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Reproductive Technology |
a Drug Discovery Program,
b Departments of Reproductive Biology,
c Chemistry,
d Structural Biology,
e Bioinformatics,
f Virology, Parker Hughes Institute, St. Paul, Minnesota 55113
h Paradigm Pharmaceuticals, LLC, St. Paul, Minnesota 55113
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, sperm motility and transport, vagina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Design of potent inhibitors of HIV-1 reverse transcriptase (RT), an enzyme responsible for the reverse transcription of the retroviral RNA to proviral DNA, has been a focal point in translational AIDS research [12–14]. Promising inhibitors include a diverse group of nonnucleoside inhibitors (NNIs) that induce allosteric changes in the HIV-1 RT, thus rendering the enzyme incapable of converting viral RNA to DNA [15–17]. There are currently three approved NNIs: nevirapine (dipyridodiazepinone derivative) [18], delavirdine (bis(heteroaryl)piperazine derivative) [19], and efavirenz (a benzoxazinone) [20]. Unfortunately, the high and erroneous replication rate of the virus leads to genetic variants, especially when selective pressure is introduced in the form of drug treatment [21, 22]. These mutants are resistant to the previously used anti-HIV agents. Switching agents or using combination therapy may decrease or delay resistance, but because viral replication is not completely suppressed, drug-resistant viral strains ultimately emerge [23]. Moreover, these drugs have significant side effects [24]. Consequently, the development of new, nontoxic broad-spectrum spermicidal and nonspermicidal anti-HIV agents that are antiviral at subnanomolar concentrations has become the focal point in translational anti-HIV microbicide research [9].
NNI bind to an allosteric site of HIV-1 RT [17, 25, 26], which is 
10 Å away from the catalytic site. NNI binding induces rotamer conformation changes in some residues (Y181 and Y188) and makes the thumb region more rigid. Both events consequently would alter the substrate binding mode and/or affect the translocation of the double strand, which are probably critical for the polymerase function, thereby leading to a noncompetitive inhibition of the enzyme. Most mutations conferring resistance to NNI are directly in contact with the NNI molecule and thus are associated with changes in the binding of NNI to RT [13, 27]. Dozens of mutant strains have been characterized as resistant to NNIs, including L100I, K103N, V106A, E138K, Y188I/C, and Y188H. In particular, the Y181C and K103N mutants may be the most difficult to treat because they are resistant to most of the NNIs that have been examined [13, 28]. For example, primary mutations associated with resistance to currently used NNIs involve residues K103, V106, V108, Y181, Y188, and G190. The mutations of these residues lead to the weakening of the inhibitor binding to RT. Delavirdine, nevirapine, and efavirenz are less effective against RT with primary mutations [13].
A comparison of nine RT-NNI crystal structures, the analysis of surface complementarity between these NNIs and RT, and estimation of their inhibition constant (Ki values) combined with a docking procedure revealed several potential ligand derivatization sites for the generation of more potent NNIs [13]. The spacious wing 2 region of the butterfly-shaped NNI-binding pocket contains multiple aromatic residues, including Y181 and Y188, which occupy a substantial volume. Y181C, Y188C, and Y188H mutations in drug-resistant HIV strains result in larger unoccupied volume in the binding pocket and a different interaction environment [13]. Therefore, preferred inhibitors should maximize the occupancy in the wing 2 region of the NNI binding site of RT. We hypothesized that an efficient use of this space by strategically designing functional groups should yield more potent anti-HIV agents with higher affinity for the NNI binding pocket of HIV RT with gain of spermicidal function. Therefore, we replaced the planar pyridyl ring of phenethyl thiazolyl thiourea (PETT) NNI, trovirdine, with other planar rings such as phenyl, thiazolyl, and naphthyl, nonplanar rings such as cyclohexenyl, adamantyl, and piperidinyl, as well as cis-myrtanyl groups [29–36]. In addition, we also introduced various substituents on the phenyl ring at various positions [13, 37]. We also introduced for the first time chiral centers in PETT compounds to understand the effect of stereochemistry on the potency of anti-HIV activity of these NNIs [38, 39].
The cyclohexenyl thiourea (CHET) NNIs can structurally be segmented into four quadrants: 1) the cyclohexenyl ring, 2) the ethyl linker, 3) the thiourea moiety, and 4) the heterocycle. In our previous structure-activity relationship studies, we investigated variations in quadrants 1 and 4 on anti-HIV and sperm-immobilizing activity (SIA) [40]. In these studies, we systematically replaced the planar pyridyl ring of trovirdine (N-(2-pyridylethyl)-N'-(5-bromo-2-pyridyl)thiourea) with phenyl, heterocyclic, and alicyclic rings and also substituted the pyridyl ring with ortho, meta, or para functional groups such as a methyl group, fluorine, bromine, or a chlorine atom. In this study, the third and fourth quadrants were varied. Our first goal was to examine how heterocycle rings and their functionalization as well as thiourea moieties affect the potency of anti-HIV and SIA of CHET compounds. We examined this question using thiazolyl, pyridyl, or benzopyridyl-substituted 15 cyclohexenyl ring-containing compounds with functionalization of electron donating and electron-withdrawing groups on the heterocycle ring. The role of thiourea moiety was investigated by replacing the sulfur atom with an oxygen atom. Our second goal was to examine the in vivo mucosal toxicity and contraceptive activity of the lead cyclohexenyl thiourea and urea compounds either alone or in combination using the rabbit model.
The results of our structure-activity relationship studies of 15 heterocyclic ring-substituted NNIs as well as in vivo contraceptive efficacy studies of the lead compounds clearly demonstrated the potent and broad-spectrum anti-HIV activity, rapid spermicidal activity, and lack of mucosal toxicity as well as the marked ability of thiourea/urea compounds to reduce in vivo fertility. These attributes are particularly attractive for the clinical development of CHET-NNIs as potent dual-function spermicidal microbicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CHET compounds were synthesized according to the synthetic scheme shown in Figure 1. In brief, 2-amino-5-substituted pyridine was condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative. Further reaction with cyclohexenyl ethylamine in dimethylformamide (DMF) at 100°C gave the target compounds in good yields. Additional substitutions on the pyridyl ring included chloro, bromo, or methyl groups at ortho (compounds 2, 5), meta (compound 3), or para (compound 4) positions. The urea- and thiourea-substituted CHET derivatives were prepared by using carbodiimidazole or thiocarbodiimidazole derivatives of the respective amines. The names of the 15 heterocyclic cyclohexenyl thiourea/urea compounds synthesized and tested in this study are listed in Table 1. Purity was determined by proton (1H), carbon (13C), and fluorine (19F) nuclear magnetic resonance spectroscopy (Varian Oxford 300-MHz spectrometer; Varian Associates, Palo Alto, CA), Fourier transform infrared spectroscopy (FT-Nicolet Model Protege 460 instrument; Nicolet Instrument Corp., Madison, WI), mass spectroscopy (Hewlett Packard matrix-assisted laser desorption spectrometer model G2025A; Hewlett Packard, Wilmington, DE), ultraviolet spectrophotometry (Beckmann Model 3DU 7400 UV-Visible spectrophotometer; Beckmann Instruments, Fullerton, CA), and elemental analysis. Compounds with a purity of >99% were used for the biological activity and efficacy studies.
