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Antimicrobial Agents and Chemotherapy, January 2008, p. 225-236, Vol. 52, No. 1
0066-4804/08/$08.00+0     doi:10.1128/AAC.00972-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Comparative Evaluation of the Inhibitory Activities of a Series of Pyrimidinedione Congeners That Inhibit Human Immunodeficiency Virus Types 1 and 2{triangledown}

Robert W. Buckheit Jr.,1* Tracy L. Hartman,1 Karen M. Watson,1 Sun-Gan Chung,2 and Eui-Hwan Cho2

ImQuest BioSciences, Inc., 7340 Executive Way, Suite R, Frederick, Maryland 21704,1 Samjin Pharmaceutical Company, Ltd., Seoul, South Korea2

Received 26 July 2007/ Returned for modification 14 September 2007/ Accepted 22 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Seventy-three analogs of SJ-3366 (1-(3-cyclopenten-1-ylmethyl)-5-ethyl-6-(3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione) were synthesized and comparatively evaluated for their ability to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2 and for their ability to suppress virus entry and reverse transcription. These studies were performed to identify inhibitors with activity greater than that of the current lead molecule (SJ-3366) and to utilize structure-activity relationships (SAR) to define the chemical features of the pyrimidinedione congeners responsible for their efficacy, toxicity, and dual mechanism of action against HIV. The results of our SAR evaluations have demonstrated that the addition of the homocyclic moiety at the N-1 of the pyrimidinedione results in acquisition of the ability to inhibit virus entry and extends the range of action of the compounds to include HIV-2. In addition, the results demonstrate that analogs with a methyl linker between the homocyclic substitution and the N-1 of the pyrimidinedione had a greater number of highly active molecules than those analogs possessing ethyl linkers. Six molecules were identified with activity equivalent to or greater than that of SJ-3366, and five additional molecules with highly potent inhibition of reverse transcriptase and virus entry and possessing high efficacy against both HIV-1 and HIV-2 were identified. Six molecules exhibited significant inhibition of viruses with the highly problematic nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance engendering amino acid change K103N in the reverse transcriptase. These evaluations indicate that a new class of NNRTIs has been identified and that these NNRTIs possess highly potent inhibition of HIV-1 with an extended range of action, which now includes HIV-2.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over 25 million people have died since the first case of AIDS was identified in 1981, and the number of people living with human immunodeficiency virus (HIV) worldwide continues to expand—from 35 million in 2001 to 39.5 million in 2006 (46). Approximately 4.3 million people worldwide became newly infected with HIV in 2006, and an estimated 2.9 million human deaths were attributed to AIDS in 2006 (46). The rate of HIV infection and AIDS-related deaths is projected to increase over the course of the next decade with rapid expansion in Asia, Africa, and Eastern Europe. The epidemic is not limited to underdeveloped and low- to middle-income countries, as the rate of HIV infection has also risen in the United States and Western Europe (45).

Currently 27 antiviral therapies have been approved for use in HIV-infected patients (47), including nucleoside, nucleotide, and nonnucleoside reverse transcriptase inhibitors, protease inhibitors, a chemokine receptor-specific entry inhibitor, and a fusion inhibitor. The first drugs approved to treat HIV infection inhibited the specific activity of the virally encoded reverse transcriptase (RT), the viral enzyme essential for conversion of the viral RNA genome into a DNA provirus that integrates itself into the host genome (24, 28, 29). Two classes of RT inhibitors are currently marketed—nonnucleoside RT inhibitors (NNRTIs) and nucleoside or nucleotide RT inhibitors (NRTIs or NtRTIs) (15, 16, 19, 22, 27). Another approved and marketed class of HIV antiviral therapeutics inhibits the HIV protease, a viral enzyme required to process newly synthesized viral polyproteins into the mature viral gene products, enabling the virus to assemble itself into new infectious virus particles (18). A third class of HIV therapeutics inhibits infection by the virus at the stage of virus entry (21) and virus fusion to a target host cell (49). Clinical experience with all HIV agents has clearly demonstrated the ability of HIV to easily evade the antiviral effects of any monotherapeutic drug administration strategy through the rapid accumulation of amino acid changes in the targeted proteins—RT, protease, or envelope glycoproteins gp120 and gp41 (5). The highly error-prone HIV RT, with its lack of proofreading capability, generates significant heterogeneity within the highly related but nonidentical populations (or quasispecies) of viruses circulating in a patient (31). It is widely accepted that most drug-resistant viruses preexist within the population of viruses and are selected from within this heterogeneous environment upon application of selective drug pressure (reviewed in reference 5). In addition to the high levels of resistance possible to single therapeutic agents, each of the anti-HIV drugs employed thus far has had significant dose-limiting and long-term toxicities that render successful long-term therapy for HIV-associated disease difficult to achieve.

