PCTA-based contrast agent

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 invention This invention generally relates to the field of magnetic resonance imaging (MRI). More specifically, it relates to isomers of PCTA-based contrast agents and MRI contrast agents rich in these isomers. [Background technology]

[0002] A typical example of an MRI contrast agent used in routine diagnostic work is a gadolinium complex compound, characterized by a high stability constant that ensures the in vivo release of free metal ions (which are known to be highly toxic to living organisms).

[0003] Another important parameter defining the tolerability of gadolinium-based contrast agents is the kinetic inertness (or kinetic stability) of the Gd(III) complex, which is the half-life (t) of the complex's dissociation (i.e., decomposition). 1 / 2 This is estimated from the following.

[0004] In particular, high inertness is important in complex compounds with low thermodynamic stability and / or long retention times before elimination, in order to avoid or minimize possible decomplexation or metal exchange reactions.

[0005] EP1931673 (Guerbet) contains the following formula [ka] The PCTA derivatives shown and the synthetic routes for their production are disclosed.

[0006] EP 2988756 (same applicant) discloses a pharmaceutical composition comprising the above derivative together with a calcium complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. According to EP2988756, this calcium complex compensates for the weak thermodynamic stability observed in PCTA-based gadolinium complexes by forming a strong complex with free lanthanide ions through 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, which are preferentially selected from RRS, RSR, and RSS diastereomers.

[0008] In all of the above patents, among specific 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 recently the following formula [ka] It is identified as the gadolinium chelate (CAS registry number: 933983-75-6) of 2,2',2''-(3,6,9-triaza-1(2,6)-pyridinacyclodecafane-3,6,9-triyl)tris(5-((2,3-dihydroxypropyl)amino)-5-oxopentanoic acid), or as P03277 or gadopicrenol.

[0009] Regarding gadopicrenol, EP1931673 has a relaxing activity of 11 mM. -1 s -1 Gd -1 It is stated that the conditions are (in water, 0.5 T, 37°C), and EP2988756 has a thermodynamic equilibrium constant of 10 -14.9(log K term = 14.9) is described.

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

[0011] The precursor for the preparation of the PCTA derivatives (including gadopiclenol) disclosed in EP1931673 is a Gd complex of 3,6,9,15 - tetraazabicyclo - [9.3.1] pentadeca - 1(15),11,13 - triene - tri(α - glutamic acid) having the following formula [Chemical formula] and is identified herein as "Gd(PCTA - tris - glutamic acid)". In particular, gadopiclenol is obtained by amidating the above compound with isocerinol.

[0012] As the applicant has observed, Gd(PCTA - tris - glutamic acid) has three stereocenters in the glutamic acid moiety (indicated by an asterisk (*) in the above structure) that give rise to 2 3 = 8 possible stereoisomers. More specifically, the above structure can give rise to the 4 pairs of enantiomers shown in Table 1 below. [Table 1]

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

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

[0015] Regarding gadopicrenol, 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 isocerinol portions attached to it (indicated by asterisks (*) and hollow circles (〇) in the structural formula below). [ka]

[0016] Therefore, the theoretical total number of stereoisomers of this compound is 2 6 = 64. [Overview of the project] [Problems that the invention aims to solve]

[0017] However, neither EP1931673 nor EP2988756 provides a detailed description of the composition of the isomer mixture obtained according to the described synthetic route, nor does it teach the separation and characterization of these isomers, nor does it disclose the stereospecific synthesis of gadopicrenol. [Means for solving the problem]

[0018] The applicant has discovered that certain isomers of the above-mentioned precursor Gd(PCTA-tris-glutaric acid) and its derivatives (particularly gadopicrenol) possess improved physicochemical properties, particularly with respect to relaxability and kinetic inertness.

[0019] One embodiment of the present invention relates to a compound selected from the group consisting of: Equation (Ia): [ka] 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] 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), and Mixtures of such RRR and SSS enantiomers, and their pharmaceutically acceptable salts.

[0020] Another embodiment of the present invention is at least 50% of the RRR isomer represented by 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 represented by formula (Ib) [( This relates to a mixture of isomers of Gd(PCTA-tris-glutaric acid) 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 mixtures thereof, or pharmaceutically acceptable salts thereof.

[0021] Another aspect of the present invention relates to an amide obtained by bonding one of the above-mentioned compounds or isomer mixtures with an amino group, for example, preferably selinol or isocerinol.

[0022] One embodiment of the present invention is given by formula (II A): F(NR1R2)3(II A) [In the formula, F is: Formula IIIa [ka] RRR enantiomer residues indicated by Formula IIIb [ka] The SSS enantiomer residues indicated by, or These are mixtures of RRR and SSS enantiomer residues; Each of the three -NR1R2 groups is bonded to the open bond of the carboxyl portion of F, indicated by the black circle (●) in the above structural formula; R1 is a C1-C6 alkyl group substituted with H or, in some cases, 1-4 hydroxyl groups; R2 is a C1-C6 alkyl group substituted with 1 to 4 hydroxyl groups, preferably a C1-C3 alkyl group substituted with 1 or 2 hydroxyl groups. is This relates to the amide derivative shown.

[0023] One embodiment of the present invention is given by formula (II B): F'(NR1R2)3(II B) [In the formula, F' is: Formula (III) [ka] It is a mixture of isomers of the Gd(PCTA-tris-glutaric acid) residue shown by, The isomer mixture of the Gd(PCTA-tris-glutaric acid) residue comprises at least 50% of the enantiomer residue of formula (IIIa), the enantiomer residue represented by formula (III B), or a mixture thereof; -The NR1R2 group is bonded to the open bond of the carboxyl portion of F', indicated by the black circle (●) in the above structural formula, as defined above for the compound shown in formula (II A). This invention relates to an isomer mixture of amide derivatives of Gd(PCTA-tris-glutaric acid) having the following properties.

[0024] Further aspects of the present invention relate to Gd(PCTA-tris-glutaric acid), or preferably a mixture thereof of RRR / SSS, or a mixture of isomers of Gd(PCTA-tris-glutaric acid) comprising at least 50% of either an enantiomer or a mixture of RRR / SSS enantiomers thereof, or a pharmaceutically acceptable salt of an amide derivative represented by formula (IIA) or (IIB) above, particularly suitable for use as an MRI contrast agent and for imaging of organs or tissues of the human or animal body using MRI technology.

[0025] Further embodiments relate to pharmaceutically acceptable compositions comprising, in combination with one or more physiologically acceptable carriers or excipients, at least one compound or isomer mixture of the present invention, or a pharmaceutically acceptable salt or amide derivative thereof as defined above.

