Amino Acid Phosphoramidate Nucleosides: Potential ADEPT/GDEPT Substrates Edward J. McIntee and Carston R. Wagner* Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA Received 16 May 2001; accepted 18 July 2001 Abstract—A series of aromatic, serum-stable, water soluble and nontoxic amino acid phosphoramidate monoesters of 5-fluoro-2 0 - deoxyuridine (FUdR) and 1-b-arabinofuranosylcytosine (Ara-C) was shown to inhibit the cellular growth of the human leukemia cell line CCRF-CEM in the presence of human prostatic acid phosphatase (hPAP). # 2001 Elsevier Science Ltd. All rights reserved. The chemotherapeutic nucleosides 5-fluoro-2 0 -deoxy- uridine (FUdR) and 1-b-arabinofuranosylcytosine (Ara-C) both function as antimetabolites. They possess potent antitumor activity and have been used since the 1960s for the treatment of acute myelogenous leukemia and various types of carcinomas. Unfortunately, like most anticancer agents, associated toxic side effects make their use dose limiting. Consequently, the ability to site specifically deliver nucleosides could dramatically enhance their therapeutic index by reducing the potential for subsequent systemic toxicities. Over the last decade, several attempts have been made to generate an active drug from an inactive precursor, through the action of an enzyme present predominantly at the tumor site, with the goal of developing a new, less cytotoxic strategy for the treatment of cancer. Antibody and gene-directed enzyme prodrug therapies are two targeting strategies designed to improve the selectivity of antitumor agents. The approaches are based on the activation of a prodrug by an antibody–enzyme con- jugate targeted to a tumor-associated antigen (ADEPT) or by an enzyme expressed specifically by tumor cells (GDEPT). 1,2 The ADEPT approach involves two steps. The first step is the administration of an antibody directed at a tumor-associated antigen that is used to vector a specific enzyme to the tumor site. The enzyme- conjugated antibodies are allowed to localize at the tumor site over a period of time (i.e., 24 h). Next, the tumor-localized enzyme can then convert the subse- quently administered prodrug into an active cytotoxic agent in the immediate extracellular fluid surrounding the tumor. The activated drug can then diffuse through the tumor cell membrane or be carried to tumorgenic cells within close proximity that might fail to express the target antigen. Several ADEPT approaches have been developed and clinically studied thus far, and are reviewed in the lit- erature. 2 5 In particular, alkaline phosphatase con- jugated antibodies have been employed to activate phosphorylated prodrugs. 6,7 With this in mind, we con- sidered the use of phosphoramidate prodrugs as possi- ble substrates in an ADEPT or GDEPT approach. Previously, we have shown that amino acid phosphor- amidate monoesters of nucleosides are nontoxic com- pounds that are stable in human sera with t 1/2 > 2 days. 8,9 They have excellent pharmacokinetic para- meters as demonstrated in a rat model, with longer half- lives, longer total body clearance rates, and a larger volume of distribution than the parent nucleosides. 9 Furthermore, we have shown that a subset of amino acid phosphoramidate nucleosides are potent antiviral and anticancer agents in their own right with their mechanism of action correlating with the appearance of the corresponding phosphorylated metabolites. 8,10 Pre- viously, it was found that phosphoramidate monoesters of AZT could undergo P–O bond cleavage in the pre- sence of sweet potato acid phosphatase type XA to yield AZT. 11 For ADEPT/GDEPT approaches, a human enzyme is recommended to ensure that the host will not elicit an immumogenic response to the enzyme. 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00513-3 Bioorganic & Medicinal Chemistry Letters 11 (2001) 2803–2805 *Corresponding author. Tel.: +1-612-625-2614; fax: +1-612-624- 0139; e-mail: wagne003@tc.umn.edu