Nucleoside and nonnucleoside RT inhibitors and protease inhibitors have been effectively used in highly active antiretroviral therapies (HAART) to significantly reduce viral load in infected individuals for prolonged periods of time (33, 36). The utilization of HAART has dramatically changed the therapeutic landscape of HIV treatment, and the application of cocktails of antiretroviral agents is now the standard of care for HIV patients (50). The dramatic reduction in viral load and clinical improvements achieved with HAART are rigorous validation of the abilities of anti-HIV drugs to contain and manage HIV-associated disease and demonstrate that combinations of three or more anti-HIV agents—even when directed against only 2 of the putative 10 viral targets—is superior to single- or dual-drug therapy. Thus, the prevailing belief is that the addition of new anti-HIV agents to HAART regimens will provide additional clinical benefits. Despite its success, HAART suffers from the emergence of multidrug-resistant virus strains, toxicity, difficult treatment regimens, and inadequate pharmacology (bioavailability and tissue distribution) (12, 40, 44).

The initial discovery of the significant antiviral activity of the 2,4(1H, 3H)-pyrimidinedione analogs was obtained upon evaluation of SJ-3366 (1-(3-cyclopenten-1-ylmethyl)-5-ethyl-6- (3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione) (11). This molecule was reported as a highly potent and novel NNRTI with an extended range of action which included HIV type 2 (HIV-2), suggesting a new class of NNRTIs had been defined. SJ-3366 is a member of the HEPT class of molecules which originated with the discovery of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) (3, 32). HEPT, a 6-substituted acyclouridine derivative, is a novel, potent, and highly specific nonnucleoside inhibitor of HIV-1 RT. A number of HEPT analogs have been reported, such as 6-(3,5-dimethylbenzyl)-1-(ethoxymethyl)-5-ethyluracil (1) and 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)-thymine (7) which inhibit HIV-1 replication at nanomolar concentrations. 6-Benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442) (2, 17, 43) has been tested clinically but was subsequently withdrawn due to insufficient reduction in plasma viral load in phase III studies. Other HEPT analogs have also been described, including 6-[(3,5-dimethylbenzyl)-5-ethyl-1-(ethylthio)-methyl]-uracil (14), 6-[(3,5-dimethylphenyl)-selenyl]-1-(ethoxymethyl)-5-isopropyl- uracil (25), 3,4-dihydro-2-sec-butoxy-6-(3,5-dimethylbenzyl)-4-oxo-pyrimidine (30), and 5-ethyl-6-methyl-3-carbetoxy-4-[(3', 5'-dimethylphenyl)-thio]-pyridine-2(1H)-one (20).

Based on the high potency of SJ-3366, detailed antiviral evaluations have been performed to specifically examine the antiviral structure-activity relationships (SAR) of substituting a homocyclic moiety at N-1 of the pyrimidinedione. A number of compounds with cyclic substitutions were synthesized and analyzed, including those with cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, and 1-, 2-, or 3-cyclopenten-1-yl modifications. Medicinal chemistry resulted in the synthesis of 74 congeners, and the detailed synthetic pathways and HIV-1 inhibitory activity of the molecules have been recently reported (8). The results of these initial studies identified a series of potent 2,4(1H,3H)-pyrimidinedione analogs with subnanomolar antiviral activity against HIV-1 and no apparent cytotoxicity up to the limit of solubility in aqueous solution. In addition, all of these new pyrimidinediones with cyclic substitutions have an extended range of action which includes HIV-2, but not the closely related simian immunodeficiency virus (SIV). The results presented herein extend the initial evaluation of the pyrimidinedione series to include comparative evaluation in assays measuring activity against HIV-1 and HIV-2, as well as the abilities of the compounds to inhibit virus entry and reverse transcription. The results suggest that multiple compounds in the series possess biological activity similar to that of SJ-3366 (11), the lead compound in the pyrimidinedione series which is currently undergoing advanced preclinical development directed by U.S. Food and Drug Administration Investigational New Drug guidelines.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and viruses. The established human cells CEM-SS (23, 34, 35), HeLa-CD4-LTR-β-galactosidase (26), 174xCEM (41), and HL2/3 (13) and the laboratory-derived virus isolates HIV-1RF (38), HIV-1IIIB (38, 39, 42), and SIVMAC251 used in these evaluations were obtained from the NIAID AIDS Research and Reference Reagent Program. HIV-2ROD was obtained from Luc Montagnier (Pasteur Institut). The cells were maintained in RPMI 1640 medium or Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml).

Materials. Seventy-four pyrimidinedione congeners were obtained as dry powders from Samjin Pharmaceutical Co., Ltd. (Seoul, South Korea). The general structure of the compound is provided in Fig. 1. Crystalline stock materials were stored at –70°C and solubilized in 100% dimethyl sulfoxide. All stocks were diluted at least 400-fold prior to performing drug susceptibility assays. Wild-type HIV-1 RT was purchased from ChimerX, and the reverse transcriptase possessing the K103N mutation was obtained from Steven Hughes (4). Other materials required for the performance of RT inhibition assays and anti-HIV assays and for the growth and maintenance of established and fresh human cells have been described previously (4, 9).