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

[0027] One embodiment of the present invention is a method for synthesizing an amide derivative represented by formula (II A): F(NR1R2)3(II A) [In the formula, F, R1, and R2 are as described above and include the following: a) Obtain RRR or SSS isomers of the Gd(PCTA-tris-glutaric acid) complex, or mixtures thereof; and b) Convert the isomer or isomer mixture obtained in step a) into its amide derivative; Furthermore A method for preparing an isomer mixture of the amide derivative represented by formula (II B) above, including the following: a') Obtain a mixture of isomers of Gd(PCTA-tris-glutaric acid) containing at least 50% enantiomer RRR or SSS, or a mixture thereof; b') Convert the isomer mixture of Gd(PCTA-tris-glutaric acid) obtained in step a') into the corresponding isomer mixture of each amide derivative. Regarding. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 shows the HPLC chromatogram of Gd(PCTA-tris-glutaric acid) recovered as an isomer mixture from Example 1, which was carried out according to the synthesis procedure disclosed in the prior art ([GdL]=0.2mM, 25℃). [Figure 2]The HPLC chromatogram of the RRR / SSS enantiomer pair C of Gd(PCTA-tris-glutaric acid) obtained from Example 3 is shown. [Figure 3] The MS spectra of the main peaks in Figure 2 are shown. The m / z ratio Gd(H4L)+ is 752.14 m / z. [Figure 4] HPLC chromatograms are shown: a) Isomer mixture of Gd (PCTA-tris-glutaric acid) 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). [Figure 5] Chiral HPLC chromatograms are shown: a) enantiomer pair C (compound VI from Example 3), b) RRR enantiomer (compound XII from Example 5), and c) SSS enantiomer (compound XVII from Example 6) of Gd(PCTA-tris-glutaric acid). [Figure 6] The following are HPLC chromatograms of amide derivatives obtained by the reaction of Gd(PCTA-tris-glutaric acid) with isocerinol. a): Amide derivative obtained as an isomer mixture from Example 2 (for convenience, the four main peaks are shown as A', B', C', and D'). b): Amide derivative obtained by the reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with R-isocerinol; c): Amide derivative obtained by the reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with S-isocerinol; d): Amide derivative obtained by the reaction of RRR / SSS Gd(PCTA-tris-glutaric acid) with racemic isocerinol. [Figure 7] Referring to the test in Example 7, the changes in HPLC area values ​​for peaks A(◇), B(□), C(△), and D(〇) over time are shown ([GdL] = 0.2 mM, [HCl] = 1.0 M, 25℃). [Figure 8]Referring to the test in Example 8, the change in HPLC area values ​​over time is shown: Isomer mixture (◇); RRR / SSS Gd(PCTA-tris-glutaric acid) + R isoselinol (□); RRR / SSS Gd(PCTA-tris-glutaric acid) + S isoselinol (△); RRR / SSS Gd(PCTA-tris-glutaric acid) + racemic isoselinol (〇) total area. ([GdL] = 0.2 mM, [HCl] = 1.0 M, 25℃). [Figure 9] The X-ray structure of the single crystal of the ternary complex of Gd(PCTA-tris-glutaric acid)-oxalate with guanidine counterion, represented by the formula {(C(NH2)3)2[Gd(PCTA-tris-glutaric acid)(C2O4)]}·1H2O, is shown (showing the chirality RRR of the (indicated) chiral carbon atom of the glutaric acid group). [Figure 10] The unit cell of the crystal shown in Figure 9 contains the 2RRR + 2SSS complex. [Figure 11] The X-ray structure of the single crystal obtained from the ternary complex formed between the carbonate anion and the amide compound D' by the coupling reaction of RRR / SSS Gd (PCTA-tris-glutaric acid) and racemic isocerinol, and the statistical analysis of the recovered crystal are shown. [Modes for carrying out the invention]

[0029] Detailed description of the present invention Gd(PCTA-tris-glutaric acid) can be obtained as a mixture of isomers (referred herein as a mixture of isomers of "Gd(PCTA-tris-glutaric acid)") by the synthetic method disclosed in the prior art (see US6,440,956, cited by EP1931673), which can be confirmed as several peaks by HPLC.

[0030] Using preparative HPLC, it became possible to separate four peaks with the same m / z ratio (Gd(H4L)+: 752.14 m / z) from the mixture.

[0031] A representative chromatogram of the separated isomer mixture is shown in Figure 1. Here, each peak is denoted by the letters A, B, C, and D for convenience, and is reasonably attributed to one of the enantiomer pairs shown above. More precisely, each peak is associated with an enantiomer pair, characterized by the same m / z ratio in the MS spectrum, which is indistinguishable by conventional reversed-phase HPLC.

[0032] Surprisingly, the enantiomer pair associated with peak C in the HPLC chromatogram (or, hereafter referred to interchangeably as enantiomer pair C) was found to exhibit optimal characteristics, particularly in terms of reducing kinetic inactivity and the tendency to release Gd.

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

[0034] Furthermore, the relaxation values ​​associated with the enantiomer pair related to peak C are significantly higher than those reported in 1931673B1 for an isomer mixture of Gd(PCTA-tris-glutaric acid) tested under the same conditions.

[0035] This enantiomer pair C is the RRR isomer of [(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., Gd(PCTA-tris-glutaric acid) represented by formula (Ia). [ka] and each of 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, i.e., Gd(PCTA-tris-glutaric acid) represented by formula (Ib) [ka] It was shown that it includes [something].

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

[0037] For example, coupling reactions of RRR / SSS Gd(PCTA-tris-glutaric acid), and similarly its individual RRR or SSS enantiomers, with isocerinol ultimately yield amide derivatives having the same molecular formula as gadopicrenol. In this regard, it is interesting that regardless of the type of isocerinol used, whether it is an R or S isomer or a racemic isocerinol, conjugation with the RRR / SSS enantiomer of Gd(PCTA-tris-glutaric acid) yields each amide derivative with the same retention time (and therefore indistinguishable by normal reversed-phase HPLC).

[0038] Thus, the different isomers (or more commonly, amine derivatives) of the added isocerinol do not affect the key properties of the final conjugate compound, which are essentially determined by the stereochemistry of the Gd(PCTA-tris-glutaric acid) precursor.

[0039] In fact, the improved properties shown by the enantiomer pair associated with peak C for the isomer mixture of Gd(PCTA-tris-glutaric acid) are substantially maintained after conjugation with isoselinol, regardless of the conformation of the conjugated isoselinol.

[0040] In particular, regardless of the stereochemistry of isocerinol, coupling it with the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutaric acid) yields an amide compound with greater kinetic inertness and relaxation ability compared to gadopicrenol obtained as an isomer mixture by conventional synthesis procedures.

[0041] In this specification, and unless otherwise specified, the expression “isomer mixture” (with respect to a particular compound) includes, in 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 “isomer mixture” refers to a mixture in which at least two of the eight diastereomers (or diastereoisomers as also referred herein) are not separated, more precisely, to the four enantiomer pairs shown in Table 1, resulting from the three stereocenters contained within the molecule. On the other hand, when used with respect to the amide derivatives of Gd(PCTA-tris-glutaric acid) (e.g., gadopicrenol), the expression “isomer mixture” refers to the above (possibly four) undefined, unseparated mixture of at least two amide derivatives of each enantiomer pair of the Gd(PCTA-tris-glutaric acid) residue.

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

[0043] In this specification and in the claims, the expressions "isomer mixture of Gd(PCTA-tris-glutaric acid) amide derivatives" and "amid derivative of Gd(PCTA-tris-glutaric acid) amide derivatives" are interchangeable.

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

[0045] The expression “RRR / SSS enantiomer pair” (or “RRR / SSS enantiomer” generally refers to a mixture of the enantiomer RRR of the compound in question and its respective enantiomer SSS, including racemic mixtures thereof. In this specification, this expression is typically used in reference to Gd(PCTA-tris-glutaric acid) and refers to a mixture of the enantiomers RRR and SSS of this compound (or its RRR / SSS mixture). More specifically, “RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid)” (or “RRR / SSS” as used interchangeably herein). The expression "Gd(PCTA-tris-glutaric acid)") is, for example, shown below: [(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 [(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). [ka]

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

[0047] The term “stereoselective synthesis” (or “asymmetric synthesis” as interchangeably used herein) encompasses a chemical reaction (or reaction sequence) in which one or more new elements of chirality are formed within a substrate molecule, resulting in unequal amounts of stereoisomers (enantiomers or diastereomers). In this specification, the term “stereoselective synthesis” is used particularly with respect to the RRR of Gd(PCTA-tris-glutaric acid) and its respective enantiomer SSS, and refers to a synthesis that enables obtaining a complex containing 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 salt” refers to a derivative of the compound of the present invention, which is appropriately modified by converting the parent compound, if a free acidic or basic group is present, into a corresponding addition salt with any base or acid that is conventionally intended to be pharmaceutically acceptable.

[0049] Preferred inorganic base cations that can be appropriately used to prepare the salts of the present invention include, for example, alkali or alkaline earth metal ions such as potassium, sodium, calcium, or magnesium.

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

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

[0052] Furthermore, the terms “part” and “residue” are intended to define the remaining portion of a molecule after it has been properly bound or conjugated to the rest of the molecule, either directly or via any appropriate linker.

[0053] For example, when used in reference to amide derivatives of Gd(PCTA-tris-glutaric acid) (either as an isomer mixture of RRR or SSS isomers, or as an RRR / SSS enantiomer mixture or enantiomer pair), the term "residue" refers to the portion of Gd(PCTA-tris-glutaric acid) that is bonded to the amine group in order to produce the corresponding amide derivative.

