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JAC Advance Access originally published online on November 22, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):80-86; doi:10.1093/jac/dkl454
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Pronounced in vitro and in vivo antiretroviral activity of 5-substituted 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy] pyrimidines

Jan Balzarini1,*, Dominique Schols1, Kristel Van Laethem1, Erik De Clercq1, Dana Hocková2, Milina Masojidkova2 and Antonin Holy2

1 Rega Institute for Medical Research, K.U.Leuven B-3000 Leuven, Belgium 2 Centre for New Antivirals and Antineoplastics, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic 166 10 Praha, Czech Republic

*Corresponding author. Tel: +32-16-33-73-52; Fax: +32-16-33-73-40; E-mail: jan.balzarini{at}rega.kuleuven.be

Received 29 June 2006; returned 12 September 2006; revised 14 September 2006; accepted 10 October 2006


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Objectives: To discover new potent and selective anti-human immunodeficiency virus (HIV) acyclic nucleoside phosphonate (ANP) drugs with in vivo antiretroviral activity.

Methods: New acyclic pyrimidine nucleoside phosphonate derivatives that mimic the structure of the anti-HIV purine nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA, tenofovir) were designed by linking the acyclic side chain of the ANPs through an ether bond to the C-6 position instead of the N-1 position of the pyrimidine ring. The compounds were evaluated against HIV and Moloney murine sarcoma virus (MSV) in cell culture, including a broad variety of HIV-1 clade clinical isolates and relevant mutant (drug-resistant) HIV-1 isolates. Their antiviral activities were correlated and investigated in an in vivo model consisting of MSV-infected newborn mice. MSV-induced tumour formation and associated death were recorded in drug-treated animals.

Results: Several 5-substituted 6-[2-(phosphonomethoxy)ethoxy]-2,4-diaminopyrimidine (PMEO-DAPy) analogues were found to inhibit a broad variety of HIV-1 clinical isolates. They showed a more favourable cross-resistance profile to mutant virus isolates than adefovir and tenofovir. There was a close correlation between inhibition of MSV in C3H/3T3 cells and inhibition of HIV-1 in CEM cells. The PMEO-DAPy derivatives potently inhibited MSV-induced tumour cell formation in newborn mice. The 5-methyl analogue PMEO-5-Me-DAPy proved markedly more inhibitory to MSV-induced tumour cell formation and associated animal death than its unsubstituted parent PMEO-DAPy derivative. When compared with adefovir, PMEO-5-Me-DAPy was less toxic and more antivirally active in MSV-infected mice.

Conclusions: PMEO-5-Me-DAPy deserves further (pre)clinical investigations as a candidate anti-HIV drug.

Keywords: HIV , MSV , acyclic nucleoside phosphonates , ANP , antiretroviral drugs , PMEO-DAPy


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There are currently three acyclic nucleoside phosphonate (ANP) drugs approved for clinical use against cytomegalovirus [cidofovir or (S)HPMPC], hepatitis B virus (adefovir or PMEA) and human immunodeficiency virus (HIV) [tenofovir or (R)PMPA] (the latter two compounds in their oral prodrug forms, adefovir dipivoxil and tenofovir disoproxil fumarate).1 In these drugs, the aliphatic 3-hydroxy-2-phosphonomethoxypropyl (HPMP), 2-phosphonomethoxyethyl (PME) and 2-phosphonomethoxypropyl (PMP) side chain is linked to the N-1 atom of cytosine (C) [as in (S)HPMPC] or the N-9 atom of adenine (A) [as in PMEA and (R)PMPA]. More recently, a new class of ANP analogues has been designed where the base moiety is a pyrimidine such as 2,4-diaminopyrimidine (DAPy) linked to the aliphatic phosphonate via an ether linkage through an oxygen atom at the C-6 position of the pyrimidine ring (Figure 1). Such compounds (designated HPMPO-DAPy, PMEO-DAPy and PMPO-DAPy, depending on the nature of the acyclic alkyl phosphonate side chain) have proven to possess an antiviral activity spectrum that closely resembles that of the corresponding (S)HPMP-, PME- and (R)PMP-adenine derivatives.2–4 The PMEO and (R)PMPO derivatives represent the first pyrimidine ANPs in the PME and PMP series of ANPs that show pronounced antiviral activity, including anti-HIV and anti-hepatitis B virus (HBV) activity. Interestingly, these novel pyrimidine ANPs may mimic an incomplete purine ring due to the fact that the aliphatic phosphonate side chain is linked to the C-6 position (instead of the N-1 position) through an ether linkage (Figure 1). The premise that the 2,4-diaminopyrimidine in the PMEO series of compounds should be considered as an open-ring analogue of the 2,6-diaminopurine in the PME series has been confirmed through molecular modelling.5 Compounds that contain a substituent at the C-5 position of the pyrimidine base may even better mimic the purine skeleton. Therefore, a series of 5-substituted PMEO-pyrimidine derivatives, including the methyl, formyl, cyano, chloro and bromo analogues, were synthesized6,7 and evaluated for their antiretroviral activity in cell culture.


