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Biology of Reproduction 63, 196-205 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Novel Thiourea Compounds as Dual-Function Microbicides

Osmond J. D'Cruza, Taracad K. Venkatachalamb, and Fatih M. Uckun1,c

a Drug Discovery Program, Departments of Reproductive Biology, b Chemistry, and c Virology, Parker Hughes Institute, St. Paul, Minnesota 55113

ABSTRACT

Sexually active women represent the fastest growing human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we previously reported the synthesis of novel nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase with sperm-immobilizing activity (SIA). To gain further insight into the structure-function relationship controlling these two properties of NNIs, we have rationally designed and synthesized 30 novel thiourea compounds and examined them for dual-function, anti-HIV and spermicidal activity. Twelve of the 30 thiourea compounds exhibited potent anti-HIV activity in the nanomolar range (IC50 = <1–9 nM). Nine of the 30 thiourea derivatives exhibited both anti-HIV and spermicidal activity. Among the phenyl ring-containing thioureas, the 2-fluoro (HI-240) -substituted and 2-chloro (HI-253) -substituted derivatives exhibited potent anti-HIV activity (IC50 = <1 nM) with SIA (EC50 = 70 µM and 147 µM). Among the alicyclic ring-containing thioureas, the 5-bromo (HI-346) and 5-chloro (HI-445) functionalized cyclohexenyl ring-substituted thioureas were the most potent dual-function spermicides (EC50 = 42 and 57 µM), with anti-HIV activity at nanomolar range (IC50 = 3 nM). Unlike nonoxynol-9 (N-9), none of the potent dual-function thiourea compounds were cytotoxic to normal human vaginal, ectocervical, and endocervical epithelial cells at spermicidal concentrations. We conclude that as potent anti-HIV agents with SIA and reduced cytotoxicity when compared with N-9, the phenyl-substituted and cyclohexenyl-substituted thiourea derivatives, especially compounds HI-253 (N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea), and HI-445 (N-[2-(5-chloropyridinyl)]-N'-[2-(1-cyclohexenyl)ethyl-thiourea) show unique clinical potential to become the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring HIV by heterosexual vaginal transmission.

sperm, sperm motility

INTRODUCTION

Human immunodeficiency virus (HIV)—the etiologic agent of acquired immunodeficiency syndrome (AIDS)—is the fastest growing cause of death in women of reproductive age [1–3].

Worldwide, heterosexual transmission accounts for 90% of all HIV infections in women [2, 4]. Currently, an estimated 14.1 million women worldwide are infected with HIV, representing 44% of all adult infections. Considering that the AIDS pandemic is still in its infancy on a global scale, this evolving demographic situation warrants urgent attention, particularly for the adolescent population. Therefore, effective strategies are needed to reduce heterosexual and perinatal HIV transmission. In the absence of an effective prophylactic anti-HIV therapy or vaccine, new emphasis has been placed on the development of intravaginal microbicidal agents capable of reducing the transmission of HIV [5, 6]. In addition, prophylactic contraception is fundamentally important in HIV-infected women for prevention of HIV transmission and pregnancy, especially because 80% of women with AIDS are of childbearing age [7].

At present, all commercially available spermicidal microbicides have detergent ingredients that disrupt cell membranes [8, 9]. The most widely used vaginal spermicide, nonoxynol-9 (N-9) has been shown because of its membrane-disruptive properties to damage the cervicovaginal epithelium [10–12], cause an acute inflammatory tissue response [13], alter vaginal microflora [14, 15], and enhance the risk of promoting opportunistic infections in the genitourinary tract [16]. Such opportunistic infections are known to enhance the susceptibility of the ectocervical epithelium and the endocervical mucosa to HIV infection [17–19]. Despite its ability to inactivate HIV in vitro, the reported failure of N-9 to prevent heterosexual vaginal transmission of HIV in clinical settings in addition to its adverse effects on the cervicovaginal epithelium and vaginal microflora has prompted the search for new female-controlled microbicides that are both more effective and safer than N-9 [20–24]. Unlike the detergent-based microbicides that target cell membranes, the intravaginal or intrarectal use of topical formulations of anti-HIV drugs such as nonnucleoside inhibitors (NNIs) might be an effective approach for preventing the sexual transmission of HIV. These inhibitors of viral replication have been proposed by the World Health Organization as candidates for intravaginal microbicides to inhibit HIV replication in mucosal cells [25, 26]. Inasmuch as physiological fertilization is dependent on the ability of ejaculated sperm to swim, bind the zona pellucida, and penetrate the egg, all of which are primarily dependent on sperm motility, adding spermicidal function to potent anti-HIV drugs could be an effective way to curb heterosexual HIV transmission as well as prevent conception.