[0054] In particular, the term "residues of a mixture of isomers of Gd(PCTA-tris-glutaric acid)" refers to compounds having the following formula (III). [ka]

[0055] This residue can conjugate, for example, to the amino residue shown in formula -NR1R2 via an open bond in the carboxyl portion indicated by the black circle (●) in the above structural formula, to produce the corresponding amide derivative shown in the following formula. [ka]

[0056] Similarly, the terms "residues of the RRR and SSS enantiomers of Gd(PCTA-tris-glutaric acid)" are given by the following formula (IIIA), respectively. [ka] and equation (IIIb) [ka] This refers to compounds that possess [a certain characteristic].

[0057] The term "residue" also applies to the corresponding residues of an RRR / SSS enantiomer pair, or more generally, an enantiomer mixture.

[0058] A preparative HPLC method was needed to separate four peaks (referred to as A, B, C, and D for convenience) with the same m / z ratio (Gd(H4L)+: 752.14 m / z) of Gd(PCTA-tris-glutaric acid) obtained by non-stereoselective synthesis disclosed in the prior art. By taking into account the three stereocenters present in the molecule (indicated by asterisks in the molecular structure above), the four signals in the HPLC chromatogram of the Gd(PCTA-tris-glutaric acid) complex were assigned to four enantiomer pairs formed by optical isomers with different glutaric acid residues, as shown in Table 1 above.

[0059] To investigate the kinetic inertness of a racemic mixture of Gd(PCTA-tris-glutaric acid), particularly its four enantiomer pairs separated by HPLC, their dissociation reactions under acidic conditions were examined. To ensure pseudo-first-order kinetic conditions, a large excess of H was added. + We particularly utilized (HCl = 1.0 M).

number

[0060] A solution of Gd(PCTA-tris-glutaric acid) (isomer mixture) in 1M HCl was prepared and analyzed over time as described in Example 7.

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

[0062] As expected, the acid-catalyzed dissociation of the complex led to a decrease in the integrated areas of peaks A, B, C, and D, while new signals were formed and increased in response to the free ligand (m / z: 597.24). Interestingly, however, the rates of decrease in the areas of signals A, B, C, and D were not equal (for example, the decrease in the areas of peaks A and B was significantly faster than the decrease in the areas of peaks C and D).

[0063] Therefore, we evaluated the decrease in the integral area values ​​of signals A, B, C, and D and plotted them as a function of time. The obtained results are shown graphically in Figure 7 (the observed differences present among the behavior of the four peaks are highlighted).

[0064] Dissociation rate and half-life (t) of different enantiomer pairs of Gd(PCTA-tris-glutaric acid) complexes 1 / 2 = ln2 / k X k characterizing ) X Pseudo-first-order velocity constant (where k X Each of these is =k A , k B , k C and k D The mean half-life of the isomer mixture of Gd(PCTA-tris-glutaric acid) was calculated by fitting area-time data pairs, as described in detail in Example 7. Furthermore, the mean half-life of the isomer mixture of Gd(PCTA-tris-glutaric acid) was also obtained by considering the percentage composition of the mixture. The results obtained are summarized in Table 2, and several reference contrast agents (e.g., Gd-DOTA(Dotarem)) were used. TM Compare the values ​​of ) and Eu(PCTA) (a europium complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) with the corresponding values ​​referenced in the literature.

[0065] These results confirm that the dissociation rates of the four enantiomer pairs differ considerably from one another.

[0066] In particular, the enantiomer pair associated with peak C surprisingly exhibits the highest kinetic reactivity and the lowest tendency to release Gd among all other possible isomers.

[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 of the enantiomer pair associated with peak C 1 / 2 The value is significantly higher than the value measured for Eu(PCTA) with q=2 (see, for example, Tircso, G. et al. Inorg Chem 2006, 45(23), 9269-80), for example, Gd-DOTA (Dotarem), a commercially available contrast agent with the highest stability and inertness. TM Regarding t 1 / 2 It is exactly equivalent to the value.

[0068] Next, the fraction enriched with this compound was recovered by flash chromatography as described in detail in Example 3, and the enantiomer pair associated with peak C was obtained with a purity of at least approximately 90% (as HPLC area %), see Figure 2.

[0069] Surprisingly, the relaxation value obtained for the recovered enantiomeric pair was r1 = 9.3 ± 0.1 mM -1 s -1 This was significantly higher than r1 = 7.2 recorded for the isomer mixture of Gd(PCTA-tris-glutaric acid) in EP1931673B1 (under the same conditions).

[0070] The unexpected combination of high relaxation ability and higher inertness (resulting in greater tolerability) demonstrated by this enantiomeric pair is particularly interesting.

[0071] Thus, we expended our efforts to identify the set of enantiomers associated with peak C.

[0072] In particular, the stereoselective synthesis of the RRR and SSS isomers of Gd(PCTA-tris-glutaric acid) described in Examples 5 and 6, respectively, was established, and the same HPLC retention time t of peak C in normal reversed-phase HPLC was achieved. r Crude products containing the main compound were obtained. By using the related isomer (R)-(-)-5-oxotetrahydrofuran-2-carboxylic acid as an important intermediate, the corresponding SSS isomer of Gd(PCTA-tris-glutaric acid) with the same HPLC retention time was also obtained (Figure 4).

[0073] Alternatively, the synthesis of Gd(PCTA-tris-glutaric acid) using methyl(2S)-bromoglutarate can yield the complex as an isomer mixture that is substantially indistinguishable from that recovered together with the racemic methylbromoglutarate disclosed in the prior art.

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

[0075] Furthermore, the crystal was formed from the enantiomer pair C and 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 of the glutaric acid arms of the molecule, the presence of equimolar ratios of RRR and SSS isomers within each unit cell of the crystal, and the RRR / SSS racemic nature of this pair (Figure 10).

[0076] These results are all consistent, demonstrating that the compound associated with peak C actually consists of an RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid) (RRR / SSS Gd(PCTA-tris-glutaric acid), as used interchangeably herein).

[0077] More specifically, from the above results, the compound corresponding to peak C identified by the present invention is given 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-glutaric acid)) and the following formula [ka] A mixture of each enantiomer represented by [(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)) (or the mixture is represented by the following formula Ic [ka] (represented by) It could be defined as including.

[0078] Accordingly, one aspect of the present invention is the RRR / SSS pair of enantiomers of Gd(PCTA-tris-glutaric acid), single enantiomers of this pair, mixtures thereof, pharmaceutically acceptable salts thereof, amide derivatives thereof, and compositions comprising them.

[0079] In particular, one embodiment of the present invention relates to a compound preferably selected from the group consisting of: an individual 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); 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); RRR / SSS enantiomer pair; and their pharmaceutically acceptable salts.

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

[0081] Another aspect of the present invention relates to Gd(PCTA-tris-glutaric acid) or mixtures thereof that are rich in any of the above enantiomers.

[0082] The expression “enriched” as used with respect to the isomers, enantiomers, or enantiomer pairs of the present invention (in particular when referring to Gd(PCTA-tris-glutaric acid) or its amide derivatives) includes, in its meaning, mixtures of isomers in which such isomers, enantiomers, or enantiomer pairs are present in amounts higher than those typically found in mixtures obtained by non-stereoselective synthetic procedures of the prior art.

[0083] Such enrichment (with respect to isomers or enantiomer pairs of Gd(PCTA-tris-glutaric acid)) corresponds, for example, to at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, for example at least 90% of such isomers or enantiomer pairs in the mixture.

[0084] In particular, another aspect of the present invention is a mixture of Gd(PCTA-tris-glutaric acid) isomers (i.e., at least 50% of the Gd(PCTA-tris-glutaric acid) complex is 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 The terms relating to -triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium; or the SSS isomer [(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 is represented by an indiscriminate mixture of other possible isomers).

[0085] Therefore, one embodiment of the present invention is Gd(PCTA-tris-glutaric acid) or a salt thereof, wherein each of these enantiomers, enantiomer RRR or enantiomer SSS, or a mixture of these enantiomers, is composed of at least 50% (e.g., in moles) of the isomer mixture of the acid or salt.

