PCTA-based contrast agents

Abstract

To provide specific isomers of Gd(PCTA-tris-glutamic acid) and its derivatives that possess improved physico-chemical properties in terms of relaxivity and kinetic inertness.SOLUTION: The present invention relates to the RRR / SSS pair of enantiomers of Gd(PCTA-tris-glutamic acid), single enantiomers of the pair, pharmaceutically acceptable salts thereof, their amide derivatives, and compositions comprising at least 50% of these compounds.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates generally to the field of magnetic resonance imaging (MRI), and more specifically to isomers of PCTA-based contrast agents and MRI contrast agents enriched in these isomers. [Background technology]

[0002] Typical examples of MRI contrast agents used in routine diagnostic practice are gadolinium complex compounds, characterized by high stability constants that ensure the release in vivo of the free metal ion, which is known to be highly toxic to living organisms.

[0003] Another important parameter that defines the tolerability of gadolinium-based contrast agents is the kinetic inertness (or kinetic stability) of the Gd(III) complex, which is defined as the half-life (t 1 / 2 ) is estimated from

[0004] Particularly in complex compounds with low thermodynamic stability and / or long retention times before discharge, high inertness is important to avoid or minimize possible decomplexation or transmetallation reactions.

[0005] EP1931673 (Guerbet) describes the following formula: [ka] Disclosed are PCTA derivatives represented by the formula: and synthetic routes for their preparation.

[0006] EP 2988756 (by the same applicant) discloses pharmaceutical compositions containing the above derivatives together with calcium complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, which, according to EP 2988756, compensate for the poor thermodynamic stability observed with PCTA-based gadolinium complexes by forming strong complexes with free lanthanide ions by metal exchange, thereby improving the tolerability of the contrast agent.

[0007] Furthermore, both EP1931673 and EP2988756 refer to enantiomers or diastereomers of the claimed compounds, or mixtures thereof, preferentially selected from the RRS, RSR and RSS diastereomers.

[0008] In both of the above mentioned patents, among other derivatives, (α3,α6,α9)-tris(3-((2,3-dihydroxypropyl)amino)-3-oxopropyl)-3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-(κN3,κN6,κN9,κN15,κO3,κO6,κO9) gadolinium is disclosed, and more recently [ka] It has been identified as the gadolinium chelate of 2,2',2''-(3,6,9-triaza-1(2,6)-pyridinacyclodecaphane-3,6,9-triyl)tris(5-((2,3-dihydroxypropyl)amino)-5-oxopentanoic acid) (CAS Registry Number: 933983-75-6), alternatively as P03277 or gadopiclenol.

[0009] Regarding gadopiclenol, EP1931673 states that the relaxivity is 11 mM -1 s -1 Gd -1 (in water, 0.5 T, 37°C), and EP2988756 states that the thermodynamic equilibrium constant is 10 -14.9(log K term = 14.9).

[0010] Furthermore, for this same compound, the relaxivity in human serum was 12.8 mM -1 s -1 (37℃, 1.41 T), stability (log K term ) is reported to be 18.7, and the dissociation half-life is approximately 20 days (pH 1.2; 37°C) (Investigative Radiology 2019, Vol 54,(8), 475-484).

[0011] The precursors for the preparation of PCTA derivatives (including gadopiclenol) disclosed in EP 1931673 are represented by the formula: [ka] The Gd complex of 3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-tri(α-glutaric acid) (identified herein as "Gd(PCTA-tris-glutaric acid)") having the formula: In particular, gadopiclenol can be obtained by amidating the above compound with isoserinol.

[0012] As the applicants have observed, Gd(PCTA-tris-glutarate) is 3 There are three stereocenters in the glutaric acid moiety (indicated by an asterisk (*) in the above structure) resulting in 8 possible stereoisomers. More specifically, the above structure can give rise to four pairs of enantiomers as shown in Table 1 below. [Table 1]

[0013] The isomer RRR is the mirror image of the isomer SSS, which is why they are called enantiomers (or enantiomeric pairs). As is known, enantiomers exhibit the same physicochemical properties and can only be distinguished using chiral methodologies such as chiral chromatography or polarized light.

[0014] On the other hand, isomer RRR is neither equivalent nor mirror image of any of the other six isomers mentioned above, and these other isomers are identified as diastereomers of the RRR (or SSS) isomer. Diastereoisomers may exhibit different physicochemical properties (e.g., melting point, water solubility, relaxivity, etc.).

[0015] Regarding gadopiclenol, its chemical structure contains a total of six stereocenters: three in the glutaric acid portion of the precursor mentioned above and one in each of the three isoserinol moieties attached to it (indicated by an asterisk (*) and a hollow circle (〇), respectively, in the structure below). [ka]

[0016] This brings the theoretical total number of stereoisomers of this compound to 2 6 =64. Summary of the Invention [Problem to be solved by the invention]

[0017] However, neither EP 1 931 673 nor EP 2 988 756 provide any detailed description of the composition of the isomeric mixtures obtained following the synthetic routes described, nor do they teach the separation and characterization of these isomers, nor do they disclose a stereospecific synthesis of gadopiclenol. [Means for solving the problem]

[0018] Applicants have discovered that certain isomers of the above-mentioned precursor Gd(PCTA-tris-glutarate) and its derivatives (particularly gadopiclenol) have improved physicochemical properties, particularly with regard to relaxivity and kinetic inertness.

[0019] One embodiment of the present invention relates to a compound selected from the group consisting of: Formula (Ia): [ka] having the enantiomer [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR enantiomer); Formula (Ib): [ka] and the enantiomers [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomers), Mixtures of such RRR and SSS enantiomers, and pharma- ceutically acceptable salts thereof.

[0020] Another embodiment of the present invention is a compound having at least 50% of the RRR isomer of formula (Ia) [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium or the SSS isomer of formula (Ib) [( The present invention relates to an isomeric mixture of Gd(PCTA-tris-glutarate) comprising: αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, or a mixture thereof, or a pharma- ceutically acceptable salt thereof.

[0021] Another aspect of the invention relates to amides obtained by combining one of the above compounds or isomer mixtures with an amino group, such as, preferably, serinol or isoserinol.

[0022] One embodiment of the present invention is a compound of formula (II A): F(NR1R2)3(II A) [In the formula, F is: Formula IIIa [ka] RRR enantiomeric residues, denoted by Formula IIIb [ka] or They are a mixture of RRR and SSS enantiomeric residues; Each of the three -NR1R2 groups is bonded to an open bond of the carboxyl moiety of F, which is shown as a black circle (●) in the above structural formula; R1 is H or C1-C6 alkyl optionally substituted with 1 to 4 hydroxyl groups; R2 is a C1-C6 alkyl optionally substituted with 1 to 4 hydroxyl groups, preferably a C1-C3 alkyl substituted with 1 or 2 hydroxyl groups; is] The present invention relates to an amide derivative represented by the formula:

[0023] One embodiment of the present invention is a compound of formula (II B): F'(NR1R2)3(II B) [In the formula, F' is: Formula (III) [ka] is an isomeric mixture of Gd(PCTA-tris-glutarate) residues represented by The isomeric mixture of Gd(PCTA-tris-glutarate) residues comprises at least 50% of the enantiomeric residue of formula (IIIa) above, the enantiomeric residue of formula (III B) above, or a mixture thereof; Each -NR1R2 group is attached to the open bond of the carboxyl moiety of F', indicated by a filled circle (●) in the above structural formula, and is as defined above for compounds of formula (II A). The present invention relates to an isomeric mixture of amide derivatives of Gd(PCTA-tris-glutaric acid) having the formula:

[0024] A further aspect of the present invention relates to Gd(PCTA-tris-glutarate), or preferably its RRR / SSS mixture, or an isomeric mixture of Gd(PCTA-tris-glutarate) comprising at least 50% of any of these enantiomers or the RRR / SSS enantiomeric mixture, or a pharma- ceutically acceptable salt of an amide derivative of formula (IIA) or (IIB) above, for use as an MRI contrast agent, in particular suitable for diagnostic imaging of organs or tissues of the human or animal body using MRI techniques.

[0025] A further embodiment relates to a pharma- ceutically acceptable composition comprising at least one compound or isomeric mixture of the invention, or a pharma- ceutically acceptable salt or amide derivative thereof as defined above, in admixture with one or more physiologically acceptable carriers or excipients.

[0026] In another aspect, the present invention relates to the stereoselective synthesis of the RRR or SSS isomer of Gd(PCTA-tris-glutarate) or its salts.

[0027] One embodiment of the present invention relates to a method for the synthesis of an amide derivative of formula (II A): F(NR1R2)3(II A) [In the formula, F, R1 and R2 are as described above and comprise: a) obtaining the RRR or SSS isomer of the Gd(PCTA-tris-glutarate) complex, or a mixture thereof; and b) converting the isomer or mixture of isomers obtained in step a) into its amide derivative; And A process for preparing an isomeric mixture of amide derivatives of the above formula (II B) comprising: a') obtaining an isomeric mixture of Gd(PCTA-tris-glutarate) containing at least 50% of the enantiomers RRR or SSS, or a mixture thereof; b') Converting the isomeric mixture of Gd(PCTA-tris-glutaric acid) obtained in step a') into the corresponding isomeric mixture of the respective amide derivatives Regarding. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows the HPLC chromatogram of Gd(PCTA-tris-glutarate) recovered as an isomer mixture from Example 1, carried out according to the synthesis procedure disclosed by the prior art ([GdL]=0.2 mM, 25° C.). [Diagram 2]FIG. 1 shows the HPLC chromatogram of the RRR / SSS enantiomer pair C of Gd(PCTA-tris-glutarate) obtained from Example 3. [Diagram 3] The MS spectrum of the main peak in Figure 2 is shown. m / z ratio Gd(H4L)+: 752.14 m / z. [Figure 4] HPLC chromatograms of: a) isomeric mixture of Gd(PCTA-tris-glutarate) from Example 1; b) enantiomer pair C (compound VI from Example 3); c) RRR enantiomer (compound XII from Example 5), and d) SSS enantiomer (compound XVII from Example 6). [Diagram 5] 1 shows chiral HPLC chromatograms of: a) enantiomer pair C (compound VI of Example 3), b) RRR enantiomer (compound XII of Example 5), and c) SSS enantiomer (compound XVII of Example 6) of Gd(PCTA-tris-glutarate). [Figure 6] HPLC chromatograms of amide derivatives obtained by reaction of Gd(PCTA-triglutaric acid) with isoserinol. a): Amide derivative obtained as isomer mixture from Example 2 (for convenience, the four main peaks are shown as A', B', C' and D'). b): Amide derivative obtained by reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with R-isoserinol; c): Amide derivative obtained by reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with S-isoserinol; d): Amide derivative obtained by reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with racemic isoserinol. [Figure 7] Referring to the test in Example 7, the changes over time in the HPLC area values ​​of peaks A (◇), B (□), C (△), and D (◯) are shown ([GdL] = 0.2 mM, [HCl] = 1.0 M, 25°C). [Figure 8]The HPLC area values ​​change with time with reference to the test in Example 8: total area of ​​isomer mixture (◇); RRR / SSS Gd(PCTA-tris-glutaric acid) + R-isoserinol (□); RRR / SSS Gd(PCTA-tris-glutaric acid) + S-isoserinol (△); RRR / SSS Gd(PCTA-tris-glutaric acid) + racemic isoserinol (◯). ([GdL]=0.2 mM, [HCl]=1.0 M, 25°C). [Figure 9] The single crystal X-ray structure of the ternary complex of Gd(PCTA-tris-glutarate)-oxalate with guanidine counterions, with the formula {(C(NH2)3)2[Gd(PCTA-tris-glutarate)(C2O4)]}·1H2O, is shown, showing the chirality RRR of the (labeled) chiral carbon atom of the glutarate group. [Figure 10] 10 shows the unit cell of the crystal of FIG. 9, which comprises a 2RRR + 2SSS complex. [Figure 11] The X-ray structure of a single crystal obtained from the ternary complex formed between carbonate anion and amide compound D', obtained by coupling reaction of RRR / SSS Gd(PCTA-tris-glutarate) with racemic isoserinol, and the statistical analysis of the recovered crystals are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The synthesis methods disclosed in the prior art (see US Pat. No. 6,440,956 cited by EP 1931673) afford Gd(PCTA-tris-glutarate) as a mixture of isomers (referred to herein as "isomeric mixture of Gd(PCTA-tris-glutarate)"), which can be seen by HPLC as several peaks.

