Contrast agents based on PCTA bearing gadolinium

Abstract

The present invention relates to contrast agents based on PCTA bearing gadolinium. The present invention relates to an RRR / SSS enantiomer pair of Gd (PCTA-tris-glutamic acid), a single enantiomer of the pair, a pharmaceutically acceptable salt thereof, an amide derivative thereof and a composition comprising at least 50% of these compounds.

Description

[0001] This application is a divisional application of the parent case, Chinese invention patent application 201980052546.2. Technical Field

[0002] The present invention relates generally to the field of magnetic resonance imaging (MRI). More particularly, the present invention relates to isomers of PCTA-based contrast agents, and to MRI contrast agents enriched with these isomers. Background Art

[0003] MRI contrast agents used in daily diagnostic practice typically comprise gadolinium complex compounds, which are characterized by high stability constants that ensure resistance against the release of free metal ions (known to be highly toxic to living organisms) in vivo.

[0004] Another key parameter in the definition of the tolerability of gadolinium-based contrast agents is the kinetic inertness (or kinetic stability) of the Gd(III)-complex, which is measured by the half-life (t 1 / 2 ) estimate.

[0005] High inertness is particularly important for those coordination compounds with lower thermodynamic stability and / or longer retention times before excretion, in order to avoid or minimize possible decomplexation or transmetallation reactions.

[0006] EP1931673 (Guerbet) discloses PCTA derivatives of the formula

[0007]

[0008] and the synthetic route for its preparation.

[0009] EP 2988756 (same applicant) discloses a pharmaceutical composition comprising the above derivatives and a calcium complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. According to EP 2988756, the calcium complex compensates for the poor thermodynamic stability of the gadolinium complex based on PCTA by forming a strong complex with free lanthanide ions via transmetallation, thereby improving the tolerability of the contrast agent.

[0010] Both EP1931673 and EP2988756 further relate to enantiomers or diastereomers of the claimed compounds or mixtures thereof, preferably selected from RRS, RSR and RSS diastereomers.

[0011] The above patents all disclose that, among the 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, more recently identified as the gadolinium chelate of 2,2′,2″-(3,6,9-triaza-1(2,6)-pyridiniumcyclodecaphane-3,6,9-triyl)tris(5-((2,3-dihydroxypropyl)amino)-5-oxopentanoate) (CAS Reg. No. 933983-75-6), which has the formula

[0012]

[0013] Also identified as P03277 or gadolinium polycyclic phenol.

[0014] For gadolinium polycyclic phenol, EP1931673 reported 11mM -1 s -1 G -1 The relaxivity (in water, at 0.5T, 37°C) of EP 2988756 is 10 -14.9 The thermodynamic equilibrium constant (log K term =14.9).

[0015] In addition, for this same compound, the patentee has reported 12.8 mM in human serum. -1 s -1 The relaxation rate value (37℃, 1.41T), stability (log K term ) and a dissociation half-life of approximately 20 days (at pH 1.2; 37°C) (Investigative Radiology 2019, Vol. 54, (8), 475-484).

[0016] The precursor for preparing PCTA derivatives (including gadolinium polycyclic phenol) disclosed in EP1931673 is a Gd complex of 3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-tri(α-glutaric acid) having the formula

[0017]

[0018] Identified herein as "Gd(PCTA-tris-glutaric acid)." In particular, the gadolinium polycyclic phenol is obtained by amidation of the above compound with isoserinol.

[0019] As observed by the applicants, Gd(PCTA-tri-glutaric acid) possesses three stereogenic centers on the glutaric acid moiety (identified by asterisks (*) in the above structure), which yield 2 3 = 8 possible stereoisomers. More specifically, the above structure can generate 4 pairs of enantiomers, which are shown in Table 1 below.

[0020] Table 1

[0021] RRR SSS RSR SRS RRS SSR RSS SRR

[0022] Isomer RRR is the mirror image of isomer SSS and this is why they are called enantiomers (or enantiomeric pairs). As is known, enantiomers exhibit identical physicochemical properties and can only be distinguished using chiral methodologies, such as chiral chromatography or polarized light.

[0023] On the other hand, isomer RRR is neither equal to nor a mirror image of any of the other six isomers mentioned above; these other isomers are therefore identified as diastereomers of the RRR (or SSS) isomer. Diastereomers may exhibit different physicochemical properties (e.g., melting point, water solubility, relaxivity, etc.).

[0024] With regard to gadolinium polycyclic phenol, its chemical structure contains a total of 6 stereocenters, 3 present on the glutaric acid portion of the precursor as described above, and 1 present in each of the 3 isoserinol portions attached thereto, identified by an asterisk (*) and an open circle (°), respectively, in the following structure:

[0025]

[0026] For this compound, this yields a total of 2 6 = Total theoretical number of 64 stereoisomers.

[0027] However, neither EP1931673 nor EP2988756 describes the exact composition of the isomeric mixture obtained by following the reported synthetic routes, nor does either of them provide any teaching on the separation and characterization of any of these isomers, nor disclose any stereospecific synthesis of gadolinium polycyclic phenols. Summary of the invention

[0028] The Applicant has now discovered that specific isomers of the above-mentioned precursor Gd (PCTA-tri-glutaric acid) and its derivatives (especially gadolinium polycyclic phenol) have improved physicochemical properties, especially with regard to relaxivity and kinetic inertness.

[0029] One embodiment of the present invention relates to a compound selected from the group consisting of:

[0030] Enantiomers having 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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR enantiomer):

[0031]

[0032] Enantiomers having formula (Ib) [(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-triacetate (3-)κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomer):

[0033]

[0034] Such mixtures of RRR and SSS enantiomers and pharmaceutically acceptable salts thereof.

[0035] Another embodiment of the present invention relates to an isomeric mixture of Gd(PCTA-tri-glutaric acid) comprising at least 50% of the RRR isomer of formula (Ia) [(αR,α'R,α"R)-α,α',α"-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetate (3-)-κN3,κN6,κN9,κN15,κN2] 3,κO6,κO9]-gadolinium, or the SSS isomer of formula (Ib) [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

[0036] Another aspect of the invention relates to amides obtained by conjugating one of the above compounds or the isomeric mixture with an amino group, such as, preferably, serinol or isoserinol.

[0037] One embodiment of the present invention relates to an amide derivative of formula (II A)

[0038] F(NR1R2)3(II A)

[0039] in:

[0040] F is:

[0041] RRR enantiomeric residues of formula IIIa

[0042]

[0043] The SSS enantiomer residue of formula IIIb

[0044]

[0045] or a mixture of such RRR and SSS enantiomeric residues;

[0046] and each of the three -NR1R2 groups is bound to an open bond of the corresponding carboxyl portion of F, identified by a solid circle (·) in the above structure;

[0047] R1 is H or C1-C6 alkyl optionally substituted by 1-4 hydroxyl groups;

[0048] R2 is a C1-C6 alkyl group optionally substituted by 1 to 4 hydroxyl groups, and preferably a C1-C3 alkyl group substituted by one or two hydroxyl groups.

[0049] Another embodiment of the present invention relates to an isomeric mixture of an amide derivative of Gd(PCTA-tri-glutaric acid) having the formula (II B)

[0050] F'(NR1R2)3(II B)

[0051] in:

[0052] F' is a mixture of isomers of the residue of Gd(PCTA-tri-glutaric acid) of formula (III)

[0053]

[0054] The isomeric mixture of Gd(PCTA-tri-glutaric acid) residues comprises at least 50% of the enantiomeric residues of the above formula (IIIa), the enantiomeric residues of the above formula (IIIb) or a mixture thereof; and

[0055] Each of the -NR1R2 groups is bound to an open bond of the corresponding carboxyl moiety of F', identified in the above structure by a solid circle (·), and is as defined above for the compound of formula (II A).

[0056] Another aspect of the present invention relates to the following pharmaceutically acceptable salts: RRR or SSS enantiomers of Gd(PCTA-tri-glutaric acid) or preferably RRR / SSS mixtures or isomeric mixtures of Gd(PCTA-tri-glutaric acid), wherein the isomeric mixtures of Gd(PCTA-tri-glutaric acid) contain at least 50% of any of these enantiomers or RRR / SSS enantiomer mixtures or their amide derivatives of the above-mentioned formula (II A) or (II B), which are used as MRI contrast agents, in particular for diagnostic imaging of organs or tissues of the human or animal body by the use of MRI technology.

[0057] Another aspect of the invention relates to pharmaceutically acceptable compositions comprising at least one compound or isomeric mixture according to the invention or a pharmaceutically acceptable salt or amide derivative thereof (as defined above) in admixture with one or more physiologically acceptable carriers or excipients.

[0058] In another aspect, the present invention relates to the stereoselective synthesis of the RRR or SSS isomers of Gd(PCTA-tri-glutaric acid) or their salts.

[0059] One embodiment of the present invention relates to: a method for synthesizing an amide derivative of formula (II A),

[0060] F(NR1R2)3(II A)

[0061] Wherein F, R1 and R2 are as described above, the method comprises:

[0062] a) obtaining the RRR or SSS isomer of the Gd(PCTA-tri-glutaric acid) complex or a mixture thereof; and

[0063] b) converting the isomer or isomer mixture obtained in step a) into its amide derivative;

[0064] And a method for preparing an isomer mixture of the amide derivative of the above formula (II B), the method comprising:

[0065] a′) obtaining an isomeric mixture of Gd(PCTA-tri-glutaric acid) comprising at least 50% of the enantiomer RRR or SSS or a mixture thereof;

[0066] b') converting the isomeric mixture of Gd(PCTA-tri-glutaric acid) obtained in step a') into the corresponding isomeric mixture of the corresponding amide derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Shown is the HPLC chromatogram of Gd(PCTA-tris-glutaric acid) collected as a mixture of isomers from Example 1 carried out by following a synthetic procedure disclosed in the prior art ([GdL] = 0.2 mM, 25°C).

[0068] Figure 2 The HPLC chromatogram of the RRR / SSS enantiomer pair C of Gd(PCTA-tris-glutaric acid) from Example 3 is shown.

[0069] Figure 3 show Figure 2 MS spectrum of the main peak. m / z ratio of Gd(H4L) + :752.14m / z.

[0070] Figure 4 show HPLC chromatograms of the following: a) isomeric mixture of Gd (PCTA-tri-glutaric acid) of Example 1; b) enantiomeric pair C (Compound VI of Example 3); c) RRR enantiomers (Compound XII of Example 5); and d) SSS enantiomers (Compound XVII of Example 6).

[0071] Figure 5 The following chiral HPLC chromatograms are shown: a) enantiomeric pair C (Compound VI of Example 3); b) RRR enantiomers (Compound XII of Example 5); and c) SSS enantiomers of Gd(PCTA-tri-glutaric acid) (Compound XVII of Example 6).

[0072] Figure 6 HPLC chromatograms showing amide derivatives obtained by reacting Gd (PCTA-tri-glutaric acid) with isoserinol. a): amide derivative obtained as a mixture of isomers from Example 2, wherein the 4 main peaks are identified as A', B', C' and D' for convenience; b): amide derivative obtained by reacting RRR / SSS Gd (PCTA-tri-glutaric acid) with R-isoserinol; c): amide derivative obtained by reacting RRR / SSS Gd (PCTA-tri-glutaric acid) with S-isoserinol; and d): amide derivative obtained by reacting RRR / SSS Gd (PCTA-tri-glutaric acid) with racemic isoserinol.