[0086] Preferably, the enrichment of Gd(PCTA-tris-glutaric acid) (either the enantiomer or a mixture thereof) is at least 60%, more preferably at least 70%, most preferably at least 80%, for example, at least 90%.

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

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

[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-glutaric acid) enriched with this isomer, can be prepared by using a stereoselective synthesis comprising alkylating 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]pentanedioate dimethyl ester, for example, as described in detail in Example 5.

[0090] Similarly, by substituting (2R)-2-[(trifluoromethylsulfonyl)oxy]pentanedioate dimethyl ester (for example, as described in Example 6), it is possible to obtain Gd(PCTA-tris-glutaric acid) that is appropriately enriched with each SSS isomer or its child isomer.

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

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

[0093] More specifically, further embodiments of the present invention relate to pharmaceutically acceptable salts of the compound, preferably RRR / SSS enantiomer mixtures, for use as contrast agents, particularly suitable for magnetic resonance imaging (MRI) analysis, selected from the individual enantiomers RRR, SSS, or RRR / SSS enantiomer pairs, or Gd(PCTA-tris-glutaric acid) enriched by at least 50% of any of these individual enantiomers.

[0094] Suitable examples of pharmaceutically acceptable salts include salts with cations of inorganic bases selected from alkali or alkaline earth metals such as potassium, sodium, calcium, or magnesium; cations of organic bases selected from ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, or N,N-dimethylglucamine; or cations of amino acids selected from lysine, arginine, ornithine.

[0095] In another aspect, the present invention relates to a conjugate of one of the above-mentioned compounds or isomer mixtures with an amine preferably represented by the formula NHR1R2.

[0096] One embodiment of the present invention is given by formula (II A): F(NR1R2)3(II A) This relates to amide derivatives of the RRR enantiomer, SSS enantiomer, or mixture of two such enantiomers of Gd(PCTA-tris-glutaric acid), as shown by [formula].

[0097] Another embodiment of the present invention is formula (II B): F'(NR1R2)3(II B) This relates to an isomer mixture of amide derivatives of Gd(PCTA-tris-glutaric acid) containing at least 50% of the individual enantiomer RRR, or enantiomer SSS, or a mixture thereof.

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

[0099] Preferred examples include amide derivatives represented by formula (II A) above, where F is a mixture of RRR and SSS enantiomer residues of Gd(PCTA-tris-glutaric acid) (or a pair of RRR / SSS enantiomer residues), or amide derivatives represented by formula (II B), where F' is a mixture of isomers of Gd(PCTA-tris-glutaric acid) residues containing at least 50% of the mixture of RRR and SSS enantiomer residues.

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

[0101] Preferably, in these amide derivatives, F' is enriched with at least 60% (i.e., contains at least 60%), more preferably at least 70%, most preferably at least 80%, and especially preferably at least 90%, a mixture of RRR and SSS enantiomer residues. A suitable example is the amide derivative represented by formula (II B) above, where R1 is H and R2 is a 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 isomer mixture of an amide derivative represented by formula (II B) above, wherein 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 selinol residue, or even more preferably, an isoselinol residue selected from, for example, R isoselinol, S isoselinol, or racemic isoselinol. Most preferably, the amide compound has racemic isoselinol.

[0103] For example, non-limiting and representative examples of the above compounds include the following: The following formula: [ka] [(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 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 isomer RRR-RRR); The following formula: [ka] [(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 isomer RRR-SSS); The following formula: [ka] [(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 isomer SSS-RRR); Alternatively, the isomers in which the isocerinol portion has the stereoconfiguration RSR, SSR, SRS, RSS, or RRS.

[0104] The amide represented by formula (II B) having isocerinol has the same molecular formula as gadopicrenol, but contains a central portion F' enriched with at least 50% RRR isomer or SSS isomer residues of Gd(PCTA-tris-glutaric acid), or more preferably a mixture of RRR and SSS enantiomer residues.

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

[0106] Remarkably, the improved properties of RRR / SSS Gd(PCTA-tris-glutaric acid) are substantially maintained after conjugation with isoselinol, regardless of the stereochemistry of the conjugated isoselinol.

[0107] In particular, the conjugation of isocerinol with RRR / SSS Gd (PCTA-tris-glutaric acid) yields an amide derivative that has the same molecular formula but exhibits greater kinetic inertness and relaxation ability than gadopicrenol, which is obtained as an isomer mixture by conventional synthesis procedures.

[0108] In fact, the same tests performed to evaluate the kinetic inertness of four different enantiomer pairs isolated from the isomer mixture of Gd(PCTA-tris-glutaric acid) were repeated using the gadopicrenol (isomer mixture) obtained in Example 2 and amide derivatives obtained by conjugation of RRR / SSS Gd(PCTA-tris-glutaric acid) with i) R-isocerinol, ii) S-isocerinol, and iii) racemic isocerinol, respectively.

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

[0110] The results obtained are summarized in Table 3, and several reference contrast agents (e.g., Gd-DOTA (Dotarem)) are used. TMCompare the values ​​for ) and Eu(PCTA)) with the corresponding values ​​referenced in the literature.

[0111] The data on one side of Table 3 is estimated from the area values ​​of the complexes obtained by coupling RRR / SSS Gd (PCTA-tris-glutaric acid) with R, S, and racemic isocerinol. 1 / 2 The value and t calculated by fitting area-time dynamic data 1 / 2 It shows a very good agreement with the value.

[0112] On the other hand, the data in Table 3 shows that all t1 / 2 values ​​of the amide compounds obtained by the reaction of RRR / SSS Gd (PCTA-tris-glutaric acid) with i) R-isocerinol, ii) S-isocerinol, and iii) racemic isocerinol were approximately 8 times that of gadopicrenol (isomer mixture), confirming that the high kinetic inertness exhibited by RRR / SSS Gd (PCTA-tris-glutaric acid) is substantially maintained even after coupling with isocerinol.

[0113] The overall consistency of half-life values ​​obtained for different complex compounds resulting from the conjugation of RRR / SSS Gd(PCTA-tris-glutaric acid) with R, S, or racemic isocerinol indicates that the chirality of the isocerinol pendant does not affect the kinetic inertness of the final complex. Furthermore, the r1 relaxation ability of compounds obtained from the conjugation of RRR / SSS Gd(PCTA-tris-glutaric acid) with R, S, and racemic isocerinol was measured under the same conditions used for gadopicrenol in the literature.

[0114] The results obtained are compared in Table 5. Here again, regardless of the stereochemistry of the added isocerinol, the r1 relaxation ability of the conjugation compound obtained from RRR / SSS Gd(PCTA-tris-glutaric acid), measured in both water and HSA, is higher than that reported for gadopicrenol in the relevant literature.

[0115] Thus, the amide derivatives are obtained by the conjugation of RRR / SSS Gd(PCTA-tris-glutaric acid) and isocerinol according to the present invention, and are characterized by improved kinetic inertness and higher relaxation ability despite having the same structure as the gadopicrenol compound.

[0116] Next, as described in detail in Example 10, crystals were obtained from the amide derivative resulting from the conjugation of RRR / SSS Gd (PCTA-tris-glutaric acid) and racemic isocerinol. X-ray diffraction analysis of the single crystal obtained from the ternary complex formed between the carbonate anion and the amide derivative confirmed that the glutaric acid arm of the core molecule C was in an RRR / SSS configuration.

[0117] The X-ray structures and statistical analyses of all recovered crystals are shown in Figure 11.

[0118] The stereoselective synthesis results of the individual RRR or SSS isomers, as well as the (crystalline) structures recorded for both the enantiomer pair C and its conjugate with isocerinol, all match each other, proving that the compound at peak C in the HPLC in Figure 1 corresponds, in fact, to the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid).

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

[0120] In particular, other embodiments of the present invention are: a) The following formula [ka] To obtain (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioate dimethyl ester represented by; and b) The recovered (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioate dimethyl ester is given by the following formula: [ka] Alkylation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene as shown by This invention relates to a stereoselective method for preparing Gd(PCTA-tris-glutaric acid) 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), which comprises the above.

