Gd-Complexes of New Arylpiperazinyl Conjugates of DTPA-Bis(amides): Synthesis, Characterization and Magnetic Relaxation Properties

Two new DTPA-bis(amide) based ligands conjugated with the arylpiperazinyl moiety were synthesized and subsequently transformed into their corresponding Gd(III) complexes 1 and 2 of the type [Gd(L)H2O] nH2O. The relaxivity (R1) of these complexes was measured, which turned out to be comparable with that of Omniscan®, a commercially available MRI contrast agent. The cytotoxicity studies of these complexes indicated that they are non-toxic, which reveals their potential and physiological suitability as MRI contrast agents. All the synthesized ligands and complexes were characterized with the aid of analytical and spectroscopic methods, including elemental analysis, 1H-NMR, FT-IR, XPS and fast atom bombardment (FAB) mass spectrometry.


Introduction
Magnetic Resonance Imaging (MRI) is at present one of the most powerful and efficient non-invasive imaging modalities available for clinical diagnosis. MRI is considered the safest diagnostic technique compared to competing radio-diagnostic methods due to the fact it does use harmful high-energy radiation [1][2][3]. With an enormous diagnostic potential, MRI can be used to assess anatomical changes and for monitoring of organ functions, for instance following functions of the human brain on a real time-scale by functional-MRI (fMRI). In cranial abnormalities or multiple sclerosis, MRI is considered the only reliable diagnostic method [4,5]. MRI contrast agents (CAs) usually involve low molecular weight Gd(III) chelates with an acyclic or macrocyclic ligand [6]. They are diagnostic magneto-pharmaceuticals used to enhance the image contrast by increasing the water proton relaxation rate in the body. The efficacy, known as relaxivity, of a CA is measured by its ability to transmit the paramagnetic properties into the bulk water proton and thereby shorten the longitudinal relaxation (T1) time of water protons, which in turn provides impressive anatomical information [2]. Some representative advantages of employing the Gd(III) ion in most MRI CAs are due to its favorable combination of a large magnetic moment (spin-only μeff = ¼ 7.94 BM, from seven half-filled f orbitals) and long electron spin relaxation time (10 −8 to 10 −9 s, from symmetric S electronic state) [7].
In general, anionic Gd-complexes, for instance Gd(DTPA) 2− , suffer from limitations such as hyperosmolality under physiological conditions and limited utility in focal lesion detection, leading to adverse effects [8]. To overcome the inherent limitations of anionic Gd-complexes and to improve the tissue and/or organ-specificity, the preparation of neutral Gd-macrocyclic analogues for the development of efficient ("optimized") CAs is highly desirable [9][10][11][12]. Earlier reports have suggested that incorporation of alkyl and aromatic groups in the side arm of diethylenetriamine pentaacetic acid (DTPA) rendered excellent relaxivity and water solubility [13][14][15]. In the light of the above, we have designed novel ligands from the reaction of DTPA-bis(anhydride) with suitably modified arylpiperazines and subsequently transformed them into their corresponding Gd(III)-complexes 1 and 2. Herein, we wish to disclose the synthesis, characterization, relaxivity measurements and cytotoxic studies of these new complexes.

Results and Discussion
The development of an optimum MRI contrast agent would necessitate consideration of the high relaxivity, non-cytotoxicity and high water solubility of the targeted complexes. As suggested by earlier reports, modification of the ligand, for instance by the introduction of polar groups on the alkyl substituents of the amide N-atoms of DTPA-bis(amide), can lead to the formation of water soluble Gd-complexes [16]. Therefore, we intended to synthesize Gd-complexes with different arylpiperazine ligands bearing different functionalities in the aryl moiety.
To this end, we first planned to synthesize ligand 7 (Scheme 1), possessing a nitrile group in the aryl moiety. Condensation of benzonitrile 3 [17] with piperazine 4 in DMF generated the coupled product 5 in excellent yield. Reduction of the nitro group of intermediate 5, using Pd-C or Ra-Ni as catalysts, proved problematic and led to the formation of complex mixtures of products, which were difficult to resolve by column chromatography purification. Hence, the reduction of nitro group via transfer hydrogenation with ammonium formate in methanol, using Pd-C as catalyst was employed to produce the corresponding intermediate 6 in high yield. Finally, reaction of aryl amine 6 with diethylene triamine pentaacetic acid dianhydride (DTPAA) [18] in DMF produced the desired ligand 7. With 7 available, we turned to the synthesis of Gd-complex formation. Unfortunately, the reaction of 7 either with GdCl3 or Gd(OAc)3 in pyridine was unsuccessful, unreacted 7 being recovered from these reactions. The decreased reactivity of 7 towards complexation could be attributed to both steric and electronic factors. The substitution position and electron withdrawing ability of nitrile group on the aryl moiety generated congestion and reduced the nucleophilicity of the amine moiety (Scheme 1).  Consequently, we decided to synthesize ligands 16 and 17, which bear methoxy and methoxymethyl substituents in the aryl moiety (Scheme 2). Thus, phenol 9 was reacted with the appropriate alkyl halide in DMF to produce known compounds 10 [19] and 11 [20], respectively. Treatment of intermediates 10 and 11 with piperazine 4 produced 12 and 13 in good yields (Scheme 2). Reduction of the nitro group of intermediates 12 and 13 with ammonium formate in methnaol, using Pd-C as catalyst, generated the corresponding aryl amines 14 and 15 in high yields. Condensation of amines 14 and 15 with DTPAA produced the desired ligands 16 and 17, respectively. Finally, heating of ligands 16 and 17 with GdCl3 in pyridine produced the desired complexes 1 and 2 in good yield (Scheme 2). The structures of complexes 1 and 2 were established by their infrared (IR) spectra, elemental analysis, fast atomic bombardment (FAB) mass spectrometry and X-ray photoelectron spectroscopy (XPS) measurements. Complexes 1 and 2 are highly hygroscopic and were isolated as hydrated solids. The appearance of the ν (OH) band from the water of crystallization at 3426 and 3480 cm −1 , supported this observation [13]. The disappearance of the carbonyl stretching bands of the free ligands at 1728 and 1733 cm −1 in both complexes 1 and 2 indicated the participation of the carbonyl groups in coordination [21]. The chemical composition of compounds 1 and 2 was further confirmed by XPS ( Figure 1). In compound 1, the C 1s region showed three peaks (eV) at 284.8 (C-C), 285.8 (C-N, C-O) and 287.7 (C=C, C=O) [4], whereas the Gd 4d region had four peaks, showing a multiplet structure, the N 1s region displayed one peak at 399.7 eV. Likewise, the O 1s region has peaks (eV) at 531.0 (C-O) and 532.7 (C=O) (Figure 1) [22]. The XPS measurement of compound 2 showed a similar peak pattern as 1 ( Figure 1).   After structural characterization of 1 and 2, we next moved on to measure their relaxvities. Whereas the commercially available MRI contrast agent Omniscan ® is freely soluble in water and methanol; compounds 1 and 2 have moderate water solubility. Relaxvities were calculated as the inverse of the relaxation times per mM based on the earlier report [23]. The R1 data (Table 1) revealed that 2 had a higher relaxivity than 1, but lower than Omniscan ® . This relatively lower relaxivity of 1 and 2 compared Omniscan ® could be attributed to their lower solubility in water. In vitro cell toxicity of compounds 1 and 2 was studied on adherent 3T3 mouse embryonic fibroblastic cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [25]. This assay is used as a quantitative colorimetric method to measure cytotoxicity as well as cell viability. The study revealed that compounds 1 and 2 were non-toxic (Table 1), which warrants their physiological suitability as potential contrast agents for MRI.

