Ionizable Calixarene-Crown Ethers with High Selectivity for Radium over Light Alkaline Earth Metal Ions

Introduction

α particle emitters are of increasing interest as the radionuclide attached to monoclonal antibodies or other targeting mechanisms for applications in cell-directed therapy of cancer. R particles are more effective than β⁻ particles for cell-killing and promise a more effective treatment of cancer than other forms of radiation. This is because α particles have high initial energy (4–8 MeV), short path lengths (40–80 μm, or several cell diameters), and consequently greater energy dissipation per unit length.¹ Cell-directed immunotherapy can help improve irradiation of tumor cells while sparing normal tissues.² The success of this approach will require effective chemistry for attaching the radionuclide to the antibody.³ Therefore, a concerted effort has been directed toward the design of chelating agents capable of holding the desired α-emitting radionuclide, both selectively and with high stability, to the antibody. This stability must be maintained in the body under physiological conditions and challenged by metal cations (at much higher concentration) that might otherwise compete for binding with the chelate. Bifunctional chelating agents such as tetraaza macrocycles have been used for this purpose to specifically bind the β emitters ⁹⁰Y and ⁶⁷Cu to antibodies.⁴ One of the R-emitting radionuclides considered suitable for radioimmunotherapy of cancer is the 11.4 d half-life ²²³Ra, which decays through a rapid chain of daughter products to ²⁰⁷Pb, emitting four R particles, two β particles, and several γ rays, with a combined energy of about 28 MeV.^1b

Although ²²³Ra has desirable decay properties for radioimmunotherapy, bifunctional radium-selective ligands together with effective linkers to the protein antibody have not been reported. A few radium-complexing agents reported in the literature either lack selectivity or do not have sufficient binding stability to serve as cancer-therapeutic agents, and none have linkers attached.⁵

It is known that the 1,3-alkoxycalix[4]arene-crown-6 cavity has a high selectivity for Cs⁺ over K⁺.⁶ Since Ra²⁺ has an ionic radius (r = 1.62 Å) that is similar to that of Cs⁺ (r = 1.67 Å),⁷ the size of this crown cavity would presumably be suitable for Ra²⁺. However, neutral calixarene-crowns usually have relatively weak coordination with alkaline earth metal ions. Attaching sidearm functional groups to macrocyclic hosts can enhance their binding ability with alkaline earth metal ions. For example, proton-ionizable crowns with carboxylate sidearms have been shown to exhibit higher stability constants with alkaline earth metal ions relative to the nonionizable counterparts.^5c We report here the binding characteristics of two new ionizable calixarene-crowns, p-tert-butylcalix[4]arene-crown-6-dicarboxylic acid (3) and p-tert-butylcalix[4]arene-crown-6-dihydroxamic acid (4), which show high selectivity for Ra²⁺ over light alkaline earth metal ions. The two proton-ionizable groups and the rigid calixarene-crown cavity apparently provide a strong and selective binding environment for Ra²⁺. This type of ionizable calixarene–crown appears promising for complexing ²²³Ra and linking the complex to monoclonal antibodies or other cell-specific proteins for radioimmunotherapy of cancer.

Experimental Section

General Procedures and Techniques

All reactions were carried out in a nitrogen atmosphere. All reagents were of reagent grade and were used without further purification. Tetrahydrofuran (THF) was freshly distilled from sodium benzophenone ketyl; N,N′-dimethylformamide (DMF) was distilled from KOH pellets and kept over molecular sieves (4 Å). ¹H NMR (200 MHz) spectra were recorded with a Bruker AC200 in the absence of internal standard. J values are given in Hertz. FAB mass spectra were obtained with a Finnigan MAT90 mass spectrometer using m-nitrobenzyl alcohol (NBA) as a matrix. For reasons of clarity and to reduce space, the name calix[4]arene was used instead of the original IUPAC name: pentacyclo[19.3.1.1³,⁷.1⁹,¹³.1¹⁵,¹⁹]octacosa-1(25),3,5,7(28),9,11,13(27),15,17,19(26),21,23-dodecane. Compound 1⁸ was prepared according to a literature procedure, and 2⁹ was prepared according to a modified literature procedure. We used Cs₂-CO₃ instead of KtBuO as base and template for the synthesis of p-tert-butylcalix[4]arene-crown-6 with a yield similar to that reported but easier to purify. Standard workup means that the organic layers were finally washed with water, dried over sodium sulfate (Na₂SO₄), filtered, and concentrated in vacuo.

