic Synthesis, Wiley, NY 1999, 3rd Edition].

[0058] One embodiment of the present invention relates to a method for preparing a dimer compound 5, substantially as shown in the following general synthetic scheme 2.

[0059] Option 2

[0060]

[0061] As the main steps, it includes:

[0062] 1) Neutralize the hydrobromide of DO3A tritert-butyl ester in an organic solvent to obtain a solution of DO3A tritert-butyl ester 1A in an organic solvent;

[0063] 2) Reaction of D-glucosamine with epichlorohydrin yields a solution of compound 2 in an organic solvent such as DMAC; moreover, product separation is not required.

[0064] 3) In the presence of a base, react the compound of formula 2 from step 2) with the tri-tert-butyl DO3A from step 1), and optionally concentrate the solution to obtain a crude organic product. Dilute the crude organic product with water, a water / organic solvent mixture, and / or optionally an aqueous solution to obtain a water / organic crude product. Purify the water / organic crude product and optionally remove any organic solvent to obtain an aqueous solution or an aqueous mixture of the protected ligand of formula 3; and, product separation is not required.

[0065] 4) Acidify the solution of the protected ligand of formula 3 in step 3) to obtain an acidic aqueous solution or aqueous mixture of the corresponding deprotected ligand 4. Neutralize the acidic solution and purify the resulting neutral solution to obtain an aqueous solution or aqueous mixture of the deprotected ligand 4. Furthermore, it is not necessary to separate the latter.

[0066] 5) Gadolinium metal ions were added to the solution of ligand 4 to obtain a solution of the corresponding complex compound 5; and

[0067] 6) Separate the complex.

[0068] Step 1

[0069] The first step of this method involves preparing a solution of DO3A tritert-butyl ester 1A in an organic solvent such as MeCN by directly converting the hydrobromide of DO3A tritert-butyl ester into the corresponding free base in an organic solvent in the presence of a base or basic salt.

[0070] In a preferred embodiment, step 1) of the method includes:

[0071] i) The hydrobromide of DO3A tritert-butyl ester is suspended together with a base or basic salt such as K2CO3 in an organic solvent such as MeCN to obtain a suspension containing the formed salt.

[0072] ii) Filter the suspension; and

[0073] iii) Collect and optionally concentrate the filtered suspension to obtain a solution of DO3A tritert-butyl ester 1A in an organic solvent, which is suitable for direct use in subsequent condensation reactions without the need for any purification or separation of the ester.

[0074] Preferably, the ester hydrobromide and K2CO3 are suspended in an organic solvent at room temperature to obtain a mixture, which is then maintained at a temperature of 20 to 30°C, preferably about 25°C, for 16 to 30 hours, more preferably 18 to 20 hours, with stirring. The mixture is then treated to remove the salts formed, preferably by filtration.

[0075] 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 with a final concentration of 55-65%, preferably about 60% (w / w), which is suitable for direct use in subsequent condensation steps of the method.

[0076] Step 2

[0077] This step involves preparing a solution of the intermediate compound of formula 2 by reacting D-glucosamine with epichlorohydrin. In one embodiment, the reaction is carried out in a solvent mixture, preferably in water / DMAC, using a slightly stoichiometric excess of epichlorohydrin, for example, 2 to 3 moles per mole of D-glucosamine, more preferably about 2.2 moles of epichlorohydrin. Preferably, this reaction in step 2) is carried out using a slightly stoichiometric excess of epichlorohydrin, for example, 2 to 3 moles per mole of D-glucosamine, more preferably 2.05 to 2.5 moles, and even more preferably about 2.2 moles of epichlorohydrin.

[0078] In a preferred embodiment, step 2) of the method includes:

[0079] i) Adding an aqueous solution of D-glucosamine to a solution of epichlorohydrin in DMAC yields the intermediate compound of formula 2 in a water / DMAC solvent mixture; and

[0080] ii) Remove water from the solvent mixture to obtain a solution of compound 2 in an organic solvent.

[0081] D-glucosamine is added to the epichlorohydrin solution, preferably at room temperature for about 2 hours, to obtain a mixture. The mixture is then stirred at a temperature of 15 to 30°C, preferably 15 to 25°C, more preferably 20 to 25°C, for 16 to 24 hours, preferably 16 to 20 hours, more preferably about 17 hours.

[0082] The mixture is then distilled to remove water and any optional epichlorohydrin residue. Distillation is preferably carried out under reduced pressure at a temperature preferably 40-65°C to obtain a solution of the desired Formula 2 intermediate compound in DMAC, with a residual water content preferably <2% w / w. The resulting solution is then used directly for subsequent condensation reactions without any separation or purification of the product.

[0083] Step 3

[0084] This step essentially involves the condensation of DO3A tritert-butyl ester 1A with intermediate 2 in the presence of a base such as Et3N or more preferably DIPEA. The condensation is preferably performed by mixing the base and a solution of ester 1A from step 1) in MeCN with a solution of intermediate 2 collected directly from step 2) in DMAC to obtain a crude solution (or crude product) containing the protected ligand of formula 3 in a mixture of MeCN / DMAC organic solvents, which is then purified.

[0085] In one embodiment, the crude organic solution produced by the condensation reaction is diluted or partially concentrated with water, and then diluted with water or an aqueous solvent mixture (preferably water / MeCN) to obtain a water / organic crude product or an aqueous crude product, as may be used interchangeably herein.

[0086] In a preferred embodiment, the resulting water / organic crude product, or preferably the organic crude product obtained by condensation reaction, is added to an aqueous solution that promotes the precipitation of a reaction-promoting salt (including DO3A tri-tert-butyl hydrochloride), and then removed by filtration to provide a water / organic filtrate solution. The water / organic crude product or the water / organic filtrate solution is then preferably purified by chromatography.

[0087] In one embodiment, the aqueous solution used to promote the precipitation of hydrochloride is ammonia.

[0088] More specifically, step 3) of the method preferably includes:

[0089] i) In the presence of a base (preferably DIPEA), the intermediate compound of formula 2 in step 2) is condensed with DO3A tritert-butyl ester 1A in step 1) to obtain an organic crude solution in an organic solvent mixture containing the condensation product of formula 3 and the reactant salt, and optionally the organic crude is concentrated.

[0090] ii) Dilute the crude organic product from step i) with water or a water / organic solvent mixture (preferably water / MeCN) to obtain a water / organic crude product;

[0091] iii) Optionally, add an aqueous solution to the water / crude organic product to promote the precipitation of the reaction salts, which are then removed by filtration to obtain an aqueous / organic filtrate solution; or

[0092] iv) Dilute the crude organic product from step i) with an aqueous solution of the precipitate of the reaction-promoting salt (removed by filtration) to obtain an aqueous / organic filtration solution;

[0093] v) Purify the water / organic crude product from step ii) or the water / organic filtrate from step iii) or iv) to obtain a solution of the protected ligand of Formula 3 in an aqueous / organic solvent mixture. This solution can be used in the subsequent deprotection step of the method without any separation or further purification of the protected product; and

[0094] vi) Optionally remove any organic solvent from the mixture to obtain a solution of the protected ligand of Formula 3 in water, which is used in the subsequent deprotection step of the method without any separation or further purification of the protected product.

[0095] The condensation reaction is preferably carried out by adding a base and a solution of ester 1A collected from step 1) in MeCN to a solution of the intermediate compound of formula 2 collected from step 2) in DMAC.

