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Antimicrobial Agents and Chemotherapy, February 2007, p. 611-615, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00444-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antiviral Activities of Novel 5-Phosphono-Pent-2-en-1-yl Nucleosides and Their Alkoxyalkyl Phosphonoesters

Hyunah Choo,^1, James R. Beadle,¹ Earl R. Kern,² Mark N. Prichard,² Kathy A. Keith,² Caroll B. Hartline,² Julissa Trahan,¹ Kathy A. Aldern,¹ Brent E. Korba,³ and Karl Y. Hostetler^1*

Department of Medicine, Division of Infectious Disease, University of California, San Diego, La Jolla, California 92093-0676, and the Veterans Medical Research Foundation, San Diego, California 92161,¹ Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama 35233,² Division of Molecular Virology and Immunology, Georgetown University Medical Center, Rockville, Maryland 20850³

Received 10 April 2006/ Returned for modification 26 May 2006/ Accepted 31 October 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Three acyclic nucleoside phosphonates are currently approved for clinical use against infections caused by cytomegalovirus (Vistide), hepatitis B virus (Hepsera), and human immunodeficiency virus type 1 (Viread). This important antiviral class inhibits viral polymerases after cellular uptake and conversion to their diphosphates, bypassing the first phosphorylation, which is required for conventional nucleoside antivirals. Small chemical alterations in the acyclic side chain lead to marked differences in antiviral activity and the spectrum of activity of acyclic nucleoside phosphonates against various classes of viral agents. We synthesized a new class of acyclic nucleoside phosphonates based on a 5-phosphono-pent-2-en-1-yl base motif in which the oxygen heteroatom usually present in acyclic nucleoside phosphonates has been replaced with a double bond. Since the intrinsic phosphonate moiety leads to low oral bioavailability and impaired cellular penetration, we also prepared the hexadecyloxypropyl esters of the 5-phosphono-pent-2-en-1-yl nucleosides. Our earlier work showed that this markedly increases antiviral activity and oral bioavailability. Although the 5-phosphono-pent-2-en-1-yl nucleosides themselves were not active, the hexadecyloxypropyl esters were active against DNA viruses and hepatitis B virus, in vitro. Notably, the hexadecyloxypropyl ester of 9-(5-phosphono-pent-2-en-1-yl)-adenine was active against hepatitis B virus mutants resistant to lamivudine, emtricitabine, and adefovir.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The acyclic nucleoside phosphonates (ANPs) are an important class of antiviral drugs that are approved for treatment of viral infections, including infections with cytomegalovirus (cidofovir [Vistide]), hepatitis B virus (adefovir dipivoxil [Hepsera]), and human immunodeficiency virus type 1 (HIV-1; tenofovir disoproxil fumarate [Viread]) (11, 12). To show antiviral activity, ANPs must (i) undergo intracellular activation to the diphosphate, (ii) compete with endogenous deoxynucleotide triphosphates for binding to the viral polymerase, (iii) incorporate into the nascent DNA, and (iv) terminate viral replication. ANPs possess an enzymatically stable phosphonomethyl ether which, unlike conventional nucleoside antivirals, makes them independent of the first intracellular phosphorylation step.

In general, antiviral nucleosides and ANPs consist of a base moiety and a carbohydrate mimic. Structural features of the carbohydrate mimic influence the ability of the nucleoside analog to participate in the antiviral mechanism. For example, the 5'-triphosphates of stavudine (2',3'-didehydro-3'-deoxythymidine) and abacavir [Ziagen; (1S,4R)-cis-4-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol] are potent inhibitors of HIV replication, and the presence of a double bond in the sugar moiety appears to play a role in binding to the HIV reverse transcriptase (9). Likewise, potent antiviral activity is found in ANPs possessing a phosphonomethoxyethyl side chain; changes in the acyclic side chain or in the nucleoside base modulate activity and the antiviral spectrum against various virus classes (15).