Figure 1
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  		FIG. 1. General chemical structure of pyrimidinedione congeners. The general chemical structure of the pyrimidinedione congeners evaluated denotes the chemical modifications made in the compound at sites X, R1, R2, and R3, which were defined as highly active substitutions in the HEPT compound literature, and the specific modifications made at site R which involved the homocyclic substitutions evaluated herein. A methyl or ethyl linker was incorporated at site X2.

 
Antiviral assays. The HIV-1 and HIV-2 inhibitory activity of the compounds was evaluated in CEM-SS cells as described previously (6) in microtiter anti-HIV assays, which quantify the ability of a compound to inhibit HIV-induced cell killing. Quantification was performed using the tetrazolium dye 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT), which is metabolized to a colored formazan product by viable cells. Antiviral and toxicity data are reported as the quantity of drug required to inhibit 50% of virus-induced cell killing (50% effective concentration [EC50]) and the quantity of drug required to reduce cell viability by 50% (toxic concentration of an agent at which 50% of the target cells are nonviable [TC50]). The therapeutic index (TI) has been defined as the TC50/EC50 ratio.

RT inhibition assay. The abilities of test compounds to inhibit the enzymatic activity of recombinant HIV-1 RT (p66/51 dimer) were evaluated as previously described (4). Briefly, inhibition of purified HIV-1 RT was determined by the amount of 32P-labeled GMP incorporated into a nascent DNA strand, with a poly(rC)·oligo(dG)·oligo(rC-dG) homopolymer primer, in the presence of increasing concentrations of the target compounds. Identical methods were used to evaluate the activity of the test compounds against enzyme possessing the K103N mutation (6, 37, 48).

Inhibition of virus entry (MAGI assay) and cell-to-cell fusion. Serially diluted compound and virus (at a predetermined titer) were added to HeLa-CD4-LTR-β-galactosidase cells that had been placed in a 96-well flat-bottomed plate 24 h prior to assay initiation. Cells, compound, and virus were allowed to incubate for 2 h at 37°C and 5% CO2, and then the cells were washed to remove any unbound virus and test compound. Following the addition of tissue culture medium to the wells and a 48 h incubation, the cells were lysed and evaluated for β-galactosidase expression using a chemiluminescent substrate (Gal-Screen; Tropix). Controls for the multinuclear-activation galactosidase indicator (MAGI) assay included the attachment inhibitor Chicago Sky Blue (CSB) (Sigma-Aldrich, St. Louis, MO), the nucleoside RT inhibitor zidovudine (AZT) (Sigma-Aldrich), and the nonnucleoside RT inhibitor efavirenz (NIAID AIDS Research and Reference Reagent Program). These control compounds were evaluated in parallel with the test compounds to quantify antiviral activity from residual compound within the target cells following the washing steps. The control assays included a 2 h pretreatment of the target cells with compound, followed by washing prior to infection as well as evaluation in parallel with the test compounds. The fusion inhibition assay was performed identically except that HL2/3 cells expressing viral gp120 and gp41 and constitutively expressing Tat were cocultivated with the HeLa-CD4-LTR-β-galactosidase cells and test or control compounds were not washed away after the initial 2 h incubation. Chemiluminescence was evaluated 48 h after coculture as described previously (10).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Structure-activity relationship evaluations. Based on the results of in vitro evaluations performed on the pyrimidinedione SJ-3366 (1-(3-cyclopenten-1-ylmethyl)-5-ethyl-6-(3,5-dimethylbenzoyl)-2,4(1H,3H)-pyrimidinedione), a series of 73 additional congeners was synthesized and evaluated for efficacy against both HIV-1 and HIV-2. These studies were designed to identify more potent inhibitors than SJ-3366 for continued development, and SAR evaluations were performed to further understand the molecular and structural features of the pyrimidinediones responsible for their activity and ability to inhibit both RT and virus entry. The general chemical structure of the pyrimidinedione molecules is presented in Fig. 1 with the sites of chemical modification denoted as R, R1, R2, R3, and X. Most of the previous analog synthesis for members of the HEPT family has entailed incorporation of acyclic moieties at N-1 of the pyrimidinedione, and these previously reported studies have identified a strong SAR for substitutions in the HEPT molecule. Position 6 of the pyrimidinedione and positions 3 and 5 of the benzoyl, phenoxy, or phenylthio group appear to be of particular importance for realization of antiviral activity. Thus, we limited the modifications at R1, R2, R3, and X (Fig. 1) to those which have been previously associated with favorable geometry or antiviral activity. Our primary interest was to examine the effects of introduction of various homocyclic moieties at the N-1 position of the pyrimidinedione on the antiviral activities of the compounds. Two groups of congeners were synthesized; they differed with regard to the presence of a methyl or ethyl linker (at position X2 in Fig. 1) between the N-1 of the pyrimidinedione and the homocyclic substitution (R group in Fig. 1). Each compound was then evaluated in a variety of biological screening assays to quantify the relative therapeutic activity of each. The assays used to characterize the biological activity of the compounds included in vitro cytopathic effect (CPE) inhibition assays in CEM-SS cells infected with either HIV-1 or HIV-2, inhibitory activity in biochemical RT inhibition assays, and cell-based virus entry inhibition assays. In addition, the entire series of molecules was evaluated for the ability to inhibit cell-to-cell fusion using the coculture of HeLa-CD4-LTR-β-galactosidase cells with HL2/3 cells and for their ability to inhibit SIV in cell-based CPE inhibition assays in 174xCEM cells. All of the congeners failed to inhibit fusion and SIV replication. The data obtained upon evaluation of the entire series of molecules in each of the biological assays are presented in Table 1 (compounds with a methyl linker) and Table 2 (compounds with an ethyl linker).