[0030] The preparative HPLC method allowed the separation of four peaks with the same m / z ratio (Gd(H4L)+: 752.14 m / z) from the mixture.

[0031] A representative chromatogram of the separated isomeric mixture is shown in Figure 1, where each peak, for convenience, is designated by the letters A, B, C, and D, respectively, and can reasonably be attributed to one of the enantiomeric pairs indicated above. More precisely, each peak is associated with a pair of enantiomers, characterized by the same m / z ratio in the MS spectrum that would be indistinguishable by conventional reversed-phase HPLC.

[0032] Surprisingly, it was found that the enantiomer pair associated with peak C in the HPLC chromatogram (or hereinafter interchangeably referred to as enantiomer pair C) exhibited optimal properties, particularly with respect to kinetic inertness and reduced tendency to release Gd.

[0033] For example, the dissociation half-life (in 1 M HCl) of enantiomeric pair C was found to be several tens of times longer than that of the enantiomeric pair related to peak B and more than 10 times longer than the average half-life of the Gd(PCTA-tris-glutarate) isomer mixture.

[0034] Furthermore, the relaxivity values ​​associated with the enantiomer pair associated with peak C are significantly higher than those reported in 1931673B1 for the isomeric mixture of Gd(PCTA-tris-glutarate) tested under the same conditions.

[0035] The enantiomeric pair C is [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, i.e., the RRR isomer of Gd(PCTA-tris-glutarate) of formula (Ia). [ka] and its respective enantiomers [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, i.e., Gd(PCTA-tris-glutarate) represented by formula (Ib). [ka] It has been shown that the compound comprises:

[0036] Surprisingly, the improved properties exhibited by the individual enantiomers (RRR and SSS) of Gd(PCTA-tris-glutarate) and the RRR / SSS enantiomer pair (or hereinafter also referred to as RRR / SSS Gd(PCTA-tris-glutarate)) are unexpectedly substantially maintained after their conjugation (e.g., formation of their amide derivatives).

[0037] For example, the coupling reaction of RRR / SSS Gd(PCTA-tris-glutarate), and likewise its individual RRR or SSS enantiomers with isoserinol, ultimately gives amide derivatives with the same molecular formula as gadopiclenol. In this regard, it is interesting to note that regardless of the type of isoserinol used, whether it is the R or S isomer or racemic isoserinol, the conjugation of Gd(PCTA-tris-glutarate) with the RRR / SSS enantiomers gives the respective amide derivatives with the same retention times (and therefore indistinguishable by conventional reversed-phase HPLC).

[0038] Thus, the addition of different isomers of isoserinol (or more generally amine derivatives) does not affect the key properties of the final conjugate compound, which are essentially determined by the stereochemistry of the Gd(PCTA-tris-glutarate) precursor.

[0039] Indeed, the improved properties exhibited by the enantiomeric pair associated with peak C for the isomeric mixture of Gd(PCTA-tris-glutarate) are substantially maintained after conjugation with isoserinol, regardless of the configuration of the bound isoserinol.

[0040] In particular, regardless of the configuration of isoserinol, its coupling with the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutaric acid) affords amide compounds with greater kinetic inactivity and relaxivity compared to gadopiclenol obtained as an isomeric mixture by prior art synthetic procedures.

[0041] In the present specification, and unless otherwise stated, the expression "isomeric mixture" (with respect to a particular compound) includes within its meaning a mixture containing at least two stereoisomers of that compound. In particular, when used with respect to Gd(PCTA-tris-glutaric acid), the expression "isomeric mixture" refers to an unseparated mixture of at least two of the eight diastereomers (also referred to herein as diastereoisomers), more precisely the four enantiomeric pairs shown in Table 1, resulting from the three stereocenters contained within the molecule. On the other hand, when used with respect to amide derivatives of Gd(PCTA-tris-glutaric acid) (e.g. gadopiclenol), the expression "isomeric mixture" refers to an undefined unseparated mixture of at least two of the above (four possible) of the respective amide derivatives of the enantiomeric pairs of the Gd(PCTA-tris-glutaric acid) residue.

[0042] In this regard, since each of the amine groups of the amide derivative may contain one or more stereocenters, the total number of possible stereoisomers of the amide derivative may increase accordingly. For example, the conjugation of three molecules of Gd(PCTA-tris-glutaric acid) and isoserinol (each having a stereocenter) results in a total of six stereocenters on each molecule, bringing the number of possible stereoisomers of the corresponding amide derivative up to 64 (32 enantiomeric pairs).

[0043] In the present description and claims, the terms "isomeric mixture of amide derivatives of Gd(PCTA-tris-glutaric acid)" or "amide derivatives of an isomeric mixture of Gd(PCTA-tris-glutaric acid)" are used interchangeably.

[0044] The expression "enantiomer C" refers to the pair of enantiomers associated with peak C, as in Figure 1. This enantiomer C corresponds to the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutarate).

[0045] The term "RRR / SSS enantiomer pair" (or RRR / SSS enantiomer) generally refers to a mixture of the enantiomer RRR of a compound of interest and its respective enantiomer SSS, including racemic mixtures thereof. As used herein, the term is typically used with respect to Gd(PCTA-tris-glutarate) to refer to a mixture of the enantiomers RRR and SSS of this compound (or the RRR / SSS mixture thereof). More specifically, the term "RRR / SSS enantiomer pair of Gd(PCTA-tris-glutarate)" (or "RRR / SSS enantiomer" as used interchangeably herein) refers to a mixture of the enantiomers RRR and SSS of this compound (or the RRR / SSS mixture thereof). The expression "Gd(PCTA-tris-glutaric acid)") means, for example, [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium This refers to a mixture of gadolinium (RRR enantiomers) and [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomers). [ka]

[0046] The expression "compound D" refers to the amide derivative obtained by the coupling reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with isoserinol.

[0047] The expression "stereoselective synthesis" (or "asymmetric synthesis" as used interchangeably herein) encompasses within its meaning a chemical reaction (or a sequence of reactions) in which one or more new elements of chirality are formed in a substrate molecule, resulting in the generation of unequal amounts of stereoisomers (enantiomers or diastereomers). In this specification, the expression "stereoselective synthesis" is used in particular with respect to RRR of Gd(PCTA-tris-glutarate) and its respective enantiomer SSS, and refers to a synthesis that makes it possible to obtain a complex that contains at least 55%, preferably 65%, more preferably 75% and most preferably at least 85% of one of the two enantiomers.

[0048] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of compounds of the present invention which are suitably modified by converting the parent compound, if any, of a free acid or basic group is present, into the corresponding addition salt with any base or acid that is conventionally intended to be pharmaceutically acceptable.

[0049] Preferred cations of inorganic bases which can be suitably used to prepare the salts of the invention include, for example, ions of alkali or alkaline earth metals, such as potassium, sodium, calcium or magnesium.

[0050] Preferred cations of organic bases include those of primary, secondary and tertiary amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine, and the like.

[0051] Preferred cations and anions of amino acids include, for example, those of taurine, glycine, lysine, arginine, ornithine or aspartic acid and glutamic acid.

[0052] Furthermore, the terms "moiety" and "residue" are intended to define the remaining portion of a molecule after it has been appropriately attached or conjugated to the remainder of the molecule, either directly or through any suitable linker.

[0053] For example, when used with respect to amide derivatives of Gd(PCTA-tris-glutaric acid) (either in the form of an isomeric mixture of RRR or SSS isomers, or an RRR / SSS enantiomeric mixture or enantiomeric pair), the term "residue" refers to the portion of Gd(PCTA-tris-glutaric acid) that is bound to an amine group to yield the corresponding amide derivative.

[0054] In particular, the term "residue of an isomeric mixture of Gd(PCTA-tris-glutarate)" refers to a compound having the following formula (III): [ka]

[0055] This residue may, for example, be conjugated to an amino residue of the formula -NR1R2 via an open bond in the carboxyl moiety indicated by a filled circle (●) in the above structural formula to give the corresponding amide derivative of the formula: [ka]

[0056] Similarly, the term "residue of the RRR and SSS enantiomers of Gd(PCTA-tris-glutarate)" refers to the residues of the following formula (IIIA): [ka] and formula (IIIb) [ka] It refers to a compound having the formula:

[0057] The term "residue" similarly applies to the corresponding residues of the RRR / SSS enantiomeric pair, or in general, the enantiomeric mixture.

[0058] A preparative HPLC method was required that allows the separation of four peaks (for convenience, designated as A, B, C and D, respectively) of Gd(PCTA-tris-glutarate) with the same m / z ratio (Gd(H4L)+: 752.14 m / z) obtained according to the non-stereoselective synthesis disclosed in the prior art. Taking into account the three stereocenters present in the molecule (indicated by asterisks in the molecular structure above), the four signals of the HPLC chromatogram of the Gd(PCTA-triglutarate) complex were assigned to the four enantiomeric pairs formed with the different optical isomers of the glutaric acid residue, as shown in Table 1 above.

