WO2006051142A1

WO2006051142A1 - 1-pyrazolylethyl-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acids and application of the gadolinium (iii) complexes thereof in clinical diagnosis

Info

Publication number: WO2006051142A1
Application number: PCT/ES2005/000601
Authority: WO
Inventors: Paloma BALLESTEROS GARCÍA; Elena PÉREZ MAYORAL
Original assignee: Universidad Nacional De Educación A Distancia (U.N.E.D.)
Priority date: 2004-11-08
Filing date: 2005-11-07
Publication date: 2006-05-18

Abstract

The invention relates to macrocyclic compounds having general formula A and the paramagnetic complexes of lanthanides thereof which are used as contrast agents for magnetic resonance imaging. More specifically, the invention relates to compounds having general formula A, wherein R1 and/or R2 are hydrogens, methyl groups, nitre groups and amino groups. The invention further relates to a method of obtaining said compounds from the corresponding bromoethylpyrazoles, comprising the following steps: 1) monoalkylation of the cyclen; 2) alkylation of the remaining amino groups with methyl bromoacetate in a basic medium; and, finally, 3) basic hydrolysis of the methyl esters in order to produce the corresponding tetrasodium salt. The invention relates to complexes of Gd(III) and of other lanthanides obtained from compounds having general formula A, to the method of obtaining said complexes and to the experimental and clinical use of same in the production of contrast agents for clinical diagnosis by means of magnetic resonance imaging (General formula A).

Description

ACIDS 1 -PIRAZOLILETIL-1, 4,7,10- TETRAAZACICLODODECANO-4,7,10- TRIACETICS. APPLICATION OF YOUR GADOLIN COMPLEX (III) EN

THE CLINICAL DIAGNOSIS

TECHNICAL FIELD OF THE INVENTION

The synthesis and characterization of a series of organic ligands with pyrazolylethyl groups of General Formula A is presented, which is indicated below:

B

R ₁ ; R ₂ = H, Me, NO ₂ or NH ₂ x = H, alkali ions or Λ / -methylglucosamine

General Formula

A new series of macrocyclic complexes of Gd (III) and other lanthanides are obtained, which are used as contrast agents for Magnetic Resonance Imaging (MRI).

STATE OF THE PREVIOUS TECHNIQUE

Magnetic Resonance Imaging (MRI) is the modality chosen for the evaluation of most intracranial and spinal pathologies. The use, increasingly frequent, of this technique (MRI) as a tool in biomedical research and in non-invasive clinical diagnosis, has promoted the development and the search for a new class of drugs called contrast agents (CA). CAs are substances designed to significantly reduce the times of longitudinal and transverse relaxation (Ti and T ₂ ) of water protons in the tissues in which they are distributed. The main determinants of contrast in an MRI image are the relaxation times of water protons, Ti and T ₂ . Thus, when the difference in contrast between healthy and pathological regions of a tissue is very small, due to small variations in relaxation times, the use of CA is highly beneficial. Thus, the effectiveness of an AC is assessed, firstly, by the determination of its relaxivity, which is defined as the longitudinal or transverse relaxation rate of water protons at 1 mM concentration of paramagnetic complex.

The most used and researched CAs are the lanthanide complexes, mainly gadolinium, derived from polyamopolycarboxylic acids. Today, they are still being studied by most of the research groups that, worldwide, are dedicated to the design and development of new CAs. The compounds that are generally used as CA in clinical practice are the Gd (III) complexes derived from diethylenetriaminepentaacetic acids [DTPA-Gd (III)] and 1,4,7,10-tetraazacyclododecane-1, 4,7,10- tetraacetic [DOTA-Gd (III)], which contain aminoacetic acid units and macrocyclic ligands in their structure, respectively.

