A gadolinium(III) complex based on the thymine nucleobase with properties suitable for magnetic resonance imaging ap- plications

The paramagnetic gadolinium(III) ion is used as contrast agent in magnetic resonance (MR) imaging to improve the lesion detection and characterization. It generates a signal by changing the relaxivity of protons from associated water molecules and creates a clearer physical distinction between the molecule and the surrounding tissues. New gadolinium-based contrast agents displaying larger relaxivity values and specifically targeted might provide higher resolution and better functional images. We have synthesized the gadolinium(III) complex of formula [Gd(thy)2(H2O)6](ClO4)3·2H2O (1) [thy = 5-methyl-1H-pyrimidine-2,4-dione or thymine], which is the first reported compound based on gadolinium and thymine nucleobase. 1 has been characterized through vis-IR, SEM-EDAX and single-crystal X-ray diffraction techniques, and its magnetic and relaxometric properties have been investigated by means of SQUID magnetometer and MR imaging phantom studies, respectively. On the basis of its high relaxivity values, this gadolinium(III) complex can be considered a suitable candidate for contrast enhanced magnetic resonance imaging.


Introduction
Thymine is one of the four natural nitrogen bases that are precursors and part of the structure of the deoxyribonucleic acid (DNA) macromolecule [1,2]. This pyrimidine base has been widely studied, in part because of the common mutations of DNA caused when adjacent thymines are irradiated by UV light and are dimerized, generating the well-known thymine dimers [2,3]. Besides, considerable effort has also been devoted to the rational design of drugs that might selectively inhibit thymine biosynthesis, thereby blocking DNA replication, especially in rapidly dividing malignant cells.
In comparison with other natural nucleobases, the coordination chemistry of thymine-based metal complexes has been much less investigated. Most of the thymine-containing complexes have been prepared with the nucleobase in form of thyminate anion, that is, releasing one or two protons of its N-H groups, whereas the reported examples obtained with the thymine molecule acting through its carbonyl groups as a neutral ligand toward the metal are much scarcer [4][5][6][7]. In that respect, theoretical studies have been performed on metal clusters to investigate preferential binding sites of the thymine molecule [8].
There exist some published thymine-based complexes that exhibit singular properties, as for instance the Ru II -thymine complex [Ru(PPh3)2(thy)(bpy)]PF6 [where PPh3 = triphenylphosphine and bpy = 2,2′-bipyridine], which is a potent cytotoxic agent with ability to bind to DNA inducing apoptotic cell death in human colon carcinoma [6,7]. Thymine has also shown to be a highly specific ligand towards Hg II metal ion [9,10]. The discovery of the linear thymine-Hg II -thymine structure, which affords a high stabilization of thymine-thymine pairs in DNA, has led to the designing and development of recent thymine-based sensors [11][12][13].
Regarding lanthanide complexes, only two structures based on thyminate anion have been published so far, namely, the heteropolynuclear complexes of general formula [{Cp*Rh III (µ-thym)}4{Ln III (NO3)2}] + , where Ln = Dy III and Er III , which form cationic metallacalix [4]arene-type systems [14]. Hence, no crystal structure of gadolinium complex based on thymine has been reported up to date.

Reagents and Instruments
All of the manipulations were performed under aerobic conditions. All the commercial chemicals were used as received. CAUTION!: Although no problems were encountered in this work, care should be taken when using the potentially explosive perchlorate anion (ClO4 -).
Elemental analyses (C, H, N) and X-ray microanalysis were performed by the Central Service for the Support to Experimental Research (SCSIE) at the University of Valencia. Scanning electron microscopy (SEM) images and results were obtained from a Hitachi S-4800 field emission scanning electron microscope. Infrared spectrum (IR) of 1 was recorded with a PerkinElmer Spectrum 65 FT-IR spectrometer in the range of 400 to 4000 cm −1 . Variable-temperature, solid-state direct current (DC) magnetic susceptibility data down to 2.0 K were collected on a Quantum Design MPMS-XL SQUID magnetometer (Quantum Design, Inc., San Diego, CA, USA) equipped with a 5 T DC magnet. The experimental magnetic data were corrected for the diamagnetic contributions of the constituent atoms and also for the sample holder. Data were collected on a Philips Achieva 3T clinical MR scanner with a volumetric head 8 channels SENSE coil at La Fe University and Polytechnic Hospital.

X-ray Data Collection and Structure Refinement
X-ray diffraction data from a single crystal of 1 with dimensions 0.34 × 0.12 × 0.05 mm 3 was collected on a Bruker D8 Venture diffractometer with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å). The structures were solved by standard direct methods and subsequently completed by Fourier recycling by using the SHELXTL software packages. The obtained model was refined with version 2018/1 of SHELXL against F 2 on all data by full-matrix least squares [21]. All non-hydrogen atoms were anisotropically refined, whereas the hydrogen atoms of the thymine molecules were set in calculated positions and refined isotropically by using the riding model. The graphical manipulations were performed with the DIAMOND program [22]. The CCDC code for 1 is 2009091.

Synthetic Procedure
Compound 1 is prepared from a mixture of Gd2O3 and thymine, which reacts in an aqueous suspension acidulated with perchloric acid, and is stirred at 60 °C for 1h. The resulting solution is left to evaporate at room temperature for 1 week, thus generating colorless parallelepipeds of 1. Once synthesized, 1 is air-stable over a period of several days. In order to study its stability further, electronic absorption spectra of 1 were collected both in solid state and in aqueous solution. Remarkably, the two spectra show the same absorption bands, this fact indicating its stability in aqueous solution (Fig. 2a). SEM-EDAX analysis gave a molar ratio of 1:3 for the Gd/Cl relation in 1 (Fig. 2b).
To further analyze the coordination environment and geometry of the Gd III ion in 1, the SHAPE program was used [27]. In 1, the single Gd III ion exhibits a coordination number (CN) equal to 8. For Gd(1) a value of 0.307 was obtained and associated with a square antiprism (SAPR) geometry, the next and closer computed value being 1.733, which was assigned to a biaugmented trigonal prism (BTPR) geometry (Table 3). Hence, these calculated values allow us to assign the D4d symmetry to the Gd(1) ion in complex 1 (Table 3).