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  		TABLE 1. Structure-activity relationship evaluations for compounds possessing a methyl linkera

 

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  		TABLE 2. Structure-activity relationship evaluations for compounds possessing an ethyl linkera

 
Discovery of highly active inhibitors of HIV-1 and HIV-2. Of the pyrimidinedione congeners synthesized, all 74 molecules were determined to inhibit HIV-1 and all but 6 compounds inhibited HIV-2, albeit with variable relative potency. Activity against HIV-1 ranged from 0.0003 µM to 5.9 µM among compounds with the methyl linker and from 0.01 µM to 1.49 µM among the compounds with the ethyl linker. For HIV-2, activity of the compounds ranged from 0.1 to 26 µM among the compounds with the methyl linker and from 0.2 to 28.2 µM among the compounds possessing an ethyl linker. Among the entire series of compounds, 17 congeners were determined to be highly active inhibitors of HIV-1, defined as those molecules exhibiting a therapeutic index greater than 50,000. Of these 17 compounds, 16 possessed a methyl linker and only 1 possessed an ethyl linker. Of the 17 highly active compounds, 10 possessed TI values between 50,000 and 200,000, 7 possessed TIs ranging from 200,000 to 2,000,000, and one compound possessed a TI greater than 2,000,000. An overview of the data obtained from this detailed SAR evaluation is presented in Table 3 and includes the range of activities observed in each of the screening assays for the entire series of molecules.


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  		TABLE 3. Summary of antiviral activities of pyrimidinedione congeners

 
Of the 74 molecules evaluated, the compounds highlighted in bold font in Table 4 were defined as being the most potent inhibitors of HIV-1 replication based on their overall TI. These congeners include compounds 19, 38, 49, 62, and 63 among those with the methyl linker and compound 78 with the ethyl linker. These six compounds have TIs ranging from 491,237 to 2,551,345. In each case, the "toxic" concentration of the compound was dictated by the precipitation of the compound in the aqueous tissue culture medium employed in the assays rather than by overt cell killing in the in vitro assays performed.


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  		TABLE 4. Summary of the most active compounds in the pyrimidinedione seriesa

 
Although not among the most potent of the pyrimidinedione inhibitors based on TI values, five additional congeners exhibited significantly high levels of activity in each of the biological inhibition assays performed. These compounds are indicated by underlining in Table 4. The five compounds (compounds 14, 15, 16, 40, and 45) inhibited HIV-1 (at 0.8, 0.3, 6.0, 5.0, and 5.0 nM, respectively), HIV-2 (at 0.1, 0.5, 0.6, 0.3, and 0.5 µM, respectively), virus entry (at 9.0, 9.0, 6.0, 80, and 9.0 nM, respectively), and wild-type RT (18, 3, 16, 95, and 79 nM, respectively). Compounds 16 and 40 remained active when tested against either the wild-type or K103N-possessing RT. The five compounds exhibited TIs of 25,455, 37,607, 214,954, 165,294, and 5,924, respectively.

Evaluation of the activity of the compounds against HIV-1 and HIV-2 based on direct comparison of their respective EC50s, rather than TIs, resulted in the identification of the most active inhibitors against each virus without consideration for cellular toxicity or compound precipitation in aqueous solution (highlighted in italic font in Table 4). Against HIV-1, compound 49 was found to be the most active agent with an EC50 of 0.1 nM, while compounds 21, 15, 19, 23, 14, 47, 39, 17, 62, and 63 had EC50s below 2 nM. Against HIV-2, compounds 14, 19, 41, 43, and 49 were found to be the most active inhibitors (EC50 of 100 nM for each) (Table 4).

