Discovery of substituted isoxazolecarbaldehydes as potent spermicides, acrosin inhibitors and mild anti-fungal agents

Discovery of substituted isoxazolecarbaldehydes as potent spermicides, acrosin inhibitors and mild anti-fungal agents G.Gupta R.K.Jain J.P.Maikhuri P.K.Shukla 1 M.Kumar 1 A.K.Roy 0 A.Patra 0 V.Singh 0 S.Batra 0 0 Medicinal Chemistry, Central Drug Research Institute , Lucknow 226 001 , India 1 Fermentation Technology BACKGROUND: The continued endeavour to design novel, non-detergent molecules that can be useful as topical, prophylactic contraceptives has led to the discovery of substituted isoxazolecarbaldehydes as a new class of compounds exhibiting both spermicidal and acrosin inhibitory activities simultaneously. METHODS: Normal human semen samples were used to detect the spermicidal and acrosin inhibitory activities of the new compounds. Lactobacillus, HeLa and Candida cultures were used to determine the safety of compounds towards normal vaginal flora, their cytotoxicity and anti-fungal activity. Supravital staining and the hypo-osmotic swelling test (HOST) were used to detect the effect on sperm membrane integrity. Nonoxynol-9 (N-9) was used as a reference standard. RESULTS: The 5- and 3-substituted isoxazolecarbaldehydes showed significant spermicidal [minimum effective concentration (MEC) 5 0.005 - 2.5%] and acrosin inhibitory (IC50 5 3.9 - 58 3 1024 mol/l) activities in several molecules along with weak fungicidal activity against Candida albicans. Lineweaver - Burk and Dixon plot analysis of a representative structure showed non-competitive inhibition of human acrosin enzyme, and the most potent acrosin inhibitors also considerably diminished the induction of the acrosome reaction by Ca21 ionophore. Some compounds were found to be significantly safer than N-9 towards Lactobacillus acidophilus in vitro at their respective spermicidal MECs. In the cytotoxicity assay, the IC50 of these compounds towards the HeLa cell line was of the same order as N-9 (0.9 - 0.1 mmol/l); however, in contrast, the compounds exhibited only a moderate effect on sperm membrane integrity. CONCLUSIONS: This study indicates that 5- and 3-substituted isoxazolecarbaldehydes are 'first generation' multifunctional, spermicidal molecules that hold promise for development as topical contraceptives with useful associated activities that can add considerably to their effectiveness, safety and prophylaxis. acrosin inhibition/anti-fungal/isoxazolecarbaldehydes/spermicide/topical microbicide Introduction HIV/AIDS makes dual protection a must for contraceptives, which is only possible with topical microbicidal spermicides (World Health Organization, 2002). Oral contraceptives, intrauterine devices (IUDs), sterilization, injectables, implants, etc. do not offer any protection against sexually transmitted diseases (STDs) and AIDS. Condoms do provide dual protection if used correctly and consistently, but they are not women controlled. Thus, safe, effective, acceptable and self-administered topical preparations with both microbicidal and spermicidal activity are likely to have a major positive impact on reproductive health, especially in areas with a high prevalence of STDs including HIV (World Health Organization, 2002). Nonoxynol-9 (N-9), a non-ionic detergent with demonstrable in vitro toxicity to a number of bacteria and viruses including HIV-1 (Grimes and Cates, 1990), is widely used as an over-the-counter spermicide. However, studies have shown that frequent use of N-9 may cause vaginal irritation and ulceration (Rekart, 1992; Stafford et al., 1998) that may actually increase the risk of HIV-1 transmission (Stephenson, 2000). N-9 has been shown to cause a interleukin-1-induced pro-inflammatory response, involving NF-kB activation in cervicovaginal epithelium leading to recruitment of HIV-1 host cells and increased HIV-1 replication in infected cells (Fichorova et al., 2001). In addition, poor contraceptive efficacy of N-9 and its total failure as a microbicide has suddenly augmented the need for a new, better spermicidal molecule that is lacking detergent-type membrane toxicity and has clinical advantage over currently available vaginal preparations (DCruz and Uckun, 1999). Such spermicides will form an inevitable ingredient in all future microbicidal preparations. Synthesis and evaluation of novel, non-detergent structures at this institute has led to the discovery of several prototypes with potent spermicidal activity. These include acrylophenones (Kumaria et al., 2002; Maikhuri et al., 2003), dithiocarbamates (Tripathi et al., 1996), quinines (Maikhuri et al., 2003), anysylidines, thymols and isoxazoles/isoxazolines (Srivastava et al., 1999). In isoxazoline and isoxazole derivatives reported by us previously, we have found a potent spermicidal response including anti-HIV activity in a few derivatives (Srivastava et al., 1999). In our continued efforts to discover spermicidal efficacy in new isoxazole derivatives, we undertook the evaluation of 5-, 4- and 3-substituted isoxazolecarbaldehydes (Patra et al., 2001; Roy and Batra, 2003a,b). It was discovered that a few of the derivatives belonging to 5- and 3-isoxazolecarbaldehydes show significant spermicidal activity. A spermicidal agent also possessing an acrosin inhibitory property can considerably increase the contraceptive efficacy of the compound besides concurrently reducing the application dose. On the other hand, protection against opportunistic fungal infections such as Candida, which is the most commonly encountered fungal pathogen in the human vagina, is highly desirable in topical preparations for vaginal use. In view of these considerations, these compounds were simultaneously evaluated for acrosin inhibition and anti-fungal activity. The details of our studies with respect to the biological evaluation as a spermicide, acrosin inhibitor and fungicide, the structure activity relationship, cytotoxicity towards a human cervical cell line and sperm plasma membrane, and safety towards normal vaginal flora for these compounds are presented herein. Materials and methods Substituted isoxazolecarbaldehydes Suitably substituted isoxazolecarbaldehydes (28 compounds) were synthesized by the chemist authors in the Medicinal Chemistry Division of the Institute as per the molecular structures shown in Figure 1. All compounds were . 99.5% pure [analytical high-performance liquid chromatography (HPLC)] and were characterized by mass spectroscopy, infrared spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, and by chemical analysis (Patra et al., 2001; Roy and Batra, 2003a,b) All other chemicals/biochemicals were procured from Sigma-Aldrich Inc., MO. Human spermatozoa Fresh human semen samples obtained by masturbation into a sterile vial from healthy, young, fertile donors were liquefied for 45 min at 378C and used for in vitro spermicidal and acrosin inhibitory assays. Samples having . 60 106/ml sperm count with . 65% motility and normal morphology were used in the study. Significant acrosomal defects were very rarely encounte (...truncated)