We now describe the antiviral evaluation of a new series of acyclic nucleoside phosphonate analogs, the 5-phosphono-pent-2-en-yl (PPen) nucleosides, which incorporate a double bond into the phosphonomethyl ether side chain (Fig. 1) (9a). Since ANPs, as a class, are not readily taken up by cells because of their anionic character, we also prepared and evaluated hexadecyloxypropyl esters of each PPen nucleoside, because this strategy has been shown to increase the antiviral activities of cidofovir (4, 22, 23, 36), 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] (5), and several other classes of ANPs (33, 35). The compounds and their hexadecyloxypropyl esters were evaluated for in vitro activity against cytomegalovirus (CMV), herpes simplex virus (HSV), vaccinia virus (VV), cowpox virus (CV), HIV-1, varicella-zoster virus (VZV), wild-type and drug-resistant hepatitis B virus (HBV), and Epstein-Barr virus (EBV).

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FIG. 1. Structure of PPen nucleosides compared with stavudine. The 5-phosphono-pent-2-en-1-yl analogs of the bases (B) adenine, thymine, guanine, cytosine, and uracil and their hexadecyloxypropyl esters were synthesized and evaluated for antiviral activity.

(Portions of this paper were presented in abstract form at the International Conference on Antiviral Research, 11 to 14 April 2005, Barcelona, Spain, and the 2005 Frontiers in Drug Development for Viral Hepatitis meeting, 11 to 15 December 2005, Kohala Coast, Hawaii.)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synthesis of PPen nucleosides. The PPen nucleosides and their hexadecyloxypropyl esters were synthesized as reported previously (9a). Proof of structure and purity (>98%) of all compounds was confirmed by ¹H and ³¹P nuclear magnetic resonance, electrospray ionization mass spectrometry, and thin-layer chromatography (silica gel plates with visualization by UV light Phospray [Supelco, Bellefonte, PA] and charring at 400°C).

Cells and viruses. Human foreskin fibroblast (HFF) cells were prepared as primary cultures and used in the CMV, VZV, VV, and CV assays. CMV strain AD-169 and VZV strain Ellen were propagated using standard virological techniques as reported previously (27, 38). VV strain Copenhagen and CV strain Brighton were kindly provided by John W. Huggins (Department of Viral Therapeutics, Virology Division, U.S. Army Medical Research Institute of Infectious Disease, Frederick, MD). Working stocks of these viruses were propagated in Vero cells obtained from the American Type Culture Collection (Manassas, VA). MRC-5 human lung fibroblast cells were propagated as described previously (4, 19).

Plaque reduction assays in HFF cells. The plaque reduction assays for CMV, VV, CV, and VZV were performed as described previously (5, 27, 37, 38), and standard methods were used to determine the compound concentration required to reduce plaque formation by 50% (EC₅₀).

ELISA for EBV. EBV assays were done as described previously (38). An enzyme-linked immunosorbent assay (ELISA) was performed on cells fixed with 95% ethanol-acetic acid, rinsed with phosphate-buffered saline, and incubated with a monoclonal antibody to EBV viral capsid antigen (Chemicon, Temecula, CA) followed by an incubation with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G1 (Southern Biotechnology, Birmingham, AL), and the EC₅₀ and 50% cytotoxic concentration (CC₅₀) values were determined (38).

DNA reduction antiviral assays for activity against HSV-1 in vitro. The antiviral activities were determined against HSV type 1 (HSV-1) by DNA reduction with MRC-5 human lung fibroblast cells using HSV-1 DNA probes as described previously (19).

Neutral red uptake assay for cytotoxicity. HFF or MRC-5 cells were seeded into 96-well tissue culture plates. MRC-5 cells were plated sparsely and allowed to expand during drug treatment. With MRC-5 cells, the medium was replaced after 24 h with minimal essential medium containing 2% fetal bovine serum (FBS), and drug was added to the first row and then diluted serially fivefold from 100 µM to 0.03 µM. With HFF cells, the wells were allowed to become confluent prior to addition of drug-containing medium. The assays were done, and the CC₅₀ values were determined as described previously (4, 5, 36-38).