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In Vitro Assays of Anti-HIV Activity
Purified RT assays Each of the 15 cyclohexenyl thiourea/urea compounds were tested for RT inhibitory activity against purified recombinant HIV-1 RT using the cell-free Quan-T-RT assay system (Amersham Corp., Arlington Heights, IL), which utilizes the scintillation proximity assay (SPA) principle, as previously described in detail [41, 42]. In the assay, a DNA/RNA template is bound to SPA beads via a biotin/streptavidin linkage. The primer DNA is a 16-mer oligo(T), which has been annealed to a poly(rA) template. The primer-template is bound to a streptavidin-coated SPA bead. [3H]TTP (thymidine 5' triphosphate) is incorporated into the primer by reverse transcription. In brief, [3H]TTP, at a final concentration of 0.5 µCi/sample, was diluted in RT assay buffer (49.5 mM Tris-HCl, pH 8.0, 80 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, 2.5 mM EGTA, 0.05% Nonidet P-40) and added to annealed DNA/RNA bound to SPA beads. The compound being tested was added to the reaction mixture at 0.001–100 µM concentrations. Addition of 10 mU of recombinant HIV RT and incubation at 37°C for 1 h resulted in the extension of the primer by incorporation of [3H]TTP. The reaction was stopped by addition of 0.2 ml of 120 mM EDTA. The samples were counted in an open window using a Beckman LS 7600 instrument and IC50[RT] values (concentration at which the compound inhibits recombinant RT by 50%) were calculated by comparing the measurements to untreated samples.
The p24 assays for anti-HIV activity The anti-HIV activity of the CHET compounds was measured by determining their ability to inhibit the replication of the NNI-sensitive HIV-1 strain HTLVIIIB, multidrug-resistant HIV-1 strain RT-MDR, and the NNI-resistant RT Y181C and K103N mutant strain A17 variant in peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors, as described previously [43, 44]. Normal human PBMCs from HIV-negative donors were cultured for 72 h in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 20% (v/v) heat-inactivated fetal calf serum, 3% interleukin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaHCO3, 50 µg/ml gentamycin, and 4 µg/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1-h adsorption period at 37°C in a humidified 5% CO2 atmosphere. Subsequently, cells were cultured for 7 days in 96-well microtiter plates (100 µl/well; 2 x 106 cells/ml, triplicate wells) in the presence and absence of various concentrations (0.001–100 µM) of the CHET compounds. Cells from noninfected controls were handled in the same way except the virus was omitted from the preparation. Aliquots of culture supernatants were removed from the wells on the seventh day after infection for measurement of p24 antigen levels as previously described [42–44]. The p24 enzyme immunoassay was the unmodified kinetic assay available commercially (Coulter Corporation/Immunotech, Inc., Westbrook, ME). The assay uses a murine monoclonal antibody to the HIV core protein coated onto microwell strips to which the antigen present in the test culture supernatant sample binds. The plates were read on an ELISA reader (Molecular Devices, Sunnyvale, CA) at 650 nm and p24 levels, expressed as ng/ml, were calculated against known standards supplied by Coulter/Immunotech, Inc. Percent viral inhibition was calculated by comparing the p24 values for the test substance-treated infected cells with the p24 values for untreated infected cells (i.e., virus controls). The anti-HIV activity was expressed as IC50[p24] values calculated from the dose-response curves and defined as the drug concentration that decreased p24 antigen production in HIV-infected PBMCs by 50%. In parallel, the effects of various treatments on cell viability were also examined, as described previously [43]. In brief, noninfected normal PBMCs were treated with each compound for 7 days under identical experimental conditions. A microculture tetrazolium assay (MTA), using 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide (XTT), was performed to quantify cellular proliferation.
Assays of Sperm Immobilizing Activity
The SIA of 15 cyclohexenyl thiourea/urea compounds was determined by computer-assisted sperm analysis (CASA) using a Hamilton Thorne Integrated Visual Optical System, version 10.9i instrument (Hamilton Thorne Research Inc., Danvers, MA) as described previously [45, 46]. Highly motile fractions of sperm were prepared from donor semen (n = 11) by discontinuous (90–45%) gradient centrifugation using Enhance Plus medium (Conception Technologies, San Diego, CA) and the swim-up method [47, 48]. All donor semen specimens were obtained after informed consent and in compliance with the guidelines of the Parker Hughes Institute Institutional Review Board. For each experiment, pooled motile sperm (
107/ml; >95% progressively motile with curvilinear velocity >100 µm/sec) prepared from 4–6 donors were suspended in 1 ml of Biggers, Whitten, and Whittingam medium (BWW) containing 25 mM HEPES and 0.3% BSA in the presence and absence of serial twofold dilutions of test substance (500–7.8 µM) in 0.5% dimethyl sulfoxide (DMSO). The corresponding volume of DMSO (0.5%) was added to control tubes. Sperm suspensions with and without the cyclohexenyl thiourea/urea compounds were incubated at 37°C and the percentage of motile sperm was evaluated by CASA at 3 h.
For CASA, 5 µl of each sperm suspension was loaded into a 20-µm Microcell slide (Conception Technologies) in a counting chamber at 37°C. At least 5–8 fields per slide were scanned for analysis. Each field was recorded for 30 sec. The computer calibrations were set at 30 frames at a frame rate of 30/sec. Other settings were as follows: minimum contrast, 8; minimum size, 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 µm/sec; threshold straightness, 80%; and magnification factor, 1.95.
The sperm kinematics parameters that were determined included numbers of motile and progressively motile sperm; curvilinear velocity (VCL); average path velocity (VAP), straight-line velocity (VSL), beat cross frequency, the amplitude of lateral head displacement, and the derivatives straightness (straightness = VSL/VAP x 100) and linearity (linearity = VSL/VCL x 100). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled. The percent motilities were compared with vehicle-treated control suspensions of motile sperm. The SIA of the compounds was expressed as the mean EC50 values (the final concentration of the compound in medium that decreased the proportion of motile sperm by 50%). The mean EC50 value for each compound was obtained from the dose-response curves of three to five independent experiments.