[0059] To investigate the kinetic inactivity of the racemic mixture of Gd(PCTA-tris-glutarate), especially its four enantiomeric pairs separated by HPLC, their dissociation reactions under acidic conditions were investigated. To ensure pseudo-first-order kinetic conditions, a large excess of H + ([HCl] = 1.0 M) was specifically utilized.

number

[0060] Solutions of Gd(PCTA-tris-glutarate) (mixture of isomers) in 1M HCl were prepared and analyzed over time as described in Example 7.

[0061] In particular, the area values ​​of the peaks A, B, C and D were evaluated over time by HPLC.

[0062] Upon acid-catalyzed dissociation of the complex, as expected, the integrated areas of peaks A, B, C, and D decreased, while a new signal corresponding to the free ligand formed and increased (m / z: 597.24). Interestingly, however, it was found that the rates of decrease in the areas of signals A, B, C, and D were not equal to each other (e.g., the decrease in the areas of peaks A and B was significantly faster than that of peaks C and D).

[0063] Therefore, the decrease in the integrated area of ​​the signals A, B, C, and D was evaluated and plotted as a function of time, and the results obtained are shown graphically in Figure 7 (highlighting the observed differences between the behavior of the four peaks).

[0064] Dissociation rates and half-lives (t 1 / 2 =ln2 / k X ) characterizes k X The pseudo first-order rate constant (where k X are respectively = k A , k B , k C and k D ) was calculated by fitting the area-time data pairs as described in detail in Example 7. In addition, the average half-life values ​​of the isomeric mixtures of Gd(PCTA-tris-glutarate) were also obtained by taking into account the percentage composition of the mixture. The obtained results are summarized in Table 2 and compared with those of some reference contrast agents (e.g., Gd-DOTA (Dotarem TM ) and Eu(PCTA), the europium complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid.

[0065] These results confirmed that the dissociation rates of the four enantiomeric pairs differed significantly from each other.

[0066] In particular, the enantiomeric pair associated with peak C is surprisingly the most kinetically inactive of all other possible isomers and has the lowest propensity to release Gd.

[0067] In fact, the t measured for this pair of enantiomers C 1 / 2 The value is, for example, about 68 times that of B. Furthermore, the t 1 / 2 This value is significantly higher than that measured for Eu(PCTA), which also has q=2 (see, for example, Tircso, G. et al. Inorg Chem 2006, 45(23), 9269-80) and is comparable to that measured for e.g. Gd-DOTA (Dotarem), the most stable and inert commercially available contrast agent. TM ) reported in the literature 1 / 2 Is exactly equal to the value.

[0068] Fractions enriched in this compound were then collected by flash chromatography as detailed in Example 3, achieving a purity of at least about 90% (as HPLC area %, see Figure 2) of the enantiomeric pair associated with peak C.

[0069] Surprisingly, the relaxivity values ​​obtained for the recovered enantiomeric pairs were r1 = 9.3 ± 0.1 mM -1 s -1 which is significantly higher than the r1 = 7.2 recorded (under the same conditions) for the isomeric mixture of Gd(PCTA-tris-glutarate) in EP1931673B1.

[0070] The unexpected combination of high relaxivity and greater inertness (leading to better tolerability) exhibited by this enantiomeric pair is particularly interesting.

[0071] Thus, efforts were expended to identify the pair of enantiomers associated with peak C.

[0072] In particular, we set up the stereoselective synthesis of the RRR and SSS isomers of Gd(PCTA-tris-glutarate), described in Examples 5 and 6, respectively, and obtained the same HPLC retention time t r The crude product was obtained with the major compound having the formula: ##STR00013## By using the related isomer (R)-(-)-5-oxotetrahydrofuran-2-carboxylic acid as a key intermediate, the corresponding SSS isomer of Gd(PCTA-tris-glutarate) with the same HPLC retention time was also obtained (FIG. 4).

[0073] Alternatively, the synthesis of Gd(PCTA-tris-glutarate) carried out using methyl (2S)-bromoglutarate can yield the complex as an isomeric mixture that is virtually indistinguishable from that recovered with racemic methyl bromoglutarate as disclosed in the prior art.

[0074] The enantiomer pair associated with peak C was then analyzed by a specific chiral HPLC method (capable of separating single enantiomers of the pair) by comparing it with the synthesized SSS and RRR isomers of Gd(PCTA-tris-glutarate). The resulting chromatogram (see Figure 5) confirmed that the two enantiomers associated with peak C have the same retention times as the synthesized RRR and SSS isomers of Gd(PCTA-tris-glutarate).

[0075] Additionally, crystals were formed from the enantiomeric pair C with guanidinium oxalate, as disclosed in detail in Example 10. As can be seen in Figure 9, X-ray diffraction of the single crystal confirmed the RRR configuration of the chiral centers in the glutaric acid arms of the molecule, the presence of an equimolar ratio of RRR and SSS isomers within each unit cell of the crystal, and confirmed the RRR / SSS racemic nature of this pair (Figure 10).

[0076] These results are all consistent and demonstrate that the compound associated with peak C is indeed composed of the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutarate) (used interchangeably herein, RRR / SSS Gd(PCTA-tris-glutarate).

[0077] More specifically, from the above results, it is found that the compound corresponding to peak C identified by the present invention is represented by the following formula: [ka] [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or RRR-Gd(PCTA-tris-glutarate)) of the formula [ka] and its enantiomers [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or SSS-Gd(PCTA-tris-glutaric acid)) represented by the following formula Ic [ka] (represented by It can be defined as including

[0078] Thus, one aspect of the present invention is the enantiomeric RRR / SSS pair of Gd(PCTA-tris-glutarate), single enantiomers of this pair, mixtures thereof, pharma- ceutically acceptable salts thereof, amide derivatives thereof, and compositions comprising them.

[0079] In particular, one embodiment of the present invention relates to compounds preferably selected from the group consisting of: the individual enantiomers [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR enantiomer); Each enantiomer, i.e., the individual enantiomer [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomer); the RRR / SSS enantiomer pair; and pharma- ceutically acceptable salts thereof.

[0080] According to a preferred embodiment, the present invention relates to the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutarate) (or more simply referred to herein as "RRR / SSS Gd(PCTA-tris-glutarate)"), e.g., comprising a mixture of the two individual RRR and SSS enantiomers of the complex, a racemic mixture thereof, or a salt thereof, according to one embodiment of the present invention.

[0081] Another aspect of the invention relates to Gd(PCTA-tris-glutarate) enriched in any of the above enantiomers, or mixtures thereof.

[0082] The term "enriched" as used with respect to an isomer, or enantiomer, or enantiomeric pair of the present invention (particularly when referring to Gd(PCTA-tris-glutaric acid) or its amide derivatives) includes within its meaning mixtures of isomers in which such isomer, enantiomer, or enantiomeric pair is present in an amount higher than would normally be found in a mixture obtained following prior art non-stereoselective synthetic procedures.

[0083] Such enrichment (with respect to an isomer, or enantiomer pair, of Gd(PCTA-tris-glutarate)) corresponds, for example, to an amount of such isomer or enantiomer pair in the mixture of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, e.g., at least 90%.

[0084] In particular, another aspect of the invention relates to an isomeric mixture of Gd(PCTA-tris-glutarate) that contains at least 50% of any of the above isomers (in terms of its isomeric composition, i.e., relative to the sum of the individual isomers that make up the isomeric mixture of Gd(PCTA-tris-glutarate)) (i.e., at least 50% of the Gd(PCTA-tris-glutarate) complexes are the RRR isomer [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9 or the SSS isomers [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium; or mixtures thereof; or salts thereof (the remaining amount of the complex being represented by an indiscriminate mixture of the other possible isomers).

[0085] Thus, one embodiment of the present invention is Gd(PCTA-tris-glutarate), or a salt thereof, where the respective enantiomers RRR, or the enantiomers SSS, or the RRR / SSS mixture of these enantiomers, are such that the acid or salt constitutes at least 50% (e.g., on a molar basis) of the isomeric mixture.

[0086] Preferably, the enrichment in Gd(PCTA-tris-glutarate) (any one of the above enantiomers or mixtures thereof) is at least 60%, more preferably at least 70%, most preferably at least 80%, for example at least 90% enrichment.

[0087] More preferably, the enrichment is in the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutarate).

[0088] In a preferred embodiment, the present invention relates to the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutarate) or an isomeric mixture of Gd(PCTA-tris-glutarate) containing at least 50% of the RRR / SSS enantiomer pair, i.e., at least 50% of the complex is composed of the RRR / SSS enantiomer pair, Gd(PCTA-tris-glutarate).

[0089] The RRR isomer [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, or Gd(PCTA-tris-glutarate) enriched in this isomer, can be prepared by using a stereoselective synthesis involving alkylation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (or "picrene" as used interchangeably herein) with (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester, as described in more detail, for example, in Example 5.

[0090] Similarly, the alternative use of (2R)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester (e.g., as described in Example 6) can provide either the SSS isomer of Gd(PCTA-tris-glutarate), or Gd(PCTA-tris-glutarate) appropriately enriched for the SSS isomer.

[0091] The stereoselective synthesis of the RRR and SSS isomers of Gd(PCTA-tris-glutarate) is novel and constitutes a further embodiment of the present invention.

[0092] In another embodiment, the present invention relates to the above enantiomers, enantiomer pairs, or enriched Gd(PCTA-tris-glutarate), in the form of a pharma- ceutically acceptable salt, for use as a contrast agent particularly suitable for magnetic resonance imaging (MRI) analysis.

[0093] More specifically, a further embodiment of the present invention relates to a pharma- ceutically acceptable salt of a compound selected from the individual enantiomer RRR of Gd(PCTA-tris-glutarate), or the individual enantiomer SSS, or the RRR / SSS enantiomer pair, or Gd(PCTA-tris-glutarate) enriched in any of these individual enantiomers by at least 50%, preferably the RRR / SSS enantiomer mixture, for use as a contrast agent, particularly suitable for magnetic resonance imaging (MRI) analysis.

[0094] Suitable examples of pharma- ceutically acceptable salts include salts with a cation of an inorganic base selected from an alkali or alkaline earth metal, such as potassium, sodium, calcium or magnesium, or a cation of an organic base selected from ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine, or a cation of an amino acid selected from lysine, arginine, or ornithine.

[0095] According to another aspect, the invention relates to a conjugate of any one of the above compounds or isomeric mixtures with an amine, preferably of formula NHR1R2.

