ES2277760B1 - COMPLEXING AGENTS DERIVED FROM PIRAZOLILETILDIETILENTRIAMINOTETRAACETICOS ACIDS. GADOLINIUM COMPLEXES (III) WITH APPLICATIONS IN CLINICAL DIAGNOSIS BY MAGNETIC RESONANCE. - Google Patents

Abstract

Acid-derived complexing agents pyrazolylethyldiethylenetriaminetetraacetic agents. Gadolinium complexes (III) with applications in clinical diagnosis by Resonance Magnetic

Compounds of General Formula A whose complexes Paramagnetic Gd (III) and other lanthanides are used as contrast agents for Magnetic Resonance Imaging.

Compounds of General Formula A wherein R 1 and / or R 2 and / or R 3 are hydrogens, alkyl chains up to 12 carbon atoms, halogens (F, Cl, Br, and I), acids carboxylics and derivatives, nitro groups and amino groups.

A procedure for obtaining these compounds starting from the corresponding bromoethylpyrazoles, synthesized from the corresponding pyrazole, which involves the following stages:

1) alkylation of the starting amine; 2) groups check out tert-butoxycarbonylamino; 3) alkylation of amino groups with methyl bromoacetate and finally, 4) the basic hydrolysis leading to the tetrasodium salt.

The complexes of Gd (III) and other lanthanides derived from the compounds of General Formula A, the procedure for obtaining the complexes, and their experimental and clinical use in the manufacture of contrast agents for clinical diagnosis by Magnetic Resonance Imaging .

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Description

Acid-derived complexing agents pyrazolylethyldiethylenetriaminetetraacetic agents. Gadolinium complexes (III) with applications in clinical diagnosis by Resonance Magnetic

Technical field of the invention

The synthesis and characterization of a series of ligands with pyrazolylethyl groups of General Formula A listed below:

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A new series of complexes of Gd (III) and other lanthanides derived from structure A that are used as contrast agents for Resonance Imaging Magnetic (MRI).

Prior state of the art

The great differentiation and spatial resolution of soft tissues has made Magnetic Resonance Imaging (MRI) is one of the most used techniques in the diagnosis clinical.

The main determinants of contrast in an MR image are the relaxation times of the protons of the water, T1 and T2. Thus, when the contrast difference between healthy and pathological regions of a tissue is very small, Due to small variations in relaxation times, use of contrast agents is highly beneficial. Agents contrast are substances capable of considerably altering the relaxation times of water protons in tissues in where they are distributed. The use of these agents is a great improvement in clinical diagnosis in terms of high specificity, better characterization of tissues, reduction of artifacts in the image and functional information thereof. The diagnosis cancer and vascular disease, along with monitoring effective therapy can be done using new probes capable of informing us about events specific that occur at both a cellular and molecular level. For all these reasons, contrast agents can be considered as substances capable of giving information about the biological environment of tissues.

The most widely used contrast agents today are paramagnetic gadolinium chelates. The Gd (III) ion is the metal most used in Diagnostic Imaging because it has seven unpaired electrons that make it the most stable paramagnetic metal. In addition to having a high electron spin, it has a relatively small electronic relaxation time, an intrinsic requirement that makes it an effective contrast agent for MRI diagnostics. However, the free Gd (III) ion is very toxic and cannot be used in in vivo experimentation, so the development of complexing agents is absolutely necessary in order to reduce its toxicity and maintain, as far as possible, the properties. which make it a good contrast agent. The effectiveness of a contrast agent is assessed, first of all, by determining its relaxivity, that is, by increasing the relaxation of the water protons.

In the last 15 years, numerous articles aimed at studying the structure and dynamics of the Gd (III) complexes, which has meant a breakthrough in understanding structural parameters, dynamic and electronic determinants in the relaxation of these complex.