Direct comparison of the compounds highlighted in Table 4 shows that the possession of a cyclopropyl, cyclobutyl, phenyl, or 1- or 3-cyclopenten-1-yl group were associated with active compounds with the methyl linker and that only the 2- or 3-cyclopenten-1-yl was associated with activity among the compounds with the ethyl linker. Compounds with cyclopentyl or cyclohexyl were less active compounds.

Most active inhibitors of virus entry and reverse transcription. Of the 74 molecules evaluated, the compounds presented in Tables 1 and 2 could be ranked in order of relative activity in the MAGI-based entry and biochemical RT inhibition assays in addition to their overall ability to inhibit HIV-1 and HIV-2.

In the entry inhibition assays utilizing HIV-1, compounds possessing a methyl linker had entry inhibitory activity which ranged from 0.006 µM to 2.93 µM, while those with the ethyl linker had activity ranging from 0.009 µM to 2.62 µM. With regard to their ability to inhibit the entry of HIV-1 into target cells in the MAGI assay, compounds 14, 15, 16, 18, 33, 41, 44, 45, 46, 49, 77, and 79 exhibited inhibitory concentrations below 10 nM (range, 6 to 12 nM). To address the possibility that the results obtained in the MAGI assay might be due in part to the presence of residual internalized compound following the 2-hour exposure to compound and virus prior to washing, Chicago Sky Blue, AZT, and efavirenz were evaluated in parallel as control compounds. In these experiments, cells were exposed to the control compounds, washed to remove extracellular compound, and then infected with HIV-1 in the absence of compound, and the antiviral activity due to the residual compound was quantified at 48 h. In these assays (data not shown), CSB and AZT were inactive (>10 µg/ml and >0.5 µM, respectively), while efavirenz had limited antiviral activity (0.2 to 0.4 µM) at a concentration approximately 1,000 times higher than its antiviral EC50 value. Evaluation of SJ-3366 (compound 62) in the same pretreatment control assay yielded results similar to those found with CSB and AZT with no antiviral activity defined at a high test concentration of 0.5 µM. The activity of efavirenz is much greater if the compound is present during the 2-hour attachment inhibition period, increasing 10-fold to 0.02 to 0.04 µM. If efavirenz is not washed away in this MAGI assay, the EC50 is defined as approximately 0.0001 µM. The activity of SJ-3366 (compound 62) is much greater in the MAGI assay, indicating that entry inhibition is likely occurring as opposed to residual activity involving inhibition of HIV-1 reverse transcription. SJ-3366 becomes more active, like efavirenz, if the compound is left on continuously during the MAGI assay. Conversely, CSB is equally active when present during the 2-hour attachment inhibition assay or if left on continuously in the MAGI assay. Thus, in light of the high potency of the pyrimidinedione congeners against HIV-1, it should be noted that the MAGI assay results presented in Tables 1 and 2 using live HIV-1 may include both entry inhibition and RT inhibition activities, since HIV-1 is sensitive to both means of inhibition. In our hands, HIV-2 was not able to infect the MAGI cells, and thus, the entry inhibitory activity of the pyrimidinediones must be quantified directly from the CPE assay results. In our mechanistic assays, including a single round of replication, time of compound addition assays, the pyrimidinediones act to inhibit HIV-2 at an early time point in a manner similar to attachment and fusion inhibitors and lose activity when the compound is added at time points after entry has occurred and prior to the onset of HIV-2 reverse transcription (R. W. Buckheit, Jr., unpublished data). Thus, these assays demonstrate that the pyrimidinediones act only as inhibitors of HIV-2 entry with no effect on HIV-2 RT. In similar assays with HIV-1 and a single round of replication, the addition of the pyrimidinediones to the cultures can be delayed identically to the HIV-1 RT inhibitors (AZT, efavirenz, and nevirapine) without any loss of activity, confirming that the primary mechanism of action against HIV-1 is inhibition of reverse transcription.

In the biochemical HIV-1 RT inhibition assays (Tables 1 and 2), the activity of the methyl series of inhibitors ranged from 0.003 to 15.6 µM against wild-type enzyme in assays using the poly(rC)·oligo(dG) template primer system. Compounds with the ethyl linker possessed activity ranging from 0.011 to 2.36 µM in the wild-type enzyme assay. Compound 15 exhibited the highest level of activity in the HIV-1 RT inhibition assay (3 nM), while compounds 37, 43, 17, 16, 14, 20, 21, and 49 exhibited activity in the RT inhibition assay at concentrations below 20 nM. As expected based on assays with a single round of replication, no inhibitory activity was detected against purified HIV-2 RT up to the highest concentration tested in the assays for any of the test molecules (data not shown), confirming that inhibition of HIV-2 occurs solely through entry inhibition.