[0096] One embodiment of the present invention is a compound of formula (II A): F(NR1R2)3(II A) The present invention relates to amide derivatives of the RRR enantiomer, the SSS enantiomer, or a mixture of two such enantiomers of Gd(PCTA-tris-glutarate), represented by the formula:

[0097] Another embodiment of the present invention is a compound of formula (II B): F'(NR1R2)3(II B) The present invention relates to isomeric mixtures of amide derivatives of Gd(PCTA-tris-glutaric acid) containing at least 50% of the individual enantiomer RRR, or the enantiomer SSS, or a mixture of these enantiomers, represented by the formula:

[0098] In the above formulae (II A) and (II B), the meanings of F, F', R1 and R2 are as defined above.

[0099] Preferred examples include amide derivatives of formula (II A) above, in which F is a mixture of RRR and SSS enantiomeric residues (or a pair of RRR / SSS enantiomeric residues) of Gd(PCTA-tris-glutarate), or amide derivatives of formula (II B), in which F' is an isomeric mixture of Gd(PCTA-tris-glutarate) residues containing at least 50% of a mixture of RRR and SSS enantiomeric residues.

[0100] In a preferred embodiment, the present invention relates to an amide derivative of formula (II B) above, wherein F' is an isomeric mixture of Gd(PCTA-tris-glutarate) residues of formula III above, comprising at least 50% of a mixture of RRR and SSS enantiomeric residues of formulas (IIIA) and (IIIB), respectively.

[0101] Preferably, in these amide derivatives, F' is enriched in the mixture of RRR and SSS enantiomeric residues (i.e. contains at least 60%), more preferably at least 70%, most preferably at least 80%, e.g., particularly preferably at least 90%. Suitable examples include amide derivatives of formula (II B) above, in which R1 is H and R2 is C1-C3 alkyl substituted with one or more, preferably one or two, more preferably two hydroxyl groups.

[0102] In a preferred embodiment, the present invention relates to an isomeric mixture of amide derivatives of the above formula (II B), in which F' is a residue of formula (III) as defined above, R1 is H and R2 is a C1-C3 alkyl substituted with one or two hydroxyl groups. More preferably, R2 is a serinol residue, or even more preferably an isoserinol residue, for example selected from R isoserinol, S isoserinol or racemic isoserinol. Most preferably, the amide compound has racemic isoserinol.

[0103] For example, non-limiting representative examples of the above compounds include: The following formula: [ka] Denoted by, [(αS,α'S,α''S)-α,α',α''-tris[3-[(2(S),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or the isomer SSS-SSS); The following formula: [ka] -[(αR,α'R,α''R)-α,α',α''-tris[3-[(2(R),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or the isomer RRR-RRR); The following formula: [ka] Denoted by, [(αR,α'R,α''R)-α,α',α''-tris[3-[(2(S),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or the isomer RRR-SSS); The following formula: [ka] Denoted by, [(αS,α'S,α''S)-α,α',α''-tris[3-[(2(R),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or the isomer SSS-RRR); or the respective isomers in which the isoserinol moiety is in the RSR, SSR, SRS, RSS, or RRS configuration.

[0104] The amide of formula (II B) with isoserinol has the same molecular formula as gadopiclenol, but contains a central moiety F' enriched by at least 50% with the RRR or SSS isomeric residues, or more preferably a mixture of RRR and SSS enantiomeric residues, of Gd(PCTA-tris-glutaric acid).

[0105] Notably, regardless of the type of isoserinol used (i.e., R or S isomer, or racemic isoserinol), the conjugation of Gd(PCTA-tris-glutarate) with the RRR / SSS enantiomer pair is indistinguishable by HPLC since the retention times are the same, as shown in Figure 6. Figure 6 further shows that this retention time is the same as that of peak D' separated by HPLC from gadopiclenol obtained as an isomer mixture in Example 2.

[0106] Surprisingly, the improved properties of RRR / SSS Gd(PCTA-tris-glutarate) are substantially maintained after conjugation with isoserinol, regardless of the configuration of the bound isoserinol.

[0107] In particular, conjugation of isoserinol with RRR / SSS Gd(PCTA-tris-glutaric acid) leads to amide derivatives with the same molecular formula but greater kinetic inactivity and relaxivity than gadopiclenol, which is obtained as an isomeric mixture in prior art synthetic procedures.

[0108] Indeed, the same tests carried out to evaluate the kinetic inactivity of the four different enantiomeric pairs isolated from the isomeric mixture of Gd(PCTA-tris-glutarate) were repeated by using the amide derivatives obtained by conjugation of gadopiclenol (isomer mixture) obtained in Example 2 and RRR / SSS Gd(PCTA-tris-glutarate) with i) R-isoserinol, ii) S-isoserinol; and iii) racemic isoserinol, respectively.

[0109] The average half-lives of the resulting conjugated compounds and gadopiclenol (mixture of isomers) were calculated taking into account the decrease in the total HPLC area over time, as described in detail in Example 8. For the amide derivatives obtained by reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with i) R-isoserinol; ii) S-isoserinol; iii) racemic isoserinol, k was calculated by fitting the area-time data pairs performed in Example 7 for RRR / SSS Gd(PCTA-tris-glutaric acid). X Pseudo first-order rate constant and half-life (t 1 / 2 =ln2 / k X ) was also calculated.

[0110] The obtained results are summarized in Table 3 and compared with those of some reference contrast agents (e.g. Gd-DOTA (Dotarem TM) and Eu(PCTA)) are compared with the corresponding values ​​referenced in the literature.

[0111] The data on one side of Table 3 are the t values ​​estimated from the area values ​​of the complexes obtained by coupling RRR / SSS Gd(PCTA-tris-glutarate) with R, S and racemic isoserinol. 1 / 2 values ​​and t calculated by fitting the area-time kinetic data 1 / 2 It shows that there is very good agreement between the values.

[0112] On the other hand, the data in Table 3 show that the t1 / 2 values ​​of all the amide compounds obtained by the reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with i) R-isoserinol, ii) S-isoserinol, and iii) racemic isoserinol are approximately 8 times higher than that of gadopiclenol (mixture of isomers), confirming that the high kinetic inactivity of RRR / SSS Gd(PCTA-tris-glutaric acid) is substantially maintained after coupling with isoserinol.

[0113] The overall consistency of the half-life values ​​obtained for the different complexes resulting from the conjugation of RRR / SSS Gd(PCTA-tris-glutarate) with R,S or racemic isoserinol indicates that the chirality of the isoserinol pendant does not affect the kinetic inactivity of the final complexes. Furthermore, r1 relaxivities were measured for the compounds obtained by conjugation of RRR / SSS Gd(PCTA-tris-glutarate) with R,S and racemic isoserinol under the same conditions used in the literature for gadopiclenol.

[0114] The results obtained are compared in Table 5. Again, irrespective of the configuration of the added isoserinol, the r1 relaxivities measured for the conjugated compounds obtained from RRR / SSS Gd(PCTA-tris-glutarate) both in water and in HSA are higher than those reported in the relevant literature for gadopiclenol.

[0115] Thus, the amide derivatives obtained by conjugation of the RRR / SSS Gd(PCTA-tris-glutaric acid) of the present invention with isoserinol are characterized by improved kinetic inactivity and higher relaxivity, despite having the same structure of the gadopiclenol compound.

[0116] Crystals were then obtained from the amide derivative resulting from the conjugation of RRR / SSS Gd(PCTA-tris-glutarate) with racemic isoserinol, as described in detail in Example 10. X-ray diffraction studies of single crystals obtained from the ternary complex formed between carbonate anion and the amide derivative confirmed the RRR / SSS configuration of the glutarate arms of the core molecule C.

[0117] The X-ray structures and statistical analysis of all recovered crystals are shown in FIG.

[0118] The results of the stereoselective synthesis of the individual RRR or SSS isomers, as well as the (crystal) structures recorded for both the enantiomeric pair C and its conjugate with isoserinol, are all in agreement with each other, proving that the compound attributed to HPLC peak C in Figure 1 does in fact correspond to the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutarate).

[0119] The synthesis of the RRR and SSS isomers of Gd(PCTA-tris-glutarate) represents a further embodiment of the present invention.

[0120] In particular, another embodiment of the present invention comprises: a) The following formula [ka] to obtain (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester represented by the formula: b) The recovered (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester, [ka] By alkylating 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15), 11,13-triene represented by the formula: The present invention relates to a stereoselective process for the preparation of Gd(PCTA-tris-glutarate) enriched with the isomer [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR isomer), comprising:

[0121] In one embodiment, the process can yield Gd(PCTA-tris-glutarate) enriched in the desired RRR isomer of the complex by at least 55%, preferably at least 60%, more preferably at least 70%, and most preferably at least 80%, e.g., about 85%.

[0122] Similarly, by substituting (2R)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester in step a) of the process, it is possible to obtain the respective SSS isomer of Gd(PCTA-tris-glutarate), or Gd(PCTA-tris-glutarate) suitably enriched in this isomer.

[0123] In yet another embodiment, the present invention relates to an RRR / SSS pair of enantiomers of Gd(PCTA-tris-glutarate), or Gd(PCTA-tris-glutarate) enriched at least 50% in said enantiomeric pair, preferably for use as an intermediate in the preparation of derivatives thereof, such as amide derivatives.

[0124] Yet another embodiment of the present invention comprises: a) obtaining the RRR or SSS isomer of Gd(PCTA-tris-glutarate), or a mixture thereof; and b) converting the isomer or mixture of isomers obtained in step a) into the desired amide derivative. Formula (II A) F(NR1R2)3(II A) [In the formula, F, R1, and R2 are as defined above] This invention relates to a method for the synthesis of an amide derivative of Gd(PCTA-tris-glutaric acid) represented by the formula:

[0125] Step a) of the process for obtaining the RRR or SSS isomer of Gd(PCTA-tris-glutarate) is, for example, carried out as described above and in detail, for example, in Examples 5 and 6.

[0126] Alternatively, step b) of this process can be carried out according to conventional procedures, for example as reported in the prior art cited above.

[0127] A further aspect of the present invention is a') obtaining an isomeric mixture of Gd(PCTA-tris-glutarate) containing at least 50% of the individual enantiomers RRR, or SSS, or preferably a mixture thereof; and b') converting the isomeric mixture of Gd(PCTA-tris-glutaric acid) obtained in step a') into the corresponding isomeric mixture of the desired amide derivatives; The compound according to formula (II B) F(NR1R2)3(II B) [In the formula, F', R1, and R2 are as defined above] This invention relates to a process for the synthetic preparation of a mixture of isomers of amide derivatives of Gd(PCTA-tris-glutaric acid) represented by the formula:

[0128] Step a' of this process, for example obtaining an isomeric mixture of Gd(PCTA-tris-glutarate) containing at least 50% of the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutarate), can be obtained by chromatography, including for example preparative HPLC or flash chromatography, starting from Gd(PCTA-tris-glutarate) obtained as an isomeric mixture by known procedures, for example as disclosed in Example 3.

