Synthesis of New Bis(3-hydroxy-4-pyridinone) Ligands as Chelating Agents for Uranyl Complexation

Five new bis(3-hydroxy-4-pyridinone) tetradentate chelators were synthesized in this study. The structures of these tetradentate chelators were characterized by 1H-NMR, 13C-NMR, FT-IR, UV-vis, and mass spectral analyses. The binding abilities of these tetradentate chelators for uranyl ion at pH 7.4 were also determined by UV spectrophotometry in aqueous media. Results showed that the efficiencies of these chelating agents are dependent on the linker length. Ligand 4b is the best chelator and suitable for further studies.


Introduction
Uranium is introduced into the body by ingestion, inhalation, or through wounds. The risk of uranium contamination has considerably magnified because of the extensive use of uranium as nuclear fuel in fission reactors and as weapon-grade nuclear material. The hexavalent uranyl ion [UO 2 2+ , U(VI)] is the most stable form in vivo [1] and is complexed in the blood by chelating agents, such as proteins or carbonates. Meanwhile, tissues, especially the kidney and bones, accumulate uranium for months to years, which will induce cancer and chemical intoxication [2][3][4]. Thus, uranium should be eliminated from the body by administration of nontoxic chelating agents that can form stable complexes with the uranyl ion. Among the different chelators, 3-Hydroxy-4-pyridinones (3, have emerged as one of the hotspots in studies that focus on heavy metal chelators because of their special bidentate structure, highly selective chelating capacity, and significant physiological activities [5][6][7][8][9][10][11][12]. To date, three kinds of 3,4-HOPO derivatives are available, namely bidentate hydroxypyridinones [5,6], tetradentate hydroxypyridinones [7][8][9], and hexadentate hydroxypyridinones [10,11]. The ideal design for chelators is to synthesize the hydroxypyridinones, which have excellent chelating efficacy and high selectivity of interaction, with special biological receptors in one molecular unity. The chelating capacity of bidentate hydroxypyridinones is usually inferior to that of hexadentate desferrioxamine [13,14]. Hexadentate hydroxypyridinones have a higher chelating ability than hexadentate desferrioxamine, but the poor absorption caused by their high molecular weight limits their application [15]. Thus, tetradentate hydroxypyridinones have been one of most extensively investigated compounds among heavy metal chelators [16]. Recent studies have reported that the tetradentate hydroxypyridinones exhibit better assays in vivo such as high chelating efficacy for Fe and Ga as well as excellent hydrophilic character [17][18][19][20]. However, few studies have examined the hydroxypyridinone chelating uranyl ions. In this paper, a series of new tetradentate hydroxypyridinone chelators is reported, and the binding affinities towards a uranyl cation (UO 2 2+ ) were examined with UV spectrophotometry.

Characterization
All products were purified and characterized by FT-IR, NMR, UV-vis and MS, and all characterizations were in accordance with the structures of the products.
The 1 H-NMR spectra of compounds 2-4a are shown in Figure 1. Compared with 2a, the signal of NHCH 2 CH 2 -in 3a shifted to the low chemical field (from 2.75 to 3.31) because of the electrophilic effect of the -CONH, and the singlet of the -COCH 2 CO-in 3a appeared at δ = 3.20 ppm. Compound 4a was obtained after the hydroxyls of 3a were deprotected. The peaks at 7.34 ppm of the benzene ring and 5.07 ppm of the methylene disappeared when the benzyl group was removed, and the signal of the CH 3 -Pys shifted from 2.16 ppm to 2.42 ppm.
The 1 H-NMR spectra of 2b-e, 3b-e, and 4b-e have similar results. The 1 H-NMR spectra of 4b and 4c exhibit two doublets at 7.6 ppm-7.7 ppm and 6.45 ppm-6.55 ppm with J AB = 7.5 Hz for the two nonequivalent protons in the pyridine ring. Compared with 4d and 4e, the signals of the pyridine ring protons appeared in the higher field because the oxygen atom is more electronegative than the nitrogen atom. The signals of the two nonequivalent protons in the pyridine rings of 4d and 4e appeared at 8.30 ppm-8.33 ppm (d, J = 7.2 Hz, 1H) and 7.23 ppm-7.25 ppm (d, J = 7.2 Hz, 1H), respectively. Additionally, the signals of -COCH 2 CO-in 4d and 4e appeared in a lower field than in 4b and 4c under the same condition. The singlet of -COCH 2 CO-in 4b and 4c appeared at 3.0 ppm-3.1 ppm, whereas that of 4d and 4e appeared at 3.6 ppm-3.8 ppm, respectively.