In light of the fact that the pyrimidinediones appear to be highly novel nonnucleoside inhibitors of viral RT, evaluations were next performed to determine whether any of the congeners possessed activity against resistant enzyme possessing the highly problematic K103N mutation (K103N-RT). In general, the activity of the methyl linker- and ethyl linker-substituted compounds tested against the K103N-RT ranged from 0.067 to >306 µM and 1.5 to >271 µM, respectively. Inhibitory activity against the K103N-RT was generally greater with the methyl series of compounds compared with the ethyl series, where the majority of the compounds became essentially inactive against the mutant enzyme. Six molecules were found to inhibit this resistant enzyme at levels only slightly reduced from their level of activity against wild-type enzyme (less than a twofold change in 50% inhibitory concentration [IC50]). Compounds 16, 22, 23, 28, 40, and 46 all remained active against the K103N-RT compared to their activity against the wild-type enzyme. Compound 40 had the greatest inhibitory potential against the K103N-RT with an IC50 of 0.19 µM.

Categorization of functional activities of SAR compounds. The activity of molecules within the series of compounds in the various antiviral assays yielded a variety of antiviral phenotypes as shown in Table 5. Although it must be kept in mind that all of the congeners were active in all of the assays performed, albeit with different relative potencies, cutoff values for "active" compounds were selected to differentiate the most active compounds in each cell-based or biochemical assay. The threshold values used to define the "active" compounds for these assays are provided in Table 5, footnote b. Active molecules, represented by plus signs in Table 5, exhibited activity greater than the threshold values. Compounds with activity that fell below the threshold value are denoted by minus signs in Table 5. These evaluations clearly show the inability to associate the molecular features of the congeners with activity in specific assays and demonstrate that comparative evaluation of activity in each of the assays results in a different rank order for the compounds. Interestingly, the overall level of activity of a compound against HIV-1 is independent of their rank order ability to inhibit virus entry and/or reverse transcription as can be seen with the results for compounds 38 and 63. Compounds with high-level inhibitory potential against HIV-1 may be among the most active in all assays performed (compound 16), some of the assays performed (compounds 49, 19, and 38), or none of the assays performed (compound 63).


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  		TABLE 5. Functional categories of anti-HIV inhibitors

 
Comparative evaluation of the EC99s of the most potent congeners. Based on the wide range of antiviral properties of the series of pyrimidinediones and the inability to associate specific structural features of the molecules with overall activity in the various in vitro assays, additional evaluations were performed in CEM-SS cells to define the concentration of the compound which reduced the replication of HIV-1 and HIV-2 to below detectable levels. Dose-response curves were generated over a range of concentrations, and the percent protection from HIV-induced cytopathic effects was quantified at each concentration to define the lowest concentration of the compound which was able to reduce virus replication to this undetectable level. Compounds 16, 17, 18, 19, 20, 22, 40, 45, 49, 56, 62, and 63 were evaluated in this study, and the data obtained are presented in Table 6. These evaluations suggest that EC99s ranged from 1 nM to 1 µM concentration levels with compounds 18, 19, 45, and 49 reducing virus replication to undetectable levels in these cell cultures at the lowest concentrations (0.01, 0.001, 0.0003, and 0.001 µM, respectively). The remaining compounds reached the undetectable limit at approximately 1 µM. Compounds 17, 40, 45, and 49 achieved these EC99s against HIV-2 at concentrations of 1, 0.3, 1.0, and 1.0 µM, respectively.


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  		TABLE 6. Concentrations of selected pyrimidinediones required for reduction of virus replication below detectable limits in cell culturea

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently described the synthesis and HIV-1 and HIV-2 activities of a series of pyrimidinedione inhibitors related to HEPT (8). Based on the activity of the lead pyrimidinedione, SJ-3366 (compound 62) (11), analogs were synthesized in which the moiety at the N-1 of the pyrimidinedione was modified with homocyclic groups while maintaining the characteristics of the most active reported HEPT analogs at other critical residues (position 6 of the pyrimidinedione and position 3 and 5 of the benzoyl, phenoxy, or phenylthiol group). Results of HIV testing of these novel pyrimidinediones indicate that a new class of NNRTIs has been identified which possess highly potent inhibition of HIV-1 with an extended range of action which now includes HIV-2. The results that were initially obtained on the series of pyrimidinedione congeners suggested that the activity of the compounds against HIV-1 and HIV-2 was most pronounced with the cyclopropyl, phenyl, and 1- or 3-cyclopenten-1-yl modifications at the N-1 site of the pyrimidinedione, but the SAR evaluations were not able to differentiate the specific structural moieties required for activity against HIV-1 versus HIV-2. It can be assumed based on the data provided in the literature that the expansion of the range of action to include significant HIV-2 inhibition, and hence introduction of the second entry inhibition mechanism of antiviral action against both HIV-1 and HIV-2, is a direct result of the homocyclic addition at the N-1 site of the pyrimidinedione. The anti-HIV-2 and entry inhibition activity of all reported HEPT analogs is minimal to nonexistent. Herein we have expanded our evaluations to look at the antiviral properties of the entire series of pyrimidinedione congeners in assays relevant to their antiviral activity against HIV-1 and HIV-2 and have prioritized the most potent and broadly acting members of the series for further development.