[0129] On the other hand, step b') of the process, which involves coupling the enriched isomeric mixture of Gd(PCTA-tris-glutarate) recovered from step a') with the amine of interest, can be carried out according to conventional procedures, e.g. as reported in the prior art cited above.

[0130] For example, the product recovered from step a') can be reacted with isoserinol, for example by using the synthetic procedure detailed in Example 4.

[0131] A further embodiment of the present invention relates to amides of formula (II A) or (II B) as defined above for use as contrast agents, in particular suitable for Magnetic Resonance Imaging (MRI) analysis.

[0132] In particular, in another embodiment, the present invention relates to a compound selected from the group consisting of the individual RRR or SSS enantiomers of Gd(PCTA-tris-glutarate), mixtures of such RRR / SSS enantiomers, isomeric mixtures of Gd(PCTA-tris-glutarate) enriched by 50% in either the individual RRR or SSS enantiomers or mixtures thereof, pharma- ceutically acceptable salts thereof, amide derivatives thereof of formula (IIA) or (II B), for the preparation of a pharmaceutical formulation for use in diagnostic imaging, either in vivo, in vitro, or ex vivo, by using MRI techniques, of organs, tissues or regions of the human or animal body, as well as biological samples including cells, biological fluids and biological tissues obtained from a living mammalian patient, preferably a human patient.

[0133] A further aspect of the present invention relates to a pharmaceutical composition for diagnosis comprising at least one of the above-mentioned isomeric compounds or isomeric mixtures of the present invention, or a pharma- ceutically acceptable salt or amide derivative as described above, in admixture with one or more physiologically acceptable excipients, diluents or solvents.

[0134] Preferably, the pharmaceutical composition comprises an amide derivative of formula (II A) above. (wherein F is a residue of the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutarate); or More preferably, the isomer mixture of the amide derivative represented by the above formula (II B) In formula (II B), F' is at least 50% enriched in the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutarate), and -NR1R2 is an isoserinol residue. The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0135] In a preferred embodiment, the pharmaceutical composition comprises a compound of formula [ka] The present invention also includes amide compounds of formula (II B) conjugated with isoserinol, wherein F' in formula (II B) is a residue of Gd(PCTA-tris-glutaric acid) enriched in the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutaric acid) by at least 60%, preferably at least 70%, preferably at least 80%, and most preferably at least 90%.

[0136] In a further aspect, the present invention relates to an MRI contrast medium comprising an effective amount of at least one isomeric compound or isomeric mixture of the present invention as described above, or a pharma- ceutically acceptable salt thereof, or an amide derivative thereof, in combination with one or more pharma- ceutically acceptable excipients, diluents or solvents.

[0137] In this scope, and unless otherwise specified, the term "effective amount" or "effective dose" as used herein means any amount of the Gd(PCTA-tris-glutarate) of the present invention or a pharma- ceutically acceptable salt thereof, or its amide derivatives represented by formula (II A) or (II B) or a pharmaceutical composition thereof, sufficient for its intended diagnostic purpose: i.e., for visualizing biological elements including cells, biological fluids and biological tissues ex vivo, or for achieving in vivo diagnostic imaging of an organ, tissue or region of a patient's body.

[0138] Unless otherwise indicated, the term "individual patient" or "patient" as used herein refers to a living human or animal patient, preferably a human undergoing an MR diagnostic evaluation.

[0139] Details concerning dosage amounts, dosage forms, modes of administration, pharma- ceutically acceptable carriers, excipients, diluents, adjuvants, etc. are known in the art.

[0140] Non-limiting examples of preferred compounds of the present invention, procedures for their preparation, and their characterization are described in the following sections with the aim of explaining the invention in more detail without limiting its scope.

[0141] Experimental Part HPLC characterization of the obtained compounds General Procedure Procedure 1: HPLC characterization of Gd(PCTA-tris-glutarate) (isomer mixture and individual / enriched isomers) HPLC characterization of Gd(PCTA-tris-glutarate) obtained as an isomer mixture from Example 1 was carried out using an Agilent 1260 Infinity II system. The experimental setup for the HPLC measurements is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, PDA detector HPLC (Agilent 1260 Infinity II system) Stationary phase: Phenomenex Gemini (registered trademark) 5μm C18 110Å Mobile phase: H2O / HCOOH 0.1%:methanol [Table 2]

[0142] Step 2: HPLC characterization of gadopiclenol (isomeric mixture) and compounds obtained by coupling of enantiomer pair C with R, S, or racemic isoserinol The HPLC characterization of gadopiclenol as the isomer mixture obtained from Example 2 or as the compound obtained by communication of the enantiomeric pair C of Gd(PCTA-tris-glutarate) with R, S, or racemic isoserinol was carried out using a Thermo Finnigan LCQ DECA XPPlus system. The experimental setup for the HPLC measurements is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, PDA detector HPLC equipped with LCQ Deca XP-Plus-Thermo Finnigan Stationary phase: Phenomenex Gemini (registered trademark) 5μm C18 110Å Mobile phase: H2O / TFA 0.1%: Acetonitrile / 0.1% TFA [Table 3] The resulting HPLC chromatogram is shown in FIG.

[0143] Step 3: Chiral HPLC method for separating the enantiomers of compound C A specific chiral HPLC method was set up to separate the RRR and SSS enantiomers of enantiomeric pair C (compound VI) prepared as described in Example 3. Separation and characterization of the enantiomers was performed using an Agilent 1200 system or a Waters Alliance 2695 system. The experimental setup for the HPLC measurements is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, PDA detector Equipped with HPLC Stationary phase: SUPELCO Astec CHIROBIOTIC 5 μm 4.6x250mm Mobile phase: H2O / HCOOH 0.025% : acetonitrile Elution: Isocratic 2% acetonitrile (30 min) Flow rate 1 mL / min Column temperature: 40℃ Detection 210-270 nm. The resulting HPLC chromatogram is shown in Figure 5a. Comparison with the chromatograms of the pure RRR enantiomer (compound XII of Example 5, Tr. 7.5 min) and the pure SSS enantiomer (compound XVII of Example 6, Tr. 8.0 min) is shown in Figures 5b) and 5c), respectively.

[0144] Example 1: Synthesis of Gd(PCTA-tris-glutarate) (mixture of isomers) Gd(PCTA-tris-glutarate) as a random mixture of stereoisomers was prepared by using the procedures reported in the prior art above, according to the following synthetic scheme 1. [ka]

[0145] a) Preparation of Compound II Racemic glutamic acid (33.0 g, 0.224 mol) and sodium bromide (79.7 g, 0.782 mol) were suspended in 2M HBr (225 mL). The suspension was cooled to -5 °C and NaNO2 (28.0 g, 0.403 mol) was added slowly in portions over 2.5 h while maintaining the internal temperature below 0 °C. The yellow mixture was stirred at -5 °C for an additional 20 min, after which concentrated sulfuric acid (29 mL) was added dropwise to the mixture. The resulting dark brown mixture was allowed to warm to room temperature and extracted with diethyl ether (4x150 mL). The combined organic phase was washed with brine, dried over Na2SO4, concentrated to a brown oil (21.2 g) and used in the next step without further purification. The oil was dissolved in ethanol (240 mL), the resulting solution was cooled in ice and thionyl chloride (14.5 mL, 0.199 mol) was added slowly. The slightly yellow solution was stirred at room temperature for 2 days. The solvent was then removed in vacuo and the crude oil was dissolved in dichloromethane (200 mL) and washed with 5% aqueous NaHCO3 (4x50 mL), water (1x50 mL) and brine (1x50 mL). The organic phase was concentrated and purified on silica eluting with petroleum ether-ethyl acetate 3:1 to give 19.5 g of pure product. (Yield 33%).

[0146] b) Preparation of Compound IV A solution of compound II (17.2 g, 0.0645 mol) in acetonitrile (40 mL) was added to a suspension of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (picrene) compound (III) (3.80 g, 0.018 mol) and K2CO3 (11.2 g, 0.0808 mol) in acetonitrile (150 mL). The yellow suspension was heated at 65 °C for 24 h, after which the salts were filtered off and the organic solution was concentrated. The orange oil was dissolved in dichloromethane and the product was extracted with 1 M HCl (4x50 mL). The aqueous phases were combined, cooled in ice and brought to pH 7-8 with 30% aqueous NaOH. The product was then extracted with dichloromethane (4x50 mL) and concentrated to give a brown oil (10.1 g, 73% yield). This compound was used in the next step without further purification.

[0147] c) Preparation of Compound V Compound IV (9.99 g, 0.013 mol) was dissolved in ethanol (40 mL) and 5M NaOH (40 mL). The brown solution was heated at 80° C. for 23 h. The ethanol was concentrated, the solution was cooled in ice and brought to pH 2 with concentrated hydrochloric acid. The ligand was purified on resin Amberlite XAD 1600 (eluted with water-acetonitrile mixtures) to give 5.7 g (73% yield) as a white solid after lyophilization. The product was characterized by several peaks on HPLC.

[0148] d) Preparation of Compound VI Compound V (5.25 g, 0.0088 mol) was dissolved in deionized water (100 mL) and the solution was brought to pH 7 with 2 M NaOH (20 mL). GdCl3 solution (0.0087 mol) was slowly added at room temperature and the pH was adjusted to 7 with 2 M NaOH to confirm the complexation with xylenol orange. Once the complexation was complete, the solution was concentrated to remove salts and impurities and purified on resin Amberlite XAD1600 eluted with a water-acetonitrile gradient. After lyophilization, the pure compound was obtained as a white solid (6.79 g, 94% yield). The product was characterized by HPLC. The resulting HPLC chromatogram, in which several peaks can be characterized, is shown in Figure 1.

[0149] Use of (S)-methyl α-bromoglutarate obtained from L-glutamic acid also affords a compound completely equivalent to compound VI, consisting of an isomeric mixture having an HPLC chromatogram substantially superimposable to that of FIG.