[0073] Figure 7 Refers to the test of Example 7 and shows the HPLC area values ​​of peaks A (◇), B (□), C (Δ) and D (○) as a function of time ([GdL]=0.2 mM, [HCl]=1.0 M, 25° C.).

[0074] Figure 8 Refers to the test of Example 8, and shows the HPLC area values ​​as a function of time: total area of ​​the isomer mixture (◇); RRR / SSS Gd(PCTA-tri-glutaric acid)+R isoserinol (□); RRR / SSS Gd(PCTA-tri-glutaric acid)+S isoserinol (Δ); RRR / SSS Gd(PCTA-tri-glutaric acid)+racemic isoserinol (○). ([GdL]=0.2mM, [HCl]=1.0M, 25°C).

[0075] Fig. 9 The X-ray structure of a single crystal of a ternary complex of Gd(PCTA-tri-glutaric acid)-oxalate with a guanidine counterion of formula {(C(NH2)3)2[Gd(PCTA-tri-glutaric acid)(C2O4)]}·1H2O is shown, showing the chirality RRR of the (identified) chiral carbon atom of the glutaric acid side group.

[0076] Fig.10 show Fig. 9 Unit cell of a crystal containing the 2RRR+2SSS complex.

[0077] Fig.11 Shown is the X-ray structure of a single crystal obtained from a ternary complex formed between a carbonate anion and an amide compound D' obtained by coupling reaction of RRR / SSS Gd(PCTA-tri-glutaric acid) with racemic isoserinol and statistical analysis of the collected crystals. DETAILED DESCRIPTION OF THE INVENTION DETAILED DESCRIPTION

[0079] The synthesis method disclosed in the prior art (see US Pat. No. 6,440,956 cited in EP1931673) allows obtaining Gd(PCTA-tris-glutaric acid) as a mixture of isomers (otherwise identified herein as "isomer mixture of Gd(PCTA-tris-glutaric acid)"), which can be seen as several peaks in HPLC.

[0080] A preparative HPLC method has been proposed allowing the separation of 4 peaks with identical m / z ratios (Gd(H4L)+: 752.14 m / z) from the mixture.

[0081] Figure 1 A representative chromatogram of the resolved isomeric mixture is shown, wherein for convenience, each peak identified by letters A, B, C and D, respectively, is reasonably attributed to one of the enantiomer pairs identified above. More precisely, each peak is associated with a pair of enantiomers, characterized by identical m / z ratios in the MS spectrum, which cannot be further distinguished by normal reverse phase HPLC.

[0082] We have now surprisingly found that the enantiomeric pair associated with peak C of the HPLC chromatogram (or enantiomeric pair C, used interchangeably hereinafter) shows the best performance, in particular in terms of kinetic inertness and reduced tendency to release Gd.

[0083] For example, we found that enantiomeric pair C has a dissociation half-life (in 1 M HCl) that is some tens of times higher than that of the enantiomeric pair associated with peak B and more than ten times higher than the average half-life of the Gd(PCTA-tris-glutaric acid) isomer mixture.

[0084] Furthermore, the relaxivity values ​​associated with the enantiomeric pair involved in Peak C are significantly higher than those reported in 1931673B1 for an isomeric mixture of Gd(PCTA-tris-glutaric acid) tested under the same conditions.

[0085] This enantiomeric pair C was confirmed to include: [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, i.e., the isomer RRR of Gd(PCTA-tri-glutaric acid) of formula (Ia)

[0086]

[0087] and the corresponding mirror image 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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium, i.e., the SSS isomer of Gd(PCTA-tri-glutaric acid) of formula (Ib)

[0088]

[0089] Surprisingly, the improved properties exhibited by the individual enantiomers (RRR and SSS) and the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid) (or RRR / SSS Gd(PCTA-tri-glutaric acid), hereinafter used interchangeably) are surprisingly still substantially maintained even after their conjugation, for example resulting in the obtaining of their amide derivatives.

[0090] For example, the coupling reaction of RRR / SSS Gd(PCTA-tri-glutaric acid) and likewise each of its individual RRR or SSS enantiomers with isoserinol leads to final amide derivatives having the same molecular formula of gadolinium polycyclic phenol. In this regard, it is interesting to note that, regardless of the type of isoserinol used, whether the R or S isomer, or racemic isoserinol, its conjugation with the RRR / SSS enantiomers of Gd(PCTA-tri-glutaric acid) leads to the corresponding amide derivatives, which have the same retention time and are thus difficult to distinguish by normal reverse phase HPLC.

[0091] Therefore, the different isomeric forms of the additional isoserinol (and more generally of the amide derivatives) do not affect the main properties of the final conjugate compound, which are essentially determined by the stereochemistry of the Gd(PCTA-tris-glutaric acid) precursor.

[0092] Indeed, the improved properties shown for the enantiomeric pair associated with peak C of the isomeric mixture of Gd(PCTA-tris-glutaric acid) are substantially maintained after conjugation with isoserinol, regardless of the configuration of the coupled isoserinol.

[0093] In particular, regardless of the configuration of isoserinol, its coupling with the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutaric acid) leads to amide compounds with greater kinetic inertness and relaxivity compared to the gadolinium polycyclic phenols obtained as isomer mixtures using prior art synthetic procedures.

[0094] In the present specification, and unless otherwise provided, the expression "mixture of isomers" (referring to a specific compound) includes within its meaning a mixture comprising at least two stereoisomers of the compound. In particular, when used in relation to Gd(PCTA-tri-glutaric acid), the expression "mixture of isomers" refers to an unseparated mixture of at least two of the following: 8 diastereoisomers (or diastereomers, which can be used interchangeably herein), and more precisely 4 enantiomeric pairs generated by the 3 stereocenters contained in the molecule and identified in Table 1. On the other hand, when used in relation to amide derivatives of Gd(PCTA-tri-glutaric acid), such as gadolinium polycyclic phenol, the expression "mixture of isomers" refers to an undefined and unseparated mixture of the corresponding amide derivatives of the above-mentioned at least two (out of the possible 4) enantiomeric pairs of the Gd(PCTA-tri-glutaric acid) residue.

[0095] To this extent, since each amine group of the amide derivative may in turn contain one or more stereocenters, the total number of possible stereoisomers of the amide derivative may be increased accordingly. For example, conjugation of Gd(PCTA-tri-glutaric acid) with 3 isoserinol molecules each having a corresponding stereocenter brings the number of possible stereoisomers of the corresponding amide up to 64 (32 enantiomeric pairs), resulting from the presence of a total of 6 stereocenters on the molecule.

[0096] In the present specification and claims, the expressions "an isomeric mixture of amide derivatives of Gd(PCTA-tri-glutaric acid)" or "an amide derivative of an isomeric mixture of Gd(PCTA-tri-glutaric acid)" can be used interchangeably.

[0097] The expression "enantiomer C" refers to the Figure 1 The enantiomeric pair is related to peak C. The enantiomer C corresponds to the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid).

[0098] The expression "RRR / SSS enantiomer pair" (or RRR / SSS enantiomers) generally refers to a mixture of the enantiomer RRR and the corresponding mirror image isomer SSS of the desired compound, including a racemic mixture thereof. In the present specification, this expression is typically used in relation to Gd (PCTA-tri-glutaric acid) and refers to a mixture of the enantiomers RRR and SSS of the compound (or its RRR / SSS mixture). More specifically, the expression "RRR / SSS enantiomer pair of Gd (PCTA-tri-glutaric acid)" (or "RRR / SSS enantiomers" as used interchangeably herein) Gd(PCTA-tri-glutaric acid)") refers to [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR enantiomer) 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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomers), for example, as shown in the figure below:

[0099]

[0100] The expression "Compound D'" refers to an amide derivative obtained by the coupling reaction of RRR / SSS Gd (PCTA-tri-glutaric acid) with isoserinol.

[0101] The expression "stereoselective synthesis" (or "asymmetric synthesis" as used interchangeably herein) comprises within its meaning a chemical reaction (or reaction sequence) in which one or more new elements of chirality are formed in the substrate molecule and unequal amounts of stereoisomers (enantiomers or diastereoisomers) of the products are produced. In the present specification, the expression "stereoselective synthesis" is particularly used for RRR and the corresponding mirror image isomer SSS involving Gd (PCTA-tri-glutaric acid) and refers to a synthesis that allows obtaining a complex containing at least 55%, preferably 65%, more preferably 75% and most preferably at least 85% of either of the two enantiomers.

[0102] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present invention wherein the parent compound is suitably modified by converting any free acid or basic groups (if present) into the corresponding addition salts with any base or acid that is generally considered pharmaceutically acceptable.

[0103] Preferred cations of the inorganic bases which may be suitably used to prepare the salts of the invention include, for example, ions of alkali metals or alkaline earth metals, such as potassium, sodium, calcium or magnesium.

[0104] Preferred cations of organic bases include, for example, those of primary, secondary and tertiary amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine.

[0105] Preferred cations and anions of amino acids include, for example, those of taurine, glycine, lysine, arginine, ornithine or aspartic acid and glutamic acid.

[0106] Furthermore, the terms "moiety" or "moieties", "residue" or "residues" are intended herein to define the remaining portion of a designated molecule once suitably linked or conjugated to the rest of the molecule, either directly or through any suitable linker.

[0107] For example, when used in relation to an amide derivative of Gd(PCTA-tri-glutaric acid) (in the form of either an isomeric mixture or the RRR or SSS isomers or a RRR / SSS enantiomeric mixture or an enantiomeric pair thereof), the term "residue" refers to the portion of Gd(PCTA-tri-glutaric acid) that is attached to the amine group to give the corresponding amide derivative.

[0108] In particular, the term "residue of an isomeric mixture of Gd(PCTA-tri-glutaric acid)" refers to a compound having formula (III)

[0109]

[0110] This residue can be conjugated, for example, to an amino residue of formula -NR1R2 via the open bond of the carboxyl moiety, which is identified in the above structure by a solid circle (·), to give the corresponding amide derivative of formula

[0111]

[0112] Similarly, the term "the residues of the RRR and SSS enantiomers of Gd(PCTA-tri-glutaric acid)" refers to residues having formula (IIIA)

[0113]

[0114] And formula (IIIb)

[0115] of compounds.

[0116] The term "residue" applies analogously to the corresponding residue of the RRR / SSS enantiomeric pair or generally to a mixture of enantiomers.

[0117] A preparative HPLC method has been proposed which allows the separation of four peaks, identified for convenience as A, B, C and D, respectively having the same m / z ratio (Gd(H4L)+: 752.14 m / z), from Gd(PCTA-tri-glutaric acid) obtained by a non-stereoselective synthesis disclosed in the prior art. The four signals in the HPLC chromatogram of the Gd(PCTA-tri-glutaric acid) complex have been assigned respectively to the four enantiomeric pairs formed by the different optical isomers of the glutaric acid residue previously identified in Table 1, taking into account the three stereocenters present in the molecule (identified as asterisks in the above molecular structure).