The results of our SAR studies with the 74 pyrimidinedione inhibitors has resulted in the identification of the following general properties of the series of inhibitors. (i) The analogs with methyl linkers (compounds 14 through 63) had a greater number of highly active molecules (17 active compounds) than the analogs possessing ethyl linkers (1 active compound among compounds 64 though 87). (ii) Six molecules were identified with activity equivalent to or greater than the lead compound SJ-3366 (compound 62). (iii) Five molecules were identified with highly potent inhibition of RT and virus entry and possessing high efficacy against both HIV-1 and HIV-2. (iv) Six molecules exhibited significant inhibition of viruses with the highly problematic NNRTI resistance engendering amino acid change K103N in the RT. (v) Analogs have been identified in which relative potency in the various assays (anti-HIV-1, anti-HIV-2, RT inhibition, and virus entry inhibition) could be disassociated from one another with various effects on the overall level of anti-HIV-1 activity.

The most active compounds in the pyrimidinedione series were identified in each functional assay, and the results of these comparative studies have provided significant additional insight regarding the structural requirements of the compounds for activity in each of the assays. As mentioned, compounds with the methyl linker exhibited greater levels of activity than those with the ethyl linker, suggesting that the distance and spatial positioning of the homocyclic substitution from the pyrimidinedione is an important determinant of activity. Compound 78, however, was found to be one of the most potent inhibitors of the entire series and possesses the ethyl linker, suggesting that the nature of other structural features of the molecule are also highly important or can compensate for the increased distance of the homocyclic substituent from the pyrimidinedione. Substituents at N-1 of the pyrimidinedione had the greatest impact on the activity of the molecules. Based on the lack of HIV-2 activity with acyclic substitutions at N-1, the homocyclic substitutions resulted in the extension of the range of action of these compounds to include HIV-2, but not the closely related SIV. Therefore, the homocyclic substitution must also be responsible for introduction of the ability of the pyrimidinediones to inhibit the entry of both HIV-1 and HIV-2. Among the most active agents, the preferred substitutions at the N-1 site include cyclopropyl, phenyl, and 1- or 3-cyclopenten-1-yl. Cyclobutyl, cyclopentyl, cyclohexyl, and 2-cyclopeneten-1-yl substitutions generally resulted in a loss of activity of the compounds, with the exception of compound 78, which possesses the 2-cyclopenten-1-yl change along with the ethyl linker.

Two subsets of compounds within the pyrimidinedione series appear to be especially informative regarding the effects of substitutions at the various sites of the molecule. These subsets include compounds 14 through 19 and compounds 51 through 63. Compounds 14 through 19 possess the cyclopropyl modification at N-1 with the methyl linker. Compound 19 is by far the most active with a TI of greater than 2.5 million, but it has the same EC50 versus HIV-1 as does compound 15 (0.5 nM versus 0.3 nM, respectively), which possesses a TI of 37,607. The significant toxicity of compound 15 appears to be explained by the phenylthio substituent. Comparison of compounds 17 and 18 to compound 19 suggests that the presence of an ethyl versus an isopropyl at R1 of the pyrimidinedione and a phenoxy versus a benzoyl has a dramatic effect on antiviral activity. Compounds 17 and 18 have TIs that are, respectively, 200-fold and 20-fold less than that of compound 19, resulting from reduced efficacy (compound 18 [ethyl at R1]) and increased toxicity (compound 17 [phenoxy]). The complex interplay between the chemical modifications in the molecules is particularly evident when comparing compounds 17, 18, and 19 to the activity of compound 16. Though less efficacious compared to compound 19 and more efficacious compared to compound 18, the nontoxic nature of compound 16 results in a relatively high TI of 214,954. Thus, efficacy and toxicity determinants cannot be simply defined from the SAR data. In comparatively evaluating activity observed in other assays, the phenylthio group in compounds 14 and 15 seems to lead to compounds with better RT inhibitory activity and the greatest inhibition of HIV-2 and virus entry. The phenylthio group, however, confers much greater cytotoxicity, yielding narrow TIs for these compounds. Although highly active against both HIV-1 and HIV-2, compound 19 is the least active against RT and is an intermediate inhibitor of virus entry. These results suggest that the mechanistic activity against RT and virus entry may act synergistically to yield higher overall levels of antiviral activity in cell-based virus replication assays.