[0150] Example 2: Synthesis of Gadopiclenol (Isomeric Mixture) Gadopiclenol as a random mixture of stereoisomers was prepared by coupling the isomeric mixture of Gd(PCTA-tris-glutarate) obtained from Example 1 with racemic isoserinol according to the following synthetic scheme 2, as disclosed in EP11931673B1. [ka]

[0151] Preparation of Compound VII Compound VI (0.90 g, 0.0011 mol) obtained from Example 1 was added to a solution of racemic isoserinol (0.40 g, 0.0044 mol) in water adjusted to pH 6 with concentrated HCl. N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (1.0 g, 0.0055 mol) and hydroxybenzotriazole (HOBT) (0.12 g, 0.00088 mol) were then added and the resulting solution was stirred at room temperature and pH 6 for 24 h. The product was then purified by preparative HPLC on silica C18, eluting with a water / acetonitrile gradient. The fractions containing the pure compound were concentrated and lyophilized to give a white solid (0.83 g, 78% yield). The product was characterized by HPLC. The resulting HPLC chromatogram is shown in Figure 4a.

[0152] Example 3: Isolation of the enantiomeric pair related to peak C Compound VI (1.0 g, 0.0013 mol), obtained as described in Example 1 (step d), was dissolved in water (4 mL) and the solution was acidified to pH 2-3 with concentrated hydrochloric acid. The resulting solution was loaded onto a column of pre-packed silica C18 (Biotage® SNAPULTRAC18 120 g, HP-sphere C18 25 μm) and purified on an automated flash chromatography system eluting with a slow gradient of deionized water (4 CV) followed by acetonitrile. The fractions enriched in the enantiomeric pair associated with peak C were combined, concentrated and lyophilized to give a white solid (200 mg). The HPLC chromatogram of the resulting enriched enantiomeric pair C is shown in FIG. The corresponding MS spectrum (Gd(H4L) + :752.14 m / z) is shown in Figure 3.

[0153] Example 4: Coupling of enantiomeric pair C with isoserinol a) Coupling of enantiomeric pair C with R-isoserinol The enriched enantiomeric pair C (34 mg, titer 90%, 0.040 mmol), recovered for example as in Example 3, was dissolved in deionized water (5 mL), R-isoserinol (16 mg, 0.17 mmol) was added, and the pH was adjusted to 6 with HCl 1M. Then, EDCI·HCl (39 mg, 0.20 mmol) and HOBT (3 mg, 0.02 mmol) were added, and the solution was stirred at room temperature at pH 6 for 48 h. The solution was concentrated and loaded onto a pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) and eluted with a water / acetonitrile gradient using an automated flash chromatography system. Fractions containing pure product or showing a major peak with an area of ​​more than 90% on HPLC were combined, concentrated, and lyophilized to give a white solid (21 mg, 54% yield). The HPLC chromatogram of the obtained product is shown in Figure 6b.

[0154] b) Coupling of enantiomeric pair C with S-isoserinol For example, the enriched enantiomeric pair C (55 mg, titer 90%, 0.066 mmol), recovered as described in Example 3, was dissolved in deionized water (5 mL) and S-isoserinol (34 mg, 0.29 mmol) adjusted to 6 with 1 M HCl was added. Then, EDCI·HCl (64 mg, 0.33 mmol) and HOBT (4.5 mg, 0.033 mmol) were added and the solution was stirred at room temperature at pH 6 for 48 h. The solution was concentrated and loaded onto a pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) and eluted with a water / acetonitrile gradient using an automated flash chromatography system. Fractions containing pure product or showing a major peak with an area of ​​more than 90% on HPLC were combined, concentrated and lyophilized to give a white solid (52 mg, 81% yield). The HPLC chromatogram of the obtained product is shown in FIG. 6c.

[0155] c) Coupling of enantiomeric pair C with racemic isoserinol For example, the enriched enantiomeric pair C (54 mg, titer 90%, 0.065 mmol), recovered as described in Example 3, was dissolved in deionized water (5 mL) and racemic isoserinol (27 mg, 0.29 mmol) was added, adjusted to pH 6 with 1 M HCl. EDCI·HCl (62 mg, 0.32 mmol) and HOBT (4.3 mg, 0.032 mmol) were then added and the solution was stirred at room temperature at pH 6 for 24 h. The solution was concentrated and loaded onto a pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) and eluted with a water / acetonitrile gradient using an automated flash chromatography system. Fractions containing pure product or showing a major peak with an area of ​​more than 90% on HPLC were combined, concentrated and lyophilized to give a white solid (60 mg, 95% yield). The HPLC chromatogram of the obtained product is shown in Figure 6d.

[0156] Example 5: Stereoselective synthesis of RRR Gd(PCTA-tris-glutarate) (compound XII) RRR-enriched Gd(PCTA-tris-glutarate) acid was prepared according to synthetic scheme 3, which comprises the following steps: [ka]

[0157] a) Preparation of Compound VIII The preparation was carried out as reported in Tetrahedron 2009, 65, 4671-4680. Specifically, to a solution of (S)-(+)-5-oxotetrahydrofuran-2-carboxylic acid (2.48 g, 0.019 mol) (commercially available) in anhydrous methanol (20 mL) was added 37% aqueous HCl (50 μL). The solution was refluxed under N2 atmosphere for 24 h. After cooling in ice, NaHCO3 was added and the suspension was filtered, concentrated, and purified on silica gel with hexane / ethyl acetate 1:1. Fractions containing pure product were combined and concentrated to give a colorless oil (2.97 g, 89% yield).

[0158] b) Preparation of Compounds IX and X Compound VIII (445 mg, 2.52 mmol) obtained in step a) was dissolved in anhydrous dichloromethane (6 mL) and triethylamine (0.87 mL, 6.31 mmol) was added. After the solution was cooled to -40 °C, (trifluoromethanesulfonic acid) trifluoromethanesulfonic anhydride (0.49 mL, 2.91 mmol) was added slowly. After stirring the dark solution at -40 °C for 1 h, a solution of compound III (104 mg, 0.506 mmol) in anhydrous dichloromethane (3 mL) and triethylamine (1 mL, 7.56 mmol) was added and the solution was allowed to slowly warm to room temperature and stirred at room temperature overnight. The organic solution was then washed with 2 M HCl (4 × 10 mL) and the aqueous phase was extracted again with dichloromethane (3 × 10 mL). The combined organic phases were concentrated in vacuo to give 400 mg of a brown oil, which was used in the next step without further purification.

[0159] c) Preparation of Compound XI Compound X (400 mg, 0.59 mmol) was dissolved in methanol (2.5 mL) and 5 M NaOH (2.5 mL). To ensure complete hydrolysis, the brown solution was heated at 80° C. for 22 h. The methanol was concentrated, the solution was brought to pH 1 with concentrated HCl, and purified using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) eluted with a gradient of deionized water / acetonitrile. Fractions containing pure product were combined, concentrated, and lyophilized (64 mg, 18% yield). HPLC showed a major peak.

[0160] d) Compound XII Compound XI (32 mg, 0.054 mmol) was dissolved in deionized water (4 mL) and the pH was adjusted to 7 with 1 M NaOH. GdCl3·6H2O (20 mg, 0.054 mmol) was added and the pH was adjusted to 7 with 0.1 M NaOH. The clear solution was stirred at room temperature overnight and the completion of complexation was checked by xylenol orange and HPLC. HPLC of the crude product showed the desired RRR isomer as the major peak: about 80% by area. The mixture was brought to pH 2 with concentrated HCl and purified using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) eluted with a deionized water / acetonitrile gradient. The fractions containing the pure product were combined, concentrated and lyophilized (36 mg, 90% yield). For example, by using the procedure of Example 2, the recovered compound can be reacted with isoserinol to give the corresponding RRR amide derivative.

[0161] Example 6: Stereoselective synthesis of SSS Gd(PCTA-tris-glutarate) (compound XVII) SSS-enriched Gd(PCTA-tris-glutarate) acid was similarly prepared according to synthetic scheme 4, which comprises the following steps: [ka]

[0162] a) Preparation of Compound XIII To a solution of (R)-(-)-5-oxotetrahydrofuran-2-carboxylic acid (5.0 g, 0.038 mol) (commercially available) in anhydrous methanol (45 mL) was added 37% aqueous HCl (100 μL). The solution was refluxed under N2 atmosphere for 24 h. After cooling in ice, NaHCO3 was added and the suspension was filtered, concentrated and purified on silica gel with hexane / ethyl acetate 1:1. Fractions containing pure product were combined and concentrated to give a colorless oil (6.7 g, 99% yield).

[0163] b) Preparation of Compounds XIV and XV Compound XIII (470 mg, 2.67 mmol) was dissolved in anhydrous dichloromethane (6 mL) and trimethylamine (0.93 mL, 6.67 mmol) was added. After the solution was cooled to -40 °C, trifluoromethanesulfonic anhydride (0.50 mL, 3.07 mmol) was slowly added dropwise. After stirring the dark solution at -40 °C for 1 h, compound III (140 mg, 0.679 mmol) and trimethylamine (0.93 mL, 6.67 mmol) were added and the solution was slowly brought to room temperature overnight. The organic solution was then washed with water (3 x 5 mL) and 2 M HCl (4 x 5 mL). The aqueous phase was extracted again with dichloromethane (3 x 10 mL). The combined organic phases were concentrated in vacuo to give 350 mg of a brown oil, which was used in the next step without further purification.

[0164] c) Preparation of compound XVI Compound XV (350 mg, 0.514 mmol) was dissolved in methanol (4.5 mL) and 5M NaOH (4.5 mL). The resulting brown solution was heated at 80° C. for 16 h to achieve complete hydrolysis. The methanol was concentrated, the solution was brought to pH 2 with concentrated HCl, and purified using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 μm) eluted with a water / acetonitrile gradient. Fractions containing the pure product were combined, concentrated, and lyophilized (52 mg, 17% yield). HPLC showed a major peak.

[0165] d) Preparation of compound XVII Compound XVI (34 mg, 0.057 mmol) was dissolved in deionized water (5 mL) and the pH was adjusted to 7 with 1 M HCl. GdCl3·6H2O (20 mg, 0.0538 mmol) was added and the pH was adjusted to 7 with 0.1 M NaOH. The solution was stirred overnight at room temperature and the completion of complexation was checked by xylenol orange and HPLC. HPLC of the crude product showed the desired SSS isomer as the major peak: about 85% by area. The solution was brought to pH 2.5 with concentrated HCl and purified using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 μm) eluted with a water / acetonitrile gradient. Fractions containing the pure product SSS were combined, concentrated and lyophilized (39 mg, 87% yield).