[0118] In order to investigate the kinetic inertness of the racemic mixture of Gd(PCTA-tri-glutaric acid) separated by HPLC and in particular of its four enantiomeric pairs separated by HPLC, we investigated their dissociation reactions under acidic conditions. In order to ensure pseudo-first order kinetic conditions, a large excess of H + ([HCl]=1.0M).

[0119]

[0120] where L is protonated PCTA-tris-glutaric acid (free ligand) and y is the number of protons attached to the ligand.

[0121] Solutions of Gd(PCTA-tris-glutaric acid) (mixture of isomers) in 1 M HCl were prepared and analyzed over time as explained in Example 7.

[0122] Specifically, the area values ​​of each of the A, B, C and D peaks were estimated over time by HPLC.

[0123] Due to the acid-catalyzed dissociation of the complex, we verified that as expected, the integrated areas of peaks A, B, C and D decreased, while a new signal was formed and grew, which was equivalent to the free ligand (m / z: 597.24). However, it is interesting to find that the area reduction rates of signals A, B, C and D are not equal to each other; for example, the areas of peaks A and B decreased significantly faster than those of peaks C and D.

[0124] Therefore, the decrease in the integrated area values ​​of the A, B, C and D signals was evaluated and plotted as a function of time. The results obtained are shown graphically in Figure 7 , which highlights the observed differences between the behavior of the 4 peaks.

[0125] k X The pseudo-first-order rate constant (k X Correspondingly, = k A , k B , k C and k D ), which characterizes the dissociation rates of different enantiomeric pairs of the Gd(PCTA-tri-glutaric acid) complex, and the half-life (t 1 / 2 =ln2 / k X ), as explained in detail in Example 7. Mean half-life values ​​for mixtures of Gd(PCTA-tri-glutaric acid) isomers were also obtained by taking into account the percentage composition of the mixture. Table 2 summarizes the results obtained and compares them with some reference contrast agents cited in the literature, such as Gd-DOTA (Dotarem TM ) and Eu(PCTA) (europium complex of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) were compared.

[0126] These results demonstrate that the dissociation rates of the four enantiomeric pairs are quite different from each other.

[0127] In particular, the enantiomeric pair associated with Peak C surprisingly has the highest kinetic inertness and lower tendency to release Gd compared to all other possible isomers.

[0128] In fact, for example, the t measured for the pair of enantiomers C 1 / 2The value of is about 68 times higher than that of B, for example. In addition, the t of the enantiomeric pair associated with peak C is 1 / 2 The values ​​are also significantly higher than those measured for Eu(PCTA), which also has q=2 (see, for example, Tircso, G. et al. Inorg Chem 2006, 45(23), 9269-80), and are consistent with those reported in the literature for Gd-DOTA (Dotarem TM ) reported 1 / 2 The value is completely equivalent, Gd-DOTA (Dotarem TM ) is the commercially available contrast agent with the best stability and inertness.

[0129] Fractions enriched in this compound are then collected by flash chromatography (e.g. as explained in detail in Example 3), resulting in the enantiomeric pair associated with Peak C having a degree of purity of at least about 90% (expressed as HPLC area %, see Figure 2 ).

[0130] Surprisingly, a relaxivity value of r1 = 9.3 ± 0.1 mM was obtained for the collected enantiomeric pair -1 s -1 , which is significantly higher than the r1 value = 7.2 recorded for the isomer mixture of Gd(PCTA-tri-glutaric acid) in EP1931673B1 (under the same conditions).

[0131] The unexpected combination of higher relaxivity and increased inertness (resulting in higher tolerance) displayed by this enantiomer is of particular interest.

[0132] Therefore, efforts have been made to identify the enantiomeric conjugate associated with Peak C.

[0133] In particular, the stereoselective synthesis of the RRR and SSS isomers of Gd(PCTA-tri-glutaric acid) described in Examples 5 and 6, respectively, has been established, resulting in a crude product with a major compound having an HPLC retention time t of peak C identical to that of peak C using conventional reverse phase HPLC. r By using the related isomer (R)-(-)-5-oxotetrahydrofuran-2-carboxylic acid as a key intermediate, the corresponding SSS isomer of Gd(PCTA-tri-glutaric acid) was also obtained with the same HPLC retention time ( Figure 4 ).

[0134] The synthesis of Gd(PCTA-tris-glutaric acid) by using (2S)-methyl bromoglutarate allows obtaining complexes which are essentially isomeric mixtures indistinguishable from the isomeric mixtures collected using racemic methyl bromoglutarate (as disclosed in the prior art).

[0135] The enantiomeric pair associated with peak C was then analyzed by comparison with the synthetic SSS and RRR isomers of Gd(PCTA-tri-glutaric acid) using a specific chiral HPLC method (capable of separating the individual enantiomers of the pair). Figure 5 The chromatogram in confirms that the 2 enantiomers associated with peak C have the same retention time as the RRR and SSS isomers synthesized with Gd(PCTA-tri-glutaric acid).

[0136] Furthermore, crystals were formed from enantiomeric pair C with guanidinium oxalate as disclosed in detail in Example 10. X-ray diffraction studies of single crystals allowed the establishment of the RRR configuration of the chiral center of the glutaric acid arm of the molecule, such as Fig. 9 It can be seen that there are equimolar ratios of RRR and SSS isomers in each unit cell of the crystal and thus the RRR / SSS racemic nature of the pair ( Fig.10 ).

[0137] All of these results confirm the establishment that the compounds associated with Peak C are actually composed of the RRR / SSS enantiomeric pair of Gd(PCTA-tris-glutaric acid) or RRR / SSS Gd(PCTA-tris-glutaric acid) as used interchangeably herein.

[0138] More specifically, the above results allow to define that the compound corresponding to the peak C identified in the present invention comprises [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium or RRR-Gd(PCTA-tri-glutaric acid) of the following formula:

[0139]

[0140] and the corresponding mirror image 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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium or SSS-Gd(PCTA-tri-glutaric acid) of the following formula

[0141]

[0142] wherein the mixture is further represented by formula Ic

[0143]

[0144] Thus, one aspect of the present invention is the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid), the individual enantiomers of the pair, mixtures thereof, pharmaceutically acceptable salts thereof, amide derivatives thereof, and compositions comprising the same.

[0145] In particular, one embodiment of the present invention relates to compounds which are preferably selected from: single enantiomers [(αR,α'R,α"R)-α,α',α"-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (RRR enantiomer); the corresponding mirror images thereof; Like isomers, i.e., single enantiomers [(αS,α'S,α"S)-α,α',α"-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (SSS enantiomer); its RRR / SSS enantiomeric pair; and pharmaceutically acceptable salts thereof.

[0146] According to a preferred embodiment, the present invention relates to the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid), more simply identified as "RRR / SSS Gd(PCTA-tri-glutaric acid)" in other parts of this document, which comprises a mixture of two individual RRR and SSS enantiomers of the complex, for example, according to one embodiment of the present invention, a racemic mixture thereof or a salt thereof.

[0147] Another aspect of the present invention relates to Gd(PCTA-tri-glutaric acid) enriched in any of the above enantiomers or mixtures thereof.

[0148] The expression "enriched" used to refer to an isomer or enantiomer or enantiomeric pair according to the invention (in particular when referring to Gd(PCTA-tri-glutaric acid) or its amide derivatives) includes within its meaning isomeric mixtures in which such isomer, enantiomer or enantiomeric pair is present in a higher amount with respect to the amount typically contained in the mixture obtained according to the prior art non-stereoselective synthesis operations.

[0149] This enrichment (relating to Gd(isomers or enantiomeric pairs of PCTA-tri-glutaric acid)) corresponds, for example, to an amount of at least 50% of such isomers or enantiomeric pairs in the mixture, preferably at least 60%, more preferably at least 70% and even more preferably at least 80%, for example at least 90%.

[0150] In particular, another aspect of the present invention relates to an isomeric mixture of Gd(PCTA-tri-glutaric acid) comprising at least 50% (relative to its isomeric composition, i.e. relative to the sum of the individual isomers constituting the isomeric mixture of Gd(PCTA-tri-glutaric acid)) of any of the above isomers, i.e. wherein at least 50% of the isomeric mixture of the Gd(PCTA-tri-glutaric acid) complex is composed of 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-triacetate (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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium; or a mixture thereof; or a salt thereof (the remainder of the complex is represented by an indistinguishable mixture of any other possible isomers thereof).

[0151] Thus, one embodiment of the present invention relates to Gd(PCTA-tri-glutaric acid) or a salt thereof, wherein the corresponding enantiomer RRR or enantiomer SSS or a RRR / SSS mixture of these enantiomers represents at least 50% of the isomer mixture constituting the acid or salt (e.g. on a molar basis).

[0152] Preferably, the enrichment of Gd(PCTA-tri-glutaric acid) (in one of the above enantiomers or a mixture thereof) is at least 60%, more preferably at least 70%, most preferably at least 80%, such as at least 90%.

[0153] More preferably, the enrichment is in the RRR / SSS enantiomer pair of Gd(PCTA-tris-glutaric acid).

[0154] In a preferred embodiment, the present invention relates to the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid), or to an isomeric mixture of Gd(PCTA-tri-glutaric acid) comprising at least 50% of its RRR / SSS enantiomeric pair, i.e. in other words, to Gd(PCTA-tri-glutaric acid), wherein at least 50% of the complexes consist of its RRR / SSS enantiomeric pair.

[0155] For example, 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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium or Gd(PCTA-tri-glutaric acid) enriched in 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 "pyclen" as used interchangeably herein, with dimethyl (2S)-2-[(trifluoromethylsulfonyl)oxy]glutarate as better described in detail in Example 5.

[0156] Likewise, alternative use of dimethyl (2R)-2-[(trifluoromethylsulfonyl)oxy]glutarate (eg, as described in Example 6) allows access to the corresponding SSS isomers of Gd(PCTA-tris-glutaric acid) or Gd(PCTA-tris-glutaric acid) appropriately enriched therewith.

[0157] The stereoselective synthesis of the RRR and SSS isomers of Gd(PCTA-tri-glutaric acid) is novel and constitutes another embodiment of the present invention.

[0158] In another embodiment, the present invention relates to the above enantiomer, enantiomeric pair or enriched Gd(PCTA-tri-glutaric acid) in the form of a pharmaceutically acceptable salt for use as a contrast agent particularly suitable for magnetic resonance imaging (MRI) analysis.

[0159] More specifically, another embodiment of the present invention relates to a pharmaceutically acceptable salt of a compound selected from a single enantiomer RRR or a single enantiomer SSS or a RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid) or a Gd(PCTA-tri-glutaric acid) at least 50% enriched in one of these single enantiomers, or preferably a RRR / SSS enantiomeric mixture, which is used as a contrast agent, particularly suitable for magnetic resonance imaging (MRI) analysis.