In experiments designed to examine the kinetics of sperm immobilization by CHET compounds, 1-ml aliquots of a highly motile (107/ml; >95% progressively motile with curvilinear velocity >100 µm/sec) prepared from donor semen (n = 6–8) in assay medium were mixed with 5 µl of a 100 mM solution of each of the 15 CHET compounds to yield a final concentration of 500 µM. The corresponding volume of DMSO (0.5%) was added to control tubes. Samples were analyzed by CASA just prior to and following addition of the test agents (30 sec) and at timed intervals of 1 min for up to 10 min for compounds 1, 5b, and 6b; every 10 min for up to 180 min for compounds 2–4, 5a, 6a, and 7–13. To eliminate any time lapse during the sample transfer to CASA, the actual analysis time recorded during CASA was used to plot the time response curves. The time required for 50% sperm motility loss (T1/2) was calculated by nonlinear regression analysis of time-response curves from 3–5 independent experiments. Representative compounds (5b and 6b) were also tested for the duration of SIA using 0.5-ml semen aliquots (72 ± 6% progressive motility) obtained from eight donors, and T1/2 values were obtained by nonlinear regression analysis.
In experiments designed to confirm the irreversible nature of SIA, motile fraction of sperm (>107/ml) prepared from donor semen were resuspended in 1-ml aliquots of BWW-0.3% BSA in the presence and absence of 5b (500 µM). Corresponding volume (0.5%) of DMSO was added to control sperm containing assay medium. Following a 5-min incubation at 37°C, duplicate aliquots of control and test sperm suspensions were used for sperm motility assessment using CASA. The remaining sperm suspension was washed by addition of fresh assay medium and centrifugation (500 x g for 5 min). The supernatants were discarded, and the pellets were resuspended in 1 ml each of BWW-0.3% BSA medium (without test agents or vehicle) at 37°C. Duplicate aliquots were reassessed for sperm motion parameters by CASA at 10-min intervals for up to 60 min. The results were compared with the sperm motion parameters of similarly processed sperm suspensions of motile sperm suspended in assay medium. In parallel, the viability of sperm suspension was quantitated by the eosin-Y dye exclusion method. An aliquot of the sperm suspension was mixed with an equal volume of 0.5% eosin-Y dye solution in saline and the sperm suspension was enumerated for dye uptake by light microscopy under bright field optics (Olympus BX40; Olympus Corporation, Lake Success, NY). For each treatment, 300–400 sperm were evaluated.
Assay for Epithelial Cell Viability
The potential cytotoxicity of lead CHET NNIs, compounds 5a and 6a, in comparison with N-9 against normal human vaginal, ectocervical, and endocervical epithelial cells (Clonetics Corporation, San Diego, CA) was measured using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay as described previously [49–51]. In brief, exponential growing vaginal, ectocervical, and endocervical epithelial cells were seeded into 96-well plates at a density of 2 x 104 cells/well and incubated for 24 h at 37°C prior to drug exposure. On the day of treatment, culture medium was aspirated from the wells and replaced with fresh medium containing serial twofold drug concentrations ranging from 7.8 to 1000 µM or vehicle (0.1% DMSO). Nonoxynol-9 (N-9; IGEPAL CO-630; Rhone Poulenc, Cranbury, NJ) was diluted in PBS. Triplicate wells were used for each treatment. Culture plates were then incubated for 3 h before adding 10 µl of MTT solution (5 mg/ml in PBS) to each well. Wells containing only medium and MTT were used as control for each plate. The tetrazolium/formazan reaction was allowed to proceed for 4 h at 37°C, and then 100 µl of the solubilization buffer (10% SDS in 0.1% HCl) was added to all wells and mixed thoroughly to dissolve the dark-blue formazan crystals. After an overnight incubation at 37°C, the optical density (OD) at 540 nm was measured using a 96-well multiscanner autoreader with the solubilization buffer serving as blank. To translate the OD540 values into the number of live cells in each well, the OD540 values were compared with those of standard OD540-versus-cell number curves generated for each cell line. The percent survival was calculated using the formula % survival = live cell number [test]/live cell number [control] x 100. The results were expressed as mean IC50 values for three independent experiments. The IC50[MTT] was defined as the concentration required for 50% reduction in cell survival.
Sperm Motility Using Bovine Cervical Mucus> Penetration Assay
The ability of CHET compounds (1, 2, 4, 5a, 5b, 6a, 6b, 7, and 9) to immobilize sperm in semen was tested using the bovine cervical mucus penetration assay [52]. The bovine cervical mucus penetration test was performed using the Penetrak kit (Biochem Immunosystems, Inc., Allentown, PA) essentially according to the manufacturer's instructions [48]. Because of the variable nature of human sperm in cervical mucus penetration tests as well as the decline of sperm motility in human semen over time, the functional motility of sperm exposed to CHET compounds was evaluated using preselected donor semen that was found to be compatible with the assay conditions. Briefly, flat capillary tubes were thawed at room temperature for 30 min and snapped above the mucus meniscus. Vials containing 0.2-ml aliquots of liquefied donor semen were treated with and without 500 µM each of the nine spermicidal cyclohexenyl thiourea/urea compounds in 0.5% DMSO and the cut end of two capillary tubes were placed vertically within 2 min of addition of test agents. Following a 2-h incubation period at room temperature, the capillary tubes were then placed on a calibrated microscope slide and the distance traveled by the vanguard sperm was recorded to the nearest millimeter. Each compound was tested twice with semen obtained from different donors. The values of the four capillary tubes were averaged for the reported result.
Molecular Modeling Analysis
A quantitative structure-activity relationship (QSAR) analysis model was developed to find a multivariate mathematical relationship between a set of 20 physicochemical properties (descriptors) describing topological, electronic, and structural features with observed T1/2 values of 15 heterocyclic NNIs. All NNIs were first built and energy minimized using the Open Force Field within Cerius 2 program (Molecular Simulation, Inc., San Diego, CA) and then read into a study table and subjected to the calculations of all 20 descriptors within the QSAR module [53]. These descriptors, including molecular refractivity (MolRef) index, hydrophobicity (AlogP), atomic polarizability (Apol), and the X-component of principal moment of inertia (PMI-X), etc., were compared with the T1/2 values in a search for the best correlation. A stepwise regression method was used to search for and select the model that significantly predicted the T1/2 values. All 4 regressors and 11 interaction terms were included to search for the linear regression equation.