Design of potent inhibitors of HIV-1 reverse transcriptase (RT) has been a focal point in translational AIDS research [27–30]. The NNIs are a diverse set of compounds that include tetrahydroimidazobenzodiazepinethione, also known as TIBO, compounds [31]; 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine, also known as HEPT, derivatives [32–35]; bis(heteroaryl)piperazine, also known as BHAP, analogues [30]; 2'–5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'', 2''-oxathiole-2'', 2''-dioxide) pyrimidine, also known as TSAO [36]; dihydroalkoxybenzyloxopyrimidine, also known as DABO [37, 38]; and phenethylthiazolylthiourea, also known as PETT, derivatives [39–42]. They interfere with the activity of viral RT by binding to a specific allosteric site of HIV-1 RT near the polymerase site and severely limit the conformational flexibility needed for its function, rendering the viral protein inactive [43–49]. A number of crystal structures of RT complexed with NNIs have been reported, and such structural information has provided the basis for further derivatization of NNI aimed at maximizing binding affinity to RT [44–50].

The success of NNIs for the clinical treatment of AIDS has led us to the computer-aided design and chemical synthesis of mechanism-based antiretroviral agents that also exhibit spermicidal activity. Recently, we described the structure-based design and synthesis of novel NNIs by generating a novel computer model in which a composite binding pocket was constructed from nine individual crystal structures of RT-NNIs complexes [51, 52]. This computer-docking procedure revealed abundant sterically allowed usable space surrounding the pyridyl ring of the thiourea compound, trovirdine. Therefore, we strategically designed functional groups to obtain more potent anti-HIV agents with higher affinity for the NNI-binding pocket of HIV-1 RT that also exhibit spermicidal activity [53]. In an attempt to gain further insight into the structural determinants controlling these two properties, we have synthesized 30 novel thiourea compounds and examined them for anti-HIV activity as well as for spermicidal activity. Here, we report the identification of several dual-function thiourea compounds with potent anti-HIV activity and sperm-immobilizing activity (SIA). The discovery of the potent dual-function properties of phenyl- and cyclohexenyl-substituted thiourea compounds is particularly relevant for curbing vaginal transmission of HIV by leukocytes and sperm as the infectious cells in the semen [54, 55].

MATERIALS AND METHODS

Reagents and Instrumentation

All chemicals were purchased from Aldrich Chemical Corporation (Milwaukee, WI). Anhydrous acetonitrile and N,N-dimethylformamide were transferred to reaction vessels via cannula under nitrogen. All reactions were carried out under nitrogen. Proton (1H), carbon (13C), and fluorine (19F) nuclear magnetic resonance spectra (NMR) were recorded on a Varian Oxford 300-MHz spectrometer (Varian Associates, Palo Alto, CA) using an automated broadband probe. All NMR spectra were recorded in CDCl3. 13C NMR spectra were recorded using the proton-decoupling technique. 19F NMR spectra were recorded in CDCl3, and a 1% solution of trifluoroacetic acid in water was used as an internal standard in a fused capillary tube. Fourier transform infrared (FT-IR) spectra were recorded with a FT-Nicolet Model Protege 460 instrument (Nicolet Instrument Corp., Madison, WI). Mass spectra analyses were performed by using a model G2025A Hewlett Packard (Wilmington, DE) matrix-assisted laser desorption spectrometer in the molecular ion detection mode. The matrix used was cyanohydroxycinnamic acid. Ultraviolet (UV) spectra were recorded from Beckmann model 3DU 7400 UV-Visible spectrophotometer (Beckmann Instruments, Fullerton, CA) using a cell-path length of 1 cm. Elemental analysis was performed by MicroLab Inc. (Atlanta, GA). Column chromatography was performed using silica gel. The solvents used for elution varied depending on the compound and included one of the following: ethyl acetate, methanol, chloroform, hexane, methylene chloride, and ether.

