Synthetic Approaches to Heterocyclic Ligands for Gd-Based MRI Contrast Agents

Magnetic Resonance Imaging (MRI) methods are currently used in the clinic for the non invasive detection and characterization of a wide variety of pathologies. Increases in the diagnostic efficiency of MRI have been helped by both the design of dedicated MR sequences revealing specific aspects of the pathology and by the development of more sensitive and selective Contrast Agents (CAs), capable of more precisely delineating the borderline regions. In the present review we focus on the synthetic strategies used to obtain MRI CAs containing heterocyclic rings.


Introduction
Cardiovascular and neurodegenerative diseases, as well as tumors, are often clinically diagnosed using non invasive methods such as PET (Positron Emission Tomography) [1,2], SPECT (Single Photon Emission Tomography) [2], MRI (Magnetic Resonance Imaging) [3], Optical Imaging [4], ultrasound methods [5] or their multimodal combinations [6].In many cases all of these methods involve the additional use of specific probes (known as Contrast Agents, CAs) to increase image resolution and discrimination between healthy and pathological areas.Therefore, the development of more sensitive, selective and efficient CAs is an important task of strategic interest due to their potential applications in many biomedical imaging procedures.Classically the widespread presence of heterocyclic rings in Nature and their very favorable coordinating properties have prompted the use of a variety of nitrogen-based heterocycles in the manufacture of many CAs.As a more recent example, Fu et al. [7] have described a new family of benzoylpiperidines 1 (Figure 1) as serotonin 5-HT 2A ligands for PET or SPECT Imaging of the brain.Notably, in spite of the considerable progress of MRI and PET protocols, X-Ray Imaging still accounts for 75-80% of all diagnostic imaging procedures.In this respect, non-ionic X-Ray CAs based on the attachment of heterocyclic moieties to the 5-position of diatrizoic acid (2) or iohexol (3) have been reported [8].Most of these new X-Ray contrast agents consist of sterically congested lactams 4, derived from the 2,4,6-triiodoisophthalamide, which exhibit water solubility, stability and osmolality, depending on the heterocycle included.Approaches based on optical methods are fast emerging as alternatives to conventional X-Ray Imaging.Near Infra-Red light (NIR) is increasingly being considered nowadays as a powerful non-invasive biomedical imaging tool.It is specially recommended as a complementary method to X-ray mammography for examinations of young women with dense breast tissues or patients with scars and implants, often employing NIR absorbing dyes.Indocyanine green (ICG, 5) is a clinically approved NIR dye used for testing of hepatic function and fluorescence angiography in ophthalmology and even for detection of breast tumors [9].NIR dye 6, a modification of ICG, overcomes many of its limitations with regards to spatial resolution and sensitivity [10].
In the following sections we address the various synthetic strategies developed to produce heterocyclic ligands useful for preparing Gadolinium based CAs for MRI [11].The present work complements these contributions by addressing in more detail the heterocyclic chemistry involved in these processes.

Contrast Agents (CAs) for MRI: Gd-based complexes
As mentioned above, the diagnostic efficiency by many MRI methods frequently relies on the use of a new type of drugs, referred to as contrast agents (CAs), able to discriminate between normal and pathological tissues due to their different MR properties [11][12][13].The role of the CAs is to enhance the MRI signal by shortening the relaxation times of water protons in those tissues in which they distribute.Generally, the most investigated paramagnetic CAs are lanthanide complexes, with particular emphasis on the corresponding Gd(III)-chelates.Mn(II) and Fe(III) salts have also been investigated as paramagnetic metals, but they often become weakly chelated and dissociate spontaneously under in vivo conditions [14].
The modification of both linear and macrocyclic parental structures, Gd-Dtpa and Gd-Dota, is currently found to be an essential part of the investigations generating new CAs with improved magnetic properties.In this respect, new Gd-based CAs must exhibit sufficiently high thermodynamic and kinetic stabilities as important determinants for their use in MRI diagnosis.In addition, the CA´s must have improved molecular relaxivity properties, r 1 or r 2 (s -1 mM -1 ), defined as the longitudinal or transversal relaxation rates of the water protons in a 1 mM aqueous solution of the Gd(III)-chelate.
The following sections cover the synthetic approaches used to prepare these ligands, emphasizing the synthetic methodologies implemented to obtain heterocyclic chelators resulting progressively in more stable Gd complexes.