Rabbits
Fifty-seven female and 18 male sexually mature, specific-pathogen-free, New Zealand White (NZW) rabbits were obtained from Charles River Laboratories (Wilmington, MA) and acclimated for a minimum of 3 wk. The bucks (mean age = 9 mo, range = 8–10 mo, and mean body weight = 4.3 kg) were trained for 1–2 mo to provide semen via an artificial vagina. Virgin does were 6–7 mo old (mean body weight = 3.8 kg). Animals were housed individually in stainless steel cages (dimensions, 20 x 25 x 30 inches) under standard conditions (temperature, 68 ± 2°F, 50 ± 10% relative humidity, and 12-h fluorescent light cycle). Food (2031 Global High Fiber Rabbit Diet; Harlan Teklad, Madison, WI) and water were available ad libitum. All animal husbandry operations were conducted under current U.S. Department of Agriculture Guidelines. The rabbits were isolated for a minimum of 4 wk. All animal procedures were approved by the Parker Hughes Institute Animal Use and Care Committee.
In Vivo Contraceptive Efficacy in the Rabbit Model
For each contraceptive test, the does were divided into four subgroups of 5 or 10: i) vehicle group, ii) compound 5a group, iii) compound 5b group, and iv) compound 5a + 5b group. Does were superovulated by an i.m. injection of 75 IU of eCG (Gestyl; Diosynth B.B., Oss, Holland) 96 h prior to an i.v. injection of 100 IU of hCG (Sigma Chemical Co., St. Louis, MO), which was administered at the time of artificial insemination. Semen was obtained from trained bucks (n = 18) of proven fertility via a prewarmed (45°C) artificial vagina immediately before use [54]. Sperm count and motility were assessed to ensure that the males were ejaculating good-quality semen. Prior to artificial insemination, semen samples without the contamination of urine or gel were pooled and 0.6-ml (>30 x 107 sperm/ml) aliquots were transferred to 5-ml polystyrene tubes and mixed with the test substances (compounds 5a, 5b, or 5a + 5b) to a final concentration of 1 mM in 0.5% DMSO. The corresponding volume of DMSO (0.5%) was added to control semen. The treated semen (0.5 ml; >15 x 107 sperm) was immediately transferred to a tuberculin syringe and was deposited within 1 min of adding the test compound by inserting the tuberculin syringe into the vagina to a depth of 6 cm. Presence of progressively motile sperm in compound 5a- and/or compound 5b-treated semen was established by CASA analysis using the same batch of semen under identical conditions used for insemination. On postinsemination Day 8, inseminated does were killed, the uteri and the ovaries were excised, and the total numbers of implantations in each uterine horn and the number of corpora lutea in each ovary indicative of ovulated eggs were determined. The ratio of embryos to corpora lutea was used as a measure of fertility.
Rabbit Vaginal Irritation Test
Due to the lipophilic nature of CHET-NNIs, a microemulsion-based formulation strategy was developed. A novel, submicron (30–80 nm) particle size microemulsion-based system with high solubilizing capacity for CHET-NNIs was identified with systemic mapping of ternary phase diagrams and a drug solubilization study [55]. The gel microemulsion optimized for intravaginal drug delivery of CHET-NNIs by providing good spreadability and retention over the vaginal mucosa was used for the in vivo mucosal toxicity. For the vaginal irritation study, 27 NZW rabbits in subgroups of three were treated intravaginally with 1 ml of gel microemulsion with and without increasing concentrations (0.5%, 1.0%, and 2.0%) of compounds 5a and 5b or a single concentration (1.0%) of 5a + 5b for 10 consecutive days. A gel formulation containing 4% N-9 was used as a positive control [56]. Animals were killed on Day 11, and the reproductive tract was examined grossly and microscopically after completion of the study. The vaginal tissues were rapidly removed and parts of the upper (cervico-vagina), middle, and lower (uro-vagina) regions of each vagina were fixed in 10% neutral-buffered formalin. Tissues were embedded in paraffin, sectioned at 4–6 µm, stained with hematoxylin and eosin, and examined under 200x and 400x magnification using a Leica light microscope (Milton Keynes, Buckinghamshire, UK) interfaced with an image analysis system. The images were captured using the ImagePro Plus program (Media Cybernetics, Silver Spring, MD) in conjunction with a 3CCD camera (DAGE-MTI, Inc., Michigan City, IN), and images were transferred to Adobe Photoshop 6.0 software (Adobe Systems, Inc., San Jose, CA) for observation and analysis. Each of the three regions of vagina was examined for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion. The scores were assigned based on the scoring system of Eckstein et al. [57], which was as follows: individual score: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = intense irritation; total score: <8 = acceptable, 9–10 = marginal, and 11 = unacceptable. Results were expressed as the mean ± SD values. The histopathologic scoring was performed by two veterinarian pathologists with extensive expertise in toxicology research.
Statistical Analysis
Nonlinear regression analysis was used to find the IC50, EC50, and T1/2 values from the concentration- and time-dependent curves using GraphPad Prism (version 3.0) software (San Diego, CA). Linear regression analysis and Pearson correlation tests were employed to determine the relationship between two variables. A stepwise regression method was used to search for and select the model that reliably predicted the T1/2 values. Four regressors (MolRef, AlogP, Apol, and PMI-X) and the 11 interaction terms were included to search for the linear regression equation. A forward selection procedure was adopted that included the most significant regression terms in the model (JMP Software; SAS, Cary, NC). The final linear model included the intercept, the significant regressors, and the significant interactions between the regressors, and this was fitted to determine the parameter coefficients of each of these regression terms. A multiple regression model was developed to describe the relationship of T1/2 values with four regressors. The statistical significance of differences in fertility between the control and treatment groups was analyzed by Fisher exact test. The statistical significance of differences in mean number of corpora lutea and implants between the groups was analyzed by a one-way analysis of variance, followed by a Dunnett multiple comparison test. Differences were considered statistically significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The structure-activity relationship studies of CHET compounds were performed by varying the heterocycle ring, thiourea moiety, and substitutions on the heterocycle ring. All 15 thiourea/urea compounds in this study were tested for their RT inhibitory activity in cell-free assays using purified recombinant HIV-1 RT (reported as IC50[rRT]), and the active derivatives and the lead compounds were also tested in cell culture for their ability to inhibit the replication of the NNI-sensitive HIV-1 strain HTLVIIIB, multidrug-resistant HIV-1 strain RT-MDR, and the NNI-resistant double mutant HIV-1 strain, A17 variant, in PBMCs from healthy volunteer donors (reported as IC50[p24]).