Synthetic Scheme for Substituted Thiourea Derivatives

The names of the 31 thiourea compounds synthesized and tested in this study are listed in Table 1. The synthesis of 30 novel thiourea compounds as inhibitors of HIV-1 RT was based on a computer model in which a composite binding pocket was constructed from 9 individual crystal structures of RT-NNI complexes [51, 52]. Modeling studies for rational drug design included the construction of a composite NNI-binding pocket for nine RT-NNI crystal structures, the analyses of surface complementarity between NNIs and RT, and application of inhibitory constants (Ki values) combined with a docking procedure involving the novel thiourea compounds [51, 52]. Our computational approach allowed the identification of several ligand derivatization sites for the generation of more potent dual-function thiourea compounds. Our detailed analysis of trovirdine [51] revealed multiple sites that can be used for the incorporation of larger functional groups. In our composite binding pocket, the docked trovirdine molecule showed abundant sterically allowed usable space surrounding the pyridyl ring and the ethyl linker and near the 5'-bromo position. We hypothesized that addition of strategically designed functional groups should yield more potent anti-HIV agents with SIA. Therefore, we synthesized a series of thiourea compounds in which one of the nitrogen atoms of the thiourea was attached to a phenyl, heterocyclic, or alicyclic moiety through a ethyl bridge, and the other nitrogen atom was attached to a 5'-bromo- or 5'-trifluoromethyl-substituted or an unsubstituted pyridyl ring.


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  		TABLE 1. Thiourea compounds synthesized and tested in this study

For the synthesis of novel 5'-substituted pyridyl thiourea compounds, additional substitutions on the phenyl ring included either fluoro, chloro, bromo or methoxy groups at ortho (2,5), meta (3), or para (4) positions on the phenyl ring. Figure 1 shows the general synthetic scheme followed as detailed in previous reports [51, 52, 56, 57]. Trovirdine (N-[2-(pyridyl)]-N'-[2–5-bromopyridinyl)-thiourea) was synthesized according to literature procedure [42].



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  		FIG. 1. Synthetic scheme for phenyl, heterocyclic, and alicyclic ring-containing substituted thiourea compounds

Determination of the Partition Coefficients (Pa)

The partition of the test compounds between n-octanol and water as a measure of their lipophilicity was determined by taking the UV absorption of each compound in n-octanol, water-saturated n-octanol (1:1), and n-octanol-saturated water (1:1). The UV absorption maxima were determined by selecting a wavelength with the maximum absorption peak in the water-saturated octanol layer and the corresponding peak in the octanol-saturated water. Three absorption spectra were obtained for each compound. The partition coefficient (Pa) was calculated as the ratio of the compound concentration present in the lipid phase to the compound concentration present in the aqueous phase.

In Vitro Assays of Anti-HIV Activity

The HTLVIIIB strain of HIV-1 was propagated in CCRF-CEM cells, and the virus stocks obtained from cell-free supernatants of infected cells were titered using MT-2 cells [58, 59]. Cell-free supernatants were harvested and frozen in 1-ml aliquots at -70°C. Titration of the stock virus was performed using MT-2 cells, and the cytopathic effect of the virus used in this study was typical of HIV-1 on MT-2 cells [58, 59].

For in vitro assays of the anti-HIV-1 activities of phenyl, heterocyclic, and alicyclic ring-substituted thiourea compounds, normal peripheral blood mononuclear cells (PBMCs) from HIV-1 negative donors were cultured for 72 h in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) 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 gentamicin, 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. Stock solutions (10 mM) of thiourea compounds were prepared in dimethylsulfoxide (DMSO). Cells were cultured for 7 days in 96-well microtiter plates (100 µl/well; 2 x 106 cells per milliliter, triplicate wells) in the presence and absence of various concentrations (0.001 to 100 µM) of the thioureas. 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 p24 antigen, as previously described [59, 60]. 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 [56–58]. The plates were read on an ELISA reader (Molecular Devices, Sunnyvale, CA) at 650 nm, and p24 levels, expressed in nanograms per milliliter, were calculated against known standards supplied by Coulter/Immunotech, Inc. Percentage 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).