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  		Figure 1. Structural formulae of the 5-substituted PMEO-DAPy analogues and the reference compounds adefovir and tenofovir.

 

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Antiretroviral activity of test compounds in cell culture

Human lymphocyte CEM cell cultures (2–3 x 105 cells/mL) were seeded in 200 µL wells of 96-well microtitre plates in the presence of a series of compound concentrations (5-fold dilutions) in cell culture medium, and infected with ~100 CCID50 HIV-1(IIIB) or HIV-2 (ROD). After 4 days, the inhibition of HIV-induced giant cell formation was microscopically recorded. To determine the inhibitory effect of the test compounds against murine sarcoma virus (MSV)-induced transformation of murine fibroblast C3H/3T3 cells, the cells were seeded in 1 mL wells of 48-well plates and grown to confluency prior to infection with 80 focus forming units/well in the presence of several (5-fold) dilutions of the test compounds. After 6 days, the percentage of transformed cells in the infected cell cultures was microscopically recorded.

The antiviral activity of the PMEO derivatives against a variety of different clinical HIV-1 clade isolates and laboratory strains in peripheral blood mononuclear cells (PBMC) cultures were also examined as follows. Primary clinical isolates representing different HIV-1 clades and an HIV-2 isolate were all kindly provided by Dr L. Lathey from BBI Biotech Research Laboratories, Inc., Gaithersburg, MD, and their co-receptor use (R5 or X4) was determined by us on the astroglioma U87.CD4 cell line transfected with either CCR5 or CXCR4. The following clinical isolates were included in the study: UG273 (clade A, R5), US2 (clade B, R5), ETH2220 (clade C, R5), UG270 (clade D, X4), ID12 (clade A/E, R5), BZ163 (clade F, R5), BCF-DIOUM (clade G, R5), BCF06 (group O, X4) and HIV-2 BV-5061W (X4). Antiviral testing of these isolates in PBMC was as follows: PBMC from healthy donors were isolated by density gradient centrifugation and stimulated with phytohaemagglutinin (PHA) at 2 µg/mL (Sigma, Bornem, Belgium) for 3 days at 37°C. The PHA-stimulated blasts were washed twice with PBS and counted after staining with Trypan Blue. The cells were then seeded at 0.5 x 106 cells per well into a 48-well plate containing varying concentrations of compound in cell culture medium (RPMI 1640) containing 10% fetal calf serum (FCS) and interleukin-2 (25 U/mL, R&D Systems Europe, Abingdon, UK). The virus stocks were diluted in medium and added at a final dose of 250 pg of p24 or p27/mL as determined by a viral core antigen (Ag)-specific ELISA. Cell supernatant was collected at day 12 and HIV-1 core Ag in the culture supernatant was analysed by a p24 Ag ELISA kit (Perkin Elmer, Zaventem, Belgium).

Several clinical HIV-1 isolates (a gift from Kristel Van Laethem, Rega Institute for Medical Research)8,9 that contain reverse transcriptase inhibitor (RTI)-related mutations were investigated for their sensitivity to the test compounds in CEM cell cultures as described above. The mutations in the RT of the clinical isolates are indicated in the footnote to Table 3.