HBV antiviral analysis. Confluent cultures of 2.2.15 cells were maintained on 96-well flat-bottom tissue culture plates in RPMI 1640 medium with 2% FBS as previously described (26). Cultures were treated with nine consecutive daily doses of the test compounds (six for each test concentration on two replicate plates). The culture medium was changed every day with medium containing the indicated concentrations of the test compounds. HBV DNA levels were assessed by quantitative blot hybridization 24 h after the last treatment. Cytotoxicity was assessed by uptake of neutral red dye and semiquantitative analysis of the absorbance of internalized dye at 510 nM (A₅₁₀) 24 h following the last treatment (three cultures per test concentration) (26). Activity against lamivudine-resistant (2) and adefovir-resistant (3) HBV mutants was determined in a 5-day assay using a transient-transfection method in Huh7 cells, and cytotoxicity was determined as previously described (21).

HIV assays in MT-2 cells. MT-2 cells (AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health) were maintained in RPMI 1640 supplemented with 10% FBS (JRH Biosciences, Lenexa, Kans.) The antiviral activity of each compound was determined by inoculating MT-2 cells with HIV-1_LAI at a multiplicity of infection of 0.001 50% tissue culture infective doses/cell, followed by incubation in the presence of threefold serial drug dilutions (three wells per dilution) as previously described (13). The antiviral activity of each compound is expressed as the EC₅₀, which is the concentration required to inhibit p24 antigen production by 50%.

MT-2 cytotoxicity. Cytotoxicity was assessed in rapidly dividing MT-2 cells incubated with drug for 72 h and harvested. Flow count beads (Beckman Coulter, Miami, FL) were added to the cell suspension followed by propidium iodide staining and analysis using an Epics Elite flow cytometer (Beckman Coulter). The CC₅₀ was calculated from the cell counts and viability (13).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The PPen nucleosides were tested against CMV, HSV, VV, CV, VZV, and EBV (Table 1). The unmodified PPen nucleosides were inactive, except for PPen-G, which showed low but measurable activity against CMV and VZV. However, when esterified with the hexadecyloxypropyl (HDP) group, substantial activity was noted in most cases. HDP-PPen-G was the most broadly active PPen-N, showing low micromolar EC₅₀ values ranging from 1.8 to 12.2 µM against CMV, HSV-1, VV, CV, and VZV. In a single experiment, HDP-PPen-A inhibited replication of EBV with an EC₅₀ of 0.1 µM. HDP-PPen-T had submicromolar EC₅₀s against HCMV and HSV-1 and an EC₅₀ of 3 µM against VZV but was not active against cowpox and vaccinia viruses in vitro. HDP-PPen-U did not inhibit any of the viruses against which it was tested.

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TABLE 1. Antiviral activities and cytotoxicities of PPen nucleosides and their hexadecyloxypropyl esters in vitroa

In MT-2 cells infected with HIV-1, significant activity was noted only with HDP-PPen-C (EC₅₀, 2.5 µM) and HDP-PPen-T (EC₅₀, 1.0 µM). However, there was little antiviral selectivity, as the 50% cytotoxic concentration for these two compounds was 3.3 and 1.3 µM, respectively (data not shown).

We also evaluated the HDP-PPen nucleosides against HBV in 2.2.15 cells (Table 2). As noted with the DNA viruses, the PPen nucleosides were without activity against HBV, but their HDP esters had antiviral activity, with EC₅₀ values ranging from 1.8 to 3.9 µM. Although the HDP esters were more cytotoxic, their selectivity indexes were substantial, ranging from 61 to >417. Neither PPen-U nor its HDP ester showed antiviral activity against HBV.