Each of the 31 thiourea compounds was tested for RT inhibitory activity against purified, recombinant HIV-1 RT by the cell-free Quan-T-RT system (Amersham Corp., Arlington Heights, IL), which utilizes the scintillation proximity assay principle [61]. The anti-HIV activity was expressed as the IC50 value, calculated from the dose-response curves, and defined as the drug concentration that decreases p24 antigen production in HIV-1-infected PBMCs or the HIV-1 rRT activity by 50%.

Assays of SIA

To evaluate the effect of each of the 31 thiourea compounds on human sperm motility and kinematics, highly motile fractions of sperm were prepared from donor semen (n = 8) by discontinuous (90–45%) gradient centrifugation using Enhance Plus medium (Conception Technologies, San Diego, CA) and the swim-up method [53, 62]. The swim-up method was used after density gradient centrifugation, washing, and resuspension of sperm in Biggers, Whitten, and Whittingham medium (BWW)-3% BSA. Pooled swim-up fraction was washed once prior to spermicidal assay. All donor semen specimens were obtained after informed consent and in compliance with the guidelines of the Parker Hughes Institute Institutional Review Board. Pooled motile sperm (>=10 x 106 per milliliter) prepared from three to six donors were suspended in 1 ml of BWW containing 25 mM Hepes (Irvine Scientific, Santa Ana, CA), and 0.3% BSA in the presence and absence of serial twofold dilutions of test substance (500 to 15.6 µM) in 0.5% DMSO. The stock solutions of thioureas were prepared in DMSO (100 mM) and diluted in assay medium to yield the desired concentrations (up to 1000 µM). Corresponding volume of DMSO (either 0.5% or 1%) was added to control tubes. After 3 h incubation at 37°C, the percentage of motile sperm was evaluated by CASA [53, 62–64]. The motility percentages were compared with those in sham-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 decreases the proportion of motile sperm by 50%), calculated from three independent experiments.

Sperm Kinematic Parameters

For computer-assisted sperm analysis (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 five to eight fields per slide were scanned for analysis using a Hamilton Thorne Integrated Visual Optical System, version 10, instrument (Hamilton Thorne Research Inc., Danvers, MA). Each field was recorded for 30 sec. The computer calibrations were set at 30 frames at a frame rate of 30 per second. 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; and threshold straightness at 80%; and magnification factor, 1.95.

The sperm kinematics parameters that were determined included numbers of motile and progressively motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head), straight-line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken), beat-cross frequency (BCF; frequency of sperm head crossing sperm average path), the amplitude of lateral sperm-head displacement (ALH; the mean width of sperm head oscillation), and the derivatives straightness (STR = VSL/VAP x 100) and linearity (LIN = VSL/VCL x 100; departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled.

Assay for Cell Viability

The potential cytotoxicity of two phenyl ring- (HI-240 and HI-253), one heterocyclic ring- (HI-207) and two cyclohexenyl (HI-346 and 445) ring-containing, most potent dual-function thiourea compounds, in comparison to N-9, against normal human vaginal, ectocervical, and endocervical epithelial cells (Clonetics Corporation, San Diego, CA), was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay [53, 65]. Briefly, exponentially growing vaginal, ectocervical, and endocervical epithelial cells were seeded into 96-well plates at a density of 2 x 104 cells per 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. 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% sodium dodecyl sulfate 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 at 540 nm were measured with 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 percentage survival was calculated using the following formula: % survival = live cell number in test group/live cell number in control group 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.

Statistical Analysis

Results are presented as the mean or as mean ± SD values from independent measurements. Nonlinear regression analysis was used to find IC50 and EC50 values from the concentration-effect curves using the PRISM, version 2.0, software program (GraphPad, San Diego, CA).