CN
In conclusion, two new Gd(III) complexes 1 and 2 of the type [Gd(L)H2O]·nH2O have been synthesized The relaxivity of these complexes was slightly lower compared to Omniscan ® , a commercially available MRI contrast agent. The lower relaxivity of 1 and 2 was attributed to their lower solubility in water. The cytotoxicity studies of these complexes revealed that they are non-toxic which warrants their potential and physiological suitability as MRI contrast agents. The water solubility of these compounds could be increased by introducing polar functions in the aromatic ring, which in turn, may improve their relaxivity. Hence, these compounds may serve as a starting point to obtain optimized structures to produce more efficient MRI contrast agents.

General Information
Melting points were determined on a Büchi apparatus (Büchi, Flawil, Switzerland) and were uncorrected. Elemental analysis was carried out on a Perkin Elmer Elemental Analyzer Series 11 Model 2400 (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on a Perkin Elmer 16F PC FTIR spectrophotometer. 1 H-and 13 C-NMR spectra were measured in CDCl3 and DMSO-d6 using TMS as internal standard on a LA 500 MHz spectrometer (JEOL, Peabody, MA, USA). Mass spectra were recorded on a 6890 N GC-MS system (Agilent Technologies, Santa Clara, CA, USA). Analytical TLC was carried out on silica gel 60 F254 plates (catalog #-5554-7, Merck, Darmstadt, Germany); column chromatography was carried out on Merck silica gel (200-400 mesh, catalog # 61860805001730). All chemicals and reagents were obtained from Sigma-Aldrich (Buchs, Switzerland) in reagent grade and were used without further purification.

Relaxivity Measurements
Longitudinal relaxation times measurements were performed in a 3T MRI machine (3T Imaging, Morton Grove, IL, USA) The magnetic resonance images were taken at 17 different TR values ranging from 20 to 1500 ms and the T1 were obtained from non-linear least square fit measured at each T1 values. R1 were calculated as an inverse of relaxation times per mM.

In Vitro Cell Toxicity
Toxicity for compounds 1 and 2 was analyzed on adherent 3T3 mouse embryonic fibroblastic cells by MTT assay following Mesaik et al. [25] with some modification. In brief, cells were incubated at 6 × 104 mL −1 concentration in 96 well flat bottom plate in 5% CO2 and 37 °C for 24 h. After adherence of cells, compounds were added at various concentrations for further 48 h incubation. On day 3 the tetrazolium dye MTT was added. After 4 h of incubation media was removed and organic solvent DMSO was added to dissolve insoluble purple formazan. Absorbance was taken at 540 nm using a spectrophotometer and the % viability of the cells was calculated.

Conclusions
In conclusion, two new Gd(III) complexes 1 and 2 of the type [Gd(L)H2O]·nH2O have been synthesized The relaxivity of these complexes were slightly lower compare to Omniscan ® , a commercially available MRI contrast agent. The lower relaxivity of 1 and 2 was attributed to their lower solubility in water. The cytotoxicity studies of these complexes revealed that they are non-toxic which warrant their potential and physiological suitability as MRI contrast agents. The water solubility of these complexes may be increased by introducing polar functions in the aromatic ring, which in turn, may improve their relaxivity. Hence, these complexes may serve as a starting point to get optimized compound to achieve more efficient MRI contrast agent.