In vivo antiretroviral activity of ANPs

The inhibitory effects of the 5-substituted PMEO derivatives on the initiation of MSV-induced tumour formation and associated death of the MSV-infected mice were evaluated by subcutaneously injecting MSV in the left hind leg of 2- to 3-day-old NMRI mice, and treating the mice intraperitoneally with different doses of the test compounds for 5 subsequent days starting 4 h prior to virus inoculation. Adefovir and tenofovir were included for comparative reasons. The time of appearance of visible MSV-induced tumour formation and animal death were used as parameters to estimate the antiviral potential of the test compounds.

We adhered to ethical standards for manipulating the animals. An approval from the Ethics Committee of the K.U.Leuven has been obtained.


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Inhibitory activity of 5-substituted PMEO-DAPy derivatives against retroviruses in cell culture

Five 5-substituted PMEO-pyrimidine derivatives have been evaluated against laboratory HIV-1(IIIB) and HIV-2(ROD) strains in CEM cell cultures, and against a variety of clinical HIV-1 clade isolates in PBMC cultures (Tables 1 and 2). Whereas most of the 5-substituted PMEO derivatives showed comparable antiviral activity against HIV-1 and HIV-2 in CEM cell cultures when compared with the parent PMEO-DAPy and adefovir, and (R)PMPO-DAPy and tenofovir, PMEO-5-Me-DAPy was one order of magnitude more inhibitory against HIV-1 and HIV-2 than all other 5-substituted PMEO-DAPy derivatives evaluated (Table 1). Its anti-HIV activity (EC50) was as low as 0.06 µg/mL. Also, PMEO-5-Me-DAPy was up to 4-fold more inhibitory than PMEO-DAPy and 4- to 10-fold more inhibitory than adefovir against a wide variety of clinical HIV-1 clade isolates in PBMC cultures (Table 2). The EC50s were in the range of 0.04–0.6 µg/mL and the EC90 values were generally 2- to 3-fold higher. In this respect, the PMEO-5-Me-DAPy derivative proved to be among the most potent anti-HIV ANP ever reported in cell cultures and in freshly prepared PBMC cultures.


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  		Table 1. Antiretroviral activity of test compounds in cell culture

 


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  		Table 2. Inhibitory activity of test compounds against a variety of HIV-1 clade isolates in PBMC cultures

 
Interestingly, the PMEO derivatives were also markedly inhibitory against MSV-induced C3H/3T3 cell transformation. Again, PMEO-5-Me-DAPy, but also (R)PMPO-DAPy, was ~4- to 130-fold more inhibitory to MSV than the parent PMEO-DAPy, and the other 5-substituted PMEO-DAPy derivatives (Table 1). In fact, there was a close correlation between the anti-HIV activity of the PMEO pyrimidine derivatives and their anti-MSV activity in cell culture (r = 0.86). None of the 5-substituted PMEO-pyrimidines were markedly toxic in cell culture at 100 µg/mL, except PMEO-5-Me-DAPy that showed a CC50 of 3.4 µg/mL, which was comparable to the CC50 of parental PMEO-DAPy, but 5- and 37-fold lower than noted for adefovir and tenofovir in CEM cell cultures. As a consequence, PMEO-5-Me-DAPy is 4- to 20-fold more selective than parental PMEO-DAPy against HIV-1, HIV-2 or MSV in cell culture.

Inhibitory activity of 5-substituted PMEO-DAPy derivatives against drug-resistant clinical HIV-1 isolates

The test compounds have also been investigated for their inhibitory activity against a number of clinical virus isolates that had emerged in drug-treated HIV-1-infected individuals. The isolates display patterns of reverse transcriptase (RT) mutations that are known to be associated with resistance to all clinically approved nucleoside and nucleotide RT inhibitors (indicated in the footnote to Table 3). The selected virus strains contain either thymidine analogue-associated mutations (TAMs) with and without the lamivudine-resistance mutation M184V (HIV-1/C20 and HIV-1/C19, respectively), or multi-nucleoside reverse transcriptase inhibitor (NRTI) resistance (MNR) mutations (HIV-1/L6.5) in association with the adefovir/tenofovir-characteristic K65R (HIV-1/DE434.4) or the lamivudine-characteristic M184V (HIV-1/HA20.17) mutation.