[0160] For example, suitable examples of pharmaceutically acceptable salts include salts with cations of an inorganic base selected from an alkali metal or alkaline earth metal (such as potassium, sodium, calcium or magnesium) or an organic base cation selected from ethanolamine, diethanolamine, morpholine, glucosamine, N-methylglucamine, N,N-dimethylglucamine or a cation of an amino acid selected from lysine, arginine and ornithine.

[0161] According to another aspect, the invention relates to a conjugate of one of the above compounds or the isomeric mixture, preferably with an amine of formula NHR1R2.

[0162] One embodiment of the present invention relates to an amide derivative of the RRR enantiomer, the SSS enantiomer or a mixture of these two enantiomers of Gd(PCTA-tri-glutaric acid) of formula (II A)

[0163] F(NR1R2)3(II A).

[0164] Another embodiment of the present invention relates to an isomeric mixture of an amide derivative of Gd(PCTA-tri-glutaric acid) of formula (II B) comprising at least 50% of a single enantiomer RRR or an enantiomer SSS or a mixture of these enantiomers

[0165] F'(NR1R2)3(II B).

[0166] In the above-mentioned formulae (II A) and (II B), F, F', R1 and R2 have the same meanings as defined above.

[0167] Preferred examples include amide derivatives of the above formula (II A), wherein F is a mixture of RRR and SSS enantiomeric residues of Gd(PCTA-tri-glutaric acid) (or a pair of RRR / SSS enantiomeric residues); or amide derivatives of the above formula (II B), wherein F' is an isomeric mixture of Gd(PCTA-tri-glutaric acid) residues, which contains at least 50% of a mixture of RRR and SSS enantiomeric residues.

[0168] In a preferred embodiment, the present invention relates to an amide derivative of the above-mentioned formula (II B), wherein F' is an isomeric mixture of the Gd (PCTA-tri-glutaric acid) residue of the above-mentioned formula III, which contains at least 50% of a mixture of the corresponding RRR and SSS enantiomeric residues of formula (IIIA) and (IIIB).

[0169] Preferably, in these amide derivatives, F' is a mixture of residues enriched in RRR and SSS enantiomers by at least 60% (i.e. comprising at least 60%), more preferably at least 70%, most preferably at least 80%, for example particularly preferably at least 90%. Suitable examples include amide derivatives of formula (II B) above, wherein R1 is H, and R2 is C1-C3 alkyl, which is substituted by one or more, preferably one or two and more preferably two hydroxyl groups.

[0170] In a preferred embodiment, the present invention relates to an isomeric mixture of an amide derivative of the above formula (II B), 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 serinol residue, even more preferably an isoserinol residue, for example selected from R isoserinol, S isoserinol or racemic isoserinol. Most preferably, the amide compound has racemic isoserinol.

[0171] For example, non-limiting representative examples of the above compounds include:

[0172] - gadolinium of the formula [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9] (or the isomer SSS-SSS);

[0173]

[0174] - [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or isomer RRR-RRR) of the following formula

[0175]

[0176] - [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or isomer RRR-SSS) of the following formula

[0177]

[0178] - [(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-triacetate (3-)-κN3,κN6,κN9,κN15,κO3,κO6,κO9]-gadolinium (or isomer SSS-RRR) of the following formula

[0179]

[0180] or the corresponding isomers, wherein the isoserinol moiety is in RSR, SSR, SRS, RSS or RRS configuration.

[0181] The amide of formula (II B) with isoserinol has the same molecular formula as the gadolinium polycyclic phenol, but comprises a central part F', an RRR isomer or SSS isomer residue enriched with at least 50% of Gd(PCTA-tri-glutaric acid), or more preferably a mixture of RRR and SSS enantiomeric residues enriched with Gd(PCTA-tri-glutaric acid).

[0182] Notably, regardless of the type of isoserinol used (i.e., whether it was the R or S isomer, or racemic isoserinol), its conjugation with the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid) yielded conjugates such as Figure 6 As shown in , the conjugates have the same retention time and are thus indistinguishable by HPLC. Figure 6 It was further shown that the retention time was the same as that of the peak D' separated from the gadolinium polycyclic phenol obtained as an isomer mixture as in Example 2 by HPLC.

[0183] Surprisingly, the improved properties of RRR / SSS Gd(PCTA-tri-glutaric acid) were essentially maintained after its conjugation to isoserinol, regardless of the configuration of the coupled isoserinol.

[0184] In particular, conjugation of isoserinol with RRR / SSS Gd(PCTA-tri-glutaric acid) leads to amide derivatives that have the same molecular formula as the gadolinium polycyclic phenols obtained as isomer mixtures using prior art synthetic procedures, but are kinetically inert and have higher relaxivity.

[0185] In fact, the same test performed to evaluate the kinetic inertness of 4 different enantiomeric pairs isolated from an isomeric mixture of Gd(PCTA-tri-glutaric acid) was repeated by using gadolinium polycyclic phenol (isomer mixture) as obtained in Example 2 and amide derivatives obtained by conjugation of RRR / SSS Gd(PCTA-tri-glutaric acid) with i) R-isoserinol; ii) S-isoserinol; and iii) racemic isoserinol, respectively.

[0186] The average half-life of the conjugated compound and the gadolinium polycyclic phenol (mixture of isomers) was calculated taking into account the decrease in the total HPLC area over time as described in detail in Example 8. For the amide derivatives obtained by the reaction of RRR / SSS Gd (PCTA-tri-glutaric acid) with i) R-isoserinol; ii) S-isoserinol; and iii) racemic isoserinol, k was also calculated by fitting the area-time data pairs performed by RRR / SSS Gd (PCTA-tri-glutaric acid) as in Example 7. X Pseudo-first-order rate constant and half-life (t 1 / 2 =ln2 / k X ).

[0187] The obtained results are summarized in Table 3 and compared with some reference contrast agents in the literature such as Gd-DOTA (Dotarem TM ) and Eu(PCTA) involved in the comparison.

[0188] On the one hand, the data from Table 3 show the estimated t values ​​of the complexes obtained by coupling RRR / SSS Gd(PCTA-tri-glutaric acid) with R, S and racemic-isoserinol. 1 / 2 The values ​​were compared with the t values ​​calculated by fitting the area-time kinetic data. 1 / 2 There is very good agreement between the values.

[0189] On the other hand, the data in Table 3 highlight that all t values ​​of the amide compounds obtained by the reaction of RRR / SSS Gd(PCTA-tri-glutaric acid) with i) R-isoserinol; ii) S-isoserinol; and iii) racemic isoserinol are 1 / 2 The values ​​are all about 8 times higher than those of gadolinium polycyclic phenol (isomer mixture), thus confirming that the higher kinetic inertness shown by RRR / SSS Gd(PCTA-tri-glutaric acid) is substantially maintained even after its coupling with isoserinol.

[0190] The overall consistency of the half-life values ​​obtained for the different coordination compounds resulting from the conjugation of RRR / SSS Gd(PCTA-tri-glutaric acid) with R, S or racemic isoserinol also suggests that the chirality of the isoserinol side chain has no effect on the kinetic inertness of the final complex. The r1 relaxation rates of the compounds obtained by conjugation of RRR / SSS Gd(PCTA-tri-glutaric acid) with R, S and racemic isoserinol were also measured under the same conditions as used for gadolinium polycyclic phenols in the literature.

[0191] The obtained results are compared in Table 5. Furthermore, regardless of the configuration of the attached isoserinol, the r1 relaxivities of the conjugated compounds obtained from RRR / SSS Gd(PCTA-tri-glutaric acid) measured in water and HSA were higher than those reported in the related art for gadolinium polycyclic phenols.

[0192] Thus, by conjugating the RRR / SSS Gd(PCTA-tri-glutaric acid) of the present invention with isoserinol, an amide derivative is obtained, which, although having the same structure as the gadolinium polycyclic phenol compound, is characterized by improved kinetic inertness and higher relaxivity.

[0193] Crystals were then obtained from the amide derivative obtained by conjugation of RRR / SSS Gd(PCTA-tri-glutaric acid) with racemic isoserinol as described in detail in Example 10. X-ray diffraction studies of single crystals obtained from the ternary complex formed between the carbonate anion and the amide derivative confirmed the RRR / SSS configuration of the glutaric acid arm of the core molecule C.

[0194] The X-ray structures and statistical analyses of all collected crystals are as follows Fig.11 as shown in .

[0195] Thus, the results of the stereoselective syntheses of the individual RRR or SSS isomers and the (crystal) structures recorded from the enantiomeric pair C and its conjugate with isoserinol are consistent with each other and allow the establishment of the Figure 1 The compound of peak C in the HPLC is actually equivalent to the RRR / SSS enantiomer pair of Gd(PCTA-tri-glutaric acid).

[0196] The synthesis of RRR and SSS isomers of Gd(PCTA-tri-glutaric acid) represents another embodiment of the present invention.

[0197] In particular, another embodiment of the present invention relates to a stereoselective process for preparing Gd(PCTA-tri-glutaric acid) enriched in isomers [(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), the process comprising:

[0198] a) obtaining dimethyl (2S)-2-[(trifluoromethylsulfonyl)oxy]glutarate of the following formula

[0199]

[0200] b) Alkylation of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene of the following formula with the collected (2S)-2-[(trifluoromethylsulfonyl)oxy]pentanedioic acid dimethyl ester

[0201]

[0202] In one embodiment, the process is able to obtain Gd(PCTA-tri-glutaric acid) which is enriched in at least 55%, preferably at least 60%, more preferably at least 70% and most preferably at least 80%, for example about 85% of the desired RRR isomer of the complex.

[0203] Similarly, the alternative use of dimethyl (2R)-2-[(trifluoromethylsulfonyl)oxy]glutarate in step a) of the process allows for the corresponding SSS isomer of Gd(PCTA-tri-glutaric acid) or Gd(PCTA-tri-glutaric acid) suitably enriched in this isomer.

[0204] In still another embodiment, the present invention relates to the RRR / SSS pair of Gd(PCTA-tri-glutaric acid) alcohol enantiomers or Gd(PCTA-tri-glutaric acid) enriched with at least 50% of this same enantiomeric pair, which is used as an intermediate for the preparation of its derivatives, such as preferably amide derivatives.

[0205] In still another embodiment, the present invention relates to a process for the synthetic preparation of an amide derivative of Gd(PCTA-tri-glutaric acid) of formula (II A),

[0206] F(NR1R2)3(II A)

[0207] Wherein F, R1 and R2 are as described above, the method comprises:

[0208] a) obtaining the RRR or SSS isomer of Gd(PCTA-tri-glutaric acid) or a mixture thereof; and

[0209] b) converting the isomer or isomer mixture obtained in step a) into the desired amide derivative.

[0210] For example, step a) of the process leading to the RRR or SSS isomer of Gd(PCTA-tri-glutaric acid) is carried out as described above and is provided, for example, as described in detail in Examples 5 and 6.

[0211] On the other hand, step b) of the method is performed according to conventional procedures, such as reported in the above-mentioned prior art.