[0121] In one embodiment, this method can produce Gd(PCTA-tris-glutaric acid) enriched with at least 55%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%, for example about 85%, of the desired RRR isomer of the complex.

[0122] Similarly, by using (2R)-2-[(trifluoromethylsulfonyl)oxy]pentanedioate dimethyl ester instead in step a) of this manufacturing method, each SSS isomer of Gd(PCTA-tris-glutaric acid), or Gd(PCTA-tris-glutaric acid) in which this isomer is appropriately enriched, can be obtained.

[0123] In yet another embodiment, the present invention preferably relates to an RRR / SSS pair of the enantiomer of Gd(PCTA-tris-glutaric acid) or Gd(PCTA-tris-glutaric acid) enriched with at least 50% of this enantiomer pair, for use as an intermediate in the preparation of its derivatives, such as amide derivatives.

[0124] Another embodiment of the present invention is: a) Obtaining the RRR or SSS isomer of Gd(PCTA-tris-glutaric acid), or mixtures thereof; and b) Converting the isomer or mixture of isomers obtained in step a) into the desired amide derivative. Formula (II A) includes F(NR1R2)3(II A) [In the formula, F, R1, and R2 are as described above.] This invention relates to a method for producing amide derivatives of Gd(PCTA-tris-glutaric acid) represented by the formula shown.

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

[0126] On the other hand, step b) of this manufacturing method can be carried out, for example, by following the conventional procedure reported in the prior art cited above.

[0127] Further aspects of the present invention include: a') To obtain an isomer mixture of Gd(PCTA-tris-glutaric acid) containing at least 50% of the individual enantiomers RRR or SSS, or preferably a mixture thereof; and b') Convert the isomer mixture of Gd(PCTA-tris-glutaric acid) obtained in step a') into the corresponding isomer mixture of the desired amide derivative. The above formula (II B) includes F(NR1R2)3(II B) [In the formula, F', R1, and R2 are as described above.] This invention relates to a method for the synthesis and preparation of isomer mixtures of amide derivatives of Gd(PCTA-tris-glutaric acid) represented by [formula].

[0128] For example, step a' of this production method, which involves obtaining an isomer mixture of Gd(PCTA-tris-glutaric acid) containing at least 50% RRR / SSS enantiomer pairs of Gd(PCTA-tris-glutaric acid), can be obtained by chromatography, including preparative HPLC or flash chromatography, using Gd(PCTA-tris-glutaric acid) obtained as an isomer mixture by known procedures as a starting material, as disclosed in Example 3, for example.

[0129] On the other hand, step b') of the present manufacturing method, which includes coupling the enriched isomer mixture of Gd(PCTA-tris-glutaric acid) recovered from step a') with the amine of the target, can be carried out, for example, according to the conventional procedure reported in the prior art cited above.

[0130] For example, the product recovered from step a') can be reacted with isocerinol by using the synthesis procedure described in detail in Example 4, for example.

[0131] Further embodiments of the present invention relate to an amide represented by formula (II A) or (II B) above, for use as a contrast agent, particularly suitable for magnetic resonance imaging (MRI) analysis.

[0132] In particular, in another embodiment, the present invention relates to compounds selected from the group consisting of individual RRR or SSS enantiomers of Gd(PCTA-tris-glutaric acid), mixtures of such RRR / SSS enantiomers, mixtures or mixtures of isomers of Gd(PCTA-tris-glutaric acid) enriched by 50% of either the individual RRR or SSS enantiomer, pharmaceutically acceptable salts thereof, and amide derivatives thereof represented by formula (IIA) or (IIB), for the preparation of pharmaceutical formulations for use in vivo or in vitro, or ex vivo, imaging diagnostics of organs, tissues or regions of the human or animal body, and biological samples including cells, biological fluids and biological tissues obtained from living mammalian patients (preferably human patients) using MRI technology.

[0133] Further aspects of the present invention relate to a pharmaceutical composition for diagnostic purposes comprising, in combination with one or more physiologically acceptable excipients, diluents, or solvents, at least one of the above-described isomer compounds or isomer mixtures of the present invention, or a pharmaceutically acceptable salt or amide derivative thereof.

[0134] Preferably, the pharmaceutical composition is an amide derivative represented by the above formula (II A). (In formula (II A), F is a residue of the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid); or, More preferably, an isomer mixture of the amide derivative represented by formula (II B) above. (In formula (II B), F' is enriched with at least 50% of the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid), and -NR1R2 is an isocerinol residue.) It consists of including.

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

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

[0137] Within this scope, and unless otherwise specified, the terms “effective amount” or “effective dose” as used herein mean any amount of Gd(PCTA-tris-glutaric acid) or any pharmaceutically acceptable salt thereof, or its amide derivative represented by formula (II A) or (II B), or any pharmaceutical composition thereof, sufficient for its intended diagnostic purpose: namely, for example, to visualize biological elements including cells, biological fluids, and biological tissues ex vivo, or to achieve in vivo imaging of organs, tissues, or regions of a patient’s body.

[0138] Unless otherwise specified, the terms “individual patient” or “patient” as used herein refer to a living human or animal patient, preferably a human being undergoing MRI diagnostic evaluation.

[0139] Details regarding dosage, dosage form, mode of administration, pharmaceutically acceptable carriers, excipients, diluents, and adjuvants are known in this field.

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

[0141] Experiment Part HPLC characterization of the obtained compounds General procedure Step 1: HPLC Characterization of Gd (PCTA-tris-glutaric acid) (Isomer mixture and individual / enriched isomers) The HPLC characterization of Gd(PCTA-tris-glutaric acid) obtained as an isomer mixture from Example 1 was performed using an Agilent 1260 Infinity II system. The experimental setup for the HPLC measurement is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, and PDA detector. Equipped with 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 compounds obtained by coupling gadopicrenol (isomer mixture) with enantiomer pair C with R, S, or racemic isocerinol. Gadopicrenol, as an isomer mixture obtained from Example 2, or as a compound obtained by communication of the enantiomer pair C of Gd(PCTA-tris-glutaric acid) with R, S, or racemic isocerinol, was HPLC characterized using a Thermo Finnigan LCQ DECA XPPlus system. The experimental setup for the HPLC measurement is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, and PDA detector. Equipped with HPLC (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 obtained HPLC chromatogram is shown in Figure 6.

[0143] Step 3: Chiral HPLC method for separating the enantiomer of compound C A specific chiral HPLC method was set up to separate the RRR and SSS enantiomers of enantiomer pair C (compound VI) prepared as described in Example 3. Enantiomer separation and characterization were performed using an Agilent 1200 system or a Waters Alliance 2695 system. The experimental setup for the HPLC measurement is summarized below. Analysis conditions HPLC system with quaternary pump, degasser, autosampler, and 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 minutes) Flow rate 1 mL / min Column temperature: 40°C Detection range: 210-270 nm. The obtained HPLC chromatogram is shown in Figure 5a). A comparison with the chromatograms of the pure RRR enantiomer (compound XII from Example 5, Tr. 7.5 min) and the pure SSS enantiomer (compound XVII from Example 6, Tr. 8.0 min) is shown in Figures 5b) and 5c), respectively.