p-tert-Butylcalix[4]arene-crown-6-dicarboxylic Acid (3)

To a solution of p-tert-butylcalix[4]arene-crown-6 (2) (1.70 g, 2 mmol) in THF–DMF (9:1 v/v) (50 cm³) was added sodium hydride (0.25 g, 10 mmol), and the mixture was heated under reflux for 0.5 h. Subsequently ethyl bromoacetate (1.34 g, 8 mmol) was added. The reaction mixture was heated under reflux for 5 h, and after cooling, the mixture was cautiously poured into ice-cold HCl (6 M, 20 cm³). The solvent was evaporated under reduced pressure, and the residue was partitioned between 100 cm³ of chloroform and 100 cm³ of 0.5 N HCl. The organic layer was followed by standard workup. The crude reaction product was triturated with 80% ethanol (20 cm³) to give the diester (1.49 g, 73%). The diester thus obtained (1.02 g, 1 mmol) was dissolved in THF (10 cm³), tetramethylammonium hydroxide (Me₄NOH) (25% solution in methanol, 3 cm³, 7 mmol), and water (10 cm³) were added, and the mixture was heated under reflux for 24 h. The cooled mixture was concentrated to dryness and treated with 10% hydrochloric acid (10 cm³) to form a precipitate which was filtered off and redissolved in chloroform, followed by standard workup to afford p-tert-butylcalix[4]arene-crown-6-dicarboxylic acid (3) as a colorless solid (0.79 g, 82%). IR (KBr, cm⁻¹): 3200–3600 (OH), 1750 (C=O). ¹H NMR (CDCl₃, 200 MHz): δ 0.70–1.50 (m, 36H, C(CH₃)₃), 3.00–4.10 (m, 24H, OCH₂CH₂O and ArCH₂Ar), 4.30–4.70 (m, 4H, ArCH₂Ar), 5.27 (s, 4H, OCH₂), 6.50–7.20 (m, 8H, ArH), 8.95 (br s, 2H, COOH). Anal. Calcd for C₅₈H₇₈O₁₂: C, 72.05; H, 8.07. Found: C, 71.92; H, 8.17. Mass spectrum: m/e 967.2 (M⁺, calcd 967.2).

p-tert-Butylcalix[4]arene-crown-6-dihydroxamic Acid (4)

Compound 3 (0.97 g, 1 mmol) and oxalyl chloride (5 cm³, 57 mmol) in carbon tetrachloride (50 cm³) were refluxed for 5 h. After being cooled, the solution was concentrated under a stream of nitrogen. The residual acid chloride (confirmed by IR spectroscopy: absence of ν_O−H) was dissolved in THF (50 cm³). The solution was added dropwise to a THF solution containing O-benzylhydroxylamine hydrochloride (2 g, 12.5 mmol) and pyridine (1 g, 12.7 mmol). The solution was heated at 35 °C for 24 h. After being cooled, the precipitate was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in chloroform (100 cm³) and followed by standard workup. The residue left was triturated with CHCl₃–hexanes (1:5 v/v) to give the pure hydroxamic acid precursor (0.76 g, 65%). The benzyl group was deprotected by catalytic hydrogenation with palladium on activated charcoal at room temperature under atmospheric pressure in acetic acid–methanol (1:3 v/v) (0.60 g, 92%). IR (KBr, cm⁻¹): 2400–3600 (OH and NH), 1665 (CdO). ¹H NMR (CDCl₃, 200 MHz): δ 0.84 (s, 18H, C(CH₃)₃), 1.34 (s, 18H, C(CH₃)₃), 3.23 (d, 4H, Jax,eq) 12.8 Hz, ArCH₂Ar), 3.40–4.00 (m, 20H, OCH₂CH₂O), 4.48 (d, 4H, J_ax,eq = 12.8 Hz, ArCH₂Ar), 5.10 (s, 4H, OCH₂), 6.55 (s, 4H, ArH), 7.14 (s, 4H, ArH), 8.05 (s, 2H, NH). Anal. Calcd for C₅₈H₈₀N₂O₁₂: C, 69.85; H, 8.09; N, 2.81. Found: C, 69.97; H, 8.18; N, 2.75. Mass spectrum: m/e 997.2 (M⁺, calcd 997.2).

General Procedure for the Preparation of Barium Complexes

To a desired diacid ligand (0.1 mmol) dissolved in CH₃CN (50 cm³) was added Ba(OH)₂ (0.17 g, 1 mmol), and the mixture was refluxed for 2 h and concentrated to dryness. Chloroform (50 cm³) was added and filtered to remove an extra amount of Ba(OH)₂. The filtrate was followed by standard workup to give the barium complex.

