Synthesis, characterization and preclinical formulation of a dual-action phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities

In a systematic effort to develop a microbicide contraceptive capable of pr eventing transmission of human immunodeficiency virus (HIV), as well as pro viding fertility control, we have previously identified novel phenyl phosph ate derivatives of zidovudine (ZDV) with 5-halo 6-alkoxy substitutions in t he thymine ring and halo substitution in the phenyl moiety respectively. He re, we describe the synthesis, characterization, and successful preclinical formulation of our lead compound, 5-bromo-6-methoxy-3'-azidothymidine-5'-( p-bromophenyl) methoxyalaninyl phosphate (WHI-07), which exhibits potent an ti-HIV and sperm immobilizing activities. The anti-HIV activity of WHI-07 w as tested by measuring viral p24 antigen production and reverse transcripta se activity as markers of viral replication in HIV-1 infected human periphe ral blood mononuclear cells (PBMC). WHI-07 inhibited replication of HIV in a concentration-dependent fashion with nanomolar IC50 values. The effects o f WHI-07 on human sperm motion kinematics were analysed by computer-assiste d sperm analysis (CASA), and its effects on sperm membrane integrity were e xamined by confocal laser scanning microscopy (CLSM), and high-resolution l ow-voltage scanning electron microscopy (HR-LVSEM). WHI-07 caused cessation of sperm motility in a concentration- and time-dependent fashion, The in-v itro cytotoxicities of WHI-07 and nonoxynol-9 (N-9) were compared using nor mal human ectocervical and endocervical epithelial cells by the MTT cell vi ability assay. Unlike N-9, WHI-07 had no effect upon sperm plasma and acros omal membrane integrity. N-9 was cytotoxic to normal human ectocervical and endocervical cells at spermicidal doses, whereas WHI-07 was selectively sp ermicidal. The in-vivo vaginal absorption and vaginal toxicity of 2% gel-mi croemulsion of WHI-07 was studied in the rabbit model. The sperm immobilizi ng activity of WHI-07 was 18-fold more potent than that of N-9. Over a 10 d ay period, there was no irritation or local toxicity to the vaginal epithel ia or systemic absorption of WHI-07. Therefore, as a potent anti-HIV agent with spermicidal activity, and lack of mucosal toxicity, WHI-07 may have th e clinical potential to become the active ingredient of a vaginal contracep tive for women who are at high risk for acquiring HIV by heterosexual vagin al transmission.