[0096] The appropriate amounts of base and ester 1A can be conveniently determined relative to the amount of D-glucosamine undergoing the reaction. In one embodiment, the condensation reaction is carried out by using 1.6 to 2.4 moles, preferably about 1.8 moles of ester 1A, and 2 to 4 moles, preferably about 2.3 moles of DIPEA per mole of initial D-glucosamine undergoing the reaction.

[0097] The addition is preferably carried out at a temperature of 40-50°C. Then, the condensation reaction is carried out at a temperature of 50 to 80°C, preferably 65 to 75°C, for a period of time, for example 60-80 hours, preferably 70-75 hours, to obtain a crude solution containing the desired condensation product of Formula 3 and the hydrochloride in a MeCN / DMAC solvent mixture.

[0098] In one embodiment, the crude solution is then diluted with water to obtain a water / organic crude product, preferably at a concentration of about 25-30%, more preferably about 25% (w / w). In a preferred embodiment, the amount of water contained in the aqueous crude product is at least equal by weight to the amount of organic solvent, particularly MeCN, in the mixture; more preferably, the crude product has a water:MeCN ratio of about 60:40.

[0099] Then, preferably by chromatography, more preferably on a resin, and even more preferably on an adsorption resin, such as Amberlite. Purified water / crude organic product at 1600. In a preferred embodiment, water / MeCN mixture is used as the eluent on an adsorption resin (e.g., Amberlite). The aqueous crude product was purified at 1600 to obtain unreacted DO3A tritert-butyl ester 1A and pure condensation product as the separation fraction in a water / MeCN solvent mixture.

[0100] In another embodiment, the crude solution produced by the condensation reaction is first concentrated by removing at least a portion of the MeCN, for example, through distillation. The concentrated solution is then diluted with water or a water:MeCN mixture to obtain a water / organic crude product having the aforementioned water:MeCN ratio, which is then purified by chromatography as described above.

[0101] Optionally, the water / organic crude product obtained as described above is added to an aqueous solution, such as ammonia, and the aqueous solution is cooled to promote the precipitation of unreacted DO3A tritert-butyl ester as hydrochloride, which is then removed by filtration and optionally recycled. Then, it is passed through an adsorption resin (such as Amberlite as described above). Chromatographic purification on 1600 resin removed most of the hydrochloride from the filtrate, yielding residual DO3A tritert-butyl ester 1A and pure condensation product, which were then separated in water / organic solvents (such as water / MeCN solvent mixtures).

[0102] In a preferred embodiment, an aqueous solution, for example comprising ammonia, is added directly to the crude organic solution produced by the condensation reaction. Cooling the mixture promotes the precipitation of unreacted DO3A tritert-butyl ester as hydrochloride. The precipitate is then removed by filtration and optionally recycled. Subsequently, the precipitate is removed by adsorption with a resin (such as Amberlite as described above). Chromatographic purification on 1600 resin removed most of the hydrochloride from the filtrate, yielding residual DO3A tritert-butyl ester 1A and pure condensation product, which were then separated in water / organic solvents (such as water / MeCN solvent mixtures).

[0103] The optional final distillation of the organic solvent, for example, under reduced pressure, from the pure fraction, yields an aqueous solution of the condensation product of Formula 3 with a final concentration of 5-15% (w / w), preferably about 10% (w / w), which is suitable for direct use in the subsequent deprotection step without any separation or additional purification of intermediates.

[0104] Interestingly, the above process steps allow for the acquisition of a protected condensation product 3 in an aqueous solvent or a mixture of aqueous solvents, thereby enabling its deprotection and coordination to the final complex 5 by using water, and more generally an aqueous solution or a mixture of aqueous solvents, as one of the sole or primary reaction solvents. The protected condensation product 3 in the aqueous solvent can be obtained according to various methods known to those skilled in the art. For example, as described above, a 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, an organic solvent mixture, or an aqueous solution to obtain a water / organic crude product. Prior to removal of the organic solvent, the water / organic crude product can be purified by chromatography, preferably by a resin, more preferably by an adsorption resin, such as Amberlite. 1600. The organic solvent can then be removed to obtain an aqueous solution of the compound of formula 3, for example by vacuum distillation. The protected condensation product 3 in the aqueous solvent mixture can also be obtained according to various methods known to those skilled in the art. For 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 tritert-butyl ester 1A from step 1) can be diluted with water, an organic solvent mixture, or an aqueous solution to obtain an aqueous / organic solution to be used in subsequent steps without removing product 3.

[0105] Therefore, according to a preferred aspect of the invention, the invention includes the further step of converting a solution of the compound of formula 3 into an aqueous solution or 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, thereby obtaining an aqueous solution.

[0106] Step 4

[0107] This step involves deprotecting the protected ligand of Formula 3 by removing the carboxyl protecting group, thereby obtaining an aqueous solution or mixture of water containing the corresponding free ligand 4. This reaction is preferably carried out by acidification of an aqueous solution or mixture of the protected ligand of Formula 3 collected directly from step 3) of the method.

[0108] In one implementation, step 4) of the method includes:

[0109] i) Add acid to the aqueous solution or mixture of water of the compound of formula 3 collected in step 3) to obtain an acidic solution of free ligand 4.

[0110] ii) Add a base to the acidic solution to obtain a solution that is substantially neutralized by ligand 4;

[0111] iii) Purify the neutralized solution and then optionally concentrate it to obtain an aqueous solution or mixture of water of free ligand 4 suitable for use in the subsequent coordination reaction, without the need for any ligand separation.

[0112] In one embodiment, a solution of the protected compound of formula 3 is acidified by adding an acid (e.g., a 34% aqueous HCl solution). The acidification is carried out by using a large excess of HCl, for example, 30 to 100, preferably 30 to 80, more preferably 40 to 50 times the molar amount of the protected compound 3.

[0113] The acid is added at 20-35°C, preferably 30-35°C. The resulting solution is then kept at 30-40°C with stirring for 10-36 hours, preferably 25-30 hours, followed by deprotection of the ligand by, for example, chromatography.

[0114] The acidic solution is then cooled at, for example, 25°C and then neutralized by adding a base (preferably NaOH) to obtain a crude solution with a final pH of 6.5 to 7.5, which is then purified.

[0115] The purification steps preferably include: i) distilling the neutralized solution to remove the formed tert-butanol, ii) desalting the distillation residue, and iii) chromatographic purification of the desalted solution.

[0116] In particular, in one embodiment, the solution produced by adding alkali is first distilled, preferably at a temperature of 40 to 60°C, to remove the formed tert-butanol. The distillation residue is then desalted, preferably by nanofiltration, and the collected solution is purified.

[0117] In one embodiment, the solution obtained by nanofiltration is first concentrated under vacuum to a concentration preferably 23-27% (w / w) at a temperature of, for example, 40 to 60°C, preferably about 50°C, and then passed through a resin (more preferably on Amberlite). Elution and purification at 1600 rpm. Optionally, use... The eluent is treated and concentrated under vacuum at about 50°C to obtain an aqueous solution or water mixture of the Formula 4 ligand with a final concentration preferably 8-25%, which can be used directly in the subsequent coordination reaction without separating the ligand.

[0118] Advantageously, the above steps involve using water as one of the sole or primary reaction solvents, thereby avoiding or reducing the use of organic solvents, particularly the harsh solvents required in the prior art methods described above, such as DCM and harsh reaction reagents such as TFA and TIPS, which are very difficult to handle and therefore unsuitable for large-scale production. Furthermore, this step results in obtaining the desired ligand in an aqueous solution or water mixture ready for use in the complexation reaction, without the need for separation.

