Bhavana Kushwaha

Dithiocarbamate-thiourea hybrids useful as vaginal microbicides also show reverse transcriptase inhibition: design, synthesis, docking and pharmacokinetic studies.

Prophylactic prevention is considered as the most promising strategy to tackle STI/HIV. Twenty-five dithiocarbamate-thiourea hybrids (14-38) were synthesized as woman controlled topical vaginal microbicides to counter Trichomonas vaginalis and sperm along with RT inhibition potential. The four promising compounds (18, 26, 28 and 33) were tested for safety through cytotoxic assay against human cervical cell line (HeLa) and compatibility with vaginal flora, Lactobacillus. Docking study of most promising vaginal microbicide (33) revealed that it docked in a position and orientation similar to known reverse transcriptase inhibitor Nevirapine. The preliminary in vivo pharmacokinetics of compound 33 was performed in NZ-rabbits to evaluate systemic toxicity in comparison to Nonoxynol-9.

A unique dithiocarbamate chemistry during design & synthesis of novel sperm-immobilizing agents.

1-Substituted piperazinecarbodithioates were obtained by an unusual removal of CS2 in benzyl substituted dithiocarbamate derivatives under acid and basic conditions during design and synthesis of 1,4-(disubstituted)piperazinedicarbodithioates as double edged spermicides. A plausible mechanism for CS2 removal has been proposed. All synthesized compounds were subjected to spermicidal, antitrichomonal and antifungal activities. Twenty-one compounds irreversibly immobilized 100% sperm (MEC, 0.06-31.6 mM) while seven compounds exhibited multiple activities. Benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-1-(carbodithioate) (18) and 1-benzyl 4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbodithioate) (24) exhibited appreciable spermicidal (MEC, 0.07 and 0.06 mM), antifungal (MIC, 0.069-0.14 and >0.11 mM) and antitrichomonal (MIC, 1.38 and 0.14 mM) activities. The probable mode of action of these compounds seems to be through sulfhydryl binding which was confirmed by fluorescence labeling of sperm thiols.

Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents: design, synthesis and docking studies.

Azole and carbodithioate hybrids were synthesized as alkyl 1H-azole-1-carbodithioates (7-27) and evaluated for spermicidal/microbicidal activities against human sperm, Trichomonas vaginalis and Candida species. Seventeen compounds (7-14, 16-18 and 20-25) showed spermicidal activity at MEC 1.0% (w/v) and permanently immobilized 100% normal human spermatozoa within ∼30 s. Seventeen compounds (7-11, 13-18 and 20-25) exhibited anti-Candida activity (IC50 1.26-47.69 μg/mL). All compounds were devoid of bactericidal activity against four bacterial strains (50.00 μg/mL) and antiprotozoal activity against Trichomonas vaginalis (200.00 μg/mL). Four promising compounds (10, 17, 20 and 22) have better safety profile as compared to Nonoxynol-9 (N-9). Docking study was done to visualize the possible interaction of designed scaffold with prospective receptor (Cyp51) of Candida albicans.

Ammonium salts of carbamodithioic acid as potent vaginal trichomonacides and fungicides

Chemical attenuation of the reactive oxygen species (ROS)-sensitive anaerobes Trichomonas vaginalis, which is the most prevalent non-viral sexually transmitted infection, and two often coexisting vaginal infections, namely Candida albicans and Staphylococcus aureus, which are opportunistic reproductive tract infections, was attempted with novel ammonium salts of carbamodithioic acid through inhibition of free thiols. In vitro and in vivo efficacies of the designed compounds were evaluated as topical vaginal microbicides. Five compounds showed exceptional activity against drug-resistant and -susceptible strains with negligible toxicity to host (HeLa) cells in vitro in comparison with the standard vaginal microbicide nonoxynol-9 (N-9), without disturbing the normal vaginal flora (i.e. Lactobacillus). The compounds significantly inhibited the cytopathic effects of Trichomonas on HeLa cells in vitro with efficacies comparable with metronidazole (MTZ); however, their efficacy to rescue host cells from co-infection (protozoal and fungal) was greater than that of MTZ. The compounds inhibited β-haemolysis of red blood cells caused by Trichomonas and were found to be active in vivo in the mouse subcutaneous abscess assay. Some compounds rapidly immobilized human sperm. A mechanism involving inhibition of free thiols and consequently the cysteine proteases of T. vaginalis by the new compounds has been proposed. Thus, a unique scaffold of antimicrobial agents has been discovered that warrants further investigation for development as contraceptive vaginal microbicides.