A greater diversity of changes in the chemical structure are present in the series of compounds ranging from compound 50 through 63 with the 3-cyclopenten-1-yl modification at the N-1 of the pyrimidinedione and the methyl linker. Similar to what was found with compound 19, a benzoyl or phenoxy group yielded compounds with the greatest activity (compounds 61 to 63 and 55 to 57, respectively). Among these six compounds, those with the ethyl rather than isopropyl substitution at R1 were the most active of the series (compound 56 and compound 62). Compounds 58 through 60 provide evidence that substituting H at the R2 and R3 sites or a methyl or H at the R1 site eliminated activity completely. Compound 53 shows that the addition of an isopropyl group at R1 also is not tolerated as an active compound. This series again demonstrates that the presence of the phenylthio group yields more toxic compounds but that a complex interplay between EC50 and TC50 values occurs among the molecules. Further evaluation of the effect of the ethyl and methyl linkers can be directly evaluated within this series, where compounds were synthesized with the 3-cyclopenten-1-yl group. The addition of the ethyl linker essentially eliminates the activity of all directly comparable molecules in the series (compounds 52, 53, 56, 57, 62, and 63 directly relate to compounds 82 through 87).

The complete evaluation of the activities of the test compounds presented herein suggests a complex interplay of chemical groups in that each of the sites has direct impact on the activity of the compound against HIV-1 or HIV-2, on the toxicity of the compound to human cells, and on the functional activity in the entry and RT inhibition assays. Thus, compounds within the series may be selected for further development based on overall antiviral activity (with five compounds being highly active in all assays performed) or based on their prioritized level of activity in inhibiting virus entry or reverse transcription. Additionally, several molecules in the series were determined to have equivalent inhibitory activity when tested against enzyme with the highly problematic K103N mutation which confers resistance to the NNRTIs, suggesting a therapeutic strategy of targeting resistant virus in addition to wild-type virus. The results of our studies and prioritization suggest that a small number of these pyrimidinediones could be selected for further evaluation and potential clinical development based on their efficacy, toxicity, and solubility properties, as well as their antiviral profiles as described in the assays performed here. These compounds would include compounds 19, 49, 62, and 63. Each of these molecules possesses anti-HIV activity that is greater than that observed for other reported HEPT-like inhibitors which have been tested and reported (2, 17, 43). One of the most active HEPT-like compounds evaluated, MKC-442, progressed to phase III human clinical trials (43).

We have also evaluated data obtained in cell culture to define the concentration of each of the highly active compounds that would suppress virus replication and virus-induced CPEs below detectable limits as another means of prioritizing these compounds for further development. In these evaluations, compounds 18, 19, and 49 appear well suited on the basis of these estimated concentrations, suggesting that one of these three may be a better lead compound than compound 62. As inhibitors of both virus entry and reverse transcription and with potency in the subnanomolar range, these inhibitors would likely be strong clinical candidates.

Though the pyrimidinedione congeners clearly act as nonnucleoside inhibitors of HIV-1 RT, their second mechanism of action is being characterized further. Our data (unpublished data) have demonstrated that the primary mechanism of inhibition of HIV-2 occurs early in assays with single round of replication, time of compound addition assays (similar to other attachment and fusion inhibitors), and the compounds have no inhibitory activity whatsoever against purified HIV-2 RT. However, the lack of activity of the compounds against SIV or in fusion inhibition assays indicates that the compounds act via a more specific entry inhibitory mechanism which must be elucidated. We have shown that the pyrimidinediones do not directly inhibit the interaction of gp120 with CD4 and do not prevent the attachment of virus to target cells and that activity is equivalent with CCR5- and CXCR4-tropic viruses. Cell-based studies with both HIV-1 and HIV-2 suggest that the pyrimidinediones recognize a conformational structure that is formed upon culture of the virus with target cells at 4°C (a prefusion conformational target) and that inhibition by this secondary mechanism may involve both envelope and Gag determinants (unpublished data). The selection and genetic characterization of drug-resistant viruses to the highly active pyrimidinediones provide additional mutational evidence of a role for both envelope and Gag (unpublished data). These additional development activities, including studies of the detailed mechanism of action, resistance selection evaluations, combination interactions with other approved HIV compounds, and studies to evaluate their potential utility as topical anti-HIV microbicides are in progress. Clinically, the successful development of these compounds through pharmacology and toxicology and human clinical trials directed by U.S. Food and Drug Administration Investigational New Drug guidelines could potentially result in a single small molecule of significant potency that could replace two classes of inhibitors currently being used as primary or salvage therapy (NNRTI plus Fuzeon) and thus may offer a significant benefit to clinical treatment adherence.


   ACKNOWLEDGMENTS
 
This study was supported by a grant (HMP-97-D-1-0003) to Samjin Pharmaceuticals Co., Ltd., from the Good Health R&D Project, Ministry of Health & Welfare, Republic of Korea.

Some components of the research were performed by us in the laboratories of Southern Research Institute (Frederick, MD) and TherImmune Research Corporation (Gaithersburg, MD).


   FOOTNOTES
 
* Corresponding author. Mailing address: ImQuest BioSciences, Inc., 7340 Executive Way, Suite R, Frederick, MD 21704. Phone: (301) 696-0274. Fax: (301) 696-0381. E-mail: rbuckheit{at}imquest.com Back

{triangledown} Published ahead of print on 29 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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