The most widely used contrast agents in diagnostic imaging are gadolinium complexes derived from diethylenetriaminepentaacetic acid (DTPA) and acid 1,4,7,10-tetraaza-1,4,7,10-cyclododecanetetraacetic acid (DOTA).

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Currently, research in this field is focused on the design and synthesis of new chelating agents that have a high affinity for the corresponding paramagnetic metal, as well as a high relaxivity. In this sense, a family of ligands that include pyrazole rings and an iminodiacetic acid unit was described, with affinity for the Gd (III) ion (Ballesteros García, Paloma et al ., Complexons with the structure of N- [2- [ azol-1 (2) -yl] ethyl] iminodiacetic acids, synthesis, analytical study, and biological applications. PCT Int. Appl. (1996), 43, WO9641797; P. López et al , N-2- (Azol-1 (2) -yl) ethyliminodiacetic acids: a novel series of Gd (III) chelators as T2 relaxation agents for magnetic resonance imaging. Bioorg. Med. Chem ., 1999 , 7 , 517). These chelating agents have low affinity for the metal ion, but nevertheless, they have relaxation properties that are superior to those of commercial complexes used in clinical practice. Recently, a new series of mixed ligands with bi- or bispyrazole structure has been described (P. Ballesteros and S. Cerdán, New Gd (III) Ligands with bi- and bis-azole structures. PCT Int. Appl., WO 0259097, 2002; E. Pérez Mayoral et al . A novel series of complexones with bis- or biazole structure as mixed ligands of paramagnetic contrast agents for MRI. Bioorg. Med. Chem ., 2003 , 11 , 5555). In this case, the stability constant with the metal has been slightly improved, with respect to the aforementioned complexones, although it is equally low because these compounds form double tetradentate complexes with the metal, which means that they cannot be used for diagnostic purposes. due to its toxicity. However, as in the previous cases, they have relaxation properties (r1 and r2) much higher than those described for low molecular weight complexes. The magnetic properties of these complexes are a great advance over commercial complexes in terms of their effectiveness as contrast agents. In principle, this improvement in relaxation properties could be due, in addition to a high hydration number, to the incorporation of azoles in the structure of these ligands.

More recently in 2004, it has applied for a patent (P. Ballesteros García, E. Pérez Mayoral, Heterocyclic ligands and their gadolinium (III) complexes with biomedical applications, P200402679) in which a new series of pyrazolylethyldiethylenetriaminetetraacetic acids with the structure represented below:

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Gd (III) complexes derived from 1-3 are more effective, in terms of relaxation, r_ {1 (2)}, than dtpa-Gd (III) used as reference compound used in non-invasive clinical diagnosis by Resonance Magnetic (Table 1).

TABLE 1 Values of the longitudinal and transverse relaxation times, T1 (2)}, and of relaxivity, r_ {1 (2)}, of the Gd (III) complexes of 1-3 and dtpa-Gd (III)

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The present invention describes a series of organic ligands derived from pyrazolylethyldiethylenetriaminetetraacetic acids and their complexes of Gd (III) and other lanthanides, with a structure similar to 1-3 . Surprisingly, these complexes are more efficient than Gd (III) -1-3 to act as contrast agents. Furthermore, the complexes of this invention show an efficacy far superior to that of the contrast agents currently used in clinical diagnosis, such as dtpa-Gd (III) .

Detailed description of the invention

This invention presents a new family of pyrazolylethyldiethylenetriaminetetraacetic acids ( A ) and their synthesis, with the General Formula indicated below:

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The compounds of the present invention include a pyrazolylethyl group in its structure, substituted in turn with radicals R1, R2, and R3 which are hydrogens, chains alkyl of up to 12 carbon atoms, halogens (F, Cl, Br, and I), carboxylic acids and derivatives such as amides, esters and nitriles, nitro groups and amino groups.

A study of Magnetic Resonance is presented Detailed MRI that allows us to evaluate the effectiveness of your complexes of Gd (III) in solution as contrast agents for MRI.