RESULTS

Anti-HIV and SIA of Phenyl Thiourea Compounds

Thiourea compounds were assayed for their ability to inhibit HIV-1 replication in normal human PBMCs infected with the HIV-1 strain HTLVIIIB (reported as IC50[p24]), as well as for their RT-inhibitory activity in cell-free assays using purified recombinant HIV-1 RT (reported as IC50[rRT]). The SIA of thiourea compounds was evaluated by CASA (reported as EC50). The lipophilicity of the compounds was measured by their partition coefficients between n-octanol and water (reported as Pa).

We tested the effect of ring substitution and functionalization of the pyridyl moiety of the thiourea derivative, trovirdine. Table 2 lists the anti-HIV and SIA profiles of 18 phenyl ring-substituted thiourea compounds, in which one of the nitrogen atoms of the thiourea is attached to one of 16 different substituted phenyl rings (R1) through an ethyl bridge, and the other nitrogen atom is attached to a 5'-bromo- or 5'-trifluoromethyl-substituted (R2) pyridyl ring. Among the 16 substituted-phenylthiourea derivatives, functionalization of the phenyl ring with 2,5-dimethoxy (HI-236), 2-fluoro (HI-240), 3-fluoro (HI-241), and 2-chloro (HI-253) was associated with enhanced anti-HIV activity (IC50[p24]) = <1 nM; IC50[rRT] = 0.10 to 0.70 µM) when compared with the unsubstituted phenylthiourea compound (HI-275) (IC50[p24]) = 4 nM, and IC50[rRT] = 1.25 µM. In addition, phenyl ring-containing thiourea compounds substituted with 3-methoxy (HI-239), 4-fluoro (HI-242), and 4-chloro (HI-255) functional groups were also potent inhibitors of HIV-1 (IC50[p24]) = 1 to 9 nM; IC50[rRT] = 0.4 to 6.4 µM. By comparison, the unsubstituted 5'-trifluoromethyl phenylthiourea (HI-308) was inactive, and its alkoxy- (HI-310) and halo- (HI-311) substituted derivatives were less potent (IC50[p24]) values of 0.08 and 0.09 µM and IC50[rRT] values of 2 and 0.6 µM, respectively. Thus, the substitution of the phenyl ring with various functional groups had a major impact on the anti-HIV activity of unsubstituted parent compound (HI-275). Specifically, functionalization of 5-bromo pyridyl-substituted thiourea derivatives with alkoxy or halo substitutions on the phenyl ring led to superior anti-HIV activity.


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  		TABLE 2. Effect of 18 phenyl-ring substituted thiourea compounds on p24 antigen production in HIV-infected PBMC, enzymatic activity of HIV-1 rRT, and human sperm motility.

Exposure of the highly motile fraction of human sperm to 12 of the 18 substituted phenylthiourea derivatives listed in Table 2 did not affect sperm motility, even at concentrations as high as 500 µM. Notably, halo-functionalized, phenyl-substituted thiourea derivatives with 2-fluoro (HI-240), 2-chloro (HI-253), and 4-chloro (HI-255) functional groups showed, in addition to their potent anti-HIV activity (IC50[p24] = <1 nM), also spermicidal activity, with EC50 values of 147, 70, and 160 µM, respectively (Table 2). Thus, halo substitution of the phenyl ring was essential for the SIA of phenylthiourea compounds.

The SIA of these dual-function thiourea derivatives was not related to their partition coefficient values (Table 2). The spermicidal compounds HI-240, HI-253, HI-255, and HI-275 had low Pa values (range, 1.04 to 3.50). The lack of a correlation between the Pa values and the biological activity of phenyl-substituted thiourea derivatives demonstrates that these properties are not dependent on the lipophilicity of the compounds.