Chart 1 lists the anti-HIV and SIA profiles of 15 cyclohexenyl thiourea/urea compounds in which one of the nitrogen atoms of the thiourea/urea is attached to the cyclohexenyl ring through an ethyl bridge and the other nitrogen atom is attached to a thiazolyl (compounds 1–3), pyridyl (compounds 4–9), or benzothiazolyl (compounds 10–13) ring (R). Among the thiazolyl-ring-containing CHET compounds, both the unsubstituted (compound 1) and the 4-acetyl-substituted thiazolyl-ring-containing thiourea (compound 2) were devoid of anti-HIV activity (IC50[RT] values of >100 µM), whereas its 4-methyl-substituted derivative (compound 3) exhibited significant anti-HIV activity (IC50[RT] = 3 µM and IC50[p24] = 0.008 µM). Among the eight N-pyridyl ring-containing cyclohexenyl thiourea/urea compounds, functionalization of the pyridyl ring with 5-bromo atom (compound 5a), 5-chloro atom (compound 6a), or a 5-methyl group (compound 7) was associated with a marked increase in anti-HIV activity with submicromolar IC50[RT] values (0.5–1.2 µM) and nanomolar IC50[p24] values (0.003–0.005 µM) when compared with the unsubstituted pyridyl CHET compound 4 (IC50[RT] = 22 µM and IC50[p24] = 0.03 µM). The sulfur atom in the thiourea moiety was essential for the anti-HIV activity of pyridyl CHET compounds because its replacement by an oxygen atom either abolished (compound 5b) or markedly reduced (compound 6b) the anti-HIV activity. A change from pyridyl ring to benzothiazole ring (compound 10) resulted in diminished anti-HIV-1 activity (IC50[RT] = 2 µM and IC50[p24] = 0.02 µM), and further substitution of the benzothiazole ring with 6-fluoro (compound 11), 4-methyl (compound 12), or 6-methoxy (compound 13) groups led to a complete loss of anti-HIV activity (IC50[RT] = >100 µM). Thus, the substitution of the pyridyl ring with electron-withdrawing groups such as Br or Cl and electron-donating group such as CH3 led to potent anti-HIV activity of CHET compounds.
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The dose (reported as EC50) and time-dependent (reported as T1/2) SIA of each of the 15 cyclohexenyl thiourea/urea compounds was determined with the use of a computer-assisted sperm analyzer using a highly motile fraction of human sperm (Chart 1). Analysis of sperm-motion kinematics using CASA established that the incubation alone did not alter the sperm-motion parameters, such as progressive velocity, straightness of the swimming pattern, linearity of the sperm tracks, beat-cross frequency, and amplitude of sperm-head displacement (data not shown), whereas, of the 15 NNIs evaluated for SIA, 12 (viz., compounds 1, 2, 4–7, 9–11, and 13) exhibited a gain of spermicidal function at micromolar concentrations (EC50[SIA] values = 45–194 µM). The unsubstituted thiazolyl thiourea (compound 1), which lacked significant anti-HIV activity, was spermicidal, with a mean IC50[SIA] value of 80 µM. Substitution on the thiazolyl ring with acetyl at the 4-position (compound 2) led to a decrease in SIA activity, while substitution with a methyl group (compound 3) led to a complete loss of SIA and gain of potent anti-HIV activity. The N-pyridyl CHET compounds exhibited variable SIA (mean IC50[SIA] values = 45 to >500 µM). Notably, the N-pyridyl thiourea compounds with a bromine atom (compound 5a), chlorine atom (compound 6a), or a methyl group (compound 7) at the 5-position of the pyridyl ring led to an increase in potency of anti-HIV activity (IC50 = 0.003–0.005 µM) as well as SIA (EC50 = 45–96 µM). The greatest effect was exhibited by compounds 5a and 6a. Interestingly, structural analogs of compounds 5a and 6a where the sulfur atom of the thiourea moiety (X) was replaced with an oxygen atom (compounds 5b and 6b) were virtually devoid of anti-HIV activity but retained SIA. The concentration-dependent SIA of thiourea/urea compounds was associated with a parallel decline in sperm kinematic parameters, particularly track speed (VCL), path velocity (VAP), and straight-line velocity (VSL) (data not shown). The thiazolyl, pyridyl, and benzopyridyl CHET compounds with methyl group substitutions either at the 4- or 6-position were nonspermicidal even at 500 µM. Thus, pyridyl-ring-containing CHET compounds with electron-withdrawing groups (Br or Cl) attached at the 5-position of the pyridyl ring showed marked SIA in addition to their superior anti-HIV activity.
Differential Kinetics of Sperm Immobilization by Thiourea and Urea Analogs
Chart 1 also lists the corresponding times required for 50% motility loss (T1/2) of progressively motile sperm exposed to each of the 15 cyclohexenyl thiourea/urea compounds. The T1/2 values ranged from 0.9 min to >180 min. The comparative time-dependent effects of cycohexenyl-pyridyl thiourea versus urea compounds with a bromine atom (compounds 5a and 5b) or a chlorine atom (compounds 6a and 6b) on sperm motility analyzed by CASA is shown in Figure 2, A and B. The time kinetics of sperm immobilization was fast with spermicidal urea analogs (compounds 5b and 6b) when compared with thiourea derivatives (compounds 5a and 6a). The mean T1/2 values for thiourea compounds 5a and 6a (at 500 µM concentrations) were 65 and 34 min, respectively, whereas the corresponding mean T1/2 values for the urea compounds 5b and 6b were 1.5 and 0.9 min. Thus, the urea analogs immobilized sperm 38-fold faster than the thiourea derivatives. The kinetics of sperm immobilization by compounds 5b and 6b were unaffected when SIA was assessed in undiluted semen samples (T1/2 = <2 min). A stepwise regression method was used to search for and select the model that significantly predicted the T1/2 values. Regression analysis of the relationship between log10-transformed observed T1/2 values and predicted T1/2 values for 11 spermicidal cyclohexenyl thiourea/urea compounds showed a significant fit of the regression model (R = 0.93; P = 0.0018) (Fig. 3). The time-dependent sperm motility loss induced by thiourea/urea compounds was also associated with concomitant changes in the movement characteristics of the surviving sperm, particularly with respect to track speed (VCL), path velocity (VAP), and straight-line velocity (VSL) (data not shown). Interestingly, the unsubstituted thiazolyl CHET compound 1 also exhibited rapid SIA with a mean T1/2 of 1.2 min. By comparison, the sperm motility in control samples remained stable (94% ± 3% compared with baseline) during the 180-min monitoring period.