As an example, this specification describes the synthesis of ligand 4 (figure 1).

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Compound 4 has been synthesized from bis ( tert -butoxycarbonylamino) diethylenetriamine ( 5 ) as indicated in Scheme 1. The first synthesis step consists of the alkylation of 4-iodopyrazole (commercial) using 1,2-dibromoethane to obtain the corresponding bromoethylpyrazole 6 , which by reaction with amine 5 leads to compound 7 . Treatment of 7 in an acid medium and subsequent alkylation with methyl bromoacetate gives the amino ester 8 . Finally the basic hydrolysis of 8 leads to the ligand 4 object of the invention.

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Scheme 1

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The corresponding gadolinium complex is obtained by reaction between equimolecular quantities of GdCl 3 · 6H 2 O and the corresponding organic ligand in aqueous solution at room temperature.

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Synthesis of the compound 4

Tert-butyl ester of the acid (2 - {(2- tert - butoxycarbonylaminoethyl) - [2- (4-iodo- pyrazol-1-yl) ethyl] amino} ethyl) ester (7)

A mixture of 2-bromoethyl-4-iodopyrazole ( 6 ) (749 mg, 2.48 mmol) and amine 5 (1.5 g, 4.95 mmol) is heated at 80 ° C for 5 h. It is then allowed to cool, 20 mL of dichloromethane are added and the solid formed is filtered. The solvent is then removed under reduced pressure and the reaction crude is purified by column chromatography on silica gel (CH2Cl2 / EtOH 98: 2). Amine 7 (1.1 g; 71%) is obtained in the form of a yellow oil; IR (ATR): ν3341, 1690, 1505, 1247, 1165 cm -1; 1 H-NMR (400 MHz, CDCl 3): δ 7.55, 5.54 (2 H, s, H 3, H 4), 4.97 (2 H, broad s, NH), 4.12 (2 H, t apparente, J = 5.6, 5.3 Hz, CH2 -N (Azol)), 3.02 (4 H, m, CH2 NH-BOC), 2.85 (2 H, t apparente, J = 5.5, 5.3 Hz, CH2 -N), 2.52 (4H, t, J = 5.6 Hz, N-CH2), 1.47 (18H, s, CH3) ppm; 13 C-NMR (100 MHz, CDCl 3): δ182, 144.7, 137.2, 134.4, 79.7, 54.1, 53.4, 51.0, 38.3, 28.5 ppm.

Acid methyl ester [(2 - {[2- (bis-methoxycarbonylmethylamino) ethyl] - [2- (4- iodopyrazol-1-yl) -ethyl] amino} ethyl) methoxycarbonylmethylamino] acetic (8)

A solution of carbamate 7 (1 g; 1.91 mmol) in HCl / MeOH (6N; 20 mL) is kept under stirring at room temperature for 1 h. The solvent is then removed under reduced pressure, obtaining a residue in the form of a white solid. Then, the solid obtained together with methyl bromoacetate (1.5 g; 10.98 mmol), K 2 CO 3 (2.74 g, 20 mmol) in acetonitrile (20 mL) is heated under reflux for 22 h. Subsequently, it is allowed to cool, the salts formed are filtered and the solvent is removed under reduced pressure. The reaction crude is purified by column chromatography on silica gel (CH2Cl2 / EtOH 98: 2) to obtain ester 8 (732 mg; 63%) as a yellow oil.

IR (ATR): ν 1734, 1435, 1198, 1175, 734 cm -1; 1 H-NMR (400 MHz, CDCl 3): δ 7.64 (1 H, s, H 3), 7.47 (1 H, s, H 5), 4.19 (2 H, t, J = 6.2 Hz, CH 2 -N (Azol)), 3.70 (12 H, s, CO 2 Me), 3.52 (8 H, s, C H 2 CO 2 Me ), 2.88 (2 H, t apparente, J = 6.2, 5.9 Hz, CH 2 -N), 2.70 (4 H, m, NC H 2 CH 2 -N), 2.60 (4 H , m, N-CH 2 C H 2 -N) ppm; 13 C-NMR (100 MHz, CDCl 3): δ171.2, 143.7, 134.5, 54.8, 54.03, 53.02, 51.7, 51.1, 50.8 ppm.

