Synthesis of Ionizable Calix[4]arenes for Chelation of Selected Divalent Cations

Two sets of functionalised calix[4]arenes, either with a 1,3-crown ether bridge or with an open-chain oligo ether moiety in 1,3-position were prepared and further equipped with additional deprotonisable sulfonamide groups to establish chelating systems for selected cations Sr2+, Ba2+, and Pb2+ ions. To improve the complexation behaviour towards these cations, calix[4]arenes with oligo ether groups and modified crowns of different sizes were synthesized. Association constants were determined by UV/Vis titration in acetonitrile using the respective perchlorate salts and logK values between 3.2 and 8.0 were obtained. These findings were supported by the calculation of the binding energies exemplarily for selected complexes with Ba2+.

In this regard, the calix [4]arene skeleton can be seen as an ideal platform to build an optimized chelator. Two out of the four hydroxy groups on the lower rim are possible to be functionalized with proton-ionizable groups, which leads to the formation of neutral complexes with divalent cations. The remaining two can be furnished with oligo ether groups, either open-chain or bridged, leading to the calixcrown scaffold, which is easily accessible. Using this concept, both the advantages of the electrostatic, macrocyclic, and cryptate effect are unified.
Three divalent cations Sr 2+ , Ba 2+ , and Pb 2+ are in the focus of our interest, because they all possess radioisotopes with useful nuclear properties for various diagnostic or therapeutic applications in nuclear medicine [36], and are therefore suitable to prepare radiopharmaceuticals. The beta-emitter 89 Sr is applied as "bone seeker" [37], and 90 Sr is used for superficial brachytherapy of some cancers [38,39]. 131 Ba (t 1/2 = 11.5 d) is a γ-emitter for possible diagnostic uses and is discussed as a bone-scanning agent in scintigraphy [40][41][42]. Furthermore, Ba 2+ functions as a non-radioactive surrogate [43][44][45] for both alpha-emitters 223/224 Ra, because of their analogous chemical properties and their radii of similar range [46]. Radium-223 and radium-224, have suitable half-lives ( 223 Ra: 11.4 d, 224 Ra: 3.6 d) and nuclear decay properties (decay chain with four alpha and two beta particles) [47] that make them useful tools for alpha-particle therapy [48].
[ 223 Ra]RaCl 2 is known as Xofigo ® and is applied in clinics for the treatment of bone metastases. Furthermore, Pb 2+ was part of our research. On the one hand, it is the stable end product ( 207 Pb and 208 Pb) of both radium decay chains. On the other hand, 212 Pb is a promising β --emitter, and a feasible candidate for radiopharmaceutical applications, since it can also be used as an in vivo generator for 212 Bi, which is a strong alpha emitter [49,50]. No indications were found regarding radiopharmaceutical applications of the light alkaline earth metals beryllium, magnesium, and calcium for therapy or diagnosis.
To provide the ideal cavity for heavy group 2 metals for stable complexation, it is essential to choose a suitable oligo ether length or crown size, respectively, combined with an adequate number of donor sites. Recently, the impact of the crown-6-functionalization of two simple calix [4]crown-6 derivatives has been elaborated [29,30]. The group of R. A. Bartsch focused on extraction of alkaline earth metal cations using functionalized calixcrowns [51][52][53] and found the calix [4]arene-1,3-crown-6 derivatives to be very effective group 2 metal ion extraction agents. The additional impact of two trifluoromethylsulfonylamide groups as proton-ionizing residues was corroborated by them and our research group [26]. Furthermore, these compounds showed high selectivity for Ba 2+ over the lighter alkaline earth metal or alkali metal ions. However, Ra 2+ was not investigated. There are only a handful of reports dealing with Ra 2+ and the efficiency of various ligands including calixarenes as ionophores in nuclear waste management [28,54,55]. Particularly in radiopharmacy, high stability of the complex is urgently important so that a M 2+ -release and the following accumulation in bone tissues is minimized [42,44].
The objective of this research was to evaluate and compare different open-chain and bridged p-tert-butylcalix [4]arene derivatives as possible leading compounds that could, upon further modification, yield viable chelators for the selected divalent metal ions Sr 2+ , Ba 2+ , and Pb 2+ in radiopharmaceutical applications and provide information about comparable stability constants. The existing literature about group 2 metal ligands specifically with radium, is focused mainly on extraction studies. Therefore, the UV titration as reliable and constant method for the calculation of stability constants was used to determine association constants for the respective ions. Additionally, theoretical calculations involving Ba 2+ as a surrogate for Ra 2+ were accomplished to underline the results.