CAs derived from acyclic ligands
Miyake et al. [15] have reported a new type of optically active acyclic ligands 7 and 8 derived from (S)-aspartic acid and (S)-histidine (Scheme 1).Their Gd(III)-complexes exhibited a high relaxivity (r 1 = 9.4 and 9.9 s -1 mM -1 , respectively, at 300 MHz and 295 K), but their thermodynamic stability was not sufficient to be clinically relevant.Inclusion of pyridine ring was carried out by reductive amination between the corresponding amino acid and pyridine 2-carbaldehyde, affording compounds 9 and 10.Reaction of 9 with 2,2-dimethoxyacetaldehyde in the presence of NaBH 3 CN, followed by treatment in acidic medium yielded compound 11, which by condensation with 9 or 10 under the mentioned conditions and subsequent basic hydrolysis, afforded the chelating ligands 7 and 8.
Pyridine derivatives 12a-c, also based on ethylendiamine backbone, were recently reported by Platas-Iglesias et al. (Scheme 2) [16].Compounds 12a-c were synthesized from 2,6-pyridinedicarboxylic acid dimethyl ester by reduction of one of the ester groups using NaBH 4 in MeOH, followed by oxidation of the corresponding alcohol yielding compound 13.Condensation of 13 with ethylendiamine and subsequent reduction of the imine derivative gave compounds 14a-b.Alkylation of 14a with tert-butyl bromoacetate and deprotection of the ester moieties in acidic medium afforded 12a.Compound 12b was synthesized from precursor 14b by Mannich-type reaction with paraformaldehyde and phosphorous acid in 6 M HCl [17].Finally, chelate 12c, containing the same structural skeleton with the ethyl bridge substituted by a more rigid cyclohexyl moiety, was prepared using the same synthetic sequence used to prepare 12b (Scheme 2) [18].Gd-12a induced an r 1 value 5.0 s -1 mM -1 measured at 20 MHz and 25 ºC.
These Gd(III)-complexes were considered as T 2 relaxation agents for MRI (r 1 4.6-5.9s -1 mM -1 ; r 2 7.4-13.9s -1 mM -1 at 360 MHz and 25 ºC).Their synthesis was carried out starting from the corresponding heterocyclic rings by alkylation, using phase transfer conditions, to give compounds 16.Reaction of 16 with methyl iminodiacetate and subsequent acid or basic hydrolysis of the corresponding methyl ester afforded the chelating ligands 15 (Scheme 3).

Scheme 3.
N In a similar fashion, Mayoral et al. [20] described a novel family of chelating agents 17 containing bi-and bis-pyrazole structure, which form double tetradentate complexes with Gd(III).They exhibited a larger relaxivity in a range of 13.8-37.0s -1 mM -1 (60 MHz and 37 ºC), even compared to dendrimers with numerous metallic centers (Figure 3).Ligands 17a-d were obtained by alkylation of the corresponding bi or bispyrazole, reaction with methyl iminodiacetate and followed by the basic hydrolysis as mentioned above for the preparation of 15 (Scheme 3).
Compounds 18 were extremely reactive species, reacting with the corresponding nucleophiles to give different products, depending on the starting substrate.When compounds 18 reacted with water, complexones 17e-f were isolated after basic hydrolysis of the corresponding ester, while compounds 17g-i were prepared by reaction of 18 with the nucleophile shown in Scheme 4.
Heptadentate ligand 19 containing three pyridine rings was synthesized in two steps according to Scheme 5. Alkylation of 21 with the pyridylmethyl chloride 20 lead to compound 22, which when treated with NaOH/EtOH afforded the ligand 19.Gd(III)-19 showed an unusual and higher relaxivity with respect to the other heptadentate complexes (13.3 s -1 mM -1 measured at 60 MHz and 298 K) [22].
The Gd(III)-complex of ligand 23 with a pyridine ring as part of the triamine skeleton was reported by Aime et al. [23].This complex showed a high relaxivity (9.1 s -1 mM -1 at 20 MHz and 25 ºC) in comparison with other heptacoordinating complexes, probably due to the presence of the hydroxyl groups that contribute to an increase of the second coordination sphere of the complex.Compound 23 was synthesized in two steps through a double-Mannich reaction, as shown in Scheme 6.Thus, reaction of 3,5-dyhydroxypyridine with paraformaldehyde and ethyl iminodiacetate gave compound 24, whose treatment with 6M ClH afforded 23 in good overall yield (56%).Scheme 6.