DTPA DOTA

In this regard, examples of mixed ligands including iminodiacetic acid groups and azolic rings, capable of forming tetradentate complexes with Gd (III) (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; 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 compounds have greater relaxation than the commercial complexes currently used as CA in diagnostic imaging. However, the complexing capacity of these complexes is not high enough to allow clinical use. More recently, in 2003, a new series of bi and bisazóliocas complexones with both magnetic and affinity properties with the improved metal center have been described, with respect to the Gd (III) complexes discussed above, but not enough to that these complexes may be viable AC in the clinical diagnosis (P. Ballesteros and S. Cerdán, New Gd (III) Ligands with bi- and bis-azolic 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). These results also extend to a new family of Gd (III) complexes derived from bispyrazoles, described very recently, in which the heterocyclic centers are connected by units of ethylene glycol and thioethylene glycol (Pérez-Mayoral, E. et al. Synthesis of a new family of ligands with bispyrazole structure. reactivity of bispyrazolylmethyl ethers. Heterocycles, 2005, 65, 1691). The magnetic properties of these complexes represent a great advance, with respect to commercial complexes, in terms of effectiveness as contrast agents. However, they have a low affinity with Gd (III), which means that they cannot be used for diagnostic purposes due to their toxicity. In principle, this improvement of the relaxation properties could be due, in addition to a high hydration number, to the incorporation of azoles in the structure of these ligands. For this reason, in this invention a series of new organic ligands and their Gd (III) complexes are presented which have a greater stability constant with the metal ion and therefore a lower toxicity.

DETAILED DESCRIPTION OF THE INVENTION

This invention presents a new family of macrocyclic ligands with structure of 1-pyrazolylethyl-1, 4,7,10-tetraazacyclododecane-4,7, 10-triacetic acids (A) and their synthesis, with the General Formula indicated below. :

TO

General Formula

A detailed Magnetic Resonance (MRI) study is presented that allows establishing the participation of the azol in the complexation with the metal center.

This Report describes, as an example, the synthesis of ligand 1 (Figure 1) and the study of the magnetic properties of the corresponding complex of Gd (III).

Figure 1

The compound 1 obtained in the form of its sodium salt has been synthesized by the following reaction scheme (scheme 1).

2 H ₂ SO ₄

Scheme 1 The first stage of synthesis consists in the alkylation of cyclic-2 H2SO ₄ with 2-bromoethyl-3,5-d-methylpyrazole (2) leading to compound 3, which followed by alkylation with methyl bromoacetate gives rise to 4 Finally, the basic hydrolysis of 4 leads to compound 1. The gadolinium complexes are synthesized by reaction between equimolecular amounts of GdCb-ΘHaO and the organic ligand 1 in aqueous solution at room temperature.

Synthesis of compound 1

Trisodium salt of {7- [2- (3,5-dimethylpyrazol-1-l) ethyl] -4,10-b-s-methoxycarbonylmethyl-1, 4,7,10-tetraazacyclododec-1-yl} acetic acid (one )