[0212] Another aspect of the present invention relates to a method for synthesizing an isomeric mixture of an amide derivative of Gd(PCTA-tri-glutaric acid) of formula (II B) above,

[0213] F(NR1R2)3(II B)

[0214] Wherein F', R1 and R2 are as described above, the method comprises:

[0215] a') obtaining an isomeric mixture of Gd(PCTA-tri-glutaric acid) comprising at least 50% of the corresponding enantiomer RRR or SSS, or preferably a mixture thereof; and

[0216] b') converting the isomeric mixture of Gd(PCTA-tri-glutaric acid) obtained in step a') into an isomeric mixture of the corresponding amide derivative of interest.

[0217] For example, step a') of the method resulting in an isomeric mixture of Gd(PCTA-tri-glutaric acid) containing at least 50% of its RRR / SSS enantiomeric pair can be obtained, for example, by a chromatographic method comprising preparative HPLC or flash chromatography, starting from Gd(PCTA-tri-glutaric acid) obtained as an isomeric mixture using known procedures (stating), for example as disclosed in Example 3.

[0218] On the other hand, step b') of the process, which consists of coupling the isomer-enriched mixture of Gd(PCTA-tri-glutaric acid) collected from step a') with the amine of interest, can be performed according to conventional procedures, such as the methods cited in the above-mentioned prior art.

[0219] For example, the product recovered from step a') can be reacted with isoserinol by using the synthetic procedure provided in detail in Example 4.

[0220] Another embodiment of the present invention relates to the amides of formula (II A) or (II B) as described above for use as contrast agents, particularly suitable for magnetic resonance imaging (MRI) analysis.

[0221] In particular, in another embodiment, the present invention relates to compounds selected from: a single RRR or SSS enantiomer of Gd(PCTA-tri-glutaric acid), a mixture of such RRR / SSS enantiomers, an isomeric mixture of Gd(PCTA-tri-glutaric acid) (which is enriched in at least 50% of one of such single RRR or SSS enantiomers, or a mixture thereof), a pharmaceutically acceptable salt thereof, an amide derivative thereof of formula (II A) or (II B), which are used for the preparation of pharmaceutical preparations for diagnostic imaging of human or animal organs, tissues or regions or biological samples using MRI technology, the biological samples including cells, biological fluids and biological tissues, which are derived from living mammalian patients, and preferably human patients.

[0222] Another aspect of the present invention relates to a pharmaceutical composition for diagnostic use, comprising at least one of the above-mentioned isomeric compounds or isomeric mixtures of the present invention as described above or their pharmaceutically acceptable salts or amide derivatives thereof, mixed with one or more physiologically acceptable excipients, diluents or solvents.

[0223] Preferably, the pharmaceutical composition comprises an amide derivative of the above formula (II A), wherein in the formula (II A):

[0224] F is the residue of the RRR / SSS enantiomeric pair of Gd(PCTA-tri-glutaric acid);

[0225] Or more preferably, an isomeric mixture of the amide derivative of the above formula (II B), wherein in formula (II B), F' is an isomeric mixture of Gd(PCTA-tri-glutaric acid) enriched in at least 50% of the RRR / SSS enantiomeric pair, and -NR1R2 is an isoserinol residue.

[0226] In a preferred embodiment, the pharmaceutical composition comprises an amide compound of formula (II B), wherein F' is a residue of Gd(PCTA-tri-glutaric acid), which is enriched by at least 60%, preferably at least 70%, more preferably at least 80%, for example most preferably at least 90% of the RRR / SSS enantiomer pair of Gd(PCTA-tri-glutaric acid), which is conjugated to isoserinol and has the following formula:

[0227]

[0228] In another aspect, the present invention relates to an MRI contrast agent comprising an effective amount of at least one isomeric compound or isomeric mixture of the present invention as described above or a pharmaceutically acceptable salt or amide derivative thereof in combination with one or more pharmaceutically acceptable excipients, diluents or solvents.

[0229] In this regard, unless otherwise indicated, the term "effective amount" or "effective dose" as used herein refers to any amount of Gd (PCTA-tri-glutaric acid) or a pharmaceutically acceptable salt thereof or an amide derivative thereof of formula (II A) or (II B) of the present invention, or a pharmaceutical composition thereof, which is sufficient to meet its intended diagnostic purpose: i.e., for example, ex vivo visualization of biological elements, including cells, biological fluids and biological tissues, or in vivo diagnostic imaging of a patient's body organ, tissue or region.

[0230] As used herein, unless otherwise indicated, the term "subject patient" or "patient" refers to a living human or animal patient, and preferably a human undergoing MR diagnostic evaluation.

[0231] Details concerning dosage, dosage form, mode of administration, pharmaceutically acceptable carriers, excipients, diluents, adjuvants, etc. are well known in the art.

[0232] The following section reports non-limiting examples of preferred compounds of the invention, the procedures allowing their preparation and their characterization, with the purpose of illustrating the invention in more detail without limiting its scope.

[0233] Experimental Section

[0234] HPLC characterization of obtained compounds

[0235] General Operation

[0236] Run 1: HPLC characterization of Gd(PCTA-tri-glutaric acid) (isomer mixture and single / enriched isomer).

[0237] An Agilent 1260 Infinity II system was used to perform HPLC characterization of Gd(PCTA-tris-glutaric acid) obtained as a mixture of isomers from Example 1. The experimental setup for the HPLC measurements is outlined below.

[0238] Analysis conditions

[0239]

[0240]

[0241] The obtained HPLC chromatogram is as follows Figure 1 as shown in .

[0242] The HPLC chromatogram of the enriched enantiomer pair C is shown in Figure 2 as shown in .

[0243] Procedure 2: HPLC characterization of gadolinium polycyclic phenol (mixture of isomers) and compounds obtained by coupling of enantiomeric pairs C with R, S or racemic isoserinol.

[0244] HPLC characterization of gadolinium polycyclic phenol as isomeric mixture from Example 2 or as compounds obtained by conjugation of enantiomeric pair C of Gd(PCTA-tris-glutaric acid) with R, S or racemic isoserinol was performed using a Thermo Finnigan LCQ DECA XPPlus system. The experimental setup for HPLC measurements is outlined below.

[0245] Analysis conditions

[0246]

[0247]

[0248] The obtained HPLC chromatogram is as follows Figure 6 as shown in .

[0249] Procedure 3: Chiral HPLC method for enantiomer separation of compound C

[0250] A specific chiral HPLC method was established to separate the RRR and SSS enantiomers of enantiomeric pair C (Compound VI) prepared as described in Example 3. Separation and characterization of enantiomers was performed using an Agilent 1200 system or a Waters Alliance 2695 system. The experimental setup for HPLC measurements is outlined below.

[0251] Analysis conditions

[0252]

[0253] The obtained HPLC chromatogram is as follows Figure 5 ), compared with the pure RRR enantiomer (Compound XII of Example 5, Tr.7.5min.) and the pure SSS enantiomer (Compound XVII of Example 6, Tr.8.0min), respectively. Figure 5 ) as shown.

[0254] Example 1: Synthesis of Gd(PCTA-tri-glutaric acid) (isomer mixture)

[0255] Gd(PCTA-tri-glutaric acid) has been prepared as an indistinguishable mixture of stereoisomers according to the following Synthesis Scheme 1 by using the procedures reported in the above prior art:

[0256] Solution 1

[0257]

[0258] a) Preparation of Compound II

[0259] Racemic glutamic acid (33.0 g, 0.224 mol) and sodium bromide (79.7 g, 0.782 mol) were suspended in 2M HBr (225 mL). The suspension was cooled to -5°C and NaNO2 (28.0 g, 0.403 mol) was added slowly in small portions over 2.5 hours, maintaining the internal temperature below 0°C. The yellow mixture was stirred at -5°C for another 20 minutes; then concentrated sulfuric acid (29 mL) was added dropwise to the mixture. The resulting dark brown mixture was warmed to RT and then extracted with ether (4x150 mL). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated to give a brown oil (21.2 g) which was used in the next step without further purification.

[0260] 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 pale yellow solution was stirred at RT for 2 days. The solvent was then removed in vacuo, and the crude oil was dissolved in dichloromethane (200 mL), washed with 5% NaHCO3 aqueous solution (4x50 mL), water (1x50 mL) and brine (1x50 mL). The organic phase was concentrated and purified on silica, eluted with petroleum ether-ethyl acetate 3:1, to give 19.5 g of pure product (yield 33%).

[0261] b) Preparation of Compound IV

[0262] 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 (pyclen) compound (III) (3.80 g, 0.018 mol) and K2CO3 (11.2 g, 0.0808 mol) in acetonitrile (150 mL). The yellow suspension was heated at 65°C for 24 h, then the salts were filtered off and the organic solution was concentrated. The orange oil was dissolved in dichloromethane and the product was extracted with 1M HCl (4 x 50 mL). The organic phases were combined, cooled with ice, and brought to pH 7-8 with 30% aqueous NaOH. The product was then extracted with dichloromethane (4 x 50 mL) and concentrated to give a brown oil (10.1 g, 73% yield). The compound was used in the next step without further purification.

[0263] c) Preparation of Compound V

[0264] 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 h. The ethanol was concentrated; the solution was cooled with ice and brought to pH 2 with concentrated HCl. The ligand was purified on the resin Amberlite XAD 1600, eluted with a water-acetonitrile mixture, and 5.7 g was obtained after freeze drying as a white solid (yield 73%). The product was characterized by several peaks in HPLC.

[0265] d) Preparation of Compound VI

[0266] Compound V (5.25 g, 0.0088 mol) was dissolved in deionized water (100 mL) and the solution was brought to pH 7 with 2 M NaOH (20 mL). GdCl3 solution (0.0087 mol) was added slowly at RT, the pH was adjusted to 7 with 2 M NaOH, and the complexation was checked with xylenol orange. Once the complexation was complete, the solution was concentrated and purified on the resin Amberlite XAD 1600, eluting with a water-acetonitrile gradient to remove salts and impurities. After freeze drying, the pure compound was obtained as a white solid (6.79 g, yield 94%). The product was characterized by HPLC; the obtained HPLC chromatogram characterized by several peaks is shown in Figure 1 as shown in .

[0267] Even by using (S)-methyl α-bromoglutarate obtained starting from L-glutamic acid, a compound completely equivalent to compound VI can be obtained, which is composed of Figure 1 The HPLC chromatograms consisted of a mixture of isomers with essentially overlapping patterns.

[0268] Example 2: Synthesis of Gadolinium Polycyclic Phenol (Isomeric Mixture)

[0269] As disclosed in EP11931673 B1, gadolinium polycyclic phenol was prepared as an indistinguishable mixture of stereoisomers by coupling the isomeric mixture of Gd(PCTA-tri-glutaric acid) obtained from Example 1 with racemic isoserinol according to the following synthetic scheme 2:

[0270] Solution 2

[0271]

[0272] Preparation of Compound VII

[0273] Compound VI (0.90 g, 0.0011 mol) obtained from Example 1 was added to a solution of racemic isoserinol (0.40 g, 0.0044 mol) in water adjusted to pH 6 with concentrated HCl. N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (1.0 g, 0.0055 mol) and hydroxybenzotriazole (HOBT) (0.12 g, 0.00088 mol) were then added, and the resulting solution was stirred at pH 6 and RT for 24 h. The product was then purified by preparative HPLC on silica C18, eluting with a water / acetonitrile gradient. The fractions containing the pure compound were concentrated and freeze-dried to give a white solid (0.83 g, yield 78%). The product was characterized by HPLC; the HPLC chromatogram obtained is shown in Figure 4 As shown in a.