High stability constants and high relaxivity values are the essential requirements of a Gd-complex to be a potential CA for MRI.Consequently, design of octadentate chelating ligands is pursued in most investigations of new CAs with high stability constants.Dtpa analogues derived from piperidine and azepane 25 and 26 have been reported as potential CAs for MRI [24].The synthetic routes used to prepare them firstly involved the functionalised heterocycles synthesis and subsequent alkylation (Scheme 7).The synthesis of 25 and 26 started from the piperidine 27, which after benzylation of amine group and subsequent ester reduction afforded the compound 28.Scheme 7. Dtpa analogues derived from piperidine and azepane.Treatment of 28 with thionyl chloride followed by reaction with sodium azide induces the ring expansion giving the corresponding azide, which was reduced to give the diamine 29.On the other hand, reduction of 27 using LiBH 4 lead to compound 30, which by reaction with p-toluensulfonyl chloride followed by treatment with sodium azide gave compound 31.Reduction of the azide groups in compound 31 and subsequent treatment with concentrated H 2 SO 4 afforded diamine 32.Finally, alkylation of 29 and 32 using tert-butyl bromoacetate and subsequent treatment with an acidic medium lead to 25 and 26, respectively.
In the same way, Cheng T.-H.et al. [25] reported the Gd(III)-complex of N'-2-pyridylmethyl derivative 33 showing similar relaxivity values (4.2 s -1 mM -1 at 20 MHz and 310 K) and stability constants to Gd(III)-dtpa.This ligand was synthesized from diethylenetriamine with protection of the terminal amine groups according to the route shown in Scheme 8. Alkylation of the central nitrogen of the amine backbone gave compound 34, which was sequentially treated with acid, alkylated with tertbutyl bromoacetate and finally hydrolysed in acidic medium yielding ligand 33.Scheme 8.
i) Phathalic anhydride, MeOH; ii) 2-picolyl chloride, NaOH; iii) HCl, reflux; iv) tert-butyl bromoacetate, K 2 CO 3 , acetonitrile; iv) HCl, rt Recently, we reported an experimental and theoretical study of lanthanide complexes of the modified dtpa derivative 35 that includes a 3,5-dimethylpyrazolylethyl arm, corroborating the effective azole coordination with the metal center [26].This compound was synthesized starting from N,N'-Boc-diethylenetriamine using a similar synthetic approach as for 33, which gave 35 in 83% overall chemical yield (Scheme 9).The corresponding Gd-complex of 35 exhibited an r 1 value of 5.1 s -1 mM -1 measured at 60 MHz and 310 K.
i) protected triamine, K 2 CO 3 , acetonitrile; ii) 6M ClH; iii) tert-butyl bromoacetate, DIEA, KI, DMF Parac-Vogt et al. [29] reported a stable dinuclear Gd(III)-complex of compound 38 exhibiting a r 1 value of 13.6 s -1 mM -1 (20 MHz, 25 ºC).Compound 38 was prepared from the bis-indole derivative 39, which was attached to two units of dtpa using TBTU/TEA in DMSO, according to Scheme 11.Gd-complexes have been also produced to recognize molecules such as sialic acids, a generic term for the N-or O-substituted derivatives of neuraminic acid, a nine-carbon monosaccharide containing a carboxyl group at the anomeric carbon.Glycoproteins or glycolipids containing sialic acids as terminal residues are often present in the cell surface or intracellular membranes.
Frullano et al. [30] synthesized a Gd-complex of 40 able to recognize and reversibly bind to sialic acid residues (Scheme 12), often proposed as diagnostic indicators in several diseases.Ligand 40 was synthesized from tri(ethylamine)amine (TREN) with two amine groups protected as tert-butoxycarbonyl (Boc) moieties and subsequent treatment with 2-methyl-2-thioimidazoline hydroiodide in refluxing ethanol.Removal of the Boc protecting groups in acidic medium afforded compound 41, which reacted with 2-formylphenylboronic acid giving the corresponding imine.Reduction of the latter and subsequent separation leaded to the monoamine 42.Finally, 40 was prepared by condensation between compound 42 and the dtpa-bis-anhydride (Scheme 12).Scheme 12. Gd(III)-40 recognizing and reversibly binding to the sialic acid residues.One of the first CAs that included pyrimidine rings in its structure was the Mn(II) complex of N,N´-dipyridoxylethylenediamine-N,N´-diacetate 5,5´-bis(phosphonate) (DPDP, 43), considered the active component of Teslascan ® , a contrast medium for hepatic MRI [31].This complex was synthesized in 1989 from pyridine 44 by condensation with ethylenediamine and subsequent reduction of the corresponding imine affording compound 45.Alkylation of amine groups in 45 with bromoacetic acid in basic medium gave 43 (Scheme 13) [32].Scheme 13.Synthesis of DPDP as chelating ligand for Mn(II).