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SIA of Novel NNIs Is Irreversible
In order to determine whether the SIA of CHET compounds was reversible, highly motile fraction of sperm exposed to 500 µM of compound 5b for 5 min were washed and resuspended in fresh sperm motility assay medium and sperm motility was reassessed by CASA at 10-min time intervals for up to 60 min. In parallel, sperm viability was quantitated by the eosin-Y dye exclusion method. No recovery in sperm motility was observed by CASA (91% ± 3% for control versus 0% at all time points), indicating that the drug-induced sperm immobilization was irreversible. Greater than 97% of drug-treated sperm were eosin-Y positive, indicating that the loss of sperm motility was concomitantly associated with loss of sperm viability.
Spermicidal CHET Compounds Inhibit Functional Sperm Motility in Cervical Mucus
To be an effective contraceptive, the cyclohexenyl thiourea/urea compounds should retain SIA in genital tract secretions. Therefore, we tested the ability of spermicidal cyclohexenyl thiourea/urea compounds in semen to inhibit sperm transport into a standardized midcycle bovine cervical mucus column (Penetrak). The bovine cervical mucus penetration test has been used to evaluate the in vitro fertilizing capacity of human sperm [52]. Within 2 min of addition of the compounds (500 µM), the capillary tubes filled with cervical mucus were incubated with semen for 2 h and the distance traveled by the vanguard sperm was recorded. The mean penetration distance of the vanguard sperm from vehicle-treated control specimens (n = 4) in bovine cervical mucus was 47.5 mm after 2 h of migration, whereas the corresponding distances for sperm treated with spermicidal CHET compounds ranged from 2 to 21 mm (P < 0.01; Table 2). The most rapid spermicidal CHET compounds, compounds 1, 5b, and 6b, induced 83–96% inhibition of functional motility in cervical mucus when compared with the vehicle control. A significant positive correlation (R = 0.845; P = 0.004, n = 9) was noted between the mean penetration distance of the vanguard sperm in the cervical mucus test and the observed mean T1/2 values as assessed by means of the Pearson test. The ability of NNIs to inhibit the functional motility in cervical mucus indicated that SIA of these NNIs is unaffected by genital tract secretions.
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Correlation of SIA of NNIs by Quantitative> Structure-Activity Relationships
To determine if the SIA correlated with the physicochemical properties of the NNIs, the mean T1/2 values were analyzed against 20 physicochemical parameters (viz., topological, electronic, and structural descriptors) of each NNI using QSAR descriptors in the Cerius 2 program. Figure 4, A–D, shows significant positive correlations between T1/2 values and four QSAR descriptors. The T1/2 values of spermicidal NNIs significantly correlated with their lower molecular refractivity index (MolRef: r = 0.883; P = 0.0003), lower hydrophobicity (AlogP: r = 0.722; P = 0.012), lower atomic polarizability (Apol: r = 0.707; P = 0.014), and lower principal moment of inertia (PMI-X: r = 0.638; P = 0.034) values as assessed by means of the Pearson test (n = 11). Multiple regression analyses were, therefore, performed on log10-transformed data in order to describe the relationship of T1/2 values with four regressors that reflected the physicochemical properties of the NNIs. The stepwise regression method yielded four regression terms that most significantly reduced the residual sum of squares (Intercept, Apol, MolRef, and the interaction term Apol x MolRef) of the T1/2 values. Through this model, we developed the following prediction equation: T1/2 = 4.769 x MolRef - 0.005163 x Apol - 0.000841 x (Apol - 11987.2) x (MolRef - 88.291) - 308.93. A linear equation that included these regression terms explained a significant proportion of the variation in T1/2 values (R2 = 0.87, R = 0.93, F3.7 = 15.46, P = 0.0018). The parameter estimates were -308.9 ± 52.5 for the intercept (P = 0.0006), 4.769 ± 1.161 (P = 0.0045) for MolRef, -0.00516 ± 0.00619 (P = 0.432) for Apol, and -0.000841 ± 0.00047 (P = 0.1167) for the interaction term Apol x MolRef (Fig. 5), the most significant parameter effect being MolRef. Thus, these descriptors allowed a physical explanation of electronic and molecular properties contributing to rapid SIA.
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Anti-HIV Activity of CHET Compounds Against Mutant HIV-1 Strains
Based on their excellent antiviral activity, the dual-function lead CHET compounds 5a and 6a were further evaluated for their activity against NNI-resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving RT residues 74V, 41L, and 215L) and A17 variant with a Y181C plus K103N mutations in RT (Chart 2). The anti-HIV activities of these NNIs were compared with known NNIs, trovirdine, delavirdine, and nevirapine. Our lead compounds 5a and 6a were three times more effective against the multidrug resistant HIV-1 strain RT-MDR than they were against HTLVIIIB with wild-type RT. The ranking order of potency against RT-MDR was 5a (IC50 = 1 nM) = compound 6a (IC50 = 1 nM) > trovirdine (IC50 = 20 nM) > delavirdine (IC50 = 400 nM) > nevirapine (IC50 = 5000 nM). Compounds 5a and 6a were 20 times more potent than trovirdine, 400 times more potent than delavirdine, and 5000 times more potent than nevirapine against the multidrug-resistant HIV-1 strain RT-MDR. Similarly, both compounds 5a and 6a were more effective than trovirdine, delavirdine, and nevirapine against the problematic NNI-resistant HIV-1 strain A17 variant with both Y181C and K103N mutations in RT. Neither compounds exhibited significant cytotoxicity at effective concentrations (CC50 = >100 µM). These findings established the lead dual-function compounds 5a and 6a as potent NNI of drug-sensitive as well as multidrug-resistant strains of HIV-1.