Complexation
Although present metal-complexation studies are focused on a set of three-charged hard metal ions [21,22] (e.g., Fe, Al, and Ga), the current study on decorporation [23] for UO 2 2+ is based on the extreme damage to the environment caused by uranyl cations (UO 2 2+ ) in biological systems. In this paper, the complexation behavior of tetradentate hydroxypyridinones 4a-e and the uranyl cation was evaluated by the spectrophotometric method [24,25]. Then ligands 4a-e complex with the uranyl, and the formation constants (log K) for the ligands could be calculated as follows: Based on the Lambert-Beer law: The complex stability constant of 4a and UO 2 2+ at pH 7.4 was calculated by Equation (1), and the corresponding logK cond U-L 4a at 7.4 pH levels was 21.7. The corresponding complex stability constants of other ligands were also calculated by Equation (1), and the results are shown in Table 1.  Although the structures of Ligands 4a-e are similar, some consistent differences are apparent. The change of the linker length has a great influence on the their U(VI) chelation efficiency [26][27][28]. As the length of the linker increases, the angle formed between the uranium and two phenolic oxygen donors also becomes greater, and it leads to an increase of the strain in this complex. [29] As a result of the strain imposed by the linker, that two carbon atoms may be considered the optimal length is consistent with the high efficacy of ligands 4b and 4e for in vivo uranyl chelation. Ligands 4b and 4e exhibit the highest K cond at pH 7.4, and the corresponding logK cond are 22.7 and 22.2. The modest reduction of body uranium in animals by the injection of bidentate Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid, disodium salt) and its U(VI)-catechol complex (log KML ) is 15.9 [29][30][31], which suggests the U(VI)-Ligands 4b complex and U(VI)-Ligands 4e complex Ligands 4b and 4e exhibit higher stability than the U(VI)-Tiron complex. Thus, Ligands 4b and 4e are most suitable for further studies.

General
The organic reagents used were pure commercial products from Aladdin. The solvents were purchased from Chengdu Kelong Chemical Reagents Co. (Sichuan, China). Anhydrous CH 2 Cl 2 was distilled prior to use. The 300-400 mesh silica gel was purchased from Qingdao Hailang. The 1 H-NMR, 13 C-NMR spectra were recorded on Bruker Avance 300, Avance 400, or Avance 600 spectrometer (Carlsruhe, Germany). The FTIR spectra were obtained from Nicolet 380 FTIR spectrophotometer (Thermo Fisher Nicolet, Madison, WI, USA) with a resolution of 4 cm´1 from 400 cm´1 to 4000 cm´1. UV-vis spectrophotometer (Thermo Scientific Evolution 201, Waltham, MA, USA) used had a double-beam light source from 190 nm to 1100 nm. Mass spectral analysis was conducted using Varian 1200 LC/MS (Palo Alto, CA, USA).

Metal Complexation Solutions
In all the complexation studies in aqueous solution, the water was distilled three times and the atmospheric CO 2 was excluded from the system with a purging steam of N 2 under 80˝C. The UO 2 (NO 3 ) 2¨6 H 2 O is analytical grade, the buffered solution were NaAc/HAc (pH = 5.5) and Tris-HCl (pH = 7.4, 9.0). All the ligands were synthesized and dissolved into the water, except the solvent of the ligand 4c was DMSO:H 2 O = 1:9 to ensure all the ligands was dissolved. The ligands concentration (C L ) was 2.0ˆ10´3 mol/L and the UO 2 2+ (C M ) concentration was 2.0ˆ10´3 mol/L, then added the solutions into the 10 mL comparison tubes by different volume, the total concentration (C L + C M ) was 2.0ˆ10´3 mol/L for all complexation samples.

Conclusions
In summary, five new ligands of bis(3-hydroxy-4-pyridinone) tetradentate ligands were synthesized and characterized. The stability constants determined in this study provide evidence for the extremely high affinities of the ligands for UO 2 2+ complexes.