[0119] Step 5

[0120] This step involves coordinating the dimer ligand of Formula 4 with gadolinium ions to obtain an aqueous solution or mixture of water of the desired chelate complex 5.

[0121] More specifically, this step preferably includes:

[0122] i) Add a gadolinium salt, such as GdCl3, to the ligand solution collected from step 4) to obtain a mixture containing the dimerized chelate complex 5;

[0123] ii) Add alkali to obtain a mixture with a pH of about 5 to about 7;

[0124] iii) Purify the mixture to obtain a solution of the dimer complex of Formula 5; and

[0125] iv) Concentrate the collected solution.

[0126] The reaction is preferably carried out by adding GdCl3 directly to the ligand solution collected from the previous step of the method. The addition is preferably carried out at a temperature of 25-45°C. The amount of GdCl3 required for complete ligand coordination is determined by titrating the ligand solution using known methods, such as using copper sulfate as a titrant.

[0127] In one embodiment, the ratio between the ligand of Formula 4 and the added GdCl3 is 1:1.98 to 1:2.02 (mol / mol); more preferably 1:2.00, to ensure the depletion of the added lanthanide ions.

[0128] After addition, the pH of the resulting mixture is adjusted to approximately 5 to approximately 7.5 by adding an alkali, preferably NaOH.

[0129] For example, in one embodiment, GdCl3 is added to the ligand solution at a temperature of 20-25°C. The pH of the resulting mixture is adjusted to 7-7.5, for example, about 7, by adding NaOH, and then stirred at 20-25°C for about 25 hours to achieve complete ligand coordination.

[0130] In an alternative embodiment, GdCl3 and the required amount of NaOH to maintain the pH at the desired neutral value can be added simultaneously, and the resulting mixture can then be maintained with stirring for about 25 hours as described above.

[0131] In a preferred embodiment, the mixture obtained by adding GdCl3 is adjusted to a pH of about 5 to about 6, preferably 5 to 5.6, more preferably about 5.3, and then kept at about 40°C with stirring for 1-4 hours, for example about 2 hours. Then, the optionally present residual free species, such as free Gd, are evaluated, for example by titration and / or HPLC methods. 3+ The ligand or partially ligand is added, and the calculated amount of ligand or GdCl3 is added to obtain an aqueous solution or water mixture of the dimer complex of Formula 5, which is then purified.

[0132] The purification is preferably carried out by chromatography, and preferably by resin.

[0133] In one embodiment, purification includes purification using a polymer resin (preferably Amberlite). The mixture produced by elution and coordination reaction on 1600 resin.

[0134] In another embodiment, purification includes a first elution of the mixture resulting from the coordination reaction on a chelating resin (e.g., selected from Hi-Trap IMAC FF, Lewatit MonoPlus TP 260, Lewatit-TP 208, IRC748I, DIAION CR11, SILIAMets AMPA, and SILIAMetDOTA, preferably selected from DIAION CR11 and Amberlite IRC748), thereby allowing for the minimization of any optional free gadolinium content, as well as the polymerization resin (e.g., Amberlite). The collected eluent was further purified on 1600 resin.

[0135] According to the actual implementation plan, through Amberlite Elute on 1600 resin and properly purify the mixture by adjusting the pH to approximately neutral.

[0136] In other aspects, the mixture resulting from adjusting the solution pH to a lower value (e.g., 5-5.6) is preferably first eluted on a chelating resin (e.g., Amberlite IRC748 or Diaion CR11 resin). The collected eluent is then preferably readjusted to a pH of approximately 5.5-6 and preferably concentrated under vacuum at 50°C to obtain an aqueous solution or water mixture of the dimer complex, preferably at a concentration of about 25% (w / w), which is then processed by Amberlite. Purification on 1600 resin.

[0137] The collected fractions are then optionally treated with charcoal and filtered. The resulting filtrate is then preferably concentrated, for example by distillation under vacuum at 45-55°C, to obtain a solution of dimer complex 5 with a final concentration of approximately 25% (w / w).

[0138] Step 6

[0139] The dimer complex of formula 5 is then separated. This complex can be separated from the aqueous solution or water mixture of step 5, for example, by freeze-drying or spray drying. In a preferred embodiment, the desired dimer is obtained by spray drying from the solution collected directly from step 5 of the method, as a white solid.

[0140] The overall yield of this method, determined from the limiting reactant DO3A tritert-butyl ester 1A as a free base, is at least 20%, preferably 25%, more preferably about 28-30%, or even >30%.

[0141] Interestingly, the above methods include one-pot steps suitable for large-scale implementation and do not require the separation of any prepared precursors (e.g., compounds of formula 1A) or reaction intermediates. As a result, the synthetic method of the present invention aims to obtain the final product with an overall yield at least about 18% higher than that disclosed in WO2017098044.

[0142] Furthermore, the lack of intermediate separation allows for a reduction in the overall process time.

[0143] Furthermore, the proposed method includes the use of water, or more generally, an aqueous solvent or a mixture of aqueous solvents, as the reaction solvent in all steps following the preparation of coupling product 3. Specifically, when preparing compound 3 in an organic solvent in step 3), for example, by reacting compound 2 from step 2) with DO3A tri-tert-butyl ester 1A from step 1), the organic solvent can be replaced with an aqueous solvent or a mixture of aqueous solvents by methods known to those skilled in the art, such as first diluting the organic solution of compound 3 with water, a water / organic solvent mixture, or an aqueous solution, and then optionally obtaining an aqueous solution of compound 3 by removing the organic solvent. Using an aqueous solvent or a mixture of aqueous solvents as the reaction solvent in all steps following the coupling preparation is highly advantageous, particularly from the viewpoints of cost, environmental impact, and ease of industrial-scale implementation. In fact, the method disclosed in WO2017098044 uses solvents such as DCM and materials such as TFA and TIPS, which are not only expensive but also difficult to handle, especially when scaling up the method industrially, and are unsafe from a worker health perspective. Because the method of the present invention avoids or greatly reduces the use of organic solvents by using an aqueous solvent or a mixture of aqueous solvents in all steps after the preparation of the Formula 3 compound, the present invention solves the problems of prior art methods, such as those used in industrial production. Furthermore, the method of the present invention surprisingly provides very high yields of isolated dimer complexes, particularly yields higher than those of prior art methods, even if they only involve the use of an aqueous solvent or a mixture of aqueous solvents after the preparation of the Formula 3 compound.

[0144] All solvents and starting materials, including reaction reagents such as epichlorohydrin, D-glucosamine, and hydrobromide of DO3A tritert-butyl ester, are commercially available or can be obtained by known methods.

[0145] In a preferred embodiment, the hydrobromide of DO3A tritert-butyl ester, which is used as a starting material for preparing a solution of the corresponding ester 1A, is prepared by the manufacturing method described in the common pending patent application EP19215900.2 (by the same applicant as this application) and stored until use.

[0146] Non-limiting examples of preferred embodiments of the method of the present invention are reported in the following sections, which are intended to illustrate the invention in more detail without limiting its scope.