Design and synthesis of substituted morpholin/piperidin-1-yl-carbamodithioates as promising vaginal microbicides with spermicidal potential.

A series of seventeen morpholin/piperidin-1-yl-carbamodithioate (3-19) were synthesized as topical vaginal microbicidal spermicides. The synthesized compounds were evaluated for their anti-Trichomonas activity against MTZ susceptible and resistant strains along with their spermicidal and antifungal potential. All the synthesized compounds were assessed for their safety through cytotoxic assay against human cervical cell line (HeLa) and compatibility with vaginal flora, Lactobacillus. The study identified eleven dually active compounds with apparent safety. The plausible mode of action of these compounds was through sulfhydryl binding, confirmed via reduction in available free thiols on human sperm. The most promising compound 9 significantly inhibited (P<0.001) thiol-sensitive sperm hexokinase. The stability of compound 9 in simulated vaginal fluid (SVF) was performed via HPLC-PDA method, which supported its utility for vaginal administration.

Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach.

In an ongoing effort to discover an effective, topical, dual‐function, non‐surfactant contraceptive vaginal microbicide, a novel series of 2,2′‐disulfanediylbis(3‐(substituted‐1‐yl)propane‐2,1‐diyl) disubstituted‐1‐carbodithioates were designed by using a bioisosterism approach. Thirty‐three compounds were synthesized, and interestingly, most demonstrated multiple activities: they were found to be spermicidal at a minimal effective concentration of 1–0.001 %, trichomonacidal against drug‐susceptible and resistant Trichomonas strains at minimal inhibitory concentration (MIC) ranges of 10.81–377.64 and 10.81–754.14 μM, respectively, and fungicidal at MIC 7.93–86.50 μM. These compounds were also found to be non‐cytotoxic to human cervical (HeLa) epithelial cells and vaginal microflora (Lactobacilli) in vitro. The most promising compound, 2,2′‐disulfanediylbis(3‐(pyrrolidin‐1‐yl)propane‐2,1‐diyl)dipyrrolidine‐1‐carbodithioate (5), exhibited spermicidal activity 15‐fold higher than that of the marketed spermicide Nonoxynol‐9 (N‐9) and also demonstrated microbicidal potency. To identify common structural features required for spermicidal activity, a 3D‐QSAR analysis was carried out, as well as in vivo efficacy studies and fluorescent labeling studies to determine the biological targets of compound 5.

Energy Utilization for Survival and Fertilization--Parsimonious Quiescent Sperm Turn Extravagant on Motility Activation in Rat

ABSTRACT Quiescent sperm survive in cauda epididymis for long periods of time under extreme crowding conditions and with a very limited energy substrate, while after ejaculation, motile sperm live for a much shorter period with an unlimited energy resource and without crowding. Thus, the energy metabolism in relation to the energy requirement of the two may be quite different. A simple physiological technique was evolved to collect viable quiescent sperm from rat cauda epididymis to compare its energy metabolism with motile sperm. Quiescent sperm exhibited 40%–60% higher activities of mitochondrial electron transport chain complexes I–IV and ATP synthase in comparison to motile sperm and accumulated Ca2+ in the midpiece mitochondria to enhance oxidative phosphorylation (OxPhos). In contrast, motile sperm displayed up to 75% higher activities of key glycolytic enzymes and secreted more than two times the lactate than quiescent sperm. Quiescent sperm phosphorylated AMPK and MAPK-p38, while motile sperm phosphorylated AKT and MAPK/ERK. Glycolytic inhibitor iodoacetamide prevented motility activation of quiescent rat sperm and inhibited conception in rabbits more effectively than OxPhos uncoupler 2,4-dinitrophenol. Apparently, quiescent sperm employ the most energy efficient OxPhos to survive for extended periods of time under extreme conditions of nutrition and crowding. However, on motility initiation, sperm switch predominantly to glycolysis to cater to their high- and quick-energy requirement of much shorter periods. This study also presents a proof of concept for targeting sperm energy metabolism for contraception.

Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ⩽0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC50=961 μg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC50=269 μg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.

Designed Chemical Intervention with Thiols for Prophylactic Contraception

Unlike somatic cells, sperm have several-fold more available-thiols that are susceptible to redox-active agents. The present study explains the mechanism behind the instant sperm-immobilizing and trichomonacidal activities of pyrrolidinium pyrrolidine-1-carbodithioate (PPC), a novel thiol agent rationally created for prophylactic contraception by minor chemical modifications of some known thiol drugs. PPC, and its three derivatives (with potential active-site blocked by alkylation), were synthesized and evaluated against live human sperm and metronidazole-susceptible and resistant Trichomonas vaginalis, in vitro. Sperm hexokinase activity was evaluated by coupled enzyme assay. PPC irreversibly immobilized 100% human sperm in ∼30 seconds and totally eliminated Trichomonas vaginalis more efficiently than nonoxynol-9 and metronidazole. It significantly inhibited (P<0.001) thiol-sensitive sperm hexokinase. However, the molecule completely lost all its biological activities once its thiol group was blocked by alkylation. PPC was subsequently formulated into a mucoadhesive vaginal film using GRaS excipients and evaluated for spermicidal and microbicidal activities (in vitro), and contraceptive efficacy in rabbits. PPC remained fully active in quick-dissolving, mucoadhesive vaginal-film formulation, and these PPC-films significantly reduced pregnancy and fertility rates in rabbits. The films released ∼90% of PPC in simulated vaginal fluid (pH 4.2) at 37°C in 5 minutes, in vitro. We have thus discovered a common target (reactive thiols) on chiefly-anaerobic, redox-sensitive cells like sperm and Trichomonas, which is susceptible to designed chemical interference for prophylactic contraception. The active thiol in PPC inactivates sperm and Trichomonas via interference with crucial sulfhydryl-disulfide based reactions, e.g. hexokinase activation in human sperm. In comparison to non-specific surfactant action of OTC spermicide nonoxynol-9, the action of thiol-active PPC is apparently much more specific, potent and safe. PPC presents a proof-of-concept for prophylactic contraception via manipulation of thiols in vagina for selective targeting of sperm and Trichomonas, and qualifies as a promising lead for the development of dually protective vaginal-contraceptive.

Design and synthesis of coumarin–glyoxal hybrids for spermicidal and antimicrobial actions: a dual approach to contraception

Today there is an urgent need for safe and effective dual-purpose contraceptive agents, which can simultaneously prevent unintended pregnancies and sexually transmitted infections (STI). There are several naturally occurring antimicrobial and antibiotic drugs (novobiocin, coumermycin and chlorobiocin) reported in the literature, which are based on 4-hydroxy coumarins as the active pharmacophore. Based on these interesting reports, we designed and synthesized a library of new 4-hydroxy coumarin derivatives and evaluated them for spermicidal activity. Among the tested compounds, two compounds (2a and 2d) displayed better activity (greater than 95% sperm immobilization at 0.5 mM concentration) than the positive control nonoxynol-9 (N-9). Furthermore, all the compounds were screened for antimicrobial activity against different strains of Trichomonas vaginalis and two compounds (2c and 2h) exhibited potent activity as compared to the reference drug metronidazole. The cytotoxicity assay showed that most of these compounds were safer than the N-9 against the human cervical HeLa cell line and normal vaginal flora Lactobacillus jensenii strains. The studies have demonstrated that compound (2a) is a potential lead to develop a dually active vaginal contraceptive.