[0274] Example 3: Separation of enantiomeric pairs associated with Peak C.

[0275] 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 HCl. The resulting solution was loaded into a pre-packed silica C18 column ( SNAP ULTRA C18 120 g, HP-sphere C18 25 μm), purified by automated flash chromatography system, eluted with deionized water (4CV) and then with a very slow acetonitrile gradient. The enriched fractions of the enantiomeric pair associated with peak C were combined, concentrated, and freeze-dried to give a white solid (200 mg).

[0276] The HPLC chromatogram of the obtained enriched enantiomer pair C is as follows Figure 2 as shown in .

[0277] The corresponding MS spectra (Gd(H4L) + :752.14m / z) Figure 3 as shown in .

[0278] Example 4: Coupling of enantiomeric pair C with isoserinol.

[0279] a) Coupling of enantiomeric pair C with R-isoserinol.

[0280] The enriched enantiomer pair C (34 mg, titer 90%, 0.040 mmol) collected, for example, as in Example 3, was dissolved in deionized water (5 mL), R-isoserinol (16 mg, 0.17 mmol) was added, and the pH was adjusted to 6 with HCl 1 M. Then EDCI·HCl (39 mg, 0.20 mmol) and HOBT (3 mg, 0.02 mmol) were added, and the solution was stirred at pH 6 for 48 h at RT. The solution was concentrated and loaded onto a pre-packed silica C18 column ( SNAP ULTRA C18 12 g, HP-sphere C18 25 μm), using an automated flash chromatography system with a water / acetonitrile gradient elution. Fractions containing pure product or showing a main peak with an area greater than 90% on HPLC were combined, concentrated, and freeze-dried to give a white solid (21 mg, yield 54%).

[0281] The HPLC chromatogram of the obtained product is as follows Figure 6 As shown in b.

[0282] b) Coupling of enantiomeric pairs C and S-isoserinol

[0283] The enriched enantiomer pair C (55 mg, titer 90%, 0.066 mmol) collected as in Example 3 was dissolved in deionized water (5 mL), S-isoserinol (34 mg, 0.29 mmol) was added, and the pH was adjusted to 6 with 1 M HCl. Then EDCI·HCl (64 mg, 0.33 mmol) and HOBT (4.5 mg, 0.033 mmol) were added, and the solution was stirred at pH 6 for 48 h at RT. The solution was concentrated and loaded onto a pre-packed silica C18 column ( SNAP ULTRA C18 12 g, HP-sphere C18 25 μm), eluted with water / acetonitrile gradient using an automated flash chromatography system. Fractions containing pure product or a main peak showing an area greater than 90% on HPLC were combined, concentrated, and freeze-dried to give a white solid (52 mg, yield 81%).

[0284] The HPLC chromatogram of the obtained product is as follows Figure 6 As shown in c.

[0285] c) Coupling of enantiomeric pair C with racemic isoserinol.

[0286] The enriched enantiomer pair C (54 mg, titer 90%, 0.065 mmol) collected as in Example 3 was dissolved in deionized water (5 mL), racemic isoserinol (27 mg, 0.29 mmol) was added, and the pH was adjusted to 6 with 1 M HCl. Then EDCI·HCl (62 mg, 0.32 mmol) and HOBT (4.3 mg, 0.032 mmol) were added, and the solution was stirred at pH 6 for 24 h at RT. The solution was concentrated and loaded onto a pre-packed silica C18 column ( SNAP ULTRA C18 12 g, HP-sphere C18 25 μm), eluted with water / acetonitrile gradient using an automated flash chromatography system. Fractions containing pure product or a main peak showing an area greater than 90% on HPLC were combined, concentrated, and freeze-dried to give a white solid (60 mg, yield 95%).

[0287] The HPLC chromatogram of the obtained product is as follows Figure 6 As shown in d.

[0288] Example 5: Stereoselective synthesis of RRR Gd(PCTA-tri-glutaric acid) (Compound XII).

[0289] RRR-enriched Gd(PCTA-tris-glutaric acid) acid was prepared by following Synthesis Scheme 3.

[0290] Solution 3

[0291]

[0292] include:

[0293] a) Preparation of Compound VIII

[0294] The preparation was carried out as reported in Tetrahedron 2009, 65, 4671-4680.

[0295] In particular: 37% HCl aqueous solution (50 μL) was added to a solution of (S)-(+)-5-oxotetrahydrofuran-2-carboxylic acid (2.48 g, 0.019 mol) (commercially available) in anhydrous methanol (20 mL). The solution was refluxed in an N2 atmosphere for 24 h. After ice cooling, 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 give a colorless oil (2.97 g, 89% yield).

[0296] b) Preparation of compounds IX and X

[0297] 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. The solution was cooled at -40 °C and trifluoromethanesulfonic anhydride (0.49 mL, 2.91 mmol) was added slowly. The dark solution was stirred at -40 °C for 1 h and then a solution of compound III (104 mg, 0.506 mmol) in anhydrous dichloromethane (3 mL) and triethylamine (1 mL, 7.56 mmol) was added. The solution was allowed to reach RT and stirred at RT overnight. The organic solution was then washed with 2M HCl (4 x 10 mL) and the aqueous phase was extracted with dichloromethane (3 x 10 mL). The organic phases were combined and concentrated in vacuo to give 400 mg of a brown oil which was used in the next step without further purification.

[0298] c) Preparation of Compound XI

[0299] Compound X (400 mg, 0.59 mmol) was dissolved in methanol (2.5 mL) and 5 M NaOH (2.5 mL). The brown solution was heated at 80 °C for 22 h to ensure complete hydrolysis. The methanol was concentrated and the solution was brought to pH 1 with concentrated HCl and purified by an automated flash chromatography system ( SNAP ULTRA C18 12g, HP-sphere C18 25μm) was purified by gradient elution with deionized water / acetonitrile. The fractions containing the pure product were combined, concentrated, and freeze-dried (64mg, yield 18%). HPLC showed a main peak.

[0300] d) Compound XII

[0301] 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 RT overnight and the coordination was checked by xylene orange and HPLC. HPLC of the crude product showed the desired RRR isomer as the main peak: about 80% area%. The mixture was brought to pH 2 with concentrated HCl and purified by an automated flash chromatography system with a silica C18 pre-packed column ( SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) was purified by gradient elution with deionized water / acetonitrile. The fractions containing the product were combined, concentrated, and freeze-dried (36 mg, yield 90%).

[0302] By reacting the collected compounds with isoserinol, for example by using the procedure of Example 2, the corresponding RRR amide derivatives can then be obtained.

[0303] Example 6: Stereoselective synthesis of SSS Gd(PCTA-tri-glutaric acid) (Compound XVII).

[0304] SSS-enriched Gd(PCTA-tri-glutaric acid) acid has been similarly prepared by the following synthetic scheme 4

[0305] Solution 4

[0306]

[0307] include:

[0308] a) Preparation of Compound XIII

[0309] 37% HCl aqueous solution (100 μL) was added to a solution of (R)-(-)-5-oxotetrahydrofuran-2-carboxylic acid (5.0 g, 0.038 mol) (commercially available) in anhydrous methanol (45 mL). The solution was refluxed for 24 h in a N2 atmosphere. After ice cooling, NaHCO3 was added, the suspension was filtered, concentrated, and purified on silica with hexane / ethyl acetate 1:1. The fractions containing the pure product were combined and concentrated to give a colorless oil (6.7 g, 99% yield).

[0310] b) Preparation of Compounds XIV and XV

[0311] Compound XIII (470 mg, 2.67 mmol) was dissolved in anhydrous dichloromethane (6 mL) and triethylamine (0.93 mL, 6.67 mmol) was added. The solution was cooled to -40 °C and trifluoromethanesulfonic anhydride (0.50 mL, 3.07 mmol) was slowly added dropwise. The dark solution was stirred at -40 °C for 1 h and then compound III (140 mg, 0.679 mmol) and triethylamine (0.93 mL, 6.67 mmol) were added and the solution was slowly allowed to reach RT overnight. The organic solution was then extracted with water (3 x 5 mL) and 2M HCl (4 x 5 mL). The aqueous phase was further extracted with dichloromethane (3 x 10 mL). The organic phases were combined and concentrated in vacuo to give 350 mg of a brown oil which was used in the next step without further purification.

[0312] c) Preparation of Compound XVI

[0313] Compound XV (350 mg, 0.514 mmol) was dissolved in methanol (4.5 mL) and 5 M NaOH (4.5 mL). The brown solution was heated at 80 °C for 16 h to ensure complete hydrolysis. The methanol was concentrated and the solution was brought to pH 2 with concentrated HCl and purified by an automated flash chromatography system ( SNAP ULTRA C18 12g, HP-sphere C18 25μm) was purified by water / acetonitrile gradient elution. The fractions containing the pure product were combined, concentrated and freeze-dried (52mg, yield 17%). HPLC showed a main peak.

[0314] d) Preparation of Compound XVII

[0315] 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 RT and the coordination was checked by xylene orange and HPLC. HPLC of the crude product showed the desired SSS isomer as the main peak: about 85% area%. The solution was brought to pH 2.5 with concentrated HCl and purified by an automated flash chromatography system using a C18 pre-packed column ( SNAP ULTRA C18 12 g, HP-sphere C18 25 μm) was purified by gradient elution with water / acetonitrile. The fractions containing the pure product SSS were combined, concentrated, and freeze-dried (39 mg, yield 87%).

[0316] Example 7: Kinetic Study of the Dissociation Reaction of Gd(PCTA-tri-glutaric acid) (Isomeric Mixture) in 1.0 M HCl Solution (25°C)

[0317] The kinetic inertness of Gd(III)-complexes is characterized by the dissociation rate measured in 0.1-1.0 M HCl or by the rate of transmetallation reactions occurring in solutions with Zn(II) and Cu(II) or Eu(III) ions. However, the dissociation of lanthanide(III)-complexes formed with macrocyclic ligands is very slow and usually proceeds via a proton-assisted pathway without involving endogenous metal ions such as Zn 2+ and Cu 2+ .

[0318] We characterized the kinetic inertness of the complex Gd (PCTA-tri-glutaric acid) by the dissociation reaction rate occurring in 1.0M HCl solution. The complex (mixture of isomers from Example 1) (0.3 mg) was dissolved in 2.0 mL of 1.0M HCl solution and the solution kept at 25°C was tracked by HPLC over time. HPLC measurements were performed using an Agilent 1260 Infinity II system using analytical method 1.

[0319] There is a large excess of H + ([HCl] = 1.0 M) to ensure pseudo-first-order kinetic conditions.

[0320]

[0321] Where L is the protonated PCTA-tri-glutaric acid free ligand, and y is the number of protons attached to the ligand.