Recently, 4-benzyloxy-5-pyrimidinone carboxylic acid 46 was reported as a building block to prepare several ligands through coupling with the corresponding amine.Two synthetic routes leading to 46 were reported (Scheme 14) [33].One of them starts with self-condensation of 47 and subsequent in situ reaction with acetamidine, giving compound 48.Nitrogen methylation followed by hydroxyl group deprotection in an acidic medium and benzylation of the freed hydroxyl moiety at the 5-position yielded compound 49.Finally, oxidation of 49, using phase transfer conditions, afforded the acid 46.
The second approach was a shorter one starting from diethyl oxalate and benzyl benzyloxyacetate.Condensation of both, in the same conditions mentioned above, leaded to ester 50, which by nitrogen methylation and basic hydrolysis of the ester group yielded the desired acid 46.Coupling of 46 with amines may produce a large series of hydroxypyrimidinones. Particularly, the Gd-complex of 51 exhibited a relaxivity value of 9.0 s -1 mM -1 measured at 20 MHz and 25 ºC.Scheme 14.Two synthetic strategies to prepare 51.Activation of 54 using N-hydroxy-succinimide (NHS), followed by amidation with TREN and subsequent benzyloxy group deprotection afforded compound 52.The corresponding Gd(III)-complex showed improved relaxivity (6.6 s -1 mM -1 at 20 MHz and 298 K), compared to the CAs normally used in clinical diagnosis.However, its stability constant decreased slightly with respect to the analogous 51 without the glycine unit.Bis-hydroxypyridonate chelates were investigated, the MR imaging investigations and biodistribution being reported [35].
More recently, Pierre et al. [36] described a new dendrimeric Gd(III) chelator 55, including 12 hydroxyl groups to ensure the overall solubility in water, and exibiting a r 1 values of 1.6 and 1.8 times greater than its corresponding monomer at 20 and 90 MHz, respectively (14.3 and 18.0 s -1 mM -1 25 ºC, respectively ).Ligand 55 derived from hydroxypyridonate was synthesized according to Scheme 16.Reaction of tris-benzyloxyethanolamine and N-tert-butoxycarbonylaminoaspartic acid in the presence of the coupling reagent, HATU (N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), and subsequent deprotection of the amino group in an acidic medium led to compound 56.Coupling of 56 with an additional aspartic acid unit and subsequent deprotection of the amino group afforded 57.Amidation reaction between 2,3-benzyloxyterephtalic chloride and 57, followed by coupling with compound 58, and hydrogenolysis of benzyl moieties yielded ligand 55.This chelating ligand coordinated one Gd(III) ion showing two water molecules in the inner-sphere.
Ligand 62 containing a tetrazolylmethyl arm was prepared from compound 61 according to Scheme 17 [41].Compound 61 was alkylated with freshly distilled chloroethylnitrile under heterogeneous conditions followed by treatment with TMS-N 3 , in the presence of Bu 2 -SnO, yielding the corresponding tetrazole derivative, which by acid hydrolysis afforded 62 (r 1 of Gd-62 was 4.8 s -1 mM -1 measured at 60 MHz and 37 ºC).