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TABLE 2. Antiviral activities of PPen and HDP-PPen nucleosides against HBVa

The active compounds were also evaluated against a panel of drug-resistant hepatitis B viruses (Table 3). In transient infections in Huh7 cells, HDP-PPen-A retained full activity against several lamivudine-resistant HBV polymerase mutants (L180M, M204V, M204I, and L180M/M204V), while L180M and the double mutant L180M/M204V were resistant to HDP-PPen-G. The pyrimidine analogs HDP-PPen-C and HDP-PPen-T were resistant to L180M, M204V, M204I, and the LM/MV double mutant. All of the HDP-PPen nucleosides showed activity equal to wild type against an adefovir-resistant mutant (N236T); however, only HDP-PPen-A exhibited full activity against all five of the drug-resistant HBV mutants.

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TABLE 3. Activities of HDP-PPen nucleosides against drug-resistant hepatitis B virus

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nucleoside and nucleotide analogs are two important classes of drugs that selectively target viral polymerases and inhibit viral replication (11, 12). Nucleoside analogs require conversion to the 5' mono-, di-, and finally the triphosphates before incorporation into viral DNA and include compounds such as acyclovir, ganciclovir, stavudine, abacavir, and lamivudine. The acyclic nucleoside phosphonates bypass the initial phosphorylation and are converted to their mono- and diphosphate derivatives by nucleoside kinases (12). Studies by Holy et al. of 9-[2-(phosphonmethoxy)ethyl], 9-(R)-[2-(phosphonmethoxy)propyl, and (S)-9-(3-hydroxy-2-phosphono-methoxy)propyl nucleotide analogs established the potent broad-spectrum antiviral activities available from ANPs and led to three FDA-approved phosphonates: cidofovir (Vistide), adefovir dipivoxil (Hepsera), and tenofovir disoproxil fumarate (Viread) (16). Other groups have focused on the design of phosphonate isosteres in which the 5'-oxygen of an antiviral nucleotide is either removed or replaced by an enzymatically stable methylene group. This strategy led to phosphonate analogs of acyclovir (20), ganciclovir (8), stavudine (24), dideoxynucleosides (34), tetrahydrofuran derivatives (6), cyclopropane nucleosides (30, 39), and anti-HCV nucleosides (25). Acyclic phosphonates incorporating unsaturated acyclosugar side chains have also been evaluated (14, 32).

In our own efforts to identify selective virus inhibitors, we prepared a novel family of nucleotide analogs using a phosphonopentenyl group as the acyclic sugar moiety. The general structure of the new compounds, 5-phosphono-pent-2-en-1-yl nucleosides, is shown in Fig. 1. The phosphonopentenyl side chain design was chosen so that the resulting analogs would resemble the corresponding portion of 2',3'-didehydro-2',3'-dideoxynucleosides, such as stavudine, 2',3'-didehydro-2',3'-dideoxyadenosine, and abacavir, nucleosides with potent anti-HIV activity. Choo et al. (9) found previously that the 2',3' double bond of these unsaturated nucleosides interacts with the aromatic moiety of Tyr115 of HIV-1 reverse transcriptase by hydrophobic interaction. We hypothesized that the unsaturated acyclosugar side chain of the PPen structure might improve binding of the diphosphate to the target viral polymerases. The acyclic PPen side chain should also be more flexible than related cyclic analogs (24), which might increase binding within the polymerase complex and facilitate approach of the -phosphorous to the 3'-OH of the replicating viral DNA.

The PPen nucleosides were evaluated in various antiviral assays, and the results are summarized in Table 1. No inhibitory activity was observed up to 100 µM for PPen-A, -C, -U, -C, or -T, and PPen-G was only weakly active against CMV (68.4 µM) and VZV (7.2 µM). The PPen nucleosides also were not antiproliferative or cytotoxic in MRC-5 or MT-2 cells up to 100 µM (Tables 1 and 2).