[0322] The HPLC chromatogram of Gd (PCTA-tri-glutaric acid) is characterized by the presence of 4 signals (A, B, C and D) with the same m / z ratio in the MS spectrum (Gd (H4L) +: 752.14 m / z). Each of these peaks can be reasonably attributed to one of the 4 pairs of enantiomers generated by the three stereocenters on the three glutaric acid arms of the molecule, previously identified in Table 1. In the presence of 1.0 M HCl, the HPLC chromatogram of the complex changes with time: in particular, the areas of the A, B, C and D peaks decrease, although in different ways for the different peaks, while new signals corresponding to the uncoordinated diastereomers are formed and grow with time. The differences in the reduction of the peak integrated areas can be explained by the different dissociation rates of the enantiomeric pairs associated with the different peaks.

[0323] In the presence of excess [H+], the dissociation reaction of the enantiomeric pair of Gd(PCTA-tri-glutaric acid) can be regarded as a pseudo-first-order reaction, and the reaction rate can be expressed by the following equation 2, where k A , k B , k C and k D is the pseudo-first-order rate constant calculated by fitting the area-time data pairs, and [A] t , [B] t , [C] t and [D] t is the total concentration of compounds A, B, C and D at time t.

[0324]

[0325] The decrease in the area values ​​of the A, B, C and D signals has been evaluated and plotted over time. The area values ​​of the A, B, C and D signals as a function of time are shown in FIG. Figure 7 as shown in .

[0326] The area value at time t is expressed by the following equation:

[0327]

[0328] Among them A t , A0 and A e are the area values ​​at time t, at the beginning and at the end of the reaction, respectively. k characterizes the dissociation rate of different enantiomer pairs of Gd(PCTA-tri-glutaric acid) complex X The pseudo-first-order rate constant (k X =k A , k B , k C and k D ) by fitting Figure 7 The area-time data is then applied to the above equation 3. This gives k X Rate constant and half-life (t 1 / 2 =ln2 / k X ), and the average half-life values ​​of the isomeric mixture of Gd (PCTA-tri-glutaric acid) calculated by taking into account the percentage composition of the mixture. The obtained values ​​are summarized in the following Table 2 and compared with some reference contrast agents (Gd-DOTA or DOTAREM TM ) involves a comparison of corresponding values.

[0329] Table 2. Characterization of different stereoisomers of Gd(PCTA-tri-glutaric acid). The rate constant (k) for the acid-catalyzed dissociation of Eu(PCTA) in 1.0 M HCl (pH 0) (25 °C) X ) and half-life (t 1 / 2 =ln2 / k X )

[0330]

[0331] a)Inorg.Chem.1992,31,1095-1099.

[0332] b) Tircso, G. et al. Inorg Chem 2006, 45(23), 9269-80.

[0333] The results in Table 2 clearly show that the k characterizing the acid-catalyzed dissociation of the enantiomeric pair associated with peak C is XThe rate constants are significantly smaller than the k of the stereoisomers of the Gd(PCTA-tri-glutaric acid) complex associated with peaks A, B, and D. X Rate constant. The half-life values ​​(t 1 / 2 ) show that the t 1 / 2 Value (t of Gd(PCTA-tri-glutaric acid) 1 / 2 The values ​​are t values ​​associated with peaks A, B, and D, respectively. 1 / 2 The values ​​were about 28, 68 and 8 times higher. In addition, the t 1 / 2 The value is slightly higher than that of Gd(DOTA) 1 / 2 Values ​​(in 1.0M HCl t 1 / 2 =23h).

[0334] Example 8: Kinetic study of the dissociation reaction of gadolinium polycyclic phenol (mixture of isomers from Example 2) and the coordination compound obtained by coupling the enantiomeric pair C with R, S and racemic isoserinol in 1.0 M HCl solution (25°C)

[0335] The kinetic inertness of all complexes was characterized by the dissociation reaction rate occurring in 1.0 M HCl solution. For each batch, the complex (0.4 mg) was dissolved in 2.0 mL of 1.0 M HCl solution and then the dissociation reaction was followed over time at 25° C. by HPLC. HPLC determinations were performed using a Thermo Finnigan LCQ DECA XPPlus system according to analytical procedure 2.

[0336] The HPLC chromatogram of the gadolinium polycyclic phenol collected as a mixture of isomers from Example 2 is characterized by the presence of four major peaks (identified as A', B', C' and D' for convenience) with identical MS and UV-Vis spectra. However, in the HPLC chromatogram of the coordination compound obtained by coupling the enantiomeric pair C with isoserinol, there is only one signal ( Figure 6 As observed, since the chirality of the isoserinol side group has no effect on the retention time of the coupled diastereomers, the presence of 4 signals in the HPLC chromatogram of gadolinium polycyclic phenol (mixture of isomers) can be explained by the presence of 4 enantiomeric pairs formed with the stereocenter of the glutaric acid residue: 1) RRR-SSS (signal D'), 2) RSR-SRS, 3) RRS-SSR, and 4) RSS-SRR.

[0337] In order to obtain information about the kinetic inertness of all the above complexes, in a large excess of H +([HCl] = 1.0M) to study their dissociation reaction to ensure that pseudo-first-order reaction conditions occur. As discussed above in Example 7 for Gd (PCTA-tri-glutaric acid) isomers, the progress of the reaction over time was checked by HPLC, and the peak area value of the complex was plotted as a function of time.

[0338] As expected, the integrated values ​​of A', B', C' and D' decrease over time, while the peak of the free ligand increases. Since the area value in the HPLC chromatogram is proportional to the concentration of gadolinium polycyclic phenol (mixture of isomers), the half-life of the dissociation reaction of gadolinium polycyclic phenol (mixture of isomers) can be estimated based on half of the sum of the area values. The half-life of gadolinium polycyclic phenol (mixture of isomers) was found to be 5.2 hours at 25°C and pH 0 (1.0M HCl). The half-life of the complex obtained by coupling the enantiomer pair C with R, S and racemic-isoserinol was also calculated based on half of the area value in the HPLC chromatogram. The half-life of the complex obtained by coupling the enantiomer pair C with R, S and racemic-isoserinol was 41, 43 and 44 hours at 25°C and pH 0 (1.0M HCl). The pseudo-first-order rate constant (k) characterizing the dissociation reaction rate of the complex obtained by coupling the enantiomeric pair C with R, S and racemic-isoserinol x ) can also be calculated by fitting the area-time kinetic data using Equation 3, as described above:

[0339]

[0340] Among them A t , A0 and A e The area values ​​at time t, at the start and at the end of the reaction are the k values ​​for the acid-catalyzed dissociation of the complex obtained by coupling the enantiomeric pair C with R, S and racemic-isoserinol. X Pseudo-first-order rate constant (half-life: t 1 / 2 ,t 1 / 2 =ln2 / k X ). The k obtained by fitting the kinetic data is x and t 1 / 2 The values ​​are summarized in Table 3 below and compared with corresponding values ​​from the literature for some reference contrast agents.

[0341] The t of the complex obtained by coupling the enantiomeric pair C with R, S with racemic-isoserinol was estimated from the area values ​​and calculated by fitting the area-time kinetic data. 1 / 2 The comparison of the values ​​is very consistent. The t 1 / 2The 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 isoserinol is approximately the same and 8 times higher than that measured for gadolinium polycyclic phenol (isomer mixture), which confirms that RRR-SSS Gd(PCTA-tri-glutaric acid) remains essentially kinetically inert even after coupling with isoserinol. In addition, the t 1 / 2 The values ​​also indicate that the chirality of the isoserinol side group does not affect the kinetically inert nature of the final complex.

[0342] Table 3. Rate constants (k) characterizing the acid-catalyzed dissociation of gadolinium polycyclic phenol (mixture of isomers), the coordination complex obtained by coupling the enantiomeric pair C with R, S and racemic-isoserinol, Dotarem and Eu(PCTA) in 1.0 M HCl (25 °C). X ) and half-life (t 1 / 2 =ln2 / k x )

[0343]

[0344] *Calculated based on half of the total area

[0345] **Calculated based on the fitting equation

[0346] Example 9: Relaxation Characteristics

[0347] The relaxation properties of the inventive PCTA-based coordination compounds were measured at 37°C and in different media (water and human plasma) at different magnetic field strengths, namely 0.47 and 1.41 T, and compared with the relaxivity values ​​measured under the same conditions for Gd-complexes with similar coordination clathrates.

[0348] Material

[0349] instrument

[0350] Longitudinal water proton relaxation rates (R1 = 1 / T1) were measured at 0.47 T using a Minispec MQ-20 spectrometer (Bruker Biospin, Germany) operating at a proton Larmor frequency of 20 MHz; MR experiments were performed at 1.41 T using a Minispec MQ-60 spectrometer (Bruker Biospin, Germany) operating at a proton Larmor frequency of 60 MHz.

[0351] method

[0352] Sample preparation

[0353] All test articles were used as received and diluted in the chosen medium (water or human plasma) by weighing the required amount of paramagnetic chelate complex to give a 5 or 10 mM starting solution.

[0354] Relaxation rate measurement

[0355] Five different concentration samples (0.1, 0.25, 0.5, 0.75 and 1 mM) have been prepared for each culture medium by further diluting the starting 5 or 10 mM solution.

[0356] Relaxation measurement

[0357] Relaxation rate measurements were performed at 0.47T and 1.41T with a preset temperature sample of 37°C, kept constant by means of a thermostatic bath connected to the spectrometer sample holder. The 5 sample solutions had been preliminarily preheated to 37°C in an external thermostatic bath and then placed in the internal bath for 10 minutes to ensure temperature stabilization. The longitudinal relaxation time T1 was measured by means of a standard inversion recovery sequence, where the inversion time (TI) was varied in 15 steps from 10 ms to at least 5 times T1. Statistical analysis (single exponential fit for T1 measurements, linear fit for evaluation of longitudinal relaxation rates) was performed using (Wolfram, USA). The errors of the calculated parameters were evaluated by fitting procedures.

[0358] result

[0359] Table 4 below shows the relaxivity values ​​r1 values ​​for Gd(PCTA-tri-glutaric acid) (isomeric mixture) reported in EP 1931673 B1 and the corresponding r1 values ​​obtained for the purified fractions of RRR / SSS Gd(PCTA-tri-glutaric acid) under the same conditions.

[0360] Table 4

[0361]

[0362] *Values ​​reported in EP 1931673 B1

[0363] Table 5 below summarizes the relaxivity values ​​r1 of the amide derivatives obtained by conjugation of Gd(PCTA-tri-glutaric acid) with isoserinol measured in H2O and HSA at 37°C and the stereochemistry of the serinol used for the conjugation, compared with the corresponding values ​​described in the cited prior art for gadolinium polycyclic phenol (mixture of isomers).

[0364] Table 5

[0365]

[0366]

[0367] *Value reported in EP 1931673; otherwise, 11-12 mM -1 s -1 The values ​​of are as described in EP 2988756 for the same compounds;

[0368] **Values ​​reported in Invest.Radiol.2015,50,835-842.

[0369] On the one hand, the results obtained show that the higher relaxivities measured for the corresponding isomer mixtures by RRR / SSS Gd(PCTA-tri-glutaric acid) are substantially maintained for the corresponding conjugated derivatives. On the other hand, these results are consistent with the fact that the stereochemistry of the isoserinol moiety does not affect the main properties of the final compound, which are related to the stereochemistry of the glutaric acid arm.

