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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Synlett. Author manuscript; available in PMC 2010 July 21.
Published in final edited form as:
Synlett. 1999 September 1; 11: 1784–1786.
doi:  10.1055/s-1999-2944
PMCID: PMC2907548
NIHMSID: NIHMS214567

Monofunctionalization of Calix[4]arene Tetracarboxylic Acid at the Upper Rim with Isothiocyanate Group: First Bifunctional Chelating Agent for Alpha-Emitter Ac-225

Abstract

A procedure is reported for synthesizing a novel, water-soluble bifunctional chelating agent derived from calix[4]arene. This chelate features tetracarboxylic acid groups at the lower rim as an actinium-225 ionophore, and an isothiocyanate functional group at the upper rim for labeling of the N-terminus of monoclonal antibodies through thiourea linkage.

Keywords: actinium-225, bifunctional chelating agents, calixarenes, isothiocyanate

For radioimmunotherapy of cancer, it is well established that alpha (α) particles from radionuclide labels are more effective cytotoxic agents than beta (β) particles. This is because α particles dissipate a larger amount of energy over a shorter range (few cell diameters), and thus are more effective in cell killing than are β particles.1,2 Among the α-emitting radionuclides considered suitable for radioimmunotherapy of cancer is actinium-225 (t1/2 = 10 d), which decays through a rapid chain of daughter products to a stable product and emits four α particles, two β particles, and several gamma-ray (γ) photons with a combined energy of about 28 MeV.3 Applications in radioimmunotherapy require Ac-225 to be bound to a protein that specifically targets cancer cells. This application also requires a bifunctional complexing agent that tightly binds Ac-225 on one side and can be linked to protein on the other side. This bifunctional complexing agent must be stable under physiological conditions. We have recently reported4 that t-butylcalix[4]arene tetracarboxylic acid and t-butylcalix[6]arene hexacarboxylic acid showed high efficiency for binding actinium-225 under neutral and weak acid conditions. The calix[4]arene platform exhibited better selectivity and higher kinetic stability than calix[6]arene analog with respect to acid and serum abundant cations (such as Na+, K+, Mg2+, Ca2+, and Zn2+) promoted pathways. Calix[4]arene tetracarboxylic acid appeared to be a suitable candidate as an actinium-225 complexing agent for applications in immunotherapy. Medicinal applications require a water soluble immunoconjugate. Modeling studies reported recently by Reinhoudt group5 revealed that in the Ac3+ complex of calix[4]arene tetracarboxylic acid, the cation was bound inside the cavity with average coordination distances of Ac---Ophenol (2.78 Å) and Ac---O2C (2.47 Å), the carboxylate groups behave as monodentate. We report herein the first design of a water-soluble bifunctional chelating agent (Compound 6 in Scheme 1) for binding actinium-225 for labeling of monoclonal antibodies. The four carboxylic acid groups, fixed in conic conformation at the lower rim of the calix[4]arene platform, act as an actinium ionophore. The monofunctionalized isothiocyanato group of the upper rim is designed to facilitate the combination of this C-functionalized bifunctional chelator with N-terminus of desired monoclonal antibody via thiourea linkage.

The synthetic protocol is summarized in Scheme 1. Reaction of calix[4]arene (1) with 3 equivalent of ethyl bromoacetate in the presence of excess amount of CaH2 or BaO as base and template yielded only the hydrolytic decomposition product instead of triester (2) reported in the literature.6,7 Conversion of triacid to triester derivative was accomplished by refluxing in EtOH with catalytic amount of p-toluenesulfonic acid. Nitration was conducted with the treatment of 70% HNO3 in the presence of HOAc at 0°C. Under these conditions, only one nitro group was introduced at the para position of the phenol ring of calix[4]arene without ester group. Further esterification of the remaining phenol group of 3 kept the tetraester in conic conformation. Hydrolysis of the mononitro-tetraester derivative, using NaOH or KOH as base, gave a mixture of free acid 4 and Na+ or K+ complexes. Only a bulky base, such as tetramethylammonium hydroxide (Me4NOH), gave a satisfactory NMR spectrum with exclusion of cation complexation. Reduction of 4 using the standard pressurized hydrogenation with Pd-C or Raney-Ni as a catalyst, gave virtually no reduction product; however, in the presence of palladized charcoal, hydrazine hydrate8,9 reduced 4 to amine 5 in nearly-quantitative yield within 2 hours. Reaction of the amino compound with thiophosgene10 gave the title compound, 5-isothiocyanato-25,26,27,28-tetrakis-[(hydroxycarbonyl)methoxy]-calix[4]arene (6).