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  		Table 3. Antiviral activity of test compounds against a variety of drug-resistant HIV-1 isolates

 
Whereas adefovir and tenofovir showed a 17- to 30-fold decreased activity against HIV-1/C19 and HIV-1/C20, PMEO-DAPy and PMEO-5-Me-DApy lost only 4- to 6-fold and 9- to 16-fold antiviral activity, respectively.

Also, the MNR virus strains kept more pronounced sensitivity to the inhibitory effects of the PMEO derivatives than to adefovir and tenofovir. The HIV-1/L6.5 and HIV-1/HA20.17 strains kept pronounced sensitivity to the PMEO derivatives (at most a loss of activity that was 1.7- to 5-fold) but a 16- to 40-fold loss of sensitivity to adefovir and tenofovir. Particularly striking was the >50- to 100-fold loss of activity of adefovir/tenofovir against the K65R-containing MNR isolate (HIV-1/DE434.4) whereas only a 7- to 16-fold decreased inhibitory activity was noticed for the PMEO derivatives (Table 3).

Inhibitory activity of PMEO-DAPy derivatives against MSV-induced tumour formation in newborn NMRI mice

Since adefovir and tenofovir have been proven inhibitory to MSV-induced tumour cell formation in newborn NMRI mice, we evaluated the antiretroviral potential of the 5-substituted PMEO-DAPy derivatives in the MSV/NMRI mouse model and compared their antiviral potential with that obtained for the parental PMEO-DAPy, adefovir and tenofovir (Table 4). The drug doses were 50, 20, 8, 5 or 2 mg/kg/day. The prototype drugs adefovir and tenofovir and the unsubstituted parent PMEO-DAPy compound were included for comparative purposes. In the control mice that received only vehicle (placebo), 50% of the mice developed a tumour after 4–5 days (4.88 ± 0.66) (visible tumour formation in all mice after 6 days) and 50% of the mice died at ~12 days (11.9 ± 1.74) after initiation of the experiment (all mice died within 13 days) (Table 4). None of the mice was tumour-free when treated with PMEO-5-CN-DAPy (all doses), and only 5% of the mice did not develop a tumour within 20 days post virus inoculation upon the administration of PMEO-5-Cl-DAPy at 20 mg/kg. However, initiation of tumour formation was significantly and dose-dependently delayed when compared with control mice (Table 4). When administering PMEO-5-Me-DAPy at 50 mg/kg/day, none of the mice developed any sign of tumour formation before toxicity-related death (days 9–12). Also, none of the MSV-infected mice developed tumours upon treatment with PMEO-5-Me-DAPy at a dose of 20 mg/kg, and 90% of the animals remained tumour-free if treated with the drug at 5 mg/kg. The unsubstituted parent PMEO-DAPy derivative was able to suppress tumour formation in 84%, 80% and 44% of the mice that were exposed to 50, 20 and 8 mg/kg of drug, respectively. Also, adefovir was inferior to PMEO-5-Me-DAPy since at 20 mg/kg tumours developed in 11% of the mice (0% under PMEO-5-Me-DAPy treatment) and 35–80% at 5–8 mg/kg (10% under PMEO-5-Me-DAPy treatment). In this respect, PMEO-5-Me-DAPy was also superior to tenofovir (Table 4).


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  		Table 4. Inhibitory activity of test compounds against MSV-induced tumour cell formation in newborn NMRI mice

 
The mean day of death of the MSV-exposed animals was also significantly delayed for all PMEO derivatives tested (Table 4). Again, PMEO-5-Me-DAPy proved to be the most potent drug resulting in long-term (20 day) survivors of the experiment at 20 and 5 mg/kg. It should be noted that at 50 mg/kg/day, PMEO-5-Me-DAPy caused premature death of the animals after day 11. Under similar experimental conditions, all animals died after day 9 upon treatment with adefovir at 50 mg/kg. Such animal toxicity at the highest dose tested (50 mg/kg) was not seen with any of the other compounds, including the parent drug PMEO-DAPy, the 5-CN- and 5-Cl-substituted PMEA-DAPy derivatives and tenofovir.