Tetrasodium salt of acid acid [(2 - {[2- (bis-methoxycarbonylmethylamino) ethyl] - [2- (4-iodopyrazol-1-yl) -ethyl] amino} ethyl) methoxycarbonylmethylamino] acetic (4)

A suspension of 8 (274 mg; 0.44 mmol), NaOH (70.4 mg; 1.76 mmol) in distilled H 2 O (0.6%) is kept with stirring at room temperature until the total disappearance of the starting compound. The reaction mixture is then washed with CH2Cl2 and the solvent is removed under reduced pressure. 4 (260 mg, 93%) is obtained as a yellow solid; IR (ATR): ν 1581, 1401, 1327 cm -1; 1 H-NMR (400 MHz, D 2 O): δ 7.80 (1 H, s, H 3), 7.59 (1 H, s, H 5), 4.27 (2 H , t, J = 6 Hz, CH2 -N (Azol)), 3.27 (8 H, s, CH2CO2 Na), 2.91 (2 H, t, J = 6 Hz, CH_ 2 -N), 2.75 (4 H, m, NC H 2 CH 2 -N), 2.67 (4 H, m, NC H 2 CH 2 -N) ppm; 13 C-NMR (100 MHz, D 2 O): δ177.8, 146.4, 137.8, 59.8, 57.4, 54.4, 52.9, 52.0, 51.1 ppm.

General procedure for the synthesis of the Gd (III) complex of ligand 4 : A solution of one equivalent of the tetrasodium salt 4 and one equivalent of GdCl 3 · 6H 2 O in 5 mL of water (MQ) at pH ~ 5-7, keep stirring at room temperature for 4 h. The solvent is then removed under reduced pressure.

Magnetic Resonance Study: Relaxation

The Magnetic Resonance studies presented in this invention have been carried out in a 60 MHz (1.5 T) spectrometer. Longitudinal and transverse relaxation times have been measured at a concentration of 1 mM of organic ligand 4 or of the corresponding Gd (III) complex, 150 mM of NaCl (ionic strength) and 100 mM of TRIS / HCl using water (MQ) as a solvent, at different temperatures and pH.

Relaxation has been calculated according to equation that is represented below:

r_ {1 (2)} =? [1 / T1 (2)] / [LGd]

where r_ {1 (2)} is the longitudinal (transverse) relaxivity,? [1 / T1 (2)}] is the difference between the inverse of the longitudinal (transverse) relaxation times of the corresponding complex of Gd (III) and of the ligand and, [LGd] is the concentration of the Gd (III) complex used (equal to the concentration of ligand).

Table 2 shows the values of r_ {1 (2)} of the complexes synthesized at T 25 ° C and pH ~ 7.

TABLE 2 Relaxation values, r_ {1 (2)}, of the Gd (III) complex of 4 and dtpa-Gd (III)

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As can be seen, both r1 and r2 are much higher than the values obtained for dtpa-Gd (III) , taken as a reference compound.

The present invention includes graphs in which the dependence of r, with the temperature and the pH of the complexes of Gd (III) of 1-4 and dtpa-Gd (III) is represented .

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Figure 2 represents the variation of r1 with the temperature at a pH of approximately 7. It is observed that the Relaxation increases as the temperature decreases, this being typical behavior of complexes derived from acids polyaminopolycarboxylics with a water molecule in its first coordination sphere. According to the Theory of Relaxation and according to the Solomon-Bloembergen and Morgan equations It may happen that: a) the relaxation time of the protons of the water from the first coordination sphere (T_ {1 (2) M}) is less than the residence time of the water molecule (\ tau_ {M}) with what the relaxivity decreases as the temperature drops, or conversely, b) that T_ {1 (2) M} is greater than \ tau_ {M} increasing the Relaxation as the temperature decreases. This last case is the which justifies the increase in r1 at low temperatures in the complexes presented in this invention.