Barium Complex with 3

¹H NMR (CDCl₃, 200 MHz): δ 0.92 (s, 18H, C(CH₃)₃), 1.26 (s, 18H, C(CH₃)₃), 3.27 (d, 4H, J_ax,eq = 12.3 Hz, ArCH₂Ar), 3.50–4.30 (m, 20H, OCH₂CH₂O), 4.38 (d, 4H, J_ax,eq = 12.3 Hz, ArCH₂Ar), 4.58 (br s, 4H, OCH₂), 6.84 (s, 4H, ArH), 7.18 (s, 4H, ArH). Anal. Calcd for C₅₈H₇₆O₁₂Ba: C, 63.20; H, 6.90; Ba, 12.45. Found: C, 63.02; H, 6.97; Ba, 12.29. Mass spectrum: m/e 1102.5 (M⁺, calcd 1102.5).

Barium Complex with 4

¹H NMR (CDCl₃, 200 MHz): δ 0.93 (s, 18H, C(CH₃)₃), 1.27 (s, 18H, C(CH₃)₃), 3.28 (d, 4H, J_ax,eq = 12.3 Hz, ArCH₂Ar), 3.50–4.30 (m, 20H, OCH₂CH₂O), 4.42 (d, 4H, J_ax,eq = 12.3 Hz, ArCH₂Ar), 4.57 (br s, 4H, OCH₂), 6.85 (s, 4H, ArH), 7.18 (s, 4H, ArH), 7.96 (s, 2H, NH). Anal. Calcd for C₅₈H₇₈N₂O₁₂Ba: C, 61.51; H, 6.94; N, 2.47; Ba, 12.12. Found: C, 61.29; H, 7.02; N, 2.39; Ba, 12.03. Mass spectrum: m/e 1132.5 (M⁺, calcd 1132.5).

Solvent Extraction Study

A standard Ra²⁺ solution (75 μg of ²²³Ra in 3 N hydrochloric acid solution) was obtained from the Pacific Northwest National Laboratory. For solvent extraction studies, the solutions were prepared using deionized water obtained from a Milli-Q reagent water system. Measurements of pH were made with an Orion Research Model 701A pH meter with an Orion 91-03 glass semimicroelectrode. Radium concentrations were measured by γ spectrometry using a high-resolution Ge(Li) detector (EG&G Ortec) with a multi-channel analyzer.

For solvent extraction experiments, 1.5 cm³ of a chloroform solution containing a desired ligand, 1.5 cm³ of the aqueous phase containing radium (0.031 mg/L), and buffer solution were placed in stoppered glass vials. Samples were shaken vigorously for a period of 30 min, which was shown to give constant values of distribution coefficients (D). After phase equilibration, 1-octanol (0.5%) was added to suppress the formation of an emulsion. Aliquots of 1.0 cm³ of each phase were then taken by pipets and placed into 5 cm³ polyethylene vials for radioactivity measurements. The area of the peak corresponding to ²²³-Ra (269 keV) was taken as the measure of its concentration. The typical standard deviation of the radioactivity measurements was less than 3%. The concentration of other alkaline earth metal ions such as Ba²⁺,Sr²⁺, Ca²⁺, and Mg²⁺ was detected by the inductively coupled plasma spectrometer (ICP-AES) (IRIS Model, Thermo Jarrell Ash Co., Franklin, MA). Solvent extraction experiments were also performed with a crown ether monocarboxylic acid ligand, 5,^5e known to have affinity for Ra²⁺, and an acyclic crown ether dicarboxylic acid, 6,¹⁰ for comparison (Figure 2).

Figure 2.

Structures of the crown ether monocarboxylic acid 5 and the acyclic crown ether dicarboxylic acid 6.

Results and Discussion

Compounds 3 and 4 were synthesized according to the reaction route shown in Figure 1. The ¹H NMR spectra of the free ligands indicated a mixture of conformations for ligand 3, and a “conic” structure for ligand 4. Treatment of the ionizable calixarene-crowns with a suspension of Ba(OH)₂ in acetonitrile resulted in a 1:1 complex between the ligand and the Ba²⁺ ion. For the barium complex with ligand 3, the two carboxylic acid protons disappeared, and the conformation was fixed in a conic structure as observed with ¹H NMR. The Ba²⁺ complex of 4 showed little conformational change during the complexation.

Figure 1.