Anti-HIV and SIA of Heterocyclic Thiourea Compounds

Next, we synthesized a series of thiourea derivatives in which one of the nitrogen atoms of the thiourea is attached to one of five different heterocyclic substituents (R1) through an ethyl bridge and the other nitrogen atom is attached to a 5-bromo or 5-trifluoromethyl substituted pyridyl ring. Table 3 lists the anti-HIV and SIA profiles of five heterocyclic ring-substituted thiourea derivatives with and without 5-bromo or 5-trifluoromethyl functionalization (R2) on the pyridine ring. When compared with trovirdine (listed as HI-142), both the piperdil (HI-172) and piperazinyl (HI-258)-containing thiourea derivatives were more potent in inhibiting the replication of HIV-1 strain HTLVIIIB in human PBMC, with IC50 values of <1 nM and 2 nM, respectively, but they lacked SIA (EC50 values = > 500 µM). Among the eight heterocyclic thiourea derivatives tested, the 5-bromo-functionalized thioureas (HI-172, HI-258 and trovirdine) were more potent than trifluoromethyl-substituted (HI-309) or unsubstituted thioureas (HI-207). Unlike trovirdine, which lacked SIA, the unsubstituted pyridyl thiourea (HI-207) exhibited anti-HIV activity (IC50[p24] = 0.27 µM), as well as SIA (EC50 = 279 µM). In addition, the more lipophilic piperidinyl derivative of trifluoromethyl functionalized thiourea (HI-344) exhibited SIA (EC50 = 131 µM), but it lacked anti-HIV activity (IC50[rRT] = >100 µM; Table 3).


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  		TABLE 3. Effect of eight heterocylic-ring-substituted thiourea compounds on p24 antigen production in HIV-infected PBMC, enzymatic activity of HIV-1 rRT, and human sperm motility.

Anti-HIV and SIA of Alicyclic Thiourea Compounds

In addition to the compounds described above bearing phenyl and heterocyclic rings with and without 5' functionalization, we also examined the effect of alicyclic ring-containing thiourea compounds. Table 4 lists the anti-HIV and SIA profiles of five alicyclic-substituted thiourea derivatives. Replacement of the pyridyl ring of trovirdine with a cis-myrtanyl (HI-444) resulted in loss of anti-HIV activity, whereas replacement with a cyclohexenyl ring did not. The unsubstituted cyclohexenyl ring-containing inhibitor (HI-541) lacked SIA (EC50 = >500 µM). However, functionalization at the 5'-position of the pyridyl ring of cyclohexenyl ring-containing thioureas with a bromine or a chlorine atom led to a significant increase in anti-HIV activity as well as gain of spermicidal function. Cyclohexenyl ring-substituted thiourea derivatives with either 5-bromo (HI-346) or 5-chloro (HI-445) functionalization were the most potent dual-function agents, with IC50[p24] values of 3 nM and EC50 values of 42 µM and 57 µM, respectively. Both of these compounds also inhibited HIV-1 RT activity with IC50 values of 0.6 µM and 0.5 µM, respectively. By comparison, the anti-HIV activity of the trifluoromethyl-substituted cyclohexenyl thiourea derivative (HI-347) was 26-fold less active, and its SIA was twofold to threefold less active than those of halo-functionalized thiourea derivatives. Thus, the potency order for SIA of cyclohexenyl substituted thiourea derivatives was as follows: bromo > chloro > trifluoromethyl. Spermicidal cyclohexenyl ring-containing thiourea derivatives had higher Pa values (6.3–11.5) than the inactive cis-myrtanyl-substituted (2.1) thiourea derivative (HI-444), suggesting that the biological activity of cyclohexenyl thiourea derivatives may in part be related to their increased lipophilicity.


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  		TABLE 4. Effect of four alicyclic-ring-substituted PETT derivatives on p24 antigen production in HIV-infected PBMC, enzymatic activity of HIV-1 rRT, and human sperm motility.

All three cyclohexenyl ring-containing thioureas with additional substitutions on the 5'-position of the pyridyl ring induced a concentration-dependent SIA. (Fig. 2). The effective 100% inhibitory concentration (EC100) was obtained at 0.25 mM for compounds HI-346 and HI-445 and at 1 mM for HI-347. The concentration-dependent sperm motility loss induced by cyclohexenyl-substituted thiourea derivatives was also associated with significant changes in the movement characteristics of the surviving sperm, particularly with respect to VCL, VAP, and VSL. The representative sperm kinematic parameters of cyclohexenyl-substituted, thiourea-treated sperm versus concentration is shown in Figure 3. The decreases in VSL and VCL or VSL and VAP were similar in magnitude. Therefore, values for LIN of the sperm tracks and the STR of the swimming pattern remained relatively constant. Also, the BCF and the ALH were relatively stable as the proportion of motile sperm declined during the linear phase of motility loss. The sperm motion parameters of control sperm showed no significant changes (data not shown).