[0147] Experimental Section

[0148] Abbreviations and Terminology Definitions

[0149] DO3A tri-tert-butyl ester: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester

[0150] DO3A tri-tert-butyl ester HBr: 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester hydrobromide

[0151] TAZA: 1,4,7,10-Tetraazacyclododecane

[0152] tBuOK Potassium tert-Butoxide

[0153] DMAC N,N-dimethylacetamide

[0154] DMC dichloromethane

[0155] DIPEA N,N-Diisopropylethylamine

[0156] HCl hydrochloric acid

[0157] MeCN Acetonitrile

[0158] NaOH (sodium hydroxide)

[0159] NH3 ammonia

[0160] MRI (Magnetic Resonance Imaging)

[0161] MeCN Acetonitrile

[0162] NMM N-methylmorpholine

[0163] K2CO3 (potassium carbonate)

[0164] TFA (trifluoroacetic acid)

[0165] TIPS: Triisopropylsilane

[0166] FLD fluorescence detector

[0167] UV / Vis

[0168] HPLC characterization of the obtained compounds.

[0169] General Procedure

[0170] Step 1: HPLC characterization and determination of DO3A tritert-butyl ester

[0171] Chromatographic conditions

[0172]

[0173] Step 2: HPLC method for monitoring the formation of intermediate 2

[0174] This method is used to monitor the mixture at the end of D-glucosamine alkylation and after water distillation.

[0175] Chromatographic conditions

[0176]

[0177] Sample preparation: Add 200 μL of 5 mM ammonium acetate solution to 75 μL of the mixture, and dilute to 5 mL with diluent.

[0178] Step 3: HPLC method for monitoring the formation and purification of intermediate 3

[0179] General Procedure

[0180] This method is used to monitor the formation and purification steps of intermediate 3.

[0181] Analysis conditions

[0182]

[0183] Accurately weigh 2.0 g of ammonium acetate into a 1000 mL volumetric flask, then dilute to the mark with water. Transfer 600 mL of the ammonium acetate solution and 300 mL of methanol into the 1000 mL volumetric flask. Sonicate for half an hour.

[0184] Step 4: HPLC method for monitoring the formation and purification of chelate ligand 4.

[0185] General Procedure

[0186] The formation and purification of dimer ligand 4 were monitored by reversed-phase HPLC with UV detection at 210 nm.

[0187] Analysis conditions

[0188]

[0189] Example 1: Synthesis of DO3A tri-tert-butyl hydrobromide

[0190] The synthesis of the initial DO3A tritert-butyl hydrobromide was carried out using the steps constituting the purpose of co-pending patent application EP19215900.2 (by the same applicant as this application). Specifically:

[0191] A solution of tert-butyl bromoacetate (51.32 kg; 263.12 mol) in DMA (50.72 kg; 53.96 L) was added to a suspension of commercially available TAZA (14.39 kg; 83.53 mol) and sodium acetate (21.58 kg; 263.12 mol) in DMAC (98.07 kg; 104.33 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 the mixture was centrifuged and washed with water (2 x 57 kg) after 2 hours. The wet solid was dried under vacuum to give 36.62 kg; 61.48 mol of DO3A tritert-butyl hydrobromide (73.6% yield). The product was identified as 100% w / w by HPLC (reference standard) and 99.86% w / w by NMR (reference standard).

[0192] Example 2: Preparation of dimer compound 5

[0193] Dimer compound 5 was obtained by using the synthetic steps illustrated below.

[0194]

[0195] include:

[0196] a) Neutralization of hydrobromide yields DO3A tritert-butyl ester 1A

[0197] The DO3A tritert-tert-butyl ester HBr (36.62 kg; 61.5 mol) and potassium carbonate (16.99 kg; 122.9 mol) obtained as described in Example 1 were suspended in MeCN (72.29 kg; 91.50 L) at room temperature and the mixture was stirred at 24 °C for 20 hours. The resulting salt was then filtered off, and the filtrate was partially distilled under vacuum at 50 °C to obtain a MeCN solution of DO3A tritert-tert-butyl ester 1A (30.68 kg; 59.6 mol) with a final concentration of approximately 62% w / w, which could be used for the next step without further separation. The concentration of 1A in the solution was determined by HPLC-UV.

[0198] b) Synthesis of intermediate 2

[0199] At room temperature, a solution of D-glucosamine (6 kg; 33.1 mol) in water (15 kg) was added dropwise over 2 hours to a solution of epichlorohydrin (6.74 kg; 72.8 mol) in DMAC (6 kg; 6.38 L). The mixture was stirred for 17 hours. The mixture was then diluted with DMAC (12 kg; 12.77 L), and the water was vacuum distilled at 45–50 °C to give a solution of intermediate 2 in DMAC with a residual water content of <2.0%, which was used for the next step without further purification.

[0200] c) Alkylation of intermediate 2 with DO3A tritert-butyl ester 1A to obtain protected ligand 3

[0201] DIPEA (9.79 kg; 75.8 mol) and a solution of substrate 1A collected in step a were added to a solution of intermediate 2 heated at 50 °C. The resulting mixture was then stirred at 70 °C for 72 hours, and the conversion was monitored by HPLC-UV. The mixture was then cooled to... Dilute with 16% w / w ammonia solution (36 L). Stir the resulting mixture at 25°C for 15 hours to obtain a salt precipitate, which was removed by filtration. Then, pass through Amberlite... The filtrate was purified by chromatography on a 1600 (450 L; eluent: water / MeCN gradient) scale. The pure fraction (HPLC area % ≥ 90) was then collected, the organic solvent was distilled, and the aqueous residue was concentrated under vacuum at about 50 °C to give a solution of protected ligand 3 in water (concentration approximately 10% w / w), suitable for direct use in the next step without any further separation.

[0202] d Deprotection

[0203] A 34% aqueous hydrochloric acid solution (64.72 kg; 603.5 mol) was added to the solution of protected ligand 3 collected from step c, while maintaining the temperature at approximately 30 °C. At the end of the addition, the mixture was heated to... Continue stirring for 30 hours. Cool the solution to... The mixture was neutralized by adding a 30% sodium hydroxide aqueous solution (final pH approximately 7), tert-butanol (formed as a reaction byproduct) was removed by distillation, and the mixture was desalted by nanofiltration. The desalted mixture was then partially concentrated under vacuum at 50°C to a final concentration of 25% (w / w) and filtered through Amberlite. Purification was performed by chromatography on 1600 (150 L; eluent water). The pure fraction was treated with charcoal (HPLC area % ≥ 90) and concentrated under vacuum at 45–55 °C to obtain a 10% (w / w) aqueous solution of the desired ligand 4 (11.4 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0204] e) Cooperate

[0205] The solution of ligand 4 was heated to 40°C, and an aqueous solution of gadolinium chloride (21.35 kg solution; 6.01 kg gadolinium chloride; 22.8 mol) was added, maintaining the temperature at approximately 40°C. After addition, the pH of the solution was adjusted to 5.3 by adding 10% sodium hydroxide aqueous solution, and the mixture was kept at 40°C for 2 hours to form the corresponding paramagnetic complex 5. The presence of any free species was assessed, for example, by titration. The final solution of the dimer complex was thus obtained, which was purified on Diaion CR11 (6L) to reduce any free gadolinium content. After loading, the resin was washed with water; the collected solution was adjusted to pH 5.5 and then concentrated under vacuum at 50°C to obtain a 25% w / w aqueous solution, which was then loaded into Amberlite at pH 6.2. The selected fraction was treated with charcoal and then vacuum distilled at 50 °C to obtain a mixture with a final concentration of approximately 25% (w / w). The gadolinium complex was separated by spray drying as a white powder (11.74 kg; 10.80 kg, anhydrous alkali; 8.3 mol), and the final titration result was 99% (w / w, anhydrous alkali).