Preparation of the proton-ionizable calixarene-crown ethers p-tert-butylcalix[4]arene-crown-6-dicarboxylic acid (3) and p-tert-butylcalix[4]arene-crown-6-dihydroxamic acid (4). Reagents: (a) Ts(OCH₂CH₂)₅OTs, Cs₂CO₃, CH₃CN, reflux 24 h, 32%; (b) (i) NaH, BrCH₂COOEt, THF, reflux 5 h, 73%; (ii) Et₄NOH, THF–H₂O, reflux 24 h, 82%; (c) (i) (COCl)₂; (ii) O-benzylhydroxamine, pyridine, 35 °C, 24 h, 65%; (iii) H₂/Pd–C, HOAc–MeOH, 92%.

Figure 3 shows the percent extraction (Ex%) of ²²³Ra with different ligands plotted against the pH of the aqueous phase. The percentage of extracted Ra²⁺ became appreciable for both ligands 3 and 4 at about pH 4.5, reached a maximum at about pH 8.0, and declined sharply at pH > 9.5. At higher pH values, radium existed as the Ra(OH)⁺ species, which was probably too large to enter the calixarene-crown cavity. Ligand 6, an acyclic polyether with two carboxylic acid groups, also exhibited high extraction efficiency for Ra²⁺ in the pH range studied. Ligand 5, a crown ether monoacetic acid, showed limited extraction of Ra²⁺.

Figure 3.

Two-phase solvent extraction of Ra²⁺ with different ligands. The extraction conditions are given in the text. The pH of the aqueous phase was adjusted with succinic acid–NH₄OH for pH 4–6, Tris–HCl for pH 7–9, and Tris–Me₄NOH for pH over 10. (O) 3, (●) 4, (■) 5, and (□) 6.

The selectivity of ligands 3 and 4 for Ra²⁺ versus other alkaline earth metal ions was evaluated by competition experiments between water containing five alkaline earth metal ions (5 cm³, [Ra²⁺] = 0.03 mg/L, [Mg²⁺] = [Ca²⁺] = [Sr²⁺] = [Ba²⁺] = 0.20 mM) and chloroform containing a specific ligand (5 cm³, [ionophore] = 1.0 mM). The competition results at pH 8.9 are given in Table 1. In the competition experiments, Ra²⁺ was measured by γ spectroscopy and the other alkaline earth metal ions in the aqueous phase were determined by ICP-AES. As shown in Table 1, ligands 3 and 4 were able to extract Ra²⁺ almost quantitatively. The extractability of the alkaline earth metal ions followed the order Ra²⁺ > Ba²⁺ > Sr²⁺ > Ca²⁺ ⪢ Mg²⁺. Nearly zero amount of Mg²⁺ was extracted in the competition experiments with either ligand 3 or ligand 4. Ligand 4 with two hydroxamic acid groups showed a slightly higher selectivity for Ra²⁺ than its carboxylic acid counterpart, ligand 3. In terms of the distribution coefficient D (metal content in the organic phase/aqueous phase), the values for Ra²⁺, Ba²⁺, Sr²⁺, Ca²⁺, and Mg²⁺ were found to be about 1000, 120, 3.4, 0.3, and 0.001, respectively. Both ligands 5 and 6 showed no selectivity for Ra²⁺ versus the other alkaline earth metal ions. The reliability of ICP-AES in measuring extraction efficiency was confirmed by using a ¹³³Ba tracer technique. For this purpose, the above-mentioned competition experiment was modified by adding a trace amount of ¹³³Ba (Isotopes Products Laboratories, Burbank, CA), and the concentrations of barium in the CHCl₃ and aqueous phases were measured by counting the areas of the peak at 356 keV. The D value thus obtained was about 100 for ligand 4, which was comparable to the value obtained from ICP-AES.

Table 1.

Percent Extraction of Alkaline Earth Metal Ions from Water to CHCl₃ at pH 8.90 and 25 °C

	percent extraction
ionophore	Mg2+	Ca2+	Sr2+	Ba2+	Ra2+
3	<0.1	15.6	73.5	98.7	>99.9
4	<0.1	12.7	78.3	99.2	>99.9
5	12.4	17.8	13.8	12.5	13.4
6	93.8	92.5	90.3	87.0	89.0