1H NMR11 of the monofunctionalized calixarene 4 clearly showed cone conformation. Because of its asymmetric structure, four pairs of doublets for the ArCH2Ar methylene protons (two AX systems) and 3 singlets in the ratio of 2:1:1 for OCH2COOH groups were found in the spectrum. Amino- and isothiocyanato- derivatives showed simplified pattern for OCH2COOH groups (2 singlets in the ratio of 3:1). Upon reduction, the singlet at 7.36 ppm for a benzene ring containing a nitro group disappeared, and a new singlet at upper field (6.59 ppm) attributed to the two protons on the aniline ring was found to merge. An upfield shift was also observed for ArCHaxAr methylene protons, which might be due to small change in conformation.

In summary, monofunctionalization of calix[4]arene-tetracarboxylic acid with an isothiocyanato group can be conducted with an excellent yield according to scheme 1 (total yield up to 22% based on compound 2). Further work to conjugate this actinium-225 ionophore to monoclonal antibodies and to evaluate the stability, affinity, and specificity of the radiolabeled antibody are currently in progress and will be reported elsewhere. Calixarene-based actinium ionophores will also have other potential biomedical applications such as contrast agents (CA) in magnetic resonance imaging (MRI)1214 and as luminescent labels for bioassay purpose (with chromophoric antenna).1517

Acknowledgments

This work was supported by the Pacific Northwest National Laboratory (PNNL) and US Department of Energy (DOE) under contract No. DE-AC06-76RLO 1830. The authors wish to thank Dr. D. Scott Wilbur of the Department of Radiation Oncology at the University of Washington Medical Center (Seattle) for many helpful discussions.