Scheme 17
The pH environment of a complex can modify some of the determinant factors that contribute to the relaxivity of CAs, such as the number of water molecules (q) in the inner-sphere of the complex, or even the second coordinating sphere.Pyridylmethyltetraamides 63 derived from Dota were studied as pH dependent CAs [42].Gd-63b showed a r 1 values of 5.6 and 4.1 s -1 mM -1 at pH of 8.5 and 3.3, respectively (20 MHz).Compounds 63 were prepared by alkylation of cyclen with the corresponding chloroacetamide 64 (Scheme 18).Ligands 64a-b were obtained by reaction of chloroacetyl chloride and the corresponding amine.Compound 64b was synthesized from 2-hydroxypicolinic acid by esterification with methanol, transformation of the ester in its amide by reaction with ammonia and subsequent reduction with borane giving amine 65.The protection of the hydroxy group as benzyloxy moiety in 65 was carried out in three steps: i) protection of the amine as tert-butylcarbamate, ii) benzoylation of hydroxyl group and, iii) deprotection of amine group in acidic medium affording compound 66.Scheme 18. Pyridylmethyltetraamides as pH responsive CAs.Another example of a CA displaying pH dependent relaxivity is the Gd(III)-complex of ATPconjugated DO3A 67, reported by Ratnakar and Alexander (6.5 s -1 mM -1 measured at 24 MHz, 308 K and pH of 8.5) [43].Compound 67 was prepared from cyclen by trialkylation using bromoacetic acid and subsequent reaction with 1,3-bromopropane in basic medium leading to compound 68.Treatment of 68 with ATP disodium salt in water afforded 67 (Scheme 19).Scheme 19.ATP-conjugated DO3A 67 which its Gd(III)-complex showing pH dependence relaxivity.Several contributions have reported novel series of tetraazamacrocycles containing one pyridine ring as part of the macrocycle.Their lanthanide complexes generally showed high stability and improved relaxivity.These heptadentate ligands form stable complexes with lanthanide ions, allowing the coordination of two water molecules in the inner-sphere and consequently providing higher relaxivity than Dota chelates.In general these complexes are characterized by a relatively fast water exchange rate as compared to the octacoordinated ligands.Scheme 20 shows the synthetic approach to prepare 69 [44].Ligand 69a was synthesized by reaction of 1,4,7-tritosyl-1,4,7-triazaheptane with bis(2,6-chloromethyl)pyridine, followed by deprotection of the amine groups in an acidic medium yielding compound 70a.Alkylation of 70a with chloroacetic acid in the presence of sodium carbonate, and subsequent treatment in acidic medium yielded 69a (Gd-69 showed a r 1 of 6.9 s -1 mM -1 at 20 MHz and 25 ºC).Compounds 69b-c were synthesized from the corresponding triprotected amine via the macrocycles 70b-c.The latter reacted with methyl chloroacetate as alkylating agent yielding the corresponding esters, which were hydrolyzed using KOH in methanol.Complexes Gd-69b and Gd-69c exhibited a r 1 values of 5.9 and 6.3 s -1 mM -1 , respectively (20 MHz and 25 ºC).Analogous systems 71 with substitutents on a pyridine ring were prepared using a similar synthetic approach to that employed to obtain 69, starting from the corresponding bis(2,6-chloromethyl)pyridine derivative (Figure 6) [45].Their corresponding Gd(III)-complexes depicted improved relaxivity with respect to 69 (r 1 = 8.25 s -1 mM -1 measured at 20 MHz and 25 ºC, for Gd-71a).Studies on the binding of the Gd(III)-71a to biomacromolecules such as the human serum albumin (HSA), were described and even the formation of the inclusion compounds using β-cyclodextrins and poly-β-cyclodextrins were reported.These non covalent adducts showed higher relaxivity due to the reduced motion of the corresponding Gd(III)complexes, becoming well tolerated in animal tests.Gd(III)-71b was considered as micelar CAs, its relaxivity depending on concentration (maximum value of r 1 was 29.2 s -1 mM -1 at 20 MHz, 25 ºC and 1.5 mM).