Equations of Solomon-Bloembergen and Morgan:

r_ {1 (2)} = q / 55. 5 (T_ {1 (2) M}) + \ tau M)

1 / T_ {1 (2) M} = k / r 6 f (? c)

where, q is the number of hydration of the complex, T_ {1 (2) M} is the time of longitudinal (transverse) relaxation of the water molecule directly attached to the metal, \ tau_ {M} is the residence of water in the first sphere of coordination with the metal, K is a constant, r is the distance between the protons of the water and metal and finally, \ tau_ {c} is the time of correlation cash.

Figure 3 represents the variation of r1 with the pH measured at 60 MHz and 37 ° C and it is observed that, while the relaxivity of dtpa-Gd (III) remains constant in a pH range from 9 to approximately 4.5, the relaxivity of the complex exemplified in this invention is pH dependent. It can be observed that the relaxivity of 4-Gd (III) increases as the pH decreases, being much higher than that observed in the case of Gd (III) -dtpa and, r_ {1} reaches its maximum value in an interval pH close to physiological pH.

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Finally it is highlighted that taking into account the previously detailed magnetic resonance study,
4-Gd (III) shows, surprisingly, an efficiency much superior to that of the Gd (III) complexes of 1-3 and that of dtpa-Gd (III) , a commercial Gd (III) complex currently used in the clinical diagnosis by Magnetic Resonance.

Claims (8)

1. A compound of General Formula A, eleven Where the radicals R1, R2, and R3 they are hydrogens, alkyl chains of up to 12 carbon atoms, halogens (F, Cl, Br, and I), carboxylic acids and derivatives such such as amides, esters and nitriles, nitro groups and amino groups, X is hydrogen, alkali ions or N-methylglucosamine and, whose complexes of Gd (III) and other lanthanides are used as contrast agents for Magnetic Resonance Imaging (IRM). 2. The compound of claim 1 wherein the radicals R1 and R3 are hydrogens and R2 is a iodine atom. 3. A process for obtaining the compounds of General Formula A of claim 1 in which the starting point is the corresponding distinctly substituted pyrazoles, and which involves the steps indicated below: 1) alkylation of the corresponding pyrazole with 1, 2-dibromoethane under phase transfer conditions; 2) alkylation at the central nitrogen of the protected diethylenetriamine using the synthesized bromoethylpyrazoles; 3) deprotection of tert -butoxycarbonylamino (BOC) groups in an acid medium; 4) alkylation of the terminal amino groups with methyl bromoacetate in a basic medium and finally, 5) the basic hydrolysis of the methyl esters to obtain the tetrasodium salt. 4. The Gd (III) complexes of the compounds of claim 1 wherein the radicals R1 and R 2 and R 3 are hydrogens, alkyl chains of up to 12 carbon atoms, halogens (F, Cl, Br, and I), carboxylic acids and derivatives, nitro groups and amino groups. 5. The Gd (III) complex of the compound of claim 2 wherein the radicals R1 and R3 are hydrogens and R 2 is an iodine. 6. The synthesis of paramagnetic complexes of Gd (III), and other lanthanides, of the ligands of the claims 1 to 2 by means of a single synthetic step, which consists of reacting equimolar amounts of the ligand organic and the corresponding lanthanide chloride in water deionized (MQ) at room temperature. 7. Use of the compounds of claim 1 to 2 in the manufacture of contrast agents for Resonance Magnetics in clinical diagnosis. 8. Use of the complexes of claim 4 to 5 in the manufacture of contrast agents for Resonance Magnetics in clinical diagnosis.