References and Notes

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6. Iwamoto K, Fujimoto K, Matsuda T, Shinkai S. Tetrahedron Lett. 1990;31:7169.
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11. 5-Nitro-26,27,28-tris[(ethoxycarbonyl)methoxy]calix-[4]arene (3): To a solution of 2 (3.41g, 5 mmol) in CH2Cl2 (100 cm3) was added a mixture of 70% HNO3 (5 cm3, 52 mmol) and glacial acetic acid (7 cm3). The color of the reaction mixture immediately turned to dark purple. Within 30 min. the color of the mixture changed to yellowish at which point water (50 cm3) was added and the organic layer was evaporated and the residue recrystallized from CHCl3-C2H5OH (1/5, v/v) to gave 3. (2.04 g, 56%). Mp: 157–159°C. 1H NMR (CDCl3): 1.1–1.4 (m, 9H, CH3), 3.32 (d, J = 13.4 Hz, 2H, ArCH2Ar eq), 3.41 (d, J = 13.8 Hz, ArCH2Ar eq), 4.1–4.6 (m, 6H, OCH2CH3), 4.72 (s, 2H, ArOCH2), 4.77 (d, J = 13.8 Hz, ArCH2Ar, ax), 4.91 (d, J = 13.4 Hz, ArCH2Ar, ax), 4.95 (s, 4H, ArOCH2), 6.5–7.0 (m, 9H, ArH), 7.94 (s, 2H, ArH meta), 8.03 (s, 1H ArOH). m/z (FAB): 727 (100%, M+). Anal. Calcd for C40H41NO12: C, 66.02; H, 5.64; N, 1.93. Found: C, 66.15; H, 5.48; N, 1.87%.
5-Nitro-25,26,27,28-tetrakis[hydroxycarbonyl)methoxy]-calix[4]arene (4): To a solution of 3 (1.45 g, 2 mmol) in THF-DMF (5:1 v/v, 50 cm3) was added NaH (0.2 g, 8 mmol) and the mixture was heated under reflux for 30 min, after which BrCH2COOEt (0.84 g, 5 mmol) was added and the reaction mixture was refluxed for further 5 h. After cooling, the mixture was cautiously poured over ice-cold HCl (6 M, 30 cm3). The solvent was evaporated and the residue was partitioned between CH2Cl2 (100 cm3) and HCl (0.2 M, 100 cm3). The organic layer was evaporated and the residue was crystallized from 90% EtOH to afford the tetraester as yellow crystals. (1.09 g, 67%). To a solution of tetraester (1 g, 1.23 mmol) in THF (20 cm3) was added Me4NOH (25% solution in methanol 6 cm3, 14 mmol) and water (20 cm3). The mixture was refluxed for 12 h and was concentrated to dryness under reduced pressure. The residue was treated with 10% HCl (10 cm3) to form a precipitate which was filtered and recrystallized from 50% methanol to afford 4 (0.70 g, 81 %). Mp: 297–298°C. 1H NMR (CD3OD) 3.26 (2H, d, J = 13.8 Hz, ArCH2Ar eq), 3.38 (2H, d, J = 13.8 Hz, ArCH2Ar eq), 4.59 (2H, s, ArOCH2), 4.76 (2H, s, ArOCH2), 4.80 (4H, s, ArOCH2), 4.91 (2H, d, J = 13.8 Hz, ArCH2Ar ax), 5.04 (2H, d, J = 13.8 Hz, ArCH2Ar ax), 6.35 (1H, t, J = 7.5 Hz, ArH), 6.48 (2H, d, J = 7.5 Hz, ArH), 6.88 (2H, t, J = 7.7 Hz, ArH), 7.03 (4H, d, J = 7.7 Hz, ArH), 7.36 (s, 2H, ArH). HPLC: (C18 reversed phase column, binary linear gradient of 0–100% B/35 min; solvent A = 0.05 mol dm−3 Et3N/HOAc, pH = 5.5; solvent B = MeOH) 21.7 min. m/z (FAB): 724 (100%, M+Na). Anal. Calcd for C36H31NO14: C, 61.63; H, 4.42; N, 2.00. Found: C, 61.78; H, 4.25; N, 2.11%. 5-amino-25,26,27,28-tetrakis-[(hydroxycarbonyl)methoxy]calix[4]arene (5): To a solution of 4 (0.5 g, 0.71 mmol) in EtOH (50 cm3) was added 10% palladium on activated carbon (50 mg) under argon atmosphere, the resulting mixture was refluxed for 2 hours after which the solution was almost colorless. Pd-C catalyst filtered and washed with EtOH, the combined filtrate was evaporated to give almost pure 5 (0.44 g, 92%). Mp: 323°C (decomp.). 1H NMR (D2O) 3.31 (2H, d, J = 12.6 Hz, ArCH2Ar eq), 3.47 (2H, d, J = 12.6 Hz, ArCH2Ar eq), 4.33 (s, 2H, ArOCH2), 4.40 (s, 6H, ArOCH2), 4.50 (2H, d, J = 13.8 Hz, ArCH2Ar ax), 5.64 (2H, d, J = 13.8 Hz, ArCH2Ar ax), 6.59 (s, 2H, ArH), 6.8 (m, 3H, ArH), 7.17 (d, br, 6H, ArH). HPLC: 19.1 min. m/z (FAB): 694 (100%, M+Na). Anal. Calcd for C36H33NO12: C, 64.38; H, 4.92; N, 2.09. Found: C, 64.59; H, 4.84; N, 2.11%.
5-isothiocyanato-25,26,27,28-tetrakis[(hydroxycarbonyl)- methoxy]calix[4]arene (6): To a solution of 5 (0.3 g, 0.45 mmol) in H2O (10 cm3) was added thiophosgene (0.5 cm3, 2.88 mmol) in CHCl3 (10 cm3) and the mixture was stirred overnight at r.t. The organic phase was evaporated and solid filtered to give 6 (0.25 g, 78%). Mp: > 370°C. IR νmax/cm−1 2100 (NCS) and 1660 (C=O). 1H NMR ([2H6]DMSO) 3.3–3.6 (4H, m, ArCH2Ar eq), 4.1–4.4 (4H, m, ArCH2Ar ax), 4.61 (6H, s, ArOCH2), 4.82 (2H, s, ArOCH2), 6.1–7.2 (11H, m, ArH). HPLC: 13.4 min. m/z (FAB): 736 (100%, M+Na). Anal. Calcd for C37H31NO12S: C, 62.27; H, 4.35; N, 1.96. Found: C, 62.14; H, 4.32; N, 2.05%.
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