Preparation of the Functionalized Calix[4]arenes
For a better understanding of the complexation ability, two sets of calix [4]arene derivatives either with open-chain or bridged oligoethers were evaluated. The first set of chelators containing the open-chain oligo ether functions was prepared to start from the basic compound 1, which contains the same number of oxygen donor atoms as calix [4]crown-6 14a. The complete synthesis path is described in Scheme 1. Calix 1 is proposed to form complexes with Na + and K + [56]. For the introduction of proton-ionizable groups to improve the complexation behavior, the two remaining free OH groups were modified by alkylation with ethyl bromoacetate to yield calix 2. In the next step, calix 2 was saponified under basic conditions to yield diacid 3 in quantitative yield without further purification after precipitation with HCl. To introduce the amide functions, compound 3 was then treated with oxalyl chloride to form the dichloride 4, which was instantly reacted with morpholine or 1,4,7-trioxa-10-azacyclododecane to yield amides 5 and 6, respectively. Both amines were used for modification to raise the number of donors for complexation and the steric demand. The introduction of proton-ionizable sulfonamides to yield 7a-c follows the same procedure by using trifluoromethyl sulfonamide, perfluoroisopropyl sulfonamide, and perfluorophenyl sulfonamide, respectively, which were deprotonated prior to the reaction with dichloride 4. To check the influence of the resulting cavity, the flexibility of the functionalized crown ether bridge on the association constant and the possibility to introduce further functional groups at the crown, the second set of chelators is prepared based on the bridging moiety like a simple crown, benzocrown or aza crown. For this purpose, the respective bridging compounds 3,6,10,13-tetraoxapentadecane-1,15-diyl ditosylate (8b) [57] and the catechol derivatives 12a and 12b [58] were prepared according to literature procedures. Additionally, ditosylates 12c,d resulting from functionalized catecholes 9b,c were prepared to allow a later functionalisation of the calix-bridge using conventional ligation reactions. The preparation procedure is outlined in Scheme 2. For the connection of the functionalized crown ethers with the calix [4]arene skeleton, the resulting ditosylates 8a,b and 12a-d of the oligo ethers were reacted with t Bu-calix [4]arene (13) under basic conditions using K 2 CO 3 in dichloromethane (DCM) to prepare the calix-crown-6 derivatives 14a,b and the calix-benzocrown-6 derivatives 14c-f in yields of 36-81%. The complete synthesis path is described in Scheme 3. The two remaining free OH groups of the calix-crowns 14a-f were alkylated with ethyl bromoacetate to yield 15a-f. In the next step, they were saponified under basic conditions to yield the respective diacid derivatives 16a-f in mostly quantitative yields without further purification after precipitation with HCl. To introduce the proton-ionizable fluorinated sulfonamide functions, an amide coupling strategy using EDC and (Cl-)HOBt was applied. Thus, compounds 16a-f were dissolved in anhydrous acetonitrile and reacted with the respective perfluorinated sulfonamides 17-19 at ambient temperature using the aforementioned coupling agents to form calix-crowns 20-22 and the modified calix-benzocrowns 23-27 in yields of 27-97%. Scheme 3. Preparation of the proton-ionizable calix [4]crown derivatives 20-27 with perfluorosulfonamide functions.

The Influence of the Crown Type
The influence of lower-rim crown modifications on the metal-calixcrown-coordination was next checked. For this purpose, reaction binding energies of Ba 2+ with four selected crown-bearing calixarenes were calculated ( Figure 1). That involved calix [4]crown-6 20 and additionally benzo-crown derivative 23 with the modification on adding an aromatic six-membered ring at the bottom of the crown, propylene derivative 22 with an extended crown by a CH 2 group, and calix [4]crown-5 28 lacking an ethoxy unit (CH 2 CH 2 O). As the result of the calculated binding energies (corrected for basis set superposition error) shown in Table 1, the energetically most favored environment to host the metal ion is found for calix [4]crown-6 20, while in the other three compounds, the metal-crown binding is weakened up to about 10%. Furthermore, the lowest binding energy is found for the barium ion and calix [4]crown-5. At first glance, the crown-5 might appear to feature an optimal size to host ions like Ba 2+ , as the size of the macrocycle cavity relative to the ionic radius is often used as a common parameter to rationalize and design new ligands in host-guest chemistry [59]. Considering the geometrical parameters given in Table S1, the crown-5 cavity seems to enclose the Ba 2+ ion better than the crown-6; however, the calculated binding energies show a reversed trend. Our calculations are in accordance with previous studies in the field of host-guest chemistry of crown ethers [60]. Islam et al., for instance, showed that Na + binds more tightly to a crown-6 body although the crown-5 hole's size matches the sodium ion radius better compared to the one of crown-6 [61].