[0144] Example 1: Synthesis of Gd(PCTA-tris-glutaric acid) (isomer mixture) Gd(PCTA-tris-glutaric acid) as an indiscriminate mixture of stereoisomers was prepared by following the synthetic scheme 1 below, using the procedure reported in the prior art described above. [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 2 M HBr (225 mL). The suspension was cooled to -5°C, and NaNO2 (28.0 g, 0.403 mol) was slowly added over 2.5 hours while maintaining the internal temperature below 0°C. The yellow mixture was stirred at -5°C for a further 20 minutes, after which concentrated sulfuric acid (29 mL) was added dropwise. The resulting dark brown mixture was warmed to room temperature and extracted with diethyl ether (4 x 150 mL). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated into a brown oil (21.2 g), which was used in the next step without further purification. The oil was dissolved in ethanol (240 mL), the resulting solution was cooled with ice, and thionyl chloride (14.5 mL, 0.199 mol) was slowly added. The slightly yellowish solution was stirred at room temperature for 2 days. Next, the solvent was removed under vacuum, and the crude oil was dissolved in dichloromethane (200 mL) and washed with 5% NaHCO3 (4 x 50 mL) aqueous solution, water (1 x 50 mL), and brine (1 x 50 mL). The organic phase was concentrated and purified by elution with petroleum ether-ethyl acetate 3:1 on silica to obtain 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 hours, after which the salt was filtered off and the organic solution was concentrated. The orange oil was dissolved in dichloromethane, and the product was extracted with 1 M HCl (4 x 50 mL). The aqueous phase was combined, cooled on ice, and the pH was adjusted to 7-8 with 30% NaOH aqueous solution. The product was then extracted with dichloromethane (4 x 50 mL) and concentrated to obtain a brown oil (10.1 g, yield 73%). 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 5 M NaOH (40 mL). The brown solution was heated at 80°C for 23 hours. The ethanol was concentrated, the solution was cooled on ice, and the pH was adjusted to 2 with concentrated hydrochloric acid. The ligand was purified using Amberlite XAD 1600 resin (eluted with a water-acetonitrile mixture), and 5.7 g was obtained as a white solid after lyophilization (73% yield). The product was characterized by several peaks by 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 adjusted 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 complex formation with xylenol orange. After complex formation was complete, the solution was concentrated to remove salts and impurities, and purified by elution on Amberlite XAD1600 resin using 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 obtained HPLC chromatogram, which characterizes several peaks, is shown in Figure 1.

[0149] By using (S)-methyl α-bromoglutarate obtained from L-glutamic acid, a compound completely equivalent to compound VI, which consists of an isomer mixture having an HPLC chromatogram that is substantially superimposed on Figure 1, can also be obtained.

[0150] Example 2: Synthesis of gadopicrenol (isomer mixture) Gadopicrenol as an indiscriminate mixture of stereoisomers was prepared by coupling the isomer mixture of Gd(PCTA-tris-glutaric acid) obtained from Example 1 with racemic isocerinol according to the following synthesis 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 isocerinol (0.40 g, 0.0044 mol) in water adjusted to pH 6 with concentrated HCl. Next, 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 added, and the resulting solution was stirred at room temperature at pH 6 for 24 hours. The product was then purified by preparative HPLC using a water / acetonitrile gradient and silica C18. The fraction containing the pure compound was concentrated and lyophilized to obtain a white solid (0.83 g, 78% yield). The product was characterized by HPLC. The obtained HPLC chromatogram is shown in Figure 4a.

[0152] Example 3: Isolation of enantiomer pairs associated with 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 pre-packed silica C18 column (Biotage® SNAPULTRAC18 120 g, HP-sphereC18 25 μm) and purified using an automated flash chromatography system with elution using a slow gradient of deionized water (4 CV) followed by acetonitrile. The fraction enriched with the enantiomer pair associated with peak C was combined, concentrated, and lyophilized to obtain a white solid (200 mg). The HPLC chromatogram of the resulting enriched enantiomer pair C is shown in Figure 2. Corresponding MS spectrum (Gd(H4L)) + The frequency (752.14 m / z) is shown in Figure 3.

[0153] Example 4: Coupling of enantiomeric pair C with isocerinol a) Coupling of enantiomer pair C and R-isoserinol For example, as in Example 3, the recovered enriched enantiomeric pair C (34 mg, 90% potency, 0.040 mmol) was dissolved in deionized water (5 mL), R-isocerinol (16 mg, 0.17 mmol) was added, and the pH was adjusted to 6 with 1 M HCl. Next, 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 hours. The solution was concentrated and loaded onto a pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphereC18 25 μm), and eluted with a water / acetonitrile gradient using an automated flash chromatography system. The fraction containing the pure product, or the fraction showing a major peak with an area greater than 90% on HPLC, was combined, concentrated, and lyophilized to obtain a white solid (21 mg, 54% yield). The HPLC chromatogram of the obtained product is shown in Figure 6b.

[0154] b) Coupling of enantiomer pair C and S-isoserinol For example, enriched enantiomeric pair C (55 mg, 90% potency, 0.066 mmol) recovered as described in Example 3 was dissolved in deionized water (5 mL), and S-isocerinol (34 mg, 0.29 mmol) was added after adjusting the pH to 6 with 1 M HCl. Next, 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 hours. 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. The fraction containing the pure product, or the fraction showing a major peak with an area greater than 90% on HPLC, was combined, concentrated, and lyophilized to obtain a white solid (52 mg, 81% yield). The HPLC chromatogram of the obtained product is shown in Figure 6c.

[0155] c) Coupling of enantiomer pair C with racemic isocerinol For example, enriched enantiomeric pair C (54 mg, 90% potency, 0.065 mmol) recovered as described in Example 3 was dissolved in deionized water (5 mL), and racemic isocerinol (27 mg, 0.29 mmol) was added after adjusting the pH to 6 with 1 M HCl. Next, EDCI·HCl (62 mg, 0.32 mmol) and HOBT (4.3 mg, 0.032 mmol) were added, and the solution was stirred at room temperature at pH 6 for 24 hours. 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. The fraction containing the pure product, or the fraction showing a major peak with an area greater than 90% on HPLC, was combined, concentrated, and lyophilized to obtain 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-glutaric acid) (compound XII) RRR-rich Gd(PCTA-tris-glutaric acid) was prepared according to synthetic scheme 3, which comprises the following steps. [ka]

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

[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 cooling the solution to -40°C, trifluoromethanesulfonic acid anhydride (0.49 mL, 2.91 mmol) was slowly added. After stirring the dark-colored solution at -40°C for 1 hour, the 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 slowly returned to room temperature and stirred overnight at room temperature. Next, the organic solution was washed with 2 M HCl (4 × 10 mL), and the aqueous phase was extracted again with dichloromethane (3 × 10 mL). The organic phases were combined and concentrated under vacuum to obtain 400 mg of 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 adjusted to pH 1 with concentrated HCl, and purified by elution with a gradient of deionized water / acetonitrile using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm). Fractions containing the 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 complex formation was checked by xylenol orange and HPLC. HPLC of the crude product showed the desired RRR isomer as the major peak: approximately 80% in area %. The mixture was adjusted to pH 2 with concentrated HCl and purified by elution with a deionized water / acetonitrile gradient using an automated flash chromatography system equipped with a silica C18 prepacked column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 μm). Fractions containing the pure product were combined, concentrated, and lyophilized (36 mg, 90% yield). For example, by using the procedure of Example 2, the corresponding RRR amide derivative can be obtained by reacting the recovered compound with isocerinol.

[0161] Example 6: Stereoselective Synthesis of SSS Gd(PCTA-tris-glutaric acid) (Compound XVII) The SSS-enriched Gd(PCTA-tris-glutaric acid) acid was similarly prepared according to Synthesis Scheme 4 comprising the following steps.

Chemical Structure

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

[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 cooling the solution to -40 °C, trifluoromethanesulfonic anhydride (0.50 mL, 3.07 mmol) was slowly added dropwise. The dark-colored solution was stirred at -40 °C for 1 hour, then 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. Next, the organic solution was washed with water (3 × 5 mL) and 2 M HCl (4 × 5 mL). The aqueous phase was extracted again with dichloromethane (3 x 10 mL). The organic phases were combined and concentrated in vacuo to obtain 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 5 M NaOH (4.5 mL). The resulting brown solution was heated at 80°C for 16 hours to completely hydrolyze it. The methanol was concentrated, the solution was pH 2 with concentrated HCl, and purified by elution with a water / acetonitrile gradient using an automated flash chromatography system equipped with a silica C18 prepack column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 μm). The 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 complex formation was checked by xylenol orange and HPLC. HPLC of the crude product showed the desired SSS isomer as the major peak: approximately 85% in area %. The solution was adjusted to pH 2.5 with concentrated HCl and purified by elution with a water / acetonitrile gradient using an automated flash chromatography system equipped with a silica C18 prepack column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 μm). The fractions containing the pure product SSS were combined, concentrated, and lyophilized (39 mg, 87% yield).

