Biotin Reagents for Antibody Pretargeting. Synthesis, Radioiodination, and in Vitro Evaluation of Water Soluble, Biotinidase Resistant Biotin Derivatives

Abstract

As part of our development of antibody pretargeting for cancer therapy, an investigation has been conducted to examine the stability of water solubilized, radioiodinated biotin derivatives toward biotinidase degradation in mouse and human serum. Eight new biotin derivatives were synthesized to conduct the study. The biotin derivatives synthesized contained (1) the biotin moiety, (2) a water solubilizing linker moiety, (3) p-iodobenzoate or p-tri-n-butylstannylbenzoate moieties, and (4) in some of the compounds, N-methyl or α-methyl containing moieties were added to block biotinidase activity. The linker moiety, 4,7,10-trioxa-1,13-tridecanediamine, 5, was included in the biotin derivatives to improve their water solubility, and it also functioned as a 17 Å spacer between the biotin and the benzoyl moieties. Four of the new biotin derivatives (12, 14, 22, and 23) contained a p-tri-n-butylstannylbenzoyl moiety as precursors which could be radioiodinated in the last synthetic step. The other four biotin derivatives (13, 15, 24, and 25) contained p-iodobenzoyl moieties and were used as HPLC reference standards. Initial studies involved radioiodination of 12 to yield [¹²⁵I]13. Radioiodinated 13, which did not contain a moiety for blocking biotinidase activity, was found to be rapidly degraded in both mouse and human serum at 37 °C. Derivatives which were designed to be stable to biotinidase incorporated N-methyl and α-methyl moieties adjacent to the biotin carboxylate group. In one set of biotin derivatives (14 and 15), the N-methyl moiety was obtained by incorporating N,N-dimethyl-4,7,10-trioxa-1,13-tridecanediamine, 9, as a linker in the place of 5. In the second set of biotin derivatives (22 and 24), the N-methyl moiety was introduced by incorporating a sarcosine (N-methylglycine) moiety between biotin and 5. The radioiodinated N-methyl containing biotin derivatives [¹²⁵I]15 and [¹²⁵I]24 were found to be very stable to biotinidase degradation. An α-methyl group was obtained in a pair of biotin derivatives (23 and 25) by incorporating a 3-aminobutyric acid moiety between biotin and 5. The radioiodinated α-methyl containing derivative, [¹²⁵I]25, was found to have an intermediate stability with regards to biotinidase degradation.