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There are currently 20 clinically approved drugs for treatment of HIV-infected individuals, among them, seven NRTIs and tenofovir, the first nucleotide reverse transcriptase inhibitor (NtRTI). The latter drug differs from the NRTIs in that the intact dideoxyribose sugar ring is replaced by an aliphatic side chain at which a phosphonate moiety is linked. In this respect, the NtRTI tenofovir mimics a nucleoside 5'-monophosphate derivative and circumvents the first obligatory phosphorylation step of NRTIs which is often the bottleneck in the activation of NRTIs.

Interestingly, the PMEO-DAPy compounds contain a pyrimidine base (2,4-diaminopyrimidine, DAPy) mimicking an incomplete purine ring, and therefore, may mimic the corresponding PME-2,6-diaminopurine (PMEDAP) derivative which is a potent anti-HIV compound.10,11 In this respect, the PMEO-DAPy derivatives represent the very first pyrimidine ANPs that are endowed with antiretroviral activity in cell culture.2,3 The structure of PMEO-5-Me-DAPy entirely fits in the purine ring mimic, in that the methyl group may occupy the space of the N-7 position in purines and therefore, may resemble the intact purine ring even more closely than the parental PMEO-DAPy. Since PMEO-5-Me-DAPy shows the same toxicity profile as the parental PMEO-DAPy but proved more inhibitory against HIV and MSV in cell culture, it is clearly more selective an antiretroviral agent than PMEO-DAPy. It is, however, somewhat intriguing why the 5-methyl-substituted PMEO-DAPy derivative kept similar cytostatic activity as the unsubstituted parent PMEO-DAPy (3.4–3.9 µg/mL) whereas the other 5-substituted PMEO derivatives were devoid of marked toxicity (CC50 ≥ 100 µg/mL). It may not be co-incidence that the non-toxic compounds invariably contain an electron-withdrawing substituent at the C-5 position (i.e. Cl, Br, CN, CHO), whereas the 5-methyl group in PMEO-5-Me-DAPy has opposite (electron-donating) properties. Most likely, the electronic properties of the 5-substituent may determine differences in the affinity of the compounds against either their phosphorylating enzymes and/or their eventual antiviral target (i.e. HIV reverse transcriptase).

The MSV/mouse model has for a long time been considered as a relevant (small) animal model to evaluate ANPs in vivo.12,13 Adefovir and tenofovir are exquisitely active in this animal model, and also the potential of ANPs to be effectively administered to the MSV-infected animals by infrequent dosing was initially discovered and demonstrated in this animal model.14 These properties of the ANPs have led to the development of a once daily pill of tenofovir for the treatment of HIV-infected individuals. Since this small animal model has been shown to be useful to study ANPs in the in vivo setting and requires small amounts of available experimental drugs, we decided to compare the in vivo antiretroviral properties of novel 5-substituted PMEO-DAPy derivatives with those of the parental PMEO-DAPy and the clinically established adefovir and tenofovir. From these investigations, it turned out that particularly the 5-methyl-substituted PMEO-DAPy derivative proved exquisitely inhibitory, both in cell culture and in vivo. With the exception of tenofovir, the observed pronounced in vivo antiretroviral potency of this compound has been unprecedented for the ANPs. Moreover, the demonstration of its superior suppression of a wide variety of clinical HIV-1 clade isolates (Table 2), shows that PMEO-5-Me-DAPy may hold interesting antiviral promise to justify further preclinical investigations. However, it should be kept in mind that ANPs, in particular adefovir, have proven to be increasingly toxic for the animals (mice) at younger ages. For example, adefovir is significantly embryotoxic (10–40 mg/kg) in pregnant mice,15 moderately toxic (50 mg/kg) in newborn mice [ref. (12) and Table 4] and virtually non-toxic (100 mg/kg) in adult mice.13 Whether this is also the case for PMEO-5-Me-DAPy needs further investigation.