[0206] Overall yield determined by DO3A tri-tert-butyl ester 1A: 28%.

[0207] Example 3: Preparation of Compound 5

[0208] a) Neutralization of DO3A 3tBu HBr

[0209] DO3A tri-tert-butyl ester HBr (241.6 g; 0.406 mol) and potassium carbonate (112.1 g; 0.811 mol) were suspended in MeCN (477.2 g; 0.604 L), and the mixture was stirred at 25 °C for 20 hours. The formed salt was then filtered off, and the filtrate was partially distilled under vacuum at 50 °C to obtain a solution of DO3A tri-tert-butyl ester 1A (204.5 g; 0.397 mol) in MeCN with a final concentration of 61% w / w, which was used for the next step without further separation. The 1A in the solution was determined by HPLC-UV.

[0210] b) Synthesis of intermediate 2

[0211] At 24°C, D-glucosamine (40.0 g; 0.221 mol) / water (99.3 g) was added dropwise to a solution of epichlorohydrin (44.9 g; 0.486 mol) in DMAC (40.0 g; 0.043 L) over 2 hours. The mixture was stirred for 17 h, then DMAC (80.0 g; 0.085 L) was added, and water was removed by vacuum distillation at 50°C to obtain a solution of intermediate 2 in DMAC with a residual water content <2.0% w / w.

[0212] c) Alkylation of compound 1A with intermediate 2

[0213] At 50°C, a solution of DIPEA (65.3 g; 0.505 mol) and 1A was added to the solution of intermediate 2 collected from step b). The mixture was further stirred at 70°C for 72 hours, with the conversion monitored by HPLC-UV. After cooling to 25°C, it was diluted with 16% w / w ammonia solution (0.240 L). The resulting mixture was stirred at 20°C for 15 hours to obtain a salt precipitate, which was removed by filtration. The precipitate was then processed using Amberlite. The filtrate was purified by chromatographic purification at 1600 (3 L; eluent: water / MeCN gradient). The pure fraction (HPLC area % ≥ 90) was collected and distilled to remove the organic solvent. The resulting residue was then concentrated under vacuum at 50 °C to give an aqueous solution of protected ligand 3 with a final concentration of approximately 10% w / w, which was used directly in the next step of the method.

[0214] d) Deprotection of intermediate 3

[0215] A 34% aqueous hydrochloric acid solution (431.5 g; 4.023 mol) was added to the solution of intermediate 3 in step c) while maintaining the temperature at 30 ÷ 35 °C. At the end of the addition, the mixture was heated to 37 °C and maintained with stirring for 36 hours. The solution was then cooled to 25 °C and neutralized by adding a 30% aqueous sodium hydroxide solution. Tert-butanol, which formed as a byproduct, was removed by distillation, and the mixture was desalted by nanofiltration. The mixture was then partially concentrated to a concentration of 24% w / w under vacuum at 50 °C and purified by Amberlite. HPLC purification was performed at 1600 (1 L; eluent: water). The selected fraction was evaluated by HPLC-UV, treated with charcoal, and concentrated under vacuum at 50 °C to obtain a 10% w / w aqueous solution of the desired ligand 4 (0.075 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0216] e) Cooperate

[0217] The solution of deprotected ligand 4 was heated to 37°C, and then an aqueous solution of gadolinium chloride (140.5 g solution; 39.5 g gadolinium chloride; 0.150 mol) was added, maintaining the temperature in the range of 37–43°C. At the end of the addition, the pH was adjusted to 5.3 by adding a 10% sodium hydroxide aqueous solution. The mixture was kept at 40°C for 2 hours to form the corresponding paramagnetic complex 5. The presence of any free species was assessed, for example, by titration. The solution was then purified on Diaion CR11 chelating resin (0.16 L) to reduce any free gadolinium content. After loading, the resin was washed with water, the pH was adjusted to 5.5, and the solution was concentrated under vacuum at 50°C to obtain a 25% w / w aqueous solution. This solution was loaded into Amberlite at pH 6. 1600 (3.3 L; eluent: water / MeCN gradient). Selected fractions were evaluated by HPLC-FLD and UV treatment with charcoal, and the resulting solution was vacuum distilled at 50 °C. The final solution (25% w / w) was spray-dried to separate the gadolinium complex as a white powder (82.0 g, corresponding to 74.6 g as anhydrous product; titration determination: 99% w / w, anhydrous alkali).

[0218] Overall yield of DO3A tri-tert-butyl ester 1A: 29%.

[0219] Example 4: Preparation of Compound 5

[0220] a) Neutralization of DO3A, 3tBu, and HBr

[0221] DO3A 3tBu HBr (241.6 g; 0.406 mol) and potassium carbonate (112.1 g; 0.811 mol) were suspended in MeCN (480.0 g; 0.608 L), and the mixture was stirred at 27 °C for 18 hours. The formed salt was then filtered off, and the filtrate was partially distilled under vacuum at 50 °C to obtain a solution of DO3A tri-tert-butyl ester 1A (205.0 g; 0.398 mol) in MeCN at a final concentration of 58% w / w, which was used for the next step without further separation. The 1A in the solution was determined by HPLC-UV.

[0222] b) Synthesis of intermediate 2

[0223] At 25°C, D-glucosamine (40.0 g; 0.221 mol) / water (100.0 g) was added dropwise to a solution of epichlorohydrin (44.9 g; 0.486 mol) in DMAC (40.0 g; 0.043 L) over 2 hours. The mixture was kept stirred for 16 hours, then DMAC (80.0 g; 0.085 L) was added, and water was removed by vacuum distillation at 50°C to obtain a solution of intermediate 2 in DMAC with a residual water content <2.0% w / w.

[0224] c) Alkylation of compound 1A with intermediate 2

[0225] At 50 °C, DIPEA (65.3 g; 0.505 mol) and solution 1A were added to the solution of intermediate 2 collected from step b). The mixture was further stirred at 75 °C for 70 h, and the conversion was monitored by HPLC-UV. The mixture was then partially concentrated under vacuum at 60 °C. After cooling to 23 °C, a mixture of previously prepared water (80.0 g) and MeCN (126.4 g; 0.160 L) was added, followed by a 25% w / w ammonia solution (144.5 g; 0.16 L). The resulting mixture was stirred at 22 °C for 14 h. The mixture was filtered and passed through Amberlite. 1600 (3 L; eluent: water / MeCN gradient) chromatography purification. After elution, fractions with sufficient purity (HPLC area % ≥ 90) were collected, the organic solvent was distilled, and the resulting solution was concentrated under vacuum at 50 °C to obtain an aqueous solution of protected ligand 3 (concentration: 13% w / w), which was used directly in the next step.