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Treatment of Semen with Thiourea/Urea NNIs Leads to Inhibition of In Vivo Fertility
Because compound 5a is a broad-spectrum anti-HIV agent with SIA, whereas its structural analog, compound 5b, is an extremely rapid spermicidal agent, we evaluated in vivo fertilizing ability of sperm exposed to the lead dual-function CHET compound 5a either alone or in combination with its structural analog, compound 5b, in the rabbit model. Aliquots of freshly pooled rabbit semen obtained from fertile bucks (n = 18) were mixed with vehicle alone or an optimal concentration of compounds 5a, 5b, or 5a + 5b at the time of artificial insemination of superovulated NZW does in subgroups of 5 or 10. Artificial insemination was performed immediately after the addition of vehicle or vehicle containing the test compounds (<60 sec) to minimize ex vivo sperm immobilization. Presence of progressively motile sperm (>90% at 10 min for compound 5a and >70% for compound 5b at 1 min when compared with vehicle control) was established by microscopic evaluation of the treated samples. Fertility was assessed based on the proportion of does with uterine implants on postinsemination Day 8 as well as by the ratio of number of uterine implants in both uterine horns per number of corpora lutea in the ovaries. The pregnancy rates and Day 8 implantation data are provided in Table 3. Treatment of semen with compound 5a, 5b, or 5a + 5b at the time of artificial insemination caused a marked or complete reduction in fertility as assessed by the number of implants (compound 5a-treated, 28/226; compound 5b-treated, 0/147; compound 5a + 5b-treated, 0/126; vehicle control, 82/245; P < 0.0001, Fisher exact test) or the percent implants (12.3%, 0%, 0%, and 33.4%, respectively) based on number of implants to corpora lutea. Conception was completely inhibited after insemination with semen treated with compound 5b or 5a + 5b. However, the mean numbers of ovarian corpora lutea on postovulation/insemination Day 8 were similar in all four subgroups. These studies indicated that the marked decrease in fertility caused by exposure of semen to compounds 5a and 5b was a result of SIA in semen inhibiting the in vivo sperm transport.
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Lack of Epithelial Cell Toxicity of Spermicidal CHET-NNIs
The MTT assay measuring cell viability was used to assess the in vitro cytotoxicity of lead CHET-NNIs, compounds 5a and 6a, in comparison with the detergent-type vaginal virucidal spermicide N-9 against confluent monolayers of normal human vaginal, ectocervical, and endocervical epithelial cells. The IC50 values obtained from the concentration-response cell survival curves measured by the MTT assays were compared with spermicidal EC50[SIA] values measured by CASA. In MTT assays, N-9 exhibited significant cytotoxicity to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations (EC50 = 80 µM), with mean IC50[MTT] values of 64 µM, 58 µM, and 32 µM, respectively (selectivity index: SI = 0.8, 0.7, and 0.4; Fig. 6). By comparison, the IC50[MTT] values for CHET-NNIs 5a and 6a against normal human vaginal, ectocervical, and endocervical epithelial cells were well above their spermicidal EC50 values (IC50[MTT] values >500–1000 µM; SI range = >9–24). Thus, unlike N-9, which was spermicidal only at cytotoxic concentrations, CHET-NNIs showed high selectivity indices against these genital tract epithelial cells.
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Gel Formulations of Thiourea/Urea NNIs Lack In Vivo Mucosal Toxicity
To confirm the lack or reduced toxicity of CHET-NNIs when compared with N-9 against female genital tract epithelial cells, we tested compounds 5a and 5b either alone or in combination for their ability to induce mucosal toxicity in the rabbit model. Compounds 5a and 5b were formulated in a submicron particle-size gel-microemulsion comprising oil-in-water microemulsion and polymeric hydrogels that exhibit suitable spreading and retention and good compatibility with vaginal mucosa for intravaginal delivery of lipophilic dual-function CHET-NNIs. Table 4 summarizes the combined scores of histologic changes in three different regions of rabbit vaginal tissue (cervico-vagina, midvagina, and uro-vagina) after 10 days of intravaginal application of gel formulation containing 0.5–2.0% of compounds 5a and 5b or 1% 5a + 5b. The irritation observed was compared with vaginal tissues from rabbits given 4% N-9 gel for 10 days. Intravaginal administration of 0.5–2.0% of compound 5a, which is approximately 350–1400 times higher than its spermicidal EC50 and nearly 5 x 106 to 2 x 107 times higher than its in vitro anti-HIV IC50, did not cause significant vaginal irritation (mean individual scores 0–2; total scores 3–4; range 3–5, acceptable range for clinical trial) in any of the nine rabbits evaluated (Table 4). Similarly, repeated intravaginal administration of 0.5–2.0% of compound 5b, which is approximately 150–600 times higher than its spermicidal EC50, did not cause significant vaginal irritation in the rabbit model (mean individual scores 0–1; total score 1–3; range 1 to 4, acceptable range for clinical trial; Table 4). Notably, combination of gel formulation containing 1% compounds 5a and 5b had minimal effect on the vaginal mucosa (mean individual scores 0–1; total scores 1, acceptable for clinical trial; Table 4). Unlike N-9, gel microemulsions of lead thiourea/urea NNIs did not cause epithelial cell disruption in any of the 21 NZW rabbits examined. Only minimal to mild leukocyte influx, edema, or vascular congestion that could normally be expected to be seen following repeated intravaginal instillation of test agents was evident in the cervico-vagina, mid, and uro-vagina. Under identical experimental conditions, a 4% N-9 gel used as a standard control for evaluation of intravaginal microbicides [56] caused marginal vaginal irritation and inflammation characterized by extensive epithelial ulceration, edema, and leukocyte influx in all three regions of vaginal mucosa (total score 9; range 7–11, unacceptable range).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies on structure-based drug design by use of a computer docking procedure for the NNI binding pocket obtained from nine RT-NNI crystal structures revealed abundant sterically allowed usable space surrounding the pyridyl ring of NNIs [13, 27, 29, 32, 37]. Therefore, we strategically designed functional groups to obtain more potent anti-HIV agents with higher affinity for the NNI binding pocket of HIV RT. The use of our composite binding pocket led to the synthesis of novel thiourea NNIs active against HTLVIIIB and drug-resistant HIV-1 mutants A17 (Y181C), A17-variant (Y181C + K103N mutant RT), and RT-MDR (74V + 41L + 106A + 215Y mutant RT) at nanomolar concentrations [29–39] with the gain of spermicidal function at micromolar concentrations [40, 49]. Our earlier docking studies using the computer-generated model of the NNI binding pocket suggested that the replacement of the planar pyridyl ring of the PETT derivative, trovirdine, with a nonplanar cyclohexenyl ring, which occupies larger volumes, would better fit the spacious wing 2 region of the NNI binding pocket [29, 31]. As expected, the cyclohexenyl-substituted thiourea derivatives were more potent than trovirdine. When compounds 5a and 6a were docked into the NNI binding site of RT, they fit into the butterfly-shaped binding region, with one part of the molecule residing in wing 1 and the other in wing 2. The docking results indicated that the cyclohexenyl group of compound 5a or 6a is situated in the wing 2 region of the NNI binding pocket, providing contact with RT residues, including Y181 [31]. In addition, the cyclohexenyl group contains more ring hydrogens than the heterocyclic pyridyl ring and therefore has more hydrogen atom-mediated contacts and fewer carbon atom-mediated contacts with RT residues than trovirdine. Because the cyclohexenyl rings are conformationally more flexible than aromatic ring-containing thiourea derivatives, they have an added advantage by being able to fit an uncompromising binding pocket more effectively than conventional NNIs [13]. The observed potencies of compounds 5a and 6a against drug-resistant strains of HIV-1 are consistent with our hypothesis that a larger group that can favorably interact with the wing 2 region of the binding site is a desirable feature for inhibition of these mutants because Y181C and Y188C mutations result in a larger binding pocket [13].