A mixture of cyclone-2 H ₂ SO ₄ (460 mg; 2.67 mmol), 2 (542 mg; 2.67 mmol) and Na ₂ CO ₃ (1.42 g; 13.35 mmol) in CHCI ₃ (50 mL) is heated at reflux for 16 h. Allow to cool, the white solid formed is filtered and the solvent is evaporated under reduced pressure. Then, the residue 3 obtained together with methyl bromoacetate (1.37 g; 8.98 mmol) and Na ₂ CO ₃ (952 mg; 8.98 mmol) in acetronitrile (20 mL) is heated at reflux for 15 h. It is allowed to cool, the white solid formed is filtered and the solvent is evaporated under reduced pressure. The obtained residue is purified by column chromatography on silica gel (CH ₂ CI ₂ / EtOH, 95: 5) to obtain 4 as a yellow oil (752 mg, 55%). ¹ H-NMR (400 MHz, DMSO-Ci ₆ ): 5.79 (1 H, s, H ₄ ), 4.03 (2 H, apparent t, J = 7.0, 6.5 Hz, CH ₂ -N (azol)) , 3.66 (9 H, s, OCH ₃ ), 3.30 (8 H, s, N-CH ₂ CO ₂ Me), 2.75 (2 H, wide s, CH ₂ -N), 2.74-2.18 (16 H, m , N-CH ₂ -CH ₂ -N), 2.21 (3 H, s, CH ₃ ), 2.10 (3 H, s, CH ₃ ) ppm. Finally, basic hydrolysis of 4 is carried out by reaction with three equivalents of NaOH in water (MQ; 0.6%) to obtain compound 1; IR (ATR): v 1578, 1435, 1285 cm ^" ¹ ; ¹ H-NMR (400 MHz, D ₂ O): 5.88 (1 H, s, H ₄ ), 4.05 (2 H, apparent t, J = 7.6, 7.2 Hz, CH ₂ -N (azo!)), 3.11-2.97 (6 H, m, N-CH ₂ ), 2.80 (2 H, apparent t, J = 7.6, 7.2 Hz, CH ₂ -N) , 2.65-2.34 (10 H, m, CH ₂ -N), 2.22 (3 H, s, CH ₃ ), 2.12 (3 H, s, CH ₃ ) ppm; ¹³ C-NMR (100 MHz, CDCI ₃ ) : 181.5, 166.5, 149.9, 142.8, 106.8, 60.4, 54.2, 53.0-21.2, 45.9, 13.8, 11.8 ppm. Gd (III) complex of ligand 1: A solution of 1 equivalent of 1 and an equivalent of GdCla-bhbO in 5 mL of water (MQ) at pH ~ 5-7 is maintained with stirring at room temperature for 24 h. Then, the final solution is brought to pH = 8 and the gadolinium hydroxide formed is filtered. The solvent is removed under reduced pressure.

Magnetic Resonance Study: relaxivity

The Magnetic Resonance studies presented in this invention have been performed on a 60 MHz (1.5 T) spectrometer. Longitudinal and transverse relaxation times have been measured at a concentration of 1 mM of the organic ligand or the corresponding Gd (III) complex, 150 mM of NaCI (ionic strength) and 100 mM of TRIS / HCI using water (MQ) as solvent, at different temperatures and pH. The relajativity has been calculated according to the equation represented below: r 1 (2) = Δ [1 / τi ( ₂ )] / [LGd] where,

is the longitudinal (transversal) relaxivity, Δ [1 / τi (2)] is the difference between the inverse of the longitudinal (transversal) relaxation times of the corresponding complex of Gd (III) and of the ligand and, [LGd] is Ia concentration of the Gd (III) complex used (equal to the concentration of the ligand).

Table 1 shows the values of Ti @) and r-iβ) of the synthesized complex 1-Gd (III) at T 25 ⁰ C and pH -7.

Complex T ₁ (S) T ₂ (s) T ₁ (mM ^' V ¹ ) T ₂ (InMV ¹ )

1-Gd (III) Ó46 O39 1.90 ± 0.01 2.00 ± 0.01

DOTA-Gd 0.43 0.35 2.00 ± 0.01 2.31 ± 0.03

Ti: inversion-recovery sequence. T ₂ Carr-Purcell-Maiboom-Gill sequence, both in a 60 MHz Spectrometer (1.5 Tesla).

As can be seen in Table 1, 1-Gd (III) has relativity values of the same order of magnitude as those obtained in the case of DOTA-Gd, taken as the reference complex. The present invention includes the graphs that represent the dependence of the rhein with the temperature and the pH. ιϊ pH = 7

285 290 295 300 305 310 315 T (K)

R2 pH = 7

285 290 295 300 305 310 315 T (K)

Figure 2. Variation of the relaxivity with the temperature. 1 mM solutions of the ligand (complex), 150 mM NaCI and 100 mM TRIS / HCI.

