Structural requirements for potent human spermicidal activity of dual-function aryl phosphate derivative of bromo-methoxy zidovudine (Compound WHI-07)

WHI-07, a novel bromo-methoxy-substituted aryl phosphate derivative of zido vudine (ZDV), is a potent dual-function contraceptive agent. Although the b romo-methoxy functional groups in the thymine ring of ifs ZDV ape very impo rtant for its sperm-immobilizing activity (SIA), the importance of the este rification of the phosphate group with an amino acid side chain and the ide ntity of the para substituent in the aryl moiety remain unclear. In the pre sent study, we have synthesized 23 new analogues of WHI-07 by replacing the alanine (Ala) side chain with different amino acids containing nonpolar si de chains, namely tryptophan (Trp), proline (Pro), phenylalanine (Phe), leu cine (Leu), methionine (Met), valine (Val), or glycine (Cly). The pam subst ituents on the aryl moiety included bromo, chloro, fluoro, nitro, or methox y groups. The SIA of each of the 23 WHI-07 analogues was evaluated by compu ter assisted sperm analysis. The potential cytotoxicity of these compounds against normal human ectocervical and endocervical epithelial cells was eva luated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro mide) cell viability assays. The replacement of the Ala side chain of WHI-0 7 with Val, Leu, or Phe led to a complete loss of SIA (EC50 values > 500 mu M), whereas replacement with Trp reduced the SIA by 4-fold. The presence o f para substituents on the phenyl moiety led to significant alterations in SIA. The anti-human immunodeficiency virus (HIV) activity of Trp-containing WHI-07 analogues was also diminished. Our finding highlights the necessity of Ala side chain and the presence of electron-withdrawing para-bromo subs tituent on the phenyl moiety in addition to bromo-methoxy functionalization groups on the thymine ring in order for the phosphoramidate derivatives of ZDV to be effective dual-function spermicidal agents. Unlike the detergent -type microbicide, nonoxynol-9, which was cytotoxic to normal human ectocer vical and endocervical epithelial cells (IC50 values of 22 mu M and 16 mu M , respectively) at spermicidal concentrations (EC50 = 81 mu M), WHI-07 and its active analogues were selectively spermicidal without cytotoxicity agai nst female genital tract epithelial cells. WHI-07 and its Trp analogues hol d particular clinical promise for the development of novel, nondetergent-ty pe prophylactic contraceptives for the prevention of heterosexual HIV/acqui red immunodeficiency syndrome transmission.