Because of its short half-life and high radioactivity, it was not possible for us to obtain stability constants of the ²²³Ra complexes using common spectroscopic or potentiometric titration methods. A competition method was used to obtain the relative extraction constants of ²²³Ra by ligands 3 and 4 with respect to EDTA at pH 8.5.¹¹ Using an EDTA solution, the new distribution ratio D′ was assumed to be [RaL]_org/ [RaEDTA]_aq. Assuming that the solubility of RaEDTA in the CHCl₃ phase and RaL in the aqueous phase was negligible, the following simplified equations were obtained:

$$\begin{array}{cc}\hfill & {\mathrm{EDTA}}^{2-}+{\mathrm{Ra}}^{2+}\rightleftarrows \mathrm{RaEDTA}{K}_1=\left[\mathrm{RaEDTA}\right]∕\left[{\mathrm{Ra}}^{2+}\right]\left[{\mathrm{EDTA}}^{2-}\right]\hfill \\ \hfill & {\mathrm{L}}^{2-}+{\mathrm{Ra}}^{2+}\rightleftarrows \mathrm{RaL}{K}_2={\left[\mathrm{RaL}\right]}_{\mathrm{org}}∕\left[{\mathrm{Ra}}^{2+}\right]\left[{\mathrm{L}}^{2-}\right]\hfill \\ \hfill & \log (K_2∕K_1)=\log ({\left[\mathrm{RaL}\right]}_{\mathrm{org}}∕\left[\mathrm{RaEDTA}\right])+\log (\left[{\mathrm{EDTA}}^{2-}\right]∕\left[{\mathrm{L}}^{2-}\right])=\log D^{\prime }+\log (\left[{\mathrm{EDTA}}^{2-}\right]∕\left[{\mathrm{L}}^{2-}\right])\hfill \end{array}$$

A linear relationship was observed between log D′ and log([EDTA²⁻]/[L²⁻]) for both ligands (Figure 4), with a slope close to unity, suggesting that the above assumptions were reasonable.

Figure 4.

log D′ vs log([EDTA²⁻]/[L]) at pH 8.50 for ligands 3 (○) and 4 (●).

From the intercept we obtained the extraction constants of the Ra²⁺ complexes with the calixarene-crown ligands relative to that of EDTA. Ligand 3 had a value of K₂ = 0.51K₁, and ligand 4 had a value of K₂ = 12.6K₁, where K₁ is the extraction constant of Ra²⁺ with EDTA at pH 8.5. Ligand 4 had an extraction constant that appeared to be about an order of magnitude higher than that of ligand 3. This could be explained from the following two reasons: (1) the stronger hydroxamic acid group¹² was more effective in radium binding relative to the carboxylic acid group in the ionizable calixarene-crowns; (2) from the proton NMR study of the Ba²⁺ complexes, ligand 4 showed less conformational change during the complexation than ligand 3, thus minimizing the unfavorable entropy and enthalpy change during the complexation.

Finally, we investigated the kinetic stability of the radium complexes of ligands 3 and 4 in the presence of serum-abundant metal ions including Na⁺, K⁺, Mg²⁺, Ca²⁺, and Zn²⁺. Radium was first extracted into the CHCl₃ phase at pH 8.5. The organic phase was then back-extracted with a pH 7.4 buffer containing 10⁻² M each of Na⁺, K⁺, Mg²⁺, Ca²⁺, and Zn²⁺. After the organic phase was shaken for 24 h, <5% of ²²³Ra was removed. The ²²³Ra coordinated with ligands 3 and 4 showed high kinetic stability in the presence of other metal ions at relatively high concentrations and at a near neutral pH.

Conclusion

For the first time, we were able to design ionophores derived from calix[4]arene to have selectivity for Ra²⁺ over the lighter alkaline earth metal ions and high kinetic stability for ²²³Ra complex in the presence of serum-abundant metal ions. Calix[4]arene-crown-6 offers a more rigid cavity than common crown ethers to fit the size of radium. Two ionizable groups neutralize the charge of the complex and act as lariat arms to prevent radium from escaping from the cavity. The extraction stability constant of carboxylate derivative 3 with Ra²⁺ falls into the same order of magnitude as that of EDTA. The hydroxamate derivative 4 with Ra²⁺ is 1 order of magnitude higher than that of 3. Although the thermodynamic stability constants of these two ligands with Ra²⁺ are not extremely high, the kinetic stability is more attractive. Once radium was included in the pseudo-three-dimensional cage, proton- and cation-promoted pathways show little effect on the loss of radium from the complexes. Further work to make water–soluble bifunctional chelating agents, which have ionizable calixarene-crown moieties at the lower rim as radium ionophore and an isothiocyanate group at the upper rim for linkage to monoclonal antibodies, is now in progress.