Figure 2 shows the variation of both r1 and r2 with the temperature measured at a pH of approximately 7. It is observed that the relaxivity increases as the temperature drops, this behavior being typical of complexes derived from polyamopolopolycarboxylic acids with a molecule of water in its first sphere of coordination. According to the Theory of relaxivity and according to the equations of Solomon-Bloembergen and Morgan it can happen that: a) the relaxation time of the water protons of the first coordination sphere (T ^ M) is less than the residence time of The water molecule (TM) with which the relaxivity decreases when the temperature decreases, or on the contrary, b) that T _{1 (2} ) M is greater than TM increasing the relaxivity when the temperature decreases. This last case is the one that justifies the increase of r1 (2) at low temperatures in the complexes presented in this invention. Solomon-Bloembergen and Morgan equations: ri (2) = q /55.5 (Ti (2) M) ⁺ τ _M )

1 / T _{1 (2) M} = k / r ⁶ f (τ _c )

where, q is the number of hydration of the complex, TI (2) M is the longitudinal (transverse) relaxation time of the water molecule directly attached to the metal, τ _M is the residence time of the water in the first sphere of coordination with the metal, K is a constant, r is the distance between the water and metal protons and finally, τ _c is the effective correlation time. ιi T 25 C

10

PH

R2 T 25 C

5 4 5 6 7 8 9 10 PH

Figure 3. Variation of the relaxivity with the pH. 1 mM solutions of the ligand (complex), 150 mM NaCI and 100 mM TRIS / HCI.

Figure 3 shows the variation of r1 (2) with the pH and it is observed that, while the DOTA-Gd (III) relaxivity remains constant in a pH range of 9 to about 4.5, the relaxivity of the complex that is presented As an example in this invention it is pH dependent. In the case of 1-Gd (III), r1 (2) increases at acidic pHs while maintaining constant at basic pHs. Taking into account the magnetic resonance study described above, the 1-Gd (III) complex of the invention meets the requirements for use as a contrast agent in diagnostic imaging. 1-Gd (III) has an efficacy equal to DOTA-Gd (III) at pH 7 (commercial complex currently used in clinical diagnosis). However, 1-Gd (III) presents greater relaxivity (r1 and r2) as the pH decreases (Figure 3), which translates into a greater efficacy of 1-Gd (III), at acidic pHs, with respect to to DOTA-Gd (III). This increase in the relaxivity in the complex is due to the acceleration, catalyzed at acidic pH, of the water molecule located in the first sphere of coordination with the Gd (III) ion.

Claims

1.- A compound of General Formula A,

Where Ri and R ₂ radicals are hydrogen, methyl groups, nitro groups and amino groups and whose complexes of Gd (III) and other lanthanides are used as contrast agents for Magnetic Resonance Imaging (MRI).

2. The compound of claim 1 wherein the radicals Ri and R ₂ are methyl groups.

3.- A process for obtaining the compounds of General Formula A of claim 1, in which part of the corresponding bromoethylpyrazoles, previously synthesized, and involving the steps indicated below: 1) monoalkylation of cyclic; 2) alkylation of the rest of the amino groups with methyl bromoacetate in basic medium and finally, 4) the basic hydrolysis of the methyl esters to obtain the corresponding trisodium salt.

4. The complexes of Gd (III) of the compounds of claim 1 wherein the radicals Ri and R ₂ are hydrogen, methyl groups, nitro groups and amino groups.

5. The Gd (lll) of the compound of claim 2 wherein Ri and R ₂ radicals are methyl groups.

6.- The synthesis of the paramagnetic complexes of Gd (III), and other lanthanides, of the ligands of claim 1 and 2 by a single synthetic step, which it consists in reacting equimolecular amounts of the organic ligand and the corresponding lanthanide chloride in deionized water (MQ) at room temperature.

7. The use of the compounds of claims 1 and 2 in the manufacture of contrast agents for Magnetic Resonance in clinical diagnosis.

8. The use of the complexes of claims 4 and 5 in the manufacture of contrast agents for Magnetic Resonance in clinical diagnosis.