[0166] Example 7: Kinetic study of the dissociation reaction of Gd(PCTA-tris-glutaric acid) (mixture of isomers) in 1.0 M HCl solution (25° C.) The kinetic inertness of Gd(III) complexes is characterized either by the rate of dissociation measured in 0.1-1.0 M HCl or by the rate of transmetallation reactions occurring in solution with Zn(II) and Cu(II) or Eu(III) ions. However, dissociation of lanthanide(III) complexes formed with macrocyclic ligands is very slow, generally with Zn(II) complexes.2+ or Cu 2+ It proceeds via a proton-assisted pathway without the participation of endogenous metal ions such as The kinetic inertness of the complex Gd(PCTA-tris-glutarate) was characterized by the rate of dissociation reaction occurring in 1.0 M HCl solution. The complex (isomer mixture of Example 1) (0.3 mg) was dissolved in 2.0 mL of 1.0 M HCl solution and the evolution of the solution kept at 25° C. was followed over time by HPLC. The HPLC measurements were performed on an Agilent 1260 InfinityII system using analytical procedure 1.

[0167] Large excess of H + The presence of ([HCl] = 1.0 M) ensures pseudo first-order kinetic conditions.

number

[0168] The HPLC chromatogram of Gd(PCTA-tris-glutarate) is characterized by the presence of four signals (A, B, C, and D) with the same m / z ratio in the MS spectrum (Gd(H4L)+: 752.14 m / z). Each of these peaks can be reasonably attributed to one of the four pairs of enantiomers generated by the three stereocenters on the three glutarate arms of the molecule, as shown in Table 1. The HPLC chromatogram of this complex in the presence of 1.0 M HCl changes over time, in particular the areas of peaks A, B, C, and D decrease, although not equally in the different peaks, while new signals corresponding to the uncomplexed diastereoisomers are formed and increase over time. The differences in the decrease in the integrated areas of the peaks can be interpreted by the different dissociation rates of the enantiomeric pairs associated with the different peaks.

[0169] [H +When k is present in excess, the dissociation reaction of the enantiomeric pair of Gd(PCTA-tris-glutarate) can be treated as a pseudo-first-order process, and the reaction rate can be expressed as follows (Eq. 2, where k A , k B , k C , and k D is the pseudo first-order rate constant calculated by fitting the area vs. time data pairs, [A] t , [B] t , [C] t , and [D] t is the total concentration of A, B, C, and D compounds at time t).

number

[0170] The area value at time t can be expressed as follows:

number

[0171] Example 8: Kinetic study of the dissociation reaction of gadopiclenol (isomer mixture of Example 2) and the complex compounds obtained by coupling the enantiomer pair C with R, S, and racemic isoserinol, respectively, in 1.0 M HCl solution (25° C.) The kinetic inertness of all complexes was characterized by the rate of dissociation reaction occurring in 1.0 M HCl solution. For each batch, the complex (0.4 mg) was dissolved in 2.0 mL of 1.0 M HCl solution, and the dissociation reaction at 25 °C was followed over time by HPLC. HPLC measurements were performed using a Thermo Finnigan LCQ DECA XPPlus system according to analytical procedure 2.

[0172] The HPLC chromatogram of gadopiclenol recovered as an isomer mixture from Example 2 is characterized by the presence of four major peaks (for convenience, designated as A', B', C' and D') with identical MS and UV-Vis spectra. However, the HPLC chromatogram of the complex compound obtained by coupling the enantiomeric pair C with isoserinol only had one signal, whether it was R, S isoserinol or racemic isoserinol (Figure 6). As observed, the chirality of the isoserinol pendant does not affect the retention time of the coupled diastereoisomers, the presence of four signals in the HPLC chromatogram of gadopiclenol (isomeric mixture) can be interpreted by the presence of four enantiomeric pairs formed at the stereocenter of the glutaric acid residue: 1) RRR-SSS (signal D'), 2) RSR - SRS, 3) RRS - SSR, 4) RSS - SRR.

[0173] To obtain information on the kinetic inactivity of all the above complexes, a large excess of H was added to ensure the occurrence of pseudo-first-order conditions. + Their dissociation reactions were investigated in the presence of ([HCl] = 1.0 M). The progress of the reaction was monitored over time by HPLC, and the area values ​​of the complex peaks were plotted as a function of time, as described above in Example 7 for the Gd(PCTA-tris-glutarate) isomers.

[0174] As expected, the integrals of A', B', C' and D' decrease with time, while the peaks of the free ligand increase. Since the area values ​​of the HPLC chromatograms are directly proportional to the concentration of gadopiclenol (mixed isomers), the half-life of the dissociation reaction of gadopiclenol (mixed isomers) can be estimated from half the sum of the area values. The half-life of gadopiclenol (mixed isomers) was found to be 5.2 hours at 25 °C and pH 0 (1.0 M HCl). The half-lives of the complexes obtained by coupling the enantiomeric pair C with R, S and racemic isoserinol were also calculated from half the area values ​​of the HPLC chromatograms. The half-lives of the complexes obtained by coupling the enantiomeric pair C with R, S and racemic isoserinol were found to be 41, 43 and 44 hours at 25 °C and pH 0 (1.0 M HCl). The pseudo-first-order rate constants (k x ) is calculated by dividing the area-time kinetic data by the equation 3

number

[0175] The t was estimated from the area values ​​of the complexes obtained by coupling the enantiomeric pair C with R, S and racemic isoserinol and calculated by fitting the area-time kinetic data. 1 / 2 The comparison of values ​​shows very good agreement. 1 / 2 The values ​​clearly show that the dissociation half-life of the D' isomer obtained by coupling the enantiomeric pair C isomers with R, S, and racemic isoserinol is almost the same as that measured for gadopiclenol (mixture of isomers) and is 8 times longer, confirming that the higher kinetic inactivity of RRR-SSS Gd(PCTA-tris-glutarate) is substantially maintained after coupling with isoserinol. Moreover, the t values ​​of the complexes obtained by coupling the enantiomeric pair C with R, S, and racemic isoserinol are almost the same as those measured for gadopiclenol (mixture of isomers) and are 8 times longer, confirming that the higher kinetic inactivity of RRR-SSS Gd(PCTA-tris-glutarate) is substantially maintained after coupling with isoserinol. 1 / 2 The values ​​also show that the chirality of the isoserinol pendants does not affect the kinetic inertness of the final complex. [Table 5]

[0176] Example 9: Relaxation properties The relaxometric properties of the PCTA-based complex compounds of the present invention were measured at different magnetic field strengths, i.e., 0.47 and 1.41 T, at 37° C., in different media (water and human plasma) and compared with the relaxometric values ​​measured for Gd-complexes with similar coordination cages under the same conditions.

[0177] material Device Longitudinal water proton relaxation rates (R1 = 1 / T1) were measured at 0.47 T using a Minispec MQ-20 spectrometer (Bruker Biospin, Germany) operating at a proton Larmor frequency of 20 MHz. MR experiments at 1.41 T were performed using a Minispec MQ-60 spectrometer (Bruker Biospin, Germany) operating at a proton Larmor frequency of 60 MHz. method Sample preparation All test articles were used as supplied and the required amount of paramagnetic chelate complex was weighed and diluted with the medium of choice (water or human plasma) to give a starting solution of 5 or 10 mM. Relaxation capacity measurement Five different concentration samples (0.1, 0.25, 0.5, 0.75, and 1 mM) for each vehicle were prepared by further diluting the 5 or 10 mM starting solutions. Measurement of relaxivity Relaxivity measurements were performed at 0.47T and 1.41T with the sample at a preset temperature of 37°C kept constant by a thermostatic bath connected to the sample holder of the spectrometer. The five sample solutions were preheated to 37°C in an external thermostatic bath and then left for 10 min in the internal bath to allow the temperature to stabilize. The longitudinal relaxation times T1 were measured using a standard inversion recovery sequence, where the inversion time (TI) was varied from 10 ms to at least 5 times T1 in 15 steps. Statistical analysis (monoexponential fitting for T1 measurements, linear fitting for evaluation of longitudinal relaxivity) was performed by Mathematica® (Wolfram, USA). The errors of the estimated parameters were evaluated by the fitting procedure.

[0178] result Table 4 below shows the relaxivity r1 values ​​reported in EP 1931673 B1 for Gd(PCTA-tris-glutarate) (isomeric mixture) and the corresponding relaxivity r1 values ​​obtained under the same conditions for purified fractions of RRR / SSS Gd(PCTA-tris-glutarate). [Table 6]

[0179] Table 5 below compares the relaxivity values ​​r1 measured at 37°C in both HO and HSA for the amide derivatives obtained by communication of isoserinol with RRR / SSS Gd(PCTA-Tris-glutaric acid) with the corresponding values ​​cited in the prior art for gadopiclenol (mixture of isomers), together with the stereochemistry of the serinol used for the conjugation. [Table 7]

[0180] On the one hand, the obtained results show that the higher relaxivities measured for RRR / SSS Gd(PCTA-tris-glutarate) relative to the corresponding isomeric mixture are substantially maintained for the respective conjugate derivatives. On the other hand, these results are in agreement with the fact that the stereochemistry of the isoserinol moiety does not affect the main properties of the final compounds, which are mainly related to the stereochemistry of the glutarate arm.

[0181] Example 10: X-ray diffraction Enantiomeric Pair C Crystal preparation Single crystals of formula {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O, where Gd(H3L) is the tris-protonated RRR / SSS Gd(PCTA-tris-glutarate), suitable for X-ray diffraction studies, were grown by slow evaporation of water from an aqueous solution of the RRR / SSS-rich compound C recovered from Example 3. To facilitate crystallization, two inner sphere water molecules of the Gd(PCTA-tris-glutarate) complex were replaced by oxalate anions, and the associated guanidinium salt was crystallized from water. The starting solution consisted of 49.6 mg of (C(NH2)3)2(C2O4) (2.5.0×10 -4 mol) was added to an aqueous solution of enriched Compound C recovered from Example 3 (1.0 mL; 5.0 × 10 -5 The pH was adjusted to 3.3 by stepwise addition of solid H2C2O4.

[0182] Crystals were isolated and XRD data were collected from at least five crystals at the X-ray diffraction beamline (XRD1) of Elettra Synchrotron, Trieste (Italy) by the procedure disclosed, for example, in Lausi A. et al., The European Physical Journal Plus, 2015, 130, 1-8. Specifically, the collected crystals were immersed in NHV oil (Jena Bioscience, Jena, Germany), frozen in liquid nitrogen, and attached to the goniometer head using a Kapton loop (MiTeGen, Ithaca, USA). When different crystal shapes were available, all of them were tested. The complete data set was collected at 100 K (Oxford Cryostream 700 - nitrogen stream supplied by Oxford Cryosystems Ltd., Oxford, United Kingdom) by the rotating crystal method. Data were acquired on a Pilatus 2M hybrid pixel area detector (DEC Tris Ltd., Baden-Daettwil, Switzerland) using a monochromatic wavelength of 0.700 Å.