[0166] Example 7: Kinetic test of the dissociation reaction of Gd(PCTA-tris-glutaric acid) (isomer mixture) in 1.0 M HCl solution (25°C) The kinetic inertness of Gd(III) complexes is characterized by either the dissociation rate measured in 0.1–1.0 M HCl, or the rate of the metal exchange reaction between Zn(II) and Cu(II) or Eu(III) ions in solution. However, the dissociation of lanthanide(III) complexes formed with macrocyclic ligands is very slow, and generally Zn(II) is the most common.2+ Ya Cu 2+ This process proceeds via proton-assisted pathways without the involvement of endogenous metal ions. The kinetic inertness of the complex Gd(PCTA-tris-glutaric acid) was characterized by the rate of its dissociation reaction in a 1.0 M HCl solution. 0.3 mg of the complex (isomer mixture from Example 1) was dissolved in 2.0 mL of 1.0 M HCl solution, and the reaction of the solution, maintained at 25°C, was tracked over time by HPLC. HPLC measurements were performed using analytical procedure 1 on an Agilent 1260 Infinity II system.

[0167] A huge excess of H + The presence of ([HCl] = 1.0 M) guarantees a pseudo-first-order kinetic condition.

number

[0168] The HPLC chromatogram of Gd(PCTA-tris-glutaric acid) is characterized by the presence of four signals (A, B, C, and D) with the same m / z ratio (Gd(H4L)+: 752.14 m / z) in the MS spectrum. Each of these peaks is reasonably attributable to one of four pairs of enantiomers resulting from the three stereocenters on the three glutaric acid 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, particularly the decrease in the area of ​​peaks A, B, C, and D, although not in the same way as the different peaks, while new signals corresponding to uncomplexed diastereoisomers are formed and increase over time. The differences in the decrease in the integrated area of ​​the peaks can be interpreted by the different dissociation rates of the enantiomer pairs associated with the different peaks.

[0169] [H +When ] is present in excess, the dissociation reaction of the enantiomer pair of Gd(PCTA-tris-glutaric acid) can be treated as a pseudo-primary manufacturing method, and the reaction rate can be expressed by the following equation 2 (where k A , k B , k C , and k D This is a pseudo-first-order velocity constant calculated by fitting area-time data pairs, [A] t [B] t [C] t , and [D] t (where is the total concentration of compounds A, B, C, and D at time t).

number

[0170] The area value at time t can be expressed by the following formula.

number

[0171] Example 8: Kinetic test of the dissociation reaction of a complex compound obtained by coupling gadopicrenol (a mixture of isomers from Example 2) and enantiomeric pair C with R, S, and racemic isocerinol, respectively, in a 1.0 M HCl solution (25°C). The kinetic inertness of all complexes was characterized by the rate of dissociation reaction 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 tracked over time by HPLC. HPLC measurements were performed using the 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 (shown for convenience as A', B', C' and D') having the same MS and UV-Vis spectra. However, in the HPLC chromatogram of the complex compound obtained by coupling enantiomer pair C with isocerinol, there was only one signal regardless of whether it was R, S isocerinol or racemic isocerinol (Figure 6). As observed, the chirality of the isocerinol pendant does not affect the retention time of the coupled diastereoisomers. The presence of four signals in the HPLC chromatogram of gadopiclenol (isomer mixture) can be interpreted by the presence of four enantiomer pairs formed at the stereocenters 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 inertness of all the above complexes, in order to ensure the occurrence of pseudo-first-order conditions, the dissociation reactions of these complexes were investigated in the presence of a large excess of H + ([HCl]= 1.0 M). The progress of the reaction was checked by HPLC over time, and the peak area values of the complexes were plotted as a function of time as described in Example 7 above for the Gd(PCTA-tris-glutaric acid) isomers.

[0174] As expected, the integral values ​​of A', B', C', and D' decrease over time, while the peak for the free ligand increases. Since the area value of the HPLC chromatogram is directly proportional to the concentration of gadopicrenol (isomer mixture), the half-life of the dissociation reaction of gadopicrenol (isomer mixture) can be estimated from half of the sum of the area values. The half-life of gadopicrenol (isomer mixture) 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 enantiomeric pair C with R, S, and racemic-isocerinol were also calculated from half of the area values ​​of the HPLC chromatogram. The half-lives of the complexes obtained by coupling enantiomeric pair C with R, S, and racemic-isocerinol were found to be 41, 43, and 44 hours at 25°C and pH 0 (1.0 M HCl). The pseudo-first-order rate constant (k) characterizes the rate of the dissociation reaction of the complex obtained by coupling the enantiomeric pair C with R, S, and racemic-isocerinol. x ) The area-time velocity data is shown in Equation 3 above.

number

[0175] The area values ​​of the complexes obtained by coupling the enantiomeric pair C with R, S, and racemic-isocerinol were estimated, and the t value was calculated by fitting area-time kinetic data. 1 / 2 The comparison of values ​​is in very good agreement. The t values ​​shown in Table 3 are... 1 / 2 The values ​​clearly show that the dissociation half-life of the D' isomer obtained by coupling the enantiomeric pair C isomer with R, S, and racemic isocerinol is almost the same as that measured for gadopicrenol (isomer mixture), and is 8 times longer. From this, it is confirmed that the higher dynamic inertness of RRR-SSS Gd (PCTA-tris-glutaric acid) is substantially maintained even after coupling with isocerinol. Furthermore, the t of the complex obtained by coupling the enantiomeric pair C with R, S, and racemic isocerinol 1 / 2 The values ​​also indicate that the chirality of the isocerinol pendant does not affect the dynamic inertness of the final complex. [Table 5]

[0176] Example 9: Relaxation Characteristics The relaxation measurement characteristics 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 relaxation values ​​measured under the same conditions for Gd- complexes having similar coordination cages.

[0177] material Device The longitudinal water proton relaxation rate (R1 = 1 / T1) was 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 pretreatment All test samples were used as supplied, and the required amount of paramagnetic chelate complex was weighed out and diluted with a selected medium (water or human plasma) to obtain a 5 or 10 mM starting solution. Relaxation capacity measurement Five different concentration samples (0.1, 0.25, 0.5, 0.75, and 1 mM) were prepared for each medium by further diluting a 5 or 10 mM starting solution. Measurement of relaxation Relaxation levels were measured at 0.47T and 1.41T with preset temperature samples at 37°C, maintained constant by a constant temperature bath connected to the spectrometer's sample holder. Five sample solutions were preheated to 37°C in an external constant temperature bath and then left in the internal bath for 10 minutes to allow the temperature to stabilize. The longitudinal relaxation time T1 was 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 (simple exponential fitting for T1 measurement, linear fitting for evaluation of longitudinal relaxation ability) was performed using Mathematica® (Wolfram, USA). Errors in the estimated parameters were evaluated by the fitting procedure.

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

[0179] Table 5 below compares the relaxation degree values ​​r1, measured at 37°C in both H2O and HSA, for amide derivatives obtained by communication of RRR / SSS Gd (PCTA-tris-glutaric acid) and isocerinol, along with the stereochemistry of the serinol used in conjugation, with the corresponding values ​​cited in the prior art for gadopicrenol (isomer mixture). [Table 7]

[0180] On the one hand, the results obtained indicate that the higher relaxation ability measured for RRR / SSS Gd(PCTA-tris-glutaric acid) relative to the corresponding isomer mixture is substantially maintained for each conjugate derivative. On the other hand, these results are consistent with the fact that the stereochemistry of the isocerinol moiety does not affect the major properties of the final compound, which are mainly related to the stereochemistry of the glutaric acid arm.

[0181] Example 10: X-ray diffraction Enantiomer C Crystal preparation A single crystal of the compound C represented by the formula {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O, suitable for X-ray diffraction testing (wherein Gd(H3L) is trisprotonated RRR / SSS Gd(PCTA-tris-glutaric acid)), was grown by slow evaporation of water from an aqueous solution of RRR / SSS-rich compound C recovered from Example 3. To promote crystallization, two internal spherical water molecules of the Gd(PCTA-tris-glutaric acid) complex were replaced with 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 × 10⁻⁶). -4 A 1.0 mL aqueous solution of enriched compound C (0.0483 M GdH3L aqueous solution) was recovered from Example 3 (mol) (5.0 × 10 -5 It was prepared by dissolving (in mol) of solid H2C2O4. The pH was adjusted to 3.3 by adding solid H2C2O4 in stages.