[0226] d) Deprotection of intermediate 3

[0227] A 34% aqueous hydrochloric acid solution (435.0 g; 4.057 mol) was added to the solution of intermediate 3 in step c) while maintaining the temperature at 30–35 °C. At the end of the addition, the mixture was maintained at 35 °C with stirring for 32 hours. Afterward, the solution was cooled to 23 °C and neutralized by adding a 30% aqueous sodium hydroxide solution. Tert-butanol, formed as a byproduct, was removed by distillation, and the mixture was desalted by nanofiltration. The mixture was then partially concentrated to a concentration of 22% w / w under vacuum at 50 °C and purified by Amberlite. HPLC purification was performed at 1600 (1 L; eluent: water). The selected fraction was evaluated by HPLC-UV, treated with charcoal, and concentrated under vacuum at 50 °C to obtain a 25% w / w aqueous solution of the desired ligand 4 (0.076 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0228] e) Cooperate

[0229] At 25°C, gadolinium chloride aqueous solution (142.4 g solution; 40.1 g gadolinium chloride; 0.152 mol) was added to the deprotected ligand 4 solution, and the temperature was maintained in the range of 23 ÷ 27°C. The pH was adjusted to 7.0 ÷ 7.5 by adding 30% sodium hydroxide aqueous solution. The mixture was kept at 25°C for 24 hours to form the corresponding paramagnetic complex 5. The presence of any free species can be assessed, for example, by titration. The resulting solution was analyzed in Amberlite. Purification was performed at 1600 (3.3 L; eluent: water / MeCN gradient). Selected fractions were evaluated by HPLC-FLD and UV treatment with charcoal, and the resulting solution was vacuum distilled at 50 °C. The final solution (25% w / w) was spray-dried to separate the gadolinium complex as a white powder (73.5 g, corresponding to 66.9 g as anhydrous product; titration determination: 99% w / w, anhydrous alkali).

[0230] Overall yield of DO3A tri-tert-butyl ester 1A: 26%.

[0231] Example 5: Preparation of Compound 5

[0232] a) Neutralization of DO3A 3tBu HBr

[0233] DO3A 3tBu HBr (724.9 g; 1.217 mol) and potassium carbonate (336.4 g; 2.434 mol) were suspended in MeCN (1431.7 g; 1.812 L), and the mixture was stirred at 25 °C for 18 hours. The formed salt was then filtered off, and the solution was partially distilled under vacuum at 50 °C to obtain a solution of DO3A tri-tert-butyl ester 1A (613.9 g; 1.193 mol) in MeCN at a final concentration of 56% w / w, which was used for the next step without further separation. The determination of 1A in the solution was performed by HPLC-UV.

[0234] b) Synthesis of intermediate 2

[0235] At 24°C, D-glucosamine (120.0 g; 0.662 mol) / water (298.0 g) was added dropwise to a solution of epichlorohydrin (134.8 g; 1.457 mol) in DMAC (120.0 g; 0.128 L) over 2 hours. The mixture was kept stirred for 18 hours, then DMAC (240.0 g; 0.255 L) was added, and water was removed by vacuum distillation at 50°C to obtain a solution of intermediate 2 in DMAC with a residual water content <2.0% w / w.

[0236] c) Alkylation of compound 1A with intermediate 2

[0237] A solution of DIPEA (195.9 g; 1.516 mol) and substrate 1A was added to a solution of intermediate 2 at 45 °C. The mixture was then stirred at 70 °C for 75 h, and the conversion was monitored by HPLC-UV. The mixture was then partially concentrated under vacuum at 60 °C. The residue was cooled to 25 °C and diluted with a previously prepared mixture of water (720.0 g) and MeCN (379.2 g; 0.480 L). The solution was then analyzed by Amberlite. The solution was purified by chromatographic purification at 1600 (9 L; eluent: water / MeCN gradient). Fractions with sufficient purity (HPLC area % ≥ 90) were collected, and the resulting solution was distilled to remove organic solvents. The resulting aqueous residue was concentrated under vacuum at 50 °C to give an aqueous solution of protected ligand 3 with a final concentration of 15% w / w for the next step.

[0238] d) Deprotection of intermediate 3

[0239] A 34% aqueous hydrochloric acid solution (1294.4 g; 12.070 mol) was added to the collected intermediate 3 solution, and the temperature was maintained at 30 ÷ 35 °C. At the end of the addition, the mixture was heated to approximately 40 °C and maintained at this temperature for 28 hours with stirring. The solution was then cooled to 25 °C and neutralized by adding a 30% aqueous sodium hydroxide solution. The generated tert-butanol was removed by distillation, and the residual mixture was first desalted by nanofiltration and then partially concentrated to a concentration of 26% w / w under vacuum at 50 °C. It was then processed using Amberlite. 1600 (3 L; eluent water) chromatographic purification of concentrated solution. The pure fraction was collected, treated with charcoal, and concentrated under vacuum at 50 °C to obtain a 12% w / w aqueous solution of deprotected ligand 4 (0, 228 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0240] e) Cooperate

[0241] The solution of deprotected ligand 4 was heated to 37°C, and then an aqueous solution of gadolinium chloride (427.1 g solution; 120.2 g gadolinium chloride; 0.456 mol) was added, maintaining the temperature in the range of 37 ÷ 43°C. At the end of the addition, the pH was adjusted to 5.5 by adding 10% sodium hydroxide aqueous solution, and the resulting mixture was maintained at 42°C for 2 hours to form the corresponding paramagnetic complex 5. The presence of any free species was assessed, for example, by titration. The solution was purified at 25°C with Diaion CR11 chelating resin (0.48 L) to reduce any free gadolinium content. After loading, the resin was washed with water, the pH was adjusted to 5.8, and the solution was concentrated under vacuum at 50°C to obtain a 26% w / w aqueous solution. This solution was loaded into Amberlite at pH 6.2. On 1600 (10 L; eluent: water / MeCN gradient). The selected fraction was evaluated by HPLC-FLD and UV treatment with charcoal, and the resulting solution was vacuum distilled at 50 °C. The final solution (22% w / w) was spray-dried to separate the gadolinium complex as a white powder (226.5 g, corresponding to 208.3 g, anhydrous product; titration determination: 99% w / w, anhydrous alkali).

[0242] Overall yield of DO3A tri-tert-butyl ester 1A: 27%.

[0243] Example 6: Preparation of Compound 5

[0244] a) Neutralization of DO3A 3tBu HBr

[0245] DO3A 3tBu HBr (724.9 g; 1.217 mol) and potassium carbonate (336.4 g; 2.434 mol) were suspended in MeCN (1435.5 g; 1.817 L), and the mixture was stirred at 25 °C for 18 hours. The formed salt was then filtered off, and the solution was partially distilled under vacuum at 50 °C to obtain a solution of DO3A tri-tert-butyl ester 1A (607.9 g; 1.181 mol) in MeCN with a final concentration of 62% w / w, which was used for the next step without further separation. The determination of 1A in the solution was performed by HPLC-UV.

[0246] b) Synthesis of intermediate 2

[0247] At 25°C, D-glucosamine (120.0 g; 0.662 mol) / water (300.0 g) was added dropwise to a solution of epichlorohydrin (134.8 g; 1.457 mol) in DMAC (120.0 g; 0.128 L) for 2 hours. The mixture was kept stirred for 18 hours, then DMAC (240.0 g; 0.255 L) was added, and water was removed by vacuum distillation at 50°C to obtain a solution of intermediate 2 in DMAC with a residual water content <2.0% w / w.

[0248] c) Alkylation of compound 1A with intermediate 2

[0249] At 50°C, a solution of DIPEA (196.1 g; 1.517 mol) and substrate 1A was added to the solution in step 2. The mixture was then stirred at 70°C for 70 hours, and the conversion was monitored by HPLC-UV. After cooling to 25°C, it was diluted with water (720.0 g). The final product was then analyzed using Amberlite. The mixture was purified by 1600 (9 L; eluent: water / MeCN gradient) chromatography. After elution, fractions with sufficient purity (HPLC area % ≥ 90) were collected, the organic solvent was distilled, and the resulting solution was concentrated under vacuum at 50 °C to obtain an aqueous solution of protected ligand 3 with a final concentration of 14% w / w for the next step.