NMR Investigations
To determine the stability constants for the complexation of Ba 2+ , Sr 2+ , and Pb 2+ , a reliable method was developed in the past by our group using 1 H NMR spectroscopy. Due to the different 1 H NMR spectra, which were recorded for the respective complexes in comparison with the ligands, a 1 H NMR titration method was established [26,29,30]. For this purpose, the compounds were dissolved in acetonitrile-d 3 and treated in portions with an acetonitrile-d 3 solution containing Ba(ClO 4 ) 2 . It was observed, that after addition of 0.5 equivalents of Ba salt, a new set of signals appeared, belonging to the respective Ba-complex exemplarily shown for calix 24 in Figure 2. This leads to two separate species: ligand 24 and complex Ba-24 in the 1 H NMR spectrum (see, for instance, the difference of the methylene protons of the calix skeleton in the box of Figure 3). After the addition of 1 eq. of Ba(ClO 4 ) 2 , no ligand was detectable anymore, which leads to the assumption of a complex with 1:1 stoichiometry. The situation in Figure 2 is a consequence of a slow exchange on the NMR time scale and is therefore not suitable for a logK determination by NMR titration for our calix compounds [62]. Next, a more reliable titration method based on UV/vis spectroscopy was used instead to determine the association constants.

UV Titration Studies
To determine the association constants by UV titration, the open-chain calix [4]arenes 7a,b as well as the calix [4]    Additionally, a high value is even found for calix 5 with morpholine modification and its respective complexes with Ba 2+ and Pb 2+ possibly due to the higher number of donor atoms. The constants for the open-chain derivatives Ba-7a,b are lower or equal to the value found for 14a in contrast to the values found for Pb-7a,b, which were higher. Differences between the M 2+ complexes from the same ligand arose from the different ion radii as well as from the chemical behavior (HSAB concept), as Sr 2+ and Ba 2+ belong to the alkaline earth metals (group 2) and Pb 2+ is a group 4 metal ion and therefore, show a softer ion character.

General
All chemicals were purchased from commercial suppliers and used without further purification unless otherwise specified. Anhydrous THF was purchased from Acros, anhydrous Ba(ClO 4 ) 2 was purchased from Alfa Aesar, and deuterated solvents were purchased from deutero GmbH. Compounds 8b [57], 9c [63], 11a, 12a [59], 11b, 12b [64], 14a, 15a, 16a, and 20 [26] were prepared according to the literature. NMR spectra of all compounds were recorded on an Agilent DD2-400 MHz NMR or an Agilent DD2-600 MHz NMR spectrometer with ProbeOne. Chemical shifts of the 1 H, 19 F, and 13 C spectra were reported in parts per million (ppm) using TMS as an internal standard for 1 H/ 13 C and CFCl 3 for 19 F spectra. Mass spectrometric (MS) data were obtained on a Xevo TQ-S mass spectrometer (Waters) by electron spray ionization (ESI). The melting points were determined on a Galen III melting point apparatus (Cambridge Instruments & Leica) and are uncorrected. TLC detections were performed using Silica Gel 60 F 254 sheets (Merck, Darmstadt, Germany). TLCs were developed by visualization under UV light (λ = 254 nm). Chromatographic separations were accomplished by using an automated silica gel column chromatography system Biotage Isolera Four and appropriate Biotage KP-SIL SNAP columns. UV/Vis-measurements were realized at a Specord 50 by Analytik Jena. The calculation of the stability constants was accomplished using HypSpec 1.1.18.

The Computational Methodology
The calculations of calixcrowns were performed using Kohn-Sham DFT. The general gradient approximation functional BP86 [65,66] was used for geometry optimizations and the B3LYP [67,68] to calculate single point energies. With the latter settings, we have used a continuum solvation COSMO model for acetonitrile (dielectric constant ζ = 37.5) in order to approximately include the solvent environment used in the experiment. Ahlrichs' triple zeta valence polarized (def2-TZVP) [69] basis set was employed as well as the resolution of identity (RI) approach and corresponding auxiliary basis sets [70], along with Grimme's D3 dispersion correction [71]. Since the def2-TZVP was not available for radium, we have used the split valence polarized (def-SVP) basis set [72] for both Ra 2+ and Ba 2+ in the calculations that involved a comparison between their binding energies. Effective core potentials have been used for the heavy metals in order to account for scalar relativistic effects. The Turbomole 7.3 package [73] was used for all calculations in this study. To prevent the over-stabilization of final energies, basis set superposition error (BSSE) was considered. Binding energies were calculated as differences of electronic energies E via E(complex)-[E(calixcrown)+E(M 2+ )].