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

[0183] result The structure was elucidated using 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 using F 2 This was performed by a full matrix least squares method based on [the specified formula]. Anisotropic thermal motion refinement was used for all atoms. For hydrogen atoms, isotropic U [the specified formula] was used for the hydroxyl group. factors = 1.2·U eq or U factors = 1.5·U eq (U eq(where is the equivalent isotropic thermal coefficient of the bonded non-hydrogen atom). Hydrogen atoms in solvent water molecules were not included in the refined model because their positions could not be clearly identified in the electron density peaks of the Fourier difference map. The essential crystal and refinement data are shown 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 a 2RRR + 2SSS complex. This means that each crystal contains equimolar (50-50%) isomers of SSS and RRR.

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

[0185] Fifteen single crystals were isolated and X-ray diffraction data collection for seven crystals was performed on the X-ray diffraction beamline (XRD1) at the Elettra Synchrotron, Trieste (Italy) by procedures disclosed, for example, in Lausi A. et al., The European Physical Journal Plus, 2015, 130, 1-8. Specifically, the recovered crystals were immersed in NHV oil (Jena Bioscience, Jena, Germany), frozen in liquid nitrogen, and attached to a 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 (nitrogen stream supplied from Oxford Cryostream 700 - Oxford Cryosystems Ltd., Oxford, United Kingdom) by the rotating crystal method. Data were acquired using a Pilatus 2M hybrid pixel area detector (Dectris Ltd., Baden-Daettwil, Switzerland) with a monochromatic wavelength of 0.700 Å.

[0186] The structure was solved by direct methods. Fourier analysis and refinement were carried out by full-matrix least-squares methods based on F 2 Anisotropic thermal motion refinement was used for all atoms. Hydrogen atoms were included at positions calculated with U factors = 1.2·U eq or U factors = 1.5·U eq (U eq is the equivalent isotropic thermal parameter of the bonded non-hydrogen atoms). The hydrogen atoms of the solvent water molecules were not included in the refined model because their positions could not be clearly identified from the electron density peaks of the Fourier difference map. Table 7. Crystallographic data and stereochemistry of the stereocenters for 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 indices [I > 2σ(I)] a R1 = 0.0554, wR2 = 0.1496 Stereochemistry at the stereogenic centers of the ASU 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 the procedures and details used, see, for example, Lausi A., Polentarutti M., Onesti S., Plaisier J. R., Busetto E., Bais G., Barba L., Cassetta A., Campi G., Lamba D., Pifferi A., Mande S. C., Sarma D. D., Sharma S. M., 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 Figure 11.

Claims

1. Formula (IIB): 【Chemistry 1】 A method for producing an isomer mixture of amide derivatives of Gd (PCTA-tris-glutaric acid), wherein the isomer mixture is (1) In equation (II B), F' is equation (IIIa) 【Chemistry 2】 This is the RRR enantiomer residue shown by, Three -NR １ R ２ Each group is bonded to an open bond (●) in the carboxyl portion of F'; R １ C is either substituted with H or 1 to 4 hydroxyl groups, or unsubstituted. １ -C ６ It is alkyl; R ２ C is either substituted with 1 to 4 hydroxyl groups or is unsubstituted. １ -C ６ It is alkyl. Containing at least 50% of an amide derivative of Gd(PCTA-tris-glutaric acid), (2) In equation (II B), F' is equation (III b) 【Transformation 3】 This is an SSS enantiomer residue represented by , Three - NR １ R ２ The groups are each bonded to the open bond (●) of the carboxyl moiety of F'; R １ C is either substituted with H or 1 to 4 hydroxyl groups, or unsubstituted. １ -C ６ It is alkyl; R ２ C is either substituted with 1 to 4 hydroxyl groups or is unsubstituted. １ -C ６ It is alkyl. It contains at least 50% of an amide derivative of Gd(PCTA-tris-glutaric acid), or (3) A mixture comprising at least 50% of the Gd(PCTA-tris-glutaric acid) amide derivative of (1) and the Gd(PCTA-tris-glutaric acid) amide derivative of (2), The manufacturing method is a') [(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), [(αS,α'S,α''S)-α,α',α''-tri To obtain an isomer mixture of Gd(PCTA-tris-glutaric acid) containing at least 50% of su(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), or a mixture thereof; and b') Convert the isomer mixture of Gd (PCTA-tris-glutaric acid) obtained in step a') into the corresponding isomer mixture of each amide derivative. A manufacturing method comprising the following.

2. R １ H is R ２ C is substituted with one or two hydroxyl groups. １ -C ３ The manufacturing method according to claim 1, wherein the alkyl group is alkyl.

3. R ２ The method for producing a product according to claim 1 or 2, wherein is a selinol residue or isoselinol.

4. R ２ The manufacturing method according to any one of claims 1 to 3, wherein is selected from R isoselinol, S isoselinol, or racemic isoselinol.

5. The production method according to any one of claims 1 to 4, wherein the isomer mixture of Gd(PCTA-tris-glutaric acid) in step a') is obtained by chromatography using the Gd(PCTA-tris-glutaric acid) obtained as an isomer mixture as a starting material.

6. The method for producing the product recovered from step a') with isocerinol, according to any one of claims 1 to 5.

7. Formula (IIB): 【Transformation 7】 The use of a mixture of isomers of an amide derivative of Gd(PCTA-tris-glutaric acid), as shown in the above, for the preparation of a pharmaceutical formulation for use in vivo imaging of organs, tissues, or regions of the human or animal body using MRI technology, The mixture of isomers (1) In equation (II B), F' is equation (IIIa) 【Transformation 8】 This is the RRR enantiomer residue shown by, Three -NR １ R ２ Each group is bonded to an open bond (●) in the carboxyl portion of F'; R １ C is either substituted with H or 1 to 4 hydroxyl groups, or unsubstituted. １ -C ６ It is alkyl; R ２ C is either substituted with 1 to 4 hydroxyl groups or is unsubstituted. １ -C ６ It is alkyl. Containing at least 50% of an amide derivative of Gd(PCTA-tris-glutaric acid), (2) In equation (II B), F' is equation (III b) 【Chemistry 9】 This is an SSS enantiomer residue represented by , Three -NR １ R ２ Each group is bonded to an open bond (●) in the carboxyl portion of F'; R １ C is either substituted with H or 1 to 4 hydroxyl groups, or unsubstituted. １ -C ６ It is alkyl; R ２ C is either substituted with 1 to 4 hydroxyl groups or is unsubstituted. １ -C ６ It is alkyl. It contains at least 50% of an amide derivative of Gd(PCTA-tris-glutaric acid), or (3) A mixture comprising at least 50% of the Gd(PCTA-tris-glutaric acid) amide derivative of (1) and the Gd(PCTA-tris-glutaric acid) amide derivative of (2), use.

8. R １ H is R ２ C is substituted with one or two hydroxyl groups. １ -C ３ The use according to claim 7, wherein the alkyl group is used.

9. R １ H is R ２ ga-CH ２ CH(OH)CH ２ The use described in claim 7, which is OH.

10. The isomer mixture is an amide derivative selected from the group consisting of the following: [(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 (isomer 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 (isomer 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); or mixtures thereof The use according to claim 7, comprising at least 50% of the above.

11. The use according to claim 7, wherein in formula (II B), F' comprises at least 60% RRR and SSS enantiomer residues.

12. The use according to any one of claims 7 to 11, wherein in formula (II B), F' comprises at least 70% RRR and SSS enantiomer residues.

13. The use according to any one of claims 7 to 11, wherein in formula (II B), F' comprises at least 80% RRR and SSS enantiomer residues.

14. The use according to any one of claims 7 to 11, wherein in formula (II B), F' comprises at least 90% RRR and SSS enantiomer residues.

Images