It was interesting to observe that the PMEO derivatives generally retained more of their antiviral activity against several drug-resistant clinical virus isolates than adefovir and tenofovir. Indeed, in comparison with adefovir and tenofovir, the PMEO derivatives retained higher antiviral activities against the MNR clinical isolates included in this study. In general, the highest levels of resistance were observed with the mutant virus strain that contained the MNR complex in association with K65R. K65R is known to be an important resistance mutation towards tenofovir16,17 and this might also explain the higher levels of resistance towards the new ANPs, although at a lesser extent than being the case for adefovir and tenofovir. The virus strain carrying M41L, L210W and T215Y in its RT showed also a reduced susceptibility towards the PMEO compounds, but again at lower fold-resistance values than observed for adefovir and tenofovir. A similar phenomenon was found for the TAM-containing HIV-1 isolates that are generally associated with a reduced phenotypic susceptibility and a reduced clinical response to not only thymidine analogues, but to all NRTIs. The level of drug resistance is dependent on the number of TAMs and the extent is known to differ depending on the nature of the drug. In this respect, the antiviral activities of the PMEO and PMPO derivatives are clearly less compromised than those of adefovir and tenofovir in the presence of TAMs. Our observations add to the potential clinical value of this novel subclass of ANPs, and warrant further in-depth investigations on this group of test compounds. Nevertheless, our results confirm the importance of the tenofovir-characteristic key mutation K65R in the decreased inhibitory activity of the ANPs, including the PMEO-subclass of drugs, albeit for the latter compounds at a lower extent than the parental adefovir and tenofovir. It is now imperative to perform drug resistance selection experiments in cell culture with the PMEO and PMPO derivatives to reveal the speed of drug resistance development and to determine the nature of the mutations that may appear in the reverse transcriptase of such drug-resistant virus strains.

In conclusion, the 5-substituted PMEO-DAPy derivatives consistently suppress a variety of clinical HIV-1 clade isolates in cell culture and retained more of their activity against MNR and TAM-containing HIV-1 isolates than adefovir and tenofovir. In vivo antiretroviral data indicate that among the PMEO-DAPy derivatives the 5-methyl-substituted PMEO-DAPy derivative was at least as efficient (compared with adefovir), if not markedly more potent (compared with PMEO-DAPy) an antiretroviral agent in MSV-infected mice. These observations warrant further in vivo antiretroviral efficacy studies for the PMEO derivatives, in particular PMEO-5-Me-DAPy, and should stimulate the design and synthesis of additional 5-substituted PMEO-DAPy derivatives. Given the fact that adefovir and tenofovir are effective chemotherapeutics in HBV- and HIV-infected patients, 5-substituted (R)PMPO-DAPy analogues, similar to the PMEO-DAPy derivatives, should also be prepared and compared with tenofovir to explore their anti-HIV and anti-HBV potential in both the in vitro and in vivo setting.


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None to declare.


   Acknowledgements
 
The excellent technical assistance of Mrs Béla Nováková, Mr Willy Zeegers, Mrs Lizette van Berckelaer, Mrs Sandra Claes and Mrs Ann Absillis is gratefully acknowledged. We thank Mrs Christiane Callebaut and Mrs Inge Aerts for dedicated editorial help. This study was performed as part of a research project of the Institute of Organic Chemistry and Biochemistry #Z 055 0506 and of the Centre of New Antivirals and Antineoplastics 1M6136896301, by the programmes of targeted projects of the Academy of Sciences of the Czech Republic (#S4055109, 1QS400550501), by Gilead Sciences (Foster City, CA, USA), the Fifth Framework Programme of the European Commission, ISEP/FORTIS, and the Flemish ‘Geconcerteerde Onderzoeksacties’ Contract GOA/2005/19.