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  		FIG. 2. Concentration-dependent inhibition of human sperm motility by cyclohexenyl ring-containing substituted thiourea derivatives. Highly motile fractions of sperm were incubated with increasing concentrations (15.6–1000 µM) of HI-346, HI-347, and HI-445 or 1% DMSO in the assay medium, and the percentage of motile sperm were evaluated by CASA. Each data point represents the mean from three independent experiments



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  		FIG. 3. Concentration-dependent effect of cyclohexenyl ring-containing substituted thiourea derivatives on human sperm motion kinematics. Highly motile fractions of sperm were incubated with increasing concentrations of substituted cyclohexenyl thioureas or with 1% DMSO in the assay medium, and their effects on VAP, VSL, and VCL were evaluated by CASA. Results are expressed as the mean ± SD for three separate experiments

Selective Spermicidal Activity of Dual-Function Thiourea Compounds

In order to determine whether the observed SIA was due to nonspecific membrane damaging effect of dual-function thiourea derivatives, we tested the potential cytotoxicity of five most potent dual-function thiourea derivatives versus N-9 against confluent monolayers of normal human vaginal, ectocervical, and endocervical epithelial cells using the MTT assay. Cells were exposed to these compounds at concentrations ranging from 7.8 µM to 1000 µM for 3 h. The concentration-dependent cell survival curves for dual-function thiourea derivatives versus N-9 for these cells measured by the MTT assay were compared with the SIA measured by CASA (Table 5). In MTT assays, N-9 exhibited significant cytotoxicity to normal human vaginal, ectocervical and endocervical epithelial cells at spermicidal concentrations (EC50 = 81 µM) with mean IC50[MTT] values of 64 µM (selectivity index [SI] = 0.7), 58 µM (SI = 0.7), and 32 µM (SI = 0.3), respectively. By comparison, the IC50[MTT] values for the most potent spermicidal dual-function thiourea derivatives against normal human vaginal, ectocervical, and endocervical epithelial cells were well above their spermicidal EC50 values (SI range, >6.8 to >23.8). Thus, unlike N-9, which was spermicidal only at cytotoxic concentrations, dual-function spermicidal thiourea derivatives showed high selectivity indices against these cells. Thus, spermicidal dual-function thiourea derivatives were significantly less active against reproductive-tract epithelial cells.


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  		TABLE 5. Effect of spermicidal thiourea compounds on the cell viability of normal human vaginal, ectocervical, and endocervical epithelial cells as quantitated by MTT assay

DISCUSSION

We identified several thiourea compounds as dual-function spermicidal agents with anti-HIV activity. Among the 30 novel thiourea derivatives that were rationally designed, synthesized, and evaluated for anti-HIV activity and SIA, the ortho-fluoro, ortho-chloro, and para-chloro-substituted phenyl ring-containing derivatives and cyclohexenyl ring-containing derivatives were the most potent dual-function agents. In particular, thiourea compounds HI-253 (N-[2-(2-chlorophenethyl)] N'-[2-(5-bromopyridyl)-thiourea), HI-346 (N-[2-(5-bromopyridil)]-N'-[2-(1-cyclohexenyl)ethyl]-thiourea), and HI-445 (N-[2-(5-chloropyridil)]-N'-[2-cyclohexenyl)ethyl-thiourea) were identified as potential lead compounds for the development of clinically useful dual-function anti-HIV spermicides.