In the present study, the dual-function structure-activity relationship studies of cyclohexenyl thiourea/urea compounds were performed by varying the heterocycle ring, thiourea moiety, and substitutions on the heterocycle ring. In addition to the CASA analysis, the inhibition of sperm transport within the cervical mucus provided a physiologically relevant indicator of SIA. In general, the pyridyl ring was preferred over thiazolyl or benzothiazolyl rings and substitution at the ortho position was preferred over meta or para substitution. The electronic nature of the ortho substituent was equally important for both antiviral and SIA. The data obtained with compounds 4–11 showed that the presence of electron-withdrawing groups at the ortho position was associated with marked increase in anti-HIV as well as SIA. The preferred groups were bromo, chloro, and methyl. Interestingly, the corresponding urea derivatives, although devoid of anti-HIV activity, were extremely rapid spermicidal agents, being 38-fold faster than thiourea NNIs. Obviously, the sulfur atom in the thiocarbonyl group is essential for antiviral activity whereas the oxygen atom contributes to a rapid SIA. The differences in the effects of thioureas and ureas can be attributed to the stronger electron-donor properties of the sulfur atom, facilitating the formation of associative, coordinative, and other intermolecular bonds [58]. The rapid SIA of urea analogs indicated that a combination of thiourea and urea derivatives could result in superior contraceptive activity of the broad-spectrum and dual-function thiourea NNIs. The anti-HIV activity of compound 5a was 700-fold more potent than the currently used detergent-based virucidal spermicide, nonoxynol-9. The SIA was twofold more potent than that of N-9 [45]. Results of our in vivo contraceptive efficacy studies in the rabbit demonstrated that coadministration of thiourea and urea compounds during artificial insemination significantly affected sperm fertilizing ability in vivo in hormonally primed female rabbits, indicative of the inability of cyclohexenyl thiourea/urea compound-exposed sperm to reach the site of fertilization. We hypothesize that coadministration of these two highly active antiviral/spermicidal microbicides, which act by two different mechanisms, should generate potent synergism for in vivo antiviral and contraceptive activities.
Although the structural requirement for the broad-spectrum anti-HIV activity of CHET compounds has been elucidated, the mechanism of SIA of these NNIs is less clear. Therefore, we developed a multivariate mathematical relationship (QSAR model) between a set of 20 physical and chemical properties (viz., topological, geometric, electronic, and polar surface descriptors) and SIA of each of the 15 NNIs. QSAR relationships of the 15 heterocyclic ring substituted NNIs revealed that, of the 20 physicochemical parameters that were evaluated using QSAR descriptors in the Cerius 2 program, four descriptors positively correlated with SIA. These parameters included the molecular refractivity index, hydrophobicity, atomic polarizability, and principal moment of inertia. The molecular refractivity index is a combined measure of molecular size and polarizability. In order to examine the relation between SIA and these physicochemical properties, multiple linear regression models were developed. A suitable QSAR model was obtained, showing not only statistical significance but also predictive ability. These descriptors allowed a physical explanation of electronic and molecular properties contributing to SIA of cyclohexenyl thiourea/urea compounds. The lower molar refractivity clearly reflects smaller steric effect that compounds 1 and 4 as well as compounds 5b and 6b have due to their unsubstituted thazolyl/pyridyl rings or substitution with an oxygen atom. These two factors contribute to the overall low polarizability and principal moment of inertia. Thiourea and bulkier substituted rings result in higher hydrophobicity (AlogP) values that contribute negatively to the SIA. Our results suggest that QSAR analysis is valuable in predicting the SIA of NNIs and will be of major importance for the design of new and more potent ant-HIV spermicides of this class.
We hypothesize that the dramatic reduction in time required for SIA by urea analogs versus thiourea NNIs is due to their metabolic oxidation to the spermicidal urea derivative by sperm. On the basis of the differential kinetics of sperm immobilization, the reactions of thioureas with reactive oxygen species [59–61], and our ability to generate the urea analog from thiourea NNIs by chemical oxidation, we propose three probable in vivo pathways for the conversion of thiourea NNIs to spermicidal urea analogs, as shown in Figure 7. In the nucleophilic substitution reaction pathway, the enol form of compound 5a first gets protonated either at the thiol group or on the electron-rich nitrogen center to yield a cation intermediate. In the second step, the nucleophilic attack by the hydroxyl anion on the cation leads to the elimination of hydrogen sulfide and the formation of urea derivative. In the free-radical pathway, the thiol group is susceptible to attack by a hydroxyl radical to form a thiol radical, which reacts with water molecules to continuously generate thiol radicals until the substrate is consumed to form the final urea derivative. In the singlet oxygen pathway, the singlet oxygen generated by sperm superoxidase dismutases can combine with the sulfur atom of thiourea to give a sulfoxide or sulfinic acid intermediate together with the formation of sulfur, sulphur dioxide, and ammonium sulfate. The free-radical pathway is the preferred pathway for the conversion of thiourea to urea by sperm. The urea analog of compound 5a can be generated by chemical oxidation via the free-radical pathway using N-bromosuccinamide and dioxane/water mixture (data not shown).
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The second objective of these studies was to determine the toxic effects, if any, resulting from repeated intravaginal application of lead thiourea/urea NNIs. The lead dual-function cyclohexenyl thiourea (5a) and urea (5b) compounds were formulated via a submicron-particle-size gel-microemulsion comprising oil-in-water microemulsion and polymeric hydrogels and tested in the rabbit vaginal irritation test for potential mucosal toxicity. In contrast with 4% N-9 gel, gel formulations of both compounds 5a and 5b either alone or in combination lacked mucosal toxicity after daily application for 10 days. Our results clearly demonstrated that the lead thiourea/urea compounds are not damaging to vaginal mucosa of the rabbit despite the fact that they are potent spermicidal agents when added to human or rabbit semen. Furthermore, the observed reduction in RT activity and p24 antigen production as well as SIA by these NNIs were not due to cytotoxic effects because cell viability of PBMC or normal human female genital tract epithelial cells was not affected adversely (IC50[MTA/MTT]