Analysis of the Hirshfeld surfaces
Hirshfeld surfaces of the cationic [Gd(thy)2(H2O)6] 3+ complex were calculated and its closer intermolecular interactions were analyzed through the CrystalExplorer program [28,29]. These surfaces were mapped having into account the distance from a point on the surface to the nearest atom outside (de) and inside (di) the surface. To overcome limitations relate to the size of atoms, a normalized contact distance (dnorm) was also considered [28,29]. For compound 1, Hirshfeld surfaces are shown in Fig. 4, where the shorter contacts are displayed using red color [30]. Intermolecular O···H contacts between water molecules and between water molecules and carbonyl groups of the thymine molecules are the main interactions detected on the Hirshfeld surface (Fig. 4). The most important O···H contacts are those involving H-bonds between non-coordinated and coordinated water molecules, which are approximately 48% of the complete fingerprint plot, whereas the O···H interactions involving non-coordinated water molecules and carbonyl groups are highlighted from the full fingerprint as ca. 19% of the plot. Finally, additional N···H contacts involving non-coordinated water molecules and N-H groups of the thymine molecules only cover approximately 2% of the fingerprint plot (Fig. 4).

Magnetic Properties
Dc magnetic susceptibility measurements were carried out on a freshly prepared microcrystalline sample of 1 in the 2-300 K temperature range and under an external magnetic field of 0.5 T. The MT versus T plot (M being the molar magnetic susceptibility per Gd III ion) for compound 1 is given in Fig. 5. At room temperature, the MT value is ca. 7.89 cm 3 mol -1 K, which is very close to that expected for a magnetically isolated Gd III ion (4f 7 ion with gGd = 2.0, SGd = 7/2 and LGd = 0). Upon cooling, the MT value approximately follows the Curie law to ca. 25 K with decreasing temperature, before MT decreases reaching a minimum value of ca. 7.21 cm 3 mol -1 K at 2 K. The decrease of the MT value observed for complex 1 would likely be assignable to intermolecular interactions and/or small zero-field splitting (ZFS) effects [31].
To analyze the magnetic behavior and fit the experimental data of the χMT versus T plot for complex 1, the theoretical expression for the magnetic susceptibility of a single and isotropic SGd = 7/2 center was used [M = (NμB 2 gGd 2 /3kB)SGd(SGd + 1)/(T -)] [22,29]. Due to possible intermolecular interactions that can take place in 1, a  parameter was also included in this expression. The best least-squares fit gave the parameters gGd = 2.008(1) and  = -0.039(1) K with R = 1.9 × 10 −5 for 1 {R being the agreement factor defined as Σi[(χMT)i obs − (χMT)i calcd ] 2 /[(χMT)i obs ] 2 }. Field dependence of the molar magnetization (M) plot for 1 at 2 K is given in the inset of Fig. 5. This plot exhibits a continuous increase of M with the applied magnetic field, showing a maximum value of M obtained for 1 (ca. 6.92 μB) at 5.0 T that is as expected for a mononuclear Gd III complex [24]. The experimental data of the M versus H plot were close to the Brillouin curve generated with values of g and S of 2.0 and 7/2, respectively ( Figure 5) [31].

MR Imaging Phantom Studies
The relaxometric properties of compound 1 as a potential high-field MR imaging contrast agent were evaluated [32]. 13 samples of 1 were prepared in physiological serum with concentrations covering the range of 0.0-3.2 mM and were measured on a clinical MR scanner (Philips Achieva 3T) by using the volumetric head 8 channels SENSE coil (Fig. 6). The relaxation rate, denoted by R (and expressed in s -1 ), was obtained for each concentration through the calculation of the corresponding relaxation time T. R1 was obtained by calculating the T1 time from FFE sequences with 2°, 5°, 10°, 15°, 25° and 45° flip angles, whereas r2 and r2* values were obtained after calculating T2 and T2* relaxation times from TSE and GRE sequences with 32 echo times each, TE1 = 10 ms, ∆TE = 10 ms and TE1 = 0.9 ms, ∆TE = 0.7 ms, respectively [33]. Thus, the longitudinal relaxivity (r1) of 1 at 3 T was determined to be 16.1 mM -1 s -1 , whereas the transversal relaxivities r2 and r2* values were 13.5 and 14.5 mM -1 s -1 , respectively (Figs. 7 and 8). These results show relaxivity values for 1 that are much higher than those of commercial MR imaging contrast agents currently employed on 3 T equipments [32], which make 1 potentially useful.

Conclusions
In summary, the preparation, crystal structure, magnetic properties and MR imaging phantom studies of a novel Gd III complex based on the thymine nucleobase, of formula [Gd(thy)2(H2O)6](ClO4)3·2H2O (1) [thy = thymine], have been reported. 1 is the first example of compound based on gadolinium and thymine. Given that most of the thymine-containing complexes have been reported with this ligand in form of thyminate anion, we presented in this work a singular strategy to prepare lanthanide systems where the thymine molecule acts as a neutral ligand towards the metal ion. Besides, 1 displays high relaxivity values and, therefore, can be considered as a suitable candidate for further developments and MR analysis.
Supplementary Materials: X-ray crystallographic data in CIF format for compound 1 are available online at www.mdpi.com/xxx/s1.