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1 De Clercq E. (2003) Clinical potential of the acyclic nucleoside phosphonates cidofovir, adefovir and tenofovir in treatment of DNA virus and retrovirus infections. Clin Microbiol Rev 16:569–96.[Abstract/Free Full Text]

2 Holy A, Votruba I, Masojídková M, et al. (2002) 6-[2-(Phosphonomethoxy) alkoxy]pyrimidines with antiviral activity. J Med Chem 45:1918–29.[CrossRef][Web of Science][Medline]

3 Balzarini J, Pannecouque C, De Clercq E, et al. (2002) Antiretrovirus activity of a novel class of acyclic pyrimidine nucleoside phosphonates. Antimicrob Agents Chemother 46:2185–93.[Abstract/Free Full Text]

4 De Clercq E, Andrei G, Balzarini J, et al. (2005) Antiviral potential of a new generation of acyclic nucleoside phosphonates, the 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidines. Nucleosides Nucleotides Nucleic Acids 24:331–41.[CrossRef][Web of Science][Medline]

5 Ying C, Holy A, Hocková D, et al. (2005) Novel acyclic nucleoside phosphonate analogues with potent anti-hepatitis B virus activities. Antimicrob Agents Chemother 49:1177–80.[Abstract/Free Full Text]

6 Hocková D, Holy A, Masojídková M, et al. (2003) 5-Substituted-2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines-acyclic nucleoside phosphonate analogues with antiviral activity. J Med Chem 46:5064–73.[Web of Science][Medline]

7 Hocková D, Holy A, Masojídková M, et al. (2004) Synthesis and antiviral activity of 2,4-diamino-5-cyano-6-[2-(phosphonomethoxy)ethoxy]pyrimidine and related compounds. Bioorg Med Chem 12:3197–202.[CrossRef][Medline]

8 Schmit J-C, Martinez-Picado J, Ruiz L, et al. (1998) Evolution of HIV drug resistance in zidovudine/zalcitabine- and zidovudine/didanosine-experienced patients receiving lamivudine-containing combination therapy. Antivir Ther 3:81–8.

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10 Pauwels R, Balzarini J, Schols D, et al. (1988) Phosphonylmethoxyethyl purine derivatives, a new class of anti-human immunodeficiency virus agents. Antimicrob Agents Chemother 32:1025–30.[Abstract/Free Full Text]

11 Naesens L, Balzarini J, Rosenberg I, et al. (1989) 9-(2-Phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP): a novel agent with anti-human immunodeficiency virus activity in vitro and potent anti-Moloney murine sarcoma virus activity in vivo. Eur J Clin Microbiol Infect Dis 8:1043–7.[CrossRef][Web of Science][Medline]

12 Balzarini J, Naesens L, Herdewijn P, et al. (1989) Marked in vivo antiretrovirus activity of 9-(2-phosphonylmethoxyethyl)adenine, a selective anti-human immunodeficiency virus agent. Proc Natl Acad Sci USA 86:332–6.[Abstract/Free Full Text]

13 Naesens L, Balzarini J, Bischofberger N, et al. (1996) Antiretroviral activity and pharmacokinetics in mice of oral bis(pivaloylmethyl)-9-(2-phosphonylmethoxyethyl)adenine, the bis(pivaloyloxymethyl) ester prodrug of 9-(2-phosphonylmethoxyethyl)adenine. Antimicrob Agents Chemother 40:22–8.[Abstract]

14 Balzarini J, Naesens L, De Clercq E. (1990) Anti-retrovirus activity of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in vivo increases when it is less frequently administered. Int J Cancer 46:337–40.[Web of Science][Medline]

15 Lee JS, Mullaney S, Bronson R, et al. (1991) Transplacental antiretroviral therapy with 9-(2-phosphonylmethoxyethyl)adenine is embryotoxic in transgenic mice. J Acquir Immune Defic Syndr 4:833–8.

16 Kagan RM, Merigan TC, Winters MA, et al. (2004) Increasing prevalence of HIV-1 reverse transcriptase mutation K65R correlates with tenofovir utilization. Antivir Ther 9:827–8.[Web of Science][Medline]

17 Leon A, Mallolas J, Martinez E, et al. (2005) High rate of virological failure in maintenance antiretroviral therapy with didanosine and tenofovir. AIDS 19:1695–7.[Web of Science][Medline]


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