Rational design of bifunctional chelating agents for alpha-emitters radium-223 and actinium-225 in radioimmunotherapy of cancer

There has been increasing interest in developing radioisotope labeled immunoconjugates in radioimmunodiagnosis and radioimmunotherapy of cancer tumors. Central to the success of the application of radionuclides in nuclear medicine is the development of bifunctional chelating agents which will hold the desired radioisotope with high thermodynamic and kinetic stabilities, while at the same time will have a second functional group to covalently bind the targeting molecules. Alpha-emitters with much higher cytotoxicity than beta-emitters in radioimmunotherapy remain virtually untouched due to the lack of proper bifunctional chelators for these radionuclides. Two isotopes, radium-223 (t1/2 = 11.4 d) and actinium-225 (t1/2 = 10 d), are the most promising alpha-emitters for radioimmunotherapy applications. Bifunctional chelating agents for Ra2+ and Ac3+ have been rationally designed.; Ra2+ is the largest divalent cation in the periodic table. The high hydration energy and low charge density of this ion make it almost impossible to have any ionophore to form thermodynamically very stable complexes with Ra2+. Proton-ionizable calixcrowns, such as p-tert -butylcalix[4]arene-crown-6-diacetic acid and p-tert-butylcalix[4]arene-crown-6-dihydroxamic acid, can bind Ra2+ strongly and selectively under basic conditions through both ion-dipole and electrostatic interactions. Rigid calix[4]arene-crown-6 with a cavity size between 18-crown-6 and 21-crown-7 is selective for Ra 2+ (r = 1.62 A) over light alkali and alkaline earth metal ions; the two acid groups after deprotonation at high pH values increase drastically the strength of complexation. The pseudo three-dimensional cage-like Ra 2+ complexes thus formed showed high in vitro kinetic stability in serum. Two protocols were developed to design bifunctional radium chelating agents: (1) ionophores with ionizable calixcrown moieties at the lower rim as radium chelating sites and monofunctionalization at the lipophilic upper rim with an isothiocyanate group for further linking to monoclonal antibodies via stable thiourea linkage; (2) ionophores with calix[4]-benzo-18-crown-6 and two acid groups on both sides of the calixcrown as radium coordinating sites and introduction of an isothiocyanate group on the para position of the benzoether.; Ac3+ is the largest trivalent cation in the periodic table. Calix[4]arene derivatives with four carboxylic acid groups or three carboxylates plus one amide group at the lower rim form Ac3+ complexes with both high thermodynamic stability and in vitro serum stability. The high stability under neutral and acidic conditions has been confirmed by means of solvent extraction, potentiometric titrations and instant thin layer chromatography. Molecular modeling studies showed that Ac3+ was bound inside the ionophoric cavity and was excluded from contact with environmental solvent molecules or competing cationic species. The high selectivity of these ligands for Ac3+ over alkali and alkaline earth metal ions is governed by the preorganization effect of the ligands and the fact that electrostatic interaction favors the complexation of high valent cations with anionic ligands. Water soluble bifunctional chelating agents with actinium selective ionophores at the lower rim and a mono-isothiocyanate group at the upper rim of the calix[4]arene parent molecule to be connected to the N-terminus of desired proteins were also designed.