[0370] Example 10: X-ray diffraction

[0371] Enantiomeric pairs C

[0372] Crystal preparation

[0373] Single crystals of the formula {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O, where Gd(H3L) is a triprotonated RRR / SSS Gd(PCTA-tri-glutaric acid) suitable for X-ray diffraction studies, were grown by slow evaporation of water from an aqueous solution of RRR / SSS-enriched Compound C collected from Example 3. To promote crystallization, two inner-sphere water molecules of the Gd(PCTA-tri-glutaric acid) complex were replaced by oxalate anions, and the relevant guanidinium salt was then crystallized from water. 49.6 mg of (C(NH2)3)2(C2O4) (2.5.0×10 -4 mol) was dissolved in the aqueous solution of the enriched compound C collected from Example 3 to prepare a starting solution (1.0 mL; 0.0483 M GdH3L aqueous solution 5.0 × 10 -5 mol). The pH was adjusted to 3.3 by gradual addition of solid H2C2O4.

[0374] Crystals were isolated and XRD data were collected from at least 5 crystals at the X-ray diffraction beamline (XRD1) of the Elettra Synchrotron, Trieste (Italy) using a procedure such as that disclosed by Lausi A. et al., The European Physical Journal Plus, 2015, 130, 1-8. In particular: the collected crystals were immersed in NHV oil (Jena Bioscience, Jena, Germany), frozen in liquid nitrogen, and mounted on the goniometer head with a kapton ring (MiTeGen, Ithaca, USA). When different crystal shapes were available, all crystals were tested. A complete data set was collected by the rotating crystal method at 100 K (nitrogen flow provided by Oxford Cryostream 700 - Oxford Cryosystems Ltd., Oxford, United Kingdom). The XRD data were obtained using a Pilatus 2M hybrid pixel area detector (DECTRIS Ltd., Baden-Daettwil, Switzerland). The data are acquired at a single wavelength.

[0375] result

[0376] The structure was solved by the dual space algorithm implemented in the SHELXT direct method (Sheldrick GM (2015). "SHELXT-Integrated space-group and crystal-structure determination", Acta Crystallographica Section A, 71, 3-8). Fourier analysis and refinement were performed by F-based 2 The full matrix least squares method was used. Anisotropic thermal motion refinement was used for all atoms. Hydrogen atoms were included in the calculated positions, where the isotropic U 因子 =1.2·U eq or U 因子 =1.5·U eq (For hydroxyl group)(U eq is the equivalent isotropic thermal factor for bonded non-hydrogen atoms). Hydrogen atoms of solvent water molecules were not included in the refined model because it was not possible to locate them unambiguously in the electron density peaks of the Fourier difference map.

[0377] Basic crystal and purification data are reported in the table below.

[0378] Table 6. Crystallographic data and stereocenter configurations of the {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O dataset

[0379] Crystal system: monoclinic

[0380] Space group P 21 / c

[0381] Unit Cell

[0382]

[0383]

[0384] α=90°

[0385] β=90.80(3)°

[0386] γ=90°

[0387] volume 4127.9(14)

[0388] Final R index [I>2σ(I)] R1=0.0281,wR2=0.0700

[0389] R1=Σ||Fo|-|Fc|| / Σ|Fo|,wR2={Σ[w(Fo2-Fc2)2] / Σ[w(Fo2)2]} 1 / 2

[0390] Chiral center configuration

[0391]

[0392] The X-ray structures of the unit cell of the {(C(NH2)3)2[Gd(H3L)(C2O4)]}·5H2O complex and the resulting crystal are shown in Fig. 9 and 10 Available in.

[0393] Fig.10 The display indicates that each unit cell contains 2RRR+2SSS complexes, where this means that each crystal contains the isomers SSS and RRR in an equimolar (50-50%) ratio.

[0394] Amide derivatives with racemic isoserinol

[0395] The formula [GdC 35 H 54 N7O 15Single crystals of ][CH6N3]2[CO3]·18H2O(GdL) (where GdL is RRR / SSS Gd(PCTA-tri-glutaric acid) conjugated with racemic isoserinol) were grown from an aqueous solution of the amide derivative of Gd(PCTA-tri-glutaric acid) and racemic isoserinol collected from Example 4c). To promote crystallization, the two inner-sphere water molecules of the final complex were replaced by carbonate anions, and the associated guanidinium salt was crystallized from water by slow diffusion of ethanol and ether at 4°C. Specifically, 1.0 molar equivalents (97 mg of GdL complex and 9 mg of guanidine carbonate {C(NH2)3}2CO3) were dissolved in 1 mL of water at pH = 10.5, in which a mixture of EtOH and Et2O was slowly diffused.

[0396] Fifteen single crystals were isolated and a set of XRD data for seven crystals was performed at the X-ray diffraction beamline (XRD1) of the Elettra Synchrotron, Trieste (Italy) using, for example, the procedure disclosed by Lausi A. et al., The European Physical Journal Plus, 2015, 130, 1-8. In particular: the collected crystals were immersed in NHV oil (Jena Bioscience, Jena, Germany), frozen in liquid nitrogen, and mounted on the goniometer head with a kapton ring (MiTeGen, Ithaca, USA). All crystals were tested when different crystal shapes were available. The complete data set was collected by the rotating crystal method at 100 K (nitrogen flow provided by Oxford Cryostream 700—Oxford Cryosystems Ltd., Oxford, United Kingdom). The XRD data were obtained on a Pilatus 2M hybrid pixel area detector (DECTRIS Ltd., Baden-Daettwil, Switzerland) using The data are acquired at a single wavelength.

[0397] The structure was solved by direct methods. Fourier analysis and refinement were performed by Fourier-based full-matrix least squares. Anisotropic thermal motion refinement has been used for all atoms. Hydrogen atoms are included in the calculated positions, where for methyl and hydroxyl groups, the isotropic U factor = 1.2·Ueq or the U factor = 1.5·Ueq (Ueq is the equivalent isotropic thermal factor for bonded non-hydrogen atoms). Hydrogen atoms of solvent water molecules are not included in the refined model because it is impossible to clearly locate them in the electron density peaks of the Fourier difference map.

[0398] Table 7. Crystallographic data and stereocenter configurations of the GdL data set.

[0399] Crystal triangle

[0400] Space group R-3

[0401] Unit Cell

[0402]

[0403]

[0404] α=90°

[0405] β=90°

[0406] γ=120°

[0407] volume 31997(11)

[0408] Final R index [I>2σ(I)]a R1=0.0554,wR2=0.1496

[0409] Stereocenter configuration in ASU

[0410] C7 R C28A R-54 (1) % occupancy C28B S-45 (1) % occupancy)

[0411] C14 R C31A R-62(1)% Occupancy C31B S-38(1)% Occupancy

[0412] C21 R C34A R-50(1)% occupancy C34B S-50(1)% occupancy

[0413] R1=Σ||Fo|-|Fc|| / Σ|Fo|,wR2={Σ[w(Fo2-Fc2)2] / Σ[w(Fo2)2]}1 / 2

[0414] For methods and details of use, see, e.g., Lausi A., Polentarutti M., Onesti S., Plaisier JR, Busetto E., Bais G., Barba L., Cassetta A., Campi G., Lamba D., Pifferi A., Mande SC, Sarma DD, Sharma SM, Paolucci G., The European Physical Journal Plus, 2015, 130, 1-8.

[0415] D'-CO3 2-The X-ray structures of the complexes and the statistical analysis of the collected crystals are shown in Fig.11 as shown in .

Claims

1. Amide derivatives of formula (II A) F(NR1R2)3(II A) in: F is: RRR enantiomeric residues of formula IIIa The SSS enantiomer residue of formula IIIb or a mixture of these RRR and SSS enantiomeric residues; and three -NR1R2 groups are each bound to an open bond of the corresponding carboxyl portion of F, which is identified by a solid circle (·) in the above structure; R1 is H or C1-C6 alkyl optionally substituted by 1-4 hydroxyl groups; R2 is a C1-C6 alkyl group optionally substituted by 1 to 4 hydroxyl groups, and preferably a C1-C3 alkyl group substituted by one or two hydroxyl groups.

2. An isomeric mixture of an amide derivative of Gd(PCTA-tri-glutaric acid) having the formula (II B) F'(NR1R2)3(II B) in: F' is an isomeric mixture of the Gd(PCTA-tri-glutaric acid) residue of formula III, The isomeric mixture of Gd(PCTA-tri-glutaric acid) residues contains at least 50% of the RRR enantiomeric residues of formula IIIa The SSS enantiomer residue of formula IIIb or a mixture thereof; and -NR1R2 groups are each bound to an open bond of the corresponding carboxyl moiety of F', which is identified by a solid circle (·) in the above structure; R1 is H or C1-C6 alkyl optionally substituted by 1-4 hydroxyl groups; R2 is a C1-C6 alkyl group optionally substituted by 1 to 4 hydroxyl groups, and preferably a C1-C3 alkyl group substituted by one or two hydroxyl groups.

3. The isomeric mixture according to claim 2, wherein F' comprises at least 60% of a mixture of RRR and SSS enantiomeric residues.

4. The isomeric mixture according to claim 3, wherein in formula (IIB), F' comprises at least 70% of a mixture of RRR and SSS enantiomeric residues.

5. The isomeric mixture according to claim 4, wherein in formula (IIB), F' comprises at least 80% of a mixture of RRR and SSS enantiomeric residues.

6. The isomeric mixture according to claim 5, wherein in formula (IIB), F' comprises at least 90% of a mixture of RRR and SSS enantiomeric residues.

7. The amide derivative or isomer mixture according to any one of claims 1 to 6, wherein in formula (IIA) or (IIB), R1 is H, and R2 is a C1-C3 alkyl group substituted by one or two hydroxyl groups.

8. An amide derivative or isomeric mixture according to any one of claims 1 to 7 for use in the preparation of a pharmaceutical preparation for diagnostic imaging of organs, tissues or regions of the human or animal body or biological samples by using MRI techniques.

9. The amide derivative or isomeric mixture according to claim 8, wherein the diagnostic imaging is in vivo, in vitro or ex vivo.

10. The amide derivative or isomer mixture according to claim 8 or 9, wherein the biological sample is selected from cells, biological fluids and biological tissues derived from a living mammalian patient.

11. The amide derivative or isomeric mixture according to claim 10, wherein the living mammalian patient is a human patient.

12. Use of an amide derivative or isomeric mixture according to any one of claims 1 to 7 for the preparation of a pharmaceutical preparation for diagnostic imaging of organs, tissues or regions of the human or animal body or biological samples by using MRI techniques.

13. Use according to claim 12, wherein the diagnostic imaging is in vivo, in vitro or ex vivo.

14. The use according to claim 12 or 13, wherein the biological sample is selected from cells, biological fluids and biological tissues derived from a living mammalian patient.

15. The use according to claim 14, wherein the living mammalian patient is a human patient.

16. A preparative HPLC method which allowed the separation of 4 peaks with identical m / z ratios from a mixture (Gd(H4L)+: 752.14 m / z).

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