[0250] d) Deprotection of intermediate 3

[0251] A 34% hydrochloric acid aqueous solution (1290.0 g; 12.030 mol) was added to the solution of intermediate 3, and the temperature was maintained at 30 ÷ 35 °C. At the end of the addition, the mixture was heated to 38 °C and maintained with stirring for 30 hours. The solution was then cooled to 25 °C and neutralized by adding a 30% sodium hydroxide aqueous solution. The generated tert-butanol was removed by distillation, and the residual mixture was first desalted by nanofiltration and then partially concentrated to a concentration of 22% w / w under vacuum at 50 °C. It was then processed using Amberlite. 1600 (3 L; eluent: water) chromatographic purification of the concentrated solution. The selected fraction was evaluated by HPLC-UV, treated with charcoal, and concentrated under vacuum at 50 °C to obtain a 23% w / w aqueous solution of the desired ligand 4 (0, 226 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0252] d) Cooperate

[0253] At 25°C, gadolinium chloride aqueous solution (423.3 g solution; 119.2 g gadolinium chloride; 0.452 mol) was added to the deprotected ligand 4 solution, maintaining the temperature between 23°C and 27°C. The pH was adjusted to 7.0–7.5 by adding 30% sodium hydroxide aqueous solution. The mixture was kept at 25°C for 24 hours. The resulting solution was tested in Amberlite. Purification was performed at 1600 (10 L; eluent: water / MeCN gradient). Selected fractions were evaluated by HPLC-FLD and UV treatment with charcoal, and the resulting solution was distilled under vacuum at 50 °C. The final solution (25% w / w) was spray-dried to separate the gadolinium complex as a white powder (201.3 g, corresponding to 185.2 g, anhydrous product; titration determination: 99% w / w, anhydrous alkali).

[0254] Overall yield of DO3A tri-tert-butyl ester 1A: 24%.

[0255] Example 7: Preparation of Compound 5

[0256] a) Neutralization of compound 1.

[0257] The DO3A, 3tBu, HBr 1 (724.9 g; 1.217 mol) and potassium carbonate (336.4 g; 2.434 mol) were then suspended in MeCN (1431.7 g; 1.812 L), and the mixture was stirred at 25 °C for 18 hours. The salt was then filtered off, and the solution was partially distilled under vacuum at 50 °C, retaining compound 1A (613.6 g; 1.192 mol, 98% conversion) in the MeCN solution (final concentration: 56% w / w) until the next step, without further separation.

[0258] b) Synthesis of intermediate 2.

[0259] At 24°C, D-glucosamine (120.0 g; 0.662 mol) / water (298.0 g) was added dropwise to a solution of epichlorohydrin (134.8 g; 1.457 mol) in DMAC (120.0 g; 0.128 L) over 2 hours. The mixture was kept stirred for 18 hours, then DMAC (240.0 g; 0.255 L) was added, and water was removed by vacuum distillation at 50°C to obtain a solution of intermediate 2 in DMAC with a residual water content <2.0% w / w.

[0260] c) Alkylation of compound 1A with intermediate 2

[0261] At 50 °C, a solution of DIPEA (195.9 g; 1.516 mol) and substrate 1A was added to a solution of intermediate 2. The mixture was then stirred at 70 °C for 75 h, and the conversion was monitored by HPLC-UV (HPLC area %: 75). The mixture was then partially concentrated under vacuum at 60 °C. The residue was cooled to 25 °C, diluted with a previously prepared mixture of water (240.0 g) and MeCN (379.2 g; 0.480 L), and then diluted with a 25% ammonia solution (433.4 g; 0.480 L). The resulting mixture was kept under stirring for 16 h to obtain a hydrochloride precipitate, which was then filtered. The filtrate was then collected and passed through Amberlite. HPLC purification was performed at 1600 rpm (9 L; eluent: water / MeCN gradient). Fractions with sufficient purity (HPLC A% ≥ 90%) were collected, and the resulting solution was distilled to remove the organic solvent. The resulting aqueous residue was concentrated under vacuum at 50 °C to give an aqueous solution of protected ligand 3 with a final concentration of 14% w / w for the next step.

[0262] d) Deprotection of intermediate 3.

[0263] A 34% aqueous hydrochloric acid solution (1294.4 g; 12.070 mol) was added to the collected intermediate 3 solution, and the temperature was maintained at 30 ÷ 35 °C. At the end of the addition, the mixture was heated to approximately 40 °C and maintained at this temperature for 30 hours with stirring. The solution was then cooled to 25 °C and neutralized by adding a 30% aqueous sodium hydroxide solution. The generated tert-butanol was removed by distillation, and the residual mixture was first desalted by nanofiltration and then partially concentrated to a concentration of 26% w / w under vacuum at 50 °C. It was then processed using Amberlite. 1600 (3 L; eluent water) chromatographic purification concentrate. Collect the pure fraction, treat with charcoal, and concentrate under vacuum at 50 °C to obtain a 25% w / w aqueous solution of deprotected ligand 4, which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0264] e) Cooperate

[0265] The solution of deprotected ligand 4 was heated to 37°C, and then an aqueous solution of gadolinium chloride (427.1 g; 120.2 g gadolinium chloride; 0.456 mol) was added, maintaining the temperature in the range of 37–43°C. At the end of the addition, the pH was adjusted to 5.5 by adding 10% sodium hydroxide aqueous solution, and the resulting mixture was maintained at 42°C for 2 hours. The solution was purified at 25°C with Diaion CR11 chelating resin (0.48 L) to reduce any free gadolinium content. After elution, the resin was washed with water, the pH was adjusted to 5.8, and the solution was concentrated under vacuum at 50°C to obtain a 26% w / w aqueous solution. This solution was then loaded into Amberlite at pH 6.2. 1600 (10 L; eluent: water / MeCN gradient). The selected fraction was evaluated by HPLC-FLD and UV treatment with charcoal, and the resulting solution was vacuum distilled at 50 °C. The final solution (22% w / w) was spray-dried to separate the gadolinium complex as a white powder (251.6 g, corresponding to 231.5 g, anhydrous product; titration determination: 99% w / w, anhydrous alkali).

[0266] Overall yield of DO3A tritert-butyl ester 1A: 30%.

[0267] Example 8: Preparation of Dimer Compound 5

[0268] a) Neutralization of hydrobromide yields DO3A tritert-butyl ester 1A

[0269] At room temperature (i.e., 25°C), DO3A tritert-tert-butyl ester HBr (36.60 kg; 61.4 mol) and potassium carbonate (16.99 kg; 122.9 mol) obtained as described in Example 1 were suspended in MeCN (72.50 kg; 91.77 L), and the mixture was stirred at 24°C for 19 hours. The resulting salt was then filtered off, and the filtrate was partially distilled under vacuum at 50°C to obtain a MeCN solution of DO3A tritert-tert-butyl ester 1A (31.07 kg; 60.4 mol) with a final concentration of approximately 56% w / w, which could be used for the next step without further separation. The 1A in the solution was determined by HPLC-UV.