The molecular mechanism of the spermicidal activity exhibited by thiourea compounds is unknown. We previously reported that aryl phosphate derivatives of zidovudine with bromo-methoxy functionalization on the thymine ring also acquire potent SIA in addition to their potent anti-HIV activity [65–70]. Because the ethyl linker and the bromo substitutions on the pyridyl ring are present in 21 of 31 thiourea derivatives investigated, the unique SIA of halo-substituted phenylthiourea derivatives is most likely due to their halo substituents in the phenyl ring. It was demonstrated that substituting ortho-fluoro group on the phenyl ring with a chloro group further enhanced the spermicidal potency of phenyl-substituted thiourea derivatives. Similarly, the unsubstituted cyclohexenyl ring-containing thiourea lacked SIA, whereas 5' functionalization on the pyridyl ring of the cyclohexenyl ring-substituted thiourea derivatives resulted in potent dual-function SIA irrespective of the 5'-substitutions on the pyridyl ring. The lack of SIA of unsubstituted cyclohexenyl ring-containing thiourea clearly demonstrated that in addition to the presence of a cyclohexenyl ring, further functionalization of the pyridyl ring is essential for the gain of spermicidal function, the potency of which can be modulated by various functional groups.

In order to determine whether the biological activities of the thiourea compounds vary with their lipophilicities, their partition coefficients between n-octanol and water were determined as an index of the rate of transfer of the molecule across the cell membrane and into the cell. The observed lack of a correlation between the Pa values and the biological activity of phenyl-substituted thiourea derivatives suggests that the dual-function properties of phenyl- and heterocyclic-ring-substituted thiourea derivatives are not dependent on the partition coefficient of the molecule. On the other hand, the cyclohexenyl substitution enhances the lipophilic character that might contribute to an increased penetrability of cyclohexenyl ring-substituted thiourea derivatives through the plasma membrane of sperm.

The ectocervical epithelium and endocervical mucosa are highly susceptible to HIV-1 infection because of lesions or hormonal liquefaction and other changes that occur before, during, and after ovulation [14–18]. Our studies clearly demonstrated that unlike the detergent spermicide, N-9, the dual-function thiourea derivatives were selectively spermicidal when compared with N-9, which was cytotoxic to human vaginal, ectocervical, as well as endocervical epithelial cells at spermicidal concentrations. Thus, the cervicovaginal epithelial cells appear to be vulnerable to cytotoxic insults from the detergent-type spermicides. Therefore, the dual-action spermicidal thiourea derivatives that do not elicit any detergent-type membrane toxicity may offer significant clinical advantages. The lack or reduced toxicity of spermicidal thiourea derivatives to human vaginal, ectocervical, and endocervical epithelial cells when compared with N-9 warrant the further development of these dual-action compounds as a new class of vaginal contraceptives. In the acidic environment of the vagina, these spermicidal thiourea compounds would be stable as protonated species because of the presence of a pyridine ring with a basic nitrogen atom. On the basis of the lack or reduced cytotoxicity of these spermicidal thioureas, we believe that the dual-function thiourea compounds, when administered intravaginally, will not have adverse systemic side effects. Future preclinical studies will carefully examine the effects of intravaginally applied dual-function thiourea compounds on the short-term and long-term safety and reproductive health of test animal species.

The synthesis of novel thiourea compounds as dual-function anti-HIV agents with spermicidal function represents a significant step forward in the development of new microbicides for curbing heterosexual HIV transmission. In addition to their potent anti-HIV activity against the wild-type RT, compounds HI-346 and HI-445 exhibited potent antiviral activity against NNI-resistant as well as multidrug-resistant strains of HIV-1 [52, 57]. Virucidal spermicides that are also active against NNI-resistant and multidrug resistant strains of HIV-1 will be a tremendous advantage for the development of broad-spectrum microbicides because a high percentage of newly infected individuals in the United States harbor NNI-resistant mutants. The promising results reported herein illustrate that dual-function NNIs, particularly HI-253, HI-346, and HI-445, show unique clinical potential to become the active ingredients of new, female-controlled, topical virucidal vaginal contraceptives for women who are at high risk for acquiring HIV by heterosexual transmission. The further development of these dual-function NNIs as novel vaginal virucidal spermicides may provide the basis for a new strategy aimed at prevention of the sexual transmission of HIV while providing effective fertility control for women.

FOOTNOTES

First decision: 11 February 2000.

1 Correspondence: Fatih M. Uckun, Parker Hughes Institute, 2665 Long Lake Rd., Suite 330, St. Paul, MN 55113. FAX: 651 697 1042; fatih_uckun{at}mercury.ih.org Back

Accepted: February 25, 2000.

Received: December 31, 1999.

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