[0183] result The structure was solved by a dual space algorithm implemented in the SHELXT direct method (Sheldrick GM(2015). “SHELXT -Integrated space-group and crystal-structure determination”, Acta Crystallographica Section A, 71, 3-8). Fourier analysis and refinement were performed by F 2 The full-matrix least-squares fit was performed based on the anisotropic thermal refinement. Hydrogen atoms were fitted with isotropic U for the hydroxyl groups. factors = 1.2 U eq or U factors = 1.5 U eq was included in the calculated position (U eq(where is the equivalent isotropic thermal coefficient for the bound non-hydrogen atoms.) Hydrogen atoms of the solvent water molecules were not included in the refined model because they could not be clearly located in the electron density peaks of the Fourier difference maps. Essential crystallographic and refinement data are given in the table below. [Table 8] The X-ray structures of the {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O complex and the unit cell of the formed crystal are shown in Figures 9 and 10, respectively. Figure 10 shows that each unit cell contains 2RRR + 2SSS complexes, which means that each crystal contains the isomers SSS and RRR in equimolar (50-50%) ratios.

[0184] Amide derivatives with racemic isoserinol The formula [GdC 35 H 54 N7O 15 Single crystals of [CH6N3]2[CO3]·18H2O(GdL) (GdL is the conjugated RRR / SSS Gd(PCTA-tris-glutaric acid) of racemic isoserinol) were grown in an aqueous solution of the amide derivative of Gd(PCTA-tris-glutaric acid) with racemic isoserinol recovered from Example 4c). To facilitate crystallization, the two inner sphere water molecules of the final complex were replaced by carbonate anions, and the associated guanidinium salt was crystallized from water by slow diffusion of ethanol and diethyl ether at 4 °C. Specifically, 1.0 molar equivalent (97 mg of GdL complex and 9 mg of guanidine carbonate {C(NH2)3}2CO3) was dissolved in 1 mL of H2O, pH = 10.5, and slowly diffused with a mixture of EtOH and Et2O.

[0185] Fifteen single crystals were isolated and XRD data collection of seven crystals was performed at the X-ray diffraction beamline (XRD1) of Elettra Synchrotron, Trieste (Italy) using the procedure disclosed, for example, in Lausi A. et al., The European Physical Journal Plus, 2015, 130, 1-8. Specifically, the collected crystals were immersed in NHV oil (Jena Bioscience, Jena, Germany), frozen in liquid nitrogen, and mounted on the goniometer head using (MiTeGen, Ithaca, USA). When different crystal shapes were available, all of them were tested. The complete data set was collected at 100 K (Oxford Cryostream 700 - nitrogen stream supplied by Oxford Cryosystems Ltd., Oxford, United Kingdom) by the rotating crystal method. Data were acquired on a Pilatus 2M hybrid pixel area detector (DEC Tris Ltd., Baden-Daettwil, Switzerland) using a monochromatic wavelength of 0.700 Å.

[0186] The structure was solved by direct methods. Fourier analysis and refinement were performed by F 2 The full-matrix least-squares fit was performed based on the anisotropic thermal motion refinement. Hydrogen atoms were fitted with isotropic U for the methyl and hydroxyl groups. factors = 1.2 U eq or U factors = 1.5 U eq was included in the calculated position (U eq (where is the equivalent isotropic thermal coefficient of the bound non-hydrogen atom.) Hydrogen atoms of the solvent water molecules were not included in the refined model because they could not be clearly located in the electron density peaks of the Fourier difference map. Table 7. Crystallographic data and stereocenter configurations of the GdL data set Crystal system: trigonal space group R-3 Unit cell a = 53.395(8)Å b = 53.395(8)Å c = 12.959(3)Å α = 90° β = 90° γ = 120° Volume(Å3) 31997(11) Final R-index [I>2σ(I)]aR1= 0.0554, wR2= 0.1496 Configuration of ASU stereocenters C7 R C28A R-54(1)% occupancy C28B S-45(1)% occupancy C14 R C31A R-62(1)% occupancy C31B S-38(1)% occupancy C21 R C34A R-50(1)% occupancy C34B S-50(1)% occupancy a R1=Σ|| Fo | - | Fc || / Σ| Fo |, wR2= {Σ[w(Fo2 - Fc2)2] / Σ[w(Fo2)2]} 1 / 2 For details and the procedures used, see, for example, Lausi A., Polentarutti M., Onesti S., Plaisier JR, Busetto E., Bais G., Barba L., Cassetta A., Campi G., Lamba D., Pifferi A., Mande SC, Sarma DD, Sharma SM, Paolucci G., The European Physical Journal Plus, 2015, 130, 1-8. D'-CO3 2- The X-ray structure of the complex and the statistical analysis of the recovered crystals are shown in FIG.

Claims

1. A compound selected from the group consisting of: Formula Ia: 【Chemistry 1】 the enantiomer [(αR,α'R,α''R)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (enantiomer RRR); Formula Ib 【Chemistry 2】 and the enantiomer [(αS,α'S,α''S)-α,α',α''-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomer), Mixtures of such RRR and SSS enantiomers; and pharma- ceutically acceptable salts thereof.

2. A compound of formula (I) containing at least 50% of the compound of claim 1 or a pharma- ceutically acceptable salt thereof. 【Chemistry 3】 The isomeric mixture of Gd(PCTA-tris-glutaric acid) shown below.

3. 3. The isomeric mixture of claim 2, wherein the compound of claim 1 is a mixture of the RRR and SSS enantiomers of claim 1, or a pharma- ceutically acceptable salt thereof.

4. 4. The compound according to claim 1 or the isomeric mixture according to any of claims 2-3, wherein the pharma- ceutically acceptable salt is a salt with a cation of an inorganic base selected from alkali or alkaline earth metals, such as potassium, sodium, calcium, magnesium, or a cation of an organic base selected from ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine, or a cation of an amino acid selected from lysine, arginine and ornithine, or optionally with an anion of an inorganic acid selected from haloacids, such as chloride, bromide or iodide, and other suitable ions, such as acetate, succinate, citrate, fumarate, maleate or oxalate.

5. Formula (IIA) 【Chemistry 4】 [In the formula, F is of formula IIIa: 【Chemistry 5】 RRR enantiomeric residue represented by Formula IIIb: 【Chemistry 6】 or They are a mixture of RRR and SSS enantiomeric residues; 3 -NR １ R ２ Each group is attached to an open bond (●) in the carboxyl moiety of F; R １ is H or C optionally substituted with 1 to 4 hydroxyl groups １ -C ６ is alkyl; R ２ is optionally substituted with 1 to 4 hydroxyl groups; １ -C ６ Alkyl, preferably C substituted with 1 or 2 hydroxyl groups １ -C ３ Alkyl is] 2. An amide derivative of the compound of claim 1 having the formula:

6. Formula (IIB): 【Chemistry 7】 [In the formula, F' is: Formula (III): 【Chemistry 8】 is an isomeric mixture of Gd(PCTA-tris-glutaric acid) residues represented by The isomeric mixture of Gd(PCTA-tris-glutarate) residues comprises at least 50% of the enantiomeric residue of formula (IIIa), the enantiomeric residue of formula (III B), or a mixture thereof; -NR １ R ２ is as defined in claim 5. A mixture of isomers of amide derivatives of Gd(PCTA-tris-glutaric acid) having the formula:

7. 7. The isomeric mixture of claim 6, wherein in formula (II B), F' is an isomeric mixture of residues of formula III comprising at least 50% of a mixture of RRR and SSS enantiomeric residues.

8. In formula (II A) or (II B), R １ is H, and R ２ C substituted with one or two hydroxyl groups １ -C ３ 8. The amide derivative according to claim 5 or the isomeric mixture according to claim 6 or 7, wherein the aryl group is alkyl.

9. In formula (II A) or (II B), R １ is H, and R ２ Ga-CH ２ CH(OH)CH ２ 9. The amide derivative or isomer mixture according to claim 8, wherein R is 0-OH.

10. The amide derivative according to claim 5, selected from the group consisting of: [(αS,α'S,α''S)-α,α',α''-tris[3-[(2(S),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (isomers SSS-SSS); [(αS,α'S,α''S)-α,α',α''-tris[3-[(2(R),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (isomer SSS-RRR); [(αR,α'R,α''R)-α,α',α''-tris[3-[(2(R),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (isomers RRR-RRR); [(αR,α'R,α''R)-α,α',α''-tris[3-[(2(S),3-dihydroxypropyl)amino]-oxopropyl]-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (isomer RRR-SSS); and mixtures thereof.

11. The isomeric mixture according to any one of claims 7 to 9, wherein in formula (II B), F' comprises at least 60% of a mixture of RRR and SSS enantiomeric residues.

12. A pharmaceutical composition comprising a compound or isomeric mixture according to any one of claims 1 to 4, or an amide derivative or isomeric mixture according to any one of claims 5 to 10, in admixture with one or more pharma- ceutically acceptable carriers, diluents or excipients.

13. 13. The pharmaceutical composition according to claim 12, comprising an isomeric mixture of the amide derivative of formula (II B) according to claim 6.

14. The pharmaceutical composition according to claim 13, wherein in formula (II B), F' is as defined in claim 7.

15. In formula (II B), R １ is H, and R ２ Ga-CH ２ CH(OH)CH ２ The pharmaceutical composition of claim 14, wherein said compound is OH.

16. In formula (II A), R １ is H, and R ２ Ga-CH ２ CH(OH)CH ２ 6. The amide derivative of claim 5, wherein F is OH and F is a mixture of RRR and SSS enantiomeric residues.

17. formula 【Chemistry 9】 17. A pharmaceutical composition comprising a compound represented by the formula: wherein at least 50% of the compound is an amide derivative according to claim 16.

18. 10. A pharma- ceutically acceptable salt of a compound according to claim 1, or an RRR / SSS mixture according to claim 2 or 3, or an amide derivative according to claim 5, or an isomeric mixture according to claim 6, for use as an MRI contrast agent.

19. The isomeric mixture according to any one of claims 7 to 9, wherein in formula (II B), F' comprises at least 70% of a mixture of RRR and SSS enantiomeric residues.

20. The isomeric mixture according to any one of claims 7 to 9, wherein in formula (II B), F' comprises at least 80% of a mixture of RRR and SSS enantiomeric residues.

21. 10. The isomeric mixture according to any one of claims 7 to 9, wherein in formula (II B), F' comprises at least 90% of a mixture of RRR and SSS enantiomeric residues.

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