Optimization of Alkylating Agent Prodrugs Derived from Phenol and Aniline Mustards: A New Clinical Candidate Prodrug (ZD2767) for Antibody-Directed Enzyme Prodrug Therapy

Abstract

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.

Induction of apoptosis by the ADEPT agent ZD2767: comparison with the classical nitrogen mustard chlorambucil and a monofunctional ZD2767 analogue

“…The prodrug ZD2767P was synthesized as described previously (Springer et al, 1995), as was the monofuntional ZD2767D analogue (Monks et al, submitted). Chlorambucil was purchased from Sigma Chemical Co. (Poole, Dorset, UK) and CPG2 was provided by CAMR (Salisbury, Wiltshire, UK).…”

“…LoVo colorectal tumour cells were chosen for these studies as they are sensitive to, and have been extensively used in in vitro and in vivo studies with, the ZD2767 ADEPT system (Blakey et al, 1996;Springer et al, 1995). Apoptosis was determined using 3 distinct methods which assess apoptosis-related morphological changes.…”

“…), via the cleavage of the carbamate bond releasing the glutamate moiety (Springer et al, 1995;Blakey et al, 1996).…”

A Phase I Study of Single Administration of Antibody-Directed Enzyme Prodrug Therapy with the Recombinant Anti–Carcinoembryonic Antigen Antibody-Enzyme Fusion Protein MFECP1 and a Bis-Iodo Phenol Mustard Prodrug

“…In contrast, 268.8 mg/m 2 Â3 prodrug given 13 hours (median; range, 11.6-15 hours) after 3,000 units/m 2 MFECP1 (patients 26-28) was safe and was established as the maximum tolerated dose. Grade 1 and 2 toxicity for all doses included leukopenia (1), neutropenia (2), thrombocytopenia (8), anemia (1), raised bilirubin (2), raised alanine aminotransferase (4), raised aspartate aminotransferase (2), raised g-glutamyl aminotransferase (1), coagulopathy (1), nausea (13), vomiting (7), diarrhea (1), fatigue (11), and anorexia (1). Toxicity of ADEPT with MFECP1 and BIP prodrug was found to depend on the serum enzyme level, dose of prodrug, and time interval between fusion protein and prodrug.…”

“…CPG2 converts BIP prodrug to active drug by cleavage of a glutamate moiety. The BIP prodrug was chosen due to the short half-life of its activated drug, which is expected to prevent leak back from tumor (13).…”