The relaxivity of pyridine-based complexes can be improved through the introduction of polar moieties supported over the heterocyclic ring, a circumstance probably improving the effects of the second coordination sphere.An example of that is the Gd(III)-complex of macrocycle 72, containing two free hydroxyl groups, with r 1 of 8.5 s -1 mM -1 at 20 MHz and 25 ºC [23].Ligand 72 was prepared by double Mannich reaction between 3,5-dihydroxypyridine, paraformaldehyde and amine 73 followed by treatment with neat TFA-anisole (Scheme 21).On the other hand, inclusion of the methylenephosphonic arm on triamine backbone of these macrocycles induced higher relaxivity of the corresponding Gd(III)-complexes.Thus, the complex of 74 showed two water molecules in its inner-sphere and another water molecule bound to the phosphate group providing an important contribution to the higher relaxivity (r 1 = 8.3 s -1 mM -1 at 20 MHz and 25 ºC) [46].Compound 74 was prepared from N-tosylaziridine and diethyl aminomethylphophonate leading to the amine 75 (Scheme 22).Cyclization of 75 with bis(2,6-chloromethyl)pyridine in basic medium, deprotection of amine groups and subsequent alkylation of them afforded compound 74 in high overall chemical yield.A similar synthetic route (Scheme 23) was used to obtain the macrocycles 76 starting from the corresponding amino acids [47].Relaxivity of their Gd(III)-76a-c was 8.3, 10.5, and 8.1 s -1 mM -1 at 20 MHz and 25 ºC, respectively, and in the range of the major of the heptadentate complexes.However, the inclusion of two phosphonate groups in 76c caused a decrease of the number of water molecules in the inner-sphere (q = 1) remaining two of those in second coordinating sphere (q 2nd = 2).Esterification of the corresponding amino acid followed by reaction with two units of the N-tosylaziridine yielded compounds 77, which gave the corresponding macrocycles under the conditions mentioned above; deprotection of amine groups in 77a using acidic medium leaded to compound 78.While alkylation of the free amine groups in compound 78 using chloroacetic acid gave 76a, treatment of 78 with phosphoric acid and paraformaldehyde yielded 76c.Ligand 76b was synthesized from 77b in five steps as follow: i) protection of hydroxyl group, ii) formation of macrocycle, iii) deprotection of amine groups, iv) alkylation of those and hydroxyl using methyl chloroacetate and, v) basic hydrolysis.

Scheme 23.
CO Zheng et al. [48] have developed new lipophilic macromolecular chelators of lanthanide ions 79 for in vivo applications.Some types of cells can be labeled with these complexes in order to visualize cell migration in different biological systems.Compounds 79 were prepared by reaction of bis(2,6bromomethyl)pyridine and the corresponding amines and subsequent reaction with (CF 3 CO) 2 O to give compound 80, this last additional step being necessary to purify the corresponding amines (Scheme 24).Finally, treatment of 80 in basic medium and reaction of those with DTPA-bisanhydride afforded compounds 79.Scheme 24.Lipophilic macromolecular chelators of lanthanide ions 79.Multinuclear complexes based on 5,6-dihydro-1,10-phenanthrolin-5-yl-DO3A 81 were reported as complexes with higher molecular weights and consequently reduced motion of the complex and increased relaxivity [49] (Figure 7).Complex Gd(III)-81 spontaneously forms highly stable triscomplexes with Fe(II) and Ni(II) characterized because of their relaxivity is not dependent on the temperature in a 5-30 ºC range.Relaxivity values reported for Gd-81 and Fe[Gd-81] 3 were 3.7 and 36.6 s -1 mM -1 at 20 MHz and 37 ºC, respectively.

Concluding Remarks and Future Perspectives
We have described above the main synthetic strategies used to produce heterocyclic CAs, a family of ligands depicting very appropriate stability and relaxivity properties for MRI.Basically, most of them are produced through two general organic reactions, namely amine alkylations or amidation.In general heterocycles provide good electron donor ligands suitable for improving the chelating capacity of the earlier complexones.The present review provides an adequate frame to analyze the importance of the heterocyclic ring in determining the coordination chemistry, the relaxivity and the stability properties of the resulting complexes.
On these grounds, the use of heterocyclic CA´s is expected to increase in the future.The possibilities to obtain physiologically responsive agents, reflecting tissue properties beyond anatomy has already started [50].Further improvements are expected from the combination of novel synthetic approaches and updated MR imaging techniques, as Magnetization Transfer [51].The combination of both, synthetic and MRI approaches will certainly exceed the capabilities of their independent use.As a complementary field, the development of heterocyclic contrast agents useful for in vivo spectroscopy and spectroscopic imaging of pH and pO 2 in healthy and pathological tissues, constitutes an area of growing interest [52][53][54][55].The combination of spectroscopic and imaging approaches in multiparametric studies will further enhance the diagnostic potential of these new methods [56].Finally, the development of multimodal heterocyclic probes, active in different imaging modalities (MRI, MRS, PET), is currently envisioned as one of the most attractive goals for the immediate future.

Figure 5 .
Figure 5. Cyclen derivatives used to synthesize the most useful macrocyclic CAs.