[0270] b) Synthesis of intermediate 2

[0271] At room temperature, D-glucosamine (6 kg; 33.1 mol) / water (15.1 kg) was added dropwise to a solution of epichlorohydrin (6.75 kg; 73.0 mol) in DMAC (6.2 kg; 6.60 L) over 2 hours. The mixture was maintained under stirring for 16 hours. The mixture was then diluted with DMAC (12.2 kg; 12.98 L), and the water was vacuum distilled at 55–60 °C to give a solution of intermediate 2 in DMAC with a residual water content of <2.0%, which was used for the next step without further purification.

[0272] c) The alkylation of intermediate 2 with DO3A tritert-butyl ester 1A yields protected ligand 3.

[0273] DIPEA (9.80 kg; 75.8 mol) and a solution of substrate 1A collected in step a were added to a solution of intermediate 2 heated at 50 °C. The resulting mixture was then stirred at 70 °C for 80 h, and the conversion was monitored by HPLC-UV. The mixture was then partially concentrated under vacuum at 60 °C. The residue was cooled to 25 °C and diluted with a pre-prepared mixture of water (12.2 kg) and MeCN (19.0 kg; 24.05 L), followed by dilution with a 25% ammonia solution (21.7 kg; 24.03 L). The resulting mixture was kept under stirring for 15 h. The obtained hydrochloride precipitate was filtered off. The filtrate was then collected and passed through Amberlite. Purification was performed by chromatography at 1600 (450 L; eluent: water / MeCN gradient). The pure fraction (HPLC area % ≥ 90) was then collected, the organic solvent was distilled, and the aqueous residue was concentrated under vacuum at about 50 °C to give a solution of protected ligand 3 in water (concentration approximately 10% w / w), suitable for direct use in the next step without any further separation.

[0274] d) Deprotection

[0275] A 34% hydrochloric acid aqueous solution (64.75 kg; 603.8 mol) was added to the solution of protected ligand 3 collected from step c, and the temperature was maintained at approximately 30 °C. At the end of the addition, the mixture was heated to... Continue stirring for 30 hours. Cool the solution to... The mixture was neutralized by adding a 30% sodium hydroxide aqueous solution (final pH approximately 7), tert-butanol (formed as a reaction byproduct) was removed by distillation, and the mixture was desalted by nanofiltration. The desalted mixture was then partially concentrated under vacuum at 50°C to a final concentration of 25% (w / w) and filtered through Amberlite. Purification was performed by chromatography at 1600 (150 L; elution water). The pure fraction was treated with charcoal (HPLC area % ≥ 90) and concentrated under vacuum at 45–55 °C to obtain a 12% (w / w) aqueous solution of the desired ligand 4 (15.3 mol), which was quantified by potentiometric titration using copper sulfate solution as the titrant.

[0276] e) Cooperate

[0277] The solution of ligand 4 was heated to 40°C, and an aqueous solution of gadolinium chloride (27.79 kg solution; 8.10 kg gadolinium chloride; 30.7 mol) was added, maintaining the temperature at approximately 40°C. After addition, the pH of the solution was adjusted to 5.5 by adding 10% sodium hydroxide aqueous solution, and the mixture was maintained at 40°C for 2 hours to form the corresponding paramagnetic complex 5. The presence of any free substance was assessed, for example, by titration. The final solution of the dimer complex was thus obtained, which was purified on Diaion CR11 (6L) to reduce any free gadolinium content. After loading, the resin was washed with water; the collected solution was adjusted to pH 5.5 and then concentrated under vacuum at 50°C to obtain a 23% w / w aqueous solution, which was then loaded into Amberlite at pH 5.7. 1600 (500 L; eluent: gradient water / MeCN). The selected fraction was treated with charcoal and then vacuum distilled at 50 °C to give a mixture with a final concentration of about 28% (w / w). The gadolinium complex was separated from it by spray drying as a white powder (13.38 kg; 12.58 kg anhydrous alkali; 9.7 mol), and the final titration result was 99% (w / w; anhydrous alkali).

[0278] The overall yield determined by DO3A tri-tert-butyl ester 1A was 33%.

Claims

1. A method for preparing a dimer complex compound 5 of the following formula. The method includes: 1) Prepare a solution of DO3A tritert-butyl ester of formula 1A in an organic solvent; ； 2) A solution of compound 2 in an organic solvent was prepared by reacting D-glucosamine with epichlorohydrin. include: i) Adding an aqueous solution of D-glucosamine to a solution of epichlorohydrin in dimethylacetamide yields a water / organic solvent mixture of compound 2; and ii) Remove water from the mixture. To obtain a solution of compound 2 in an organic solvent; 3) Mix the solutions prepared according to steps 1) and 2) to obtain a solution of compound 3. ; pass a. React the compound of formula 2 in step 2) with the tri-tert-butyl DO3A 1A in step 1) in the presence of a base to obtain a crude organic solution; b. Dilute the obtained crude organic solution with water or a water / organic solvent mixture to obtain a water / organic crude product; c. Purify the water / organic crude product to obtain a water / organic solvent mixture of compound 3, and d. Remove the organic solvent from the mixture to obtain an aqueous solution of compound 3. 4) Without separating the compound from the solution of step 3, remove the tert-butyl protecting group from the compound of formula 3 to obtain a solution of the corresponding free ligand of formula 4. ; pass i) Add acid to the aqueous solution of the compound of formula 3 from step 3 to remove the protected tert-butyl group, and obtain an acidic aqueous solution of the corresponding free ligand of formula 4. ii) Add a base to the acidic aqueous solution to obtain a neutral aqueous solution containing the free ligands; iii) Purify, and then optionally concentrate the resulting neutral aqueous solution to obtain an aqueous solution of the free ligand of Formula 4. 5) In the absence of a free ligand of separable 4, i) Add the gadolinium salt to the solution of ligand 4 from step 4 to obtain a mixture containing the dimer complex of formula 5; ii) Purify the mixture to obtain a solution of the dimer complex of Formula 5; and iii) Concentrate the solution; and 6) Separate the dimer complex, In all steps following the preparation of compound 3, the reaction solvent is an aqueous solvent.

2. The method of claim 1, wherein the solution of DO3A tritert-butyl ester 1A in step 1 is prepared from the hydrobromide of DO3A tritert-butyl ester.

3. The method of claim 1 or 2, wherein step 1) comprises: i) Suspend the hydrobromide of DO3A tritert-butyl ester in an organic solvent together with a base or basic salt to obtain a suspension; ii) Filter the suspension; and iii) Collect and optionally concentrate the filtered suspension to obtain a solution of DO3A tritert-butyl ester 1A in an organic solvent.

4. The method of claim 1, comprising adding an aqueous solution to the water / organic crude product of step b), wherein the water / organic crude product of step b) is obtained by diluting the organic crude solution with a mixture of water and organic solvent.

5. The method of claim 1, wherein the purification of the water / organic crude product in step c) is performed by chromatography.

6. The method of claim 1, wherein step iii) comprises: The neutral solution is distilled to remove the formed tert-butanol; The distillation residue is desalted; The desalted solution was purified by chromatography to obtain an aqueous solution of the ligand of formula 4; And optionally, the aqueous solution can be concentrated.

7. The method of claim 6, wherein the concentration of the obtained aqueous solution of formula 4 ligand is 8-25% (w / w).

8. The method of claim 1, wherein step 5.ii) comprises purifying the mixture by chromatography.

9. The method of claim 1, wherein step 6) comprises separating the dimer complex of formula 5 directly from the solution collected in step 5) by spray drying, as a white solid.

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