We have observed that ANPs frequently fail to exhibit biological activity in vitro because the double negative charge associated with the phosphonate group impairs transport of drug through the cell membrane (1). In earlier work we prepared ANPs modified with lipophilic groups to create mimics of lysophosphatidylcholine, a dietary lipid which is absorbed intact from the gastrointestinal tract and readily metabolized in cells. We showed that cidofovir modified by esterification with a hexadecyloxypropyl group (HDP-CDV) enters cells rapidly and is metabolized to give levels of CDV-diphosphate, the active metabolite, that are 100-fold higher relative to unmodified CDV (1). The in vitro activity of (S)-HPMPA was also enhanced using this strategy (5), and some weakly active ANPs displayed significant activity after lipid modification (33, 35). Esterification with alkoxyalkyl groups also makes ANPs orally bioavailable (10) and orally effective against lethal poxvirus infections (7, 31). This was the rationale for the synthesis of the hexadecyloxypropyl esters of the PPen nucleosides. We evaluated them for antiviral effects against various viruses and found that several now showed significant activity (Table 1). The broadest antiviral activity was found in the guanine analog, HDP-PPen-G, which had EC₅₀s below 12 µM for all viruses except HIV-1 and EBV. HDP-PPen-A was a slightly stronger inhibitor of HBV (EC₅₀, 1.5 µM) and showed substantial activity against EBV (EC₅₀, 0.1 µM). Against HIV-1, only the two pyrimidine analogs HDP-PPen-C and HDP-PPen-T showed significant activity but exhibited no antiviral selectivity. As expected, HDP-PPen-U was inactive in all the antiviral assays (VZV and EBV were not assayed).

Since four of the five HDP-PPen nucleoside compounds inhibited HBV replication and our previous work demonstrated that alkoxyalkyl esters of nucleoside phosphates and phosphonates are targeted to the liver (10, 17, 18), we evaluated the active analogs against a panel of drug-resistant HBV (Table 2). Hepatitis B virus is one of the 10 leading causes of mortality worldwide, and the emergence of strains resistant to the approved drugs, lamivudine and adefovir dipivoxil, is a major clinical concern (29). Resistance to lamivudine emerges rapidly during monotherapy and after 4 years affects approximately 70% of treated patients (28). Adefovir dipivoxil is effective in vitro and in vivo against lamivudine-resistant HBV (40). We found that like adefovir, HDP-PPen-A retains full activity against the lamivudine-resistant HBV polymerase mutants (L180M, M204V, M204I, and L180M/M204V) in transient infections in Huh7 cells. HDP-PPen-G was effective against M204V and M204I, but L180M and the double mutant, L180M/M204V, were resistant. All of the lamivudine-resistant strains exhibited resistance to the pyrimidine PPen analogs HDP-PPen-C and HDP-PPen-T.

Adefovir resistance has been associated with the selection of the mutant N236T, but its emergence is less frequent and delayed relative to lamivudine resistance (3). Interestingly, all of the HDP-PPen nucleosides retained activity equal to the wild type against the adefovir-resistant mutant (N236T). Among the compounds, only HDP-PPen-A retained full activity against all five drug-resistant HBV mutants, suggesting that this nucleotide analog should be evaluated as a salvage therapy in patients infected with drug-resistant HBV strains or used in combination with other drugs to prevent the emergence of resistance.

In summary, the PPen nucleosides are a new class of acyclic nucleoside phosphonates, possessing significant antiviral activity when esterified with the hexadecyloxypropyl group. Further work will be required both to design analogs with improved antiviral activity and to investigate their mechanism of action.

 
The studies were supported by NIH grants AI-66499, AI-64615, and AI-29164 from NIAID and EY-11832 and EY-07366 from the National Eye Institute (K.Y.H.), by NIAID contracts NO1-AI-85347 and NO1-AI-30049 from the Antiviral Research Branch (E.R.K.), and by NIAID contract NO1-AI-45179 (B.A.K.).

 
* Corresponding author. Mailing address: Department of Medicine (0676), University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0676. Phone: (858) 552-8585, ext. 2616. Fax: (858) 534-6133. E-mail: khostetl{at}ucsd.edu.

Published ahead of print on 27 November 2006.

Present address: Life Sciences Division, Korea Institute of Science and Technology, Seoul 136-791, Korea.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, February 2007, p. 611-615, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00444-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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